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https://ieeexplore.ieee.org/ielx7/6287639/8948470/09201377.pdf
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Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and its Control Strategy
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Received August 10, 2020, accepted August 25, 2020, date of publication September 21, 2020, date of current version October 1, 2020.
Digital Object Identifier 10.1109/ACCESS.2020.3025343
Three-Phase Flexible Transformer Based on
Bipolar Direct AC/AC Chopper and
Its Control Strategy
YIBO WANG , (Member, IEEE), GUO-WEI CAI , CHUANG LIU , (Member, IEEE),
BINGDA ZHU , DONGBO GUO , AND HANWEN ZHANG
Northeast Electric Power University, Jilin 132012, China
Corresponding authors: Yibo Wang (469682939@qq.com) and Chuang Liu (victorliuchuang@163.com)
This work was supported by the National Natural Science Foundation of China under Grant 51877035.
ABSTRACT Dynamic states (e.g., voltage sags and swells) in the power grid can cause faults and defects
in sensitive loads and in consequence financial losses. This paper deals with a flexible transformer based
on bipolar direct AC/AC chopper (BD-AC). It is a combination of BD-AC and traditional power frequency
transformer, and it can realize the flexible regulation of voltage amplitude and phase angle respectively
according to voltage requirements. Firstly, the topology and modulation strategy of the selected BD-AC
are analyzed. On this basis, the flexible transformer system topology based on BD-AC and transformer is
proposed. Then, based on the structure of flexible transformer system, its working principle is analyzed in
detail, and the regulation range of voltage amplitude and phase angle are quantitatively studied. Furthermore,
considering the principle of voltage regulation, a triangle angle theory based control strategy is proposed.
Finally, the correctness and effectiveness of the theoretical analysis of the proposed flexible transformer
system are carried out by detailed computer simulation.
INDEX TERMS Flexible transformer, flexible ratio, voltage amplitude control, voltage phase angle control,
bipolar direct AC-AC chopper (BD-AC).
I. INTRODUCTION
With the rapid development of social economy, the
electrification of daily life is growing and the demand for
power quality of the end-users is also increasing [1], [2].
The improvement of power quality can not only reduce the
adverse impact on sensitive load, ensure the normal quality
of industrial products and scientific experiments, but also
play an important role in the safe and economic operation of
power grid and reduce energy losses [3], [4]. Voltage quality
is one of the basic indicators for power quality [5]–[7] and
it has a great influence on the normal operation of electrical
equipment, e.g., the voltage sags will increase the winding
current of the motor, reduce the efficiency and service life of
the motor, and make sensitive electronic equipment unable to
work normally, etc., and the voltage swells will accelerate the
aging of the insulation of electrical equipment, increase the
loss of electric energy and the cost of electricity. Therefore,
The associate editor coordinating the review of this manuscript and
approving it for publication was Alexander Micallef
173336
.
the parameters of voltage, its quality, are very important from
both the viewpoint of the power grid and end-users [8]–[10].
At present, the reasons affecting the voltage quality are
various, and the factors caused are more complicated, e.g.,
dynamic loads, switching effects, uncontrolled power flow,
faults/accidents and adverse weather conditions. In order to
mitigate the adverse effects of the above reasons on the
voltage quality, there are two paths leading to the protection
of sensitive loads against voltage perturbation and the reduction of energy losses [11], i.e., to improve the ride-through
capability of electrical devices during voltage perturbation
and install voltage stabilizers to reduce voltage fluctuation.
Both of the means should be developed simultaneously and
independently. For the latter, the ac voltage stabilizers based
on mechanical or solid-state on-load tap-changers (OLTC) is
of such kind of equipment [12], [13]. The OLTC realizes the
voltage regulation of the power grid through mechanical or
solid-state tap in static conditions. Besides, with the development of power electronic technology in recent years, there
are various types of hybrid AC compensators used in modern
This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/
VOLUME 8, 2020
Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
AC network to alleviate voltage disturbance. And a common
design method of such devices is to combine the characteristics of traditional transformer and power electronic converter.
The advantages of this design are that the transformer has the
superiority of electromagnetic coupling - galvanic separation,
and the power electronic converter has good dynamic performance and can realize the dynamic regulation of voltage.
There are numerous different solutions and topologies of
voltage stabilizers, also called hybrid AC voltage compensators. The research of hybrid AC voltage compensators
is mainly focused on installations that replace transformer
in distribution network, i.e., shaping flexible transformer.
In [14], a smart distribution transformer based on bidirectional power electronic switches is proposed, and it
has a step-wise characteristics and narrow control range.
A hybrid transformer based on matrix converter is presented
in [15]–[17], and its structure of the proposed solutions needs
a more advanced control system, which increases complexity
and costs and decreases reliability. Moreover, because of the
untypical transformer voltage ratio, the hybrid transformer
cannot operate after system fault occurs [15], and the voltage
control range is limited to ±20% [16]. Additionally, in [18],
the voltage regulation range can be increased to ±50%, however as, the proposed solution cannot operate after a fault in
the power electronic unit, either. Furthermore, the gain of the
solution proposed in [18] has non-linear characteristic and
dependent on matching conditions. Besides, solid state transformer (SST) is another strongly developed group of hybrid
AC voltage compensators [19]–[21], which is constituted by
AC/DC, DC/DC, DC/AC converters and medium frequency
transformer. However, as with [15], [17] and [18], SST cannot
operate with power electronic module damaged, thus, the
reliability of SST is reduced. And even if the reliability is
improved by increasing system redundancy, the complexity
of the whole system will be improved [22], [23]. Additionally,
because of the electric energy from the power supply to the
power load is transferred through the power electronic unit,
the rated power of converters must be equal to the power of
the load, and in view of the structural characteristics of SST,
energy transfer is multi-level, which increases the energy cost
of the whole system [22].
A solution based on bipolar direct AC/AC choppers
(BD-AC) is proposed to build a flexible transformer in this
paper, which can improve voltage parameters in the AC
distribution system. The presented flexible transformer is a
combination of BD-AC and transformer, and it can realize
the flexible regulation of voltage amplitude and phase angle
respectively according to voltage requirements. Furthermore,
with the three-phase topology, voltage of each phase can be
flexibly adjusted by means of other two phases voltage. Thus,
yielding the possibility to control the RMS value and phase
shift of output voltages. Moreover, unlike the solutions presented above, the proposed flexible transformer can operate
after a fault in the electronic unit. In addition, it has a simple
control strategy, a wider range and a continuously variable
voltage control character. On the other hand, one point needs
VOLUME 8, 2020
to be emphasized that the power in distribution network has
the magnitude of MW and the BD-AC for medium voltage
and high power scenarios is difficult to realize in engineering
with the present state of the IGBT art. The main purpose of
this paper is not considered matching the power rating of the
proposed solution with the transmission line parameters in
large-scale power systems. Instead, this article is a demonstration of a forward-looking solution.
This paper is organized as follows: in section II, the topology and PWM modulation principle of the BD-AC is
described; the structure, operation principle and control strategy of the BD-AC based flexible transformer system are
proposed in section III; in section IV and section V, some
simulation results are presented; finally, in section VI, conclusions are provided.
II. TOPOLOGY AND PWM MODULATION PRINCIPLE
OF THE BD-AC
Non-differential AC/AC chopper (ND-AC) shown in
Fig. 1 (a) is currently used, and it consists of four insulated
gate bipolar transistors (IGBTs) and a capacitor (C) used
to absorb the energy stored in the stray inductance of the
line [24]. Fig. 2 (a) and (c) are the modulation principles
and switching signals corresponding to the ND-AC. It can
be seen that the control of the ND-AC is similar to the one
used for a DC/DC buck converter with the main difference
that it takes into account the sign of the input voltage (vin ).
Thus, if vin is positive, the S1 and S1C power devices follow
a complementary PWM pattern, while the others are turned
on. Similarly, if vin is negative, the S2 and S2C power devices
follow a complementary PWM pattern, while the others are
turned on. The advantage of this control is that the current
path is always available regardless of the direction of inductance current. Since each IGBT has only half a cycle of PWM
control, the total switching loss is reduced. On the other hand,
according to the working principle of ND-AC, the polarity of
vin is the same as that of the output voltage (vout ), i.e., the
ND-AC has unipolar output characteristics. Then, in order
to obtain voltage bipolar output, the BD-AC is proposed
in [25], and the bipolar means that the vout of the AC/AC
chopper could be ‘in-phase’ or in ‘anti-phase’ in relation to
the vin . The topology and principle of PWM modulation of the
BD-AC are shown in Fig. 1 (b) and Fig. 2.
As can be seen from Fig. 1 (b), the BD-AC consists of
two ND-AC parallel connection, thus forming H bridge structure. For convenience of description, the two ND-AC used
are defined as P-Leg (Positive Leg) and N-Leg (Negative
Leg) respectively. With the connection type, the common
ground between the input and output ports of the two legs are
retained. Moreover, the BD-AC achieves the output of continuously controllable bipolar voltage. In addition, due to the
common sharing ground of the input and output, the feature
that output can reverse or maintain phase angle with input is
supported well.
According to the principle of PWM modulation shown
in Fig. 2, the relationship between vout and vin is shown in
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Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
FIGURE 1. Topology of ND-AC and BD-AC.
FIGURE 2. Modulation strategy of BD-AC.
FIGURE 3. BD-AC based three-phase flexible transformer.
equation (1).
vout = (d1 − d2 ) · vin = D · vin
(1)
where d1 and d2 are defined as the time interval when
switches are turned on during one switching period of P-Leg
and N-Leg, respectively. Obviously, the range of values of
d1 and d2 is [0, 1]. D is defined as modulation ratio of BD-AC,
and its value is within the range of [−1, 1].
III. BD-AC BASED FLEXIBLE TRANSFORMER SYSTEM
A. TOPOLOGY OF THE FLEXIBLE TRANSFORMER SYSTEM
Considering the characteristics of BD-AC, the topology of
flexible transformer system based on BD-AC is proposed,
which is shown in Fig. 3.
As can be seen from Fig. 3, the topology of the proposed
flexible transformer system is composed of transformer,
BD-AC modules (AC/AC-A, AC/AC-B, AC/AC-C) and its
control system. The primary side of transformer consists
of three windings: WA , WB and WC . The secondary side
consists of two parts: main winding (Wa1 , Wb1 and Wc1 ) and
auxiliary winding (Wa2 , Wb2 and Wc2 ). Need to be pointed
out that the three-phase auxiliary windings (Wa2 , Wb2 and
173338
Wc2 ) in Fig. 3 are redrawn for ease of representation. From
the connection relation of the electric power devices, the main
parts of the presented flexible transformer system are BD-AC
modules operating in each phase. The AC/AC-A is operating
on A-phase, however, it is supplied from two other phases
(B-phase and C-phase). Analogously, B-phase and C-phase
have the same structure. Each BD-AC module consists of
two BD-AC, and the outputs of BD-AC are connected to the
main winding (Wa1 , Wb1 and Wc1 ) respectively. That means
A-phase, B-phase and C-phase have mutual support ability.
Overall, the secondary main windings (Wa , Wb , Wc ) of transformer and the BD-AC modules constitute a series system.
Furthermore, in order to improve the reliability of the whole
system, bypass switches (Sbp1 , Sbp2 , Sbp3 and Sbp−a , Sbp−b ,
Sbp−c ) are installed at the input and output ports of the BD-AC
modules respectively. Similar to other high power and high
voltage applications of bypass switches, in normal working
condition, Sbp1, Sbp2 , Sbp3 are in closed state, Sbp−a , Sbp−b ,
Sbp−c are in open state. On the contrary, in overhaul/fault
state, Sbp1 , Sbp2 , Sbp3 are in open state, on the contrary, Sbp−a ,
Sbp−b , Sbp−c are in closed state.
VOLUME 8, 2020
Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
B. OPERATION PRINCIPLE OF THE FLEXIBLE
TRANSFORMER SYSTEM
Based on the analysis of flexible transformer topology,
the operation principle of flexible transformer system is analyzed as follows. From the structure of flexible transformer
system, it can be seen that the output voltage of flexible
transformer system (v̇La , v̇Lb , v̇Lc ) is equal to the sum of the
main winding voltage of transformer (v̇Ta , v̇Tb , v̇Tc ) and the
output voltage of BD-AC modules (1v̇bc , 1v̇ca , 1v̇ab ). And
the relationship can be described in equation (2).
v̇La
v̇Ta
1v̇bc
v̇Lb = v̇Tb + 1v̇ca
(2)
v̇Lc
v̇Tc
1v̇ab
The main winding voltage of transformer (v̇Ta , v̇Tb , v̇Tc )
depends on the turn ratio of the primary winding to the
secondary main winding (nTa1 = nTb1 = nTc1 = nT1 ).
The output voltage of the BD-AC module (1v̇bc , 1v̇ca , 1v̇ab )
depends on the turns ratio of the primary winding to the
secondary auxiliary winding of the transformer (nTa2 =
nTb2 = nTc2 = nT2 ) and the duty cycle of the converter.
That is, the flexible transformer system satisfies the following
equation (3) and (4).
v̇Sa
nTa1
0
0
v̇Ta
v̇Sb = 0
nTb1
0 v̇Tb
(3)
v̇Sc
0
0
nTc1
v̇Tc
1v̇bc
0
DA1 DA2
v̇Ca
1v̇ca = DB2
0
DB1 v̇Cb
(4)
1v̇ab
DC1 DC2
0
v̇Cc
where (v̇Sa , v̇Sb , v̇Sc ) are the input voltage of flexible transformer system, i.e., the primary side voltage of transformer.
(v̇Ca , v̇Cb , v̇Cc ) stand for auxiliary winding voltage of the
transformer, and can be expressed as (4). (DA1 , DA2 ), (DB1 ,
DB2 ), (DC1 , DC2 ) are the duty cycle of BD-AC installed in Aphase, B-phase and C-phase respectively, their values satisfy
the equation (5) and (6).
1
0
0
n
v̇Ca
v̇Sa
Ta2
1
v̇Cb = 0
(5)
0 v̇Sb
n
Tb2
v̇
v̇Cc
Sc
1
0
0
nTc2
DA1
dAb1
dAb2
DB1 = dBc1 − dBc2
(6)
DC1
dCa1
dCa2
DA2
dAc1
dAc2
DB2 = dBa1 − dBa2
(7)
DC2
dCb1
dCb2
where (dAb1 , dAb2 ), (dAc1 , dAc2 ), (dBc1 , dBc2 ), (dBa1 , dBa2 ),
(dCa1 , dCa2 ), (dCb1 , dCb2 ) are the duty ratios of P-Leg and
N-Leg in each BD-AC of A-phase, B-phase and C-phase
respectively.
Based on the above analysis, the input-output relationship
of flexible transformer system, i.e., the relationship between
VOLUME 8, 2020
(v̇La , v̇Lb , v̇Lc ) and (v̇Sa , v̇Sb , v̇Sc ) can be expressed as equation (8).
1
v̇Sa
NTA
v̇La
0
0
1
NTB = 0
v̇Lb
0
v̇
Sb
NTC
0
0
v̇Lc
1
v̇
Sc
v̇Ta
0
0
v̇Tb
0
= 0
0
0
v̇Tc
1
v̇Sa
1v̇bc
0
0
1
(8)
1v̇ca
0
+ 0
v̇
Sb
0
0
1v̇ab
1
v̇Sc
where (NTA , NTB , NTC ) represent the transformer ratio of
flexible transformer system.
Furthermore, the primary voltage of flexible transformer
system can be expressed as equation (9).
j0
e
v̇Sa
j −2π
v̇Sb = VS
(9)
e 3
2π
v̇Sc
ej 3
where VS represents the primary voltage amplitude of flexible
transformer system.
Then, formula (2), (3), (4) and (9) can be brought into
formula (8), and it can be rewritten as follows.
1
DA1
DA2
nT1
nT2
nT2
NTA
1
D
D
B1
B2
NTB =
(10)
n
n
n
T1
T2
T2
NTC
1
DC1
DC2
nT1
nT2
nT2
Thus,
DA1
DA2
1
nT1
nT2
nT2
v̇Sa
v̇La
1
D
D
B1
B2
v̇Lb =
(11)
n
v̇Sb
n
n
T1
T2
T2
v̇Sc
v̇Lc
1
DC1
DC2
nT1
nT2
nT2
Furthermore, equation (12) can be obtained with taking (9)
into (11).
DA1
DA2
1
j0
nT1
nT2
nT2
e
v̇La
DB2
1
DB1 j −2π
v̇Lb = VS
e 3 (12)
n
nT1
nT2
T2
j 2π
v̇Lc
DC1
DC2
1 e 3
nT2
nT2
nT1
It can be seen from (10) to (12) that flexible transformer
system can not only adjust the amplitude of output voltage,
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Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
but also has the ability of adjusting the phase angle. And
whether the three phases are balanced or not, the load voltage
is supported by corresponding phase and regulated by the
other two phases, so as to ensure the balance of three-phase
voltage. The amplitude and phase angle regulation range of
output voltage of flexible transformer system depend on the
transformer ratio (NTA , NTB , NTC ). Taking A-phase as an
example, the relation surface between the transformer ratio
of flexible transformer system and the duty cycle of BD-AC
under different transformer ratio of transformer can be drawn
in Fig. 4, based on this, the voltage regulation ability of the
flexible transformer system is analyzed.
FIGURE 5. Phasor diagram of voltage regulation range of flexible
transformer system.
A-phase as an example, the regulation range diagram of
equation (13) is given in Fig. 5.
j0
e
1
DA1 DA2
v̇La
−2π
j
v̇Lb = VS DB2
1
DB1 e 3 (13)
2π
v̇Lc
DC1 DC2
1
ej 3
FIGURE 4. Relation between ratio of flexible transformer system and duty
cycle of BD-AC.
As can be seen from Fig. 4, the voltage regulation range of
flexible transformer system is different with various ratio of
transformer. No matter in NT1 = NT2 or NT1 6 = NT2 scene,
the voltage regulation range of flexible transformer system is
smaller with the increase of transformer ratio, and vice versa.
On the other hand, when the demand of voltage regulation
is high, the pressure bearing demand of AC/AC chopper
increases, and vice versa. When ratio is designed, i.e., as a
constant, the relationship between duty cycle of BD-AC and
flexible transformer system ratio is a corresponding surface
in Fig. 4. The transformer ratio of transformer needs to be
optimized according to grid voltage and voltage demand of
the load. On the other hand, the regulation range of the voltage
phase is [−π/3, π/3] with the range [−1, 1] of duty cycle of
BD-AC installed in flexible transformer system.
In order to facilitate further analysis, the voltage regulation
range of flexible transformer system when transformer ratio
equals NT1 = NT2 = 1 is quantitatively analyzed, then,
equation (12) can be simplified to equation (13). Then, taking
173340
When the duty cycle of the BD-AC satisfies the relation
DA1 = DA2 ∈ [−1, 1], the output voltage v̇La and the input
voltage v̇Sa of the flexible transformer system are collinear,
that is to say, the flexible transformer system can only realize
the voltage amplitude regulation, and the regulation range is
[0, 2]; Moreover, when the duty cycle of the BD-AC satisfies
the relation DA1 6= DA2 , the flexible transformer system has
the ability to adjust the amplitude and phase angle of voltage
at the same time, and the regulation range is [0, 2] and [−π/3,
π/3], respectively.
From another point of view, the transformation ratio
of transformer is optimized with the flexible transformer
installed in the power system. And if the input voltage of the
flexible transformer system fluctuates at this time, the flexible
transformer system has the function of maintaining the voltage stability at the output, i.e., realizing the voltage flexible
regulation.
C. CONTROL STRATEGY OF THE FLEXIBLE
TRANSFORMER SYSTEM
Based on the analysis of the operation principle of the proposed flexible transformer system above, it can be known
that in order to meet the load demand for voltage, the realtime sampling value of load voltage needs to be obtained.
Then, the control strategy based on voltage control signal is
generated by the control circuit and pulse drive of IGBT is
formed. Moreover, the regulating voltage is generated and
superimposed to the main winding voltage of transformer.
The protection control circuit starts the relevant equipment
to bypass the secondary side auxiliary winding of the transformer under the case of system failure, i.e., the BD-AC modular is bypassed until the system recovers. Thus, the control
strategy of flexible transformer system is constructed, and in
VOLUME 8, 2020
Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
order to facilitate the elaboration, the A-phase voltage is taken
as the control object for analysis and the details are as follows.
In the voltage regulation process of the flexible transformer system, the A-phase regulated voltage is constructed
by AC/AC-A. The AC/AC-A voltage synthesis principle is
shown in Fig. 6.
FIGURE 7. Control block of the flexible transformer system.
FIGURE 6. A-phase voltage regulation schematic.
In Fig. 6, A-phase voltage drops from vaN to v0aN , and the
flexible transformer system needs to provide a compensation
voltage 1vA to maintain the voltage at the target value,
i.e., flexible transformer system uses B-phase and C-phase
voltage to adjust and control the voltage with AC/AC-A, and
the voltage demand is DA1 · v̇bN +DA2 · v̇cN . At the same time,
based on the triangle angle theory, the relationship is satisfied
equation (14) in the process of voltage regulation.
1vA
DA1 · vbN
DA2 · vcN
=
=
(14)
sin (π/3)
sin (π/3 + θA )
sin (π/3 − θA )
Furthermore,
√
v̇LA = vSA 6 (ωt) + 2MA · vSA 6 (ωt + θA )
(15)
where MA and θA can be calculated by (16) and (17), respectively.
q
1
MA =
(16)
(DA1 + DA2 )2 + 3 (DA1 − DA2 )2
2n h
.
i
√
3 (DA1 − DA2 ) (DA1 + DA2 ) (17)
θA = tan−1
system is activated, and voltage regulation is performed by
comparing the voltage sample value with its target value until
it is satisfied.
IV. SIMULATION RESULTS
Through the analysis of the working principle of flexible
transformer system above, it can be known that the proposed
flexible transformer system makes it possible to improve
energy quality. When the duty cycle of BD-AC in each phase
takes different values, the flexible transformer system has
different voltage regulation capabilities, i.e., it is working
in different modes. In this paper, two scenarios (voltage
amplitude and phase angle regulation mode) are simulated
and verified, respectively. And the main specification and
components of the analyzed flexible transformer is shown
in Table 1.
TABLE 1. Simulation parameters.
where n = nT2 is the turns ratio of the primary winding to the
secondary auxiliary winding of the transformer.
And then, it has the following relations in the three-phase
system:
√
v̇LA = vSA 6 (ωt) + √ 2MA · vSA 6 (ωt + θA )
(18)
v̇ = vSB 6 (ωt) + √2MB · vSB 6 (ωt + θB )
LB
v̇LC = vSC 6 (ωt) + 2MC · vSC 6 (ωt + θC )
As can be seen that the flexible transformer system can
not only realize the regulation of the voltage amplitude, but
also has the voltage phase angle regulation capability. Based
on the analysis above, the control block of the flexible transformer system is shown in Fig. 7.
It can be seen from Fig. 7 that when the load voltage meets
the requirements or the converter in the flexible transformer
system fails, the bypass switch SP is turned on. When the sampling circuit detects that the voltage does not meet the requirements, the regulation capability of the flexible transformer
VOLUME 8, 2020
A. VOLTAGE AMPLITUDE REGULATION
Keeping the phase angle of the voltage as a constant, and
setting symmetric scene (voltage amplitude symmetrical
sags/swells 30%) and asymmetric scene (A-phase voltage
amplitude sag 40%, B-phase voltage amplitude sag 30% and
C-phase voltage amplitude swell 30%) to verify the voltage regulation capability of the flexible transformer system,
respectively. And the simulation results are shown in Fig. 8-9.
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Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
FIGURE 8. Simulation results: flexible transformer voltage time
waveforms during symmetric scene without phase jump.
FIGURE 10. Simulation results: flexible transformer voltage time
waveforms during source voltage phase shift only.
remain stable. Similarly, in order to maintain the voltage
of the secondary side of flexible transformer, compensation
voltage (1vbc , 1vca , 1vab ) is provided with the asymmetric
voltage sags/swells occurs, and the simulation results are
shown in Fig. 9.
B. VOLTAGE PHASE ANGLE REGULATION
FIGURE 9. Simulation results: flexible transformer voltage time
waveforms during asymmetric scene without phase jump.
In the above two scenarios, flexible transformer has a
good compensation effect. In Fig. 8, the primary side voltage
amplitude (vSa , vSb , vSc ) sags 30% at 0.05s, and after 0.05s,
a three-phase voltage amplitude (vSa , vSb , vSc ) swells occurs
(swells 30%). During this process, compensation voltage
(1vbc , 1vca , 1vab ) is provided and the target voltages of
the output side of the flexible transformer (vLa , vLb , vLc )
173342
When the phase angle of the primary side voltage of flexible
transformer changes and the amplitude is a constant, the simulation results are shown in Fig. 10.
As can be seen, in Fig. 10, the A-phase primary side voltage
vSa phase angle lags 45◦ at 0.05s, and after 0.05s, B-phase
voltage vSb phase angle lags 30◦ occurs. During this process,
in order to ensure that the voltage on the output side of
the flexible transformer (vLa , vLb , vLc ) are not affected, a
compensation voltage is provided. Moreover, the simulation
results of voltage fluctuations with phase angle jump are also
given in Fig. 11. As can be seen from Fig. 11 that A-phase primary side voltage amplitude vSa sags 30% with phase angle
lags 15◦ at 0.05s, and after 0.05s, the phase angle leads 15◦
and hold for 0.05s. In the above process, the use of flexible
transformer makes it possible to improve energy quality. And
with the regulation of power electronic equipment, the flexible transformer works well. Besides, owing to the adopted
AC/AC chopper has the same buck/boost operation process
for non-inverting and inverting modes [25], the proposed flexible transformer can realize bidirectional regulation of power.
Thus, it is suitable for the active distribution network with
bidirectional power regulation requirements with distributed
power access.
VOLUME 8, 2020
Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
FIGURE 11. Simulation results: flexible transformer voltage time
waveforms during source voltage swell with phase jump.
V. CONCLUSION
Voltage fluctuation will affect the normal operation of the
sensitive load, and even lead to its breakdown, resulting in
unnecessary economic losses. To solve this problem, a flexible transformer based on bipolar direct AC/AC chopper for
flexible regulation of supply voltage, and additionally operating as a power shifter transformer has been presented in
this paper. Based on the analysis of topology and modulation strategy of bipolar direct AC/AC chopper, the structure
and operation principle of flexible transformer system are
analyzed in detail. Then, the voltage regulation range of the
flexible transformer system is studied with voltage amplitude
and phase angle as the analysis object. And the relationship
between transformer ratio of the flexible transformer system
and duty cycle of the BD-AC is acquired in different scenarios. Finally, in order to test and verify the operation and
circuit described in this paper, the exemplary time waveforms
of voltage are shown in the simulation investigation. And the
obtained result validate that the proposed system is able to
realize the flexible regulation of voltage.
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173343
Y. Wang et al.: Three-Phase Flexible Transformer Based on Bipolar Direct AC/AC Chopper and Its Control Strategy
YIBO WANG (Member, IEEE) was born in
Shandong, China, in 1989. He received the B.S.
and M.S. degrees in electrical engineering from
Northeast Electric Power University, Jilin, China,
in 2010 and 2016, respectively, where he is currently pursuing the Ph.D. degree. His current
research interests include renewable energy integration into power networks, power systems, and
power quality.
GUO-WEI CAI was born in Jilin, China, in 1968.
He received the B.S. and M.S. degrees in electrical engineering from Northeast Electric Power
University, Jilin, in 1990 and 1993, respectively,
and the Ph.D. degree in electrical engineering from
the Harbin Institute of Technology, Harbin, China,
in 1999. Since 2004, he has been a Professor with
the School of Electrical Engineering, Northeast
Electric Power University. His current research
interests include power system transient stability
analysis and smart grid with renewable power generation.
CHUANG LIU (Member, IEEE) received the M.S.
degree in electrical engineering from Northeast
Electric Power University, Jilin, China, in 2009,
and the Ph.D. degree in electrical engineering from
the Harbin Institute of Technology, Harbin, China,
in 2013.
From 2010 to 2012, he was with the Future
Energy Electronics Center, Virginia Polytechnic
Institute, and State University, Blacksburg, VA,
USA, as a Visiting Ph.D. Student, supported by the
Chinese Scholarship Council. He was an Associate Professor with the School
of Electrical Engineering, Northeast Electric Power University, in 2013,
where he has been a Professor, since 2016. His research interests include
power-electronics-based on ac and dc transformers for future hybrid ac–dc
power grids, flexible operation and control of power grid based on ac–ac
transformation, and power-electronics-based power system stability analysis
and control.
173344
BINGDA ZHU was born in Henan, China, in 1996.
He received the B.S. degree from Northeast
Electric Power University, Jilin, China, in 2018,
where he is currently pursuing the M.S. and
Ph.D. degrees in electrical engineering. His current
research interests include direct PWM ac–ac converters, control strategy, power quality optimization in distribution networks, and the application
of high-power electronic conversion technology in
smart grid.
DONGBO GUO was born in Shandong, China,
in 1990. He received the B.S. degree from
Northeast Electric Power University, Jilin, China,
in 2016, where he is currently pursuing the
M.S. degree in electrical engineering. His current
research interests include flexible operation and
control of power grid based on ac–ac conversion,
direct PWM ac–ac converters, and the application
of high-power electronic conversion technology in
smart grid.
HANWEN ZHANG was born in Jilin, China,
in 1994. He received the B.S. degree from Northeast Electric Power University, Jilin, in 2017,
where he is currently pursuing the M.S. degree in
electrical engineering. His current research interests include power quality control in distribution networks, direct PWM ac–ac converters, and
energy efficient integrated conversion technology.
VOLUME 8, 2020
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Simulation and mental health outcomes: a scoping review
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Advances in simulation
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cc-by
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Simulation and mental health outcomes: a
scoping review Williams1*
, Priya Reddy1, Stuart Marshall2, Bronwyn Beovich1 and Lesley McKarney3 Brett Williams1*
, Priya Reddy1, Stuart Marshall2, Bronwyn Beovich1 and Lesley McKarney3 Abstract Background: A scoping review was conducted in order to map and determine the gaps in literature on the impact
of simulation as an educational approach to improve mental health care outcomes. As it became apparent that no
literature existed on this topic, the study aimed to examine the educational impact of simulation on mental health
education. Methods: An established five-stage scoping methodology was used: (1) identification of the research question, (2)
identification of relevant studies, (3) study selection, (4) charting the data and (5) collation, summarising and
reporting of results. CINAHL, ProQuest, PubMed, MEDLINE, EMBASE and PsychINFO databases were searched. These
databases were deemed to represent a majority of the literature while accommodating for the particular search
strategy used for this review. Websites that provide grey literature were also searched for articles of relevance. Results: A total of 48 articles were included in this review, with a considerable portion of studies conducted in the
USA and UK. Others were conducted in an array of locations including Australia, Canada, Iran and Taiwan. Of the
included articles, seven groups of simulation methods (including standardised patients, virtual reality and manikins
as patients) were evident, with standardised patients being most prominent. Conclusions: Literature is lacking to evidence the benefit of simulation on mental health patient outcomes. However, the available literature suggests a variety of simulation-based education, and training methods are
currently being used within mental healthcare education. The findings do suggest some methods of simulation,
such as the use of standardised patients, are more commonly used in education and have been deemed as
effective to assist in mental health education. As no article specifically examining the mental health outcomes of
patients treated by health professionals taught by simulation was identified, the educational outcomes outlined in
this paper may be used to inform further research, incorporating mental health patient outcomes. Keywords: Students, Health occupations, Patient simulation, Mental health, Manikin Williams et al. Advances in Simulation (2017) 2:2
DOI 10.1186/s41077-016-0035-9 Williams et al. Advances in Simulation (2017) 2:2
DOI 10.1186/s41077-016-0035-9 Open Access * Correspondence: brett.williams@monash.edu
1Department of Community Emergency Health & Paramedic Practice,
Monash University, Peninsula Campus, PO Box 527McMahons Road,
Frankston 3199, Victoria, Australia
Full list of author information is available at the end of the article Background
h
l h multiple
settings
including
the
classroom,
clinical
consulting room or hospital ward, simulation laboratory
or the virtual world [2]. Many mental healthcare compe-
tencies are well-suited to SBE, and it can be used to
expose students to clinical situations or events that
occur infrequently or pose a high risk in terms of safety
or liability [3]. However, some authors believe that simu-
lation could be more widely adopted in the field of
mental health education [4–6]. In healthcare education, simulation is used for both the
teaching and assessment of students and staff. There are
a variety of simulation-based education (SBE) and train-
ing
methods,
sometimes
delivered
in
combination,
dependent on the content and learning outcomes. The
levels of difficulty, complexity and challenge can be
tailored to suit the context, learning or assessment
objectives and the experience level of the students [1]. The versatility of simulation allows it to take place in A preliminary literature search by the research team
suggested very little information on the impact of simu-
lation on mental health patient outcomes existed. Thus,
a scoping study of the literature was performed to
identify the extent of this literature gap and map what * Correspondence: brett.williams@monash.edu
1Department of Community Emergency Health & Paramedic Practice,
Monash University, Peninsula Campus, PO Box 527McMahons Road,
Frankston 3199, Victoria, Australia
Full list of author information is available at the end of the article Williams et al. Advances in Simulation (2017) 2:2 Page 2 of 8 Page 2 of 8 1. Identify the research question
2. Identify relevant studies
3. Study selection
4. Charting the data
5. Collating, summarising and reporting results 1. Identify the research question outcomes have been reported. At present, in the general
medical literature, there are relatively few studies on the
impact of simulation on patient outcomes and the collat-
eral effects at a population level. This is especially true
in the mental health sector [7, 8]. 2. Identify relevant studies 3. Study selection 4. Charting the data 5. Collating, summarising and reporting result There is however significant research to show that
simulation-based health education promotes knowledge ac-
quisition and maintenance of clinical knowledge, attitude
and skills [7, 9, 10]. A variety of simulation methodologies
have been used in healthcare education including; actors
trained to portray a person with a particular health concern
(a simulated patient (SP)) [11–13], manikins and computer-
generated scenarios [3]. Identify relevant studies y
A comprehensive literature search of six online peer-
reviewed
databases
was
conducted. These
included
CINAHL,
ProQuest,
PubMed,
Ovid
MEDLINE,
EMBASE and PsychINFO. No restriction was placed on
dates or locations of publications. The search strategy
and results can be seen in Table 1 and Fig. 1. The full
search strategies for each database are presented in
Additional file 1. Three grey literature sites were also ex-
amined for non-peer-reviewed articles, these included
http://www.greylit.org/, Google Scholar and http://www. tripdatabase.com/. Hand searches were also undertaken
on full-text articles. The authors were unable to identify any literature on
simulation as an educational approach affecting mental
health patient outcomes. Therefore, this study was
broadened to examine the educational impact of simula-
tion on mental health education outcomes. Background
h
l h These methods can range from
“low-fidelity” where the level of realism is low to “high-fi-
delity” where there is a high degree of realism such as
human patient simulators or manikins that are able to
replicate a growing range of physiological signs [14]. Methods Scoping reviews aim to identify the nature and extent of
existing literature on a selected topic and can indicate
the value of venturing into a full systematic review [16,
17]. A scoping review was chosen for this study as it had
been identified in a preliminary literature search that
little information existed in this area, and this method-
ology allowed for a broader body of literature which may
underpin the topic to be investigated. Due to the appar-
ent lack of pre-existing information, a systematic review
was deemed as an unsuitable approach. With the use of
Levac, Colquhoun and O’Brien’s [16] five-stage method-
ology for scoping reviews, a range of literature sources
including grey literature sites were incorporated to
complete a comprehensive review. These stages are as
follows: Identify the research question With the incidence of mental illness increasing globally,
health occupations require innovative and improved
mental health educational methods [18]. The breadth of
literature available on the use of simulation in medical
education has led to the proposal of the research ques-
tion “Can simulation improve mental health outcomes?”
The topic was deemed to be focused yet broad enough
to conduct an effective scoping review to determine the
gaps in the literature around the use of simulation and
mental health outcomes. As our preliminary investiga-
tion into the literature identified a lack of information
regarding patient outcomes, this broad approach also
enabled information to be captured around related is-
sues such as the types of simulations being used in men-
tal health care, as well as skills and knowledge outcomes
of students and clinicians. Due to the lack of literature
identified examining patient outcomes, the research
question evolved to examine educational outcomes as a
platform to inform future patient outcome studies. Simulation offers many opportunities for the develop-
ment of skills, knowledge and behaviours for students and
clinicians working in mental health settings. Simulation
also provides opportunities to address the challenges re-
lated to stigma, safety and liability present in the psychi-
atric clinical setting [3]. For people living with a mental
illness, there are often compounding social, cultural, eco-
nomic, family or other factors that may form part of their
presentation or care. Well-trained and briefed simulated
patients in well-designed simulation scenarios can portray
the complexities of mental illness with high fidelity [15]. Simulation can help familiarise students with mental ill-
nesses before they encounter them in a clinical setting, in-
creasing
the
student’s
ability
to
appropriately
and
confidently respond to patient needs. Study selection To be included, articles were required to satisfy the three
following inclusion criteria: 1. Articles incorporating mental health 2. Articles including simulation educational methods/
outcomes 3. Articles relating to health professionals and/or
health students. Articles were excluded if they were not written in
English or did not meet the above inclusion criteria
in terms of population, context or concept [19]. The
search was conducted by one author and ratified by 3. Articles relating to health professionals and/or
health students. Articles were excluded if they were not written in English or did not meet the above inclusion criteria
in terms of population, context or concept [19]. The
search was conducted by one author and ratified by Williams et al. Advances in Simulation (2017) 2:2 Page 3 of 8 Table 1 Key search terms
Main concepts
Simulation
Mental health
Health occupations
Search
Terms
“mannequin” MH “Patient Simulation”
MH “Simulations+” “simulation, medical”
“Health Personnel as Patients+” “virtual patient”
MH “Clinical Competence”
exp Mental Health/exp Mental
Disorders/“mental conditions”
Health Occupations/Allied Health Personnel/ exp
Primary Health Care/ exp Students, Health Occupations
“MH” refers to Mesh Heading and “exp” refers to Explode “MH” refers to Mesh Heading and “exp” refers to Explode training or study [20]. It also allows educators, re-
searchers and teachers to assess and objectively evaluate
the effectiveness of the intervention [20]. It aims to
guide the users on ways and areas of improvement based
on participant involvement, achievement and growth
[20, 21]. The model was utilised in this study as it has
been widely cited in the educational literature and was
developed to enable the evaluation of a variety of inter-
ventions in many environments [21–23]. Thus, it was
deemed a more appropriate model for our purposes
compared with others which were developed for use in
more specific situations, such as within continuing med-
ical education [24]. Furthermore, while Kirkpatrick’s
model may not be useful in determining individual study
quality, it is a useful tool to measure the progress of an
emerging body of research aiming to eventually impact
patient outcomes [25], such as SBE. Based on its means
of
utilisation,
this
model
was
included
to
further
strengthen the review and assess the studies included. The levels, where level 4 is generally the most desired
outcome for intervention, are as follows (Table 2): an expert librarian; screening and full-text reviews
were completed by two authors. Consensus was
reached on all full-text papers. Charting the data Once the studies were selected, the spreadsheet program
Excel was used to enter relevant information about each
study such as intervention type, population studied and
outcomes. This information was then synthesised by sort-
ing and grouping the literature according to common
themes. Analysis of the included articles determined
several different simulation methods which are used in
mental health education, and these interventions were
examined to determine their effectiveness in improving
student/clinician knowledge and skill development. To assist with condensing the literature, the simulation
methods were grouped based on key components; these
included the following: 1. Simulation utilising people as patients 2. Simulation utilising virtual reality 3. Simulation utilising manikins as patients Classification of educational outcomes: Kirkpatrick’s
model This stage of the scoping study involves organisation of
the identified information to provide an overview of the
existing literature on the topic. Thematic analysis of in-
formation from the included studies is performed to Results This initial search yielded a total of 1460 articles from
the online peer-reviewed databases (after eliminating du-
plicates) while the grey literature sites and hand searches
produced no new results. A title and abstract screening
located 77 articles that were to be included for the next
stage in sorting. A further full article review yielded 48
results which are included in this review. This current scoping review was conducted in order to
determine the existing evidence regarding the use of
simulation and its effect in improving mental health
patient outcomes. However, the scoping review did not
uncover any literature reporting on patient mental
health outcomes related to SBE. As a consequence of
the broad literature search strategy, current information
was also gathered on other outcomes of SBE within
mental
health
such
as
learner
reactions
to
SBE
(Kirkpatrick model, level 1), knowledge and educational
outcomes (Kirkpatrick model, level 2) and application of
this education (Kirkpatrick model, level 3). In an envir-
onment where no patient outcomes have been reported,
knowledge, skill enhancement and skill application are
important outcomes which may inform future studies of
SBE in relation to patient mental health outcomes. Among the 48 articles included for this review, 29
were conducted in the United States of America (USA),
seven in the United Kingdom (UK), five in Australia,
two in Canada and one each in the countries Taiwan,
Italy, Malaysia, Iran and Germany. Furthermore, seven
different types of simulation methods were identified,
including the following: Simulated/standardised patients and actors
[1, 11–13, 26–38]
Virtual reality [6, 39–46]
Role play [47–52]
Manikins [14, 53–55]
Computer simulation [56–59]
Objective structured clinical examination (OSCE)
[60–63]
Voice simulation (which refers to the use of sounds Simulated/standardised patients and actors Virtual reality [6, 39–46] Role play [47–52] Manikins [14, 53–55] Computer simulation [56–59] Objective structured clinical examination (OSCE)
[60–63] The seven simulation types identified by the scoping
review were SPs (n = 17), virtual reality (n = 9), role play
(n = 6), manikins as patients (n = 4), computer simula-
tion (n = 4), OCSE (n = 4) and voice simulation (n = 4). These results suggest that although SBE is being used, it
has received very little empirical examination in the
mental health sector. Results The following discussion will focus
on the skill enhancement and efficacy of various forms
of simulation used in mental health education, and
potential links to mental health patient outcomes. Voice simulation (which refers to the use of sounds
and voice through an electronic medium to portray
the sounds encountered by a schizophrenic patient)
[18, 64–66] From these articles, 39 suggested improvements in
mental health education outcomes while the remaining
nine articles either suggested no benefit or was used to
assess clinician knowledge. Furthermore, 26 of the 48 ar-
ticles focussed on undergraduate students, seven on
postgraduates and 17 on clinicians. As some articles in-
cluded more than one category of participant, the total
is greater than 48. A full list of 48 included publications
can be found in Additional file 2. address the research question as well as to identify gaps
in the literature. address the research question as well as to identify gaps
in the literature. model The Kirkpatrick model is a framework created in order
to determine the efficacy of a particular intervention, Fig. 1 Search strategy methodology Page 4 of 8 Williams et al. Advances in Simulation (2017) 2:2 Table 2 Four levels of Kirkpatrick’s model
Level 1 = participants react favourably to the learning or intervention. Level 2 = participants acquired knowledge, skills and attitudes based on
the intervention or study. Level 3 = participants applied what they learnt into practice. Level 4 = once applied, there was an outcome to that application of
skills learnt from the intervention. the vast majority of articles scoring 2 or 3 on Kirkpa-
trick’s model. The current literature on mental health
education using simulation lacks the patient outcome
data and therefore provides abundant opportunities for
future research and evaluation. Level 2 = participants acquired knowledge, skills and attitudes based on
the intervention or study. Level 3 = participants applied what they learnt into practice. Level 3 = participants applied what they learnt into practice. Level 4
once applied there was an outcome to that application of Level 3 = participants applied what they learnt into practice. Level 4 = once applied, there was an outcome to that application of
skills learnt from the intervention. Discussion Education for mental healthcare is an important topic as
there is a high incidence of mental illness worldwide,
and it is projected to be an increasing cause of burden
of
disease
in
the
future
[67,
68]. Research
and
technology is constantly improving, as such the under-
standing of mental illness is also improving, creating
new treatment and education methods for clinicians and
healthcare students [69]. SBE has been viewed as an
educational method with the potential to improve the
care of individuals with mental illness [49]. The use of
simulation
has
been
implemented
in
medical
and
nursing education for decades with evidence suggesting
its benefits, to both learners and patients [52]. Simulated standardised patients (SPs) The use of individuals to portray patients has been sug-
gested as the most effective way to educate healthcare
professionals in communication and other clinical skills
[13]. Communication skills are vital for any healthcare
workers with the need for developing effective inter-
personal techniques a central component in all patient/ The Kirkpatrick model [20] assisted in providing
strength to the review (see Additional file 2 for the Kirk-
patrick ranking of each study). The lack of patient out-
come data is evident in the results of this review, with Williams et al. Advances in Simulation (2017) 2:2 Page 5 of 8 Page 5 of 8 general consensus between the main findings of the
studies is that these methods are effective in mental
health education [6, 41, 56]. client interactions, especially within mental healthcare
[70]. SPs are a valuable modality when there is a high
degree of emotion and/or communication required, as
they can provide non-verbal as well as verbal informa-
tion and responses [1]. VR: skill enhancement Satter’s [44] study of 14 practicing primary care physi-
cians (PCPs) found that they were slightly better at
diagnosing major depressive disorder and post-traumatic
stress disorder with the use of avatars, as compared to
those who used paper-based scenarios. In another study,
Heiser [56] found that psychiatrists had the same chance
of
correctly
diagnosing
paranoia
for
a
computer-
simulated patient or a real patient, which suggests that
this method may be suitable for use in mental healthcare
education. Given the technology capability in 1979,
Heiser’s results may not be necessarily comparable with
modern day technology but the general method of simu-
lation used is still relevant and transferable today. VR is
proving to be a more prominent method for mental
health
education
in
today’s
society,
particularly
as
technology advances. Its effectiveness is yet to be fully
determined; however, the indication from the majority of SPs: skill enhancement Hall [12] noted that the use of actors to portray psychi-
atric patients was effective in enhancing the assessment
and
therapeutic
communication
skills
of
nursing
students. Of the 112 students that were a part of the
pilot study, 80% agreed that the SP accurately portrayed
depression and 100% of the cohort reflected improved
communication [12]. A mixed methods study by Lewy [28] reported that
paediatric residents (n = 34) working with SPs attained
an increased confidence in patient treatment, with 69%
stating
the
intervention
was
“extremely
helpful”. Shahabudin [30] trained medical students to act as
patients presenting with various mental illnesses in order
to assess the knowledge and diagnostic abilities of 42
general practitioners (GPs). The findings were grouped
into three categories based on the performance of the
doctors: group A—where 11.9% of the GPs informed the
SPs of their anxiety diagnosis, group B—28.6% of the
GPs prescribed medication for anxiety but did not in-
form the SP of their diagnosis and group C—where
59.5% of GPs did not diagnose nor treat the SP [30]. This study highlighted the lack of consistent training,
assessment and treatment in the GP management of
mental health and highlights an opportunity for future
research and investigation. Voice simulation is effective in portraying the experi-
ence of schizophrenia, and as Weiland’s [66] qualitative
study suggested, it is a valuable tool in increasing
patience and empathy in nursing students. Seventy-four
students listened to audio recordings of common voices
heard in schizophrenia while attempting to complete
certain tasks such as a job application. Based on the
reflective evaluations completed by the students post-
intervention, common themes emerged included feelings
of “frustration” and “feeling overwhelmed”. However, the
experience had positive outcomes, with reporting of
increased
levels
of
patience
and
empathy
towards
schizophrenic patients [66]. VR: simulation efficacy Guise [6] found that virtual patients can be very effective
in teaching clinical decision-making skills to nursing stu-
dents, especially those parts of distance learning groups. The reduced risk of negative consequences for incorrect
diagnosis and treatment-assisted students in learning
mental health clinical skills. Lambert [41] found that the
use of a simulated virtual patient, or avatar, portraying a
person living with a mental illness was effective in edu-
cating nursing students on appropriate communication
methods. The study investigated 85 mental health nurs-
ing students who followed the in-hospital journey of the
fictional avatar for a 2-week period. Although the study
did not focus on any form of assessment, it found that
the students became more understanding and ethical
practitioners at the completion of the fortnight and
urged other organisations to follow suit in their training
methods [41]. SPs: simulation efficacy Fussell’s study in [38] suggested that actors portraying
people with substance abuse provided a reliable and ef-
fective learning modality in the education and assess-
ment of substance abuse counsellors. Role play was
suggested by Roberts’ [50] randomised controlled trial to
be neither effective nor ineffective in improving the
assessment skills and views of undergraduate medical
students regarding people living with a mental illness. Virtual reality (VR) Virtual reality is a computer-generated scenario or envir-
onment with which an individual can actively interact
[43]. The concept is becoming increasingly common in
healthcare education with the concomitant decrease in
risk to patient safety [71]. VR technologies can include
the computer-generated scenarios of virtual environ-
ments, voice simulation and virtual patients. Seventeen
articles included the use of virtual reality technologies
with
occupations
incorporating
a
mix
of
nursing,
medical and psychiatric students and practitioners. The Williams et al. Advances in Simulation (2017) 2:2 Page 6 of 8 studies is that VR is a positive way to educate healthcare
professionals regarding mental illness. outcomes. The measurable patient outcomes in mental
health interventions may be less tangible compared with
that of physical disorders. This may present one of the
challenges in taking the evaluation of simulation in men-
tal healthcare beyond educational outcomes. Recommendations for future research The most significant gap in the current research base is
the lack of evidence that mental health SBE directly im-
pacts patient outcomes. This scoping review has found
several gaps in the current literature that may provide
researchers, policymakers and educators with a “road-
map” of future research opportunities. These include
interdisciplinary research, patient outcomes, different
methods of simulation (such as pre-recorded DVDs
which may use simulated patients portraying various
clinical situations), prehospital care and SPs. g
g
y
In an earlier study, Kameg [14] noted that the use of
manikins was an effective approach in teaching commu-
nication skills to nursing students. Similarly, Grant’s [53]
review paper noted that high-fidelity manikins, when in
combination with OSCE’s, were a viable training source
to improve therapeutic communication skills. Unsworth’s
study in [55] utilised the SimMan (a medium-fidelity
manikin)
and
found
that
the
use
of
manikins
demonstrated to students where their areas of weaknesses
were
and
what
needed
to
be
improved
for
their
prospective healthcare careers. The qualitative measures
of the study revealed the students thought of the interven-
tion as “bridging the gap” between developing vital skills
that are rarely seen in practice but are necessary to
understand [55]. Although there are a number of articles in the litera-
ture reporting on simulation in mental healthcare educa-
tion, there is a lack of patient outcome measures linked
to SBE. This was supported in the evaluation using
Kirkpatrick’s model as nil level 4 studies were located in
any of the 48 articles. Further research needs to be con-
ducted into how simulation in mental healthcare relates
to patients’ outcomes in terms of successful or unsuc-
cessful treatment, including the quality of life of the
patient and their family. It is the authors’ view that fur-
ther research is also needed for professions other than
medicine and nursing. As a large portion of the existing
research focussed on these professions, the findings may
not be generalisable to other disciplines. In addition,
more focus needs to be given to out-of-hospital care, in-
cluding emergency paramedic clinicians and GPs work-
ing with people experiencing acute mental health issues. A significant gap in the mental health literature relates
to
indigenous
populations
including
Aboriginal
Australians. Similarly, there is a dearth of literature on
the use of SBE in the mental healthcare of paediatric
patients, young adults and the elderly population. Limitations Articles not in English were excluded from this review;
this is an important limitation and publication-bias as
significant data may have been missed due to the inabil-
ity to appropriately categorise the articles. The search of
grey literature sites yielded only peer-reviewed articles;
this suggests the depth of included articles does not in-
corporate non-peer-reviewed literature which could have
brought strength to the findings. Also, with only three
articles reporting on impacts in non-westernised coun-
tries [30, 32, 42], care must be taken when generalising
results to the wider population. Manikins as patients This scoping review only located four articles that uti-
lised manikins in the mental health setting. All of these
articles focussed on nursing students, suggested a posi-
tive response and portrayed the effectiveness of the use
of manikins in mental health education. Skill enhancement A study by Kameg [54] found that students who were
previously at risk of failing were no longer at risk after
completing training with the use of high-fidelity mani-
kins. The quasi-experimental study used a cohort of 35
nursing
students
to
complete
a
30-item
Health
Education Systems Incorporated (HESI™) custom exam
pre- and post-simulation [54]. There was a statistically
significant improvement in the student risk profile post-
simulation, with 10 of the 13 “at risk” students improv-
ing their category to the “non-risk” level [54]. Consent for publication We are not reporting any data obtained from any individual; therefore,
consent is not required. Conclusions 1. Doolen J, et al. An evaluation of mental health simulation with standardized
patients. Int J Nurs Educ Scholarsh. 2014;11(1):55–62. 1. Doolen J, et al. An evaluation of mental health simulation with standardized
patients. Int J Nurs Educ Scholarsh. 2014;11(1):55–62. Although SBE has been demonstrated to be beneficial in
many aspects of healthcare education, to the best of the
authors’ knowledge, there is no existing evidence regard-
ing the effect of SBE on mental health patient outcomes. The literature suggests a variety of simulation methods
are currently being used within mental healthcare educa-
tion. As evidenced by the number of Kirkpatrick level 2
and 3 findings in research papers included in this scop-
ing study, simulation has proved generally beneficial in
terms
of
educational
and
clinical
skill
outcomes. However, the results remain variable and therefore not
necessarily
generalisable. Many
attitudes,
skills
and
competencies vital to mental healthcare practice are seen
as well-suited to SBE methodologies. Therefore, further
research would be valuable to comprehensively examine
the effects of SBE, including that of patient outcomes. This research progress is necessary to add to the
evidence base of mental healthcare. 2. Gaba DM. The future vision of simulation in health care. Qual Saf Health
care. 2004;13 suppl 1:i2–10. 2. Gaba DM. The future vision of simulation in health care. Qual Saf Health
care. 2004;13 suppl 1:i2–10. 3. Brown J. Applications of simulation technology in psychiatric mental health
nursing education. J Psychiatr Ment Health Nurs. 2008;15(8):638–44. 4. Gorrindo T, et al. Web-based simulation in psychiatry residency training: a
pilot study. Acad Psychiatry. 2011;35(4):232–7. 5. Thomson AB, et al. How we developed an emergency psychiatry training
course for new residents using principles of high-fidelity simulation. Med
Teach. 2013;35(10):797–800. 6. Guise V, Chambers M, Välimäki M. What can virtual patient simulation offer
mental health nursing education? J Psychiatr Ment Health Nurs. 2012;19(5):410–8. 7. McGaghie WC, et al. Evaluating the impact of simulation on translational
patient outcomes. Simul Healthc. 2011;6(Suppl):S42. 7. McGaghie WC, et al. Evaluating the impact of simulation on translational
patient outcomes. Simul Healthc. 2011;6(Suppl):S42. 8. Zendejas B, et al. Patient outcomes in simulation-based medical education:
a systematic review. J Gen Intern Med. 2013;28(8):1078–89. 9. Lapkin S, et al. Effectiveness of patient simulation manikins in teaching
clinical reasoning skills to undergraduate nursing students: a systematic 8. Zendejas B, et al. Patient outcomes in simulation-based medical education:
a systematic review. J Gen Intern Med. 2013;28(8):1078–89. 9. Conclusions Lapkin S, et al. Effectiveness of patient simulation manikins in teaching
clinical reasoning skills to undergraduate nursing students: a systematic
review. Clin Simul Nurs. 2010;6(6):e207–22. 10. Cook DA, et al. Technology-enhanced simulation for health professions
education: a systematic review and meta-analysis. JAMA. 2011;306(9):978–88. 11. Brown R, Doonan S, Shellenberger S. Using children as simulated patients in
communication training for residents and medical students: a pilot
program. Acad Med. 2005;80(12):1114–20. 12. Hall MJ, et al. Use of standardized patients
clerkship. Acad Med. 2004;79(1):28–31. 12. Hall MJ, et al. Use of standardized patients to enhance a psychiatry
clerkship. Acad Med. 2004;79(1):28–31. Additional files 13. Keltner NL, Grant JS, McLernon D. Use of actors as standardized psychiatric
patients: facilitating success in simulation experiences. J Psychosoc Nurs
Ment Health Serv. 2011;49(5):34–40. Additional file 1: Search strategy for each database. (DOCX 15 kb)
Additional file 2: Data summary of publications. (DOCX 23 kb) 14. Kameg K, et al. The impact of high fidelity human simulation on self-efficacy
of communication skills. Issues Ment Health Nurs. 2010;31(5):315–23. 15. Kidd LI, Knisley SJ, Morgan KI. Effectiveness of a Second Life® simulation as a
teaching strategy for undergraduate mental health nursing students. J
Psychosoc Nurs Ment Health Serv. 2012;50(7):28–37. We did not collect any data or materials in this research. We did not collect any data or materials in this research. Received: 5 October 2016 Accepted: 31 December 2016 Received: 5 October 2016 Accepted: 31 December 2016 Funding 17. Arksey H, O’Malley L. Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005;8(1):19–32. This project was possible due to funding made available by the Department
of Health and Human Services, Victorian State Government. 18. Kidd LI, et al. Mindful teaching practice: lessons learned through a hearing
voices simulation. Issues Ment Health Nurs. 2015;36(2):112–7. 19. The Joanna Briggs Institute. Joanna Briggs Institute Reviewers’ Manual: 2015
edition/Supplement. 2015 18/11/2016]; 2015. http://joannabriggs.org/assets/
docs/sumari/Reviewers-Manual_Methodology-for-JBI-Scoping-Reviews_
2015_v2.pdf. 19. The Joanna Briggs Institute. Joanna Briggs Institute Reviewers’ Manual: 2015
edition/Supplement. 2015 18/11/2016]; 2015. http://joannabriggs.org/assets/
docs/sumari/Reviewers-Manual_Methodology-for-JBI-Scoping-Reviews_
2015_v2.pdf. Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. Acknowledgements The authors would like to acknowledge the Department of Health and
Human Services for their funding to undertake this study. 16. Levac D, Colquhoun H, O’Brien KK. Scoping studies: advancing the
methodology. Implement Sci. 2010;5(1):1. Authors’ contributions 20. Kirkpatrick DL, Kirkpatrick JD. Implementing the four levels: a practical guide
for effective evaluation of training programs. California: Berrett-Koehler;
2007. 20. Kirkpatrick DL, Kirkpatrick JD. Implementing the four levels: a practical guide
for effective evaluation of training programs. California: Berrett-Koehler;
2007. BW, PR, SM and LM designed and developed the study. BW and PR
completed the literature review and screening of articles. BW and PR
co-wrote the first draft. All authors contributed to the critical review and
approval of the final paper. 21. Steinert Y, et al. A systematic review of faculty development initiatives
designed to improve teaching effectiveness in medical education: BEME
Guide No. 8. Med Teach. 2006;28(6):497–526. 21. Steinert Y, et al. A systematic review of faculty development initiatives
designed to improve teaching effectiveness in medical education: BEME
Guide No. 8. Med Teach. 2006;28(6):497–526. Author details
1 1Department of Community Emergency Health & Paramedic Practice, 1Department of Community Emergency Health & Paramedic Practice,
M
h U
P
l C
PO B
M M h
R
d 1Department of Community Emergency Health & Paramedic Practice,
Monash University, Peninsula Campus, PO Box 527McMahons Road,
Frankston 3199, Victoria, Australia. 2Monash University, HealthPEER, Claytoria
Victoria, Australia. 3Latrobe University, Melbourne, Australia. 1Department of Community Emergency Health & Paramedic Practice,
Monash University, Peninsula Campus, PO Box 527McMahons Road, Department of Community Emergency Health & Paramedic Practice,
Monash University, Peninsula Campus, PO Box 527McMahons Road,
2 Frankston 3199, Victoria, Australia. 2Monash University, HealthPEER, Claytoria,
Victoria, Australia. 3Latrobe University, Melbourne, Australia. Frankston 3199, Victoria, Australia. 2Monash University, HealthPEER, Claytoria,
Victoria, Australia. 3Latrobe University, Melbourne, Australia. Received: 5 October 2016 Accepted: 31 December 2016 Mental health patient outcomes Although much evidence exists to support the use of
SBE in medical and nursing education, both in terms of
educational and patient outcomes, there is far less evi-
dence regarding its use in the healthcare category of
mental health. Moreover, to the best knowledge of the
authors, no studies have been published that report on
patient mental health outcomes in relation to SBE. Many
of the attitudes, skills and competencies necessary for ef-
fective mental healthcare are well-suited to SBE, and
there appears to be abundant opportunities for the de-
velopment and evaluation of this teaching methodology
within mental health [3]. However, the mental health
educational outcomes reported in this paper including
knowledge, skill enhancement and skill application have
relevance and may be used to inform future studies in
the
area,
including
those
concerned
with
patient y
g
y
The
evidence
suggests
that
robust
evaluation
of
simulation programs needs to be undertaken to provide Page 7 of 8 Williams et al. Advances in Simulation (2017) 2:2 Page 7 of 8 evidence of the impact of simulation in mental health-
care education beyond educational outcomes. SBE holds
many opportunities for curricula improvement and
development. Ideally, evaluation plans would be incorpo-
rated at the design phase of new programs and intro-
duced into programs which already exist. That is,
planning backwards and teaching forward, that consider
Kirkpatrick’s model. Further research should consider
and focus on designs that are both qualitative and quan-
titative to obtain both narrative and objective data. Consideration should also be given to improve reporting
where SP and role play approaches are involved [72]. Competing interests
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https://www.frontiersin.org/articles/10.3389/fsurg.2021.799405/pdf
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Olfactory Neuroblastoma: Surgical Treatment Experience of 42 Cases
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Frontiers in surgery
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cc-by
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ORIGINAL RESEARCH
published: 01 February 2022
doi: 10.3389/fsurg.2021.799405 ORIGINAL RESEARCH
published: 01 February 2022
doi: 10.3389/fsurg.2021.799405 ORIGINAL RESEARCH
published: 01 February 2022
doi: 10.3389/fsurg.2021.799405 ORIGINAL RESEARCH
published: 01 February 2022
doi: 10.3389/fsurg.2021.799405 Xiao Cai 1,2,3,4, Zhouying Peng 1,2,3,4, Hua Zhang 1,2,3,4, Ruohao Fan 1,2,3,4, Yan Fang 1,2,3,4 and
Zhihai Xie 1,2,3,4* Xiao Cai 1,2,3,4, Zhouying Peng 1,2,3,4, Hua Zhang 1,2,3,4, Ruohao Fan 1,2,3,4, Yan Fang 1,2,3,4 and
Zhihai Xie 1,2,3,4* 1 Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China,
2 Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha, China, 3 National Clinical Research
Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China, 4 Anatomy Laboratory of Division
of Nose and Cranial Base, Clinical Anatomy Center of Xiangya Hospital, Central South University, Changsha, China Objective: Our purpose was to estimate the safety and effectiveness of the endoscopic
endonasal approach (EEA) in olfactory neuroblastoma (ONB) and determine whether
preservation of the dura and olfactory bulb could be considered in selected patients. Keywords: olfactory neuroblastoma, endoscopic endonasal surgery, skull base surgery, survival rate, prognosis Methods:
We retrospectively reviewed patients diagnosed with ONBs between
July 2010 and June 2021 at our institution, and collected data on demographic,
disease stage, surgical approach, overall survival (OS), disease-free survival (DFS), and
postoperative complications. Edited by:
Xiaobiao Zhang,
Fudan University, China Edited by:
Xiaobiao Zhang,
Fudan University, China
Reviewed by:
A. B. Zulkiflee,
University Malaya Medical
Centre, Malaysia
Jianfeng Liu,
China-Japan Friendship
Hospital, China
*Correspondence:
Zhihai Xie
xiedoctor@csu.edu.cn Reviewed by:
A. B. Zulkiflee,
University Malaya Medical
Centre, Malaysia
Jianfeng Liu,
China-Japan Friendship
Hospital, China Results:
The study sample included 42 patients (8 treated for recurrence and 34
initial cases), 28 of which were men and 14 were women with a median age of 47.19
years. The mean duration from the beginning of treatment and follow-up time was
8.91 and 51 months, respectively. Among the 42 patients, 32 had unilateral lesions,
and the rest had bilateral lesions. Patient symptoms were predominantly nasal, and
four patients presented without any symptoms. The modified Kadish staging was A in
three patients, B in 14 patients, C in 17 patients, and D in 8 patients. According to
the preoperative examinations, five patients had cervical lymph node metastasis, and
no patients had distant metastases. EEA was used in 38 patients, cranioendoscopic
approach in 3, and open craniofacial approach in 1. The 5-year OS and DFS rates were
89.1 and 79.2%, respectively. The 2-year OS and DFS rates were both 89.1%. The
overall surgical complication incidence was 9.52% (one cerebrospinal fluid rhinorrhea,
one cervical hematoma, and two epileptic seizures). *Correspondence:
Zhihai Xie
xiedoctor@csu.edu.cn Specialty section:
This article was submitted to
Surgical Oncology,
a section of the journal
Frontiers in Surgery
Received: 21 October 2021
Accepted: 29 December 2021
Published: 01 February 2022
Citation:
Cai X, Peng Z, Zhang H, Fan R,
Fang Y and Xie Z (2022) Olfactory
Neuroblastoma: Surgical Treatment
Experience of 42 Cases. Front. Surg. 8:799405. doi: 10.3389/fsurg.2021.799405 Specialty section:
This article was submitted to
Surgical Oncology,
a section of the journal
Frontiers in Surgery Received: 21 October 2021
Accepted: 29 December 2021
Published: 01 February 2022 Conclusion:
The present results support the importance of earlier treatment for
advanced ONB and the fact that it is safe and efficacious to treat ONBs via EEA. The
preservation of the dura can be considered for select patients—specifically those without
skull base involvement and who underwent postoperative comprehensive therapy. ORIGINAL RESEARCH
published: 01 February 2022
doi: 10.3389/fsurg.2021.799405 Keywords: olfactory neuroblastoma, endoscopic endonasal surgery, skull base surgery, survival rate, prognosis INTRODUCTION TABLE 1 | The clinical and demographic data of the patients. Number of patients Percentage (%)
Sex
Males
28
66.67
Females
14
33.33
Age
>45 years
27
64.29
≤45 years
15
35.71
Initial cases
34
80.95
Unilateral lesion
32
76.19%
Symptom
Epistaxis
21
Nasal obstruction
20
Hyposmia or anosmia
5
Headache
5
Ocular symptoms
5
Pain of nose
3
No symptom
4
Kadish
A
3
7.14
B
14
33.33
C
17
40.48
D
8
19.05
NLN* metastasis (before the treatment)
5
11.9
*NLN, neck lymph node. Number of patients Percentage (%) Olfactory neuroblastoma is an extremely rare malignant tumor
of the nasal cavity that arises from the olfactory neuroepithelium. It accounts for 3–6% of the nasal cavity and nasal sinus
malignancies; although, as contemporary histologic techniques
are likely to increase detection, it is difficult to determine the
true incidence (1). Although olfactory neuroblastoma (ONB) is
an uncommon disease, a portion of its characteristics has been
identified. Its incidence does not differ significantly according
to gender distribution. It affects a wide range of age groups,
but most commonly occurs between 50 and 60 years of age. The tumor can involve peripheral parts such as the paranasal
sinuses, cribriform plate, and orbits (2). The most common site
of metastasis is the cervical lymph nodes (10–33% of patients),
with relatively few distant metastases. Kadish et al. developed the
most referenced staging system. This system divides tumors into
three groups: group A tumors are limited to the nasal cavity,
group B tumors involve the nasal cavity and paranasal sinuses,
and group C tumors extend beyond the nasal cavity and paranasal
sinuses (3). The modification of this staging system by Morita
et al. established group D for tumors with regional (neck lymph
nodes) or distant metastases (4). The standard treatment for ONB is a comprehensive therapy
that includes surgical resection and postoperative radiotherapy. En-block resection via craniofacial approach (CFA) has been
the gold standard surgical modality for ONB previously
(5). However, the treatment modalities have changed. Based
on remarkable progress in technology, endoscopic endonasal
approaches (EEAs) have gained acceptance and become an
alternative standard for the surgical treatment of ONB (1, 6–9). *NLN, neck lymph node. sino-nasal component. The lamina papyracea, cribriform plate,
fovea ethmoidalis, planum sphenoidale, dura, brain, olfactory
bulbs, and tracts were resected depending on the extent of
tumor involvement. Statistical Methods We studied epidemiological data, treatment options, histologic
outcomes, postoperative complications, disease-related or other
outcomes, and the course of the disease. Descriptive statistics
for scaled values and frequencies of study patients within
the categories for each of the parameters of interest were
enumerated. OS and DFS rates were determined using the
Kaplan-Meier method. The statistical significance of differences
between the actuarial curves was evaluated using the log-rank
test. Follow-up time was defined as the time from the end date
of treatment for the original disease to first recurrence, death,
or last contact. For all tests, the significance was set at p <
0.05. Statistical tests were performed with the assistance of the
Statistical Product and Service Solutions (SPSS) Statistics 24
statistical software application (International Business Machines
Corporation, USA). Citation: Cai X, Peng Z, Zhang H, Fan R,
Fang Y and Xie Z (2022) Olfactory
Neuroblastoma: Surgical Treatment
Experience of 42 Cases. Front. Surg. 8:799405. doi: 10.3389/fsurg.2021.799405 February 2022 | Volume 8 | Article 799405 Frontiers in Surgery | www.frontiersin.org Endoscopic Resection of ONB Cai et al. TABLE 1 | The clinical and demographic data of the patients. Number of patients Percentage (%) TABLE 1 | The clinical and demographic data of the patients. Patient Characteristics The study included all patients with ONB who underwent surgery
between July 2010 and June 2021 at our institution. Each case was
diagnosed via histopathological examination. Seven patients who
had been treated for recurrence were identified among 42 patients
with ONB. Each patient underwent a preoperative endoscopy,
a sino-nasal CT scan, MRI, and a CT scan or X-ray of the
chest. Tumor staging was based on the Kadish staging system,
which was initially based on imaging data and then corrected
after surgery based on histological data. All patients underwent
surgery by the same surgeon. INTRODUCTION The skull base is reconstructed in multiple
layers, including the fascia lata of the thigh or mucosa flap,
when available. Our purpose was to estimate the safety and effectiveness
of EEA as an ONB surgical treatment standard. We also
strove to determine whether preservation of the dura and
olfactory bulb could be considered in select patients without skull
base involvement and if outcomes were similar to those who
underwent resection of the dura and olfactory bulbs. RESULTS There are three surgical methods for treating ONB: EEA,
CFA, and the cranioendoscopic approach. The first step is
tumor resection of the nasal cavity and sinuses. It is vital to
identify the attachment of the tumor origin and resection of the Patients FIGURE 2 | Kaplan-Meier graph of DFS of 42 cases of ONB. Patients The
clinical
and
demographic
data
of
the
patients
are
summarized in Table 1. The study sample included 8 patients February 2022 | Volume 8 | Article 799405 Frontiers in Surgery | www.frontiersin.org 2 Endoscopic Resection of ONB Cai et al. TABLE 2 | Treatment modality. Number of patients
Percentage (%)
Surgery methods
EEA*
38
90.48
Cranioendoscopic approach
3
7.14
CFA**
1
2.38
Resection including dura,
part of brain, and olfactory
bulbs, and tracts
31
73.81
Surgery only
14
33.33
Comprehensive therapy***
28
66.67
*EEA, endoscopic endonasal approach; **CFA, open craniofacial approach. ***Comprehensive therapy, Surgery and radiotherapy or chemotherapy or both. TABLE 3 | Treatment strategies used for each resection range. Group
A*
B*
Total
11
31
Kadish
A
1
2
B
7
7
C
1
16
D
2
8
Comprehensive therapy
100%
54.84%
Hymans
1–2
3
17
3–4
3
8
Surgery methods
EEA
100%
87.10%
*A, Resection without dura, part of the brain, and olfactory bulbs, and tracts; B, Resection
including dura, part of the brain, and olfactory bulbs, and tracts. TABLE 2 | Treatment modality. Number of patients
Percentage (%)
Surgery methods
EEA*
38
90.48
Cranioendoscopic approach
3
7.14
CFA**
1
2.38
Resection including dura,
part of brain, and olfactory
bulbs, and tracts
31
73.81
Surgery only
14
33.33
Comprehensive therapy***
28
66.67
*EEA, endoscopic endonasal approach; **CFA, open craniofacial approach. ***Comprehensive therapy, Surgery and radiotherapy or chemotherapy or both. patients who used EEA (90.48%), 3 used the cranioendoscopic
approach (7.14%), and 1 used CFA (2.38%). Of the 42 patients,
31 (73.81%) underwent resection including the dura, part of
the brain, olfactory bulbs, and tracts (Table 3). We decided
the resection range according to the preoperative images
(preoperative endoscopy, a skull-base HRCT scan, MRI) and
intraoperative observation which could help us to estimate
that if there were skull base involvement. Some patients
without skull base involvement estimated by the preoperative
images and intraoperative observation also underwent resection
including the dura, part of the brain, olfactory bulbs, and
tracts, for example, the patient No. 27 (Supplementary Figure 1). Postoperative radiotherapy was performed in 27 patients
(64.29%), of whom 14 (51.85%) underwent chemotherapy at the
same time. Postoperative radiotherapy was not performed in 15 *EEA, endoscopic endonasal approach; **CFA, open craniofacial approach. ***Comprehensive therapy, Surgery and radiotherapy or chemotherapy or both. TABLE 3 | Treatment strategies used for each resection range. TABLE 3 | Treatment strategies used for each resection range. Patients Group
A*
B*
Total
11
31
Kadish
A
1
2
B
7
7
C
1
16
D
2
8
Comprehensive therapy
100%
54.84%
Hymans
1–2
3
17
3–4
3
8
Surgery methods
EEA
100%
87.10%
*A, Resection without dura, part of the brain, and olfactory bulbs, and tracts; B, Resection
including dura, part of the brain, and olfactory bulbs, and tracts. TABLE 3 | Treatment strategies used for each resection range. Group
A*
B*
Total
11
31
Kadish
A
1
2
B
7
7
C
1
16
D
2
8
Comprehensive therapy
100%
54.84%
Hymans
1–2
3
17
3–4
3
8
Surgery methods
EEA
100%
87.10%
*A, Resection without dura, part of the brain, and olfactory bulbs, and tracts; B, Resection
including dura, part of the brain, and olfactory bulbs, and tracts. FIGURE 1 | Kaplan-Meier graph of OS of 42 cases of ONB. FIGURE 2 | Kaplan-Meier graph of DFS of 42 cases of ONB. FIGURE 1 | Kaplan-Meier graph of OS of 42 cases of ONB. *A, Resection without dura, part of the brain, and olfactory bulbs, and tracts; B, Resection
including dura, part of the brain, and olfactory bulbs, and tracts. who had been treated for recurrence (19.05%) and 34 initial
cases (80.95%). Included in this study were 28 males (66.67%)
and 14 females (33.33%). The average age at presentation was
47.19 years (range = 16–79 years). The mean duration from
the beginning of treatment and follow-up time was 8.91 months
(range = 5 days−72 months) and 51 months (range = 2–127
months), respectively. Among the 42 patients, 32 (76.19%) had
only unilateral lesions and the remainder (23.81%) had bilateral
lesions. The order of symptom sequence was epistaxis (n =
21), nasal obstruction (n = 20), hyposmia or anosmia (n = 5),
headache (n = 5), ocular symptoms (n = 5), and pain in the
nose (n = 3). There were four patients without any symptoms. The modified Kadish staging was A in 3 patients (7.14%), B
in 14 patients (33.33%), C in 17 patients (40.48%), and D in
8 patients (19.05%). Five patients (11.90%) had cervical lymph
node metastasis, while no patients had distant metastasis at
presentation according to the preoperative examination. FIGURE 1 | Kaplan-Meier graph of OS of 42 cases of ONB. FIGURE 1 | Kaplan-Meier graph of OS of 42 cases of ONB. Operative Findings and Additional
Treatment All patients underwent surgery with curative intent. The
treatment modalities are shown in Table 2. There were 38 FIGURE 2 | Kaplan-Meier graph of DFS of 42 cases of ONB. February 2022 | Volume 8 | Article 799405 Frontiers in Surgery | www.frontiersin.org 3 Endoscopic Resection of ONB Cai et al. FIGURE 3 | (A) Kaplan-Meier graph of OS according to resection; (B)
Kaplan-Meier graph of DFS according to resection (a, including dura, part of
the brain, and olfactory bulbs, and tracts; b, not). FIGURE 4 | Kaplan-Meier graph of DFS according to Ka
according to the modified Kadish stage of t
or “C and D”). Although the 5-year DFS of
advanced tumors (71.5%) was lower than th
early-stage tumors (94.1%), the differences w
significant (p = 0.7) (Figure 4). Complications
The overall surgical complication incidence w
One patient had cerebrospinal fluid rhinorrh
cervical hematoma. Both patients underwen
Two patients had epileptic seizures. Representative Case
A 41-year-old man with persistent nasal obs
nasal mass (Figure 5). The patient underwent
resection. Dural resection was performed, and
uninvolved. Radiotherapy and chemotherapy FIGURE 3 | (A) Kaplan-Meier graph of OS according to resection; (B)
Kaplan-Meier graph of DFS according to resection (a, including dura, part of
the brain, and olfactory bulbs, and tracts; b, not). FIGURE 4 | Kaplan-Meier graph of DFS according to Kadish stage. FIGURE 4 | Kaplan-Meier graph of DFS according to Kadish stage. FIGURE 4 | Kaplan-Meier graph of DFS according to Kadish stage. according to the modified Kadish stage of tumors (“A and B”
or “C and D”). Although the 5-year DFS of patients with more
advanced tumors (71.5%) was lower than that of patients with
early-stage tumors (94.1%), the differences were not statistically
significant (p = 0.7) (Figure 4). Complications The overall surgical complication incidence was 9.52% (4 of 42). One patient had cerebrospinal fluid rhinorrhea, and one had a
cervical hematoma. Both patients underwent another surgery. Two patients had epileptic seizures. Representative Case A 41-year-old man with persistent nasal obstruction had a left
nasal mass (Figure 5). The patient underwent an EEA for tumor
resection. Dural resection was performed, and the brain appeared
uninvolved. Radiotherapy and chemotherapy were administered
postoperatively. No postoperative complications were observed. The patient remained disease-free 29 months postoperatively. FIGURE 3 | (A) Kaplan-Meier graph of OS according to resection; (B)
Kaplan-Meier graph of DFS according to resection (a, including dura, part of
the brain, and olfactory bulbs, and tracts; b, not). patients (35.71%), but one of them underwent chemotherapy
(6.67%). All symptoms were relieved after the surgery. Oncological Outcomes (D–F) The patient remained
toperatively These findings emphasize the importance of earlier treatment of
advanced ONB. studies are required to validate this finding, particularly for
local recurrence. The gold standard treatment for sino-nasal tumors since
it was first described by Ketcham et al. (14) was open
craniofacial resection and radiotherapy. Recently, EEA has been
accepted as a common surgical method as an alternative to
CFA. Previous reports have indicated that endoscopic resection
can replace traditional craniofacial resection in select patients
with ONBs (1, 6–9). Rimmer et al. reviewed 95 patients
with ONB for a mean follow-up of 89 months, who were
treated with endoscopic or craniofacial resection and reported
no significant difference in outcomes between endoscopic
and craniofacial resection (2). A recent meta-analysis of 609
patients with ONBs concluded that endoscopic resection has
a comparable control rate to craniofacial resection (10). In
our study, there was no significant difference in the 5-year
DFS between endoscopic and craniofacial resection. Functional
preservation and fewer complications should be noted as
advantages of EEA in comparison to traditional CFA. There
was only one patient who had a cervical hematoma and
one of two patients in our study, who had epileptic seizures
after surgery, underwent CFA. Of the possible post-operative
complications, CSF leak is the most relevant and was found in
∼10% of cases in previous studies (2, 15–17). In the present
study, only one CSF leak was observed, suggesting the safety
of our surgical modality. However, long-term observational Although postoperative radiotherapy is standard, there are
some exceptions like the small tumors with good prognosis
and extensive surgical resection both visually and histologically
(including some ONBs) (18). We advised all the patients to
go to the oncology department and ask for comprehensive
therapy like radiation therapy. And the oncologists evaluated
if the patients need and are able to endure comprehensive
therapy according to our surgery and patients’ physical condition. In our study, comprehensive therapy was not performed
in 14 patients. Traditionally, resection of the cribriform, dura, part of the
brain, olfactory bulbs, and tracts, regardless of tumor stage,
has been the standard for all ONB treatments. This is due
to two reasons. First, the gold standard treatment for sino-
nasal tumors is open craniofacial resection. Oncological Outcomes The Kadish staging system was established in 1976 with three
grades (A, B, and C) based on the extent of the primary tumor. This was modified to include a new grade (D) for patients with
lymph nodes or distant metastases. However, the prognostic
value of the Kadish staging system was not constant. Several
studies have found that the early Kadish stages (A or B) have
favorable survival rates (2, 10, 11) whereas having not (12, 13). In our study, the 5-year DFS of patients with higher grades (C,
D) was lower than that of patients with lower grades (A, B). The differences were not statistically significant. All 3 patients
who had neck lymph node metastasis after the therapy had the
highest grades (C, D) at first, and only 1 of 5 patients who had
distant metastasis after therapy had the lower grade (B) at first. The 5-year OS and DFS rates were 89.1 and 79.2%, respectively. The 2-year OS and DFS rates were 89.1% (Figures 1, 2). The
incidence of local and regional recurrence was 11.9% (5 of 42),
and the average recurrence time was 51.6 months (range = 11–
102 months). Three patients (7.14%) had cervical lymph node
metastasis, while 5 patients (11.9%) had distant metastasis. The
5-year cumulative OS and DFS in patients treated with resection
of the dura, part of the brain, and olfactory bulbs, and tracts was
86.5 and 71% compared to 100 and 100% for those who were not
by Kaplan Meier (Figure 3). There was no significant difference
in DFS between patients who underwent open and endoscopic
surgery (p = 0.44). The DFS of patients was separately assessed February 2022 | Volume 8 | Article 799405 Frontiers in Surgery | www.frontiersin.org 4 Cai et al. Endoscopic Resection of ONB FIGURE 5 | (A–C) MRI imaging in this 41-year-old male patient with persistent nasal obstruction revealed a large, left nasal mass. (D–F) The patient remained
disease-free 29 months postoperatively. FIGURE 5 | (A–C) MRI imaging in this 41-year-old male patient with persistent nasal obstruction revealed a large, left nasal mass. (D–F) The patient remained
disease free 29 months postoperatively FIGURE 5 | (A–C) MRI imaging in this 41-year-old male patient with persistent nasal obstruction revealed a large, left nasal mass. (D–F) The patient remained
disease-free 29 months postoperatively. ing in this 41-year-old male patient with persistent nasal obstruction revealed a large, left nasal mass. Oncological Outcomes The development of
endoscopic surgery allows for resection of the intranasal tumor
and cribriform plate bone while avoiding unnecessary resection
of the dura and olfactory bulb, and it is now the treatment for
select sino-nasal tumors. Second, the theory that the origin of the
tumor is the olfactory neuroepithelium in the superior nasal vault
and cribriform is widely held, and many surgeries are considered
incomplete unless the dura and olfactory apparatus are resected. The olfactory neuroepithelium is also dispersed throughout the
superior turbinate, middle turbinate, nasal cavity, and numerous February 2022 | Volume 8 | Article 799405 Frontiers in Surgery | www.frontiersin.org 5 Cai et al. Endoscopic Resection of ONB AUTHOR CONTRIBUTIONS ZX, HZ, and RF contributed to conception and design of the
study. HZ, RF, and YF organized the database. XC performed
the statistical analysis and wrote the first draft of the manuscript. ZP and ZX wrote sections of the manuscript. All authors
contributed to manuscript revision, read, and approved the
submitted version. SUPPLEMENTARY MATERIAL The present results support the importance of earlier treatment
of advanced ONB and the thesis that it is safe and efficacious
to treat ONBs via EEA. The present results also suggested that
preservation of the dura may be considered in select patients
without skull base involvement and who were able to undergo
postoperative comprehensive therapy. This should be reassessed
following long-term observations of oncologic outcomes in
our series. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fsurg. 2021.799405/full#supplementary-material Supplementary Figure 1 | All the pictures were from patient No. 27. (A) HES ×5:
Pathological picture of the olfactory bulbs. And the olfactory bulbs were not
involved. (B) HES ×20: Pathological picture of the olfactory bulbs. (C) HES ×5:
Pathological picture of tumor tissues. (D) The tumor cells were positive for Ki-67. Supplementary Figure 1 | All the pictures were from patient No. 27. (A) HES ×5:
Pathological picture of the olfactory bulbs. And the olfactory bulbs were not Supplementary Figure 1 | All the pictures were from patient No. 27. (A) HES ×5:
Pathological picture of the olfactory bulbs. And the olfactory bulbs were not
involved. (B) HES ×20: Pathological picture of the olfactory bulbs. (C) HES ×5:
Pathological picture of tumor tissues. (D) The tumor cells were positive for Ki-67. involved. (B) HES ×20: Pathological picture of the olfactory bulbs. (C) HES ×5:
Pathological picture of tumor tissues. (D) The tumor cells were positive for Ki-67. ETHICS STATEMENT The study was reviewed and approved by The Medical Ethics
Committee of Xiangya Hospital of Central South University. Ethical review and approval was not required for the study on
human participants, in accordance with the local legislation and
institutional requirements. Long-term observation is critical to determine the oncological
outcomes of ONBs. The mean follow-up period in the present
study was 51 months, which is not sufficient to evaluate the effect
of the intervention on prognosis. Rimmer et al. reported that after
an average of 49 months, local and regional recurrence occurred
(2) and Nalavenkata et al. reported an average of 60 months (21). Therefore, continuous and careful observation is necessary for
our study. DATA AVAILABILITY STATEMENT reports of ectopic origins of ONB have been reported in recent
studies (19, 20). Besides the low rates of complications, there was
no difference in survival in our study in patients treated with or
without resection of the dura and olfactory bulb, suggesting that
without skull base involvement, the morbidity of dural resection
could have been avoided in selected patients. In our study,
we decided the resection range according to the preoperative
images (preoperative endoscopy, a sino-nasal CT scan, MRI)
and intraoperative observation to estimate the presence of skull
base involvement. All patients in whom no resection of the
dura and olfactory bulb(s) was performed had no skull base
involvement, and they underwent postoperative comprehensive
therapy (radiotherapy, chemotherapy, or both). The original contributions presented in the study are included
in the article/Supplementary Materials, further inquiries can be
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Synergistic Effects of Traffic-Related Air Pollution and Exposure to Violence on Urban Asthma Etiology
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
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nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4892366 Citation Citation
Clougherty, Jane E., Jonathan I. Levy, Laura D. Kubzansky, P. Barry Ryan, Shakira Franco Suglia,
Marina Jacobson Canner, and Rosalind J. Wright. 2007. Synergistic effects of traffic-related air
pollution and exposure to violence on urban asthma etiology. Environmental Health Perspectives
115(8): 1140-1146. Clougherty, Jane E., Jonathan I. Levy, Laura D. Kubzansky, P. Barry Ryan, Shakira Franco Suglia,
Marina Jacobson Canner, and Rosalind J. Wright. 2007. Synergistic effects of traffic-related air
pollution and exposure to violence on urban asthma etiology. Environmental Health Perspectives
115(8): 1140-1146. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Synergistic effects of traffic-related air pollution
and exposure to violence on urban asthma etiology
Citation
Clougherty, Jane E., Jonathan I. Levy, Laura D. Kubzansky, P. Barry Ryan, Shakira Franco Suglia,
Marina Jacobson Canner, and Rosalind J. Wright. 2007. Synergistic effects of traffic-related air
pollution and exposure to violence on urban asthma etiology. Environmental Health Perspectives
115(8): 1140-1146. Synergistic effects of traffic-related air pollution
and exposure to violence on urban asthma etiology
Citation
Clougherty, Jane E., Jonathan I. Levy, Laura D. Kubzansky, P. Barry Ryan, Shakira Franco Suglia,
Marina Jacobson Canner, and Rosalind J. Wright. 2007. Synergistic effects of traffic-related air
pollution and exposure to violence on urban asthma etiology. Environmental Health Perspectives
115(8): 1140-1146. Synergistic Effects of Traffic-Related Air Pollution and Exposure to Violence
on Urban Asthma Etiology Of multiple exposure periods, year-of-diagnosis NO2 was most predictive of
asthma outcomes. CONCLUSIONS: We found an association between traffic-related air pollution and asthma solely
among urban children exposed to violence. Future studies should consider socially patterned sus-
ceptibility, common spatial distributions of social and physical environmental factors, and potential
synergies among these. Prospective assessment of physical and social exposures may help determine
causal pathways and critical exposure periods. Few studies have examined the influence
of stress on pollution susceptibility, though
some findings suggest differential susceptibil-
ity by SEP, possibly mediated by life stress
(Morello-Frosch and Shenassa 2006). Time-
series studies indicate effect modification of
short-term pollution exposures by SEP
(Jerrett et al. 2004; Lin et al. 2004; Martins
et al. 2004), though others found no signifi-
cant modification (Zanobetti and Schwartz
2000). Fewer studies have considered long-
term exposures, though some indicate greater
associations between long-term air pollution KEY WORDS: childhood asthma, exposure to violence (ETV), geographic information systems (GIS),
intraurban variability, nitrogen dioxide (NO2), social–environmental synergy, stress. Environ
Health Perspect 115:1140–1146 (2007). doi:10.1289/ehp.9863 available via http://dx.doi.org/
[Online 22 March 2007] The gradient of socioeconomic position (SEP)
on health may be explained partly by a combi-
nation of increased contaminant exposures
and greater susceptibility to their effects. Air
pollution, for instance, may be higher near
major roadways, power plants, and industrial
sites, where property values are lower and
lower-income populations reside (Graves
1988). Increased life stress among lower-SEP
populations has also been proposed as a pri-
mary pathway through which SEP affects
health (Gee and Payne-Sturges 2004;
Morello-Frosch and Shenassa 2006). the resultant influence on asthma exacerbation
patterns (Maantay 2007). However, fewer
studies have considered disproportionate
susceptibility among lower-SEP populations. Exposure to violence (ETV) has been
conceptualized as a chronic urban stressor,
potentially elevated in communities where
pollution is higher. Chronic stress effects of
episodic violence are grounded in trauma the-
ory (e.g., post-traumatic stress), detailed else-
where (Wright 2006). Episodic violence,
post-traumatic stress (Augustyn et al. 2002;
Overstreet and Braun 2000), and hyper-
vigilance (Gordon and Riger 1991)—more
prevalent in lower-SEP urban communities
(Sampson et al. 1997)—may negatively influ-
ence health though physiologic alterations,
including immune dysregulation, and behav-
ioral pathways. Many urban caregivers, for
example, restrict children’s behavior, keeping
them indoors due to fear of violence (Levy
et al. 2004; Wright et al. Synergistic Effects of Traffic-Related Air Pollution and Exposure to Violence
on Urban Asthma Etiology Jane E. Clougherty,1 Jonathan I. Levy,1 Laura D. Kubzansky,2 P. Barry Ryan,3 Shakira Franco Suglia,1,4
Marina Jacobson Canner,5 and Rosalind J. Wright2,5 1Department of Environmental Health, and 2Department of Society, Human Development, and Health, Harvard School of Public Health,
Boston, Massachusetts, USA; 3Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, Georgia,
USA; 4Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts, USA; 5Channing Laboratory, Department
of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA Chronic stress has been linked to asthma
exacerbations in cross-sectional (Oh et al. 2004) and prospective (Sandberg et al. 2004)
population studies. Other evidence suggests a
role for stress in the onset of asthma (Wright
et al. 2002, 2004a). Chronic stress may influ-
ence hypothalamic–pituitary–adrenal (HPA)
axis and cortisol dysregulation (Hellhammer
et al. 1997; Ockenfels et al. 1995), glucocor-
ticoid resistance (Miller et al. 2002), sympa-
thetic–adrenal–medullary (SAM) activation,
catecholamine production (Glaser and
Kiecolt-Glaser 2005), immune mediator
function, inflammation (Umetsu et al. 2002),
and cytokine production (Chen et al. 2003;
Wright et al. 2004a). Stress and pollution
affect some common physiologic systems,
facilitating synergistic effects; for example,
psychological stress (Epel et al. 2004) and
ozone (Fugisawa 2005) both affect oxidative
stress pathways. BACKGROUND: Disproportionate life stress and consequent physiologic alteration (i.e., immune
dysregulation) has been proposed as a major pathway linking socioeconomic position, environ-
mental exposures, and health disparities. Asthma, for example, disproportionately affects lower-
income urban communities, where air pollution and social stressors may be elevated. OBJECTIVES: We aimed to examine the role of exposure to violence (ETV), as a chronic stressor, in
altering susceptibility to traffic-related air pollution in asthma etiology. METHODS: We developed geographic information systems (GIS)–based models to retrospectively
estimate residential exposures to traffic-related pollution for 413 children in a community-based
pregnancy cohort, recruited in East Boston, Massachusetts, between 1987 and 1993, using monthly
nitrogen dioxide measurements for 13 sites over 18 years. We merged pollution estimates with
questionnaire data on lifetime ETV and examined the effects of both on childhood asthma etiology. RESULTS: Correcting for potential confounders, we found an elevated risk of asthma with a 1-SD
(4.3 ppb) increase in NO2 exposure solely among children with above-median ETV [odds ratio
(OR) = 1.63; 95% confidence interval (CI), 1.14–2.33)]. Among children always living in the same
community, with lesser exposure measurement error, this association was magnified (OR = 2.40;
95% CI, 1.48–3.88). Analysis was supported by American Lung
Association Lung Health Dissertation grant (J.E.C.).
Data collection and analysis for the nitrogen dioxide
passive network and violence exposure assessment
were supported by the Massachusetts Port Authority
(P.B.R.) and K08 HL 04187 (R.J.W.), respectively.
J.I.L. was supported by Health Effects Institute grant
4727-RFA04-5/05-1 and National Institutes of
Health grant R03 ES013988. S.F.S. was supported
by National Research Service Award F31 HD049317
and T32ES007142. Address correspondence to J.E. Clougherty, Harvard
School of Public Health; Department of
Environmental Health, Landmark Center Room
404, P.O. Box 15677, Boston, MA 02215 USA.
Telephone: (617) 384-8811. Fax: (617) 384-8859.
E-mail: jcloughe@hsph.harvard.edu Address correspondence to J.E. Clougherty, Harvard
School of Public Health; Department of
Environmental Health, Landmark Center Room
404, P.O. Box 15677, Boston, MA 02215 USA.
Telephone: (617) 384-8811. Fax: (617) 384-8859.
E-mail: jcloughe@hsph.harvard.edu Accessibility Research Materials and Methods land-use regression (LUR) models (Brauer
et al. 2002; Brunekreef et al. 1997). LUR
shows strong predictive power for intraurban
nitrogen dioxide variability (Hochadel et al. 2006; Sahsuvaroglu and Jerrett 2004), using
traffic and land use characteristics (i.e., popu-
lation density, major sources). and mortality among less-educated adults
(Hoek et al. 2002; Krewski et al. 2000). Pregnant women were recruited from
East Boston Neighborhood Health Center
(EBNHC), Boston, Massachusetts, between
1987 and 1993, as described elsewhere
(Hanrahan et al. 1992). East Boston is a
working-class urban neighborhood bisected
by major highways and access roads to Logan
International Airport (Figure 1). Following
888 live births originally enrolled, caregivers
of 417 children completed questionnaires in
1997 detailing the child’s lifetime violence
exposure. Loss to follow-up was attributed
largely to families moving out of the neigh-
borhood; those who moved but continued to
participate are included. Written informed
consent was obtained from participants
(mothers) before study initiation, in accor-
dance with both Brigham and Women’s
Hospital and Beth Israel Deaconess Medical
Center Human Subjects Committees. In urban settings, traffic-related air pollu-
tion may be elevated along with ETV, and pre-
vious studies have linked traffic-related air
pollution to asthma exacerbation and respira-
tory outcomes. In the United States and
Europe, children living or attending school
near truck routes and highways show increased
asthma and allergy symptoms (Brauer et al. 2002), hospitalizations (Edwards et al. 1994;
Lin et al. 2002), allergic rhinitis (Duhme et al. 1996), and reduced lung function (Brunekreef
et al. 1997). Traffic-related pollutants have also
been associated with asthma development
(Gordian et al. 2006; Zmirou et al. 2004). y
j
In this study, we explore the hypothesis
that a chronic stressor (here, lifetime ETV)
predicts stronger associations between traffic-
related air pollution exposure and childhood
asthma development. We employ data from
the Maternal-Infant Smoking Study of East
Boston (MISSEB), a community-based
prospective pregnancy cohort examining
asthma, respiratory, and cognitive develop-
ment. Questionnaires were administered
detailing violence exposures (both witnessing
and victimization) and avoidance behaviors
(staying in at night, avoiding certain areas,
keeping children indoors). Because the MIS-
SEB did not examine air pollution, we used
geographic information systems (GIS) and
LUR at the neighborhood scale to retrospec-
tively estimate pollution exposures, using
monthly NO2 data collected over 18 years in
the surrounding neighborhoods. Incorporating traffic-related air pollution
into large-scale epidemiologic studies requires
models linking traffic and ambient concentra-
tions. Synergistic Effects of Traffic-Related Air Pollution and Exposure to Violence
on Urban Asthma Etiology 2004b), making chil-
dren more sedentary, increasing indoor expo-
sures, and decreasing spatial autonomy that is
important to development (Katz 1991). Because of this potential spatial covariance
across exposures, and because stress and pollu-
tion may influence common physiologic path-
ways (i.e., oxidative stress) and health outcomes
(i.e., respiratory disease), stronger methods are
needed to disentangle their effects and investi-
gate synergies (Gee and Payne-Sturges 2004;
O’Neill et al. 2003; Weiss and Bellinger 2006). The environmental justice literature has docu-
mented significant disproportionate contami-
nant exposures in minority and lower-SEP
communities (Brulle and Pellow 2006), and The authors declare they have no competing
financial interests. Received 27 October 2006; accepted 22 March
2007. 1140 VOLUME 115 | NUMBER 8 | August 2007 • Environmental Health Perspectives Air pollution and violence in asthma etiology Materials and Methods Passive NO2 samples were collected con-
temporaneously 1 week each month from
January 1987 through December 2004, using
Palmes tubes and analyzed by spectrophotom-
etry. Samples were collected at 28 unob-
structed locations, 1 m above ground, across
the Logan Airport grounds and surrounding
communities (Ayres 2006). We used monthly
averages for 13 sites within community
spaces, geocoded by hand using aerial photog-
raphy. Geocoding (identifying residential
locations on an active map embedded in a
GIS) allowed for the analysis of spatial charac-
teristics of each location. Missing concentra-
tions were imputed using weighted average
concentrations for surrounding months at the
same site. Violence exposure assessment. Exposure
to violence was assessed using the My Child’s
ETV scale (Selner-O’Hagan et al. 1998). At
interview, children ranged from 4 to 11.5 years
of age; those > 8 years of age also answered the
questionnaire themselves. The questionnaire
includes items on witnessing events: a) hitting,
slapping, punching, b) a shooting, c) a stab-
bing, or d) hearing gunshots. We added one
question on witnessing domestic verbal abuse. Respondents indicate the event frequency,
over their lifetime and previous year, on a scale
of 1 (0–1 time) to 4 (more than 10 times). Caregivers reported child’s approximate age at
first witnessing. Residential retrospective exposure esti-
mates. Using the predictive NO2 model, we
created residential exposure indices. Each
child’s address at enrollment (during preg-
nancy) and follow-up were geocoded using the
ESRI StreetMap Address Locator (ESRI,
Redlands, CA), and spatial variables derived in
GIS, to apply Equation 1 to each address. NO2 exposure estimates were defined for each
year of follow-up, for both reported resi-
dences. NO2 exposures for years following
1997 were derived using the questionnaire
address; between 1990 and 1997, lacking resi-
dential information, we created time-weighted
annual exposures using the two known
addresses. For example, estimated exposure in
1991 for a child enrolled in 1990 is: [(6/7) ×
(estimated NO2 in 1991 at residence reported
at enrollment) + (1/7) × (estimated NO2 in
1991 for residence reported in 1997)]. Rasch modeling summarized responses
into a continuous score, modeling the condi-
tional probabilities for each “yes” response,
given the presumed event severity and each
child’s true-but-unobserved exposure (a latent
construct) (Kindlon et al. 1996). Materials and Methods Relationships between traffic and
health have been examined using several dif-
ferent traffic indicators, with no consensus on
which indictors best capture variability in
traffic-related pollution or health outcomes in
different settings. Prior studies have success-
fully extrapolated traffic exposures from sam-
pling homes to larger cohorts using predictive Measures. Traffic-related pollution expo-
sures. NO2 has been shown to be a reliable
indicator for traffic-related primary air pollu-
tion (Hochadel et al. 2006; Nieuwenhuijsen
2000). We used a long-term spatially resolved
NO2 data set, explored temporal trends in Figure 1. Distribution of East Boston cohort and NO2 sampling sites. (A) Residential addresses at enrollment (about 1990). (B) Residential addresses at violence
questionnaire date (1997). 21.2–25.0
25.0–30.0
30.0–33.0
33.0–36.0
36.0–49.0
NO2 sampling sites
Cohort addresses 1997
NO2 estimate (ppb)
22.5–26.0
26.0–28.0
28.0–31.0
31.0–35.0
35.0–43.7
NO2 sampling sites
Cohort addresses intake
NO2 estimate (ppb)
A
B
0
445
890
1,780
2,670
3,560
Meters
N
N
0
445
890
1,780
2,670
3,560
Meters Figure 1. Distribution of East Boston cohort and NO2 sampling sites. (A) Reside
questionnaire date (1997). 22.5–26.0
26.0–28.0
28.0–31.0
31.0–35.0
35.0–43.7
NO2 sampling sites
Cohort addresses intake
NO2 estimate (ppb)
A
0
445
890
1,780
2,670
3,560
Meters
N ntial addresses at enrollment (about 1990). (B) Residential addresses at violence
21.2–25.0
25.0–30.0
30.0–33.0
33.0–36.0
36.0–49.0
NO2 sampling sites
Cohort addresses 1997
NO2 estimate (ppb)
B
N
0
445
890
1,780
2,670
3,560
Meters A A B Figure 1. Distribution of East Boston cohort and NO2 sampling sites. (A) Residential addresses at enrollment (about 1990). (B) Residential addresses at violence
questionnaire date (1997). Figure 1. Distribution of East Boston cohort and NO2 sampling sites. (A) Residential addresses at enrollment (about 1990). (B) Residential addresses at violence
questionnaire date (1997). 1141 Environmental Health Perspectives •
VOLUME 115 | NUMBER 8 | August 2007 Environmental Health Perspectives •
VOLUME 115 | NUMBER 8 | August 2007 Clougherty et al. distribution in NO2 could not be rejected
(Shapiro-Wilk p < 0.0001), concentrations
were not transformed. Models were run
in PROC GLM, corroborated in PROC
MIXED (SAS Institute Inc., Cary, NC) with
random effects by site, to account for within-
site autocorrelation. pollution concentrations, compared multiple
traffic indicators as predictors of concentra-
tions, and developed retrospective exposure
indices. optimal exposure interval, which is applied
going forward. Sensitivity analyses on the
final epidemiologic models assessed whether
interval selection affected results. Materials and Methods Expanding
on this approach, the model was generalized
to account for features theoretically influenc-
ing severity—frequency, whether events
occurred at home, whether the child knew the
victim(s) or perpetrator(s)—and included
parent and child’s report, wherever available,
as detailed elsewhere (Franco Suglia 2006). To explain variability in NO2, we consid-
ered 25 traffic indicators derived from
Massachusetts Highway Department (MHD)
1990 data (Table 1) and site characteristics
(land use, elevation, proximity to industrial
areas, population density) derived from U.S. Census 2000 data and aerial photography
(U.S. Census Bureau 2001). We created this lifetime NO2 exposure
trajectory for each child, and calculated expo-
sures during seven intervals potentially influ-
encing asthma etiology: a) life course through
diagnosis (or end of follow-up); b) year of
birth; c) before 5 years of age (median age of
diagnosis); d) year of ETV questionnaire
(known residential address); e) years between
first violent event and diagnosis; f ) year of
diagnosis; and g) 1 year before diagnosis. [NO2]ij = β1j × Yearj + β2 × (traffici)
+ β3 × (land usei) + eij ,
[1] [NO2]ij = β1j × Yearj + β2 × (traffici)
+ β3 × (land usei) + eij ,
[1] [NO2]ij = β1j × Yearj + β2 × (traffici)
+ β3 × (land usei) + eij ,
[1] [NO2]ij = β1j × Yearj + β2 × (traffici)
+ β3 × (land usei) + eij ,
[1] [1] As a validity check, we assessed the
relationship between the Rasch ETV and
Checklist of Children’s Distress Symptoms
(CCDS) (Richters and Martinez 1990),
administered contemporaneously. CCDS is a
parental report of 24 post-traumatic stress
symptoms over the prior 6 months (e.g., irri-
tability, inability to fall asleep, nightmares,
fear of attending school). The lifetime Rasch
ETV and 6-month CCDS were significantly
correlated (Spearman r = 0.21, p < 0.0001),
corroborating an association between violence
and distress symptoms reported elsewhere
(Martinez and Richters 1993). where Yearj is a categorical indicator for each
sampling year j and capturing secular pollu-
tion trends; traffici is a suite of traffic charac-
teristics for site i (candidate variables listed in
Table 1); and land usei is a suite of candidate
site characteristics (listed above). For intervals ending at diagnosis, median
diagnosis age was used for nonasthmatics. Some exposures after diagnosis (i.e., at ETV
questionnaire) are considered to be attributed
to the measure’s relative stability. Air pollution and violence in asthma etiology Air pollution and violence in asthma etiology logit(asthma diagnosis) =
intercept + β1 × (IHighETV)
+ β2 × (NO2) + β3 × (IHighETV × NO2)
+ (potential confounders). [3] Traffic-related pollution and LUR models. Over 18 years, 2,291 monthly NO2 samples
were collected and analyzed, averaging 24.0
ppb (0.78–69.4 ppb). Some overall NO2
decline occurred over time, with some re-
ordering of sites (Figure 2). LUR models
explained significant variability as a function
of secular trends, traffic density within 500 m,
distance from major roads, and block group
population density (R2 = 0.83; Table 3). More
variability in NO2 was explained by spatial
(R2 = 0.53) than temporal terms (R2 = 0.29). Residential estimates averaged 27.5 ppb
(18.7–42.6 ppb) overall, higher than meas-
ured concentrations, because several NO2
samplers were located in open spaces (i.e.,
parks, backyards), whereas cohort homes
clustered near major roads (Figure 1). follow-up visits. Maternal education was cate-
gorized as less than high school, high school
or technical school graduate, or some college. At each clinic visit during pregnancy, mothers
were asked about smoking status. A urine
specimen was obtained for determination of a
creatinine-corrected cotinine level as detail,
and mothers were classified as never-smokers
during pregnancy if they always reported
never smoking and their urine cotinine levels
were consistently < 200 ng cotinine/mg crea-
tinine (Hanrahan et al. 1992). Maternal
asthma status was based on report of physi-
cian-diagnosed asthma using the standardized
American Thoracic Society respiratory ques-
tionnaire (Ferris 1978). [3] Equation 3 produces the statistical test of the
interaction; if β3 differs significantly from
zero, ETV significantly modifies the associa-
tion of NO2 on asthma. Because NO2 data were collected only in
East Boston and adjoining Winthrop, we
expect NO2 estimates to be more accurate for
children always living in these neighborhoods. Likewise, the Rasch ETV likely better reflects
chronic stress among children always living in
the same community where exposure occurred. Therefore, all statistical analyses are performed
for the entire cohort, and repeated using only
lifetime residents of East Boston and Winthrop. Data analysis. We examined independent
associations of air pollution and ETV on
asthma diagnosis using univariate odds ratios
(ORs). To examine the modifying effect of
violence on the NO2–asthma relationship, we
constructed two interaction models, of the
forms used elsewhere (Tsaih et al. 2004): j
g
Effects of NO2 and ETV on asthma. Results We were able to geocode home addresses for
409 children at follow-up, and 369 at enroll-
ment. Demographics of those children and
their caregivers are presented in Table 2. Caregivers of 100 children (24%) reported
asthma diagnoses during follow-up; caregivers
of an additional seven children (2%) retro-
spectively reported asthmatic bronchitis. Only
8% of mothers reported ever having asthma. ETV varied significantly; at least one violent
event was reported for 46% of children, at
least two for 18%, and Rasch scores ranged
from –1.00 to 2.93. Lifetime residents did
not significantly differ from the full cohort in
ETV, asthma rates, or other demographic
factors or confounders. logit(asthma diagnosis) = intercept + β1 × (ILowETV × NO2) intercept + β1 × (ILowETV × NO2) + β2 × (IHighETV) + β3 × (IHighETV × NO2) + β2 × (IHighETV) + β3 × (IHighETV × NO2)
+ (potential confounders),
[2] For the full cohort, we found no indepen-
dent effect of ETV on asthma (OR = 0.98;
95% CI, 0.78–1.22). Stratified analyses, how-
ever, indicated that year-of-diagnosis NO2
was significantly associated with asthma solely
among children with above-median ETV
(OR = 1.65; 95% CI, 1.16–2.34). For chil-
dren with below-median ETV, there was no
association between NO2 and asthma (OR =
0.94; 95% CI, 0.70–1.26). + β2 × (IHighETV) + β3 × (IHighETV × NO2)
+ (potential confounders),
[2] + (potential confounders), where ILowETV = 1 if below-median ETV (ref-
erence group), 0 otherwise. IHighETV = 1 if
above-median ETV, 0 otherwise. NO2 is a
centered continuous variable with SD = 1.0;
ORs thus refer to a 1-SD increase above the
mean. Potential confounders included mater-
nal asthma, education, smoking before and
after pregnancy, child’s sex and age. Equation
2 produces the slopes by ETV group and
their significance. Among lifetime resident children, with
less expected exposure measurement error, we 1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
50
40
30
20
10
0
NO2 (ppb)
Webster St. (11)
East Boston Health Center (12)
Day Square (13)
Coleridge and Short Sts. (14)
Bayswater St. (15, 16, 17)
Winthrop (18, 19, 20)
Marginal St. (26)
Playing fields (29)
Lamson and Sumner Sts. (30)
Year Figure 2. Annual average NO2 (ppb) across 13 neighborhood sampling sites. For legibility, average annual
values at three clustered sites on Bayswater Street and in adjacent Winthrop have been combined. Air pollution and violence in asthma etiology Univariate ORs for the seven candidate NO2
exposure periods indicated that a 1-SD increase
only in year-of-diagnosis NO2 (4.3 ppb)
showed near-significant associations with
asthma for the full cohort [OR = 1.17; 95%
confidence interval (CI), 0.94–1.46], and was
used going forward as our key exposure metric. Materials and Methods We used
univariate logistic regression for each NO2
interval against asthma diagnosis to select an Candidate variables were selected with
p < 0.05 in univariate nonparametric associa-
tions with NO2. Backward elimination pro-
duced a parsimonious model with p < 0.1 for
all terms. Because the assumption of normal Table 1. Traffic indicators explored as predictors of monthly NO2 concentrations at sampling sites. Table 1. Traffic indicators explored as predictors of monthly NO2 concentrations at sampling sites. Indicator type
Unit
Cumulative density scores
Unweighted traffic density within 50-, 100-, 200-, 300-, 500-m buffers
Vehicle-meters per day/m2
Kernel (inverse-distance)-weighted density: 50-, 100-, 200-, 300-, 500-m buffers
Vehicle-meters per day/m2
Density of urban roads (> 8,500 cars/day) within 200 m
Vehicle-meters per day/m2
Summary measures
Total road length within 50, 100, 200, 300, 500 m
Meters
Total average daily traffic × length within 200 m
Vehicle-meters per day/m2
Distance-based measures
Distance to nearest urban road (> 8,500 cars/day)
Meters
To nearest major road (> 13,000 cars/day)
Meters
To nearest highway (> 19,000 cars/day)
Meters
To nearest MHD-designated truck route
Meters
Characteristics of nearest major road
Average daily traffic
Vehicles/day
Average daily traffic/distance to major road
(Vehicles/day)/m
Diesel fraction
Percent
Trucks per day
Vehicles/day
Trucks/distance to major road
(Vehicles/day)/m Child’s asthma diagnosis. During MISSEB
follow-up, parental reports of child’s asthma
diagnosis were ascertained through bi-
monthly telephone or face-to-face interviews
over the child’s first 2 years, every 6 months
through 4 years of age, and annually there-
after. Parents were asked “Since we last spoke
to you, have you been told by a doctor or
nurse that your child has asthma?” and like-
wise for asthmatic bronchitis. The same ques-
tions were asked on ETV questionnaire
administration. Children were considered to
have asthma if the parent reported any diag-
nosis of asthma or asthmatic bronchitis. Potential confounders. We also ascer-
tained demographic characteristics, smoking,
and medical history through standardized
questionnaires administered during MISSEB 1142 VOLUME 115 | NUMBER 8 | August 2007 • Environmental Health Perspectives VOLUME 115 | NUMBER 8 | August 2007 • Environmental Health Perspectives Values are percent or mean ± SD. Environmental Health Perspectives •
VOLUME 115 | NUMBER 8 | August 2007 Results Lifetime
Full cohort
residents
Characteristic
(n = 413)
(n = 255)
Child
Sex [female (%)]
50
53
Race (%)
Hispanic
52
52
Caucasian
44
44
Asthma diagnosis (%)
Overall
26
24
Low ETV–Low NO2
24
22
Low ETV–High NO2
26
21
High ETV–Low NO2
16
11
High ETV–High NO2
36
43
Age in 1997
6.8 ± 1.6
6.6 ± 1.6
Rasch ETV
0.60 ± 0.97
0.60 ± 0.96
NO2 year of diagnosis
27.5 ± 4.3
27.6 ± 4.3
Mother (%)
Asthma diagnosis
7.7
7.0
Less than high school education 41
43
Smoker
25
22
Values are percent or mean ± SD Results Numbers in the key correspond to sampling sites in Figure 1. 1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
50
40
30
20
10
0
NO2 (ppb)
Webster St. (11)
East Boston Health Center (12)
Day Square (13)
Coleridge and Short Sts. (14)
Bayswater St. (15, 16, 17)
Winthrop (18, 19, 20)
Marginal St. (26)
Playing fields (29)
Lamson and Sumner Sts. (30)
Year g
A second regression model contained
main effects for ETV and NO2, and their
interaction: A second regression model contained
main effects for ETV and NO2, and their
interaction:
Environmental Health Perspectives •
VOLUME 115 | NUMBER 8 | August 2007
1143
Table 2. Characteristics of cohort participants. Lifetime
Full cohort
residents
Characteristic
(n = 413)
(n = 255)
Child
Sex [female (%)]
50
53
Race (%)
Hispanic
52
52
Caucasian
44
44
Asthma diagnosis (%)
Overall
26
24
Low ETV–Low NO2
24
22
Low ETV–High NO2
26
21
High ETV–Low NO2
16
11
High ETV–High NO2
36
43
Age in 1997
6.8 ± 1.6
6.6 ± 1.6
Rasch ETV
0.60 ± 0.97
0.60 ± 0.96
NO2 year of diagnosis
27.5 ± 4.3
27.6 ± 4.3
Mother (%)
Asthma diagnosis
7.7
7.0
Less than high school education 41
43
Smoker
25
22
Values are percent or mean ± SD. Figure 2. Annual average NO2 (ppb) across 13 neighborhood sampling sites. For legibility, average annual
values at three clustered sites on Bayswater Street and in adjacent Winthrop have been combined. Numbers in the key correspond to sampling sites in Figure 1. 1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
40
30
20
10
0
NO2 (ppb)
Webster St. (11)
East Boston Health Center (12)
Day Square (13)
Coleridge and Short Sts. (14)
Bayswater St. (15, 16, 17)
Winthrop (18, 19, 20)
Marginal St. (26)
Playing fields (29)
Lamson and Sumner Sts. (30)
Year Table 2. Characteristics of cohort participants. Table 2. Characteristics of cohort participants. Table 2. Characteristics of cohort participants. Table 2. Characteristics of cohort participants. Lifetime
Full cohort
residents
Characteristic
(n = 413)
(n = 255)
Child
Sex [female (%)]
50
53
Race (%)
Hispanic
52
52
Caucasian
44
44
Asthma diagnosis (%)
Overall
26
24
Low ETV–Low NO2
24
22
Low ETV–High NO2
26
21
High ETV–Low NO2
16
11
High ETV–High NO2
36
43
Age in 1997
6.8 ± 1.6
6.6 ± 1.6
Rasch ETV
0.60 ± 0.97
0.60 ± 0.96
NO2 year of diagnosis
27.5 ± 4.3
27.6 ± 4.3
Mother (%)
Asthma diagnosis
7.7
7.0
Less than high school education 41
43
Smoker
25
22
Values are percent or mean ± SD. Figure 2. Annual average NO2 (ppb) across 13 neighborhood sampling sites. For legibility, average annual
values at three clustered sites on Bayswater Street and in adjacent Winthrop have been combined. Numbers in the key correspond to sampling sites in Figure 1. 1143 Environmental Health Perspectives •
VOLUME 115 | NUMBER 8 | August 2007 Clougherty et al. significance (adjusted OR = 1.52; 95% CI,
1.05–2.19) for children with above-median
ETV, in the full cohort multivariate model. We repeated analyses removing 10 children
whose diagnosis occurred before first violent
event, with little bearing on results. Last, sen-
sitivity analyses indicated that median-
dichotomized My ETV, My Child’s ETV,
and CCDS scores could not be substituted
for the Rasch indicator with significance. found similar effects with greater magnitude. There was a near-significant effect of NO2 on
asthma (OR = 1.28; 95% CI, 0.97–1.69),
with no independent effect of ETV (OR =
1.12; 95% CI, 0.84–1.48). Stratified analyses
indicated that NO2 was significantly associ-
ated with asthma solely among children with
above-median ETV (OR = 2.33; 95% CI,
1.47–3.71, vs. OR = 0.87; 95% CI, 0.59–1.28
with below-median ETV). interactions across numerous potential risk
factors. The pollution model relies on NO2
data, commonly used as a marker of primary
vehicular emissions, but not necessarily repre-
sentative of all pollutants of interest. Road
construction and airplane technology changes
made it unclear a priori whether NO2 could
be predicted by spatial indicators in this neigh-
borhood, particularly using 1990 traffic data,
though our model proved robust. We lacked
complete residential history, likely creating
some misclassification in a cohort with signifi-
cant residential instability. Discussion We found an association between traffic-related
pollution (NO2) and asthma diagnosis only
among children with elevated ETV, after con-
trolling for potential confounders. Of multiple
exposure intervals, year-of-diagnosis NO2 best
predicted asthma (with some evidence for NO2
after first violence exposure), supporting a theo-
retical model wherein individuals may become
susceptible or “primed” through social path-
ways to some environmental triggers, including
traffic-related pollutants. Despite limitations of
our data sets, these results agree with evidence
elsewhere that chronic stress may shape biologic
response in early life (Wright et al. 2004a) and
potentiate effects of air pollution through
common physiologic systems. g
y
The interpretation of our interaction
model is complicated by the fact that behav-
iors may differ in violent neighborhoods,
where parents often keep children indoors
due to fear of violence. Therefore, children
may be more exposed to indoor NO2, which
is generally higher than outdoor NO2, and is
influenced both by infiltration from outdoors
and by smoking, gas stoves, and other sources
which may be more prevalent in lower-
income communities (Baxter et al. 2006;
Spengler et al. 1983). Future studies should
examine differential susceptibility to a wider
range of pollution exposures, including
indoor allergens and environmental tobacco
smoke; although we accounted for maternal
smoking, we did not examine the effect of
other smokers in the home. Likewise, ETV
may proxy for other social exposures responsi-
ble for the susceptibility effect we found; chil-
dren witnessing violence may have greater
family instability (Overstreet and Braun
2000) or may be directly victimized, poten-
tially leading to injury-related susceptibilities. Among the lifetime residents, in multivari-
ate logistic regression, we found similar associa-
tions with greater magnitude. Stratified
analyses indicated elevated odds of asthma with
increased NO2 solely among children with
above-median ETV (adjusted OR = 2.40; 95%
CI, 1.48–3.88). The NO2–ETV interaction
term was statistically significant (p = 0.0009),
and robust to inclusion of main effects. Our findings contribute to the existing
environmental justice literature by identifying
potential changes in pollution susceptibility
within communities affected by violence and
other stressors. We also indicate ancillary
effects of violence on children in urban com-
munities, in addition to direct injury and
post-traumatic stress. Sampson et al. Table 2. Characteristics of cohort participants. However, we found
no difference in asthma prevalence or expo-
sures between movers and nonmovers; thus we
expect misclassification to be nondifferential,
biasing results toward the null and making our
significant results more noteworthy. We used multivariate logistic regression to
formally test the observed interaction, adjust-
ing for potential confounders. In stratified
analysis across the full cohort, we found ele-
vated odds of asthma with increased NO2
solely among children with above-median
ETV (Table 4), and the magnitude was unaf-
fected by addition of potential confounders
(adjusted OR = 1.63; 95% CI, 1.14–2.33). The interaction between NO2 and ETV was
statistically significant (p = 0.03), and robust
to inclusion of both main effects. Discussion (1997)
identified associations among neighborhood
deterioration, violence, and low collective effi-
cacy (social control of neighborhoods through
residents’ collective effort); we observed that a
heightened susceptibility to pollution, associ-
ated with violence exposures or fear thereof,
may lead to synergistic health effects of social
and physical environmental conditions. Sensitivity analyses indicated that only
average NO2 for the years between first vio-
lence exposure and asthma diagnosis could be
substituted for year-of-diagnosis NO2 with Table 3. Land use regression modeling results for
annual average NO2 at 13 sampling sites (R 2 = 0.83). We aimed to investigate whether a psy-
chosocial stressor modifies pollution effects on
asthma, and would prefer a long-term stress
measure to corroborate the ETV scale. The
CCDS elicits distress using a 6-month symp-
toms recall, inappropriate to our goal of cap-
turing life-course stress. The correlation
between CCDS and violence does, however,
corroborate an association, supporting the Although our findings are biologically
plausible and highly suggestive, there are
potential limitations in interpreting our mod-
els. A clear limitation is our small sample size
for investigating multiplicative effects; despite
the relatively high asthma prevalence (26%), a
larger cohort would be required to consider Table 4. Multivariate model for asthma diagnosis [OR (95% CI)]. Table 4. Multivariate model for asthma diagnosis [OR (95% CI)]. VOLUME 115 | NUMBER 8 | August 2007 • Environmental Health Perspectives 1144 Air pollution and violence in asthma etiology plausibility of stress pathways to pollution sus-
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hood asthma: results of the Vesta case-control study. J Epidemiol Commun Health 58:18–23. Wright RJ, Cohen S, Carey V, Weiss ST, Gold DR. 2002. Parental
stress as a predictor of wheezing in infancy: A prospective
birth-cohort study. Am J Resp Crit Care Med 165:358–365. Tsaih S-W, Korrick S, Schwartz J, Amarasiriwardena C, Aro A,
Sparrow D, et al. 2004. Lead, diabetes, hypertension, and 1146 VOLUME 115 | NUMBER 8 | August 2007 • Environmental Health Perspectives
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BEMOR PARVARISHI. YATROGENIYA. YATROGENIK KASALLIKLAR. Toshkent Davlat Stomatalogiya Instituti 1-Bosqich Talabasi
Hayrullayeva Mahzumabonu Muzaffarbekovna Ilmiy Raxbar: Axmadaliyeva Dono Taganovna. Annotatsiya:Bemorlarning parvarishi bu - bеmorning ahvolini еngillatish uchun,
tibbiy profilaktika va sanitariya chora-tadbirlarini, shifokor tomonidan tayinlangan
muolajalarni o‘z vaqtida amalga oshirish va diagnostika tartibidagi muolajalarni bеlgilash
va o‘tkazish, bеmorlarni nazorat qilish va uning holatini monitoringini amalga oshirish-
birinchi tibbiy yordam ko‘rsatish va unga mos bo‘lgan tibbiy xujjatlarni
rasmiylashtirishdir. Kalit sõzlar:Bemor parvarishi, tibbiy sir, yatrogeniya, yatrogenik kasalliklar,umumiy
parvarish, xususiy parvarish, tibbiy xatolar. Tibbiyot sohasidagi har qanday ishchi shafqat, mеhribonlik, sеzgirlik va javobgarlik,
bеmorga g‘amxo‘rlik va ehtiyotkorlik bilan munosabatda bo‘lishi kеrak. Ibn Sino bеmorga
alohida yondashuv haqida: "Siz har bir kishining shaxsan o‘ziga xos tabiati borligini
bilishingiz kеrak. Hech kimga o‘xshamaydigan noyob bеmor sifatida qarash kеrak‖. Bu
so‘z nafaqat nutq madaniyatini, balki hushmuomalalik hissi, bеmorning kayfiyatini
ko‘tarish, uni ehtiyotkorona ifodalash bilan jarohatlamaslik ma'nosini anglatadigan
so‘zdir.Bеmorlarni parvarish qilishni amalga oshirishda ikkita asosiy yo‘nalish mavjud:
umumiy parvarish va maxsus parvarish. • Umumiy parvarish - kasallikning tabiatidan qat‘i nazar (umumiy tеkshiruv, tana har
oratini o‘lchash va boshqalar) umumiy parvarishlash tadbirlarini amalga oshirish. • Umumiy parvarish - kasallikning tabiatidan qat‘i nazar (umumiy tеkshiruv, tana har
oratini o‘lchash va boshqalar) umumiy parvarishlash tadbirlarini amalga oshirish. • Maxsus parvarish - kasallikning tashxisiga (masalan, xolеtsistografiya uchun kasal
tayyorlash, siydik pufagi katеtеrizatsiyasiga) qarab spеtsifik parvarish qilish tadbirlarini
amalga oshirish. • Maxsus parvarish - kasallikning tashxisiga (masalan, xolеtsistografiya uchun kasal
tayyorlash, siydik pufagi katеtеrizatsiyasiga) qarab spеtsifik parvarish qilish tadbirlarini
amalga oshirish. Yatrogеnik kasalliklar. Bеmor bilan muloqotning dеontologik printsiplarini buzish,
kasallikning rivojlanishiga olib kеlishi mumkin, bu yatrogеnik kasalliklar dеb ataladi
(yunoncha -iatros - vrach, -genesis - hosil bo‘ladi, paydo bo‘ladi). Yatrogеn kasalliklar
(yatrogеniya) bеmorning patologik axvolidir, bu kasallik shifokor yoki boshqa tibbiy
xodimning bеparvoligi yoki nojo‘yaharakatlaridan kеlib chiqadi. Bеmorga og‘zaki
muloqotlarning kamligi, zararliligi turli xil psixogеn shikastlanishlarga olib kеlishi
mumkin.Ammo 300 yildan ortiq vaqt oldin «Ingliz Gipokrati» Tomas Sidеnxеm (1624-
1689) bеmor uchun nafaqat bеmorning ruxini jaroxatlaydigan tibbiy xodimning harakati,
balki boshqa mumkin bo‘lgan omillarni - tibbiy manipulatsiyalarning noto‘g‘ri
oqibatlarining xavfini ta'kidladi. Shuning uchun hozirgi vaqtda yatrogеnik kasallikning
kеlib chiqishi tibbiyot xodimlarining muayyan harakatlariga bog‘liq bo‘lgan har qanday
kasallikka taalluqlidir. Tibbiy sir. Bеmorlarni dеontologik parvarish qilish tibbiy sirlarni birgalikda saqlash
zarurligiga bog‘liq. Tibbiy xodimlar chuqur shaxsiy xaraktеrdagi kasallik haqida ma'lumot
bеrishga xaqli emaslar. Biroq, bu talab boshqa shaxslarga xavf tug‘diradigan xolatlarga 32 2023 MAY Infeksion yatrogeniya – bu be‘morda tibbiyot xodimlarining noto‘g‘ri xarakatlari tufayli
kelib chiquvchi infeksiya. Infeksion yatrogeniyaning sababi diagnostik, organizatsion va
davolash – profilaktik tadbirlarda aseptika konunlariga rioya qilmaslik. Bundan tashqari
infeksion yatrogeniya sababi gemokomponent terapiya va emlashlarni o‘tkazishda ehtiyot
qoidalarini buzish. Faqatgina jarrohlik amaliyotining barcha bosqichlarida aseptika
qoidalariga qat‘iy rioya qilishgina infeksion yatrogeniyaning oldini olish imkonini
beradi.Barcha sanalgan yatrogeniyalarning jarrohlikka aloqasi bor. Ayniqsa somatik
yatrogeniyalar guruxidan eng xavflisi bo‘lib diagnostik yatrogeniyalar xisoblanadi, u esa
o‘z ortidan noto‘g‘ri davolash, invaziv tekshirish usullaridan keyin infeksion asoratlar
kelib chiqishi mumkin. Tibbiyotda turli yatrogen patologiyalar kelib chiqish sabablari xar
xil bo‘lib, ular jarroxlik bo‘limi shifokori ishining turli bosqichlarida kelib chiqishi
mumkin.Diagnostik bosqichda:- Be‘mor axvolini tavsiflovchi anamnez va ob‘yektiv
ma‘lumotlar, laborator, apparat – instrumental va boshqa ko‘rsatkichlarni yetarli darajada
bo‘lmasligi.- Diagnostik va jarroxlik muolajalari natijasida a‘zo va to‘qimalarning
jarohatlanishi. - Turli xil reaksiya va asoratlarga olib keluvchi diagnostik va medikamentoz preparatlarni
buyurish. - Turli xil reaksiya va asoratlarga olib keluvchi diagnostik va medikamentoz preparatlarni
buyurish. - Be‘morning tashqi ko‘rinishi, xidi, harakatlariga bo‘lgan salbiy muomila - Be‘morga bo‘lgan professional muomila chegaralaridan chiquvchi hulq, be‘mor
―shaxsiy hayotiga‖ aralashish. Davolash – profilaktika bosqichida: - Be‘morga bo‘lgan professional muomila chegaralaridan chiquvchi hulq, be‘mor
―shaxsiy hayotiga‖ aralashish. Davolash – profilaktika bosqichida: - Operatsiyaga tayyorgarlikni noto‘g‘ri va yetarli bo‘lmasligi, operatsiyadan oldingi va
keyingi profilaktika asoratlari - Operatsiyaga tayyorgarlikni noto‘g‘ri va yetarli bo‘lmasligi, operatsiyadan oldingi va
keyingi profilaktika asoratlari - Operatsiyani noto‘g‘ri bajarish(operatsiya ko‘lamini asossiz ravishda kengaytirish yoki
kamaytirish, tavsiya etilmagan jarrohlik aralashuvlarini bajarish, a‘zo va to‘qimalarning
jaroxatlanishi, yot jismni qoldirish) - Operatsiyani noto‘g‘ri bajarish(operatsiya ko‘lamini asossiz ravishda kengaytirish yoki
kamaytirish, tavsiya etilmagan jarrohlik aralashuvlarini bajarish, a‘zo va to‘qimalarning
jaroxatlanishi, yot jismni qoldirish) Foydalanilgan adabiyotlar: Foydalanilgan adabiyotlar: 2023 MAY taalluqli emas: vеnеrik kasalliklar, yuqumli kasalliklar, inson immunitеt tanqisligi virusi
(OITV) infеktsiyasi, zaharlanish va hokazolar. Bunday xolatlarda sog‘liqni saqlash
xodimlari tеgishli tashkilotlarga zudlik bilan ma‘lumot bеrishlari kеrak. Yuqumli kasallik,
oziq-ovqatdan zaharlanish yoki pеdikulyoz aniqlangan xollarda epidеmiologiya va
sanitariya-еpidеmiologiya tadbirlarini o‘tkazish uchun, sanitariya-еpidеmiologiya
stantsiyasiga tashxis qo‘yilgan ondayoq tеlеfon orqali ma'lumot bеrish va shu bilan birga
tugallangan favqulodda xabarnoma shaklini yuborish zarur . Tibbiy xatoliklar. Tibbiy xodimning ma'naviy-axloqiy mе'yorlariga rioya etishi nafaqat
ularning vazifalarini bajarishga, balki o‘z vazifalarini bajarishdan qochish yoki
profеssional bo‘lmagan tarzda bajarganligi uchun javobgarlikni ham ta'minlaydi. Tibbiy
xodimning faoliyatida ham xatolar, ham tibbiy huquqbuzarlik sodir bo‘lishi
mumkin.Tibbiy amaliyotda xatolar adashishlar bilan bog‘liq. Tibbiy qonunbuzarliklar o‘z
kasbiy majburiyatlariga bеfarq munosabatda bo‘lishdan kеlib chiqadi. Xuddi shunday
jinoyat, masalan, dori vositalarini noto‘g‘riqo‘llash, ayniqsa, kuchli dori-darmonlarni
ko‘plab ko‘llash fojiali oqibatlarga olib kеlishi mumkin.Yatrogeniya- bu shifokorning
noto‘g‘ri va to‘g‘ri xarakatlari asosida rivojlanib, natijada organizm funksiyalari buzilishi,
odatiy faoliyatini cheklashi, nogironlik yoki o‘limga olib keluvchi profilaktik, diagnostik,
davolash aralashuvi va muolajalarning noxush asoratlaridir. ―Yatrogeniya‖ termini yunon
tilidan olingan bo‘lib, iatros – shifokor + genes – keltirib chiqaruvchi, ya‘ni ―shifokor
tomonidan keltirib chikarilgan kasallik‖ degan ma‘noni anglatadi.Yatrogeniya asosida xulq
atvor me‘yorlarini buzilishi, bilim va malakaning yetishmasligi va sovuqqonlik yotadi. Bunda asosiy rolni, shubxasiz,shifokorlik etikasini buzilishi o‘ynaydi. SHuning uchun
deontologiya va yatrogeniya muammolari o‘zaro bog‘liqdir.Yatrogeniya sabablari ko‘lami
keng. Bugungi kunda yatrogen patologiyasi e‘tibor berilmaydigan yoki sezmaslik mumkin
bo‘lmagan jiddiy tibbiy-ijtimoiy muammoga aylandi. SHuni qayd etish kerakki
yatrogeniyaning o‘sib borishi, shifokorning be‘morga bo‘lgan salbiy psixologik va psixik
ta‘siriga bog‘liq. Adabiyotlarda yana yatrogeniyani diagnostik va davolash patologiyalari,
davolash va tashxis asoratlari, tibbiyotdagi noxush xolatlar, dori kasalligi, dorilarning
nojo‘ya ta‘siri, ―ikkilamchi kasalliklar‖, gospitalizm dab xam ataladi.Xirurgiyada
yatrogen patologiyaning quyidagi turlari ajratiladi: Psixogen, somatik, dori, infeksion va aralash. Psixogen yatrogeniya – bu salbiy psixogen omillar ta‘sirida rivojlanuvchi kasallik. Psixogen yatrogeniya nevroz, psixoz, nevrasteniya, isteriya, fobiya, depressiya, ko‘rquv
xissi, depressiv va ipoxondrik buzilishlar shaklida namoyon bo‘ladi. Ular tibbiyot
xodimining be‘mor salomatligi to‘g‘risida noto‘g‘ri va extiyotsizlik bilan ma‘lumot
berishi, be‘morni o‘z kasallik tarixi va maxsus tibbiy adabiyotlar bilan tanishishi,
ommaviy ma‘ruzalarni eshitish, ayniqsa televizor orqali noto‘g‘ri tushunchaga ega
bo‘lishidan kelib chiqadi. Shifokor be‘mor bilan muloqotga kirishganda shuni esda tutishi
kerakki, u be‘mor uchun katta ma‘lumot manbaidir va unga qarab be‘mor o‘z xolati
haqidagi ma‘lumotga ega bo‘ladi. Dori yatrogeniyasi – bu be‘morda shifokor buyurgan dori moddalarini qabul qilingandan
keyin kelib chiquvchi buzilishlardir. Dori yatrogeniyasi – bu be‘morda shifokor buyurgan dori moddalarini qabul qilingandan
keyin kelib chiquvchi buzilishlardir. 33 profilaktik muolajalar) natijasida kelib chiquvchi kasallik. Foydalanilgan adabiyotlar: ГРАМОТА.РУ — справочно-информационный интернет-портал «Русский язык» |
Словари | Проверка слова Schwarz O. Psychogenese und Psychoterapie körperlicher Symptome. — Wien, 1925. (Сборник работ Шильдера, Бауера, Брауна, Гейера, Штраудберга, Майера (Schilder,
Bauer, Braun, Heyer, Straudberg, Mayer).) Schwarz O. Psychogenese und Psychoterapie körperlicher Symptome. — Wien, 1925. (Сборник работ Шильдера, Бауера, Брауна, Гейера, Штраудберга, Майера (Schilder,
Bauer, Braun, Heyer, Straudberg, Mayer).) Bumke O. Der Arzt als Ursache seelischer Störungen // Deutsche Medizinische
Wochenschrift, 1925; 51 (1): 3 Bumke O. Der Arzt als Ursache seelischer Störungen // Deutsche Medizinische
Wochenschrift, 1925; 51 (1): 3 Каннабих Ю. В. К профилактике одной из форм реактивной (иатрогенной)
депрессии // Профилактика нервных и психических заболеваний / Ред. Давыденков Каннабих Ю. В. К профилактике одной из форм реактивной (иатрогенной)
депрессии // Профилактика нервных и психических заболеваний / Ред. Давыденков 34 34 2023 MAY
С. Н., Розенштейн Л. М. — Москва: Издательство Мосздравотдела, 1929. — С. 75—
88 2023 MAY
С. Н., Розенштейн Л. М. — Москва: Издательство Мосздравотдела, 1929. — С. 75—
88 88 Платонов К. И. Слово как физиологический и лечебный фактор // Психотерапия. Сборник статей под ред. проф. К. И. Платонова. Труды Государственного
психоневрологического института Народного комиссариата здравоохранения УССР. — Харьков: Государственное издательство Украины, 1930. ВОЗ призывает к принятию неотложных мер для снижения вреда, наносимого Платонов К. И. Слово как физиологический и лечебный фактор // Психотерапия. Сборник статей под ред. проф. К. И. Платонова. Труды Государственного
психоневрологического института Народного комиссариата здравоохранения УССР. — Харьков: Государственное издательство Украины, 1930. ВОЗ призывает к принятию неотложных мер для снижения вреда, наносимого
пациентам при оказании медицинской помощи“. Всемирная организация
здравоохранения (13-sentabr 2019-yil). Avdeev A. I., Kozlov S. V. Yatrogen patologiya (sud-tibbiyot ko'rinishi) // „Sud tibbiyoti
va ekspert amaliyotining dolzarb masalalari“. - Novosibirsk, 2009 yil. Avdeev A. I., Kozlov S. V. Yatrogen patologiya (sud-tibbiyot ko'rinishi) // „Sud tibbiyoti
va ekspert amaliyotining dolzarb masalalari“. - Novosibirsk, 2009 yil. Лурия Р. А.. Внутренняя картина болезней и иатрогенные заболевания, 4-е изд
15.000 экз, М.: Медицина, Lua xatosi: bad argument #2 to 'formatDate': invalid
timestamp 'Yanvar'.. Whitfield CL. Psixiatrik dorilar travma agenti sifatida // Tibbiyotda xavf va xavfsizlik
xalqaro jurnali. - 2010 yil. — Yoʻq. 22. — P. 195–207. - doi : 10.3233/JRS-2010-0508. Whitfield CL. Psixiatrik dorilar travma agenti sifatida // Tibbiyotda xavf va xavfsizlik
xalqaro jurnali. - 2010 yil. — Yoʻq. 22. — P. 195–207. - doi : 10.3233/JRS-2010-0508. 35 35
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The Challenges of Chagas Disease— Grim Outlook or Glimmer of Hope?
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The Challenges of Chagas Disease—
Grim Outlook or Glimmer of Hope? Rick L. Tarleton*, Richard Reithinger, Julio A. Urbina, Uriel Kitron, Ricardo E. Gürtler T
hrough its impact on worker productivity, premature
disability, and death, Chagas disease accounts for
670,000 disability-adjusted life years per annum [1]. This makes it the most important parasitic disease of the
Americas. It is both a disease of poverty (Figures 1 and 2)
and, like other neglected tropical diseases, also “poverty
promoting” [2]. Traditionally confined to Latin America,
Chagas disease is becoming an important health issue in
the United States and Europe. First, due to the continuous
influx of immigrants from disease-endemic countries in Latin
America, a proportion of whom are infected with Trypanosoma
cruzi, an increasing number of infected subjects are seen
in clinical practice, whether, for example, through routine
screening of US blood and organ banks [3] or physicians’
offices in Europe [4]. The appearance of T. cruzi in US
blood banks led to the implementation of the first Food and
Drug Administration–approved diagnostic blood screening
test for Chagas disease earlier this year [5]. Second, an
increasing number of autochthonous Chagas disease cases
have been reported in the US [6,7], which may mirror the
increased reporting of T. cruzi infection in domestic animals
and wildlife. Recognizing that Chagas disease can no longer
be considered an “exotic” disease in the US, the American
Society of Tropical Medicine and Hygiene held a clinical
course in Chagas disease prior to its 2007 annual meeting
(http://www.astmh.org/meetings/premeeting.cfm#clinical). wild animals (e.g., raccoons and opossums), with human
cases being relatively rare [6,7]. T. cruzi is mainly transmitted
through blood-feeding triatomine bugs, but can also occur
congenitally [7,11], through blood transfusion [12] or
organ transplantation [13], and through the ingestion of
contaminated food or fluids [14]. The complex life cycle
involves different parasite life stages in both vector and host,
all highly adapted to their respective environments, which
maximizes transmission potential and/or host immune
evasion and, hence, long-term parasite survival (http://www. dpd.cdc.gov/dpdx/HTML/TrypanosomiasisAmerican. htm#Life%20Cycle). T Most infected people do not know that they have become
infected, as—in the disease’s acute stage—the symptoms are
benign (e.g., fever, swollen lymph glands, and, occasionally,
an inflammatory reaction at the biting site or a swollen eye)
or very rare (severe myocarditis or meningoencephalitis)
[15]. Symptoms of acute infection may last up to a few weeks
or months, and parasites may be found in the blood during
this stage. Neglected Diseases The Challenges of Chagas Disease—
Grim Outlook or Glimmer of Hope? Infections then remain largely asymptomatic
(with few or no parasites found in the circulation), often for
years or even decades, until up to 30% of patients develop
chronic Chagas disease, i.e., cardiac or gastrointestinal
complications, which if left untreated are severely debilitating Funding: The ideas discussed in this article have benefited from the support to
the authors over the years by a number of research-funding bodies, including the
US National Institutes of Health (awards R01 AI-022070, R01 AI-033106, and P01
AI-044979 to RLT); The Burroughs Wellcome Fund (RLT); the Sir Halley Stewart Trust
(RR and REG); the UNICEF/UNDP/World Bank/WHO Special Program for Research
and Training in Tropical Diseases, the European Commission’s INCO-DEV Program,
and the Howard Hughes Medical Institute (JAU); the Fogarty International Center
and the National Institute of Environmental Health Sciences (US National Institutes
of Health / National Science Foundation Ecology of Infectious Disease Program
award R01 TW05836 to UK and REG); and the Agencia Nacional de Promoción
Científica y Técnica and the University of Buenos Aires (REG). The authors received
no specific funding for this article. p
g
g
p
g
Despite estimates of up to 15 million existing cases and
50,000–200,000 new infections per year, funding for research,
prevention, and control has been limited, and therapeutic
options remain unsatisfactory. Consequently, several
editorials and perspectives have recently drawn attention
to Chagas disease and T. cruzi [8–10]. While these papers
highlighted the impact of this disease on public health in the
Americas, and rightly pointed out that major achievements
have been made in its control, they failed to emphasize
several key challenges that are currently undermining these
achievements and that must be urgently addressed in order
to move to the next stage: ensuring the long-term and
sustainable control of this devastating disease. Competing Interests: The authors have declared that no competing interests exis mpeting Interests: The authors have declared that no competing int Citation: Tarleton RL, Reithinger R, Urbina JA, Kitron U, Gürtler RE (2007) The
challenges of chagas disease—Grim outlook or glimmer of hope? PLoS Med 4(12):
e332. doi:10.1371/journal.pmed.0040332 Copyright: © 2007 Tarleton et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited. Etiology, Distribution, and Clinical Manifestations gy
Chagas disease (also known as American trypanosomiasis)
is named for the Brazilian physician Carlos Chagas, who
discovered the disease exactly a century ago and published
its first description in 1909. It is caused by the protozoan
parasite T. cruzi and is found in wildlife, domestic animals,
and humans throughout much of rural as well as peri-urban
areas of Mexico, Central America, and South America. In
the US T. cruzi has been reported in dogs and a range of Rick L. Tarleton is at the Center for Tropical and Emerging Global Diseases and
Department of Cellular Biology, University of Georgia, Athens, Georgia, United
States of America. Richard Reithinger is with the Clinical Trials Area, Westat,
Rockville, Maryland, United States of America, the School of Medicine and Health
Sciences, George Washington University, Washington, D. C., United States of
America, and the London School of Hygiene and Tropical Medicine, London,
United Kingdom. Julio A. Urbina is at the Instituto Venezolano de Investigaciones
Científicas, Caracas, Venezuela, and the Drugs for Neglected Diseases Initiative,
Geneva, Switzerland. Uriel Kitron is at the Center for Zoonoses Research, University
of Illinois, Champaign, Illinois, United States of America. Ricardo E. Gürtler is with
the Department of Ecology, Genetics and Evolution, Universidad de Buenos Aires,
Buenos Aires, Argentina. The Neglected Diseases section focuses attention either on a specific disease or
describes a novel strategy for approaching neglected health issues in general. * To whom correspondence should be addressed. E-mail: tarleton@cb.uga.edu PLoS Medicine | www.plosmedicine.org December 2007 | Volume 4 | Issue 12 | e332 1852 December 2007 | Volume 4 | Issue 12 | e332 doi:10.1371/journal.pmed.0040332.g001
Figure 1. Chagas Disease Is Closely Linked to Poverty and Socioeconomic Development, and Poor Housing In Particular
Example of a typical household environment in Chagas disease–endemic areas: mud-and-thatch house with a front veranda (in the background) and
storage area (in the front), located in the dry Chaco forest around Amamá village, northern Argentina. doi:10.1371/journal.pmed.0040332.g001 Figure 1. Chagas Disease Is Closely Linked to Poverty and Socioeconomic Development, and Poor Housing In Particular
Example of a typical household environment in Chagas disease–endemic areas: mud-and-thatch house with a front veranda (in the background) and
storage area (in the front), located in the dry Chaco forest around Amamá village, northern Argentina. and in many cases, fatal [15]. Grim Outlook 1: The Challenge of Vector Control Tools to manage Chagas disease are numerous but are
severely limited with regard to crucial aspects of prevention,
detection, and treatment. Current vector control methods
and strategies have significant limitations, diagnostics are
variable and of unknown reliability, drugs for treatment are
inadequate, and vaccines are nonexistent. Yet some bright
spots exist, such as the decrease in transmission that has been
achieved through control of insect vectors and screening of
blood and blood products. PLoS Medicine | www.plosmedicine.org Etiology, Distribution, and Clinical Manifestations Chagas cardiomyopathy,
which may result in cardiac arrhythmias, apical aneurysm,
congestive heart failure, thromboembolism, and sudden
cardiac death (Figures 3 and 4), is the most common form
of cardiomyopathy in South and Central America and the
leading cause of cardiovascular death in disease-endemic
areas [16]. In people with suppressed immune systems (e.g.,
due to HIV/AIDS or chemotherapy), Chagas disease can
reactivate with abundant parasites found in the blood and
tissues. Because of its potentially long asymptomatic phase,
however, Chagas disease is often considered a “silent killer”
[9], which is one of several reasons why it fails to attract
media attention. countries, notably Mexico, no control programs for this
disease have ever been established. Among the many problems
with exclusive reliance on insecticides is that prevention of
re-infestation of houses by the insect vectors requires repeated
spraying, which also promotes the development of insecticidal
resistance—an outcome that is already documented in
Argentina [18] and is increasingly detected in Bolivia. There
are alternative insecticides, but their use is accompanied by
added problems of higher costs or increased toxicity. The decentralization of health services in many endemic
countries combined with declining funding for control
efforts has transferred the burden of control to provinces
or local communities that are ill-equipped to supply these
services. Even if vector control were better coordinated and
fully funded, eradication of either the many domestic vector
species or the parasites is not feasible for this zoonosis. Potentially, improvements in housing could permanently
reduce vectorial transmission. However, at a minimum
cost of US$200 to more than US$2,000 per house [19],
such improvements will have to await significant economic
development in endemic areas or specific investments for
housing improvements on the order of US$10 billion. Grim Outlook 2: The Challenge of Chemotherapies and
Vaccines The “Southern Cone” initiative [17] has knocked down
transmission rates dramatically in the southern tier of South
America and is credited with interrupting vector-borne
transmission via Triatoma infestans in Uruguay, Chile, and
Brazil, primarily through insecticidal spraying of houses. However, elimination of T. infestans (one of the program’s
stated objectives) has not been achieved in the core of its
distribution throughout the hyperendemic Gran Chaco region
spanning northern Argentina, Paraguay, and Bolivia. Success
may even be more difficult elsewhere in Latin America where
there are many different vector species, each with distinct
feeding and infestation behaviors, while in some endemic The current chemotherapies for T. cruzi infection and
Chagas disease have many shortcomings, as do those in drug
discovery and development. The available compounds for
treatment, benznidazole (Rochagan, Roche Pharmaceuticals)
and nifurtimox (Lampit, Bayer HealthCare), both have
severe side effects, require long courses of treatment, and
exhibit variable efficacy [20]. At present, the World Health
Organization and other sources indicate that these drugs are
active only in the acute and short-term (up to a few years)
chronic phase (http://www.who.int/tdr/diseases/chagas/
direction.htm). A number of studies now provide reason to December 2007 | Volume 4 | Issue 12 | e332 1853 doi:10.1371/journal.pmed.0040332.g002
Figure 2. Interior of House Showing the Type of Wall Construction
Often Found in Households in Many Endemic Areas
Cracks in walls are commonly found, inhabited by insects, as evidenced
by the insect feces on wall and wall hangings. question this recommendation [21–27]. Although some of
these studies were non-controlled and used cure parameters
that may be debatable because of the absence of a diagnostic
gold standard (see below), they have been remarkably
consistent in showing moderate to significant efficacy in long-
term chronic infections. Thus, whilst waiting for the results
of, for example, the BENEFIT trial (http://clinicaltrials. gov/ct/show/NCT00123916?order=1) evaluating the efficacy
of benznidazole in chronic Chagas disease patients (expected
to be completed in 2011), and unless or until better drugs are
available, benznidazole and nifurtimox should be more widely
used, based on the published evidence that such compounds
may reduce the parasite burden and moderate disease
progression in all stages of the disease [21–28]. It would
therefore be ethically questionable to restrict the drugs’
use to only patients with a defined duration of (acute and
short-term chronic) infection, as currently recommended. Grim Outlook 2: The Challenge of Chemotherapies and
Vaccines It is standard practice to use test sera that
are positive on multiple other serological tests to assess the
sensitivity of new tests, proving that any new test is no worse,
but not necessarily any better, than existing ones. However,
it is well documented that individuals with confirmed
infection are typed as inconclusive or negative on multiple
existing serological tests [34–36]. The design of tests to
detect these inconclusive or “conventional seronegative”
subjects has not been a priority. Likewise, the development of
technologies that can more rapidly assess treatment efficacy,
diagnose congenital infections, or determine the impact of
transmission control methods has been slow. Fortunately, the
development of sensitive, accurate, and practical diagnostic
methods is a highly tractable problem, given an appropriate
level of investment and interest. The development of highly
sensitive and specific diagnostic field and laboratory tools
to determine active infection is a crucial requirement for
moving the entire field forward in the research, clinical, and
public health arenas. The success of drug treatment in arresting disease
progression in subjects who have been infected for more than
20 years [21,22], coupled with a wealth of other data, has
recently prompted a welcome shift toward studying Chagas
disease as a problem of parasite persistence, rather than
primarily as a problem of an inappropriate or imbalanced
immune response [30]. This shift has also generated more
interest in vaccines as a vehicle for control or treatment. Even
so, vaccine development for T. cruzi infection has had a very
slow start and remains nonexistent as a strategy for control
or prevention. There are substantial potential problems with
vaccines for Chagas disease, not the least of which include
how one would test a conventional prophylactic vaccine for an
infectious illness that is rarely detected until years or decades
after the initial infection. However, given the cost-effective
nature of vaccines and potential innovative applications (e.g.,
in transmission blocking or as therapeutics), more research
is warranted, and vaccines should be included in the overall
long-term strategy for control, prevention, and treatment of
T. cruzi infection. Grim Outlook 2: The Challenge of Chemotherapies and
Vaccines However, the decision to treat chronic patients with these
drugs should be made on a case-by-case basis, only after
thorough clinical assessment and with continuous monitoring
of potential side effects. doi:10.1371/journal.pmed.0040332.g002 doi:10.1371/journal.pmed.0040332.g002 Figure 2. Interior of House Showing the Type of Wall Construction
Often Found in Households in Many Endemic Areas
Cracks in walls are commonly found, inhabited by insects, as evidenced
by the insect feces on wall and wall hangings. Figure 2. Interior of House Showing the Type of Wall Construction
Often Found in Households in Many Endemic Areas
Cracks in walls are commonly found, inhabited by insects, as evidenced
by the insect feces on wall and wall hangings. Academic and other noncommercial drug discovery efforts
have yielded an increasing number of targets and new drug
candidates, but surprisingly few of these promising leads have
moved beyond the discovery/candidate stage. This stalemate
is due in part to limited funding for further research and
development, but even with drugs that have been available
for more than 25 years, such as benznidazole, definitive
preclinical evidence of cure in animal models is strikingly
absent. A recent report from the Wellcome Trust–supported
Pharmaceutical Research and Development Policy Project
concluded that there is increasing industrial interest and
progress in drug development for neglected diseases
[29]—but this is certainly not the case for Chagas disease. Indeed, promising compounds such as Schering-Plough’s
posaconazole have yet to be further pursued as anti–T. cruzi
drugs, despite extensive and promising preclinical data [20]. cannot be identified and thus treated, and the effectiveness
of treatment cannot be efficiently assessed. Moreover, the
effectiveness of any control campaign, whether targeted
at insect vectors, blocking of transmission, or vaccination
of individuals, cannot be measured without competent
diagnostics. Transmission via blood transfusion or tissue
transplantation has been a point of concern for many years
in Latin America but has only come to the fore in the US and
Europe as the number of immigrants unknowingly carrying
T. cruzi has increased. Most current serological tests, whether
developed in-house or purchased commercially, employ
crude antigen preparations from inappropriate parasite life-
cycle stages (i.e., epimastigotes—which are present in the
insect vector but not in mammalian hosts). Development
of tests using one or more recombinant proteins/peptides
may be an improvement, but even these tests often provide
inconsistent and/or unreliable results [31–33]. Grim Outlook 2: The Challenge of Chemotherapies and
Vaccines The absence
of a true gold standard (i.e., a method to consistently detect
the presence of parasites in those individuals with T. cruzi
infection) makes evaluation of the sensitivity of serological
tests difficult. It is standard practice to use test sera that
are positive on multiple other serological tests to assess the
sensitivity of new tests, proving that any new test is no worse,
but not necessarily any better, than existing ones. However,
it is well documented that individuals with confirmed
infection are typed as inconclusive or negative on multiple
existing serological tests [34–36]. The design of tests to
detect these inconclusive or “conventional seronegative”
subjects has not been a priority. Likewise, the development of
technologies that can more rapidly assess treatment efficacy,
diagnose congenital infections, or determine the impact of
transmission control methods has been slow. Fortunately, the
development of sensitive, accurate, and practical diagnostic
methods is a highly tractable problem, given an appropriate
level of investment and interest. The development of highly
sensitive and specific diagnostic field and laboratory tools
to determine active infection is a crucial requirement for
moving the entire field forward in the research, clinical, and
public health arenas. cannot be identified and thus treated, and the effectiveness
of treatment cannot be efficiently assessed. Moreover, the
effectiveness of any control campaign, whether targeted
at insect vectors, blocking of transmission, or vaccination
of individuals, cannot be measured without competent
diagnostics. Transmission via blood transfusion or tissue
transplantation has been a point of concern for many years
in Latin America but has only come to the fore in the US and
Europe as the number of immigrants unknowingly carrying
T. cruzi has increased. Most current serological tests, whether
developed in-house or purchased commercially, employ
crude antigen preparations from inappropriate parasite life-
cycle stages (i.e., epimastigotes—which are present in the
insect vector but not in mammalian hosts). Development
of tests using one or more recombinant proteins/peptides
may be an improvement, but even these tests often provide
inconsistent and/or unreliable results [31–33]. The absence
of a true gold standard (i.e., a method to consistently detect
the presence of parasites in those individuals with T. cruzi
infection) makes evaluation of the sensitivity of serological
tests difficult. December 2007 | Volume 4 | Issue 12 | e332 Key Needs and Opportunities: Diagnostics and
Integrated Vector Control One of the key issues concerning Chagas disease is that of
diagnosis. Without effective diagnostics, infected individuals PLoS Medicine | www.plosmedicine.org December 2007 | Volume 4 | Issue 12 | e332 December 2007 | Volume 4 | Issue 12 | e332 1854 doi:10.1371/journal.pmed.0040332.g003
Figure 3. Cineventriculogram of a 49-Year-Old Male with Chagas Heart Disease
The picture shows the apical aneurysm (sacular) with normal coronary arteries. doi:10.1371/journal.pmed.0040332.g003
Figure 3. Cineventriculogram of a 49-Year-Old Male with Chagas Heart Disease
The picture shows the apical aneurysm (sacular) with normal coronary arteries. doi:10.1371/journal.pmed.0040332.g003 doi:10.1371/journal.pmed.0040332.g003 Figure 3. Cineventriculogram of a 49-Year-Old Male with Chagas Heart Disease
The picture shows the apical aneurysm (sacular) with normal coronary arteries. Figure 3. Cineventriculogram of a 49-Year-Old Male with Chagas Heart Disease
The picture shows the apical aneurysm (sacular) with normal coronary arteries. Although vector management has been the foundation of
the overall strategy for prevention of T. cruzi infection, this
area is also ripe for improvements. Despite the long-held
promises of vector suppression through residual spraying
with pyrethroid insecticides, current procedures often fail
to eliminate triatomine bugs, especially in semiarid rural
areas and in peridomestic habitats [37]. The lack of simple,
sensitive tools for early detection of low-density populations
of triatomine bugs that reappear after insecticide spraying
undermines the effectiveness of control and elimination
programs. Traditional high-risk settings require integrated
control programs that are tailored to local environmental
and sociocultural characteristics and employ a long-term
perspective. The bust-and-boom cycles of the recent past,
largely dependent on available funding, demonstrate that
the effectiveness of Chagas disease vector control as currently
practiced is limited. tools have been shown to effectively reduce the contact
of humans or reservoir dogs with the triatomine vector of
T. cruzi. Bed-nets and collars would become particularly
attractive options if they were used in an integrated approach
aimed at controlling other vector-borne diseases such as
malaria and leishmaniasis, respectively. The key issue is
whether or not these new tools can be applied successfully at
geographic scales ranging from individual villages to entire
regions where current methods are not sufficient for the
desired objective (control versus elimination). PLoS Medicine | www.plosmedicine.org Glimmer of Hope 6. Beard CB, Pye G, Steurer FJ, Rodriguez R, Campman R, et al. (2003)
Chagas disease in a domestic transmission cycle, southern Texas, USA. Emerg Infect Dis 9:103-105. g
7. Dorn P, Perniciaro L, Yabsley MJ, Roellig DM, Balsamo G, et al. (2007)
Autochthonous transmission of Trypanosoma cruzi, Louisiana. Emerg Infect
Dis 13: 605-507. 8. [No authors listed] (2006) Chagas’ disease—An epidemic that can no
longer be ignored. Lancet 368: 619. doi:10.1371/journal.pmed.0040332.g004 g
g
9. Maguire JH (2006) Chagas’ disease—Can we stop the deaths? N Engl J Med
355: 760-761. 10. Schofield CJ, Jannin J, Salvatella R (2006) The future of Chagas disease
control. Trends Parasitol 22: 583-588. 11. Gürtler RE, Segura EL, Cohen JE (2003) Congenital transmission of
Trypanosoma cruzi infection in Argentina. Emerg Infect Dis 9: 29-32. 12. Young C, Losikoff P, Chawla A, Glasser L, Forman E (2007) Transfusion-
acquired Trypanosoma cruzi infection. Transfusion 47: 540-547. 13. Centers for Disease Control and Prevention (2006) Chagas disease after
organ transplantation—Los Angeles, California, 2006. MMWR Morb Mortal
Wkly Rep 55: 798-800. y
p
14. Benchimol Barbosa PR (2006) The oral transmission of Chagas’ disease:
An acute form of infection responsible for regional outbreaks. Int J Cardiol
112:132-133. 15. Prata A (2001) Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis 1: 92-100. 16. Rassi A Jr, Rassi A, Little WC, Xavier SS, Rassi SG, et al. (2006)
Development and validation of a risk score for predicting death in
Chagas’heart disease. N Engl J Med 355: 799-808. doi:10.1371/journal.pmed.0040332.g004 Figure 4. Chest X-Ray of 4 Different Patients with Chagas Heart
Disease
A: normal; B: mild cardiomegaly; C: moderate cardiomegaly; D: severe
cardiomegaly with pulmonary congestion. g
g
17. Dias JC, Silveira AC, Schofield CJ (2002) The impact of Chagas disease
control in Latin America: A review. Mem Inst Oswaldo Cruz 97: 603-612. 18. Picollo M, Vassena C, Santo Orihuela P, Barrios S, Zaidemberg M, et al. (2005) High resistance to pyrethroid insecticides associated with ineffective
field treatments in Triatoma infestans (Hemiptera: Reduviidae) from
Northern Argentina. J Med Entomol 42: 637-642. g
J
19. Briceno-Leon R (1987) Rural housing for control of Chagas disease in
Venezuela. Parasitol Today 3: 384-387. and, via the sequencing of the T. cruzi genome and proteomes
[42,43], has presented multiple leads for drug targets and
diagnostic/vaccine candidates. However, neither for-profit
nor nonprofit companies have taken on the challenges
of developing these leads further. Glimmer of Hope The problems of Chagas disease are many, but they are
not insurmountable. There are numerous partial solutions
already at hand that, if used in a coordinated manner,
and with consideration of the unique characteristics of
endemic areas (e.g., rural underdevelopment, poverty, lack
of adequate housing, and increasingly decentralized health
services), could have a significant impact. The entities that
will fund and coordinate such an integrated effort remain
to be identified, but clearly the involvement of the public
sector is essential. Better diagnostics, drugs, and improved
approaches to vector control programs will require more
and better research, sharper focus, and greater rigor on the
part of the research community. In addition, contributions
from governments, the private sector, and nongovernmental
organizations will be needed to establish the infrastructure
for testing drugs and control methods as well as the platforms
to develop and implement effective and affordable diagnostic
tests. Fortunately, the research community has provided the
majority of potential drug, vaccine, and diagnostic candidates On the other hand, progress in related research can help
counter these shortcomings in vector control strategies. Our
understanding of the eco-epidemiology of infection has
significantly improved in recent years, especially with the
application of geospatial analytical tools. These advances
have allowed the development of infection and disease
transmission models [38] as well as the more targeted
planning of prevention and control strategies coupled with
continued surveillance (e.g., identification of so-called
transmission “hotspots” or loci of bug re-infestation) [39]. Several tools to prevent infection and to control (peri-)
domestic bug populations have been evaluated, including
insecticide-treated bed-nets [40] and dog collars [41], and
optimum insecticide doses in peridomestic habitats. These P December 2007 | Volume 4 | Issue 12 | e332 December 2007 | Volume 4 | Issue 12 | e332 1855 doi:10.1371/journal.pmed.0040332.g004
Figure 4. Chest X-Ray of 4 Different Patients with Chagas Heart
Disease
A: normal; B: mild cardiomegaly; C: moderate cardiomegaly; D: severe
cardiomegaly with pulmonary congestion. 3. Centers for Disease Control and Prevention (2007) Blood donor screening
for chagas disease—United States, 2006-2007. MMWR Morb Mortal Wkly
Rep 56: 141-143. p
4. Riera C, Guarro A, Kassab HE, Jorba JM, Castro M, et al. (2006) Congenital
transmission of Trypanosoma cruzi in Europe (Spain): A case report. Am J
Trop Med Hyg 75: 1078-1081. 5. Food and Drug Administration (2006) Product approval information
licensing action. ORTHO T. cruzi ELISA Test System. Available: http://
www.fda.gov/cber/products/tryorth121306.htm. Accessed 19 November
2007. Glimmer of Hope Effective scientific,
philanthropic, and political leadership and forward-thinking
coordination of a community effort in this realm, at both
local and regional levels, is badly needed if we want to make
significant inroads into this devastating disease. Ultimately,
the success of such efforts will be heavily dependent on the
long-term stability and prospects for economical, societal, and
political development in the Americas. y
20. Urbina JA, Docampo R (2003) Specific chemotherapy of Chagas disease:
Controversies and advances. Trends Parasitol 19: 495-501. 21. Viotti R, Vigliano C, Lococo B, Armenti H, LaPuente A, et al. (1998)
Chronic chagasic cardiomyopathy: Clinical and serologic evolution with
and without benznidazole in long-term follow-up. XIII World Congress of
Cardiology; 26–30 April 1998; Rio de Janeiro, Brazil. pp. 697-701. 22. Viotti R, Vigliano C, Lococo B, Bertocchi G, Petti M, et al. (2006) Long-
term cardiac outcomes of treating chronic Chagas disease with benznidazole
versus no treatment: A nonrandomized trial. Ann Intern Med 144: 724-734. 23. Andrade AL, Martelli CM, Oliveira RM, Silva SA, Aires AI, et al. (2004)
Short report: Benznidazole efficacy among Trypanosoma cruzi-infected
adolescents after a six-year follow-up. Am J Trop Med Hyg 71: 594-597. 24. Pereira-Chioccola VL, Fragata-Filho AA, Levy AM, Rodrigues MM,
Schenkman S (2003) Enzyme-linked immunoassay using recombinant trans-
sialidase of Trypanosoma cruzi can be employed for monitoring of patients
with Chagas’ disease after drug treatment. Clin Diagn Lab Immunol 10:
826-830. Supporting Information Alternative Language Text S1. Translation of the article into Spanish
by JAU Alternative Language Text S1. Translation of the article into Spanish
by JAU 25. de Andrade AL, Zicker F, de Oliveira RM, Almeida Silva S, Luquetti A, et
al. (1996) Randomised trial of efficacy of benznidazole in treatment of early
Trypanosoma cruzi infection. Lancet 348: 1407-1413. Found at doi:10.1371/journal.pmed.0040332.sd001 (71 KB DOC). 26. Sosa Estani S, Segura EL, Ruiz AM, Velazquez E, Porcel BM, et al. (1998) Efficacy of chemotherapy with benznidazole in children in the
indeterminate phase of Chagas’ disease. Am J Trop Med Hyg 59: 526-529 Acknowledgements p
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27. Sosa-Estani S, Segura EL (2006) Etiological treatment in patients infected
by Trypanosoma cruzi: Experiences in Argentina. Curr Opin Infect Dis 19:
583-587. We are grateful to Drs. Anis Rassi and Julio Lazzari for critical
comments on the manuscript; we also thank Dr. Carla Cecere for
providing us with the picture for Figure 2 and Dr. Rassi for providing
us with the pictures for Figures 3 and 4; REG provided Figure 1. The
opinions expressed in this article are those of the authors and may
not reflect the positions of their employing organizations. 28. de Castro AM, Luquetti AO, Rassi A, Chiari E, Galvao LM (2006) Detection
of parasitemia profiles by blood culture after treatment of chronic
Trypanosoma cruzi infection. Parasitol. Res 99: 379-383. 29. Moran M, Ropars AL, Guzman J, Diaz J, Garrison C (2005) The new
landscape of neglected disease drug development. The Wellcome Trust. Available: http://www.thegeorgeinstitute.org/shadomx/apps/fms/
fmsdownload.cfm?file_uuid=F2B06396-EEA0-851E-3049-C9A030AEDE0F&si
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2004. Changing history. Available: http://www.who.int/whr/2004/en/.
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validity of serologic tests for Trypanosoma cruzi and the effectiveness of
transfusional screening strategies in a hyperendemic region. Transfusion
45: 554-561. PLoS Medicine | www.plosmedicine.org PLoS Medicine | www.plosmedicine.org December 2007 | Volume 4 | Issue 12 | e332 December 2007 | Volume 4 | Issue 12 | e332 1856 peridomestic populations of Triatoma infestans in rural western Argentina: A
district-wide randomized trial. Bull World Health Organ 82: 196-205. peridomestic populations of Triatoma infestans in rural western Argentina: A
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38. Cohen JE, Gürtler RE (2001) Modeling household transmission of
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Reinfestation sources for Chagas disease vector, Triatoma infestans,
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Prevention of the transmission of Chagas’ disease with pyrethroid-
impregnated materials. Am J Trop Med Hyg 68: 307-311. 34. Wincker P, Bosseno MF, Britto C, Yaksic N, Cardoso MA, et al. (1994) High
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42. Atwood JA 3rd, Weatherly DB, Minning TA, Bundy B, Cavola C, et al. (2005) The Trypanosoma cruzi proteome. Science 309: 473-476. (2005) The Trypanosoma cruzi proteome. Science 309: 473-476 43. El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, et al. (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of
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37. Gürtler RE, Canale DM, Spillmann C, Stariolo R, Salomon OD, et al. (2004)
Effectiveness of residual spraying with deltamethrin and permethrin on December 2007 | Volume 4 | Issue 12 | e332 1857 PLoS Medicine | www.plosmedicine.org
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Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia
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Supplementary Figure S3. IL3Rα P248 at the IL3R assembly interface is critical for cell
differentiation. A, B, C, IL3 induced proliferation of FDH cells expressing: A, IL3Rα and βc
WT (black filled circle) or βc mutants of the “assembly interface”, T348A (red filled circle),
T348F (blue filled circle), T348W (black open circle), T348L (red open circle) and T348R
(blue open circle); B, βc and IL3Rα WT (black filled circle) or IL3Rα mutants of the “assembly
interface”, M246K/P248K (black open circle), M246W/P248W (blue filled circle),
M246F/P248F (blue open circle) or M246L/P248L/V249L (red filled circle); C, βc and IL3Rα
WT (black filled circle) or IL3Rα P248 mutants, P248K (black open circle), P248W (blue filled
circle), P248F (blue open circle) or P248L (red filled circle). Proliferation of FDH cells
expressing IL3Rα WT or mutant is normalized to the maximum proliferation of FDH cells
expressing WT IL3Rα and βc in 100 ng/mL IL3 for 48 hours. Data are the mean of triplicate
determinations from a representative experiment ± standard deviation of the mean (SD). Similar
results were obtained in 3 independent experiments. D, Flow cytometric analysis of cell surface
βc and IL3Rα in FDH cells expressing βc and IL3Rα P248L or WT. Representative histograms
are shown with IL3Rα (blue), βc (red) and isotype control (black). E, The binding affinity of
IL3 for IL3Rα WT or IL3Rα P248L in the absence or presence of βc was measured in saturation
binding assays using radio-iodinated IL3 and indicated as KD ± standard error of the mean
(SEM). P values are expressed relative to IL3Rα WT in respective cell lines. F, Stability of
IL3Rα P248L compared to IL3Rα WT at the cell surface as assessed by biotinylation of all cell
surface proteins followed by streptavidin (strep) pulldown enrichment and immunoblotting of
pulldowns for IL3Rα. G, Raw IL3Rα-IL3Rα’ FLIM data from a representative experiment (of
n=4 independent experiments) with cells expressing IL3Rα-mScarlet-I or IL3Rα-SYPF2 fusion
proteins and βc. FLIM donor lifetimes were converted into %FRET efficiencies (shown in Fig. 2D, 2E) as indicated in Methods. H, as for (G) but for IL3Rα P248L. I, Raw IL3Rα-βc FLIM
data from a representative experiment (of n=3 independent experiments) with cells expressing Supplementary Figure S3. IL3Rα P248 at the IL3R assembly interface is critical for cell
differentiation. A, B, C, IL3 induced proliferation of FDH cells expressing: A, IL3Rα and βc
WT (black filled circle) or βc mutants of the “assembly interface”, T348A (red filled circle),
T348F (blue filled circle), T348W (black open circle), T348L (red open circle) and T348R
(blue open circle); B, βc and IL3Rα WT (black filled circle) or IL3Rα mutants of the “assembly
interface”, M246K/P248K (black open circle), M246W/P248W (blue filled circle),
M246F/P248F (blue open circle) or M246L/P248L/V249L (red filled circle); C, βc and IL3Rα
WT (black filled circle) or IL3Rα P248 mutants, P248K (black open circle), P248W (blue filled
circle), P248F (blue open circle) or P248L (red filled circle). Proliferation of FDH cells
expressing IL3Rα WT or mutant is normalized to the maximum proliferation of FDH cells
expressing WT IL3Rα and βc in 100 ng/mL IL3 for 48 hours. Data are the mean of triplicate
determinations from a representative experiment ± standard deviation of the mean (SD). Similar
results were obtained in 3 independent experiments. D, Flow cytometric analysis of cell surface
βc and IL3Rα in FDH cells expressing βc and IL3Rα P248L or WT. Representative histograms
are shown with IL3Rα (blue), βc (red) and isotype control (black). E, The binding affinity of
IL3 for IL3Rα WT or IL3Rα P248L in the absence or presence of βc was measured in saturation
binding assays using radio-iodinated IL3 and indicated as KD ± standard error of the mean
(SEM). P values are expressed relative to IL3Rα WT in respective cell lines. F, Stability of
IL3Rα P248L compared to IL3Rα WT at the cell surface as assessed by biotinylation of all cell
surface proteins followed by streptavidin (strep) pulldown enrichment and immunoblotting of
pulldowns for IL3Rα. G, Raw IL3Rα-IL3Rα’ FLIM data from a representative experiment (of
n=4 independent experiments) with cells expressing IL3Rα-mScarlet-I or IL3Rα-SYPF2 fusion
proteins and βc. FLIM donor lifetimes were converted into %FRET efficiencies (shown in Fig. IL3Rα-mScarlet-I or IL3Rα P248L-mScarlet-I and βc-SYFP2 fusion proteins. J, Expression of
monocytic differentiation genes in FDH cells after 5 days in IL3 normalized to RPLP0
expression. n=4 independent experiments. Blue: IL3Rα P248L (hexamer); red: IL3Rα WT
(dodecamer). K, Cumulative number of FDH cells cultured with IL3 quantified at specific time
points up to 20 days. The cell number from n=3 independent experiments are shown at each
time point and the connecting line indicates the mean from independent experiments. L, M, N,
Flow cytometric analysis of %CD11b+Gr1+ (L), mature lineage cocktail (M) and CD117
expression (N) in FDH cells expressing βc and various IL3Rα P248 mutants (n=3). O, P, Fetal
liver cells from βc-/-βIL3-/- mice were transduced to express βc and IL3Rα P248L (hexamer,
blue) or WT (dodecamer, red) and assessed for % caspase-3 negative cells after IL3 withdrawal
(n=3) (O) and serial replating colony numbers in cytokine cocktail (n=3) (P). IL3Rα-mScarlet-I or IL3Rα P248L-mScarlet-I and βc-SYFP2 fusion proteins. J, Expression of
monocytic differentiation genes in FDH cells after 5 days in IL3 normalized to RPLP0
expression. n=4 independent experiments. Blue: IL3Rα P248L (hexamer); red: IL3Rα WT
(dodecamer). K, Cumulative number of FDH cells cultured with IL3 quantified at specific time
points up to 20 days. The cell number from n=3 independent experiments are shown at each
time point and the connecting line indicates the mean from independent experiments. L, M, N,
Flow cytometric analysis of %CD11b+Gr1+ (L), mature lineage cocktail (M) and CD117
expression (N) in FDH cells expressing βc and various IL3Rα P248 mutants (n=3). O, P, Fetal
liver cells from βc-/-βIL3-/- mice were transduced to express βc and IL3Rα P248L (hexamer,
blue) or WT (dodecamer, red) and assessed for % caspase-3 negative cells after IL3 withdrawal
(n=3) (O) and serial replating colony numbers in cytokine cocktail (n=3) (P).
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The Effects of Nutrition on Chronic Conditions
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Nutrients
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The Effects of Nutrition on Chronic
Conditions Ojo, O. & Adegboye, A. R. A. Published PDF deposited in Coventry University’s Repository Ojo, O. & Adegboye, A. R. A.
Published PDF deposited in Coventry University’s Repository Original citation:
Ojo, O & Adegboye, ARA 2023, 'The Effects of Nutrition on Chronic Conditions',
Nutrients, vol. 15, no. 5, 1066. https://doi.org/10.3390/nu15051066 DOI 10.3390/nu15051066
ISSN 2072-6643 Publisher: MDPI Editorial
The Effects of Nutrition on Chronic Conditions
Omorogieva Ojo 1,*
and Amanda Rodrigues Amorim Adegboye 2,3 Omorogieva Ojo 1,*
and Amanda Rodrigues Amorim Adegboye 2,3 Omorogieva Ojo 1,*
and Amanda Rodrigues Amorim Adegboye 2,3 School of Health Sciences, University of Greenwich, Avery Hill Campus, London SE9 2UG, UK
Centre for Agroecology Water and Resilience Coventry University Coventry CV8 3LG UK 1
School of Health Sciences, University of Greenwich, Avery Hill Campus, London SE9 2UG, UK 3
Centre for Healthcare Research, Coventry University, Coventry CV1 5FB, UK 3
Centre for Healthcare Research, Coventry University, Coventry *
Correspondence: o.ojo@greenwich.ac.uk The effects of nutrition on chronic conditions, such as diabetes, obesity, heart disease,
and stroke, continue to generate interest among researchers. This is based on the fact that
diet is a modifiable risk factor [1]. The composition of diet, including the proportions and
types of macronutrients and micronutrients, is a major contributor to chronic diseases [1]. The beneficial effects of nutritional interventions on chronic conditions have been
well documented, although differences remain among researchers concerning their overall
impact [1]. Evaluations of the role of nutrition in chronic conditions draw on diet’s effects on
body weight, body composition, glycemic and insulin excursions, and vascular remodelling. The effect of diet in modulating gut microbiota dysbiosis is also an evolving area of
research [2]. This Special Issue, entitled “Nutrition in Chronic Conditions”, aims to examine the
effect of nutrition in the development, care, and management of chronic conditions. This
Special Issue includes 11 original studies conducted in high- and middle-income countries,
3 systematic reviews with meta-analysis, and 3 literature reviews. This editorial provides
an overview of the key findings of the papers published in this Special Issue. These
papers are broadly divided into seven topics: the effects of diet on (1) insulin and glucose
metabolism; (2) gut health; (3) brain and cognitive impairment; (4) infections, chronic
conditions, malnutrition, and all-cause mortality; (5) obesity and dietary variables in
postmenopausal women; (6) non-alcoholic fatty liver disease in mice, specifically the
consumption of coffee; and (7) chronic conditions and COVID-19 infection. 1. Insulin and Glucose Metabolism The literature on the use of diets with a low glycemic index (GI) and a low glycemic
load (GL) in the management of diabetes in adult populations is vast. However, little
is known if GI and GL peaks are related to glycemic control, particularly in young and
healthy populations. Using a representative national school-based sample of students
(12–17 years old) without diabetes, da Rocha et al. [3] investigated the association between
dietary indicators of the quality of carbohydrate intake and markers of glycemic control. The authors found the GI of diet was better at predicting insulinemia, regardless of weight
status, compared to the GL [3]. The authors argued that guidance on food consumption
based on carbohydrate quality should be provided to adolescents as a measure of glycemic
control, as higher GIs are highly associated with the intake of refined carbohydrates. Encouraging healthy lifestyle habits combined with a diet with low GI and low GL can also
help control obesity and reduce the risk of developing type 2 diabetes [3]. Editorial Editorial Publisher: MDPI Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is
an open access article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/
4.0/). nutrients nutrients nutrients Citation: Ojo, O.; Adegboye, A.R.A. The Effects of Nutrition on Chronic
Conditions. Nutrients 2023, 15, 1066. https://doi.org/10.3390/nu15051066 Citation: Ojo, O.; Adegboye, A.R.A. The Effects of Nutrition on Chronic
Conditions. Nutrients 2023, 15, 1066. https://doi.org/10.3390/nu15051066 Received: 1 February 2023
Accepted: 8 February 2023
Published: 21 February 2023 Received: 1 February 2023
Accepted: 8 February 2023
Published: 21 February 2023 Apart from the impact of low GI diets on metabolism, postprandial insulin, glucose,
and triglyceride responses have been investigated. Louca et al. [4] found that individuals
with hypertension had higher postprandial insulinemic and lipemic responses to two
standardized test meals compared to the normotensive controls after adjustments for sex,
age, and BMI. This effect was partially mediated by visceral fat mass. No significant
difference was observed for postprandial glucose. These findings corroborate existing
literature on the key role of visceral fat in metabolic syndrome [4]. Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/nutrients Nutrients 2023, 15, 1066. https://doi.org/10.3390/nu15051066 Nutrients 2023, 15, 1066 2 of 6 A drastic and prolonged reduction in carbohydrate intake leads to the exhaustion of
glucose reserves in the body, shifting metabolism into ketogenesis and inducing hepatic
oxidation of fatty acids. This process produces ketone bodies, an important alternative
to glucose as the body’s source of energy. Dilliraj et al. [5] conducted a review to explore
current evidence regarding ketone bodies in relation to nutrition, metabolic pathways,
signalling functions, and effects on clinical conditions. Based on the studies reviewed,
ketone bodies, which are formed under normal metabolism in the absence of glucose,
play a key role in controlling oxidative stress and inflammation, resulting in improved
mitochondrial function and growth, energy rescue, and adaptative epigenetic control. However, clinical trials are needed to validate the results obtained from in vitro and in vivo
studies as well as from animal models [5]. 2. Gut Health Ojo et al. [6] conducted a systematic review and meta-analysis of randomised con-
trolled trials to evaluate the effects of almonds on gut microbiota, glycometabolism, and
inflammatory markers in patients with type 2 diabetes. This review was conducted against
the backdrop of rising global prevalence of type 2 diabetes and the recognition that nutri-
tional interventions, including the use of almonds, which are rich sources of dietary fibre,
essential minerals, protein, and monounsaturated fatty acids, may be effective in managing
symptoms of type 2 diabetes. y
p
yp
Ojo et al. [6] found that an almond-based diet was effective in promoting the growth
of short-chain fatty acid-producing bacteria and lowering glycated haemoglobin and body
mass index in patients with type 2 diabetes. The nutrient composition of almond, such as
high fibre content and low glycemic index, may be involved in the biological mechanism
of its effect. However, almonds did not appear to have a significant effect (p > 0.05) on
fasting blood glucose, postprandial blood glucose, inflammatory parameters, glucagon-like
peptide 1, and Homeostatic Model Assessment of Insulin Resistance [6]. p p
In a separate systematic review, Ojo et al. [7] carried out a network meta-analysis of
randomised controlled trials. This review aimed to evaluate the effect of prebiotics and
oral antidiabetic agents on the gut microbiome in patients with type 2 diabetes. Prebiotics
are substrates (non-viable) that are resistant to gastric acid and intestinal absorption and
are used selectively by host microorganisms, which leads to benefits [7]. Prebiotics may
promote eubiosis of the gut microbiome and ensure glucose homeostasis in patients with
type 2 diabetes. The network meta-analysis found that prebiotics significantly reduced
(p < 0.05) glycated haemoglobin, compared to the control, in patients with type 2 diabetes. However, prebiotics and oral antidiabetic agents did not have a significant effect (p > 0.05)
on the gut microbiome, body mass index, fasting blood glucose, and postprandial blood
glucose [7]. 4. Infections, Chronic Conditions, Malnutrition, and All-Cause Mortality Helicobacter pylori (H. pylori) infection is the most common cause of gastritis and other
gastrointestinal disorders worldwide. Habbash et al. [10] investigated whether there is
an association between dietary habits and H. pylori infections among 200 Bahraini adults. The authors found that among H. pylori-infected individuals, the consumption of coffee,
green tea, and honey was significantly lower compared to non-infected individuals. They
also found that vitamin D deficiency was a risk factor for H. pylori infection (OR = 1.1;
95% CI: 1.05, 1.18; p < 0.001). The authors suggested that coffee, green tea, and honey
intake might be protective against H. pylori infection [10]. However, given the retrospective,
cross-sectional study design, no causal relationship between dietary factors and H. pylori
infection could be inferred. Zupo et al. [11] conducted a systematic review and meta-analysis of the prevalence
of zinc deficiency among patients suffering from inflammatory bowel disease (IBD). Zinc
is essential for cell growth, tissue repair, and immune function. The authors included 17
studies and estimated an overall pooled prevalence of 50% (95% CI 0.48–0.52). However,
the reviewed studies showed high heterogeneity, I2 = 96% [11]. These studies were further
divided into two groups: Crohn’s disease (CD) (n = 9) and ulcerative colitis (UC) (n = 8). The prevalence of zinc deficiency was higher in patients with CD (54%) compared to those
with UC (41%). The results point out that one in two patients with IBD has zinc deficiency,
which can play a role in the severity of the disease. Therefore, clinicians should monitor
zinc levels and other trace elements in patients with IBD. p
Naber and Purohit [12] conducted a review to explore the role of diet in the manage-
ment of chronic kidney disease (CKD). The authors focused on the Dietary Approaches
to Stop Hypertension, the Mediterranean diet, and the whole-food, plant-based diet for
their potential role in delaying CKD progression. They found strong evidence supporting
the relevance of diets, which meet the daily nutritional requirement of patients, in the
prevention and progression of CKD, particularly the whole-food, plant-based diet without
the inclusion of animal products. Malnutrition is prevalent among patients with chronic heart failure (CHF) due to
the lack of appetite, unintentional weight loss, impaired intestinal function, catabolic
metabolism, and other comorbidities. Schuetz et al. [13] investigated the cost-effectiveness
of an individualised nutritional therapy in 645 hospitalised patients with CHF. 3. Brain and Cognitive Impairment Metabolic syndrome (MS) is a prevalent condition worldwide and is characterised
by a cluster of conditions, including central obesity, hyperglycemia, insulin resistance,
hypertension, and dyslipidemia. Insulin resistance, believed to be a key underlying mecha-
nism responsible for MS, affects multiple tissues and organs, including the central nervous
system, leading to cognitive impairment and Alzheimer’s disease (AD). However, the
inverse relationship between MS and cognitive impairment has not been fully explored. Rojas et al. [8] reviewed studies investigating a new hypothesis suggesting that cognitive
impairment plays a role in the development of insulin resistance and, consequently, the
appearance of MS. The authors concluded that a bidirectional relationship between MS
and cognitive impairment seems to exist. However, large-scale longitudinal studies are still
required to establish a causal relationship between these two factors [8]. In another study, Sochocka et al. [9] investigated the effect of Ginkgo biloba extract (Egb)
as an alternative therapy on the mechanisms of innate immune response of peripheral
blood leukocytes (PBLs) in patients with AD. The authors found that EGb has advantageous Nutrients 2023, 15, 1066 3 of 6 properties for health management in older adults and AD sufferers, especially in women
with AD [9]. properties for health management in older adults and AD sufferers, especially in women
with AD [9]. 5. Obesity and Dietary Variables in Postmenopausal Women There are controversial results regarding the relationship between obesity and bone
metabolism. López-Gómez et al. [16] investigated the differences in bone turnover among
250 postmenopausal women with and without obesity and compared their risk of fracture at
five years of follow-up. The authors found that a bone formation marker (P1NP) was higher
in women without obesity compared to women with obesity. However, postmenopausal
women with obesity showed lower marker levels of bone formation, especially at younger
ages. On the other hand, older women with obesity showed higher markers of bone
resorption. This might be due to a decrease in vitamin D levels in women with obesity
irrespective of age, which is associated with a high parathyroid hormone (PTH) level. However, no significant difference in the risk of fracture based on BMI was observed
(OR = 0.90; 95% CI 0.30–2.72; p = 0.85). The authors concluded that the potential protective
effect of obesity on bone mass and osteoporosis needs to be further investigated in other
studies [16]. 7. Chronic Conditions and COVID-19 Infection In 2020, healthcare systems around the world were challenged by the COVID-19 pan-
demic. During the pandemic, it was observed that age and pre-existing health conditions
(e.g., cancer, asthma, cancer, obesity, and diabetes) were risk factors for negative COVID-19
infection outcomes. Several micronutrient deficiencies were also associated with a higher
risk for severe clinical symptoms. Voelkle et al. [18] found a heightened prevalence of
micronutrient deficiencies (e.g., selenium, vitamin D, vitamin A, and zinc), particularly
in older patients hospitalised for COVID-19. These deficiencies were also associated with
more severe COVID-19 infection. The authors highlighted the need for further research
regarding the effect of micronutrient supplementation on the treatment and prevention of
COVID-19 infection [18]. The lockdown policies adopted by many countries to control the spread of the virus
had a major impact on people’s lifestyles. Arayess et al. [19] observed that a personalised
lifestyle intervention in children with overweight and obesity was less successful in de-
creasing BMI z-score during the COVID-pandemic compared to the same intervention one
year prior to the first lockdown in the Netherlands [19]. 6. The Influence of Coffee Consumption on Non-Alcoholic Fatty Liver Disease in Mice Di Mauro et al. [17] examined the effect of coffee consumption on non-alcoholic fatty
liver disease in mice. In particular, this study aimed to establish if the intake of coffee might
influence the expression of long non-coding ribonucleic acid (IncRNAs) in the liver. In this
study, 24 four-week-old male mice were housed randomly in cages. Following one week of
acclimation, the mice were randomly assigned to 1 of the 3 diets for 12 weeks, including
a standard diet, a high-fat diet, and a high-fat diet plus decaffeinated coffee solution. This study found that decaffeinated coffee was effective in modulating the expression
of IncRNAs, which are involved in the key pathways in the onset and progression of
non-alcoholic fatty liver disease [17]. 4. Infections, Chronic Conditions, Malnutrition, and All-Cause Mortality The authors
found that the overall incremental cost-effectiveness ratio for the individualised nutritional
therapy vs. no nutritional therapy was 2625 Swiss Francs per life day gained. They
concluded that the intervention increased life expectancy at an acceptable incremental
cost-effectiveness ratio [13]. Malnutrition and loss of muscle mass are also prevalent among patients with cancer. In clinical assessments, handgrip strength (HGS) is used as a proxy of overall muscle
strength. However, there are no population-specific values for HGS, particularly among
oncology patients. Tribolet et al. [14] proposed sex-specific values for HGS stratified by age
and tumour entity and tested their prognostic ability. The authors validated the prognostic
value of HGS with respect to long-term mortality in hospitalised undernourished patients
with cancer [14], which might aid clinical decisions. Kwon et al. [15] examined the association between the intake of dietary fibres and
CVD and all-cause mortality in the general population and among those with hypertension,
diabetes, and dyslipidemia in a 10-year longitudinal study. After adjustments for con-
founders, the authors found that a higher intake of fibres reduced the risk of both all-cause
mortality and CVD mortality [15]. Nutrients 2023, 15, 1066 4 of 6 References [CrossRef] [PubMed]
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Evolution of Ketosis: Potential Impact on Clinical Conditions. Nutrients 2022, 14, 3613. [CrossRef] [PubMed] lliraj, L.N.; Schiuma, G.; Lara, D.; Strazzabosco, G.; Clement, J.; Giovannini, P.; Trapella, C.; Narducci,
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R f] [P bM d] lliraj, L.N.; Schiuma, G.; Lara, D.; Strazzabosco, G.; Clement, J.; Giovannini, P.; Trapella, C.; Narducci,
volution of Ketosis: Potential Impact on Clinical Conditions. Nutrients 2022, 14, 3613. [CrossRef] [PubMed] 6. Ojo, O.; Wang, X.-H.; Ojo, O.O.; Adegboye, A.R.A. The Effects of Almonds on Gut Microbiota, Glycometabolism, and Inflammatory
Markers in Patients with Type 2 Diabetes: A Systematic Review and Meta-Analysis of Randomised Controlled Trials. Nutrients
2021, 13, 3377. [CrossRef] [PubMed] 7. Ojo, O.; Wang, X.; Ojo, O.O.; Brooke, J.; Jiang, Y.; Dong, Q.; Thompson, T. The Effect of Prebiotics and Oral Anti-Diabetic Agents on
Gut Microbiome in Patients with Type 2 Diabetes: A Systematic Review and Network Meta-Analysis of Randomised Controlled
Trials. Nutrients 2022, 14, 5139. [CrossRef] [PubMed] [
] [
]
8. Rojas, M.; Chávez-Castillo, M.; Pirela, D.; Parra, H.; Nava, M.; Chacín, M.; Angarita, L.; Añez, R.; Salazar, J.; Ortiz, R.; et al. Metabolic Syndrome: Is It Time to Add the Central Nervous System? Nutrients 2021, 13, 2254. 17.
Mauro, S.; Salomone, F.; Scamporrino, A.; Filippello, A.; Morisco, F.; Guido, M.; Lembo, V.; Cossiga, V.; Pipitone, R.M.; Grimaudo,
S.; et al. Coffee Restores Expression of lncRNAs Involved in Steatosis and Fibrosis in a Mouse Model of NAFLD. Nutrients 2021,
13, 2952. [CrossRef] 8. Conclusions Based on the above research findings, it is clear that nutrition plays an important role
in the development and severity of chronic conditions in children, adults, and older adults. Therefore, healthy dietary patterns should be promoted, and further research should be
conducted to fully understand the biological pathways regarding how diet may influence
chronic diseases. Nutrients 2023, 15, 1066 5 of 6 Author Contributions: Conceptualization, O.O. and A.R.A.A.; methodology O.O. and A.R.A.A.;
validation, O.O. and A.R.A.A.; formal analysis, O.O. and A.R.A.A.; writing—original draft prepara-
tion, O.O. and A.R.A.A.; writing—review and editing, O.O. and A.R.A.A. All authors have read and
agreed to the published version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 18.
Voelkle, M.; Gregoriano, C.; Neyer, P.; Koch, D.; Kutz, A.; Bernasconi, L.; Conen, A.; Mueller, B.; Schuetz, P. Prevalence of
Micronutrient Deficiencies in Patients Hospitalized with COVID-19: An Observational Cohort Study. Nutrients 2022, 14, 1862.
[CrossRef] [
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19.
Arayess, L.; Knockaert, N.; Winkens, B.; Lubrecht, J.W.; Verweij, M.; Vreugdenhil, A.C.E. The Side-Effects of the COVID-19
Pandemic: Increased BMI z-Score in Children with Overweight and Obesity in a Personalised Lifestyle Intervention One Year
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In situ observations of the Swiss periglacial environment using GNSS instruments
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Earth system science data
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In situ observations of the Swiss periglacial
environment using GNSS instruments Alessandro Cicoira1,2,3,⋆, Samuel Weber1,4,5,6,7,⋆, Andreas Biri4, Ben Buchli4, Reynald Delaloye2,
Reto Da Forno4, Isabelle Gärtner-Roer1, Stephan Gruber8, Tonio Gsell4, Andreas Hasler9, Roman Lim4,
Philippe Limpach10,18, Raphael Mayoraz11, Matthias Meyer4, Jeannette Noetzli6,7, Marcia Phillips6,7,
Eric Pointner12, Hugo Raetzo13, Cristian Scapozza14, Tazio Strozzi15, Lothar Thiele4, Andreas Vieli1,
Daniel Vonder Mühll16, Vanessa Wirz1,19, and Jan Beutel17 Alessandro Cicoira1,2,3,⋆, Samuel Weber1,4,5,6,7,⋆, Andreas Biri4, Ben Buchli4, Reynald Delaloye2,
Reto Da Forno4, Isabelle Gärtner-Roer1, Stephan Gruber8, Tonio Gsell4, Andreas Hasler9, Roman Lim4,
Philippe Limpach10,18, Raphael Mayoraz11, Matthias Meyer4, Jeannette Noetzli6,7, Marcia Phillips6,7,
Eric Pointner12, Hugo Raetzo13, Cristian Scapozza14, Tazio Strozzi15, Lothar Thiele4, Andreas Vieli1,
Daniel Vonder Mühll16, Vanessa Wirz1,19, and Jan Beutel17
1Department of Geography, University of Zurich, Zurich, Switzerland
2Department of Geosciences, University of Fribourg, Fribourg, Switzerland
3School of Architecture, Civil and Environmental Engineering, Swiss Federal Institute of Technology,
Lausanne, Switzerland
4Computer Engineering and Networks Laboratory, ETH Zurich, Zurich, Switzerland
5Chair of Landslide Research, Technical University of Munich, Munich, Germany
6WSL Institute for Snow and Avalanche Research SLF, Davos, Switzerland
7Climate Change, Extremes and Natural Hazards in Alpine Regions Research Center CERC,
Davos Dorf, Switzerland
8Department of Geography and Environmental Studies, Carleton University, Ottawa, Canada
9SensAlpin GmbH, Davos, Switzerland
10Terradata AG, Zurich, Switzerland
11Ct. Valais, Sion, Switzerland
12Rovina und Partner AG, Visp, Switzerland
13Federal Office for the Environment FOEN, Ittigen, Switzerland
14Institute of Earth Sciences, University of Applied Sciences and Arts of Southern Switzerland (SUPSI),
Mendrisio, Switzerland
15GAMMA Remote Sensing and Consulting AG, Gümlingen, Switzerland
16Personalized Health and Related Technologies, ETH Zurich, Zurich, Switzerland
17Department of Computer Science, University of Innsbruck, Innsbruck, Austria
18Institute of Geodesy and Photogrammetry, ETH Zurich, Zurich, Switzerland
19Pro Natura Schaffhausen, Schaffhausen, Switzerland
⋆These authors contributed equally to this work. Correspondence: Alessandro Cicoira (alessandro.cicoira@geo.uzh.ch) and Samuel Weber
(samuel.weber@slf.ch)
Received: 21 May 2021 – Discussion started: 11 June 2021
Revised: 13 July 2022 – Accepted: 19 August 2022 – Published: 18 November 2022 Abstract. Monitoring of the periglacial environment is relevant for many disciplines including glaciology, nat-
ural hazard management, geomorphology, and geodesy. Since October 2022, Rock Glacier Velocity (RGV) is
a new Essential Climate Variable (ECV) product within the Global Climate Observing System (GCOS). How-
ever, geodetic surveys at high elevation remain very challenging due to environmental and logistical reasons. During the past decades, the introduction of low-cost global navigation satellite system (GNSS) technologies
has allowed us to increase the accuracy and frequency of the observations. Earth Syst. Sci. Data, 14, 5061–5091, 2022
https://doi.org/10.5194/essd-14-5061-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License. Published by Copernicus Publications. 1
GNSS monitoring in high mountain environments sensor stations, their technical specifications, an overview of
the local geodetic network, and of the data communication
system used to collect the data in near real time. The ob-
servation positions are distributed throughout the Swiss Alps
and range in elevation from 2304 to 4003 ma.s.l.. The data
cover sites with differing geomorphological characteristics
including rock glaciers (ice-rich creeping landforms indicat-
ing the occurrence of permafrost), permafrost-affected land-
slides, and steep rock walls. The field sites are presented in
Sect. 3 and in more detail in Appendix A. The primary data
of this paper are the raw GNSS observables of all GNSS sta-
tions, accompanied by two-axis inclinometer measurements
as well as weather station data for some of the investigated
sites. The published dataset contains the complete raw data
at full sampling rates of all instruments (primary dataset; see
Sect. 4) as well as a selection of derived data products (sec-
ondary dataset; see Sect. 5). The derived data products are
down-sampled and cleaned time series of weather station and
inclinometer data as well as post-processed GNSS daily posi-
tions obtained by double-difference processing of the GNSS
data. A short discussion about validity and uncertainty of pri-
mary and derived data products is given in Sect. 6. In Ap-
pendix B, we briefly describe the different research projects
that contributed to the consortium, to the development of the
sensors, and to the evolution of the field-site monitoring net-
work. The history of the consortium is tightly linked to the
success of the method, and it can not be illustrated without
mentioning its practical applications in the fields of natu-
ral hazard management, e.g. in the Swiss Permafrost Mon-
itoring Service (PERMOS). A toolset, originally created for
the data describing the Matterhorn Hörnligrat field-site data
(Weber et al., 2019a), allows us to (re-)create and indepen-
dently update (living data process) the data documented in
this paper. The code, first presented in Weber et al. (2019b),
is updated and available at https://git.uibk.ac.at/informatik/
neslab/public/permasense/permasense_datamgr (last access:
21 October 2022, Weber et al., 2022). A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment and steep rock walls) at altitudes ranging from 2304 to 4003 ma.s.l. and spread across the Swiss Alps. The
primary data products consist of raw GNSS observables in RINEX format, inclinometers, and weather station
data. Additionally, cleaned and aggregated time series of the primary data products are provided, including daily
GNSS positions derived through two independent processing tool chains. and steep rock walls) at altitudes ranging from 2304 to 4003 ma.s.l. and spread across the Swiss Alps. The
primary data products consist of raw GNSS observables in RINEX format, inclinometers, and weather station
data. Additionally, cleaned and aggregated time series of the primary data products are provided, including daily
GNSS positions derived through two independent processing tool chains. The observations documented here extend beyond the dataset presented in the paper and are cur-
rently continued with the intention of long-term monitoring. An annual update of the dataset, available at
https://doi.org/10.1594/PANGAEA.948334 (Beutel et al., 2022), is planned. With its future continuation, the
dataset holds potential for advancing fundamental process understanding and for the development of applied
methods in support of e.g. natural hazard management. In situ observations of the Swiss periglacial
environment using GNSS instruments Today, permanent GNSS instruments
enable continuous surface displacement observations at millimetre accuracy with a sub-daily resolution. In this paper, we describe decennial time series of GNSS observables as well as accompanying meteorological
data. The observations comprise 54 positions located on different periglacial landforms (rock glaciers, landslides, Published by Copernicus Publications. 5062 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 2.1
GNSS sensor instruments Each location is equipped with a station consisting of an inte-
grated GNSS sensor, a 12 V solar power system mounted on
and housed inside a fibreglass reinforced tubular mast, and
anchored to the underlying rock (see left panel in Fig. 1). The sensor systems consist of an active GNSS antenna, a
GNSS receiver, a two-axis inclinometer, a data logger, and in
many cases, a low-power wireless transmission system (see
right panel in Fig. 1). Typically, the sensor is mounted on
top of a rock outcrop or a boulder large enough for a sta-
ble positioning (see Sect. 3.3 for more details). Power for all
instruments is provided by a standard 12 V photovoltaic sys-
tem: a solar panel, a charge controller, and a 12 V absorbed
glass mat (AGM) sealed lead-acid battery housed inside a
Pelicase. In order to guarantee maximum exposure to solar
radiation, facilitate operation with extended snow cover in
winter, and protect the instrument, the instrument is mounted
inside the top of the fibreglass mast. All wires are routed in-
side the tubular mast. 2.2
Two-axis inclination sensors Two-axis inclinometers (Murata SCA830) are integrated in
the sensor systems that are aligned with a marking on the
mast mount. This allows a direct determination of the rota-
tion along two axes and subsequently allows us to correct
the GNSS measurements for the tilt of the mast, i.e. cor-
rect for rotational movements, common in the active layer
of permafrost landforms (Wirz et al., 2013; Cicoira et al.,
2021). Experience has shown that this correction can yield
favourable results and provides the coordinates of the mount-
ing point (Wirz et al., 2014a), but in most cases, the exact
point of rotation still remains unclear because the geometry
of the rotating mass (where the station is mounted) is un-
known. 1
GNSS monitoring in high mountain environments We expect and en-
courage the use of this dataset to further develop process-
ing methods, provide atmospheric (Hurter et al., 2012) and
ground-based (Henkel et al., 2018) observations as well as
for educational purposes, but we also foresee future research Observations based on permanent in situ global navigation
satellite system (GNSS) instruments have been investigated
intensively over the past decade in pursuit of a better pro-
cess understanding of the periglacial environment and its
related mass movements (Wirz et al., 2013; Ravanel and
Deline, 2014; Cicoira et al., 2019b). The advantage of this
method for assessing surface displacements over traditional
field surveying and remote-sensing techniques is the level
of detail that can be obtained at different temporal scales. Today it is possible to monitor at millimetre accuracy with
a temporal resolution of minutes, using double-difference
processing techniques (Teunissen and Montenbruck, 2017)
and commodity receiver hardware similar to those found in
consumer products, e.g. mobile phones or automotive sys-
tems (Paziewski et al., 2021). Depending on the level of de-
tail required, chiefly characterised by the accuracy of coor-
dinate tuples, their temporal resolution is achieved by op-
erating GNSS receiver pairs continuously and subsequently
post-processing the observation data derived. While the cost
footprint of the required hardware is several orders of mag-
nitude lower than that required for traditional geodetic sur-
veying equipment (Wirz et al., 2013), the power and com-
munication bandwidth required for operating the sensors re-
motely remain high. However, it has been shown that by se-
lectively duty cycling receivers and transmitting the GNSS
observation data on a low-power wireless sensor network
(WSN) (Buchli et al., 2012), a trade-off between energy/data
volumes and the fidelity required can be obtained. In this
way, near-real-time permanent monitoring in remote loca-
tions, over large timescales, and in adverse meteorological
conditions become possible. In this paper, we document a dataset of continuous GNSS
observations of 54 positions obtained in the periglacial en-
vironment of the Swiss Alps over the past 14 years in the
framework of the X-Sense Project and the PermaSense con-
sortium. PermaSense has been a large interdisciplinary re-
search consortium targeted to custom-design low-cost wire-
less sensors for the detection and analysis of temporal and
spatial variability of high-alpine slope movements (Beutel
et al., 2011). In Sect. 2, we provide a description of the GNSS https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5063 A. 1
GNSS monitoring in high mountain environments Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment applications in the fields of geomorphology, engineering ge-
ology, and natural hazard management. SCA830), an ambient temperature/humidity sensor (Sen-
sirion SHT31), and power supervisory circuits are integrated. In order to reduce the necessary power footprint, e.g. at
times of reduced solar radiation for energy harvesting, a
data-acquisition schedule can be set on each device individu-
ally. This sampling schedule has hourly granularity, and time
windows when the system is running are typically centred
at 12:00:00 UTC to facilitate overlapping time intervals for
double-difference GNSS processing. 2
Instrumentation technology and data management The measurement network consists of distributed GNSS sta-
tions that are permanently installed on locations of interest,
i.e. on the surface of a landform under investigation. The
GNSS sensor and the fix installations were designed in the
framework of the PermaSense Project; both are depicted in
Fig. 1. In addition, a MEMS two-axis inclinometer has been
fully integrated into the sensor system. At selected locations,
automatic weather stations provide auxiliary ambient data
that are valuable for the analysis of the GNSS observations. In the following paragraphs, we provide a brief description of
the different sensors (GNSS, inclinometers, weather stations)
as well as the data communication and management infras-
tructure. All sensor types, including their indicative period
of operation, unit system, and key characteristics are synthe-
sised in Table 1. p
g
In 2010, a proof-of-concept study lead to the development
of the first generation of these instruments, which were ca-
pable of logging data to internal SD-card storage only (Wirz
et al., 2013). This first prototype was subsequently refined
into two wireless systems: one focused on experimentation
(Beutel et al., 2011), the second being a fully integrated wire-
less GNSS sensor (Buchli et al., 2012). This two-step ap-
proach allowed for a fast startup of the project, which was
initially independent of infrastructure and network coverage. Over time, most of the sensor locations (initially equipped
with data loggers only) have been equipped with wireless
data transmission, facilitating sensor operation and near-real-
time data retrieval (see Tables A1–A6). The data availability
is visualised in Fig. A1. 2.1
GNSS sensor instruments https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5064 Figure 1. Structure and components of the GNSS sensor developed in the PermaSense Project. (Left) Structure of the GNSS station. (Right)
Detail of the embedded GNSS antenna, data logger, and wireless transmitter. Figure 1. Structure and components of the GNSS sensor developed in the PermaSense Project. (Left) Structure of the GNSS station. (Right)
Detail of the embedded GNSS antenna, data logger, and wireless transmitter. Table 1. Overview list of the sensors used, ordered by sensor type. n.a. denotes not available. Table 1. Overview list of the sensors used, ordered by sensor type. n.a. denotes not available. Sensor type
Sensor
Unit
Interval
Accuracy
L1/L2-GNSS;
Leica GRX1200+, AR10/25 antenna
m
30 s
n.a. L1-GPS;
u-blox LEA-6T, Trimble Bullet III
m
5, 30 s
n.a. Inclination
Murata SCA830-D07 Inclinometer
◦
120 s
±30 mg
Air temperature
Vaisala WXT520
◦C
120 s
±0.3 ◦C
Barometric pressure
Vaisala WXT520
hPa
120 s
±1 hPa
Relative humidity
Vaisala WXT520
% RH
120 s
±3–5 % RH
Wind speed
Vaisala WXT520
kmh−1
120 s
±3 % at 10 ms−1
Wind direction
Vaisala WXT520
◦
120 s
±3 ◦at 10 ms−1
Precipitation
Vaisala WXT520
mm
120 s
Resolution 0.01 mm
Radiation
Kipp & Zonen CNR4
Wm−2
120 s
Non-linearity < 1 % plied in the data-conversion procedures as advised by the
manufacturer. LAN (WLAN) backbone and a fine distribution using a low-
power WSN was sought. The topology of the wireless data-
communication backbone in the Matter Valley is illustrated
in Fig. A6. Using this approach, it is possible to reach back
into areas in the back of side valleys that are interesting from
the monitoring perspective, but not covered with regards to
connectivity. All locations serviced by the WLAN backbone
have Internet Protocol (IP) connectivity facilitating the use of
standard sensing equipment. For ease of maintenance, a local
WLAN hotspot is also available at these sites for all kinds of
activities that require an internet connection. In selected lo-
cations, standard 3G/4G modems were further implemented
as a backup to the network communication system. plied in the data-conversion procedures as advised by the
manufacturer. 2.3
Auxiliary weather instruments A GNSS sensor has been installed on stable terrain at se-
lect positions in order to provide a stable reference for the
double-difference GNSS post-processing. These additional
positions ensure a shorter baseline than what would be ob-
tained by using the national reference network. Moreover,
these reference stations act as the sink of the local WSN
and host the instrumentation for the data collection from all
GNSS measurement points. More information is provided in
Sect. 3.2. A Vaisala WXT520 compact all-in-one weather station was
installed at select locations to obtain a more detailed record
of local weather data. The measured variables include am-
bient air temperature, air pressure, relative humidity, wind
(speed and direction), and liquid precipitation. At two lo-
cations, a four-component net radiometer (Kipp & Zonen
CNR4) was installed without capabilities for ventilation and
heating. The WXT520 is capable of heating the rain and
wind sensor; but for practical reasons, this feature is only en-
abled when enough power is available, which typically corre-
sponds to good weather periods and turned off especially in
prolonged bad-weather periods. Both instruments have been
vendor-calibrated and the respective calibration data are ap- The GNSS sensor instruments are based on commodity
L1-GNSS receivers with the extended capability to output
the raw satellite observables (u-blox LEA-6T) and an ac-
tive antenna (Trimble Bullet III) (Wirz et al., 2013; Buchli
et al., 2012). Furthermore, a two-axis inclinometer (Murata https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 3.1.2
Steep rock walls Three field sites are focussing on steep bedrock. The Matter-
horn Hörnligrat (MH), the Randa Grossgufer (RA), and the
Sattelspitz (SA) north-west ridge are all located in the Matter
Valley. All sites are particularly relevant from the perspective
of natural hazard management. The Hörnligrat, despite its re-
moteness, has an important social and economic component
due to all the tourist activities that take place on the Matter-
horn. The Grossgufer is the remaining lip of the large and 2.4
Near-real-time data communication The locations monitored in the scope of the research and the
data presented in this paper are located in high-alpine areas,
where cellular network coverage is limited or unavailable. Some field sites have been instrumented following a long-
term monitoring strategy, where retrieval of the data once
or twice a year is sufficient for the continuation of the time
series and their analysis. However, many of the GNSS sta-
tions, especially in the Mattertal, are particularly relevant
from a natural hazard perspective, and timely data commu-
nication is essential for their applicability to management
strategies. For this reason, large effort was put into the de-
sign and implementation of a data-communication system in
near real time. A unique solution comprising of a wireless Since WLAN equipment serving the communication
backbone is energy-consuming, the actual sensors are inte-
grated with a custom low-power WSN operating on ISM-
band radio transceivers and a specialised communication
protocol for data gathering (Beutel et al., 2009). Using a hier- https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5065 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment are located in the periglacial environment or in its proximity. We classify the instrumented landforms into three types: (i)
rock glaciers, (ii) landslides, and (iii) steep rock walls. In this
section, we present an overview of the landform types and
instrumented field sites. A more detailed description of the
investigated landforms, accompanied by some scientific ref-
erences which can be used as a starting point for additional
information, is given in Appendix A. All the landforms and
their types are illustrated in Fig. 2 alongside the number of
the GNSS sensors installed (details can be investigated in the
Supplement of the paper). An example of a small-scale local
geodetic network and the wireless communication geometry
are provided in Sect. 3.2. archical system of commodity components for the backbone
and a highly optimised and custom network for the fine distri-
bution to spatially separated sensing locations, it is possible
to operate on a more reduced resource footprint, over long
periods of time, and minimising the number of interventions. 3.1.1
Rock glaciers Eleven rock glaciers across the Swiss Alps have been instru-
mented with permanent GNSS sensors. A cluster of five rock
glaciers is concentrated in the right orographic side of the
Matter Valley: Dirru (DI), Distelhorn (DIS), Gugla-Bielzug
(BH) (positions 10 and 13), Ritigraben (RIT), Steintälli (ST). In the neighbouring Saas Valley, above the village of Saas-
Baalen, the rock glaciers Gruben (GRU) and Jaegi (JAE)
host two more GNSS stations. These valleys are rich in rock
glaciers, many of which move relatively fast, with average
displacement rates of up to several metres per year (Delaloye
et al., 2013; Strozzi et al., 2020). In order to consistently manage and document the field site
and all the sensors with homogeneous metadata, a set of rules
has been defined: – An individual protocol sheet is used for each interven-
tion (field work day) where all noteworthy items are
recorded (installation, maintenance, removal). Some rock glaciers are being monitored under the auspices
of the PERMOS (Noetzli et al., 2019, kinematics data are
available online http://www.permos.ch, last access: 21 Oc-
tober 2022). They include Ritigraben (RIT1) and Gruben
(GRU1) in the Valais, the Stabbio di Largario (LAR1 and
LAR2) in Val Blenio, and a second large cluster in the upper
Engadine, consisting of the rock glaciers Murtèl-Corvatsch
(COR1), Muragl (MUA1), and Schafberg (SCH1). Engadine
and the Matter Valley are the two driest valleys in Switzer-
land and are characterised by high mountain peaks. For these
reasons, they host the majority of rock glaciers in the country
(Haeberli and Vonder Mühll, 1996). Many of the sites, espe-
cially the PERMOS locations, are also monitored by terres-
trial geodetic surveys. The relative data can be obtained from
the responsible institutions and from the PERMOS office. – Sensor interventions on site take place at different times
for each position. To simplify things, the whole day of
an intervention is typically assumed to be invalid data. – All sensor devices are mapped to a distinct position ID. The mapping contains start and finish time stamps, the
device ID of the sensor, sensor type, and calibration
data. – All data from a specific data source (sensor type) are
kept in an individual data structure. Queries are typi-
cally made per data type and position ID. – Detailed circumstances (orientation angles, status of the
station) are recorded using auxiliary data formats: text
files, Excel files or photographs. https://doi.org/10.5194/essd-14-5061-2022 2.5
Data-management infrastructure All data collected are organised and stored so that repro-
ducible research and reuse of the data in different contexts
and in future projects are possible. The data infrastructure
must also allow for flexibility with respect to extensions (e.g. integrating new sensor types), support of different sampling
rates, metadata integration and life-cycle management. The
data back-end is implemented using a data-streaming mid-
dleware, where a dedicated processing structure called a vir-
tual sensor is responsible for processing a specific data type,
e.g. one virtual sensor for temperature measurements and an-
other virtual sensor for GNSS positions. Complete process-
ing chains can be implemented by concatenating virtual sen-
sors, either within the same instance of the Global Sensor
Network (GSN; Aberer et al., 2006) or also across multiple
instances of the GSN. More details about data processing and
storing are given in Weber et al. (2019a). 3.1
Landforms 3.1.3
Landslides Five permafrost-affected landslides have been instrumented
in the Matter Valley with the goal of monitoring unstable
and potentially dangerous slopes: (i) the south-west flank
of the Breithorn (BH), (ii) one of the culminating peaks of
the Mischabel ridge, (iii) the west flank of the Gugla sum-
mit, locally called Längenschnee (LS), (iv) the Grabengufer
(GG) landslide above the village of Randa, and (v) the Wisse
Schijen (WYS) on the eastern flank of the Weisshorn, di-
rectly facing it on the other side of the valley. All these sites
are well-known by the local population and the cantonal au-
thorities for a long-lasting history of displacement and in
some cases for hazard phenomena. The Längenschnee and
the Grabengufer landslides are particularly interesting from
a historical and geomorphological perspective, respectively. More details can be found in Appendices A and B. Based on the aforementioned factors, a set of local refer-
ence positions has been defined and equipped with continu-
ously operating GNSS receivers as well as high-bandwidth
data transmission (see Table 2) forming a local geodetic ref-
erence network. In cases where only few or single observa-
tion points have been deployed, stations from the permanent
GNSS network in Switzerland (AGNES) serve as a refer-
ence. Periodic checks against the national GNSS network have
shown that all but two (Längenschnee/RL01 and Hörn-
ligrat/HOGR) reference locations are stable and do not ex-
hibit local movements (see Fig. A2). The station RL01 was
abandoned after the discovery that the ridge where it is lo-
cated is actually moving at a rate of a few centimetres per
year, contrary to the assumptions made at an initial site sur-
vey. The reference station HOGR represents a special case
since it is located on a moving buttress on the Matterhorn
Hörnligrat Ridge. In this case however, due to the extreme
nature of the environment in which the station is installed, no
stable terrain at the same elevation is available. Therefore, the 3
Field sites The dataset described can be divided into five main geo-
graphical areas. Moving from west to east, they are (i) the
Matterhorn Hörnli Ridge, (ii) the main trunk of the Matter
Valley, (iii) the upper Saas Valley in the Canton of Valais,
(iv) Stabbio di Largario in Ticino, and (v) the upper Engadin
Valley in Canton Graubünden. All of the field sites monitored https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5066 Figure 2. Overview map of the field sites. The field sites are divided into five major geographical and geomorphological areas, for which
a panel provides a more detailed sight of the sensor network. Further details can be investigated in the Supplement of the paper. Satellite
imagery from the Swiss Federal Office of Topography, swisstopo. Figure 2. Overview map of the field sites. The field sites are divided into five major geographical and geomorphological areas, for which
a panel provides a more detailed sight of the sensor network. Further details can be investigated in the Supplement of the paper. Satellite
imagery from the Swiss Federal Office of Topography, swisstopo. famous Randa rockslide which took place in 1991. The Sat-
telspitz north-west ridge is a currently accelerating buttress,
potentially endangering some constructions and activities in
the Täsch-Alp. (Wirz et al., 2013; Bu et al., 2021). For the chosen approach
using single-frequency GNSS receivers, a short baseline on
the order of hundreds of metres to a few kilometres maxi-
mum, is favourable. Furthermore, the non-moving reference
position and the rover receiver should be situated in the same
altitude regime, exhibit similar shading by topographic ob-
stacles (sky view), and need to be operated in overlapping
time windows in order to generate the best results. https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 3.2
Local geodetic network The most accurate position data can be computed using dif-
ferential GNSS processing techniques (see Sect. 5) when cor-
rection data from a GNSS receiver on a non-moving position
are available in close proximity to the rover (short baseline) https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5067 ,
p
g
Table 2. Local geodetic network: reference stations: RD01 – Reference Dirru 01, RL01 – Reference Längenschnee 01, RG01 – Reference
Grabengufer 01, RAND – Reference Randa, HOGR – Hörnligrat Reference. Reference
Area
Topographic Feature
Sensor
Period
RD01
Next to Dirru rock glacier
Large bedrock feature
u-blox LEA-6T, Trimble Bullet III
March 2011–ongoing
RL01
Between Längenschnee and Breithorn
Fractured ridge
u-blox LEA-6T, Trimble Bullet III
August 2011–May 2013
RG01
Above Grabengufer rock glacier
Bedrock on ridge
u-blox LEA-6T, Trimble Bullet III
September 2011–ongoing
RAND
Top of Grossgufer
Bedrock above rockfall
Leica GRX1200+, AR25 antenna
May 2011–ongoing
HOGR
Hörnligrat ridge
Rock buttress on ridge
Leica GRX1200+, AR10 antenna
December 2010–ongoing Table 2. Local geodetic network: reference stations: RD01 – Reference Dirru 01, RL01 – Reference Länge
Grabengufer 01, RAND – Reference Randa, HOGR – Hörnligrat Reference. ii. From a geomorphological perspective, the chosen point
must be representative of the investigated process. As
explained in Wirz et al. (2014b) and later in Cicoira
et al. (2021), the velocity measured at the surface of
a periglacial landform is typically the result of three
super-imposed signals. On bedrock, the representatives
of the selected point is directly connected with the struc-
tural geology of the site, making the selection less prone
to personal interpretation. In order to minimise the noise
due to stochastic movement within the active layer and
at the terrain surface, it is favourable to choose large
boulders well embedded in the active layer (with two
metaphors: a stable boat in water or a tooth with a
deep root; see Fig. 3). The additional two-axis incli-
nation measurements allow us to retrieve critical infor-
mation about the three-dimensional nature of the roto-
translation of the surveyed point (not known a priori)
and with the use of proper post-processing analysis, re-
duce the relative error associated in the measurements
(Wirz et al., 2014a). 3.3
Field site selection and prerequisites for in situ
GNSS sensor installation by a rockfall (see
Fig. A7b) or a snow avalanche. In this case, the posi-
tion has to be replaced. A location as close as possible
and with similar characteristics has to be chosen in or-
der to minimise the influence of spatial variability and
guarantee (as much as possible in this unwanted condi-
tion) data homogeneity and the continuation of the time
series. Within the PermaSense project, the installation of an in
situ GNSS sensor on an observation point had to com-
ply with minimum technical and geomorphological require-
ments, which can be met fairly easy, depending on the field
site. i. From a technical perspective, good visibility to the hori-
zon in direction of the Equator (southern sky when
working in the Northern Hemisphere) is required both
for good and persistent satellite visibility as well as
for maximum solar radiation for power generation (see
Fig. 3). Where different positions are equivalent with
regard to the other criteria, the technical feasibility of
the measurements can be used as a discriminant for a
decision. In case of abundant snow cover or large peri-
ods without sunlight, a tall mast and conservative power
set-ups are critical. 3.2
Local geodetic network reference position is maintained, but the observations have to
be corrected for the movement of the reference position be-
fore further analysis, as explained in more detail in Weber
et al. (2019a). 3.3
Field site selection and prerequisites for in situ
GNSS sensor installation 3.3
Field site selection and prerequisites for in situ
GNSS sensor installation The variety of the field sites presented in this paper is the re-
sult of the historical development of the PermaSense project
(a short summary is given in Appendix B). The multitude
of measuring points and field sites presented embodies the
variety of applications that the project has served over time. Natural hazard management applications are often very well
constrained in the selection of the site and the single loca-
tion of the measurement positions, as these are the relevant
ones from the hazard perspective. On the other hand, long-
term climatic monitoring requires more thorough thinking
and preliminary investigations to select the best-suited field
sites first and then the single measurement points. It is impor-
tant to note that the measurements provide a detailed descrip-
tion of the kinematic behaviour of surface boulder, which
are only a proxy of deep-seated geomorphological processes. The results have to be evaluated with care and expert knowl-
edge; see e.g. Cicoira et al. (2021) for the components of
rock glacier velocities. In this paragraph, we provide a brief
overview of three guiding criteria (technical and geomorpho-
logical) that all the GNSS positions have to respect. iii. The longevity of the measuring point must be antici-
pated prior to installing the GNSS station. In extreme
cases with blocks sliding on the surface or strongly ro-
tating above the surface, it may be required to re-level
the GNSS instrument back to vertical after some time
(see Fig. A7a). Finally, it may happen that a station is
severely damaged or destroyed, e.g. by a rockfall (see
Fig. A7b) or a snow avalanche. In this case, the posi-
tion has to be replaced. A location as close as possible
and with similar characteristics has to be chosen in or-
der to minimise the influence of spatial variability and
guarantee (as much as possible in this unwanted condi-
tion) data homogeneity and the continuation of the time
series. iii. The longevity of the measuring point must be antici-
pated prior to installing the GNSS station. In extreme
cases with blocks sliding on the surface or strongly ro-
tating above the surface, it may be required to re-level
the GNSS instrument back to vertical after some time
(see Fig. A7a). Finally, it may happen that a station is
severely damaged or destroyed, e.g. 4
Primary data products In this section, we present the primary data products pro-
vided within the scope of this paper. The primary data prod-
ucts are the raw files as provided by the sensors. They com-
prise GNSS observables in the form of compressed RINEX
files, as well as inclinometer and weather station data (CSV
files). Technical specifications about the sensors are given https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5068 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure 3. Two GNSS stations mounted atop large boulders on the Breithorn landslide 2982 ma.s.l., Herbriggen (a) and on the Largario Rock
Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a field deployment including all sensor setup and anchoring appears. Figure 3. Two GNSS stations mounted atop large boulders on the Breithorn landslide 2982 ma.s.l., Herbriggen (a) and on the Largario Rock
Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a field deployment including all sensor setup and anchoring appears. Glacier 2355 ma.s.l., Val di Blenio (b) illustrate how a field deployment including all sensor setup and anchoring appears. Figure 4. A decade of air temperature and precipitation from weather station DH13, as well as displacement, inclination, and azimuth for
Gugla Bielzug Rock Glacier (position BH13) and displacements for reference station RD01. Figure 4. A decade of air temperature and precipitation from weather station DH13, as well as displacement, inclination, and azimuth for
Gugla Bielzug Rock Glacier (position BH13) and displacements for reference station RD01. Figure 4. A decade of air temperature and precipitation from weather station DH13, as well as displacement, inclination, and azimuth for
Gugla Bielzug Rock Glacier (position BH13) and displacements for reference station RD01. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5069 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment in Sect. 2 and in Table 1. More comprehensive information
about the data availability, operation window of all sensors,
their instrumentation, and additional plots are given in the
appendix. Tables A1–A6 provide a detailed list of all GNSS
and automatic weather stations, along with some basic infor-
mation about them. The label of the station is indicated on
the very left. 4.2
Inclinometer data Almost all L1-GPS sensors are equipped with an integrated
two-axis inclinometer based on a MEMS component (Mu-
rata SCA830-D07). An example of the inclinometer data for
station BH13 is given in Fig. 4. These are sampled at 2 min
intervals during periods at which the GNSS sensor is run-
ning. The schedule for the inclinometer data acquisition is
the same as for the GNSS sensor. Data are provided in CSV
files. The data can be used to constrain the rotational move-
ment of the sensor around a centre of rotation, which in prin-
ciple is not known. Wirz et al. (2014b) provided a framework
to use this information to correct the position measurement
on the ground for the component of the rotation between the
anchor and the sensor (given by the mast height). For some
applications (e.g. Leinauer et al., 2022), the inclinometer data
have been proven to be a valuable proxy of more expensive
position/displacement measurements. The sensor labels are given due to historical reasons
and follow a geographical and chronological order. Each
sensor is given a four-character alphanumeric label, with
two or three letters indicating the field site (as illus-
trated in Sect. 3) and the number identifying a given
sensor on a field site. The labelling of the data files
consists of this four-character alphanumeric label prefix-
ing a string for the sensor type, and a string indicat-
ing the time period of the measurements included in the
file: e.g. BH10_gps_inclinometer_2018.csv con-
tains GPS inclinometer data for the year 2018 for the position
Breithorn 10. More information on the structure of the data
repository is provided in Sect. 7 and Table 3. 4
Primary data products Thereafter, we indicate the period of operation,
from the date of deployment to the end date of the operations,
or an indication if it is currently ongoing. Next, the name
of the reference station (as in Table 2) used for the double-
difference GNSS processing is given. The mast height indi-
cates the height of the antenna above the rock instrumented. Additional to this basic information, the field “online data”
allow us to identify all the sensors that are currently con-
nected with the online data-management infrastructure via
the near-real-time WSN (see Sect. 2.4 for more details). Un-
der the field “location”, the coordinates of the station at the
time of deployment are indicated in the EPSG:2056 – Swiss
CH1903+ / LV95 coordinate system. The current coordinates
are available in the dataset. Finally, two fields are used to il-
lustrate the sensors installed at each station. We distinguish
between kinematics for all GNSS stations, including optional
inclinometers, and automatic weather stations. A more de-
tailed overview of the data availability for all sensors is given
in Fig. A1, where all operating periods, data gaps, and dis-
continued sensors are visualised. allows for more flexible analysis, especially for the explo-
ration of real-time kinematic applications. As explained in
Sect. 2.1, the GNSS rovers employed (Wirz et al., 2013;
Buchli et al., 2012) are not continuously sampling data at
the given rate. In order to reduce energy consumption, the
data generation duration can be configured based on a user-
defined daily schedule. The schedule is defined using hourly
granularity with a minimum duration of 1 h per day. 4.3
Weather station data The Dirruhorn and Matterhorn sites are instrumented with
automatic weather stations. The data comprise ambient air
temperature, air pressure, relative humidity, wind (speed and
direction), and precipitation as well as four-component net
radiation. The time series for the Dirruhorn field site is given
in Fig. 4. The raw weather station data sampled at 2 min in-
tervals are provided in the form of CSV files. 4.1
GNSS raw observation data national coordinate system by using the online REFRAME
conversion service (REST API) by swisstopo. The geode-
tic datum of all daily position data is EPSG:2056 – Swiss
CH1903+ / LV95 coordinate system with the reference frame
Bessel (ellipsoidal). After post-processing, the derived data
are uploaded again to the PermaSense database for conve-
nient online data access. A custom-developed set of wrapper
scripts to be used with RTKLIB is described in Appendix D. The processing using the Bernese GNSS software is a
similar process and follows the same steps presented in
Fig. 5. However, further details on this tool are omitted due
to its license-only availability. Because the two processing
tools use different algorithms and parameter sets, the output
data can show differences. Most noticeably, due to different
parameter-threshold settings, it may happen that a daily com-
puted position is available for a given tool, but not for the
other. Given the long-term nature of the observations, these
often spurious missing values can be safely interpolated in a
further processing step if deemed necessary by the applica-
tion. 5.1
GNSS processing The main data-transformation step performed within this
paper is the transformation from the raw primary GNSS
data to calculate daily static GNSS positions using double-
difference GNSS post-processing. Double-difference GNSS
processing (Teunissen and Montenbruck, 2017) is based on
data obtained in a common observation interval from a sta-
tion pair. The workflow is illustrated in Fig. 5. Positions for
the so-called “rover” station can be calculated with high pre-
cision under the assumption that the location of the “refer-
ence” station is quasi-stationary and that observations from
both stations are subject to similar perturbations. Care should
be taken that the baseline distance between any station pair
is short, the field of view to the satellites (horizon) is similar,
and a station pair is located in the same altitude regime. The
main quality indicators of the input data (GNSS observables)
are the number of visible satellites, the signal-to-noise ratio,
and the observation duration. For the derived data products,
the ratio of fixed ambiguities as well as the standard devia-
tions per coordinate axis are key quality indicators. Double-
difference processing achieves best accuracy when utilising
the precision final GNSS data products from the International
GNSS Service (IGS), although other GNSS data products
can be used as well, e.g. if near-real-time solutions are re-
quired for practical applications in landslide-monitoring pro-
cedures. 4.1
GNSS raw observation data In order to favour direct usage of the data presented in this pa-
per, we provide data products derived from post-processing
of the raw sampled data presented above. This process con-
sists of three main steps: data transformations, cleaning, and
aggregation. The only transformation needed for the pre-
sented dataset is from the raw GNSS observables to daily
positions; more details are discussed in the next subsection. In the second step, artefacts that have been identified man-
ually or are known a priori from the metadata (e.g. due to
field interventions) are corrected in the data-cleaning step. Available cleaning operations are to delete, offset, or replace
single or multiple data points. Finally, for all data, aggregates
are computed. For inclinometer and weather data, the aggre-
gates are given in hourly values. Different aggregation func- Raw GNSS observables are the key primary data product of
this paper. The raw observables are provided in the form of
industry standard daily RINEX 2.11 files for each station pre-
sented. They include carrier-phase measurements, pseudo-
range values as well as Doppler shift and raw signal strength. Furthermore, the two positions, RAND and HOGR, equipped
with a reference grade L1-/L2-GNSS receiver, and used as
reference stations, contain both GPS and GLONASS obser-
vation data. For both receiver types employed, signals are
tracked at an interval of 30 s with the exception of the L1-
GPS reference stations that were changed from 30 to 5 s mid-
way through the project. By using a higher sampling rate,
a very large amount of data is obtained, but the approach https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5070 tions are used. In most cases, the arithmetic mean is applied. For the cumulative precipitation, we used sum. Peak rain in-
tensity is obtained with the maximum. From the GNSS data,
the daily positions are calculated with the GNSS processing
routine. The aggregation function in the toolchain can further
provide aggregation over longer time periods, e.g. to weekly,
monthly, or yearly values. In the following discussion, we
provide a brief explanation of the methods used, which rep-
resent operational best practices emerged from the context of
the PermaSense project. A more detailed description of the
processing steps and the related algorithm (also provided in
the paper) is given in Appendices C and D. 4.1
GNSS raw observation data The GNSS processing flow employed uses two dif-
ferent post-processing toolchains, namely the Bernese GNSS
Software (Dach et al., 2015) and the open-source RTK-
LIB toolchain (http://www.rtklib.com, last access: 31 De-
cember 2021). A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure 5. Typical GNSS post-processing workflow using the Bernese GNSS software or RTKLIB. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has
severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense
Data Manager. Figure 5. Typical GNSS post-processing workflow using the Bernese GNSS software or RTKLIB. Figure 5. Typical GNSS post-processing workflow using the Bernese GNSS software or RTKLIB. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has
severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense
Data Manager. Figure 6. Data cleaning example: Inclinometer west-component time series data for location BH13 on the Gugla-Bielzug rock glacier has
severe outliers and jumps in the raw data that can be removed, compensated for, and aggregated to smooth time series using the PermaSense
Data Manager. 5.2.2
Inclinometer data All GNSS-derived daily positions are converted to rel-
ative coordinates starting with a zero value at the on-
set of the measurement period and have undergone a
rudimentary data-cleaning step: outliers and jumps per-
taining to device changes on field service days have
been removed. All cleaned daily files showing displace-
ments from the beginning are available in the folder
gnss_derived_data_products. Such cleaned time
series easily enable us to visualise and compare the displace-
ment rates measured at the several locations and to distin-
guish different patterns linked to landforms (see Fig. 7). Similarly, the raw data from inclinometer and weather sta-
tion sensors are available at sampling intervals of 120 s. Based on the example of the Gugla-Bielzug rock glacier
described earlier (see Sect. 5.2 and Fig. 6), all time-series
data from inclinometer and weather stations are cleaned,
offset-compensated, and aggregated to hourly data prod-
ucts. All of these data files are available in the folder
timeseries_derived_data_products. https://doi.org/10.5194/essd-14-5061-2022 5.2
Data cleaning and aggregation A generic toolchain to access, compile, clean, aggregate,
and validate both primary as well as derived data from the
online PermaSense database has been developed by Weber
et al. (2019b) (DOI: http://doi.org/10.5281/zenodo.2542715)
and an update is available at https://git.uibk.ac.at/informatik/
neslab/public/permasense/permasense_datamgr (last access:
21 October 2022, Weber et al., 2022). This tool allows us
to query the database based on location and sensor type/data
type, compile raw CSV files, apply metadata, data cleaning
and correction functions, aggregate time-series data to pre-
selectable aggregation intervals, and produce intuitive vali-
dation plots. The data cleaning and correction step allows us
to delete outliers based on time intervals or value thresholds
as well as perform simple mathematical corrections, e.g. to
apply a constant factor or an offset for a given interval. Us-
ing this latter feature allows us to correct artefacts introduced
during site maintenance, e.g. from changing sensor equip-
ment or from re-levelling a GNSS mast that is required when
the tilt angle changes very much. An example for such a cor-
rection is shown in Fig. 6 for the north component of the in-
clinometer data of station BH13 that is located on the Gugla-
Bielzug rock glacier. Additionally, outliers from days with
sensor-device changes are removed using the del operation,
and offsets due to differences between two sensor devices are
corrected using the offset operation. The filtered original
data (with a number of artefacts) are plotted in orange, the
cleaned data are shown in blue, and the aggregated data over
1400 min (daily values) are shown in red in Fig. 6. A quick
tutorial for this tool is given in Appendix C. The post-processing with RTKLIB starts from the daily
observation files, compiled using the online database as one
file per day per position. To constitute a baseline pair between
a rover position and a reference station, in most cases we use
a sensor from the local geodetic network. Differently, we use
reference positions from AGNES for determining the coordi-
nates of the reference stations, and in cases where the base-
line distance is too high for the calculation of accurate posi-
tions. All rover–reference pairs are indicated in Tables A1–
A6. Thereafter, coordinates are calculated. In a final step, the
daily coordinates are re-projected from the WGS84 to Swiss https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5071 5.3
Standardised analysis plots For each position, a number of standardised graphs are
generated (see Figs. A3, A4 and A5 ). These plots
are available for all derived data products in the folder https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5072 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5072
A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment
Figure 7. Time series of all GNSS positions presented in this paper, classified by landform type and range of displacement rates. The outlier
in the Landslides Mattertal (LS05, Summer 2020) was due to a superficial roto-translatory movement of the boulder on which the sensor was
placed, as confirmed by a 24◦rotation visible in the inclinometer data. Figure 7. Time series of all GNSS positions presented in this paper, classified by landform type and range of displacement rates. The outlier
in the Landslides Mattertal (LS05, Summer 2020) was due to a superficial roto-translatory movement of the boulder on which the sensor was Figure 7. Time series of all GNSS positions presented in this paper, classified by landform type and range of displacement rates. The outlier
in the Landslides Mattertal (LS05, Summer 2020) was due to a superficial roto-translatory movement of the boulder on which the sensor was
placed, as confirmed by a 24◦rotation visible in the inclinometer data. timeseries_sanity_plots of the data repository and
also as a supplement to this paper. hindering direct comparison to other sensors, typically used
for validation. The processing of the derived data products is completely
transparent: the complete processing workflow from raw sen-
sor data to the derived data products applying cleaning and
aggregation is described in Sect. 5. Standardised visualisa-
tions for every data channel (an example is given in Fig. 6)
are provided at https://doi.org/10.1594/PANGAEA.948334
(Beutel et al., 2022) and allow to check the data cleaning
and aggregation on a per-channel basis. Note that data offset
were corrected only when field site interventions included
in the meta data provided an objective reason. No arbitrary
stylistic correction is performed. https://doi.org/10.5194/essd-14-5061-2022 6
Validation and uncertainty of data products The dataset presented in this manuscript comprises primary
(raw) and derived data products for a series of sensors. In
Table 1 we provide an overview of the sensor types, their
sampling intervals and their nominal accuracy. For most of
the sensor systems used, the work presented here represents
a novel application domain, with custom developed sensor
systems applied in a challenging operating regime. In many
cases, the extreme environmental conditions and demanding
logistics make so that our measurements are the only ones
performed in the area over such extended periods of time, The processing accuracy of the GNSS observables (input
data), to daily coordinates (primary data product) heavily de- Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5073 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment
5073
Table 3. Structure, description, formats, and sizes of the dataset components. File
Data description
Format
# Data points
# Files
Size
gnss_data_raw.zip
Raw daily position data
csv
231’136
885
61.3 MB
gnss_data_raw_observables.zip
GNSS raw observations
RINEX 2.11
199’551’511
126’395
38.1 GB
timeseries_data_raw.zip
Raw primary sensor data
csv
60’622’944
472
10.4 GB
gnss_derived_data_products.zip
Daily position data
csv
229’669∗
774
23 MB
timeseries_derived_data_products.zip
Sensor data after cleaning/aggregation
csv
3’290’599
416
161.3 MB
timeseries_sanity_plots.zip
Standard plots for all data
png
–
694
174.1 MB
dirruhorn_nodepositions.xlsx
General metadata file
xlsx
–
1
98 kB
matterhorn_nodepositions.xlsx
General metadata file
xlsx
–
1
58 kB
permos_nodepositions.xlsx
General metadata file
xlsx
–
1
37 kB
README.md
–
md
–
1
4 kB
Total
–
–
263’925’859
129’636
48.9 GB
∗In part the data have been processed by two different toolchains, RTKLIB and Bernese. Table 3. Structure, description, formats, and sizes of the dataset components. this as noise. This noise can be considered irrelevant, because
as the feature under investigation usually exhibit a yearly dis-
placement 2–3 orders of magnitude higher than the seasonal,
instrumental noise itself (displacement in metres, noise in
millimetres). Nevertheless, short-term outliers in the obser-
vations should be interpreted carefully, as they can be an am-
plified result of noise in the reference station (e.g. spikes in
RAND and HOGR in Fig. A2). A last caveat should be given
on the interpretation of the data, which despite hourly to daily
resolution, should be interpreted with care at such shorter
timescales, critically distinguishing between noise and sig-
nals in the data. pends on the quantity and quality of the input data. 6
Validation and uncertainty of data products This in
turn depends on the satellite availability, the meteorological
and atmospheric conditions, on the duration of the data ac-
quisition intervals that in turn depend on the available power
as well as the ground kinematics observed. In effect, the ac-
curacy of the primary GNSS data products varies over time
and space (between different positions). For this reason, no
average/typical values are given for the whole dataset. In-
stead, quality indicators are available for each daily position
fix calculated. For the GNSS data the standard deviation for
each axis (E, N, vertical) as well as the ratio of fixed am-
biguities is given, the ratio is a percentage that specifies how
well the GNSS solver was able to resolve integer ambiguities
for fixing a solution – a commonly used quality indicator in
GNSS post-processing. For the rtklib solution a static param-
eter set is chosen for each reference position, i.e. all baseline
pairs to the same reference are processed in the same way. The details are in the respective documentation of the tools
used. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 8
Conclusions and outlook This paper documents the data resulting from a multiple
field-site monitoring effort using in situ GNSS sensors on dif-
ferent landforms in the Swiss Alps. This dataset constitutes
one of the largest and highest fidelity datasets document-
ing mass movement by means of permanent ground mea-
surement points, a method that has been developed and put
into practice by the authors. The data are obtained mainly at
mass movement sites in the periglacial field with a few place-
ments at lower altitudes at or beyond the fringe of permafrost. Most of these sites are subject to further investigations using
a multitude of methods, e.g. terrestrial surveys, seismome-
ters, time-lapse photography, UAV surveys, InSAR. A basic
overview of these field sites, methods employed, and data
available are documented. As such, this dataset provides an
important step for future work and development of novel
methods, further process understanding, and help for miti-
gating natural hazards as well as adaptation strategies. Since
October 2022, Rock Glacier Velocity (RGV) is a new Essen-
tial Climate Variable (ECV) product within the Global Cli-
mate Observing System (GCOS) (WMO, 2022). The Gugla-Bielzug rock glacier (BH 10/13) is situated to-
wards the north above the village of Herbriggen be-
tween the peaks of the Gugla and Breithorn. The frontal
part of the rock glacier opens up into a very steep gully
with frequent and high-volume discharge of loose ma-
terial. The Gugla-Bielzug rock glacier and Bielzug de-
bris flow are serious hazard sources in the area. Signifi-
cant efforts have been undertaken to (i) study the details
and (ii) implement protective measures for the safety
of the valley habitat and infrastructure. Specifically, a
debris catchment, several damming structures, and an
early warning system using geophones have been im-
plemented (Kummert et al., 2018a; Kummert and De-
laloye, 2018; Kummert et al., 2018b; Wirz et al., 2014b;
Guillemot et al., 2021; Oggier et al., 2016). The Stabbio di Largario rock glacier (LAR) is located
in the Adula/Rheinwaldhorn massif and therefore adds
to the monitoring concept in a different weather zone
(Scapozza et al., 2014). 7
Code and data availability western flank of the Distelhorn, above the municipality
of Grächen in the Matter Valley. The two instrumented
GNSS positions are located on two fronts. The upper
one (at 2500 ma.s.l.) shows a bulgy morphology, while
the lower one (at 2420 ma.s.l.) is more distinct. Ac-
cess to the rock glacier is facilitated by the presence of
nearby ski slopes. western flank of the Distelhorn, above the municipality
of Grächen in the Matter Valley. The two instrumented
GNSS positions are located on two fronts. The upper
one (at 2500 ma.s.l.) shows a bulgy morphology, while
the lower one (at 2420 ma.s.l.) is more distinct. Ac-
cess to the rock glacier is facilitated by the presence of
nearby ski slopes. – https://git.uibk.ac.at/informatik/neslab/public/
permasense/rtklib_processing
(last
access:
26
Oc-
tober
2022)
(DOI:
https://doi.org/10.5281/zenodo. 7251288, Beutel, 2022). The Gruben rock glacier (GRU) and Jaegi rock glacier
(JAE) are located in the adjacent Saas Valley, near Saas
Grund, (Switzerland). They are among the first rock
glaciers investigated in Switzerland (Haeberli et al.,
1979; Haeberli, 1996, 1985; Haeberli and Schmid,
1988). The Jaegi rock glacier consists of two over-
riding fronts at around 2550 ma.s.l. The rock glacier
has shown signs of destabilisation since the 1950s
(Ghirlanda et al., 2016). Currently, the upper lobe –
between 2670 and 2550 ma.s.l. – has to be considered
destabilised, with large displacement rates and geomor-
phological signs of degradation. The GNSS position is
located close to this fast-moving front. The dataset (structure is described in Table 3) and its an-
nual update are available from the following website: – Dataset
https://doi.org/10.1594/PANGAEA.948334
(Beutel et al., 2022) – Dataset
https://doi.org/10.1594/PANGAEA.948334
(Beutel et al., 2022) 8
Conclusions and outlook This rock glacier is known as
an important source of debris, reworked by debris flow
descending from the Soi Valley, and having caused sev-
eral damages at the village of Dangio-Torre in the past,
destroying the chocolate plant located at the conflu-
ence of Soi Valley into the Blenio Valley on 28–29 Au-
gust 1908. Appendix A: Detailed description of the field sites 7
Code and data availability The dataset published with this paper contains data from
1 March 2011 until 31 December 2021. An overview of
the structure, file types, and size of the datasets, for both
the raw primary data and derived data products, is given
in Table 3. Furthermore, the dataset also contains the key
metadata files for the field sites. Annual updates of this
dataset are planned (living data process). Using the toolset
described in Appendix C and using the online repository at
http://data.permasense.ch, last access: 26 October 2022 (see
Weber et al., 2019a for details), the data user can also create
custom updates of the dataset independently. Furthermore, a
set of wrapper scripts for GNSS post-processing using the
open-source RTKLIB toolchain (http://www.rtklib.com, last
access: 26 October 2022) are described in Appendix D. This
toolchain allows us to compute the differential GNSS daily
positions from both the RINEX files contained in this dataset
as well as the online data from the PermaSense database. Furthermore, the accuracy of the daily coordinate fixes de-
pends also on the physical behaviour observed (the kinematic
of the measured landforms). If the observation point moves
significantly during one observation interval (epoch) an aver-
age position is computed. For static (non-moving positions)
this effect can be easily neglected. For fast motion, or stick-
slip events on the contrary, this effect can negatively impact
the accuracy of the measurements that basically results in a
time offset for the observed displacement. If required a de-
tailed analysis using finer grained temporal scales for the
GNSS post-processing can be applied to the input data. On
longer temporal scales, a seasonal period in noise can be ob-
served for most stations and especially notable for the refer-
ence station. In Figs. 4 and B2, we show two examples of the
seasonal variability in the position of a reference station. This
pattern could be explained by the physical behaviour of the
boulder on which the sensor is mounted. However, because
no validation measurements are present, we have to consider The toolset (code) for preparing, processing, validating,
and updating the data contained in this publication is avail-
able from the following providers and links: Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5074 – https://git.uibk.ac.at/informatik/neslab/public/
permasense/permasense_datamgr (last access: 26 Oc-
tober
2022)
(DOI:
https://doi.org/10.5281/zenodo. 7251255, Weber et al., 2022). A1
Rock glaciers The Dirru Rock Glacier (DI) is a well-studied landform
for this peculiar kinematic behaviour (Delaloye et al.,
2010). Continuous kinematic measurements have been
available since 2011 for three positions in the upper
part and the first steep flank of the rock glacier. In year
2018, two more positions were instrumented on the fast-
moving tongue, with the goal of creating a spatially re-
solved network for the analysis of the spatial variability
of rock glacier velocities at a daily scale (Cicoira et al.,
2019b, 2021). The Muragl rock glacier (MUR) is located in the homonym
valley in the municipality of Samedan (Switzerland). The rock glacier has already been investigated 20 years
ago in some of the first detailed photogrammetric stud-
ies from aerial and satellite imagery in the periglacial
environment (Kääb et al., 1997, 1998). On site, one
fixed GNSS sensor is located a few hundred metres up
from the PERMOS borehole (Cicoira et al., 2019a). The Distelhorn rock glacier (DIS) is the northernmost rock
glacier of the Matter Valley cluster. It is located on the The Distelhorn rock glacier (DIS) is the northernmost rock
glacier of the Matter Valley cluster. It is located on the The Distelhorn rock glacier (DIS) is the northernmost rock
glacier of the Matter Valley cluster. It is located on the https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5075 The Randa Grossgufer (RA) is a 30 × 106 m3 rockslide site
on the orographic left side of the Matter Valley, just
opposite from the Dirru rock glacier. It has been thor-
oughly investigated by numerous researchers (Willen-
berg et al., 2008b, a; V. Gischig et al., 2011; Fäh et al.,
2012-12; S. Gischig et al., 2011; Moore et al., 2011;
Burjánek et al., 2010; Eberhardt et al., 2004). Moni-
toring activity has increased in the past years with one
GNSS reference and 3× GNSS sensor rovers situated at
the lip of the detachment. The Murtèl-Corvatsch rock glacier (COR) is one of the first
rock glaciers to be studied intensively worldwide. Its
displacement rates are 1–2 orders of magnitude smaller
than most other rock glaciers presented in this dataset. A3
Landslides The creep
rates and the seasonal variability of this rock glacier are
less pronounced and are more related to the general pat-
terns observed in the Alps, rather than its steeper neigh-
bours in the Matter Valley (Noetzli et al., 2019). A1
Rock glaciers It
hosts the longest time series of permafrost temperatures
in an alpine rock glacier, dating back to 1987 (Vonder
Mühll and Haeberli, 1990; Hoelzle et al., 2002). Dur-
ing the same period, vertical inclinometer profiles were
also measured manually, providing a unique dataset. Recent detailed process-oriented studies are based on
the dataset presented (Cicoira et al., 2019a, 2021). The Sattelspitz buttress (SA) is a minor buttress on the west
ridge above Täsch (Switzerland). Due to slope instabil-
ities and hazard potential for the fresh water supply of
the village of Täsch, a kinematic monitoring has been
set up at this site. A fixed GNSS sensor has been in-
stalled on top of a rock tower that is currently showing
the largest displacement rates on the rock face. The data
have not been analysed in a scientific publication. The Ritigraben rock glacier (RI) is a well-studied landform
in the vicinity of the ski slopes of Grächen (Switzer-
land). In addition to the kinematics data, borehole tem-
peratures and inclinometers data are available (Kenner
et al., 2017, 2018; Cicoira et al., 2019a). The Schafberg rock glacier (SCH) is located above the mu-
nicipality of Pontresina (Switzerland). The rock glacier
delivers debris to steep slopes prone to snow avalanches
and debris flows that endanger the infrastructure and
the village in the valley bottom. Therefore, it has been
the object of investigations for this and other projects
related to natural hazards. The kinematic measure-
ments are complemented by borehole temperatures and
past inclinometer profile measurements (Arenson et al.,
2002), available through PERMOS. Detailed studies
about rock glacier dynamics have investigated this well-
monitored site (Cicoira et al., 2019a; Kenner et al.,
2020). 1Geologisches Gutachten, Dr. R. Urs Winterhalter, Zurich,
1959, personal communication, Rosie Allmendinger, 2018, Her-
briggen, various news reports from 1959. A3
Landslides The Breithorn landslide (BH) is a landslide situated
on the south-west slope of the Breithorn summit
(3176 m a.s.l.). It is located in close proximity, a
few hundred metres north, of the Gugla-Bielzug rock
glacier, which holds the same label. Positions 3, 7, 9,
12, and 68 are/were located on the landslide. Positions
10 and 13 are located on the rock glacier (see below). The Längenschnee (LS) and Gugla (GU) landslides can be
found one ridge line further south in the valley. This area
features a large, relict landslide that surged at least once
in 1959, when the village of Herbriggen was evacuated
for a number of days in mid-winter1. The lower parts of
the slopes leading down from the Gugla are known as
Längenschnee, a sediment and debris-rich area charac-
terised by sharp steepening drop, leading into the valley
and towering above the village of Herbriggen. Here, the
downslope movement of the whole area poses a signif-
icant and real risk for the habitat in the valley. Impor-
tant countermeasures apart from observation and mon-
itoring have been undertaken recently, e.g. a protective
dam is currently being constructed and a large boulder
(Grosse Stei) in the area of Längenschnee has been fixed
by anchoring and concrete under-filling. This boulder
has been monitored since 2018 using a GNSS sensor
(position LS11). The Steintälli rock glacier (ST) is located a few hundred
metres higher in the same catchment as the Dirru rock
glacier. It is not directly connected to the valley bottom
and is characterised by gentler slope gradients in com-
parison to the other rock glaciers in the valley. How-
ever, it carries two exemplary steep frontal zones above
which the two GNSS sensors are positioned. The creep
rates and the seasonal variability of this rock glacier are
less pronounced and are more related to the general pat-
terns observed in the Alps, rather than its steeper neigh-
bours in the Matter Valley (Noetzli et al., 2019). The Steintälli rock glacier (ST) is located a few hundred
metres higher in the same catchment as the Dirru rock
glacier. It is not directly connected to the valley bottom
and is characterised by gentler slope gradients in com-
parison to the other rock glaciers in the valley. How-
ever, it carries two exemplary steep frontal zones above
which the two GNSS sensors are positioned. A2
Steep rock walls The Matterhorn Hörnli ridge (MH) is the east ridge of the
Matterhorn. The measurements are mainly clustered in
and around a prominent high-alpine rockfall (at an el-
evation of about 3500 ma.s.l.) that took place in year
2003 (Hasler et al., 2008, 2011, 2012). The highest po-
sition at this site and in the entire dataset is located
at the Solvey Hut at 4003 ma.s.l. Apart from the ther-
mal and kinematic measurements documented in Weber
et al. (2019a), recent years have also seen experimenta-
tion with seismic sensors (Weber et al., 2018b, c). The Matterhorn Hörnli ridge (MH) is the east ridge of the
Matterhorn. The measurements are mainly clustered in
and around a prominent high-alpine rockfall (at an el-
evation of about 3500 ma.s.l.) that took place in year
2003 (Hasler et al., 2008, 2011, 2012). The highest po-
sition at this site and in the entire dataset is located
at the Solvey Hut at 4003 ma.s.l. Apart from the ther-
mal and kinematic measurements documented in Weber
et al. (2019a), recent years have also seen experimenta-
tion with seismic sensors (Weber et al., 2018b, c). The Grabengufer landslide (GG) is a complex landform
comprising of a landslide (sagging) originating at ap-
prox. 2800 ma.s.l. as well as a very fast-moving rock The Grabengufer landslide (GG) is a complex landform
comprising of a landslide (sagging) originating at ap-
prox. 2800 ma.s.l. as well as a very fast-moving rock Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5076 glacier releasing its debris into a hazard debris flow
ending just north of the village of Randa (Delaloye
et al., 2013). It has seen mitigation measures for many
decades with the earliest on record dating back to 1945,
when a protective wall was constructed to fence off a
lateral part of the moving rock glacier in the area of the
“Grüne Garten” above the village of Randa2. The Wisse Schijen landslide (WYS) is situated on the
orographic left side of the Matter Valley at around
3100 m a.s.l., to the west of Randa. A large landslide
is affecting the top of the permafrost slope equipped
with avalanche protection structures (eight rows of snow
nets). 2Protocol of the Swiss Federal Council, 15 February 1945. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A2
Steep rock walls These nets retain snow in the Wisse Schijen
avalanche release area and protect numerous rows of
steel snow bridges below. A GNSS sensor is situated
in the centre of the landslide to monitor slope displace-
ments parallel to borehole inclinometer measurements. https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5077 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Data availability for all primary data products. The time periods when data are available are indicated in green. Figure A1. Data availability for all primary data products. The time periods when data are available are indicated in green. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5078 5078
A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment
Figure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given in
Table 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. 50 8
C co a, S
ebe et a
G SS obse
at o s o t e S
ss pe g ac a e
o
e
Figure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given
Table 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. Figure A2. Time series (weekly mean) the three components of all reference stations used. A detailed description of each station is given in
Table 2. For a discussion of the spike in HOGR that also appears in the nearby RAND, see Sect. 6. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment
5079
Figure A3. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A4. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5079 Figure A3. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A3. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acce BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A4. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A4. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 https://doi.org/10.5194/essd-14-5061-2022 a Location coordinates are given for the first day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor location
detached in a rockfall ending the time series. a Location coordinates are given for the first day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor loc
detached in a rockfall ending the time series. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5080 Figure A5. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A5. Station BH13 on the Breithorn/Bielzug rock glacier above Herbriggen; VS exhibits a steady acceleration downslope. Figure A6. Wireless LAN (WLAN) backbone and fine distribution of wireless connectivity to all sensor locations in the Matter Valley. The
basis for such connection is a WLAN access point located at the cable car station of the Klein Matterhorn 3883 ma.s.l. about 6.5 km away,
where the network is attached to a local Internet service provider using glass fibre. In a previous publication, Weber et al. (2019a) provided
a more comprehensive and detailed description of the technology of the WSN. Figure A6. Wireless LAN (WLAN) backbone and fine distribution of wireless connectivity to all sensor locations in the Matter Valley. The
basis for such connection is a WLAN access point located at the cable car station of the Klein Matterhorn 3883 ma.s.l. about 6.5 km away,
where the network is attached to a local Internet service provider using glass fibre. In a previous publication, Weber et al. (2019a) provided
a more comprehensive and detailed description of the technology of the WSN. Figure A7. In very active movement zones finding a single block to fix the measurement instrument can be challenging, both w.r.t. longevity
and the representative motion of the investigated landform. The two locations shown in this figure are very active zones in steep parts of
two rock glaciers above Herbriggen (Switzerland): Dirru rock glacier (a) and Breithorn/Bielzug rock glacier (b). Photos from PermaSense
archive. Figure A7. In very active movement zones finding a single block to fix the measurement instrument can be challenging, both w.r.t. longevity
and the representative motion of the investigated landform. The two locations shown in this figure are very active zones in steep parts of
two rock glaciers above Herbriggen (Switzerland): Dirru rock glacier (a) and Breithorn/Bielzug rock glacier (b). Photos from PermaSense
archive. https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5081 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment oira, S. Weber et al.: GNSS observations of the Swiss periglacial environment
A1. Per position overview of the sensors in the Matter Valley field site (see Fig. 2): Part 1. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
BH03
2015-10-07–ongoing
RD01
1.0
X
2629378
1109934
2754
X
X
BH07
2011-05-18–ongoing
RD01
1.0
X
2629787
1110178
2982
X
X
BH09
2011-05-18–ongoing
RD01
1.5
X
2630158
1110218
3159
X
X
BH10
2011-08-17–2018-01-08b
RD01
1.5
X
2629255
1109755
2662
X
X
BH12
2012-02-24–ongoing
RD01
1.0
X
2629596
1110114
2872
X
X
BH13
2012-02-24–ongoing
RD01
1.0
X
2629332
1109779
2701
X
X
DI02
2011-05-02–ongoing
RD01
1.5
X
2629569
1107710
2770
X
X
DI03
2017-06-09–ongoing
RD01
1.5
X
2629354
1107798
2676
X
X
DI04
2017-06-09–ongoing
RD01
1.0
X
2629241
1107861
2599
X
X
DI07
2010-12-16–ongoing
RD01
1.5
X
2629355
1107810
2673
X
X
DI55
2011-03-09–ongoing
RD01
1.0
X
2629457
1107876
2694
X
DI57
2011-03-01–2014-09-11
RD01
1.5
2629354
1107816
2673
X
DIS1
2012-07-19–ongoing
RD01
1.0
2632748
1115588
2426
X
X
DIS2
2012-07-19–ongoing
RD01
1.0
2632911
1115403
2501
X
X
GG01
2011-09-29–ongoing
RG01
1.0
X
2628937
1104474
2906
X
X
GG02
2011-09-29–2020-05-17c
RG01
1.0
X
2628872
1104513
2894
X
X
GG52
2011-10-03–2016-12-30
RG01
1.0
2628872
1104514
2894
X
GG66
2012-10-26–2016-12-30
RG01
1.3
X
2628817
1104430
2905
X
GG67
2012-11-20–2016-12-30
RG01
1.3
X
2628782
1104448
2889
X
GU02
2011-05-18–2013-05-24
RD01
1.0
2629715
1109034
2969
X
X
GU03
2011-05-18–2013-05-24
RD01
1.5
2629761
1108988
2996
X
X
GU04
2011-08-17–2013-05-24
RD01
1.5
2629946
1108966
3130
X
X
LS01
2011-05-18–2013-08-19
RD01
1.5
2629465
1109281
2804
X
X
LS04
2011-05-18–2013-08-19
RD01
1.5
2629455
1109179
2802
X
X
LS05
2012-02-24–ongoing
RD01
1.0
X
2629059
1109378
2611
X
X
LS06
2018-04-17–ongoing
RD01
1.0
X
2628942
1109255
2524
X
X
LS11
2014-11-21–2020-10-03c
RD01
1.0
X
2629018
1109451
2588
X
X
LS12
2014-11-21–ongoing
RD01
1.0
X
2629053
1109268
2604
X
X
a Location coordinates are given for the first day of deployment. b Sensor was destroyed in an avalanche ending the time series. c Sensor location
detached in a rockfall ending the time series. Table A1. Per position overview of the sensors in the Matter Valley field site (see Fig. 2): Part 1. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5082 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Table A2. a Location coordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Per position overview of the sensors in the Matter Valley field site (see Fig. 2): Part 2. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
RAND
2011-05-28–ongoing
ZERMb
X
2625632
1107181
2415
X
RA01
2015-10-08–ongoing
RAND
1.0
X
2625797
1107096
2326
X
X
RA02
2015-10-08–ongoing
RAND
1.0
X
2625772
1107086
2336
X
X
RA03
2016-05-27–ongoing
RAND
1.0
X
2625736
1107044
2325
X
X
RD01
2011-03-01–ongoing
RAND
0.5
X
2629577
1108071
2706
X
RG01
2011-09-29–ongoing
RAND
0.5
X
2628984
1104371
2974
X
RIT1
2012-07-19–ongoing
RD01
1.0
2631650
1113771
2605
X
X
RL01
2011-08-17–2013-05-24
RD01
1.0
2629491
1109569
2873
X
X
SA01
2018-08-06–ongoing
RAND
1.0
X
2628397
1099584
3079
X
X
SATT
2018-08-29–ongoing
RAND
X
2628357
1099535
3127
X
X
ST02
2011-05-18–ongoing
RD01
1.0
2630157
1108556
2997
X
X
ST05
2011-05-18–ongoing
RD01
1.5
2630237
1108650
3029
X
X
WYS1
2014-11-20–ongoing
RAND
1.0
X
2624011
1105068
3056
X
X
DH13
2011-03-08–ongoing
X
2629563
1108035
2690
X
DH42
2011-08-17–ongoing
X
2628985
1104370
2827
X
DH68
2013-08-20–ongoing
X
2629598
1110119
2870
X
DH69
2018-07-10–2020-06-03
X
2629385
1107919
2644
X
DH73
2018-07-10–2020-06-03
X
2629385
1107919
2644
X
a Location coordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5083 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment e A3. Per position overview of the sensors in the Matterhorn field site. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
HOGR
2011-02-03–ongoing
ZERMb
X
2618012
1092200
3463
X
MH33
2014-08-16–ongoing
HOGR
X
2617961
1092175
3487
X
X
MH34
2014-08-14–ongoing
HOGR
X
2618001
1092197
3463
X
X
MH35
2015-06-02–ongoing
HOGR
X
2617961
1092175
3487
X
X
MH40
2015-06-03–ongoing
HOGR
X
2617957
1092175
3489
X
MH43
2018-08-15–2020-07-08
HOGR
X
2617957
1092175
3489
X
MH15
2015-06-02–ongoing
X
2618019
1092200
3402
X
MH25
2010-12-17–ongoing
X
2618019
1092200
3402
X
MH51
2019-06-26–ongoing
X
2617392
1091918
4003
X
a Location coordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Table A3. Per position overview of the sensors in the Matterhorn field site. ordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Table A4. Per position overview of the sensors in the Saas Tal field site. A4. Per position overview of the sensors in the Saas Tal field site. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
GRU1
2012-07-25–ongoing
RD01
0.5
2640436
1113468
2823
X
X
JAE1
2012-07-26–ongoing
RD01
0.5
2639856
1111235
2585
X
X
a Location coordinates are given for the first day of deployment. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 5084 Appendix B: Project context and history X-Sense, the initial project, conducted from 2010 to 2013,
focused on a study area on the orographic right side of the
Matter Valley above the municipalities of Randa and Her-
briggen (Switzerland). This area is dominantly situated in
permafrost, exhibits several features (Wirz et al., 2013) (see
Fig. 2), and has a rich history w.r.t. mass movement-related
natural hazards. Specifically, the earliest known records for
hazard mitigation efforts date back to 1945 (subsidies by the
Swiss federal government for rock-wall protection measures
near the Grabengufer) and February 1959 (evacuation of the
village of Herbriggen due to an excessive landslide sponta-
neously developing on the Längenschnee/Gugla area). y
p g
g
g
)
Apart from obtaining sensor data and working on geosci-
entific process studies, this project also focussed on devel-
oping and proving the utility of low-powered wireless GNSS
sensors in the scope of the application described. As men-
tioned earlier in Sect. 2, a new set of sensors was developed
based on commodity L1-GPS receivers and ubiquitous wire-
less data access based on previous work on the Matterhorn
(Talzi et al., 2007; Hasler et al., 2008, 2012; Weber et al.,
2017) and Jungfraujoch (Hasler et al., 2011; Girard et al.,
2012). The main challenge, besides designing a robust and
long-lived sensing system suitable for year-round operation
in a high-alpine setting, lies in the fact that the GNSS sen-
sors employed are characterised by (i) large data volumes and
(ii) a significant power consumption compared to many other
in situ sensors used in this domain. This is due to the fact
that the GNSS receiver needs to be operated continuously
over large periods of time (typically hours) and without us-
ing any low-power operating modes and also using an active
antenna in order to obtain sufficient observation data from
the satellite constellation w.r.t. both quality and quantity. For
the detection of very small displacements, such as in compact
bedrock or the ability to react to changing displacement dy-
namics quickly, e.g. in natural hazard scenarios, a 24/7 oper-
ation of the sensors is required. The resulting energy and data
footprint of the GNSS sensor alone (without data logging and
data transmission) is on the order of watts and megabytes per
station and per day. Therefore, it significantly exceeds typical
requirements of geoscientific data-acquisition systems, e.g. a
typical data logger with sensors attached. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5085 decision-making by the Swiss cantonal and federal author-
ities. A selection of the most noteworthy events and mea-
sures are described in the following: In spring 2013, exces-
sive discharge from the Gugla-Bielzug rock glacier caused
severe debris flow in the Bielzug torrent, causing a partial
evacuation of the village of Herbriggen. Subsequently, a new
catchment with dam as well as geophone-based monitoring
was projected and erected. In order to protect hikers crossing
the Grabengufer, a hanging bridge spanning the upper part of
the discharge gully was constructed in 2010. Due to the rapid
evolution of the Grabengufer rock glacier and the landslide
above it, the bridge was hit by discharged debris multiple
times, subsequently closed and dismantled. In 2017, a new
bridge with a span of 494 m was erected further downslope
in the gully. In 2018, a large boulder on the order of 2000 m3
was blasted in a 2-month effort to protect the village of Randa
below (see Figs. B1 and B2). This freestanding boulder was
located at the front of the landslide feeding into the Graben-
gufer rock glacier and was gradually revealed due to continu-
ous erosion happening because of the excessive slope move-
ments in the area. Figure B2 shows the long-term evolution
of position GG02. The seasonal accelerations throughout the
time series and the anomalous behaviour (exponential accel-
eration) during 2020 are notable; this lead to the detachment
of the boulder from the slope and a consequent rockfall. The
process is visible in both the displacement and inclinome-
ter data. Another relevant case interests the area of Längen-
schnee, where a large rock boulder (2524 ma.s.l.) endanger-
ing the municipality of Herbriggen was stabilised with an-
chors and concrete under-filling in 2014. Here, the monitor-
ing of slope movement using 3× GPS on unstable masses
and 1× GNSS sensor on the stabilised rock serve as inte-
gral parts of the protection measures for the village of Her-
briggen. Due to the recent evolution of the landslide, the vil-
lage has received a new hazard zonation in 2018, and four
large protective dams have recently been erected on the upper
limit of the village. In the Ritigraben area, the rock glacier
has repeatedly led to severe debris flow with impact on the
road, railway track, and Matter Vispa river below. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment The most
notable event was in 2018 when the debris discharged by the
Ritigraben Rock Glacier obstructed the river and caused se-
vere flooding all the way into the central sewage treatment
plant of the valley (Kenner et al., 2017, 2018). The method devised in this initial project (Buchli et al.,
2012; Wirz et al
2013) has proven to be very successful Appendix B: Project context and history A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environme Table A5. Per position overview of the sensors in the Val Blenio field site. ble A5. Per position overview of the sensors in the Val Blenio field site. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
LAR1
2014-09-26–ongoing
SANBb
1.0
2718881
1148509
2355
X
X
LAR2
2014-09-28–ongoing
SANBb
1.0
2718731
1148483
2304
X
X
a Location coordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Table A6. Per position overview of the sensors in the Engadine field site. A6. Per position overview of the sensors in the Engadine field site. General
Locationa
Kinematics
Weather
Station
Period of operation (yyyy-mm-dd)
Reference
Mast height [m]
Online data
East
North
Altitude
L1/L2-GNSS
L1-GPS
Inclination
Radiation
Weather data
COR1
2015-12-17–ongoing
SAMEb
0.8
2783147
1144727
2669
X
X
MUA1
2012-08-04–ongoing
SAMEb
1.0
2791144
1153620
2609
X
X
SCH1
2012-08-04–ongoing
SAMEb
1.0
2791062
1152725
2809
X
X
a Location coordinates are given for the first day of deployment. b Data from the permanent GNSS network in Switzerland (AGNES) are used here. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Figure B1. The Grabengufer rock glacier in winter. Panel (a) shows an overview of the catchment with the Grabenhorn on top, the Graben-
gufer landslide, and the Grabengufer rock glacier above the Dorfbächji channel, with exposed eroded debris. Panel (b) shows the zoomed-in
area highlighted with the yellow box, the Grabengufer landslide in summer. The different measurement positions, the reference station, and
the freestanding block (approx. 2000 m3) blasted in summer of 2018 are labelled. Photos from PermaSense archive. Figure B1. The Grabengufer rock glacier in winter. Panel (a) shows an overview of the catchment with the Grabenhorn on top, the Graben-
gufer landslide, and the Grabengufer rock glacier above the Dorfbächji channel, with exposed eroded debris. Panel (b) shows the zoomed-in
area highlighted with the yellow box, the Grabengufer landslide in summer. The different measurement positions, the reference station, and
the freestanding block (approx. 2000 m3) blasted in summer of 2018 are labelled. Photos from PermaSense archive. Figure B2. A decade of displacement, inclination, and azimuth for the Grabengufer rock glacier (position GG02). In 2020, the boulder
where the GNSS station was installed showed an anomalous behaviour and strong acceleration until falling into the gully. The data show this
process until the last day. Figure B2. A decade of displacement, inclination, and azimuth for the Grabengufer rock glacier (position GG02). In 2020, the boulder
where the GNSS station was installed showed an anomalous behaviour and strong acceleration until falling into the gully. The data show this
process until the last day. Appendix C: PermaSense data manager Appendix B: Project context and history The method devised in this initial project (Buchli et al.,
2012; Wirz et al., 2013) has proven to be very successful
and was thus expanded to other locations and applications of
monitoring (Kenner et al., 2018; Cicoira et al., 2021) as well
as natural hazard mitigation (Kenner et al., 2020) in collab-
oration with partners of PERMOS, the Swiss cantonal and
federal authorities (Randa Grossgufer, Wisse Schijen, PER-
MOS GNSS sites) (Noetzli et al., 2019). The deployment activities of GNSS sensors started in the
summer of 2010 on the central orographic right side of the
Matter Valley above the village of Herbriggen. From then on,
the newly developed GNSS sensors were tested and put to
use to survey kinematics across different landforms and haz-
ard areas (Wirz et al., 2014b). Further extensions took place
at the Steintälli rock glacier, the Gugla/Bielzug rock glacier,
the Längenschnee, Breithorn, Gugla landslide areas as well
as the Grabengufer above Randa. During the study period, further hazard and mitigation
events took place where the data documented by and sup-
plementing this paper served as integral components for Earth Syst. Sci. Data, 14, 5061–5091, 2022 5086 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment Earth Syst. Sci. Data, 14, 5061–5091, 2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment the online PermaSense database at http://data.permasense.ch
(last access: 21 October 2022) into a local file system. Specifically, the PermaSense data manager allows us to Appendix D: GNSS processing using the
open-source RTKLIB The output format of the position data CSV files produced
by RTKLIB is described in Table D1. A set of scripts automates the computation of static double-
difference GNSS solutions using the open-source toolchain
RTKLIB (http://www.rtklib.com, last access: 21 Octo-
ber 2022). The scripts are configurable for each baseline pair
w.r.t. input data, configuration parameter, and the toolchain to
be used. The configuration files and especially paths are set
up specifically for processing and producing the PermaSense
GNSS data contained in this dataset, but they can be adapted
to other processing needs accordingly. This allows flexibility
for individual processing needs for each position, should that
be required. The scripts are designed to run on x86 Linux,
but porting this to other platforms is straightforward. D1
Prerequisites In order to run these scripts, the following prerequisites must
be installed: – query data from the PermaSense GSN server and save
them locally as CSV files – RTKLIB
processing
scripts
https://git.uibk.ac.at/
informatik/neslab/public/permasense/rtklib_processing
(last access: 21 October 2022, Beutel, 2022) – load the locally store CSV files – filter according to reference values if available
– RT
(las – RTKLIB can be obtained from http://www.rtklib.com/
(last access: 21 October 2022) or alternatively https://
github.com/rtklibexplorer/RTKLIB (last access: 21 Oc-
tober 2022) – filter according to reference values if available – clean data manually if needed – generate aggregates using an arithmetic mean (excep-
tions for weather data) – RINEX file compression tools https://terras.gsi.go.jp/ja/
crx2rnx.html (last access: 21 October 2022) – RINEX file compression tools https://terras.gsi.go.jp/ja/
crx2rnx.html (last access: 21 October 2022) – generate per-year CSV files for each position and data
type Some of the double-difference baselines are configured us-
ing reference positions from the Permanent GNSS network in
Switzerland (AGNES) and therefore these observation data
must be obtained directly from swisstopo as they are not con-
tained in this dataset. – generate standard plots for all positions as an intuitive
sanity check – query images from the PermaSense database server,
convert to JPEG and save them locally. python manage_GSNdata.py By default, data are generated in the directory ./data. https://doi.org/10.5194/essd-14-5061-2022 D2
Processing sequence and configuration setup A README.md with this software package explains its us-
age. In short, a suitable Python environment is required. Us-
ing anaconda, you can install the requirements by executing
the following command: A single processing job always computes a single daily posi-
tion for a given baseline pair. There are two kinds of configu-
ration files that are required for each processing job: (i) a pa-
rameter file that specifies a baseline pair, data down-/upload
location, starting dates, default directories, and (ii) tools to
use for processing as well as a RTKLIB tool-configuration
file. The script package first collects the necessary data from
either the online server at http://data.permasense.ch (last ac-
cess: 21 October 2022) from the IGS service or from a lo-
cal file repository from disk and copies these files together
with the necessary configuration files into a local tempo-
rary directory. If required, RINEX files are produced using
the conv2bin tool from the RTKLIB toolchain. Thereafter,
the post-processing tool rnx2rtkp is called, producing an
output file with the coordinate data (see Table D1). Lastly,
the coordinates are transformed from WGS84 to the Swiss
national coordinate system by using the online REFRAME
conversion service (REST API) by swisstopo. conda env create -f condaEnvironment.yaml conda env create -f condaEnvironment.yaml
conda activate permasense_datamgr conda env create -f condaEnvironment.yaml onda activate permasense_datamgr Individual positions can be enabled/disabled in the main
Python file manage_GSNdata.py, the metadata for filter-
ing and cleaning is contained in the folder ./metadata. Finally the tool is run by python manage_GSNdata.py Appendix C: PermaSense data manager (last access: 21 October 2022, Weber et al., 2022). It con-
tains both a Python toolbox for downloading and processing
primary as well as secondary data. The toolbox contains
routines for the compilation, cleaning, aggregation, and vali-
dation of both primary as well as derived data products from Codes for the management and processing of data associ-
ated with this paper are available at https://git.uibk.ac.at/
informatik/neslab/public/permasense/permasense_datamgr https://doi.org/10.5194/essd-14-5061-2022 Earth Syst. Sci. Data, 14, 5061–5091, 2022 5087 D3
Processing wrapper script syntax The main processing wrapper script is responsible for down-
loading all required data, creating a local temporary com-
puted space, executing the post-processing tool rnx2rtkp,
uploading the resulting data to the PermaSense database, and
archiving input and output data as well as cleaning up the
temporary file space. Earth Syst. Sci. Data, 14, 5061–5091, 2022 5088 5088 Table D1. GNSS computed daily position data CSV file format. Value
Unit
Description
time
[–]
Timestamp of the position data
position
[–]
Inventory position number from the database
label
[–]
Station label
processing_time
ms
Processing time in Unix time in milliseconds
device_type
[–]
Sensor type
version
[–]
Processing framework version
reference_label
[–]
Reference station label
e
m
Easting
n
m
Northing
h
m
Altitude
sd_e
m
Standard deviation easting
sd_n
m
Standard deviation northing
sd_h
m
Standard deviation altitude
ratio_of_fixed_ambiguities
[–]
Quality metric from post-processing Acknowledgements. We are extremely grateful for the extraor-
dinary local support that we received during our research activi-
ties in the Matter and Saas valleys, specifically from the munici-
palities of Zermatt (Romy Biner-Hauser), St. Niklaus (Gaby Fux),
Herbriggen, Randa, Taesch (Klaus Tscherrig), and Saas Grund;
the whole team of Air Zermatt (Gerold Biner); Kurt Lauber,
Stephanie Mayor, Martin Lehner, Edith Lehner, and the Hörnlihütte
team; Europahütte (Marcel Brantschen); Kinnhütte (Victor Imbo-
den); Alpin Center Zermatt; Zermatter Bergbahnen (Kurt Guntli);
Sprengtechnik-GFS (Willy Gitz and Angelo Gruber); Hotel Bahn-
hof, Zermatt (Fabi Lauber); and the local mountain guides (Her-
mann Biner, Robert Andenmatten, Willy Taugwalder, Urs Lerjen,
Benedikt Perren, Bruno Jelk, Hannes Walser, Simon Anthamat-
ten, Yann Dupertuis, and Anjan Truffer). Without this strong pos-
itive welcome, this work would not have been possible. We do not
know what we would have done without our “home base” at Ho-
tel Bergfreund in Herbriggen (CH). A big thank you to all gener-
ations of the Rosi and Rudi Allmendinger families for their gen-
erous support. Furthermore, we are thankful for technical support
and consultancy from the Art of Technology (Rolf Schmid), Zurich,
CH, as well as Swisstopo (Elmar Brockmann), Wabern, CH, and
to the many friends and helpers who were involved in supporting
the field work: Lucas Girard, Stephanie Gubler, Christoph Walser,
Robert Kenner, Johann Müller, Jeff Moore, and Valentin Gischig. Finally, we acknowledge the two anonymous referees as well as
Ken Mankoff and Inga Beck for handling the peer-review process
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formance
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https://doi.org/10.1109/ACCESS.2020.3048352, 2021. Girard, L., Beutel, J., Gruber, S., Hunziker, J., Lim, R., and Weber,
S.: A custom acoustic emission monitoring system for harsh en-
vironments: Application to freezing-induced damage in alpine
rock walls, Geosci. Instrum. Method. Data Syst., 1, 155–167,
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sor Network Node for High-accuracy Positioning Applications,
Lecture Notes on Computer Science 7158. Proc. of 9th Euro-
pean Conference on Wireless Sensor Networks (EWSN 2012),
Trento, Italy, 15–17 February 2012, https://link.springer.com/
chapter/10.1007/978-3-642-28169-3_12 (last access: 31 Octo-
ber 2022), 2012. Gischig, S., Moore, J. R., Evans, K. F., Amann, F., and Loew, S.:
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and Stempfhuber, W.: Composite rock slope kinematics at
the current Randa instability, Switzerland, based on remote
sensing and numerical modeling, Eng. Geol., 118, 37–53,
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D.: Ambient vibration analysis of an unstable mountain slope,
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246X.2009.04451.x, 2010. A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment 5089 Review statement. This paper was edited by Inga Beck and
Kenneth Mankoff and reviewed by two anonymous referees. Review statement. This paper was edited by Inga Beck and
Kenneth Mankoff and reviewed by two anonymous referees. through heat conduction on rock glacier dynamics: a nu-
merical modelling approach, The Cryosphere, 13, 927–942,
https://doi.org/10.5194/tc-13-927-2019, 2019a. through heat conduction on rock glacier dynamics: a nu-
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the seasonal rhythm of rock glacier flow, Earth Planet. Sc. Lett.,
528, 115844, https://doi.org/10.1016/j.epsl.2019.115844, 2019b. D3
Processing wrapper script syntax compute_solution.sh -p
[parameter_file] -d -b -r -c -f -u YYYY MM DD
#
-d:
igs data download
#
-b:
no data download and no conversion
for the basestation
#
-r:
no data download and no conversion
for the roverstation
#
-c:
no conversion
#
-f:
use IGS final data product
#
-u:
upload to GSN database Supplement. The supplement related to this article is available
online at: https://doi.org/10.5194/essd-14-5061-2022-supplement. Supplement. The supplement related to this article is available
online at: https://doi.org/10.5194/essd-14-5061-2022-supplement. Author contributions. JB, AC, and SW developed the concept
and prepared the manuscript. JB, SG, AH, SW, BB, AB, MM,
RL, TG, and RDF developed the sensor technology, the data-
management architecture as well as the tools for managing the data. VW, SG, HR, and JB conceived the initial GNSS sensor deploy-
ments with the help of LT, TS, AV, and DVM that jointly had instru-
mental roles in launching and executing the initial X-Sense project. PL implemented the first GNSS post-processing prototype. JB, AV,
SW, AC, AH, HR, LT, TS, RD, IGR, RM, JN, MP, EP, CS, and
DVM contributed to the scaling and application of the technology
to further field sites and applications, especially in the domain of
long-term monitoring, natural hazard mitigation and early warning. All the authors contributed to the article and approved the submitted
manuscript. Financial support. This research has been supported by fund-
ing from the Swiss National Science Foundation NCCR-MICS,
the ETH Zurich Competence Center Environment and Sustainabil-
ity (CCES), the Swiss Federal Office of the Environment (FOEN),
nano-tera.ch (grant no. 530659), and the Swiss Permafrost Moni-
toring Network (PERMOS). Support in the form of equipment has
been given by Hilti Schweiz AG, Arc’teryx, Petzl, and Beal. The
technical workshops at ETHZ and UniZH as well as Art of Tech-
nology, Zurich, contributed to the successful development and im-
plementation of various pieces of equipment. Competing interests. The contact author has declared that none
of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Earth Syst. Sci. Data, 14, 5061–5091, 2022 https://doi.org/10.5194/essd-14-5061-2022 A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment A. Cicoira, S. Weber et al.: GNSS observations of the Swiss periglacial environment References Guillemot, A., Baillet, L., Garambois, S., Bodin, X., Helm-
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Enhanced flood hazard assessment beyond decadal climate cycles
based on centennial historical data (Duero basin, Spain)
Gerardo Benito1, Olegario Castillo2, Juan A. Ballesteros-Cánovas3, Maria Machado1, and Mariano Barriendos4 3Climatic Change Impacts and Risks in the Anthropocene (C-CIA), Institute for Environmental Sciences,
University of Geneva, Geneva, Switzerland 4Dpt. d’Història i Arqueologia, Universitat de Barcelona, Montalegre 6, 08001 Barcelona, Spain Correspondence: Gerardo Benito (benito@mncn.csic.es) Received: 11 June 2021 – Discussion started: 26 July 2021
Revised: 17 October 2021 – Accepted: 23 October 2021 – Published: 2 December 2021 Abstract. Current climate modelling frameworks present
significant uncertainties when it comes to quantifying flood
quantiles in the context of climate change, calling for new in-
formation and strategies in hazard assessments. Here, state-
of-the-art methods on hydraulic and statistical modelling are
applied to historical and contemporaneous flood records to
evaluate flood hazards beyond natural climate cycles. A com-
prehensive flood record of the Duero River in Zamora (Spain)
was compiled from documentary sources, early water-level
readings and continuous gauge records spanning the last
500 years. Documentary evidence of flood events includes
minute books (municipal and ecclesiastic), narrative descrip-
tions, epigraphic marks, newspapers and technical reports. We identified 69 flood events over the period 1250 to 1871,
of which 15 were classified as catastrophic floods, 16 as ex-
traordinary floods and 38 as ordinary floods. Subsequently,
a two-dimensional hydraulic model was implemented to re-
late flood stages (flood marks and inundated areas) to dis-
charges. The historical flood records show the largest floods
over the last 500 years occurred in 1860 (3450 m3 s−1), 1597
(3200 m3 s−1) and 1739 (2700 m3 s−1). Moreover, at least
24 floods exceeded the perception threshold of 1900 m3 s−1
during the period (1500–1871). Annual maximum flood
records were completed with gauged water-level readings
(pre-instrumental dataset, PRE: 1872–1919) and systematic
gauge records (systematic dataset, SYS: 1920–2018). The
flood frequency analyses were based on (1) the expected mo-
ments algorithm (EMA) and (2) the maximum likelihood estimator (MLE) method, using five datasets with different
temporal frameworks (historic dataset, HISTO: 1511–2018;
PRE–SYS: 1872–2018; full systematic record, ALLSYS:
1920–2018; SYS1: 1920–1969; and SYS2: 1970–2018). The
most consistent results were obtained using the HISTO
dataset, even for high quantiles (0.001 % annual exceedance
probability, AEP). PRE–SYS was robust for the 1 % AEP
flood with increasing uncertainty in the 0.2 % AEP or 500-
year flood, and ALLSYS results were uncertain in the 1 %
and 0.2 % AEP floods. Enhanced flood hazard assessment beyond decadal climate cycles
based on centennial historical data (Duero basin, Spain)
Gerardo Benito1, Olegario Castillo2, Juan A. Ballesteros-Cánovas3, Maria Machado1, and Mariano Barriendos4 Since the 1970s, the frequency of ex-
traordinary floods (>1900 m3 s−1) declined, although floods
on the range of the historical perception threshold occurred
in 2001 (2075 m3 s−1) and 2013 (1654 m3 s−1). Even if the
future remains uncertain, this bottom-up approach addresses
flood hazards under climate variability, providing real and
certain flood discharges. Our results can provide a guide on
low-regret adaptation decisions and improve public percep-
tion of extreme flooding. 1
Introduction There are major challenges in dealing with flood hazards on
a global scale (UNISDR, 2015). Climate warming is part
of the problem, but the challenges will continue to be great
as a result of population growth and human occupation of
flood risk zones (Kundzewicz et al., 2014). Most “top-down”
approaches based on downscaling of nested climate models Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021
https://doi.org/10.5194/hess-25-6107-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. G. Benito et al.: Enhanced flood hazard assessment with hydrological approaches (deterministic and statistical)
typically produce uncertain results, with a wide range of sce-
narios which are difficult to implement on a local scale (Gar-
cía et al., 2014; IPCC, 2012). Given the uncertainty in cli-
mate model projections, a new focus on other risk variables
(demography, land use, urbanisation) has led to supporting
decision-making processes (Döll et al., 2015). This “bottom-
up” approach is based on reducing exposure and vulnerabil-
ity but still fails to solve the probability assessment of flood
hazards due to the stochastic nature of weather (Kundzewicz
et al., 2010). In this regard, the information from past flood
events has become an important data source to quantify the
links between the occurrence of extreme events and natural
climate variability that provide expectations of future climate
change. al., 2019) shows a growing interest to harness past flood in-
formation to evaluate flood hazards and risks (St. George et
al., 2020). In the European Union, several countries have pro-
posed the use of past flood data on the probability of future
floods as part of low-regret actions to solve the uncertainty in
downscaling climate model results on a local scale (García
et al., 2014; European Commission, 2021). Going forward,
technical guidance and new protocols on the use of past flood
archives are needed to reach engineers and stakeholders so it
can become a far-reaching and smart tool to cope with flood-
ing (EXCIMAP, 2007; England et al., 2019). In this paper, we firstly implement a holistic methodology
combining historical flood evidence and a two-dimensional
hydraulic model to reconstruct centennial registers of peak
discharges and their relationship with natural climate vari-
ability and triggers. Secondly, we apply two flood frequency
statistical methods to different types and series lengths of
flood data sources (historic, pre-instrumental and gauged
records) to evaluate the robustness of flood discharges for
low-probability events. Thirdly, we consider the historical
flood imprint on local communities as a basic tool to improve
public risk awareness on urban space, heritage buildings and
cultural landscapes. Our results based on this bottom-up ap-
proach show a direct guide on flood possibilities beyond
decadal climate cycles that can be used to provide a portfolio
of low-regret solutions suitable for climate change adapta-
tion. G. Benito et al.: Enhanced flood hazard assessment Over recent decades, palaeoflood and documentary flood
archives have been used to quantify flood discharge and fre-
quency over centennial to millennial timescales with appli-
cations for engineering design and risk estimation (Aldrete,
2007; Baker, 2008; Wetter et al., 2011; Elleder et al., 2013). A recent pan-European historical archive analysis has iden-
tified nine flood-rich periods (30–40-year interval) over the
last 500 years, all except the last one (1992–2016) occur-
ring during intervals of colder air temperatures than the inter-
flood period before and after (Blöschl et al., 2020). The exis-
tence of flood-rich periods was also demonstrated at millen-
nial timescales for the Mediterranean basin and at a European
scale using palaeoflood sedimentary evidence (Benito et al.,
2015c). These studies reveal that flood-producing mecha-
nisms, on a local to regional scale, are not necessarily driven
by temperature anomalies but are controlled by the behaviour
process of ocean and atmospheric circulation (Woollings et
al., 2010; Ballesteros-Cánovas et al., 2019). Thus, long-term
flood registers, produced by multiple atmosphere–ocean in-
teractions, contain a wide range of weather anomalies to ad-
vise on expected flood extremes in a changing climate, and,
more importantly, it reveals what flood dimension is actually
possible on a local scale (Elleder et al., 2013; Macdonald,
2013). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 6108 2.1
Geographical and physical setting (a) The Duero River basin (green) in the Iberian Peninsula together with Miño (light blue), Tagus (yellow), Guadiana (orange) and
Guadalquivir (dark blue) catchments draining towards the Atlantic Ocean; (b) relief map of NW Iberia with the Duero catchment boundary,
main drainage network and major cities. The rectangle at Zamora shows the location of (c); (c) hillshade map with extension of the floodplain
between Toro and Zamora. Map shows location of gauge stations in the Duero and Valderaduey rivers. Figure 1. (a) The Duero River basin (green) in the Iberian Peninsula together with Miño (light blue), Tagus (yellow), Guadiana (orange) and
Guadalquivir (dark blue) catchments draining towards the Atlantic Ocean; (b) relief map of NW Iberia with the Duero catchment boundary,
main drainage network and major cities. The rectangle at Zamora shows the location of (c); (c) hillshade map with extension of the floodplain
between Toro and Zamora. Map shows location of gauge stations in the Duero and Valderaduey rivers. The Duero River east of Zamora flows along a 2–3 km
wide floodplain (Fig. 1c). In Zamora the floodplain is asym-
metric with a 300 m wide floodplain at the southern mar-
gin, whereas on the northern side the channel is cut on
silicified sandstone and conglomerates dating back to Early
Cretaceous–Palaeocene (Areniscas de Salamanca; Delgado-
Iglesias and Alonso-Gavilán, 2008). West of Zamora, the
river is incised in granite and metamorphic rocks of Palaeo-
zoic age, forming a confined bedrock canyon, with punctu-
ated valley expansions. The combination of a narrow and
steep valley floor has been optimal for the development
of hydroelectric dam facilities along the Arribes del Duero
(narrows of the Duero), an impressive 800 m deep bedrock
canyon formed by the Duero at the Spanish–Portuguese bor-
der. turies (Santo Tomé, Santiago el Viejo, San Claudio de Oli-
vares, San Cebrián and Santa Maria la Nueva; Fig. 2a). Other
Romanesque churches bearing marks corresponding to his-
toric flooding were built during the first half of the 13th
century (Santa Maria de la Horta, San Frontis, Dueñas de
Cabañales, San Leonardo and Santa Lucia). The first known
bridge (Puente Viejo), located near the Olivares mill, was de-
stroyed by the 1310 flood event (Marquina, 1949b), though
several basal piers remain visible (site 24, Fig. 2b). 2.1
Geographical and physical setting The Duero River drains the northern Spanish Plateau and
flows east–west into the Atlantic Ocean at Porto (Portugal)
(Fig. 1a). It is one of the longest rivers of the Iberian Penin-
sula (897 km) and the largest in catchment area (98 073 km2),
of which 78 859 km2 is in Spain and 19 214 km2 in Portugal. The flood records studied are located in Zamora, in the lower
part of the Spanish Duero basin (Fig. 1b), draining a catch-
ment area of 46 137 km2. Historical and palaeoflood hydrology has demonstrated its
potential to determine the quantitative information of specific
flood events over centennial to millennial time spans (Ben-
ito et al., 2015a; Cœur and Lang, 2008; Wetter et al., 2011;
Elleder et al., 2013; Wilhelm et al., 2019). Major advances
are related to (1) digitalisation of archival sources, facilitat-
ing the search and screening tasks; (2) lidar data used for
digital terrain models, buildings and urban spaces; (3) appli-
cation of sophisticated two- or even three-dimensional hy-
draulic modelling to estimate peak discharges, local flow ve-
locity and depth; and (4) new statistical procedures and soft-
ware to feed historical and palaeoflood data into quantitative
frequency analysis (Macdonald, 2013; Benito et al., 2020). Although in the past the use of historical flood archives was
mainly considered in academic research circles, a recent pub-
lication by the US federal government guidelines (England et The Duero basin is surrounded by the Cantabrian Moun-
tains to the north, the Iberian Range to the east and the Cen-
tral Range (Gredos and Guadarrama Mountains) to the south. Geologically, the Duero catchment area comprises two ma-
jor zones: (1) the eastern side covers the Cenozoic endorheic
continental basin (∼50000 km2) composed of detritic, car-
bonate and evaporitic units (Alonso-Zarza et al., 2002), over-
lain by Quaternary alluvial fans and fluvial staircase terraces
developed by fluvial dissection related to the onset of ex-
oreic basin conditions (Martin Serrano, 1991; Rodríguez-
Rodríguez et al., 2020), and (2) the west is composed of
Palaeozoic granitic and a metamorphic basement, where the
fluvial network is deeply incised, forming confined river val-
leys. https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6109 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 1. 2.1
Geographical and physical setting The me-
dieval Stone Bridge, still in use, was finished during the 13th
century, although with a first written reference in 1167 as
“Ponten Novum” or New Bridge (Enríquez de Salamanca,
1998; site 8, Fig. 2). The number of reported floods increases in parallel with
the demographic growth during the 14th century that brought
the third major urban expansion and new commercial and ar-
tisanal activities (Fig. 2a). These activities were carried out
in the new suburbs next to the Stone Bridge (Horta neigh-
bourhood) as well as in the areas surrounding the three wa-
ter mills. The Olivares mill and its neighbourhood sit on the
right bank, the activity of which, linked to the wool, cloth
and tannery industry, was already referred to at the end of
the 11th century (Gutiérrez González, 1993). The water mills
of La Pinilla (12th century) and Cabañales (15th–16th cen-
turies) on the left bank, traditionally an area of meadows,
were known for their tannery and pottery manufacture. A
later economic expansion took place during the 18th century
under the protection of the Spanish crown. 2.2
Historical urban development and flood
documentation (b) Orthophoto of Zamora (year 2017) showing
the same urban expansion areas as (a). Legend: (1) Las Dueñas convent, (2) San Frontis church, (3) Olivares mills, (4) San Claudio de
Olivares church, (5) Campo de la Verdad (likely location of old Santa Clara convent), (6) Santiago de los Caballeros church, (7) old San
Francisco convent, (8) Stone Bridge, (9) Gate of Pescado dated to the 14th century (a new one was built in 1849; Fig. 4b), (10) Santa
Lucia church, (11) Santa Maria de la Horta, (12) Santo Tomé church, (13) San Leonardo, (14) cavalry headquarters, (15) Mengue Avenue,
(16) Iron Bridge, (17) Railway Bridge, (18) Cabañales mills, (19) San Jerónimo, (20) survey monument N. P. 1482 (IGN 1925), (21) IGN
survey mark 305004, (22) IGN survey mark 305005, (23) IGN survey mark 305003. Source: (a) Biblioteca virtual Ministerio de Defensa
(https://bibliotecavirtual.defensa.gob.es/, last access: 24 November 2021), (b) National Geographical Institute. Figure 2. Historic flood landmarks and GPS point locations. (a) Historic map of Zamora (1 : 2900 in scale) published on 11 March 1766
by Juan Martín Cermeño. Old graphic scale of 200 toes (1 toe = 13.5 cm). Black polygons illustrate the timing of urban development
through time in the old city (9th–13th centuries) and suburbs (14th century and beyond). The city of Zamora was surrounded by walls
that in the old city had defence purposes and in the suburbs protected against flooding. (b) Orthophoto of Zamora (year 2017) showing
the same urban expansion areas as (a). Legend: (1) Las Dueñas convent, (2) San Frontis church, (3) Olivares mills, (4) San Claudio de
Olivares church, (5) Campo de la Verdad (likely location of old Santa Clara convent), (6) Santiago de los Caballeros church, (7) old San
Francisco convent, (8) Stone Bridge, (9) Gate of Pescado dated to the 14th century (a new one was built in 1849; Fig. 4b), (10) Santa
Lucia church, (11) Santa Maria de la Horta, (12) Santo Tomé church, (13) San Leonardo, (14) cavalry headquarters, (15) Mengue Avenue,
(16) Iron Bridge, (17) Railway Bridge, (18) Cabañales mills, (19) San Jerónimo, (20) survey monument N. P. 1482 (IGN 1925), (21) IGN
survey mark 305004, (22) IGN survey mark 305005, (23) IGN survey mark 305003. Source: (a) Biblioteca virtual Ministerio de Defensa
(https://bibliotecavirtual.defensa.gob.es/, last access: 24 November 2021), (b) National Geographical Institute. 2.2
Historical urban development and flood
documentation The old city of Zamora, still partially walled, is located on a
prominent bedrock hill on the right side of the Duero River
and treasures a rich architectural ensemble formed by the
24 Romanesque churches and monasteries (10th–13th cen-
turies). The urban expansion towards the east, parallel to the
riverside area, took place mainly in three phases during the
11th, 12th and 14th centuries (Fig. 2a), coinciding with peri-
ods of economic and population growth (Gutiérrez González,
1993; Larrén, 1999). The five oldest Romanesque churches located on the
Duero River were built during the late 11th and 12th cen- Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 G. Benito et al.: Enhanced flood hazard assessment
c flood landmarks and GPS point locations. (a) Historic map of Zamora (1 : 2900 in scale) published on 11 March 1766
Cermeño. Old graphic scale of 200 toes (1 toe = 13.5 cm). Black polygons illustrate the timing of urban development
he old city (9th–13th centuries) and suburbs (14th century and beyond). The city of Zamora was surrounded by walls
y had defence purposes and in the suburbs protected against flooding. (b) Orthophoto of Zamora (year 2017) showing
xpansion areas as (a). Legend: (1) Las Dueñas convent, (2) San Frontis church, (3) Olivares mills, (4) San Claudio de
5) Campo de la Verdad (likely location of old Santa Clara convent), (6) Santiago de los Caballeros church, (7) old San
, (8) Stone Bridge, (9) Gate of Pescado dated to the 14th century (a new one was built in 1849; Fig. 4b), (10) Santa
) Santa Maria de la Horta, (12) Santo Tomé church, (13) San Leonardo, (14) cavalry headquarters, (15) Mengue Avenue,
(17) Railway Bridge (18) Cabañales mills (19) San Jerónimo (20) survey monument N P 1482 (IGN 1925) (21) IGN 6110 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 2. Historic flood landmarks and GPS point locations. (a) Historic map of Zamora (1 : 2900 in scale) published on 11 March 1766
by Juan Martín Cermeño. Old graphic scale of 200 toes (1 toe = 13.5 cm). Black polygons illustrate the timing of urban development
through time in the old city (9th–13th centuries) and suburbs (14th century and beyond). The city of Zamora was surrounded by walls
that in the old city had defence purposes and in the suburbs protected against flooding. G. Benito et al.: Enhanced flood hazard assessment 2a), which still offered not
only military protection (Larrén, 1999) but also protection
against flood risk in part of the city of Zamora (south and east
of the Horta neighbourhood). However, the greatest change
took place in the mid and late 19th century, with the moderni-
sation of the city (Segundo Viloria’s project in 1880) as well
as in the mid-20th century, with the construction of the Vigo
road along the Duero River right margin, from Olivares to
the confluence with the Valderaduey River (Fig. 2b, connect-
ing sites 9, 15, 16 and 17). In this urban growth, a significant
portion of the remaining city walls were removed or incorpo-
rated within new buildings (Gutiérrez González, 1993). This
city expansion brought a narrowing of the river section at the
floodplain and changes in the location and growth of fluvial
bars, mainly next to the bridges and weirs. Furthermore, the
limited natural space of the riverside area was highly trans-
formed during the construction of the sewage system and
two new bridges: (1) the old railway bridge built in 1895,
reformed in 1933 and without traffic since 1986, and (2) the
“Iron Bridge” (1892–1900), still in use for road traffic circu-
lation (Fig. 2b, site 16). Other infrastructures are diversion
weirs related to water mills historically used for grinding
wheat (Fig. 2). The most recent bridge was opened in 2013,
connecting the suburbs of San Frontis and Olivares. The Portuguese Douro catchment area is more likely to
generate catastrophic floods than the Spanish side due to
the convergence of significant tributaries with a much larger
runoff contribution (Vehlas, 1997). For instance, the largest
gauged flood of the Douro River in Régua (91 119 km2 in
catchment area; Fig. 1) was 16 700 m3 s−1 in 1909 (Silva
and Oliveira, 2002), whereas this event in Zamora recorded
2155 m3 s−1. Among the reasons of this flood discharge
disparity are a high instability and advection of humid air
masses towards Portugal, high relief that decreases concen-
tration time, and more impervious igneous and metamorphic
bedrock. In contrast, the Spanish Duero River flows on wide
valleys, detrital bedrock and well-developed floodplains with
a lack of flood peak convergence since the headwater flows
reach the peaks with a delay with respect to those coming
from the middle and lower valley. 2.3
Climate and flood hydrology characteristics The climate is continental Mediterranean in most of the
catchment area, with a strong temperate oceanic influence to-
wards the mouth of the Duero River in the Atlantic Ocean at
the city of Porto. The temperature and precipitation regimes
are characterised by a marked seasonal and monthly vari-
ability. Summers are hot and dry, and winters are typically
mild and relatively wet. Rainfall is mainly produced by cold
Atlantic frontal systems crossing the Iberian Peninsula from
November to April. There is a strong west–east rainfall con-
trast due to the relief effect, with annual rainfall at the lower
Duero in Porto of 1175 mm, whereas at the Spanish Duero
basin it is 580 mm, with a wide inter-annual variability rang-
ing from 350 and 800 mm. Similarly, daily maximum rainfall
on the Portuguese side may reach 200 mm, whereas for the
Spanish Duero it ranges between 60–100 mm. G. Benito et al.: Enhanced flood hazard assessment For example, the 1909
flood peak was recorded in Régua and Porto on the morn-
ing of 24 December (from the Portuguese catchment area),
whereas on the Spanish side in Valladolid the peak occurred
on the afternoon of 25 December, with the flood wave reach-
ing Zamora during the night. As a result, the ranking of
the years in which the largest floods occur in Zamora may
differ from that observed in Régua and Porto at the lower
Douro basin (Silva and Oliveira, 2002). G. Benito et al.: Enhanced flood hazard assessment sionally combined with snowmelt at mountain ranges sur-
rounding the catchment area. Extreme flood discharges may
be 30 times greater than the mean discharge, yielding one
of the largest specific peak discharges compared to simi-
lar European catchment areas (Pardé, 1953; Benito et al.,
2015a). Although the number of hazardous floods has de-
creased over recent decades, the Duero River Watershed Au-
thority reported for the Zamora Province flood damages of
EUR 270 000 per year over the period 2009–2013 (DHD,
2016). distribution of buildings and infrastructures associated with
flood damages. At the beginning of the 19th century, with the
French military invasions, large parts of the city walls were
demolished (eastern walls in Fig. 2a), which still offered not
only military protection (Larrén, 1999) but also protection
against flood risk in part of the city of Zamora (south and east
of the Horta neighbourhood). However, the greatest change
took place in the mid and late 19th century, with the moderni-
sation of the city (Segundo Viloria’s project in 1880) as well
as in the mid-20th century, with the construction of the Vigo
road along the Duero River right margin, from Olivares to
the confluence with the Valderaduey River (Fig. 2b, connect-
ing sites 9, 15, 16 and 17). In this urban growth, a significant
portion of the remaining city walls were removed or incorpo-
rated within new buildings (Gutiérrez González, 1993). This
city expansion brought a narrowing of the river section at the
floodplain and changes in the location and growth of fluvial
bars, mainly next to the bridges and weirs. Furthermore, the
limited natural space of the riverside area was highly trans-
formed during the construction of the sewage system and
two new bridges: (1) the old railway bridge built in 1895,
reformed in 1933 and without traffic since 1986, and (2) the
“Iron Bridge” (1892–1900), still in use for road traffic circu-
lation (Fig. 2b, site 16). Other infrastructures are diversion
weirs related to water mills historically used for grinding
wheat (Fig. 2). The most recent bridge was opened in 2013,
connecting the suburbs of San Frontis and Olivares. distribution of buildings and infrastructures associated with
flood damages. At the beginning of the 19th century, with the
French military invasions, large parts of the city walls were
demolished (eastern walls in Fig. 2.2
Historical urban development and flood
documentation The flood references between the 14th and 18th centuries
are concentrated in the riverside areas of new commercial
and artisanal expansion to the east of the Stone Bridge (in the
streets of San Julian, La Plata-Balborraz, Baños, Horta and
Cuartel de abajo). The frequent flood references are linked
not only to its geomorphological and hydraulic settings but to a greater exposure and vulnerability. On the left river mar-
gin, flood reports are related to ecclesiastic buildings (Las
Dueñas, San Francisco, San Frontis) and their surrounding
orchards. The urban development that took place from the 19th cen-
tury onwards is also reflected in the increase and spatial https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 6111 3.1
Instrumental records The Duero River hydrological gauge stations next to Zamora
(Fig. 1c) comprise (i) Carrascal (Station N.2066, pe-
riod 1918–2021) located 8 km downstream of Zamora, (ii)
Zamora (Station N.2121, 2002–2021), (iii) Villachica (Sta-
tion N.2096, 1929–1967) and (iv) Toro (Station N.2062,
2011–2021); the latter two are located 25 and 28 km up-
stream of Zamora, respectively. In Zamora, the Duero River mean annual discharge is
99 m3 s−1 (period 2002–2017), with flow partially regulated
by reservoirs. General hydrological characteristics are (i)
maximum discharge from December to May, (ii) a peak be-
tween February and March, and (iii) minimum discharge
from July to September. This seasonal pattern is influenced
by a mix of snowmelt and rainwater from tributaries drain-
ing the Gredos and Cantabrian mountains. Most of the largest
floods are related to persistent winter rainfall (several weeks)
associated with successive passage of Atlantic fronts, occa- The oldest gauged records correspond to water-level read-
ings taken on a daily basis in the El Porvenir gauge sta-
tion, covering the period 1880 and 1943. The gauged sec-
tion is located ca. 23 km downstream of Zamora at the San
Roman hydroelectric station (operating since 1903). Accord-
ing to Marquina (1949b), the El Porvenir rating curve was
well established to a stage of 5 m (1450 m3 s−1) using the
Villachica gauge data, whereas for higher discharges extrap- 3.2
Historical data sources Our historical flood database was collected from published
compilations, unpublished documents, epigraphic marks,
historical maps, photos and newspapers (listed in the Sup-
plement). The documentary flood records in Zamora essen-
tially comprise a continuous series from 1545 to 1860 and
a non-continuous dataset between 1250 and 1545 collected
from ecclesiastic and municipal archives. In the Cathedral of
Zamora archives, the Extracts of ecclesiastical agreements
comprises two volumes over the period 1601 to 1745, and
the Books of ecclesiastic agreements includes 34 volumes
over the period 1601 to 1913. The municipal books (Libros
de Actas) comprise 259 volumes over the period 1500–1899,
although there are some missing documents over the periods
1503–1507, 1521–1530 and 1576–1585. Many references of
those books and local chronicles were collected by historio-
graphic collections, namely the historical memoirs of the city
of Zamora (four volumes) by Fernández Duro (1882). Ex-
treme climate and hydrological event descriptions with ref-
erence to flooding in Zamora were also compiled in local ec-
clesiastic chronicles, namely by Zataraín-Fernández (1898),
and in the geological description of the Zamora Province by
Puig y Larraz (1883). Large floods typically affected his-
toric buildings, such as churches, convents, bridges, walls
and gates, the traces of which are recorded in architectural
catalogues that describe inscriptions and flood marks, repair
work to flood damages, or changes in the location of ec-
clesiastical communities due to the effects of major floods
(Gomez-Moreno, 1927; Antón, 1927). (
)
The reported floods were compared with historical series
in the lower Douro River in Portugal (Loureiro, 1904; Aires
et al., 2000; Amorim et al., 2017; Alcoforado et al., 2021). Flood severity was classified according to Barriendos and
Martín-Vide (1998) into three flood categories: (1) ordinary,
causing overbank flows of low to moderate intensity and tem-
porary disruption of the human activities; (2) extraordinary,
causing overbank flows of moderate intensity, with limited
damages to crops, houses and river dykes; and (3) catas-
trophic, causing extensive overflow with significant damage
to agriculture, mills, and/or destruction of houses and infras-
tructures. G. Benito et al.: Enhanced flood hazard assessment The most detailed historical maps belong to the collection
of plans of the municipal architect Segundo Viloria in 1880
(provincial historical archive of Zamora) and Zamora’s mu-
nicipal maps (19th–20th centuries). A final source of flood
data, mainly over the last 4 decades, was obtained from lo-
cal and regional newspapers (Heraldo de Zamora, La Voz
de Zamora, La Opinión, Impero, among others) and national
press (Ahora, La Correspondencia de España, ABC, La Van-
guardia). Regarding more general reference to the Duero
River and its tributaries, an outstanding compilation of flood
dates from historical data sources was compiled by Fontana
Tarrats (1971–1977). olation of the curve may result in errors of ca. ±10 %. The
flood record was completed with the Carrascal station (1920–
present) managed by the Iberdrola hydropower company,
which allows robust estimations of historic floods in Zamora. The Toro and Villachica stations were used to test the gauged
data at Carrascal, particularly those corresponding to flood
peaks. 3.3
Two-dimensional hydraulic modelling Flood water level (stage) related to historical floods includes
(i) flood marks and observed water depth measures at sites
reached by the flow (e.g. monastery, bridge, chapel, etc.),
(ii) description of flooded areas (e.g. floodplain sectors, or-
chards, mills), (iii) description of non-flooded areas and (iv)
comparative flood level of subsequent historical floods (e.g. said flood reached lower levels than previous one). The doc-
umented flood evidence can be used to estimate exact or rel-
ative (minimum or maximum) flood discharges associated
with observed water levels. The conversion of flood level
to discharge is obtained by matching a modelled water sur-
face elevation for a given modelled discharge to the surveyed
elevation of the known historical flood level (Benito et al.,
2020). The most outstanding historical flood compilation effort
was carried out by Rodríguez Marquina (1941–1949), who
analysed all available historical and hydrological reports on
flooding for the construction of the dams in the Duero and
Esla rivers. Marquina’s manuscripts provided a highly de-
tailed description and height survey of epigraphic marks,
including some flood marks that later disappeared during
restoration works. Marquina also compiled original water-
level gauge readings from the Duero and Esla rivers at dif-
ferent locations (e.g. El Porvenir gauge). The temporal evo-
lution of floodplain areas, buildings and riverine structures
(weirs, bridges, mills, orchards, etc.) were evaluated using
historic maps, drawings and etchings from historic times,
namely by Anton van der Wyngaerde in 1570 (Kagan, 2008;
Rodríguez-Méndez and García-Gago, 2014), Josep Auguier
in 1756 (Museum of Zamora) and Juan Martín Cermeño in
1766 (digital archive of the Ministry of Defence; Fig. 2a). Discharge estimation by hydraulic modelling was carried
out using a two-dimensional hydraulic model (Iber) which
solves the depth-averaged shallow water (2D Saint-Venant)
equations using a finite volume method with a second-order
roe scheme (Bladé et al., 2014; http://www.iberaula.com, last
access: 24 November 2021). This two-dimensional hydraulic
model is particularly suitable for flow in alluvial floodplains
with secondary currents. The model uses a non-structured
mesh consisting of triangles or quadrilateral elements whose
spatial resolution was set to 20 m for the channel bed, 10 m
for the channel margins and floodplains, 1 m for river bars,
and 1 to 5 m in the city streets and at major infrastructures
(Fig. S1). The 13 km length modelled reach extends from the
Carrascal gauge station to the Duero River junction with the https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. https://doi.org/10.5194/hess-25-6107-2021 https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6112 G. Benito et al.: Enhanced flood hazard assessment Valderaduey River (at the highway A-11; Fig. S1a). The Car-
rascal section is situated at a narrow bedrock canyon pro-
ducing a backwater effect upon the upstream alluvial reach
where the city of Zamora is located. The mesh elements were
built from a cloud of lidar data with an average distance of
1.4 m (some are below 1 m) supplied by the Spanish National
Geographic Institute (IGN; https://www.ign.es, last access:
24 November 2021). The topography of the river channel
bottom and banks was obtained from field surveys with an
echo-sounder device at cross-sections of 40 to 500 m dis-
tance (LINDE project, Ministry for Ecological Transition). The surveyed points from 268 cross-sections were extracted
and integrated with the lidar cloud data using the spatial an-
alyst tools in ArcGIS v.10. The bridges were introduced to
model historical flood discharges considering their construc-
tion date. ber of floods above a threshold or censored level is calcu-
lated. Stationary flood series are those that remain within
the 95 % tolerance interval (Naulet et al., 2005). The flood
frequency analysis was performed with two computer pro-
grams: PeakFQ Version 7.2 (Flynn et al., 2006; Veilleux et
al., 2014) and AFINS (Botero and Francés, 2006, 2010). The
PeakFQ program applies a generalised method-of-moments
estimator denoted the expected moments algorithm (EMA;
Cohn et al., 1997), whereas AFINS uses the maximum likeli-
hood method (Frances, 2004). Flood frequency analysis was
carried out with different combinations of three datasets: (1)
documentary floods using minimum flow and/or peak dis-
charge estimated from flood marks and reported flood de-
scriptions (historic dataset, HISTO); (2) water-level readings
on a scale gauge transformed into discharge at El Porvenir
station (pre-instrumental dataset; PRE); and (3) continuous
systematic records at Carracal station (systematic dataset:
SYS), which was analysed first over the whole gauged period
1920–2018 (ALLSYS) and, secondly, subdivided on early
(1920–1969; SYS1) and late (1970–2018; SYS2) datasets. Manning’s n values were assigned from land use map
classes (Corine Land Cover map) following the methodolog-
ical guide of the National Flood Inundation Hazard Map
(MMA, 2011). In our study, the initial set of n values was de-
fined as 0.04 for the main channel and between 0.045 and 0.1
for the floodplains. G. Benito et al.: Enhanced flood hazard assessment Model calibration was performed using
flow discharge-stage records at two gauge stations, namely
the Carrascal one located at the farthest downstream cross-
section and Zamora station in the upper sector of the mod-
elled reach. For this calibration, the outflow stage for suc-
cessive increments of inflow discharges in the upper section
in Zamora was compared to the rating curve at the Carras-
cal gauge station. The difference in stage between the model
and the gauge station for a discharge of 1100 m3 s−1 was
2 cm, and for a discharge of 3100 m3 s−1 it was 12 cm. Af-
ter the model calibration, Manning’s n at the channel was set
at 0.035. The documentary flood information is non-systematic data
of censored type since only flows over a particular magnitude
(commonly producing damages) are reported in documen-
tary records. The minimum flood stage (perception thresh-
old) is set according to flood magnitude spilling over urban
areas, disrupting human activities (communication, manu-
facture works) and/or producing damage (orchards, bridge,
houses and ecclesiastic goods). The flooding of sensitive
urban areas, or perception threshold, may change through
time according to the progressive human occupation of the
riverine areas and the socio-economic context (Benito et al.,
2004). In the case of Zamora, the perception threshold did
not register significant changes through historical time. The
minimum discharge (1900 m3 s−1) is required to flow over-
bank in the low urban neighbourhoods of Horta, Olivares and
Frontis and for cutting the main communication infrastruc-
tures next to the Duero River. The reconstructed historical
flood discharges were added to the continuous gauged an-
nual maximum flow records at the Porvenir and Carrascal
stations. The flood marks and flooded sites mentioned in documen-
tary flood evidence were surveyed using a Trimble GPS, sup-
ported by four geodetic survey monuments of the Spanish
National Geographical Institute (numbers 305003, 305004,
601005 and 601006; Table S1). We modelled successive in-
crements of inlet water discharges at the upstream reach
to simulate a steady flow. The hydraulic model provided a
peak discharge vs. water stage relationship for 30 sites with
known historical flood evidence (epigraphic marks, docu-
mented heights, description of flooded/non-flooded areas). We used the mean and standard deviation to estimate aver-
age peak discharge for each historical flood or used the mini-
mum or maximum flood values according to the mean of the
documentary flood evidence. G. Benito et al.: Enhanced flood hazard assessment The PeakFQ software uses the mentioned expected mo-
ments algorithm and a generalised version of the Grubbs–
Beck test for identifying multiple potential influential low
flows (PILFs; Cohn et al., 2013). Low annual discharge val-
ues may have excessive influence on the estimated frequency
of large floods (Veilleux et al., 2014), and their identifica-
tion of PILFs improve estimated frequency of large floods. PeakFQ is well designed to treat both historical and sys-
tematic data but only allows fitting of a log Pearson type
III (LP3) distribution. A Gumbel distribution, commonly
used in Spain, was fitted using the AFINS software that
applies a maximum likelihood estimation method (MLE). This method has demonstrated a high capacity to incorporate
in the estimation process any non-systematical data (Leese,
1973; Stedinger and Cohn, 1986). Visual matching of the 3.3
Two-dimensional hydraulic modelling Sci., 25, 6107–6132, 2021 6113 3.4
Flood frequency analysis Flood data stationarity for censored samples (historical and
systematic flooding) was confirmed using Lang’s test (Lang
et al., 1999). This test assumes that stationary flood series
can be described by a homogeneous or stationary Poisson
process. The 95 % tolerance interval of the cumulative num- https://doi.org/10.5194/hess-25-6107-2021 4.1
Flood variability at decadal and multi-decadal
timescales The same analysis ex-
panded towards the gauged period (since 1920) provides a
flow discharge higher than 1900 m3 s−1, leading to similar
inundation extent of reported overflows. The moving average
analysis of temporal distribution using a 31-year filter identi-
fies flood-rich periods at 1600–1640, 1730–1750, 1770–1790
and 1880–1910 (Fig. 3). Over the 20th century, floods over
1900 m3 s−1 increased their frequency in the period 1935–
1966. Later, some large scattered floods occurred in 1978–
1979 and 2001. IVT =
v
u
u
u
u
t
1
g
300 hPa
Z
1000 hPa
q u dp
2
+
1
g
300 hPa
Z
1000 hPa
q v dp
2
, (1) IVT =
v
u
u
u
u
t
1
g
300 hPa
Z
1000 hPa
q u dp
2
+
1
g
300 hPa
Z
1000 hPa
q v dp
2
, (1) where g is the gravitational constant (m s−2), q is the spe-
cific humidity (kg kg−1), u and v are the zonal and merid-
ional wind components (m s−1), and p is the pressure (hPa). The IVT (kg m−1s−1) is estimated to be between the sea
level pressure and 300 hPa. To account for the rainfall–runoff
transformation time in the Duero basin, we computed the av-
eraged IVT over the 10 preceding days to the maximum peak
discharge. Moreover, we used the Katalog Der Grosswet-
terlagen Europas (1881–2004) to identify the predominant
circulation pattern associated with each flood (Gestengabe
and Werner, 2005). Since this catalogue starts in 1881, we
assigned a likely circulation type pattern for floods that
took place before 1880 based on the geopotential field from
20CRV3 (Table S2, Fig. S3). Finally, we also used the North
Atlantic Oscillation (NAO) index to characterise the regional
influence of the North Atlantic atmospheric circulation vari-
ability (Hurrell, 1995; Brönnimann et al., 2008) on the his-
torical flood events. The NAO index reflects the difference in
anomalies of the sea level pressure between Gibraltar (south-
western Iberian Peninsula) and Reykjavik (Iceland) stations,
as has been used as a surrogate of temperature and precipita- Large floods were produced mainly during the winter pe-
riod (DJF; 66 %), followed by spring (MAM; 28 %), autumn
(SON; 4 %) and summer (JJA; 2 %). 4.1
Flood variability at decadal and multi-decadal
timescales To provide a general context of the climate triggers of
the larger floods exceeding the perception threshold of
1900 m3 s−1, we investigated the related atmospheric circu-
lation based on the 20th century reanalysis climate data (Ver-
sion 3 and 2c, 20CRV3 and 2c) from NOAA/CIRES. These
datasets cover the period 1836–2015 at a sub-daily scale (3 h)
from around the globe at 2◦× 2◦and provides relevant me-
teorological fields at different pressure levels (Compo et al.,
2011). Thus, the climate analysis is restricted to floods occur-
ring during this period. First, we extracted several ensemble
mean fields at a daily scale from the surface and troposphere
level, such as geopotential height, wind components, diver-
gence and specific moisture up to 250 hPa. This information
was used to carry out composite analyses of mean monthly
anomalies (with regard to 1980–2010) to describe the gen-
eral situation during flood events. Then, we investigated the
source of moisture triggering floods at a daily scale. To this
end, we compute the vertically integrated water vapour trans-
port (IVT) for the region 0–90◦N, −100–20◦E, as suggested
by Lavers et al. (2012) (Eq. 1), and plot the wind vector. Documentary flood descriptions of the Duero River in
Zamora can be traced back to the 13th century, although con-
tinuous records started in the mid-16th century. At that time,
the main configuration of Zamora neighbourhoods, weirs,
mills and bridges was similar to those of the late 19th cen-
tury and early 20th century (Fig. 2a, b). This long-standing
urban configuration allows a precise analysis of the sites and
flooded areas as well as a qualitative reference of flood mag-
nitudes over the last millennia. The morphological changes
in river channel and banks are minor and mostly related to
fluvial islands and lateral bar stability by vegetation mainly
over the last 30 years. For instance, the historical and present
orthophotos show stabilisation of a lateral bar next to the
Cabañales mills, upstream of the Stone Bridge. The fluvial
banks have remained at a similar position according to the
historical maps, at least over the last 300 years (Fig. 2a, b). The documentary flood dataset comprises 69 flood entries
over the period 1250–1871 CE. These entries include mainly
floods within catastrophic and extraordinary categories as
they were registered due to bank overflow and damages in
orchards, infrastructures and houses. G. Benito et al.: Enhanced flood hazard assessment tion winter pattern in the Iberian Peninsula (López-Moreno
et al., 2011). plotting positions to the distribution curve and their statis-
tical parameters were used to test the goodness of fit. Confi-
dence intervals of the fitted distribution indicate the range of
discharges statistically possible based on the available data. https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6114 4.2
Composite series of flood discharges 1264, 1310, 1485 and 1586 with references to high flood
levels and moderate damage. For instance, in San Claudio
church an inscription at the arch (Fig. 4f) refers to times
of “bad years” during the Kingdom of Alfonso X dated
at 1259 CE. Marquina (1941–1944) relates that inscription
with a blurred mark located at the base of the arch, on the
upper-right doorpost (Fig. 4f), that is attributed to the 30 De-
cember 1258 flood (628.7 m a.s.l.) matching a discharge of G. Benito et al.: Enhanced flood hazard assessment Table 1. Reconstructed discharges of the major historical floods in Zamora. CAT: catastrophic flood; EXT: extraordinary flood; ORD
ordinary flood. Year
Date
Discharge
Minimum discharge
Flood
Comments
(m3 s−1)
(m3 s−1)
categorya
1258
12 December
3700
3540
CAT
Elevation of mark on the upper-right doorpost in San Claudio
1264
–
2700
2400
CAT
Dueñas convent flooded
1310
24 January
>2000
CAT
Severe damage to old bridge
1485
11 November
>2500
CAT
Floods in Esla and Pisuerga
1545
20 January
>2000
CAT
Bridge arch destroyed
1556
–
>2000
CAT
Bridge arch and towers severely damaged
1586
–
2800
2800
CAT
Old St. Clara flooded, archive destroyed
1597
16 January
3200
3000
CAT
1.5 m depth at St. M. Horta, larger than 1739 flood
1611
28 February
>2000
CAT
Arch and tower in bridge damaged
1626
2 January
>2000
CAT
Medieval bridge damaged
1636
4 February
2500
2300
CAT
2.5 m long gap open at Cabañales wall
1739
March
2700
>2500
CAT
Damaged 248 houses; flood marks in Las Dueñas; reported
flooding in S. Claudio, S. Frontis, Santiago el Viejo
1788
25 January
2550
2500
EXT
Horta, Cabañales and Olivares zones flooded; 1.5 m depth at the
infantry barracks
1839
December
1800
EXT
Mark in Olivares
1843
18 February
2500
2300
EXT
Cabañales and infantry barracks in Horta flooded, similar to
1788 flood
1860
25 December
3450
>3200
CAT
Flood marks in S. Frontis, Dueñas and Gate of Pescado, among
others
1872
30 January
1864∗
ORD
Villachica station, Marquina (1941–1944)
1873
17 January
2200
1860b
EXT
Stage description at Iron Bridge,
Porvenirb
1880
17 February
2370∗
EXT
El Porvenir staff gauge
1881
14 January
2210∗
EXT
El Porvenir staff gauge
1895
27 February
2380∗
EXT
El Porvenir staff gauge
1900
13 February
2098∗
EXT
El Porvenir staff gauge
1909
25 December
2155∗
EXT
El Porvenir staff gauge
1911
11 March
1542∗
ORD
El Porvenir staff gauge
1919
19 February
1620
ORD
El Porvenir staff gauge
a Flood category according to Barriendos and Martín-Vide (1998) classification. b Discharge calculated from observations at El Porvenir (Marquina, 1941–1944). ∗Historic
discharges estimated at the scale of El Porvenir since 1880. Table 1. Reconstructed discharges of the major historical floods in Zamora. CAT: catastrophic flood; EXT: extraordinary flood; ORD:
ordinary flood. Table 1. Reconstructed discharges of the major historical floods in Zamora. CAT: catastrophic flood; EX
ordinary flood. 4.2.1
Historical flood peak levels and discharge
determination Over the last 500 years, the largest historical floods occurred
in 1597, 1739 and 1860, exceeding 2800 m3 s−1. Previous
large floods producing severe damage occurred in 1258, 4.1
Flood variability at decadal and multi-decadal
timescales This seasonal distribu-
tion was maintained in the different flood-rich periods es-
tablished, with over 50 % of floods concentrated in winter,
except for the period 1770–1790, where the highest number
of floods occurred in spring. The highest concentration of
severe winter events occurred during the 1630s, 1730s and
1960s, with a lack of reported large floods in the second
half of the 17th century and beginning of the 19th century. The analysis of flood causes points to persistent winter rain
episodes, occasionally enhanced by snowmelt. https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6115 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment 6116 Figure 3. Normalised flood frequency distribution of the docu-
mented number of floods (only catastrophic + extraordinary cate-
gories) of the Duero River in Zamora using moving average, tak-
ing 11-year and 31-year data intervals. The anomalies for 1500–
1880 CE were estimated from documentary records and after 1980
include gauged floods (staff gauge and continuous gauge) with dis-
charge higher than 1900 m3 s−1. ference of 1.05 m (Fig. 4e). The San Frontis flood mark also
refers to the 1860 flood as the largest compared to the pre-
vious 1597 and 1739 floods, although the description at the
plate mentions a “1592 flood”, which seems to be a tran-
scription error as noted by Marquina (1941–1944). The 1739
flood marks at Las Dueñas (627.45 m a.s.l.) and two mini-
mum flood stages at Santiago El Viejo (626.6 m a.s.l.) and
San Frontis-Cuesta de San Jerónimo (626.5 m a.s.l.) are as-
sociated with a discharge of 2700 m3 s−1. These flood stages
are lower than the 1597 flood reference reported at St. Maria
de la Horta (628.35 m a.s.l.; Fig. 4c) and the minimum flood
stages at Los Descalzos (628.2 m a.s.l.) and San Juan de las
Monjas (628.3 m a.s.l.). In the medieval bridge the spillway
holes were coved by flood waters. The 1597 flood evidence
matches a stage associated with a discharge of 3200 m3 s−1,
meaning the second-largest over the last 500 years (Fig. 6). Figure 3. Normalised flood frequency distribution of the docu-
mented number of floods (only catastrophic + extraordinary cate-
gories) of the Duero River in Zamora using moving average, tak-
ing 11-year and 31-year data intervals. The anomalies for 1500–
1880 CE were estimated from documentary records and after 1980
include gauged floods (staff gauge and continuous gauge) with dis-
charge higher than 1900 m3 s−1. A second-rank flooding corresponds to peak magni-
tudes exceeding 2200 m3 s−1 that commonly produced over-
bank flow and damages in the Horta and Olivares suburbs
(Fig. 6). In this second magnitude rank, floods occurred in
1586, 1636, 1788, 1843, 1853, 1880, 1881, 1895 and 1909
(2155 m3 s−1). The 1586 event flooded the old Santa Clara
convent (Fig. 5, site 5) and destroyed the archive that, as-
suming a minimum 0.5–1 m water depth, gives discharges
between 2600–3000 m3 s−1. These flood magnitudes have
caused damages in piers and towers of the Stone Bridge (e.g. G. Benito et al.: Enhanced flood hazard assessment 1636, 1880). The 1880 flood caused such major damages to
the bridge that it was reformed in the early 20th century, re-
ducing the number of arches from 22 to 15 and enlarging
the lightening arches or spillways (Rodríguez-Méndez et al.,
2012). As a reference, these floods cover the bridge piers and
spilled water through the lightening arches. In this magnitude
rank, the flood cluster occurring during the late 19th century
recorded at the Porvenir gauge (Fig. 6) is worthy of mention. 3700 m3 s−1. During the 1310 flood the old bridge (early me-
dieval) was destroyed, although no references to flood marks
indicating flood stage were found. g
g
The 1860 flood is the largest, at least over the last
500 years, with evidence of flood stage on three epigraphic
marks and five precise reports from sites at both sides of the
Duero River (Fig. 4b, d, e, f). The flood peak occurred on
the night of 29–30 December, damaging 441 buildings in the
city and another 263 in the suburbs. Fortunately, telegraphs
received in the evening from cities upstream alerted of the
flood severity, and people living in risk areas were evacuated. In the village of Peleagonzalo (30 km upstream; Fig. 1c) 154
houses out of 160 were destroyed and later rebuilt in 1862
on a nearby hill. Downstream of Zamora’s medieval bridge,
the most reliable flood evidence is found in Dueñas Convent,
San Claudio, Olivares water mill and Puerta del Pescado,
all pointing to a water stage of ca. 628.5 m a.s.l. (Fig. 5a,
sites 1, 4, 3 and 9). Upstream of the medieval bridge, flood
elevation was 628.9 m a.s.l. at Santa Lucia (site 10) and at
Iron Bridge (Zamora-Salamanca road, site 16), reaching San
Leonardo and La Plata Street (site 13). The epigraphic flood
mark in San Frontis (627.8 m a.s.l.; site 2; Fig. 4d) is slightly
below flood elevation reached in the Olivares flood stage ref-
erences at the opposite river bank. These 1860 flood high
water marks fit a water surface elevation generated by the
two-dimensional model of 3450 m3 s−1 (±100 m3 s−1). A third set of documented floods was reported to pro-
duce flooding in low city neighbourhoods and orchards sur-
rounding convents and monasteries but without any descrip-
tion allowing any sort of discharge estimation. G. Benito et al.: Enhanced flood hazard assessment Modern flood
analogues producing occasional inundation of low city areas
(Cabañales and Olivares) and minor disruption of traffic ac-
tivity are associated with discharges exceeding 1900 m3 s−1
(Figs. 5b, 7). https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 G. Benito et al.: Enhanced flood hazard assessment 4.2.2
Modern flood records In the Carrascal station daily flows exceeding 2200 m3 s−1
occurred
in
1959–1960,
1961–1962,
1979
and
2001
(2140 m3 s−1) (Table S3). Intriguingly, the January 1962
flood recorded a daily discharge of 3071 m3 s−1 that, trans-
formed to peak discharge, results in 3200–3300 m3 s−1. Pub-
lished photographs and descriptions in newspapers (Fig. 7a,
b) show ca. 1.5 m water depths in the Horta neighbourhood
(Mengue Avenue), ca. 1.7 m from the Cabañales mill and
in the lower Cabañales neighbourhood (Table S1). The doc-
umented flood stages agree with a discharge of ca. 1900– Several documentary descriptions and epigraphic marks
allow comparison of the 1860 flood with previous flooding. For instance, in the Las Dueñas convent the 1860 flood epi-
graphic mark in the refectory is 1.80 m above floor level,
whereas the 1739 flood is 0.75 m, meaning a water stage dif- Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 6117 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 4. Photos illustrating flood epigraphic marks and sites referred to in written flood reports. (a) View towards the cathedral (centre
uphill) with remnants of the old bridge destroyed by a flood in 1310. At the left opposite river margin are the Olivares mills and the
tower of the San Claudio church (photo by António Passaporte, 1927–1936; source: Loty Archive-02471, historic heritage photo, http:
//www.mcu.es/fototeca_patrimonio, last access: 24 November 2021). (b) View of the Gate of Pescado, where an epigraphic mark was 1.5 m
from the gate base (source: Historical Provincial Archive). (c) View of Santa Maria de la Horta reached by floods at least in 1597 and 1788
(photo by António Passaporte, 1927–1936; source: Loty Archive-02479, historic heritage photo, http://www.mcu.es/fototeca_patrimonio,
last access: 24 November 2021). (d) Epigraphic mark of the 1860 flood in San Frontis church. The description states that the 1860 flood was
the largest of the last 3 centuries. (e) Epigraphic flood mark of the 1860 flood in Las Dueñas convent at the refectory of the convent: “The
magnificent Duero River reached here in 1860, and the community had moved for 24 hours to San Frontis”. Inset: epigraphic mark of the
1739 flood in Las Dueñas convent in the same dining room but ca. 1 m lower than the 1860 mark. (f) View of San Claudio church between
1927 and 1936. G. Benito et al.: Enhanced flood hazard assessment dimensional hydraulic model results. (a) Flood extension and depth for 3450 m3 s−1 estimated for the 1860 flood and flood
riptions of flooded sites (number in blue square). (b) Flood extension and depth for a discharge of 1900 m3 s−1. Legend with
marks and flooded sites in Fig. 2. Elevation (metres above sea level) reached by 1860 flood in Table S1. Aerial orthophoto
sh National Geographic Institute, IGN (https://www.ign.es, last access: 24 November 2021). Figure 5. Two-dimensional hydraulic model results. (a) Flood extension and depth for 3450 m3 s−1 estimated for the 1860 flood and flood
marks and descriptions of flooded sites (number in blue square). (b) Flood extension and depth for a discharge of 1900 m3 s−1. Legend with
names of flood marks and flooded sites in Fig. 2. Elevation (metres above sea level) reached by 1860 flood in Table S1. Aerial orthophoto
from the Spanish National Geographic Institute, IGN (https://www.ign.es, last access: 24 November 2021). with the estimated peak based on the photographic flow stage
evidence. 2000 m3 s−1 that suggests operative problems of the gauge
station during this flood. The seasonal hydrograph shows the
January 1962 flood as the second peak of a sequence of five
maxima that occurred from December 1961 to April 1962
(Fig. 8d). At the Villachica and Toro gauge stations (30 km
upstream) this flood recorded daily discharges of 1729
(4 January, 1 d earlier) and 1531 m3 s−1 (5 January), re-
spectively. A linear regression was fitted to daily discharges
recorded at the Carrascal and Villachica stations over the pe-
riod December 1959–May 1960 (the largest peak in Carras-
cal of 2343 m3 s−1) and for the Carrascal and the Toro sta-
tions for the 1959–1960 and 1978–1979 periods. The cal-
culated daily discharge for the 1962 flood at the Carrascal
station varied between 2100 and 1940 m3 s−1, which agrees Recent large floods are commonly produced during
anomalously wet winters whose atmospheric conditions may
persist for weeks or even months, producing hydrographs
with multiple flow peaks (Fig. 8a, c). One of the most severe
winters occurred in 1935–1936, with rains starting in late De-
cember and extending unit April, giving rise to 12 peaks and
a high-flow stage over the whole season. Other flood sea-
son types (e.g. 1946–1947) show shorter frontal rain passage
during the late winter and early spring (Fig. 8b). 4.2.2
Modern flood records The inscription in the arc refers to the “bad years of 1258” contemporaneous with the one at the upper doorpost pointing
to a flood (Marquina, 1949a). At the pedestal of the cross was an inscription to the 1839 flood, which disappeared after restoration work
(photo by António Passaporte; source: Loty Archive-02464, historic heritage photo http://www.mcu.es/fototeca_patrimonio, last access:
24 November 2021). Figure 4. Photos illustrating flood epigraphic marks and sites referred to in written flood reports. (a) View towards the cathedral (centre
uphill) with remnants of the old bridge destroyed by a flood in 1310. At the left opposite river margin are the Olivares mills and the
tower of the San Claudio church (photo by António Passaporte, 1927–1936; source: Loty Archive-02471, historic heritage photo, http:
//www.mcu.es/fototeca_patrimonio, last access: 24 November 2021). (b) View of the Gate of Pescado, where an epigraphic mark was 1.5 m
from the gate base (source: Historical Provincial Archive). (c) View of Santa Maria de la Horta reached by floods at least in 1597 and 1788
(photo by António Passaporte, 1927–1936; source: Loty Archive-02479, historic heritage photo, http://www.mcu.es/fototeca_patrimonio,
last access: 24 November 2021). (d) Epigraphic mark of the 1860 flood in San Frontis church. The description states that the 1860 flood was
the largest of the last 3 centuries. (e) Epigraphic flood mark of the 1860 flood in Las Dueñas convent at the refectory of the convent: “The
magnificent Duero River reached here in 1860, and the community had moved for 24 hours to San Frontis”. Inset: epigraphic mark of the
1739 flood in Las Dueñas convent in the same dining room but ca. 1 m lower than the 1860 mark. (f) View of San Claudio church between
1927 and 1936. The inscription in the arc refers to the “bad years of 1258” contemporaneous with the one at the upper doorpost pointing
to a flood (Marquina, 1949a). At the pedestal of the cross was an inscription to the 1839 flood, which disappeared after restoration work
(photo by António Passaporte; source: Loty Archive-02464, historic heritage photo http://www.mcu.es/fototeca_patrimonio, last access:
24 November 2021). Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 6118 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment In this case,
flow peaks are enhanced by rain on snow and snowmelt pro-
cesses. In general, the largest flow peaks are produced by the https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment 6119 G. Benito et al.: Enhanced flood hazard assessment duce persistent rainfall events. This situation is characteris-
tic of negative NAO-like phases found during flood events,
with a mean 10 d composite NAO index of −0.9 ± 1.9. Look-
ing at the specific moisture fields from the 10 d before the
peak discharge in Zamora city allows us to identify the
source of the moisture responsible for each event. In com-
bination with the wind fields, the responsible moisture is
transported from low to mid altitude, along long and nar-
row bands from subtropical latitudes to the Duero basin. The
shape and the intensity of the integrated water vapour trans-
ported therefore suggest the existence of atmospheric rivers
(ARs; IVT >250 kg m−1 s−1; wind velocity >12.5 m s−1). This has been the case of 82 % of the floods >1900 m3 s−1
since the largest flood occurred in 1860. Thus, except for
the flood which took place in 1962, an AR-like structure has
been detected over the Duero basin (Fig. S3). The example
with the largest, the lowest (above 1900 m3 s−1 threshold)
and more recent flood events is shown in Fig. 10, with max-
imum 10 d composite IVT >550, 475 and 700 kg m−1 s−1. Although landfall produced by the arrival of ARs is primor-
dially related to orographic conditions, rainfall seems to be
enhanced by divergence in altitude (250 hPa) over Portu-
gal and convergences at surface level over the Duero basin
(Fig. 9b). Thus, according to the 20th-century reanalysis
records, the mean precipitation rate anomaly during flood
months in the Duero basin was +2.78 ± 1.3 (Fig. 9b; values
presented in Table S2). This precipitation mostly occurred
during slightly-warmer-than-normal months, as suggested by
the mean temperature anomaly of +0.12 ± 1.0 (Fig. S2 and
Table S2), which is consistent with the advection of moisture
from tropical latitudes. Figure 6. The 1500–2018 Duero River flood discharges based on
documentary (historic) water-level readings on staff gauges (El
Porvenir) and continuous gauging (El Carrascal). 4.4
Flood frequency analysis The stationarity tests (Lang et al., 1999) of the combined doc-
umentary and instrumental (natural regime) flood series with
discharge equal to or above 1900 m3 s−1 show stationarity
conditions over the period 1511–2018 (Fig. S2). The number
of floods decreased the frequency in the mid-18th century,
overlapping the lower tolerance interval, but the overall pe-
riod is stationary for the perception threshold that includes
extraordinary and catastrophic flood categories. G. Benito et al.: Enhanced flood hazard assessment Documentary
floods with damaging overflows (catastrophic and extraordinary cat-
egories) exceeded a threshold discharge of 1900 m3 s−1. Discharge
reconstruction was performed based on different description details
(from vague to high-quality) and flow depth reported at specific
flooded sites and with epigraphic marks (types described in leg-
end). Reported ordinary floods were estimated below the discharge
threshold. The flood season is indicated by colours as in the legend. Figure 6. The 1500–2018 Duero River flood discharges based on
documentary (historic) water-level readings on staff gauges (El
Porvenir) and continuous gauging (El Carrascal). Documentary
floods with damaging overflows (catastrophic and extraordinary cat-
egories) exceeded a threshold discharge of 1900 m3 s−1. Discharge
reconstruction was performed based on different description details
(from vague to high-quality) and flow depth reported at specific
flooded sites and with epigraphic marks (types described in leg-
end). Reported ordinary floods were estimated below the discharge
threshold. The flood season is indicated by colours as in the legend. passage of the second or third of those cold fronts, once the
soils are saturated after previous rains. 4.3
Synoptic analysis and moisture transport (c) Horta suburb (Mengue Avenue) and Stone Bridge (background) during
the 1959 flood (source: Memoria gráfica de Zamora). (d) Flood level reached by the 1959 flood at La Horta (source: Memoria gráfica de
Zamora). (e) The Stone Bridge during the 1959 flood with the water flowing through the bridge spillways (source: Memoria gráfica de
Zamora). (f) The Olivares suburb during the 1948 flood (source: Memoria gráfica de Zamora; La Opinión-El Correo de Zamora, 2000) Figure 7. The city of Zamora during flood episodes. (a) Upstream view of the Duero river during the 1962 flood. On the left, the su Figure 7. The city of Zamora during flood episodes. (a) Upstream view of the Duero river during the 1962 flood. On the left, the submerged
buildings are the Olivares mill and the San Claudio church (source: Archivo Gerardo Pastor Olmedo, vol. III, La Historia Contemporánea). (b) A detailed view of the 1962 flood at San Claudio (background) published by the newspaper Imperio (5 January 1962) (available at
https://prensahistorica.mcu.es, last access: 24 November 2021). (c) Horta suburb (Mengue Avenue) and Stone Bridge (background) during
the 1959 flood (source: Memoria gráfica de Zamora). (d) Flood level reached by the 1959 flood at La Horta (source: Memoria gráfica de
Zamora). (e) The Stone Bridge during the 1959 flood with the water flowing through the bridge spillways (source: Memoria gráfica de
Zamora). (f) The Olivares suburb during the 1948 flood (source: Memoria gráfica de Zamora; La Opinión-El Correo de Zamora, 2000) sis, the 5 % (lower) confidence interval is well constrained by
the cluster of observed floods (Fig. 11a) plotted within the
1 %–5 % annual exceedance probability (AEP) (Fig. 11c). The subdivided systematic datasets (SYS1 and SYS2) show
a poor performance at the upper tail of the distribution with
a wide range of discharges for the confidence intervals, par-
ticularly the upper 95 %. atic data provide realistic quantile values for return intervals
lower than the 1 % AEP, with unreasonable discharge val-
ues for the higher quantiles. The 2 % AEP quantile (T =
50 years) of the early systematic dataset (1920–1969) gave
a higher flood magnitude than the one from the latter sys-
tematic set (1970–2018), suggesting a decrease in the annual
flood discharges towards the late 20th century. 4.3
Synoptic analysis and moisture transport The meteorological predisposition and atmospheric circula-
tion pattern analyses are focused on the major historical flood
events since the early 19th century. The cyclonic western
and south-western circulations are the main patterns related
to intense floods, accounting for ∼71 % of the cases (Ta-
ble S2). South-meridional-type circulation seems to be less
linked to floods (∼17 %), while through- (∼5 %) or high-
similarity patterns (∼5 %) are marginal. Figure 9 shows the
composite monthly geopotential at mid-levels (500 hPa) as
well as the composite seasonal precipitation rate, wind vec-
tor and vorticity, displaying negative and positive values (sur-
face level and 250 hPa, respectively). Major flood events in
the Duero basin are linked to an intense cyclonic anomaly
over the north-west of the Iberian Peninsula which extends
to the mid-Atlantic Ocean (Fig. 9a). This pattern is well rep-
resented at surface level (Fig. S2), allowing the arrival of a
frontal system moving inland from easterly and warmer At-
lantic positions. The result is the advection of relative warm
moisture mass, favoured by stronger zonal winds that pro- The flood frequency analysis (FFA) was carried out with
two independent methods, EMA and MLE, combining non-
systematic flood data (historic and pre-instrumental) and sys-
tematic continuous flood records (gauge station records). The
Gumbel (two parameters) and log Pearson type III distri-
bution (three parameters) functions were applied to differ-
ent datasets. Discharges calculated for different return peri-
ods (T) are shown in Table 2. Figure 11 shows the plotting
positions and the frequency curve fitted with LP3 distribu-
tion for different datasets showing good visual matching and
within the confidence intervals. The confidence intervals in
the upper tail of the distribution are narrower for the HISTO
and ALLSYST datasets; moreover, in the PRE–SYS analy- https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6120 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 7. The city of Zamora during flood episodes. (a) Upstream view of the Duero river during the 1962 flood. On the left, the submerged
buildings are the Olivares mill and the San Claudio church (source: Archivo Gerardo Pastor Olmedo, vol. III, La Historia Contemporánea). (b) A detailed view of the 1962 flood at San Claudio (background) published by the newspaper Imperio (5 January 1962) (available at
https://prensahistorica.mcu.es, last access: 24 November 2021). G. Benito et al.: Enhanced flood hazard assessment Figure 8. Daily discharges showing multiple peaks during selected water years: (a) 1935–1936, (b) 1946–1947, (c) 1959–1960. (c) Daily
discharge recorded at the Villachica, Toro and Carrascal stations (see location in Fig. 1c). The recorded peak on 5 January 1962 in Carrascal
was anomalously higher compared to Villachica and Toro records, and the peak was corrected using a linear regression. Figure 8. Daily discharges showing multiple peaks during selected water years: (a) 1935–1936, (b) 1946–1947, (c) 1959–1960. (c) Daily
discharge recorded at the Villachica, Toro and Carrascal stations (see location in Fig. 1c). The recorded peak on 5 January 1962 in Carrascal
was anomalously higher compared to Villachica and Toro records, and the peak was corrected using a linear regression. Figure 9. (a) Composite monthly anomaly of the geopotential at 500 hPa. (b) Composite seasonal precipitation rate retrieved from 20CRV3
(blue-coloured) and composite divergence for the 10 d prior to the peak of each flood at 250 hPa (dotted) and convergence at sea level (purple
line). L: low pressure; HL: high pressure. Figure 9. (a) Composite monthly anomaly of the geopotential at 500 hPa. (b) Composite seasonal precipitation rate retrieved from 20CRV3
(blue-coloured) and composite divergence for the 10 d prior to the peak of each flood at 250 hPa (dotted) and convergence at sea level (purple
line). L: low pressure; HL: high pressure. tion functions (LP3 and Gumbel) and two independent fitting
methods (EMA and MLE). The PRE–SYS dataset showed
good agreement in middle-term quantiles (T = 100 years and
200 years), whereas the SYS data analysis results in 10 %–
20 % discharge differences that increased to 40 %–50 % in
the systematic sub-datasets (SYS1 and SYS2) for the higher
quantiles (T = 500 years). 70 years), but differences are wider towards frequent floods
(e.g. 210 m3 s−1 for the 25-year flood) and in the upper quan-
tiles, with 300 m3 s−1 in the 500-year flood. In the ALLSYS
dataset, the discharge range (DD) is wide over all quantiles
and slightly increasing towards the 500-year flood. The diver-
gence on discharge calculated by the Gumbel and LP3 distri-
butions is higher when applied to the subdivided systematic
datasets within differences between 370–810 m3 s−1 in the
100-year and 500-year flood, typically used for flood haz-
ard mapping. 4.3
Synoptic analysis and moisture transport The FFA analysis performed with the Gumbel distribution
using the MLE method gives different discharge values to
those obtained by the LP3 distribution, as expected. The dif-
ferential performance can be evaluated in terms of discharge
difference (DD in m3 s−1) obtained by the two distributions
for a given quantile among different datasets. The most con-
sistent results are obtained for the HISTO dataset with dif-
ferences of 20–75 m3 s−1 for quantiles less than 0.2 % AEP,
reaching 155 m3 s−1 in the 0.001 % AEP flood. In the PRE–
SYS dataset, the discharge calculated with Gumbel and LP3
distribution is similar for 1 % and 0.5 % AEP floods (T ∼ The HISTO dataset fitted with LP3 distribution also pro-
vided the more realistic quantile values compared to other
datasets. For instance, the largest flood during the 508-year
record length, namely the 1860 event (3450 m3 s−1), is asso-
ciated with a 500-year recurrence interval (T ) in the HISTO
analysis and with T >1000 years and ∼200 years in the
PRE–SYS and ALLSYS frequency curves. The incorporation of historical and pre-instrumental data
into the FFA results in a slight decrease in the magnitude
of the flood quantiles when compared with those calculated
with the systematic record. In general terms, the system- https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6121 Peak discharges in m3 s−1 Figure 10. Integrated water vapour transported averaged on the 10 d
prior. Examples for historical floods took place in 1860 and 1936,
and the recent flood took place in 2001. G. Benito et al.: Enhanced flood hazard assessment In summary, the FFA analysis using the histor-
ical dataset provided the most consistent results in discharges
calculated for all flood quantiles using two different distribu- https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6122 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 10. Integrated water vapour transported averaged on the 10 d
prior Examples for historical floods took place in 1860 and 1936 Table 2. Flood quantiles for different exceedance annual probabilities of the Duero River in Zamora calculated for different datasets applying th
log Pearson type III distribution and the maximum likelihood estimator methods to fit a Gumbel distribution. DD represents the difference in year
for each quantile by the aforementioned distributions. T : average recurrence interval (in years). e 2. Flood quantiles for different exceedance annual probabilities of the Duero River in Zamora calculated for different datasets applying the expected moments algorithm to fit a
Pearson type III distribution and the maximum likelihood estimator methods to fit a Gumbel distribution. DD represents the difference in years of the calculated discharge obtained
ach quantile by the aforementioned distributions. T : average recurrence interval (in years). Peak discharges in m3 s−1
ceedance annual
T
Historic documentary,
Staff gauge (El Porvenir)
All systematic
Systematic data
Systematic data
probability
staff gauge and systematic
and systematic
data only
1920–1969
1970–2018
(%/value)
(years)
LP 3
Gumbel
DD years
LP 3
Gumbel
DD years
LP 3
Gumbel
DD years
LP 3
Gumbel
DD years
LP 3
Gumbel
DD years
20/0.2
5
1240
1275
35
1450
1325
125
1390
1345
45
1760
1590
170
1080
1070
10
10/0.1
10
1650
1625
25
1875
1675
200
1810
1700
110
2160
1980
180
1445
1350
95
4/0.04
25
2140
2070
70
2330
2120
210
2335
2140
195
2520
2475
45
1960
1700
260
2/0.02
50
2475
2400
75
2610
2450
160
2720
2470
250
2700
2840
140
2380
1970
410
1/0.01
100
2790
2720
70
2840
2775
65
3090
2800
290
2830
3200
370
2825
2230
595
0.5/0.005
200
3070
3050
20
3030
3100
70
3450
3125
325
2920
3575
655
3300
2490
810
0.2/0.002
500
3410
3475
65
3230
3530
300
3910
3550
360
3000
4045
1045
4000
2830
1170
0.1/0.001
1000
3640
3795
155
3360
3855
495
4250
3880
370
3050
4405
1355
4550
3090
1460 . G. Benito et al.: Enhanced flood hazard assessment Flood quantiles for different exceedance annual probabilities of the Duero River in Zamora calculated for different datasets applying the expected moments algorithm to fit a
rson type III distribution and the maximum likelihood estimator methods to fit a Gumbel distribution. DD represents the difference in years of the calculated discharge obtained
h quantile by the aforementioned distributions. T : average recurrence interval (in years). G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment Figure 11. (a) Temporal representation of documentary and gauged floods. Grey shaded areas indicate censored flood records during the
pre-instrumental period. The coloured horizontal lines are perception thresholds over different time periods; i.e. only floods exceeding the
perception threshold discharge were recorded. The yellow diamond dots show discharges for documentary floods including a range of
discharge uncertainty (vertical lines). The pink triangles are reported discharge from staff gauge observations at El Porvenir. The white dots
are annual flows from the Carrascal gauge station. (b) Log Pearson type III distribution fitted with historic flood events and gauged discharge
(staff and continuous gauged). (c) Idem fitted with staff and continuous gauge records. (d) Idem with all continuous gauge records. (e) Idem
with gauged records over the period 1920–1969. (f) Idem with gauged discharges over the period 1970–2018. Figure 11. (a) Temporal representation of documentary and gauged floods. Grey shaded areas indicate censored flood records during the
pre-instrumental period. The coloured horizontal lines are perception thresholds over different time periods; i.e. only floods exceeding the
perception threshold discharge were recorded. The yellow diamond dots show discharges for documentary floods including a range of
discharge uncertainty (vertical lines). The pink triangles are reported discharge from staff gauge observations at El Porvenir. The white dots
are annual flows from the Carrascal gauge station. (b) Log Pearson type III distribution fitted with historic flood events and gauged discharge
(staff and continuous gauged). (c) Idem fitted with staff and continuous gauge records. (d) Idem with all continuous gauge records. (e) Idem
with gauged records over the period 1920–1969. (f) Idem with gauged discharges over the period 1970–2018. bers of large floods. Climatically, this period corresponds to
the Little Ice Age (1500–1850) that in Iberia is characterised
by cold conditions with alternating wet–dry phases (Oliva et
al., 2018). Flood-rich periods were identified between 1600
and 1640, 1730 and 1750, 1770 and 1790, 1850 and 1880,
1924 and 1948, and 1960 and 1980 (Fig. 3), coinciding with
flood episodes in Atlantic Iberian rivers (Benito et al., 1996,
2003; Barriendos and Rodrigo, 2006) and overlapping in
time with flood periods described in western European re-
gions (Blöschl et al., 2020). The temporal pattern of these
flood clusters suggests a multi-decadal natural variability in
the atmospheric circulation, affecting both flood frequency
and magnitude (Nobre et al., 2017). 5.1
Multi-decadal flood patterns and climate
variability Documentary flood records in Zamora are scarce during the
early medieval period, coincident with the late Climatic Me-
dieval Anomaly that is characterised by warm temperatures
and high hydrological variability. The limited flood data sug- DD years Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 https://doi.org/10.5194/hess-25-6107-2021 6123 G. Benito et al.: Enhanced flood hazard assessment In western Iberia, ex-
cess of winter precipitation is related to a southern position gest, however, the occurrence of exceptional floods such as
the 1258 and 1264 events, the former with a magnitude sim-
ilar to or exceeding the 1860 flood as it is interpreted from
inscription at the doorpost in St. Claudio church. The 1258
flood was exceptional in terms of peak flow but also in exten-
sion affecting other Iberian Atlantic basins, at least the Tagus
and Guadalquivir rivers (Benito et al., 2003). The lack of in-
formation of catastrophic flooding between 1270 and 1500 is
common to other Iberian rivers, although it is likely related to
the discontinuity of written reports and preservation of doc-
umentary archives prior to the 14th century (Barriendos and
Rodrigo, 2006). The temporal distribution of floods over the past 500 years
shows at least six flood-rich periods of 20–40-year duration,
commonly separated by ∼60-year periods with scarce num- 5.2
Atmosphere–ocean interaction leading to
catastrophic flooding During the 18th century the largest flood occurred on 5 De-
cember 1739, with an estimated peak of 2700 m3 s−1. In
this winter, a previous peak flow was already reported on
1 November, with a likely discharge of 2000 m3 s−1 and at
least another peak documented in April 1740 with lower
magnitude (∼1800 m3 s−1). The reconstructed monthly
NAO index (Luterbacher et al., 1999) shows negative val-
ues in the month previous to peaks, namely −1.27 (Oc-
tober), −1.47 (November), and −0.38 and −0.55 (March
and April), that, together with −2.38 and −1.14 during Jan-
uary and February, show persistent meridional atmospheric
zonal flow during that exceptional winter. Other years with
severe flood peaks associated with high negative NAO in-
dex were January and March 1931 (−2.08 and −1.16 NAO)
and January and May 1881 (−3.6 and −1.42). The 29 De-
cember 1860 flood was preceded by a negative NAO index
in November (−3.44) and December (−2.14) and contin-
ued into January 1861 (−0.56). In the Tagus River, similarly
large floods have been associated with a very high frequency
of negative NAO mode during the initial 20–25 d before the
flood peak (Salgueiro et al., 2013). In western Iberia, the relationship between temperature
and precipitation tends to be negative as cyclonic conditions
are related to moist and relatively warm air masses from
the Atlantic. Indeed, there is a total absence of large floods
during the Maunder minimum period (1645–1715), a period
with lower air temperatures, that intriguingly reveal a link be-
tween low solar activity and decreased flood frequency in the
Iberian Peninsula (Vaquero, 2004). In contrast, periods with
more frequent floods in the western Iberian region coincide
with transitions to cool and wet conditions associated with a
southward migration of westerlies (Benito et al., 2015b). p
(
g
,
)
The negative NAO-like phases allow the arrival to the
Iberian Peninsula of a frontal system bringing warmer and
enriched moisture air masses. Our analyses highlighted that
the source of this moisture is the Caribbean Sea, and that is
the so-called atmospheric river structures (Ralph et al., 2017;
Dacre et al., 2015; Waliser and Guan, 2017). Thus, most of
the major floods recorded in Zamora (88 %) were linked to
the occurrence of these phenomena. G. Benito et al.: Enhanced flood hazard assessment of Atlantic storm tracks occurring during the negative mode
of NAO (Trigo et al., 2014). of Atlantic storm tracks occurring during the negative mode
of NAO (Trigo et al., 2014). of Atlantic storm tracks occurring during the negative mode
of NAO (Trigo et al., 2014). traordinary flooding has decreased and mostly occurred in
late winter–early spring, which confirms the delay of flood
peaks under a warming climate in Europe (Blöschl et al.,
2019). A detailed hydroclimatic analysis driving these flood-
rich periods reveals complexities and dissimilarities among
them. The first two periods (1600–1640, 1730–1750) were
also identified as decades with frequent dry years in spring
(needed for agriculture) according to reported prayers and
novenas in churches and processions for rain usually under-
taken in March and early April (Álvarez-Vázquez, 1986). The second two periods (1770–1790, 1850–1880) were dom-
inated by overall wet winter years, although the total of
rainy years was never more than a quarter of the drought
years (Álvarez-Vázquez, 1986). In the Douro River in Porto
these two periods were also identified by their anoma-
lous frequency and severity of floods (Alcoforado et al.,
2021; Amorim et al., 2017) that include the largest flood
on record (4–6 December 1739), which reached a stage
of 12 m in a bedrock section at the right margin just up-
stream of the Dom Luiz I Bridge (Loureiro, 1904; Taborda,
2006). In Régua the 1739 flood peak was estimated at
18 000 m3 s−1, and in Porto (∼105 km downstream) it likely
reached 20 000 m3 s−1 (Silva and Oliveira, 2002). https://doi.org/10.5194/hess-25-6107-2021 https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6124 G. Benito et al.: Enhanced flood hazard assessment 30 floods exceeding the perception threshold (1900 m3 s−1),
whereas 8 floods exceeded that discharge over the gauged
period (1920–2018). plate air mass, especially with a south-west orientation (Trigo
et al., 2014). Thus, our results suggest that at least 70 % of
the major floods recorded were related to either western or
southern cyclonic circulation patterns and at least in 14 %
of the cases were related to southern and western merid-
ional circulation patterns. This implies that snowmelt and/or
rain on snow from the surrounding mountains could have
contributed to additional runoff (Stewart, 2009). Therefore,
these mechanisms were related to main triggering mecha-
nisms of torrential floods in mountain streams in the Duero
basin (Ballesteros-Cánovas et al., 2015; Morán-Tejeda et al.,
2019). Moreover, during wet winters, characteristic of neg-
ative NAO-like phases (López-Moreno et al., 2007), a high
soil moisture content prevalence in a large portion of the
basin could have enabled a recharge of the groundwater sys-
tem and therefore favoured the direct rainfall–runoff trans-
formation (Berghuijs et al., 2019; Benito et al., 2010, 2011). plate air mass, especially with a south-west orientation (Trigo
et al., 2014). Thus, our results suggest that at least 70 % of
the major floods recorded were related to either western or
southern cyclonic circulation patterns and at least in 14 %
of the cases were related to southern and western merid-
ional circulation patterns. This implies that snowmelt and/or
rain on snow from the surrounding mountains could have
contributed to additional runoff (Stewart, 2009). Therefore,
these mechanisms were related to main triggering mecha-
nisms of torrential floods in mountain streams in the Duero
basin (Ballesteros-Cánovas et al., 2015; Morán-Tejeda et al.,
2019). Moreover, during wet winters, characteristic of neg-
ative NAO-like phases (López-Moreno et al., 2007), a high
soil moisture content prevalence in a large portion of the
basin could have enabled a recharge of the groundwater sys-
tem and therefore favoured the direct rainfall–runoff trans-
formation (Berghuijs et al., 2019; Benito et al., 2010, 2011). p
More important are the consequences of possible climate-
related non-stationarity for estimating flood quantiles (Milly
et al., 2008). The alternation of flood-rich and flood-poor
periods identified during the last 500 years implies differ-
ences in the statistic values over time. 5.3
The significance of past floods in flood hazard
analysis The historic city of Zamora is highly sensitive to flood haz-
ards and weather extremes as both have direct impacts on
architectural and patrimonial assets and cultural landscapes. Reported evidence of flood incidence provides a rich event
catalogue with descriptions of more than 88 historical floods
over a period of 760 years as well as 17 pre-instrumental
observations on a gauged water-level scale and 99 years of
gauged continuous records. Due to the extensive historical records of the area, flood
hazard assessment can be performed by integrating differ-
ent flood sources, datasets and timescales. Moreover, the im-
plementation of two independent methods (EMA and MLE)
for fitting regression models to censored data together with
two distribution functions (LP3 and Gumbel) allows the ro-
bustness of results to be tested for low-probability quantiles. In the case of the 1 % AEP flood (T = 100 years), both the
HISTO dataset (511 years) and the PRE–SYST (147 years)
result in a similar discharge (2800 m3 s−1), whereas the SYS
dataset (99 years) provides a range of 2800–3100 m3 s−1. In
the upper tail of the distribution, the 0.2 % AEP flood (T =
500 years) based on HISTO data is well constrained to 3410–
3475 m3 s−1, whereas a wide discharge range is obtained
with PRE–SYST (3230–3530 m3 s−1), and it is even wider
with the SYST dataset (3550–3910 m3 s−1). The historical
flood record indicates that, at least over the last 511 years,
only one flood reached a discharge of 3450 m3 s−1 (1860
flood), which is within the range of the 0.2 % AEP flood,
whereas the SYST dataset overestimates the quantile dis-
charge (3910 m3 s−1). In even rarer floods, the 0.1 % AEP
based on the HISTO dataset was estimated in the range of
3640–3795 m3 s−1, which fits the 3700 m3 s−1 estimated for
the 1258 flood, although that historical flood was not in-
cluded in the frequency analysis. g
g
The basic hypothesis in flood frequency analysis (FFA) in-
corporating historical information (non-systematic) is that a
certain perception threshold of water level exists, and all the
exceedances of this level over a specific time interval have
been recorded (Benito et al., 2015a). Perception thresholds
are typically related to river morphological elements (e.g. G. Benito et al.: Enhanced flood hazard assessment However, the prob-
lem affects both historic and modern gauge records with
the advantage that the former includes a longer-term flood
dataset (rich and poor flood periods), whereas there may
be a bias in gauged data for specific flood patterns. For
instance, seven catastrophic floods were recorded over the
period 1920–1969, whereas only two occurred over 1970–
2018, which results in strong differences in the 25-year flood,
with 2500 m3 s−1 and 1650 m3 s−1, respectively. The gauged
registers indicate a trend to decrease the magnitude of ex-
treme floods since the 1970s; however, flood events such as
those in 2001 (2075 m3 s−1) and 2013 (1654 m3 s−1) illus-
trate the occurrence of extreme flooding despite the peak dis-
charge attenuation by reservoirs. The overall gauge record
(1920–2018) estimates the 25-year flood to be at an interme-
diate value of 2105–2300 m3 s−1. In the case of long-term
historical records, multiple flood-rich and flood-poor periods
are combined, giving averaged estimates for the flood quan-
tile discharges (e.g. T 25 years: 2140–2070 m3 s−1). 5.2
Atmosphere–ocean interaction leading to
catastrophic flooding A similar mechanism for
moisture input has been associated with intense floods in Por-
tugal (Trigo et al., 2014) and to other large European rivers
draining to the Atlantic Ocean (Lavers and Villarini, 2015;
Ballesteros-Cánovas et al., 2019). Rainfall events linked to
the arrival of the ARs are generally related to the uplift
forced by orography (Ralph et al., 2006), which is consis-
tent with the mountain reliefs surrounding the Duero basin. Although the frequency, position and magnitude of ARs de-
pends on planetary-scale phenomena (Ralph et al., 2011),
the moisture transport capacity may be enhanced under cli-
mate change conditions as a consequence of an increase in
the water-holding capacity of the atmosphere (Lavers et al.,
2013), which could have consequences on climate predispo-
sition for floods in the Duero basin. In terms of flood magnitude, the Duero data suggest that
the largest floods occurred at the onset and final stages of the
Little Ice Age (LIA). In both instances, these floods are re-
lated to persistent rainfalls occurring during winter months. Interestingly, during the 16th and early 17th centuries, catas-
trophic floods occurred mainly in January and extraordinary
floods in early spring (March), whereas at the final stage of
the LIA the largest floods tend to occur between February
and March (with the exception of the 1860 flood). The flood magnitudes over the 20th century may be biased
by the environmental changes (agricultural transformation in
the early part of the century) and reservoir construction (since
1950s). The 1924–1948 flood-rich period took place dur-
ing the early-20th-century warming (ETCW; 1910s–1940s),
a period of strong internal variability in the climate sys-
tem, which featured an anomalous warming of the Arctic
region impacting climate in northern Europe (Brönnimann,
2009). Two periods (1924–1927, 1935–1941) of extraordi-
nary winter floods (Q>1250 m3 s−1) that took place dur-
ing the ETCW could arguably make this event relevant also
for future analogous warming phases (Ballesteros-Cánovas
et al., 2019). Over the late 20th century, the frequency of ex- Our analyses also pointed out that floods were linked to
warmer-than-normal air temperatures, as identified in the
composite analyses in the Duero basin (Fig. S2). This is con-
sistent with the cyclonic circulation and the advection of tem- https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6125 G. Benito et al.: Enhanced flood hazard assessment G. Benito et al.: Enhanced flood hazard assessment 6126 non-stationary approach, historical flood frequency analysis
over the last 300 years shows fluctuations in the estimated
flood quantiles at a decadal scale responding to a combi-
nation of multi-decadal cold–warm cycles and interannual
ocean–atmosphere interactions. However, such analysis is
not easy to implement (e.g. López and Francés, 2013), and it
is beyond the aim of the present study. Alternative options us-
ing climate models and scenarios may generate even higher
uncertainties on flood hazard and risk planning studies (Seri-
naldi and Kilsby, 2015). Lins and Cohn (2011) suggested that
it is preferable to continue with simple (stationary) statistical
models with well-understood limitations than use sophisti-
cated models whose correspondence to reality is uncertain. In this debate, historical hydrology informs about timing and
persistence of flood clusters and identifies the relationships
of the largest floods with low-frequency atmospheric circu-
lation and other environmental drivers, providing robust data
of real floods that occurred at a centennial scale. In sum-
mary, a long-term FFA framework under stationary models
provides good average values supported by flood extremes
beyond century-scale climate cycles. ness and social learning and enhances adaptation (Di Bal-
dassarre et al., 2015). Conversely, over the last 40 years, only
the 2001 flood exceeded 2000 m3 s−1, and since the 1980s
there is strong socio-economic pressure to expand urban oc-
cupation in the San Frontis, Olivares and Cabañales suburbs. Fortunately, the remarkable work on historical flood data col-
lection done by Marquina (1949a, b) preserved the details on
the location and position of epigraphic marks no longer at
their original sites. g
Within the framework of the European Flood Directive
(2007/60/EC), EU member states are requested to map flood
hazards (typically 100-year and 500-year floods) as well as
consider the effects of climate change on flood risks. The cli-
mate effect on flood hazards is complex and not ubiquitous
within a world that increases population, exposure and vul-
nerability (Kundzewicz et al., 2019). Recent developments
on climate change science and adaptation actions focus on
win–win strategies for sustainable climate action. The study
of past flooding, either historic or palaeofloods, can be a di-
rect guide to flood possibilities for adaptation actions to ex-
treme flooding under climate change as it reveals what is ac-
tually possible under conditions of centennial climate vari-
ability (Brázdil et al., 2012; Macdonald, 2013). 5.4
Public perception and risk culture Flooding of the Duero River has been a recurrent problem for
the city of Zamora, with most reported incidents related to
the medieval bridge, water mill facilities, ecclesiastic build-
ings, farms and houses. Despite the abundant flood docu-
mentation, details on the relative importance of each historic
flood are not always available, and only for the most catas-
trophic events are there precise references to flood levels
on epigraphic marks. The best-preserved epigraphic marks
are located inside San Frontis Church and Dueñas Convent,
which became a cloistered convent with difficult access for
the public. Epigraphic marks are important elements for pub-
lic perception in central Europe (Brázdil et al., 2005; Her-
get and Meurs, 2010; Wetter et al., 2011; Elleder et al.,
2013; Glaser et al., 2010) and in Mediterranean regions (Al-
drete et al., 2007; Cœur and Lang, 2008; Barriendos et al.,
2019), which are maintained as part of the historical asset. In
Zamora flood marks are not well known to the public, and,
in the modern urban expansion, some flood marks were re-
moved during restoration works of architectural assets. For
instance, the 1860 flood mark disappeared from the old city
gate (Puerta del Pescado) when, due to traffic problems, the
old monument was moved to a new location in 1909. Cu-
riously, in a municipal council meeting (29 January 1908),
a city councillor (Eusebio Calonge Sansó, cited in Heraldo
de Zamora, 30 January 1908) proposed to preserve the 1860
flood mark at its place for the public memory of that ex-
treme flood. In that time, five floods over 2000 m3 s−1 were
recorded in a 30-year period (1880 to 1909) that kept alive
the public perception of flood risk and its socio-economic
consequences. It also corroborates a well-established obser-
vation that occurrence of a damaging flood improves aware- G. Benito et al.: Enhanced flood hazard assessment The flood
magnitudes result neither from complex climate models nor
from probabilistic language, difficult to understand for the
general public. Common sense holds that what has really
happened can happen again (Baker, 2008). This approach
based on past flood occurrences also serves to increase public
confidence in any proposed solution that ultimately involves
a large economic or social expense for hazard mitigation. 5.3
The significance of past floods in flood hazard
analysis river banks, dikes) above which overflow produces damages
reported in municipal and ecclesiastic act books (Stedinger
and Cohn, 1986; Frances et al., 1994; Macdonald, 2013). For instance, in the Duero’s documented floods, the percep-
tion threshold is the flood discharge overflowing riverbanks
at Olivares, Horta, Frontis and Cabañales neighbourhoods
that is ∼1900 m3 s−1. Each year, therefore, the Duero River
was characterised as having a peak discharge either exceed-
ing or not exceeding that perception threshold. In the qual-
itative historical classification without any further damage
descriptions, such events correspond to extraordinary floods
(Barriendos and Martín-Vide, 1998). Catastrophic floods in-
volve higher damages that are typically recorded in epi-
graphic marks and/or reports of impacts on historic build-
ings. The scarce urban development of Zamora until the sec-
ond half of the 19th century allows a temporal stability (from
1511 to 1872) of this perception threshold, which was con-
firmed for modern flood analogues of historical extraordinary
and even catastrophic floods. Over the 408 years prior to the
continuous gauged record (1511–1919) there is evidence of Previous studies of FFA under the non-stationary frame-
work have been applied to documentary flood datasets using
climatic (e.g. NAO) and environmental indices (reservoir in-
dex) as external covariates (Machado et al., 2015). In such a https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 Supplement. The supplement related to this article is available on-
line at: https://doi.org/10.5194/hess-25-6107-2021-supplement. Supplement. The supplement related to this article is available on-
line at: https://doi.org/10.5194/hess-25-6107-2021-supplement. – Over the historical time, flood-rich periods were identi-
fied in the periods 1600–1640, 1730–1750, 1770–1790
and 1880–1910, the first two in decades of frequent
drought and the latter in overall wet winter years. In the
20th century flood frequency increased between 1935
and 1960, overlapping the early-20th-century warm pe-
riod. Later, the flooding occurred punctuated on time,
namely in 1978–1979 and 2001. Author contributions. GB and MM designed the research and ap-
plied for funding acquisition. OC carried out the two-dimensional
hydraulic modelling. JABC performed the analysis of the atmo-
spheric circulation in relation to historical floods and its hydrome-
teorological interpretation. MB and MM collected and analysed the
documentary flood database. MM collected and analysed the urban
history and its relation to flood perception through time. All authors
interpreted results. GB, JABC and MM wrote the first draft, and all
authors contributed to reviewing and editing the paper. – Floods in the Duero basin mostly occur during negative-
like NAO phases that force frontal systems to travel in
meridional latitudes, bringing moisture and warmer air
masses from the tropical latitudes in the so-called atmo-
spheric river structures. Competing interests. The contact author has declared that neither
they nor their co-authors have any competing interests. – We demonstrate that the temporal extent of the flood
dataset is a critical factor in the quality of the flood
frequency results. The most consistent results, indepen-
dently of fitting methods and distribution functions, cor-
respond to the historical dataset (also including pre-
instrumental and systematic records), showing calcu-
lated discharges within a narrow range even for high
quantiles (1000-year flood). Combined pre-instrumental
(water-level readings) and systematic gauge data pro-
vided robust results for the 100-year flood, with increas-
ing uncertainty in the 500-year flood. In the systematic
dataset results were uncertain in the 100-year and 500-
year floods, despite the almost 100-year-long datasets. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Acknowledgements. We acknowledge the hydrological data and
documentary support provided by Jose Angel Martinez Pérez,
head of operation and management service at Iberdrola (hy-
dropower company) in Carbajosa de la Sagrada (Salamanca), and
by Yolanda Diego Martín, director of documentation manage-
ment of the Iberdrola historical archive in Ricobayo (Zamora). 6
Conclusions Climate change effect on local flood magnitudes is becoming
a major problem in flood hazard mapping due to model un-
certainty and multiple possible scenarios. There is a need for
better identification and guidance for tools for a robust adap-
tation to future flood risk. In this work, we demonstrate that
the reconstruction of flood histories under climate variability
beyond the temporal length of observations provides realistic
insight into future floods under worst-case scenarios. In Zamora (Spain), a long-term flood record of the Duero
River was collated from documentary archives (1256–1871),
early water-level observations (1872–1919) and gauged data
(1920–2018). Early flood records were discontinuously re-
ported, but since ca. 1500, flood events have been well docu-
mented. Documentary flood information includes narrative
descriptions (annals, chronicles and memory books), epi-
graphic marks, newspapers and technical reports. The urban
configuration at the riverside area has been stable since the
14th century, which implies continuous, homogeneous and
comparable data over the last 500 years. Our key findings are
as follows. https://doi.org/10.5194/hess-25-6107-2021 Hydrol. Earth Syst. Sci., 25, 6107–6132, 2021 6127 G. Benito et al.: Enhanced flood hazard assessment – Although the likelihood of future floods is uncertain
with conventional downscaling climate models, the ex-
tending of flood records beyond cycles of past climate
variability provides further understanding of possible
flood sizes and calculated magnitudes of flood quantiles
to guide low-regret adaptation decisions and to improve
public perception of extreme flooding. – Although the likelihood of future floods is uncertain
with conventional downscaling climate models, the ex-
tending of flood records beyond cycles of past climate
variability provides further understanding of possible
flood sizes and calculated magnitudes of flood quantiles
to guide low-regret adaptation decisions and to improve
public perception of extreme flooding. – There is documentary evidence of 69 flood registers dur-
ing the period 1511–1871, with 15 catastrophic floods,
16 extraordinary floods and 38 ordinary floods. Catas-
trophic and extraordinary floods typically produced
overflow of urban areas, typically exceeding a discharge
of 1900 m3 s−1, based on a two-dimensional hydraulic
model calibrated with gauge records. – The largest floods over the last 500 years occurred
in 1860 (3450 m3 s−1), 1597 (3200 m3 s−1) and 1739
(2700 m3 s−1). Historic flood magnitudes were greater
than the ones recorded in the early water readings
(largest in 1895 with 2380 m3 s−1) and in the gauging
station (largest in 1960 with 2360 m3 s−1). Code and data availability. The data used in this article can be ob-
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English
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Efficacy and safety of patients with chronic kidney disease undergoing left atrial appendage closure for atrial fibrillation
|
PloS one
| 2,023
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cc-by
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PLOS ONE PLOS ONE Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Background The relative safety and efficacy of left atrial appendage closure (LAAC) for atrial fibrillation
(AF) in patients with chronic kidney disease (CKD) have not been well defined. To evaluate
the results in this cohort, we conducted a systematic review and meta-analysis of observa-
tional studies. OPEN ACCESS We searched the PubMed, EMBASE, Web of Science, and Cochrane Library databases
from inception to January 2023 for all relevant studies. Our inclusion criteria were met by
twelve observational studies that included 61324 patients altogether. Citation: Liu C, Han S, Cui K, Wang F (2023)
Efficacy and safety of patients with chronic kidney
disease undergoing left atrial appendage closure
for atrial fibrillation. PLoS ONE 18(10): e0287928. https://doi.org/10.1371/journal.pone.0287928 Abstract a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Chaofan Liu1☯, Shaojie Han2☯, Kaijun Cui2*, Fang WangID3* Chaofan Liu1☯, Shaojie Han2☯, Kaijun Cui2*, Fang WangID3*
1 School of Nursing, Chengdu University of Traditional Chinese Medicine, Chengdu, China, 2 Department of
Cardiology, West China Hospital, Sichuan University, Chengdu, China, 3 Guang’an Shi Zhongyi Yiyuan:
Guang’an Hospital of Traditional Chinese Medicine, Beijing, China 1 School of Nursing, Chengdu University of Traditional Chinese Medicine, Chengdu, China, 2 Department of
Cardiology, West China Hospital, Sichuan University, Chengdu, China, 3 Guang’an Shi Zhongyi Yiyuan:
Guang’an Hospital of Traditional Chinese Medicine, Beijing, China 1 School of Nursing, Chengdu University of Traditional Chinese Medicine, Chengdu, China, 2 Department of
Cardiology, West China Hospital, Sichuan University, Chengdu, China, 3 Guang’an Shi Zhongyi Yiyuan:
Guang’an Hospital of Traditional Chinese Medicine, Beijing, China ☯These authors contributed equally to this work. * cuikaijunscu@163.com (KC); fangwang202302@163.com (FW) Results Editor: Fateen Ata, Hamad Medical Corporation,
QATAR Compared with no CKD group, in-hospital mortality (OR: 2.84, 95% CI: 2.12–3.81, p<0.01,
I2 = 0%), acute kidney injury (AKI) (OR: 4.39,95% CI:4.00–4.83, P<0.01, I2 = 3%), major
bleeding events (OR: 1.44, 95% CI: 1.29–1.60, p<0.01 I2 = 0%), and pericardial effusion/
tamponade (OR 1.30; 95% CI 1.13–1.51, p < 0.01; I2 = 0%) were more common in the CKD
group, especially in patients with end-stage renal disease (ESRD). No significant difference
was observed in the occurrence of stroke (OR: 1.24, 95% CI: 0.86–1.78, P = 0.25, I2 = 0%),
LAAC success rates (OR: 1.02, 95% CI: 0.33–3.16, p = 0.97, I2 = 58%) and vascular access
complications (OR: 1.13, 95% CI: 0.91–1.39, p = 0.28, I2 = 0%) between the two groups. During the follow-up, there was no difference in the risk of stroke between the two groups. Published: October 26, 2023 Copyright: © 2023 Liu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. Efficacy and safety of patients with chronic
kidney disease undergoing left atrial
appendage closure for atrial fibrillation Chaofan Liu1☯, Shaojie Han2☯, Kaijun Cui2*, Fang WangID3* Conclusions Funding: The authors received no specific funding
for this work. CKD patients who receive LAAC have a greater risk of in-hospital mortality, AKI, pericardial
effusion/tamponade, and major bleeding events than those without CKD, especially in
patients with ESRD. No significant difference in the risk of stroke was found in the long-term Competing interests: The authors have declared
that no competing interests exist. 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE chronic kidney disease and left atrial appendage closure follow-up after LAAC between the two groups, demonstrating a similar efficacy of LAAC to
prevent stroke in CKD patients. follow-up after LAAC between the two groups, demonstrating a similar efficacy of LAAC to
prevent stroke in CKD patients. Introduction Atrial fibrillation (AF) is a common cardiac arrhythmia in patients with end-stage renal dis-
ease (ESRD) or chronic kidney disease (CKD) [1]. CKD and ESRD are regarded as indepen-
dent risk factors for the development of AF. Patients with CKD or ESRD have a prevalence of
nonvalvular atrial fibrillation (NVAF) that ranges from 13% to 27% [2, 3]. Large prospective
studies have revealed that people with CKD may be more susceptible to developing AF [4]. Stroke and systemic embolism are complications closely related to the prognosis of atrial fibril-
lation. For this reason, in patients with an elevated risk of thrombosis, as typically determined
by the CHA2DS2-Vasc score, current guidelines recommend oral anticoagulants for the pre-
vention of embolic events [1]. Patients with impaired renal function not only had a higher inci-
dence of AF but also had a higher risk of embolism than patients with normal renal function
[5, 6]. In addition to the increased thromboembolic risk, patients with AF and concurrent
CKD are more likely to experience bleeding events, especially if they are using anticoagulants
[5, 7]. Moreover, due to a lack of data, novel oral anticoagulant (NOAC) administration should
be avoided in patients with significantly impaired renal function, and the use of warfarin is
linked to contradictory outcomes [1]. Over the past ten years, a non-pharmacologic method of
preventing ischemic stroke in patients with AF who are not anticoagulant candidates has
gained widespread acceptance as an alternative to NOACs. This method involves percutaneous
left atrial appendage closure (LAAC) using the Watchman, Amplatzer Cardiac Plug, Lambre
device and so on [8]. Several studies have evaluated the effectiveness and safety of LAAC in CKD patients, but
these are mostly small-sample studies, and the results of different studies are conflicting. To
better understand the safety and long-term efficacy of LAAC for patients with AF and CKD,
we conducted a meta-analysis of the literature. Methods The review was carried out in accordance with the Preferred Reporting Items for Systematic
Reviews and Meta-Analyses standards [9]. Search strategy To discover all published clinical studies that reported the effects of LAAC in CKD or ESRD
and no CKD or ESRD. A thorough review of the literature was performed in the PubMed,
Web of Science, Cochrane Library, and Embase databases up to January 2023. Each term
included “atrial fibrillation” and “left atrial appendage”, and (“renal Insufficiency” or “renal
failure” or "end-stage renal disease" or "chronic kidney disease" or "dialysis" or "hemodialysis"). We have no restrictions on the type of research. A manual search of secondary materials,
including the references of initially found articles, reviews, and comments, was used to find
relevant studies. All references were downloaded in order to consolidate, get rid of duplicates,
and conduct additional research. 2 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE chronic kidney disease and left atrial appendage closure Data extraction C.L. and S.H. separately searched the articles and retrieved the data. When the two authors
were unable to agree, the third author (F.W.) was approached to make a decision. Each study’s
data were thoroughly extracted to yield the following information: title, name of the first
author, publication year, year of the study, country, demographic and characteristic data of
subjects, total numbers of participants, and complication rates in each study. Study selection and outcomes The search results were examined by two blinded, independent writers (C.L. and S.H.), who
included papers if they followed the following standards: (1) peer-reviewed journals published
the research, (2) compared patients receiving LAAC for AF who had CKD or ESRD to those
who had not, and (3) at least one LAAC-related complication should be mentioned. No limita-
tions on sample size, follow-up time, or language existed for us. We talked until we came to an
agreement when there were differences between the reviewers. Cohort study quality was evalu-
ated using the Newcastle-Ottawa quality assessment scale (NOS). Interested procedural endpoints included in-hospital mortality, stroke, LAAC success rate,
major bleeding events, vascular access complications, acute kidney injury, and pericardial effu-
sion/tamponade. Long-term endpoints included all-cause mortality, stroke, and major bleeding
events. Four of the included studies reported composite endpoint, but the definition of these
composite endpoints was not exactly the same. We did not include them in the final analysis. Procedural success was defined as successful implantation and a peri-device leakage less than 5
mm based on our included studies. This is the definition given by the current guidelines and
the studies we included, and this definition is the result of numerous previous studies [10]. Based on the studies we included, patients with estimated glomerular filtration rate
(eGFR)<60 mL/min/1.73 m2 were categorized as having a chronic kidney disease (CKD), and
end-stage renal disease (ESRD) was defined as the eGFR < 15 ml/min/1.73 m2 or chronic
hemodialysis treatment [11, 12]. Statistical analysis Means and standard deviations for continuous variables and the quantity and percentages for
categorical variables are how descriptive statistics are presented. The Cochrane Collaboration’s
and PRISMA guidelines’ recommendations for statistical analysis were followed, using Review
Manager, version 5.4. The effects on procedural and long-term outcomes were presented as
odds ratios (ORs) with 95% confidence intervals (CI). Regardless of heterogeneity among the
studies, inverse variance weighted random-effects approach was adopted for each outcome
since it allowed for a more conservative evaluation of the pooled effect size. A sensitivity analy-
sis was conducted by removing one study at a time to analyze the impact of each study on
overall heterogeneity and to assess the study’s overall robustness. According to Cochrane, pub-
lication bias was evaluated by looking at the funnel plot’s symmetry. The cutoff for statistical
significance was p <0.05. PLOS ONE chronic kidney disease and left atrial appendage closure Table 1. Characteristics of the included studies. Study
Country
Study design
Patient selection
Follow-up
Type of Occluder
Kefer et al. (2016)
Europe and North America
Prospective multicenter study
2008,12–2013,11
498d
1
Xue et al. (2018)
Germany
Single-centre, retrospective study
2012,2–2017,1
637d
2
Luani et al. (2019)
Germany
Single-centre, prospective study
NR
1.6y
3
Brockmeyer et al. (2020)
Germany
Single-centre, retrospective study
2012,3–2016,3
391.2d
4
Ahuja et al. (2021)
America
Retrospective multicenter study
2016,1–2017,12
90d
NR
Faroux et al. (2021)
Europe and North America
Retrospective multicenter study
NR
2y
5
Fastner et al. (2021)
Germany
Prospective multicenter study
2014,7–2016,1
More than 1y
NR
Munir et al. (2021)
America
Retrospective multicenter study
2015–2018
NR
6
Benini et al. (2022)
Spain
Single-centre, retrospective study
2011,1–2019,7
567d
7
Michlicka-Klys et al. (2022)
Poland
Single-centre, prospective study
2009–2019
25.56m
8
Ueno et al. (2022)
Japan
Single-centre, retrospective study
2020,6–2022,4
45d
9
Fink et al. (2023)
Germany
Retrospective multicenter study
2006,4–2019,12
405d
10
1 Amplatzer Cardiac Plug; 2 Watchman; 3 Watchman; 4. Amplatzer Cardiac Plug, Amulet, and Watchman, 5. Amplatzer Cardiac Plug, Amulet, Watchman, Lambre,
and Ultrasea device, 6 Watchman 7 ACP, Amulet, Watchman, and Others;8 ACP, Amulet, and Watchman; 9 Watchman; 10 Watchman, Watchman FLX, AMPLATZER
Amulet, Lambre, Wavecrest Device; NR:not report. Table 1. Characteristics of the included studies. 1 Amplatzer Cardiac Plug; 2 Watchman; 3 Watchman; 4. Amplatzer Cardiac Plug, Amulet, and Watchman, 5. Amplatzer Cardiac Plug, Amulet, Watchman, Lambre,
and Ultrasea device, 6 Watchman 7 ACP, Amulet, Watchman, and Others;8 ACP, Amulet, and Watchman; 9 Watchman; 10 Watchman, Watchman FLX, AMPLATZER
Amulet, Lambre, Wavecrest Device; NR:not report. https://doi.org/10.1371/journal.pone.0287928.t001 group was more likely to present with coronary heart disease (60.23% vs. 46.20%, p < 0.01),
hypertension (61.37% vs. 58.27%, p < 0.01), diabetes (32.01% vs. 24.02%, p < 0.01), and
stroke/ transient ischemic attack (TIA) (34.36% vs. 26.94%, p < 0.01). In addition, in the CKD
group, there were higher CHA2DS2-VASc scores and HAS-BLED scores. The patient’s base-
line characteristics are summarized in Table 2. Study characteristics The initial results from our search method returned 647 potentially useful articles (415 articles
from Embase, 151 articles from Web of Science,76 articles from PubMed, and 5 from
Cochrane Library (Fig 1). 458 papers were subjected to title and abstract screening after 189
duplicate articles were eliminated. Twenty-two articles received full text analysis, and 436 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE chronic kidney disease and left atrial appendage closure Fig 1. Flow chart of literature screening. https://doi.org/10.1371/journal.pone.0287928.g001 Fig 1. Flow chart of literature screening. Fig 1. Flow chart of literature screening. https://doi.org/10.1371/journal.pone.0287928.g001 https://doi.org/10.1371/journal.pone.0287928.g001 papers were excluded. 4 studies were single-arm studies, 1 study was disregarded since it used
the same database. 5 of the 22 publications were disregarded because their results were unin-
teresting. Finally, our research included 12 observational studies enrolling a total of 61324
patients who underwent LAAC [10–21]. Four studies utilized the Watchman device, one uti-
lized the Amplatzer Cardiac Plug device, and 7 applied more than 3 types of devices, including
the Watchman, Amplatzer cardiac plug, Amplatzer Amulet, Lambre, or Wavecrest device. The
research’s characteristics are summarized in Table 1. The NOS scores of the studies varied
from 6 to 8 and are shown in S1 Table in S1 File. Compared with the no CKD group, the CKD papers were excluded. 4 studies were single-arm studies, 1 study was disregarded since it used
the same database. 5 of the 22 publications were disregarded because their results were unin-
teresting. Finally, our research included 12 observational studies enrolling a total of 61324
patients who underwent LAAC [10–21]. Four studies utilized the Watchman device, one uti-
lized the Amplatzer Cardiac Plug device, and 7 applied more than 3 types of devices, including
the Watchman, Amplatzer cardiac plug, Amplatzer Amulet, Lambre, or Wavecrest device. The
research’s characteristics are summarized in Table 1. The NOS scores of the studies varied
from 6 to 8 and are shown in S1 Table in S1 File. Compared with the no CKD group, the CKD 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE PLOS ONE chronic kidney disease and left atrial appendage closure Table 2. Characteristics of the included patients. Study
Group
Sample, N
Male, N
(%)
Mean,
Age
CHA2DS2-VASc
score
HAS-BLED
score
HTN, N
(%)
DM, N
(%)
Stroke/TIA, N
(%)
CAD, N
(%)
Kefer et al. (2016)
Non-
CKD
639
425(66.5)
74.9±8.4
4.2±1.6
2.9±1.1
536(83.9)
152(23.8)
264(41.3)
183(28.6)
CKD
356
194(54.5)
78.0±7.4
4.9±1.5
3.4±1.2
321(90.2)
135(37.9)
111(31.2)
162(45.5)
ESRD
19
11(57.9)
76.9±6.6
4.7±1.7
4.3±1.3
13(68.4)
8(42.1)
9(47.4)
9(47.4)
Xue et al. (2018)
Non-
CKD
149
111(74.5)
73.2±7.8
3.4±1.4
3.0±1.0
115(77.2)
29(19.5)
22(14.8)
NR
CKD
151
92(60.9)
77.0±7.2
4.3±1.5
4.0±1.0
123(81.5)
56(37.1)
17(11.3)
NR
Luani et al. (2019)
Non-
CKD
116
74(63.8)
72.1±8.0
3.4±1.6
3.1±0.9
98(84.5)
40(34.5)
21(17.5)
NR
CKD
73
42(57.5)
75.9±6.7
4.5±1.4
3.7±1.0
66(90.4)
45(61.6)
12(16.4)
NR
Brockmeyer, et al. (2020)
Non-
CKD
65
42(64.6)
74.4±7.1
4.1±1.4
3.7±0.8
55(84.6)
19(29.2)
14(21.5)
42(64.6)
CKD
81
42(51.9)
78.2±7.3
4.7±1.3
3.9±0.9
76(93.8)
33(40.7)
14(17.3)
63(77.8)
Ahuja et al. (2021)
Non-
CKD
16749
10214
(61.0)
73.6±9.4
3.6±1.5
NR
10391
(62.0)
4733
(28.3)
NR
7816(56.7)
CKD
3954
2555(64.6)
76.1±8.1
4.2±1.4
NR
2027(51.3)
1855
(46.9)
NR
2426(61.4)
ESRD
571
375(65.7)
69.5±20.9
3.8±1.4
NR
351(61.6)
333(58.3)
NR
358(62.8)
Faroux et al. (2021)
Non-
CKD
794
486(61.2)
75.1±8.4
4.4±1.5
3.5±1.0
669(84.3)
245(30.9)
303(38.2)
238(30.0)
CKD
300
184(61.3)
77.8±8.2
4.9±1.5
4.0±1.1
283(94.3)
134(44.7)
95(31.7)
124(41.3)
Fastner et al. (2021)
Non-
CKD
324
218(67.3)
76(71–80)
4.2±1.5
3.5±1.0
301(92.9)
84(25.9)
105(32.4)
123(38.0)
CKD
284
150(52.8)
NR
4.9±1.4
4.3±1.0
265(93.3)
120(42.3)
75(26.4)
155(54.6)
ESRD
15
12(80.0)
75(69–79)
5.1±1.7
4.6±1.1
14(93.3)
10(66.7)
4(26.7)
11(73.3)
Munir et al. (2021)
Non-
CKD
31405
18005
(57.3)
77(71–82)
4(3–4)
NR
17060
(54.3)
6735
(21.4)
NR
14690
(46.8)
CKD
3545
2285(64.5)
77.5(73–
83)
4(3–4)
NR
2127(60.0)
185(5.2)
NR
2130(60.1)
ESRD
1115
755(67.7)
71(65–78)
3(2–4)
NR
724(65.0)
40(3.6)
NR
730(65.5)
Benini et al. (2022)
Non-
CKD
53
32(60.4)
73(65–78)
4(3–5)
3(3–4)
47(88.3)
11(21.8)
25(59.9)
NR
CKD
71
45(63.4)
77(69–81)
4(3–6)
4(3–4)
65(91.6)
23(32.4)
24(43.6)
NR
Michlicka-Klys, et al. (2022)
Non-
CKD
167
102(61.1)
71.0±9.1
3.7±1.4
2.9±0.7
NR
47(28.1)
37(22.2)
NR
CKD
105
49(46.7)
75.5±7.7
4.9±1.3
3.2±0.9
NR
41(39.0)
27(25.7)
NR
Ueno et al. (2022)
Non-
ESRD
93
61(65.6)
80(75–84)
5(4–6)
3(2–3)
71(76.3)
26(28.0)
39(41.9)
NR
ESRD
25
20(80.0)
73(71–79)
5(4–6)
4(3–5)
16(64.0)
12(48.0)
16(64.0)
NR
Fink et al. Procedural outcomes The LAAC success rates were reported in five studies, and they all failed to discover a connec-
tion between CKD and LAAC success rates (OR: 1.02, 95% CI: 0.33–3.16, p = 0.97 Fig 2). Mod-
ern heterogeneity was present across the analyses (I2 = 58%, p = 0.05). The outcomes are
displayed in Fig 2. In addition, we separately analyzed in-hospital mortality, stroke, pericardial
effect/tamponade, vascular access complications, and major bleeding events associated with
the procedure. As shown in Fig 3, data on in-hospital mortality were provided by eight studies, totaling
60650 patients. CKD increased the risk of in-hospital mortality by 2.84 times according to the
overall meta-analysis (OR: 2.84, 95% CI: 2.12–3.81, p < 0.01). Low heterogeneity was present
across the studies (I2 = 0%, p = 0.71). In addition, we discovered that individuals with ESRD
had an 8.61-fold higher rate of in-hospital mortality than those without ESRD (OR: 8.61, 95%
CI: 5.94–12.48, p < 0.01 I2 = 0%, Fig 5). Seven studies reported stroke cases, and the two groups did not differ significantly (OR:
1.24, 95% CI: 0.86–1.78, p = 0.25, I2 = 0%, Fig 3). In three investigations, acute kidney injury
(AKI) was mentioned. The CKD group had a greater incidence (OR: 4.39, 95% CI: 4.00–4.83,
p <0.01, I2 = 3%, Fig 3). Periprocedural major bleeding events were documented in eight
investigations. The meta-analysis showed a 1.44-fold increased incidence of major bleeding
events in people with CKD (OR: 1.44, 95% CI: 1.29–1.60, p < 0.01, Fig 4). No evidence of het-
erogeneity was found among the studies (I2 = 0%, p = 0.32). In the ESRD group, patients had a
1.6-fold risk of bleeding (OR: 1.63, 95% CI: 1.33–2.01, p < 0.01, I2 = 3%, Fig 5). Vascular access
complications were reported in 5 studies. Between the CKD group and the no CKD group,
there was no statistically significant difference in the frequency of vascular access 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 https://doi.org/10.1371/journal.pone.0287928.g003 chronic kidney disease and left atrial appendage closure Fig 4. Forest plots showing the effect of major bleeding events, pericardial effusion/tamponade and vascular access complications in patients with or
without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g004 Long-term outcomes
Seven studies, with an average follow-up period of 1–2 years, described the outcomes of long-
term follow-up following LAAC. During the follow-up, there was no discernible difference in
the risk of stroke between the CKD group and the no CKD group (OR 1.33; 95% CI 0.53–3.34;
p = 0.55; I2 = 45%; Fig 6). The CKD group had higher major bleeding events during follow-up
than the no CKD group (OR: 1.67; 95% CI: 1.45–1.92; p < 0.01; I2 = 0%; Fig 6). During the fol-
low-up, the CKD group’s all-cause mortality was greater than that of the group without CKD
(OR 3.45; 95% CI 2.01–5.92; p < 0.01; I2 = 69%, Fig 6). Fig 4. Forest plots showing the effect of major bleeding events, pericardial effusion/tamponade and vascular access complications in patients with or
without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g004
PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023
8 / 15 Fig 4. Forest plots showing the effect of major bleeding events, pericardial effusion/tamponade and vascular access complications in patients with or
without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g004 he effect of major bleeding events, pericardial effusion/tamponade and vascular access complications in patients with or
se Fig 4. Forest plots showing the effect of major bleeding events, pericardial effusion/tamponade and vascular access complications in patients with or
without chronic kidney disease. PLOS ONE (2023)
Non-
ESRD
57
34(59.6)
73.5±8.6
4.8±1.6
3.0±1.1
53(93.0)
35(61.4)
14(24.6)
NR
ESRD
57
40(70.2)
73.9±7.4
4.6±1.7
3.5±1.0
54(94.7)
30(52.6)
10(17.5)
NR
HTN: hypertension; DM: diabetes; TIA: transient ischemic attack; CAD: coronary artery disease; CKD: chronic kidney disease; ESRD: end-stage renal diseas Table 2. Characteristics of the included patients. complications (OR: 1.13, 95% CI: 0.91–1.39, p = 0.28 I2 = 0%, Fig 4). Additionally, our research
revealed that pericardial effusion/tamponade in the CKD group was significantly higher than
in the no CKD group (OR 1.30; 95% CI 1.13–1.51, p < 0.01; I2 = 0%, Fig 4). Likewise, patients
with ESRD showed a greater incidence of pericardial effusion/tamponade (OR: 1.54, 95% CI:
1.17–2.03, p < 0.01, I2 = 0% Fig 5). PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 6 / 15 PLOS ONE chronic kidney disease and left atrial appendage closure Fig 2. Forest plots showing the effect of LAAC success rate in patients with or without chronic kidney disease. Fig 2. Forest plots showing the effect of LAAC success rate in patients with or without chronic kidney disease. Fig 2. Forest plots showing the effect of LAAC success rate in patients with or without chronic kidney disease. Fig 2. Forest plots showing the effect of LAAC success rate in patients with or without chronic kidney disease. Fig 3. Forest plots showing the effect of in-hospital mortality, stroke and acute kidney injury in patients with or without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g003 Fig 3. Forest plots showing the effect of in-hospital mortality, stroke and acute kidney injury in patients with or without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g003 Fig 3. Forest plots showing the effect of in-hospital mortality, stroke and acute kidney injury in patients with or without chronic kidney disease. https://doi.org/10.1371/journal.pone.0287928.g003 Fig 3. Forest plots showing the effect of in-hospital mortality, stroke and acute kidney injury in patients with or without chronic kidney disease. https://doi org/10 1371/journal pone 0287928 g003 7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE chronic kidney disease and left atrial appendage closure chronic kidney disease and left atrial appendage closure Fig 5. Forest plots showing the effect of stroke, in-hospital mortality, major bleeding events and pericardial effusion/tamponade in
patients with or without end-stage renal disease. https://doi.org/10.1371/journal.pone.0287928.g005 Fig 5. Forest plots showing the effect of stroke, in-hospital mortality, major bleeding events and pericardial effusion/tamponade in
patients with or without end-stage renal disease. Fig 5. Forest plots showing the effect of stroke, in-hospital mortality, major bleeding events and pericardial effusion/tamponade in
patients with or without end-stage renal disease. https://doi.org/10.1371/journal.pone.0287928.g005 https://doi.org/10.1371/journal.pone.0287928.g005 Long-term outcomes Seven studies, with an average follow-up period of 1–2 years, described the outcomes of long-
term follow-up following LAAC. During the follow-up, there was no discernible difference in
the risk of stroke between the CKD group and the no CKD group (OR 1.33; 95% CI 0.53–3.34;
p = 0.55; I2 = 45%; Fig 6). The CKD group had higher major bleeding events during follow-up
than the no CKD group (OR: 1.67; 95% CI: 1.45–1.92; p < 0.01; I2 = 0%; Fig 6). During the fol-
low-up, the CKD group’s all-cause mortality was greater than that of the group without CKD
(OR 3.45; 95% CI 2.01–5.92; p < 0.01; I2 = 69%, Fig 6). Seven studies, with an average follow-up period of 1–2 years, described the outcomes of long-
term follow-up following LAAC. During the follow-up, there was no discernible difference in
the risk of stroke between the CKD group and the no CKD group (OR 1.33; 95% CI 0.53–3.34;
p = 0.55; I2 = 45%; Fig 6). The CKD group had higher major bleeding events during follow-up
than the no CKD group (OR: 1.67; 95% CI: 1.45–1.92; p < 0.01; I2 = 0%; Fig 6). During the fol-
low-up, the CKD group’s all-cause mortality was greater than that of the group without CKD
(OR 3.45; 95% CI 2.01–5.92; p < 0.01; I2 = 69%, Fig 6). 8 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE chronic kidney disease and left atrial appendage closure PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 chronic kidney disease and left atrial appendage closure chronic kidney disease and left atrial appendage closure Fig 6. Forest plot showing the effect of left atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. https://doi.org/10.1371/journal.pone.0287928.g006 Discussion
The following are the key conclusions of our study: 1) the current study shows that patients
with and without CKD experience similar procedure success rates. In the perioperative period,
the CKD and non-CKD groups had similar risks of stroke and vascular access complications. 2) patients with CKD tended to have more periprocedural pericardial effect/tamponade, major
bleedings, in-hospital mortality, and AKI after LAAC. 3) the data also revealed that after a long
follow-up, the stroke rates for patients with and without CKD were the same. Fig 6. Forest plot showing the effect of left atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. https://doi.org/10.1371/journal.pone.0287928.g006
PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023
10 / 15 Fig 6. Forest plot showing the effect of left atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. Fig 6. Forest plot showing the effect of left atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. https://doi.org/10.1371/journal.pone.0287928.g006 Fig 6. Forest plot showing the effect of left atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. eft atrial appendage closure on the overall risk of stroke, bleeding, and mortality in the long-term follow up. Sensitivity analysis To determine how each study’s removal influenced the results, we carried out a sensitivity
analysis. An overview of the outcomes of the sensitivity analysis is given in S2 Table in S1 File. The result of pericardial effusion/tamponade between the CKD and no CKD groups exhibited
poor stability in the sensitivity analysis, which was done by deleting the greatest sample size. After excluding the study by Munir et al., the pericardial effusion/tamponade rates were com-
parable between CKD and no CKD (OR: 1.44, 95% CI: 0.69–3.00, p = 0.34). The remaining
endpoints showed good stability in the sensitivity analysis. Given the number of studies, we
included in our analysis, we did not identify publication bias. 9 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 PLOS ONE Discussion The following are the key conclusions of our study: 1) the current study shows that patients
with and without CKD experience similar procedure success rates. In the perioperative period,
the CKD and non-CKD groups had similar risks of stroke and vascular access complications. 2) patients with CKD tended to have more periprocedural pericardial effect/tamponade, major
bleedings, in-hospital mortality, and AKI after LAAC. 3) the data also revealed that after a long
follow-up, the stroke rates for patients with and without CKD were the same. PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 10 / 15 PLOS ONE chronic kidney disease and left atrial appendage closure It is still challenging to treat patients with AF who have CKD or ESRD. Compared to those
without CKD, patients with AF and CKD had a higher risk of stroke and bleeding incidents
[5]. Patients with ESRD and AF provide additional clinical challenges because there is little evi-
dence to show a reduction in thromboembolic events associated with OACs while bleeding
incidents are still common [22, 23]. Our research revealed that the CKD group had an almost 3-fold higher in-hospital mortality
rate, while the risk of in-hospital mortality increased 8-fold in patients with ESRD. In the gen-
eral population, CKD is independently linked to death [24]. AF and CKD are closely related dis-
eases with a shared set of risk factors such as coronary artery disease and hypertension [25, 26]. Additionally, CKD and ESRD have been linked to higher mortality and morbidity rates in
patients undergoing percutaneous coronary intervention and transcatheter aortic valve replace-
ment [27, 28]. Prior small-scale studies found no differences between patients with and without
CKD in in-hospital mortality. These studies, however, had very few or no patients with ESRD
and their sample sizes were constrained [13, 15, 18, 20]. According to our findings, in-hospital
mortality was significantly higher in CKD/ESRD patients than in patients without CKD/ESRD
patients. Based on other studies of structural heart interventions, these people have a higher
chance of dying from cardiac interventions because they are sicker at the start of treatment [27,
28]. Regrettably, no specific cause of death was given in our included studies. After LAAC, patients with CKD experienced AKI more frequently than patients without
CKD. To see the LAA before and after closure during LAAC, contrast is routinely used. Approximately 10% to 13% of patients undergoing LAAC have reported AKI [29–31]. Discussion AKI
after LAAC has previously been reported to be independently correlated with CKD [31]. Fur-
thermore, AKI after LAAC may cause thromboembolic effects, such as stroke. To prevent
AKI, efforts should be made to maximize protect renal function, reduce contrast volume, and
prevent sudden hemodynamic changes like hypotension during surgery. Patients with CKD
experienced a greater incidence of bleeding events in our study during the perioperative
period. Patients with AF and concurrent CKD are more likely to experience bleeding events. A
significant retrospective cohort study with 516197 patients indicated that for every 10 mL/
min/1.73 m2 decrease in eGFR, the chance of hemorrhaging increased by 9% [32]. Compared
to patients without CKD, those with CKD had a slightly higher HAS-BLED score. The higher
overall bleeding risk may help to explain some of the higher bleeding rates. Therefore, our
study underscores the significance of the link between renal disorders and stroke risk in
patients with AF and extends these findings to patients with CKD receiving LAAC. In addi-
tion, we found a higher incidence of pericardial effusion/ tamponade in CKD patients, but
after removing the largest study by sensitivity analysis, the results were found to change, and
the incidence between the two groups was not significantly different. Further studies may be
needed in the future. However, in patients with ESRD, we found a higher incidence of pericar-
dial effusion/ tamponade than in no ESRD patients. CKD patients are more likely to experience thromboembolic events [33]. Despite patients
with CKD having a considerably increased risk of stroke as indicated by their CHA2DS2-VASc
score compared to those without CKD, we observed comparable rates of stroke in both groups
following the effective LAAC. Furthermore, we discovered a 96.4% decrease in the risk of
ischemic stroke in the CKD group and a 91.8% decrease in the no CKD group when compared
to the projected risk based on the data released by Lip et al.[34] These results all indicate that
LAAC is effective in preventing stroke in patients with CKD. During long-term follow-up,
CKD patients had more bleeding events. More than half of the research we included was gas-
trointestinal hemorrhage. In fact, regardless of other variables like antithrombotic medication,
the risk of gastrointestinal bleeding rises as eGFR declines [35]. PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 Discussion In individuals with unex-
plained gastrointestinal bleeding, CKD is also linked to a higher risk of recurrent bleeding PLOS ONE | https://doi.org/10.1371/journal.pone.0287928
October 26, 2023 11 / 15 PLOS ONE chronic kidney disease and left atrial appendage closure [36]. The greater rate of bleeding seen in these patients, particularly at the start of follow-up,
may be related to the fact that LAAC requires anticoagulant medication for at least three
months following the LAAC [37]. According to several studies, three months after LAAC,
approximately 50% of bleeding incidents occur [20, 38]. During the follow-up period, the
CKD group had a higher incidence of all-cause mortality. This could be explained in part by
the fact that CKD patients were older and had more comorbid conditions than non-CKD
patients, particularly cardiovascular disorders. Furthermore, a study has shown, that patients
with CKD already have a lower life expectancy than the general population [24]. The studies we included spanned from 2016 to 2023, and there is no uniform regulation on
anticoagulation after LAAC. Among the studies we included, 6 studies gave detailed anticoa-
gulation recommendations after LAAC [10–12, 14, 20, 21]. But only one study gave the num-
ber of people using each anticoagulant or antiplatelet drug. In Fink et al. ’s study, there was a
higher rate of use of dual antiplatelet (84.2% vs. 64.9%) and a lower rate use of anticoagulant
(12.3% vs.35.1%) in the end-stage renal disease (ESRD) group compared with the non-ESRD
group [12]. Supporting information
S1 Checklist.
(DOCX) S1 File. Search strategies. (DOCX) S1 File. Search strategies. (DOCX) Limitations First, because the included studies are observational, confounders and bias risk do exist. Despite having the most reliable data, any conclusions reached depend greatly on the bias risk
of the individual research and should be treated with caution. Second, we lacked access to indi-
vidual patient data, and our use of the available summary data from published research was
constrained. Third, we found a higher incidence of pericardial effusion in CKD patients, but
after removing the largest study by sensitivity analysis, the results were found to change, and
the incidence between the two groups was not significantly different. Further studies may be
needed in the future. Conclusions In conclusion, although the success rates of LAAC are comparable between the two groups,
CKD patients who receive LAAC have a greater risk of in-hospital mortality, AKI, and bleed-
ing events than those without CKD, especially in patients with ESRD. No significant difference
in the risk of stroke was found in the long-term follow-up after LAAC between the two groups,
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Liquid Crystal Phase Behaviour of Attractive Disc-Like Particles
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International journal of molecular sciences
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Int. J. Mol. Sci. 2013, 14, 16414-16442; doi:10.3390/ijms140816414 Int. J. Mol. Sci. 2013, 14, 16414-16442; doi:10.3390/ijms140816414 OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms Article
Liquid Crystal Phase Behaviour of Attractive Disc-Like Particles
Liang Wu, George Jackson and Erich A. M¨uller *
Department of Chemical Engineering, Imperial College London, South Kensington Campus,
London SW7 2AZ, UK; E-Mails: l.wu09@imperial.ac.uk (L.W.); g.jackson@imperial.ac.uk (G.J.)
* Author to whom correspondence should be addressed; E-Mail: e.muller@imperial.ac.uk;
Tel.: +44-0-20-7594-1569. Received: 25 June 2013; in revised form: 24 July 2013 / Accepted: 25 July 2013 /
Published: 8 August 2013 1. Introduction Liquid crystals [1–3] are intermediate (meso) phases with characteristics that lie between the fully
positionally- and orientationally-ordered crystal and the disordered liquid states. Since their discovery
by Reinitzer in 1883 [4], liquid crystals (LCs) have attracted a longstanding research interest, because of
their unique thermodynamic, structural, optical and electronic properties. An essential requirement for
the stabilization of an LC phase is that molecules be highly anisometric in shape. A wide variety of oblate
disc-shaped LC particles with length scales ranging from tens of ˚Angstr¨om (molecular dimensions) to
hundreds of nanometers (macromolecular/colloidal dimensions) have been observed to exhibit rich phase
behaviour, including isotropic, nematic and columnar phases. Discotic LCs tend to comprise poly-aromatic cores of oblate geometry, e.g., hexa-alkanoyloxy
benzenes,
or metal organic complexes of phenyl pyridines,
porphyrazines,
phthalocyanines,
triphenylenes, etc. [2,3,5–7]. These relatively low molecular weight compounds are commonly
referred to as thermotropic liquid crystals, owing to the leading role temperature plays in the phase
transformations between the various states. Colloidal particles of discotic geometry have also been
extensively studied in recent years, including nickel hydroxide theophrastite sheets, aluminum hydroxide
gibbsite platelets and nontronite or laponite mineral clays [8–16]. The stability of the colloidal
anisotropic phases are commonly governed by the solute concentration, a characteristic that leads to
the classification of such systems as lyotropic LCs. An interesting intermediate class of naturally
occurring discotic particles includes asphaltenes, polyaromatic compounds of intermediate molecular
weight (500 to 10,000 g/mol) found in heavy crude oils, which aggregate and precipitate from solution
over particular ranges of composition, temperature and pressure [17]. As a result of their oblate rigid
cores, these asphaltenic systems exhibit anisotropic LC phases with both thermotropic and lyotropic
characteristics [18–22]. In computer simulation studies [23–36] and theoretical treatments [37–46], simple disc-like particles
are often taken as prototypical models of the oblate LC molecules; the discs are usually characterized
by their aspect ratio, D/L, where L is the thickness of the disc and D is its diameter. Hard disc-like
particles have proven to be appropriate models for lyotropic colloidal LCs. The theoretical treatment of hard non-spherical fluids dates back to the 1940s [8]. The Onsager
view that repulsive (excluded volume) interactions are of key importance in the formation of
orientationally ordered phases is now widely accepted. Liang Wu, George Jackson and Erich A. M¨uller * Department of Chemical Engineering, Imperial College London, South Kensington Campus,
London SW7 2AZ, UK; E-Mails: l.wu09@imperial.ac.uk (L.W.); g.jackson@imperial.ac.uk (G.J.)
* Author to whom correspondence should be addressed; E-Mail: e.muller@imperial.ac.uk;
Tel.: +44-0-20-7594-1569. Received: 25 June 2013; in revised form: 24 July 2013 / Accepted: 25 July 2013 /
Published: 8 August 2013 Received: 25 June 2013; in revised form: 24 July 2013 / Accepted: 25 July 2013 /
Published: 8 August 2013 Abstract:
We employ a generalized van der Waals-Onsager perturbation theory to
construct a free energy functional capable of describing the thermodynamic properties and
orientational order of the isotropic and nematic phases of attractive disc particles. The
model mesogen is a hard (purely repulsive) cylindrical disc particle decorated with an
anisotropic square-well attractive potential placed at the centre of mass. Even for isotropic
attractive interactions, the resulting overall inter-particle potential is anisotropic, due to the
orientation-dependent excluded volume of the underlying hard core. An algebraic equation
of state for attractive disc particles is developed by adopting the Onsager trial function
to characterize the orientational order in the nematic phase. The theory is then used to
represent the fluid-phase behaviour (vapour-liquid, isotropic-nematic, and nematic-nematic)
of the oblate attractive particles for varying values of the molecular aspect ratio and
parameters of the attractive potential. When compared to the phase diagram of their athermal
analogues, it is seen that the addition of an attractive interaction facilitates the formation of
orientationally-ordered phases. Most interestingly, for certain aspect ratios, a coexistence
between two anisotropic nematic phases is exhibited by the attractive disc-like fluids. Keywords:
equation of state; discotics; attractive cylindrical disc; nematic-nematic
equilibria; anisotropic square well; phase diagrams Int. J. Mol. Sci. 2013, 14 16415 1. Introduction In his pioneering work on nematic LCs [8],
Onsager proposed the now well-accepted free energy functional for nematic states and demonstrated that
the isotropic-nematic phase transition can be driven by entropy alone. Entropy-driven phase transitions
have now been studied extensively by computer simulation of hard-body anisometric particles, where
both nematic (discotic) and columnar ordering has been identified in hard-disc models upon increasing
the density of the system [23–25,27,28,33–36,47]. For a proper description of the temperature dependence of the thermodynamic properties of a system,
both repulsive and attractive inter-molecular interactions have to be considered on an equal footing. This
is in line with a van der Waals description of fluids, where the hard core is treated as the reference in
determining the fluid structure [48,49], and a mean-field attraction is included to account for the cohesive
interactions and fluid-phase equilibria. A simple hard-core model which incorporates the attractive Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 16416 interactions therefore offers promise in representing the phase behaviour of thermotropic LCs. A number
of studies have extended the early work of Kimura [50], who combined Onsager’s hard-rod model with
anisotropic dispersion forces (e.g., see [51–60]). Some further progress has been made in understanding
the effect of generalized attractive potentials [60–64] and dipolar [65–70] or chiral [71–73] interactions
on the stability of the various LC phases. As has already been mentioned, purely repulsive discotic particles have been shown to exhibit
columnar phases in the higher density region. This was first demonstrated by Veerman and
Frenkel [25,74] with simulations of the hard cut-sphere system; columnar order was then subsequently
found in systems of oblate hard spherocylinders [32]. In a previous paper [46], we coupled a generic
equation of state for the nematic phase of hard disc-like particles with a cell theory [39,44] to describe
the boundaries of the isotropic, nematic, and columnar phases of the hard cut spheres. The transition
from a nematic to a columnar state is observed at packing fractions of about 40%, and the coexistence
densities are found to be relatively insensitive to the aspect ratio of the discs (apart, perhaps, in the
limit of infinitely thin particles). This means that for hard discs in dense states above a ∼40% packing
fraction, the columnar phase (and other positionally ordered phases such as the crystalline solid) will
be stable relative to the nematic state. 1. Introduction A spherical attractive interaction of range
λD is represented as the dashed sphere. Figure 1. The attractive cylindrical disc (ACD) model. The model is characterized by the
thickness, L, and diameter, D, of the hard core. A spherical attractive interaction of range
λD is represented as the dashed sphere. D
L
D
2. Theory
2.1. Generalized van der Waals-Onsager Free Energy Functional D 1. Introduction In the case of discs with attractive interactions, one may also
expect an additional stabilization of the columnar phase, depending on the nature of the attractions; e.g.,
oblate Gay-Berne particles exhibit a deep energetic minimum when the particles are close together in a
face-to-face parallel relative orientation, leading to an enhanced stability of the columnar structure [75]
compared to the lack of a columnar phase for purely repulsive oblate ellipsoids [76]. For our model hard
discs with square-well attractions, however, the nature of the enveloping square well is such that multiple
energetic contacts are possible when the particles are not directly over one another, so that the energetic
gain from a columnar geometry is not as evident (cf. the work of del Rio et al. [63] for disc particles
with large ranges of the attractive well). As a consequence, we would not expect discs with enveloping
attractive wells to exhibit columnar phases for states of low to moderate density, 0 < η < 0.35, though
caution has to be exercised in assessing the stability of the nematic phase for the higher density states. It
would be interesting to simulate such systems to confirm this, but such an analysis is beyond the scope
of the current work. Of particular importance here is the closed-form algebraic equation of state (EOS) developed for
attractive hard spherocylinders [77,78], where the attractive potential is expanded in spherical harmonics
representing different contributions to the anisotropic attractions [79]. We develop a model for attractive
oblate (cylindrical-disc) mesogens by adding an anisotropic square-well (SW) potential, which mediates
the intermolecular attractions, to a hard-cylindrical disc. The model of attractive discs is depicted in
Figure 1, where the parameter λ is introduced to quantify the attractive range. A perturbation approach
is applied to develop a free energy functional for the attractive disc system. By using the Onsager trial
function to describe the degree of orientational order in the nematic phase, the free energy and equation of
state for the nematic state can be expressed in closed algebraic form, which also allows a straightforward
superimposition of contributions from intermolecular interactions such as chiral, dipolar and associating
interactions, or the extension to other types of dispersive intermolecular potentials (e.g., Lennard-Jones,
Mie, Yukawa). Int. J. Mol. Sci. 2013, 14 16417 Figure 1. The attractive cylindrical disc (ACD) model. The model is characterized by the
thickness, L, and diameter, D, of the hard core. 2. Theory 2.1. Generalized van der Waals-Onsager Free Energy Functional In this section, we describe the perturbation theory [49,80] employed to construct the free energy
for the nematic and isotropic phases of attractive non-spherical particles. A system of N particles in a
volume V at a temperature T is considered, where the total Helmholtz free energy, A, is divided into two
contributions, a reference term and a perturbation term. The former is assumed to be known precisely,
and the latter represents the contribution from the attractive potential perturbation: A
NkT = Aref
NkT + Aatt
NkT
(1) (1) Here, k is the Boltzmann constant, and the reference contribution, Aref, is taken from an expression for
the hard-core particles. Following the Parsons-Lee approach (PL) [81–83], the reference repulsive term
can be expressed in compact form as: Here, k is the Boltzmann constant, and the reference contribution, Aref, is taken from an expression for
the hard-core particles. Following the Parsons-Lee approach (PL) [81–83], the reference repulsive term
can be expressed in compact form as: Aref
NkT = Aid
iso
NkT +
Z
f(⃗ω) ln {4πf(⃗ω)}d⃗ω + G(η)
ZZ
Vexc(⃗ω1, ⃗ω2)f(⃗ω1)f(⃗ω2)d⃗ω1d⃗ω2
(2) (2) where Aid
iso is the ideal contribution to the free energy of the isotropic state (incorporating the translational
and rotational kinetic terms), ⃗ω is the orientation vector of a particle, and f(⃗ω) is the single-particle
orientational distribution function. In the Parsons-Lee approach, the contributions from the high-order
virial terms are incorporated in an effective manner by scaling the corresponding hard-sphere equation of
state [84,85]. The density-dependent function, G(η), represents the residual free energy of the equivalent
hard-sphere system: G(η) =
4η −3η2
8Vm(1 −η)2
(3) (3) where η = ρVm is the packing fraction, ρ is the single-particle density and Vm is the molecular volume. Alternatively, a improved generic equation of state for hard disc-like particles developed in [46] can be
chosen to describe the reference system. where η = ρVm is the packing fraction, ρ is the single-particle density and Vm is the molecular volume. Alternatively, a improved generic equation of state for hard disc-like particles developed in [46] can be
chosen to describe the reference system. Int. J. Mol. Sci. 2. Theory 2013, 14 16418 In
the
high-temperature
limit,
the
attractive
free
energy
can
be
approximated
at
first
order [49,77,80] by: In
the
high-temperature
limit,
the
attractive
free
energy
can
be
approximated
at
first
order [49,77,80] by: Aatt =
U att
ref
(4) (4) where < · · · >ref represents an ensemble average over all configurations of the reference system. In the
canonical ensemble, the mean-attractive energy is formally written as: where < · · · >ref represents an ensemble average over all configurations of the reference system. In the
canonical ensemble, the mean-attractive energy is formally written as: Aatt
≈
U att
ref
=
1
ZNV T
ZZ
d⃗rNd⃗ωNU att(⃗rN, ⃗ωN) exp
−U ref(⃗rN, ⃗ωN)
kT
(5) ref
=
1
ZNV T
ZZ
d⃗rNd⃗ωNU att(⃗rN, ⃗ωN) exp
−U ref(⃗rN, ⃗ωN)
kT
(5) (5) with the configurational integral defined as: ZNV T =
ZZ
d⃗rNd⃗ωN exp
−U ref(⃗rN, ⃗ωN)
kT
(6) (6) which is a function of the positions, ⃗rN, and orientations, ⃗ωN, of all N particles. Further
simplification is achieved by assuming pairwise additive interactions, such that U att(⃗rN, ⃗ωN) =
PN
i=1
PN
j>i uatt
ij (⃗rij, ⃗ωi, ⃗ωj), where the vector ⃗rij = ⃗ri−⃗rj, and introducing the pair distribution function
of reference system, gref (⃗r12, ⃗ω1, ⃗ω2), defined as: gref(⃗r12, ⃗ω1, ⃗ω2)
=
ρref
12 (⃗r12, ⃗ω1, ⃗ω2)
ρref
1 (⃗r1, ⃗ω1)ρref
1 (⃗r2, ⃗ω2)
=
N(N −1)
ρref
1 (⃗r1, ⃗ω1)ρref
1 (⃗r2, ⃗ω2)
×
1
ZNVT
Z
d⃗rN−2d⃗ωN−2 exp
−U ref(⃗rN, ⃗ωN)
kT
(7) ρ1 ( 1,
1)ρ1 ( 2,
2)
×
1
ZNVT
Z
d⃗rN−2d⃗ωN−2 exp
−U ref(⃗rN, ⃗ωN)
kT
(7) (7) The attractive term can thus be re-written as a sum of pair contributions. When considering a nematic
phase where the distribution of particle positions is uniform, the single-particle density of the system can
be factorised as ρref(⃗ri, ⃗ωi) = ρf(⃗ωi), where the single-particle orientation distribution function, f(⃗ω),
has been introduced. On inserting Equation (7) into Equation (5) and using the nematic form of the
single-particle density, one can write: The attractive term can thus be re-written as a sum of pair contributions. When considering a nematic
phase where the distribution of particle positions is uniform, the single-particle density of the system can
be factorised as ρref(⃗ri, ⃗ωi) = ρf(⃗ωi), where the single-particle orientation distribution function, f(⃗ω),
has been introduced. 2. Theory On inserting Equation (7) into Equation (5) and using the nematic form of the
single-particle density, one can write: Aatt
=
1
2
ZZZZ
uatt
12 (⃗r12, ⃗ω1, ⃗ω2) gref (⃗r12, ⃗ω1, ⃗ω2)
×
ρref(⃗r1, ⃗ω1)ρref(⃗r2, ⃗ω2) d⃗r1d⃗ω1d⃗r2d⃗ω2
=
ρ2V
2
ZZZ
uatt
12 (⃗r12, ⃗ω1, ⃗ω2)
×
f(⃗ω1)f(⃗ω2)gref (⃗r12, ⃗ω1, ⃗ω2) d⃗r12d⃗ω1d⃗ω2
(8) (8) Since the particles of interest are non-spherical, both the attractive interaction and the excluded
volume are complicated functions of the relative positions and orientations of particles [79,86]. It is thus
useful to expand the pair potential as a series in spherical harmonics. Here, we consider an anisotropic
square-well (ASW) potential of the form [77,79]: Since the particles of interest are non-spherical, both the attractive interaction and the excluded
volume are complicated functions of the relative positions and orientations of particles [79,86]. It is thus
useful to expand the pair potential as a series in spherical harmonics. Here, we consider an anisotropic
square-well (ASW) potential of the form [77,79]: uatt(⃗r12, ⃗ω1, ⃗ω2)
=
−s(r12)[ϵ0 + ϵ2P2(cos γ)]
(9)
s(r12)
=
1
σ(ˆ⃗r12, ⃗ω1, ⃗ω2) ≥r12 < λD
0
r12 ≥λD
(10) (9) (10) Int. J. Mol. Sci. 2013, 14 16419 Int. J. Mol. Sci. 2013, 14 where λ is the range parameter, D is the reference diameter of the disc, and P2(cos γ) is the second
Legendre polynomial for the relative orientation, γ = arccos(⃗ω1 · ⃗ω2), between the principle axes of the
two discs. In order to make the approach tractable, we use the low-density limit to approximate the pair
distribution function of the reference system: lim
ρ→0 gref (⃗r12, ⃗ω1, ⃗ω2) = exp
−uref (⃗r12, ⃗ω1, ⃗ω2)
kT
(11) (11) where uref (⃗r12, ⃗ω1, ⃗ω2) is the pair interaction of the reference system. Since the reference system
corresponds to the hard-core particles, uref (⃗r12, ⃗ω1, ⃗ω2) is a purely repulsive interaction, which is infinity
when hard cores overlap, and zero otherwise. 2. Theory Introducing the ASW attractive potential in Equation (8)
with the approximation of Equation (11) and integrating out the centre-to-centre distance between
particle 1 and 2, one can express the free energy perturbation as [77,78]: Aatt
NkT
=
−ρϵ0
2kT
4π
3 λ3D3 −⟨Vexc(γ)⟩⃗ω1,⃗ω2
−
ρϵ2
2kT
4π
3 λ3D3 ⟨P2(cos γ)⟩⃗ω1,⃗ω2 −⟨Vexc(γ)P2(cos γ)⟩⃗ω1,⃗ω2
(12) (12) where the double orientational average of a quantity J(⃗ω1, ⃗ω2) is defined as: ⟨J(⃗ω1, ⃗ω2)⟩⃗ω1,⃗ω2 =
ZZ
J(⃗ω1, ⃗ω2)f(⃗ω1)f(⃗ω2)d⃗ω1d⃗ω2
(13) (13) Combining the separate contributions, the free energy of the full system is obtained in terms of angle
averages of the configurational contributions: A[f(⃗ω)]
NkT
=
Aid
iso
NkT +
Z
f(⃗ω) ln [Ωf(⃗ω)] d⃗ω + G(η) ⟨Vexc(γ)⟩⃗ω1,⃗ω2
−
ρϵ0
2kT
4π
3 λ3D3 −⟨Vexc(γ)⟩⃗ω1,⃗ω2
−
ρϵ2
2kT
4π
3 λ3D3 ⟨P2(cos γ)⟩⃗ω1,⃗ω2 −⟨Vexc(γ)P2(cos γ)⟩⃗ω1,⃗ω2
(14) (14) In this expression for the free energy, one can recognize the coupling between the repulsive and attractive
contributions of the pair potential. The excluded volume is seen in both the terms corresponding to
the isotropic and the anisotropic attractive contributions; the last term in ⟨Vexc(γ)P2(cos γ)⟩⃗ω1,⃗ω2 also
constitutes a direct coupling of the two contributions. The single-particle orientational distribution
function in the isotropic state is constant, fiso(⃗ω) = 1/4π, which simplifies the orientational integrals of
Equation (14). In the anisotropic phases, the equilibrium orientational distribution of the molecules can
be found by minimizing the total free energy functional, A [f(⃗ω)]. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential We start by examining the angle averages of the purely repulsive contributions, i.e., the excluded
volume term. The excluded volume, Vexc(γ), of hard cylinders can be expressed conveniently as a power
series in sin γ:
∞ Vexc(γ) =
∞
X
i=0
Ci sini γ
(15) (15) Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 16420 where the coefficient, Ci, depends on the specific geometry of molecules. It had been shown that an
approximate expression, which is obtained by truncation of the series at the fourth-order term in sin4 γ,
is sufficient to faithfully reproduce exact numerical results [46]. The excluded volume of two hard
cylinders can be accurately represented as [46,87]: Vexc(γ)
Vm
= C∗
0 + C∗
1 sin γ + C∗
2 sin2 γ + C∗
4 sin4 γ
(16) (16) ere the coefficients are given by: where the coefficients are given by: where the coefficients are given by: C∗
0
=
8,
C∗
1 = 2D
L + 8
π
L
D
C∗
2
=
3π
4 + 8
π −7, C∗
4 = (4 −3π)/16
(17) (17) and the molecular volume of a cylindrical disc is Vm = πLD2/4. Using the notation of Equation (13),
the double orientational average of the excluded volume can be written as: Vexc(γ)
Vm
⃗ω1,⃗ω2
= C∗
0 + C∗
1 ⟨sin γ⟩⃗ω1,⃗ω2 + C∗
2
sin2 γ
⃗ω1,⃗ω2 + C∗
4
sin4 γ
⃗ω1,⃗ω2
(18) (18) The equilibrium state corresponds to the minimum of the total free energy, A[f(⃗ω)], with respect to
the orientational distribution function under the additional constraint that the orientational distribution
function is normalised:
δ
Z The equilibrium state corresponds to the minimum of the total free energy, A[f(⃗ω)], with respect to
the orientational distribution function under the additional constraint that the orientational distribution
function is normalised:
δ
Z δ
δf(⃗ω)(A[f(⃗ω)] −λL
Z
f(⃗ω)d⃗ω) = 0
(19) (19) where λL is a Lagrange undetermined multiplier, which ensures that
R
f(⃗ω)d⃗ω = 1. The minimum
condition leads to a self-consistent integral equation for f(⃗ω), which can be solved by various numerical
methods, including the expansion of the orientational distribution function as a spherical harmonic
series [88,89] or use of Monte Carlo annealing techniques [90]. These approaches do not, however,
provide an analytical solution for the equilibrium orientational distribution function. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential In his seminal
publication [8], Onsager proposed a functional form for f(⃗ω) in terms of a parameter α, which
characterizes the degree of orientational order. This so-called Onsager trial function (OTF) fOTF(⃗ω)
is written in hyperbolic form: fOTF(⃗ω) = α cosh(α cos θ)
4π sinh(α)
(20) (20) and is seen to depend on the parameter α, and the polar angle θ = arccos(⃗ω · ⃗ω0), where ⃗ω0 is the
director of the nematic phase. One should note that when α →0, fOTF(⃗ω) naturally reduces to 1/4π,
corresponding to the expected distribution in the isotropic phase. Large values of α (∼10) correspond to
a nematic phase. On integrating the OTF over all possible orientations, one can show that it is correctly
normalised; thus, it is not necessary to include the term in the multiplier, λL, in Equation (19) when
incorporating the OTF. The details of calculations of the orientational averages using the OTF have been
discussed in detail in earlier publications [78,91]. The introduction of the OTF to describe the degree Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential 2013, 14 16421 of orientational order of the nematic phase allows the orientational averages to be rendered in algebraic
form, which is more tractable in practice [77,78,91]: ⟨sin γ⟩⃗ω1,⃗ω2
≈
√π
1
α1/2 −
15
16α3/2
+ O(1/α5/2)
(21)
sin2 γ
⃗ω1,⃗ω2
≈
4
α + O(1/α2)
(22)
sin3 γ
⃗ω1,⃗ω2
≈
√π 6
α3/2 + O(1/α5/2)
(23)
sin4 γ
⃗ω1,⃗ω2
≈
0 + O(1/α2)
(24) (21) (22) sin3 γ
⃗ω1,⃗ω2
≈
√π 6
α3/2 + O(1/α5/2)
(23)
sin4 γ
⃗
⃗
≈
0 + O(1/α2)
(24) (23) α
sin4 γ
⃗ω1,⃗ω2
≈
0 + O(1/α2)
(24) (24) Using Equations (21)–(24) in Equation (18) for the excluded volume and P2(sin γ) = 1 −3 sin2 γ/2
for the anisotropic attractive term, the contributions to configurational free energy can be expressed as: Using Equations (21)–(24) in Equation (18) for the excluded volume and P2(sin γ) = 1 −3 sin2 γ/2
for the anisotropic attractive term, the contributions to configurational free energy can be expressed as: ⟨P2(sin γ)⟩⃗ω1,⃗ω2
≈
1 −6
α
(25)
Vexc(γ)
Vm
⃗ω1,⃗ω2
≈
C∗
0 + C∗
1
√π
1
α1/2 −
15
16α3/2
+ C∗
2
4
α
(26)
Vexc(γ)
Vm
P2(sin γ)
⃗ω1,⃗ω2
≈
C∗
0 + C∗
1
√π
1
α1/2 −
15
16α3/2
+
C∗
2 −3
2C∗
0
4
α −
9
α3/2C∗
1
√π
(27) (25) (26) (27) Collecting all of the terms, one obtains the Helmholtz free energy for the system of attractive cylinder
disc (ACD) in terms of the orientational parameter α: Anem(α)
NkT
=
Aid
iso
NkT + ln α −1 + G(η)
C∗
0 + C∗
1
√π
1
α1/2 −
15
16α3/2
+ C∗
2
4
α
−
ρVm
2
ϵ0
kT
( 4π
3
D3
Vm
−C∗
0
1 + ϵ2
ϵ0
−C∗
1
√π
1 + ϵ2
ϵ0
α−1/2
−
4C∗
2
1 + ϵ2
ϵ0
+ 6ϵ2
ϵ0
4π
3
D3
Vm
−C∗
0
α−1
+
15
16C∗
1
√π
1 + 53
5
ϵ2
ϵ0
α−3/2
) (28) Using the OTF to represent the orientational order of the nematic phase, the free energy is expressed
in an explicit algebraic form. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential As a result, the functional variation of A[f(⃗ω)] with respect to f(⃗ω) can be
simplified to a derivative of the free energy (Equation (28)) with respect to the orientational parameter
α. Taking the derivative of the free energy with respect to α results in a cubic expression for x = √α: ∂
∂α
A(α)
NkT = 1
x5
x3 + a2x2 + a1x + a0
= 0
(29) (29) where the coefficients are given by: a0
=
45
32C∗
1
√π
G(η) + ρVm
2
ϵ0
kT
1 + 53
5
ϵ2
ϵ0
(30)
a1
=
−4C∗
2G(η) −ρVm
2
ϵ0
kT
4C∗
2
1 + ϵ2
ϵ0
+ 6ϵ2
ϵ0
4π
3
D3
Vm
−C∗
0
(31)
a2
=
−C∗
1
√π
2
G(η) + ρVm
2
ϵ0
kT
1 + ϵ2
ϵ0
(32) (30) (31) (32) Int. J. Mol. Sci. 2013, 14 16422 The equilibrium value of α is determined by solving the cubic equation, the roots of which can be cast
in a trigonometric form: The equilibrium value of α is determined by solving the cubic equation, the roots of which can be cast
in a trigonometric form: αj = 1
9
(
a2 −2
q
a2
2 −3a1 cos
2jπ
3
+ 1
3 arccos −27
a0 −1
3a1a2 + 2
27a3
2
2 [a2
2 −3a1]3/2
!)2
(33) (33) where j = 0, 1, 2 represent the three solutions to Equation (29). The value of αj, describing the
equilibrium orientational distribution of the nematic phase, corresponds to the largest root, (j = 0):
αeq = α0. The expressions for the chemical potential, µnem, and the compressibility factor (equation of state),
Znem = PV/(NkT), of the nematic phase can be derived from standard thermodynamic relationships,
µ = (∂A/∂N)V,T and P = −(∂A/∂V )N,T: µnem
kT
=
µid
iso
kT + ln ρ + ln αeq −1 + 1
8
µres
hs
kT
"
C∗
0 + C∗
1
√π
1
α1/2
eq
−
15
16α3/2
eq
! 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential + C∗
2
4
αeq
#
−
ρVm
ϵ0
kT
( 4π
3
D3
Vm
−C∗
0
1 + ϵ2
ϵ0
−C∗
1
√π
1 + ϵ2
ϵ0
α−1/2
eq
−
4C∗
2
1 + ϵ2
ϵ0
+ 6ϵ2
ϵ0
4π
3
D3
Vm
−C∗
0
α−1
eq + 15
16C∗
1
√π
1 + 53
5
ϵ2
ϵ0
α−3/2
eq
)
(34) and and
Znem
=
1 + Zres
hs
8
"
C∗
0 + C∗
1
√π
1
α1/2
eq
−
15
16α3/2
eq
! + C∗
2
4
αeq
#
−
ρVm
2
ϵ0
kT
( 4π
3
D3
Vm
−C∗
0
1 + ϵ2
ϵ0
−C∗
1
√π
1 + ϵ2
ϵ0
α−1/2
eq
−
4C∗
2
1 + ϵ2
ϵ0
+ 6ϵ2
ϵ0
4π
3
D3
Vm
−C∗
0
α−1
eq + 15
16C∗
1
√π
1 + 53
5
ϵ2
ϵ0
α−3/2
eq
)
(35)
where µres
hs /kT = (3η3 −9η2 + 8η) / (1 −η)3 and Zres
hs = (4η −2η2)/(1 −η)3 are the residual chemical
potential and compressibility factor. and
Znem
=
1 + Zres
hs
8
"
C∗
0 + C∗
1
√π
1
α1/2
eq
−
15
16α3/2
eq
! + C∗
2
4
αeq
#
−
ρVm
2
ϵ0
kT
( 4π
3
D3
Vm
−C∗
0
1 + ϵ2
ϵ0
−C∗
1
√π
1 + ϵ2
ϵ0
α−1/2
eq
−
4C∗
2
1 + ϵ2
ϵ0
+ 6ϵ2
ϵ0
4π
3
D3
Vm
−C∗
0
α−1
eq + 15
16C∗
1
√π
1 + 53
5
ϵ2
ϵ0
α−3/2
eq
)
(35) (35) where µres
hs /kT = (3η3 −9η2 + 8η) / (1 −η)3 and Zres
hs = (4η −2η2)/(1 −η)3 are the residual chemical
potential and compressibility factor. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential An important quantity that is used to characterize the degree of orientational order of the nematic
phase is the nematic order parameter, S2, which is commonly defined as the orientational average of the
second Legendre polynomial, P2(cos(θ)): S2 =
Z
P2(cos(θ))f(θ)d⃗ω
(36) (36) Because the OTF is used to represent the orientational distribution in nematic phase, f(θ) = fOTF(⃗ω),
S2 can be expressed as a function of the orientational parameter α [91]: S2 = 1 −3 coth αeq
αeq
+ 3
α2
eq
(37) (37) Int. J. Mol. Sci. 2013, 14 16423 In the case of the isotropic phase, where no orientational order is exhibited, the orientational averages
of Equation (14) are evaluated using the isotropic value, f(⃗ω) = 1/4π. 2.2. Equation of State for Hard-Cylindrical Disc Particles with an Anisotropic Square-Well Potential The Helmholtz free energy
expression for the isotropic phase is then given by: Aiso
NkT
=
Aid
iso
NkT + G(η)
Vexc(γ)
Vm
iso
⃗ω1,⃗ω2
−ρVm
2
ϵ0
kT
"
4π
3
D3
Vm
−
Vexc(γ)
Vm
iso
⃗ω1,⃗ω2
#
+
ρVm
2
ϵ2
kT
Vexc(γ)
Vm
P2(sin γ)
iso
⃗ω1,⃗ω2
(38) (38) The orientational averages in the isotropic phase can be evaluated as: Vexc(γ)
Vm
iso
⃗ω1,⃗ω2
≈
C∗
0 + π
4 C∗
1 + 2
3C∗
2 + 8
15C∗
4
(39)
Vexc(γ)
Vm
P2(sin γ)
iso
⃗ω1,⃗ω2
≈
−π
32C∗
1 −2
15C∗
2 −16
105C∗
4
(40) (39) (40) From the expressions for free energy of the isotropic phase (cf., Equation (38)), we obtain the
corresponding chemical potential and compressibility factor: µiso
kT
=
µid
iso
kT + ln ρ + 1
8
µres
hs
kT
C∗
0 + π
4 C∗
1 + 2
3C∗
2 + 8
15C∗
4
−
ρVm
ϵ0
kT
4π
3
D3
Vm
−C∗
0 −π
4 C∗
1 −2
3C∗
2 −8
15C∗
4
+
ρVm
ϵ2
kT
−π
32C∗
1 −2
15C∗
2 −16
105C∗
4
(41 +
ρVm
ϵ2
kT
−π
32C∗
1 −2
15C∗
2 −16
105C∗
4
(41) (41) and Ziso
=
1 + Zres
hs
8
C∗
0 + π
4 C∗
1 + 2
3C∗
2 + 8
15C∗
4
−
ρVm
2
ϵ0
kT
4π
3
D3
Vm
−C∗
0 −π
4 C∗
1 −2
3C∗
2 −8
15C∗
4
+
ρVm
2
ϵ2
kT
−π
32C∗
1 −2
15C∗
2 −16
105C∗
4
(42) (42) Before proceeding, we should note that the PL approach for the isotropic-nematic phase of hard
disc-like particles has been compared with a generic equation of state, which accounts for both
negative and positive contributions of the higher-body virial coefficients [46]; only positive virial
coefficients are possible with the PL approximation. Though an improved quantitative description of the
isotropic-nematic coexistence densities when compared with the exact simulation data is obtained by
incorporating the negative virial coefficients (particularly for the very thin discs), both approaches
provide the same overall qualitative fluid-phase behaviour. A comparison of the differences in the two
approaches for our systems of attractive hard discs is made in the following section. Int. J. Mol. Sci. 2013, 14 16424 3. Results and Discussion The fluid-phase diagrams of attractive cylindrical discs of various aspect ratios with square-well
attractive interaction are calculated by equating the pressure and chemical potential of the coexisting
phases at a given temperature. For the ACD model, the range of the attractive potential is
characterized by the parameter λ, which is chosen to be λ
≥
1 in order to ensure that the
resulting integrals for the contributions from the repulsive and attractive interactions remain separable. Dimensionless units are adopted throughout:
pressure, P ∗
=
PVm/ϵ0, and packing fraction,
η = ρVm. Two dimensionless scales for the temperature have been used in our study: the dimensionless
temperature, T ∗= kT/ϵ0, in terms of the isotropic square-well depth, ϵ0; and the reduced van der
Waals-like temperature, T ∗
vdw = kT/λ3. Though the dimensionless form, T ∗, is commonly used
to denote the temperature in simulations of such systems, the reduced van der Waals temperature,
T ∗
vdw, is useful to allow for direct comparisons in terms of the strength of the attractive interactions
(corresponding states representation). 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials First, we focus on discs with a spherically symmetric SW potential, so that the orientation-dependent
attractive contribution is inactive, i.e., ϵ2 = 0. Although the attractive interaction is isotropic, the
orientation-dependent excluded volume gives rise to an overall interaction between the discs, which
is anisotropic. The fluid-phase behaviour of ACDs of aspect ratio D/L = 5 and attractive range λ = 1
is presented in Figure 2. It is clear from the phase diagram that the system exhibits three fluid phases:
vapour (V), liquid (L), and nematic (N). The vapour-liquid equilibrium (VLE), seen at relatively low
densities, which terminates at the VLE critical point, (T ∗
c = 0.37720, ηc = 0.10362 and P ∗
c = 0.01383),
is determined by the free energy of the isotropic phase of the ACD particles. Oblate hard cores with
square-well attractive interactions have been studied by Meneses-Ju´arez et al. [92], but in this case the
range of the attractive interaction is smaller than the diameter of the molecule, so a direct comparison is
not possible. At moderate to high densities, an isotropic liquid-nematic transition is observable. In the
low temperature region, the isotropic-nematic coexistence becomes a broad region of vapour-nematic
equilibrium (VNE), which is bounded by a vapour-liquid-nematic (V-L-N) triple line. Above the triple
temperature, the nematic phase coexists with an isotropic liquid phase. It is worthwhile noting that the
width of liquid-nematic equilibrium (LNE) coexistence curve broadens markedly when T approaches
the triple point. Int. J. Mol. Sci. 2013, 14 16425 Figure 2. (a) The temperature-density and (b) pressure-temperature representations of the
fluid-phase equilibria for attractive cylindrical discs (ACDs) with an aspect ratio of D/L = 5
and an isotropic attractive interaction of range λ = 1. The generic disc-like molecules are
modelled as hard cylinders of length L and diameter D, enveloped by an attractive square
well of depth −(ϵ0 + ϵ2P2(cos γ)) and range λD; in the case of isotropic attractions, ϵ0 ̸= 0
and ϵ2 = 0. The dimensionless properties are defined in terms of D and ϵ0, as T ∗= kT/ϵ0
for the temperature, P ∗= PD3/ϵ0 for the pressure and η = ρVm for the packing fraction,
where Vm is the volume of the cylindrical core. The stable phases are indicated as vapour (V),
isotropic liquid (L) and nematic (N), and T ∗
c denotes the vapour-liquid equilibrium (VLE)
critical point. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials The dashed curves represent the results obtained using a generic reference
EOS for hard-cylindrical discs (HCDs) [46]. The dot-dashed line in (a) represents the V-L-N
three-phase coexistence line. 0.0
0.1
0.2
0.3
0.4
0.5
0.30
0.32
0.34
0.36
0.38
0.40
0.42
0.44
V-N
N
L-N
L
V
V-L
D/L !, " #
$%&Į&, %' &
*
T
(
PL
HCD EoS
(a)
0.30
0.32
0.34
0.36
0.38
0.40
0.000
0.005
0.010
0.015
0.020
0.025
0.030
*
cT
V
L
N
D/L !, !"
#$%Į%, !"#
*
T
*
P
PL
HCD EoS
(b)
model developed in our current work allows one to assess the effect of the anisotropy of
ing hard disc on the phase behaviour of the ACD system. The phase diagram of hard discs
ratio D/L = 10 with attractive interactions of range λ = 1 is shown in Figure 3. The syst
a single region of coexistence between isotropic (vapour or liquid) and nematic phases wit 0.0
0.1
0.2
0.3
0.4
0.5
0.30
0.32
0.34
0.36
0.38
0.40
0.42
0.44
V-N
N
L-N
L
V
V-L
D/L !, " #
$%&Į&, %' &
*
T
(
PL
HCD EoS
(a) 0.30
0.32
0.34
0.36
0.38
0.40
0.000
0.005
0.010
0.015
0.020
0.025
0.030
*
cT
V
L
N
D/L !, !"
#$%Į%, !"#
*
T
*
P
PL
HCD EoS
(b) The model developed in our current work allows one to assess the effect of the anisotropy of the
underlying hard disc on the phase behaviour of the ACD system. The phase diagram of hard discs of
aspect ratio D/L = 10 with attractive interactions of range λ = 1 is shown in Figure 3. The system
shows a single region of coexistence between isotropic (vapour or liquid) and nematic phases within Int. J. Mol. Sci. 2013, 14 16426 the temperature range considered. For the attractive disc systems with an aspect ratio of D/L > 5,
the VLE becomes metastable with respect to isotropic-nematic (I-N) coexistence. The region of I-N
coexistence is broad at low temperatures, with the difference between the coexisting densities of the
isotropic and nematic phases becoming narrow at higher temperatures. The coexisting densities of the
two phases is also seen to gradually shift to higher packing fractions when the temperature is increased. The contribution of the attractive interactions stabilizes the orientational order in the low-density and
low-temperature region. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials The I-N transition of the attractive disc model approaches the behaviour of the
corresponding purely repulsive discs in the high-temperature region. Figure 3. Temperature-density representation of the fluid-phase equilibria for attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 10 and an isotropic attractive
interaction of range λ = 1. The stable phases are indicated as isotropic (I) and nematic
(N). See the caption of Figure 2 for further details. 0.0
0.1
0.2
0.3
0.4
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
*
T
N
I-N
I
D/L !", !"
#$%Į%, !"#
PL
HCD EoS
When the aspect ratio is increased to D/L = 80, a different type of phase behaviour is exhibited
by the system. As shown in Figure 4, the isotropic-nematic coexistence is located at low densities
(η ∼0.1), while a region of nematic-nematic equilibrium (NNE) appears at moderate to high densities. The N1-N2 coexistence is bounded by the I-N1-N2 triple line and nematic-nematic critical point (T ∗
NN). An examination of the temperature dependence of the nematic order parameter is shown in Figure 5:
the degree of order of the high-density nematic state (N2) decreases slightly, while that of the low-
density nematic state (N1), though still highly ordered, behaves as a monotonically increasing function
of temperature up to T ∗
NN. At T ∗
NN, by definition, the differences in density and orientational order
between two nematic phases become indistinguishable. 0.0
0.1
0.2
0.3
0.4
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
*
T
N
I-N
I
D/L !", !"
#$%Į%, !"#
PL
HCD EoS 0.0
0.1
0.2
0.3
0.4
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
*
T
N
I-N
I
D/L !", !"
#$%Į%, !"#
PL
HCD EoS When the aspect ratio is increased to D/L = 80, a different type of phase behaviour is exhibited
by the system. As shown in Figure 4, the isotropic-nematic coexistence is located at low densities
(η ∼0.1), while a region of nematic-nematic equilibrium (NNE) appears at moderate to high densities. The N1-N2 coexistence is bounded by the I-N1-N2 triple line and nematic-nematic critical point (T ∗
NN). 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials An examination of the temperature dependence of the nematic order parameter is shown in Figure 5:
the degree of order of the high-density nematic state (N2) decreases slightly, while that of the low-
density nematic state (N1), though still highly ordered, behaves as a monotonically increasing function
of temperature up to T ∗
NN. At T ∗
NN, by definition, the differences in density and orientational order
between two nematic phases become indistinguishable. As the aspect ratio of the discs is made larger (corresponding to thinner particles), the
isotropic-nematic moves to lower densities. For particles that are moderately thin (D/L = 10 to 50),
the vapour-liquid transition between the two isotropic fluid phases becomes metastable with respect to
a transition between an isotropic liquid and a nematic phase. For larger aspect ratios (e.g., D/L > 80),
the isotropic-nematic transition moves to very low densities (packing fractions below 10%). As a
consequence, the usual van der Waals “vapour-liquid” phase transition is now exhibited in the anisotropic Int. J. Mol. Sci. 2013, 14 16427 region of the phase diagram, corresponding to nematic-nematic coexistence with its associated critical
point. A similar progression from V-L-N, through I-N, to I-N1-N2 phase behaviour with increasing aspect
ratio has been observed for attractive rod-like LC molecules [77,93–98]. The coexistence between two
nematic phases has been found experimentally in studies of hexa-alkylbenzene derivatives of discotic
mesogens [99] and in solutions of the calamitic polypeptides [95], both of which are characterized
by extreme oblate and prolate aspect ratios. It is also interesting to note that an analogous transition
in the vapour-liquid-solid phase equilibria is exhibited by attractive spherical particles as the range of
interaction is decreased: the VLE is found to become metastable relative to the isotropic fluid-solid
transition, and on further decreasing the attractive range, an iso-structural coexistence between two solid
phases is observed [100,101]. Figure 4. (a) The temperature-density and (b) pressure-temperature representations of
the fluid-phase equilibria for attractive cylindrical discs (ACDs) with an aspect ratio of
D/L = 80 and an isotropic attractive interaction of range λ = 1. The stable phases are
indicated as isotropic liquid (I), low-density nematic (N1) and high-density nematic (N2),
and T ∗
NN is the nematic-nematic critical point. The dot-dashed line in (a) represents the
I-N1-N2 three-phase coexistence line. See the caption of Figure 2 for further details. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials 9.7
9.9
10.1
10.3
10.5
0.92
0.94
0.96
0.98
1.00
D/L !", !"
#$%Į%, !"#
*
NN
T
2
S
*
T
N1
N2 Also included in Figures 2–4 are calculations using an improved representation of the reference
EOS [46] of the isotropic and nematic states of hard-cylindrical discs HCDs. It is clear that the qualitative
features of the fluid-phase behaviour are similar to those obtained with the PL approach for the hard-core
reference system, so we retain the standard PL treatment for simplicity, and the HCD reference EOS is
not employed in the subsequent analysis. By varying the parameter λ, different ranges of the attractive interaction may be explored. The
temperature-density projections of the fluid-phase equilibria are shown in Figure 6 for discs with an
aspect ratio of D/L = 10 and attractive ranges of λ = 1, 2, 3 and 5 in a representation where the
temperature is reduced by the attractive range, T ∗
vdw = T ∗/λ3. As the attractive range is increased, the
phase diagrams for the D/L = 10 discs with λ = 3 and 5 exhibit vapour, liquid, and nematic phases with
corresponding triple points, the same features as seen with the less anisotropic discs (D/L = 5, λ = 1). The fluid-phase coexistence of ACDs with λ = 3 and 5 converge onto a universal van der Waalsian
curve. However, it is apparent that the phase coexistence curve for λ = 1 does not follow this type of
corresponding state behaviour. Presumably, the VLE coexistence is not observed for attractive discs with
the same aspect ratio (D/L = 10), but a smaller attractive range (λ = 1), because the average excluded
volume of a pair of discs is of comparable size to the enveloping attractive sphere of the isotropic SW
interaction. The fluid-phase behaviour of discs characterized by an aspect ratio D/L = 50 is shown in Figure 7
for various values of the attractive range. It is noted that the large attractive range widens the isotropic-
nematic coexistence and shifts the I-N transition to higher densities. Increasing the attractive range is
not seen to promote a change in phase behaviour, supporting the view that the hard-body interaction
(free-volume entropy) is the dominant feature of highly anisometric particles. The coexistence between
two nematic phases is maintained in attractive disc systems of even larger aspect ratio, D/L = 80. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials 0.0
0.1
0.2
0.3
0.4
0.5
5
7
9
11
13
15
I-N2
N2
N1-N2
N1
I-N1
I
D/L !", # $
%&"Į", &' "
(
*
T
PL
HCD EoS
(a)
5
7
9
11
13
15
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
*
NN
T
N1
N2
I
D/L !", !"
#$%Į%, !"#
*
P
*
T
PL
HCD EoS
(b) 0.0
0.1
0.2
0.3
0.4
0.5
5
7
9
11
13
15
I-N2
N2
N1-N2
N1
I-N1
I
D/L !", # $
%&"Į", &' "
(
*
T
PL
HCD EoS
(a) (b) 5
7
9
11
13
15
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
*
NN
T
N1
N2
I
D/L !", !"
#$%Į%, !"#
*
P
*
T
PL
HCD EoS
(b) Int. J. Mol. Sci. 2013, 14 16428 Figure 5. The temperature dependence of the nematic order parameter, S2, of the coexisting
nematic phases for attractive cylindrical discs (ACDs) of aspect ratio D/L = 80 with an
isotropic attractive interaction of range λ = 1 (cf., Figure 4) The nematic-nematic critical
point, T ∗
NN, is also indicated. See the caption of Figure 2 for further details. Figure 5. The temperature dependence of the nematic order parameter, S2, of the coexisting
nematic phases for attractive cylindrical discs (ACDs) of aspect ratio D/L = 80 with an
isotropic attractive interaction of range λ = 1 (cf., Figure 4) The nematic-nematic critical
point, T ∗
NN, is also indicated. See the caption of Figure 2 for further details. 9.7
9.9
10.1
10.3
10.5
0.92
0.94
0.96
0.98
1.00
D/L !", !"
#$%Į%, !"#
*
NN
T
2
S
*
T
N1
N2
Also included in Figures 2–4 are calculations using an improved representation of the reference
EOS [46] of the isotropic and nematic states of hard-cylindrical discs HCDs. It is clear that the qualitative
features of the fluid-phase behaviour are similar to those obtained with the PL approach for the hard-core
reference system, so we retain the standard PL treatment for simplicity, and the HCD reference EOS is
not employed in the subsequent analysis. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio D/L = 80 and isotropic attractive interaction
of varying range, λ. The reduced temperature is defined in a van der Waals-corresponding
states form as T ∗
vdw = T ∗/λ3. The dot-dashed lines correspond to the I-N1-N2 three-phase
coexistence lines in each case. See the caption of Figure 2 for further details. Figure 8. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio D/L = 80 and isotropic attractive interaction
of varying range, λ. The reduced temperature is defined in a van der Waals-corresponding
states form as T ∗
vdw = T ∗/λ3. The dot-dashed lines correspond to the I-N1-N2 three-phase
coexistence lines in each case. See the caption of Figure 2 for further details. 0.0
0.1
0.2
0.3
0.4
8
9
10
11
12
N2
N1
I
I-N2
N1-N2
I-N1
*
vdw
T
D/L=80, !"Į"#$!%&"
'&(
'&%
'&)
'&* 0.0
0.1
0.2
0.3
0.4
8
9
10
11
12
N2
N1
I
I-N2
N1-N2
I-N1
*
vdw
T
D/L=80, !"Į"#$!%&"
'&(
'&%
'&)
'&*
Figure 9. The temperature dependence of the nematic order parameter, S2, of the coexisting
nematic phases for attractive cylindrical discs (ACDs) of aspect ratio D/L = 80 with an
isotropic attractive interaction of varying range, λ (cf., Figure 8) The nematic-nematic critical
point, T ∗
NN, is also indicated. See the caption of Figure 2 for further details. Figure 9. The temperature dependence of the nematic order parameter, S2, of the coexisting
nematic phases for attractive cylindrical discs (ACDs) of aspect ratio D/L = 80 with an
isotropic attractive interaction of varying range, λ (cf., Figure 8) The nematic-nematic critical
point, T ∗
NN, is also indicated. See the caption of Figure 2 for further details. 10.0
10.1
10.2
10.3
10.4
10.5
10.6
0.94
0.95
0.96
0.97
0.98
0.99
1.00
D/L !", !Į!, !"#
N2
N1
*
vdw
T
2
S
!"
!#
!$ 10.0
10.1
10.2
10.3
10.4
10.5
10.6
0.94
0.95
0.96
0.97
0.98
0.99
1.00
D/L !", !Į!, !"#
N2
N1
*
vdw
T
2
S
!"
!#
!$
These examples indicate that the long-ranged isotropic SW attraction slightly destabilizes the
orientationally-ordered phases. In the case of the thicker discs (D/L = 10), the long-range attractions N1 These examples indicate that the long-ranged isotropic SW attraction slightly destabilizes the
orientationally-ordered phases. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials In
Figure 8, systems of D/L = 80 attractive discs of varying attractive range are seen to exhibit the same Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 16429 type of phase diagram, and all appear to obey a corresponding state principle in terms of the reduced
temperature. Although a larger range of the isotropic attraction does not qualitatively affect the phase
behaviour of discs with D/L = 80, the degree of orientational order of the coexisting low-density
nematic phase (N1) becomes lower as λ increases, as can be seen in Figure 9. Figure 6. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio D/L = 10 and isotropic attractive interaction
of varying range, λ. The dot-dashed line corresponds to the V-L-N three-phase coexistence
line for the systems with λ = 3 and 5. The reduced temperature is defined in a van
der Waals-corresponding states form as T ∗
vdw = T ∗/λ3. See the caption of Figure 2 for
further details. 0.0
0.1
0.2
0.3
0.4
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
D/L=10, !Į!"# $%! &
*
vdw
T
'%(
'%$
'%)
'%* 0.0
0.1
0.2
0.3
0.4
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
D/L=10, !Į!"# $%! &
*
vdw
T
'%(
'%$
'%)
'%*
Figure 7. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio D/L = 50 and isotropic attractive interaction
of varying range, λ. The reduced temperature is defined in a van der Waals-corresponding
states form as T ∗
vdw = T ∗/λ3. See the caption of Figure 2 for further details. Figure 7. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio D/L = 50 and isotropic attractive interaction
of varying range, λ. The reduced temperature is defined in a van der Waals-corresponding
states form as T ∗
vdw = T ∗/λ3. See the caption of Figure 2 for further details. 0.0
0.1
0.2
0.3
0.4
0.5
4
5
6
7
8
9
10
N
I
I-N
D/L=50, !Į!"# $%! &
*
vdw
T
'%(
'%$
'%)
'%* 0.0
0.1
0.2
0.3
0.4
0.5
4
5
6
7
8
9
10
N
I
I-N
D/L=50, !Į!"# $%! &
*
vdw
T
'%(
'%$
'%)
'%* Int. J. Mol. Sci. 2013, 14 16430 Figure 8. 3.1. Attractive Cylindrical Discs with Isotropic SW Potentials In the case of the thicker discs (D/L = 10), the long-range attractions Int. J. Mol. Sci. 2013, 14 16431 Int. J. Mol. Sci. 2013, 14 (λ = 3 and 5) promote the coexistence between gas and isotropic liquid phases with no orientational
order (S2 = 0), while attractive discs with short-ranged attractive interactions (λ = 1) exhibit an
isotropic-nematic transition. For the systems with large aspect ratios (D/L = 50 and 80), where the
phase behaviour is dominated by the excluded volume interactions, the long-range isotropic attractions
do not change the type of phase behaviour, but weaken the degree of the orientational order of the
coexisting nematic phases. 3.2. Attractive Cylindrical Discs with Anisotropic SW Potentials For a system of discs with a large
aspect ratio, e.g., D/L = 50 (cf., Figure 12), the isotropic-nematic region becomes much broader
at lower temperatures as ϵ2 is increased, while the system converges into the hard-disc limit in the
high-temperature limit. For highly anisometric systems, the anisotropic shape and orientation-dependent
attractions both contribute to the stabilization of the nematic phases. Hence, for an anisotropic strength
of ϵ2 = 0.7ϵ0, which is comparable to the isotropic attraction, the D/L = 50 discs with anisotropic
attractions exhibit a nematic-nematic coexistence, which is equivalent to that seen for D/L = 80 discs
with isotropic attractions. I
ddi i
i
i
i
i h
i i
i
f
l
l
ld 0.0
0.1
0.2
0.3
0.4
100
120
140
160
180
200
220
240
260
280
300
D/L=10, !", !Į!"
#
*
T
!"#$
!"#$%&!$
!"#0.3 ! It is clear that a positive anisotropic attractive interaction stabilizes the nematic phase and enhances
the propensity of the system to form orientationally-ordered states. For a system of discs with a large
aspect ratio, e.g., D/L = 50 (cf., Figure 12), the isotropic-nematic region becomes much broader
at lower temperatures as ϵ2 is increased, while the system converges into the hard-disc limit in the
high-temperature limit. For highly anisometric systems, the anisotropic shape and orientation-dependent
attractions both contribute to the stabilization of the nematic phases. Hence, for an anisotropic strength
of ϵ2 = 0.7ϵ0, which is comparable to the isotropic attraction, the D/L = 50 discs with anisotropic
attractions exhibit a nematic-nematic coexistence, which is equivalent to that seen for D/L = 80 discs
with isotropic attractions. It is clear that a positive anisotropic attractive interaction stabilizes the nematic phase and enhances
the propensity of the system to form orientationally-ordered states. For a system of discs with a large
aspect ratio, e.g., D/L = 50 (cf., Figure 12), the isotropic-nematic region becomes much broader
at lower temperatures as ϵ2 is increased, while the system converges into the hard-disc limit in the
high-temperature limit. For highly anisometric systems, the anisotropic shape and orientation-dependent
attractions both contribute to the stabilization of the nematic phases. Hence, for an anisotropic strength
of ϵ2 = 0.7ϵ0, which is comparable to the isotropic attraction, the D/L = 50 discs with anisotropic
attractions exhibit a nematic-nematic coexistence, which is equivalent to that seen for D/L = 80 discs
with isotropic attractions. 3.2. Attractive Cylindrical Discs with Anisotropic SW Potentials In real systems, the dispersion forces are associated with the functional groups distributed at
various points in the molecule. The assignment of a central isotropic attraction is but a simplistic
first-order approximation. Recognizing the morphological anisotropy of discotic LCs, one can postulate
the existence of additional orientation-dependent (anisotropic) attractions. The effect of including
anisotropic attractions on the fluid-phase diagram of discs with D/L = 10 and λ = 1 is shown in
Figure 10: increasing the anisotropy of the attractions does not alter the phase behaviour qualitatively,
but drives the I-N transition to lower densities. The VLE in the low-density region is very sensitive to the
incorporation of anisotropic attractive interactions. As shown in Figure 11, the VLE is destabilized
on increasing the strength of the positive anisotropic attractions; for a sufficient large value of the
anisotropy, ϵ2 = 0.3ϵ0, the VLE becomes metastable with respect to the isotropic-nematic coexistence. A similar phenomena was also reported for the systems of attractive spherical and rod-like particles with
anisotropic attractions [77]. Figure 10. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 10, and a positive anisotropic
attractive interaction range λ = 1 and varying strength ϵ2 > 0. See the caption of Figure 2
for further details. 0.0
0.1
0.2
0.3
0.4
0.5
0.5
0.8
1.1
1.4
1.7
2.0
D/L=10, !", !Į!"
#
*
T
!"#
!"#$% #
& !"#$' #
I
N
I-N 0.0
0.1
0.2
0.3
0.4
0.5
0.5
0.8
1.1
1.4
1.7
2.0
D/L=10, !", !Į!"
#
*
T
!"#
!"#$% #
& !"#$' #
I
N
I-N Int. J. Mol. Sci. 2013, 14 16432 Figure 11. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 10 and an positive anisotropic
attractive interaction range, λ = 5, and varying strength, ϵ2 > 0. The dot-dashed lines
corresponds to the V-L-N three-phase coexistence line in each case. See the caption of
Figure 2 for further details. 0.0
0.1
0.2
0.3
0.4
100
120
140
160
180
200
220
240
260
280
300
D/L=10, !", !Į!"
#
*
T
!"#$
!"#$%&!$
!"#0.3 ! It is clear that a positive anisotropic attractive interaction stabilizes the nematic phase and enhances
the propensity of the system to form orientationally-ordered states. 3.2. Attractive Cylindrical Discs with Anisotropic SW Potentials In addition to attractive interactions with a positive anisotropy, for some molecules one would expect
interactions with a negative anisotropy favouring a perpendicular configuration: e.g., the quadrupolar
interactions between aromatic moieties give rise to both parallel (side-by-side) and perpendicular (T-
shaped) relative orientations of the cores. These kinds of interactions can be represented in our ACD
models using negative values for the parameter ϵ2. In Figures 13 and 14, we present the phase behaviour
of D/L = 10, λ = 1 and D/L = 80, λ = 3 disc systems, respectively. In the case of the D/L = 10, λ =
1 system, increasing the negative anisotropic attractions leads to a destabilization of the isotropic-nematic
region and promotes the appearance of VLE when ϵ2 = −0.3ϵ0. For discs with a larger aspect ratio of
D/L = 80, the orientation-dependent attractive interaction is seen to destabilize the nematic-nematic
equilibrium and to drive the isotropic phase boundary to higher densities. For sufficiently large values of
the negative anisotropic interaction, ϵ2 = −0.5ϵ0, the system no longer exhibits the region of nematic- Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 16433 nematic coexistence seen for the equivalent system with less negative anisotropic interaction, ϵ2 > −0.3
(cf., Figure 14). −0.3 Figure 12. (a) Temperature-density representation of the fluid-phase diagram for ACD with
an aspect ratio of D/L = 50, attractive range λ = 3 and varying anisotropic interaction
strength ϵ2. The dot-dashed line represents th I-N1-N2 three-phase coexistence line; (b) The
nematic-nematic coexistence region for the case of ϵ2 = 0.7ϵ0. See the caption of Figure 2
for further details. Figure 12. (a) Temperature-density representation of the fluid-phase diagram for ACD with
an aspect ratio of D/L = 50, attractive range λ = 3 and varying anisotropic interaction
strength ϵ2. The dot-dashed line represents th I-N1-N2 three-phase coexistence line; (b) The
nematic-nematic coexistence region for the case of ϵ2 = 0.7ϵ0. See the caption of Figure 2
for further details. 0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
400
0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
400
D/L=50, !", !Į!"
*
T
!"#$%
!"#$%&'!%
!"#$0.3 ! 3.2. Attractive Cylindrical Discs with Anisotropic SW Potentials I
N
I-N
(a)
!"# $%&!$
!"# $%'!$
0 1
0 2
0 3
290
295
300
305
310
315
320
0.04
0.08
0.12
0.16
0.20
0.24
0.28
290
295
300
305
310
315
320
290
295
300
305
310
315
N1-N2
N1
I-N1
D/L=50, !", !Į!"
*
T
!"#$%
!"#$%&'!%
!"#$0.3 ! I
N2
I-N2
(b)
!"# $%&!$
!"# $%'!$ 0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
400
0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
400
D/L=50, !", !Į!"
*
T
!"#$%
!"#$%&'!%
!"#$0.3 ! I
N
I-N
(a)
!"# $%&!$
!"# $%'!$ 0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
0.0
0.1
0.2
0.3
0.4
0.5
100
150
200
250
300
350
*
T
!"#$%&'!%
!"#$0.3 ! I
N
I-N
!"# $%&!$
!"# $%'!$
0.1
0.2
0.3
290
295
300
305
310
315
320
0.04
0.08
0.12
0.16
0.20
0.24
0.28
290
295
300
305
310
315
320
0.1
0.2
0.3
290
295
300
305
310
315
N1-N2
N1
I-N1
D/L=50, !", !Į!"
*
T
!"#$%
!"#$%&'!%
!"#$0.3 ! I
N2
I-N2
(b)
!"# $%&!$
!"# $%'!$ 290
295
300
305
310
315
320
0.04
0.08
0.12
0.16
0.20
0.24
0.28
N1-N2
N1
I-N1
D/L=50, !", !Į!"
*
T
!"#$%
!"#$%&'!%
!"#$0.3 ! I
N2
I-N2
(b)
!"# $%&!$
!"# $%'!$ Int. J. Mol. Sci. 2013, 14 16434 Figure 13. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 10 and a negative anisotropic
attractive interaction of range λ = 1 and varying strength, ϵ2 < 0. The dot-dashed line
corresponds to the V-L-N three-phase coexistence line. See the caption of Figure 2 for
further details. Figure 13. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 10 and a negative anisotropic
attractive interaction of range λ = 1 and varying strength, ϵ2 < 0. The dot-dashed line
corresponds to the V-L-N three-phase coexistence line. See the caption of Figure 2 for
further details. 0.0
0.1
0.2
0.3
0.4
0.5
0.4
0.6
0.8
1.0
1.2
1.4
D/L=10, !", !Į! "#! "#$!%& ! "#$!%' ! (
*
T
VLE
Figure 14. 3.2. Attractive Cylindrical Discs with Anisotropic SW Potentials Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 80 and an negative anisotropic
attractive interaction of range λ = 3 and varying strength, ϵ2 < 0. The dot-dashed lines
correspond to the I-N1-N2 three-phase coexistence lines in each case. See the caption of
Figure 2 for further details. 0.0
0.1
0.2
0.3
0.4
0.5
0.4
0.6
0.8
1.0
1.2
1.4
D/L=10, !", !Į! "#! "#$!%& ! "#$!%' ! (
*
T
VLE Figure 14. Temperature-density representation of the fluid-phase equilibria of attractive
cylindrical discs (ACDs) with an aspect ratio of D/L = 80 and an negative anisotropic
attractive interaction of range λ = 3 and varying strength, ϵ2 < 0. The dot-dashed lines
correspond to the I-N1-N2 three-phase coexistence lines in each case. See the caption of
Figure 2 for further details. 0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
100
150
200
250
300
*
T
!"#$%&' %
!"#$%&( %
!"#$%&) %
D/L=80, !"
!"#$
. Conclusions
In this work, we present a closed-form equation of state for the description of the thermodynamic
roperties and orientational ordering of attractive hard-core cylindrical-disc fluids, which serves as a 0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
100
150
200
250
300
*
T
!"#$%&' %
!"#$%&( %
!"#$%&) %
D/L=80, !"
!"#$ Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 16435 basic model for thermotropic discotic liquid crystals. With the aid of the Onsager trial function to
represent the single-particle orientational distribution function in the nematic phase, the free energy is
expressed in algebraic form and the functional variation of free energy reduced to a simple derivative
with respect to the Onsager orientational parameter α. A cubic solution of αeq is then obtained
when higher-order terms in the expansion are neglected and ordered states (α ∼10) are considered. Using this approach, we provide a qualitative description of the fluid-phase diagrams of ACD particles. basic model for thermotropic discotic liquid crystals. With the aid of the Onsager trial function to
represent the single-particle orientational distribution function in the nematic phase, the free energy is
expressed in algebraic form and the functional variation of free energy reduced to a simple derivative
with respect to the Onsager orientational parameter α. A cubic solution of αeq is then obtained
when higher-order terms in the expansion are neglected and ordered states (α ∼10) are considered. Using this approach, we provide a qualitative description of the fluid-phase diagrams of ACD particles. The separate effects of the shape anisotropy of the hard discs and the orientation-dependent attractive
interactions are examined in detail. The hard core of the particle is primarily responsible for determining
the type of phase behaviour. The contribution of attractive interactions, though secondary, cannot be
neglected in thermotropic LC systems. For the ACD model, the attractive range, λ, of the enveloping
square well determines the formation of the isotropic liquid state. The anisotropic term in the ASW
potential, which is controlled by the coefficient ϵ2 (or its ratio ϵ2/ϵ0 relative to the isotropic term),
is key to the stabilization/destabilization of orientationally-ordered phases. The attractive disc model
studied in the current work is a simple prototypical coarse-grained representation of real molecular
interactions in discotic liquid crystals: neither will the repulsive interactions be purely repulsive in
real systems nor will the attractive interactions be of the simple van der Waals square-well form. This
having been said, many common discotic thermotropic particles comprise large fused aromatic cores,
which will have lower energetic overlap volumes at the edges of the particles than in the central region,
which is captured volumetrically at least with our square-well model. Int. J. Mol. Sci. 2013, 14 We are therefore confident that,
qualitatively, at least, our model will describe the isotropic and nematic ordering behaviour of discotic
thermotropic mesogens. A simple square-well hard-spherocylinder model of this generic form has been
used to successfully represent the ordering behaviour of solutions of rod-like polypeptide (poly γ-benzyl
L-glutamate) macromolecules with a quantitative description of the phase boundaries [98] basic model for thermotropic discotic liquid crystals. With the aid of the Onsager trial function to
represent the single-particle orientational distribution function in the nematic phase, the free energy is
expressed in algebraic form and the functional variation of free energy reduced to a simple derivative
with respect to the Onsager orientational parameter α. A cubic solution of αeq is then obtained
when higher-order terms in the expansion are neglected and ordered states (α ∼10) are considered. Using this approach, we provide a qualitative description of the fluid-phase diagrams of ACD particles. The separate effects of the shape anisotropy of the hard discs and the orientation-dependent attractive
interactions are examined in detail. The hard core of the particle is primarily responsible for determining
the type of phase behaviour. The contribution of attractive interactions, though secondary, cannot be
neglected in thermotropic LC systems. For the ACD model, the attractive range, λ, of the enveloping
square well determines the formation of the isotropic liquid state. The anisotropic term in the ASW
potential, which is controlled by the coefficient ϵ2 (or its ratio ϵ2/ϵ0 relative to the isotropic term),
is key to the stabilization/destabilization of orientationally-ordered phases. The attractive disc model
studied in the current work is a simple prototypical coarse-grained representation of real molecular
interactions in discotic liquid crystals: neither will the repulsive interactions be purely repulsive in
real systems nor will the attractive interactions be of the simple van der Waals square-well form. This
having been said, many common discotic thermotropic particles comprise large fused aromatic cores,
which will have lower energetic overlap volumes at the edges of the particles than in the central region,
which is captured volumetrically at least with our square-well model. We are therefore confident that,
qualitatively, at least, our model will describe the isotropic and nematic ordering behaviour of discotic
thermotropic mesogens. 4. Conclusions In this work, we present a closed-form equation of state for the description of the thermodynamic
properties and orientational ordering of attractive hard-core cylindrical-disc fluids, which serves as a In this work, we present a closed-form equation of state for the description of the thermodynamic
properties and orientational ordering of attractive hard-core cylindrical-disc fluids, which serves as a Int. J. Mol. Sci. 2013, 14 A simple square-well hard-spherocylinder model of this generic form has been
used to successfully represent the ordering behaviour of solutions of rod-like polypeptide (poly γ-benzyl
l
l
l
i h
i
i
d
i i
f h
h
b
d i L-glutamate) macromolecules with a quantitative description of the phase boundaries [98]. It is important to point out that in liquid state theory [49], any perturbation approach requires
an accurate description of the reference system. The reference adopted here for the ACDs is the
hard-cylindrical disc system described with a Parsons-Lee approach. Because of anomalous negative
contributions from higher-order virial coefficients[42], the Parsons-Lee approach does not provide an
accurate description of hard-disc fluids, when compared with the exact simulation data, particularly in
the limit of infinitely thin discs. An improved equation of state for oblate disc-like particles has been
developed [46], which incorporates these contributions and provides a better description for the isotropic
and nematic phases of hard-disc systems. In the current work, we have not treated the columnar states
that are expected in the high density region of the phase diagram. The methodology developed here allows other interactions to be incorporated within the attractive
hard-disc to provide a more realistic free energy functional. The molecular-based statistical associating
fluid theory (SAFT) [102,103], and its recent extensions [104–108], have been shown to be a powerful
methodology, allowing accurate descriptions of the phase behaviour of complex fluids and fluid mixtures. By building on the SAFT formalism, it is possible to explicitly include additional perturbation terms,
such as those related to chain (or ring) geometries, hence paving the path towards a more sophisticated
model for disc-like LC molecules. The use of the attractive disc model to describe the LC phase
behaviour of real disc-like molecular fluids and the extension of the current methodology to other
anisotropic phases (e.g., columnar ordering) will be the subject of future studies. Int. J. Mol. Sci. 2013, 14 16436 Acknowledgments L.W. thanks the Department for Business Innovation and Skills of the UK, and the China
Scholarship Council for funding a PhD studentship. Funding to the Molecular Systems Engineering
Group from the Engineering and Physical Sciences Research Council (EPSRC) of the UK (grants
GR/T17595, GR/N35991, EP/E016340 and EP/J014958), the Joint Research Equipment Initiative
(JREI) (GR/M94426) and the Royal Society-Wolfson Foundation refurbishment scheme is also
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Fc gamma RIIb expression levels in human liver sinusoidal endothelial cells during progression of non-alcoholic fatty liver disease
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RESEARCH ARTICLE RESEARCH ARTICLE
Fc gamma RIIb expression levels in human
liver sinusoidal endothelial cells during
progression of non-alcoholic fatty liver
disease Tomoko IshikawaID1, Hiroshi Yokoyama2,3, Tomokazu Matsuura3, Yoko Fujiwara1,4* 1 Institute for Human Life Innovation, Ochanomizu University, Bunkyo-ku, Tokyo, Japan, 2 Division of
Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine,
Minato-ku, Tokyo, Japan, 3 Department of Laboratory Medicine, The Jikei University School of Medicine,
Minato-ku, Tokyo, Japan, 4 Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-
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a1111111111 * fujiwara.yoko@ocha.ac.jp * fujiwara.yoko@ocha.ac.jp OPEN ACCESS Liver sinusoidal endothelial cells (LSECs) play a pivotal role in hepatic function and homeo-
stasis. LSEC dysfunction has been recognized to be closely involved in various liver dis-
eases, including non-alcoholic steatohepatitis (NASH), but not much is known about the fate
of the scavenger receptors in LSECs during NASH. Fc gamma receptor IIb (FcγRIIb),
known as a scavenger receptor, contributes to receptor-mediated endocytosis and immune
complexes clearance. In this study, to elucidate the fate of FcγRIIb in the progression of
non-alcoholic fatty liver disease (NAFLD), we examined FcγRIIb levels in NAFLD biopsy
specimens by immunohistochemistry, and investigated their correlation with the exacerba-
tion of biological indexes and clinicopathological scores of NASH. The FcγRIIb expression
levels indicated significant negative correlations with serum levels of blood lipids (triglycer-
ide, total cholesterol, high-density lipoprotein-cholesterol), type 4 collagen and hyaluronic
acid, which are involved in hepatic lipid metabolism disorder, fibrosis, and inflammation,
respectively. However, there was no significant difference of FcγRIIb expression levels
among the pathological grades of NAFLD. During NAFLD progression, inflammation and
fibrosis may influence the expression of FcγRIIb and their scavenger functions to maintain
hepatic homeostasis. Citation: Ishikawa T, Yokoyama H, Matsuura T,
Fujiwara Y (2019) Fc gamma RIIb expression
levels in human liver sinusoidal endothelial cells
during progression of non-alcoholic fatty liver
disease. PLoS ONE 14(1): e0211543. https://doi. org/10.1371/journal.pone.0211543 Editor: Yu Wang, The University of Hong Kong,
HONG KONG Received: March 27, 2018
Accepted: January 16, 2019
Published: January 29, 2019 Copyright: © 2019 Ishikawa et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This work was supported by the Japan
Society for the Promotion of Science, Grant
Number: 15K00810 (https://www.jsps.go.jp/
english/index.html) to TI. The funder had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Introduction Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome
and is categorized into non-alcoholic fatty liver (NAFL), with simple fatty liver, and non-alco-
holic steatohepatitis (NASH), with inflammation and fibrosis. NAFL is a benign disease, but
NASH can lead to severe chronic hepatic diseases, such as advanced fibrosis [1], cirrhosis [2],
and hepatocellular carcinoma (HCC) [3, 4]. In 1998, a two-hit model [5] was proposed as a Competing interests: The authors have declared
that no competing interests exist. 1 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Fc gamma RIIb in human NASH mechanism of NASH pathogenesis. The first hit is fat accumulation in the liver parenchyma
caused by obesity and insulin resistance, and subsequent second hits are several cellular
stresses including oxidative stress, mitochondrial dysfunction, and apoptosis stress. In recent
years, a multiple parallel-hit hypothesis [6] has been attracting a lot of attention. Namely, the
multiple factors (e.g., inflammation from gut-derived endotoxin or adipocytokines, endoplas-
mic reticulum stress, and innate immunity) may act in parallel and lead to the progression of
steatosis, hepatocellular ballooning, inflammation, and fibrosis. There are many cases in which
patients are not obese or diabeti, and they are asymptomatic until they reach a severe period of
NAFLD. So it is necessary to elucidate the details of clinical phenotypes in the early stages of
NASH. Recent reports suggest that liver sinusoidal endothelial cells (LSECs) may play a pivotal role
in the onset and progression of chronic hepatitis and liver cancer [7].It is well known that the
major functions of LSECs are [1] to control the transport of molecules in blood to the hepato-
cytes via fenestrae, and [2] to remove foreign or unwanted materials by means of scavenger
receptors. LSECs are the most permeable endothelial cells due to the presence of fenestrae and lack of
basement membranes, and play a fundamental role in maintaining hepatic homeostasis. Therefore, the loss of these specific structural features is responsible for causing dysfunction of
LSECs, and disrupting hepatic homeostasis [8].Capillarization, characterized by the loss of
fenestrae, the formation of tight junctions, and the expression of vascular endothelium-associ-
ated markers CD31, CD34, and von Willebrand factor in LSECs, occurs in human cirrhosis
and chronic hepatitis [9], rat chronic liver disease induced by thioacetamide administration
[10], and several pathological models in vivo and in vitro [7]. PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Routine histological and biochemical analysis For routine histology, liver biopsy samples were fixed with 4% paraformaldehyde in phos-
phate-buffered saline (PBS) and embedded in paraffin blocks. Sections, 3 μm thick, were
stained with hematoxylin-eosin (H-E) and Masson’s trichrome for routine examination. The
pathological investigation was performed by a board-certified pathologist of the Japanese Soci-
ety of Pathology, and the histological grade of steatosis, inflammation, ballooning, fibrosis
stage and NAFLD activity score (NAS) were determined using NASH Clinical Research Net-
work criteria proposed by Kleiner et al [33]. Basic and diagnostic biochemical tests were obtained by routine methods. In this study, we
analyzed the number of blood platelets (Ptl), serum levels of aspartate aminotransferase (AST),
alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase
(γ-GTP), total bilirubin (TB), total protein (TP), albumin (Alb), triglyceride (TG), total choles-
terol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol
(HDL-C), fasting blood sugar (FBS), hemoglobin A1c (HbA1c), VICol, and hyaluronic acid
(Hyal). Patients We analyzed 26 patients with NAFLD who had undergone liver biopsy for definitive diagnosis
at Jikei University Hospital between 2011 and 2015 and were histologically diagnosed with
NAFL or NASH. Fasting blood samples were collected from patients before liver biopsy. Patients who had been clinically diagnosed with other liver diseases or had consumed more
than 20 g of alcohol per day were excluded from this study. Two patients with hepatitis B sur-
face antigen (HBsAg) positive were included. This study was carried out with the approval of
the Ethics Committee of the Jikei University School of Medicine, and the Ethics Committee of
Ochanomizu University. Informed consent was obtained from all patients. Introduction As a ligand for FcγRIIb, fibrinogen-like protein 2 (FGL2), which is known
to have immunomodulatory function, has been reported to have an affinity for FcγRIIb [30]. Interestingly, the plasma level of FGL2 is higher in patients with NAFLD [31] and liver cirrho-
sis [32]. clearance of small immune complexes in hepatic sinusoids [27].Moreover, the expression of
FcγRIIb in endothelial cells has been suggested to be associated with hepatic and cardiovascu-
lar diseases [28, 29]. As a ligand for FcγRIIb, fibrinogen-like protein 2 (FGL2), which is known
to have immunomodulatory function, has been reported to have an affinity for FcγRIIb [30]. Interestingly, the plasma level of FGL2 is higher in patients with NAFLD [31] and liver cirrho-
sis [32]. These scavenger receptors on LSECs share the clearance task, and their downregulation
may lead to increased blood concentrations of their specific ligands: hyaluronan, oxLDL, type
IV collagen (IVCol), IgG and perhaps FGL2. However, not much is known about the fate of
the endocytic receptors during human NAFLD. In the present study, we aimed to elucidate
the correlations of FcγRIIb expression levels in LSECs of NAFL and NASH patients with clini-
cal biochemical and pathological phenotypes. Furthermore, we examined another scavenger
receptor, stabilin-2 expression, in LSECs of NAFLD patients. Introduction Capillarization of LSECs plays a
key role even in the onset and progression of NAFLD/NASH [11]. Capillarization and sinusoi-
dal communication are implicated in hepatic fibrosis, and the protection of LSECs may be an
effective strategy to avoid fibrosis initiation and progression [10, 12]. The other noteworthy
role of LSECs is the clearance function by means of scavenger receptor (SR) from the periph-
eral blood. The term “scavenger receptor” was originally coined to denote a cell surface recep-
tor on the macrophage that mediates endocytosis and degradation of waste materials [13]. Thereafter, however, the expression of SRs has been reported on several cell types. PrabhuDas
M et al. proposed the nomenclature and classification of SRs into 10 classes termed SR-A to
SR-H [14]. The LSECs express SR-A (macrophage SR), SR-B (CD36), and SR-H (stabilin-1,
stabilin-2), but do not express SR-D, SR-E, SR-F, and SR-G [15]. In physiological conditions,
LSECs express three types of receptors that mediate the endocytosis of waste materials, hyalur-
onan receptors (stabilin-2, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)),
mannose receptors (CD206), and a low-affinity gamma immunoglobulin Fc region receptor
IIb (FcγRIIb) [15–17]. Both stabilin-1 and stabilin-2, which are hepatic hyaluronan clearance receptors, have been
purified from rat livers and were characterized by Polits et al. [18]. Serum hyaluronan is a rec-
ognized non-invasive marker of liver fibrosis, and can accurately and independently predict
mortality in patients with liver disease [19, 20]. Stabilin-1 and stabilin-2 are involved in the
LSEC endocytosis of oxidized low-density lipoprotein (oxLDL), but not in the Kupffer cell
endocytosis via the clathrin-mediated pathway [21]. In the human HCC and a murine HCC
model, loss of stabilin-2, LYVE-1, and FcγRIIb in LSECs is noted in the majority of liver
tumours [22]. The FcγRIIb is the sole inhibitory FcγR, which contains an immunoreceptor tyrosine based
inhibitory motif (ITIM) in its cytoplasmic domain [23, 24]. FcγRIIb is mainly present on the
surface of all leukocytes except NK cells and T cells, and is involved in regulating immunoreac-
tivity. FcγRIIb is also expressed in hepatic sinusoids [25, 26]. FcγRIIb contributes to the PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 2 / 13 Fc gamma RIIb in human NASH clearance of small immune complexes in hepatic sinusoids [27].Moreover, the expression of
FcγRIIb in endothelial cells has been suggested to be associated with hepatic and cardiovascu-
lar diseases [28, 29]. Immunohistochemistry and morphometry Immunohistochemical staining of FcγRIIb was performed on liver biopsy samples. The paraf-
fin sections were deparaffinized and treated with 10 mM citric buffer (pH6.0) for 10 min at
95˚C for antigen unmasking. Endogenous peroxidase activity was exhausted by 3% hydrogen
peroxide. The sections were then pre-incubated with 5% normal goat serum-1%BSA in PBS,
and incubated with a primary rabbit anti-human FcγRIIb antiserum (Peptide Institute, Osaka, 3 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Fc gamma RIIb in human NASH JAPAN) or a rabbit anti-human stabilin 2 antiserum (abcam). Secondary antiserum from
Histo Fine simple stain (MAX-PO Multi, Nichirei, Tokyo, JAPAN) was used, and detected
with diaminobenzidine (DAB) and then counterstained with hematoxylin. The FcγRIIb
expression level was morphometrically quantified on microphotographs taken with a 20 x
objective lens on a BZ-X710 microscope and Hybrid Cell Count software (KEYENCE, Tokyo,
JAPAN) as follows. For all specimens, as much fields as possible of the hepatic parenchymal
area was photographed (12–35 fields per patient). The procedures to extract the whole tissue
area and FcγRIIb positive area were determined, respectively, as shown in S1 Fig. Then the
areas were automatically measured under the same conditions. To avoid observer-related bias,
morphological observation and evaluation were blindly performed, i.e. the observer was
unaware of the diagnosis and pathological scores. FcγRIIb distribution Representative light microscopic images of patients who were diagnosed as NASH (a: NAS, 2;
b: NAS, 8) are shown in Fig 1. As it is well known, the FcγRIIb signals were found only in
LSECs [25]. In a case with low NAS (= 2), FcγRIIb signals were strong and surrounded the
entire circumference of the sinusoids (Fig 1A), whereas in a case with the highest NAS (= 8)
among the cases we analyzed, FcγRIIb signals were very weak and were only irregularly
expressed in LSECs (Fig 1B). Patient characteristics Table 1 summarized the age, sex, BMI, and pathological characteristics of the 26 patients ana-
lyzed in this study. The number of patients was 13 for both males and females, and the mean
ages for males and females were 48.5 and 56.6 years, respectively. With the biopsy-proven
pathological examination, two patients were diagnosed as NAFL and 24 patients as NASH. In
Table 1, the two patients diagnosed with NAFL are listed from the top, followed by the 24 diag-
nosed as NASH, in ascending order of NAS and then ascending ALT value. This suggests that
the serum ALT level is not a sufficient indicator of NASH progression, though it is used as one
of the screening criterions for NAFLD. Statistical analyses Statistical analyses were performed using SPSS Statistics 23 software (IBM, NY, USA). Com-
parisons of FcγRIIb expression levels with sex, diagnosis, and clinicopathological scores were
performed by the Mann-Whitney or the Kruskal-Wallis nonparametric test. A P-value <0.05
was considered to be significant. Investigation of associations between FcγRIIb expression and
body mass index (BMI) or serum biochemical values were performed by the Pearson correla-
tion coefficient test. R-values(±) >0.7, >0.4, >0.3 were considered to have a strong, medium,
and small correlation, respectively. FcγRIIb expression levels For morphometric analysis, the FcγRIIb expression level was calculated as the ratio of the posi-
tive area to whole tissue area, using the morphometric procedure described in the materials
and methods section (S1 Table). We also analyzed the FcγRIIb expression level as the intensity
of positive signals (S1 Table), and this showed a similar tendency to the measurement results
stated above. Hereinafter, the ratio of the FcγRIIb positive area is denoted as the FcγRIIb
expression level in this paper. FcγRIIb expression levels 4 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Fc gamma RIIb in human NASH Diagnosis
Age
Sex
BMI
Blood Test
Pathological Stage
AST
ALT
ALP
γ-
GTP
TB
TP
Alb
Plt
TG
TC
LDL-C
HDL-C
FBS
HbA1c
IV
Col
Hyal
Steatosis
Inflammation
Ballooning
Fibrosis
NAS
(U /
L)
(U /
L)
(U /
L)
(U /
L)
(mg /
dL)
(g /
dL)
(g /
dL)
(104 /
μL)
(mg /
dL)
(mg /
dL)
(mg /
dL)
(mg /
dL)
(mg /
dL)
(%)
(ng /
mL)
(ng /
mL)
NAFL
54
F
23.6
21
14
334
30
0.4
7.0
3.8
530
197
227
160
67
103
5.8
117
<10
1
1
0
0
2
NAFL
43
F
31.3
78
141
249
663
0.8
6.1
3.5
89
316
304
192
112
4.7
3
0
0
0
3
NASH
73
M
23.0
46
50
265
40
0.9
7.3
4.1
220
98
209
153
56
6.0
127
83
1
0
1
1B
2
NASH
54
M
28.4
33
64
306
51
0.8
7.5
4.6
257
241
216
183
33
101
5.8
115
38
2
0
0
1A
2
NASH
66
F
30.2
43
23
318
18
0.6
6.9
3.5
247
185
163
120
43
117
5.8
162
1
1
1
2
3
NASH
65
F
23.0
34
148
440
1871
0.5
6.1
3.7
415
158
293
170
123
139
8.6
1
1
1
1B
3
NASH
46
M
32
54
150
27
0.9
6.9
4.2
269
116
192
137
55
5.8
23
3
1
0
1A
4
NASH
33
F
18.4
140
179
213
30
0.7
8.6
4.9
362
709
355
218
137
161
7.6
148
25
1
1
2
1A
4
NASH
38
M
89
205
147
109
1.6
6.0
4.2
163
252
174
149
25
301
10.6
86
44
2
1
1
3
4
NASH
58
F
22.4
170
206
220
70
0.7
8.0
4.9
225
111
179
130
49
114
5.7
196
64
2
1
1
3
4
NASH
34
M
34.6
153
259
241
352
1.6
7.0
4.5
171
206
204
167
37
214
8.9
149
45
2
1
1
3
4
NASH
65
F
30.0
34
17
243
35
0.6
7.4
2.8
199
78
155
94
61
93
5.6
320
1
2
2
3
5
NASH
57
M
25.3
25
32
382
34
0.4
7.3
3.8
77
108
168
102
66
138
6.4
218
203
1
2
2
3
5
NASH
53
F
29.0
40
50
391
28
0.8
7.3
3.9
230
152
180
144
36
100
6.7
147
258
1
2
2
3
5
NASH
58
M
26.4
64
61
344
106
0.7
7.6
4.5
207
196
212
148
64
100
5.4
1
2
2
3
5
NASH
68
F
31.6
60
78
407
26
0.6
7.4
4.0
119
126
172
93
54
100
6.1
151
78
1
2
2
3
5
NASH
46
M
30.5
59
93
202
182
0.8
7.2
4.6
209
216
197
98
99
126
6.7
2
2
1
1B
5
NASH
47
M
28.7
88
156
372
49
1.2
7.8
4.9
186
148
262
181
81
104
5.6
124
25
3
1
1
1A
5
NASH
47
M
36.7
148
183
411
86
1.4
7.6
4.4
196
205
215
174
41
117
6.1
206
20
2
2
1
3
5
NASH
31
M
29.0
221
267
191
119
1.0
7.8
5.0
163
364
249
176
73
145
6.4
297
72
2
1
2
1B
5
NASH
36
M
36.9
181
327
231
104
1.6
7.3
4.6
263
134
180
141
39
7.1
167
24
3
1
1
2
5
NASH
67
F
23.4
73
51
209
18
0.7
8.3
4.4
235
76
162
117
45
90
6.1
176
2
2
2
3
6
NASH
50
F
37
66
211
29
1.0
7.9
4.3
215
163
185
147
38
101
5.8
113
14
2
2
2
1B
6
NASH
64
M
25.8
67
77
242
47
1.2
7.0
3.8
165
431
124
92
32
123
5.9
162
26
2
2
2
3
6
NASH
60
F
23.7
70
90
481
84
0.7
7.9
4.3
232
81
250
188
62
108
5.7
128
83
1
3
2
3
6
NASH
54
F
23.5
105
126
249
108
1.0
7.9
4.2
166
269
188
113
75
127
7.5
187
153
3
3
2
3
8
For the 26 patients, the two patients diagnosed with NAFL are listed from the top, followed by the 24 diagnosed as NASH, listed in ascending order of NAS and then ascending ALT value. FcγRIIb expression levels https //doi org/10 1371/journal pone 0211543 t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 5 / 13 Fc gamma RIIb in human NASH Fig 1. FcγRIIb expression and localization in liver tissues of patients. Representative light microscopic images of
patients who were diagnosed as NASH (a: NAS, 2; b: NAS, 8). FcγRIIb signals detected with DAB (brown) are
distributed in LSECs. In b, arrowheads indicate LSECs that do not detect FcγRIIb signals. Nuclei were counterstained
with hematoxylin (blue). Scale bars represent 100 μm. h
//d i
/10 1371/j
l
0211543 001 Fig 1. FcγRIIb expression and localization in liver tissues of patients. Representative light microscopic images of
patients who were diagnosed as NASH (a: NAS, 2; b: NAS, 8). FcγRIIb signals detected with DAB (brown) are
distributed in LSECs. In b, arrowheads indicate LSECs that do not detect FcγRIIb signals. Nuclei were counterstained
with hematoxylin (blue). Scale bars represent 100 μm. Fig 1. FcγRIIb expression and localization in liver tissues of patients. Representative light microscopic images of
patients who were diagnosed as NASH (a: NAS, 2; b: NAS, 8). FcγRIIb signals detected with DAB (brown) are
distributed in LSECs. In b, arrowheads indicate LSECs that do not detect FcγRIIb signals. Nuclei were counterstained
with hematoxylin (blue). Scale bars represent 100 μm. https://doi.org/10.1371/journal.pone.0211543.g001 https://doi.org/10.1371/journal.pone.0211543.g001 https://doi.org/10.1371/journal.pone.0211543.g001 Correlation with biological indexes and serum biochemical markers The correlations of FcγRIIb expression levels with BMI, Plt, and serum biochemical markers
are shown in Fig 2. The Pearson correlation test indicated that there was no correlation
between FcγRIIb levels and BMI. The regression lines of serum AST, ALT, ALP, and γ-GTP
showed negative slopes, but there was no significant correlation. The regression lines showed
positive slopes for TB, Alb, and Plt, which decreased with liver disease deterioration, though
there was no significant correlation. On the other hand, in the factors related to lipid dynam-
ics, it was shown that the FcγRIIb levels had a small negative correlation (R = -0.357, -0.300)
with TG and TC, and a medium negative correlation (R = -0.409) with HDL-C. With LDL-C,
there was no significant correlation, but the regression line also showed a negative slope, sug-
gesting that it is similar to other lipid-related factors. In FBS and HbA1c, there was no correla-
tion between FcγRIIb levels, meaning there was no correlation of FcγRIIb levels with short-
and medium-term blood glucose levels. Both IVCol and hyaluronan, which increase with pro-
gression of hepatic fibrosis, showed medium negative correlation (R = - 0.490, -0.525). Comparisons of diagnosis, sex, and pathological scores The comparisons of diagnosis, sex, and pathological values are shown in Fig 3. FcγRIIb levels
were significantly higher in females compared with in males (Mann-Whitney test, P = 0.009),
whereas there was no significant difference in FcγRIIb expression levels of the liver biopsy
specimen among the pathological grades of NAFLD. The mean FcγRIIb levels tended to be
higher with the progression of steatosis, but no significant difference was observed. FcγRIIb
levels became lower with the progression of inflammation and ballooning. Especially, it was
significantly lower in patients with ballooning stage 3 as compared with those in stage 2 (Krus-
kal-Wallis test, P = 0.022). Interestingly, in the comparison between the fibrosis stages, which
are closely related to the exacerbation of NASH, FcγRIIb levels tended to be higher at the early
stage of fibrosis and became slightly lower at the 1B, 2 and 3 fibrosis stages. This is consistent
with the findings that there were medium negative correlations between FcγRIIb levels and
IVCol or hyaluronan. It is also suggested that the decline of FcγRIIb levels in the progression
of NAFLD correlates with the exacerbation of fibrosis. Stabilin-2 distribution High amounts of HDL-C, IVCol, and hyaluronan in peripheral blood coincided with low
expression of FcγRIIb. Interestingly, some of these molecules are ligands for LSEC receptors PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 6 / 13 Fc gamma RIIb in human NASH PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 7 / 13 Fc gamma RIIb in human NASH Fig 2. Correlation with biological indexes and biochemical data. Scatter plots show the correlation of FcγRIIb expression
levels with BMI, Plt, and serum biochemical data. Statistical correlation was analyzed by the Pearson correlation coefficient
test. #: one outlier (indicated as in γ-GTP diagram) removed. : small correlation by the Pearson correlation coefficient test
(R<-0.3). : medium correlation by the Pearson correlation coefficient test (R<-0.4). Fig 2. Correlation with biological indexes and biochemical data. Scatter plots show the correlation of FcγRIIb expression
levels with BMI, Plt, and serum biochemical data. Statistical correlation was analyzed by the Pearson correlation coefficient
test. #: one outlier (indicated as in γ-GTP diagram) removed. : small correlation by the Pearson correlation coefficient test
(R<-0.3). : medium correlation by the Pearson correlation coefficient test (R<-0.4). Fig 2. Correlation with biological indexes and biochemical data. Scatter plots show the correlation of FcγRIIb expression
levels with BMI, Plt, and serum biochemical data. Statistical correlation was analyzed by the Pearson correlation coefficient
test. #: one outlier (indicated as in γ-GTP diagram) removed. : small correlation by the Pearson correlation coefficient test
(R<-0.3). : medium correlation by the Pearson correlation coefficient test (R<-0.4). https://doi.org/10.1371/journal.pone.0211543.g002 https://doi.org/10.1371/journal.pone.0211543.g002 other than FcγRIIb. Then, we examined the distribution of stabilin-2, a hyaluronan receptor in
liver from patients with NASH. Under the microscopic observation, stabilin-2 positive LSECs
tended to be more frequent in patients with low level of blood hyaluronan (S2 Fig), but non-
specific signals were detected in some biopsy specimen. Thus, we were unable to elucidate the
expression levels of stabilin-2 during NASH. PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Fc gamma RIIb in human NASH hepatic regeneration and healing [35, 36]. In a rodent NASH model, capillarization arises at an
early stage of NASH and exacerbates with NASH progression [11]. In human LSECs, the distribution pattern of FcγRIIb has recently been shown in intrahepatic
lobules of normal livers [37]. In relation to hepatic diseases, the expression levels of LSEC markers,
including FcγRIIb, of HCC patients were sequentially lost during tumor progression [22], and it
disappeared in 63% of cases in the peritumoral tissue samples of a tissue microarray of HCC [7]. Interestingly, several of the serum components that showed negative correlations with the
expression of FcγRIIb are ligands for LSEC receptors: hyaluronan, which is the first molecule
shown to be endocytosed via stabilin in LSECs, HDL-C, which binds to a receptor in LSEC,
and IVCol, which probably binds to the Mannose receptor in LSEC [17, 38]. These serum
components are well known to increase with the progression of NASH. The increase of indi-
vidual serum components indicates the progression of hepatic injury in the NASH patients
and probably reflects the functional status of scavenger receptors expressed in LSECs. Our
results indicate that the decrease of FcγRIIb expression may also be closely related to its scav-
enger function, involving several LSEC receptors. g
g
p
Hyaluronic acid synthesized by mesenchymal cells is generally eliminated by LSECs. Serum
hyaluronic acid contents increase in alcoholic liver disease, chronic hepatitis C, and NAFLD
[39–41]. Recently, it was demonstrated that the index of hyaluronic acid in patients can accu-
rately predict the survival rate of liver diseases of varying severity [20].Here, we attempted to
elucidate the expression of stabilin-2 proteins, one of the hyaluronan receptors, in liver biopsy
tissue of NASH patients, but we were unable to elucidate its relationship with NASH progres-
sion. However, there are several reports indicating the relationship between stalibin-2 and
FcγRIIb expression in HCC. Geraud and colleagues examined the expression of FcγRIIb and
stabilin-2 by using a tissue microarray of HCC patients and revealed the loss of both receptor
proteins in the majority of tumorous tissues [22]. In the peri-tumorous liver tissues, loss of sta-
bilin-2 expression was significantly less likely to occur (38%) than loss of FcγRIIb expression
(63%), and the loss of stabilin-2 in peri-tumorous tissues was associated with survival. PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 Discussion In this study, we determined the expression levels of FcγRIIb, which is a scavenger receptor on
the LSEC, in human NAFL and NASH biopsy specimens, and elucidated their correlations
with individual biological indexes and pathological grades. The FcγRIIb expression levels indi-
cated significant negative correlations with serum TG, TC, HDL-C, VICol, and hyaluronan
concentrations, respectively. Capillarization of LSECs is one of the basic pathological features of hepatic cirrhosis and
chronic hepatitis [34]. LSECs of cirrhosis patients produce IVCol, fibronectin, and laminin,
lose their characteristic fenestrations, and the expression levels of LSEC receptors LYVE-1,
FcγRIIb and stabilin are decreased [9]. The microenvironment caused by the transformed
LSECs is involved in the onset and progression of hepatic fibrosis, and instructively dictates Fig 3. Comparisons of diagnosis, sex, and pathological scores. Box plots show FcγRIIb expression levels. Lines inside the boxes denote medians, the boxes represent
the interquartile range, and whiskers extend to the minimum and maximum. The statistical differences among groups are analyzed by the Mann-Whitney or the
Kruskal-Wallis test. A P-value <0.05 was considered to be significant. https://doi org/10 1371/journal pone 0211543 g003 Fig 3. Comparisons of diagnosis, sex, and pathological scores. Box plots show FcγRIIb expression levels. Lines inside the boxes denote medians, the boxes represent
the interquartile range, and whiskers extend to the minimum and maximum. The statistical differences among groups are analyzed by the Mann-Whitney or the
Kruskal-Wallis test. A P-value <0.05 was considered to be significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 8 / 13 Conclusions In summary, the present study contributes to support the notion that the changes in LSEC
phenotype are one of crucial events in human NASH progression. Our results suggest that pro-
gression of inflammation, hepatic fibrosis, and hepatic lipid metabolism disorder during
NASH may influence the expression and scavenger functions of FcγRIIb in LSECs. Supporting information S1 Fig. Morphometric imaging for determining FcγRIIb expression levels. Representative
images of patients who were diagnosed as NASH (a: NAS, 2; b: NAS, 8). Whole tissue area
(other than the area shown in black) and FcγRIIb expression area (pink) were automatically
extracted under a determined procedure. In b, arrowheads indicate LSECs that do not detect
FcγRIIb signals. FcγRIIb signals detected in LSECs were accurately extracted. Scale bars repre-
sent 100 μm. S2 Fig. Stabilin-2 detection in NASH. Representative light microscopic images of liver tissues
in NASH patients with (a) blood hyaluronan: 23 ng/ml; FcgRIIb level: 8.97%; fibrosis stage:
1A; NAS: 4, (b) blood hyaluronan: 258 ng/ml; FcgRIIb level: 3.87%; fibrosis stage: 3; NAS: 5. Immunohistochmical signals of stabilin-2 in LSECs are detected (arrowheads). Unfortunately,
nonspecific signals are also detected in b. Nuclei were counterstained with hematoxylin (blue). Scale bars represent 100 μm. S1 Table. FcgR2b expression level. For the 26 patients, the two patients diagnosed with NAFL
are listed from the top, followed by the 24 diagnosed as NASH, listed in ascending order of
NAS and then ascending ALT value. The order of patients in this table is the same as Table 1. A comparison with the Fibrosis score and FcgR2b expression level is stated in Discussion.
FcgR2b positive area / whole tissue area. They
described that loss of stabilin-2 may enhance survival by preventing endothelial-tumor cell
adhesive interactions and microvascular invasion. Schledzewski and colleagues revealed that
serum hyaluronic acid levels were elevated in stabilin-1 and stabilin-2 double KO mice, but the
liver showed only mild perisinusoidal fibrosis without dysfunction [42]. They also indicated
that the expression levels of FcγRIIb and LYVE-1 in double KO mice were not different from
WT mice. From these previous observations, the attenuation pattern of individual LSEC recep-
tors appears to vary depending on the type of liver disease and the degree of progression. The
attenuation of FcγRIIb in LSECs seems to be involved in inflammation and fibrosis in NASH
patients, at the least. Platelets are also closely involved in inflammation and fibrosis, and the
platelet count of NASH patients decreases with progressive fibrosis [43]. Platelets mediate
binding of endothelial cells to T cells during hepatic injury [44], and promote various pro-
cesses such as inflammation, fibrosis and liver repair by release of biological substances [45]. In this study, although there was no correlation between platelet count and FcγRIIb expression
levels, the regression line indicates a positive slope. In other words, patients with low FcγRIIb
expression levels exhibit a tendency to have a low platelet count (Fig 2), a high inflammation
grade, or a high fibrosis stage (Fig 3). FcγRIIb expression levels may be related to the enhance-
ment of inflammation and fibrosis in NASH patients. The phenotypic changes of LSECs associated with various liver diseases have been clarified
in detail. In the LSEC studies, contradictory results may be shown depending on species differ-
ences, phenotypic markers used for identification, and culture conditions [46]. Here, we
showed that FcγRIIb expression may coincide with scavenger functions of several LSEC
ligands related to inflammation, hepatic fibrosis, and hepatic lipid metabolism. These findings PLOS ONE | https://doi.org/10.1371/journal.pone.0211543
January 29, 2019 9 / 13 Fc gamma RIIb in human NASH are useful to clarify details of transformation and dysfunction in LSECs during NAFLD. Fur-
ther investigations are required to assess the significance and to address the role of scavenger
receptors, including FcγRIIb, in LSECs during NASH progression. are useful to clarify details of transformation and dysfunction in LSECs during NAFLD. Fur-
ther investigations are required to assess the significance and to address the role of scavenger
receptors, including FcγRIIb, in LSECs during NASH progression. Acknowledgments The authors are grateful to Ms. Misako Shirai, who offered continuing support for this study,
and to Ms. Yuka Moriya for correcting grammatical errors and other problems with the lan-
guage of the manuscript. We also thank Dr. T. Takizawa for providing rabbit anti-human
FcγRIIb antisera. References 1. Wong RJ, Liu B, Bhuket T. Significant burden of nonalcoholic fatty liver disease with advanced fibrosis
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January 29, 2019 Fc gamma RIIb in human NASH Investigation: Tomoko Ishikawa. Methodology: Tomoko Ishikawa, Tomokazu Matsuura. Project administration: Tomoko Ishikawa. Resources: Hiroshi Yokoyama, Tomokazu Matsuura. Software: Tomoko Ishikawa. Supervision: Tomokazu Matsuura, Yoko Fujiwara. Validation: Tomoko Ishikawa, Hiroshi Yokoyama, Tomokazu Matsuura, Yoko Fujiwara. Visualization: Tomoko Ishikawa, Hiroshi Yokoyama. Writing – original draft: Tomoko Ishikawa. Writing – review & editing: Tomoko Ishikawa, Hiroshi Yokoyama, Tomokazu Matsuura,
Yoko Fujiwara. Methodology: Tomoko Ishikawa, Tomokazu Matsuura. Project administration: Tomoko Ishikawa. Resources: Hiroshi Yokoyama, Tomokazu Matsuura. Supervision: Tomokazu Matsuura, Yoko Fujiwara. Supervision: Tomokazu Matsuura, Yoko Fujiwara. Validation: Tomoko Ishikawa, Hiroshi Yokoyama, Tomokazu Matsuura, Yoko Fujiwara. Visualization: Tomoko Ishikawa, Hiroshi Yokoyama. Visualization: Tomoko Ishikawa, Hiroshi Yokoyama. Writing – original draft: Tomoko Ishikawa. Writing – review & editing: Tomoko Ishikawa, Hiroshi Yokoyama, Tomokazu Matsuura,
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THz Pulsed Imaging in Biomedical Applications
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1. Introduction The increasing request for diagnosis techniques in the biomedical area, able to provide
morphological, chemical and/or functional information for early, noninvasive and label-free detection,
has led to hard cooperation in different scientific fields. Driven by technological advances,
a plethora of new modern diagnostics systems is investigated and validated, as complementary
biomedical techniques to the conventional ones, covering various scales, from macromolecules to
tissues/organs. Among them, worthy of special mention are: infrared (IR) imaging [1,2], scanning
near-field microscopy [3,4], photoacoustic microscopy [5], ultrasonic imaging [6], optical coherence
tomography [7,8], digital holography microscopy [9–11], Raman scattering microscopy [12], Coherent
Raman Scattering (CRS) spectroscopic imaging [13–23], two-photon fluorescence (TPF) [19,24] and
second-harmonic generation (SHG) imaging [25–27], and super-resolved imaging techniques [28–31]. Many efforts were done in THz technology,
improving THz sources and detectors’
responses [32–38], and ensuring devices’ flexibility and portability, in recent decades. This has
stimulated a wide diffusion of THz systems for spectroscopic and imaging purposes, applicable in
various science fields like biology and medicine [39–44], gas sensing [45], chemical analysis [46,47],
new materials characterization in low-frequency range and non-destructive evaluation of composite
materials and constructions [48,49], astronomy [50], microelectronics and security [51–53], agri-food
industry [54], art conservation [55], etc. Thus, alongside these techniques, TPI has been developed and
applied in the field of biomedicine. The increasing request for diagnosis techniques in the biomedical area, able to provide
morphological, chemical and/or functional information for early, noninvasive and label-free detection,
has led to hard cooperation in different scientific fields. Driven by technological advances,
a plethora of new modern diagnostics systems is investigated and validated, as complementary
biomedical techniques to the conventional ones, covering various scales, from macromolecules to
tissues/organs. Among them, worthy of special mention are: infrared (IR) imaging [1,2], scanning
near-field microscopy [3,4], photoacoustic microscopy [5], ultrasonic imaging [6], optical coherence
tomography [7,8], digital holography microscopy [9–11], Raman scattering microscopy [12], Coherent
Raman Scattering (CRS) spectroscopic imaging [13–23], two-photon fluorescence (TPF) [19,24] and
second-harmonic generation (SHG) imaging [25–27], and super-resolved imaging techniques [28–31]. Many efforts were done in THz technology,
improving THz sources and detectors’
responses [32–38], and ensuring devices’ flexibility and portability, in recent decades. Received: 13 December 2019; Accepted: 24 March 2020; Published: 1 April 2020 Received: 13 December 2019; Accepted: 24 March 2020; Published: 1 April 2020 Abstract: Recent advances in technology have allowed the production and the coherent detection of
sub-ps pulses of terahertz (THz) radiation. Therefore, the potentialities of this technique have been
readily recognized for THz spectroscopy and imaging in biomedicine. In particular, THz pulsed
imaging (TPI) has rapidly increased its applications in the last decade. In this paper, we present a short
review of TPI, discussing its basic principles and performances, and its state-of-the-art applications
on biomedical systems. Keywords: terahertz spectroscopy; terahertz time-domain spectroscopy; terahertz pulsed imaging;
near-field imaging; biomedical imaging; cancer diagnosis; endoscopy THz Pulsed Imaging in Biomedical Applications Annalisa D’Arco 1,*
, Marta Di Fabrizio 2
, Valerio Dolci 1,2,3, Massimo Petrarca 1,3 and
Stefano Lupi 2,4 1
INFN-Section of Rome ‘La Sapienza’, P.le Aldo Moro 2, 00185 Rome, Italy; valerio.dolci@roma1.infn.it
massimo.petrarca@uniroma1.it (M.P.) 1
INFN-Section of Rome ‘La Sapienza’, P.le Aldo Moro 2, 00185 Rome, Italy; valerio.dolci@roma1.infn.it (V.D.)
massimo.petrarca@uniroma1.it (M.P.) p
(
)
2
Physics Department, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Rome, Italy;
martadifabrizio@gmail.com (M.D.F.); stefano.lupi@roma1.infn.it (S.L.) 2
Physics Department, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Rome, Italy;
martadifabrizio@gmail.com (M.D.F.); stefano.lupi@roma1.infn.it (S.L.)
3
SBAI-Department of Basic and Applied Sciences for Engineering, Physics, University of Rome ‘La Sapienza
Via Scarpa 16, 00161 Rome, Italy
4 Physics Department, University of Rome La Sapienza , Piazzale Aldo Moro 5, 00185 Rome, Italy;
martadifabrizio@gmail.com (M.D.F.); stefano.lupi@roma1.infn.it (S.L.)
3
SBAI-Department of Basic and Applied Sciences for Engineering, Physics, University of Rome ‘La Sapienza’,
Via Scarpa 16, 00161 Rome, Italy g
(
);
p
(
)
3
SBAI-Department of Basic and Applied Sciences for Engineering, Physics, University of Rome ‘La Sapienza’,
Via Scarpa 16, 00161 Rome, Italy 4
INFN-LNF Via E. Fermi 40, 00044 Frascati, Italy *
Correspondence: annalisa.darco@roma1.infn.it www.mdpi.com/journal/condensedmatter Condens. Matter 2020, 5, 25; doi:10.3390/condmat5020025 Review 1. Introduction This has
stimulated a wide diffusion of THz systems for spectroscopic and imaging purposes, applicable in
various science fields like biology and medicine [39–44], gas sensing [45], chemical analysis [46,47],
new materials characterization in low-frequency range and non-destructive evaluation of composite
materials and constructions [48,49], astronomy [50], microelectronics and security [51–53], agri-food
industry [54], art conservation [55], etc. Thus, alongside these techniques, TPI has been developed and
applied in the field of biomedicine. pp
THz radiation has a variety of properties that make this spectral region a viable spectroscopic
imaging technique. First of all, (i) THz radiation has low photon energy (4 meV at 1 THz), coinciding with
the energy levels related to rotational and vibrational molecule modes and intermolecular vibrations, Condens. Matter 2020, 5, 25; doi:10.3390/condmat5020025 www.mdpi.com/journal/condensedmatter 2 of 28 Condens. Matter 2020, 5, 25 such as hydrogen bonds. These low-frequency motions allow the identification of biomolecules,
characterizing their spectral features in THz region [56,57]. (ii) In addition, the low photon energy is
insufficient to cause atoms ionization. (iii) For this reason, it is suitable and attractive for noninvasive
biomedical imaging, has a great potentiality and could be applied in vivo real-time studies without
causing biological ionizing damage, unlike X- and γ-rays [42,43]. Whereas extremely low frequency
electromagnetic fields have been long studied in relation to their possible human health effects,
the biological effects of radiofrequency and THz signals have come under scrutiny. In particular,
the effects of THz radiation are of interest because of the expanding THz technologies in biomedical
applications. The prevailing view is that THz radiation, if thermal effects are not considered, does not
damage directly the DNA, but it could act as a co-inductor for non-thermal effects [58]. Recent
studies show evidence of potential thermal and non-thermal effects induced by exposure to THz
radiation [59,60]. The biological response to this stimulus is due to different parameters: the physical
settings of the THz radiation (frequency, mean and peak power, radiation intensity, continuous or
pulsed radiation, the degree of coherence, etc.,); the physical and biological properties of the irradiated
biological object (refractive index, absorption features, type of cells, tissues, organs) and the design
of the exposure [60]. (iv) In addition, THz radiation is very sensitive to polar molecules, like water. 1. Introduction (v) That is why the strong absorption, due to water molecules (220 cm−1 for pure water at 1 THz
and room temperature) [43], limits THz penetrability into the fresh tissues from tens to hundreds
microns [42] and the capability of diagnostic only for superficial layers in vivo. (vi) The high sensitivity
of water content can be used like an endogenous marker for the differentiation between fresh healthy
and pathological tissues [42,43]. (vii) In particular, the time-domain spectroscopy (TDS) and imaging
are insensitive to the thermal background and show high signal-to-noise ratio (SNR) [61]. (viii) TPI
coherent detection ensures the record of temporal profile of the THz electric field; so, both the THz
amplitude and phase can be estimated. From these, the broadband absorption coefficient and refractive
index of a medium are determined, without using Kramers–Kronig relations [57,61]. The absorption is
generally due to the chemical constituents of the material; thus, its spectral signatures are measured,
providing useful information. As consequence, TPI has the potential to realize images with both
morphological and functional information, in this frequency range. For all these reasons, THz radiation
is considered very important and deserves special attention. In this review, we introduce TPI, discussing its basic principles and performances, and the
state-of-the-art applications on biomedical systems. The review is organized as follows: in the first section, we outline the main features of pulsed
THz systems and their key role in THz imaging setups, showing the equipment needed and some
application ideas. Later, in the following section, after summarizing the main properties of the THz
contrast image, we also focus on TPI challenges and relevant results achieved in the biomedical field. 2. THz Pulsed System/Imaging Over the past two decades, THz generation and detection technology has considerably advanced,
and several devices are commercially available now [33–37,62,63]. Their development has led to various
spectroscopic and imaging techniques in the THz spectral window. According to THz sources used,
the systems are divided into two general categories: the continuous wave (CW) systems, which produce
a single or several discrete frequencies, and pulsed systems, characterized by a broadband frequency
output, introduced for the first time as potential imaging tool in 1995 by Hu and Nuss [64]. All of them
feature a large variety of properties, as well as, frequencies, power and sensitivity. In this paragraph,
we briefly discuss the most common TPI systems that are the aim of our review, with a strong emphasis
on THz pulsed sources and detectors. 2.1. Generation and Detection of THz Pulsed Radiation 2.1. Generation and Detection of THz Pulsed Radiation In TPI systems, a broadband frequency emission, from tens or hundreds of GHz up to several
THz, is obtained. Currently, various modalities can be listed for generating and detecting pulsed 3 of 28 Condens. Matter 2020, 5, 25 THz radiation [65,66]: biased photoconductive antennas (PCAs) [67–69], optical rectification (OR)
in nonlinear optical (NLO) crystals [70–72], THz production by plasma in air [73–75] and carrier
tunneling in coupled double quantum well structures [76–78]. They exhibit different physical properties
concerning powers, used materials, covered spectral range, SNR and dynamic range (DR). Among them,
the most established approaches, based on PCAs and on OR, require pumping made by expensive
infrared femtosecond lasers, often emitting in the near-infrared (NIR) region. For the first mentioned,
a pulsed laser beam illuminates PCA gap, made of high resistive semiconductor thin film with two
electrical contact pads. Thus, in the presence of the applied bias voltage and laser beam, photocurrent
is generated and the static bias field accelerates the free carriers producing broadband frequency THz
waves into the free space. OR ensures the THz broadband generation through NLO centrosymmetric crystals [70–72],
extended from 0.1 THz and beyond 40 THz [57,79,80],
such as organic NLO crystals. 4-N,N-dimethylamino -4’-N’-methyl -stilbazolium 2,4,6-trimethylbenzenesulfonate (DSTMS) [81–84]
and 4-N,N-dimethylamino-4’-N’-methyl-stilbazolium tosylate (DAST) show efficient generation from
0.3 to >16 THz in the phase-matching condition between 720 nm and 1650 nm. The NLO materials need
to have high second-order NLO susceptibility, high transparency at both pump and THz frequency,
and high optical damage threshold, in order to satisfy the phase-matching condition between the
fundamental optical pump and generated THz waves [85,86]. When intense NIR laser beams propagate
through some crystals, second order nonlinear effects occur, developing a DC or low frequency
polarization [38]. This leads to the radiation of an electromagnetic single-cycle pulse with a wide
frequency spectrum, ranging roughly from zero frequency to some maximum value [38]. Another way to produce THz radiation is by charges acceleration. As consequence, the charges
radiate electromagnetic waves, that under particular conditions, lie in the THz region of the
electromagnetic spectrum. 2.1. Generation and Detection of THz Pulsed Radiation The processes can be done either within semiconductors, in vacuum
or in air [87–90]: via electrons acceleration by the intense laser pulse [87,88], by an applied bias voltage
across an air gap, similarly to PCA, or by nonlinear four-wave mixing of the fundamental and the
second harmonic frequencies of the laser beam, in air or in various gases [89,90]. Besides OR, photoconduction or photocurrent, surge is another primary mechanism for THz
generation from semiconductor materials [57,59]. When semiconductor quantum wells (QWs) are
under a bias, the mechanism for THz generation is attributed to the creation of polarized electron–hole
pairs [91–93]. In its turn, two schemes are ideal to THz pulse detection, based on incoherent and coherent
techniques. In terms of incoherent techniques, power-to-signal transducers, suitable for incoming
energy measurements, are used. For coherent ones instead, the detectors give information concerning
the power, frequency spectral range and phase of the incoming electrical signals [57]. Among THz
incoherent detectors commercially available, we can mention bolometers [94]; Golay-cells, providing
stable performances and high sensitivity; and pyroelectric detectors [95] and thermopiles with low
sensitivity. Commercial bolometers, resistive temperature sensors, widely used in THz spectral
region, are cryogenic ones. The cooling of the devices ensures high sensitivity, with noise equivalent
powers (NEPs) ranging from 10−12 W/Hz1/2 to 10−14 W/Hz1/2. Golay-cell detectors [96] measure
the thermal energy of THz propagating through a window [61]. Their spectral performances are
strongly dependent on the material used for the window. In the case of diamond and crystal quartz
windows, the spectral range covers from millimeters-wave to visible frequencies [61], while for HDPE
(High-Density Polyethylene) window the spectral range is upper bounded at ~20 THz. Power DR can
vary from hundreds nW up to 1 mW, with power DR from hundreds nW up to 1 mW. These devices can
reach excellent NEPs at room temperature—around ~10−10 W/Hz1/2 [61]. Instead, pyroelectric devices
show lower sensitivity (NEPs ~10−9–10−10 W/Hz1/2) compared to bolometers and Golay performances. Table 1 summarizes some performances of the most used incoherent THz detectors. 4 of 28 Condens. Matter 2020, 5, 25 Table 1. Main incoherent detectors suitable for THz spectral range with their performances:noise
equivalent powers NEPs, THz coverage and detector bandwidth, linked to the detector response time
(the higher the bandwidth, the faster the response time). Bolometer, Golay cell and Pyroelectric are pure
incoherent detectors, while Schottky diode is used both for coherent and incoherent measurements. 2.1. Generation and Detection of THz Pulsed Radiation Detector
NEP (W/ Hz1/2)
THz Coverage (THz)
Bandwidth (Hz)
Cryogenic Bolometer
10−14–10−12
0.1–30
102–103
Golay cell
10−10
0.01–20 1
0.01–700 2
10–102
Pyroelectric
10−10–10−9
0.1–10
10–108
Schottky diode
10−14–10−11
0.1–2
102–106
1 High-Density Polyethylene (HDPE) window; 2 Diamond window. THz cameras were the object of intense research activities in recent years. Actually, various THz
cameras, based either on cooled and uncooled bolometers or semiconductor devices, are commercially
available. Among the THz thermal cameras, the Golay cells are slow detectors and are difficult to
integrate into array detectors. Consequently, the imaging scheme for them is a single-pixel detector [97]. The pyroelectric cameras, like the Pyrocam series sold by Ophir Optics, can operate with pulsed and
CW sources, as well as thermopile cameras [98,99]. Generally, they are widely commercially available,
and their low sensitivity makes them less attractive for THz imaging. The microbolometric cameras
are arrays of bolometers; because of the relatively higher sensitivity they are considered the prime
candidates for THz imaging. When cooled at cryogenic temperatures, they can achieve NEPs around
10−16 W/Hz1/2. With proper design modifications, microbolometric cameras at room temperature also
achieve high enough sensitivity. Many such imaging techniques cannot be realized with existing THz cameras, but require
a coherent detection scheme. Among commercial coherent or heterodyne detectors, largely used,
we mention: Schottkly diodes [100,101], PCAs and NLO crystals or via electro-optical (EO) effect
in nonlinear crystals [70–72]. Schottkly diodes [100,101] show limited spectral bandwidth around
1–2 THz and NEPs up to 10−14 W/Hz1/2, and actually they are the object of research. PCAs (frequency
range limited <10 THz and NEPs around 10–12 W/Hz1/2) can detect a THz electric field profile with
a coherent detection scheme. The probe beam, derived from the same laser used to generate the
THz radiation, irradiates receiver PCA, which has no external bias voltage. The laser pulses gate the
detector acting as a photocurrent switch by generating charge carriers. The incoming THz radiation,
on the receiver, induces the bias voltage, thus the photocarriers are accelerated [67,69]. The transient
current is measured by a low-current amplifier and a lock-in technique, that are referenced against
a modulation placed on the PCA emitter bias voltage. In EO rectification NLO crystals, instead, the THz field induces birefringence in them; at a given
instant, the polarization state of the probe laser pulse changes proportionally to the THz field
amplitude [25,102]. 2.1. Generation and Detection of THz Pulsed Radiation Then, a balanced photodiode is used for measuring these changes in birefringence,
reconstructing the THz pulse. In recent years, great interest in THz sensors and cameras is focused
on the development of devices with high sensitivity, which operate at room temperature and fast
operation speed [103], such as optoelectronic sensors and detectors [104–107]. 2.2. TPI Equipment The switch from the spectroscopy system to the imaging one is based on the possibility to collect
spectroscopic information on large sample areas. For this purpose, the point-by-point signal collection
requires a mechanism for scanning: moving the beam or the sample holder. The first case is realized
with a beam motion or oscillating objective; using for example, galvo-mirrors and/or piezoelectric
rotator or stages. In the second case, the lateral translation of the specimen stage is combined with
a stationary illumination; for this purpose, a 3D linear translation stage is used, allowing sample holder
motion along the THz beam (1D motion) and perpendicularly to it (2D motion). 5 of 28
of 26 Condens. Matter 2020, 5, 25
Condens. Matter 2020, 5, x FO The acquisition speed is one of the most critical challenges when using THz radiation for biomedical
imaging, and it requires more attention for the implementation of clinical devices. The imaging speed
depends on the specific image scheme chosen: using a point source and detector, the acquisition is slow,
due to the point-by-point sample scanning. Although, the two-dimensional galvo-scanner, combined
with a fast detector or sophisticated signal processing techniques [108–110], improves the scanning
speed and the acquisition on a large field of view. motion). The acquisition speed is one of the most critical challenges when using THz radiation for
biomedical imaging, and it requires more attention for the implementation of clinical devices. The
imaging speed depends on the specific image scheme chosen: using a point source and detector, the
acquisition is slow, due to the point‐by‐point sample scanning. Although, the two‐dimensional
galvo‐scanner, combined with a fast detector or sophisticated signal processing techniques [108–
110] improves the scanning speed and the acquisition on a large field of view The object surface is illuminated by the THz beam, and it is sampled on a discrete grid, scanned
in continuous or pixel-by-pixel raster modes. The information collected in each pixel is digitalized,
transferred to PC from a data acquisition card (DAQ) and quantized in a finite number of bits for pre-
and post-image processing. 110], improves the scanning speed and the acquisition on a large field of view. The object surface is illuminated by the THz beam, and it is sampled on a discrete grid, scanned
in continuous or pixel‐by‐pixel raster modes. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems A beamsplitter splits the
itial laser beam into two parts: the pump and the probe beams. The pump beam is modulated
a an optical or mechanical chopper or by modulating the THz emitter bias voltage, and then is
cused on the THz emitter. The generated THz radiation is then collimated and focused onto a target
mple. When a THz pulse illuminates a target, a train of THz pulses will be reflected from the various
terfaces or transmitted. The transmitted (Figure 1b) and/or the reflected (Figure 1b) electric fields
e, then, recollimated and refocused onto THz detector using THz transparent lenses or parabolic
irrors. The electrical signal is filtered and detected by lock-in amplifier detection. The analog output
the lock-in amplifier is collected and digitalized by a dedicated DAQ. A delay line is used in order
temporally sample the THz electric field. Thus, for each individual pulse in the detected signal,
e amplitude and phase are different and are measured too. Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of common
TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond laser, BS for
beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock‐in amplifier, data
acquisition card and desktop computer, respectively. (b) The schematic layout of two potential
sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low‐absorption material plates or standing
in free space. The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz
signal illuminates one sample surface and it is reflected by the same surface
Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of
common TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond
laser, BS for beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock-in
amplifier, data acquisition card and desktop computer, respectively. (b) The schematic layout of two
potential sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low-absorption material plates or standing
in free space. 2.2. TPI Equipment The information collected in each pixel is digitalized,
transferred to PC from a data acquisition card (DAQ) and quantized in a finite number of bits for
pre‐ and post‐image processing. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems A typical pulsed THz wave generation and detection system is a pump-probe setup. It can be
considered as an extended THz time-domain spectroscopy (THz-TDS) method. The basic idea of
THz-TDS and TPI systems is illustrated in Figure 1a. y
A typical pulsed THz wave generation and detection system is a pump‐probe setup. It can be
considered as an extended THz time‐domain spectroscopy (THz‐TDS) method. The basic idea of
THz‐TDS and TPI systems is illustrated in Figure 1a. Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of common
TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond laser, BS for
beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock‐in amplifier, data
acquisition card and desktop computer, respectively. (b) The schematic layout of two potential
sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low‐absorption material plates or standing
in free space. The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz
signal illuminates one sample surface and it is reflected by the same surface
Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of
common TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond
laser, BS for beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock-in
amplifier, data acquisition card and desktop computer, respectively. (b) The schematic layout of two
potential sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low-absorption material plates or standing
in free space. The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz signal
illuminates one sample surface and it is reflected by the same surface. Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of common
TPI system based on photoconductive switches. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems Here, Fs laser stands for femtosecond laser, BS for
beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock‐in amplifier, data
acquisition card and desktop computer, respectively. (b) The schematic layout of two potential
sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low‐absorption material plates or standing
in free space. The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz
signal illuminates one sample surface and it is reflected by the same surface. TPI system, shown in Figure 1a, is powered by a femtosecond laser. A beamsplitter splits the
nitial laser beam into two parts: the pump and the probe beams. The pump beam is modulated via
n optical or mechanical chopper or by modulating the THz emitter bias voltage, and then is focused
n the THz emitter. The generated THz radiation is then collimated and focused onto a target
ample. When a THz pulse illuminates a target, a train of THz pulses will be reflected from the
arious interfaces or transmitted. The transmitted (Figure 1b) and/or the reflected (Figure 1b) electric
elds are, then, recollimated and refocused onto THz detector using THz transparent lenses or
arabolic mirrors. The electrical signal is filtered and detected by lock‐in amplifier detection. The
nalog output of the lock‐in amplifier is collected and digitalized by a dedicated DAQ. A delay line
Figure 1. This is an example of TPI system switched by PCAs. (a) The schematic layout of
common TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond
laser, BS for beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock-in
amplifier, data acquisition card and desktop computer, respectively. (b) The schematic layout of two
potential sample orientation: transmission and reflection modes. The standard transmission imaging
configuration involves the specimen placed between THz low-absorption material plates or standing
in free space. The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz signal
illuminates one sample surface and it is reflected by the same surface. TPI system, shown in Figure 1a, is powered by a femtosecond laser. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems Our system exhibits
performances comparable with the commercial systems, and being a home-made spectrometer, it can
switch on the other two spectroscopic configurations using metallic flip mirrors, inserted in the THz
collimated region. We can select between THz–TDS refection spectroscopy and attenuated total
reflection (ATR) spectroscopy, see Figure 2a. plemented in transmission configuration. It generates broadband THz radiation (0.1–3.5 THz) via photoconductive switches, used both
r THz emission and detection. We use a mode‐locked ultrafast laser (FemtoFiber NIRpro, Toptica),
780 nm with a 100 fs temporal pulse width and a repetition rate of 80 MHz to illuminate twin
0620‐11 Hamamatsu PCAs. The femtosecond laser is split into two beams by a 50:50
amsplitter—each beam has 15 mW average power. Dielectric mirrors deflect the beams towards
mitter and detector PCAs. TPX lenses are used to collimate and focalize THz radiation. The object is
gned in the focus of the THz beam, perpendicularly to the propagating THz radiation. The
int‐by‐point spectral signal collection is obtained by scanning a large area of the object with 3D
es stage (Thorlabs). After transmission through the sample, THz pulses are collimated and
focused on the THz‐detector PCA by TPX lenses. Simultaneously, the probe beam is used to gate
e THz‐detector PCA. The THz electric field as a function of time is measured with a delay line
12]. The output signal from the THz‐detector, extracted by a lock‐in amplifier (Stanford, SR830), is
ansferred to a National Instrument acquisition card (NI 6361‐BNC connector) allowing the data
llection. Our system exhibits performances comparable with the commercial systems, and being a
me‐made spectrometer, it can switch on the other two spectroscopic configurations using metallic
p mirrors, inserted in the THz collimated region. We can select between THz–TDS refection In Figure 2b, the temporal electric field profile, in free-space, as a function of time, is reported
with relative power spectrum, for the scheme in transmission. ectroscopy and attenuated total reflection (ATR) spectroscopy, see Figure 2a. In Figure 2b, the temporal electric field profile, in free‐space, as a function of time, is reported
ith relati e power spectrum for the scheme in transmission gure 2. (a) Schematic layout of our TPI system. In the scheme, Fs laser stands for femtosecond laser,
S for beamsplitter, DL for delay line, LS is referred to lenses, THz det. and THz emit. are THz
etector and THz emitter respectively. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems Matter 2020, 5, x FOR P If the pump and probe optical paths are equal, the THz electric field is measured at one fixed
instant in time. In order to sample the whole THz pulse, a delay line is introduced in the optical
scheme, to delay pump and probe beams. Generally, this can be performed using a mechanical
translation stage, that moves at fixed steps; but rapid-scanning delay lines can also be used to speed
the acquisition [111,112]. tected signal, the amplitude and phase are different and are measured too. If the pump and probe optical paths are equal, the THz electric field is measured at one fixed
stant in time. In order to sample the whole THz pulse, a delay line is introduced in the optical
heme, to delay pump and probe beams. Generally, this can be performed using a mechanical
anslation stage, that moves at fixed steps; but rapid‐scanning delay lines can also be used to speed For example, we reported our home-made TPI system based on THz-TDS (see Figure 2a). It was
lemented in transmission configuration. uisition [111,112]. r example, we reported our home‐made TPI system based on THz‐TDS (see Figure 2a). It was It generates broadband THz radiation (0.1–3.5 THz) via photoconductive switches, used both
for THz emission and detection. We use a mode-locked ultrafast laser (FemtoFiber NIRpro, Toptica),
at 780 nm with a 100 fs temporal pulse width and a repetition rate of 80 MHz to illuminate twin G10620-11
Hamamatsu PCAs. The femtosecond laser is split into two beams by a 50:50 beamsplitter—each
beam has 15 mW average power. Dielectric mirrors deflect the beams towards emitter and detector
PCAs. TPX lenses are used to collimate and focalize THz radiation. The object is aligned in the focus
of the THz beam, perpendicularly to the propagating THz radiation. The point-by-point spectral
signal collection is obtained by scanning a large area of the object with 3D axes stage (Thorlabs). After transmission through the sample, THz pulses are collimated and refocused on the THz-detector
PCA by TPX lenses. Simultaneously, the probe beam is used to gate the THz-detector PCA. The THz
electric field as a function of time is measured with a delay line [112]. The output signal from
the THz-detector, extracted by a lock-in amplifier (Stanford, SR830), is transferred to a National
Instrument acquisition card (NI 6361-BNC connector) allowing the data collection. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems The generated THz signal travels at a normal incidence to the sample surface and the
transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz signal
illuminates one sample surface and it is reflected by the same surface. TPI system, shown in Figure 1a, is powered by a femtosecond laser. A beamsplitter splits the
initial laser beam into two parts: the pump and the probe beams. The pump beam is modulated via
an optical or mechanical chopper or by modulating the THz emitter bias voltage, and then is focused
on the THz emitter. The generated THz radiation is then collimated and focused onto a target
sample. When a THz pulse illuminates a target, a train of THz pulses will be reflected from the
various interfaces or transmitted. The transmitted (Figure 1b) and/or the reflected (Figure 1b) electric
fields are, then, recollimated and refocused onto THz detector using THz transparent lenses or
parabolic mirrors. The electrical signal is filtered and detected by lock‐in amplifier detection. The
analog output of the lock‐in amplifier is collected and digitalized by a dedicated DAQ. A delay line
TPI system, shown in Figure 1a, is powered by a femtosecond laser. A beamsplitter splits the
initial laser beam into two parts: the pump and the probe beams. The pump beam is modulated
via an optical or mechanical chopper or by modulating the THz emitter bias voltage, and then is
focused on the THz emitter. The generated THz radiation is then collimated and focused onto a target
sample. When a THz pulse illuminates a target, a train of THz pulses will be reflected from the various
interfaces or transmitted. The transmitted (Figure 1b) and/or the reflected (Figure 1b) electric fields
are, then, recollimated and refocused onto THz detector using THz transparent lenses or parabolic
mirrors. The electrical signal is filtered and detected by lock-in amplifier detection. The analog output
of the lock-in amplifier is collected and digitalized by a dedicated DAQ. A delay line is used in order
to temporally sample the THz electric field. Thus, for each individual pulse in the detected signal,
the amplitude and phase are different and are measured too. 6 of 28
f 26 Condens. Matter 2020, 5, 25
ndens. 2.2.1. TPI Far-Field Systems
2 2 1 TPI Far‐Field Systems With two metal flip mirrors, introduced in the collimated THz
eam region, one can select the scheme for THz spectroscopic imaging in transmission (B) or
flection (C) or ATR (A) spectroscopy. (b) The temporal electric field profile, in free‐space, as a
nction of time, is reported with relative power spectrum. Figure 2. (a) Schematic layout of our TPI system. In the scheme, Fs laser stands for femtosecond laser,
BS for beamsplitter, DL for delay line, LS is referred to lenses, THz det. and THz emit. are THz detector
and THz emitter respectively. With two metal flip mirrors, introduced in the collimated THz beam
region, one can select the scheme for THz spectroscopic imaging in transmission (B) or reflection (C)
or ATR (A) spectroscopy. (b) The temporal electric field profile, in free-space, as a function of time,
is reported with relative power spectrum. gure 2. (a) Schematic layout of our TPI system. In the scheme, Fs laser stands for femtosecond laser,
for beamsplitter, DL for delay line, LS is referred to lenses, THz det. and THz emit. are THz
tector and THz emitter respectively. With two metal flip mirrors, introduced in the collimated THz
am region, one can select the scheme for THz spectroscopic imaging in transmission (B) or
lection (C) or ATR (A) spectroscopy. (b) The temporal electric field profile, in free‐space, as a
nction of time is reported with relative power spectrum
Figure 2. (a) Schematic layout of our TPI system. In the scheme, Fs laser stands for femtosecond laser,
BS for beamsplitter, DL for delay line, LS is referred to lenses, THz det. and THz emit. are THz detector
and THz emitter respectively. With two metal flip mirrors, introduced in the collimated THz beam
region, one can select the scheme for THz spectroscopic imaging in transmission (B) or reflection (C)
or ATR (A) spectroscopy. (b) The temporal electric field profile, in free-space, as a function of time,
is reported with relative power spectrum. 7 of 28 7 of 28 Condens. Matter 2020, 5, 25 2.2.2. TPI Near-Field Systems This means that NF resolution is not limited by diffraction so that, in this way it
becomes independent of the wavelength. The direct-contact approach, instead, is generally employed with EO sampling using THz-TDS. The sample could be then in contact with the detection crystal, leading to a direct measurement of
the THz electric field in NF region. In [130], the deposition of various metal structures onto a gallium
phosphite (GaP) crystal can improve the resolution by imaging at 20 µm. 2.2.2. TPI Near-Field Systems In this way, high resolutions are achieved (even λ/100), with the only limitations imposed by the tip
diameter [129]. This means that NF resolution is not limited by diffraction so that, in this way it
becomes independent of the wavelength. at the wavelength λ and numerical aperture NA. The minimal resolvable feature corresponds to the
minimum size, in the object plane, of the smallest object that the optical system can resolve: as the
resolution is directly proportional to the wavelength and inversely proportional to the numerical
aperture of the optical components, at 1 THz the resolution turns out to be around 0.5 mm [113]. In fact, 1 THz corresponds to 0.3 mm and one must consider the loss of resolution caused mostly by
lenses on the beam path. For this reason, effective and wide spreading of THz images in biomedical
research and diagnostics is difficult. The wavelength-limited resolution is caused by diffraction of
propagating waves, so this problem can be overcome by collecting THz pulses in near-field (NF),
namely at a distance with the sample comparable to the wavelength. In this way, evanescent waves,
that propagate only very close to the sample and then rapidly decay, can be detected. That is the reason
why THz detector must be placed in proximity of the sample. Several techniques have been explored
to enable sub-wavelength THz imaging [37,114,119,120]. With this purpose, detectors and sources of
different shapes can be used; in particular, Hunsche et al. [121] proposed and demonstrated, for the
first time, a method deriving from the confocal microscopy. It uses pinholes that act as spatial filter,
reducing the THz beam size to sub-wavelength dimensions and blocking outside the cone of light. This results in increased lateral and depth resolutions [122,123]. However, the aperture introduces
an immense loss of THz light intensity at deep sub-wavelength resolutions (<λ/10) [124], caused by
the low frequency cut-off[125] and optical coupling loss. Following, it is possible to place a small
detector or emitter directly behind the aperture [126–128]. The approaches without apertures, instead,
are better set to achieve deep sub-wavelength resolutions, because the low-frequency cut-offis avoided. The sharp metallic tips for local field enhancement, tip apex, small detectors and/or sources elements
with sub-wavelength dimensions are placed in proximity of the sample surface or in direct-contact. In this way, high resolutions are achieved (even λ/100), with the only limitations imposed by the tip
diameter [129]. 2.2.2. TPI Near-Field Systems The major limitation of THz far-field imaging systems is due to the problem of the
diffraction-limited spatial resolution central to THz wavelengths, compared, for example, to visible
radiation [113,114]. The optical resolution R in the object plane is generally defined by the Rayleigh
criterion [115–118], described as: R = 0.61·λ
NA
≈
λ
2NA R = 0.61·λ
NA
≈
λ
2NA at the wavelength λ and numerical aperture NA. The minimal resolvable feature corresponds to the
minimum size, in the object plane, of the smallest object that the optical system can resolve: as the
resolution is directly proportional to the wavelength and inversely proportional to the numerical
aperture of the optical components, at 1 THz the resolution turns out to be around 0.5 mm [113]. In fact, 1 THz corresponds to 0.3 mm and one must consider the loss of resolution caused mostly by
lenses on the beam path. For this reason, effective and wide spreading of THz images in biomedical
research and diagnostics is difficult. The wavelength-limited resolution is caused by diffraction of
propagating waves, so this problem can be overcome by collecting THz pulses in near-field (NF),
namely at a distance with the sample comparable to the wavelength. In this way, evanescent waves,
that propagate only very close to the sample and then rapidly decay, can be detected. That is the reason
why THz detector must be placed in proximity of the sample. Several techniques have been explored
to enable sub-wavelength THz imaging [37,114,119,120]. With this purpose, detectors and sources of
different shapes can be used; in particular, Hunsche et al. [121] proposed and demonstrated, for the
first time, a method deriving from the confocal microscopy. It uses pinholes that act as spatial filter,
reducing the THz beam size to sub-wavelength dimensions and blocking outside the cone of light. This results in increased lateral and depth resolutions [122,123]. However, the aperture introduces
an immense loss of THz light intensity at deep sub-wavelength resolutions (<λ/10) [124], caused by
the low frequency cut-off[125] and optical coupling loss. Following, it is possible to place a small
detector or emitter directly behind the aperture [126–128]. The approaches without apertures, instead,
are better set to achieve deep sub-wavelength resolutions, because the low-frequency cut-offis avoided. The sharp metallic tips for local field enhancement, tip apex, small detectors and/or sources elements
with sub-wavelength dimensions are placed in proximity of the sample surface or in direct-contact. 3. Basics of THz Imaging TPI can be viewed as an extension of the THz-TDS method. The classic way to obtain THz imaging
from THz-TDS is illustrated above. In order to image the objects, a 2D point-by-point raster scanning
of them, combined with a coherent detection, is performed; and the individual temporal profiles are
recorded for each point-pixel. The raw one-dimensional time or extracted frequency data are converted
into physical values [57,61,64,131,132]. The normalization is chosen to enhance the image contrast,
and it can be summarized in the diagram (Figure 3). 8 of 28
f Condens. Matter 2020, 5, 25 Figure 3. Schematic summary concerning the image contrast in THz‐TDS imaging
Figure 3. Schematic summary concerning the image contrast in THz-TDS imaging Figure 3. Schematic summary concerning the image contrast in THz‐TDS imaging. Figure 3. Schematic summary concerning the image contrast in THz-TDS imaging. In time domain, when there is interaction with the sample, the THz pulse can be attenuated,
elayed or broadened, compared to the reference without the sample, generally measured in air. The
ectric field amplitude, at a fixed time, represents a contrast modality to normalize the image. In
ddition, the normalized amplitude of the main peak, as the ratio between maxima of the sample
nd reference electric fields, provides image information regarding the absorption, reflection or
cattering losses in the object. Finally, the knowledge of time delay of the main peak with respect to
he reference allows mapping the optical path changes, providing material/thickness contrast [133]. or example, the time‐of‐flight (TOF) technique permits to estimate the depth information about
arget internal dielectric profiles. A THz pulse is launched to the sample and the reflected echo is
measured in amplitude or/and phase. The TOF information of the echo pulse indicates the presence
f the boundaries, or inner structures along the THz propagation path, which extracts the
ne‐dimensional depth profile. Thus, by performing a 2D scan, a 3D image of the object can be
isualized, viewing into a layered structure or inside the optically opaque materials. 3D imaging
otentiality is suitable for the implementation of various imaging techniques, largely used in
iomedical contexts. Applying the Fourier transform on the temporal electric pulse, one gets the
mplitude and phase of the spectrum. The first gives a general indication of the losses; the second
ne is related to the refractive index. 3. Basics of THz Imaging Fixing the frequency, one can visualize the object in the
equency domain, the amplitude and the phase images [134]. This offers more contrast, because of
he sufficiently different refractive indexes of the materials than the losses, which provide a
In time domain, when there is interaction with the sample, the THz pulse can be attenuated,
delayed or broadened, compared to the reference without the sample, generally measured in air. The electric field amplitude, at a fixed time, represents a contrast modality to normalize the image. In addition, the normalized amplitude of the main peak, as the ratio between maxima of the sample
and reference electric fields, provides image information regarding the absorption, reflection or
scattering losses in the object. Finally, the knowledge of time delay of the main peak with respect to
the reference allows mapping the optical path changes, providing material/thickness contrast [133]. For example, the time-of-flight (TOF) technique permits to estimate the depth information about target
internal dielectric profiles. A THz pulse is launched to the sample and the reflected echo is measured
in amplitude or/and phase. The TOF information of the echo pulse indicates the presence of the
boundaries, or inner structures along the THz propagation path, which extracts the one-dimensional
depth profile. Thus, by performing a 2D scan, a 3D image of the object can be visualized, viewing into
a layered structure or inside the optically opaque materials. 3D imaging potentiality is suitable for the
implementation of various imaging techniques, largely used in biomedical contexts. Applying the
Fourier transform on the temporal electric pulse, one gets the amplitude and phase of the spectrum. The first gives a general indication of the losses; the second one is related to the refractive index. Fixing
the frequency, one can visualize the object in the frequency domain, the amplitude and the phase
images [134]. This offers more contrast, because of the sufficiently different refractive indexes of the
materials than the losses, which provide a meaningful contrast. he sufficiently different refractive indexes of the materials than the losses, which provide a
meaningful contrast. Summarizing, the THz‐TDS measurements allow the extraction of both amplitude and phase of
Hz waveform. The complex refractive index and absorption coefficient can be obtained using the
resnel coefficients [135,136], and from them the complex permittivity [137,138]. 3. Basics of THz Imaging The knowledge of
ample spectral behavior ensures to extract information concerning its morphological structure and
he optical properties: the variation of refractive indexes could differentiate tissues, and could be
elated to the pathological status. Moreover, many materials are relatively transparent to THz
adiation, and are used in the development of 3D THz imaging modalities that constitute an active
esearch field [107,139]. Tomographic slices are employed for object/tissue 2D or 3D reconstructions,
n order to obtain their internal properties and features [111]. The method tried a natural extension
or the object rotation towards THz computer tomography (CT) [140,141]. In addition to THz CT,
here are several other tomographic techniques based on THz radiation, including THz interaction
Summarizing, the THz-TDS measurements allow the extraction of both amplitude and phase
of THz waveform. The complex refractive index and absorption coefficient can be obtained using
the Fresnel coefficients [135,136], and from them the complex permittivity [137,138]. The knowledge
of sample spectral behavior ensures to extract information concerning its morphological structure
and the optical properties: the variation of refractive indexes could differentiate tissues, and could
be related to the pathological status. Moreover, many materials are relatively transparent to THz
radiation, and are used in the development of 3D THz imaging modalities that constitute an active
research field [107,139]. Tomographic slices are employed for object/tissue 2D or 3D reconstructions,
in order to obtain their internal properties and features [111]. The method tried a natural extension for
the object rotation towards THz computer tomography (CT) [140,141]. In addition to THz CT, there are
several other tomographic techniques based on THz radiation, including THz interaction tomography,
THz tomosynthesis, time-of-flight pulsed imaging and 3D THz holography [139]. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications . THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications
As briefly discussed in the introduction and in the previous paragraph, the unique spectral
eatures of THz radiation make this technology particularly interesting in the biomedical field—for
As briefly discussed in the introduction and in the previous paragraph, the unique spectral
features of THz radiation make this technology particularly interesting in the biomedical field—for
ex vivo and in vivo experiments and/or analyses. During the past decades, systematic spectroscopic Condens. Matter 2020, 5, 25 9 of 28 investigations carried out in the THz frequency region on biological targets, as biomacromolecule,
cells and tissues, demonstrated differences in their optical properties. In the last five years only, the rate
of imaging experiments in the THz domain is increasing. An unavoidable issue, when talking about THz imaging, is water: THz is particularly sensitive to
water content, exhibiting a strong absorption [142] and consequently limiting THz penetration depth. In fact, the strong absorption of water limits the penetration depth in fresh tissues between tens
and hundreds of microns. For example, into the human skin THz waves can penetrate only a few
hundred microns [143]. In vivo clinical measurements, it limits the probing on superficial layers
of the target and the reflection mode becomes the suitable choice [144,145]. The limitation could
be overcome in the case of ex vivo examinations, where histopathological evaluations are required. Researchers overcome these limitations using thin and/or fixed samples and appropriate geometry. The transmitted TPI is performed on thin prepared tissues, suitable for in vitro, and reflected TPI
allows the investigation of surface features, reliable in vivo and ex vivo imaging. However, difficulties in extrapolating measurements on tissue to ex vivo include, for example,
saline uptake from the sample storage environment, changes in hydration level during the measurement,
temperature- and humidity-dependent and scattering effects. Considering that, the human body
contains a large amount of water (~60%) and THz is heavily absorbed by water [142], fresh tissue can
vastly alter THz spectroscopy measurements. As fresh tissue dehydrates when left exposed to the
air, many contrast features are reduced during the data acquisition. Therefore, for biomedical studies,
sample conservation is crucial. Several research groups have investigated excised and fixed tissues considering numerous
approaches; among them are dehydration [146,147], alcohol perfused [36], formalin fixed [148–150],
gelatin embedding [151,152], lyophilized [153], freezing [154,155] and fixation in paraffin emulsion
and embedding [156]. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications Formalin fixing, in order to preserve and fix excised tissues, is a common histopathological routine
for diagnosis. The formalin reduces the sample variation due to the dehydration process [149,154]—the
tissues’ water content is replaced by formalin. In addition, lyophilization could represent an interesting alternative to fresh tissue for THz
spectroscopic measurements [153]; its pros are fast and effective water removal, and structural
preservation. The problems with handling fresh tissue, such as time variability in the THz bandwidth,
dynamic range and sample thickness, are therefore overcome [153]. Several studies proposed the
freezing of biological sample, as an alternative technique to increase the penetration depth of THz waves
in the tissues, as the absorption coefficient of ice is one order of magnitude lower than the absorption
coefficient of water [143]. The discussed technique has a limitation in clinical use; the process of
freezing might cause necrosis. Some suggestions are proposed by using a penetration-enhancing agent
(PEA) to increase the THz radiation delivery depth. The treatment with PEAs, that are cost-effective
and easy-to-find, biocompatible and nontoxic to the human body, could also ensure long preservation
time for ex vivo tissues and the application in vivo. Oh et al. [157] used glycerol as PEA, easily absorbed
by human skin and tissue and with an absorption coefficient much lower than that of water in the THz
spectral region. They treated abdominal mouse skin with glycerol and demonstrated the enhancement
of penetration depth with the reconstruction of a metal blade hidden under the tissue by the second
pulse reflection of it [157]. Various alternative approaches are suggested. Oleic acid is proposed by Wang et al. [158] for
trapping moisture inside thin tissue slices, in transmission acquisition mode. They realized a sandwich
configuration, by placing a frozen layer of oleic acid above and below the frozen tissue sample,
between tsurpica windows and waited for complete thawing, before starting the measurements at
room temperature. Oleic acid is highly transparent to THz radiation and its refractive index matched
tsurpica. The oleic acid layers were used to keep up the tissue hydration, showing good performances
for 70 min. Fan et al. [151] used gelatin embedding for porcine skin slices reflection measurements. By looking at the THz image data and computing the optical parameters of the gelatin-embedded Condens. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications Matter 2020, 5, 25 10 of 28 sample, they found a successful method to preserve the sample for at least 35 h, both for imaging and
spectroscopy purposes. Condens. Matter 2020, 5, x FOR PEER REVIEW
10 of 26 Since the first demonstration of THz imaging [64], TPI and spectroscopy show their ability to
differentiate human tissues [141,159], with clear differences between the tissue properties, in particular
regarding absorption coefficients [160]. Fatty tissue consists of mainly hydrocarbon chains, largely
nonpolar molecules, thus its absorption coefficient and refractive index are much smaller than the ones
of muscle, kidney and/or liver tissues [141]. Since the first demonstration of THz imaging [64], TPI and spectroscopy show their ability to
differentiate human tissues [141,159], with clear differences between the tissue properties, in
particular regarding absorption coefficients [160]. Fatty tissue consists of mainly hydrocarbon
chains, largely nonpolar molecules, thus its absorption coefficient and refractive index are much
ll
h
h
f
l
k d
d/
l Concerning the optical characterization at the low frequency region, many spectroscopic works
are present in the literature [161–167]. One significant work about the ability in distinguishing and
classifying the THz response of different types of tissues is Ref. [131], which is mentioned for the
chirped probe pulse technique. It offers a significant improvement concerning the slow acquisition
time, and it could have great potential in imaging acquisition. smaller than the ones of muscle, kidney and/or liver tissues [141]. Concerning the optical characterization at the low frequency region, many spectroscopic works
are present in the literature [161–167]. One significant work about the ability in distinguishing and
classifying the THz response of different types of tissues is Ref. [131], which is mentioned for the
chirped probe pulse technique. It offers a significant improvement concerning the slow acquisition
ti
d it
ld h
t
t
ti l i
i
i
i iti In addition, the high sensitivity of THz waves to water content in the tissues becomes the key
contrast factor in many biomedical applications [168,169]; the water content evaluation allows label-free
differentiation for various types of tissues as an endogenous marker, distinguishing between healthy
and pathological tissues [152,168], see Figure 4. time, and it could have great potential in imaging acquisition. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications In addition, the high sensitivity of THz waves to water content in the tissues becomes the key
contrast factor in many biomedical applications [168,169]; the water content evaluation allows
label‐free differentiation for various types of tissues as an endogenous marker, distinguishing
between healthy and pathological tissues [152 168] see Figure 4 y
p
g
g
Figure 4. Refractive index n and absorption coefficient α in THz spectral region, observed by
Gavdush et al. [152], and H&E‐stained histology of gelatin‐embedded ex vivo human tissues: intact
tissue, gliomas of grade I to IV, and edema. (a)–(c) grade I; (d)–(f) grade II; (g)–(i) grade III; and (j)–(l)
grade IV. The THz optical properties of gliomas are compared with equal data for the intact and
edematous tissues, averages within the entire set of brain tissues specimens [152]. Figure 4. Refractive index n and absorption coefficient α in THz spectral region, observed by Gavdush
et al. [152], and H&E-stained histology of gelatin-embedded ex vivo human tissues: intact tissue,
gliomas of grade I to IV, and edema. (a)–(c) grade I; (d)–(f) grade II; (g)–(i) grade III; and (j)–(l) grade
IV. The THz optical properties of gliomas are compared with equal data for the intact and edematous
tissues, averages within the entire set of brain tissues specimens [152]. Figure 4. Refractive index n and absorption coefficient α in THz spectral region, observed by
Gavdush et al. [152], and H&E‐stained histology of gelatin‐embedded ex vivo human tissues: intact
tissue, gliomas of grade I to IV, and edema. (a)–(c) grade I; (d)–(f) grade II; (g)–(i) grade III; and (j)–(l)
grade IV. The THz optical properties of gliomas are compared with equal data for the intact and
edematous tissues, averages within the entire set of brain tissues specimens [152]. Figure 4. Refractive index n and absorption coefficient α in THz spectral region, observed by Gavdush
et al. [152], and H&E-stained histology of gelatin-embedded ex vivo human tissues: intact tissue,
gliomas of grade I to IV, and edema. (a)–(c) grade I; (d)–(f) grade II; (g)–(i) grade III; and (j)–(l) grade
IV. The THz optical properties of gliomas are compared with equal data for the intact and edematous
tissues, averages within the entire set of brain tissues specimens [152]. In the case of cancer, the contrast between healthy and malign tissue is originated from the
structural variation and different water content [170]. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications Both refractive index
and absorption coefficient in the tumor region are higher than in normal tissue due to the higher water
content in cancer tissue. TPI has been successfully applied to liver [164,173,174], colon [165,175,176], intestine [161],
brain [39,156,177], skin [178], ovarian [167], oral [154] and breast cancers [132,163,179]. TPI has been successfully applied to liver [164,173,174], colon [165,175,176], intestine [161],
brain [39,156,177], skin [178], ovarian [167], oral [154] and breast cancers [132,163,179]. TPI has been successfully applied to liver [164,173,174], colon [165,175,176], intestine [161],
brain [39,156,177], skin [178], ovarian [167], oral [154] and breast cancers [132,163,179]. TPI has been successfully applied to liver [164,173,174], colon [165,175,176], intestine [161],
brain [39,156,177], skin [178], ovarian [167], oral [154] and breast cancers [132,163,179]. For instance, a particular attention deserves the oral‐, gastric‐ and intestinal‐ neoplasia, because
an early/rapid diagnosis is required. However, it may be quite difficult due to oral‐gastric‐intestinal
apparatus anatomy. Sim et al. [154] used a TPI reflection system in the frequency range of 0.2–1.2
THz to assess oral carcinoma. Seven oral tissues from four patients were analyzed, and then the
authors compared the optical, frozen and room temperature THz, histopathological images. They
found that THz images at −20 °C showed better contrast between healthy and cancer regions
compared to the room temperature ones (20 °C). Additionally, frozen temperature images
demonstrated better area correlation than the histopathological images [154]. The recent and
detailed review of Danciu et al. [180] takes an overview of diagnostics methods and current available
data on THz‐based detection for digestive cancers. Some current in vitro and ex vivo progress is
highlighted for identifying specific digestive neoplasia [180]. For instance, a particular attention deserves the oral-, gastric- and intestinal- neoplasia, because
an early/rapid diagnosis is required. However, it may be quite difficult due to oral-gastric-intestinal
apparatus anatomy. Sim et al. [154] used a TPI reflection system in the frequency range of 0.2–1.2 THz
to assess oral carcinoma. Seven oral tissues from four patients were analyzed, and then the authors
compared the optical, frozen and room temperature THz, histopathological images. They found that
THz images at −20 ◦C showed better contrast between healthy and cancer regions compared to the
room temperature ones (20 ◦C). Additionally, frozen temperature images demonstrated better area
correlation than the histopathological images [154]. The recent and detailed review of Danciu et
al. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications [180] takes an overview of diagnostics methods and current available data on THz-based detection
for digestive cancers. Some current in vitro and ex vivo progress is highlighted for identifying specific
digestive neoplasia [180]. Instead, TPI has revealed the contrast between normal and neoplastic tissue regions [159,181]. One of the first applications on human ex vivo wet tissue involved imaging in excised basal cell
carcinoma [182]; the regions had recognition of healthy skin and basal cell carcinoma (BBC), both in
vitro [96] or in vivo and ex vivo [183,184]. Instead, TPI has revealed the contrast between normal and neoplastic tissue regions [159,181]. One of the first applications on human ex vivo wet tissue involved imaging in excised basal
cell carcinoma [182]; the regions had recognition of healthy skin and basal cell carcinoma (BBC),
both in vitro [96] or in vivo and ex vivo [183,184]. According to Wallace et al. [184], TPI could differentiate between basal cell carcinoma (BCC)
and normal tissue both in vivo and ex vivo, and it is under test whether TPI can facilitate the tumor
margins delineation, prior to surgery. The BCC’s areas have different THz properties compared to
healthy tissue. The general clinical image does not identify the skin cancer distribution with
accuracy in depth, under the skin surface. However, THz images, as shown in [184], clearly display
the cancer distribution on the skin and also the extent of the neoplasm invasion into the skin, with a
depth of 250 μm. According to Wallace et al. [184], TPI could differentiate between basal cell carcinoma (BCC)
and normal tissue both in vivo and ex vivo, and it is under test whether TPI can facilitate the tumor
margins delineation, prior to surgery. The BCC’s areas have different THz properties compared to
healthy tissue. The general clinical image does not identify the skin cancer distribution with accuracy
in depth, under the skin surface. However, THz images, as shown in [184], clearly display the cancer
distribution on the skin and also the extent of the neoplasm invasion into the skin, with a depth of
250 µm. Comparing ex vivo and in vivo images of the same tumors, similar contrast levels can be found,
however tissue deformation and shrinkage after excision leads to an exact match in the contrast
pattern. Nevertheless, the contrast level, present in the THz images, was enough to map diseased
tissue margins, when compared to histology. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications In fact, the presence of cancer induces, in
many cases, increased blood supply to the affected tissues and a local increase in tissue water
content, which suggests water contrast for THz cancer imaging [171]. As a result, it involves a higher
refractive index and absorption coefficient [39,169,172]. As proof, Figure 5 shows refractive indexes
and absorption coefficients of normal and cancer regions of a fresh rat brain tissue sample, as a
function of frequency [39]. In the case of cancer, the contrast between healthy and malign tissue is originated from the
structural variation and different water content [170]. In fact, the presence of cancer induces, in many
cases, increased blood supply to the affected tissues and a local increase in tissue water content,
which suggests water contrast for THz cancer imaging [171]. As a result, it involves a higher refractive
index and absorption coefficient [39,169,172]. As proof, Figure 5 shows refractive indexes and
absorption coefficients of normal and cancer regions of a fresh rat brain tissue sample, as a function of
frequency [39]. 11 of 28 11 of 28 Condens. Matter 2020, 5, 25 Figure 5. Optical characterization of a fresh rat brain tissue sample [39]. (a) Refractive index and (b)
Absorption coefficient (cm‐1) of normal and tumor regions, from 0.8 to 1.5 THz. Both refractive index
and absorption coefficient in the tumor region are higher than in normal tissue due to the higher
water content in cancer tissue. Figure 5. Optical characterization of a fresh rat brain tissue sample [39]. (a) Refractive index and (b)
Absorption coefficient (cm−1) of normal and tumor regions, from 0.8 to 1.5 THz. Both refractive index
and absorption coefficient in the tumor region are higher than in normal tissue due to the higher water
content in cancer tissue. Figure 5. Optical characterization of a fresh rat brain tissue sample [39]. (a) Refractive index and (b)
Absorption coefficient (cm‐1) of normal and tumor regions, from 0.8 to 1.5 THz. Both refractive index
and absorption coefficient in the tumor region are higher than in normal tissue due to the higher
water content in cancer tissue. Figure 5. Optical characterization of a fresh rat brain tissue sample [39]. (a) Refractive index and (b)
Absorption coefficient (cm−1) of normal and tumor regions, from 0.8 to 1.5 THz. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications Reese et al. [185], using THz pulsed imaging for
studying freshly excised colon cancer, have differentiated normal and diseased colon tissues, with a
good contrast for their recognition. Moreover, Wahaia et al. [186] measured colon tissue samples
both fixed in formalin and embedded in paraffin. In the first case, water is still present, and in the
second one, it is eliminated. They still obtained good image contrast between healthy and cancerous
regions. As a result, water is not the only factor contributing to the contrast between the two
Comparing ex vivo and in vivo images of the same tumors, similar contrast levels can be found,
however tissue deformation and shrinkage after excision leads to an exact match in the contrast
pattern. Nevertheless, the contrast level, present in the THz images, was enough to map diseased
tissue margins, when compared to histology. Reese et al. [185], using THz pulsed imaging for studying
freshly excised colon cancer, have differentiated normal and diseased colon tissues, with a good
contrast for their recognition. Moreover, Wahaia et al. [186] measured colon tissue samples both fixed
in formalin and embedded in paraffin. In the first case, water is still present, and in the second one,
it is eliminated. They still obtained good image contrast between healthy and cancerous regions. As a result, water is not the only factor contributing to the contrast between the two different tissue 12 of 28 Condens. Matter 2020, 5, 25 areas. Thus, the tumor boundaries in THz images can be recognized, and these are in accordance with
the visible images, indicating that the THz imaging technique could be useful for diagnosing cancers. Potentially, this technique could be employed as a complementary label-free technique, allowing
surgeons to determine tumor margins in real time. While different kinds of cancer can be differentiated from healthy tissue according to the high
sensitivity of THz radiation to water content, the strong potential of TPI in cancer assessment is not
limited to fresh tissues, as it has been efficiently demonstrated for discrimination of pathological and
healthy dehydrated tissue as well [132,174,177]. Afterwards, TPI has been used to identify tumor borders on excised breast carcinoma [187]. While THz imaging has proven potential contrast between breast cancer and healthy tissue, both in
fresh and formalin-fixed, paraffin-embedded (FFPE) tumors [154,179,188,189], all published works
were performed on flat sections of the tumor. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications The application of THz imaging to a three-dimensional
sample can be exploited to produce cross section images by in-depth scanning, without slicing the
tumor. Bowman et al. [190], in their work, illustrated the powerful THz pulsed imaging applications to
two different carcinoma samples: infiltrating ductal carcinoma and lobular carcinoma, both embedded
in paraffin blocks. THz pulsed images showed clear definition of the cancer boundaries in the
block. The results were correlated in 3D with histopathology sections sliced throughout the blocks. THz images highlighted the cancer regions only when there were interfaces inside the tissue, thus near
to the sample surface. In all the histopathology images, instead, the infiltrating ductal carcinoma and
lobular carcinoma were clearly visible, at any section. Furthermore, the THz 3D block images could be
sectioned into planar (x-y, x-z, and y-z) images in order to produce in-plane and in-depth tissue images,
thus successfully identifying cancer regions, without slicing the tissue. These results demonstrate the
effectiveness of THz pulsed imaging for tumor edge identification, when diseased tissue is near the
surgical excision. Reid et al. [176] conducted a study on normal, tumor and dysplastic freshly excised colon samples
from 30 patients, using a conventional THz-TDS system, in reflection mode. The study showed
a sensitivity of 82% and a specificity of 77% in discriminating between normal and pathological tissues,
while a sensitivity of 89% and a specificity of 71% in differentiating normal and dysplastic samples [176]. Another study [191], using diverse intelligent analysis methods (neural networks, decision trees,
and support vector machines) re-evaluated the data provided by [191]. This method increased the
sensitivity to 90%–100% and the specificity to 86%–90%, improving the overall diagnosis precision. With the aim of reducing patient re-operation rates and improving the ability of resection margin
in breast conserving surgery, Santaolalla et al. [192] applied a multivariate Bayesian classifier to the
samples waveform produced by TPI probe system. They can discriminate tumor from benign breast
tissue, obtaining a sensitivity of 96% and a specificity of 95%. Nowadays, in vivo THz detection investigates mostly superficial normal and diseased tissues,
close to the epithelial layer (i.e., breast, skin). Observing the significant contrast in THz dielectric
permittivity responses of healthy skin, dysplastic and non-dysplastic skin nevi, Zaytsev et al. [193,194]
distinguished the precursor of melanoma, with a non-invasive in vivo analysis, proving the efficiency
of THz pulsed radiation in the early diagnosis of the melanomas. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications To facilitate the use of TPI to scan tumor resection margins intraoperatively, Teraview Ltd. (Cambridge, UK) has proposed and developed a handheld probe system [195]. TPI handheld probe
measures tissue sample positioned in histology cassette, in reflection mode. The TPI handheld probe
ensures to discriminate benign from malignant breast tissue in an ex vivo setting. The purpose of
the THz device is to give valid support for rapid histopathological evaluation of excised tissues in
surgery [195]. Despite this, in the last ten years, researchers began to extend THz applications to inner tissues,
organs or hollow cavities of the human body. The request is for endoscopic access, achievable using
highly bendable waveguides with low transmission loss [196–198]. In 2009, Ji et al. [199] fabricated
and developed a miniaturized fiber-coupled THz endoscope apparatus. It emits and collects THz 13 of 28 13 of 28 Condens. Matter 2020, 5, 25 radiation using an optical fiber linked to a femtosecond laser. The measurement is performed close
to the reflective surface of a profound tissue or an organ [199]. The researchers tested the device
by looking at THz reflections from mouth, tongue and palm skin tissues and demonstrated that the
moisture of internal organs is a significant confounding issue. Some years later, an innovative THz
prototype, with single-channel detection based on flexible metal-coated THz waveguides [197,198]
and a specific polarization exposure method, was integrated into a commercial optical endoscope
and demonstrated its potentiality, successfully differentiating between normal and cancer colonic
tissues [175]. This also makes it a suitable tool for investigating the water content and hydration profile
of the skin. By using some basic image processing techniques like intensity windowing, histogram
manipulation, edge detection and region growing [200–202], the discrimination of healthy and cancer
regions by THz images can be considerably improved. Condens. Matter 2020, 5, x FOR PEER REVIEW
13 of 26
moisture of internal organs is a significant confounding issue. Some years later, an innovative THz
prototype, with single‐channel detection based on flexible metal‐coated THz waveguides [197,198]
and a specific polarization exposure method, was integrated into a commercial optical endoscope
and demonstrated its potentiality, successfully differentiating between normal and cancer colonic
tissues [175]. This also makes it a suitable tool for investigating the water content and hydration
profile of the skin. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications By using some basic image processing techniques like intensity windowing,
histogram manipulation, edge detection and region growing [200–202], the discrimination of healthy
d
i
b
TH i
b
id
bl
i
d The same group also developed THz otoscope that enables the detection of otitis media [203],
a disease that causes fluid and purulence in the posterior part of the tympanic membrane within
the middle ear. The THz otoscope is able to measure the change in water content on the tympanic
membrane; therefore, it is applicable as a medical device for otitis media diagnosis [203]. and cancer regions by THz images can be considerably improved. The same group also developed THz otoscope that enables the detection of otitis media [203], a
disease that causes fluid and purulence in the posterior part of the tympanic membrane within the
middle ear. The THz otoscope is able to measure the change in water content on the tympanic
b
th
f
it i
li
bl
di
l d
i
f
titi
di di
i [203] In [204] and in Figure 6, an imaging device was proposed as a potential tool for the detection of
deterioration in the feet of diabetic patients. Assuming that dehydration of the feet skin of diabetics,
owing to peripheral vascular disease, is a central element of their deterioration process, they take
advantage of THz pulsed imaging in order to obtain a promising diagnostic tool. The results are
encouraging and provide key elements that will allow the design of a clinical trial in the future. membrane; therefore, it is applicable as a medical device for otitis media diagnosis [203]. In [204] and in Figure 6, an imaging device was proposed as a potential tool for the detection of
deterioration in the feet of diabetic patients. Assuming that dehydration of the feet skin of diabetics,
owing to peripheral vascular disease, is a central element of their deterioration process, they take
advantage of THz pulsed imaging in order to obtain a promising diagnostic tool. The results are
encouraging and provide key elements that will allow the design of a clinical trial in the future g
g
p
y
g
Figure 6. Platform for early screening of diabetic foot syndrome [204]. (a) THz setup design. It
consists of an elevated surface where two high‐density polyethylene windows are used to place the
patient’s feet. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications A chair is also provided in order to maintain the patient’s position and avoid motion of
the feet during the diagnostic image acquisition. The space under the patient sitting area is used to
place the THz‐TDS spectrometer and a raster scanning system [204]. (b) Photograph of the assembled
setup during the testing. (c) Schematic layout of the raster‐scanning imaging system, placed on the
platform under the windows as indicated in (a). Figure 6. Platform for early screening of diabetic foot syndrome [204]. (a) THz setup design. It consists
of an elevated surface where two high-density polyethylene windows are used to place the patient’s
feet. A chair is also provided in order to maintain the patient’s position and avoid motion of the feet
during the diagnostic image acquisition. The space under the patient sitting area is used to place the
THz-TDS spectrometer and a raster scanning system [204]. (b) Photograph of the assembled setup
during the testing. (c) Schematic layout of the raster-scanning imaging system, placed on the platform
under the windows as indicated in (a). Figure 6. Platform for early screening of diabetic foot syndrome [204]. (a) THz setup design. It
consists of an elevated surface where two high‐density polyethylene windows are used to place the
patient’s feet. A chair is also provided in order to maintain the patient’s position and avoid motion of
the feet during the diagnostic image acquisition. The space under the patient sitting area is used to
place the THz‐TDS spectrometer and a raster scanning system [204]. (b) Photograph of the assembled
setup during the testing. (c) Schematic layout of the raster‐scanning imaging system, placed on the
platform under the windows as indicated in (a). Figure 6. Platform for early screening of diabetic foot syndrome [204]. (a) THz setup design. It consists
of an elevated surface where two high-density polyethylene windows are used to place the patient’s
feet. A chair is also provided in order to maintain the patient’s position and avoid motion of the feet
during the diagnostic image acquisition. The space under the patient sitting area is used to place the
THz-TDS spectrometer and a raster scanning system [204]. (b) Photograph of the assembled setup
during the testing. (c) Schematic layout of the raster-scanning imaging system, placed on the platform
under the windows as indicated in (a). Concerning brain tumors, Ji et al. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications (f) THz
reflectometry imaging (TRI). (g) 5‐ALA‐induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with
GFP and H&E modalities, while in white light images, cancer regions are not identified. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractiv
dexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh
Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. (f) THz
reflectometry imaging (TRI). (g) 5-ALA-induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with GFP
and H&E modalities, while in white light images, cancer regions are not identified. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive indexe
d absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat brain Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. (f) THz
reflectometry imaging (TRI). (g) 5‐ALA‐induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with
GFP and H&E modalities, while in white light images, cancer regions are not identified. Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. (f) THz
reflectometry imaging (TRI). (g) 5-ALA-induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with GFP
and H&E modalities, while in white light images, cancer regions are not identified. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive
indexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh
rat brain tissue sample, see Figure 8. Yamaguchi et al. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications in Figure 7 and in [205] demonstrated the effectiveness of THz
reflectometry imaging (TRI) in distinguishing cancer and normal regions within a brain tissue
i
R l
i
l
hi h i
i
i
(d
i
d i
d)
i ll
ll
l
d
h
Concerning brain tumors, Ji et al. in Figure 7 and in [205] demonstrated the effectiveness of THz
reflectometry imaging (TRI) in distinguishing cancer and normal regions within a brain tissue section. 14 of 28 14 of 28 Condens. Matter 2020, 5, 25 Relatively high intensity regions (depicted in red) are spatially well correlated to the tumor areas found
with green fluorescent protein (GFP) and Hematoxylin and eosin (H&E) techniques. Condens. Matter 2020, 5, x FOR PEER REVIEW
14 of 26 Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. (f) THz
reflectometry imaging (TRI). (g) 5‐ALA‐induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with
GFP and H&E modalities, while in white light images, cancer regions are not identified. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractiv
dexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fre
t brain tissue sample, see Figure 8. Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. (f) THz
reflectometry imaging (TRI). (g) 5-ALA-induced ppIX fluorescence imaging. TRI images show red
regions (relatively high intensity) that are in agreement with tumor regions images obtained with GFP
and H&E modalities, while in white light images, cancer regions are not identified. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive index
d absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat bra
sue sample, see Figure 8. Figure 7. Brain tumor discrimination with different imaging techniques [205]. (a) MRI (magnetic
resonance imaging). (b) White light imaging. (c) GFP (green fluorescent protein) imaging. (d) H&E
(Hematoxylin and eosin) stained imaging. (e) OCT (optical coherence tomography) imaging. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications The THz image has
been realized by computing a Tumor probability, where zero value corresponds to healthy tissue,
based on the different refractive indexes of tumor and normal tissue regions. The red area in (a)
shows the tumor region and is well in agreement with the dark purple area in HE‐stained image (b). Figure 8. Brain tumor images of rat fresh tissue using different techniques: (a) THz spectroscopy and
(b) HE-stained (hematoxylin and eosin) image of the same tissue section [39]. The THz image has been
realized by computing a Tumor probability, where zero value corresponds to healthy tissue, based on
the different refractive indexes of tumor and normal tissue regions. The red area in (a) shows the tumor
region and is well in agreement with the dark purple area in HE-stained image (b). Figure 8a shows the THz image based on the refractive index information (expressed as tumor
probability), that results higher in the tumor than in the normal region (see also Figure 5). The red area
locates the diseased tissue with great accordance to the HE-stained (hematoxylin and eosin) image of
the same tissue section, where a darker purple region is visible (Figure 8b) [39]. Table 2 summarizes the main kind of tumor studied with TPI, with their relative references. Most works are ex vivo, while some papers for skin cancer are available in vivo and in vitro too. Table 2. Different kind of tumors investigated with TPI and their references. Tumor
Sample Status
References
Liver
Ex vivo
[173,174]
Brain-cervical
Ex vivo
[39,156,157,177,205,206]
Breast
Ex vivo
[132,163,172,179,181,187–190,192,195]
Oral-gastric-intestinal
Ex vivo
[154,161,162,164–166,176,180,185,186,207–210]
Skin
Ex vivo
In vivo
In vitro
[159,182–184,193,194]
Ovarian
Ex vivo
[167] Table 2. Different kind of tumors investigated with TPI and their references. Additionally, THz radiation is [211] a non-ionizing one, such that it is biologically safe for in vivo
applications [212]. Moreover, the limited penetration depth of THz radiation has focused medical
imaging research into the dentistry area [213]. TPI has been found to be an interesting technique for
dental tissue (enamel, dentine and pulp) discrimination because refractive index differences enable the
three tissue regions to be identified [66,148,214–217]. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications [39], instead, take advantage of different optical properties (like refractive indexes
and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat brain
tissue sample, see Figure 8. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive
indexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh
rat brain tissue sample, see Figure 8. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive indexes
and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat brain
tissue sample, see Figure 8. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive
indexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh
rat brain tissue sample, see Figure 8. Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive indexes
and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat brain
tissue sample, see Figure 8. 15 of 28 Condens. Matter 2020, 5, 25
rat brain tissue sample, Figure 8. Brain tumor images of rat fresh tissue using different techniques: (a) THz spectroscopy and
(b) HE‐stained (hematoxylin and eosin) image of the same tissue section [39]. The THz image has
been realized by computing a Tumor probability, where zero value corresponds to healthy tissue,
based on the different refractive indexes of tumor and normal tissue regions. The red area in (a)
shows the tumor region and is well in agreement with the dark purple area in HE‐stained image (b). Figure 8. Brain tumor images of rat fresh tissue using different techniques: (a) THz spectroscopy and
(b) HE-stained (hematoxylin and eosin) image of the same tissue section [39]. The THz image has been
realized by computing a Tumor probability, where zero value corresponds to healthy tissue, based on
the different refractive indexes of tumor and normal tissue regions. The red area in (a) shows the tumor
region and is well in agreement with the dark purple area in HE-stained image (b). Figure 8. Brain tumor images of rat fresh tissue using different techniques: (a) THz spectroscopy and
(b) HE‐stained (hematoxylin and eosin) image of the same tissue section [39]. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications TPI is not the only possible technique for dental
disease monitoring [218]: (i) visual caries examination, for example, loss of enamel translucency in the
region between the contacting proximal surfaces of two adjacent teeth, (ii) X-ray imaging, (iii) electrical
impedance measurements, (iv) ultrasound and (v) fluorescence-based methods [218,219] can also
be adopted. However, many disadvantages can be assessed to some of these techniques: (i) is not
possible at early detection stages, (i) and (ii) are difficult for posterior teeth, (ii) is potentially harmful
for patients’ health because it uses ionizing radiation. For these reasons, additional strategies for dental
health monitoring, with a particular emphasis on diagnosis at an earlier stage of formation and the use
of non-ionizing radiations, are widely requested [218]. Condens. Matter 2020, 5, 25 16 of 28 16 of 28 Arnone et al. used TPI in order to distinguish different animal and human tissues, in particular
enamel, dentine and pulp, in human teeth. They realized two different kind of images with the
same data set: TOF and absorption images. Being enamel ~99% mineral and dentine ~70% mineral,
they show different refractive indexes, that result in different THz TOFs. The produced TOF image
shows enamel only and enamel and dentine regions. In the absorption image, instead, the inner pulp
region of tooth is visible, because it shows a strong absorption, thanks to the additional material in that
tooth area [214]. Zinov’eva et al. [215] did some transmission images of human permanent molar tooth
slices, at different THz frequencies, ranging from 0.2 to 1.5 THz. The teeth used in the experiments
were processed to produce artificial lesions by chemical demineralization. The images clearly resolved
enamel and dentine areas. In addition, demineralized tooth regions have shown an increase of THz
transmission signal in comparison with healthy tooth tissue. This difference can be used to trace
demineralization development in dental tissues [215]. Artificial demineralization detection has been
the first step towards dental caries detection because caries regions are the result of mineral loss from
enamel, causing a change in enamel refractive indexes and absorption coefficients. These changes can
be exploited to identify lesions not visible to the naked eye. Early detection is important because initial
stages of demineralization are reversible [66,148]. Crawley et al. [148] calculated enamel, dentine and
caries absorption coefficients and refractive indexes in the range between 0.3 and 2.0 THz, in THz
transmission geometry. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications Like [214], they produced a THz absorption map in a 210 µm thick tooth
slice and a TOF image, but with more accurate spatial resolution. Caries are correctly detected by the
absorption image because average absorption coefficient of carious enamel is typically 35% larger than
the one associated with healthy enamel, in that frequency range, using TPI. Enamel and dentine are
precisely identified using TOF data because they are related to different refractive indexes. In reflection
geometry, instead, some images have been created by plotting the change in THz pulse height at
a specific time delay after reflection from the sample surface. In these images, both caries are detected,
and dentine and enamel are differentiated. Moreover, they investigated a hypomineralization region of
a 200 µm thick tooth slice and demonstrated that TPI images can distinguish hypomineralization from
enamel caries (demineralization) [216]. Finally, they demonstrated that even a TPI image of a tooth
hemisection (much thicker with respect to previous slices) can discriminate caries inside it. A 3D study of dental tissue has been reported in [217]. The enamel-dentine junction in 12 human
incisor teeth has been detected in 91% of the cases. A series of ~100 µm deep steps were chemically
produced in order to alter enamel thickness. They were imaged and the authors demonstrated that they
accurately and reliably make direct measurements of enamel thickness; this is necessary for monitoring
enamel erosion, a common dental disorder. Finally, in order to verify TPI validity and effectiveness in dental caries detection, Kamburo˘glu et
al. [218] compared TPI (static images and movie video) with common radiological techniques: intraoral
photostimulable phosphor late (PSP) and cone beam CT (CBCT) for the detection of dental caries
ex vivo [218–220]. They demonstrated that TPI shows good performances for caries identification,
compared to the most used techniques, see Figure 9. 17 of 28
dental
caries 17 of 28
dental
caries Condens. Matter 2020, 5, 25
intraoral photostimula
caries ex vivo [218 ,
p
q
,
g
Figure 9. Tooth caries identification with different imaging techniques [218]. (a) White light image. (b) THz static image at 0.35 THz. (c) THz image from video movie. (d) CBCT (cone beam CT) image. (e) PSP (photostimulable phosphor late) image. Red arrows indicate dental caries in tooth tissue. The
five images are in agreement in identifying caries regions. Figure 9. Tooth caries identification with different imaging techniques [218]. (a) White light image. omography, THz tomosynthesis, time‐of‐flight pulsed imaging and 3D THz holography [139
4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications (b) THz static image at 0.35 THz. (c) THz image from video movie. (d) CBCT (cone beam CT) image. (e) PSP (photostimulable phosphor late) image. Red arrows indicate dental caries in tooth tissue. The five images are in agreement in identifying caries regions. q
g Figure 9. Tooth caries identification with different imaging techniques [218]. (a) White light image. (b) THz static image at 0.35 THz. (c) THz image from video movie. (d) CBCT (cone beam CT) image. (e) PSP (photostimulable phosphor late) image. Red arrows indicate dental caries in tooth tissue. The
five images are in agreement in identifying caries regions. Figure 9. Tooth caries identification with different imaging techniques [218]. (a) White light image. (b) THz static image at 0.35 THz. (c) THz image from video movie. (d) CBCT (cone beam CT) image. (e) PSP (photostimulable phosphor late) image. Red arrows indicate dental caries in tooth tissue. The five images are in agreement in identifying caries regions. References 1. Cao, Q.; Zhegalova, N.; Wang, S.; Akers, W.; Berezin, M. Multispectral imaging in the extended near-infrared
window based on endogenous chromophores. J. Biomed. Opt. 2013, 18, 101318. [CrossRef] [PubMed] 1. Cao, Q.; Zhegalova, N.; Wang, S.; Akers, W.; Berezin, M. Multispectral imaging in the extended near-infrared
window based on endogenous chromophores. J. Biomed. Opt. 2013, 18, 101318. [CrossRef] [PubMed] 2. Wilson, R.; Nadeau, K.; Jaworski, F.; Rowland, R.; Nguyen, J.; Crouzet, C.; Saager, R.; Choi, B.; Tromberg, B.;
Durkin, A. Quantitative short-wave infrared multispectral imaging of in vivo tissue optical properties. J. Biomed. Opt. 2014, 19, 086011. [CrossRef] [PubMed] 3. Bazylewski, P.; Ezugwu, S.; Fanchini, G. A review of Three-Dimensional Scanning Near-Field Optical
Microscopy (3D-SNOM) and Its Applications in Nanoscale Ligth Management. Appl. Sci. 2017, 7, 973. [CrossRef] 4. Weng, Q.; Panchai, V.; Lin, K.T.; Sun, L.; Kajihara, Y.; Tzalenchuk, A.; Komiyama, S. Comparison of active
and passive methods for the infrared scanning near-field microscopy. Appl. Phys. Lett. 2019, 114, 153101. [CrossRef] 5. Jeon, S.; Kim, J.; Lee, D.; Baik, J.W.; Kim, C. Review on pratical photoacoustic microscopy. Photoacoustics
2019, 15, 100141. [CrossRef] 6. Guo, R.; Lu, G.; Qin, B.; Fe, B. Ultrasound Imaging Technologies for Breast Cancer Detection and Management:
A Review. Ultrasound Med. Biol. 2018, 44, 37–70. [CrossRef] 7. Xu, C.; Carney, P.S.; Boppart, S.A. Wavelength-dependent
tomography. Opt. Express 2005, 13, 450–462. [CrossRef] 7. Xu, C.; Carney, P.S.; Boppart, S.A. Wavelength-dependent scattering in spectroscopic optical coherence
tomography. Opt. Express 2005, 13, 450–462. [CrossRef] 8. Xu, C.; Vinegoni, C.; Ralston, T.S.; Luo, W.; Tan, W.; Boppart, S.A. Spectroscopic spectral-domain optical
coherence microscopy. Opt. Lett. 2006, 31, 1079–1081. [CrossRef] 9. Kim, M.K. Principles and techniques of digital holographic microscopy. SPIE Rev. 2010, 1, 018005. [Cr 9. Kim, M.K. Principles and techniques of digital holographic microsco 9. Kim, M.K. Principles and techniques of digital holographic microscopy. SPIE Rev. 2010, 1, 018005. [CrossRef]
10. Marquet, P.; Depeursinge, C.; Magistretti, P.J. Review of quantitative phase-digital holographic microscopy: 10. Marquet, P.; Depeursinge, C.; Magistretti, P.J. Review of quantitative phase-digital holographic microscopy:
Promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of
psychiatric disorders. Neurophotonics 2014, 1, 020901. [CrossRef] 11. Pitkäaho, T.; Manninen, A.; Naughton, T.J. Focus prediction in digital holographic microscopy using deep
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5. Conclusions In summary, the rapid THz technological advances have led THz radiation to emerge as a promising
and useful tool in medicine. We have outlined the detection techniques of THz pulsed radiation and
biomedical applications, based on the use of TPI systems. In summary, the rapid THz technological advances have led THz radiation to emerge as a promising
and useful tool in medicine. We have outlined the detection techniques of THz pulsed radiation and
biomedical applications, based on the use of TPI systems. In this perspective, we have summarized the prominent advantages in the use of THz radiation,
with particular attention to THz pulsed radiation. Briefly, we have illustrated THz pulsed sources,
and subsequently incoherent and coherent detectors, that are commercially available and/or of interest
for technological research. The common schematic layouts for reflection and transmission imaging
modes are discussed too. Concerning the THz radiation properties, we have discussed the major application of TPI
in the field of biomedicine, reporting many examples of ex vivo and in vivo studies, including
cases of histopathological imaging of cancers, which are suitable targets. The enhancement of the
sensing capabilities and penetration depth within tissues, in medical applications, were addressed. This represents a delicate point to question, and in particular the use of PEAs should require more
attention, and many extended trials should be carried out to allow the application of TPI systems in
clinical fields and hospitals. Although THz technology is rapidly increasing as a medical imaging modality, and THz imaging
applications in biomedical fields have drawn extensive interest, the technologies are maturing and the
principles have been demonstrated in order to direct efforts towards the realization that THz clinical
applications are viable in the real world. 18 of 28 18 of 28 Condens. Matter 2020, 5, 25 Author Contributions: Writing-Original Draft Preparation, A.D. and M.D.F.; Writing-Review & Editing, all authors;
Formal Analysis, M.D.F. and V.D.; Supervision, S.L. and M.P.; Project Administration, S.L.; Funding Acquisition,
S.L. All authors have read and agreed to the published version of the manuscript. Funding: We acknowledge the financial support of the Bilateral Cooperation Agreement between Italy and Japan
of the Italian Ministry of Foreign Affairs and of the International Cooperation (MAECI), in the framework of the
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obtained by five different imaging modalities. J. Digit. References Imaging 2011, 24, 804–813. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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Environmental and meteorological factors linked to malaria transmission around large dams at three ecological settings in Ethiopia
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Kibret et al. Malar J (2019) 18:54
https://doi.org/10.1186/s12936-019-2689-y Kibret et al. Malar J (2019) 18:54
https://doi.org/10.1186/s12936-019-2689-y Malaria Journal Open Access © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: A growing body of evidence suggests that dams intensify malaria transmission in sub-Saharan Africa. However, the environmental characteristics underpinning patterns in malaria transmission around dams are poorly
understood. This study investigated local-scale environmental and meteorological variables linked to malaria trans-
mission around three large dams in Ethiopia. Methods: Monthly malaria incidence data (2010–2014) were collected from health centres around three dams
located at lowland, midland and highland elevations in Ethiopia. Environmental (elevation, distance from the res-
ervoir shoreline, Normalized Difference Vegetation Index (NDVI), monthly reservoir water level, monthly changes in
water level) and meteorological (precipitation, and minimum and maximum air temperature) data were analysed to
determine their relationship with monthly malaria transmission at each dam using correlation and stepwise multiple
regression analysis. Results: Village distance to reservoir shoreline (lagged by 1 and 2 months) and monthly change in water level
(lagged by 1 month) were significantly correlated with malaria incidence at all three dams, while NDVI (lagged by 1
and 2 months) and monthly reservoir water level (lagged by 2 months) were found to have a significant influence at
only the lowland and midland dams. Precipitation (lagged by 1 and 2 months) was also significantly associated with
malaria incidence, but only at the lowland dam, while minimum and maximum air temperatures (lagged by 1 and
2 months) were important factors at only the highland dam. Conclusion: This study confirmed that reservoir-associated factors (distance from reservoir shoreline, monthly aver-
age reservoir water level, monthly water level change) were important predictors of increased malaria incidence in
villages around Ethiopian dams in all elevation settings. Reservoir water level management should be considered as
an additional malaria vector control tool to help manage malaria transmission around dams. Keywords: Malaria, Climate, Environment, Reservoir shoreline, Water level, Dam, Ethiopia instance, while rainfall limits the availability of breeding
habitats for mosquito vectors, temperature determines
the length of mosquito larvae development and the rate
of growth of the malaria parasites inside the vector [2,
3]. In addition, environmental modifications, such as the
construction of dams and irrigation schemes, also affect
the type and distribution of mosquito breeding habitats
[4, 5]. Environmental and meteorological factors
linked to malaria transmission around large
dams at three ecological settings in Ethiopia Solomon Kibret1,2* , G. Glenn Wilson1,3, Darren Ryder1, Habte Tekie4 and Beyene Petros5 Background Kesem Dam (hereafter referred as the lowland dam) is
located on the Awash River in the lowlands of the Ethio-
pian Rift Valley, 225 km east of Addis Ababa, the capital
of Ethiopia. Its crest height of 25 m stores a maximum of
500 million cu m of water, covering an area of 200 sq km. The maximum length of the shoreline at full capacity is
55.4 km. The primary purpose of the dam is to irrigate
20,000 ha of land for sugarcane production downstream. The area is characterized as semi-arid with a mean daily
temperature of 27 °C. The hottest month is May (mean
daily temperature is 38 °C) and the coldest is December
(average daily temperature is 18 °C). The area receives an
average annual total rainfall of 600 mm; the main rainy
season (June to August) accounts for 80% of the total
rainfall. An estimated population of 35,000 lives within a
5-km radius of Kesem reservoir [20]. Climatic variables such as precipitation and air tem-
perature are important determinants of the spatial distri-
bution and relative abundance of malaria vector species
in Africa [16]. For instance, in Africa, Anopheles gam-
biae is the predominant species in high rainfall environ-
ments, while Anopheles arabiensis is more common in
arid areas [17, 18]. However, climatic conditions are also
inter-related with elevation. For example, air temperature
decreases as elevation increases, and consequently the
abundance and species composition of malaria vectors
may change significantly with elevation [16]. Koka Dam (hereafter referred as the midland dam)
is located in the Ethiopian Rift Valley in Central Ethio-
pia, 100 km south of Addis Ababa. It has a crest height
of 42 m, and a full water storage capacity of 1188 mil-
lion m3. The surface area of the reservoir at full capacity
is 236 sq km and the length of the reservoir at full stor-
age capacity is 86 km. The primary purpose of the dam
is to generate 43.2 MW of electricity from three turbines
(approximately 6% of the current total grid-based gen-
erating capacity of the country). Currently, the Wonji
sugarcane irrigation scheme (6000 ha), located approxi-
mately 12 km downstream of the dam, is also dependent
on releases from Koka Dam. In addition, the dam is used
for flood control. Background Malaria is a serious public health challenge in sub-Saha-
ran Africa, with an estimated 200 million cases of malaria
in 2017 alone [1]. This region accounts for 92% of the
global malaria burden [1]. A number of environmental,
climatic, seasonal, and ecological factors determine the
occurrence and intensity of malaria transmission. For In Africa, dams have been demonstrated to enhance
rates of malaria transmission in areas of unstable trans-
mission [5, 6]. Increased malaria incidence following dam © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. In Africa, dams have been demonstrated to enhance
rates of malaria transmission in areas of unstable trans-
mission [5, 6]. Increased malaria incidence following dam
*Correspondence: s.kibret@gmail.com
2 Present Address: Program in Public Health, University of California, Irvine,
CA 92697, USA
Full list of author information is available at the end of the article *Correspondence: s.kibret@gmail.com
2 Present Address: Program in Public Health, University of California, Irvine,
CA 92697, USA
Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kibret et al. Malar J (2019) 18:54 Page 2 of 16 analysed to determine factors linked to malaria transmis-
sion at each dam setting. construction was reported around several African dams
[7–14]. Overall, dams have been shown to contribute
to over 1 million malaria cases annually in sub-Saharan
Africa [15]. However, the extent to which various envi-
ronmental and climatic factors may have contributed
to enhanced rates of malaria transmission around these
sites remains poorly understood. Background The area receives a total annual rain-
fall of 850 mm and the mean daily temperature is 22 °C
(National Meteorological Agency, unpublished report). The hottest month is May (mean daily temperature is
29 °C) and the coldest is December (average daily tem-
perature is 12 °C). An estimated population of 29,000
lives within 5 km of the reservoir [20]. i
In Ethiopia, local increases in malaria rates have been
blamed on the establishment of new dams [9, 10, 12–14]. A new era of dam construction currently underway in
Ethiopia [19] has also elevated concerns for the pub-
lic health impact of these infrastructures. Yet, dams are
important contributors to Ethiopia’s economic develop-
ment and food security. However, a poor understanding
of their effects on malaria transmission in different eco-
logical settings represents a critical barrier to the sustain-
ability of water storage infrastructures.f Understanding how different environmental and cli-
matic factors affect rates of malaria transmission is
required to develop appropriate disease control tools. A
recent review suggested that the relationship between
dams and malaria incidence varies across ecological set-
tings [15]. However, the study did not investigate how
environmental (other than the presence of dams) and cli-
matic factors vary across these ecological settings and, in
turn, affect rates of malaria incidence around dams. The
present study aims to investigate relationships among a
number of environmental and meteorological factors
associated with malaria transmission around Ethiopian
dams in three ecological settings: highland, midland and
lowland elevations. Koga Dam (hereafter referred as the highland dam)
is located on the Koga River, one of the major tributar-
ies of the Blue Nile River, 560 km northwest of Addis
Ababa. The dam has a storage capacity of 83.1 million
m3 and surface area of 175 sq km. It was commissioned
in 2009 to irrigate 7000 ha of wheat, corn and teff crops. The rainy season (June to August) generates about 70%
of the run-off feeding the Koga River [21]. The length
of the reservoir shoreline at full capacity is 120 km. The
area is characterized as highland with a mean daily tem-
perature of 19 °C and receives an average annual rainfall
of 1500 mm. The hottest month is May (average daily
temperature is 26 °C) and the coldest is January (average
daily temperature is 10 °C). An estimated 32,680 people
live within 5 km of the reservoir [20]. Retrospective malaria case data Five years (January 2010 to December 2014) of weekly
malaria data were obtained from the health facilities
at each of the dam sites. Inhabitants of the study area
visit these health facilities to receive medical attention. At each health centre, each febrile case was tested by a Methodsh The present study was conducted around three dams in
Ethiopia: Kesem Dam [975 m above sea level (asl)], Koka
Dam (1551 m asl) and Koga Dam (1980 m asl) (Fig. 1). At each dam, six villages within a 5-km radius of the res-
ervoir shoreline were randomly selected for this study. Only villages located upstream of the dam were included
to avoid potentially confounding influences of the down-
stream river environment. Time series malaria case data
as well as environmental and meteorological data were Kibret et al. Malar J (2019) 18:54 Page 3 of 16 Fig. 1 Map of the study area and location of study villages in relation to the reservoir shorelines Fig. 1 Map of the study area and location of study villages in relation to the reservoir shorelines Malaria is the leading public health challenge in all
study villages, peaking between September and Novem-
ber following the months of the rainy season (June to
August). The inhabitants of the study villages are agrar-
ians, and cattle herding is also common. Plasmodium
falciparum is the predominant malaria-causing para-
site, accounting for 70–80% of malaria infections (Oro-
mia Health Bureau, unpublished report). The remaining
malaria infections are due to Plasmodium vivax. Anoph-
eles arabiensis is the major malaria vector species in the
study area while Anopheles pharoensis plays a second-
ary role [12]. Potential mosquito breeding habitats in the
study area include shoreline puddles, irrigation canals,
rain pools, and man-made pools [22]. trained laboratory technician for malaria using micro-
scopic blood screening to distinguish between P. fal-
ciparum and P. vivax. Test results were recorded in the
laboratory registry, along with data of outpatient name,
age, gender, and residency. These data were de-identified
and exported to Microsoft Excel and SPSS for analysis. These data were checked for completeness and correct-
ness by cross-referencing the laboratory registry with the
outpatient registry at each clinic. The completeness of the
registry ranges from 75 to 82% across the health facilities. A recent external quality assessment of the skills of the
microscopists in these health facilities identified that 80%
of slides were correctly recorded with correct parasite
quantification [23]. For quality control, five positive and
five negative slides were randomly selected from health
facilities each month, and taken to the District Labora-
tory for re-checking. Statistical analysis To satisfy the assumptions of individual statistical anal-
yses, first the normality in the distribution of monthly
malaria incidence, environmental and meteorological
data sets was tested using SPSS. Temperatures (both
minimum and maximum), reservoir water level (and
change in water level) and NDVI values were nor-
mally distributed and analysed as explanatory variables. Malaria incidence (dependent variable) and precipitation
data were found to have a skewed distribution and thus
were log-transformed accordingly. Monthly NDVI data for the study villages were acquired
from the US National Oceanic and Atmospheric Admin-
istration (NOAA) that documents data of the Moderate
Resolution Imaging Spectroradiometer (MODIS) instru-
ments on-board the Terra and Aqua Satellites. These
satellites provide a vegetation survey at a 250-m spatial
resolution every 16 days [25]. The MODIS NDVI prod-
ucts are computed from atmospherically corrected, bi-
directional surface reflectances that have been masked
for water, clouds, heavy aerosols, and cloud shadows. NDVI is a measure of vegetation condition, used here as
a proxy for mosquito habitat availability [26]. NDVI val-
ues vary between + 1.00 and − 1.00; the higher the NDVI
value, the denser the green vegetation. For each village, malaria incidence was calculated as
the number of cases per 1000 population. One-way Anal-
ysis of Variance (ANOVA), followed by a Tukey’s test,
was used to test for the differences in malaria incidence
between the three dam sites. Average monthly meteorological data (precipitation,
minimum and maximum air temperature) were calcu-
lated and lagged by one and 2 months to allow time for
mosquitoes and malaria parasites to complete their life
cycle prior to the expression of any malaria incidence. Similarly, monthly NDVI data were also lagged by one
and 2 months to allow time for mosquito development. Monthly relative humidity data were not included in the
analysis due to there being too many missing values for
the duration of the study period. To determine any cor-
relation between meteorological/environmental vari-
ables and malaria incidence at each dam site, univariate
associations were first examined by regressing single
explanatory factors (i.e., environmental and meteoro-
logical variables) against malaria incidences for each
dam site. Since there might be cross-correlation between
independent variables over time, cross-correlation analy-
ses were conducted. When the correlation coefficient
for the association between the independent variables
was greater than 0.5, these variables were analysed in
Autoregressive Integrated Moving Averages (ARIMA) to
avoid multicollinearity [27]. Environmental datah The environmental data used for this study comprised vil-
lage elevation, village distance from reservoir shoreline, Kibret et al. Malar J (2019) 18:54 Page 4 of 16 monthly averages and exported to Microsoft Excel and
SPSS for analysis. monthly averages and exported to Microsoft Excel and
SPSS for analysis. Normalized Difference Vegetation Index (NDVI) and res-
ervoir water level. Village elevation was recorded using
a handheld Geographical Positioning System receiver
(GPSMAP 60CSx, Garmin International Inc., USA). For
each study village, data on monthly distance from reser-
voir shoreline were acquired from the European Space
Agency image repository [24]. These images had a reso-
lution of 150 × 150 m, were geo-referenced, and taken in
the first week of each month between January 2010 and
December 2014. These were then imported to ArcGIS 9.2
to estimate the distance between the centre of each study
village and the nearest reservoir shoreline for each month
of the study period. Statistical analysis After the effect of any auto-
correlation had been removed by the ARIMA procedure,
stepwise forward multiple regression analyses were used
to identify the meteorological/environmental factors that
best explained malaria incidence at each dam site. Only
those variables with a significant correlation (P < 0.05)
with malaria incidence were added in the multiple regres-
sion models. Among lagged variables, only those with the
highest correlation (r2 > 0.5) were included to these analy-
ses. All analyses were performed using Microsoft Excel
and SPSS Version 21 software. g
g
Daily reservoir water level data were obtained for each
dam from the Ethiopian Electricity and Power Corpora-
tion, and the Ministry of Water Resources for the dura-
tion of the study period (January 2010 to December
2014). These were then exported to Microsoft Excel and
SPSS for analysis. The data were aggregated to monthly
averages and monthly changes in water level (i.e., calcu-
lated by subtracting the amount of the reservoir water
level (m) at the end of a month from that at the beginning
of the month; negative values indicate receding water
level while positive values indicate increasing water lev-
els) for each of the three dams. The objective of includ-
ing monthly changes in water level was to determine how
the magnitude of change in water level correlates with
malaria incidence as it directly affects the nature of the
shoreline for mosquito breeding habitats. pact of environmental factors on malaria incidence Village proximity to a reservoir shoreline was negatively
correlated with malaria incidence in all the three dam
sites: the shorter a village’s distance to the shoreline,
the higher the malaria incidence in the following month
(Fig. 4). Indeed, approximately 69% (annual average from
51 to 86%) of annual malaria cases occurred when a vil-
lage’s distance was less than 2 km from the shoreline. This trend was consistent across the three dam sites. Mean monthly malaria incidence was 1.7- and 5.6-times
higher at the lowland dam (mean = 96.3; 95% CI = 81.5–
111.0; ANOVA: F = 54.7; P < 0.001) than the midland
(mean = 56.7; 95% CI = 45.9–67.4) and highland dam
(mean = 17.2; 95% CI = 13.9–20.4) dams, respectively
(Table 1). The temporal variation in malaria incidence
at the three dams showed a seasonal peak between Sep-
tember and November at all study dams (Fig. 2). Differ-
ences in malaria incidence between villages and years
at each dam site, however, were not statistically signifi-
cant (ANOVA, P > 0.05). Malaria incidence was gener-
ally strongly correlated with elevation (r2 = 0.97; P < 0.05):
malaria incidence decreased as elevation increased
(Fig. 3). h
Malaria incidence peaked following the months of
high reservoir water level (Fig. 5). There was gener-
ally a 2-month lag-time between peak water level and
peak malaria incidence, which was consistent across
the dams. Similarly, malaria peaks also followed peaks
in positive water level change at each dam (Fig. 6),
with a lag-time ranging from 1 month (highland dam)
to 2–3 months (midland and lowland dams). Likewise,
high NDVI levels were associated with peaks in malaria
incidence either 1–2 (lowland and midland dams) or
3 months (highland dam) later (Fig. 7).i Table 1 Summary of monthly mean malaria incidence
at the three study dams in Ethiopia, 2010–2014
* ANOVA test. The difference in malaria incidence between midland and
highland dam was also significant (Tukey test, P < 0.01)
Dam location
Mean malaria
incidence
95% CI
Odds ratio
P*
Lowland
96.3
81.5–111.0
1
–
Midland
56.7
45.9–67.4
1.7
< 0.01
Highland
17.2
13.9–20.4
5.6
< 0.01 Table 1 Summary of monthly mean malaria incidence
at the three study dams in Ethiopia, 2010–2014 Univariate analysis detected significant relationships
between environmental variables and malaria inci-
dence across the three dams (Table 2). Meteorological data Five years (January 2010 to December 2014) of daily
meteorological data, including total rainfall (mm), and
mean daily minimum and maximum air temperature
(°C), were obtained from three meteorological stations
at each of the three study dam sites. Any missing val-
ues were replaced with daily average data from the clos-
est neighbouring station. Data were then aggregated to Kibret et al. Malar J (2019) 18:54 Kibret et al. Malar J (2019) 18:54 Page 5 of 16 Results Impact of environmental factors on malaria incidence pact of environmental factors on malaria incidence NDVI (lagged
by 1 and 2 months; r = 0.567 and 0.669, respectively),
village distance from the reservoir shoreline (lagged
by 1 and 2 months; r = − 0.598 and − 0.441, respec-
tively), monthly average reservoir water level (lagged by * ANOVA test. The difference in malaria incidence between midland and
highland dam was also significant (Tukey test, P < 0.01) Fig. 2 Temporal variation in monthly malaria incidence in reservoir communities at the lowland, midland and highland dams in Ethiopia,
2010–2014 Fig. 2 Temporal variation in monthly malaria incidence in reservoir communities at the lowland, midland and highland dams in Ethiopia,
2010–2014 Kibret et al. Malar J (2019) 18:54 Page 6 of 16 Fig. 3 Relationship between malaria incidence and village elevation at the lowland, midland and highland dams in Ethiopia peaks in minimum air temperature at the lowland and
highland dams, but from 1 to 2 months following the
same peaks at the midland dam (Fig. 9). However, the
relationship between seasonal malaria incidence and
variation in maximum air temperature was less clear. At
the lowland dam, malaria peaks occurred 2 to 4 months
following late-summer troughs in maximum air tempera-
ture, but from 0 to 3 months following similar troughs at
the midland dam. The same pattern was less consistent
at the highland dam, with malaria incidence peaks tend-
ing to follow minor September peaks in maximum air
temperature.l 2 months; r = 0.362) and monthly change in reservoir
water level (lagged by 1 month; r = − 0.616) were sig-
nificantly associated with monthly malaria incidence
at the lowland dam. At the midland dam, distance
from reservoir shoreline (lagged by 1 and 2 months;
r = − 0.455 and − 0.368, respectively), NDVI (lagged
by 2 months; r = 0.452), monthly average reservoir
water level (lagged by 2 month; r = 0.408) and monthly
change in reservoir water level (lagged by 1 and
2 months; r = − 0.481 and − 0.366, respectively) were
significantly associated with malaria incidence. At the
highland dam, a strong correlation was found between
monthly malaria incidence and distance from reser-
voir shoreline (lagged by 1 and 2 months; r = 0.487 and
− 0.377, respectively) and monthly changes in reservoir
water level (lagged by 1 month; r = − 0.301). pact of environmental factors on malaria incidence Univariate analysis of the influence of meteorological
variables on seasonal malaria incidence indicated differ-
ences in variables that were significantly associated with
malaria incidence between the three dam sites (Table 3). At the lowland dam, monthly total precipitation lagged
by 1 and 2 months (r = 0.414; r = 0.672, respectively)
were the only variables with a significant correlation with
monthly malaria incidence. At the midland dam, monthly
total precipitation lagged by 2 months (r = 0.329) and
monthly mean minimum temperature lagged by 1 and
2 months (r = 0.501; r = 0.612, respectively) were sig-
nificantly correlated with monthly malaria incidence. At
the highland dam, monthly mean minimum (r = 0.419; Impact of meteorological variables on malaria incidence Impact of meteorological variables on malaria incidence
A peak in malaria incidence followed mid-year peaks
in rainfall at each of the three dams (Fig. 8). Lag times
were relatively consistent between dams, ranging from
an average of 2.4 or 2.6 months at the lowland and mid-
land dams to 2.0 months at the highland dam. A peak
in malaria incidence tended to occur a month following Kibret et al. Malar J (2019) 18:54 Page 7 of 16 Fig. 4 Temporal variation in monthly malaria incidence and villages distance from reservoir shoreline at a lowland, b midland and c highland dams
in Ethiopia. NB: Y-axis scales vary between the three plots Fig. 4 Temporal variation in monthly malaria incidence and villages distance from reservoir shoreline at a lowland, b midland and c highland dams
in Ethiopia. NB: Y-axis scales vary between the three plots Table S1). For instance, maximum temperature was sig-
nificantly correlated with NDVI, monthly reservoir water
level and reservoir water level change at each of the three
dams. r = 0634) and maximum (r = 0.364; r = 0.451) air temper-
ature lagged by 1 and 2 months were significantly corre-
lated with monthly malaria incidence. Stepwise multiple regression analyses selected few
environmental and meteorological variables as factors
most explaining malaria incidence across the three study
dams (Table 4). At the low land dam, village distance Regression models Cross-correlation analysis showed that a number of envi-
ronmental and meteorological variables were signifi-
cantly correlated with each other (see Additional file 1: Kibret et al. Malar J (2019) 18:54 Page 8 of 16 Fig. 5 Temporal variation in monthly malaria incidence and monthly average reservoir water level at a the lowland, b midland and c highland
study dams in Ethiopia, 2010–2014. NB: Y-axis scales vary between the three plots Fig. 5 Temporal variation in monthly malaria incidence and monthly average reservoir water level at a the lowland, b midland and c highland
study dams in Ethiopia, 2010–2014. NB: Y-axis scales vary between the three plots Fig. 5 Temporal variation in monthly malaria incidence and monthly average reservoir water level at a the lowland, b midland and c highland
study dams in Ethiopia, 2010–2014. NB: Y-axis scales vary between the three plots Fig. 5 Temporal variation in monthly malaria incidence and monthly average reservoir water level at a the lowland, b midland and c highland
study dams in Ethiopia, 2010–2014. NB: Y-axis scales vary between the three plots (r2 = 0.221; P < 0.001) explained 71.1% of variation in
monthly malaria incidence. At the highland dam, vil-
lage distance to reservoir shoreline lagged by 1 month
(r2 = 0.324; P < 0.001), monthly change in reservoir water
level lagged by 2 months (r2 change = 0.374; P < 0.001)
and monthly mean minimum temperature lagged by
2 months (r2 change = 0.068; P < 0.001) explained 76.5%
of variation in monthly malaria incidence. Overall, dam-
associated factors, such as distance to shoreline or the to reservoir shoreline lagged by 1 month (r2 = 0.468;
P < 0.001), monthly average change in reservoir water
level lagged by 2 months (r2 change = 0.189; P < 0.001)
and monthly total precipitation lagged by 1 month (r2
change = 0.156; P < 0.001) together explained 81% of the
monthly variability in malaria incidence. At the midland
dam, village distance to reservoir shoreline lagged by
1 month (r2 = 0.398; P < 0.001), monthly reservoir water
level lagged by 2 months (r2 change = 0.266; P < 0.001)
and monthly total precipitation lagged by 2 months Kibret et al. Malar J (2019) 18:54 Page 9 of 16 Fig. 6 Temporal variation in monthly malaria incidence and monthly change in reservoir water level at the a lowland, b midland and c highland
study dams, Ethiopia. Regression models NB: Y-axis scales vary between the three plots. Negative water level changes refer to receding water levels Fig. 6 Temporal variation in monthly malaria incidence and monthly change in reservoir water level at the a lowland, b midland and c highland
study dams, Ethiopia. NB: Y-axis scales vary between the three plots. Negative water level changes refer to receding water levels magnitude of water level changes, were found to be the
most important variables contributing to malaria inci-
dence in nearby villages. shoreline was the most important variable at all three
dams, explaining 47, 40 and 32% of the monthly vari-
ation in malaria incidence at the lowland, midland and
highland dams, respectively. This indicates the role that
dams play in malaria transmission by providing favour-
able breeding habitats for malaria mosquitoes. Previ-
ous studies indicated that An. arabiensis, the primary
malaria vector mosquito in Ethiopia, breeds along res-
ervoir shorelines [12, 14]. Discussionh This study revealed that dam-associated environmen-
tal factors and local meteorological drivers influence
malaria transmission around large dams in Ethiopia. The importance of these factors, however, varied across
lowland, midland and highland dam sites. Interest-
ingly, a village’s distance from the nearest reservoir Monthly reservoir water level (lagged by 2 months) was
also positively correlated with monthly malaria incidence Kibret et al. Malar J (2019) 18:54 Page 10 of 16 Fig. 7 Relationship between monthly malaria incidence and monthly NDVI at a lowland, b midland and c highland dams in Ethiopia. NB: Y-axis
scales vary between the three plots Fig. 7 Relationship between monthly malaria incidence and monthly NDVI at a lowland, b midland and c highland dams in Ethiopia. NB: Y-axis
scales vary between the three plots at the lowland and midland dams. This suggests that dur-
ing periods of high water level, reservoir shorelines get
closer to villages (as shown by distance) and contribute
to increased mosquito abundance as the result of mos-
quito breeding in reservoir shoreline habitats. The pre-
sent results also indicate that the shorter the distance
between villages and reservoir shoreline the higher the
malaria incidence. This is in agreement with the find-
ings of a recent study that documented enhanced lar-
val abundance of An. arabiensis and An. pharoensis, the major malaria vectors in Ethiopia, in lowland and mid-
land dam areas [14]. Similar observations were also made
around Lake Victoria in Kenya where the abundance of
An. gambiae complex (of which An. arabiensis is a mem-
ber) substantially increased during high water levels [28]. In southwest Ethiopia, Sena et al. [29] found that eleva-
tion and distance from reservoir were important factors
determining malaria transmission around Gilgel-Gibe. Generally, the significant association between village
distance from reservoir shoreline and malaria incidence Kibret et al. Discussionh Malar J (2019) 18:54 Page 11 of 16 Table 2 Correlation between environmental variables and monthly malaria incidence at the lowland, midland
and highland dams in Ethiopia
* Significant correlation at P < 0.05
Environmental variable
Pearson’s correlation with mean monthly malaria incidence
Lowland dam
Midland dam
Highland dam
NDVI
0.341
0.255
0.127
NDVI lagged by 1 month
0.567*
0.303
0.239
NDVI lagged by 2 months
0.669*
0.452*
0.302
Village distance from reservoir shoreline
− 0.252
− 0.132
− 0.103
Village distance from reservoir shoreline lagged by 1 month
− 0.598*
− 0.455*
− 0.487*
Village distance from reservoir shoreline lagged by 2 months
− 0.441*
− 0.368*
− 0.377*
Monthly average reservoir water level
0.231
0.312
0.121
Monthly average reservoir water level lagged by 1 month
0.296
0.324
0.209
Monthly average reservoir water level lagged by 2 months
0.362*
0.408*
0.299
Monthly change in reservoir water level
− 0.124
− 0.235
− 0.191
Monthly change in reservoir water level lagged by 1 month
− 0.616*
− 0.481*
− 0.694*
Monthly change in reservoir water level lagged by 2 months
− 0.244
− 0.366*
− 0.301 Table 2 Correlation between environmental variables and monthly malaria incidence at the lowland, midland
and highland dams in Ethiopia earson’s correlation with mean monthly malaria incidence confirms the role of dams in malaria transmission at all
three dam settings. by temperature—an increase in temperature leads to
increased proportions of infective mosquitoes [37]. However, the effect of temperature largely depends on
elevation: as elevation increases, temperature decreases,
which affects both mosquito and malaria parasite devel-
opment [38]. The minimum temperature required for
the development of P. falciparum and P. vivax is approxi-
mately 18 °C and 15 °C, respectively, limiting the spread
of malaria at higher altitudes [39]. There is also a rela-
tionship between increasing altitude and decreasing
mosquito abundance in African highlands [38]. In light
of future climate change, higher temperatures could also
facilitate faster desiccation of breeding habitats, compro-
mising larval development. These effects of minimum
temperature might explain the significance of this factor
in determining malaria transmission rates around the
highland dam in the present study. Whilst precipitation was the most important mete-
orological factors associated with malaria incidence at
the lowland and midland dams, minimum tempera-
ture appeared to be a significant driver of malaria inci-
dence around the highland dam. Discussionh In fact, precipitation is
strongly correlated with reservoir water level as periods
of high water level follow heavy rains between June and
August. Teklehaimanot et al. [30] indicated that precipi-
tation is the most important factor for malaria transmis-
sion in the lowlands of Ethiopia as mosquito breeding is
largely limited by water availability. Peak malaria trans-
mission often follows the main rainy season in Ethiopia
[31]. Precipitation has a direct and indirect effect on
malaria transmission around dams: it increases reservoir
water level which creates potential mosquito breeding
habitats along the shorelines closer to reservoir villages,
and forms rain pools that mosquitoes use for breeding.hf g
y
Monthly NDVI (lagged by 1 and 2 months) was sig-
nificantly correlated with malaria incidence, particularly
around the lowland dam. Several studies have shown
a positive significant correlation between NDVI in the
preceding month and malaria in West, Central and East
Africa [40–42]. However, it should be noted that tempo-
ral variation in NDVI is often highly correlated with rain-
fall particularly in semi-arid lowlands, as shown in the
present study and others [43, 44]. In the Sudanese Savan-
nah region of Mali, Gaudart et al. [42] reported NDVI
to be an important predictor of the total surface area of
breeding sites, as NDVI values increase with soil mois-
ture. In Eritrea, Graves et al. [45] found that NDVI is a
better predictor of malaria incidence than rainfall. In the
absence of rainfall data, NDVI can thus be used to pre-
dict malaria risk in lowland areas. The effect of minimum temperature on malaria trans-
mission in the highlands has long been recognized
[32–35]. Temperature is a key determinant of the length
of mosquito and malaria parasite life cycle [34, 36]. For
instance, at 16 °C, larval development may take more
than 45 days (reducing the number of mosquito genera-
tions and putting the larvae at increased risk of preda-
tors), compared to only 10 days at 30 °C [30]. However,
temperature increases above 30 °C have been regarded
as detrimental to parasite and mosquito development
[36]. By affecting the duration of the aquatic stage of the
mosquito life cycle, temperature determines the timing
and abundance of mosquitoes following adequate rain-
fall. The feeding frequency of mosquitoes is also affected Kibret et al. Malar J (2019) 18:54 Page 12 of 16 Fig. Discussionh Malar J (2019) 18:54 Page 14 of 16 Table 3 Correlation between climatic variables and monthly malaria incidence at the lowland, midland and highland
dams in Ethiopia
* Significance (P < 0.05)
Climate variable
Pearson’s correlation with mean monthly malaria incidence
Lowland dam
Midland dam
Highland dam
Monthly total precipitation
0.213
0.107
0.042
Monthly total precipitation lagged by 1 month
0.414*
0.268
0.211
Monthly total precipitation by 2 months
0.672*
0.376*
0.282
Monthly mean minimum temperature
0.231
0.184
0.302
Monthly mean minimum temperature lagged by 1 month
0.195
0.342*
0.419*
Monthly mean minimum temperature lagged by 2 months
0.232
0.399*
0.634*
Monthly mean maximum temperature
0.161
0.103
0.147
Monthly mean maximum temperature lagged by 1 month
0.208
0.198
0.364*
Monthly mean maximum temperature lagged by 2 months
0.279
0.233
0.451* able 3 Correlation between climatic variables and monthly malaria incidence at the lowland, midland and highland
dams in Ethiopia etween climatic variables and monthly malaria incidence at the lowland, midland and highland Table 4 Optimum stepwise multiple regression models relating monthly malaria incidence with environmental
and climatic factors at the three dam sites
a The non-standardized coefficient of a variable is also referred as ‘effect size’ which indicates the result of a single unit increase in this variable on malaria incidence
Model
Predictors
Non-
standardized
coefficienta
Adjusted R2
Sig. Discussionh 8 Temporal variation in monthly malaria incidence and monthly total precipitation at the a lowland, b midland and c highland study dams,
Ethiopia. NB: Y-axis scales vary between the three plots Monthly change in reservoir water level (lagged by
h )
f
h
d
previously been shown to determine availability of shore-
l
h b
f
b
d
d [
]
Fig. 8 Temporal variation in monthly malaria incidence and monthly total precipitation at the a lowland, b midland and c highland study dams,
Ethiopia. NB: Y-axis scales vary between the three plots Fig. 8 Temporal variation in monthly malaria incidence and monthly total precipitation at the a lowland, b midland and c highland study dams,
Ethiopia. NB: Y-axis scales vary between the three plots previously been shown to determine availability of shore-
line habitats for mosquito breeding around [12]. Faster
water level drawdown rates, determined by the magni-
tude in water level change between consecutive months, Monthly change in reservoir water level (lagged by
2 months) was one of the most important determi-
nants of monthly malaria incidence around the lowland
and midland dams. The rate of water level change has Kibret et al. Malar J (2019) 18:54 Page 13 of 16 Fig. 9 Temporal variation in malaria incidence and minimum and maximum air temperatures at the a lowland, b midland and c highland study
dams, Ethiopia. NB: Y-axis scales vary between the three plots Fig. 9 Temporal variation in malaria incidence and minimum and maximum air temperatures at the a lowland, b midland and c highland study
dams, Ethiopia. NB: Y-axis scales vary between the three plots Increasing water levels, which also shorten the distance
from villages to shorelines, were positively correlated
with increasing malaria incidence. This also explains the
seasonality of malaria around dam villages which peaks were associated with low larval mosquito abundance and
fewer shoreline puddles [46]. Similarly, the present study
showed that a rapidly receding reservoir shoreline was
associated with lower malaria incidence rates (Fig. 6). Kibret et al. Discussionh Lowland dam
1
Village distance from reservoir shoreline (lagged by 1 month)
− 9.46
0.468
< 0.001
2
Village distance from reservoir shoreline (lagged by 1 month), monthly change in reservoir water
level (lagged by 1 month)
4..61
0.657
< 0.001
3
Village distance from reservoir shoreline (lagged by a month), monthly change in reservoir water
level (lagged by 1 month), monthly total precipitation (lagged by 2 months)
2.49
0.813
< 0.001
Midland dam
1
Village distance from reservoir shoreline (lagged by 1 month)
− 5.47
0.398
< 0.001
2
Village distance from reservoir shoreline (lagged by 1 month), monthly reservoir water level (lagged
by 2 month)
3.89
0.532
< 0.001
3
Village distance from reservoir shoreline (lagged by 1 month), monthly reservoir water level (lagged
by 2 months), monthly total precipitation (lagged by 2 month)
0.66
0.711
< 0.001
Highland dam
1
Village distance from reservoir shoreline (lagged by 1 month)
− 1.98
0.324
< 0.001
2
Village distance from reservoir shoreline (lagged by 1 month), monthly change in reservoir water
level
2.04
0.698
< 0.001
3
Village distance from reservoir shoreline (lagged by 1 month), monthly change in reservoir water
level (lagged by 2 months), monthly minimum temperature precipitation (lagged by 1 month)
2.75
0.765
< 0.001 Table 4 Optimum stepwise multiple regression models relating monthly malaria incidence with environmental
and climatic factors at the three dam sites pwise multiple regression models relating monthly malaria incidence with environmental
the three dam sites ultiple regression models relating monthly malaria incidence with environmental
d
it dams have been less clear. The present study has for the
first time identified environmental and meteorological
factors associated with increased malaria transmission
around dams at different ecological settings. Its findings
underscore the role of reservoir water levels in malaria
transmission nearby, and also allow the potential of using
reservoir water level management for malaria vector con-
trol to be assessed. Reservoir water level management
was effectively implemented to disrupt malaria vector
breeding in habitats in the Tennessee Valley, USA [47]. immediately after the rainy season when reservoirs
fill up. Reservoir water level is thus an important fac-
tor underpinning the production of shoreline mosquito
breeding habitats. Understanding the various factors that contribute
to malaria transmission is crucial in order to forecast
malaria risk and devise disease control tools. References 1. WHO. World Malaria Report 2018. Geneva: World Health Organization;
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lished maps and institutional affiliations. Received: 15 November 2018 Accepted: 21 February 2019 Received: 15 November 2018 Accepted: 21 February 2019 Conclusion Dams intensify malaria transmission in Ethiopia. The rate
of reservoir water level change and village distance from
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determinants around dams. As many dams are currently
planned in sub-Saharan Africa, understanding the factors
underlying increased malaria transmission is crucial to
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Not applicable. Ethics approval and consent to participate
Not applicable. Consent for publication
Not applicable. Consent for publication
Not applicable. Acknowledgements The authors wish to thank Kefyalew Girma for his support with satellite data
analysis. Discussionh Although
evidence for the general impact of dams on malaria is
well documented in sub-Saharan Africa [5, 6, 15], spe-
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approach under field experiments and found that faster
drawdown rates suppress larval development [46]. How-
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existing African dams. Funding Funding
The study was funded by the University of New England, Australia, and the
International Foundation for Science (IFS, Grant #W/4752-2). Funding
The study was funded by the University of New England, Australia, and the
International Foundation for Science (IFS, Grant #W/4752-2). Consent for publication
Not applicable. Competing interests Competing interests
The authors declare that they have no competing interests. Availability of data and materials Availability of data and materials
All data used for this study are presented here. Raw data can be obtained by
contacting the corresponding author. Availability of data and materials
All data used for this study are presented here. Raw data can be obtained by
contacting the corresponding author. This study has three main limitations. First, the malaria
data used were retrospective data with a 75–82% level
of completeness. Active case detection would have
improved confidence in the present findings relative to
retrospective datasets. Second, the difference in malaria
control use (e.g., bed nets) among the study dams was
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Anopheles abundance in Ethiopia. BMC Infect Dis. 2013;13:161. 14. Kibret S, Wilson GG, Ryder D, Tekie H, Petros B. Malaria impact of large
dams at different eco-epidemiological settings in Ethiopia. Trop Med
Health. 2017;45:4. 14. Kibret S, Wilson GG, Ryder D, Tekie H, Petros B. Malaria impact of large
dams at different eco-epidemiological settings in Ethiopia. Trop Med
Health. 2017;45:4. Kibret et al. Malar J (2019) 18:54 Kibret et al. Malar J (2019) 18:54 Can water-level manage-
ment reduce malaria mosquito abundance around large dams in sub-
Saharan Africa? PLoS ONE. 2018;13:e0196064. 30. Teklehaimanot HD, Lipsitch M, Teklehaimanot A, Schwartz J. Weather-
based prediction of Plasmodium falciparum malaria in epidemic-prone
regions of Ethiopia. I. Patterns of lagged weather effects reflect biological
mechanisms. Malar J. 2004;3:41. 47. Hess AD, Kiker CC. Water level management for malaria control on
impounded waters. J Natl Malar Soc. 1944;3:181–96. 47. Hess AD, Kiker CC. Water level management for malaria control on
impounded waters. J Natl Malar Soc. 1944;3:181–96. •
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English
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Campylobacter jejuni Fatal Sepsis in a Patient with Non-Hodgkin’s Lymphoma: Case Report and Literature Review of a Difficult Diagnosis
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International journal of molecular sciences
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Campylobacter jejuni Fatal Sepsis in a Patient wit
Non-Hodgkin’s Lymphoma: Case Report and
Literature Review of a Difficult Diagnosis Maria Teresa Gallo 1,†, Enea Gino Di Domenico 1,*,†, Luigi Toma 2, Francesco Marchesi 3,
Lorella Pelagalli 4, Nicola Manghisi 1, Fiorentina Ascenzioni 5, Grazia Prignano 1,
Andrea Mengarelli 3 and Fabrizio Ensoli 1 1
Department of Clinical Pathology and Microbiology, San Gallicano Institute, IRCCS, Rome 00144, Italy
gallo@ifo.it (M.T.G.); n.manghisi@gmail.com (N.M.); prignano@ifo.it (G.P.); ensoli@ifo.it (F.E.) 1
Department of Clinical Pathology and Microbiology, San Gallicano Institute, IRCCS, Rome 00144, Italy
gallo@ifo.it (M.T.G.); n.manghisi@gmail.com (N.M.); prignano@ifo.it (G.P.); ensoli@ifo.it (F.E.) 1
Department of Clinical Pathology and Microbiology, San Gallicano Institute, IRCCS, Rome 00144, Italy;
gallo@ifo.it (M.T.G.); n.manghisi@gmail.com (N.M.); prignano@ifo.it (G.P.); ensoli@ifo.it (F.E.)
2
Department of Infectious Disease, San Gallicano Institute, IRCCS, Rome 00144, Italy; toma@ifo.it
3
Department of Hematology, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy;
marchesi.francesco@tiscali.it (F.M.); mengarelli@ifo.it (A.M.) p
gy
gy
y
gallo@ifo.it (M.T.G.); n.manghisi@gmail.com (N.M.); prignano@ifo.it (G.P.); ensoli@ifo.it (F.E.)
2
Department of Infectious Disease, San Gallicano Institute, IRCCS, Rome 00144, Italy; toma@ifo.it
3
Department of Hematology, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy;
marchesi.francesco@tiscali.it (F.M.); mengarelli@ifo.it (A.M.) p
,
,
,
,
y;
3
Department of Hematology, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy;
marchesi.francesco@tiscali.it (F.M.); mengarelli@ifo.it (A.M.) 4
Intensive Care Medicine, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy; pelagalli@ifo.it
5
Department of Biology and Biotechnology “Charles Darwin”, University of Rome Sapienza, Rome 00185, 4
Intensive Care Medicine, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy; pelagalli@ifo.it
5 4
Intensive Care Medicine, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy; pelagalli@ifo.it
5
Department of Biology and Biotechnology “Charles Darwin”, University of Rome Sapienza, Rome 00185,
Italy; fiorentina.ascenzioni@uniroma1.it 4
Intensive Care Medicine, Regina Elena National Cancer Institute IRCCS, Rome 00144, Italy; pelagalli@ifo.it
5
Department of Biology and Biotechnology “Charles Darwin”, University of Rome Sapienza, Rome 00185,
Italy; fiorentina.ascenzioni@uniroma1.it ,
g
,
,
y; p
g
5
Department of Biology and Biotechnology “Charles Darwin”, University of Rome Sapienza, Rome 00185
Italy; fiorentina.ascenzioni@uniroma1.it y
*
Correspondence: e.didomenico@ifo.it; Tel.: +39-06-5266-2956; Fax: +39-06-5266-5396 †
These authors contributed equally to this work. Academic Editor: Susanna Esposito Received: 25 January 2016; Accepted: 7 April 2016; Published: 12 April 2016 Abstract: Campylobacter jejuni (C. jejuni) bacteremia is difficult to diagnose in individuals with
hematological disorders undergoing chemotherapy. The cause can be attributed to the rarity
of this infection, to the variable clinical presentation, and to the partial overlapping symptoms
underlying the disease. Campylobacter jejuni Fatal Sepsis in a Patient wit
Non-Hodgkin’s Lymphoma: Case Report and
Literature Review of a Difficult Diagnosis Here, we report a case of a fatal sepsis caused by C. jejuni in a 76-year-old
Caucasian man with non-Hodgkin’s lymphoma. After chemotherapeutic treatment, the patient
experienced fever associated with severe neutropenia and thrombocytopenia without hemodynamic
instability, abdominal pain, and diarrhea. The slow growth of C. jejuni in the blood culture
systems and the difficulty in identifying it with conventional biochemical phenotyping methods
contributed to the delay of administering a targeted antimicrobial treatment, leading to a fatal
outcome. Early recognition and timely intervention are critical for the successful management
of C. jejuni infection. Symptoms may be difficult to recognize in immunocompromised patients
undergoing chemotherapy. Thus, it is important to increase physician awareness regarding the clinical
manifestations of C. jejuni to improve therapeutic efficacy. Moreover, the use of more aggressive
empirical antimicrobial treatments with aminoglycosides and/or carbapenems should be considered
in immunosuppressed patients, in comparison to those currently indicated in the guidelines for
cancer-related infections supporting the use of cephalosporins as monotherapy. Keywords: Campylobacter jejuni; non-Hodgkin’s lymphoma; chemotherapy; skin lesion International Journal of
Molecular Sciences International Journal of
Molecular Sciences 2. Case Presentation
A 76-year-old ma A 76-year-old man was hospitalized in our Department of Hematology of the “Regina Elena”
National Cancer Institute in Rome on 13 March, 2014. He suffered from a Diffuse Large B-Cell
Lymphoma that had evolved from a previously diagnosed indolent non-Hodgkin Lymphoma (NHL)
which was refractory to three chemo-immunotherapeutic lines of treatment and was characterized by
cerebral and meningeal involvement at the time of last progression. National Cancer Institute in Rome on 13 March, 2014. He suffered from a Diffuse Large B-Cell
Lymphoma that had evolved from a previously diagnosed indolent non-Hodgkin Lymphoma
(NHL) which was refractory to three chemo-immunotherapeutic lines of treatment and was
characterized by cerebral and meningeal involvement at the time of last progression. Upon admission, the patient had evening fever and severe dysarthria (Figure 1). On March 14, Upon admission, the patient had evening fever and severe dysarthria (Figure 1). On March 14,
he received an urgent salvage treatment based on a chemo-immunotherapeutic regimen containing
Rituximab 375 mg/m2 on day 1, Methotrexate 1 g/m2 on day 2, and Cytarabine 1 g total dose twice
daily, for days 3 and 4. Given the presence of evening fevers and a moderate increase in procalcitonin
levels (mini VIDAS system, bioMérieux, Florence, Italy) to 2.62 ng/mL (normal, <0.5 ng/mL),
an empirical antibiotic therapy was administered including Ceftriaxone (2 g daily) at the beginning
of the salvage chemo-immunotherapy, even in the absence of any microbiological evidence from the
blood cultures and surveillance swabs. After 48 h, a complete regression of fever and a decrease in
procalcitonin levels to 1.69 ng/mL were observed. Serial blood cultures, taken on March 18, were
incubated in an automated, noninvasive culture system (BacT/ALERT, bioMérieux, Florence, Italy). he received an urgent salvage treatment based on a chemo-immunotherapeutic regimen containing
Rituximab 375 mg/m2 on day 1, Methotrexate 1 g/m2 on day 2, and Cytarabine 1 g total dose twice
daily, for days 3 and 4. Given the presence of evening fevers and a moderate increase in
procalcitonin levels (mini VIDAS system, bioMérieux, Florence, Italy) to 2.62 ng/mL (normal,
<0.5 ng/mL), an empirical antibiotic therapy was administered including Ceftriaxone (2 g daily) at
the beginning of the salvage chemo-immunotherapy, even in the absence of any microbiological
evidence from the blood cultures and surveillance swabs. After 48 h, a complete regression of fever
and a decrease in procalcitonin levels to 1.69 ng/mL were observed. 1. Introduction Campylobacter jejuni represents one of the most common worldwide causes of bacterial
gastroenteritis with over 190,000 cases occurring annually in the 27 member states of the European
Union (www.efsa.europa.eu/efsajournal). Clinical manifestations include abdominal pain, fever, and
diarrhea [1]. Unlike other enteric infections, C. jejuni is only rarely associated with extraintestinal localization
and systemic invasive illness [1,2]. Bacteremia caused by C. jejuni has been detected in less than 1% of Int. J. Mol. Sci. 2016, 17, 544; doi:10.3390/ijms17040544 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 544
Unlike other ente
localization and system 2 of 9
stinal
ed in patients with gastroenteritis and it has been mainly reported in elderly and in immunocompromised
patients [1,2]. immunocompromised patients [1,2]. In this study, we describe a case of C. jejuni sepsis in a patient with non-Hodgkin’s lymphoma
h
l
d i
f
l
Th
l
i
id
f C j j
i b
i
d
h
i
f In this study, we describe a case of C. jejuni sepsis in a patient with non-Hodgkin’s lymphoma
that resulted in a fatal outcome. The low incidence of C. jejuni bacteremia and the paucity of associated
symptoms make this infection difficult to detect in patients with hematological disorders where
selecting the appropriate antibiotic treatment is crucial, and at present, early and distinctive clinical
features have not yet been fully elucidated. that resulted in a fatal outcome. The low incidence of C. jejuni bacteremia and the paucity of
associated symptoms make this infection difficult to detect in patients with hematological disorders
where selecting the appropriate antibiotic treatment is crucial, and at present, early and distinctive
clinical features have not yet been fully elucidated. 2 Case Presentation 2. Case Presentation
A 76-year-old ma Serial blood cultures, taken on
March 18, were incubated in an automated, noninvasive culture system (BacT/ALERT, bioMérieux,
Florence, Italy). Figure 1. The patient’s clinical course. Procalcitonin (PCT-Q) levels were expressed as ng/mL. Antimicrobial susceptibility testing (AST) was performed by Etest®, according to the Clinical and
Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae. Figure 1. The patient’s clinical course. Procalcitonin (PCT-Q) levels were expressed as ng/mL. Antimicrobial susceptibility testing (AST) was performed by Etest®, according to the Clinical and
Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae. Figure 1. The patient’s clinical course. Procalcitonin (PCT-Q) levels were expressed as ng/mL. Antimicrobial susceptibility testing (AST) was performed by Etest®, according to the Clinical and
Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae. Figure 1. The patient’s clinical course. Procalcitonin (PCT-Q) levels were expressed as ng/mL. Antimicrobial susceptibility testing (AST) was performed by Etest®, according to the Clinical and
Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae. On March 19, the hemocytometric assessment showed severe neutropenia and thrombocytopenia
(hemoglobin 75 g/liter, platelet count 6 × 109/liter, white blood cell count 0.06 × 109/liter). On March
20, the stool culture exam gave negative results. Nevertheless, on March 21, the patient had a relapse
(fever > 39 °C) in the absence of symptoms indicating hemodynamic instability as well as abdominal
pain or diarrhea. Based on the assumption that the patient was undergoing a sepsis, the patient was
On March 19, the hemocytometric assessment showed severe neutropenia and thrombocytopenia
(hemoglobin 75 g/liter, platelet count 6 ˆ 109/liter, white blood cell count 0.06 ˆ 109/liter). On March
20, the stool culture exam gave negative results. Nevertheless, on March 21, the patient had a relapse
(fever > 39 ˝C) in the absence of symptoms indicating hemodynamic instability as well as abdominal
pain or diarrhea. Based on the assumption that the patient was undergoing a sepsis, the patient
was empirically treated with intravenous Piperacillin-Tazobactam (4.5 g three times a day), without 3 of 9 Int. J. Mol. Sci. 2016, 17, 544 clinical improvement. The abdominal echography revealed a severe circumferential thickening of
the cecum wall with submucosal edema, whereas procalcitonin levels increased to 3.64 ng/mL. Meanwhile, on March 22, the blood cultures were positive revealing curved gram-negative rods at the
microscopic analysis. The organism was subcultured onto chocolate agar (bioMérieux, Florence,
Italy) and then incubated at 36 ˝C in a microaerophilic environment with 5% CO2. 2. Case Presentation
A 76-year-old ma Thus, on
March 23, based on the abdominal echography (suggestive for ileotiphlitis), and the patient’s general
clinical conditions and increased procalcitonin levels, even in the absence of microbiological data
(blood cultures were negative, so far), a different antibiotic therapeutic regimen was implemented. The patient was administered Tygeciclin 50 mg intravenously twice a day after a loading dose of
100 mg, Metronidazole 500 mg four times a day, and Caspofungin 50 mg daily after a loading dose
of 70 mg. Despite implementing this type of antibiotic treatment, a rapid clinical deterioration in the
patient was observed. Additionally, on March 23, cellulitis in the patient’s left leg was observed during
a dermatological consultation. However, a skin biopsy was not advised due to the general health
condition of the patient. p
After 48 h of incubation, on March 24, irregular shaped grey and flat colonies appeared on the
chocolate agar plates. The isolate was initially identified as C. jejuni by distinct colony morphology
and by conventional biochemical tests resulting in oxidase- and hippurate-positive results. Despite the microbiology laboratory promptly notifying the possible or likely infection of
C. jejuni and the immediate implementation of empirical intravenous treatment with Gentamicin
6 mg/kg/Die, a further worsening of the patient’s clinical condition was observed on March 24. Surprisingly, microbiological testing by VITEK 2 system (bioMérieux, Florence, Italy) initially identified
the microorganism as Francisella Tularensis (96% of identification confidence) whereas repeated
testing yielded Moraxella spp. (95% of identification confidence), thereby creating uncertainty in
the identification of the microorganism present. Thus, the poor health condition of the patient
and severe cytopenia (hemoglobin 69 g/liter, platelet count 3 ˆ 109/liter, and white blood cell
count 0.5 ˆ 109/liter) contributed to a rapid fatal outcome on March 26. Further identification of
the microorganism was performed by sequence analysis (ABI PRISM 3130xl Genetic Analyzer) of the
16S rRNA gene [3]. The sequence showed 99.9% similarity and 100% coverage for the strains of C. jejuni
subsp. jejuni ATCC 700819. The sequences were deposited in the European Nucleotide Archive (ENA)
with accession number LN864495. Antimicrobial susceptibility testing (AST) was performed by Etest®, according to the Clinical and
Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae as follows: ciprofloxacin,
ď1 µg/mL (Sensitive); doxycycline, ď4 µg/mL (Sensitive); gentamicin, ď4 µg/mL (Sensitive);
meropenem, ď4 µg/mL (Sensitive) (Table 1). Table 1. Antibiotic susceptibility testing of the isolated bacteria. 3. Discussion Infections caused by C. jejuni are only rarely complicated by extraintestinal localization or
bacteremia [1]. In immunocompetent patients, C. jejuni bacteremia can be transient and resolved
without antimicrobial therapy [1]. Conversely, individuals with immune deficiency or another
serious underlying condition (cardiovascular disorders, hematological malignancies, liver disease,
hypogammaglobulinemia, and human immunodeficiency virus infection) are exposed to an increased
risk of bacteremia due to C. jejuni [2,4]. In these individuals, an effective antimicrobial treatment has
been significantly associated with an improved outcome [2]. In a large number of cases, a timely
identification of the pathogen and appropriate empirical antimicrobial therapy are hampered by the
atypical presentation of the symptoms caused by C. jejuni [1,2]. The clinical signs of Campylobacter
bacteraemia are generally accompanied by an acute-onset febrile illness of a transient nature
with self-limiting enteritis. Nevertheless, in a large percentage of cases the clinical presentation
of Campylobacter bacteraemia may show a febrile illness without gastrointestinal symptoms [4]. Other typical manifestations observed in severe sepsis caused by Campylobacter may include, skin
lesions, cytopenia, and diarrhea, however, these symptoms also occur frequently in patients with
aggressive lymphomas undergoing chemotherapy [2]. Moreover, the absence of consensus on the
optimal antibiotic regimen and the lack of studies comparing different empirical treatments for C. jejuni
bacteraemia make it difficult for the clinician to select an appropriate antimicrobial therapy. Different
strategies were adopted, including fluoroquinolones (ciprofloxacin), macrolides (erythromycin), and
aminoglycosides (gentamicin) [5]. Fluoroquinolones (e.g., ciprofloxacin) were largely used for the treatment of Campylobacter
infection and, in general, are considered the drugs of choice for the empirical treatment of diarrheal
illnesses [6–8]. Campylobacter and other organisms, such as Salmonella or Shigella species, were
generally susceptible to fluoroquinolones, thus empirical treatment with these drugs is used
without waiting for the stool culture results. However, since the early 1990s a growing number
of fluoroquinolone-resistant Campylobacter strains have been registered in Asia as well as in several
European countries. This increase of resistant strains is not only the result of the excessive use of these
antimicrobials in clinical practice, but it is also the consequence of the use of fluoroquinolones in food
producing animals and in veterinary species [9–11]. Thus, the possibility of fluoroquinolone-resistant
strains must be considered in all cases of Campylobacter bacteraemia. In the presence of confirmed Campylobacter infections, macrolides (erythromycin, or alternatively
clarithromycin or azithromycin) represent the frontline agents [12], with tetracycline, doxycycline,
and chloramphenicol considered alternative drugs [8]. 2. Case Presentation
A 76-year-old ma Antibiotic Tested
MIC
Test Result
Ciprofloxacin
ď1 µg/mL
Sensitive
Doxycycline
ď4 mcg/mL
Sensitive
Gentamicin
ď4 mcg/mL
Sensitive
Meropenem
ď4 mcg/mL
Sensitive
MIC: Minimal Inhibitory Concentration performed by Etest® (bioMérieux, Florence, Italy), according to the
Clinical and Laboratory Standards Institute (CLSI) breakpoints for non-Enterobacteriaceae. Table 1. Antibiotic susceptibility testing of the isolated bacteria. The Central Ethics Committee I.R.C.C.S. Lazio, section of the Istituti Fisioterapici Ospitalieri in
Rome, in compliance with the Helsinki Declaration, approved this case report (Prot. CE/1016/15—4
December 2015). Data and relevant scientific articles were identified via specific PubMed database searches from
January 1980 and December 2015. The terms included in the search comprised: “Campylobacter jejuni” 4 of 9 Int. J. Mol. Sci. 2016, 17, 544 and “bacteremia” or “Campylobacter” and “bacteremia” or “non-Hodgkin’s lymphoma”. Research
was restricted to English language articles. and “bacteremia” or “Campylobacter” and “bacteremia” or “non-Hodgkin’s lymphoma”. Research
was restricted to English language articles. 3. Discussion However, recent evidence suggests that
Campylobacter is also becoming increasingly resistant to macrolides, which represents a rising concern
for public health [13]. The use of macrolides at subtherapeutic levels in chickens is considered a major
factor influencing the emergence of resistant strains [13–15]. Thus, for serious systemic infections
it has been demonstrated that aminoglycoside, gentamicin, or carbapenems are the most efficient
antimicrobials [2,4,8,16,17]. In our case, the absence of clear clinical signs of a possible infection with enteric pathogens
suggested that the patient be treated with ceftriaxone in accordance with the guidelines for
cancer-related infections in immunosuppressed patients that support the use of cephalosporins in
monotherapy [18]. Third-generation cephalosporins are largely used for the empirical treatment of
community-acquired infectious diarrhea. However, these antimicrobial agents have not been proven
effective for treating bacteremia due to Campylobacter species other than Campylobacter fetus [2,19]. Moreover, the use of third-generation cephalosporins and fluoroquinolones in the treatment of
Campylobacter bacteraemia has shown poor prognosis and a high frequency of resistant strains has
resulted in a general discouragement towards using this class of antibiotics [4,20], particularly Int. J. Mol. Sci. 2016, 17, 544 5 of 9 in hospitals and communities with a high prevalence of extended-spectrum beta-lactamases
(ESBLs)-producing bacteria. After the first antimicrobial treatment, the patient presented neutropenia and fever, and therapy
was then subsequently changed. In the absence of relevant microbiological data, the guidelines for the
empirical therapy of febrile neutropenic cancer patients receiving chemotherapy recommend the use
of pipercillin-tazobactam as first line monotherapy for the treatment of bloodstream infections [21]. However, Campylobacter isolates are not regularly susceptible to penicillins [22–24] and the b-lactamase
enzyme found in C. jejuni is preferentially inhibited by clavulanic acid, but not by tazobactam or
sulbactam [22,24]. In our case, only after having diagnosed sepsis caused by C. jejuni, the patient was
empirically treated with gentamicin and the subsequent susceptibility drug profile indicated that this
strain was in fact susceptible to this antimicrobial (Table 1). Nevertheless, the patient died because of
complications due to a septic status and multiorgan failure. It is important to note that the treatment
with gentamicin in this patient had started long after the appearance of initial enteric symptoms
(diarrhea) and the first signs of sepsis. The delayed start of the targeted antimicrobial treatment was
due to the difficulty in identifying C. 3. Discussion jejuni bacteraemia, which, in turn, was the consequence of the very
slow growth of this bacterium in standard automatic blood culture systems [25]. In fact, blood cultures
are only rarely performed in patients presenting an apparently simple diarrhea symptom. This, as
well as the slow growth of the characteristics of C. jejuni and the self-limited nature of this infection
may represent a contributing cause to underestimating the real incidence of C. jejuni bacteraemia [26]. Additionally, the inability of the automated biochemical phenotyping system to promptly and correctly
identify C. jejuni further deferred the recognition of the pathogen. The slow growth of C. jejuni in
the BacT/ALERT and the repeated unsuccessful attempts in identifying the bacteria reported for the
VITEK 2 system made the recognition of this pathogen particularly elusive. Indeed, previous studies
have demonstrated that despite the Neisseria-Haemophilus (NH) identification card for VITEK 2
correctly identifying most C. jejuni ssp. Jejuni, misclassifications occur at a rate of more than 10% [27]. In this case, the diagnosis, and consequently the start of an appropriate therapy, was further delayed
by the negative result of the stool cultures after the first episodes of diarrhea. Diarrheal illnesses in
patients with neoplasia and immunosuppressive therapy are rarely perceived as a necessity to perform
blood cultures, even when there is a fever present. On the other hand, it should be considered that
blood stream infections caused by C. jejuni might occur without evidence of diarrhea, suggesting that
this bacterium can access the intestinal mucosa without causing local inflammation [28]. A retrospective study suggested that a diagnostic clue for the presence of C. jejuni infection might
be represented by leukopenia or thrombocytopenia, particularly when associated with an acute febrile
diarrheal illness [29]. However, in neoplastic and immune suppressed patients, such as in our case, the
marked cytopenia might be interpreted as a result of the immunosuppressed status of the patient who
underwent a chemo-immunotherapeutic program. In addition, three days before the fatal outcome, the patient also experienced the occurrence
of cellulitis of the left leg. It has been reported that, although less recognized, skin lesions may
represent a complication of Campylobacter bacteraemia that occurs particularly in patients with
immune-related problems [30]. Again, the presence of NHL and chemotherapy made it difficult
to recognize cellulitis as a sign of C. jejuni infection since lymphomas can be also characterized by
an initial skin presentation [31]. 4. Conclusions In summary, although C. jejuni bacteraemia is uncommon, it may develop either primarily or
secondarily from gastroenteritis, and thereby may represent a severe disease for immunocompromised
individuals [30]. Occasionally, both NHLs and C. jejuni sepsis may intertwine; in cases such as
this, it may create difficulties in being able to make a plain distinction between the root cause(s) of
a patient’s symptoms. Many hematological disorders, especially lymphoid neoplasms, have a high
risk for infection, thus when dealing with immunocompromised patients a septic disease should be 6 of 9 Int. J. Mol. Sci. 2016, 17, 544 suspected even in the presence of mild symptoms. In aggressive lymphoma, and in patients undergoing
chemotherapy, fatigue, fever, diarrhea, as well as skin lesions and cytopenia may occur frequently, but
these symptoms may occur also in severe sepsis caused by C. jejuni. Recognizing the early symptoms
of a C. jejuni bacteraemia in hematological patients is key to initiate an effective antimicrobial therapy. From our experience, and from the data reported in the literature [2,4], blood cultures should always
be performed in febrile patients with gastroenteritis. Therapy with appropriate antimicrobial agents
is an important component in the management of immunocompromised patients with C. jejuni
bacteraemia. Guidelines for cancer-related infections in immunosuppressed patients support the use of
cephalosporins in monotherapy [18], whereas for the treatment of febrile neutropenic cancer patients
receiving chemotherapy the use of pipercillin-tazobactam is recommended [21]. From our study, and
from the data reported in the literature, it emerged that immunosuppressed patients with suspected
Campylobacter sepsis should receive a more aggressive antimicrobial treatment—possibly combining
aminoglycosides and/or carbapenems with cephalosporins in the first line antimicrobial empirical
treatment. Nevertheless, the risk caused by the rise in antibiotic resistance among bacteria, particularly
with Campylobacter spp. should also be considered where an increase in the administration of multiple
antibiotics is likely to lead to colonization and infection with antibiotic-resistant organisms [32]. antibiotics is likely to lead to colonization and infection with antibiotic-resistant organisms [32]. In this case, the unequivocal identification of C. jejuni was not obtained in time, and only by
sequence analysis of the 16S rRNA gene. This further suggested that diagnostic systems, other than
those based on the biochemical identification (i.e., molecular techniques and Mass Spectrometry—MS)
should be preferred for a prompt and unequivocal laboratory identification of C. jejuni. References 1. Young, K.T.; Davis, L.M.; Dirita, V.J. Campylobacter jejuni:
Molecular biology and pathogenesis. Nat. Rev. Microbiol. 2007, 5, 665–679. [CrossRef] [PubMed] 2. Pacanowski, J.; Lalande, V.; Lacombe, K.; Boudraa, C.; Lesprit, P.; Legrand, P.; Trystram, D.; Kassis, N.;
Arlet, G.; Mainardi, J.L.; et al. Campylobacter bacteremia: Clinical features and factors associated with fatal
outcome. Clin. Infect. Dis. 2008, 47, 790–796. [CrossRef] [PubMed] 3. Di Domenico, E.G.; Toma, L.; Prignano, G.; Pelagalli, L.; Police, A.; Cavallotti, C.; Torelli, R.; Sanguinetti, M.;
Ensoli, F. Misidentification of Streptococcus uberis as a human pathogen: A case report and literature review. Int. J. Infect. Dis. 2015, 33, 79–81. [CrossRef] [PubMed] 4. Nielsen, H.; Hansen, K.K.; Gradel, K.O.; Kristensen, B.; Ejlertsen, T.; Østergaard, C.; Schønheyder, H.C. Bacteraemia as a result of Campylobacter species: A population-based study of epidemiology and clinical risk
factors. Clin. Microbiol. Infect. 2010, 16, 57–61. [CrossRef] [PubMed] 5. Hagensee, M.E.; Benyunes, M.; Miller, J.A.; Spach, D.H. Campylobacter jejuni bacteremia and Guillain-Barre´
syndrome in a patient with GVHD after allogeneic BMT. Bone Marrow. Transplant. 1994, 13, 349–351. [PubMed] 6. Guerrant, R.L.; van Gilder, T.; Steiner, T.S.; Thielman, N.M.; Slutsker, L.; Tauxe, R.V.; Hennessy, T.; Griffin, P.M.;
DuPont, H.; Sack, R.B.; et al. Practice guidelines for the management of infectious diarrhea. Clin. Infect. Dis. 2001, 32, 331–351. [CrossRef] [PubMed] 7. Aarestrup, F.M.; McDermott, P.F.; Wegener, H.C. Transmission of antibiotic resistance from food animals to
humans. In Campylobacter; Nachamkin, I., Szymanski, C.M., Blaser, M.J., Eds.; ASM Press: Washington, DC,
USA, 2008; pp. 645–665. 8. Ge, B.; Wang, F.; Sjölund-Karlsson, M.; McDermott, P.F. Antimicrobial resistance in campylobacter:
Susceptibility testing methods and resistance trends. J. Microbiol. Methods. 2013, 95, 57–67. [CrossRef]
[PubMed] 9. Endtz, H.P.; Ruijs, G.J.; van Klingeren, B.; Jansen, W.H.; van der Reyden, T.; Mouton, R.P. Quinolone
resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolones in
veterinary medicine. J. Antimicrob. Chemother. 1991, 27, 199–208. [CrossRef] [PubMed] y
10. Sam, W.I.C.; Lyons, M.M.; Waghorn, D.J. Increasing rates of ciprofloxacin resistant Campylobacter. J. Clin. Pathol. 1999, 52, 709–710. [CrossRef] [PubMed] 11. Luangtongkum, T.; Jeon, B.; Han, J.; Plummer, P.; Logue, C.M.; Zhang, Q. Antibiotic resistance in
Campylobacter: Emergence, transmission and persistence. Future Microbiol. 2009, 4, 189–200. [CrossRef]
[PubMed] 12. Blaser, M.J.; Engberg, J. Clinical aspects of Campylobacter jejuni and Campylobacter coli infections. In Campylobacter; Nachamkin, I., Szymanski, C.M., Blaser, M.J., Eds.; ASM Press: Washington, DC, USA,
2008; pp. 99–121. pp
13. 4. Conclusions Combined
molecular protocols (such as 16S rRNA PCR, DNA sequencing, and Multilocus Sequence Typing
(MLST) analysis) revealed the successful identification of C. jejuni strains from stool and from blood
cultures, even in patients where traditional culture protocols failed [33–36]. These results demonstrate
the potential of molecular methods in improving the diagnosis of bacterial infections caused by C. jejuni. Numerous PCR-based techniques (real-time PCR and pyrosequencing) have also been developed
for the rapid detection and identification of bacteria in clinical blood specimens [37]. Commercially
available real-time PCR for the direct detection of bacteria in blood has been introduced [38], but the
use of these tools has not become routine in clinical microbiology laboratories. Indeed, molecular
techniques are rather costly, and require people with high levels of technical expertise, and therefore
these techniques are consequently not suitable for routine identification, particularly in institutes with
limited financial resources or in developing countries. Moreover, the high sensitivity of PCR-based
methods and DNA sequencing that have the potential to detect all bacterial DNA present in a clinical
sample may cause serious problems in clinical interpretation. Background levels of bacterial DNA
might be detected in the blood of patients in the absence of any signs of bacteremia [39]. Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) MS is a reliable tool
for a rapid, precise, and cost-effective classification of a broad spectrum of bacteria and yeast [40]. MALDI-TOF MS analysis was in complete agreement with molecular tests identifying C. jejuni and
C. coli [41]. Moreover, changes in protein biomarkers, such as those caused by an amino acid
substitution, have been used to differentiate between C. jejuni ssp. jejuni and subsp. doylei, and
to assess phylogenetic relationships in different isolates [42]. Several studies have evaluated the
contribution of MALDI-TOF MS towards identifying microorganisms in positive blood culture [40,43]. Results showed that MALDI-TOF MS accurately identified blood-borne organisms in more than 80%
of cases. Nevertheless, the ability of MALDI-TOF MS to correctly identify microorganisms in blood
cultures clearly depends on the bacteria concentration [44,45]. Novel application of MALDI-TOF
MS has increased its potential in the detection of blood-borne organisms and thus may allow faster
bacterial identification than the conventional automated blood cultures systems in the near future [46]. However, the efficacy of MALDI-TOS MS technology in reducing the time for identifying positive
blood cultures, particularly for slow growing bacteria such as C. jejuni, remains to be evaluated. Int. J. 4. Conclusions Mol. Sci. 2016, 17, 544 7 of 9 Since individuals with hematological disorders, especially lymphoid neoplasms, have a high
risk for infection, the close cooperation between the hematologist, infectious disease specialist, and
microbiologist can be of primary importance in providing a timely and effective intervention. Acknowledgments: This work was supported by L’Associazione Nazionale Contro le Infezioni Ospedaliere
(L’ANCIO). We would like to thank Tania Merlino, who kindly edited the English language used in our manuscript. Author Contributions:
Maria Teresa Gallo; Enea Gino Di Domenico; Luigi Toma; Francesco Marchesi;
Lorella Pelagalli; Nicola Manghisi; Fiorentina Ascenzioni; Grazia Prignano; Andrea Mengarelli; Fabrizio Ensoli
participated in the study conception and design; Maria Teresa Gallo, Grazia Prignano, Nicola Manghisi contributed
to the acquisition of literature data; Enea Gino Di Domenico, Luigi Toma, Francesco Marchesi, Andrea Mengarelli,
Fabrizio Ensoli drafted the manuscript. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest. [CrossRef] [PubMed] 16. Blaser, M.J. Campylobacter jejuni and related species. In Mandell, Douglas and Bennett’s Principles and Practice
of Infectious Diseases, 6th ed.; Mandell, G.L., Bennett, J.E., Dolin, R., Eds.; Churchill Livingstone: New York,
NY, USA, 2006; pp. 2548–2557. 17. Okada, H.; Kitazawa, T.; Harada, S.; Itoyama, S.; Hatakeyama, S.; Ota, Y.; Koike, K. Combined treatment
with oral kanamycin and parenteral antibiotics for a case of persistent bacteremia and intestinal carriage
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein
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Open Access Open Access * Correspondence: adam.rash@aht.org.uk
1Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK
Full list of author information is available at the end of the article Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 An efficient genome sequencing method for
equine influenza [H3N8] virus reveals a new
polymorphism in the PA-X protein Adam Rash1*, Alana Woodward1, Neil Bryant1, John McCauley2 and Debra Elton1 Adam Rash1*, Alana Woodward1, Neil Bryant1, John McCauley2 and Debra Elton1 © 2014 Rash et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Background for HA, which makes it difficult to study the evolution of
either the other gene segments or the virus as a whole. It is
also known that reassortment between the different line-
ages of EIV has occurred [4-8,15,16] but the full extent is
unknown due to a lack of data available. Avian H3N8 influ-
enza viruses have been shown to frequently exchange in-
ternal gene segments, and it has been suggested that the
extensive reassortment within the H3 subtype poses a
threat to human and animal health [17]. Next generation
sequencing technologies have made it easier to sequence
whole viral genomes, however these technologies are not
readily available to all as considerable investment in equip-
ment and bioinformatics expertise are needed. We aimed
to develop a simple and robust method to sequence whole
EIV genomes from all H3N8 lineages using Sanger dideoxy-
nucleotide sequencing technology. Equine influenza virus (EIV) is an influenza A virus
belonging to the Orthomyxoviridae family. These viruses
have a negative sense, single-stranded RNA genome
consisting of eight viral gene segments [1]. Originally
thought to have transmitted from birds, H3N8 EIV was
first isolated during a widespread outbreak in the United
States in 1963 [2], and has since spread worldwide caus-
ing multiple major outbreaks of disease in horses. Dur-
ing the 1980s the virus diverged into two antigenically
distinct lineages [3], American and Eurasian, and since
then the American lineage has further evolved into the
Florida sublineage clades 1 and 2, which continue to co-
circulate today [4]. These lineages have historically been
based on antigenic and genetic data for HA. A phylogen-
etic study by Murcia et al. [5] showed that phylogenetic
trees produced for each of the viral gene segments also
supported division into the American and Eurasian
lineages, and all but segment 7 divided into the two
clades of the Florida sublineage. However, less than 100
complete viral genomes covering the 46 years from 1963
to 2008 were available at the time of the study. Each of the eight influenza virus gene segments con-
tains two non-coding regions (NCRs), one at the 5′
terminus containing 13 conserved nucleotides, and the
other at the 3′ terminus, which contains 12 nucleotides
[18]. Unlike the 5′ end, the 3′ terminus exhibits vari-
ation at the fourth nucleotide. Abstract Background: H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its
first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data
available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA),
one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and
determine the level of reassortment occurring between sub-lineages. Methods: To facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and
efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then
sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published
method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral
RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the
design of novel primers for an N8 neuraminidase segment. Results: Two field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07,
representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length
from 400–600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient
overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the
non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes
sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates. Conclusions: These methods can be used to determine the genome sequences of EIV, including the NCRs, from
both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR
products than previously reported methods for influenza A viruses, the techniques used are affordable and the
equipment required is available in most research laboratories. The adoption of these methods will hopefully allow
for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better
understanding of EIV evolution. Keywords: Equine influenza virus, H3N8, Genome sequencing, Non-coding regions, M13, PA-X Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Page 2 of 9 Background This variation in the
fourth nucleotide has been shown to affect the rescue of
virus from a reverse genetics system [19]. A second ob-
jective, to implement a method previously described for
sequencing the NCRs of influenza viruses [20], was
adapted and carried out on an EIV, as well as an N8 sub-
type neuraminidase, for the first time. Other groups have studied the evolution of individual
influenza A virus genes. H3N8 EIV PB2 [6] and matrix
proteins [7] were found to belong to the same lineage as
North American avian strains, whilst PB1, PA, HA and
NP were found to have evolved independently from
other influenza A viruses [8-10]. Equine NS was sug-
gested as being restricted to subtype [11], as the NS seg-
ments of the H3N8 viruses were close to one another
but not to that of the H7N7 viruses, however very lim-
ited numbers of EIV genomes were available at the time
these studies were performed, and in some cases only
two different EIV strains were used. More recently a
study found that the internal genes of the 1963 EIV pan-
demic virus were of western hemispheric avian influenza
origin [12]. This study also showed that the virus shared
a most recent common ancestor with avian influenza vi-
ruses from South America shortly before its emergence. The time to most recent common ancestor for avian/
equine NP was calculated as being 1954, which agreed
with the hypothesis that the virus emerged in South
America prior to its introduction into the USA in 1963
by horses imported by air from Argentina [12]. Here we describe the genome sequencing method and
highlight the sequence differences found between repre-
sentatives of the two circulating clades of the Florida
sublineage. Results q
g
q
At the time of writing, only 81 full genome sets were
available from the NCBI Influenza Virus Resource for
EIV and only one or two gene segment sequences had
been published for the majority of strains. To address
the lack of available genomes, a method to sequence the ge-
nomes of equine influenza viruses belonging to both clades
of the currently circulating Florida sublineage, using an
EIV specific primer set for PCR and M13 primers for
sequencing, was developed. A/equine/Richmond/1/07
was selected as a representative of recent Florida sublineage
clade 2 (FC2) viruses, as well as being a current OIE recom-
mended vaccine strain. A/equine/Lincolnshire/1/07 was
chosen because it was the first virus belonging to clade 1 of
the Florida sublineage isolated in the UK [4]. Published
nucleotide sequences were aligned for each segment and
primers were designed to conserved regions (data not
shown)
to amplify
products of 400–600 nucleotides
(Figure 1). Each specific primer was elongated at the 5′ end
by adding either M13 forward or M13 reverse primer Following an extensive outbreak in 1989 affecting a
highly vaccinated population of racehorses in the UK, it be-
came clear that, like human influenza virus, EIV undergoes
antigenic drift and therefore vaccine strains need to be kept
up to date [13,14]. A formal process for vaccine strain se-
lection, overseen by the World Organisation for Animal
Health (OIE) was put in place. This process relies heavily
on surveillance data collected from the field, of which most
is focussed solely on the HA gene and the protein it en-
codes. Therefore the majority of published sequences are Page 3 of 9 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 PB2, PB1, PA & HA:
M13
AF
M13
BF
M13
CF
M13
DF
M13
AR
M13
BR
M13
CR
M13
DR
M13
BF
M13
BR
NP & NA:
A
B
C
D
A
B
C
A
B
M & NS:
M13
AF
M13
AR
M13
CF
M13
CR
M13
AF
M13
AR
M13
BF
M13
BR
Figure 1 Schematic representation of PCR primer design for genome sequencing EIV. Primers were tagged with M13 forward or reverse
sequences for use in the sequencing stage. Results Segments 1–4 (PB2, PB1, PA and HA) were divided into four sections, segments 5 & 6 (NP and NA)
into three sections and segments 7 & 8 (M and NS) into two sections, each of approximately 400–600 nucleotides in length. B NP & NA: B M & NS: B A Figure 1 Schematic representation of PCR primer design for genome sequencing EIV. Primers were tagged with M13 forward or reverse
sequences for use in the sequencing stage. Segments 1–4 (PB2, PB1, PA and HA) were divided into four sections, segments 5 & 6 (NP and NA)
into three sections and segments 7 & 8 (M and NS) into two sections, each of approximately 400–600 nucleotides in length. There were a total of 279 nucleotide differences between
the two viruses, which resulted in 65 amino acid changes
(Table 2). Approximately 45% (29) of the total amino
acid changes were observed within the polymerase and
nucleoprotein segments, with 45% (13) of these occur-
ring within PA alone. The two glycoprotein segments,
HA and NA, contained 40% (26) of the total amino acid
differences, of which 54% (14) were within HA and 46%
(12) within NA. sequences (Table 1), as described in a method for sequen-
cing swine influenza genomes [21] which facilitated effi-
cient sequencing. Four amplicons were produced for the
larger genome segments, 1 to 4, three amplicons for seg-
ments 5 and 6, and two amplicons for segments 7 and 8
(Figure 2). A total of 26 PCR products were amplified
successfully from RNA extracted from allantoic fluid for
both virus strains, A/equine/Richmond/1/07 and A/equine/
Lincolnshire/1/07. The nucleotide sequences of the PCR
products were determined on both strands and the com-
plete viral genome was assembled successfully without the
need for further specific primers to complete gaps. The
PCR products overlapped by approximately 100 nucleotides
at each end and sequences were edited, so that the final
coding sequence contained no primer-derived sequences. This method has since been applied to a further 17 strains
of EIV, all primers worked well for Florida clade 1 and clade
2 strains from 2009–2013, and sequences were made avail-
able on the GISAID (Global Initiative on Sharing Avian In-
fluenza Data) EpiFlu database [22] (see Additional file 1). Assembled sequences for each segment of A/equine/
Richmond/1/07 and A/equine/Lincolnshire/1/07 were also
uploaded onto GenBank, accession numbers indicated in
additional material (see Additional file 2). Results The two smaller segments, M and NS, contained a
total of 10 amino acid changes within the four predicted
polypeptides (M1, M2, NS1 and NS2/NEP) that they
encode. Nucleotide changes in three segments resulted in dif-
ferent lengths for their predicted polypeptides. A dupli-
cation of six nucleotides in A/equine/Richmond/1/07
resulted in a two amino acid insertion within the puta-
tive signal peptide of the precursor HA protein extend-
ing its length from 15 to 17 amino acids, as observed in
recent FC2 isolates [4]. In contrast, when compared to
earlier EIV isolates from 1963–2000 the predicted amino
acid sequences for the NS1 protein from both viruses
were truncated by 11 amino acids, as seen for other re-
cent isolates [4]. This was caused by a premature stop
codon at position 220, resulting in a predicted polypep-
tide length of 219 amino acids. The open reading frame The gene segment sequences for each of the two vi-
ruses were aligned against one another for comparison. Rash et al. Results Virology Journal 2014, 11:159
Page 5 of 9
http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Page 5 of 9 Page 5 of 9 Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV (Continued) Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV (Continued)
NA/CR
GC AACAGCTATGACCATG AGTAGAAACAAGGAGTT
M/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGTAGATATTTAAAG
1-654
50
M/AR
GC AACAGCTATGACCATG CTAGCCTTACTAGCAAC
M/BF
GC GTAAAACGACGGCCAGT CAGTACCACGGCTAAAG
571-1027
50
M/BR
GC AACAGCTATGACCATG AGTAGAAACAAGGTAGTTTTTTAC
NS/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTGACAAAAAC
1-492
50
NS/AR
GC AACAGCTATGACCATG CTGCTCCTTCTTCGGTG
NS/BF
GC GTAAAACGACGGCCAGT CATCATACTTAAAGCAAAC
407-890
50
NS/BR
GC AACAGCTATGACCATG AGTAGAAACAAGGTAGTGTTTTTTAT Table 1 Primer sequences and annealing temperatures used to sequence the genome o (252 amino acids) or are truncated by 19 amino acids. To investigate further and to study the evolution of the
truncation, the PA-X region of segment 3 from an add-
itional 29 EIV isolated in the UK between 2005 and
2013, including 9 from 2007, were sequenced using the
method described here (see Additional file 3). The 42
amino acid truncation in A/equine/Richmond/1/07 PA- of NS2/NEP however, was unaffected by this nucleotide
substitution. In addition, a novel truncation in the re-
cently discovered PA-X gene was identified in A/equine/
Richmond/1/07, caused by an early stop codon at pos-
ition 20 of the +1 reading frame. The truncation of PA-
X by 42 amino acids has not been described before, with
the majority of strains having either a full length version 600bp
800bp
1kb
400bp
A
B
C
D
A
B
C
D
A
B
C
D
Segment 1 (PB2)
Segment 2 (PB1)
Segment 3 (PA)
600bp
800bp
1kb
400bp
Segment 4 (HA)
A
B
C
D
A
B
C
Segment 5 (NP)
A
B
C
Segment 6 (NA)
600bp
800bp
1kb
400bp
A
B
Segment 7 (M)
A
B
Segment 8 (NS)
Figure 2 Agarose gel electrophoresis analysis of genome segment PCR products. Agarose gel (1%) showing PCR fragments A, B,
C & D of segments 1–4 (PB2, PB1, PA and HA), A, B & C of segments 5 and 6 (NP and NA), and A & B of segments 7 and 8 (M and NS) of
EIV Northamptonshire/1/13. Figure 2 Agarose gel electrophoresis analysis of genome segment PCR products. Results Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Page 4 of 9 Page 4 of 9 Table 1 Primer sequences and annealing temperatures used to sequence the genome
Primer name
Primer sequence (5′-3′)
Approximate
coverage (5′-3
PB2/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG
1-655
PB2/AR
GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT
PB2/BF
GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA
569-1335
PB2/BR
GC AACAGCTATGACCATG CCTCAAGAGTTGATG
PB2/CF
GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC
1216-1874
PB2/CR
GC AACAGCTATGACCATG ATTATTTGAGCAGTATC
PB2/DF
GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT
1793-2341
PB2/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC
PB1/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG
1-719
PB1/AR
GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG
PB1/BF
GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA
577-1301
PB1/BR
GC AACAGCTATGACCATG GGTTTAATATGGATACACC
PB1/CF
GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC
1222-1863
PB1/CR
GC AACAGCTATGACCATG CATTTTAAACAAACTTC
PB1/DF
GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG
1789-2341
PB1/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC
PA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG
1-615
PA/AR
GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC
PA/BF
GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG
529-1255
PA/BR
GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC
PA/CF
GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG
1149-1785
PA/CR
GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC
PA/DF
GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG
1700-2233
PA/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG
HA/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT
1-515
HA/AR
GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG
HA/BF
GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G
428-1032
HA/BR
GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC
HA/CF
GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC
939-1336
HA/CR
GC AACAGCTATGACCATG GAGCCACCAGCAATTCT
HA/DF
GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA
1251-1733
HA/DR
GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC
NP/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC
1-570
NP/AR
GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC
NP/BF
GC GTAAAACGACGGCCAGT GACACCACATACCAAAC
480-1075
NP/BR
GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT
NP/CF
GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG
1012-1569
NP/CR
GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC
NA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT
1-508
NA/AR
GC AACAGCTATGACCATG GCCCTATTTTGACACTC
NA/BF
GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC
417-1049
NA/BR
GC AACAGCTATGACCATG CCGAAACCTTTTACACCG
NA/CF
GC GTAAAACGACGGCCAGT CACAGTTGGATATTTGTG
951-1461 Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV
Primer name
Primer sequence (5′-3′)
Approximate nucleotide
coverage (5′-3′)
Annealing temperature
used (°C)
PB2/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG
1-655
50
PB2/AR
GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT
PB2/BF
GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA
569-1335
60
PB2/BR
GC AACAGCTATGACCATG CCTCAAGAGTTGATG
PB2/CF
GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC
1216-1874
45
PB2/CR
GC AACAGCTATGACCATG ATTATTTGAGCAGTATC
PB2/DF
GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT
1793-2341
50
PB2/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC
PB1/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG
1-719
50
PB1/AR
GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG
PB1/BF
GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA
577-1301
50
PB1/BR
GC AACAGCTATGACCATG GGTTTAATATGGATACACC
PB1/CF
GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC
1222-1863
50
PB1/CR
GC AACAGCTATGACCATG CATTTTAAACAAACTTC
PB1/DF
GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG
1789-2341
50
PB1/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC
PA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG
1-615
50
PA/AR
GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC
PA/BF
GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG
529-1255
50
PA/BR
GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC
PA/CF
GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG
1149-1785
50
PA/CR
GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC
PA/DF
GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG
1700-2233
50
PA/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG
HA/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT
1-515
50
HA/AR
GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG
HA/BF
GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G
428-1032
50
HA/BR
GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC
HA/CF
GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC
939-1336
50
HA/CR
GC AACAGCTATGACCATG GAGCCACCAGCAATTCT
HA/DF
GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA
1251-1733
50
HA/DR
GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC
NP/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC
1-570
50
NP/AR
GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC
NP/BF
GC GTAAAACGACGGCCAGT GACACCACATACCAAAC
480-1075
45
NP/BR
GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT
NP/CF
GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG
1012-1569
55
NP/CR
GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC
NA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT
1-508
45
NA/AR
GC AACAGCTATGACCATG GCCCTATTTTGACACTC
NA/BF
GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC
417-1049
45
NA/BR
GC AACAGCTATGACCATG CCGAAACCTTTTACACCG Table 1 Primer sequences and annealing temperatures used to sequence the genome
Primer name
Primer sequence (5′-3′)
Approximate
coverage (5′-
PB2/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG
1-655
PB2/AR
GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT
PB2/BF
GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA
569-1335
PB2/BR
GC AACAGCTATGACCATG CCTCAAGAGTTGATG
PB2/CF
GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC
1216-1874
PB2/CR
GC AACAGCTATGACCATG ATTATTTGAGCAGTATC
PB2/DF
GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT
1793-2341
PB2/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC
PB1/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG
1-719
PB1/AR
GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG
PB1/BF
GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA
577-1301
PB1/BR
GC AACAGCTATGACCATG GGTTTAATATGGATACACC
PB1/CF
GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC
1222-1863
PB1/CR
GC AACAGCTATGACCATG CATTTTAAACAAACTTC
PB1/DF
GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG
1789-2341
PB1/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC
PA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG
1-615
PA/AR
GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC
PA/BF
GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG
529-1255
PA/BR
GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC
PA/CF
GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG
1149-1785
PA/CR
GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC
PA/DF
GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG
1700-2233
PA/DR
GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG
HA/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT
1-515
HA/AR
GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG
HA/BF
GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G
428-1032
HA/BR
GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC
HA/CF
GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC
939-1336
HA/CR
GC AACAGCTATGACCATG GAGCCACCAGCAATTCT
HA/DF
GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA
1251-1733
HA/DR
GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC
NP/AF
GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC
1-570
NP/AR
GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC
NP/BF
GC GTAAAACGACGGCCAGT GACACCACATACCAAAC
480-1075
NP/BR
GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT
NP/CF
GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG
1012-1569
NP/CR
GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC
NA/AF
GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT
1-508
NA/AR
GC AACAGCTATGACCATG GCCCTATTTTGACACTC
NA/BF
GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC
417-1049
NA/BR
GC AACAGCTATGACCATG CCGAAACCTTTTACACCG
NA/CF
GC GTAAAACGACGGCCAGT CACAGTTGGATATTTGTG
951-1461 eratures used to sequence the genome of H3N8 EI Rash et al. Results Agarose gel (1%) showing PCR fragments A, B,
C & D of segments 1–4 (PB2, PB1, PA and HA), A, B & C of segments 5 and 6 (NP and NA), and A & B of segments 7 and 8 (M and NS) of
EIV Northamptonshire/1/13. Page 6 of 9 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07
Segment
Nucleotide changes
Protein
Amino acid changes
Amino acid changes from Richmond/1/07 to Lincolnshire/1/07
1
35
PB2
4
A105T, K251R, I398V, K660R
2
48
PB1
8
F94L, M119V, V149I, M179I, R329Q, E377D,D618E, K621R
3
47
PA
13
D64E, I86M, M210A, K237E, G240E, P259S, N321S, L348I, S409N, I465V,
T476A, I500V, R626K
4
47a
HA1†
11b
K-14T, F-11L, I-9a1, F-9b1, N7G, R62K, V78A, D104N, A138S, N159S, E291D
HA2
3
T43A, E50G, I198V
5
30
NP
4
I131M, T257I, A359T, S450N
6
37
NA‡
12
T9A, S12F, V35A, E40K, G42D, H66Y, P78S, I191V, N235D, S337N, I410V,
G416E
7
24
M1
3
I15V, I80V, K95R
M2
2
S86D, 290G
8
11
NS1
4
I48S, I84V, Y207H, G210R
NS2/NEP
1
M52I
aIncludes a 6 nucleotide duplication resulting in btwo additional amino acids in A/equine/Richmond/1/07. †Numbering starting after the putative signal sequence,
‡numbering starting from start codon 1Two amino acid insertion in Richmond/1/07 HA, not present in Lincolnshire/1/07. Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07
Segment
Nucleotide changes
Protein
Amino acid changes
Amino acid changes from Richmond/1/07 to Lincolnshire/1/07 Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07 aIncludes a 6 nucleotide duplication resulting in btwo additional amino acids in A/equine/Richmond/1/07. †Numbering starting after the putative signal sequence,
‡numbering starting from start codon 1Two amino acid insertion in Richmond/1/07 HA, not present in Lincolnshire/1/07. segment 6, and both the 5′ and 3′ of segment 7 were
not visible by gel electrophoresis, as shown in Figure 3. Despite bands of the correct size not being visible for
these products, sequence covering the NCR regions of
interest were successfully determined for all three. Sub-
sequent sequencing of the amplified NCRs revealed that
the 13 nucleotides of the 5′ end were identical in all 8
viral gene segments, as well as the 12 bases of the 3′
end of the vRNA except for the fourth nucleotide. Results The
three polymerase segments (PB2, PB1 and PA) all con-
tained a cytosine, whilst the remaining segments (HA,
NP, NA, M and NS) contained a uracil at the fourth nu-
cleotide position. X was identified in three of the isolates, one from an-
other horse in the same outbreak that A/equine/Rich-
mond/1/07 was isolated from, and the other two from a
separate outbreak in 2007. The remaining isolates did
not share the truncation, and all had a full length PA-X
of 252 amino acids (61 amino acids following the +1
frameshift) (see Additional file 3). Sequencing of the non-coding regions of equine influenza
viruses Previous studies have shown that there are discrepancies
in the segments that contain a cytosine at nucleotide
position four of the 3′ NCR, and that a cytosine at this
position is not restricted to the polymerase segments
[23]. This variation has been implicated in the differing
levels of vRNA and mRNA synthesis observed during
the virus replication cycle whereby a uracil at this pos-
ition increased mRNA production and delayed vRNA
synthesis [23]. Another study showed that the fourth nu-
cleotide of the NCR at the 3′ end of influenza vRNA
segments could influence rescue of viruses using reverse
genetics [19]. As one of our future aims was to generate
a reverse genetics system for A/equine/Richmond/1/07,
we determined the sequence of the NCRs for each vRNA
segment for this virus strain. Viral RNA was self-ligated,
then each of the NCRs amplified using a universal
primer complementary to the opposite NCR and a seg-
ment specific primer, as described by de Wit et al. [20]. Modifications were made to all but two of the published
primer sequences to ensure that they were complemen-
tary to equine influenza viruses sequenced previously
(Table 3). The NCRs from each segment were amplified
successfully, but with varying degrees of efficiency. In
particular the 3′ of segment 8 was amplified to a high
level, whereas products of the correct size for the 5′ of Discussion We and others have previously shown that reassortment
has occurred between different EIV [4-8,15,16], but a
lack of full genome sequences for EIV makes it difficult
to ascertain the extent of reassortment between them,
and whether reassortment has occurred between EIV
and influenza A viruses from other species. We therefore
developed a simple method for sequencing viral ge-
nomes that included primers with M13 sequence tags to
improve the sequencing efficiency. This was based upon
the approach recommended by the WHO for sequen-
cing swine influenza virus isolates in 2009 [21], however
our method used only 26 PCR fragments to cover the
segment-specific regions of EIV, rather than 46 frag-
ments. Alternative methods have been employed for
sequencing influenza A viruses, such as using universal
primers to simultaneously amplify all eight genome seg-
ments, or segment specific primers to amplify entire seg-
ments; however, in our hands such protocols result in
poor amplification of the three largest genome segments
(data not shown). Other methods based on amplification Page 7 of 9 Page 7 of 9 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Table 3 Primer sequences used for sequencing 3′ and 5′
NCRs of H3N8 EIV
Primer
Primer sequence†
Universal 3′
5′-CCTTGTTTCTACTAGC-3′
Universal 5′
5′-CCTGCTTTTGCTAGT-3′
PB2 3′
5′-GGGTATTTCATTGCCATCATCC-3′
PB2 5′
5′-GACTCTAGCATACTTACTGACAG-3′
PB1 3′
5′-GACAGTATCCATGGTGTATCCTGT-3′
PB1 5′
5′-GTATGGTTGAGGCCATGGTGTCC-3′
PA 3′
5′-ATCCCTGTGATTTCAAATCTTTCTTC-3′
PA 5′
5′-GAATGCCTGATTAATGATCCCTG-3′
HA 3′
5′-AATGTTCCATTTGCTACTGCATG-3′
HA 5′
5′-GGATTTCATTCGCCATATCATG-3′
NP 3′
5′-TCAAATGCCGAAAGT-3′
NP 5′
5′-CCGATCGTGCCTTCCTTTGACAT-3′
NA 3′
5′-GTGGAGTAGATCATAAAATTGCC-3′
NA 5′
5′-CAGACCTGTTTCATTGTTATTGAG-3′
M 3′
5′-ATAAAGCGTCTACGCTGCAGTCC-3′
M 5′
5′-AAAGAGGGCCTTCTACGGAAGG-3′
NS 3′
5′-CCGTATTATCATTCCATTTAAG-3′
NS 5′
5′-TGATAATACGGTTAGAATCTCT-3′
†Sequences from de Wit et al., [20] shown in italics with specific individual
nucleotide changes in bold text, plus novel primer sequences for the N8
NA segment. Table 3 Primer sequences used for sequencing 3′ and 5′
NCRs of H3N8 EIV
Primer
Primer sequence†
Universal 3′
5′-CCTTGTTTCTACTAGC-3′
Universal 5′
5′-CCTGCTTTTGCTAGT-3′
PB2 3′
5′-GGGTATTTCATTGCCATCATCC-3′
PB2 5′
5′-GACTCTAGCATACTTACTGACAG-3′
PB1 3′
5′-GACAGTATCCATGGTGTATCCTGT-3′
PB1 5′
5′-GTATGGTTGAGGCCATGGTGTCC-3′
PA 3′
5′-ATCCCTGTGATTTCAAATCTTTCTTC-3′
PA 5′
5′-GAATGCCTGATTAATGATCCCTG-3′
HA 3′
5′-AATGTTCCATTTGCTACTGCATG-3′
HA 5′
5′-GGATTTCATTCGCCATATCATG-3′
NP 3′
5′-TCAAATGCCGAAAGT-3′
NP 5′
5′-CCGATCGTGCCTTCCTTTGACAT-3′
NA 3′
5′-GTGGAGTAGATCATAAAATTGCC-3′
NA 5′
5′-CAGACCTGTTTCATTGTTATTGAG-3′
M 3′
5′-ATAAAGCGTCTACGCTGCAGTCC-3′
M 5′
5′-AAAGAGGGCCTTCTACGGAAGG-3′
NS 3′
5′-CCGTATTATCATTCCATTTAAG-3′
NS 5′
5′-TGATAATACGGTTAGAATCTCT-3′
†Sequences from de Wit et al., [20] shown in italics with specific individual
nucleotide changes in bold text, plus novel primer sequences for the N8
NA segment. Table 3 Primer sequences used for sequencing 3′ and 5′
NCRs of H3N8 EIV differences between the two viruses. Discussion This was expected
as these two proteins are under constant immune-driven
selection pressure to undergo antigenic drift. Interest-
ingly a high number of amino acid differences were
found in PA, especially when compared to the other two
polymerase subunits PB2 and PB1, and a similar finding
was observed by Murcia et al. [5]. The other internal
segments contained fewer changes, which is not surpris-
ing as they are both smaller and may be under less im-
mune pressure than the surface proteins. Interestingly, a mutation in the +1 reading frame of
PA, causing a premature stop codon in the translated
amino acid sequence of PA-X, was observed in A/
equine/Richmond/1/07. PA-X is a recently discovered
protein containing the N-terminal 191 amino acids of
PA and, in the majority of strains, a further 61 amino
acids derived from a frameshift to the +1 reading frame
of PA [24]. PA-X has been implicated in the modulation
of influenza virus pathogenicity and virulence in a
mouse model, whereby PA-X deficient viruses caused
greater clinical signs and were less able to shut off host
cell responses compared to wild-type viruses with full
length PA-X [24]. The premature stop codon in A/
equine/Richmond/1/07 would lead to a truncation of the
protein by 42 amino acids. Truncated forms of PA-X
have been described previously, however the majority of
these are due to a nonsense mutation at codon 42 in
the +1 reading frame [25]. Sequencing of PA, as de-
scribed here, revealed that several other virus isolates
from different outbreaks in 2007 as well as from the
same yard as A/equine/Richmond/1/07, had the same
truncated form of PA-X, however the truncated form did
not persist in the UK. †Sequences from de Wit et al., [20] shown in italics with specific individual
nucleotide changes in bold text, plus novel primer sequences for the N8
NA segment. of small PCR fragments do not include the M13 se-
quences in the primers, which makes the method de-
scribed here simple and efficient. A method previously
described for sequencing the NCRs of the influenza gene
segments was also modified and successfully used for
the first time on an equine influenza virus, with novel
primers designed for an N8 subtype NA. Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Page 8 of 9 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 PCR amplification of gene segments polymerase segments, as found in other influenza viruses,
and uracil at this position in the remaining 5 segments. This is the same pattern seen in the majority of other influ-
enza A viruses for which the promoter sequences have been
determined, including the prototype avian influenza virus,
A/chicken/Rostock/34 (H7N1) [26]. Viral gene segments were amplified in 50 μl PCR reac-
tions consisting of 2 μl cDNA (representing 10% of the
reverse transcription reaction), dNTP mix (0.2 mM each
final concentration) (Qiagen), 1 × Pfu buffer, 2.5U Native
Pfu DNA polymerase (Stratagene), water and oligonucleo-
tide pairs (final concentration each of 0.2 μM) as listed in
Table 1. The cycling conditions were as follows: initial
denaturation at 96°C for 1 minute, followed by 25 cycles of
denaturation at 96°C for 15 seconds, primer annealing at
50-60°C (see Table 1) for 10 seconds and elongation at 60°C
for 5 minutes. PCR reactions were analysed on a 1% agar-
ose gel containing GelRed nucleic acid stain (Biotium)
according to manufacturer’s directions. PCR products were
purified using a QIAquick PCR purification kit (Qiagen) ac-
cording to manufacturer’s directions. The methods outlined here can be used to determine
the genome sequences of EIV, including the NCRs, from
both clade 1 and clade 2 of the Florida sublineage. The
techniques described here are affordable, and the equip-
ment required is available in most research laboratories. The sequence assembly process is simple and does not
require in depth bioinformatics, unlike next generation
sequencing methodology. Due to the small genome size
and small sample numbers usually associated with EIV, this
method is therefore highly cost effective and straightfor-
ward. Amplicon sequencing has also been shown to be less
labour intensive and more affordable than plasmid cloning
methods [27]. This method also permits the sequencing of
individual gene segments with relative ease, as was the case
with PA described here to investigate the frequency of the
truncated form of PA-X. PCR amplification of non-coding regions
The method described by de Wit et al. [20] was used
with modifications, as detailed in Table 2. Novel primers
were designed to amplify the N8 subtype NA segment. Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Briefly, following an initial denaturation at 65°C for 5 mi-
nutes in the presence of T4 RNA ligase buffer and 20U
RNAsin RNase inhibitor (Promega), 15 μl RNA was li-
gated using 40U T4 RNA ligase (New England Biolabs)
at 37°C for 1 hour. The ligation reaction was stopped by
heat inactivation at 65°C for 10 minutes. cDNA was
transcribed from 4 μl of the ligated RNA by incubating
the RNA in a mixture consisting of 0.5 μg random pri-
mer (Promega), dNTP mix (0.5 mM each final concen-
tration) (Qiagen) and 20U RNasin at 65°C for 5 minutes,
then cooling to 4°C, before adding 20U RNasin, 5 mM
DTT, 200U Superscript II (Invitrogen) and 1 × First
Strand buffer in a total reaction volume of 20 μl. The re-
action mixture was subsequently incubated at 25°C for
5 minutes, followed by 50°C for 1 hour. 50 μl PCR reac-
tions consisting of 4 μl cDNA (representing 20% of the
reverse transcription reaction), dNTP mix (20 mM final
concentration), universal primer (3′- or 5′- final concen-
tration 0.2 μM) (Table 2), gene segment specific primer
(3′- or 5′- final concentration 0.2 μM) (Table 2), 1 × Pfu
buffer and 2.5U Native Pfu DNA polymerase (Strata-
gene) were made. The cycling conditions were as fol-
lows: initial denaturation at 96°C for 6 minutes, followed
by 40 cycles of denaturation at 96°C for 30 seconds, pri-
mer annealing at 37°C for 1 minute and elongation at
72°C for 2 minutes. PCR reactions were analysed on a
2.5% agarose gel containing GelRed nucleic acid stain
(Biotium) according to manufacturer’s directions. PCR
products were purified using a QIAquick PCR purifica-
tion kit (Qiagen) according to manufacturer’s directions. Where multiple bands were present in the gel, bands
of the correct size were excised and purified using a Methods
Vi EIV A/equine/Richmond/1/07 and A/equine/Lincolnshire/
1/07 had previously been isolated and passaged twice
in embryonated chicken eggs [4]. RNA was isolated
from 140 μl virus stocks containing ~107 EID50/ml
using a QIAamp Viral RNA Mini Kit (Qiagen), accord-
ing to manufacturer’s directions. RNA was eluted in
50 μl elution buffer. Conclusions We have developed a simple, efficient and affordable
method for sequencing whole genomes of EIV that of-
fers an improvement compared with previously pub-
lished methods. The adoption of these methods should
facilitate an increase in the number of full genome se-
quences available for EIV. This will benefit surveillance
programmes for EIV and improve understanding of the
evolutionary paths taken by the virus, including the level
of reassortment. Discussion Sequence analysis of the NCRs from each segment
showed that EIV strain A/equine/Richmond 1/07 had
cytosine at position 4 of the 3′ vRNA in the three The two segments encoding the surface glycoproteins,
HA and NA, contained a large number of amino acid 200bp
100bp
400bp
600bp
3’
3’
3’
3’
5’
5’
5’
5’
Seg. 1 (PB2)
Seg. 2 (PB1)
Seg. 3 (PA)
Seg. 4 (HA)
200bp
100bp
400bp
600bp
3’
3’
3’
3’
5’
5’
5’
5’
Seg. 5 (NP)
Seg. 6 (NA)
Seg. 7 (M)
Seg. 8 (NS)
Figure 3 Agarose gel electrophoresis analysis of non-coding region PCR products. Agarose gel (2.5%) showing PCR fragments for the
non-coding regions of A/equine/Richmond/1/07 influenza virus gene segments. The positions of molecular weight markers are indicated by
black arrows. Bands of the expected size were visible in all lanes except for NA 5′, M 3′ and 5′. Figure 3 Agarose gel electrophoresis analysis of non-coding region PCR products. Agarose gel (2.5%) showing PCR fragments for the
non-coding regions of A/equine/Richmond/1/07 influenza virus gene segments. The positions of molecular weight markers are indicated by
black arrows. Bands of the expected size were visible in all lanes except for NA 5′, M 3′ and 5′. Sequencing Sequencing reactions were performed using the BigDye
terminator sequencing kit version 3.1 (Applied Biosys-
tems). M13 forward and reverse primers were used for
gene segment PCR products, whilst for the non-coding
regions the primers used for the PCR stage were reused,
both at a final concentration of 80nM. The sequencing
reactions were run on a 3130xl genetic analyzer (Applied
Biosystems), and the resulting nucleotide sequences were
visualised, assembled and edited using SeqMan II ver-
sion 5.03 (DNAStar, Inc) and BioEdit version 7.0.5.3 (Ibis
Pharmaceuticals Inc.). p
8. Gorman OT, Bean WJ, Kawaoka Y, Webster RG: Evolution of the
nucleoprotein gene of influenza A virus. J Virol 1990, 64:1487–1497. p
8. Gorman OT, Bean WJ, Kawaoka Y, Webster RG: Evolution of the
nucleoprotein gene of influenza A virus. J Virol 1990, 64:1487–1497. 9. Kawaoka Y, Krauss S, Webster RG: Avian-to-human transmission of the PB1
gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol
1989, 63:4603–4608. 9. Kawaoka Y, Krauss S, Webster RG: Avian-to-human transmission of the PB1
gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol
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of influenza A viruses. Virology 1989, 172:601–608. 11. Nakajima K, Nobusawa E, Nakajima S: Evolution of the NS genes of the
influenza A viruses. I. The genetic relatedness of the NS genes of animal
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influenza A viruses. I. The genetic relatedness of the NS genes of animal
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equine influenza (H3N8) in the United Kingdom in 1989: diagnostic use
of an antigen capture ELISA. Vet Rec 1993, 133:515–519. 14. Binns MM, Daly JM, Chirnside ED, Mumford JA, Wood JM, Richards CM,
Daniels RS: Genetic and antigenic analysis of an equine influenza H3
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Fouchier RA: Efficient generation and growth of influenza A/PR/8/34
from eight cDNA fragments. Virus Res 2004, 103:155–161. from eight cDNA fragments. Virus Res 2004, 103:155–161. cDNA synthesis Using a UNI-12 primer, as described by Hoffmann et al. [28], cDNA was transcribed by denaturing 2 μl RNA in
the presence of UNI-12 (1 μM final concentration) and
7 μl water at 70°C for 10 minutes and then cooling on
ice. Following this, dNTP mix (final concentration each
0.5 μM), 1 × First Strand buffer, 200U Superscript II
reverse transcriptase (Invitrogen) and water to a final
volume of 20 μl were added. The reaction mixture was
incubated at 42°C for 45 minutes. Page 9 of 9 Page 9 of 9 Rash et al. Virology Journal 2014, 11:159
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http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159
http://www.virologyj.com/content/11/1/159 QIAquick gel extraction kit (Qiagen) according to
manufacturer’s directions. 6. Gorman OT, Donis RO, Kawaoka Y, Webster RG: Evolution of influenza A
virus PB2 genes: implications for evolution of the ribonucleoprotein complex
and origin of human influenza A virus. J Virol 1990, 64:4893–4902. 7. Ito T, Gorman OT, Kawaoka Y, Bean WT, Webster RG: Evolutionary analysis
of the influenza A virus M gene with comparison of the M1 and M2
proteins. J Virol 1991, 65:5491–5498. Acknowledgements
h
k This work was supported by the Horserace Betting Levy Board, EIP 2011–2012. Authors’ contributions 20. de Wit E, Bestebroer TM, Spronken MIJ, Rimmelzwaan GF, Osterhaus ADME,
Fouchier RAM: Rapid sequencing of the non-coding regions of influenza
A virus. J Virol Methods 2007, 139:85–89. This study is a result of collective work. DE, NB and JMC conceived this study
and participated in its design. AR and AW participated in the design of and
carried out the experimental work and sequence analysis. AR drafted the
manuscript. DE and JMC participated in the discussion and modification of
the manuscript. All authors read and approved the final manuscript. 21. WHO: Sequencing primers and protocol. [www.who.int/csr/resources/
publications/swineflu/sequencing_primers/en/] 22. GISAID EpiFlu database. [http://platform.gisaid.org] 23. Lee K, Seong BK: The position 4 nucleotide at the 3′ end of influenza virus
neuraminidase vRNA is involved in temporal regulation of transcription and
replication of neuraminidase RNAs and affects the repertoire of influenza
virus surface antigens. J Gen Virol 1998, 79:1923–1934. Author details
1 24. Jagger BW, Wise HM, Kash JC, Walters K-A, Wills NM, Xiao Y-L, Dunfee RL,
Schwartzman LM, Ozinsky A, Bell GL, Dalton RM, Lo A, Efstathiou S, Atkins
JF, Firth AE, Taubenberger JK, Digard P: An overlapping protein-coding
region in influenza A virus segment 3 modulates the host response. Science 2012, 337:199–204. 1Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK. 2MRC National Institute for Medical Research, Mill Hill, London, UK. Published: 2 September 2014 25. Shi M, Jagger BW, Wise HM, Digard P, Holmes EC, Taubenberger JK:
Evolutionary conservation of the PA-X open reading frame in segment 3
of influenza A virus. J Virol 2012, 86:12411–12413. References
1. McGeoch D, Fellner P, Newton C: Influenza virus genome consists of eight
distinct RNA species. Proc Natl Acad Sci 1976, 73:3045–3049. 2. Waddell GH, Teigland MB, Sigel MM: A new influenza virus associated
with equine respiratory disease. J Am Vet Med Assoc 1963, 143:587–590. 3. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA:
Antigenic and genetic evolution of equine H3N8 influenza A viruses. J Gen Virol 1996, 77:661–671. 4. Bryant NA, Rash AS, Russell CA, Ross J, Cooke A, Bowman S, MacRae S,
Lewis NS, Paillot R, Zanoni R, Meier H, Griffiths LA, Daly JM, Tiwari A,
Chambers TM, Newton JR, Elton DM: Antigenic and genetic variations in
European and North American equine influenza virus strains (H3N8)
isolated from 2006 to 2007. Vet Microbiol 2009, 138:41–52. 5. Murcia PR, Wood JL, Holmes EC: Genome-scale evolution and
phylodynamics of equine H3N8 influenza A virus. J Virol 2011,
85:5312–5322. Additional files Additional file 1: GISAID EpiFlu database [19] accession numbers
for PA-X sequences. Additional file 2: GenBank accession numbers for A/equine/
Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu
database [19] accession numbers for other strains. Additional file 3: Alignment of predicted amino acid sequences for
PA-X from EIV isolated in the UK between 2005 and 2013. Amino
acids of the C-terminal PA-X domain only, following the +1 frameshift,
are shown. Additional file 1: GISAID EpiFlu database [19] accession numbers
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influenza virus genes: application to the reassortment of equine
influenza virus genes. Virus Res 1994, 32:391–399. Additional file 2: GenBank accession numbers for A/equine/
Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu
database [19] accession numbers for other strains. 16. Bean WJ: Correlation of influenza A virus nucleoprotein genes with host
species. Virology 1984, 133:438–442. 17. Dong BB, Xu CL, Dong LB, Cheng HJ, Yang L, Zou SM, Chen M, Bai T, Zhang
Y, Gao RB, Li XD, Shi JH, Yuan H, Chen T, Zhu Y, Xiong Y, Yang S, Shu YL:
A novel reassortant H3N8 influenza virus isolated from drinking water
for duck in a domestic farm in Poyang Lake area. Biomed Environ Sci
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PA-X from EIV isolated in the UK between 2005 and 2013. Amino
acids of the C-terminal PA-X domain only, following the +1 frameshift,
are shown. Additional file 3: Alignment of predicted amino acid sequences for
PA-X from EIV isolated in the UK between 2005 and 2013. Amino
acids of the C-terminal PA-X domain only, following the +1 frameshift,
are shown. 18. Skehel JJ, Hay AJ: Nucleotide sequences at the 5′ termini of influenza
virus RNAs and their transcripts. Nucleic Acids Res 1978, 5:1207–1219. Additional file 1: GISAID EpiFlu database [19] accession numbers
for PA-X sequences.
Additional file 2: GenBank accession numbers for A/equine/
Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu
database [19] accession numbers for other strains.
Additional file 3: Alignment of predicted amino acid sequences for
PA-X from EIV isolated in the UK between 2005 and 2013. Amino
acids of the C-terminal PA-X domain only, following the +1 frameshift,
are shown. References 1. McGeoch D, Fellner P, Newton C: Influenza virus genome consists of eight
distinct RNA species. Proc Natl Acad Sci 1976, 73:3045–3049. 26. Robertson JS: 5′ and 3′ terminal nucleotide sequences of the RNA
genome segments of influenza virus. Nucleic Acids Res 1979, 6:3745–3757. 27. Lee HK, Tang JW-T, Kong DH-L, Koay ES-C: Simplified large-scale Sanger
genome sequencing for influenza A/H3N2 virus. PLoS One 2013, 8:e64785. 2. Waddell GH, Teigland MB, Sigel MM: A new influenza virus associated
with equine respiratory disease. J Am Vet Med Assoc 1963, 143:587–590. 3. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA:
Antigenic and genetic evolution of equine H3N8 influenza A viruses. J Gen Virol 1996, 77:661–671. g
g
28. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR: Universal primer set
for the full-length amplification of all influenza A viruses. Arch Virol 2001,
146:2275–2289. 4. Bryant NA, Rash AS, Russell CA, Ross J, Cooke A, Bowman S, MacRae S,
Lewis NS, Paillot R, Zanoni R, Meier H, Griffiths LA, Daly JM, Tiwari A,
Chambers TM, Newton JR, Elton DM: Antigenic and genetic variations in
European and North American equine influenza virus strains (H3N8)
isolated from 2006 to 2007. Vet Microbiol 2009, 138:41–52. doi:10.1186/1743-422X-11-159
Cite this article as: Rash et al.: An efficient genome sequencing method
for equine influenza [H3N8] virus reveals a new polymorphism in the
PA-X protein. Virology Journal 2014 11:159. 5. Murcia PR, Wood JL, Holmes EC: Genome-scale evolution and
phylodynamics of equine H3N8 influenza A virus. J Virol 2011,
85:5312–5322.
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Treatment of Epulis Fissuratum with carbon dioxide laser in a patient with antithrombotic medication
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Braz Dent J (2012) 23(1): 77-81 Braz Dent J (2012) 23(1): 77-81 77
ISSN 0103-6440 Treatment of Epulis Fissuratum with Carbon Dioxide
Laser in a Patient with Antithrombotic Medication Luís Silva MONTEIRO1,2
João MOUZINHO1
Ana AZEVEDO1,2
Marco Infante da CÂMARA1
Marco André MARTINS2,3
José Maria La Fuente4 1Oral Surgery and Oral Medicine Department, Higher Institute of Health Sciences, Paredes, Portugal and Dental
Sciences Group, Health Sciences Investigation Center (CICS), Paredes, Portugal
2Stomatology Department, Nossa Senhora da Conceição de Valongo Hospital, Porto, Portugal
3Physiology Department, Higher Institute of Health Sciences, Paredes, Portugal
4Oral Implantology Institute of Alicante, Alicante, Spain Epulis fissuratum is a pseudotumor growth located over the soft tissues of the vestibular sulcus caused by chronic irritation from poorly
adapted dentures. Treatment indication for these lesions is surgical excision with appropriate prosthetic reconstruction. The hemostatic
capacity of carbon dioxide (CO2) laser is well described in the literature as a useful tool in oral surgery, especially in patients with clotting
disorders. This paper presents a case of a 72-year-old female patient referred to the ‘Nossa Senhora da Conceição de Valongo Hospital’
at Porto, Portugal, with a massive growth of vestibular oral mucosa in the mandible and maxilla associated with ill-fitting dentures,
suggestive of epulis fissuratum. The patient was taking antithrombotic medication. The lesions were excised with CO2 laser, and no
significant complications, such as hemorrhage, pain, swelling or infection, were recorded. Twenty days after surgery, both areas were
completely reepithelizaded. Prosthetic rehabilitation and function were achieved with the fabrication of new maxillary and mandibular
dentures. Follow-up 1 month and 1 year after treatment revealed the areas free of recurrence. The use of CO2 lasers is currently the
gold standard in the excision of this type of lesion, especially in patients with hemorrhagic diathesis or under antithrombotic therapy. Key Words: epulis fissuratum, CO2 laser, antithrombotic medication, oral cavity, oral pathology. Correspondence: Dr. Luís Silva Monteiro, Departamento de Medicina e Cirurgia Oral, Instituto Superior de Ciências da Saúde - Norte, Rua Central
de Gandra, 1317, 4585-116 Gandra PRD, Portugal. Tel:+351-1919120226. email: lmonteiro.md@gmail.com Correspondence: Dr. Luís Silva Monteiro, Departamento de Medicina e Cirurgia Oral, Instituto Superior de Ciências d
de Gandra, 1317, 4585-116 Gandra PRD, Portugal. Tel:+351-1919120226. email: lmonteiro.md@gmail.com INTRODUCTION As a result, the risk of a severe bleeding during or after
oral surgical procedures is increased (3). Lasers have been used in dentistry for different
purposes and have be the subject on different fields
of dental research (4-10). The use of laser in oral and
maxillofacial surgery has become more and more
widespread over the last decades with favorable
experiences (11,12). Carbon dioxide (CO2) laser emits
energy at a 10.6 µm wavelength (infrared zone), which
is absorbed by water. The high water content of the oral
soft tissues makes this laser a useful tool in oral soft
surgery with several advantages including excellent
hemostasis, high precision in tissue destruction, no need
for sutures, non-contact surgery, wound sterilization
and minimal postoperative pain and edema (11-16). The hemostatic capacity of CO2 laser is reported as an Epulis fissuratum is a pseudotumor growth located
over the soft tissues of the vestibular sulcus caused by
chronic irritation from poorly adapted prostheses with
variable degrees of hypertrophy and hyperplasia. The
treatment of choice is surgical excision with appropriate
prosthetic reconstruction (1,2). In modern societies, there is an increasing number
of older patients with common systemic diseases such
as cardiovascular diseases, especially those treated
with anticoagulation therapy because of cardiologic
indications. In the last years, some guidelines of dental
management of patients using antithrombotic drugs have
recommended not to routinely discontinue anti-platelet
and anti-coagulation medication before dental surgery. Braz Dent J 23(1) 2012 L.S. Monteiro et al. 78 additional benefit in oral surgery for patients that suffer
from clotting disorders (17). Oral Medicine and Surgery Department of the ‘Nossa
Senhora da Conceição de Valongo’ Hospital at Porto,
Portugal, for a maxillary/mandibular gingival mass with
12 months of evolution. She had arterial hypertension
and congestive heart failure. Habitual medication
included ticlopidine 250 mg and captopril 25 mg. On oral
examination, a fibrous mass of 6 x 2 cm with multiple
folds was located on the mandibular vestibular sulcus and
two other similar fibrous masses, with 2 x 1 cm each, were
found in the maxillary vestibular sulcus (Fig. 1A-C). The This paper presents the treatment of massive and
simultaneous maxillary and mandibular epulis fissuratum
with CO2 laser surgery and the subsequent prosthetic
rehabilitation in a patient under antithrombotic therapy. Braz Dent J 23(1) 2012 CASE REPORT A 72-year-old female patient was referred to the B
D
t J 23(1) 2012
Figure 1. Clinical images of the case. A and B = Initial presentation of a mandibular epulis fissuratum; C = Initial presentation of
the maxillary epulis fissuratum; D and E = Exeresis of the maxillary epulis fissuratum with CO2 laser; F = Operatory specimen of
the maxillary lesions; G = Operatory specimen of the mandibular lesion; H and I = Aspect of the treated areas 1 month after surgery Figure 1. Clinical images of the case. A and B = Initial presentation of a mandibular epulis fissuratum; C = Initial presentation of
the maxillary epulis fissuratum; D and E = Exeresis of the maxillary epulis fissuratum with CO2 laser; F = Operatory specimen of
the maxillary lesions; G = Operatory specimen of the mandibular lesion; H and I = Aspect of the treated areas 1 month after surgery. Braz Dent J 23(1) 2012 Treatment of epulis fissuratum with CO2 laser 79 for renewal of dentures (18). patient had ill-fitted maxillary and mandibular complete
dentures. Epulis fissuratum presumptive diagnosis was
made. Complete blood count and general biochemistry
were within normal values with an INR of 2.3. Habitual
medication was not discontinued for surgery. The lesions
were treated under local anesthesia (2% lidocaine with
1:100,000 epinephrine) with CO2 laser (DEKA™ Smart
US20D, Firenze, Italy), pulse mode, 0.9-mm focus, 5-6
W power, focalizing the beam for cutting of the mucosa
and defocalizing the beam when tissue vaporization
was required (Fig. 1D-G). Usual safety precautions of
protecting the operator, patient, and assistant were strictly
followed. First, hyperplastic tissue was peripherally
delimited using the CO2 laser in a focused mode. Then,
using a suture, tension was applied on each area of the
lesion and the surrounding tissue to obtain a clean cut
in the excision procedure also using the CO2 laser in a
focused mode. At the end of the surgery the beam was
used on a defocused mode to promote better hemostasis. Additionally, a partial vestibuloplasty was performed. Immediately after surgery, each old denture was relined
with a tissue conditioner (Viscogel; Dentsply, Konstanz,
Germany). Neither sutures nor dressings were used and
the wounds were allowed to repair by second intention. Excised tissues were submitted for routine histological
examination with indication of a CO2 laser excision. Paracetamol 1 g every 12 h during 3 days and 0.12%
chlorhexidine mouthwashes were prescribed. CASE REPORT The patient returned after 1 month and 1 year
later, without any signs of lesion recurrence (Fig. 1H-I). Surgical excision is the definitive treatment of
epulis fissuratum, always with appropriate prosthetic
reconstruction. The treatment is usually performed
with conventional surgery excision with scalpel. This
technique, however, is associated with significant loss
of sulcus depth, sometimes with full elimination of the
vestibule (2,13). This could be reduced performing
a vestibuloplasty with vestibular deepening without
union of surgical borders. A denture covered with
tissue conditioner is adapted and reinserted over the
surgical bed, permitting the maintenance of vestibular
sulcus. However, without suture of the wound borders,
hemostasis could be difficult especially for patients with
hemorrhagic diathesis or under antithrombotic therapy. Morimoto et al. (19) observed that, a surgical procedure
such as tooth extraction was associated with a significantly
increased incidence of postoperative hemorrhage in
patients receiving antithrombotic therapy. Therefore, this
technique could be problematic for these individuals. Moreover, based on recent guidelines, cardiologists rather
prefer not to suspend any antithrombotic medication
before oral surgery procedures (3). In this sense, CO2 laser excision appears a useful
tool in this type of surgery. One of its main advantages
over conventional surgeries is that CO2 laser surgery
provides an excellent hemostasis (20,21). Blood vessels
smaller than 0.5-mm diameter are spontaneously sealed
(1), allowing excellent visibility (bloodless operating
field) and precision when dissecting through the tissue
planes (1,20). Compared with scalpel surgery, a clot
of denatured collagen is formed on the surface and the
acute inflammation reaction is delayed and minimal after
laser disinfection of wound, with few myofibroblasts and
hence little wound contraction (11). All these advantages
minimize possible postoperative hemorrhage. For these
reasons there is no need for suture and the wound is
allowed to repair by second intention. Over the past years,
laser hemostasis has been established as an alternative to
conventional techniques (11,17). Gáspár and Szabó (17)
found no significant differences between the group of
patients with hemorrhagic diathesis and control patients
regarding the duration of operation, degree of bleeding
and healing of the wound and complication using laser
surgery. In the present case, there was a good bleeding
control in both mandibular and maxillary epulis. Braz Dent J 23(1) 2012 CASE REPORT After 20
days, wound healing was completed uneventfully. A
3-mm-deep extension was gained in the maxillary sulcus,
increasing denture retention. No postoperative pain or
edema was reported. On histopathological report, both
lesions revealed fibrous tissue with some lymphocytic
infiltration, limited by a stratified epithelium with
acantosis. There were no signs of malignancy. A final
diagnosis of epulis fissuratum was established for both
lesions. Appropriate new prosthetic rehabilitation was
provided. The patient returned after 1 month and 1 year
later, without any signs of lesion recurrence (Fig. 1H-I). DISCUSSION patient had ill-fitted maxillary and mandibular complete
dentures. Epulis fissuratum presumptive diagnosis was
made. Complete blood count and general biochemistry
were within normal values with an INR of 2.3. Habitual
medication was not discontinued for surgery. The lesions
were treated under local anesthesia (2% lidocaine with
1:100,000 epinephrine) with CO2 laser (DEKA™ Smart
US20D, Firenze, Italy), pulse mode, 0.9-mm focus, 5-6
W power, focalizing the beam for cutting of the mucosa
and defocalizing the beam when tissue vaporization
was required (Fig. 1D-G). Usual safety precautions of
protecting the operator, patient, and assistant were strictly
followed. First, hyperplastic tissue was peripherally
delimited using the CO2 laser in a focused mode. Then,
using a suture, tension was applied on each area of the
lesion and the surrounding tissue to obtain a clean cut
in the excision procedure also using the CO2 laser in a
focused mode. At the end of the surgery the beam was
used on a defocused mode to promote better hemostasis. Additionally, a partial vestibuloplasty was performed. Immediately after surgery, each old denture was relined
with a tissue conditioner (Viscogel; Dentsply, Konstanz,
Germany). Neither sutures nor dressings were used and
the wounds were allowed to repair by second intention. Excised tissues were submitted for routine histological
examination with indication of a CO2 laser excision. Paracetamol 1 g every 12 h during 3 days and 0.12%
chlorhexidine mouthwashes were prescribed. After 20
days, wound healing was completed uneventfully. A
3-mm-deep extension was gained in the maxillary sulcus,
increasing denture retention. No postoperative pain or
edema was reported. On histopathological report, both
lesions revealed fibrous tissue with some lymphocytic
infiltration, limited by a stratified epithelium with
acantosis. There were no signs of malignancy. A final
diagnosis of epulis fissuratum was established for both
lesions. Appropriate new prosthetic rehabilitation was
provided. REFERENCES 1. Tamarit-Borrás M, Delgado-Molina E, Berini-Aytés L, Gay-
Escoda C. Removal of hyperplastic lesions of the oral cavity. A
retrospective study of 128 cases. Med Oral Patol Oral Cir Bucal
2005;10:151-162. The healing process was completed after 20 days
without scaring and with anatomic sulcus integrity. Fisher and Frame (25) suggested treatment of epulis
fissuratum with CO2 laser without first intention, since
second intention healing was seen to cause scant tissue
alteration and little loss of vestibular depth. In the
present case, a 3-mm-deep extension was gained in the
maxillary sulcus, increasing denture retention. Dentures
must be readjusted and placed back into the mouth as
soon as possible (2,23,24). Recurrences are rare as long
as the sources of trauma and/or the patient’s habits are
eliminated and the appropriate prosthetic reconstruction
is provided. Tamarit-Borrás et al. (1) observed relapse of
lesion after epulis fissuratum excision with CO2 laser in
patients who had failed to replace or re-fit their dentures. 2. Niccoli-Filho W, Neves AC, Penna LP, Seraidarian PI, Riva R. Removal of epulis fissuratum associated to vestibuloplasty with
carbon dioxide laser. Lasers Med Sci 1999;14:203-206. 3. van Diermen DE, Aartman IH, Baart JA, Hoogstraten J, van der Waal
I. Dental management of patients using antithrombotic drugs: critical
appraisal of existing guidelines. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 2009;107:616-624. 4. Estrela C, Ribeiro RG, Estrela CR, Pécora JD, Sousa-Neto
MD. Antimicrobial effect of 2% sodium hypochlorite and
2% chlorhexidine tested by different methods. Braz Dent J
2003;14:58-62. 5. Tramontina VA, Machado MA, Nogueira Filho GR, Kim SH,
Vizzioli MR, Toledo S. Effect of bismuth subgallate (local
hemostatic agent) on wound healing in rats. Histological and
histometric findings. Braz Dent J 2002;13:11-16. 6. Carvalho TL, Bombonato KF, Brentegani LG. Histometric
analysis of rat alveolar wound healing. Braz Dent J 1997;8:9-12. In conclusion, given the intrinsic qualities of CO2
laser when used for oral tissue surgery, such as bloodless
operating field, cutting precision, non-contact technique,
no need for sutures, and reduction of postoperative
pain, edema, infection and hemorrhage, it is reasonable
to assume that this treatment option should become
the gold standard in the treatment of denture-related
hyperplasias, especially in patients with hemorrhagic
diathesis or under antithrombotic therapy. 7. Basso FG, Oliveira CF, Fontana A, Kurachi C, Bagnato VS,
Spolidório DM, et al.. In vitro effect of low-level laser therapy on
typical oral microbial biofilms. Braz Dent J 2011;22:502-510. 8. DISCUSSION Most cases of epulis fissuratum occur in the
anterior region of the jaws (1,13,18). Simultaneous
maxillary and mandibular occurrence, as in the present
case, is less frequent. This pathology is more frequent in
females and in elderly patients (1,18). The most common
complaints are a fibrous mass in the mouth, as happened
in the present case, disuse of dentures, pain or the need Additional and important advantages of CO2
lasers, as observed in the present case, are the cutting
precision, the uniqueness of its non-contact technique Braz Dent J 23(1) 2012 Braz Dent J 23(1) 2012 L.S. Monteiro et al. 80 and the reduction of postoperative complaints such as
pain, infection and edema (2,20,22). Regarding the size
of the lesions of this case, it is remarkable that pain
was absent during the intraoperative and postoperative
periods. This is an important advantage of CO2 laser
treatment reported by many authors. Pogrel et al. (23) attributed this reduction in pain to the fact that
the inflammatory reaction associated with CO2 laser
application is reduced because of blood and lymphatic
vessel sealing, with prevention of the extravasation of
fluids responsible for inflammation and pain. Moreover,
laser irradiation cause sealing of the nerve endings in
the surgical contact area and the denaturalized collagen
layer formed on the surface of the surgical wound serves
to isolate from the oral fluids (24,25). ao Hospital Nossa Senhora da Conceição de Valongo, Porto,
Portugal, que apresentou-se com crescimentos exuberantes das
mucosas vestibular maxilar e mandibular associados a próteses
mal adaptadas. A paciente estava sob uso de medicação anti-
trombótica. As lesões foram excisadas com laser de CO2. Não
foram reportadas complicações significativas como hemorragia,
dor, tumefação ou infecção. Vinte dias após a cirurgia, ambas
as áreas encontravam-se completamente reepitelizadas. A
reabilitação protética foi promovida com a produção de novas
próteses superior e inferior. O acompanhamento após 1 mês e
1 ano não mostrou evidências de recidiva. A utilização do laser
CO2 é nos nossos dias a técnica de eleição na excisão deste tipo
de lesões especialmente em pacientes com diáteses hemorrágicas
ou terapia anti-trombótica. REFERENCES Moura-Netto C, Guglielmi C de A, Mello-Moura AC, Palo RM,
Raggio DP, Caldeira CL. Nd:YAG laser irradiation effect on apical
intracanal dentin - a microleakage and SEM evaluation. Braz Dent
J 2011;22:377-381. 9. Faria MI, Souza-Gabriel AE, Alfredo E, Messias DC, Silva-Sousa
YT. Apical microleakage and SEM analysis of dentin surface after
980 nm diode laser irradiation. Braz Dent J 2011;22:382-387. 10. Lino MD, Carvalho FB, Oliveira LR, Magalhães EB, Pinheiro AL,
Ramalho LM. Laser phototherapy as a treatment for radiotherapy-
induced oral mucositis. Braz Dent J 2011;22:162-165. Braz Dent J 23(1) 2012 Epulis fissuratum é um crescimento pseudotumoral localizado
sobre os tecidos do sulco vestibular causada por irritação crônica
de próteses mal adaptadas. O tratamento indicado para estas lesões
é a sua excisão cirúrgica com reabilitação protética apropriada.
A capacidade hemostática do laser de dióxido de carbono (CO2)
está amplamente descrita na literatura como instrumento útil
em procedimentos cirúrgicos especialmente em pacientes que
sofrem de distúrbios de coagulação. Este artigo apresenta um
caso de uma paciente do sexo feminino de 72 anos, enviada RESUMO 11. Gama SK, de Araújo TM, Pinheiro AL. Benefits of the use of the CO2
laser in orthodontics. Lasers Med Sci 2008;23:459-465. Epulis fissuratum é um crescimento pseudotumoral localizado
sobre os tecidos do sulco vestibular causada por irritação crônica
de próteses mal adaptadas. O tratamento indicado para estas lesões
é a sua excisão cirúrgica com reabilitação protética apropriada. A capacidade hemostática do laser de dióxido de carbono (CO2)
está amplamente descrita na literatura como instrumento útil
em procedimentos cirúrgicos especialmente em pacientes que
sofrem de distúrbios de coagulação. Este artigo apresenta um
caso de uma paciente do sexo feminino de 72 anos, enviada 12. Işeri U, Ozçakir-Tomruk C, Gürsoy-Mert H. Treatment of epulis
fissuratum with CO2 laser and prosthetic rehabilitation in patients
with vesiculobullous disease. Photomed Laser Surg 2009;27:675-
681. 13. Keng SB, Loh HS. The treatment of epulis fissuratum of the oral
cavity by CO2 laser surgery. J Clin Laser Med Surg 1992;10:303-306. 14. Tuncer I, Ozçakir-Tomruk C, Sencift K, Cöloğlu S. Comparison
of conventional surgery and CO2 laser on intraoral soft tissue
pathologies and evaluation of the collateral thermal damage. Treatment of epulis fissuratum with CO2 laser 81 Photomed Laser Surg 2010;28:75-79. 2009;64:108-113. 15. de Magalhaes-Junior EB, Aciole GT, Santos NR, dos Santos JN,
Pinheiro AL. Removal of oral lichen planus by CO2 laser. Braz
Dent J 2011;22:522-526. 21. Kesler G. Clinical applications of lasers during removable
prosthetic reconstruction. Dent Clin North Am 2004;48:963-969. 22. Aciole GT, Aciole JM, Soares LG, Santos NR, Santos JN, Pinheiro
AL. Surgical treatment of oral lymphangiomas with CO2 laser:
report of two uncommon cases. Braz Dent J 2010;21:365-369. 16. Aciole GT, Aciole JM, Soares LG, Santos NR, Santos JN, Pinheiro
AL. Surgical treatment of oral lymphangiomas with CO2 laser:
report of two uncommon cases. Braz Dent J 2010;21:365-369. 23. Pogrel MA, Yen ChK, Hansen LS. A comparison of carbon dioxide
laser, liquid nitrogen cryosurgery and scalpel wounds in healing. Oral Surg Oral Med Oral Pathol 1990;69:269-273. 17. Gáspár L, Szabó G. Significance of the hemostatic effect of lasers
in oral surgery. Orv Hetil 1989;130:2207-2210. 24
Pogrel MA. The carbon dioxide laser in soft tissue preprosthetic
surgery. J Prosthet Dent 1989;61:203-208. 18. Canger EM, Celenk P, Kayipmaz S. Denture-related hyperplasia:
A clinical study of a Turkish population group. Braz Dent J
2009;20:243-248. 25. Fisher SE, Frame JW. The effects of the carbon dioxide surgical
laser on oral tissues. RESUMO Br J Oral Maxillofac Surg 1984;22:414-425. 25. Fisher SE, Frame JW. The effects of the carbon dioxide surgical
laser on oral tissues. Br J Oral Maxillofac Surg 1984;22:414-425. 19. Morimoto Y, Niwa H, Minematsu K. Risk factors affecting
postoperative hemorrhage after tooth extraction in patients
receiving oral antithrombotic therapy. J Oral Maxillofac Surg
2011;69:1550-1556. Received February 3, 2011 Received February 3, 2011
Accepted November 17, 2011 20. Mahler P, Pouyssegur V, Rocca JP, De Moor R, Nammour S. Preprosthetic surgery of the edentulous maxilla: vestibular
deepening with the aid of the CO2 laser. Rev Belge Med Dent Braz Dent J 23(1) 2012
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تحرير المراد بالقرن الآخرين في قوله تعالى: ( ثُمَّ أَنْشَأْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِينَ) من خلال الآية رقم (31) إلى الآية رقم (41) في سورة المؤمنون
|
مجلة الجامعة الإسلامية للدراسات الإسلامية (عقيدة - تفسير - حديث)
| 2,021
|
cc-by
| 20,959
|
IUGJIS
Vol 29, No 4, 2021, pp 682 -707
ISSN 2410-
5201 مجلة الجامعة للدراسات اإلسالمية (عقيدة
–
تفسير
–
حديث)
تاريخ اإلرسال(
21
-
11
-
2020
،)
تاريخ قبول النشر(
22
-
03
-
2021
)
https://doi.org/10.33976/IUGJIS.29.4/2021/28
1 اسم الجامعة والبلد:
2 اسم الجامعة والبلد
)(للثاني
3
اسم الجامعة
والبلد
)(للثالث
*
:البريد االلكتروني للباحث املرسل
E-mail address:
قسم القران وعلومه-
كلية الشريعة وأصول
الدين-
جامعة الملك خالد
:كلمات مفتاحية
(تحرير–
القرن–
اآلخرين–
سورة–
)المؤمنون
اسم الباحث:
1.*
:تحرير المراد بالقرن اآلخرين في قوله تعالى(
َّثُم
َأَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين ) من خالل اآلية رقم
(
31
( ) إلى اآلية رقم41
) في سورة المؤمنون
.د
نبيل محمد مرعي
Exploring What is Meant by the 'Generation of Others' in the Qur'anic Verse: "THEN WE
PRODUCED AFTER THEM A GENERATION OF OTHERS" (23:31), Taking into Consideration the
Interpretation of Verses 31-41 of Surah Al-Mu'minūn "The Believers"
Abstract:
In this study the researcher dealt with exploring what is meant by the 'generation of
Nabiel-2009@hotmail.com
:الملخص
تناول الباحث في هذه الدراسة تحرير المراد بالقرن اآلخرين في قوله تعالى: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا َآخَرِين ) من سورة المؤمنون
بعد أن تناول تفسير اآليات من اآلية رقم31
إلى اآلية رقم41
، وقد سلك الباحث في هذا البحث المنهج االستقرائي االستنباطي
والوصفي التحليلي، متناوالً دراسته ألقوال المفسرين الواردة فيها، وأدلتها، وأوجه الداللة، وإمكانية االعتراض عليها من خالل
سياق اآلي ات، ودالالت األلفاظ ومعانيها في المقطع، وبقية نظائرها في القصص ذات الصلة، بحيث توصل الباحث إلى أن المراد
.من هذا القرن هم (عاد) قوم هود عليه السالم، محرراً للعديد من األدلة التي تؤيد اختياره
وهللا ويل التوفيق. IUGJIS
Vol 29, No 4, 2021, pp 682 -707
ISSN 2410-
5201 مجلة الجامعة للدراسات اإلسالمية (عقيدة
–
تفسير
–
حديث)
تاريخ اإلرسال(
21
-
11
-
2020
،)
تاريخ قبول النشر(
22
-
03
-
2021
)
https://doi.org/10.33976/IUGJIS.29.4/2021/28
1 اسم الجامعة والبلد:
2 اسم الجامعة والبلد
)(للثاني
3
اسم الجامعة
والبلد
)(للثالث
*
:البريد االلكتروني للباحث املرسل
E-mail address:
قسم القران وعلومه-
كلية الشريعة وأصول
الدين-
جامعة الملك خالد
:كلمات مفتاحية
(تحرير–
القرن–
اآلخرين–
سورة–
)المؤمنون
اسم الباحث:
1.*
:تحرير المراد بالقرن اآلخرين في قوله تعالى(
َّثُم
َأَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين ) من خالل اآلية رقم
(
31
( ) إلى اآلية رقم41
) في سورة المؤمنون
.د
نبيل محمد مرعي
Exploring What is Meant by the 'Generation of Others' in the Qur'anic Verse: "THEN WE
PRODUCED AFTER THEM A GENERATION OF OTHERS" (23:31), Taking into Consideration the
Interpretation of Verses 31-41 of Surah Al-Mu'minūn "The Believers"
Abstract:
In this study the researcher dealt with exploring what is meant by the 'generation of
Nabiel-2009@hotmail.com
:الملخص
تناول الباحث في هذه الدراسة تحرير المراد بالقرن اآلخرين في قوله تعالى: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا َآخَرِين ) من سورة المؤمنون
بعد أن تناول تفسير اآليات من اآلية رقم31
إلى اآلية رقم41
، وقد سلك الباحث في هذا البحث المنهج االستقرائي االستنباطي
والوصفي التحليلي، متناوالً دراسته ألقوال المفسرين الواردة فيها، وأدلتها، وأوجه الداللة، وإمكانية االعتراض عليها من خالل
سياق اآلي ات، ودالالت األلفاظ ومعانيها في المقطع، وبقية نظائرها في القصص ذات الصلة، بحيث توصل الباحث إلى أن المراد
.من هذا القرن هم (عاد) قوم هود عليه السالم، محرراً للعديد من األدلة التي تؤيد اختياره
وهللا ويل التوفيق. IUGJIS
Vol 29, No 4, 2021, pp 682 -707
ISSN 2410-
5201 مجلة الجامعة للدراسات اإلسالمية (عقيدة
–
تفسير
–
حديث)
تاريخ اإلرسال(
21
-
11
-
2020
،)
تاريخ قبول النشر(
22
-
03
-
2021
)
https://doi.org/10.33976/IUGJIS.29.4/2021/28
1 اسم الجامعة والبلد:
2 اسم الجامعة والبلد
)(للثاني
3
اسم الجامعة
والبلد
)(للثالث
*
:البريد االلكتروني للباحث املرسل
E-mail address:
قسم القران وعلومه-
كلية الشريعة وأصول
الدين-
جامعة الملك خالد
اسم الباحث:
1.*
:تحرير المراد بالقرن اآلخرين في قوله تعالى(
َّثُم
َأَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين ) من خالل اآلية رقم
(
31
( ) إلى اآلية رقم41
) في سورة المؤمنون
.د
نبيل محمد مرعي
Nabiel-2009@hotmail.com مجلة الجامعة للدراسات اإلسالمية (عقيدة
–
تفسير
–
حديث) وهللا ويل التوفيق. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 الحمد هلل رب العالمين الرحمن الرحيم مالك يوم الدين وبه نستعين، و
ال
ة صال سال الو م على المبعوث رحمة للعالمين، وإمام
:المتقين، رسول الهدى ومصباح الدجى محمد بن عبد هللا المصطفى، وعلى آله وصحبه أجمعين، أما بعد فقد أنزل هللا القرآن على نبيه بلسان عربي مبين، وجعله شرفًا له ولقومه، ومن تبعه إلى يوم الدين، وجعل حظ المرء من
:كنوز القرآن بقدر تدبره آياته ومعانيه، واستفراغ جهده ووقته فيه، فعجائب القرآن ال تنقضي، وألجل ذلك ندبنا إلى تدبر آياته فقال
:{أَفَالَ يَتَدَب رُونَ الْقُرْآنَ أَمْ عَلَى قُلُوبٍ أَقْفَالُهَا} [محمد24
]
، وبي
ن أنه تبيان لكل شيء فقا:ل
}ٍ{وَنَز لْنَا عَلَيْكَ الْكِتَابَ تِبْيَانًا لِكُل ِ شَ يْ ء
:[النحل89
]
،
:وعر فنا بأنه ما فرط فيه من شيء فقال
:{ مَا فَر طْنَا فِي الْكِتَابِ مِنْ شَ يْ ءٍ} [األنعام38
]. وسورة المؤمنون من السور المكية التي تناولت قضية التوحيد واإليمان باهلل، وإرسا ل الرسل ومواقف أقوامهم منهم، وحال
األمم التي كذبت رسلهم، وسوء عاقبتهم، وعند قراءتي قصص هذه السورة، لفت انتباهي ذكره
القصة الثانية دون بيان اسم القوم
:أو رسولهم، واكتفى باإلشارة إليهم في مطلع قصتهم بقوله
َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين :} [المؤمنون31
]
، ولم يذكر هللا من
هم، علمًا بأن من جاء بعد نوح
هو نبي هللا هود
، ثم جاء بعده صالح
، وبعد العودة إلى كتب التفسير لمعرفة المقصود
ب
هذا القرن، وما قيل فيهم، وجدتُ أقواالً عدة، فمنهم من يرى أنهم عاد قوم هود
، ومنهم من يرى أنهم ثمود قوم صالح ، ومنهم من
يجمع بين القولين، ومنهم من ذكر قوالً آخر غير عاد وثمود، ولكل قول أدلته من خالل سياق اآليات، فرأيتُ أن أسعى جاهدًا في
تحرير المراد بهذا القرن، والمقصودِين فيه، ثم نظرتُ في كتب قصص األنبياء ؛ فاتضح أن شأنهم شأن المفسرين في نسبتها، وهذا
ما دفعني ل كتابة بحث( :بعنوان تحرير المراد بالقرن اآلخر
ين
:في قوله تعالى
} َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
من خالل اآلية رقم
(
31
( ) إلى اآلية رقم41
.) في سورة المؤمنون)، مستمدًا العون من هللا، طالبًا إياه أن يهديني لما اختلف فيه من الحق بإذنه IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 Abstract: In this study, the researcher dealt with exploring what is meant by the 'generation of
others' in the Holy Qur'an verse: "Then We produced after them a generation of others"
(23:31), taking into consideration the interpretation of verses 31-41 of the same Surah. The researcher used the inductive, deductive, descriptive, analytical approach. This is
done through the study of the sayings of the commentators in regards to the concerned
verses, their evidence and meaning aspect, the possibility of objecting to them using
the context of the verses, their meanings in the extract, and the rest of their counterparts
in the related stories. The researcher concluded that what is meant by this "generation"
is the people of Hud, peace be upon him, (Aad), providing many proofs that support
his choice 682
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
Keywords: Exploring, Generation, Others, Surah, Al-Mu'minūn "The Believers". Keywords: Exploring, Generation, Others, Surah, Al-Mu'minūn "The Believers". 682 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد د. نبيل بن محمد مرعي سعيد ًأوال :
:أهمية البحث وأسباب اختياره ،تُعرف أهمية الموضوع من خالل النظر في مضمونه، وذلك أن شرف العلم بشرف معلومه، وقيمة الشيء في محتواه
وهذا البحث موضوعه القرآن الكريم، ومحتواه أخبار األنبياء والمرسلين وحال األمم السابقة، يسعى فيه الباحث إلى معرفة القول
الراجح من بين األقوال الواردة في اآلية؛ ألهميته؛ وأهمية اإلضافة العلمية التي تعتمد على االستقراء والتحليل والنظر في سياق
:اآليات، وربطها بنظائرها في بقية السور، وتعود أسباب اختيار الموضوع إلى اآلتي 1
-
.أهميته كما سبق بيانه 1
-
.أهميته كما سبق بيانه 2
-
إيراد ابن كثير هذه اآليات في كتابه " قصص األنبياء"، في قصة هود عليه السالم، مع أن العذاب المنصوص عليه
،في جميع مواضع قصص هود عليه السالم هو الريح، بخالف هذا الموضع، فقد نص على أن العذاب هو الصيحة
مما لفت انتباهي ألهمية دراسة اآليات، والتأمل فيها، واستقراء ما ورد فيها، والتأكد من صحة إيراد ا بن كثير، ومحاولة
.الوصول لتحرير المراد منها 3
-
الرغبة في.معرفة القول الراجح من بين األقوال مما تعم الحاجة إليه 3
-
الرغبة في.معرفة القول الراجح من بين األقوال مما تعم الحاجة إليه IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 683 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. :ًخامسا:منهج البحث سلكتُ في هذا البحث المنهج االستقرائي االستنباطي والوصفي التحليلي، وذلك باستقراء واستنباط دالئل اآليات التي
تفيد في
تحديد المراد بالقرن اآلخرين، وتحليلها
من خالل سورة المؤمنون
والسور األخرى . :سادسً ا:إجراءات البحث
.ذكر نص اآلية واسم السورة ورقمها في متن البحث
.االلتزام بتوثيق مادة البحث وشواهده، مع تدوين نتائج ما توصل إليه الباحث في الخاتمة
أوثق النقل وأعزوه إلى من نقلت عنه في الهامش، بذكر اسم المؤلف و
الكتاب والجزء والصفحة، وفي حالة النقل بال معنى: يصدر
كل ة: انظ
ال ز
.ذكر نص اآلية واسم السورة ورقمها في متن البحث
.االلتزام بتوثيق مادة البحث وشواهده، مع تدوين نتائج ما توصل إليه الباحث في الخاتمة
الهامش، بذكر اسم
من نقلت عنه ف
أوثق النقل وأعزوه إل
المؤلف و
حالة
الكتاب والجزء والصفحة، وف :ًرابعا الدراسات:السابقة بعد االطالع على عناوين رسائل وفهارس العديد من الجامعات، كجامعة الملك خالد، وجامعة أم القرى، وجامعة اإلمام
محمد بن سعود، وجامعة القاهرة، ومكتبة المصطفى اإللكترونية، والشبكات العنكبوتية، ومنتدى الكتب المصورة وغيرها؛ لم يجد
الباحث فيها ما كُتب في.تحديد المراد من القوم في هذا الموضوع IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 ًأوال :
:أهمية البحث وأسباب اختياره نبيل بن محمد مرعي سعيد ًأوال :
:أهمية البحث وأسباب اختياره نبيل بن محمد مرعي سعيد 4
-
.دراسة الحكمة من تحديد نوع العقوبة بالصيحة لقوم عاد في هذا الموضع في حال صحة نسبة هذه اآليات إلى زمنهم
5
-
إضافة جديدة للمكتبة القرآنية تتناول دراسة:مستقلة في تحرير المراد بالقرن اآلخرين في قوله تعالى
ْ{ثُم أَنْشَ أْنَا مِن
} َبَعْدِهِمْ قَرْنًا آخَرِين .وتستند على أقوى األدلة ما أمكن ذلك
6
-
الحرص على الوقوف على لون من ألوان اإلعجاز القرآني في استخدام األلفاظ المشتركة، وتنوعها في التعبير عن
.العقوبة ثان :ًيا:األهداف :تهدف هذه الدراسة إلى اآلتي 1
-
الوصول إلى الرأي الراجح من أقوال المفسرين
:في المراد بالقرن اآلخرين في قوله تعالى {ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا
} َآخَرِين.من وجهة نظر الباحث 1
-
الوصول إلى الرأي الراجح من أقوال المفسرين
:في المراد بالقرن اآلخرين في قوله تعالى {ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا
} َآخَرِين.من وجهة نظر الباحث 4
-
.بيان أن كل لفظة لها داللة، تُمكِن المتأمل فيها من استخراج الكثير من كنوز القرآن وفوائده ًثالث :ا :مشكلة البحث المتأمل في ترتيب القصص القرآني عادة ؛ يجد أن قصة عاد تأتي دائمًا عقب قصة نوح ؛ وذلك في سور األعراف
وهود والشعراء، وقد نص هللا على أنه أهلكهم بالريح، بينما القصة الواردة في سورة المؤمنون عقب قصة نوح ذكر هللا بأنه أخذهم
بالصيحة، وعذاب الصيحة يُذكر في قصة ثمود وقوم مدين، وألجل هذا حصل اإلشكال لدى المفسرين، فمن نظر للترتيب قال بأنها
نزلت في عاد قوم هود
، ومن نظر للعاقبة قال بأنها نزلت في ثمود قوم صالح ، وبعض المفسرين ذكر القولين دون اختيار، وبعضهم
أشار إلى أنها تحتمل القولين، والبعض ذكر بأنها تعنى بقوم آخرين غير عاد و،ثمود
مما تترتب عليه إشكاال للناظر في كتب
،التفسير، وحيرة في بيان المراد
،وهذا البحث سيحاول إيجاد حلٍ لهذا اإلشكال من خالل االستقراء والنظر في قوة األدلة ووجوهها
والسياق القرآني، وغيرها مما يمكن الباحث بإذن هللا إلى اختيار الرأي األقرب للصواب، وسوف تجيب هذه الد راسة عن األسئلة
:اآلتية :ما المقصود بالقرن اآلخرين في قوله تعالى
} َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
من سورة المؤمنون؟
إلى أي مدى يمكن االحتجاج بترتيب القصص القرآني للترجيح بين األقوال؟
هل نستطيع الوصول للرأي الراجح من خالل النظر في سياقات قصتي عاد وثمود في سور القرآن مع موازنة هذه القصة في سورة
المؤمنون؟ :ما المقصود بالقرن اآلخرين في قوله تعالى
} َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
من سورة المؤمنون؟
إلى أي مدى يمكن االحتجاج بترتيب القصص القرآني للترجيح بين األقوال؟ إلى أي مدى يمكن االحتجاج بترتيب القصص القرآني للترجيح بين األقوال؟
هل نستطيع الوصول للرأي الراجح من خالل النظر في سياقات قصتي عاد وثمود في سور القرآن مع موازنة هذه القصة في سورة
المؤمنون؟ هل يمكن أللفاظ العقوبة أن تتناوب عن بعضها حسب سياق المقطع أو موضوع السورة؟
ن ال ذا ؟
ز
أ
كل ال ذا
ة ه
هل ال هل يمكن أللفاظ العقوبة أن تتناوب عن بعضها حسب سياق المقطع أو موضوع السورة؟ IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 684 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 قال
:تعالى :{ثُمَّ أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِينَ } [المؤمنون31
]. يخبر هللا تعالى في هذه اآليات بعد أن ذكر قصة:نوح في المقطع السابق فقال
} َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
:[المؤمنون31
]
، و
لم يذكر هللا في هذه اآليات
من هم هذا القرن، وال اسم النبي الذي أرسله عليهم، سوى اإلشارة إلى أنه أنشأهم
بعد نوح عليه السالم، ومعنى(ﭳ :)
:النشئ إيجاد الشيء وتربيته،... وقال
{قُلْ هُوَ ال َذِي أَنْشَ أَكُمْ وَجَعَلَ لَكُمُ الس مْعَ وَاألَْبْصَ ارَ وَاألَْفْئِدَة
:قَلِيالً مَا تَشْ كُرُونَ } [الملك23
،]
:وقال
:{هُوَ أَعْلَمُ بِكُمْ إِذْ أَنْشَ أَكُمْ مِنَ األَْرْضِ } [النجم32
]
،
:وقال
{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا
،} َآخَرِين وقا:ل
:{ثُم أَنْشَ أْنَاهُ خَلْقًا آخَرَ } [المؤمنون14
:]، {وَنُنْشِ ئَكُمْ فِي مَا الَ تَعْلَمُونَ } [الواقعة61
}]، {وَلَقَدْ عَلِمْتُمُ الن شْ أَةَ األُْولَى
:[الواقعة62
]، فهذه كلها في اإليجاد المختص باهلل(
1
)، وقول
ه( :
ْمِنْ بَعْدِهِم ) المقصود بهم قوم نوح؛ ،ألنها ذُكِرتْ عقبهم مباشرة
( :وقوله َقَرْنًا آخَرِين
) قيل: قوم عاد، وقيل: قوم ثمود، وقيل:
غيرهم، وهذا هو محور بحثنا، ونهدف من خالله إلى تحرير المراد بهذا
ًالقرن، وقد أفردنا له مبحثًا مستقال(
2
). بعًا اس :
:هيكل البحث ويشتمل على المقدمة
:وفيها
،أهمية البحث، وأسباب اختياره، وأهداف البحث، ومشكلته، والدراسات السابقة، ومنهج البحث
وتمهيد ومبحثين
:وخاتمة على النحو التالي ويشتمل على المقدمة
:وفيها
،أهمية البحث، وأسباب اختياره، وأهداف البحث، ومشكلته، والدراسات السابقة، ومنهج البحث
وتمهيد ومبحثين
:وخاتمة على النحو التالي
التمهيد: تفسير اآليات من31
إلى41
.من سورة المؤمنون
المبحث األول: أقوال المفسرين في المراد بالقرن اآلخر
ين
.) َفي قوله: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا آخَرِين
المبحث الثاني: تحرير المراد بالقرن اآلخر
ين
.) َفي قوله: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا آخَرِين
والخاتمة، وفيها أهم النتائج والتوصيات .، وقائمة بثبت مصادر مراجع البحث المبحث األول: أقوال المفسرين في المراد بالقرن اآلخر
ين
.) َفي قوله: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا آخَرِين
المبحث الثاني: تحرير المراد بالقرن اآلخر
ين
.) َفي قوله: (ثُمَّ أَنشَ أْنَا مِن بَعْدِهِمْ قَرْنًا آخَرِين
والخاتمة، وفيها أهم النتائج والتوصيات .، وقائمة بثبت مصادر مراجع البحث التمهيد: تفسير اآليات من31
إلى41
من سورة المؤمنون IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 685 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد قال
:تعالى
ْ{فَأَرْسَ لْنَا فِيهِمْ رَسُ والً مِنْهُمْ أَنِ اعْبُدُوا َّللاََّ مَا لَكُمْ مِنْ إِلَهٍ غَي :رُهُ أَفَالَ تَتَّقُونَ } [المؤمنون32
.] :وقوله
ً(فَأَرْسَ لْنَا فِيهِمْ رَسُ وال)
قيل: هود، وقيل صالح، كما سيأتي معنا في المبحث
األول(
3
)؛ ألنه أساس البحث، قال
القرطب :ي"
:تعدية اإلرسال في قوله )(ﭺ
بفي مع أنه يتعدى بإلى للداللة على أن هذا الرسول المرسل إليهم نشأ فيهم بين أظهرهم"(
4
)،
( :وقوله ْمِنْهُم:) قال القرطبي "أي من عشيرتهم، يعرفون مولده ومنشؤه؛ ليكون سكونهم إلى قوله أكثر"(
5
). :وقوله تعالى (
ََّأَنِ اعْبُدُوا َّللا ُمَا لَكُمْ مِنْ إِلَهٍ غَيْرُه )
:قال السعدي"
،فكلهم اتفقوا على هذه الدعوة، وهي أول دعوة يدعون بها أممهم
( :األمر بعبادة هللا، واإلخبار أنه المستحق لذلك، والنهي عن عبادة ما سواه، واإلخبار ببطالن ذلك وفساده، ولهذا قال َأَفَالَ تَتَّقُون )
ربكم، فتجتنبوا هذه
األوثان واألصنام" (
6
). قال
:تعالى
ْ{وَقَالَ الْمََلَُ مِنْ قَوْ مِهِ الَّذِينَ كَفَرُ وا وَكَذَّبُوا بِلِقَاءِ اآلْ خِرَةِ وَأَت ُرَفْنَاهُمْ فِي الْحَيَاةِ الدُّنْيَا مَا هَذَا إِالَّ بَشَ رٌ مِثْل َكُمْ يَأْكُلُ مِمَّا تَأْكُلُون
َمِنْهُ وَيَشْ رَبُ مِمَّا تَشْ ر :بُونَ } [المؤمنون33
.] :قال القرطبي "(
َُوَقَالَ الْمََل :) أي
األشراف والقادة والرؤساء، (
ِمِنْ قَوْ مِهِ الَّذِينَ كَفَرُ وا وَكَذَّبُوا بِلِقَاءِ اآلْ خِرَة )
يريد بالبعث
والحساب، (وَأَتْرَفْنَاهُمْ فِي الْحَيَاةِ الدُّنْيَا )
أي: وسعنا عليهم نعم الدنيا حتى
بطروا"(
7
). وقيل: وأغنياهم في الحياة الدنيا، ... واإلتراف
هو التنعم بمالذ العيش(
8
). قال
:تعالى :{وَلَئِنْ أَطَعْتُمْ بَشَ رًا مِثْلَكُمْ إِنَّكُمْ إِذًا لَخَاسِ رُ ونَ } [المؤمنون34
]. (
(1
-
:ينظر األصفهاني، المفردات في غريب القرآن ص494
. 686 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد يريد لمغبونون بترككم آلهتكم واتباعكم إياه من غير فضيلة له عليكم(
1
)،
:قال أبو السعود"وقوله( :
ْمِثْلَكُم )
ِأي في الص ِ فات
ِواألحوال،
وإيثارُ مثلكم على مثلنا للمبالغة في تهوين أمره عليه السالم وتوهينِه ... وإذا وقع بين اسمِ إن وخبرِها لتأكيد مضمون
ِالش رطِ والجملةُ جوابٌ لقسمٍ محذوفٍ قبل إنِ الش رطيةِ المصد رةِ بالالم؛ الموطئة، أي:
َوباهلل لئن أطعتمُ بشرًا مثلَكم إن كم إذًا لخاسرون"(
2
.)
قال
:تعالى
} َ{أَيَعِدُكُمْ أَنَّكُمْ إِذَا مِتُّمْ وَكُنْتُمْ تُرَابًا وَعِظَامًا أَنَّكُمْ مُخْرَجُون
:[المؤمنون35
]. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 قال
:تعالى
ْ{فَأَرْسَ لْنَا فِيهِمْ رَسُ والً مِنْهُمْ أَنِ اعْبُدُوا َّللاََّ مَا لَكُمْ مِنْ إِلَهٍ غَي :رُهُ أَفَالَ تَتَّقُونَ } [المؤمنون32
.] :قوله(
ْأَيَعِدُكُم) استفهام بمعنى التوقيف على جهة االستبعاد وبمعنى الهزء بهذا الوعد(
3
)،
:وقال أبو السعود "{
ْأَيَعِدُكُم
}
{ استئنافٌ مَسوقٌ لتقرير ما قبله من زجرهم عن ات ِباعِه عليه الس المُ بإنكار وقوع ما يدعُوهم إلى اإليمان به واستبعاده
ْأَنَّكُمْ إِذَا مِتُّم ،}
وصرتم نخرة مجردة عن اللحوم واألعصاب، وتقديم التراب لعراقته في االستبعاد، وانقالبه من األجزاء البادية، أو كان متق دموكم ترابًا
صرفًا ومتأخروكم عظامًا"(
4
)، {
َمُخْرَجُون } أي أحياء من قبوركم بعد موتكم(
5
.) (
(1
-
:ينظر القرطبي، الجامع ألحكام القرآن12
/
122
. f G
) / CC BY 4 0
(
(1
-
:ينظر القرطبي، الجامع ألحكام القرآن12
/
122
.
(
(2
-
أبو السعود، إرشاد العقل السليم6
/
133
.
(
(3
-
:ينظر
ابن
عطية، المحرر الوجيز4
/
143
.
(
(4
-
أبو السعود: إرشاد العقل السليم6
/
134
.
(
(5
-
:ينظر الجزائري، أيسر التفاسير3
/
516
.
(
(6
-
ابن عباس، تفسيره ص287
.
(
(7
-
:ينظر ابن عطية، المحرر الوجيز4
/
143
.
(
(8
-
:ينظر الشوكاني، فتح القدير3
/
572
.
(
(9
-
ينظر: طنطاوي، التفسير
الوسيط للقرآن الكريم10
/
3
(
(10
-
:ينظر المراغي، تفسيره18
/
23
. قال
:تعالى
:{هَيْهَاتَ هَيْهَاتَ لِمَا تُوعَدُونَ } [المؤمنون36
.] قال ابن عباس
:
"بَعيدًا بَعيدًا (
َلِمَا تُوعَدُون )الَ يكون هَذَا"(
6
،)
،وهذه كلمة لها معنى الفعل التقدير بعد كذا، فطورا تلي
الفاعل دون الم، تقول: هيهات مجيء زيد، أي بعد ذلك(
7
)
.، والتكرير للتأكيد قال
:تعالى {إِنْ هِيَ إِالَّ حَيَاتُنَا الدُّنْيَا نَمُوتُ وَنَحْيَا وَمَا نَحْنُ بِمَبْعُوثِينَ } [الم :ؤمنون37
]. أي: ما الحياة إال حياتنا الدنيا، ال الحياة اآلخرة التي تعدنا بها، وجملة: نموت ونحيا، مفسرة لما ادعوه من قصرهم حياتهم
على حياة الدنيا، ثم صرحوا بنفي البعث، وأن الوعد به منه افتراء على هللا فقالوا: ما نحن بمبعوثين(
8
)
، وفي هذه اآلية بيان لتماديهم
في جحودهم وجهلهم وغرورهم، فلم يكتفوا باستبعاد حصول البع ث والجزاء يوم القيامة، بل أضافوا إلى ذلك اإلنكار الشديد
لحصولهما(
9
)
. قال
:تعالى
[ } َ{إِنْ هُوَ إِالَّ رَجُلٌ افْتَرَى عَلَى َّللاَِّ كَذِبًا وَمَا نَحْنُ لَهُ بِمُؤْ مِنِين :المؤمنون38
.] قال
:تعالى
[ } َ{إِنْ هُوَ إِالَّ رَجُلٌ افْتَرَى عَلَى َّللاَِّ كَذِبًا وَمَا نَحْنُ لَهُ بِمُؤْ مِنِين :المؤمنون38
.] أي ما هود إال رجل يختلق الكذب على هللا، فتارة يقول: مالكم من إله غير هللا خالق السماوات واألرض وأخرى يقول: إنكم
إذا متم وكنتم ترابًا وعظامًا إنكم مخرجون، وما نحن بمصدقيه فيما يد عى ويزعم من التوحيد والبعث(
10
)
. قال
:تعالى
:{قَالَ رَب ِ انْصُ رْنِي بِمَا كَذَّبُونِ } [المؤمنون39
. قال
:تعالى
:{قَالَ رَب ِ انْصُ رْنِي بِمَا كَذَّبُونِ } [المؤمنون39
. قال
:تعالى
:{قَالَ رَب ِ انْصُ رْنِي بِمَا كَذَّبُونِ } [المؤمنون39
. (
(2
-
أبو السعود، إرشاد العقل السليم6
/
133
. (
(3
-
:ينظر
ابن
عطية، المحرر الوجيز4
/
143
. (
(4
-
أبو السعود: إرشاد العقل السليم6
/
134
. (
(5
-
:ينظر الجزائري، أيسر التفاسير3
/
516
. (
(6
-
ابن عباس، تفسيره ص287
. (
(7
-
:ينظر ابن عطية، المحرر الوجيز4
/
143
. (
(8
-
:ينظر الشوكاني، فتح القدير3
/
572
. (
(9
-
ينظر: طنطاوي، التفسير
الوسيط للقرآن الكريم10
/
33
. (
(10
-
:ينظر المراغي، تفسيره18
/
23
. 687 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 قال
:تعالى {إِنْ هِيَ إِالَّ حَيَاتُنَا الدُّنْيَا نَمُوتُ وَنَحْيَا وَمَا نَحْنُ بِمَبْعُوثِينَ } [الم :ؤمنون37
]. نبيل بن محمد مرعي سعيد :أي: قال نبيهم لما علم بأنهم ال يصدقونه البتة
رب انصرني عليهم وانتقم لي منهم بسبب تكذيبهم إياي(
1
)
:، وفي قوله
(رَب ِ انْصُ رْنِي بِمَا كَذ بُونِ ) وجوه: أحدها: أن في نصره إهالكهم فكأنه قال أهلكهم بسبب تكذيبهم إياي، وثانيها: انصرني بدل م ا
كذبوني كما تقول هذا بذاك؛ أي بدل ذاك ومكانه، والمعنى أبدلني من غم تكذيبهم سلوة النصر عليهم، وثالثها: انصرني بإنجاز ما
وعدتهم من العذاب(
2
)،
:َفاستجاب هللا دعاءه وقَال(
َعَم ا قَلِيلٍ لَيُصْ بِحُن نَادِمِين)
. قال
:تعالى
:{ قَالَ عَمَّا قَلِيلٍ لَيُصْ بِحُنَّ نَادِمِينَ } [المؤمنون40
]. قال
:تعالى
:{ قَالَ عَمَّا قَلِيلٍ لَيُصْ بِحُنَّ نَادِمِينَ } [المؤمنون40
]. :ما زائدة، وقيل صفة للزمان والتقدير
عن زمان قليل يندمون، كقديم وحديث، في قولك: ما رأيته قديمًا وال حديثًا،
وفي
معناه: عن قريب(
3
)، ومعنى ليصبحن: ليصيرن، وأصبح فالن عالمًا أي: صار(
4
)،
:قال الخازن"
يعني ليصيرن نادِمِينَ على كفرهم
وتكذيبه"(
5
). (
(1
-
:ينظر الشوكاني، فتح القدير3
/
572
.
(
(2
-
:ينظر الرازي، مفاتيح الغيب23
/
272
.
(
(3
-
ابن جزي، التسهيل لعلوم التنزيل2
/
51
والزمخشري، الكشاف3
/
187
.
(
(4
-
:ينظر ابن منظور، لسان العرب2
/
502
.
(
(5
-
الخازن، تفسيره3
/
272
.
(
(6
-
ينظر: ابن منظور، لسان العرب2
/
521
-
522
.
(
(7
-
ابن الجوزي، زاد المسير في علم التفسير3
/
262
.
(
(8
-
الزمخشري، الكشاف3
/
187
.
(
(9
-
ابن الجوزي، زاد المسير في علم التفسير3
/
262
.
(
(10
-
:ينظر النسفي، مدارك التنزيل وحقائق التأويل2
/
469
. (
(1
-
لم أجد هذا القول البن عباس صراحة عند تفسير هذه اآلية، وإنما قال: "(ثم أنشأنا من بعدهمْ ) خلقًا من بعد هَالَ ك قوم نوح (قَرْن ،"اً آخَرِينَ ) قومًا آخَرين
تفسير ابن عباس ص286
. المبحث األول أقوال المفسرين في المراد بالقرن اآلخر
ين
:في قوله تعالى
ً{ثُمَّ أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْن} َا آخَرِين 689
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 قال
:تعالى
ِ{فَأَخَذَتْهُمُ الصَّ يْحَةُ بِالْحَق ِ فَجَعَلْنَاهُمْ غُثَاءً فَبُعْدًا لِلْقَوْ مِ الظَّال :مِينَ } [المؤمنون41
]. :قال ابن منظور"وصييييح: صيييوت بأقصيييى طاقته، والصييييحة: العذاب، أو الغارة إذا فوجئ الحي بها"(
6
.)
:قال المفسيييرون
صيياح بهم جبريل صيييحة رجفت لها األرض من ًتحتهم، فصيياروا لشييد تها غُثاء(
7
)
، قال الزمخشييري: شييبههم في دمارهم بالغثاء: وهو
حميل السييييييييل مما
ب لي وأسيييييييود من العيدان والورق، ومنه قوله تعالى:
:{ فَجَعَلَهُ غُثَاءً أَحْوَى} [األعلى5](
8
)،
:وقال الزجاج"
:الغُثاء
الهالك والبالي من ورق الشيييييييييييجر الذي إِذا جرى
يل رأيته مخالطًا زَبَده
السييييييييييي"(
9
)، { ﰆ }
فهالكًا يقال: بعد بعدًا، وأبعدَ هلك، وهو من
{ المصادر المنصوبة بأفعال ال يستعمل إظهارها ﰆ ﰈ
}بيان لمن دعي عليه بالبعد نحو هيت لك(
10
). (
(1
-
:ينظر الشوكاني، فتح القدير3
/
572
. (
(2
-
:ينظر الرازي، مفاتيح الغيب23
/
272
. (
(3
-
ابن جزي، التسهيل لعلوم التنزيل2
/
51
والزمخشري، الكشاف3
/
187
. (
(4
-
:ينظر ابن منظور، لسان العرب2
/
502
. (
(5
-
الخازن، تفسيره3
/
272
. (
(6
-
ينظر: ابن منظور، لسان العرب2
/
521
-
522
. (
(7
-
ابن الجوزي، زاد المسير في علم التفسير3
/
262
. (
(8
-
الزمخشري، الكشاف3
/
187
. (
(9
-
ابن الجوزي، زاد المسير في علم التفسير3
/
262
. (
(10
-
:ينظر النسفي، مدارك التنزيل وحقائق التأويل2
/
469
. 688 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد :القول األول: عاد قوم هود، وأصحاب هذا القول، هم :القول األول: عاد قوم هود، وأصحاب هذا القول، هم منسوب البن عباس
(
1
)، وقال به: مقاتل بن سليمان(
2
)، ويحيى بن سالم(
3
)، والسمرقندي(
4
)، والواحدي(
5
)، والثعلبي(
6
)،
والزمخشري(
7
)، والنسفي(
8
)
، والخازن(
9
) ،والعليمي(
10
) ،وصاحبا تفسير الجاللين(
11
) ،والمراغي(
12
)، وأبو العباس الصوفي(
13
)
، وصديق
خان(
14
)، والقطان(
15
)
،
وإبراهيم اإلبياري(
16
)
،
ومحمد المكي الناصري(
17
)،
ومحمد األمين الهرري(
18
)، وفضل حسن عباس(
19
). (
(1
-
لم أجد هذا القول البن عباس صراحة عند تفسير هذه اآلية، وإنما قال: "(ثم أنشأنا من بعدهمْ ) خلقًا من بعد هَالَ ك قوم نوح (قَرْن ،"اً آخَرِينَ ) قومًا آخَرين
تفسير ابن عباس ص286
. تفسير ابن عباس ص286
. (
(2
-
:ينظر مقاتل بن سليمان، تفسيره3
/
156
. ا (
(3
-
:ينظر يحيى بن سالم، تفسيره1
/
400
. (
(4
-
:ينظر السمرقندي، بحر العلوم2
/
479
. (
(5
-
:ينظر الواحدي، الت فْسِ يرُ البَسِ يْط15
/
564
. (
(6
-
:ينظر الثعلبي، الكشف والبيان عن تفسير القرآن7
/
45
. (
(7
-
:ينظر ،الزمخشري الكشاف3
/
185
. (
(8
-
:ينظر النسفي، مدارك التنزيل وحقائق التأويل2
/
467
. (
(9
-
:ينظر
الخازن، لباب التأويل في معاني التنزيل
3
/
271
. (
(9
-
:ينظر
الخازن، لباب التأويل في معاني التنزيل
3
/
271
. (
(10
-
:ينظر ،العليمي فتح الرحمن في تفسير القرآن4
/
469
.ا (
(10
-
:ينظر ،العليمي فتح الرحمن في تفسير القرآن4
/
469
.ا (
(10
-
:ينظر ،العليمي فتح الرحمن في تفسير القرآن4
/
469
.ا (
(11
-
:ينظر المحلي والسيوطي، تفسير الجاللين1
/
449
. (
(11
-
:ينظر المحلي والسيوطي، تفسير الجاللين1
/
449
. (
(13
-
:ينظر األنجري، البحر المديد في تفسير القرآن المجيد3
/
573
. (
(13
-
:ينظر األنجري، البحر المديد في تفسير القرآن المجيد3
/
573
. (
(14
-
:ينظر
صديق خان، فتحُ البيان في مقاصد القرآن
9
/
111
. (
(14
-
:ينظر
صديق خان، فتحُ البيان في مقاصد القرآن
9
/
111
. (
(15
-
،القطان
تيسير التفسير
2
/
476
. (
(16
-
:ينظر مجموعة من العلماء، الموسوعة القرآنية10
/
373
. (
(16
-
:ينظر مجموعة من العلماء، الموسوعة القرآنية10
/
373
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 :القول األول: عاد قوم هود، وأصحاب هذا القول، هم 689 IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد :ورجح هذا القول
السمعاني(
1
)،
والبغوي(
2
)،
والخازن(
3
) ،وأبو حيان األندلسي(
4
)، وابن كثير(
5
) ،والبقاعي(
6
) ،وأبو السعود(
7
)،
ومجموعة من العلماء بإشراف مجمع البحوث اإلسالمية باألزهر في التفسير الوسيط للقرآن الكريم(
8
)،
ومحمد سيد طنطاوي(
9
)،
)والباحث في موسوعة التفسير الموضوعي بعنوان: (عاد(
10
). أدلتهم: استدل أصحاب هذا
:القول باآلتي ًأوال
: أنه كالم ابن عباس، حيث قال: "وكان بين نوح وهود ثمانمائة سنة، وعاش هود أربعمائة وأربعًا وستين سنة،
وكان بين هود
"وصالح مئة سنة(
11
)
،
ففُهم منه أن بين هود وصالح مئة سنة، وبالتالي فإن المقصود بالقرن الذي أنشأه هللا عقب نوح
هو القرن
الذي عاش فيه عاد قوم هود
، وهو دليلهم اآلتي
-الثاني-
. ثانيًا
: يشهد له قول هود
:
ِ {وَاذْكُرُوا إِذْ جَعَلَكُمْ خُلَفَاءَ مِنْ بَعْدِ قَوْ مِ نُوحٍ وَزَادَكُمْ فِي الْخَلْقِ بَسْ طَةً فَاذْكُرُوا آالَ ءَ ّللا
} َلَعَل كُمْ تُفْلِحُون
:[األعراف69
]
:، وهو يوافق قوله تعالى :{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِينَ } [المؤمنون31
]
،ورجحه محمود حجازي لهذا الدليل(
12
). ثالثًا
: الترتيب التاريخي لقصة هود
على إثر قصة نوح
كما في سورة األعراف وهود والشعراء والذاريات والقمر،
قال به
النسفي(
13
)
،والثعالبي(
14
)، وأبو السعود(
15
). :ًأوال
أن ابن عباس
لم يقل به صراحة، وإنما استنبط المفسرون قوله من األثر الوارد عنه كما سبق معنا(
16
). :ًأوال
أن ابن عباس
لم يقل به صراحة، وإنما استنبط المفسرون قوله من األثر الوارد عنه كما سبق معنا(
16
). 690
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4 0
(
(1
-
:ينظر السمعاني، تفسيره3
/
473
. (
(2
-
:ينظر
البغوي، معالم التنزيل في تفسير القرآن
5
/
416
. (
(3
-
:ينظر الخازن، لباب التأويل في
معاني التنزيل
3
/
271
. (
(4
-
:ينظر أبو حيان، البحر المحيط في التفسير7
/
558
. (
5
(
-
:ينظر ابن كثير، البداية والنهاية1
/
288
. (
(6
-
:ينظر
البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 ا 691
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
1(-
ينظر: ص10
-
11
.
(
(2
-
:ينظر مجاهد، تفسيره485
،
وابن جرير، جامع البيان17
/
47
.
(
(3
-
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ابن جرير، جامع البيان
19
/
28
.
(
(4
-
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/
51
.
(
(5
-
:ينظر
ابن عاشور، ا لتحرير والتنوير18
/
49
.
(
(6
-
:ينظر
السعدي، تيسير الكريم الرحمن في تفسير كالم المنان
ص551
. (
(6
-
:ينظر
السعدي، تيسير الكريم الرحمن في تفسير كالم المنان
ص551
. (Islamic University of Gaza) / CC BY 4 0
(
1(-
ينظر: ص10
-
11
.
(
(2
-
:ينظر مجاهد، تفسيره485
،
وابن جرير، جامع البيان17
/
47
.
(
(3
-
:ينظر
ابن جرير، جامع البيان
19
/
28
.
(
(4
-
:ينظر ابن جزي، التسهيل لعلوم التنزيل2
/
51
.
(
(5
-
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ابن عاشور، ا لتحرير والتنوير18
/
49
.
(
(6
-
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السعدي، تيسير الكريم الرحمن في تفسير كالم المنان
ص551
. (
(6
-
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السعدي، تيسير الكريم الرحمن في تفسير كالم المنان
ص551
. (
(2
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:ينظر مجاهد، تفسيره485
،
وابن جرير، جامع البيان17
/
47
. (
(4
-
:ينظر ابن جزي، التسهيل لعلوم التنزيل2
/
51
. :القول األول: عاد قوم هود، وأصحاب هذا القول، هم 7
)
-
:ينظر أبو السعود، إرشاد العقل السليم6
/
132
. (
(8
-
:ينظر مجموعة من العلماء، التفسير الوسيط للقرآن الكريم6
/
1287
. (
(9
-
:ينظر سيد طنطاوي، التفسير الوسيط للقرآن الكريم10
/
30
. 10
)
–
:ينظر مجموعة من الباحثين، موسوعة التفسير الموضوعي23
/
17
. (
(11
-
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/
29
-
30. وذكره البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
، وأبو السعود ، إرشاد العقل السليم
6
/
132
، واأللوسي، روح المعاني9
/
230
، والقاسمي ، محاسن التأويل7
/
289
.، وآخرون
(
(12
-
:ينظر حجازي، التفسير الواضح2
/
623
. (
(13
-
:ينظر النسفي، مدارك التنزيل وحقائق التأويل2
/
467
. (
(14
-
:ينظر
الثعالبي، الجواه ر الحسان في تفسير القرآن4
/
148
. 15
)
-
:ينظر أبو السعود، إرشاد العقل السليم6
/
132
. (
16
(-
ينظر: ص9
. (
(2
-
:ينظر
البغوي، معالم التنزيل في تفسير القرآن
5
/
416
. (
(3
-
:ينظر الخازن، لباب التأويل في
معاني التنزيل
3
/
271
. (
(4
-
:ينظر أبو حيان، البحر المحيط في التفسير7
/
558
. (
5
(
-
:ينظر ابن كثير، البداية والنهاية1
/
288
. آ (
(6
-
:ينظر
البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
. (
(6
-
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البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
. 7
)
-
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/
132
. 7
)
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/
132
. (
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/
1287
. (
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-
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/
1287
. (
(9
-
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/
30
. (
(9
-
:ينظر سيد طنطاوي، التفسير الوسيط للقرآن الكريم10
/
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. 10
)
–
:ينظر مجموعة من الباحثين، موسوعة التفسير الموضوعي23
/
17
. 10
)
–
:ينظر مجموعة من الباحثين، موسوعة التفسير الموضوعي23
/
17
. (
(13
-
:ينظر النسفي، مدارك التنزيل وحقائق التأويل2
/
467
. (
(14
-
:ينظر
الثعالبي، الجواه ر الحسان في تفسير القرآن4
/
148
. :القول األول: عاد قوم هود، وأصحاب هذا القول، هم 690 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد د. نبيل بن محمد مرعي سعيد ثانيًا:: التشيييييييابه الكبير بين القصيييييييتين من ناحية الترف والعقوبة، فالترف كقوله عن ثمود
َ{أَتُتْرَكُون
ُفِي مَا هَاه( َنَا آمِنِين146
) فِي
( ٍجَنَّاتٍ وَعُيُون147
( ٌيم
ِ
) وَ زُرُ وعٍ وَنَخْ لٍ طَلْعُهَ ا هَضِِِِِِِِِ148
ُ) وَتَنْحِتُونَ مِنَ الْجِ بَالِ بُي :وتًا فَارِهِينَ } [الشِِِِِِِِِعراء146
-
149
]
،
:والعقوبة كقوله تعالى {فَإِنْ أَعْرَضييييييي ُ وا فَقُلْ أَنْذَرْتُكُمْ صييييييي َ اعِقَةً مِثْلَ صييييييي َ اعِقَةِ عَادٍ وَثَمُودَ} [فصيييييييلت :
13
]،
وال يمكن الجزم بالقول بأنها
.خاصة بعاد قوم هود :ثالثًا
قوله:
:{قَالَ عَمَّا قَلِيلٍ } [المؤمنون40
]
،فيه إشيييييارة إلى قرب العقوبة، وهو ما يناسيييييب قوله تعالى ع
لى ل
سيييييان صيييييالح
:
:{فَقَالَ تَمَتَّعُوا فِي دَارِكُمْ ثَالَ ثَةَ أَيَّامٍ ذَلِكَ وَعْدٌ غَيْرُ مَكْذُوبٍ } [هود65
]
،
فهي
أ.يام قليلة رابعًا : ذكرت
لفظة
:ثمود قبل عاد في سيييييييورة ق عند قوله تعالى
ِ حَابُ الر س
{ كَذ بَتْ قَبْلَهُمْ قَوْ مُ نُوحٍ وَأَصييييييي ْ( ُ وَثَمُود12
َ) و ُعَادٌ وَفِرْعَوْن
:وَإِخْوَانُ لُوطٍ } [ق12
،
13
،]
:وسيييييورة الحاقة عند قوله تعالى {كَذ بَتْ ثَمُودُ وَعَادٌ بِالْقَارِعَةِ } [الحاق :ة4]
، وهذا يرد على من يسيييييتدل
.بالترتيب التاريخي ًخامس
ا
: أدلة أصحاب القول الثاني بأن ثمود أُهلكوا بالصيحة، وأخذتهم في الصباح كما سيأتي معنا(
1
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ًخامس
ا
: أدلة أصحاب القول الثاني بأن ثمود أُهلكوا بالصيحة، وأخذتهم في الصباح كما سيأتي معنا(
1
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم (
(3
-
:ينظر
ابن جرير، جامع البيان
19
/
28
. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم مجاهد(
2
)
، والطبري(
3
)
،
ورجح هذا القول: ابن جزي(
4
)، وابن عاشور(
5
)، والسعدي(
6
). مجاهد(
2
)
، والطبري(
3
)
،
ورجح هذا القول: ابن جزي(
4
)، وابن عاشور(
5
)، والسعدي(
6
). :أدلتهم:استدل أصحاب هذا القول بدليلين
:ًأوال أن قوم ثمود أُهلكوا بالصيحة، وهذا ما يناسبه في آخر القصة عند
قوله تعالى:
ِ{فَأَخَذَتْهُمُ الصَّ يْحَةُ ب الْحَق ِ فَجَعَلْنَاهُمْ غُثَاءً فَبُعْدًا
:لِلْقَوْمِ الظَّالِمِينَ } [المؤمنون41
]. :ثانيًا:أن قوم ثمود أخذتهم الصيحة في الصباح، فقال هنا {قَالَ عَمَّا قَلِيلٍ لَيُصْ بِحُنَّ نَادِمِينَ } [ا :لمؤمنون40
]
،
وفي سورة الحجر
قال :
:{فَأَخَذَتْهُمُ الصَّ يْحَةُ مُصْ بِحِينَ } [الحجر83
]
،
.فكان هالكهم في الصباح :قال ابن عاشور"واألظهر أن المراد به هنا ثمود
؛
:ألنه الذي يناسبه قوله في آخر القصة
}ِ {فَأَخَذَتْهُمُ الصَّ يْحَةُ بِالْحَق
:[المؤمنون41
]
،، وألن ثمود أهلكوا بالصاعقة:ولقوله
:{قَالَ عَم ا قَلِيلٍ لَيُصْ بِحُن نَادِمِينَ } [المؤمنون40
]، مع قوله في سورة الحجر
:{فَأَخَذَتْهُمُ الص يْحَةُ مُصْ بِحِينَ } [الحجر83
]
،فكان هالكهم في الصباح،
ولعل تخصيصهم بالذكر هنا دون عاد خالفًا لما تكرر في
:غير هذه اآلية؛ ألن العبرة بحالهم أظهر لبقاء آثار ديارهم بالحجر كما قال تعالى
( َ{وَإِن كُمْ لَتَمُرُّ ونَ عَلَيْهِمْ مُصْ بِحِين137
ِ) وَبِالل يْل 691 د. نبيل بن محمد مرعي سعيد ( َأَفَالَ تَعْقِلُون138
:)} [الصافات137
،
138
]"(
1
). قال ا :لسعدي"
الظاهر أنهم " ثمود " قوم صالح عليه السالم، ألن هذه القصة
تشبه قصتهم"(
2
). ويمكن الرد على الدليل األول بما ذكره الباحث في موسوعة التفسير الموضوعي عند بحثه بعنوان (عاد): إن الصيحة
ليست مختصة بثمود حتى تكون دليالً إلخراج السياق عن ظاهره، فقد أهلك هللا:بها أقوامًا غير ثمود قال تعالى
{وَأَخَذَتِ ال ذِينَ ظَلَمُوا
:الص يْحَةُ فَأَصْ بَحُوا فِي دِيَارِهِمْ جَاثِمِينَ } [هود94
]
،، ففي هذه اآلية بيان أن هالك قوم شعيب بالصيحة، وقوم لوط أهلكوا بالصيحة
:قال تعالى {فَأَخَذَتْهُمُ الص يْحَةُ مُشْ رِقِينَ } [الحج :ر73
]
:، وأصحاب القرية المذكورون في سورة يس أهلكوا بها، قال تعالى
ْ{ إِنْ كَانَت
:إِال صَ يْحَةً وَاحِدَةً فَإِذَا هُمْ خَامِدُونَ } [يس29
](
3
)
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
ي :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم كما يمكننا الرد على استداللهم الثاني
بأن الصيحة أخذتهم في الصباح
:في قوله
َّ{ قَالَ عَمَّا قَلِيلٍ لَيُصْ بِحُن
} َنَادِمِين
:[المؤمنون40
]
:، بقوله تعالى حاكيًا عن عاد في سورة األحقاف
}ْ{فَأَصْ بَحُوا الَ يُرَى إِالَّ مَسَ اكِنُهُم ، وهذا ما استشهد به ابن الجوزي
حيث قال: "وذكر أهل التفسير أن أصبح في القرآن على وجهين: أحدهما: إدراك الصباح للصبح، ومنه قوله تعالى في األحق:اف
}ْ{فَأَصْ بَحُوا الَ يُرَى إِال مَسَ اكِنُهُم(
4
)
. :كما أن هناك فرقًا بين قوله في سورة المؤمنون
} َّ{لَيُصْ بِحُن :، وقوله في سورة الحجر } َ{مُصْ بِحِين ،التي استشهدوا بها
وجعلوها تفسيرًا لها، فاألولى تحتمل عدة معانٍ منها: إدراك الصباح، وهو تفسير ابن الجوزي
كما سبق، ومنها: صار، وهو تفسير
:الخازن في قوله
} {لَيُصْ بِحُن :بقوله"
ليصيرن"(
5
)
،
بخالف الثانية-
} َ{مُصْ بِحِين
-
ال تحتمل إال معنىً واحدًا وهو الصباح، ومثله
:قوله تعالى في قوم لوط
:{وَإِن كُمْ لَتَمُرُّ ونَ عَلَيْهِمْ مُصْ بِحِينَ } [الصافات137
]
،
لاو
يمكن
:الجزم بأن قوله
} َ{مُصْ بِحِين ٌتفسير
:لقوله
} {لَيُصْ بِحُن ،، بل يضعف عند االستدالل بوجود االحتمال .وهذا ملحظ مهم وسيأتي مزيد من التفصيل في المبحث الثاني بالفقرتين الثامنة والتاسعة عند بيان القول الراجح(
6
). ،القول الثالث: عاد وثمود اصو :حب هذا القول
ع
بدالكريم الخطيب(
7
). :أدلته
استدل صاحب هذا القول بأن قوم عاد وثمود كانوا على شاكلة واحدة، وكالهما جاءوا بعد نوح، وقد جمعهم ال قرآن
.في قرن واحد :قال الخطيب"
وهذا هو السر في التعبير القرآني بلفظ«أنشأنا »
،بدالً من لفظ أقمنا، أو خلقنا.. ونحوهما، والقرن اآلخرون
الذين جاءوا بعد قوم نوح، هم قوم عاد وقوم ثمود، وقد جمعهما القرآن الكريم في قرن واحد؛ ألنهم كانوا على شاكلة واحدة، وقد جاء
قوم ثمود، خلفًا لقوم عاد، في ديارهم ومساكنهم"(
8
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم :قال القرطبي "وممن أخذ بالصيحة أيضً ا أصحاب مدين قوم شعيب
، فال يبعد"أن يكونوا هم، وهللا أعلم(
16
)
، وقال
:الشوكاني"وقيل: هم أصحاب مدين قوم شعيب؛ ألنهم ممن أهلك بالصيحة"(
17
). :قال القرطبي "وممن أخذ بالصيحة أيضً ا أصحاب مدين قوم شعيب
، فال يبعد"أن يكونوا هم، وهللا أعلم(
16
)
، وقال
:الشوكاني"وقيل: هم أصحاب مدين قوم شعيب؛ ألنهم ممن أهلك بالصيحة"(
17
). (Islamic University of Gaza) / CC BY 4.0
(
(1
-
سبق تخريجه. (
(2
-
:ينظر
الماتريدي، تأويالت أهل السنة
7
/
466
. (
(3
-
:ينظر المظهري، تفسيره6
/
379
. (
(4
-
:ينظر ابن الجوزي، زاد المسير في علم التفسير3
/
261
. (
(5
-
:ينظر البيضاوي، أنوار التنزيل وأسرار التأويل4
/
86
. (
(6
-
:ينظر النيسابوري، غرائب القرآن ورغائب الفرقان5
/
118
. (
(7
-
:ينظر ،القاسمي محاسن التأويل7
/
289
. (
(8
-
:ينظر ،ابن عادل
اللباب في علوم الكتاب
14
/
202
. (
(9
-
:ينظر
اإليجي، جامع البيان في تفسير القرآن
3
/
81
. (
(10
-
:ينظر البقاعي، نظم الدرر في تناسب اآليات والسور5
/
197
. (
(11
-
:ينظر
البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
. (
(12
-
:ينظر ابن عطية، المحرر الوجيز
4
/
142
. (
(13
-
:ينظر ،الرازي
مفاتيح الغيب الكبير
23
/
274
. (
(14
-
:ينظر القرطبي، الجامع ألحكام القرآن12
/
120
. (
(15
-
:ينظر الشوكاني، فتح القدير3
/
571
. (
(16
-
القرطبي، الجامع ألحكام القرآن12
/
120
. (
(17
-
الشوكاني، فتح القدير3
/
571
. وإيراد الشوكاني لهذا القول؛ ألنه ينقل عن ال IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
سبق تخريجه. (
(2
-
:ينظر
الماتريدي، تأويالت أهل السنة
7
/
466
. (
(3
-
:ينظر المظهري، تفسيره6
/
379
. (
(4
-
:ينظر ابن الجوزي، زاد المسير في علم التفسير3
/
261
. (
(5
-
:ينظر البيضاوي، أنوار التنزيل وأسرار التأويل4
/
86
. (
(6
-
:ينظر النيسابوري، غرائب القرآن ورغائب الفرقان5
/
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. (
(7
-
:ينظر ،القاسمي محاسن التأويل7
/
289
. (
(8
-
:ينظر ،ابن عادل
اللباب في علوم الكتاب
14
/
202
. (
(9
-
:ينظر
اإليجي، جامع البيان في تفسير القرآن
3
/
81
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ويمكننا الرد عليه بأن الجمع بين أمتين في قرن واحد
غير محتمل
:لما يأتي 692 IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون ًأوال : أثر
ابن عباس
"حيث بي ن أن بين عاد وثمود مئة عام فقال: " وكان بين هود وصالح مئة سنة(
1
.)
ثانيًا:: قوله تعالى
:{ فَأَرْسَ لْنَا فِيهِمْ رَسُ والً } [المؤمنون32
]
، ولم يقل: رسولين، فهو رسول واحد لقومٍ مخصو صين، وبهذا ال يصح
.االعتداد بهذا القول وتجدر اإلشارة إلى
أن
بعض المفسرين عند تفسيرهم لهذه اآلية ذكروا أشهر القولين–
األول والثاني-
، دون ترجيح أو
اختيار، مع اختالف عباراتهم، ويشير بعضهم إلى أن اآلية تحتملهما، ومن هؤالء: الماتريدي(
2
)،
والمظهري(
3
)،
وابن الجوزي(
4
)،
والبيضاوي(
5
)،
والنيسابوري(
6
)، والقاسمي(
7
)، وابن عادل(
8
)، واإليجي(
9
)، البقاعي (
10
). :قال البقاعي "ويترجح إرادة عاد لما أعطوا مع ذلك من قوة األبدان وعظم األجسام، وبذلك قال ابن عباس
، وإرادة ثمود
لما في الشعراء والقمر مما يشابه بعض قولهم هنا، وللتعبير عن عذابهم بالصيحة، ولموافقتهم لقوم نوح في تعليل ردهم بكونه بشرًا"
(
11
). وتجدر اإلشارة إلى
أن
بعض المفسرين عند تفسيرهم لهذه اآلية ذكروا أشهر القولين–
األول والثاني-
، دون ترجيح أو
اختيار، مع اختالف عباراتهم، ويشير بعضهم إلى أن اآلية تحتملهما، ومن هؤالء: الماتريدي(
2
)،
والمظهري(
3
)،
وابن الجوزي(
4
)،
والبيضاوي(
5
)،
والنيسابوري(
6
)، والقاسمي(
7
)، وابن عادل(
8
)، واإليجي(
9
)، البقاعي (
10
). :قال ابن عطية وفي جل الروايات ما يقتضي أن قوم عاد أقدم إال أنهم لم يهلكوا بصيحة، وفي هذا احتماالت كثيرة وهللا
أعلم(
12
)
ُ. وعنون الرازي في كتابه عند تفسير اآلية بقوله: الْقِص ةُ الث انِيَة-
ٍقِص ةُ هُود
ْأَو
ٍصَ الِح
(
13
.)
:القول الرابع: أصحاب مدين، وذكر هذا القول
لم يستبعده القرطبي(
14
)، ونقله الشوكاني(
15
). :أدلتهم
استدل صاحب هذا القول بأن قوم شعيب
:ممن أُخذوا بالصيحة، قال تعالى {وَلَم ا جَاءَ أَمْرُنَا نَج يْنَا
شُ عَيْبًا وَال ذِينَ آمَنُوا
مَعَهُ بِرَحْمَةٍ مِن ا وَأَخَذَتِ ال ذِينَ ظَلَمُوا الص يْحَةُ فَأَصْ بَحُوا فِي دِيَارِهِمْ جَاثِمِينَ } [هود :
94
]
. أ
دير3
/
571
.
.وإيراد الشوكاني لهذا القول؛ ألنه ينقل عن القرطبي في كتابه الجامع 693
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(مأ
ع
ي/
(
(17
-
الشوكاني، فتح القدير3
/
571
.
.وإيراد الشوكاني لهذا القول؛ ألنه ينقل عن القرطبي في كتابه الجامع :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم (
(10
-
:ينظر البقاعي، نظم الدرر في تناسب اآليات والسور5
/
197
. (
(11
-
:ينظر
البقاعي، نظم الدرر في تناسب اآليات والسور
5
/
197
. (
(12
-
:ينظر ابن عطية، المحرر الوجيز
4
/
142
. (
(13
-
:ينظر ،الرازي
مفاتيح الغيب الكبير
23
/
274
. (
(14
-
:ينظر القرطبي، الجامع ألحكام القرآن12
/
120
. (
(15
-
:ينظر الشوكاني، فتح القدير3
/
571
. (
(16
-
القرطبي، الجامع ألحكام القرآن12
/
120
. (
(17
-
الشوكاني، فتح القدير3
/
571
. .وإيراد الشوكاني لهذا القول؛ ألنه ينقل عن القرطبي في كتابه الجامع (
(2
-
:ينظر
الماتريدي، تأويالت أهل السنة
7
/
466
. (
(3
-
:ينظر المظهري، تفسيره6
/
379
. (
(4
-
:ينظر ابن الجوزي، زاد المسير في علم التفسير3
/
261
. (
(5
-
:ينظر البيضاوي، أنوار التنزيل وأسرار التأويل4
/
86
. (
(6
-
:ينظر النيسابوري، غرائب القرآن ورغائب الفرقان5
/
118
. (
(7
-
:ينظر ،القاسمي محاسن التأويل7
/
289
. (
(8
-
:ينظر ،ابن عادل
اللباب في علوم الكتاب
14
/
202
. إ (
(9
-
:ينظر
اإليجي، جامع البيان في تفسير القرآن
3
/
81
. آ أ
(
(15
-
:ينظر الشوكاني، فتح القدير3
/
571
. أ
(
(17
-
الشوكاني، فتح القدير3
/
571
. .وإيراد الشوكاني لهذا القول؛ ألنه ينقل عن القرطبي في كتابه الجامع 693 IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد م
:قال دروزة محمد عزت "لم تتضمن هذه اآليات أسماء أنبياء وأقوام، ولكن عبارتها تلهم أنها تعني أقوام هود وصالح
"وشعيب وغيرهم ممن ذكرتهم سالسل القصص في السور األخرى(
2
). :ويمكننا الرد على القولين الرابع والخامس بما يأتي
ًأوال : قول هود
:
ْ{وَاذْكُرُوا إِذْ جَعَلَكُمْ خُلَفَاءَ مِن
َبَعْدِ قَوْ مِ نُوحٍ وَزَادَكُمْ فِي الْخَلْقِ ب
طَةً فَاذْكُرُوا آالَ ءَ ّللا ِ لَعَل كُمْ تُفْلِحُونَ } [األعرا
سييييييييي ْ :ف
69
]. :ووجه الرد أن قوم هود هم الذين كانوا بعد قوم نوح، وقوم صييييييييييالح بعدهم بدليل قوله تعالى على لسييييييييييان صييييييييييالح {وَاذْكُرُو ْا إِذ
جَعَلَكُمْ خُلَفَا :ءَ مِنْ بَعْدِ عَادٍ} [األعراف74
]. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 (
(3
-
مجموعة من الباحثين، موسوعة التفسير الموضوعي34
/
247
-
248
. 694
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
:ينظر عزة، التفسير الحديث5
/
313
.
(
(2
-
عزة، التفسير الحديث5
/
313
.
(
(3
-
مجموعة من الباحثين، موسوعة التفسير الموضوعي34
/
247
-
248
. IUG J
l f I l
i St di
(I l
i U i
it
f G
) / CC BY 4 0
(
(3
-
مجموعة من الباحثين، موسوعة التفسير الموضوعي34
/
247
-
248
. (
(1
-
:ينظر عزة، التفسير الحديث5
/
313
. (
(1
-
مجموعة من الباحثين، موسوعة التفسير الموضوعي34
/
249
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم نبيل بن محمد مرعي سعيد والباحث في موسوعة التفسير الموضوعي عند دراسته قصة هود يميل إلى ترجيح هذا القول حيث يقول: "ولشدة التشابه
بين هذه األمة وكل من عاد وثمود وقعت الحيرة عند المفسرين بأنها هذه أو هذه؛ ألن القرآن لو أراد أن يحدد هذه األمة على وجه
التخصيص لنصب من العالمات م ا يقطع ببيان هُويتها لو كان الغرض من إيرادها ال يتحقق إال بذلك، كما أن هذه القصة انفردت
"بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في قصة كل من عاد وثمود (
1
). :أدلته :استدل صاحب هذا القول بما يأتي
ًأوال : عدم وجود دليل قطعي يرجح
أحدهما على اآلخر، ولداللة القرآن على وجود أمم كثيرة لم تذكر أسماؤها في القرآن من تشرين على
مر
.الزمان
ثانيًا:: استدالله بقوله تعالى
:{وَال ذِينَ مِنْ بَعْدِهِمْ الَ يَعْلَمُهُمْ إِال ّللا ُ } [إبراهيم9]
. ثالثًا : شدة التشابه بين هذه األمة وكل من عاد.وثمود
رابعًا : انفراد هذه القصة بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في قصة كل من عاد وثمو.د والباحث في موسوعة التفسير الموضوعي عند دراسته قصة هود يميل إلى ترجيح هذا القول حيث يقول: "ولشدة التشابه
بين هذه األمة وكل من عاد وثمود وقعت الحيرة عند المفسرين بأنها هذه أو هذه؛ ألن القرآن لو أراد أن يحدد هذه األمة على وجه
التخصيص لنصب من العالمات م ا يقطع ببيان هُويتها لو كان الغرض من إيرادها ال يتحقق إال بذلك، كما أن هذه القصة انفردت
"بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في قصة كل من عاد وثمود (
1
). والباحث في موسوعة التفسير الموضوعي عند دراسته قصة هود يميل إلى ترجيح هذا القول حيث يقول: "ولشدة التشابه
بين هذه األمة وكل من عاد وثمود وقعت الحيرة عند المفسرين بأنها هذه أو هذه؛ ألن القرآن لو أراد أن يحدد هذه األمة على وجه
التخصيص لنصب من العالمات م ا يقطع ببيان هُويتها لو كان الغرض من إيرادها ال يتحقق إال بذلك، كما أن هذه القصة انفردت
"بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في قصة كل من عاد وثمود (
1
). :أدلته :استدل صاحب هذا القول بما يأتي
ًأوال : عدم وجود دليل قطعي يرجح
أحدهما على اآلخر، ولداللة القرآن على وجود أمم كثيرة لم تذكر أسماؤها في القرآن من تشرين على
مر
.الزمان
ثانيًا:: استدالله بقوله تعالى
:{وَال ذِينَ مِنْ بَعْدِهِمْ الَ يَعْلَمُهُمْ إِال ّللا ُ } [إبراهيم9]
. 695
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4 0
(
(1
-
مجموعة من الباحثين، موسوعة التفسير الموضوعي34
/
249
. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثانيًا : أشارت اآليات إلى أن هؤالء القوم كانوا مترفين، وهو ما يناسبه في قوم عاد أو قوم ثمود، إال أن قوم عاد كانو ا أكثر ترفًا، ولم
يُنص في القرآن على أن قوم مدين كانوا من المترفين، بل الظاهر من خالل دعوة شعيب لهم بأال يبخ سوا الناس شيئًا، ويأمر هم بإيفاء
المكيال والميزان؛ أنهم كانوا يمارسون التطفيف والبخس ومشغوفين بهما، ويتواطئون عليهما أكثر من أي شيء آخر، وهذا الس لوك
ال يقوم به المترفون فيما يتصوره العاقل؛ لكثرة مالهم وعدم حاجتهم لحقوق الغير، بخالف البخالء، فقد تلجأهم الحاجة وا لفقر إلى
ظل ه
النا
بخ ثالثًا:: قولهم بأن هللا نص على عذاب قوم شعيب بالصيحة في قوله تعالى
ً{وَلَم ا جَاءَ أَمْرُنَا نَج يْنَا شُ عَيْب ا وَال ذِينَ آمَنُوا مَعَهُ بِرَحْمَةٍ مِن ا
:وَأَخَذَتِ ال ذِينَ ظَلَمُوا الص يْحَةُ فَأَصْ بَحُوا فِي دِيَارِهِمْ جَاثِمِينَ } [هود94
]، فقد ذكر هللا في آي:ة األعراف بأنه أهلكهم بالرجفة قال تعالى
:{فَأَخَذَتْهُمُ الر جْفَةُ فَأَصْ بَحُوا فِي دَارِهِمْ جَاثِمِينَ } [األعراف91
]
، ومثلها قال عن قو:م صالح
َ{فَأَخ ْذَتْهُمُ الر جْفَةُ فَأَصْ بَحُوا فِي دَارِهِم
:جَاثِمِينَ } [األعراف78
]
:، وذكر كذلك بأنهم أُهلكوا بالصيحة في سورة هود قال تعالى
{وَأَخَذَ ال ذِينَ ظَلَمُوا
ا لص يْحَةُ فَأَصْ بَحُوا فِي
:دِيَارِهِمْ جَاثِمِينَ } [هود67
]
،فلو سلمنا بأن دلي
لهم مقبول فهو أقرب إلى قوم صالح
من قوم شعيب؛ للترتيب الت.اريخي ورد هذا القول في موسوعة التفسير الموضوعي حيث قال الباحث في
قصة هود
: "جائز أن يكونوا قومًا آخرين غير
عاد وثمود، وذلك لعدم وجود دليل قطعي يرجح أحدهما على اآلخر، ولداللة القرآن على وجود أمم كثيرة لم تذكر أسماؤها في القرآن
منتشرين على مر
الزمان من لدن نوح
إلى بعثة محمد
:قال تعالى
َ{أَلَمْ يَأْتِكُمْ نَبَأُ ال ذِينَ مِنْ قَبْلِكُمْ قَوْ مِ نُوحٍ وَعَادٍ وَثَمُودَ وَال ذِين
ِمِنْ بَعْدِهِمْ الَ يَعْلَمُهُمْ إِال ّللا ُ جَاءَتْهُمْ رُسُ لُهُمْ بِالْبَي ِنَاتِ فَرَدُّوا أَيْدِيَهُمْ فِي أَفْوَاهِه
مْ وَقَالُوا إِن ا كَفَرْنَا بِمَا
أُرْسِ لْتُمْ بِهِ وَإِن ا لَفِي شَ ك ٍ مِم ا تَدْعُونَنَا
:إِلَيْهِ مُرِيبٍ } [إبراهيم9]
... :{وَلَقَدْ أَرْسَ لْنَا رُسُ الً مِنْ قَبْلِكَ مِنْهُمْ مَنْ قَصَ صْ نَا عَلَيْكَ وَمِنْهُمْ مَنْ لَمْ نَقْصُ صْ عَلَيْكَ} [غافر78
]
حيث
"ذكر أممًا كثيرة لم يقص خبرها (
3
)
. 694 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد د. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم نبيل بن محمد مرعي سعيد 2
-
قوله تعالى:
:{فَأَرْسَ لْنَا فِيهِمْ رَسُ والً مِنْهُمْ } [المؤمنون32
،]
ولم يكن من غيرهم، وهود
من قوم
ه، قال عنه هللا :
:{عَلَى رَجُلٍ مِنْكُمْ لِيُنْذِرَكُمْ} [األعراف69
]. 3
-
قوله تعالى:
:{وَأَتْرَفْنَاهُمْ فِي الْحَيَاةِ الدُّنْيَا} [المؤمنون33
]، وأعظم قوم عاشوا في ت رف ونعيم
ه
م قوم عاد بدليل قوله:
:{ال تِي لَمْ يُخْلَقْ مِثْلُهَا فِي الْبِالَ دِ} [الفجر8]. 4
-
وغيرها من العالمات كما سيأتي معنا في تحرير القول الراجح عند المو
ا .زنة 2
-
قوله تعالى:
:{فَأَرْسَ لْنَا فِيهِمْ رَسُ والً مِنْهُمْ } [المؤمنون32
،]
ولم يكن من غيرهم، وهود
من قوم
ه، قال عنه هللا :
:{عَلَى رَجُلٍ مِنْكُمْ لِيُنْذِرَكُمْ} [األعراف69
]. 3
-
قوله تعالى:
:{وَأَتْرَفْنَاهُمْ فِي الْحَيَاةِ الدُّنْيَا} [المؤمنون33
]، وأعظم قوم عاشوا في ت رف ونعيم
ه
م قوم عاد بدليل قوله:
:{ال تِي لَمْ يُخْلَقْ مِثْلُهَا فِي الْبِالَ دِ} [الفجر8]. 4
-
وغيرها من العالمات كما سيأتي معنا في تحرير القول الراجح عند المو
ا .زنة خامسً ا
: وأما قوله: "كما أن هذه القصة انفردت بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في
قصة
:كل من عاد وثمود" فمردود عليه كذلك، حيث ورد الحديث عن عالمات الترف في قصة عاد في أكثر من موضع، كقوله تعالى
ُّ{وَأَتْرَفْنَاهُمْ فِي الْحَيَاةِ الد :نْيَا} [المؤمنون33
]، وأعظم قوم عاشوا في ترف ونعيم هم قوم عاد بدليل قوله:
{ال تِي لَمْ يُخْلَقْ مِثْلُهَا فِي
:الْبِالَ دِ} [الفجر8
.] المبحث الثاني
تحرير المراد بالقرن اآلخر
ين
:في قوله تعالى
} َ{ثُمَّ أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
بعد
النظر في األقوال الواردة، وأدلتها، واالعتراضات عليها، وبعد التأمل في اآليات من اآلية رقم
31
إلى اآلية
رقم41
،
ودراسة السياق العام، والنظر في أقرب األقوال إليها، ظهر لي أن المراد بالقرن اآلخر
ين
في هذا الموضع هم عاد قوم
هود،
وهو
:القول األول لآلتي
ًأوال : أنه مما يستفاد من أثر ابن عباس
ويستأنس به
، ويقويه؛ لكونه مروي عن صحابي(
1
). ًثانيا: قال به جمهور المفسرين(
2
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثالثًا : شدة التشابه بين هذه األمة وكل من عاد.وثمود
رابعًا : انفراد هذه القصة بالكشف عن منهج المترفين من دعوة اإلصالح، الذين لم يرد التصريح به في قصة كل من عاد وثمو.د :ويمكننا الرد عليه بما يأتي
:ًأوال
ال يلزم في كل أمر مبهم في القرآن أن نجد له دليالً قطعيًا لبيانه، فوجود الدليل يخرجه من اإلبهام، وتنتفي ا لحكمة من إبهامه؛
وعدم ذكر اسييييييييييييم الرسييييييييييييول أو القوم لحكمة يعلمها هللا، إال أنه ندبنا إلى التفكر والتأمل في كتابه فقال في ذات السييييييييييييورة-
سييييييييييييو رة
المؤمنون-
:
ْ{أَفَلَم : يَِِ دَّبَّرُ وا الْقَوْ لَ } [المؤمنون68
]، ولعيييل من حكمييية هيييذا حتى تتفييياوت منيييازل ا لنييياس في فهم القرآن، ويعرف
.الراسخون في العلم من غيرهم .الراسخون في العلم من غيرهم ثانيًا:: اسيييتدالله بقوله تعالى
:{وَال ذِينَ مِنْ بَعْدِهِمْ الَ يَعْلَمُهُمْ إِال ّللا ُ } [إبراهيم9
]
، بع ،يد؛ ألن اآلية نصيييت على ذكر عاد بعد نوح
:وثمود بعد عاد، ثم أبهمت البقية، بينما آية المؤمنون {ثُم أَنْشيييي َ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِينَ } [المؤم :نون31
]
، جاءت عقب قصيييية نوح
مباشرة:، والمعروف أنه لم يتبق أمم كثيرة بعد نوح بدليل قوله تعالى
َ{وَجَعَلْن
ا ذُر ِي تَهُ هُمُ الْبَاقِينَ } [ال :صافات77
]. ثالثًا:: قوله: شيييييييدة التشيييييييابه بين هذه األمة وبين عاد وثمود مردود بصيييييييريح قوله تعالى
ُ{ وَاذْكُرُوا إِذْ جَعَلَكُمْ خ }ٍلَفَاءَ مِنْ بَعْدِ قَوْ مِ نُوح
:[األعراف69
]
:، وقوله
ِ{وَاذْكُرُوا إِذْ جَعَلَكُمْ خُلَفَاءَ م :نْ بَعْدِ عَادٍ } [األعراف74
]، فأين تكون هذه األ ،مة التي يقصيييييييييييييييدها الباحث
ولماذا لم تذكر في بقية سور القرآن؟ رابعًا : هذا القول بعيد عن الصييييييييييييييحة من حيث التعليل، حيث قال: "ألن القرآن لو أراد أن يحدد هذه األمة على وجه التخصيييييييييييييييص
لنصييييب من العالمات ما يقطع ببيان هُويت ها"، ومما ال شييييك فيه
أ ن القرآن قد نصييييب عدة عالمات في قصييييص عاد لبيان هُوية هذه
:األمة منها 1
-
قوله تعالى:
:{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِينَ } [المؤمنون31
،]
والضمير يعود إلى نو ح
، والذي جاء بعد نوح
.عليه السالم هو هود عليه السالم 695 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. 696
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
1(-
سبق ذكره، ينظر: ص9
.
(
2(-
ينظر: ص9
-
10
.
(
(3
-
مجموعة من الباحثين، موسوعة الصحيح المسبور من التفسير بالمأثور3
/
495
. 696
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4 0
(
(3
-
مجموعة من الباحثين، موسوعة الصحيح المسبور من التفسير بالمأثور3
/
495
. 696
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(
2(-
ينظر: ص9
-
10
.
(
(3
-
مجموعة من الباحثين، موسوعة الصحيح المسبور من التفسير بالمأثور3
/
495
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثالثًا،: أن قوم عاد هم خلفاء قوم نوح بنص القرآن كما سبق معنا، وهو أحد أدلة أصحاب القول األول
قال تعالى على لسان
هود
:
ْ{وَاذْكُرُوا إِذ :جَعَلَكُمْ خُلَفَاءَ مِنْ بَعْدِ قَوْ مِ نُوحٍ } [األعراف69
]،
:وقوله تعالى} َ{ثُم أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين ، فالمقصود
بالقرن هنا: هو األمة من الناس، قال حكم
ت
:بشير"
واألظهر أن القرن هم األمة المتعاصرون في الزمن الواحد؛ فإذا ذهبوا وخلفهم
جيل آخر فهم قرن ثانٍ ، كما ثبت في الصحيحين عن رسول هللا-
َصَ ل ى ّللا ُ عَلَيْهِ وَسَ ل م-
أنه قال: "خير القرون قرني، ثم الذين
"يلونهم، ثم الذين يلونهم(
3
). ًرابعا:: في قوله تعالى
:{فَأَرْسَ لْنَا فِيهِمْ رَسُ والً مِنْهُمْ} [المؤمنون32
]، كما قال تع الى على لسان هود
: {
}ْعَلَى رَجُلٍ مِنْكُمْ لِيُنْذِرَكُم
:[األعراف69
]. المبحث الثاني
تحرير المراد بالقرن اآلخر
ين
:في قوله تعالى
} َ{ثُمَّ أَنْشَ أْنَا مِنْ بَعْدِهِمْ قَرْنًا آخَرِين
بعد
النظر في األقوال الواردة، وأدلتها، واالعتراضات عليها، وبعد التأمل في اآليات من اآلية رقم
31
إلى اآلية
رقم41
،
ودراسة السياق العام، والنظر في أقرب األقوال إليها، ظهر لي أن المراد بالقرن اآلخر
ين
في هذا الموضع هم عاد قوم
هود،
وهو
:القول األول لآلتي ا خامسً ا:: قوله تعالى
:{وَقَالَ الْمََلَُ مِنْ قَوْمِهِ ال ذِينَ كَفَرُوا} [المؤمنون33
]، كقوله تعالى عن قوم عاد:
ْ{قَالَ الْمََلَُ ال ذِينَ كَفَرُوا مِن
:قَوْمِهِ} [األعراف66
]. 696 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم نبيل بن محمد مرعي سعيد ًسادس
ا:: قوله تعالى
:{وَأَتْرَفْنَاهُمْ فِي الْحَيَاةِ الدُّنْيَا} [المؤمنون33
]
، هذا الترف له أدلة عديدة من قصة هود
:، منها قوله تعالى
( َ{أَتَبْنُونَ بِكُل ِ رِيعٍ آيَةً تَعْبَثُون128
( َ) وَتَت خِذُونَ مَصَ انِعَ لَعَل كُمْ تَخْلُدُون129
:)} [الشعراء128
،
129
]
:، إلى قوله َ{أَمَد كُمْ بِأَنْعَامٍ وَبَنِين
(
133
( ٍ) وَجَن اتٍ وَعُيُون134
:)} [الشعراء133
،
134
]
:، قال ابن كثير"
أتبنون بكل ريع آية؛ أي معلمًا بناء مشهورًا تعبثون أي
وإنما تفعلون ذلك عبثًا ال لالحتياج إليه، بل لمجرد اللعب واللهو وإظهار القوة، و لهذا أنكر عليه نبيهم عليهم السالم ذلك، ألنه تضييع
للزمان وإتعاب لَلبدان في غير فائدة، واشتغال بما ال يجدي في الدنيا وال في اآلخرة"(
1
)، وهذا دليل الترف المعماري، وأ قوى األدلة
على شدة ترفهم، أو سبقهم في الترف
:قوله تعالى َ{أَلَمْ تَرَ كَيْفَ فَعَلَ رَبُّك( ٍبِعَاد6
( ِ) إِرَمَ ذَاتِ الْعِمَاد7
ِ) ال تِي لَمْ يُخْلَقْ مِثْلُهَا فِي الْبِالَ د
(
8
:)} [الفجر6
-
8]
.، فال يوجد ترف أعظم من هذا، بعد أن أكد هللا بأنها بلدة لم يُخلق مثلها، وقد يعود الضمير فيها إلى القبيلة سا ًا بع:: قوله تعالى
ٌ{إِنْ هُوَ إِال رَجُل[ } َافْتَرَى عَلَى ّللا ِ كَذِبًا وَمَا نَحْنُ لَهُ بِمُؤْمِنِين :المؤمنون38
]
:، كقوله تعالى على لسان قوم هود
:{وَإن ا لَنَظُنُّكَ مِنَ الْكَاذِبِينَ } [األعراف66
]
:، وقوله تعالى
:{مَا نَحْنُ لَكَ بِمُؤْمِنِينَ } [هود53
]. سا ًا بع:: قوله تعالى
ٌ{إِنْ هُوَ إِال رَجُل[ } َافْتَرَى عَلَى ّللا ِ كَذِبًا وَمَا نَحْنُ لَهُ بِمُؤْمِنِين :المؤمنون38
]
:، كقوله تعالى على لسان قوم هود
:{وَإِن ا لَنَظُنُّكَ مِنَ الْكَاذِبِينَ } [األعراف66
]
:، وقوله تعالى
:{مَا نَحْنُ لَكَ بِمُؤْمِنِينَ } [هود53
]. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 أ 3
)
-
ابن الجوزي، نزهة األعين النواظر في علم الوجوه والنظائر1
/
91
. /
(
(ير
وزي ز
بن/
3
)
-
ابن الجوزي، نزهة األعين النواظر في علم الوجوه والنظائر1
/
91
. 1
)
-
ابن كثير، تفسير القرآن العظيم6
/
137
. (
(2
-
ابن الجوزي، زاد المسير5
/
110
. 697
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)
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ابن كثير، تفسير القرآن العظيم6
/
137
.
(
(2
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ابن الجوزي، زاد المسير5
/
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.
3
)
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ابن الجوزي، نزهة األعين النواظر في علم الوجوه والنظائر1
/
91
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
/ y of Gaza) / CC BY 4 0
(
(1
-
ابن فارس، مقاييس اللغة3
/
324
.
(
(2
-
:ينظر ابن منظور، لسان العرب2
/
521
-
.
522
(
(3
-
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/
107
.
(
(4
-
عزة، التفسير الحديث5
/
313
.
(
(5
-
ابن فارس، مقاييس اللغة3
/
285
.
(
(6
-
:ينظر ابن منظور، لسان العرب10
/
198
.
(
(7
-
:ينظر
األزهري، تهذيب اللغة1
/
122
. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم :وكذلك ال يمنع أن يكون العذاب النازل على عاد بدأ بالصيييييباح، وانتهى صيييييباحاً فقال َ{سييييي َ خ رَهَا عَلَيْهِمْ سييييي َ بْعَ لَيَالٍ وَثَمَانِيَة
:أَي امٍ حُسييييييي ُ ومًا } [الحاقة7]
،
:وتجدر اإلشِِِِارة إلى أن ر ذكر عاد وثمود وفرعون في سِِِِورة الفجر، فقال
َ{أَلَمْ تَرَ كَيْفَ فَعَلَ رَبُّك
( ٍبِعَاد6
( ِ) إِرَمَ ذَاتِ الْعِمَاد7
( ِ) الَّتِي لَمْ يُخْلَقْ مِثْلُهَ ا فِي الْبِالَ د8) وَثَمُودَ الَّذِينَ جَابُوا ا( ِخْرَ بِالْوَاد
َّ
لصِِِِِِِِِ9
ِ) وَفِرْعَوْ نَ ذِي األَْوْ تَاد
(
10
( ِ) الَّذِينَ طَغَوْا فِي الْبِالَ د11
( َ) فَأَكْثَرُ وا فِيهَا الْفَسِِ َ اد12
( ٍ) فَصِِ َ بَّ عَلَيْهِمْ رَبُّكَ سِِ َ وْطَ عَذَاب13
:)} [الفجر6
-
13
]،
ونص 697 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون فيها على صِِب العذاب عليهم، ولعل فيها إشِِارة إلى أن هال ًهذه األمم كانت جميعها فجرا ، ؛ ألن الفجر مؤذن بدخول الصِِبا
وهذه من المناسبات بين اسم السورة وموضوع
ات.ها فيها على صِِب العذاب عليهم، ولعل فيها إشِِارة إلى أن هال ًهذه األمم كانت جميعها فجرا ، ؛ ألن الفجر مؤذن بدخول الصِِبا
وهذه من المناسبات بين اسم السورة وموضوع
ات.ها تاسعًا:: وأما ما ورد في قوله تعالى
ْ{فَأَخَذَتْهُمُ الص يْحَةُ بِالْحَق ِ فَجَعَلْنَاهُمْ غُثَاءً فَبُعْدًا لِل :قَوْ مِ الظ الِمِينَ } [المؤمنون41
]، بأن
الصيحة
:كانت عذابًا على قوم صالح أو قوم شعيب، بينما قوم عاد أُهلكوا بالريح، فنود أن نفصل في اآلتي
أوالً: معنى الصيحة :: قال ابن فارس"الصاد والياء والحاء أصل صحيح، وهو الصوت العالي، منه الصياح"(
1
)
:. وقال ابن منظور
"وصيح: صوت بأقصى طاقته، يكون ذلك في الناس وغيرهم؛ ... والصيحة: العذاب، ويقال: صيح في آل فالن إذا هلكوا،
:والصيحة
الغارة إذا فوجئ الحي بها"(
2
). :وقال هللا }ُ{فَأَخَذَتْهُمُ الص يْحَة
أي الهلكة،
وصيحة الغارة إذا فاجأتهم الخيل المغيرة... ويقال: ما ينتظرون إال مثل صيحة
الحبلى أي شرًا يفجؤهم(
3
)،
:قال دروزة محمد عزت عن الصيحة"كناية عن عذاب هللا وهي عامة المعنى"(
4
). nal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
ابن فارس، مقاييس اللغة3
/
324
.
(
(2
-
:ينظر ابن منظور، لسان العرب2
/
521
-
.
522
(
(3
-
:ينظر األزهري، تهذيب اللغة5
/
107
.
(
(4
-
عزة، التفسير الحديث5
/
313
.
(
(5
-
ابن فارس، مقاييس اللغة3
/
285
.
(
(6
-
:ينظر ابن منظور، لسان العرب10
/
198
.
(
(7
-
:ينظر
األزهري، تهذيب اللغة1
/
122
. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثامنًا:: قوله تعالى
:{لَيُصْ بِحُن نَادِمِينَ } [المؤمنون40
]
، استدل بها أصحاب القول الثاني على أن المقصود قوم صالح؛ ألن العذاب
:نزل بهم في الصباح كما قال هللا
:{فَأَخَذَتْهُمُ الص يْحَةُ مُصْ بِحِينَ } [الحجر83
]
:، ولعله فاتهم قول هللا في قوم عاد
{فَأَصْ بَحُوا الَ يُرَى
ِإ :ال مَسَ اكِنُهُمْ} [األحقاف25
]
:، وهو ما يناسب قول هللا في سورة المؤمنون
} َ{لَيُصْ بِحُن نَادِمِين ، ولعل الندم أصابهم مع رؤيتهم ألول
مراحل العذاب حيث استمر سبع ليالٍ وثمانية أيام فأصبحوا ال يُرى إال مساكنهم، فقيل: أصبحوا وقد غطت هم الر ِيح بالر مْل ف ال يُرَ وْ ن(
2
)
،
.وهذا ما استشهد به ابن الجوزي حيث قال: "وذكر أهل التفسير أن أصبح في القرآن على وجهين: أحدهما: إدراك الصباح للصبح
:ومنه قوله تعالى في الكهف
} ِ{فَأَصْ بَحَ يُقَل ِبُ كَف يْه:، وفي األحقاف
}ْ{فَأَصْ بَحُوا الَ يُرَى إِال مَسَ اكِنُهُم
:، وفي نون
} َ{لَيَصْ رِمُن هَا مُصْ بِحِين ،
وفيه
ا :
} ِ{فَأَصْ بَحَتْ كَالص رِيم ،واآلخر: بمعنى صار،
:ومنه قوله تعالى في آل عمران
}{ فَأَصْ بَحْتُمْ بِنِعْمَتِهِ إِخْوَانًا:، وفي المائدة
{
} َفَأَصْ بَحَ مِنَ الْخَاسِ رِين
"(
3
)
. وكذلك ال يوجد دليل صريح على أ:ن معنى قوله
} {لَيُصْ بِحُن ، أي الصباح، فقد تحتمل كذلك المعنى اآلخر ألصبح
:بمعنى: صار، فيكون المعنى: ليصيرن نادمين، ومثلها كذلك قوله في األحقاف
} {فَأَصْ بَحُوا. :كما أن هناك فرقًا بين قوله في سورة المؤمنون
} {لَيُصْ بِحُن:، وقوله في سورة الحجر
ِ{ مُصْ ب} َحِين التي اس ،تشهدوا بها
وجعلوها تفسيرًا لها، فاألولى تحتمل عدة معانٍ كما سبق، بخالف الثانية
فهي أصرح في نص العذاب بالصباح. (
(1
-
ابن فارس، مقاييس اللغة3
/
324
. 699
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
الشنقيطي، أضواء البيان في إيضاح القرآن بالقرآن7
/
16
.
(
(2
-
الفراهيدي، كتاب العين7
/
82
.
3
)
-
:ينظر مجموعة من الباحثين، موسوعة التفسير
الموضوعي-
، بحث عاد23
/
17
.
(
(4
-
ابن كثير، تفسير القران العظيم5
/
413
.
(
5
(
-
الشيخ أحمد حطيبة ،دروس صوتية قام بتفريغها موقع الشبكة اإلسالمية
http://www.islamweb.net
(
(6
-
السمين الحلبي، عمدة الحفاظ في تفسير أشرف األلفاظ3
/
54
. :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثالثًا
: ما ذكره الباحث في موسوعة التفسير الموضوعي عند بحثه في عاد: إن الصيحة ليست مختص ة بهم حتى تكون دليالً إلخراج
:السياق عن ظاهره، فقد أهلك هللا بها أقوامًا غير ثمود قال تعالى
:{وَأَخَذَتِ ال ذِينَ ظَلَمُوا الص يْحَةُ فَأَصْ بَحُوا فِي دِيَارِهِمْ جَاثِمِينَ } [هود
94
]، ففي هذه اآلية بيان أن هالك قوم شعيب بالصيحة، وقوم لوط أهلكوا بالصيحة:، قال تعالى
:{فَأَخَذَتْهُمُ الص يْحَةُ مُشْ رِقِينَ } [الحجر
73
]
:، وأصحاب القرية المذكورون في سورة يس أهلكوا بها، قال تعالى
:{إِنْ كَانَتْ إِال صَ يْحَةً وَاحِدَةً فَإِذَا هُمْ خَامِدُونَ } [يس29
](
3
)
. قال ابن كثير :
"
والظاهر أنه اجتمعت عليهم الصيحة، مع
الريح الصرصر العاصفة القوية الباردة"(
4
)
، وقال الشيخ أحمد
:حطيبة"
وال مانع أن يكونوا هم قوم عاد، فقد أخذهم هللا
بالريح التي أرسلها عليهم، وبصحية صاح فيهم جبريل،
فأهلكهم هللا عز
وجل وأبادهم بذلك، فال مانع من أن يكونوا هم؛ ألنهم الذين كانوا من بعد قوم نوح"(
5
). :ويتضح مما سبق أن الصيحة كانت عذابًا كذلك على عاد كما في قوله
} ٍ{فَأَرْسَ لْنَا عَلَيْهِمْ رِيحًا صَ رْصَ رًا فِي أَي امٍ نَحِسَ ات
:[فصلت16
]
،، فهي مصاحبة للريح في هبوبها
وبناءً عليه ف يجوز أن يعبر عن نوع العذاب في هذا المقطع بالصيحة وفي بقية
المقاطع بالر يح الصرصر؛ ألن الصيحة جزء منها، كما عبر عن عذاب قوم صالح بالرجفة، وفي بقية المقاطع بالصيحة، ولعل
الصيحة كانت مالزمة للرجفة، ومثلها الصيحة المقصودة في سورة المؤمنون، فهي عبارة عن الصوت الشديد المصاحب لهبوب
الريح من بعيد كما مر معنا في تعريف الصيحة، ثم اقتلع تهم الرياح، فجعلتهم غثاء، وهذه ما تُعرف بالصاعقة، وذكرنا بأن من
:معانيها: أصوات الرعد ووقعها الشديد، ومما يؤيد هذا قوله تعالى
ْ{فَلَم ا رَأَوْهُ عَارِضً ا مُسْ تَقْبِلَ أَوْ دِيَتِهِمْ قَالُوا هَذَا عَارِضٌ مُمْطِ رُنَا بَل
ٌهُوَ مَا اسْ تَعْجَلْتُمْ بِهِ رِيح
:فِيهَا عَذَابٌ أَلِيمٌ} [األحقاف24
]
،
:قال السمين الحلبي"
والعارض: البادي عرضه؛ فتارًة تختص بالسحاب
:كقوله تعالى
} {هَذَا عَارِضٌ مُمْطِ رُنَا أي سحاب قد عرض في األفق"(
6
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم :ثانيًا
:ذكر هللا بأنه أهلك قوم عاد وثمود بالصاعقة فقال تعالى
ً{فَإِنْ أَعْرَضُ وا فَقُلْ أَنْذَرْتُكُمْ صَ اعِقَة : مِثْلَ صَ اعِقَةِ عَادٍ وَثَمُودَ } [فصلت
13
]، مما يدل على أن هناك مرحل.ة من العذاب متشابهة بين قوم عاد وقوم ثمود :وقد وصف هللا الريح التي أرسلها على قوم عاد بأنها صرصر، أي: ذات صوت شديد، فقال
ْ{فَأَرْسَ لْنَا عَلَيْهِم ِر يحًا صَ رْصَ رًا
:فِي أَي امٍ نَحِسَ اتٍ } [فصلت16
]
:، وقال ٌ{وَأَم ا عَاد :فَأُهْلِكُوا بِرِيحٍ صَ رْصَ رٍ عَاتِيَةٍ} [الحاقة6]
. :قال الشنقيطي"
:قوله تعالى :{فَأَرْسَ لْنَا عَلَيْهِمْ رِيحًا صَ رْصَ رًا فِي أَي امٍ نَحِسَ اتٍ } [فصلت16
]
، الصرصر: وزنه بالميزان
الصرفي«فعفل»
:، وفي معنى الصرصر، لعلماء التفسير وجهان معروفان أحدهما : أن الريح الصرصر هي الريح العاصفة الشديدة
:الهبوب التي يسمع لهبوبها صوت شديد، وعلى هذا، فالصرصر من الصرة التي هي الصيحة المزعجة، ومنه قوله تعالى ِ{فَأَقْبَلَت (
(2
-
:ينظر ابن منظور، لسان العرب2
/
521
-
. 522
أ (
(3
-
:ينظر األزهري، تهذيب اللغة5
/
107
. (
(5
-
ابن فارس، مقاييس اللغة3
/
285
. (
(6
-
:ينظر ابن منظور، لسان العرب10
/
198
. (
(7
-
:ينظر
األزهري، تهذيب اللغة1
/
122
. 698 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد :امْرَأَتُهُ فِي صَ ر ةٍ } [الذاريات29
]أي في صيحة، ومن هذا المعنى صرير الباب والقلم، أي صوتهما ،الوجه الثاني : أن الصرصر
:من الصر الذي هو البرد الشديد المحرق، ومنه على أصح التفسيرين قوله تعالى
:{كَمَثَلِ رِيحٍ فِيهَا صِ رٌّ } [آل عمران117
]
،
أي فيها برد شديد محرق"(
1
). :قال الخليل الفراهيدي"وريح صرصر: ذات صر، ويقال: ذات صوت، والصرصر نعت لها من البرد،
والصر: البرد الذي
:يضرب كل شيء ويحسه، ومنه قوله تعالى
:{رِيحٍ فِيهَا صِ رٌّ } [آل عمران117
]
،
وصر
الباب، وصرت اآلذان إذا سمعت
لها صوتًا ودويًا، والصرة: شدة الصياح"(
2
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم :وهذا الوصف :{فَجَعَلْنَاهُمْ غُثَاءً } [المؤمنون41
]
ْ، يتفق تمامًا مع وص فه تعالى لعذاب قوم عاد
في قوله تعالى:
{إِال
:جَعَلَتْهُ كَالر مِيمِ } [الذاريات42
]، قال مقاتل: إال جعلته باليًا كالتراب بعد ما كانوا مثل نخل منقعر صاروا رميمًا(
3
)
:، وقال ابن كثير
"أي كالشيء البالي"(
4
)، وهو قول المراغي(
5
)،
وابن الجوزي(
6
)،
والخازن(
7
)، والشوكاني(
8
)
:، وقال الخطيب"
هو بيان لما تترك هذه
الريح العقيم من آثار ومخل فات وراءها، إنها ال تترك شيئًا تمر عليه إال دم رته، وحطمته، وأتت على كل صالحة فيه، فيتحول إلى
كيان بال متفتت، والرميم: العظام البالية، والر مة: الحبل البالي، والر م : إصالح الشيء البالي"(
9
)،
:كقوله تعالى في سورة يس َ{قَال
:مَنْ يُحْيِ الْعِظَامَ وَهِيَ رَمِيمٌ } [يس78
]، أي: بالية،
:كما يتفق مع وصف المعذبين من قوم عاد في بقية المواضع
َ{تَنْزِعُ الن اس
:كَأَن هُمْ أَعْجَازُ نَخْلٍ مُنْقَعِرٍ } [القمر20
]
:، وقوله
:{كَأَنَّهُمْ أَعْجَازُ نَخْلٍ خَاوِيَةٍ} [الحاقة7
]
:، قال ابن عاشور"
وذلك يحصل لعود
النخل إذا طال مكثه مطروحًا"(
10
)
،
.ومعروف أن الشيء إذا طال مكثه يصبح باليًا كالغثاء الحادي عشر:: ما ذكرته من الردود على القول األول، فيمكن الرد عليه بما يأتي
:ًأوال أثر
ابن عباس
يستأنس به، واستنباط المفسرين من
هذا األ .ثر له وجاهة
ثانيًا
: التشابه الكبير بين القصتين ال يعني تجاوز عاد والقول بأنها في ثمود أو غيرهم مع وجود أدلة صريحة على أن عا
د عقب نوح
.في التاريخ مباشرة الحادي عشر:: ما ذكرته من الردود على القول األول، فيمكن الرد عليه بما يأتي
:ًأوال أثر
ابن عباس
يستأنس به، واستنباط المفسرين من
هذا األ .ثر له وجاهة ثانيًا
: التشابه الكبير بين القصتين ال يعني تجاوز عاد والقول بأنها في ثمود أو غيرهم مع وجود أدلة صريحة على أن عا د عقب نوح
.في التاريخ مباشرة :ثالثًا
قوله :
:{قَالَ عَمَّا قَلِيلٍ } [المؤمنون40
]
،
:هذا من قول هللا لرسيييييوله اسيييييتجابة لدعائه إذ قال
َ{ قَال
} ِرَب ِ انْصييييي ُ رْنِي بِمَا كَذ بُون
:[المؤمنون39
]
،وليس من كالم الرسول حتى نفسره بقول صالح:
َّ{فَقَالَ تَمَتَّعُوا فِي دَارِكُمْ ثَالَ ثَةَ أَي :امٍ} [هود65
]
. IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ثالثًا
: ما ذكره الباحث في موسوعة التفسير الموضوعي عند بحثه في عاد: إن الصيحة ليست مختص ة بهم حتى تكون دليالً إلخراج
:السياق عن ظاهره، فقد أهلك هللا بها أقوامًا غير ثمود قال تعالى
:{وَأَخَذَتِ ال ذِينَ ظَلَمُوا الص يْحَةُ فَأَصْ بَحُوا فِي دِيَارِهِمْ جَاثِمِينَ } [هود
94
]، ففي هذه اآلية بيان أن هالك قوم شعيب بالصيحة، وقوم لوط أهلكوا بالصيحة:، قال تعالى
:{فَأَخَذَتْهُمُ الص يْحَةُ مُشْ رِقِينَ } [الحجر
73
]
:، وأصحاب القرية المذكورون في سورة يس أهلكوا بها، قال تعالى
:{إِنْ كَانَتْ إِال صَ يْحَةً وَاحِدَةً فَإِذَا هُمْ خَامِدُونَ } [يس29
](
3
)
. قال ابن كثير :
"
والظاهر أنه اجتمعت عليهم الصيحة، مع
الريح الصرصر العاصفة القوية الباردة"(
4
)
، وقال الشيخ أحمد
:حطيبة"
وال مانع أن يكونوا هم قوم عاد، فقد أخذهم هللا
بالريح التي أرسلها عليهم، وبصحية صاح فيهم جبريل،
فأهلكهم هللا عز
وجل وأبادهم بذلك، فال مانع من أن يكونوا هم؛ ألنهم الذين كانوا من بعد قوم نوح"(
5
). م ح
مأ
م
عا
م
:ويتضح مما سبق أن الصيحة كانت عذابًا كذلك على عاد كما في قوله
} ٍ{فَأَرْسَ لْنَا عَلَيْهِمْ رِيحًا صَ رْصَ رًا فِي أَي امٍ نَحِسَ ات
:[فصلت16
]
،، فهي مصاحبة للريح في هبوبها
وبناءً عليه ف يجوز أن يعبر عن نوع العذاب في هذا المقطع بالصيحة وفي بقية
المقاطع بالر يح الصرصر؛ ألن الصيحة جزء منها، كما عبر عن عذاب قوم صالح بالرجفة، وفي بقية المقاطع بالصيحة، ولعل
الصيحة كانت مالزمة للرجفة، ومثلها الصيحة المقصودة في سورة المؤمنون، فهي عبارة عن الصوت الشديد المصاحب لهبوب
الريح من بعيد كما مر معنا في تعريف الصيحة، ثم اقتلع تهم الرياح، فجعلتهم غثاء، وهذه ما تُعرف بالصاعقة، وذكرنا بأن من
:معانيها: أصوات الرعد ووقعها الشديد، ومما يؤيد هذا قوله تعالى
ْ{فَلَم ا رَأَوْهُ عَارِضً ا مُسْ تَقْبِلَ أَوْ دِيَتِهِمْ قَالُوا هَذَا عَارِضٌ مُمْطِ رُنَا بَل
ٌهُوَ مَا اسْ تَعْجَلْتُمْ بِهِ رِيح
:فِيهَا عَذَابٌ أَلِيمٌ} [األحقاف24
]
،
:قال السمين الحلبي"
والعارض: البادي عرضه؛ فتارًة تختص بالسحاب
:كقوله تعالى
} {هَذَا عَارِضٌ مُمْطِ رُنَا أي سحاب قد عرض في األفق"(
6
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم 699 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد ًعاشرا:: وأما قوله تعالى
:{فَجَعَلْنَاهُمْ غُثَاءً } [المؤمنون41
]
،، فهو وصف حالهم بعد العقاب، والغثاء: هو الشيء البالي، قاله قتادة
:وقال البخاري"هو الزبد وما ارتفع على الماء"(
1
)، وقيل: عبارة عن حميل السيل مما بلي وأسود من العيدان(
2
). ًعاشرا:: وأما قوله تعالى
:{فَجَعَلْنَاهُمْ غُثَاءً } [المؤمنون41
]
،، فهو وصف حالهم بعد العقاب، والغثاء: هو الشيء البالي، قاله قتادة
:وقال البخاري"هو الزبد وما ارتفع على الماء"(
1
)، وقيل: عبارة عن حميل السيل مما بلي وأسود من العيدان(
2
). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم رابعًا : ذكرت
لفظة
:ثمود قبل عاد في سيييييييييورة ق عند قوله تعالى
( ُحَابُ الر س ِ وَثَمُود
ْ
{كَذ بَتْ قَبْلَهُمْ قَوْ مُ نُوحٍ وَأَصييييييييي12
ُ) وَعَادٌ وَفِرْعَوْن
( ٍوَإِخْوَانُ لُوط13
:)} [ق12
،
13
]
:، وسييييييييييييييورة الحاقة عند قوله تعالى
:{كَذ بَتْ ثَمُودُ وَعَادٌ بِالْقَارِعَةِ } [الحاقة4]
،
و الجواب على هذا
بأنه ليسييت عادة القرآن أن يكون التقديم بسييبب الترتيب الزمني ، فعلى سييبيل المثال نجد القصيية األولى في سييورة الشييعراء هي قصيية
ًموسيييى مع فرعون، ثم تحدث عن قصييية نوح وهود وصيييالح مع أن موسيييى متأخرا
،عنهم :قال السيييهيلي"
والمعاني تتقدم بأحد خمسييية (
(1
-
ابن حجر، فتح الباري بشرح صحيح البخاري1
/
161
. 700
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
ابن حجر، فتح الباري بشرح صحيح البخاري1
/
161
. (
(2
-
:ينظر عباس، قصص القران الكريم ص221
. (
(3
-
:ينظر مقاتل بن سليمان، تفسيره3
/
279
. (
(4
-
ابن كثير، تفسير القران العظيم7
/
263
. (
5(-
:ينظر المراغي، تفسيره26
/
30
. (
6(-
:ينظر
ابن الجوزي، زاد المسير4
/
172
. (
7(-
:ينظر الخازن، تفسيره6
/
246
. (
(8
-
:ينظر الشوكاني، فتح القدير5
/
108
. (
(9
-
الخطيب، التفسير القرآني للقرآن14
/
527
. (
(10
-
ابن عاشور، التحرير والتنوير27
/
194
. (
(2
-
:ينظر عباس، قصص القران الكريم ص221
. (
(3
-
:ينظر مقاتل بن سليمان، تفسيره3
/
279
. (
(4
-
ابن كثير، تفسير القران العظيم7
/
263
. (
5(-
:ينظر المراغي، تفسيره26
/
30
. (
6(-
:ينظر
ابن الجوزي، زاد المسير4
/
172
. (
(8
-
:ينظر الشوكاني، فتح القدير5
/
108
. (
(9
-
الخطيب، التفسير القرآني للقرآن14
/
527
. (
(10
-
ابن عاشور، التحرير والتنوير27
/
194
. 700 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد أشيييييياء: إما بالزم
ان، وإما بالطبع، وإما بالرتبة، وإما بالسيييييبب وإما بالفضيييييل والكمال، فإذا سيييييبق معنى من المعاني إلى الخلد والفكر
بأحد هي األسباب الخمسة، أو بأكثرها سبق اللفظ الدال على ذلك المعنى السابق، وكان ترتيب األلفاظ بحسب ذلك"(
1
). IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0
(
(1
-
السهيلي، نتائج الفكر في النحو1/
209
(
(2
-
البقاعي، نظم الدرر في تناسب اآليات والسور8/
121
(
3
(-
ابن عاشور، التحرير والتنوير29
/
116 المصادر و
المراجع القاهرة :دار إحياء الكتب
.العربية
ل
الخازن
ن
د ن
إ اه
ن
الش(
1415ه)ل ا
التأ ل
ف
ان
التنز ل ت ق ق
ت
د
ل
شاه ن ر ب
.ي
،الواحدي
( أبو الحسن علي بن أحمد بن محمد بن علي الواحدي، النيسابوري، الشافعي
ت :
468
)هـ التَّفْسِيرُ البَسِيْط ،
أصل تحقيقه في
(
15
،) رسالة دكتوراة بجامعة اإلمام محمد بن سعود
ط1. عمادة البحث العلمي-
.جامعة اإلمام محمد بن سعود اإلسالمية
،المحلي جالل الدين محمد بن
،أحمد والسيوطي، جالل الدين عبد الرحمن بن أبي بكر. (د.ت). تفسير الجاللين. ط1. القاهرة: دار
.الحديث
،دروزة محمد
( .عزت1383هـ). التفسير الحديث. (د.ط). القاهرة :دار إحياء الكتب
.العربية
الخازن، علي بن محمد بن إبراهيم بن عمر
.الشيحي(
1415هـ). لباب التأويل في معاني التنزيل. تحقيق :تصحيح محمد علي
.شاهين
ط1. بيروت: دار الكتب
.العلمية
،السمعاني منصور بن محمد بن عبد الجبار المروزى، أبو
.المظفر (
1997م). تفسير القرآن. تحقيق: ياسر بن
،إبراهيم وآخرين. ط1
. الرياض: دار
.الوطن
)د (د ت
طيبة، أ
د
الشيخ أ
تف ي
طيبة
تية قا بتف يغ ا
د:الشبكة اإل ال ية
ق:ق
(د ط) (د ) ال ر
ي
،الواحدي
( أبو الحسن علي بن أحمد بن محمد بن علي الواحدي، النيسابوري، الشافعي
ت :
468
)هـ التَّفْسِيرُ البَسِيْط ،
أصل تحقيقه في
(
15
،) رسالة دكتوراة بجامعة اإلمام محمد بن سعود
ط1. عمادة البحث العلمي-
.جامعة اإلمام محمد بن سعود اإلسالمية
ل
ال
الل
ال
أ
الل
ط
ال
ال
ال
أ
ف
)
(
ك
ط
ال الل
ا
القا ،الواحدي
( أبو الحسن علي بن أحمد بن محمد بن علي الواحدي، النيسابوري، الشافعي
ت :
468
)هـ التَّفْسِيرُ البَسِيْط ،
أصل تحقيقه في
(
15
،) رسالة دكتوراة بجامعة اإلمام محمد بن سعود
ط1. عمادة البحث العلمي-
.جامعة اإلمام محمد بن سعود اإلسالمية
،المحلي جالل الدين محمد بن
،أحمد والسيوطي، جالل الدين عبد الرحمن بن أبي بكر. (د.ت). تفسير الجاللين. ط1. القاهرة: دار
.الحديث ،دروزة محمد
( .عزت1383هـ). التفسير الحديث. (د.ط). القاهرة :دار إحياء الكتب
.العربية
الخازن، علي بن محمد بن إبراهيم بن عمر
.الشيحي(
1415هـ). لباب التأويل في معاني التنزيل. تحقيق :تصحيح محمد علي
.شاهين
ط1. بيروت: دار الكتب
.العلمية
،السمعاني منصور بن محمد بن عبد الجبار المروزى، أبو
.المظفر (
1997م). تفسير القرآن. تحقيق: ياسر بن
،إبراهيم وآخرين. ط1
. الرياض: دار
.الوطن
.)حطيبة، أحمد. (د.ت تفسير الشيخ أحمد
حطيبة. دروس صوتية قام بتفريغها: موقع الشبكة اإلسالمية:. (د.ط). (د.م). :القول الثاني: ثمود قوم صالح، وأصحاب هذا القول، هم ًوفي هذا الموضييييييع نجد كالم ا لبعض المفسييييييرين عن
سيييييير التقديم والتأخير ،
:قال البقاعي"
وتقديمهم أيضيييييياً من حيث
إ ن
بالدهم أقرب إلى قريش، وواعظ القرب أكبر"(
2
)، و
قال ابن عاشِِور: "
ابتدئ بذكر ثمود ألن العذاب الذي أصييابهم من قبيل القرع إذ
أصابتهم الصواعق المسماة في بعض اآليات بالصيحة، والطاغية: الصاعقة في قول ابن عباس
وقتادة: نزلت عليهم صاعقة أو
:صواعق فأهلكتهم، ألن منازل ثمود كانت في طريق أهل مكة إلى الشام في رحلتهم فهم يرونها، قال تعالى
{فَتِلْكَ بُيُوتُهُمْ خَاوِيَةً بِمَا
:ظَلَمُوا } [النمل52
]
،وألن الكالم على مهلك عاد أنسب فأخر لذلك أيضا" (
3
). ويتضح مما سبق أن القول باحتمال اآلية لقوم صالح له وجه، وخاصة أن الرسول يُطلق ويراد به الجنس أحي ،ًانا
وللتشابه الكبير بين القصتين، والقول بأنها في عاد وثمود محتمل كذلك، ولعل هذا من اإلبهام الذي تحتمله اآل يات لغرض أخذ
العظة والعبرة، وال يلزم بيان أسماء األشخاص أو األماكن، إال أن
القول
األول
هو الراجح من وجهة نظ
ر الباحث
كما سبق بيانه
،في إحدى عشرة فقرة
.ور تعالى أعلى وأعلم، وصلى ر وسلم على نبينا محمد وآله وصحبه أجمعين إن من :أهم النتائج لهذه الدراسة تتلخص فيما يلي 1
-
أن الراجح في
.المراد بالقرن اآلخرين في اآلية المذكورة هم عاد قوم هود عليه السالم
2
-
أن العذاب بالصيحة ليس مختصاً بثمود، بل وقع كذلك على عاد قوم هود بداللة اآليات في سورة فصلت والقمر والحاقة. 3
-
أن الصيحة جزء من العذاب أو مقدمة له، وقد يكون معها الرجفة أو الريح أو الصاعقة ، فقد تتناوب ألفاظ العقوبة
ف ي كل
مقطع حسب سياق اآليات. 4
-
.أن معنى قوله (ليصبحن) في اآلية: ليصيرن 4
-
.أن معنى قوله (ليصبحن) في اآلية: ليصيرن يوصي الباحث بالمزيد من دراسة القصص القرآني وأخبار األمم السابقة، وتحرير مواضع الخالف عند المفسرين في
بع ،ض آياتها
من خالل النظر في القصص المناظرة، ودراسة األلفاظ وسياق المقاطع بغية الوصول إلى الرأي.الراجح 701 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد المصادر و
المراجع 702
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 المصادر و
المراجع أبو السعود، محمد بن محمد العمادي. (د.ت). إرشاد العقل السليم إلى مزايا القرآن الكريم. (د.ط). بيروت: دار إحياء التراث العربي،
ت
بي بيروت. الشنقيطي، محمد األمين بن محمد
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ت :
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أصل تحقيقه في
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عل
بن
محمد
بن
إبراهي
بن
عمر(الشيح1415ه ) لباب
التأ يل
ف
معان
التنزيل تحقيق:تصحيح
محمد ،الزمخشري محمود بن عمرو بن
،أحمد جار
.الله(
1407هـ). الكشاف عن حقائق غوامض التنزيل. ط3. بيروت: دار الكتاب
.العربي
الثعلبي، أحمد بن محمد بن إبراهيم ، أبو
.إسحاق (
2002م)
.الكشف والبيان عن تفسير القرآن. تحقيق :اإلمام أبي محمد بن عاشور. ط1
. بيروت: دار إحياء التراث
.العربي
،الخازن عالء الدين علي بن محمد بن إبراهيم بن عمر
( .الشيحي1415هـ). لباب التأويل في معاني التنزيل. تحقيق :تصحيح محمد
علي شاهين. ط1. بيروت: دار الكتب
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ض ط1ا
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لإ
ابن الجوزي، عبد الرحمن بن علي بن
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الراضي. (د.ط). بيروت: مؤسسة
.الرسالة إاآ )
ي (
البقاعي، إبراهيم بن عمر بن حسن
.الرباط(د.ت). نظم الدرر في تناسب اآليات والسور. (د.ط). القاهرة :دار الكتاب
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،السهيلي أبو القاسم عبد الرحمن بن عبد :هللا بن أحمد السهيلي (المتوفى581
)هـ ،نتائج الفكر في النَّحو للسُّهَيلي ، ط1
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الكتب العلمية. )
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)هـ ،نتائج الفكر في النَّحو للسُّهَيلي ، ط1
:، بيروت دار
الكتب العلمية IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 المصادر و
المراجع تحقيق :تصحيح محمد
علي شاهين. ط1. بيروت: دار الكتب
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ض ط1ت دا
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.صادر 703
IUG Journal of Islamic Studies (Islamic University of Gaza) / CC BY 4.0 703 تحرير المراد
بالقرن اآلخرين في قوله تعالى ( :
ثُم أَنْشَ أْنَا
ْمِن
ْبَعْدِهِم قَرْنًا
َآخَرِين )من خالل اآلية رقم (
31
)إلى اآلية رقم (
41
)في سورة المؤم نون د. نبيل بن محمد مرعي سعيد ،القاسمي محمد جمال الدين بن محمد سعيد بن قاسم
.الحالق(
1418هـ). محاسن التأويل. تحقيق :محمد باسل عيون السود
. ط1
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دار الكتب
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.تمام (
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السالم عبد الشافي محمد. ط1. بيروت: دار الكتب
.العلمية هلل ما
ي
البغوي، الحسين بن مسعود، محيي
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1997م). معالم التنزيل في تفسير القرآن. تحقيق :محمد عبد هللا
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ط4. (د.م): دار طيبة للنشر
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1420هـ). مفاتيح الغيب. ط3. بيروت: دار إحياء التراث
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ا
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OS EMPRESÁRIOS E A POLÍTICA EDUCACIONAL: COMO O PROCLAMADO DIREITO À EDUCAÇÃO DE QUALIDADE É NEGADO NA PRÁTICA PELOS REFORMADORES EMPRESARIAIS
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Luiz Carlos de Freitas1 Resumo: A despeito de que os empresários sempre estiveram tentando interferir com os processos
educacionais desde os tempos da teoria do capital humano, o que pode estar havendo de novo que esteja
motivando um redobrado interesse do empresariado pela educação? É possível que modificações no
processo de desenvolvimento econômico-social dos países, ou as próprias crises do capital, estejam
mobilizando os empresários? Acreditamos que sim. O atual interesse dos empresários tem aspectos
específicos que merecem ser examinados. Não é recomendável que acreditemos que “a história está se
repetindo”. Tal linearidade de análise nos desarmaria para o enfrentamento local das contradições que
estão postas por esta nova escalada do capital sobre a educação. A política educacional dos reformadores é
produzida para articular a necessidade de se qualificar para as novas formas de organização do trabalho
produtivo, ao mesmo tempo que preserva e amplifica as funções sociais clássicas da escola: exclusão e
subordinação. Está em jogo o controle político e ideológico da escola, em um momento em que algum
grau a mais de acesso ao conhecimento é exigido pelas novas formas de organização do trabalho
produtivo, novas exigências de consumo do próprio sistema capitalista e novas pressões políticas por
ascensão social via educação. ç
Palavras-chave: Política educacional; reformas educacionais; avaliação; trabalho produtivo. Resumen: Apesar de que los empresarios siempre estaban tratando de interferir con el proceso educativo
desde el momento de la teoría del capital humano, lo que puede estar ocurriendo de nuevo que está
impulsando un mayor interés en la iniciativa empresarial a través de la educación? Es posible que los
cambios en el proceso de desarrollo socio-económico de los países o de sus propias crisis del capital, los
empresarios se están movilizando? Nosotros creemos que sí. El interés actual de los empresarios tiene
aspectos específicos que merecen ser examinados. Se recomienda que cree que “la historia se repite”. Tal
análisis linealidad nos desarma al lugar estamos enfrentando las contradicciones que plantea esta nueva
escalada del capital en la educación. La política educativa de los reformadores se produce para articular la
necesidad de calificar para las nuevas formas de organización del trabajo productivo, al tiempo que
preserva y amplía las funciones sociales clásicas de la escuela, la exclusión y la subordinación. Debate Debate Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Luiz Carlos de Freitas1 En juego
está el control político e ideológico de la escuela, en un momento en algún grado más el acceso al
conocimiento requerido por las nuevas formas de organización del trabajo productivo, las nuevas
demandas de consumo del sistema capitalista y las nuevas presiones políticas para la movilidad social a
través de la educación. Palabras clave: Política de la educación; las reformas educativas; evaluación; el trabajo productivo Abstract: Despite that businessmen were always trying to interfere with the educational process from the
times of theory of human capital, what can be happening of new that is motivating an increased interest of 48 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Debate the business community through education? Is it possible that changes in the socio-economic
development process of countries or their own crises of capital, are mobilizing the businessmen? We think
so. The current interest of businessmen has specific aspects that deserve to be examined. It is not
recommended that we believe that “history is repeating itself”. This linearity of analysis disarm us for the
local confrontations of contradictions placed by this new escalation of capital on education. The
educational policy of the reformers is produced to articulate the need to qualify for the new forms of
organization of productive work, at the same time that it preserves and amplifies the classical social
functions of the school: exclusion and subordination. At stake is the political and ideological control of
the school, at a time when some greater degree of access to knowledge is required by new forms of
organization of productive work, new consumption demands of the capitalist system and new political
pressures for social mobility through the education. p
y
g
Keywords: Educational policy; educational reforms; evaluation; productive work. Em vários países os empresários aparecem no cenário da educação local como promotores de
reformas educacionais. Diane Ravitch (2011) os chama, nos Estados Unidos, de reformadores
empresariais da educação (corporate reformers). A despeito de que os empresários sempre estiveram tentando interferir com os processos
educacionais desde os tempos da teoria do capital humano, o que pode estar havendo de novo que esteja
motivando um redobrado interesse do empresariado pela educação? É possível que modificações no
processo de desenvolvimento econômico-social dos países, ou as próprias crises do capital, estejam
mobilizando os empresários? Acreditamos que sim. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014.
49 Luiz Carlos de Freitas1 O atual interesse dos empresários tem aspectos específicos que merecem
ser examinados. Não é recomendável que acreditemos que “a história está se repetindo”. Tal linearidade
de análise nos desarmaria para o enfrentamento local das contradições que estão postas por esta nova
escalada do capital sobre a educação. É fato notório que as corporações estão vagando pelo mundo procurando por mão de obra
barata. Países populosos como China, Rússia, Índia e Brasil estão na mira dos processos de intensificação
da exploração da força de trabalho, pois ainda possuem tais bolsões. No caso do Brasil, as corporações fizeram uso da exploração de bolsões de mão de obra barata
como a população do campo e as força de trabalho feminina, entre outros. Nesta fase, os empresários não
necessitaram de uma boa estrutura educacional. Hoje, no entanto, tais bolsões já não dão conta de
abastecer as necessidades de mão de obra. Temos somente 15% aproximadamente da população no
campo e 56,1% da mão de obra feminina já está incorporada ao mercado de trabalho contra 71,5% dos
homens2. Quando tais bolsões diminuem, continua-se a necessitar de mais mão de obra. Entram em cena
os estrangeiros desocupados em seus países. Chegam, só em São Paulo, 30 por dia3. A contínua
necessidade de mão de obra pode fazer com que a renda média paga aos trabalhadores de setores inteiros
da economia comece a crescer. Salários pagos são um componente fundamental na definição do lucro. Usualmente, os processos de fabricação também tendem a se sofisticar para intensificar a força de
trabalho, exigindo tais processos mais educação. No caso da área de serviços a dependência da mão de 49 49 Debate Debate obra “educada” é ainda maior. Há ainda mudanças globais na divisão internacional do trabalho fruto da
própria mobilidade do capital pelo mundo. Quando um país aumenta o salário médio de sua força de trabalho sem ampliar sua
produtividade4, os empresários ficam “desestimulados” pela queda de rentabilidade em seus
investimentos. Se há uma ampliação da renda média isso começa a derrubar o lucro. Daí o termo
“armadilha”. Ocorre que o aumento da produtividade é dependente de fatores importantes e que sofrem o
efeito de passivos históricos: é o caso da educação. Portanto, não são de fácil e rápida solução. Mas para
os empresários, os quais só agora se interessaram pela qualidade da educação, isso não conta. Se não se colhe o bônus neste momento, não se colhe nunca mais. Se não se enriquece
o país neste momento, é muito difícil enriquecê-lo depois. Para colher o bônus é
preciso ter emprego e educação de qualidade. Os europeus, EUA, Japão, Cingapura,
Coreia, China, Taiwan, todos esses países aproveitaram. O Brasil precisaria crescer ao
menos 4%. Se o PIB fica abaixo de 2%, não é possível. Além do que deixamos a desejar
em educação. Estamos presos à armadilha da renda média.7 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Luiz Carlos de Freitas1 É preciso
resolver de imediato o problema educacional para puxar o aumento de produtividade de imediato. Sem
isso, dizem, perde-se competitividade internacional – ou seja, os lucros não são os esperados. O
argumento é oportunista, pois eles bem sabem que o aumento da produtividade não depende apenas da
educação. O conflito que aparece entre educadores profissionais e os empresários diz respeito ao que se
entende por uma boa educação: para os empresários é saber ler, escrever, contar e algumas competências
mais que estão sendo esperadas na porta da fábrica, medidas em um teste padronizado. Se as notas
aumentam, então houve melhoria. Se há mais formandos, houve melhoria. Para os educadores, isso é
apenas uma pequena parte da tarefa. Nota alta não é sinônimo de boa educação5. O conflito se amplia porque para os empresários – à imagem e semelhança de sua empresa –
tudo é uma questão de gerenciamento e competição. Portanto, se os educadores não dão conta de “seu
pedaço” (leia-se atender às necessidades das empresas) é porque não sabem gerenciar os recursos que são
dados. E se as empresas já desenvolveram métodos de administração bem sucedidos para elas, porque não
transplantá-los para as escolas e redes de ensino? Se eles têm a solução, os governos é claro agradecem. Eles também precisam mostrar à população que as escolas estão melhorando. Esta urgência, que é assumida também pelos governos, ocorre porque um país que pague renda
média menor a seus trabalhadores pode produzir bens e serviços mais baratos e permitir maiores lucros
aos empresários: melhora a competitividade. Sem educação de “qualidade” não se amplia o número de
formandos e com poucos formandos o salário médio sobe ao invés de descer. Lei da oferta e procura. A
armadilha da renda média consiste no aumento do salário médio dos trabalhadores sem que se consiga
alterar fatores de infraestrutura como educação, responsáveis pelo aumento da produtividade e
simultaneamente, dispor de uma maior quantidade de formandos que reduza pressões salariais6. Esta questão é tratada abaixo: Esta questão é tratada abaixo: 50 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Debate Neste quadro, os empresários estão se organizando para atacar estas questões de infraestrutura
que afetam o aumento da produtividade. Luiz Carlos de Freitas1 Vários movimentos estão se organizando há algum tempo: LIDE
(Grupo de Líderes Empresariais)8, Movimento Todos pela Educação9 e seus adjacentes, disfarçados de
“especialistas em educação” organizados em Organizações Não Governamentais (ONG) e empresas de
assessoramento educacional. Há um grupo mais ideológico e há outro mais operacional, o pessoal do
faturamento10. Não acreditam na escola pública e querem sua privatização – seja por concessão11, seja por
vouchers12. Na verdade ganham bem para desqualificar a escola pública – mesmo sem evidência de que suas
receitas são melhores13. Há, portanto, uma disputa pelo conceito de educação e pelos métodos de formação da
juventude. Os empresários e seus apoiadores defendem uma versão instrumentalizada de educação a qual
disfarçam muito bem com bandeiras como “primeiro o básico”, “os direitos da criança têm que vir
primeiro”, etc. Coisas com as quais nós até podemos concordar, mas sob outra concepção. Os educadores
querem uma educação de qualidade social, voltada para os valores, para a formação humana ampla e
entendem que a educação não é matéria para ser privatizada, pois é um bem público. Como tal, não pode
ser entregue ao controle de um setor da sociedade, os empresários. Isso não é democrático – mesmo no
quadro de dificuldades pelas quais passa a escola pública. Neste quadro, as pressões sobre a área da educação partem agora de entidades organizadas pelos
empresários com esta finalidade, como indicamos antes, e também de ações organizadas por estas junto
aos governos e junto ao Congresso Nacional. O caminho seguido é o mesmo que os empresários americanos seguiram nos últimos 30 anos: 1. Enfatizar a crise da educação e a necessidade de reformar a política educacional; 2. Uma ênfase no direito
à aprendizagem com dupla limitação: a) fala-se de direito à aprendizagem e não de direito à formação
humana, à educação; b) e restrita ao ambiente da escola, portanto isolada de importantes ligações com a
vida. Neste particular, é importante assinalar que a própria forma escolar atual já foi concebida com o
intuito de isolar as crianças da vida, vale dizer das contradições sociais. A proximidade com estas, levaria a
juventude a pensar sobre a nossa forma de organização social e seus limites, ensejando desejos de
mudança ou revolta. Isolados no interior das salas de aulas, restritos à aprendizagem do básico, lhes é
prometido um dia, chegar aos níveis mais avançados e complexos de educação, que de fato nunca
chegarão a ver. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014.
51 Luiz Carlos de Freitas1 Historicamente, a escola sempre sonegou seu conteúdo para a classe trabalhadora. Com o discurso do direito restrito à aprendizagem do básico, perpetua-se por um lado a
exclusão dos processos de formação humana e ao mesmo tempo libera-se a conta gota o conhecimento
necessário para que a juventude dê conta de atender às demandas das novas formas de organização da
produção. Acesso a um pouco mais de letramento, leitura e matemática. O estreitamento curricular impede que outras áreas de desenvolvimento da criança sejam
exercitadas (artística, criativa, afetiva, corporal). Com a escola focada em português e matemática, as
demais disciplinas são abordadas em “projetos interdisciplinares” que conduzem à banalização do
conteúdo destas. 51 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. 51 Debate O apelo ao básico é visto como politicamente correto, pois tem um sabor de distribuição do
conhecimento básico a todos, dando a impressão de uma política de garantia de direitos para todos. Porém, ao se examinar os sistemas voltados para a aprendizagem do básico proposto pelos reformadores
empresariais, o que se verifica é que tal política não garante a aprendizagem de todos e de cada um. A
escola tem a sua roupagem atualizada, mas as suas funções sociais são mantidas intactas: exclusão e
subordinação. Novas formas de exclusão são adicionadas como a especialização de escolas que assumem a
função de receber uma população que sabidamente não aprenderá, liberando as outras para a tarefa de
tentar ensinar o básico; a criação de trilhas diferenciadas de progressão (às vezes em salas exclusivamente
destinadas à recuperação do aluno, segregadas) que são destinadas a garantir a passagem do tempo até a
exclusão do aluno ao final de algum ciclo ou período de tempo; o deslocamento do aluno para formas de
atendimento alternativas como a Educação de Jovens e Adultos, onde são certificados precariamente à
margem do sistema. As formas de organização do trabalho no interior da escola não são alteradas, pelo contrário, a
ordem e a obediência são reforçadas com o apelo às famílias para que ajudem a controlar seus filhos, às
vezes com contratos escritos que responsabilizam a famílias. O conservadorismo – inclusive moral –
amplia-se. A disciplina fica cada vez mais rígida, cada vez mais voltada para instalar processos de
subordinação. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014.
52 Luiz Carlos de Freitas1 As estatísticas mostram que não há avanço no fechamento das brechas que distanciam a
aprendizagem de ricos e pobres, brancos e negros e a população que necessita de atendimento especial é
submetida à segregação em escolas ou em ambientes dentro das escolas14. A pobreza vai sendo confinada
nas escolas públicas, a classe média vai sendo retirada para escolas administradas por concessão ou por
meio de vouchers e os mais ricos continuam em suas escolas próprias, privadas de alto nível15. Dessa forma,
abre-se uma linha de acesso ao ensino para a classe média emergente e os mais ricos ficam protegidos do
convívio com os mais pobres. As práticas de avaliação que já dominavam a escola devido a seu isolamento em relação à vida
tomam o controle de todo o processo. Mais tempo para a avaliação e testes frequentes roubam tempo da
aprendizagem do aluno. A política educacional dos reformadores é produzida para articular a necessidade de se qualificar
para as novas formas de organização do trabalho produtivo, ao mesmo tempo que preserva e amplifica as
funções sociais clássicas da escola: exclusão e subordinação. Está em jogo o controle político e ideológico da escola, em um momento em que algum grau a
mais de acesso ao conhecimento é exigido pelas novas formas de organização do trabalho produtivo,
novas exigências de consumo do próprio sistema capitalista e novas pressões políticas por ascensão social
via educação. A matriz de controle mundial das políticas educacionais é hoje a Organização para a Cooperação
e Desenvolvimento Econômico (OCDE)16, um organismo internacional destinado à cooperação e
desenvolvimento econômico das nações desenvolvidas, que associa-se às estruturas anteriormente 52 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. 52 Debate existentes de Bancos de financiamento (Banco Mundial, Banco Internacional para Reconstrução e
Desenvolvimento (BIRD)). Ela é responsável pela avaliação em nível mundial da qualidade da educação
dos países ricos nas disciplinas de leitura, matemática e ciências pelo exame do Programme for International
Student Assessment (PISA). Somam-se ainda a estes, uma plêiade de fundações e bilionários que resolveram
“doar recursos para a educação”. Uma parte deles está voltada para a criação de estruturas de controle
ideológico e influência em governos e legislativos; outra, está mais interessada em abrir mercado que até
agora esteve sob controle do Estado – por exemplo: a educação – e faturar. Têm forte apoio da mídia. Luiz Carlos de Freitas1 No
caso brasileiro, a organização que mais cumpre esta função é o Movimento Todos pela Educação. Neste quadro, as avaliações de larga escala nacionais e internacionais emergem como um
instrumento político de promoção da internacionalização da política educacional. O padrão de qualidade é
o padrão PISA – um programa internacional de avaliação de estudantes promovido pela OCDE17. 53
Sua ação de controle passa por várias formulações que podem ser utilizadas em conjunto ou
separadamente: conscientes da importância do professor o foco de controle dos reformadores
empresariais é o professor. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014.
53 escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. Reunimos esta longa lista de ações mais recentes do capital no campo da educação porque
alguns incrédulos das novas configurações de controle sobre a escola argumentam que isso não seria uma
novidade já que os empresários sempre tiveram interesse no controle político e ideológico da escola, desde
os tempos da teoria do capital humano. É verdade. Entretanto mudaram a forma e o empenho. Agora, a questão educacional tem outra
posição no quadro das condições que são responsáveis pela valorização do capital, como resumidamente
no início. Depende também dela o aumento da produtividade. No entanto, cumpram-se ou não as expectativas de desenvolvimento econômico formuladas
pelo capital, a educação será sempre responsabilizada – independentemente dos resultados. Se forem
positivos, a educação será cobrada por aumentar a qualidade para elevar a produtividade, se forem
negativos, será responsabilizada por não ter produzido a elevação da qualidade, travado o avanço da
produtividade e ter derrubado a competitividade internacional do país. Vale lembrar que há outras funções esperadas da escola e da educação: aumento de formandos
nas profissões como forma de reduzir a pressão por salário que venha a elevar a renda média acima do que
já avançou. Trata-se do fenômeno já observado tanto nos Estados Unidos como no Brasil e que diz respeito
à queda do salário médio pago à medida que a “eficiência” do setor educacional gera mais profissionais e
os coloca no mercado. Tal situação faz com que havendo uma oferta maior de profissionais, se reduza o
salário pago: lei da oferta e procura. Enfim, seja para derrubar o salário médio por superpopulação de formandos, seja para aumentar
a produtividade, seja ainda para promover o controle ideológico e político de uma instituição pela qual
passa toda a juventude, seja para qualificar de acordo com as suas necessidades de produção, seja por
todos estes motivos, os reformadores empresariais resolveram – em escala mundial – controlar mais de
perto o que ocorre na educação garantindo um relativo aumento de qualificação da força de trabalho ao
mesmo tempo em que não perdem e ampliam o controle político e ideológico da escola e garantem as
suas funções clássicas: exclusão e subordinação. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Luiz Carlos de Freitas1 Centram sua ação na pessoa do professor propondo que deixem de ter
estabilidade no emprego, tenham salário variável cujo componente está ligado aos resultados dos testes
dos alunos; procuram estabelecer processos de avaliação personalizados dos professores e, com isso,
controlar as ênfases de formação que desejam, além de controlar igualmente as agências formadoras;
querem controlar a formação do professor difundindo que ela é muito teórica e precisa ser mais prática
colocando a formação numa perspectiva pragmatista; apostilam as redes de forma a controlar o conteúdo
que é passado para os estudantes, bem como a sua forma; enfatizam a formação do gestor de forma a
torná-lo um controlador dos profissionais da educação no interior da escola responsabilizando-o pelos
resultados esperados nos testes; favorecem processos de privatização de forma a abrir mercado e a colocar
a educação diretamente sob controle do empresariado que atua no mercado educacional (gestão por
concessão e vouchers); provocam o sentimento de que a educação está em crise e que o direito à
aprendizagem está em jogo como forma de sensibilizar a população, através da mídia, para suas soluções
miraculosas; centram a concepção da qualidade da educação nas notas altas, estabelecendo uma identidade
entre notas altas (às vezes em uma ou duas disciplinas que mais lhe interessam) e qualidade da educação;
reduzem a formação da juventude à ideia de direito à aprendizagem, estreitando a concepção de educação
e reduzindo-a à aprendizagem no interior da escola; fortalecem os processos de aprendizagem que isolam
a criança da vida e, portanto, das contradições sociais existentes na vida, difundindo a meritocracia como
base explicativa do funcionamento social; exercitam processos meritocráticos com alunos, professores e
gestores que ajudam a fixar a meritocracia como forma de progredir na vida via empreendedorismo;
desmoralizam o magistério como forma de fragilizar a sua articulação política e apresentam os sindicatos
como responsáveis pelo atraso da educação, defensores dos direitos dos professores e não defensores do
direito de aprender do aluno; desenvolvem processos de avaliação em larga escala censitários com a
finalidade de alavancar processos de responsabilização da escola ignorando os fatores sociais que
dificultam a ação da escola; propõem e influenciam a elaboração de leis que responsabilizem as escolas e
os gestores; financiam fortemente as suas ideias via fundações e iniciativa privada; ampliam o tempo 53 Debate Debate escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. Focando no direito à aprendizagem tenta-se apagar a importância de outros direitos que são
fundamentais para o exercício do direito à educação: o direito à alimentação, o direito à habitação, ao
trabalho, à moradia, à renda, etc. Não há como defender um direito isolado dos outros, pois um depende
do outro como mostram os estudos que correlacionam desempenho na escola e nível socioeconômico. Os
testes não medem só aprendizagem, medem simultaneamente nível socioeconômico. Com isso, causas sociais são camufladas em causas escolares via avaliações de larga escala
baseadas em testes. A sociedade menos avisada, pelo menos a princípio, acredita. Há quem proponha que
placas com o Índice de Desenvolvimento da Educação Básica (Ideb) devem ser levantadas nas portas das
escolas para denunciar a falta de qualidade e a identificação dos culpados: os professores. 54 erminal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Debate O proclamado direito à educação vira direito à aprendizagem e nos limites da escola, para em
seguida virar direito ao básico, limitado à aprendizagem de leitura e matemática. Transmutado em direito à
aprendizagem, ficam igualmente de fora todas as outras dimensões da formação que não seja a cognitiva,
privilegiadamente leitura e matemática, e as demais disciplinas e áreas de formação assumem formas
aligeiradas (por exemplo, projetos, áreas) onde o conteúdo é secundarizado para que o aluno possa focar
na aprendizagem de leitura e matemática, ou seja, as disciplinas que caem nas provas. As práticas escolares não valorizam as artes, a afetividade, o desenvolvimento do corpo, da
criatividade entre outros aspectos que favorecem exatamente os processos de criação que são básicos para
a implementação de inovações. Como advertem os estudos, são estas as características que devem ser
fortalecidas se queremos ser competitivos internacionalmente, já que a capacidade de inovar, de criar é que
define a posição dos países no cenário internacional. Como afirma Levin (2012)18: Em todo o mundo ouvimos falar bastante sobre a criação de escolas de classe mundial. Normalmente, o termo refere-se a escolas cujos alunos recebem pontuações muito
elevadas em comparações internacionais de desempenho de estudantes como o PISA
ou o TIMSS. A prática de restringir o significado de escolas modelos ao critério estreito
de pontuação de desempenho é normalmente premissa da visão de que os resultados
dos testes estão intimamente ligados à formação de uma força de trabalho capaz e a
uma economia competitiva. escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. Na verdade, as relações entre os resultados medidos em
testes e os ganhos de produtividade são modestos e explicam uma parcela relativamente
pequena da maior ligação entre nível educacional e os resultados econômicos. O que é
omitido em tais avaliações estreitas são os efeitos que a educação tem sobre o
desenvolvimento das capacidades e habilidades interpessoais e intrapessoais e que
afetam a qualidade e a produtividade da força de trabalho [...] a busca por escolas de
classe mundial deve abranger uma série de características do desenvolvimento humano
que se estendem muito além de resultados dos testes. As consequências desta pressão sobre o sistema escolar baseada em responsabilização
(accountability) estão bastante documentadas na literatura internacional19. 1. Estreitamento curricular. Avaliações geram tradições. Dirigem o olhar de professores,
administradores e estudantes. Se o que é valorizado em um exame são leitura e matemática, a isso eles
dedicarão sua atenção privilegiada, deixando os outros aspectos formativos de fora20. 1. Estreitamento curricular. Avaliações geram tradições. Dirigem o olhar de professores,
administradores e estudantes. Se o que é valorizado em um exame são leitura e matemática, a isso eles
dedicarão sua atenção privilegiada, deixando os outros aspectos formativos de fora20. 2. Competição entre profissionais. A colocação dos profissionais de educação em processos de
competição entre si e entre escolas levará à diminuição da possibilidade de colaboração entre estes. A
educação, entretanto, tem que ser uma atividade colaborativa. A ação de um professor, não se esgota
apenas no tempo em que ele passa com o aluno. Afeta outros professores, pois o aluno é o mesmo. Se um
deles destrói a autoestima do aluno, todos serão atingidos por este fato. 2. Competição entre profissionais. A colocação dos profissionais de educação em processos de
competição entre si e entre escolas levará à diminuição da possibilidade de colaboração entre estes. A
educação, entretanto, tem que ser uma atividade colaborativa. A ação de um professor, não se esgota
apenas no tempo em que ele passa com o aluno. Afeta outros professores, pois o aluno é o mesmo. Se um
deles destrói a autoestima do aluno, todos serão atingidos por este fato. 3. Pressão sobre o desempenho dos alunos e preparação para os testes. Premidos pela necessidade de
assegurar um salário variável na forma de bônus, os professores pressionarão seus alunos aumentando a
tensão entre estes. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014.
55 escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. Premidos pela necessidade de apresentar sua escola como uma boa escola à
comunidade, reproduzirão práticas que tenderão a afastar de suas salas e de suas escolas alunos com
dificuldades para a aprendizagem. Além disso, proliferam os simulados e a utilização do tempo escolar
para preparar o aluno para os testes21. 55 55 Debate 4. Fraudes. Por esta mesma linha de pressão, chega-se à fraude. As variáveis que afetam a
aprendizagem do aluno não estão todas sobre controle do professor e nem as mais relevantes podem estar
sob seu controle. Esta realidade produz um sentimento de impotência que associada à necessidade de
sobreviver tem levado à fraude. Multiplicam-se os casos de ajuda do próprio professor durante a
realização de exames, quando não a simples alteração da nota obtida pelo aluno em exames22. 5. Aumento da segregação socioeconômica no território. Estudo do Centro de Estudos e Pesquisas em
Educação, Cultura e Ação Comunitária (Cenpec) mostra que com a pressão por desempenho, as escolas
podem especializar-se em determinadas clientelas de estudantes, sendo deixadas no conjunto do território
para a destinação de alunos de baixo desempenho23. As escolas vão travando a entrada de alunos de risco
e dirigindo-as a outras escolas. 5. Aumento da segregação socioeconômica no território. Estudo do Centro de Estudos e Pesquisas em
Educação, Cultura e Ação Comunitária (Cenpec) mostra que com a pressão por desempenho, as escolas
podem especializar-se em determinadas clientelas de estudantes, sendo deixadas no conjunto do território
para a destinação de alunos de baixo desempenho23. As escolas vão travando a entrada de alunos de risco
e dirigindo-as a outras escolas. 6. Aumento da segregação socioeconômica dentro da escola. Não é diferente dentro das escolas. Estas
serão levadas a fazer turmas de estudantes que se destaquem no desempenho para que “segurem” a média
da escola e o acesso a benefícios. Os alunos com dificuldades vão ser segregados em turmas separadas. A
experiência americana não revela que houve uma maior equidade, por exemplo, entre os desempenhos
médios dos negros e brancos24. 7. Precarização da formação do professor. O apostilamento das redes contribui para que o professor
fique dependente de materiais didáticos estruturados retirando dele a qualificação necessária para fazer a
adequação metodológica segundo requer cada aluno25. Além disso, uma visão pragmatista cada vez mais se
instala nas agências formadoras do professor, diminuindo sua formação aos aspectos práticos das
metodologias. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Referências: ADRIÃO, T. et al. Uma modalidade peculiar de privatização da educação pública: a aquisição de sistemas
de ensino por municípios paulistas. Educação e Sociedade, Campinas, v. 30, n. 108, p. 799-818, 2009. ADRIÃO, T. et al. Uma modalidade peculiar de privatização da educação pública: a aquisição de sistemas
de ensino por municípios paulistas. Educação e Sociedade, Campinas, v. 30, n. 108, p. 799-818, 2009. AU, W. High-stakes testing and curricular control: a qualitative metasynthesis. Educational Research,
Washington, v. 36, n. 5, p. 258-267, 2007. AU, W. High-stakes testing and curricular control: a qualitative metasynthesis. Educational Research,
Washington, v. 36, n. 5, p. 258-267, 2007. ______. Unequal by design: high-stakes testing and the standardization of inequality. New York: Routledge,
2009. BOWERS, M. J.; WILSON, R. E.; HYDE, R. L. Atlanta Public Schools: investigative report (v. 1-3). Atlanta:
Office of the Governor, jun. 2011. Disponível em:
<http://www.calameo.com/books/0001070442388e8a1b081>. Acesso em: 28 jan. 2011. BOWERS, M. J.; WILSON, R. E.; HYDE, R. L. Atlanta Public Schools: investigative report (v. 1-3). Atlanta:
Office of the Governor, jun. 2011. Disponível em: j
p
<http://www.calameo.com/books/0001070442388e8a1b081>. Acesso em: 28 jan. 2011. BRAUN, H.; CHUDOWSKY, N.; KOENIG, J. Getting Value Out of Value-Added: report of a workshop. Washington:
The
National
Academies
Press,
2010. Disponível
em:
<http://www.nap.edu/catalog/12820.html>. Acesso em: 07 jan. 2011. CREDO. Charter School Performance in New York City. New York: CREDO, 2010. Disponível em:
<http://credo.stanford.edu/reports/NYC%202009%20_CREDO.pdf>. Acesso em: 28 jan. 2011. ERNICA, M.; BATISTA, A. A. Educação em territórios de alta vulnerabilidade social na metrópole: um caso na
periferia de SãoPaulo (Informe de Pesquisa n. 3). São Paulo: CENPEC, 2011. FRANKENBERG, E.; SIEGEL-HAWLEY, G. S.; WANG, J. (2011) Choice without equity: charter
school segregation. Education Policy Analysis Archives, Tempe, v. 19, n. 1, p. 1-96, 2011. FREITAS, L. C. de. Os reformadores empresariais da educação: da desmoralização do magistério à
destruição do sistema público de educação. Educação e Sociedade, Campinas, v. 33, n. 119, p. 379-404, 2012. GUISBOND, L.; NEILL, M.; SCHAEFFER, B. A década de progresso educativo perdida sob a NCLB:
que lições tirar desse fracasso político? Educação e Sociedade, Campinas, v. 33, n. 119, p. 405-430, 2012. LEUNG, R. The “Texas Miracle”. 22 ago. 2004.Disponível em:
<http://www.cbsnews.com/stories/2004/01/06/60II/main591676.shtml>. Acesso em: 22 fev. 2011. MARSH, J. et al. A big apple for educators: New York City’s Experiment with Schoolwide Performance
Bonuses. Santa Monica: Rand Education, 2011. Disponível em:
<http://www.rand.org/pubs/monographs/MG1114.html>. Acesso em: 28 jan. 2011. NEAL, D.; SCHANZENBACH, D. W. Ela conclui que os exames e avaliações institucionalizaram: [...] não apenas como lidamos com dados, mas também, e mais importante, o que conta
como dado. A lei [NCLB29] exige que as escolas dependam de base científica, de
investigação, mas, como se vê, estudos de caso, etnografias, entrevistas e outras formas
de pesquisa qualitativa parecem cair fora desta definição - e, portanto, são considerados
inaceitáveis, como base para a tomada de decisões. [...] Nossa fé cega em números
acabou causado empobrecimento em como (e quais) informações são usadas para
ajudar a resolver problemas do mundo real. Nós agora aparentemente acreditamos que
os números não são apenas necessários, mas suficientes para as decisões baseadas em
pesquisa (p. 1, grifos da autora).30 As consequências destas políticas estão claras na literatura internacional, a pressa dos
empresários em resolver seus problemas de rentabilidade poderá nos levar a uma década perdida na
educação brasileira. escolar destinado a ensino à distância on line nas escolas como forma de melhor estabelecer controle sobre
o ensino. ONG como a Teach For América, nos Estados Unidos, formam professores em cinco
semanas. O braço internacional desta organização faz ensaios no Rio de Janeiro26. 8. Destruição moral do professor e do aluno. As pressões sobre o professor terminam obrigando-o a
segregar os alunos que estão nas pontas dos desempenhos (mais altos e mais baixos) e concentrar-se no
centro, em especial naqueles que estão próximos da média, para não caírem abaixo dela e para subirem
acima dela. Esta concentração em torno da média penaliza seriamente os mais necessitados27. As pressões
também vão segregando os professores28. Estas consequências resultam em um ataque frontal ao protagonismo dos professores na sala de
aula, nas escolas e na própria condução da escola pública. O magistério é submetido a um controle
refinado. Estas são as consequências mais recorrentes das políticas educacionais dos reformadores
empresariais. Evitando o debate qualitativo e a análise mais profunda, elas tentam se legitimar pela
apresentação de uma grande quantidade de “números”. Para Quintero (2012), há um desejo insaciável dos políticos por dados. Ela escreve: Em um nível básico, [os dados] parecem sinalizar uma orientação geral para a tomada
de decisões com base na melhor informação que temos, o que é uma coisa muito boa. Mas há dois problemas aqui. Primeiro, tendem a ter uma visão extremamente estreita
da informação que é relevante, isto é, [focam] dados que podem ser quantificados
facilmente; e segundo lugar, parece que estamos operando sob a ilusão de que os dados,
em si mesmos, podem contar histórias e revelar a verdade (p. 1). 56 56 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Debate QUINTERO, E. The data-driven education movement. Shanker Blog, Washington, 22 out. 2012.
Disponível em: <http://shankerblog.org/?p=7015>. Acesso em: 28 jan. 2011. QUINTERO, E. The data driven education movement. Shanker Blog, Washington, 22 out. 2012. Disponível em: <http://shankerblog.org/?p=7015>. Acesso em: 28 jan. 2011. RAVITCH, D. The death and life of the great American school system. 2. Ed. Rev. Amp. New York: Brasic
Books, 2011. RAVITCH, D. The death and life of the great American school system. 2. Ed. Rev. Amp. New York: Brasic
Books, 2011. ______. “Nota mais alta não é educação melhor. Estadão, São Paulo, 02 ago. 2010a. Disponível em:
<http://www.estadao.com.br/noticias/impresso,nota-mais-alta-nao-e-educacao-melhor,589143,0.htm>. Acesso em: 28 jan. 2011. ______. “Nota mais alta não é educação melhor. Estadão, São Paulo, 02 ago. 2010a. Disponível em:
<http://www.estadao.com.br/noticias/impresso,nota-mais-alta-nao-e-educacao-melhor,589143,0.htm>. Acesso em: 28 jan. 2011. ______. New York education officials are lying to the state’s schoolkids. 2010b. Disponível em:
<http://www.nydailynews.com/opinions/2010/03/31/2010-03- ______. New York education officials are lying to the state’s schoolkids. 2010b. Disponível em:
<http://www.nydailynews.com/opinions/2010/03/31/2010-03-
31
k
d
i
ffi i l
l i
h
lkid h
l#i
0
XGNI U
A ______. New York education officials are lying to the state’s schoolkids. 2010b. Disponível em:
<http://www.nydailynews.com/opinions/2010/03/31/2010-03-
31_new_york_state_education_officials_are_lying_to_schoolkids.html#ixzz0npXGNIgU>. Acesso em:
20 j l 2010 p
y
y
p
31_new_york_state_education_officials_are_lying_to_schoolkids.html#ixzz0npXGNIgU>. Acesso em:
20 jul. 2010. 31_new_york_state_education_officials_are_lying_to_schoolkids.html#ixzz0npXGNIgU>. Acesso em:
20 jul. 2010. ROTHSTEIN, R. “A Nation at Risk” Twenty-Five Years Later. Cato Unbound, Washington, 07 abr. 2008. Disponível em: <http://www.cato-unbound.org/2008/04/07/richard-rothstein/a-nation-at-risk-twenty-
five-years-later/>. Acesso em: 25 jan. 2011. SETUBAL, M. A. Os melhores professores para as piores escolas. Folha de São Paulo, São Paulo, p. 3, 26
abr. 2012. SETUBAL, M. A. Os melhores professores para as piores escolas. Folha de São Paulo, São Paulo, p. 3, 26
abr. 2012. TUCKER, C. Beverly Hall needs to retire. ago. 2010. Disponível em: <http://blogs.ajc.com/cynthia-
tucker/2010/08/26/beverly-hall-needs-to-retire/>. Acesso em: 29 jan. 2011. ZASTROW, C. von. In Teachers We Trust: An Interview with Finnish Education Expert Reijo
Laukkanen. Learning First Alliance, Alexandria, 29 set. 2008. Disponível em:
<http://www.learningfirst.org/teachers-we-trust-interview-finnish-education-expert-reijo-laukkanen>. Acesso em: 28 jan. 2011. Referências: Left Behind by Design: proficiency counts and test-based
accountability. Review of Economics and Statistics, Cambridge, v. 92, n. 2, p. 263-283, mai. 2010. NICHOLS, S. L.; BERLINER, D. C. Collateral Damage: how high-stakes testing corrupts America’s
schools. Cambridge: Harvard Educational Press, 2007. 57 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Debate Notas: 1 Professor Titular da Faculdade de Educação da UNICAMP. E-mail: freitas.lc@uol.com.br. 2 Cf. disponível em: <http://www.oeconomista.com.br/cresce-numero-de-mulheres-no-mercado-de-trabalho-diz-dieese/>. Acesso em: 30 abr. 2014. 3
Cf. disponível
em:
<http://www1.folha.uol.com.br/mercado/2013/08/1324745-aumenta-o-registro-de-trabalhadores-
imigrantes-em-sp.shtml>. Acesso em: 30 abr. 2014. 4 Cf. em disponível em: <http://economia.estadao.com.br/noticias/economia-geral,produtividade-do-brasil-tem-queda-
dramatica-diz-estudo,144537,0.htm>. Acesso em: 30 abr. 2014. 5 Ravitch (2010a). 6 Cf. disponível em: <http://veja.abril.com.br/noticia/educacao/ensino-medio-brasileiro-era-ruim-e-esta-pior>. Acesso em: 30
abr. 2014. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. 6 Cf. disponível em: <http://veja.abril.com.br/noticia/educacao/ensino-medio-brasileiro-era-ruim-e-esta-pior>. Aces
abr. 2014. 7
Cf. disponível
em:
<http://oglobo.globo.com/economia/se-nao-for-neste-momento-nao-se-colhe-mais-diz-demografo-
9465694#ixzz2bfVgxFjS>. Acesso em: 05 mai. 2014. 8 Cf. disponível em: <http://www.lidebr.com.br/>. Acesso em: 05 mai. 2014. 8 Cf. disponível em: <http://www.lidebr.com.br/>. Acesso em: 05 mai. 2014. 9 Cf. disponível em: <http://www.todospelaeducacao.org.br/>. Acesso em: 05 mai. 2014. 10 Cf. disponível em: <http://avaliacaoeducacional.com/2013/08/10/quem-ganha-com-a-responsabilizacao/>. Aces
abr. 2014. 11 Sistema em que empresas privadas de gestão assumem a escola e a administram recebendo financiamento público para tal. 12 Sistema em que os pais recebem um cheque que cobre as despesas escolares de seus filhos e escolhem a escola que quiserem
para educar seus filhos. 13 Cf. disponível em: <http://avaliacaoeducacional.com/2013/08/08/charters-significativamente-insignificantes/>. Acesso em:
30 abr. 2014; CREDO (2010) e Marsh e colaboradores (2011). 14 Cf. Freitas (2012). Cf. Guisbond, Neill e Schaeffer (2012). 58 Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. Germinal: Marxismo e Educação em Debate, Salvador, v. 6, n. 1, p. 48-59, jun. 2014. 58 Debate Recebido em: 06/2014
Publicado em: 12/2014. 15 Cf. disponível em: <http://dianeravitch.net/2013/08/12/chile-the-most-pro-market-school-system-in-the-world-part-1/>.
Acesso em: 05 mai. 2014. 15 Cf. disponível em: <http://dianeravitch.net/2013/08/12/chile-the-most-pro-market-school-system-in-the-world-part-1/>. Acesso em: 05 mai. 2014. 16 A OCDE é uma organização com sede na Europa nascida depois da segunda guerra mundial, tendo como pano de fundo o
Plano Marshall de reconstrução da Europa. Passou por várias reformulações e tem hoje a função de monitorar as condições de
operação dos países considerados ricos (cerca de 35) e indicar ações de cooperação econômica entre eles. No caso da realização
de avaliações internacionais aceita que participe alguns países mesmo não sendo considerados do clube dos ricos. O Plano Nacional de Educação no Brasil tem incorporado nele as metas do PISA. 17 O Plano Nacional de Educação no Brasil tem incorporado nele as metas do PISA. disponível em: <http://roundtheinkwell.files.wordpress.com/2012/09/more-than-just-test-scores-sept2012-2.pdf>. Aces
05 mai. 2014. 19 Este resumo também apareceu na Revista Educação e Sociedade, n. 119, e é atualizado com novas pesquisas que
disponibilizadas. e resumo também apareceu na Revista Educação e Sociedade, n. 119, e é atualizado com novas pesquisas que vão sen
onibilizadas. 20 Cf. Au (2007; 2009). 21 Cf. Nichols e Berliner (2007) e Braun, Chudowsky e Koenig (2010). 22 Cf. Tucker (2010), Bowers, Wilson e Hyde (2011), Leung (2004) e Ravitch (2010b). 22 Cf. Tucker (2010), Bowers, Wilson e Hyde (2011), Leung (2004) e Ravitch (2010b). 23 Cf. Ernica e Batista (2011). 24 Cf. Rothstein (2008) e Frankenberg, Siegel-Hawley e Wang (2011). 24 Cf. Rothstein (2008) e Frankenberg, Siegel-Hawley e Wang (2011). 25 Cf. Adrião (2009). 26 Ensina! No mundo (20 de abril de 2012). Disponível em: <http://www.ensina.org.br/ensina/no-mundo/>. Acesso em: 05
mai. 2014. 26 Ensina! No mundo (20 de abril de 2012). Disponível em: <http://www.ensina.org.br/ensina/no-mundo/>. Acesso em: 05
mai. 2014. 27 Cf. Neal e Schanzenbach (2010). 27 Cf. Neal e Schanzenbach (2010). 28 Cf. Setubal (2012) e Zastrow (2008). 29 Lei de responsabilidade educacional americana. 30 Cf. Quintero (2012). 30 Cf. Quintero (2012). 30 Cf. Quintero (2012). Recebido em: 06/2014
Publicado em: 12/2014. 59
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https://openalex.org/W2950192628
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English
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A Single Nucleotide Resolution Model for Large-Scale Simulations of Double Stranded DNA
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bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
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1 Introduction 1 of outstanding biological problems, and also to assist the advance
of DNA-based nanotechnology. Since the discovery of the structure of the deoxyribonucleic acid
(DNA),1–3 the geometry of the double-helix and its topological
implications
have
engaged
and
fascinated
the
scientific
community.4,5 It is becoming more and more evident that not
only is the genetic information encoded in the DNA sequence of
primary importance, but also that changes in its three-
dimensional structure can alter crucial biological functions,
such as gene expression and replication.6–10 At the same time,
the rapid improvement of techniques using DNA functionalised
colloids,11,12
DNA-origami13
and,
more
generally,
supra-
molecular DNA assembly14 is setting new standards for DNA-
based nano-technology. This has far-reaching applications,
ranging from materials science (to create new DNA-based and
possibly biomimetic materials), to medicine (to be used in, e.g.,
gene-therapy and drug delivery). In light of this, the formulation
of accurate theoretical and computational models that can
efficiently capture the behaviour of DNA, either in vivo or
in vitro, is of great importance in order to understand a number Several fully atomistic models for double-stranded (ds) DNA
are available in the literature.15–17 While these give an accurate
description of the dynamics of DNA molecules and their
interaction with single proteins, the complexity of the all-atom
approach places severe limits on the size (up to about a hundred
base-pairs) and time scales (of the order of ms) which can be
probed.18 Coarse-graining, where large collections of atoms or
molecules are represented by single units, allows larger systems
to be simulated for longer at the expense of molecular detail. One of the most challenging aspects in designing a computational
model is to retain the key microscopic details necessary to answer
a given question while ‘‘trimming’’ the rest. At the large scale limit,
entire eukaryotic chromosomes can be modelled using simple
bead-and-spring polymer models,7,19 where each monomer can
represent up to 3000 base-pairs (bp) and the simulated time can
reach time-scales spanning minutes19 or even several hours;20
similar chains of beads can also be used to model naked DNA,
though clearly such an approach neglects microscopic details such
as the base-pair specificity or the double-stranded structure. While in some cases these models can still capture the essential
physics,21 in others they are only a crude approximation of
the real systems. a School of Physics and Astronomy, University of Edinburgh,
Peter Guthrie Tait Road, Edinburgh EH9 3FD, Scotland, UK
b Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit,
University of Edinburgh, Western General Hospital, Crewe Road,
Edinburgh EH4 2XU, UK
c EPCC, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD,
Scotland, UK Soft Matter View Article Online
View Journal | View Issue Y. A. G. Fosado,a D. Michieletto,a J. Allan,b C. A. Brackley,a O. Henrichac and
D. Marenduzzo*a Y. A. G. Fosado,a D. Michieletto,a J. Allan,b C. A. Brackley,a O. Henrichac and
D. Marenduzzo*a The computational modelling of DNA is becoming crucial in light of new advances in DNA nano-
technology, single-molecule experiments and in vivo DNA tampering. Here we present a mesoscopic
model for double stranded DNA (dsDNA) at the single nucleotide level which retains the characteristic
helical structure, while being able to simulate large molecules – up to a million base pairs – for time-scales
which are relevant to physiological processes. This is made possible by an efficient and highly-parallelised
implementation of the model which we discuss here. The model captures the main characteristics of DNA,
such as the different persistence lengths for double and single strands, pitch, torsional rigidity and the
presence of major and minor grooves. The model constitutes a starting point for the future implementation
of further features, such as sequence specificity and electrostatic repulsion. We show that the behaviour of
the presented model compares favourably with single molecule experiments where dsDNA is manipulated
by external forces or torques. We finally present some results on the kinetics of denaturation of linear DNA
and supercoiling of closed dsDNA molecules. Received 12th August 2016,
Accepted 8th November 2016
DOI: 10.1039/c6sm01859a
www.rsc.org/softmatter Received 12th August 2016,
Accepted 8th November 2016
DOI: 10.1039/c6sm01859a www.rsc.org/softmatter This journal is ©The Royal Society of Chemistry 2016 Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The faded red spheres represent the steric interaction volume of the
red beads. Neither the interacting beads nor the ghost beads along the blue
chain are shown to ease visualisation. g
Motivated by this goal, in this paper we introduce a single
nucleotide resolution coarse-grained model for dsDNA which
retains several biologically-relevant DNA features, while being
capable of delivering large-scale simulations. The model is
implemented in the LAMMPS molecular dynamics engine30
which allows us to comfortably study molecules on the order
of thousands of bp (kbp). Because the code is fully parallel and
highly scalable, it is portable to supercomputers to reach the
length and time scales needed for some of the biological applications
just mentioned. The scope of this work is to present the construction
of the model, starting from the known geometry of DNA4 (Section 2),
and to discuss the validation of its main physical features, i.e. helical pitch, persistence length and torsional rigidity (Section 3). These properties are traditionally addressed via single-molecule
experiments31,32 in vitro, and we here provide an indirect validation
via simulated single-molecule experiments, obtaining a remark-
ably good agreement with the experimentally observed values
(Section 4). Finally, we present an application of this model to
the dynamics of DNA denaturation, and discuss further future
applications. These range from the study of DNA denaturation
to that of supercoil dynamics in the presence of topological
proteins (Section 6). The flexibility of the model and the scalability
provided by the LAMMPS engine means it provides a solid frame-
work on which to base further studies of the topological properties
of DNA and DNA–protein interactions. hereafter, and shown in blue), while the other represents the
nitrogenous base (‘‘patch’’ hereafter, shown in pink), and is
placed at a distance of 0.5 nm from the bead centre. Beads have
an excluded volume so that they cannot overlap, whereas patches
have no associated excluded volume. In order to see the resolution
of the system, a nucleotide structure lying inside the bead is shown
in black in Fig. 1(a). Depending on the relevant features of the system to be
modelled, a second patch representing the phosphate group
may also be included explicitly to more accurately represent the
DNA steric hindrance. 1 Introduction Several successful mesoscopic models have
recently been proposed which aim at bridging the gap between
the ‘‘all-atom’’ and ‘‘bead-spring’’ limits.22–25 Nevertheless, a
coarse grained model able to retain the necessary physical
microscopic details while allowing simulations of the several 9458 | Soft Matter, 2016, 12, 9458--9470 This journal is ©The Royal Society of Chemistry 2016 Paper Fig. 1
(a) The level of coarse-graining of the model is here summarised by
encapsulating the atoms forming one nucleotide into one bead-patch
complex. The small yellow sphere represents the position of the phosphate
with respect to the complex, while the pink sphere denotes the position of
the hydrogen bond between bases. The blue sphere approximates the
excluded volume of the nucleotide. (b) This panel shows the main inter-
action sites between consecutive beads in the same strand. The equilibrium
distance between patches (E–F) is set to 0.34 nm while the one between
beads centres (A–B) to 0.46 nm. This leads to an equilibrium distance of
0.7 nm between the external edge of the backbone (C–D). These distances
are set so that the correct pitch of 10 bp is recovered. (c) Two nucleotides
are bonded via a breakable harmonic spring. Their distance is set so that the
full chain thickness is around 2 nm, as that of B-DNA. (d) Representation of
the double-stranded DNA model. The red chain also shows the beads
which interact sterically (solid red) as well as the phantom beads (solid
grey). The faded red spheres represent the steric interaction volume of the
red beads. Neither the interacting beads nor the ghost beads along the blue
chain are shown to ease visualisation. tens or hundreds of kilo-base pairs (kbp) that would be needed
to address many biologically relevant questions, is still currently
needed. Examples of biological processes for which such a mesoscopic
approach would be highly valuable can be classified in two broad
categories: processes where DNA is mechanically manipulated
by enzymatic machines (for example during replication or
transcription which require opening of the double-helical
structure), or processes where interactions between DNA and
proteins depend more subtly on the topological and geometrical
properties of the double-helix. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. When this second patch is not included,
we imply that the phosphate is sitting 0.5 nm from the bead
centre but slightly away from the antipodal point to the patch,
marked in yellow in Fig. 1(a) for clarity. Each bead-patch complex thus represents a single nucleotide,
and acts as a rigid body; we connect a chain of these bodies via
FENE bonds of length dbp = 0.46 nm between the beads to
represent one strand of DNA. We set the distance between two
consecutive patches along the strand (E–F in Fig. 1(b)) at 0.34 nm
by means of a Morse potential; the difference between the lengths This journal is ©The Royal Society of Chemistry 2016 1 Introduction An example of the latter class of
problems is the so-called ‘‘linking number paradox’’, where it
has been observed that the unbinding of DNA from a nucleo-
some releases only one unit of linking number, rather than the
1.7 units of writhe which were stored;26,27 the resolution of the
paradox is that the nucleosome also stores some twist (the terms
twist and writhe are explained below). To complicate the picture
even more, there are several proteins which operate to alter the
DNA topology, whose collective actions may sometimes trigger
complex feedback mechanisms that are crucial for biological
functions.28,29 For a model to be applicable to such problems, it
must possess both a good accuracy in mimicking the geometry
of the double-helix, and the ability to consider long molecules
on which many proteins may act simultaneously, so that cooperative
effects can be investigated. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1
(a) The level of coarse-graining of the model is here summarised by
encapsulating the atoms forming one nucleotide into one bead-patch
complex. The small yellow sphere represents the position of the phosphate
with respect to the complex, while the pink sphere denotes the position of
the hydrogen bond between bases. The blue sphere approximates the
excluded volume of the nucleotide. (b) This panel shows the main inter-
action sites between consecutive beads in the same strand. The equilibrium
distance between patches (E–F) is set to 0.34 nm while the one between
beads centres (A–B) to 0.46 nm. This leads to an equilibrium distance of
0.7 nm between the external edge of the backbone (C–D). These distances
are set so that the correct pitch of 10 bp is recovered. (c) Two nucleotides
are bonded via a breakable harmonic spring. Their distance is set so that the
full chain thickness is around 2 nm, as that of B-DNA. (d) Representation of
the double-stranded DNA model. The red chain also shows the beads
which interact sterically (solid red) as well as the phantom beads (solid
grey). The faded red spheres represent the steric interaction volume of the
red beads. Neither the interacting beads nor the ghost beads along the blue
chain are shown to ease visualisation. Fig. 1
(a) The level of coarse-graining of the model is here summarised by
encapsulating the atoms forming one nucleotide into one bead-patch
complex. The small yellow sphere represents the position of the phosphate
with respect to the complex, while the pink sphere denotes the position of
the hydrogen bond between bases. The blue sphere approximates the
excluded volume of the nucleotide. (b) This panel shows the main inter-
action sites between consecutive beads in the same strand. The equilibrium
distance between patches (E–F) is set to 0.34 nm while the one between
beads centres (A–B) to 0.46 nm. This leads to an equilibrium distance of
0.7 nm between the external edge of the backbone (C–D). These distances
are set so that the correct pitch of 10 bp is recovered. (c) Two nucleotides
are bonded via a breakable harmonic spring. Their distance is set so that the
full chain thickness is around 2 nm, as that of B-DNA. (d) Representation of
the double-stranded DNA model. The red chain also shows the beads
which interact sterically (solid red) as well as the phantom beads (solid
grey). Soft Matter, 2016, 12, 9458--9470 | 9459 Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. While the pitch of the chain is set by the ratio of the base
pairing distance and the distance between successive phosphate
groups on a DNA strand, the right-handedness is imposed using
a dihedral potential between the quadruplets of monomers
forming two consecutive nucleotides (A, E, F and B in Fig. 1(b)). This potential regulates the angle between the planes A–E–F and
E–F–B. The minimum of this potential is set equal to 361, so as to
match the geometry of a regular dsDNA helix. Fig. 2
An example of an equilibrated configuration of a 1000 bp double-
stranded DNA molecule, as simulated with the model presented in Section 1. In order to limit the splay of consecutive nucleotides (also
called ‘‘roll’’4) we used a stiffharmonic potential so as to keep
the angle between particles E, F and B (two patches and one
bead) at 901 (Fig. 1(b)). This interaction imposes the planarity
between consecutive bases in the same strand. Finally, the last
ingredient of this model is a Kratky–Porod potential regulating
the angle between three consecutive patches along one strand. This allows us to finely regulate the chain stiffness. that the model (as presented up to now) does not include. Some
notable examples are: (i) the distinction between minor and
major groove; (ii) the description of electrostatic effects due to
charges on the DNA, and to the variation of the density of
counter-ions in solution (our parameters are tuned assuming
room temperature and a physiological buffer, 0.15 M NaCl, see
Appendix A); and (iii) the effect of sequence heterogeneity, or
sequence specificity (in this simplest description, our dsDNA
is viewed as a homopolymer). Such effects will be important,
for instance, when one needs to more faithfully describe inter-
actions between DNA and DNA-binding proteins. It is in principle
possible to include these effects in a modular fashion in our
framework, and in the Discussion (see also Appendix B) we describe
how we can account for (i) in a simple way, and how (ii) and (iii)
might be implemented in the future. The excluded volume around each bead depicted in Fig. 1(d)
(faded red spheres) has diameter 1 nm. Since we use spherical
beads rather than asymmetrically shaped ones (this is important for
the speed of the algorithm), the geometry of the double-strand
depicted in Fig. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1(b and d) would involve a large degree of over-
lapping which would lead to a large steric repulsion. To avoid this
we consider two types of beads in each strand: sterically interacting
beads (shown as small solid red spheres for one strand in Fig. 1(d))
are intercalated by two ghost beads (depicted as small grey spheres)
which do not interact sterically along the same strand but they do
interact with all the beads on the complementary strand with an
excluded volume of 0.5 nm. This choice ensures that only non-
overlapping beads sterically interact with one another. In addition,
this allows us to preserve the correct thickness of the chain (2 nm
for B-DNA), to maintain the desired distance between contiguous
nucleotides and avoid the strands crossing through one another. In
Fig. 2 we show a typical equilibrated configuration using the
presented model for a 1000 bp molecule. It is worth mentioning that in the current version of the
model, hydrogen bonding is the only interaction responsible
for holding the two DNA strands together. The rest of the
potentials are defined independently for each strand. As a
consequence, when the model was tested for single stranded
DNA (ssDNA), and the persistence length was computed, its value
(30 nm) was higher than expected from experiments33 (E1–2 nm). To model ssDNA and reproduce this dramatic change in flexibility,
we set that, once the hydrogen bond keeping the two strands
together breaks, both the dihedral and the Kratky–Porod potentials
acting on each individual strand should be turned-off. In this way,
we effectively take into account of the larger flexibility of single
DNA strands and, in particular, we observe a persistence length of
about 1 nm (see Section 5). We should stress here that the model as presented in this
section should be thought of as a simple, starting point, which
is based on some crucial geometric constraints of double-stranded
DNA. One of the main strength of the model is the ability to deliver
large-scale simulations, which is achieved by using spherical
monomers that interact via standard potentials. These are effi-
ciently implemented in LAMMPS and ensure a highly scalable
performance in large scale parallel simulations (see Appendix A for
more details). From now on, we only show results for the model
without the explicit presence of the phosphates unless otherwise
noted. At the same time, there are several characteristics of dsDNA 9460 | Soft Matter, 2016, 12, 9458--9470 This journal is ©The Royal Society of Chemistry 2016 2 The model We start by considering a complex made of two spherical
monomers (see Fig. 1(a)), one of which represents the sugar-
phosphate backbone of the DNA (this is referred to as a ‘‘bead’’ This journal is ©The Royal Society of Chemistry 2016 Soft Matter, 2016, 12, 9458--9470 | 9459 Fig. 2
An example of an equilibrated configuration of a 1000 bp double-
stranded DNA molecule, as simulated with the model presented in Section 1. Paper
View Article Online View Article Online Soft Matter Soft Matter Paper A–B and E–F implies that the distance between the implicit
phosphates at the external edge of the beads (C–D in Fig. 1(b))
is dph = 0.7 nm. The ratio between dbp and dph is well known to
crucially regulate the correct pitch of the chain4 (for details about
the potentials used see Appendix A). Nucleotides belonging to different strands are bonded together
via breakable harmonic springs acting between two patches and
representing hydrogen bonds (see Fig. 1(c)). The equilibrium bond
distance is set to zero; if the extent of the bond increases beyond
a critical value rc = 0.3 nm, the bond breaks, modelling the
denaturation of the chain. 3.1
Persistence length The persistence length of dsDNA is a well-studied physical
property that plays an important role in the wrapping of dsDNA
around histone octamers to form the chromatin fibre, as well as
in many other biological processes. In physical terms it gives a
measure of the length-scale over which the direction of the
chain is no longer correlated with itself. Following the description
of an elastic rod by Moroz and Nelson37 one can define the
bending rigidity via the elastic energy functional Following Moroz and Nelson37 once again, we first define
the torsional stiffness of an elastic rod C via the elastic energy
functional Etors
kBT ¼ C
2
ðL
0
O3ðsÞ2ds;
(3) (3) (3) Ebend
kBT ¼ lp
2
ðL
0
dt
ds
2
ds;
(1) (1) (1) where O3(s) is the rate of rotation of a local reference frame
along the curve around the tangent t(s), defined as in the
previous section. where lp is the bending persistence length, s the arclength
parameter and t(s) = dr/ds the tangent to the chain (at s) whose
location in space is described by r(s). This quantity can also be
readily measured by computing the tangent–tangent correlator: Analogous to the measurement for the bending persistence
length via the tangent–tangent correlator, we here measure the
torsional persistence length by computing the decorrelation of the
twist angle. This correlator can be quantified by defining a local
reference frame for each base pair, and tracking the rotation of the
frames from one base pair to the next via their Euler angles. Each
local frame is specified by the tangent vector t(n) as defined above, a
normal vector f(n), defined as the projection of the vector connecting
two beads in a base-pair onto the plane perpendicular to t(n), and a
third vector v(n) = t(n) f(n), perpendicular to both t(n) and f(n). ht(s)t(s0)i = e|ss0|/lp. (2) (2) In our model, we use the position of the patches to extract the
centreline of the dsDNA molecule, where the tangent at the nth
patch at position r(n) is t(n) (r(n + 1) r(n))/|r(n + 1) r(n)|. One
can compute the tangent–tangent correlator along this curve and
obtain the persistence length by extracting the exponent of the
exponential decay. In order to avoid finite-size effects due to the
presence of ends, we neglect the two terminal segments (B5 bp
at each end). 3.1
Persistence length The resulting curve (shown in Fig. 3) is adjusted by
tuning the parameters of the model and until the exponential fit
returns a persistence length of lp C 143 7 bp, in agreement
with the experimentally observed values. The Euler angles between the frames at n and n + 1 can be
used to obtain the twist increment between those base-pairs,
and the correlation function for the total twist between m
consecutive base-pairs O(m) calculated. Since dsDNA has an
equilibrium twist angle y0 = 361 per bp, we subtract this out,
and calculate the correlation for the residual twist DO(m) =
O(m) my0. It can be in fact shown38 (see also Appendix C) that
the average cosine of the residual total twist between any two
reference frames separated by m bases exhibits an exponential
decay as: Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3
The tangent–tangent correlator ht(n)t(n0)i computed for a chain
300 bp long; it shows an exponential decay as in eqn (2) with a correlation
length lp = 143 7 bp. Points show correlations measured from the
simulations (average over time), and the line shows a fit to eqn (2). Error
bars give the standard error of the mean. This journal is ©The Royal Society of Chemistry 2016 3 Parameterisation Our model has several parameters which can be varied to
control the pitch, bending and torsional properties of the
simulated DNA molecule. Nonetheless, we are interested in 9460 | Soft Matter, 2016, 12, 9458--9470 This journal is ©The Royal Society of Chemistry 2016 Fig. 3
The tangent–tangent correlator ht(n)t(n0)i computed for a chain
300 bp long; it shows an exponential decay as in eqn (2) with a correlation
length lp = 143 7 bp. Points show correlations measured from the
simulations (average over time), and the line shows a fit to eqn (2). Error
bars give the standard error of the mean. Soft Matter
View Article Online View Article Online Soft Matter
View Article Online Paper Soft Matter modelling the B form of dsDNA, of which two main physical
properties are: the persistence length lp = 50 nm C 150 bp, and
the torsional rigidity C/kBT C 60–80 nm C 177–235 bp.34–36
Due to the interplay between the potentials presented in the
previous section, there is no simple mapping between individual
simulation parameters and the resulting physical properties;
instead we obtain a simulated molecule with the correct values
of lp and C via a systematic tuning of the parameters. In this
section we measure these properties from the microscopic
positions of the beads in equilibrated DNA molecule configurations. Then in the following section, we use the parametrised force field to
simulate single-molecule experiments, showing that the DNA
molecules show the correct macroscopic response to mechanical
manipulations. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4
The average of the cosine of the total twist angle DO(m) is
computed for a chain 300 bp long; in this figure we show the correlator
to decay exponentially as in eqn (4) with a characteristic length lt = 512
18 bp. Data points are obtained from simulations while the line is an
exponential fit. Error bars give standard error of the mean. Fig. 4
The average of the cosine of the total twist angle DO(m) is
computed for a chain 300 bp long; in this figure we show the correlator
to decay exponentially as in eqn (4) with a characteristic length lt = 512
18 bp. Data points are obtained from simulations while the line is an
exponential fit. Error bars give standard error of the mean. Fig. 5
In order to simulate single-molecule experiments the model for
dsDNA is anchored to a surface at the bottom end while being stretched
with a constant force F from the top end. We then monitor the end-to-end
elongation along the z-direction, Rz, and report its equilibrium value for a
given force in Fig. 6. so that the curve displays a characteristic decay length lt = 512
18 bp C 174 6 nm, which is consistent with experimental
estimates valid for the B-form of dsDNA. where excluded volume effects are neglected (a good approximation
when L is not much larger than lp, as in our case). In order to test if
our model can reproduce this result, we performed simulations in
which a constant pulling force directed along ez and acting on the
last base pair of the dsDNA was applied, while the other end of the
molecule was anchored at a surface (see Fig. 5). 4 Validation through single molecule
experiments Many cellular processes, such as replication and transcription,
are carried out by proteins acting on single DNA segments. In
light of this, recent years have seen an increasing interest in
experimental techniques such as optical tweezers and atomic
force microscopy, that can probe the response of DNA to
external stresses (modelling the effect of DNA-binding enzymes)
at the single-molecule level. In particular, the stretching and
twisting behaviour of DNA under external forces and torques
has been thoroughly investigated.31,34–36,39–41 The force–extension curve31 measured for a chain 300 bp
long is reported in the inset of Fig. 6 as data points, while the
solid curve is the fit to eqn (5). The fitting results in values for
both L and lp, that we can compare with the values obtained in
the previous sections and set by the parameters of our model. In particular for a 300 bp chain we obtain L = 100.3 1.7 nm Fig. 6
Force–extension curve from the simulation (data points) of two
different length chains: 300 bp (blue) and 10 kbp (red). The inset data (low
force regime) is fitted by the function in eqn (5) (solid line) and the data
above 10 pN is fitted with eqn (6). For the WLC the free parameters for the
fitting are the total polymer length L and the persistence length lp, both of
which are in agreement with the fixed parameters of the model (see text). For the EWL in addition to the previous parameters the stretching modulus
S is found. In this section we reproduce the conditions of two different
experiments, in order to test the response of our model DNA to
stretching and twisting. This also provides us with an independent
method to evaluate its persistence length and torsional rigidity. In
the following, we therefore keep the parameters of the model fixed
at the values used in the previous section, and do not further tune
them to achieve the experimentally known behaviours but simply
validate the model as it is through its response to mechanical stress. 3.2
Torsional rigidity The behaviour of DNA when twisted is regulated by its torsional
rigidity. There are several well known examples in which this
property is crucial for important biological processes, such as
transcription and gene expression.28,29 Furthermore, the high
torsional stiffness of DNA molecules implies that, when placed
under torsion, they preferentially bend, thereby creating writhe
and plectonemes.4 In order to take this feature correctly into
account, it is therefore crucial to accurately model the competition
between bending and torsional rigidities.36 hcos DO(m)i = em/2C,
(4) (4) where we define lt = 2C the characteristic torsional correlation
length. We obtain hcos DO(m)i by simulating a 300 bp long
dsDNA molecule and by averaging the value over time. The
curve obtained is shown in Fig. 4 on top of which we show the
fitted exponential. Again, we tune the parameters of the model This journal is ©The Royal Society of Chemistry 2016 Soft Matter, 2016, 12, 9458--9470 | 9461 Fig. 5
In order to simulate single-molecule experiments the model for
dsDNA is anchored to a surface at the bottom end while being stretched
with a constant force F from the top end. We then monitor the end-to-end
elongation along the z-direction, Rz, and report its equilibrium value for a
given force in Fig. 6. Paper
View Article Online View Article Online Fig. 4
The average of the cosine of the total twist angle DO(m) is
computed for a chain 300 bp long; in this figure we show the correlator
to decay exponentially as in eqn (4) with a characteristic length lt = 512
18 bp. Data points are obtained from simulations while the line is an
exponential fit. Error bars give standard error of the mean. Soft Matter Soft Matter Paper Soft Matter 4.1
Response to stretching The classic elastic response of DNA to an external stretching
force F is known to be well described by the inextensible worm-
like chain (WLC) for forces up to 10 pN. Using this framework,
the force required to induce an end-to-end distance Rz = [r(L)
r(0)]ez for a chain of length L and persistence length lp can be
approximated by:42,43 Fig. 6
Force–extension curve from the simulation (data points) of two
different length chains: 300 bp (blue) and 10 kbp (red). The inset data (low
force regime) is fitted by the function in eqn (5) (solid line) and the data
above 10 pN is fitted with eqn (6). For the WLC the free parameters for the
fitting are the total polymer length L and the persistence length lp, both of
which are in agreement with the fixed parameters of the model (see text). For the EWL in addition to the previous parameters the stretching modulus
S is found. Fig. 6
Force–extension curve from the simulation (data points) of two
different length chains: 300 bp (blue) and 10 kbp (red). The inset data (low
force regime) is fitted by the function in eqn (5) (solid line) and the data
above 10 pN is fitted with eqn (6). For the WLC the free parameters for the
fitting are the total polymer length L and the persistence length lp, both of
which are in agreement with the fixed parameters of the model (see text). For the EWL in addition to the previous parameters the stretching modulus
S is found. Flp
kBT ¼ Rz
L þ
1
4 1 Rz
L
2 1
4;
(5) (5) This journal is ©The Royal Society of Chemistry 2016 9462 | Soft Matter, 2016, 12, 9458--9470 Fig. 7
The model DNA is anchored to a surface at the bottom end while
being stretched with a constant force F, and a torque C is applied at the top
end. We then monitor the linking number and report its equilibrium value
for a given torque. With this information is possible to compute the
superhelical density. Soft Matter
View Article Online View Article Online Paper (which gives a bp step size of 0.33 0.01 nm) and lp = 47
2 nm C 140 7 bp. In the previous section, we obtained a value
for lp of 49 nm. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. At forces of 65 pN or more, dsDNA changes its form
dramatically33 and it has been observed to stretch up to 70%
beyond the canonical contour length shown by its B-form. This
is not currently reproduced in our model and it would require a
change in the structure of how the base-pairs are arranged and
stacked together (i.e. the distance between base-pairs would no
longer be 0.34 nm). We also performed simulations of a stretching experiment on a
chain ten thousand base-pairs long (comparable to viral P4 DNA). The results for this case are reported as red data-points in Fig. 6. These measurements are in agreement with the behaviour of the
300 bp-long chain within error-bars, although they systematically
show a slightly larger extension, possibly due to finite-size effects. At forces of 65 pN or more, dsDNA changes its form
dramatically33 and it has been observed to stretch up to 70%
beyond the canonical contour length shown by its B-form. This
is not currently reproduced in our model and it would require a
change in the structure of how the base-pairs are arranged and
stacked together (i.e. the distance between base-pairs would no
longer be 0.34 nm). By measuring the deviation of twist DTw from the equilibrium
value Tw0 = N/p, i.e. the number of base-pairs divided by the pitch
p = 10 bp, we can then readily obtain s. With this information it is
possible to map out the response curve of the molecule to an
external torque. A feature of this is a linear regime for small |s|
which we recover (see Fig. 8). The torsional rigidity, C, can finally
be calculated (in the limit of large stretching forces25) as37 Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. x ¼ L 1 1
2
kBT
Flp
1=2
þ F
S
"
#
:
(6) (6) The force–extension curve from the simulations of a 300 bp
chain in this regime, corresponds to the data points above 10 pN
shown in blue in Fig. 6. Fitting these points with eqn (6) gives
L = 99.7 0.5 nm, lp = 60.2 2 nm and S = 2086 23 pN. This
value of L is the one expected for a chain made by 300 bp. The
value for the persistence length lp is slightly bigger than the one
observed in the WLC regime; on the other hand, this apparent
increase of lp is also observed in experiments.33 Finally, the
stretching modulus S is found to be twice the one expected for
real dsDNA (B1000 pN), although this difference should not be
critical to the processes we are interested in the following. Fig. 7
The model DNA is anchored to a surface at the bottom end while
being stretched with a constant force F, and a torque C is applied at the top
end. We then monitor the linking number and report its equilibrium value
for a given torque. With this information is possible to compute the
superhelical density. about the axis, and the writhe (Wr), i.e. the number of self-
crossings of the ribbon centreline. Although the chain we use is
not closed into a loop, and therefore it is not possible to
formally define a linking between the strands, it is possible to
compute the linking number between two ‘‘artificially’’ closed
strands48,49 which follow the paths of the DNA strands along
the chain backbone and then join the respective ends far away
from the molecule (see Appendix D). Furthermore, the molecule
is kept straight (writhe-free) by applying a constant stretching
force which ensures that the twist is very close to the computed
linking number. We also performed simulations of a stretching experiment on a
chain ten thousand base-pairs long (comparable to viral P4 DNA). The results for this case are reported as red data-points in Fig. 6. These measurements are in agreement with the behaviour of the
300 bp-long chain within error-bars, although they systematically
show a slightly larger extension, possibly due to finite-size effects. This journal is ©The Royal Society of Chemistry 2016 4.1
Response to stretching The results are therefore in good agreement
with the calculation and the tuning of the persistence length
performed in the previous section. When the applied force is greater than 10 pN the existence of
a finite stretching modulus (S) has to be taken into account. The Extensible WLC (EWLC) has proven to be the most adequate
model to describe this particular case.33 This model assumes that
the contour length of the molecule increases linearly with the
applied force,44 and the following formula can be used between 5
and 50 pN:45 5 DNA denaturation and supercoiling experimentally.69 We then observed that the denatured region
spreads to the middle of the molecule, finally melting the whole
chain when K2 t 1.2 kBT and producing two single strands. DNA denaturation is the separation and unwinding of the two
strands, transforming a DNA duplex into two isolated and
unbound single strands.53 This process can be driven by heating
a solution of dsDNA molecules, and a critical ‘‘melting’’ temperature
Tm can be defined as the temperature at which 50% of a long
dsDNA molecule is denatured. This critical temperature commonly
depends on the genetic sequence, pH and salt concentration.50,54,55
Localised, temporary, and dynamic denatured segments are often
referred to as ‘‘bubbles’’. Single stranded DNA (ssDNA) is much more flexible than its
bound counterpart. In order to mimic this behaviour in our
model, we eliminate both the dihedral and the Kratky–Porod
interactions between nucleotides which are part of a ‘‘bubble’’
larger than two base-pairs. This results in single strands with a
persistence length of around 2 bp which are extremely flexible,
as one can appreciate from the snapshots in Fig. 9. As previously mentioned, another way to denature DNA is
by increasing the temperature of the system. We performed
simulations where this pathway was adopted to denature our model
DNA and found that the melting temperature is approximately 70 1C
which is somewhat close but below the experimentally observed
melting temperature.70 It is well known that local denaturation has several biological
implications such as favouring transcription initiation, DNA repair
or recombination,28,56,57 and that the dynamics of these bubbles
can be affected by torsional stress, which is itself often regulated by
enzymes, such as RNA polymerases.58–60 This fascinating interplay
between the elasticity and biology of DNA has received much
theoretical and experimental attention,50,57,60–64 but there have
been remarkably few attempts to address it from a computational
point of view.65,66 We should point out here two limitations of the current
model. First, while it can be used to study the reverse of partial
denaturation by, for instance, non-monotonically tuning the
value of K2 or temperature, the current model cannot create
hybridised molecules in which nucleotides partner up with
nucleotides other than those to which they were bonded to
start with. In other words, ‘‘secondary’’ structures and hairpins
cannot be formed at this stage. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8
Response to torque experiment for a chain 600 bp long pulled
with an external stretching force of 16 pN. Here we show the linear regime
for small |s| which gives the torsional rigidity C by a linear fit with eqn (8). Fig. 9
This figure shows the fraction of denatured base pairs f as a
function of time and for different bond energies connecting the patches
of paired bases, for a chain 300 bp long. Snapshots from simulations are
also shown. The energies used range between K2 = 1.0 kBT and K2 = 1.4 kBT. We always observe that in linear dsDNA the denaturation process nucleates
from the ends, as suggested by experiments.69 lt = 2C B 176 nm, very close to the measurement performed in the
previous section (yielding lt = 174 6 nm). 4.2
Response to twisting The torsional stiffness of DNA can be calculated by computing
the twist response of dsDNA to an imposed external torque, for
instance applied by a magnetically controlled macroscopic
bead36,46 (see Fig. 7). For different magnitudes of the applied
torque, |C|, we compute the superhelical density, s. The level of
supercoiling is determined by the linking number Lk, which is
the number of times one DNA strand wraps round the other. C ¼
1
kBT
a0
y0
DG
Ds;
(8) C ¼
1
kBT
a0
y0
DG
Ds;
(8) (8) where a0 = 0.34 nm and y0 = 361 are respectively the double
helical rise and the equilibrium twist angle across a base-pair
step for a relaxed dsDNA molecule. The data points shown in Fig. 8 are obtained from simulations
of a 600 bp long chain anchored at a surface to one end, while the
other end was pulled by a constant force of 16 pN and different
applied torques, G = Cez. From the fit we get the value of torsional
persistence length C B 88 nm C 260 bp in good agreement with
experimental results.50–52 One can finally use the relation between
the torsional persistence length lt and the torsional stiffness C
obtained from the twistable worm-like chain theory,38 which gives Since a dsDNA chain has a preferred equilibrium linking number
Lk0, the superhelical density may be defined as s = (Lk Lk0)/Lk0. The well-known White–Fuller theorem47 Lk = Tw + Wr,
(7) (7) relates the linking number of the edges of a ribbon (Lk) to the
twist (Tw), i.e. the extent of rotation of the two ribbon edges Soft Matter, 2016, 12, 9458--9470 | 9463 This journal is ©The Royal Society of Chemistry 2016 Fig. 9
This figure shows the fraction of denatured base pairs f as a
function of time and for different bond energies connecting the patches
of paired bases, for a chain 300 bp long. Snapshots from simulations are
also shown. The energies used range between K2 = 1.0 kBT and K2 = 1.4 kBT. We always observe that in linear dsDNA the denaturation process nucleates
from the ends, as suggested by experiments.69
Paper
View Article Online View Article Online lt = 2C B 176 nm, very close to the measurement performed in the
previous section (yielding lt = 174 6 nm). Fig. 9464 | Soft Matter, 2016, 12, 9458--9470 This journal is ©The Royal Society of Chemistry 2016 4.2
Response to twisting 8
Response to torque experiment for a chain 600 bp long pulled
with an external stretching force of 16 pN. Here we show the linear regime
for small |s| which gives the torsional rigidity C by a linear fit with eqn (8). Fig. 9
This figure shows the fraction of denatured base pairs f as a
function of time and for different bond energies connecting the patches
of paired bases, for a chain 300 bp long. Snapshots from simulations are
also shown. The energies used range between K2 = 1.0 kBT and K2 = 1.4 kBT. We always observe that in linear dsDNA the denaturation process nucleates
from the ends, as suggested by experiments.69
Soft Matter
Paper
View Article Online lt = 2C B 176 nm, very close to the measurement performed in the
previous section (yielding lt = 174 6 nm). Fig. 8
Response to torque experiment for a chain 600 bp long pulled
with an external stretching force of 16 pN. Here we show the linear regime
for small |s| which gives the torsional rigidity C by a linear fit with eqn (8). Soft Matter Soft Matter Soft Matter Paper Fig. 9
This figure shows the fraction of denatured base pairs f as a
function of time and for different bond energies connecting the patches
of paired bases, for a chain 300 bp long. Snapshots from simulations are
also shown. The energies used range between K2 = 1.0 kBT and K2 = 1.4 kBT. We always observe that in linear dsDNA the denaturation process nucleates
from the ends, as suggested by experiments.69 Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This model can also be extended to include base-pair specificity,
and variable salt or pH concentration, while allowing the user to
reach biologically relevant time and length scales. In this paper we
have shown that this model is capable of reproducing DNA melting
and, more importantly, of tracking the dynamics of supercoiled
molecules B1000 bp long for up to B2 ms. In the near future, we
aim to use this model to investigate further the interplay between
denaturation and supercoiling, especially in light of its connection
to gene expression.28,29 Fig. 10
This figure shows the relaxation of a negatively supercoiled
circular dsDNA. (left) The molecule (500 bp long) is initialised as a perfect
ring from which three full turns are removed. (right) As the system evolves,
the twist deficit is converted into writhe, and the molecule assumes stable
buckled configurations. This behaviour is expected for a real dsDNA
molecule because the torsional stiffness is larger than the bending rigidity. Fig. 10
This figure shows the relaxation of a negatively supercoiled
circular dsDNA. (left) The molecule (500 bp long) is initialised as a perfect
ring from which three full turns are removed. (right) As the system evolves,
the twist deficit is converted into writhe, and the molecule assumes stable
buckled configurations. This behaviour is expected for a real dsDNA
molecule because the torsional stiffness is larger than the bending rigidity. will be of use to investigate the dynamics of denaturation of long
dsDNA molecules, whether torsionally relaxed or supercoiled. We should also highlight that the presented model has
some notable limitations which arise from the compromise
between accuracy and scalability. For instance, our model lacks
the ability of reproducing realistic hybridisation events where
distant parts of the chains can become bonded forming an
intermediate hairpin. It also lacks sequence specificity, and a
detailed description of counterion-mediated electrostatic inter-
actions. While the choice of neglecting such events renders the
modelling faster, it will be possible in principle to include them
in the future, in cases where we are interested in hybridisation. As a preliminary step to show that our model can readily
take into account supercoiling, in Fig. 10 we present an
example of simulated closed (ring) dsDNA. 5 DNA denaturation and supercoiling Second, as previously mentioned,
the model does not include sequence specificity, which is known
to affect the local dynamics of denaturation. We aim to address
both these aspects in the future. In regard to sequence specificity,
this can be accounted for straightforwardly by defining two
types of harmonic bonds connecting patches in the com-
plementary strands and by using springs with different stiffness
such that K2(AT) o K2(CG). Since stacking is also sequence-
dependent we could, in a similar way, define different types of
stacking (Morse) potentials with distinct parameters which can
depend on the local sequence. In light of this, we expect that
this model, thanks to its high scalability when run in parallel, Although theoretical models can capture the thermodynamics of
a ‘‘stress-induced DNA-duplex destabilisation’’ (SIDD),67 elucidating
the kinetics of such a process, under both equilibrium and out-
of-equilibrium conditions, is an important question that can be
addressed using computer simulations. In this section we show that our model can readily recapitulate
DNA denaturation upon decreasing the stiffness, K2, of the spring
connecting patches in the two strands (Uhb). While the most
common strategy to denature DNA consists in increasing the
solution temperature, this pathway instead mimics a change in
solution pH.68 In Fig. 9 we show the fraction of denatured base-pairs as a
function of time for three different choices of K2. As the energy
of the bond is decreased, we observe the unbinding of two
strands, which starts from the ends of the chain, as observed This journal is ©The Royal Society of Chemistry 2016 Soft Matter
View Article Online View Article Online Paper Paper Soft Matter Fig. 10
This figure shows the relaxation of a negatively supercoiled
circular dsDNA. (left) The molecule (500 bp long) is initialised as a perfect
ring from which three full turns are removed. (right) As the system evolves,
the twist deficit is converted into writhe, and the molecule assumes stable
buckled configurations. This behaviour is expected for a real dsDNA
molecule because the torsional stiffness is larger than the bending rigidity. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In particular, we
consider a molecule 500 bp long and we initialise it with a
linking number deficit of DLk = Lk0 Lk = 3 (47 turns instead
of the usual 50 for a pitch of 10 bp). In a linear molecule this
deficit would be quickly washed out by the free motion of the
ends, whereas in a closed molecule the difference creates a
negative supercoiling s = DLk/Lk0 C 0.06 which is conserved
throughout the dynamics. The supercoiling can then be distributed
into the torsional or bending degrees of freedom as long as the
White–Fuller theorem47 is satisfied (see eqn (7)). Since the torsional
stiffness of DNA is larger than the bending rigidity, much of the
twist is quickly converted to writhe, as can be readily seen in Fig. 10. One of the improvements that can be readily made to the
model is to account for the presence of major and minor
grooves. Distinguishing between major and minor grooves
may be important, for example, to capture the correct inter-
action of the chain with DNA-binding proteins, since these
often bind selectively to one of the grooves. To address this
issue, the model can be extended to include explicitly a phosphate
group by means of a third monomer per nucleotide (see Appendix
B for details). We note that without additional parametrization, the
model is able to display the presence of asymmetric grooves with a
total length of 1.22 nm (for the minor groove) and 2.18 nm (for the
major groove). This journal is ©The Royal Society of Chemistry 2016 6.1
Future improvements and limitations Our model aims at bridging the gap between all-atoms and
coarse-grained models for dsDNA; while it is currently less
sophisticated than other mesoscopic models, such as oxDNA
and 3SPN.2, most notably in the treatment of sequence specificity
or hybridisation, this model exploits the scalability of LAMMPS,
and is ideally suited to study problems such as DNA–protein
interactions, or the denaturation of supercoiled DNA, where it is
essential to consider long molecules, as well as to simultaneously
model double-stranded and denatured regions. Soft Matter, 2016, 12, 9458--9470 | 9465 6 Discussion The interplay between the physics and biology of DNA is one of
the most intriguing topics in biophysics. While computational
models can strongly aid the understanding of this fascinating
open problem, the computational resources for such an expensive
task have traditionally been limited. Researchers often use either
very detailed and accurate all-atoms models, which can only cover
short time and length scales, or coarse-grained models, which can
follow the evolution of the system for much longer times, but at
the expense of neglecting key physical properties of dsDNA. Mesoscopic models have been recently proposed to fill in the
gap between these two approaches: very successful and recent
examples are the oxDNA code introduced in ref. 22, and the
3SPN.1 and 3SPN.2 codes.24 However, such software has not yet
been exported to a highly efficient and parallel environment. Here, we have proposed a mesoscopic coarse grained model that
can be readily implemented at minimal cost into LAMMPS, one
of the most popular molecular dynamics engines for atomistic
and mesoscopic simulation. Another important aspect worth considering in the future is
the electrostatic interaction of DNA, either with itself or the
environment. This is neglected in the current formulation of
the model for the sake of efficient scalability of the parallelised
code. Therefore the parameter tuning in Appendix A was
carried out by implicitly assuming that the salt concentration
corresponds to physiological conditions (0.15 M NaCl) and that
the system is at room temperature (27 1C); under these conditions
as previously mentioned the persistence length is lp E 50 nm. This, of course, will limit the range of applications of our model to
systems where electrostatic properties are screened. Different
approaches could be tested to address this aspect where needed. In ref. 71, for example, a Debye–Hu¨ckel potential is used to
model DNA–DNA interactions, with an effective charge located This journal is ©The Royal Society of Chemistry 2016 Soft Matter, 2016, 12, 9458--9470 | 9465 View Article Online View Article Online Soft Matter Paper at the backbone sites and an interaction radius depending on
the salt concentration. This additional force field can be easily
added to our model. A sligthly different approach could be
to capture the effects of screened electrostatic repulsion by
modulating the effective thickness of the chain in a similar way
to that proposed in ref. 72 and 73. 7 Conclusions In summary, we have introduced a coarse-grained single-
nucleotide model for dsDNA, which can be readily implemented
in computationally efficient and parallelised engines, such as
LAMMPS. We tuned the model in order to reproduce the crucial
physical features of dsDNA such as bending and torsional rigidities. We then tested our model by simulating single-molecule experi-
ments so as to independently check the parameterisation and
the response of our model to external manipulation. Finally, we
studied denaturation and the dynamics of supercoiled DNA. We
have shown that this implementation can comfortably reach length
and time scales that are relevant to both single molecule and
biological experiments, therefore making our model interesting
for applications. In the future we intend to refine this model
and to extend it in order to study biologically-inspired out-of-
equilibrium scenarios. Fig. 11
This plot shows the scaling behaviour of the model and it is
expressed as the simulation time achieved in a day of running time as a
function of the number of parallel processes (MPI-tasks). Two different
benchmarks were used to test the scalability: a small sample consisting of
60 kbp and a 16-times larger one with 960 kbp. The results are compared
with the timings taken for a run with 24 processes for each benchmark,
corresponding to one fully occupied node on the ARCHER XC30 architecture. This leads to parallel efficiencies (see inset) in excess of 100% for 1 and 8
processes. Total simulation times of up to 2 ms per day are feasible. 6.2
Computational scalability We extensively tested the scalability of the model (Fig. 11) by
employing two benchmarks. They consisted both of linear,
double-stranded DNA molecules of length of 600 bp each. The strands were initialised as a regular array of 10 10 or
40 40 strands, respectively to form a total system of 60 kbp
and 960 kbp. The daily simulation times were derived from the
loop timings of runs with 30 000 timesteps (60 kbp) and 10 000
timesteps (960 kbp) and were compared with those of a run
with 24 processes (MPI-tasks), corresponding to one fully
occupied node on ARCHER (see below). We made use of the
‘‘shift’’ load-balancing algorithm in LAMMPS, which re-positions
the cutting planes between the single processes in order to
mitigate a potential load imbalance between the individual pro-
cesses (further details and full input files are available upon
request). The model displays a very good speed-up up to hundreds
of processes when deployed in parallel. These results are for
so-called ‘‘strong scaling’’ where the number of processes is
increased while the total problem size, in our case the number
of nucleotides, is kept constant. The scaling tests were performed
on ARCHER, a Cray XC30 supercomputer with 4920 compute
nodes, each consisting of two 2.7 GHz 12-core Intel Ivy Bridge
processors and Aries Interconnect (Dragonfly topology). Finally, exploiting the ability of LAMMPS to function as a library
coupled to external programs, it is possible to design systems in
which ATP-driven proteins interact with the model dsDNA. This
paves the way to the attractive avenue of molecular dynamics
simulation of large-scale out-of-equilibrium and biologically inspired
systems, which are appealing to a broad range of researchers. 6 Discussion For the model we present
here, it is possible to moderately modify the thickness of the
chain by adjusting the excluded volume interaction between
phosphates, when these are explicitly considered. In particular, for the smaller problem size of 60 kbp we
observe a parallel efficiency of about 50% at 512 MPI-tasks,
allowing it to run for about 2 ms per day. More processes do not
lead to a further speed-up and the parallel efficiency decreases
rapidly due to the relatively small number of ‘‘atoms’’ per
process (LAMMPS requires several hundred atoms per process
to show good scaling behaviour). The larger benchmark of
960 kbp shows a parallel efficiency of about 50% at 2048 MPI-
tasks, which permits simulation times of about 0.4 ms per day. Compared to the smaller benchmark the performance degrades
more slowly in this case, making simulation times of up to
1 ms per day at 8192 MPI-tasks feasible. These results strongly
encourage its use on a larger scale. Other existing models22,23
might therefore be more suitable for studies of short DNA–DNA
hybridisation leading to DNA origami and synthetic DNA
assemblies. The model we presented here is instead more apt
to study denaturation, supercoiling and DNA–protein interactions
on larger length and time-scales as previously discussed. This journal is ©The Royal Society of Chemistry 2016 Appendix A: details of the model The dynamics of the system are evolved using the Large-scale
Atomic/Molecular Massively Parallel Simulator (LAMMPS) for
which the scripts and input files employed in this work are
available upon request. The position of the ith atom in the
system, xi, obeys the Langevin equation Fig. 11
This plot shows the scaling behaviour of the model and it is
expressed as the simulation time achieved in a day of running time as a
function of the number of parallel processes (MPI-tasks). Two different
benchmarks were used to test the scalability: a small sample consisting of
60 kbp and a 16-times larger one with 960 kbp. The results are compared
with the timings taken for a run with 24 processes for each benchmark,
corresponding to one fully occupied node on the ARCHER XC30 architecture. This leads to parallel efficiencies (see inset) in excess of 100% for 1 and 8
processes. Total simulation times of up to 2 ms per day are feasible. md2xi
dt2 ¼ g dxi
dt =Ui þ gi;
(9) (9) where g is the friction coefficient and gi is a stochastic noise
term which satisfies hZa(t)Zb(t0)i = 2gkBTdabd(t t0). The term 9466 | Soft Matter, 2016, 12, 9458--9470 This journal is ©The Royal Society of Chemistry 2016 Table 1
Parameter values in the model and expressed in simulation units
Interaction
Parameters
Backbone: Ubb
K1 = 30, R0 = 0.6825, e = 1 and ss = 0.4430
Hydrogen bond: Uhb
K2 = 6, r0 = 0 and rc = 0.3
Steric: ULJ
e = 1 and ss = 1
Dihedral: Udihedral
K3 = 50, n = 1, and d = 1441
Morse: Umorse
K4 = 30, l = 8 and r0 = 0.34
Planarity: Uharmonic
K5 = 200 and a0 = 901
Bending: Ubending
K6 = 52
Soft Matter
View Article Online Paper Paper =Ui is the gradient of the total potential Ui affecting bead i,
whose contributions are described below. Table 1
Parameter values in the model and expressed in simulation units A.2
Non-bonded interactions The excluded volume between beads is modelled via a trun-
cated and shifted Lennard-Jones (LJ) potential. This potential
acts between all possible pairs of beads so as to avoiding
overlapping, and has the following form: Dt = 0.005tBr. (17) (17) ULJðrÞ ¼ 4e
ss
r
12
ss
r
6
þ 1
4
;
(12) A.3
Simulation units if r r rc, and 0 otherwise. Here r represents the distance
between patches i and i0, r0 the equilibrium bond distance, K2
the spring constant, and rc is the critical distance above which the
bond breaks. The minimum of this potential is located at r = r0. Mapping the simulation units to physical ones can be done by
setting the fundamental units: distance, energy and time. These are shown in Table 2. The chosen system of reference
is a bath at room temperature T = 300 K and with the viscosity
of water Z = 1 cP. Finally, the numerical integration is performed
in an NVT ensemble by a standard velocity-Verlet algorithm with
integration time-step Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (10) the vector joining a bead to its patch, imposes the planarity
between consecutive patches. the vector joining a bead to its patch, imposes the planarity
between consecutive patches. if r R0: UharmonicðaÞ ¼ K5
2
a a0
ð
Þ2:
(15) (15) where R0 is the maximum bond length, K1 is the spring
constant and r is the Euclidean distance between bead i and
bead i + 1. When summed to the Lennard-Jones potential
(acting between any two beads), the minimum of this potential
is located at rmin = 0.96 ss. As described in Section 1 the minimum of this potential is set
to a0 = 901. As described in Section 1 the minimum of this potential is set
to a0 = 901. Finally, the bending rigidity is given by a potential on the
angle y formed by three consecutive patches that reads The ‘‘hydrogen bond’’ is mimicked by a truncated harmonic
potential between the patches along the two strands (i and i0). This potential reads Ubending(y) = K6[1 + cos(y)]. (16) (16) The parameters for each potential are reported in simulation
units in Table 1. UhbðrÞ ¼
K2
2 r0 rc
ð
Þ2
r r0
ð
Þ2 rc r0
ð
Þ2
h
i
;
(11) (11) This journal is ©The Royal Society of Chemistry 2016 A.1
Bonded interactions The interactions between two consecutive beads in the same
strand i and i + 1 are modelled by the Finite Extensible Non-
linear Elastic (FENE) potential: UbbðrÞ ¼
K1R02
2
ln 1
r
R0
2
"
#
if r o R0
1;
if r R0:
8
>
>
<
>
>
:
(10) Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 12
Sketch of a base-pair with the phosphate-group included explicitly. The bead-patch-phosphate complex (blue–pink–green for the first
strand and red–cyan–yellow for the second strand) acts as a rigid body
representing one nucleotide. Fig. 13
(a) Representation of the double-stranded DNA model with
phosphates. Interactive and ghost beads are shown in red for one of the
strands with the corresponding phosphates in yellow. The complementary
strand and its phosphates are shown in blue and green respectively. The
hydrogen bond sites (patches) are not depicted in this picture. (b) The top
graphic shows (blue dots) the average distance between one phosphate
chosen randomly from the red strand and the ten consecutive phosphates
in the blue strand. The width of the minor groove can then be extracted
from the interpolation curve (in black) at a distance of 3.62 bp, giving a value
of 1.22 nm. This is when the green phosphate is on top of the previously
chosen phosphate in the red strand, as depicted in (a). In a similar way the
bottom graphic shows the average distance between one phosphate chosen
randomly from the blue strand and the ten consecutive phosphates in the
complementary strand. This time the major groove is extrapolated from the
black curve at a distance of 6.38 bp, giving a width of 2.18 nm. shown in red, the patch in cyan and the phosphate in yellow. All
the interactions mentioned in Section 2 remain unchanged. Including the phosphate in the model will add an additional
degree of freedom per nucleotide, which is regulated by a
harmonic angle interaction between two consecutive patches
and a phosphate in the same strand, similar to the one imposing
the planarity between consecutive nucleotides (see eqn (15)) and
with the same value of the involved parameters (see Table 1). The
smallest angle between the two phosphates in a base-pair and the
helix axis (shown in light grey in Fig. 12) is f = 1301 and results in
the minor groove when following the helical path of the dsDNA,
as can be seen in Fig. 13(a). The conjugate angle is 2301 and it
gives rise to the major groove. If the pitch of the chain is 10 bp
and therefore the helical angle between two consecutive base
pairs is 361, then the minor groove is made by 130/36 = 3.62 bp
with a total length of around 3.62 0.34 = 1.2 nm. Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017 11:55:33.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Correspondingly,
the total length of the major groove is 2.2 nm. Appendix C: computing the torsional
persistence length To obtain the torsional properties of the DNA molecule described
in Section 3.2 we consider a discrete elastic rod. As described in
ref. 38, eqn (3) is an integral over the rate of rotation of the
Darboux frame (or material frame) of reference with respect to the
distance along the rod. We first find the discrete approximation to
this in terms of the Euler angles an, bn, gn which describe the
rotation which generates the frame at segment n + 1 from that at
segment n. To do this we make the approximation that the step
size between segments is constant and denote it a; this gives One way of determining the presence of grooves in our
model is by comparing the average distance between one fixed
phosphate chosen randomly from one of the strands and
the ten consecutive phosphates in the complementary strand
(including the one in the same base-pair). Since the grooves
have a different size, the resulting plots differ from one another
depending on the position of the fixed phosphate, whether it is
on the first or the second, strand. In Fig. 13(b) we show these
plots where the top graphic displays a global minimum that is
related to the presence of the minor groove. In this plot, the
blue dots represent the distance (averaged over time and base-
pairs) between phosphates, measured from the simulation of a
chain made by 300 bp. The spline interpolation of the blue dots
is shown in black and the red point represents the inferred
position of the minor groove, located at 3.62 bp with a distance
of 1.22 nm. The bottom graphic in Fig. 13(b) is related to the
major groove, in this case the interpolation gives a total width
of 2.18 nm. The length of the grooves can be modified by
changing the angle (f) between the phosphates, but as the pitch
remains constant the sum of the total widths of the grooves will
always be 3.4 nm. Etors
kBT ¼ C
a 1 cos an þ gn
ð
Þ
½
; where twist angle between the frames is given by an + gn, so the total
angle between m consecutive beads is given by OðmÞ ¼ P
m
n¼1
an þ gn
ð
Þ. Appendix B: major and minor grooves (12) As mentioned in Section 6, the presence of grooves can be
incorporated into our model by adding a third spherical
monomer per nucleotide, representing the phosphate group. In Fig. 12 a top view of a base-pair is sketched. For one of the
nucleotides, the excluded volume of the bead is shown in blue
(with diameter of 1 nm), the patch is marked with pink (with no
excluded volume) and its corresponding phosphate is shown in
green at a distance of 1.02 nm from the center of the helix axis
and with an excluded volume of 0.2 nm. Similarly, for the
complementary nucleotide the excluded volume of the bead is for r o 21/6ss, and 0 otherwise. Here ss represents the diameter
of a spherical bead, e parametrises the strength of the repulsion
and r is the Euclidean distance between the beads. The minimum
of this potential is located at r = rc = 21/6ss. The dihedral interaction which regulates the handedness of
the chain is given by: Udihedral(f) = K3[1 + cos(f d)],
(13) (13) where f is the angle between planes formed by the triplets
described in Section 1 and d is a phase angle related to the
equilibrium helical pitch. Soft Matter, 2016, 12, 9458--9470 | 9467
Table 2
Mapping between simulation and physical units
Parameter
Experimental units
Distance (ss)
1 nm C 3 bp
Energy (e = kBT)
4.1419 1021 J
Force (F = e/ss)
4.1419 1012 N
Mobility (m = 1/(3pZss))
1.06 1011 m Ns1
Diffusion (D = mkBT)
4.39 1010 m2 s1
Time (tBr = ss
2/D)
2.28 109 s Table 2
Mapping between simulation and physical units
Parameter
Experimental units Table 2
Mapping between simulation and physical units The stacking of consecutive base-pairs is set by a combi-
nation of a Morse potential constraining the distance between
consecutive patches Umorse(r) = K4[1 el(rr0)]2. (14) (14) where r0 is the equilibrium distance. A stiffharmonic potential
setting the angle a between the tangent along one strand and This journal is ©The Royal Society of Chemistry 2016 Appendix C: computing the torsional
persistence length
To obtain the torsional properties of the DNA molecule described
in Section 3.2 we consider a discrete elastic rod. As described in
ref. Appendix B: major and minor grooves 38, eqn (3) is an integral over the rate of rotation of the
Darboux frame (or material frame) of reference with respect to the
distance along the rod. We first find the discrete approximation to
this in terms of the Euler angles an, bn, gn which describe the
rotation which generates the frame at segment n + 1 from that at
d
hi
k
h
i
i
h
h
Fig. 13
(a) Representation of the double-stranded DNA model with
phosphates. Interactive and ghost beads are shown in red for one of the
strands with the corresponding phosphates in yellow. The complementary
strand and its phosphates are shown in blue and green respectively. The
hydrogen bond sites (patches) are not depicted in this picture. (b) The top
graphic shows (blue dots) the average distance between one phosphate
chosen randomly from the red strand and the ten consecutive phosphates
in the blue strand. The width of the minor groove can then be extracted
from the interpolation curve (in black) at a distance of 3.62 bp, giving a value
of 1.22 nm. This is when the green phosphate is on top of the previously
chosen phosphate in the red strand, as depicted in (a). In a similar way the
bottom graphic shows the average distance between one phosphate chosen
randomly from the blue strand and the ten consecutive phosphates in the
complementary strand. This time the major groove is extrapolated from the
black curve at a distance of 6.38 bp, giving a width of 2.18 nm. Paper
View Article Online View Article Online Soft Matter Fig. 12
Sketch of a base-pair with the phosphate-group included explicitly. The bead-patch-phosphate complex (blue–pink–green for the first
strand and red–cyan–yellow for the second strand) acts as a rigid body
representing one nucleotide. Soft Matter Paper p This journal is ©The Royal Society of Chemistry 2016 References 1 J. D. Watson and F. H. C. Crick, Nature, 1953, 171, 737–738. 2 M. Wilkins, A. Stokes and H. Wilson, Nature, 1953, 171,
738–740. 3 R. Franklin and R. Gosling, Nature, 1953, 172, 156–157. Acknowledgements This work was funded by ERC (Consolidator Grant 648050
THREEDCELLPHYSICS). OH acknowledges support from the EPSRC
Reseach Software Engineer Fellowship Scheme (EP/N019180/1). This
work used the ARCHER UK National Supercomputing Service. Y. A. G. F. acknowledges support from CONACyT PhD Grant 384582. Fig. 14
The ‘‘closure’’ procedure can be performed on a pair of linear
open curves to construct a closed pair whose linking number can be
formally defined through the Gauss’ integral (see eqn (18)). In this case the
curves are linked once. See text for further details. energy functional, so this must be subtracted from O(m) so that the
ensemble average is a simple exponential decay. The Darboux frame
at each DNA base-pair is given by the tangent vector, and the normal
vector defined as the projection of the vector connecting the two
beads onto the plane perpendicular to the tangent. Appendix D: closure procedure
for linear DNA 4 C. R. Calladine, H. Drew, F. B. Luisi and A. A. Travers,
Understanding DNA: the molecule and how it works, Elsevier
Academic Press, 1997. In this section we review the procedure to compute the linking
number of an open segment of dsDNA. For clarity we report a
schematic in Fig. 14. Given two curves CR and CB mapping the
interval I = [0 : 1] - R3, it is possible to formally compute their
linking number only if closed, i.e. CR(0) = CR(1) and CB(0) =
CB(1). For a linear open segment of dsDNA, a pair of closed
strands can be defined by considering the vectors tangent to
the terminal pair of beads of the two single strands forming the
dsDNA segment and extending the curves away from the pair of
strands. After reaching a certain distance by following, for
instance, t1R and t2R, one can close the contour by defining a
vector fR that joins the two new terminal beads (see Fig. 14). By
following this procedure one can finally construct a pair of
closed oriented curves gR and gB, for instance ‘‘stitching’’ CR,
t1R, fR, t2R, and similarly for the blue curve. Their linking
number can be computed through the numerical evaluation of
the double integral 5 A. Bates and A. Maxwell, DNA topology, Oxford University
Press, 2005. 6 G. Cavalli and T. Misteli, Nat. Struct. Mol. Biol., 2013, 20, 290–299. 7 C. A. Brackley, S. Taylor, A. Papantonis, P. R. Cook and
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E3605–E3611. 8 P. R. Cook and D. Marenduzzo, J. Cell Biol., 2009, 186, 825–834. 9 P. R. Cook, Nat. Genet., 2002, 32, 347–352. 8 P. R. Cook and D. Marenduzzo, J. Cell Biol., 2009, 186, 825–834. 9 P. R. Cook, Nat. Genet., 2002, 32, 347–352. 10 B. Alberts, A. Johnson, J. Lewis, D. Morgan and M. Raff,
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D. J. Pine and P. M. Chaikin, Soft Matter, 2009, 5, 2422. 12 L. Di Michele and E. Eiser, Phys. Chem. Chem. Phys., 2013,
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persistence length An appropriate measure of the thermal fluctuations about the
equilibrium twist is given by the mean of the cosine of this angle;
since this quantity will decrease with m, and we identify the decay
constant as the torsional persistence length hcosO(m)i = ema/lt. The
ensemble average is found in the usual way by taking the integral
over the phase space of the system; in the small a limit this gives lt = 2C. This is the case for an elastic rod; for the DNA molecule, the non-
zero equilibrium twist between each base-pair will appear in the 9468 | Soft Matter, 2016, 12, 9458--9470 9468 This journal is ©The Royal Society of Chemistry 2016 View Article Online Paper Paper Soft Matter Fig. 14
The ‘‘closure’’ procedure can be performed on a pair of linear
open curves to construct a closed pair whose linking number can be
formally defined through the Gauss’ integral (see eqn (18)). In this case the
curves are linked once. See text for further details. Paper backbones by contours more finely interspersed with points,
i.e. enhancing the resolution of the integral by decreasing the
infinitesimal element dr. Clearly, this can slow down the com-
putation of Lk. We found a good compromise between precision
and speed by adding three intermediate points every pair of beads
for which we consistently measured the correct linking number
during topology-preserving simulations (for instance by considering
circular dsDNA). Open Access Article. Published on 08 November 2016. Downloaded on 07/08/2017
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ð
Þ ¼ 1
4p
ð
gR
ð
gB
rR rB
j
j
rR rB
j
j3 drR drB
ð
Þ;
(18) (18) 16 E. Fadrna´, N. Sˇpacˇkova´, J. Sarzyn˜ska, J. Kocˇa, M. Orozco,
T. E. Cheatham III, T. Kulinski and J. Sˇponer, J. Chem. Theory Comput., 2009, 5, 2514–2530. where rR and rB are the vectors defining the position of the
segments along the curves gR and gB, respectively. If the centreline
running through the pair of curves has no self-intersections (null
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Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography
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Scientific reports
| 2,015
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cc-by
| 10,552
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Morphological Characterisation of
Unstained and Intact Tissue Micro-
architecture by X-ray Computed
Micro- and Nano-Tomography
Lucy A. Walton1, *, Robert S. Bradley2, *, Philip J. Withers2, Victoria L. Newton3,
Rachel E. B. Watson3, Clare Austin1, 4, * & Michael J. Sherratt3, * received: 24 November 2014
accepted: 27 March 2015
Published: 15 May 2015 Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and
by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the
absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography
(nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue
regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in
turn can be segmented due to their different X-ray opacities and morphologies. We then establish,
using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure
not only increases lumen cross-sectional area and straightens medial elastic lamellae but also
induces profound remodelling of the adventitial layer. Finally we apply microCT to another human
organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show
that conventional histological and immunohistochemical staining protocols are compatible with
prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical
microscopy to characterise the 3D structure and composition of archival paraffin embedded biological
materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons. Characterising tissue sub-structure by conventional histological sectioning (microtomy) frequently
induces tears, fractures, compressions and folds1 and, in the specific case of large arteries, often causes
detachment of the adventitial layer. Crucially, in order to accurately characterise the structure of tis-
sues such as elastic arteries, it will also be necessary to image sufficiently large regions of the vessel in
three-dimensions (3D)2,3. Although recent advances in serial block face scanning electron microscopy
and confocal laser microscopy have been employed to visualise the 3D structure of the aortic medial
layer3 and adventitial collagen4, these techniques can only be applied to relatively small tissue volumes. However, X-ray computed microtomography (microCT) which is conventionally used for determining
the structure of hard calcified tissues such as bone, can also be used to visualise the 3D morphology of
intact non-calcified organs and organisms in the presence of X-ray contrast agents5,6. In this study we
aimed to determine if sub-μm tissue-structures (in large arteries and skin) could be visualised in the
absence of exogenous X-ray contrast agents.h g
y
g
Large conduit arteries protect smaller vessels from damaging variations in blood pressure7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Figure 1. Sectioning-induced artefacts in paraffin embedded vascular and cutaneous tissues. Light
microscopy image of H&E stained rat CCA. Figure 1. Sectioning-induced artefacts in paraffin embedded vascular and cutaneous tissues. Light
microscopy image of H&E stained rat CCA. life-threatening cardiovascular events including heart failure and aortic aneurysm9–11. In order therefore
to characterise both the pathological progression of these disorders and the efficacy of potential treat-
ments it is important to visualise arterial wall structure. In practice this is commonly achieved by two
dimensional sectioning of unpressurised vessels and subsequent characterisation of the medial layer only. In pioneering work published in 1964, Wolinsky and Glagov documented acute reductions in elastic
lamellae “waviness” and medial layer thickness with increasing intra-luminal pressure12. In their model
system, the New Zealand White Rabbit, the majority of these acute tissue remodelling events took place
at sub-physiological pressures13 of less than 80 mm Hg. Despite this clear evidence that the structure of
the pressurised vessel differs profoundly from the structure of the unpressurised vessel (a state which
only occurs after death) and that the outer adventitial layer also plays a vital role in limiting vessel dis-
tension14 and preventing aneurysms15, with few exceptions3,4,16,17 arterial morphology is still routinely
characterised in 2D from sectioned, unpressurised, tissues. p
Here, we characterise the structure of intact Wistar rat large arteries (preserved in either an unpressur-
ised or pressurised state) using microCT. We demonstrate that this technique can differentiate between
sub-tissue level layers and that intra-luminal pressure results in profound remodelling of both the medial
and adventitial layers. In future studies, it will be important to progress from the static application of
intra-luminal pressure (in which water flow may influence tissue architecture and volume over long time
scales) to dynamic x-ray imaging of native arteries subjected to physiological cyclic loading regimes. We
also demonstrate that X-ray micro-tomography is applicable to other organs such as human skin and
that sufficient contrast can be achieved without the use of X-ray contrast agents (which can interfere
with subsequent histological and immunohistochemical analyses). As a consequence the technique will
be applicable to most archival paraffin embedded biological material. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Morphological Characterisation of
Unstained and Intact Tissue Micro-
architecture by X-ray Computed
Micro- and Nano-Tomography
Lucy A. Walton1, *, Robert S. Bradley2, *, Philip J. Withers2, Victoria L. Newton3,
Rachel E. B. Watson3, Clare Austin1, 4, * & Michael J. Sherratt3, * This
mechanical dampening function is mediated primarily by cross-linked elastin in the medial elastic lamel-
lae and by collagen fibrils in the outer adventitial layer8. Aberrations in this wall structure, as a conse-
quence of heritable connective tissue disorders, ageing and diseases such as diabetes, are associated with 1Institute of Cardiovascular Sciences. 2School of Materials. 3Institute of Inflammation and Repair, University of
Manchester, Manchester, United Kingdom. 4Faculty of Health and Social Care, Edge Hill University, Ormskirk,
United Kingdom. *These authors contributed equally to this work. Correspondence and requests for materials
should be addressed to M.J.S. (email: michael.sherratt@manchester.ac.uk) 1 Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 www.nature.com/scientificreports/ Figure 1. Sectioning-induced artefacts in paraffin embedded vascular and cutaneous tissues. Light
microscopy image of H&E stained rat CCA. Results and discussion (B) Major arterial sub-structures
are readily identifiable in the vessel wall. (C and D) The resolution achievable with a 4x objective lens (C:
voxel size 0.75 μm, 2D resolution 1.2 μm) can be improved using a 20x objective (D: voxel size 0.50 μm, 2D
resolution 0.7 μm). (E) Virtual slice extracted from an X-ray tomogram at nanoscale resolution (150 nm)
of an intact rat aorta. The tomogram was taken with Zernike phase contrast which gives contrast to edge
features and resolves fibrous structures within the inter-lamellar regions. (F) Elastic lamellae and inter-
lamellar regions can be readily segmented and gold particles (Au) can be used to locate the same tissue
region on differing instruments. Figure 2. Sub-micron X-ray tomography resolves discrete tissue regions and components in paraffin
embedded arteries. (A) Virtual slice extracted from an X-ray tomogram of an intact rat CCA (yellow box
indicates magnified region in panel B). Even in the absence of exogenous X-ray contrast agents, the X-ray
density of native soft tissues is higher than that of both paraffin and air. (B) Major arterial sub-structures
are readily identifiable in the vessel wall. (C and D) The resolution achievable with a 4x objective lens (C:
voxel size 0.75 μm, 2D resolution 1.2 μm) can be improved using a 20x objective (D: voxel size 0.50 μm, 2D
resolution 0.7 μm). (E) Virtual slice extracted from an X-ray tomogram at nanoscale resolution (150 nm)
of an intact rat aorta. The tomogram was taken with Zernike phase contrast which gives contrast to edge
features and resolves fibrous structures within the inter-lamellar regions. (F) Elastic lamellae and inter-
lamellar regions can be readily segmented and gold particles (Au) can be used to locate the same tissue
region on differing instruments. Until recently, x-ray tomography has been limited in its ability to reveal features within non-calcified,
and hence weakly x-ray absorbing, tissues. Whilst x-ray absorption and therefore contrast can be
enhanced using high Z elements22 difficulties remain with ensuring that these agents: i) penetrate larger
tissues, ii) differentiate between tissue components and iii) are compatible with subsequent histologi-
cal techniques23. Alternatively, x-ray phase contrast approaches can enhance contrast in non-calcified
tissues including cartilage24, tendon25, plaque containing blood vessels26, coronary artery27, ligaments
and the intervertebral disc28. However, such approaches may require complex imaging set-ups29 and
post-processing30, and image acquisition times can be long for laboratory instruments. Results and discussion Microtomography Preserves Arterial Structure and Resolves Compositionally Distinct Tissue
Regions and Components. Histological analysis of tissue structure is most commonly performed
on thin (<10 μm) sections cut from formalin fixed and paraffin embedded samples18. Although the
formalin-induced amine cross-links and paraffin wax act in concert to chemically strengthen and physi-
cally support the tissue19, the sectioning process invariably disrupts the structure of organs such as large
arteries. As a consequence vascular tissue sections are commonly characterised by folds, holes (primarily,
although not exclusively, located in the adventitia) and by separation of the collagen-rich adventitia from
the medial external elastic lamina (Fig. 1). By avoiding mechanical sectioning, X-ray microCT is capable
of preserving the 3D structure of whole organisms and organs5,6. Here we show that X-ray microCT can
also visualise structurally intact and compositionally distinct regions and macro-molecular assemblies in
blood vessels at an approximate spatial resolution of 1.2 μm (Fig. 2A,B) when using a 4x objective lens or
0.7 μm when using a higher power 20x lens (Fig. 2C,D). Due to the highly organised architecture of large
arteries we can show that common tissue components, the fibrillar collagen-rich adventitia, elastin-rich
elastic lamellae and cell-rich inter-lamellar regions, differ in their relative X-ray opacities and can there-
fore be visualised by this technique. Although in isolation, individual virtual X-ray tomogram slices (such
as those depicted in Fig. 2A–D) contain a wealth of structural information, each slice represents only a
fraction of the 3D X-ray density data which is encoded in the complete reconstruction. By viewing the
3D data as sequential 2D projections in an animated Video (Video S1) new structural information, such
as the topography of the luminal surface, becomes clear. Crucially, although perturbations in the rough-
ness of this surface are thought to play an important role in promoting localised vascular damage (and
hence cardiovascular diseases such as atherosclerosis20,21), conventional sectioning methods are unable
to visualise the 2D micro-topography of the luminal intima/blood interface. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Sub-micron X-ray tomography resolves discrete tissue regions and components in paraffin
embedded arteries. (A) Virtual slice extracted from an X-ray tomogram of an intact rat CCA (yellow box
indicates magnified region in panel B). Even in the absence of exogenous X-ray contrast agents, the X-ray
density of native soft tissues is higher than that of both paraffin and air. Results and discussion In this paper, we
demonstrate that excellent differential contrast can be achieved in non-calcified tissues with a laboratory
microCT system using absorption contrast in conjunction with phase contrast enhancement. We attrib-
ute this improved contrast to both our specimen preparation techniques and the instrumentation. Tissue Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 3 www.nature.com/scientificreports/ samples were prepared using a standard histological procedure which involves chemical fixation, alcohol
dehydration and infiltration with paraffin wax. Differential dehydration of tissue components (such as
low-density, hydrated cells and densely packed hydrophobic elastic lamellae in large arteries) in com-
bination with localised paraffin infiltration may increase internal contrast. Instrumental improvements
include the use of thin scintillators which absorb fewer higher energy x-rays than conventional flat panel
detectors31. These high energy x-rays do not contribute significantly to contrast of low Z materials but
rather to the background noise. Furthermore, flat panel detectors may contain an Al filter which would
further reduce the relative contribution from low energy x-rays to image formation, making them suit-
able for x-ray energies greater than ~40 keV32. Finally, in-line phase contrast enhances contrast at the
multiple boundaries (such as lumen-wall and elastic lamellae-interlamellar regions) which are found in
tissues such as arteries thereby providing additional edge-enhancement30. The Complex Architecture of Medial Layer Interlamellar Spaces is Evident in Nanotomography
Reconstructions. Whilst the X-ray micro-tomograms depicted in panels A-D of Fig. 2 are highly
informative, most organs and tissue are organised at multiple length scales. In order therefore to resolve,
for example, fine elastic fibres, collagen fibril bundles and vascular smooth muscle cell structure within
the arterial medial lamellar unit3,33 it is necessary to improve on the 0.5 μm resolution which can be
achieved by using the Carl Zeiss XRM Xradia Versa microCT. Sub-100 nm resolutions can be achieved
with both synchrotron and laboratory X-ray sources34 and by using concepts pioneered in optical micros-
copy (such as Fresnel zone plates) X-rays can be focused with phase contrast achieved by propagation
phase contrast or Zernike phase contrast phase rings35. These techniques have previously been used
to image unstained bacteria, insects and mineralised tissues36,37. Here we demonstrate using Zernike
phase contrast on a Carl Zeiss XRM Xradia Ultra-810 (with a resolution of 150 nm) that, compared
with the amorphous appearance which characterises medial interlamellar regions in micro-tomograms,
these same areas appear structurally heterogeneous by nanotomography (nanoCT) (Fig. 2B,D). Results and discussion This
dense fibrous network may be composed of discrete, small-diameter, elastic fibres and collagen fibril
bundles3,33. If necessary, tissue structure could be studied across multiple length scales by labelling a
region of interest with gold spheres (as depicted by the area of bright contrast in Fig. 2F) hence enabling
cross-correlative tomographic imaging of the same sample region by microCT and nanoCT. In order to
fully exploit the wealth of 3D structural data which is present in both this nanoCT reconstruction and
microCT data sets, we have developed semi-automatic image analysis approaches to segment structurally
and compositionally distinct, yet low-contrast, tissue regions. Tissue-Specific Segmentation Strategies are Required to Quantitatively Characterise 3D tissue
Structure. Large arteries are highly structured tissues in which the major ECM components, elastin
and fibrillar collagens, are both X-ray opaque (compared with the paraffin support) and largely confined
to the medial elastic lamellae and adventitial layers respectively (Fig. 1,2). Additionally, the elastic lamel-
lae confer an ordered structure on the medial layer which contrasts with the less-ordered adventitial
layer. We have developed a sequential image processing protocol which exploits differences in X-ray
opacity in order to segment the vessel wall from the supporting paraffin38,39 and then uses the mor-
phological characteristics40 of the elastic lamellae to segment the media from the adventitia (Fig. 3). By
automatically segmenting 3D X-ray tomography volumes, we have been able to: i) avoid potential sam-
pling errors and gain insight into variations in morphology by rapidly characterising these morphological
parameters at all points around the vessel cross-section in each of the 450 virtual slices (4x objective),
ii) characterise the morphology of the intact adventitial layer and iii) assess the effects of intra-luminal
pressure on the topography of clinically important interfaces such as that formed between the luminal
surface and the blood stream41 (Figs. 4 and 5). A similar approach can be used to segment distinct 3D
structures within the medial layer of nanoCT reconstructions (Fig. 2E,F). Having established methods for
visualising and measuring the 3D volume and topography of discrete tissue regions and surfaces we can
use these approaches to quantify the effects of intra-luminal pressure on the structure of large arteries. MicroCT Preserves Arterial Structure and Allows for the Measurement of Novel Morphological
Characteristics. In 1964 Wolinsky and Glagov reported on the effects of intra-luminal pressure on the
2D architecture of the aortic media12. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Results and discussion (A) Virtual trans-axial slice through an X-ray tomogram of a rat CCA obtained by micro CT. (B) Segmentation of the medial and adventitial layers was performed based on differences in grey level
texture using the following steps: i) unwrapping to straighten out the wall, ii) morphological opening and
island removal so that only features which are aligned mostly along the vertical direction are retained
(most features within the adventitia are removed) and iii) extraction of the contour between the medial
and adventitial layers. This is based on an optimization method to find the geodesic path through a weight
image. (C) The contour is re-wrapped onto the original slice. (D) Rendering showing the output of the
segmentation process which enables the medial and adventitial layers to be virtually dissected. abundance in the pressurised vessel (Fig. 4E,F). As a consequence of this remodelling, the cross-sectional
area (CSA) of the adventitia is significantly reduced in the pressurised vessel (medial CSA: 0 mm Hg:
65,258 ± 53 μm2, 110 mm Hg: 66,922 ± 76 μm2, p < 0.0001; adventitial CSA: 0 mm Hg: 72,658 ± 228 μm2,
110 mm Hg: 56,844 ± 82 μm2, p < 0.0001). Crucially however, it remains to be determined if comparable
adventitial remodelling would be evident either between physiological intra-luminal pressures and/or
in dynamic, rather than static, loading conditions. In order to understand the function of healthy and
diseased arteries and to design appropriate tissue engineered replacements it will be necessary to model
the mechanical behaviour of arterial tissues42. MicroCT has the potential to contribute to these models
by identifying localised remodelling events in mechanically deformed cardiovascular tissues and in other
organs such as aged and hence fragile skin43. MicroCT can Visualise Tissue Sub-Structures in Other ECM-Rich Organs. Skin is an organ which
is composed of two main tissue layers: an outer cell-rich and avascular epidermis and a supporting
ECM-rich dermis which also contains an extensive microvasculature and discrete structures such as hair
follicles. As with large arteries, skin is prone to sectioning induced artefacts (Fig. 6A). Whilst in skin, the
epidermis rarely separates from the dermis, both tissue regions are subject to sectioning-induced tears
and the separation and delamination of the outer stratum corneum. In contrast, microCT reconstructions
clearly show a closely adhered stratum corneum (Fig. 6B,C). Results and discussion Here we show that, compared with the unpressurised vessel, the
pressurised (at 110 mm Hg) rat common carotid artery (CCA) also exhibits significant structural remod-
elling (Fig. 4). From the segmented volume, it is apparent that both medial (0 mm Hg: 29.99 ± 0.02 μm,
110 mm Hg: 25.62 ± 0.69 μm, p < 0.0001) and adventitial (0 mm Hg: 31.12 ± 0.11 μm, 110 mm Hg:
20.55 ± 0.03 μm, p < 0.0001) layer thickness are significantly lower in the pressurised vessel (Fig. 5A,B). By segmenting the 3D X-ray tomogram data we can additionally measure the cross-sectional area (CSA)
of the lumen and medial and adventitial layers in each of the 450 slices in the tomogram (Fig. 5C–E). Wolinksy and Glagov12 reported a pressure-induced non-linear increase in vessel radius and a decrease in
medial layer thickness, here we show that intra-luminal pressure is associated with a greater lumen CSA
(0 mm Hg: 270,030 ± 256 μm2, 110 mm Hg: 404,043 ± 122 μm2, p < 0.0001). By preserving both major struc-
tural layers of the artery we additionally show that the adventitial layer is less uniform in morphology
than the medial layer and that the numerous pores (identified by their low X-ray density compared with
paraffin wax) which characterise the unpressurised adventitia are considerably reduced in volume and Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Semi-automatic segmentation of medial and adventitial layers and elastic lamellae in large
arteries. (A) Virtual trans-axial slice through an X-ray tomogram of a rat CCA obtained by micro CT. (B) Segmentation of the medial and adventitial layers was performed based on differences in grey level
texture using the following steps: i) unwrapping to straighten out the wall, ii) morphological opening and
island removal so that only features which are aligned mostly along the vertical direction are retained
(most features within the adventitia are removed) and iii) extraction of the contour between the medial
and adventitial layers. This is based on an optimization method to find the geodesic path through a weight
image. (C) The contour is re-wrapped onto the original slice. (D) Rendering showing the output of the
segmentation process which enables the medial and adventitial layers to be virtually dissected. Figure 3. Semi-automatic segmentation of medial and adventitial layers and elastic lamellae in large
arteries. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Results and discussion This densely packed barrier layer (which
is composed primarily of extracellular lipids and disulphide-bonded intracellular keratins), the cellular
epidermis, the ECM-rich dermis, hair follicles and potentially a branched micro-vasculature can all be
resolved by virtue of their relative X-ray density and architecture (Fig. 6B,C). The 3D architecture of each
reconstruction may also be examined by virtual sectioning of the volume at arbitrary angles to visualise,
for example, the structure of skin perpendicular (Video S2) and parallel with the surface (Video S3). In
this latter video the rhomboidal organisation of the stratum corneum, which is not evident in histological
sections, can be clearly seen as X-ray dense circles formed by cross-linked keratin-rich hair shafts. To our
knowledge this is the first demonstration that microCT can visualise such skin sub-structures. Previously, Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 5 www.nature.com/scientificreports/ entificreports/
microCT has been employed to either visualise skin in relation to other organs (whole limb m
Figure 4. Intra-luminal pressure-induced remodelling of rat common carotid arteries. (A and
Sections of vessels preserved unpressurised (A) and pressurised (B) states. (C and D) X-ray tomog
reconstructions of unpressurised (C) and pressurised (D) vessels. The medial and adventitial layers
segmented in yellow and in grey respectively. (E and F) Segmented pores (with an X-ray density lo
than paraffin: depicted as orange 3D volume projections) from unpressurised (E) and pressurised a
(F) mapped onto a 2D circumferential slice. (G and H) 3D projections of unwrapped luminal surf
extracted from reconstructions of unpressurised (G) and pressurised (H) vessels. The unpressurise
is characterised by a rippled appearance which is oriented along the axis of the vessel. Figure 4. Intra-luminal pressure-induced rem
Sections of vessels preserved unpressurised (A)
reconstructions of unpressurised (C) and pressu
segmented in yellow and in grey respectively. (E
than paraffin: depicted as orange 3D volume pro
(F) mapped onto a 2D circumferential slice. (G
extracted from reconstructions of unpressurised
is characterised by a rippled appearance which i Figure 4. Intra-luminal pressure-induced remodelling of rat common carotid arteries. (A and B)
Sections of vessels preserved unpressurised (A) and pressurised (B) states. (C and D) X-ray tomography
reconstructions of unpressurised (C) and pressurised (D) vessels. The medial and adventitial layers are
segmented in yellow and in grey respectively. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Results and discussion (E and F) Segmented pores (with an X-ray density lower
than paraffin: depicted as orange 3D volume projections) from unpressurised (E) and pressurised arteries
(F) mapped onto a 2D circumferential slice. (G and H) 3D projections of unwrapped luminal surfaces
extracted from reconstructions of unpressurised (G) and pressurised (H) vessels. The unpressurised surface
is characterised by a rippled appearance which is oriented along the axis of the vessel. microCT has been employed to either visualise skin in relation to other organs (whole limb microCT in
mice)44 or to localise foreign particles such as gunshot residues45. Histological and Immunohistochemical Procedures are Compatible with microCT. It is clear from
the data presented in Fig. 2–6 that even in the absence of exogenous X-ray contrast agents X-ray tomog-
raphy can resolve sub-structures in chemically fixed, paraffin embedded samples. By combining X-ray Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 6 www.nature.com/scientificreports/ Figure 5. Morphological characterisation of medial and adventitial layer dimensions in unpressurised
and pressurised arteries. Frequency distribution histograms of mean medial layer thickness (A) and
adventitial layer thickness (B). The cross-sectional area (CSA) of the medial (C) and adventitial (D) layers
and the lumen (E) can be rapidly calculated from segmented X-ray tomograms. Thicknesses and areas are
derived from a large tomographic volume (450 axial slices, axial length 338 μm). Figure 5. Morphological characterisation of medial and adventitial layer dimensions in unpressurised
and pressurised arteries. Frequency distribution histograms of mean medial layer thickness (A) and
adventitial layer thickness (B). The cross-sectional area (CSA) of the medial (C) and adventitial (D) layers
and the lumen (E) can be rapidly calculated from segmented X-ray tomograms. Thicknesses and areas are
derived from a large tomographic volume (450 axial slices, axial length 338 μm). tomography imaging with subsequent histological46 and immunohistochemical analyses it should be
possible to confirm the identity of discrete structures in X-ray tomograms. Exposure to synchrotron-
derived X-rays has been shown to induce thermal and/or oxidative damage to structural protein such as
collagen47. In this study we examined the effects of X-ray exposure alone (in our microCT system) and
X-ray exposure in combination with X-ray contrast agents on the binding of common histological stains
to skin sections. Haematoxylin and eosin (H&E) staining is widely used to distinguish between cellular
epidermis and ECM-rich dermis of mammalian skin. Results and discussion Here we used human skin biopsies to character-
ise: i) the ability of commonly used X-ray contrast agents (phosphotunstic acid [PTA] and inorganic
iodine [Lugol’s solution; I2KI]) to enhance differentiation of these tissue regions and ii) the effects of
X-ray tomography (of both unexposed and PTA- or I2KI-exposed biopsies) on subsequent H&E staining. Using both PTA and I2KI contrasting agents, good image contrast was achieved between the epidermis
and dermis in scans of less than 4.5 hours duration (Fig. 7A). However, as previously reported5 tissue
penetration of PTA was slow. Full penetration of a 3 mm diameter skin biopsy was not achieved after
40 hours exposure (Fig. 7B). Furthermore, whilst PTA exposure inhibited subsequent H&E staining, both
epidermal and dermal cells and the dermal matrix were clearly stained following X-ray and I2KI + X-ray
exposure. This inhibition of H&E staining may be due to blockage of aluminium binding sites (which
are utilised by Harris’ Haematoxylin). The success of H&E staining post-I2KI exposure could be due to
a lack of interaction between iodine and aluminimum binding sites or the loss of iodine ions prior to
H&E staining. tomography imaging with subsequent histological46 and immunohistochemical analyses it should be
possible to confirm the identity of discrete structures in X-ray tomograms. Exposure to synchrotron-
derived X-rays has been shown to induce thermal and/or oxidative damage to structural protein such as
collagen47. In this study we examined the effects of X-ray exposure alone (in our microCT system) and
X-ray exposure in combination with X-ray contrast agents on the binding of common histological stains
to skin sections. Haematoxylin and eosin (H&E) staining is widely used to distinguish between cellular
epidermis and ECM-rich dermis of mammalian skin. Here we used human skin biopsies to character-
ise: i) the ability of commonly used X-ray contrast agents (phosphotunstic acid [PTA] and inorganic
iodine [Lugol’s solution; I2KI]) to enhance differentiation of these tissue regions and ii) the effects of
X-ray tomography (of both unexposed and PTA- or I2KI-exposed biopsies) on subsequent H&E staining. Using both PTA and I2KI contrasting agents, good image contrast was achieved between the epidermis
and dermis in scans of less than 4.5 hours duration (Fig. 7A). However, as previously reported5 tissue
penetration of PTA was slow. Full penetration of a 3 mm diameter skin biopsy was not achieved after
40 hours exposure (Fig. 7B). Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Results and discussion Furthermore, whilst PTA exposure inhibited subsequent H&E staining, both
epidermal and dermal cells and the dermal matrix were clearly stained following X-ray and I2KI + X-ray
exposure. This inhibition of H&E staining may be due to blockage of aluminium binding sites (which
are utilised by Harris’ Haematoxylin). The success of H&E staining post-I2KI exposure could be due to
a lack of interaction between iodine and aluminimum binding sites or the loss of iodine ions prior to
H&E staining. g
Whilst it is important to show that a widely used stain such as H&E is compatible with prior exposure
to X-rays and X-ray contrast agents. This stain has low tissue specificity. The dermal ECM in human skin
is composed primarily of fibrillar collagens and elastic fibres48. These supra-molecular assemblies can be
specifically visualised by picrosirius red (PSR) staining and polarised light microscopy and by bright-field
light microscopy of Weigert’s resorcin fuchin stained tissue respectively49. Fibrillar collagens were readily
identified in sections cut from X-ray exposed biopsies and the binding of PSR to basic groups in the
collagen triple helix50 appeared unaffected by prior exposure to PTA or I2KI (Fig. 8A). However, the
staining of elastic fibres using Weigert’s (resorcin fuchin) stain which relies on the binding of iron, was
again disrupted by the presence of PTA (Fig. 8B). It appears therefore that although X-ray exposure alone
has little or no discernible effect on subsequent histological analysis the compatibility of specific combi-
nations of X-ray contrast agent and histological stain may need to be established on a case-by-case basis. 7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Sectioning induced artefacts and sub-micron X-ray tomography of human skin. (A) H&E
stained human skin biopsy. The dermal-epidermal junction is denoted by a red dotted line. (B and C)
Virtual slices extracted from an X-ray tomogram of a human skin biopsy. The dermal and epidermal layers
and large structures such as hair follicles are clearly visible and outer stratum corneum remains densely
packed and closely adhered to the underlying tissue. Figure 6. Sectioning induced artefacts and sub-micron X-ray tomography of human skin. (A) H&E
stained human skin biopsy. The dermal-epidermal junction is denoted by a red dotted line. (B and C)
Virtual slices extracted from an X-ray tomogram of a human skin biopsy. Results and discussion The dermal and epidermal layers
and large structures such as hair follicles are clearly visible and outer stratum corneum remains densely
packed and closely adhered to the underlying tissue. Crucially however, we also build on the observations of Chen and colleagues, who showed that microCT
is compatible with immunohistochemical analyses in frog cartilage51, to demonstrate that an anti-keratin
14 antibody can successfully and specifically bind to epidermal epitopes in human skin following expo-
sure to either X-rays alone or to both X-rays and X-ray contrast agents (Fig. 8C). Crucially however, we also build on the observations of Chen and colleagues, who showed that microCT
is compatible with immunohistochemical analyses in frog cartilage51, to demonstrate that an anti-keratin
14 antibody can successfully and specifically bind to epidermal epitopes in human skin following expo-
sure to either X-rays alone or to both X-rays and X-ray contrast agents (Fig. 8C). Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Conclusions Here we show that major tissue structures may be resolved by sub-micron X-ray tomography of native
tissues and organs and that histological and immunohistochemical analysis of tissue structure and com-
position is compatible with prior exposure to X-rays and some X-ray contrast agents. In the specific case
of the pressurised artery we demonstrate that static intra-luminal pressure has a differential effect on the
morphology and hence volume of the medial and adventitial layers. gy
y
It is clear therefore that, with little user intervention, microCT and nanoCT in combination can
rapidly visualise the 3D structure of relatively large tissue volumes (up to ~10 mm3) at sub-μm spatial
resolutions. Compared with complementary 3D visualisation approaches, such as serial-sectioning com-
bined with light or electron microscopy, or more recently developed technologies such as serial block
face / SEM approaches, X-ray tomography not only maintains the same spatial resolution in all three
axes but avoids artefacts induced by the loss, distortion and miss-alignment of sections52. In addition, as
X-ray tomography is non-destructive, it can be used to identify volumes of interest for subsequent com-
plementary microscopical and analytical analysis within a correlative tomography framework53. A key 8 Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 www.nature.com/scientificreports/ Figure 7. The use of contrast agents in the skin with X-ray tomography. Using the Carl Zeiss Versa-510
system unstained skin may be visualised. However, contrast is low and the differentiation of structures
within the skin is difficult. The use of PTA or I2KI contrasting agents improves the differentiation obtained
between structures in the skin. PTA provides the best contrast when looking at structures within the
skin, however, due to its molecular size, its rate of staining is slow and full penetration of the tissue is not
achieved, even after many hours (A). Further to this, if tissue that has been imaged using X-ray tomography
is then sectioned and stained with H&E to visualise skin structure, the expected staining pattern is disrupted
by the presence of PTA. However, X-ray exposure and the use of I2KI contrasting agent does not prevent
successful H&E staining of skin sections (B). Figure 7. The use of contrast agents in the skin with X-ray tomography. Using the Carl Zeiss Versa-510
system unstained skin may be visualised. However, contrast is low and the differentiation of structures
within the skin is difficult. Conclusions The use of PTA or I2KI contrasting agents improves the differentiation obtained
between structures in the skin. PTA provides the best contrast when looking at structures within the
skin, however, due to its molecular size, its rate of staining is slow and full penetration of the tissue is not
achieved, even after many hours (A). Further to this, if tissue that has been imaged using X-ray tomography
is then sectioned and stained with H&E to visualise skin structure, the expected staining pattern is disrupted
by the presence of PTA. However, X-ray exposure and the use of I2KI contrasting agent does not prevent
successful H&E staining of skin sections (B). area for improving the technique would be to image native tissues. Although, in the current study, the
use of chemical fixatives and a mechanical paraffin support provide a stable medium for the collection
of high quality (without movement artefacts) 3D data, chemical fixation can induce both micro- and
non-structural remodelling54,55. Previously microCT has been employed to characterise short-term (mil-
liseconds) mechanical behaviours of metals and long-term (days) developmental remodelling in living
insects56,57. It remains to be determined if microCT of native mammalian tissues is capable of resolving
acute structural remodelling events in tissue sub-structure(s) as a consequence of dynamic physiological
strains. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Materials and Methods Tissue preparation, X-ray contrast agent exposure and histological and immunohistochemi-
cal staining. All procedures accorded to the UK Animals (Scientific Procedures) Act 1986 and were
approved by the University of Manchester ethical review process. Pressure myography is conventionally
used to study small arteries58–60. In this study we adapted the technique by using stronger sutures, larger
cannulas and a surgical knotting technique to secure the rat CCA61. Left and right CCAs were dissected
from 250-300 g male Wistar rats (Charles River, UK). The right CCA was mounted onto a pressure
myograph (CH-1-QT, Living Systems: Vermont, USA). The left CCA was chemically fixed in the unpres-
surised state but the right CCA was subjected to an intra-luminal pressure of 110 mm Hg (equivalent to
mean arterial pressure)62 which was maintained during fixation. All arteries (left and right CCA and
descending thoracic aorta) were chemically fixed in 4% paraformaldehyde for two hours prior to etha-
nol dehydration and paraffin wax embedding using tygon tubing as a mould (Internal diameter 1.6 mm,
Harvard Apparatus, Kent, UK)63,64. The dimensions of the tubing were chosen to minimise the thickness
of the external paraffin wax. Finally wax cylinders were glued onto the head of metal pins so that the
vessel axis could be mounted perpendicular to the X-ray source.hf The 3D structure of human skin and the effects of X-ray exposure and X-ray contrast agents on
subsequent histological and immunohistochemical staining procedures were characterised using but-
tock skin punch biopsies (obtained with informed consent and local ethical approval from the North
West Research Ethics Committee [REC] reference 07/Q1409/9) and breast skin samples donated to the
Manchester Skin Health Biobank (REC reference 09/H1010/10). All experiments were carried out in
accordance with the approved local REC guidelines. Following fixation in Bouin’s solution, skin samples
were either: i) stained with Lugol’s solution (I2KI) for 16 hours followed by washing in water; ii) stained Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 9 www.nature.com/scientificreports/ t
Figure 8. The effects of X-ray tomography on subsequent histology and immunofluorescence of
structural components of the skin. Staining of tissue sections, from skin exposed to X-ray, for collagen
show that prior staining with PTA or I2KI does not prevent the visualisation of collagen fibres (A). However,
Weigert’s staining of skin for elastin fibres is disrupted by the presence of PTA (B). with phosphotungstic acid (PTA) for 40 hours followed by washing in 70% ethanol, or iii) left unstained
and submerged in 70% ethanol for 16 hours5. Materials and Methods Following data acquisition, volumetric data was reconstructed from the scan data using the FDK
algorithm66. Segmentation and morphological characterisation of 3D arterial reconstructions. Prior to
segmentation, noise in the data was reduced by applying a 1D bilateral filter in the axial direction67. The
vessel wall was then segmented in Avizo 8 using a conventional thresholding approach which relied on
differences in X-ray density (Fig. 3). However, as the component medial and adventitial layers exhibited
very similar X-ray densities, we developed a novel semi-automatic segmentation technique which
exploited differences in grey-level texture in the two layers. Specifically, the elastic lamellae in the medial
layer form concentric rings in the trans-axial plane, which become approximately straight lines when the
vessel is digitally ‘unwrapped’; whereas the structures in the adventitial layer are less well ordered. This
segmentation protocol was applied to each trans-axial slice in the 3D data set and comprised the follow-
ing stages (as depicted in Fig. 3). First, the vessel wall was unwrapped by applying a smoothing spline
(fitted to the image coordinates of the lumen edge) and resampling lines perpendicular to the spline
curve at equal intervals. Next, the wall was segmented from the supporting paraffin background by
applying a grey level threshold. The main adventitial features (radial connection and small groups of
connected pixels) were then removed by applying a 1D morphological opening operation along the
vertical axis (Fig. 3). Finally, the boundary between the medial and adventitial layers was established. The
outer internal elastic lamina was located by finding, for each row, the outer most segmented pixel (with
coordinate xe). In order to deal with residual adventitial features (which can cause deviations in the
boundary position) an optimisation approach was adopted, based on the fast marching method68,69,
which penalises large deviations from a vertical path. This method is related to Dijktra’s method for
computing the shortest path in a network, and in this case was used to find the geodesic path through a
weighted image. The weighted image was constructed by first determining the mean edge position along
the horizontal axis, x by fitting a Gaussian mixture model to the distribution of xe + 1. Pixels with coor-
dinates xe + 1 that were close to x were given extra weight; whereas segmented pixels from the initial
thresholded image were given a lower weight. Materials and Methods Unlike Weigert’s,
exposure to X-ray and PTA or I2KI does not prevent the binding of Keratin 14 antibody to its epitope in
skin, confirming that immunofluorescence is possible following X-ray tomography (C). Figure 8. The effects of X-ray tomography on subsequent histology and immunofluorescence of
structural components of the skin. Staining of tissue sections, from skin exposed to X-ray, for collagen
show that prior staining with PTA or I2KI does not prevent the visualisation of collagen fibres (A). However,
Weigert’s staining of skin for elastin fibres is disrupted by the presence of PTA (B). Unlike Weigert’s,
exposure to X-ray and PTA or I2KI does not prevent the binding of Keratin 14 antibody to its epitope in
skin, confirming that immunofluorescence is possible following X-ray tomography (C). with phosphotungstic acid (PTA) for 40 hours followed by washing in 70% ethanol, or iii) left unstained
and submerged in 70% ethanol for 16 hours5. with phosphotungstic acid (PTA) for 40 hours followed by washing in 70% ethanol, or iii) left unstained
and submerged in 70% ethanol for 16 hours5. Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 10 www.nature.com/scientificreports/ X-ray tomography and data processing. CCAs were imaged using a Carl Zeiss Xradia Versa-510
system (Carl Zeiss: California, USA) with the X-ray source voltage and current set to 60 kV and 83 mA,
respectively. The detector and source were positioned ~35 mm and ~10 mm respectively from the sample
to achieve a small amount of X-ray phase contrast using the inline method with the 4x objective30,65. Two
successive scans were performed for each sample: i) a low resolution data collection scan of the complete
artery cross-section using the 4x objective which achieved a voxel size of 0.75 μm and; ii) a higher reso-
lution region-of-interest scan of a smaller tissue volume which employed the 20x objective and achieved
a voxel size of 0.50 μm (detector to sample and sample to sources distances: 6.5 mm and 18 mm respec-
tively). This improved resolution using the 20x objective (Fig. 2D) would be necessary to measure the
dimensions of key structures such as the medial elastic lamellae. Exposure times per radiograph were
in the range 6-10 s for the 4x scan, and 26-35 s for the 20x scan. Approximately 2000 projections were
collected for each scan as the sample rotated over 360o. Materials and Methods Further steps were taken to ensure good data
quality; prior to scanning, each sample was placed within the scanner chamber overnight to ensure the
sample and mount had attained thermal equilibrium (28 °C). Furthermore, the source was switched on
for at least 30 minutes prior to each scan to improve stability. y
Multiscale X-ray tomography imaging of the artery samples was carried out using Carl Zeiss Xradia
Versa-510 (microCT) and Carl Zeiss Xradia Ultra-810 (nanoCT) systems. Gold beads of diameter 1-3 μm
were placed on the sample to enable the same region to be identified from all scans. The aorta sample
was scanned at two resolutions with the Versa XRM. The first, using the 4x objective, enabled the whole
vessel cross-section to be imaged at 0.94 μm voxel size. A second, higher resolution region-of-interest,
scan was carried out using the 20x objective yielding a voxel size of 0.5 μm. For both scans, the source
voltage was set to 60 kV and 2001 projections were taken over 360° with and exposure time of 4 s and
30 s respectively. Nano-scale imaging of the elastic lamellae and the inter-lamellar regions was then car-
ried out using the Ultra-810. This system is an X-ray microscope based on zone plate optics and has
two magnification modes giving 150 nm and 50 nm resolution respectively. The system uses X-rays with
energy 5.4 keV and can achieve Zernike phase contrast via the use of a phase ring35. A 65 × 65 × 65 μm
region-of-interest of the sample was scanned at 150 nm resolution using Zernike phase contrast and 1851
projections were taken over 180° with an exposure time of 40 s/projection. p
j
p
p
j
Buttock skin biopsies were imaged on a Carl Zeiss Xradia MicroXCT-400 system using the 4x objec-
tive, with a source voltage of 40 kV, a source current of 250 μA and an exposure time of 30 s/projection. The sample was positioned 8 mm from the source, and 45 mm from the detector. X-ray contrast-enhanced
(I2KI or PTA) breast skin samples were imaged on a Carl Zeiss Xradia Versa-510 system also using the
4x magnification. I2KI stained tissue was imaged with 3201 projections at an exposure time of 1.5 s/
projection, whilst PTA-stained tissue required only 601 projections at 1 s/projection (due to greater con-
trast). Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 Materials and Methods A geodesic path (which is attracted to large weights and
equates to the contour followed by the medial adventitial layer) can then be extracted. This contour can
then be wrapped onto the original trans-axial slices by applying the inverse of the coordinate transform
found in the first step. Finally, the surface of the medial layer boundary in the 3D stack was smoothed
to remove noisy features. The complete process was implemented in Matlab 2013a (Mathworks, Scientific Reports | 5:10074 | DOI: 10.1038/srep10074 11 www.nature.com/scientificreports/ Massachuesetts, USA). Some manual adjustments to the segmentation were necessary in regions where
the contrast was poorer, and this was achieved using the segmentation tools in Avizo 8. Despite this need
for occasional manual intervention, in general, segmentation of wall layers within a single microCT data
set can be achieved within 2-3 hours. Massachuesetts, USA). Some manual adjustments to the segmentation were necessary in regions where
the contrast was poorer, and this was achieved using the segmentation tools in Avizo 8. Despite this need
for occasional manual intervention, in general, segmentation of wall layers within a single microCT data
set can be achieved within 2-3 hours. Histological and immunohistochemical analysis. The effects of sectioning and intra-luminal
pressure on the rat CCA was characterised by hematoxylin and eosin (H&E: Sigma Aldrich, Milton
Keynes, UK) staining of 5 μm thick paraffin wax sections. Bright field optical images were captured using
a Biozero-8000 fluorescence microscope (Keyence; Osaka, Japan). Subsequent to X-ray tomography (of
both native and I2KI- or PTA- exposed), breast skin samples were sectioned, dewaxed, rehydrated and
stained for cells and ECM (by H&E; Harris’ Haematoxylin, Eosin Y, Sigma Aldrich, Dorset, UK), fibril-
lar collagens (picrosirius red; 0.1% Sirius red F3BA (Sigma Aldrich) in saturated aqueous picric acid
for 1 hour followed by a brief rinse in 0.1% acetic acid ) and elastic fibres (Weigert’s resorcin fuchsin;
(Clin-Tech Ltd, Guilford, UK; 30 minutes in Weigerts and then rinsed in IMS, and then in water)49. Sections were dehydrated and mounted with DPX mounting media (Sigma-Aldrich) before imaging. Picrosirius red staining was visualised using polarised light microscopy (Leitz DMRB microscope: Leica,
Milton Keynes, UK) with an Infinity X camera (DeltaPix: Maalov, Denmark), other stains were visual-
ised using the All-in-one Type Fluorescence Microscope Biozero-8000 (Keyence; Osaka, Japan). References Hemodynamic alterations in hypertensive obese rabbits. Hypertension 26, 465–470 (1995).i yp
4. Gasser, T. C., Ogden, R. W. & Holzapfel, G. A. Hyperelastic modelling of arterial layers with distributed collagen fibre orientations
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The authors are grateful to the staff of the Manchester X-ray Imaging Facility and the wish to acknowledge
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Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography. Sci. Rep. 5, 10074;
doi: 10.1038/srep10074 (2015). How to cite this article: Walton, L. A. et al. Morphological Characterisation of Unstained and Intact
Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography. Sci. Rep. 5, 10074;
doi: 10.1038/srep10074 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The
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Arsenic elevated groundwater irrigation: Farmers’ perception on rice and vegetables contamination in a naturally arsenic endemic area
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Arsenic elevated groundwater irrigation: Farmers’
perception on rice and vegetables contamination in a
naturally arsenic endemic area MD Rokonuzzaman
The Education University of Hong Kong
Ye ZH
Sun Yat-sen University
Wu C
The Education University of Hong Kong
LI WC
(
waichin@eduhk.hk
)
The Education University of Hong Kong The Education University of Hong Kong Research Article Keywords: groundwater arsenic, perception, rice and vegetables contamination, scalp hair, PCA
Posted Date: December 1st, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2320514/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
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License Page 1/21 Page 1/21 Abstract Arsenic (As) elevated groundwater irrigation for rice and vegetable cultivation, and its associated health threat is a
significant concern worldwide. Researchers are endeavoring to invent As mitigating strategies to combat this
terrible hazard; all their striving have ensued without adequate grassroots information about farmers' perception
of the As accumulation scenario in their crops. This study investigates Farmers’ perception and current status of
crop and body loading in naturally arsenic endemic regions. Results reveal that one fourth of the farmers giving
the positive message regarding the As contamination scenario in rice and vegetables. Although ten of farmers’
socioeconomic characteristics were positively significant, distinctive emphasize should be given to five predictor
variables explaining 88 percent variances such as knowledge, direct participation in farming, information sources
used, participant education, and organizational participation. Path analysis depicts that direct participation in
farming presents the highest positive total effect (0.855) and direct effect (0.503), whereas information sources
show the highest positive indirect effect (0.624). The mean As content in all five locations was statistically
significant at the 5%, 5%, 0.1%, 1%, and 1% probability levels in scalp hairs, rice, vegetables, soils, and irrigation
water, respectively. Ninety-two point five percent of the variation is explained by the first principal component
(PC1). Significant variations were primarily explained by As levels in irrigation water, rice grain, and soil. Farmers’
perception is far behind the actual field status of As level and its transfer. Therefore, intensified priorities should be
administered on the farmers' characteristics contributing to the variances in perception. 1. Introduction Groundwater arsenic (As) exceeding the permissible limit set by the WHO (< 10 µg/L) and FAO (100 µg/L) for
irrigation and its application for rice and vegetable production posed a potential health concern worldwide
(Akinbile and Haque, 2012; Chakraborti et al., 2018; Sarkar and Paul, 2016; WHO, 2004). While more than a
hundred countries worldwide are involved in the rice-growing practice, 90% of the world's rice is grown in just 14
Asian countries, with groundwater serving as the primary irrigation water supply in most (Elert, 2014; Mondal and
Polya, 2008; Yu et al., 2020). This problem is even more significant in the top five rice-producing countries such as
China, India, Bangladesh, Vietnam, and Indonesia (Ginting et al., 2018; Meharg et al., 2008; Nookabkaew et al.,
2013; Williams et al., 2007). Like rice, Half of the value of vegetable exports worldwide comes from Asia
(FAO/WITS, 2017). Therefore, these contributed to a higher risk of As poisoning from food consumption to both
the As endemic and non-endemic populations, regardless of where they live. Several research studies have already
proved that the consumption of rice and vegetables cultivated with As elevated groundwater is a potential
Contributor to the human body globally (Al Rmalli et al., 2005; Kumar et al., 2016; Roychowdhury et al., 2003;
Williams et al., 2006). Meghna River basin, the eastern and northern region of the active deltaic plain of the southern coast, and the old
deltaic plain of southwestern Bangladesh have all experienced high As occurrences; however, pollution is less
severe in the southeastern and northwestern part of Bangladesh (Paul, 2004). People living in high-risk areas knew
what to look for in terms of warning signs of As poisoning and ailments induced by drinking As-contaminated
groundwater (Akmam & Higano, 2002; Caldwell et al., 2003; Paul, 2004). Despite the fact that most of the people
in the endemic area of Bangladesh opted for As safe drinking, still, As related ailments are prevailing there (Huq et
al., 2006; Joseph et al., 2015). Consumption of rice and vegetables grown with As elevated groundwater is
supposed to cause this health concern (Kumar et al., 2016; Williams et al., 2006). To date, the majority of scientific
interest has been devoted to determining the sources and causes of arsenic contamination and inventing cost- Page 2/21 Page 2/21 Page 2/21 effective methods for removing arsenic from irrigation water (Das et al., 2008; Singh et al., 2015). 2.1 Study area The high groundwater contamination in the district of Chandpur in Bangladesh's southeast has made it a well-
known As endemic zone (Rahman, 2009). Chakraborti et al. (2010) found 100 to 1318 g/L As, while Jakaria et al. (2007) and Mishra et al. (2021) found that 80–90% of the tubewells in the area have As concentrations above 50
µg/L. According to estimates, more than 90% of the locals in this area rely on water from tubewells for both
drinking and irrigation needs (Jakariya et al., 2007; Rahman, 2009). Hajiganj, Chandpur Sadar (Sadar), Faridganj,
Matlab North, and Kachua, five of the most well-known and heavily As-elevated Upazila (sub-district) in Chandpur,
have been chosen as the study region (more information on the research site has been supplemented in Appendix
2.1A). 1. Introduction While
determining the source of the contamination and developing technologies to remediate As from groundwater are
critical in combatting the problem, research efforts should go far beyond these efforts to ensure the sustainability
of the technologies engaging farmers' perspectives (Khan et al., 2009; Kumar and Popat, 2010; Paul, 2004;
Pearson et al., 2018). Furthermore, all efforts to minimize arsenic pollution have been made without adequate
grassroots knowledge bases regarding the prime stakeholders, i.e. the farmers' perception of As accumulation in
rice and vegetables due to As contaminated groundwater irrigation. A recent scenario demonstrated that the
farmers' cooperation even seriously affected the implementation of the "policy of remediation during fallow (PRF)"
to tackle soil fertility deterioration due to heavy metal pollution by the government of China, which finally saw the
light after the survey of farmers' perspectives and associated recommendations (Yu et al., 2020). Therefore a
comprehensive approach for evaluating As perception is essential for establishing research priorities, ensuring
development strategies, and designing pertinent stakeholder engagement to combat the As-induced concern. This research aims to explore how rice and vegetable farmers in Bangladesh perceive As accumulation in their rice
and vegetables, its subsequent health consequences, alleviation possibility with mitigation strategies, and
investigate if there is an association between their socio-economic status and their level of perception. This
endeavor also attempts to compare farmers’ perception based on the actual field based As accumulation data. The study's findings should be useful in pinpointing particular socioeconomic and demographic features where
further steps need to be intensified for sustainable As mitigation and farmers' adoption of the same based on the
actual As status of irrigation water and its subsequent transfer to the rice and vegetables. The purpose of this
study is not to investigate whether farmers had heard of As pollution problem in general, but to explore if they are
knowledgeable and have the correct perception about crucial aspects of arsenic poisoning, such as the source of
As in rice and vegetables, its symptoms, diseases caused by poisoning, and how to prevent and mitigate As
poisoning. 2.2 Collection of data Multistage purposive sampling has been applied to select study locations and the respondents. Purposive
sampling has the advantage of allowing researchers to gain a better understanding of the study's research
problem and study sites (Palinkas et al., 2015). Furthermore, identifying and choosing the right people or groups is
a necessary step in this process who are remarkably experienced or knowledgeable regarding the subject matter
(Cresswell & Plano Clark, 2011). In addition to experience and knowledge, Bernard (2002) and Spradley (1979) Page 3/21 Page 3/21 worth noting the significance of willingness, availability to participate, and the capability of experience and
opinions communication in an articulate, reflective, and expressive manner. Forty (40) farmers from each of the
locations (totaling 200) fulfilling specific criteria, such as actively participating in farming, irrigating groundwater
for rice and vegetable cultivation, consuming rice and vegetables produced in their fields, and drinking As safe
water purposively sampled in this study. 2.3 Samples collection and preparation Ten farmers were purposively selected from each location to collect their scalp hair samples as well as four sub-
samples of vegetables, rice, soils, and irrigation water from their fields to construct composite samples (Khan et
al., 2008) (Appendix 2.3A includes a supplementary page with information on the plant species and the procedure
for collecting samples). Rice samples were separated from the chaff. After being cleaned for 5 minutes in running
water and rinsed twice with deionized water, the vegetables sample were patted dry with filter paper, and then dried
in an oven at 60°C for 24 hours (Liu et al., 2010). Prior to chemical analysis, the vegetable and rice samples were
ground using a carnelian mortar. Male farmers in the area often get regular haircuts, making it possible to
measure As concentrations in hairs from recent exposure, while women's long hair is better suited to chronicling
more extended periods of exposure; therefore, only samples from male farmers' scalps were taken using stainless-
steel scissors (Bang et al., 2009). To standardize As levels across the scalp, 1 gram of hair was obtained from
each scalp site on the same person (Hindmarsh, 2000; Mazumder, 2000). Aluminum foil-wrapped samples were
transported to the laboratory and stored at -20°C in zip lock bags pending chemical analysis (Liu et al., 2015). To
get rid of any debris that might have been stuck to the sample, the samples were double rinse with 5 mL of
deionized water and methanol (Hinwood et al., 2003). 2.4.1 Farmers socioeconomic Characteristics Farm size = A+{1/2(B + C) + E + F}-D Farm size = A+{1/2(B + C) + E + F}-D Where A = land used for own farming; B = land given to others on borga; C = land appropriation from others on
borga; D = land given to others on the lease; E = land taken from others on the lease; F = homestead area. Where A = land used for own farming; B = land given to others on borga; C = land appropriation from others on
borga; D = land given to others on the lease; E = land taken from others on the lease; F = homestead area. Income from farming and other ethically right sources on a regular basis over a year was regarded as annual
income and is expressed in thousand (1000.00) taka. Similarly, agricultural credit use was expressed in thousand
takas. An adapted version of the Subramanium (1986) scale was used to assess social/organizational
participation. The scale comprised of ten statements that indicated the respondent's involvement with
organizations both within and outside his living community (Kumar and Popat, 2010). The score given for no
membership = 0; membership in one organization = 1; and office-bearer in one organization = 2. Accordingly,
attending meetings 'Never,' 'Occasionally,' and 'Regularly' received 1, 2, and 3 points. To obtain a respondent's final
scores, the scores obtained as a member or office bearer were multiplied with the score received for attendance to
meetings. The cosmopoliteness item was predicated on an individual's orientation outside of his social structure. A 6 item (4-point scale) statement was prepared for this purpose. Each participant was required to mention how
many times he traveled to each of the six places with the frequency of visit such as 'often,' 'occasionally,' 'rarely,'
and 'never,' and weights assigned to these responses were 3, 2, 1, and 0, respectively. A respondent's score of
cosmopoliteness was determined by summing the points allotted for the six different types of locations he has
visited. Opinionatedness of a farmer was measured through a four items scale prepared for the study. A Score of
3, 2, 1, and 0 was assigned for high, medium, low, and no opinionatedness. A respondent's innovativeness was
assessed based on the relative earliness in adopting new ideas (Rogers, 1995); here, 13 improved ideas on As
reducing agricultural practices. 2.4.1 Farmers socioeconomic Characteristics Scores were provided based on how long it took a farmer to adopt each technique,
such as 5 = within one year, 4 = within two years, 3 = within three years, 2 = within four years, and 1 = within five
years, however, 0 = do not use. A farmer's innovativeness score was calculated by aggregating his scores for all 13
improved agricultural techniques. Risk orientation was assessed using a scale modified from Samantha's (1977)
scale. The scale comprised ten statements, four positive and the rest negative, based on Edwards' (1957)
screening guidelines. Respondents' opinions were recorded using a 5-point Likert scale (Likert, 1932) with positive
statements assigned values of 5, 4, 3, 2, and 1 and negative statements assigned values of 1, 2, 3, 4, and 5
representing 'strongly agree,' 'agree,' 'Undecided, 'disagree,' and 'strongly disagree', respectively. For calculating the
ownership score of farm power and machinery (FPM), seven items of farming and irrigation management tools
were selected, and the score was assigned for the possession of each country plow = 1, hand sprayer = 2, rice
weeder = 1, shallow tubewell (STW) (joint ownership) = 3, power tiller = 4, shallow tubewell (STW) (single
ownership) = 4, and harvester = 4. The number of tools was multiplied by the assigned score to obtain the final
score. Farmers' knowledge was assessed based on the method used by Paul (2004) with little modification. For
each participant, a composite score was computed based on their responses to 11 questions (into six groups)
about the source, symptoms, and As induced diseases, as well as potential preventive approaches and remedies
to the arsenic accumulation problem in crops. One focus group discussion (FGD) was held in each Upazila to Income from farming and other ethically right sources on a regular basis over a year was regarded as annual
income and is expressed in thousand (1000.00) taka. Similarly, agricultural credit use was expressed in thousand
takas. An adapted version of the Subramanium (1986) scale was used to assess social/organizational
participation. The scale comprised of ten statements that indicated the respondent's involvement with
organizations both within and outside his living community (Kumar and Popat, 2010). The score given for no
membership = 0; membership in one organization = 1; and office-bearer in one organization = 2. Accordingly,
attending meetings 'Never,' 'Occasionally,' and 'Regularly' received 1, 2, and 3 points. 2.4.1 Farmers socioeconomic Characteristics To obtain a respondent's final
scores, the scores obtained as a member or office bearer were multiplied with the score received for attendance to
meetings. The cosmopoliteness item was predicated on an individual's orientation outside of his social structure. A 6 item (4-point scale) statement was prepared for this purpose. Each participant was required to mention how
many times he traveled to each of the six places with the frequency of visit such as 'often,' 'occasionally,' 'rarely,'
and 'never,' and weights assigned to these responses were 3, 2, 1, and 0, respectively. A respondent's score of
cosmopoliteness was determined by summing the points allotted for the six different types of locations he has
visited. Opinionatedness of a farmer was measured through a four items scale prepared for the study. A Score of
3, 2, 1, and 0 was assigned for high, medium, low, and no opinionatedness. A respondent's innovativeness was
assessed based on the relative earliness in adopting new ideas (Rogers, 1995); here, 13 improved ideas on As
reducing agricultural practices. Scores were provided based on how long it took a farmer to adopt each technique,
such as 5 = within one year, 4 = within two years, 3 = within three years, 2 = within four years, and 1 = within five
years, however, 0 = do not use. A farmer's innovativeness score was calculated by aggregating his scores for all 13
improved agricultural techniques. Risk orientation was assessed using a scale modified from Samantha's (1977)
scale. The scale comprised ten statements, four positive and the rest negative, based on Edwards' (1957)
screening guidelines. Respondents' opinions were recorded using a 5-point Likert scale (Likert, 1932) with positive
statements assigned values of 5, 4, 3, 2, and 1 and negative statements assigned values of 1, 2, 3, 4, and 5
representing 'strongly agree,' 'agree,' 'Undecided, 'disagree,' and 'strongly disagree', respectively. For calculating the
ownership score of farm power and machinery (FPM), seven items of farming and irrigation management tools
were selected, and the score was assigned for the possession of each country plow = 1, hand sprayer = 2, rice
weeder = 1, shallow tubewell (STW) (joint ownership) = 3, power tiller = 4, shallow tubewell (STW) (single
ownership) = 4, and harvester = 4. The number of tools was multiplied by the assigned score to obtain the final
score. 2.4.1 Farmers socioeconomic Characteristics With great care, a structured interview schedule was constructed and translated into Bangla to facilitate
information-gathering from the native speakers (Kumar and Popat, 2010). The data were collected through face-
to-face interviews from June 2019 to August 2020. In order to characterize the socioeconomic backgrounds of
farmers, sixteen variables were assessed, such as age, family education, participant education, farm size,
knowledge, annual income, family size, information sources, direct participation in farming, agricultural credit use,
cosmopoliteness, opinionatedness, innovativeness, risk orientation, farm power and machinery (FPM),
organizational participation. The number of years from the farmer's birth to the interview was used to compute his age and was rounded to the
nearest whole number. Regarding farmer's education, a score of 1 was assigned for each class passed. In order to
calculate the family education, the overall score on education was recorded and then divided by the 'effective
family size' (Pareek and Trivedi, 1964). The 'effective family size' was calculated by subtracting the number of
children under the age of four from the total number of family members. The following formula was used to
generate the Index of family education, which was used to quantify family education. Index of Family Education = Total educational score/Effective family size Family size was determined as the total number of individual farmers' family members. Direct participation in
farming was measured by how a farmer performs agricultural work by himself rather than others. An individual Page 4/21 Page 4/21 farmer could get a score of 0 to 3 for each agricultural operation. The score of the participants could range from 0
to 18, where 0 indicates 'no direct participation' and 18 'high direct participation' in farming. A 5-point scale
checking any of the responses- most often, often, sometimes, rarely, and never with scores 4, 3,2,1 and 0
respectively were provided against each item to measure the degree to which the farmers used information
sources. The total rank score for each item was obtained by multiplying the frequencies with the respective
weights and adding them up. Farm size was computed using the following formula and expressed in hectare (ha). 2.4.3 Sample analysis and quality control Atomic fluorescence spectrophotometry (AF-610A, manufactured in Beijing, China) was used to measure the
content of As in vegetables, grains, soils and water adopting the protocol followed by Huang et al. (2006). Exactly
0.25 g of soil was weighed and a few drops of deionized water were added to a 100 ml Erlenmeyer flask to
quantify soil total As. The soil was mixed with 2 ml of concentrated HClO4, 5 ml of HNO3, and 6 ml of HCl. Over
the course of about 1.5 hours, the reaction died down while the digestion continued at 150 degrees Celsius in the
flask covered with a little glass filter heated by an electric heater. A 50 A volumetric flask (50 ml) was used to
combine 5 ml of sulfourea solution (50 g/L) with the digest, and the remaining volume was filled with double
deionized water. not more than 0.5g of rice grain or 0.5g of vegetables (0.5mm) was placed in an Erlenmeyer flask
(100 ml) with 1.25 ml of concentrated H2SO4 and 20 ml of HNO3 to measure As in rice or vegetables, separately. A
gentle boiling digestion was performed using an electric heater after an overnight reaction. Once again, 5 ml
concentrated HNO3 was added to nearly 10 ml digested brown solution for repeated digestion. When the digestion
was still not complete after 2/3 additional HNO3 digestions, concentrated HClO4 was added (2 ml) for further
digestion. However, 3–4 hours was needed for the entire digestion process. The digest was transferred to a 25 ml
measuring flask, spiked with 2.5 ml of a sulfourea solution (50 g/L), and the volume was made with double
deionized water. The As level in hair samples was assessed with a hydrogen generation-atomic fluorescence
spectrometer (AFS-820, Beijing Titan Instruments Co., China) after the samples were digested with a 1:4 mixture of
HClO4 and HNO3 based on the procedure used of Liu et al. (2010). The samples were digested, and then 2% HCl
was used to dissolve the remnants. All the reagents used were of an analytical grade or better. Tomato leaves
(NIST 1573a) were used as a certified reference material during the quality assurance process. The range of
values found was 0.109 µg/g to 0.120 µg/g, against the certified value 0.112 0.004 µg/g. 2.4.2 Farmers’ perception assessment According to Hodgetts (1979), no two people will have the same perception of life, and no two people will see
things in the same way. For recording farmers’ perception, appropriate statements were prepared with the
cooperation of researchers, farmer leaders, available rice and vegetable growers, and agriculture officer and
validated with data from a field survey (Kumar and Popat, 2010). After subjecting these statements to judges’
rating (Rekha and Ambujam, 2010), the interview schedule contained 43 statements under six groups and was
administered to the respondents for expressing their perceptions on the use of As contaminated or safe water for
rice and vegetable production. To avoid acquiescence, the propensity of participants to agree or disagree with
statements irrespective of the item content, the interview schedule was constructed with both negative and
positive statements. According to Schweizer et al. (2011), using negative and positive statements when replying to
questions helps to avoid phrasing problems and responder personal bias. However, the statements were rated on a
five-point Likert scale (Likert, 1932) where ‘strongly agree,’ ‘agree,’ ‘undecided, ‘disagree,’ and ‘strongly disagree’
were scored with 1, 2, 3, 4, and 5, for negative statements and 5, 4, 3, 2, and 1 for positive statements, respectively. 2.4.1 Farmers socioeconomic Characteristics Farmers' knowledge was assessed based on the method used by Paul (2004) with little modification. For
each participant, a composite score was computed based on their responses to 11 questions (into six groups)
about the source, symptoms, and As induced diseases, as well as potential preventive approaches and remedies
to the arsenic accumulation problem in crops One focus group discussion (FGD) was held in each Upazila to Page 5/21 Page 5/21 establish the scores for anticipated answers consisting of farmer leaders, available rice and vegetable growers,
and Agriculture officers. The participants' recommendations were considered to provide various points for correct
answers and a zero for incorrect ones. 3.1 Farmers’ socioeconomic characteristics The summary of farmers' socioeconomic characteristics have been supplemented in Table 1 (supplementary
sheet). Almost two-thirds of the participants were under middle-aged to old-aged group while 34 percent could be
categorized into young age group in this study. The rural youth's paradigm shift is clearly articulated in terms other
than agriculture (Rekha & Ambujam, 2010). Education is the process by which desired changes in human behavior
takes place. It is primarily supposed that a higher level of education should influence farmers to be aware of and
critically evaluate the consequences of As contaminated groundwater irrigation. Two-thirds of the respondents (66
percent) and slightly over fifty percent of their family members had primary and low to medium education,
respectively, while 26 percent of participants passed secondary to above secondary classes. It could be seen that
only 8 percent of respondents and 22 percent of the family members were illiterate. Less than half (42 percent) of
participants had small families, while 31 percent had large families. On the other hand, The knowledge status of
the respondents showed that no less than 50 percent of farmers lack adequate knowledge of As and its impact on
rice and vegetable cultivation with contaminated groundwater, while 34% possess high knowledge. All the
participants in the study area had basic knowledge regarding the groundwater contamination with As used for
drinking water due to substantial awareness-building circulation from government and non-government
organizations in the past decades. However, the knowledge differences were created with the advanced aspect
regarding the crop contamination due to As elevated groundwater irrigation. The family size also influences the
farmers' perception of groundwater irrigation. More than half (58 percent) of the farmers had small, 29 percent
had medium, and only 4 percent possessed large (3.01-6.00 ha) farm holdings, which are the collective
possession from own and others land in borga. Farmers with larger farms are predicted to be more eager to
convert their land to irrigated fields to minimize their loss rather than keeping the land barren (Rekha and
Ambujam, 2010). The result also revealed that the farm size largely determined the annual income of the
participants. Nearly 60 percent of the respondents had very low to medium-income mainly derived from
agriculture, particularly rice and vegetables. Of the rest, 19 percent had high, and 20 percent had very high annual
income from some business in addition to agriculture. 2.5 Statistical analysis Prior to analysis, collected data were encoded, entered into a Microsoft Excel 2019 spreadsheet, and double-
checked for mistakes. SPSS 20.0 was used to analyze the data. Cross-tabulation in Excel was used to calculate Page 6/21 Page 6/21 Page 6/21 descriptive statistics such as percentages and frequencies (Kumar and Popat, 2010). Mean, median, and standard
deviation (SD) had been used to categorize farmers into low, medium, and high groups (Kumar and Popat, 2010). The Pearson correlation coefficient (r) was employed to analyze the correlation between dependent and
independent variables (Adam et al., 2015). This study included a stepwise multiple regression analysis to
determine the socioeconomic parameters influencing perception in the research area (Udayakumara et al., 2010). Path analysis was carried out to determine independent variables' influence and path effect on farmers' perception
(Netuveli and Bartley, 2012). Using R Statistics Software, version 3.5.3, two-way analysis of variance (ANOVA) and
a least-significant-differences (LSD) test were conducted. Principle component analysis (PCA) was carried out
using Minitab 18 statistical software (Shakoor et al., 2018). 3.1 Farmers’ socioeconomic characteristics Cosmopoliteness influences farmers' perception since it enables them to be introduced to the latest technologies
by exploring neighboring localities, towns, and abroad. Nearly half (48 percent) of the participants had low
cosmopoliteness, followed by 32 percent with high cosmopoliteness. Similar to the cosmopoliteness, distribution
of the farmers based on the information sources exposure showed less than half (48 percent) of the participants
had a low level of information sources exposure, followed by 52 percent had medium to a high level to get the Page 7/21 latest agriculture information. The farmers' educational status would have influenced the exposure to information
sources. In addition, the information technology revolution had a profound impact on the farming communities. All the farmers in this study had active participation in the agricultural and farm management activities; however,
they were categorized based on their extent of involvement. More direct participation in farming enhances the
actual field-based knowledge and experience and increases farm productivity due to the close observation and
management possibility. Over half (57 percent) of the participants had medium to high direct participation in
farming in their crop production, and the rest required some support from others for cultivation activities. Opinionatedness allows a farmer to exercise leadership capacity for the fellow crop growers regarding several
decision-making processes, including crop variety selection, irrigation management, and intercultural operations. Nearly 50% of participants had low opinionatedness, 27 percent had medium, and 24 percent had high
opinionatedness. Regarding agricultural credit use, mostly half (49 percent) of the farmers did not use any credits;
only 7 percent had low use, while 22 percent received medium and high credits for rice and vegetable production. Different banks, NGOs, cooperative organizations, and businessmen provide the credits. Although presumed as
the financial support for the initial period, the higher interest finally captures them into the trap for most cases. The organizational participation based farmers’ distribution depicts that approximately half (49 percent) of the
participants had low, one-third had high, and 18 percent had medium participation with different organizations. Organizational participation facilitates social networks to promote the information flow, which stimulates farmers'
perceptions and decision-making on agricultural management (Bouma et al. 2008; Kilelu, 2004; Owusu et al.,
2012). Farmers' innovativeness in the adoption of As mitigation irrigation management and other practices in the
study area was evaluated. 3.1 Farmers’ socioeconomic characteristics It elucidates that almost half (49%) of the respondents have no innovativeness,
followed by 26 percent have medium level, and 25 percent have high innovativeness. The ownership of
agricultural machinery largely determines the freedom of production management, especially the irrigation
practice with a specific strategy. The respondents mainly had similar agricultural machinery where 43% and 31%
possessed a medium and higher number of irrigation management tools. Table A.1 (supplementary sheet) also
demonstrates that almost one-third of farmers had individual low, medium, and high-risk orientations. Those who
had higher educational status, information sources used, and high organizational participation had a higher level
of risk orientation (Rekha and Ambujam, 2010). In addition to the above, this study revealed that higher ownership
of FPM also influences farmers' risk orientation. However, this psychological character influenced farmers'
perception and the adoption of the As mitigating strategy. 3.2.1 Perception on As-contaminated or groundwater (AsW) or
As free water (AsFW) use Nearly two-thirds (62 percent) of the respondents strongly agree, and one-fourth agrees that no AsW means no
rice/vegetable cultivation (Table 3 in supplementary sheet). They opined that AsW is available throughout the year
for crop cultivation in their locality while AsFW is seasonal. Apart from this, an overwhelming (89 percent) of
respondents still debated not using the AsFW in their fields. This might be because although they are aware of the
drinking water As contamination, the majority of them still lack proper knowledge regarding the possible crop
contamination with As. On the other hand, only 19 percent of farmers believe in the possibility of rice and
vegetable cultivation with AsFW. The explanation for such a stance is that they possess comparatively larger farm
holdings with adequate irrigation management tools. 3.2 Farmers’ perception According to McGraw-Hill (2004), perception is the process by which sensory stimuli are registered as meaningful
experiences, while Epstein et al. (2018) understand perception as the way of dispersing stimulation through
structured experiences. Perceptions are more sophisticated constructs made up of simple pieces connected by
association and are therefore more susceptible to the influence of learning. Though the senses of taste, hearing,
touch, and smell have all been investigated, the vision has garnered the most interest. Perception is the process of
becoming aware of or comprehending sensory information in psychology, philosophy, and cognitive science
(McGraw-Hill, 2004). Table 2 demonstrates that 25 percent of the farmers possess high perception in the study
area regarding As contamination in rice and vegetables due to contaminated groundwater irrigation, drivers of
irrigating As elevated groundwater, it's possible mitigation strategies and health impact. Page 8/21 Table 2
Farmers’ perception on arsenic contaminated groundwater irrigation for rice
and vegetables production (N = 200)
Category
Percent
Mean
Standard Deviation
Low perception (129–136)
39
Medium perception (137–155)
36
146.6
14.16
High perception (157–178)
25
Total
100 Table 2
Farmers’ perception on arsenic contaminated groundwater irrigation for rice
and vegetables production (N = 200) Table 2 On the other hand, 36 percent of them have a medium, and 39 percent have low perception levels. After a
comprehensive assessment of farmers’ awareness regarding As in drinking water and foods, Mishra et al. (2021)
reported that Bangladeshi farmers have comparatively high awareness regarding As in drinking water rather than
in the foods they consume. A total of 43 statements under seven groups were administered to get a detailed
understanding of farmers' perceptions (Table 3 in supplementary sheet). All the farmers responded to each of the
statements from their learned experiences. A brief overview has been presented under seven subsections below. 3.2.2 Drivers for irrigating AsW Easy accessibility is the prime cause for AsW use, is unequivocally declared by all the participants in this study. Nearly 98 percent of the respondents claimed that they prefer irrigating their crop fields with some shareholders to
reduce the production cost. This prevalent scenario of field irrigation practice threatens the choosy irrigation
management in this study area. The scarcity of the AsFW (e.g., surface water), particularly during the winter
season, compels them to go for groundwater irrigation. Another reason for using AsW is the saving purpose of the
AsFW for household use, as reported by 26 percent of the respondents. Only 3 percent of the farmers are self-
sufficient to irrigate with their own pump and manage irrigation as per their choice. 3.2.7 Farmers' practiced As mitigation strategy Nearly one-third of farmers perceive that alternate wetting and drying (AWD) and surface water irrigation can
reduce As accumulation in rice and vegetables. Seven percent of the participants believe that raised bed rice
cultivation would limit As loading in rice grains. A very insignificant part (1–2 percent) of the participants perceive
fertilizer management, such as supplementing with more urea, MoP, gypsum, zinc sulphate, cow dung, and
intercultural operations such as mulching in vegetable fields or spreading Ash would limit As accumulation. 3.2.5 Impact of fertilizers and pesticides on As addition Application of pesticides (Campos, 2002) and fertilizers, especially Phosphate fertilizer, (Jayasumana et al., 2015)
may escalate As levels in the crop fields. Almost all the respondents were undecided since they did not get such
information from any media or social networking. 3.2.6 Health impact From their knowledge of groundwater As contamination and knowledge about the As related health impact from
the drinking water exposure, 7 percent agreed, and 35 percent of the farmers highly agreed with the possible As
transfer to the human body due to As elevated rice and vegetables consumption. However, more than fifty percent
of the respondents remained undecided. Similarly, 45 percent of the participants perceive As may cause cancers,
while 39 percent agreed on the development of skin lesions. participants observed their irrigation channel became red, 40 percent reported yield loss near the channel, and lan
became hard.
3.2.4 Effect of AsW irrigation on rice & vegetables participants observed their irrigation channel became red, 40 percent reported yield loss near the channel, and land
became hard. participants observed their irrigation channel became red, 40 percent reported yield loss near the channel, and land
became hard. 3.2.4 Effect of AsW irrigation on rice & vegetables Only 19 percent of the respondents believe in the As accumulation in rice & vegetables upon As contaminated
groundwater application. The level of education, organizational participation, information source exposure, and
cosmopoliteness enhanced their knowledge regarding this issue and influenced their perception. More than 95
percent of farmers were undecided about the other parameters such as the impact on tillering, influence on plants'
height, uniformity of flowering, plant growth and grains maturity, grains filling percentage, or yield reduction. However, only 2–4 percent of participants agree with those advanced symptoms. 3.2.3 Effect of AsW irrigation on crop fields While demonstrating the impact of AsW irrigation on crop fields from their experiences, two-thirds of the farmers
remained undecided whether the AsW led to add additional As in their crop fields or not, although the rest one-third
believed in As addition. Similarly, four-fifth of the farmers were undecided regarding the fertility loss of their crop
fields with As incorporation due to groundwater irrigation. On the other hand, slightly over 50 percent of the Page 9/21 Page 9/21 3.3 Correlations Correlation coefficients between the independent and dependent variables has been estimated (Table 4 in
supplementary sheet); and Table 5 (in supplementary sheet) shows the correlation matrix representing the overall
interaction between the variables. According to Table 4 (supplementary sheet), among the socioeconomic
characteristics, farmers' age, annual income, family education, family size, farm size, and agricultural credit use
were non-significant. In contrast, farmers' age and family size were negatively correlated with their perception of
As elevated groundwater irrigation for rice and vegetable production. The study of Alam (2001) and Kabir (2002)
revealed a negative correlation of family size with perception, while Majlish (2007) reported a non-significant
correlation. Afique (2006), Pal (2009), and Adeola (2012) revealed that farm size had no discernible effect on
farmers' perceptions. Friedler et al. (2006) argued that there was no correlation between the age or income of
farmers and their perceptions. Islam (2000) observed no association between farmers' utilization of credit and
their perception. Page 10/21 On the other hand, farmers' education, knowledge, information sources, direct participation in farming,
cosmopoliteness, opinionatedness, innovativeness, risk orientation, farm power and machinery (FPM), and
organizational participation were positively significant with perception at a 1% significance level (shown in Table 4
in supplementary sheet). Pal (2009) revealed that farmers' education positively correlates with their perception. Kabir & Rainis (2012) and Adeola (2012) also found that education significantly affects farmers' perceptions in
Bangladesh and Nigeria. Individuals with higher education levels usually perceive risks and understand mitigation
necessity in a very advanced way (Dosman et al., 2001). In their survey in Gujarat province in India, Kumar & Popat
(2010) exposed that knowledge, a psychological characteristic of the farmer, had a significant positive association
with their perception. The study of Adeola (2012) reported similar findings in Nigeria. The farmers' information
sources can play a crucial role in building positive or negative perceptions of any phenomenon. Rezaei et al. (2017) claimed a significant relationship between farmers' exposure to the information sources and their
perception. Farmers engaged in farming activities helps determining their decision-making capacity in any
circumstance (Larsen et al., 2002; Rahaman et al., 2018). Therefore, direct farming participation had a significant
relationship with farmers' perceptions (Rokonuzzaman, 2016). Islam (2000) revealed a significant positive
correlation between farmers' perception and annual income. Regarding the association between farmers' ownership of FPM and their perception, through their study in the
water markets in Bangladesh, Mottaleb et al. 3.3 Correlations (2019) demonstrate that irrigation pump ownership largely
determines farmers' perception. However, they concluded that since the irrigation system in Bangladesh is mainly
based on pumping underground water, pump ownership significantly influences the structure and choice of
irrigation practices. Regarding the relationship between organizational participation and perception, Keshavarz
and Karami (2013) reported that membership in social organizations positively influences farmers' perceptions. Membership in formal or informal organizations helps the farmers get benefits and social support (Fuller-Iglesias
et al., 2009). Segnestam (2009) argued that organizational participation helps disseminate innovations and
develop mutual trust among the farmers, which eventually shapes farmers' perceptions. While studying
cosmopoliteness, Alam (2001) noted a significant positive association between farmers' cosmopoliteness and
their perception. According to Hamid (1995), there is a significant relationship between cosmopoliteness and
farmers' use of the recommended level of plant protection practices. Farmers' opinionatedness and perception
were found to have a significant positive association in the study of Islam (2000). Londhe et al. (2018) discovered
a substantial positive relationship between perception and participants' risk orientation and innovativeness. The
study of Rekha & Ambujam (2010) in Tamil Nadu, India, about the farmers' perception of contaminated water
irrigation revealed a significant positive correlation between farmers' perception and their educational status,
information sources, annual income, farm size, risk orientation, and innovativeness. 3.4 Regression results Predictor variables (independent variables) that explain farmers' perceptions (the dependent variable) were
determined using a stepwise multiple regression analysis. Table 6 illustrates the findings of stepwise regression. The total variance explained by the five independent variables is 0.884 (R = .889, R2 = 0. 884), as seen in this table. Of the total variance, participants' knowledge explained 74.6%, direct participation in farming 8.2%, information
sources 4.5%, participant education 0.7%, and organizational participation 0.8%. The F value for participants'
knowledge, direct participation in farming, and information sources are significant at 0.1% level, while for
participants' education and organizational participation are significant at 5% level. This means that the five
recognized predictor variables account for 88 percent of the variance in the dependent variables. Page 11/21 Page 11/21 Page 11/21 Table 6
Regression of the estimated perception on the independent variables (N = 200)
Variables
R
R
Square
Adjusted R
Square
Std. Error of the
Estimate
R Square
Change
F
Change
Sig. F
Change
Participants
knowledge
.865
.748
.746
7.140
.748
291.373
.000
Direct participation
in farming
.911
.830
.826
5.899
.082
46.587
.000
Information sources
.935
.875
.871
5.089
.045
34.297
.000
Participant
education
.939
.882
.877
4.973
.007
5.533
.021
Organizational
participation
.943
.889
.884
4.832
.008
6.638
.012
3 5 P
h
l
i 3.5 Path analysis With the path analysis, the total effects are broken down into indirect and direct effects on certain independent
variables. Direct participation in farming presents the highest positive total effect (0.855) and direct effect (0.503),
whereas information sources show the highest positive indirect effect (0.624) (Fig. 1). Organizational participation
(0.796, 0.226) and participant education (0.716, 0.196) represent the second and third highest total and positive
direct effect, respectively, both with positive impact (Table 7 in supplementary sheet). Risk orientation (0.593)
ranked second and organizational participation (0.570) ranked third in terms of positive indirect effect. Out of the
eight independent variables, four variables [participant education (X1), knowledge (X2), information sources (X3),
and cosmopoliteness (X5)], each of these, has a highest indirect effect on farmers' perceptions directing towards transformation through direct participation in farming and organizational participation. On the other
hand, another three [innovativeness (X6), risk orientation (X7), organizational participation (X8)] have the highest
indirect effect through direct participation in farming and participant education which are depicted in Table 7
(supplementary sheet). However, path analysis revealed that just a few variables directly impacted farmers'
perception levels. However, interconnected variables were principally involved for the effect of several variables on
farmers' perceptions. 3.6 Arsenic content in collected samples The study revealed As content in irrigation water (ranges from 0.108–0.356, 0.111–0.338, 0.110–0.371, 0.041–
0.364, and 0.065–0.356), soils (ranges from 15.645–30.675, 14.325–29.612, 16.327–32.1, 11.895–32.667, and
11.375–32.262), vegetables (ranges from 0.83–3.56, 0.26–3.25, 0.45–3.8, 0.21–3.9, and 0.23–3.84), rice grains
(ranges from 0.192–0.75, 0.22–0.69, 0.18–0.89, 0.09–0.86, and 0.117–0.74), and farmers’ scalp hair (ranges
from 0.34–2.21, 0.4–2.36, 0.42–2.38, 0.32–2.44, and 0.35–2.17) for Sadar, Faridganj, Matlab north, Kachua, and
Hajiganj, respectively. Table 8 demonstrates the probability level of As content in all five items. Arsenic in irrigation
water is significantly different at a 1% probability level in the study sites. Page 12/21 Table 8
Comparison of As concentration in different components collected from five (05) different location of Chandpur
districts of Bangladesh
Locations
As in irrigation water
(mg/L) (against
background value 0.1
mg/L by FAO and 0.01
mg/L by WHO;
Chakraborti et al.,
2018; WHO, 2004)
As in soil
(mg/kg)
(against global
average 10 and
FAO limit 50
mg/kg; FAO,
1992; Rahman
et al., 2013)
As in vegetable
(mg/kg)
(against
permissible
limit 0.5 to 1.0
mg/kg; Liu et
al., 2010;
MAFF, 1997)
As in Grain
(mg/kg)
(against
permissible
limit 0.37
mg/kg;
WHO,
2016)
As in hair
(mg/kg) (against
background
value 0.08–
0.250 and
toxicity indicator
1.0 mg/kg;
Arnold et al.,
1990)
Hajiganj
0.227ab
21.90b
2.03ab
0.459a
1.24ab
Kachua
0.204bc
20.69c
1.82cd
0.418b
1.08c
Matlab
north
0.192c
21.10b
1.61d
0.367c
1.00c
Faridganj
0.234a
23.00a
2.21a
0.472a
1.28a
Sadar
0.217b
23.08a
1.93c
0.399bc
0.96cd
LS
**
**
***
*
*
CV (%)
6.81
8.81
5.51
6.28
6.70
SE (±)
1.17
0.93
1.24
1.15
0.96 In column, means followed by different letters are significantly different. LS means level of significance, CV means
co-efficient of variance, SE means standard error, ***means at 0.1% level of probability, **means at 1% level of
probability and * means at 5% level of probability In column, means followed by different letters are significantly different. LS means level of significance, CV means
co-efficient of variance, SE means standard error, ***means at 0.1% level of probability, **means at 1% level of
probability and * means at 5% level of probability The lowest As content in irrigation water is revealed from Matlab north while the highest is found in Faridganj. Similarly, As level in the study sites' soil is significantly different (p ≤ 0.01), where Matlab north and Hajiganj's soil
contain statistically similar As to Sadar and Faridganj. 3.6 Arsenic content in collected samples significantly (p ≤ 0.05) higher As is found in grains from
Faridganj and Hajiganj compared with that from Kachua, Matlab north, and Sadar. In contrast, the lowest and
highest grain As is recorded in Matlab north and Faridganj, respectively. Vegetables As in all the five study areas
differs significantly at a 1% probability level. Vegetables As level from Hajiganj is pretty close to Faridganj, and the
same for Sadar is very close to Kachua. Similar to the grain As content, the lowest and highest vegetables As is
recorded in Matlab north and Faridganj, respectively. Faridganj and Hajiganj have been found to have significantly
((p ≤ 0.001)) higher and closely resemble hair As concentration. Again, hair As level observed from Matlab north
and Kachua is also statistically similar. Hair As content of Sadar is also at immediate proximity to Matlab north
and Kachua. At all five locations, the mean As concentration in vegetables and irrigation water is much higher
comparing with the permissible limit (Chakraborti et al., 2018; WHO, 2004), while the As level in soil is higher than
the As level on the global scale but below the FAO proposed limit for agriculture (FAO, 1992; Rahman et al., 2013). Except for Matlab north, grain As content surpassed the safe limit (WHO, 2016) at all places. On the other hand,
scalp hair As is recorded above the toxicity limit for four locations except for Sadar revealed below toxicity level
but above the background value. Results suggest significant As transfer from irrigation water to rice and
vegetables and subsequent body loading. In column, means followed by different letters are significantly different. LS means level of significance, CV means
co-efficient of variance, SE means standard error, ***means at 0.1% level of probability, **means at 1% level of
probability and * means at 5% level of probability 3.7 Principal component analysis (PCA) Lengthwise, Cluster II was the lowest, and Cluster IV was the
highest, suggesting the lowest and highest variations, respectively. It's clear that there's a strong relationship
between categories (I) and (II) among the four options. Table 9 (in the supplementary sheet) displays the PCA
results for As concentration of various parameters. Table 9 shows that the first principal component PC has an
eigenvalue greater than 1, indicating that it adequately describes the variances. As for irrigation water (0.458),
grain (0.448), and soil (0.446), these three factors account for the vast majority (92.5%) of the total variance
explained by the first PC (Table 9). Values highlighted in bold in the Table are particularly relevant to
understanding the PC, as a higher numerical value denotes a more substantial contribution. Thus, the PC1 loading
values were largely influenced by the parameters of irrigation water As, grain As, and soil As. 4. Conclusion The most concerning health issue in naturally As endemic regions is the high concentration of As in groundwater
and its subsequent transfer to human body via rice and vegetable consumption. The level of farmers' perception
about the source of As contamination, As-induced ailment, its symptoms, and potential measures to minimize
crop loading with As was investigated in this study. Arsenic in scalp hairs, vegetables, rice, and irrigation water
exceeded the permissible limit. Statistically significant (at the 5%, 5%, 0.1%, 1%, and 1% probability levels) mean
As content in scalp hairs, rice, vegetables, soils, and irrigation water, respectively, was observed in all the study
sites. Ninety-two point five percent of the variation is explained by the first principal component (PC1) and the
irrigation water, grains and soil- As are the dominating parameters. However, only 25% farmers have high
perceptions regarding this transfer pathway and health impacts. The study explored the association between
farmers' perception and their socioeconomic status and identified the predictor variables responsible for
perception variances. These are crucial aspects in formulating policies for As mitigation and education programs
in all the As endemic nations. This study clearly show that As perception is not widespread among the farmers,
the primary stakeholder, at this moment although there significant As contamination and transfer to the crops is
evident. While most participants had a little perception about the As problem in irrigation water and its uptake by
rice and vegetables, their knowledge gap is notably prominent regarding the mitigation measures available to
prevent the contamination. Additionally, it has been found that perception is related to direct participation in
farming, farmers' cosmopoliteness, opinionatedness, innovativeness, risk orientation, ownership of farm power
and machinery (FPM), and organizational participation. Public awareness programs should expand and target the
regular participants in any organization, farmers' leaders, middle to old-aged groups, and innovators in all areas. 3.7 Principal component analysis (PCA) Page 13/21 Figure 2 depicts four unique clusters are produced by the varying lengths of the eigenvectors. Correlations
between items are represented by the angle between eigenvectors, and the length of each eigenvector is
proportional to the variance of the corresponding data item. Hair As, grain As, water As, soil As, and vegetable As
are all examples of factors that fall into one of the five categories denoted by clusters (I), (II), (III), or (IV). Parameters with identical values are observed to cluster together in Fig. 2. This divergence can be explained by the
fact that the As in irrigation water and soil (cluster III) contribute to a similar variance, while the As in scalp hair
(clusters I), grain Figure 2 depicts four unique clusters are produced by the varying lengths of the eigenvectors. Correlations
between items are represented by the angle between eigenvectors, and the length of each eigenvector is
proportional to the variance of the corresponding data item. Hair As, grain As, water As, soil As, and vegetable As
are all examples of factors that fall into one of the five categories denoted by clusters (I), (II), (III), or (IV). Parameters with identical values are observed to cluster together in Fig. 2. This divergence can be explained by the
fact that the As in irrigation water and soil (cluster III) contribute to a similar variance, while the As in scalp hair
(clusters I), grain (clusters II), and vegetables (cluster IV) do not. Lengthwise, Cluster II was the lowest, and Cluster IV was the
highest, suggesting the lowest and highest variations, respectively. It's clear that there's a strong relationship
between categories (I) and (II) among the four options. Table 9 (in the supplementary sheet) displays the PCA
results for As concentration of various parameters. Table 9 shows that the first principal component PC has an
eigenvalue greater than 1, indicating that it adequately describes the variances. As for irrigation water (0.458),
grain (0.448), and soil (0.446), these three factors account for the vast majority (92.5%) of the total variance
explained by the first PC (Table 9). Values highlighted in bold in the Table are particularly relevant to
understanding the PC, as a higher numerical value denotes a more substantial contribution. Thus, the PC1 loading
values were largely influenced by the parameters of irrigation water As, grain As, and soil As. (clusters II), and vegetables (cluster IV) do not. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval This work included human subjects and received the ehithical approval from the ethical review committee of the
Education University of Hong Kong and also have verified the consent from subjects participating in the study was
received prior to conducting the study. Author Contributions Rokonuzzaman MD: Conceptualization, Methodology, Field survey, Data collection, Sample collection, Statistical
analysis, Software, Investigation, Writing original draft, Review & editing. Ye ZH: Analysis, Methodology, literature
background development, Visualization, Investigation, Review & editing. Wu C: methodological framework,
Resources, Investigation, review & editing. LI WC: Conceptualization, Methodology, Statistical analysis, Software,
Project administration, Supervision, Funding acquisition, Writing original draft, review & editing. Declarations Funding Acknowledgments: Page 14/21 This work was supported by grants from Seed Funding Grant (Ref: RG53/19-20R), Funding support to GRF
Proposal (Ref: RG21/2020-2021R), Dean's Research Fund (Ref: IRS-10-2020) and SES Grant for Collaborative
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deployment in Asia and Africa. Theoretical and Applied Genetics, 133(5), 1427–1442. Figure 2 Principle component analysis (PCA) (IrriAs = Irrigaiton water As; SoilAs = Soil As; GrainAs= Grain As; VegAs=
Vegetables As; ScalpAs= Scalp hair As) Supplementaryfile.pdf Figures Figure 1 Page 20/21 Path analysis Path analysis Figure 2
Principle component analysis (PCA) (IrriAs = Irrigaiton water As; SoilAs = Soil As; GrainAs= Grain As; VegAs=
Vegetables As; ScalpAs= Scalp hair As) Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementaryfile.pdf Page 21/21
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Pengaruh Motivasi Belajar Dan Manajemen Kelas terhadap Hasil Belajar Siswa
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Abstrak Tujuan dari penelitian untuk mengetahui pengaruh motivasi belajar dan manajemen kelas terhadap hasil
belajar siswa. Penelitian menggunakan metode kuantitatif dengan teknik korelasi metode. Pengumpulan data
instrumen berupa angket. Populasi penelitian adalah 117 orang dan sampelnya 43 orang. Penelitian
menggunakan beberapa uji regresi dengan uji hipotesis pada taraf signifikansi 0,05. Hasil penelitian
menunjukkan bahwa terdapat pengaruh yang signifikan berdasarkan hasil uji Annova nilai signifikansi 0,000
dapat dinyatakan bahwa variabel pengelolaan kelas dan pembelajaran secara simultan atau bersama-sama
berpengaruh signifikan terhadap variabel hasil belajar. Berdasarkan uji R diperoleh 0,971 menunjukkan bahwa
korelasi atau hubungan antara variabel hasil belajar dan nilai variabel motivasi belajar dan pengelolaan kelas
termasuk dalam kategori sangat kuat, dapat diartiak 94,3% variasi pembelajaran hasil dapat dijelaskan oleh
variabel dependen manajemen kelas dan motivasi belajar yang digunakan dalam persamaan regresi. Sisanya
4,7% dijelaskan oleh variabel lain di luar penelitian ini. j
p
Kata kunci : Motivasi Belajar, Manajemen Kelas dan Hasil Belajar. Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa Agung Hidayatullah
Magister Manajemen Pendidikan, Universitas Jambi, Indonesia
E-mail : agunghidayatullahagutri@gmail.com Edukatif : Jurnal Ilmu Pendidikan Volume 3 Nomor 4 Tahun 2021 Halm 1451 - 1459
EDUKATIF: JURNAL ILMU PENDIDIKAN
Research & Learning in Education
https://edukatif.org/index.php/edukatif/index Abstract The purpose of the study was to determine the effect of learning motivation and classroom management on
student learning outcomes. The research uses quantitative methods with method correlation techniques. Instrument data collection is in the form of a questionnaire. The research population was 117 people and the
sample was 43 people. The study used several regression tests with hypothesis testing at a significance level of
0.05. The results showed that there was a significant effect based on the results of the Annova test, a
significance value of 0.000. It can be stated that the classroom management and learning variables
simultaneously or jointly have a significant effect on the learning outcome variables. Based on the R test, it
was obtained 0.971 indicating that the correlation or relationship between the learning outcomes variables
and the value of the learning motivation and classroom management variables was included in the very strong
category, it could be interpreted that 94.3% of the variation in learning outcomes could be explained by the
dependent variable class management and learning motivation used in regression equation. The remaining
4.7% is explained by other variables outside this study. Keywords: Learning Motivation, Class Management and Learning Outcomes. Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071
Copyright (c) 2021 Agung Hidayatullah
Corresponding author
Email
: agunghidayatullahagutri@gmail.com
ISSN 2656-8063 (Media Cetak)
DOI
: https://doi.org/10.31004/edukatif.v3i4.620
ISSN 2656-8071 (Media Online) Copyright (c) 2021 Agung Hidayatullah Copyright (c) 2021 Agung Hidayatullah Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071
Copyright (c) 2021 Agung Hidayatullah
Corresponding author
Email
: agunghidayatullahagutri@gmail.com
ISSN 2656-8063 (Media Cetak)
DOI
: https://doi.org/10.31004/edukatif.v3i4.620
ISSN 2656-8071 (Media Online) Corresponding author
Email
: agunghidayatullahagutri@gmail.com
DOI
: https://doi.org/10.31004/edukatif.v3i4.620 PENDAHULUAN Peserta didik yang sedang mengikuti proses pembelajaran baik pada tingkat dan jenjang pendidikan
tertentu, tentunya menginginkan hasil belajar yang baik (Harahap, et al., 2021). Pendidikan sebagai usaha
membina dan mengembangkan pribadi manusia dari aspek-aspek rohaniah dan jasmaniah juga harus
berlangsung secara bertahap. Oleh karena suatu kematangan yang bertitik akhir pada optimalisasi
perkembanga atau pertumbuhan, baru dapat tercapai bilamana berlangsung melalui proses demi proses kearah
tujuan akhir perkembangan atau pertumbuhan (Enkoswara, 2011). Di madrasah Tsanawiyah ini sebagai tempat riset oleh peneliti tidak sedikit siswa yang memiliki
motivasi belajar rendah. Untuk membantu siswa yang memiliki motivasi belajar rendah perlu dilakukan suatu
upaya dari guru agar siswa yang bersangkutan untuk dapat meningkatkan motivasi belajarnya. Salah satu
penghambat kesuksesan remaja adalah kurangnya motivasi. Untuk mengembangkan pemikiran kreatif, kita
harus mempunyai motivasi yang cukup. Motivasi akan membuat kita bersemangat untuk merealisasikan apa
yang ada dalam imajinasi kreatif kita. Menurut Mawarsih (Harahap, et al., 2021) keberhasilan belajar sering disebabkan adanya motivasi yang
kuat. Dalam penelitian ini guru yang sangat disorot adalah wali kelas yang bertugas sebagai manajer di dalam
kelas yang dalam hal ini merupakan salah satu aspek yang ikut diteliti dalam karya tulis ilmiah. Peran seorang
wali kelas dalam manajemen kelas berperan penting dalam proses belajar mengajar seorang manajer kelas
harus mengetahui tentang bagaimana kelas tersebut masuk ke jenis kelas yang dapat diamati oleh manager. Pada mata pelajaran aqidah akhlak merupakan mata pelajaran yang akan peneliti ambil sebagai sampel
dari beberapa mata pelajaran yang ada, dikarenakan pada mata pelajran ini siswa banyak yang lebih merasa
pelajaran yang tidak terlalu penting padahal sabagai lembaga pendidikan agama mata pelajaran aqidah akhlak
ini sangatlah penting. Bertitik tolak dari uraian diatas, penulis tertarik untuk melakukan penelitian tentang
pengaruh motivasi belajar dan manajemen kelas terhadap hasil belajar siswa. Rumusan masalah yang akan di bahas dalam karya ilmiah yaitu adakah pengaruh motivasi belajar yang
signifikan terhadap hasil belajar siswa, adakah pengaruh manajemen kelas yang signifikan terhadap hasil
belajar siswa, adakah pengaruh motivasi belajar dan manajemen kelas yang signifikan terhadap hasil belajar
siswa. Tujuan yang ingin dicapai dalam penelitian ini adalah untuk mengetahui pengaruh motivasi belajar
terhadap hasil belajar siswa, untuk mengetahui pengaruh manajemen kelas terhadap hasil belajar, untuk
mengetahui pengaruh motivasi belajar dan manajemen kelas terhadap hasil belajar. ISSN 2656-8063 (Media Cetak)
ISSN 2656-8071 (Media Online) Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1452 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 PENDAHULUAN Hasil penelitian dapat dipergunakan peneliti lanjut sebagai bahan referensi untuk meningkatkan kualitas
pendidikan bila dihubungkan dengan motivasi belajar dan manajemen kelas serta hasil belajar siswa dan dapat
melakukan penelitian lanjutan yang lebih luas dan mendalam. Pentingnya penelitian ini dilakukan karena
mengingat pentingnya peranan motivasi bagi siswa dalam belajar, maka guru diharapkan dapat
membangkitkan dan meningkatkan motivasi belajar siswa-siswanya. Agar siswa dapat mencapai hasil belajar
yang optimal, maka siswa harus memiliki motivasi belajar yang tinggi, walaupun pada kenyataannya tidak
semua siswa memiliki motivasi belajar yang tinggi dalam belajar. Hipotesis dalam penelitian ini peneliti jabarkan sebagai berikut: Hipotesis dalam penelitian ini peneliti jabarkan sebagai berikut: Hipotesis dalam penelitian ini peneliti jabarkan sebagai berikut: Ha1 : Adanya pengaruh yang signifikan antara motivasi belajar denganHasil belajar di Madrasah
Tsanawiyah X Kabupaten Tebo Ha1 : Adanya pengaruh yang signifikan antara motivasi belajar denganHasil belajar di Madrasah
Tsanawiyah X Kabupaten Tebo Ho1 : Tidak ada pengaruh yang signifikan antara motivasi belajardengan hasil belajar di Madrasah
Tsanawiyah X Kabupaten Tebo2. Ho1 : Tidak ada pengaruh yang signifikan antara motivasi belajardengan hasil belajar di Madrasah
Tsanawiyah X Kabupaten Tebo2. PENDAHULUAN Ha2 : Adanya pengaruh antara manajemen kelas terhadap hasil belajarsiswa di Madrasah Tsanawiyah X
Kabupaten Tebo Ha2 : Adanya pengaruh antara manajemen kelas terhadap hasil belajarsiswa di Madrasah Tsanawiyah X
Kabupaten Tebo Ho2 : Tidak ada pengaruh antara menajemen kelas terhadap hasilbelajar siswa di Madrasah Tsanawiyah
X Kabupaten Tebo3 Ha3 : Terdapat korelasi atau hubungan serta pengaruh motivasi belajardan manajemen kelas terhadap Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1453 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 1453 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 hasil belajar siswa di Madrasah TsanawiyahX Kabupaten Tebo Ho3 : Tidak terdapat korelasi atau hubungan serta pengaruh motivasibelajar dan manajemen kelas
terhadap hasil belajar siswa di MadrasahTsanawiyah X Kabupaten Tebo Ho3 : Tidak terdapat korelasi atau hubungan serta pengaruh motivasibelajar dan manajemen kelas
terhadap hasil belajar siswa di MadrasahTsanawiyah X Kabupaten Tebo Ho3 : Tidak terdapat korelasi atau hubungan serta pengaruh motivasibelajar dan manajemen kelas
terhadap hasil belajar siswa di MadrasahTsanawiyah X Kabupaten Tebo Menurut Hintzman (Muhibin, 2014) Belajar adalah sesuatu perubahan yang terjadi dalam diri organisasi
manusia atau hewan disebabkan oleh perubahan pengalaman yang dapat mempengaruhi tingkah laku
organisme tersebut. Hasil belajar merupakan hasil dari suatu interaksi tindak belajar dan tindak mengajar. Dari
sisi guru, tindak mengajar diakhiri dengan proses evaluasi hasil belajar. Dari sisi siswa, hasil belajar
merupakan berakhirnya penggal dan puncakproses belajar (Dimyati & Mudjiono, 2019). Menurut Egok
(Riyanti, Wahyudi, & Suhartono, 2021) ada dua faktor yang memengaruhi hasil belajar yaitu faktor internal
dan faktor eksternal. Faktor internal merupakan faktor yang berasal dari dalam diri siswa itu sendiri, seperti
kecerdasan, kemampuan berpikir kritis, motivasi, kesehatan, dan cara belajar, serta kemandirian belajar,
sedangkan faktor eksternal merupakan faktor yang datang dari luar diri siswa, seperti lingkungan keluarga,
lingkungan sekolah, dan lingkungan masyarakat. Kemandirian belajar merupakan salah satu faktor internal
yang memengaruhi hasil belajar. Adapun menurut Makki & Aflahah (Hae, Tantu, & Widiastuti, 2021) mengatakan bahwa motivasi
belajar sangat menentukan tingkat pencapaian hasil belajar anak. Motivasi adalah suatu perubahan energi
didalam pribadi seseorang yang ditandai dengan timbulnya efektif (perasaan) dan reaksi untuk mencapai
tujuan (Harahap, et al., 2021). Menurut (Zulfiana, 2014) perubahan energi dapat berupa perubahan kegiatan
fisik. METODE PENELITIAN Pendekatan pada penelitian ini yaitu kuantitatif korelasi digunakan untuk menghitung, menggambarkan,
dan menggali tentang pengaruh motivasi ekstern terhadap hasil belajar siswa (Dantes & Nyoman, 2012). Populasi adalah suatu kumpulan menyeluruh dari suatu objek yang merupakan perhatian dalam penelitian
(Martono & Nanang, 2010). Populasi penelitian adalah seluruh siswa Madrasah Tsanawiyah X kabupaten
Tebo yang berjumlah 117 orang. Sampel adalah bagian dari jumlah dan karekteristik yang dimiliki oleh
populasi tersebut (Martono & Nanang, 2010). Sampel yang digunakan dalam penelitian ini adalah siswa kelas
VIII A dan VIII B MTs X Rimbo Bujang yang berjumlah 43 orang. Metode sampling yang digunakan dalam penelitian ini adalah Non Probability sampling. Menurut
(Marchali, 2018) Non probability sampling yaitu teknik pengambilan sampel yang tidak memberi peluang
atau kesempatan samabagi setiap unsur atau anggota populasi untuk dipilih menjadi anggota sampel. Sampel
yang digunakan dalam penelitian ini yaitu kelas VIII MTs X Kabupaten Tebo. Dalam pengumpulan data
peneliti melakukannya dengan langkah pertama dan utama yaitu dengan melakukan observasi pada madrasah
yang di tujusebagai landasan utama mengapa di pelukannya penelitian ini. Setelah melakukan obsevasi denganhasil yang di peroleh maka peneliti melanjutkannya dengan proses
penelitian langsung di lapangan dengan menggunakan metode angket, dimana dalam metode ini peneliti
mengukur hal-hal yang telah dipaparkan sebelumnya dalam latar belakang penelitian ini. Setelah
menggunakan angket nantinya peneliti akan melakukan anasisi hasil dari angket yang tadinya sudah di isi oleh
siswa melalui perhitungan secara langsung maupun perhitungan menggunakan bantuan aplikasi SPSS
(Statistical Product and Service Solutions). Setelah hasil didapatkan maka peneliti nantinya dapat
memaparkan apakah hipotesis dari penelitian ini terjawab. Dan natinya dapat menajadi sebagai bahan
masukan terhadap madrasah guna meningkatkan pemebelajran yang lebih baik lagi. Adapun instrumen yang digunakan dalam penelitian menggunakan pedoman observasi, angket serta
dokumentasi. Pedoman observasi digunakan sebagai alat bantu yang digunakan peneliti ketika mengumpulkan
data melalui pengamatan dan pencatatan terhadap variabel yang diteliti. Sedangkan angket merupakan alat
bantu yang dipakai peneliti untukmendapatkan data tentang korelasi. Angket terdiri dari 35 butir pertanyaan
yang diajukan kepada siswa dengan rincian 20 soal bersifat positif dan 15 soal bersifat negatif dan yang
terakhir adalah dokumentasi yaitu alat bantu peneliti berupa data foto kegiatan dalam penelitian serta data
hasil belajar siswa pada mata pelajaran Aqidah Akhlak. Pengujian Validitas dan reabilitas instrumen angket dilakukan menggunakan program aplikasi SPSS 16. Uji normalitas dilakukan terlebih dahulu dengan menggunakan One sample kolmogorv-Smirnov Tes. PENDAHULUAN Karena seseorang mempunyai tujuan tertentu dari aktifitasnya, maka seseorang mempunyai motivasi
yang kuat untuk mencapainya dengan segala upaya yang dapat dia lakukan untuk mencapainya. Sebelum adanya penelitian ini, sudah ada beberapa penelitian atau tulisan yang telah dilakukan oleh
beberapa peneliti terdahulu yaitu tentang pengaruh motivasi terhadap hasil belajar siswa. Penelitian yang
dilakukan oleh (Zulfiana, 2014), dengan judul Korelasi Motivasi Belajar Pendidikan Agama Islam terhadap
Hasil Belajar Siswa di Sekolah Menengah Pertama Negeri 5 Muara Bungo dengan hasil bahwa prestasi
pelajaran PAI ditentukan oleh motivasi sebesar 20,25% sedangkan 79,75% ditentukan oleh hal lain, hal
tersebut disebabkan karena Sekolah Menengah Pertama Negeri 5 Muara Bungo hanya menyediakan waktu 2
jam untuk mempelajari bidang studi pendidikan agama Islam. Penelitian oleh (Purnama, 2013) dengan judul
motivasi belajar dari Sekolah Ilmu Kesehatan Insan Cendikia Husada Bojonegoro. Dengan hasil bahwa
motivasi dalam belajar mempengaruhi pada hasil dari proses belajar,motivasi berperang sebagai pendorong
semangat dalam proses belajar.Penelitian oleh (Stevany, 2012) dengan judul pengaruh Motivasi terhadap
prestasi belajar siswa kelas VIII SMP Negeri Sekecamatan Bantul dengan hasil bahwa prestasi belajar siswa
ditentukan oleh motivasi sebesar 40,25% sedangkan 59,75% ditentukan oleh hal lain. Kerangka Pemikiran pada penelitian ini yaitu Kerangka Pemikiran pada penelitian ini yaitu Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071
Gambar 1. Kerangka Pemikiran
X1
Manajemen Kelas
Y
Motivasi Belajar
X2
Hasil Belajar Gambar 1. Kerangka Pemikiran
X1
Manajemen Kelas
Y
Motivasi Belajar
X2
Hasil Belajar Manajemen Kelas Hasil Belajar Gambar 1. Kerangka Pemikiran Gambar 1. Kerangka Pemikiran Gambar 1. Kerangka Pemikiran Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1454 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 1454 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 METODE PENELITIAN untuk
dapat mendeteksi ada atau tidaknya multikolinieritas di dalam model regresi adalah melihat nilaiTolerance
dan VIF ( Variance Inflation Factor) melalui program SPSS. Uji heteroskedastisitas dilakukan untuk menguji
apakah dalam sebuah model regresi telah terjadi ketidaksamaan varian dari residual suatu pengamatan ke
pengamatan yang lain. Uji Goodness of Fit atau uji kelayakan model digunakan untuk mengukur ketepatan
fungsi regresi sampel dalam menaksir nilai aktual. Secara statistik uji Goodness of Fit dapat dilakukan melalui
pengukuran nilai koefisien determinasi, nilai statistik F dan nilai statistik t. Uji signifikasi simultan F dilakukan untuk mengetahui pengaruh variable independen (Motivasi Belajar
dan Manajemen Kelas) terhadap variabel dependen (Hasil belajar). Uji t dikenal dengan uji parsial, yaitu
untuk menguji bagaimana pengaruh masing-masing variabel bebasnya secara sendiri-sendiri terhadap variable
terikatnya. HASIL DAN PEMBAHASAN PENELITIAN Pembahasan hasil penelitian ini dilakukan agar dapat memberikan penjelasan dan gambaran sehingga
dapat memberikan pemahaman mengenai hasil dari penelitian. Pembahasan ini berisi kajian mengenai hasil
temuan yang berhubungan dengan penelitian sehingga dapat diketahui terdapat pengaruh atau tidaknya antara
motivasi dan manajemen kelas terhadap hasil belajar. Sehubungan dengan penelitian yang telah dilakukan, Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1455 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 1455 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 maka akan dikemukakan beberapa hasil penelitian. Dalam penelitian ini menunjukan hasil bahwasannya hasil
belajar siswa di pengaruhi oleh motivasi manajemen kelas. Hasil dari penelitian tersebut akan dibahas secara
tepat dan sesuai dengan hasil yang telah didapatkan oleh peneliti dari penelitian yang telah dilakukan pada
madrasah tersebut. Tabel 1. Hasil Uji Reabilitas
Variabel
Cronbach’s
Alpha
Keterangan
Motivasi (X1)
0.826
Reliabel
Manajemen Kelas (X2)
0.826
Reliabel
Hasil Belajar (Y)
0.921
Reliabel Berdasarkan hasil uji reliabilitas didapatkan hasil berupa variabel motivasi belajar (X1) dengan nilai
0,826. Nilai ini lebih besar dari pada 0,60 sehingga untuk variabel motivasi belajar (X1) dinyatakan reliabel,
sedangkan variabel manajemen kelas (X2) memiliki hasil pengujian dengan nilai 0,826 sehingga juga
dikatakan reliabel karena nilainya diatas 0,60 dan variabel hasil belajar (Y) memiliki hasil pengolahan data
sebesar 0,921 sehingga nilainya juga lebih dari 0,60 dan dinyatakan reliabel. Hasil ini menunjukan bahwa
variable motivasi belajar (X1), motivasi belajar (X2) Dan hasil belajar(Y) memiliki kesamaan yaitu reliabel
yang artinya instrumen penelitian yang digunakan pada penenelitian ini dinyatakan konsisten. Tabel 2. Hasil Uji Normalitas
One-Sample Kolmogorov-Smirnov Test
Unstandardize
d Residual
N
22
Normal Parametersa
Mean
.0000000
Std. Deviation
3.00215562
Most
Extreme
Differences
Absolute
.115
Positive
.109
Negative
-.115
Kolmogorov-Smirnov Z
.539
Asymp. Sig. (2-tailed)
.933
a. Test distribution is Normal. Tabel 2. Hasil Uji Normalitas
One-Sample Kolmogorov-Smirnov Test
Unstandardize
d Residual
N
22
Normal Parametersa
Mean
.0000000
Std. Deviation
3.00215562
Most
Extreme
Differences
Absolute
.115
Positive
.109
Negative
-.115
Kolmogorov-Smirnov Z
.539
Asymp. Sig. (2-tailed)
.933
a. Test distribution is Normal. Berdasarkan tabel hasil uji normalitas diperoleh nilai Asymp.Sig (2 tailed) lebih besar dari pada taraf
kepercayaan 5% yaitu 0,933 hal ini lebih besar nilainya 0,005 sehingga data penelitian dikategorikan
berdistribusi normal. HASIL DAN PEMBAHASAN PENELITIAN Pada pengujian asumsi multikolinearista didapatkan hasil bahwa nilai tolerance dari
motivasi belajar (X1) dan menajemen kelas (X2) sebesar 0,369 artinya nilainya lebih dari 0,1. Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071
Tabel 3. Uji Asumsi Multikolinearista
Coefficientsa
Model
Collinearity Statistics
Tolerance VIF
1
(Constant) Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1456 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620
Motivasi Belajar
.369
2.714
Manajemen Kelas .369
2.714
a. Dependent Variable: Hasil Belajar 1456 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 1456 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 Dari table uji asumsi multikolinearista nilai VIF (Variace Inflation Factor) sebesar 2,714, nilai ini
kurang dari 10. Oleh sebab itu maka dinyatakan tidak terjadi gejala multikolenieritas karena nilai tolerance
>0,01 dan nilai VIF <10. Tabel 4. Uji Heteroskedastisitas Tabel 4. Uji Heteroskedastisitas Tabel 4. Uji Heteroskedastisitas
Coefficientsa
Model
Unstandardized Coefficients Standardized Coefficients
t
Sig. B
Std. Error
Beta
1 (Constant)
3.084
4.083
.755 .459
Motivasi Belajar .002
.118
.005
.013 .990
Manajemen Kelas -.012
.106
-.042
-.110 .914
a. Dependent Variable: Absres Pada hasil pengujian heteroskedastisitas yang bertujuan untuk menguji apakah dalam sebuah model
regresi terjadi ketidaksamaan variasi dari residual suatu pengamatan kepengamatan yang lain didapatkan
bahwa hasil variable motivasi belajar (X1) sebesar 0,990. Dan nilai dari variabel manajemen kelas (X2) di
dapatkan jumlah nilai sebesar 0,914. Dari hasil ini dapat dikatakan bahwa variabel motivasi belajar (X1) dan
manajemen kelas (X2) tidak berpengaruh signifikan terhadap absolut residu dengan nilai signifikan 0,05 yang
artinya tidak mengalami gejala heteroskasdisitas. Tabel 5. Uji Goodness Of Fit
Model Summaryb
Model R
R Square Adjusted R Square Std. Error of the Estimate
1
.971a .943
.937
3.15621
a. Predictors: (Constant), Manajemen Kelas, Motivasi Belajar
b. Dependent Variable: Hasil Belajar
Dari pengujian Goodness Of Fit didapatkan hasil berupa R sebesar 0,971, hal ini menunjukan bahwa
korelasi atau hubungan antara variabel dependen (hasil belajar) dengan nilai variabel independen (motivasi
belajar dan manajemen kelas) termasuk dalam kategori sangat kuat. Angka koefisien determinasi R2 yang
dihasilkan sebesar 0,943 artinya 94,3 % variasi dari hasil belajar mampu dijelaskan oleh variabel motivasi
belajar dan manajemen kelas yang digunakan dalam persamaan regresi, sedangkan sisanya sebesar 4,7%
dijelaskan oleh variabel lain diluar penelitian ini. HASIL DAN PEMBAHASAN PENELITIAN Tabel 5. Uji Goodness Of Fit
Model Summaryb
Model R
R Square Adjusted R Square Std. Error of the Estimate
1
.971a .943
.937
3.15621
a. Predictors: (Constant), Manajemen Kelas, Motivasi Belajar
b. Dependent Variable: Hasil Belajar Tabel 5. Uji Goodness Of Fit Dari pengujian Goodness Of Fit didapatkan hasil berupa R sebesar 0,971, hal ini menunjukan bahwa
korelasi atau hubungan antara variabel dependen (hasil belajar) dengan nilai variabel independen (motivasi
belajar dan manajemen kelas) termasuk dalam kategori sangat kuat. Angka koefisien determinasi R2 yang
dihasilkan sebesar 0,943 artinya 94,3 % variasi dari hasil belajar mampu dijelaskan oleh variabel motivasi
belajar dan manajemen kelas yang digunakan dalam persamaan regresi, sedangkan sisanya sebesar 4,7%
dijelaskan oleh variabel lain diluar penelitian ini. Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1457 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 Tabel 6. Daftar Hasil Signifikasi Simultan F
ANOVAb
Model
Sum of Squares df
Mean Square F
Sig. 1
Regression 3105.683
2
1552.841
155.882 .000a
Residual
189.272
19
9.962
Total
3294.955
21
a. Predictors: (Constant), Manajemen Kelas, Motivasi Belajar
b. Dependent Variable: Hasil Belajar Tabel 6. Daftar Hasil Signifikasi Simultan F Hasil uji F didapatkan nilai signifikasi 0,000, karena nilai signifikasinya lebih kecil dari 0,05
(0,000<0,05) maka dapat dikatakan bahwa variabel motivasi belajar (X1) dan manajemen kelas (X2) secara
simultan atau bersama-sama berpengaruh secara signifikan terhadap variabel hasil belajar (Y). p
g
g
p
j
Hal ini diperkuat kembali menggunakan hasil uji T parsial dengan hasil bahwa variabel motivasi belajar
(X1) memiliki nilai signifikasi sebesar 0,000 lebih kecil dari 0,05, sehingga variabel motivasi belajar (X1)
dinyatakan berpengaruh secara signifikan terhadap variabel hasil belajar (Y) hasil penelitian ini sejalan
dengan hasil penelitian yang dilakukan oleh (Zulfiana, 2014) tentang korelasi motivasi belajar Pendidikan
Agama Islam terhadap hasil belajar siswa di Sekolah Menengah Pertama Negeri 5 Muara Bungo dengan hasil
penelitian motivasi belajar berpengaruh secara signifikan terhadap hasil belajar siswa. Variabel manajemen
kelas (X2) memiliki nilai signifikasi sebesar 0,000 lebih kecil dari pada 0,05 sehingga variabel manajemen
kelas juga berpengaruh secara signifikan terhadap hasil belajar (Y) hasil penelitian ini sejalan dengan hasil
penelitian yang dilakukan oleh (Husnul, 2017) tentang manajemen kelas dalam pembelajaran Matematika Di
SMA Negeri Yogyakarta hasil temuan manajemen kelas berpengaruh secara signifikan terhadap hasil belajar
siswa. HASIL DAN PEMBAHASAN PENELITIAN Adapun makna sumbangan terhadap kemajuan ilmu pengetahuan diharapkan dapat bermanfaat terutama
dalam hal dapat menjadi bahan acuan untuk kegiatan pendidikan, terutama dalam upaya perbaikan dan
peningkatan mutu pendidikan yang mengarah pada peningkatan kinerja guru, digunakan sebagai sumbang
saran dalam meningkatkan dan mengembangkan peran kepemimpinan kepala sekolah khususnya yang
berpengaruh langsung pada peningkatan kinerja guru, sehingga guru dapat bekerja dengan penuh kerelaan,
bersemangat, dan siap bersaing dengan sekolah lain. Diharapkan dapat bermanfaat bagi kepala sekolah
dijadikan pertimbangan dalam menentukan wali kelas agar terciptanya guru bekerja dengan nyaman, aman,
kreatif, dan menyenangkan, serta memberdayakan potensi yang dimiliki sekolah, dalam rangka menunjang
kualitas pendidikan di sekolah. Bagi guru dapat meningkatkan kinerjanya dalam pengelolaan pembelajaran
dan pengelolaan kelas yang dimotivasi oleh pimpinan sekolah agar dapat bekerja dengan efektif, efesien,
nyaman, aman, berinovasai kerja tinggi, dan siap bersaing dengan sekolah lain. Batasan temuan penelitian ini yaitu pada kelas VIII Madrasah Tsanawiyah X Kecamatan Rimbo Bujang
dan pada mata pelajaran Aqidah Akhlak pada tahap kelas inilah tahap dimana siswa sangat memerlukan
adanya motivasi dalam belajar. Pada penelitian ini membatasi pada motivasi belajar, peran guru dalam
menajemen kelas dalam upaya mencapai hasil dari belajar siswa. KESIMPULAN Dari hasil penelitian dapat disimpulkan bahwa variabel motivasi belajar (X1) berpengaruh signifikan
terhadap variable hasil belajar (Y). hal ini di buktikan dengan adanya hasil uji T Parsial dengan hasil nilai
signifikasi sebesar 0,000 lebih kecil dari 0,05. Hal tersebut menjawab pertanyaan peneliti pada rumusan Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 1458 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 masalah tentang adakah pengaruh motivasi belajar yang signifikan terhadap hasil belajar siswa di madrasah
tsanawiyah X kabupaten tebo. Variabel manajemen kelas (X2) berpengaruh signifikan terhadap variable hasil
belajar (Y), hal ini dibuktikan dengan adanya hasil uji T Parsial dengan hasil nilai signifikasi sebesar 0,000
lebih kecil dari 0,05. Hal tersebut menjawab pertanyaan peneliti pada rumusan masalah tentang adakah
pengaruh manajemen kelas yang signifikan terhadap hasil belajar siswa di madrasah tsanawiyah X kabupaten
tebo. Variabel motivasi belajar (X1) dan manajemen kelas (X2) berpengaruh signifikan terhadap variabel hasil
belajar (Y). Hal ini di kuatkan dengan bukti adanya hasil uji Simultan F dengan hasil nilai signifikasi
0,000,nilai ini lebih kecil dari 0,05 maka dapat dikatakan bahwa variable indenpenden ( motivasi belajar dan
manajemen kelas) secara simultanatau bersama-sama berpengaruh signifikan terhadap variabel dependen
(hasil belajar). UCAPAN TERIMA KASIH Dalam penyelesaian Tesis ini penulis telah banyak mendapatkan bantuan dan bimbingan dari berbagai
pihak, sebagai ucapan terimakasih yang tiada terhingga di tujukan kepada yang terhormat bapak Prof. Drs. H. Sutrisno, M. Sc., PH. D selaku Rektor Universitas Jambi, bapak Prof. Dr. H. Haryadi, SE, MMS selaku
Direktur Pascasarjana Universitas Jambim Ibu Dr. Hj. Muazza, M.Si selaku Ketua Program Pendidikan
Magister Manajemen Pendidikan Universitas Jambi, bapak Dr. K. A Rahman, M. Pd. I selaku pembimbing I
yang telah memberikan pemikiran, koreksi, bimbingan arahan serta motivasi dalam penyelesaian Tesis ini. Ibu
Dr. Masbirorotni, M. Sc. Ed selaku pembimbing II yang telah memberikan pemikiran, koreksi, bimbingan
arahan serta motivasi dalam penyelesaian Tesis ini, bapak dan ibu dosen serta seluruh civitas akademik
Pascasarjana Megister Manajemen Pendidikan Universitas Jambi, bapak kepala Madrasah Tsanawiyah bapak
Budiarto, SH, bapak guru mata pelajaran Aqidah Akhlak Bapak Muhammad Ali Mu’min, S. Pd. I. Semoga
bantuan dan dorongan serta bimbingan yang telah diberikan kepada penulis dapat diterima sebagai amal
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p-ISSN 2656-8063 e-ISSN 2656-8071 1459 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
DOI: https://doi.org/10.31004/edukatif.v3i4.620 1459 Pengaruh Motivasi Belajar dan Manajemen Kelas terhadap Hasil Belajar Siswa – Agung Hidayatullah
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Buddhadi Sekolah Minggu Buddhis Bodhisattva. EDUKATIF: JURNAL ILMU PENDIDIKAN, 3,
1261 - 1270. Retrieved from https://edukatif.org/index.php/edukatif/index Purnama, N. I. (2013). Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071 is, K. (2015). Manajemen Kelas Classroom Management. Bandung: Alfabeta. Motivasi Belajar. Jurnal Penelitian. Riyanti, Y., Wahyudi, & Suhartono. (2021). Pengaruh Kemandirian Belajar Terhadap Hasil Belajar
Matematika Siswa Sekolah Dasar. EDUKATIF: JURNAL ILMU PENDIDIKAN, 3(4). Stevany. (2012). Pengaruh Motivasi Terhadap Prestasi Belajar Siswa Kelas Vlll Smp Negeri Sekecamatan
Bantul. Jurnal Penelitian. Supradyani, d. (2013). Kontribusi Kemampuan Manajemen Kelas, Etos Kerja Dan Pemanfaatan Media
Belajar Terhadap Efektifitas Pembelajaran. e-Journal Program Pascasarjana Universitas Pendidikan
Ganesha Program Studi Administrasi Pendidikan, 4. Suyono. (2012). Belajar dan Pembelajaran. Bandung: PT Remaja Rosdakarya. Suyono. (2012). Belajar dan Pembelajaran. Bandung: PT Remaja Rosdakarya. Wahid, I. A. (2016). Pengaruh Motivasi, Etos Kerja Dan Disiplin Kerja Terhadap Kinerja Pegawai Negeri
Sipil (Pns) Pada Dinas Kehutanan Dan Perkebunan Daerah Kabupaten Morowali. Jurnal Katalogis,
Volume 4 Nomor 8, Agustus 2016 hlm 156-163, 4. Retrieved from https://media.neliti.com › media ›
publications Wiyani, & Ardy, N. (2013). Manajemen Kelas. Yogyakarta: Ar-Ruzz Media. Wiyani, & Ardy, N. (2013). Manajemen Kelas. Yogyakarta: Ar-Ruzz Media. Yusuf, R. N., Musyadad, V. F., Iskandar, Y. Z., & Widiawati, D. (2021). Implikasi Asumsi Konsep Diri
Dalam Pembelajaran Orang Dewasa. EDUKATIF : JURNAL ILMU PENDIDIKAN, 3(4). doi:https://doi.org/10.31004/edukatif.v3i4.513 Zulfiana. (2014). Korelasi Motivasi Belajar Agama Islam Terhadap Hasil Belajar Siswa Disekolah Menengah
Pertama Negeri 5 Muara Bungo. Jurnal Penelitian. Edukatif : Jurnal Ilmu Pendidikan Vol 3 No 4 Tahun 2021
p-ISSN 2656-8063 e-ISSN 2656-8071
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Awkward Just-In-Time (JIT) Compilation: A Developer’s Experience
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EPJ web of conferences
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cc-by
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© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). ∗e-mail: iosborne@princeton.edu A Developer’s Experience Abstract. Awkward Array is a library for performing NumPy-like computa-
tions on nested, variable-sized data, enabling array-oriented programming on
arbitrary data structures in Python. However, imperative (procedural) solutions
can sometimes be easier to write or faster to run. Performant imperative pro-
gramming requires compilation; JIT-compilation makes it convenient to com-
pile in an interactive Python environment. Various functions in Awkward Ar-
rays JIT-compile a user’s code into executable machine code. They use several
different techniques, but reuse parts of each others’ implementations. We dis-
cuss the techniques used to achieve the Awkward Arrays acceleration with JIT-
compilation, focusing on RDataFrame, cppyy, and Numba, particularly Numba
on GPUs: conversions of Awkward Arrays to and from RDataFrame; stan-
dalone cppyy; passing Awkward Arrays to and from Python functions compiled
by Numba; passing Awkward Arrays to Python functions compiled for GPUs
by Numba; and header-only libraries for populating Awkward Arrays from C++
without any Python dependencies EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 1 Introduction Awkward Array library [1] offers its users an array-oriented programming style in a dynam-
ically typed language. The advantage of the array-oriented calculations is that they fit the
data analysis workflow better, considering the large amounts of data which a physicist typi-
cally loads into an interactive environment. Moreover,the code that performs one operation
on all data is easy enough to write, then observe the intermediate results on distributions,
and reiterate. The imperative code, on the other hand, is better for the stage after the inter-
active exploration, but the performant imperative programming requires compilation. The
loops may take longer to write, especially when fitting into a Just-In-Time (JIT)-compiler
and getting the compile-time types correctly, but are often self-explanatory and faster. It is
not uncommon for a user analysis code to be expressed in a traditional high-performance,
statically typed language - C++, and a statically typed language requires compilation. The JIT-compilation makes it convenient to compile in an interactive Python environ-
ment. Several JIT-compilation techniques are used to achieve the desired acceleration. The
Awkward Array functions JIT-compile a user’s code into executable machine code. They use
different techniques, but reuse parts of each others’ implementations. The techniques discussed in this article are focusing on integrating RDataFrame [3], cp-
pyy [4], and Numba [5], particularly Numba on GPUs. These include Awkward Arrays to EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 and from RDataFrame conversions, the standalone cppyy integration, passing Awkward Ar-
rays to and from Python functions compiled by Numba, passing Awkward Arrays to Python
functions compiled for GPUs by Numba, and populating Awkward Arrays from C++ without
any Python dependencies using a header-only library. and from RDataFrame conversions, the standalone cppyy integration, passing Awkward Ar-
rays to and from Python functions compiled by Numba, passing Awkward Arrays to Python
functions compiled for GPUs by Numba, and populating Awkward Arrays from C++ without
any Python dependencies using a header-only library. 2 Awkward Arrays and RDataFrame The Awkward Arrays can be converted to the RDataFrame columns and the RDataFrame
columns can be converted to Awkward Arrays as described in detail in [6] and [7]. The user benefits from a faster execution of both the ROOT C++ functions and the user-
defined pure C++ functions. Here is an example of such conversion shown beneath. import awkward as ak df = ak.to_rdataframe({"Events": array}) df = ak.to_rdataframe({"Events": array}) rdf = (
df.Filter("Events.nMuon() == 2")
.Filter("Events.Muon_charge()[0] != Events.Muon_charge()[1]")
.Define("dimuon_mass", """
// this is C++
return std::sqrt(2 * Events.Muon_pt()[0] * Events.Muon_pt()[1]
* (std::cosh(Events.Muon_eta()[0] - Events.Muon_eta()[1])
- std::cos(Events.Muon_phi()[0] - Events.Muon_phi()[1])));
""")) rdf = (
df.Filter("Events.nMuon() == 2")
.Filter("Events.Muon_charge()[0] != Events.Muon_charge()[1]")
.Define("dimuon_mass", """
// this is C++
return std::sqrt(2 * Events.Muon_pt()[0] * Events.Muon_pt()[1]
* (std::cosh(Events.Muon_eta()[0] - Events.Muon_eta()[1])
- std::cos(Events.Muon_phi()[0] - Events.Muon_phi()[1])));
""")) The ak.to_rdataframe function presents a view of an Awkward Array as an
RDataFrame source. The ak.from_rdataframe function converts the selected columns as
native Awkward Arrays. The implementation leverages on a zero-copy Awkward Array view
and implements all for-loops on data in C++. The handle to this Array view is a lightweight 40-byte C++ object allocated on the stack. The generated RDataSource takes pointers into the original array data via this view. Next, the
column readers are generated based on the run-time type of the views. Finally, the readers
are passed to a generated source derived from ROOT::RDF::RDataSource. The ak.from_rdataframe function converts the selected columns as native Awkward
Arrays. The templated C++ header-only implementation and the dynamically generated C++
code are used to extract the columns’ types and data. import ROOT array = ak.from_rdataframe(
df, columns=("Muon_charge", "Muon_eta", "Muon_mass",
"Muon_phi", "Muon_pt", "nMuon")
) import ROOT df = ROOT.RDataFrame("Events",
"root://eospublic.cern.ch//eos/opendata/cms/derived-data/"
"AOD2NanoAODOutreachTool/Run2012BC_DoubleMuParked_Muons.root") f = ROOT.RDataFrame("Events",
"root://eospublic.cern.ch//eos/opendata/cms/derived-data/"
"AOD2NanoAODOutreachTool/Run2012BC_DoubleMuParked_Muons.root" array = ak.from_rdataframe(
df, columns=("Muon_charge", "Muon_eta", "Muon_mass",
"Muon_phi", "Muon_pt", "nMuon")
) ) This array has type 2 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 type: 61540413 * {
Muon_charge: var * int32,
Muon_eta: var * float32,
Muon_mass: var * float32,
Muon_phi: var * float32,
Muon_pt: var * float32,
nMuon: uint32
}
d
f it
l
l
k lik type: 61540413 * {
Muon_charge: var * int32,
Muon_eta: var * float32,
Muon_mass: var * float32,
Muon_phi: var * float32,
Muon_pt: var * float32,
nMuon: uint32 } and some of its values look like [{Muon_charge: [-1, -1], Muon_eta: [1.07, -0.564], ...},
{Muon_charge: [1, -1], Muon_eta: [-0.428, 0.349], ...},
{Muon_charge: [1], Muon_eta: [2.21], Muon_mass: [0.106], ...},
...,
{Muon_charge: [-1, 1], Muon_eta: [-2.15, 0.291], ...}] On the one hand, the users who do their analysis entirely in Python ecosystem can benefit
from faster execution using ROOT C++ functions, or pure C++, in an otherwise Awkward
analysis at full speed. The performance cost of converting Awkward Arrays into RDataFrame
is negligible, since it is a zero-copy view. On the other hand, those who prefer C++, ROOT, and RDataFrame, have an ability to
convert their data into Awkward Arrays in order to leverage the tools available in the wider
Scientific Python ecosystem. 3 Standalone cppyy Like ROOT and RDataFrame, cppyy allows a user to write C++ and JIT-compile it to use it
from Python. The C++ code can be included to Python from a C++ file or written directly as
a Python string. But cppyy can be installed without the entire ROOT package and Awkward
Array’s interface to it is more low-level: users can write C++ functions that operate on whole
arrays, multiple arrays, and return arrays. The ak.Array, the Python class for all Awkward Arrays, implements a magic function
__cast_cpp__ that is called by cppyy to determine a C++ type of the array. The Numba
implementation [8] is reused here to generate a C++ array view on demand. The generated
ArrayView C++ class hashed type is registered as a cpp_type Python string attribute of the
ak.Array class. The cppyy maps the C++ class type as a string to a Python type. The down
side is that the user cannot redefine the function. Each function must have a unique name. This is similar to the PyROOT interpreter, because an earlier version of cppyy is used to bind
Python and C++ in PyROOT. The user does not need to know what cpp_type is - the cpp_type is generated on de-
mand when the array needs to be passed to the C++ function. array = ak.Array([
[{"x": 1, "y": [1.1]}, {"x": 2, "y": [2.2, 0.2]}],
[],
[{"x": 3, "y": [3.0, 0.3, 3.3]}],
]) array = ak.Array([
[{"x": 1, "y": [1.1]}, {"x": 2, "y": [2.2, 0.2]}],
[],
[{"x": 3, "y": [3.0, 0.3, 3.3]}],
]) source_code_cpp = """ source_code_cpp = """ 3 3 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 template<typename T>
double go_fast_cpp(T& awkward_array) {
double out = 0.0;
for (auto list : awkward_array) {
for (auto record : list) {
for (auto item : record.y()) {
out += item; cppyy.cppdef(source_code_cpp) out = cppyy.gbl.go_fast_cpp[array.cpp_type](array)
assert out == ak.sum(array["y"]) The cppyy version that is used must be 3.1 or later. The cppyy library can construct an
object of a previously declared type based on an arguments. This new feature is not available
in the earlier versions. cppyy implements an implicit instantiation from __cast_cpp__
returning a tuple. 4 Awkward Arrays and Numba Numba is a JIT-compiler of functions with Python syntax, and Awkward Arrays can be passed
to and from Numba-compiled functions with a similar interface as cppyy. Numba can be
used in contexts in which acceleration is needed, but C++ is not—it allows users to write
accelerated code in the same Python language as the rest of their code, albeit in a subset of
that language (not all Python code can be compiled). Numba infers the argument types at call time, and generates optimized code based on this
information. Numba also compiles separate specializations depending on the input types. import numba as nb, numpy as np @nb.jit
def path_length(array):
result = np.zeros(len(array), dtype=np.float32)
for i, row in enumerate(array):
result[i] = 0
for j, val in enumerate(row):
result[i] += val
return result def path_length(array): result = np.zeros(len(array), dtype=np.float32) for j, val in enumerate(row): return result The implementation to pass Awkward Arrays to and from Python functions compiled by
Numba defines a numba_type property of an ak.Array that is a type of a generated C++
Array view, as discussed before. Awkward Arrays can be iterated over in Numba-compiled
functions using zero-copy views. 4 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 import cupy as cp import cupy as cp # make an Awkward Array of variable-length lists on the GPU with CuP
N = 2**20 # make an Awkward Array of variable-length lists on the GPU with CuPy
N = 2**20
counts = cp.random.poisson(1.5, N).astype(np.int32)
content = cp.random.normal(0, 45, int(counts.sum())).astype(np.float32)
array = ak.unflatten(content, counts) @nb.cuda.jit(extensions=[ak.numba.cuda]) @nb.cuda.jit(extensions=[ak.numba.cuda]) blocksize = 256
numblocks = (N + blocksize - 1) // blocksize result = cp.empty(len(array), dtype=np.float32)
path_length[numblocks, blocksize](result, array) 5 Python functions compiled for GPUs by Numba Numba can also compile code for Nvidia GPUs through LLVM’s CUDA backend, and Awk-
ward Arrays can be passed as arguments into such functions. Currently, the extension needs
to be defined by a user in a function decorator (as extensions=[ak.numba.cuda] below),
but this requirement will be removed in a future version of Numba. 6 Header-only libraries To create Awkward Arrays in C++ without any dependence on Python, we provide a separate
set of header-only libraries that implement an awkward::layoutbuilder [9] that builds up
an array by appending elements and then shares that array through basic C types (raw array
buffers and a JSON-formatted string the convey structure). #include "awkward/LayoutBuilder.h"
using awkward::layoutbuilder;
// instantiating layoutbuilder types (type aliases omitted for brevity)
enum Field : std::size_t {one, two};
using UserDefinedMap = std::map<std::size_t, std::string>;
UserDefinedMap fields_map({
{Field::one, "one"},
{Field::two, "two"}
});
RecordBuilder<
RecordField<Field::one, NumpyBuilder<double>>,
RecordField<Field::two, ListOffsetBuilder<int64_t,
5 #include "awkward/LayoutBuilder.h"
using awkward::layoutbuilder; #include "awkward/LayoutBuilder.h"
using awkward::layoutbuilder; #include "awkward/LayoutBuilder.h" using awkward::layoutbuilder; // instantiating layoutbuilder types (type aliases omitted for brevity)
enum Field : std::size_t {one, two};
using UserDefinedMap = std::map<std::size_t, std::string>;
UserDefinedMap fields_map({
{Field::one, "one"},
{Field::two, "two"}
});
RecordBuilder<
RecordField<Field::one, NumpyBuilder<double>>,
RecordField<Field::two, ListOffsetBuilder<int64_t, NumpyBuilder<int32_t>>>
> builder(fields_map); NumpyBuilder<int32_t>>>
> builder(fields_map); // executable code to create and fill the layoutbuilder
auto& one_builder = builder.field<Field::one>();
auto& two_builder = builder.field<Field::two>(); // executable code to create and fill the layoutbuil
auto& one_builder = builder.field<Field::one>();
auto& two_builder = builder.field<Field::two>(); one_builder.append(1.1);
auto& two_subbuilder = two_builder.begin_list();
two_subbuilder.append(1);
two_builder.end_list(); one_builder.append(1.1);
auto& two_subbuilder = two_builder.begin_list(); auto& two_subbuilder = two_builder.begin_list(); two_builder.begin_list(); two_subbuilder.append(1); two_subbuilder.append(2); two_builder.end_list(); 7 Summary and future plans The Awkward Array library was designed around array-oriented programming, in part be-
cause imperative programming in Python is impractical at large scales. However, this library
also provides many ways to access the same data—without copying it—in JIT-compiled con-
texts, enabling imperative computing at large scales. While these bridges may be used directly by users, they are also pathways to future fea-
tures. For example, automatic bindings from Kaitai Struct [10] to Awkward Arrays are in
development, and these rely heavily on the header-only awkward::layoutbuilder. JIT-
compilation in Julia [11] is also in development, borrowing from the patterns set by C++ and
Numba integration. 8 Acknowledgment This work is supported by NSF cooperative agreement OAC-1836650 (IRIS-HEP) and NSF
cooperative agreement PHY-2121686 (US-CMS LHC Ops). [4] cppyy [Online] https://cppyy.readthedocs.io/en/latest/ RecordBuilder< RecordBuilder< RecordField<Field::two, ListOffsetBuilder<int64_t, 5 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 NumpyBuilder<int32_t>>>
> builder(fields_map);
// executable code to create and fill the layoutbuilder
auto& one_builder = builder.field<Field::one>();
auto& two_builder = builder.field<Field::two>();
one_builder.append(1.1);
auto& two_subbuilder = two_builder.begin_list();
two_subbuilder.append(1);
two_builder.end_list();
one_builder.append(2.2);
two_builder.begin_list();
two_subbuilder.append(1);
two_subbuilder.append(2);
two_builder.end_list(); [2] Harris, C.R., Millman, K.J., van der Walt, S.J. et al. Array programming with NumPy.
Nature 585, 357–362 (2020). DOI: 10.1038/s41586-020-2649-2 References [1] Pivarski, J., Osborne, I., Ifrim, I., Schreiner, H., Hollands, A., Biswas, A., Das, P., Roy
Choudhury, S., Smith, N., & Goyal, M. (2018). Awkward Array [Computer software]. https://doi.org/10.5281/zenodo.4341376 [1] Pivarski, J., Osborne, I., Ifrim, I., Schreiner, H., Hollands, A., Biswas, A., Das, P., Roy
Choudhury, S., Smith, N., & Goyal, M. (2018). Awkward Array [Computer software]. https://doi.org/10.5281/zenodo.4341376 [2] Harris, C.R., Millman, K.J., van der Walt, S.J. et al. Array programming with NumPy. Nature 585, 357–362 (2020). DOI: 10.1038/s41586-020-2649-2 [3] RDataFrame: Easy Parallel ROOT Analysis at 100 Threads Danilo Piparo, Philippe
Canal, Enrico Guiraud, Xavier Valls Pla, Gerardo Ganis, Guilherme Amadio, Axel
Naumann,
Enric Tejedor EPJ Web Conf. 214 06029 (2019) DOI: 10.1051/epj-
conf/201921406029 [4] cppyy [Online] https://cppyy.readthedocs.io/en/latest/ [4] cppyy [Online] https://cppyy.readthedocs.io/en/latest/ 6 EPJ Web of Conferences 295, 06003 (2024)
CHEP 2023 https://doi.org/10.1051/epjconf/202429506003 [5] Siu Kwan Lam, stuartarchibald, Antoine Pitrou, Mark Florisson, Stan Seibert, Graham
Markall, esc, Todd A. Anderson, rjenc29, Guilherme Leobas, luk-f-a, Michael Collison,
Jay Bourque, Aaron Meurer, Travis E. Oliphant, Nick Riasanovsky, Kaustubh, Michael
Wang, densmirn, ... James Bourbeau. (2022). numba/numba: Version 0.56.4 (0.56.4). Zen-
odo. https://doi.org/10.5281/zenodo.7289231 [6] Osborne, I., Pivarski, J. 2022 Awkward to RDataFrame and back [arXiv 2302.09860]
DOI: 10.48550/arXiv.2302.09860 [7] Osborne, I., Pivarski, J., (2022, September 15). Awkward RDataFrame Tutorial. PyHEP
2022 (virtual) Workshop. Zenodo. https://doi.org/10.5281/zenodo.7081586 [8] Jim Pivarski, Peter Elmer and David Lange, (16 November 2020). Awkward Ar-
rays in Python, C++, and Numba, EPJ Web of Conferences 245, 05023 (2020). https://doi.org/10.1051/epjconf/202024505023 [9] Goyal, M., Osborne, I., Pivarski, J. The Awkward World of Python and C++. ACAT 2022
Workshop. https://doi.org/10.48550/arXiv.2303.02205 [10] Kaitai Struct [Online]. https://kaitai.io [11] Julia [Online] https://julialang.org 7
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English
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Indium-catalysed ring opening of 2-hydroxybutyrolactone through the cleavage of C(sp<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mrow /> <mml:mn>3</mml:mn> </mml:msup></mml:math>)–O bond
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Comptes rendus. Chimie
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cc-by
| 5,883
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Indium-catalysed ring opening of
2-hydroxybutyrolactone through the cleavage of
C(sp3)–O bond Sameh Aouna, Joe Massouha, Noémie Scorneta, Laurent Giordanoa,
Alphonse Tenagliaa, Gérard Buono
a, Patrick Reyb, Virginie Bellière-Baca∗, b
and Damien Hérault
∗, a a Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France b Adisseo France SAS, Antony Parc 2, 10 Place du Général de Gaulle, 92160 Antony,
France E-mails: sameh.aoun@gmail.com (S. Aoun), joe.massouh@centrale-marseille.fr
(J. Massouh), noemie.scornet@gmail.com (N. Scornet),
laurent.giordano@centrale-marseille.fr (L. Giordano), tenaglia.alfonso@orange.fr
(A. Tenaglia), gerard.buono@centrale-marseille.fr (G. Buono),
Patrick.Rey@adisseo.com (P. Rey), virginie.belliere-baca@adisseo.com
(V. Bellière-Baca), damien.herault@centrale-marseille.fr (D. Hérault) (V. Bellière-Baca), damien.herault@centrale-marseille.fr (D. Hérault) Abstract. The ring opening by the cleavage of the C(sp3)–O bond of 2-hydroxybutyrolactone with
(ethylthio)trimethylsilane was effectively catalysed by indium triiodide. Screening the reaction con-
ditions allowed optimizing the ring opening. The effect of the nucleophile/catalyst ratio on the reac-
tion efficiency has also been demonstrated. After optimization, 2-hydroxy-4-(ethylthio)butyric acid
was isolated with an excellent yield (85%), thus making this method useful for the preparation of me-
thionine analogues. Keywords. Methionine, Lactone, Sulfur nucleophiles, Indium, Thiol. . Methionine, Lactone, Sulfur nucleophiles, Indium, Thio Funding. This work was supported by Adisseo (SA post-doc grant, NS internship grant), the Région
PACA and Centrale Marseille (JM Ph.D. grant), the CNRS and AMU. Manuscript received 5 July 2023, accepted 18 July 2023. Funding. This work was supported by Adisseo (SA post-doc grant, NS internship grant), the Région
PACA and Centrale Marseille (JM Ph.D. grant), the CNRS and AMU. Manuscript received 5 July 2023, accepted 18 July 2023. ∗Corresponding authors. Chimie Sameh Aoun, Joe Massouh, Noémie Scornet, Laurent Giordano, Alphonse
Tenaglia, Gérard Buono, Patrick Rey, Virginie Bellière-Baca and Damien
Hérault Sameh Aoun, Joe Massouh, Noémie Scornet, Laurent Giordano, Alphonse
Tenaglia, Gérard Buono, Patrick Rey, Virginie Bellière-Baca and Damien
Hérault Volume 26 (2023), p. 89-98
Published online: 11 September 2023
https://doi.org/10.5802/crchim.245 Volume 26 (2023), p. 89-98 This article is licensed under the
Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ Les Comptes Rendus. Chimie sont membres du
Centre Mersenne pour l’édition scientifique ouverte
www.centre-mersenne.org
e-ISSN : 1878-1543 Les Comptes Rendus. Chimie sont membres du
Centre Mersenne pour l’édition scientifique ouverte
www.centre-mersenne.org
e-ISSN : 1878-1543 Les Comptes Rendus. Chimie sont membres du
Centre Mersenne pour l’édition scientifique ouverte
www.centre-mersenne.org
e-ISSN : 1878-1543 Comptes Rendus
Chimie
2023, Vol. 26, p. 89-98
https://doi.org/10.5802/crchim.245 Comptes Rendus
Chimie
2023, Vol. 26, p. 89-98
https://doi.org/10.5802/crchim.245 1. Introduction amino acid in human and animal organisms. It plays
an important role in cellular survival and protein
biosynthesis. Added to food supplements, it im-
proves the quantity and quality of products (meat,
milk) from herbivorous animals [1–5]. Methionine
can be obtained synthetically by cyclisation of ho-
moserine followed by an opening of the lactone us-
ing mercaptan salts [6]. Methionine may be given as Considerable developments are underway to de-
velop alternative sustainable and competitive path-
ways for methionine and hydroxy analogue pro-
duction. Indeed, methionine is the most essential ISSN (electronic) : 1878-1543 https://comptes-rendus.academie-sciences.fr/chimie/ https://comptes-rendus.academie-sciences.fr/chimie/ Sameh Aoun et al. 90 Figure 1. Structures of methionine, HMB and
KMB derivatives. Scheme 1. Ring opening of 1a catalysed by
Lewis acid. Scheme 1. Ring opening of 1a catalysed by
Lewis acid. Here we describe a general route to the desired
product, in which a direct coupling between 1a and
(ethylthio)trimethylsilane was effectively catalysed
by InI3 in the presence of K2CO3 under drastic con-
ditions (InI3 is stored and weighted in glove-box,
Schlenck techniques are used, see Chapter 4: exper-
imental section). Our method was inspired by the
methodology initially developed by Baba and co-
workers [21] (Scheme 2). They first studied the for-
mation of thioether starting from ester. Among sev-
eral Lewis acid catalyst, the best result was achieved
with InI3 (77%), which showed a better ability to pro-
mote the substitution than InBr3 or InCl3. Then, they
applied their best catalytic conditions to the ring-
opening reaction of γ-butyrolactone (1b), which
gave the corresponding thioethercarboxylic acid in
73% yield, using InI3 as a catalyst and (phenylth-
iol)trimethylsilane as a nucleophile. Better yields can
be obtained with larger lactones which highlights
the relative difficulty to open butyrolactones by the
cleavage of the C(sp3)–O bond. Figure 1. Structures of methionine, HMB and
KMB derivatives. a racemic compound as well as one of its analogue,
the 2-hydroxy-4-(methylthio)butyric acid (HMB). In-
deed the latter and 2-keto-4-(methylthio)butyric acid
(KMB) (Figure 1) are biotransformed in the animal
organisms to form methionine in vivo [7–10]. The
synthesis of these derivatives is therefore an alter-
native to the preparation of methionine at industrial
level. 1In
the
classical
synthesis
of
methionine
analogues,
methanethiol is used. But for practical reasons (odor and han-
dling), we used EtSH and more convenient TMSSEt in this study. 1. Introduction KMB is synthetized by a 1,4 Michael addition on
an α,β-unsaturated ketone [1–5,11] while the open-
ing of 2-hydroxybutyrolactone (1a) using mercaptan
salts is the classic pathway to HMB [12], which are en-
ergy consuming and generates a significant amount
of solid waste (notably sodium salts). To overcome
this problem, the replacement of mercaptan salts by
soft nucleophiles in the presence of Lewis acids is
promising. In this context, several methods of (α-
substituted) lactones ring opening have been carried
out, in particular with the use of a aluminium halides
as Lewis acids [13,14], but they still have a limitation
in the application scope in terms of the nature of the
substitution [13–17]. The ring-opening reaction of 2-
hydroxy-substituted lactones such as 1a remains rel-
atively underdeveloped despite its great usefulness
to lead to HMB with minimum waste. To our knowl-
edge, scarce reports of 1a opening reactions have ap-
peared where the reaction occurs at the carbon atom
C(sp3)–O with various strong or soft nucleophiles like
nitrogen [15], selenium [18–20], sulphur [1,16,21], or
iodide atoms [22,23]. 2. Results and discussion We first began our investigations by reproducing
the ring-opening reaction of 1b, but by replacing
(phenylthiol)trimethylsilane
with
ethanethiol
or
(ethylthio)trimethylsilane (Table 1, entries 1 and
2).1 Ethanethiol was stirred in toluene with 1b and
a catalytic amount of InI3 at 90 °C to obtain 2b in
only 15% yield after 20 h of stirring. However, the
use of (ethylthio)trimethylsilane as a nucleophile
under the same conditions led to a full conversion
after only 3 h of stirring (Entry 2). When 1a was used,
no ring opening was observed with EtSH even after In
this
work,
we
developed
a
ring-opening
methodology of 2-hydroxybutyrolactone (1a) catal-
ysed by a Lewis acid, with soft sulphur nucleophile to
obtain 2-hydroxy-4-(ethylthio)butyric acid (2a), an
HMB analogue (Scheme 1). 91 Scheme 2. Formation of thioether from ester and γ-butyrolactone (1b) catalysed by InI3 developed by
Baba and coworkers [21]. Scheme 2. Formation of thioether from ester and γ-butyrolactone (1b) catalysed by InI3 developed by
Baba and coworkers [21]. Scheme 2. Formation of thioether from ester and γ-butyrolactone (1b) catalysed by InI3 developed by
Baba and coworkers [21]. Table 1. InI3-catalysed ring opening of 1a and 1b
Entry
Substrate
Nucleophile
t (h)
Isolated yields (%)
2
3
4
1
1b
EtSH
20
2b: 15a
0
0
2
1b
EtSSiMe3
3
2b: 99
0
0
3
1a
EtSHb
48
0
0
0
4
1a
EtSSiMe3
22
2a: 30
3a: 7
0
5
1a
EtSSiMe3c
22
2a: 42
0
4a: 15
aNMR yields. bReaction performed with 3.8 equiv. of EtSH and 20% of InI3. cReaction in the presence of
1.1 equiv. of K2CO3. Table 1. InI3-catalysed ring opening of 1a and 1b aNMR yields. bReaction performed with 3.8 equiv. of EtSH and 20% of InI3. cReaction in the presence of
1.1 equiv. of K2CO3. aNMR yields. bReaction performed with 3.8 equiv. of EtSH and 20% of InI3. cReaction in the presence of
1.1 equiv. of K2CO3. increasing the amounts of both nucleophile and cat-
alyst, as well as the reaction time (Entry 3). So, we
leaned towards the use of EtSSiMe3 because it gave
an excellent result with 1b (Entry 2) and its boiling
point is higher than the reaction temperature. To our
delight, the ring opening of 1a carried out with this
soft nucleophile led to the desired product 2a in 30%
yield along with 3a in 7% yield (ring opening and
thioesterification product, entry 4). 2. Results and discussion At this point, we
concluded that our system could be different from
Baba’s one due to the presence of the hydroxy group
at the α-position of the lactone. Indeed, the addition
of a stoichiometric amount of K2CO3 to the reaction
mixture allowed to increase the yield of 2a, although
the formation of by-product 4a, resulting from the nucleophilic attack on the C-1 position (ester), was
also observed (Entry 5). EtSH has a boiling point about 35 °C, which is be-
low the reaction temperature. Its volatility can lead
to a loss of reagent while heating at 90 °C. To this
end, reactions with EtSH were performed in an auto-
clave system (closed Schlenk tube of 5 mL) contain-
ing only 2/5 of dead volume instead of a conventional
reflux system. No real improvement was observed
with this nucleophile. But when reactions were car-
ried out with EtSSiMe3 (boiling point about 134 °C,
above the reaction temperature) using the same con-
ditions, the yields have been improved (Table 2). First, a control experiment in the absence of the
catalyst did not lead to the formation of products 92 Sameh Aoun et al. Table 2. InI3-catalysed ring opening of 1a in an autoclave system
Entry
Nucleophile
InI3 (%)
K2CO3 (equiv.)
Isolated yields (%)
2a
3a
4a
1
EtSSiMe3
-
-
0
0
0
2
EtSSiMe3
-
1.1
0
0
0
3
EtSH
-
1.1
Traces
0
0
4
EtSSiMe3
10
-
35
4
Traces
5
EtSSiMe3
10
1.1a
66
0
9
6
EtSH
10
-
0
0
0
7
EtSH
10
1.1a
18
0
0
8
EtSH
10
1.1b
26
0
0
9
EtSSiMe3
10
1.1b
85
0
11
10
EtSSiMe3
10
1.1c
16
0
9
aWithout preliminary drying of 1a and K2CO3. bWith preliminary drying of 1a and K2CO3. cWith addition
of 0.25 mL of water in the reaction mixture. Table 2. InI3-catalysed ring opening of 1a in an autoclave system aWithout preliminary drying of 1a and K2CO3. bWith preliminary drying of 1a and K2CO3. cWith addition
of 0.25 mL of water in the reaction mixture. With optimal reaction conditions in hand, based
on the Baba’s study [21], further screening of cat-
alysts in autoclave systems was realized (Table 3).2
As expected, InI3 provided the best result for the ring
opening of 1a (Entry 1). But In(OTf)3 (less hard LA
than InI3) also showed good activity (Entry 2). 2In comparison to the study of Yamakasi and Shibuya [22,23],
the Brönsted acid HI (1 equiv.) was used in EtSH as solvent (C
= 0.5 M), at 35 °C without base. Mixture of iodide substituted
compounds were detected by mass spectroscopy, but no desired
SEt substituted products were observed. 2. Results and discussion Ring opening of 1a with various loadings of InI3
Entry
Nucleophile
InI3 (%)
Isolated yields (%)
2a
3a
4a
1
EtSH
10
26
0
0
2
EtSH
20
23
0
0
3
EtSSiMe3
5
59
6
0
4
EtSSiMe3
10
85
0
11
5
EtSSiMe3 (1.1 equiv)
10
24a
15a
0
6
EtSSiMe3 (0.3 equiv)
10
Tracesa
Tracesa
0
7
EtSSiMe3
20
91
Traces
3
8
EtSSiMe3
33
61
6
0
9
EtSSiMe3b
33
47
2
0
10
EtSSiMe3 (3 equiv)
33
90
0
0
aNMR observations. bThe putative catalytic intermediate InI(3−n)(SEt)n was heated to 90 °C (2 h) then
cooled to r.t. before the addition of 1a and K2CO3. Formation of InI(3−n)(SEt)n: InI3 + n EtSSiMe3 →
InI(3−n)(SEt)n +n ISiMe3. The InI3 quantity has no influence on the yield
when EtSH was used (Table 4, entries 1 and 2). Nev-
ertheless, with the EtSSiMe3 and 5% of InI3 (Entry 3),
the desired product 2a was isolated with 59% yield
try 8), which could indicate either an insufficient
activation of plausible intermediates InI(3−n)(SEt)n
formed in situ, or an insufficient amount of nu-
cleophile The second hypothesis was confirmed by Table 3. Catalysts screening for the ring opening of 1a
Entry
Catalyst
Isolated yields (%)
2a
3a
4a
1
InI3
85
0
11
2
In(OTf)3
58
8
0
3
Bi(OTf)3
4
7
0
4
Al(OTf)3
0
0
0
5
AlBr3
0
0
10 Table 3. Catalysts screening for the ring opening of 1a
Entry
Catalyst
Isolated yields (%)
2a
3a
4a
1
InI3
85
0
11
2
In(OTf)3
58
8
0
3
Bi(OTf)3
4
7
0
4
Al(OTf)3
0
0
0
5
AlBr3
0
0
10 Table 3. Catalysts screening for the ring opening of 1a Table 4. Ring opening of 1a with various loadings of InI3
Entry
Nucleophile
InI3 (%)
Isolated yields (%)
2a
3a
4a
1
EtSH
10
26
0
0
2
EtSH
20
23
0
0
3
EtSSiMe3
5
59
6
0
4
EtSSiMe3
10
85
0
11
5
EtSSiMe3 (1.1 equiv)
10
24a
15a
0
6
EtSSiMe3 (0.3 equiv)
10
Tracesa
Tracesa
0
7
EtSSiMe3
20
91
Traces
3
8
EtSSiMe3
33
61
6
0
9
EtSSiMe3b
33
47
2
0
10
EtSSiMe3 (3 equiv)
33
90
0
0
aNMR observations. bThe putative catalytic intermediate InI(3−n)(SEt)n was heated to 90 °C (2 h) then
cooled to r.t. before the addition of 1a and K2CO3. 2. Results and discussion How-
ever, other Lewis acids based on metal triflate have
been tested. The soft and larger Bi(OTf)3 was found
to be unsatisfactory (Entry 3), as well as the unreac-
tive oxophile Al(OTf)3 (Entry 4). Also, AlBr3 allowed
the formation of only a small quantity of the thioester
4a (10%). Therefore, the reactivities are clearly de-
pendent on the nature of the Lewis acid as observed
by Baba. 2–4a (Entries 1–3). On the other hand, with 10% of
InI3 and in the absence of K2CO3, there was no sig-
nificant efficiency compared to a conventional re-
flux system (Entry 4, compared to entry 4 in Table 1). When the base was present (Entry 5), the yield of 2a
was increased significantly (66%). As expected, EtSH
instead of EtSSiMe3 was less efficient in producing 2a
(18% yield) in the presence of K2CO3 (Entry 7) while
it did not generate the desired product without a
base (Entry 6). In addition, the presence of a residual
amount of water in 1a or K2CO3 is suspected to de-
crease the reactivity of the nucleophile reagent, and
consequently, of leading to lower yields. Drying 1a
and K2CO3 before their use confirmed this hypoth-
esis (Entries 8 and 9). Finally with optimised condi-
tions, EtSSiMe3 gave the desired product. Indeed, 2a
was successfully isolated with 85% yield along with
by-product 4a in 11% yield, which corresponds to an
overall conversion of 96%. The introduction of addi-
tional water into the reaction mixture logically led to
a dramatic decrease in yield (Entry 10). We studied in more detail the loading of InI3 on
the outcome of the reaction. As shown in Table 4,
the ring opening of 1a was examined with different
catalytic amounts of this Lewis acid. 93 Sameh Aoun et al. Table 3. Catalysts screening for the ring opening of 1a
Entry
Catalyst
Isolated yields (%)
2a
3a
4a
1
InI3
85
0
11
2
In(OTf)3
58
8
0
3
Bi(OTf)3
4
7
0
4
Al(OTf)3
0
0
0
5
AlBr3
0
0
10
Table 4. 2. Results and discussion Formation of InI(3−n)(SEt)n: InI3 + n EtSSiMe3 →
InI(3−n)(SEt)n +n ISiMe3. Table 4. Ring opening of 1a with various loadings of InI3 Table 4. Ring opening of 1a with various loadings of InI3 aNMR observations. bThe putative catalytic intermediate InI(3−n)(SEt)n was heated to 90 °C (2 h) then
cooled to r.t. before the addition of 1a and K2CO3. Formation of InI(3−n)(SEt)n: InI3 + n EtSSiMe3 →
InI(3−n)(SEt)n +n ISiMe3. The InI3 quantity has no influence on the yield
when EtSH was used (Table 4, entries 1 and 2). Nev-
ertheless, with the EtSSiMe3 and 5% of InI3 (Entry 3),
the desired product 2a was isolated with 59% yield,
while the yield reached 91% with 20% of Lewis acid
(Entry 7). Surprisingly, a remarkable decrease in yield
was observed with 33% of the catalyst loading (En- try 8), which could indicate either an insufficient
activation of plausible intermediates InI(3−n)(SEt)n
formed in situ, or an insufficient amount of nu-
cleophile. The second hypothesis was confirmed by
a rise in the yield with 3 equivalents of EtSSiMe3
(Entries 9 and 10). This suggests that likely at least
(3 ∗n (catalyst) + 1) equivalents of the nucleophile 94 Sameh Aoun et al. Table 5. Reactivity study of different α-substituted-γ-butyrolactones in InI3-catalysed ring-opening
reactions Table 5. Reactivity study of different α-substituted-γ-butyrolactones in InI3-catalysed ring-opening
reactions Entry
Substrate
Nucleophile
Base (equiv.)
Isolated yields (%)
2
3
4
1
1a
EtSH
K2CO3 (1.1)
2a: 26
0
0
2
1c
EtSH
K2CO3 (1.1)
2a: 21
3a: 4
4a: Traces
3
1d
EtSH
K2CO3 (1.1)
2d: 39
0
0
4
1a
EtSSiMe3
-
2a: 35
3a: 4
4a: Traces
5a
1a
EtSSiMe3
-
2a: 34
3a: 4
4a: Traces
6
1c
EtSSiMe3
-
2a: 55
3a: 5
4a: 17
7
1d
EtSSiMe3
-
2d: 2
3d: 17
4d: 39
8
1a
EtSSiMe3
K2CO3 (1.1)
2a: 85
0
4a: 11
9
1c
EtSSiMe3
K2CO3 (1.1)
2a: 47
0
4a: Traces
10
1d
EtSSiMe3
K2CO3 (1.1)
2d: 11b
0
4d: 53b
11
1a
EtSSiMe3
DBU (1.1)
2a: 40
0
4a: Traces
aReaction performed with the addition of molecular sieves. bNMR yields. Entry
Substrate
Nucleophile
Base (equiv.)
Isolated yields (%)
2
3
4 aReaction performed with the addition of molecular sieves. bNMR yields. are required to achieve the maximum conversion. This hypothesis was also confirmed by the experi-
ment with 0.3 equiv. 2. Results and discussion of EtSSiMe3 and 10% of InI3 (En-
try 6), where only traces (instead of expected 20–30%)
of expected products were observed. This lack of
conversion suggests a plausible interaction between
the catalyst and the nucleophile. The ratio nucle-
ophile/catalyst appears to be important and the pu-
tative InI(3−n)(SEt)n species could be formed before
acting as a catalyst. It should be noted that Baba and
co-workers used an InI3/ester/PhSSiMe3 = 1/10/20
ratio in their experiments, so more than (3*n (cata-
lyst) +1) equivalents of nucleophile. (Table 5, entries 1–3). On the other hand, when the
nucleophile is EtSSiMe3 and in the absence of a
base, 1c was found to be the most reactive sub-
strate with a yield of 55% of 2a (Entries 4–7). More-
over, the addition of K2CO3 promoted the reactivity
of 1a to 2a, while 1d mainly provided the thioester 4d
(Entries 8–10). The presence of the -OTMS group on
the substrate 1c (Entries 2, 6 and 9) did not seem to
have a beneficial influence in the selective formation
of the compound 2a in these conditions. It is impor-
tant to note that this type of K2CO3 base is not crip-
pling for industrial purposes due to the possibility of
regeneration [27]. Based on the above results, we de-
cided to keep a base, but we replaced K2CO3 with the
more soluble DBU (Entry 11), which resulted in a dis-
appointing loss of yield. Finally, the outcome of the ring opening of sev-
eral α-substituted-γ-butyrolactones was examined
under various reaction conditions (Table 5). 1c [24] and 1d [25,26] were prepared from 1a ac-
cording to the protocols of the literature and iso-
lated with 96% and 86% yields respectively. Of the
two compounds, the ether 1d was found to be the
most reactive with EtSH in the presence of K2CO3 4.2.1. 3-(Trimethylsilyl)oxydihydrofuran-2(3H)-one
(1c) This compound was prepared according to a pro-
cedure reported elsewhere [24]. To a stirred solution
of 1a (0.074 mL, 1 mmol, 1.0 equiv.), in CH3NO2
(1 mL) under argon was added freshly distilled HMDS
(0.21 mL, 1 mmol, 1.0 equiv.). The reaction mix-
ture was stirred for 30 min at room temperature. Af-
ter concentration, the crude product was purified
by column chromatography using a gradient eluent
(from 100% petroleum ether to 90:10 = petroleum
ether:ethylacetate). 1c was recovered as colorless oil
(167 mg, 96%). These data are consistent with those
described in the literature [29]. 3. Conclusions In conclusion, we have developed an efficient and
practical synthesis of one methionine analogue, the 95 Sameh Aoun et al. HMB, by the InI3-catalysed ring-opening reaction
between 2-hydroxybutyrolactone (1a) and a thiosi-
lane in the presence of a base (K2CO3) which can be
regenerated. This could be advantageous for further
industrial developments. The reaction occurred se-
lectively at the carbon atom C(sp3)–O. The method
allows easy access to the desired product 2a in a high
isolated yield (85%) with 10% InI3 catalyst, when the
reaction is carried out in an autoclave and in the ab-
sence of water. To our knowledge, the current one-
pot reaction system is the only one, among the re-
ported procedures, that produces HMB analogues in
excellent yields from 1a using catalytic electrophilic
activation. with an Atmospheric Pressure Ionization (API) source
or on MaXiS 4G (Bruker) equipped with an Electron
Spray Ionization (ESI) source. Mass spectra were ob-
tained using a Time Of Flight (TOF) analyser. Ele-
mentary analyses were recorded on a Thermo Finni-
gan EA 1112 apparatus. 4.1. General methods All reagents were obtained from commercial sources
and used as received unless otherwise noted. K2CO3
was dried at 85 °C under vacuum in a Büchi glass
oven B-585. THF and toluene were purified and
dried over Braun solvent purification system (MB-
SPS-800). InI3 and the other Lewis acids were stored
and weighted in glove-box in oven-dried glassware. All reactions were performed in oven-dried glass-
ware under an atmosphere of argon using Schlenk
techniques unless otherwise noted. Analytical Thin
Layer Chromatography (TLC) was carried out on
Merck silica gel60 F254. Products were stained with
potassium permanganate dyeing reagent solution
followed by gentle heating. Flash chromatography
was performed on Combiflash® Companion or with
Merck silica gel 60 (230–400 mesh). 1H and 13C NMR
spectra were recorded in CDCl3, at ambient temper-
ature on Bruker Avance III 300 or 400 spectrometers
operating at 300 and 400 MHz respectively for 1H. 13C
nuclei were observed with 1H decoupling. Solvent
residual signals were used as internal standard [28]. Chemical shifts (δ) and coupling constants (J) are
given in ppm and Hz respectively. The peaks patterns
are indicated as the following format multiplicity (s:
singlet; d: doublet; t: triplet; q: quartet; qt: quintu-
plet; m: multiplet; dd: doublet of doublet). The pre-
fix br. indicates a broadened signal. IR spectra were
obtained using a Bruker Alpha Platinum ATR. HRMS
were recorded on SYNAPT G2 HDMS (Waters) or
on QStar Elite (Applied Biosystems SGIEX) equipped 1H NMR (400 MHz, CDCl3) δ 4.43–4.36 (m, 2H,
CH2OC(O)), 4.23–4.16 (m,1H, CHOSi), 2.51–2.43 (m,
1H, CH2CHOSi), 2.28–2.18 (m, 1H, CH2CHOSi), 0.20
(s, 9H, Si(CH3)3). 4.2.2. 3-Methoxydihydrofuran-2(3H)-one (1d) 4.2. Starting materials The 2-hydroxybutyrolactone 1a is commercially
available and was given by our industrial partner
Adisseo. It is dry over 3 Å MS for 72 h. The 2-hydroxybutyrolactone 1a is commercially
available and was given by our industrial partner
Adisseo. It is dry over 3 Å MS for 72 h. 4.2.1. 3-(Trimethylsilyl)oxydihydrofuran-2(3H)-one
(1c) 4.2.1. 3-(Trimethylsilyl)oxydihydrofuran-2(3H)-one
(1c) 4.2.2. 3-Methoxydihydrofuran-2(3H)-one (1d) NaH (60% in oil, 302 mg, 7.54 mmol, 1.5 equiv.)
was introduced in a double-neck round bottom flask. The oil is removed by several washings with pentane. Then, dry THF (10 mL) under argon atmosphere is
added. At 0 °C, 1a (0.37 mL, 5.02 mmol, 1.0 equiv.)
is added dropwise to the stirred solution. After 1 h,
while warming to room temperature, freshly distilled
MeI (0.47 mL, 7.54 mmol, 1.5 equiv.) is added. Sub-
sequently, the reaction mixture was stirred for 22 h
and then, 20 mL of saturated NH4Cl aqueous solution
is added. The layers were separated, and the aque-
ous layer was extracted with 3 × 25 mL of Et2O. The
combined organic layer was dried over Na2SO4, fil-
tered and concentrated. The crude product was puri-
fied by column chromatography using a gradient elu-
ent (from 100% petroleum ether to 60:40 = petroleum 96 Sameh Aoun et al. ether:ethylacetate). 1d (501 mg, 86%) was obtained
as yellow oil. and stirred for 22 h. After cooling to room temper-
ature, the resulting mixture was filtered, and then
was poured into 20 mL of HClaq (1M) and the layers
were separated. After extraction of the aqueous phase
with 3×20 mL of ethylacetate, the combined organic
layer was washed with brine, and dried over Na2SO4,
filtered and concentrated. The crude product was
purified by column chromatography using a gradi-
ent eluent (from 100% petroleum ether to 100% ethyl
acetate). R f = 0.55 (AcOEt/petroleum ether 8:2). 1H NMR
(400 MHz, CDCl3) δ 4.39–4.34 (m, 1H, CH2OC(O)),
4.23–4.17 (m,1H, CH2OC(O)), 4.00 (t, J = 7.8 Hz,
1H, CHOCH3), 3.51 (s, 3H, OCH3), 2.53–2.46 (m,
1H, CH2CHOCH3), 2.23–2.14 (m, 1H, CH2CHOCH3). 13C NMR (100 MHz, CDCl3): δ 174.8, 75.0, 65.4, 58.2,
29.4. These data are consistent with those described
in the literature [25,26]. 4.3. Experimental procedures and characteriza-
tions data This compound was prepared following the gen-
eral procedure starting from EtSSiMe3 (0.83 mL,
4.64 mmol, 1.9 equiv. (90% purity)), InI3 (242 mg,
0.48 mmol, 0.2 equiv.), 1a (0.18 mL, 2.44 mmol, 1.0
equiv.) and anhydrous K2CO3 (371 mg, 2.68 mmol,
1.1 equiv.). The crude product was purified by col-
umn chromatography using a gradient eluent (from
100% petroleum ether to 100% ethyl acetate). 4.3.1. 4-(Ethylthio)butyric acid (2b) 4.3.1. 4-(Ethylthio)butyric acid (2b) To a stirred suspension of InI3 (100 mg, 0.2 mmol,
0.1 equiv.) in anhydrous toluene (2 mL) was added
EtSSiMe3 [0.680 mL (90% purity), 3.8 mmol, 1.9
equiv.]. The reaction mixture was stirred for a few
minutes under N2 atmosphere at room temperature
before the addition of γ-butyrolactone 1b (0.15 mL,
2 mmol, 1.0 equiv.). The mixture was then heated
to 90 °C and stirred for 3 h. After cooling to room
temperature, the resulting mixture was poured into
20 mL of HClaq (1M) and the layers were separated. The aqueous layer was extracted with 3 × 20 mL of
ethylacetate, and the combined organic layers were
washed with brine, dried over Na2SO4 and filtered. After concentration in vacuo, carboxylic acid 2b was
obtained as a yellow liquid (295 mg, 99%) without fur-
ther purification. R f = 0.63 (AcOEt/petroleum ether:
8:2). 1H NMR (400 MHz, CDCl3) δ 10.66 (CO2H), 2.59–
2.47 (m, 6H, CH2SCH2 and CH2CO2H), 1.91 (qt, J =
7.19, 7.25 Hz, 2H, CH2CH2CH2), 1.24 (t, J = 7.4 Hz,
3H, CH3). 13C NMR (100 MHz, CDCl3): δ 179.7, 32.9,
30.8, 25.8, 24.4, 14.8. MS (ESI+): m/z = 155 [M+Li]+;
171 [M+Na]+. These data are consistent with those
described in the literature [14]. 2a (365 mg, 91%) was obtained as yellowish oil. R f = 0.52 (AcOEt/ petroleum ether: 8:2). 1H NMR
(400 MHz, CDCl3) δ 5.52 (br s, 2H, OH and CO2H),
4.43 (dd, J = 8.0, 3.8 Hz,1H, CHOH), 2.70 (t, J =
6.7 Hz, 2H, SCH2CH2), 2.56 (q, J = 7.4 Hz, 2H,
CH3CH2S), 2.18–2.09 (m, 1H, CH2CHOH), 2.01–1.93
(m, 1H, CH2CHOH), 1.26 (t, J = 7.4 Hz, 3H, CH3). 13C
NMR (100 MHz, CDCl3): δ 179.1, 69.4, 33.6, 27.3, 25.9,
14.8. IR (film): 3400, 2966, 2926, 1718, 1261, 1213,
1166, 1089 cm−1. HRMS (ESI): m/z [M+H]+ Calcd for
C6H12O3S 165.0580; Found 165.0579. 4.3.4. S-Ethyl
4-(ethylthio)-2-hydroxybutanethioate
(3a) 4.3.8. S-ethyl 4-hydroxy-2-methoxybutanethioate
(4d) This compound is another by-product formed
during the synthesis of 2d and was isolated during
the chromatography. 4d (174 mg, 39%) was obtained
as brown oil. R f = 0.65 (AcOEt/petroleum ether 8:2). 1H NMR
(400 MHz, CDCl3) δ 3.93–3.85 (m, 1H, CHOCH3),
3.73–3.65 (m, 2H, CH2OH), 3.42 (s, 3H, OCH3), 2.81
(q, J = 7.4 Hz, 2H, CH2CH3), 1.91–1.76 (m, 2H,
CH2CH2OH), 1.20 (t, J = 7.4 Hz, 3H, CH2CH3). 13C
NMR (101 MHz, CDCl3): δ 174.7, 75.0, 65.3, 58.1, 29.4,
19.5, 19.0. IR (film): 3408, 2931, 2880, 1417, 1376,
1177, 1105 cm−1. HRMS (ESI+): m/z [M+Na]+ Calcd
for C7H14O3SNa+ 201.0556; found 201.0562. The authors declare no conflict of interest. The authors declare no conflict of interest. 4.3.6. 4-(ethylthio)-2-methoxybutanoic acid (2d) This compound was prepared following the gen-
eral procedure starting from 1d (290 mg, 2.5 mmol,
1.0 equiv.), EtSH (0.355 mL, 4.75 mmol, 1.9 equiv.)
and 0.1 equiv. of InI3 (124 mg, 0.25 mmol). The crude
product was purified by column chromatography us-
ing DCM, then DCM with 5% MeOH as the eluent. 2d
(172 mg, 39%) was obtained as yellowish oil. Acknowledgments We thank Dr. Christophe Chendo and Dr. Valérie
Monnier for mass spectrometry analyses (Spec-
tropole, Fédération des Sciences Chimiques de
Marseille), Arnaud Treuvey (Centrale Marseille) for
IR spectroscopy and Yoann Cotelle and Maxime
Huchede for reading the manuscript. This compound is a by-product formed during the
synthesis of 2d. This compound was prepared follow-
ing the general procedure starting from 1d (290 mg,
2.5 mmol, 1.0 equiv.), 0.1 equiv. of InI3 (124 mg,
0.25 mmol) and no base was added. The crude prod-
uct was purified by column chromatography using
a gradient eluent (from 100% petroleum ether to
100% ethylacetate). 3d (93 mg, 17%) was obtained as
yellow oil. Conflicts of interest R f = 0.56 (DCM/MeOH 9:1). 1H NMR (400 MHz,
CDCl3) δ 3.98 (dd, J = 7.0, 5.0 Hz, 1H, CHOCH3),
3.45 (s, 3H, OCH3), 2.68–2.60 (m, 2H, SCH2CH2), 2.53
(q, J = 7.5 Hz, 2H, CH3CH2S), 2.08–2.00 (m, 2H,
CH2CHOCH3), 1.24 (t, J = 7.4 Hz, 3H, CH3CH2S). 13C NMR (100 MHz, CDCl3): δ 177.7, 78.7, 58.6, 32.4,
27.1, 25.8, 14.8. IR (film): 2928, 1718, 1450, 1193,
1117 cm−1. HRMS (ESI+): m/z [M+H]+ Calcd for
C7H14O3S 179.0736; Found 179.0737. Supplementary data Supporting information for this article is available on
the journal’s website under https://doi.org/10.5802/
crchim.245 or from the author. [1] P. Deck, K. M. Exner, B. Buschhaus, 2008, Patent WO
2008/022953A1.
[2] P. Rey, G. Blanchard, 2010, Patent EP 2 229 821 A2.
[3] T. Willke, Appl. Microbiol. Biotechnol., 2014, 98, 9893-9914. 4.3.5. S-Ethyl 2,4-dihydroxybutanethioate (4a) 4.3.5. S-Ethyl 2,4-dihydroxybutanethioate (4a) This compound is a by-product formed during the
synthesis of 2a and was isolated during the chro-
matography. 4a (10 mg, 3%) was obtained as color-
less oil. R f = 0.55 (AcOEt/ petroleum ether 8:2). 1H NMR
(500 MHz, CDCl3) δ 4.41 (dd, J = 9.3, 3.0 Hz,1H,
CHOH), 3.83 (t, J = 4.1 Hz, 2H, CH2OH), 2.84 (q,
J = 7.5 Hz, 2H, CH3CH2S(O)), 2.10–2.04 (m, 1H,
CH2CHOH), 1.85–1.79 (m, 1H, CH2CHOH), 1.22 (t,
J = 7.3 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3):
δ 206.3, 75.9, 59.4, 35.8, 22.8, 14.6. IR (film): 3328,
2973, 1417, 1086, 1044, 879 cm−1. HRMS (ESI+): m/z
[M+Na]+ Calcd for C6H12O3SNa+ 187.0399; found
187.0398. 4.3.8. S-ethyl 4-hydroxy-2-methoxybutanethioate
(4d) 4.3.4. S-Ethyl
4-(ethylthio)-2-hydroxybutanethioate
(3a) This compound is a by-product formed during the
synthesis of 2a and was isolated during the chro-
matography. 3a (traces, <1%) was obtained as color-
less oil. R f = 0.37 (AcOEt/ petroleum ether 1:9). 1H NMR
(300 MHz, CDCl3) δ 4.41 (dd, J = 8.3, 3.6 Hz, 1H,
CHOH), 2.92 (q, J = 7.4 Hz, 2H, CH3CH2S(O)), 2.84
(br s, 1H, OH), 2.70–2.65 (m, 2H, SCH2CH2), 2.56
(q, J = 7.4 Hz, 2H, CH3CH2SCH2), 2.17–2.06 (m,
1H, CH2CHOH), 1.98–1.87 (m, 1H, CH2CHOH), 1.29–
1.23 (m, 6H, 2CH3). 13C NMR (100 MHz, CDCl3): δ
203.8, 76.9, 34.5, 27.2, 25.9, 23.1, 14.8, 14.7. IR (film):
3420, 2966, 2929, 1678, 1263, 1088 cm−1. HRMS (ESI):
m/z [M+Na]+ Calcd for C8H16O2S2 231.0484; Found
231.0484. 4.3.2. General procedure for the ring opening of com-
pounds 1 In an autoclave system (5 mL closed Schlenk tube)
with a minimised dead volume, the nucleophile was
added to a stirred suspension of Lewis acid in an-
hydrous toluene (2.4 mL). The reaction mixture was
stirred for a few minutes under N2 atmosphere at
room temperature before the addition of dried 1
(2.44 mmol, 1.0 equiv.) followed by the eventual ad-
dition of base. The mixture was then heated to 90 °C 97 Sameh Aoun et al. [1] P. Deck, K. M. Exner, B. Buschhaus, 2008, Patent WO
2008/022953A1. [2] P. Rey, G. Blanchard, 2010, Patent EP 2 229 821 A2. [3] T. Willke, Appl. Microbiol. Biotechnol., 2014, 98, 9893-9914. References R f = 0.75 (AcOEt/ petroleum ether 1:9). 1H NMR
(400 MHz, CDCl3) δ 3.89 (dd, J = 7.8, 4.8 Hz, 1H,
CHOCH3), 3.46 (s, 3H, OCH3), 2.86 (q, J = 7.4 Hz, 98 Sameh Aoun et al. [18] J.-G. Boiteau, P. Van de Weghe, J. Eustache, Org. Lett., 2001, 3,
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Insect egg size and shape evolve with ecology but not developmental rate
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Church, Samuel H., Seth Donoughe, Bruno A. S. De Medeiros, and Cassandra G. Extavour. 2019. Insect Egg Size and Shape Evolve with Ecology but Not Developmental Rate. Nature 571, no. 7763: 58-62. Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42241195 http://nrs.harvard.edu/urn-3:HUL.InstRepos:42241195 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Insect egg size and shape evolve with ecology but not
developmental rate Samuel H. Church*,1, Seth Donoughe*,1,2, Bruno A. S. de Medeiros1, Cassandra G. Extavour1,3 1 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA
02138, United States 1 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA
02138, United States
2 Current address: Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago, IL 60637, United States
3 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138,
United States
* These authors contributed equally to the work. * These authors contributed equally to the work. * These authors contributed equally to the work. Over the course of evolution, the size of life changes dramatically. These changes are thought
to be explained by developmental, morphological, and ecological pressures. To perform
phylogenetic tests of these hypotheses, we generated a dataset of more than ten thousand
descriptions of insect eggs, and combined these with genetic and life-history datasets. We
show that across eight orders of magnitude in egg volume variation, the relationship between
size and shape itself evolves, such that predicted global patterns of scaling do not adequately
explain egg shape diversity. We show that size is not correlated with developmental rate, and
that for many insects, egg size is not correlated with adult body size either. Instead, we find
that the evolution of parasitoidism and aquatic oviposition help to explain the size and shape
diversification. Our study suggests that where eggs are laid, rather than universal allometric constants, underlies size and shape evolution. Size is a fundamental factor in many biological processes. Size may impact ecological
interactions1, 2, it scales with features of morphology and physiology3, and larger animals often
have higher fitness4. Previous studies have aimed to identify the macroevolutionary forces that
explain observed size distributions1, 5, 6, but limited data on the phylogenetic distribution of size
has precluded robust tests of these predicted forces4, 7. Here we address this problem by assembling
a dataset of insect egg phenotypes with sufficient taxon sampling to rigorously test hypotheses
about the causes and consequences of size evolution in a phylogenetic framework. Insect eggs are a compelling system in which to test these macroevolutionary hypotheses. Egg
morphologies are extraordinarily diverse8, yet they can be readily compared across distant lineages
using quantitative traits. Changes in egg size have been studied in relation to changes in other
aspects of organismal biology9, including adult body size10–12, features of adult anatomy13, and
offspring fitness via maternal investment14. Eggs must also withstand the physiological challenges
of being laid in diverse microenvironments, including in water, air, or internal to plants or
animals15. Furthermore, because the fertilized egg is the homologous, single-cell stage in the
lifecycle of multicellular organisms, egg size diversity is relevant to both cell size and organism
size evolution8, 14. Three classes of hypotheses have been proposed to explain the evolution of egg size and shape:
[1] geometric constraints due to the physical scaling of size and shape13, 16–19, [2] the interaction
between size and the rate of development20–22, and [3] the diversification of size and shape
response to ecological or life-history changes10, 13, 15, 23. We use a phylogenetic approach to test all three of these hypotheses, and show that many presumed universal patterns in size, shape, and
embryonic development are not supported across insects. Instead, we find that models accounting
for ecological change best explain extant egg morphological diversity. Using custom bioinformatic tools, we assembled a dataset of 10,449 published descriptions of
eggs, comprising 6,706 species, 526 families, and every extant hexapod order (Figs. 1a, S1)24. We
combined this dataset with backbone hexapod phylogenies25, 26 that we enriched to include taxa in
the egg morphology dataset (Fig. S2), and used it to describe the distribution of egg shape and size
(Fig. 1b). Our results showed that insect eggs span more than eight orders of magnitude in volume
(Figs. constants, underlies size and shape evolution. 1a, 1c, S3), and revealed new candidates for the smallest and largest described insect eggs:
respectively, a parasitoid wasp Platygaster vernalis27 (volume = 7 x 10-7 mm3), and an earth-boring
beetle, Bolboleaus hiaticollis28 (volume = 5 x 102 mm3) (Fig. 1c). Plotting eggs by morphology revealed that some shapes evolved only in certain clades (Fig. 1a,
S4-S7). Oblate ellipsoid eggs (aspect ratio <1) are found only in stoneflies, moths and butterflies
(Plecoptera, Fig. S4, and Lepidoptera, Fig. 1c image 4, S5). Egg cases (oothecae) have evolved in
multiple lineages29. To test whether oothecae constrain shape or size, we measured individual eggs
within cases, and found that these are morphologically similar to freely laid relatives (Fig. S8). The most prominent pattern was that distantly related insects have converged on similar
morphologies many times independently (Fig. 1a, S7). This high degree of morphological
convergence allowed us to robustly test trait associations across independent evolutionary events. Evolutionary allometry of insect eggs To test which aforementioned scaling relationship best describes insect egg evolution, we
compared support for Hypotheses A and B using a phylogenetic generalized least-squares
approach (PGLS) to determine the scaling exponent of length and width (the slope of the regression
of log-length and log-width). A slope less than one would support Hypothesis A, while a slope
greater than one would support Hypothesis B33. An alternative, Hypothesis C, is that egg shape
remains the same as size changes, which would result in a slope near one (an isometric relationship;
hypotheses shown in Fig. 2a-d). We found that Hypothesis B is best supported across all insects:
larger eggs have higher aspect ratios than smaller eggs (0 < p-value < 0.005, slope = 0.78, Fig. 2e,
Table S6), even when controlling for adult body size (Fig. S14, Table S8). We found no support
for Hypothesis A, suggesting that future hypotheses of egg shell evolution may need to account
for additional factors such as chorion composition and thickness when considering cost. However,
the allometric relationship between size and shape evolves dynamically across the phylogeny, as
has also been shown for metabolic scaling in mammals34. Hypothesis C could not be rejected for
beetles and their relatives, nor for butterflies, moths, and caddisflies (respectively Neuropteroidea,
p-value of alternative hypothesis test = 0.04, and Amphiesmenoptera, p-value = 0.01; Fig. 2f, S12,
Table S7). Calculating the scaling relationship on lineage subgroups revealed that additional
clades, including mayflies, crickets, and shield bugs, also show an isometric relationship (Fig. S13). The marked differences in scaling exponents are evidence that egg evolution was not
governed by a universal allometric constant. Instead, evolutionary forces beyond the constraints To test which aforementioned scaling relationship best describes insect egg evolution, we
compared support for Hypotheses A and B using a phylogenetic generalized least-squares
approach (PGLS) to determine the scaling exponent of length and width (the slope of the regression
of log-length and log-width). A slope less than one would support Hypothesis A, while a slope
greater than one would support Hypothesis B33. An alternative, Hypothesis C, is that egg shape
remains the same as size changes, which would result in a slope near one (an isometric relationship;
hypotheses shown in Fig. 2a-d). We found that Hypothesis B is best supported across all insects:
larger eggs have higher aspect ratios than smaller eggs (0 < p-value < 0.005, slope = 0.78, Fig. Evolutionary allometry of insect eggs Two opposing hypotheses based on predicted geometric constraints have been proposed to explain the evolutionary relationship between egg shape and size. Hypothesis A: When eggs evolve to be
larger, they get wider (increases in egg size are associated with decreases in aspect ratio)17, 18. This
hypothesis predicts a reduction in relative surface area as size increases, which has been proposed
as a solution to the presumed cost of making eggshell material18. Hypothesis B: When eggs evolve
to be larger, they get longer (increases in egg size are associated with increases in aspect ratio)13,
18, 19. This hypothesis predicts a reduction in relative cross-sectional area as eggs get larger, which
has been proposed as a solution to the need for eggs to pass through a narrow opening during
oviposition13, 19. To test these hypotheses about the physical scaling of size and shape, we began by modeling the
individual evolutionary history of each morphological trait. This allowed us to first determine
whether distributions of extant shape and size have been shaped by phylogenetic relationships, or
if these traits are instead stochastically distributed. For egg volume, aspect ratio, asymmetry, and
angle of curvature (Fig. 1d), we compared four models of evolution: Brownian motion (BM),
Brownian motion with evolutionary friction (Ornstein-Uhlenbeck, OU), Brownian motion with a
decreasing rate of evolution (Early Burst, EB), and a non-phylogenetic model of stochastic motion
(white noise, WN). We found strong evidence that models accounting for phylogenetic covariance
fit our data better than a non-phylogenetic model (WN); in other words, insect egg morphology
tends to be similar in closely related insects (Table S5). For egg size and aspect ratio, an ‘Early-
Burst’ (EB) model in which evolutionary rate decreases over time, best describes the observed
data. This change in rate is distributed non-uniformly over the insect phylogeny, with some clades
evolving faster than others (Figs. S9-S11). In earlier studies EB models were rarely detected30;
however our findings are consistent with recent work evaluating datasets that, like ours, comprise many taxa and orders of magnitude in morphological variation31, 32. Having established the
phylogenetic models that best describe egg trait evolution, we used these results to test hypotheses
about the relationship between egg shape and size. Evolutionary allometry of insect eggs 2e,
Table S6), even when controlling for adult body size (Fig. S14, Table S8). We found no support
for Hypothesis A, suggesting that future hypotheses of egg shell evolution may need to account
for additional factors such as chorion composition and thickness when considering cost. However,
the allometric relationship between size and shape evolves dynamically across the phylogeny, as
has also been shown for metabolic scaling in mammals34. Hypothesis C could not be rejected for
beetles and their relatives, nor for butterflies, moths, and caddisflies (respectively Neuropteroidea,
p-value of alternative hypothesis test = 0.04, and Amphiesmenoptera, p-value = 0.01; Fig. 2f, S12,
Table S7). Calculating the scaling relationship on lineage subgroups revealed that additional
clades, including mayflies, crickets, and shield bugs, also show an isometric relationship (Fig. S13). The marked differences in scaling exponents are evidence that egg evolution was not
governed by a universal allometric constant. Instead, evolutionary forces beyond the constraints of physical scaling (e.g. development or ecology) are required to explain egg morphological
diversification. of physical scaling (e.g. development or ecology) are required to explain egg morphological
diversification. Developmental traits and egg evolution The egg is the starting material for embryogenesis, and the size of the hatchling is directly related
to the size of the egg at fertilization35. It was reported that embryogenesis takes longer in species
with larger eggs22, and that this relationship could influence size evolution20, 21. This would be
consistent with the observation that larger adult species have lower metabolic rates than smaller
species36. To test this prediction across our egg dataset, we assembled published embryological
records, and found that indeed, simply comparing egg volume and duration of embryogenesis
yields the previously reported positive relationship22 (Fig. S17). However, a linear regression that
does not account for phylogenetic relationships is inappropriate for this analysis due to the
covariance of traits on an evolutionary tree37. When we accounted for phylogenetic covariance, we
found that there is no significant relationship between egg size and duration of embryogenesis
across insects, such that eggs of very different sizes develop at a similar rate and vice versa (0.02
< p-value < 0.10; Fig. 3b, Table S11). These results suggest that the often-invoked trade-off
between size and development20–22 does not hold across insects. We also tested the hypothesis that the size of the egg has a positive relationship with adult body
size. Previous work reported this relationship in subsets of insects, and moreover suggested that
smaller insects lay proportionally larger eggs for their bodies11, 35, 38. Such a relationship between
egg size and body size would result in an allometric scaling exponent less than one. We combined our dataset of egg size with published adult body length data for insect families39, and found that
this relationship was not generalizable across all insect lineages. For example, in flies and their
relatives (Antliophora) and mayflies and odonates (Palaeoptera), egg size is not predicted by body
size, meaning insects of similar body size lay eggs of different sizes (p-value, Antliophora = 0.02,
Palaeoptera = 0.19; Fig. 3c, 3d, Table S12). In Polyneoptera, thrips and true bugs (Condylognatha),
and bees, ants, and wasps (Hymenoptera), an isometric relationship between egg size and body
size cannot be rejected (p-value of alternative hypothesis test, Polyneoptera = 0.02, Hymenoptera
= 0.01, Condylognatha = 0.01, Fig. S18, Table S13). In general, the predictive power of the
relationship between body size and egg size is low: average egg volume can vary by up to four
orders of magnitude among species with similar body size (Fig. 3c). Developmental traits and egg evolution At the time of fertilization an egg is a single cell. Therefore we tested whether the size of this cell
evolved with the size of the genome, as has been observed for other cell types40, using a database
of genome size for hexapods41. Although the data appeared to show a positive relationship between
egg size and genome size (Table S14), we found that this relationship was driven entirely by the
lineage Polyneoptera (and specifically grasshoppers, Acrididae). This lineage has evolved genome
sizes an order of magnitude larger than other insects and has relatively large eggs (Fig. S19). Across other insect lineages, egg volume and genome size are not significantly related (0 < p-
value < 0.08, Table S14), and egg volume can range over six orders of magnitude for species with
similar genome size (Fig. S19c). This indicates that genome size is not a general driver of egg size. The decoupling of genome size, body size, and developmental rate from egg size evolution
suggests that insect egg diversification has not been universally constrained by development. Oviposition ecology explains egg morphology Egg size and shape have been predicted to evolve in response to changes in life-history and
ecology. Recent work in birds has highlighted one such relationship, suggesting that birds with
increased flight capability have more elliptical and asymmetrical eggs13. We asked whether an
analogous relationship exists between insect flight capability and egg shape. Unlike birds, insects
have undergone hundreds of evolutionary shifts to flightless and even wingless forms42. We
focused on two clades in which flight evolution has been extensively studied. Stick insects
(Phasmatodea) have flightless and wingless species43, 44 (Fig. S22), and many butterflies
(Lepidoptera) show migratory behavior45, a proxy for increased flight capability relative to non-
migratory taxa (Fig. S22). We found that, in contrast to birds, evolutionary changes in flight ability
in these two insect clades were not associated with changes in egg shape (Ornstein-Uhlenbeck
model with multiple optima per regime, OUM; ∆AICc < 2, exact values in Tables S18, S19). Like flight capacity, the microenvironment that insect eggs experience varies widely, including
being exposed to air, submerged or floating in water, or contained within a host animal8 (Fig. 4a). Each microenvironment places different demands on the egg, such as access to oxygen and water
during development15. Preliminary studies in small groups of insects suggested that evolutionary
changes in oviposition ecology and life history may drive the evolution of egg size and shape10, 23. Like flight capacity, the microenvironment that insect eggs experience varies widely, including
being exposed to air, submerged or floating in water, or contained within a host animal8 (Fig. 4a). Each microenvironment places different demands on the egg, such as access to oxygen and water
during development15. Preliminary studies in small groups of insects suggested that evolutionary
changes in oviposition ecology and life history may drive the evolution of egg size and shape10, 23. To test this prediction across all insects, we compiled records on two oviposition ecology modes
that have been extensively studied: oviposition within an animal host (internal parasitic
oviposition) and oviposition in or on water. For each mode we reconstructed ancestral changes
along the insect phylogeny, and found that both aquatic and internal parasitic oviposition have
been gained and lost multiple times independently (Fig. 4a, 4b, S20, S21). Oviposition ecology explains egg morphology This extensive To test this prediction across all insects, we compiled records on two oviposition ecology modes
that have been extensively studied: oviposition within an animal host (internal parasitic
oviposition) and oviposition in or on water. For each mode we reconstructed ancestral changes
along the insect phylogeny, and found that both aquatic and internal parasitic oviposition have
been gained and lost multiple times independently (Fig. 4a, 4b, S20, S21). This extensive convergent evolution allowed us to perform a strong test of whether egg size and shape evolution
are explained by the evolution of oviposition ecology. We found that the evolution of new oviposition environments is linked to changes in egg size and
shape. Models that accounted for shifts to either aquatic or internal parasitic oviposition better
explained size and shape distributions than models that did not (OUM, ∆AICc > 2, exact values
in Tables S15, S16, S17). In this analysis we compared model fit for each ecology-trait pair
separately, and found that these two ecological states were correlated with different egg
morphologies. Specifically, shifts to aquatic oviposition were significantly associated with the
evolution of smaller eggs with a lower aspect ratio (Fig. 4c, 4d, Table S15), while shifts to internal
parasitic oviposition are significantly associated with smaller, more asymmetric eggs (Figs. 4c, 4e,
Table S17). Moreover, we note that the smallest eggs are from parasitoid wasps that develop
polyembryonically (i.e. multiple embryos form from a single egg46; Fig. S23). Neither oviposition
mode is associated with consistent changes in the allometric relationship between size and shape
(Fig. S24). Given that OU models can be favored when dataset size and measurement error are large47, we
repeated these analyses 100 times using simulated ecological states independent of egg
morphological traits. The results of this bootstrap analysis showed that our observed result
favoring ecological models of morphological evolution is unlikely to be caused by dataset size
alone (p-value = 0.01, Table S20). Additionally, these results were robust to uncertainty in
phylogenetic relationships and uncertainty in how taxa were classified for oviposition ecology
(Tables S16). These findings are evidence that the microenvironment experienced by the egg played an important role in morphological evolution. played an important role in morphological evolution. played an important role in morphological evolution. played an important role in morphological evolution. played an important role in morphological evolution. Oviposition ecology explains egg morphology Insect eggs present an ideal case for testing the predictability of macroevolutionary patterns in size
and shape. By comparing insect egg size and shape, we found that previous hypotheses about
evolutionary trade-offs with developmental time, body size, or the presumed cost of egg shells do
not hold. Although we showed that developmental time is not linked to egg size, we suggest that
other features of development (e.g. cell number and distribution) may scale in predictable ways
across eight orders of magnitude in egg size. Finally, we provide evidence that the ecology of
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paper at www.nature.com/nature. Acknowledgements This work was supported by the National Science Foundation (NSF) under
Grant No. IOS-1257217 to CGE, and NSF Graduate Research Training Fellowship No. DGE1745303 to SHC, and by a Jorge Paulo Lemann Fellowship to BdM from Harvard University. We thank the Extavour lab and Brian Farrell for discussion, and Casey Dunn, Dakota McCoy, Dan
Rice, Albert Kao, Elena Kramer, Jack Boyle, Leonora Bittleston, Mansi Srivastava, Milo Johnson,
Peter Wilton, Richard Childers, and Sofia Prado-Irwin for suggestions on initial versions of this
manuscript. We acknowledge the Ernst Mayr Library at the Museum of Comparative Zoology at
Harvard, and specifically Mary Sears, for assistance in gathering the references used in this study. Author contributions SHC and SD contributed equally to dataset generation, experimental
design, data analysis, writing, and figure preparation. SHC wrote all code to perform statistical
analyses. Oviposition ecology explains egg morphology BdM performed phylogenetic analyses. BdM and CGE contributed to experimental
design, interpretation, and writing. Author information Reprints
and
permissions
information
is
available
at
www.nature.com/reprints. The authors declare no competing interests. Correspondence and
requests for materials should be addressed to SHC (church@g.harvard.edu) and CGE
(extavour@oeb.harvard.edu). at Figure Legends Figure Legends 1. The shapes and sizes of hexapod eggs. a, Eggs plotted in a morphospace defined by volume
(mm3) and aspect ratio (unitless), log scale. Points are colored by clades in b, with relationships
according to Misof et al. 201425, one of the backbone phylogenies used in this study. Numbered
points correspond to six eggs in c, selected to show a range of sizes and shapes, arranged by aspect
ratio27, 28, 48–51. d, Size and shape are described using six features, calculated as shown. 1. The shapes and sizes of hexapod eggs. a, Eggs plotted in a morphospace defined by volume
(mm3) and aspect ratio (unitless), log scale. Points are colored by clades in b, with relationships
according to Misof et al. 201425, one of the backbone phylogenies used in this study. Numbered
points correspond to six eggs in c, selected to show a range of sizes and shapes, arranged by aspect
ratio27, 28, 48–51. d, Size and shape are described using six features, calculated as shown. 2.
The allometric relationship of egg shape and size evolves across insects. 2. The allometric relationship of egg shape and size evolves across insects. Hypothesized relationships between size and shape: a, larger eggs are proportionally wider, (solid
line); b, larger eggs are proportionally longer (dotted line); c, shape and size scale isometrically
(dashed line). d, Each hypothesis predicts a different scaling exponent—the slope of the regression
between log-length and log-width. e, Egg length and width plotted in log-log space. Dashed line
represents a hypothetical 1:1 relationship (c). Solid lines are clade-specific PGLS regressions;
points are randomly selected representatives per genus. n = genera: Palaeoptera = 104,
Polyneoptera = 262, Condylognatha = 202, Hymenoptera = 356, Neuropteroidea = 265,
Amphiesmenoptera = 76, Antliophora = 199. f, The distribution of scaling exponents from PGLS
regressions of seven monophyletic clades, calculated over the posterior distribution. White lines,
boxes, bars, and dots represent median, 25-to-75th percentiles, 5-to-95th percentiles, and outliers. Asterisks indicate a significant relationship (p-value < 0.01, exact values in Table S6), and double-
daggers indicate the relationship is not distinguishable from isometry (p-value > 0.01, exact values
in Table S7). n = 100 PGLS regressions. Colors correspond to Fig. 1b. Shifts in oviposition ecology are associated with changes in egg morphology. a, Two modes of oviposition ecology: laying eggs within an animal host (orange; e.g. parasitoid wasps),
and in water (blue; e.g. mosquitoes). Other oviposition substrates (e.g. terrestrial, within plants)
shown in gray. b, Ancestral state reconstruction of oviposition mode reveals both evolved multiple
times (see Fig. S17, S18). c-f, The distribution of egg features, colored by ecology: c, volume
(mm3, log scale); d, aspect ratio (unitless, log scale); e, asymmetry (unitless); and f, angle of
curvature (degrees). Asterisks indicate that the model accounting for ecology fits the data better
(OUM, ∆AICc > 2, exact values in Table S14-S19) than a non-ecological model. 3.
Developmental features do not co-vary with egg size. a, Mature eggs undergo
embryonic development, hatch, and grow into adults. b, Egg volume (mm3) compared to duration 3. Developmental features do not co-vary with egg size. a, Mature eggs undergo
embryonic development, hatch, and grow into adults. b, Egg volume (mm3) compared to duration of embryogenesis, defined as time from egg-laying to hatching (hours), adjusted for incubation
temperature. When phylogeny is accounted for, there is no significant relationship. c, Egg volume
(mm3) compared to adult body volume, calculated as body length cubed (mm3). Dashed line
represents a hypothetical 1:1 relationship (isometry). Solid lines are clade-specific PGLS
phylogenetic regressions; points are family- or order-level average egg size and median adult size. n = family- or order-level averages: Palaeoptera = 15, Polyneoptera = 31, Condylognatha = 36,
Hymenoptera = 44, Neuropteroidea = 36, Amphiesmenoptera = 31, Antliophora = 39. d, The
distribution of scaling exponents from PGLS regressions of seven monophyletic clades. White
lines, boxes, bars, and dots represent median, 25-to-75th percentiles, 5-to-95th percentiles, and
outliers. Asterisks indicate a significant relationship (p-value < 0.01, exact values in Table S12),
and double-daggers indicate the relationship is not distinguishable from isometry (p-value > 0.01,
exact values in Table S13). n = 100 PGLS regressions. Colors correspond to Fig. 1b. Methods Data availability The dataset of insect eggs is publicly available at Dryad https://datadryad. org/review?doi=doi:10.5061/dryad.pv40d2r and described in Church et al. 201924. The phylogenetic posterior distributions are provided as supplemental files ‘phylogeny_posterior
distribution_misof_backbone.nxs’
and The phylogenetic posterior distributions are provided as supplemental files ‘phylogeny_posterior
distribution_misof_backbone.nxs’
and ‘phylogeny_posterior_distribution_rainford_backbone.nxs’. Code availability All code required to reproduce the analyses and figures shown here is available
at https://github.com/shchurch/Insect_Egg_Evolution. Creating the insect egg dataset A list of the 1,756 literature sources used to generate the egg
dataset is provided as a supplemental file, ‘bibliography egg dataset.pdf’. A full description of the
methods used to assemble the insect egg dataset have been published separately24. Egg descriptions
were collected from published accounts of insect eggs using custom software to parse text from
PDFs and measure published images (Fig. 1b), followed by manual verification. Each entry in the
egg dataset includes a reference to an insect genus and, when reported, species name. Scientific
names were validated using TaxReformer24, which relies on online taxonomic databases52–56. Measuring egg features Full trait definitions are described in the Supplementary Information and
summarized briefly below. To resolve ambiguous cases and to measure published images, we used
the definitions below. Egg length: We defined egg length as the distance in millimeters (mm) of the axis of rotational
symmetry. Egg length: We defined egg length as the distance in millimeters (mm) of the axis of rotational
symmetry. Egg width: We defined egg width as the widest diameter (mm), measured perpendicular to the axis
of rotational symmetry of the egg. For eggs described in published records as having both a width and breadth or depth (i.e. the egg is a flattened ellipsoid57), we defined width as the wider of the
two diameters, and breadth as the diameter perpendicular to both the width and length. and breadth or depth (i.e. the egg is a flattened ellipsoid57), we defined width as the wider of the
two diameters, and breadth as the diameter perpendicular to both the width and length. Egg volume: Volume (mm3) was calculated using the equation for the volume of an ellipsoid:
⅙πlw2, following previous workers12, 58. Egg aspect ratio: Aspect ratio was calculated as the ratio of length to width. Egg aspect ratio: Aspect ratio was calculated as the ratio of length to width. Methods Egg asymmetry: Asymmetry was calculated as the ratio between the two egg diameters at the first
and third quartile of the length axis, minus one. The first quartile was always defined as the larger
of the two diameters. Angle of egg curvature: The angle of curvature was measured as the angle (degrees) of the arc
created by the endpoints and midpoint of the length axis. Phylogenetic methods A genus-level phylogeny was built by combining mitochondrial 18S and
28S sequence data from the SILVA database59–62 with phylogenetic constraints from published
higher-level insect phylogenies25, 26. To account for phylogenetic uncertainty in comparative
analyses, trees were estimated using a hierarchical approach63, 64. Separate phylogenies for each
insect order were inferred in a Bayesian framework using MrBayes v. 3.2.665 and 100 post-burn-
in trees were randomly chosen for each order using the order-level backbone trees of Rainford et
al. 201426 and Misof et al. 201425. See Supplementary Information, section “Sequence alignment
and phylogenetic methods” for more details. Annotating the egg dataset with life-history trait data For each of the ecological features of Annotating the egg dataset with life-history trait data For each of the ecological features of
interest (internal parasitic oviposition, aquatic oviposition, flightlessness, and migratory behavior)
taxonomic descriptions from the literature were matched to taxa in the insect egg dataset. For some
taxonomic groups it was not possible to classify all members unambiguously. In these cases, the
ecological state was coded “uncertain”, and the potential impact of this uncertainty on results was
tested. For each trait the ancestral state reconstruction was estimated using an equal-rates model
(R package corHMM67, function rayDISC, node.states=‘marginal’). For a full list of sources and
methods used see the Supplementary Information, section “Evolutionary history of ecological
traits”. Annotating the egg dataset with developmental trait data For developmental traits, a set of Annotating the egg dataset with developmental trait data For developmental traits, a set of
references were assembled from the embryological and ecological literature, and then used to
compile data on interval between syncytial mitoses, time to cellularization, and duration of embryogenesis. Developmental rate observations were rescaled to approximate rates at a
standardized temperature of 20°C following previous work66. For a full list of sources, methods
used in this calculation, and further discussion of developmental trait definitions, see the
Supplementary Information, section “Collecting developmental time data”. Data analysis and evolutionary model comparison Egg length, width, volume, and aspect ratio were log10 transformed. Angle of curvature and
asymmetry were square root transformed. Models of evolution were compared using the R package geiger68. For each trait (egg length, width,
volume, aspect ratio, asymmetry, and angle of curvature), the model fit of a Brownian Motion
(BM), Ornstein-Uhlenbeck (OU), and Early-Burst (EB) model was compared against a null
hypothesis of a white noise (WN) model that assumes no evolutionary correlation. See
Supplementary Information, section ‘Evolutionary model fitting’ for details. The performance of
the best fitting model was further analyzed by comparing expected values of parameters from simulations under the model to observed parameters, using the R package arbutus69. simulations under the model to observed parameters, using the R package arbutus69. The ancestral state of volume, aspect ratio, and angle of curvature were mapped on the summary
phylogeny using the R package phytools70 (version 0.6-44, function contMap). Evolutionary rate
regimes of volume, aspect ratio, and the angle of curvature were fit on the summary phylogeny
using the program BAMM71, 72 (version 2.5.0, R package BAMMtools version 2.1.6,
setBAMMpriors, prior for expected number of shifts set to 10, for 10,000,000 generations). All evolutionary regression analyses were performed using a phylogenetic generalized least
squares (PGLS) approach in the R packages ape73 (version 5.0, correlation structure =
corBrownian) and nlme74 (version 3.1-131.1). Given that the EB models best fit the data, we also
tested a corBlomberg correlation structure, which invokes an accelerating-decelerating model of
evolution, with the decelerating rate of trait change fixed at 1.3. For comparisons performed at the genus level, each regression was repeated over 100 trees
randomly drawn from the posterior distribution randomly selecting a representative egg dataset
entry per genus. For comparisons performed at the family level, each regression was repeated 100
times calculating the family level average egg data from 50% of entries per family. For phylogenetic regressions controlling for a third variable, we calculated the phylogenetic
residuals of each variable against the dependent variable, and then calculated the phylogenetic
regression of the residuals75. To test alternative hypotheses, new data were simulated using a fixed
scaling exponent and the parameters of the best fitting model with the R package phylolm76
(version 2.5, function ‘rTrait’). Allometric regressions were performed over all insect taxa as well as for seven monophyletic groups of insects individually (Palaeoptera, Polyneoptera, Condylognatha, Hymenoptera,
Neuropteroidea, Amphiesmenoptera, Antliophora). 52. Patterson, D., Mozzherin, D., Shorthouse, D. P. & Thessen, A. Challenges with using Data analysis and evolutionary model comparison In addition, the scaling exponent between egg
length and width was calculated for each monophyletic group of taxa with more than 20 tips but
fewer than 50. Following ancestral state reconstruction of ecological regimes, for each ecology-trait pair (internal
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1Department of Agribusiness Faculty of Agriculture Universitas Halu Oleo Kendari 93232 Indonesia *Corresponding author: fofosesario123@gmail.com *Corresponding author: fofosesario123@gmail.com To cite this article:
Sesario, F., Abdi, A., & Yusria, W. O. (2023). Financial Feasibility Analysis (Case Study of Tunas Maru Catfish
Enlargement Business in Poasia District Kendari City). International Journal of Agricultural Social Economics and
Rural Development (Ijaserd), 3(2), 59–64. https://doi.org/10.37149/ijaserd.v3i2.1034 To cite this article:
Sesario, F., Abdi, A., & Yusria, W. O. (2023). Financial Feasibility Analysis (Case Study of Tunas Maru Catfish
Enlargement Business in Poasia District Kendari City). International Journal of Agricultural Social Economics and
Rural Development (Ijaserd), 3(2), 59–64. https://doi.org/10.37149/ijaserd.v3i2.1034 Received: July 26, 2023; Accepted: September 26, 2023; Published: October 01, 2023 Received: July 26, 2023; Accepted: September 26, 2023; Published: October 01, 2023 ABSTRACT This research aims to determine the financial feasibility and sensitivity of the catfish
enlargement business in the Poasia District of Kendari City from a financial perspective. This research
was conducted in the Poasia District of Kendari City from August 2021 to April 2022. The object of
this study is the Tunas Maru catfish enlargement business. The study uses case study methods with
quantitative data analysis. This research aims to determine the feasibility of catfish enlargement
businesses in terms of financial aspects. Financial aspects are analyzed using the present net
method value. Net B/C Ratio, Internal Rate Of Return, Payback Period, and Sensitivity Analysis. The
data used in this study are primary and secondary. This study's results obtained the value of NPV
=52.364.673, Net B / C = 39,3, IRR = 52%, and Payback Period = 2 years five months. This business
is worth working on financially. In addition, based on the calculation of sensitivity analysis, the effort of
catfish enlargement remains feasible for a 10% decrease in production. Keywords: catfish; enlargement business; financial. Keywords: catfish; enlargement business; financial. INTRODUCTION Catfish is a type of freshwater fish cultivated and used as food for the people of Indonesia. Consumption of catfish in recent years has increased. In the past, catfish was only consumed by
farming families. Now, its consumers are increasingly widespread and are loved by various circles of
society. The distinctive taste of the meat and various cooking methods make catfish more attractive;
even many large restaurants make it a mainstay of the menu. Previously, people only relied on
catches from nature to get these fish. Now, catfish have been cultivated on a large scale, namely in
earthen ponds, tarpaulin ponds, and permanent ponds.(Estellita & Andriani, 2014) In Indonesia, especially in Kendari City, most people perceive catfish as not sound; catfish is
considered a cheap fish and synonymous with dirty rearing places. However, the demand for catfish
in the city of Kendari is increasing, as evidenced by the increasing number of food stalls selling catfish
parcels. (Ngadiyo et al., 2017) Poasia sub-district is one of the sub-districts in the city of Kendari, which is the destination for
catfish rearing. Based on the results of field interviews, catfish farming in Kendari City is still lacking. A
lack of public understanding about catfish farming causes this. The Tuna's maru catfish rearing
business has been running for 12 years, using 7 ponds as soil media. The production of catfish every
year is erratic. This is caused by pests and diseases that cause many catfish to die. Poasia sub-district is one of the sub-districts in the city of Kendari, which is the destination for
catfish rearing. Based on the results of field interviews, catfish farming in Kendari City is still lacking. A
lack of public understanding about catfish farming causes this. The Tuna's maru catfish rearing
business has been running for 12 years, using 7 ponds as soil media. The production of catfish every
year is erratic. This is caused by pests and diseases that cause many catfish to die. This is an open-access article under a Creative Commons Attribution-ShareAlike 4.0 International License. International Journal of Agricultural Social
Economics and Rural Development (Ijaserd)
2023:3(2):59-64
https://ejournal.agribisnis.uho.ac.id/index.php/ijaserd
doi: http://doi.org/10.37149/ijaserd.v3i2.1034 International Journal of Agricultural Social
Economics and Rural Development (Ijaserd)
2023:3(2):59-64
https://ejournal.agribisnis.uho.ac.id/index.php/ijaserd
doi: http://doi.org/10.37149/ijaserd.v3i2.1034 INTRODUCTION Furthermore, research conducted
by Rahayu & Farid (2018) focuses on the analysis of the catfish farming business, where the analysis
results show that catfish rearing is feasible with an R/C value > 1. Research conducted by Wajdi et al. (2018) only focuses on a feasibility study of catfish farming businesses. The result of the analysis
shows that the business is feasible but does not calculate the sensitivity analysis, so it does not know
if there is an increase in what percentage of the business is still feasible or not, while this study does
not analyze the feasibility of the business but also performs sensitivity analysis of fish rearing. catfish
so that they know the tolerance limit for the increase in what percentage of the business is still
feasible to run. Research conducted by (Wardana et al., 2014) considerations can be obtained through a study of various aspects regarding the feasibility of a
business to run so that the results of the study can be used. To decide whether a project or business
should be implemented. Feasible or delayed or even canceled. Therefore, it is essential to perform a
sensitivity analysis. Research that focuses on the cultivation of catfish rearing was conducted by
Jatnika et al. (2014), who researched the development of catfish farming on dry land. The analysis
results showed that the feasibility of a business in a pond area of 12-16 m2, a pond area of 20-25 m2,
and a pond area of 30-45 m2 is feasible and can provide benefits. Furthermore, research conducted
by Rahayu & Farid (2018) focuses on the analysis of the catfish farming business, where the analysis
results show that catfish rearing is feasible with an R/C value > 1. Research conducted by Wajdi et al. (2018) only focuses on a feasibility study of catfish farming businesses. The result of the analysis
shows that the business is feasible but does not calculate the sensitivity analysis, so it does not know
if there is an increase in what percentage of the business is still feasible or not, while this study does
not analyze the feasibility of the business but also performs sensitivity analysis of fish rearing. catfish
so that they know the tolerance limit for the increase in what percentage of the business is still
feasible to run. INTRODUCTION Research conducted by (Wardana et al., 2014) with an analysis of financial feasibility and income contribution to a household income of
catfish farming, further research conducted by Sudana et al. (2013) analyzed the feasibility of African
catfish farming and its effect on the income level of fish farmers, the result of this business is that the
catfish is feasible to be cultivated in terms of financial, marketing and social aspects, these three
aspects have a significant effect on farmers' income. Furthermore, Astari et al. (2021) researched
business feasibility analysis and development strategies for catfish farming from financial and non-
financial aspects to determine the feasibility of the business from all aspects and not focus on
financial aspects from a financial and non-financial perspective. Furthermore, research conducted by
Mahyuddin et al. (2014), which analyzed the feasibility and sensitivity of input prices in catfish
farming, showed that the business was feasible to develop. If there was an increase of 20%, then the
business was still feasible to operate, as for the problem, namely the price. Expensive fish feed, low
selling price, cannibalistic nature of catfish and tarpaulin replacement costs. The phenomena in the field show that the opportunities and potential for enlargement of
Tunas Maru catfish are in the midst of ignorance of the financial aspects in the form of profits and
threats they face and the production of their products. So, the researchers aimed to study the financial
feasibility and sensitivity of the business of raising catfish tunas maru in the Poasia sub-district,
Kendari city. MATERIALS AND METHODS This research was conducted in the Poasia District of Kendari City. The location is determined
purposively, considering that Tunas Maru is a catfish enlargement business that meets the criteria for
researching its feasibility in terms of long-term effort. This research was carried out for 8 months, from
August 2021 to April 2022. The data analysis in this study is quantitative. Namely, the analysis used
to analyze financial feasibility agricultural criteria and sensitivity analyses were also performed. To
review the research objectives, NPV, Net B/C, IRR, Payback Period, and Sensitivity Analysis
according to (Pandangaran, 2008) INTRODUCTION Given the future conditions that are full of uncertainty, specific considerations are needed in starting a
business or maintaining it, as well as the business of raising catfish tunas maru where the
opportunities and potential for raising catfish amid ignorance of the financial aspects in the form of
profits and threats they face as well as the production of its products in the future, it is necessary to
determine the feasibility of the business from a financial point of view and perform a sensitivity
analysis. This is in line with the opinion of Sulastri (2016), who says that given the uncertain future
conditions, specific considerations are needed in starting a business, where the basis for these year is erratic. This is caused by pests and diseases that cause many catfish to die. Given the future conditions that are full of uncertainty, specific considerations are needed in starting a
business or maintaining it, as well as the business of raising catfish tunas maru where the
opportunities and potential for raising catfish amid ignorance of the financial aspects in the form of
profits and threats they face as well as the production of its products in the future, it is necessary to
determine the feasibility of the business from a financial point of view and perform a sensitivity
analysis. This is in line with the opinion of Sulastri (2016), who says that given the uncertain future
conditions, specific considerations are needed in starting a business, where the basis for these International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 considerations can be obtained through a study of various aspects regarding the feasibility of a
business to run so that the results of the study can be used. To decide whether a project or business
should be implemented. Feasible or delayed or even canceled. Therefore, it is essential to perform a
sensitivity analysis. Research that focuses on the cultivation of catfish rearing was conducted by
Jatnika et al. (2014), who researched the development of catfish farming on dry land. The analysis
results showed that the feasibility of a business in a pond area of 12-16 m2, a pond area of 20-25 m2,
and a pond area of 30-45 m2 is feasible and can provide benefits. Cost Analysis Overall costs are grouped into investment costs and operating costs. Investment costs are
transaction costs that must be paid by Mr. Amrin when starting a Tunas Maru catfish-rearing
business. In contrast, operational costs are incurred from the first to the eleventh year of running a
catfish-rearing business. Table 1. Total cost of catfish rearing business in 2010-2021
No
Cost Component
Cost (IDR)
1
Investment Fee
39.740.000
2
Operating Costs
61.359.000
Total Cost
101.099.000
Source: Primary Data Processed, 2022 Investment costs incurred at the beginning of the implementation of the tunas maru catfish
rearing business include land, buildings, ponds, nets, and various equipment used in the amount of
IDR 39,740,000. Financially, all investment costs are charged at the beginning of the business year. The tools invested in this business have different economic lives, which, after passing the economic
life of the item, will experience damage or sub-optimal performance, so investment tools that can no
longer be used must be replaced with new ones and will require reinvestment costs, which has an
economic life. Based on Table 1, the total investment costs incurred for raising catfish are
IDR39,740,000. This can be a guide for someone who wants to cultivate catfish. The total operating
costs incurred amounted to IDR61,359,000, consisting of fixed and variable costs. Variable costs are
costs incurred by the business of raising catfish shoots maru, whose amount varies depending on the
amount of production. The total cost incurred was IDR101,099,000. Table 2. Total income of catfish rearing business in 2021
No
Description
Total (IDR)
1
Reception
32.500.000
2
Total cost
4.993.272
Total Income
27.506.727
Source: Primary Data Processed, 2022 In Table 2, it can be seen that the revenue obtained from Tunas Maru catfish is IDR. 32,500,000/year is obtained from the number of catfish production times and the selling price of
catfish. The revenue obtained from the beginning of the business until now has always fluctuated,
which is influenced by the quantity of the product and the selling price. This is in line with the research
of (Kusumastuti et al., 2016), which says that the revenue from the catfish processing agroindustry
comes from products sold at a selling price, the revenue obtained is influenced by the quantity of the
product and the selling price. The amount of income received is IDR. 27,506,727/year is obtained
from revenue after deducting the total cost. International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 closely related to mindset and can affect technological knowledge. In 2010, Mr. Amrin established a
catfish-rearing business with an initial capital of around IDR39,740,000, which was obtained from a
bank loan and used as investment capital for land and equipment purchases. closely related to mindset and can affect technological knowledge. In 2010, Mr. Amrin established a
catfish-rearing business with an initial capital of around IDR39,740,000, which was obtained from a
bank loan and used as investment capital for land and equipment purchases. Cost Analysis This is in line with the research of (Ramdhani et al., 2021),
which states that income is the total revenue (TR) per harvest minus the total cost (TC) per harvest. Income is also called profit or net income; the smaller the total costs incurred and the greater the
amount of production obtained, the greater the income obtained. This is also in line with Sumardani et
al. (2017), which says that the revenue of catfish farmers is obtained from the amount of catfish
production, the selling price of catfish in the market, and the income of catfish farmers, the total
revenue of catfish is reduced by the total cost of catfish production incurred. Business Overview/ History y
The maru catfish farming business was founded in 2010 and is managed by Mr. Amrin. This
business already has a business license. Based on the research results, Mr. Amrin is 50 years old. This shows that the respondents are in the productive age range, in line with the opinion of Suratiyah
(2015), which states that the productive age range is between 15 and 54 years old, and the rest are in
the non-productive age category. Cost is a sacrifice made by producers in managing their business to
get maximum results. The catfish rearing business is located in the Poasia sub-district, Kendari City. This business was initially established to open a business, and also because the location is suitable
for fish farming, Mr. Amrin made a catfish enlargement business. According to (Purwanto & Taftazani,
2018) which say that the more significant number of dependents a family has will usually affect the
level of family expenditure, and the allocation of funds will decrease if it is not followed by sufficient
income; therefore, the number of dependents is a reason for someone to work. Based on the results
of interviews with respondents, it is known that the respondent's last education was junior high school,
which shows that the respondent has sufficient education. (Nurkholis, 2013) argues that education is
necessary to achieve balance and perfection in individual development. The level of education is Sesario et al eISSN: 2774-9126 eISSN: 2774-9126 60 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 Financial Feasibility Analysis Analysis of the feasibility of catfish rearing business in Poasia District to determine the
financial feasibility analysis used the analysis of Net Present Value (NPV), Internal Rate of Return
(IRR), Net Benefit Cost Ratio (NBCR), and Payback Period (PP). (
)
(
)
y
(
)
Results Based on the NPV calculations presented in Table 3, the results of the NPV
calculations on Tunas Maru catfish are IDR. 52,364,673 NPV obtained, or greater than 0, indicates
that the tuna fish business is feasible. The results of the NPV found by Wardana et al. (2014) are
greater, namely IDR. 130,113,461. This aligns with research conducted by Mahyuddin et al. (2014), Sesario et al eISSN: 2774-9126 61 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 who found that if the NVP is greater than 0, this business is financially feasible because it can return
or exceed the capital used. nd that if the NVP is greater than 0, this business is financially feasible because it can return
ed the capital used. Table 3. Feasibility of Catfish Raising Business in Poasia District
No
Business Feasibility Analysis
Results
Description
1
Net Present Value (NPV)
IDR52.364.673
Feasible
2
Internal Rate of Return (IRR)
52%
Feasible
3
Net Benefit Cost Ratio
39,3
Feasible
4
Payback Period
2,5 years
Feasible
Source: Primary Data Processed, 2022 Table 3. Feasibility of Catfish Raising Business in Poasia District
N
B
i
F
ibilit A
l
i
R
lt
D Furthermore, Kusmiati & Wati (2020)say farming is feasible if the NVP is more favorable
because the NPV is greater than 0. It can be interpreted that the value to be obtained and the benefits
obtained are more significant than the costs incurred. Based on Table 3, the IRR value obtained is
52%. Because the IRR value obtained exceeds the current interest rate of 14%, catfish can be
cultivated. Research conducted by Mahyuddin et al. (2014) shows that the IRR value obtained is
23.24% because the interest rate obtained is greater than the current interest rate. This figure shows
that agricultural operations are still profitable. Based on Net B/C, a value of 39.3 was obtained. This
indicates that the Tunas Maru catfish game is feasible because it meets the requirements for a value
greater than 1, which means a business can be feasible if the Net B/C value is greater than 1. Financial Feasibility Analysis Net B
value /C of 39.3 means cultivating catfish tunas maru will provide 39.3 times the total cost incurred. This is in line with the research of Wardana et al. (2014), Gusnawati et al. (2014), and Abidin et al. (2019), where the Net B/C value is greater than 1, which is 2.29, which can be said to be feasible,
meaning that the Net B/C value of 2.29 will provide a net benefit of 2.29 times that of total costs
incurred. The payback period is a specific period that shows the flow of income with the investment
amount in the form of present value in maru tuna catfish. The value of the payback period in Table 3
is 2.5, showing how long the investment capital will return, expressed in years. The value of 2.5 in
several periods indicates that a tuna catfish is suitable for cultivation considering that the investment
capital can be returned within two years and five months. In contrast, research shows that a payback period value of 2 months means the investment
will return in 2 months. This value is relatively small because the investment used is relatively low so
that capital can be obtained faster. Considerations based on four appropriate considerations: NPV of
IDR 52,364,673; IDR 52%; Net B/C 39.3; PP 2.5 years.The result of the analysis is that tuna catfish is
suitable for cultivation. International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 constant, the catfish farming business in tarpaulin ponds can still survive. Hence, it is feasible to be
developed further in the future, which will come. constant, the catfish farming business in tarpaulin ponds can still survive. Hence, it is feasible to be
developed further in the future, which will come. CONCLUSIONS Based on the study's results, raising catfish shoots maru is feasible to cultivate when viewed
from the financial aspect based on the assessment of the four investment feasibility criteria. The
results of the sensitivity analysis with changes in the form of a 10% increase in costs and a 10%
decrease in production on the four investment eligibility criteria, namely the results of calculations on
NPV of IDR 47,128,205, IRR value of 51%, Net B/C 38.5 and a payback period of 2.5 years. Therefore, the enlargement of the catfish shoots maru is still feasible to be cultivated if there is a
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Kesejahteraan Ekonomi Keluarga Pekerjaan K3L Universitas Padjadjaran. Pekerjaan Sosial,
1(2), 33-43. https://doi.org/10.24198/focus.v1i2.18255 Rahayu, A. P., & Farid, M. (2018). Analisa Usaha Budidaya Ikan Lele Masamo ( Clarias Gariepinus )
Kecamatan
Kembangbahu
Kabupaten
Lamongan. Grouper,
9(2),
45-55. https://doi.org/10.30736/grouper.v9i1.27 Ramdhani, I., Darwis, & Ariefi, H. (2021). Analisis Usaha Budidaya Ikan Lele (Clarias Sp) Pada
Kelompok Budidaya di Kampung buana bakti Kecamatan Kerinci Kanan Kabupaten Siak. Sosial Ekonomi Pesisir, 2(4), 17-25. https://doi.org/10.24843/BUM.2018.v17.i03.p12 p
g
p
Sudana, S., Arga, I., & Suparta, N. (2013). Kelayakan Usaha Budidaya Ikan Lele Dumbo (Clarias
Gariepinus) dan Pengaruhnya Terhadap Tingkat Pendapatan Petani Ikan Lele di Kabupaten
Tabanan. Manajemen Agribisnis, 1 (1), 27-32. j
g
( )
Sulastri, L. (2016). Studi Kelayakan Bisnis Untuk Wirausaha. La Goods Publishing. Sulastri, L. (2016). Studi Kelayakan Bisnis Untuk Wirausaha. La Goods Publishing. Sumardani, uranjaya, Soniari, & Radiawan. (2017). Sensitivity Analysis Table 4. Sensitivity Analysis of Catfish Cultivation Business in Poasia District with a 10% Decrease
No
Investment appraisal criteria
Results
Description
1
Net Present Value (NPV)
IDR47.128.205
Feasible
2
Internal rate of return (IRR)
51%
Feasible
3
Benefit-cost Ratio
38,5
Feasible
4
Payback period
2,5 year
Feasible
Source: Primary Data Processed, 2022 Table 4. Sensitivity Analysis of Catfish Cultivation Business in Poasia District with a 10% D
No
Investment appraisal criteria
Results
Desc The sensitivity analysis results on the financial feasibility analysis in Table 4 show that if there
is a 10% increase in costs and a 10% decrease in production, the NPV obtained is IDR. Maru is still
worth cultivating. At the IRR, if there is a 10% decrease in cost increase and a 10% decrease in
production, the IRR value obtained is 51% because the IRR value is greater than the current interest
rate of 14%, then catfish rearing is still feasible. At the Net B/C value, if there is a 10% increase in
costs and a 10% decrease in production, the result is 38.5 because the Net B/C value is greater than
1, then the enlargement of catfish tunas maru is still feasible. Suppose there is an increase in costs of
10% and a decrease in production of 10% in the payback period. The results were obtained for 2.5
years, where there was no change in the payback period. In that case, this indicates that the
enlargement of the tunas maru catfish is feasible to be cultivated. The sensitivity analysis results
show a decrease in the value of the business feasibility criteria, but it still meets all the investment
feasibility criteria. There is a 10% increase in costs and a 10% decrease in production in the future. While other factors are considered constant, the business of raising catfish tunas maru in an earthen
pond is worth developing in the future. This aligns with research conducted by Mahyuddin et al. (2014). If there is an increase in feed prices by 20% in the future while other factors are considered Sesario et al eISSN: 2774-9126 eISSN: 2774-9126 62 Wardana, M. F. P., Suwandari, A., & Hartadi, R. (2014). Analisis Kelayakan Finansial Dan Kontribusi
Pendapatan Terhadap Pendapatan Rumah Tangga Pembudidaya Ikan Lele Dumbo. Agritrop
Jurnal Ilmu-Ilmu Pertanian, 7(9), 22-37. REFERENCES Aplikasi Teknologi Budidaya Ikan Lele Kombinas Sumardani, uranjaya, Soniari, & Radiawan. (2017). Aplikasi Teknologi Budidaya Ikan Lele Kombinasi
Sistem Sirkulasi Air Tertutup Dan Teknologi Bioflok Di Desa Ketewel Kecamatan Sukawati
Kabupaten Gianyar. Buletin Udayana Mengabdi, 16(1), 166-170. p
y
y
g
Suratiyah, K. (2015). Ilmu Usahatani Penebar Swadaya. Wajdi, M. F., Qomariyati, N., K, P. W., & Laily, D. W. (2018). Studi Kelayakan Usaha Budidaya Ikan
Lele di Desa Karanggeneng Kecamatan Karanggeng Kabupaten Lamongan. Litbang Pemas,
7(8). https://doi.org/10.30736/grouper.v9i1.27 Sesario et al Sesario et al 63 eISSN: 2774-9126 International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64
Wardana, M. F. P., Suwandari, A., & Hartadi, R. (2014). Analisis Kelayakan Finansial Dan Kontribusi
Pendapatan Terhadap Pendapatan Rumah Tangga Pembudidaya Ikan Lele Dumbo. Agritrop
Jurnal Ilmu-Ilmu Pertanian, 7(9), 22-37. International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64
Wardana, M. F. P., Suwandari, A., & Hartadi, R. (2014). Analisis Kelayakan Finansial Dan Kontribusi
Pendapatan Terhadap Pendapatan Rumah Tangga Pembudidaya Ikan Lele Dumbo. Agritrop
Jurnal Ilmu-Ilmu Pertanian, 7(9), 22-37. International Journal of Agricultural Social Economics and Rural Development (Ijaserd)
2023: 3(2):59-64 Wardana, M. F. P., Suwandari, A., & Hartadi, R. (2014). Analisis Kelayakan Finansial Dan Kontribusi
Pendapatan Terhadap Pendapatan Rumah Tangga Pembudidaya Ikan Lele Dumbo. Agritrop
Jurnal Ilmu-Ilmu Pertanian, 7(9), 22-37. Wardana, M. F. P., Suwandari, A., & Hartadi, R. (2014). Analisis Kelayakan Finansial Dan Kontribusi
Pendapatan Terhadap Pendapatan Rumah Tangga Pembudidaya Ikan Lele Dumbo. Agritrop
Jurnal Ilmu-Ilmu Pertanian, 7(9), 22-37. 64 eISSN: 2774-9126 Sesario et al Sesario et al
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https://hal.archives-ouvertes.fr/hal-02306813/file/Scientific_Reports_9_2428.pdf
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English
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Tailing miniSOG: Structural Bases of the Complex Photophysics of a Flavin-Binding Singlet Oxygen Photosensitizing Protein
| null | 2,018
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cc-by
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To cite this version: Joaquim Torra, Céline Lafaye, Luca Signor, Sylvain Aumonier, Cristina Flors, et al.. Tailing miniSOG:
structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing pro-
tein. Scientific Reports, 2019, 9 (1), pp.2428-1-2428-10. 10.1038/s41598-019-38955-3. hal-02306813 Distributed under a Creative Commons Attribution 4.0 International License Tailing miniSOG: structural bases
of the complex photophysics of
a flavin-binding singlet oxygen
photosensitizing protein Received: 2 August 2018
Accepted: 11 January 2019
Published: xx xx xxxx Received: 2 August 2018
Accepted: 11 January 2019
Published: xx xx xxxx miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving
as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its
1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization
using an integrated approach combining spectroscopic and structural methods. Our results provide
a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between
oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway
has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the
photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the
accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of
magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich
residues that are progressively photo-oxidized, further contributing to facilitate the production of
1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent
performance in correlative light and electron microscopy experiments. miniSOG (for mini Singlet Oxygen Generator)1 is a 106 amino acid flavin-binding protein that generates 1O2
under exposure to blue light. It was originally developed by Shu and coworkers for correlative light and electron
microscopy (CLEM) as it both fluoresces and catalyzes the photo-oxidation of diaminobenzidine (DAB), provid-
ing high-resolution images1. Novel applications are being actively developed since2–5. miniSOG was engineered
from the LOV2 (Light, Oxygen and Voltage) domain of Arabidopsis thaliana phototropin 21. Proteins based on
LOV domains are blue-light photoreceptors that form a light-induced and reversible flavin-cysteine covalent
adduct that consumes the energy of the excited state6. Replacement of the cysteine residue by an alanine or glycine
avoids the formation of the covalent bond and leads to a fluorescent protein7,8. miniSOG contains six mutations
as compared to its precursor, two of them involving residues surrounding the chromophore. Its cofactor FMN is
ubiquitously found in nature9,10 and generates 1O2 with high quantum yield (Φ∆)11, but also other reactive oxygen
species (ROS)12. HAL Id: hal-02306813
https://hal.science/hal-02306813v1
Submitted on 23 Oct 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.nature.com/scientificreports www.nature.com/scientificreports Received: 2 August 2018
Accepted: 11 January 2019
Published: xx xx xxxx Tailing miniSOG: structural bases
of the complex photophysics of
a flavin-binding singlet oxygen
photosensitizing protein Close inspection of the photophysical and photosensitizing properties of miniSOG reveals a number of strik-
ing observations: (1) its Φ∆ is much lower than that of FMN (0.03 vs. 0.51)11,13,14; (2) the lifetime (τT) of triplet
miniSOG (3miniSOG*) is much shorter than that of FMN in nitrogen-saturated solutions (33.6 µs15 vs 200 µs16);
(3) oxygen quenching is less efficient than for FMN (τT
air = 31.3 µs15 vs 3.1 µs in air-saturated solutions); (4) in 1Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, Barcelona, 08017, Spain. 2Univ. Grenoble Alpes,
CNRS, CEA, IBS (Institut de Biologie Structurale), F-38000, Grenoble, France. 3European Synchrotron Radiation
Facility, F-38043, Grenoble, France. 4Madrid Institute for Advanced Studies in Nanoscience (IMDEA Nanoscience),
Ciudad Universitaria de Cantoblanco, C/Faraday 9, 28049, Madrid, Spain. 5Nanobiotechnology Unit Associated to
the National Center for Biotechnology (CNB-CSIC-IMDEA), Ciudad Universitaria de Cantoblanco, 28049, Madrid,
Spain. 6Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, California,
94158-9001, United States. 7Cardiovascular Research Institute, University of California-San Francisco, San Francisco,
California, 94158-9001, United States. Correspondence and requests for materials should be addressed to S.N. (email: santi.nonell@iqs.url.edu) or G.G. (email: guillaume.gotthard@esrf.fr) or A.R. (email: antoine.royant@ibs.fr) Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 1 www.nature.com/scientificreports/ Figure 1. Model of oxygen photosensitization by miniSOG. ΦF: fluorescence quantum yield, ΦT: triplet state
quantum yield, kP: protein quenching rate constant, kPhot: photoproduct formation rate constant, kO2: oxygen
quenching rate constant, ΦΔ: singlet oxygen quantum yield. I, II, III, and IV indicate the three deactivation and
one oxidation pathways discussed in the main text. Figure 1. Model of oxygen photosensitization by miniSOG. ΦF: fluorescence quantum yield, ΦT: triplet state
quantum yield, kP: protein quenching rate constant, kPhot: photoproduct formation rate constant, kO2: oxygen
quenching rate constant, ΦΔ: singlet oxygen quantum yield. I, II, III, and IV indicate the three deactivation and
one oxidation pathways discussed in the main text. addition to 1O2 it also produces superoxide (O2
•−)12,14; (5) it undergoes a remarkable transformation upon expo-
sure to light, whereby Φ∆ increases 10-fold (to ~0.3) and τT
air shortens by 10-fold (to ~3 µs)13,14. The absence of a
structure of miniSOG so far had prevented to rationalize these observations, which we have attempted here using
a combined structural and photophysical approach. Tailing miniSOG: structural bases
of the complex photophysics of
a flavin-binding singlet oxygen
photosensitizing protein p
p y
pp
Based on the extensive data present in the literature and the photophysical and structural results presented
herein, a mechanism of excited-state deactivation of miniSOG can be proposed that involves three main path-
ways (Fig. 1). The shorter lifetime of 3miniSOG* compared to 3FMN* indicates that protein quenching is a major
mechanism of triplet decay (pathway I). Its rate constant kP is largely determined by electron transfer with nearby
electron-rich residues17. Quenching of the singlet state can be safely ruled out since no shortening of the fluores-
cence lifetime or decrease in the fluorescence quantum yield are observed relative to free FMN. In the presence
of oxygen, a second decay pathway (pathway II) is possible, namely oxygen quenching to produce 1O2 (energy
transfer) or O2
•− (electron transfer), as observed for FMN in solution12. It is also possible to produce O2
•− by reac-
tion of oxygen with a radical anion formed during protein quenching in pathway I. Finally, miniSOG undergoes a
photoinduced transformation (pathway III, rate constant kPhot), for which we provide here a detailed description
for the first time. Results and Discussion High resolution crystal structure of miniSOG. We have solved the structure of miniSOG at 1.17 Å res-
olution (Fig. 2a and Supporting Information), which shows an increase in rigidity of the environment of the
chromophore compared to that in the LOV2 domain, the location of potential quenchers of the excited states
of FMN, and the phosphoribityl tail of FMN lying in a tunnel bridging the bulk solvent and the chromophore
encased in the core of the protein (Fig. 2b). The latter hinders oxygen access to the isoalloxazine ring. The pres-
ence halfway through the tunnel of a chloride ion, which can be a good mimic of molecular oxygen18,19, suggests
that oxygen diffusion can occur. Deactivation mechanism of miniSOG triplet excited state (Pathways I and II). The values of the
relevant rate constants for pathways I and II can be inferred from the 3miniSOG* lifetime measurements. Comparison of the decay rate constant (1/τT) of miniSOG and SOPP3, the miniSOG mutant with the longest τT
reported so far (3.3 ms in nitrogen-saturated solutions)17 allows us to estimate the rate constant for protein
quenching (kP = kT
N2 − kT
N2
,SOPP3, Table 1). SOPP3 is a miniSOG variant, which encases the same chromophore
FMN and, most importantly, lacks most of the electron-rich residues present in the vicinity of the flavin in mini-
SOG. Hence, protein quenching of the triplet chromophore in SOPP3 is essentially suppressed, which makes
SOPP3 a convenient model for the study of the contribution of protein quenching in miniSOG. Likewise, the
pseudo-first order rate constant for oxygen quenching (kO2 = kT
Air − kT
N
2) can be estimated from τT data in air-
and nitrogen-saturated solutions (Table 1). Comparison of kp and kO2 in Table 1 reveals that protein quenching (pathway I) is the main triplet deactivation
pathway, removing 93% of the triplets in air-saturated solutions kP/(kP + kO2). Oxygen only quenches 7% of the
triplets, which limits Φ∆ to 0.6 × 0.07 = 0.042 (Eq. 1), in excellent agreement with the experimental value. It can
therefore be concluded that the modest Φ∆ of miniSOG is due to an unfavorable combination of low kO2 and high
kP values, as proposed previously17. Results and Discussion Φ = Φ
×
+
+
≈Φ
×
+
Δ
k
k
k
k
k
k
k
(1)
T
O
p
Phot
O
T
O
p
O
2
2
2
2 (1) Our structural results above suggest that the low value of kO2 is due to the steric hindrance of the ribityl tail within
the tunnel which provides oxygen access to the FMN. Regarding kP, the miniSOG structure shows that six
electron-rich residues are positioned within 8.2 to 10.2 Å from the isoalloxazine ring, namely Tyr30, Tyr73, Trp81,
His85, Met89 and Tyr98, and are thus close enough to the chromophore to act as electron-transfer quenchers of
3miniSOG*20. In addition, four hydrophilic residues, Glu44, Asp72, Asp82 and Glu103, form hydrogen bonds Our structural results above suggest that the low value of kO2 is due to the steric hindrance of the ribityl tail within
the tunnel which provides oxygen access to the FMN. Regarding kP, the miniSOG structure shows that six
electron-rich residues are positioned within 8.2 to 10.2 Å from the isoalloxazine ring, namely Tyr30, Tyr73, Trp81,
His85, Met89 and Tyr98, and are thus close enough to the chromophore to act as electron-transfer quenchers of
3miniSOG*20. In addition, four hydrophilic residues, Glu44, Asp72, Asp82 and Glu103, form hydrogen bonds Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 2 www.nature.com/scientificreports/ Figure 2. High-resolution crystallographic structure of miniSOG. (a) Secondary structure (white) represented
with FMN (green), chloride (yellow), magnesium (green) and water molecules (red). Represented residues:
mutations from LOV2 (orange), residues hydrogen-bonded to the FMN ring (magenta), and potential
quenchers of 3FMN* (cyan). (b) Topology of the FMN-binding site. Figure 2. High-resolution crystallographic structure of miniSOG. (a) Secondary structure (white) represented
with FMN (green), chloride (yellow), magnesium (green) and water molecules (red). Represented residues:
mutations from LOV2 (orange), residues hydrogen-bonded to the FMN ring (magenta), and potential
quenchers of 3FMN* (cyan). (b) Topology of the FMN-binding site. Parameter
in D2O
in H2O
Pathway
References
τS
5.0 ns
4.9–5.5 ns
13,15,43
ΦF
0.43
0.37–0.44
1,14,15,43,44
ΦT
0.6
0.6
15
ΦΔ
0.03–0.04
0.03–0.05
13,14,23,31,44
k T
N2
2.41 × 104 s−1
2.98 × 104 s−1
15
kP
2.38 × 104 s−1
2.95 × 104 s−1b
I
This work
kT
Air
2.59 × 104 s−1
3.19 × 104 s−1
15
kO2
1.8 × 103 s−1
2.3 × 103 s−1
II
This work
kPhot
6.0 s−1
—
III
This work
Table 1. Results and Discussion Photophysical properties of miniSOG in D2O- and H2O-based phosphate buffer. D2O was used
to increase the singlet oxygen lifetime, thus boosting the reactions and processes in which singlet oxygen is
involved and facilitating its detection45. aAssuming the same value of kT
N2
,SOPP3 in H2O and D2O. Parameter
in D2O
in H2O
Pathway
References
τS
5.0 ns
4.9–5.5 ns
13,15,43
ΦF
0.43
0.37–0.44
1,14,15,43,44
ΦT
0.6
0.6
15
ΦΔ
0.03–0.04
0.03–0.05
13,14,23,31,44
k T
N2
2.41 × 104 s−1
2.98 × 104 s−1
15
kP
2.38 × 104 s−1
2.95 × 104 s−1b
I
This work
kT
Air
2.59 × 104 s−1
3.19 × 104 s−1
15
kO2
1.8 × 103 s−1
2.3 × 103 s−1
II
This work
kPhot
6.0 s−1
—
III
This work
Table 1. Photophysical properties of miniSOG in D2O- and H2O-based phosphate buffer. D2O was used
to increase the singlet oxygen lifetime, thus boosting the reactions and processes in which singlet oxygen is
involved and facilitating its detection45. aAssuming the same value of kT
N2
,SOPP3 in H2O and D2O. Table 1. Photophysical properties of miniSOG in D2O- and H2O-based phosphate buffer. D2O was used
to increase the singlet oxygen lifetime, thus boosting the reactions and processes in which singlet oxygen is
involved and facilitating its detection45. aAssuming the same value of kT
N2
,SOPP3 in H2O and D2O. Table 1. Photophysical properties of miniSOG in D2O- and H2O-based phosphate buffer. D2O was used
to increase the singlet oxygen lifetime, thus boosting the reactions and processes in which singlet oxygen is
involved and facilitating its detection45. aAssuming the same value of kT
N2
,SOPP3 in H2O and D2O. with FMN, and may thus enhance protein quenching and O2
•− formation21. Replacing selectively these residues
should lead to a lengthening of the triplet lifetime of miniSOG22 and hence to a higher fraction of triplets being
trapped by oxygen, thus to a higher Φ∆ value. In fact, some of these positions have already been mutated in light
of their capacity of direct electron transfer from the FMN: such miniSOG mutants show considerably longer τT
values (e.g., 196 µs for miniSOG Q103L (SOPP)23, 1.1 ms for miniSOG W81F (Supplementary Fig. S2), and 3.3 ms
for SOPP317 in oxygen-free solutions) and larger Φ∆ values (0.25, 0.33 and 0.6, respectively), in agreement with with FMN, and may thus enhance protein quenching and O2
•− formation21. Results and Discussion Replacing selectively these residues
should lead to a lengthening of the triplet lifetime of miniSOG22 and hence to a higher fraction of triplets being
trapped by oxygen, thus to a higher Φ∆ value. In fact, some of these positions have already been mutated in light
of their capacity of direct electron transfer from the FMN: such miniSOG mutants show considerably longer τT
values (e.g., 196 µs for miniSOG Q103L (SOPP)23, 1.1 ms for miniSOG W81F (Supplementary Fig. S2), and 3.3 ms
for SOPP317 in oxygen-free solutions) and larger Φ∆ values (0.25, 0.33 and 0.6, respectively), in agreement with Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 3 www.nature.com/scientificreports/ Figure 3. Blue-light induced structural changes on the chromophore of miniSOG. (a) 2.0 Å resolution
difference Fourier map calculated between non-irradiated and irradiated parts of a miniSOG crystal contoured
at a −3.0 σ level (magenta) superimposed on the FMN molecule (green). (b) ESI-TOF mass spectra acquired in
the low mass range (m/z < 500) of miniSOG progressively irradiated with blue-light. Figure 3. Blue-light induced structural changes on the chromophore of miniSOG. (a) 2.0 Å resolution
difference Fourier map calculated between non-irradiated and irradiated parts of a miniSOG crystal contoured
at a −3.0 σ level (magenta) superimposed on the FMN molecule (green). (b) ESI-TOF mass spectra acquired in
the low mass range (m/z < 500) of miniSOG progressively irradiated with blue-light. Eq. 1. It is worth noting also that miniSOG produces more O2
•− than free FMN12, which indicates that the radical
anion pathway contributes to the production of O2
•−. Indeed, SOPP shows an 8-fold higher Φ∆ value than mini-
SOG but only a 1.3 higher yield of O2
•− 23. Thus, removal of hydrophilic side chains in the vicinity of the chromo-
phore should strongly reduce the relative formation of O2
•− vs. 1O2. Consequences of blue-light irradiation of miniSOG on its FMN chromophore (Pathway III). In
light of Eq. 1, the observed 10-fold decrease in τT and similar increase in Φ∆ upon extended photolysis suggest
severe changes in both kP and kO2. Blue-light (440 nm) irradiation of a miniSOG crystal at 10 W·cm−2 led to a
five-fold decrease of the fluorescence signal over a 30 min course (Supplementary Fig. S3) and was gentle enough
to keep diffraction around 2.0 Å resolution while affecting a sufficient fraction of molecules so that structural
alterations could be visualized in electron density maps. Results and Discussion A difference Fourier map calculated from non-irradiated
and irradiated parts of a crystal revealed the loss of electron density all along the ribityl tail of the FMN (Fig. 3a),
strongly suggesting its cleavage. Besides, Electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry
performed on irradiated protein samples show (Fig. 3b) the progressive disappearance of the FMN peak at
m/z = 457.1 in favor of a peak at m/z = 243.1. p
To get further insights into the photoconversion, we performed additional photophysical investigations. Besides the already-known shortening of τT and increase in Φ∆, exposure of miniSOG samples to light induces
photobleaching of the FMN chromophore and appearance of new absorption and fluorescence bands (Fig. 4a,b). The leaching out of FMN from miniSOG was routinely checked and could be safely ruled out. The quantum yield
and rate constant of pathway III could be estimated (Table 1, Supplementary Fig. S1). Noteworthy, the Φ∆ value
increases when the photoconverted miniSOG is excited at 355 nm, but remains essentially constant when probed
at 473 nm (Fig. 4c,d). g
Phototransformation of FMN to lumichrome (LC) is consistent with all of the above observations: (1) LC is a
photodegradation product of flavins in aqueous solutions24; (2) the observed mass loss upon irradiation matches
the molar mass difference between FMN (456.3 Da) and LC (242.2 Da); (3) LC absorbs and fluoresces at shorter
wavelengths than FMN, (Fig. 5); (4) LC lacks the phosphoribityl tail of FMN, which facilitates the access of
molecular oxygen to the isoalloxazine ring, resulting in the increase of kO2 and the decrease of τT; (5) LC is a worse
electron acceptor than FMN, hence protein quenching is less favored. The ΔrG° value for quenching of 3ribofla-
vin* by tryptophan is −86.5 kJ·mol−1 (riboflavin is analogous to FMN except for the phosphate group) while is
more positive for 3LC*, −67.2 kJ·mol−1 25; (6) finally, LC is also an excellent 1O2 photosensitizer25–27, hence the
combination of a higher kO2 and a lower kP yield a higher Φ∆ value (Eq. 1) when excited at 355 nm but not at
473 nm, where LC barely absorbs. Consequences of blue-light irradiation of miniSOG on its amino acid residues (Pathway IV). We investigated if our structural data could also support a decrease in kP. Indeed, the 2Fobs − Fcalc electron density
map of blue-light irradiated miniSOG reveals the unambiguous oxidation of three surface residues during irradi-
ation (Fig. 6a, Supplementary Fig. Results and Discussion S4). Tyr73 has been partially converted to a γ-peroxotyrosine. The loss of elec-
tron density on Trp81 is compatible with the formation of N-formylkynurenine (NFK), a well-known tryptophan
oxidation product28,29. Finally, His85 can be modeled by either a singly, or a doubly oxidized histidine, namely
2-oxo-histidine and 2,4-dioxo-histidine. Mass spectrometry analysis of blue-light irradiated miniSOG samples
reveals sequential additions of +16 mass units to the native protein mass of 13882.0 Da, consistent with increasing
oxidation steps of the protein (Fig. 6b). All three structural modifications account for six of the eight additional Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 4 www.nature.com/scientificreports/ Figure 4. Spectroscopic characterization of photoconverted miniSOG. Evolution of miniSOG’s absorbance
(a) and fluorescence (b) upon laser irradiation at 355 nm. Insets show the difference absorption spectra before
and after irradiation, and a zoomed-in image of the new fluorescence bands. (c) Time-resolved NIR 1O2
phosphorescence decays of native (green) and photoconverted miniSOG excited at 473 nm (blue) or 355 nm
(magenta). (d) Observed ΦΔ enhancement at 473 nm (blue) and 355 nm (magenta). Figure 4. Spectroscopic characterization of photoconverted miniSOG. Evolution of miniSOG’s absorbance
(a) and fluorescence (b) upon laser irradiation at 355 nm. Insets show the difference absorption spectra before
and after irradiation, and a zoomed-in image of the new fluorescence bands. (c) Time-resolved NIR 1O2
phosphorescence decays of native (green) and photoconverted miniSOG excited at 473 nm (blue) or 355 nm
(magenta). (d) Observed ΦΔ enhancement at 473 nm (blue) and 355 nm (magenta). oxygen atoms evidenced in the mass spectrometry analysis. The two non-assigned additions could correspond
to oxidation of Tyr30, Met89 or Tyr98, although we did not observe unambiguous oxidation of these residues. Oxidation of Tyr73, His85 and Trp81 eliminates potential quenchers of 3miniSOG*, thereby decreasing the value
of kp. According to Eq. 1, this should contribute to an increase in Φ∆. However, since protein oxidation (pathway
IV) occurs simultaneously to FMN → LC transformation, which also increases Φ∆, it is not possible to ascertain
the individual contribution of both effects. f
Finally, oxidation of tryptophan into NFK could contribute to the increased Φ∆ value observed at 355 nm since
NFK is a potent singlet oxygen photosensitizer (Φ∆ = 0.17)30. However, the W81F mutant shows a doubled Φ∆ (=0.33)
already before photolysis on account of its lower kp value (Eq. Results and Discussion 1), indicating that the potential benefits of producing
NFK as secondary photosensitizer are of minor value as compared to the effect of eliminating a protein quencher. Conclusion We have performed an extensive structural characterization of miniSOG in the dark and its photoproduct formed
in the presence of molecular oxygen, which led us to explain in structural terms the details of its complex photo-
physical behavior. miniSOG is initially moderately efficient towards 1O2 generation because of a combination of
limited oxygen accessibility and 3FMN quenching by electron-rich side chains. Prolonged irradiation to blue light
leads to several structural alterations of miniSOG, which include photodegradation of FMN into LC and oxida-
tion of the quenching side chains. All this results in an increase of Φ∆ when photoconverted miniSOG is excited
at the wavelengths where the formed LC absorbs. Formation of LC liberates the access of molecular oxygen to the
alloxazine ring and reduces protein quenching of the triplet state, while oxidized electron-rich side chains cannot
quench the triplet state of the chromophore. The competition between oxygen quenching and protein quenching
of flavin triplet state seems to be a general feature of flavin-binding proteins31, hence our results will be useful to
guide the evolution of such a protein towards retaining or gaining a specific function. Finally, our results explain
the apparent discrepancy between the poor level of singlet oxygen generation by miniSOG, which had been
consistently measured at low light fluences, and its efficiency in CLEM experiments, in which the singlet oxygen
generation capability of miniSOG is exploited over its whole lifetime. Methods
Ch
i
l Chemical compounds. Riboflavin-5′-monophosphate sodium salt hydrate (FMN) (Chemochroma),
Lumichrome (Santa Cruz Biotechnology), tris(hydroxymethyl)aminomethane (Merck), sodium chloride
(Panreac Applichem), imidazole (Merck), L-arabinose (Sigma Aldrich) and ampicillin (Sigma Aldrich) were
used as received. Phosphate-buffered saline (PBS) or deuterated dPBS solutions were prepared by dissolving the
required amount of a PBS tablet (Sigma Aldrich) in milliQ water or deuterium oxide (Sigma-Aldrich). Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 5 www.nature.com/scientificreports/ www.nature.com/scientificreports
entificreports/
Figure 5. FMN is photoconverted to lumichrome. (a) Photoconversion of FMN (green) to LC (magenta). Normalized absorption and fluorescence spectra of (b) FMN and (c) LC. (d) Topology of blue-light irradiated
miniSOG showing the increased access to the alloxazine ring. Figure 5. FMN is photoconverted to lumichrome. (a) Photoconversion of FMN (green) to LC (magenta). Normalized absorption and fluorescence spectra of (b) FMN and (c) LC. (d) Topology of blue-light irradiated
miniSOG showing the increased access to the alloxazine ring. Expression and purification. Genes coding for a C-terminal 6xHis-tagged recombinant miniSOG and
miniSOG W81F were inserted in a pBad expression vector and over-expressed in Escherichia coli CodonPlus
(DE3) RIL Cells (Stratagene) or in TOP10 cells (Invitrogen). Bacterial cells were grown in LB broth medium
containing 1 mM Ampicillin. At an OD600 of approximately 0.6, expression of recombinant protein was induced
by the addition of L-arabinose and cells were grown for an additional 24 h at 25 °C. Cells were pelleted by cen-
trifugation (4000 g, 4 °C, 30 min), re-suspended in buffer A (20 mM Tris-Hcl pH 8.0, 500 mM NaCl), comple-
mented with complete protease inhibitors-EDTA (Roche) and disrupted using a micro-fluidizer. The soluble
fraction was recovered by centrifugation (40,000 g, 4 °C, 30 min), and loaded on a 1 mL Ni-NTA superflow col-
umn (Qiagen) pre-equilibrated with buffer A. The His-tagged protein was eluted with 150 mM imidazole in buffer
A. Fractions containing purified proteins were pooled and concentrated to a volume of 0.5 mL using Centricon
devices (Amicon 10 kDa cut-off) and loaded onto a size-exclusion chromatography column (Hiload Superdex75
10/300, GE Healthcare) for the final step of the purification procedure. The column was equilibrated with 20 mM
Tris-HCl pH 8.0 and the pooled peak fractions were concentrated to 4 mg·mL−1. Protein expression and purifi-
cation was always performed in the dark or under red light. The purity of the protein solutions was confirmed by
SDS-PAGE. Methods
Ch
i
l The final concentration was determined by UV-vis absorption spectroscopy using a molar absorption
coefficient of 14 mM−1·cm−1 at 448 nm. Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 6 www.nature.com/scientificreports/ Figure 6. Identification of protein residue oxidation in blue-light irradiated miniSOG. (a) 2Fobs − Fcalc electron
density maps contoured at a 1.0, 0.6 and 1.0 σ level superimposed on the refined model of residues Tyr73,
Trp81, and His85, respectively. (b) Deconvoluted ESI-TOF mass spectra of miniSOG progressively irradiated
with blue-light. The peak at 13882.0 Da corresponds to the native protein. Figure 6. Identification of protein residue oxidation in blue-light irradiated miniSOG. (a) 2Fobs − Fcalc electron
density maps contoured at a 1.0, 0.6 and 1.0 σ level superimposed on the refined model of residues Tyr73,
Trp81, and His85, respectively. (b) Deconvoluted ESI-TOF mass spectra of miniSOG progressively irradiated
with blue-light. The peak at 13882.0 Da corresponds to the native protein. Figure 6. Identification of protein residue oxidation in blue-light irradiated miniSOG. (a) 2Fobs − Fcalc electron
density maps contoured at a 1.0, 0.6 and 1.0 σ level superimposed on the refined model of residues Tyr73,
Trp81, and His85, respectively. (b) Deconvoluted ESI-TOF mass spectra of miniSOG progressively irradiated
with blue-light. The peak at 13882.0 Da corresponds to the native protein. Figure 6. Identification of protein residue oxidation in blue-light irradiated miniSOG. (a) 2Fobs − Fcalc electron
density maps contoured at a 1.0, 0.6 and 1.0 σ level superimposed on the refined model of residues Tyr73,
Trp81, and His85, respectively. (b) Deconvoluted ESI-TOF mass spectra of miniSOG progressively irradiated
with blue-light. The peak at 13882.0 Da corresponds to the native protein. Figure 6. Identification of protein residue oxidation in blue-light irradiated miniSOG. (a) 2Fobs − Fcalc electron
density maps contoured at a 1.0, 0.6 and 1.0 σ level superimposed on the refined model of residues Tyr73,
Trp81, and His85, respectively. (b) Deconvoluted ESI-TOF mass spectra of miniSOG progressively irradiated
with blue-light. The peak at 13882.0 Da corresponds to the native protein. Spectroscopic measurements. All spectroscopic measurements were performed using quartz cuvettes
(Hellma) under magnetic stirring and at room temperature. Absorption spectra were recorded on a double beam
Cary 6000i spectrophotometer (Varian). Fluorescence spectra were measured on Fluoromax-4 spectrofluorom-
eter (Horiba). Time-resolved near-infrared (NIR) phosphorescence signals at 1275 nm were measured using a
customized PicoQuant Fluotime 200 lifetime system. Methods
Ch
i
l Briefly, an AO-Z-473 solid state AOM Q-switched laser
(Changchun New Industries Optoelectronics Technology Co., China) was used for excitation at 473 nm, working
at 1.0 kHz repetition rate at 473 nm. The average power that reached the sample was conveniently modulated by
neutral density filters. For excitation at 355 nm, the frequency-tripled output of a diode-pumped pulsed Nd:YAG
laser (FTSS355-Q, Crystal Laser, Berlin, Germany) was used, working at 1 kHz repetition (0.5 mW, or 5 mW,
1 ns pulse width). An uncoated SKG-5 filter (CVI Laser Corporation, Albuquerque, U.S.A.) was placed at the
exit port of the laser to remove any NIR component. The luminescence exiting from the sample was filtered
by a 1100 nm long-pass filter (Edmund Optics, York, U.K.) and a narrow bandpass filter at 1275 nm (bk-1270-
70-B, bk Interfernzoptik, Germany) to remove any scattered laser radiation and isolate the 1O2 emission. A Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 7 www.nature.com/scientificreports/ TE-cooled near-IR sensitive photo multiplier tube assembly (H9170-45, Hamamatsu Photonics Hamamatsu
City, Japan) in combination with a multichannel scaler (NanoHarp 250, PicoQuant Gmbh, Germany) was used
as photon-counting detector. The time-resolved 1O2 emission decays were analyzed by fitting Eq. 2 32 to the data
using GraphPad Prism 5. (
)
S
S
e
e
(2)
t
t
t
( )
(0)
T
T
τ
τ
τ
=
−
−
τ
Δ
Δ
−
−
τ
Δ (2) τT and τ∆ are the lifetimes of the photosensitizer triplet state and of 1O2, respectively, and S(0) is a quantity
proportional to Φ∆. Φ∆ values were determined by comparing the S(0) values of optically-matched solutions of the
corresponding flavoprotein and FMN at 473 nm (Eq. 3)32. τT and τ∆ are the lifetimes of the photosensitizer triplet state and of 1O2, respectively, and S(0) is a quantity
proportional to Φ∆. Φ∆ values were determined by comparing the S(0) values of optically-matched solutions of the
corresponding flavoprotein and FMN at 473 nm (Eq. 3)32. φ
φ
=
Δ
Δ
S
S
(3)
protein
protein
FMN
FMN
,
(0)
(0)
, φ
φ
=
Δ
Δ
S
S
protein
protein
FMN
FMN
,
(0)
(0)
, (3) MN was taken as reference photosensitizer with Φ∆ = 0.51 in PBS11 and 0.57 in dPBS33. FMN was taken as reference photosensitizer with Φ∆ = 0.51 in PBS11 and 0.57 in dPBS33. Methods
Ch
i
l b
d b
d l
fl
h
h
l
h d Transient absorption spectra were monitored by nanosecond laser flash photolysis using a Q-switched
Nd-YAG laser (Surelite I-10, Continuum) operating at the 3rd harmonic. The luminescence exiting from the sam-
ple was filtered by a 610 nm long-pass filter (CVI Laser Corporation, NM, USA). Changes in the sample absorb-
ance were detected at 715 nm using a Hamamatsu R928 photomultiplier to monitor the intensity variations of an
analysis beam produced by a 75 W short arc Xe lamp (USHIO) and spectral discrimination was obtained using a
PTI 101 monochromator. The signal was fed to a Lecroy Wavesurfer 454 oscilloscope for digitizing and averaging
(typically 10 shots) and finally transferred to a PC for data storage and analysis. The system was controlled using
the in-house-developed LKS software (LabView, National Instruments). Determination of kPhot. The rate constant for the photoproduct formation has been determined measuring
the progressive loss of miniSOG in solution as a function of the absorbed light dose at 473 nm using Eq. 4. The
slope of the resulting plot yielded the photobleaching quantum yield ΦPhot, from which the rate constant for pho-
tobleaching was calculated as: τ
= Φ
k
(4)
Phot
Phot
T
Air τ
= Φ
kPhot
Phot
T
Air (4) X-ray crystallography. Crystallization procedures. miniSOG was concentrated to 4 mg·mL−1. The crys-
tallization condition consisted of 100 mM Tris-HCl pH 8.0, 20 mM MgCl2, 28% PEG 4000, 0 or 15 mM CoCl2 at
20 °C. Crystals appeared and grew to final size after 1–2 days. Data collection and processing. X-ray data were collected on beamlines ID23-134 and ID2935 of the ESRF and
were indexed, integrated, merged and scaled using the XDS software package36. Molecular replacement was car-
ried out using the model structure of LOV2 (PDB ID: 4eep) with the program Phaser MR37. Structure refinement
was performed using Refmac538 and manual improvement of the model with Coot39. The native structure of
miniSOG was used as a starting model for model building of bleached-miniSOG. Data collection and refinement
statistics are presented in Supplementary Table S1. Structure analysis and representation were performed with
Pymol40. Preparation of photobleached miniSOG samples. miniSOG crystals. A single miniSOG crystal was
soaked in a cryoprotectant solution containing 20% of glycerol then harvested with a nylon loop. Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 Methods
Ch
i
l The crystal
was exposed to 440 nm laser (10 W·cm−2) on the ID29S-Cryobench setup41 at room temperature using a HC1
humidity control device42. Spectra were recorded at a 1 Hz rate. After 30 min of total exposure, the crystal was
flashcooled in liquid nitrogen. miniSOG solutions. Fresh miniSOG or miniSOG W81F solutions in air-saturated deuterated PBS were illumi-
nated at 355 nm (~5 mW·cm−2) or 473 nm (~15 mW·cm−2) for elapsed irradiation times. Absorption and fluo-
rescence spectra as well as time-resolved 1O2 phosphorescence decays were recorded at different time intervals of
cumulative irradiation. Liquid chromatography-mass spectrometry (LC-MS). Liquid Chromatography Electrospray
Ionization Mass Spectrometry (LC/ESI-MS) was carried out on a 6210 LC/ESI-TOF mass spectrometer interfaced
with a binary HPLC pump system (Agilent Technologies). The mass spectrometer was calibrated in the positive
ion mode with ESI-L (low concentration tuning mix, Agilent Technologies) before each series of measurements,
the calibration providing mass accuracy <1 ppm in the 100–3200 m/z range. All solvents used were HPLC grade:
water and acetonitrile (LC-MS Chromasolv, Sigma-Aldrich); formic acid was from Acros Organics (puriss., p.a.).i Liquid chromatography-mass spectrometry (LC-MS). Liquid Chromatography Electrospray
Ionization Mass Spectrometry (LC/ESI-MS) was carried out on a 6210 LC/ESI-TOF mass spectrometer interfaced
with a binary HPLC pump system (Agilent Technologies). The mass spectrometer was calibrated in the positive
ion mode with ESI-L (low concentration tuning mix, Agilent Technologies) before each series of measurements,
the calibration providing mass accuracy <1 ppm in the 100–3200 m/z range. All solvents used were HPLC grade:
water and acetonitrile (LC-MS Chromasolv, Sigma-Aldrich); formic acid was from Acros Organics (puriss., p.a.). Data acquisition was carried out in the positive ion mode with spectra in the profile mode and mass spectra
were recorded in the 130–2000 m/z range. The mass spectrometer was operated with the following experimental
settings: ESI source temperature was set at 325 °C; nitrogen was used as drying gas (5 L/min) and as nebulizer
gas (30 psi); the capillary needle voltage was set at 3500 V. Fragmentor value was of 250 V and skimmer of 65 V. The instrument was operated in the 2 GHz (extended dynamic range) mode and spectra acquisition rate was of
1 spectrum/s. Data acquisition was carried out in the positive ion mode with spectra in the profile mode and mass spectra
were recorded in the 130–2000 m/z range. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Before analysis, miniSOG samples were diluted to a final concentration of 10 µM in acetonitrile/water/formic
acid (50:50:0.1, v/v/v) and infused directly in the mass spectrometer by a syringe pump at a flow rate of 10 μl/min. A blank run was carried out infusing only protein buffer diluted at the same ratio as the protein sample in the
same solvent system.ht y
The MS data were acquired and processed with the MassHunter workstation software (Data acquisition
v.B.04.00, Qualitative analysis with Bioconfirm v.B.07.00, Agilent Technologies). Protein Data Bank accession codes. The structures of non-irradiated and blue-light irradiated miniSOG
have been deposited in the Protein Data Bank under entry codes 6GPU and 6GPV, respectively. Protein Data Bank accession codes. The structures of non-irradiated and blue-light irradiated miniSOG
ave been deposited in the Protein Data Bank under entry codes 6GPU and 6GPV, respectively. References References
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2. Sanchez-Weatherby, J. et al. Improving diffraction by humidity control: A novel device compatible with X-ray beamlines. Acta
Crystallogr. D Biol. Crystallogr. 65, 1237–1246 (2009).hl y
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43. Wingen, M. et al. The photophysics of LOV-based fluorescent proteins – new tools for cell biology. Photochem. Photobiol. Sci. 13,
875–883 (2014).l y
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3. Wingen, M. et al. The photophysics of LOV-based fluorescent proteins – new tools for cell biology. Photochem. Photobiol. Sci. 13
875–883 (2014).l 875 883 (2014). 44. Rodríguez-Pulido, A. et al. Fluorescent flavoprotein heterodimers: combining photostability with singlet oxygen generation. ChemPhotoChem 2, 571–574 (2018). (
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44. Rodríguez-Pulido, A. et al. Fluorescent flavoprotein heterodimers: combining photostability with singlet oxygen generation. ChemPhotoChem 2, 571–574 (2018). 44. Rodríguez-Pulido, A. et al. Fluorescent flavoprotein heterodimers: combining photostability with singlet oxygen genera
ChemPhotoChem 2, 571–574 (2018). 5. Foote, C. S. & Clennan, E. L. Properties and reactions of singlet dioxygen. In Active Oxygen in Chemistry (eds Foote, C.S., Valentine
J., Greenberg, A. & Liebman, J.F.) 105-140 (Springer Netherlands, 1995). Acknowledgements We thank Rubén Ruiz-González, Marjolaine Noirclerc-Savoye and David von Stetten for their contribution at an
early stage of the project. The ESRF is acknowledged for access to beamlines and facilities for molecular biology
via its in-house research program. AR acknowledges funding from the French Agence Nationale de la Recherche
(project SOxygen, ANR-11-JSV5-0009 and project CrystalBall, ANR-14-CE06-0005-02), from the Spanish
Ministerio de Economía y Competitividad (CTQ2016-78454-C2-1-R, MAT2015-66605-P and SEV-2016-0686)
and the Fundació la Marató de TV3 (grant No. 20133133). This work used the platforms of the Grenoble Instruct-
ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and
GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). Author Contributions J.T. performed and analyzed the spectroscopic and photophysical investigations. C.L., S.A., G.G. and A.R. performed and analyzed the structural work. L.S. performed and analyzed the mass spectrometry experiments. C.F. and X.S. participated to the discussion of the data. S.N., G.G. and A.R. designed the study, discussed the data
and wrote the manuscript with J.T. and C.F. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-38955-3 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:2428 | https://doi.org/10.1038/s41598-019-38955-3 10
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Review of: "Analytical Study and Amelioration of Plastic Pavement Material Quality"
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Review of: "Analytical Study and Amelioration of Plastic
Pavement Material Quality" Farida El-Dars1
1 Helwan University Farida El-Dars1
1 Helwan University Farida El-Dars1 Potential competing interests: No potential competing interests to declare. 1. The idea is novel. This should be reflected in the title as: “Evaluation of the use of plastic waste material as pavement
construction substitutes.” The study is physical/mechanical and not chemical, even though it uses heat treatment. 2. The abstract must indicate the importance of the work in the country, i.e., the extent of plastic waste, and a brief of the
findings of the work. Limit the amount of experimental data in this section and stress the results. 1. The idea is novel. This should be reflected in the title as: “Evaluation of the use of plastic waste material as pavement
construction substitutes.” The study is physical/mechanical and not chemical, even though it uses heat treatment. 1. The idea is novel. This should be reflected in the title as: “Evaluation of the use of plastic waste material as pavement
construction substitutes.” The study is physical/mechanical and not chemical, even though it uses heat treatment. construction substitutes. The study is physical/mechanical and not chemical, even though it uses heat treatment. 2. The abstract must indicate the importance of the work in the country, i.e., the extent of plastic waste, and a brief of the
findings of the work. Limit the amount of experimental data in this section and stress the results. 2. The abstract must indicate the importance of the work in the country, i.e., the extent of plastic waste, and a brief of the
findings of the work. Limit the amount of experimental data in this section and stress the results. 3. Introduction: The first paragraph can be removed, and the focus on the problem at hand starts in paragraph 2. 4. Do not go into details concerning previous studies and their results, but only mention them and their outcomes. It is
important to use these details in the discussion of the work's results. 5. In the aim, state what the purpose of the work is (remove first sentence). 5. In the aim, state what the purpose of the work is (remove first sentence). 6. In the preparation of specimens, it is better to insert a table with the % of each plastic waste and % sand used while
indicating names such as (sample 1, sample 2, etc.). 7. Qeios, CC-BY 4.0 · Review, April 13, 2024 Qeios ID: R8K2BJ · https://doi.org/10.32388/R8K2BJ Review of: "Analytical Study and Amelioration of Plastic
Pavement Material Quality" In the results and discussion, do not re-mention the % of samples and their composition, but it's easier to state
“Prepared plastic waste formulations in table .” 8. Figure 6 is crowded; rearrange to clarify important results of the test. 9. If possible, can the results be combined in a table and figures be used to explain the main property changes in
composites formulated? 10. At the end of the discussion, an economic evaluation of the use of plastic waste must be stated. The purpose of using
waste is more than environmentally viable; it must be also economically viable as well as durable. For example, the
prices should be compared, as well as the durability of each material, and a plan on how to start a pilot project to
gather plastic waste should be outlined. Think environmental and economic—this is what leads to sustainability. Qeios ID: R8K2BJ · https://doi.org/10.32388/R8K2BJ 1/1
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Probing unified theories with reduced couplings at future hadron colliders
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European physical journal. C, Particles and fields
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Eur. Phys. J. C (2021) 81:185
https://doi.org/10.1140/epjc/s10052-021-08966-4 Regular Article - Theoretical Physics Probing unified theories with reduced couplings at future hadron
colliders In turn the discovery potential
of the 100 TeV FCC-hh is investigated and found that large
parts of the predicted spectrum of these models can be tested, but the higher mass regions are beyond the reach even of the
FCC-hh. Probing unified theories with reduced couplings at future hadron
colliders S. Heinemeyer1,2,3,a, J. Kalinowski4,b, W. Kotlarski5,c, M. Mondragón6,d, G. Patelli
G. Zoupanos7,8,9,g 1 Instituto de Física Teórica (UAM/CSIC), Universidad Autónoma de Madrid Cantoblanco, 28049 Madrid, Spain
2 Campus of International Excellence UAM + CSIC, Cantoblanco, 28049 Madrid, Spain
3 Instituto de Física de Cantabria (CSIC-UC), 39005 Santander, Spain
4 Faculty of Physics, University of Warsaw, ul. Pasteura 5, 02-093 Warsaw, Poland
5 Technische Universität Dresden, Institut für Kern- und Teilchenphysik (IKTP), 01069 Dresden, Germany
6 Instituto de Física, Universidad Nacional Autónoma de México, A.P. 20-364, 01000 Mexico, CDMX, Mexico
7 Physics Department, Nat. Technical University, 157 80 Zografou, Athens, Greece
8 Max-Planck Institut für Physik, Föhringer Ring 6, 80805 München, Germany
9 Theoretical Physics Department, CERN, Geneva, Switzerland Received: 23 November 2020 / Accepted: 7 February 2021 / Published online: 23 February 2021
© The Author(s) 2021 Received: 23 November 2020 / Accepted: 7 February 2021 / Published online: 23 February 2021
© The Author(s) 2021 but the higher mass regions are beyond the reach even of the
FCC-hh. Abstract
The search for renormalization group invariant
relations among parameters to all orders in perturbation the-
ory constitutes the basis of the reduction of couplings con-
cept. Reduction of couplings can be achieved in certain
N = 1 supersymmetric grand unified theories and few of
them can become even finite at all loops. We review the basic
idea, the tools that have been developed as well as the result-
ing theories in which successful reduction of couplings has
been achieved so far. These include: (i) a reduced version
of the minimal N = 1 SU(5) model, (ii) an all-loop finite
N = 1 SU(5) model, (iii) a two-loop finite N = 1 SU(3)3
model and finally (vi) a reduced version of the Minimal
Supersymmetric Standard Model. In this paper we present
a number of benchmark scenarios for each model and inves-
tigate their observability at existing and future hadron collid-
ers. The heavy supersymmetric spectra featured by each of
the above models are found to be beyond the reach of the 14
TeV HL-LHC. It is also found that the reduced version of the
MSSM is already ruled out by the LHC searches for heavy
neutral MSSM Higgs bosons. 1 Introduction The reduction of couplings method [1–4] (see also [5–8])
is a promising method which relates originally seemingly
independent parameters to a single, “primary” coupling. The method requires the original theory to which it is applied
to be a renormalizable one, and the resulting relation among
the parameters to be valid at all energy scales, i.e. renormal-
ization group invariant (RGI). A next (natural) step, after the introduction of a novel
symmetry through a grand unified theory (GUT) [9–14]), in
order to achieve reduction of free parameters of the SM is
the relation of the gauge sector to the Yukawa sector (gauge
Yukawa unification, GYU). This was the central characteris-
tic of the reduction of couplings approach in the first period
of searches, applied in N = 1 GUTs [15–28]. According to
that approach, being in a GUT environment, RGI relations
are set between the unification scale and the Planck scale. One-loop consideration can guarantee the all-loop validity of
those relations. Moreover, RGI relations can be found which
guarantee all order finiteness of a theory. The method has pre-
dicted the top quark mass in the finite N = 1 SU(5) model
[15,16] as well as in the minimal N = 1 SU(5) one [17]
before its experimental measurement [29]. a e-mail: Sven.Heinemeyer@cern.ch
b e-mail: kalino@fuw.edu.pl
c e-mail: wojciech.kotlarski@tu-dresden.de
d e-mail: myriam@fisica.unam.mx
e e-mail: patellis@central.ntua.gr (corresponding author)
f e-mail: ntrac@central.ntua.gr
g e-mail: George.Zoupanos@cern.ch a e-mail: Sven.Heinemeyer@cern.ch
b e-mail: kalino@fuw.edu.pl
c e-mail: wojciech.kotlarski@tu-dresden.de
d e-mail: myriam@fisica.unam.mx
e e-mail: patellis@central.ntua.gr (corresponding author)
f e-mail: ntrac@central.ntua.gr
g e-mail: George.Zoupanos@cern.ch Since SuperSymmetry (SUSY) seems an essential ingre-
dient for the reduction of couplings method, we have to 12 3 185
Page 2 of 20 185
Page 2 of 20 Eur. Phys. J. C (2021) 81 :185 include a supersymmetry breaking sector (SSB), which
involves dimension-1 and -2 couplings. The supergraph
method and the spurion superfield technique played an
important role for the progress in that sector, leading to com-
plete all-loop finite models, i.e. including the SSB sector. The all-loop finite N = 1 SU(5) model [30] has given a pre-
diction for the Higgs mass compatible with the experimental
results [31–33] and a heavy SUSY mass spectrum, consistent
with the experimental non-observation of these particles. The
reduction of couplings method has been applied to several
other cases. 1 Introduction The full analysis of the most successful models,
that includes predictions in agreement with the experimen-
tal measurements of the top and bottom quark masses for
each model, can be found in a recent work [44]. RGI relations. Such a relation has, in general, the form
(g1, . . . , gA) = const. which should satisfy the following
partial differential equation (PDE) μ d
dμ = ⃗∇ · ⃗β =
A
a=1
βa
∂
∂ga
= 0,
(1) (1) where βa is the β-functions of ga. The above PDE is equiv-
alent to the following set of ordinary differential equations
(ODEs), which are called reduction equations (REs) [2–4], βg
dga
dg = βa,
a = 1, . . . , A −1,
(2) (2) In this paper we address the question to what extent the
reduction of couplings idea, as applied in the so far phe-
nomenologically successful models, can be experimentally
tested at HL-LHC and future FCC hadron collider. To this end
we propose a number of benchmark points for each model. We present the SUSY breaking parameters used as input in
each benchmark to calculate the corresponding Higgs boson
and supersymmetric particles masses. Then we compute the
expected production cross sections at the 14 TeV (HL-)LHC
and the 100 TeV FCC-hh and investigate which production
channels can be observed. where now g and βg are the primary coupling and its corre-
sponding β-function. There are obviously A −1 relations in
the form of (g1, . . . , gA) = const. in order to express all
other couplings in term of the primary one. The crucial demand is that the above REs admit power
series solutions ga =
n
ρ(n)
a
g2n+1,
(3) (3) which preserve perturbative renormalizability. Without this
requirement, we just trade each “dependent” coupling for an
integration constant. The power series, which are a set of spe-
cial solutions, fix that constant. It is very important to point
out that the uniqueness of such a solution can be already
decided at the one-loop level [2–4]. In supersymmetric the-
ories, where the asymptotic behaviour of several parameters
are similar, the use of power series as solutions of the REs
are justified. But, usually, the reduction is not “complete”,
which means that not all of the couplings can be reduced
in favor of the primary one, leading to the so called “partial
reduction” [45,46]. 1 Introduction The present work is organized as follows. In Sect. 2 we
review the basic idea of the reduction of couplings. In Sect. 3
we list the phenomenological constraints used in our analy-
ses, wile in Sect. 4 we explain the computational setup. In
Sects. 5–8 wereview four interesting models, namely (i) the
minimal N = 1 supersymmetric SU(5), (ii) the finite N = 1
supersymmetric SU(5), (iii) the finite SU(3)3 and (iv) the
MSSM, in which the reduction of couplings has been suc-
cessfully applied. We briefly review some earlier results of
our phenomenological analysis. In this context the new ver-
sion of the FeynHiggs [34–43] code plays a crucial role,
which was used to calculate the Higgs-boson predictions, in
particular the mass of the lightest CP-even Higgs boson. The
improved predictions of FeynHiggs are compared with the
LHC measurements and the Beyond Standard Model (BSM)
Higgs boson searches. As a new part of the analysis we exam-
ine in each model the discovery potential of the Higgs and
SUSY spectrum at approved future and hypothetical future
hadron colliders. Finally, Sect. 8 is dedicated to brief con-
clusive remarks. We proceed to the reduction scheme for massive param-
eters, which is far from being straightforward. A number of
conditions is required (see for example [47]). Nevertheless,
progress has been achieved, starting from [48], and finally we
can introduce mass parameters and couplings carrying mass
dimension [49,50] in the same way as dimensionless cou-
plings. Consider the superpotential Consider the superpotential W = 1
2 μi j i j + 1
6 Ci jk i j k,
(4) (4) and the SSB sector Lagrangian 2 Theoretical basis −LSSB = 1
6 hi jk φiφ jφk + 1
2 bi j φiφ j
+ 1
2 (m2) j
i φ∗iφ j + 1
2 M λiλi + h.c.,
(5) Here we will briefly review the core idea of the reduction of
couplings method. The target is to single out a basic parame-
ter (which we will call the primary coupling), where all other
parameters can be expressed in terms of this one through (5) 123 Eur. Phys. J. C (2021) 81 :185 Page 3 of 20
185 185 it can be proven [70,71] that the following relations are
all-loop RGI where φi’s are the scalar fields of the corresponding super-
fields i’s and λi are the gauginos. where φi’s are the scalar fields of the corresponding super-
fields i’s and λi are the gauginos. Let us write down some well known relations: M = M0
βg
g ,
(12) M = M0
βg
g ,
(12)
hi jk = −M0 βi jk
C ,
(13)
bi j = −M0 βi j
μ ,
(14)
(m2)i
j = 1
2 |M0|2 μdγ i j
dμ ,
(15) (12) (i) The β-function of the gauge coupling at one-loop level
is given by [51–55] (i) The β-function of the gauge coupling at one-loop level
is given by [51–55] (14) β(1)
g
= dg
dt =
g3
16π2
i
T (Ri) −3 C2(G)
,
(6) (15) (6) where M0 is an arbitrary reference mass scale to be spec-
ified and Eq. (12) is the Hisano–Shifman relation [66]
(note that in both assumptions we do not rely on specific
solutions of these equations). where M0 is an arbitrary reference mass scale to be spec-
ified and Eq. (12) is the Hisano–Shifman relation [66]
(note that in both assumptions we do not rely on specific
solutions of these equations). where T (Ri) is the Dynkin index of the rep Ri where the
matter fields belong and C2(G) is the quadratic Casimir
operator of the adjoint rep G. p
j
p
(ii) The anomalous dimension γ (1) i
j, at a one-loop level, of
a chiral superfield is As a next step we substitute the last equation, Eq. 2 Theoretical basis (15), by a
more general RGI sum rule that holds to all orders [72] m2
i + m2
j + m2
k
= |M|2
1
1 −g2C2(G)/(8π2)
d ln Ci jk
d ln g
+ 1
2
d2 ln Ci jk
d(ln g)2
+
l
m2
l T (Rl)
C2(G) −8π2/g2
d ln Ci jk
d ln g ,
(16) γ (1) i
j =
1
32π2 [ Cikl C jkl −2 g2 C2(Ri)δi
j]. (7) (7) (iii) The β-functions of Ci jk’s, at one-loop level, following
the N = 1 non-renormalization theorem [56–58], are
expressed in terms of the anomalous dimensions of the
fields involved (16) which leads to the following one-loop relation m2
i + m2
j + m2
k = |M|2. (17) (17) βi jk
C
= dCi jk
dt
= Ci jl γ l
k + Cikl γ l
j + C jkl γ l
i . (8) (8) Finally, note that in the case of product gauge groups, Eq. (12)
takes the form WeproceedbyassumingthattheREsadmitpowerseriessolu-
tions: Mi = βgi
gi
M0,
(18) (18) Ci jk = g
n=0
ρi jk
(n)g2n. (9) (9) where i denotes the group of the product. This will be used
in the Reduced MSSM case. Consider an N = 1 globally supersymmetric gauge the-
ory, which is chiral and anomaly free, where G is the gauge
group and g the associated gauge coupling. The theory has
the superpotential of Eq. (4), while the one-loop gauge and
Ci jks β-functions are given by Eqs. (6) and (8) respectively
and the one-loop anomalous dimensions of the chiral super-
fields by Eq. (7). Trying to obtain all-loop results we turn to relations
among β-functions. The spurion technique [58–62] gives
all-loop relations among SSB β-functions [63–69]. Then,
assuming that the reduction of Ci jk is possible to all
orders Trying to obtain all-loop results we turn to relations
among β-functions. The spurion technique [58–62] gives
all-loop relations among SSB β-functions [63–69]. Then,
assuming that the reduction of Ci jk is possible to all
orders Demanding the vanishing of all one-loop β-functions, Eqs. (6, 7) lead to the relations dCi jk
dg
= βi jk
C
βg
,
(10) (10)
i
T (Ri) = 3C2(G),
(19)
CiklC jkl = 2δi
jg2C2(Ri). 2 Theoretical basis (20) (19) as well as for hi jk as well as for hi jk (20) hi jk = −M dCi jk
d ln g ,
(11)
The finiteness conditions for an N = 1 supersymmetric the-
ory with SU(N) associated group is found in [73] while dis- hi jk = −M dCi jk
d ln g ,
(11)
The finite
ory with hi jk = −M dCi jk
d ln g , The finiteness conditions for an N = 1 supersymmetric the-
ory with SU(N) associated group is found in [73] while dis- hi jk = −M dCi jk
d ln g , The finiteness conditions for an N = 1 supersymmetric the-
ory with SU(N) associated group is found in [73] while dis- (11) 12 3 185
Page 4 of 20 185
Page 4 of 20 Eur. Phys. J. C (2021) 81 :185 (iv) When considered as solutions of vanishing Yukawa β-
functions (at one-loop order), i.e. βi jk = 0, the above
solutions are isolated and non-degenerate;
then, each of the solutions in Eq. (24) can be extended
uniquely to a formal power series in g, and the asso-
ciated super Yang–Mills models depend on the single
coupling constant g with a vanishing, at all orders, β-
function. cussion of the no-charge renormalization and anomaly free
requirementscanbefoundin[74].Itshouldbenotedthatcon-
ditions (19) and (20) are necessary and sufficient to ensure
finiteness at the two-loop level [51–55]. The requirement of finiteness, at the one-loop level, in
softly broken SUSY theories demands additional constraints
among the soft terms of the SSB sector [75], while, once
more, these one-loop requirements assure two-loop finite-
ness, too [76]. These conditions impose restrictions on the
irreducible representations Ri of the gauge group G as well
as on the Yukawa couplings. For example, since U(1)s are
not compatible with condition (19), the MSSM is excluded. Therefore, a GUT is initially required with the MSSM being
its low energy theory. Also, since condition (20) forbids the
appearance of gauge singlets (C2(1) = 0), F-type spon-
taneous symmetry breaking [77] are not compatible with
finiteness. Finally, D-type spontaneous breaking [78] is also
incompatible since it requires a U(1) group. While the validity of the above cannot be extended to
non-SUSY theories, it should be noted that reduction of cou-
plings and finiteness are intimately related. 3 Phenomenological constraints In this section we briefly review several experimental con-
straints that were applied in our phenomenological analysis. The used values do not correspond to the latest experimen-
tal results, which, however, has a negligible impact on our
analysis. The nontrivial point is that the relations among couplings
(gauge and Yukawa) which are imposed by the conditions
(19) and (20) should hold at any energy scale. The neces-
sary and sufficient condition is to require that such relations
are solutions to the REs (see Eq. (10)) In our models we evaluate the pole mass of the top quark
while the bottom quark mass is evaluated at the MZ scale
(to avoid uncertainties to its pole mass). The experimental
values, taken from Ref. [86] are: βg
dCi jk
dg
= βi jk
(21) βg
dCi jk
dg
= βi jk (21) mexp
t
= 173.1 ± 0.9 GeV,
mb(MZ) = 2.83 ± 0.10 GeV. (25) holdingatallorders.Wenote,oncemore,thattheexistenceof
one-loop level power series solution guarantees the all-order
series. (25) We interpret the Higgs-like particle discovered in July
2012 by ATLAS and CMS [31,32] as the light CP-even
Higgs boson of the MSSM [87–89]. The Higgs boson exper-
imental average mass is [86]1 Thereexistthefollowingtheorem[79,80]whichpointsdown
which are the necessary and sufficient conditions in order
for an N = 1 SUSY theory to be all-loop finite. In Refs. [79–
85] it was shown that for an N = 1 SUSY Yang–Mills theory,
based on a simple gauge group, if the following four condi-
tions are fulfilled: Mexp
h
= 125.10 ± 0.14 GeV. (26) (26) The theoretical uncertainty [34,35], however, for the pre-
diction of Mh in the MSSM dominates the total uncer-
tainty, since it is much larger than the experimental one. In
our following analyses we shall use the new FeynHiggs
code [34–42] (Version 2.16.0) to predict the Higgs mass.2
FeynHiggs evaluates the Higgs masses based on a combi-
nation of fixed order diagrammatic calculations and resum-
mation of the (sub)leading logarithmic contributions at all
orders. This provides a reliable Mh even for a large SUSY
scale. This new version gives a downward shift on the Higgs
mass Mh of O(2 GeV) for large SUSY masses and in partic-
ular gives a reliable point-by-point evaluation of the Higgs- (i) No gauge anomaly is present. (i) No gauge anomaly is present. (i) No gauge anomaly is present. 2 An analysis of the impact of the improved Mh calculation in various
SUSY models can be found in [90]. 1 This is the latest available LHC combination. More recent measure-
ments confirm this value. 1 This is the latest available LHC combination. More recent measure-
ments confirm this value.
2 An analysis of the impact of the improved Mh calculation in various
SUSY models can be found in [90]. 3 Phenomenological constraints (ii) The β-function of the gauge coupling is zero at one-loop
level β(1)
g
= 0 =
i
T (Ri) −3 C2(G). (22) (22) (iii) The condition of vanishing for the one-loop anomalous
dimensions of matter fields, γ (1)i
j = 0 =
1
32π2 [ Cikl C jkl −2 g2 C2(R)δi
j],
(23) (23) admits solution of the form admits solution of the form Ci jk = ρi jkg,
ρi jk ∈C. (24) (24) 123 3 3 Eur. Phys. J. C (2021) 81 :185 Page 5 of 20
185 Page 5 of 20
185 185 boson mass uncertainty [43]. The theoretical uncertainty cal-
culated is added linearly to the experimental error in Eq. (26). Furthermore, recent results from the ATLAS experiment
[91] set limits to the mass of the pseudoscalar Higgs boson,
MA, in comparison with tan β. For models with tan β ∼
45–55, as the ones examined here, the lowest limit for the
physical pseudoscalar Higgs mass is boson mass uncertainty [43]. The theoretical uncertainty cal-
culated is added linearly to the experimental error in Eq. (26). scale, the physical top quark mass mt as well as the physical
pseudoscalar boson mass MA as input. The first two values
are calculated by the private code while MA is calculated
only in DR scheme. This single value is obtained from the
SPheno output where it is calculated at the two-loop level
in the gaugeless limit [117,118]. The flow of information
between codes in our analysis is summarised in Fig. 1. boson mass uncertainty [43]. The theoretical uncertainty cal-
culated is added linearly to the experimental error in Eq. (26). Furthermore, recent results from the ATLAS experiment
[91] set limits to the mass of the pseudoscalar Higgs boson,
MA, in comparison with tan β. For models with tan β ∼
45–55, as the ones examined here, the lowest limit for the
physical pseudoscalar Higgs mass is Furthermore, recent results from the ATLAS experiment
[91] set limits to the mass of the pseudoscalar Higgs boson,
MA, in comparison with tan β. For models with tan β ∼
45 55 as the ones examined here the lowest limit for the MA, in comparison with tan β. For models with tan β ∼
45–55, as the ones examined here, the lowest limit for the
physical pseudoscalar Higgs mass is between codes in our analysis is summarised in Fig. 1. MA ≳1900 GeV. We also consider the following four flavor observables
where SUSY has non-negligible impact. For the branch-
ing ratio BR(b →sγ ) we take a value from [92–97],
while for the branching ratio BR(Bs →μ+μ−) we use
a combination of [98–105]: BR(b →sγ )exp
BR(b →sγ )SM = 1.089 ± 0.27,
BR(Bs →μ+μ−) = (2.9 ± 1.4) × 10−9. (28) (28) For the Bu decay to τν we use [97,106–108] and for
MBs
we use [109,110]: For the Bu decay to τν we use [97,106–108] and for
MBs
we use [109,110]: BR(Bu →τν)exp
BR(Bu →τν)SM = 1.39 ± 0.69,
Mexp
Bs
MSM
Bs
= 0.97 ± 0.2. (29) (29) 5 The minimal N = 1 supersymmetric SU(5) model We start with the partial reduction of the N = 1 SUSY
SU(5) model [17,48]. Our notation is as follows: I(10)
and I(5) refer to the three generations of leptons and quarks
(I = 1, 2, 3), (24) is the adjoint which breaks SU(5)
to SU(3)C × SU(2)L × U(1)Y and H(5) represent the
two Higgs superfields for the electroweak symmetry break-
ing (ESB) [124,125]. The choice of using only one set of
(5 + 5) for the ESB renders the model asymptotically free
(i.e. βg < 0 ). The superpotential of the model is described
by Finally, weconsider ColdDarkMatter (CDM) constraints. Finally, weconsider ColdDarkMatter (CDM) constraints. Since the Lightest SUSY Particle (LSP), which in our case is
the lightest neutralino, is a promising CDM candidate [111,
112], we examine if each model is within the CDM relic
density experimental limits. The current bound on the CDM
relic density at 2 σ level is given by [113] g
p
g
[
112], we examine if each model is within the CDM relic
density experimental limits. The current bound on the CDM
relic density at 2 σ level is given by [113] CDMh2 = 0.1120 ± 0.0112. (30) (30) In the following sections we will apply these constraints
to each model and discuss the corresponding collider phe-
nomenology. W = gt
4 ϵαβγ δτ (3)
αβ (3)
γ δ Hτ +
√
2gb (3)α(3)
αβ Hβ
+ gλ
3 β
αγ
β α
γ + g f Hαβ
α Hβ
+ μ
2 γ
α α
γ + μH Hα Hα,
(31) 3 Phenomenological constraints At this point both codes contain a consistent set of all
required parameters. SM-like Higgs boson mass as well as
low energy observables mentioned in Sect. 3 are evaluated
using FeynHiggs. To obtain collider predictions we use
SARAH to generate UFO [119,120] model for MadGraph
event generator. Based on SLHA spectrum files gener-
ated by SPheno, we use MadGraph5_aMC@NLO [121]
to calculate cross sections for Higgs boson and SUSY
particle production at the HL-LHC and a 100 TeV FCC-
hh. Processes are generated at the leading order, using
NNPDF31_lo_as_0130 [122] structure functions inter-
faced through LHAPDF6 [123]. Cross sections are computed
using dynamic scale choice, where the scale is set equal to the
transverse mass of an event, in 4 or 5-flavor scheme depend-
ing on the presence or not of b-quarks in the final state. The
results are given in Sects. 5–7. (27) MA ≳1900 GeV. (27) MA ≳1900 GeV. 4 Computational setup (31) The setup for our phenomenological analysis is as follows. Starting from an appropriate set of MSSM boundary condi-
tions at the GUT scale, parameters are run down to the SUSY
scale using a private code. Two-loop RGEs are used through-
out, with the exception of the soft sector, in which one-loop
RGEs are used. The running parameters are then used as
inputs for both FeynHiggs [34–43] and a SARAH [114]
generated, custom MSSM module for SPheno [115,116]. It should be noted that FeynHiggs requires the mb(mb) where only the third generation Yukawa couplings are
taken into account. The indices α, β, γ, δ, τ are SU(5) ones. A detailed presentation of the model can be found in [17] as
well as in [126,127]. Our primary coupling is the gauge coupling g. In this
modelthegauge-Yukawaunificationcanbeachievedthrough
two sets of solutions which are asymptotically free [17]: 123 123 123 185
Page 6 of 20 Eur. Phys. J. C (2021) 81 :185 Fig. 1 Flow of information between used computer codes (see text for details) Fig. 1 Flow of information between used computer codes (see text for details) a : gt =
2533
2605g + O(g3),
gb =
1491
2605g + O(g3),
gλ = 0,
g f =
560
521g + O(g3),
b : gt =
89
65g + O(g3),
gb =
63
65g + O(g3),
gλ = 0,
g f = 0,
(32) independent ones, since they cannot be reduced in a suitable
form. The lowest-order reduction for the parameters of the
SSB Lagrangian are given by: BH = 1029
521 μH M,
B = −3100
521 μ M,
(34)
ht = −gt M,
hb = −gb M,
h f = −g f M,
hλ = 0,
m2
Hu = −569
521 M2,
m2
Hd = −460
521 M2,
m2
= 1550
521 M2,
m2
3 = 436
521 M2,
m2
1,2 = 8
5 M2,
m2
3 = 545
521 M2,
m2
1,2 = 12
5 M2. (35) (34) (32) where the higher order terms denote uniquely computable
power series in g. Let us note that the reduction of the dimen-
sionless sector is independent of the dimensionful one. These solutions describe the boundaries of a RGI surface
in the parameter space which is AF and where g f and gλ
could be different from zero. 3 gλ = 0 is inconsistent, but gλ ≲0.005 is necessary in order for the
proton decay constraint [21] to be satisfied. A small gλ is expected to
not affect the prediction of unification of SSB parameters. 4 Computational setup C (2021) 81 :185 Page 7 of 20
185 Fig. 2 Scatter plots for the minimal N = 1 SU(5) model. Left: the lightest Higgs mass, Mh, as a function of M. The B-physics constraints allow
(mostly) higher scale points (with green color). Right: the theoretical uncertainty of the light Higgs mass [43] Fig. 2 Scatter plots for the minimal N = 1 SU(5) model. Left: the lightest Higgs mass, Mh, as a function of M. The B-physics constraints allow
(mostly) higher scale points (with green color). Right: the theoretical uncertainty of the light Higgs mass [43] The prediction for Mh as a function of the unified gaugino
mass M with μ < 0 is given in Fig. 2 (left). The
MBs
channel is responsible for the gap at the B-physics allowed
points (green points). The scattered points come from the
fact that for each M we vary the free parameters μ and μH. Figure 2 (right) gives the theoretical uncertainty of the Higgs
mass for each point, calculated with FeynHiggs 2.16.0
[43]. There is substantial improvement to the Higgs mass
uncertainty compared to past analyses, since it has dropped
by more than 1 GeV. our case is the lightest neutralino, is strongly Bino-like. Combined with the heavy mass it acquires (1-2 TeV), it
cannot account for a relic density low enough to agree
with experimental observation. Thus, we need a mechanism
that reduces this CDM abundance. This could be related to
the problem of neutrino masses, which cannot be gener-
ated naturally in this particular model. However, one could
extend the model by considering bilinear R-parity violat-
ing terms and thus introduce neutrino masses [128,129]. R-
parity violation [130–133] would have a small impact on
the collider phenomenology, but remove the CDM bound of
Eq. (30) completely. Other mechanisms, not involving R-
parity violation, that could be invoked if the amount of CDM
appears to be too large, concern the cosmology of the
early universe. For example, “thermal inflation” [134] or
“late time entropy injection” [135] can bring the CDM den-
sity into agreement with Planck measurements. For the orig-
inal discussion see [44]. Large parts of the predicted particle spectrum are in agree-
ment with the B-physics observables and the lightest Higgs
boson mass measurement and its theoretical uncertainty. 4 The analysis presented in [136,137] only reaches MA ≤2000 GeV,
where an exclusion down to tan β ∼30 is expected. An extrapolation
to tan β ∼50 reaches Higgs-boson mass scales of ∼2500 GeV. 4 Computational setup We
choose three benchmarks in the low-mass region, mark-
ing the points with the lightest SUSY particle (LSP) above
1200 GeV (MINI-1), 1500 GeV (MINI-2) and 2200 GeV
(MINI-3), respectively. The mass of the LSP can go as high
as ∼3800 GeV, but the cross sections calculated below
will then be negligible and we restrict ourselves here to
the low-mass region. The values presented in Table 1 were
used as input to get the full supersymmetric spectrum from
SPheno 4.0.4 [115,116]. Mi are the gaugino masses and
the rest are squared soft sfermion masses which are diagonal
(m2 = diag(m2
1, m2
2, m2
3)), and soft trilinear couplings (also
diagonal Ai = 13×3Ai). Table 3 shows the expected production cross section for
selected channels at the 100 TeV future FCC-hh collider. We
do not show any cross sections for √s = 14 TeV, since the
prospects for discovery of MINI scenarios at the HL-LHC
are very dim. SUSY particles are too heavy to be produced
with cross sections greater that 0.01fb. Concerning the heavy
Higgs bosons, the main search channels will be H/A →
τ +τ −. Our heavy Higgs-boson mass scale shows values ≳
2500 GeV with tan β ∼50. The corresponding reach of the
HL-LHC has been estimated in [136,137]. In comparison
with our benchmark points we conclude that they will not be
accessible at the HL-LHC. 4 The resulting masses of all the particles that will be rel-
evant for our analysis can be found in Table 2. The three
first values are the heavy Higgs masses. The gluino mass
is M ˜g, the neutralinos and the charginos are denoted as
M ˜χ0
i and M ˜χ±
i , while the slepton and sneutrino masses for
all three generations are given as M˜e1,2,3, M˜ν1,2,3. Similarly,
the squarks are denoted as M ˜d1,2 and M˜u1,2 for the first two
generations. The third generation masses are given by M˜t1,2
for stops and M ˜b1,2 for sbottoms. The situation changes for the FCC-hh [138]. Theory anal-
yses [139,140] have shown that for large tan β heavy Higgs-
boson mass scales up to ∼8 TeV may be accessible, both
for neutral as well as for charged Higgs bosons. The relevant 1,2
At this point there is an important remark. No point ful-
fills the strict bound of Eq. (30), since we have overpro-
duction of CDM in the early universe. 4 Computational setup Therefore, a partial reduction is
possible where gλ and g f are independent (non-vanishing)
parameters without endangering asymptotic freedom (AF). The proton decay constraints favor solution a, therefore we
choose this one for our discussion.3 (35) We choose the gaugino mass M for characterizing the SUSY
breaking scale. Finally, we note that (i) B and BH are
treated as independent parameters without spoiling the one-
loop reduction solution of Eq. (35) and (ii) the soft scalar
mass sum rule still holds despite the specific relations among
the gaugino mass and the soft scalar masses. The SSB Lagrangian is We analyze the particle spectrum predicted for μ < 0
as the only phenomenologically acceptable choice (in the
μ > 0 the quark masses do not match the experimental mea-
surements). Below MGUT all couplings and masses of the
theory run according to the RGEs of the MSSM. Thus we
examine the evolution of these parameters according to their
RGEs up to two-loops for dimensionless parameters and
at one-loop for dimensionful ones imposing the correspond-
ing boundary conditions. −Lsoft = m2
Hu ˆH∗α ˆHα + m2
Hd
ˆH
∗
α ˆH
α
+ m2
ˆ† α
β
ˆβ
α
+
I=1,2,3
[m2
I ˆ∗(I)
α
ˆ(I)α + m2
I ˆ† (I)αβ ˆ(I)
βα ]
+
1
2 Mλλ + BH ˆH
α ˆHα + B ˆα
β ˆβ
α
+ h f ˆH
α ˆβ
α ˆHβ
+ hλ
3
ˆβ
α ˆγ
β ˆα
γ + ht
4 ϵαβγ δτ ˆ(3)
αβ ˆ(3)
γ δ ˆHτ
+
√
2hb ˆ(3)α ˆ(3)
αβ ˆH
β
+ h.c.
,
(33) As presented in [44], the pole top mass mt is predicted
within 2σ of Eq. (25). Concerning the mb(MZ) prediction
(also in [44]), we take into account a theoretical uncertainty
of ∼3%. But even taking theoretical and experimental uncer-
tainties into account in combination, we find agreement with
the experimental value only at the 4σ level. However, since
there additional uncertainties of a few percent on the quark
Yukawa couplings at the SUSY-breaking scale, that were not
fully included (see [21]) into the evaluation of the bottom
mass, we still consider the model as viable and proceed with
its analysis. (33) where the hat denotes the scalar components of the chiral
superfields. The parameters M, μ and μH are treated as 12 3 Eur. Phys. J. 4 Computational setup 0.07
0.02
˜χ0
2 ˜χ0
2
0.06
0.02
˜qi ˜χ0
1 , ˜q∗
i ˜χ0
1
0.38
0.14
0.02
˜χ0
2 ˜χ0
3
0.03
0.01
˜qi ˜χ0
2 , ˜q∗
i ˜χ0
2
0.51
0.17
0.02
˜χ0 ˜χ0
0 02
0 01
˜νi ˜e∗˜ν∗˜e j
0 06
0 02 Table 2 Masses of Higgs bosons and some of the SUSY particles for each benchmark of the Minimal N = 1 SU(5) (in TeV)
MH
MA
MH±
M ˜g
M ˜χ0
1
M ˜χ0
2
M ˜χ0
3
M ˜χ0
4
M ˜χ±
1
M ˜χ±
2
MINI-1
2.660
2.660
2.637
5.596
1.221
2.316
4.224
4.225
2.316
4.225
MINI-2
3.329
3.329
3.300
6.717
1.500
2.827
5.076
5.077
2.827
5.078
MINI-3
8.656
8.656
8.631
9.618
2.239
4.176
7.357
7.358
4.176
7.359
M˜e1,2
M˜ν1,2
M˜τ
M˜ντ
M ˜d1,2
M˜u1,2
M ˜b1
M ˜b2
M˜t1
M˜t2
MINI-1
3.729
3.728
2.445
2.766
5.617
6.100
4.332
4.698
4.312
4.704
MINI-2
4.539
4.538
2.968
3.356
6.759
7.354
5.180
5.647
5.197
5.652
MINI-3
6.666
6.665
4.408
4.935
9.722
10.616
7.471
8.148
7.477
8.151
Table 3 Expected production cross sections (in fb) for SUSY particles in the MINI scenarios. There are no channels with cross sections exceeding
0.01 fb at √s = 14 TeV
Scenarios √s MINI 1 100 TeV MINI 2 100 TeV MINI 3 100 TeV Scenarios √s
MINI 1 100 TeV MINI 2 100 TeV MINI 3 100 TeV Table 3 Expected production cross sections (in fb) for SUSY particles in the MINI scenarios. There are no channels with cross sections exceeding
0 01 fb at √s = 14 TeV 0.01 fb at √s = 14 TeV
Scenarios √s MINI-1 100 TeV MINI-2 100 TeV MINI-3 100 TeV Scenarios √s
MINI-1 100 TeV MINI-2 100 TeV MINI-3 100 TeV
˜χ0
1 ˜χ0
1
0.04
0.02
˜ui ˜χ−
1 , ˜di ˜χ+
1 + h.c. 1.00
0.35
0.03
˜χ0
1 ˜χ0
3
0.02
0.01
˜ui ˜χ−
2 , ˜di ˜χ+
2 + h.c. 4 Computational setup The LSP, which in 12 3 3 3 Eur. Phys. J. C (2021) 81 :185 185
Page 8 of 20 Table 1 Minimal N = 1 SU(5) predictions that are used as input to SPheno. Mass parameters are in GeV and rounded to 1 GeV
M1
M2
M3
|μ|
b
Au
Ad
Ae
tan β
m2
Q1,2
MINI-1
1227
2228
5310
4236
4012
4325
4772
1732
50.3
61712
MINI-2
1507
2721
6376
5091
4962
5245
5586
2005
52.0
74452
MINI-3
2249
4019
9138
7367
12462
7571
8317
3271
50.3
107622
m2
Q3
m2
L1,2
m2
L3
m2
u1,2
m2
u3
m2
d1,2
m2
d3
m2
e1,2
m2
e3
MINI-1
45482
37142
27672
59742
41812
54782
41772
41602
24912
MINI-2
54692
45212
33582
72062
50392
54782
49942
50702
30192
MINI-3
78902
66392
49342
104122
72332
94952
72112
74592
44642
Table 2 Masses of Higgs bosons and some of the SUSY particles for each benchmark of the Minimal N = 1 SU(5) (in TeV)
MH
MA
MH±
M ˜g
M ˜χ0
1
M ˜χ0
2
M ˜χ0
3
M ˜χ0
4
M ˜χ±
1
M ˜χ±
2
MINI-1
2.660
2.660
2.637
5.596
1.221
2.316
4.224
4.225
2.316
4.225
MINI-2
3.329
3.329
3.300
6.717
1.500
2.827
5.076
5.077
2.827
5.078
MINI-3
8.656
8.656
8.631
9.618
2.239
4.176
7.357
7.358
4.176
7.359
M˜e1,2
M˜ν1,2
M˜τ
M˜ντ
M ˜d1,2
M˜u1,2
M ˜b1
M ˜b2
M˜t1
M˜t2
MINI-1
3.729
3.728
2.445
2.766
5.617
6.100
4.332
4.698
4.312
4.704
MINI-2
4.539
4.538
2.968
3.356
6.759
7.354
5.180
5.647
5.197
5.652
MINI-3
6.666
6.665
4.408
4.935
9.722
10.616
7.471
8.148
7.477
8.151
Table 3 Expected production cross sections (in fb) for SUSY particles in the MINI scenarios. There are no channels with cross sections exceeding
0.01 fb at √s = 14 TeV
Scenarios √s MINI-1 100 TeV MINI-2 100 TeV MINI-3 100 TeV Scenarios √s
MINI-1 100 TeV MINI-2 100 TeV MINI-3 100 TeV
˜χ0
1 ˜χ0
1
0.04
0.02
˜ui ˜χ−
1 , ˜di ˜χ+
1 + h.c. 1.00
0.35
0.03
˜χ0
1 ˜χ0
3
0.02
0.01
˜ui ˜χ−
2 , ˜di ˜χ+
2 + h.c. 4 Computational setup The finite SU(5) group is broken to the MSSM, which
of course in no longer a finite theory [15–18,22,25]. The energy of 100 TeV is big enough to produce SUSY
particles in pairs. However, the cross sections remain rela-
tively small. Only for the MINI-1 scenario the squark pair and
squark-gluino (summed over all squarks) production cross
sections can reach tens of fb. For MINI-2 and MINI-3 scenar-
ios the cross sections are significantly smaller. In these sce-
narios squarks decay preferentially into a quark+LSP (with
BR ∼0.95), gluino into ˜t ¯t and ˜b ¯b +h.c with BR ∼0.33
each. In order for this finite to all-orders SU(5) model to achieve
gauge Yukawa unification (GYU), it should have the follow-
ing characteristics: The SUSY discovery reach at the FCC-hh with 3 ab−1 was
evaluated in [141] for a certain set of simplified models. In
the following we will compare these simplified model lim-
its with our benchmark points to get an idea, which part of
the spectrum can be covered at the FCC-hh. A more detailed
evaluation with the future limits implemented into proper
recasting tools would be necessary to obtain a firmer state-
ment. However, such a detailed analysis goes beyond the
scope of our paper and we restrict ourselves to the simpler
direct comparison of the simplified model limits with our
benchmark predictions. (i) The one-loop anomalous dimensions are diagonal i.e.,
γ (1) j
i
∝δ j
i . (i) The one-loop anomalous dimensions are diagonal i.e.,
γ (1) j
i
∝δ j
i . i
i
(ii) The fermions of the 5i and 10i (i = 1, 2, 3) are not
coupled to the 24. i
i
(ii) The fermions of the 5i and 10i (i = 1, 2, 3) are not
coupled to the 24. (iii) The pair of the MSSM Higgs doublets are mostly com-
posed from the 5 and ¯5 Higgs that couple to the third
generation (iii) The pair of the MSSM Higgs doublets are mostly com-
posed from the 5 and ¯5 Higgs that couple to the third
generation The superpotential of the model, with an enhanced sym-
metry due to the reduction of couplings, is given by [26,28]: Concerning the scalar tops, the mass predictions of MINI-
1 and MINI-2 are well within the anticipated reach of the
FCC-hh, while MINI-3 predicts a too heavy stop mass. 4 Computational setup 0.07
0.02
˜χ0
2 ˜χ0
2
0.06
0.02
˜qi ˜χ0
1 , ˜q∗
i ˜χ0
1
0.38
0.14
0.02
˜χ0
2 ˜χ0
3
0.03
0.01
˜qi ˜χ0
2 , ˜q∗
i ˜χ0
2
0.51
0.17
0.02
˜χ0
2 ˜χ0
4
0.02
0.01
˜νi ˜e∗
j, ˜ν∗
i ˜e j
0.06
0.02
˜χ0
3 ˜χ0
4
0.05
0.02
Hb ¯b
84.04
30.10
0.17
˜χ0
2 ˜χ+
1
2.20
0.98
0.18
Ab ¯b
84.79
29.79
0.18
˜χ0
3 ˜χ+
2
0.10
0.04
0.01
H+b¯t + H−t ¯b
33.24
12.76
0.1
˜χ0
4 ˜χ+
2
0.10
0.04
0.01
H−b ¯b
0.04
0.02
˜g ˜g
7.76
2.02
0.11
Ht ¯t
0.03
0.01
˜g ˜χ0
1
0.28
0.11
0.01
At ¯t
0.02
0.01
˜g ˜χ0
2
0.34
0.12
0.01
Htb
0.01
˜g ˜χ+
1
0.70
0.27
0.03
H A
0.03
0.01
˜qi ˜q j, ˜qi ˜q∗
j
21.15
7.44
0.74
H H+
0.06
0.02
˜χ+
1 ˜χ−
1
1.19
0.54
0.09
H+W −
6.50
2.96
0.03
˜χ+
1 ˜χ−
2
0.05
0.02
HW +
0.02
0.01
˜χ+
2 ˜χ−
1
0.05
0.02
H+H−
0.04
0.01
˜χ+
2 ˜χ−
2
0.06
0.02
AH+
0.06
0.02
˜ei ˜e∗
j
0.16
0.08
0.01
AW +
0.02
0.01
˜qi ˜g, ˜q∗
i ˜g
30.57
9.33
0.66
H Z
1.38
0.58
0.01
˜νi ˜ν∗
j
0.04
0.02
AZ
1.20
0.52
0.01 INI-2 100 TeV MINI-3 100 TeV Scenarios √s
MINI-1 100 TeV MINI-2 100 TeV MINI-3 100 TeV 12 Eur. Phys. J. C (2021) 81 :185 Page 9 of 20
185 185 decay channels are H/A →τ +τ −and H± →τντ, tb. This
places our three benchmark points well within the covered
region (MINI-1 and MINI-2) or at the border of the parameter
space that can be probed (MINI-3). decay channels are H/A →τ +τ −and H± →τντ, tb. This
places our three benchmark points well within the covered
region (MINI-1 and MINI-2) or at the border of the parameter
space that can be probed (MINI-3). mass in the correct range. As discussed below, improved
Higgs calculations predict a somewhat different interval that
is still in agreement with current experimental data. The par-
ticle content of the model has three (5 + 10) supermultiplets
for the three generations of leptons and quarks, while the
Higgs sector consists of four supermultiplets (5+5) and one
24. 4 Computational setup On
the other hand, even for MINI-1 and MINI-2 no 5 σ discovery
can be expected. The situation looks more favorable for the
first and second generation squarks. All the predicted masses
can be excluded at the FCC-hh, whereas a 5 σ discovery will
be difficult, but potentially possible (see Fig. 19 in [141]). Even more favorable appear the prospects for gluino searches
at the FCC-hh. All three benchmark points may lead to a 5 σ
discovery (see Fig. 13 in [141]). On the other hand, chances
for chargino/neutralino searches are slim at the FCC-hh. The
Next-to LSP (NLSP) can only be accessed for M ˜χ0
1 ≲1 TeV
(see Fig. 21 in [141]), where all our benchmark points have
M ˜χ0
1 > 1 TeV. Taking into account that our three benchmark
points represent only the lower part of the possible mass
spectrum (with LSP masses of up to ∼1.5 TeV higher), we
conclude that even at the FCC-hh large parts of the possible
SUSY spectrum will remain elusive. W =
3
i=1
[ 1
2gu
i 10i10i Hi + gd
i 10i5i Hi ]
+ gu
23 102103H4
+ gd
23 10253 H4 + gd
32 10352 H4 + g f
2 H2 24 H2
+ g f
3 H3 24 H3 + gλ
3 (24)3. (36 (36) Discussion of the model with a more detailed descrip-
tion can be found in [15–17]. The non-degenerate and iso-
lated solutions to the vanishing of γ (1)
i
are: (gu
1)2 = 8
5 g2,
(gd
1)2 = 6
5 g2,
(gu
2)2 = (gu
3)2 = 4
5 g2,
(gd
2)2 = (gd
3)2 = 3
5 g2,
(gu
23)2 = 4
5 g2,
(gd
23)2 = (gd
32)2 = 3
5 g2,
(gλ)2 = 15
7 g2,
(g f
2 )2 = (g f
3 )2 = 1
2 g2,
(g f
1 )2 = 0,
(g f
4 )2 = 0. (37 6 The finite N = 1 supersymmetric SU(5) model We proceed now to the finite to all-orders SU(5) gauge the-
ory, where the reduction of couplings is restricted to the
third generation. An older examination of this specific finite
unified theory (FUT) was shown to be in agreement with
the experimental constraints at the time [30] and has pre-
dicted, almost five years before its discovery, the light Higgs (37) 3 3 Eur. Phys. J. C (2021) 81 :185 185
Page 10 of 20 185
Page 10 of 20 Fig. 3 Scatter plot for the finite N = 1 SU(5) model. Left: Mh as a function of M. Green points comply with B-physics constraints. Right: the
lightest Higgs mass theoretical uncertainty calculated with FeynHiggs 2.16.0 [43] Fig. 3 Scatter plot for the finite N = 1 SU(5) model. Left: Mh as a function of M. Green points comply with B-physics constraints. Right: the
lightest Higgs mass theoretical uncertainty calculated with FeynHiggs 2.16.0 [43] We have also the relation h = −MC, while the sum rules
lead to: symmetric spectrum (which is in agreement with all exist-
ing experimental constraints). In particular, very heavy col-
ored SUSY particles are favored (nearly independent of the
Mh uncertainty), in agreement with the non-observation of
those particles at the LHC [157]. m2
Hu + 2m2
10 = M2,
m2
Hd −2m2
10 = −M2
3 ,
m2
5 + 3m2
10 = 4M2
3
. (38) We choose three benchmarks, each featuring the LSP above
2100GeV,2400GeVand2900GeVrespectively.Again,they
are chosen from the low-mass region. Although the LSP can
be as heavy as ∼4000 GeV, but in such cases the production
cross sections even at the FCC-hh would be too small. The
input and output of SPheno 4.0.4 [115,116] can be found
in Tables 4 and 5 (with the notation as in Sect. 5). (38) Therefore, we only have two free parameters, namely m10
and M in the dimensionful sector. When SU(5) breaks down to the MSSM, a suitable rota-
tion in the Higgs sector [15,16,142–145], permits only a
pair of Higgs doublets (coupled mostly to the third family)
to remain light and acquire vev’s. Avoiding fast proton decay
is achieved with the usual doublet-triplet splitting, although
different from the one applied to the minimal SU(5) due
to the extended Higgs sector of the finite model. 6 The finite N = 1 supersymmetric SU(5) model Therefore,
below the GUT scale we get the MSSM where the third gen-
eration is given by the finiteness conditions while the first
two remain unrestricted. Concerning DM, the model exhibits a high relic abun-
dance for CDM, as the lightest neutralino (which is the LSP)
is again strongly Bino-like (see [44]). The CDM alternatives
proposed for the Minimal SU(5) model can also be applied
here. It should be noted that the bilinear R-parity violating
terms proposed in the previous section preserve finiteness, as
well. The expected production cross sections for various final
states are listed in Table 6. At 14 TeV HL-LHC none of
the Finite N = 1 SU(5) scenarios listed in Table 4 has a
SUSY production cross section above 0.01 fb, and thus will
(likely) remain unobservable. All superpartners are too heavy
to be produced in pairs. Also the heavy Higgs bosons are far
outside the reach of the HL-LHC [136,137]. Conditions set by finiteness do not restrict the renormal-
ization properties at low energies, so we are left with bound-
ary conditions on the gauge and Yukawa couplings (37),
the h = −MC relation and the soft scalar-mass sum rule
at MGUT. The quark masses mb(MZ) and mt are predicted
within 2σ and 3σ uncertainty, respectively of their experi-
mental values (see [44] for details). The only phenomeno-
logically viable option is to consider μ < 0, as shown in
[44,146–152]. At the FCC-hh the discovery prospects for the heavy
Higgs-boson spectrum is significantly better. With tan β ∼
50 the first two benchmark points, FUTSU5-1 and FUTSU5-
2, are well within the reach of the FCC-hh. The third point,
FUTSU5-3, however, with MA ∼16 TeV will be far outside
the reach of the FCC-hh. Prospects for detecting production
of squark pairs and squark-gluino pairs are also very dim
since their production cross section is also at the level of a few
fb. This is as a result of a heavy spectrum in this class of mod-
els (see [141] with the same Figures as discussed in Sect. 5). Concerning the stops, the lighter one might be accessible The scatter plot of the light Higgs boson mass is given
in Fig. 3 (left), while its theory uncertainty [43] is given in
Fig. 3 (right), with the same color coding as in Fig. 2. This
point-by-point uncertainty (calculated with FeynHiggs)
drops significantly (w.r.t. 123 6 The finite N = 1 supersymmetric SU(5) model Charginos and neutralinos will
remain unobservable due to the heavy LSP. As in the previ-
ous section, since only the lower part of the possible mass
spectrum has been considered (with LSP masses higher by
up to ∼1 TeV), we have to conclude that again large parts
of the possible mass spectra will not be observable at the
FCC-hh. q =
⎛
⎝
d u D
d u D
d u D
⎞
⎠∼(3, 3∗, 1),
qc =
⎛
⎝
dc dc dc
uc uc uc
Dc Dc Dc
⎞
⎠∼(3∗, 1, 3),
λ =
⎛
⎝
N Ec ν
E N c e
νc ec S
⎞
⎠∼(1, 3, 3∗)
(40) q =
⎛
⎝
d u D
d u D
d u D
⎞
⎠∼(3, 3∗, 1), qc =
⎛
⎝
dc dc dc
uc uc uc
Dc Dc Dc
⎞
⎠∼(3∗, 1, 3), 6 The finite N = 1 supersymmetric SU(5) model past analyses) to 0.65 −0.70 GeV. The scattered points come from the free parameter m10. Compared to our previous analyses [44,146–156], the
improved evaluation of Mh and its uncertainty prefer a heav-
ier (Higgs) spectrum and thus allows only a heavy super- 123 Page 11 of 20
185 Eur. Phys. J. C (2021) 81 :185 Page 11 of 20
185 185 Table 4 Finite N = 1 SU(5) predictions that are used as input to SPheno. Mass parameters are in GeV and rounded to 1 GeV
M1
M2
M3
|μ|
b
Au
Ad
Ae
tan β
m2
Q1,2
FUTSU5-1
2124
3815
8804
4825
8542
7282
7710
2961
49.9
81122
FUTSU5-2
2501
4473
10198
5508
10482
8493
9023
3536
50.1
93872
FUTSU5-3
3000
5340
11996
6673
23612
10086
10562
4243
49.9
110302
m2
Q3
m2
L1,2
m2
L3
m2
u1,2
m2
u3
m2
d1,2
m2
d3
m2
e1,2
m2
e3
FUTSU5-1
66342
38692
31202
76842
50532
76352
41772
30842
22412
FUTSU5-2
76692
45212
37472
88872
68652
88262
68932
36022
25512
FUTSU5-3
91162
53552
37452
104192
81702
103622
77082
43292
34032
Table 5 Masses for each benchmark of the Finite N = 1 SU(5) (in TeV)
MH
MA
MH±
M ˜g
M ˜χ0
1
M ˜χ0
2
M ˜χ0
3
M ˜χ0
4
M ˜χ±
1
M ˜χ±
2
FUTSU5-1
5.688
5.688
5.688
8.966
2.103
3.917
4.829
4.832
3.917
4.833
FUTSU5-2
7.039
7.039
7.086
10.380
2.476
4.592
5.515
5.518
4.592
5.519
FUTSU5-3
16.382
16.382
16.401
12.210
2.972
5.484
6.688
6.691
5.484
6.691
M˜e1,2
M˜ν1,2
M˜τ
M˜ντ
M ˜d1,2
M˜u1,2
M ˜b1
M ˜b2
M˜t1
M˜t2
FUTSU5-1
3.102
3.907
2.205
3.137
7.839
7.888
6.102
6.817
6.099
6.821
FUTSU5-2
3.623
4.566
2.517
3.768
9.059
9.119
7.113
7.877
7.032
7.881
FUTSU5-3
4.334
5.418
3.426
3.834
10.635
10.699
8.000
9.387
8.401
9.390 Demandingthevanishingofthegaugeone-loopβ-function,i.e. b = 0, we are led to the choice n f = 3. Phenomenological
reasons lead to the choice of the SU(3)C ×SU(3)L×SU(3)R
model, discussed in Ref. [158], while a detailed discussion of
the general well known example can be found in [159–162]. The leptons and quarks transform as: in FUTSU5-1. For the squarks of the first two generations
the prospects of testing the model are somewhat better. All
three benchmark models could possibly be excluded at the
2 σ level, but no discovery at the 5 σ can be expected. The
same holds for the gluino. 7 The finite SU(N)3 model (40) We proceed now to a FUT based on a product gauge
group. Consider an N = 1 SUSY theory with SU(N)1 ×
SU(N)2×· · ·×SU(N)k having n f families transforming as
(N, N ∗, 1, . . . , 1)+(1, N, N ∗, . . . , 1)+· · ·+(N ∗, 1, 1, . . . , N). Then, the first order coefficient of the β-function, for each
SU(N) group is: where D are down-type quarks acquiring masses close to
MGUT. A cyclic Z3 symmetry is imposed on the multiplets
to achieve equal gauge couplings at the GUT scale and in that
case the vanishing of the first-order β-function is satisfied. where D are down-type quarks acquiring masses close to
MGUT. A cyclic Z3 symmetry is imposed on the multiplets
to achieve equal gauge couplings at the GUT scale and in that
case the vanishing of the first-order β-function is satisfied. Continuing to the vanishing of the anomalous dimension of
all the fields (see Eq. (20)), we note that there are two trilinear
invariant terms in the superpotential, namely: b =
−11
3 + 2
3
N+n f
2
3 + 1
3
1
2
2N = −3N+n f N. g
Continuing to the vanishing of the anomalous dimension of
all the fields (see Eq. (20)), we note that there are two trilinear
invariant terms in the superpotential, namely: (39) 12 3 Eur. Phys. J. C (2021) 81 :185 185
Page 12 of 20 Table 6 Expected production cross sections (in fb) for SUSY particles in the FUTSU5 scenarios
Scenarios
√s
FUTSU5-1
100 TeV
FUTSU5-2
100 TeV
FUTSU5-3
100 TeV
Scenarios
√s
FUTSU5-1
100 TeV
FUTSU5-2
100 TeV
FUTSU5-3
100 TeV
˜χ0
2 ˜χ0
3
0.01
0.01
˜νi ˜ν∗
j
0.02
0.01
0.01
˜χ0
3 ˜χ0
4
0.03
0.01
˜ui ˜χ−
1 , ˜di ˜χ+
1 + h.c. 0.15
0.06
0.02
˜χ0
2 ˜χ+
1
0.17
0.08
0.03
˜qi ˜χ0
1 , ˜q∗
i ˜χ0
1
0.08
0.03
0.01
˜χ0
3 ˜χ+
2
0.05
0.03
0.01
˜qi ˜χ0
2 , ˜q∗
i ˜χ0
2
0.08
0.03
0.01
˜χ0
4 ˜χ+
2
0.05
0.03
0.01
˜νi ˜e∗
j, ˜ν∗
i ˜e j
0.09
0.04
0.01
˜g ˜g
0.20
0.05
0.01
Hb ¯b
2.76
0.85
˜g ˜χ0
1
0.03
0.01
Ab ¯b
2.73
0.84
˜g ˜χ0
2
0.03
0.01
H+b¯t + h.c. 5 [163,164] and Refs. therein discuss in detail the spontaneous breaking
of SU(3)3. 7 The finite SU(N)3 model 1.32
0.42
˜g ˜χ+
1
0.07
0.03
0.01
H+W −
0.38
0.12
˜qi ˜q j, ˜qi ˜q∗
j
3.70
1.51
0.53
H Z
0.09
0.03
˜χ+
1 ˜χ−
1
0.10
0.05
0.02
AZ
0.09
0.03
˜χ+
2 ˜χ−
2
0.03
0.02
0.01
˜ei ˜e∗
j
0.23
0.13
0.05
˜qi ˜g, ˜q∗
i ˜g
2.26
0.75
0.20 Table 6 Expected production cross sections (in fb) for SUSY particles in the FUTSU5 scenarios f Tr(λqcq) + 1
6 f ′ ϵi jkϵabc
× (λiaλ jbλkc + qc
iaqc
jbqc
kc + qiaq jbqkc),
(41) to the third generation. The FUT breaking leaves its mark in
the form of Eq. (43), i.e. boundary conditions on the gauge
and Yukawa couplings, the relation among the soft trilinear
coupling, the corresponding Yukawa coupling and the unified
gaugino mass and finally the soft scalar mass sum rule at
MGUT. In this specific model the sum rule takes the form: (41) with f and f ′ thecorrespondingYukawacouplings.Thesuper-
fields ( ˜N, ˜N c) obtain vev’s and provide masses to leptons and
quarks m2
Hu + m2
˜tc + m2
˜q = M2 = m2
Hd + m2
˜bc + m2
˜q. (45) (45) md = f ⟨˜N⟩,
mu = f ⟨˜N c⟩,
me = f ′⟨˜N⟩,
mν = f ′⟨˜N c⟩. (42) The model is finite to all-orders if the solution of Eq. (43)
is both isolated and unique. Then, f ′ = 0 and we have the
relations Having three families, 11 f couplings and 10 f ′ couplings
are present in the most general superpotential. Demanding
the vanishing of all superfield anomalous dimensions, 9 con-
ditions are imposed Having three families, 11 f couplings and 10 f ′ couplings
are present in the most general superpotential. Demanding
the vanishing of all superfield anomalous dimensions, 9 con-
ditions are imposed f 2 = f 2
111 = f 2
222 = f 2
333 = 16
9 g2. (46) (46) Since all f ′ vanish, at one-loop order, the lepton masses
vanish. Since these masses, even radiatively, cannot be pro-
duced because of the finiteness conditions, we are faced
with a problem which needs further study. If the solution of
Eq. (43) is unique but not isolated (i.e. parametric), we can
have non zero f ′ leading to non-vanishing lepton masses and
at the same time achieving two-loop finiteness. 7 The finite SU(N)3 model In that case
the set of conditions restricting the Yukawa couplings read: Since all f ′ vanish, at one-loop order, the lepton masses
vanish. Since these masses, even radiatively, cannot be pro-
duced because of the finiteness conditions, we are faced
with a problem which needs further study. If the solution of
Eq. (43) is unique but not isolated (i.e. parametric), we can
have non zero f ′ leading to non-vanishing lepton masses and
at the same time achieving two-loop finiteness. In that case
the set of conditions restricting the Yukawa couplings read:
j,k
fi jk( fljk)∗+ 2
3
j,k
f ′
i jk( f ′
ljk)∗= 16
9 g2δil,
(43) (43) where where fi jk = f jki = fki j,
f ′
i jk = f ′
jki = f ′
ki j = f ′
ikj = f ′
kji = f ′
jik. (44) f 2 = r
16
9
g2,
f ′2 = (1 −r)
8
3
g2,
(47) (47) The masses of leptons and quarks are acquired from the vev’s
of the scalar parts of the superfields ˜N1,2,3 and ˜N c
1,2,3. 3
5 where r parametrises the different solutions and as such is a
free parameter. It should be noted that we use the sum rule
as boundary condition for the soft scalar masses. , ,
At MGUT the SU(3)3 FUT breaks5 to the MSSM, where as
was already mentioned, both Higgs doublets couple mostly In our analysis we consider the two-loop finite version of
the model, where again below MGUT we get the MSSM. We 3 Eur. Phys. J. C (2021) 81 :185 Page 13 of 20
185 Page 13 of 20
185 185 Fig. 4 Scatter plot for the finite N = 1 SU(3)3 model. Left: Mh as a function of M. Right: the Higgs mass theoretical uncertainty [43] Fig. 4 Scatter plot for the finite N = 1 SU(3)3 model. Left: Mh as a function of M. Right: the Higgs mass theoretical uncertainty [43] in the previous sections can be applied in this case as well,
preserving finiteness. take into account two new thresholds for the masses of the
new particles at ∼1013 GeV and ∼1014 GeV resulting in a
wider phenomenologically viable parameter space [156]. 7 The finite SU(N)3 model It should be noted that in this model the scale of the heavy
Higgs bosons does not vary monotonously with M ˜χ0
1 , as in
the previously considered models. This can be understood
as follows. The Higgs bosons masses are determined by a
combination of the sum rule at the unification scale, and the
requirement of successful electroweak symmetry breaking
at the low scale. Like in the finite scenario of the previous
section, there are no direct relations between the soft scalar
masses and the unified gaugino mass, but they are related
through the corresponding sum rule and thus vary corre-
latedly, a fact that makes the dependence on the boundary
values more restrictive. Furthermore (and even more impor-
tantly), the fact that we took into account the two thresholds at
∼1013 GeV and ∼1014 GeV (as mentioned above), allows
the new particles, mainly the Higgsinos of the two other fam-
ilies (that were considered decoupled at the unification scale
in previous analyses) and the down-like exotic quarks (in a
lower degree), to affect the running of the (soft) RGEs in a
non-negligible way. Thus, since at low energies the heavy
Higgs masses depend mainly on the values of m2
Hu, m2
Hd, |μ|
and tan β, they are substantially less connected to M ˜χ0
1 than
in the other models, leading to a different exclusion potential,
as will be discussed in the following. Looking for the values of the parameter r which com-
ply with the experimental limits, we find that both the top
and bottom masses are in the experimental range (within
2σ) for the same value of r between 0.65 and 0.80 (we sin-
gled out the μ < 0 case as the most promising). The inclu-
sion of the above-mentioned thresholds gives an important
improvement on the top mass from past versions of the model
[158,165–167]. Figure 4 (left) shows the scatter plot of the light Higgs
boson mass (green points satisfy the B-physics constraints),
while the point-by-point calculated theoretical uncertainty
is presented in Fig. 4 (right). The scattered points are due
to the fact that we vary five parameters, namely r and four
of the parameters that form the sum rule. The uncertainty
is found in the range between 0.6 GeV and 1.0 GeV. All
constraints regarding quark masses, the light Higgs boson
mass and B-physics are satisfied, rendering the model very
successful. 7 The finite SU(N)3 model The prediction of the SUSY spectrum results in
relatively heavy particles, in full agreement with the current
experimental searches. Again, we choose three benchmarks, each featuring the
LSP above 1500 GeV, 2000 GeV and 2400 GeV respectively
(but the LSP can go as high as ∼4100 GeV, again with too
small cross sections). The input and output of SPheno 4.0.4
[115,116] can be found in Tables 7 and 8 respectively (with
the notation as in Sect. 5). Scenarios of finite SU(3)3 are beyond the reach of the
HL-LHC. Not only superpartners are too heavy, but also
heavy Higgs bosons with a mass scale of ∼7 TeV cannot be
detected at the HL-LHC. At 100 TeV collider (see Table 9),
on the other hand, all three benchmark points are well within
the reach of the H/A →τ +τ −as well as the H± →τντ, tb
searches [139,140], despite the slightly smaller values of
tan β ∼45. This is particularly because of the different
dependence of the heavy Higgs-boson mass scale on M ˜χ0
1 , as
discussed above. However, we have checked that MA can go
up to to ∼11 TeV, and thus the heaviest part of the possible
spectrum would escape the heavy Higgs-boson searches at
the FCC-hh. Like the previous models, the CDM relic density fails to
comply with the experimental bounds (see Eq. (30)). The
lightest neutralino is the LSP and considered as a CDM can-
didate, but its relic density does not go below 0.15, since it
is strongly Bino-like and would require a lower scale of the
particlespectrum.ItshouldbenotedthatiftheB-physicscon-
straints allowed for a unified gaugino mass ∼0.5 TeV
lower, then agreement with the CDM bounds as well could
be achieved (see [44]). However, the alternatives proposed 12 3 3 Eur. Phys. J. C (2021) 81 :185 185
Page 14 of 20 Table 7 Finite N = 1 SU(3)3 predictions that are used as input to SPheno. 8 The reduced MSSM Interesting are also the prospects for production of squark
pairs and squark-gluino, which can reach ∼20 fb for the
FSU33-1 case, going down to a few fb for FSU33-2 and
FSU33-3 scenarios. The lightest squarks decay almost exclu-
sively to the third generation quark and chargino/neutralino,
while gluino enjoys many possible decay channels to quark-
squark pairs each one with branching fraction of the order of
a percent, with the biggest one ∼20% to t ˜t1 + h.c.. We finish our phenomenological analyses with the applica-
tion of the method of coupling reduction to a version of the
MSSM, where a covering GUT is assumed. The original par-
tial reduction can be found in Refs. [168,169] where only
the third fermionic generation is considered. Following this
restriction, the superpotential reads: We briefly discuss the SUSY discovery potential at the
FCC-hh, referring agian to [141] with the same Figures as
discussed in Sect. 5. Stops in FSU33-1 and FSU33-2 can be
tested at the FCC-hh, while the masses turn out to be too
heavy in FSU33-3. The situation is better for scalar quarks,
where all three scenarios can be tested, but will not allow for
a 5 σ discovery. Even more favorable are the prospects for
gluino. Possibly all three scenarios can be tested at the 5 σ
level. As in the previous scenario, the charginos and neutrali-
nos will not be accessible, due to the too heavy LSP. Keeping
in mind that only the lower part of possible mass spectrum
is represented by the three benchmarks (with the LSP up to
∼1.5 TeV heavier), we conclude that as before large parts of
the parameter space will not be testable at the FCC-hh. The
only partial exception here is the Higgs-boson sector, where
only the the part with the highest possible Higgs-boson mass
spectra would escape the FCC-hh searches. W = Yt H2Qtc + YbH1Qbc + Yτ H1Lτ c + μH1H2 ,
(48) (48) where Yt,b,τ refer only to the third family, and the SSB
Lagrangian is given by by (with the trilinear couplings ht,b,τ
for the third family) −LSSB =
φ
m2
φ ˆφ∗ˆφ +
m2
3 ˆH1 ˆH2 +
3
i=1
1
2 Miλiλi + h.c
+
ht ˆH2 ˆQ ˆtc + hb ˆH1 ˆQ ˆbc + hτ ˆH1 ˆL ˆτ c + h.c.
. 7 The finite SU(N)3 model Mass parameters are in GeV and rounded to 1 GeV
M1
M2
M3
|μ|
b
Au
Ad
Ae
tan β
m2
Q1,2
FSU33-1
1522
2758
6369
6138
10022
4520
4413
1645
46.2
55742
FSU33-2
2070
3722
8330
7129
10832
5841
5734
2357
45.5
72552
FSU33-3
2500
4484
10016
6790
9722
7205
7110
2674
49.7
8709
m2
Q3
m2
L1,2
m2
L3
m2
u1,2
m2
u3
m2
d1,2
m2
d3
m2
e1,2
m2
e3
FSU33-1
47052
23822
37542
52342
55482
51972
70432
15582
30952
FSU33-2
72552
31362
41312
67492
72252
67452
85232
22382
33422
FSU33-3
90742
38312
54832
81522
72072
25582
86002
25072
40002
Table 8 Masses for each benchmark of the finite N = 1 SU(3)3 (in TeV)
MH
MA
MH±
M ˜g
M ˜χ0
1
M ˜χ0
2
M ˜χ0
3
M ˜χ0
4
M ˜χ±
1
M ˜χ±
2
FSU33-1
7.029
7.029
7.028
6.526
1.506
2.840
6.108
6.109
2.839
6.109
FSU33-2
6.484
6.484
6.431
8.561
2.041
3.817
7.092
7.093
3.817
7.093
FSU33-3
6.539
6.539
6.590
10.159
2.473
4.598
6.780
6.781
4.598
6.781
M˜e1,2
M˜ν1,2
M˜τ
M˜ντ
M ˜d1,2
M˜u1,2
M ˜b1
M ˜b2
M˜t1
M˜t2
FSU33-1
2.416
2.415
1.578
2.414
5.375
5.411
4.913
5.375
4.912
5.411
FSU33-2
3.188
3.187
2.269
3.186
7.026
7.029
6.006
7.026
6.005
7.029
FSU33-3
3.883
3.882
2.540
3.882
8.334
8.397
7.227
8.334
7.214
7.409 8 The reduced MSSM where D, Nt and Nb are known quantities which can be found
in Ref. [171]. Proceeding to the the SSB Lagrangian, Eq. (49), and the
dimension-one parameters, i.e the trilinear couplings ht,b,τ,
we first reduce ht,b and we get We assume that the ratios of the top and bottom Yukawa
to the strong coupling are constant at the GUT scale, i.e. they
have negligible scale dependence, hi = ciYi M3 = ciGi M3g3,
where ci = −1 i = t, b, hi = ciYi M3 = ciGi M3g3,
where ci = −1 i = t, b, d
dg3
Y 2
t,b
g2
3
= 0. d
dg3
Y 2
t,b
g2
3
= 0. where M3 is the gluino mass. Adding the corrections from
the gauge and the tau couplings we have Then,includingthecorrectionsfromthe SU(2),U(1)andtau
couplings, at the GUT scale, the coefficients G2
t,b become: Then,includingthecorrectionsfromthe SU(2),U(1)andtau
couplings, at the GUT scale, the coefficients G2
t,b become: ct = −AA Abb + AtbBB
Abt Atb −Abb Att
,
cb = −AA Abt + Att BB
Abt Atb −Abb Att
. G2
t = 1
3 + 71
525ρ1 + 3
7ρ2 + 1
35ρτ,
G2
b = 1
3 + 29
525ρ1 + 3
7ρ2 −6
35ρτ,
(50) Again, Att, Abb and Atb can be found in Ref. [171]. Again, Att, Abb and Atb can be found in Ref. [171]. We end up with the soft scalar masses m2
φ of the SSB We end up with the soft scalar masses m2
φ of the SSB
Lagrangian Assuming the relations m2
c M2 (i We end up with the soft scalar masses m2
φ of the SSB
Lagrangian. Assuming the relations m2
i
= ci M2
3 (i
=
Q, u, d, Hu, Hd), and adding the corrections from the gauge,
the tau couplings and hτ, we get (50) (50) where where cQ = −cQNum
Dm
,
cu = −1
3
cuNum
Dm
,
cd = −cdNum
Dm
,
cHu = −2
3
cHuNum
Dm
,
cHd = −cHdNum
Dm
,
(52) ρ1,2 =
g2
1,2
g2
3
= α1,2
α3
,
ρτ = g2
τ
g2
3
=
Y 2
τ
4π
α3
. (51) (51) (52) We shall treat Eq. (50) as boundary conditions at the GUT
scale. where Dm,cQNum,cuNum,cdNum,cHuNum,cHdNum andthecom-
plete analysis are again given in Ref. [171]. 8 The reduced MSSM (49)
(49) We start with the dimensionless sector and consider
initially the top and bottom Yuakwa couplings and the
strong gauge coupling. The rest of the couplings will be
treated as corrections. If Y 2
(t,b)/(4π) ≡α(t,b), the REs and 12 3 123 Page 15 of 20
185 Eur. Phys. J. C (2021) 81 :185 Page 15 of 20
185 185 Table 9 Expected production cross sections (in fb) for SUSY particles in the FSU33 scenarios
Scenarios √s
FSU33-1
100 TeV
FSU33-2
100 TeV
FSU33-3
100 TeV
Scenarios √s
FSU33-1
100 TeV
FSU33-2
100 TeV
FSU33-3
100 TeV
˜χ0
1 ˜χ0
1
0.04
0.01
0.01
˜qi ˜g, ˜q∗
i ˜g
22.12
3.71
1.05
˜χ0
2 ˜χ0
2
0.04
0.01
˜νi ˜ν∗
j
0.10
0.03
0.01
˜χ0
2 ˜χ+
1
0.58
0.16
0.07
˜ui ˜χ−
1 , ˜di ˜χ+
1 + h.c. 1.22
0.25
0.08
˜χ0
3 ˜χ+
2
0.02
0.01
0.01
˜qi ˜χ0
1 , ˜q∗
i ˜χ0
1
0.55
0.13
0.05
˜χ0
4 ˜χ+
2
0.02
0.01
0.01
˜qi ˜χ0
2 , ˜q∗
i ˜χ0
2
0.60
0.13
0.04
˜g ˜g
2.61
0.30
0.07
˜νi ˜e∗
j, ˜ν∗
i ˜e j
0.36
0.12
0.04
˜g ˜χ0
1
0.20
0.05
0.02
Hb ¯b
0.71
1.23
1.19
˜g ˜χ0
2
0.20
0.04
0.01
Ab ¯b
0.72
1.23
1.18
˜g ˜χ+
1
0.42
0.09
0.03
H+b¯t + h.c. 0.37
0.75
0.58
˜qi ˜q j, ˜qi ˜q∗
j
25.09
6.09
2.25
H+W −
0.10
0.25
0.19
˜χ+
1 ˜χ−
1
0.37
0.10
0.04
H Z
0.02
0.04
0.04
˜ei ˜e∗
j
0.39
0.12
0.06
AZ
0.02
0.04
0.04 Table 9 Expected production cross sections (in fb) for SUSY particles in the FSU33 scenarios the Yukawa RGEs give Then, the two-loop coefficients, Ji, including the corrections
from the gauge and the tau Yukawa couplings, are: the Yukawa RGEs give αi = G2
i α3,
where G2
i = 1
3,
i = t, b. J 2
t = 1
4π
Nt
D ,
J 2
b = 1
4π
Nb
5D , If the tau Yukawa is included in the reduction, the corre-
sponding G2 coefficient for tau turns negative [170], explain-
ing why this coupling is treated also as a correction (i.e. it
cannot be reduced). where D, Nt and Nb are known quantities which can be found
in Ref. [171]. 8 The reduced MSSM These values do
not obey any soft scalar mass sum rule. Going to the two-loop level, we assume that the correc-
tions take the following form: If only the reduced system was used, i.e. the strong, top
and bottom Yukawa couplings as well as the ht and hb, the αi = G2
i α3 + J 2
i α2
3,
i = t, b. 12 3 Eur. Phys. J. C (2021) 81 :185 185
Page 16 of 20 185
Page 16 of 20 Fig. 5 Left: the lightest Higgs boson mass, Mh in the Reduced MSSM. The green points is the full model prediction. Right: the lightest Higgs
mass theoretical uncertainty [43] Fig. 5 Left: the lightest Higgs boson mass, Mh in the Reduced MSSM. The green points is the full model prediction. Right: the lightest Higgs
mass theoretical uncertainty [43] coefficients turn to be coefficients turn to be tively. The input of SPheno 4.0.4 [115,116] can be found
in Table 10 (notation as in Sect. 5). cQ = cu = cd = 2
3,
cHu = cHd = −1/3, Table 11 shows the resulting masses of Higgs bosons and
some of the lightest SUSY particles. The lightest neutralino
(LSP) is Wino-like, as imposed by the Hisano–Shifman rela-
tion, Eq. (12), and thus the CDM relic density is below the
boundaries of Eq. (30). This renders this model viable if
Eq. (30) is applied only as an upper limit and additional
sources of CDM are allowed. An additional DM component
could be, e.g., a SUSY axion [172], which would then bring
the total DM density into agreement with the Planck mea-
surement of CDMh2. This is in contrast to the other three
models discussed above. which clearly obey the sum rules which clearly obey the sum rules m2
Q + m2
u + m2
Hu
M2
3
= cQ + cu + cHu = 1,
m2
Q + m2
d + m2
Hd
M2
3
= cQ + cd + cHd = 1. (53) (53) There is an essential point for the gaugino masses that
shouldbementioned.TheapplicationoftheHisano–Shifman
relation, Eq. (12), is made for each gaugino mass as a bound-
ary condition with unified gauge coupling at MGUT. Then,
at one-loop level, the gaugino mass depends on the one-loop
coefficient of the corresponding β-function and an arbitrary
mass M0,
Mi = bi M0. 8 The reduced MSSM This fact permits, with a suitable
choice of M0, to have the gluino mass equal to the unified
gaugino mass, while the gauginos masses of the other two
gauge groups are given by the gluino mass multiplied by the
ratio of the appropriate one-loop β coefficient. In addition, there is one more point that should be stressed. We find MA ≲1.5 TeV (for large values of tan β as in the
other models), values substantially lower than in the previ-
ously considered models. This can be understood as follows. In this model, we have direct relations between the soft scalar
masses and the unified gaugino mass, which receive correc-
tions from the two gauge couplings g1 and g2 and the Yukawa
coupling of the τ lepton. As mentioned above, in the absence
of these corrections the relations obey the soft scalar mass
sum rule. However, unlike all the previous models, these cor-
rections make the sum rule only approximate. Thus, these
unique boundary conditions result in very low values for the
masses of the heavy Higgs bosons (even compared to the
minimal SU(5) case presented above, which also exhibits
direct relations which however obey the sum rule). A rela-
tively light spectrum is also favored by the prediction for the
light CP-even Higgs boson mass, which turns out to be rel-
atively high in this model and does not allow us to consider
heavier spectra. Thus, in this model, contrary to the models
analyzed before, because of the large tan β ∼45 found here,
the physical mass of the pseudoscalar Higgs boson, MA, is
excluded by the searches H/A →ττ at ATLAS with 139/fb
[91] for all three benchmarks. One could try considering a
heavierspectrum,inwhichwewouldhave MA ≳1900 GeV, For our analysis we choose the unification scale to apply
the corrections to all these RGI relations. The full discussion
on the selection of the free parameters of the model can be
found in [44]. In total, we vary ρτ, ρhτ , M and μ. This results
in the scattered points of the next figure. The model’s predictions for the bottom and top mass lie
within 2σ of Eq. (25). The scatter plot of the light Higgs
boson mass Mh is shown in Fig. 5 (left),while the theory
uncertainty given in Fig. 5 (right) has dropped below 1 GeV. 123 8 The reduced MSSM Particularly, it would be above 128
GeV, a value that is clearly excluded, especially given the
improved (much smaller) uncertainty calculated by the new
FeynHiggs code). Thus, the current version of this model
has been ruled out experimentally. Consequently, we do not
show any SUSY or Higgs production cross sections. new results have been obtained using the updated Higgs-
boson mass calculation of FeynHiggs. In each case bench-
mark points in the low-mass regions have been chosen for
which the SPheno code has been used to calculate the spec-
trum of SUSY particles and their decay modes. Finally the
MadGraph event generator was used to compute the pro-
duction cross sections of relevant final states at the 14 TeV
(HL-)LHC and 100 TeV FCC-hh colliders. The first three (unified) models were found to be in com-
fortable agreement with LHC measurements and searches,
with the exception of the bottom quark mass in the Reduced
Minimal SU(5), for which agreement with measurements
can be achieved only at the 4σ level. In addition it was
found that all models predict relatively heavy spectra, which
evade largely the detection in the HL-LHC. We found one
noticeable exception. The reduced MSSM features a rela-
tively light heavy Higgs-boson mass spectrum. Together with
the relatively high value of tan β this spectrum is excluded
already by current searches at ATLAS and CMS for in the
pp →H/A →τ +τ −mode. We also analyzed the accessi-
bility of the SUSY and heavy Higgs spectrum at the FCC-hh
with √s = 100 TeV. We found that the lower parts of the
parameter space will be testable at the 2 σ level, with only
an even smaller part discoverable at the 5 σ level. However,
the heavier parts of the possible SUSY spectra will remain
elusive even at the FCC-hh. One exception here is the heavy 8 The reduced MSSM The Higgs mass predicted by the model lies perfectly in the
experimentally measured range. The Mh limits set a limit on the low-energy supersym-
metric masses, which we briefly discuss. The three selected
benchmarks correspond to DR pseudoscalar Higgs boson
masses above 1900 GeV, 1950 GeV and 2000 GeV respec- 123 Page 17 of 20
185 Eur. Phys. J. C (2021) 81 :185 Page 17 of 20
185 185 Table 10 Reduced MSSM predictions that are used as input to SPheno mass parameters are in GeV and rounded to 1 GeV
M1
M2
M3
|μ|
b
Au
Ad
Ae
tan β
m2
Q1,2
RMSSM-1
3711
1014
7109
4897
2842
5274
5750
20
44.9
59852
RMSSM-2
3792
1035
7249
4983
2942
5381
5871
557
44.6
61032
RMSSM-3
3829
1045
7313
5012
2982
5427
5942
420
45.3
61612
m2
Q3
m2
L1,2
m2
L3
m2
u1,2
m2
u3
m2
d1,2
m2
d3
m2
e1,2
m2
e3
RMSSM-1
55452
21062
20692
62772
53862
59892
51142
30512
44912
RMSSM-2
56562
21222
22902
63852
54762
61102
52192
31532
41812
RMSSM-3
57082
21062
22792
64272
55062
61722
52692
32292
35042
Table 11 Masses for each benchmark of the reduced MSSM (in TeV)
MH
MA
MH±
M ˜g
M ˜χ0
1
M ˜χ0
2
M ˜χ0
3
M ˜χ0
4
M ˜χ±
1
M ˜χ±
2
RMSSM-1
1.393
1.393
1.387
7.253
1.075
3.662
4.889
4.891
1.075
4.890
RMSSM-2
1.417
1.417
1.414
7.394
1.098
3.741
4.975
4.976
1.098
4.976
RMSSM-3
1.491
1.491
1.492
7.459
1.109
3.776
5.003
5.004
1.108
5.004
M˜e1,2
M˜ν1,2
M˜τ
M˜ντ
M ˜d1,2
M˜u1,2
M ˜b1
M ˜b2
M˜t1
M˜t2
RMSSM-1
2.124
2.123
2.078
2.079
6.189
6.202
5.307
5.715
5.509
5.731
RMSSM-2
2.297
2.139
2.140
2.139
6.314
6.324
5.414
5.828
5.602
5.842
RMSSM-3
2.280
2.123
2.125
2.123
6.376
6.382
5.465
5.881
5.635
5.894 but in that case the light Higgs mass would be well above
its acceptable region. Particularly, it would be above 128
GeV, a value that is clearly excluded, especially given the
improved (much smaller) uncertainty calculated by the new
FeynHiggs code). Thus, the current version of this model
has been ruled out experimentally. Consequently, we do not
show any SUSY or Higgs production cross sections. but in that case the light Higgs mass would be well above
its acceptable region. 9 Conclusions The reduction of couplings scheme consists in searching for
RGE relations among parameters of a renormalizable the-
ory that hold to all orders in perturbation theory. In cer-
tain N = 1 theories such a reduction of couplings indeed
appears to be theoretically realised and therefore it devel-
oped to a powerful tool able to reduce the parameters and
increasethepredictivityof thesetheories. Inthepresent paper
first we briefly reviewed the ideas concerning the reduction
of couplings of renormalizable theories and the theoretical
methods which have been developed to confront the prob-
lem. Then we turned to the question of testing experimen-
tally the idea of reduction of couplings. Four specific mod-
els, namely the reduced minimal N = 1 SU(5), the all-loop
finite N = 1 SU(5), the two-loop Finite N = 1 SU(3)3
and the Reduced MSSM, have been considered for which 12 3 3 Eur. Phys. J. C (2021) 81 :185 185
Page 18 of 20 185
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vide a link to the Creative Commons licence, and indicate if changes
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English
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Handshakes and Fights: The Regulatory Interplay of RNA-Binding Proteins
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Frontiers in molecular biosciences
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MINI REVIEW
published: 29 September 2017
doi: 10.3389/fmolb.2017.00067 MINI REVIEW published: 29 September 2017
doi: 10.3389/fmolb.2017.00067 Handshakes and Fights: The
Regulatory Interplay of RNA-Binding
Proteins Erik Dassi*
Centre for Integrative Biology, University of Trento, Trento, Italy Erik Dassi* Centre for Integrative Biology, University of Trento, Trento, Italy What drives the flow of signals controlling the outcome of post-transcriptional regulation
of gene expression? This regulatory layer, presiding to processes ranging from splicing
to mRNA stability and localization, is a key determinant of protein levels and thus cell
phenotypes. RNA-binding proteins (RBPs) form a remarkable army of post-transcriptional
regulators, strong of more than 1,500 genes implementing this expression fine-tuning
plan and implicated in both cell physiology and pathology. RBPs can bind and control
a wide array of RNA targets. This sheer amount of interactions form complex regulatory
networks (PTRNs) where the action of individual RBPs cannot be easily untangled from
each other. While past studies have mostly focused on the action of individual RBPs
on their targets, we are now observing an increasing amount of evidence describing
the occurrence of interactions between RBPs, defining how common target RNAs are
regulated. This suggests that the flow of signals in PTRNs is driven by the intertwined
contribution of multiple RBPs, concurrently acting on each of their targets. Understanding
how RBPs cooperate and compete is thus of paramount importance to chart the wiring of
PTRNs and their impact on cell phenotypes. Here we review the current knowledge about
patterns of RBP interaction and attempt at describing their general principles. We also
discuss future directions which should be taken to reach a comprehensive understanding
of this fundamental aspect of gene expression regulation. Edited by:
Ulf Andersson Ørom,
Max Planck Institute for Molecular
Genetics, Germany Reviewed by:
Igor Ulitsky,
Whitehead Institute for Biomedical
Research, United States
Barbara Bardoni,
Centre National de la Recherche
Scientifique UMR7275 Institut de
Pharmacologie Moléculaire et
Cellulaire, France *Correspondence:
Erik Dassi
erik.dassi@unitn.it Keywords: RNA-binding proteins, post-transcriptional regulation, regulatory networks, regulatory elements,
cooperation, competition, autoregulation Specialty section: Specialty section:
This article was submitted to
Protein and RNA Networks,
a section of the journal
Frontiers in Molecular Biosciences The regulatory interplay phenomenon occurs when two RNA-binding proteins (RBPs) concur to
regulate a common RNA target. Their combined action thus defines the steady-state levels of this
RNA, its availability for translation (mRNA) or ability to exert its role (non-coding RNA). The
two interacting RBPs can either be synergistic (cooperate), aim for a different regulatory outcome
(compete), or regulate one another to tune their action (mutual control). Received: 29 August 2017
Accepted: 20 September 2017
Published: 29 September 2017 Received: 29 August 2017
Accepted: 20 September 2017
Published: 29 September 2017 RNA-binding proteins are major players in post-transcriptional control of gene expression
(PTR), the regulatory layer responsible for fine-tuning protein synthesis and thus determining
cell phenotypes (Gerstberger et al., 2014). PTR is involved in a wide range of processes, from
splicing and alternative polyadenylation to mRNA localization, storage, and degradation, ultimately
leading to translational control (Glisovic et al., 2008). The action exerted by PTR has profound
implications for both cell physiology and pathologies such as cancer (Wurth and Gebauer, 2015)
and neurodegenerative diseases (De Conti et al., 2017). COOPERATIVE TARGET REGULATION The interplay of two RNA-binding proteins having a common
regulatory goal, and whose action is synergistic, is said to
be cooperative. Occurrences of this phenomenon have been
observed at the various steps of post-transcriptional regulation
and can be mediated by the physical interaction of the RBPs Abbreviations: RBP, RNA-binding protein; PTR, post-transcriptional regulation
of gene expression; PTRN, post-transcriptional regulatory network; ADAR,
adenosine deaminase, RNA specific; ADARB1, adenosine deaminase, RNA specific
B1; AGO2, argonaute 2, RISC catalytic component; ANXA2R, annexin A2
receptor; BRF1, RNA polymerase III transcription initiation factor subunit;
CAPRIN1, cell cycle associated protein 1; CCND1, cyclin D1; CDKN1A, cyclin
dependent kinase inhibitor 1; CELF1/2, CUGBP Elav-like family member 1/2;
CHRNA1, cholinergic receptor nicotinic alpha 1 subunit; CSTF3, cleavage
stimulation factor subunit 3; CPEB1, cytoplasmic polyadenylation element
binding protein 1; DDX3X, DEAD-box helicase 3, X-linked; DDX6, DEAD-
box helicase 6; DHX9, DExH-box helicase 9; EIF4EBP2, eukaryotic translation
initiation factor 4E binding protein 2; ELAVL1, ELAV like RNA binding
protein 1; FUS, FUS RNA binding protein; HDAC6, histone deacetylase 6;
HNRNPA0,A1,A2B1,C,D,F,L,LL,M,U, heterogeneous nuclear ribonucleoprotein
A0,A1,A2B1,C,D,F,L,LL,M,U; IGF2BP1/3, insulin like growth factor 2 mRNA
binding protein 1/3; LARP4, La ribonucleoprotein domain family member 4;
MATR3, matrin 3; MSI1/2, musashi RNA binding protein 1/2; MYC, MYC
proto-oncogene, bHLH transcription factor; OCLN, occludin; PABPC1, poly(A)
binding protein cytoplasmic 1; PAN3, PAN3 poly(A) specific ribonuclease
subunit; PARN, poly(A)-specific ribonuclease; PDCD4, programmed cell death 4;
PTBP1, polypyrimidine tract binding protein 1; PUM1/2, pumilio RNA binding
family member 1/2; QKI, QKI, KH domain containing RNA binding; RBFOX2,
RNA binding protein, fox-1 homolog 2; RBM5/10/38, RNA binding motif
protein 5/10/38; SECISBP2, SECIS binding protein 2; SMN1, survival of motor
neuron 1, telomeric; SRSF1/2, serine and arginine rich splicing factor 1/2; SSB,
sjogren syndrome antigen B; SYNCRIP, synaptotagmin binding cytoplasmic RNA
interacting protein; TARDBP, TAR DNA binding protein; TIA1, TIA1 cytotoxic
granule associated RNA binding protein; TIAL1, TIA1 cytotoxic granule associated
RNA binding protein like 1; TRA2B, transformer 2 beta homolog; U2AF2, U2
small nuclear RNA auxiliary factor 2; YBX1, Y-box binding protein 1; ZFP36,
ZFP36 ring finger protein; ZNF385A, zinc finger protein 385A. The control of messenger RNA stability is another process
in which cooperation between RBPs plays an important role. Among the cis-elements involved, AU-rich elements (AREs)
are the best-characterized, and several studies uncovered ARE-
mediated interplay occurrences. Citation: Dassi E (2017) Handshakes and
Fights: The Regulatory Interplay of
RNA-Binding Proteins. Front Mol Biosci 4:67 September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org The Regulatory Interplay of RNA-Binding Proteins Dassi or by them sharing targets without direct contact, as shown in
Figure 1A. or by them sharing targets without direct contact, as shown in
Figure 1A. RBPs represent a formidable “army” of post-transcriptional
regulators, including over 1,500 human genes geared at
implementing the PTR expression fine-tuning plan. Their
structure is highly modular, often including multiple domains
dictating the binding specificity by independently making contact
with the RNA (Lunde et al., 2007). RBPs control a wide array
of targets, as revealed by techniques based on crosslinking
and immunoprecipitation (CLIPs; Wheeler et al., 2017). These
interactions, happening in the context of ribonucleoprotein
complexes, give rise to complex post-transcriptional regulatory
networks (PTRNs), whose output eventually determines cell
phenotypes. Efforts to identify both RBP targets and the RBPs
regulating specific RNAs have revealed several cases of RBP-
RBP interplay under different patterns. Also, an early large-scale
analysis of a few yeast RBPs suggested the pervasive occurrence
of combinatorial regulation (Hogan et al., 2008). g
RNA processing has been observed to rely on RBP
cooperation
at
its
various
stages. The
heterogeneous
nuclear ribonucleoproteins (hnRNPs) are a large family of
multifunctional RBPs, mainly involved in alternative splicing. HNRNPL, a member of this family, cooperates with PTBP1 to
exclude exon P3A of CHRNA1 via a protein-protein interaction
mediating HNRNPL binding to the region upstream of that exon
in the pre-mRNA (Rahman et al., 2013). Genome-wide analyses
found HNRNPM, C, and H as part of a larger RBP complex
also including RBFOX2, MATR3, and other proteins. HNRNPM
binding sites are enriched in the proximity of RBFOX2 ones, and
RBFOX2 was shown to stimulate HNRNPM-mediated splicing
repression, suggesting a widespread cooperation of the two
RBPs (Damianov et al., 2016). MATR3 instead represses splicing
by interacting with PTBP1 (Coelho et al., 2015). Eventually,
hnRNPs homodimers were also observed. Multiple HNRNPA1
proteins bind cooperatively by spreading on target transcripts
to unwind the RNA secondary structure and allow splicing to
occur (Okunola and Krainer, 2009). Another important group
of splicing regulators is the serine/arginine (SR) proteins. These
RBPs control their targets combinatorially through distinct and
overlapping binding sites mostly in a synergistic fashion (Bradley
et al., 2015). Citation: Also nuclear export and alternative polyadenylation
exploit RBP cooperation. ZNF385A (Hzf) and ELAVL1 (HuR)
co-regulate p53 export in an RNA-dependent fashion (Nakamura
et al., 2011). MSI1 remodels the RNA structure to guide the
polyadenylation site choice by CPEB1 in Xenopus (Weill et al.,
2017), while PABPC1 recruits HNRNPLL to the 3′end of
immunoglobulin mRNA to induce isoform switching through
alternative polyadenylation (Peng et al., 2017). Given the role these mechanisms can have in shaping the
wiring and the output of PTRNs, it is essential to understand
them and characterize their role in physiology and pathology. Here we review our current knowledge of RBP interplay patterns,
discuss the various forms they take and their role in shaping
post-transcriptional regulatory networks. Frontiers in Molecular Biosciences | www.frontiersin.org COOPERATIVE TARGET REGULATION HNRNPF is a required cofactor,
through an RNA-independent interaction, for mRNA decay
induced by two known ARE-binding proteins (AREBPs), ZFP36,
and BRF1 (Reznik et al., 2014). Another AREBP, AUF1, promotes
mRNA silencing by binding near AGO2 sites and contributing
to its loading with miRNAs (Wu et al., 2013; Min et al.,
2017). Genome-wide analyses using RBP binding and gene
expression data have also found TIA1 and ELAVL1 to cooperate
through AREs in a distance-dependent way. TIA1 and PUM1/2
destabilize their target transcripts, while MSI1 and ELAVL1
stabilize theirs (HafezQorani et al., 2016). ELAVL1 is a well-
known regulator of mRNA stability, and further works have
shown synergism with other RBPs such as RBM38, which
increases ELAVL1 binding through physical interaction at ARE
sites (Cho et al., 2010). Also ADAR1, a key player of A-to-I RNA
editing, forms RNA-dependent complexes with ELAVL1. The
complex controls the degradation rate of ADAR1 editing targets,
thus coupling the two processes by a cooperative mechanism
(Wang et al., 2013). ADAR1 also coordinates with ADAR2 to
modulate editing and stability of the Ctn nuclear RNA through
an RNA-dependent interaction, showing that RBP interplay is September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org Frontiers in Molecular Biosciences | www.frontiersin.org 2 The Regulatory Interplay of RNA-Binding Proteins Dassi A
B
C
FIGURE 1 | RBPs regulatory interplay modes. The figure shows the different patterns of regulatory interaction observed between RNA-binding proteins. We use a
hypothetical mRNA 3′UTR as the interaction substrate to illustrate these mechanisms. (A) Describes the cooperative interplay mode. We have proximal cooperative
binding (up) when two RBPs physically interact through nearby binding sites (or distant sites brought in proximity by the RNA secondary structure conformation), thus
shaping their action through this interaction. RBPs can also cooperate through distant binding sites and without interacting directly (down), exerting their synergistic
activity independently from one another. (B) Represents the competitive interplay mode. This pattern consists of two RBPs (RBP1 and RBP2), contending for binding
to one or more overlapping binding sites on an RNA species. This competition results in a balance of RBP1 and RBP2 bound to these molecules, which determines
the outcome of the regulation. (C) Describes the mutual interplay mode. Here, two RBPs (RBP1 and RBP2) can control the expression of one another to constrain
and fine-tune the outcome of the regulation of their target RNAs. COMPETITIVE TARGET REGULATION The impact of cooperative RBP interactions eventually reaches
out to translational control, as suggested by some recent
works. A complex mechanism sees HNRNPA0, HNRNPA2B1,
and ELAVL1 concurrently binding to the 5′UTR of ANXA2R
mRNA. They focus the translation on its first uORF, thus tightly
inhibiting the synthesis of the downstream coding sequence
(Zhang et al., 2016). Another 5′UTR-mediated RNA-dependent
interaction occurs between DDX3X and CAPRIN1. These RBPs
exploit structured UTRs to control translation through PABPC1
and promote cell migration and spreading (Figure 2A) (Copsey
et al., 2017). Interactions in translational control also occur
within the 3′UTR. The synthesis of selenoproteins relies on the
direct interaction between SECISBP2 and the SMN complex. This
interaction allows to assemble and translate the selenoprotein
mRNP through the selenocysteine insertion sequence element,
found in the 3′UTR of these mRNAs (Gribling-Burrer et al.,
2017). ZFP36 and TTP mediate a further role of 3′UTR ARE
elements in translational control. Indeed, the interaction between
ZFP36 and PABPC1 leads to translational repression of ZFP36
target mRNAs in the inflammatory response (Zhang et al., 2017),
while ZFP36 and DDX6 inhibit the translation of target mRNAs
by shifting them to lighter polysomes (Qi et al., 2012). Eventually,
the cooperation of SYNCRIP and MSI2 is critical for myeloid The interplay of two RNA-binding proteins having different
regulatory goals, and whose action is antagonistic, is called
competitive. This pattern, seen in action at the various steps
of post-transcriptional control, involves the formation of a
balance of the competing RBPs on the target RNA species, which
eventually defines the outcome of the regulation (Figure 1B). RNA processing offers several examples of competitive
patterns, mainly involving alternative splicing regulation by the
hnRNPs family. Within this family, HNRNPL and HNRNPLL
antagonistically modulate the splicing of CHRNA1, with
HNRNPL supporting the exclusion of exon P3A and HNRNPLL
favoring its inclusion (Rahman et al., 2013). hnRNPs also
compete with the other major family of splicing regulators, the
SR proteins. In particular SRSF1 blocks cooperative HNRNPA1
binding on exonic splicing silencers. As SRSF2 is unable to do so,
exon inclusion levels are defined by the balance of the three RBPs
(Zhu et al., 2001). HNRNPC instead prevents the exonization of
Alu elements, and the resulting disruption of transcript function,
by blocking U2AF2 from binding at cryptic splice sites found in
Alu-containing exons (Zarnack et al., 2013). COOPERATIVE TARGET REGULATION This mechanism can be heterogeneous (up), with RBP2 binding to RBP1 mRNA (or vice-versa) or
autogenous (down), where an RBP binds to its cognate mRNA. A
B
C FIGURE 1 | RBPs regulatory interplay modes. The figure shows the different patterns of regulatory interaction observed between RNA-binding proteins. We use a
hypothetical mRNA 3′UTR as the interaction substrate to illustrate these mechanisms. (A) Describes the cooperative interplay mode. We have proximal cooperative
binding (up) when two RBPs physically interact through nearby binding sites (or distant sites brought in proximity by the RNA secondary structure conformation), thus
shaping their action through this interaction. RBPs can also cooperate through distant binding sites and without interacting directly (down), exerting their synergistic
activity independently from one another. (B) Represents the competitive interplay mode. This pattern consists of two RBPs (RBP1 and RBP2), contending for binding
to one or more overlapping binding sites on an RNA species. This competition results in a balance of RBP1 and RBP2 bound to these molecules, which determines
the outcome of the regulation. (C) Describes the mutual interplay mode. Here, two RBPs (RBP1 and RBP2) can control the expression of one another to constrain
and fine-tune the outcome of the regulation of their target RNAs. This mechanism can be heterogeneous (up), with RBP2 binding to RBP1 mRNA (or vice-versa) or
autogenous (down), where an RBP binds to its cognate mRNA. leukemia cells survival, with the two RBPs physically interacting
to promote translation of their targets (Vu et al., 2017). leukemia cells survival, with the two RBPs physically interacting
to promote translation of their targets (Vu et al., 2017). not limited to mRNAs (Anantharaman et al., 2017a). Other
cis-elements also use such control patterns. Two examples are the
stability-promoting interaction of LARP4 with PABPC1 (Yang
et al., 2010) and the collaborative control of c-MYC mRNA
stabilization by IGF2BP1 and HNRNPU, SYNCRIP, YBX1, or
DHX9 (Weidensdorfer et al., 2009). COMPETITIVE TARGET REGULATION This binding induces higher steady-state levels of MSI1 mRNA by exerting a stabilizing effect. In turn, this enhances MSI1
translation, ultimately leading to upregulation of its protein levels (Vo et al., 2012). 2017b). Globally, 3′UTR regions containing AREs have been
theorized as “hotspots” for multiple RBPs binding overlapping
sites, fostering competition between these factors and influencing
target mRNA stability (Plass et al., 2017). Accordingly, the
degradation of most mRNAs starts with deadenylation. Two
PAN3 isoforms, PAN3S and PAN3L, compete for PABPC1 to
regulate deadenylation with opposite effects. Their interaction
sees PAN3S enhancing deadenylation and PAN3L repressing it
(Chen et al., 2017). pre-mRNAs with opposing consequences on exons inclusion
(Gazzara et al., 2017). This mechanism is also used by QKI and
PTBP1, competing for the splicing of specific exons on almost
200 common targets (Hall et al., 2013). Aside from splicing, other
processing steps are also affected by RBP competition. TARDBP
(TDP43) and FUS are functionally redundant on HDAC6 mRNA,
as they compete for overlapping binding sites but produce the
same effect, i.e., tuning its processing and nuclear export (Kim
et al., 2010). Eventually, also viral RNA can be controlled by RBP
competition. ELAVL1 was indeed shown to compete with PTBP1
to facilitate SSB (La) binding on the 3′UTR of hepatitis C RNA,
thus enhancing its replication (Shwetha et al., 2015). Eventually,
several
works
have
shown
occurrences
of
translational control mediated by RBP competition, with
translation
initiation
as
the
most
targeted
step. LARP1
competes with eIF4E to control terminal oligopyrimidine
(TOP) mRNAs
translation. LARP1 binds
their
cap and
adjacent 5′TOP motif, impeding access of eIF4E to the cap
and thus blocking eIF4F assembly (Hopkins et al., 2016). MSI1
can instead inhibit the translation of its target mRNAs by
competing with eIF4G for PABPC1. MSI1 thus blocks the 80S
assembly and leads to stress granules recruitment of stalled
preinitiation complexes (Kawahara et al., 2008). Another
mechanism involving PABPC1 sees it contending with YBX1
for binding to a regulatory element in YBX1 3′UTR. While
PABPC1 stimulates YBX1 translation, YBX1 itself seeks to
repress it (Figure 2B) (Lyabin et al., 2011). Also ARE-binding
proteins are antagonistic regulators of translation. CELF1
and ELAVL1 compete for a 3′UTR element in MYC mRNA. CELF1 represses MYC translation, without affecting its mRNA
levels, by decreasing ELAVL1 association with the mRNA
(Liu et al., 2015). CELF1 and ELAVL1 also bind to the same
3′UTR element on OCLN mRNA. COMPETITIVE TARGET REGULATION Outside the hnRNP
and SR families, genome-wide analyses found a conserved
functional antagonism in splicing regulation between CELF2
and RBFOX2. These RBPs bind to overlapping sites on several September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org 3 The Regulatory Interplay of RNA-Binding Proteins Dassi A
B
C A
B
C
FIGURE 2 | Examples of RBP interplay mechanisms. The figure illustrates an occurrence of each RBP interaction mode. (A) Shows the 5′UTR-mediated cooperation
of DDX3X and CAPRIN1 which controls the translation of RAC1 mRNA. This RNA-dependent association exploits a further interaction with PABPC1 at the leading
edge of the cell to promote fibroblasts migration and spreading (Copsey et al., 2017). (B) Depicts the antagonistic interaction of YBX1 and PABPC1 on the 3′UTR of
YBX1 mRNA. This two RBPs target overlapping binding sites within the same regulatory element (the YBX1 binding sequence is shown). While PABPC1 stimulates
YBX1 mRNA translation in a poly(A)-tail-independent manner, YBX1 attempts to repress it by inhibiting translation initiation (Lyabin et al., 2011). (C) Portrays the
mutual heterogeneous interaction between the ELAVL1 and MSI1 RBPs. ELAVL1 binds to an AU-rich element in the distal part of the 3′UTR of MSI1 mRNA (the
ELAVL1 binding sequence is shown). This binding induces higher steady-state levels of MSI1 mRNA by exerting a stabilizing effect. In turn, this enhances MSI1
translation, ultimately leading to upregulation of its protein levels (Vo et al., 2012). B FIGURE 2 | Examples of RBP interplay mechanisms. The figure illustrates an occurrence of each RBP interaction mode. (A) Shows the 5′UTR-mediated cooperation
of DDX3X and CAPRIN1 which controls the translation of RAC1 mRNA. This RNA-dependent association exploits a further interaction with PABPC1 at the leading
edge of the cell to promote fibroblasts migration and spreading (Copsey et al., 2017). (B) Depicts the antagonistic interaction of YBX1 and PABPC1 on the 3′UTR of
YBX1 mRNA. This two RBPs target overlapping binding sites within the same regulatory element (the YBX1 binding sequence is shown). While PABPC1 stimulates
YBX1 mRNA translation in a poly(A)-tail-independent manner, YBX1 attempts to repress it by inhibiting translation initiation (Lyabin et al., 2011). (C) Portrays the
mutual heterogeneous interaction between the ELAVL1 and MSI1 RBPs. ELAVL1 binds to an AU-rich element in the distal part of the 3′UTR of MSI1 mRNA (the
ELAVL1 binding sequence is shown). COMPETITIVE TARGET REGULATION CELF1 represses OCLN
translation through mRNA translocation to P-bodies while The evidence for antagonistic control of messenger RNA
stability by RBPs is diverse and, as for cooperative patterns,
involves AU-rich elements (AREs) and other mechanisms. Suggesting an ancient origin for RBP competition patterns,
Puf proteins were found to form a competitive network of
interactions in yeast, regulating target mRNAs decay and
translational repression (Lapointe et al., 2017). In human cells,
AUF1 and ELAVL1 compete for binding to CDKN1A and
CCND1 mRNAs by targeting overlapping sites with opposing
effects on mRNA stability (enhancing for ELAVL1 and decay-
promoting for AUF1; Lal et al., 2004). ELAVL1 competes likewise
with TIA1 for binding to the 3′UTR of PDCD4 mRNA, with
the RBPs being functionally redundant as they both induce
mRNA stabilization (Wigington et al., 2015). Similarly, ELAVL1
and ZFP36 are broad antagonistic regulators of stability. They
bind to overlapping sites, with ZFP36 acting as a destabilizing
factor (Mukherjee et al., 2014). AREBPs also interact with other
RBPs, as shown by ADAR2 modifying access of ELAVL1 and
PARN to Ctn RNA to enhance its stability (Anantharaman et al., Frontiers in Molecular Biosciences | www.frontiersin.org September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org 4 The Regulatory Interplay of RNA-Binding Proteins Dassi Dassi ELAVL1 enhances it by displacing CELF1 (Yu et al., 2013). TIA1 and ELAVL1 instead antagonize for cytochrome c mRNA
translation by concurrently binding distinct 3′UTR sites (Kawai
et al., 2006). et al., 2011). Autogenous regulation occurs throughout post-
transcriptional processes. RBM10 negatively autoregulates its
mRNA by promoting AS-NMD (Sun et al., 2017), as also
suggested for one RBM10 isoform by a second work (Loiselle
et al., 2017). Similar AS-NMD patterns are also employed by FUS
(Zhou et al., 2013), PTBP1 (Wollerton et al., 2004), HNRNPA2B1
(McGlincy et al., 2010), HNRNPL (Rossbach et al., 2009), TIA1
and TIAL1 (Le Guiner et al., 2001), SRSF2 (Sureau et al., 2001),
and TRA2B (Stoilov, 2004). QKI isoforms control nuclear RNA
stability, splicing, and translation to cross-regulate themselves
and fine-tune the isoform balance of this key developmental
regulator (Fagg et al., 2017). ADAR edits its mRNA to alter
localization and reduce its efficiency (Drosophila) or protein
levels (Mouse) (Savva et al., 2012). Alternative polyadenylation
is targeted by ELAVL1 to produce a longer 3′UTR including
a destabilizing element (Dai et al., 2012). COMPETITIVE TARGET REGULATION Similarly, CSTF3 can
use an intronic poly(A) site to attenuate its expression (Luo
et al., 2013). An unusual mechanism sees DGCR8 guiding the
Microprocessor complex to cleave an hairpin in its 5′UTR,
thus negatively regulating its expression (Triboulet et al., 2009). Eventually, YBX1 and PABPC1 autoinhibit their translation
initiation by respectively targeting the 3′UTR (Lyabin et al., 2011)
or the 5′UTR (Kini et al., 2015). MUTUAL RBP REGULATION The RBP interaction pattern in which one regulates the
expression of the other is said to be mutual. The two RNA-
binding proteins can be different (heterogeneous interplay), or a
single RBP can control its cognate mRNA, a mechanism called
autogenous regulation (Figure 1C). Accumulating evidence
suggests the occurrence of these patterns throughout post-
transcriptional regulation. Autogenous regulation, in particular,
is increasingly credited to be a general behavior. While it is not
competitive or cooperative per se, since a single RBP is involved,
autogenous regulation is fundamental in shaping RBP interaction
patterns and is thus discussed here. p
The heterogeneous interactions observed so far hint at the
presence of an extended RBP regulatory hierarchy, realizing the
regulator-of-regulators concept (Keene, 2007). This is exemplified
by our discovery of a network of 23 RBPs hierarchically
controlled by ELAVL1 (Dassi et al., 2013), and by two large-
scale analyses of RBP interactions, although including only a
fraction of all RBPs (Mittal et al., 2011; Dassi et al., 2016). Further works on ELAVL1 have shown it promoting the stability
and translation of MSI1 mRNA (Figure 2C) (Vo et al., 2012). ELAVL1 is itself regulated via alternative polyadenylation by the
neuronal ELAV RBPs during neuronal differentiation (Mansfield
and Keene, 2012). Splicing is one widely used means for
RBPs to control other RBPs expression. RBFOX2 regulates
70 RBPs by modulating alternative splicing-coupled nonsense-
mediated decay (AS-NMD) of their mRNA. As these RBPs are
frequently under autogenous regulation, RBFOX2 represent a
global controller of such behavior (Jangi et al., 2014). The stability
of RBFOX2 mRNA is, in turn, decreased by CELF2 to tune the
outcome of their splicing antagonism (Gazzara et al., 2017). AS-
NMD is also used by RBM10 to repress RBM5 mRNA (Sun
et al., 2017), with RBM5 in turn controlling the expression
of one splicing variant of RBM10 to reduce its pro-oncogenic
role (Loiselle et al., 2017). Eventually, these heterogeneous
interactions produce global effects on cell phenotypes. This is
shown by the destabilization of EIF4EBP2 mRNA by IGF2BP3,
which positively regulates eIF4E to promote translational
activation and enhance proliferation of lung adenocarcinoma
cells (Mizutani et al., 2016). Frontiers in Molecular Biosciences | www.frontiersin.org REFERENCES Fagg, W. S., Samuel Fagg, W., and Ares, M. (2017). Autogenous cross-regulation
of quaking mRNA processing and translation balances quaking functions in
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families tunes mRNA splicing outcomes. Genome Res. 27, 1360–1370. doi: 10.1101/gr.220517.117 Anantharaman, A., Tripathi, V., Khan, A., Yoon, J. H., Singh, D. K.,
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modifying access of decay-promoting RNA-binding proteins. Nucleic Acids Res. 45, 4189–4201. doi: 10.1093/nar/gkw1304 Gerstberger, S., Hafner, M., and Tuschl, T. (2014). A census of human RNA-
binding proteins. Nat. Rev. Genet. 15, 829–845. doi: 10.1038/nrg3813 Glisovic, T., Bachorik, J. L., Yong, J., and Dreyfuss, G. (2008). RNA-binding
proteins and post-transcriptional gene regulation. FEBS Lett. 582, 1977–1986. doi: 10.1016/j.febslet.2008.03.004 Ayala, Y. M., De Conti, L., Avendaño-Vázquez, S. E., Dhir, A., Romano, M.,
D’Ambrogio, A., et al. (2011). TDP-43 regulates its mRNA levels through
a negative feedback loop. EMBO J. 30, 277–288. doi: 10.1038/emboj.20
10.310 Gribling-Burrer, A.-S., Leichter, M., Wurth, L., Huttin, A., Schlotter, F., Troffer-
Charlier, N., et al. (2017). SECIS-binding protein 2 interacts with the
SMN complex and the methylosome for selenoprotein mRNP assembly
and translation. Nucleic Acids Res. 45, 5399–5413. doi: 10.1093/nar/
gkx031 Bradley, T., Cook, M. E., and Blanchette, M. (2015). SR proteins control
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Menichelli, E., Williamson, J. R., et al. (2012). Cooperativity in RNA-protein
interactions: global analysis of RNA binding specificity. Cell Rep. 1, 570–581. doi: 10.1016/j.celrep.2012.04.003 HafezQorani, S., Lafzi, A., de Bruin, R. G., van Zonneveld, A. J., van der Veer, E. P.,
Son, Y. A., et al. (2016). Modeling the combined effect of RNA-binding proteins
and microRNAs in post-transcriptional regulation. Nucleic Acids Res. 44:e83. doi: 10.1093/nar/gkw048 Chen, C.-Y. A., Zhang, Y., Xiang, Y., Han, L., and Shyu, A. B. (2017). Antagonistic
actions of two human Pan3 isoforms on global mRNA turnover. RNA 23,
1404–1418. doi: 10.1261/rna.061556.117 Hall, M. P., Nagel, R. J., Fagg, W. PERSPECTIVES Regulatory interactions between RNA-binding proteins occur, in
their various forms, throughout all processes composing post-
transcriptional control of gene expression. The pervasiveness
of these mechanisms suggests that they represent an important
additional layer of regulation, providing precision and flexibility
to PTR networks. But where do we stand in their discovery and characterization? As seen in the previous sections, most evidence derives from
low-throughput assays, rather than by large-scale analyses of the
interacting potential of two RBPs. Our current toolbox is the
limiting factor. CLIPs (Wheeler et al., 2017) can only analyze
single RBPs in isolation, and few RBPs have been assayed by
CLIP yet. SEQRS instead analyses the sequence specificity of RBP
complexes by in vitro selection and high-throughput sequencing
(Campbell et al., 2012). However, being in vitro implies it ignores
the effect of other factors on mRNA accessibility. Eventually,
Loregic (Wang et al., 2015) computationally characterizes the
cooperative logic of regulatory factors, requiring both binding
and gene expression data. So, to allow large-scale analyses to
become commonplace, future experimental work should focus
on developing methods to analyze pairs of RBPs concurrently,
in vivo, and at a genome-wide scale. Further efforts should
also include computational tools detecting RBP interactions
from binding data, even when suitable gene expression data is
unavailable or does not allow detecting such interactions. Autogenous interactions have been observed for at least 57
human RBPs (as per the AURA2 database; Dassi et al., 2014), and
are thus increasingly considered to be a mechanism of general
significance. Analyzing the human proteome with the catRAPID
algorithm highlighted an enrichment of autogenous associations
within aggregation-prone disordered proteins (Zanzoni et al.,
2013). This suggests autoregulation as a mean to reduce
protein expression and prevent the accumulation of toxic
aggregates. An example of this phenomenon is TARDBP, which
represses its mRNA by binding to a 3′UTR element (Ayala The evidence described above suggests that the healthy cell
requires finely balanced RBP interactions. Even slight alterations
to RBPs, impacting their binding specificity and interaction
potential, could indeed favor the development of diseases such as
cancer. This makes RBP interplay patterns an attractive target for September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org 5 The Regulatory Interplay of RNA-Binding Proteins Dassi Dassi In conclusion, RBP interactions provide precision, as they
fine-tune the expression of their targets through a balanced
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hypothesis on the autogenous association of alpha-synuclein. Nucleic Acids Res. 41, 9987–9998. doi: 10.1093/nar/gkt794 Copyright © 2017 Dassi. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Zarnack, K., König, J., Tajnik, M., Martincorena, I., Eustermann, S., Stévant, I.,
et al. (2013). Direct competition between hnRNP C and U2AF65 protects
the transcriptome from the exonization of Alu elements. Cell 152, 453–466. doi: 10.1016/j.cell.2012.12.023 September 2017 | Volume 4 | Article 67 Frontiers in Molecular Biosciences | www.frontiersin.org 8
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O Impacto da Crise de 2008 na Estrutura Temporal de Correlação Condicional da BM&FBovespa
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Revista Brasileira de Gestão de Negócios
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REVISTA BRASILEIRA DE GESTÃO DE NEGÓCIOS
ISSN 1806-4892
Review of Business Management
© FECAP
DOI: 10.7819/rbgn.v16¡50.1534
Subject Area: Finance and Economics
RBGN
The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of
Conditional Correlations
O impacto da crise de 2008 na estrutura temporal de correlação condicional da
BM&FBovespa
El impacto de la crisis de 2008 en la estructura temporal de correlación condicional
de BM&FBOVESPA
Mauro Mastella1
Rodrigo Coster2
R
i
d
J
17 2013 / A
d
F b
21 2014 ISSN 1806-4892 El impacto de la crisis de 2008 en la estructura temporal de correlación condicional
de BM&FBOVESPA Mauro Mastella1
Rodrigo Coster2 Received on January 17, 2013 / Approved on February 21, 2014
Responsible editor: André Taue Saito, Dr. Evaluation process: Double Blind Review 1. Doctoral student at the Federal University of Rio Grande do Sul (UFRGS). Assistant professor at the State University of
Rio Grande do Sul (UERGS). [mauro.mastella@ufrgs.br]
2. Master in Business Administration at the Federal University of Rio Grande do Sul (UFRGS). [rodrigo.coster@ufrgs.br]
Authors’ address: Rua 7 de Setembro, 1156 – Centro – Porto Alegre – RS – CEP: 90.010-191 – Brazil RESUMEN correlations higher during high volatility periods? (LAURENT; BAUWENS; ROMBOUTS, 2006.) En este artículo se utiliza un modelado BEKK-
MGARCH para identificar el comportamiento
histórico de la estructura de covarianza temporal
de la BM&FBOVESPA en relación a otros
mercados del continente americano. El objetivo
de la investigación es analizar el impacto de la
crisis de 2008 sobre la cohesión del mercado de
valores de Brasil en comparación con otras bolsas
de muestra. Para ello, los datos históricos se han
obtenido de cinco diferentes índices del mercado
de valores que van desde el período precrisis hasta
2011. Los resultados del modelo bivariado indican
la presencia de una mayor cohesión entre los
índices bursátiles durante la crisis y el no retorno
a la cohesión de estos niveles de precrisis. También
indican que el par de índices IBOV x IPSA es la
opción más adecuada para la diversificación de la
cartera entre los pares analizados. En este artículo se utiliza un modelado BEKK-
MGARCH para identificar el comportamiento
histórico de la estructura de covarianza temporal
de la BM&FBOVESPA en relación a otros
mercados del continente americano. El objetivo
de la investigación es analizar el impacto de la
crisis de 2008 sobre la cohesión del mercado de
valores de Brasil en comparación con otras bolsas
de muestra. Para ello, los datos históricos se han
obtenido de cinco diferentes índices del mercado
de valores que van desde el período precrisis hasta
2011. Los resultados del modelo bivariado indican
la presencia de una mayor cohesión entre los
índices bursátiles durante la crisis y el no retorno
a la cohesión de estos niveles de precrisis. También
indican que el par de índices IBOV x IPSA es la
opción más adecuada para la diversificación de la
cartera entre los pares analizados. (LAURENT; BAUWENS; ROMBOUTS, 2006.)
This research aims at analyzing changes in
correlations between markets over time, especially
at the time of the 2008-2009 subprime crisis. Since volatilities between different assets move
together in different markets, recognizing this
characteristic by using multivariate modeling
leads to more relevant empirical models than
the use of univariate models separately. From a
financial standpoint, this paves the way for better
decision-making tools, such as asset pricing,
portfolio selection, option pricing, hedging and
risk management. For diversification of portfolios
to be effective, it is necessary, for example, that
the covariance between assets in the portfolio be
fairly constant over time. RESUMEN Otherwise, changes
in the term structure of covariances would lead
to the need to readjust assets’ weights. Another
desirable behavior is that correlations among
assets or markets be low, for effective risk dilution. Palabras clave: GARCH multivariante. Correlación Condicional. Volatilidad. Palabras clave: GARCH multivariante. Correlación Condicional. Volatilidad. f
This is no different in the Brazilian
case. The volatility of Brazil’s stock market is
interrelated to the volatility of other markets. In this sense, the objective of this research is
to investigate changes brought about by the
outbreak of the subprime crisis on the volatility
relationships of BM&FBovespa with other
exchanges in the Americas. To this end, historical
series from five different stock market indexes
ranging from the pre-crisis period to 2011 were
collected. By means of a MGARCH model using
a BEKK formulation proposed by Engle and
Kroner (1995), the conditional covariances and
correlation between the different stock markets
were estimated and analyzed. ABSTRACT Este artigo utiliza uma modelagem BEKK-
-MGARCH para identificar o comportamento
histórico da estrutura temporal de covariância
da BM&FBovespa em relação às outras bolsas do
continente americano. O objetivo da pesquisa é
analisar o impacto da crise de 2008 sobre a co-
esão da Bolsa brasileira relativamente às demais
bolsas da amostra. Para isso, foram colhidas séries
históricas de cinco diferentes índices bursáteis
abrangendo desde o período pré-crise até 2011. Os resultados da modelagem bivariada indicam
a ocorrência de um aumento da coesão entre os
índices bursáteis durante o período de crise e o não
retorno dessa coesão aos níveis pré-crise. Também
indicam que par de índices IBOV x IPSA repre-
senta a opção mais adequada para diversificação
de portfólio entre os pares analisados. This article uses a BEKK-MGARCH model
to identify the historical behavior of the
term structure of covariance of the Brazilian
BM&FBovespa stock exchange when compared
to other exchanges in the American continent. The purpose of this research is to analyze the
impact of the 2008 crisis on the cohesion of
the Brazilian stock exchange when compared to
the other exchanges in the sample. To this end,
historical series were collected from five different
stock market indexes ranging from the pre-crisis
period until 2011. The bivariate modeling results
indicate the presence of increased cohesion in the
stock market indexes during the crisis period and
the non-return of this cohesion to pre-crisis levels. They also indicate that, among the pairs analyzed,
the pair of indexes IBOV x IPSA are the most
appropriate choice for portfolio diversification. Keywords: Multivariate GARCH. Conditional
Correlation. Volatility. Keywords: Multivariate GARCH. Conditional
Correlation. Volatility. Palavras-chave: GARCH multivariado. Correla-
ção condicional. Volatilidade. 110 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 1 INTRODUCTION Volatility modeling in financial term series
has gained a lot of attention since the emergence
of seminal ARCH model in Engle’s article (1982). Since then, vast literature on the univariate
models derived from ARCH has been published. Although the volatility of returns is at the center
of this attention, understanding comovements
in financial returns is also extremely important;
in this way, Multivariate GARCH (MGARCH)
models were reached. The Brazilian stock market has received
a great deal of attention from investors and
researchers, due to the gradual and sustained
growth of the Brazilian economy over recent
years and to the increase in trade relationships
with different countries. Therefore, the study
of the term behavior of its covariance structure
is relevant for both academics interested in
model checking as well as international investors
interested in the adequate allocation of capital
between different assets in a portfolio. The study One application for MGARCH models is
the study of the relationships between different
markets’ volatilities and covolatilities. In this way,
answers to general questions about the behavior
of markets are sought for. Does the volatility
of a market cause volatility in other markets? Is the volatility of assets transmitted directly
(through its conditional variance) or indirectly
(through conditional covariances)? Does a shock
in one market increase volatility in others? Are 111 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster is also timely because it indirectly addresses the
subject of decoupling, that is, the assumption that,
in the post-crisis period, emerging economies’
financial markets would be separating themselves
from major world markets, acquiring greater
independence. The analysis of the term behavior
of the Brazilian stock market’s correlations
alongside the others may provide evidence
regarding this hypothesis. National Bureau of Economic Research. We
intend, in this way, to find clues concerning the
influence of the 2008 crisis on the relationships
between the BM&FBovespa and the other stock
markets that make up the sample. In the following section we present the
main concepts referring to MGARCH modeling. Next, we describe the research method and the
data series used. Subsequently, the main results
are discussed and the final considerations of this
research are presented. Recent publications seek to analyze
the contagion effect. 2 THE MULTIVARIATE GARCH MODEL The MGARCH model is a natural
evolution of the univariate GARCH models and
is an option when you have two or more series
and want to model not only their conditional
variances, but also the cross effects (spillovers) of
volatilities. However, one of its disadvantages are
its many parameters, which increase exponentially
as the number of series also increases. In an
attempt to avoid this problem, several MGARCH
models have been proposed, in which the main
differences occur in the restrictions made to the
model at time of estimation. 1 INTRODUCTION A multivariate GARCH
approach is used by Frank and Hesse (2009) to
analyze the spillover in emerging markets during
the 2008 crisis, drawing on various financial
variables such as banking spreads, sovereign
risk and stock market indexes in developed
and developing economies. The results brought
evidence that contradicts the hypothesis of
decoupling between markets. The work of Fenn
et al. (2011) offers a comprehensive analysis of
the correlation between 98 financial products
between 1999 and 2010 using two different
methodologies: the random matrix theory, to
demonstrate that the correlation matrices are
incompatible with random price changes; and
principal component analysis, to demonstrate that
a small number of components is responsible for
a large proportion of variance within the analyzed
markets. The authors demonstrate that there was
an increase in the relationship between different
markets following the 2008 crisis. Recently,
Bouaziz, Selmi and Boujelbene (2012) analyzed
the international transmission of the subprime
crisis between the exchanges of the United States,
France, Germany, Italy, the UK and Japan, using
an MS-GARCH approach, finding evidence of
volatility spillover only in the total analysis period
and during the crisis. 2.1 Definition Introduced by Bollerslev and Wooldridge
(1992), an M-varied GARCH (p,q) model (also
known as vech Model) can be described as follows: (1) (1) in which
corresponds to the conditional variance of series
i over time. Since the models also allows for
covariance estimation,
will be defined as this
covariance, that is,
. Values for
can also be obtained according to the equation However, it is beyond the scope of this
research to study the contagion mechanism’s
dominant factors. This work aims at analyzing the
impact of the 2008 crisis on the term behavior
of the correlation between American markets,
focusing research on the Brazilian stock market’s
correlations with other exchanges in the Americas,
using a dynamic multivariate GARCH approach
and timing of the crisis officially released by the 112 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations represents the operator stacking the lower triangular
part of a symmetric matrix
in a
vector
and
is a vector
When we open the matrix operation for a
GARCH(1,1) bivatiate case, we obtain represents the operator stacking the lower triangular
part of a symmetric matrix
in a
vector
and
is a vector
When we open the matrix operation for a
GARCH(1,1) bivatiate case, we obtain (2) where
and
are matrixes with
d i m e n s i o n s
, (3)
(4)
(5) (3)
(4)
(5) (3) (4) (5) but the use of this model is unworkable
since the number of parameters is
, according to Bollerslev (2008). . Thus, a criticism of this model is that
past values of
affect only its own conditional
variance and its covariances, not interfering in
the variance of the other series. Because of this
restriction, the number of parameters of bivariate
GARCH (1,1) is reduced from 21 to 9. 2.2 Types of models The CCC model, on the other hand,
has no restriction as to the diagonalization of
matrices, but states that the correlations should
be constant over time, substituting equation (4)
for
, with the remaining
equations showing no changes. This restriction
reduces the num ber of required parameters for
the covariance of a bivariate model to only 1
(p 12), reducing the total number of parameters in
the model to 15. In an attempt to avoid the problem of
the high number of parameters, various models
based on MGARCH were proposed, and among
them must be mentioned the diagonal vech and
the CCC (constant conditional correlation). The
restriction of the diagonal vech model – used in
the original MGARCH article – forces matrixes
Ai and Bj to become diagonal, so that, for the
bivariate model with p = 1 = 1 model, the model
obtained is: 2.3 Bekk Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 2.3 Bekk (6)
(7)
(8) (6) (6) Presented by Engle and Kroner (1995),
the BEKK model (Baba, Engle, Kraft and Kroner)
introduces a way to calculate the covariance
matrix (Ht). Instead of imposing restrictions on
the VECH model, it suggests that the covariance
matrix of an m-variate BEKK model (p,q) follow (8) The use of this restriction makes
become a function only of its lagged p-terms and
of the crossed product of past q-lags of (9) Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster where C is an inferior triangular matrix
and
and
,
are
matrixes. For the BEKK-MGARCH (1,1) bivariate model we
would obtain the following equations:
(10)
(11)
(12) where C is an inferior triangular matrix
and
and
,
are
matrixes. For the BEKK-MGARCH (1,1) bivariate model we where C is an inferior triangular matrix
and
and
,
are
matrixes. For the BEKK-MGARCH (1,1) bivariate model we where C is an inferior triangular matrix
and
and
,
are
matrixes. For the BEKK-MGARCH (1,1) bivariate model we
would obtain the following equations:
(10)
(11)
(12) (10) (11) (12) The same as in the VECH model, the
BEKK model has a large number of parameters
. One solution to this problem is
to restrict matrices B and A from being diagonal
(LAURENT; BAUWENS; ROMBOUTS, 2006). the period of analysis included only dates between
09/22/2003 and 12/29/2011. Moreover, the dates
on which the values of certain indexes were not
available were eliminated, leaving a total of 1,858
trading days in which the values were available
for all indexes. Although its number of parameters is large, the
model has a very attractive feature, which is
non-restriction in its parameters. This freedom
is due to the fact that the parameters enter the
equation quadratically, thus avoiding negative
variance estimates, as pointed out by Enders
(2009). However, by forcing parameters to enter
the equation quadratically, interpretability is lost,
because the same parameter appears multiplying
more than one factor. Next, the daily log returns for each index
were calculated. These returns were then separated
into three periods: the Pre-Crisis Period, between
01/10/2003 and 11/30/2007, with 943 returns for
each index; the Crisis Period, between 12/01/2007
and 06/30/2009, with 354 returns for each index;
and the Post-Crisis Period, between 07/01/2009
and 12/31/2011, with 560 returns for each
index. 2.3 Bekk These periods were chosen according to
the Business Cycle dating Dating Committee of
the National Bureau of Economic Research. This
segregation is not obligatory to estimating the
number of covariances, since the BEKK model
is dynamic. An alternative to this separation into
three periods could be intervention analysis – the
name given to the methodology that suggests
the use of proxy variables to indicate events that
are external to the series. However, intervention
analysis is useful only for modeling variations
in the series’ average. Therefore, as the aim of
this study is to examine whether there were also
changes in the correlation structures of the series,
we chose to model the three periods separately. 3 DATA DESCRIPTION For this research, we used the daily series
of four Latin American market indexes: Índice
Bovespa (IBOV) from Brazil, Índice de Precios y
Cotizaciones (IPC) from Mexico, Índice Merval
(Merval) from Argentina and Índice de Precio
Selectivo de Acciones (IPSA) from Chile. We
chose Standard & Poor’s 500 Index (S&P500)
to represent the U.S. market. Data was collected in the Yahoo Finance
site and series were formed based on closing values,
with the necessary adjustments for dividends and
splits, on each trading day. Initially index values
from the 01/01/1998 to 12/31/2011 period
were searched for. Daily rates were found for all
indexes, except for the IPSA, whose quotes were
only available after September 2003. Therefore, Descriptive statistics of the indexes
and their respective returns for each period
are presented in Table 1, below. The standard
deviation of variables in levels is not displayed
because it does not add relevant information to
the analysis. 114 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations TABLE 1 – Descriptive statistics of the indexes in levels and their returns
Variables in levels
Log-Returns
Pre-crisis
Crisis
Post-crisis
Pre-crisis
Crisis
Post-crisis
IBOV
Standard deviation
-
-
-
0.017440
0.030957
0.015011
Maximum
65,318.00
73,517.00
72,996.00
0.073090
0.154728
0.057473
Average
33,472.00
52,481.00
63,642.00
0.001422
- 0.000572
0.000175
Minimum
15,806.00
29,435.00
48,668.00
- 0.068565
- 0.120961
- 0.084307
IPC
Standard deviation
-
-
-
0.012790
0.022912
0.011652
Maximum
32,836.12
32,095.04
38,696.24
0.071187
0.111115
0.045387
Average
17,765.00
25,308.00
33,504.00
0.001418
- 0.000566
0.000755
Minimum
7,771.93
16,891.03
23,359.94
- 0.063715
- 0.072661
- 0.059853
IPSA
Standard deviation
-
-
-
0.009711
0.017988
0.011267
Maximum
3,499.50
3,294.40
5,040.97
0.033257
0.150250
0.057322
Average
2,162.00
2,771.00
4,145.00
0.000910
- 0.000102
0.000536
Minimum
1,359.18
2,101.10
3,061.45
- 0.060318
- 0.062146
- 0.072363
MERVAL
Standard deviation
-
-
-
0.017652
0.029219
0.018484
Maximum
2,351.44
2,244.97
3,664.82
0.060860
0.124891
0.069183
Average
1,563.00
1,635.00
2,661.00
0.001084
- 0.000930
0.000784
Minimum
794.26
828.99
1,477.84
- 0.101537
- 0.129516
- 0.113521
SP500
Standard deviation
-
-
-
0.007850
0.024462
0.013210
Maximum
1,565.15
1,515.96
1,363.61
0.028678
0.104236
0.046317
Average
1,260.00
1,120.00
1,172.00
0.000393
- 0.001347
0.000567
Minimum
995.97
676.53
879.56
- 0.035343
- 0.094695
- 0.068958
Source: the authors. 3 DATA DESCRIPTION TABLE 1 – Descriptive statistics of the indexes in levels and their returns of models that are more complete than GARCH
(1,1) did not provide more accurate predictions. For data analysis, “R” version 2.13.2
software was used, complemented by the
“mgarchBEKK” package, version 0.07-8. Through
the use of package “mgarchBEKK”, parameters
estimation is carried out by maximizing the quasi-
maximum probability function and the Broyden-
Fletcher-Goldfarb-Shanno (BFGS) numerical
optimization method. The data shows that the average returns
for the indexes in the crisis periods returns are
lower than in the pre-and post-crisis periods. Considering the crisis period as a high volatility
period, in certain indexes it is in this time interval
that we find maximum and minimum values for
returns. Moreover, the only index that had not yet
reached again the maximum value of the pre-crisis
period was the SP500. In the series of returns we applied the
Augmented Dickey-Fuller test to verify the
existence of a unity root, yielding p-values
smaller than 0.01 for all series, indicating their
stationarity. This verification is important because
the GARCH family models assume the series’
stationarity. With this result, for all series we chose
to adjust a MGARCH (1,1) model. Hansen and
Lunde’s (2005) research analyzed the efficiency in
generating forecasts from econometric models at
different time lags and pointed out that the results Next, we will discuss main research results. Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 4 RESULTS Analysis of the term structure of the
covariance between the rates of stock exchanges
was carried out in pairs, always in conjunction
with IBOV. This generated four GARCH (1,1)
bivariate models (IBOV × SP500, IBOV × IPC, 115 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster IBOV × IPSA and IBOV × Merval), for each of
the three periods, to allow comparison between
the different estimates for the parameters and
also between covariances. We chose to use the
bivariate model facing a pentavariate model,
due to the smaller number of parameters to be
estimated (only 7 in each bivariate model, against
65 parameters in each pentavariate model). Each
bivariate model was tested within each of the three
periods, with cut-off dates obeying the official
dating of the crisis according to the National
Bureau of Economic Research. adapted from Laurent, Bauwens and Rombouts
(2006), based on the seminal work of Engle and
Kroner (1995). The Tables that present the results
of parameter estimations were structured so that
the IBOV is always represented by index 1 and the
other exchange in the analyzed pair by index 2. 4.1 IBOV X SP500 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations using the model shown in Equation 9, in each of
the three periods. The correlation is an alternative
to covariance representation, correcting it with the
standard deviations of the two series and always
varying between 1 and -1. To make visualization
of the graph easier, the moving Average of the
correlation values was represented with term
window size equal to 1. Analyzing Figure 1, we observe that the
correlation between IBOV and SP500 indexes
presented values closer to zero, with less variability,
during the pre-crisis period. That is, if an investor
wanted to hold portfolio diversification using
the stocks that make up these indexes, he would
find it harder in periods following the crisis. One can see that there was a change in the level
of correlation over the crisis period, in which
the average correlation increased from 0.649 to
0.783, indicating that markets varied in a more
conjugated over the high volatility period. The
average correlation remained virtually unchanged
in the post-crisis period, when compared to the
previous period, with an average of 0.759. FIGURE 1 – Behavior of the conditional correlation
between ibov x sp500 over time * Indicates a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). 4.1 IBOV X SP500 The first pair of indexes assessed were
IBOV and SP500. Table 2, below, presents the
parameter estimates and the standard errors (in
brackets). The BEKK-MGARCH (1,1) equation
used for modeling was described previously, TABLE 2 – Bekk-Mgarch(1,1) results for IBOV X S TABLE 2 – Bekk-Mgarch(1,1) results for IBOV X SP500
Pre-crisis
Estimate (standard deviation)
During
Estimate (standard deviation)
Post-crisis
Estimate (standard deviation)
C
c11
-0.0145 (0.0013)*
0.0155 (0.0028)*
0.0136 (0.0005)*
c21
-
-
-
c12
-0.0034 (0.0007)*
0.0161 (0.0034)*
0.0092 (0.0012)*
c22
-0.0052 (0.0003)*
0.0119 (0.0028)*
-0.0049 (0.0017)*
A
a11
0.1084 (0.1329)
-1.1439 (0.119)*
0.6334 (0.0863)*
a21
-0.2693 (0.2386)
0.9041 (0.1417)*
-0.4065 (0.0996)*
a12
0.071 (0.043)
-0.6266 (0.0939)*
0.4778 (0.0862)*
a22
0.166 (0.0893)
0.3261 (0.115)*
-0.5361 (0.1028)*
B
b11
0.26 (0.1245)*
0.2867 (0.1308)*
-0.0616 (0.2535)
b21
-1.5139 (0.2417)*
0.3305 (0.221)
0.0354 (0.13)
b12
0.1008 (0.0695)
0.1061 (0.0746)
0.5084 (0.2778)
b22
-0.636 (0.1924)*
0.1214 (0.0917)
-0.2414 (0.2445)
Source: the authors. * Indicates a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). TABLE 2 – Bekk-Mgarch(1,1) results for IBOV X SP500 statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). In general, the ARCH parameters (matrix
A) were not significant in the pre-crisis period. That is, the volatility of the return of the indexes
was little influenced by its last values. During
this period, there is evidence that volatility had
a minimum level (significant estimates in matrix
C), and a persistence in variance and covariance
structures (GARCH parameters, represented by
matrix B). another index (SP500 or IBOV). The evidence
that volatility maintained a minimum level
remained. Analysis of the GARCH part points to
the persistence of IBOV’s volatility only. After the crisis, the indexes also showed
a degree of influence established by their own
returns and the returns of other index, as well as
evidence of a constant level of volatility – however,
no influence referring to their past variances and
covariances. During the crisis, the ARCH components
became significant, indicating an immediate
response to past returns of the index itself (IBOV
or SP500) and also as to the past returns of In Figure 1 we can observe the term
behavior of the correlation between indexes. We
should remember that the results were obtained 116 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. Source: the authors.
* Indicates a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). Source: the authors.
* Indicates a statistically significant coefficient at 5% The coefficients presented here follow the form of the equation (9) Source: the authors.ifihfi es a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). 4.2 IBOV X IPC The analysis of the second pair of indexes
showed a slightly different ratio compared to
the first pair analyzed. Table 3, below, presnets
parameter estimates and their standard errors (in
brackets). FIGURE 1 – Behavior of the conditional correlation
between ibov x sp500 over time TABLE 3 – Bekk-Mgarch(1,1) results for IBOV X IPC
Pre-crisis
Estimate
(standard deviation)
During
Estimate (standard deviation)
Post-crisis
Estimate (standard deviation)
C
c11
-0.0138 (0.0009)*
0.008 (0.0036)*
0.0072 (0.0023)*
c21
-
-
-
c12
-0.0012 (0.0012)
0.0174 (0.0017)*
-0.0011 (0.0021)
c22
0.0024 (0.0025)
0.0009 (0.0072)
-0.0003 (0.0011)
A
a11
-0.086 (0.0565)
-0.9182 (0.1287)*
0.2277 (0.1156)*
a21
-0.4705 (0.0886)*
0.6568 (0.198)*
0.0771 (0.1525)
a12
0.1076 (0.0432)*
-0.511 (0.1947)*
0.1765 (0.0917)
a22
-0.5449 (0.0563)*
0.3522 (0.2717)
-0.3393 (0.1234)*
B
b11
0.3661 (0.0979)*
0.3671 (0.1548)*
-1.21 (0.1419)*
b21
0.2187 (0.0791)*
0.5885 (0.2146)*
1.3348 (0.3596)*
b12
0.4619 (0.0912)*
0.1746 (0.0786)*
-1.0973 (0.0433)*
b22
0.296 (0.1108)*
0.2778 (0.1425)
1.214 (0.1431)* TABLE 3 – Bekk-Mgarch(1,1) results for IBOV X IPC 117 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster In this pair of indexes, the post-crisis
period showed a decrease in average correlation
(0.724), still higher than the pre-crisis levels and
with greater variability than in the crisis period. That is, an investor who wanted to hold a portfolio
diversification between these two markets would
find it hard to do so, because of the high variability
of correlations (pre- and post-crisis periods) or of
the high value of the correlation (crisis period). For this pair of indexes, the GARCH
component was significant during the
three periods analyzed, indicating a strong
persistence in volatility, both crossed (IBOV
× IPC) and not-crossed (IBOV x IBOV e IPC
× IPC). Over time, the ARCH components
were partly significant. We observed that
the cross-relationship between the returns
of the indexes was not relevant in the post-
crisis period, that is, markets failed to give
immediate responses referring to the returns
of the other exchange, responding only to
their own returns. Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 4.3 IBOV X IPSA The analysis of the third combination
of markets does not identify obvious cross-
relationships between their volatilities, as shown
in Table 4, in which are presented the parameter
estimates and their respective standard errors (in
brackets). No specific relationship can be identified
referring to the minimum levels of volatility,
because the only parameter that remained
significant over time was c11, indicating that
only the volatility of IBOV had a non-zero level
support. In the pre-crisis period, both the estimates
of the ARCH and the GARCH parameters were,
in general, statistically significant. However,
in the three periods observed, no specific and
lasting relationship can be identified referring
to the minimum levels of volatility, because
the only parameter that remained significant
over time was c11, indicating that only IBOV’s
volatility possessed a level with nonzero support,
a behavior previously observed in other pairs of
analyzed series. It is noteworthy that we expected a
decrease in the correlation between the series over
the post-crisis period, a fact that is demonstrated
in Figure 2. This reduction can be explained by the
absence of evidence of cross-relationships between
returns and volatility. Another relationship that is
visible in Figure 2 is the increase in the correlation
average over the crisis period (from 0.633 to
0.810) and the reduction of variability over this
same period. FIGURE 2 – Behavior of conditional correlation IBOV
X IPC over time Values found for the C matrix indicates
that volatility had a constant nonzero level during
the post-crisis period. Over that period, in general,
the ARCH and GARCH parameters were also
statistically significant, although it is not possible
to identify a clear cross-relationship between the
indexes. FIGURE 2 – Behavior of conditional correlation IBOV
X IPC over time 118 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 4.3 IBOV X IPSA 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations TABLE 4 – Bekk-Mgarch(1,1) results for IBOV X IPSA
Pre-crisis
Estimate (standard deviation)
During
Estimate (standard deviation)
Post-crisis
Estimate (standard deviation)
C
c11
-0.0137 (0.0023)*
0.0158 (0.0021)*
0.011 (0.0012)*
c21
-
-
-
c12
0.0013 (0.0018)
-0.0015 (0.0014)
0.0017 (0.0009)*
c22
0.0003 (0.0011)
0 (0.0027)
0.0065 (0.001)*
A
a11
0.166 (0.091)
-0.2578 (0.07)*
0.2685 (0.0747)*
a21
-0.3424 (0.1297)*
-0.6527 (0.1295)*
0.2182 (0.0978)*
a12
0.1453 (0.0418)*
0.0556 (0.0536)
0.0183 (0.0502)
a22
-0.6243 (0.0717)*
-0.3063 (0.1114)*
0.6249 (0.071)*
B
b11
-0.6036 (0.1848)*
-0.5534 (0.1189)*
-0.3391 (0.1587)*
b21
1.0352 (0.3759)*
-0.2553 (0.1375)
-0.3504 (0.1589)*
b12
-0.4799 (0.0517)*
-0.4338 (0.0607)*
-0.2374 (0.095)*
b22
0.7673 (0.1223)*
-0.217 (0.1219)
-0.2472 (0.1464)
Source: the authors. * Indicates a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). TABLE 4 – Bekk-Mgarch(1,1) results for IBOV X IPSA Indicates a statistically significant coefficient at 5%. The coefficients presented here follow the form of the equation (9). Figure 3, below, demonstrates another
behavior that had already been observed in the
pairs belonging to previous series: there is a clear
change in the average correlation level during the
crisis (from 0.477 to 0.666); in this case, however,
the level shift comes with an increase in variability. Among the pairs of indexes analyzed, this was the
one with the lowest average correlation over the
three periods. In this case, in the post-crisis period, the
behavior not previously observed in the previous
series was the reduction of the correlation’s
resilience, that is, apparently, the correlation does
not sustain a constant average over time. This
represents an additional problem to investors who
are interested in holding portfolio diversification
across markets represented by these indexes,
because it makes it even harder to maintain the
assumption that the correlation matrices are fairly
constant over time. FIGURE 3 – Behavior of conditional correlation IBOV
X IPSA over time Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 4.4 IBOV X MERVAL The last pair of indexes in the sample
shows the Brazilian and the Argentinian stock
exchanges. Table 5, below, presents the parameter
estimates and their standard deviation errors (in
brackets). FIGURE 3 – Behavior of conditional correlation IBOV
X IPSA over time 119 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster TABLE 5 – Bekk-Mgarch(1,1) results for IBOV X MERVAL
Pre-crisis
Estimate
(standard deviation)
During
Estimate
(standard deviation)
Post-crisis
Estimate
(standard deviation)
C
c11
0.0151 (0.0019)*
0.0152 (0.0017)*
0.0115 (0.0024)*
c21
-
-
-
c12
0.0061 (0.0031)*
0.0022 (0.002)
0.0114 (0.0012)*
c22
-0.0125 (0.0014)*
0 (0.0034)
0.0104 (0.0009)*
A
a11
-0.1141 (0.0727)
-0.059 (0.0854)
0.5771 (0.1242)*
a21
0.412 (0.1038)*
0.8812 (0.1122)*
-0.2477 (0.0848)*
a12
0.213 (0.1102)
0.1339 (0.0906)
1.0244 (0.1355)*
a22
-0.031 (0.1266)
0.2887 (0.1405)*
-0.5943 (0.1073)*
B
b11
0.4291 (0.2712)
-0.4311 (0.1159)*
0.0097 (0.319)
b21
-0.3431 (0.3091)
-0.1999 (0.09)*
-0.3937 (0.3967)
b12
0.6351 (0.1803)*
-0.5862 (0.1241)*
-0.0238 (0.0708)
b22
-0.4709 (0.2065)*
-0.2718 (0.1337)*
-0.0636 (0.2166)
Source: the authors. * Indicates statistical significance for 5% coefficient value. Coefficients presented here follow the equation format (9). TABLE 5 – Bekk-Mgarch(1,1) results for IBOV X MERVAL * Indicates statistical significance for 5% coefficient value. Coefficients presented here follow the equation format (9). In general, over the three periods analyzed,
the components of the model (level, ARCH part
and GARCH part) were not significant for two
consecutive periods. We observed that in the
pre-crisis period the most obvious relationship
between the indexes occurred at a level and that
no obvious cross-relationship between the indexes
can be identified. identify two different sublevels of correlation in
the crisis period (approximately 0.753 and 0.846),
a characteristic that can only be observed because
of the use of a dynamic model. We would also like to add that, in the post-
crisis period, there was a decrease in the average
correlation to 0.718, still above the pre-crisis
period. Compared to the previous pairs of indexes,
this one showed high resilience during this period. In the crisis period, on the other hand,
there was a clear persistence in volatilities,
as proven by the significance of all matrix B
parameters. However, no evidence that this
relationship was maintained during the period
immediately following were obtained. 4.4 IBOV X MERVAL We noticed
that in the post-crisis period we could again
identify a relationship at minimum levels of
volatility, as well as the emergence of a relationship
in response to lagged returns, both crossed as well
as not crossed. These changes in the characteristics
of relationships between series hinder decision
making by investors interested in portfolio
diversification across these markets. FIGURE 4 – Behavior of conditional correlation IBOV
X MERVAL OVER TIME FIGURE 4 – Behavior of conditional correlation IBOV
X MERVAL OVER TIME Figure 4 shows the term behavior of the
correlation between IBOV and Merval. The pre-
crisis period presented an average correlation of
0.531 and a gradually smaller variability over
time. In the crisis period, the average correlation
level rose to 0.798, a behavior already observed
in previous series. In this case, however, we can Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 5 FINAL CONSIDERATIONS The Ljung-Box test for standardized
residuals rejected the hypothesis of no
autocorrelation in exchanges SP500 and CPI
(highlighted with *), with a significance level of
5%, only in the period during the crisis. Other
models with higher lags were tested for these
exchanges; there was, however, no change the
results of this test. This result does not invalidate
the model, because, as demonstrated earlier,
in these cases the parameter estimates were
significant in most cases. The analysis of a group of term series using
multivariate models allows for the identification
of cross-relationships between the series, which
is not possible using various univariate models
separately. Accordingly, the scope of this research
was to evaluate the impact of the 2008 crisis on
the term structure of BM&FBovespa’s conditional
correlation when compared to other exchanges in
the Americas. This research yields results that do not
support the hypothesis of decoupling, since the
correlations continued to be high in the post-crisis
period when compared to the pre-crisis period. The change in the term behavior of the correlation
between BM&FBovespa compared to the other
exchanges in the sample can be regarded as an
indication of the occurrence of the contagion
effect. The results found for the relationship
between the U.S. exchanges are consistent with
the results of other surveys conducted with data
from other stock markets, such as Kim and Kim
(2011), for the contagion effect between the U.S. market and Asian exchanges, and Fenn et al (2011)
for the same effect in emerging markets, although
the variables and the period of analysis are slightly
different. The results of this study are also similar
to Bouaziz, Selmi and Boujelbene (2012), who
found a significant increase in the coefficients
of dynamic correlation in the following pairs of The results found in this analysis are
consistent with the results of Lin and Chen
(2010), in which the authors, to investigate
the term behavior of the correlation between
the markets of Tokyo and Hong Kong, also
detected autocorrelation and found no significant
differences in certain parameters in their model. However, since the structure of the model adopts
a matrix form, we cannot rule out only a few
estimated parameters. Certain studies agree with this article’s
main results. In this sense, Borland (2009)
identified the occurrence of a marked increase in
stock returns’s comovements, in volatilities and in
changes in volatility in periods of panic. 4.5 Analysis of residuals So as to validate these analyzes, we
proceeded to carry out a test to check for the
presence of serial correlation in the model
residuals. The results are shown in Table 6, below: 120 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations TABLE 6 – ljung-box test for standardized residuals (LAG = 20)
Model
Exchange
Pre-crisis
During
Post-crisis
test stat
p-value
test stat
p-value
test stat
p-value
IBOV × SP500
IBOV
25.8655
0.1703
17.0546
0.6494
25.6993
0.176
SP500
24.3204
0.2287
66.1374
<0.001*
12.5802
0.8947
IBOV × IPC
IBOV
20.7689
0.4108
11.0224
0.9456
17.3346
0.6311
IPC
33.6626
0.0285
31.7359
0.0462*
26.9989
0.1353
IBOV × IPSA
IBOV
23.2867
0.2749
17.0546
0.6494
19.2287
0.507
IPSA
33.237
0.0318
26.8476
0.1396
39.2545
0.0062
IBOV × MERV
IBOV
24.705
0.213
22.5484
0.3115
17.2228
0.6385
MERV
18.2339
0.572
26.2513
0.1577
37.8948
0.0091
Source: the authors. * Indicates statistical significance for coefficient value at 5 TABLE 6 – ljung-box test for standardized residuals (LAG = 20) * Indicates statistical significance for coefficient value at 5 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 5 FINAL CONSIDERATIONS Moldovan
and Medrega (2011) found a strengthening of
relationships between the analyzed indexes during
the crisis. Recently, Sandoval and Franca (2012)
found evidence that markets tend to behave more
similarly during periods of high volatility. 121 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 Mauro Mastella / Rodrigo Coster exchanges: U.S.-France, U.S.-Germany, U.S.-Italy
and U.S.-U.K.. In the Brazilian case, Perobelli,
Vidal and Securato (2013) used an approach
based on the stability of factor charges and also
found signs of contagion for the subprime crisis. cases, when studying correlations between
markets, they are treated as constants. However,
it has become a stylized finance fact that the
correlations between the returns of assets or
indexes are not constant over time, as previously
documented by Erb, Harvey and Viscanta (1994),
Longin and Solnik (1995) and Engle (2002). In the present study, it is interesting to
note that the 2008 crisis, even though it had its
origins in the U.S. mortgage market, was able
to change the term correlation structure of the
Brazilian exchange as to the other exchanges
in the Americas. Once the financial markets
become increasingly integrated, the international
mobility of capital allows investors to carry out
the diversification of their investment portfolios
in an international way, looking for assets that
are little correlated so as to minimize risks. One
consequence of this increasing internationalization
of investments and interconnectivity of world
trade is that, in times of turmoil for a certain
economy, markets in other countries are also
impacted. In the case of the 2008 crisis, this is
no different, also generating impacts throughout
the Americas. Finally, the regulatory authorities of the
Brazilian capital market should keep in mind the
conditional correlations between international
markets, so as to enhance the regulations and to
structure effective ways of decreasing the risk of
contagion by the Brazilian stock market. In this
way, the quality of this capital market can be
improved and further developed, aiming to attract
non-speculative investments. In other words, the
increase of international economic integration
makes studies of contagion events relevant to
the establishment of effective political-economic
interventions by monetary authorities. Taking the increased interconnection
between American economies and Asian and
European markets into account, future research
can be carried out using a set of indexes that
represents the overseas markets, in order to also
examine the correlation of the Brazilian market
with them. 5 FINAL CONSIDERATIONS Moreover, a multivariate model
that takes into account more than two levels
simultaneously can be implemented to analyze
the dynamic connections between a greater
number of indexes, comparing the result with the
modeling carried out here, although the number
of parameters to be estimated will increase
considerably. In this sense, the use of BEKK-MGARCH
(1,1) modeling, also used by Kim and Kim
(2011), was adequate for achieving the proposed
objectives. An important result found in every
pair of series was the increased cohesion between
stock market indexes during the crisis and the
fact that this cohesion did not return to pre-crisis
levels. That is, an investor who sought to diversify
his portfolio would have advantages when doing
so in the Brazilian and Chilean markets, because
the pair of indexes IBOV × IPSA was the one that
presented the lowest average correlation over the
three periods. On the other hand, the use of the same
dates to establish the beginning and the end of the
crisis period for all markets analyzed is a limitation
in this study, since in some cases (e.g.: IBOV×
IPC) the term behavior of the correlation seems
to indicate that there was a change to the level of
crisis in the days preceding the specified period,
as observed in Figure 2. REFERences Bollerslev, T. Glossary to ARCH (GARCH). Sept. 2008. CREATES Research Papers 2008-49. Bollerslev, T. Glossary to ARCH (GARCH). Sept. 2008. CREATES Research Papers 2008-49. ______; WOOLDRIDGE, J. M. Quasi-
maximum likelihood estimation and inference in
dynamic models with time-varying covariances. Econometric Reviews, Philadelphia, v. 11, n. 2,
p. 143-172, 1992. ______; WOOLDRIDGE, J. M. Quasi-
maximum likelihood estimation and inference in
dynamic models with time-varying covariances. Moreover, many of our analyzes and
conclusions were possible only because of the
use of dynamic multivariate modeling. In certain Econometric Reviews, Philadelphia, v. 11, n. 2,
p. 143-172, 1992. 122 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014 The Impact of the 2008 Crisis on BM&FBovespa’s term Structure of Conditional Correlations BORLAND, L. Statistical signatures in times of
panic: markets as a self-organizing system. 2009. Available at: <http://arxiv.org/abs/0908.0111>. Acesso em: 03 jan. 2013. beat a GARCH(1,1)? Journal of Applied
Econometrics, Chicester, v. 20, n. 7, p. 873– 889,
Dec. 2005. BORLAND, L. Statistical signatures in times of
panic: markets as a self-organizing system. 2009. Available at: <http://arxiv.org/abs/0908.0111>. Acesso em: 03 jan. 2013. KIM, B. H.; KIM, H. Spillover effects of the
US financial crisis on financial markets in
emerging Asian countries. Apr. 2011. Auburn
Economics Working Paper Series, with number
auwp2011-04. BOUAZIZ, M. C.; SELMI, N.; BOUJELBENE,
Y. Contagion effect of the subprime financial
crisis: evidence of DCC multivariate GARCH
models. European Journal of Economics,
Finance & Administrative Sciences, [S.l], n. 44,
p. 66, Jan. 2012. Laurent, S.; Bauwens, L.; Rombouts,
J. V. K. Multivariate GARCH models: a survey. Journal of Applied Econometrics, Chicester, v. 21, n. 1, p. 79-89, Jan./Feb. 2006. ENDERS, W. Applied econometric time series. Hoboken, NJ: John Wiley and Sons, 2009. LIN, Y.; CHEN, Y. Study on time varying
conditional correlations of stock market returns
based on multivariate GARCH model. Advanced
Management Science (ICAMS), 2010 IEEE
International Conference, July 2010. Available
at: <http://ieeexplore.ieee.org/xpl/login.jsp?t
p=&arnumber=5553092&url=http%3A%2F
%2Fieeexplore.ieee.org%2Fxpls%2Fabs_all. jsp%3Farnumber%3D5553092>. Acesso em:
03 jan. 2013. ENGLE, R. F. Autoregressive conditional
heteroscedasticity with estimates of the variance
of the United Kingdom inflation. Econometrica,
Oxford, v. 50, n. 4. p. 987-1007, July 1982. ______. Dynamic conditional correlation: a simple
class of multivariate generalized autoregressive
conditional heteroskedasticity models. Journal of
Business and Economic Statistics, Alexandria,
v. 20, n. 3, p. 339-350, July 2002. ______; KRONER, K. F. Multivariate
simultaneous generalized arch. Econometric
Theory, Cambridge, v. 11, n. 1. p. 122-150,
Mar. REFERences 1995. LONGIN, F.; SOLNIK, B. Is the correlation in
international equity returns constant: 1960-1990. Journal of International Money and Finance,
Oxford, v. 14, n. 1, p. 3-26, 1995. ERB, C. B.; HARVEY, C. R.; VISKANTA, T. E. Forecasting international equity correlations. Financial Analysts Journal, Charllottesville, v. 50, n. 6, p. 32-45, Nov./Dec. 1994. MOLDOVAN, I.; MEDREGA, C. Correlation
of international stock markets before and during
the subprime crisis. The Romanian Economic
Journal, [S. l.], v. 14, n. 40, p. 173-193, June
2011. Financial Analysts Journal, Charllottesville, v. 50, n. 6, p. 32-45, Nov./Dec. 1994. FENN, D. J. et al. Temporal evolution of financial
market correlations. Physical Review E, New
York, v. 84, n. 2, p. 1-15, Aug. 2011. PEROBELLI, F. F. C.; VIDAL, T. L.; SECURATO,
J. R. Avaliando o efeito contágio entre economias
durante crises financeiras. Estudos Econômicos,
São Paulo, v. 43, n. 3, p. 557-594, jul./set. 2013. FRANK, N.; HESSE, H. Financial spillovers
to emerging markets during the global financial
crisis, Czech Journal of Economics and Finance,
Prague, v. 59, n. 6, p. 507-521, Dec. 2009. SANDOVAL JÚNIOR, Leonidas; FRANCA,
I. P. Correlation of financial markets in times
of crisis. Physica A, Amsterdam, v. 391, n. 1-2,
p. 187-208, Jan. 2012. HANSEN, P. R.; LUNDE, A. A forecast
comparison of volatility models: does anything 123 Rev. bus. manag., São Paulo, Vol. 16, No. 50, pp. 110-123, Jan./Mar. 2014
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https://openalex.org/W2271805741
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https://cardiovascular.elpub.ru/jour/article/download/255/257
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Russian
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WHY CARDIOVASCULAR MORTALITY IN MOSCOW IS LOWER THAN IN OTHER REGIONS OF THE RUSSIAN FEDERATION?
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Kardiovaskulârnaâ terapiâ i profilaktika
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Почему в Москве смертность от сердечно-сосудистых
заболеваний ниже, чем в других регионах Российской
Федерации? Погосова Н. В., Оганов Р. Г., Суворов С. В. ФГБУ Государственный научно-исследовательский центр профилактической медицины Минздрава
России. Москва, Россия Погосова Н. В., Оганов Р. Г., Суворов С. В. Погосова Н. В., Оганов Р. Г., Суворов С. В. ФГБУ Государственный научно-исследовательский центр профилактической медицины Минздрава
России. Москва, Россия о осо а
. ., О а о
. ., Су оро С. . ФГБУ Государственный научно-исследовательский центр профилактической медицины Минздрава
России. Москва, Россия России. Москва, Россия Начиная с 2003г в РФ отмечается снижение смертности от сердеч-
но-сосудистых заболеваний (ССЗ), которое с 2006г приобрело
более устойчивый и выраженный характер среди мужчин и женщин. С 2003 по 2013гг общий коэффициент смертности от болезней сис-
темы кровообращения (число умерших на 100 тыс. населения)
снизился на 25% (698,1 vs 927,5), тем не менее, он остается более
высоким, чем в начале 90-х годов (621,0 на 100 тыс. населения
в 1991г). Отмечаются существенные различия между регионами РФ
по показателям заболеваемости и смертности от ССЗ. За период
с 2006 по 2013гг стандартизованный показатель смертности
от ишемической болезни сердца (ИБС) в Москве снизился на 35,7%,
что в 1,5 раза больше, чем в РФ, в 1,3 раза больше, чем в Санкт-
Петербурге, и в 2,6 раз больше, чем в Московской области. В 2012г
показатель ожидаемой продолжительности жизни (ОПЖ) москви-
чей достигает 76,0 лет (в среднем по России — 70,0 лет), и всего
4 года разделяет москвичей и жителей Евросоюза. ОПЖ мужчин
в Москве в 2013г достигла 72,3 лет. Существенно более низкие показатели смертности от ССЗ и более высокие показатели ОПЖ
в Москве, очевидно, обусловлены более высоким социально-эко-
номическим уровнем жизни населения, большей психологической
устойчивостью и большей доступностью психологической (психо-
терапевтической) помощи, более высоким уровнем потребления
рыбы, фруктов и ягод, большей доступностью амбулаторно-поли-
клинической и высокотехнологичной медицинской помощи для
жителей столицы. Ключевые слова: сердечно-сосудистые заболевания, смертность,
заболеваемость, факторы риска. Кардиоваскулярная терапия и профилактика, 2015; 14(2): 4–12
http://dx.doi.org/10.15829/1728-8800-2015-2-4-12 Кардиоваскулярная терапия и профилактика, 2015; 14(2): 4–12
http://dx.doi.org/10.15829/1728-8800-2015-2-4-12 Кардиоваскулярная терапия и профилактика, 2015; 14(2): 4–12
http://dx.doi.org/10.15829/1728-8800-2015-2-4-12 Поступила 03/02-2015
Принята к публикации 10/03-2015 Pogosova N. V., Oganov R. G., Suvorov S. V. FSBI State Scientific-Research Centre for Preventive Medicine of the Healthcare Ministry. Moscow, Russia Pogosova N. V., Oganov R. G., Suvorov S. V. FSBI State Scientific-Research Centre for Preventive Medicine of the Healthcare Ministry. Moscow, Russia Since 2003 there is a decline of cardiovascular (CVD) mortality of the
RF, that established in 2006 in women and in men. From 2003 to 2013
y. Кардиоваскулярная терапия и профилактика, 2015; 14(2) Кардиоваскулярная терапия и профилактика, 2015; 14(2) БСК — болезни системы кровообращения, ВОЗ — Всемирная организация здравоохранения, ИБС — ишемическая болезнь сердца, МИ — мозговой инсульт, ОИМ — острый инфаркт миокарда, ОПЖ — ожида-
емая продолжительность жизни, ССЗ — сердечно-сосудистые заболевания, ЦВЗ — цереброваскулярные заболевания. Почему в Москве смертность от сердечно-сосудистых
заболеваний ниже, чем в других регионах Российской
Федерации? total coefficient of cardiovascular mortality (number of died per 100
thousand of population) decreased by 25% (698,1 vs. 927,5), although
still it is higher than in the beginning of the nineties (621,0 per 100
thous. of population in 1991 y.). The significant differences in RG
regions are noted by the values of morbidity and mortality from CVD. For the period 2006-2013 y. the standardized value of mortality from
coronary heart disease (CHD) in Moscow decreased by 35,7% that is
1,5 more than in RF and 1,3 more higher than in St-Petersburg, and 2,6
times more than in Moscow region. In 2012 the value of the suspected
life duration (SLD) of Moscow citizens was 76,0 y. (mean in Russia — 70,0 y), and just 4 years is the gap between then and EU citizens. In
2013 SLD in Moscow reached 72,3 y. Significantly lower values of
mortality from CVD and higher values of SLD can be explained by
higher socio-economic level, higher psychological endurance and
better availability of psychological (psychotherapeutic) help, higher
level of fish, fruits and berries consumption, better availability of
outpatient and high technology medical care for Moscow citizens. Key words: cardiovascular diseases, mortality, morbidity, risk
factors. Cardiovascular Therapy and Prevention, 2015; 14(2): 4–12
http://dx.doi.org/10.15829/1728-8800-2015-2-4-12 Cardiovascular Therapy and Prevention, 2015; 14(2): 4–12
http://dx.doi.org/10.15829/1728-8800-2015-2-4-12 e-mail: npogosova@gnicpm.ru [Погосова Н. В.* — д. м.н., профессор, руководитель отдела вторичной профилактики ХНИЗ, Оганов Р. Г. — д. м.н., профессор, академик РАН, руководитель отдела профилактики коморбидных состояний,
Суворов С. В. — н. с. отдела вторичной профилактики ХНИЗ]. ессор, руководитель отдела вторичной профилактики ХНИЗ, Оганов Р. Г. — д. м.н., профессор, академик РАН, руководитель отдела профилактики коморбидных состояний,
ичной профилактики ХНИЗ]. [Погосова Н. В.* — д. м.н., профессор, руководитель отдела вторичной профилактики ХНИЗ, Оганов Р. Г. — д. м.н., профессор, академик РАН, руководитель отдела профил
Суворов С. В. — н. с. отдела вторичной профилактики ХНИЗ]. Передовая статья Сердечно-сосудистые заболевания (ССЗ) оста-
ются ведущей причиной смертности и инвалидиза-
ции населения в большинстве стран мира,
в т. ч. в России [1]. Они являются сложно решаемой
проблемой для национальных систем здравоохране-
ния и наносят серьезный экономический ущерб. ССЗ вносят существенный вклад в заболеваемость
населения в национальном и международном мас-
штабах [2]. Характеристика заболеваемости явля-
ется не простой задачей ввиду разных подходов к ее
оценке, в отличие от показателей смертности,
но и констатация причин смерти не во всех случаях
может оказаться корректной. Почему в Москве смертность от сердечно-сосудистых
заболеваний ниже, чем в других регионах Российской
Федерации? 383,6
365,6
368,5
351,2
354,7
312,5
289,0
306,1
284,2
280,0
263,1
282,6
212,8
196,9
433,8
385,2
374,3
423,7
429,4
412,2
429,7
358,2
351,4
362,1
333,7
287,1
258,9
333,8
150,0
200,0
250,0
300,0
350,0
400,0
450,0
2006
2007
2008
2009
2010
2012
2013
МО
СПБ
Москва
-24,7%
-13,7%
-28,5%
-35,7%
РФ
Россия
Москва
Московская область
Санкт-Петербург
40,0
40,1
40,8
41,1
40,1
38,3
37,2
50,3
49,7
48,5
45,8
41,3
42,3
40,0
46,2
47,6
47,6
46,6
48,4
49,4
50,0
68,2
64,5
66,1
61,9
52,3
45,2
59,9
35
45
55
65
2006
2007
2008
2009
2010
2012
2013
РФ
МО
СПБ
Москва
-7,0%
+3,0%
-31,6%
-20,5%
Россия
Москва
Московская область
Санкт-Петербург Рис. 2 Стандартизованные показатели смертности от ИБС
(на 100 тыс. населения). 383,6
365,6
368,5
351,2
354,7
312,5
289,0
306,1
284,2
280,0
263,1
282,6
212,8
196,9
433,8
385,2
374,3
423,7
429,4
412,2
429,7
358,2
351,4
362,1
333,7
287,1
258,9
333,8
150,0
200,0
250,0
300,0
350,0
400,0
450,0
2006
2007
2008
2009
2010
2012
2013
МО
СПБ
Москва
-24,7%
-13,7%
-28,5%
-35,7%
РФ
Россия
Москва
Московская область
Санкт-Петербург Ежегодно в странах Европы умирает от ССЗ
4,1 млн. человек (46% всех смертей), в т. ч. 1,8 млн. человек от ИБС (20% в структуре смертности),
1,1 млн. человек от МИ (12% в структуре смертно-
сти) и 1,2 млн. человек (14%) от других ССЗ [3, 5]. В структуре смертности в РФ ССЗ занимают
большую долю, чем в среднем по Европе — 55,7% vs
46% по данным ВОЗ [5], при этом в стране 29,4%
смертей приходится на ИБС и 17,6% — на МИ. И хотя россияне очень сильно опасаются онкологи-
ческих заболеваний, последние в 3,9 раза реже ста-
новятся причиной смерти, чем ССЗ. Рис. 2 Стандартизованные показатели смертности от ИБС
(на 100 тыс. населения). Рис. 2 Стандартизованные показатели смертности от ИБС
(на 100 тыс. населения). Рис. 3 Стандартизованные показатели смертности от ОИМ,
повторного ИМ (на 100 тыс. населения). 40,0
40,1
40,8
41,1
40,1
38,3
37,2
50,3
49,7
48,5
45,8
41,3
42,3
40,0
46,2
47,6
47,6
46,6
48,4
49,4
50,0
68,2
64,5
66,1
61,9
52,3
45,2
59,9
35
45
55
65
2006
2007
2008
2009
2010
2012
2013
РФ
МО
СПБ
Москва
-7,0%
+3,0%
-31,6%
-20,5%
Россия
Москва
Московская область
Санкт-Петербург От ССЗ умирает больше женщин, чем мужчин:
51% и 42%, соответственно, в структуре смертности
европейского населения соответствующего пола [1],
при этом женщины умирают в более поздние воз-
растные периоды. Почему в Москве смертность от сердечно-сосудистых
заболеваний ниже, чем в других регионах Российской
Федерации? В связи с разными
индикаторами оценки сравнительный анализ пока-
зателей сердечно-сосудистой заболеваемости между
странами не всегда возможен, а качественные дан-
ные по частоте новых случаев ССЗ и уровню заболе-
ваемости в большинстве стран отсутствуют. Многие
европейские страны и Всемирная организация
здравоохранения (ВОЗ) ориентируются в этом
вопросе на результаты и содержимое выписок боль-
ных из стационаров. Согласно этим данным в 80-е
и 90-е гг в Европе наблюдался рост заболеваемости
ишемической болезнью сердца (ИБС) и мозговыми
инсультами (МИ), однако, начиная с 2000-х годов,
произошла стабилизация, и рост прекратился [3]. С 2006г усредненный европейский показатель
госпитализаций по поводу ИБС составляет 800 чело-
век на 100 тыс. населения, а усредненный показа-
тель госпитализаций по поводу МИ колеблется
от 400 до 450 на 100 тыс. населения, начиная с 2004г
[4]. Необходимо учитывать, что показатели заболе-
ваемости не стандартизуются по возрасту. среди мужчин, так и среди женщин [8]. В 2005г стар-
товал приоритетный национальный проект “Здоро-
вье”, в рамках которого с 2008 по 2011гг была реали-
зована
масштабная
“сосудистая
программа”,
направленная на достижение качественно нового
современного уровня оказания помощи пациентам
с ССЗ: создание 55 региональных сосудистых цент- Рис. 1 Стандартизованные коэффициенты смертности населения
от БСК (на 100 тыс. населения). 547,1
585,9
613,3
683,8
681,6
722,3
779,1
735,3
833,2
362,0
387,6
391,3
508,8
473,5
502,8
516,2
547,4
570,3
640,1
757,1
824,4
795,7
787,2
663,4
650,7
776,2
852,3
470,9
606,0
513,4
520,5
608,9
640,1
744,5
657,7
706,3
350,0
550,0
750,0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
Москва
Московская область
Санкт-Петербург
МО
СПБ
Москва
-34,3%
-24,9%
-36,7%
-36,5%
РФ 547,1
585,9
613,3
683,8
681,6
722,3
779,1
735,3
833,2
362,0
387,6
391,3
508,8
473,5
502,8
516,2
547,4
570,3
640,1
757,1
824,4
795,7
787,2
663,4
650,7
776,2
852,3
470,9
606,0
513,4
520,5
608,9
640,1
744,5
657,7
706,3
350,0
550,0
750,0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
Москва
Московская область
Санкт-Петербург
МО
СПБ
Москва
-34,3%
-24,9%
-36,7%
-36,5%
РФ Рис. 1 Стандартизованные коэффициенты смертности населения
от БСК (на 100 тыс. населения). Рис. 2 Стандартизованные показатели смертности от ИБС
(на 100 тыс. населения). Рис. 3 Стандартизованные показатели смертности от ОИМ,
повторного ИМ (на 100 тыс. населения). Кардиоваскулярная терапия и профилактика, 2015; 14(2) Рис. 4 Стандартизованные показатели смертности от ЦВЗ
(на 100 тыс. населения). РФ
МО
СПБ
Москва
-39,0%
-35,9%
-36,5%
-33,6%
272,9
241,6
251,1
220,6
218,3
176,0
166,4
177,3
165,9
158,5
149,2
163,9
122,4
117,7
256,0
172,3
164,0
236,3
232,5
215,6
220,6
188,8
183,7
222,4
175,9
151,4
141,3
174,1
100,0
150,0
200,0
250,0
300,0
2006
2007
2008
2009
2010
2012
2013
Россия
Москва
Московская область
Санкт-Петербург ров и 146 первичных сосудистых отделений с общим
объемом финансирования >16 821 млн. рублей. К 2012г на 24% увеличилось число пациентов ССЗ,
которым была оказана высокотехнологичная
помощь — 335 vs 255 тыс. человек в 2009г [7]. С 2009г
приоритетное внимание уделяется формированию
здорового образа жизни населения и профилактике
неинфекционных заболеваний, в первую очередь
ССЗ, в учреждениях первичной медико-санитарной
помощи. С 2003 по 2013гг общий коэффициент смертно-
сти от БСК (число умерших на 100 тыс. населения)
снизился в РФ на 25% — 698,1 vs 927,5, тем не менее,
он остается более высоким, чем в начале 90-х
годов — 621,0 на 100 тыс. населения в 1991г. В 2012г
средний возраст смерти от ССЗ составил в России
среди мужчин 70,4 года, среди женщин 83 года [6]. 3456,9
2622,9
2964,2
3037,4
3018,3 3004,2 2983,0 3023,5
3040,8
1697,6
1682,5
1814,61819,5
1782,1
1881,7
1809,0
1734,4 1695,4
2659,7
1769,7
2688,0
2602,1
2687,8
2021,2
2115,6
1951,8
2456,6
2363,0
2607,8
2679,9
2326,1
3010,0
2517,3
2691,2
1994,1
2870,4
1500,0
1750,0
2000,0
2250,0
2500,0
2750,0
3000,0
3250,0
3500,0
2005
2006
2007
2008
2009
2010
2011
2012
2013
РФ
МО
СПБ
Москва
+31,8%
+50,3%
+1,6%
+0,9%
Россия
Москва
Московская область
Санкт-Петербург
Болезни системы кровообращения
Рис. 5 Зарегистрировано больных: взрослые с диагнозом, установ-
ленным впервые в жизни (на 100 тыс. населения). Необходимо отметить наличие существенных
различий между регионами РФ по показателям
заболеваемости и смертности от ССЗ. Различия
среднего возраста смерти от ССЗ между регионами
достигают 20 лет. В 2011г средний возраст смерти
от ССЗ составил в Москве 76 лет у мужчин и 83 года
у женщин, а в Ненецком и Ханты-Мансийском
автономных округах — 56 и 59 лет, соответственно,
у мужчин и 79 лет у женщин [9]. В статье представлен анализ динамики некото-
рых показателей, характеризующих ситуацию
с ССЗ, в двух мегаполисах — Москве и Санкт-
Петербурге, а также в Московской области и в сред-
нем по России за период 2005-2013гг. Показатели, представленные на рисунках 1-4,
9 и таблицах 2-4, основаны на данных Росстата
(“Демографический ежегодник России” соответ
ствующих лет). Почему в Москве смертность от сердечно-сосудистых
заболеваний ниже, чем в других регионах Российской
Федерации? В 2013г в РФ от болезней системы
кровообращения (БСК) на 100 тыс. населения соот-
ветствующего пола умерло 716,1 женщин (60,2%
в структуре смертности) и 677,2 мужчин (47,2%
в структуре смертности) [6]. Начиная с 2003г, в России отмечается снижение
смертности от ССЗ, которое с 2006г приобрело
более устойчивый и выраженный характер, как Рис. 3 Стандартизованные показатели смертности от ОИМ,
повторного ИМ (на 100 тыс. населения). 5 Кардиоваскулярная терапия и профилактика, 2015; 14(2) Таблица 1 Таблица 1
Зарегистрировано больных: взрослые с диагнозом, установленным впервые в жизни на 100 тыс. населения
2009
2010
2011
2012
2013
Инфаркт мозга
РФ
МО
СПБ
Москва
188,4
154,9
101,6
54,4
197,5
166,6
93,3
49,7
197,5
153,6
87,4
53,3
207,4
154,0
92,8
57,3
235,9
148,3
74,1
61,4
Субарахноидальное кровоизлияние
РФ
МО
СПБ
Москва
29,5
28,8
17,2
3,6
17,4
14,2
13,5
3,4
13,4
12,0
10,8
2,4
11,0
9,4
6,0
1,8
12,7
8,0
5,6
1,4
Внутримозговое или внутричерепное кровоизлияние
РФ
МО
СПБ
Москва
33,0
28,0
26,7
7,7
32,6
29,0
24,3
6,0
32,8
27,0
22,6
6,0
32,6
27.1
17,7
5,1
38,2
23,3
21,6
4,6
Инсульт неуточненный как кровоизлияние или инфаркт
РФ
МО
СПБ
Москва
232,1
130,1
101,7
51,5
195,5
105,6
87,0
40,5
143,3
86,5
69,4
27,8
105,0
92,4
59,1
22,4
103,6
73,6
51,5
16,2 ровано больных: взрослые с диагнозом, установленным впервые в жизни на 100 тыс. населения 6 Передовая статья
Рис. 6 Зарегистрировано больных: взрослые с диагнозом, установленным впервые в жизни (на 100 тыс. населения). 134,7
130,6
130,4
133,6
140,2
139,3
141,4
140,2
143,4
80,5
85,2
85,2
90,6
97,8
93,7
95,4
97,8
108,4
140,4
148,3
146,2
144,2
149,9
133,7
142,0
145,7
151,0
74,8
121,9
80,3
93,0
106,7
125,5
157,7
137,7
154,2
50,0
100,0
150,0
200,0
2005
2006
2007
2008
2009
2010
2011
2012
2013
ОИМ
27,8
24,7
25,2
25,5
21,2
20,6
20,2
20,3
20,8
12,1
10,8
11,7
11,9
9,5
10,9
12,0
12,2
13,4
26,1
25,8
22,6
22,2
23,1
23,6
25,0
24,8
22,3
10,4
19,1
11,3
15,2
18,0
28,8
26,1
23,2
23,6
0,0
10,0
20,0
30,0
2005
2006
2007
2008
2009
2010
2011
2012
2013
Повторный ИМ
РФ
МО
СПБ
Москва
-6,1%
-7,0%
-52,6%
-25,7%
РФ
МО
СПБ
Москва
+33,7%
+17,0%
-60,2%
-9,7%
Россия
Москва
Московская область
Санкт-Петербург Рис. 6 Зарегистрировано больных: взрослые с диагнозом, установленным впервые в жизни (на 100 тыс. населения). Таблица 1 Таблица 2
Динамика выборочных социально-экономических показателей
Среднедушевые денежные доходы населения в РФ и городе Москве* (руб.)
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
5170,4
6410,3
8111,9
10196,0
12602,7
14940,6
16856,9
18950,8
20754,9
23058,0
25646,6
Москва
16826,6
20899,1
24957,5
29802,6
35489,7
34207,4
41890,8
44051,5
47318,9
48621,6
55100,2
Среднемесячная номинальная начисленная заработная плата в РФ и городе Москве (руб.)
2000
2005
2007
2008
2009
2010
2011
Россия
2223,4
8554,9
13593,4
17290,1
18637,5
20952,2
23369,2
Москва
3229,3
14424,6
23623,3
30552,1
33358,0
38410,5
44898,7
Уровень безработицы в РФ и в городе Москве (%)
2000
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
10,6
7,2
7,2
6,1
6,3
8,4
7,3
6,5
5,5
5,5
Москва
3,9
0,8
1,6
0,8
0,9
2,7
1,8
1,4
0,8
Примечание: *Денежные доходы населения включают доходы лиц, занятых предпринимательской деятельностью, выплаченную
заработную плату наемных работников, социальные выплаты (пенсии, пособия, стипендии, страховые возмещения и прочие выплаты),
доходы от собственности в виде процентов по вкладам, ценным бумагам, дивидендов и другие доходы. Среднедушевые денежные доходы
(в месяц) исчисляются делением годового объема денежных доходов на 12 и на численность населения. Федеральная служба государственной
статистики РФ, октябрь 2014г. Динамика выборочных социально-экономических показателей
Среднедушевые денежные доходы населения в РФ и городе Москве* (руб.) Динамика выборочных социально-экономических показателе
жные доходы населения в РФ и городе Москве* (руб.) Примечание: *Денежные доходы населения включают доходы лиц, занятых предпринимательской деятельностью, выплаченную
заработную плату наемных работников, социальные выплаты (пенсии, пособия, стипендии, страховые возмещения и прочие выплаты),
доходы от собственности в виде процентов по вкладам, ценным бумагам, дивидендов и другие доходы. Среднедушевые денежные доходы
(в месяц) исчисляются делением годового объема денежных доходов на 12 и на численность населения. Федеральная служба государственной
статистики РФ, октябрь 2014г. Кардиоваскулярная терапия и профилактика, 2015; 14(2) 9 Потребление овощей, бахчевых, фруктов, ягод, рыбы
и рыбопродуктов в РФ и Москве (в кг в год на душу насе-
ления). 90,0100,0
103,0
101,0
106,0
109,0
109,0
77,0
78,077,0
85,0
82,084,086,0
88,0
94,0
74,0
79,0
79,0
81,0
80,0
84,0
85,0
87,0
76,0
74,0
76,0
68,0
71,0
86,0
77,0
89,0
71,0
67,0
64,0
64,0
61,0
60,0
59,0
62,0
61,0
61,0
62,0
65,0
70,0
90,0
89,0
122,0
50,0
100,0
150,0
13,1 14,6
15,0
19,4
21,1
22,6
23,7
30,1
17,1
13,9
9,89,9
10,4
10,5
11,0
11,3
11,9
12,6
9,3
9,5
9,7
10,4
11,9
15,9
12,3
20,4
25,4
18,4
17,4
15,6
14,6
13,8
13,3
12,5
12,9
12,3
13,4
14,7
16,4
17,5
18,7
23,2
24,3
5,0
15,0
25,0
35,0
Рыба и рыбопродукты
Овощи и бахчевые
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
Россия
Москва
Россия
Москва Рис. 8 ОПЖ мужчин, годы. учесть имевшие место здесь исходно более высокие
показатели смертности (рисунок 3). Смертность от цереброваскулярных заболева-
ний (ЦВЗ) — второй важнейший показатель смерт-
ности от ССЗ, снизилась существенно, причем наи-
более выражено в целом по РФ — на 39%. Во всех
трех рассматриваемых субъектах ЦВЗ стали уносить
более чем на треть меньше жизней (рисунок 4). Таким образом, в 2013г самые низкие стандар-
тизованные показатели смертности из всех рассма-
триваемых зафиксированы в Москве: от БСК —
362,0 на 100 тыс. населения, от ИБС — 196,9
на 100 тыс. и от ЦВЗ — 117,7 на 100 тыс. В отличие от показателей смертности, которые
в период 2005-2013гг в большинстве случаев снижа-
лись, показатели заболеваемости ССЗ демонстриро-
вали различную направленность и зависимость от орга-
низационных аспектов (рисунок 5). Число зарегистри-
рованных больных с диагнозом ССЗ, установленным
впервые в жизни, в РФ к 2012г (в сравнении с 2005г)
возросло на 15,9%. В 2013г, благодаря масштабной про-
грамме диспансеризации населения, число пациентов
с впервые выявленными ССЗ всего за один год (в срав-
нении с 2012г) увеличилось в России на 13,7%. Рис. 9 Потребление овощей, бахчевых, фруктов, ягод, рыбы
и рыбопродуктов в РФ и Москве (в кг в год на душу насе-
ления). ственная позитивная динамика имела место
в Санкт-Петербурге, где число больных ИМ, уста-
новленным впервые в жизни, сократилось наполо-
вину, а число больных повторным ИМ — на 60,2%
(рисунок 6). Обращает на себя внимание слабо
выраженная положительная динамика в отношении
числа случаев повторных ИМ в городе Москве. Кардиоваскулярная терапия и профилактика, 2015; 14(2) 70,8
76,4
80,0
80,1
78,7
77,5
76,1
74,3
75,2
73,6
70,0
69,0
65,4
65,4
64,7
69,3
68,3
67,5
69,8
65,4
69,7
76,0
74,2
71,6
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва
65,1
72,3
77,2
77,2
75,6
74,1
72,5
70,8
71,6
70,2
64,6
63,1
59,0
59,2
58,3
63,8
62,8
61,4
64,6
59,0
64,8
71,6
69,9
66,7
50,0
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва
56,0
60,0
61,0
64,0
66,0
70,0
69,0
75,0
58,0
35,0
39,0
39,043,046,0
48,0
51,0
54,0
32,0
27,0
30,0
33,0
31,0
28,0
29,0
29,0
71,0
62,0
61,0
62,0
57,0
52,0
46,0
45,0
43,0
41,0
37,0
42,0
45,0
42,0
43,0
47,0
49,0
25,0
35,0
45,0
55,0
65,0
75,0
1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013
Фрукты и ягоды
90,0100,0
103,0
101,0
106,0
109,0
109,0
94,0
74 0
79,0
79 0
81,0
80,0
84,0
85,0
87,0
76,0
74 0
76,0
71,0
90,0
89,0
122,0
100,0
150,0
Овощи и бахчевые
Овощи, бахчевые, фрукты и ягоды
138
139
173
159159166
154
89
77
86
100
96
105
105
109
133
128
123
119
116
111
106
104
145
170
163
157
152
139
139
122
89
90
119112105
103106
104
96
101
104
109
110119128
153
151
50
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
100
150
Россия
Москва
Россия
Москва и профилактика, 2015; 14(2)
ственная позитивная динамика имела место
в Санкт-Петербурге, где число больных ИМ, уста-
новленным впервые в жизни, сократилось наполо-
вину, а число больных повторным ИМ — на 60,2%
(рисунок 6). Обращает на себя внимание слабо
выраженная положительная динамика в отношении
числа случаев повторных ИМ в городе Москве. Рис. 9 Потребление овощей, бахчевых, фруктов, ягод, рыбы
и рыбопродуктов в РФ и Москве (в кг в год на душу насе-
ления). Кардиоваскулярная терапия и профилактика, 2015; 14(2) 56,0
60,0
61,0
64,0
66,0
70,0
69,0
75,0
58,0
35,0
39,0
39,043,046,0
48,0
51,0
54,0
32,0
27,0
30,0
33,0
31,0
28,0
29,0
29,0
71,0
62,0
61,0
62,0
57,0
52,0
46,0
45,0
43,0
41,0
37,0
42,0
45,0
42,0
43,0
47,0
49,0
25,0
35,0
45,0
55,0
65,0
75,0
1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013
Фрукты и ягоды
90,0100,0
103,0
101,0
106,0
109,0
109,0
77,0
78,077,0
85,0
82,084,086,0
88,0
94,0
74,0
79,0
79,0
81,0
80,0
84,0
85,0
87,0
76,0
74,0
76,0
68,0
71,0
86,0
77,0
89,0
71,0
67,0
64,0
64,0
61,0
60,0
59,0
62,0
61,0
61,0
62,0
65,0
70,0
90,0
89,0
122,0
50,0
100,0
150,0
13,1 14,6
15,0
19,4
21,1
22,6
23,7
30,1
17,1
13,9
9,89,9
10,4
10,5
11,0
11,3
11,9
12,6
9,3
9,5
9,7
10,4
11,9
15,9
12,3
20,4
25,4
18,4
17,4
15,6
14,6
13,8
13,3
12,5
12,9
12,3
13,4
14,7
16,4
17,5
18,7
23,2
24,3
5,0
15,0
25,0
35,0
Рыба и рыбопродукты
Овощи и бахчевые
Овощи, бахчевые, фрукты и ягоды
138
139
173
159159166
154
89
77
86
100
96
105
105
109
133
128
123
119
116
111
106
104
145
170
163
157
152
139
139
122
89
90
119112105
103106
104
96
101
104
109
110119128
153
151
50
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
100
150
Россия
Москва
Россия
Москва
Россия
Москва
Россия
Москва 56,0
60,0
61,0
64,0
66,0
70,0
69,0
75,0
58,0
35,0
39,0
39,043,046,0
48,0
51,0
54,0
32,0
27,0
30,0
33,0
31,0
28,0
29,0
29,0
71,0
62,0
61,0
62,0
57,0
52,0
46,0
45,0
43,0
41,0
37,0
42,0
45,0
42,0
43,0
47,0
49,0
25,0
35,0
45,0
55,0
65,0
75,0
1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013
Фрукты и ягоды
Овощи и бахчевые
Овощи, бахчевые, фрукты и ягоды
138
139
173
159159166
154
89
77
86
100
96
105
105
109
133
128
123
119
116
111
106
104
145
170
163
157
152
139
139
122
89
90
119112105
103106
104
96
101
104
109
110119128
153
151
50
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
100
150
Россия
Москва
Россия
Москва Овощи, бахчевые, фрукты и ягоды
138
139
173
159159166
154
89
77
86
100
96
105
105
109
133
128
123
119
116
111
106
104
145
170
163
157
152
139
139
122
89
90
119112105
103106
104
96
101
104
109
110119128
153
151
50
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
100
150 Фрукты и ягоды Рис. Кардиоваскулярная терапия и профилактика, 2015; 14(2) 56,0
60,0
61,0
64,0
66,0
70,0
69,0
75,0
58,0
35,0
39,0
39,043,046,0
48,0
51,0
54,0
32,0
27,0
30,0
33,0
31,0
28,0
29,0
29,0
71,0
62,0
61,0
62,0
57,0
52,0
46,0
45,0
43,0
41,0
37,0
42,0
45,0
42,0
43,0
47,0
49,0
25,0
35,0
45,0
55,0
65,0
75,0
1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013
Фрукты и ягоды
90,0100,0
103,0
101,0
106,0
109,0
109,0
77,0
78,077,0
85,0
82,084,086,0
88,0
94,0
74,0
79,0
79,0
81,0
80,0
84,0
85,0
87,0
76,0
74,0
76,0
68,0
71,0
86,0
77,0
89,0
71,0
67,0
64,0
64,0
61,0
60,0
59,0
62,0
61,0
61,0
62,0
65,0
70,0
90,0
89,0
122,0
50,0
100,0
150,0
13,1 14,6
15,0
19,4
21,1
22,6
23,7
30,1
17,1
13,9
9,89,9
10,4
10,5
11,0
11,3
11,9
12,6
9,3
9,5
9,7
10,4
11,9
15,9
12,3
20,4
25,4
18,4
17,4
15,6
14,6
13,8
13,3
12,5
12,9
12,3
13,4
14,7
16,4
17,5
18,7
23,2
24,3
5,0
15,0
25,0
35,0
Рыба и рыбопродукты
Овощи и бахчевые
Овощи, бахчевые, фрукты и ягоды
138
139
173
159159166
154
89
77
86
100
96
105
105
109
133
128
123
119
116
111
106
104
145
170
163
157
152
139
139
122
89
90
119112105
103106
104
96
101
104
109
110119128
153
151
50
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012
100
150
Россия
Москва
Россия
Москва
Россия
Москва
Россия
Москва Кардиоваскулярная терапия и профилактика, 2015; 14(2)
Рис. 7 ОПЖ при рождении, годы. Рис. 8 ОПЖ мужчин, годы. Таблица 3 Таблица 3
Случаи самоубийства в РФ и г. Москве (на 100 тыс. населения)
2006
2007
2008
2009
2010
2011
2012
2013
Россия
29,8
28,8
26,9
26,2
23,5
21,4
20,2
19,6
Москва
8,9
8,0
8,0
7,6
7,7
5,5
5,2
4,5 Случаи самоубийства в РФ и г. Москве (на 100 тыс. населения) в отношении снижения смертности от ИБС (рису-
нок 2). За период 2006-2013гг стандартизованный
показатель смертности от ИБС в Москве снизился
на 35,7%, что в 1,5 раза больше, чем в РФ, в 1,3 раза
больше, чем в Санкт-Петербурге, и в 2,6 раз больше,
чем в Московской области. в отношении снижения смертности от ИБС (рису-
нок 2). За период 2006-2013гг стандартизованный
показатель смертности от ИБС в Москве снизился
на 35,7%, что в 1,5 раза больше, чем в РФ, в 1,3 раза
больше, чем в Санкт-Петербурге, и в 2,6 раз больше,
чем в Московской области. Показатели заболеваемости и ОПЖ, представ-
ленные на рисунках 5-8 и в таблицах 1 и 6, основаны
на ежегодных Статистических материалах Мин-
здрава России “Медико-демографические показа-
тели Российской Федерации” и данных ВОЗ соот-
ветствующих лет. Смертность от острого инфаркта миокарда
(ОИМ) и повторного ИМ за обозначенный период
демонстрирует не столь однозначную тенденцию:
она снижалась в Санкт-Петербурге, Москве и РФ,
тогда как в Московской области показатель даже
повысился, хотя и незначительно. Особенно выра-
женная положительная динамика имела место
в Санкт-Петербурге (-31,6%), однако необходимо Стандартизованные в соответствии с Европей-
ским стандартом возрастной структуры показатели
смертности от БСК за рассматриваемый период
снижались, и в 2013г усредненный показатель по РФ
был на 34,3% ниже по сравнению с 2005г — 547,1 vs
833,2 на 100 тыс. населения (рисунок 1). В Москве
снижение было несколько более выраженным,
но еще более существенная разница имела место 7 Кардиоваскулярная терапия
Рис. 7 ОПЖ при рождении, годы. Рис. 8 ОПЖ мужчин, годы. 70,8
76,4
80,0
80,1
78,7
77,5
76,1
74,3
75,2
73,6
70,0
69,0
65,4
65,4
64,7
69,3
68,3
67,5
69,8
65,4
69,7
76,0
74,2
71,6
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва
65,1
72,3
77,2
77,2
75,6
74,1
72,5
70,8
71,6
70,2
64,6
63,1
59,0
59,2
58,3
63,8
62,8
61,4
64,6
59,0
64,8
71,6
69,9
66,7
50,0
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва Кардиоваскулярная терапия и профилактика, 2015; 14(2) Кардиоваскулярная терапия и профилактика, 2015; 14(2)
учесть имевшие место здесь исходно более высокие
показатели смертности (рисунок 3). Смертность от цереброваскулярных заболева-
ний (ЦВЗ) — второй важнейший показатель смерт-
ности от ССЗ, снизилась существенно, причем наи-
более выражено в целом по РФ — на 39%. Во всех
трех рассматриваемых субъектах ЦВЗ стали уносить
более чем на треть меньше жизней (рисунок 4). Таким образом, в 2013г самые низкие стандар-
тизованные показатели смертности из всех рассма-
триваемых зафиксированы в Москве: от БСК —
362,0 на 100 тыс. населения, от ИБС — 196,9
на 100 тыс. и от ЦВЗ — 117,7 на 100 тыс. В отличие от показателей смертности, которые
в период 2005-2013гг в большинстве случаев снижа-
лись, показатели заболеваемости ССЗ демонстриро-
вали различную направленность и зависимость от орга-
низационных аспектов (рисунок 5). Число зарегистри-
рованных больных с диагнозом ССЗ, установленным
впервые в жизни, в РФ к 2012г (в сравнении с 2005г)
возросло на 15,9%. В 2013г, благодаря масштабной про-
грамме диспансеризации населения, число пациентов
с впервые выявленными ССЗ всего за один год (в срав-
нении с 2012г) увеличилось в России на 13,7%. В то же время больных с диагнозом первого
ОИМ стало меньше и в среднем по России на 6,1%,
и в субъектах РФ (на четверть уменьшилось число
таких больных в Москве). При этом наиболее суще-
ственная позитивная динамика имела место
в Санкт-Петербурге, где число больных ИМ, уста-
новленным впервые в жизни, сократилось наполо-
вину, а число больных повторным ИМ — на 60,2%
(рисунок 6). Обращает на себя внимание слабо
выраженная положительная динамика в отношении
числа случаев повторных ИМ в городе Москве. Рис. 7 ОПЖ при рождении, годы. Рис. 8 ОПЖ мужчин, годы. 70,8
76,4
80,0
80,1
78,7
77,5
76,1
74,3
75,2
73,6
70,0
69,0
65,4
65,4
64,7
69,3
68,3
67,5
69,8
65,4
69,7
76,0
74,2
71,6
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва
65,1
72,3
77,2
77,2
75,6
74,1
72,5
70,8
71,6
70,2
64,6
63,1
59,0
59,2
58,3
63,8
62,8
61,4
64,6
59,0
64,8
71,6
69,9
66,7
50,0
60,0
70,0
80,0
1980
1985
1990
1995
2000
2005
2010
2012
2013
Евросоюз
РФ
Москва
Рис. 9 Потребление овощей, бахчевых, фруктов, ягод, рыбы
и рыбопродуктов в РФ и Москве (в кг в год на душу насе-
ления). Кардиоваскулярная терапия и профилактика, 2015; 14(2) в год на душу населения)
Водка и ликероводочные изделия
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
14,7
15,2
14,6
14,3
14,5
15
14,4
14,2
13,8
12,9
12,4
11,6
11
10,9
10,7
9,8
Москва
18,9
18,7
18,8
19,7
20,8
23
24
24,2
23,8
22,9
20,2
19,3
16,6
16,9
16,5
16
Вино и винодельческая продукция (без шампанских и игристых вин)
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
3,5
3,4
3,6
3,9
4,3
5
5,4
5,9
5,7
6,6
7,2
7,2
7,2
6,8
6,5
6,4
Москва
6,8
6,6
6,3
5
4,8
6,2
6,1
6,4
5,9
7,3
8,4
9,4
10,1
9,9
9,7
8,9
Пиво
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
27,6
30,6
35,8
43,5
48,7
52,7
58,4
62,2
70,1
80,9
79,7
71,8
70,3
70,8
71,1
70,2
Москва
30
56
61,8
78,9
85,7
89,1
99,1
108,5
111
101,4
95,8
79,7
87,8
91,3
89,9
92,5
Федеральная служба государственной статистики РФ, июнь 2014г. Таблица 4
Потребление алкоголя в РФ и Москве (в л. в год на душу населения) Потребление алкоголя в РФ и Москве (в л. в год на душу населения) Таблица 5
Удельный вес населения, систематически занимающегося
физической культурой и спортом в РФ и Москве (%)
2007
2008
2009
2010
2011
2012
Россия
14,83
15,90
17,32
18,38
20,58
22,49
Москва
12,30
12,42
17,42
18,42
22,37
23,01
Министерство спорта РФ, октябрь 2014г. Удельный вес населения, систематически занимающегося
физической культурой и спортом в РФ и Москве (%) В РФ в 80-е годы ОПЖ постепенно увеличива-
лась, однако в период 1990-1995гг. ОПЖ резко сни-
зилась — за пять лет на 4,6 года [8]. Только начиная
с 2005г данный показатель начал расти, и в 2011г
превысил ОПЖ 1990г [6]. Однако различия в ОПЖ
между РФ и странами Европейского союза остаются
существенными и составляют ~10 лет. В то же время
существенные региональные различия, имеющиеся
в России, касаются и этого важного показателя. ОПЖ населения в городе Москве в 1995-2000гг
резко увеличивается, достигает докризисного
уровня, и в дальнейшем демонстрирует тенденцию
к неуклонному повышению. В 2012г показатель
ОПЖ москвичей достигает 76 лет, и всего 4 года
разделяет москвичей и жителей Евросоюза. Таким
образом, за два последних десятилетия ОПЖ моск-
вичей увеличилась на 6,7 года. Необходимо отме-
тить, что отчетливая положительная динамика
показателей ОПЖ за этот период отмечалась
в т. ч. у мужчин (рисунок 8). Кардиоваскулярная терапия и профилактика, 2015; 14(2) Необходимо отме-
тить, что отчетливая положительная динамика
показателей ОПЖ за этот период отмечалась
в т. ч. у мужчин (рисунок 8). ОПЖ мужчин в Москве
достигла в 2013г 72,3 лет. В том же году ОПЖ моск-
вичек составила 80,2 года, россиянок — 76,3 лет. Какие факторы могли обеспечить более низкие
показатели смертности от ССЗ и большую ОПЖ
жителей столицы? Известна сопряженность показателей здоровья
населения с социально-экономическими факто-
рами. В этом отношении показательными представ-
ляются различия в социально-экономическом
уровне обеспеченности населения. Таблица 2
наглядно показывает, что среднедушевые денежные
доходы населения на всем протяжении рассматри-
Таблица 4
Потребление алкоголя в РФ и Москве (в л. в год на душу населения)
Водка и ликероводочные изделия
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
14,7
15,2
14,6
14,3
14,5
15
14,4
14,2
13,8
12,9
12,4
11,6
11
10,9
10,7
9,8
Москва
18,9
18,7
18,8
19,7
20,8
23
24
24,2
23,8
22,9
20,2
19,3
16,6
16,9
16,5
16
Вино и винодельческая продукция (без шампанских и игристых вин)
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
3,5
3,4
3,6
3,9
4,3
5
5,4
5,9
5,7
6,6
7,2
7,2
7,2
6,8
6,5
6,4
Москва
6,8
6,6
6,3
5
4,8
6,2
6,1
6,4
5,9
7,3
8,4
9,4
10,1
9,9
9,7
8,9
Пиво
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
27,6
30,6
35,8
43,5
48,7
52,7
58,4
62,2
70,1
80,9
79,7
71,8
70,3
70,8
71,1
70,2
Москва
30
56
61,8
78,9
85,7
89,1
99,1
108,5
111
101,4
95,8
79,7
87,8
91,3
89,9
92,5
Федеральная служба государственной статистики РФ, июнь 2014г. Таблица 5
Удельный вес населения, систематически занимающегося
физической культурой и спортом в РФ и Москве (%)
2007
2008
2009
2010
2011
2012
Россия
14,83
15,90
17,32
18,38
20,58
22,49
Москва
12,30
12,42
17,42
18,42
22,37
23,01
Министерство спорта РФ, октябрь 2014г. Таблица 4
Потребление алкоголя в РФ и Москве (в л. Кардиоваскулярная терапия и профилактика, 2015; 14(2) В то же время больных с диагнозом первого
ОИМ стало меньше и в среднем по России на 6,1%,
и в субъектах РФ (на четверть уменьшилось число
таких больных в Москве). При этом наиболее суще- 8 Передовая статья Передовая статья
В таблице 1 представлена динамика впервые
диагностированных случаев МИ за период 2009-
2013гг. Очевидно, благодаря модернизации здра-
воохранения, неуклонно снижалось число боль-
ных с неуточненными МИ (как кровоизлияние
или инфаркт): более чем в 2 раза в среднем по РФ
и в Санкт-Петербурге, в 1,8 раз в Московской
области и в 3 раза в Москве. По состоянию
на 2013г в Москве больных с неуточненным диаг-
нозом МИ оказалось более чем в 3 раза меньше,
чем в Санкт-Петербурге, и в 4,5 раза меньше, чем
в Московской области. Обращает на себя внима-
ние существенно меньшее число больных с впер-
вые установленными МИ в Москве. Больных
ишемическим инсультом в Москве было более
чем в 3,5 раз меньше, чем в среднем по России,
причем за весь период 2009-2013гг. Аналогичная
ситуация по геморрагическому инсульту, который
диагностировался за рассматриваемый период
в Москве более чем в 4-8 раз реже, чем в среднем
по РФ. Одним из важнейших параметров оценки
состояния здоровья являются показатели ожидае-
мой продолжительности жизни (ОПЖ) с рождения. На рисунке 7 представлена динамика ОПЖ в стра-
нах Евросоюза, РФ и Москве за 30-летний период. ОПЖ в странах Евросоюза на протяжении послед-
них десятилетий неуклонно росла, и в 2012г соста-
вила 80,0 лет: у мужчин — 77,2 лет и у женщин —
83,1 лет. ОПЖ жителей Евросоюза возросла
за 3 последние десятилетия на 6,7 лет у мужчин
и на 5,8 лет у женщин [10]. В РФ в 80-е годы ОПЖ постепенно увеличива-
лась, однако в период 1990-1995гг. ОПЖ резко сни-
зилась — за пять лет на 4,6 года [8]. Только начиная
с 2005г данный показатель начал расти, и в 2011г
превысил ОПЖ 1990г [6]. Однако различия в ОПЖ
между РФ и странами Европейского союза остаются
существенными и составляют ~10 лет. В то же время
существенные региональные различия, имеющиеся
в России, касаются и этого важного показателя. ОПЖ населения в городе Москве в 1995-2000гг
резко увеличивается, достигает докризисного
уровня, и в дальнейшем демонстрирует тенденцию
к неуклонному повышению. В 2012г показатель
ОПЖ москвичей достигает 76 лет, и всего 4 года
разделяет москвичей и жителей Евросоюза. Таким
образом, за два последних десятилетия ОПЖ моск-
вичей увеличилась на 6,7 года. Кардиоваскулярная терапия и профилактика, 2015; 14(2) Таблица 2
наглядно показывает, что среднедушевые денежные
доходы населения на всем протяжении рассматри- 9 Кардиоваскулярная терапия и профилактика, 2015; 14(2) Кар
Таблица 6
Некоторые показатели работы системы здравоохранения в РФ и Москве
Показатели
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
Москва
Россия
Москва
Россия
Москва
Россия
Москва
Россия Москва
Россия
Москва Россия
Москва
Россия
Москва
Россия Москва
Обеспеченность койками
на 100 000 населения
98,5
89,8
96,4
91,0
95,1
94,5
92,4
94,1
90,1
93,2
88,1
93,2
85,8
85,1
83,9
81,0
81,5
78,0
Средняя занятость койки
в год (в днях)
318,0
287,0
317,0
290,0
318,0
288,0
321,0
294,0
325,0
299,0
325,0
300,0
324,0
302,0
323,0
298,0
322,0
293,0
Число врачей на 100 000
населения
42,9
64,9
43,0
66,8
43,4
67,1
43,8
68,8
44,1
70,3
44,1
71,9
44,0
66,3
44,6
64,1
44,3
65,9
АПУ
й
5 1
7 4
5 1
7 8
5 3
8 3
5 3
8 5
5 3
8 6
5 3
8 8
6 0
9 7
6 9
10 9
6 6
11 3 Таблица 6
Некоторые показатели работы системы здравоохранения в РФ и Москве
Показатели
2005
2006
2007
2008
2009
2010
2011
2012
2013
Россия
Москва
Россия
Москва
Россия
Москва
Россия
Москва
Россия Москва
Россия
Москва Россия
Москва
Россия
Москва
Россия Москва
Обеспеченность койками
на 100 000 населения
98,5
89,8
96,4
91,0
95,1
94,5
92,4
94,1
90,1
93,2
88,1
93,2
85,8
85,1
83,9
81,0
81,5
78,0
Средняя занятость койки
в год (в днях)
318,0
287,0
317,0
290,0
318,0
288,0
321,0
294,0
325,0
299,0
325,0
300,0
324,0
302,0
323,0
298,0
322,0
293,0
Число врачей на 100 000
населения
42,9
64,9
43,0
66,8
43,4
67,1
43,8
68,8
44,1
70,3
44,1
71,9
44,0
66,3
44,6
64,1
44,3
65,9
АПУ
число посещений
на человека
в год по поводу
заболевания
5,1
7,4
5,1
7,8
5,3
8,3
5,3
8,5
5,3
8,6
5,3
8,8
6,0
9,7
6,9
10,9
6,6
11,3
число посещений
на человека в год
профилактических
2,1
1,9
2,1
1,9
2,4
2,2
2,5
2,4
2,5
2,5
2,5
2,5
2,5
2,5
2,6
2,5
2,9
3,1
Число лиц, которым ока-
зана скорая медицинская
помощь амбулаторно и при
выездах (на 1000 населения)
362,0
258,0
361,0
270,0
365,0
284,0
360,0
296,0
360,7
305,2
357,3
324,2
352,0
304,5
332,1
320,2
344,0
314,0 Таблица 6 ваемого периода в Москве были в 2-3 раза выше,
чем в среднем по РФ. Кардиоваскулярная терапия и профилактика, 2015; 14(2) ОПЖ мужчин в Москве
достигла в 2013г 72,3 лет. В том же году ОПЖ моск-
вичек составила 80,2 года, россиянок — 76,3 лет. В таблице 1 представлена динамика впервые
диагностированных случаев МИ за период 2009-
2013гг. Очевидно, благодаря модернизации здра-
воохранения, неуклонно снижалось число боль-
ных с неуточненными МИ (как кровоизлияние
или инфаркт): более чем в 2 раза в среднем по РФ
и в Санкт-Петербурге, в 1,8 раз в Московской
области и в 3 раза в Москве. По состоянию
на 2013г в Москве больных с неуточненным диаг-
нозом МИ оказалось более чем в 3 раза меньше,
чем в Санкт-Петербурге, и в 4,5 раза меньше, чем
в Московской области. Обращает на себя внима-
ние существенно меньшее число больных с впер-
вые установленными МИ в Москве. Больных
ишемическим инсультом в Москве было более
чем в 3,5 раз меньше, чем в среднем по России,
причем за весь период 2009-2013гг. Аналогичная
ситуация по геморрагическому инсульту, который
диагностировался за рассматриваемый период
в Москве более чем в 4-8 раз реже, чем в среднем
по РФ. Одним из важнейших параметров оценки
состояния здоровья являются показатели ожидае-
мой продолжительности жизни (ОПЖ) с рождения. На рисунке 7 представлена динамика ОПЖ в стра-
нах Евросоюза, РФ и Москве за 30-летний период. ОПЖ в странах Евросоюза на протяжении послед-
них десятилетий неуклонно росла, и в 2012г соста-
вила 80,0 лет: у мужчин — 77,2 лет и у женщин —
83,1 лет. ОПЖ жителей Евросоюза возросла
за 3 последние десятилетия на 6,7 лет у мужчин
и на 5,8 лет у женщин [10]. Какие факторы могли обеспечить более низкие
показатели смертности от ССЗ и большую ОПЖ
жителей столицы? Известна сопряженность показателей здоровья
населения с социально-экономическими факто-
рами. В этом отношении показательными представ-
ляются различия в социально-экономическом
уровне обеспеченности населения. Кардиоваскулярная терапия и профилактика, 2015; 14(2) населения)
Операция
Регион
Годы
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
АКШ
Россия
0,07
0,09
0,11
0,13
0,15
0,18
0,20
0,22
0,24
2,43
Москва
0,28
0,34
0,34
0,27
0,28
0,34
0,43
0,49
0,47
Санкт-Петербург
0,20
0,27
0,27
0,37
0,45
0,60
0,58
0,66
0,70
ЧКВ
Россия
0,08
0,10
0,15
0,19
0,25
0,31
0,37
0,46
0,56
0,68
Москва
0,51
0,64
0,74
0,96
1,10
1,21
1,35
1,45
1,53
Санкт-Петербург
0,16
0,27
0,41
0,52
0,70
0,98
1,12
1,42
1,88
ЧКВ+АКШ
Россия
0,16
0,19
0,26
0,33
0,41
0,49
0,57
0,68
0,80
3,10
Москва
0,78
0,98
1,08
1,23
1,38
1,55
1,78
1,94
2,00
Санкт-Петербург
0,36
0,53
0,68
0,89
1,15
1,58
1,70
2,08
2,59
Примечание: взято из Л. А. Бокерия, Р. Г. Гудкова Ежегодные сборники “Сердечно-сосудистая хирургия” 2004-2013гг; АКШ —
аортокоронарное шунтирование, ЧКВ — чрескожное коронарное вмешательство. Число вмешательств по реваскуляризации миокарда
в РФ, Москве и Санкт-Петербурге (на 1 тыс. населения) жения показателей смертности. Для достижения
дальнейшего устойчивого снижения смертности
от ССЗ и повышение продолжительности жизни
населения требуется реализация целого комплекса
профилактических мероприятий, направленных
на формирование здорового образа жизни, сниже-
ние уровней факторов риска, осуществление пер-
вичной профилактики ССЗ в сочетании с оптими-
зацией медицинской помощи, направленной
на достижение целевых показателей вторичной
профилактики ССЗ. жения показателей смертности. Для достижения
дальнейшего устойчивого снижения смертности
от ССЗ и повышение продолжительности жизни
населения требуется реализация целого комплекса
профилактических мероприятий, направленных
на формирование здорового образа жизни, сниже-
ние уровней факторов риска, осуществление пер-
вичной профилактики ССЗ в сочетании с оптими-
зацией медицинской помощи, направленной
на достижение целевых показателей вторичной
профилактики ССЗ. Согласно данным 2005-2013гг, представленным
в таблице 6, при сходной обеспеченности койками
среднегодовая занятость койки в Москве несколько
ниже, чем в среднем по России. Москвичи реже
госпитализируются и реже вызывают скорую меди-
цинскую помощь, но при этом чаще посещают вра-
чей амбулаторно-поликлинических учреждений
по поводу своих заболеваний (в 2013г в ~2 раза
чаще). Очевидно, не последнюю роль здесь играет
существенно более высокая обеспеченность вра-
чами на 100 тыс. населения (в 1,5-1,6 раз большая,
чем в среднем по России). В 2011-2013гг в Москве
был реализован важный этап модернизации столич-
ного здравоохранения, в рамках которого было
закуплено >60 тыс. единиц современного оборудо-
вания, включая аппаратуру для ультразвуковой
диагностики, компьютерные и магнитно-резонанс-
ные томографы, ангиографы, значительная часть
которого поступила в городские поликлиники. На базе стационаров Москвы были открыты 11
региональных сосудистых центров и 23 первичных
сосудистых отделения. Многие жители столицы
имеют возможность обращаться в целый ряд феде-
ральных учреждений кардиологического профиля. Кардиоваскулярная терапия и профилактика, 2015; 14(2) Среднедушевые денежные
доходы включают выплаченную заработную плату
наемных работников, социальные выплаты (пен-
сии, пособия, стипендии, страховые возмещения
и др.), доходы от собственности в виде процентов
по вкладам, ценным бумагам, дивидендам, другие
доходы, и исчисляются делением годового объема
денежных доходов на 12 и на численность населе-
ния. При этом если в 2000-х годах различия
по уровню номинальной начисленной заработной
платы были небольшими, то в последующем разрыв
увеличивался, и в последние годы среднемесячная
номинальная заработная плата в Москве была в 1,7-
1,9 раз выше, чем в среднем по стране, при этом
уровень безработицы в столице был ниже в 4-9 раз. Мониторинг психологических факторов риска
ССЗ на регулярной основе не проводился, однако
стабильно более низкий (в 3-4,4 раза) уровень числа
самоубийств в Москве в сравнении с РФ (таблица 3)
может указывать на большую психологическую
устойчивость населения и большую доступность как
экстренной, так и плановой психологической/пси-
хотерапевтической помощи. Все больше число россиян следует принципам
здорового питания: начиная с 2000г, отмечается ста-
бильный рост потребления овощей, фруктов, бахче-
вых и ягод как в целом в РФ, так и в столице (рису-
нок 9). При этом москвичи едят больше фруктов
и ягод, но меньше овощей и бахчевых. Потребление
рыбы и рыбопродуктов в годы перестройки сущест-
венно снизилось, но, начиная с 2000г начало расти. В Москве рыбы и рыбопродуктов за весь представ-
ленный период потреблялось существенно больше,
чем в среднем по России, особенно существенная
разница отмечалась в последние годы: в 2012г потре-
бление рыбы в столице на 43% превысило средне-
российский показатель. При этом в сравнении
с 2000г потребление рыбы в Москве выросло более
чем в 2 раза. Потребление пива, вина и особенно водки
и ликероводочных изделий в период 1998-2013гг
в Москве было выше в сравнении со среднероссий-
скими показателями (таблица 4). Нельзя исклю-
чить, что большие различия в потреблении алкоголя
в определенной степени объясняются лучшим уче-
том потребляемого алкоголя в столице и большим
количеством неучтенного алкоголя в целом по Рос-
сии. Тем не менее, представленные данные кос-
венно указывают на несостоятельность достаточно
часто высказываемой точки зрения о том, что алко-
голь является определяющей причиной высоких
показателей смертности населения в России. Удельный вес населения, систематически зани-
мающегося физкультурой и спортом, начиная
с 2009г в Москве и РФ, был примерно одинаковым
(таблица 5). 10 Передовая статья Таблица 7
Число вмешательств по реваскуляризации миокарда
в РФ, Москве и Санкт-Петербурге (на 1 тыс. Кардиоваскулярная терапия и профилактика, 2015; 14(2) Все это существенно повысило доступность высо-
котехнологичной помощи для жителей столицы. Закономерно, что число вмешательств по реваску-
ляризации миокарда (на 1 тыс. населения) в Москве
превышает среднероссийский уровень в 2,5-3 раза
(таблица 7). В то же время, начиная с 2009г, в Санкт-
Петербурге операций по реваскуляризации мио-
карда выполняется примерно столько же или
больше (в разные годы), чем в Москве, при этом
показатели смертности от ИБС в Санкт-Петербурге
на ~30% выше, чем в столице. Очевидно, что соот-
ветствие медицинской помощи современным стан-
дартам, включая доступность высокотехнологичной
помощи, играет большую роль, тем не менее, эта
роль не является определяющей в отношении сни- Boytsov SA, Samorodskaya IV. Dynamics of Cardiovascular Mortality Among Men
and Women in Subjects of Russian Federation (2002 to 2011) Kardiologiia 2014; 4:
4-18. Russian (Бойцов С. А., Самородская И. В. Динамика сердечно-сосудистой
смертности среди мужчин и женщин в субъектах Российской Федерации (2002-
2011 гг.). Кардиология 2014; 4: 4-18). 3.
Nichols M, Townsend N, Scarborough P and Rayner M. European Cardiovascular
Disease Statistics. 2012 (European Heart Network and European Society of
Cardiology) клинической и высокотехнологической медицин-
ской помощи для жителей столицы. клинической и высокотехнологической медицин-
ской помощи для жителей столицы. клинической и высокотехнологической медицин-
ской помощи для жителей столицы. Для достижения дальнейшего устойчивого сни-
жения смертности от ССЗ и повышения продолжи-
тельности жизни населения требуется реализация
целого комплекса профилактических мероприятий, 8.
Oganov RG, Maslennikova GYa. Demographic trends in the Russian Federation: the
impact of cardiovascular disease. Cardiovascular Therapy and Prevention 2012;
1: 5-10. Russian (Оганов Р. Г., Масленникова Г. Я. Демографические тенден-
ции в Российской Федерации: вклад болезней системы кровообращения.
Кардиоваская терапия и профилактика 2012; 1: 5-10). Кардиоваскулярная терапия и профилактика, 2015; 14(2) направленных на формирование здорового образа
жизни, снижение уровней факторов риска, осу-
ществление первичной профилактики ССЗ в соче-
тании с оптимизацией медицинской помощи,
направленной на достижение целевых показателей
вторичной профилактики ССЗ. World Health Organization. Global Health Observatory Data Repository, Mortality
and global health estimates: Life expectancy. http://apps.who.int/gho/data/node.
main.687?Lang=en (30 August 2013): WHO; 2013. Yakovleva TV. Improving the system of providing medical care to patients with
cardiovascular diseases in the Russian Federation. 20 March, 2013. http://old.
rosminzdrav.ru/health/cardiovascular/97 Russian (Яковлева Т. В. Совершен
ствование системы оказания медицинской помощи больным с сердечно-сосу-
дистыми заболеваниями в Российской Федерации. 20 марта 2013 г. http://old.
rosminzdrav.ru/health/cardiovascular/97). 4.
World Health Organization Regional Office for Europe. European Health for All
Database (HFA-DB). http://data.euro.who.int/hfadb/ (30 August 2013):WHO
Regional Office for Europe, Copenhagen, Denmark; 2013. Заключение Начиная с 2003г, в РФ отмечается снижение
смертности от ССЗ, которое с 2006г приобрело
более устойчивый и выраженный характер как среди
мужчин, так и среди женщин. С 2003 по 2013гг
общий коэффициент смертности от БСК снизился
на 25% — 698,1 vs 927,5, тем не менее, он остается
более высоким, чем в начале 90-х годов — 621,0
на 100 тыс. населения в 1991г. Отмечаются сущест-
венные различия между регионами РФ по показате-
лям заболеваемости и смертности от ССЗ. За период
2006-2013гг стандартизованный показатель смерт-
ности от ИБС в Москве снизился на 35,7%, что
в 1,5 раза больше, чем в РФ, в 1,3 раза больше, чем
в Санкт-Петербурге, и в 2,6 раз больше, чем в Мос-
ковской области. В 2012г показатель ОПЖ москви-
чей достигает 76 лет, и всего 4 года разделяет моск-
вичей и жителей Евросоюза. ОПЖ мужчин в Москве
достигла в 2013г 72,3 лет. Существенно более низкие
показатели смертности от ССЗ в Москве в сравне-
нии со среднероссийскими, очевидно, обусловлены
более высоким социально-экономическим уровнем
жизни населения, большей психологической устой-
чивостью и большей доступностью психологиче-
ской (психотерапевтической) помощи, более высо-
ким уровнем потребления рыбы, фруктов и ягод,
а также большей доступностью амбулаторно-поли- 11 Кардиоваскулярная терапия и профилактика, 2015; 14(2) Prevention in Clinical Practice (constituted by representatives of nine societies
and by invited experts). Developed with the special contribution of the European
Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J
2012; 33: 1635-1701.
3.
Nichols M, Townsend N, Scarborough P and Rayner M. European Cardiovascular
Disease Statistics. 2012 (European Heart Network and European Society of
Cardiology)
4.
World Health Organization Regional Office for Europe. European Health for All
Database (HFA-DB). http://data.euro.who.int/hfadb/ (30 August 2013):WHO
Regional Office for Europe, Copenhagen, Denmark; 2013.
5.
World Health Organization. WHO Mortality Database: 1st May 2013 update. http://
www.who.int/healthinfo/statistics/mortality_rawdata/en/index.html
(30August
2013): World Health Organization, Department of Health Statistics and Information
Systems, Geneva, Switzerland, 2013.
6.
Demographic Yearbook of Russia 2012: Statistical handbook. Rosstat. M.: 2012; 535
p. Russian (Демографический ежегодник России 2012. Стат сборник. Росстат.
M.: 2012; 535 с). 5.
World Health Organization. WHO Mortality Database: 1st May 2013 update. http://
www.who.int/healthinfo/statistics/mortality_rawdata/en/index.html
(30August
2013): World Health Organization, Department of Health Statistics and Information
Systems, Geneva, Switzerland, 2013. Yakovleva TV. Improving the system of providing medical care to patients with
cardiovascular diseases in the Russian Federation. 20 March, 2013. http://old.
rosminzdrav.ru/health/cardiovascular/97 Russian (Яковлева Т. В. Совершен
ствование системы оказания медицинской помощи больным с сердечно-сосу-
дистыми заболеваниями в Российской Федерации. 20 марта 2013 г. http://old.
rosminzdrav.ru/health/cardiovascular/97).
Oganov RG, Maslennikova GYa. Demographic trends in the Russian Federation: the
impact of cardiovascular disease. Cardiovascular Therapy and Prevention 2012;
1: 5-10. Russian (Оганов Р. Г., Масленникова Г. Я. Демографические тенден-
ции в Российской Федерации: вклад болезней системы кровообращения.
Кардиоваская терапия и профилактика 2012; 1: 5-10).
Boytsov SA, Samorodskaya IV. Dynamics of Cardiovascular Mortality Among Men
and Women in Subjects of Russian Federation (2002 to 2011) Kardiologiia 2014; 4:
4-18. Russian (Бойцов С. А., Самородская И. В. Динамика сердечно-сосудистой
смертности среди мужчин и женщин в субъектах Российской Федерации (2002-
2011 гг.). Кардиология 2014; 4: 4-18).
World Health Organization. Global Health Observatory Data Repository, Mortality
and global health estimates: Life expectancy. http://apps.who.int/gho/data/node.
main.687?Lang=en (30 August 2013): WHO; 2013. Литература 1. European Mortality Database. Mortality indicators by 67 causes of death, age, sex. HFA-MDB. UpdatedJuly 2011. 1. European Mortality Database. Mortality indicators by 67 causes of death, age, sex. HFA-MDB. UpdatedJuly 2011. 1. European Mortality Database. Mortality indicators by 67 causes of death, age, sex. HFA-MDB. UpdatedJuly 2011. 2. Perk J, de Backer BG, Gohlke H. European Guidelines on cardiovascular disease
prevention in clinical practice (version 2012). The Fifth Joint Task Force of the
European Society of Cardiology and Other Societies on Cardiovascular Disease
Prevention in Clinical Practice (constituted by representatives of nine societies
and by invited experts). Developed with the special contribution of the European
Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J
2012; 33: 1635-1701. 3. Nichols M, Townsend N, Scarborough P and Rayner M. European Cardiovascular
Disease Statistics. 2012 (European Heart Network and European Society of
Cardiology) 4. World Health Organization Regional Office for Europe. European Health for All
Database (HFA-DB). http://data.euro.who.int/hfadb/ (30 August 2013):WHO
Regional Office for Europe, Copenhagen, Denmark; 2013. 5. World Health Organization. WHO Mortality Database: 1st May 2013 update. http://
www.who.int/healthinfo/statistics/mortality_rawdata/en/index.html
(30August
2013): World Health Organization, Department of Health Statistics and Information
Systems, Geneva, Switzerland, 2013. 6. Demographic Yearbook of Russia 2012: Statistical handbook. Rosstat. M.: 2012; 535
p. Russian (Демографический ежегодник России 2012. Стат сборник. Росстат. M.: 2012; 535 с). 12 12
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https://openalex.org/W3044216553
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https://europepmc.org/articles/pmc7384859?pdf=render
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English
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Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity
|
eLife
| 2,020
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cc-by
| 17,223
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Competing interest: See
page 17 Funding: See page 17
Received: 21 April 2020
Accepted: 01 July 2020
Published: 27 July 2020 RESEARCH ARTICLE Interrogating the recognition landscape
of a conserved HIV-specific TCR reveals
distinct bacterial peptide cross-reactivity Introduction Cross-reactivity represents an intrinsic feature of immunity that allows post-thymically selected T cell
receptors (TCRs) to recognize distinct peptides originating from diverse microbial origins when
bound by a single MHC. This considerable immune redundancy exists to cover the deficit between
the predicted number of MHC-bound antigenic peptide epitopes that humans encounter during
their lifetime (~1012) versus the number of unique TCRs available in the periphery (<108)
(Mason, 1998; Wucherpfennig, 2004; Sewell, 2012). A biologically relevant consequence of cross-
reactivity where infection with an initial microbe alters immunity to subsequent, non-related infecting
pathogen - termed heterotypic immunity (Gil et al., 2015) - has been reported to influence the
course of natural infections in humans (Aslan et al., 2017), and can also affect vaccine-mediated
immunity and immune-mediated checkpoint therapy outcomes in vivo (Sioud, 2018). Where such
cross-reactivity narrows the immune response and/or skews the enrichment of non-protective
immune responses, sub-optimal
immunity following infection
or post-vaccination can
result
(Clute et al., 2005; Aslan et al., 2017). The potential to elicit immune pathology also exists, for Interrogating the recognition landscape
of a conserved HIV-specific TCR reveals
distinct bacterial peptide cross-reactivity Juan L Mendoza1†, Suzanne Fischer1, Marvin H Gee1, Lilian H Lam2,3,
Simon Brackenridge4, Fiona M Powrie2,3, Michael Birnbaum1,5,
Andrew J McMichael4, K Christopher Garcia1,6*, Geraldine M Gillespie4* Juan L Mendoza1†, Suzanne Fischer1, Marvin H Gee1, Lilian H Lam2,3,
Simon Brackenridge4, Fiona M Powrie2,3, Michael Birnbaum1,5,
Andrew J McMichael4, K Christopher Garcia1,6*, Geraldine M Gillespie4* Juan L Mendoza1†, Suzanne Fischer1, Marvin H Gee1, Lilian H Lam2,3, 1Department of Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, United States; 2Kennedy Institute of Rheumatology, University
of Oxford, Oxford, United Kingdom; 3Translational Gastroenterology Unit, Nuffield
Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom;
4Nuffield Department of Medicine, University of Oxford, NDM Research Building,
Old Road Campus, Headington, Oxford, United Kingdom; 5Koch Institute at MIT,
Cambridge, United States; 6Howard Hughes Medical Institute, Stanford University
School of Medicine, Stanford, United States Abstract T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered
during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where
previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has
been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to
consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell
exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a ‘public’,
HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via
screening of MHC-restricted libraries comprising ~2108 sequence-diverse peptides, AGA1 TCR
specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides
to probe the non-redundant protein database, bacterial peptides that elicited functional responses
by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC
class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is
discussed. *For correspondence:
kcgarcia@stanford.edu (KCG);
geraldine.gillespie@ndm.ox.ac.uk
(GMG) *For correspondence:
kcgarcia@stanford.edu (KCG);
geraldine.gillespie@ndm.ox.ac.uk
(GMG) *For correspondence:
kcgarcia@stanford.edu (KCG);
geraldine.gillespie@ndm.ox.ac.uk
(GMG) Present address: †Pritzker
School of Molecular Engineering
and Department of Biochemistry
and Molecular Biology,
University of Chicago, Chicago,
United States *For correspondence:
kcgarcia@stanford.edu (KCG);
geraldine.gillespie@ndm.ox.ac.uk
(GMG) Reviewing editor: Pamela J
Bjorkman, California Institute of
Technology, United States Reviewing editor: Pamela J
Bjorkman, California Institute of
Technology, United States Reviewing editor: Pamela J
Bjorkman, California Institute of
Technology, United States
Copyright Mendoza et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. Copyright Mendoza et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 1 of 21 Research article Research article Research article Immunology and Inflammation example,
via
the
amplification
of
T
cells
with
autoimmune
potential
(Rist
et
al.,
2009;
ImMaDiab Study Group et al., 2018). However, beneficial effects could also materialize if a cross-
reactive response driven by the primary infecting microbe is protective against a subsequent infec-
tion involving a non-related pathogen (Su et al., 2013). g
p
g
The possibility that cross-reactive T cells could influence immunity to non-related pathogens
exists but has not been extensively explored – this is particularly relevant in the setting of HIV infec-
tion, where viral-induced gut-intestinal (GI) barrier dysbiosis could potentially allow commensal or
pathogenic microbes to influence virus-specific T cell populations. Although this has been addressed
to some extent in the context of HIV infection for MHC class II and CD4 T cells (Su et al., 2013;
Campion et al., 2014), this topic has received only limited attention in relation to CD8+ T cells and
MHC class I restricted epitopes (Pohlmeyer et al., 2018). To explore this, we chose a specific exam-
ple corresponding to a ‘public’ HIV-specific TCR, namely, the AGA1 TCR, that recognizes the immu-
nodominant, HLA-B*57:01-restricted gag-derived KAFSPEVIPMF (KF11) peptide epitope (Stewart-
Jones et al., 2012). The KF11 peptide regularly elicits a set of closely related ‘public’ TCRs utilizing
highly conserved TCR V alpha (AV) 5, V beta (BV) 19 and CDR3 A/B hypervariable chain motifs that
were originally identified in non-related, HIV-infected B*57:01+ patients who progress slowly to
AIDS (Gillespie et al., 2006; Yu et al., 2007). Reviewing editor: Pamela J
Bjorkman, California Institute of
Technology, United States More recently, KF11-specific T cells utilizing near iden-
tical AGA1-related BV19-CDR3-BJ1-2 chains, or cells carrying conserved CDR3-BJ1-2 motif (x-Y-G-Y-
T, where x = polar residues) segments on diverse BV chain backgrounds, have been reported in
both slow and normal progressor HIV+ patients (Mendoza et al., 2012; Simons et al., 2008), sug-
gesting that the x-Y-G-Y-T motif is frequently selected in response to the KF11 epitope. Although
AGA1 TCR-mediated cross-reactive responses against broad clade variants, including C clade
escape variants of KF11 have been described (Gillespie et al., 2002), there are no documented
cases of cross reactivity against non-HIV peptides. Using
the
yeast
display-based
approach
(Adams
et
al.,
2011;
Birnbaum
et
al.,
2014a; Gee et al., 2018) where ~ 2108 random peptides were linked to and presented by HLA-
B*57 on the surface of yeast cells, we interrogated the cross-reactivity of the AGA1 TCR. This strat-
egy revealed a number of important features of AGA1 TCR-mediated specificity, including character-
ization of the minimal, central dipeptide motif within the 11 amino acid peptide sequences required
to facilitate AGA1 TCR binding. In addition, by using TCR-retrieved peptide libraries to interrogate
the non-redundant sequence databases, a number of bacterial peptides that elicited strong func-
tional responses by AGA1-expressing T cell clones were identified. The implications of these findings
are discussed. Results HLA-B57 yeast display library design and validation
Yeast cell libraries displaying HLA-B*57:03-b2m linked to a randomized peptide library were gener-
ated to screen the breadth of AGA1 TCR binding (see Figure 1A (i) for overview of strategy). The
peptide-b2m-MHC library construct was based on previous designs (Hansen et al., 2009;
Birnbaum et al., 2014a; Gee et al., 2018) and comprised a single peptide test reagent (KF11) or
randomized peptide libraries linked to human b2m and the heavy chain of HLA-B*57:03 encoding a
Y84A mutation. A myc-tag incorporated between the MHC a3 domain and the Aga2 yeast protein
allows
the
monitoring
of
peptide-b2m-HLA-B*57:03
expression
on
the
yeast
cell
surface
(Figure 1A (ii)). The functionality of yeast displayed HLA-B*57:03 was confirmed initially by staining a
control KF11-b2m-HLA-B*57:03 yeast display with fluorescently conjugated AGA1 TCR tetramers
(Figure 1B). Following validation, mutagenized peptide libraries were produced. For the preparation
of these libraries, peptide residues at positions 2 (Ala/Ser/Thr) and 11 (Phe/Trp/Tyr) (CW) were
restricted to retain the preferred HLA-B*57:03 anchor binding motifs (Barber et al., 1997), whilst
the remaining nine amino acids were randomized using degenerate NNK nucleotide addition as
described previously (Adams et al., 2011; Figure 1A (ii)). The theoretical diversity was 1.06E15
unique nucleotide sequences (4.61E12 unique protein sequences), of which the experimental library
contained 2 108 unique transformants. To evaluate the peptide repertoire that allowed AGA1 TCR binding in the context of HLA-
B*57:03, streptavidin-coated magnetic beads (MACS) conjugated to biotinylated AGA1 TCR were Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 2 of 21 Research article Immunology and Inflammation Figure 1. Schematic overview of the peptide-b2m-HLA-B*57:03 yeast display platform. (A) (i) Plasmid libraries encoding semi-randomized peptide
sequences generated by polymerase chain reaction amplification using rationally designed degenerate oligos and linked to the HLA-B*57:03 heavy
chain and b2m were (1) transfected into yeast cells to generate libraries (2). Biotinylated AGA1 TCR reagents conjugated to streptavidin-coated
magnetic beads were subsequently used to purify TCR-reactive peptide-MHC complexes expressed on yeast cells (3). The identities of peptides
allowing TCR binding were confirmed via plasmid extraction and next generation sequencing analyses (4). This process was repeated up to four times
under successively stringent rounds of selection. (ii) Outline of the peptide-b2m-HLA-B*57:03-Aga2 yeast single chain construct illustrating the peptide-
b2m-HLA-B*57:03 fusion chain, interspersed with repeating Gly-Ser linker sequence motifs (upper image). Results Individual peptide sequences (X-axis) versus their frequencies (number of sequence reads) in the naı¨ve, Round 1,
Round 2 and Round 3 AGA1 TCR-selected libraries are denoted (Y-axis) in (i), with the three dominant peptides following Round 3 enrichment
illustrated in (ii) as exploded pie chart slices. Percentage (%) peptide frequencies are indicated. The corresponding peptide legend is color-coded
according to RasMol amino schema. Figure 1. Schematic overview of the peptide-b2m-HLA-B*57:03 yeast display platform. (A) (i) Plasmid libraries encoding semi-randomized peptide
sequences generated by polymerase chain reaction amplification using rationally designed degenerate oligos and linked to the HLA-B*57:03 heavy
chain and b2m were (1) transfected into yeast cells to generate libraries (2). Biotinylated AGA1 TCR reagents conjugated to streptavidin-coated
magnetic beads were subsequently used to purify TCR-reactive peptide-MHC complexes expressed on yeast cells (3). The identities of peptides
allowing TCR binding were confirmed via plasmid extraction and next generation sequencing analyses (4). This process was repeated up to four times
under successively stringent rounds of selection. (ii) Outline of the peptide-b2m-HLA-B*57:03-Aga2 yeast single chain construct illustrating the peptide-
b2m-HLA-B*57:03 fusion chain, interspersed with repeating Gly-Ser linker sequence motifs (upper image). HLA-B*57:03-preferred anchor binding
residues were fixed at position 2 (p2) to Ala/Ser/Thr and position 11 (p11, CW) to Phe/Trp/Tyr, whereas non-anchor residues were allowed to express
any amino acid (X) throughout the selection process (lower image). (B) Staining of a KF11-b2m-HLA-B*57:03 yeast display test platform with AGA1 TCR
fluorescent tetramers. cMyc staining denotes cell-surface expression of the KF11-b2m-HLA-B*57:03 construct, with SA-647 staining employed to
monitor non-specific binding of the fluorescent label to transfected yeast cells. (C) Frequency of top 20 Round 3 peptide sequences, pre-(naı¨ve) and
post-AGA1 TCR-mediated selection. Individual peptide sequences (X-axis) versus their frequencies (number of sequence reads) in the naı¨ve, Round 1,
Round 2 and Round 3 AGA1 TCR-selected libraries are denoted (Y-axis) in (i), with the three dominant peptides following Round 3 enrichment
illustrated in (ii) as exploded pie chart slices. Percentage (%) peptide frequencies are indicated. The corresponding peptide legend is color-coded
according to RasMol amino schema. The online version of this article includes the following source data for figure 1: Source data 1. AGA1 TCR-recovered top 20 peptides_Naı¨ve to Round 3 screens. Source data 2 AGA1 TCR recovered top 20 peptides Round 3 frequencies Source data 1. AGA1 TCR-recovered top 20 peptides_Naı¨ve to Round 3 screens
Source data 2. Results HLA-B*57:03-preferred anchor binding
residues were fixed at position 2 (p2) to Ala/Ser/Thr and position 11 (p11, CW) to Phe/Trp/Tyr, whereas non-anchor residues were allowed to express
any amino acid (X) throughout the selection process (lower image). (B) Staining of a KF11-b2m-HLA-B*57:03 yeast display test platform with AGA1 TCR
fluorescent tetramers. cMyc staining denotes cell-surface expression of the KF11-b2m-HLA-B*57:03 construct, with SA-647 staining employed to
monitor non-specific binding of the fluorescent label to transfected yeast cells. (C) Frequency of top 20 Round 3 peptide sequences, pre-(naı¨ve) and
post-AGA1 TCR-mediated selection. Individual peptide sequences (X-axis) versus their frequencies (number of sequence reads) in the naı¨ve, Round 1,
Round 2 and Round 3 AGA1 TCR-selected libraries are denoted (Y-axis) in (i), with the three dominant peptides following Round 3 enrichment
illustrated in (ii) as exploded pie chart slices. Percentage (%) peptide frequencies are indicated. The corresponding peptide legend is color-coded
according to RasMol amino schema. The online version of this article includes the following source data for figure 1: Figure 1. Schematic overview of the peptide-b2m-HLA-B*57:03 yeast display platform. (A) (i) Plasmid libraries encoding semi-randomized peptide
sequences generated by polymerase chain reaction amplification using rationally designed degenerate oligos and linked to the HLA-B*57:03 heavy
chain and b2m were (1) transfected into yeast cells to generate libraries (2). Biotinylated AGA1 TCR reagents conjugated to streptavidin-coated
magnetic beads were subsequently used to purify TCR-reactive peptide-MHC complexes expressed on yeast cells (3). The identities of peptides
allowing TCR binding were confirmed via plasmid extraction and next generation sequencing analyses (4). This process was repeated up to four times
under successively stringent rounds of selection. (ii) Outline of the peptide-b2m-HLA-B*57:03-Aga2 yeast single chain construct illustrating the peptide-
b2m-HLA-B*57:03 fusion chain, interspersed with repeating Gly-Ser linker sequence motifs (upper image). HLA-B*57:03-preferred anchor binding
residues were fixed at position 2 (p2) to Ala/Ser/Thr and position 11 (p11, CW) to Phe/Trp/Tyr, whereas non-anchor residues were allowed to express
any amino acid (X) throughout the selection process (lower image). (B) Staining of a KF11-b2m-HLA-B*57:03 yeast display test platform with AGA1 TCR
fluorescent tetramers. cMyc staining denotes cell-surface expression of the KF11-b2m-HLA-B*57:03 construct, with SA-647 staining employed to
monitor non-specific binding of the fluorescent label to transfected yeast cells. (C) Frequency of top 20 Round 3 peptide sequences, pre-(naı¨ve) and
post-AGA1 TCR-mediated selection. Cross-reactivity of AGA1+ T cell clones to library- and database-derived
peptide hits The top library peptide sequences from Round 3 enrichments were used as input to generate a posi-
tion probability matrix, used previously to predict antigens to score peptide sequences using a slid-
ing window along proteins from the ‘non-redundant’ (nr)-database containing both human and
microbial proteins (Gee et al., 2018; Birnbaum et al., 2014b). From this search, six KF11-like hits
were returned (Supplementary file 1 - Table 1), implying that the yeast display selection results
could uncover the cognate specificity of this TCR. However, a substantial number of microbial pep-
tide hits that do not resemble the KF11 peptide dominated the sequence homology searches
(Supplementary file 1 -Table 2). From the top 20 peptides hits retrieved, six microbial peptide hits,
(Mic 1 to 6), were selected for follow-up in T cell functional studies (Table 1); their selection was
biased to include sequence from microbes potentially encountered by humans - either those associ-
ated with the environment (e.g. soil-derived), or those previously isolated from, or linked to infec-
tions in humans (Cha´vez de Paz et al., 2004; Chhour et al., 2005; Wolfgang et al., 2012). Peptides
identified from the yeast display library (Lib 1 to 6) and individual ‘nr’-derived microbial peptides
predicted to be recognized by the AGA1 TCR were synthesized and tested for their ability to induce
IFNg secretion from an AGA1-expressing T cell clone (Figure 3A (i-ii) and Table 1 for the sequence
identity of peptides). All responses were compared to the index KF11 gag peptide epitope. In terms
of the yeast display-derived peptides, five of the six tested resulted in a measurable response. These
peptides elicited IFNg production that varied significantly across the peptide titration range, and
one library peptide (Lib 2) failed to induce IFNg production despite its selection by the AGA1 TCR in
yeast display enrichment screens. Of the ‘nr’-mined microbial peptides, three activated the AGA1+
T cell clone, of which two (Mic 1 and 3) peptides mapped to microbes previously linked with gingival
(Cha´vez de Paz et al., 2004; Chhour et al., 2005) and gastrointestinal (GI) infections in humans
(Wolfgang et al., 2012). In terms of IFNg production, responses to these peptides approached the
magnitude elicited by the index KF11 peptide. The remaining three ‘nr’-derived microbial peptides
drove weak or no T cell responses. Results AGA1 TCR-recovered top 20 peptides_ Round 3 frequencies. 3 of 21 Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 Research article Research article Research article Immunology and Inflammation employed to perform iterated rounds of library selection. The enrichment of library peptides was
tracked via deep sequencing analysis. By Round 3 of selection, clear evidence of peptide sequence
enrichment emerged for variants that resembled the known KAFSPEVIPMF (KF11) peptide. Of the
roughly 2.1 106 reads detected from deep sequencing runs, the top 20 selected peptides repre-
sented 89.4% of recovered reads at Round 3, and three peptide sequences accounted for approxi-
mately 35% of these reads (Figure 1C). Distinct patterns of individual amino acid enrichment/fixation within the peptide sequences had
also emerged by Round 3 (Figure 2, Figure 2—figure supplement 1). The most striking distribution
of amino acids included the near exclusive selection of peptides with a conserved central p5Pro-
p6Glu motif, which contrasted with the greater diversity tolerated at auxiliary positions along the
remaining peptide sequences (Figure 2A). Given that the majority of interactions are formed
between CDR3 alpha chain amino acids and the KF11 peptide Glu6 side chain in the published
HLA*57:03-KF11-AGA1 TCR structure (Stewart-Jones et al., 2012; Figure 2B), fixation of this cen-
tral motif most likely reflects its critical importance for AGA1 TCR binding. Amino acid selection out-
side the p5Pro-p6Glu di-motif was variable, although patterns of conservation specific to the
physiochemistry of residues were apparent; the strong enrichment of hydrophobic amino acids at p3
and p7, and to a lesser extent at p10, for example, reflected the chemistries of these residues in
the KF11 peptide (p3Phe, p7Val, p10Met, respectively). Similarly, p4 position - a solvent-exposed
Ser and an important AGA1 TCR contact residue in the KF11 peptide - displayed a specific enrich-
ment of polar residues in library-derived peptides. Selection patterns were less obvious at positions
1, 8 and 9, where mixtures of both polar and non-polar residues were tolerated. Cross-reactivity of AGA1+ T cell clones to library- and database-derived
peptide hits We questioned if the differential ability of the various library and microbial peptides to elicit T cell
responses primarily reflected binding differences to HLA-B*57:01. This is particularly relevant for the
Lib-derived peptides, which were bound to b2m as part of the HLA-B*57:03 yeast display single
chain trimer construct but were presented in cellular assays as non-linked peptide epitopes in com-
plex with HLA-B*57:01 on antigen presenting B cells. To address this, we performed a sandwich
ELISA-based UV exchange peptide binding assay to gauge the ability of the various peptides to
bind and therefore ‘rescue’ HLA-B*57:01 pre-refolded with UV-labile peptide upon photo- Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 4 of 21 Research article Research article Research article
Immunology and Inflammation Immunology and Inflammation Figure 2. Fixation of specific peptide amino acids residues following Round 3 AGA1 TCR–mediated selection
(A) Amino acid enrichment along the Figure 2. Fixation of specific peptide amino acids residues following Round 3 AGA1 TCR–mediated selection. (A) Amino acid enrichment along the
length of AGA1 TCR selected 11mer peptides are illustrated (i) in bar chart format for approximately 2.0 106 selected peptide sequences, with
individual amino acid enrichment at each position of the 11mer peptide represented on the X-axis and sequence frequency on the Y-axis. K1, A2, F3
refers to amino acid positions 1, 2, 3, etc, of the HIV KF11 epitope which is included for reference. Enrichment data for pre-(naı¨ve) and post-AGA1 TCR
selected peptides from all libraries are provided in Figure 2—figure supplement 1). (ii) The top 1000 peptide sequences are reported in pie-chart
format, with the original KF11 (KAFSPEVIPMF) peptide amino acids included above the relevant pie-charts for reference. K1, A2, F3 refers to amino acid
positions 1, 2, 3, etc, of the HIV KF11 peptide sequence. (iii) A Heat map of the entire Round 3 peptide sequence dataset (~2.0106 peptides)
demonstrating the near absolute dominance of Pro and Glu at positions 5 and 6, respectively, in AGA1 TCR-selected peptide datasets. The amino acid
residues corresponding to the KF11 peptide are outlined in black. Heatmap scale = 0 – 100%, 10% increments. (B) Structural overview of the primary
contacts formed between the AGA1 TCR and KF11 when restricted by HLA-B*57:03. (i) Slide view of the HLA-B*57:03 alpha 1 (a1) helix in cartoon form
(grey), with the alpha 2 (a2) helix removed for clarity. Cross-reactivity of AGA1+ T cell clones to library- and database-derived
peptide hits The peptide is depicted in stick format (blue) with the KF11 position 2 Ala (A2) and Position 11 Phe
(F11) anchor resides highlighted in green. The TCR CDR1a (deep teal), CDR3a (pale cyan), CDR3b (deep salmon) and CDR1b (light pink) loops that
form the main contacts with HLA-B*57:03-KF11 are illustrated. (ii) Barrel view of HLA-B*57:03-KF11, oriented from the peptide’s N terminus, with HLA-
B*57:03 a1 and a2 helices display in cartoon format (grey) and KF11 amino acids 3–6 illustrated in stick format (blue). The remainder of the KF11
peptide sequence is omitted for clarity. The primary polar contacts (~3 a˚ngstro¨ ms (A˚ )) between TCR CDR1a (deep teal) and CDR3a (pale cyan) amino
acids and the KF11 peptide at positions 4 to 6 are illustrated (yellow dash lines). CDR1b residue D30 (light pink) and CDR3b amino acids S96 and Y97
(deep salmon) that reportedly form weaker peptides mediated contacts are also displayed. TCR amino acids positions are number according to Arden
nomenclature (see Ref 37). Structural images were generated in PyMOL four using the Protein Data Bank coordinates 2YPL. The online version of this article includes the following source data and figure supplement(s) for figure 2: Figure 2. Fixation of specific peptide amino acids residues following Round 3 AGA1 TCR–mediated selection. (A) Amino acid enrichment along the
length of AGA1 TCR selected 11mer peptides are illustrated (i) in bar chart format for approximately 2.0 106 selected peptide sequences, with
individual amino acid enrichment at each position of the 11mer peptide represented on the X-axis and sequence frequency on the Y-axis. K1, A2, F3
refers to amino acid positions 1, 2, 3, etc, of the HIV KF11 epitope which is included for reference. Enrichment data for pre-(naı¨ve) and post-AGA1 TCR
selected peptides from all libraries are provided in Figure 2—figure supplement 1). (ii) The top 1000 peptide sequences are reported in pie-chart
format, with the original KF11 (KAFSPEVIPMF) peptide amino acids included above the relevant pie-charts for reference. K1, A2, F3 refers to amino acid
positions 1, 2, 3, etc, of the HIV KF11 peptide sequence. (iii) A Heat map of the entire Round 3 peptide sequence dataset (~2.0106 peptides)
demonstrating the near absolute dominance of Pro and Glu at positions 5 and 6, respectively, in AGA1 TCR-selected peptide datasets. The amino acid
residues corresponding to the KF11 peptide are outlined in black. Research article Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 3 selection. Figure supplement 4—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 3 selection. Figure supplement 5. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 4 selection. g
Figure supplement 5—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 3 selection. illumination. In terms of the library-derived peptides, there was a strong concordance between the
magnitude of T cell responses generated and the extent to which the peptides stabilised HLA-
B*57:01 (Figure 3B). Lib 2 peptide, for example, demonstrated the weakest binding to HLA-B*57:01
and represented the only library-derived peptide that failed to elicit functional responses by AGA1-
expressing T cell clones. A similar pattern existed for the microbial peptides, where the largest T cell
responses were generated by the strongest HLA-B*57:01 binding peptides. Interestingly, four pepti-
des exhibited enhanced binding relative to the index KF11 peptide, including the Mic 1 peptide,
which corresponds to a Sporosarcina newyorkensis-derived halodehydrogenase (HdH) peptide, and
its closest library sequence match, Lib 1. illumination. In terms of the library-derived peptides, there was a strong concordance between the
magnitude of T cell responses generated and the extent to which the peptides stabilised HLA-
B*57:01 (Figure 3B). Lib 2 peptide, for example, demonstrated the weakest binding to HLA-B*57:01
and represented the only library-derived peptide that failed to elicit functional responses by AGA1-
expressing T cell clones. A similar pattern existed for the microbial peptides, where the largest T cell
responses were generated by the strongest HLA-B*57:01 binding peptides. Interestingly, four pepti-
des exhibited enhanced binding relative to the index KF11 peptide, including the Mic 1 peptide,
which corresponds to a Sporosarcina newyorkensis-derived halodehydrogenase (HdH) peptide, and
its closest library sequence match, Lib 1. We re-analyzed responses to the database-derived microbial peptides using a second T cell clone
isolated from a different donor that expressed an AGA1-homologous TCR, namely clone 1.2
(Figure 3C and Supplementary file 1 - Table 3). The functional screens were extended to assess
both IFNg production and CD107a up-regulation in response to the microbial peptides. This T cell
clone also demonstrated a clear preference for the ‘nr’-mined Olsenella uli, Candida orthopsilosis
peptides and in particular, the S. Research article newyorkensis peptide, both in terms of IFNg production and up-
regulation of the LAMP protein, CD107a (Figure 3C (i) and (ii), respectively). Cross-reactivity of AGA1+ T cell clones to library- and database-derived
peptide hits Heatmap scale = 0 – 100%, 10% increments. (B) Structural overview of the primary
contacts formed between the AGA1 TCR and KF11 when restricted by HLA-B*57:03. (i) Slide view of the HLA-B*57:03 alpha 1 (a1) helix in cartoon form
(grey), with the alpha 2 (a2) helix removed for clarity. The peptide is depicted in stick format (blue) with the KF11 position 2 Ala (A2) and Position 11 Phe
(F11) anchor resides highlighted in green. The TCR CDR1a (deep teal), CDR3a (pale cyan), CDR3b (deep salmon) and CDR1b (light pink) loops that
form the main contacts with HLA-B*57:03-KF11 are illustrated. (ii) Barrel view of HLA-B*57:03-KF11, oriented from the peptide’s N terminus, with HLA-
B*57:03 a1 and a2 helices display in cartoon format (grey) and KF11 amino acids 3–6 illustrated in stick format (blue). The remainder of the KF11
peptide sequence is omitted for clarity. The primary polar contacts (~3 a˚ngstro¨ ms (A˚ )) between TCR CDR1a (deep teal) and CDR3a (pale cyan) amino
acids and the KF11 peptide at positions 4 to 6 are illustrated (yellow dash lines). CDR1b residue D30 (light pink) and CDR3b amino acids S96 and Y97
(deep salmon) that reportedly form weaker peptides mediated contacts are also displayed. TCR amino acids positions are number according to Arden
nomenclature (see Ref 37). Structural images were generated in PyMOL four using the Protein Data Bank coordinates 2YPL. The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. All Round 3 AGA1 TCR recovered library peptide amino acid frequencies. Source data 2. Top 1000 Round 3 AGA1 TCR recovered library peptide amino acid frequencies. Source data 3. Heatmap of all Round 3 AGA1 TCR recovered library peptides. Figure 2 continued on next page Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 5 of 21 Research article Research article Research article Immunology and Inflammation Figure 2 continued
Figure supplement 1. Individual amino acid frequencies in the naı¨ve (pre-AGA1 TCR-selected) HLA-B*57:03-restricted yeast display peptide r
Figure supplement 1—source data 1. Amino acid signatures of Yeast display libraries-Naı¨ve. Figure supplement 2. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 1 selection. Figure supplement 2—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 1 selection. Figure supplement 3. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 2 selection. Figure supplement 3—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 2 selection. Figure supplement 4. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 3 selection. Figure supplement 4—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 3 selection. Figure supplement 5. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 4 selection. Figure supplement 5—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 3 selection. Figure 2 continued Figure 2 continued Figure supplement 1. Individual amino acid frequencies in the naı¨ve (pre-AGA1 TCR-selected) HLA-B*57:03-restricted yeast display peptide repertoire. Figure supplement 1—source data 1. Amino acid signatures of Yeast display libraries-Naı¨ve. Figure supplement 2. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 1 selection. Figure supplement 2—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 1 selection. Figure supplement 3. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptid
following Round 2 selection. Figure supplement 2—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 1 selection. Figure supplement 3. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 2 selection. Figure supplement 2—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 1 selection. Figure supplement 3. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 2 selection. Figure supplement 3—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 2 selection. Figure supplement 4. Individual amino acid frequencies of the AGA1 TCR-selected, HLA-B*57:03-restricted yeast display peptide repertoire
following Round 3 selection. Figure supplement 3—source data 1. Amino acid signatures of Yeast display libraries-AGA1 TCR Round 2 selection. Figure supplement 4. Recognition HdH peptides by AGA1-expressing T cell clones Recognition HdH peptides by AGA1-expressing T cell clones
As the Mic 1 peptide, which mapped to the haloacid dehydrogenase (HdH) enzyme of S. newyorken-
sis, elicited the strongest T cell-mediated responses at concentrations comparable to the KF11 epi-
tope, we chose this peptide for follow-up studies. We re-interrogated the ‘nr’ database using this
peptide motif to hunt for related peptides. A number of highly homologous peptides were identified
of which the majority mapped to HdH enzymes specific to distinct microbial genera. Ten additional
HdH peptides (Table 2) were synthesized and tested in T cell functional assays. The majority of these
peptides elicited IFNg production at titrations comparable to the S. newyorkensis (HdH1/Mic1) pep-
tide, and only marginally weaker than responses induced by the index KF11 epitope (Figure 4A). One peptide (HdH2), was recognized at the highest peptide concentration only, whereas two addi-
tional peptides (HdH5 and HdH6) failed to elicit T cell responses. To address if this related to HLA-
B*57:01 binding or recognition by the AGA1 TCR, we tested all HdH peptides in the sandwich
ELISA-based UV exchange peptide binding assay (Figure 4B). When the HdH and KF11 peptides
were ranked according to the strength of peptide binding ELISA data, specific amino acids were
prominent in the stronger binding peptides: position (p)2 Thr/Ser (preferred anchors), p3Iso, p7Iso,
a Pro at p8 or 9 (but not both) and p10Try. Peptides in the medium- and low-binding categories,
although incorporating some of these residues, had either single or combined differences at these
positions (Figure 4B and Supplementary file 1 - Table 4). We also compared UV peptide-exchange
peptide binding ELISA data to NetMHCPan4.1 (http://www.cbs.dtu.dk/services/NetMHCpan/) pre-
dicted binding affinities for KF11 and the HdH peptides (Figure 4, Figure 4—figure supplement 1). Although a negative correlation was observed, this association was very weak (R = 0.18), and most Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 6 of 21 Research article Research article Immunology and Inflammation igure 3. Recognition of library-derived peptides and their closest peptide sequence matches identified from the ’nr’ database by AGA1-expressing T
ell clones. (A) Recognition of six library peptides (Lib) and their closest ’nr’-derived microbial peptides (Mic) matches were compared to the index
KF11 in IFN-g based ELISpot assay screens using an AGA1-expressing T cell clone, summarized in (i) with individual Lib and Mic peptide responses plus
omparison to KF11 denoted in (ii). Recognition HdH peptides by AGA1-expressing T cell clones Peptide concentration (Molar (M )) is denoted on the X-axis, with numbers of Spot Forming Cells (SFC) per 500 cell
nput on the Y-axis. Initial screens were performed with one replica per screen, and were screened on two separate occasions following re-stimulation
f T cell clone 1.1 (biological repeat, n = 2), with one representative shown here. Test peptide IDs, their origins and sequence identities are specified in
able 1. (B) Binding of library derived and the sequence-mined microbial peptides to HLA-B*57:01, assessed by UV-mediated peptide exchange
andwich ELISA. The Y-axis denotes average absorbance readings at 450 nm, with the peptides tested reported on the X-axis. The background
orresponding to the no peptide rescue (nr) control is denoted in grey (also illustrated across the samples by grey hatching). Assays were performed in
uplicate (technical repeats, n = 2) on two separate occasions using different peptide stock dilutions (biological repeats, n = 2), with one representative
hown here. Error bars corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, their origins and sequence identities are
pecified in Table 1. (C) Recognition of the six nr-derived microbial peptides (Mic 1–6) by the closely related AGA1-like T cell clone 1.2, assessed
y IFN-g based ELISpot assay and summarized in (i) with comparison to the KF11 index peptide reported. Peptide concentration (Molar (M)) is denoted
n the X-axis with the numbers of Spot Forming Cells (SFC) per 500 cell input on the Y axis. Assays were performed in duplicate (technical repeats,
= 2) on two separate occasions using different peptide stock dilutions and following re-stimulation and resting of T cell clone 1.2 (biological repeats,
2)
i h
i
h
h
E
b
di
h
S
d
d E
f h
M
(SEM)
d T
id
ID
i i Figure 3. Recognition of library-derived peptides and their closest peptide sequence matches identified from the ’nr’ database by AGA1-expressing T
cell clones. (A) Recognition of six library peptides (Lib) and their closest ’nr’-derived microbial peptides (Mic) matches were compared to the index
KF11 in IFN-g based ELISpot assay screens using an AGA1-expressing T cell clone, summarized in (i) with individual Lib and Mic peptide responses plus
comparison to KF11 denoted in (ii). Peptide concentration (Molar (M )) is denoted on the X-axis, with numbers of Spot Forming Cells (SFC) per 500 cell
input on the Y-axis. Recognition HdH peptides by AGA1-expressing T cell clones Initial screens were performed with one replica per screen, and were screened on two separate occasions following re-stimulation
of T cell clone 1.1 (biological repeat, n = 2), with one representative shown here. Test peptide IDs, their origins and sequence identities are specified in
Table 1. (B) Binding of library derived and the sequence-mined microbial peptides to HLA-B*57:01, assessed by UV-mediated peptide exchange
sandwich ELISA. The Y-axis denotes average absorbance readings at 450 nm, with the peptides tested reported on the X-axis. The background
corresponding to the no peptide rescue (nr) control is denoted in grey (also illustrated across the samples by grey hatching). Assays were performed in
duplicate (technical repeats, n = 2) on two separate occasions using different peptide stock dilutions (biological repeats, n = 2), with one representative
shown here. Error bars corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, their origins and sequence identities are
specified in Table 1. (C) Recognition of the six nr-derived microbial peptides (Mic 1–6) by the closely related AGA1-like T cell clone 1.2, assessed
by IFN-g based ELISpot assay and summarized in (i) with comparison to the KF11 index peptide reported. Peptide concentration (Molar (M)) is denoted
on the X-axis with the numbers of Spot Forming Cells (SFC) per 500 cell input on the Y axis. Assays were performed in duplicate (technical repeats,
n = 2) on two separate occasions using different peptide stock dilutions and following re-stimulation and resting of T cell clone 1.2 (biological repeats,
n = 2), with one representative shown here. Error bars corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, origins
and sequences are specified in Table 1. (ii) Up-regulation of CD107 on T cell clones in response to 1 and 10 mM Mic peptides compared to the index
KF11 epitope was assessed by flow cytometry. Negative (no added peptide - purple dashed line) and positive controls (10 ng/mL PMA stimulation) are
reported for reference. Figure 3. Recognition of library-derived peptides and their closest peptide sequence matches identified from the ’nr’ database by AGA1-expressing T
cell clones. Recognition HdH peptides by AGA1-expressing T cell clones (A) Recognition of six library peptides (Lib) and their closest ’nr’-derived microbial peptides (Mic) matches were compared to the index
KF11 in IFN-g based ELISpot assay screens using an AGA1-expressing T cell clone, summarized in (i) with individual Lib and Mic peptide responses plus
comparison to KF11 denoted in (ii). Peptide concentration (Molar (M )) is denoted on the X-axis, with numbers of Spot Forming Cells (SFC) per 500 cell
input on the Y-axis. Initial screens were performed with one replica per screen, and were screened on two separate occasions following re-stimulation
of T cell clone 1.1 (biological repeat, n = 2), with one representative shown here. Test peptide IDs, their origins and sequence identities are specified in
Table 1. (B) Binding of library derived and the sequence-mined microbial peptides to HLA-B*57:01, assessed by UV-mediated peptide exchange
sandwich ELISA. The Y-axis denotes average absorbance readings at 450 nm, with the peptides tested reported on the X-axis. The background
corresponding to the no peptide rescue (nr) control is denoted in grey (also illustrated across the samples by grey hatching). Assays were performed in
duplicate (technical repeats, n = 2) on two separate occasions using different peptide stock dilutions (biological repeats, n = 2), with one representative
shown here. Error bars corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, their origins and sequence identities are
specified in Table 1. (C) Recognition of the six nr-derived microbial peptides (Mic 1–6) by the closely related AGA1-like T cell clone 1.2, assessed
by IFN-g based ELISpot assay and summarized in (i) with comparison to the KF11 index peptide reported. Peptide concentration (Molar (M)) is denoted
on the X-axis with the numbers of Spot Forming Cells (SFC) per 500 cell input on the Y axis. Assays were performed in duplicate (technical repeats,
n = 2) on two separate occasions using different peptide stock dilutions and following re-stimulation and resting of T cell clone 1.2 (biological repeats,
n = 2), with one representative shown here. Error bars corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, origins
and sequences are specified in Table 1. (ii) Up-regulation of CD107 on T cell clones in response to 1 and 10 mM Mic peptides compared to the index
KF11 epitope was assessed by flow cytometry. Recognition HdH peptides by AGA1-expressing T cell clones Negative (no added peptide - purple dashed line) and positive controls (10 ng/mL PMA stimulation) are
reported for reference. The online version of this article includes the following source data for figure 3: Source data 1. AGA1+ T cell clone 1.1 recognition of top 6 Lib and closest ’nr’ mined microbial (Mic) peptide hits_all combined_ELISpot data. Source data 2. AGA1+ T cell clone 1.1 recognition of top 6 Lib and closest ’nr’ mined microbial (Mic) peptide hits_all combined_ELISpot data. Source data 3. UV-exchange HLA-B*57:01 peptide binding ELISA data for Top 6 Lib and closest ’nr’ mined microbial (Mic) peptides. Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 7 of 21 Source data 4. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_individual plots_ELISpot data. Source data 5. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_CD107 data. Research article
Immunology and Inflammatio Source data 4. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_individual plots_ELISpot data. Source data 5. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_CD107 data. Research article
Immun Source data 4. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_individual plots_ELISpot data. Source data 5. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_CD107 data. Research article
Immunology and Inflam Immunology and Inflammation Source data 4. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_individual plots_ELISpot data. Source data 5. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_CD107 data. Source data 4. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_individual plots_ELISpot data. Source data 5. AGA1+ T cell clone 1.2 recognition of 6 ’nr’ mined microbial (Mic) peptide hits_CD107 data. likely reflects a general lack of data-informed algorithm training for MHC class I with longer peptide
using artificial neural network analysis-based systems such as NetMHC. In terms of the individual peptides, HdH6 demonstrated weaker HLA-B*57:01 binding, potentially
owing to the presence of a non-favored (Glu) anchor residues at p2. Recognition of processed antigens Although antigen presenting cells pulsed with exogenous ‘nr’ mined microbial peptides elicited
strong cross-reactive T cell responses, we next wanted to ascertain if T cell-mediated recognition of
these peptides occurred under conditions that more accurately reflect the physiological processing
of microbial antigens in vivo. To test this, we generated lysates of the three microbial species that
elicited AGA1 TCR-specific responses in the peptide-based functional assays. The lysates were incu-
bated with HLA-B*57:01-expressing HL60 cells (Zorn et al., 2002) pre-treated overnight with cal-
cium ionophore and IFNg to induce DC-myeloid-like cellular differentiation and enhance MHC class I
expression (Wu et al., 2004a). Following 7 hr of incubation, the cells were washed extensively and
their ability to stimulate AGA1-expressing clone 1.1 and the related clone, 1.2, was assessed by ELI-
Spot to evaluate IFNg secretion. As demonstrated in Figure 4D, T cell clones 1.1 and 1.2 specifically
responded to lysates generated from S. newyorkensis whereas reactivities to the other lysates,
including the negative control Ruminococcus gnavus lysate, were not detected. Recognition HdH peptides by AGA1-expressing T cell clones In contrast, HdH2 and HdH5
produced strong ELISA binding signals, and their impact was potentially mediated at the level of
TCR recognition, either via interactions that directly abrogate the peptide’s central ‘p5Pro-p6Glu’
TCR recognition hotspot (via replacement of p5Pro with Glu in HdH5, for example) or mutations dis-
tal to the hotspot that potentially distorts the normal trajectory of the peptide required to facilitate
AGA1 TCR binding (p9Pro to Ser mutation in HdH2). To compare the overall sequence commonality between the canonical KF11 peptide, the top 20
library-derived peptides and the panel of HdH peptides recognized by the AGA1-expressing T cell
clones, Seq2Logo motifs were generated. As illustrated in Figure 4C, the AGA1 TCR-selected
yeast display peptide library logo reflected tolerance to a broad array of amino acids beyond the
conserved, central dipeptide ‘p5Pro-p6Glu’ motif. The HdH-based logo allowed further refinement
of the motif based on T cell functionality and demonstrated that enhanced recognition by AGA1
TCR-expressing T cell clones required further fixation of amino acids outside the central ‘dipeptide’
motif; most notably, with a preference for p9Pro in HdH peptides. This logo also illustrated that
despite low KF11 sequence homology (40–50%), the majority of HdH peptides elicited functional T
cell responses at magnitudes approaching that generated by the KF11 peptide. Discussion The background corresponding to the no
peptide rescue (nr) control is denoted in grey (also illustrated across the samples by grey hatching). Assays were performed in duplicate (technical
repeats) on two separate occasions using different peptide stock dilutions (biological repeats, n = 2), with one representative shown here. Error bars
corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, their origins and sequence identities are specified in Table 2. (C) Summary of evolved peptide motifs recognised by the AGA1 TCR. Recognition beyond the original KAFSPEVIPMF (KF11) index motif is exemplified
initially by the diverse peptide sequences retrieved during repeated rounds of AGA1 TCR-mediated peptide selection, with a Seq2Logo motif reported
for the top 20 Round 3 evolved peptide libraries. Following evaluation of ’nr’-database derived peptides in T cell functional assays, peptides that
elicited the strongest functional responses - in this case, a S. newyorkensis-derived haloacid dehydrogenase peptide -allowed further refinement of
database-led search motifs and identification of related peptide that were functionally recognized by AGA1 TCR-expressing T cell clones. Amino acids
shared between KF11 and the S. newyorkensis-derived Mic1/HdH1 peptide is illustrated in the smaller right panel (pink shading). (D) Recognition of
bacterial cell lysates from S. newyorkensis (Mic 1), C. orthopsilosis (Mic 2), O. uli (Mic 3) and R. gnavus (control) by AGA1-expressing T cell clones 1.1
and 1.2 was tested using an IFN-g based ELISpot assay. Bacterial cell lysates (20mg/mL) were incubated with cytokine-matured HLA-B*57:01 positive
HL60 cells for 7 hours, following which T cell responses were evaluated. PMA (10ng/mL) was included as a positive control, and the background control
comprised HL60 cells incubated with T cells only Lysate identity is denoted on the X axes and the numbers of Spot Forming Cells (SFC) per 500 cell Figure 4. Recognition of haloacid dehydrogenase (HdH) peptides and bacterial lysate-derived antigen by AGA1-expressing T cell clones. (A) Ten non-
redundant (’nr’) database-mined HdH peptides with close homology to S. newyorkensis peptide (Mic 1/HdH1) were tested in an IFN-g based ELISpot
assays using an AGA1-expressing T cell clone. Peptide concentration (Molar (M)) is denoted on the X-axis and the numbers of Spot Forming Cells (SFC)
per 500 cell input is displayed on the Y-axis. Responses to the KF11 epitope are noted in red. Discussion Cross-reactivity represents an essential component of T cell-mediated immunity that allows a rela-
tively small repertoire of TCRs to mount effective immune responses against the larger number of
pathogenic peptide epitopes encountered during a lifetime. A physiologically relevant outcome of
cross-reactivity, where previous exposure to one microbe alters the immune response to a subse-
quent, non-related pathogen (heterotypic immunity) has the potential to drive either efficacious or
pathogenic outcomes in vivo (Gil et al., 2015). This interesting phenomenon has been almost exclu-
sively explored in the context of viral immune responses, and whether peptides of non-viral origin,
such as those originating from bacteria, yeast or fungi can influence viral-specific CD8+ T cell immu-
nity
by
mechanisms
involving
MHC
class
I
restriction
has
received
limited
exploration
(Pohlmeyer et al., 2018). This is especially relevant in the setting of HIV infection, where viral-
induced gut-intestinal (GI) barrier dysfunction (Brenchley et al., 2006a; Brenchley et al., 2006b)
and microbiome dysbiosis (Dillon et al., 2016; Mudd and Brenchley, 2016; Nowak et al., 2015;
Lozupone et al., 2013) could potentially allow commensal or pathogenic microbes to influence the
frequencies/functions of resident or infiltrating virus-specific T cells. Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 8 of 21 Research article Immunology and Inflammation Figure 4. Recognition of haloacid dehydrogenase (HdH) peptides and bacterial lysate-derived antigen by AGA1-expressing T cell clones. (A) Ten non-
redundant (’nr’) database-mined HdH peptides with close homology to S. newyorkensis peptide (Mic 1/HdH1) were tested in an IFN-g based ELISpot
assays using an AGA1-expressing T cell clone. Peptide concentration (Molar (M)) is denoted on the X-axis and the numbers of Spot Forming Cells (SFC)
per 500 cell input is displayed on the Y-axis. Responses to the KF11 epitope are noted in red. Assays were performed in duplicate (technical repeats) on
two separate occasions using different peptide stock dilutions and following re-stimulation and resting of T cell clone 1.2 (biological repeats), with one
representative shown here. Error bars corresponding to the Standard Error of the Mean (SEM) reported. Test peptide IDs, their origins and sequence
identities are specified in Table 2. (B) Binding of the HdH peptides to HLA-B*57:01, assessed in the UV-mediated peptide exchange sandwich ELISA
assay. The Y-axis denotes average absorbance readings at 450 nm and the X-axis denotes test peptides. Discussion Assays were performed in duplicate (technical repeats) on
two separate occasions using different peptide stock dilutions and following re-stimulation and resting of T cell clone 1.2 (biological repeats), with one
representative shown here. Error bars corresponding to the Standard Error of the Mean (SEM) reported. Test peptide IDs, their origins and sequence
identities are specified in Table 2. (B) Binding of the HdH peptides to HLA-B*57:01, assessed in the UV-mediated peptide exchange sandwich ELISA
assay. The Y-axis denotes average absorbance readings at 450 nm and the X-axis denotes test peptides. The background corresponding to the no
peptide rescue (nr) control is denoted in grey (also illustrated across the samples by grey hatching). Assays were performed in duplicate (technical
repeats) on two separate occasions using different peptide stock dilutions (biological repeats, n = 2), with one representative shown here. Error bars
corresponding to the Standard Error of the Mean (SEM) are reported. Test peptide IDs, their origins and sequence identities are specified in Table 2. (C) Summary of evolved peptide motifs recognised by the AGA1 TCR. Recognition beyond the original KAFSPEVIPMF (KF11) index motif is exemplified
initially by the diverse peptide sequences retrieved during repeated rounds of AGA1 TCR-mediated peptide selection, with a Seq2Logo motif reported
for the top 20 Round 3 evolved peptide libraries. Following evaluation of ’nr’-database derived peptides in T cell functional assays, peptides that
elicited the strongest functional responses - in this case, a S. newyorkensis-derived haloacid dehydrogenase peptide -allowed further refinement of
database-led search motifs and identification of related peptide that were functionally recognized by AGA1 TCR-expressing T cell clones. Amino acids
shared between KF11 and the S. newyorkensis-derived Mic1/HdH1 peptide is illustrated in the smaller right panel (pink shading). (D) Recognition of
bacterial cell lysates from S. newyorkensis (Mic 1), C. orthopsilosis (Mic 2), O. uli (Mic 3) and R. gnavus (control) by AGA1-expressing T cell clones 1.1
and 1.2 was tested using an IFN-g based ELISpot assay. Bacterial cell lysates (20mg/mL) were incubated with cytokine-matured HLA-B*57:01 positive
HL60 cells for 7 hours, following which T cell responses were evaluated. PMA (10ng/mL) was included as a positive control, and the background control
comprised HL60 cells incubated with T cells only. Lysate identity is denoted on the X-axes and the numbers of Spot Forming Cells (SFC) per 500 cell
Figure 4 continued on next page Mendoza et al. eLife 2020;9:e58128. Immunology and Inflammation Immunology and Inflammation Figure 4 continued input are displayed on the Y-axes. Assays were performed in duplicate (technical repeats, n = 2) on two separate occasions (biological repeats, n = 2)
using fresh lysate stock dilutions and following re-stimulation and resting of T cell clones 1.1 and 1.2. One representative is shown. input are displayed on the Y-axes. Assays were performed in duplicate (technical repeats, n = 2) on two separate occasions (biological repeats, n = 2)
using fresh lysate stock dilutions and following re-stimulation and resting of T cell clones 1.1 and 1.2. One representative is shown. The online version of this article includes the following source data and figure supplement(s) for figure 4: using fresh lysate stock dilutions and following re-stimulation and resting of T cell clones 1.1 and 1.2. One representative is shown. The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. AGA1+ T cell clone 1.2 recognition of ’nr’ mined HdH peptides_ELISpot data. Source data 2. UV-exchange HLA-B*57:01 peptide binding ELISA data for HdH peptides. urce data 3. Recognition of S. newyorkensis bacterial lysates by AGA1+ T cell clones 1.1 and 1.2_ELISpot data. ure supplement 1. Weak, negative correlation between HLA-B*57:01 UV exchange-peptide binding data and N Source data 3. Recognition of S. newyorkensis bacterial lysates by AGA1+ T cell clones 1.1 and 1.2_ELISpot data. Figure supplement 1. Weak, negative correlation between HLA-B*57:01 UV exchange-peptide binding data and Net MHCpan 4.1 predicted affinities
for KF11 and the HdH peptides. gative correlation between HLA-B*57:01 UV exchange-peptide binding data and Net MHCpan 4.1 predicted affin 1. Correlation between UV exchange HLA-B*57:01 peptide binding data and NetMHC pan4.1 predicitons. Figure supplement 1—source data 1. Correlation between UV exchange HLA-B*57:01 peptide binding data and NetMHC pan4.1 predicitons. Figure supplement 1—source data 1. Correlation between UV exchange HLA-B*57:01 peptide binding data a To explore cross-reactivity beyond the normal scope of viral-derived peptide epitopes we chose
the well-characterized and commonly selected HLA-B*57:01-restricted AGA1 TCR that recognizes
the HIV gag-derived KF11 epitope. Although specific features, including the short and convergent
nature of both the TCR CDR3alpha/beta regions (Venturi et al., 2006), in addition to the unusual
nature of the KF11 peptide when bound by HLA-B*57:01 (Stewart-Jones et al., 2005), may drive
preferential usage of this TCR, external influences could additionally contribute to its enhanced fre-
quency or common selection in vivo. Immunology and Inflammation To evaluate the cross-reactive breadth of the AGA1 TCR, we
employed previously developed MHC class I yeast display technology (Adams et al., 2011;
Birnbaum et al., 2014a; Gee et al., 2018). This approach permits a high-throughput, non-biased
screen of cross-reactivity via TCR-mediated sampling of a repertoire approximating ~2108 distinct
peptides bound by MHC on the yeast cell surface. Data obtained using this well-validated approach
illustrated key features of AGA1 TCR-mediated specificity. The first and most notable included fixa-
tion of the peptide’s central p5Pro-p6Glu di-motifs in TCR-selected libraries following 3 rounds of
yeast display selection. As illustrated by our previous analysis of the HLA-B*57:03-KF11-AGA1 TCR
co-crystal complex, KF11 peptide residues 4 to 6 primarily contribute to extensive H bonding and
van der Waals interactions, the majority of which are formed between CDR3 alpha chain amino acids
and the KF11 peptide’s p6Glu residue (Stewart-Jones et al., 2012). The importance of p6Glu for
AGA1 TCR-mediated recognition of library peptides was also reflected here by its absolute conser-
vation in peptide sequences retrieved following repeated rounds of selection. The near equivalent
requirement for p5 Pro in library-enriched peptides may instead reflect its indirect role facilitating
the precise peptide conformation to generate a preferred AGA1 TCR recognition landscape akin to
that described for KF11 in complex with HLA-B*57:03. Despite the substantial involvement of KF11
p4Ser in the published AGA1 TCR-B*57:03-KF11 binding interface, this residue was not completely
conserved in all peptides following selection. However, as library peptides with p4 polar residues
were specifically enriched, this may reflect pressure to fulfil H bonding requirements analogous to
those described previously for the interaction between KF11p4Ser and the AGA1 TCR CDR3 alpha
chain region in the AGA1 TCR-B*57:03-KF11 co-complex structure (Stewart-Jones et al., 2012). Although the library design generated in this study allowed for the KF11 sequence to be present,
it was not found in either the naı¨ve or in subsequent rounds of AGA1 TCR-enriched libraries. Hence,
the yeast clone was unlikely to be a transformant in these screens. This, to some extent, reflects one
small limitation of the yeast display screens where the functional library cannot fully cover the theo-
retical diversity of all potential cross-reactive peptides. However, the real strength of the method is
reflected by its ability to identify the restricting peptide when absent, in addition to related pepti-
des, from available databases using algorithms and statistical-based approaches. Discussion DOI: https://doi.org/10.7554/eLife.58128 9 of 21 9 of 21 Research article Research article Immunology and Inflammation Immunology and Inflammation This is what we
observed for the KF11 peptide which emerged as the top database-mined peptide hit using
library peptide-led database screens. Following Round 3 peptide sequence acquisition, data-informed algorithms were developed to
identify similar peptides in the non-redundant database. Next to KF11-mined peptides and related
variants, microbial peptides comprised the top peptide hits. Of the small panel of bacterial peptides
included in follow-up T cell functional studies, three peptides were recognised. One peptide that eli-
cited weak responses was derived from O. uli, a gram-positive member of the Coriobacteriaceae
family previously associated with orthodontic infections in humans (Vieira Colombo et al., 2016;
Dewhirst et al., 2001). A second peptide mapped to a hypothetical protein (CORT_0A05310) from
C. orthopsilosis, an invasive yeast strain associated primarily with blood-borne infections linked to Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 10 of 21 Research article Research article Research article Immunology and Inflammation catheter-delivered nutrients (hyperalimentation) during hospitalization (Tavanti et al., 2005;
Yong et al., 2008; Silva et al., 2009; Merseguel et al., 2015). However, the largest responses were
driven by a peptide sequence (LTISPEIPPYF) that mapped to a haloacid dehydrogenase (HdH)
enzyme from S. newyorkensis. Although normally associated with terrestrial environments and food
production/processing facilities, these endospore-forming Firmicutes have also been isolated from
plants, in addition to humans and animals specimens (blood and fecal) (Oliver et al., 2018),
(Wolfgang et al., 2012), most likely as a consequence of exposure to contaminated soil. Although
KF11 and the S. newyorkensis-derived HdH peptide share only 50% sequence homology, keys amino
acids were conserved; these included peptide amino acids comprising a central TCR recognition
‘hotspot’ (p4Ser and p6 Glu) in addition to residues that shaped the solvent-exposed peptide arch
(p5Pro and p9Pro) that helped both stabilize interactions with HLA-B*57:01 and allowed AGA1 TCR
binding (Stewart-Jones et al., 2012). Through further rounds of database interrogation, a panel of
closely related HdH peptide variants were identified from distinct bacterial genera, of which the
majority were also recognised by AGA1-expressing T cell clones. Interestingly, one of these peptides
(LAITPEIAPYF) mapped to Bacillus massiliosenegalensis, a species previously isolated from the gut
microbiome in humans (Forster et al., 2019; Ramasamy et al., 2013). Collectively, our findings raise
the question that if encountered in a HLA-B*57:01-restricted context, could haloacid dehydrogenase
peptides originating from these diverse microbes influence the frequencies and/or functionality of
AGA1 TCR expressing T cells in vivo? Immunology and Inflammation p
g
It is known that certain bacteria, either directly via bacterial-derived LPS (Tripathy et al., 2017),
CpG DNA (Speiser et al., 2005) and metabolites generated by specific microbiome-resident popu-
lations (Luu et al., 2018) or indirectly, via the induction of cytokine expression (Wong and Pamer,
2001), can affect the functionality or non-specifically drive the expansion of CD8+ T cells. Whether
virus-specific CD8+ T cell populations are occasionally expanded by bacterial-derived antigens in an
MHC-restricted context is less well understood. Although this question remains unanswered here for
the AGA1 TCR, CD8+ T cells that specifically cross-react with both self-peptide and commensal
microbes have been reported, mainly in the context of autoimmune disease. In the murine NOD
MyD88-/- model of type I diabetes, the disease-associated enrichment of fusobacteria encoding a
peptide with homology to the islet-specific glucose-6-phosphate catalytic subunit-relate (IGRP) pro-
tein, reportedly drove activation of IGRP-reactive CD8+ T cells that promoted development of dia-
betes (Tai et al., 2016). A subset of autoreactive CD8+ T cells with specificity for an islet-derived
peptide epitope that cross-reacts with a peptide derived from the commensal organism Bacteroides
stercoris, has also been reported in human type I diabetic patients (ImMaDiab Study Group et al.,
2018). Evidence that virus-specific CD8+ T cell frequencies are influenced by bacterial-derived pepti-
des is less clear. In a murine study, elevated numbers of lung-derived MCMV-specific CD8 T cells
was attributed to indigenous microbiota (Sprent et al., 2000), and although subclinical MCMV reac-
tivation in lung tissue as a contributing factor was not fully excluded, heterologous recognition of
peptides from endogenous microbes sharing consensus motifs with MCMV was proposed as a
potential driver of these cells. HIV-specific CD8+ T cells are abundant in the GI tract (Ferre et al., 2010), an environment that
represents a major site of HIV replication and reservoir of viral persistence, thus providing a constant
source of cognate antigen in vivo. Hence, the extent, if any, to which bacterial-derived MHC class I
restricted peptides would influence HIV-reactive T cell frequencies in this environment is unclear. However, T cells infiltrating the Lamina Propria and intestinal intraepithelial encounter diverse patho-
genic and commensal microbes in healthy donors (Isakov et al., 2009). Immunology and Inflammation With exposure to bacterial
antigen exacerbated in HIV infection via translocation and systemic dissemination of microbial prod-
ucts following mucosal barrier disruption, it remains unknown if this setting allows specific HIV-reac-
tive CD8+ T cell populations to be influenced by microbes in an MHC-restricted context, and
specifically, if MHC class I proteins can present microbial peptides that inflate specific subsets of
memory HIV-specific T cell populations during active disease. Additionally, determining if specific cir-
cumstances, for example, during gastrointestinal infections in healthy individuals or in patients with
chronic Inflammatory Bowel Disease, allow MHC-restricted microbial peptides to influence the pre-
cursor frequencies of viral-specific CD8+ T cells would be of interest. Although proposed in this
study, definitive evidence that specific bacteria carrying KF11-related HdH peptides pre-select and
expand AGA1-expressing CD8 positive T cells in vivo is lacking, hence further studies are required
to investigate this hypothesis. Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 11 of 21 Immunology and Inflammation Materials and methods Key resources table
Reagent type
(species) or resource
Designation
Source or reference
Identifiers
Additional
information
Cell line
(S. cerevisiae)
EBY100
D. Wittrup,
PMID:9181578
MATa AGA1::GAL1-
AGA1::URA3 ura3-52
trp1 leu2-delta200
his3-delta200
pep4::HIS3 prbd1. Immunology and Inflammation 6R can1 GAL
Strain, strain
background
(Escherichia coli)
Rosetta 2
Novagen
71400–3
F- ompT hsdSB(rB
- mB
- )
gal dcm (DE3)
pRARE2 (CamR)
Genetic reagent
(Homo sapiens)
AGA1 TCR biotin tag,
PET22b+ construct
This paper
disulfide bridge was
introduced
into both the alpha and beta
TCR chains, hexahistidine tag
and a BirA biotinylation
substrate motif
Recombinant
DNA reagent
Peptide-HLA-B*5703,
PYAL vector
This paper
a single chain fusion display
where HLA-B*57:03 heavy
chain domains were
included downstream of the
KF11 epitope sequence
and the b2m gene
Commercial
assay or kit
streptavidin
microbeads
Miltenyi Biotec
130-048-102
Commercial
assay or kit
LS column
Miltenyi Biotec
130-042-401
Commercial
assay or kit
Zymoprep II kit
Zymo Research
D2004
Commercial
assay or kit
MiSeq Reagent V2 kit
Illumina
MS-102–2002
Antibody
anti-myc 488
(mouse monoclonal,
9B11)
Cell Signaling
2279
RRID:AB_2151849
Peptide,
recombinant
protein
Streptavidin Alexa 647
PMID:17187819
Software, algorithm
PandaSeq
PMID:22333067
Software, algorithm
Geneious, version 6
Biomatters, Inc
Software, algorithm
Perl scripts for data prep,
analysis and prediction
PMID:24855945
Software, algorithm
Matlab, R2015b
Mathworks, Inc
Cell line
(Homo sapiens)
HL60
ATCC
RRID:CVCL_0002
Antigen processing
experiments
Antibody
Anti-human CD8-APC
(mouse monoclonal,
clone RPA-T8)
BD Bioscience
555369
RRID:AB_398595
Functional T cell
assay (2.5 mL/test)
Antibody
Anti-human CD107a PE
(mouse monoclonal,
clone H4A3)
BD Bioscience
555801
RRID:AB_396135
Functional T cell assay
(3 mL/test)
Chemical
compound, drug
BD Golgi Stop
BD Bioscience
554724
Protein transport inhibitor
for T cell functional
studies (1:1500)
Chemical
compound, drug
BD Cytofix
BD Bioscience
554655
Cell fixation prior to
flow cytometric analysis
(150 mL/tube)
Continued on next page 12 of 21 Immunology and Inflammation Continued
Reagent type
(species) or resource
Designation
Source or reference
Identifiers
Additional
information
Software, algorithm
FlowJo
FlowJo-BD
Analysis of flow
cytometry data
Commercial assay or kit
Human IFNg ELISpotPLUS
kit-ALP strips
MABTECH
3420-4AST
T cell functional ELISpot
assays to assess
IFN-g production. Software, algorithm
AID ELISpot Classic
resolving IMAGES
Peptide,
recombinant
protein
synthetic peptides
This paper
KF11, HdH1-11,
Mic1-6, Lib1-6
Synthetic KF11, Mic, Lib
and HdH peptides for
functional T
cell studies – as
per concentrations and
titrations in Materials
and methods. Immunology and Inflammation Others
Interferon g
PeproTech
300-02-100mg
Maturation of antigen
presenting cells
(1000 U/mL)
Chemical
compound, drug
Calcium Ionophore
Sigma
A23187
Maturation of antigen
presenting cells
(100 ng/mL)
Chemical
compound, drug
PMA
Sigma
P1585
Positive control for T
cell functional assays
(10 ng/mL)
Strain, strain
background
(Sporosarcina
newyorkensis)
Sporosarcina
newyorkensis
DSMZ-German Collection
of Microorganisms and
Cell Cultures GmbH
DSM-23544
Bacterial cultures for
generation of lysates
(20 mg/mL)
Strain, strain
background
(Olsenella uli)
Olsenella uli
DSMZ-German Collection
of Microorganisms and
Cell Cultures GmbH
DSM 7084
Bacterial cultures for
generation of lysates
(20 mg/mL)
Strain, strain
background
(Candida
orthopsilosis)
Candida orthopsilosis
DSMZ-German Collection
of Microorganisms and
Cell Cultures GmbH
DSM 24508
Bacterial cultures for
generation of lysates
(20 mg/mL)
Strain, strain
background
(Ruminococcus
gnavus)
Ruminococcus gnavus
DSMZ-German Collection
of Microorganisms and
Cell Cultures GmbH
CC55_001C
Bacterial cultures for
generation of lysates
(20 mg/mL)
Commercial assay, kit
Pierce BCA
Protein Assay Kit
Thermo
Fisher Scientific
23225
Quantification of bacterial
cell lysates. Genetic reagent
(Homo sapiens)
HLA-B*57:01
biotin tagged
PMID:11953462
Expression plasmid for
HLA-B*57:01 protein
expression. Peptide,
recombinant
protein
9MT4 UV peptide
PMID:21430058
UV exchange peptide
for HLA-B*57:01 protein
refold (1:100)
Antibody
anti-human ABC
(mouse monoclonal,
clone W6/32)
Biolegend
311402
Coating antibody for
UV-peptide exchange
ELISA
(10 mg/mL)
Antibody
anti-human B2M biotin
(mouse, monoclonal,
clone 2M2)
Biolegend
316308
RRID:AB_493689
Detection antibody for
UV-peptide exchange
ELISA
(1 mg/mL)
Chemical
compound, drug
ExtrAvidin-peroxidase
Sigma
E2886
Assay development step
(1) for UV-peptide
exchange ELISA
(1:1000)
Continued on next page
Research article
Immunology and Inflammation R Continued
Reagent type
(species) or resource
Designation
Source or reference
Identifiers
Additional
information
Chemical
compound, drug
TMB High sensitivity
Substrate
Biolegend
421501
Assay development step
(2) for UV-peptide exchange
ELISA
(100 mL/well)
Chemical
compound, drug
Stop Solution
Biolegend
423001
Assay development stop
step (3) for UV-peptide
exchange ELISA
(100 mL/well)
Software, algorithm
FLUOstar
BMG LABTECH
ELISA plate
absorbance
reading@450 nm
Research article
Immunology and Inflammation Design and construction of HLA-B*57:03 yeast display library Design and construction of HLA-B*57:03 yeast display library
A random peptide library displayed by the human MHC class I HLA-B*57:03 subtype, was used to
generate the MHC class I expressing yeast display platform tested in this study. Although the AGA1
TCR is restricted by the KF11 peptide in complex with HLA-B*57:01, this TCR also binds with high
affinity (Kd ~5 mM) to the near-identical HLA-B*57:03-KF11 complex (Stewart-Jones et al., 2012). The peptide-b2m-MHC heavy chain single chain trimer construct design was based on previously
developed MHC class I yeast display scaffolds (Adams et al., 2011; Birnbaum et al., 2014a;
Gee et al., 2018) and incorporated a single chain fusion display where HLA-B*57:03 heavy chain
domains were included downstream of the KF11 epitope sequence and the b2m gene. Flexible
GSSS linkers interconnected KF11, b2m ([GSSS]3) and the B*57:03 heavy chain regions ([GSSS]4). To
minimize linker protrusions that could affect TCR binding, mutagenesis of position 84 (Tyr > Ala) in
the HLA-B*57:03 alpha 1 (a1) region was performed to generate a larger linker-accommodating cav-
ity (Hansen et al., 2009). A DNA construct library encoding an 11 amino acid (11mer) peptide-MHC
library was generated where all peptide residues were fully randomized except for the preferred
HLA-B*57:01/57:03 anchor binding residues, which were restricted to Ser, Thr and Ala at position
two and to Trp, Phe and Tyr at position 11 to preserve binding to HLA-B*57:03 (Barber et al.,
1997). All positions except for the anchor residues were generated using an NNK codon library. Position two utilized a DCK codon and position 11 used either a TGG codon for Trp or a TWY codon
for Phe and Tyr. Two separate library inserts were generated to encode for the codon library
NNKDCKNNKNNKNNKNNKNNKNNKNNKNNK(TGG/TWY) with theoretical nucleotide diversities of
2.1E14 and 8.4E14 for a total diversity of 1.06E15. The peptide-HLA-B*57:03 construct library was used to generate a yeast display library detailed
above as previously described (Gee et al., 2018; Birnbaum et al., 2014b). Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 13 of 21 Continued
Reagent type
(species) or resource
Designation
Source or reference
Identifiers
Additional
information
Chemical
compound, drug
TMB High sensitivity
Substrate
Biolegend
421501
Assay development step
(2) for UV-peptide exchange
ELISA
(100 mL/well)
Chemical
compound, drug
Stop Solution
Biolegend
423001
Assay development stop
step (3) for UV-peptide
exchange ELISA
(100 mL/well)
Software, algorithm
FLUOstar
BMG LABTECH
ELISA plate
absorbance
reading@450 nm
Design and construction of HLA-B*57:03 yeast display library
A random peptide library displayed by the human MHC class I HLA-B*57:03 subtype, was used to
generate the MHC class I expressing yeast display platform tested in this study. Although the AGA1
TCR is restricted by the KF11 peptide in complex with HLA-B*57:01, this TCR also binds with high
affinity (Kd ~5 mM) to the near-identical HLA-B*57:03-KF11 complex (Stewart-Jones et al., 2012). The peptide-b2m-MHC heavy chain single chain trimer construct design was based on previously
developed MHC class I yeast display scaffolds (Adams et al., 2011; Birnbaum et al., 2014a;
Gee et al., 2018) and incorporated a single chain fusion display where HLA-B*57:03 heavy chain
domains were included downstream of the KF11 epitope sequence and the b2m gene. Flexible
GSSS linkers interconnected KF11, b2m ([GSSS]3) and the B*57:03 heavy chain regions ([GSSS]4). To
minimize linker protrusions that could affect TCR binding, mutagenesis of position 84 (Tyr > Ala) in
the HLA-B*57:03 alpha 1 (a1) region was performed to generate a larger linker-accommodating cav-
ity (Hansen et al., 2009). A DNA construct library encoding an 11 amino acid (11mer) peptide-MHC
library was generated where all peptide residues were fully randomized except for the preferred
HLA-B*57:01/57:03 anchor binding residues, which were restricted to Ser, Thr and Ala at position
two and to Trp, Phe and Tyr at position 11 to preserve binding to HLA-B*57:03 (Barber et al.,
1997). All positions except for the anchor residues were generated using an NNK codon library. Position two utilized a DCK codon and position 11 used either a TGG codon for Trp or a TWY codon
for Phe and Tyr. Two separate library inserts were generated to encode for the codon library
NNKDCKNNKNNKNNKNNKNNKNNKNNKNNK(TGG/TWY) with theoretical nucleotide diversities of
2.1E14 and 8.4E14 for a total diversity of 1.06E15. The peptide-HLA-B*57:03 construct library was used to generate a yeast display library detailed
above as previously described (Gee et al., 2018; Birnbaum et al., 2014b). R Briefly, freshly prepared
EBY-100 electrocompetent yeast were incubated with a 5:1 ratio of pMHC insert to linearized pYAL
vector by mass (~1 ug vector and ~5 ug insert per electroporation) and transformed to generate a
randomized peptide library. The insert contained an equal mass mixture of the polymerase chain
reaction product containing the two libraries detailed above. The final library, which was generated
via 20 electroporations, was then quantified as containing ~2108 unique transformants via limiting
dilution. Selection and sequencing of HLA-B*57:03 peptide library Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 Briefly, freshly prepared
EBY-100 electrocompetent yeast were incubated with a 5:1 ratio of pMHC insert to linearized pYAL
vector by mass (~1 ug vector and ~5 ug insert per electroporation) and transformed to generate a
randomized peptide library. The insert contained an equal mass mixture of the polymerase chain
reaction product containing the two libraries detailed above. The final library, which was generated
via 20 electroporations, was then quantified as containing ~2108 unique transformants via limiting
dilution. Selection and sequencing of HLA-B*57:03 peptide library
Each step described below was performed essentially as previously reported and was conducted
with enough yeast to ensure >10 fold coverage of the previous step’s theoretical diversity The yeast
Research article
Immunology and Inflammation Selection and sequencing of HLA-B*57:03 peptide library Each step described below was performed essentially as previously reported and was conducted
with enough yeast to ensure >10 fold coverage of the previous step’s theoretical diversity. The yeast
display library was seeded at an optical density of 1 (corresponding to 1 107 yeast/mL) for growth
in SDCAA media, pH 5.5, at 30˚C and grown while shaking at 250 rpm until the culture reached satu-
ration overnight. The library was then induced in SGCAA media, pH 5.5, and allowed to incubate
while shaking at 250 rpm at 20˚C for 48 hr. After induction, 1 106 yeast were assayed for induction by staining for a C-terminal Myc epi-
tope tag placed between the MHC and Aga2. Next, yeast corresponding to >10 fold diversity of the
initial library or the yeast from the previous round of selection were incubated with streptavidin
microbeads (Miltenyi Biotec 130-048-102), washed in PBS + 0.5% BSA, and passed through a Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 14 of 21 Research article Research article Immunology and Inflammation Miltenyi LS column (Miltenyi Biotec 130-042-401) to remove non-specific binders. Yeast were then
incubated with streptavidin beads co-incubated with 400 nM biotinylated TCR, washed in PBS+BSA,
and then run through an LS column. Retained yeast that were then eluted from the column, washed
into SDCAA, and re-cultured. Selections were repeated for an additional two rounds. Finally, a more
stringent final round of selection was conducted by staining yeast with 400 nM TCR streptavidin tet-
ramers, incubating with anti-fluorophore microbeads, and sorting via magnetic selection. Protein production and purification Soluble AGA1 TCR was expressed and refolded as described previously (Boulter et al., 2003). To
facilitate stable chain pairing, an extracellular C region disulfide bridge was introduced into both the
alpha and beta TCR chains by site-directed mutagenesis. The modified TCR alpha chain was cloned
upstream of a hexahistidine tag and a BirA biotinylation substrate motif was inserted downstream of
the modified TCR beta chain. Both chains were cloned into the PET22b+ vector (Novagen), and
were expressed in the E. coli strain Rosetta-2 (Novagen), isolated as inclusion bodies, purified, re-
solubilized, and refolded as described (Stewart-Jones et al., 2012; Boulter et al., 2003). Refolded
TCR complexes were purified by anion exchange, Ni+2 and size exclusion chromatography. The func-
tionality of the AGA1 TCR was confirmed by cellular and SPR-based studies prior to inclusion in
HLA-B*57:03 yeast display screening runs. Analysis via next-generation sequencing y
g
q
g
Peptide enrichment and overall motifs were analyzed via next-generation sequencing as previously
described. Briefly, plasmids from 5 107 yeast per round of selection were recovered from selected
yeast (Zymoprep II kit, Zymo Research). Peptide sequences were then amplified via PCR. During the
course of PCR, individual barcode sequences (to deconvolve rounds of selection) and random 8mer
sequences (to increase sequence diversity) were added to the amplicons. The product of these PCRs
were used in a second PCR to affix Illumina sequencing adapters. Final PCR products were gel puri-
fied, pooled, quantitated, and sequenced on an Illumina MiSeq using a v2 2 150 nt kit with PhiX
DNA spiked in to ensure sufficient sequence diversity for high-quality reads. As detailed previously
data processing, analysis, and predictions were as previously described (Birnbaum et al., 2014b). The forward and reverse sequencing data were used to create a single sequence of the paired-end
reads using PandaSeq (Masella et al., 2012), deconvoluted into each round of selection by barcode,
and trimmed for analysis using Geneious version 6 (Biomatters Inc). In order to correct for potential
PCR or sequencing errors, sequences differing by one nucleotide from the most abundant were coa-
lesced. Amino acid frequencies for each round were determined after translation and removal of
sequences containing frame shifts or stop codons. Predictions from the profile-based search of the
NCBI non-redundant protein data database (downloaded June 21, 2013) was preformed using the
statistics from the third round using an amino acid frequency cut-off of 0.01. The custom scripts for
data processing and analysis are available on GitHub (https://github.com/jlmendozabio/NGSpepti-
deprepandpred;
copy
archived
at
https://github.com/elifesciences-publications/
NGSpeptideprepandpred; Mendoza, 2020). Peptide generation Conventional peptide epitopes that included a panel of library-derived peptides (Lib 1–6) and their
closest non-redundant (nr) database-mined microbial sequence hits (Mic 1–6) were synthesized com-
mercially using Fmoc chemistry (Genscript, USA). A photo-labile version of the ISPRTLNAW epitope
(9MT4), where a UV sensitive 3-amino-3-(2-nitrophenyl)-propionic acid residue replaced the C-termi-
nal penultimate Ala residue, and validated previously (Brackenridge et al., 2011), was sourced from
LUMC, Leiden, The Netherlands. Growth of microbial strains and preparation of lysates p
p
y
The following microbes were grown aerobically in the specified culture conditions: Adherent-Invasive
Escherichia coli LF82 in Luria Broth at 37˚C; Candida orthopsilosis DSM 24508 in potato dextrose
medium at 30˚C; Sporosarcina newyorkensis DSM-23544 in Caso medium at 37˚C. Strict anaerobes
were cultured in a Whitley DG250 workstation at 37˚C with 10% H2, 10% CO2, and 80% N2: Olse-
nella uli DSM 7084 in DSMZ Medium 104 and Ruminococcus gnavus CC55_001C in filtered Rein-
forced Clostridial Medium (RCM). Filtered RCM was generated by centrifugation of the media at
10,000 x g for 10 min to remove agar components, followed by filtration through 0.2 mm filters. Optical density (OD600) to colony-forming unit (CFU) conversions were calculated from overnight
cultures. Individual microbes were cultured for 24–48 hr in liquid media. Bacterial cells were har-
vested by centrifugation at 10,000 x g for 10 min, followed by three washes in sterile PBS. Cells
were re-suspended in sterile PBS and heat-inactivated at 70˚C for 1 hr, followed by two freeze-thaw
cycles. Protein concentration was determined by BCA assay (Pierce BCA Protein Assay Kit, Thermo
Fisher Scientific). IFNg ELISpot assay g
p
y
The standard ELISpot assay was used to detect IFNg release by AGA1 CD8+ T cells clones, but with
modifications (Stewart-Jones et al., 2012). 5000 peptide-pulsed HLA-B*57:01 homozygous immor-
talized (mycoplasma negative) B cell lines (BCLs) were incubated with 500 CD8+ T cell from two
clones expressing the AGA1 TCR in the presence of 10-fold serial dilutions of KF11 and test pepti-
des ranging from 10 5 to 10 9M final concentration. Assays were harvested following 15 hr incuba-
tion at 37˚C. Results are reported as spot forming cells (SFCs) per 500 T cell input, and SFCs double
that observed with media alone were considered positive. UV-mediated peptide exchange and sandwich ELISA assay UV-mediated peptide exchange and sandwich ELISA assay
Refolding of HLA-B*57:01, b2m and 9MT4, and subsequent UV-photocleavage/peptide exchange
were performed as outlined previously (Brackenridge et al., 2011; Toebes et al., 2009). 0.5 mM
(~25 mg/mL) of UV-sensitive HLA-B*57:01-b2m-9MT4 refolded monomers were incubated with 50
mM of the various test peptides in polypropylene 96 (V-shaped) well plates (Greiner Bio-one), and
the final volume was adjusted to 125 mL by adding UV exchange buffer (20mMTris pH7, 150 mM 15 of 21 15 of 21 Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 Research article Research article Research article Immunology and Inflammation NaCl). Samples were incubated on ice in a CAMAG UV cabinet and photo-illumination at 366 nm
was performed for 60 min. Following centrifugation at 4000 g for 10 min to remove aggregated
material, 90% of the sample volumes were transferred to a fresh plate, of which 3 mL was subse-
quently diluted 1:50 in 2% bovine serum albumin (BSA)-PBS. 50 mL of this mix was transferred (in
duplicate) into an ELISA plate pre-coated overnight at 4˚C with 10 mg/mL of the anti-human MHC
monoclonal antibody W6/32 (Biolegend) and blocked in 2% BSA-PBS prior to sample addition. Fol-
lowing immobilization for 2 hr at 4˚C, and extensive washing in 0.01%Tween-PBS (referred to herein
as ELISA wash buffer), the plates were incubated with 50 mL of biotinylated anti-human b2-microglo-
bulin (2M2) antibody (BioLegend, RRID:AB_493689) diluted 1:100 in 2% BSA-PBS for 1.5 hr at 4˚C. Plates were washed for a further six times in ELISA wash buffer, prior to the addition of Extravidin
peroxidase (Sigma) diluted 1:1000 in 2%-BSA PBS. Following 30 min at RT and a subsequent round
of plate washings, 100 mL of tetramethyl benzidinesubstrate (TMB) (BioSource) was added, and
plates were developed for 10 min. 100 mL of H2SO4 STOP solution (BioLegend) was added to termi-
nate the reactions and the plates were read immediately at 450 nm absorbance using a FLUOstar
OMEGA plate reader. Research article Acknowledgements The authors thank the support staff at both Stanford and Oxford University for their technical assis-
tance. We also thank Dr. Dris Elatmioui at LUMC, The Netherlands who synthesized the UV-sensitive
peptide. This work was supported by the following grants: MRC_MR/M019837/1(GMG), NIH
5R01AI103867 (KCG), U19AI057229 (KCG) and HHMI (KCG). T cell functional assays using bacterial cell lysates T cell functional assays using bacterial cell lysates
2 106 HL60 cells (ATCC CLL-240/RRID:CVCL_0002, STR profiled and mycoplasma negative) were
treated overnight with calcium ionophore and IFNg at 37˚C to induce DC/myeloid-like differentiation
and to enhance MHC class I expression (Wu et al., 2004a; Wu et al., 2004b). The cells were subse-
quently divided into 48 well NUNC plates and 20 mg/mL of the test bacterial cell lysates were added
to individual wells. Following a further 7 hr of incubation, the cells were washed extensively and their
ability to stimulate AGA1-expressing T cell clones was assessed by ELISpot assay. 15,000 lysate-
pulsed HL60 cells were incubated with 500 CD8+ T cell from two clones expressing the AGA1 TCR
for 15 hr prior to development. Positive control included non-lysate-pulsed HL60 and T cells incu-
bated with 10ng/mL PMA. Results are reported as spot forming cells (SFCs) per 500 T cell input, and
SFCs double that observed with media alone were considered positive. Funding
Funder The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication. The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication. CD107a expression CD107a expression following T cell stimulation was evaluated as previously described, but with slight
protocol alterations (Stewart-Jones et al., 2012). 40,000 T cell clones were incubated with alloge-
neic HLA-B*57:01+ BCLs (mycoplasma negative) at a ratio of 4:1, pulsed with KF11 and the ‘nr’-
derived Mic peptides at final concentrations of 1 and 10 mM. Cells incubated with media alone, or in
the presence of 10 ng/mL PMA were included as negative and positive controls, respectively. 5 mL
of CD107a PE labelled monoclonal antibody (BD Biosciences, clone H4A3, RRID:AB_396135) was
added to each well and the samples were incubated at 37˚C. Golgi-Stop (BD Biosciences) was added
to individual samples 1 hr post incubation at a concentration recommended by the manufacturer. Following a total of 5 hr, cells were stained with anti-human CD8 APC (BD Biosciences, clone RPA-
T8, RRID:AB_398595) and fixed in BD Cell Fix prior to flow cytometry analysis using a CyAn. Data
analysis was performed using FlowJo software. 16 of 21 Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 Research article Research article Immunology and Inflammation Additional information Competing interests
Marvin H Gee: MHG is a co-founder of 3T Biosciences. K Christopher Garcia: KCG is a co-founder of
3T Biosciences. The other authors declare that no competing interests exist. Funding
Funder
Grant reference number
Author
Medical Research Council
MR/M019837/1
Geraldine Martina Gillespie
National Institutes of Health
5R01AI103867
K Christopher Garcia
National Institutes of Health
U19AI057229
K Christopher Garcia
Howard Hughes Medical Insti-
tute
K Christopher Garcia
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication. Author contributions Author contributions
Juan L Mendoza, Marvin H Gee, Data curation, Software, Formal analysis, Validation, Investigation,
Methodology, Writing - original draft, Writing - review and editing; Suzanne Fischer, Data curation,
Software, Formal analysis, Validation, Investigation, Methodology; Lilian H Lam, Methodology, Writ-
ing - review and editing; Simon Brackenridge, Fiona M Powrie, Methodology; Michael Birnbaum,
Conceptualization, Resources, Software, Formal analysis, Validation, Investigation, Methodology,
Writing - review and editing; Andrew J McMichael, Conceptualization, Investigation, Writing - origi-
nal draft, Writing - review and editing; K Christopher Garcia, Funding acquisition, Conceptualization,
Data curation, Investigation, Methodology, Writing - review and editing; Geraldine M Gillespie, Con-
ceptualization, Data curation, Investigation, Methodology, Writing - original draft, Writing - review
and editing Author ORCIDs
Lilian H Lam
http://orcid.org/0000-0003-3582-9209
Simon Brackenridge
https://orcid.org/0000-0002-0587-7560
K Christopher Garcia
https://orcid.org/0000-0001-9273-0278
Geraldine M Gillespie
https://orcid.org/0000-0002-1075-870X
8128. DOI: https://doi.org/10.7554/eLife.58128
17 of 21 Author ORCIDs
Lilian H Lam
http://orcid.org/0000-0003-3582-9209
Simon Brackenridge
https://orcid.org/0000-0002-0587-7560
K Christopher Garcia
https://orcid.org/0000-0001-9273-0278
Geraldine M Gillespie
https://orcid.org/0000-0002-1075-870X
Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128
17 of 21 Author ORCIDs
Lilian H Lam
http://orcid.org/0000-0003-3582-9209
Simon Brackenridge
https://orcid.org/0000-0002-0587-7560
K Christopher Garcia
https://orcid.org/0000-0001-9273-0278
Geraldine M Gillespie
https://orcid.org/0000-0002-1075-870X 17 of 21 17 of 21 Mendoza et al. eLife 2020;9:e58128. DOI: https://doi.org/10.7554/eLife.58128 Research article Research article Immunology and Inflammation Immunology and Inflammation Immunology and Inflammation Decision letter and Author response
Decision letter https://doi.org/10.7554/eLife.58128.sa1
Author response https://doi.org/10.7554/eLife.58128.sa2 Additional files Supplementary files
. Supplementary file 1. Non-redundant (‘nr’) database recovered KF11-related peptides, TCR
sequence similarity of T cell clones and HLA-B*57:01 HdH peptide binding hierarchies. Table 1:
Round 3 of the AGA-1 TCR yeast display selection results were used to identify sequence related
peptides from the non-redundant (’nr’) database. Listed are the prediction results for KF11-related
peptides derived from the gag HIV protein. GI identification and blast scores are included. Table 2:
AGA-1 TCR yeast display selection results from Round 3 screens were used to identify sequence
related peptides from the non-redundant (’nr’) database. Listed are the prediction results of
sequence-related, non-KF11 peptides. GI identification, blast scores, % identity to the KF11 peptide
and to ’nr’ database mined peptide hits are included. Table 3: TCR alpha chain amino acid sequence
identity of clone 1.2 and the AGA1 (clone 1.1) TCR. Clones 1.1 and 1.2 both utilize the V alpha 5
(AV5) chain segments, encode identical CDR3 motifs and carry one amino acid sequences difference
that maps to the J alpha (AJ) region (underlined). This residue is outside the TCR:peptide-MHC bind-
ing interface described previously for the AGA1 TCR-B*57:03-KF11 co-complex (red) (Stewart-
Jones et al., 2012). AGA1 TCR clone 1.1 and the related clone 1.2 are 100% sequence identical
across the CDR3 and J regions of their V beta19 (BV19) TCR chain sequences (CASTGSY-
GYTFGSGTRLTVT) (Stewart-Jones et al., 2012). The TCR V region, CDR3 and J region boundaries
as defined by ImMunoGeneTics (IMGT), http://www.imgt.org/IMGTrepertoire/. Table 4: Peptide
binding strength hierarchy ranked on the basis of UV exchange HLA-B*57:01 peptide binding ELISA
data. The index KF11 epitope and the HdH peptides are ranked in descending order according to
their peptide binding strength as determined using the UV exchange peptide binding assay. High,
medium and low binders are color-coded from dark to light shades of grey, respectively. Amino
acids differences specific to low/medium binders outside the p4-p6 TCR recognition interface are
highlighted (bold squared). p1 = peptide position 1, etc. . Source data 1. Library peptide-based returned ’nr’ hits_KF11 variant peptide . Source data 1. Library peptide-based returned ’nr’ hits_KF11 variant peptides. . Source data 2. Library peptide-based returned ’nr’ hits_non-KF11 related peptides. . Source data 3. Correlation between ELISA data and NetMHC Pan4.1 binding predictions for HdH
peptides. . Transparent reporting form Data availability Next-generation sequencing data has been deposited in the Sequence Read Archive under acces-
sion numbers SAMN14376837 and SAMN14376838. Accession Sample Name SPUID Organism Tax
ID Strain SAMN14376837 AGA1_255 AGA1_255 synthetic construct 32630 EBY100 SAMN14376838
AGA1_177 AGA1_177 synthetic construct 32630 EBY100 https://www.ncbi.nlm.nih.gov/biosample/
14376837 https://www.ncbi.nlm.nih.gov/biosample/14376838. The following datasets were generated: References DOI: https://doi.org/10.1038/nm1511, PMID: 17115046 Brenchley JM, Price DA, Douek DC. 2006b. HIV disease: fallout from a mucosal catastrophe? Nature
Immunology 7:235–239. DOI: https://doi.org/10.1038/ni1316, PMID: 16482171 Campion SL, Brodie TM, Fischer W, Korber BT, Rossetti A, Goonetilleke N, McMichael AJ, Sallusto F. 2014. Proteome-wide analysis of HIV-specific naive and memory CD4(+) T cells in unexposed blood donors. The
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Author(s)
Year Dataset title
Dataset URL
Database and
Identifier
Gee M
2020
BioSample: SAMN14376837;
Sample name: AGA1_255; SRA:
SRS6329182
https://www.ncbi.nlm. nih.gov/biosample/
14376837
NCBI BioSample,
SAMN14376837
Gee M
2020
BioSample: SAMN14376838;
Sample name: AGA1_177; SRA:
SRS6329183
https://www.ncbi.nlm. nih.gov/biosample/
14376838
NCBI BioSample,
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Info-gap management of public health Policy for TB with HIV-prevalence and epidemiological uncertainty
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RESEARCH ARTICLE Open Access Abstract Background: Formulation and evaluation of public health policy commonly employs science-based mathematical
models. For instance, epidemiological dynamics of TB is dominated, in general, by flow between actively and latently
infected populations. Thus modelling is central in planning public health intervention. However, models are highly
uncertain because they are based on observations that are geographically and temporally distinct from the
population to which they are applied. Aims: We aim to demonstrate the advantages of info-gap theory, a non-probabilistic approach to severe uncertainty
when worst cases cannot be reliably identified and probability distributions are unreliable or unavailable. Info-gap is
applied here to mathematical modelling of epidemics and analysis of public health decision-making. Methods: Applying info-gap robustness analysis to tuberculosis/HIV (TB/HIV) epidemics, we illustrate the critical role
of incorporating uncertainty in formulating recommendations for interventions. Robustness is assessed as the
magnitude of uncertainty that can be tolerated by a given intervention. We illustrate the methodology by exploring
interventions that alter the rates of diagnosis, cure, relapse and HIV infection. Results: We demonstrate several policy implications. Equivalence among alternative rates of diagnosis and relapse
are identified. The impact of initial TB and HIV prevalence on the robustness to uncertainty is quantified. In some
configurations, increased aggressiveness of intervention improves the predicted outcome but also reduces the
robustness to uncertainty. Similarly, predicted outcomes may be better at larger target times, but may also be more
vulnerable to model error. Conclusions: The info-gap framework is useful for managing model uncertainty and is attractive when uncertainties
on model parameters are extreme. When a public health model underlies guidelines, info-gap decision theory
provides valuable insight into the confidence of achieving agreed-upon goals. Keywords: TB management, HIV-AIDS, Public health, Epidemiology, Uncertainty, Robustness, Info-gap ywords: TB management, HIV-AIDS, Public health, Epidemiology, Uncertainty, Robustness, Info-gap Info-gap management of public health
Policy for TB with HIV-prevalence and
epidemiological uncertainty Yakov Ben-Haim1, Clifford C Dacso2 and Nicola M Zetola3* Yakov Ben-Haim1, Clifford C Dacso2 and Nicola M Zetola3* © 2012 Ben-Haim et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. © 2012 Ben-Haim et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. *Correspondence: nzetola@gmail.com
3Division of Infectious Diseases, Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania
Full list of author information is available at the end of the article Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Background in infectious disease epidemiology results also from inter-
dependency among individuals. When prospective studies
or randomized controlled trials are available, they usually
represent selected groups with as little variance as pos-
sible and may not apply to other populations [2]. Such
lack of generalizability may be more problematic for the
recommendations developed by international organiza-
tions. Those guidelines use the best available information
and expert opinion. Nonetheless the yield, effective-
ness and cost of the interventions vary significantly due
to heterogeneity of the populations in which they are
implemented [1,3]. Public health policies affect millions of people and deter-
mine the allocation of health care funds. However, select-
ing an intervention for a given population at a given time
is highly uncertain. Data supporting public health deci-
sions are scarce, of poor quality, not fully generalizable
and lack appropriate controls [1]. The high uncertainty *Correspondence: nzetola@gmail.com
3Division of Infectious Diseases, Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania
Full list of author information is available at the end of the article Page 2 of 17 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 compartment models are the most common, and we use
a slightly modified version of the widely used Murray-
Salomon model [17-19] to describe the evolution of
TB/HIV epidemics under various scenarios. The details
of the model appear in Appendix “The Murray-Salomon
model” section. Science-based mathematical models commonly support
public health decisions [4-7]. Many models were devel-
oped to explain or predict the course of an epidemic for
specific interventions. However, these models are lim-
ited by the uncertainty of the data and assumptions they
employ [5,7]. Despite severe uncertainty in public health decision-
making, actions must be timely and cost-effective. Analy-
sis of uncertainty is central in responsible decision making
using uncertain data and models. Info-gap theory
h
b
f The robustness function is the basic decision-support tool
in an info-gap analysis. If our dynamic model were accu-
rate we could evaluate any proposed intervention in terms
of the outcome of that intervention that is predicted by the
model. An intervention with low predicted TB prevalence
is preferred over an intervention with higher predicted
prevalence. Information-gap (info-gap) theory [8] was developed for
decision making when knowledge gaps are substantial,
worst cases cannot be reliably identified, and probabil-
ity distributions are unreliable or unavailable. An info-gap
model is the disparity between what is known and what
needs to be known in order to achieve an acceptable
outcome. The focus is on robustly achieving satisfactory
outcomes, thus making this technique suitable for pub-
lic health policy decision making [9]. Info-gap theory has
been applied in engineering, biological conservation, eco-
nomics, project management, medicine and homeland
security (see http://info-gap.com). The problem is great model uncertainty. This means
that predicted outcomes are unreliable and it is unrealis-
tic to prioritize interventions in terms of their predicted
outcomes. Using the model to find the intervention whose
predicted outcome is best, is not suited to planning with
highly uncertain models. Model-based predictions are useful, but when decid-
ing which public health intervention to implement, we
should also ask: how wrong could the model be, and an
acceptable outcome is still guaranteed? For any speci-
fied intervention we ask: what is the largest error in the
model, up to which all realizations of the model would
yield acceptable outcomes? Equivalently, what outcomes
can reliably be anticipated from this intervention, given
the unknown disparity between the model and reality? Answers to these questions lie in the robustness func-
tion, specified in Appendix
“Definition of robustness”
section. The robustness is dimensionless, and equals the
greatest fractional error in the model parameters that is
consistent with a specified outcome requirement. We use
the robustness function to prioritize the interventions in
terms of their robustness against uncertainty for achieving
the required outcome. We develop a framework for the practical use of info-gap
theory in public health for controlling infectious diseases. We focus on tuberculosis (TB) in the context of pandemic
HIV as an example. Epidemiological background The World Health Organization reported 9.4 million inci-
dent TB cases and 1.7 million TB deaths in 2009 and
estimated that only 63% of annual incident TB cases
were detected and reported; of these, 86% were success-
fully treated [10,11]. Given the disease burden, the United
Nations Millennium Development Goals include targets
and indicators related to TB control. The targets include
decreasing TB incidence by 2015, halving TB prevalence
and mortality by 2015 (compared with 1990), and diag-
nosing 70% of new smear-positive cases and curing 85%
of these cases by 2015. However, despite current efforts,
many countries will not achieve these targets [10-14]. Knight [20] recognized that probability distributions
are sometimes unknown and that severe uncertainty may
be non-probabilistic. Wald [21], Ben-Tal and Nemirovski
[22] and others developed tools for robustly managing
non-probabilistic uncertainty by minimizing the worst
outcome on a set of possibilities. Info-gap theory is non-
probabilistic and handles situations where worst cases are
unknown. The HIV-AIDS pandemic is the major worldwide chal-
lenge to TB control [11,13,15,16]. HIV creates a situation
of serious uncertainty for public health interventions
based on pre-HIV era models [10,11,13]. This is reflected
in population distribution, spread, control, and recur-
rence. Latently and actively infected individuals con-
tribute differently to spread of disease. It is necessary to
consider infectivity, rapidity of progression, re-infection,
individuals with higher susceptibility for infection and
reinfection resulting from HIV coinfection, etc. in order
to produce refined models of diagnosis and treatment. We summarize here the main attributes of the info-gap
robustness function: a plot of robustness-to-uncertainty
versus required performance. This is the basic info-gap
tool for prioritizing available options. Info-gap implementation Info-gap methodology requires three main elements: a
system model, a performance measure, and a model of
uncertainty. The system model is a mathematical repre-
sentation of a system and its influence on the variables
of interest, for which management aspirations (perfor-
mance criteria) are set. A performance measure assesses
value or utility of outcomes. The model of uncertainty is
a non-probabilistic representation of the degree to which
the value of parameters, the form of a function, or the
structure of a model may deviate from nominal estimates. When models are highly uncertain, it is unrealistic to pri-
oritize one’s options based on predicted outcomes of those
options, because those predictions have no robustness to
errors in the underlying models. Options must be eval-
uated in terms of the level of performance that can be
reliably achieved; this is expressed by robustness. Combining the trade offand zeroing properties yields
realistic prioritization of options. Info-gap models of uncertainty are non-probabilistic Info-gap robustness analysis is implementable even
when probability distributions are unknown, and thus
is suited to severe uncertainty. In contrast, Monte
Carlo simulation, Bayesian analysis, or probabilistic risk
assessment require knowledge of probabilities. Other
non-probabilistic tools include interval analysis, fuzzy set
theory [27], possibility theory [28] and Robust Decision
Making (RDM). A comparison of info-gap and RDM has
recently been published [29]. Grassly et al [32] note, in discussing epidemiology of
HIV/AIDS, that “not all sources of error are amenable to
statistical analysis” (p.i37), due to biased, inaccurate or
unavailable data. The basic idea of info-gap model uncer-
tainty is that we do not know how wrong our estimates are,
we have no reliable knowledge of worst cases, and we do
not know probability distributions for the estimates. The
info-gap model uncertainty model is a non-probabilistic
quantification of uncertainties. Info-gap is operationally distinct from the min-max or
worst-case decision strategy [9] A dominant uncertainty in TB dynamics with HIV
prevalence is in model parameter values, though HIV
causes significant uncertainties in model structure. Struc-
tural uncertainty refers to missing terms in the equations,
missing equations, or unknown nonlinearities. Structural
uncertainty is dealt with much less frequently than param-
eter uncertainty because of technical challenges. We focus
on parameter uncertainty in this paper because of its
importance and to facilitate the presentation of this first
application of info-gap theory to public health. Info-gap robustness does not require knowledge of a worst
case. When even typical scenarios are poorly character-
ized, it is usually impractical to characterize worst cases,
which is required by the min-max strategy. Info-gap the-
ory does require specifying acceptable outcomes. Thus it
is well suited to policy making, because preferences on
outcomes are the driving force. Robustness trades offagainst performance [23,24] Robustness trades offagainst performance [23,24] More
demanding
performance
requirements
are
less robust against uncertainty than less demanding Many different epidemiological models have been
used to evaluate treatment strategies. Deterministic Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 3 of 17 survival in economic [8], biological [26] and other com-
petitive environments [31]. requirements. This trade offis quantified and expressed
graphically by the monotonic robustness curve. Prioritization of options depends on performance
requirements The system model in our example is summarized in two
functions. C(t) is the variation over time of the total num-
ber of TB cases, untreated and treated, HIV-positive and
HIV-negative, as a fraction of the initial population. R(t) is
the total number of relapses, fast and slow, HIV-positive
and HIV-negative, as a fraction of the initial population. (See eqs.(23) and (24) in Appendix “The Murray-Salomon
model” section.) Prioritization of options may change as requirements
change. This is called “preference reversal” and is
expressed by the intersection of the robustness curves of
different options. Preference reversal provides insight to
anomalous behavior such as the Ellsberg and Allais para-
doxes in human decision making [8], the equity premium
puzzle in economics [8], and animal foraging [26]. We will
show that preference reversal occurs when selecting pub-
lic health interventions because priorities are time- and
context-dependent. The public health practitioner wishes to control the
total number of TB cases: the fewer the better. How-
ever, trying to minimize this prevalence depends on model
predictions that are highly uncertain. The performance
requirement is to keep the total fraction of TB cases at
a specified time, tm, below a critical value, Cm, eq.(25) in
Appendix “Performance requirements” section. Interpreting robustness curves: trade offand zeroing p
g
g
All info-gap robustness curves have two properties,
mentioned earlier: trade offbetween performance and
robustness, and zeroing of the robustness curve. These
properties are central in using robustness curves to evalu-
ate public health policy. The coefficients of the epidemiological models are spec-
ified in Tables 1 and 2. Thoughout our examples, the initial
conditions correspond to low TB and low HIV prevalence
(the first data-column of Table 3) unless specified other-
wise. The control variables specified in Appendix “Control
variables” section are themselves model parameters. The
robustness curve in Figure 1 is evaluated for the nominal
values of the control variables specified in Tables 1 and 2. This set of control variables is the “baseline intervention”. The uncertain variables specified in Appendix “Uncer-
tainty” section are also model parameters. Their nominal
values and uncertainty estimates are specified in Table 4. These nominal values are the same as appear in Tables 1
and 2 for these variables. The total case load is evaluated
at time tm = 10 years after initiation unless indicated
otherwise. We can interpret the numerical values along the robust-
ness curve as follows. The prevalence, C(t), and its critical
value, Cm, are normalized to the initial population size. For instance, Cm = 0.025 means that the prevalence at
time tm must not exceed 2.5% of the initial population
size. The robustness corresponding to this value of Cm, is
0.1 as seen in Figure 1. This means that the performance
requirement is guaranteed if the uncertain model param-
eters vary from their nominal values by no more than
10% of their error estimates. (The model parameters are
constrained to be positive since they are first-order rate
constants.) The public health practitioner may feel that robust-
ness to 10% uncertainty in the model parameters is rather
small, given the substantial uncertainty in the epidemio-
logical dynamics of TB with HIV prevalence. If we want
robustness to, say, 25% uncertainty in the model parame-
ters we must accept a larger final case load, namely, Cm =
0.033 as seen in Figure 1. Greater robustness is obtained
only by accepting poorer outcome; this is an irrevocable
trade offthat is quantified by the robustness curve. Info-gap robustness may proxy for the probability of
satisfying the performance requirement [8,30,31] We use info-gap theory [8] to model and manage uncer-
tainties in the following parameters: slow and fast relapse
rates for HIV positives and negatives, TB infection rates
for HIV positives and negatives, and the HIV infection
rate. Much literature suggests these parameters for their
impact on the course of epidemics and the difficulty A more robust option is often more likely to achieve
the required outcome. By prioritizing the options using
info-gap robustness, one maximizes the probability of sat-
isfying the requirement, without knowing probability dis-
tributions. The proxy property is central to understanding Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 4 of 17 starts at about 4.2% of the initial population and decays
to about 3% in the first 1.5 years, thereafter decaying
more slowly, reaching 2.1% of the initial population size
after 10 years. The relapse population starts very small,
rises rapidly in the first year and thereafter decays grad-
ually. The reduction in the rate of decrease of the TB
cases after 1.5 years, Figure 2, results from the influx
of relapses which have built up since initiation of the
intervention. in measuring them [10,11,16,33-36]. Other uncertain-
ties could also be investigated, depending on the pur-
pose of the analysis. We use estimated values for each
uncertain parameter, and estimated errors typically cho-
sen as half of an interval estimate of the parameter. The
info-gap model of uncertainty is specified in Appendix
“Uncertainty” section. We aim to achieve the performance requirement by
judicious choice of control variables, defined in Appendix
“Control variables” section. Eligible control variables are
any coefficients of the dynamic model that can be influ-
enced by public health or related medical intervention. We use the diagnosis rate, cure rate, relapse rate, and HIV
infection rate. We define an intervention in terms of the
values of these variables [15,34,37-40]. Results: robustness and policy evaluation We use the info-gap robustness function to evaluate
alternative interventions aimed at controlling the rela-
tive TB prevalence, C(t), at a specified target time, tm,
in the future. An intervention is specified by the values
of the control variables. The evaluation leads to realis-
tic assessment of outcomes and preferences among the
interventions. uncertainty in attaining that outcome. The robustness curve in Figure 1 is based on satisficing
the relative TB prevalence: requiring that the prevalence
not exceed the critical value, Cm. Figure 1 shows the
robustness vs. the critical prevalence. The positive slope
of the robustness curve in Figure 1 expresses the trade
offbetween robustness and performance: large robust-
ness entails large prevalence at the specified target time
(10 years). Equivalently, requiring low relative prevalence
entails low robustness to uncertainty in the epidemiolog-
ical model. The robustness curve quantifies the intuition
that more demanding outcomes (small prevalence) are
more vulnerable to model uncertainty (small robustness). We can interpret the numerical values along the robust-
ness curve as follows. The prevalence, C(t), and its critical
value, Cm, are normalized to the initial population size. For instance, Cm = 0.025 means that the prevalence at
time tm must not exceed 2.5% of the initial population
size. The robustness corresponding to this value of Cm, is
0.1 as seen in Figure 1. This means that the performance
requirement is guaranteed if the uncertain model param-
eters vary from their nominal values by no more than
10% of their error estimates. (The model parameters are
constrained to be positive since they are first-order rate
constants.) The robustness curve in Figure 1 is based on satisficing
the relative TB prevalence: requiring that the prevalence
not exceed the critical value, Cm. Figure 1 shows the
robustness vs. the critical prevalence. The positive slope
of the robustness curve in Figure 1 expresses the trade
offbetween robustness and performance: large robust-
ness entails large prevalence at the specified target time
(10 years). Equivalently, requiring low relative prevalence
entails low robustness to uncertainty in the epidemiolog-
ical model. The robustness curve quantifies the intuition
that more demanding outcomes (small prevalence) are
more vulnerable to model uncertainty (small robustness). Trade off Key to understanding the trade offexpressed by the
robustness curve is the concept of satisficing. In contrast
to optimizing, satisficing asks for an outcome that meets
minimal needs but may not be the best imaginable. The
satisficing strategy is not merely “accepting second best.”
Satisficing is aspirational, setting a goal just like opti-
mization, but also requiring robustness to uncertainty. The satisficing strategy induces a trade offbetween the
aspiration for good outcome and the robustness against
uncertainty in attaining that outcome. Interpreting robustness curves: trade offand zeroing cTable A5, p.63, [18]. dFootnote c, Table A5, p.63, [18]. eFootnote ‡, Table A5, p.63, and Figure A5, p.57 [18]. fFootnote b, Table A5, p.63,[18]. gRate: per person per year. In Botswana the average is 477 cases per year per 100,000 people. 62% of them are HIV infected. hDepends on HIV prevalence. In areas with HIV and without preventive treatment, 25% of babies born from HIV mothers are infected. iIn Botswana. j[Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA. 1999 Aug 18;282(7):677–86.]. kNot needed since λ is a primary datum. ℓDecision variable. mCan also be treated as a decision variable. eFootnote ‡, Table A5, p.63, and Figure A5, p.57 [18]. gRate: per person per year. In Botswana the average is 477 cases per year per 100,000 people. 62% of them are HIV infected. hDepends on HIV prevalence. In areas with HIV and without preventive treatment, 25% of babies born from HIV mothers are infected. iIn Botswana. j[Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA. 1999 Aug 18;282(7):677–86.]. kNot needed since λ is a primary datum. ℓDecision variable. mCan also be treated as a decision variable. mCan also be treated as a decision variable. is precisely the nominal prediction of the prevalence at
time tm as seen by the right end-point in Figure 2. That
is, the value of C(tm), evaluated with the best estimates of
the model parameters, equals 0.021. The horizontal inter-
cept in Figure 1 is an example of the property of zeroing
that holds for all info-gap robustness curves: The outcome Interpreting robustness curves: trade offand zeroing Figures 2 and 3 show the temporal evolution of the rel-
ative prevalence of TB cases, C(t), and relative relapses,
R(t), based on the nominal estimates of the model param-
eters, with moderately low initial TB and HIV prevalence. C(t) and R(t) are fractions of the initial total population
size. Figure 2 shows that the total number of TB cases Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 5 of 17 Table 1 Model parameters in the Murray-Salomon basic model
Symbol
Definition
HIV neg
HIV pos
Birth rate g
births/year/person
0.03c
0c,h
N
population size
0.821i
0.179i
T
births per year = birth rate×Nc
λ g
infection rate
1.81 × 10−3 m
2.96 × 10−3 m
Kk
# respiratory contacts with infected/person/year
Lk
probability that respir. contact with infectious source leads to infection
KL
5–15a
Yk
# infectious cases in population
μ g
non-TB death rate
0.009c
0.05c
p
proportion of new infections entering slow breakdown
0.9 (0.85–0.95)a
0.4 (0.3–0.5a)
βF g
fast breakdown rate
2c
3
βS
g
slow breakdown rate
0.001 (5–15 × 10−4 a)
0.075 (0.05–0.10a)
χ g
rate of application of INH to infected individuals
0.75 (ℓ)
ν
protection from superinfection conferred by primary infection
0.75 (0.5–1a)
w
short-term INH effectiveness
0.7
h
long-term INH effectiveness
0.7
di,j
proportion of pre-diagnosed cases in clinical category i entering diagnosis category j
d1,1
0.45 (0.4–0.5)j
0.35 (0.3–0.4)j, e
d2,1
0.55 (0.5–0.6)
0.65 (0.6–0.7) e
d3,1
e
di,2
di,2 = 1 −di,1
f
si
proportion of new cases in clinical category i
s1, s2
proportion of new cases in clinical category i
0.45 (0.4–0.5a)
s3
proportion of new cases in clinical category i
s3 = 1 −s1 −s2 d
δj g
diagnosis rate for category j
0.6 ℓ
0.6 ℓ
σ g
smear conversion rate
0.03 c
Model parameters in the Murray-Salomon basic model Table Two p 42 in ref [18] except for K L Y and N which are defined in footnote 2 on p 21 of [18] Where a Table 1 Model parameters in the Murray-Salomon basic model Model parameters in the Murray-Salomon basic model, Table Two, p.42, in ref. [18], except for K, L, Y and N which are defined in footnote 2 on p.21 of [18]. Where a
value is specified only for HIV-negative, the same value is used for HIV-positive. aFootnote 8, p.24, [18]. bFootnote e, Table A5, p.63, [18]. Zeroing We note that the robustness curve in Figure 1 reaches the
horizontal axis at the value Cm = 0.021. This means that
requiring the prevalence not to exceed 2.1% of the initial
population has no robustness against model uncertainty. The value of Cm at which the robustness becomes zero Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 6 of 17 Table 2 Model parameters in the Murray-Salomon basic model
Symbol
Definition
HIV neg
HIV pos
εU f
spontaneous cure rate for untreated cases
0.2 (0.14–0.25)g
μi
U
f
TB death rate for untreated cases in clinical category i
μ1
U
0.12 (0.075–0.20a)
0.45 (0.3–0.6a)
μ2
U, μ3
U
0.7μ1
U
b
0.7μ1
U
b
gi,k
proportion of treated cases in clinical category i and treatment category k
g1,1
0.5 d
g2,1
0.28 d
g3,1
d
gi,2
gi,2 = 1 −gi,1 e
εk
T
f
cure rate for treated case in treatment category k
ε1
T
0.8
ε2
T
0.5c
μi,k
T
f
TB death rate for treated cases in clinical category i and treatment category k
μ1,1
T
0.075c
0.16c
μ1,2
T
0.12c
0.24c
μ2,k
T , μ3,k
T
0.7μ1,k
T
b
0.7μ1,k
T
b
rU
proportion of spontaneously recovered cases entering the slow relapse category
0.009c
rk
T
proportion of recovered cases from treatment category k entering the slow relapse category
r1
T
0.0096c
r2
T
0.0094c
ρF f
fast relapse rate
2c
3
ρS f
slow relapse rate
0.001 (5–15 × 10−4 a)
γ f
rate of HIV infection
0.075 (0.011–0.95)h
Model parameters in the Murray-Salomon basic model, Table Two, p.42, in ref. [18], except for K, L, Y and N which are defined in footnote 2 on p.21 of [18]. Where a
value is specified only for HIV-negative, the same value is used for HIV-positive. aFootnote 8, p.24, [18]. bFootnote e Table A5 p 63 [18] Table 2 Model parameters in the Murray-Salomon basic model predicted by the model, when adopted as the performance
requirement, has no robustness against uncertainty in
the model. the performance of these control variables. Due to the
trade offproperty, only larger prevalence can reliably be
expected to result from this choice of the control vari-
ables. Predicted outcomes are not reliable for prioritizing
the interventions. It is not surprising that the predicted outcome is
extremely vulnerable to error in the model upon which the
prediction is based. Zeroing However, the zero-robustness of pre-
dicted outcomes has an important implication for policy
selection. Equivalent interventions The robustness curve in Figure 1 is for a particular
choice of values of the control variables: the baseline
intervention. The zeroing property—no robustness of the
predicted outcome of these control values—implies that
we should not assess these control values in terms of
their predicted outcome. The predicted prevalence of
0.021 at time tm = 10 years does not reliably reflect Different combinations of interventions can yield essen-
tially equivalent results, as in Figure 4. The baseline
intervention (solid), is characterized by low diagnosis rate
and high relapse rate. The other intervention (dash) has
higher diagnosis rate and lower relapse rate as specified in
Table 5. (Interventions are specified by the values of con-
trol variables presented in Table 5). The robustness curves Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 7 of 17 Table 3 Initial conditions
Symbol
Low TB Prevalence
Medium TB Prev. High TB Prev. Equivalent interventions Low
Med
High
Low
Med
High
Low
Med
High
HIV
HIV
HIV
HIV
HIV
HIV
HIV
HIV
HIV
1
U
0.9
0.8
0.6
0.8
0.650
0.500
0.6
0.5
0.4
2
IF
0.0075
0.015
0.03
0.011
0.018
0.028
0.03
0.0375
0.045
3
IS
0.03
0.06
0.12
0.05
0.088
0.125
0.12
0.15
0.18
4
SF
0.003
0.006
0.012
0.01
0.018
0.025
0.012
0.015
0.018
5
HS
0.009
0.018
0.036
0.02
0.035
0.050
0.036
0.045
0.054
Ci,j
U
6
(i, j) = (1, 1)
0.005
0.01
0.02
0.008
0.014
0.020
0.02
0.025
0.03
7
(i, j) = (2, 1)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
8
(i, j) = (3, 1)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
9
(i, j) = (1, 2)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
10
(i, j) = (2, 2)
0.001
0.002
0.004
0.002
0.004
0.005
0.004
0.005
0.006
11
(i, j) = (3, 2)
0.001
0.002
0.004
0.002
0.004
0.005
0.004
0.005
0.006
Ci,k
T
12
(i, k) = (1, 1)
0.01
0.02
0.04
0.02
0.035
0.050
0.04
0.05
0.06
13
(i, k) = (2, 1)
0.005
0.01
0.02
0.01
0.018
0.025
0.02
0.025
0.03
14
(i, k) = (3, 1)
0.005
0.01
0.02
0.01
0.018
0.025
0.02
0.025
0.03
15
(i, k) = (1, 2)
0.005
0.01
0.02
0.02
0.035
0.050
0.02
0.025
0.03
16
(i, k) = (2, 2)
0.002
0.004
0.008
0.005
0.009
0.013
0.008
0.01
0.012
17
(i, k) = (3, 2)
0.0025
0.005
0.01
0.003
0.005
0.008
0.01
0.0125
0.015
18
RF
0.002
0.004
0.008
0.005
0.009
0.013
0.008
0.01
0.012
19
RS
0.006
0.012
0.024
0.015
0.025
0.038
0.024
0.03
0.036
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000 Symbol
Low TB Prevalence
Medium TB Prev. High TB Prev. Equivalent interventions Low
Med
High
Low
Med
High
Low
Med
High
HIV
HIV
HIV
HIV
HIV
HIV
HIV
HIV
HIV
1
U
0.9
0.8
0.6
0.8
0.650
0.500
0.6
0.5
0.4
2
IF
0.0075
0.015
0.03
0.011
0.018
0.028
0.03
0.0375
0.045
3
IS
0.03
0.06
0.12
0.05
0.088
0.125
0.12
0.15
0.18
4
SF
0.003
0.006
0.012
0.01
0.018
0.025
0.012
0.015
0.018
5
HS
0.009
0.018
0.036
0.02
0.035
0.050
0.036
0.045
0.054
Ci,j
U
6
(i, j) = (1, 1)
0.005
0.01
0.02
0.008
0.014
0.020
0.02
0.025
0.03
7
(i, j) = (2, 1)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
8
(i, j) = (3, 1)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
9
(i, j) = (1, 2)
0.002
0.004
0.008
0.003
0.005
0.008
0.008
0.01
0.012
10
(i, j) = (2, 2)
0.001
0.002
0.004
0.002
0.004
0.005
0.004
0.005
0.006
11
(i, j) = (3, 2)
0.001
0.002
0.004
0.002
0.004
0.005
0.004
0.005
0.006
Ci,k
T
12
(i, k) = (1, 1)
0.01
0.02
0.04
0.02
0.035
0.050
0.04
0.05
0.06
13
(i, k) = (2, 1)
0.005
0.01
0.02
0.01
0.018
0.025
0.02
0.025
0.03
14
(i, k) = (3, 1)
0.005
0.01
0.02
0.01
0.018
0.025
0.02
0.025
0.03
15
(i, k) = (1, 2)
0.005
0.01
0.02
0.02
0.035
0.050
0.02
0.025
0.03
16
(i, k) = (2, 2)
0.002
0.004
0.008
0.005
0.009
0.013
0.008
0.01
0.012
17
(i, k) = (3, 2)
0.0025
0.005
0.01
0.003
0.005
0.008
0.01
0.0125
0.015
18
RF
0.002
0.004
0.008
0.005
0.009
0.013
0.008
0.01
0.012
19
RS
0.006
0.012
0.024
0.015
0.025
0.038
0.024
0.03
0.036
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
0.02
0.025
0.03
0.035
0.04
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
Figure 1 Robustness of relative TB prevalence. Run 8. for these two control strategies, at 10 years, are nearly the
same, suggesting that the public health practitioner may
choose freely between them, perhaps employing addi-
tional criteria such as cost or ease of implementation. Equivalence may be lost if parameters are changed. For
instance, we will see later (Figure 5) that these interven-
tions evaluated at 10, 20 or 30 years have very different
robustness curves. Equivalent interventions Table 4 Nominal values and error weights of uncertain
variables
Symbol
Nominal value, ui
Error weight, si
λ
1.81 × 10−3
0.0009
λ
2.96 × 10−3
0.0018
γ
0.075
0.2
ρS
0.001
0.0005
ρS
0.001
0.001
ρF
2
1
ρF
3
1.5 for these two control strategies, at 10 years, are nearly the
same, suggesting that the public health practitioner may
choose freely between them, perhaps employing addi-
tional criteria such as cost or ease of implementation. Equivalence may be lost if parameters are changed. For
instance, we will see later (Figure 5) that these interven-
tions evaluated at 10, 20 or 30 years have very different
robustness curves. for these two control strategies, at 10 years, are nearly the
same, suggesting that the public health practitioner may
choose freely between them, perhaps employing addi-
tional criteria such as cost or ease of implementation. Equivalence may be lost if parameters are changed. For
instance, we will see later (Figure 5) that these interven-
tions evaluated at 10, 20 or 30 years have very different
robustness curves. 0.02
0.025
0.03
0.035
0.04
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
Figure 1 Robustness of relative TB prevalence. Run 8. 0.02
0.025
0.03
0.035
0.04
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
Figure 1 Robustness of relative TB prevalence. Run 8. Table 4 Nominal values and error weights of uncertain
variables
Symbol
Nominal value, ui
Error weight, si
λ
1.81 × 10−3
0.0009
λ
2.96 × 10−3
0.0018
γ
0.075
0.2
ρS
0.001
0.0005
ρS
0.001
0.001
ρF
2
1
ρF
3
1.5 Table 4 Nominal values and error weights of uncertain
variables Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 8 of 17 0
2
4
6
8
10
0.02
0.025
0.03
0.035
0.04
0.045
Time (yr)
C(t)
Figure 2 Relative TB prevalence vs. time. Run 8. 0
2
4
6
8
10
0.02
0.025
0.03
0.035
0.04
0.045
Time (yr)
C(t)
Figure 2 Relative TB prevalence vs. time. Run 8. 0
2
4
6
8
10
0.02
0.025
0.03
0.035
0.04
0.045
Time (yr)
C(t)
Figure 2 Relative TB prevalence vs. time. Run 8. 0.02
0.025
0.03
0.035
0.04
0
0.05
0.1
0.15
0.2
0.25
Critical Prevalence
Robustness
Figure 4 Equivalent robustness for two interventions. Run 8: —,
run 15: – –. Impact of initial TB and HIV prevalence We now consider higher initial prevalences. The overall
shape of the dynamic response is very similar in each case,
except that the prevalence increases significantly as the
initial prevalence increases. As in Figures 2 and 3, in each
scenario the initial TB prevalence decreases rapidly dur-
ing the first 2 years, and thereafter decreases more slowly
as the new relapse population—which peaks around the
end of the first year—flows back into active cases. 0
2
4
6
8
10
0.008
0.009
0.01
0.011
0.012
Time (yr)
R(t)
Figure 3 Relative relapses vs. time. Run 8. Figure 7 shows robustness curves for a target time 10
years after initiation, for low (solid), medium (dash) and
high (dot-dash) initial prevalence of TB and HIV. The
low-prevalence curve (solid) is the same as Figure 1. The
robustness curves shift dramatically to the right as the
baseline prevalence of TB and HIV increases, indicat-
ing poorer estimated outcome and lower robustness to
uncertainty. Equivalent interventions 0.02
0.025
0.03
0.035
0.04
0
0.05
0.1
0.15
0.2
0.25
Critical Prevalence
Robustness
Figure 4 Equivalent robustness for two interventions. Run 8: —,
run 15: – –. Figure 4 Equivalent robustness for two interventions. Run 8: —,
run 15: – –. Figure 6 shows a different aspect of the equivalence
of interventions. The figure shows robustness curves for
two strategies specified in Table 5. Both strategies aim to
control the relative prevalence of TB, but one (solid) is
geared for a 10-year target time, while the other (dash)
considers a 30-year target. The estimated outcomes—
prevalence—are very nearly the same for these two
strategies, each at its respective target time, as shown by
their shared horizontal intercept at Cm = 0.018. These
predictions result from estimated model parameters, so
one might be inclined to conclude that TB prevalence of
0.018 can be achieved at either 10 or 30 years by using the
corresponding intervention. However, the epidemiological model is highly uncertain,
and the robustness curves in Figure 6 of these two strate-
gies are quite different. Not surprisingly, the 30-year target
is much less robust to uncertainty. It would be erroneous
to treat these two strategies as outcome-equivalent since
their performances at positive robustness are quite dif-
ferent. Nominal equivalence (equivalence of the predicted
outcome) does not imply robustness equivalence. Intervention aggressiveness Figure 8 shows robustness curves for low initial TB and
HIV prevalence with interventions specified in Table 5. The solid curve is the baseline intervention, against which
the other curves entail more aggressive intervention in
either or both the active cases and the relapse population. Ben-Haim et al. Intervention aggressiveness BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 9 of 17 Table 5 Control variables for robustness curves
Run
Init
tm
δj = δ
j
εk
T = εk
T
ρF
ρF
λ
λ
γ
βF
βF
Prev a
(yr)
8
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
9
1
10
(0.65, 0.65)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
10
1
10
(0.65, 0.65)
(0.88, 0.55)
2
3
0.00181
0.00296
0.075
2
3
11
1
10
(0.65, 0.65)
(0.88, 0.55)
1.5
2.25
0.00181
0.00296
0.075
2
3
12
1
10
(0.65, 0.65)
(0.88, 0.55)
1
1.5
0.00181
0.00296
0.075
2
3
15
1
10
(0.85, 0.85)
(0.8, 0.5)
1.2
2
0.00181
0.00296
0.075
2
3
19
5
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
21
5
10
(0.65, 0.65)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
22
5
10
(0.65, 0.65)
(0.88, 0.55)
2
3
0.00181
0.00296
0.075
2
3
23
5
10
(0.65, 0.65)
(0.88, 0.55)
1
1.5
0.00181
0.00296
0.075
2
3
20
9
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
24
9
10
(0.65, 0.65)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
25
9
10
(0.65, 0.65)
(0.88, 0.55)
2
3
0.00181
0.00296
0.075
2
3
26
9
10
(0.65, 0.65)
(0.88, 0.55)
1
1.5
0.00181
0.00296
0.075
2
3
27
1
20
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
28
1
30
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
29
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.0375
2
3
30
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.05
2
3
31
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.06
2
3
32
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.0009
0.00148
0.075
2
3
33
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.0003
0.0005
0.075
2
3
38
1
30
(0.85, 0.85)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
2
3
39
1
10
(0.6, 0.6)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
1
1.5
41
1
10
(0.65, 0.65)
(0.8, 0.5)
2
3
0.00181
0.00296
0.075
1
1.5
aData-column in Table 3. Intervention aggressiveness Table 5 Control variables for robustness curves Figure 5 shows robustness curves at target times, tm,
of 10, 20 and 30 years (solid, dash, dot-dash respec-
tively). The initial prevalences of TB and HIV are low. The
interventions are all at the baseline. The progression from solid to dash to dot-dash in
Figure 8 represents increasingly aggressive intervention
in the active TB case population. We see that increasing
aggressiveness, in this specific parameter configuration,
results in increasing prevalence and decreasing robust-
ness to model error at the target time. The explanation
is that aggressive treatment of active cases enlarges the
relapse population which flows back into the active case
population. The predicted prevalence decreases as the target time
increases, as shown by the horizontal intercepts in
Figure 5. The baseline intervention is predicted to reduce
the prevalence, (in units of initial population size), as
the time horizon increases. However, the zeroing prop-
erty means that these predictions have no robustness to
uncertainty in the model used for prediction. Only higher
prevalence has positive robustness. The top curve in Figure 8 modifies the most aggressive
case (dot-dash) by also including more aggressive inter-
vention in the TB relapse population. This reduction in
relapse reduces the predicted prevalence after 10 years,
and increases the robustness to uncertainty. From Figure 5 we see that, for critical TB prevalence Cm
less than 3%, the 30-year TB prevalence is more robust
than the 20-year prevalence which is more robust than
the 10 year prevalence. For instance, at critical TB preva-
lence of Cm = 0.02, the robustnesses for 10, 20 and 30
year horizons are 0, 0.08 and 0.12, respectively. This inter-
vention has no robustness to uncertainty when requiring a Different target times Most of the results discussed so far evaluated the robust-
ness for a target time 10 years after initiation. We now
consider the implications of different target times. Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 10 of 17 0.02
0.04
0.06
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
10 yr
30 yr
20 yr
Figure 5 Robustness curves at 10, 20 and 30 years. Run 8: —, run
27: – –, run 28: ·–. 0
.05
0.1
.15
0.2
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
Med
High
Low
Figure 7 Robustness curves for low, medium and high initial TB
and HIV prevalence. Run 8: —, run 19: – – run 20: ·–. 0.02
0.04
0.06
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
10 yr
30 yr
20 yr
Figure 5 Robustness curves at 10, 20 and 30 years. Run 8: —, run
27: – –, run 28: ·–. 0.02
0.04
0.06
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
10 yr
30 yr
20 yr
Figure 5 Robustness curves at 10, 20 and 30 years. Run 8: —, run
27: – –, run 28: ·–. 0
.05
0.1
.15
0.2
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
Med
High
Low
Figure 7 Robustness curves for low, medium and high initial TB
and HIV prevalence. Run 8: —, run 19: – – run 20: ·–. Figure 7 Robustness curves for low, medium and high initial TB
and HIV prevalence. Run 8: —, run 19: – – run 20: ·–. Suppose we are willing to aim at a final TB prevalence of
3.7%. We see from Figure 5 that now the 10-year horizon
is more robust than 20 years which is more robust than 30
years. The robustnesses are now 30%, 24% and 22% for 10,
20 and 30 years. The robustness curves have intersected
one another and the robustness rankings are reversed. As
the target time decreases, the predicted outcome becomes
worse (horizontal intercept moves right) but the cost of
robustness improves. This causes the robustness curves to
cross one another. More intuitively, we can say that pre-
diction of TB prevalence is more reliable for short time 2% prevalence after 10 years; in fact, the estimated preva-
lence at 10 years is greater than 2%. Different target times The prevalence at 20
years will be no worse than 2% provided that the model
coefficients err by no more than 8%, and at 30 years the
robustness to error is 12%. The practitioner may feel that even 12% robustness
against model-coefficient error is rather small, given the
severe uncertainty of TB epidemiology in the context of
epidemic HIV. This means that, even at a 30-year hori-
zon, this intervention cannot reliably achieve a relative
prevalence as low as 2%. 0.02
0.03
0.04
0
0.05
0.1
0.15
0.2
0.25
0.3
Critical prevalence
Robustness
Figure 8 Robustness with varying aggressiveness. Run 8: —, run
9: – –, run 10:·–, run 12: · · ·. 0.02
0.03
0.04
0
0.05
0.1
0.15
0.2
0.25
0.3
Critical prevalence
Robustness
Figure 8 Robustness with varying aggressiveness. Run 8: —, run
9: – –, run 10:·–, run 12: · · ·. 0
0.025
0.05
0.075
0.1
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
30 yr
10 yr
Figure 6 Nominal equivalence of two interventions. Run12: —,
run 38: – –. 0.02
0.03
0.04
0
0.05
0.1
0.15
0.2
0.25
0.3
Critical prevalence
Robustness
Figure 8 Robustness with varying aggressiveness. Run 8: —, run
9: – –, run 10:·–, run 12: · · ·. 0
0.025
0.05
0.075
0.1
0
0.05
0.1
0.15
0.2
0.25
Critical prevalence
Robustness
30 yr
10 yr Critical prevalence p
Figure 8 Robustness with varying aggressiveness. Run 8: —, run
9: – –, run 10:·–, run 12: · · ·. p
Figure 6 Nominal equivalence of two interventions. Run12: —,
run 38: – –. Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 11 of 17 increase TB prevalence unless compensating measures are
taken. Significantly, the cost of robustness (slope of the
robustness curve) does not change as a result of decreased
HIV infection rate. Reducing HIV infection rate shifts
the robustness curve to the right, with almost no change
in slope. horizon than for long times. But since a long time is
required to overcome the relapse effect, we observe the
intersection of the robustness curves and the consequent
reversal of their robust dominance. Results like Figure 5 have important policy implications
for TB control over long time periods. The policy maker
may be tempted to choose one option that is predicted to
yield better short term results. Different target times However, that choice might
be wrong when one opts to satisfice the outcome with
robustness to uncertainty. Predictions of mathematical
models (horizontal intercepts) are not sufficiently reliable
for comparing and prioritizing interventions; the cost of
robustness (slope) must also be considered. In the exam-
ple in Figure 5 one might conclude that prevalence less
than 3% is not achievable at any target time, that 3.7%
is feasible at 10-years but not beyond, and that other
interventions are needed for longer-term outcomes. Conclusion
d We demonstrated a generic info-gap framework for
managing model uncertainty in public health decision
making. By applying it to a mathematical model of
TB/HIV epidemics, we illustrated specific recommenda-
tions for interventions in the control of TB with HIV in
various settings. The complicated multi-dimensional epidemiological
dynamics are dominated by the flow back and forth
between the actively and latently infected TB populations
and the different rates of progression of different subpopu-
lations between these compartments. Counter-intuitively,
the total TB case load even decades after initiation can
increase as a result of increased diagnosis and cure rates,
and it can increase as the control of HIV becomes more
aggressive. These findings highlight the critical impor-
tance of modeling in the assessment and planning of
public health intervention. Model predictions are often
used to choose interventions. However, model predic-
tions must be interpreted in light of model uncertain-
ties. Predicted outcomes have zero robustness to model
error. Only worse-than-predicted outcomes (higher rel-
ative prevalence) have positive robustness against model
error. This means that predicted outcomes are not reliable
for prioritizing the interventions. The trade offbetween
robustness and outcome is quantified by the info-gap
model analysis and is a critical component of the decision-
making process. The basic Murray-Salomon model: No HIV in terms of their capacity for achieving specified out-
comes, with robustness to uncertainty. Failure to quantify
the uncertainty inherent in public health interventions
leads to disappointment from unrealized expectations,
and failed policy. Where a public health model under-
lies guidelines, info-gap decision theory provides valu-
able insight into the confidence of achieving agreed-upon
goals. The basic M-S model is the following 19 differential
equations (eqs.(6) and (7) occur in 6 different forms each)
appearing on pp.19–20 of Murray and Salomon [18]: dU
dt = T −λU −μU
(1) dU
dt = T −λU −μU
(1)
dIF
dt = (1 −p)λU −βFIF −wχIF −μIF
(2)
dIS
dt = pλU −βSIS −χIS −(1−p)λ(1−ν)IS −μIS (3)
dSF
dt = (1−p)λ(1−ν)(IS+HS+RS)−βFSF−wχSF−μSF
(4) (1) dIF
dt = (1 −p)λU −βFIF −wχIF −μIF
(2) (2) Appendices dIS
dt = pλU −βSIS −χIS −(1−p)λ(1−ν)IS −μIS (3) aAs fraction of total population at start of simulation. Impact of HIV mortality Figure 9 shows 10-year robustness curves for various HIV
infection rates, with low initial TB and HIV prevalence,
as specified in Table 5. The HIV infection rate decreases
in the progression from solid, dash, dot-dash to dot-dot. As the HIV infection rate decreases, the estimated 10-year
TB prevalence increases and the robustness decreases. The explanation lies in the high mortality rate of the
HIV population. As the HIV infection rate decreases, the
size of the relapse population decays more slowly, allow-
ing greater flow back into the active TB case population. Interventions that decrease HIV infection rates or restore
immunity to HIV patients, will counter-intuitively tend to 0.02
.025
0.03
.035
0.04
0
0.05
0.1
0.15
0.2
0.25
0.3
Critical prevalence
Robustness
Figure 9 Robustness for various HIV infection rates. Run 8: —, run
31: – –, run 30: ·–, run 29: · · ·. We explore the performance of interventions that alter
the rate constants of diagnosis, cure, relapse and HIV
infection. Some interventions have quite similar predicted
outcomes and robustness curves. This enables the policy
maker to choose between these interventions based on
additional criteria, such as ease or cost of implementation. It is not true, however, that interventions with the same
estimated outcomes necessarily have the same robustness
against model error. We demonstrate the policy implications of initial TB
and HIV prevalence, of HIV mortality, of degree of treat-
ment aggressiveness, and of the target time at which
outcomes are evaluated. Public health policies are evalu-
ated in terms of confidence—expressed as robustness to
modeling error—in achieving specified TB prevalence at
the target time. Predicted outcomes have zero robustness
and thus are not reliable for evaluating and comparing
interventions. Instead, interventions must be prioritized Figure 9 Robustness for various HIV infection rates. Run 8: —, run
31: – –, run 30: ·–, run 29: · · ·. Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 12 of 17 The basic Murray-Salomon model: No HIV bLow prevalence. Introduction Our model does not delay transfer from the HIV-
negative sub-model. Sub-models Each of the two sub-populations—HIV-
negative and HIV-positive—is divided into the 19 groups
represented by the state variables in Table 6. Each state
variable has a differential equation in eqs.(1)–(9). Let us denote the HIV-negative state variables as before,
and the HIV-positive state variables with the same letters
but with an over-bar. For compactness we represent these
two sets of variables with two vectors: However, the ‘vice versa’ is a mistake. The correct
equations for Ci,j
U (with analogs for Ci,j
T ) are: However, the ‘vice versa’ is a mistake. The correct
equations for Ci,j
U (with analogs for Ci,j
T ) are: x =
U, IF, IS, SF, HS, C1,1
U , C2,1
U , C3,1
U , C1,2
U , C2,2
U , C3,2
U ,
C1,1
T , C2,1
T , C3,1
T , C1,2
T , C2,2
T , C3,2
T , RF, RS
(14)
x =
U, IF, IS, SF, HS, C
1,1
U , C
2,1
U , C
3,1
U , C
1,2
U , C
2,2
U , C
3,2
U ,
C
1,1
T , C
2,1
T , C
3,1
T , C
1,2
T , C
2,2
T , C
3,2
T , RF, RS
(15) x =
U, IF, IS, SF, HS, C1,1
U , C2,1
U , C3,1
U , C1,2
U , C2,2
U , C3,2
U ,
C1,1
T , C2,1
T , C3,1
T , C1,2
T , C2,2
T , C3,2
T , RF, RS
(14) dC1,j
U
dt
= · · · + σC2,j
U + · · ·
(10)
dC2,j
U
dt
= · · · −σC2,j
U + · · ·
(11)
dC3,j
U
dt
= · · · + 0Ci⋆,j
U + · · ·
(12) (10) (14) (11) (15) (12) The model parameters listed in Tables 1 and 2 take
different values for HIV-negative and HIV-positive pop-
ulations (as specified in the tables). Let us denote the
model parameters as before for the HIV-negative popula-
tion, and use the same symbols with an over-bar for the
HIV-positive population. Eq.(10) states that smear-negative individuals join the
smear-positive population at rate σ. Eq.(11) states that
smear-negative individuals leave the smear negative pop-
ulation at rate σ. Introduction We will now formulate the extended dynamic model to
include a differentiation between HIV-positive and HIV-
negative populations. M-S do this also, and state [18], p.4
that they use “two sub-models—one for the HIV sero-
negative population, and one for the HIV sero-positive
population. Each sub-model follows the structure” which
is presented here as eqs.(1)–(9). They write that dRF
dt
= (1 −rU)εU
i=1,2,3
j=1,2
Ci,j
U +
k=1,2
⎡
⎣
1 −rk
T
εk
T
i=1,2,3
Ci,k
T
⎤
⎦
−ρFRF −μRF
(8)
dRS
dt = rUεU
i=1,2,3
j=1,2
Ci,j
U +
k=1,2
⎡
⎣rk
Tεk
T
i=1,2,3
Ci,k
T
⎤
⎦
−ρSRS −(1 −p)λ(1 −ν)RS −μRS
(9) dRF
dt
= (1 −rU)εU
i=1,2,3
j=1,2
Ci,j
U +
k=1,2
⎡
⎣
1 −rk
T
εk
T
i=1,2,3
Ci,k
T
⎤
⎦
−ρFRF −μRF
(8) Individuals move from each category in the
HIV-negative sub-model to the corresponding
category in the HIV-positive sub-model at the HIV
infection rate, which varies over time. Because the
effects of HIV on immune function are not marked
with respect to tuberculosis until the CD4 count has
dropped below 500, we actually move individuals
from the HIV-negative to the HIV-positive
sub-model after they have been infected with HIV for
3 years. The two sub-models are also linked through
the annual risk of infection, as HIV-negative
tuberculosis cases can infect HIV-positive
individuals, and vice versa [18], pp.4–5 (8) dRS
dt = rUεU
i=1,2,3
j=1,2
Ci,j
U +
k=1,2
⎡
⎣rk
Tεk
T
i=1,2,3
Ci,k
T
⎤
⎦
−ρSRS −(1 −p)λ(1 −ν)RS −μRS
(9) (9) The term ‘±σ’ appears in eqs.(6) and (7). M-S write:a The term ‘±σ’ appears in eqs.(6) and (7). M-S write:a It should be noted in the equations for Ci,j
U and Ci,j
T
that the smear rate σ is multiplied by the number of
individuals in the respective category i⋆, where i⋆= 2
(smear-negative) for i = 1 (smear-positive) and vice
versa, and i⋆= ∅for i = 3 (extra-pulmonary). The
term including σ is added for i = 1, subtracted for
i = 2, and equal to 0 for i = 3. The result of this
formulation is that smear-negative patients convert
to smear-positive at a rate of σ. Our model does not delay transfer from the HIV-
negative sub-model. The Murray-Salomon model BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 13 of 17 dCi,j
U
dt
= {βF(IF + SF) + βS[ IS + (1 −h)HS]
+ρSRS + ρFRF} di,jsi
−δjCi,j
U ± σCi⋆,j
U −εUCi,j
U −(μ + μi
U)Ci,j
U,
(6)
d The instantaneous rate of infection, λ in eq.(1), is
defined by Murray and Salomon [18], p.21, as: dCi,j
U
dt
= {βF(IF + SF) + βS[ IS + (1 −h)HS] λ = KL Y
N
(13) +ρSRS + ρFRF} di,jsi
−δjCi,j
U ± σCi⋆,j
U −εUCi,j
U −(μ + μi
U)Ci,j
U,
(6)
λ = KL Y
N (13) (6) −δjCi,j
U ± σCi⋆,j
U −εUCi,j
U −(μ + μi
U)Ci,j
U,
(6)
for i = 1, 2, 3 and j = 1, 2
dCi,k
T
dt
= gi,k
j=1,2
δjCi,j
U ± σCi⋆,k
T
−εk
TCi,k
T −(μ + μi,k
T )Ci,k
T , (7)
for i = 1, 2, 3 and k = 1, 2 The HIV-Extended model for i = 1, 2, 3 and j = 1, 2 for i = 1, 2, 3 and j = 1, 2 Introduction The Murray-Salomon model The Murray-Salomon (M-S) model [17,18] is a set of
coupled differential equations that describe the time evo-
lution of TB. A modification deals with TB infecteds in a
population containing HIV smear-positive individuals. In
section “The basic Murray-Salomon model: No HIV” we
define the basic non-HIV model. In section “The HIV-Ex-
tended model” we present the M-S extension to include
an HIV sub-population. The state variables are defined in
Table 6 and the parameters are defined in Table 1. dSF
dt = (1−p)λ(1−ν)(IS+HS+RS)−βFSF−wχSF−μSF (4) dHS
dt
= χ(wIF + wSF + IS) −(1 −p)λ(1 −ν)HS
−(1 −h)βSHS −μHS
(5) (5) Table 6 State variables—sizes of sub-populations—in the Murray-Salomon basic model, Table One, p.41, in ref. [18]
Index
Symbol
Definition
Initial valuea
HIV Neg
HIV pos
Ref. 1
U
Uninfected
0.9b
0.2c
[10]
2
IF
Infected subject to fast breakdown
0.05–0.1
0.1
[33,34]
3
IS
Infected subject to slow breakdown
0.1
0.1
[33,34]]
4
SF
Superinfected subject to fast breakdown
0
5
HS
INH recipient subject to slow breakdown
0.01
0.01–0.03
6–11
Ci,j
U
Untreated cases, of 6 types:
0
i = 1: smear-positive pulmonary
i = 2: smear-negative pulmonary
i = 3: extra-pulmonary
j = 1: fast diagnosis category
j = 2: slow diagnosis category
i⋆= 2 if i = 1 (eqs.(6) and (7))
i⋆= 1 if i = 2 (eqs.(6) and (7))
i⋆= 0 if i = 3 (eqs.(6) and (7))
12–17
Ci,k
T
Treated cases, of 6 types:
0
i as above
k = 1: good treatment category
k = 2: bad treatment category
18
RF
Recovered cases subject to fast relapse
0.4 (0.28–0.52)
[33,34]
19
RS
Recovered cases subject to slow relapse
0.050 (0.035–0.065)
[33,34]
The definition of superscript i⋆is in [18] on p.20. s of sub-populations—in the Murray-Salomon basic model, Table One, p.41, in ref. [18] Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. Introduction Finally, we write our info-gap model of uncertainty as a
family of nested sets of uncertain vectors: Finally, we write our info-gap model of uncertainty as a
family of nested sets of uncertain vectors: M-S introduce further highly structured coupling
between eqs.(17) and (18) through the TB infection rate,
[18], p.23, λ. We do not employ the M-S differentiation
between the infection rates for HIV-negative and HIV-
positive populations. Instead we simply use λ and λ for the
TB infection rates in the HIV-negative and HIV-positive
populations. U(α) =
u : ui > 0,
ui − ui
si
≤α, for all i
, α ≥0
(22) (22) α is called the ‘horizon of uncertainty’. When α = 0
there is no uncertainty and the set U(0) contains only
the estimated values, u. As α increases, the sets U(α)
become more inclusive. These sets are unbounded in the
space on which the parameters are defined. The info-gap
model embodies the information we have—estimates and
errors—without committing to any meaningful worst case
(other than the limits which are imposed by the definition
of the variables). Introduction That way all individuals are accounted
for. Page 14 of 17 Page 14 of 17 Ben-Haim et al. BMC Public Health 2012, 12:1091
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http://www.biomedcentral.com/1471-2458/12/1091 Eqs.(1)–(9) are 1st-order linear inhomogeneous differ-
ential equations. Only eq.(1) has an inhomogeneous term:
T births per year. Let F(t) and F(t) denote the matri-
ces of coefficients (model parameters) in the differential
equations for HIV-negative and HIV-positive populations,
respectively. Let e1 denote the 19-vector with a 1 in the
first element and zeros elsewhere. We can now compactly
denote eqs.(1) as: For each uncertain parameter, ui, we have an estimated
value, denoted ui, and an error term si typically cho-
sen as half of an interval estimate of the parameter. The
error estimate may be derived from a statistical confi-
dence interval, or from a plausible extension of a confi-
dence interval as discussed by Grassly et al [32], or from
other professional judgment. The basic idea of an info-gap
model of uncertainty is that we don’t know how wrong our
estimate is; we have no reliable estimate of a worst case. In
fact, since the typical values are poorly known, worst-case
estimates are even less reliable. dx
dt = F(t)x + e1T
(16) (16) More precisely, the fractional error of the estimate, ui,
in units of the error, si, is unknown. That is, this fractional
error is bounded by a number, α, whose value is unknown: Let γ denote the HIV infection rate, per person per
year. Following M-S, we will move individuals from each
HIV-negative category to the corresponding HIV-positive
category at rate γ . Thus, instead of eq.(16), we have the
following coupled sets of equations: ui − ui
si
≤α,
α ≥0
(20) (20) dx
dt = F(t)x + e1T −γ x
(17) (17) But this must be further refined to reflect the fact
that the uncertain parameters are 1st-order removal-rate
constantsb, which means that they cannot be negative. Thus we adjoin these constraints to the inequality as: dx
dt = F(t)x + e1T + γ x
(18) (18) ui > 0,
ui − ui
si
≤α,
α ≥0
(21) (21) The term ‘−γ x’ in eq.(17) removes individuals from the
HIV-negative population at the HIV infection rate, and
the term ‘+γ x’ in eq.(18) introduces them into the HIV-
positive population at the same rate. Uncertainty Many uncertainties accompany the dynamic model. We
concentrate on uncertainty in the values of some of the
model parameters, as this is the dominant impact of HIV
prevalence. We use info-gap theory to model and manage
these uncertainties [8]. Many different types of info-gap
models of uncertainty are available. We employ a model
particularly suited to severe lack of information. In some situations one may not be able to estimate error
weights, si. In such situations the fractional error in eq.(20)
can be replaced by a fractional error relative to the esti-
mate, |(ui− ui)/ ui|. The info-gap model is then formulated
as in eq.(22) with this new fractional error. The dominant uncertain parameters are: ρS, ρS, slow relapse rates. ρF, ρF, fast relapse rates. λ, λ, TB infection rates. Robustness: formulation
Performance requirements γ , HIV infection rate. Definition of robustness
A i
i
i A relation such as eq.(25) is called a “satisficing” require-
ment, as opposed to an optimization requirement. We do
not aim to minimize the aggregate prevalence, C(tm). Our
goal is to make the TB prevalence adequately small: no
greater than the critical value Cm, as stated in eq.(25). Note that the satisficing requirement includes optimiza-
tion as a special case. Satisficing and optimizing are the
same when Cm is chosen as the predicted minimal value. An intervention is specified by specifying the values of the
control variables, q. If our dynamic model were accurate
we could evaluate any proposed intervention in terms of
the outcome of that intervention that is predicted by the
model. An intervention whose predicted outcome entails
low TB prevalence is preferred over an intervention with
larger predicted prevalence. The problem is that the dynamic model is highly uncer-
tain. This means that it is unrealistic to prioritize inter-
ventions in terms of their predicted outcomes. Since those
predictions are highly uncertain, it is unwise to eval-
uate interventions only in terms of their model-based
predictions. Performance requirements Let us denote uncertain variables generically as ui, com-
piled in a vector u. This vector is: We will consider an aggregated variable for monitoring
the TB status of the population. Our goal is to keep
the value of this variable acceptably small. The variable
we consider is the total number of cases, untreated and u = (ρS, ρF, λ, ρS, ρF, λ, γ )
(19) (19) u = (ρS, ρF, λ, ρS, ρF, λ, γ ) Page 15 of 17 Page 15 of 17 Ben-Haim et al. BMC Public Health 2012, 12:1091
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http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 coefficients and can meaningfully take any positive value. These coefficients combine with several other coefficients
to determine the fraction of new untreated cases that
move into the treated category, as seen from eqs.(6) and
(7). In other words, the control variables combine to pro-
duce aggregate effects such as the proportion of new cases
that are diagnosed. One can “calibrate” a set of control
variables in terms of aggregate properties, for instance by
keeping track of how many cases are created (new mem-
bers of CU(t)) and how many are treated (new members
of CT(t)). Unless the population is at steady state (and
the intervention tries to prevent this), the calibration in
terms of the proportion diagnosed depends on the time
after initiation of intervention and on the duration dur-
ing which the accounting is done. We do not calibrate our
model since we focus on a different challenging problem:
prioritizing alternative interventions. treated, HIV-positive and HIV-negative, as a fraction of
the initial population: C(t) =
i,j
Ci,j
U(t) + Ci,j
T (t) + Ci,j
U(t) + Ci,j
T (t)
( (23) There are other variables that one could consider. For
instance, one could consider the total number of relapses,
fast and slow, HIV-positive and HIV-negative, as a fraction
of the initial population: R(t) = RF(t) + RS(t) + RF(t) + RS(t)
(24) (24) One could also consider the instantaneous or the average
rates of change of C(t) and R(t). Returning to the aggregate prevalence, C(t), our goal is
to keep it below a specified maximum acceptable value at
a specified target time tm. Thus the performance require-
ment is: C(tm) ≤Cm
(25) (25) C(tm) ≤Cm Control variables We aim to achieve this goal by judicious choice of control
variables that we denote generically as qi, combined in a
vector q. Eligible control variables are any coefficients of
the dynamic model that can be influenced by public health
or related medical intervention. When a control variable
is also an info-gap uncertain variable we will refer to the
estimated value as the control variable. The uncertainty is
then in whether the specified value—the estimate—will be
realized in practice. We will consider the following control
variables: The model-based predictions are useful, but we also
ask: how wrong could the model be, and the predicted
outcome is still acceptable? That is, for any specified inter-
vention, q, we ask: what is the largest fractional error in
the uncertain parameters, up to which all realizations of
the model would yield acceptable outcomes? The answer
to that question is the robustness function, which we will
soon specify. We use the robustness function to priori-
tize the interventions in terms of their robustness against
uncertainty for achieving the required outcomes. δj, δj, diagnosis rates (same for HIV negative and
positive populations). k
k εk
T, εk
T, cure rates for treateds (same for HIV negative
and positive populations). εk
T, εk
T, cure rates for treateds (same for HIV negative
and positive populations). The robustness function for the performance require-
ment in eq.(25) is: ρF, ρF, estimated fast relapse rates. α(q, Cm) = max
α :
max
u∈U(α) C(tm, u)
≤Cm
(26) λ, λ, estimated TB infection rates. (26) γ , estimated HIV infection rate. βF, βF, fast breakdown rates. We can “read” this relation from left to right as follows. The robustness, α, of intervention q, with performance
requirement Cm, is the maximum horizon of uncertainty,
α, up to which the maximum aggregate prevalence, C(t),
for all realizations of the uncertain coefficients u in the
info-gap model U(α), does not exceed the critical value,
Cm. We are not ameliorating a worst case; the worst We can “read” this relation from left to right as follows. We can “read” this relation from left to right as follows. Competing interests The authors have no competing interests. Control variables The robustness, α, of intervention q, with performance
requirement Cm, is the maximum horizon of uncertainty,
α, up to which the maximum aggregate prevalence, C(t),
for all realizations of the uncertain coefficients u in the
info-gap model U(α), does not exceed the critical value,
Cm. We are not ameliorating a worst case; the worst We define an intervention in terms of the values of these
variables. None of these control variables corresponds
directly to any of the standard performance measures such
as the incidence, prevalence, and death rates associated
with TB. For instance, the coefficients δj and δj, while
called “diagnosis rates”, are in fact 1st-order kinetic rate Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 Page 16 of 17 Ben-Haim et al. BMC Public Health 2012, 12:1091
http://www.biomedcentral.com/1471-2458/12/1091 case is unknown because the horizon of uncertainty, α, is
unbounded. Instead, we are asking how large an uncer-
tainty can be tolerated by the intervention, q. In choosing
the intervention to enhance the robustness, we attempt to
protect against the unbounded uncertainty of the impact
of HIV/AIDS on the TB dynamics. 9. Ben-Haim Y, Dacso CC, Carrasco J, Rajan N: Heterogeneous
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Reasoning 2009, 50:1046–1065. 10. Dye C: Global epidemiology of tuberculosis. Lancet 2006,
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1 1Yitzhak Moda’i Chair in Technology and Economics, Technion—Israel
Institute of Technology, Haifa 32000, Israel. 2Molecular & Cell Biology and
Medicine, Baylor College of Medicine, Houston, Texas. 3Division of Infectious 1Yitzhak Moda’i Chair in Technology and Economics, Technion—Israel
Institute of Technology, Haifa 32000, Israel. 2Molecular & Cell Biology and
Medicine, Baylor College of Medicine, Houston, Texas. 3Division of Infectious
Diseases, Department of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania. 26. Carmel Y, Ben-Haim Y: Info-gap robust-satisficing model of foraging
behavior: Do foragers optimize or satisfice? Am Naturalist 2005,
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MurraySalomon ModelingTBControlStrategies HCPDS1998.pdf] Acknowledgements Financial Support: This work was supported in part by NIH grant R01AI097045. The funders had no role in study design, data collection and analysis, decision
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experience from western Kenya. AIDS 2006, 20:41–48. 37. Vynnycky E, Nagelkerke N, BorgdorffMW, van Soolingen D, van Embden
JD, Fine PE: The effect of age and study duration on the relationship
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proportion of tuberculosis disease attributable to recent
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implications of age-dependent risks of disease and the role of
reinfection. Epidemiol Infect 1997, 119(2):183–201. 39. Vynnycky E, BorgdorffMW, Leung CC, Tam CM, Fine PE: Limited impact
of tuberculosis control in Hong Kong: attributable to high risks of
reactivation disease. Epidemiol Infect 2008, 136(7):943–952. 40. Verver S, Warren RM, Beyers N, Richardson M, van der Spuy GD, Borgdorff
MW, Enarson DA, Behr MA, van Helden PD: Rate of reinfection
tuberculosis after successful treatment is higher than rate of new
tuberculosis. Am J Respir Crit Care Med 2005, 171(12):1430–1435. doi:10.1186/1471-2458-12-1091
Cite this article as: Ben-Haim et al.: Info-gap management of public health
Policy for TB with HIV-prevalence and epidemiological uncertainty. BMC
Public Health 2012 12:1091. 40. Verver S, Warren RM, Beyers N, Richardson M, van der Spuy GD, Borgdorff
MW, Enarson DA, Behr MA, van Helden PD: Rate of reinfection
tuberculosis after successful treatment is higher than rate of new
tuberculosis. Am J Respir Crit Care Med 2005, 171(12):1430–1435. doi:10.1186/1471-2458-12-1091
Cite this article as: Ben-Haim et al.: Info-gap management of public health
Policy for TB with HIV-prevalence and epidemiological uncertainty. BMC
Public Health 2012 12:1091. Submit your next manuscript to BioMed Central
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English
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Effect of Hypoproteic and High-Fat Diets on Hippocampal Blood-Brain Barrier Permeability and Oxidative Stress
|
Frontiers in nutrition
| 2,019
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cc-by
| 8,145
|
ORIGINAL RESEARCH
published: 09 January 2019
doi: 10.3389/fnut.2018.00131 Effect of Hypoproteic and High-Fat
Diets on Hippocampal Blood-Brain
Barrier Permeability and Oxidative
Stress Cristhyane Costa de Aquino 1†, Ricardo A. Leitão 2,3,4†, Luís A. Oliveira Alves 1,
Vanessa Coelho-Santos 2,3, Richard L. Guerrant 5, Carlos F. Ribeiro 2,3,4, João O. Malva 2,3,4*,
Ana P. Silva 2,3,4 and Reinaldo B. Oriá 1* Cristhyane Costa de Aquino 1†, Ricardo A. Leitão 2,3,4†, Luís A. Oliveira Alves 1,
Vanessa Coelho-Santos 2,3, Richard L. Guerrant 5, Carlos F. Ribeiro 2,3,4, João O. Malva 2,3,4*,
Ana P. Silva 2,3,4 and Reinaldo B. Oriá 1* 1 Laboratory of Tissue Healing, Ontogeny and Nutrition, Department of Morphology, School of Medicine, Institute of
Biomedicine, Federal University of Ceara, Fortaleza, Brazil, 2 Faculty of Medicine, Institute of Pharmacology and Experimental
Therapeutics, University of Coimbra, Coimbra, Portugal, 3 Coimbra Institute for Clinical and Biomedical Research, Faculty of
Medicine, University of Coimbra, Coimbra, Portugal, 4 CNC.IBILI, University of Coimbra, Coimbra, Portugal, 5 Division of
Infectious Diseases and International Health, Center for Global Health, School of Medicine, University of Virginia,
Charlottesville, VA, United States Keywords: high-fat diet, regional basic diet, blood-brain barrier, neuroinflammation, oxidative stress, malnutrition Edited by:
Francisco Ciruela,
University of Barcelona, Spain Oriá
reinaldo70.oria@gmail.com
†These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Neuroenergetics, Nutrition and Brain
Health, Specialty section:
This article was submitted to
Neuroenergetics, Nutrition and Brain
Health, a section of the journal
Frontiers in Nutrition Received: 30 July 2018
Accepted: 06 December 2018
Published: 09 January 2019 Edited by:
Francisco Ciruela,
University of Barcelona, Spain Edited by:
Francisco Ciruela,
University of Barcelona, Spain Francisco Ciruela,
University of Barcelona, Spain Worldwide, millions of people are exposed to dietary imbalance that impacts in health
and quality of life. In developing countries, like in Brazil, in poor settings, dietary habits,
traditionally hypoproteic, are changing rapidly to western-type high-fat foods. These
rapidly changing dietary habits are imposing new challenges to human health and
there are many questions in the field that remain to be answered. Accordingly, we
currently do not know if chronic consumption of hypoproteic (regional basic diet, RBD)
or high-fat diets (HFD) may impact the brain physiology, contributing to blood-brain
barrier (BBB) dysfunction and neuroinflammatory events. To address this issue, mice
were challenged by breastfeeding from dams receiving standard, RBD or HFD from
suckling day 10 until weaning. Immediately after weaning, mice continued under the
same diets until post-natal day 52. Herein, we show that both RBD and HFD cause not
only a peripheral but also a consistent central neuroinflammatory response, characterized
by an increased production of Reactive Oxygen Species (ROS) and pro-inflammatory
cytokines. Additionally, BBB hyperpermeability, accounted by an increase in hippocampal
albumin content, a decrease in claudin-5 protein levels and collagen IV immunostaining,
was also observed together with an upregulation of vascular cell adhesion molecule
1 (VCAM-1). Interestingly, we also identified a significant astrogliosis, manifested by
upregulation of GFAP and S100β levels and an intensification of arbor complexity of
these glial cells. In sum, our data show that dietary imbalance, related with hypoproteic
or high-fat content, impairs BBB properties potentially favoring the transmigration of
peripheral immune cells and induces both a peripheral and central neuroinflammatory
status. Noteworthy, neuroinflammatory events in the hippocampus may cause neuronal
malfunction leading to cognitive deficits and long-term persistence of this phenomenon
may contribute to age-related neurodegenerative diseases. Reviewed by:
Yinghua Yu,
Xuzhou Medical University, China
Mercedes G. López,
Centro de Investigación y de Estudios
Avanzados (CINVESTAV), Mexico Reviewed by:
Yinghua Yu,
Xuzhou Medical University, China
Mercedes G. López,
Centro de Investigación y de Estudios
Avanzados (CINVESTAV), Mexico Reviewed by:
Yinghua Yu,
Xuzhou Medical University, China
Mercedes G. López,
Centro de Investigación y de Estudios
Avanzados (CINVESTAV), Mexico *Correspondence:
João O. Malva
jomalva@fmed.uc.pt
Reinaldo B. Oriá
reinaldo70.oria@gmail.com
†These authors have contributed
equally to this work *Correspondence:
João O. Malva
jomalva@fmed.uc.pt
Reinaldo B. Oriá
reinaldo70.oria@gmail.com *Correspondence:
João O. Malva
jomalva@fmed.uc.pt
Reinaldo B. Abbreviations: WHO, World Health Organization; BBB, Blood-brain barrier;
RBD, Regional Basic Diet; HFD, High Fat Diet; PFA, Paraformaldehyde;
PBS, Phosphate buffered saline; PVDF, Polyvinylidene difluoride membrane;
DEEPPD, Diethyl-pera-phenylenediamine; H2O2, Hydrogen Peroxide; TBAR,
Thiobarbituric Acid Reactive; MDA, Malondialdehyde; PND, Pos-natal day; GFAP,
Glial Fibrillary Acidic Protein; MPO, Myelloperoxidase. Animals In this study, C57BL/6J pregnant female mice from Charles
River, Inc. were acclimated and fed with a standard chow diet
for 6 days at the Faculty of Medicine, University of Coimbra’s
vivarium, with free access to water and food. Thereafter, pregnant
females were housed individually in nursery cages to give birth. Newborn suckling mice (standardized to 6–8 litters/dam) were
randomized to three experimental groups, with challenged dams
receiving either regional basic diet (RBD) or high fat diet (HFD),
starting 10 days after birth delivery. The controls received the
chow diet. The RBD is moderately deficient in proteins and fats
(82% carbohydrates, 6% protein, 2% fat), while the HFD contains
approximately 60% fat (21% carbohydrates, 18% proteins, 60%
fat). On day 21, pups were weaned from their mothers and
continued with the initial diet until 52th day of age. After
anesthesia, mice were transcardially perfused with phosphate
buffered saline (PBS) or with 4% paraformaldehyde (PFA) in
PBS, and the hippocampi harvested for western blotting or total
brain removed for immunofluorescence, respectively. In order
to assess weight gain, mice were weighed 3 times per week. The vivarium Ethics Committee of the Faculty of Medicine
of the University of Coimbra approved all the experimental
protocols. The effects of the diets on BBB permeability
were evaluated by assessing the presence of albumin in the
brain parenchyma and the expression of hippocampal vascular
unit-related claudin-5, an endothelial tight-junctional protein,
collagen IV and VCAM-1 immunoreactivity. Additionally,
systemic inflammation-related serum cytokines, hippocampal
oxidative stress, neuroinflammation and associated astrocytic
response were further investigated. The effects of malnutrition may be endemic in low-income
families. Many people cope with poor education, hygiene and
precarious access to adequate health care, which could create a
poor environment for optimal brain development (5). During
the last decades obesity has reached epidemic proportions in
developing countries, causing deaths and permanent sequelae
(6). The magnitude of this burden, especially on children’s
cognitive development, is still mostly unknown. Blood-brain barrier (BBB) is a dynamic and specialized
structure composed of endothelial cells, connected with each
other by tight (TJs) and adherens (AJs) junctions, and associated
with pericytes, basement membrane and astrocyte endfeet, that
together with microglia and neurons comprise the neurovascular
units. The brain endothelium, with the lack of fenestrations
and low fluid-phase endocytosis (pinocytosis), is the first
“physical barrier” where intercellular complexes confer low
paracellular permeability and high electrical resistance to the
BBB (7). Citation: de Aquino CC, Leitão RA,
Oliveira Alves LA, Coelho-Santos V,
Guerrant RL, Ribeiro CF, Malva JO,
Silva AP and Oriá RB (2019) Effect of
Hypoproteic and High-Fat Diets on
Hippocampal Blood-Brain Barrier
Permeability and Oxidative Stress. Front. Nutr. 5:131. January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 1 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. INTRODUCTION observations, there is still scarcity of studies evaluating the impact
of high-fat and low-protein diets (especially the latter one) on
the BBB integrity and neuroinflammatory responses. Therefore,
in this study, we bring novel data on mice BBB challenged by
two models of malnutrition, based upon chronic feeding with a
Brazilian northeast regional basic diet [RBD; (15)], and a high-fat
content-diet (HFD) (16). According to the World Health Organization (WHO), about
45% of deaths, among children under 5 years old, are directly
related to various degrees of malnutrition in poor settings of
the middle- and low-income countries (1). Interestingly, in the
same settings, overweight and obesity rates are escalating during
early childhood, a scenario particularly evident in Brazilian
favelas [shantytowns; (2)]. Characteristics of this dual burden
are also seen largely elsewhere in low resource countries (3). Worldwide, surveillance data from WHO shows that 155 million
pre-school children suffer from impairment in physical and
cognitive development, while 41 million are overweight or obese
(4). Obesity is particularly worrisome, since the impact in human
health is expected to greatly increase in the coming years. This
scenario raises public health concerns related to malnutrition,
short and long-term illness and their increasing costs. Animals The core importance of the BBB is demonstrated
by its role in the maintenance of brain homeostasis and
protection against toxic compounds and blood composition
fluctuations, but simultaneously providing essential nutrients for
the normal brain function (7, 8). In fact, the presence of selective
transporters allows the passage of specific molecules into the
brain parenchyma that are essential for its function. Malnutrition (hypoproteic diets) and obesity (high-fat diets)
may be both associated with systemic inflammation (9,
10). Moreover, these effects may mount an environmental
enteropathy vicious cycle (with microbiota and epigenetics
changes) (11) that may lead to different levels of central nervous
system (CNS) dysfunction, including BBB disruption (12) that
is the gatekeeper of the brain. In addition, oxidized-LDL,
which may be increased by malnutrition states (13), can induce
apoptosis to brain endothelial cells (14). Despite these interesting Determination of Oxidative Stress Markers
Reactive oxygen species (ROS) levels in the hippocampus were
measured by N, N-diethyl-pera-phenylenediamine (DEPPD)
assay as previously described (17). In brief, 5 µL of hippocampi
lysates were added to 140 µL of 0.1 M sodium acetate buffer (pH
4.8) at 37◦C in a 96-well plate. Samples were taken in triplicate
and 100 µL of the mixed DEPPD solution and ferrous sulfate
at a ratio of 1:25 was added to each well to initiate reaction. The microtiter plate was then incubated at 37◦C, for 5 min, and
absorbance was measured by a spectrophotometer plate reader
(Biotek, Synergy HT), at 505 nm. ROS levels were calculated from
a calibration curve and expressed as hydrogen peroxide (H2O2)
equivalent (1 unit = 1.0 mg H2O2/L). The calibration curve for
standard solution was obtained by calculating slopes from an
optical density graph. January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 2 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. (GE-Healthcare Biosciences) and quantified using Image Studio
software (version 5.2, Licor). The thiobarbituric acid reactive-species (TBARs) assay
was
used
to
assess
products
of
lipid
peroxidation,
via
malondialdehyde (MDA) (17). Briefly, 100 µL of tissue
supernatant were incubated at Room Temperature (RT) in the
dark for 1 h in a TBA solution together with butylhydroxytoluene
(BHT; Sigma-Aldrich) and a catalyzer (Iron III chloride; Sigma-
Aldrich). Afterwards, samples were incubated at 95–100◦C for
60 min and followed by butanol extraction. Animals The supernatants
were read spectrophotometrically at 532 nm (Biotek, Synergy
HT) and the concentration of MDA was calculated with respect
to
a
calibration
curve
using
1,1,3,3-tetramethoxypropane
(Sigma-Aldrich) (range: 0.1–83.5 µM). Results were expressed as
µM/mg of hippocampal tissue. Enzyme-Linked Immunosorbent Assay Blood samples were withdrawn by cardiac puncture into BD
Vacutainer SST Tubes (BD Bioscience, Franklin Lakes, NJ, USA). Serum was separated by centrifugation at 1,100 × g for 15 min
and stored at −80◦C until analysis. The released levels of IL-1β,
TNF-α, and IL-10 from the three animal groups were quantified
by using an ELISA Ready-SET-Go kit (eBioscience, San Diego,
CA, United States), as specified in the datasheet. The results were
expressed as pg/mL. Morphological Analysis of Astrocytic
Processes GFAP-labeled astrocytes were analyzed as previously described
(19). Briefly, images were uploaded to the Simple Neurite Tracer
plugin of FIJI Software to measure the length (µm) and count the
number of astrocytic processes. Further, to infer about the arbor
complexity of astrocytes a Sholl analysis was performed. A total
of 30 cells were analyzed (10 cells/animal from a total of three
different animals for each experimental group). Western Blotting g
Hippocampal tissue was processed for western blotting according
to standard procedures (17, 18). Briefly, the tissue was
homogenized in lysis buffer (0.15 M sodium chloride, 0.05 M
Tris-base, 0.005 M ethyleneglycoltetraacetic acid, 0.5% sodium
deoxycholate, 0.1% SDS and 1% X-Triton, pH 7.5) supplemented
with protease inhibitor cocktail tablets (Roche Applied Sciences). Total protein content was quantified using the bicinchoninic
acid method (BCA; Pierce) and stored at −20◦C until further
use. Afterwards, the processed samples were boiled (at 95◦C)
in sample buffer (Tris-HCl 0.5 M, 10.4% SDS pH 6.8) for
5 min. Different concentrations of sample, containing specific
proteins, have been analyzed. Each sample was submitted
to electrophoresis in polyacrylamide gel (10% and 12%),
running at 130 V. Then, sample proteins were transferred to
a polyvinylidene difluoride membrane (PDVF) (Millipore) for
1 h 30 min at 110 V. Following membrane blocking with BSA
(5%), samples were incubated with primary antibodies (goat anti-
TNF-α, 1:500, Millipore; rabbit anti-IL-1β; 1:200, Santa Cruz
Biotechnology; goat anti-albumin, 1:20,000, Bethyl Laboratories;
mouse anti-claudin-5, 1:100, Invitrogen; rabbit anti-caveolin-
1, 1:200, Santa Cruz Biotechnology, rabbit anti-p-caveolin-1,
1:200, Santa Cruz Biotechnology; mouse anti-VCAM-1, 1:200,
Santa Cruz Biotechnology; rabbit anti-GFAP, 1:2,000, Sigma-
Aldrich; mouse anti-S100β, 1:500, Sigma-Aldrich), overnight,
at 4◦C, followed by incubation with alkaline phosphatase-
conjugated secondary antibody anti-goat IgG (1:10,000, GE
Healthcare), anti-rabbit (1:20,000, GE Healthcare), or anti-
mouse (1:10,000, GE Healthcare), 1h at RT. Internal control
for protein content in each sample was evaluated by mouse
anti-GAPDH (1:10,000, Sigma-Aldrich). Targeted protein bands
were visualized following membrane incubation with enhanced
chemifluorescence (ECF, Amersham) using a Typhoon FLA 9000 Statistics Data represent the mean value ± standard error of the mean
(SEM) a minimum of 6 animals per group. Statistical analysis
of weight gain and Sholl analysis was performed using two-
way ANOVA followed by Dunnett’s multiple comparisons test. Regarding all the other experimental approaches, the statistical
analysis was performed using Kruskal-Wallis test, followed by
Dunn’s post-test for multiple comparisons. All statistics were
calculated using GraphPad Prism 6.0. The level of significance
was P < 0.05. Immunofluorescence Following perfusion with PFA, brains were removed and
incubated overnight with 4% PFA, followed by 30% sucrose
(in PBS 0.01 M) for 24 h, at 4◦C, and then stored at −80◦C,
until further use. Brains were cryosectioned at 20 µm thick
coronal slices and mounted on glass immunoslides, washed
with PBS solution, then permeabilized with Triton X-100 (1%),
and blocked with 3% bovine serum albumin for 1 h at RT. Primary antibodies incubation was performed during 24 h, at
4◦C, using a monoclonal mouse anti-GFAP-Cy3 conjugated
antibody (1:500, Sigma-Aldrich), and rabbit anti-collagen IV
(1:200, Abcam). After that, brain slices were incubated with
5 µg/mL Hoechst 33342 (Sigma-Aldrich) for 5 min in the dark
at RT, for nuclei staining. Sample preparations were mounted
in Dako fluorescence medium (Dako, Glostrup, Denmark) and
images acquired and processed using a Carl Zeiss LSM 710
Meta confocal microscope (Carl Zeiss; Oberkochen, Germany). Quantification of GFAP and collagen IV immunoreactivity was
accomplished using the NIH ImageJ 1.47 analysis software. In
brief, all photograph area was considered as well as three different
zones without staining (black) to be used for background
subtraction. To determine the corrected total fluorescence,
we used the following formula: correct total fluorescence =
(integrated intensity) – (area of picture × mean background). The results are expressed as the mean of fluorescence intensity
(arbitrary units) of five brain slices obtained from three different
animals for each experimental group. RESULTS Mice were grouped in three cohorts submitted to control diet (n
= 7), RBD (n = 8) and HFD (n = 8). Body weight was monitored
every 2 days for a total of 42 days, starting at postnatal day 10 January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 3 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. (mean body weight = 4.4 ± 0.3 g), until day 52 of age. Results
showed that until post-natal day (PND) 25, all groups present a
similar development. After that, we observed that control group
developed, as expected, with a slow and sustained weight gain,
reaching a mean value of 14.9 ± 0.2 g (332.7 ± 4.9% of control). Regarding RBD group, the body weight slightly increased over
time reaching 10.6 ± 0.05 g (248.5 ± 1.2% of control) at 52 days of
age, whereas there was a marked increase in body weight gain for
the HF diet group reaching 26.2 ± 0.2 g (605 ± 6.1% of control;
Figure 1). 153 ± 7.6% of control, P < 0.01) by both diets. Regarding IL-
1β of its protein levels with HFD and only a tendency, yet not
significant, by RBD diet (Figure 3C, 119 ± 3.5% of control, P <
0.01). Overall, we concluded that both diets triggered a peripheral
inflammatory state that was accompanied by a concomitant
hippocampal neuroinflammation. The (neuro)inflammatory processes have been described as
key players in BBB disruption and transmigration of peripheral
immune cells into brain parenchyma (7, 20). To evaluate BBB
alterations induced by RBD and HFD, we measured the protein
levels of albumin in brain tissue, since this blood-borne protein
does not cross the BBB under normal conditions. Interestingly,
albumin content in the hippocampus was significantly increased
in mice chronically exposed to RBD (Figure 4A; 139.3 ± 10.5%;
P < 0.05) and HFD (Figure 4A; 179.4 ± 25.3%; P < 0.01),
indicating a hyperpermeability of BBB triggered by both diets. To further unravel the transport across the BBB that is being
altered, we analyzed the protein levels of endothelial claudin-5
and caveolin-1 (Cav-1), the latter has been associated with brain
injury-related BBB breakdown (21). The transport at the BBB can
occur through paracellular and transcellular pathways, with the
first being controlled by intercellular complexes present between Afterwards, we evaluated several inflammatory mediators in
blood serum of these animals. RESULTS First, we observed a significant
increase in lipid peroxidation products (measured by MDA
reaction products) in HFD animals (Figure 2A, 15.3 ± 0.2
µM/mg; P < 0.001), with no effect on RBD diet animals
(Figure 2A; 2.18 ± 0.1 µM/mg). Regarding pro-inflammatory
cytokines, there was an increase in both TNF-α and IL-1β serum
levels not only in RBD (Figures 2B,C; 16 ± 2.5 pg/mL TNF-
α, P < 0.001; 8.9 ± 1.5 pg/mL IL-1β, P < 0.05; respectively),
but also in HFD animals (Figures 2B,C; 12.5 ± 1.6 pg/mL TNF-
α P < 0.01; 11 ± 1.6 pg/mL IL-1 β, P < 0.01, respectively). Moreover, the serum levels of the anti-inflammatory cytokine
IL-10 decreased with both diets (Figure 2D; RBD 7.8 ± 0.2
pg/mL IL-10, P < 0.05; HFD 6.5 ± 0.4 pg/mL IL-10, P < 0.01). Such results clearly show that both diets induce a peripheral
pro-inflammatory profile. FIGURE 2 | Effect of regional basic (RBD) and high-fat (HFD) diets on
inflammatory mediators. (A) Malondialdehyde (MDA) formation, a marker of
oxidative stress in hippocampal tissue, is only increased in HFD animals. ELISA quantification on blood serum protein levels of pro-inflammatory
cytokines (B) TNF-α and (C) IL-1β shows an upregulation after chronic
exposure to both diets. Additionally, ELISA quantification of the
anti-inflammatory cytokine (D) IL-10 shows a significant decrease of its protein
levels in both diet feeding animals. Data were analyzed by using Kruskal-Wallis
test, followed by Dunn’s post-test for multiple comparisons, n = 5–8. *P <
0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The
results are shown as mean ± SEM. It has already been demonstrated that blood composition
fluctuations can have an impact on brain function. Therefore,
we also evaluated the inflammatory status of hippocampi of the
animals feed with both diets. In fact, we observed a significant
increase in ROS production (Figure 3A; RBD 648.5 ± 4.7, P
< 0.001; HFD 964.3 ± 23.3, P < 0.001) and TNF-α protein
levels (Figure 3B; RBD 137 ± 10% of control, P < 0.05, HFD FIGURE 1 | Effect of regional basic (RBD) and high-fat (HFD) diets feeding on
mice body weight from postnatal day 10 to postnatal day 52. Data were
analyzed by using two-way ANOVA followed by Dunnett’s multiple
comparisons test. *P < 0.0001 and #P < 0.0001 vs. nourished control group
(CTR). The results are shown as mean ± SEM. RESULTS FIGURE 2 | Effect of regional basic (RBD) and high-fat (HFD) diets on
inflammatory mediators. (A) Malondialdehyde (MDA) formation, a marker of
oxidative stress in hippocampal tissue, is only increased in HFD animals. ELISA quantification on blood serum protein levels of pro-inflammatory
cytokines (B) TNF-α and (C) IL-1β shows an upregulation after chronic
exposure to both diets. Additionally, ELISA quantification of the
anti-inflammatory cytokine (D) IL-10 shows a significant decrease of its protein
levels in both diet feeding animals. Data were analyzed by using Kruskal-Wallis
test, followed by Dunn’s post-test for multiple comparisons, n = 5–8. *P <
0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The
results are shown as mean ± SEM. FIGURE 2 | Effect of regional basic (RBD) and high-fat (HFD) diets on
inflammatory mediators. (A) Malondialdehyde (MDA) formation, a marker of
oxidative stress in hippocampal tissue, is only increased in HFD animals. ELISA quantification on blood serum protein levels of pro-inflammatory
cytokines (B) TNF-α and (C) IL-1β shows an upregulation after chronic
exposure to both diets. Additionally, ELISA quantification of the
anti-inflammatory cytokine (D) IL-10 shows a significant decrease of its protein
levels in both diet feeding animals. Data were analyzed by using Kruskal-Wallis
test, followed by Dunn’s post-test for multiple comparisons, n = 5–8. *P <
0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The
results are shown as mean ± SEM. FIGURE 2 | Effect of regional basic (RBD) and high-fat (HFD) diets on
inflammatory mediators. (A) Malondialdehyde (MDA) formation, a marker of
oxidative stress in hippocampal tissue, is only increased in HFD animals. ELISA quantification on blood serum protein levels of pro-inflammatory
cytokines (B) TNF-α and (C) IL-1β shows an upregulation after chronic
exposure to both diets. Additionally, ELISA quantification of the
anti-inflammatory cytokine (D) IL-10 shows a significant decrease of its protein
levels in both diet feeding animals. Data were analyzed by using Kruskal-Wallis
test, followed by Dunn’s post-test for multiple comparisons, n = 5–8. *P <
0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The
results are shown as mean ± SEM. January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 4 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. FIGURE 3 | Effect of regional basic (RBD) and high-fat (HFD) diets on hippocampal neuroinflammatory response. RESULTS Both diets induced an (A) oxidative stress observed
by an increase in reactive oxygen species (ROS) and (B) an increase of TNF-α protein levels in mice hippocampal homogenates. (C) Only HFD feeding animals show a
significant upregulation of IL-1β protein levels. Above the bars, representative western blot images of TNF-α (19 kDa), IL-1β (17 kDa) and GAPDH (37 kDa) are shown. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons, n = 5–7. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The results are shown as mean ± SEM. FIGURE 3 | Effect of regional basic (RBD) and high-fat (HFD) diets on hippocampal neuroinflammatory response. Both diets induced an (A) oxidative stress observed
by an increase in reactive oxygen species (ROS) and (B) an increase of TNF-α protein levels in mice hippocampal homogenates. (C) Only HFD feeding animals show a
significant upregulation of IL-1β protein levels. Above the bars, representative western blot images of TNF-α (19 kDa), IL-1β (17 kDa) and GAPDH (37 kDa) are shown. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons, n = 5–7. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). The results are shown as mean ± SEM. immunoreactivity (Figures 5A,C; 47,997 ± 5,101, P < 0.001,
when compared with the control 23,784 ± 2,412) in the
hippocampus. RBD had also an effect on both parameters
analyzed, but not so significant when compared to HFD
(Figure 5B, 135 ± 8.2% of control, P < 0.05; Figure 5C, 36,195
± 3,758, P < 0.05, when compared with the control 23,784 ±
2,412). Moreover, when the protein levels of S100β, a specific
marker of astrocytes reactivity, were measured, an upregulation
was observed after exposure to both diets (Figure 5D; RBD
131 ± 4.79% of control, P < 0.05, HFD 149 ± 11.9% of
control, P < 0.01). Taking into consideration the importance of a
simultaneously analysis of cellular morphological alterations, we
further concluded that both diets induced an increase in the total
length of astrocytic processes (Figure 5F; CTR 70.2 ± 3.6 µm;
RBD 88.8 ± 5 µm, P < 0.05; HFD 88.6 ± 5; P < 0.05), and
promoted an arbor complexity of these cells (Figure 5G; P <
0.05). RESULTS Curiously, only in HFD animals was possible to observe an
increase of the total number of astrocytic processes (Figure 5E;
CTR 4.1 ± 0.1; RBD 4.3 ± 0.1; HFD 5.1 ± 0.3; P < 0.01). adjacent endothelial cells, such claudin-5 a tight junction (TJ)
protein, and the second pathway occurs through the endothelial
cell and involves the formation of vesicles, including caveolin-1-
coated vesicles. Herein, we observed a significant downregulation
of claudin-5 protein levels (Figure 4B) in both RBD (63.5 ± 6.7%,
P < 0.01) and HFD animals (56.8 ± 6.5%, P < 0.01), suggesting
an increase in paracellular permeability of the BBB. After that,
we investigated the effect of diets on Cav-1 and concluded that
only HFD was able to induce a significant upregulation of its
protein levels (Figure 4C, 131 ± 6.5%, P < 0.05). Further, to
corroborate the weakening of BBB we also evaluated alterations
in collagen IV (coll IV) immunoreactivity. This protein is one
of the most abundant in basal membrane, that gives support to
brain microvessels. In fact, there was a clear reduction in the
staining induced by both diets (Figures 4D,E; CTR 162,180 ±
28,098; RBD 46,959 ± 7,285, P < 0.001; HFD 82,098 ± 17,780,
P < 0.05). Since BBB has also a key role in restrain the transmigration
of peripheral immune cells into brain parenchyma, we further
analyzed the protein levels of vascular cell adhesion protein 1
(VCAM-1), an adhesion molecule that promotes the passage
of peripheral immune cells across BBB. Interestingly, chronic
exposure to both diets induced an upregulation of VCAM-1
(Figure 4F; RBD 113 ± 2.7%, P < 0.05; HFD 126 ± 4.9%,
P < 0.001). Frontiers in Nutrition | www.frontiersin.org DISCUSSION In recent years, a westernization of diets, with an increase
in fat content, on developing countries has been observed,
and has a huge socio-economic impact (24). Despite several
preclinical studies exploring this issue, there is a scarcity of
information regarding the impact of protein malnutrition
on BBB properties. The BBB is known as the gatekeeper
of the brain, being extremely selective and so protecting
the brain from variations in blood composition and toxins. Nevertheless, it has also a role in providing essential nutrients
for proper brain function. Thus, BBB dysfunction may lead Data presented herein shows that malnutrition related with
RBD or HFD triggers hallmarks of oxidative stress, (neuro)
inflammation, and BBB permeability. Importantly, astrocytes
have a crucial role in BBB structure and function (22), as well
as in neuroinflammatory processes (23). Thus, we investigated
the effect of both diets on astrocytes and concluded that
HFD induced a significant increase in GFAP protein levels
(Figures 5A,B; 188 ± 17.3% of control, P < 0.001) and January 2019 | Volume 5 | Article 131 5 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. FIGURE 4 | Effect of regional basic (RBD) and high-fat (HFD) diets on blood-brain barrier properties. An increase of albumin (A) and a decrease of claudin-5 (B)
protein levels were observed with both RBD and HFD diets. (C) Regarding caveolin-1 (Cav1) protein levels, there was also a significant increase of its total levels in
HFD group. (D) Representative immunohistochemistry images for Collagen IV (green, brain vessels), and Hoechst 33342 (blue, nuclei). Scale bar = 40 µm. (E) DRB
and HFD induced a significant decrease in collagen IV immunoreactivity. Moreover, both diets induced a significant upregulation in (F) VCAM-1 (vascular cell adhesion
molecule-1). Above the bars, representative western blot images of Albumin (66 kDa), claudin-5 (25 kDa), caveolin-1 (20 kDa), VCAM-1 (110 kDa), and GAPDH (37
kDa) are shown. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons, n = 5–7. *P < 0.05, **P < 0.01, ***P <
0.001, ****P < 0.0001 vs. nourished control group (CTR). The results are shown as mean ± SEM. |
(
)
(
)
( )
( ) FIGURE 4 | Effect of regional basic (RBD) and high-fat (HFD) diets on blood-brain barrier properties. An increase of albumin (A) and a decrease of claudin-5 (B)
protein levels were observed with both RBD and HFD diets. Frontiers in Nutrition | www.frontiersin.org DISCUSSION (C) Regarding caveolin-1 (Cav1) protein levels, there was also a significant increase of its total levels in
HFD group. (D) Representative immunohistochemistry images for Collagen IV (green, brain vessels), and Hoechst 33342 (blue, nuclei). Scale bar = 40 µm. (E) DRB
and HFD induced a significant decrease in collagen IV immunoreactivity. Moreover, both diets induced a significant upregulation in (F) VCAM-1 (vascular cell adhesion
molecule-1). Above the bars, representative western blot images of Albumin (66 kDa), claudin-5 (25 kDa), caveolin-1 (20 kDa), VCAM-1 (110 kDa), and GAPDH (37
kDa) are shown. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons, n = 5–7. *P < 0.05, **P < 0.01, ***P <
0.001, ****P < 0.0001 vs. nourished control group (CTR). The results are shown as mean ± SEM. with BBB function (12, 17, 18, 25). In fact, a chronic long-
term inflammatory environment in the brain usually leads
to chronic impairment of the BBB, which will contribute to
long-term neurodegeneration and neuropathology, including
neurodegenerative conditions like Alzheimer’s disease (26). to brain damage and indeed is known to be a common
feature in all neurodegenerative diseases. Moreover, under
aggression related with trauma, ischemia, metabolic disorders
or malnutrition, neuroinflammatory cascades are initiated
involving glial cells and molecular pathways that may interfere January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 6 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. FIGURE 5 | Effect of regional basic (RBD) and high-fat (HFD) diets on astrocytes properties. Representative immunohistochemistry images for (A) GFAP (red,
astrocytes), and Hoechst 33342 (blue, nuclei). Scale bar = 40 µm. Both diets induced an increase in (B) GFAP protein levels, (C) immunoreactivity, and (D) S10
protein levels. Yet, HFD had a more pronounced effect than RBD. Regarding morphological alterations, only HFD increased the (E) total number of processes, w
effect on RBD animals. However, both diets induced a significant increase in (F) the total length of such processes and (G) on arbor complexity. Results are expr
as mean + S.E.M., n = 5–7 [for (B,D)], n = 10 [for (C,G)], n = 30 [for (E,F)]. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for m
comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). Frontiers in Nutrition | www.frontiersin.org DISCUSSION A two-way ANOVA followed by Bonferroni’s Multiple comparison test
used for Sholl analysis, where *P < 0.05 and **P < 0.01 vs. CTR. n
addition,
long-term
undernutrition
(27),
similar
to
hippocampal physiology and cognitive performance asso FIGURE 5 | Effect of regional basic (RBD) and high-fat (HFD) diets on astrocytes properties. Representative immunohistochemistry images for (A) GFAP (red,
astrocytes), and Hoechst 33342 (blue, nuclei). Scale bar = 40 µm. Both diets induced an increase in (B) GFAP protein levels, (C) immunoreactivity, and (D) S100β
protein levels. Yet, HFD had a more pronounced effect than RBD. Regarding morphological alterations, only HFD increased the (E) total number of processes, with no
effect on RBD animals. However, both diets induced a significant increase in (F) the total length of such processes and (G) on arbor complexity. Results are expressed
as mean + S.E.M., n = 5–7 [for (B,D)], n = 10 [for (C,G)], n = 30 [for (E,F)]. Data were analyzed by using Kruskal-Wallis test, followed by Dunn’s post-test for multiple
comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. nourished control group (CTR). A two-way ANOVA followed by Bonferroni’s Multiple comparison test was
used for Sholl analysis, where *P < 0.05 and **P < 0.01 vs. CTR. In
addition,
long-term
undernutrition
(27),
similar
to
our RBD and HFD feeding protocol (28, 29), induces a
systemic inflammation with brain deleterious effects, being
the hippocampus one of the most susceptible brain regions
(29). Curiously, calorie intake, meal frequency, texture and
content seem to be associated with modification in hallmarks of hippocampal physiology and cognitive performance associated
with neurogenesis (30). It was recently shown that a preexisting
protein malnutrition worsens the motor deficits observed after
stroke (31). Moreover, post-ischemia protein malnutrition
can
aggravate
neuroinflammatory
processes
and
inhibits
neuroplasticity in the hippocampus (32). Accordingly, in our January 2019 | Volume 5 | Article 131 Frontiers in Nutrition | www.frontiersin.org 7 Diet Imbalance Triggers BBB Dysfunction de Aquino et al. chronic model of malnutrition or high-fat content diet we also
observed both peripheral and central inflammatory markers,
with an increase in both ROS production and pro-inflammatory
cytokines. associated with increased oxidative stress and GFAP in the
cerebral cortex and in the hippocampal dentate gyrus with
impaired recognition memory (42). DISCUSSION These data support our
observation that particularly HFD chronic consumption causes a
significant astrocyte reactivity in the hippocampus, as shown by
increased expression of GFAP and S100β. Nevertheless, RBD had
also an effect but not so strong as with HFD. Curiously, Feoli et al. (43) did not detect alterations in hippocampal GFAP and S100β
immune contents on the 60th postnatal day in undernourished
rats. Such contradiction can be explained by differences related
with diet feeding protocols and animal strains. Still, the authors
observed an increase of S100β levels in the cerebrospinal fluid
(43). Long-term feeding with RBD (27, 33) and HFD (34)
may lead to intestinal barrier leakage with increased gut-
to-blood bacterial translocation, and continued LPS systemic
circulation. Alterations in the gut microbiota toward more LPS-
releasing Gram-negative bacteria by both diets may contribute
to low-grade systemic inflammation with brain deleterious
effects (27, 28), including BBB disruption and oxidative stress
(35). In addition, systemic inflammation could lead to BBB
dysfunction by increased myeloperixodase (MPO)-releasing
neutrophil activity (36). This potential MPO release from
circulating blood neutrophils may be due to increased LPS
binding to the endothelial surface of the brain capillaries (37). Interestingly, rats challenged with cecal ligation and puncture
(as a model of sepsis) showed increased BBB permeability to
Evans Blue brain staining and reduced endothelial claudin-3 and
claudin-5 levels, effects that were ameliorated by the cholesterol-
lowering drug simvastatin (38). The evaluation of astrocytic morphological alterations is of
crucial importance to understand the role of these glial cells on
the brain function. Thus, in order to stratify the possible different
types of astrocytes, a classification of A1 and A2 astrocytes has
been arisen by Ben Barres lab based on different gene expression
profiles (44–46). Both A1 and A2 astrocytes are reactive glial cells,
in which A1 type seems to be induced by exposure to IL-1α, TNF-
α and complement proteins (45). Therefore, we can hypothesize
that our malnutrition models are shifting astrocytes to an A1
profile. Accordingly, we observed a significant astrogliosis proved
by an increase of GFAP and S100β expression, and by significant
morphological
alterations
of
astrocytes. Still,
a
genomic
analysis is necessary to correctly classified the type of reactive
astrocytes. It is also known that systemic inflammation can lead to
BBB dysfunction by increasing the activity of proinflammatory
cytokine-releasing neutrophils (36), white blood cells that
are an essential part of the innate immune system. DISCUSSION In
fact, it has been reported that HFD contributes to BBB
disruption either in young and aged mice, as shown by
increased permeability of brain microvessels to IgG and to
sodium fluorescein and changes in tight junction proteins
including occludin and claudin-5 (39, 40). Further, the same
authors concluded that HFD triggers neuroinflammation, as
evaluated by reactive microglia and release of pro-inflammatory
cytokines (40). Moreover, in an animal model of HFD for
9 weeks, an increase in leukocyte transmigration into the
brain parenchyma was observed (41). Accordingly, in the
present study we showed a downregulation of collagen IV,
the most important protein of the BBB basement membrane,
and the tight junction protein, claudin-5, which is strong
indicator of BBB dysfunction. Moreover, there was an increase
in VCAM-1 protein levels, an adhesion molecule specifically
expressed in endothelial cells and necessary to immune
cells transmigration into the brain parenchyma. Therefore,
hippocampal Cav1 immunoreactivity was increased, suggesting
active endothelial transcytosis, further supporting the BBB
hyperpermeability induced by the diets. Thus, we showed
that both RBD and HFD feeding protocols induced a very
robust (neuro) inflammatory process as well as a BBB
hyperpermeability. In summary, the present report shows that RBD and HFD
chronic feeding impacts BBB permeability, and leads to brain
oxidative stress and neuroinflammation with a prominent role
of astrocytes. Systemic inflammation was also observed and may
underly central alterations. However, more studies are warranted
to dissect the mechanisms associated with these effects. AUTHOR CONTRIBUTIONS CdA, RL, LO, and VC-S: performed the experiments and wrote
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terms of the Creative Commons Attribution License (CC BY). The use, distribution
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English
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Lyapunov Inequalities for One-Dimensional p-Laplacian Problems with a Singular Weight Function
|
Journal of inequalities and applications
| 2,010
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Hindawi Publishing Corporation
Journal of Inequalities and Applications
Volume 2010, Article ID 865096, 9 pages
doi:10.1155/2010/865096 Hindawi Publishing Corporation
Journal of Inequalities and Applications
Volume 2010, Article ID 865096, 9 pages
doi:10.1155/2010/865096 Academic Editor: Yeol Je Cho Copyright q 2010 I. Sim and Y.-H. Lee. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. We estimate Lyapunov inequalities for a single equation, a cycled system and a coupled system of
one-dimensional p-Laplacian problems with weight functions having stronger singularities than
L1 We estimate Lyapunov inequalities for a single equation, a cycled system and a coupled system of
one-dimensional p-Laplacian problems with weight functions having stronger singularities than
L1. one dimensional p Laplacian problems with weight functions having stronger singularities than
L1. Research Article
Lyapunov Inequalities for
One-Dimensional p-Laplacian Problems with
a Singular Weight Function Inbo Sim1 and Yong-Hoon Lee2
1 Department of Mathematics, University of Ulsan, Ulsan 680-749, South Korea
2 Department of Mathematics, Pusan National University, Pusan 609-735, South Korea Inbo Sim1 and Yong-Hoon Lee2
1 Department of Mathematics, University of Ulsan, Ulsan 680-749, South Korea
2 Department of Mathematics, Pusan National University, Pusan 609-735, South Korea Inbo Sim1 and Yong-Hoon Lee2 2 Department of Mathematics, Pusan National University, Pusan 609-735, South Ko Correspondence should be addressed to Yong-Hoon Lee, yhlee@pusan.ac.kr Received 8 October 2009; Accepted 24 November 2009 Academic Editor: Yeol Je Cho 1. Introduction The Lyapunov inequality for linear ordinary differential equation −u′′ rtu,
t ∈a, b,
ua 0 ub,
L L where r ∈Ca, b, 0, ∞, gives a necessary condition for the existence of a positive solution
as follows: 4
b −a ≤
b
a
rtdt. 1.1 1.1 Lyapunov 1 initiated to estimate the above inequality. Since then, there have been several
results to generalize the above linear ordinary differential equation in many directions. 2
Journal of Inequalities and Applicatio Journal of Inequalities and Applications 2 Before stating many efforts, it is worth to mention Hartman and Pinasco’s work. Hartman
2 obtained the generalized inequality by using Green’s function: Before stating many efforts, it is worth to mention Hartman and Pinasco’s work. Hartman
2 obtained the generalized inequality by using Green’s function: b −a ≤
b
a
t −ab −trtdt. 1.2 1.2 In fact, for a ≤t ≤b, by the inequality t −ab −t ≤b −a2
4
,
1.3 1.3 condition 1.2 is a generalization of condition 1.1. condition 1.2 is a generalization of condition 1.1. Pinasco 3 extended linear ordinary differential equations to the following one-
dimensional p-Laplacian problem: −ϕp
u′t′ rtϕput,
t ∈a, b,
ua 0 ub,
P P where ϕpx |x|p−2x, p > 1 and r ∈Ca, b, 0, ∞. He obtained Lyapunov inequality for
P as follows: 2p
b −ap/q ≤
b
a
rtdt,
1.4 1.4 where 1/p 1/q 1. There have been many studies for various types of equations. Among others, one may
refer to de N´aploi and Pinasco 4 for the case of monotone quasilinear operators which
include one-dimensional p-Laplacian as a special case, Parhi and Panigrahi 5 for the case
of third order differential equations, Ca˜nada et al. 6 for the case of partial differential
equations which have a weight function in L1, and Clark and Hinton 7 for the case of
Hamiltonian systems. Until now, the most general class of weight functions for the Lyapunov inequalities is
L1a, b. The purpose of this paper is to get Lyapunov inequalities for single equations as well
as systems of one-dimensional p-Laplacian problems with singular weight functions which
have a stronger singularities than those of L1a, b. For this purpose, we first give three specific classes of weight functions. The first class
can be given as A ≜
r ∈Ca, b, 0, ∞ :
ab/2
a
ϕ−1
p
ab/2
s
rτdτ
ds
b
ab/2
ϕ−1
p
s
ab/2
rτdτ
ds <∞
. 1.5 1.5 Journal of Inequalities and Applications 3 Journal of Inequalities and Applications It comes naturally from the study of the existence of positive solutions for p-Laplacian
problems. The second one is just the extension of Hartman’s condition to p-Laplacian
problems given as follows: B ≜
r ∈Ca, b, 0, ∞ :
b
a
s −ap−1b −sp−1rsds < ∞
. 1.6 1.6 It is easy to see that L1a, b ⊂A ∩B and classes A and B are equivalent when p 2. It is also
known 8 that B ⫋A for p > 2 and A ⫋B for 1 < p < 2. The third one can be given as C ≜
r ∈Ca, b, 0, ∞ : there are α, β>0 such that α, β < p −1 and b
a
s −aαb −sβrsds < ∞
. 1.7 1.7 It is obvious to see that C ⊂B and C ⊂A see 8. It is obvious to see that C ⊂B and C ⊂A see 8. It is obvious to see that C ⊂B and C ⊂A see 8. It is obvious to see that C ⊂B and C ⊂A see 8. This paper is organized as follows. In Section 2, we show Lyapunov inequality for
one-dimensional p-Laplacian problem when a weight function is r ∈A ∩B. where 1/p 1/q 1. In Section 3, we
estimate Lyapunov inequality for a cycled system of one-dimensional p-Laplacian problem
when a weight function is r ∈C. Finally in Section 4, we have Lyapunov inequality for a
strongly coupled system of one-dimensional p-Laplacian problem when a weight function is
r ∈C. 2. Single Equation Let us consider problem P. By a solution of P we mean that u ∈Ca, b ∩C1a, b, ϕpu′
is absolutely continuous in any compact subinterval of a, b, and u satisfies the first equation
in P in a, b and ua 0 ub. We assume r ∈A ∩B. It is known that all solutions for
P are of class C1
0a, b see 9. Theorem 2.1. Assume r ∈A ∩B. If u is a positive solution for P, then one has b −ap−1
2p−2
≤
b
a
t −ap−1b −tp−1rtdt. 2.1 2.1 By H¨older’s inequality, we get Proof. By H¨older’s inequality, we get |ut| ≤
t
a
u′s
ds ≤t −ap−1/p
t
a
u′ pds
1/p
. 2.2 2.2 Journal of Inequalities and Applications 4 For a ≤t ≤a b/2, noting t −a ≤2/b −at −ab −t, we have |ut| ≤
2
b −at −ab −t
p−1/pab/2
a
u′ pds
1/p
. 2.3 2.3 Thus, we have Thus, we have |ut|p ≤
2
b −at −ab −t
p−1ab/2
a
u′ pds
. 2.4 2.4 Similarly, by H¨older’s inequality, we get Similarly, by H¨older’s inequality, we get Similarly, by H¨older’s inequality, we get |ut| ≤
b
t
u′s
ds ≤b −tp−1/p
b
t
u′ pds
1/p
. 2.5 2.5 For a b/2 ≤t ≤b, noting b −t ≤2/b −at −ab −t, we get For a b/2 ≤t ≤b, noting b −t ≤2/b −at −ab −t, we get |ut|p ≤
2
b −at −ab −t
p−1b
ab/2
u′ pds
. 2.6 2.6 Adding 2.4 and 2.6, we have Adding 2.4 and 2.6, we have 2|ut|p ≤
2
b −at −ab −t
p−1b
a
u′ pds
. 2.7 2.7 Multiplying both sides of 2.7 by rt and rewriting, we get Multiplying both sides of 2.7 by rt and rewriting, we get b −ap−1
2p−2
rt|ut|p ≤rtt −ab −tp−1
b
a
u′ pds
. 2.8 2.8 Since u is a solution for P, we have Since u is a solution for P, we have b
a
u′ pdt
b
a
rt|ut|pdt. 2.9 2.9 We note that the right-hand side makes sense because u is in C1
0a, b. Integrating 2.8 on
a, b and using 2.9, we have We note that the right-hand side makes sense because u is in C1
0a, b. 2. Single Equation Integrating 2.8 on
a, b and using 2.9, we have b −ap−1
2p−2
b
a
u′ pdt
b
a
b −ap−1
2p−2
rt|ut|pdt
≤
b
a
rtt −ab −tp−1
b
a
u′ pds
dt. 2.10 2.10 of Inequalities and Applications
5 Journal of Inequalities and Applications Journal of Inequalities and Applications 5 Therefore, we get b −ap−1
2p−2
≤
b
a
rtt −ab −tp−1dt. 2.11 2.11 Remark 2.2. i When p 2, the above result coincides with Hartman’s estimate. But
Hartman’s argument does not work here by lack of Green’s function for p-Laplacian. Remark 2.2. i When p 2, the above result coincides with Hartman’s estimate. But
Hartman’s argument does not work here by lack of Green’s function for p-Laplacian mark 2.2. i When p 2, the above result coincides with Hartman’s estimate. But
artman’s argument does not work here by lack of Green’s function for p-Laplacian. Hartman s argument does not work here by lack of Green s function for p Laplacian. ii If r ∈L1a, b, for a ≤t ≤b, since t −ab −t ≤b −a2/4 and p/q p −1, we
have Pinasco’s estimate 1.4. Thus our estimate generalizes Pinasco’s. g
y
p
p
ii If r ∈L1a, b, for a ≤t ≤b, since t −ab −t ≤b −a2/4 and p/q p −1, we
have Pinasco’s estimate 1.4. Thus our estimate generalizes Pinasco’s. For 0 ≤r ∈Ca, b, Pinasco 3 also estimated the lower bounds for eigenvalues {λn}
of For 0 ≤r ∈Ca, b, Pinasco 3 also estimated the lower bounds for eigenvalues {λn} of of −ϕp
u′t′ λrtϕput,
t ∈a, b,
ua 0 ub. Pλ Pλ The proof mainly makes use of the nodal property of its corresponding eigenfunctions {un};
that is, un has n−1 interior zeros in a, b. Recently, when r ∈A∩B, Kajikiya et al. 9 showed
the existence of eigenvalues {λn} for Pλ and its corresponding eigenfunctions also have the
nodal property. Employing Pinasco’s argument 3, Theorem 1.1 with 2.1, for each n ∈N,
we have λn ≥
b −ap−1
2np−2 b
artt −ab −tp−1dt
. 2.12 2.12 3. Cycled System Let us consider a cycled system: ϕp
u′
1t′ r1tϕpu2t 0,
t ∈a, b,
ϕp
u′
2t′ r2tϕpu3t 0,
t ∈a, b, ϕp
u′
1t′ r1tϕpu2t 0,
t ∈a, b,
ϕp
u′
2t′ r2tϕpu3t 0,
t ∈a, b, CS ϕp
u′
n−1t′ rn−1tϕpunt 0,
t ∈a, b,
ϕp
u′
nt′ rntϕpu1t 0,
t ∈a, b,
u1a · · · una 0 u1b · · · unb. CS We say that u1, u2, . . . , un is a solution of CS if ui ∈Ca, b ∩C1a, b, ϕpu′
i is absolutely
continuous in any compact subinterval of a, b, each ui satisfies the equations in CS in
a, b, and u1a · · · una 0 u1b · · · unb. We assume that ri ∈C. We note that
all solutions for CS are of class C1
0a, b see 10. We say that u1, u2, . . . , un is a solution of CS if ui ∈Ca, b ∩C1a, b, ϕpu′
i is absolutely
continuous in any compact subinterval of a, b, each ui satisfies the equations in CS in
a, b, and u1a · · · una 0 u1b · · · unb. We assume that ri ∈C. We note that
all solutions for CS are of class C1
0a, b see 10. 6
Journal of Inequalities and Applicati Journal of Inequalities and Applications 6 Theorem 3.1. Assume ri ∈C. If u1, u2, . . . , un is a positive solution of CS, then Theorem 3.1. Assume ri ∈C. If u1, u2, . . . , un is a positive solution of CS, then b
a
t −ab −tp−1r1tdt · · ·
b
a
t −ab −tp−1rntdt ≥
b −ap−1n
2p−2n
. 3.1 3.1 Proof. We only show the case n 2. For the general case, we can prove it by repeating this
procedure. As in 2.7, for i 1, 2, we have |uit|p−1 ≤
2p−2p−1/p
b −ap−12/p t −ab −tp−12/p
b
a
u′
i
pds
p−1/p
3.2 3.2 or or |uit| ≤
2p−2/p
b −ap−1/p t −ab −tp−1/p
b
a
u′
i
pds
1/p
. 3. Cycled System 3.3 3.3 Multiplying the first equation of CS by u1 and integrating on a, b, we have by 3.2 and
3.3 that p y
3.3 that b
a
u′
1
pdt ≤
b
a
r1t|u2|p−1|u1|dt
≤
b
a
2p−2
b −ap−1 t −ab −tp−1r1tdt
×
b
a
u′
2
pds
p−1/p b
a
u′
1
pds
1/p
. 3.4 3.4 ×
b
a
u′
2
pds
p−1/p b
a
u′
1
pds
1/p
. Thus, we have b
a
u′
1
pdt
p−1/p
≤
2p−2
b −ap−1
b
a
t −ab −tp−1r1tdt
b
a
u′
2
pds
p−1/p
. 3.5 3.5 Similarly, for the second equation in CS, we have Similarly, for the second equation in CS, we have b
a
u′
2
pdt
p−1/p
≤
2p−2
b −ap−1
b
a
t −ab −tp−1r2tdt
b
a
u′
1
pds
p−1/p
. 3.6 3.6 Thus, we have have
b
a
t −ab −tp−1r1tdt
b
a
t −ab −tp−1r2tdt ≥
b −ap−12
2p−22
. 3.7 Journal of Inequalities and Applications
7 7 Journal of Inequalities and Applications Journal of Inequalities and Applications Corollary 3.2. Assume ri r ∈C, for i 1, 2, . . . , n. If u1, u2, . . . , un is a positive solution of
CS, then one has Corollary 3.2. Assume ri r ∈C, for i 1, 2, . . . , n. If u1, u2, . . . , un is a positive solution of
CS, then one has b
a
t −ab −tp−1rtdt ≥b −ap−1
2p−2
. 3.8 3.8 4. Strongly Coupled System Let us consider a strongly coupled system: Let us consider a strongly coupled system: ϕp
u′
1t′ r1tϕpu1t ϕpu2t · · · ϕpunt 0,
t ∈a, b,
ϕp
u′
2t′ r2tϕpu1t ϕpu2t · · · ϕpunt 0,
t ∈a, b, · · ·
SCS SCS ϕp
u′
nt′ rntϕpu1t ϕpu2t · · · ϕpunt 0,
t ∈a, b,
u1a · · · una 0 u1b · · · unb, where ri ∈C. We can give a definition for a solution of SCS as the definition for a solution
of CS and it is known that all positive solutions for SCS are of class C1
0a, b see 10. We emphasize that it is only shown for a positive solution so far. Theorem 4.1. Assume ri ∈C. If u1, u2, . . . , un is a positive solution of SCS, then one has b
a
t −ab −tp−1r1tdt · · ·
b
a
t −ab −tp−1rntdt ≥1
n
b −ap−1
2p−2
. 4.1 4.1 Proof. As in the proof of Theorem 3.1, we only show the case n 2. Multiplying u1 to the first
equation in SCS and integrating on a, b and using 2.7, 3.2, and 3.3, we have Proof. As in the proof of Theorem 3.1, we only show the case n 2. Multiplying u1 to the first
equation in SCS and integrating on a, b and using 2.7, 3.2, and 3.3, we have b
a
u′
1
pdt ≤
b
a
r1t|u1|pdt
b
a
r1t|u2|p−1|u1|dt
≤
2p−2
b −ap−1
b
a
t −ab −tp−1r1tdt
b
a
u′
1
pds
2p−2
b −ap−1
b
a
t −ab −tp−1r1tdt
×
b
a
u′
2
pds
p−1/p b
a
u′
1
pds
1/p
. 4.2 4.2 Journal of Inequalities and Applications Journal of Inequalities and Applications 8 Similarly, from the second equation of SCS, we have Similarly, from the second equation of SCS, we have b
a
u′
2
pdt ≤
b
a
r2t|u2|pdt
b
a
r2t|u1|p−1|u2|dt
≤
2p−2
b −ap−1
b
a
t −ab −tp−1r2tdt
b
a
u′
2
pds
2p−2
b −ap−1
b
a
t −ab −tp−1r2tdt
×
b
a
u′
1
pds
p−1/p b
a
u′
2
pds
1/p
. 4. Strongly Coupled System If u1, u2, . . . , un is a positive solution of
SCS, then one has b
a
t −ab −tp−1rtdt ≥1
n2
b −ap−1
2p−2
. 4.10 4.10 Acknowledgment The first author was supported by the 2009 Research Fund of the University of Ulsan. The first author was supported by the 2009 Research Fund of the University of Ulsan. 4. Strongly Coupled System 4.3 4.3 Let us denote X
b
a|u′
1|pdt, Y
b
a|u′
2|pdt, C1 2p−2/b −ap−1
b
at −ab −tp−1r1tdt,
and C2 2p−2/b −ap−1
b
at −ab −tp−1r2tdt. Then from 4.2 and 4.3, we have
X ≤C1X C1X1/pY p−1/p,
Y ≤C2Y C2Y 1/pXp−1/p,
4.4 Let us denote X
b
a|u′
1|pdt, Y
b
a|u′
2|pdt, C1 2p−2/b −ap−1
b
at −ab −tp−1r1tdt,
and C2 2p−2/b −ap−1
b
at −ab −tp−1r2tdt. Then from 4.2 and 4.3, we have
X ≤C1X C1X1/pY p−1/p,
Y ≤C2Y C2Y 1/pXp−1/p,
4.4 X ≤C1X C1X1/pY p−1/p, X ≤C1X C1X1/pY p−1/p,
Y ≤C2Y C2Y 1/pXp−1/p,
4.4 4.4 respectively. Equation 4.4 implies respectively. Equation 4.4 implies X ≤C1X Y C1
X1/pY p−1/p Y 1/pXp−1/p
,
Y ≤C2X Y C2
X1/pY p−1/p Y 1/pXp−1/p
,
4.5 4.5 respectively. Therefore, we have X Y ≤C1 C2X Y C1 C2
X1/pY p−1/p Y 1/pXp−1/p
. 4.6
Since X1/pY p−1/p Y 1/pXp−1/p ≤X Y 11, page 38, we get X Y ≤C1 C2X Y C1 C2
X1/pY p−1/p Y 1/pXp−1/p
. 4.6
Since X1/pY p−1/p Y 1/pXp−1/p ≤X Y 11, page 38, we get X Y ≤C1 C2X Y C1 C2
X1/pY p−1/p Y 1/pXp−1/p
. 4 4.6 Since X1/pY p−1/p Y 1/pXp−1/p ≤X Y 11, page 38, we get X Y ≤2C1 C2X Y. 4.7 4.7 Hence, we have
C1 C2 ≥1
2. 4.8 Hence, we have C1 C2 ≥1
2. 4.8 4.8 1
2 ≥2
That is,
b
a
t −ab −tp−1r1tdt
b
a
t −ab −tp−1r2tdt ≥1
2
b −ap−1
2p−2
. 4.9 That is, That is, s,
b
a
t −ab −tp−1r1tdt
b
a
t −ab −tp−1r2tdt ≥1
2
b −ap−1
2p−2
. 4.9 4.9 Journal of Inequalities and Applications Journal of Inequalities and Applications 9 Corollary 4.2. Assume ri r ∈C, for i 1, 2, . . . , n. If u1, u2, . . . , un is a positive solution of
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Genetic polymorphisms of interleukin-16 in Egyptian patients with primary knee osteoarthritis
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Egyptian Rheumatology and Rehabilitation
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
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licence, visit http://creativecommons.org/licenses/by/4.0/. Hafez et al.
Egyptian Rheumatology and Rehabilitation (2023) 50:12
https://doi.org/10.1186/s43166-023-00176-y Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12
https://doi.org/10.1186/s43166-023-00176-y Egyptian Rheumatology
and Rehabilitation Hafez et al. Open Access Open Access Background *Correspondence:
Eman Abdel Razek Hafez
emanhafez@mans.edu.eg; emanhafez22@gmail.com
1 Department of Rheumatology and Rehabilitation, Faculty of Medicine,
Mansoura University, Mansoura, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Mansoura
University, Mansoura, Egypt
3 Department of Forensic and Toxicology, Faculty of Medicine, Mansoura
University, Mansoura, Egypt
4 Department of Microbiology, Faculty of Medicine, Mansoura University,
Mansoura, Egypt *Correspondence:
Eman Abdel Razek Hafez
emanhafez@mans.edu.eg; emanhafez22@gmail.com
1 Department of Rheumatology and Rehabilitation, Faculty of Medicine,
Mansoura University, Mansoura, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Mansoura
University, Mansoura, Egypt
3 Department of Forensic and Toxicology, Faculty of Medicine, Mansoura
University, Mansoura, Egypt
4 Department of Microbiology, Faculty of Medicine, Mansoura University,
Mansoura, Egypt KOA is a complex disease that affects nearly 10% of indi-
viduals aged 55 years old or more, with the character-
istic degradation of articular cartilage, often ending as
joints disability [1]. OA has many associated risk factors,
including age, obesity, trauma, diet, smoking habits, and
hormone therapy [2–4]. The pathogenicity of OA is still
unrecognized and requires more explanation. Abstract Background The pro-inflammatory cytokine, interleukin 16 (IL-16), has been shown to be secreted in low levels in
knee osteoarthritis (KOA). The aim of the study was to examine the relationship between IL-16 polymorphisms and
the risk of KOA in the Egyptian population, as well as the clinical and radiographic severity of KOA. Results IL16 rs11556218 thymidine triphosphate (T) T G (guanosine triphosphate), GG, TG + GG genotypes, and G
allele (odd ratio (OR) = 0.315; 95% confidence interval (CI) = 0.191–0.518; P < 0.001; OR = 0.363; 95% CI = 0.162–0.815,
P = 0.014; OR = 0.323; 95% CI = 0.202–0.519, P < 0.001; OR = 0.480; 95% CI = 0.338–0.683, P < 0.001 respectively);
rs4778889 cytidine triphosphate (C) T,CC, TC + CC genotypes, and C allele (OR = 0.519, 95% CI = 0.319–0.844,
P = 0.008; OR = 0.309, 95% CI = 0.105–0.916, P = 0.034; OR = 0.485, 95% CI = 0.304–0.775, P = 0.002; OR = 0.537, 95%
CI = 0.365–0.791, P = 0.001 respectively); and rs4072111 CT, TT, CT + TT genotypes, and T allele (OR = 0.537, 95%
CI = 0.323–0.893, P = 0.017, OR = 0.316, 95% CI = 0.096–0.843, P = 0.049, OR = 0.502, 95% CI = 0.309–0.816, P = 0.005;
OR = 0.534, 95% CI = 0.353–0.809, P = 0.004 respectively) were associated with a decreased KOA risk, and they were
significantly associated with decreased the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and
the Kellgren-Lawrence (K/L) scores. Neither IL-16 serum levels nor IL-16 polymorphisms were associated with the susceptibility to KOA. Low KOA risk was
associated with the haplotypes GTC and TCT. Conclusion There was no correlation between serum IL-16 levels and KOA susceptibility or IL-16 polymorphisms. GTC and TCT haplotypes were associated with low KOA risk. The variant alleles rs11556218GG, TG + GG; rs4778889 CC,
TC + CC; and rs4072111 TT, CT + TT were associated with a reduced risk of KOA. Keywords Knee osteoarthritis, IL16, Single nucleotide polymorphism Genetic polymorphisms of interleukin‑16
in Egyptian patients with primary knee
osteoarthritis Eman Abdel Razek Hafez1*, Reham Magdi Shaat1, Ola Mohamed Gharbia1, Shereen Aly Machaly1,
Ola Ali El‑ Emam2, Nermin Youssef Abo El –Kheir2, Narmin Saied2, Alaa Abo Nour2, Sherif Elkhanishy3,
Rasha Hassan4 and Heba El Shehawy2 Exclusion criteria
A Autoimmune or systemic inflammatory diseases such
as rheumatoid arthritis, systemic lupus erythematosus,
spondyloarthropathies, gouty arthritis, and septic arthri-
tis, patients with previous traumatic knee injury, and all
secondary KOA were excluded from the study. IL-16 is a CD4-specific ligand. Thus, it selectively
activates CD4 + T cells, macrophages, monocytes, and
eosinophils by binding with the molecule CD4 [12]. In
addition, IL-16 can increase the production of inflam-
matory cytokines, such as TNF-α, IL-1β, IL-6, and IL-15,
leading to inflammatory response [19, 20]. sInclusion criteriah The diagnosis of KOA was assessed according to the
American College of Rheumatology clinical criteria (clin-
ical and radiographic) [23]. Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 Page 2 of 9 Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 as on clinical and radiologic severity in patient with KOA
in the Egyptian population. Pathogenesis of synovitis and cartilage destruction
related to OA can be attributed to the role of inflamma-
tory cytokines [5, 6]. Individual variability of cytokine
concentrations can explain the susceptibility variations
and disease severity which in turn are basically attributed
to single nucleotide polymorphisms (SNPs) in cytokine
encoding genes [7].l Study subjectsh This is a case–control study conducted on 150 patients
with the diagnosis of primary KOA consecutively
selected from the Rheumatology and Rehabilitation
outpatient clinic at the Mansoura University hospital,
between March 2016 and April 2017, and 150 controls
selected from healthy volunteers without evidence of OA,
visiting the same hospital for regular check-up. Approval
of the study by the institutional research board, Man-
soura University, Faculty of Medicine, was received (code
R/16.05.39). A written informed consent was obtained
from all participants. Pro-inflammatory cytokines, including TNF, IL-1β,
IL-6, IL-8, IL-15, IL-17, IL-18, and IL-21, were previ-
ously involved in OA pathophysiology [8]. For example,
in a study in 2019, IL-15 has been shown to be related to
the severity of KOA [9]. In addition, another study inves-
tigated the level of IL 33 in RA in comparison with OA
but concluded that there was no relation between IL33
level and OA [10]. IL-16 had not been considered to be
involved in OA until 2015, when two Chinese papers
reported that some IL-16 gene polymorphisms had an
impact on KOA susceptibility [11, 12].l IL-16 is a cytokine having a pro-inflammatory features
including chemoattraction and modulation of T cell acti-
vation [13] and is a crucial mediator in inflammatory
and autoimmune diseases in addition to growth and pro-
gression of tumor [14, 15]. The IL-16 gene is located on
chromosome 15q26.3 [16] and is initially translated into
a precursor protein consisting of 631 amino acids, which
is cleaved by caspase-3 to make the active C-terminal
domain containing 121 amino acids [17, 18].ih Data collection and clinical examination l
Many IL-16 gene SNPs have been thoroughly studied. A prevalent SNP in IL-16 gene is rs4778889 T/C, situ-
ated 295 bp upstream from the start site of transcription
and linked with modified levels of gene expression. Two
additionally SNPs, rs11556218 T/G and rs4072111 C/T,
are situated in an exon region, and their single-nucleotide
changes would result in an amino acid replacement; the
first leads to an asparagine (Asn) to lysine (Lys) replace-
ment in exon 6 of the IL-16 gene, and the second results
in a serine (Ser) to proline (Pro) replacement [21].i Baseline clinical data was achieved through interview-
ing the included participants, demographic characteris-
tics, history of associated medical conditions, review of
systems, and history of knee trauma or surgery. Full gen-
eral and local musculoskeletal examination with stress
on local knee examination for diagnosis and assessment
of KOA was performed. Weight (kg) measurement was
performed on a calibrated scale, and a stadiometer was
used to measure standing height. Body mass index (BMI)
was calculated as weight in kilograms divided by height
in meters squared (kg/m2). IL16 has been identified to be secreted from OA syno-
vial fibroblasts at low concentrations [22]. For that and
during the OA inflammatory process, IL-16 might be a
prospective mediator. Since the IL-16 production could
be genetically controlled, it is reasonable to hypothesize
a potential relationship between IL-16 gene polymor-
phisms and KOA risk.h Restriction digestion and gel electrophoresis [26] Restriction digestion and gel electrophoresis [26]
Restriction endonucleases BsmAI, NdeI, and AhdI
were used for rs4072111C/T, rs11-556218 T/G, and
rs4778889T/C, respectively. 1.2 μL of the restriction
endonuclease was required for the digestion of 10 μL of
PCR amplification product, and each product was treated
in a 37° C water bath for 16 h. The resultant product was
subjected to electrophoresis through running on 2% aga-
rose gel and then imaged. For genotype verification, Gen-
eray Biotech sequenced the amplified and digested DNA
products. Primer design and PCR amplification [25] Primer design and PCR amplification [25]
Primer sequences for each SNP were as follows:
rs4778889T/C:
5′-CCATGTCAAAACGGTAGCCT-
CAAGC-3′ and 5′-CTCCACA CTCAAAGCCTTTTG-
TTCCTATGA-3′ rs4072111C/T: 5′-TTCAGGTAC-AAACC
CAGCCAGC-3′ and 5′-CAC TGTG ATC CCGGTC
CAGTC-3′
rs11556218T/G:
5′-TGTGA-CAATCACAG
CTTGCCTG-3′ and 5′-GCTCAGGTTC-ACAGAGTGTTT
CCATA-3′ Primer design and PCR amplification [25]
Primer sequences for each SNP were as follows:
rs4778889T/C:
5′-CCATGTCAAAACGGTAGCCT-
CAAGC-3′ and 5′-CTCCACA CTCAAAGCCTTTTG-
TTCCTATGA-3′ rs4072111C/T: 5′-TTCAGGTAC-AAACC
CAGCCAGC-3′ and 5′-CAC TGTG ATC CCGGTC
CAGTC-3′
rs11556218T/G:
5′-TGTGA-CAATCACAG
CTTGCCTG-3′ and 5′-GCTCAGGTTC-ACAGAGTGTTT
CCATA-3′ Evaluation of OA Evaluation of OA
For the evaluation of pain, stiffness, and function
from the Western Ontario and Mc-Master University
(WOMAC), OA index scoring was used in patients with
OA [24]. Total WOMAC is the sum of the results of the
three subscales. WOMAC of high rating indicates more
pain and stiffness and serious restriction of function. The objective of this research was to investigate the
association of IL-16 polymorphisms with susceptibility to
KOA and the impact of SNPs on IL16 serum levels as well Page 3 of 9 Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 DNA extraction and measurement [11] For analyzing DNA, a peripheral 2 mL of venous blood
was obtained from each participant, and an anticoagu-
lant (EDTA-Na2) was added; the sample was stored at
– 20 °C. DNA extraction was done by QIAGEN kit for
extraction, and the OD 260/280 ratio was measured by
spectrophotometer. Good DNA purity could be consid-
ered with an OD 260/280 ratio between 116 and 210,
with the inclusion of the sample in the study. A tempera-
ture of – 70 °C was used for DNA storage. Sample size consideration For the evaluation of the statistical power of the sam-
ple, the CaTS software (Center for Statistical Genetics,
University of Michigan [29]) was used. The power of the
sample size was calculated to be 80% regarding the next
circumstances: 150 patients, 150 healthy controls, the
prevalence of disorder is 12%, average allelic frequency of
39.7%, significance level of 0.05. A volume of 25 μL was used in PCR amplification reac-
tions. The total volume contains 1.0 μL of template DNA,
12.5 μL hot start master mix, 1.0 μL of forward primers,
1.0 μL of reverse primer, and 11.3 μL ionized water.h Laboratory assessment storage of the total serum was carried out at – 20 °C until
its further use. For detection of serum IL-16 concentra-
tions, a sandwich enzyme-linked immunosorbent assay
(ELISA) was used. ELISA with the same batch of reagents
was used according to the manufacturer’s instructions. The minimum concentration of detection for IL-16 was
5 pg/mL with 10% intra-assay coefficients of variation. Statistical analysis
ll
d d y
Collected data was reviewed, coded, tabulated, and intro-
duced to a PC using the Statistical Package for the Social
Sciences (IBM SPSS Version 20.0.). Data was presented
and analyzed according to the nature of data obtained for
each parameter. For parametric numerical data, mean and
standard deviation (± SD) were selected, while median
and range were used for non-parametric numerical data
and frequency and percentage of non-numerical data. Normality of data distribution was tested by using the
Kolmogorov–Smirnov test. The Student T test was car-
ried out for assessing the statistical significance of the
mean difference between two study groups. The statistical
significance of the difference of a non-parametric variable
between two study groups was assessed by Mann–Whit-
ney test (U test). For comparison between more than two
study group ordinal variables, the Kruskal–Wallis test was
used to assess the statistical significance. Chi-square test
was carried out for examining the relationship between
two qualitative variables. Significance of the results
obtained was judged at P ≤ 0.05. Fisher’s exact test was
used when the expected count is less than 5 in more than
20% of cells for examining the relationship between two
qualitative variables. Deviations from Hardy–Weinberg
equilibrium expectations were determined using the chi-
square test. Odds ratio and 95% confidence interval were
calculated. Linear regression analysis was conducted for
prediction of confounders. The HaploView program (ver-
sion 4.2) was applied for the estimation of the haplotypes The conditions of polymerase chain reaction-restric-
tion fragment length (PCR) reaction at rs11556218T/G
were as follows: denaturation of samples was at 95 °C for
5 min, then its procession for 30 denaturation cycles at
95 °C for 45 s, annealing at 60 °C for 45 s and its exten-
sion at 72 °C for 1 min, and ending with a final cycle of
extension at 72 °C for 5 min. The rs4072111C/T and
rs4778-889 T/C annealing temperatures were 67 °C and
63 °C, respectively. Radiological assessment Plain radiographs of both knees were obtained for every
patient in a semiflexed weight bearing anteroposterior
and lateral radiographs views. For grading of OA, the
Kellgren-Lawrence (KL) score was used [28]. Results Demographic data, IL16, WOMAC, and K/L score of
KOA patients are shown in Table 1. The mean age of OA
cases was 49.5 years; they were 19 males and 131 females. Healthy controls had younger age and matched gender
and BMI compared to OA cases. The serum level of IL16
was insignificantly different between KOA cases and
healthy controls. The median WOMAC was 60; median
K/L score was 2. The association between rs11556218 genotypes with
clinical and radiographic features of KOA patients is pre- The association between rs11556218 genotypes with
clinical and radiographic features of KOA patients is pre-
sented in Table 4. rs11556218GG, TG + GG; rs4778889 CC, TC + CC;
and rs4072111 TT, CT + TT genotypes were signifi-
cantly associated with lower WOMAC and Kellgren. Serum IL16 did not differ significantly between different
genotypes. rs11556218GG, TG + GG; rs4778889 CC, TC + CC;
and rs4072111 TT, CT + TT genotypes were signifi-
cantly associated with lower WOMAC and Kellgren. Serum IL16 did not differ significantly between different
genotypes. The comparison of IL16 SNPs between KOA patients
and the control group is presented in Table 2. This sample
of participants was randomly selected from a population
in Dakahleya Governorate in lower Delta, Egypt. Apply-
ing Hardy–Weinberg equation indicated that rs11556218,
rs4778889, and rs4072111 genotypes in the control group
as well as in the case group were in the Hardy–Weinberg
equilibrium. rs11556218 TG, GG, TG + GG genotypes,
and G allele; rs4778889 CT, CC, CT + CC genotypes, and
C alleles; and rs4072111 CT, TT, CT + TT genotypes,
and T allele showed significant lower frequency in KOA
when compared to the control groups, with significantly
protective effect against OA development among healthy
control subjects. Regression analysis was conducted for prediction of
factors affecting IL16 level, using age, gender, and BMI
as confounders. None was associated with prediction of
IL16 level in Table 5. Linkage disequilibrium (LD) between rs11556218 and
rs4778889 in the control was 40%; in OA, it was 78%;
LD between rs11556218 and rs4072111 in the control
was 19%; in OA, it was 69%; LD between rs4778889
and rs4072111 in the control was 1%; in OA, it was 67%
(Fig. 1). Serum IL‑16 levels [27] Serum samples were collected from both patients and
healthy controls. After blood sampling, clotting of the
serum was allowed for 30 min at 4 °C before being cen-
trifugated at 3000 rpm for 10 min at 4 °C. Isolation and Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 Page 4 of 9 and linkage disequilibrium (LD), through using the expec-
tation maximization (EM) algorithm. The code of colors
in the HaploView plot follows the standard color scheme
for HaploView: white, |D′|< 1, LOD < 2; shades of pink/
red, |D′|< 1, LOD ≥ 2; blue, |D′|= 1, LOD < 2; red, |D′|= 1,
LOD ≥ 2. and linkage disequilibrium (LD), through using the expec-
tation maximization (EM) algorithm. The code of colors
in the HaploView plot follows the standard color scheme
for HaploView: white, |D′|< 1, LOD < 2; shades of pink/
red, |D′|< 1, LOD ≥ 2; blue, |D′|= 1, LOD < 2; red, |D′|= 1,
LOD ≥ 2. chromosome 15. The application of the HaploView pro-
gram (version 4.2) was performed to estimate the hap-
lotypes and linkage disequilibrium (LD), which uses the
expectation maximization (EM) algorithm. TTC hap-
lotype showed highest frequency, while TCC showed
lowest frequency in cases and control. Taking TTC hap-
lotype as a reference, GTC and TCT haplotypes showed
significantly lower frequency in OA patients when com-
pared to the control group, with significantly protective
effect against OA development within healthy control
subjects. NB. (Logarithm of likelihood odds ratio [LOD]; (a
measure of confidence in the value of D′). Discussion Table 2 Comparison of IL16 SNPs between OA patients and controls
HW p Hardy–Weinberg p value, OR Odds ratio, CI Confidence interval
* Significance P ≤ 0.05
Genotype
Groups
Control
N = 150
Cases
N = 150
P
OR
95% CI
N
%
N
%
rs11556218
TT
49
32.7
90
60.0
-
1
(reference)
TG
83
55.3
48
32.0
* < 0.001
0.315
0.191–0.518
GG
18
12.0
12
8.0
*0.014
0.363
0.162–0.815
TG + GG
101
67.3
60
40.0
* < 0.001
0.323
0.202–0.519
T
181
60.3
228
76.0
-
1
(reference)
G
119
39.7
72
24.0
* < 0.001
0.480
0.338–0.683
HW p
0.056
0.133
rs4778889
TT
75
50.0
101
67.3
-
1
(reference)
CT
63
42.0
44
29.3
*0.008
0.519
0.319–0.844
CC
12
8.0
5
3.3
*0.034
0.309
0.105–0.916
TC + CC
75
50.0
49
32.7
*0.002
0.485
0.304–0.775
T
213
71.0
246
82.0
-
1
(reference)
C
87
29.0
54
18.0
*0.001
0.537
0.365–0.791
HW p
0.807
0.938
rs4072111
CC
87
58.0
110
73.3
-
1
(reference)
CT
53
35.3
36
24.0
*0.017
0.537
0.323–0.893
TT
10
6.7
4
2.7
*0.049
0.316
0.096–0.843
CT + TT
63
42.0
40
26.7
*0.005
0.502
0.309–0.816
C
227
75.7
256
85.3
-
1
(reference)
T
73
24.3
44
14.7
*0.004
0.534
0.353–0.809
HW p
0.619
0.614
Table 3 Analysis of rs11556218, rs4778889, and rs4072111 haplotypes in OA cases and controls
OR Odds ratio, CI Confidence interval
* Significance P ≤ 0.05
Haplotype
rs11556218
rs4778889
rs4072111
Control
Cases
p
OR
95% CI
TTC
T
T
C
0.401
0.688
-
1
(reference)
GTC
G
T
C
0.267
0.092
* < 0.001
0.282
0.177–0.449
TCT
T
C
T
0.155
0.044
* < 0.001
0.250
0.132–0.474
GCT
G
C
T
0.047
0.062
0.398
1.381
0.679–2.809
GCC
G
C
C
0.046
0.058
0.519
1.227
0.594–2.537
GTT
G
T
T
0.037
0.028
0.558
0.810
0.331–1.983
TCC
T
C
C
0.043
0.016
*0.051
0.374
0.132–1.063 Table 2 Comparison of IL16 SNPs between OA patients and controls
HW p Hardy–Weinberg p value, OR Odds ratio, CI Confidence interval
* Significance P ≤ 0.05
Genotype
Groups
Control
N = 150
Cases
N = 150
P
OR
95% CI
N
%
N
%
rs11556218
TT
49
32.7
90
60.0
-
1
(reference)
TG
83
55.3
48
32.0
* < 0.001
0.315
0.191–0.518
GG
18
12.0
12
8.0
*0.014
0.363
0.162–0.815
TG + GG
101
67.3
60
40.0
* < 0.001
0.323
0.202–0.519
T
181
60.3
228
76.0
-
1
(reference)
G
119
39.7
72
24.0
* < 0.001
0.480
0.338–0.683
HW p
0.056
0.133
rs4778889
TT
75
50.0
101
67.3
-
1
(reference)
CT
63
42.0
44
29.3
*0.008
0.519
0.319–0.844
CC
12
8.0
5
3.3
*0.034
0.309
0.105–0.916
TC + CC
75
50.0
49
32.7
*0.002
0.485
0.304–0.775
T
213
71.0
246
82.0
-
1
(reference)
C
87
29.0
54
18.0
*0.001
0.537
0.365–0.791
HW p
0.807
0.938
rs4072111
CC
87
58.0
110
73.3
-
1
(reference)
CT
53
35.3
36
24.0
*0.017
0.537
0.323–0.893
TT
10
6.7
4
2.7
*0.049
0.316
0.096–0.843
CT + TT
63
42.0
40
26.7
*0.005
0.502
0.309–0.816
C
227
75.7
256
85.3
-
1
(reference)
T
73
24.3
44
14.7
*0.004
0.534
0.353–0.809
HW p
0.619
0.614 Table 2 Comparison of IL16 SNPs between OA patients and controls HW p Hardy–Weinberg p value, OR Odds ratio, CI Confidence interval
* Significance P ≤ 0.05 Table 3 Analysis of rs11556218, rs4778889, and rs4072111 haplotypes in OA cases and controls
OR Odds ratio, CI Confidence interval
* Significance P ≤ 0.05
Haplotype
rs11556218
rs4778889
rs4072111
Control
Cases
p
OR
95% CI
TTC
T
T
C
0.401
0.688
-
1
(reference)
GTC
G
T
C
0.267
0.092
* < 0.001
0.282
0.177–0.449
TCT
T
C
T
0.155
0.044
* < 0.001
0.250
0.132–0.474
GCT
G
C
T
0.047
0.062
0.398
1.381
0.679–2.809
GCC
G
C
C
0.046
0.058
0.519
1.227
0.594–2.537
GTT
G
T
T
0.037
0.028
0.558
0.810
0.331–1.983
TCC
T
C
C
0.043
0.016
*0.051
0.374
0.132–1.063 layers of the cartilage, allowing the degradation imbal-
ance to continue near the synovial border in the deep
layer. Discussion Different inflammatory elements are engaged in OA
pathologically. In OA, there is a loss of enhanced
chondrocyte anti-inflammatory and synthetic activ-
ity due to enhanced degrading activity [30, 31]. The
enhanced synthetic activity is restricted to the far deep A comparison between rs11556218, rs4778889, and
rs4072111 haplotypes in OA cases and controls is shown
in Table 3. We performed the current analyses with
data derived from the chromosomal region 15q25.1 on Table 1 Demographic data, IL16, and clinical and radiological features in OA cases and control groups
BMI Body mass index, WOMAC Western Ontario and McMaster Universities Arthritis Index, K/L score Kellgren-Lawrence
* Significance P ≤ 0.05
Data
Groups
Control
N = 150
Cases
N = 150
P
Age
Mean ± SD
39.6 ± 8.7
49.5 ± 9.1
* < 0.001
Sex
Males
N (%)
20 (13.3%)
19 (12.7%)
0.864
Females
N (%)
130 (86.7%)
131 (87.3%)
BMI (kg/m2)
Mean ± SD
31.2 ± 4.7
31.8 ± 4.8
0.327
IL16 (pg/mL)
Median (range)
213.5 (29.3–455.7)
200.5 (23.7–484.5)
0.147
WOMAC
Median (range)
-
60 (8–96)
-
K/L score
Median (range)
-
2 (1–4)
- Table 1 Demographic data, IL16, and clinical and radiological features in OA cases and control groups Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 Page 5 of 9 layers of the cartilage, allowing the degradation imbal-
ance to continue near the synovial border in the deep
layer. In the end, malfunction and apoptosis of chon-
drocyte restrict the capacity for reaction and acceler-
ate OA progression [30, 31]. Therefore, we postulated that the susceptibility to OA
may be modulated by IL-16 polymorphisms. In our study, there were no significant differences
between OA cases and healthy controls as regards sex
and BMI, but the mean age was significantly older in
OA cases (P < 0.001). However, none of these factors was
associated with prediction of IL16 level as demonstrated
by multivariate regression analysis. This makes the rela-
tion between OA and IL16 more reliable. Discussion Egyptian Rheumatology and Rehabilitation (2023) 50:12 Page 6 of 9 Table 4 Association of IL16 level, WOMAC, and K/L score with IL16 genotypes
P1 comparison between rs11556218 TT, TG, GG, P2 comparison between rs11556218 TG + GG and TT, P3 comparison between rs4778889 TT, TC, CC, P4 comparison
between rs4778889 TC + CC and TT, P5 comparison between rs4072111 CC, CT, TT, P6 comparison between CT + TT and CC
* Significance P ≤ 0.05
Genotypes
Variables
IL16
WOMAC
Kellgren
Median
Range
Median
Range
Median
Range
rs11556218
TT (N = 90)
210.8
55.3–484.5
71
53–96
3
2–4
TG (N = 48)
155.9
23.7–418.4
44
30–53
2
1–2
GG (N = 12)
274
55.7–460.4
25
8–30
1
1–1
TG + GG (N = 60)
155.9
23.7–460.4
43
8–53
2
1–2
P1
0.328
* < 0.001
* < 0.001
P2
0.338
* < 0.001
* < 0.001
rs4778889
TT (N = 101)
207
23.7–484.5
69
8–96
2.5
11–4
TC (N = 44)
189.1
29.6–424.4
43
22–56
2
1–2
CC (N = 5)
291.9
96.9–460.4
21
8–53
1
1–2
TC + CC (N = 49)
194
29.6–460.4
43
8–56
2
1–2
P3
0.593
* < 0.001
* < 0.001
P4
0.890
* < 0.001
* < 0.001
rs4072111
CC(N = 110)
203.7
23.7–484.5
67.5
29–96
2.5
1–4
CT(N = 36)
227.1
29.6–424.4
36.5
19–56
2
1–2
TT(N = 4)
123.9
96.9–150.8
30.5
8–53
1.5
1–2
CT + TT(N = 40)
155.9
29.6–424.4
36.5
8–56
2
1–2
P5
0.298
* < 0.001
* < 0.001
P6
0.971
* < 0.001
* < 0.001 Table 4 Association of IL16 level, WOMAC, and K/L score with IL16 genotypes P1 comparison between rs11556218 TT, TG, GG, P2 comparison between rs11556218 TG + GG and TT, P3 comparison between rs4778889 TT, TC, CC, P4 comparison
between rs4778889 TC + CC and TT, P5 comparison between rs4072111 CC, CT, TT, P6 comparison between CT + TT and CC
*i In this study, the prevalent IL-16 SNP namely
rs4778889 and two other SNPs (rs4072111 and
rs11556218) were chosen for the assessment of their
association with KOA patients and healthy controls. Discussion In the end, malfunction and apoptosis of chon-
drocyte restrict the capacity for reaction and acceler-
ate OA progression [30, 31].h layers of the cartilage, allowing the degradation imbal-
ance to continue near the synovial border in the deep
layer. In the end, malfunction and apoptosis of chon-
drocyte restrict the capacity for reaction and acceler-
ate OA progression [30, 31].h In our study, there were no significant differences
between OA cases and healthy controls as regards sex
and BMI, but the mean age was significantly older in
OA cases (P < 0.001). However, none of these factors was
associated with prediction of IL16 level as demonstrated
by multivariate regression analysis. This makes the rela-
tion between OA and IL16 more reliable. Therefore, we postulated that the susceptibility to OA
may be modulated by IL-16 polymorphisms. Hafez et al. Discussion Table 5 Regression analysis for prediction of factors affecting
IL16 level
Β regression coefficient
* Significance P ≤ 0.05
Variables
Β
P
Age
1.426
0.141
Sex
3.500
0.521
BMI
1.586
0.289 Our study revealed that participants carrying the
rs11556218 G allele showed lower risk to KOA devel-
opment in comparison with those carrying T allele. Also, the participants carrying rs4778889 C allele
were less prone to develop OA than those carrying T Fig. 1 Linkage disequilibrium between (1) rs11556218, (2) rs4778889, and (3) rs4072111 in A control and B OA groups Fig. 1 Linkage disequilibrium between (1) rs11556218, (2) rs4778889, and (3) rs4072111 in A control and B OA groups Hafez et al. Egyptian Rheumatology and Rehabilitation (2023) 50:12 Page 7 of 9 Page 7 of 9 Page 7 of 9 Page 7 of 9 allele. Individuals carrying rs4072111 T allele were less
prone to develop KOA than those carrying C allele. These results goes with the study of Luo and his col-
leagues [12], who studied the same genes in a Chinese
population. In our study, the serum levels of IL-16 did not show any
significant differences between OA patients with differ-
ent genotypes and the control group. Similarly, in a study
by Gupta et al. [31] on 90 Malaysian patients, no signifi-
cant difference was found between the serum levels of
IL16 in the healthy controls and KOA patients, suggest-
ing the probability of other influences on IL-16 concen-
trations apart from genetics. This can be explained by the
relatively small sample size; more data from additional
patients will provide further confirmation which would
be provided by additional patient data. allele. Individuals carrying rs4072111 T allele were less
prone to develop KOA than those carrying C allele. These results goes with the study of Luo and his col-
leagues [12], who studied the same genes in a Chinese
population. In contrast to our study, Liu et al. [11] reported an
association between rs4072111 CT and the increased risk
of OA, but rs11556218 TC genotype was associated with
reduced risk. This discrepancy could be explained by the
differences in ethnic origins of the studied populations,
the presence of other SNPs within IL-16 gene associ-
ated with the susceptibility to OA, or variable exposure
to other risk factors, such as tobacco smoking and heavy
drinking in the former two studies. Luo et al. Discussion [12] revealed that serum IL16 levels were
higher in KOA patients than in controls but not associ-
ated with IL16 gene polymorphisms. Those results go
with our study except for serum levels of IL16 which
were not significantly different from the control group. This discrepancy in the results might be attributed to the
lower exposure to risk factors, such as tobacco smoking
and heavy alcohol drinking in the former two studies. A haplotype is recognized as a collection of SNPs on a sin-
gle chromatid that is probably considered inherited together
more frequently than previously proposed by chance in a
block pattern due to existing linkage disorder [32].hi This study revealed that there is a significant associa-
tion between GTC and TCT haplotypes and the reduced
risk of KOA. The limitation of the studyh In contrast to our study, Luo et al. [12] found that GCC
and TTT haplotypes were significantly associated with
elevated risk of KOA. Also, Liu and his colleagues [11]
found that TTT haplotype was associated with increased
risk, while the GCC haplotype was associated with
decreased risk of KOA. Three limitations of this study should be addressed. First,
the relatively small size of our patients makes the power
of study statistics insufficient. Therefore, further studies
on larger samples are needed. Second is the study pop-
ulation confined to the Mansoura city population; thus,
the findings may not be universal to other populations. More studies on other ethnic groups would be valuable. Third, only three SNPs in the IL-16 gene were investi-
gated. Identifications of more SNPs and studying their
association with KOA would be interesting. When we studied linkage disequilibrium, there was LD
between rs11556218 and rs4778889 which was higher in
KOA patients; LD between rs11556218 and rs4072111
was higher in KOA, but LD between rs4778889 and
rs4072111was higher in the control group. Liu et al. [11]
found linkage disequilibrium between rs4778889 SNP
and rs11556218 which were similar to our results. Conclusionh There is a relationship between IL16 polymorphisms and
the risk of KOA. We found that IL16 rs11556218 TG,
GG, TG + GG genotypes, and G allele; rs4778889 CT,
CC, CT + CC genotypes, and C alleles; and rs4072111
CT, TT, CT + TT genotypes, and T allele are associated
with significant lower risk of KOA. Serum levels of IL-16
is not associated with the susceptibility to KOA. The hap-
lotypes GTC and TCT are associated with low KOA risk. The variant alleles rs11556218GG, TG + GG; rs4778889
CC, TC + CC; and rs4072111 TT, CT + TT are associated
with lower WOMAC and K/L score. There is no asso-
ciation between IL-16 polymorphisms and IL-16 serum
levels. When we studied the association between IL16 SNPs
and clinical and radiographic data, only rs11556218 GG,
TG + GG, rs4072111 TT, and CT + TT were significantly
associated with lower WOMAC and K/L scores which
denote that individuals carrying these genotypes could
be at less susceptibility to develop severe KOA than other
genotypes. In the study of Luo et al. [12] for evaluation of the
impact of IL-16 polymorphisms on KOA risk stratified
by sex, they found that there is a clear strong associa-
tion in female patient subgroups, but they could not find
an explanation and revealed that this finding was unex-
pected, which could be explained by higher number of
female patients compared to males, those genes located
on chromosome 15 and not linked to sex chromosomes,
or might also be the result of estrogen-related effects;
estrogen can interact with IL-16 and reduce the possibil-
ity of developing KOA. Abbreviations
KOA
Knee osteoarthritis
IL-16
Interleukin 16
WOMAC
Western Ontario and McMaster Universities Arthritis Index
K/L scores Kellgren-Lawrence
PCR
Polymerase chain reaction-restriction fragment length
ELISA
Enzyme-linked immunosorbent assay Abbreviations
KOA
Knee osteoarthritis
IL-16
Interleukin 16
WOMAC
Western Ontario and McMaster Universities Arthritis Index
K/L scores Kellgren-Lawrence
PCR
Polymerase chain reaction-restriction fragment length
ELISA
Enzyme-linked immunosorbent assay Abbreviations
KOA
Knee osteoarthritis
IL-16
Interleukin 16
WOMAC
Western Ontario and McMaster Universities Arthritis Index
K/L scores Kellgren-Lawrence
PCR
Polymerase chain reaction-restriction fragment length
ELISA
Enzyme-linked immunosorbent assay Consent for publication
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Adenosine triphosphate
C
Cytidine triphosphate
G
Guanosine triphosphate
T
Thymidine triphosphate
LD
Linkage disequilibrium
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doi.org/10.1371/journal.pone.0184802 31. Publisher’s Note
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USO DE SEMENTES DE ACEROLA COMO BIOSSORVENTE PARA REMOÇÃO DE Cr(VI) DE SOLUÇÕES AQUOSAS
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1Universidade Federal de Sergipe, Departamento de Engenharia Química
E-mail para contato: jessik_cristian@hotmail.com 1Universidade Federal de Sergipe, Departamento de Engenharia Química
E-mail para contato: jessik_cristian@hotmail.com RESUMO – Este trabalho teve como objetivo avaliar o potencial de utilização de
sementes de acerola (Malpighia emarginata) na adsorção de Cr(VI) de soluções aquosas. Na obtenção do biossorvente foi utilizado tratamento térmico à temperatura de 170 °C e,
em seguida, o biossorvente foi triturado. A determinação de Cr(VI) foi feita por
espectrofotometria UV-visível através da complexação com 1,5-difenilcarbazida, medindo
a absorbância no comprimento de onda de 540 nm. Foram realizados experimentos
variando o tempo de contato e o pH inicial. A eficiência da remoção de íons de Cr(VI) foi
avaliada por espectroscopia de infravermelho. A remoção média do biossorvente utilizado
foi de 66%. 2.1. Preparação do biossorvente As sementes de polpas de frutas foram cedidas pela indústria Pomar do Brasil Indústria e
Comércio de Alimentos Ltda, localizada no bairro Distrito Industrial, em Aracaju-SE. As sementes
foram coletadas em sacos plásticos e levadas ao Laboratório de Química Industrial (LQI) da
Universidade Federal de Sergipe, onde foram devidamente lavadas com água para eliminação de
restos de polpa. As sementes foram secas em mufla (GP Científica) a 170 °C, sendo em seguida
trituradas em um processador de alimentos. A granulometria do biossorvente foi feita utilizando
peneiras de 4, 9, 12, 32, 100 e 200 mesh e classificador granulométrico da Bertel. 2.2. Determinação de acidez 1,0 g do pó do biossorvente 12 mesh foi misturado em 50 mL de água destilada. A mistura foi
agitada utilizando agitador magnético durante 24 horas. Logo após, a mistura foi filtrada, sendo 10
mL do filtrado titulado com NaOH 0,1 mol L-1, utilizando fenolftaleína como indicador segundo
procedimento do Instituto Adolfo Lutz (2008). através da Resolução N° 20, de 18 de julho de 1986, estabeleceu os valores máximos permissíveis de
alguns metais em que para Cr(III) e Cr(VI) são, respectivamente, 1,0 mg L-1 e 0,5 mg L-1. através da Resolução N° 20, de 18 de julho de 1986, estabeleceu os valores máximos permissíveis de
alguns metais em que para Cr(III) e Cr(VI) são, respectivamente, 1,0 mg L-1 e 0,5 mg L-1. Diante do exposto, este trabalho buscou avaliar a potencialidade do uso de sementes de acerola
na remoção de Cr(VI). O estudo foi realizado em batelada visando identificar o percentual de
remoção e o comportamento cinético da adsorção de Cr(VI) pelo biossorvente. 1. INTRODUÇÃO A acerola (Malpighia emarginata) tem grande importância nutricional por ser fonte natural de
vitamina C. Aliado ao aspecto nutricional e funcional do fruto, a acerola apresenta uma elevada
produção e é bastante utilizada na região Nordeste do Brasil na fabricação de polpa de fruta, sorvete e
suco, apresentando forte potencial para industrialização. Ressalta-se que as cascas e as sementes
possuem pouco aproveitamento, contribuindo desta forma para a geração de resíduos sólidos (MELO
et al., 2009). Os processos convencionais para remoção de metais pesados apresentam custos elevados o que
vem favorecendo o aparecimento de novas tecnologias e métodos de redução/eliminação dos
poluentes. Uma dessas alternativas é a biossorção que consiste na remoção de poluentes dos efluentes
utilizando materiais de origem biológica, que após podem ser utilizados, por exemplo, em fornos de
cimentos como forma de tratamento do resíduo. Este processo pode torna-se vantajoso, uma vez que
combina boa eficiência de remoção dos poluentes com custos de implantação e operação reduzidos
(PINA, 2011). O cromo hexavalente, Cr(VI), é tóxico e carcinogênico por causa do elevado potencial de
oxidação e por sua elevada capacidade de penetrar em membranas biológicas. Excessiva exposição ao
Cr(VI) pode causar várias doenças, incluindo danos ao fígado, rins, sistema circulatório, tecidos
nervosos e sistema sanguíneo (MELO, 2008). Atualmente, existem fortes restrições governamentais
relativamente à deposição de águas residuais. O Conselho Nacional do Meio Ambiente (CONAMA) 1 1 Área temática: Engenharia Ambiental e Tecnologias Limpas através da Resolução N° 20, de 18 de julho de 1986, estabeleceu os valores máximos permissíveis de
alguns metais em que para Cr(III) e Cr(VI) são, respectivamente, 1,0 mg L-1 e 0,5 mg L-1. O estudo do efeito do tempo de contato na remoção de Cr(VI) foi realizado na concentração
inicial de Cr(VI) 5 mg L-1, quantidade de biossorvente 0,1 g (12 mesh); temperatura 25 ± 1°C e pH 2. A concentração de Cr(VI) adsorvido foi medida na faixa de tempo de 10 a 140 min. O estudo da influência do pH na remoção do Cr(VI) foi realizada com ajuste do pH inicial da
solução em 2, 5, 7 e 8, concentração de Cr(VI) de 5 mg L-1 , massa do biossorvente 0,1 g (12 mesh) e
tempo de equilíbrio de 120 min. 2.3. Determinação de Cr(VI) A solução concentrada de Cr(VI) foi preparada a partir de dicromato de potássio (Merk)
dissolvendo 2,829 g em 1000 mL de água destilada, formando assim, uma solução de 1000 mg L-1 de
Cr(VI). Foram obtidas, por diluições, soluções nas concentrações padrões de 0,2; 0,4; 0,6; 0,8 e 1,0
mg L-1 para determinação da curva de calibração, segundo procedimento de determinação de Cr(VI)
por complexação 1,5-difenilcarbazida em meio ácido (Morita e Assumpção, 2007). A absorbância foi
medida no espectrofotômetro (UV-VIS da VARIAN) no comprimento de onda igual a 540 nm. A
concentração de Cr(VI) após a adsorção foi determinada usando o valor da absorbância da amostra. A
porcentagem de cromo hexavalente removida foi determinada pela Equação 1. (1)
100
x
C
)
C
(C
Cr(VI)
de
Remoção
%
i
f
i
(1) Área temática: Engenharia Ambiental e Tecnologias Limpas
2 2 Área temática: Engenharia Ambiental e Tecnologias Limpas
2 Área temática: Engenharia Ambiental e Tecnologias Limpas 2 2.5. Espectroscopia de absorção na região do infravermelho (FTIR) As amostras foram analisadas na forma de pastilhas de KBr utilizando o equipamento Nicolet,
modelo i-10 FTIR, com acessório SMART-OMNI, na região compreendida entre 400 e 4000 cm-1
com resolução de 4 cm-1. 2.4. Cinética de adsorção Integrando a Equação 4 nos moldes da Equação 2 e linearizado-a tem-se a Equação 5: Integrando a Equação 4 nos moldes da Equação 2 e linearizado-a tem-se a Equação 5: Área temática: Engenharia Ambiental e Tecnologias Limpas
3 Área temática: Engenharia Ambiental e Tecnologias Limpas 3 (5)
Q
1
1
t
e
2
2
e
t
Q
k
Q (5) De acordo com Weber e Moris se a difusão intrapartícula for fator determinante da velocidade
de adsorção, os dados cinéticos experimentais devem seguir a Equação 6: t
k
Q
2
1
dif
t
C
(6) sendo Qt a quantidade de Cr(VI) adsorvidade (mmols g-1), t o tempo de adsorção (min), C (mmols g-1)
uma constante relacionada coma resistência à difusão intrapartícula e kdif é o coeficiente de difusão
intrapartícula (mmols g-1 min-1/2). Este modelo fornece informações sobre os mecanismos de
transferência de massa envolvidos no processo de adsorção, por exemplo, se ocorre difusão
intrapartícula , se há ou não formação de camada limite externa. 2.4. Cinética de adsorção Vários modelos cinéticos são utilizados para examinar o mecanismo controlador do processo de
adsorção, tais como, reação química, controle da difusão e transferência de massa. Contudo, os
modelos empregados com maior frequência são o pseudoprimeira ordem e o pseudosegunda ordem. O
modelo pseudoprimeira ordem é representado pela Equação 2:
Q
k
dt
dQ
e
1
t
t
Q
(2) em que k1 é a constante de adsorção de pseudoprimeira ordem (min-1), Qe e Qt são as quantidades
adsorvidas por grama de biossorvente no equilíbrio e no tempo t, respectivamente (mmols g-1). Integrando a Equação 1 de Qt = 0 em t=0 a Qt=Qe em t=t obtém-se a Equação 3: em que k1 é a constante de adsorção de pseudoprimeira ordem (min-1), Qe e Qt são as quantidades
adsorvidas por grama de biossorvente no equilíbrio e no tempo t, respectivamente (mmols g-1). Integrando a Equação 1 de Qt = 0 em t=0 a Qt=Qe em t=t obtém-se a Equação 3:
(3)
t
k
lnQ
Q
Q
ln
1
e
t
e
t
k
lnQ
Q
Q
ln
1
e
t
e
(3) O valor de k1 e de Qe não experimental podem ser obtidos do gráfico ln(Qe-Qt) versus t. O valor de k1 e de Qe não experimental podem ser obtidos do gráfico ln(Qe-Qt) versus t. O modelo pseudosegunda ordem é representado na Equação 4: O modelo pseudosegunda ordem é representado na Equação 4:
(4)
Q
k
dt
dQ
2
e
2
t
t
Q
Q
k
dt
dQ
2
e
2
t
t
Q
(4) (4) em que k2 é a constante da taxa de adsorção de pseudosegunda ordem (g mg-1 min-1). em que k2 é a constante da taxa de adsorção de pseudosegunda ordem (g mg-1 min-1). 3. RESULTADOS E DISCUSSÃO A acidez média de 12% obtida para as sementes de acerola é decorrente de ácidos carboxílicos. A Figura 1 do espectro FTIR do pó de sementes de acerola indicam a presença destes ácidos no
biomaterial. As análises FTIR foram realizadas no biossorvente antes e depois da adsorção. Nota-se
pelo espectro que provavelmente todos os grupos da superfície do biomaterial estiveram envolvidos
na adsorção, havendo aumento na transmitância no comprimento de onda dos grupos identificados,
talvez em decorrência da interação com a espécie metálica. As bandas largas e fortes em 3450,2 e
3416,1 cm-1 foram atribuídas ao grupo hidroxila (-OH), provavelmente de ácidos carboxílicos, o que
pode indicar que este último grupo foi fortemente envolvido na biossorção (BARBOSA, 2008). 4 4 Área temática: Engenharia Ambiental e Tecnologias Limpas Figura 1: Espectro FTIR para o biossorvente (pó de acerola), sendo (a) antes da adsorção
e (b) após à adsorção. Figura 1: Espectro FTIR para o biossorvente (pó de acerola), sendo (a) antes da adso Figura 1: Espectro FTIR para o biossorvente (pó de acerola), sendo (a) antes da adsorção
e (b) após à adsorção. O pico em 2927,9 cm-1 indica o estiramento de ligações C-H de alcanos ou grupo alquila
(BARBOSA, 2008). As bandas de 1634,8 e 1626,6 cm-1 foram atribuídas à vibração da carbonila
(C=O) de ácidos carboxílicos (BARBOSA, 2008). Os picos em 1078,4 e 1044,2 cm-1 são referentes ao
estiramento de ligações C-O, provavelmente de ácidos carboxílicos (SILVERSTEIN et al., 2005). Nota-se que, após à adsorção, surgiu um novo pico de 2362,6 cm-1 que pode indicar a presença de
interações Cr-CO (SHUKLA, 2012). A Figura 2 mostra que o tempo de equilíbrio foi de 120 minutos, sendo este o tempo de contato
utilizado para se determinar qual o melhor pH inicial para a remoção de Cr(VI). O equilíbrio na
remoção de Cr(VI) pode ser explicado pela utilização de todo os sítios ativos do biomaterial. Durante
a adsorção, o cromo hexavalente se impregna na superfície do adsorvente até que toda a superfície
esteja coberta de íons do metal. Neste ponto a concentração de Cr(VI) deixa de diminuir e se mantém
em equilíbrio com o adsorvente em solução. Com isso, o tempo de equilíbrio da adsorção varia de
acordo com a superfície do adsorvente utilizado (RAMOS et al., 2013). 5 5 Área temática: Engenharia Ambiental e Tecnologias Limpas Figura 2: Efeito do tempo de contato na remoção de Cr(VI). 3. RESULTADOS E DISCUSSÃO Condições: concentração inicial
de Cr(VI) 5 mg L-1;volume do meio, 100 mL; quantidade de adsorvente, 0,1 g; temperatura
25 ± 1 °C; velocidade de agitação de 150 rpm; pH 2 para o pó da semente de acerola. Figura 2: Efeito do tempo de contato na remoção de Cr(VI). Condições: concentração inicial
de Cr(VI) 5 mg L-1;volume do meio, 100 mL; quantidade de adsorvente, 0,1 g; temperatura
25 ± 1 °C; velocidade de agitação de 150 rpm; pH 2 para o pó da semente de acerola. O pH é um dos parâmetros que interferem no potencial de adsorção. O pH afeta não só a carga
superficial de biossorvente, mas também o grau de ionização durante a reação. No pH inicial 2,0
houve remoção de 66% de Cr(VI) da solução aquosa, enquanto no pH inicial 5, 7 e 8 não houve
remoção. A literatura mostra resultados semelhantes na remoção de metais em pH ácido, a exemplo
de folhas de Araucária secas (SHUKLA, 2012), casca de nozes (ALTUN e PEHLIVAN, 2012),
carvão ativado granular (SOUZA et al., 2009), casca de coco (PINO, 2005), algas marinhas (PINA,
2012), raízes de jacinto e folhas de nim (SINGHA e DAS, 2010), pó de quiabo (CONCEIÇÃO et al.,
2014), casca e polpa de laranja Osage (PEHLIVAN, 2012). No estudo cinético, verificou-se que o melhor ajuste foi utilizando o modelo pseudosegunda
ordem (Figura 3), em que se obteve R2= 0,9823, valor de k2 de 0,3958 g mmols-1 min-1 e Qe calculado
de 0,0787 mmols g-1. Utilizando o modelo de adsorção de Weber e Moris obteve-se R2 igual a 0,9200,
kdif de 0,0063 mmols g-1 min-1/2 e C igual a 0,0018 mmols g-1. Como o valor de C foi diferente de
zero e os dados experimentais pouco se ajustaram ao modelo intrapartícula de Weber e Morris,
sugere-se que o mecanismo de transferência de massa durante a adsorção do Cr(VI) provavelmente é 6 Área temática: Engenharia Ambiental e Tecnologias Limpas 6 dominado pela formação de uma camada limite externa (Rocha et al., 2012), não excluindo contudo
outros mecanismos. dominado pela formação de uma camada limite externa (Rocha et al., 2012), não excluindo contudo
outros mecanismos. dominado pela formação de uma camada limite externa (Rocha et al., 2012), não excluindo contudo
outros mecanismos. Figura 3: Cinética de adsorção pseudosegunda ordem (Equação 5). Figura 3: Cinética de adsorção pseudosegunda ordem (Equação 5). Figura 3: Cinética de adsorção pseudosegunda ordem (Equação 5). Área temática: Engenharia Ambiental e Tecnologias Limpas 5. REFERÊNCIAS ALTUN, T.; PEHLIVAN, E.: Removal of Cr(VI) from aqueous solutions by modified walnuts shells. Food Chemistry, Vol.132(2), pp.693-700, 2012. BARBOSA, L. C. A.; Espectroscopia no infravermelho na caracterização de compostos orgânicos. Editora UFV, 1° reimpressão. Viçosa, MG: UFV, 2008. CONAMA, Resolução N° 20, de 18 de julho de 1986, in: MOTA, S.: Preservação e Conservação de
Recursos Hídricos. Rio de Janeiro: ABES, 1995. Ã CONCEIÇÃO, J. C.; RAMOS, V. H. S.; JESUS, E.; SILVA, A. S.; COSTA, A. W. M. C.: Biosortion
of Cr(VI) from Aqueous Solutions Using Chemically Modified Okra Powder. Journal of Basic
& Applied Sciences, 10, 73-79, 2014. INSTITUTO ADOLFO LUTZ. Normas analíticas do Instituto Adolfo Lutz. v. 1: Métodos químicos e
físicos para análise de alimentos, IV edição, 1ª Edição Digital. São Paulo, 2008. P. 25-26. MELO, K. R. O.; Estudo da digestão de sedimento de curtume visando a extração de cromo por
microemulsão. Tese (Doutor em Engenharia Química). Natal, RN: UFNR, 2008. Ú MELO, E. A.; CAETANO, A. C. S.; LIMA, V. L. A. G.; MACIEL, M. I. S.; ARAÚJO, C. R.:
Extração de antioxidantes de resíduos agroindustriais de acerola. Brazilian Journal Food
Technology, v. 12, n. 2, p. 155-160, abr./jun. 2009. MORITA, T.; ASSUMPÇÃO, R. M. V.; Manual de soluções, reagentes e solventes: padronização,
preparação, purificação com indicadores de segurança e de descarte de produtos químicos. São Paulo: Editora Blucher, 2007. PEHLIVAN, E.; PEHLIVAN, E.; TUTAR, H.K.; Hexavalent chromium removal by Osage Orange. Food Chemistry, Vol.133(4), p. 1478-1484, 2012. PINA, F. D. S.; Tratamento de águas contaminadas com crómio(VI) por bioadsorção em algas
marinhas. 2011. 65f. Tese (Mestre em Engenharia do Ambiente), Faculdade de Engenharia da
Universidade do Porto, Porto, Portugal, 2011. PINO, G. A. H.; Biossorção de metais pesados utilizando pó da casca de coco cerde (Cocos
nucifera). Tese (Mestre em Engenharia Metalúrgica). Rio de Janeiro, RJ: PUC-Rio, 2005. Ã RAMOS, V. H. S.; CONCEIÇÃO, J. C.; JESUS, E.; MARQUES, J. J.; SILVA, A. S.; SILVA, D. C.:
Remoção de cromo hexavalente por biossorção usando pó de quiabo. XXXVI Congresso
Brasileiro de Sistemas Particulados. Universidade Federal de Alagoas. Maceió-AL, 2013. g
ROCHA, O. R. S.; NASCIMENTO, G. E.; CAMPOS, N. F.; SILVA, V. L.; DUARTE, M. M. M. B.:
Avaliação do processo adsortivo utilizando mesocarpo de coco verde para remoção do corante
cinza reativo BF-2R. Quim. Nova, Vol. 35, No. 7, 1369-1374, 2012. 4. CONCLUSÃO Considerando os resultados apresentados, pode ser concluído que a remoção de Cr(VI) aumenta
com o aumento do tempo de contato entre adsorvente e adsorvato até atingir um tempo de equilíbrio,
sendo este 120 minutos para as sementes de acerola. A maior remoção de Cr(VI) foi de 66%
utilizando 0,1 g de biossorvente (12 mesh) com pH inicial igual a 2. Verificou-se uma cinética
rápida, sendo que em 120 min foi atingido equilíbrio. A transferência de massa durante a adsorção foi
dominada pela formação de camada limite externa. Por se tratar de material de baixo custo, considerado como resíduo da agroindústria, as sementes
de acerola apresentam-se como uma alternativa viável no tratamento de contaminação por Cr(VI). 7 7 Área temática: Engenharia Ambiental e Tecnologias Limpas 5. REFERÊNCIAS SHUKLA, D.; VANKAR, P.; Efficient biossorption of chromium(VI) ion by dry Araucaria leaves. Environmental Science and Pollution Research, Vol.19(6), pp.2321-2328, 2012. SILVERSTEIN, R. M.; WEBSTER, F. X.; KIEMLE, D. J.: Spectrometric identification of organic
compouds. John Wilwy & Sons, Seventh Edition. State University of New York, 2005. SINGHA, M., DAS, S.; Cr(VI) adsorption – mechanistic approach for biosorption from aqueous
solutions. The Bioscan: Special issue, Vol. 2; 509-514. Índia, 2010. p
SOUZA, R. S.; CARVALHO, S. M. L.; GARCIA JÚNIOR, M. L. R.; SENA, R. S. F.; Adsorção de
cromo (VI) por carvão ativado granular de soluções diluídas utilizando um sistema batelada sob
pH controlado. ACTA Amazonica, vol. 39(3), 661 – 668, 2009. p
SOUZA, R. S.; CARVALHO, S. M. L.; GARCIA JÚNIOR, M. L. R.; SENA, R. S. F.; Adsorção de
cromo (VI) por carvão ativado granular de soluções diluídas utilizando um sistema batelada sob
pH controlado. ACTA Amazonica, vol. 39(3), 661 – 668, 2009. 8 Área temática: Engenharia Ambiental e Tecnologias Limpas 8
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Cardinal invariants of strongly porous sets
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Journal of logic and analysis
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cc-by
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Journal of Logic & Analysis 9:6 (2017) 1–16
ISSN 1759-9008 1 Cardinal Invariants of Strongly Porous Sets OSVALDO GUZM´AN
MICHAEL HRUˇS´AK
ARTURO MART´INEZ-CELIS Abstract: In this work we study cardinal invariants of the ideal SP of strongly
porous sets on ω2. We prove that add(SP) = ω1, cof(SP) = c and that it is
consistent that non(SP) < add(N ), answering questions of Hruˇs´ak and Zindulka. We also find a connection between strongly porous sets on ω2 and the Martin
number for σ-linked partial orders, and we use this connection to construct a model
where all the Martin numbers for σ-k-linked forcings are mutually different. 2010 Mathematics Subject Classification 03E17, 28A75 (primary); 03E35, 03E50
(secondary) Keywords: porous sets, cardinal invariants, ideals, Martin number 1
Introduction In 1967, Dolˇzenko [5] began the study of σ-porous sets1 and since then, many
applications have been found. One of these appears in [10], where Preiss and Zaj´ıˇcek
proved that, given a Banach space X with a separable dual and a continuous convex
function f on X, the set of points in which f is not Fr´echet differentiable is σ-porous. Other applications can be found in Belna, Evans and Humke [2], Lindenstrauss and
Preiss [7], Lindenstrauss, Preiss and Tiˇser [8], Reich and Zaslavski [11], Renfro [12]
and Zaslavski [19]. We shall study the notion of strong porosity: given a metric space ⟨X, d⟩, a subset
A ⊆X is strongly porous if there is a p > 0 such that for any x ∈X and any 0 < r < 1,
there is y ∈X such that Bp·r(y) ⊆Br(x) \ A. In this paper we will refer to strongly
porous sets as porous sets. We shall work mostly with porous sets in ω2: we say that a
set A ⊆ω2 is n-porous if for every s ∈<ω2 there is a t ∈n2 such that ⟨s⌢t⟩∩A = ∅. By s⌢t we denote the concatenation of s followed by t, and by ⟨s⟩we denote the cone
of s, that is ⟨s⟩= {f ∈ω2 : s ⊆f}. It is easy to see (see Hruˇs´ak and Zindulka [6]) that 1A σ-(upper/lower) porous set is the countable union of (upper/lower) porous sets lished: December 2017
doi: 10.4115/jla.2017.9.6 doi: 10.4115/jla.2017.9.6 Published: December 2017 2 Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis 2 a set A ⊆ω2 is porous if and only if there is an n ∈ω such that A is n-porous. Zaj´ıˇcek
[17] proved that a set A in a metric space ⟨X, d⟩is σ-porous if and only if it is σ-lower
porous, where A is lower porous if for every x ∈X there exist ρx > 0 and rx > 0 such
that for any 0 ≤r ≤rx there is y ∈X such that Bρx·r(y) ⊆Br(x) \ A. Another classical
notion of porosity is upper porosity: a set A in a metric space ⟨X, d⟩is upper porous if
for every x ∈X there is ρx > 0 and a sequence rn →0 such that for every n ∈ω there
is yn ∈X such that Bρx·rn(yn) ⊆Brn(x) \ A. It is easy to see that lower porosity implies
upper porosity. 2σ-ideals are ideals closed under countable unions of their elements. 1
Introduction We will denote the σ-ideal2 generated by porous sets on ω2 by SP, the σ-ideal
generated by n-porous sets by SPn, and the σ-ideal generated by upper porous sets
by UP. Observe that SP1 is the ideal of countable sets of ω2. Further research about
different types of porosity can be found in Rojas-Rebolledo [16], Zaj´ıˇcek [17], Zapletal
[18], Zelen´y [20] and Zelen´y and Zaj´ıˇcek [21]. Cardinal invariants of these σ-ideals have been studied in Brendle [3], Hruˇs´ak and
Zindulka [6] and Repick´y [13, 14, 15]. Recall that, given a σ-ideal I over a set X, the
following are the standard cardinal invariants of I: add(I)
=
min{|A| : A ⊆I ∧S A /∈I}
cov(I)
=
min{|A| : A ⊆I ∧S A ̸= X}
non(I)
=
min{|Y| : Y ⊆X ∧Y /∈I}
cof(I)
=
min{|A| : A ⊆I ∧∀B ∈I (∃A ∈A (B ⊆A))} In [6], Hruˇs´ak and Zindulka proved that the cardinal invariants of the σ-ideal of lower
porous sets in the real line are the same as the cardinal invariants of SP. They proved
that non(SP) < mσ-centered is consistent, that cov(SP) > cof(N ) is consistent, and that
cov(SP) < c is consistent, where mσ-centered is the smallest cardinal where the Martin’s
axiom for σ-centered forcings fails and N is the ideal of sets of Lebesgue measure
zero. In [6], Hruˇs´ak and Zindulka proved that the cardinal invariants of the σ-ideal of lower
porous sets in the real line are the same as the cardinal invariants of SP. They proved
that non(SP) < mσ-centered is consistent, that cov(SP) > cof(N ) is consistent, and that
cov(SP) < c is consistent, where mσ-centered is the smallest cardinal where the Martin’s
axiom for σ-centered forcings fails and N is the ideal of sets of Lebesgue measure
zero. There are some analogous inequalities that hold for UP. In [15], M. Repick´y proved
that non(UP) ≥mσ-centered and cov(UP) ≤cof(N ) holds. He proved in [13] that
non(UP) ≥add(N ) and in [3], J. Brendle proved that add(UP) = ω1 and cof(UP) = c
hold. In [6], Hruˇs´ak and Zindulka asked if the last three inequalities hold also for the SP
ideal. In this work we show that add(SP) = ω1, cof(SP) = c and that it is consistent
that non(SP) < add(N ). Given k ∈ω and a forcing notion P a subset A ⊆P is k-linked if for every collection
{ai : i ∈k} of k elements of A, there is an a ∈P such that for every i ∈k, a ≤ai. P 1
Introduction Given k ∈ω and a forcing notion P a subset A ⊆P is k-linked if for every collection
{ai : i ∈k} of k elements of A, there is an a ∈P such that for every i ∈k, a ≤ai. P Journal of Logic & Analysis 9:6 (2017) 3 3 Cardinal Invariants of Strongly Porous Sets is σ-k-linked if P is the countable union of k-linked subsets of P. We will denote by
mk the Martin number for σ-k-linked forcings, that is, the smallest cardinal κ such
that there is a σ-k-linked forcing P and κ many P-dense subsets of P such that no
filter of P intersects them all. In [4], Brendle and Shelah constructed a model where all
the Martin numbers of the form m2i are distinct. We will show a connection between
σ-k-linked forcings and porous sets and we will use this connection to construct a
model where all the Martin numbers mi are different all at once. If X, Y are sets, then YX is the set of all functions from Y to X and <ωX = S
n∈ω
nX. If σ, s ∈<ωX, then we will denote that σ is an initial segment of s by σ ⊑s. A set
T ⊆<ωX is a tree if it is closed under restrictions to initial segments. If T ⊆<ωX is a
tree, then by [T] we denote the set of branches of T, that is, [T] = {f ∈ωX : ∀n ∈
ω ( f↾n ∈T)}. With end(T) we will denote the end nodes of T , that is the nodes of T
without extensions. In our forcing notation, the stronger conditions are the smaller ones. In general, our notation follows Bartoszy´nski and Judah [1]. 2
Additivity and cofinality The main goal of this section is to prove that add(SP) = ω1 and cof(SP) = c. We will
use the following notion. Definition 2.1 Let k ∈ω. A tree T ⊆<ω2 is a k-porous tree if for every s ∈<ω2
there is t ∈k2 such that s⌢t /∈T . Note that A ⊆ω2 is k-porous if and only if there is a k-porous tree T such that [T]
contains A. Theorem 2.2 There is a family {Tf : f ∈ω2} of 2-porous trees such that for every
X ∈SP, the set {f ∈ω2 : [Tf ] ⊆X} is countable. Theorem 2.2 There is a family {Tf : f ∈ω2} of 2-porous trees such that for every
X ∈SP, the set {f ∈ω2 : [Tf ] ⊆X} is countable. Proof We will construct the family {Tf : f ∈ω2} as follows: For every a ⊆<ω2
such that |a| = 2n, let ϕa : a →n2 be a bijective function. For every i ∈ω, let
ψi : {a ⊆i2 : ∃k ∈ω (|a| = 2k)} →ω \ {0} be an injective function. If a ⊆i2 and
|a| = 2k, define σa = ⟨0, 1, . . . , 1
| {z }
2ψi(a) times
, 0⟩. Journal of Logic & Analysis 9:6 (2017) Journal of Logic & Analysis 9:6 (2017) Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis 4 4 For each σ ∈<ω2, we will recursively define a finite tree Tσ as follows: T∅= {∅},
and if Tσ is defined then Tσ⌢i ={s ∈<ω2 : ∃t ∈end(Tσ) (∃j ∈ω (∃a ⊆|σ|+12
(|a| = 2j ∧σ⌢i ∈a ∧s ⊑t⌢σ⌢
a ϕa(σ⌢i))))}
∪{s ∈<ω2 : ∃t ∈end(Tσ) (s ⊑t⌢⟨1, 1⟩)}. It is easy to see that, for each σ ∈<ω2, Tσ is a finite 2-porous tree and that if σ ⊑τ ,
then Tσ ⊆Tτ . For each f ∈ω2, define Tf = S
n∈ω Tf↾n. It follows easily that each Tf
is a 2-porous tree. We will show that the family {Tf : f ∈ω 2} is the family we were looking for: Let
X ∈SP. Without loss of generality we will assume that X = S
i∈ω[Ti], where Ti
is an i + 1-porous tree. 2
Additivity and cofinality We must show that the set B = {f ∈ω2 : [Tf ] ⊆X} is
countable: For each s, t ∈<ω2 and each n ∈ω, define Bs,t,n = {f ∈ω2 : t ⊑f, s ∈
Tt ∧[Tf ] ∩⟨s⟩⊆[Tn]}. We will see that B ⊆S
s,t∈<ω2,n∈ω Bs,t,n : If f is such that
f ∈B, then [Tf ] ⊆S
n∈ω[Tn]. Using the Baire Category Theorem we can find s ∈Tf
and n ∈ω such that [Tf ] ∩⟨s⟩⊆[Tn]. Find k ∈ω such that s ∈Tf↾k. It follows that
f ∈Bs,f↾k,n. To finish the proof we will see that each Bs,t,n has at most 2n+1 −1 elements:
Suppose this is not the case and let s, t ∈<ω2, n ∈ω and {fi}i<2n+1 ⊆Bs,t,n. Extend s
to σ such that σ ∈end(Tt). Let j ∈ω be such that the set a = {fi↾j : i < 2n+1} has
2n+1 elements and let 2n+1 elements and let s0 = σ⌢⟨1, . . . , 1
| {z }
2·(j−|t|−1) times
⟩⌢σa. The tree Tn is n + 1-porous, so there is a τ ∈2n+1 such that s⌢
0 τ /∈Tn. Find k < 2n+1
such that ϕa( fk↾j) = τ and observe that s⌢
0 τ = s⌢
0 ϕa( fk↾j) ∈Tfk . As a consequence,
[Tfk] ∩⟨s⟩⊈[Tn], but this contradicts the fact that fk ∈Bs,t,n. This implies that each
Bs,t,n is finite, and therefore B is countable. We can now prove the main result of this section. We can now prove the main result of this section. Corollary 2.3
add(SP) = ω1, cof(SP) = c. Corollary 2.3
add(SP) = ω1, cof(SP) = c. Proof Let {Tf : f ∈ω2} be the family given by the theorem above. If H ⊆ω2 is an
uncountable set, then S{[Tf ] : f ∈H} /∈SP. As a consequence, add(SP) = ω1. On
the other hand, if κ < c and if {Xα : α < κ} ⊆SP, then there is an f ∈ω2 such that,
for every α < κ, [Tf ] ⊈Xα and therefore cof(SP) = c. Journal of Logic & Analysis 9:6 (2017) 5 5 5 Cardinal Invariants of Strongly Porous Sets Observe that this last proof can be used to show that add(SPn) = ω1 = add(SP) and
cof(SPn) = c = cof(SP). Observe that this last proof can be used to show that add(SPn) = ω1 = add(SP) and
cof(SPn) = c = cof(SP). 3
Uniformity number In this section we will study some properties concerning the uniformity number of
porosity ideals. We will prove the consistency of non(SP) < add(N ). We will also
develop some tools that we will use later in the paper. We will need the following
concept, inspired by the concept of a k-Sacks tree in <ωk. Definition 3.1 Let k > 1. A tree T ⊆<ωk is a k-anti-Sacks tree if for every s ∈T
there is i < k such that s⌢⟨i⟩/∈T. We will denote by ASk the σ-ideal generated by
the branches of k-anti-Sacks trees. This notion is the analogue of the notion of 1-porous tree in <ωk and it is closely related
to the k-Sacks forcing. Recall that a k-Sacks tree T is a tree on <ωk such that for every
s ∈T, there is a t ∈T such that, for every i < k, t⌢i ∈T. The k-Sacks forcing Sk is
the collection of all k-Sacks trees ordered by reverse inclusion. It is well-known that the
k-Sacks forcing is equivalent to Borel(ωk)/ASk (see Newelski and Rosłanowski [9]). Using a similar argument to the one we gave in the last section, it is possible to show
that add(ASk) = ω1 and that cof(ASk) = c. Alternatively, a proof of this fact can be
found in [9]. The ideals SPk and AS2k share many properties. Many of the results in this work will
concern properties of the ideals ASk that are also valid for the ideals SPk, and the
proofs for both ideals are almost the same. Whenever we state a property about one of
these ideals that is also valid for the other one, we will only give the proof for the ideal
ASk. We shall introduce a notion that we will use to keep the uniformity number small in a
forcing extension. Definition 3.2 Let P be a forcing notion and let A ⊆ωk (A ⊆ω2) be such that
A /∈ASk (A /∈SPk). We say that P strongly preserves non(ASk) in A (P strongly
preserves non(SPk) in A) if for every P-name
. X of a k-anti-Sacks tree (k-porous tree)
there is a Y ∈ASk (Y ∈SPk) such that, for every x ∈A, if x /∈Y then P ⊩“x /∈[
. X]”. We will say that P strongly preserves non(ASk) (P strongly preserves non(SPk)) if P
strongly preserves non(ASk) in ωk (P strongly preserves non(SPk) in ω2). 3
Uniformity number Definition 3.2 Let P be a forcing notion and let A ⊆ωk (A ⊆ω2) be such that
A /∈ASk (A /∈SPk). We say that P strongly preserves non(ASk) in A (P strongly
preserves non(SPk) in A) if for every P-name
. X of a k-anti-Sacks tree (k-porous tree)
there is a Y ∈ASk (Y ∈SPk) such that, for every x ∈A, if x /∈Y then P ⊩“x /∈[
. X]”. We will say that P strongly preserves non(ASk) (P strongly preserves non(SPk)) if P
strongly preserves non(ASk) in ωk (P strongly preserves non(SPk) in ω2). Journal of Logic & Analysis 9:6 (2017) 6 Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis 6 It is easy to see that, if P strongly preserves non(ASk) in A, then P ⊩“A /∈ASk” and
if P strongly preserves non(ASk), then P strongly preserves non(ASk) in A for every
A ⊆ωk. The following lemma is easy to prove. It is easy to see that, if P strongly preserves non(ASk) in A, then P ⊩“A /∈ASk” and
if P strongly preserves non(ASk), then P strongly preserves non(ASk) in A for every
A ⊆ωk. The following lemma is easy to prove. Lemma 3.3 Suppose that a forcing notion P strongly preserves non(SPk) for every
k ∈ω, then P ⊩“ω2 ∩V /∈SP”. Proof The proof is straightforward from the definitions. Proof The proof is straightforward from the definitions. Proof The proof is straightforward from the definitions. Proof The proof is straightforward from the definitions. The next lemma shows that there is a connection between anti-Sacks trees and σ-k-linked
forcings. Lemma 3.4 Let P be a forcing notion. If P is σ-k-linked, then P strongly preserves
non(ASk). Lemma 3.4 Let P be a forcing notion. If P is σ-k-linked, then P strongly preserves
non(ASk). Proof Let {Pi : i ∈ω} be a sequence of k-linked subsets such that P = S
i∈ω Pi. Let
. A be a P-name of a k-anti-Sacks tree. Define Tn ⊆ωk as follows: Proof Let {Pi : i ∈ω} be a sequence of k-linked subsets such that P = S
i∈ω Pi. Let
. A be a P-name of a k-anti-Sacks tree. Define Tn ⊆ωk as follows: Tn = {s ∈<ωk : ∃p ∈Pn(p ⊩“s ∈
. A”)} Tn = {s ∈<ωk : ∃p ∈Pn(p ⊩“s ∈
. A”)} We claim that, for each n ∈ω, Tn is a k-anti-Sacks tree. Suppose this is not the case,
so there is an s ∈Tn such that, for every i ∈k, s⌢i ∈Tn. For every i ∈k, we can pick
a condition pi ∈Pn such that pi ⊩“s⌢i ∈
. A”. Let p ∈P be such that, for every i ∈k,
p ≤pi. Then p ⊩“∀i ∈k(s⌢i ∈
. A)”. This contradicts the fact that
. A is a P-name of a
k-anti-Sacks tree. To conclude the proof, note that for every x ∈ωk, if p ⊩“x ∈[
. A]”, then x ∈[Tn],
where n is such that p ∈Pn. The lemma above is optimal in the sense that, for each k, there is a σ-(k −1)-linked
forcing Pk such that Pk ⊩“ωk ∩V ∈ASk” and therefore Pk does not strongly preserve
ASk. This will be proved in the next section. There is also a relation between porous
sets in ω2 and σ-k-linked forcings. Lemma 3.5 Let P be a forcing notion. If P is σ-2k-linked, then P strongly preserves
non(SPk). Proof The proof is similar to the previous lemma. Proof The proof is similar to the previous lemma. Proof The proof is similar to the previous lemma. Lemma 3.6 Let A ⊆ωk and I ∈{SPn, ASk : n > 0, k > 1}. Tn is a Pn name for a k-anti-Sacks tree. Now we use that
each Pn strongly preserves non(ASk) to find a family {Tj
i : i, j ∈ω} such that, for each
n ∈ω, if x ∈A and x /∈S
i∈ω[Tn
i ], then Pn ⊩“x /∈[
. Tn]”. It is easy to see that the set
Y = S{[Tj
i] : i, j ∈ω} is the set we are looking for. We will now prove the consistency of non(SP) < add(N ). For constructing the model
we are looking for, we will use the amoeba forcing A in the following presentation: A = {B ∈Borel(ω2) : µ(B) > 1
2} Here Borel(ω2) represents the collection of Borel subsets of the Cantor space and µ is
the standard Lebesgue measure over ω2. The order is given by A ≤B if and only if
A ⊆B. The following lemma is well-known (see eg Bartoszy´nski and Judah[1]). We
include the simple proof for the sake of completeness. Here Borel(ω2) represents the collection of Borel subsets of the Cantor space and µ is
the standard Lebesgue measure over ω2. The order is given by A ≤B if and only if
A ⊆B. The following lemma is well-known (see eg Bartoszy´nski and Judah[1]). We
include the simple proof for the sake of completeness. Lemma 3.7 The amoeba forcing is σ-n-linked for every n ∈ω. Lemma 3.6 Let A ⊆ωk and I ∈{SPn, ASk : n > 0, k > 1}. Lemma 3.6 Let A ⊆ωk and I ∈{SPn, ASk : n > 0, k > 1}. (1) If P is a forcing notion such that P strongly preserves non(I) in A and
. Q is
a P-name for a forcing such that P ⊩“
. Q strongly preserves non(I) in A”, then
P ∗
. Q strongly preserves non(I) in A. (1) If P is a forcing notion such that P strongly preserves non(I) in A and
. Q is
a P-name for a forcing such that P ⊩“
. Q strongly preserves non(I) in A”, then
P ∗
. Q strongly preserves non(I) in A. (2) If ⟨Pα,
. Qα : α ≤γ⟩is a finite support iteration of c.c.c. forcings such that
Pα ⊩“
. Qα strongly preserves non(I) in A” for each α ∈γ, then Pγ strongly
preserves non(I) in A. (2) If ⟨Pα,
. Qα : α ≤γ⟩is a finite support iteration of c.c.c. forcings such that
Pα ⊩“
. Qα strongly preserves non(I) in A” for each α ∈γ, then Pγ strongly
preserves non(I) in A. Proof We will only show the case when I = ASk as the other cases are similar. The
part (1) is easy, we will proceed with part (2) by induction on γ. It is easy to see that
the lemma holds for successor ordinals, and if γ has uncountable cofinality we can
use a standard reflection argument to show that Pγ strongly preserves non(ASk) in A,
so it is enough to show that the lemma holds for γ = ω. Let
. T be a Pω-name of a
k-anti-Sacks tree. For each n ∈ω, let
. Tn be a Pn-name for the following set: . Tn = {s ∈<ωk : P(n,ω) ⊩“s ∈
. T”} . Tn = {s ∈<ωk : P(n,ω) ⊩“s ∈
. T”} It is easy to see that each
. Tn is a Pn name for a k-anti-Sacks tree. Now we use that
each Pn strongly preserves non(ASk) to find a family {Tj
i : i, j ∈ω} such that, for each
n ∈ω, if x ∈A and x /∈S
i∈ω[Tn
i ], then Pn ⊩“x /∈[
. Tn]”. It is easy to see that the set
Y = S{[Tj
i] : i, j ∈ω} is the set we are looking for. It is easy to see that each
. Proof The proof is straightforward from the definitions. Journal of Logic & Analysis 9:6 (2017) 7 7 Cardinal Invariants of Strongly Porous Sets We shall show that strong preservation of non(ASk) and non(SPk) is preserved by finite
support iterations. We shall show that strong preservation of non(ASk) and non(SPk) is preserved by finite
support iterations. Theorem 3.8 If ZFC is consistent, then ZFC + non(SP) < add(N ) is consistent. Theorem 3.8 If ZFC is consistent, then ZFC + non(SP) < add(N ) is consistent. Proof Start with a model V such that V |= CH. Let P = {Pα,
. Qα : α < ω2} be a
finite support iteration of the amoeba forcing. It follows from the lemmas above that P
strongly preserves non(SPk) for every k ∈ω and therefore P ⊩“ω2 ∩V /∈SP”. As a
consequence, we have that V[G] |= non(SP) = ω1. It is a known fact (see [1]) that
V[G] |= add(N ) = ω2, hence V[G] |= non(SP) < add(N ). Lemma 3.7 The amoeba forcing is σ-n-linked for every n ∈ω. Lemma 3.7 The amoeba forcing is σ-n-linked for every n ∈ω. Journal of Logic & Analysis 9:6 (2017) 8 8 Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis Proof Let n ∈ω. For every clopen C in ω2, define Proof Let n ∈ω. For every clopen C in ω2, define AC = {A ∈A : µ(C\A) < 1
n · (µ(C) −1
2)}. We will show that A = S{AC : C is a clopen in ω2}. Let A ∈A and let ε > 0 such
that µ(A) = 1
2 + ε. Find an open set U ⊆ω2 such that A ⊆U and µ(U\A) < ε
n . Now
find a clopen set C ⊆U such that µ(C) > 1
2 + ε. Then µ(C\A) < µ(U\A) < ε
n = 1
n · (1
2 + ε −1
2) < 1
n · (µ(C) −1
2). Therefore A ∈AC. Now we must show that, for every clopen set C ⊆ω2, the
intersection K of an arbitrary family {Aj : j ∈n} ⊆AC is an element of A. This is a
consequence of the following calculations: µ(C) ≤µ(K) +
X
j∈n
µ(C\Aj) < µ(K) + 1
n · (
X
j∈n
µ(C) −1
2) = µ(K) + µ(C) −1
2. As a consequence, 1
2 < µ(K). Therefore K ∈A. We are ready to prove the main result of this section. The method of the proof was
suggested to us by J. Brendle. We are ready to prove the main result of this section. The method of the proof was
suggested to us by J. Brendle. 4
Martin numbers of σ-k-linked forcings It is easy to see that m2 ≤m3 ≤. . . and, for each k > 1, it is possible to get the
consistency of mk < mk+1 by forcing with a finite support iteration of σ-(k + 1)-linked
forcings over a model of CH. In [4], Brendle and Shelah constructed a model where all
the cardinals of the form m2k are different. In this section we will construct a model
where all the Martin numbers mi are different at the same time. In this model, the
cardinals non(ASi) will be different all at once (so will be the cardinals non(SPi)). We
will use the following forcing notions. Given k > 2 let Journal of Logic & Analysis 9:6 (2017) 9 9 Cardinal Invariants of Strongly Porous Sets Pk = {⟨s, F⟩: (a) s is a finite k-anti-Sacks tree of height ht(s),
(b) F ∈[ωk]<ω, and ⌈F↾∆F⌉is a finite k-anti-Sacks tree, and
(c) s ⊆⌈F↾∆F + 1⌉}, Pk = {⟨s, F⟩: (a) s is a finite k-anti-Sacks tree of height ht(s),
(b) F ∈[ωk]<ω, and ⌈F↾∆F⌉is a finite k-anti-Sacks tree, and
(c) s ⊆⌈F↾∆F + 1⌉}, where F↾k = {f↾k : f ∈F}, ⌈F⌉= {σ ∈<ωk : ∃f ∈F (σ ⊆f)} and ∆F = min{n ∈
ω : |F↾n| = |F|}. The order is defined by ⟨s′, F′⟩≤⟨s, F⟩if and only if s ⊆s′ and
F ⊆F′. This forcing notion will be used to work with the ideal ASk. For the ideal SPk,
we will be using a similar forcing notion. Given k > 1 let where F↾k = {f↾k : f ∈F}, ⌈F⌉= {σ ∈<ωk : ∃f ∈F (σ ⊆f)} and ∆F = min{n ∈
ω : |F↾n| = |F|}. The order is defined by ⟨s′, F′⟩≤⟨s, F⟩if and only if s ⊆s′ and
F ⊆F′. This forcing notion will be used to work with the ideal ASk. For the ideal SPk,
we will be using a similar forcing notion. Given k > 1 let Pk = {⟨s, F⟩: (a) s is a finite k-porous tree, and
(b) F ∈[ω2]<ω, and ⌈F↾∆F⌉is a finite k-anti-Sacks tree, and
(c) s ⊆⌈F↾∆F + 1⌉}. The order is defined by ⟨s′, F′⟩≤⟨s, F⟩if and only if s ⊆s′ and F ⊆F′. We will be
using the following proposition. Proposition 4.1 Given a k > 2 and an i > 1, Pk ⊩“ωk ∩V ∈ASk” and
Pi ⊩“ω2 ∩V ∈SPi”. 4
Martin numbers of σ-k-linked forcings Proof We will only check that Pk ⊩“ωk ∩V ∈ASk” as the other part is similar. It is
easy to see that, for every f ∈ωk and n ∈ω, the following sets are dense in P: Df = {⟨s, F⟩∈Pk : ∃σ ∈<ωk (σ⌢f↾(ω \ |σ|) ∈F)}
En = {⟨s, F⟩∈Pk : ∆F > n ∧s = ⌈F↾∆F + 1⌉} If G ⊆Pk is a filter meeting all these dense sets, then, using that the sets En are dense,
it follows that T = S{s : ∃F(⟨s, F⟩∈G)} is a k-anti-Sacks tree. If σ ∈<ωk and if
C[σ] = {σ⌢x↾(ω \ |σ|) : x ∈[T]}, then, using that the Df are dense, it follows that
ωk ∩V ⊆S{C[σ] : σ ∈<ωk} ∈ASk. The last proposition together with the Lemma 3.4 implies that Pk is not σ-k-linked. In
contrast with this last observation, we have the following proposition. Proposition 4.2 For each k > 1, Pk+1 is σ-k-linked and Pk is σ-(2k −1)-linked. Journal of Logic & Analysis 9:6 (2017) 10 Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis Proof Again, we will only check that Pk+1 is σ-k-linked, the other part is done in
a similar way: For any two finite (k + 1)-anti-Sacks trees s, t of height ht(s), ht(t)
respectively, define P(s, t) = {⟨s, F⟩∈Pk+1 : ht(t) > ∆F ∧F↾ht(t) = t}. It is easy to see that Pk+1 = S{P(s, t) : s, t are finite (k + 1)-anti-Sacks trees}. We will
show that every P(s, t) is k-linked. Let {⟨s, Fi⟩: i < k} ⊆P(s, t) and let F = S
i<k Fi. We must show that ⟨s, F⟩∈Pk+1. The properties (a) and (c) are easily verified, so
the only thing left to do is to show that ⌈F↾∆F⌉is a (k + 1)-anti-Sacks tree. Let
σ ∈⌈F↾∆F⌉. If |σ| < ht(t), then, because F↾ht(t) = t, it is possible to find an i ∈k
such that σ⌢⟨i⟩/∈⌈F↾∆F + 1⌉. If |σ| ≥ht(t), then, for every i < k, σ only has
(at most) one immediate successor in Fi and therefore it is always possible to find a
j ∈k + 1 such that σ⌢⟨j⟩/∈⌈F↾∆F + 1⌉. From these last two propositions we get the following result. The
following theorem is the main tool we will use to prove the main result of this section. Theorem 4.6 If ZFC is consistent, then ZFC + ∀i > 2 (∃Li (Li is ⟨ℵi, ASi⟩-Luzin)) +
∀k > 1 (∃L′
k (L′
k is ⟨ℵ2k, SPk⟩-Luzin)) is consistent. In the lemma above, it is clear that if we replace Lα ⊩“
. Qα = Pi ∗Pk” by
Lα ⊩“
. Qα = Pi”, then, in the extension, we still have a ⟨κ, ASi⟩-Luzin set. The
following theorem is the main tool we will use to prove the main result of this section. Theorem 4.6 If ZFC is consistent, then ZFC + ∀i > 2 (∃Li (Li is ⟨ℵi, ASi⟩-Luzin)) +
∀k > 1 (∃L′
k (L′
k is ⟨ℵ2k, SPk⟩-Luzin)) is consistent. Proof Let L = ⟨Lα,
. Qα : α ∈ωω⟩be a finite support iteration of length ωω such
that, for each i > 1 and each α ∈[ωi, ωi+1), Lα ⊩“
. Qα = Pi+1 ∗Qi+1”, where
Qi+1 = Pi+1 when i + 1 is a number of the form 2k + 1 and Qi+1 = {∅} in all the
other cases (for α < ω2, Lα ⊩“
. Qα = {∅}”). We will show that the extension is the
model we are looking for. As usual in this work, we will only show that there are
⟨ℵi, ASi⟩-Luzin sets for every i > 2 (the rest can be done in a similar way). Using
the lemma above, for each i > 2, in V[Gωi] there is a ⟨ℵi, ASi⟩-Luzin set Li. The
only thing left to do is to show that Li remains ⟨ℵi, ASi⟩-Luzin in V[G]. Using that
L is c.c.c. it is easy to see that, in V[G], [Li]<ωi ⊆ASi↾Li, so we only need to
show that ASi↾Li ⊆[Li]<ωi holds in V[G]. First, using Lemma 3.4, Lemma 3.6 and
Proposition 4.2, we observe that L[ωi,ωω] strongly preserves non(ASi) in Li, so if
. T is
a L[ωi,ωω]-name of a i-anti-Sacks tree, then, in V[Gωi], there is a X ∈ASi↾Li such that
L[ωi,ωω] ⊩“[
. T] ∩Li ⊆X”. It follows that ASi↾Li ⊆[Li]<ωi holds in V[G]. The actual value of c in the model above may depend on V . For example, if V |= GCH,
then it is easy to see that V[G] |= c = ℵω+1. From these last two propositions we get the following result. Corollary 4.3 For each k > 1, mk ≤non(ASk+1) and m2k−1 ≤non(SPk). Proof This follows easily from the proof of the Proposition 4.1 and the last
proposition. For the proof of the main theorem we will need the following notion. Definition 4.4 Given a regular cardinal κ and I ∈{SPn, ASk : n > 0, k > 1}, we
will say that a set L is ⟨κ, I⟩-Luzin if |L| = κ and I↾L = [L]<κ. Definition 4.4 Given a regular cardinal κ and I ∈{SPn, ASk : n > 0, k > 1}, we
will say that a set L is ⟨κ, I⟩-Luzin if |L| = κ and I↾L = [L]<κ. Observe that the existence of a ⟨κ, I⟩-Luzin set implies that non(I) ≤κ. Recall that
Cohen reals are added at every limit step of countable cofinality of a finite support
iteration of arbitrary length. One common application of Cohen reals is that they are
used to construct Luzin sets with special properties. The following lemma is one of
those applications. Lemma 4.5 Let κ be a regular cardinal, let i > 2, k > 1 and let L = ⟨Lα,
. Qα : α ∈κ⟩
be a finite support iteration of length κ such that Lα ⊩“
. Qα = Pi ∗Pk”, then
L ⊩“There is a ⟨κ, ASi⟩-Luzin set and there is a ⟨κ, SPk⟩-Luzin set”. Journal of Logic & Analysis 9:6 (2017) 11 Cardinal Invariants of Strongly Porous Sets Proof Working in V[G], let L = {fα : α ∈κ∧α has countable cofinality} be a family
of Cohen reals such that each fα is added at the α-th stage of the iteration. Using
the Proposition 4.1, it is easy to show that V[G] |= [L]<κ ⊆ASi↾L. On the other
hand, if T ∈V[G] is such that V[G] |= T is an i-anti-Sacks tree, then, by a standard
reflection argument, there is an intermediate model such that T ∈V[G(β)]. As a
consequence, V[G] |= ∀γ > β( fγ /∈[T]). This implies that V[G] |= ASi↾L ⊆[L]<κ. The ⟨κ, SPk⟩-Luzin set is found in a similar way. In the lemma above, it is clear that if we replace Lα ⊩“
. Qα = Pi ∗Pk” by
Lα ⊩“
. Qα = Pi”, then, in the extension, we still have a ⟨κ, ASi⟩-Luzin set. From these last two propositions we get the following result. Lemma 4.7 Let κ be a regular cardinal, let I ∈{SPn, ASk : n > 0, k > 1} and let
L be a ⟨κ, I⟩-Luzin set. If P is a forcing notion such that |P| < κ, then P strongly
preserves non(I) in L. Proof We will do the case when I ∈{ASi : i > 1}; the other cases are similar. Let
. A be a P-name of an i-anti-Sacks tree and let P = {pα : α ∈µ}. For each α ∈µ Journal of Logic & Analysis 9:6 (2017) 12 Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis define Tα = {s ∈<ωk : pα ⊩“s ∈
. A”}. It follows that each Tα defines an i-anti-Sacks
tree. If Y = S{[Tα] ∩L : α ∈µ} then Y ∈ASk. If x ∈L and pα ⊩“x ∈[
. A]”, then
x ∈[Tα] ∩L ⊆Y . We are ready to prove the main result of this section. Theorem 4.8 If ZFC is consistent, then ZFC + ∀k > 1 (mk = non(ASk+1) = ℵk+1) +
∀i > 1 (m2i−1 = non(SPi) = ℵ2i) + non(SP) = ℵω+1 is consistent. Proof Start with a model V like the one constructed in Theorem 4.6 and V |= c = ℵω+1. Using a standard bookkeeping argument, it is possible to construct a finite support
iteration P of length ωω+1 of σ-k-linked forcings of size smaller than ℵk+1 (for every
k > 1), such that any partial order which appears in an intermediate model is listed
cofinally along the iteration. Now, using Lemmas 3.4, 3.6 and 4.7, it is possible to show
that, for every k > 2, P strongly preserves non(ASk) in Lk. If G ⊆P is a generic
filter over V, then V[G] |= non(ASk) ≤ℵk. We note that, as each small σ-k-linked
forcing appears in an intermediate model in the iteration, we have V[G] |= ℵk+1 ≤mk. As a consequence V[G] |= ℵk+1 = mk = non(ASk+1). Using a similar argument, it is
possible to show that, for each i > 1, V[G] |= ℵ2i = m2i−1 = non(SPi). To finish the
proof, use the fact that non(SP) does not have countable cofinality and that, for every
n ∈ω, non(SPn) ≤non(SP) to show that V[G] |= non(SP) = c = ℵω+1. It follows from SP1 ⊆SP2 ⊆SP3 ⊆. . . that ω1 = non(SP1) ≤non(SP2) ≤
non(SP3) ≤. . . From these last two propositions we get the following result. ≤non(SP) and we proved in the theorem above that each inequality
can be consistently strict. It is important to remark that none of these numbers
is comparable with mσ-centered. An argument for this can be found in Hruˇs´ak and
Zindulka [6]. 3A similar theorem for the ideals SPk can be proved using the same argument. 5
The covering number It follows from the fact that AS2 ⊆AS3 ⊆. . . that c = cov(AS2) ≥cov(AS3) ≥. . .. We can show that every pair of these numbers can be consistently different. We can show that every pair of these numbers can be consistently d Proposition 5.1 Let k > 1, if ZFC is consistent, then ZFC+cov(ASk+1) < cov(ASk)
is consistent.3 Journal of Logic & Analysis 9:6 (2017) 13 Cardinal Invariants of Strongly Porous Sets Proof Let V be a model such that V |= cov(ASk) = c = ω2. Let P be a finite
support iteration of length ω1 of the Pk+1 forcing defined above and let G ⊆P
be a generic filter over V. It follows that P is an iteration of σ-k-linked forcing
notions and therefore P strongly preserves non(ASk). In V[G], consider the family
C = {V[Gα] ∩ω(k + 1) : α < ω1}. Using Proposition 4.1, it is easy to show that
V[G] |= C ⊆ASk+1 and V[G] |= S C = ω(k + 1). As a consequence we have that
V[G] |= cov(ASk+1) = ω1. On the other hand, if {
. Tα : α ∈ω1} is a collection of
P-names for k-anti-Sacks trees, then we can use the fact that P strongly preserves
non(ASk) to show that there is a collection {Cα : α ∈ω1} ⊆ASk such that if
x ∈ωk and x /∈S{Cα : α ∈ω1}, then P ⊩“x /∈S
α∈ω1[
. Tα]”. This, together with
V |= cov(ASk) > ω1, implies that V[G] |= cov(ASk+1) < cov(ASk). An alternative proof of this proposition follows from the results proven in Newelski
and Rosłanowski [9]. If k > 1 then a tree T ⊆<ωω is a k-tree if every s ∈T has
at most k immediate successors. A forcing notion P has the k-localization property
if P ⊩“∀f ∈ωω (∃T ∈V (T is a k-tree and f ∈[T]))”. It is easy to see that if
P has the k-localization property, then P ⊩“ S(ASk+1 ∩V) = ω(k + 1)”. Let
Sk = {T ⊆<ωk : ∀s ∈T(∃t ∈T(∀i ∈k(s ⊑t ∧t⌢i ∈T)))} be the k-Sacks forcing
ordered by inclusion. It turns out that Sk is forcing equivalent to Borel(ωk)/ASk and
that if P is the countable support iteration or the countable support product of length ω2
of the forcing Sk, then P has the k-localization property (see [9]). 5
The covering number As a consequence,
in the extension cov(ASk+1) = ω1 and cov(ASk) = ω2. An alternative proof of this proposition follows from the results proven in Newelski
and Rosłanowski [9]. If k > 1 then a tree T ⊆<ωω is a k-tree if every s ∈T has
at most k immediate successors. A forcing notion P has the k-localization property
if P ⊩“∀f ∈ωω (∃T ∈V (T is a k-tree and f ∈[T]))”. It is easy to see that if
P has the k-localization property, then P ⊩“ S(ASk+1 ∩V) = ω(k + 1)”. Let
Sk = {T ⊆<ωk : ∀s ∈T(∃t ∈T(∀i ∈k(s ⊑t ∧t⌢i ∈T)))} be the k-Sacks forcing
ordered by inclusion. It turns out that Sk is forcing equivalent to Borel(ωk)/ASk and
that if P is the countable support iteration or the countable support product of length ω2
of the forcing Sk, then P has the k-localization property (see [9]). As a consequence,
in the extension cov(ASk+1) = ω1 and cov(ASk) = ω2. Obviously it is impossible to separate infinitely many of the cov(SPn) at the same time. This suggests the following: Question 5.2 How many of the cov(SPn) can be separated at the same time? Question 5.2 How many of the cov(SPn) can be separated at the same time? We do not even know how to separate three of them. Another question we have is the
following: We do not even know how to separate three of them. Another question we have is the
following: Question
5.3 Is
it
possible
to
get
the
consistency
of
ZFC +
∀k ∈ω (cov(SP) < cov(SPk))? Question
5.3 Is
it
possible
to
get
the
consistency
of
ZFC +
∀k ∈ω (cov(SP) < cov(SPk))? We are also interested in the relationship between non(SP) and cov(SP). It follows
from the fact that the Cohen forcing is σ-centered that, in the Cohen model, non(SP) <
cov(SP). However, we do not know if it is possible to construct a model where
non(SP) > cov(SP). Question 5.4 Is non(SP) ≤cov(SP)? Question 5.4 Is non(SP) ≤cov(SP)? Question 5.4 Is non(SP) ≤cov(SP)? Journal of Logic & Analysis 9:6 (2017) Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis 14 A related question, as to whether non(ASn) ≤cov(ASn) was asked in [9]. Finally, we
would like to discuss about the relation of the cardinal numbers of the ideals SPk and
AS2k . In this work we showed that these ideals share a lot of properties, however we do
not know if they share the same cardinal invariants. There is a connection between 2k-
anti-Sacks trees and k-porous sets given by the following argument. Let ϕk : 2k →k2 be
a bijective function. Let ψk : ω(2k) →ω2 defined by ψk(x) = ϕk(x(0))⌢ϕk(x(1))⌢. . . . Clearly, if ψn(A) ∈SPn, then A ∈AS2n . We do not know if this can be used to show a
relation between the cardinal invariants of the ideals SPk and AS2k . Question 5.5 Is non(SPk) = non(AS2k)? Is cov(SPk) = cov(AS2k)? Question 5.5 Is non(SPk) = non(AS2k)? Is cov(SPk) = cov(AS2k)? 6
Acknowledgements The authors would like to thank J. Brendle and J. Cancino for many helpful suggestions
and hours of stimulating conversations. The authors would also like to thank the
anonymous referees for their insightful corrections and suggestions. The authors were
supported by PAPIIT grants IN 102311 and IN 108014. The first-listed author was
supported by CONACyT scholarship 420090. The second-listed author was supported
by a CONACyT grant 177758. The third-listed author was supported by CONACyT
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Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis
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M´exico Centro de Ciencas Matem´aticas, UNAM, A.P. 61-3, Xangari, Morelia, Michoac´an, 58089,
M´exico Journal of Logic & Analysis 9:6 (2017) Osvaldo Guzm´an, Michael Hruˇs´ak and Arturo Mart´ınez-Celis 16 Centro de Ciencas Matem´aticas, UNAM, A.P. 61-3, Xangari, Morelia, Michoac´an, 58089,
M´exico
oguzman@matmor.unam.mx, michael@matmor.unam.mx, arturo@matmor.unam.mx
http://matmor.unam.mx/~michael
Received: 7 September 2016
Revised: 7 June 2017 Centro de Ciencas Matem´aticas, UNAM, A.P. 61-3, Xangari, Morelia, Michoac´an, 58089,
M´exico oguzman@matmor.unam.mx, michael@matmor.unam.mx, arturo@matmor.unam.mx
http://matmor.unam.mx/~michael Received: 7 September 2016
Revised: 7 June 2017 Journal of Logic & Analysis 9:6 (2017)
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English
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A Rights-based Assessment of the Temporary Protection Statute for Venezuelans in Colombia
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Zenodo (CERN European Organization for Nuclear Research)
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1.
See MIGRACIÓN COLOMBIA, Más de Dos Millones 477 Mil Venezolanos se Encuentran
Radicados en Colombia y de Ellos, el 96% Busca Hacer Parte del Estatuto Temporal de Protección –
Visibles (July 19, 2022), https://www.migracioncolombia.gov.co/noticias/mas-de-dos-millones-477-mil-
venezolanos-se-encuentran-radicados-en-colombia-y-de-ellos-el-96-busca-hacer-parte-del-estatuto-
temporal-de-proteccion-visibles. A Rights-based Assessment of the Temporary
Protection Statute for Venezuelans in Colombia Dr. Stefano Angeleri†
Dr. María Teresa Palacios Sanabria†† ††
Professor of Law, Universidad del Rosario, Bogotá †
Marie Sklodowska Curie Fellow at Queen’s University Belfast. This project has received
funding from the European Union’s Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreement No 101032116. ††
Professor of Law, Universidad del Rosario, Bogotá 6.
ENCOVI – Encuestas sobre Condiciones de Vida has monitored living conditions in
Venezuela since 2014. The project is conducted by three major Venezuelan universities: the University
Simón Bolívar, the University Central de Venezuela, and the University Católica Andrés Bello. See
Encovi, U. CATÓLICA ANDRÉS BELLO, https://www.proyectoencovi.com/encovi-2021. 7.
See U.N. ECON. COMM’N FOR LAT. AM. & THE CARIBBEAN, Social Panorama of Latin
America 2014, at 16 (2014), https://www.cepal.org/en/publications/37627-social-panorama-latin-
america-2014. ABSTRACT Since 2015, the multidimensional crisis in Venezuela has resulted in
massive emigration. Over 2.4 million Venezuelan refugees and migrants live in
neighboring Colombia, and, in 2021, almost one million of them were
undocumented.1 Such exceptionally high numbers of migrants in irregular,
precarious status led to the Colombian government’s concerns about migration
control, human rights, and social inclusion. In response, in March 2021, the
government launched an ambitious regularization scheme, the Temporary
Protection Statute for Venezuelan Migrants (TPSV), which is currently being
implemented. This Essay first explores the extent to which Colombia’s
international human rights law obligations contributed to the creation of the
TPSV. Second, it assesses how the scheme interacts with existing statuses used
by Venezuelan migrants in Colombia. Third, it identifies concerns that the TPSV
and its implementation raise under international law. By adopting the TPSV, the
Colombian government took a significant step towards a rights-oriented
approach to managing mixed migration. Yet, while the TPSV regulations are rife
with human rights rhetoric, we raise questions about whether the scheme, in its
totality, is compliant with international law. ABSTRACT ............................................................................................................................................... 80
INTRODUCTION ....................................................................................................................................... 81
I. A REGULARIZATION SCHEME TO COMPLY WITH INTERNATIONAL HUMAN RIGHTS LAW .................... 82
II. THE INTERACTOIN OF THE TPSV WITH EXISTING DOMESTIC STATUSES ............................................ 85
III. THE (IN)COMPLIANCE OF THE TPSV APPROACH WITH INTERNATIONAL OBLIGATIONS .................... 86
†
††
Marie Sklodowska Curie Fellow at Queen’s University Belfast. This project has received
funding from the European Union’s Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreement No 101032116. Professor of Law, Universidad del Rosario, Bogotá
1. See MIGRACIÓN COLOMBIA, Más de Dos Millones 477 Mil Venezolanos se Encuentran
Radicados en Colombia y de Ellos, el 96% Busca Hacer Parte del Estatuto Temporal de Protección –
Visibles (July 19, 2022), https://www.migracioncolombia.gov.co/noticias/mas-de-dos-millones-477-mil-
venezolanos-se-encuentran-radicados-en-colombia-y-de-ellos-el-96-busca-hacer-parte-del-estatuto-
temporal-de-proteccion-visibles. 2022] A Rights-based Assessment of the Temporary Protection Statute 2022] A Rights-based Assessment of the Temporary Protection Statute 81 A. Nondiscrimination on the Basis of Nationality ....................................................................... 87
B. Due Process of Law ................................................................................................................ 88
C. Special Measures Targeting Migrants with Special Vulnerabilities ........................................ 89
D. Creation of a Meaningful Legal Status ................................................................................... 90
E. Neglect of Domestic Refugee Protection ................................................................................ 91
CONCLUSION ........................................................................................................................................... 93 CONCLUSION .. 8.
See GRUPO INTERAGENCIAL SOBRE FLUJOS MIGRATORIOS MIXTOS EN COLOMBIA & 5.
See generally Inter-Am. Comm’n H.R., Preliminary Observations and Recommendations
of the Country Visit to Venezuela on Human Rights Implementation, No. 106/2020 (May 8, 2020),
http://www.oas.org/es/cidh/prensa/comunicados/2020/106.asp; AMNESTY INT’L, Venezuela: Hunger,
Punishment and Fear, the Formula for Repression used by Authorities under Nicolás Maduro (Feb. 20,
2019),
https://www.amnesty.org/en/latest/news/2019/02/venezuela-hunger-punishment-and-fear-the-
formula-for-repression-used-by-authorities-under-nicolas-maduro/; WORLD HEALTH ORG. & PAN-AM.
HEALTH ORG., PAHO’s Response to Maintaining an Effective Technical Cooperation Agenda in
Venezuela and Member States (Sep. 5, 2018), https://iris.paho.org/bitstream/handle/10665.2/49650/
CD56-INF-12-e.pdf?sequence=1&isAllowed=y. 2.
See R4V, Key Figures (Jan. 10, 2022), https://www.r4v.info/en/refugeeandmigrants.
3.
See Benjamin N. Gedan, Venezuelan Migration: Is the Western Hemisphere Prepared for a
Refugee Crisis? 37 SAIS REV. INT’L AFFS. 37, 57 (2017).
4.
See Global Migration Data Analysis Centre, Mixed Migration, INT’L ORG. FOR MIGRATION
(Feb. 21, 2022), https://www.migrationdataportal.org/themes/mixed-migration.
5.
See generally Inter-Am. Comm’n H.R., Preliminary Observations and Recommendations
of the Country Visit to Venezuela on Human Rights Implementation, No. 106/2020 (May 8, 2020),
http://www.oas.org/es/cidh/prensa/comunicados/2020/106.asp; AMNESTY INT’L, Venezuela: Hunger,
Punishment and Fear, the Formula for Repression used by Authorities under Nicolás Maduro (Feb. 20,
2019),
https://www.amnesty.org/en/latest/news/2019/02/venezuela-hunger-punishment-and-fear-the-
formula-for-repression-used-by-authorities-under-nicolas-maduro/; WORLD HEALTH ORG. & PAN-AM.
HEALTH ORG., PAHO’s Response to Maintaining an Effective Technical Cooperation Agenda in
Venezuela and Member States (Sep. 5, 2018), https://iris.paho.org/bitstream/handle/10665.2/49650/
CD56-INF-12-e.pdf?sequence=1&isAllowed=y.
6.
ENCOVI – Encuestas sobre Condiciones de Vida has monitored living conditions in
Venezuela since 2014. The project is conducted by three major Venezuelan universities: the University
Simón Bolívar, the University Central de Venezuela, and the University Católica Andrés Bello. See
Encovi, U. CATÓLICA ANDRÉS BELLO, https://www.proyectoencovi.com/encovi-2021.
7.
See U.N. ECON. COMM’N FOR LAT. AM. & THE CARIBBEAN, Social Panorama of Latin
America 2014, at 16 (2014), https://www.cepal.org/en/publications/37627-social-panorama-latin-
america-2014.
8.
See GRUPO INTERAGENCIAL SOBRE FLUJOS MIGRATORIOS MIXTOS EN COLOMBIA & 2.
See R4V, Key Figures (Jan. 10, 2022), https://www.r4v.info/en/refugeeandmigrants.
3.
See Benjamin N. Gedan, Venezuelan Migration: Is the Western Hemisphere Prepared for a
Refugee Crisis? 37 SAIS REV. INT’L AFFS. 37, 57 (2017).
4.
See Global Migration Data Analysis Centre, Mixed Migration, INT’L ORG. FOR MIGRATION
(Feb. 21, 2022), https://www.migrationdataportal.org/themes/mixed-migration.
5.
See generally Inter-Am. Comm’n H.R., Preliminary Observations and Recommendations
of the Country Visit to Venezuela on Human Rights Implementation, No. 106/2020 (May 8, 2020),
http://www.oas.org/es/cidh/prensa/comunicados/2020/106.asp; AMNESTY INT’L, Venezuela: Hunger,
Punishment and Fear, the Formula for Repression used by Authorities under Nicolás Maduro (Feb. 20,
2019),
https://www.amnesty.org/en/latest/news/2019/02/venezuela-hunger-punishment-and-fear-the-
formula-for-repression-used-by-authorities-under-nicolas-maduro/; WORLD HEALTH ORG. & PAN-AM.
HEALTH ORG., PAHO’s Response to Maintaining an Effective Technical Cooperation Agenda in
Venezuela and Member States (Sep. 5, 2018), https://iris.paho.org/bitstream/handle/10665.2/49650/
CD56-INF-12-e.pdf?sequence=1&isAllowed=y. 3.
See Benjamin N. Gedan, Venezuelan Migration: Is the Western Hemisphere Prepared for a
Refugee Crisis? 37 SAIS REV. INT’L AFFS. 37, 57 (2017). INTRODUCTION Over the last seven years, more than six million Venezuelans have fled the
country2 due to the severe economic, political, and social crisis.3 The reality of
the Venezuelan exodus is one of mixed migration because of the “intertwined
and multifaceted drivers of movement of [these] people [on the move],”4 among
them political persecution and systematic human rights violations, as well as the
deterioration of socioeconomic conditions in the country.5 The scale of the
economic crisis in Venezuela is staggering: To give one indication of its scope,
2021 statistics indicate that 94.5 percent of people in Venezuela live below the
poverty line,6 compared with about twenty-five percent in 2012.7 In light of these
realities, the International Organization for Migration (IOM) and the United
Nations High Commissioner for Refugees (UNCHR) explicitly acknowledge the
mixed character of Venezuelan emigration: In Colombia, they established a
coordination platform aptly named the Interagency Group for Mixed Migration
Flows (GIFMM). Of the six million Venezuelans who have left their country, over 1.8 million
people have settled in neighboring Colombia. During the first half of 2021, one
million Venezuelans were undocumented or in irregular status, about 700,000
held temporary, two-year residence permits, and only 28,800 were registered
asylum seekers.8 The scale of migration has resulted in multidimensional 82 THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 47: 80 challenges for state institutions and migrants themselves. In early 2021, in response to the presence of such exceptionally high
numbers of migrants in irregular or precarious status—and to address concerns
regarding migration control, human rights, and social inclusion—the Colombian
government launched an ambitious regularization scheme: the Temporary
Protection Statute for Venezuelan Migrants (TPSV).9 This scheme, an exercise
of the executive powers of the Colombian government, targets all Venezuelan
migrants and asylum seekers who meet certain eligibility criteria, including
irregular presence in the territory before January 31, 2021 and no pending
administrative or criminal proceedings.10 Despite the use of the word
“protection” in the scheme’s title, which invokes notions of international refugee
protection, the TPSV is essentially a mechanism of economic migrant
regularization.11 The TPSV envisages a staged regularization process: First, online
registration is required; second, the identity of applicants is verified in person;
and third, after an assessment of eligibility criteria, successful applicants are
issued a ten-year temporary protection permit (TPP), which allows its bearers to
access the formal labor market, social protection programs, and comprehensive
health care. 9.
The TPSV is regulated by the Colombian Ministry of Foreign Affairs. See Por Medio del
Cual se Adopta el Estatuto Temporal de Protección para Migrantes Venezolanos Bajo Régimen de
Protección Temporal y se Dictan otras Disposiciones en Materia Migratoria, Decree No. 216/2021 (Mar.
1, 2021); Por la Cual se Implementa el Estatuto Temporal de Protección para Migrantes Venezolanos
Adoptado por Medio del Decreto 216 de 2021, Res. No. 971/2021 (Apr. 28. 2021). p
p
( p
)
10.
See Decree No. 216/2021, supra note 9, arts. 4, 8, 12; Res. No. 971/2021, supra note 9, arts.
2, 5, 6, 15,16. 13.
See
MIGRACIÓN
COLOMBIA,
Visibles:
Estatuto
Temporal
de
Protección,
https://www.migracioncolombia.gov.co/visibles. 12.
See id., art. 11. The R visa is regulated by the Ministry of Foreign Affairs. See Res. No.
6045 (Aug. 2, 2017), art. 21. PLATAFORMA DE COORDINACIÓN INTERAGENCIAL PARA REFUGIADOS Y MIGRANTES DE VENEZUELA
(GIFMM/R4V), GIFMM Colombia: Resumen Situacional - Junio 2021, https://www.r4v.info/
es/document/gifmm-colombia-resumen-situacional-junio-2021-es; R4V, Total Pending Asylum Claims
per Country (Dec. 31, 2021), https://www.r4v.info/en/pending-asylum-claims. INTRODUCTION Importantly, time spent holding a TPP will count towards eligibility
for an R visa, a renewable residence visa that provides a pathway to indefinite
stay in Colombia.12 To date, 2.4 million Venezuelans have registered using the
online regularization platform and around 1.5 million TPPs have been approved
as of August 2022.13 Against this backdrop, this Essay first describes the extent to which
Colombia’s international human rights law obligations contributed to the
introduction of the TPSV. Second, it assesses how the TPSV interacts with other
status categories. Finally, it identifies certain major international human rights
and refugee law concerns that the TPSV regularization approach raises. 11.
See Res. No. 971/2021, supra note 9, art. 11. 14.
See Press Release, Canciller Claudia Blum, El Estatuto de Protección Temporal a Migrantes
Venezolanos Es una Decisión Histórica para la Región y el Mundo, CANCILLERÍA (Feb. 8, 2021),
https://www.cancilleria.gov.co/newsroom/news/estatuto-proteccion-temporal-migrantes-venezolanos-
decision-historica-region-mundo. Blum stated that the TPSV will contribute to safe, orderly, and regular
migration, echoing the language of the Global Compact. See G.A. Res. 73/195, Global Compact for Safe,
Orderly and Regular Migration (Dec. 19, 2018) [hereinafter Global Compact for Migration],
https://www.un.org/en/ga/search/view_doc.asp?symbol=A/RES/73/195. 16.
For the endorsement of the Global Compact for Migration, see Decree No. 216/2021, supra
note 9, at 3; Res. No. 971/2021, supra note 9, at 2. 19.
See, e.g., Corte Constitucional de Colombia [Constitutional Court of Colombia] (CCC),
Judgments T-006/2020, ¶ 1.2; C-355/2006, ¶ 8.4; SU-096/2018, ¶ 9; SU-677/2017, ¶ 18; T-239 de 2017,
¶ 90. p
g
g
p
y
15.
See Decree No. 216/2021, supra note 9, Preamble; Res. No. 971/2021, supra note 9,
Preamble. 20.
See Decree No. 216/2021, supra note 9, Preamble. 17.
See Global Compact for Migration, supra note 14, ¶ 12. 18.
See CONSTITUCIÓN POLÍTICA DE COLOMBIA (1991), art. 93. I. A REGULARIZATION SCHEME TO COMPLY WITH INTERNATIONAL HUMAN
RIGHTS LAW In February 2021, the Colombian government began publicizing the 2022] 83 A Rights-based Assessment of the Temporary Protection Statute regularization scheme, insisting that the measure was meant to ensure “safe,
orderly, and regular migration” and to guarantee the human rights of all
migrants.14 This conception of the TPSV is explicitly embedded in the preamble
of the TPSV decree and its implementing resolution.15 It is undeniable that such a massive regularization scheme has a positive
effect on the human rights of Venezuelans in Colombia. Indeed, holding regular
migratory status is a precondition for enjoying a greater share of citizens’ rights
at the domestic level, notably the right to work and to access social security. By
committing to regularize the status of Venezuelans in Colombia, the government
took a significant step towards a rights-oriented implementation of the Global
Compact for Migration.16 This commitment is also beneficial for migration
control and the creation of “conducive conditions that enable all migrants to
enrich our societies through their human, economic and social capacities.”17 Colombia’s serious legal commitment to international human rights law
may help explain the enactment of the TPSV. Article 93 of the Colombian
Constitution provides that ratified human rights treaties have legal primacy over
domestic sources of law18 and constitute standards for the interpretation of
fundamental rights.19 The primary role of international law in the domestic legal
framework has long helped fill normative protection gaps for migrants arising
from the absence of a comprehensive migration act in Colombian law. As a
result, Colombia’s national case law on migrants’ rights has relied heavily on
international treaties and jurisprudence. The emphasis on human rights law in the preamble to the TPSV is therefore
unsurprising, though there are some surprising omissions. The decree contains
thirty-seven references to the realization of fundamental or international human
rights for migrant adults and children, identifying the realization of these rights
as key goals of the regularization scheme and as principles that prompted the
adoption of the measure. In particular, the decree indicates that the TPSV is
necessary to guarantee the rights and social integration of irregular migrants, to
protect migrants from the risks of trafficking and other forms of abuse, and to
comply with international law.20 Specifically, the preamble features the
International Convention on the Protection of the Rights of All Migrant Workers THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 26.
See UNHCR/IOM, Press Release, UNHCR and IOM Welcome Colombia’s Decision to
Regularize Venezuelan Refugees and Migrants (Feb. 8, 2021), https://www.unhcr.org/news/
press/2021/2/60214cf74/unhcr-iom-welcome-colombias-decision-regularize-venezuelan-refugees-
migrants.html. 25.
See generally INT’L LABOUR ORG., Reporte Mesas de Trabajo: Inclusión de los Migrantes
Venezolanos en el Sistema de Protección Social Colombiano, 24-25 de Febrero, 2021 (March 2021),
https://www.ilo.org/wcmsp5/groups/public/---americas/---ro-lima/---sro-
lima/documents/publication/wcms_816136.pdf. 24.
See id. arts. 25-35. 21.
See Linda S. Bosniak, Human Rights, State Sovereignty and the Protection of
Undocumented Migrants Under the International Migrant Workers Convention, in IRREGULAR
MIGRATION AND HUMAN RIGHTS: THEORETICAL, EUROPEAN, AND INTERNATIONAL PERSPECTIVES 311,
316 (Barbara Bogusz et al. eds., 2004). 21.
See Linda S. Bosniak, Human Rights, State Sovereignty and the Protection of
Undocumented Migrants Under the International Migrant Workers Convention, in IRREGULAR
MIGRATION AND HUMAN RIGHTS: THEORETICAL, EUROPEAN, AND INTERNATIONAL PERSPECTIVES 311,
316 (Barbara Bogusz et al. eds., 2004).
22.
See Res. No. 971/2021, supra note 9, Preamble.
23.
See id.
24.
See id. arts. 25-35.
25.
See generally INT’L LABOUR ORG., Reporte Mesas de Trabajo: Inclusión de los Migrantes
Venezolanos en el Sistema de Protección Social Colombiano, 24-25 de Febrero, 2021 (March 2021),
https://www.ilo.org/wcmsp5/groups/public/---americas/---ro-lima/---sro-
lima/documents/publication/wcms_816136.pdf.
26.
See UNHCR/IOM, Press Release, UNHCR and IOM Welcome Colombia’s Decision to
Regularize Venezuelan Refugees and Migrants (Feb. 8, 2021), https://www.unhcr.org/news/
press/2021/2/60214cf74/unhcr-iom-welcome-colombias-decision-regularize-venezuelan-refugees-
migrants.html. 21.
See Linda S. Bosniak, Human Rights, State Sovereignty and the Protection of
Undocumented Migrants Under the International Migrant Workers Convention, in IRREGULAR
MIGRATION AND HUMAN RIGHTS: THEORETICAL, EUROPEAN, AND INTERNATIONAL PERSPECTIVES 311,
316 (Barbara Bogusz et al. eds., 2004).
22.
See Res. No. 971/2021, supra note 9, Preamble.
23.
See id.
24
See id arts 25 35 22.
See Res. No. 971/2021, supra note 9, Preamble. 31.
See Nicholas De Genova, Migrant “Illegality” and Deportability in Everyday Life, 31 ANN.
REV. ANTHROPOLOGY 419, 429 (2002). g
(
g
)
28.
See Anna Błuś, Beyond the Walls of Paper: Undocumented Migrants, the Border and
Human Rights, 15 EUROPEAN J. MIGRATION & L. 413, 441 (2013). 27.
This Essay uses the terms “irregular” or “undocumented” to refer to those foreign nationals
who do not comply with immigration law requirements for entry or stay in a country and are, therefore,
susceptible to deportation. For a discussion of these terms, see Elspeth Guild, Who Is an Irregular
Migrant?, in IRREGULAR MIGRATION AND HUMAN RIGHTS (Barbara Bogusz et al. eds., 2004). p
p
30.
Former temporary regularizations schemes included, for instance, the two-year Special
Permanence Permit, the Permit for Entry and Permanence, the Special Permanence Permit for the
Promotion of Formalization, and the Complementary Special Permeance Permit. See Por la Cual se
Establecen los Permisos de Ingreso y Permanencia, Permisos Temporales de Permanencia, y se
Reglamenta el Tránsito Fronterizo en el territorio nacional, Res. No. 1220/2016 (Aug. 12, 2016); Por
Medio de la Cual se Crea un Permiso Especial de Permanencia, Res. No. 5797/2017 (July 25, 2017); Por
Medio de la Cual se Crea un Permiso Especial Complementario de Permanencia (PECP), Res. No.
3548/2019 (July 3, 2019); Por el cual se adiciona la Sección 3 al Capítulo 8 del Título 6 de la Parte 2 del
Libro 2 del Decreto 1072 de 2015, Decreto Único Reglamentario del Sector Trabajo, en lo Relacionado
con la Creación de un Permiso Especial de Permanencia para el Fomento de la Formalización, Decree No.
117/2020 (Jan. 28, 2020). g
29.
See Decree No. 216/2021, supra note 9, art. 4; Res. No. 971/2021, supra note 9, art. 6. 27.
This Essay uses the terms “irregular” or “undocumented” to refer to those foreign nationals
who do not comply with immigration law requirements for entry or stay in a country and are, therefore,
susceptible to deportation. For a discussion of these terms, see Elspeth Guild, Who Is an Irregular
Migrant?, in IRREGULAR MIGRATION AND HUMAN RIGHTS (Barbara Bogusz et al. eds., 2004).
28.
See Anna Błuś, Beyond the Walls of Paper: Undocumented Migrants, the Border and
Human Rights, 15 EUROPEAN J. MIGRATION & L. 413, 441 (2013).
29.
See Decree No. 216/2021, supra note 9, art. 4; Res. No. 971/2021, supra note 9, art. 6.
30.
Former temporary regularizations schemes included, for instance, the two-year Special
Permanence Permit, the Permit for Entry and Permanence, the Special Permanence Permit for the
Promotion of Formalization, and the Complementary Special Permeance Permit. See Por la Cual se
Establecen los Permisos de Ingreso y Permanencia, Permisos Temporales de Permanencia, y se
Reglamenta el Tránsito Fronterizo en el territorio nacional, Res. No. 1220/2016 (Aug. 12, 2016); Por
Medio de la Cual se Crea un Permiso Especial de Permanencia, Res. No. 5797/2017 (July 25, 2017); Por
Medio de la Cual se Crea un Permiso Especial Complementario de Permanencia (PECP), Res. No.
3548/2019 (July 3, 2019); Por el cual se adiciona la Sección 3 al Capítulo 8 del Título 6 de la Parte 2 del
Libro 2 del Decreto 1072 de 2015, Decreto Único Reglamentario del Sector Trabajo, en lo Relacionado
con la Creación de un Permiso Especial de Permanencia para el Fomento de la Formalización, Decree No.
117/2020 (Jan. 28, 2020).
31
S
Ni h l
D G
Mi
“Ill
li ”
d D
bili
i E
d
Lif
31 A I. A REGULARIZATION SCHEME TO COMPLY WITH INTERNATIONAL HUMAN
RIGHTS LAW 47: 80 84 and Members of Their Families (ICMW) and the Convention on the Rights of
the Child. Yet, the ICMW is not the most equality-oriented choice; several
observers criticized it for “constitutionalizing” a rights divide between regular
and irregular migrants.21 Notably, the preamble makes no reference to the
International Covenant on Economic Social and Cultural Rights or the
Additional Protocol to the American Convention on Human Rights in the Area
of Economic, Social, and Cultural Rights—even though these treaties are
particularly relevant to the TPSV, given that the target of the scheme is the social
and economic integration of migrants and refugees. Their absence is suggestive
of the government’s willingness to treat the socioeconomic integration of
migrant adults as a desired policy objective rather than as an obligation of treaties
conferring socioeconomic rights. The implementing resolution, developed and issued by Colombia’s
migration management agency, Migración Colombia, also relies on human rights
to contextualize the adoption of the TPSV.22 The resolution focuses particularly
on the importance of protecting the rights of the child and the right to dignified
work for adults.23 The rights of the child are subject to special protections in
Colombia’s constitutional legal order, which may explain several provisions in
the implementing resolution that allow for the early regularization of particularly
vulnerable children, such as children enrolled in the school system and children
who are in special care settings and reeducation procedures.24 The many
references to the right to work share a clear link with the pragmatic, instrumental
purposes for which the TPSV was enacted. Ensuring that Venezuelan
beneficiaries of the TPSV are able to access the formal employment sector, for
example, in turn enables them to contribute to Colombian taxation and health
care systems, thereby increasing inputs and decreasing costs for Colombia’s
national and regional budgets.25 The TPSV has been praised by key international actors for facilitating the
realization of human rights and the socioeconomic integration of Venezuelan
migrants in Colombia. The IOM and the UNCHR, for example, have called the
TPSV “a model of pragmatism and humanity.”26 And, indeed, the TPSV
improves in meaningful ways upon other statuses available under Colombian
law. The next section situates the TPSV in that context. 85 2022] A Rights-based Assessment of the Temporary Protection Statute II. THE INTERACTOIN OF THE TPSV WITH EXISTING DOMESTIC STATUSES The TPSV creates a new migrant category: Venezuelans with a ten-year
TPP. This status is intended to replace asylum and the two-year residency
permits that were specially created for Venezuelans. Notably, the TPP is available not only to those Venezuelans who hold a
legal status, but also the one million Venezuelans in irregular status.27 The
regularization of migrants in irregular status is particularly rights-empowering
because access to employment, social rights, and justice are invariably linked to
citizenship and immigration status. Consequently, regularization is “the most
adequate solution to the issues faced by many of today’s undocumented
migrants.”28 One important condition on eligibility for the TPSV is that irregular
migrants must provide evidence that they were in Colombian territory on or
before January 31, 2021.29 However, this cutoff date leaves people who have
irregularly entered the territory since then without access to a regularization
scheme. While a potential amnesty for one million undocumented people is an
exceptional and ambitious plan, it is important to bear in mind that the
Venezuelan crisis and associated emigration are not expected to cease soon. Thus, while monumental in its scope, the scheme continues the national trend of
adopting temporary migration policy measures that do not address the
probability of future irregular entry.30 The trend is not unique to Colombia: As
Nicholas de Genova writes, every regularization has “an inherently episodic and
strictly partial character that never eliminates the field of ‘illegality’ but rather . . . simply refines and reconstitutes that field for the ineligible who will remain
undocumented along with all subsequent ‘illegal’ arrivals.”31 In addition to the irregular migrants who benefit from the regularization
scheme, Venezuelans currently residing in Colombia who have or are about to
renew the two-year, temporary stay permit—called a Special Permanence Permit
(SPP)—and Venezuelans who enter Colombia regularly until May 2023 are also 86 THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 47: 80 eligible for a TPP.32 The TPSV offers distinct benefits even for these groups. 33.
As we discuss in Part III, infra, however, the fact that the TPSV can, in principle, be
cancelled at any time does not grant full stability to prospective TPP holders. 37.
According to statistics from Migración Colombia, the Colombian migration agency, as of
September 2021, there were approximately 14,000 Haitian migrants in Colombian territory, with few
protection programs available to them. See MIGRACIÓN COLOMBIA, En el Último Mes, Migración
Colombia Ha Detectado Más de 34 Mil Migrantes Irregulares, una Cifra Equivalente al 51% del Total
de Detecciones de este 2021 (Sept. 10, 2021), https://www.migracioncolombia.gov.co/noticias/en-el-
ultimo-mes-migracion-colombia-ha-detectado-mas-de-34-mil-migrantes-irregulares-una-cifra- y
g
y
p
p
34.
See Decree No. 216/2021, supra note 9, art. 17, and Res. No. 971/2021, supra note 9, art. 1
35
S
i f
81 83 III. THE (IN)COMPLIANCE OF THE TPSV APPROACH WITH INTERNATIONAL g
g
g
equivalente-al-51-del-total-de-detecciones-de-este-2021. Furthermore, 27,000 irregular migrants from
Haiti, Cuba, Senegal, Ghana, Angola, Guinea, and Nepal were reported to have entered Colombia in
September 2021. DW, Colombia Reporta Ingreso Irregular de 27.000 Migrantes en un Mes (Sept. 9,
2021),
https://www.dw.com/es/colombia-reporta-ingreso-irregular-de-27000-migrantes-en-un-mes/a-
59149479. f
36.
See Part III, infra, on the (lack of) harmonization of the TPSV and the asylum system. 32.
See Decree No. 216/2021, supra note 9, art. 4(1) and (4) 35.
See infra notes 81-83. 32.
See Decree No. 216/2021, supra note 9, art. 4(1) and (4).
33.
As we discuss in Part III, infra, however, the fact that the TPSV can, in principle, be
cancelled at any time does not grant full stability to prospective TPP holders.
34.
See Decree No. 216/2021, supra note 9, art. 17, and Res. No. 971/2021, supra note 9, art. 1.
35.
See infra notes 81-83.
36.
See Part III, infra, on the (lack of) harmonization of the TPSV and the asylum system.
37.
According to statistics from Migración Colombia, the Colombian migration agency, as of
September 2021, there were approximately 14,000 Haitian migrants in Colombian territory, with few
protection programs available to them. See MIGRACIÓN COLOMBIA, En el Último Mes, Migración
Colombia Ha Detectado Más de 34 Mil Migrantes Irregulares, una Cifra Equivalente al 51% del Total
de Detecciones de este 2021 (Sept. 10, 2021), https://www.migracioncolombia.gov.co/noticias/en-el-
ultimo-mes-migracion-colombia-ha-detectado-mas-de-34-mil-migrantes-irregulares-una-cifra-
equivalente-al-51-del-total-de-detecciones-de-este-2021. Furthermore, 27,000 irregular migrants from
Haiti, Cuba, Senegal, Ghana, Angola, Guinea, and Nepal were reported to have entered Colombia in
September 2021. DW, Colombia Reporta Ingreso Irregular de 27.000 Migrantes en un Mes (Sept. 9,
2021),
https://www.dw.com/es/colombia-reporta-ingreso-irregular-de-27000-migrantes-en-un-mes/a-
59149479. y
g
y
p
p
34.
See Decree No. 216/2021, supra note 9, art. 17, and Res. No. 971/2021, supra note 9, art. 1.
35.
See infra notes 81-83. 38.
See International Convention on the Elimination of All Forms of Racial Discrimination
(CERD) art. 1(1), opened for signature Dec. 21, 1965, 660 U.N.T.S. 211; see also International Covenant
on Civil and Political Rights (ICCPR) art. 2(2), opened for signature Dec. 19, 1966, 999 U.N.T.S. 171;
International Covenant on Economic, Social and Cultural Rights, art. 2(2), opened for signature Dec. 19,
1966, 993 U.N.T.S. 3. OBLIGATIONS The implementation of the TPSV will facilitate the enjoyment of human
rights by successful applicants, but its approach raises concerns about
compliance with international human rights law and asylum obligations. This
section briefly examines some of these concerns, focusing on nondiscrimination
on the basis of nationality, due process of law, certain affirmative obligations to
promote and protect human rights of vulnerable people, and the neglect of the
right to seek asylum. 39.
See INT’L L. COMM’N, Draft Articles on the Responsibility of States for Wrongful Acts, with
Commentaries, 2 Y.B. INT’L L. COMM’N 31, 85 ¶ 5 (2001) (“[T]he peremptory norms that are clearly
accepted and recognized include the prohibitions of aggression, genocide, slavery, racial discrimination,
crimes against humanity and torture, and the right to self-determination.”). 41.
This principle was recently reiterated in the Global Compact for Migration, supra note 14,
¶ 15. For further discussion of state sovereignty over migration, see Catherine Dauvergne, Sovereignty, g
y
g
40.
See Inter-Am. Ct. H.R., Proposed Amendments to the Naturalization Provisions of the
Political Constitution of Costa Rica, Advisory Opinion OC-4/84 (Jan. 19, 1984) ¶ 57–60. II. THE INTERACTOIN OF THE TPSV WITH EXISTING DOMESTIC STATUSES The
TPP granted under the TPSV is valid for ten years, making it much more durable
than the SPP and, as a result, makes personal and economic life plans less
precarious.33 While the provision allowing Venezuelans who enter the country
regularly (that is, by using a passport at a Colombian port of entry) after the
announcement of the TPSV is valuable, obtaining a Venezuelan passport is
financially and administratively impossible for many Venezuelans—a fact that
may perpetuate illegality. Venezuelan asylum seekers who meet the requirements for the TPSV are
also eligible for a TPP. If successful in their application for a TPP, the asylum
seeker must decide either to accept the TPP and rescind their asylum application
or renounce the TPP and continue in the asylum system in the hope that their
application will be successful.34 Seeking asylum in Colombia is accompanied by
numerous constraints. Asylum seekers cannot freely move throughout the
country or obtain employment in Colombia until they are granted refugee status,
a process that can take years.35 As a result, many of the current asylum seekers
may decide to pursue a TPP, despite the fact that they will not receive formal
recognition as a refugee, opting instead to participate in a scheme that serves
Venezuelans with a variety of motivations for emigrating from Venezuela. This
reality reflects a governmental judgment: The Colombian government has
preferred to treat all Venezuelans as economic migrants fleeing a humanitarian
crisis rather than make efforts to strengthen the responsiveness of the national
asylum system.36 Of course, this special scheme applies only to Venezuelans. Accordingly,
other asylum seekers and migrants, such as Haitians or Cubans, may find
themselves in similar situations of vulnerability, but they do not have the same
access to straightforward and affordable forms of regularization in Colombia.37
The following section explores the problems this reality presents for Colombia’s
international legal obligations. 2022] A Rights-based Assessment of the Temporary Protection Statute 87 OBLIGATIONS 38.
See International Convention on the Elimination of All Forms of Racial Discrimination
(CERD) art. 1(1), opened for signature Dec. 21, 1965, 660 U.N.T.S. 211; see also International Covenant
on Civil and Political Rights (ICCPR) art. 2(2), opened for signature Dec. 19, 1966, 999 U.N.T.S. 171;
International Covenant on Economic, Social and Cultural Rights, art. 2(2), opened for signature Dec. 19,
1966, 993 U.N.T.S. 3.
39.
See INT’L L. COMM’N, Draft Articles on the Responsibility of States for Wrongful Acts, with
Commentaries, 2 Y.B. INT’L L. COMM’N 31, 85 ¶ 5 (2001) (“[T]he peremptory norms that are clearly
accepted and recognized include the prohibitions of aggression, genocide, slavery, racial discrimination,
crimes against humanity and torture, and the right to self-determination.”).
40.
See Inter-Am. Ct. H.R., Proposed Amendments to the Naturalization Provisions of the
Political Constitution of Costa Rica, Advisory Opinion OC-4/84 (Jan. 19, 1984) ¶ 57–60.
41.
This principle was recently reiterated in the Global Compact for Migration, supra note 14,
¶ 15. For further discussion of state sovereignty over migration, see Catherine Dauvergne, Sovereignty, A. Nondiscrimination on the Basis of Nationality Favoring Venezuelan nationals with a special regularization scheme,
thereby facilitating the realization of their human rights, is indeed a legitimate
policy aim. Venezuelans currently account for more than three percent of the
Colombian population and have fled dire humanitarian circumstances. Yet, the
TPSV may conflict with the principle of nondiscrimination in human rights law. Discrimination, according to the International Convention on the Elimination of
All Forms of Racial Discrimination (CERD), is defined as differential treatment
in comparable situations on prohibited grounds, such as race or nationality, that
has the effect of “impairing the recognition, enjoyment or exercise, on an equal
footing, of human rights.”38 Non-Venezuelan migrants may find themselves in
similar conditions of multidimensional vulnerability but unable to obtain regular
residence and robust enjoyment of a full suite of human rights. Whether a
preference based on Venezuelan nationality is legitimate and proportional in
international nondiscrimination law must be subject to debate and scrutiny. While the prohibition of discrimination on the grounds of race constitutes
a jus cogens norm,39 distinctions drawn between migrants of different
nationalities has been considered permissible in international law under certain
circumstances. For instance, an early decision of the Inter-American Court of
Human Rights held that a naturalization preference adopted by Costa Rica for
nationals of Central American countries “has a legitimate purpose and . . . does
not lead to situations which are contrary to justice” because nationals of these
countries have a “closer historical, cultural and spiritual bonds with the people
of Costa Rica.”40Although migration governance is internationally recognized as
an area subject to sovereign regulation,41 human rights obligations partly THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 47: 80 88 constrain that state power.42 For example, Article 1(3) of the CERD provides that
“nothing in this Convention may be interpreted as affecting in any way the legal
provisions of States Parties concerning nationality, citizenship or naturalization,
provided that such provisions do not discriminate against any particular
nationality.”43 The CERD Committee has clarified that differential treatment on
the basis of nationality may constitute discrimination if immigration criteria “are
not applied pursuant to a legitimate aim, and are not proportional to the
achievement of this aim.”44 Although the U.N. Migration and the Rule of Law in Global Times, 67 MODERN L. REV. 588, 590 (2004).
42.
See, e.g., STEFANO ANGELERI, IRREGULAR MIGRANTS AND THE RIGHT TO HEALTH 17-68
(forthcoming 2022).
43.
CERD, supra note 38, art. 1(3).
44.
Id. at ¶ 4.
45.
See E. Tendayi Achiume (Special Rapporteur on Contemporary Forms of Racism, Racial
Discrimination, Xenophobia and Related Intolerance), Rep. on Contemporary Forms of Racism, Racial
Discrimination, Xenophobia and Related Intolerance, U.N. Doc. A/HRC/38/52 (Apr. 25, 2018), ¶ 16.
46.
Liav Orgad, When Is Immigration Selection Discriminatory?, 115 AM. J. INT. L. UNBOUND
345, 349 (2021).
47.
See generally, e.g., Carolina Moreno & Gracy Pelacani, Corte Constitucional Colombiana:
¿Un Escenario Posible para el Experimentalismo Constitucional en Materia Migratoria?, 5 LAT. AM. L.
REV. 139, 139-57 (2020).
48.
CCC, Judgment C-395/2002, ¶ 5.
49
CCC Judgments C-070/2004 ¶ 5 1; T-250/2017 ¶ 147; T-956 de 2013 ¶ 15 A. Nondiscrimination on the Basis of Nationality Special Rapporteur on Racism
specifies that these types of nationality-based distinctions must be “exceptional”
to avoid constituting discrimination,45 “international law provides little guidance
to help states distinguish between permissible and impermissible goals, criteria,
and means for immigrant selection.”46 In Colombia, no constitutional judgment has been rendered on the
preferential treatment of Venezuelans to date, but—even before the adoption of
the TPSV—a number of scholars have recommended that the state take measures
targeting the needs of non-Venezuelan migrants as well.47 The Constitutional
Court of Colombia has, however, decided cases involving alleged discrimination
between nationals and nonnationals and between regular and irregular migrants
regarding the enjoyment of human rights. The Court held that differential
treatment on the basis of nationality or legal status must be reasonable and
proportionate in order to comply with the principle of equality and
nondiscrimination.48 The intensity of the equality test depends, inter alia, on the
type of right at stake, the objective and reasonable character of the measure under
scrutiny, and the potential violation of international human rights obligations
wrought by the contested treatment.49 Thus, the principle of nondiscrimination
on the basis of nationality may provide a basis for litigation over the TPSV before
the high courts. 42.
See, e.g., STEFANO ANGELERI, IRREGULAR MIGRANTS AND THE RIGHT TO HEALTH 17-68
(forthcoming 2022). g
49.
CCC, Judgments C-070/2004, ¶ 5.1; T-250/2017, ¶ 147; T-956 de 2013, ¶ 15. 43.
CERD, supra note 38, art. 1(3). 48.
CCC, Judgment C-395/2002, ¶ 5. Migration and the Rule of Law in Global Times, 67 MODERN L. REV. 588, 590 (2004). 56.
See Baena-Ricardo et al. v. Panama, Merits, Reparations, and Costs, Judgment, Inter-Am.
Ct. H.R. (ser. C) No. 72, ¶ 124 (Feb. 2, 2001); Ivcher Bronstein v. Perú, Merits, Reparations, and Costs,
Judgment, Inter-Am. Ct. H.R. (ser. C) No. 74, ¶ 102 (Feb. 6, 2001); Tribunal Constitucional v. Perú,
Merits, Reparations, and Costs, Inter-Am. Ct. H.R. (ser. C) No. 71, ¶ 69 (Jan. 31, 2001); Judicial
Guarantees in States of Emergency (Arts. 27(2), 25 and 8 American Convention on Human Rights),
Advisory Opinion OC-9/87, Inter-Am. Ct. H.R. (ser. A) No. 9, ¶ 27 (Oct. 6, 1987). B. Due Process of Law Second, inadequate opportunities to contest the denial or revocation of the
TPP raise due process concerns under international law. While the TPSV scheme
aims to regularize the status of Venezuelan migrants for ten years, the
administrative decree that regulates it mentions the ability of the government to 89 A Rights-based Assessment of the Temporary Protection Statute 2022] “terminate the effects of the [TPSV] at any time.”50 This provision may
jeopardize the legal certainty that regularized Venezuelans enjoy, potentially
violating procedural rights guaranteed under international law. Furthermore,
Migración Colombia may decide to deny or rescind individual permits for a
number of reasons, including for relatively standardless reasons like being
considered “inconvenient” or “a threat to national security.”51 No administrative
appeal of such a negative decision is possible.52 Such administrative discretion
and the lack of opportunity to submit an administrative appeal does not appear
to comply with the right to due process of law as developed by the Inter-
American Court of Human Rights, including with regard to irregular migrants.53 Due process requires that a person is enabled to effectively claim their
rights, without discrimination on any personal grounds, vis-à-vis any state action
or inaction that may affect them.54 This applies to all proceedings concerning the
“determination of [a person’s] rights and obligations of a civil, labor, fiscal, or
any other nature,”55 including administrative proceedings.56 In compliance with
international standards, the Constitutional Court of Colombia, in cases
concerning migrant deportations, has also stressed the importance of the
opportunity for review in administrative proceedings.57 58.
See INGRID NIFOSI-SUTTON, THE PROTECTION OF VULNERABLE GROUPS UNDER
INTERNATIONAL HUMAN RIGHTS LAW (2017). 59.
See SANDRA FREDMAN, DISCRIMINATION LAW 14-19 (2d ed., 2011). 55.
See American Convention on Human Rights, art. 8(1), adopted Nov. 22, 1969, 1144
U.N.T.S. 143. 50.
Decree No. 216/2021, supra note 9, art. 2.
51.
Id. at art. 15(3).
52.
Id. at arts. 15(3)-(4).
53.
Juridical Condition and Rights of the Undocumented Migrants, Advisory Opinion OC-18,
Inter-Am. Ct. H.R. (ser. A) No. 18, ¶ 121 (Sept. 17, 2003).
54.
See, e.g., The Right to Information on Consular Assistance in the Framework of the
Guarantees of the Due Process of Law, Advisory Opinion OC-16, Inter-Am. Ct. H.R. (ser. A) No. 16,
¶¶ 117, 119 (Oct. 1, 1999); Juridical Condition and Human Rights of the Child, Advisory Opinion OC-
17, Inter-Am. Ct. H.R. (ser. A) No. 17, ¶¶ 97, 115 (Aug. 28, 2002); Advisory Opinion OC-18, supra note
53, ¶¶ 121, 122.
55.
See American Convention on Human Rights, art. 8(1), adopted Nov. 22, 1969, 1144
U.N.T.S. 143.
56.
See Baena-Ricardo et al. v. Panama, Merits, Reparations, and Costs, Judgment, Inter-Am.
Ct. H.R. (ser. C) No. 72, ¶ 124 (Feb. 2, 2001); Ivcher Bronstein v. Perú, Merits, Reparations, and Costs,
Judgment, Inter-Am. Ct. H.R. (ser. C) No. 74, ¶ 102 (Feb. 6, 2001); Tribunal Constitucional v. Perú,
Merits, Reparations, and Costs, Inter-Am. Ct. H.R. (ser. C) No. 71, ¶ 69 (Jan. 31, 2001); Judicial
Guarantees in States of Emergency (Arts. 27(2), 25 and 8 American Convention on Human Rights),
Advisory Opinion OC-9/87, Inter-Am. Ct. H.R. (ser. A) No. 9, ¶ 27 (Oct. 6, 1987).
57.
See, e.g., CCC, Judgment T-143/2019, ¶ 8, 9, and 16.
58.
See INGRID NIFOSI-SUTTON, THE PROTECTION OF VULNERABLE GROUPS UNDER
INTERNATIONAL HUMAN RIGHTS LAW (2017).
59.
See SANDRA FREDMAN, DISCRIMINATION LAW 14-19 (2d ed., 2011). 52.
Id. at arts. 15(3)-(4). y
p
(
)
¶
(
57.
See, e.g., CCC, Judgment T-143/2019, ¶ 8, 9, and 16. 51.
Id. at art. 15(3). 53.
Juridical Condition and Rights of the Undocumented Migrants, Advisory Opinion OC-18,
Inter-Am. Ct. H.R. (ser. A) No. 18, ¶ 121 (Sept. 17, 2003). 50.
Decree No. 216/2021, supra note 9, art. 2. 54.
See, e.g., The Right to Information on Consular Assistance in the Framework of the
Guarantees of the Due Process of Law, Advisory Opinion OC-16, Inter-Am. Ct. H.R. (ser. A) No. 16,
¶¶ 117, 119 (Oct. 1, 1999); Juridical Condition and Human Rights of the Child, Advisory Opinion OC-
17, Inter-Am. Ct. H.R. (ser. A) No. 17, ¶¶ 97, 115 (Aug. 28, 2002); Advisory Opinion OC-18, supra note
53, ¶¶ 121, 122. y
p
(
)
(
)
57.
See, e.g., CCC, Judgment T-143/2019, ¶ 8, 9, and 16. Advisory Opinion OC-9/87, Inter-Am. Ct. H.R. (ser. A) No. 9, ¶ 27 (Oct. 6, 1987) C. Special Measures Targeting Migrants with Special Vulnerabilities Some migrants in situations of vulnerability may require special,
affirmative efforts to ensure the enjoyment of their human rights.58 The doctrine
of substantive equality requires that people in disadvantaged positions should be
prioritized in law and policymaking in order to facilitate the realization of their
human rights.59 The New York Declaration on Refugees and Migrants, for
example, recognizes categories of vulnerability like “women at risk, children,
especially those who are unaccompanied or separated from their families,
members of ethnic and religious minorities, victims of violence, older persons, 90 THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 47: 80 persons with disabilities, persons who are discriminated against on any basis,
indigenous peoples, victims of human trafficking, and victims of exploitation
and abuse in the context of the smuggling of migrants.”60 While the TPSV
regulations address the needs of some such vulnerable groups, described below,
a well-rounded approach to human rights-based policymaking should have
encompassed more categories of people in vulnerable conditions for special,
treatment under the TPSV scheme. The special treatment afforded to certain migrant children, pregnant
women, persons with disabilities, and transgender communities are evidence of
the adaptability of the TPSV scheme to Venezuelans who may experience
particular vulnerabilities.61 The special attention granted to children62 is
consistent with Colombia’s constitutional jurisprudence, which frequently
employs international law on the protection of children as a basis for enforcing
domestic protections.63 Under the TPSV, some children are eligible for early
processing of their applications, including children enrolled in the school system
and those who are about to graduate, children who are in special care settings,
and children who are in reeducation programs.64 Early processing of their
applications expedites their regularization and, consequently, the realization of
their human rights. Additionally, children in these categories are also eligible to
register for TPSV regularization until May 2031, as compared with the May 2022
and May 2023 deadlines for other Venezuelans.65 The fact that these protections
only apply to some children, however, appears to minimize the special
vulnerability experienced by all children. Other vulnerable group, are given different accommodations: People who
are transgender are entitled to register for regularization with the name they
recognize fitting their gender identity, if the latter is legally recognized by a
notary.66 Persons with disabilities, children, pregnant women, and the elderly are
eligible are prioritized for appointments to collect their biometric data.67 60.
See New York Declaration on Refugees and Migrants, G.A. Res. 71.1, U.N. Doc.
A/RES/71/1 (Sep. 16, 2016), at ¶ 23; Global Compact for Migration, supra note 14, Preamble, recital 7.
61.
See Res. No. 971/21, supra note 9, arts. 7, 8 and Tit. IV and V.
62.
See id. at Title IV.
63.
See, e.g., CCC, Judgments T-215/1996, T-090/21, T-390/20, T-530/19, T-530/19, T-530/19,
T-250/17, T-197/19; see also Beatriz Londoño, María Teresa Palacios & María Alejandra Lozano, Nuevas
Realidades de los Flujos Migratorios Hacia Colombia, in MIGRACIÓN Y DERECHOS HUMANOS: EL CASO
COLOMBIANO 2014-2018, at 11, 25 (Beatriz Londoño Toro & María Teresa Palacios Sanabria eds., 2019).
64.
See Res. No. 971/21, supra note 9, art. 33.
65.
See id. at arts. 25 and 26.
66.
See id. at Title V.
67.
See Res. No. 971/21, supra note 9, art. 7. 66.
See id. at Title V. 62.
See id. at Title IV. 63.
See, e.g., CCC, Judgments T-215/1996, T-090/21, T-390/20, T-530/19, T-530/19, T-530/19,
T-250/17, T-197/19; see also Beatriz Londoño, María Teresa Palacios & María Alejandra Lozano, Nuevas
Realidades de los Flujos Migratorios Hacia Colombia, in MIGRACIÓN Y DERECHOS HUMANOS: EL CASO
COLOMBIANO 2014-2018, at 11, 25 (Beatriz Londoño Toro & María Teresa Palacios Sanabria eds., 2019).
64
See Res No 971/21 supra note 9 art 33 65.
See id. at arts. 25 and 26. 67.
See Res. No. 971/21, supra note 9, art. 7. 60.
See New York Declaration on Refugees and Migrants, G.A. Res. 71.1, U.N. Doc.
A/RES/71/1 (Sep. 16, 2016), at ¶ 23; Global Compact for Migration, supra note 14, Preamble, recital 7. ,
,
(
64.
See Res. No. 971/21, supra note 9, art. 33. A/RES/71/1 (Sep. 16, 2016), at ¶ 23; Global Compact for Migration, supra note 14, Preamble, recital 7.
61.
See Res. No. 971/21, supra note 9, arts. 7, 8 and Tit. IV and V.
62
S
id
Ti l IV 61.
See Res. No. 971/21, supra note 9, arts. 7, 8 and Tit. IV and V.
6
d
i l 60.
See New York Declaration on Refugees and Migrants, G.A. Res. 71.1, U.N. Doc.
/RES/71/1 (Sep. 16, 2016), at ¶ 23; Global Compact for Migration, supra note 14, Preamble, recital 7.
61.
See Res. No. 971/21, supra note 9, arts. 7, 8 and Tit. IV and V.
62
See id at Title IV 68.
For instance, in informal interviews conducted by Angeleri with staff at the International
Organization for Migration and Jesuit Refugee Council in February 2022, informants reported that many
TPP holders encountered formal and administrative barriers (linked to the format of the TPP) which
prevented their enrollment in the health care system. 73.
See Cartagena Declaration on Refugees, Title 3, ¶ 3, opened for signature Nov. 22, 1984.
The Cartagena Declaration was incorporated in Decree No. 1067/2015, supra note 71, art. 2.2.3.1.1.1(a). D. Creation of a Meaningful Legal Status Additionally, although the TPSV was created in part to ensure that
Venezuelans have access to a legal status, it is possible that Venezuelans who
are granted a TPP pursuant to the TPSV may nevertheless continue to experience
the sorts of vulnerabilities typically associated with a lack of legal status. For
instance, if banks, chambers of commerce, power companies, broadband 91 2022] A Rights-based Assessment of the Temporary Protection Statute companies, or health care providers do not recognize the TPP as a valid
document with which to register for their services, the TPP itself and the person’s
legal status are deprived of significance, despite de jure recognition of the
status.68 This state of affairs would jeopardize the socioeconomic integration of
Venezuelans that the TPSV pursues and breach international obligations
guaranteed by the ICMW.69 71.
See MINISTERIO DE RELACIONES EXTERIORES, Por Medio del Cual se Expide el Decreto
Único Reglamentario del Sector Administrativo de Relaciones Exteriores, Decree No. 1067/2015 (May
26, 2015). 72.
See Convention Relating to the Status of Refugees, opened for signature July 28, 1951, 189
U.N.T.S. 137; CCC, Judgments T-250/2017, ¶¶ 24, 139; C-186/1996, ¶ 3.2; T-704/2003, ¶ 2(b). 76.
See U.N. HIGH COMM’R FOR REFUGEES, Factsheet Colombia: January-December 2020, at
1, https://www.acnur.org/op/op_fs/6058c69d4/unhcr-colombia-fact-sheet-january-december-2020.html. 75.
According to the R4V portal, 971,170 asylum applications have been filed in the countries
of the region. R4V, supra note 8. The portal indicates that 28,800 asylum applications are pending in
Colombia. Id. p
y
69.
International Convention on the Protection of the Rights of All Migrant Workers and
Members of Their Families, Part IV, opened for signature Dec. 18, 1990, 2220 U.N.T.S. 3. 74.
See Forced Migration of Venezuelans, Inter-Am. Comm’n H.R., Res. 2/2018 (Mar. 2, 2018),
https://www.oas.org/en/iachr/decisions/pdf/Resolution-2-18-en.pdf; U.N. HIGH COMM’R FOR REFUGEES,
Guidance Note on International Protection Considerations for Venezuelans (May 21, 2019),
https://www.refworld.org/docid/5cd1950f4.html. 70.
See id. at art. 36. 68.
For instance, in informal interviews conducted by Angeleri with staff at the International
Organization for Migration and Jesuit Refugee Council in February 2022, informants reported that many
TPP holders encountered formal and administrative barriers (linked to the format of the TPP) which
prevented their enrollment in the health care system.
69.
International Convention on the Protection of the Rights of All Migrant Workers and
Members of Their Families, Part IV, opened for signature Dec. 18, 1990, 2220 U.N.T.S. 3.
70.
See id. at art. 36.
71.
See MINISTERIO DE RELACIONES EXTERIORES, Por Medio del Cual se Expide el Decreto
Único Reglamentario del Sector Administrativo de Relaciones Exteriores, Decree No. 1067/2015 (May
26, 2015).
72.
See Convention Relating to the Status of Refugees, opened for signature July 28, 1951, 189
U.N.T.S. 137; CCC, Judgments T-250/2017, ¶¶ 24, 139; C-186/1996, ¶ 3.2; T-704/2003, ¶ 2(b).
73.
See Cartagena Declaration on Refugees, Title 3, ¶ 3, opened for signature Nov. 22, 1984.
The Cartagena Declaration was incorporated in Decree No. 1067/2015, supra note 71, art. 2.2.3.1.1.1(a).
74.
See Forced Migration of Venezuelans, Inter-Am. Comm’n H.R., Res. 2/2018 (Mar. 2, 2018),
https://www.oas.org/en/iachr/decisions/pdf/Resolution-2-18-en.pdf; U.N. HIGH COMM’R FOR REFUGEES,
Guidance Note on International Protection Considerations for Venezuelans (May 21, 2019),
https://www.refworld.org/docid/5cd1950f4.html.
75.
According to the R4V portal, 971,170 asylum applications have been filed in the countries
of the region. R4V, supra note 8. The portal indicates that 28,800 asylum applications are pending in
Colombia. Id.
76.
See U.N. HIGH COMM’R FOR REFUGEES, Factsheet Colombia: January-December 2020, at
1, https://www.acnur.org/op/op_fs/6058c69d4/unhcr-colombia-fact-sheet-january-december-2020.html. E. Neglect of Domestic Refugee Protection Finally, the emphasis placed on the TPSV marginalizes Colombia’s
refugee recognition procedures, potentially jeopardizing the rights of prospective
refugees and asylum seekers. The Colombian Constitution includes the right to seek asylum,70 and
regulations establish the procedure for granting of refugee status.71 Status is
granted according to the definition contained in the Refugee Convention of
1951.72 Colombia has also signed the Cartagena Declaration, which “includes
among refugees persons who have fled their country because their lives, safety
or freedom have been threatened by . . . massive violation of human rights or
other circumstances which have seriously disturbed public order.”73 If applied,
the political, social, and economic reality that Venezuelan migrants are fleeing
might well prima facie qualify many of them as refugees in Colombia, as
demanded by the Inter-American Commission.74 Despite the country’s apparently favorable legal frameworks, the number
of applications for asylum filed in Colombia is relatively low.75 The most recent
available statistics show that fewer than 700 people were granted refugee status
in the first half of 2020 (sixty-seven percent of whom were Venezuelans), of
more than 1.7 million Venezuelans who were living in Colombia.76 By April 92 THE YALE JOURNAL OF INTERNATIONAL LAW ONLINE [Vol. 47: 80 2022, more than 2.4 million Venezuelans were registered in the TPSV scheme,77
while there were only 28,800 pending asylum applications from Venezuelan
nationals.78 It is much easier to apply for a TPP pursuant to the TPSV than to
undergo a refugee status determination process. First, applications for refugee
status must be filed within sixty days of entering the country.79 Second, status
determination proceedings typically last two to three years or more, while the
average duration of the TPP application process is roughly seven months.80
Third, asylum seekers can neither freely move nor work in Colombia while their
application is pending.81 Fourth, asylum seekers must re-register every ninety
days to maintain their status as asylum seekers.82 For many Venezuelans, these
limitations make it clearly preferable to pursue the TPP and jeopardize the
implementation of the right to seek and obtain asylum as set forth in national and
international law. Despite the shortcomings of the refugee recognition system, the refugee
protection system and TPSV procedures could have been more effectively
harmonized. 86.
See generally Cathryn Costello & Colm O’Cinneide, The Right to Work of Asylum Seekers
and Refugees, ASILE Working Paper (May 10, 2021), https://www.asileproject.eu/wp-content/
uploads/2021/06/D4.1.CostelloOCinneide_RightToWorkASILE_10May2021_REVISED.pdf. 84.
See Rights and Guarantees of Children in the Context of Migration and/or in Need of
International Protection, Advisory Opinion OC-21/14, Inter-Am. Ct. H.R. (ser. A) No. 21, ¶ 225 (Aug.
19, 2014); Cartagena Declaration, supra note 73; CONSTITUCIÓN POLÍTICA DE COLOMBIA, supra note 18,
art. 93. 82.
See id. at art. 2.2.1.11.4.9. 85.
See Decree No. 216/2021, supra note 9, art. 17. 83.
Cf. Decree No. 1067/2015, supra note 71; Decree No. 216/2021, supra note 9. 81.
See Decree No. 1067/2015, supra note 71, arts. 2.2.1.11.5.1; 2.2.3.1.4.1. 77.
See MIGRACIÓN COLOMBIA, supra note 13.
78.
See R4V, supra note 8.
79.
See Decree No. 1067/2015, supra note 71, art. 2.2.3.1.6.1.
80.
See, e.g., Gracy Pelacani et al., Estatuto Temporal de Protección para Migrantes
Venezolanos: Reflexiones de una Política de Regularización Migratoria [Temporary Protection Statute
for Venezuelan Migrant: Reflections on a Migrant Regularization Policy], Rep. No. 3-2021, at 31,
CENTRO
DE ESTUDIOS
EN MIGRACIÓN, UNIVERSIDAD
DE
LOS ANDES (July 30, 2021),
https://migracionderecho.uniandes.edu.co/wp-content/uploads/Informe-CEM-3-Estatuto-Temporal-de-
Proteccion-para-Migrantes-Venezolanos-reflexiones-de-una-politica-de-regularizacion-migratoria-2.pdf;
E-mail from the Ministerio de Relaciones Exteriores to the Legal Clinic on International Human Mobility
at U. del Rosario (Mar. 1, 2021) (on file with author). 77.
See MIGRACIÓN COLOMBIA, supra note 13.
78.
See R4V, supra note 8.
79.
See Decree No. 1067/2015, supra note 71, art. 2.2.3.1.6.1.
80.
See, e.g., Gracy Pelacani et al., Estatuto Temporal de Protección para Migrantes
Venezolanos: Reflexiones de una Política de Regularización Migratoria [Temporary Protection Statute
for Venezuelan Migrant: Reflections on a Migrant Regularization Policy], Rep. No. 3-2021, at 31,
CENTRO
DE ESTUDIOS
EN MIGRACIÓN, UNIVERSIDAD
DE
LOS ANDES (July 30, 2021),
https://migracionderecho.uniandes.edu.co/wp-content/uploads/Informe-CEM-3-Estatuto-Temporal-de-
Proteccion-para-Migrantes-Venezolanos-reflexiones-de-una-politica-de-regularizacion-migratoria-2.pdf;
E-mail from the Ministerio de Relaciones Exteriores to the Legal Clinic on International Human Mobility
at U. del Rosario (Mar. 1, 2021) (on file with author).
81.
See Decree No. 1067/2015, supra note 71, arts. 2.2.1.11.5.1; 2.2.3.1.4.1.
82.
See id. at art. 2.2.1.11.4.9.
83.
Cf. Decree No. 1067/2015, supra note 71; Decree No. 216/2021, supra note 9.
84.
See Rights and Guarantees of Children in the Context of Migration and/or in Need of
International Protection, Advisory Opinion OC-21/14, Inter-Am. Ct. H.R. (ser. A) No. 21, ¶ 225 (Aug.
19, 2014); Cartagena Declaration, supra note 73; CONSTITUCIÓN POLÍTICA DE COLOMBIA, supra note 18,
art. 93.
85.
See Decree No. 216/2021, supra note 9, art. 17.
86.
See generally Cathryn Costello & Colm O’Cinneide, The Right to Work of Asylum Seekers
and Refugees, ASILE Working Paper (May 10, 2021), https://www.asileproject.eu/wp-content/
uploads/2021/06/D4.1.CostelloOCinneide_RightToWorkASILE_10May2021_REVISED.pdf. at U. del Rosario (Mar. 1, 2021) (on file with author).
81.
See Decree No. 1067/2015, supra note 71, arts. 2.2.1.11.5.1; 2.2.3.1.4.1. E. Neglect of Domestic Refugee Protection Unlike the TPSV scheme, which allows for the cancellation of
status based on “inconvenience,” a grant of refugee status has much stricter
criteria for withdrawal.83 Therefore, revocation of a TPP without the chance to
present an asylum claim could conceivably amount to a violation of the
peremptory norm of non-refoulement.84 Furthermore, once granted a TPP,
asylum seekers must either decide whether they wish to continue with the asylum
application and renounce the TPP or continue with the latter and withdraw from
the asylum process.85 Given the delay in obtaining decisions from public
authorities regarding international protection and the limitations to asylum
seekers’ rights, the TPP could have been designed as a temporary mechanism for
asylum seekers, allowing them to enjoy key human rights, such as the right to
work, while their refugee status recognition process is ongoing.86 This approach
would have allowed Venezuelans to benefit from the best aspects of both 2022] 93 A Rights-based Assessment of the Temporary Protection Statute schemes: Swift legal status and the right to work under the TPP, and the security
of a durable status under Colombian refugee law. CONCLUSION By adopting the TPSV, the Colombian government took a significant step
towards a rights-oriented approach to managing mixed migration. Yet, while the
TPSV regulations are rife with human rights rhetoric, we raise questions about
whether the scheme, in its totality, is compliant with international law. If
Colombia and other states responding to massive mixed migration flows are to
genuinely uphold human rights law, regularization schemes used to address those
flows must be substantively and procedurally consistent with human rights law
and respectful of refugee law.
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https://openalex.org/W2889479813
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https://www.frontiersin.org/articles/10.3389/fnmol.2018.00303/pdf
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English
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Dopamine Modulates Homeostatic Excitatory Synaptic Plasticity of Immature Dentate Granule Cells in Entorhino-Hippocampal Slice Cultures
|
Frontiers in molecular neuroscience
| 2,018
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cc-by
| 6,902
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Edited by:
Anja U. Bräuer,
University of Oldenburg, Germany
Reviewed by:
Ulf Strauss,
Charité Universitätsmedizin Berlin,
Germany
Fabrice R. Turpin,
The University of Queensland,
Australia Edited by:
Anja U. Bräuer,
University of Oldenburg, Germany Reviewed by:
Ulf Strauss,
Charité Universitätsmedizin Berlin,
Germany
Fabrice R. Turpin,
The University of Queensland,
Australia Reviewed by:
Ulf Strauss,
Charité Universitätsmedizin Berlin,
Germany
Fabrice R. Turpin,
The University of Queensland,
Australia *Correspondence:
Andreas Vlachos
andreas.vlachos@anat.uni-
freiburg.de
†These authors have contributed
equally to this work *Correspondence:
Andreas Vlachos
andreas.vlachos@anat.uni-
freiburg.de Keywords: homeostasis, synaptic scaling, neurogenesis, hippocampus, neuromodulation, stem cells Dopamine Modulates Homeostatic
Excitatory Synaptic Plasticity of
Immature Dentate Granule Cells in
Entorhino-Hippocampal Slice
Cultures Andreas Strehl 1†, Christos Galanis 2†, Tijana Radic 1, Stephan Wolfgang Schwarzacher 1,
Thomas Deller 1 and Andreas Vlachos 1,2* 1Neuroscience Center, Institute of Clinical Neuroanatomy, Goethe-University Frankfurt, Frankfurt, Germany, 2Department of
Neuroanatomy, Institute of Anatomy and Cell Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany Homeostatic plasticity mechanisms maintain neurons in a stable state. To what extent
these mechanisms are relevant during the structural and functional maturation of neural
tissue is poorly understood. To reveal developmental changes of a major homeostatic
plasticity mechanism, i.e., homeostatic excitatory synaptic plasticity, we analyzed
1-week- and 4-week-old entorhino-hippocampal slice cultures and investigated the
ability of immature and mature dentate granule cells (GCs) to express this form of
plasticity. Our experiments demonstrate that immature GCs are capable of adjusting
their excitatory synaptic strength in a compensatory manner at early postnatal stages,
i.e., in 1-week-old preparations, as is the case for mature GCs. This ability of
immature dentate GCs is absent in 4-week-old slice cultures. Further investigations
into the signaling pathways reveal an important role of dopamine (DA), which prevents
homeostatic synaptic up-scaling of immature GCs in young cultures, whereas it does
not affect immature and mature GCs in 4-week-old preparations. Together, these
results disclose the ability of immature GCs to express homeostatic synaptic plasticity
during early postnatal development. They hint toward a novel role of dopaminergic
signaling, which may gate activity-dependent changes of newly born neurons by
blocking homeostasis. ORIGINAL RESEARCH
published: 30 August 2018
doi: 10.3389/fnmol.2018.00303 INTRODUCTION Received: 29 March 2018
Accepted: 09 August 2018
Published: 30 August 2018 Homeostatic plasticity plays a fundamental role in maintaining neural networks in a stable state. Among the best studied forms is homeostatic synaptic plasticity, which adjusts synaptic strength
in a compensatory manner to changes in network activity (Davis, 2006; Marder and Goaillard,
2006; Turrigiano, 2008; Pozo and Goda, 2010). In recent years, numerous studies have addressed
the cellular and molecular mechanisms of homeostatic synaptic plasticity in various experimental
conditions (e.g., Marder and Goaillard, 2006; Turrigiano, 2012; Tien and Kerschensteiner, 2018). Yet, its relevance in neural development and maturation remains poorly understood. In this
context, we hypothesized that homeostatic plasticity, i.e., negative feedback mechanisms that aim
at stabilizing neural networks by preventing major changes in network structure and function,
may hinder the efficient activity-dependent maturation and network integration of neurons. Strehl A, Galanis C, Radic T,
Schwarzacher SW, Deller T
and Vlachos A (2018) Dopamine
Modulates Homeostatic Excitatory
Synaptic Plasticity of Immature
Dentate Granule Cells in
Entorhino-Hippocampal
Slice Cultures.
Front. Mol. Neurosci. 11:303.
doi: 10.3389/fnmol.2018.00303 Keywords: homeostasis, synaptic scaling, neurogenesis, hippocampus, neuromodulation, stem cells Citation: Strehl A, Galanis C, Radic T,
Schwarzacher SW, Deller T
and Vlachos A (2018) Dopamine
Modulates Homeostatic Excitatory
Synaptic Plasticity of Immature
Dentate Granule Cells in
Entorhino-Hippocampal
Slice Cultures. Front. Mol. Neurosci. 11:303. doi: 10.3389/fnmol.2018.00303 August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 1 Synaptic Scaling of Immature Granule Cells Strehl et al. Whole-Cell Patch-Clamp Recordings
Whole-cell patch-clamp recordings from dentate GCs of slice
cultures were carried out at 35◦C as described previously
(≤5 neurons per culture; Vlachos et al., 2012). The bath
solution contained (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3,
1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 glucose. Patch pipettes
contained (in mM) 126 K-gluconate, 10 HEPES, 4 KCl, 4 ATP-
Mg, 0.3 GTP-Na2, 10 PO-Creatine and 0.3% (w/v) Biocytin
(pH = 7.25 with KOH, 290 mOsm with sucrose) having a
tip resistance of 4–6 MΩ. In some experiments Alexa488 or
Alexa568 (both 10 µM) was added to the internal solution to
visualize neuronal morphology prior to recordings. Neurons
were recorded at holding potential of −70 mV in the presence of
10 µM D-APV and 0.5 µM TTX. In presence of the α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist CNQX (10 µM) no postsynaptic current events were
detected (data not shown). Series resistance was monitored in
2 min intervals, and recordings were discarded if the series
resistance and leak current changed significantly and/or reached
≥30 MΩor ≥100 pA, respectively. Data were low pass filtered at
6 kHz and digitized at 10 kHz. Indeed, in an earlier study we were able to provide evidence that
CA1 pyramidal neurons acquire homeostatic properties between
2 weeks and 4 weeks of postnatal in vitro maturation (Vlachos
et al., 2013), suggesting that homeostatic synaptic plasticity is a
property that neurons acquire during the course of maturation,
i.e., after a set-point is reached. Indeed, in an earlier study we were able to provide evidence that
CA1 pyramidal neurons acquire homeostatic properties between
2 weeks and 4 weeks of postnatal in vitro maturation (Vlachos
et al., 2013), suggesting that homeostatic synaptic plasticity is a
property that neurons acquire during the course of maturation,
i.e., after a set-point is reached. Immunostaining and Imaging Slice cultures were fixed, re-sliced and immunostained as
described previously (Lenz et al., 2016) with antibodies against
doublecortin (DCX; C-18; goat; Santa Cruz; 1:500) and
calbindin (Calb; D28-K, mouse; Swant; 1:1,000). For post hoc-
immunostainings, slice cultures were incubated with Alexa568-
conjugated streptavidin (Life Technologies; 1:500). Confocal
images were acquired using a Nikon Eclipse C1si laser-scanning
microscope equipped with a 40× oil-immersion objective lens
(NA 1.3; Nikon) and a 60× oil-immersion objective lens (NA 1.4;
Nikon). Quantification and Statistics Immunostainings were analyzed using the ImageJ software
package (Schindelin et al., 2012). To quantify the distribution
of DCX- and Calb-positive GCs, confocal image stacks of the
dentate gyrus were analyzed using the cell-counter plugin for
ImageJ. Preparation of Slice Cultures Entorhino-hippocampal slice cultures were prepared at postnatal
day 4–5 from C57BL/6J and CB6F1/J mice of either sex as
previously described (Vlachos et al., 2012). Cultures were allowed
to mature in vitro for either 5–6 days (1-week-old) or 22–25 days
(4-week-old). Discrimination Between Mature and
Immature Dentate Granule Cells All experimental procedures were performed according to the
German animal welfare legislation and approved by the local
animal welfare officers of Goethe-University Frankfurt and
Albert-Ludwigs-University Freiburg, Faculties of Medicine. Mice
were maintained in a 12 h light/dark cycle with food and water
available ad libitum. Every effort was made to minimize distress
and pain of animals. Recorded GCs were ad hoc characterized based on the position
of the soma within the GC layer (GCL) and their resting
membrane potential (RMP) and capacitance (C; Spampanato
et al., 2012). GCs with a RMP > −65 mV and C <30 pF
were considered immature. Some of the recorded cells were
post hoc identified, and their developmental stage was assessed
using immunostaining with antibodies against DCX and Calb
(Duan et al., 2008). Citation: The dentate gyrus of the hippocampus is an attractive model
to study homeostasis of immature neurons, since dentate granule
cells (GCs) are born and differentiate postnatally, both in vivo
(Altman and Das, 1965; van Praag et al., 2002; Toni et al., 2007;
Kempermann et al., 2015; Nicola et al., 2015; Cahill et al., 2017;
Radic et al., 2017a) and in vitro (Raineteau et al., 2004; Radic et al.,
2017b). Previous work has demonstrated that immature GCs
are capable of expressing Hebbian forms of synaptic plasticity,
i.e., long-term potentiation (LTP) of excitatory synaptic strength
(e.g., Schmidt-Hieber et al., 2004; Gonçalves et al., 2016). Hitherto, it has not been tested whether immature GCs also
express homeostatic synaptic plasticity. In this study entorhino-hippocampal slice cultures were
treated
with
a
well-established
pharmacological
protocol,
i.e., network activity blockade with tetrodotoxin (TTX; 2 µM;
for 3 days), to test for dentate GC excitatory synaptic up-scaling
(Turrigiano et al., 1998). Compensatory changes in excitatory
synaptic strength were assessed using whole-cell patch-clamp
recordings of immature and mature GCs in young (5–6 days
in vitro; 1-week-old) vs. old (22–25 days in vitro; 4-week-old)
slice cultures. Considering that dopamine (DA) modulates the
ability of GCs to express Hebbian plasticity (Mu et al., 2011), we
sought to test for the relevance of DA in regulating homeostatic
excitatory synaptic plasticity of dentate GCs. The D2-Like Receptor Agonist Quinpirole
Does Not Affect TTX-Induced Homeostatic
Plasticity in Immature Granule Cells of
1-Week-Old Slice Cultures The
experiments
in
which
we
combined
DA
and
the
D1/5 receptor blocker SCH23390 argued against the involvement
of other DA receptors, since these receptors are still activated
by DA in the presence of SCH23390. Yet, we decided to err on
the side of caution and hence tested for the effects of the D2-like
agonist quinpirole. As shown in Figure 2E, TTX-induced
synaptic scaling was not blocked in the presence of 20 µM
quinpirole. Although a trend toward higher mEPSC amplitudes
in the TTX + quinpirole group was detected, this effect did not
reach the level of significance when compared to the TTX +
vehicle only group (Figure 2E). As shown in Figure 1E, a robust increase in excitatory
synaptic strength was observed in immature GCs of TTX-treated
slice cultures. Based on this result, we conclude that immature
dentate GCs are capable of increasing their excitatory synaptic
strength in a compensatory manner, similar to what is observed
in mature cultured dentate GCs (Vlachos et al., 2012). Immature Dentate Granule Cells in
1-Week-Old Slice Cultures Scale Their
Excitatory Synapses Individual dentate GCs in the inner part of the GCL were
patched in 1-week-old slice cultures, and AMPA receptor-
mediated mEPSCs were recorded from TTX-treated cultures
(2 µM; 3 days) as well as age-/time-matched vehicle-treated
controls (Figure 1). Immature, i.e., DCX-positive, dentate GCs
were readily detectable in 1-week-old slice preparations in this
zone of the GCL (Figures 1A,B). In some cases, recorded
dentate GCs were post hoc immunostained for DCX (Figure 1D),
while position of the soma within the GCL and the RMP
(RMP > −65 mV; mean: −46.4 ± 2.1 mV) served as primary
criteria for immature GCs. It may be important to mention in this
context that some cells exhibited a particular high RMP and did
not show any mEPSC events, suggesting that AMPA receptor-
carrying synapses were not yet formed on these cells (Espósito
et al., 2005). mEPSCs were readily detectable in all neurons
with RMP of −65 mV to −25 mV in this set of experiments
(Figures 1C–E). Pharmacology Slice cultures were treated with TTX (sodium-channel blocker;
2
µM;
BioTrend),
DA
(20
µM;
TOCRIS),
SCH23390
hydrochloride
(D1-like
antagonist;
40
µM;
TOCRIS),
SKF81297 hydrobromide (D1-like agonist; 20 µM; TOCRIS),
quinpirole hydrochloride (D2-like agonist; 20 µM; TOCRIS)
for 3 days. Incubation medium was replaced once with fresh
drug-containing medium. Electrophysiological data were filtered using a low pass
elliptic filter with a cutoff frequency of 1 kHz and analyzed
off-line by two independent observers using pClamp 10.2 (Axon
Instruments, USA) and MiniAnalysis (Synaptosoft, Decatur,
GA, USA). 150–300 miniature excitatory postsynaptic current
(mEPSC) events were analyzed per recorded neuron. August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 2 Synaptic Scaling of Immature Granule Cells Strehl et al. 1-week-old
slice
cultures
which
were
treated
with
DA
(20 µM; 3 days). Indeed, in the presence of DA, the
TTX-induced increase in excitatory synaptic strength did
not emerge in immature dentate GCs of 1-week-old cultures
(Figures 2A,B). Statistical comparisons were performed using non-parametric
tests, i.e., Mann-Whitney test (to compare two groups) or
the Kruskal-Wallis test followed by Dunn’s post hoc test (for
multiple comparisons) using GraphPad Prism 7 (GraphPad
Software Inc.). p values smaller 0.05 were considered a
significant difference. In the text and figure values represent
mean ± standard error of the mean (SEM). ∗p < 0.05, ∗∗p < 0.01,
∗∗∗p < 0.001, and non-significant differences are indicated by
‘‘NS.’’ U-values are provided for significant results in the figure
captions. Interestingly, DA did not affect TTX-induced synaptic
up-scaling of mature, i.e., Calb-positive GCs (RMP ≤−65 mV),
which were recorded in the same set of cultures in these
experiments (Figures 2A,B). We conclude that DA prevents
homeostatic synaptic plasticity specifically in immature but not
mature dentate GCs in 1-week-old slice cultures. D1/5 Receptor Activation Blocks
Homeostatic Plasticity in Immature
Granule Cells of 1-Week-Old Cultures To confirm and extend these findings, TTX-experiments were
repeated in a different set of 1-week-old slice cultures using
the D1/5 receptor agonist SKF81297. Immature GCs did not
increase their mEPSC amplitudes when TTX (2 µM) was
applied together with 20 µM SKF81297 for 3 days (Figure 2C). TTX-induced synaptic scaling was not affected in presence of
SKF81297 in mature dentate GCs recorded from the same set of
cultures, while higher baseline mEPSC amplitudes were observed
for mature GCs in these recordings (control: 17.9 ± 0.9 pA;
TTX: 27.2 ± 2.1 pA; ncontrol = 8 neurons, nTTX = 6 neurons;
Mann-Whitney test, p < 0.001, U-value = 0). Consistent with
this result, TTX-induced synaptic scaling of immature GCs
emerged in presence of DA (20 µM), when the D1/5 receptor
antagonist SCH23390 (40 µM) was co-applied (Figure 2D). We
conclude that DA acts via D1/5 receptors to block TTX-induced
homeostatic synaptic plasticity in immature GCs of 1-week-old
cultures. Digital Illustrations Confocal image stacks were stored as TIF files. Figures were
prepared using Photoshop graphics software (Adobe, San Jose,
CA, USA). Image brightness and contrast were adjusted. Dopamine Blocks Homeostatic Plasticity
in Immature but Not Mature Granule Cells
of 1-Week-Old Cultures (C) Sample traces of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated miniature excitatory
postsynaptic currents (mEPSCs) recorded from immature GCs in tetrodotoxin TTX (2 µM, 3 days) and vehicle-only (control)-treated cultures. (D) Example of a
recorded (biocytin-filled), post hoc identified (streptavidin-Alexa568, white) immature GC in a DCX-stained slice culture. Scale bars, 10 µm and 5 µm (inset). (E) Group data of mEPSC recordings (amplitudes and frequencies) from immature GCs of vehicle-only-(Ctrl, control) and TTX-treated cultures (ncontrol = 9 cells,
nTTX = 9 cells; Mann-Whitney test; U-value = 6). Individual data points are indicated by gray dots. Values represent mean ± SEM (∗∗p < 0.01; NS, non-significant
difference). Homeostatic Synaptic Up-Scaling Is Not
Observed in Immature Granule Cells of
4-Week-Old Cultures homeostatic synaptic up-scaling response in mature GCs
recorded in the same set of cultures (Figures 3C,E). Based
on these results we conclude that immature GCs do not
scale their excitatory synaptic strength in 4-week-old slice
cultures. We next wondered whether immature GCs in a more
mature environment, i.e., 4-week-old slice cultures, behave
similarly. Immunostainings
revealed
that
the
majority
(about
80%)
of
GCs
are
Calb-positive
in
4-week-old
cultures
(Figures
3A,B). Nevertheless,
DCX-positive
neurons are readily detectable in the more mature cultures,
and
these
immature
neurons
disclose
a
significantly
higher
RMP
compared
to
their
mature
GC
neighbors
(Figure 3D). Dopamine Blocks Homeostatic Plasticity
in Immature but Not Mature Granule Cells
of 1-Week-Old Cultures In order to learn more about the signals that control
homeostatic synaptic plasticity in immature dentate GCs,
we tested for the role of DA. We speculated that signals
which promote Hebbian plasticity may hinder the ability of
immature GCs to express homeostatic synaptic plasticity,
thus favoring their activity-dependent maturation. Hence,
TTX-experiments
were
repeated
in
a
different
set
of Taken together, these results confirmed once more that
immature GCs of 1-week-old slice culture are capable of
adjusting their excitatory synaptic strength in a compensatory
manner. DA - likely acting via D1/5 receptors rather than
D2-like receptors - blocks this property of immature GCs
without affecting TTX-induced synaptic scaling in neighboring
mature GCs. August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 3 Synaptic Scaling of Immature Granule Cells Strehl et al. FIGURE 1 | Immature dentate granule cells (GCs) of 1-week-old slice cultures express homeostatic excitatory synaptic plasticity. (A) Example of a 1-week-old
entorhino-hippocampal slice culture stained for calbindin (Calb, magenta) and doublecortin (DCX, green). The GC layer (GCL) is shown at higher magnification. Scale
bars, 50 µm and 10 µm. (B) Quantification of DCX and Calb expression in 1-week-old slice cultures. Some cells expressed both or neither of the markers (n.c., not
classified; n = 17 cultures per group). (C) Sample traces of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated miniature excitatory
postsynaptic currents (mEPSCs) recorded from immature GCs in tetrodotoxin TTX (2 µM, 3 days) and vehicle-only (control)-treated cultures. (D) Example of a
recorded (biocytin-filled), post hoc identified (streptavidin-Alexa568, white) immature GC in a DCX-stained slice culture. Scale bars, 10 µm and 5 µm (inset). (E) Group data of mEPSC recordings (amplitudes and frequencies) from immature GCs of vehicle-only-(Ctrl, control) and TTX-treated cultures (ncontrol = 9 cells,
nTTX = 9 cells; Mann-Whitney test; U-value = 6). Individual data points are indicated by gray dots. Values represent mean ± SEM (∗∗p < 0.01; NS, non-significant
difference). FIGURE 1 | Immature dentate granule cells (GCs) of 1-week-old slice cultures express homeostatic excitatory synaptic plasticity. (A) Example of a 1-week-old
entorhino-hippocampal slice culture stained for calbindin (Calb, magenta) and doublecortin (DCX, green). The GC layer (GCL) is shown at higher magnification. Scale
bars, 50 µm and 10 µm. (B) Quantification of DCX and Calb expression in 1-week-old slice cultures. Some cells expressed both or neither of the markers (n.c., not
classified; n = 17 cultures per group). Dopamine Does Not Affect the Ability of
Granule Cells in 4-Week-Old Cultures to
Express TTX-Induced Synaptic Scaling Express TTX Induced Synaptic Scaling
What is the effect of DA on TTX-induced synaptic scaling in
4-week-old slice cultures? To address this question, we treated
a different set of 4-week-old cultures with TTX (2 µM) and
DA (20 µM) for 3 days and recorded mEPSCs from immature
and mature GCs within the same set of 4-week-old cultures (see
Figure 2B). As shown in Figures 4A,B, DA treatment had no Surprisingly, in the light of our observations in 1-week-
old cultures, TTX treatment (2 µM; 3 days) did not change
mEPSC amplitudes of immature GCs, while inducing a robust August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 4 Synaptic Scaling of Immature Granule Cells Strehl et al. FIGURE 2 | Dopamine (DA) prevents homeostatic synaptic plasticity of immature but not mature GCs in 1-week-old slice cultures. (A) Sample traces of
AMPA receptor-mediated mEPSCs recorded from immature and mature GCs under various conditions (TTX, tetrodotoxin; DA, dopamine). (B) Group data of mEPSC
recordings (amplitudes and frequencies). DA blocks TTX-induced excitatory synaptic scaling in immature but not mature GCs of 1-week-old slice cultures (immature:
ncontrol = 10 cells, nTTX = 14 cells; mature: ncontrol = 12 cells, nTTX = 11 cells; Mann-Whitney test; U-value = 2). (C) The D1-like agonist SKF81297 (20 µM; 3 days)
blocks TTX-induced excitatory synaptic scaling in immature GCs of 1-week-old slice cultures (ncontrol = 5 cells, nTTX = 7 cells; Mann-Whitney test). (D) In presence of
the D1/5 receptor antagonist SCH23390 (40 µM; 3 days) DA does not block TTX-induced excitatory synaptic scaling in immature GCs of 1-week-old slice cultures
(ncontrol = 11 cells, nTTX = 18 cells; Mann-Whitney test; U-value = 5). (E) The D2-like agonist quinpirole (20 µM; 3 days) does not affect TTX-induced excitatory
synaptic scaling in immature GCs of 1-week-old slice cultures (vehicle-only: ncontrol = 12 cells, nTTX = 14 cells; quinpirole: ncontrol = 5 cells, nTTX = 8 cells;
Kruskal-Wallis test followed by Dunn’s post hoc test). Individual data points are indicated by gray dots. Values represent mean ± SEM (∗∗p < 0.01; ∗∗∗p < 0.001; NS,
non-significant difference). FIGURE 2 | Dopamine (DA) prevents homeostatic synaptic plasticity of immature but not mature GCs in 1-week-old slice cultures. (A) Sample traces of
AMPA receptor-mediated mEPSCs recorded from immature and mature GCs under various conditions (TTX, tetrodotoxin; DA, dopamine). (B) Group data of mEPSC
recordings (amplitudes and frequencies). Dopamine Does Not Affect the Ability of
Granule Cells in 4-Week-Old Cultures to
Express TTX-Induced Synaptic Scaling (E) Group data of mEPSC
recordings (amplitudes and frequencies) from immature and mature GCs of vehicle-only (Ctrl, control) and TTX-treated cultures (immature: ncontrol = 8 cells,
nTTX = 8 cells; mature: ncontrol = 25 cells, nTTX = 28 cells; Mann-Whitney test; U-value = 57). Individual data points are indicated by gray dots. Values represent
mean ± SEM (∗∗∗p < 0.001; NS, non-significant difference). FIGURE 3 | Immature dentate GCs of 4-week-old slice cultures do not express homeostatic excitatory synaptic plasticity. (A) Example of a 4-week-old
entorhino-hippocampal slice culture stained for Calb (magenta) and DCX (green). The GCL is shown at higher magnification. Scale bars, 50 µm and 10 µm. (B) Quantification of DCX and Calb expression in 4-week-old slice cultures. Some cells expressed both or neither of the markers (n.c., not classified; n = 15 cultures
per group). (C) Sample traces of AMPA receptor-mediated mEPSCs recorded from GCs in TTX (2 µM, 3 days) and vehicle-only (control)-treated cultures. (D) Resting
membrane potential (RMP) and capacitance of immature and mature GCs were not changed after TTX treatment (Mann-Whitney test). (E) Group data of mEPSC
recordings (amplitudes and frequencies) from immature and mature GCs of vehicle-only (Ctrl, control) and TTX-treated cultures (immature: ncontrol = 8 cells,
nTTX = 8 cells; mature: ncontrol = 25 cells, nTTX = 28 cells; Mann-Whitney test; U-value = 57). Individual data points are indicated by gray dots. Values represent
mean ± SEM (∗∗∗p < 0.001; NS, non-significant difference). FIGURE 3 | Immature dentate GCs of 4-week-old slice cultures do not express homeostatic excitatory synaptic plasticity. (A) Example of a 4-week-old
entorhino-hippocampal slice culture stained for Calb (magenta) and DCX (green). The GCL is shown at higher magnification. Scale bars, 50 µm and 10 µm. (B) Quantification of DCX and Calb expression in 4-week-old slice cultures. Some cells expressed both or neither of the markers (n.c., not classified; n = 15 cultures
per group). (C) Sample traces of AMPA receptor-mediated mEPSCs recorded from GCs in TTX (2 µM, 3 days) and vehicle-only (control)-treated cultures. (D) Resting
membrane potential (RMP) and capacitance of immature and mature GCs were not changed after TTX treatment (Mann-Whitney test). (E) Group data of mEPSC
recordings (amplitudes and frequencies) from immature and mature GCs of vehicle-only (Ctrl, control) and TTX-treated cultures (immature: ncontrol = 8 cells,
nTTX = 8 cells; mature: ncontrol = 25 cells, nTTX = 28 cells; Mann-Whitney test; U-value = 57). Dopamine Does Not Affect the Ability of
Granule Cells in 4-Week-Old Cultures to
Express TTX-Induced Synaptic Scaling DA blocks TTX-induced excitatory synaptic scaling in immature but not mature GCs of 1-week-old slice cultures (immature:
ncontrol = 10 cells, nTTX = 14 cells; mature: ncontrol = 12 cells, nTTX = 11 cells; Mann-Whitney test; U-value = 2). (C) The D1-like agonist SKF81297 (20 µM; 3 days)
blocks TTX-induced excitatory synaptic scaling in immature GCs of 1-week-old slice cultures (ncontrol = 5 cells, nTTX = 7 cells; Mann-Whitney test). (D) In presence of
the D1/5 receptor antagonist SCH23390 (40 µM; 3 days) DA does not block TTX-induced excitatory synaptic scaling in immature GCs of 1-week-old slice cultures
(ncontrol = 11 cells, nTTX = 18 cells; Mann-Whitney test; U-value = 5). (E) The D2-like agonist quinpirole (20 µM; 3 days) does not affect TTX-induced excitatory
synaptic scaling in immature GCs of 1-week-old slice cultures (vehicle-only: ncontrol = 12 cells, nTTX = 14 cells; quinpirole: ncontrol = 5 cells, nTTX = 8 cells;
Kruskal-Wallis test followed by Dunn’s post hoc test). Individual data points are indicated by gray dots. Values represent mean ± SEM (∗∗p < 0.01; ∗∗∗p < 0.001; NS,
non-significant difference). a full-blown synaptic scaling response (thus confirming our
previous findings in the absence of DA). apparent influence on the effects of TTX: Immature GCs did
not scale their excitatory synapses, while mature GCs exhibited apparent influence on the effects of TTX: Immature GCs did
not scale their excitatory synapses, while mature GCs exhibited August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 5 Synaptic Scaling of Immature Granule Cells Strehl et al. FIGURE 3 | Immature dentate GCs of 4-week-old slice cultures do not express homeostatic excitatory synaptic plasticity. (A) Example of a 4-week-old
entorhino-hippocampal slice culture stained for Calb (magenta) and DCX (green). The GCL is shown at higher magnification. Scale bars, 50 µm and 10 µm. (B) Quantification of DCX and Calb expression in 4-week-old slice cultures. Some cells expressed both or neither of the markers (n.c., not classified; n = 15 cultures
per group). (C) Sample traces of AMPA receptor-mediated mEPSCs recorded from GCs in TTX (2 µM, 3 days) and vehicle-only (control)-treated cultures. (D) Resting
membrane potential (RMP) and capacitance of immature and mature GCs were not changed after TTX treatment (Mann-Whitney test). Dopamine Does Not Affect the Ability of
Granule Cells in 4-Week-Old Cultures to
Express TTX-Induced Synaptic Scaling (A) Sample traces of AMPA
receptor-mediated mEPSCs recorded from immature and mature GCs of 4-week-old entorhino-hippocampal slice cultures in presence of DA-only or DA + TTX. (B)
Group data of mEPSC recordings (amplitudes and frequencies) from immature and mature GCs of 4-week-old slice cultures cultures (immature: ncontrol = 7 cells,
nTTX = 7 cells; mature: ncontrol = 17 cells, nTTX = 14 cells; Mann-Whitney test; U-values ≤49.5). (C) Sample traces of AMPA receptor-mediated mEPSC recorded from
immature and mature GCs of 4-week-old entorhino-hippocampal slice cultures in presence of SCH23390 (D1/5 receptor antagonist) or SCH23390 + TTX. (D) Group
data of mEPSC recordings (amplitudes and frequencies) from immature and mature GCs of 4-week-old slice cultures cultures (immature: ncontrol = 7 cells,
nTTX = 7 cells; mature: ncontrol = 10 cells, nTTX = 10 cells; Mann-Whitney test; U-values ≤6). Individual data points are indicated by gray dots. Values represent
mean ± SEM (∗∗p < 0.01; ∗∗∗p < 0.001; NS, non-significant difference). Dopamine Does Not Affect the Ability of
Granule Cells in 4-Week-Old Cultures to
Express TTX-Induced Synaptic Scaling Individual data points are indicated by gray dots. Values represent
mean ± SEM (∗∗∗p < 0.001; NS, non-significant difference). Finally, we tested for the possibility that an endogenous,
i.e., hippocampal source of DA may have blocked the ability of
immature GCs to scale up their excitatory synapses in response to
TTX. Therefore, 4-week-old slice cultures were treated with TTX
and the D1/5 receptor antagonist SCH23390 (Figures 4C,D),
which rescued the scaling response of immature GCs in 1-
week-old cultures in the presence of DA (see Figure 2D). Again, under these experimental conditions immature GCs did not scale their excitatory synapses, while a robust increase in
mEPSC amplitudes was detected in mature GCs. No significant
difference was detected by statistically comparing mEPSC
amplitudes of mature GCs treated with TTX + SCH23390 and
mature GCs treated with TTX-only (see Figure 3E). Taken
together, we conclude that neither scaling of immature nor
scaling of mature GCs is influenced by DA in 4-week-old slice
cultures. August 2018 | Volume 11 | Article 303 Frontiers in Molecular Neuroscience | www.frontiersin.org 6 Synaptic Scaling of Immature Granule Cells Strehl et al. FIGURE 4 | Dopamine (DA) has no effect on homeostatic excitatory synaptic plasticity of GCs in 4-week-old slice cultures. (A) Sample traces of AMPA
receptor-mediated mEPSCs recorded from immature and mature GCs of 4-week-old entorhino-hippocampal slice cultures in presence of DA-only or DA + TTX. (B)
Group data of mEPSC recordings (amplitudes and frequencies) from immature and mature GCs of 4-week-old slice cultures cultures (immature: ncontrol = 7 cells,
nTTX = 7 cells; mature: ncontrol = 17 cells, nTTX = 14 cells; Mann-Whitney test; U-values ≤49.5). (C) Sample traces of AMPA receptor-mediated mEPSC recorded from
immature and mature GCs of 4-week-old entorhino-hippocampal slice cultures in presence of SCH23390 (D1/5 receptor antagonist) or SCH23390 + TTX. (D) Group
data of mEPSC recordings (amplitudes and frequencies) from immature and mature GCs of 4-week-old slice cultures cultures (immature: ncontrol = 7 cells,
nTTX = 7 cells; mature: ncontrol = 10 cells, nTTX = 10 cells; Mann-Whitney test; U-values ≤6). Individual data points are indicated by gray dots. Values represent
mean ± SEM (∗∗p < 0.01; ∗∗∗p < 0.001; NS, non-significant difference). no effect on homeostatic excitatory synaptic plasticity of GCs in 4-week-old slice cultures. (A) Sample traces of AMPA FIGURE 4 | Dopamine (DA) has no effect on homeostatic excitatory synaptic plasticity of GCs in 4-week-old slice cultures. Frontiers in Molecular Neuroscience | www.frontiersin.org DISCUSSION immature neurons in an emerging organotypic network. What
could be the relevance of such a mechanism? At this point
we speculate that homeostatic mechanisms may keep immature
neurons in a ‘‘ready and set’’ position, while regulatory signals,
which suppress homeostasis, may provide the ‘‘go’’ signal for
coordinated associative structural and functional changes. The
relevance
of
homeostatic
plasticity
during
neural
development remains a matter of debate. Considering its
stabilizing nature, it is conceivable that homeostasis hampers
the ability of new neurons to integrate into networks, e.g., by
counteracting major structural and functional changes such as
dendritic or axonal growth and associative synaptic plasticity. Consistent with the hypothesis that homeostatic synaptic
plasticity aims at keeping neurons in their current working range
(Turrigiano, 2008, 2012), mature dentate GCs show a robust
synaptic scaling response in 4-week-old entorhino-hippocampal
cultures, while in the same set of cultures immature GCs do
not adjust their excitatory synaptic strength in a compensatory
manner. This situation may facilitate network integration of
immature neurons, and after a set-point has been reached,
homeostatic mechanisms emerge to stabilize the neurons in their
mature state (see Vlachos et al., 2013). DA is an interesting candidate molecule in this context. Evidence has been provided that DA mediates dendritic/axonal
growth (Parish et al., 2001; Penrod et al., 2015; Shinohara
et al., 2017) and facilitates the induction of Hebbian synaptic
plasticity and learning and memory (Gangarossa and Valjent,
2012; Sariñana et al., 2014; Ntamati and Lüscher, 2016). Whether
DA is instructive in mediating these effects, or rather permissive
(by blocking homeostasis), requires further investigation. Interestingly, in our experiments, DA does not affect the
ability of mature GCs to express TTX-induced homeostatic
excitatory synaptic scaling. Both in 1-week- and 4-week-old
cultures DA has no apparent effect on baseline synaptic
transmission and synaptic strengthening after TTX treatment,
which may be one of the reasons why DA has not yet been
linked to homeostatic synaptic plasticity. However, in immature
GCs of 1-week-old slice cultures, DA blocks the ability of
immature neurons to express homeostatic synaptic plasticity. The situation might be different during early postnatal
development. In 1-week-old slice cultures immature GCs
increase their excitatory synaptic strength in response to TTX
treatment. To the best of our knowledge, this is the first
description of an early homeostatic synaptic scaling response of August 2018 | Volume 11 | Article 303 7 Synaptic Scaling of Immature Granule Cells Strehl et al. FUNDING This work was supported by Deutsche Forschungsgemeinschaft
(CRC 1080 to SS, TD and AV; CRC 974 to AV). AUTHOR CONTRIBUTIONS Why are immature GCs in 4-week-old slice cultures not
adjusting their excitatory synaptic strength in a compensatory
manner? In the experiments in which we used SCH23390 in
4-week-old slice cultures, we sought to test whether an
endogenous source of DA exists in mature slice preparations
which blocks homeostatic synaptic plasticity of immature GCs. Because synaptic scaling was not detected in the presence
of the D1/5
receptor antagonist, we conclude that DA,
i.e., the mechanism that blocks synaptic scaling in 1-week-
old slice cultures, is not the major reason for the absence
of synaptic scaling in immature GCs of 4-week-old slice
cultures. It should be noted in this context that SCH23390 may
also modulate serotonin receptors and inwardly rectifying
potassium channels. Hence, although SCH23390 had no effect
in 4-week-old cultures, it is interesting to hypothesize that
other (local) factors may exist, which prevent homeostasis of
immature neurons in more mature networks. Therefore, the
state of maturation of the environment and specific factors
secreted by neighboring neurons and glia cells may affect The study was conceived and supervised by AV. Experiments
were designed by AS, CG, TD and AV. AS and CG performed
experiments and analyzed the data with the help of TR. The
manuscript was written by AV with the help of AS, CG, SS and
TD. All authors were involved in data interpretation and critically
revising the manuscript. ACKNOWLEDGMENTS We thank Charlotte Nolte-Uhl and Simone Zenker for excellent
technical assistance, Nadine Zahn for help in data analysis, and
Dr. Maximilian Lenz for helpful comments and discussion. DISCUSSION Because pharmacological activation of D1/5 receptors (but not
D2/3 receptor activation) mimics the effects of DA, and based
on our experiments in which we used SCH23390 to block
D1/5 receptors in presence of DA, we conclude that DA acts
in a D1/5 receptor-dependent manner in our experimental
setting. Hence, DA signaling via D1/5 receptors may act
as a molecular switch that prevents homeostatic synaptic
plasticity of immature neurons, which in turn may support
their activity-dependent maturation during early postnatal
development. the ability of immature neurons to express this form of
plasticity. Whereas work from the past years has mainly focused on
the mechanisms that mediate homeostasis, the results of the
present study emphasize the relevance of signaling pathways
that prevent homeostasis. DA qualifies as one of the factors
that suppresses homeostasis specifically in immature neurons
during early postnatal development. Hence, specific factors may
exist which set the balance between homeostatic and Hebbian
synaptic plasticity, affecting specific populations of neurons
depending on their maturation state and the state of the network
(for a recent review on the interplay between homeostatic and
Hebbian plasticity, see Keck et al., 2017). Apparently, a better
understanding of these mechanisms will provide new important
insight on the role of homeostatic plasticity during development
and network maturation, which may also be of relevance in the
context of stem cell-based therapies and developmental brain
diseases. A recent study reported that early postnatal but not late adult
neurogenesis is impaired in an animal model of Parkinson’s
disease (Brandt et al., 2017). This observation is consistent
with our in vitro experiments which show an influence of
DA on immature neurons in young but not older slice
cultures. Whether the lack of DA during early postnatal
development and hence the inability to prevent homeostatic
synaptic plasticity also affect neurogenesis remains to be
shown. More work is required to learn about the interplay
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241–250. doi: 10.1016/j.mcn.2004.01.003 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Sariñana, J., Kitamura, T., Kunzler, P., Sultzman, L., and Tonegawa, S. (2014). Differential roles of the dopamine 1-class receptors, D1R and D5R, in
hippocampal dependent memory. Proc. Natl. Acad. Sci. U S A 111, 8245–8250. doi: 10.1073/pnas.1407395111 Copyright © 2018 Strehl, Galanis, Radic, Schwarzacher, Deller and Vlachos. This
is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T.,
et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682. doi: 10.1038/nmeth.2019 Schmidt-Hieber, C., Jonas, P., and Bischofberger, J. (2004). Enhanced synaptic
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Silicon mitigates nutritional stress in quinoa (Chenopodium quinoa Willd.)
|
Scientific reports
| 2,021
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cc-by
| 12,032
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OPEN Ana Carolina Sales1, Cid Naudi Silva Campos1, Jonas Pereira de Souza Junior2,
Dalila Lopes da Silva2, Kamilla Silva Oliveira2, Renato de Mello Prado2,
Larissa Pereira Ribeiro Teodoro1 & Paulo Eduardo Teodoro1* Ana Carolina Sales1, Cid Naudi Silva Campos1, Jonas Pereira de Souza Junior2,
Dalila Lopes da Silva2, Kamilla Silva Oliveira2, Renato de Mello Prado2,
Larissa Pereira Ribeiro Teodoro1 & Paulo Eduardo Teodoro1* Nutritional deficiency is common in several regions of quinoa cultivation. Silicon (Si) can attenuate
the stress caused by nutritional deficiency, but studies on the effects of Si supply on quinoa plants
are still scarce. Given this scenario, our objective was to evaluate the symptoms in terms of tissue,
physiological and nutritional effects of quinoa plants submitted to nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), and magnesium (Mg) deficiencies under Si presence. The experiment
consisted of a factorial scheme 6 × 2, using a complete solution (CS), -N, -P, -K, -Ca, -Mg combined with
absence and presence of Si (1.5 mmol L−1). Symptomatic, physiological, nutritional and evaluation
vegetative were performed in quinoa crop. The deficiencies of N, P, K, Ca and Mg in quinoa cultivation
caused visual symptoms characteristic of the deficiency caused by respective nutrients, hence
decreasing the plant dry mass. However, Si supply attenuated the deficiency effects by preserving the
photosynthetic apparatus, increasing the chlorophyll production, increasing the membrane integrity,
and decreasing the electrolyte leakage. Thus, the Si supply attenuated the visual effects provided
by deficiency of all nutrients, but stood out for N and Ca, because it reflected in a higher dry mass
production. This occurred because, the Si promoted higher synthesis and protection of chlorophylls,
and lower electrolyte leakage under Ca restriction, as well as decreased electrolyte leakage under N
restriction. Quinoa (Chenopodium quinoa Willd.) is a valuable food source due to its high nutritional potential1. Its seeds
have high quality proteins, vitamins, and essential amino acids, including lysine, methionine, and threonine,
which are lacking in the seeds of legumes and cereals2–4.h Quinoa (Chenopodium quinoa Willd.) is a valuable food source due to its high nutritional potential1. Its seeds
have high quality proteins, vitamins, and essential amino acids, including lysine, methionine, and threonine,
which are lacking in the seeds of legumes and cereals2–4.h g
g
The main cause of income losses in agricultural systems is abiotic stress5, which limits plant production6. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2021) 11:14665 Experimental conditions. Experimental conditions. The experiment was carried out in a greenhouse between July and September
2019. The seeds used belong to the BRS Piabiru cultivar, marketed by Embrapa (Brazilian Agricultural Research
Corporation). During the experimental period, the air humidity and maximum and minimum temperature
were recorded using a thermo-hygrometer (Fig. 1). There was a wide variation in the relative air humidity
(40.9 ± 18.9%), maximum temperature (41.4 ± 12.3 °C), and minimum temperature (31.5 ± 9.6 °C), and the tem-
peratures reached values that were above the optimal values for quinoa growth (− 4 °C and 38 °C)22. All plant
studies were carried out in accordance with relevant institutional, national or international guidelines and regu-
lation. Seeding was performed in two trays with 128 cells (volume of 26.6 mL each cell), containing washed sand
as a substrate. After sowing, the seeds were irrigated with distilled water until the emergency period. Then, six
seedlings were transplanted into 1.7 dm3 polypropylene pots filled with 1.5 dm3 of washed sand. After transplan-
tation, the plants were irrigated with a complete nutritional solution23 at 10% of the concentration, as indicated
by Hoagland and Arnon23, with a change in the iron source from Fe-EDTA to Fe-EDDHMA. The adjustment of
the pH value to 5.5 ± 0.1 was performed by adding NaOH (1 mol L−1) or HCl (1 mol L−1) solution. Experimental design. The experiment was carried out in a 6 × 2 factorial scheme, and it included the fol-
lowing treatments: control (complete nutritional solution); omission of N (4 mmol L−1 throughout the exper-
imental period); P (6 mmol L−1, from 10 days after transplanting, DAT); K, Ca, and Mg combined with Si
(1.5 mmol L−1) and without Si. Treatments were arranged in randomized blocks with nine replicates. The nutri-
ent solution for the treatments was prepared according to Hoagland and Arnon23. Starting treatments and Si supply. At 5 DAT, Si supply was started using a complete nutritional solution
(Fig. 2). The source of Si was sodium silicate (188.4 g L−1 of Si; 121 g L−1 of Na2O, and pH 12). Si was added to
the nutrient solution, and the pH value was adjusted to 5.5 ± 0.2 with HCl (1.0 mol L−1) or NaOH (1.0 mol L−1)
solutions, and it was immediately supplied to the plants. OPEN Minimum and maximum temperature (°C), and relative air humidity (%) recorded using a thermo-
hygrometer during the experimental period inside the greenhouse. Software used to create this Figure was
Microsoft Excel (v1804, https://www.microsoft.com). Figure 1. Minimum and maximum temperature (°C), and relative air humidity (%) recorded using a thermo-
hygrometer during the experimental period inside the greenhouse. Software used to create this Figure was
Microsoft Excel (v1804, https://www.microsoft.com). The beneficial effect of silicon on plants under stress conditions caused by nutrient deficiency has been attrib-
uted to the protection of the plant photosynthetic system18, increased production of chlorophylls19, stimulation
of antioxidant systems, and improved physical integrity of membranes18, in addition to maximized nutrient
uptake8,13,20,21. The beneficial effect of silicon on plants under stress conditions caused by nutrient deficiency has been attrib-
uted to the protection of the plant photosynthetic system18, increased production of chlorophylls19, stimulation
of antioxidant systems, and improved physical integrity of membranes18, in addition to maximized nutrient
uptake8,13,20,21. p
In the present study, the objective was to evaluate the symptoms of nitrogen, phosphorus, potassium, cal-
cium, and magnesium deficiencies under Si fertilization in terms of tissue, physiological, and nutritional effects
of quinoa plants. The hypotheses of the study are that: (a) in quinoa plants, the deficiency of macronutrients
would result in physiological disorders and symptoms at the tissue level according to the function of each nutri-
ent in the plant metabolism; (b) the application of Si could mitigate the stress caused by nutrient deficiency in
quinoa plants by increasing the uptake of missing nutrients as well as by increasing the synthesis of pigments
and protecting the photosynthetic apparatus.ii p
g
p
y
pp
If these hypotheses were confirmed, it would allow a better understanding of the benefits of Si for plant
nutrient deficiency, and it would consequently increase the sustainability of quinoa crop production, especially
in areas characterized by low soil fertility. In this way, our objective was to evaluate the symptoms in terms of
tissue, physiological and nutritional effects of quinoa plants submitted to stress by nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), and magnesium (Mg) deficiencies under Si fertilization. OPEN Among abiotic stresses, nutritional disorders stands out, because the capacity of plants to uptake nutrients
depends on their availability in the soil, and the lack of nutrients can affect essential components of plant
metabolism7. In the above described nutritional stress disorder scenario, the application of beneficial elements such as
silicon (Si) has been indicated as promising. Although it is not considered a nutrient, Si is responsible for plant
protection under abiotic stress conditions8–10. Still, to date, most studies have concentrated more on the attenu-
ation of ammonium, nitrate, and metal excess toxicity (e.g., in cucumber11) than on their deficiency in plants,
whereas studies indicating the benefit of Si in plant nutrient deficiency have mostly focused on nutrient deficiency
in major crops, such as nitrogen deficiency in rice12, potassium deficiency in sorghum13, magnesium deficiency
in corn14, and sulfur deficiency in barley15. i
Regarding the current knowledge about plant nutrient requirements, it is known that nutrients perform
essential and specific functions in plant metabolism. Thus, when a nutrient is not present in adequate amounts in
the soil, it becomes limiting, which in turn promotes its deficiency in the cells and certain metabolic changes in
plants. Despite recent studies involving mineral nutrition for herbaceous performance of the quinoa16, research
on mineral nutrition is still insufficient for the crop.hiil fi
p
The symptoms of deficiency or toxicity are usually specific to each nutrient and are influenced by the sever-
ity, species, and variety, besides environmental factors17. However, researches addressing nutrient deficiency in
quinoa crop, and how Si application influences nutrient deficiency, are still scarce. 1Universidade Federal do Mato Grosso do Sul, Campus de Chapadão do Sul – UFMS/CPCS, Chapadão do Sul, MS,
Brazil. 2Faculdade de Ciências Agrárias e Veterinárias – UNESP/FCAV, Universidade Estadual Paulista “Júlio de
Mesquita Filho”, Jaboticabal, SP, Brazil. *email: eduteodoro@hotmail.com Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/
Figure 1. Minimum and maximum temperature (°C), and relative air humidity (%) recorded using a thermo-
hygrometer during the experimental period inside the greenhouse. Software used to create this Figure was
Microsoft Excel (v1804, https://www.microsoft.com). www.nature.com/scientificreports/ Figure 1. Minimum and maximum temperature (°C), and relative air humidity (%) recorded using a thermo-
hygrometer during the experimental period inside the greenhouse. Software used to create this Figure was
Microsoft Excel (v1804, https://www.microsoft.com). Figure 1. Experimental conditions. The additional application of 49.8 mg L−1 of NaCl was
performed in the treatments without Si to balance the Na among treatments. As the source of Si used was sodium silicate, it was necessary to balance the sodium between treatments, due
to the importance of this element for quinoa crop24. For this purpose, 19.75 mg L−1 of Na, in the form of NaCl
was supplied via a nutrient solution during the entire experimental period, starting together with the Si supply. Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ Figure 2. Schematic representation of the experimental project. After the seeds germinated, the seedlings were
grown in different ionic forces of Hoagland solution (Hoagland and Arnon23). CS complete solution, DAS days
after sowing, DAE days after emergence. Software used to create this Figure was Microsoft Word (v1804, https://
www.microsoft.com). Figure 2. Schematic representation of the experimental project. After the seeds germinated, the seedlings were
grown in different ionic forces of Hoagland solution (Hoagland and Arnon23). CS complete solution, DAS days
after sowing, DAE days after emergence. Software used to create this Figure was Microsoft Word (v1804, https://
www.microsoft.com). At 7 DAT, it was started nutrient omission in the nutrient solution and increased its concentration to 30%
of that indicated by Hoagland and Arnon23 for 7 days, then to 40% for 10 days, 50% for 8 days, 60% for 13 days,
and 70% by the end of the experimental period. Once a week, the substrate was washed to avoid salinization. Deionized water was left to drain from the substrate, and the nutrient solution was applied again after 2 h. Phytosanitary treatments. For pest control, insecticide application (Thiamethoxam 141 g L−1 + Lamb-
dacyhalothrin 106 g L−1) at a rate of 1.5 mL L−1 was performed at 38 DAT. Furthermore, acaricide applications
(Fenpiroximato, 50 g L−1 and Abamectina, 18 g L−1) were performed twice a week from 40 DAT until the end of
the experiment, at a rate of 1 mL L−1. Green color index, photosystem II quantum efficiency, and pigment content. At 40 days of
growing and 10 days from treatment application, the symptoms of N, P, K, and Mg limitation were character-
ized at the tissue level. At 60 days of growing and 35 days from the treatment application, the symptoms of Ca
limitation were characterized. Experimental conditions. As soon as the symptoms were characterized, physiological evaluations of the
green color index (GCI), fluorescence and quantum efficiency of photosystem II (Fv/Fm), and chlorophyll and
carotenoid contents were performed. The GCI and pigment content were measured in the fourth leaflet from
the apex of the main stem. GCI was measured using a chlorophyllometer (Opti-Sciences, CCM-200), with five
readings per plant. g p
p
For pigment analysis, six leaf discs (6 mm) obtained from the middle third of the leaf limb were collected. The analyses were performed according to the methodology proposed by Lichtenthaler25 using a Beckman DU
640 spectrophotometer at the following wavelengths: 663 nm for chlorophyll a, 647 nm for chlorophyll b, and
470 nm for carotenoids. Their contents were defined in relation to the fresh mass of each disc.hll hi
The Fv/Fm was obtained from the chlorophyll fluorescence measurement using a fluorometer (Opti-Sciences,
Os30P). The measurements were carried out between 7:30 a.m. and 9:30 a.m. using one plant per pot, and we
measured the middle third of the plant, evaluating the fourth leaflet from the apex of the main stem. For this
measurement, the sampled region was placed in the dark for adaptation at least 30 min before the excitation of
the red-light pulse of 1 s. The evaluated parameters were F0 (minimum fluorescence for chlorophyll excitation)
and Fm (maximum fluorescence for chlorophyll excitation). From these parameters, F0/Fm (basal quantum
yield) and Fv/Fm (quantum efficiency of photosystem II) were obtained. The leaf area was also evaluated using
the equipment Li-Cor model L1-3000 and was measured in cm2. q p
Later, the leaves, stems, and roots were washed with water, detergent solution (Extran 0.1%), acid solution
(HCl 0.1%), and deionized water, and then packed in paper bags and placed in dry conditions in a forced air
circulation oven at 65 ± 5 °C until a constant mass was obtained. After drying, the samples were weighed on a
precision scale (0.001) to obtain the shoot dry mass (leaves + stem) and root dry mass. Electrolyte leakage index. For determining the cellular leakage index, six leaf discs (6 mm) were removed
from the middle third of the plant, which had new and fully developed leaves. The discs were packed in a beaker
with 20 mL of deionized water at ambient temperature for 2 h. Results and discussion Although all treatments were subjected to high air temperature (Fig. 1), it did not influence the development
of the quinoa plants. It is worth noting that the high temperature occurred only in 2 h during the day (between
12 and 14 pm) and at some punctual moments during the entire growth cycle. However, it is known that the
crop is tolerant to high temperatures31, and recent research shows that quinoa can complete its cycle even under
conditions of high air temperatures (> 40 °C)32. Nitrogen (N). As a consequence of N omission to quinoa plants, the symptom observed was chlorosis in
older leaves (Fig. 3i). N omission resulted in a decrease of 92% in N accumulation under absence of Si and 88%
under presence of Si compared to the N accumulation of plants grown in the complete solution; thus, the symp-
toms of deficiency appeared. As the symptoms evolved, chlorosis became apparent in the upper portion of the
plants, but was not markedly high due to the high mobility of nutrients through the phloem33 (Fig. 3b).hi y
g
g
y
g
g
The process of depigmentation of N-deficient leaves can be considered as consequence of nutrient redistri-
bution within plant metabolism. Due to N deficiency, the proteolysis of Rubisco enzyme and other chloroplast
proteins allows the release and relocation of the nutrient to the new organs34 with the aim to ensure plant growth
and development. p
Regarding the accumulation of nutrients in quinoa plants, the Si supply resulted in an increase in Si contents
in plant shoots (Fig. 4f), and it also promoted a higher accumulation of K, Ca, and Mg compared to that in
the plants that did not receive Si (Fig. 4c–e). The increased accumulation of K under Si supply was possibly a
consequence of the activation of H+ ATPase in the membranes, when Si was added to the nutrient solution35,
as Si improves the uptake and translocation of K+36–39. On the other hand, Ca accumulation was possibly a
consequence of the increased Ca inflow and outflow into the cytosol through Ca carrier channels located in the
membrane40. In a study assessing drought stress in corn, Kaya et al.40 reported an increase in Ca accumulation
in unstressed plants after Si addition. Results and discussion Likewise, our findings suggested that the Si addition resulted in higher Ca
accumulation when a complete nutrient solution was applied to quinoa plants.i p
pp
q
p
N deficiency resulted in a decrease in the GCI of quinoa plants (Fig. 5a) due to the decrease in pigment
content. The decrease in chlorophyll content a + b and cx + c was observed in quinoa plants that experienced N
deficiency regardless of the Si supply (Fig. 5b,c). The omission of the macronutrient caused a decrease of 81% and
83% in chlorophyll a + b content and 79% and 76% of cx + c, under absence and presence of Si, respectively, of
plants grown in complete solution. This effect was related to the structural role of N in the chlorophyll molecule41. In some crop species, leaf chlorophyll content is positively correlated with N concentration, and 70% of the N
contained in leaves participates in the synthesis and chemical structuring of chlorophylls42.i y
g
y
In the present study, regardless of the Si supply, N deficiency resulted in a higher electrolyte leakage index of
quinoa plants compared to that of quinoa plants grown in the complete solution. However, the Si supply in the
solution mitigated the deleterious effects of nutrient deficiency by reducing electrolyte leakage (Fig. 6). Previ-
ous reports have indicated that Si forms complexes with cell structural polymers, such as pectins and callose43,
and cross-links with lignins and carbohydrates by associations with phenolic acids or aromatic rings44, thus
contributing to cell membrane structuring. In the present study, there was an increase in F0 in plants in the N omission treatment, regardless of the Si
supply, compared to that of plants grown in the complete solution (Fig. 7a). Baker and Rosenqvist45 reported
that increased F0 indicates the destruction of the photosystem II (PSII) reaction center, chlorophyll (P680), or a
decreased capacity of energy transfer from the antenna complex to the PSII. These authors also stated that this
occurs when quinone (QA, which is a primary electron receptor) is oxidized and when the reaction center (i.e.,
chlorophyll) is in an “open” state, indicating urgency of the activation of photochemical reactions.hilhi The Si supply in the solution did not have a significant influence on Fm (Fig. 7b). The N deficiency promoted a
decrease in the Fm rate compared to that of plants grown in the complete solution. www.nature.com/scientificreports/ the plant material was digested by the wet digestion method, using hydrogen peroxide (H2O2) and sodium
hydroxide (NaOH)27, and the determination of the Si content was performed after the reaction of samples with
ammonium molybdate, followed by a spectrophotometer reading at 410 nm28. For quantifying the macronutri-
ent contents (N, P, K, Ca, Mg), the Bataglia methodology29 was adopted. The accumulation of Si, N, P, K, Ca and
Mg was calculated by multiplying dry mass and element contents, and it was expressed as mg per shoot. Statistical analysis. Data were submitted to analysis of variance by the F test, and when significant, the
mean comparison was performed by the Tukey test at 5% probability using the Sisvar software30. Later, a heat-
map was built for a view of the interrelationship between the treatments and the variables evaluated. For that,
the variables were standardized and the Euclidean distance between the treatments was used. The “pheatmap”
library of the R software was used. Experimental conditions. After this period, the initial electrical conductivity
reading (EC1) was performed using a manual conductivity meter (TDS-3 digital measurer). Afterward, samples
were taken to autoclave for 20 min at a temperature of 121 °C, and after cooling, a new conductivity reading was
performed to determine the final conductivity (EC2). To estimate the leakage, the following formula26 was used:
EC1/EC2 × 100. Nutritional assessments and Si content. After weighing the material, the samples were ground in a
Wiley type mill in order to determine the Si, N, P, K, Ca, Mg contents in plant shoots. For quantifying Si, https://doi.org/10.1038/s41598-021-94287-1 Scientific Reports | (2021) 11:14665 | www.nature.com/scientificreports/ Results and discussion Baker and Rosenqvist45 stated
that the increased maximum fluorescence intensity (Fm) denotes the state in which PSII reaction centers are
unable to increase photochemical reactions. This condition indicates that fluorescence has reached its maximum
capacity by the completely reduced condition of quinone (QA), and in which the reaction center (P680) is in a
“closed” state, thereby affecting the transport of electrons to photosystem I (PSI). yf
g
p
p
y
Regarding F0/Fm under N omission, it was observed higher rates of yield compared to that of plants cultivated
in the complete solution. The latter had a production of 0.16, regardless of Si supply, whereas the former had a
yield of 0.27 under absence of Si and 0.26 under presence of Si, indicating stress conditions (Fig. 7c). According
to Rohácek46, an increased F0/Fm ratio is indicative of stress, and normal F0/Fm ratio values close to 0.14 and
0.20 are commonly described in literature. Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ Figure 3. Symptomatology of quinoa plants submitted to Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence and presence of Si, (a) complete solution, (b) nitrogen omission, (c)
phosphorus omission, (d) potassium omission, (e) calcium omission, (f) magnesium omission, (g) quinoa leaves
under potassium omission, (h) quinoa inflorescence under calcium omission, (i) comparison of macronutrient
symptoms on quinoa leaves. Software used to create this Figure was Microsoft Word (v1804, https://www.micro
soft.com). Figure 3. Symptomatology of quinoa plants submitted to Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence and presence of Si, (a) complete solution, (b) nitrogen omission, (c)
phosphorus omission, (d) potassium omission, (e) calcium omission, (f) magnesium omission, (g) quinoa leaves
under potassium omission, (h) quinoa inflorescence under calcium omission, (i) comparison of macronutrient
symptoms on quinoa leaves. Software used to create this Figure was Microsoft Word (v1804, https://www.micro
soft.com). N deficiency affected the Fv/Fm in quinoa plants, as PSII efficiency was 0.644, which was 23% lower than that
of the plants grown in the complete solution (whose Fv/Fm ratio was 0.833; Fig. 7d). One study reported that
the Fv/Fm ratio of plants under normal conditions was between 0.85 and 0.75, and that values lower than 0.75
would indicate inhibitory PSII conditions47. Thus, our finding showed that N deficiency affects quinoa photo-
synthetic efficiency because N is an essential component for the functioning of proteins and chlorophyll, and,
consequently, photosynthesis48. Results and discussion Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by Tukey’s
test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test
at 5% probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Figure 5. (a) Green color index (GCI), (b) chlorophyll a + b, and (c) carotenoids of quinoa plants in a Hoagland
solution (Hoagland and Arnon23) under omission of macronutrients in the absence (− Si) and presence of silicon
(+ Si). Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by Tukey’s
test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test
at 5% probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Regarding the development of N-deficient plants, the lower pigment content and consequently lower photo-
synthetic rate resulted in a smaller leaf area than that of plants grown in the complete solution (Fig. 8a). However,
the Si supply had a positive effect on plant development, resulting in a leaf area 68% higher compared to that of
plants grown under the absence of Si. Regarding the development of N-deficient plants, the lower pigment content and consequently lower photo-
synthetic rate resulted in a smaller leaf area than that of plants grown in the complete solution (Fig. 8a). However,
the Si supply had a positive effect on plant development, resulting in a leaf area 68% higher compared to that of
plants grown under the absence of Si. Regarding plant development, for the shoot dry mass, it was evident that the N omission resulted in the
decrease in the biomass of plants grown with or without Si (Fig. 8b). However, the presence of Si in the nutrient
solution had beneficial effects on plant development. In the N omission treatment, higher shoot biomass was
observed in plants that were supplied with Si than in those that were not supplied with Si. This effect was possibly
because Si mitigated N nutritional stress. g
It was also evident that the N omission resulted in a decrease in root dry mass. However, the presence of Si
was not sufficient to influence root growth and mitigate severe deficiency effects. Results and discussion Photosynthetic capacity is associated with the N content in plant metabolism,
mainly because the tilacoids and proteins in the Calvin cycle account for the highest proportion of N in plant
metabolic components49. Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ 6
ts | (2021) 11:14665 |
https://doi.org/10.1038/s41598-021-94287-1
Figure 4. Nutrient accumulation in quinoa plants in a Hoagland solution (Hoagland and Arnon23) under omission of
macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) N accumulation (mg per plant), (b) P accumulation
(mg per plant), (c) K accumulation (mg per plant), (d) Ca accumulation (mg per plant), (e) Mg accumulation (mg per plant),
and (f) Si accumulation. Equal lowercase letters show a similarity between omission and complete treatments by Tukey’s
test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5%
probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Figure 4. Nutrient accumulation in quinoa plants in a Hoagland solution (Hoagland and Arnon23) under omission of
macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) N accumulation (mg per plant), (b) P accumulation
(mg per plant), (c) K accumulation (mg per plant), (d) Ca accumulation (mg per plant), (e) Mg accumulation (mg per plant),
and (f) Si accumulation. Equal lowercase letters show a similarity between omission and complete treatments by Tukey’s Figure 4. Nutrient accumulation in quinoa plants in a Hoagland solution (Hoagland and Arnon23) under omission of
macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) N accumulation (mg per plant), (b) P accumulation
(mg per plant), (c) K accumulation (mg per plant), (d) Ca accumulation (mg per plant), (e) Mg accumulation (mg per plant),
and (f) Si accumulation. Equal lowercase letters show a similarity between omission and complete treatments by Tukey’s
test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5%
probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ Figure 5. (a) Green color index (GCI), (b) chlorophyll a + b, and (c) carotenoids of quinoa plants in a Hoagland
solution (Hoagland and Arnon23) under omission of macronutrients in the absence (− Si) and presence of silicon
(+ Si). Scientific Reports | (2021) 11:14665 | Results and discussion N was one of the macronutri-
ents that was most limiting to the growth of quinoa plants, with a decrease of 82% in both shoot and root dry
mass under absence of Si, and a decrease of 76% and 81% in the shoot and root dry mass under Si presence,
respectively, compared to those values in plants without N omission. These findings suggested that the amount
of N required by a given plant, including quinoa, is a very important factor in plant growth, as this nutrient is
of great relevance for plant growth50. g
p
g
However, the Si supply improved plant performance under stress conditions, resulting in a larger leaf area
and dry matter biomass than that of plants under stress conditions and not supplied with Si. Thus, it became Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ Figure 6. Electrolyte leakage index of quinoa plants in a Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si). Equal lowercase letters
demonstrate a similarity between the omitted and complete treatments by Tukey’s test at 5% probability. Equal
uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5% probability. Software
used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Figure 6. Electrolyte leakage index of quinoa plants in a Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si). Equal lowercase letters
demonstrate a similarity between the omitted and complete treatments by Tukey’s test at 5% probability. Equal
uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5% probability. Software
used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). evident that the addition of the beneficial element, in this case Si, resulted in better performance of quinoa plants
submitted to N deficiency stress. Phosphorus (P). Regardless of the presence of Si, quinoa plants showed visual symptoms of deficiency,
such as pale green leaves and darker green upper leaves. As the symptoms progressed, the lower leaves became
necrotic and the upper leaves became pale green. The slower development of nutrient-deficient quinoa plants
than that of plants grown in the complete solution was also evident (Fig. 3c,i). Results and discussion These symptoms were the results
of lower P availability; in these plants, a 76% lower accumulation nutrient content compared to that of plants
grown in the complete solution was observed (Fig. 4b). However, the supply of Si resulted in higher shoot Si
contents than that of plants not supplied with Si (Fig. 4f).hi p
pp
g
The progress of symptoms evidenced the high P mobility in the phloem. In nutrient deficiency conditions,
P redistribution occurs in order to meet the demand of developing tissues50. Regarding the GCI, the P-deficient
plants had low intensity of coloration compared to that of plants in the complete solution, regardless of the
presence of Si (Fig. 5a).hi p
g
The decrease in chlorophyll a + b and cx + c in plants as a consequence of P omission confirmed that the
decreased pigment rate resulted in lower green color intensity (Fig. 5b,c). Pigment degradation can explain the
depigmentation of leaves under P omission conditions, and as a result, there was a decrease in photosynthetic
activity caused by lower ATP synthesis and Rubisco regeneration51. y
y
y
g
Regardless of the Si supply, P omission did not result in an increase in electrolyte leakage index; the plants
under P omission had similar electrolyte leakage index as that of the plants grown in the complete solution
(Fig. 6). By analyzing F0 regardless of the presence of Si, the P deficiency increased photosystem energy loss
compared to this loss in the plants grown in the complete solution. However, the presence of Si in P-deficient
plants decreased the energy loss by fluorescence by compared to that of plants that did not receive Si, revealing a
supposed beneficial effect of Si on the photosynthetic system of quinoa plants (Fig. 7a). The P omission resulted
in the highest Fm, but it was not statistically different from that of plants grown in the complete solution (Fig. 7b).hi g
yf
g
g
The F0/Fm in the P-deficient plants resulted in higher quantum basal yield than that in plants grown in the
complete solution. The use of Si resulted in a significant difference, as an average quantum basal yield of 0.35
was recorded under Si absence, whereas an average quantum basal yield of 0.25 was recorded under Si supply. Results and discussion However, although the mean was not standardized as normal, Si showed beneficial effects on this variable, and
it was able to mitigate possible deleterious effects of P deficiency (Fig. 7c). g
pfi
y
g
In plants grown with P omission, the Fv/Fm ratio was 0.666, which was 20% lower than that in quinoa plants
grown in the complete solution, which had the Fv/Fm ratio of 0.833 (Fig. 7d). The Fv/Fm ratio can indicate per-
turbations in the photosynthetic system as a consequence of environmental and biotic stresses52. When this ratio
decreases, it indicates inhibition of photochemical activity or deviation in absorbed luminous energy, evidencing
damage in the photosynthetic apparatus, and consequently the decrease in quantum efficiency53. g
p
y
pp
q
y
qfi
y
Regardless of the presence of Si, the leaf area of plants under P omission showed a decrease compared to
that of plants grown in the complete solution, demonstrating the importance of this macronutrient for plant
development (Fig. 8a). The slower development of the plants grown under P absence occurred due to the meta-
bolic function of P in the process of energy transfer and storage54. P deficiency affects the energy bonds in plant
metabolism, as this nutrient is a component of NADP and ATP55.ili p
In summary, the findings on the influence of P deficiency on shoot and root biomass, regardless of the Si
supply, emphasized the negative effects of P deficiency on plant metabolism, evidencing that the nutritional Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ p
Figure 7. Physiological evaluations of quinoa plants in a Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si, (a) Initial fluorescence (Fo), (b)
Maximum fluorescence (Fm), (c) basal quantum yield of Photosystem II, and (d) Photosystem II Efficiency. Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by Tukey’s test at
5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5%
probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). Figure 7. Physiological evaluations of quinoa plants in a Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si, (a) Initial fluorescence (Fo), (b)
Maximum fluorescence (Fm), (c) basal quantum yield of Photosystem II, and (d) Photosystem II Efficiency. Results and discussion Biomass production of quinoa plants submitted to Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) leaf area, (b) shoot and root
dry mass. Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by
Tukey’s test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by
Tukey’s test at 5% probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.micro
soft.com). Figure 8. Biomass production of quinoa plants submitted to Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) leaf area, (b) shoot and root
dry mass. Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by
Tukey’s test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by
Tukey’s test at 5% probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.micro
soft.com). Regardless of the presence of Si, K omission decreased the content of chlorophyll a + b and cx + c compared
to that in plants cultivated in the complete solution. Despite the Si supply, the beneficial element was unable to
favor the increased rate of these photosynthesizing pigments (Fig. 5b,c).hiif Regardless of the presence of Si, K omission decreased the content of chlorophyll a + b and cx + c compared
to that in plants cultivated in the complete solution. Despite the Si supply, the beneficial element was unable to
favor the increased rate of these photosynthesizing pigments (Fig. 5b,c).hiif p
y
g p g
g
The electrolyte leakage in plants under K deficiency showed no significant difference from that of plants
under the complete treatment, regardless of Si supply (Fig. 6). This was because unlike N and P, K does not have
a structural role in plant metabolism. Its primary function in the plant metabolism is enzymatic activation, and
it is required for the function of more than 60 enzymes41.i q
y
In K-deficient plants, regardless of the presence of Si, the F0 was higher than that in plants grown in the com-
plete solution. Despite this, the Si supply was unable to increase the performance of K-deficient plants (Fig. 7a). Results and discussion Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by Tukey’s test at
5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by Tukey’s test at 5%
probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.microsoft.com). stress limited the development of P-deficient plants compared to those grown in the complete solution (Fig. 8b). The scarcity of energy in plant metabolism compromises the biosynthesis processes41,56. Consequently, there is
a decrease in the formation of phospholipids, which are essential cell membrane components. As a result, the
synthesis of new cells decreases57, resulting in limited plant development. presence of Si, respectively, compared to that of plants cultivated in the complete solution (Fig. 4c). The Si sup
ply resulted in higher Si contents in plant shoots than that in the shoots of plants not supplied with Si (Fig. 4f). The symptoms of K deficiency included chlorosis only at the margins of older leaves, which was followed
by necrosis (Fig. 3d,g,i). Low K availability promotes the decrease in protein synthesis, which is reflected in the
accumulation of decarboxylated amino acids. This increases putrescine content, whose high concentrations
accentuate cellular imbalance and promote marginal necrosis of plant tissues58.hi p
g
p
The GCI of K-deficient plants, regardless of the presence of Si, had lower intensity than that of plants grown
in the complete solution (Fig. 5a). However, the Si supply reduced the depigmentation damage caused by K
deficiency. This may have occurred due to the beneficial effect of Si in protecting the photosynthetic apparatus,
which was corroborated by the decrease in F0/Fm the efficiency of Fv/Fm, allowing an efficient photosynthetic
metabolism even with the nutrient deficiency (Fig. 7c,d). Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ Figure 8. Biomass production of quinoa plants submitted to Hoagland solution (Hoagland and Arnon23) under
omission of macronutrients in the absence (− Si) and presence of silicon (+ Si), (a) leaf area, (b) shoot and root
dry mass. Equal lowercase letters demonstrate a similarity between the omitted and complete treatments by
Tukey’s test at 5% probability. Equal uppercase letters show a similarity between treatments (− Si and + Si) by
Tukey’s test at 5% probability. Software used to create this Figure was Microsoft Excel (v1804, https://www.micro
soft.com). Figure 8. www.nature.com/scientificreports/ In the present study, the leaf area was reduced as a consequence of K deficiency regardless of the Si supply
(Fig. 8a). Regardless of the presence of Si, the biomass of quinoa plants was affected by K deficiency, with a
decrease of 74% and 76% in the absence and presence of Si, respectively, compared to that of plants grown in
the complete solution (Fig. 8b). However, the presence of Si in K-deficient plants had positive effects on root
development compared to that of plants in the absence of Si.h p
p
p
The lack of K results in lower rates of carbohydrate translocation from shoots to roots, thus reducing root
development41. Therefore, the findings showed that K deficiency reduced the shoot biomass, and the Si supply
of K-deficient plants resulted in increased root biomass. Calcium (Ca). The quinoa plants cultivated in the Ca omission presented a decreased Ca accumulation of
86% and 83%, in the absence and presence of silicon, respectively, compared to quinoa plants cultivated in com-
plete solution (Fig. 4d). The Si supply provided greater Si accumulations in the shoot (Fig. 4f).hi h
The symptoms of Ca deficiency are characterized by poor formation of the growth points, the youngest leaves,
sprouting and inflorescence had irregularities (Fig. 3e,h,i). Studies carried out by Silva et al.61 in the jatropha crop,
and Miranda et al.62 in cowpea (Vigna unguiculata (L.) Walp) also found that calcium deficiency leads plants to
grow with disorganized leaves and wavy edges. This occurs because Ca presents structural function, conferring
pectin stability on the cell wall of the leaves63,64. y
Despite the decrease of Ca uptake by cultivated plants in the absence of the nutrient, the Si supply provided
higher uptake efficiency of the missing element. There are several mechanisms that explain the benefits of
using Si in the relief of the nutrient deficiency of plants, increasing the remobilization or uptake of the deficient
nutrient13,65–68. This reveals the beneficial effect of Si in improving the efficiency of the stressed plant for miss-
ing element uptake, resulting in better performance in using the nutrient needed. The presence of Si under Ca
restriction provided higher efficiency in K and Mg uptake, which was evidenced by the higher accumulation of
these macronutrients. www.nature.com/scientificreports/ Moreover, the presence of Si provided greater efficiency in K and Mg uptake, which was
evidenced by the higher accumulation of these nutrients under restriction.hi y
g
The GCI under absence of the Si is indicative of damages caused by the Ca deficiency, since there was a
decrease in the green coloration under these conditions. On the other hand, in Ca deficient plants under Si
supply, GCI was similar with the plants grown in complete solution (Fig. 5a). Using Si contributed to the miti-
gation of Ca deficiency by increasing the chlorophyll content a + b and cx + c (Fig. 5b,c). The findings reaffirm
the benefits of Si on stressed plants by increasing the chlorophyll69 content or by protecting the degradation of
chlorophyll70 in stress situations.i Ca deficiency regardless of the presence of Si showed higher electrolyte leakage index compared to quinoa
plants grown in complete solution (Fig. 6). This occurs because Ca is a nutrient with remarkable importance
for cellular structuring, since cation binds to the pectins, forming the calcium pectates, which are compounds
that give stability to the plasmatic membrane71, besides the presence of Ca reduce the action of the enzymes
Polymethylesterase (PME) and Polygacaracturonase (PG)67, which are enzymes that oxidize pectin. However,
the Si supply in the nutritive solution under Ca omission provided decrease in electrolyte leakage index, which
can be related to better activity of antioxidant enzymes during the oxidative stress72–74.i For Ca deficient quinoa plants, regardless of the presence of Si, no changes were found for photosynthetic
variables. The variables F0, Fm, F0/Fm and Fv/Fm did not present significant differences regarding the quinoa
plants grown in complete solution (Fig. 7a–d). This finding evidence that Ca deficient plants can present an
efficient transport of electrons by PSII in non-oxidized state75.h fi
The leaf area showed a decrease in Ca omission plants compared to plants grown in complete solution. In
plants grown in Ca deficiency, the addition of Si promoted an increase of 140.49% in the leaf area index in com-
parison to quinoa plants submitted to Ca omission in the absence of the element (Fig. 8a). Results and discussion The increase in F0 in K-deficient plants could be attributed to the damage to the PSII light receptor complex
or the decrease in the transfer of excitation energy from the light collector system to the reaction center45,59,60,
which were consequences of stress conditions. Fm did not differ statistically between K-deficient plants and
plants grown in the complete solution (Fig. 7b).hii p
g
p
g
The F0/Fm of K-deficient plants was significantly higher than that of plants grown in complete solution,
and the former had increased quantum basal yield. The use of Si in the K omission solution had a significant
positive effect; the absence of Si provided a quantum yield of 0.22, evidencing the stress condition, whereas in
the presence of Si, the observed quantum yield was of 0.20, corresponding to the range indicated as normal
(Fig. 7). These results demonstrated a possible beneficial effect of Si on preserving the photosynthetic apparatus
in abiotic stress conditions. As the Fv/Fm ratio of K-deficient plants was 0.764, there was no statistical differ-
ence between these plants and the plants grown in the complete solution (Fig. 7d), revealing a photosynthetic
efficiency of K-deficient plants. The positive effect of of Si addition on the K-deficient plants was also verified by
Chen et al.13 in sorghum, where under nutritional stress, plants treated with Si presented higher photosystem II
efficiency than that of untreated plants. The authors reported that the Si supply through the root system resulted
in improvements in plant development and photosynthetic activity rates. Scientific Reports | (2021) 11:14665 | https://doi.org/10.1038/s41598-021-94287-1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Under Mg deficiency, the Si supply in the solution promoted higher accumulation of Si in the shoots (Fig. 4f),
in addition to higher N and K accumulation (Fig. 4a,c). The increased activity of enzymes of the N metabolism77,78
resulted in increased N and K accumulation (Fig. 4a,c). The higher K accumulation under Mg deficiency with
Si supply was possibly due to the increased H+ ATPase activity.i pp y
p
y
y
Regarding GCI, regardless of the presence of Si, Mg-deficient plants had lower color intensity than that of
plants grown in the complete solution (Fig. 5a). The observed depigmentation was a consequence of the decreased
chlorophyll a + b and cx + c contents (Fig. 5b,c). Among the major roles of Mg in plant metabolism, 20% of the
Mg present in chloroplast is responsible for the chemical composition of chlorophyll, as it forms the central atom
in its molecular structure41. Thus, Mg deficiency directly affects the contents of these pigments.ii h
gi
y
yf
p g
Regardless of the presence of Si, Mg-deficient plants had a significantly higher electrolyte leakage index than
that of plants grown in the complete solution (Fig. 6). This may have occurred because a small portion of Mg (5
to 10%), along with Ca, takes part in cell wall synthesis41. Thus, the Mg deficiency may have influenced cell wall
structure, allowing higher electrolyte leakage.ii g
g
y
g
Regardless of the presence of Si, Mg deficiency resulted in significantly higher F0 than that of plants that
were grown in the complete solution. However, in the Mg-deficient plants, the Si supply was not enough to
support the photosynthetic metabolism of quinoa plants, which was evidenced by the increased F0 (Fig. 7). Fm
was reduced in Mg–deficient plants, and it was significantly differed from that of plants that were grown in the
complete solution (Fig. 7B).ifi For F0/Fm, Mg omission resulted in a significant difference, with higher average F0/Fm in Mg-deficient plants
than that in plants grown in the complete solution, highlighting the stress promoted by macronutrient deficiency. Regardless of the Si supply, Mg-deficient plants had a quantum production of 0.23, indicating that the addition
of Mg was not enough to attenuate the stress (Fig. 7c).hfih The Fv/Fm ratio was affected by stress caused by nutrient deficiency. www.nature.com/scientificreports/ The plants submitted to Mg omission
had the Fv/Fm ratio of 0.733, which was lower than the standard average which indicates normal performance
of the photosynthetic apparatus (Fig. 7d). Thus, Mg deficiency altered the photosynthetic efficiency of quinoa
plants, possibly by damaging the photosynthetic apparatus.i Regardless of the Si supply, Mg-deficient plants had smaller leaf area than that of plants grown in the complete
solution, evidencing the importance of this macronutrient for plant development (Fig. 8a). The smaller leaf area
may have resulted from the lower pigment content, which led to a lower photosynthetic rate and consequently
slower development of quinoa plants.i p
q
p
Despite the Si supply in the nutrient solution of Mg-deficient quinoa plants, the presence of Si was not
enough to influence plant development. Thus, the absence of Mg as well as the absence of N were most limit-
ing to crop biomass production. Mg omission led to a decrease of 75% and 79% in shoot dry mass under the
absence and presence of Si, respectively, in addition to the 85% decrease in the root system, regardless of the Si
supply (Fig. 8B). Relationship between treatments and evaluated variables. In order to demonstrate the similarity
between the evaluated treatments and the correlation between the variables, a heatmap was constructed (Fig. 9). It is possible to observe the formation of two groups in which the treatments in each group have high similarity
for all the evaluated variables. Group I allocated the treatments − Ca − Si, − Ca + Si, CS − Si and CS + Si. The other
treatments composed group II. The treatments contained in group I presented the highest means (cells in red)
for the variables PSII efficiency (Fv_Fm), green color index (GCI), shoot dry mass (SDM), root dry mass (RDM),
total chlorophyll (Chlorophyll a + b), carotenoids (Caro), Mg content, K content, leaf area (LA), and P content. On the other hand, the treatments of group II presented the highest means for initial fluorescence (F0) and basal
quantum yield (F0_Fm), which are variables that indicate when the plant is under stress, possibly demonstrating
that the treatments in group II were more critical for the development of quinoa plants.hi g
The information obtained by this study allowed us to better understand the Si benefits on the nutritional
deficiency of quinoa plants. www.nature.com/scientificreports/ The findings enable us to safely recommend Si to increase quinoa cultivation sustain-
ability on a global scale due to the wide occurrence of low fertility soils. www.nature.com/scientificreports/ The Si supply may
have contributed to mitigating the Ca deficiency stress by the indirect association of this beneficial element in
increasing the chlorophyll contents69, and consequently, photosynthetic rates13,76, which can collaborate with
better efficiency in the use of the absorbed Ca.ii fi
y
Results for the shoot and root dry mass corroborate the hypotheses of the Si benefit in mitigating Ca defi-
ciency in quinoa plants. Element supply under Ca deficiency provided better development of stressed plants,
attenuating the deleterious effects of nutritional deficiency, and influencing in higher uptake of the missing ele-
ment, higher green color index, and hence higher chlorophyll content, lower electrolyte leakage, larger leaf area,
and higher shoot and root dry mass (Fig. 8b). Magnesium (Mg). At 40 days of cultivation and 10 days of Mg omission, the quinoa plants showed chlo-
rosis symptoms (Fig. 3f). The omission of the nutrient caused a decrease of 91% and 92% in the absence and
presence of silicon, respectively, in the accumulation of Mg in comparison to plants submitted to the complete
solution (Fig. 4e,f), resulting in deficiency symptoms. Mg deficiency was characterized by standard chlorosis,
i.e., between the ribs (Fig. 3i). This occurred because the rib pigment structure remained unchanged for longer
periods compared to the structure of chlorophyll in the cells of the leaf limb76. https://doi.org/10.1038/s41598-021-94287-1 Scientific Reports | (2021) 11:14665 | www.nature.com/scientificreports/ Conclusionsh The N, P, K, Ca and Mg deficiencies in quinoa growing caused physiological alterations and visual symptoms
characteristic of nutritional deficiency caused by the respective nutrients. The Si supply attenuated the visual
effects of the deficiency of all nutrients.hf fi
y
The Si supply under Ca restriction mitigated the deleterious effects of stress by increasing chlorophyll synthe-
sis and protection. For N and Ca, the Si reduced the damages to the cellular wall by the lower electrolyte leakage
and consequently increased the dry mass of the quinoa plants.hf This study showed important results that are related to the effects of Si in mitigating stress caused by nutri-
tional deficiencies. These results are unprecedented and will serve for future research on the use of Si, minimizing
the deleterious effects of nutritional disorders in quinoa plants. https://doi.org/10.1038/s41598-021-94287-1 Scientific Reports | (2021) 11:14665 | www.nature.com/scientificreports/ Figure 9. Heatmap built to demonstrate the relationship between treatments and evaluated variables. The
package used of R to create this Figure was d3heatmap (v0.4.0, https://cran.r-project.org/web/packages/d3hea
tmap/index.html). Figure 9. Heatmap built to demonstrate the relationship between treatments and evaluated variables. The
package used of R to create this Figure was d3heatmap (v0.4.0, https://cran.r-project.org/web/packages/d3hea
tmap/index.html). Figure 9. Heatmap built to demonstrate the relationship between treatments and evaluated variables. The
package used of R to create this Figure was d3heatmap (v0.4.0, https://cran.r-project.org/web/packages/d3hea
tmap/index.html). Received: 7 January 2021; Accepted: 8 July 2021 Received: 7 January 2021; Accepted: 8 July 2021 Received: 7 January 2021; Accepted: 8 July 2021 References References
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em cultivares de Arroz Com Ciclo Precoce, Médio e Tardio. Rev. Bras. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota
|
Frontiers in cellular and infection microbiology
| 2,016
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cc-by
| 10,427
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Salmonella enterica Serovar
Typhimurium Exploits Inflammation
to Modify Swine Intestinal Microbiota
Rosanna Drumo 1, 2 †, Michele Pesciaroli 1, 3 †, Jessica Ruggeri 4, Michela Tarantino 1, 1 Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy, 2 Department of
Comparative Biomedicine and Food Science, University of Padua, Padua, Italy, 3 VISAVET Health Surveillance Centre,
Universidad Complutense Madrid, Madrid, Spain, 4 Department of Veterinary Diagnostic, Istituto Zooprofilattico Sperimentale
della Lombardia e dell’Emilia Romagna, Brescia, Italy, 5 Research and Development Area, Istituto Zooprofilattico Sperimentale
dell’Umbria e della Marche, Perugia, Italy, 6 Department of Experimental Medicine, University of Perugia, Perugia, Italy,
7 Department of Biology, University of Roma Tor Vergata, Rome, Italy Citation: Drumo R, Pesciaroli M, Ruggeri J,
Tarantino M, Chirullo B, Pistoia C,
Petrucci P, Martinelli N, Moscati L,
Manuali E, Pavone S, Picciolini M,
Ammendola S, Gabai G, Battistoni A,
Pezzotti G, Alborali GL, Napolioni V,
Pasquali P and Magistrali CF (2016)
Salmonella enterica Serovar
Typhimurium Exploits Inflammation to
Modify Swine Intestinal Microbiota. Front. Cell. Infect. Microbiol. 5:106. doi: 10.3389/fcimb.2015.00106 Drumo R, Pesciaroli M, Ruggeri J,
Tarantino M, Chirullo B, Pistoia C,
Petrucci P, Martinelli N, Moscati L,
Manuali E, Pavone S, Picciolini M,
Ammendola S, Gabai G, Battistoni A,
Pezzotti G, Alborali GL, Napolioni V,
Pasquali P and Magistrali CF (2016)
Salmonella enterica Serovar
Typhimurium Exploits Inflammation to
Modify Swine Intestinal Microbiota. Front. Cell. Infect. Microbiol. 5:106. doi: 10.3389/fcimb.2015.00106 Keywords: Salmonella Typhimurium, microbiota, inflammation, immune response, pig, salmonellosis ORIGINAL RESEARCH
published: 22 January 2016
doi: 10.3389/fcimb.2015.00106 Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal
pathogen responsible for foodborne disease worldwide. It is a successful enteric
pathogen because it has developed virulence strategies allowing it to survive in a
highly inflamed intestinal environment exploiting inflammation to overcome colonization
resistance provided by intestinal microbiota. In this study, we used piglets featuring an
intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an
attenuated S. Typhimurium in order to evaluate whether the modifications are correlated
with the virulence of the strain. This study showed that Salmonella alters microbiota in
a virulence-dependent manner. We found that the wild type S. Typhimurium induced
inflammation and a reduction of specific protecting microbiota species (SCFA-producing
bacteria) normally involved in providing a barrier against pathogens. Both these effects
could contribute to impair colonization resistance, increasing the host susceptibility to
wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium,
which is characterized by a reduced ability to colonize the intestine, and by a very
mild inflammatory response, was unable to successfully sustain competition with the
microbiota. Edited by:
D. Scott Merrell,
Uniformed Services University, USA Uniformed Services University, USA
Reviewed by:
Richard E. Isaacson,
University of Minnesota, USA
Johanna R. Elfenbein,
North Carolina State University, USA
*Correspondence:
Paolo Pasquali
paolo.pasquali@iss.it;
Chiara F. Magistrali
c.magistrali@izsum.it
†These authors have contributed
equally to this work. Reviewed by:
Richard E. Isaacson,
University of Minnesota, USA
Johanna R. Elfenbein,
North Carolina State University, USA *Correspondence:
Paolo Pasquali
paolo.pasquali@iss.it;
Chiara F. Magistrali
c.magistrali@izsum.it
†These authors have contributed
equally to this work. Received: 12 September 2015
Accepted: 28 December 2015
Published: 22 January 2016 Received: 12 September 2015 Accepted: 28 December 2015
Published: 22 January 2016 Accepted: 28 December 2015
P bli h d
J Keywords: Salmonella Typhimurium, microbiota, inflammation, immune response, pig, salmonellosis INTRODUCTION During the past few years, there has been an
expanding interest concerning the role played by intestinal
microbiota in the susceptibility to enteric pathogens. Microbiota
contributes to the digestion of dietary substances and to the
synthesis of essential food supplements such as vitamins, and to
the development or maintenance of the mucosal immune system
(Littman and Pamer, 2011). Moreover, it acts as a barrier against
invading bacteria both physically, blocking pathogen access to
the epithelial layer, and also by outcompeting for nutrients
reducing the survival and invasiveness of enteric pathogens
(Hallstrom and McCormick, 2011; Sassone-Corsi and Raffatellu,
2015). However, it has been known that S. Typhimurium requires
intestinal inflammation to circumvent “colonization resistance”
provided by the intestinal microbiota (Santos et al., 2009). It has
been shown that Salmonella can alter the normal composition
of the gut microbiota, and this influence is associated with
Salmonella virulence factors that induce inflammatory mucosal
host responses (Barman et al., 2008). Furthermore, animals
with disrupted microbiota have an increased susceptibility to
infection (Barman et al., 2008; Juricova et al., 2013). Most of
the studies examining salmonellosis have been carried out in
murine models that naturally do not develop gastroenteritis. To resemble the disease in humans, mice can be subjected to
antibiotic treatment in order to eliminate microbiota and to
develop colitis (Ahmer and Gunn, 2011). Therefore, due to the
lack of an intact microbiota, murine models are not suitable for
the comprehension of the mechanisms used by Salmonella to
thrive in the gastrointestinal environment (Elfenbein et al., 2013). To circumvent this limitation, we chose the pig as a model for
our study. The advantage of the pig lies in the great similarity
between humans and pigs in the gastrointestinal tract and in
the disease caused by Salmonella as well as being a natural host
of Salmonella (Zhang et al., 2013). We hypothesized that the
Salmonella virulence degree is a determining factor in influencing
the capability of the pathogen to overcome protective microbiota. To explore this, we compared the effects on the intestinal
microbiota of S. Typhimurium wild type to that of an attenuated
Salmonella strain lacking the ZnuABC transporter. Our findings through the reduction of microbiota members normally involved
in the intestinal homeostasis and in the inhibition of pathogen
growth. INTRODUCTION Nontyphoidal salmonellae (NTS) as Salmonella enterica serovar Typhimurium are a leading cause
of acute food-borne zoonoses worldwide being responsible for hundreds of millions of cases of
gastroenteritis and bacteremia annually (Hohmann, 2001). Pigs are important reservoir of infection
for humans as they are asymptomatic carriers of broad host-range serovars of Salmonella (Funk
and Gebreyes, 2004; Pires et al., 2011). The intestine is considered to be the biological niche of January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Salmonella Alters Gut Microbiota in Piglets Drumo et al. Salmonella with the intestinal mucosa having a central role
in regulating the immune response to bacteria (Hallstrom
and McCormick, 2011). However, Salmonella has developed
strategies to overcome and cope with most of the immune
defenses developed by the host (Behnsen et al., 2015). Examples
of the strategies used by Salmonella to evade mucosal innate
immunity include the ability to resist to the reactive oxygen
species generated during inflammation (Bogomolnaya et al.,
2013), in order to produce energy by an anaerobic respiration
chain which uses an electron acceptor specifically generated
in the gut under oxidative stress (Winter et al., 2010) and
to resist to the sequestration of essential nutrients such as
iron and zinc (Raffatellu et al., 2009; Liu et al., 2012). As a
matter of fact, the ability to resist to the antimicrobial host
responses characterizing gut inflammation promotes the growth
of Salmonella in the intestinal lumen over the competing
microbiota. During the past few years, there has been an
expanding interest concerning the role played by intestinal
microbiota in the susceptibility to enteric pathogens. Microbiota
contributes to the digestion of dietary substances and to the
synthesis of essential food supplements such as vitamins, and to
the development or maintenance of the mucosal immune system
(Littman and Pamer, 2011). Moreover, it acts as a barrier against
invading bacteria both physically, blocking pathogen access to
the epithelial layer, and also by outcompeting for nutrients
reducing the survival and invasiveness of enteric pathogens
(Hallstrom and McCormick, 2011; Sassone-Corsi and Raffatellu,
2015). However, it has been known that S. Typhimurium requires
intestinal inflammation to circumvent “colonization resistance”
provided by the intestinal microbiota (Santos et al., 2009). Animals and Samples Collection p
Thirty-one post weaned piglets old 28 days, from Salmonella-free
sows (routinely monitored with microbiological and serological
tests), were used in the experiment. Group A (9 piglets) received
sterile sodium bicarbonate buffer and it was used as naïve control
group. Groups B and C (11 piglets each) were orally infected
with 20 ml of sterile 10% sodium bicarbonate buffer containing
2 × 109 CFU of STM1znuABC (Group B) or 2 × 109 CFU of
STMwt (Group C). At 0, 1, 2, 7, and 12 days post infection
(dpi), rectal temperature was recorded and serum and fecal
samples were collected to evaluate TNF-α, IL1-α, haptoglobin,
and CRP production and to detect fecal excretion of Salmonella,
respectively. Four piglets of group A and 5 for groups B and C
were sacrificed at 1 dpi, while 5 piglets of group A and 6 for
groups B and C at 12 dpi. Portions of spleen, ileum, cecum, colon,
ileocecal lymph nodes, and tonsil of the soft palate were taken
for microbiological analysis, histology, and for mRNA isolation. Feces and cecal and colonic contents were collected to analyze the
microbiota composition. All the experiments were authorized by national authority
and conducted according to European Directive (2010/63/EU;
approval number 54/2012). Salmonella spp. Cultures The wild-type strain S. Typhimurium ATCC 14028 (hereafter
STMwt) and its isogenic attenuated znuABC mutant (hereafter
STM1znuABC; Ammendola et al., 2007), were used throughout
the study. Strains were grown overnight at 37◦C in Brain Heart
Infusion broth (Oxoid Ltd., Basingstoke, UK), harvested by
centrifugation and washed twice in ice-cold phosphate buffer
solution (PBS) (Sigma-Aldrich, Milan, Italy). INTRODUCTION It has
been shown that Salmonella can alter the normal composition
of the gut microbiota, and this influence is associated with
Salmonella virulence factors that induce inflammatory mucosal
host responses (Barman et al., 2008). Furthermore, animals
with disrupted microbiota have an increased susceptibility to
infection (Barman et al., 2008; Juricova et al., 2013). Most of
the studies examining salmonellosis have been carried out in
murine models that naturally do not develop gastroenteritis. To resemble the disease in humans, mice can be subjected to
antibiotic treatment in order to eliminate microbiota and to
develop colitis (Ahmer and Gunn, 2011). Therefore, due to the
lack of an intact microbiota, murine models are not suitable for
the comprehension of the mechanisms used by Salmonella to
thrive in the gastrointestinal environment (Elfenbein et al., 2013). To circumvent this limitation, we chose the pig as a model for
our study. The advantage of the pig lies in the great similarity
between humans and pigs in the gastrointestinal tract and in
the disease caused by Salmonella as well as being a natural host
of Salmonella (Zhang et al., 2013). We hypothesized that the
Salmonella virulence degree is a determining factor in influencing
the capability of the pathogen to overcome protective microbiota. To explore this, we compared the effects on the intestinal
microbiota of S. Typhimurium wild type to that of an attenuated
Salmonella strain lacking the ZnuABC transporter. Our findings
provide evidence that the microbiota modifications induced Salmonella with the intestinal mucosa having a central role
in regulating the immune response to bacteria (Hallstrom
and McCormick, 2011). However, Salmonella has developed
strategies to overcome and cope with most of the immune
defenses developed by the host (Behnsen et al., 2015). Examples
of the strategies used by Salmonella to evade mucosal innate
immunity include the ability to resist to the reactive oxygen
species generated during inflammation (Bogomolnaya et al.,
2013), in order to produce energy by an anaerobic respiration
chain which uses an electron acceptor specifically generated
in the gut under oxidative stress (Winter et al., 2010) and
to resist to the sequestration of essential nutrients such as
iron and zinc (Raffatellu et al., 2009; Liu et al., 2012). As a
matter of fact, the ability to resist to the antimicrobial host
responses characterizing gut inflammation promotes the growth
of Salmonella in the intestinal lumen over the competing
microbiota. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Microbiology gy
Fecal
shedding
and
organs
colonization
of
STMwt
and
STM1znuABC were determined according to the ISO 6579:
2002/Amendment
1:2007
protocol. Briefly,
samples
were
weighed and homogenized in nine parts of Buffered Peptone
Water (BPW) (Oxoid Ltd., UK). This initial solution was
then used to perform a decimal dilution series carried out
by systematically transferring an aliquot of 0.5 ml of each
successive dilution in 4.5 ml of BPW. All BPW-diluted samples
were incubated at 37◦C for 18 ± 3 h. 0.1 ml of cultures
were subsequently seeded on Modified Semisolid Rappaport-
Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated
at 41.5◦C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked
onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK)
and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence
incubated at 37◦C overnight. Suspect Salmonella colonies were
subjected to biochemical identification by the BBL Enterotube II January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 Salmonella Alters Gut Microbiota in Piglets Drumo et al. (BD Franklin Lakes, USA) and serological identification using
Salmonella group-specific antisera (Remel, Lenexa, USA). This
is a semi-quantitative approach that allows the quantification of
Salmonella in a sample within a tenfold band (detection limit 1
CFU/g feces). singletons and to normalize the data by sequencing depth. Alpha-
and beta- diversity were determined by QIIME using Shannon’s
and Fisher’s indices for alpha diversity, unweighted Unifrac and
Bray-Curtis for beta diversity, respectively. Histology Tissue samples of cecum were fixed in formalin, embedded in
paraffin wax and stained with hematoxylin, and eosin according
to standard procedures. q
q-PCR was performed using bacterial groups-specific 16S rRNA
primers (see Supplementary Table 2) to determine the amount
of bacteria in the study groups. However, this method is an
approximation of microbial abundance as a great amount of
bacteria features many copies of the 16S gene. Therefore, both
variation in the abundance of organisms and genomic copy
number variation can influence the quantitative prediction of 16S
gene abundances. Real time PCRs were carried out on SensiMix
SYBR Low-ROX Kit (Bioline). The amplification program started
with an initial step at 95◦C for 10 min, followed by 40 cycles
of 15 s each at 95◦C, 15 s at 55◦C-63◦C (depends on the Tm
of primers), and 15s at 72◦C. The 16S gene copy numbers
per µl of DNA, from each sample, were determined by using
standard curves generated from fragments of 16S rRNA genes
of reference bacteria specific for each group cloned into plasmid
(Promega) as templates. The plasmid was purified by using the
Wizard Plus SV Minipreps DNA purification kit (Promega) and
its concentration was quantified by using a NanoDrop R⃝ND-
1000 Spectrophotometer. With the molecular weight data of the
plasmid and insert sequences, the copy number (g/molecule)
was calculated according to the equation defined by Whelan
et al. (2003). For each microbial population, the corresponding
plasmid was chosen to create a 10-fold standard curve ranged
from 108 to 102 copies. Copy numbers of 16S rRNA genes
per µl of sample (feces, caecal, and colonic contents) were
transformed into logarithms to achieve normal distribution, and
the mean ± standard deviation was calculated. To estimate
the copy number of Enterobacteriaceae other than Salmonella,
for each sample the Salmonella 16S gene copy number was
subtracted from the total Enterobacteriaceae 16S gene copy
number. Immune Mediators Production TNF-α, IL1-α, haptoglobin, and C-reactive Protein (CRP)
production was measured in serum samples from animals bled
at 1 and 12 dpi using a sandwich ELISA (Porcine Quantikine
ELISA Kit, R&D System, Mn, USA), according to the producer’s
instructions. TNF-α, IL1-α, haptoglobin, and C-reactive Protein (CRP)
production was measured in serum samples from animals bled
at 1 and 12 dpi using a sandwich ELISA (Porcine Quantikine
ELISA Kit, R&D System, Mn, USA), according to the producer’s
instructions. Next-Generation Sequencing q
g
Bacterial genomic DNA (gDNA) was extracted from feces, cecal,
and colonic contents using QIAmp DNA Stool Mini Kit (Qiagen,
Hilden, Germany). Fifty nanograms of gDNA were used to
amplify by PCR the hypervariable V3-V4 regions of the 16S
rDNA using bacteria/archeal primers 515F/806R with Illumina
overhang adapters (Caporaso et al., 2012). One nanogram of PCR
amplicon was used for each sample to prepare the sequencing
library according to the Illumina Nextera XT DNA Sample
Preparation Kit. During this procedure, using a limited cycle
PCR, Illumina sequencing adapters, and dual-index barcodes
were added to the amplicon. All the libraries were subsequently
normalized and pooled by 24 prior to sequencing according to
manufacturer’s instructions (Illumina Nextera XT DNA Library
Preparation Guide). Each pool of 24 samples was sequenced on
Illumina MiSeq using a 2 × 250 paired-end (PE) setting on a
standard MiSeq flow cell. Sequencing reads were trimmed and
all the reads with a quality score below the Q20 parameters were
discarded from the analysis. Then, all the PE reads were joined
using the join_paired_ends scripts of QIIME utilities (Caporaso
et al., 2010) to create longer fragments. The Lederhosen pipeline
(based on UCLUST software and green genes v 13.5 16S database)
was used to create the OTU table for each sample. The OTU
tables were provided as input for the MatR package to remove Gene Expression Total RNA was extracted from sections of the cecum, colon,
and ileocecal lymph nodes using the PureLink RNA Mini Kit
(Ambion, Life Technologies). Reverse transcription of 1 µg of
RNA was performed for each individual sample using Tetro
cDNA Synthesis Kit (Bioline) and 5 µl of cDNA were used
for real-time reaction using SensiMix II Probe Kit (Bioline). Primers for cytokines (RPL-32, IL-1α, IL-1β, TNF-α, and IFN-γ)
were designed using PrimerQuest Design Tool (Integrated DNA
Technologies, IDT; see Supplementary Table 1). Fold changes
in gene expression were calculated using the 11Ct method in
comparison to the results for the reference housekeeping gene
RPL32. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Fecal 16S rDNA Metagenomics Next-Generation Sequencing RESULTS degree of colonization than piglets infected with STM1znuABC
(group B) in the gut organs (p < 0.05) at 1 dpi (Figure 3)
and in the colon (p < 0.05) at 12 dpi (Supplementary Figure
1). Organs samples taken from naïve animals (group A) were
negative. Pathogenicity of Salmonella Typhimurium
is Positively Correlated to Bacterial
Virulence g
These findings confirm that STMwt and STM1znuABC have
a differential colonization efficiency. Moreover, STM1znuABC
did not show a significant systemic inflammation. We could
infer that these results are a direct consequence of the intrinsic
incapability of STM1znuABC to induce an inflammatory response
but, in alternative, they could be due to the lower colonization
of STM1znuABC which is not sufficient to give rise to a systemic
immune response. Piglets infected with STM1znuABC (group B) and STMwt (group
C) had a transient increase in body temperature at 1 dpi
compared with naïve controls (group A). At 2 dpi, only the
group C (STMwt) continued showing a significantly higher body
temperature than group A (Figure 1A). Moreover, differences in
the levels of Salmonella fecal shedding were observed among the
study groups. Animals infected with STMwt and STM1znuABC
started to shed bacteria the day after experimental infection
and reached the peak of excretion at 2 dpi. However, unlike
group C (STMwt) that continued shedding a similar amount of
bacteria throughout the whole period of observation, group B
(STM1znuABC) showed a sharp decline over time (Figure 1B). To further assess the inflammatory response induced by STMwt
and STM1znuABC, piglets were bled at different time points
and haptoglobin, CRP, IL1-α, and TNF-α levels were measured
in sera. Group C (STMwt) had an early immune response
characterized by a significant increase of haptoglobin and IL1-α
at 2 dpi, and TNF-α at 2 and 7 dpi, followed by a late production
of CRP which reached a significant level at 12 dpi. Conversely,
group B (STM1znuABC) did not show any different production
of haptoglobin, CRP, IL1-α, and TNF-α when compared with
the naïve (group A; Figure 2). Piglets of different groups were
euthanized at 1 and 12 dpi to assess bacterial colonization of
organs. As shown in Figure 3, colonization occurred as early
as 1 dpi, either in gut or in systemic organs. However, piglets
infected with STMwt (group C) showed a significant higher Histology We compared the cecum histopathological findings from control,
STM1znuABC and STMwt-infected piglets at 1 and 12 dpi. At 1 dpi,
sections from control piglets did not show inflammatory infiltrate
(Figure 4A); conversely, piglets infected with STM1znuABC and
STMwt showed neutrophilic infiltrate in the lamina propria and
submucosa (Figures 4B,C). The neutrophilic infiltrate appeared
moderate and multifocal in the STM1znuABC (Figure 4B), with
crypt abscess formation, whereas marked and diffused in the
STMwt infected piglets (Figure 4C). On the other hand, the
neutrophil inflammation was mild at 12 dpi and present in a
multifocal pattern in piglets infected with STM1znuABC, while
inflammation was mild and diffuse in piglets infected with
STMwt (data not shown). Overall, a histological investigation
indicated the presence of inflammatory infiltrate only in STMwt
and STM1znuABC. A higher degree of inflammation was observed
in piglets infected with STMwt. FIGURE 1 | STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars
of body temperature of study groups in different time points. In the table on the bottom the levels of significance were reported among groups at different time points. Different letters at each time point represent significant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt
shed in feces. Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically significant when *P ≤0.05,
Mann–Whitney test. FIGURE 1 | STM1znuABC (group B) shows a lower virulence in piglets compared to the S
f b d t
t
f t d
i
diff
t ti
i t
I
th t bl
th b tt
th l
l FIGURE 1 | STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars
of body temperature of study groups in different time points. In the table on the bottom the levels of significance were reported among groups at different time points. Different letters at each time point represent significant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt
shed in feces. Statistical Analysis Statistical Analysis
Statistical analysis was performed using GraphPad 6.0 software
for Windows (GraphPad Software Inc.; San Diego; CA). Microbiota analysis by q-PCR were estimated using one-
way analysis of variance (One-way ANOVA). Fecal shedding,
organs colonization, and cytokines expression were analyzed
using non-parametric Mann–Whitney test. Differences in body
temperature and differences between groups in the TNF-
α, IL1-α, haptoglobin, and CRP production were estimated
using non-parametric Dunn’s test. Moreover, non-parametric
Kruskal–Wallis was used to test the presence of significant
differences among the sample groups analyzed for each different
taxonomical level considered (Phylum, Family, Genus) and
Benjamini-Hochberg FDR was applied to correct multiple
testing. A P ≤0.05 was considered statistically significant. Non-
parametric Dunn’s test was also used to estimate differences in
alfa diversity. January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 Salmonella Alters Gut Microbiota in Piglets Drumo et al. Histology Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically significant when *P ≤0.05,
Mann–Whitney test FIGURE 1 | STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars
of body temperature of study groups in different time points. In the table on the bottom the levels of significance were reported among groups at different time points. Different letters at each time point represent significant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt
shed in feces. Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically significant when *P ≤0.05,
Mann–Whitney test. January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4 Drumo et al. Salmonella Alters Gut Microbiota in Piglets FIGURE 2 | (A–D) S. Typhimurium induces an inflammatory response correlated to the virulence of the bacterial strain. Haptoglobin, TNF-α, IL1-α, and C-reactive
protein levels in serum of animals were determined by ELISA. The asterisks indicate statistical significance: *P ≤0.05 and **P ≤0.01 (multiple comparisons-Dunn’s
test). FIGURE 2 | (A–D) S. Typhimurium induces an inflammatory response correlated to the virulence of the bacterial strain. Haptoglobin, TNF-α, IL1-α, and C-reactive
protein levels in serum of animals were determined by ELISA. The asterisks indicate statistical significance: *P ≤0.05 and **P ≤0.01 (multiple comparisons-Dunn’s
test). Influence of Salmonella Infection on the
Expression of Pro-Inflammatory Cytokines Influence of Salmonella Infection on the
Expression of Pro-Inflammatory Cytokines
Pro-inflammatory (IL1-α, IL1-β, TNF-α) and regulatory (IFN-
γ) cytokines were observed so as to evaluate the early immune
response in the ileocecal lymph nodes, colon, and cecum at 1 and
12 dpi (Figures 5A–H; Supplementary Figures 2A–H, 3A,B). At
1 dpi, we observed a tendency of the pro-inflammatory cytokines
to increase in all organs analyzed; however, only the increase of
IL1-β (p < 0.05) in the cecum and in the colon, and IL1-α (p <
0.05) in the lymph nodes of group C (STMwt) were statistically
significant (Figures 5A–C; Supplementary Figures 2A–C, 3A–C). At 12 dpi, overall expression of IL1-α, IL1-β, and TNF-α returned
to baseline levels (Figures 5E–G; Supplementary Figures 2E–G,
3E–G). Histology Moreover, TNF-α (p < 0.01), IL1-β (p < 0.01), and
IL1-α (p < 0.05) were significantly down-regulated in the colon
of piglets infected with STM1znuABC (group B; Supplementary
Figures 2E–G), and IL1-α (p < 0.05) also in the lymph nodes
of group C (STMwt; Supplementary Figures 3E–G). members, we used quantitative real time PCR (q-PCR). As
depicted in Figure 6, consistent changes in the microbiota were
present primarily in the cecal contents at 1 day post-Salmonella
infection, with a significant increase of total 16S rRNA gene
copies (representative of total bacterial numbers) in piglets
infected with STMwt (group C; p < 0.05) compared to naïve
animals (group A) and piglets infected with STM1znuABC (group
B; p < 0.05). Differences in the Lactobacillus/Lactococcus group
were statistically significant between groups B and C (p < 0.05)
and very close to significance between groups A (naïve) and C
(STMwt) in the cecum (Figure 6A). In the feces (Supplementary
Figure 4), the Lactobacillus/Lactococcus group showed significant
differences at 1, 2, 7, and 12 dpi (p < 0.05) between groups
A and C, and only at 12 dpi between groups B and C (p <
0.05; Supplementary Figure 4). A decrease in the Eubacterium
rectale/Clostridium coccoides group was evident in group C (p <
0.05) at 12 dpi in the cecum and at 2 dpi in the feces (p < 0.01;
Figure 6B; Supplementary Figure 4C). No differences among the
three experimental groups were observed for Bacteroides in any
of the samples analyzed. Conversely, at 1 dpi an evident increase
in the Bifidobacterium group was observed in all the districts
investigated between groups A and C (p < 0.01 for cecal content
and p < 0.001 for colon and feces) and between groups B and C
(p < 0.01; Figure 6A; Supplementary Figures 4B, 5A). At 12 dpi,
the Bifidobacterium group showed a sharp reduction in groups S. Typhimurium Alters Composition of the
Microbiota in the Post-Weaned Piglets
Model Aiming to more specifically analyze the impact of STMwt and
STM1znuABC on some of the most representative bacterial January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5 Salmonella Alters Gut Microbiota in Piglets Drumo et al. FIGURE 3 | STMwt induces a higher organs colonization than STM1znuABC at 1 dpi. Piglets were orally infected with 2 × 109 CFU of STM1znuABC (group B)
or STMwt (group C), and bacterial burdens were determined at 1 dpi. Differences between groups B and C were estimated using non-parametric Mann–Whitney test
and were considered significant when *P ≤0.05. Organ samples taken from naïve animals (group A) were negative. Error bars represent one SD from the mean. FIGURE 3 | STMwt induces a higher organs colonization than STM1znuABC at 1 dpi. Piglets were orally infected with 2 × 109 CFU of STM1znuABC (group B)
or STMwt (group C), and bacterial burdens were determined at 1 dpi. Differences between groups B and C were estimated using non-parametric Mann–Whitney test
and were considered significant when *P ≤0.05. Organ samples taken from naïve animals (group A) were negative. Error bars represent one SD from the mean. FIGURE 4 | (A–C) Photomicrographies showing histological changes of the cecum. (A) Naïve control piglets; (B) piglets infected with STM 1znuABC: multifocal and
moderate neutrophilic infiltrate (arrows), crypt abscess formation; (C) piglets infected with STMwt: marked and diffuse neutrophilic infiltration. FIGURE 4 | (A–C) Photomicrographies showing histological changes of the cecum. (A) Naïve control piglets; (B) piglets infected with STM 1znuABC: multifocal and
moderate neutrophilic infiltrate (arrows), crypt abscess formation; (C) piglets infected with STMwt: marked and diffuse neutrophilic infiltration. Bacterial Diversity of the Fecal Microbiota
after Salmonella Infection Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA
with Bonferroni’s post-test. Significant differences between groups are indicated by *P ≤0.05, **P ≤0.01, and ***P ≤0.001. Eub, all bacteria; Lacto,
Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Bifido, Bifidobacterium; Prev, Prevotellaceae; Ent,
Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 6 | STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacrificed at 1 and 12 dp FIGURE 6 | STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacrificed at 1 and 12 dpi (A,B). Bacterial genomic DNA was
isolated from cecal content and qPCR analysis measured the abundance of specific commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA
with Bonferroni’s post-test. Significant differences between groups are indicated by *P ≤0.05, **P ≤0.01, and ***P ≤0.001. Eub, all bacteria; Lacto,
Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Bifido, Bifidobacterium; Prev, Prevotellaceae; Ent,
Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 6 | STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacrificed at 1 and 12 dpi (A,B). Bacterial genomic DNA was
isolated from cecal content and qPCR analysis measured the abundance of specific commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA
with Bonferroni’s post-test. Significant differences between groups are indicated by *P ≤0.05, **P ≤0.01, and ***P ≤0.001. Eub, all bacteria; Lacto,
Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Bifido, Bifidobacterium; Prev, Prevotellaceae; Ent,
Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. group B at 2 dpi (p < 0.05). At the same time, Fisher’s alpha
confirmed the significant lower alfa diversity in group C at 12 dpi
compared to group A (p < 0.05; Figure 7B). STMwt (group C) was significantly higher than the naïve animals
(group A), at 0 and at 2 dpi respectively (p < 0.01). However,
group C showed a significant lower alfa diversity at 12 dpi
than group A (Figure 7A). Bacterial Diversity of the Fecal Microbiota
after Salmonella Infection TNF-α, IL1-α, IL1-β,
and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and
STMwt-infected piglets (group C), respectively. The asterisk indicates statistical significance *P ≤0.05, Mann–Whitney test. FIGURE 5 | (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β,
and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and
STMwt-infected piglets (group C), respectively. The asterisk indicates statistical significance *P ≤0.05, Mann–Whitney test. FIGURE 6 | STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacrificed at 1 and 12 dpi (A,B). Bacterial genomic DNA was
isolated from cecal content and qPCR analysis measured the abundance of specific commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA
with Bonferroni’s post-test. Significant differences between groups are indicated by *P ≤0.05, **P ≤0.01, and ***P ≤0.001. Eub, all bacteria; Lacto,
Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Bifido, Bifidobacterium; Prev, Prevotellaceae; Ent,
Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 5 | (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β,
and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and
STMwt-infected piglets (group C), respectively. The asterisk indicates statistical significance *P ≤0.05, Mann–Whitney test. FIGURE 5 | (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β,
and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and
STMwt-infected piglets (group C), respectively. The asterisk indicates statistical significance *P ≤0.05, Mann–Whitney test. FIGURE 6 | STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacrificed at 1 and 12 dpi (A,B). Bacterial genomic DNA was
isolated from cecal content and qPCR analysis measured the abundance of specific commensal bacterial groups. White bars represent uninfected controls (group A). Bacterial Diversity of the Fecal Microbiota
after Salmonella Infection B (p < 0.001) and C (p < 0.001) in the cecal content and in
group B (p < 0.05) in the colonic one when compared to group
A (naïve; Figure 6B; Supplementary Figure 5B). The levels of the
Enterobacteriaceae other than Salmonella decreased significantly
in both groups of animals infected with Salmonella strains in the
cecal and colonic contents at 12 dpi (Figure 6B; Supplementary
Figure 5B). A higher level of Salmonella, consistent with the
microbiological findings, was observed in group C (STMwt)
compared to group B (STM1znuABC) in all the intestinal samples,
while Salmonella was never detected in group A (naïve; Figure 6;
Supplementary Figures 4, 5). B (p < 0.001) and C (p < 0.001) in the cecal content and in
group B (p < 0.05) in the colonic one when compared to group
A (naïve; Figure 6B; Supplementary Figure 5B). The levels of the
Enterobacteriaceae other than Salmonella decreased significantly
in both groups of animals infected with Salmonella strains in the
cecal and colonic contents at 12 dpi (Figure 6B; Supplementary
Figure 5B). A higher level of Salmonella, consistent with the
microbiological findings, was observed in group C (STMwt)
compared to group B (STM1znuABC) in all the intestinal samples,
while Salmonella was never detected in group A (naïve; Figure 6;
Supplementary Figures 4, 5). Massive parallel sequencing of the 16S rDNA hypervariable
V3-V4 region was performed on fecal samples available
from the three experimental groups A, B, and C. The
sequencing yielded a total of 177198 reads passing quality
control (median reads per sample 11030). OTU classification
yielded a median of 5742 OTUs per sample. Sequencing
reads are available at http://www.ncbi.nlm.nih.gov/bioproject/
PRJNA302126 (BioProject accession ID: PRJNA302126). We evaluated the bacterial diversity of the fecal microbiota
associated with Salmonella strains by estimating alpha- and beta-
diversity. Shannon index demonstrated that the fecal microbiota
diversity in piglets infected with STM1znuABC (group B) and These results show that S. Thyphimurium is able to alter
intestinal microbiota in pigs inducing modifications correlated to
its virulence. January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6 Drumo et al. Salmonella Alters Gut Microbiota in Piglets FIGURE 5 | (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. Bacterial Diversity of the Fecal Microbiota
after Salmonella Infection Using Fisher’s alpha, an index not
influenced by the sample size and less affected by the abundance
of the most common species than Shannon’s index, we found
a higher diversity in piglets belonging to group C compared to The beta diversity was calculated using both unweighted
Unifrac and Bray-Curtis dissimilarity; principal component
analysis (PCoA) was performed. As shown in Figure 8A, using
Unifrac, four out of five samples belonging to group C (STMwt) January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7 Salmonella Alters Gut Microbiota in Piglets Drumo et al. FIGURE 7 | Structural comparison of fecal microbiota among groups A, B, and C. The Shannon index (A) and Fisher’s alpha (B) were used to estimate
diversity of the fecal microbiota in naïve animals (group A) and in STM1znuABC- (group B) and STMwt- (group C) infected piglets. Boxes represent median, and first
and third quartiles; whiskers indicate minimum and maximum values. The asterisks indicate statistical significance *P ≤0.05 and **P ≤0.01, Dunn’s test. FIGURE 7 | Structural comparison of fecal microbiota among groups A, B, and C. The Shannon index (A) and Fisher’s alpha (B) were used to estimate
diversity of the fecal microbiota in naïve animals (group A) and in STM1znuABC- (group B) and STMwt- (group C) infected piglets. Boxes represent median, and first
and third quartiles; whiskers indicate minimum and maximum values. The asterisks indicate statistical significance *P ≤0.05 and **P ≤0.01, Dunn’s test. with naïve (group A) is that they showed an abundant presence
of lactic acid-producing bacteria and a reduction of short chain
fatty acids (SCFAs)-producing bacteria (Figures 9A,B). Analysis
of data also revealed that piglets infected with STMwt (group C)
initially showed a decrease in Prevotella at 2 dpi compared to
the naïve (group A). In addition, at 12 dpi, a more abundant
presence of Prevotella, Phascolarcobacterium, and Eubacterium
was evident in group C (STMwt) rather than in groups A and B. clustered separately along the principal coordinate 1 (PCA1) at
12 dpi. In addition, a clear separation of group B (STM1znuABC)
from the rest of the samples is noticeable along the principal
coordinate 2 (PCA2). The PCoA using Bray-Curtis dissimilarity
did not allow any clear separation of the groups, although all the
five samples belonging to group C (STMwt), at 2 dpi, clustered
at the extreme right along the principal coordinate 1 (PCA1;
Figure 8B). Bacterial Diversity of the Fecal Microbiota
after Salmonella Infection On the light of these data, it can inferred that
Shannon and Unifrac results, in which it seem to be differences
among groups at time 0, could be biased by small sample size. Therefore, the microbiota composition of the different groups
could be considered similar at time 0. Moreover, clustering analysis highlighted the differences
in the sample distributions according to the treatment type. At 2 dpi, the most represented genera displayed a perfect
clusterization of each single sample into its belonging study
group (Figure 9A). Similarly, at 12 dpi, each piglet grouped
into its belonging treatment group, except 2 samples (5 and 12)
clustered in a different study group (Figure 9B). Moreover, at 12
dpi, groups A (naïve) and B (STM1znuABC) are more similar to
each other, while group C (STMwt) featured more relevant effects
(Figure 9B). No significant differences were detected when each
single group was analyzed longitudinally according to the three
collection times. These data show that infection with different
strains of S. Typhimurium is associated with different alterations
of fecal microbiota. Salmonella Strains-Associated Alterations
in Fecal Microbiota by NGS In order to compare how the composition of the fecal bacteria
differed among treatment groups, the Kruskal–Wallis test and
the Benjamini-Hochberg FDR method were used to identify
differentially abundant taxa. Genus-level normalized data are
available in Supplementary Table 3. The distribution of reads
into phyla revealed that the bacterial communities of all samples
consisted primarily of Firmicutes and Bacteroidetes. Microbiota
analysis showed that 7 phyla, 112 families, 404 genera, and 15
phyla, 143 families, and 719 genera differed across groups A, B,
and C, respectively at 2 and 12 dpi (see Supplementary Table 4). Figures 9A,B and Supplementary Figure 6 represent heatmaps
showing the genus-level clustering according to frequency within
each sample (abundance ratio greater than 0.1) at times 0, 2, and
12 dpi; abundant genera were color coded red, and white color
coding indicated missing genera. The most remarkable difference
reported in the piglets infected with STMwt (group C) compared January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Frontiers in Cellular and Infection Microbiology | www.frontiersin.org DISCUSSION The importance of pigs as a source of Salmonella in the food
chain is well-known. However, while Salmonella pathogenicity
has been widely studied in mice, our knowledge on the modality
of interaction of this pathogen with pigs is still limited. It has
been known that different and multiple factors can influence
the dynamics of Salmonella colonization in swine, including January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 8 Salmonella Alters Gut Microbiota in Piglets Drumo et al. GURE 8 | Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota
ross the three study groups. PCA, principal coordinate. hogen features (virulence mechanisms, exposure dosage),
immune responses and the complex interplay between the
hogen and the intestinal microbiota (Bearson et al., 2013). this study, we used a post-weaned piglet model to compare
erences in the host colonization, inflammatory response, and
modification of the intestinal microbiota induced by STMwt and
STM1znuABC in order to elucidate the interplay among host,
pathogen and gut microbiota. STM1znuABC was chosen due to
the fact that our previous studies have revealed that this strain is
strongly attenuated either in mice or in pigs (Ammendola et al., Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota
three study groups. PCA, principal coordinate. FIGURE 8 | Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota
across the three study groups. PCA, principal coordinate. pathogen features (virulence mechanisms, exposure dosage),
pig immune responses and the complex interplay between the
pathogen and the intestinal microbiota (Bearson et al., 2013). In this study, we used a post-weaned piglet model to compare
differences in the host colonization, inflammatory response, and modification of the intestinal microbiota induced by STMwt and
STM1znuABC in order to elucidate the interplay among host,
pathogen and gut microbiota. STM1znuABC was chosen due to
the fact that our previous studies have revealed that this strain is
strongly attenuated either in mice or in pigs (Ammendola et al., Frontiers in Cellular and Infection Microbiology | www.frontiersin.org January 2016 | Volume 5 | Article 106 9 Salmonella Alters Gut Microbiota in Piglets Drumo et al. DISCUSSION GURE 9 | (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B)
with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red
gh abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5:
oup A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. FIGURE 9 | (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B)
or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red
(high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5:
group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. GURE 9 | (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B)
with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red
gh abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5:
oup A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. FIGURE 9 | (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B)
or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red
(high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5:
group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. DISCUSSION Hence, the reduced abundance of
SCFA-producing bacteria induced by STMwt could explain the
enhanced inflammatory status observed in the gastrointestinal
tract of piglets treated with this Salmonella strain; and it could be
of interest to investigate the mechanisms leading to a reduction
of this potentially protective component of the intestinal
microbiota. Moreover, upon infection with Salmonella strains,
microbiota composition also showed changes in Prevotella,
Phascolarcobacterium and Eubacterium. Similarly to what
elsewhere reported (Bearson et al., 2013), we observed a decrease
of Prevotella in piglets infected with STMwt at 2 dpi. However
the limitation of available information about the biological
function of such genera makes difficult to extrapolate any
significant meaning to our findings. At the same time it should
be acknowledged that the alpha and beta diversity patterns
across the three groups within the three time points analyzed
presented several discrepancies that can be attributable to the
sensitivity of the next-generation sequencing technology and
to the relative small sample size. However, both alpha-diversity
indices converge on a significant lower alpha-diversity in group C
compared to group A at dpi 12. At the same time, the significant
difference found in the whole microbiome composition at time
0 between group A and group B, highlighted by Shannon alpha
index and Unifrac beta diversity PCoA may raise the possibility 2007; Pasquali et al., 2008; Pesciaroli et al., 2013). Moreover,
studies carried out in a mouse model showed that ZnuABC-
mediated zinc uptake confers resistance to the antimicrobial
protein calprotectin and promotes Salmonella growth over the
competing intestinal microbiota (Liu et al., 2012). Here, we demonstrate that a different organs colonization,
intestinal inflammation and modification of porcine microbiota
are correlated with the different virulence of Salmonella strains. The inflammatory response evaluated through the expression
of the immune mediators, and corroborated by histological
findings, has shown that STMwt induces a prompt increase
of serum markers of inflammation during the early stage
of infection (1 dpi). Moreover, at the same time point, the
expression of tissue-associated markers showed a tendency to
increase even if only IL1-β in cecum and colon (p < 0.01)
and IL1-α in ileocecal lymph nodes (p < 0.01) reach statistical
significance. The prompt induction of host response could
be due to the rapid and high-level replication of STMwt as
showed by our microbiological data. DISCUSSION FIGURE 9 | (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B)
or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red
(high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5:
group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org January 2016 | Volume 5 | Article 106 10 Salmonella Alters Gut Microbiota in Piglets Drumo et al. to survive under conditions of increased redox potential due
to the production of reactive oxygen species by granulocytes
infiltrating the site of inflammation as occurs in a highly inflamed
gut (Videnska et al., 2013). Indeed, there is evidence that lactic
acid accumulation could contribute to impair the intestinal
defense barrier and increase the osmotic load in the intestinal
lumen (Ling et al., 2014). The utilization of next-generation
high-throughput sequencing allowed a wider description of the
intestinal microbiota. In our study, clustering analysis shows
that the microbiota composition changed after infection with
Salmonella strains and the characteristics of the modifications
were correlated with the virulence of the strain used. Our
analysis reveals a different abundance of the most represented
genera in piglets infected with STMwt when compared with
STM1znuABC and naïve piglets. In fact, microbiota of piglets
infected with STMwt was characterized by an overall reduction
of
SCFA-producing
bacteria
(Ruminococcaceae
including
Faecalibacterium,
Roseburia,
Butyrivibrio,
and
Clostridium
genera). SCFAs such as acetate and butyric acid are produced by
microbial fermentation of carbohydrates and provide beneficial
immunomodulatory and anti-inflammatory properties (Ling
et al., 2014). In particular, butyric acid contributes to the
inhibition of Salmonella in an acidic environment (Bearson et al.,
2006), decreases invasion of intestinal cells by down-regulating
expression of Pathogenicity island 1 (Gantois et al., 2006) and
reduces the Salmonella-induced proinflammatory response of
enterocytic cells in vitro (Malago et al., 2005). In line with these
observations, previous studies showed that Faecalibacterium,
which is correlated with butyrate production, also exhibits
anti-inflammatory effects counterbalancing intestinal microbiota
dysbiosis (Sokol et al., 2008). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org January 2016 | Volume 5 | Article 106 REFERENCES Chirullo, B., Pesciaroli, M., Drumo, R., Ruggeri, J., Razzuoli, E., Pistoia,
C., et al. (2015). Salmonella typhimurium exploits inflammation to its
own advantage in a porcine enteritis model. Front. Microbiol. 6:985. doi:
10.3389/fmicb.2015.00985 Ahmer, B. M. M., and Gunn, J. S. (2011). Interaction of Salmonella Spp. with the
intestinal microbiota. Front. Microbiol. 2:101. doi: 10.3389/fmicb.2011.00101 Ammendola, S., Pasquali, P., Pistoia, C., Petrucci, P., Petrarca, P., Rotilio, G., et al. (2007). High-affinity Zn2+ uptake system ZnuABC is required for bacterial zinc
homeostasis in intracellular environments and contributes to the virulence of
Salmonella enterica. Infect. Immun. 75, 5867–5876. doi: 10.1128/IAI.00559-07 Elfenbein, J. R., Johanna, R., Endicott-Yazdani, T., Porwollik, S., Bogomolnaya,
L. M., Cheng, P., et al. (2013). Novel determinants of intestinal colonization
of Salmonella enterica serotype typhimurium identified in bovine enteric
infection. Infect. Immun. 81, 4311–4320. doi: 10.1128/IAI.00874-13 Barman, M., Unold, D., Shifley, K., Amir, E., Hung, K., Bos, N., et al. (2008). Enteric
Salmonellosis disrupts the microbial ecology of the murine gastrointestinal
tract. Infect. Immun. 76, 907–915. doi: 10.1128/IAI.01432-07 Funk, J., and Gebreyes, W. A. (2004). Risk factors associated with Salmonella
prevalence on swine farms. J. Swine Health Prod. 12, 246–251. Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Hautefort, I., Thompson,
A., et al. (2006). Butyrate specifically down-regulates salmonella pathogenicity
island 1 gene expression. Appl. Environ. Microbiol. 72, 946–949. doi:
10.1128/AEM.72.1.946-949.2006 Barthel, M., Hapfelmeier, S., Quintanilla-Martínez, L., Kremer, M., Rohde,
M., Hogardt, M., et al. (2003). Pretreatment of mice with streptomycin
provides a Salmonella enterica serovar Typhimurium colitis model that allows
analysis of both pathogen and host. Infect. Immun. 71, 2839–2858. doi:
10.1128/IAI.71.5.2839-2858.2003 Hallstrom, K., and McCormick, B. A. (2011). Salmonella interaction with and
passage through the intestinal mucosa: through the lens of the organism. Front. Microbiol. 2:88. doi: 10.3389/fmicb.2011.00088 Bearson, S. M., Allen, H. K., Bearson, B. L., Looft, T., Brunelle, B. W., Kich, J. D.,
et al. (2013). Profiling the gastrointestinal microbiota in response to Salmonella:
low versus high Salmonella shedding in the natural porcine host. Infect. Genet
Evol. 16, 330–340. doi: 10.1016/j.meegid.2013.03.022 Hohmann, E. L. (2001). Nontyphoidal salmonellosis. Clin. Infect. Dis. 32, 263–269. doi: 10.1086/318457 Juricova, H., Videnska, P., Lukac, M., Faldynova, M., Babak, V., Havlickova, H.,
et al. (2013). Influence of Salmonella enterica serovar enteritidis infection on the
development of the cecum microbiota in newly hatched chicks. Appl. Environ. Microbiol. 79, 745–747. doi: 10.1128/AEM.02628-12 Bearson, S. M., Bearson, B. L., and Rasmussen, M. A. (2006). FUNDING This work was supported by ISS intramural research funds
and by Transnational Research Project EMIDA ERA-NET
“HealthyGut–Multi-focal strategies to improve gut health
and
reduce
enteritis
in
poultry
and
pigs”(MIPAF–DM
27373/7303/2010). This work was supported by ISS intramural research funds
and by Transnational Research Project EMIDA ERA-NET
“HealthyGut–Multi-focal strategies to improve gut health
and
reduce
enteritis
in
poultry
and
pigs”(MIPAF–DM
27373/7303/2010). AUTHOR CONTRIBUTIONS RD planned and performed the research, analyzed data and
wrote the manuscript. PP designed and planned the research,
participated to the interpretation and discussion of the results,
and revised the paper; CM planned and performed the research,
analyzed data and revised the manuscript. MP, BC, and MT
performed part of the research and revised the manuscript. GA, JR, and NM performed part of the research. SA and AB
revised the manuscript. GG revised the manuscript. GP and LM
performed part of the research and contributed to the analysis
of the data. EM and SP performed histological analysis. VN and SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
http://journal.frontiersin.org/article/10.3389/fcimb. 2015.00106 DISCUSSION On the contrary, at 1 dpi,
the histological and immunological analysis revealed a mild
intestinal inflammation and a poor systemic response induced by
STM1znuABC confirming characteristics of attenuation in growth
and virulence of this strain. As a whole, these observations
indicate that the host is able to mount a rapid innate immune
response following Salmonella infection within a few hours
after gut colonization. The magnitude of the response and the
severity of the clinical manifestations provide evidence that the
host response and lesions are correlated and dependent to S. Typhimurium virulence. It is known that, similarly to what happens in vitro and in
murine models of infection (Barthel et al., 2003; Stecher et al.,
2007; Barman et al., 2008), S. Typhimurium strains induce an
acute inflammatory response in the intestinal mucosa also in
piglets (Bearson et al., 2013). Several studies have proved how
S. Typhimurium takes advantages of inflammation to compete
with the resident microbiota and to colonize the inflamed gut
in mice (Lupp et al., 2007; Stecher et al., 2007; Barman et al.,
2008; Winter et al., 2010) and piglets (Chirullo et al., 2015). In
our study, we investigated the impact of S. Typhimurium on
the porcine intestinal microbial communities. We found that
S. Typhimurium infection modifies either the number or the
composition of gut resident bacteria. In particular, these changes
were associated with STMwt, while the attenuated STM1znuABC
seemed to be less fit to sustain competition with the microbiota. These observations are in agreement with the studies performed
in mice, where attenuated Salmonella mutants do not colonize
intestine as well as wild-type strains as they are not able to trigger
an efficient inflammatory response (Stecher et al., 2007; Lawley
et al., 2008; Raffatellu et al., 2009; Winter et al., 2010). The major changes in the microbiota composition are
mainly related to the significantly more abundant presence
of
Lactobacillus/Lactococcus
group
after
STMwt
infection. This observation is in agreement with the results obtained by
Videnska et al. (2013), which showed a significant increase of
Lactobacillaceae in chicken cecal microbiota after S. Enteritidis
infection. A possible explanation could be attributable to the
microaerophilic growth of Lactobacilli, which may allow them January 2016 | Volume 5 | Article 106 January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 11 Salmonella Alters Gut Microbiota in Piglets Drumo et al. MP performed next-generation sequencing and analyzed data. DISCUSSION PP and CP contributed to perform technical experiments. allow any speculation and further studies using a larger sample
size and, possibly, a more detailed time-course is warranted. Overall, our data show that the results of the interaction among
Salmonella, the intestinal microbiota and the gut response are
influenced by the specific characteristics of the three factors. The virulence of Salmonella and the alteration of microbiota
composition is crucial in determining the outcome of the
infection. ACKNOWLEDGMENTS We thank Massimiliano Francia for technical assistance. REFERENCES Identification of
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Gaynor, E. C., et al. (2007). Host-mediated inflammation disrupts the intestinal January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 12 Drumo et al. Salmonella Alters Gut Microbiota in Piglets microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host
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intestine. Cell Host Microbe 5, 476–486. doi: 10.1016/j.chom.2009.03.011 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. REFERENCES Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Santos, R. L., Raffatellu, M., Bevins, C. L., Adams, L. G., Tükel, C., Tsolis, R. M.,
et al. (2009). Life in the inflamed intestine, Salmonella Style. Trends Microbiol. 17, 498–506. doi: 10.1016/j.tim.2009.08.008 Copyright © 2016 Drumo, Pesciaroli, Ruggeri, Tarantino, Chirullo, Pistoia, Petrucci,
Martinelli, Moscati, Manuali, Pavone, Picciolini, Ammendola, Gabai, Battistoni,
Pezzotti, Alborali, Napolioni, Pasquali and Magistrali. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms. Sassone-Corsi, M., and Raffatellu, M. (2015). No Vacancy: How beneficial microbes
cooperate with immunity to provide colonization resistance to pathogens. J
Immunol. 194, 4081–4087. doi: 10.4049/jimmunol.1403169 Sokol, H., Pigneur, B., Watterlot, L., Lakhdari, O., Bermúdez-Humarán, L. G., Gratadoux, J. J., et al. (2008). Faecalibacterium prausnitzii is an anti-
inflammatory commensal bacterium identified by gut microbiota analysis of January 2016 | Volume 5 | Article 106 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 13
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Belief state representation in the dopamine system
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37160410 Published Version
doi:10.1038/s41467-018-04397-0 Published Version
doi:10.1038/s41467-018-04397-0 Citation Babayan, Benedicte M., Naoshige Uchida, and Samuel. J. Gershman. 2018. “Belief state
representation in the dopamine system.” Nature Communications 9 (1): 1891. doi:10.1038/
s41467-018-04397-0. http://dx.doi.org/10.1038/s41467-018-04397-0. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility ARTICLE ARTICLE Belief state representation in the dopamine system Optimal
state inference is stipulated by Bayes’ rule, which computes a
probability distribution over states (the belief state) conditional
on the available sensory information. This model explicitly
assumes the existence of multiple states distinguished by their
reward distributions (see methods). Thus, in spite of identical
sensory inputs, prior experience allows to probabilistically
distinguish several states (one associated to 1 μL and one to 10
μL). If mice rely on a multi-state representation, they now have
two reference points to compare the intermediate rewards to. Upon the introduction of new intermediate rewards, the
probability of being in the state s1 would be high for small water
amounts and low for large water amounts (Fig. 1c). The
subsequent reward expectation would then be a probability-
weighted combination of the expectations for s1 and s2. Consequently, smaller intermediate rewards would be better than
the expected small reward (a positive prediction error) and bigger
intermediate rewards would be worse than the expected big
reward (a negative prediction error), resulting in a non-
monotonic pattern of RPEs across intermediate rewards (Fig. 1d,
Supplementary Fig. 1c). y
Normative theories propose that animals represent their state
uncertainty as a probability distribution or belief state7–10 pro-
viding a probabilistic estimate of the true state of the environment
based on the current sensory information. Specifically, optimal
state inference as stipulated by Bayes’ rule computes a probability
distribution over states (the belief state) conditional on the
available sensory information. Such probabilistic beliefs about the
current’s state identity can be used to compute reward predictions
by averaging the state-specific reward predictions weighted by the
corresponding probabilities. Similarly to the way RL algorithms
update values of observable states using reward prediction errors,
state-specific predictions of ambiguous states can also be updated
by distributing the prediction error across states in proportion to
their probability. Simply put, standard RL algorithms compute
reward prediction on observable states, but under state uncer-
tainty reward predictions should normatively be computed on
belief states, which correspond to the probability of being in a
given state. In our paradigm, because reward amount defines states, reward
prediction and belief state are closely related. Yet with the same
reward
amount,
standard
RL
and
belief
state
RL
make
qualitatively
different
predictions
(Fig. 1b,
d). Belief state representation in the dopamine system Benedicte M. Babayan1,2, Naoshige Uchida
1 & Samuel.J. Gershman
2 Learning to predict future outcomes is critical for driving appropriate behaviors. Reinforce-
ment learning (RL) models have successfully accounted for such learning, relying on reward
prediction errors (RPEs) signaled by midbrain dopamine neurons. It has been proposed that
when sensory data provide only ambiguous information about which state an animal is in, it
can predict reward based on a set of probabilities assigned to hypothetical states (called the
belief state). Here we examine how dopamine RPEs and subsequent learning are regulated
under state uncertainty. Mice are first trained in a task with two potential states defined by
different reward amounts. During testing, intermediate-sized rewards are given in rare trials. Dopamine activity is a non-monotonic function of reward size, consistent with RL models
operating on belief states. Furthermore, the magnitude of dopamine responses quantitatively
predicts changes in behavior. These results establish the critical role of state inference in RL. 1 Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA. 2 Department
of Psychology, Center for Brain Science, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA. These authors contributed equally: Naoshige
Uchida, Samuel J. Gershman. Correspondence and requests for materials should be addressed to N.U. (email: uchida@mcb.harvard.edu)
or to Samuel.J.G. (email: gershman@fas.harvard.edu) 1 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1891 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 state st of average value 0.5 (on a scale between 0 and 1, which
would be equivalent to 4.5 μL). D D
opamine neurons are thought to report a reward predic-
tion error (RPE, or the discrepancy between observed and
predicted reward) that drives updating of predictions1–5. In reinforcement learning (RL) theories, future reward is pre-
dicted based on the current state of the environment6. Although
many studies have assumed that the animal has a perfect
knowledge about the current state, in many situations the infor-
mation needed to determine what state the animal occupies is not
directly available. For example, the value of foraging in a patch
depends on ambiguous sensory information about the quality of
the patch, its distance, the presence of predators, and other factors
that collectively constitute the environment’s state. A strikingly different pattern is predicted by an RL model that
uses state inference to compute reward expectations. Belief state representation in the dopamine system The
main
distinction between both classes of models is the following: the
standard RL model does not have distinct states corresponding to
the small and large reward states, and reward prediction is based
on the cached value learned directly from experienced reward,
whereas the belief state model has distinct states corresponding to
the small and large reward states (Supplementary Fig. 1, left
column). In the latter case, the animal or agent uses ambiguous
information to infer which state it is in, and predicts reward based
on this inferred state (i.e., belief state). This leads to the hypothesis that dopamine activity should
reflect prediction errors computed on belief states. However,
direct evidence for this hypothesis remains elusive. Here we
examine how dopamine RPEs and subsequent learning are
regulated under state uncertainty, and find that both are con-
sistent with RL models operating on belief states. To
test
whether
dopamine
neurons
in
mice
exhibited
this modulation by inferred states, we recorded dopamine
neuron population activity using fiber photometry (fluorometry)
(Fig. 1e)13–16. We used the genetically encoded calcium indicator,
GCaMP6f17, 18, expressed in the ventral tegmental area (VTA) of
transgenic mice expressing Cre recombinase under the control of
the dopamine transporter gene (DAT-cre mice)19 crossed with
reporter mice expressing red fluorescent protein (tdTomato)
(Jackson Lab). We focused our analysis on the phasic responses. Indeed, calcium imaging limits our ability to monitor long-
timescale changes in baseline due to technical limitations such as
bleaching of the calcium indicator. Moreover a majority of
previous work studying dopamine neurons has shown reward
prediction error-like signaling in the phasic responses1, 3, 12. Similarly to single-cell recordings1, 3, 12, population activity of
dopamine neurons measured by fiber photometry in the VTA20
(Supplementary Fig. 2) or in terminals of dopamine neurons
projecting to the ventral striatum16, 21 show canonical RPE
coding in classical conditioning tasks. | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Only
one odor and one sound cue were used for all blocks, making the two states perceptually similar prior to reward delivery. To test for state inference
influence on dopaminergic neuron signaling, we then introduced rare blocks with intermediate-sized rewards. Using (with reinforcement learning (RL)
operating on belief states) or not (with standard RL) the training blocks as reference state for computing the value of the novel intermediate states predicts
contrasting RPE patterns (b vs d). b RPE across varying rewards computed using standard RL. Because the same odor preceded both reward sizes, a
standard RL model with a single state would produce RPEs that increase linearly with reward magnitude. c Belief state b across varying rewards defined as
the probability of being in s1 given the received reward. d RPE across varying rewards computed using the value of the belief state b. A non-monotonic
pattern across increasing rewards is predicted when computing the prediction error on the belief state b. e (Top) Population activity of VTA dopaminergic
neurons is recorded in behaving mice using fiber photometry. (Bottom) Fiber location above the recorded cells in the VTA, which co-express the calcium
reporter GCaMP6f and the fluorescent protein tdTomato (scale bar: 200 μm) suggested that mice acquired the main features of the task: reward
on trial 1 indicates the current block type and reward is stable
within a block. (two-way ANOVA, no effect of current or previous block, p >
0.16; Fig. 2a, c). The dopamine response on cue presentation did
not show such modulation, only reflecting the activity on the
previous trial (one sample t-tests, p > 0.27, Fig. 2e; two-way
ANOVA, main effect of previous block on trial 1, p = 0.0025,
Fig. 2f), although the response on reward presentation showed
modulation by both the current and previous block (two-way
ANOVA on trial 1, main effect of current block, p < 0.001, main
effect of previous block, p = 0.038, Fig. 2h), with significant
changes in amplitude at block transitions for block s1 following s2
and blocks s2 (one sample t-tests, p < 0.01, Fig. 2g). (two-way ANOVA, no effect of current or previous block, p >
0.16; Fig. 2a, c). The dopamine response on cue presentation did
not show such modulation, only reflecting the activity on the
previous trial (one sample t-tests, p > 0.27, Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 2), every other training day we
replaced 10% of the training blocks (3) by intermediate reward
blocks, with each intermediate reward being presented no more
than once per day. Over their whole training history, each mouse
experienced 3980 ± 213 (mean ± s.e.m.) trials of each training
block and 42 ± 6 (mean ± s.e.m.) trials of each intermediate
reward (Supplementary Fig. 4). On the first trial of reward pre-
sentation, the dopamine neurons responded proportionally to
reward magnitude (Fig. 3a–c). Importantly, the monotonically
increasing response on this first trial, which informed mice about
the volume of the current block, suggested dopamine neurons
had access to the current reward. On the second trial, the
response of dopamine neurons presented a non-monotonic pat-
tern, with smaller responses to intermediate rewards (2 and 4 µL)
than to bigger intermediate rewards (6 and 8 µL) (Fig. 3e, f, g). h
d
l
d Analyzing the licking and dopamine activity at block start,
when the sound comes on, mice appeared to increase licking
following the small block s1 between sound offset and trial 1’s
odor onset (during a fixed period of 3 s) (Supplementary
Fig. 3a, b). Although this was not sufficient to actually reverse
the licking pattern on trial start, it likely contributed to the
observed change in licking between trial 5 and 1 (Fig. 2b). Dopamine activity showed the opposite tendency, with decreasing
activity following blocks s2 (Supplementary Fig. 3c, d). This
activity on block start indicated that mice partially predicted a
change in contingency, following the task’s initial training
structure (deterministic switch between blocks during the first
10 days). However, this predictive activity did not override the
effect of the previous block on dopamine activity on cue
presentation as it was most similar to the activity on the
preceding block’s last trial (Fig. 2e). Following trial 1, anticipatory
licking and dopamine activity on cue and reward presentation
reached stable levels, with lower activity in s1 compared to s2
(two-way ANOVAs, main effect of current block on trials 2 to 5,
p < 0.05, no effect of previous block, p > 0.4, nor interaction, p >
0.5; Fig. 2c, f, h). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 2e; two-way
ANOVA, main effect of previous block on trial 1, p = 0.0025,
Fig. 2f), although the response on reward presentation showed
modulation by both the current and previous block (two-way
ANOVA on trial 1, main effect of current block, p < 0.001, main
effect of previous block, p = 0.038, Fig. 2h), with significant
changes in amplitude at block transitions for block s1 following s2
and blocks s2 (one sample t-tests, p < 0.01, Fig. 2g). Dopaminergic and behavioral signature of belief states. Once
mice showed a stable pattern of licking and dopamine neuron
activity in the training states (Fig. 2), every other training day we
replaced 10% of the training blocks (3) by intermediate reward
blocks, with each intermediate reward being presented no more
than once per day. Over their whole training history, each mouse
experienced 3980 ± 213 (mean ± s.e.m.) trials of each training
block and 42 ± 6 (mean ± s.e.m.) trials of each intermediate
reward (Supplementary Fig. 4). On the first trial of reward pre-
sentation, the dopamine neurons responded proportionally to
reward magnitude (Fig. 3a–c). Importantly, the monotonically
increasing response on this first trial, which informed mice about
the volume of the current block, suggested dopamine neurons
had access to the current reward. On the second trial, the
response of dopamine neurons presented a non-monotonic pat-
tern, with smaller responses to intermediate rewards (2 and 4 µL)
than to bigger intermediate rewards (6 and 8 µL) (Fig. 3e, f, g). These monotonic and non-monotonic patterns on trials 1 and
2, respectively, were observed in our three different recording
conditions: (1) in mice expressing GCaMP6f transgenetically in
DAT-positive neurons and recorded from VTA cell bodies (n =
5), (2) in mice expressing GCaMP6f through a viral construct in
DAT-positive neurons and recorded from VTA cell bodies (n =
2); (3) in mice expressing GCaMP6f through a viral construct in
DAT-positive neurons and recorded from dopamine neuron
terminals
in
the
ventral
striatum
(n = 4)
(Supplementary
Fig. 5a–c). Although these patterns were observed in each
condition, the amplitude of the signal varied across the different
recording conditions, largely due to lower expression levels of Dopaminergic and behavioral signature of belief states. Once
mice showed a stable pattern of licking and dopamine neuron
activity in the training states (Fig. Results
T Testing prediction error modulation by belief state. We
designed a task that allowed us to test distinct theoretical
hypotheses about dopamine responses with or without state
inference. We trained 11 mice on a Pavlovian conditioning task
with two states distinguished only by their rewards: an identical
odor cue predicted the delivery of either a small (s1) or a big (s2)
reward (10% sucrose water) (Fig. 1a). The different trial types
were presented in randomly alternating blocks of five identical
trials, and a tone indicated block start. Only one odor and one
sound cue was used for all blocks, making the two states per-
ceptually similar prior to reward delivery. This task feature
resulted in ambiguous sound and odor cues, since they were
themselves insufficiently informative of the block identity, ren-
dering the two states ambiguous with respect to their identity. This feature increased the likelihood of mice relying on prob-
abilistic state inference. To test for state inference influence on dopaminergic neuron
signaling, we then introduced rare blocks with intermediate-sized
rewards. Because the same odor preceded both reward sizes, a
standard RL model with a single state would produce RPEs that
increase linearly with reward magnitude (Fig. 1b, Supplementary
Fig. 1a)11, 12. This prediction follows from the fact that the single
state’s value will reflect the average reward across blocks, and
RPEs are equal to the observed reward relative to this average
reward value. The actual value of the state will affect the intercept
of the linear RPE response, but not its monotonicity. In Fig. 1b
and Supplementary Fig 1a, we illustrated our prediction with a Behavior and dopamine neuron activity on training blocks. After training mice on the small (s1 = 1 µL) and big (s2 = 10 µL)
states, we measured their amount of anticipatory licking, a read-
out for reward expectation, and the dopamine responses (Fig. 2a,
d). At block transitions, mice had a tendency to anticipate a
change in contingency as they increased anticipatory licking in
trial 1 following a small block (one sample t-tests, p < 0.05,
Fig. 2b), leading to similar levels of anticipatory licking on trial 1 NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 e
Laser
Detector
Detector
VTA
Optic fiber
GCaMP6f, tdTomato
DAT-cre mouse b Reward (r )
= r-V(b)
RPE
–0.5
0
0.5
Reward (r)
0.5
1
d b
0
0.5
1
0
1
Reward (r )
0.5
Belief state: b = P(s = s1⏐r )
c c d c Reward (r) Reward (r) Fig. 1 Task design to test the modulation of dopaminergic RPEs by state inference. a Mice are trained on two perceptually similar states only distinguished
by their rewards: small (s1) or big (s2). The different trial types, each starting by the onset of a unique odor (conditioned stimulus, CS) predicting the
delivery of sucrose (unconditioned stimulus, US), were presented in randomly alternating blocks of five identical trials. A tone indicated block start. Only
one odor and one sound cue were used for all blocks, making the two states perceptually similar prior to reward delivery. To test for state inference
influence on dopaminergic neuron signaling, we then introduced rare blocks with intermediate-sized rewards. Using (with reinforcement learning (RL)
operating on belief states) or not (with standard RL) the training blocks as reference state for computing the value of the novel intermediate states predicts
contrasting RPE patterns (b vs d). b RPE across varying rewards computed using standard RL. Because the same odor preceded both reward sizes, a
standard RL model with a single state would produce RPEs that increase linearly with reward magnitude. c Belief state b across varying rewards defined as
the probability of being in s1 given the received reward. d RPE across varying rewards computed using the value of the belief state b. A non-monotonic
pattern across increasing rewards is predicted when computing the prediction error on the belief state b. e (Top) Population activity of VTA dopaminergic
neurons is recorded in behaving mice using fiber photometry. (Bottom) Fiber location above the recorded cells in the VTA, which co-express the calcium
reporter GCaMP6f and the fluorescent protein tdTomato (scale bar: 200 μm) Fig. 1 Task design to test the modulation of dopaminergic RPEs by state inference. a Mice are trained on two perceptually similar states only distinguished
by their rewards: small (s1) or big (s2). The different trial types, each starting by the onset of a unique odor (conditioned stimulus, CS) predicting the
delivery of sucrose (unconditioned stimulus, US), were presented in randomly alternating blocks of five identical trials. A tone indicated block start. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 1
RPE
–0.5
0
0.5
Reward (r )
0.5
b
0
0.5
1
0
1
Reward (r )
0.5
= r-V(b)
RPE
–0.5
0
0.5
Reward (r)
0.5
1
Belief state: b = P(s = s1⏐r )
s1
s2
s2
s1
? What is the current state? Sucrose
Odor
a
b
e
c
d
s1
s2
? Laser
Detector
Detector
VTA
Optic fiber
GCaMP6f, tdTomato
DAT-cre mouse
= r -V(s)
Fig. 1 Task design to test the modulation of dopaminergic RPEs by state inference. a Mice are trained on two perceptually similar states only distinguished
by their rewards: small (s1) or big (s2). The different trial types, each starting by the onset of a unique odor (conditioned stimulus, CS) predicting the
delivery of sucrose (unconditioned stimulus, US), were presented in randomly alternating blocks of five identical trials. A tone indicated block start. Only
one odor and one sound cue were used for all blocks, making the two states perceptually similar prior to reward delivery. To test for state inference
influence on dopaminergic neuron signaling, we then introduced rare blocks with intermediate-sized rewards. Using (with reinforcement learning (RL)
operating on belief states) or not (with standard RL) the training blocks as reference state for computing the value of the novel intermediate states predicts
contrasting RPE patterns (b vs d). b RPE across varying rewards computed using standard RL. Because the same odor preceded both reward sizes, a
standard RL model with a single state would produce RPEs that increase linearly with reward magnitude. c Belief state b across varying rewards defined as
the probability of being in s1 given the received reward. d RPE across varying rewards computed using the value of the belief state b. A non-monotonic
pattern across increasing rewards is predicted when computing the prediction error on the belief state b. e (Top) Population activity of VTA dopaminergic
neurons is recorded in behaving mice using fiber photometry. (Bottom) Fiber location above the recorded cells in the VTA, which co-express the calcium
reporter GCaMP6f and the fluorescent protein tdTomato (scale bar: 200 μm) 1
RPE
–0.5
0
0.5
Reward (r )
0.5
= r-V(b)
RPE
–0.5
0
0.5
Reward (r)
0.5
1
b
e
d
Laser
Detector
Detector
VTA
Optic fiber
GCaMP6f, tdTomato
DAT-cre mouse
= r -V(s) s1
s2
s2
s1
? What is the current state? Sucrose
Odor
a
s1
s2
? | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 2 Behavior and dopamine neuron activity on training blocks s1 and s2. a Licking across the five trials within a block. Anticipatory licking quantification
period during odor to reward delay is indicated by the horizontal black line. b Anticipatory licking at block transition increases when transitioning from the
small to the big block. c Anticipatory licking across trials within blocks. Anticipatory licking on trial 1 is similar across all block types then stabilizes at either
low or high rates for the following four trials. d Dopamine neuron activity across the five trials within a block. Horizontal black line indicates quantification
period for odor (CS) and reward (US) responses. e Dopamine neurons odor response across block transitions is stable. f Dopamine neurons odor response
across trials. Dopamine activity adapts to the current block value within one trial. g Dopamine neurons reward response shows an effect of the current
reward and previous block on trial 1. h Dopamine neurons reward response across trials. Dopamine activity reaches stable levels as from trial 2. Data
represents mean ± s.e.m. *p < 0.05 for t-test comparing average value to 0. n = 11 mice Odor response (CS) Odor re
At block transition
Δ lick rate (trial 1– trial 5)
–0.4
0
0.4
0.8
*
*
b 1
2
3
4
5
Trial
1
2
3
4
5
0
Lick/s
Within blocks
c b Block s1 (1 μL), previous s1
Block s1 (1 μL), previous s2
Block s2 (10 μL), previous s1
Block s2 (10 μL), previous s2 c Dopamine neurons
dF/F (%)
6
Trial 5
Odor
Odor
Trial 4
4
0
2
6
4
0
2
Odor
Trial 3
6
Trial 2
Odor
4
0
2
6
Trial 1
Odor
6
4
0
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
2
4
0
2
d d Reward
0
2
4
–2
ΔdF/F (%) (trial 1– trial 5)
At block transition
*
*
*
g esponse (CS)
0
0.2
0.4
0.6
0.8
1
2
3
4
5
Trial
dF/F (%)
Within blocks
f Odor
–0.5
0
0.5
ΔdF/F (%) (trial 1– trial 5)
At block transition
e onse (US)
1
2
3
4
5
Trial
dF/F (%)
Within blocks
0
2
4
h f h e Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Reward response (US)
Odor response (CS)
Dopamine neurons
Behavior
dF/F (%)
Lick/s
Odor response (CS)
At block transition
Within blocks
1
2
3
4
5
Trial
1
2
3
4
5
0
Lick/s
Within blocks
Block s1 (1 μL), previous s1
Block s1 (1 μL), previous s2
Block s2 (10 μL), previous s1
Block s2 (10 μL), previous s2
Trial 5
Odor
6
Trial 5
Odor
Odor
Odor
Trial 4
Trial 4
4
0
2
6
4
2
0
6
Time (s)
4
0
2
4
2
0
6
Time (s)
Trial 3
Odor
Odor
Trial 3
6
Trial 2
Trial 2
Odor
Odor
4
0
2
4
2
0
6
Time (s)
6
Trial 1
Trial 1
Odor
Odor
6
8
0
4
2
0
6
Time (s)
4
8
0
4
8
0
4
8
0
4
8
0
4
4
0
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
2
At block transition
Δ lick rate (trial 1– trial 5)
–0.4
0
0.4
0.8
*
*
At block transition
Within blocks
4
2
0
6
Time (s)
4
0
2
a
b
d
e
f
g
h
c Behavior
Lick/s
Trial 5
Odor
Odor
Trial 4
4
2
0
6
Time (s)
4
2
0
6
Time (s)
Trial 3
Odor
Trial 2
Odor
4
2
0
6
Time (s)
Trial 1
Odor
8
0
4
2
0
6
Time (s)
4
8
0
4
8
0
4
8
0
4
8
0
4
4
2
0
6
Time (s)
a a a Reward response (US)
Odor response (CS)
Dopamine neurons
dF/F (%)
Lick/s
Odor response (CS)
0
2
4
–2
ΔdF/F (%) (trial 1– trial 5)
At block transition
*
*
*
0
0.2
0.4
0.6
0.8
1
2
3
4
5
Trial
dF/F (%)
Within blocks
1
2
3
4
5
Trial
1
2
3
4
5
0
Lick/s
Within blocks
Block s1 (1 μL), previous s1
Block s1 (1 μL), previous s2
Block s2 (10 μL), previous s1
Block s2 (10 μL), previous s2
Odor
6
Trial 5
Odor
Odor
Odor
Trial 4
4
0
2
6
4
2
0
6
Time (s)
4
0
2
4
2
0
6
Time (s)
Odor
Odor
Trial 3
6
Trial 2
Odor
Odor
4
0
2
4
2
0
6
Time (s)
6
Trial 1
Odor
Odor
6
8
0
4
2
0
6
Time (s)
4
8
0
4
8
0
4
8
0
4
8
0
4
4
0
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
4
2
0
6
Time (s)
2
At block transition
Δ lick rate (trial 1– trial 5)
–0.4
0
0.4
0.8
*
*
–0.5
0
0.5
ΔdF/F (%) (trial 1– trial 5)
At block transition
1
2
3
4
5
Trial
dF/F (%)
Within blocks
0
2
4
4
2
0
6
Time (s)
4
0
2
b
d
e
f
g
h
c
Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The stability in anticipatory licking and
dopamine activity after exposure to the first trial of a block These monotonic and non-monotonic patterns on trials 1 and
2, respectively, were observed in our three different recording
conditions: (1) in mice expressing GCaMP6f transgenetically in
DAT-positive neurons and recorded from VTA cell bodies (n =
5), (2) in mice expressing GCaMP6f through a viral construct in
DAT-positive neurons and recorded from VTA cell bodies (n =
2); (3) in mice expressing GCaMP6f through a viral construct in
DAT-positive neurons and recorded from dopamine neuron
terminals
in
the
ventral
striatum
(n = 4)
(Supplementary
Fig. 5a–c). Although these patterns were observed in each
condition, the amplitude of the signal varied across the different
recording conditions, largely due to lower expression levels of NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 3 ARTICLE | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 2 Behavior and dopamine neuron activity on training blocks s1 and s2. a Licking across the five trials within a block. Anticipatory licking quantification
period during odor to reward delay is indicated by the horizontal black line. b Anticipatory licking at block transition increases when transitioning from the
small to the big block. c Anticipatory licking across trials within blocks. Anticipatory licking on trial 1 is similar across all block types then stabilizes at either
low or high rates for the following four trials. d Dopamine neuron activity across the five trials within a block. Horizontal black line indicates quantification
period for odor (CS) and reward (US) responses. e Dopamine neurons odor response across block transitions is stable. f Dopamine neurons odor response
across trials. Dopamine activity adapts to the current block value within one trial. g Dopamine neurons reward response shows an effect of the current
reward and previous block on trial 1. h Dopamine neurons reward response across trials. Dopamine activity reaches stable levels as from trial 2. Data
represents mean ± s.e.m. *p < 0.05 for t-test comparing average value to 0. n = 11 mice GCaMP in transgenic mice compared to those with viral
expression and overall variability in signal intensity across
animals within each recording condition. Therefore, for illustra-
tion purposes, we normalized the signals from each individual
mouse using trial 1’s response as reference for the minimum and
maximum values for the min–max normalization (y = (x −
mintrial1)/(maxtrial1 −mintrial1)) to rescale the GCaMP signals in
the 0 to 1 range (Supplementary Fig. 5d–f, Figs. 3 and 4). Similar results were obtained when measuring the peak response
following reward presentation instead of the average activity
over 1 s (Supplementary Fig. 6a–g). We compared the fits of linear and polynomial functions to the
dopamine responses, revealing highest adjusted r2 for a linear fit
for trial 1 (Supplementary Fig. 7a) and for a cubic polynomial fit
for trial 2 (Supplementary Fig. 7b). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Both RL models predict weaker
prediction error modulation with increasing exposure to the same
reward and we observed weaker versions of this non-monotonic
pattern in later trials (Supplementary Fig. 8a, c). It is however
interesting to note that different mice showed a non-monotonic
reward response modulation at varying degrees on distinct trials. For example, Mouse 4 showed a strong non-monotonic pattern
on trial 2, which then became shallower on the following trials,
whereas Mouse 9 showed a more sustained non-monotonic
pattern across trials 2 to 5 (Supplementary Fig. 8d). Lastly, the
pattern of dopamine responses was observed independently of the
baseline correction method we used, whether it was pre-trial, pre-
block, or using a running median as baseline (Supplementary
Fig. 9). the linear fit still provided a decent fit (adjusted r2 = 0.94). Thus,
dopamine activity and change in anticipatory licking both showed
modulation according to our prediction of the influence of belief
state on RPE (Fig. 1d). Although the average change in
anticipatory licking for transitions from trial 3 to 5 did not seem
to visibly follow the pattern of dopamine activity (Supplementary
Fig. 10a), a trial-by-trial analysis showed that dopamine responses
on reward presentation were significantly correlated with a
change of licking on following trial for all trial transitions within
blocks (trial 1 to 5, Pearson’s r, p < 2.5 × 10−3, Supplementary
Fig. 10b), suggesting that inhibition or lower activations of
dopamine neurons were more often followed by a decrease in
anticipatory licking whereas transient activations of dopamine
neurons tended to be followed by increased anticipatory licking. Belief state RL explains dopamine responses and behavior. We
next tested whether an RL model operating on belief states could
explain the dopamine recordings better than a standard RL
model. As the odor indicating trial start was identical for all
reward sizes, a standard RL model (without belief states) would
assume a single state, with prediction errors that scale linearly
with reward (Supplementary Fig. 1a). An RL model using belief
states, by contrast, differentiates the states based on the current
reward size and the history of prior reward sizes within a block
(Supplementary Fig. 1c). Belief states were defined as the pos-
terior probability distribution over states given the reward history,
computed using Bayes’ rule (Methods). Since the previous block
had an effect on the expectation of the first trial of a given block
(Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 3 Dopaminergic and behavioral signature of belief states. a–c Dopamine neurons activity on trial 1. Dopamine neurons show a monotonically increasing
response to increasing rewards (a, individual example), quantified as the mean response after reward presentation (0–1 s, indicated by a solid black line in
a) in the individual example (b) and across mice (c). d Change in anticipatory licking from trial 1 to trial 2. Mice increase their anticipatory licking after trial 1
proportionally to the increasing rewards. e–g Dopamine neurons activity on trial 2. Dopamine neurons show a non-monotonic response pattern to
increasing rewards (e, f, individual example), quantified across all mice (g). h Change in anticipatory licking from trial 2 to trial 3. Whereas mice do not
additionally adapt their licking for the known trained volumes (1 and 10 μL) after trial 2, they increase anticipatory licking for small intermediate rewards and
decrease it for larger intermediate rewards in a pattern, which follows our prediction of belief state influence on RPE. n = 11, data represent mean ± s.e.m. state influence on dopamine reward RPE (Fig. 1d). We focused
our analysis on trial 2 since, according to our model, that is the
most likely trial to show an effect of state inference with the
strongest difference from standard RL reward prediction errors
(Supplementary Fig. 8a, b). Both RL models predict weaker
prediction error modulation with increasing exposure to the same
reward and we observed weaker versions of this non-monotonic
pattern in later trials (Supplementary Fig. 8a, c). It is however
interesting to note that different mice showed a non-monotonic
reward response modulation at varying degrees on distinct trials. For example, Mouse 4 showed a strong non-monotonic pattern
on trial 2, which then became shallower on the following trials,
whereas Mouse 9 showed a more sustained non-monotonic
pattern across trials 2 to 5 (Supplementary Fig. 8d). Lastly, the
pattern of dopamine responses was observed independently of the
baseline correction method we used, whether it was pre-trial, pre-
block, or using a running median as baseline (Supplementary
Fig. 9). state influence on dopamine reward RPE (Fig. 1d). We focused
our analysis on trial 2 since, according to our model, that is the
most likely trial to show an effect of state inference with the
strongest difference from standard RL reward prediction errors
(Supplementary Fig. 8a, b). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 n = 1
n = 1
1.5
–0.5
0.5
Dopamine neurons
Behavior
1
0
1
2
0
3
Time (s)
dF/F (%)
2
n = 11
Trial 1
1 μL
2 μL
4 μL
6 μL
8 μL
10 μL
–2
–1
0
1
2
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
dF/F (%)
0
0.5
1
1.5
0
0.2
0.4
0.6
0.8
1
Normalized dF/F (%)
n = 11
Δ lick rate (trial 2 – trial 1)
a
b
c
d Behavior
n = 11
–2
–1
0
1
2
Reward (μL)
1
4
8
2
6
10
Δ lick rate (trial 2 – trial 1)
d n = 1
n = 1
1.5
–0.5
0.5
Dopamine neurons
1
0
1
2
0
3
Time (s)
dF/F (%)
2
Trial
1 μL
2 μL
4 μL
6 μL
8 μL
10 μL
Reward (μL)
1
4
8
2
6
10
dF/F (%)
0
0.5
1
1.5
a
b al 1
Reward (μL)
1
4
8
2
6
10
0
0.2
0.4
0.6
0.8
1
Normalized dF/F (%)
n = 11
c n = 1
1.5
–0.5
0.5
1
0
1
2
0
3
Time (s)
dF/F (%)
2
1 μL
2 μL
4 μL
6 μL
8 μL
10 μL
a a Reward (μL) n = 1
n = 11
Trial 2
Normalized dF/F (%)
dF/F (%)
0
0.5
1
1.5
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
0
0.2
0.4
0.6
0.8
1
f
g n = 1
n = 1
–0.5
0.5
dF/F (%)
Trial 2
1
0
1
2
0
3
Time (s)
Normalized dF/F (%)
dF/F (%)
0
0.5
1
1.5
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
2
6
0
0.2
0.4
0.6
0.8
1
e
f
g n = 11
–2
–1
0
1
2
Reward (μL)
1
4
8
2
6
10
Δ lick rate (trial 3 – trial 2)
h n = 1
–0.5
0.5
dF/F (%)
1
0
1
2
0
3
Time (s)
e n = 1
n = 1
n = 11
–0.5
0.5
dF/F (%)
n = 11
Trial 2
1
0
1
2
0
3
Time (s)
Normalized dF/F (%)
–2
–1
0
1
2
dF/F (%)
0
0.5
1
1.5
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
0
0.2
0.4
0.6
0.8
1
Δ lick rate (trial 3 – trial 2)
e
f
g
h e Fig. | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The non-monotonic pattern
observed on trial 2 was consistent with our hypothesis of belief | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS| (2018) 9:1891 4 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 n = 1
n = 1
n = 1
n = 1
n = 11
1.5
–0.5
0.5
–0.5
0.5
Dopamine neurons
Behavior
1
0
1
2
0
3
Time (s)
dF/F (%)
dF/F (%)
2
n = 11
n = 11
Trial 2
Trial 1
1
0
1
2
0
3
Time (s)
1 μL
2 μL
4 μL
6 μL
8 μL
10 μL
Normalized dF/F (%)
–2
–1
0
1
2
–2
–1
0
1
2
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
dF/F (%)
0
0.5
1
1.5
dF/F (%)
0
0.5
1
1.5
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
Reward (μL)
1
4
8
2
6
10
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
Normalized dF/F (%)
n = 11
Δ lick rate (trial 2 – trial 1)
Δ lick rate (trial 3 – trial 2)
a
b
c
d
e
f
g
h
Fig. 3 Dopaminergic and behavioral signature of belief states. a–c Dopamine neurons activity on trial 1. Dopamine neurons show a monotonically increasing
response to increasing rewards (a, individual example), quantified as the mean response after reward presentation (0–1 s, indicated by a solid black line in
a) in the individual example (b) and across mice (c). d Change in anticipatory licking from trial 1 to trial 2. Mice increase their anticipatory licking after trial 1
proportionally to the increasing rewards. e–g Dopamine neurons activity on trial 2. Dopamine neurons show a non-monotonic response pattern to
increasing rewards (e, f, individual example), quantified across all mice (g). h Change in anticipatory licking from trial 2 to trial 3. Whereas mice do not
additionally adapt their licking for the known trained volumes (1 and 10 μL) after trial 2, they increase anticipatory licking for small intermediate rewards and
decrease it for larger intermediate rewards in a pattern, which follows our prediction of belief state influence on RPE. n = 11, data represent mean ± s.e.m. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 2), we allowed for two different initial values on block start
depending on the previous block in both models, and fit RL
models to the trial-by-trial dopamine response of each trained We next analyzed whether behavior was influenced by state
inference. Anticipatory licking before reward delivery is a read-
out of mice’s reward expectation. Dopamine RPEs are proposed
to update expectations. To test whether mice’s behavioral
adaptation across trials followed the dopaminergic RPE pattern,
we measured how mice changed their anticipatory licking across
trials. From trial 1 to trial 2, mice changed their anticipatory
licking proportionally to the volume (Fig. 3d) but showed a non-
monotonic change from trial 2 to trial 3 (Fig. 3h; highest adjusted
r2 for a cubic polynomial fit, Supplementary Fig. 7d). Fits of linear
and polynomial functions to the change in anticipatory licking
revealed highest adjusted r2 for cubic polynomial fits for both
transitions from trial 1 and 2 (Supplementary Fig. 7c), although NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Trial 1
Trial 2
Normalized dopamine
response
Reward (μL)
6
4
2
10
1
8
Reward (μL)
6
4
2
10
1
8
0
0.2
0.4
0.6
0.8
1
n = 11
0
0.2
0.4
0.6
0.8
1
Dopamine neuron activity model fits
Model predictions on behaviour
Trial 2 - Mouse 1
Trial 2 - Mouse 2
All trials
Reinforcement learning
with belief state
Standard reinforcement
learning
Data
n = 11
0
0.25
0.5
0.75
1
0
0.25
0.5
0.75
1
Reinforcement learning with
belief state model correlations
Standard reinforcement
learning model correlations
1
Lick/s
0
0.5
1
Value
0
2
3
Reward (μL)
6
4
2
10
1
8
0
2
Reward (μL)
6
4
2
10
1
8
1
Lick/s
0
0.5
1
Value
a
b
c
d
Fig. 4 RL with belief states explains dopamine reward responses and behavior better than standard RL. Individual DA responses to rewards were fit using
either a standard RL model or a RL model computing values on belief states. a Fits to dopamine responses on trial 1. Both RL models fit the dopamine
response, since on trial 1 there is no evidence to infer a state on. b Fits to dopamine responses on trial 2. Only computing RPEs using belief states
reproduced the non-monotonic change in dopamine response across increasing rewards. c Model predictions on behavior. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The value functions from either
model fits were positively correlated with the mice’s anticipatory licking, but the RL model with belief state provided a better fit (signed rank test: p =
0.032), suggesting that mice’s anticipatory licking tracks the value of the belief state. d Individual examples of extracted value function from either model
and anticipatory licking across increasing rewards on trial 2. n = 11, data represent mean ± s.e.m. Trial 1
Normalized dopamine
response
Reward (μL)
6
4
2
10
1
8
0
0.2
0.4
0.6
0.8
1
Dopamine neuron
n = 11
a Trial 2
Reward (μL)
6
4
2
10
1
8
0
0.2
0.4
0.6
0.8
1
n = 11
n activity model fits
Reinforcement learning
with belief state
Standard reinforcement
learning
Data
b Reward (μL) Model predictions on behaviour All trials
0
0.25
0.5
0.75
1
0
0.25
0.5
0.75
1
Reinforcement learning with
belief state model correlations
Standard reinforcement
learning model correlations
c Model predictions on behaviour
Trial 2 - Mouse 1
s
1
Lick/s
0
0.5
1
Value
0
2
3
Reward (μL)
6
4
2
10
1
8
d Model predictions on behaviour
Trial 2 - Mouse 1
Trial 2 - Mouse 2
s
1
Lick/s
0
0.5
1
Value
0
2
3
Reward (μL)
6
4
2
10
1
8
0
2
Reward (μL)
6
4
2
10
1
8
1
Lick/s
0
0.5
1
Value
d Trial 2 - Mouse 2
0
2
Reward (μL)
6
4
2
10
1
8
1
Lick/s
0
0.5
1
Value d c Standard reinforcement
learning model correlations Fig. 4 RL with belief states explains dopamine reward responses and behavior better than standard RL. Individual DA responses to rewards were fit using
either a standard RL model or a RL model computing values on belief states. a Fits to dopamine responses on trial 1. Both RL models fit the dopamine
response, since on trial 1 there is no evidence to infer a state on. b Fits to dopamine responses on trial 2. Only computing RPEs using belief states
reproduced the non-monotonic change in dopamine response across increasing rewards. c Model predictions on behavior. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The value functions from either
model fits were positively correlated with the mice’s anticipatory licking, but the RL model with belief state provided a better fit (signed rank test: p =
0.032), suggesting that mice’s anticipatory licking tracks the value of the belief state. d Individual examples of extracted value function from either model
and anticipatory licking across increasing rewards on trial 2. n = 11, data represent mean ± s.e.m. was positively correlated with values extracted from both RL
models (one-tailed t-test, p < 1.0×10−6), but the correlations were
significantly higher with the values computed using a RL model
with belief state (Fig. 4c; Signed rank test, signed rank = 9, p =
0.032), and as shown in two individual examples (Fig. 4d). Although we only fit the model RPEs to the dopamine reward
response, the belief state values used to compute the error term
were apparent in the anticipatory licking activity. Finally, we
performed the same analysis on the dopamine response at cue
onset (Supplementary Fig. 11). Dopamine activity at cue onset
appeared to follow a step function on trials 2 to 5 across
increasing rewards (Supplementary Fig. 11a), similar to the
predicted belief state value (Supplementary Fig. 1c–f). This
activity was positively correlated with values from both models
(one-tailed
t-test,
p ≤1.0×10−3,
Supplementary
Fig. 11b),
although no model was a significantly better predictor (Signed
rank test, signed rank = 21, p = 0.32). mouse across all trials (Supplementary Fig. 1b, e). On trial 1, both
models predicted and fit the linearly increasing dopamine
response to increasing rewards (Fig. 4a). On trial 2, only RPEs
computed on belief states reproduced the non-monotonic change
in dopamine response across increasing rewards (Fig. 4b). We
additionally tested four model variants, two that did not include
influence from the previous block on the value for the standard
RL model (Supplementary Fig. 1a) or on the prior at block start
for the belief state model (Supplementary Fig. 1c), as well as two
other variants of the belief state RL model with distinct priors
based on the previous block (Supplementary Fig. 1d) or with
three states, adding a belief state for the intermediate rewards
(Supplementary Fig. 1f). Overall, only models computing pre-
diction errors on belief states could qualitatively reproduce the
non-monotonic pattern of dopamine activity on trial 2 (Supple-
mentary Fig. 1g–l). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Bayesian information criterion (BIC) and
random-effects model selection22, 23 computed on each of the six
models fit to individual mice’s dopamine activity both favored the
RL model with belief states with two initial free priors over other
models, in particular over the standard RL model with two free
initial values (Supplementary Table 1; Supplementary Fig. 8c). Similar results were obtained when fitting the peak GCaMP
response after reward presentation (Supplementary Table 2;
Supplementary Fig. 6h). mouse across all trials (Supplementary Fig. 1b, e). On trial 1, both
models predicted and fit the linearly increasing dopamine
response to increasing rewards (Fig. 4a). On trial 2, only RPEs
computed on belief states reproduced the non-monotonic change
in dopamine response across increasing rewards (Fig. 4b). We
additionally tested four model variants, two that did not include
influence from the previous block on the value for the standard
RL model (Supplementary Fig. 1a) or on the prior at block start
for the belief state model (Supplementary Fig. 1c), as well as two
other variants of the belief state RL model with distinct priors
based on the previous block (Supplementary Fig. 1d) or with
three states, adding a belief state for the intermediate rewards
(Supplementary Fig. 1f). Overall, only models computing pre-
diction errors on belief states could qualitatively reproduce the
non-monotonic pattern of dopamine activity on trial 2 (Supple-
mentary Fig. 1g–l). Bayesian information criterion (BIC) and
random-effects model selection22, 23 computed on each of the six
models fit to individual mice’s dopamine activity both favored the
RL model with belief states with two initial free priors over other
models, in particular over the standard RL model with two free
initial values (Supplementary Table 1; Supplementary Fig. 8c). Similar results were obtained when fitting the peak GCaMP
response after reward presentation (Supplementary Table 2;
Supplementary Fig. 6h). | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications Methods Most, if
not all, of in vivo calcium imaging studies assume that large
calcium influxes through voltage-gated calcium channels evoked
by spikes dominate calcium signals that they measure. None-
theless, this might not be true in some systems. With respect to
the dopamine system, there are some unique points that need to
be taken into account when we interpret calcium imaging data. First, dopamine neurons have a mechanism to maintain the
baseline, pace-making activity, which relies on calcium35. Second,
increasing evidence suggests that dopamine release is regulated at
the level of axon terminals, through cholinergic and glutamatergic
mechanisms36–38. Furthermore, cholinergic interneurons in the
dorsomedial striatum have been shown to track beliefs about
current state39. However, because our main results hold whether
we monitored the activity from cell bodies or axons of dopamine
neurons, these additional processes are unlikely to affect our
observation of state inference modulation of dopamine neuron
activity. Behavioral paradigm. After 1 week of recovery, mice were water-restricted in their
cages. Weight was maintained above 85% of baseline body weight. Animals were
head-restrained and habituated for 1–2 days before training. Odors were delivered
with a custom-made olfactometer53. Each odor was dissolved in mineral oil at 1:10
dilution. 30 μL of diluted odor was placed inside a filter-paper housing, and then
further diluted with filtered air by 1:20 to produce a 1000 mL/min total flow rate. Odors included isoamyl acetate, 1-hexanol and caproic acid, and differed for dif-
ferent animals. Licks were detected by breaks of an infrared beam placed in front of
the water tube (n.b. the licking behavior had no effect on whether water was
delivered). Trials were presented in blocks of 5 trials. A 15 kHz tone lasting 2 s signaled
block start, ending 3 s before the start of a block’s first trial. Each trial began with 1
s odor delivery (one odor per mouse), followed by a 1 s delay and an outcome (1 to
10 μL of 10% sucrose water, constant within a block). Inter-trial intervals were on
average 8.7 s, composed of an initial fixed 4 s period, to ensure GCaMP signals
went down to baseline between trials, followed by an interval drawn from an
exponential distribution (mean: 4.7 s), resulting in a flat hazard function such that
mice had constant expectation of when the next trial would begin. Mice did 30
blocks per day (150 trials). Methods y
An important question for future research is to determine the
origins of belief state inputs into the dopamine system. One
potential substrate is the orbitofrontal cortex, which has been
proposed to encode state spaces, in particular when states are
perceptually similar but conceptually different40. Dopamine
RPEs have also been shown to be influenced by inferred states
in reversal30 and sensory-preconditioning tasks41, which appear
to rely on state inference and require an intact orbitofrontal
cortex42–45. Another potential substrate for belief state infer-
ence is the hippocampus. It has been proposed to support
structure learning46–50, which would allow mice to infer the
latent causes governing the structure of a task, such as learning
the two-state representation despite ambiguous predictive cues. A recent study found that dopamine neurons alter their
responses based on changes in sensory features of reward51. In
the present study, we focused on reward prediction errors based
on reward sizes. It would be interesting to extend the present
study using different sensory features (e.g., taste or smell of
reward) that may define “states” in multiple dimensions, which
may in turn recruit distinct partners for computing beliefs
regarding their identity. Mice were trained 10 to 15 days on a deterministic training regime, with
alternating small (s1, 1 μL) and big (s2, 10 μL) blocks. The transition between blocks
then became probabilistic, with a 50% probability of block change when a block
started. Intermediate reward blocks (2, 4, 6, and 8 μl) were introduced only after
>20 days of training, every other training day. When mice were probed on
intermediate rewards, 3 (10%) of the training blocks were swapped by 3 different
intermediate reward block. The 11 mice were trained on this task. There are no
distinct experimental groups in this study, so no randomization or blinding was
required. For the classical conditioning task (Supplementary Fig. 2), one mouse was
trained to associate 3 different odors to three reward probabilities (0%, 50%, 90%). The trials were presented pseudo-randomly, interspersed with 10% of unpredicted
water delivery, performing 200 to 300 trials per day. Fiber photometry. The fiber photometry (or fluorometry) system used blue light
from a 473 nm DPSS laser (80–500 μW; Opto Engine LLC, UT, USA) filtered
through a neutral density filter (4.0 optical density, Thorlabs, NJ, USA) and cou-
pled into an optical fiber patchcord (400 µm, Doric Lenses, Quebec, Canada) using
a 0.65 NA microscope objective (Olympus). Methods Animals. Eleven adult male mice were used. All mice were heterozygous for Cre
recombinase under the control of the DAT gene (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J;
Jackson Laboratory)19, crossed to Rosa26-tdTomato reporter mice (Ai9, JAX
007909). Five mice were crossed to Ai95D (Rosa26-GCaMP6f reporter mice, JAX
024105). All mice were housed on a 12 h dark/12 h light cycle (dark from
06:00–18:00) and each performed the behavioral task at approximately the same
time of day each day. After surgery they were individually housed. All surgical and
experimental procedures were in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals and approved by the Harvard
Institutional Animal Care and Use Committee. Increasing evidence suggests that dopamine neurons that
project to the dorsal striatum signal different types of signals. Indeed dopamine neurons projecting to specific regions of the
dorsal striatum have been shown to be activated by rewarding,
aversive and novel stimuli16, 31, 32. Here we recorded from the
canonical dopamine system, involving VTA to ventral striatum
loops, which encode value prediction errors16, 33, 34. Whether
other dopamine inputs projecting to other areas of the dorsal
striatum and broadcasting different types of signals can also be
modulated by inferred states remains to be addressed. Surgery. All surgeries were performed under aseptic conditions with animals
under isoflurane (1%–2% at 1 L/min) anesthesia. Analgesia (ketoprofen, 5 mg/kg, I. P.; buprenorphine, 0.1 mg/kg, I.P.) was administered preoperatively and post-
operatively for 48 h. Mice were surgically implanted with a custom head-plate3 and
an optic fiber either above the medial VTA to record from cell bodies (Bregma
−3.1 AP, 0.6 ML, 4.3 DV; n = 7) or in the ventral striatum to record from
dopamine neurons terminals (Bregma 1.6 AP, 1.3 ML, 3.75 DV; n = 4). No dif-
ference was observed in the signal obtained from either region. The head-plate was
affixed to the skull with dental cement (C&B Metabond) and the optic fiber (200
µm diameter, Doric Lenses) was secured using UV-curing epoxy (Thorlabs,
NOA81), followed by a layer of black Ortho-Jet dental adhesive (Lang Dental). During the same surgery, the 6 mice not crossed with GCaMP6f reporter mice
received 200–400 nL of AAV9/Syn-Flex-GCaMP6f (Upenn Vector Core, diluted 4×
in HBSS) injections into the VTA (Bregma −3.1 AP, 0.6 ML, 4.3 DV). y
The exact sources of calcium signals remain unclear. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 In summary, our data provide direct support for the hypothesis
that belief states can drive dopamine RPEs, and subsequent
behavioral learning when animals are uncertain about the current
state. Although RL accounts of dopamine have typically con-
ceptualized its computational function as “model-free”52, our
data suggest that an internal model of the environment may have
a central role in governing dopamine responses. Two features of our task design allowed us to specifically test
the influence of belief states on dopamine RPE: an extended
training on two reference states, which allowed mice to build a
strong prior over reward distributions, and ambiguity in the cues
used to signal upcoming reward combined to inherent uncer-
tainty in the sensory perception of water amount. Importantly,
the intensive training on the two reference states did not alter the
ability of dopamine neurons to discern the new intermediate
reward sizes when first exposed to them, so the observed non-
monotonic pattern is unlikely to be explained by biased sensory
processing. Interestingly, both anticipatory licking and dopamine
activity appeared to predict a switch in contingency upon block
start. Although the amplitude of these pre-emptive changes were
relatively small compared to responses to the odor cue and
reward presentations, it indicated that the task structure influ-
enced both behavior and dopamine activity, as had been pre-
viously shown in macaques30. Discussion Our results suggest that mice make inferences about hidden states
based on ambiguous sensory information, and use these infer-
ences to determine their reward expectations. In our task design,
this results in a non-monotonic relationship between reward
magnitude and RPE, reflected in the response of dopamine
neurons. Although this pattern is strikingly different from the
patterns observed in classical conditioning studies12, 24, 25, it can
be qualitatively and quantitatively accommodated by a model in
which RPEs are computed on belief states. Our results comple-
ment recent studies that have provided additional evidence for
reflections of hidden-state inference in dopamine responses, for
example when animals learn from ambiguous temporal26–28 and
visual29 cues. Since anticipatory licking in the training blocks reflected the
value of each training block (Fig. 2c), we next examined the
relationship between anticipatory licking and values in the RL
models, with or without belief states, which obtained the best
model
comparison
scores
(BIC
and
protected
exceedance
probability). The models were not fit to these data and hence
this constitutes an independent test of the model predictions. For
each mouse, anticipatory licking in all trials and all reward sizes NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 6 6 ARTICLE | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 state. We set that value at 0.5, the averaged reward over mice’s reward history
(Fig. 1b, Supplementary Fig. 1a). To account for the effect of the previous block on
mice’s expectations (Fig. 2), we also explored a version of this model where the
value on trial 1 at block start could be different depending on the previous block (s1
or s2) (Fig. 4, Supplementary Fig. 1b). Chroma) and focused onto a photodetector (FDS10X10, Thorlabs) connected to a
current preamplifier (SR570, Stanford Research Systems). Acquisition from the red
(tdTomato) fluorophore was simultaneously acquired (band pass filter ET605/70
nm, Chroma). The preamplifier output voltage signal was collected by a NIDAQ
board (PCI-e6321, National Instruments) connected to a computer running Lab-
VIEW (National Instruments) for signal acquisition. RL with belief states: We used the same value learning rules as for the standard
TD model but replaced the state by a belief state b(s), which expresses the animal’s
state uncertainty as a probability distribution over states. This model assumed that
on each trial, mice computed the posterior probability of being in a state s given the
observed reward r following Bayes’ rule: g
q
We have examined whether our signals contain motion artefacts in a previous
study16. Using a set-up with 473 and 561 nm lasers to deliver light to excite
respectively GFP and tdTomato reporters, we previously observed large responses
to unpredicted reward in GCaMP, but not tdTomato, signals when mice are head-
fixed. We thus did not correct the GCaMP signals with tdTomato signals. b sð Þ ¼ P rjs
ð
ÞP sð Þ
P rð Þ
: Anatomical verification. At the end of training, mice were given an overdose of
ketamine/medetomidine, exsanguinated with saline, perfused with 4% paraf-
ormaldehyde, and brains were cut in 50 or 100 μm coronal sections. Sections were
stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Recording
sites and GCaMP6f expression were verified to be amid tdTomato expression in
dopamine neurons cell bodies or ventral striatum terminals (Fig. 1e). The likelihood P rjs
ð
Þ ¼ N r; rs; σ2
ð
Þ was defined as a normal distribution over
rewards r, centered on the average reward normally obtained in the current state rs
with a sensory noise variance σ2 that captured uncertainty about the detected
reward amount. This model thus explicitly assumed the existence of multiple states,
distinguished only by their reward distributions. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The normalization was
performed within each mouse, using the given mouse’s trial 1 response as reference
for the minimum and maximum values for the min–max normalization such that
y = (x −mintrial1)/(maxtrial1 −mintrial1)) (Fig. 3c and g, Supplementary Figs. 5–11). Of note, the models were not fit on the min–max normalized data but directly on
mice’s individual baseline-corrected GCaMP activity. l
l fi
h d
d b h
f
d All belief state models had a minimum of three free parameters: the learning
rate α, the sensory noise variance σ2, and a coefficient β, which mapped theoretical
prediction errors linearly to the measured dopamine response (i.e., the GCaMP
signal). Indeed, because of a relatively long delay between odor onset and reward
delivery (2 s), as well as timing jitter resulting from when mice first sniff after odor
onset, we expected our dopamine reward responses to be generally shifted above
03, 4, 54. This was accounted for by fitting β. Model fitting. For each mouse, we computed the average dopamine response for
each reward size and each trial (trials 1 to 5), separating the data based on the
previous blocks. We fit the free parameters to the dopamine responses using
maximum likelihood estimation. Optimization was performed using the MATLAB
function fmincon, initializing the optimization routine at 5 random parameter
values. performed within each mouse, using the given mouse’s trial 1 response as reference
for the minimum and maximum values for the min–max normalization such that
y = (x −mintrial1)/(maxtrial1 −mintrial1)) (Fig. 3c and g, Supplementary Figs. 5–11). Of note, the models were not fit on the min–max normalized data but directly on
mice’s individual baseline-corrected GCaMP activity. We used the following bounds on the parameter values: Polynomial fits to the dopamine neuron activity and behavior were performed
using the polyfit function in MATLAB. ●
the learning rate α 2 ½0; 0:3,
2 ●
the sensory noise variance σ2 2 ½0:01; 0:5, ●
initial values V 2 ½0; 1, ●
priors p 2 ½0:001; 0:999. Computational modeling. Standard RL: We used a simplified version of the
temporal difference (TD) model11, modeling stimuli and rewards at the trial level
instead of in real time. This model learned values (V) for each state (s). In our task,
states correspond to blocks (s1 = small reward block, s2 = large reward block). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 The prior P(s) expressed the
mice’s prior about the likelihood of the occurrence of a given state. The
denominator represented the marginal reward distribution across all states
P rð Þ ¼ P
s0 P rjs0
ð
ÞP s0
ð Þ. Data analysis. Lick rate was acquired at 1 kHz. Mean anticipatory licking was
calculated for each trial as the mean lick rate in the 1 s delay period between odor
presentation and water delivery. The differential lick rate (Δ lick rate) was com-
puted as the difference of mean anticipatory licking between two consecutive trials,
within a training day. ð Þ
P
s
j
ð
Þ ð Þ
Given the belief state b, the prediction error was: δt ¼ rt V bt
ð Þ; g
y
For GCaMP activity, we focused our analysis on the phasic responses. Indeed, a
majority of previous work has shown reward prediction error-like signaling in the
phasic responses of dopamine neurons and technical limitations such as bleaching
limit our ability to monitor long-timescale changes in baseline using calcium
imaging. Fluorescence data was acquired at 1 kHz. For each trial, the relative
change in fluorescence, dF/F = (F −F0)/F0, was calculated by taking F0 to be the
mean fluorescence during a 1 s period before the odor presentation, such that the
fluorescence measured at each time point within a trial is corrected by the average
fluorescence during the 1 s period before odor presentation for that given trial. We
further tested two additional baseline normalizations to verify that our conclusions
were robust with regards to the baseline normalization method (Supplementary
Fig. 9): (1) using as F0 the 1 s period before block start, i.e., before sound onset,
such that the fluorescence measured at each time point within a trial is corrected by
the average fluorescence during the 1 s period before sound presentation for that
given block (i.e., over five consecutive trials); (2) using as F0 the median over a 60 s
window, such that the fluorescence measured at each time point is corrected by the
median fluorescence over a 60 s period centered around that given time point. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 where the value function was approximated as a linear function of the belief state: V bt
ð Þ ¼ w1bt s1
ð Þ þ w2bt s2
ð Þ: Weights were then updated according to: Δw ¼ αδtbt: We tested four different versions of this model by testing different ways of setting
the prior P(s): ●
Setting P(s) = 0.5 (Fig. 1c, d, Supplementary Fig. 1c), since the mice
experienced
s1
and
s2
with
equal
probability
during
their
training
(Supplementary Fig. 4). pp
y
g
●
Allowing P(s) to be free parameters, defining p1 = P(s = s1) as the prior
following block s1, and p2 = P(s = s2) = 1 −p1 (Supplementary Fig. 1d). ●
Allowing P(s) to be free parameters, defining p1 = P(s = s1) as the prior
following block s1, and p2 = P(s = s2) = 1 −p1 (Supplementary Fig. 1d). ●
Allowing both p1 and p2 as free parameters (Fig. 4, Supplementary Fig. 1e). ●
Setting 3 priors free: p1, p2 and an additional prior for intermediate state (p3),
which corresponded to mice building an additional state for the novel rewards
(Supplementary Fig. 1f). Mean GCaMP activity during odor (CS) and reward (US) presentations was
calculated for each trial as the mean activity during the 1 s period after event onset. Data and model fitting were additionally verified with the peak GCaMP activity
following the reward response, by quantifying the maximum response in the 1 s
window after reward delivery (Supplementary Fig. 6, Supplementary Table 2). Two
types of further normalization were performed on the data, regardless of the
baseline correction used: (1) When analyzing the reward (US) response, since the
CS response did not always go back to baseline before reward presentation, US
responses were baseline-corrected by subtracting the mean dF/F over the 100 ms
period centered around US onset. This provided a measure for the actual change in
activity at reward presentation. (2) Since the absolute level of fluorescence was
variable across mice that expressed GCaMP6f through viral injection or
transgenetically (Supplementary Fig. 5), for illustration purposes to summarize the
data in one plot, each mouse’s mean US response across rewards was normalized
by min–max normalization when pooled together. Methods The patchcord connected to the
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values were updated using the RPE To compare model fits, we computed the Bayesian Information Criterion (BIC),
which allows direct comparison between models that have different numbers of
parameters, and exceedance and protected exceedance probabilities using Bayesian
model selection analysis, which measure how likely it is that any given model is
more frequent than all other models in the comparison set22, 23. δt ¼ rt V st
ð Þ; Code availability. The models were programmed in MATLAB. The code is
available on github (https://github.com/bbabayan/RL_beliefstate). Code availability. The models were programmed in MATLAB. The code is
available on github (https://github.com/bbabayan/RL_beliefstate). following an observation of rt, the reward delivered at trial t:
V Stþ1
¼ V St
ð Þ þ αδt; V Stþ1
¼ V St
ð Þ þ αδt; Quantification and statistical analysis. The values reported in the text and fig-
ures are the mean ± SEM. All data analysis was performed in MATLAB 2014b
(Mathworks). Non-parametric tests were used where appropriate. When using
parametric tests (t-test and ANOVA), we verified that data did not deviate sig-
nificantly from a normal distribution, using a χ2 goodness-of-fit test. Tests were where α is a learning rate and rt 2 0; 1
f
g, with rt = 0 for the small reward block s1
(1 μL) and rt = 1 for the big reward block s2 (10 μL). where α is a learning rate and rt 2 0; 1
f
g, with rt = 0 for the small reward block s1
(1 μL) and rt = 1 for the big reward block s2 (10 μL). The state s was defined by the sensory input at trial start, the CS odor. Since the
same odor preceded both reward sizes, a standard TD model would assume a single NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 two-tailed, except when otherwise mentioned, alpha was set at 0.05. Sample size
was not predetermined. 28. Sarno, S., de Lafuente, V., Romo, R. & Parga, N. Dopamine reward prediction
error signal codes the temporal evaluation of a perceptual decision report. Proc. Natl Acad. Sci. USA 114, E10494–E10503 (2017). 29. Lak, A., Nomoto, K., Keramati, M., Sakagami, M. & Kepecs, A. Midbrain
dopamine neurons signal belief in choice accuracy during a perceptual
decision. Curr. Biol. 27, 821–832 (2017). Data availability. The data that support the findings of this study are available
from the corresponding authors upon reasonable request. 30. Bromberg-Martin, E. S., Matsumoto, M., Hong, S. & Hikosaka, O. A pallidus-
habenula-dopamine pathway signals inferred stimulus values. J. Neurophysiol. 104, 1068–1076 (2010). Received: 1 August 2017 Accepted: 26 April 2018 Author contributions S.J.G. and B.M.B. designed the task, with help from N.U.; B.M.B. collected and analyzed
data; B.M.B. and S.J.G. constructed the computational models; and B.M.B., N.U., and
S.J.G. wrote the manuscript. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04397-0 Howard Hughes Medical Institute’s Janelia Farm Research Campus for providing the
AAV-GCaMP6f through the University of Pennsylvania Vector Core. This work was
supported by the National Institutes of Health grants R01MH095953 (N.U.),
R01MH101207 (N.U.), R01MH109177 (S.G.), Harvard Mind Brain and Behavior faculty
grant (S.G. and N.U.), and Fondation pour la Recherche Medicale grant SPE20150331860
(B.B.). Howard Hughes Medical Institute’s Janelia Farm Research Campus for providing the
AAV-GCaMP6f through the University of Pennsylvania Vector Core. This work was
supported by the National Institutes of Health grants R01MH095953 (N U ) Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. R01MH101207 (N.U.), R01MH109177 (S.G.), Harvard Mind Brain and Behavior faculty
grant (S.G. and N.U.), and Fondation pour la Recherche Medicale grant SPE20150331860
(B.B.). Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Acknowledgements 26. Starkweather, C. K., Babayan, B. M., Uchida, N. & Gershman, S. J. Dopamine
reward prediction errors reflect hidden-state inference across time. Nat. Neurosci. 20, 581–589 (2017). g
We thank members of the Gershman and Uchida labs for insightful discussions; S. Matias, Z. Mainen (Champalimaud Institute of Unknown), C. Burgess, M. Andermann
(Harvard Medical School), and M.W. Mathis for advice on fiber photometry; Edward
Soucy (CBS neuro-engineering platform) for instrumentation assistance; C. Dulac for
sharing resources; and V. Jayaraman, R.A. Kerr, D.S. Kim, L.L. Looger, and K. Svoboda
from the GENIE (Genetically-Encoded Neuronal Indicator and Effector) Project at the We thank members of the Gershman and Uchida labs for insightful discussions; S. Matias, Z. Mainen (Champalimaud Institute of Unknown), C. Burgess, M. Andermann
(Harvard Medical School), and M.W. Mathis for advice on fiber photometry; Edward
Soucy (CBS neuro-engineering platform) for instrumentation assistance; C. Dulac for
sharing resources; and V. Jayaraman, R.A. Kerr, D.S. Kim, L.L. Looger, and K. Svoboda
from the GENIE (Genetically-Encoded Neuronal Indicator and Effector) Project at the 27. Takahashi, Y. K., Langdon, A. J., Niv, Y. & Schoenbaum, G. Temporal
specificity of reward prediction errors signaled by putative dopamine neurons
in rat VTA depends on ventral striatum. Neuron 91, 182–193 (2016). NATURE COMMUNICATIONS| (2018) 9:1891 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications 9 | DOI: 10.1038/s41467-018-04397-0| www.nature.com/naturecommunications Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-04397-0. Competing interests: The authors declare no competing interests. © The Author(s) 2018 © The Author(s) 2018 Reprints and permission information is available online at http://npg.nature.com/ Reprints and permission information is available online at http://npg.nature.com/
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE
Open Access
Development and reliability assessment of
a new quality appraisal tool for cross-
sectional studies using biomarker data
(BIOCROSS)
Jan Wirsching1,2, Sophie Graßmann1,2, Fabian Eichelmann1,2, Laura Malin Harms1,2, Matthew Schenk1,2, Eva Barth1,
Alide Berndzen1,2, Moses Olalekan1,2, Leen Sarmini1,2, Hedwig Zuberer1,2 and Krasimira Aleksandrova1,2*
Wirsching et al. BMC Medical Research Methodology (2018) 18:122
https://doi.org/10.1186/s12874-018-0583-x RESEARCH ARTICLE
Open Access
Development and reliability assessment of
a new quality appraisal tool for cross-
sectional studies using biomarker data
(BIOCROSS)
Jan Wirsching1,2, Sophie Graßmann1,2, Fabian Eichelmann1,2, Laura Malin Harms1,2, Matthew Schenk1,2, Eva Barth1,
Alide Berndzen1,2, Moses Olalekan1,2, Leen Sarmini1,2, Hedwig Zuberer1,2 and Krasimira Aleksandrova1,2*
Wirsching et al. BMC Medical Research Methodology (2018) 18:122
https://doi.org/10.1186/s12874-018-0583-x Wirsching et al. BMC Medical Research Methodology (2018) 18:122
https://doi.org/10.1186/s12874-018-0583-x Open Access Development and reliability assessment of
a new quality appraisal tool for cross-
sectional studies using biomarker data
(BIOCROSS) Jan Wirsching1,2, Sophie Graßmann1,2, Fabian Eichelmann1,2, Laura Malin Harms1,2, Matthew Schenk1,2, Eva Barth1,
Alide Berndzen1,2, Moses Olalekan1,2, Leen Sarmini1,2, Hedwig Zuberer1,2 and Krasimira Aleksandrova1,2* Abstract Background: Biomarker-based analyses are commonly reported in observational epidemiological studies; however
currently there are no specific study quality assessment tools to assist evaluation of conducted research. Accounting
for study design and biomarker measurement would be important for deriving valid conclusions when conducting
systematic data evaluation. Methods: We developed a study quality assessment tool designed specifically to assess biomarker-based cross-sectional
studies (BIOCROSS) and evaluated its inter-rater reliability. The tool includes 10-items covering 5 domains: ‘Study rational’,
‘Design/Methods’, ‘Data analysis’, ‘Data interpretation’ and ‘Biomarker measurement’, aiming to assess different quality
features of biomarker cross-sectional studies. To evaluate the inter-rater reliability, 30 studies were distributed among 5
raters and intraclass correlation coefficients (ICC-s) were derived from respective ratings. Results: The estimated overall ICC between the 5 raters was 0.57 (95% Confidence Interval (CI): 0.38–0.74) indicating a
good inter-rater reliability. The ICC-s ranged from 0.11 (95% CI: 0.01–0.27) for the domain ‘Study rational’ to
0.56 (95% CI: 0.40–0.72) for the domain ‘Data interpretation’. Conclusion: BIOCROSS is a new study quality assessment tool suitable for evaluation of reporting quality
from cross-sectional epidemiological studies employing biomarker data. The tool proved to be reliable for
use by biomedical scientists with diverse backgrounds and could facilitate comprehensive review of biomarker studies
in human research. Keywords: BIOCROSS, Quality appraisal, Evaluation tool, Cross-sectional studies Keywords: BIOCROSS, Quality appraisal, Evaluation tool, Cross-sectional studies * Correspondence: krasimira.aleksandrova@dife.de
1Nutrition, Immunity and Metabolism Senior Scientist Group, Department of
Nutrition and Gerontology, German Institute of Human Nutrition
Potsdam-Rehbruecke (DIfE), Arthur-Scheunert-Allee 114-116, 14558 Nuthetal,
Germany
2University of Potsdam, Institute of Nutritional Science, Potsdam, Germany Background the presence or severity of a particular disease state; (ii)
evaluation of a therapeutic response and (iii) monitoring
disease development. Biomarkers hold great promise for
personalized medicine as information gained from diagnos-
tic or prognostic markers can be used to tailor treatment to
the individual for highly efficient interventions. The booming field of biomedical research over the last
decades resulted in an increasing number of research pa-
pers reporting biomarker information [1, 2]. Biomarkers
have been broadly defined as any measurable characteristic
of an organism that reflects a particular physiological state. These are molecules isolated from serum, urine, or other
fluids that can have multifaceted application (i) indicating While much of the new molecule discoveries are gener-
ated in experimental and laboratory research, epidemio-
logical studies greatly contribute to exploring relevance of
identified biomarkers in humans [3–6]. Among different study designs in epidemiology, cross-
sectional studies have gained much application in utilizing
biomarker data due to their high feasibility. Such studies y
2University of Potsdam, Institute of Nutritional Science, Potsdam, Germany Page 2 of 8 Page 2 of 8 Wirsching et al. BMC Medical Research Methodology (2018) 18:122 are relatively easy, fast and cheap to conduct and can
provide helpful information for hypothesis generation. In cross-sectional studies both exposures and outcome
are measured simultaneously and therefore it may be
difficult to determine whether the exposure proceeded
or succeeded the outcome. Even though no inferences
on causality can be drawn, cross-sectional studies have
proven helpful in gaining insights into potential corre-
lations between biomarkers and other factors [5]. each of the item-related questions. The raters were asked
to give feedback on the applicability of the tool within
2 weeks. Eligible raters were supposed to have a scientific
background (at least bachelor's degree in an epi-
demiological, biomedical or nutritional area). Further-
more,
a
briefing of
how
to
use
BIOCROSS
was
provided. The studies were chosen randomly from a
pool of 77 studies used for a systematic review con-
ducted within our working group [9]. Given the abundance of information created through
published studies, systematic reviews are often used to
summarize and conclusively present obtained knowledge
also on biomarkers. Guidelines like the STrengthening
the Reporting of OBservational studies in Epidemiology
(STROBE) [7] or STROBE-ME [8] have been developed to
guide researchers regarding criteria that may help in the
conduct of their own research and when evaluating the re-
search of others [7]. However, such guidelines are not
suited for obtaining a more objective rating of the study
quality. In particular, there is no specific tool to rapidly
evaluate study quality in commonly used cross-sectional
studies reporting biomarker data. As a third step, we improved and adapted the tool
following results from the pilot inter-rater reliability test-
ing. Raters’ feedbacks were analyzed, and the tool was
revised leading to the final version of BIOCROSS. g
The major changes that were implemented included
changing the dichotomous evaluation scale (“0” or “1”
points) towards an ordinal scale (“0”, “1” or “2” points)
leading to a maximum of 20 points. This was done to
allow a more gradual rating of studies to avoid unjustified
low scores as all items needed to be covered to receive a
point. Three ‘issues to consider’ (IC) are provided for each
of the 10 items. If all IC were discussed in a feasible way, a
score of “2” should be awarded, if 1 or 2 IC were dis-
cussed, 1 point otherwise 0 points should be awarded. By
allowing a gradual rating, the precision of our tool should
be improved. Additional textual edits were also made to
improve general understanding of each item. We recently published a study wherein we report data
from a meta-analysis aimed to evaluate correlations be-
tween biomarkers [9]. During the analysis, we noticed that
many of the peer-reviewed publications that describe uses
of biomarkers employ inconsistent methods of analysis and
data interpretation, and insufficient reporting on general
study procedures (e.g. sample handling, participant selec-
tion). However, no tool was suited to assist us in assessing
quality of individual studies in all relevant domains. We
therefore developed a study quality and reporting assess-
ment tool for biomarker-based cross-sectional studies and
evaluated its inter-rater reliability. As a final step, the adapted tool was handed out to 5
raters to assess 30 studies over a period of 4 weeks. These studies were chosen semi-randomly from the
same pool as the first evaluation. Results from the first
evaluation were used to assure, that studies of different
levels of reporting quality are being assessed. Briefing was not provided as it did not seem to have
added additional value at the first reliability assessment. We therefore assessed the use of the tool among raters
deploying the information provided in the written “User’s
guide to BIOCROSS” without preliminary extensive train-
ing (see Additional file 1). Development of BIOCROSS The development of BIOCROSS followed a 4-stage
approach: 1) design and development, 2) pilot reliability
assessment, 3) improvement/adaptation, 4) reliability as-
sessment of the adapted tool (Fig. 1). BIOCROSS evaluation tool The tool has been divided into 5 domains (‘Study rational’,’
Design/Methods’, ‘Data analysis’, ‘Data interpretation’ and
‘Biomarker measurement’), aiming to assess different qual-
ity features of biomarker cross-sectional studies. Detailed
explanations on how to score different items have been
provided (see Additional file 1). The forth domain, ‘data interpretation’ consists of two
items assessing the interpretation and evaluation of the
results as well as potential study limitations. The first
item addresses the issue of interpretation of the results
in the context of pre-specified research hypotheses. The
item assesses if results are interpreted in the context of
similar studies (if such studies exist) and how the bio-
logical context of the biomarkers under investigation is
described. The second item addresses the issue of how
study limitation arising from the cross-sectional study
design and the need of consistency with similar findings
were discussed. Results
BIOCROSS evaluation tool characteristics. It assesses if important information about
the study population is presented in a feasible way, if
exposures and potential confounders are named and de-
scribed and if values were excluded and what strategies
were applied to address that issue. The second item
assesses evaluation of the pertaining methods for statis-
tical analysis. Statistical analysis As an initial step, a 10-item tool with a crude rating
(“1” = positive and “0” = negative) was developed within
the work for a systematic review [9]. This tool was partly
based on a scale from the National Institutes of Health
[10]. Points from the original 14-point evaluation tool
were adapted and combined to 7 questions assessing the
cross-sectional study design. Furthermore, 3 questions
were added assessing biomarker related quality features,
namely: ‘Specimen characteristics and assay methods’, ‘La-
boratory measurements’ and ‘Biomarker data modeling’. To assess inter-rater agreement, a two-way intraclass
correlation coefficient (ICC) was used to calculate the
agreement among raters for the total BIOCROSS score
as well as for each of the evaluated domains. The
inter-rater-reliability (IRR), as proposed by Cicchetti et
al. [11], provides cutoff points for qualitative ratings of
agreement based on ICC values. An IRR is treated as
excellent for values between 1.0 and 0.75, good for
values between 0.74 and 0.60, fair for values between
0.59 and 0.40, and poor for values below 0.40. All statis-
tical analyses were performed using computing environ-
ment R (R Development Core Team, 2013) packages
“irr” [12] and “lpSolve” [13]. As a second step, pilot reliability was conducted among
7 raters assessing 15 studies to measure the inter-rater re-
liability. Written explanations were provided to explain Page 3 of 8 Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Fig. 1 Tool development process of BIOCROSS Fig. 1 Tool development process of BIOCROSS Domains description The first domain, ‘Study rational’ deals with evaluation of
the study objective and pre-specified research hypotheses. It looks into how the introduction provides important
information in order to get an idea of what to expect from
the study. The second domain, ‘Design/Methods’ consists of two
items assessing study population selection and representa-
tiveness. The first item assesses how the study population
selection was performed and how information about the se-
lection process is presented in the publication. The second
item of this domain addresses the representativeness of the
study population, evaluating sampling frame, participation
rate as well as the sample size justification which should be
provided by the authors. The fifth domain, ‘Biomarker measurement’ consists of
three items that assess how measurement, handling and
modelling of biomarkers were performed. The first item
addresses the specimen characteristics, handling and assay
methods used to perform analysis. It assesses if a reprodu-
cibility assessment was performed to evaluate biomarker
stability and the quantitation methods used in the analysis. The second item assesses the laboratory measurement The third domain, ‘Data analysis’ consists of two items
aimed to assess how data analysis was performed. The
first item assesses the description of study population Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Page 4 of 8 Page 4 of 8 29. Maximum values ranged from 10 (study 5) to 19
(study 29). Minimum values ranged from 7 (study 5) to 13
(study 21). The smallest difference between different raters
was 2 points (6 studies) and the biggest difference was 7
points (study 29). Standard deviations ranged from 0.69
(Study 6) to 2.11 (Study 29). The overall mean was 12.29. 29. Maximum values ranged from 10 (study 5) to 19
(study 29). Minimum values ranged from 7 (study 5) to 13
(study 21). The smallest difference between different raters
was 2 points (6 studies) and the biggest difference was 7
points (study 29). Standard deviations ranged from 0.69
(Study 6) to 2.11 (Study 29). The overall mean was 12.29. itself. The questions addressed are whether the place of
measurement, the quality control procedures or the coeffi-
cient of variation of biomarker measurements have been
provided in the paper. The third item assesses adequacy
of statistical analyses, outlier handling as well as pos-
sible errors resulting from biomarker measurement
inaccuracies and how these have been discussed in
the publication (Table 1). Agreement and reliability The ICCs representing agreement among raters for
the overall assessment score, as well as the agreements
for each of the 5 domains are depicted in Fig. 2. Datasets Total scores of BIOCROSS for each study used in the
evaluation process are depicted in Table 2. The average
values were between 8.2 for study 24 and 15.8 for study Table 1 BIOCROSS evaluation tool. Depicted is the BIOCROSS evaluation tool aimed at evaluating the quality of reporting of
biomarker cross sectional studies
Item
Issues to consider (IC)
Study quality feature
1st Domain: Study rational
1. 1.1 Was the biomarker under study described? 1.2 Was the rationale for the study (research question) clearly presented? 1.3 Were the study objectives/ hypothesis clearly stated? Hypothesis/Objective
2nd Domain: Design/Methods
2. 2.1 Were the characteristics of the study participants presented? 2.2 Were the disease stages or comorbidities of the included participants described? 2.3 Were the inclusion and exclusion criteria for study participation defined? Study population selection
3. 3.1 Was the sampling frame reported (study population source)
3.2 Was the participation rate reported (i.e. eligible persons at least 50%)? 3.3 Was sample size justification or power description provided? Study population representativeness
3rd Domain: Data analysis
4. 4.1 Were the study population characteristics (i.e. demographic, clinical and social) presented? 4.2 Were the exposures and potential confounders described? 4.3 Were any missing values and strategies to deal with missing data reported? Study population characteristics
5. 5.1 Did the authors clearly report statistical methods used to calculate estimates (e.g. Spearman/Pearson/
Linear regression, etc.)? 5.2 Were key potential confounding variables measured and adjusted statistically in reported analyses? 5.3 Was the raw effect size estimate (correlation coefficient, beta coefficient) or measure of study
precision provided (e.g. confidence intervals, precise (!) p-value*)? Statistical analysis
4th Domain: Data interpretation
6. 6.1 Was the data discussed in the context of study objectives/hypotheses? 6.2 Was the interpretation of the results considering findings from similar studies? 6.3 Was the biological context described? Interpretation and evaluation of results
7. 7.1 Was the cross-sectional nature of the analysis discussed? 7.2 Did the authors acknowledge restricted interpretation due to measurements at one point in
time and no statement about causality possible using cross-sectional studies? 7.3 Did the authors acknowledge need for consistency with other research? Study limitations
5th Domain: Biomarker measurement
8. 8.1 Were the measurement methods described? Domains description y
y
Table 2: Total scores for each study used in the evalu-
ation process. Mean: mean of all raters, SD: Standard de-
viation, Min: minimum rating, Max: maximum rating,
Median: median of all raters. *Reporting not significant (ns) or p > 0.05 is not precise and does not allow a judgment on precision Agreement and reliability (assay methods, preservation and storage, detailed
protocol, including specific reagents or kits used)
8.2 Were the reproducibility assessments performed for evaluating biomarker stability? 8.3 Were the quantitation methods well described? Specimen characteristics and assay
methods
9. 9.1 Was the laboratory/place of measurement mentioned? 9.2 Were any quality control procedures and results reported (e.g. reported coefficient of variation? 9.3 Were the analyses blinded for laboratory staff? Laboratory measurement
10. 10.1 Was the distribution of biomarker data reported (if non-normal how it was standardized)? 10.2 Did the authors report on methods or outlier detection and handling? 10.3 Were any possible errors resulting from measurement inaccuracies discussed? Biomarker data modeling
*Reporting not significant (ns) or p > 0.05 is not precise and does not allow a judgment on precision Interpretation and evaluation of results
Study limitations Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Page 5 of 8 Page 5 of 8 Page 5 of 8 Table 2 Total scores for each evaluated study
Studya
Ratings
Mean
SD
Min
Max
Median
1
11.3
0.75
10
12
11.5
2
12.3
1.70
9
14
12.5
3
14.0
1.29
12
16
14
4
12.3
1.70
11
16
12
5
8.7
0.94
7
10
9
6
10.8
0.69
10
12
11
7
12.8
1.34
10
14
13
8
12.8
1.07
11
14
13
9
11.8
0.90
10
13
12
10
12.3
0.94
11
14
12
11
13.8
1.46
11
15
14.5
12
13.8
1.07
12
15
14
13
13.8
1.07
12
15
14
14
12.5
0.76
11
13
13
15
9.3
0.75
8
10
9.5
16
11.8
1.46
9
13
12.5
17
9.2
0.90
8
11
9
18
14.5
1.89
11
17
14.5
19
14.0
1.15
13
16
13.5
20
14.7
1.80
12
17
15
21
15.7
2.05
13
18
15.5
22
11.2
0.90
10
12
11.5
23
9.8
1.07
8
11
10
24
8.2
1.07
7
10
8
25
11.8
2.03
9
15
12
26
13.3
0.94
12
14
14
27
10.5
1.26
9
13
10
28
13.7
1.37
12
16
13
29
15.8
2.11
12
19
16
30
11.8
1.57
9
14
12
aStudies used in the evaluation process of BIOCROSS Table 2 Total scores for each evaluated study (124% increase) could be achieved within the domain
‘data interpretation’ (ICCs range: from 0.25 to 0.56). The
ICCs for the rest of the domains were not substantially
changed. Agreement and reliability Within the domain Biomarker measurement,
the item “Specimen characteristics and assay methods”
as well as the item “Biomarker data modeling” could
only reach ICCs of 0.12 (95% CI: 0.02–0.28) and 0.23
(95% CI: 0.08–0.42) respectively. We also examined if items were not discriminatory,
meaning that they did not fully utilize the scale of our
tool. This could be proven for the items 1, 5 and 6 in
which the lowest grading of “0” was not awarded for any
of the studies under investigation. Figure 2 Intraclass correlation coefficient (ICC) scores
and inter-rater reliability ratings of BIOCROSS total
score and individual domains; BIOCROSS was divided
into 5 different domains meant to assess different as-
pects of a cross-sectional study. ‘Data rationale’ is meant
to assess how the authors presented the study objective
and how the hypothesis was defined. ‘Design/Methods’
assesses study population selection and representative-
ness. ‘Data analysis’ investigates how study population
characteristics were presented and how statistical ana-
lysis was performed. ‘Data interpretation’ deals with
evaluation and interpretation of results and the discus-
sion of study limitations, due to the cross-sectional de-
sign of the study. ‘Biomarker measurement’ assesses how
specific biomarker characteristics and assay methods
were presented and how laboratory measurements as
well as biomarker data analysis were performed. The average time needed by the reviewers to complete
one evaluation was 13.55 min. There were substantial
differences between the raters, with the fastest rater
using an average of less than 10 min per study and the
slowest rater needing on average of more than 20 min
per study. The level of experience and the time needed
to complete one evaluation were correlated. As ex-
pected, more experienced reviewers were faster in read-
ing and evaluating studies. The raters have been also asked to provide a feedback
on their experiences of using the BIOCROSS tool. The
most important point raised by the reviewers was the
need for re-formulation of several item explanations in
the ‘User’s guide’ to BIOCROSS. These have been
updated and further clarified in the revised version
of BIOCROSS. to reproduce the calculations are provided as a .txt file
(see Additional file 2). The inter-rater agreement was
good with an estimated ICC of 0.57 (95% CI: 0.38–0.74). Agreement and reliability Among individual domains, the ICCs varied such that
lowest agreement was observed for the three domains,
‘Study rational’: ICC = 0.11 (95% CI: 0.01–0.27), ‘Design/
Methods’: ICC = 0.15 (95% CI: 0.04–0.32) and ‘Data ana-
lysis’: ICC 0.24 (95% CI: 0.10–0.43). The raters seem to
agree most on two of the domains: ‘Data interpretation’:
ICC 0.56 (95% CI: 0.40–0.72) and ‘Biomarker measure-
ment’: ICC 0.39 (95% CI: 0.22–0.58). As compared with
the pilot test tool, an overall 30% increase of the ICC
(0.44 to 0.57) could be seen for the revised tool. Among
different domains, the most prominent improvement Discussion However, as compared to BIOCROSS, BEES-C mostly fo-
cuses on biomarker selection and measurement issues,
deploying 8 of 14 points to this section, while BIOCROSS
attempts to provide an overall assessment of the study
and not particularly focusing on biomarker measurements. Furthermore, BIOCROSS was developed to be easily ap-
plicable by professionals without practical training in epi-
demiology and biostatistics allowing an effective use within
a short period of time. BIOCROSS focuses on the evalu-
ation of most commonly reported biomarker association
study design in the current flow of biomedical literature:
cross-sectional studies; however, it may be also applicable
to other observational study designs. Moreover, as com-
pared to previous tools, such as the ‘BEES-C’, BIOCROSS
was deliberately validated to allow a critical evaluation of
the obtained data. score of 20 points. The tool could be suggested as a reliable
and valid method for assessing study reporting quality and
its further application could assist researchers in the con-
duct of systematic reviews and meta-analyses within the
rapidly evolving field of biomarker research. In the past years marked by the establishment of ‘evi-
dence-based medicine’, the assessment of study quality
of conducted research has turned into a subject of an
intensive effort and exchange of scientific guidelines and
recommendations. These recommendations have been
largely aimed to assist researchers and policy makers in
the decision process on which studies to include into
their analysis [14–17]. In a previous review of tools assessing the risk of bias
in observational research the lack of an easily applicable
tool to assess biomarker-based observational studies has
been acknowledged [18]. Based on it, the following rec-
ommendations have been issued: [1] to focus on the
development of tools with small number of key domains;
[2] to address particular study design and topic areas; [3]
to use simple checklist [4] to ensure that the tools
undergo a careful development phase and [5] to evaluate
the developed tools in terms of their validity and reliabil-
ity. In our approach we largely followed these recommen-
dations and adapted them specifically to assessing quality
of reporting of biomarker-based cross-sectional studies. One potential use of our tool in the future would be to
evaluate quality of reporting of biomarker-based epi-
demiological studies. Evaluation tools, assessing the
quality of reporting are necessary if the quality of studies
to be included into systematic reviews needs to be
reviewed. Discussion BIOCROSS was developed as a tool designed for use by
biomedical specialists to assess the quality and reporting
of biomarker-based cross-sectional studies. BIOCROSS
combines 10 items within 5 study evaluation domains
ranging from study rationale and design to biomarker
assessment and data interpretation scoring for a maximum Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Page 6 of 8 Fig. 2 ICC Scores (95% CI) and Inter-Rater reliability ratings of BIOCROSS Fig. 2 ICC Scores (95% CI) and Inter-Rater reliability ratings of BIOCROSS Despite guidelines like STROBE-ME [8] could be helpful in
designing future planned biomarker studies, they are not
meant for assessing the quality of previously conducted
studies. Similar to our approach was used by the Biomoni-
toring, Environmental Epidemiology, and Short-lived Che-
micals tool (BEES-C) aimed at evaluating studies dealing
with short-lived chemicals, including biomarkers [20, 21]. It
consists of 14 study assessment components which can be
scored with a three-point scale (Tier1, Tier2, and Tier3). However, as compared to BIOCROSS, BEES-C mostly fo-
cuses on biomarker selection and measurement issues,
deploying 8 of 14 points to this section, while BIOCROSS
attempts to provide an overall assessment of the study
and not particularly focusing on biomarker measurements. Furthermore, BIOCROSS was developed to be easily ap-
plicable by professionals without practical training in epi-
demiology and biostatistics allowing an effective use within
a short period of time. BIOCROSS focuses on the evalu-
ation of most commonly reported biomarker association
study design in the current flow of biomedical literature:
cross-sectional studies; however, it may be also applicable
to other observational study designs. Moreover, as com-
pared to previous tools, such as the ‘BEES-C’, BIOCROSS
was deliberately validated to allow a critical evaluation of
the obtained data. Despite guidelines like STROBE-ME [8] could be helpful in
designing future planned biomarker studies, they are not
meant for assessing the quality of previously conducted
studies. Similar to our approach was used by the Biomoni-
toring, Environmental Epidemiology, and Short-lived Che-
micals tool (BEES-C) aimed at evaluating studies dealing
with short-lived chemicals, including biomarkers [20, 21]. It
consists of 14 study assessment components which can be
scored with a three-point scale (Tier1, Tier2, and Tier3). Discussion As systematic analyses are only as good as the
studies used to derive the data, proper quality assessment
is necessary to assure a high quality of systematic analyses. We are not the first to address the quality of studies in
epidemiological research. Previously several tools have
been developed, some of which focusing on general aspects
of reporting across different disciplines (i.e. Consolidated
Standards of Reporting Trials (CONSORT) [19]), while
others focusing on specific research topics (i.e. Quality As-
sessment of Diagnostic Accuracy Studies (QUADAS) [17]). Page 7 of 8 Page 7 of 8 Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Wirsching et al. BMC Medical Research Methodology (2018) 18:122 Such quality assessments should be conducted through
validated tools and reported within meta-analyses. A lack
of quality appraisal can lead to misleading interpretations
of data. This is especially important in strongly developing
and changing fields of research, as new scientific know-
ledge and new experimental technics need to be consid-
ered in the evaluation. Such quality assessments should be conducted through
validated tools and reported within meta-analyses. A lack
of quality appraisal can lead to misleading interpretations
of data. This is especially important in strongly developing
and changing fields of research, as new scientific know-
ledge and new experimental technics need to be consid-
ered in the evaluation. more applicable approach such as the use of a ‘User’s
guide’. Furthermore, to reflect a real situation we also
chose reviewers with different scientific backgrounds
and level of education. As BIOCROSS asks for specific items to be included
into the paper, missing some of them can dramatically
reduce the obtained scores. To address this problem, a
gradual rating was introduced which allows to evaluate
the quality more precisely. On the other hand, discussing
points considered important could lead to a relatively high
score even though important points like statistical evalu-
ation of presentation of the data seem to be poor. This is
especially prominent for items 1, 5 and 6 which were not
discriminating in our analysis. To address this problem, we
suggest that authors use the information provided through
the IC to assess a potential risk of bias and then decide
if they want to use such a study for their analysis. As
any quality assessment tool BIOCROSS will be an or-
ganic item that can change if improvement is needed. Acknowledgements We would like to thank Sven Knüppel for providing theoretical and practical
advice for statistical analyses. Conclusion BIOCROSS is a new quality appraisal tool suitable for
assessment of evidence from cross-sectional epidemio-
logical studies employing biomarker data. The tool is reli-
able for use by biomedical scientists and could be applied
to facilitate comprehensive review of biomarker studies in
human research. BIOCROSS has several strengths. The most important
is that it combines the evaluation of cross-sectional study
design with specific characteristics of biomarker-based
studies. BIOCROSS is a freely accessible and ready to use
evaluation tool. No extensive training is necessary, as con-
clusive descriptions for each point are provided and freely
accessible. Furthermore, the tool has been validated mak-
ing it possible to critically evaluate obtained data from
scoring papers, e.g. for a systematic review. With an aver-
age rating time of around 13 min per study, BIOCROSS is
relatively fast to conduct and also suitable to rate a large
amount of studies. Additional files Additional file 1: Scoring system of BIOCROSS: A User’s Guide to
BIOCROSS. Point-by-point description to enable the application of the
BIOCROSS tool. (DOCX 24 kb)
Additional file 2: Dataset and R code. All executed R code and rater
results. (TXT 37 kb) Additional file 1: Scoring system of BIOCROSS: A User’s Guide to
BIOCROSS. Point-by-point description to enable the application of the
BIOCROSS tool. (DOCX 24 kb) Additional file 2: Dataset and R code. All executed R code and rater
results. (TXT 37 kb) Additional file 2: Dataset and R code. All executed R code and rater
results. (TXT 37 kb) Abbreviation BEES-C: Biomonitoring Environmental Epidemiology and Short-lived Chemicals;
CI: Confidence interval; CONSORT: Consolidated Standards of Reporting Trials;
IC: Issue to consider; ICC: Intraclass correlation coefficients; IRR: Inter-rater-
reliability; Max: Maximum rating; ME: Molecular Epidemiology; Min: Minimum
rating; NIH: National Institutes of Health; QUADAS: Quality Assessment of
Diagnostic Accuracy Studies; SD: Standard deviation; STROBE: STrengthening the
Reporting of OBservational studies in Epidemiology— There are also several limitations and weaknesses of
BIOCROSS. As ratings differed considerably especially
within certain domains, personal experience seems to
strongly influence the rater’s decision how to grade spe-
cific items. As BIOCROSS mostly assesses the quality of
reporting, it is difficult to draw conclusions on the actual
quality of obtained data. Therefore, the evaluation of ob-
tained data might be subjective, raising questions about
how to use this data in a systematic review. We decided
against an intensive training of our reviewers which may
have contributed to a lower level of agreement for some
of the domains. However, in reality organizing training
would be laborious, time-consuming and hard to imple-
ment. We have been therefore interested to evaluate a Discussion We understand the problematic of tools evaluating the
quality of research articles and are aware that no tool
can be perfectly objective. The qualitative analysis based on the overall feedback
of the raters pointed to a positive experience of tool
application. Most of the reviewers stated, that the tool
was easy and relatively fast to apply. Below we discuss
some of the issues raised by the raters. y
Three raters stated, that our rating with awarding 1
point if 1 or 2 IC were discussed, leaded to unjustified
high scorings of some studies. A change to: 0 and 1 IC =
0 points, 2 IC = 1 point and 3 IC = 2 points was therefore
suggested by the raters. The change in the scoring
may contribute to an increased discrimination within
several items and may be considered by other re-
searchers. Two raters stated, that even though the tool
was easy to conduct, it might be difficult to use, as the
quality of reporting does not necessarily provide valid
information about the actual quality of the data. Further-
more, some unclear formulations in the “User’s guide to
BIOCROSS” were mentioned. These points raised by the
raters were discussed and clarified and were taken into ac-
count in the revised version of the “User’s guide to BIO-
CROSS” (Additional file 1). Funding This research did not receive any specific funding. The positions of
Dr. Krasimira Aleksandrova, Fabian Eichelmann and Jan Wirsching have been
funded by the German Institute of Human Nutrition Potsdam-Rehbruecke. The publication of this article was funded by the Open Access Fund of the
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published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Received: 7 December 2017 Accepted: 19 October 2018 Consent for publication
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17.
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of QUADAS: a tool for the quality assessment of studies of diagnostic
accuracy included in systematic reviews. BMC Med Res Methodol. 2003;3:25.
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Sanderson S, Tatt ID, Higgins JP. Tools for assessing quality and
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LaKind JS, Sobus JR, Goodman M, Barr DB, Furst P, Albertini RJ, et al. A
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21.
LaKind JS, Goodman M, Barr DB, Weisel CP, Schoeters G. Lessons learned
from the application of BEES-C: systematic assessment of study quality of
epidemiologic research on BPA, neurodevelopment, and respiratory health.
Environ Int. 2015;80:41–71. Authors’ contributions KA planned and set up the study, developed the tool and supervised work
on evaluation, data analysis, as well as interpretation of data, writing and
critical revision of the manuscript. JW organized the study evaluation,
performed analysis and interpretation of the data, and manuscript drafting;
FE, LMH, MS, EB, AB, MO, LS, MZ, and HZ acted as raters in tool assessment
and contributed in the critical revision of the manuscript. All authors read
and approved the final version of the manuscript. 17. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development
of QUADAS: a tool for the quality assessment of studies of diagnostic
accuracy included in systematic reviews. BMC Med Res Methodol. 2003;3:25. 18. Sanderson S, Tatt ID, Higgins JP. Tools for assessing quality and
susceptibility to bias in observational studies in epidemiology: a systematic
review and annotated bibliography. Int J Epidemiol. 2007;36(3):666–76. 19. Moher D, Schulz KF, Altman DG, Consort G. The CONSORT statement:
revised recommendations for improving the quality of reports of parallel-
group randomized trials. Ann Intern Med. 2001;134(8):657–62. Ethics approval and consent to participate Ethics approval and consent to participate
Not applicable. Not applicable. 20. LaKind JS, Sobus JR, Goodman M, Barr DB, Furst P, Albertini RJ, et al. A
proposal for assessing study quality: biomonitoring, environmental
epidemiology, and short-lived chemicals (BEES-C) instrument. Environ Int. 2014;73:195–207. Consent for publication
Not applicable. References Psychol Assess. 1994;6(4):284–90. 12. Matthias Gamer JL, Ian Puspendra Singh. irr: Various Coefficients of Interrater
Reliability and Agreement. . R package version 0.84 ed2012. 13. others MBa. lpSolve: Interface to 'Lp_solve' v.5.5 to Solve Linear/Integer
Programs. . R package version 5.6.13 ed2015. 14. Alphs LD, Bossie CA. ASPECT-R-A tool to rate the pragmatic and explanatory
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The core problem of crime in society: Psychopath offenders
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Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 УДК: 343.3:159.97
Прегледни рад
Примљен: 1. 12. 2012. УДК: 343.3:159.97
Прегледни рад
Примљен: 1. 12. 2012. УДК: 343.3:159.97
Прегледни рад
Примљен: 1. 12. 2012. Danka Radulović
University of Belgrade
Faculty of Special Education and Rehabilitation THE CORE PROBLEM OF CRIME IN SOCIETY:
PSYCHOPATH OFFENDERS1 This article provides an argument for the thesis that solving the problem of psychopath-
ic crime is essential to reducing crime, in general. The author argues that psychopathy is pri-
marily psychological (not psychiatric) phenomenon that has important implications for crim-
inology. Based on the results of empirical researches, all the relevant indicators of the contri-
bution of psychopathy to crime were analyzed such as: the volume and types of crimes, offens-
es of violence, juvenile delinquency and recidivism. It was found that the most serious crimes,
early delinquency, criminal career length, the frequency of the crimes, including the dark fig-
ure of crime and recidivism, are intrinsically linked to psychopathy. Psychopaths are involved
in all forms of crime, especially the crime of violence. Their violence is instrumental and
predatory, not psychopathological as the mentally ill individuals is; particularly dangerous
are sexual psychopaths. Psychopaths are untreatable and poorly in aversive learning, but
because of the masks of non criminality and socialization they are difficult to identify. They
have a constellation of malignant properties (such as high aggressiveness and amorality) that
are, among others, influenced by sociocultural factors. We need strategic approaches in the
area of state response and prevention of crime, that will ensure reducing psychopathic crime,
but also the creation of the social context in which expression of psychopathic traits and preda-
tory lifestyle is unprofitable and unsustainable. Key words: crime, psychopathy, offenders, social predators, sociopsychology, criminol-
ogy. ogy. 1 The article is a part of the Projects of Ministry of science of Republic of Serbia No. 47008 and
47011. Introduction Acknowledging the importance of psychopathy on society and criminal law
system represents one of the biggest changes in the scientific approach to psy-
chopathy in the past several decades (Cleckey,1976, Hare, 1993, Blackburn, 1998,
Momirović, Popović, 2002, Radulović, 2006a). This was a result of numerous
empirical research which revealed that a category of psychopaths offenders, specif-
ic in terms of psychology, neurophysiology and criminology, are primary psycho- 583 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders logical phenomena (Hare,1996, Radulovic,2006a). Psychopaths could no longer be
viewed as just one of the psychiatric categories of crime, as earlier. Simply because
unlike other types, their antisocial and deviant behaviour is not part of psy-
chopathology process. It is part of their social predators way of life and it represents
an important generator of crime in general and crime of violence in particular. Psychopathy tops the list of risk factors of future crimes and must be considered not
only a central part of the problem of any punitive strategy of state, but also a strategi-
cally important issue in the field of prevention (Radulovic,2006a). Some authors even
treat criminology as applied discipline of psychology, studying psychology of crimi-
nal psychopaths (Momirovic,Popovic,2002). Others, as Hare (1996) gave empirical
arguments that psychopathy is psychological construct whose time has come. 1. Who are psychopathic offenders? The notion psychopathy was introduced by Koch (“psychopathic inferiority”)
and Kraepelin (“psychopathic personalities”) in the nineteenth century, instead of
earlier Prichard’s term “moral insanity”. It was used as a label for defects in abil-
ity to restrain the gratification of immediate egotistical desires, without sings of
insane or hallucination and completely without any disorder or defect of intellect,
and reasoning faculties. It refer as hard temper, shallow affection and lack of feel-
ings, bad habits and moral dispositions, behaviour impulsiveness (Radulović,
2006a). At the beginning of twentieth century instead of psychopathy, the term socipa-
thy has been used very often, but more recently this term has fallen out to favour;
nevertheless it was present in the first American classification DSM-I (APA,1952)
as „sociopathic personality antisocial reaction“ (with alcoholism and drug depend-
ence, sexual devitions and antisocial behavior as its subset). Some authors use
terms psychopathy and sociopathy as synonyms; some refer to sociopathy as sub-
type of psychopathy influenced by social forces and early experience (Radulovic,
2005a). Contemporary understanding of psychopathy, defined by its link with crime is
reflected on the description of psychopathy in the latest American and International
psychiatric classifications of mental disorders. In American classification DSM-IV
(APA,1994) the definition of “antisocial personality disorder” (term intend to
describe behaviour aspects of psychopathy), basically points to antisocial orienta-
tion of such individuals and the danger that they represent to the society. Criteria, used to diagnose psychopathy as Antisocial personality disorder
(301.7), are operational through open, behavioural demonstrations of deviant and
criminal conduct in adults (from the age of 18th). But, the connection to behaviour- 584 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 al problems surely existed during juvenile. That is why a diagnostic request for
recording conduct disorder in individuals under the age of 15th , categorized as anti-
social personality disorder has been made. In order to establish a diagnosis in adult
age, three out of seven listed characteristics need to be manifest: 1. 1. Who are psychopathic offenders? failure to con-
firm to social norms with respect to lawful behavior as indicated by repeated per-
forming acts that are grounds for arrest; 2.deceitfulness as indicated by repeated
lying, use of aliases, or cunning others for personal profit or pleasure; 3.impulsivi-
ty or failure to plan ahead; 4.irritability or aggressiveness, as indicated by repeated
physical fights or assaults; 5.reckless disregard for safety of self or others; 6.con-
sistent irresponsibility as indicated by repeated failure to sustain consistent work
behavior or honor financial obligations; 7.lack of remorse as indicated by being
indifferent to or rationalizing having hurt, mistreated, or stolen from another. This diagnosis excludes the occurrence of antisocial behavior during the
course of mental illness, such as Schizophrenia or Manic Episode. In International classification ICD-10 (WHO,1992) psychopathy is defined as
“dissocial personality disorder” (F60.2) usually coming to attention because of a
gross disparity between behavior and the prevailing social norms, and characterized
by at least 3 of the following: (a) callous unconcern for the feelings of others; (b)
gross and persistent attitude of irresponsibility and disregard for social norms, rules
and obligations; (c) incapacity to maintain enduring relationships, though having
no difficulty in establishing them; (d) very low tolerance to frustration and a low
threshold for discharge of aggression, including violence; (e) incapacity to experi-
ence guilt and to profit from experience, particularly punishment; (f) marked prone-
ness to blame others, or to offer plausible rationalizations, for the behavior that has
brought the patient into conflict with society. There may also be persistent irritabil-
ity as an associated feature. Conduct disorder during childhood and adolescence,
though not invariably present, may further support the diagnosis. Unlike the psychiatric description, which remains on the level of antisocial
behaviour, crime psychologists insist on stricter and less inclusive definition of psy-
chopathy, determined primarily by characteristics of an individual which is the
foundation of such behaviour and which becomes evident in early years. They warn
that a psychopath, by its personality, is naturally inclined to committing criminal
offence, so, according to Robert Hare (1993,p.83) “crime is a logical outcome of
psychopathy.” He defines psychopathy as a cluster of mutually linked affective,
interpersonal and behavioural features with two factors as its base. The first,
aggressive narcissism is characterized by egotism, insensibility and lack of
remorse. It is correlated with narcissistic and histrionic disorders of personality, low
level of anxiety, empathy, pronounced Machiavellianism and narcissism. 1. Who are psychopathic offenders? The sec- 585 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders ond factor is antisocial life style characterized by irresponsible and impulsive
behaviour, need for excitement, unconventional and antisocial conduct. This factor
is most prominently correlated with criminal conduct and undesirable socio-emo-
tional milieu during formative period and the diagnosis of “behavioural disorder”
and “antisocial personality disorder” (Harpur, at al.1989). Over the past forty years, a view has been present in the literature whereby socio-
psychobiology does not consider psychopathy a psychiatric category, not even a psy-
chological disorder, but rather a subspecies of men developed through evolution which,
based on strategy of cheating, lying and manipulation, even bloodthirsty violence, suc-
cessfully acquires resources and all that it desires (Mealey,1995). Hare (1993) himself
describes psychopaths as social predators who use charm, intimidation and violence to
control others and to satisfy personal egocentric, deviant needs. Totally unscrupulous,
lacking empathy and remorse, “they calmly take what they want and do as they please“
(Hare,1993, p.45). Because of their promiscuous and producing large number of chil-
dren, without investing in their development, beside social pathway developed psy-
chopaths, it could be expected the number of psychopath with genetic predispositions
might significantly increase. The author of this paper emphasis importance of psychological, not psychi-
atric diagnostics of psychopathy, because it has been empirically determined that a
psychopathic disorder, being on the border area between mental health and mental
illness, is primarily psychological by nature, since the same traits, evident in nor-
mal people, are also found in psychopaths but are of a different degree of expres-
sion (intensity). These also have a different structure which naturally gives mani-
fest behaviour an emphasized antisocial quality (Radulović, 2006a: p.74). According to this conception, psychopathy is defined a relatively permanent state
of psychological personality structure which is characterized by: (a) unique com-
pose of personality traits dominated by aggressiveness, (b) antisocial, egocentric
and hedonistic value orientation with a marked lack of moral code and presence of
malevolent intentions; and (c) behavioural demonstration of conduct disorder in
which committing crime stands out ( ibidem p.74). 2. Why is psychopathy the core issue in reduction of crime? How serious an issue psychopathy in crime is, we learn from Hans Eysenck
(1977: 55) who said that the psychopathy presents “the riddle of delinquency in par-
ticularly pure form, and if we could solve this riddle in relation to the psychopathy, we
might have a very powerful weapon to use on the problem of delinquency in general”. It is not surprise that over the past several decades a lot of researches, socio-psy-
chological in particular, has been dedicated to the relation between psychopathy and
crime (Cleckey, 1976, Hare, 1996, Meloy, 1992, Blackbourn, 1986). Knowledge so far
has been based on the results of such research, undoubtedly proving that psychopathy
is a nucleus of crime, and that focusing on strategically solutions in this area is neces-
sary, bearing in mind that in terms of criminal law, psychopathy is still an enigma(
Eysenck, 1998, Bartol, 2002, Lykken, 1995, Radulovic, 2006a). Evidence supporting this statement is plentiful and here only some of relevant
indicators of its importance for reduction of crime will be presented. The role of psychopathy is crime is analyzed concerning all relevant crimino-
logical parameters as: intensity of crime, duration of criminal career, incidence in
penal institution, criminal versatile, violent offences and recidivism. Beside that,
next important issues are analyzed: early juvenile delinquency as indicator of psy-
chopathy, deficit of aversive learning of psychopath offenders, unsatisfactory
effects of resocialization, failure of specialized treatments and difficulties of iden-
tification of psychopaths in criminal and noncriminal population and at last, psy-
chological characteristics of psychopaths relevant for crime. 1. Who are psychopathic offenders? The listed aspects of psychopathic profile such as distinctively heightened
aggressiveness, lack of moral (or the creation of the so called “inverse moral”
which operates as if mirrored and according to the principle “the worse the better”)
and disturbed behaviour are already evident in juvenile (Farrington et al, 1986,
Moffitt, 1993, Radulovic, 2006b). Regardless of the definition of psychopathy, one of its main features is persist-
ent and varied, deviant and antisocial behaviour which starts at an early age (Hare,
1993, Hart& Hare,1997, Robins,1978,1966, Robins at al. 1991). Regrettably, pro- 586 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 fessional circles rarely mention that delinquency at a children’s age represents one
of the serious early symptoms of psychopathy (Radulovic, 2005b). Instead, early
age when a minor commits his/her first criminal offence is regularly taken for what
it is; a valid, reliable predictor of future delinquent behaviour, unrelated to psy-
chopathy (Radulovic, 2006a). This is how a person’s psychopathic personality,
which persistently produces criminal activities and deviant behaviour at later
stages, remains completely unnoticed. 3.1. Intensity of crime Majority of criminal offences is committed by psychopaths, even when we
find them in relatively low percent in some samples (Farrington et al,1986, Hare,
1996 ) . They commit all types of offences on a much larger scale, including the 587 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders acts which do not constitute physical violence (fraud, forgery, crimes against the
property, and economic crime) and acts of violence in particular (Hart, Hare, 1997,
Radulovic,2006a). Many reports indicate that psychopaths are convicted two to
three times as often for all criminal offences than non-psychopaths and even four
times as often for acts of criminal violence ( Hare, McPherson, 1984). That is the
best proof of how intense their criminal behaviour is, although their real, illegal
activities are most probably even more intense, since many of their deeds remain
undetected. It is deemed that they are responsible for the bulk of the dark figures of
crime ( Beck,1993). 3.2. Criminal career Criminal career of a psychopathic delinquent is much longer than that of a
non-psychopathic criminals, because the former begins their career very early in
life, most likely before 15th , usually around the age of 10th , and such a career lasts
throughout their lives. That goes for all types of criminal acts, although in later years (after forty)
arrests of psychopaths involved in concealment of crime are less frequent (and are
reduced to capturing of non-psychopaths), solely for violent crimes, and not for
crime against the property (Hare, 1993). Because their criminal activities are per-
manent and persistent, criminal-law system, in fact does not cease to deal with them
for, as long as they live. At the same time, the society is inflicted enormous mate-
rial and social damage. Nevertheless they know very well that they are doing
unlawfully (in opposite to mental ill psychosis who are unable to understand rules
and consequences), psychopaths show a stunning lack of concern for the devasta-
tion effects their actions have on others, they do not care for the pain and destruc-
tion they cause; and with completely lack of guilty they continuous to be ruler bro-
ker. 3.4. Criminal versatile Criminal activities of psychopaths, unlike those of non-psychopathic crimi-
nals, are incomparably diverse (Hart, Hare, 1997). Psychopaths do not specialized
in a particular type of crime but they commit all kinds of criminal offences and also,
without fail acts of violence (85-90% of all cases). Even though they may prefer a
particular types of crime, for example serial killing, their criminal careers show
their involvement in all types of criminal activities, whether or not officially regis-
tered (Radulovic,2006a). Tenacity, flexibility and a high degree of criminal poten-
tial of psychopaths is reflected in the very fact that their criminal activities are high-
ly extensive and actually include all forms of crime, classic as well as new ones. These encompass minor theft, traffic violations, deceit, forgery, false introduction,
family violence, kidnapping, extortion, corruption, car theft, burglary, banditry,
robbery, murder, white slavery, trafficking in human organs, computer fraud, nar-
cotics trafficking, various types of sexual offences, marketing of products detri-
mental to human health, terrorism, espionage, homicide and luring to suicide with-
in sects, business crime, making and spreading criminal associations globally,
numerous varieties of criminal activities of organized crime, etc. Psychopaths are
very proud of their enormous criminal versatility (Hare1993), just as for the enor-
mous dark number of crime they are able to generate ( Radulovic, 2006a, Hare,
1996). 3.3. Incidence in penal institutions The higher the incidence of psychopathy we could find in the higher the level
of security of a penal institution. While generally psychopaths’representation with-
in the criminal population is about 20% (although they are accounted for the major-
ity of criminal activities) research shows that most of about 70% of adult offenders
that we meet in penal institutions with closed and very closed regimes, have psy-
chopathic personality(Radulovic,2006a). If we use mentioned criteria for antisocial
personality disorder the percent will be much higher in that setting, nearly 90%. As
minors, criminal psychopaths all went, unsuccessfully, through all types of sanc- 588 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 tioning of juvenile delinquency, as determined by the law, starting with education-
al measures of individual supervision, and including referral to correction institu-
tions, institutions for state supervised education, or specialized institution for treat-
ment and rehabilitation, and punishment of juvenile confinement. 3.6. Criminal recidivism Psychopathy occupies the key place in crime due to extremely high incidences
of recidivism of psychopaths, so one can ask whether a serious criminal recurrence
can happen outside psychopathy (Cornell at al., 1996, Momirović, Popovic, 2002). Psychopaths are undoubtedly the largest category of recidivists (over 70%)
(Radulovic, 2007). The rate of general recidivism of psychopaths is three times higher than gen-
eral recidivism of non-psychopaths, whereas the rate of violent recidivism is four
times higher (Hemphill, at al. 1998). This being one of the most significant indica-
tors of their chronic criminal behaviour (Skilling et al., 2002). With risk factors of
criminal recurrence all taken to one side, and psychopathy on to the other, the psy-
chopathy risk level surpasses by far all other factors together, regardless of the type
of crime (Radulovic, 2007, Skilling et al., 2002, Momirovic et al. 1996). Recidivism of psychopathic juvenile delinquents is one of the surest signs that
they will develop into most dangerous, hardest, most ruthless and unrelenting crim-
inals who take crime for a way of living (Radulovic, 2005b, 2007, Momirović,
Hošek, 1997). 3.5. Violent offences In a dynamic and very versatile criminal career of a psychopath, cases of
severe acts of violence are often found (Blackburn, 1998, 1986, Momirović,
Popovic, 2002, Radulovic, 2004, 2006a). Psychopaths are generally violent both
within their families and at work, and especially towards unfamiliar individuals but
also to various officials. Largest numbers of extremely destructive criminals, such
as serial killers, are recruited from the ranks of psychopaths (Radulović, 2004). Particularly common and dangerous form of psychopathic violence, and con-
tinually recurring, is sexual violence, whose victims are youngsters as well as adults
(Quinsey et al., 1995, Harris et al. 1991, Hare, McPherson, 1984). A psychopathic
offender with a diagnosis of being a sexual sadist is deemed the most dangerous 589 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders type of delinquent within a criminal population, in general. They are the hardest
recidivists and their sexual offenses start very early, as minors (Hart, Hare, 1997). Apart from physical forms of violent behaviour, psychopaths are also prone to psy-
chological violence, too, which sadly is not legislatively sanctioned although it
weighs differently compared to psychological violence of non-psychopaths,
because it tends to directly and unexpectedly change into a severe form of physical
violent behaviour (Radulović, 2004). 4. Early juvenile delinquency as indicator of psychopathy Juvenile delinquency of early psychopaths is a problem which many countries,
including the well developed ones, are faced with. Some authors, among which are
Lykken (1995), Reid (1998) Hare (1993) and others talk about epidemics of psy-
chopathic delinquency. In the Untied States one fifth of the gravest criminal
offences are committed by young offenders, mostly psychopaths. The rate of rise in
juvenile delinquency, with psychopathy being a large part of it, has been, for the
past fifty years, 250% (Reid, 1998, Bureau of Justice Statistics, 1998). Juvenile
psychopathic delinquents differ from juvenile non-psychopathic delinquents in
their criminological characteristics, which can be indispensable in their early detec-
tion within delinquent population. As well as starting early, young delinquents com- 590 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 mit all types of criminal acts as minors, with acts of violence being at the top
(whereas non-psychopaths are more likely to commit crimes against the property);
participating in new forms of crime but also developing them. Youngest recidivists, leaders of juvenile gangs, destructive vandals, delin-
quents who commit the most serious acts of violence impersonally, as if doing them
was their job, while sending innocent victims (usually unknown individual) mes-
sages “nothing personal”, come from their ranks. Criminal behaviour of juvenile
delinquents is persistent, while crime, deviations, addiction disorders, and prema-
ture sexual activities represent regular occurrences. In contrast, juvenile non-psy-
chopathic delinquency is often of transient character and is usually situational or
linked to adolescent crisis (Radulovic, 2005b). Differences between juvenile psy-
chopathic and non-psychopathic delinquents are particularly prominent in acts of
violence. Violence committed by juvenile psychopaths is callous, cold-blooded,
like a business, uncomplicated, and unstoppable. It is carried out without inhibi-
tions, feeling of guilt, empathy and fear, and with a huge potential for dehumaniza-
tion of victims, interspersed with brutality and sadism. It is generally planned, cal-
culated, and instrumental (Cornell at al., 1996), oriented to a particular goal such as
money, sensual pleasure, status, domination, control, or is motivated by revenge or
desire to punish the victim. This often happens when the person is a state of intox-
ication; sometimes it is connected to person’s impulsive nature or is motivated by
an abnormal need for excitement or risk, or is done simply “for the fun”. 4. Early juvenile delinquency as indicator of psychopathy In con-
trast, juvenile non-psychopathic violence is reactive and happens in states of
heightened emotional tension, rage, anger; as a reaction to an event or a misjudged
situation, in which victims are often familiar and close people. Research shows that
various types of juvenile psychopaths contribute to the worrying rise of juvenile
delinquency. Apart from the so called “frustrated” juvenile psychopaths (brought
up in an inadequate, cold, hostile or pathogen family environment) we find among
delinquents more and more of the so called “spoilt juvenile psychopaths” who
come from affluent families in which they were overprotected and whose parents
were overly permissive. Lykken (1995, 1998) determined that, along with impor-
tant, relatively unchanging presence of the so called “genotype” psychopathic
crime, generated by the temperament of a predisposed adolescent psychopath who
is free of fear and with a weak inhibition system, the world has been overwhelmed
by delinquency of the so called “phenotype” juvenile psychopaths (also called
sociopaths) whose psychopathic profile is the result of negative socialization (the
break-up of families being the main cause). Beside all similarity to genotype psy-
chopathy (egotism, aggressiveness, lack of feeling of guilt etc), phenotype psy-
chopathy also differs from it in one aspect – there is no lack of anxiety. On the con- 591 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders trary, anxiety is quiet evident here. According to Lykken (1998) delinquency of
this, most numerous categories of socially formed psychopaths can be reduced by
a serious, systemic approach by the society which is focused on prevention and par-
ticularly to the improvement of social and educational skills of parents. Lykken also
believes that, even children with genotype disposition towards crime, helped by
skilled parenting, can be redirected into a socially desirable direction. In his opin-
ion, even the deep-rooted psychopathic features such as the lack of fear, heightened
aggressiveness and need for excitement, which result in delinquency, can be ade-
quately channelled into sociable acceptable forms of behaviour but only if backed
by sound abilities of parents. With juvenile psychopathic delinquency on the rise, what is also apparent is
psychopathic bend of the system of values which results in favouring parasitism,
egotism, Machiavellianism, hedonism, emotional unresponsiveness and blatant
forcing of deviant forms of behaviour into becoming conventional, socially accept-
able (Radulović, Jugović, 2011). 5. Deficit of aversive learning of psychopath offenders Long criminal career starting from juvenile period and high rate of criminal re-
offences convincingly proves that usually punitive reactions to criminal psy-
chopaths do not produce results; so defining new specific strategy for psychopath-
ic offenders has become a necessity. Failure to apply punishment is expected, with
this category of offenders, bearing in mind that it has been experimentally proved
that psychopaths are less receptive to punishment than non-psychopaths (Lykken,
1957, Momirović i dr, 1974, 1979, Radulovic 2008b). With some definitions of
psychopathy, difficulties of aversive learning, noticeable from early childhood, is
taken as one of its defining characteristics (Radulovic, 2008c). Pointing out to the
inefficacy of punitive system, Cleckey (1976) suggested that punishing psy-
chopaths is the same as trying to teach mathematics to an imbecile. One of the bet-
ter known psychological explanations of crime, based on psychopathy and known
as “the theory of aversive learning deficit”, explains delinquent behaviour of psy-
chopaths as poor learning of passive avoidance that is weak aversive conditioning
(Radulovic, 2008b). 7. Failure of specialized treatments Failure of almost all applicable even specialized treatment programmes for
psychopaths so far, including psychotherapy, further supports the issue of the need
for quite different method of treatment of criminal psychopaths. Psychopaths are
totally resistant to treatment because they are perfectly content with themselves and
do not possess a genuine desire to change, not even when making declarative prom-
ises (Radulovic, 2012). Neither do they have an emotional potential for bonding
and transfer that is essential for therapy. Some forms of psychotherapy (such as psychodynamics and nondirective
client oriented therapy) fail not only to produce satisfactory results, but are con-
traindicated because psychopaths learn from them how to manipulate people, even
better; so that recidivism of psychopaths is even higher after application of such
therapies (Rice, at al.,1992, Radulovic,2007). Somewhat more favorable results,
but limited and short-lived, come from behavioural approaches which are based on
the leaning theory, but their effects are quickly lost as well (Radulovic, 2012). 6. Unsatisfactory effects of resocialization High incidence of recidivism clearly indicates that so far the methods of work-
ing with psychopathic criminals which rest on principles of resocialization, have
been an absolute failure. So we can agree with Hare (1993: 219) saying that “crim- 592 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 inal justice system spends billions of dollars every year in a vain attempt to reha-
bilitate or resocialize psychopaths”. We must not ignoring the fact that, neverthe-
less psychopaths could have good manner and be charming, they, in fact, never
have been socialized, so how could we expect that they could be re-socialized
(Hare, 1993, Radulovic, 2008c). We can also agree with the supporters of the psy-
chological approach saying that, thanks to our naive aspiration to humanize psy-
chopaths, we put the entire society in danger because they, psychopaths, like
manipulative social predators, transform us into their easy prey (Mealey, 1995,
Reid, 1998). Physically aggressive psychopaths, however, do not appear violent at
first glance, but rather leave an impression of kind, socialized individuals, well
mannered, friendly towards people (Cleckey, 1976). But their deeds show them in
a completely different light and clearly suggest that the process of socialization has
basically never been accomplished (Radulović, 2004). That is why prevention is the
only valid alternative (Radulovic, 2006a: 542). 8. Difficulties of identification of psychopaths
in criminal population Identification of psychopaths among adult criminals is the most difficult diag-
nostic problems of forensic work. Psychopaths are difficult to identify even when
they commit gravest criminal acts, such as serial killing. It is exceptionally hard to
do so in their adult years because their mask of health and non-criminality is 593 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders extraordinary; and co-morbidity with addiction diseases (van Limbeck idr, 1992)
very discernible which is why their criminal acts are attributed mostly to alco-
holism, drug influence and the like, and thus the psychopathic type of personality
remains completely undetected (Cleckey, 1976, Blackburn, 1985, Hart, Hare, 1997,
Cantwell, 1980). At first glance psychopaths seem ordinary or even social well inte-
grated. Besides, they appear “healthier and more stable” than normal people (Hare,
1993). They do not expose fear when most people do, externalize responsibility,
convincingly rationalize their behaviour, freely speak their mind, appear compla-
cent, non-aggressive, and polite (Bartol, 2002). Without hesitating, they seek new
opportunities which they regularly obtain from various officials, whom they meet,
because they make an impression of non-criminals and confident individuals. Hare
(1993) organized workshops for forensic experts and authorities on jurisdiction and
implementation, presenting to participants video shots of interviews conducted dur-
ing prison examinations of serious, violent delinquents. The experts were not told
who the people in the video shots were, but were expected to conclude that, based
on authentically recorded statements and behaviour of the interviewees. Majority
of delinquents made positive impression on the experts who gave them marks such
as “very impressive”, “honest and righteous”, “possesses good interpersonal
skills”, “intelligent and articulated” and the like. The knowledge about criminal his-
tory and arrests of the subjects from the video shots, which they acquired later,
came as a great surprise to them. It also served as a lesson about how aggressive
psychopaths present themselves in a positive light, thus making it very difficult to
ascertain, based solely on their appearance and stories, that they are in fact hard-
ened criminal. That is why it is essential to detect this personality profile as early
as possible. 8. Difficulties of identification of psychopaths
in criminal population Identification of psychopaths within a delinquent population is made more dif-
ficult also because of a detrimental tendency to wrongly equate or mix psychopathy
with a severe mental disorder – psychosis, which is spread not only among public
but also among experts outside psychological and psychiatric fields (lawyers, ped-
agogues, etc). It must be absolutely clear that with psychopathy it is not a question
of a pathogen process, but a relatively permanent state that is a psychological dis-
order of a person’s structure that is evident in early developmental phases. Because
of configuration of their personality traits, psychopaths are placed, rather early in
life, between the mentally sane and the mentally ill, staying permanently prone to
social deviations and crime, despite of the persons’ being aware of their own
actions. This prejudice is permanently maintained not merely because of ignorance,
but also because normal people find it difficult to perceive the existence of persons
who know exactly what they are doing, even when they are perpetrating the cru- 594 Социолошки преглед, vol. XLVI (2012), no. 4, стр. 583–600 ellest of deeds: ferocious murders, rape, felony, which they perform repeatedly,
unstoppably and lacking emotion, as is the case with psychopaths
(Radulovic,2006a). 9. Psychological characteristics of psychopaths relevant for crime Numerous psychological studies found out psychopaths patterns of personality
traits is prone to crime (Lykken, 1995, Blackburn, 1985, 1986, Eysenck, 1977,
Radulovic, 2008a). They also confirm the hypotheses that psychopaths offenders are
different from others offenders just in psychological traits that are relevant for crime. Psychopathic offenders exhibit a distinct set of personality traits, primary character
deficits that predispose them toward long term criminality (Hare et al. 1992,
Momirovic, Popovic, 2002, Cleckey, 1976, Bartol, 2002). Affective life of psychopaths
is on low level, marked with chronic anger and explosiveness, without deeply felt emo-
tions for others, without guilt or remorse. Their feelings are pre-socialized with domi-
nation of boredom, excitement, range, envy etc. Emotions related to others are com-
pletely absent as: empathy, sympathy, gratitude, guilty, etc. (Goleman, 1997). Criminal
psychopaths are self–centred, grandiose, pathological narcissistic, identified with preda-
tory objects (Meloy,1992). Their relationships are defined by dominance, not affection,
just as their violent crime is primary predatory, rather than affective violence, motivat-
ed often by hedonistic calculation and higher level of aggression. Large number of studies directly supported Eysenck`s thesis that psychoticism as
a measure of psychopathy very significantly distinguish, not only between criminals
and non-criminals, but also among chronic criminals and others who are not psycholog-
ical prone to crime (Rutter, Rutter, 1993). Traits correlation together to define psychoti-
cism are attributed as: aggressive, cold, egocentric, impersonal, impulsive, antisocial,
unempathic, creative, tough-minded (Eysenck, Zuckerman, 1976, Eysenck, 1998). In the research of Radulovic (2008a) conducted on the sample of 322 serious
offenders (with subsample of 209 psychopaths), it was found out that the psychopath
offenders were more aggressive, dissociate, hysteric and amoral, with lower level of
perceptive and verbal abilities, than non-psychopath offenders. That personality pattern
makes them very dangerous and inclined to chronic predators violent crime (Radulovic,
2008a). Conclusion Judging from the analysis of the researches results in the field of psychology
of crime, psychopath offenders are the key target category to reduction of crime;
specially part that is most worrying, and is the hardest and so far unsolvable, and 595 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders the one which many countries around the world are faced with, in their unsuccess-
ful endeavours to combat it, despite their developed economies, socio-political sys-
tems and enormous funds and efforts invested in attempts to prevent its spreading. Contribution of psychopathy to crime is substantial concerning all relevant
criminological parameters as: the extent of committed crime, early juvenile delin-
quency, durations of criminal career, criminal versatile, and commitment of vio-
lence, especially cold blooded, calculated, instrumental violence offences, severity
of criminal act, untreatability and very high rate of recidivism. Obviously, psy-
chopathy is not merely an isolated category of crime, but represents its nucleus
(Eycenck, 1977, 1998, Radulovic, 2006: 523). That is why any kind of work aimed at reduction and prevention of crime is
destined to have modest results for as long as the essential role of psychopathy in
crime is overlooked; together with the fact that psychopathic offenders are not
essentially, psychiatric phenomena (Radulovic, 2008c). Psychopathy can be best understood in the social context, just from its relation
to crime. It is a psychological phenomena which, being a deviation of normality
that lacks the quality of mental illness has attributes of a relatively permanent psy-
chological profile which contain personality traits that are interpersonally malevo-
lent (high level of aggressiveness, amoral, hysteria etc.). Constellation of such psy-
chological traits presents serious and dangerous psychological potential, predis-
posed to crime (Radulovic, 2006a). Among others, socio-cultural factors play important role in etiology of these
malevolent personality traits (ibidem: 236). That is why socio-psychology of psy-
chopathy, undoubtedly, deserves proper attention, so that efforts of reduction of
crime and preventive efforts would not end like the attempts aimed at resocializa-
tion of psychopaths did – a total failure. Different strategy approach for solving problem of criminal psychopaths as
social predators is needed, based on socio –psychological, instead of clinical
researches (ibidem: 528). In that approach we have to bear in mind that psychopaths are not only in nei-
ther jails, nor are they on society margins (Radulovic, 2008c). Conclusion Authors as Cleckey
(1976) and Hare (1993) warned us that “our failure to acknowledge the psy-
chopaths are among us, triggered a social crisis” (ibidem: p.163). Nowadays signs
of social threatening, predators way of living are everywhere, promoted and intrud-
ed as social acceptable model. Psychopathic life style became more widespread,
making social crisis and crime much worse, so we could agree with Hare’s thesis
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from the NIMH Epidemiologic Cathment Area project. Psychiatry 54: 116-132. Robins, L. (1966). Deviant Children Grown-Up. Baltimore: Williams and Wilkins. Robins, L. (1978). Sturdy childhood predictors of adult antisocial behaviour: Replications from lon-
gitudinal studies. Psychological Medicine 8: 611-622. Rutter, M., Rutter, M. REFERENCES (1993): Developing minds: Challenge and continuity across the life span. New York: Basic Books. van Limbeck, J., Wouters, L., Kaplan, C.D., Geerlings, P.J., van Alem, V. (1992). Prevalence of psy-
chopathology in drug-addicted Dutch. Journal of Supstance Abuse Treatment 9: 43-52. Skilling, T.A., Harris, G.T., Rice, M.E., Quinsey, V.L. (2002). Identifying persistently antisocial
offenders using the Hare Psychopathy Checklist and DSM antisocial personality criteria. Psychological Assessment 14: 27-34. World Health Organization (1992). ICD-10 Classification of mental and behavioural disorders:
Clinical Descriptions and diagnostic guidelines. Geneva: Author. 599 Danka Radulović, The Core Problem of Crime in Society: Psychopath Offenders Данка Радуловић
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и рехабилитацију Сажетак ГЛАВНИ ПРОБЛЕМ КРИМИНАЛА
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бина (као што су висока агресивност и аморалност) које су између осталог и под ути-
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Psychometric properties of the Danish Hospital Anxiety and Depression Scale in patients with cardiac disease: results from the DenHeart survey
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Citation for published version (APA):
Christensen, A. V., Dixon, J. K., Juel, K., Ekholm, O., Rasmussen, T. B., Borregaard, B., Mols, R. E., Thrysøe,
L., Thorup, C. B., & Berg, S. K. (2020). Psychometric properties of the Danish Hospital Anxiety and Depression
Scale in patients with cardiac disease: results from the DenHeart survey. Health and Quality of Life Outcomes,
18(1), Article 9. https://doi.org/10.1186/s12955-019-1264-0 General rights
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- You may not further distribute the material or use it for any profit-making activity or commercial gain You may not further distribute the material or use it for any profit making activity o
- You may freely distribute the URL identifying the publication in the public portal - Aalborg Universitet Psychometric properties of the Danish Hospital Anxiety and Depression Scale in
patients with cardiac disease
results from the DenHeart survey
Christensen, Anne Vinggaard; Dixon, Jane K; Juel, Knud; Ekholm, Ola; Rasmussen, Trine
Bernholdt; Borregaard, Britt; Mols, Rikke Elmose; Thrysøe, Lars; Thorup, Charlotte Brun;
Berg, Selina Kikkenborg
Published in:
Health and Quality of Life Outcomes
DOI (link to publication from Publisher):
10.1186/s12955-019-1264-0
Creative Commons License
CC BY 4.0
Publication date:
2020
Document Version
Publisher's PDF, also known as Version of record
Link to publication from Aalborg University
Citation for published version (APA):
Christensen, A. V., Dixon, J. K., Juel, K., Ekholm, O., Rasmussen, T. B., Borregaard, B., Mols, R. E., Thrysøe,
L., Thorup, C. B., & Berg, S. K. (2020). Psychometric properties of the Danish Hospital Anxiety and Depressio
Scale in patients with cardiac disease: results from the DenHeart survey. Health and Quality of Life Outcomes
18(1), Article 9. https://doi.org/10.1186/s12955-019-1264-0 Psychometric properties of the Danish Hospital Anxiety and Depression Scale in
patients with cardiac disease
results from the DenHeart survey
Christensen, Anne Vinggaard; Dixon, Jane K; Juel, Knud; Ekholm, Ola; Rasmussen, Trine
Bernholdt; Borregaard, Britt; Mols, Rikke Elmose; Thrysøe, Lars; Thorup, Charlotte Brun;
Berg, Selina Kikkenborg
Published in:
Health and Quality of Life Outcomes Citation for published version (APA):
Christensen, A. V., Dixon, J. K., Juel, K., Ekholm, O., Rasmussen, T. B., Borregaard, B., Mols, R. E., Thrysøe,
L., Thorup, C. B., & Berg, S. K. (2020). Psychometric properties of the Danish Hospital Anxiety and Depression
Scale in patients with cardiac disease: results from the DenHeart survey. Health and Quality of Life Outcomes,
18(1), Article 9. https://doi.org/10.1186/s12955-019-1264-0 Open Access Open Access Psychometric properties of the Danish
Hospital Anxiety and Depression Scale in
patients with cardiac disease: results from
the DenHeart survey Anne Vinggaard Christensen1*
, Jane K. Dixon2, Knud Juel3, Ola Ekholm3, Trine Bernholdt Rasmussen4,
Britt Borregaard5, Rikke Elmose Mols6, Lars Thrysøe7, Charlotte Brun Thorup8 and Selina Kikkenborg Berg1,3,9 Abstract Background: Anxiety and depression symptoms are common among cardiac patients. The Hospital Anxiety and
Depression Scale (HADS) is frequently used to measure symptoms of anxiety and depression; however, no study on
the validity and reliability of the scale in Danish cardiac patients has been done. The aim, therefore, was to evaluate
the psychometric properties of HADS in a large sample of Danish patients with the four most common cardiac
diagnoses: ischemic heart disease, arrhythmias, heart failure and heart valve disease. Methods: The DenHeart study was designed as a national cross-sectional survey including the HADS, SF-12 and
HeartQoL and combined with data from national registers. Psychometric evaluation included analyses of floor and
ceiling effects, structural validity using both exploratory and confirmatory factor analysis and hypotheses testing of
convergent and divergent validity by relating the HADS scores to the SF-12 and HeartQoL. Internal consistency
reliability was evaluated by Cronbach’s alpha, and differential item functioning by gender was examined using
ordinal logistic regression. Results: A total of 12,806 patients (response rate 51%) answered the HADS. Exploratory factor analysis supported
the original two-factor structure of the HADS, while confirmatory factor analysis supported a three-factor structure
consisting of the original depression subscale and two anxiety subscales as suggested in a previous study. There
were floor effects on all items and ceiling effect on item 8. The hypotheses regarding convergent validity were
confirmed but those regarding divergent validity for HADS-D were not. Internal consistency was good with a
Cronbach’s alpha of 0.87 for HADS-A and 0.82 for HADS-D. There were no indications of noticeable differential item
functioning by gender for any items. Conclusions: The present study supported the evidence of convergent validity and high internal consistency for
both HADS outcomes in a large sample of Danish patients with cardiac disease. There are, however, conflicting
results regarding the factor structure of the scale consistent with previous research. Trial registration: ClinicalTrials.gov: NCT01926145. Trial registration: ClinicalTrials.gov: NCT01926145. Keywords: Hospital anxiety and depression scale, Psychometric evaluation, Cardiac patients, Validity, Reliability * Correspondence: anne.vinggaard.christensen@regionh.dk * Correspondence: anne.vinggaard.christensen@regionh.dk p
gg
@ g
1Department of Cardiology, Rigshospitalet, Copenhagen University Hospital
Blegdamsvej 9, 2100 Copenhagen, Denmark
Full list of author information is available at the end of the article p
gg
g
1Department of Cardiology, Rigshospitalet, Copenhagen University Hospital,
Blegdamsvej 9, 2100 Copenhagen, Denmark
Full list of author information is available at the end of the article General rights - Users may download and print one copy of any publication from the public portal for the purpose of private study
- You may not further distribute the material or use it for any profit-making activity or commercial gain You may not further distribute the material or use it for any profit making activity o
- You may freely distribute the URL identifying the publication in the public portal - (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9
https://doi.org/10.1186/s12955-019-1264-0 Christensen et al. Health and Quality of Life Outcomes
https://doi.org/10.1186/s12955-019-1264-0 Data collection and sample Data was collected as part of the DenHeart study. The
design and methods have been described in the pre-
published protocol [25]. The DenHeart study was de-
signed as a national cross-sectional survey combined
with data from national registers at baseline and one
year follow-up. Over a period of one year (April 2013–
April 2014) all patients discharged or transferred from
one of five national heart centers were asked to fill out a
questionnaire at hospital discharge. Excluded were pa-
tients under the age of 18, patients without a Danish
civil registration number, patients who did not under-
stand Danish and patients who were unconscious when
transferred from a heart center. The Hospital Anxiety and Depression Scale (HADS)
was developed for patients with somatic illness admitted
to the hospital [5] and is often used as a self-rating scale
to screen for anxiety and depression symptoms across a
wide range of patient and general populations. The scale
includes two subscales, HADS-A and HADS-D measur-
ing anxiety and depression symptoms, respectively. The
scale is focused on the psychic symptoms of mood disor-
ders, leaving out physical symptoms that can be con-
fused with physical illness [5]. This is an advantage in
cardiac populations where symptoms such as palpita-
tions or dizziness might be related to the underlying
cardiac disease and not a potential mood disorder. Based on their discharge diagnosis from the Danish
National Patient Register [26], patients were divided into
diagnostic sub-groups [2]. Included in the current ana-
lyses are patients with ischemic heart disease, arrhyth-
mias, heart failure and heart valve diseases. Furthermore, co-morbidity characteristics were col-
lected from the Danish National Patient Register [26]. The Tu co-morbidity index was calculated including
congestive heart failure, cardiogenic shock, arrhythmia,
pulmonary oedema, malignancy, diabetes, cerebrovascu-
lar disease, acute/chronic renal failure and chronic ob-
structive pulmonary disease – all calculated ten years
back [27]. HADS has been extensively tested for validity and
reliability in English and other language versions, with
satisfactory results across different patient populations,
e.g. cardiac disease, cancer, psychological illness and in
general populations [6–8]. Looking at previous valid-
ation studies of HADS in cardiac populations, however,
there are differing results regarding the factor structure
of the scale, Table 1. Background most common cardiac diagnoses: ischemic heart disease,
arrhythmias, heart failure and heart valve diseases. Anxiety and depression symptoms are common among
cardiac patients with prevalence rates of up to 30 and
20%, respectively, at hospital discharge and up to three
months after hospitalization. This reflects the possible
severity of the physical illness on other aspects of health
[1, 2]. Previous studies have shown that anxiety and
depression symptoms can predict future morbidity and
mortality among cardiac patients [3, 4] underlining the
importance of identifying these symptoms in order to
initiate interventions to reduce them. A prerequisite for
this
is
having
a
valid
instrument
to
identify
the
symptoms. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 2 of 13 Page 2 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Data collection and sample The originally proposed two-factor
structure is confirmed in six studies [9–14], but eight
studies find different versions of a three-factor structure
to have the best fit depending on the analytic method
used [12, 13, 15–20]. By contrast, one study finds a one-
factor structure to have the best fit [21]. Information
on
demographic
characteristics
were
collected from the Civil Registration System [28] and the
Danish Education Register [29]. The HADS questionnaire The HADS is a 14 item questionnaire originally devel-
oped to measure anxiety and depression symptoms in
patients with somatic disease [5]. The instrument offers
two subscales, HADS-A and HADS-D, each consisting
of seven items and measuring anxiety and depression
symptoms, respectively. HADS-A is focused on symp-
toms relating to generalized anxiety and HADS-D on
symptoms relating to anhedonia, a central aspect of
depression [30]. Each item is scored on a scale of 0–3
with each subscale score ranging from 0 to 21. Eight
items are reverse scored with higher scores indicating a
better response. These are reversed when summing the
two subscales. The recommended cut-off values are 8–
10 for possible presence of a mood disorder and ≥11
for probable presence of a mood disorder [5]. It has
previously been found that among cardiac patients the
minimal clinically important difference on the HADS is
1.7 points [31]. Differential item functioning (DIF) is a form of meas-
urement error at item level by which patients from dif-
ferent groups with the same level of a construct being
measured do not have the same scores. The presence of
DIF by gender has been examined for HADS, but the
results are not consistent [22–24]. HADS has been translated into Danish and is fre-
quently used in clinical research but the psychometric
properties of the Danish version have not been evalu-
ated. Even though the scale has been found to be valid
and reliable in previous studies, this is no assurance of
equivalent validity when used in a different language,
culture or context. Therefore, the aim of the current
study was to evaluate the psychometric properties of the
Danish HADS in a large population of patients with the Christensen et al. Health and Quality of Life Outcomes (2020) 18:9
Page 3 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 3 of 13 Table 1 Previous validations of HADS in patients with cardiac disease
Cronbach’s alpha
Reference
Language
Population
Analytic
methods
Number
of factors
Sub scale content
HADS-A
HADS-D
Correlation
between
sub scales
Ayis et al. 2018 [9]
English
Stroke (n = 1443)
ML
PCA
CFA
(and IRT)
2
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
Kaur et al. The HADS questionnaire 2015 [15]
Malaysian
Coronary artery
disease (n = 189)
PCA
CFA
3
Anxiety:
1,3,5,7,9,11,13
Anhedonia:
2,4,6,14
Psychomotor retardation: 8,
10,12
0.89
0.69
Anhedonia: 0.70
Psychomotor
retardation: 0.51
Anhedonia –
psychomotor
retardation:
0.35
Anhedonia –
anxiety:
0.47
Psychomotor
retardation –
anxiety:
0.39
De Smedt
et al. 2013 [10]
22
European
countries
CABG, PCI, AMI,
myocardial
ischemia
(n = 8745)
CFA
2
Anxiety: 1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.82
0.74
0.60
Cosco et al. 2012 [21]
English
Cardiovascular
disease (n = 893)
MSA
1
1,2,3,4,5,6,7,9,10,11,12,13
Emons et al. 2012 [16]
Dutch
Cardiac patients
(n = 534)
MSA
EFA CFA
3
Anxiety: 1,3,5,9,13
Depression: 2,4,6,8,10,12
Restlessness: 7,11,14
Depression –
restlessness:
0.62
Restlessness
– anxiety:
0.68
Depression
– anxiety:
0.66
Kendel et al. 2010 [22]
German
CABG
(n = 1271)
Rasch
(HADS-D
only)
Depression: 2,4,6,12
Hunt-Shanks
et al. 2010 [17]
English
Cardiac patients
(n = 801)
CFA
3
Negative affect: 1,5,7,11
Autonomic anxiety: 3,9,13
Depression: 2,4,6,8,10,12,14
Martin et al. 2008 [18]
German
Chinese
English
Coronary heart
disease
(n = 1793)
MGCFA
3
Antonomic anxiety:
3,9,13 Negative affectivity: 1,
5,7,11Anhedonic depression:
2,4,6,8,10,12,14
Pais-Ribeiro
et al. 2007 [11]
Portuguese Mixed patients
incl. Coronary
heart disease
(n = 1322)
EFA
CFA
2
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.76
0.81
0.58
Wang et al. 2006 [12]
Chinese
Coronary heart
disease (n = 154)
CFA
2 or 3
2: Anxiety: 1,3,5,9,11
Depression: 2,4,6,7,8,10,12,14
3: Antonomic anxiety:
3,9,13
Negative affectivity: 1,5,7,11
Anhedonic depression: 2,4,6,
8,10,12,14
Barth and
Martin 2005 [13]
German
Coronary heart
disease (n = 1320)
EFA
CFA
EFA: 2
CFA: 3
EFA:
Anxiety:
1 3 5 7 9 11 13
0.82
(between
HADS A
d Table 1 Previous validations of HADS in patients with cardiac disease Ayis et al. 2018 [9]
English
Stroke (n = 1443)
ML
PCA
CFA
(and IRT)
2
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
Kaur et al. 2015 [15]
Malaysian
Coronary artery
disease (n = 189)
PCA
CFA
3
Anxiety:
1,3,5,7,9,11,13
Anhedonia:
2,4,6,14
Psychomotor retardation: 8,
10,12
0.89
0.69
Anhedonia: 0.70
Psychomotor
retardation: 0.51
Anhedonia –
psychomotor
retardation:
0.35
Anhedonia –
anxiety:
0.47
Psychomotor
retardation –
anxiety:
0.39
De Smedt
et al. 2013 [10]
22
European
countries
CABG, PCI, AMI,
myocardial
ischemia
(n = 8745)
CFA
2
Anxiety: 1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.82
0.74
0.60
Cosco et al. 2012 [21]
English
Cardiovascular
disease (n = 893)
MSA
1
1,2,3,4,5,6,7,9,10,11,12,13
Emons et al. The HADS questionnaire 2012 [16]
Dutch
Cardiac patients
(n = 534)
MSA
EFA CFA
3
Anxiety: 1,3,5,9,13
Depression: 2,4,6,8,10,12
Restlessness: 7,11,14
Depression –
restlessness:
0.62
Restlessness
– anxiety:
0.68
Depression
– anxiety:
0.66
Kendel et al. 2010 [22]
German
CABG
(n = 1271)
Rasch
(HADS-D
only)
Depression: 2,4,6,12
Hunt-Shanks
et al. 2010 [17]
English
Cardiac patients
(n = 801)
CFA
3
Negative affect: 1,5,7,11
Autonomic anxiety: 3,9,13
Depression: 2,4,6,8,10,12,14
Martin et al. German
Coronary heart
MGCFA
3
Antonomic anxiety: 0.39
De Smedt
et al. 2013 [10]
22
European
countries
CABG, PCI, AMI,
myocardial
ischemia
(n = 8745)
CFA
2
Anxiety: 1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.82
0.74
0.60
Cosco et al. 2012 [21]
English
Cardiovascular
disease (n = 893)
MSA
1
1,2,3,4,5,6,7,9,10,11,12,13
Emons et al. 2012 [16]
Dutch
Cardiac patients
(n = 534)
MSA
EFA CFA
3
Anxiety: 1,3,5,9,13
Depression: 2,4,6,8,10,12
Restlessness: 7,11,14
Depression –
restlessness:
0.62
Restlessness Depression –
restlessness:
0.62
Restlessness
– anxiety:
0.68
Depression
– anxiety:
0.66 0.66
Kendel et al. 2010 [22]
German
CABG
(n = 1271)
Rasch
(HADS-D
only)
Depression: 2,4,6,12
Hunt-Shanks
et al. 2010 [17]
English
Cardiac patients
(n = 801)
CFA
3
Negative affect: 1,5,7,11
Autonomic anxiety: 3,9,13
Depression: 2,4,6,8,10,12,14
Martin et al. 2008 [18]
German
Chinese
English
Coronary heart
disease
(n = 1793)
MGCFA
3
Antonomic anxiety:
3,9,13 Negative affectivity: 1,
5,7,11Anhedonic depression:
2,4,6,8,10,12,14
Pais-Ribeiro
et al. 2007 [11]
Portuguese Mixed patients
incl. Coronary
heart disease
(n = 1322)
EFA
CFA
2
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.76
0.81
0.58
Wang et al. 2006 [12]
Chinese
Coronary heart
disease (n = 154)
CFA
2 or 3
2: Anxiety: 1,3,5,9,11
Depression: 2,4,6,7,8,10,12,14
3: Antonomic anxiety:
3,9,13
Negative affectivity: 1,5,7,11
Anhedonic depression: 2,4,6,
8,10,12,14
Barth and
Martin 2005 [13]
German
Coronary heart
disease (n = 1320)
EFA
CFA
EFA: 2
CFA: 3
EFA:
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
CFA:
Psychomotor agitation: 1,7,
11
Psychic anxiety:
0.82
(between
HADS-A and
HADS-D) Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 4 of 13 Table 1 Previous validations of HADS in patients with cardiac disease (Continued)
Cronbach’s alpha
Reference
Language
Population
Analytic
methods
Number
of factors
Sub scale content
HADS-A
HADS-D
Correlation
between
sub scales
3,5,9,13
Depression: 2,4,6,8,10,12,14
Martin et al. 2004 [52]
Chinese
Acute coronary
syndrome
(n = 138)
CFA
3
Different models apply
0.79
0.55
Martin et al. Psychometric properties of HADS The following psychometric properties of the HADS
were evaluated. Floor and ceiling effects occur if more than 15% of the
patients select the lowest or highest possible score on an
item. Floor and ceiling effects can be an indication that
extreme items are missing in either end of the scale,
which can possibly limit its validity [39, 40]. p
y
y
Construct validity is defined as the degree to which an
instrument measures what it is intended to measure. It
is evaluated by testing hypotheses about an instrument –
for example, relationships between parts of an instru-
ment, relationships with scores of other instruments or
differences between relevant groups [41]. An aspect of
construct validity is structural validity, which is the
degree to which the sub-scale scores of an instrument
are an adequate reflection of the dimensions of the
construct to be measured [41]. Structural validity was
evaluated using exploratory factor analysis (EFA) and
confirmatory factor analyses (CFA). CFA was conducted
for
the
original
two-factor
structure
suggested
by
Zigmond and Snaith [5], and also for four three-factor
models [15, 42–44] and one one-factor model [21] found
in previous studies including cardiac patients. The HADS questionnaire 2003 [19]
English
MI
(n = 335)
CFA
3
Anhedonia: 2,4,6,8,10,12,14
Psychic anxiety:
3,5,9,13
Psychomotor agitation: 1,7,
11
0.83–0.86
(3 timepoints)
0.76–0.80
(3 timepoints)
Roberts et al. 2001 [14]
English
Female cardiac
patients (n = 167)
CFA
2
Anxiety:
1,3,5,7,9,11,13
Depression: 2,4,6,8,10,12,14
0.85
0.80
0.60
Martin and
Thompson
2000 [20]
English
MI
(n = 194)
EFA
3
1: 2,4,6,7,8,10,12,14
2: 3,9,13
3: 1,5,11
0.76
0.72
0.54
ML maximum likelihood; PCF principal component analysis; CFA confirmatory factor analysis; IRT item response theory; MSA Mokken scale analysis; EFA exploratory
factor analysis; CABG coronary artery bypass graft; PCI percutaneous coronary intervention; AMI acute myocardial infarction; MGCFA meta group confirmatory
factor analysis; MI myocardial infarction Table 1 Previous validations of HADS in patients with cardiac disease (Continued) ML maximum likelihood; PCF principal component analysis; CFA confirmatory factor analysis; IRT item response theory; MSA Mokken scale analysis; EFA exploratory
factor analysis; CABG coronary artery bypass graft; PCI percutaneous coronary intervention; AMI acute myocardial infarction; MGCFA meta group confirmatory
factor analysis; MI myocardial infarction Cronbach’s alpha of 0.87 for the emotional subscale and
0.91 for the physical one [38]. The Danish version of HADS has been frequently used
for research purposes, both in observational studies and
randomized controlled trials, as well as for screening
purposes in clinical practice [2, 3, 32–36]. Furthermore, two single items on anxiety and depres-
sion allowed patients to rate anxiety and depression on a
10-point Likert scale. The translation of the HADS from English into Danish
was evaluated by five independent assessors who were
fluent in both English and Danish. For each item the
equivalence of the translation was evaluated on a scale
from 1 to 4, with higher numbers indicating stronger
equivalence. The Translation Validity Index (TVI) was
calculated as the proportion of assessments rated posi-
tively with score of 3 or 4 [37]. Other instruments The Short-Form 12 health survey (SF-12) is a brief, gen-
eric measure of health-related quality of life that gener-
ates both a physical (PCS) and a mental component
score (MCS). Higher scores indicate better health status
[16]. The SF-12 has been validated in a population of pa-
tients with coronary heart disease from 22 European
countries with satisfactory results for construct validity
and a Cronbach’s alpha of 0.87 for PCS and 0.84 for
MCS, respectively, indicating high internal consistency
reliability [10]. HeartQoL is a disease-specific question-
naire that measures quality of life in cardiac patients and
produces a global score and two subscales: a physical
and an emotional scale ranging from 0 to 3 with higher
scores indicating better quality of life status [18–20]. The instrument has been validated in a large sample of
coronary patients with results confirming both discrim-
inative and convergent validity and high reliability with a Construct validity was also examined through hypoth-
eses testing by looking at HADS scores in relation to the Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 5 of 13 Page 5 of 13 Page 5 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 MCS on SF-12, the emotional subscale of HeartQoL and
a single item on anxiety and a single item on depression
(convergent construct validity), and in relation to the
PCS and physical subscale of HeartQoL (divergent con-
struct validity). HADS, SF-12 and HeartQoL subscales was examined by
stratifying mean scores of MCS, PCS, and HeartQoL
emotional and HeartQoL physical by HADS-A and
HADS-D scores above and below 8. HADS, SF-12 and HeartQoL subscales was examined by
stratifying mean scores of MCS, PCS, and HeartQoL
emotional and HeartQoL physical by HADS-A and
HADS-D scores above and below 8. Internal
consistency
was
evaluated
by
calculating
Cronbach’s alpha for subscales and also by corrected
item-total correlations. We hypothesized high correlations (r > 0.60) between
both HADS-A and HADS-D and the MCS score and the
HeartQoL emotional score and high correlations be-
tween HADS-A and a single item measuring anxiety,
and between HADS-D and a single item measuring de-
pression. Furthermore, we hypothesized low correlations
(r < 0.30) between HADS-A and HADS-D and PCS and
HeartQoL physical as these measures were not supposed
to be related to the HADS subscales. Demographic and clinical profile Demographic and clinical characteristics are presented
as frequencies or means with standard deviations (SD). Item score distributions are presented as means with
SD, frequencies for each response category and missing
data. Histograms and the Kolmogorov-Smirnov test were
used to determine whether item scores deviated from
the normal distribution. Out of 25,241 eligible patients, 12,806 had complete re-
sponses to the HADS questionnaire giving a response
rate of 51%. Demographic and clinical characteristics are
presented in Table 2. Item score statistics and translation validity index Exploratory factor analysis was conducted using prin-
cipal axis extraction based on eigenvalues greater than 1. Oblimin rotation was applied with a cut-off point of 0.30
as designating loading on a factor. The item score statistics are presented in Table 3. Item
8 showed markedly different scores compared to the rest
of the items, with more patients using high response cat-
egories, Table 3. There were floor effects on all items
and a ceiling effect on item 8, Table 3. Confirmatory
analyses
were
conducted
with
the
weighted least squared means and variance (WLSMV)
estimator. A Root Mean Square Error of Approximation
(RMSEA) estimate below 0.06 along with Comparative
Fit Index (CFI) and Tucker Lewis Index (TLI) estimates
above 0.95 indicated a good model fit [46]. Of the 14 items, 12 had an TVI of 100%, and two
(items 3 and 11) had TVI of 60% (both of these were a
part of HADS-A. The TVI for the total scale was 94%,
Additional file 1: Table S1. Other instruments DIF was examined using multivariate ordinal logistic
regression with items as the dependent variable and
gender and total score (HADS-A or HADS-D depend-
ing on the item) as the independent variables. Because
the proportional odds assumption was not fulfilled a
partial proportional odds model was used. DIF was
evaluated by different criteria. Uniform DIF can be con-
sidered if the odds ratio (OR) for gender is statistically
significantly different from 1 [45]. Interactions between
gender and total score were included to evaluate pos-
sible non-uniform DIF. A statistically significant inter-
action can be an indication of non-uniform DIF [45]. Because of the large sample size and the risk of finding
statistically significant results with no or very little clin-
ical meaning, DIF was also evaluated by Nagelkerke’s
R.2 A difference in R2 of more than 0.03 between
models was an indication of noticeable DIF (both uni-
form and non-uniform) [45]. Internal consistency reliability is an indicator of the
extent to which the items of an instrument are internally
correlated and therefore measure the same construct. This can be evaluated by calculating Cronbach’s alpha. A Cronbach’s alpha of between 0.70 and 0.95 is an indi-
cation of good internal consistency [40]. DIF is a form of measurement invariance at item
level. DIF means that there are items for which patients
from different groups with the same level of the con-
struct being measured do not have the same scores. This can indicate that the item measures different
things in the different groups. DIF can be uniform or
non-uniform depending on whether the differences are
present for all values of the scale or just for some values
of the scale [45]. Only patients with complete responses to the HADS
were included in the analyses. Analyses were conducted using SAS version 9.4, IBM
SPSS version 25 and Mplus version 7.4. Differential item functioning There were indications of DIF for item 3, 4 and 13
where women were more likely to have high item scores
compared to men and for items 11 and 14 where men
were more likely to have high item scores compared to
women. There were significant interactions between
item and subscale for items 1, 2, 5, 7, 8, 9 and 12, which
is an indication of non-uniform DIF. However, in ana-
lysis using Nagelkerke’s R2 there was no noticeable DIF
for any item, Table 7. The CFA indicated that the three-factor structure sug-
gested by Friedman et al. [44] showed the best fit for the
models tested, Table 5. The diagram from the CFA of
the three-factor structure suggested by Friedman et al. [44] is presented in Fig. 1. Internal consistency For HADS-A mean inter-item correlation was 0.50
(range 0.35–0.61) and Cronbach’s alpha was 0.87. The
corrected item-total correlations ranged from 0.52 to
0.71. Cronbach’s alpha would not be improved by the
deletion of any item. For HADS-D mean inter-item correlation was 0.41
(range 0.24–0.58). Cronbach’s alpha was 0.82. The cor-
rected item-total correlations ranged from 0.44 to 0.67. Cronbach’s alpha would not be improved by the deletion
of any item. For all HADS items the mean inter-item correlation
was 0.40 (range 0.24–0.61). Looking at the three-factor structure, the Cronbach’s
alpha for the psychomotor agitation subscale was 0.74
and 0.83 for the psychic anxiety subscale. The HADS-D
subscale was unchanged with a Cronbach’s alpha of
0.82. Cronbach’s alpha would not be improved by the
deletion of any item. Factor structure Both the EFA and the CFA were conducted on the
total population. Extensive previous literature exists that
provide suggestions for models to be tested in the CFA. The results from the EFA indicate that the original two-
factor structure of the HADS seems to fit in this cardiac
population. However, item 7 showed almost the same
loading on each subscale, Table 4. The correlation be-
tween HADS-A and HADS-D was 0.66. Spearman’s rank-order correlations were used to de-
termine convergent and divergent validity as data were
not normally distributed. Convergent validity between Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 6 of 13 Page 6 of 13 Table 2 Demographic and clinical characteristics
n
12,806
Male, n (%)
8953 (69.9)
Age, mean (SD)
65.1 (12.1)
Marital status (n,%)
Married
Divorced
Widowed
Unmarried
8307 (64.9)
1728 (13.5)
1533 (12.0)
1238 (9.6)
Educational level (n,%)
Basic school
Upper secondary or vocational school
Higher education
Missing
3903 (30.5)
5595 (43.7)
3018 (23.5)
290 (2.3)
Cardiac diagnosis (n,%)
Ischemic heart disease
Arrhythmias
Heart failure
Heart valve diseases
6832 (53.3)
4121 (32.2)
917 (7.2)
936 (7.3)
Co-morbidity (n,%)
Hypertension
4424 (34.6)
Ventricular arrhythmia
589 (4.6)
Ischemic heart disease
5544 (43.3)
Myocardial infarction
2408 (18.8)
Diabetes
1257 (9.8)
Heart failure
2210 (17.3)
Renal disease
426 (3.3)
Chronic obstructive pulmonary disease
837 (6.5)
Tu comorbidity score (n,%)
0
5271 (41.2)
1
4378 (34.2)
2
2062 (16.1)
≥3
1095 (8.5) HADS-A and the single item on anxiety was 0.68 and be-
tween HADS-D and the single item on depression it was
0.59. This confirmed the stated hypotheses about conver-
gent validity. However, the two single items were highly
correlated (0.76). Correlations between HADS-A and PCS and Heart-
QoL physical were 0.25 and 0.35, respectively. Correla-
tions between HADS-D and PCS and HeartQoL physical
were 0.50 and 0.55, respectively. This did not confirm
the hypotheses on divergent validity for HADS-D. Discussion Looking at MCS, PCS, HeartQoL emotional and Heart-
QoL physical scores in relation to HADS scores, patients
with scores below 8 on both HADS-A or HADS-D had
high scores on MCS and HeartQoL emotional. Con-
versely, patients with HADS-A and HADS-D scores
above 8 have the lowest scores. The same pattern is
found in PCS and HeartQoL physical scores, Table 6. In the present study the psychometric properties of the
HADS in a large sample of Danish cardiac patients were
evaluated. Floor effects were found on all items and ceiling
effect on item 8. The original two-factor structure of the
scale was confirmed in EFA, but CFA indicated a three-
factor structure. The hypotheses proposed were supported
for both subscales, providing evidence for convergent
validity. However, for HADS-D the hypotheses proposed
for divergent validity were not supported. Thus, divergent
validity is not indicated. Internal consistency was good for
both HADS-A and HADS-D. Correlations between HADS-A and MCS and HeartQoL
emotional were 0.67 and 0.75, respectively. Correlations
between HADS-D and MCS and HeartQoL emotional
were 0.66 and 0.63, respectively. The correlation between Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 7 of 13 Table 3 Item and score statistics
Score distribution, n (%)
Mean (SD)
0
1
2
3
Missing
HADS-A
n = 12,806
5.79
(4.19)
1. I feel tense or ‘wound up’*
1.05
(0.83)
3471
(25.8)
6413
(47.6)
2662
(19.8)
745
(5.5)
172
(1.3)
3. I get a sort of frightened feeling as if something awful is about to happen*
1.09
(0.90)
4050
(30.1)
4702
(34.9)
3754
(27.9)
361
(5.7)
196
(1.5)
5. Worrying thoughts go through my mind*
0.90
(0.89)
5189
(38.5)
5027
(37.3)
2244
(16.7)
790
(5.9)
213
(1.6)
7. I can sit at ease and feel relaxed
0.73
(0.73)
5673
(42.1)
5746
(42.7)
1721
(12.8)
156
(1.2)
167
(1.2)
9. I get a sort of frightened feeling like ‘butterflies’ in the stomach
0.62
(0.72)
6596
(50.0)
5354
(39.8)
1009
(7.5)
304
(2.3)
200
(1.5)
11. I feel restless as I have to be on the move*
0.88
(0.81)
4874
(36.2)
5549
(41.2)
2444
(18.2)
413
(3.1)
183
(1.4)
13. I get sudden feelings of panic*
0.52
(0.69)
7691
(57.1)
4355
(32.4)
1035
(7.7)
163
(1.2)
219
(1.6)
HADS-D
n = 12,806
4.29
(3.65)
2. Discussion I still enjoy the things I used to enjoy
0.72
(0.78)
6080
(41.2)
5332
(39.6)
1428
(10.6)
433
(3.2)
190
(1.4)
4. I can laugh and see the funny side of things
0.37
(0.64)
9403
(69.8)
2991
(22.2)
766
(5.7)
131
(1.0)
172
(1.3)
6. I feel cheerful*
0.51
(0.65)
8309
(61.7)
3358
(24.9)
1417
(10.5)
209
(1.6)
170
(1.3)
8. I feel as if I am slowed down*
1.40
(0.93)
2078
(15.4)
5912
(43.9)
3177
(23.6)
2122
(15.8)
174
(1.3)
10. I have lost interest in my appearance*
0.43
(0.69)
8983
(66.7)
3076
(22.9)
1080
(8.0)
138
(1.0)
186
(1.4)
12. I look forward with enjoyment to things
0.52
(0.74)
8119
(60.3)
3593
(26.7)
1313
(9.8)
225
(1.7)
213
(1.6)
14. I can enjoy a good book or radio or TV program
0.37
(0.69)
9654
(71.7)
2608
(19.4)
705
(5.2)
289
(2.2)
207
(1.5)
Each item is scored on a scale of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse response. The eight items highlighted
with * are reverse scored. These are reversed when summing the subscales Table 3 Item and score statistics 14. I can enjoy a good book or radio or TV program Each item is scored on a scale of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse respo
with * are reverse scored. These are reversed when summing the subscales of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse response. The eight items highlighted
se are reversed when summing the subscales The factor analyses indicate that the factor structure
of the HADS is not completely clear. The EFA con-
firmed the original two-factor structure suggested by
Zigmond and Snaith [5], but the CFA showed that the
three-factor structure as found by Friedman et al. [44] in
a French sample of patients suffering from major depres-
sion had the best model fit. The same result was found
by Barth and Martin in a German coronary heart disease
population [13]. Several other studies have found varia-
tions of a three-factor structure to have the best model
fit for the HADS as indicated in Table 5. Discussion The differences
in factor structure found across studies might be ex-
plained by different methodology such as data extraction
method, model fit criteria, translation or type of patients
included. (item 1, 7, 11), psychic anxiety (item 3, 5, 9, 13) and de-
pression (item 2, 4, 6, 8, 10, 12, 14), the division of items
from the original HADS-A into two factors can make
sense as relating to two different dimensions of anxiety
disorder. The items in the psychomotor agitation sub-
scale relate to physical feelings of restlessness and agita-
tion while the items in the psychic anxiety subscale
relate to emotional representation of anxiety with worry-
ing and nervous thoughts. Agitation is, however, also a
common symptom among patients with depressive dis-
orders and can occur as a side effect of antidepressant
medication [47]. The interrelatedness between symptoms of anxiety
and depression is further evident in the high correla-
tions between HADS-A and HADS-D. This did not
change when looking at the three-factor structure
instead. It has previously been argued that a high When considering the content of the three factors sug-
gested by Friedman et al. [44]; psychomotor agitation Christensen et al. Health and Quality of Life Outcomes (2020) 18:9
Page 8 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 8 of 13 Table 4 Exploratory factor analysis - rotated factor matrixa Table 4 Exploratory factor analysis - rotated factor matrixa
Factor
1
2
Item 9. I get a sort of frightened feeling like ‘butterflies’ in the stomach
0.81
Item 3. I get a sort of frightened feeling as if something awful is about to happen
0.80
Item 5. Worrying thoughts go through my mind
0.69
Item 13. I get sudden feelings of panic
0.71
Item 1. I feel tense or ‘wound up’
0.60
Item 7. I can sit at ease and feel relaxed
0.41
0.36
Item 11. I feel restless as I have to be on the move
0.46
Item 12. I look forward with enjoyment to things
0.79
Item 6. I feel cheerful
0.67
Item 2. I still enjoy the things I used to enjoy
0.72
Item 4. I can laugh and see the funny side of things
0.62
Item 8. I feel as if I am slowed down
0.54
Item 10. I have lost interest in my appearance
0.55
Item 14. I can enjoy a good book or radio or TV program
0.45
Cumulative % of variance explained
45.22
53.99
Eigenvalue
6.33
1.23
aExploratory factor analyses using principal axis extraction based in eigenvalues
greater than 1, Oblimin rotation and cut-off point of 0.30
Loadings> 0.40 in bold Table 5 Fit indices for confirmatory factor analyses of factor structures proposed in previous studies
RMSEA
Models
Number of factors
Sub scale content
RMSEA
90% CI
p-value
CFI
TLI
Zigmond and Snaith 1983 [5]
2
HADS-A:
1,3,5,7,9,11,13
HADS-D:
2,4,6,8,10,12,14
0.071
0.069;0.072
< 0.001
0.973
0.968
Dunbar et al. 2000 [43]
3
Negative affect:
1,5,7,11
Autonomic anxiety:
3,9,13
Depression:
2,4,6,8,10,12,14
0.061
0.059;0.062
< 0.001
0.981
0.976
Friedman et al. 2001 [44]
3
Psychomotor agitation:
1,7,11
Psychic anxiety:
3,5,9,13
Depression:
2,4,6,8,10,12,14
0.060
0.058;0.061
< 0.001
0.981
0.977
Caci et al. 2003 [42]
3
Anxiety:
1,3,5,9,13
Depression:
2,4,6,8,10,12
Restlessness:
7,11,14
0.064
0.062;0.065
< 0.001
0.979
0.974
Kaur et al. 2015 [15]
3
Anxiety:
1,3,5,7,9,11,13
Anhedonia:
2,4,6,14
Psychomotor retardation:
8,10,12
0.069
0.068;0.071
< 0.001
0.975
0.969
Cosco et al. 2012 [21]
1
1,2,3,4,5,6,7,9,10,11,12,13
0.111
0.109;0.113
< 0.001
0.945
0.932 Table 5 Fit indices for confirmatory factor analyses of factor structures proposed in previous studies Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 9 of 13 Page 9 of 13 (2020) 18:9 Fig. 1 Diagram from the confirmatory factor analysis presenting the
model with the best fit. Standardized loadings (SE). Table 4 Exploratory factor analysis - rotated factor matrixa PAn = psychic
anxiety; Dep = depression; PAg = psychomotor agitation In the EFA item 7 was found to load almost equally on
both factors. This has been found in previous studies as
well [13]. Item 7 reads ‘I can sit at ease and feel relaxed’;
this may reflect aspects of both anxiety and depression. Eight items in the HADS are reversely scored. This is
a recommended method to avoid acquiescence bias
which is the tendency for respondents of a survey to
agree with statements regardless of their content. How-
ever, research suggests that individual differences in
response styles can systematically affect the factor
structure [49]. The uncertainty of the factor structure
of the HADS is not necessarily a reason to discard the
instrument, but rather to be clear on the purpose of
using the scale. The two-factor structure may prove
useful as a simple indication of either anxiety or de-
pression. The possible presence of a third factor indi-
cates that the scale may provide more refined results
regarding different aspects of anxiety, rather than just
an indication of generalized anxiety. Because the results
regarding factor structure were not clear, the two-
factor structure originally proposed was used in the
remaining analyses for the paper. There were floor effects on all items, which may indi-
cate that the number of extreme response categories is
not sufficient. As the HADS was developed to detect in-
dications of a mood disorder, which is not present in the
majority of the population, even a population with se-
vere illness, it is not surprising that there are floor ef-
fects. Item 8 also showed a ceiling effect. The item reads
‘I feel as if I am slowed down’. In a population of elderly,
severely ill patients just discharged, it is not surprising
that this feeling would be prevalent. This item is suscep-
tible to influence from either age or disease which is a
bias in terms of validity as an indicator of mood. Fig. 1 Diagram from the confirmatory factor analysis presenting the
model with the best fit. Standardized loadings (SE). PAn = psychic
anxiety; Dep = depression; PAg = psychomotor agitation correlation between anxiety and depression is to be ex-
pected, not because of common symptoms but because
it is possible that anxiety can lead to depression and
that depression can lead to anxiety. Table 4 Exploratory factor analysis - rotated factor matrixa It is also possible
that the two disorders result from a common cause. The causality of this relationship cannot, however, be
determined from cross-sectional data [48]. y
The analyses of DIF indicated that there could be po-
tential problems with DIF for several items. Table 4 Exploratory factor analysis - rotated factor matrixa However, Table 6 HADS scores in relation to SF-12 and HeartQoL scores Table 6 HADS scores in relation to SF-12 and HeartQoL scores
HADS-A
< 8
≥8
HADS-D
n (%)
8211 (64.1)
553 (4.3)
< 8
MCS, mean (SD)
53.03 (7.96)
42.96 (8.63)
PCS, mean (SD)
44.13 (10.43)
34.11 (9.07)
HeartQoL emotional, mean (SD)
2.50 (0.56)
1.56 (0.68)
HeartQoL physical, mean (SD)
1.83 (0.83)
1.47 (0.81)
n (%)
2147 (16.8)
1895 (14.8)
≥8
MCS, mean (SD)
42.01 (9.16)
34.11 (9.07)
PCS, mean (SD)
33.44 (9.93)
35.53 (10.03)
HeartQoL emotional, mean (SD)
1.92 (0.70)
1.04 (0.69)
HeartQoL physical, mean (SD)
1.02 (0.71)
0.95 (0.70)
HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and
Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation
The cut-off of 8 is used as an indicator of possible mood disorder Table 6 HADS scores in relation to SF-12 and HeartQoL scores
HADS-A
< 8
≥8
HADS-D
n (%)
8211 (64.1)
553 (4.3)
< 8
MCS, mean (SD)
53.03 (7.96)
42.96 (8.63)
PCS, mean (SD)
44.13 (10.43)
34.11 (9.07)
HeartQoL emotional, mean (SD)
2.50 (0.56)
1.56 (0.68)
HeartQoL physical, mean (SD)
1.83 (0.83)
1.47 (0.81)
n (%)
2147 (16.8)
1895 (14.8)
≥8
MCS, mean (SD)
42.01 (9.16)
34.11 (9.07)
PCS, mean (SD)
33.44 (9.93)
35.53 (10.03)
HeartQoL emotional, mean (SD)
1.92 (0.70)
1.04 (0.69)
HeartQoL physical, mean (SD)
1.02 (0.71)
0.95 (0.70)
HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and
Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation
The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and
Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation
The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and
Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation
The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and
Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation
The cut-off of 8 is used as an indicator of possible mood disorder Christensen et al. Table 4 Exploratory factor analysis - rotated factor matrixa Health and Quality of Life Outcomes (2020) 18:9 Page 10 of 13 Table 7 Differential item functioning tested for gender
OR (95% CI)a
for item
responses
1, 2 and 3
Overall
p-value
Significant interaction
between gender and
sub scale
Nagelkerke’s R2
Step 1
Nagelkerke’s R2
Step 2
Nagelkerke’s R2
Step 3
DIF R2 b
HADS-A
Item 1. I feel tense or ‘wound up’
X
0.6712
0.6714
0.6717
0.0005
Item 3. I get a sort of frightened feeling
as if something awful is about to
happen
1: 0.948
(0.782;1.149)
2: 0.801
(0.719;0.892)
3: 0.916
(0.820;1.023)
0.0007
0.6287
0.6293
0.6295
0.0008
Item 5. Worrying thoughts go through
my mind
X
0.6924
0.6930
0.6932
0.0008
Item 7. I can sit at ease and feel relaxed
X
0.6008
0.6011
0.6018
0.0010
Item 9. I get a sort of frightened feeling
like ‘butterflies’ in the stomach
X
0.6718
0.6726
0.6730
0.0012
Item 11. I feel restless as I have to be on
the move
1:1.670
(1.315;2.122)
2: 1.385
(1.240;1.545)
3: 1.423
(1.289;1.571)
<.0001
0.4746
0.4785
0.4788
0.0042
Item 13. I get sudden feelings of panic
1: 0.667
(0.462;0.963)
2: 0.712
(0.600;0.845)
3: 0.781
(0.703;0.868)
<.0001
0.6291
0.6307
0.6308
0.0017
HADS-D
Item 2. I still enjoy the things I used
to enjoy
X
0.6107
0.6112
0.6116
0.0009
Item 4. I can laugh and see the funny
side of things
1: 0.865
(0.587;1.274)
2: 0.917
(0.765;1.099)
3: 0.805
(0.719;0.902)
0.0025
0.5739
0.5747
0.5747
0.0008
Item 6. I feel cheerful
1: 1.079
(0.750;1.551)
2: 1.089
(0.937;1.266)
3: 1.071
(0.956;1.200)
0.5055
0.6381
0.6381
0.6385
0.0004
Item 8. I feel as if I am slowed down
X
0.5660
0.5676
0.5684
0.0024
Item 10. I have lost interest in my
appearance
1: 0.813
(0.561;1.177)
2: 1.050
(0.905;1.218)
3: 0.956
(0.866;1.054)
0.3345
0.4235
0.4237
0.4239
0.0003
Item 12. I look forward with enjoyment
to things
X
0.6136
0.6142
0.6143
0.0007
Item 14. I can enjoy a good book or
radio or TV program
1: 2.132
(1.558;2.918)
2: 1.612
(1.361;1.909)
3: 1.431
(1.289;1.587)
<.0001
0.3805
0.3853
0.3855
0.0050
a P
i l
i
l
dd
d l
i h i
d
d
i bl
d
d
d
b
l
i d
d
i bl
M
f Acknowledgements To the patients who took the time to participate in the survey, the 800
cardiac nurses involved in data collection and the heart centres for
prioritizing this study in a busy clinic. And to Sangchoon Jeon, Yale School
of Nursing for statistical support. The large sample size in this study is an advantage be-
cause of statistical power and because it allows a hetero-
geneous sample. There is, however, a risk of finding
statistically significant results of minimal clinical import-
ance. Therefore, we have not only looked at p-values to
determine validity, but rather measures of strength of
correlation, internal consistency and Nagelkerke’s R2 for
analyses of DIF. Conclusions because of the risk of finding statistically significant re-
sults of minimal clinical importance in this large popula-
tion, changes in Nagelkerke’s R2 between models were
given priority. These indicated no noticeable DIF for any
items. The presence of DIF for gender has been explored
in previous studies [22–24, 50], but only one study
found substantial DIF for item 14, with men being more
likely to endorse this item [22]. The findings of this study supported the validity and re-
liability of the HADS in a sample of Danish patients with
cardiac disease. EFA supported the original two-factor
structure of the scale, while CFA supported a three-
factor structure consisting of the original depression
subscale and two anxiety subscales; psychomotor agita-
tion and psychic anxiety. The hypotheses regarding con-
vergent validity were confirmed, but those regarding
divergent validity were not confirmed for HADS-D. Internal consistency was good with a Cronbach’s alpha
of 0.87 for HADS-A and 0.82 for HADS-D. There were
no indications of noticeable DIF by gender for any
items. When considering the usefulness of the HADS in clin-
ical practice it should also be noted that HADS has been
shown to predict morbidity and mortality in this patient
population and similar patient populations [3, 4, 51]. Funding This work was supported by Helsefonden; the Heart Centre, Rigshospitalet, The
Danish Heart Association, the Novo Nordisk Foundation and Familien Hede
Nielsens Fond. The funders played no part in planning or conducting the
research. HADS-D Page 11 of 13 Page 11 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Abbreviations CFA: Confirmatory factor analysis; CFI: Comparative Fit Index; DIF: Differential
item functioning; EFA: Exploratory factor analysis; HADS: Hospital Anxiety and
Depression Scale; MCS: Mental component score; OR: Odds ratio;
PCS: Physical component score; RMSEA: Root Mean Square Error of
Approximation; SD: Standard deviation; SF-12: Short-Form 12; TLI: Tucker
Lewis Index; WLSMV: Weighted least squared means and variance Newer methods for exploring internal consistency
exist, e.g. the use of McDonalds omega. However, for
consistency with the methods chosen throughout this
paper and for comparison with other HADS validation
studies we chose to include Cronbach’s alpha. Authors’ contributions SKB conceived the overall idea for the DenHeart study and all authors
designed the study. AVC performed the statistical analyses under the
supervision of JKD and wrote the first draft of the manuscript. All revised the
manuscript critically. All have given their final approval of the version to be
published. The response rate was 51%, which is to be expected in
a population of severely ill patients on the day of hos-
pital discharge. This may raise concerns about represen-
tativeness, however, the proportions of patients in the
diagnostic sub-groups were similar to that of the entire
eligible population, and responders and non-responders
were comparable in terms of their demographic and
clinical profiles, suggesting a representative sample [2]. We did, however, find a higher mortality rate in non-
responders compared to responders [4]. Availability of data and materials Danish legislation on data security prohibits sharing of data. Danish legislation on data security prohibits sharing of data. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12955-019-1264-0. Additional file 1: Table S1. Translation Validity Index (TVI) for the
Danish translation of Hospital Anxiety and Depression Scale (HADS) Ethics approval and consent to participate The DenHeart study complies with the Declaration of Helsinki. Danish
legislation does not require surveys to be approved by an ethics committee
system but rather by the Danish Data Protection Agency (2007-58-0015/30–
0937). The Danish National Board of Health permitted the use of register
data. DenHeart is registered at ClinicalTrials.gov (NCT01926145). Patients
provided informed consent. In the present study we used a single question on anx-
iety and depression to measure convergent validity. However, the two questions were highly correlated. In-
cluding more comprehensive instruments to measure
anxiety and depression would have been optimal to
examine convergent validity. These were, however, not
available in the data. Limitations of the study There is no description of the process of how the HADS
was translated into Danish from the questionnaire
owner, so it is not clear whether the translation has
followed
the
recommended
steps
to
ensure
cross-
cultural validity [45]. The current analyses are, in fact,
the first specific investigation of the psychometric prop-
erties of the Danish language version of HADS. For the
current study, we evaluated the TVI for each item and
the total scale with satisfactory results. Items 3 and 11
(both in HADS-A) received the lowest rating (60%). References 23. Djukanovic I, Carlsson J, Årestedt K. Is the hospital anxiety and depression
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University, 400 West Campus Drive, Orange, CT 06477, USA. 3National
Institute of Public Health, University of Southern Denmark, Studiestræde 6,
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Winslows Vej 4, 5000 Odense, Denmark. 6Department of Cardiology, Aarhus
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Instrumental dialogue and the ethics of expressing solicitude for each other’s existence
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DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE Instrumental dialogue and the ethics of expressing solicitude for each other’s existence A newer version of this article has been published as:
Bencherki, N., & Sénac, C. (2023). Instrumental dialogue and the ethics of expressing solicitude
for each other’s existence. Language and Dialogue. http://doi.org/10.1075/ld.00138.ben 1 1 1 Instrumental dialogue and the ethics of expressing solicitude for each other’s existence Instrumental dialogue and the ethics of expressing solicitude for each other’s existence Abstract Dialogue is about forgoing control and possession when interacting with the Other. In Dialogue is about forgoing control and possession when interacting with the Other. In
comparison, the notion of instrumentality appears contrary to the very notion of dialogue. This
paper suggests, however, that mutual instrumentalization is necessary for dialogue to be a space
where participants express solicitude for each other and promote each other’s voice, action, and
existence. Building on the work of French philosopher Étienne Souriau, we argue that promoting
another’s existence requires taking their actions and speech into our own. This enables them to
also exist through us, as we allow them to instrumentalize us. Such a view better accounts for
what goes on in tangible dialogue situations, as we show by revisiting an empirical case. Our
proposal extends current research on the conditions of productive dialogue, invites being careful
about who or what populates the dialogical scene, and turns our attention to what they may need
to pursue their existence. Keywords: dialogue, solicitude, existence, Étienne Souriau, ethics, instrumentalization. DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 2 2 DIALOGUE, SOLICITUDE, AND EXISTENCE 3 3 constituted individuals (Weigand 2010) but also about the very ability of those individuals to
exist as such. Even situations where participants engage in persuasion entail some degree of
solicitude for others, if only to the extent that one cares about the ideas they wish to convey and
other people’s opinions towards them. Solicitude, as we suggest, amounts to jointly cultivating others in dialogue: tending to
them by ensuring that we do and say what they need to thrive (Bencherki et al. 2020). Building
on Cooren’s (2010) ventriloquist principle – we carry the voices of others when we talk – the
notion of cultivation suggests that whenever someone makes something or someone else act or
speaks, they are also made to act or speak by that thing or person. Speaking about an
organization, a value, or a colleague, for instance, then also means allowing those different
“figures” (as Cooren 2010 refers to them) to animate or move us to speak on their behalf and to
be their ventriloquist’s dummy – or their instrument (Cooren 2020). The ventriloquial approach points out that people can extend solicitude to fellow human
interlocutors and other non-human beings that populate the dialogical scene. This includes the
principles they hold dear, the values they share, or the projects they pursue together (Cooren
2016; Matte and Bencherki 2019). No matter their nature, all beings are thus relationally
constituted through dialogue (Cooren 2018). As people speak in different ways, they promote the
existence of some ideas or values, possibly at the expense of others. Failing to do so, ideas fade
away, values are trampled, and people are excluded. Such a view means that ethical dialogue is
even more critical, as it may impact the very existence of its participants. In this paper, we clarify that ethics does not pre-exist the productive power of dialogue. We do so by showing that dialogue generates ethical decisions and actions precisely as people
tend to and show concern for realities that do not yet exist and that they bring about, including
the very participants to dialogue. In other words, the figures on whose behalf we act – whether
justice, passion or a colleague – are not “upstream” entities that act on people the way social
structures have been described to constrain action and decisions (e.g., Giddens 1984). 1. Introduction The history of ethical thought could be caricatured as a search for a source from which
proper conduct stems, whether rationality (Kant [1785] 2002), virtue (MacIntyre 1981), social
contract (Rawls 1971), or authenticity (Taylor 1992). The realization that these so-called sources
cannot be reconciled has led ethics scholars to turn their attention away from discovering what
people should do and observing instead how people interact with these sources as they deal with
ethical pluralism (Hennion 2017; ten Bos 2002; van Oosterhout, Wempe, and Willigenburg
2004). As an alternative, research has suggested that dialogue is productive of ethical decisions
rather than conveying the dictates of a source or merely serving as a vehicle for interaction (e.g.,
Arnett and Arneson 2016; Buber 1958). More precisely, dialogue can be understood as a form of
practical ethics and be seen as “a co-elaboration of meaning on practical issues that requires the
participation of the involved public” (Létourneau 2012, 18). Much research has been concerned
with finding the conditions that would sustain dialogue and be conducive to ethical decisions
(Johannesen 1971; Krippendorff 1989; Stückelberger 2009). Many of these criteria boil down to
the principle of avoiding controlling, possessing, or instrumentalizing the Other (Arnett 2016a). Hence, dialogue also consists in welcoming the Other, tending to their needs, and seeking
mutual understanding (Derrida 2000; Levinas 1987). However, a contradiction may exist in the
heart of dialogue. While, for the philosopher Martin Buber, dialogue requires “an awareness that
others are unique and whole persons” (Cissna and Anderson 1998, 65), others stress that
dialogue demands attention to the fragile effort through which others maintain their personhood
(Cooren and Sandler 2014; Stam 2010). What is at stake, then, is how “whole” people are
presumed to be when they engage in dialogue. Rather than a contradiction, though, one can
suggest that any person’s – or thing’s – individuality comes with the risk of disindividuating,
which may clarify the ethics at stake in dialogue. The realization entails that dialogue is also a space where people express their solicitude
for each other and support each other’s pursuit of individuation (Arnett 2001; see also Bencherki
and Iliadis 2021). In other words, dialogue is not only about reciprocity between already- DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 4 Such an understanding of dialogue, we argue, requires researchers to review the
conditions of ethical dialogue. Scholarship so far has supposed that welcoming the Other in
dialogue is a moral obligation, for instance, by following the deontological tradition (Kant
[1785] 2002), or that it stems from the realization of one’s relationship to others that emerges
from rethinking the self as another (Ricœur 1991). Instead, we suggest that the normative
expectation that people engage in dialogue ethically results from the project shared among all
parties, which dictates whose existence must be nurtured. Conversely, conflict may correspond
to diverging projects being pursued. People need each other to be and act in the world: each of us plays a part in collective
action because we mutually solicit each other and share in each other’s actions – for instance, I
can teach because I appropriate the work of my colleague who taught the introductory class – but
also because we know, relative to others, what we have to do, i.e., our place in the project that
we hold in common – if we want students to learn, then I teach my class at that particular time,
on that particular topic, in that particular room, and my colleague has her part to play (Latour
2011). Latour’s (2011) comparison with an orchestra is best to understand the relevance of such
reciprocal “solicitude” for dialogue: a symphony only exists as each musician has, in turn, their
moment on the stage, where they must exercise their talent and show the mastery of their
instrument. Each musician has the duty to play as well as they can when their turn comes, but not
in a moral sense. Failing to do their best would deprive others of their turn and, eventually,
compromise the collective work, thus also reflexively devaluing their own contribution. In that
sense, a musician’s performance is also the instrument through which others can play and jointly
create a collective work. Adopting this view, therefore, also supposes revisiting the idea that dialogue should not
be instrumental. Following the idea of reciprocal solicitude, concern for each other supposes
some degree of mutual instrumentalization so that each voice is called upon, in turn, to
participate in a collective work that reflexively provides meaning and value to each of our
contributions. DIALOGUE, SOLICITUDE, AND EXISTENCE Instead,
people interact to create and nurture projects, ways of thinking, works of art, identities, values,
and organizations that become increasingly demanding as they gain existence and require them
to act in one way or another. This also applies to people themselves, who must also find a place
to exist in dialogue through their contributions. DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 5 5 Dialogue thus becomes a space where one’s being continues through that of others and where
participants value and promote each other’s existence and contribution. Dialogue can therefore be understood as fostering the affirmation of one’s existence
through that of others, which, according to the French philosopher Étienne Souriau ([1943]
2015), allows it to exist and persist. To make this argument, we will borrow from Souriau’s
notion of modes of existence. For him, some beings – such as fictional characters – only exist to
the extent that we express our solicitude towards them: Sherlock Holmes and Dr. Watson
continue existing to the extent that, for example, we read their stories, talk about them, draw
them in cartoons, and represent them in movies and plays. Their seemingly immaterial existence
is substantiated through our many material actions. If we stopped caring about them, they would
vanish, as was presumably the fate of many characters we have forgotten. However, solicitude is
not only about fictional beings; any being’s existence rests at least in part on others’ solicitude,
especially since any being exists in many ways at once, including as a fiction, i.e., as someone
(or something) we talk about, remember, depict in photographs and drawings, etc. In particular,
we will refer to Souriau’s ([1956] 2015) text “Of the mode of existence of the work to-be-made,”
where he shows that the artist’s activity is increasingly constrained by the requirements of the
work of art that she is gradually bringing into existence. Like the work of art, any being, as it
gains greater existence, also demands a particular kind of solicitude to pursue its existence. Recognizing that existence is a matter of degrees also entails that ethical voices can be more or
less loudly and clearly heard. Therefore, people engaging in ethical dialogue must speak on
behalf of those emerging existences. For our purpose, this means that the ethical dimension of
action is intimately tied to figuring out how beings may continue to exist together and into each
other and how, alternatively, they may prevent or divert others’ existence. While an instrumental view of dialogue may appear first and foremost as a theoretical
proposal, we came to formulate it inductively throughout our various empirical observations. It
is, therefore, very much attuned to what goes on tangibly in everyday dialogical situations. DIALOGUE, SOLICITUDE, AND EXISTENCE As playing one’s tpart at the wrong moment can deprive another musician of their
turn, mutual instrumentalization also supposes ensuring that we support each other’s right to
exist and act, especially since some of us may not be able to act and speak on their own. DIALOGUE, SOLICITUDE, AND EXISTENCE 6 solicitude for each other, for their organization’s norms and for other entities, which leads them
to favor specific courses of action over others. DIALOGUE, SOLICITUDE, AND EXISTENCE That
is why we illustrate our theoretical discussion by offering a new analysis of a conversation
excerpt we draw from an existing study (Matte and Cooren 2015). While not representing
obvious ethical dilemmas, the case has the merit of presenting how two people express their DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 7 7 prescriptive stance considers that human beings are capable of both monologic and dialogic
communication. Dialogue, in this perspective, is not only a conversation between two people but
also the difference between the monologic narratives constituting worldviews (Arnett 2015). The
philosopher’s goal is not so much to advocate for dialogue as it is to warn against the
instrumentalism that monologue may engender and to advocate for “a particular quality of
relating” that dialogue, in all its forms, captures (Stewart and Zediker 2000, 227). While
monologue can express passion, authenticity, and conviction, it may also express zeal, bigotry,
and a dichotomic outlook on the world (Arnett 2015). Following Buber (1958), the monologic
view may emphasize the “I-It” relationship between subject and object, while “I-Thou”
corresponds to the encounter between subjects. Therefore, dialogical ethics must consider the
conditions for such a dialogue encounter and avoid instrumentalizing either party. prescriptive stance considers that human beings are capable of both monologic and dialogic
communication. Dialogue, in this perspective, is not only a conversation between two people but
also the difference between the monologic narratives constituting worldviews (Arnett 2015). The
philosopher’s goal is not so much to advocate for dialogue as it is to warn against the
instrumentalism that monologue may engender and to advocate for “a particular quality of
relating” that dialogue, in all its forms, captures (Stewart and Zediker 2000, 227). While
monologue can express passion, authenticity, and conviction, it may also express zeal, bigotry,
and a dichotomic outlook on the world (Arnett 2015). Following Buber (1958), the monologic
view may emphasize the “I-It” relationship between subject and object, while “I-Thou”
corresponds to the encounter between subjects. Therefore, dialogical ethics must consider the
conditions for such a dialogue encounter and avoid instrumentalizing either party. A crucial element of the absence of instrumentality consists in refusing to think of
dialogue as control (Krippendorff 1989) or possession of the other; dialogue is a “space of non-
possession” (Arnett 2016b, 3). It is, in that sense, a radical acceptance of the Other without a
desire to appropriate or change them (Levinas 1987), along with preserving people’s freedom of
choice (Johannesen 1971). Dialogue also implies paying attention to the Other’s monologic
narrative to learn “about what is of fundamental importance to another” (Arnett 2015, 2). Thus, it
is “openness to possibility and happenstance” (Poulos 2008, 117). 2. Dialogical ethics as the absence of control and instrumentalization There is a rising tendency to resort to dialogue when studying ethics, which may be
observed, for instance, in the field of business ethics and corporate social responsibility. Studies
in that area increasingly turn to dialogue as a way of capturing the process through which
practitioners are accomplish ethics in daily situations, rather than attempting to uncover external
principles (Maclagan 1999). Dialogue, in that sense, would avoid merely “applying” an ethical
theory to particular circumstances and instead engages all concerned parties in a conversation
about how to guide their actions (Lozano and Sauquet 1999). Dialogue is often understood quite
literally as a conversation among stakeholders, during which proper conduct is collectively
decided after considering each party’s perspective (Christensen, Morsing, and Thyssen 2011;
Morsing and Schultz 2006). Dialogue then consists of “a ‘co-creation’ of shared understanding”
and of working together towards a “mutually acceptable solution” (Golob and Podnar 2014,
250). Researchers, then, have concerned themselves with finding the conditions under which
such dialogue would lead to better decisions regarding how to act together. Most studies
recommend that it consist of “collaboration or pluralistic deliberation” and that it must be open
to the ideas expressed in the process (Illia et al. 2017). Dialogue must also be “free of internal
and external pressure” because it is only “under equal participating conditions” that all people
involved can tease out their assumptions and agree on a definition of what is valid or fair in
terms of decisions and behaviors (García-Marzá 2005, 214). In that view, some suggest concrete
techniques to improve dialogue (Morrell and Anderson 2006). The trends observed in the field of business ethics resonate with the findings of dialogue
specialists. Dialogue is inherently polyphonic and, as such, capable of handling tensions among
the plurality of voices that express differing preferences (Bakhtin 1986; Cooren and Sandler
2014; Gurevitch 2000). This ability may be viewed either from a descriptive or a prescriptive
stance. The descriptive stance views dialogue as the “irreducibly social, relational, or
interactional character” of all human meaning-making (Stewart and Zediker 2000, 225). The DIALOGUE, SOLICITUDE, AND EXISTENCE 3. Ethics in dialogue: in defense of instrumentalization and cultivation A better acknowledgment of communication’s creative and pragmatic nature collapses
the distinction between instrumental and dialogical views. This is the case because mutual
instrumentalization provides us with a clear set of “felicity conditions” for an act of
communication to successfully promote dialogue (Austin 1962) by calling for the promotion of
each other’s existence in our speech acts. Indeed, communication works because people
contribute to it in a way that also ensures that others can contribute in their turn, thus allowing
the creation of a collective work that makes each contribution meaningful. It means that an
instrument not only expresses its voice – say, the musician plays his line on the bassoon – but
also helps other voices to be expressed, thus becoming, to some extent, their instrument too. We must allow each other’s contribution to instrumentalize us and to speak and act
through our speech and action, in order to pursue a collective work together – ideas, projects, and
a plethora of other things we carry out through dialogue. This may mean voicing them ourselves,
in the same way a musician can reprise a theme or offer a counterpoint, but at the very least, it
means leaving them the necessary space and time to express themselves. When observing what
people (and other beings) do when they talk, we realize that they act as each other’s organ or
instrument, if only by respecting the sequential structure of conversation and its timing (Cooren
2010). Said otherwise, in any conversation, the parties not only express their unique ideas but
also use their speech to allow others to speak. They do so, for example, by quoting absent
people, invoking rules, and mobilizing other people and things in their speech. They also refer to
what their interlocutors have just said and manage the conversation with them, to ensure that
each can exist and act within the dialogical space. In doing so, they can be the instruments of
those figures that speak through them, which they also instrumentalize to lend weight to their
actions. The same goes for the objects that surround us and that we use in our daily practices
when we grant them a particular recognition or importance by referring to them or describing
them in the ongoing dialogue. In a way, we give them a place in dialogue so they can exist more
richly than through their materiality. DIALOGUE, SOLICITUDE, AND EXISTENCE Being ready to embrace the unexpected also concerns dialogue’s own potential for
surprise. Indeed, dialogue is creative (McNamee and Shotter 2004; Sanders 2012). Its creativity
is precisely why dialogue is a space of non-possession: how could we possess what does not
exist yet, what emerges from our encounter? (see also Derrida 1998). This creativity may be
unsettling but is crucial to our ability to construct together the world we share by allowing
novelty to emerge from the encounter of what already exists (Krippendorff 1989). In what follows, we will show that instrumentalization becomes a necessary condition for
genuine communication to take place, and dialogue can finally emerge when it is considered as a
mutual concern with each other’s existence and as its promotion through one’s speech and action
(what Latour 2011 refers to as reciprocal possession). DIALOGUE, SOLICITUDE, AND EXISTENCE 8 DIALOGUE, SOLICITUDE, AND EXISTENCE 9 people and things that populate the communicative scene (Cooren 2000). It also allows agency –
i.e., the capacity to act – to circulate and become collective, as each person and thing becomes a
vehicle for the agency of others, repeating here and now what others have said and done
elsewhere and at another time (Cooren 2004; Cooren et al. 2005). Dialogue, then, allows people
to speak and act together, not only with one another but thanks to each other. In doing so, they
also constitute their collective reality by configuring how the elements of speech and action that
pass through them can be arranged relative to one another. In short, dialogue is constitutive of
people and things, as well as of the social order in which they participate (as suggested by
ethnomethodology; see Garfinkel 1967). As Latour (2011) explains, order is established when
turns to talk and act are distributed between people, and such distribution occurs not in advance
but through those very same words and actions. Thus, order does not result from the injunction
of a higher power (as if the orchestra’s conductor was imposing their will on the musicians). Instead, the instruments spring to action when, by being attentive to others’ turn, they determine
it is theirs – resulting in a harmonious melody (on order and disorder, see also Vásquez and
Kuhn 2019). Since dialogue has to do with the obligation of “countering the eclipse of the Other”
(Arnett 2016a), then we must ask ourselves how dialogical practices help promote the existence
of others, but also who (or what) those “others” are, precisely, that we want to promote. In an
encounter with others, we must undertake an inventory of whose existence is at stake in that
meeting and be mindful of what matters to others (Latour 2013). When understood this way,
dialogical ethics may amount to mutual instrumentalization: letting ourselves be instrumentalized
by others and ensuring their sustenance and existence – but also ensuring that we only
instrumentalize others in a way that does not “eclipse” their voice, agency, or existence. The
questions then become whether instrumentalization is mutual, whether all parties can
instrumentalize others and be instrumentalized, and whose existence is promoted through these
instrumentalizations. These questions must be answered empirically by looking at how each contribution and
its author are cultivated in dialogue (Bencherki et al. 2020). 3. Ethics in dialogue: in defense of instrumentalization and cultivation Thus, instrumentalization is a way of including in dialogue
and offering a fuller existence to the objects and subjects that make up our communicative world. This mutual instrumentalization could be regretted if it were not the critical process
through which communication is at all possible whereby dialogue constitutes and organizes the This mutual instrumentalization could be regretted if it were not the critical process
through which communication is at all possible, whereby dialogue constitutes and organizes the g
p
through which communication is at all possible, whereby dialogue constitutes and organizes the DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 10 indicating that we may also express concern for gods, values, and other shared principles. Culture, then, refers to the way we tend to everything that matters to us, from the things we eat to
ideology and children (as in puericulture). The cultivation metaphor thus reveals that things do
not exist unless we care for them. Crops, ideas, and children die when we stop tending to them. Our relationship with fellow humans and other-than-humans attests to and perpetuates their
existence as things or persons. Thus, dialogue is a space where people cultivate their concern for things and others,
including making an effort to leave them some space to be who or what they are. As an active
effort, the work of cultivation is empirically observable. For instance, a law only exists to the
extent that a judge speaks on its behalf (Cooren 2015) and a medical standard as long as
healthcare professionals uphold it (Matte and Bencherki 2019). So, understood as the way people
instrumentalize each other to talk and act thanks to each other, dialogue has existential
implications: when we allow others to express themselves through us, we also cultivate them and
allow them to continue their existence, at least in the current dialogical situation, but possibly
beyond it. Tribunals and hospital wards are, in that sense, places where multiple entities of
different ontologies express themselves and call for people’s attention so that they tend to them
to constitute social and biological bodies (Bencherki and Elmholdt 2022). They are sites where
regulations, standards, traditions, resources, equipment, symptoms, and emotions all compete as
they attempt to thrive and as people speak and act on their behalf more or less loudly and
faithfully. Consequently, through dialogue, instrumentalization makes it possible to ensure an
ethics of existence, thanks to which subjects and objects can take their rightful place in the world
we hold in common. Dialogue, then, combines ethical and ontological implications: it concerns our duty to
help others exist. We must ask ourselves whether we deem some existences to be expendable,
and what we believe to be a fair price to pay to save the ones we care about. Indeed, we may not
be able to be an instrument for all existences, and some of them may be incompatible. DIALOGUE, SOLICITUDE, AND EXISTENCE To cultivate, etymologically, comes
from the Latin cultus, meaning “caring.” It refers to the farmer’s work of tending the soil and
caring for plants. Interestingly, the word “cult,” in the religious sense, also has the same origin, DIALOGUE, SOLICITUDE, AND EXISTENCE 4. Étienne Souriau and the different modes of existence Mostly known for his work on aesthetics, the French philosopher Étienne Souriau was
recently rehabilitated to a broader audience with the publication of Bruno Latour’s (2013) An
inquiry into modes of existence, which finds inspiration in Souriau’s ([1943] 2015; [1956] 2015)
work. The latter suggests that things exist under different modes. This means not only that each
thing exists under its own mode – an idea does not exist in the same way as a rock – but also that
a same thing can exist in different ways at the same time – an idea can be written, sung, sculpted,
or just reside in someone’s head. As another example, it is tempting to think that I exist
biologically, while Sherlock Holmes exists as a being of fiction; however, I also exist as the
author of my work (see Foucault 1979), as a member of my family, and even possibly as a
fictional character in a friend’s creative writing piece, to name a few. To exist under more modes
of existence is to obtain a fuller existence; indeed, existence is not a binary state, as “we can only
respond to the question, ‘Does this exist?,’ with More or Less, not Yes or No” (Souriau [1956]
2015, 221). To gain further existence, each being, or part thereof, “hopes for existence together;
it hungers after a different mode; it wants to be transposed into that mode” (Souriau [1943] 2015,
188). This transposition occurs when one being takes up and continues the action of another,
existing under a different mode. These are anaphoric uptakes that repeat what came before but
add something of their own, creating something new that continues the previous action. When I
try a recipe from a cookbook, I provide it with greater existence. It is not merely a recipe
forgotten in the printed pages of an obscure book, but a live recipe embodied in my movements
in the kitchen and, soon, in the bodies of my guests who will taste it. The recipe exists in the
words printed on the page, in its author’s initial intent, in each reader’s attempt to prepare it… It
exists in each of these uptakes and all of them at once. For Souriau, each uptake involves judgment to the extent that it corresponds to a decision
regarding what must be taken up from the previous mode of existence. DIALOGUE, SOLICITUDE, AND EXISTENCE To better
understand this ethics of dialogical care, we turn to French philosopher Étienne Souriau ([1943]
2015), who wrote about the notion of modes of existence and stressed that beings need others to
continue their existence. Said otherwise, no being exists by itself but always through others,
which complements the notion that dialogical instrumentality is inevitable. DIALOGUE, SOLICITUDE, AND EXISTENCE 11 DIALOGUE, SOLICITUDE, AND EXISTENCE 12 of creativity and, second, because they give meaning to action: what an action means is the
difference it operates in its uptake (Bencherki and Iliadis 2021). of creativity and, second, because they give meaning to action: what an action means is the
difference it operates in its uptake (Bencherki and Iliadis 2021). The ability to differ, however, is not absolute. It also depends on the degree of existence
already achieved. Souriau ([1956] 2015) uses the sculptor metaphor to explain that, as long as
the future work of art is a block of granite, the artist can continue its existence in a variety of
manners. However, as it takes shape and becomes lines, volumes, and textures, the work of art
becomes increasingly demanding, and there are only so many ways in which its existence can be
further promoted. It seems to ask the artist, “And what are you going to do now? With what
actions are you going to promote or deteriorate me?” (Souriau [1956] 2015, 232). Souriau
clarifies that this demand does not come from a projected or future state of the work of art, as if
the artist knew what it would end up looking like ahead of time. It comes from the present
existence of the work – even a sketch or a blueprint is not a future state, but rather a current
resource for action (Suchman 1987) – and what it seems to permit or not, with the inherent risk
of erring... Souriau’s philosophy can therefore be understood as having ethical overtones, although
the term is absent from his writing. He draws attention to the fragile character of each mode of
existence, which needs others to sustain it. It poses several questions: whether we are ready to
take up others’ actions in our own to allow them to continue; what aspects of them we deem
worthy or relevant to be taken up; and whether we are willing to take the risk of deteriorating
them. All these questions would be moot points if each existence were self-sufficient, and
dialogue was merely an exchange of information. It is precisely because each being needs to
exist more and in more diverse ways that we are solicited to make these existences happen and
that we are faced with an ethical dilemma. 4. Étienne Souriau and the different modes of existence According to the author,
"It is also to choose, to select, to discard. And each of these actions entails a judgment, which is
at once the cause, the reason, and the experience of this anaphor, of each moment in the
progressive coming together of two modes of existence” (Souriau [1943] 2015, 219). These
judgments and the differentiation they operate are crucial, first, because they are the conditions DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE 13 Souriau’s philosophy is also highly relevant to the study of dialogue, not the least because it
extends the notion of hospitality and radical openness to the Other (Derrida 2000; Levinas 1987). Souriau specifies that hospitality is not merely welcoming someone into your home but being
willing to take up their actions and promote their existence – i.e., care for them – and allowing
them to instrumentalize you so that their existence can continue as you take up their speech and
their action. Souriau’s philosophy is also highly relevant to the study of dialogue, not the least because it
extends the notion of hospitality and radical openness to the Other (Derrida 2000; Levinas 1987). Souriau specifies that hospitality is not merely welcoming someone into your home but being
willing to take up their actions and promote their existence – i.e., care for them – and allowing
them to instrumentalize you so that their existence can continue as you take up their speech and
their action. In suggesting that existence is scattered across several modes of existence, Souriau’s
work lies at the intersection of dialogue studies and recent proposals that communication is also
an embodied and material phenomenon (Ashcraft, Kuhn, and Cooren 2009; Cooren 2018). Through the materiality of communication, people tend to the things that matter to them, even
though they may be as seemingly immaterial as an idea (Bencherki et al. 2021). This suggests
that we need to expand our understanding of the hospitality inherent to dialogue (or that we want
to see in dialogue if we adopt a prescriptive view) to include different modalities of dialogue (on
multimodality, see e.g., Mondada 2018). DIALOGUE, SOLICITUDE, AND EXISTENCE While Souriau’s writing relies heavily on parallels with language, his modes of
existence are not limited to what exists through language. Even in a representational conception
of language, which assumes the independent existence of the objects that language designates,
talking or writing about these objects still provides them with an additional existence in dialogue,
where they also gain further meaning as participants to dialogue. For instance, when referring to
“my cat,” the speaker also designates the animal as worthy of her and her interlocutor’s
dialogical attention; it gains status as a pet and is embedded in relational and affective networks. DIALOGUE, SOLICITUDE, AND EXISTENCE 5. The everyday ethics of solving a problem in a work context So far, we have described such a view of the relationship between dialogue and the ethics
of promoting others’ existence, mostly in theoretical terms. However, its relevance has appeared
to us as we conducted our various empirical work. In that sense, an instrumental view of
dialogue is well-equipped to account for what goes on tangibly in dialogical situations and to
expand the notion of ethics to everyday situations. Too often, ethics is limited to moments of
“undecidability,” where decisions are impossible to make (see Derrida 1994). However, as
evidenced in medical contexts, ethical decisions are everyday issues, which is particularly
obvious once mistakes are made (see e.g., Barnes 2020). To illustrate this quotidian character,
we revisit a brief dialogue segment from a study by Frédérik Matte and François Cooren (2015). It consists of a conversation between two men supervising the construction of a building that is
part of a health center for Médecins sans frontières / Doctors without borders (MSF) in Bunia, in
the Democratic Republic of Congo. The excerpt below is our transcription and translation from
French of the original data, which the authors kindly made available to us. DIALOGUE, SOLICITUDE, AND EXISTENCE 14 For Matte and Cooren (2015), the excerpt illustrates how men can “read” their situation
and tell what it requires. The authors show that Fred completes Luc’s reading about how posts
might collapse with details on why construction norms were not respected. He thus shifts the
reading from Luc’s apparent assumption that Fred did not know the norms for health centers,
suggesting that local workers made a mistake. Matte and Cooren’s analysis tells how tensions
can be understood as alternative readings of the same situation, and dialogue is thus the space
where those readings are shared. Figure 1: Fred (left) and Luc look at the posts they talk about (anonymized video still). Figure 1: Fred (left) and Luc look at the posts they talk about (anonymized video still). We offer a new interpretation of the same data, showing that as they note a problem with
the way posts were planted into the ground, the two men also display concern for various beings
whose existence is at stake. Rather than supposing that the protagonists’ orient to an abstract
situation, our analysis lists the different elements that can be said to matter to them and that they
seem to promote in their talk. As we will see, in addition to expressing concern for each other,
Luc and Fred also show that they care for the building they are constructing, for construction
norms, MSF procedures, and for construction workers. Excerpt 1: Luc and Fred figure out why the posts were not concreted (from Matte and Cooren 2015) Excerpt 1: Luc and Fred figure out why the posts were not concreted (from Matte and Cooren 2015) 1 Luc
And the posts over there, did you guys concrete them or not? 1 Luc
And the posts over there, did you guys concrete them or not? DIALOGUE, SOLICITUDE, AND EXISTENCE 15 2
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No, they’re not concreted, uh. Yeah, that’s it. Because, you see, there it- it won’t last, a weight like that. At some point pffft ((gesturing with hand and imitating the noise of a post
falling)), it will still collapse for the— [for the
[Yep
(0.5 second pause)
For the- (0.5) Oh, yes, but for the health centers- ((answering to
himself)). But you don’t do the same thing for the health centers, you
do a=
=But they are concreted for the health centers. Here, it’s
not been done and it’s too late. Uh
This has been planned normally, [ budgeted
[ Is this true? Yeah, yeah, we were supposed to put them uh in cement
But why didn’t you guys do it then? Uh, because the- the- this wasn’t understood, you see, when I arrived, it
was- it was almost all planted, y’know. Hmm, hmm. Several people, contributions, and things are at play in this excerpt, and participants show
concern for many of them. First, Luc may appear to show concern for the “posts over there” by
wondering how they were planted into the ground (line 1). For Matte and Cooren (2015), his
reference to “you guys” can be read as blaming Fred and his team. It is, then, perhaps out of
solicitude for Fred, whom Luc does not want to offend, that he explains in lines 5 to 7 the
reasons behind his question: it is out of concern for the building and, therefore, for their joint
project – erecting the health center – that he asked about the posts. Luc, therefore, first seemingly
expresses concern for the posts, then perceives that his concern may offend Fred, and as a result,
clarifies that his concern has to do, in fact, with the joint project that preoccupies them. DIALOGUE, SOLICITUDE, AND EXISTENCE 17 switching to a passive construction (which is also the case in the French original) to say that “this
wasn’t understood” and that “it was almost all planted” before he had arrived, which stops short
of identifying who exactly failed to understand and who did the planting wrong. Fred postfaces
his turn of talk with “y’know,” which could be simply a language tic, but paired with Luc’s
apparent approval on line 33 – “Hmm, hmm” – may indicate that both men, in fact, implicitly
know who is to blame. The way Fred structured his last intervention may thus appear to display
some concern for saving the face of the people in question, presumably construction workers. This brief analysis shows that dialogue can be understood as a space where different
beings and things, both concrete and abstract, pursue their existence thanks to each other’s
solicitude. We saw that Luc worried about sparing Fred’s feelings and that both men discovered
they shared a concern for the health center and MSF’s procedures. Fred, in the end, also seemed
to be concerned with saving the face of the construction workers who presumably made the
blunder. Luc and Fred do not only talk about those beings – in fact, in the case of the
construction workers, they intently avoid talking about them. They also let their concern for
those beings shape how they act and talk. As we can see in Figure 1, the two men orient their bodies toward the posts (and the
construction site; off-camera to our left), and Luc gestures to materialize his concern for the
solidity of the future building (Figure 2). Our analysis shows that what Luc and Fred say, but
most importantly how they say it, is shaped by their desire to promote the existence of some
beings. They thus lend their voices to them. However, in agreement with what Souriau ([1956]
2015) suggested, they must also make a judgment and choose which beings matter the most to
them, given their joint project. For instance, while Luc may appear at first to care for the posts
and raise his question to Fred accusingly, he then seems to realize that his relationship with his
colleague matters too and to recast his question in light of their shared concern for the health
center. Excerpt 1: Luc and Fred figure out why the posts were not concreted (from Matte and Cooren 2015) DIALOGUE, SOLICITUDE, AND EXISTENCE 16 Figure 2: Luc explains that the posts might fall (lines 5-7). Figure 2: Luc explains that the posts might fall (lines 5-7). Fred’s only answer is a “Yep” (on line 9), followed by a pause. Possibly feeling that his
attempt to clarify that he cares for the project failed, Luc then starts explaining the MSF rules
concerning the construction of a health center, which require posts to be concreted. However,
even before Luc can explain this, Fred interrupts him to finish the sentence and remarks that it is
now too late (line 17=18). In doing so, Fred thus expresses that he is knowledgeable about MSF
procedures and his disappointment at the fact that they have been overlooked. In other words, he
shows that he does share Luc’s solicitude for the health center construction norms. This seems to
surprise Luc, who answers with only an “Uh” (line 20), triggering Fred’s further elaboration that
“This has been planned normally, budgeted” (line 22), confirming that he did, respect the
procedures, again leading to surprise from Luc and yet more elaboration from Fred: the posts
were supposed to be cast in cement (line 26). Fred and Luc thus discover that they share the
same concern for the health center being built correctly and for respecting MSF’s procedures and
the same disappointment at noticing that both of those concerns were breached. On line 28, Luc reiterates his surprise, asking Fred why he and his team did not put the
posts in cement if he knows and cares about the procedures. Fred’s answer on line 30 starts with
some hesitation. He seems to be about to designate someone or something – “the- the-” – before DIALOGUE, SOLICITUDE, AND EXISTENCE 6. Instrumental dialogue’s ethics of care Understanding dialogue as a form of mutual instrumentalization – i.e., acting and
speaking through others while letting them act and speak through our own body and voice – may
appear counter-intuitive. Current research looking to define the conditions under which dialogue
can operate to lead to ethical decisions advocates against instrumentality. However, as we have
shown in our illustrative analysis, participants in dialogue must consider, in shaping their
contributions, others’ need to pursue their existence. In doing so, they intuitively recognize
reciprocity in using each other’s instruments, namely their actions and voices and the space
granted to them. Without dialogue’s instrumental aspect, some existences would never see the
light of day, and some voices would not express themselves. In particular, some beings only exist
to the extent that others speak for them, such as procedures, principles, values, or allegories;
these are “solicitudinary” beings for Souriau ([1943] 2015), meaning that they depend on others’
solicitude. In that sense, if dialogical ethics consist of a concern for the Other, such concern
cannot be equated with total autonomy. Expecting the Other’s autonomy may amount to holding
them responsible for their failure to pursue their existence and hiding the fact that some beings,
need our help to act and exist. This is well understood, for instance, in medical ethics, when it
comes to patient autonomy vis-à-vis the expectation of letting them make their own choices
instead of accompanying them to make the best decision for themselves (Levy 2014). Caring for
the Other may mean instrumentalizing them and letting them instrumentalize us so that they can
pursue their existence through our voice and action, as autonomy always involves a part of
heteronomy (see also Cooren 2010). Such a view of dialogue also entails opening up the dialogue scene by not reducing it to
an encounter between a handful of people (thus extending Cooren 2008). Indeed, dialogue is
more than just the expression of privately held perspectives among a group of human beings
seeking to share understanding (e.g., Golob and Podnar 2014). DIALOGUE, SOLICITUDE, AND EXISTENCE However, both men realize that “it is too late,” consistent with Souriau’s idea that the
work can be promoted once it reaches a certain degree of existence but also risks being
deteriorated. In the case of the health center, they might have to start over. DIALOGUE, SOLICITUDE, AND EXISTENCE 18 DIALOGUE, SOLICITUDE, AND EXISTENCE 19 responsibility – this may mean that so-called stakeholders may be more numerous and of a
different kind than those usually presumed (thus extending current views of stakeholders
participating in dialogue, e.g., Morsing and Schultz 2006). From a different theoretical
perspective, namely actor-network theory, the fact that non-humans may have a stake in
controversies has already been empirically documented (Callon, Lascoumes, and Barthe 2009). Second, even human participants need each other. In our case, we saw that Luc adjusted his talk
to spare Fred’s feelings. Other studies have shown, for instance, that people ensured that others
could participate in the conversation (Bencherki and Snack 2016), helped each other formulate
their ideas in a different language (Bencherki, Matte, and Pelletier 2016), and offered
substantiation for each other’s suggestions (Bencherki et al. 2021). Therefore, we need to
recognize that dialogue is not only about passive hospitality but also about active solicitude:
people must concretely do and say things to help each other out. Viewing dialogue as instrumental and oriented towards solicitude also reconnects it with
theorizing on agency and authority. Studies on agency have shown that it does not stem from a
single individual; it is “without agents” (Bencherki, Brummans, and Vézy 2020; Choukah and
Theophanidis 2016). Instead, it should be understood as a “chain of agency,” where individuals
can present their actions as motivated or constrained by a prior link. Moreover, they can also
present their actions as motivating or constraining the following link downstream to act in a
certain way (Castor and Cooren 2006; see also Brummans 2018). Agency, then, is always hybrid
(Callon and Law 1995; Meunier and Vásquez 2008). When agency is recognized as hybrid,
authority is not something one person possesses. It stems from the way each situation configures
agency, where some people and things are positioned as motivating and constraining collective
action (see Benoit-Barné and Cooren 2009). An instrumental view of dialogue, thus, acknowledges that if dialogue is the site where
collective action is decided, then it must also ensure that all participating agencies are
considered. Observing how people configure the situations where they act and that, in return,
guide their actions provides us with a normative standard that is more secure and empirically
verifiable than the presumption that some abstract norm should guide the way dialogue unfolds
and the way we behave towards each other. 6. Instrumental dialogue’s ethics of care First, more beings and things can
participate in dialogue than the people who are sitting around the table: abstractions such as
procedures and building norms, objects such as posts, and projects and things that do not yet
exist, such as a health center, absent people such as the construction workers … all participate
too, at least to the extent that they make human beings say and do things to promote their
existence. In applied situations – for instance, in business ethics and corporate social DIALOGUE, SOLICITUDE, AND EXISTENCE DIALOGUE, SOLICITUDE, AND EXISTENCE In our view, as collective action is jointly authored,
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correspond to diverging understandings about what collective action should be or how best to
compose it. Dialogue, therefore, is not about escaping authority but rather ensuring that it is not
authority over the other, but rather authority with the other (see also Follett 1926). More generally, viewing dialogue as supporting the Other’s existence through mutual
instrumentalization means we must reconsider current research on the condition of productive
and ethical dialogue. This line of research could also explore how dialogue may support all the
beings and voices involved in it without making assumptions about what each of them needs to
pursue its existence. Much of the literature has suggested that genuine dialogue would avoid
control and possession; it would also involve respecting each other’s freedom of choice (Arnett
2016b; Johannesen 1971; Krippendorff 1989). These are undoubtedly essential considerations as
general rules. However, specific situations may include different beings with different needs: as
we have seen, some may need that we intervene and speak on their behalf as they are not able to
speak for themselves. This may be the case of those people who are invisibilized and who cannot
speak on their own behalf due to lack of credibility or insufficient legitimacy (Fricker 2007);
talking about them and for them is thus essential, as Spivak (1988) did in her work concerning
subalterns, so that, through the speech of others, they may gain some existence and visibility. Such dialogue may even offer them the space to speak for themselves, albeit through others. Therefore, we may need to reorient current research on the conditions of dialogue, move away
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Rural/Urban and Socioeconomic Differentials in Quality of Antenatal Care in Ghana
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Rural/Urban and Socioeconomic Differentials in Quality of Antenatal Care in Ghana
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Rural/Urban and Socioeconomic Differentials in Quality of Antenatal Care in Ghana
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Rural/Urban and Socioeconomic Differentials in Quality of Antenatal Care in Ghana
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University of California eScholarship.org eScholarship.org RESEARCH ARTICLE OPEN ACCESS OPEN ACCESS
Citation: Afulani PA (2015) Rural/Urban and
Socioeconomic Differentials in Quality of Antenatal
Care in Ghana. PLoS ONE 10(2): e0117996. doi:10.1371/journal.pone.0117996
Academic Editor: Nyovani Janet Madise, University
of Southampton, UNITED KINGDOM
Received: September 24, 2014
Accepted: January 3, 2015
Published: February 19, 2015
Copyright: © 2015 Patience A. Afulani. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Academic Editor: Nyovani Janet Madise, University
of Southampton, UNITED KINGDOM Results Urban residence and higher SES are positively associated with higher ANC quality, but the
urban effect is completely explained by sociodemographic factors. Specifically, about half
of the urban effect is explained by education and wealth alone, with other variables account-
ing for the remainder. The effects of education are conditional on wealth and are strongest
for poor women. Starting ANC visits early and attending the recommended four visits as
well as receiving ANC from a higher level facility and from a skilled provider are associated
with higher quality ANC. These factors partially explain the SES differentials. Data Availability Statement: All data are freely
available in the manuscript and supporting document
files. Data were obtained from a third party, Measure
DHS, and the data set can be downloaded from http://
dhsprogram.com/data/available-datasets.cfm after
submitting a request to the DHS program. Funding: The author received no funding for this
work. She was supported by the following
fellowships: the Bixby Doctoral Fellowship in
Population from the UCLA Bixby Center on
Population and Reproductive Health; the Celia and
Joseph Blann Fellowship from the UCLA School of
Public Health; and the UCLA Affiliates Scholarship
from the UCLA Graduate Division. These fellowship Methods The data are from the Ghana Maternal Health Survey (N = 4,868). Analytic techniques in-
clude multilevel linear regression with mediation and moderation analysis. Copyright: © 2015 Patience A. Afulani. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Rural/Urban and Socioeconomic Differentials
in Quality of Antenatal Care in Ghana Patience A. Afulani1,2* 1 Department of Community Health Sciences, Fielding School of Public Health, University of California, Los
Angeles, Los Angeles, California, United States of America, 2 California Center for Population Research,
Los Angeles, California, United States of America * pafulani@ucla.edu * pafulani@ucla.edu Background Approximately 800 women die of pregnancy-related complications every day. Over half of
these deaths occur in sub-Saharan Africa (SSA). Most maternal deaths can be prevented
with high quality maternal health services. It is well established that use of maternal health
services vary by place of residence and socioeconomic status (SES), but few studies have
examined the determinants of quality of maternal health services in SSA. The purpose of
this study is to examine the determinants of antenatal care (ANC) quality in Ghana–focusing
on the role of place of residence and SES (education and wealth). The analysis also exam-
ines the interactions of these variables and the mediating role of ANC timing, frequency, fa-
cility type, and provider type. Introduction Approximately 800 women die of pregnancy-related complications every day. Ninety-nine per-
cent of these deaths occur in developing countries, with Sub-Saharan Africa (SSA) accounting
for over half of the total [1,2]. Most maternal deaths can be prevented with high quality mater-
nal health services—antenatal, delivery, and postnatal care [3,4]. But previous efforts in SSA
have concentrated more on increasing the proportion of women who use these services, rather
than the quality of the services provided [5,6]. Although antenatal care (ANC) coverage levels
have increased substantially, maternal mortality rates remain high. One reason is that skilled
delivery care is still low; another reason is poor quality antenatal and delivery care [7–10]. The recommendations to reduce maternal mortality range from preconception to postpar-
tum interventions. But because most maternal deaths occur during delivery, skilled attendance
at delivery is critical to reducing maternal mortality [11]. Many women in SSA, however, do
not use skilled attendants during delivery. The reasons include poor access to health facilities
and some sociocultural factors that discourage use of health facilities for delivery. Qualitative
studies also suggest that perceived poor quality of health services deters women from using
skilled birth attendants, although there is little quantitative evidence to support this hypothesis
[12–14]. In addition to skilled delivery care, the World Health Organization recommends that all
pregnant women receive ANC at least four times in the course of their pregnancy [15,16]. However, evidence for the role of ANC in reducing maternal mortality is not consistent
[17,18]. Some researchers suggest the inconsistent findings may reflect inadequate attention by
researchers to the content of ANC and to problems of endogeneity [19]—i.e., the content of
ANC may affect use of ANC services, and where ANC is available, women who choose to use it
may be those less likely to experience maternal mortality for other reasons. While ANC alone
may be insufficient to reduce maternal mortality, antenatal care visits (ANCVs) are an oppor-
tunity to reach women with interventions (e.g., blood pressure monitoring, iron supplementa-
tion, tetanus vaccination, and education about the danger signs of pregnancy), which are
essential to both maternal and fetal health. ANCVs are also an opportunity to identify women
with pregnancy complications and to start the appropriate management. Additionally, during
ANCVs women can be educated and assisted in planning ways to access skilled care during de-
livery [15,20,21]. Differentials in Quality of Antenatal Care improve ANC quality in the lower level health facilities, where the most vulnerable women
are more likely to seek ANC. improve ANC quality in the lower level health facilities, where the most vulnerable women
are more likely to seek ANC. funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. Competing Interests: The author has declared that
no competing interests exist. Competing Interests: The author has declared that
no competing interests exist. Implications Ghanaian women experience significant disparities in quality of ANC, with poor illiterate
women receiving the worst care. Targeted efforts to increase quality of ANC may significant-
ly reduce maternal health disparities in Ghana and SSA. A particularly crucial step is to 1 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Introduction For instance, only about 24 percent of the poorest women deliver
with a skilled birth attendant, compared to over 95 percent of the richest women [27,28]. The
maternal mortality for Ghana also remains high, at 380 per 100,000 live births, despite efforts
by the Ministry of Health and other organizations to reduce it [2]. Like many countries in SSA,
Ghana is not on track to achieve the Millennium Development Goal number 5—to reduce the
maternal mortality ratio by three quarters between 1990 and 2015 [2,29]. The low use (as well
as late use) of skilled birth attendants is contributing to the high maternal mortality in Ghana. The other reason is poor quality of delivery services in some health facilities [30]. Little is
known about the quality of ANC that Ghanaian women receive or the factors associated it. But
studies elsewhere have found rural/urban and SES disparities in ANC quality, though none has
examined the factors underlying the disparities [19,22–24]. Disparities in the quality ANC may
therefore be contributing to the disparities in the use of skilled birth attendants and potentially
to the high maternal mortality in Ghana. known about the quality of ANC that Ghanaian women receive or the factors associated it. But
studies elsewhere have found rural/urban and SES disparities in ANC quality, though none has
examined the factors underlying the disparities [19,22–24]. Disparities in the quality ANC may
therefore be contributing to the disparities in the use of skilled birth attendants and potentially
to the high maternal mortality in Ghana. g
y
This study has two aims. The first aim is to identify the factors associated with the quality of
ANC that Ghanaian women receive. The second aim is to examine how social factors—urban/
rural residence and SES (education and wealth)—affect quality of ANC received. For this sec-
ond aim, I examine the interactions between the social factors, and the factors potentially ex-
plaining the social differentials. Drawing on existing literature, I hypothesize that higher SES
and urban residence will be associated with higher quality ANC. But I expect that the lower
SES of rural women will partly explain the rural/urban difference in quality of ANC. I also ex-
amine the reverse of this hypothesis—whether place of residence explains the SES difference in
quality of ANC. Introduction This implies that high quality ANC can help improve maternal outcomes: di-
rectly through preventative measures and early management of complications and indirectly
through increased use of skilled birth attendants. The determinants of use of antenatal and other maternal health services in SSA are well
known. Few quantitative studies have, however, examined the determinants of quality of ANC
or other maternal health care that women in SSA receive [22–24]. The paucity of studies on the
quality of maternal health care is primarily due to lack of data. Quality of care is difficult to
measure—it has multiple dimensions that are hard to capture in a few variables. Donabedian
identified three dimensions of quality of care: (1) structure: the physical and human resources
to provide care; (2) process: the technical and interpersonal aspects of delivering care; and (3)
outcomes: the effects of care [25]. For maternity care, Hulton et al. regrouped elements under
these dimensions into two broad categories: service provision and patient experience [26]. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 2 / 28 Differentials in Quality of Antenatal Care Service provision data may be obtained from health facility assessments, but such data are diffi-
cult, if not impossible, to link to individual women; and patient experience data necessarily re-
quires that we ask individual women. These factors coupled with the weakness of routine
systems for collecting health information means that we have to rely on household surveys in
which women are respondents. The nature of surveys, however, limits the detail and reliability
of data on quality of care because women may not be able to assess and report on the technical
aspects [6]. Nonetheless, household surveys provide the best population-level source of infor-
mation on the quality of ANC women receive; but few researchers have taken advantage of
them to examine social disparities. This paper uses data on antenatal service provision in the
Ghana Maternal Health Survey (a special supplement to the Ghana Demographic and Health
Survey (DHS)) to examine factors associated with the quality of ANC that women in
Ghana receive. More than 95 percent of women in Ghana go for at least one ANCV during pregnancy, and
about 80 percent attend the recommended four or more ANCVs. But only about two thirds of
women deliver with a skilled birth attendant, with wide disparities by place of residence and so-
cioeconomic status (SES). PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Ethics statement This study was granted an exemption under the University of California, Los Angeles Institu-
tional Review Board exemption category 4 for research involving the study of existing data. Data The data for this analysis are from the 2007 Ghana Maternal Health Survey (GMHS) [31]. The
GMHS was the first (and still is the only) nationally representative population-based survey to
collect comprehensive information on maternal morbidity and mortality in Ghana. The survey
was conducted by the Ghana Statistical Service and the Ghana Health Service, with technical
assistance from Macro International. The main aim of the GMHS was to generate data on ma-
ternal health and mortality for policymakers and the research community involved in the Re-
ducing Maternal Morbidity and Mortality Program. Data collection was implemented in two
phases. First, a short household questionnaire was administered in 227,715 households, which
were randomly selected from 1600 primary sampling units in urban and rural areas in the ten
administrative regions of the country. The goal was to identify female deaths between ages 12–
49 years. In the second phase, 400 clusters were randomly selected from the 1600 clusters in-
cluded in phase I. Households with women age 15–49 years were selected at random from
these 400 clusters, stratified by region and rural/urban residence. Institutional populations and
those residing in refugee camps were excluded. Verbal autopsies were completed for maternal
deaths in the selected households. A household questionnaire and a women’s questionnaire
was also administered in the selected households to collect information on demographic and
health indicators. This yielded 10,858 completed household interviews and 10,370 individual
interviews with women aged 15–49 years. Face-to-face interviews were conducted in English
and three major local languages: Akan, Ga, and Ewe. The response rate was 99% at the house-
hold level and 98% for the individual women. The GMHS is described in detail in the published
survey report [31]. The questions on antenatal care were asked of only women who had a birth (live or still
birth) in the five years preceding the survey (N = 5,088 = 49.1% of all women interviewed); this
is the base sample for this analysis. The analytic sample is however 5,042 women (99.1% of the
base sample) because 46 observations are missing on key study variables. The analysis is further
restricted to women who had at least one ANCV during their last pregnancy, since quality of
ANC cannot be measured for women who did not have an ANCV; 97% (N = 4,868) of women
in analytic sample had at least one ANCV. Introduction If SES explains most of the rural/urban difference, it suggests that higher quali-
ty services are more accessible to the higher SES women. On the other hand, if place of resi-
dence explains most of the SES difference, it suggests general poor quality of care in rural areas,
which is not greatly affected by a woman’s SES. I also examine conditional effects: whether the
effect of education and wealth differ by place of residence, and whether the effect of education
differs by wealth. This analysis will help identify women who are least likely to receive high
quality ANC. Finally, I examine the timing and frequency of ANCVs and the type of ANC fa-
cility and provider as potential mediating factors for the rural/urban and SES effects. This part
of the analysis will assess whether the disparities in quality of ANC by place of residence and
SES are due to differences in the use of antenatal services or to use of different types of facilities
and providers. 3 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Dependent variable: Quality of antenatal care Place of residence captures the general quality of services in the
area where each respondent lives, as well as other factors including the accessibility of health
services. The other variable related to place of residence is the region where one lives. I control
for region to capture unmeasured contextual factors. There are ten administrative regions in
Ghana, with Greater Accra region being home to Accra—the national capital of Ghana. SES refers to the social rank of an individual and her family in a particular community or so-
ciety. It incorporates economic status usually measured by income and/or wealth and social
status usually measured by education and/or occupation [36]. While some researchers use a
composite measure of SES, I use separate measures, because each may have a different effect
[37]. Including the individual components of SES also makes it easier to identify plausible ex-
planatory pathways and mechanisms through which SES affects health outcomes [37]. I opera-
tionalize SES in this analysis as education and wealth. I examine education both as a categorical
variable (highest level of education attained by respondent) and as continuous variable (years
of education attained), but I use years of education in the final models because the two specifi-
cations produce similar results. Years of education is top coded at 12 years of education, as
there are very few women with more than 12 years of education in the sample; and centered at
the sample mean to reduce collinearity with the interaction terms and to aid interpretation
[38,39]. Wealth is measured in quintiles. The quintiles were calculated from a wealth index
based on principal component analysis of variables on household assets conducted by the
group that produced the data files [40]. Potential mediating variables: Antenatal care timing, frequency, facility, and provider
There are other factors that may affect the quality of ANC that a woman receives indepen-
dent of where she lives and her SES. These include the timing of the first ANCV, the number of
ANCVs, the type of ANC facility, and the type of ANC provider. Women who start ANC in
the first trimester and attend the recommended four or more times are more likely to receive
all the essential ANC services, because contact with the health system begins earlier and is
more frequent. Dependent variable: Quality of antenatal care Dependent variable: Quality of antenatal care In this analysis, quality of ANC is defined as receipt of the recommended ANC services dur-
ing pregnancy. This definition is based on the definition of quality of care proposed by Dona-
bedian: the extent to which actual care is in conformity with present criteria for good care [32]. I created an additive index of responses to nine questions on ANC services that women re-
ceived during their last pregnancy. The services are: being weighed, blood pressure checked, a
urine sample taken, a blood sample taken, education received on signs of pregnancy complica-
tions, education received on where to go in the event of a complication, received or told to buy
iron supplements, received an anthelminthic, and received tetanus vaccination. Each question
has a binary response (1 = Yes; and 0 = No). Women were also asked if they had received a teta-
nus vaccination at any time before pregnancy, and how many times. Four tetanus injections 4 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care are required for full protection [33]. Thus, women who reported receiving at least four injec-
tions prior to the index pregnancy were coded as having received a tetanus injection, even if
they did not receive it during the index pregnancy. An exploratory principal components anal-
ysis (PCA) of these variables yielded one dominant factor. I, however, decided to use the addi-
tive index because of the problems of PCA with binary variables [34] and because the sum is
easier to interpret. Observations missing on one or more of the component variables were as-
sumed to be zero; no observations were missing on all the component variables. This approach
may underestimate the quality of care because women who did not know whether they received
a service are counted as having not received it. However, the number of cases included for this
reason is very small. The index ranges from zero to nine with responses spanning the entire
range; the mean is 7.4. Key independent variables: Place of residence and socioeconomic status Place of residence refers to whether the respondent lives in a rural or urban area. Urban
areas are defined as localities with 5000 or more persons, while rural areas are localities with
less than 5000 persons [35]. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Dependent variable: Quality of antenatal care Higher-tiered facilities like hospitals may also provide better quality ANC than
lower-tiered ones like health centers, because of better infrastructure and personnel; and pri-
vate health facilities may provide better quality care than public health facilities, although this
is more likely for interpersonal than technical quality [41,42]. In addition, the quality of care
received may differ depending on whether the provider is a doctor, nurse/midwife, or other
provider, because of different skill levels and access to amenities (e.g., laboratory services) [43]. Prior studies and preliminary analysis for this study show that place of residence and SES are
associated with both quality of ANC and ANC timing, frequency, type of facility, and type of
provider [19,22,23]. Thus, these factors may account for some of the effects of both place of PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 5 / 28 Differentials in Quality of Antenatal Care residence and SES on quality of ANC. Prior studies have combined timing and frequency of
ANCVs with the content of the ANCVs (the services received) to measure quality of ANC and
adequacy of ANC. Examining these factors separately is however important in assessing the
underlying causes of disparities. For the analysis, the number of ANCVs is dichotomized to ‘one to three’ and ‘four or more
visits,’ based on the number of ANCVs recommended by the World Health Organization [16]. The trimester of the first ANCV is coded as first, second, and third trimester, and don’t know. The categories for the type of ANC facility are: government hospital or polyclinic; government
health center, health post, or other lower tiered government health facility; private facility (in-
cluding maternity homes); and ‘other,’ representing ANC outside a facility, including the home
of the provider or the woman. The categories for type of ANC provider are: doctor; nurse or
midwife; and ‘other,’ representing unskilled providers (auxiliary nurse or midwife, traditional
birth attendant, or any provider other than a doctor, nurse or midwife). If a respondent re-
ceived ANC in more than one type of facility or from more than one type of provider, the high-
est level facility and most highly trained provider are used. Other independent variables Other factors that may influence the quality of care received include women’s risk for ad-
verse pregnancy outcomes. Dependent variable: Quality of antenatal care For instance, women who had prior pregnancy complications or
complications in the index pregnancy may receive higher quality care because health workers
may pay greater attention to them[13]. Women who know they are at higher risk for adverse
outcomes may also actively seek better care. In addition, a woman’s age, gravidity (number of
pregnancies), or parity (number of births) may influence the type of care she receives. I there-
fore control for age, gravidity, parity, having a prior stillbirth or miscarriage, having a sister
who died from maternal causes, experiencing any complication during the pregnancy (i.e. re-
ported signs or symptoms of hemorrhage, preeclampsia, eclampsia, infection, obstructed labor,
etc.), experiencing a serious complication during the pregnancy (i.e., a problem for which she
sought care), and the reason for antenatal care (whether for checkup or for a problem). Famil-
iarity with the health system may also influence the quality of care received because women
learn where to seek high quality care. I include two variables—ever used contraception and
knowledge of where to get contraception—as proxies for familiarity with the health system. In
addition, women’s status and autonomy may enable them to advocate within their families and
at health facilities to receive better quality services. SES captures autonomy and status, at least
in part. But I also control for marital status, age at first union, and sex of the household head
(female headed household or not) as other proxies for women’s autonomy [13]. Finally, I con-
trol for religion and ethnicity to capture sociocultural factors that may influence the quality of
care that a woman receives. Analytic approach Initial analyses involved descriptive statistics for the sample—means for continuous variables
and proportions for categorical variables. Next I examined the bivariate associations between
the independent variables and the dependent variable. The descriptive statistics and cross tabu-
lations are all weighted using the sample weights provided with the data to account for the
complex sampling design. I used t-tests to assess statistical significance for the mean differences
in quality of care for binary independent variables; analysis of variance (ANOVA) for non-bi-
nary categorical independent variables; and correlations for continuous variables. Chi-squared
tests are used to examine for differences by place of residence, educational level, and wealth
[44–46]. To account for the hierarchical nature of the data, I estimated bivariate and multivari-
ate multilevel linear regression models. The use of multilevel regression is necessary because 6 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care clustering at various levels or groups results in underestimation of the standard errors, such
that, when clustering is not accounted for there is a higher chance of finding significant differ-
ences, when in fact the differences are not significant [47,48]. The levels included in this analy-
sis are individual (level 1), cluster (level 2), and district (level 3). The weights provided with the
datasets are only for the individual level, and there is not enough information to construct
weights for use in the multilevel analysis. Furthermore, there is no clear consensus on the use
of weights in multilevel analysis within the field of statistics [49]. I therefore estimated un-
weighted multilevel models. The “xtmixed” command in Stata was used to estimate the multi-
level linear regression models with random intercepts [48,50]. I estimated a multilevel linear regression model for quality of ANC to identify the factors as-
sociated with quality of ANC—controlling for other factors and inter-district and inter-cluster
variation. To determine whether the effects of place of residence, education and wealth are con-
ditional on each other, I examined three interactions: place of residence and education, place of
residence and wealth, and education and wealth. I show only statistically significant interac-
tions, in this case only the education and wealth interaction. Analytic approach Mediation models, using the dif-
ference of coefficients (c-c’) method, were used to determine whether SES accounts for some of
the rural/urban difference (and the reverse), and whether the effects of both are through ANC
frequency, timing, facility, and provider (approach of the mediation analysis is illustrated in S1
Fig.) [38,39,51]. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Descriptive results Table 1 shows the weighted and unweighted distributions of the key variables (distributions for
full set of variables in S1 Table). About two-thirds of the women live in rural areas. All regions
of Ghana are represented in the sample, contributing between 8% and 13% except for the
Ashanti region (the most populous region), which makes up 19%, and Upper East and Upper
West regions (the least populous regions), which make up only 5% and 3% respectively. The
average number of years of education is about 5, with a range of 0 to 18 years. About a third of
the women have no formal education; 22% have only a primary education; 37% have middle or
junior secondary school (JSS) education; and less than one in ten women (7%) have completed
secondary or senior secondary school (SSS) (Middle and JSS are equivalent and secondary and
SSS are also equivalent—the names changed with changes in the educational system). The
household wealth index places women into five quintiles based on household assets; thus, there
are about 20% in each group. About three-quarters of the women identified as Christian; 18%
identified as Moslem and 9% as traditionalist, spiritualist, other, or have no religion. Akans, the
dominant ethnic group in Ghana, make up close to half of the sample (47%), with Ewes at 12%,
and Gas, Dangmes, and Guans at 9%. The average age is about 31 years; age ranges from 15 to 49 years—the target group for the
survey. Most women are currently married (72%); 14% are cohabiting. On average, the women
have between three and four children. Twenty percent had a prior adverse pregnancy outcome
(stillbirth or miscarriage); 20% had some pregnancy complication during their last pregnancy;
and 17% had a complication for which they sought help. Two percent had a sibling who died
from maternal causes. About two-thirds (62%) have ever used some type of contraception, and
about half (53%) know where to get contraception. The sample distribution is similar for the
full sample (i.e., including the 3% (N = 174) of women who did not receive any ANC during
pregnancy). The average number of services received by women who attended at least one ANCV is
7.4 (95% CI: 7.32 to 7.49), with a range of zero to nine; 10% received five or fewer services, and 7 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Weighted bivariate results The bivariate statistics for the key study variables are shown in Table 2 (full set of bivariate sta-
tistics in S2 Table). The results show small but significant differences in ANC quality by place
of residence, education, and wealth. On average, women who live in urban areas receive higher
ANC quality than those in rural areas (7.7 vs. 7.2). ANC quality increases with education and
wealth: the average ANC quality score among women with no education is 7.0, compared to
7.7 for those with middle/JSS or higher; and 6.9 among the poorest compared to 7.8 among the
richest. The ANC quality also differs significantly by the timing and frequency of ANCVs as
well as by the type of ANC facility and provider. The average quality score among women who
had less than four ANCVs is 6.6, compared to 7.6 for those who attended four or more times;
and 6.3 for women who started ANCVs in the third trimester, compared to 7.6 for those who
started in the first trimester. Women who received ANC in a government hospital or polyclinic
received higher quality ANC than those who did so in other facilities; but there is no difference
in ANC quality between women who received ANC in private facilities and those who received
it in lower level government health facilities. Also, women who received ANC from a doctor
had a small but significantly higher ANC quality than those who received ANC from a nurse
or midwife, which is also higher than that from a provider other than a doctor, nurse, or mid-
wife. There is no significant difference in ANC quality by reason for seeking care. The Western region has the highest ANC quality (8.3), and the Northern and Volta regions
have the lowest (6.7). There are no major differences between the other regions except for the
Greater Accra region, which surprisingly has the third lowest ANC quality—only higher than
Northern and Volta regions. By religion, women who identify as traditionalists, spiritualists,
other, or have no religion received the lowest quality ANC, followed by Moslems, but the
quality score for Moslems is not significantly different from that of Christians. There are no
significant differences in ANC quality by age, marital status, age at first union, sex of house-
hold-head, gravidity, parity, prior miscarriage or stillbirth, sibling maternal death, and any
pregnancy complication. Differentials in Quality of Antenatal Care Table 1. (Continued)
Unweighted
Weighted
Variables
N
%
proportion
[95% C.I]
Never married
345
7.1
0.071
0.062
0.080
Notes: ANC = Antenatal Care; GMHS = Ghana Maternal Health Survey (2007); JSS = Junior Secondary School; SSS = Senior Secondary School; CI =
Confidence interval; SD = standard deviation; DK = don’t know. Table 1. (Continued) Table 1. (Continued) Notes: ANC = Antenatal Care; GMHS = Ghana Maternal Health Survey (2007); JSS = Junior Secondary School;
Confidence interval; SD = standard deviation; DK = don’t know. about a quarter received all nine services. About 80% had four or more ANCVs, with an aver-
age of about six visits; and 55% started ANCVs in the first trimester. Eighty-five percent re-
ceived ANC in a government health facility, with roughly half in a hospital or polyclinic and
the other half in a health center or other lower tiered facility; 14% received ANC in a private fa-
cility. Most women (79%) received ANC from a nurse or midwife; 19% received some ANC
from a doctor; and less than 3% received ANC from an unskilled provider. Most women went
for the ANCV for a checkup; 17% went because of a problem. Differentials in Quality of Antenatal Care Table 1. Distribution of key study variables, Women who attended ANC at least once, GMHS, N = 4,868. Unweighted
Weighted
Variables
N
%
proportion
[95% C.I]
Setting
Rural
2,967
61.0
0.648
0.617
0.679
Urban
1,901
39.1
0.352
0.321
0.383
Highest Education
None
1,588
32.6
0.330
0.296
0.364
Primary
1,072
22.0
0.221
0.202
0.239
Middle/JSS
1,804
37.1
0.375
0.345
0.404
Secondary/SSS/or higher
404
8.3
0.075
0.063
0.086
Mean years education (SD)
4,868
5.2 (4.39)
5.130
4.815
5.444
Household wealth index
Poorest
1,024
21.0
0.207
0.177
0.236
Poorer
943
19.4
0.210
0.186
0.235
Middle
930
19.1
0.204
0.182
0.227
Richer
976
20.1
0.203
0.181
0.224
Richest
995
20.4
0.176
0.155
0.197
ANC variables
ANC quality of care score
Mean (SD)
4,868
7.4 (1.52)
7.406
7.322
7.490
No. of ANC visits
1–3 visits
990
20.3
0.202
0.184
0.221
Four or more
3,878
79.7
0.798
0.779
0.816
Mean (SD)
4,868
5.8 (2.75)
5.756
5.626
5.885
Trimester of first ANC visit
First trimester
2,688
55.2
0.549
0.529
0.568
Second trimester
1,992
40.9
0.413
0.396
0.431
Third trimester
181
3.7
0.036
0.030
0.042
Don't know
7
0.1
0.002
0.000
0.003
Where ANC took place
Gov't health facility only/combine
4,119
84.6
0.853
0.829
0.877
Gov't hospital or polyclinic
2,200
45.2
0.453
0.413
0.492
Other Gov't facility
1,919
39.4
0.400
0.361
0.439
Only Private facility/maternity home
703
14.4
0.140
0.116
0.164
Home/other/DK
46
0.9
0.007
0.005
0.010
Highest trained ANC provider
Doctor
1,006
20.7
0.194
0.176
0.213
Nurse
3,743
76.9
0.785
0.766
0.803
All others
119
2.4
0.021
0.015
0.026
Reason for seeking ANC
For checkup
4,044
83.1
0.831
0.817
0.846
For a problem/9missing
824
16.9
0.169
0.154
0.183
Mean Age in years (SD)
4,868
30.5 (7.34)
30.426
30.183
30.670
Marital status
Currently married
3,510
72.1
0.718
0.697
0.739
Cohabiting
666
13.7
0.141
0.125
0.157
Previously married
347
7.1
0.070
0.061
0.078
(Continued) Table 1. Distribution of key study variables, Women who attended ANC at least once, GMHS, N = 4,868. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 8 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Level Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses. ICC = Intraclass Correlation, calculated from the variances from the null model. Variation at the level of the individual = 1.361/(1.361+0.626+0.352) = 1.361/2.339 = 0.58
ICC at district level = 0.626/2.339 = 0.268; and at cluster level = 0.352/2.339 = 0.15. doi:10.1371/journal.pone.0117996.t003 Other bivariate analyses show very little variation in the proportion of women who had at
least one ANCV by place of residence, sociodemographic, and reproductive health variables,
with over 90% in all categories. More educated and wealthier women are less likely to live in
rural areas than less educated and poorer women. More educated, wealthier, and urban women
are also more likely to start ANCVs earlier, go for at least four ANCVs, receive ANC in a gov-
ernment hospital or polyclinic or a private facility, and less likely to receive ANC in a lower
level facility. They are also more likely to receive ANC from doctors (S3 Table). Weighted bivariate results But women who have ever used contraception, women who know
where to get contraception, and women who experienced a serious pregnancy complication re-
ceived slightly higher quality of care than their respective reference groups. 9 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care Table 2. Cross tabulation of selected variables by mean quality of antenatal care, GMHS, N = 4,868. Variable
N
Mean
[95% C.1]
Setting
Rural
2,967
7.24
7.13
7.36
Urban
1,901
7.71
7.62
7.79
Highest Education
None
1,588
7.03
6.88
7.17
Primary
1,072
7.36
7.24
7.47
Middle/JSS
1,804
7.71
7.61
7.80
Secondary/SSS/higher
404
7.74
7.58
7.89
Household wealth index
Poorest
1,024
6.85
6.65
7.05
Poorer
943
7.26
7.12
7.39
Middle
930
7.53
7.41
7.65
Richer
976
7.63
7.50
7.75
Richest
995
7.84
7.75
7.93
No. of ANC visits
1–3 visits
990
6.60
6.41
6.79
Four or more
3,878
7.61
7.54
7.68
Trimester of first ANC visit
First trimester
2,688
7.59
7.51
7.68
Second trimester
1,992
7.26
7.16
7.36
Third trimester
181
6.31
5.97
6.66
Don't know
7
5.97
4.92
7.02
Where ANC took place
Gov't hospital or polyclinic
2,200
7.73
7.65
7.81
Other Gov't facility
1,919
7.12
6.96
7.27
Only Private facility/maternity home
703
7.33
7.16
7.49
Home/other/DK
46
4.91
3.96
5.85
Highest trained ANC provider
Doctor
1,006
7.73
7.63
7.83
Nurse
3,743
7.35
7.25
7.44
All others
119
6.59
6.10
7.08
Reason for seeking ANC
For checkup
4,044
7.37
7.28
7.46
For a problem/9missing
824
7.57
7.45
7.70
Marital status
Currently married
3,510
7.42
7.34
7.51
Cohabitating
666
7.18
6.95
7.41
Previously married
347
7.52
7.27
7.77
Never married
345
7.58
7.39
7.76
Ever used contraception
No
1,780
7.01
6.88
7.15
Yes
3,088
7.64
7.57
7.71
Know family planning source
No
2,270
7.27
7.16
7.38
Yes
2,598
7.52
7.43
7.62
Notes: See Table 1 for abbreviations. d i 10 1371/j
l
0117996 t002 Table 2. Cross tabulation of selected variables by mean quality of antenatal care, GMHS, N = 4,868. 10 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care Table 3. Random effects from multilevel linear regression of quality of antenatal care on relevant independent variables, GMHS, N = 4,868. Level
Quality of ANC: variance (se)
ICC
No. of groups
Mean observations per group
Null model
Full unconditional model
Full conditional model
District—level 3
0.626***
0.276***
0.275***
0.268
110
44.3
(0.056)
(0.039)
(0.039)
Cluster—level 2
0.352***
0.238***
0.239***
0.15
400
12.2
(0.0336)
(0.036)
(0.036)
Individual—level 1
1.361***
1.305***
1.305***
(0.014)
(0.014)
(0.014)
N
4,868
4,868
4,868
Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses. ICC = Intraclass Correlation, calculated from the variances from the null model. Variation at the level of the individual = 1.361/(1.361+0.626+0.352) = 1.361/2.339 = 0.582. ICC at district level = 0.626/2.339 = 0.268; and at cluster level = 0.352/2.339 = 0.15. l linear regression of quality of antenatal care on relevant independent variables, GMHS, N = 4,868. Table 3. Random effects from multilevel linear regression of quality of antenatal care on relevant independ
Quality of ANC: variance (se) doi:10.1371/journal.pone.0117996.t003 Multilevel linear regression results Table 3 shows the variances from the random effects part of the multilevel analysis of quality of
ANC for the null, full unconditional, and full conditional models. The results from the null
model show that, though more than half (58%) of the variation in quality of care is at the indi-
vidual level, there is large variation at the district and cluster levels—Intraclass Correlations
(ICC) of 0.27 and 0.15 respectively. The large ICC at the district and cluster levels supports
the need for multilevel analysis [39,47]. The variances from the full models show that the inde-
pendent variables in the model explain much more of the group level variation than the indi-
vidual level variation; over half of the district level variation are explained by the variables in
the model. The second column of Table 4 shows the multilevel linear regression results from the unad-
justed models. After accounting for inter-cluster and inter-district variation, the individual
factors positively associated with higher quality ANC are: living in an urban area, higher educa-
tion, higher wealth, attending four or more ANCVs, going for the first ANCV in the first tri-
mester, receiving ANC from a doctor, receiving ANC in a government hospital or polyclinic,
experiencing a serious pregnancy complication, ever used contraception, knowledge of where
to get contraception, being Akan, and living in the Western region. The multivariate analyses with all the relevant independent variables in the model are
shown in the last two columns of Table 4. The full unconditional model shows that when other
factors are accounted for there is no significant rural/urban difference in ANC quality, but
both education and wealth are still significantly associated with ANC quality, although the ef-
fects are small. Each additional year of education increases the ANC quality score by about
0.02 points. On average, women with some education (any level) receive higher quality ANC PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 11 / 28 Differentials in Quality of Antenatal Care Table 4. Multilevel linear regression of ANC quality on relevant independent variables, GMHS, N = 4,868. Quality of ANC: b (se)
Bivariate
Full Multivariate models
Independent variables
Unconditional
Conditional
Urban residence
0.36***
0.084
0.084
(0.068)
(0.066)
(0.066)
Years of sch. centered
0.040***
0.018**
0.043**
(0.0054)
(0.0059)
(0.013)
Wealth (ref = poorest)
Poorer/Middle
0.26***
0.17**
0.073
(0.062)
(0.060)
(0.074)
Rich/Richest
0.55***
0.21**
0.13
(0.073)
(0.077)
(0.086)
Wealth & Education interaction
Poorer/Middle*yrs.sch.ca
-0.031*
(0.015)
Rich/Richest* yrs.sch.c
-0.027
(0.015)
Four or more ANC visits
0.78***
0.60***
0.60***
(0.051)
(0.055)
(0.055)
First ANC (ref = first trimester)
Second trimester
-0.24***
-0.086*
-0.088*
(0.041)
(0.042)
(0.042)
Third trimester
-1.01***
-0.50***
-0.50***
(0.11)
(0.11)
(0.11)
DK trimester
-2.26***
-1.76***
-1.76***
(0.52)
(0.50)
(0.50)
ANC provider (ref = Nurse
Doctor
0.17**
0.049
0.051
(0.054)
(0.053)
(0.053)
All others
-0.58***
-0.37**
-0.36**
(0.14)
(0.13)
(0.13)
ANC facility (ref = Gov't hosp/polyclinic)
Other Gov't facility
-0.34***
-0.23***
-0.22***
(0.051)
(0.051)
(0.051)
Only Private facility/maternity home
-0.29***
-0.31***
-0.31***
(0.065)
(0.061)
(0.061)
Home/other/DK
-2.20***
-1.84***
-1.85***
(0.21)
(0.21)
(0.21)
Serious complication
0.14**
0.21
0.22
(0.053)
(0.11)
(0.11)
Marital Status (ref = Currently married)
Cohabiting
-0.25***
-0.16*
-0.16**
(0.063)
(0.062)
(0.062)
Previously married
-0.048
-0.023
-0.029
(0.079)
(0.081)
(0.081)
Never married
-0.033
-0.014
-0.011
(Continued) l linear regression of ANC quality on relevant independent variables, GMHS, N = 4,868. Four or more ANC visits
0.78***
0.60***
0.60***
(0.051)
(0.055)
(0.055)
First ANC (ref = first trimester)
Second trimester
-0.24***
-0.086*
-0.088*
(0.041)
(0.042)
(0.042)
Third trimester
-1.01***
-0.50***
-0.50***
(0.11)
(0.11)
(0.11)
DK trimester
-2.26***
-1.76***
-1.76***
(0.52)
(0.50)
(0.50)
ANC provider (ref = Nurse
Doctor
0.17**
0.049
0.051
(0.054)
(0.053)
(0.053)
All others
-0.58***
-0.37**
-0.36**
(0.14)
(0.13)
(0.13)
ANC facility (ref = Gov't hosp/polyclinic)
Other Gov't facility
-0.34***
-0.23***
-0.22***
(0.051)
(0.051)
(0.051)
Only Private facility/maternity home
-0.29***
-0.31***
-0.31***
(0.065)
(0.061)
(0.061)
Home/other/DK
-2.20***
-1.84***
-1.85***
(0.21)
(0.21)
(0.21)
Serious complication
0.14**
0.21
0.22
(0.053)
(0.11)
(0.11)
Marital Status (ref = Currently married)
Cohabiting
-0.25***
-0.16*
-0.16**
(0.063)
(0.062)
(0.062)
Previously married
-0.048
-0.023
-0.029
(0.079)
(0.081)
(0.081)
Never married
-0.033
-0.014
-0.011
(Continued) (Continued) PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 12 / 28 Independent variables Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses. Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses. a yrs.sch.c. = Years of school centered. b Orthodox refers to Catholic/Methodist/Presbyterian. Other Christian refers to Pentecostals, charismatics, protestants, and other Christians. All Models also include age, parity, miscarried/stillbirth, any pregnancy complication, female headed household, age at marriage, and reason for ANC, but
these are not significant even in the bivariate models. There were however left were left in the model because their exclusion increased the size of the
coefficient for the other variables suggesting they may be playing a role and their exclusion will increase omitted variable bias. than those with no education (b = 0.12, p = 0.04 for primary education, b = 0.21, p<0.001 for
middle/JSS and b = 0.17, p = 0.053 for secondary education or higher; the differences beyond
primary education are not significant). Also, women in the higher wealth quintiles (poorer/
middle and richer/richest) receive significantly higher quality ANC than those in the lowest
wealth quintile (poorest), but the difference between women in the higher wealth quintiles is
not significant (b = 0.043, p = 0.469). The multivariate models also show that after accounting for other factors, number of
ANCVs, trimester of the first ANCV, type of ANC facility, and type of ANC provider are still
significantly associated with ANC quality. On average, women who had four or more ANCVs
score 0.60 more points on ANC quality than those who had less than four visits. Women who
went for the first ANCV in the first trimester also received significantly higher quality ANC
than those who went later. In the adjusted model, there is no difference in the quality of ANC
received from doctors and that from nurses/midwifes, but those who received ANC from an
unskilled provider received significantly lower quality ANC. Net of other factors, women who
received ANC in a government hospital or polyclinic received significantly higher quality ANC
than those who received ANC from any other type of facility. There is no significant difference
in the quality of ANC received in private facilities and that received in lower tiered government
facilities. In general, ANC quality received in any health facility is higher than that received
outside a health facility. On average, women who are cohabiting are less likely to receive high quality ANC than
those married. Differentials in Quality of Antenatal Care Table 4. (Continued) Table 4. (Continued) Quality of ANC: b (se)
Bivariate
Full Multivariate models
Independent variables
Unconditional
Conditional
(0.080)
(0.087)
(0.087)
Ever contraception
0.33***
0.21***
0.21***
(0.046)
(0.046)
(0.046)
Know family planning source
0.14***
0.14***
0.14***
(0.041)
(0.039)
(0.039)
Religion (ref = Orthodox Christian) b
Other Christian. -0.033
-0.026
-0.026
(0.051)
(0.049)
(0.049)
Moslem
0.026
0.20*
0.19*
(0.077)
(0.079)
(0.080)
Traditionalist /other
-0.28***
-0.022
-0.013
(0.083)
(0.080)
(0.080)
Ethnicity (ref = Akan)
Ga/Dangme/Guan
-0.21*
-0.040
-0.045
(0.085)
(0.081)
(0.081)
Ewe
-0.29***
-0.096
-0.099
(0.085)
(0.082)
(0.082)
Mole-Dagbani/Hausa
-0.32***
-0.19
-0.19
(0.096)
(0.10)
(0.10)
Grussi/Gruma
-0.31**
-0.14
-0.14
(0.098)
(0.097)
(0.097)
Other/4missing
-0.33***
-0.21*
-0.21*
(0.093)
(0.099)
(0.099)
Region (ref = Greater Accra)
Central
0.60*
0.58**
0.57**
(0.26)
(0.19)
(0.19)
Western
1.32***
1.38***
1.38***
(0.27)
(0.20)
(0.20)
Volta
-0.24
0.050
0.050
(0.27)
(0.20)
(0.20)
Eastern
0.32
0.42*
0.41*
(0.25)
(0.18)
(0.18)
Ashanti
0.69**
0.65***
0.65***
(0.25)
(0.18)
(0.18)
Brong Ahafo
0.70**
0.79***
0.78***
(0.26)
(0.19)
(0.19)
Northern
-0.44
0.010
0.0099
(0.27)
(0.20)
(0.20)
Upper East
0.59
0.93***
0.95***
(0.30)
(0.23)
(0.23)
Upper West
0.39
0.81***
0.83***
(0.32)
(0.24)
(0.24)
Constant
6.37***
6.45***
(0.22)
(0.22) Quality of ANC: b (se) PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 13 / 28 doi:10.1371/journal.pone.0117996.t004 Bivariate Independent variables Differentials in Quality of Antenatal Care Table 4. (Continued) Table 4. (Continued)
Quality of ANC: b (se)
Bivariate
Full Multivariate models
Independent variables
Unconditional
Conditional
N
4868
4868
4868
Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses. a yrs.sch.c. = Years of school centered. b Orthodox refers to Catholic/Methodist/Presbyterian. Other Christian refers to Pentecostals, charismatics, protestants, and other Christians. All Models also include age, parity, miscarried/stillbirth, any pregnancy complication, female headed household, age at marriage, and reason for ANC, but
these are not significant even in the bivariate models. There were however left were left in the model because their exclusion increased the size of the
coefficient for the other variables suggesting they may be playing a role and their exclusion will increase omitted variable bias. Differentials in Quality of Antenatal Care Fig 1. Quality of antenatal care by education and wealth. This is a graph from the interaction of education and wealth on quality of antenatal care. It shows
that quality of antenatal care increases with education, but the magnitude of this change is greatest among the poorest women. The graph also shows that
effect of wealth is only significant at low levels of education. Fig Fig 1. Quality of antenatal care by education and wealth. This is a graph from the interaction of education and wealth on quality of antenatal care. It shows
that quality of antenatal care increases with education, but the magnitude of this change is greatest among the poorest women. The graph also shows that
effect of wealth is only significant at low levels of education. doi:10.1371/journal.pone.0117996.g001 Collinearity diagnostics show no evidence of multicollinearity of the remaining variables in the
models. The mean VIF is 1.48, with the highest VIF for any variable being 3.10 for parity. Ex-
cluding parity from the models does not change the results. Moderation results The full conditional model in Table 4 shows the interaction between education and wealth. The coefficient for education in the conditional model is the effect of education among the
poorest women (the reference group for wealth). This shows that among the poorest women,
each additional year of education increases the ANC quality score by about 0.04 points. The co-
efficients for wealth in the conditional model are the effects of wealth at the average level of ed-
ucation (since education is centered at the grand mean). The statistically insignificant
coefficients suggest that at the average level of education there is no difference in ANC quality
by wealth. The coefficients for the interaction terms are the differences in the slopes for educa-
tion between women in the middle and poorest wealth quintiles and that between women in
the richest and poorest wealth quintiles. They show that the magnitude of the effect of educa-
tion on ANC quality among the poorest women differs from that of the middle wealth group,
but not from the richest group of women. The plot of the interaction in Fig. 1 illustrates these
results clearly. Fig. 1 shows the significant increase in ANC quality with education among the
poorest women, and the non-significant and marginally significant increase for women in the
middle and richest wealth groups respectively. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Independent variables Women who have ever used contraception and women who know where to get
contraception are also more likely to receive higher quality ANC than those who have never
used contraception and those who do not know where to get contraception respectively. Net of
other factors, Moslems received higher quality ANC than all the other religious groups. Also,
women in most regions (except Volta and Northern region) received higher quality ANC than
those in the Greater Accra region. Women in the Western region received the highest quality
ANC (b = 0.43, p = 0.037 for Western region compared to the Upper East region, which has
the next highest coefficient), but there are no differences between the other regions with better
ANC quality than the Greater Accra region. None of the pregnancy risk factors are significantly associated with quality of ANC in the
adjusted models. Having a serious pregnancy complication is associated with higher quality of
ANC, but this is only marginally significant (b = 0.22, p = 0.05). Age and parity are also not sig-
nificant determinants of ANC quality. Gravidity is not used in the final multivariate results be-
cause of collinearity with parity; it is however not significant when used instead of parity. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 14 / 28 Differentials in Quality of Antenatal Care Table 5. Mediation Models-Multilevel linear regression of ANC quality on place of residence, socioeconomic status, mediating, & other
variables. Quality of ANC: b(se)
Bivariate
Partial unconditional models (PUMs)
Full unconditional model
Independent variables
PUM1
PUM2
PUM3
PUM4
PUM5
PUM6
Urban residence
0.36***
0.15*
0.11
0.13
0.084
(0.068)
(0.061)
(0.069)
(0.068)
(0.066)
Years of sch. centered
0.040***
0.020***
0.018**
0.022***
0.019**
0.018**
(0.0054)
(0.0058)
(0.0059)
(0.0060)
(0.0060)
(0.0059)
Wealth (ref = poorest)
Poorer/Middle
0.26***
0.18**
0.17**
0.19**
0.17**
0.17**
(0.062)
(0.059)
(0.059)
(0.061)
(0.061)
(0.060)
Rich/Richest
0.55***
0.27***
0.24***
0.30***
0.24**
0.21**
(0.073)
(0.072)
(0.072)
(0.079)
(0.079)
(0.077)
Four or more ANC visits
0.78***
0.62***
0.62***
0.61***
0.60***
0.59***
0.60***
(0.051)
(0.055)
(0.055)
(0.055)
(0.055)
(0.056)
(0.055)
First ANC (ref = first trimester)
Second trimester
-0.24***
-0.087*
-0.086*
-0.084*
-0.085*
-0.097*
-0.086*
(0.041)
(0.042)
(0.042)
(0.042)
(0.042)
(0.042)
(0.042)
Third trimester
-1.01***
-0.50***
-0.49***
-0.50***
-0.50***
-0.53***
-0.50***
(0.11)
(0.11)
(0.11)
(0.11)
(0.11)
(0.11)
(0.11)
DK trimester
-2.26***
-1.77***
-1.76***
-1.77***
-1.76***
-1.95***
-1.76***
(0.52)
(0.50)
(0.50)
(0.50)
(0.50)
(0.51)
(0.50)
ANC provider (ref = Nurse)
Doctor
0.17**
0.056
0.051
0.057
0.050
0.074
0.049
(0.054)
(0.053)
(0.053)
(0.053)
(0.053)
(0.054)
(0.053)
All others
-0.58***
-0.37**
-0.38**
-0.38**
-0.37**
-0.29*
-0.37**
(0.14)
(0.13)
(0.13)
(0.13)
(0.13)
(0.14)
(0.13)
ANC facility (ref = Gov't hosp/p.clinic)
Other Gov't facility
-0.34***
-0.25***
-0.26***
-0.24***
-0.24***
-0.23***
-0.23***
(0.051)
(0.051)
(0.050)
(0.050)
(0.050)
(0.052)
(0.051)
Only Private/maternity home
-0.29***
-0.30***
-0.31***
-0.31***
-0.31***
-0.30***
-0.31***
(0.065)
(0.061)
(0.061)
(0.061)
(0.061)
(0.063)
(0.061)
Home/other/DK
-2.20***
-1.86***
-1.86***
-1.86***
-1.85***
-1.93***
-1.84***
(0.21)
(0.21)
(0.21)
(0.21)
(0.21)
(0.21)
(0.21)
N
4868
4868
4868
4868
4868
4868
4868
4868
Notes: *p<0.05, ** p<0.01, *** p<0.001. Standard errors in parentheses; All the multivariate models include the set of independent variables shown in
Table 4 except p
PUM 1 also excludes education and wealth; PUM 2 place of residence and wealth; PUM 3 excludes place of residence and education; PUM 4 excludes
place of residence; PUM 5 excludes number of ANC visits and trimester of first visit; PUM 6 excludes ANC facility and provider. education receive the lowest quality ANC, but education is most beneficial for the poorest with
regards to quality of ANC. Mediation results The results of the mediation analysis are shown in Table 5 (approach to mediation analysis
is illustrated in S1 Fig.). We also see the difference in ANC quality by
wealth at lower levels of education, but no difference by wealth at higher levels of education, in-
cluding at the average level (0 years of education for the centered variable); the difference be-
tween the middle and richest wealth groups are not significant even at lower levels of
education. In summary, the results from the conditional model show that poor women with no PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 15 / 28 doi:10.1371/journal.pone.0117996.t005 The first sets of coefficients are from the unadjusted models. The par-
tial unconditional models (PUMs) are the models with all the independent variables except 16 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care one or two independent variables of interest—the potential mediating variables. PUM1 in-
cludes all independent variables except education and wealth, and the coefficient for place of
residence is its total effect on ANC quality. PUM1 shows that, when SES is not accounted for,
women who live in urban areas receive significantly higher quality ANC than those who live in
rural areas. The coefficient for urban, however, decreases by about 58% from the bivariate
model ((0.36–0.0.15)/0.36 = 0.583), suggesting more than half of the unadjusted urban effect is
explained by the other independent variables in the model. When education and wealth are
added to the model (full unconditional model), the coefficient for urban decreases by almost
half (0.15–0.08 = 0.07) and is no longer significant. This difference is the rural/urban effect me-
diated by SES, and it is significant at p<0.001. The proportion of the total effect that is mediat-
ed is 0.47 (0.07/0.15 = 0.467). These results imply that close to half of the total effect of rural/
urban residence on ANC quality is through SES; and rural/urban residence has no significant
direct effect on ANC quality when other factors including SES are accounted for. PUM 2 includes all the independent variables except place of residence and wealth, and
PUM3 includes all the independent variables except place of residence and education. These
models show significant effects of both education and wealth net of other variables. PUM 4 in-
cludes all independent variables except place of residence. The change in the coefficients for
both education and wealth from PUM2 and PUM3 to PUM4 (their indirect effects) are very
small, but significant (p = 0.002). These results imply, a small amount of the effect of education
is through wealth, and the reverse, but a larger amount of their effects are independent of each
other. The full unconditional model also shows a significant direct effect of education and
wealth when place of residence is added to the model. The coefficients for education and the
poorer/middle wealth quintile do not change from PUM4; and the coefficient for the richer/
richest wealth quintile decreases by 13% (0.24–0.21/0.24 = 0.125), but this change is not signifi-
cant (p = 0.206). These results imply that the SES difference in ANC quality is not mediated by
place of residence. The mediation analysis therefore supports the hypotheses that SES partially
explains the rural/urban differences in ANC quality, but the reverse is not true—place of resi-
dence does not explain the SES difference in ANC quality. In PUM5 and PUM6, I examine how much of the rural/urban and SES effects are due to the
frequency and timing of ANCVs, and to the type of ANC facility and provider, respectively. PUM 5 includes all the independent variables except number of ANCVs and trimester of the
first ANCV; and PUM 6 includes all the independent variables except the type of ANC facility
and provider. The coefficient for urban residence is not significant in both PUM 5&6. This im-
plies that even when frequency and timing of ANCVs and the type of ANC facility and provid-
er are not accounted for, urban residence has no significant direct effect on ANC quality net of
demographic and socioeconomic factors. (The coefficient for urban is still not significant when
the ANC frequency, timing, facility and provider are all excluded from the model, but it is sig-
nificant in all the models when wealth and education are not in the model.) There is a decrease
in the coefficient for urban from PUM5&6 to the full unconditional model from 0.11 and 0.13
to 0.084; implying 23.6% and 35.6% of the urban effect may be mediated by the timing and fre-
quency of ANCVs and the ANC facility and provider respectively. But because the coefficients
for urban in these models are all not significant, we cannot say (with at least 95% confidence)
that the mediated effects are not due to chance. (The effect of rural/urban residence that is me-
diated by ANC timing and frequency, and ANC facility and provider are significant when
wealth and education are not in the model.) The differences in the coefficients for education and wealth from PUM5 to the final uncon-
ditional model, are their effects mediated by the frequency and timing of ANCVs. Sensitivity analysis Prior studies based on similar ANC quality measures dichotomized the ANC quality score to
create a binary outcome, and then used logistic regression analysis [20,23]. Mediation analysis
is however more complicated for binary outcomes, because the addition of variables to a logis-
tic model changes it’s scale, which makes it inaccurate to use the change in the magnitude of
the coefficients as the mediated effects [38,52]. Therefore, since the distribution of the ANC
quality variable in this analysis permitted its use as continuous variable, it was used as such in
linear regression. Also, multilevel mediation is much more developed for continuous outcomes
than categorical outcomes. Simulation studies show that though the standard errors from mul-
tilevel linear regression tend to be larger than those from single level regression; hence, a higher
likelihood of finding non-significant mediated effects (type II errors); the differences are mini-
mal [39]. But, there is less on how multilevel mediation with binary outcomes compares to that
from single level logistic regression. Nonetheless, to check if the findings are consistent for dif-
ferent specifications of the ANC quality variable, a binary variable was created from the ANC
quality score (coded 0—received zero to seven services–lower quality; and 1– received eight or
nine services– higher quality), and the full models examined with multilevel logistic regres-
sions. The findings from this analysis are consistent with that from the multilevel linear regres-
sion in terms of statistical significance and direction of the associations. Of note, however, is
that the interaction between education and wealth is not significant in the logistic model. In addition, because the weights provided with the datasets cannot be applied for multilevel
analysis, the models were run using weighted single level regressions as a sensitivity check. The
results from this are also similar to the unweighted multilevel analysis in the direction and signifi-
cance of the associations for most variables. The effect sizes for some of the independent variables
in the weighted linear regression are, however, slightly larger than that from the multilevel analy-
sis. In addition, a few variables such as having a pregnancy complication and a serious pregnancy
complication, which are not significant in the multilevel analysis, are significant in the single
level analysis. This is expected, since single level analysis may underestimate standard errors
when there is clustering [39,47]. Frequency
and timing of ANCVs account for about 18% of the effect of each additional year of education
(0.022–0.018/ 0.022 = 0.182) on ANC quality; 11% of the quality difference between women in 17 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care the poorest and middle wealth quintiles (0.19–0.17/0.19 = 0.105); and 30% of the quality differ-
ence between women in the poorest and richest wealth quintiles (0.30–0.21/0.30 = 0.30). These
mediated effects are all significant (p<0.001). The differences in the coefficients for education
and wealth from PUM6 to the final unconditional model are their effects mediated by the type
of ANC facility and provider. The type of ANC facility and provider account for about 5% of
the effect of each additional year of education (0.019-.018/0.019 = 0.053) on ANC quality and
13% of the quality difference between women in the poorest and richest wealth quintiles (0.24–
0.21/0.24 = 0.125). These mediated effects are significant (p<0.01). In summary, these results
suggest some of the SES differentials in ANC quality are because higher SES women start
ANCVs early and have more frequent ANCVs and receive ANC in higher level facilities (gov-
ernment hospitals and polyclinics) and from skilled providers. But there is a significant direct
effect of SES on ANC quality that is not through these factors. Sensitivity analysis The results from the single level linear regression model also
shows that the independent variables in the model explain about 23% of the variation in quality
of ANC (R-squared = 0.23, p<0.001). The sensitivity analyses therefore show that the findings
presented are robust, and at worst underestimate the effect of some of the factors. Factors underlying rural/urban and SES disparities in ANC quality I expected SES and the other factors to explain some, but not all, of the rural/urban difference
in quality of ANC. The quality of ANC is expected to be lower in rural areas because these
areas have less developed health infrastructure and fewer well trained health workers. Qualita-
tive studies in Ghana also suggest rural women are more likely to be treated poorly—even
when they seek care in urban facilities [53,54]. Studies in Nepal and Zambia using ANC quality
measures similar to those used in this study also found higher quality ANC in urban areas
[20,23]. A potential reason for the disparity from prior studies is that some of these studies con-
trolled for fewer variables or did not control for important mediating variables like frequency
and timing of ANCVs. The positive association between SES and quality of ANC are consistent with findings from
other studies on quality of primary health care services and family planning [41,42,53] as well
as those from qualitative studies on quality of delivery services [54–56]. The few other quanti-
tative studies on quality of ANC also found significant positive associations between SES and
ANC quality—despite some methodological differences [19,22,23]. Possible reasons for quality
of care differentials by SES include hypotheses that women with higher SES live in areas where
quality of care is generally higher; use health facilities that offer higher quality of care; can
physically access and afford high quality care; know what type of care to seek and are able to
advocate for it; have higher expectations of care and insist on it; and are more likely have a rela-
tionship with health personnel, which helps them acquire high quality services. The narrower
social power gap between high SES women and health personnel may also allow higher status
women to assert their preferences to obtain high quality care [12,13,41,42,53,57]. For quality of
ANC based on services received, the differential in quality of care by SES may also be due to
differential use of ANC services. These hypotheses have generally not been empirically exam-
ined, or even critically evaluated, because data on most of the mediating factors are usually not
available. Nevertheless, by carefully examining the associations, and how they vary in the pres-
ence of other factors, we may be able to tell which factors are predominant. Discussion Most women in Ghana go for ANC at least once during pregnancy, but many are not (or at
least do not remember) receiving the full components of ANC. The factors significantly 18 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care associated with higher quality ANC (controlling for other factors and clustering) include:
higher SES (education and wealth); starting ANCVs in the first trimester; attending four or
more ANCVs; receiving ANC from a government hospital or polyclinic; receiving ANC from a
doctor, nurse, or midwife; living in regions other than Greater Accra, Northern, and Volta re-
gions; ever used contraception; knowledge of where to get contraception; and being Moslem. Women who are cohabiting receive lower quality ANC than those currently married. Urban
residence is associated with higher quality ANC in the bivariate and partial models, but this ef-
fect is fully explained by the variables in the model. SES accounts for a significant proportion
the rural/urban effect. The SES effect is partly due to early initiation of ANCVs, more frequent
ANCVs, use of higher-level health facilities, and use of skilled providers. But there is a signifi-
cant direct effect of SES net of these factors. Wealth and education have independent effects on
ANC quality, but there is a moderation effect, with education most beneficial for the poorest. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Factors underlying rural/urban and SES disparities in ANC quality A reason for this may be that, in some facilities and among some providers, certain
services may be offered to all wealthier women, but not to poorer women, assuming that they
cannot pay. Education becomes important in this instance because, poor educated women may
be more likely to know what to expect, and so can actively seek it. Poor women with no educa-
tion are therefore most vulnerable: they may not be offered certain services because it is as-
sumed they cannot pay for it, and they do not ask for it because they do not know they are
required to receive it. Also, because of the wide power gap between health providers and poor
women with no education, they may be unable to assert their preferences even if they know
what to ask for [53]. On the other hand, the higher effect of wealth at very low levels of educa-
tion suggests wealthier women use facilities and providers that provide higher quality care, and
so it does not require any effort on their part to receive higher quality care. In this case, educat-
ed but poor women may know where to seek high quality, but affordable care. The non-signifi-
cant interaction of education and wealth in the binary logistic regression (in sensitivity
analysis) is potentially because the buffering by education is important for small changes in analysis) is potentially because the buffering by education is important for small changes in
quality, but unimportant for bigger changes—whether one received the highest quality of care
or not—as cost may be a bigger barrier to receiving the best care. Third, the findings support the hypotheses that women of higher SES receive higher quality
of care partly because they use facilities and providers that provide higher quality care. Higher SES women are more likely to receive ANC from government hospitals and poly-
clinics, where ANC quality is higher. They are also more likely to receive ANC from doctors,
who may provide higher quality ANC. Higher SES women are also more likely to receive ANC
from private health facilities. In this study, ANC quality in private facilities was the same as
that in health centers and other lower level government facilities, but lower than that in govern-
ment hospitals and polyclinics. But other studies suggest quality of care is higher in private fa-
cilities, though this is more for interpersonal than technical quality [42,57,60,61]. Factors underlying rural/urban and SES disparities in ANC quality First, that SES explains the rural/urban difference in ANC quality, and not the reverse, re-
duces support for the hypotheses that high SES women receive better quality of care because of
where they live. This does not mean the quality of care in one’s place of residence is unimpor-
tant; it means a woman’s SES, if high, may enable her to obtain better quality of care above
what is available in her immediate community. Women of higher SES are not limited to seek-
ing care where they live: they can go outside of their communities to access higher quality care. Women are willing to travel long distances to seek better health care if it is within their means
[12,56]. The finding may also mean that the quality of care received by women in the same PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 19 / 28 Differentials in Quality of Antenatal Care communities, and potentially within the same health facilities, is not uniform; such that
women of higher SES are able to obtain higher quality care than lower SES women in the same
communities and facilities. This can be because higher SES women know what services are
needed, and ask for these services. Higher quality care may also be more financially accessible
to higher SES women. For example, women may have to pay for lab tests during ANCVs, and
poorer women in the same facility may not get this service because of costs. This was the case
in Ghana until 2007 when a free maternal health policy was introduced. Since the implementa-
tion of the policy was very slow, most women who participated in the GMHS were subject to
some financial cost for antenatal services [58,59]. Second, if we assume that most of the effect of education is through knowledge of what to
receive and being able to ask for it, and that of wealth mostly through financial access. Then
the independent effects of education and wealth on ANC quality suggest independent roles of
knowledge, assertiveness, and financial access. But the steep increase in quality of ANC with
education among the poorest women, with little effect of education among those in the higher
wealth groups, also suggest knowledge and assertiveness are more important for the poorest
women. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Factors underlying rural/urban and SES disparities in ANC quality The findings
therefore suggest some selection by education and wealth into facilities that provide, or are
thought to provide, higher quality care. The significant mediated effect of education and wealth
on ANC quality through the type of ANC facility and provider provides further support for
this hypothesis. The small size of the mediated effect may be due to the opposite effects of the
facilities (government hospitals and private facilities) that more educated and wealthier women
use. (To explore these effects further, I examined wealth and type of ANC facility and provider
interactions to see if the effects of ANC facility and provider were conditional on wealth, but
these were not significant.) 20 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care Fourth, the mediation analysis with frequency and timing of ANC shows that some, but not
all, of the SES effect is due to differential use of ANC services. Women who go for the first
ANCV in the first trimester and attend the recommended number of ANCVs receive higher
quality ANC. This may be because, even though the ANC quality measure only captures basic
services that can be provided during the first ANCV, the services may not be available at all
times, such that, those who start early and go more frequently have a higher chance of receiving
them. Higher SES women are also more likely to start ANCVs early and attend more frequent-
ly, partly because the services are more accessible to them [12,13,20,23]. Thus, if the entire SES
difference in ANC quality was explained by differential utilization, then the SES difference
could be attributed mostly to differential access. The results however show that though fre-
quency and timing of ANCVs account for some of the SES difference in ANC quality, there is a
direct effect of SES net of these factors. This implies factors other than those related to reaching
ANC sites (as discussed above) are also contributing to the SES difference in ANC quality. This
finding is important because, it suggests factors operating within the health system may be
causing the disparities in quality of ANC. The few prior studies on quality of ANC have, how-
ever, not made this distinction. Factors underlying rural/urban and SES disparities in ANC quality For example, in Nepal, Joshi et al found higher education, higher wealth, and urban resi-
dence to be associated with higher quality ANC (based on a similar measure of ANC quality)
and higher frequency of ANCVs [23]. But they did not include frequency and timing of
ANCVs as predictors of ANC quality. Tran et al in Vietnam also found a positive association
between education and wealth and ANC adequacy (created from a combination of frequency,
timing, and content of ANCVs); thus, did not account for the effect of the timing and frequen-
cy on the content of ANC [22]. This analysis was stratified by rural and urban, and so did not
assess rural/urban differences, and wealth was only significant for rural areas. Because frequen-
cy and timing of ANC were not controlled for in these studies, it is unclear how much of the
SES and rural/urban differences in quality of ANC were due to differential utilization of ANC. Also, combining frequency, timing, and content of ANCVs into a composite measure does not
enable identification of the potentially different underlying reasons for differences in each of
those factors. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Other determinants of ANC quality One other notable finding in this analysis is that, on average, women in all regions of Ghana
(except Volta and Northern regions) received higher quality ANC than those in the Greater
Accra region; and women in the Western region received the highest quality ANC. This is un-
expected because, the Greater Accra region, which houses Accra—the national capital of
Ghana—has one of the two largest teaching hospitals in the country, and has more health facil-
ities and health personnel than any other region in the country [62,63]. Utilization of maternal
health services including use of skilled birth attendants is also higher than that in the other re-
gions [28]. Furthermore, it is more urban, hence has greater ease of reaching health facilities,
and has a larger proportion of high SES women [28]. The lowest quality of ANC in Northern
region can be easily explained: it has the lowest density of health workers and health facilities
in Ghana, and tend to have the poorest maternal health indicators in the country [28,62,63]. On the other hand, that Western region has the highest quality ANC is more difficult to ex-
plain, considering that it has the second lowest density of health workers in the country [63]. A
potential reason for the higher quality ANC in the other regions than Greater Accra region is
that: because access to health services are worse in the other regions, those who go for ANCVs
in these regions are a select group who are able to overcome the barriers to receiving better
quality care. This is however not very likely since over 95% of pregnant women in all regions PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 21 / 28 Differentials in Quality of Antenatal Care go for ANC at least once. Another potential reason is the large number of private health facili-
ties in the Greater Accra region, which may be providing less than optimal ANC. This is plausi-
ble considering that Western, Upper East, and Upper West regions, which have fewer private
facilities, appear to be doing better. The lower quality of ANC in Accra may also point to poor
quality ANC in the peripheral health facilities, which are overshadowed by the presence of the
teaching hospital in the region. This is plausible considering the number of mismanaged cases
that are referred to the teaching hospital. Other determinants of ANC quality To my knowledge no study has examined regional
variations on quality of ANC in multivariate analysis for Ghana, thus there are no studies to
compare. This finding presents an area for further studies to identify the factors that account
for the regional variations in ANC quality in Ghana. The positive effects of use of contraception and knowledge of where to get contraception on
ANC quality are likely due to familiarity with the health system. But it is unclear why Moslem
women receive higher quality ANC than Christians, when other factors are controlled for, and
why women who are cohabiting receive lower quality ANC than those currently married. A
possible reason is that Moslem women are more able to advocate for themselves for better qual-
ity care, but this effect is suppressed because of their lower SES, and only emerges when we ac-
count for SES. For the effect of cohabitating, a potential reason is stigmatization in a country
where unmarried women who are pregnant are often frowned upon. This explanation is how-
ever more plausible for interpersonal quality of care, which is not adequately captured by the
measure of ANC quality used in this analysis. Prior studies on quality of ANC have not ade-
quately examined marital status and religion; those that did, found no significant effects of
marital status and religion on quality of ANC [19,64]. Further studies are needed to understand
the findings from this analysis. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Limitations and strengths There are a number of limitations to this study. First, the measure of quality of ANC only cap-
tures service provision. The questions in the GMHS are useful for evaluating whether or not
women are receiving the essential ANC services, but they do not capture the experience of
women with the health system—how they are treated and the nature of the interactions with
health providers. Until recently, quality of maternal health care, and particularly patient experi-
ence of care, has received relatively little attention in most of SSA [5]. Data on patient experi-
ence are not collected in the surveys that serve as the major sources of maternal health data in
SSA. This is potentially because there are no well validated instruments that can be easily incor-
porated into these surveys. Quantitative data on patient experience are however important, be-
cause qualitative studies suggest that poor attitudes of health workers are a major barrier to use
of maternal health services. These studies have also suggested differential patient experience of
care by SES and place of residence [53–56], which may even be bigger than those related to
services received. The measure of quality of ANC also has some limitations even as a measure service provi-
sion. For instance, we expect it to capture some dimensions of structure (human and physical
resources) and process (mostly the technical aspects), as a minimum of these is required to pro-
vide services. But the questions asked are limited in discriminating between basic and more ad-
vanced infrastructure and technical expertise. They are also insufficient to determine if women
who had various screening tests were adequately followed up and appropriately managed. For
example, there are instances where women present in labor with the following: severe anemia
because a diagnosis of mild anemia early in the pregnancy was never reassessed and managed;
sickling crises because a positive sickling test during ANC was never followed up with a hemo-
globin electrophoresis (to confirm sickle cell disease or sickling trait); or eclampsia because a 22 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care moderately high blood pressure was not reassessed and managed. A blood sample may also be
used for one test without running other essential tests (e.g., an HIV test, but not a hemoglobin
test). Limitations and strengths The current measure of quality of ANC will however group all these women as having re-
ceived high quality ANC. Thus, asking if a woman had the services listed at least once during
pregnancy is limited in discriminating between different levels of quality. These limitations are
however difficult to address in the absence of clinical assessments and documentation, paired
with maternal recall. Thus, the limitations discussed should not undermine the findings pre-
sented here, but they suggest that the high score on the quality index should not be taken as an
indication of high quality of ANC in Ghana; and the magnitude of the disparities in the quality
of ANC are potentially larger than shown in this analysis. The limitations also call for greater
efforts to develop, and validate better measures of quality of antenatal (and also delivery)
care—which capture both service provision and patient experience—and incorporate them
into the major health surveys in developing countries. Another limitation of the study is that it is based on cross-sectional data, which limits
causal inference. Recall and social desirability bias are also potential limitations, since the
data are based on self-report. The period of recall, which may be up to five years for some,
could affect the precision of reporting the services received. Other studies have however sug-
gested women have relatively good recall of maternal events in this time window [19,65]. On
the problem of social desirability, women may report they received the services because they
know they are expected to have received them, which may lead to overestimation of the qual-
ity of care. This may also be more likely for some groups of women than others. The omission
of variables that are related to the dependent and key independent variables from the analysis
may lead to omitted variable bias, hence problems of endogeneity and unobserved heteroge-
neity [38,45]. Even though several factors were controlled for, this cannot be completely
ruled out. The other source of endogeneity—simultaneity or reverse causation—is less of a
problem for the focal relationships, as it is highly unlikely that ANC quality will lead to one’s
education, wealth, or place of residence. The reverse is however more plausible, which in-
creases confidence in causal inference based on the temporal ordering of the events. PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Conclusions This study finds that majority of women interact with the health system at least once during
pregnancy, but the quality of ANC they receive is suboptimal, especially for women of low SES. Differential utilization of ANC services accounts for some of the SES disparities in ANC quali-
ty, but there is a significant effect of SES net of ANC utilization. Health system factors partly
account for the SES disparities in ANC quality, including differential quality of care in different
types of health facilities and potentially within health facilities for different groups of women. Th
f
d
h
b
f
l
ff This study finds that majority of women interact with the health system at least once during
pregnancy, but the quality of ANC they receive is suboptimal, especially for women of low SES. Differential utilization of ANC services accounts for some of the SES disparities in ANC quali-
ty, but there is a significant effect of SES net of ANC utilization. Health system factors partly
account for the SES disparities in ANC quality, including differential quality of care in different
types of health facilities and potentially within health facilities for different groups of women. These findings have a number of implications. First, efforts to encourage women to start
ANCVs early and attend the recommended number of times should be continued. But there is
greater need to increase the quality of the interaction that women have with the health system
during pregnancy: through efforts to provide high quality ANC to all pregnant women as well
as targeted efforts to ensure poor illiterate women are also receiving high quality ANC. Since
low SES women are more likely to use the health centers and other lower level health facilities,
improving the quality of care provided in these facilities will help reduce the SES disparities in
quality of care. A first step towards this will be to provide the basic equipment needed to pro-
vide the essential ANC services in these facilities. A second step is refresher trainings for pro-
viders at these facilities: to remind them of the essential components of ANC, why they need to
provide specific antenatal services, and how they can provide the services efficiently. Effective
monitoring and supervision to ensure the right things are being done should follow these activ-
ities. Monitoring and supervision should also be extended to private facilities to ensure they are
providing effective ANC. Limitations and strengths Prob-
lems of simultaneity may be bigger if one is estimating the effect of variables such as
frequency of ANCVs, as the quality of care one receives during an initial visit may influence
the decision to go for subsequent visits [19]. Further analysis on a restricted sample—women
who attended at least four ANCVs—however showed that the potential endogeneity of
ANCVs has little effect on the key relationships. This study has several strengths. First, it addresses a gap in the maternal health literature,
which is the dearth of quantitative studies on the determinants of quality of maternal health
services in SSA. Second, it uses a nationally representative sample of women who had a birth
(live or stillbirth) in the five years preceding the survey, hence has high generalizability. Unlike
analysis based on the usual DHS, which will include only women with a live birth (the group
that are asked the maternal health questions), the inclusion of all women with a birth in the
preceding five years reduces the chances of excluding women who received the worst care, as
these women may be more likely to have a stillbirth. The restriction of the sample to women
that went for at least one ANCV, though necessary for the analysis, may reduce the generaliz-
ability of the findings. But this represents over 95% of women in Ghana. In addition, most of
the factors that predict the quality of ANC also predict having at least one ANCV. Thus, the ef-
fects found in this study may be larger in a sample that also includes women who did not have
any ANC. The analysis also uses rigorous methods and the sensitivity analysis shows the find-
ings are robust. 23 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care Conclusions In addition, there should be efforts within health facilities to ensure
low SES women are not receiving lower quality of care because of cost, lack of knowledge, lack
of assertiveness, or other reasons. These interventions should be paired with efforts to attract,
retain, and motivate qualified personnel in the lower level health facilities. This analysis also
adds voice to calls to improve women’s SES, especially through education. Finally, that many
women are not receiving optimal ANC suggests that this may be contributing to the low use of
skilled birth attendants and poor maternal outcomes, despite the high antenatal attendance. The SES disparities in quality of ANC may also be contributing to the SES disparities in the use
of skilled birth attendants. These hypotheses require further studies. Nonetheless, targeted ef-
forts to increase ANC quality will help improve maternal health and reduce maternal health
disparities in Ghana and SSA. References 1. WHO, UNICEF, UNFPA, the World Bank (2012) Trends in maternal mortality: 1990 to 2010. WHO. Available: http://www.who.int/reproductivehealth/publications/monitoring/9789241503631/en/index. html. Accessed 2013 July 5. doi: 10.1007/s12070-012-0514-9 PMID: 25621271 2. WHO, UNICEF, UNFPA, The World Bank, United Nations Population Division (2014) Trends in mater-
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1091–1110. PMID: 8042057 13. Gabrysch S, Campbell OM (2009) Still too far to walk: Literature review of the determinants of delivery
service use. BMC Pregnancy Childbirth 9: 34. Available: http://www.biomedcentral.com/1471-2393/9/
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atic review. Reprod Health 10: 40. doi: 10.1186/1742-4755-10-40 PMID: 23962135 15. World Health Organization U (2003) Antenatal care in developing countries. Supporting Information S1 Fig. Illustrating approach to mediation analysis using the difference of coefficients
method. (DOCX)
S1 Table. Sample Distribution, Ghana Maternal Health Survey. (DOCX)
S2 Table. Cross tabulation of all study variables by mean quality of antenatal care
(DOCX)
S3 Table. Cross tabulation of key study variables by place of residence, education, and
wealth. (DOCX) S1 Fig. Illustrating approach to mediation analysis using the difference of coefficients
method. (DOCX) S1 Table. Sample Distribution, Ghana Maternal Health Survey. (DOCX) S2 Table. Cross tabulation of all study variables by mean quality of antenatal care
(DOCX) S3 Table. Cross tabulation of key study variables by place of residence, education, and
wealth. (DOCX) 24 / 28 PLOS ONE | DOI:10.1371/journal.pone.0117996
February 19, 2015 Differentials in Quality of Antenatal Care Acknowledgments I thank MEASURE DHS for granting me access to the data set. I am very grateful to Anne Peb-
ley for her suggestions and comments on the analysis and editing of this paper, and to Gail
Harrison and all the members of my doctoral committee for their suggestions and comments
on my dissertation proposal—from which this paper has emerged. I would also like to acknowl-
edge Joseph Asunka for editorial support, Cheryl Moyer for comments on an earlier version of
the paper, and the PLOS ONE reviewers for their comments. Author Contributions Conceived and designed the experiments: PA. Analyzed the data: PA. Contributed reagents/
materials/analysis tools: PA. Wrote the paper: PA. PLOS ONE | DOI:10.1371/journal.pone.0117996
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heapol/czp017 PMID: 19401360 28 / 28
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Supplementary Table 2: Primers used for RT-PCR
Gene name
Forward primer 5'-3'
Reverse primer 5'-3'
PPP1CA
GCTGACAGAGAACGAGAT
CTTGATCTTATAGGCCAGC
CSNK2A1
CAGTTGGTGAGGATAGCC
GTGGTCATATCGCAGCAG
ATP5G1
CCAGACGGGAGTTCCAGAC
GACGGGTTCCTGGCATAGC
AGPAT1
TGTCCCCATAGTCATGTCCTC
GGAAAACAGTGAGCATGGAGT
RPL 35A
TTAAATCCTTGAGGGGTACA
ACGGGACTTCTAAAAGGAAC
GARS
TCCTCTGTTTGAAGGGCAAG
AAATCAGGCAGCCACTGAAG Supplementary Table 2: Primers used for RT-PCR
Gene name
Forward primer 5'-3'
Reverse primer 5'-3'
PPP1CA
GCTGACAGAGAACGAGAT
CTTGATCTTATAGGCCAGC
CSNK2A1
CAGTTGGTGAGGATAGCC
GTGGTCATATCGCAGCAG
ATP5G1
CCAGACGGGAGTTCCAGAC
GACGGGTTCCTGGCATAGC
AGPAT1
TGTCCCCATAGTCATGTCCTC
GGAAAACAGTGAGCATGGAGT
RPL 35A
TTAAATCCTTGAGGGGTACA
ACGGGACTTCTAAAAGGAAC
GARS
TCCTCTGTTTGAAGGGCAAG
AAATCAGGCAGCCACTGAAG Supplementary Table 2: Primers used for RT-PCR
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PCA-based bootstrap confidence interval tests for gene-disease association involving multiple SNPs
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* Correspondence: jinghua.zhao@mrc-epid.cam.ac.uk; xuefzh@sdu.edu.cn
1Department of Epidemiology and Health Statistics, School of Public Health,
Shandong University, Jinan 250012, PR China
2MRC Epidemiology Unit, Institute of Metabolic Science, Addenbrooke’s
Hospital, Cambridge CB2 0QQ, UK © 2010 Peng et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. METHODOLOGY ARTICLE Open Access Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 PCA-based bootstrap confidence interval tests for
gene-disease association involving multiple SNPs Qianqian Peng1, Jinghua Zhao2*, Fuzhong Xue1* Abstract Background: Genetic association study is currently the primary vehicle for identification and characterization of
disease-predisposing variant(s) which usually involves multiple single-nucleotide polymorphisms (SNPs) available. However, SNP-wise association tests raise concerns over multiple testing. Haplotype-based methods have the
advantage of being able to account for correlations between neighbouring SNPs, yet assuming Hardy-Weinberg
equilibrium (HWE) and potentially large number degrees of freedom can harm its statistical power and robustness. Approaches based on principal component analysis (PCA) are preferable in this regard but their performance varies
with methods of extracting principal components (PCs). Results: PCA-based bootstrap confidence interval test (PCA-BCIT), which directly uses the PC scores to assess gene-
disease association, was developed and evaluated for three ways of extracting PCs, i.e., cases only(CAES), controls
only(COES) and cases and controls combined(CES). Extraction of PCs with COES is preferred to that with CAES and
CES. Performance of the test was examined via simulations as well as analyses on data of rheumatoid arthritis and
heroin addiction, which maintains nominal level under null hypothesis and showed comparable performance with
permutation test. Conclusions: PCA-BCIT is a valid and powerful method for assessing gene-disease association involving multiple
SNPs Conclusions: PCA-BCIT is a valid and powerful method for assessing gene-disease association involving multiple
SNPs. PCA-BCIT is a valid and powerful method for assessing gene-disease association involving multiple p
Methods of extracting PCs The ith PC of controls is calculated by The ith PC of controls is calculated by Potentially, PCA can be conducted via four distinct
extracting strategies (ES) using case-control data, i.e., 0. Calculate PC scores of individuals in cases and controls
separately (SES), 1. Use cases only (CAES) to obtain
loadings for calculation of PC scores for subjects in both
cases and controls, 2. Use controls only (COES) to
obtain the loadings for both groups, and 3. Use com-
bined cases and controls (CES) to obtain the loadings
for both groups. It is likely that in a case-control asso-
ciation study, loadings calculated from cases and con-
trols can have different connotations and hence we only
consider scenarios 1-3 hereafter. More formally, let (X1,
X2, ..., Xp) and (Y1, Y2, ..., Yp) be p-dimension vectors of
SNPs at a given candidate region for cases and controls
respectively, then we have, F
l Y
l Y
l Y
i
C
i
i
ip
p
1 1
2
2
(11) (11) Background The ith
PC for cases is calculated by where CXX is the correlation matrix of (X1, X2, ..., Xp),
l
l
l
l
i
i
i
ip
1
1
1
2
1
1
(
,
,
,
)
and l l
i
i
1 1 = 1, i = 1,2, ..., p. The ith
PC for cases is calculated by less computer-intensive than haplotype-based methods. Studies have shown that PCA-LRT is at least as power-
ful as genotype- and haplotype-based methods[7,16,17]. Nevertheless, the power of PCA-based approaches vary
with ways by which PCs are extracted, e.g., from geno-
type correlation, LD, or other kinds of metrics[17], and
in principle can be employed in frameworks other than
logistic regression[7,16,17]. Here we investigate ways of
extracting PCs using genotype correlation matrix from
different types of samples in a case-control study,
while presenting a new approach testing for gene-dis-
ease association by direct use of PC scores in a PCA-
based bootstrap confidence interval test (PCA-BCIT). We evaluated its performance via simulations and
compared it with PCA-LRT and permutation test using
real data. F
l X
l X
l X
i
D
i
i
ip
p
1
1
1
2
1
2
1
(4) F
l X
l X
l X
i
D
i
i
ip
p
1
1
1
2
1
2
1
(4) and for controls and for controls F
l Y
l Y
l Y
i
C
i
i
ip
p
1
1
1
2
1
2
1
(5) (5) C
l
l
YY i
i
0
(6) C
l
l
YY i
i
0 (6) where CYY is the correlation matrix of (Y1, Y2, ..., Yp). The ith PC for controls is calculated by F
l Y
l Y
l Y
i
C
i
i
ip
p
1 1
2
2
(7)
And for cases, the ith PC, i = 1,2, ..., p, is calculated by F
l Y
l Y
l Y
i
C
i
i
ip
p
1 1
2
2
(7) (7) Methods
PCA cases, the ith PC, i = 1,2, ..., p, is calculated by And for cases, the ith PC, i = 1,2, ..., p, is calculated by Assume that p SNPs in a candidate region of interest
have coded values (X1, X2, ..., Xp) according to a given
genetic model (e.g., additive model) whose correlation
matrix is C. PCA solves the following equation, F
l X
l X
l X
i
D
i
i
ip
p
1
1
2
2
(8) F
l X
l X
l X
i
D
i
i
ip
p
1
1
2
2
(8) Strategy 3 (CES): Strategy 3 (CES): Cl
l
i
i
0
(1) (1) Cl
l
i
i
0 Cl
l
i
i
0
(9) (9) where
l li i = 1, i = 1,2, ..., p, li = (li1, li2, ..., lip)’ are
loadings of PCs. The score for an individual subject is where C is the correlation matrix obtained from the
pooled data of cases and controls,
l
l
l
l
i
i
i
ip
(
,
,
,
)
1
2
and
l l
i
p
i
i
1
1 2
,
, ,
,
. The ith PC of cases is calcu-
lated by F
l X
l X
l X
i
p
i
i
i
ip
p
1
1
2
2
1 2
,
, ,
, ,
(2) (2) where cov (Fi, Fj) = 0, i ≠j, and var(F1) ≥var(F2) ≥... ≥var(Fp). F
l X
l X
l X
i
D
i
i
ip
p
1
1
2
2
(10) (10) Background conservative[7]. It is therefore necessary to seek alterna-
tive approaches which can utilize multiple SNPs simul-
taneously. The genotype-wise Armitage trend test is
appealing since it is equivalent to the score test from
logistic regression[8] of case-control status on dosage of
disease-predisposing alleles of SNP. However, testing for
the effects of multiple SNPs simultaneously via logistic
regression is no cure for difficulty with multicollinearity
and curse of dimensionality[9]. Haplotype-based meth-
ods have many desirable properties[10] and could possi-
bly alleviate the problem[11-14], but assumption of
HWE is usually required and a potentially large number
of degrees of freedom are involved[7,11,15-18]. Genetic association studies now customarily involve
multiple SNPs in candidate genes or genomic regions
and have a significant role in identifying and character-
izing disease-predisposing variant(s). A critical challenge
in their statistical analysis is how to make optimal use
of all available information. Population-based case-con-
trol studies have been very popular[1] and typically
involve contingency table tests of SNP-disease associa-
tion[2]. Notably, the genotype-wise Armitage trend test
does not require HWE and has equivalent power to its
allele-wise counterpart under HWE[3,4]. A thorny issue
with individual tests of SNPs for linkage disequilibrium
(LD) in such setting is multiple testing, however, meth-
ods for multiple testing adjustment assuming indepen-
dence
such
as
Bonferroni’s[5,6]
is
knowingly It has recently been proposed that PCA can be com-
bined with logistic regression test (LRT)[7,16,17] in a
unified framework so that PCA is conducted first to
account for between-SNP correlations in a candidate
region, then LRT is applied as a formal test for the
association between PC scores (linear combinations of
the original SNPs) and disease. Since PCs are orthogo-
nal, it avoids multicollinearity and at the meantime is Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Page 2 of 8 Page 2 of 8 where CXX is the correlation matrix of (X1, X2, ..., Xp),
l
l
l
l
i
i
i
ip
1
1
1
2
1
1
(
,
,
,
)
and l l
i
i
1 1 = 1, i = 1,2, ..., p. PCA-BCIT Given a sample of N cases and M controls with p-SNP
genotypes (X1, X2, ..., XN)T, (Y1, Y2, ..., YM)T, and Xi =
(X1i, X2i, ..., xpi) for the ith case, Yi = (Y1i, Y2i, ..., ypi) for
the ith control, a PCA-BCIT is furnished in three steps:
Step 1: Sampling Replicate samples of cases and controls are obtained
with replacement separately from (X1
(b, X2
(b), ..., XN
(b))T
and (Y1
(b, Y2
(b), ..., YM
(b))T, b = 1,2, ..., B (B = 1000). Step 2: PCA Replicate samples of cases and controls are obtained
with replacement separately from (X1
(b, X2
(b), ..., XN
(b))T
and (Y1
(b, Y2
(b), ..., YM
(b))T, b = 1,2, ..., B (B = 1000). Step 2: PCA Replicate samples of cases and controls are obtained
with replacement separately from (X1
(b, X2
(b), ..., XN
(b))T
and (Y1
(b, Y2
(b), ..., YM
(b))T, b = 1,2, ..., B (B = 1000). Step 2: PCA Simulation study which is mean Fk
D
b
(
)( ) and mean Fk
C
b
(
)( ) are statistically The performance of PCA-BCIT is shown in Table 1 for
the three strategies given a range of sample sizes. It can
be seen that strategies 2 and 3 both have type I error
rates approaching the nominal level (a = 0.05), but
those from strategy 1 deviate heavily. When sample size
larger than 800, the power of PCA-BCIT is above 0.8,
and strategies 2 and 3 outperform strategy 1 slightly. Applications different[19], indicating the candidate region is signifi-
cantly associated with disease at level a. Otherwise, the
candidate region is not significantly associated with dis-
ease at level a. Step 3: Evaluating performance of PCA-BCITs Repeat steps 1 and 2 for K ( K = 1000 ) times under
both null and alternative hypotheses, and obtain the fre-
quencies (Pa) of rejecting null hypothesis at level a (a =
0.05). The confidence interval of mean Fk
C
b
(
)( ) is estimated
by (
,
)
(
)
P
P
k
C
k
C
2
2
1
for control
(15) (15) Step 2: PCA For each replicate sample obtained at Step 1, PCA is
conducted and a given number of PCs retained with a Strategy 1 (CAES): C
l
l
XX i
i
1
1
0
(3) Page 3 of 8 Page 3 of 8 Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 threshold of 80% explained variance for all three strate-
gies[16], expressed as
F
F
F
D
D
K
D
b
1
2
,
,
,
( )
and
F
F
F
C
C
K
C
b
1
2
,
,
,
( )
. Step 3: PCA-BCIT
3a) For each replicate, the mean of the kth PC in cases is
calculated by threshold of 80% explained variance for all three strate-
gies[16], expressed as
F
F
F
D
D
K
D
b
1
2
,
,
,
( )
and
F
F
F
C
C
K
C
b
1
2
,
,
,
( )
. threshold of 80% explained variance for all three strate-
gies[16], expressed as
F
F
F
D
D
K
D
b
1
2
,
,
,
( )
and
F
F
F
C
C
K
C
b
1
2
,
,
,
( )
. has been established[21-24]. Nine SNPs have been
selected
from
the
PNPT22
region
(114157960-
114215857), and most of the SNPs are within the same
LD block (Figure 1). Females are more predisposed
(73.85%) and are used in our simulation to ensure
homogeneity. The corresponding steps for the simula-
tion are as follows. has been established[21-24]. Nine SNPs have been
selected
from
the
PNPT22
region
(114157960-
114215857), and most of the SNPs are within the same
LD block (Figure 1). Females are more predisposed
(73.85%) and are used in our simulation to ensure
homogeneity. The corresponding steps for the simula-
tion are as follows. 3a) For each replicate, the mean of the kth PC in cases is
calculated by mean F
N
F
k
D
b
ki
D b
i
N
(
)( )
( )
1
1
(12) (12) Step 1: Sampling Applications PCA-BCITs are applied to both the NARAC data on
PTPN22 in 1493 females (641 cases and 852 controls)
described above and a data containing nine SNPs near
μ-opioid receptor gene (OPRM1) in Han Chinese from
Shanghai (91 cases and 245 controls) with endopheno-
type of heroin-induced positive responses on first use
[25]. There are two LD blocks in the region of gene
OPRM1 (Figure 2). where
P
k
C
2
is
the
100 2
th
percentile
of
mean Fk
C
b
(
)( ) ,
and
P
k
C
(
)
1 2
is
the
100 1
2
(
)
th
percentile. where
P
k
C
2
is
the
100 2
th
percentile
of
mean Fk
C
b
(
)( ) ,
and
P
k
C
(
)
1 2
is
the
100 1
2
(
)
th
percentile. p
3c) Confidence intervals of cases and controls are
compared. The
null
hypothesis
is
rejected
if
(
,
)
(
)
P
P
k
D
k
D
2
2
1
and
(
,
)
(
)
P
P
k
C
k
C
2
2
1
do not overlap,
which is mean Fk
D
b
(
)( ) and mean Fk
C
b
(
)( ) are statistically
different[19], indicating the candidate region is signifi-
cantly associated with disease at level a. Otherwise, the
candidate region is not significantly associated with dis-
ease at level a. 3c) Confidence intervals of cases and controls are
compared. The
null
hypothesis
is
rejected
if 3c) Confidence intervals of cases and controls are
compared. The
null
hypothesis
is
rejected
if
(
,
)
(
)
P
P
k
D
k
D
2
2
1
and
(
,
)
(
)
P
P
k
C
k
C
2
2
1
do not overlap, Step 2: PCA-BCITing For each replicate sample, PCA-BCITs are conducted
through the three strategies of extracting PCs as out-
lined above on association between PC scores and dis-
ease (RA). where
P
k
D
2
is
the
100 2
th
percentile
of
mean Fk
D
b
(
)( ) ,
and
P
k
D
(
)
1 2
is
the
100 1
2
(
)
th
percentile. Simulation studies We examine the performance of PCA-BCIT through
simulations with data from the North American Rheu-
matoid Arthritis (RA) Consortium (NARAC) (868 cases
and 1194 controls)[20], taking advantage of the fact that
association between protein tyrosine phosphatase non-
receptor type 22 (PTPN22) and the development of RA Step 1: Sampling The observed genotype frequencies in the study sample
are taken to be their true frequencies in populations of
infinite sizes. Replicate samples of cases and controls of
given size (N, N = 100, 200, ..., 1000) are generated
whose estimated genotype frequencies are expected to
be close to the true population frequencies while both
the allele frequencies and LD structure are maintained. The observed genotype frequencies in the study sample
are taken to be their true frequencies in populations of
infinite sizes. Replicate samples of cases and controls of
given size (N, N = 100, 200, ..., 1000) are generated
whose estimated genotype frequencies are expected to
be close to the true population frequencies while both
the allele frequencies and LD structure are maintained. Under null hypothesis, replicate cases and controls are
sampled with replacement from the controls. Under
alternative hypothesis, replicate cases and controls are
sampled with replacement from the cases and controls
respectively. and that of the kth PC in controls is calculated by and that of the kth PC in controls is calculated by mean F
M
F
k
C
b
kj
C b
j
M
(
)( )
( )
1
1
(13) (13) 3b) Given confidence level (1 - a ), the confidence
interval of mean Fk
D
b
(
)( ) is estimated by percentile
method, with form (
,
)
(
)
P
P
k
D
k
D
2
2
1
for case
(14)
where
P
k
D
2
is
the
100 2
th
percentile
of
mean Fk
D
b
(
)( ) ,
and
P
k
D
(
)
1 2
is
the
100 1
2
(
)
th
percentile. (
,
)
(
)
P
P
k
D
k
D
2
2
1
for case
(14)
D (14) Applications For the NARAC data, Armitage trend test reveals none
of the SNPs in significant association with RA using
Bonferroni correction (Table 2), but the results of PCA-
BCIT with strategies 2 and 3 show that the first PC Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Page 4 of 8 Figure 1 LD (r2) among nine PTPN22 SNPs. The nine PTPN22 SNPs are rs971173, rs1217390, rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101. The triangle marks a single LD block within this region: (rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101). Figure 2 LD (r2) among nine OPRM1 SNPs. The nine OPRM1 SNPs are rs1799971, rs510769, rs696522, rs1381376, rs3778151, rs2075572,
rs533586, rs550014, rs658156. The triangles mark the LD block 1 (rs696522, rs1381376, rs3778151) and LD block 2 (rs550014, rs658156). Figure 1 LD (r2) among nine PTPN22 SNPs. The nine PTPN22 SNPs are rs971173, rs1217390, rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101. The triangle marks a single LD block within this region: (rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101). Figure 1 LD (r2) among nine PTPN22 SNPs. The nine PTPN22 SNPs are rs971173, rs1217390, rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101. The triangle marks a single LD block within this region: (rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101). Figure 1 LD (r2) among nine PTPN22 SNPs. The nine PTPN22 SNPs are rs971173, rs1217390, rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101. The triangle marks a single LD block within this region: (rs878129, rs11811771, rs11102703, rs7545038,
rs1503832, rs12127377, rs11485101). Figure 2 LD (r2) among nine OPRM1 SNPs. The nine OPRM1 SNPs are rs1799971, rs510769, rs696522, rs1381376, rs3778151, rs2075572,
rs533586, rs550014, rs658156. The triangles mark the LD block 1 (rs696522, rs1381376, rs3778151) and LD block 2 (rs550014, rs658156). Figure 2 LD (r2) among nine OPRM1 SNPs. The nine OPRM1 SNPs are rs1799971, rs510769, rs696522, rs1381376, rs3778151, rs2075572,
rs533586, rs550014, rs658156. The triangles mark the LD block 1 (rs696522, rs1381376, rs3778151) and LD block 2 (rs550014, rs658156). Figure 2 LD (r2) among nine OPRM1 SNPs. The nine OPRM1 SNPs are rs1799971, rs510769, rs696522, rs1381376, rs3778151, rs2075572,
rs533586, rs550014, rs658156. The triangles mark the LD block 1 (rs696522, rs1381376, rs3778151) and LD block 2 (rs550014, rs658156). Figure 2 LD (r2) among nine OPRM1 SNPs. The nine OPRM1 SNPs are rs1799971, rs510769, rs696522, rs1381376, rs3778151, rs2075572,
rs533586, rs550014, rs658156. The triangles mark the LD block 1 (rs696522, rs1381376, rs3778151) and LD block 2 (rs550014, rs658156). Peng et al. None of the P-values is significant after Bonferroni Correction. Applications BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Page 5 of 8 Table 1 Performance of PCA-BCIT at level 0.05 with
strategies 1-3†
Sample size
Type I error
Power
1
2
3
1
2
3
100
0.014
0.036
0.037
0.156
0.163
0.176
200
0.016
0.044
0.036
0.249
0.278
0.292
300
0.017
0.028
0.029
0.383
0.426
0.368
400
0.014
0.04
0.02
0.508
0.485
0.516
500
0.009
0.035
0.042
0.613
0.595
0.597
600
0.006
0.032
0.042
0.677
0.662
0.683
700
0.007
0.061
0.04
0.733
0.758
0.73
800
0.004
0.043
0.045
0.801
0.791
0.819
900
0.005
0.057
0.051
0.826
0.855
0.858
1000
0.01
0.056
0.05
0.871
0.901
0.889
†1 case-only extracting strategy (CAES), 2 control-only extracting strategy
(COES), 3 case-control extracting strategy (CES) Table 1 Performance of PCA-BCIT at level 0.05 with
strategies 1-3† extracted in region of PTPN22 is significantly associated
with RA. The results are similar to that from permuta-
tion test (Table 3). For the OPRM1 data, the sample characteristics are
comparable between cases and controls (Table 4), and
three SNPs (rs696522, rs1381376 and rs3778151) are
showed significant association with the endophenotype
(Table 5). The results of PCA-BCIT with strategies 2
and 3 and permutation test are all significant at level a
= 0.01. In contrast, result from PCA-LRT is not signifi-
cant at level a = 0.05 with strategy 2 (Table 3). The
apparent separation of cases and controls are shown in
Figure 3 for PCA-BCIT with strategy 3, suggesting an
intuitive interpretation. †1 case-only extracting strategy (CAES), 2 control-only extracting strategy
(COES), 3 case-control extracting strategy (CES) ne of the P-values is significant after Bonferroni Correction. Discussion †2 control-only extracting strategy (COES), 3 case-control extracting strategy (CES)
‡* significant at levels a = 0.05(*) and a = 0.01 (**). principle of a case-control study, it will be our method
of choice given that it has a comparable performance
with CES. Nevertheless, PCA-BCIT has the limitation
that it does not directly handle covariates as is usually
done in a regression model. based approaches. First of all, they are at least as power-
ful as genotype- and haplotype-based methods[7,16,17]. Secondly, they are able to capture LD information
between correlated SNPs and easy to compute with
needless consideration of multicollinearity and multiple
testing. Thirdly, BCIT integrates point estimation and
hypothesis testing as a single inferential statement of
great intuitive appeal[29] and does not rely on the distri-
butional assumption of the statistic used to calculate
confidence interval[19,26-29]. based approaches. First of all, they are at least as power-
ful as genotype- and haplotype-based methods[7,16,17]. Secondly, they are able to capture LD information
between correlated SNPs and easy to compute with
needless consideration of multicollinearity and multiple
testing. Thirdly, BCIT integrates point estimation and
hypothesis testing as a single inferential statement of
great intuitive appeal[29] and does not rely on the distri-
butional assumption of the statistic used to calculate
confidence interval[19,26-29]. Conclusions PCA-BCIT is both a valid and a powerful PCA-based
method which captures multi-SNP information in study
of gene-disease association. While extracting PCs based
on CAES, COES and CES all have good performances, it
appears that COES is more appropriate to use. While there have been several different but closely
related forms of bootstrap confidence interval calcula-
tions[28], we focus on percentiles of the asymptotic
distribution of PCs for given confidence levels to esti-
mate the confidence interval. PCA-BCIT is a data-
learning method[29], and shown to be valid and
powerful for sufficiently large number of replicates in
our study. Our investigation involving three strategies
of extracting PCs reveals that strategy 1 is invalid,
while strategies 2 and 3 are acceptable. From analyses
of real data we find that PCA-BCIT is more favourable
compared with PCA-LRT and permutation test. It is
suggested that a practical advantage of PCA-BCIT is
that it offers an intuitive measure of difference
between cases and controls by using the set of SNPs
(PC scores) in a candidate region (Figure 3). As extrac-
tion of PCs through COES is more in line with the Abbreviations SNP: single nucleotide polymorphism; HWE: Hardy-Weinberg Equilibrium; LD:
linkage disequilibrium; LRT: logistic regression test; PCA: principle component
analysis; PC: principle component; ES: extracting strategy; SES: separate case
and control extracting strategy (strategy 0); CAES: case-based extracting
strategy (strategy 1); COES: control-based extracting strategy (strategy 2); CES:
combined case and control extracting strategy (strategy 3); BCIT: bootstrap
confidence interval test. Discussion In this study, a PCA-based bootstrap confidence interval
test[19,26-28] (PCA-BCIT) is developed to study gene-
disease association using all SNPs genotyped in a given
region. There are several attractive features of PCA- Table 2 Armitage trend test on nine PTPN22 SNPs and RA susceptibility Table 2 Armitage trend test on nine PTPN22 SNPs and RA susceptibility
SNP
Genotype
Female
Male
Case
Control
P-value
Case
control
P-value
rs971173
CC
334
381
0.025
116
169
0.779
AC
236
363
85
134
AA
71
106
26
39
rs1217390
AA
268
319
0.333
99
112
0.108
AG
272
392
89
175
GG
98
138
38
55
rs878129
GG
338
507
0.009
131
187
0.384
AG
251
291
83
130
AA
52
54
13
25
rs11811771
AA
224
272
0.090
78
111
0.717
AG
303
411
104
168
GG
112
169
45
62
rs11102703
CC
312
469
0.024
121
174
0.418
AC
269
314
90
137
AA
60
69
16
31
rs7545038
GG
321
428
0.696
109
186
0.417
AG
265
342
98
114
AA
52
80
20
40
rs1503832
AA
324
487
0.013
129
185
0.249
AG
262
306
86
127
GG
55
59
12
30
rs12127377
AA
349
521
0.017
139
197
0.230
AG
243
282
78
121
GG
49
48
10
24
rs11485101
AA
564
738
0.656
206
305
0.430
AG
72
112
21
35
GG
5
2
0
2
N
f th
P
l
i
i
ifi
t
ft
B
f
i C
ti Table 2 Armitage trend test on nine PTPN22 SNPs and RA susceptibility
SNP
Genotype
Female Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Page 6 of 8 Table 3 PCA-BCIT, PCA-LRT and permutation test on real data
Study
Strategy†
99%CI
95%CI
P-value‡
PCA-LRT
Permutation test
PTPN22
2
(-5.4E-01,-4.7E-03)**
(-7.5E-16,6.9E-16)
(-4.8E-01,-8.6E-02)*
(-4.6E-16,4.2E-16)
0.006**
0.002**
3
(1.7E-02,3.3E-01)**
(-2.5E-01,-1.3E-02)
(4.9E-02,3.0E-01)*
(-2.2E-01,-3.7E-02)
0.007**
0.002**
OPRM1
2
(-1.2E+00,-1.1E-02)**
(-4.7E-16,5.0E-16)
(-1.1E+00,-1.8E-01)*
(-3.7E-16,3.4E-16)
0.107
0.002**
3
(5.3E-02,1.4E+00)**
(-4.9E-01,-1.7E-02)
(2.4E-01,1.2E+00)*
(-4.2E-01,-8.0E-02)
0.012*
0.004**
†2 control-only extracting strategy (COES), 3 case-control extracting strategy (CES)
‡* significant at levels a = 0.05(*) and a = 0.01 (**). Table 3 PCA-BCIT, PCA-LRT and permutation test on real data Table 3 PCA-BCIT, PCA-LRT and permutation test on real data
Study
Strategy†
99%CI †2 control-only extracting strategy (COES), 3 case-control extracting strategy (CES)
‡* significant at levels a = 0.05(*) and a = 0.01 (**). Acknowledgements This work was supported by grant from the National Natural Science
Foundation of China (30871392). We wish to thank Dr. Dandan Zhang
(Fudan University) and NARAC for supplying us with the data, and
comments from the Associate Editor and anonymous referees which greatly
improved the manuscript. Special thanks to referee for the insightful
comment that extraction of PCs with controls is line with the case-control
principles. Table 4 Sample characteristics of heroin-induced positive responses on first use
Cases (N = 91)
Controls (N = 245)
P-value
Age (yrs)
30.42 ± 7.65
30.93 ± 8.18
0.6057
Women (%)
26.4
29.8
0.5384
Age at onset (yrs)
26.29 ± 7.41
26.97 ± 7.89
0.4760
Reason for first use of heroin
0.7173
Curiousness
79.1
75.1
Peer pressure
6.6
4.9
Physical disease
7.7
10.2
Trouble
5.5
6.1
Other reasons
1.1
3.8 Table 4 Sample characteristics of heroin-induced positive responses on first use Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Page 7 of 8 Page 7 of 8 Table 5 Armitage trend tests on nine OPRM1 SNPs and heroin-induced positive responses on first use
SNP
Genotype
Count and frequency
Armitage trend test
Cases
Controls
Chi-square
P-value
rs1799971
AA
55
0.604
150
0.622
0.003
0.9537
AG
27
0.297
64
0.266
GG
9
0.099
24
0.112
rs510769
TT
56
0.667
167
0.749
2.744
0.0976
TC
24
0.286
53
0.237
CC
4
0.048
4
0.018
rs696522
AA
64
0.762
215
0.907
11.097
0.0009*
AG
19
0.226
21
0.089
GG
1
0.012
1
0.004
rs1381376
CC
70
0.769
221
0.913
13.409
0.0003*
CT
20
0.220
21
0.087
TT
1
0.011
0
0.000
rs3778151
GG
66
0.733
215
0.896
14.655
0.0001*
GA
23
0.256
25
0.104
AA
1
0.011
0
0.000
rs2075572
GG
50
0.556
149
0.642
1.574
0.2096
GC
33
0.367
82
0.353
CC
7
0.078
11
0.047
rs533586
TT
68
0.840
203
0.868
0.761
0.3830
TC
12
0.148
31
0.132
CC
1
0.012
0
0.000
rs550014
TT
78
0.857
203
0.832
0.093
0.7602
TC
12
0.132
41
0.168
CC
1
0.011
0
0.000
rs658156
GG
65
0.714
192
0.787
2.041
0.1531
GA
24
0.264
52
0.213
AA
1
0.011
0
0.000
* significant after Bonferroni Correction. * significant after Bonferroni Correction. References 1. Morton NE, Collins A: Tests and estimates of allelic association in
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1997 53 1253 1261 2. Sasieni PD: From genotypes t
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Shandong University, Jinan 250012, PR China. 2MRC Epidemiology Unit,
Institute of Metabolic Science Addenbrooke’s Hospital Cambridge CB2 0QQ 1Department of Epidemiology and Health Statistics, School of Public Health,
Shandong University, Jinan 250012, PR China. 2MRC Epidemiology Unit,
Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ,
UK. Shandong University, Jinan 250012, PR China. 2MRC Epidemiology Unit,
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tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004, 75:330-337. Authors’ contributions QQP, JHZ, and FZX conceptualized the study, acquired and analyzed the
data and prepared for the manuscript. All authors approved the final
manuscript. 22. Carlton VEH, Hu XL, Chokkalingam AP, Schrodi SJ, Brandon R, Alexander HC,
Chang M, Catanese JJ, Leong DU, Ardlie KG, et al: PTPN22 genetic
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arthritis. American Journal of Human Genetics 2005, 77:567-581. Received: 6 December 2008 Accepted: 26 January 2010 Published: 26 January 2010 23. Kallberg H, Padyukov L, Plenge RM, Ronnelid J, Gregersen PK, Helm-van
Mil van der AHM, Toes REM, Huizinga TW, Klareskog L, Alfredsson L, et al:
Gene-gene and gene-environment interactions involving HLA-DRB1,
PTPN22, and smoking in two subsets of rheumatoid arthritis. American
Journal of Human Genetics 2007, 80:867-875. Acknowledgements Table 5 Armitage trend tests on nine OPRM1 SNPs and heroin-induced positive responses on first use
SNP
Genotype
Count and frequency
Armitage trend test
Cases
Controls
Chi-square
P-value
rs1799971
AA
55
0.604
150
0.622
0.003
0.9537
AG
27
0.297
64
0.266
GG
9
0.099
24
0.112
rs510769
TT
56
0.667
167
0.749
2.744
0.0976
TC
24
0.286
53
0.237
CC
4
0.048
4
0.018
rs696522
AA
64
0.762
215
0.907
11.097
0.0009*
AG
19
0.226
21
0.089
GG
1
0.012
1
0.004
rs1381376
CC
70
0.769
221
0.913
13.409
0.0003*
CT
20
0.220
21
0.087
TT
1
0.011
0
0.000
rs3778151
GG
66
0.733
215
0.896
14.655
0.0001*
GA
23
0.256
25
0.104
AA
1
0.011
0
0.000
rs2075572
GG
50
0.556
149
0.642
1.574
0.2096
GC
33
0.367
82
0.353
CC
7
0.078
11
0.047
rs533586
TT
68
0.840
203
0.868
0.761
0.3830
TC
12
0.148
31
0.132
CC
1
0.012
0
0.000
rs550014
TT
78
0.857
203
0.832
0.093
0.7602
TC
12
0.132
41
0.168
CC
1
0.011
0
0.000
rs658156
GG
65
0.714
192
0.787
2.041
0.1531
GA
24
0.264
52
0.213
AA
1
0.011
0
0.000
* significant after Bonferroni Correction. Figure 3 Real data analyses by PCA-BCIT with strategy 3 and confidence level 0.95. The horizontal axis denotes studies and vertical axis
mean(PC1), the statistic used to calculate confidence intervals for cases and controls. PCA-BCITs with strategy 3 were significant at confidence
level 0.95. Figure 3 Real data analyses by PCA-BCIT with strategy 3 and confidence level 0.95. The horizontal axis denotes studies and vertical axis
mean(PC1), the statistic used to calculate confidence intervals for cases and controls. PCA-BCITs with strategy 3 were significant at confidence
level 0.95. Figure 3 Real data analyses by PCA-BCIT with strategy 3 and confidence level 0.95. The horizontal axis denotes studies and vertical axis
mean(PC1), the statistic used to calculate confidence intervals for cases and controls. PCA-BCITs with strategy 3 were significant at confidence
level 0.95. Figure 3 Real data analyses by PCA-BCIT with strategy 3 and confidence level 0.95. The horizontal axis denotes studies and vertical axis
mean(PC1), the statistic used to calculate confidence intervals for cases and controls. PCA-BCITs with strategy 3 were significant at confidence
level 0.95. Page 8 of 8 Page 8 of 8 Peng et al. BMC Genetics 2010, 11:6
http://www.biomedcentral.com/1471-2156/11/6 Peng et al. BMC Genetics 2010, 11:6
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Nonparametric tests of association of multiple genes with human
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Cite this article as: Peng et al.: PCA-based bootstrap confidence interval
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Kolonel LN, Henderson BE, Thomas DC: Modeling and E-M estimation of
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Submit your manuscript at
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
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• Immediate publication on acceptance
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• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit y
y
15. Clayton D, Chapman J, Cooper J: Use of unphased multilocus genotype
data in indirect association studies. Genetic Epidemiology 2004, 27:415-428. References Submit your next manuscript to BioMed Central
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between disease and multiple SNPs in a candidate gene. Genetic
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Variable disease severity in Saudi Arabian and Sudanese families with c.3924 + 2 T > C mutation of LAMA2
|
BMC research notes
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cc-by
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Di Blasi et al. BMC Research Notes 2011, 4:534 Di Blasi et al. BMC Research Notes 2011, 4:534
http://www.biomedcentral.com/1756-0500/4/534 RESEARCH ARTICLE Open Access * Correspondence: mmora@istituto-besta.it
1Division of Neuromuscular Diseases and Neuroimmunology, Fondazione
IRCCS Istituto Neurologico C. Besta, Milan, Italy
Full list of author information is available at the end of the article Abstract Background: Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene that encodes the
laminin a2 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical
spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor
achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis
in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected
based on homozygosity mapping and laminin a2 chain deficiency. Methods: We investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was
suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2
locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases. Results: A previously described mutation in the LAMA2 gene, a homozygous T > C substitution at position +2 of
the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients
from the 4 families revealed variable disease severity particularly as regards motor achievement and disease
progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases
indicating a founder effect of the mutation in all 4 families. Conclusions: Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is
heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a
reliable prognosis even in patients with identical LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to
date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain
deficiency in patients from those regions should initially prompt a search for this mutation. Keywords: MDC1A, LAMA2, gene, Laminin α2 chain, Merosin Variable disease severity in Saudi Arabian and
Sudanese families with c.3924 + 2 T > C
mutation of LAMA2 Claudia Di Blasi1, Emanuela Bellafiore1, Mustafa AM Salih2, M Chiara Manzini3, Steven A Moore4,
Mohammed Z Seidahmed5, Maowia M Mukhtar6, Zein A Karrar7, Christopher A Walsh3, Kevin P Campbell8,
Renato Mantegazza1, Lucia Morandi1 and Marina Mora1* © 2011 Mora et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Background abundant laminin in muscle, is also expressed in Schwann
cells, synaptic basal lamina of peripheral nerves, heart, epi-
dermis and fetal trophoblastic tissue [3]. MDC1A is char-
acterized by generalized hypotonia and severe muscle
weakness at birth with delayed motor development, proxi-
mal joint contractures, inability to achieve independent
walking, high CK levels and a clinically asymptomatic
abnormality of the central white matter on brain magnetic
resonance imaging (MRI) [4-6]. Several studies have also
documented respiratory insufficiency, often leading to Congenital muscular dystrophy type 1A (MDC1A) is an
autosomal recessive neuromuscular disorder caused by
mutations in the LAMA2 gene encoding the laminin a2
chain [1] a component of the skeletal muscle extracellular
matrix protein laminin-211 [2]. Laminin-211, the most Di Blasi et al. BMC Research Notes 2011, 4:534
http://www.biomedcentral.com/1756-0500/4/534 Page 2 of 6 Page 2 of 6 families. Patient 3’s muscle was analyzed using the fol-
lowing anti-human laminin a2-chain monoclonal anti-
bodies: 5H2, recognizing the G-domain (Gibco/BRL,
Gaithersburg, USA), 300 kDa NCL-merosin (Novocastra,
New Castle-upon-Tyne, UK), and 4H8-2 (gift to KPC
from L. Sorokin) recognizing the N-terminus of the pro-
tein. Patient 8’s muscle was tested only with the 300
kDa monoclonal. death in early childhood, and feeding difficulties. Clinical
and subclinical cardiomyopathy, sensory and motor
demyelinating neuropathy, and (late) external ophthalmo-
plegia, also occur [7]. Numerous mutations have now
been identified in the LAMA2 gene, resulting in either
complete or partial protein deficiency. However, the clini-
cal spectrum is more heterogeneous than previously
thought: a severe phenotype associated with partial expres-
sion of a laminin a2 chain isoform has been reported [8]
as well as clinically mild forms with total lack of laminin
a2 chain [9-11]. In addition, the expression of dystrophin [6A9 at 1:50,
Developmental Studies Hybridoma Bank (DSHB), The
University of Iowa], a-dystroglycan (VIA4-1 at 1:50,
Millipore), a-dystroglycan (7D11 at 1:100, DSHB), perle-
can (a716 at 1:1000, Millipore) and collagen VI (5 C6 at
1:20, DSHB) were investigated in patient 3. Dystrophin,
a-, b- and g-sarcoglycan (all from Novocastra) were
tested in patients 6 and 8. In the present study we report on three consangui-
neous Saudi Arabian families and a Sudanese family with
the previously described [12] homozygous mutation
c.3924 + 2 T > C in the LAMA2 gene. Haplotyping Haplotype analysis was performed on genomic DNA using
microsatellite markers provided by Genethon human
genetic linkage map [13] and flanking the LAMA2 gene:
upstream (D6S407) and downstream (D6S1620, and
D6S1705); and six intragenic polymorphisms: G1905A,
A2848G, G5515A, A5551G, C5579A and G6286A [14]. Allele frequency of polymorphisms in a reference popula-
tion are reported in Guicheney et al. [14]. Molecular analyses
A Genomic DNA was extracted from peripheral blood and
analyzed by the polymerase chain reaction touchdown
method using oligonucleotide primers flanking the
intron-exon junctions of all 65 LAMA2 exons (exon
numbering according to the Leiden muscular dystrophy
database) [10]. Aberrant conformers, identified by sin-
gle-strand conformation polymorphism (SSCP) analysis,
were sequenced using an ABI Prism 3100 analyzer
(Applied Biosystems, Foster City, CA, USA). Total RNA was prepared from skeletal muscle using
TRI Reagent (Ambion, Inc. Austin, TX). The isolated
RNA was reverse transcribed using a First-Strand cDNA
Synthesis Kit (Roche Molecular Biochemicals, Basel,
Switzerland), the resulting cDNA was amplified by
reverse transcriptase PCR (RT-PCR) and sequenced
using appropriate primers. Written, informed consent was obtained from the sub-
jects or their parents/legal guardians. Research was con-
ducted according to protocols approved by the
Institutional Review Boards of Children’s Hospital Bos-
ton, University of Iowa, King Saud University and Besta
Neurological Institute, and in compliance with the Hel-
sinki Declaration and local legislation. Background This mutation
leads to aberrant splicing of exon 26 and results in an in-
frame deletion of 63 amino acid residues from domain
IVa of the laminin a2 chain. We also performed haplo-
type analysis to investigate a hypothesized founder effect
of this mutation, and examined the relationship between
the mutation and clinical phenotype in the 4 families. Cryosections were incubated in primary antibodies
diluted in PBS for 1-2 h at room temperature or over-
night at 4°C. Slides were washed 5 min ×2 in PBS, then
secondary antibodies were applied in PBS for 30 min-1
h at room temperature. After washing again 5 min ×2 in
PBS, coverslips were mounted using ProLongGold with
DAPI (Molecular Probes Inc, Eugene, OR, USA). Sec-
ondary antibodies were either goat-anti-mouse tagged
with AlexaFluor488 or goat-anti-rat tagged with Alexa-
Fluor594 (both from Molecular Probes). Methods
Subjects Inclusion criteria for this study were a) clinical, neuroi-
maging and/or pathological findings suggestive of
MDC1A; and b) homozygosity at the LAMA2 locus if
consanguinity was confirmed or suspected. We investi-
gated 9 affected individuals from 4 families. Patients 1
and 2, a male and a female, were Sudanese; they were
born to parents from the same small village and had 7
healthy siblings. Patient 3 and 4 were males born to 3
rd cousin Saudi parents; family history revealed 2
deceased affected siblings with the same diagnosis and 5
healthy siblings. Patients 5 and 6 were both males born
to a 1st cousin Saudi union and had 5 unaffected broth-
ers. Patients 7, 8 and 9 were one male and two females
also born to a 1st cousin Saudi union with another 2
unaffected male and female offspring. All patients were
examined by a pediatric neurologist at a tertiary referral
clinic and clinical features are summarized in Additional
file 1: Table S1. A muscle biopsy was performed after
informed consent in patients 3, 5, 6 and 8. Results Clinical findings are summarized in Additional file 1:
Table S1. All patients were floppy babies and manifested
with generalized hypotonia. Severe proximal weakness
was also present from birth or developed within the first
6 months. In all cases motor milestones were delayed. Five of the 9 patients achieved independent walking at
3, 3.5 and 4 years, which was retained at least until 8,
11, 12 or 13 years respectively, when last seen. Patients
3-6, who never walked, could sit or stand with or with-
out support. CK was normal in patient 9, and slightly to
markedly increased in all others. SSCP analysis in patients 1, 4, 5 and 7 (index cases)
revealed abnormal conformers in exon 26. The exon
was sequenced and showed a T > C substitution at posi-
tion +2 of the consensus donor splice site. This muta-
tion has been described previously in two siblings from
a consanguineous Saudi family unrelated to ours [12]. Direct sequencing of the cDNA revealed a 189 bp in-
frame deletion, corresponding to aberrant skipping of
the whole of exon 26, with loss of 63 amino acids (resi-
dues 1246-1308) from domain IVa of the protein (See
Additional file 2: Figure S1). Electrocardiogram (performed in 5 patients) and echo-
cardiogram (performed in 4 patients) were normal; car-
diac signs were reported in none of the patients. Respiratory support was necessary in patient 3 who died
of respiratory failure at 7 years and in patient 5 who
also died of respiratory failure at 16 years. No mental
retardation, epilepsy or eye abnormalities were observed. MRI or CT, performed in 8 patients, revealed white
matter changes in all cases (Figure 1). Patients 3 and 6
underwent EMG, and myopathic features were found in
both cases. Because of the consanguinity in the Saudi families and
presumed consanguinity in the Sudanese family, it is
likely that all affected siblings are homozygous for this
mutation. A muscle biopsy from patient 3 (taken at 5 weeks)
showed mild dystrophic features; whereas muscle biop-
sies from patients 5 (taken at 3 years and 2 months), 6 Investigation of flanking and intragenic microsatellite
markers of the LAMA2 gene in the 4 index cases indi-
cated an identical mutation-associated haplotype (con-
taining the mutation) between marker D6S407 and Figure 1 T2-weighted MRI images of patient 7, taken at the
age of 19 months. Immunohistochemistry Immunohistochemical analyses were performed on mus-
cle biopsies from patients 3, 6 and 8, from the 3 Saudi Di Blasi et al. BMC Research Notes 2011, 4:534
http://www.biomedcentral.com/1756-0500/4/534 Di Blasi et al. BMC Research Notes 2011, 4:534
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http://www.biomedcentral.com/1756-0500/4/534 Page 3 of 6 Page 3 of 6 (taken at 5 years) and 8 (taken at 4 years) showed
marked dystrophic features. Immunofluorescence analy-
sis of the laminin a2 chain was only performed in
patient 3 (with 3 antibodies) and in patient 8 (1 anti-
body); this revealed greatly reduced staining intensity
with the 3 antibodies in patient 3 (Figure 2) and with
the single antibody in patient 8 (data not shown). Dys-
trophin and dystrophin-associated proteins were normal
in patients 3, 6 and 8. Discussion We previously reported two siblings from a consan-
guineous family with partial laminin a2 chain deficiency
due to an in-frame deletion and exceptionally mild clini-
cal manifestations [15]. The proband was a 39 year-old
man whose symptoms (difficulty in running and jump-
ing), first appeared at age 15 years and worsened very
slowly. When examined, the proband’s sister was found
to have a similar though even milder clinical picture. Siala et al. [16] reported that clinical severity differed
between two siblings with the same out-of-frame muta-
tion in the LAMA2 gene, which was unrelated to lami-
nin a2 chain expression (completely undetectable in
both cases). Such clinical variability implies the presence
of other genetic or epigenetic factors able to influence
disease phenotype (see Heydemann et al. [17]). In 3 families from Saudi Arabia and 1 from Sudan we
have identified a previously reported homozygous c.3924
+ 2 T > C mutation in the LAMA2 gene associated with
variable clinical phenotype. The main phenotypic differ-
ences among our 9 cases regard motor achievement. Patients 1 and 2 from the Sudanese family walked at 4
years; patients 7, 8 and 9 from a Saudi family walked at
3, 3.5 and 4 years, respectively. These 5 patients were
still walking at 8, 11, 12 and 13 years when last seen,
while patients 3, 4, 5, and 6 from the 2 other Saudi Ara-
bian families never walked. CK levels were also variable, being very high (5.5 -
16.8 × normal) in patients 3, 6, 7, and 8; high (1.2 - 4.4
× normal) in patients 1, 2, 4 and 5; and normal in
patient 9. The data of the present study provide further evidence
that the clinical spectrum of MDC1A is more heteroge-
neous than previously thought; motor achievement and
disease progression are particularly variable, making it
difficult to formulate prognoses even in patients with an
identical LAMA2- associated haplotype. Modifier genes,
such as those coding for proteins that interact with
laminin a2, and/or epigenetic factors, for instance those
involved in regulatory signaling functions, are likely to
contribute to the observed phenotypic variability. A
drawback of the present study is that muscle biopsies
were stained for laminin a2 in only 2 cases and exten-
sive immunohistological characterization was only possi-
ble in one case. Discussion Features common to all were floppiness in infancy,
delayed motor milestones or failure to achieve walking,
brain white matter attenuation on MRI or CT (not done
in 1 patient) and development of joint contractures/foot
deformities. Eye and cardiac abnormalities were not
observed. In patients 3 and 5 respiratory compromise
was present, and both died of respiratory failure at 7
and 16 years, respectively. In 1997 Allamand et al. [12] reported on a brother and
sister from a consanguineous Saudi family with the same
LAMA2 mutation as found in our patients (but charac-
terized as 3973 +2 T > C according to the previous
LAMA2 nucleotide sequence numbering). Both were
mildly affected: the boy at 3.5 years had muscle hypotonia
and inadequate head control, but walked at 26 months;
his younger sister also had hypotonia from early infancy
and poor head control and achieved walking at 3 years 8
months. Both had slightly reduced laminin a2 chain
expression as investigated by 2 antibodies recognizing the
G domain, and highly reduced expression using an anti-
body recognizing the N terminal. These results demon-
strated for the first time that use of more than one
antibody can provide valuable indications as to what
domain(s) of the laminin a2 chain may be affected in
CMD patients. Thereafter, this procedure became the
standard method for staining muscle of patients with
CMD and has also been used for prenatal diagnosis. We emphasize, finally, that the identical intragenic
polymorphisms and upstream microsatellite markers of
the LAMA2 gene in our patients, strongly suggest a
founder effect. The mutation probably originated in
Saudi Arabia since studies on severe childhood autoso-
mal recessive muscular dystrophy (SCARMD), the com-
mon form of muscular dystrophy in North Africa and
the Arabian Peninsula [18-21] indicate that affected
families from the same tribe migrated from central
Saudi Arabia to the Sudan - crossing the Red Sea - in
the 12th and 13th centuries [22]. Furthermore, in Saudi
Arabia the mutation may not be rare since we have
recently learned (personal communication of Dr. Tho-
mas L. Winder to SAM) of 3 Saudi patients with this
mutation, 2 of whom are homozygous and one who is
heterozygous. A few more cases, all originating from the
Middle East, are reported in the Leiden database http://
www.dmd.nl/. Results Showing abnormal periventricular and sub-
cortical white matter signal. Figure 2 H&E in control (A) and MDC1A (B) muscle (bar = 25
μm); and immunohistochemistry (C-H) in control (C,E,G) and
patient 3 muscle (D,F,H) laminin a2 chain with 5H2 (C,D), 300
kDa (E,F) and 4H8 (G,H) antibodies. Nuclei are stained with DAPI,
pseudo-colored in red (bar = 50 μm). Figure 1 T2-weighted MRI images of patient 7, taken at the
age of 19 months. Showing abnormal periventricular and sub-
cortical white matter signal. Figure 2 H&E in control (A) and MDC1A (B) muscle (bar = 25
μm); and immunohistochemistry (C-H) in control (C,E,G) and
patient 3 muscle (D,F,H) laminin a2 chain with 5H2 (C,D), 300
kDa (E,F) and 4H8 (G,H) antibodies. Nuclei are stained with DAPI,
pseudo-colored in red (bar = 50 μm). Figure 1 T2-weighted MRI images of patient 7, taken at the
age of 19 months. Showing abnormal periventricular and sub-
cortical white matter signal. Di Blasi et al. BMC Research Notes 2011, 4:534
http://www.biomedcentral.com/1756-0500/4/534 Di Blasi et al. BMC Research Notes 2011, 4:534
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http://www.biomedcentral.com/1756-0500/4/534 Page 4 of 6 intragenic polymorphism G6286A (data not shown),
suggesting remote consanguinity and a founder effect in
all 4 families. By contrast, the downstream markers
(D6S1620, and D6S1705) were not identical in the 4
cases. It is noteworthy that our cases show a much wider
clinical spectrum than suggested by the siblings
described by Allamand et al. [12]: from the very severe
patient 3 and his brother (patient 4 who never walked
or sat unaided), to patients 1, 2, 8 and 9 who were still
walking at latest examination. intragenic polymorphism G6286A (data not shown),
suggesting remote consanguinity and a founder effect in
all 4 families. By contrast, the downstream markers
(D6S1620, and D6S1705) were not identical in the 4
cases. References 1. Helbling-Leclerc A, Zhang X, Topaloglu H, Cruaud C, Tesson F,
Weissenbach J, Tomé FM, Schwartz K, Fardeau M, Tryggvason K, et al:
Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-
deficient congenital muscular dystrophy. Nat Genet 1995, 11(2):216-218. 1. Helbling-Leclerc A, Zhang X, Topaloglu H, Cruaud C, Tesson F,
Weissenbach J, Tomé FM, Schwartz K, Fardeau M, Tryggvason K, et al:
Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-
deficient congenital muscular dystrophy. Nat Genet 1995, 11(2):216-218. The c.3924 + 2 T > C mutation to date has been
found only in patients originating from the Middle East
or Sudan; therefore laminin 2 chain deficiency in
patients from those regions should initially prompt a
search for this mutation. 2. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D,
Ekblom P, Engel J, Engvall E, Hohenester E, Jones JC, et al: A simplified
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Schwann cells, striated muscle, and trophoblast, is expressed late in
nerve and muscle development. Proc Natl Acad Sci USA 1988,
85(5):1544-1548. Discussion The clinical implication is that a laminin
a2 chain deficiency in Middle Eastern or Sudanese By contrast, in our patient 3, analyzed with 3 different
antibodies, laminin a2 chain expression was markedly
reduced: clearly more so than the cases described in
Allamand et al. [12]. He was the most severely affected
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http://www.biomedcentral.com/1756-0500/4/534 patients should initially prompt a search for the c.3924
+ 2 T > C mutation in the LAMA2 gene. manuscript; LM and RM contributed to writing the clinical reports, revised
the manuscript critically and provided financial support; MM was responsible
for study design, supervised the study and manuscript completion. All
authors read and approved the final manuscript. manuscript; LM and RM contributed to writing the clinical reports, revised
the manuscript critically and provided financial support; MM was responsible
for study design, supervised the study and manuscript completion. All
authors read and approved the final manuscript. Competing interests
Th
h
d
l
h Our data provide further evidence that the clinical spec-
trum of MDC1A due to a single mutation is heteroge-
neous, particularly in terms of motor achievement and
disease progression, making it difficult to give a reliable
prognosis even in patients with identical LAMA2-asso-
ciated haplotype. The authors declare that they have no competing interests. Received: 20 October 2011 Accepted: 13 December 2011
Published: 13 December 2011 Received: 20 October 2011 Accepted: 13 December 2011
Published: 13 December 2011 Additional material 4. Tomé FM, Evangelista T, Leclerc A, Sunada Y, Manole E, Estournet B,
Barois A, Campbell KP, Fardeau M: Congenital muscular dystrophy with
merosin deficiency. C R Acad Sci III 1994, 317(4):351-357. Additional file 1: Table S1. Clinical findings. Additional file 2: Figure S1. Sequencing of genomic DNA from control
(A) and patient (B) showing the T > C transition at position +2 of the
consensus donor splice site of exon 26. Direct sequencing of the cDNA
revealing a 189 bp in-frame deletion, corresponding to aberrant skipping
of the whole of exon 26: (C) control and (D) patient. Additional file 1: Table S1. Clinical findings. Additional file 1: Table S1. Clinical findings. Additional file 2: Figure S1. Sequencing of genomic DNA from control
(A) and patient (B) showing the T > C transition at position +2 of the
consensus donor splice site of exon 26. Direct sequencing of the cDNA
revealing a 189 bp in-frame deletion, corresponding to aberrant skipping
of the whole of exon 26: (C) control and (D) patient. 5. Dubowitz V, Fardeau M: Proceedings of the 27th ENMC sponsored
workshop on congenital muscular dystrophy. 22-24 April 1994, The
Netherlands. Neuromuscul Disord 1995, 5(3):253-258. workshop on congenital muscular dystrophy. 22 24 April 1994, The
Netherlands. Neuromuscul Disord 1995, 5(3):253-258. 6. Philpot J, Sewry C, Pennock J, Dubowitz V: Clinical phenotype in
congenital muscular dystrophy: correlation with expression of merosin
in skeletal muscle. Neuromuscul Disord 1995, 5(4):301-305. 6. Philpot J, Sewry C, Pennock J, Dubowitz V: Clinical phenotype in
congenital muscular dystrophy: correlation with expression of merosin
in skeletal muscle. Neuromuscul Disord 1995, 5(4):301-305. 7. Geranmayeh F, Clement E, Feng LH, Sewry C, Pagan J, Mein R, Abbs S,
Brueton L, Childs AM, Jungbluth H, et al: Genotype-phenotype correlation
in a large population of muscular dystrophy patients with LAMA2
mutations. Neuromuscul Disord 2010, 20(4):241-50. 7. Geranmayeh F, Clement E, Feng LH, Sewry C, Pagan J, Mein R, Abbs S,
Brueton L, Childs AM, Jungbluth H, et al: Genotype-phenotype correlation
in a large population of muscular dystrophy patients with LAMA2
mutations. Neuromuscul Disord 2010, 20(4):241-50. Acknowledgements This study was partially supported by the Italian Ministry of Health (MM, LM
and RM), the NINDS (RO1 NS 35129 to CAW), the Muscular Dystrophy
Association (Development Grant to MCM). The University of Iowa Wellstone
Muscular Dystrophy Cooperative Research Center is funded by NIH U54
NS053672 (SAM and KPC). CAW and KPC are investigators of the Howard
Hughes Medical Institute. 8. Pegoraro E, Fanin M, Trevisan CP, Angelini C, Hoffman EP: A novel laminin
alpha2 isoform in severe laminin alpha2 deficient congenital muscular
dystrophy. Neurology 2000, 55(8):1128-1134. 8. Pegoraro E, Fanin M, Trevisan CP, Angelini C, Hoffman EP: A novel laminin
alpha2 isoform in severe laminin alpha2 deficient congenital muscular
dystrophy. Neurology 2000, 55(8):1128-1134. 9. Prandini P, Berardinelli A, Fanin M, Morello F, Zardini E, Pichiecchio A,
Uggetti C, Lanzi G, Angelini C, Pegoraro E: LAMA2 loss-of-function
mutation in a girl with a mild congenital muscular dystrophy. Neurology
2004, 63(6):1118-1121. The authors thank Don Ward for help with the English, Daniel Rakiec and
Jillian Felie at Children’s Hospital, Boston for help with microsatellite analysis
and sequencing, Terese Nelson at the University of Iowa for immunostaining
patient 3, and Joel Carl at the University of Iowa for assembling the
photomicrographs in Figure 2. 10. Di Blasi C, Piga D, Brioschi P, Moroni I, Pini A, Ruggieri A, Zanotti S, Uziel G,
Jarre L, Della Giustina E, et al: LAMA2 gene analysis in congenital
muscular dystrophy: new mutations, prenatal diagnosis, and founder
effect. Arch Neurol 2005, 62(10):1582-1586. Author details
1Di i i
f N 11. Vigliano P, Dassi P, Di Blasi C, Mora M, Jarre L: LAMA2 stop-codon
mutation: Merosin-deficient congenital muscular dystrophy with
occipital polymicrogyria, epilepsy and psychomotor regression. Eur J
Paediatr Neurol 2009, 13(1):72-76. 1Division of Neuromuscular Diseases and Neuroimmunology, Fondazione
IRCCS Istituto Neurologico C. Besta, Milan, Italy. 2Division of Pediatric
Neurology, Department of Pediatrics, College of Medicine, King Saud
University, Riyadh, Saudi Arabia. 3Howard Hughes Medical Institute, Division
of Genetics and Manton Center for Orphan Disease Research, Children’s
Hospital, Boston, MA 02115, USA. 4Department of Pathology, Roy J. and
Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242,
USA. 5Department of Pediatrics, Security Forces Hospital, Riyadh, Saudi
Arabia. 6Institute of Endemic Diseases, University of Khartoum, Khartoum,
Sudan. 7Department of Pediatrics and Child Health, College of Medicine,
University of Khartoum, Khartoum, Sudan. 8Howard Hughes Medical Institute
and Department of Molecular Physiology and Biophysics, Roy J. and Lucille
A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. 12. Allamand V, Sunada Y, Salih MA, Straub V, Ozo CO, Al-Turaiki MH, Akbar M,
Kolo T, Colognato H, Zhang X, et al: Mild congenital muscular dystrophy
in two patients with an internally deleted laminin alpha2-chain. Hum
Mol Genet 1997, 6(5):747-752. 13. Dib C, Fauré S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P,
Marc S, Hazan J, Seboun E, et al: A comprehensive genetic map of the
human genome based on 5264 microsatellites. Nature 1996,
380(6570):152-154. 14. Guicheney P, Vignier N, Zhang X, He Y, Cruaud C, Frey V, Helbling-Leclerc A,
Richard P, Estournet B, Merlini L, et al: PCR based mutation screening of
the laminin alpha2 chain gene (LAMA2): application to prenatal
diagnosis and search for founder effects in congenital muscular
dystrophy. J Med Genet 1998, 35(3):211-217. Di Blasi et al. BMC Research Notes 2011, 4:534
http://www.biomedcentral.com/1756-0500/4/534 Authors’ contributions CDB carried out the molecular and microsatellite analysis and drafted the
manuscript; EB performed molecular analysis; MAMS evaluated clinical and
neurologic features of the patients, collected DNA and muscle biopsies and
revised the manuscript critically for important intellectual content; MCM
carried out homozygosity mapping and linkage analysis; SAM performed
immunochemical evaluation of muscle biopsies and prepared the figures;
MZS, MMM and ZAK evaluated neurologic, neurophysiologic and MRI
features; CAW and KPC supervised the study and critically revised the 15. Di Blasi C, He Y, Morandi L, Cornelio F, Guicheney P, Mora M: Mild
muscular dystrophy due to a nonsense mutation in the LAMA2 gene
resulting in exon skipping. Brain 2001, 124(Pt 4):698-704. 16. Siala O, Kammoun Feki F, Louhichi N, Hadj Salem I, Gribaa M, Elghzel H,
Saad A, Triki C, Fakhfakh F: Molecular prenatal diagnosis of muscular
dystrophies in Tunisia and postnatal follow-up role. Genet Test 2008,
12(4):581-586. Page 6 of 6 Page 6 of 6 17. Heydemann A, Doherty KR, McNally EM: Genetic modifiers of muscular
dystrophy: implications for therapy. Biochim Biophys Acta 2007,
1772(2):216-228. 18. Salih MA, Omer MI, Bayoumi RA, Karrar O, Johnson M: Severe autosomal
recessive muscular dystrophy in an extended Sudanese kindred. Dev
Med Child Neurol 1983, 25(1):43-52. 19. Salih MA: Childhood muscular dystrophy: an African review. Ann Trop
Paediatr 1985, 5(4):167-173. 20. Salih MA, Mahdi AH, al-Jarallah AA, al Jarallah AS, al-Saadi M, Hafeez MA,
Aziz SA: Childhood neuromuscular disorders: a decade’s experience in
Saudi Arabia. Ann Trop Paediatr 1996, 16(4):271-280. 21. Salih MA, Mahdi AH, al-Rikabi AC, al-Bunyan M, Roberds SL, Anderson RD,
Campbell KP: Clinical and molecular pathological features of severe
childhood autosomal recessive muscular dystrophy in Saudi Arabia. Dev
Med Child Neurol 1996, 38(3):262-270. 22. Salih MAM: Muscular dystrophies and myopathies in Arab populations. In
Genetic Disorders among Arab Populations.. 2 edition. Edited by: Teebi AS. Berlin Heidelberg: Springer-Verlag; 2010:145-179. doi:10.1186/1756-0500-4-534
Cite this article as: Di Blasi et al.: Variable disease severity in Saudi
Arabian and Sudanese families with c.3924 + 2 T > C mutation of
LAMA2. BMC Research Notes 2011 4:534. 17. Heydemann A, Doherty KR, McNally EM: Genetic modifiers of muscular
dystrophy: implications for therapy. Biochim Biophys Acta 2007,
1772(2):216-228. 18. Salih MA, Omer MI, Bayoumi RA, Karrar O, Johnson M: Severe autosomal
recessive muscular dystrophy in an extended Sudanese kindred. Dev
Med Child Neurol 1983, 25(1):43-52. 18. Authors’ contributions Salih MA, Omer MI, Bayoumi RA, Karrar O, Johnson M: Severe autosomal
recessive muscular dystrophy in an extended Sudanese kindred. Dev
Med Child Neurol 1983, 25(1):43-52. 19. Salih MA: Childhood muscular dystrophy: an African review. Ann Trop
Paediatr 1985, 5(4):167-173. 19. Salih MA: Childhood muscular dystrophy: an African review. Ann Trop
Paediatr 1985, 5(4):167-173. 20. Salih MA, Mahdi AH, al-Jarallah AA, al Jarallah AS, al-Saadi M, Hafeez MA,
Aziz SA: Childhood neuromuscular disorders: a decade’s experience in
Saudi Arabia. Ann Trop Paediatr 1996, 16(4):271-280. 21. Salih MA, Mahdi AH, al-Rikabi AC, al-Bunyan M, Roberds SL, Anderson RD,
Campbell KP: Clinical and molecular pathological features of severe
childhood autosomal recessive muscular dystrophy in Saudi Arabia. Dev
Med Child Neurol 1996, 38(3):262-270. 22. Salih MAM: Muscular dystrophies and myopathies in Arab populations. In
Genetic Disorders among Arab Populations.. 2 edition. Edited by: Teebi AS. Berlin Heidelberg: Springer-Verlag; 2010:145-179. doi:10.1186/1756-0500-4-534
Cite this article as: Di Blasi et al.: Variable disease severity in Saudi
Arabian and Sudanese families with c.3924 + 2 T > C mutation of
LAMA2. BMC Research Notes 2011 4:534. Submit your next manuscript to BioMed Central
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TEACHING ENGLISH AS A SECOND LANGUAGE IN PRESCHOOL EDUCATION
|
Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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ПРИКЛАДНЫЕ НАУКИ В СОВРЕМЕННОМ МИРЕ:
ТЕОРИЯ И ПРАКТИКА DOI: Xoliqjonova Zulxumor
A student of Chirchik State Pedagogical Institute
Phone number: +998(94)8401049
xoliqjonovazulxumorxon@gmail.com TEACHING ENGLISH AS A SECOND LANGUAGE IN
PRESCHOOL
EDUCATION TEACHING ENGLISH AS A SECOND LANGUAGE IN
PRESCHOOL
EDUCATION Abstract: Learning English as a second language at preschools has been being
implemented for a few years in Uzbekistan. Since that so many methods have
been experienced in order to get success in teaching. Teaching children at the
age of 5-6 should be sociable and unusual. It requires creativity and tools that
interest children. However, some teachers complain of not getting concentration
of children at preschool even though the room is equipped with everything
needs. So only authentic materials is not enough to teach children suitibly. This
article reveals effective ways of teaching and teaching strategies. Key words: children at the early childhood, effective teaching, movement,
visual aids, competitive games. Nowadays, learning English is a requirement of the period and more and more
parents want their children to learn English at the early childhood. According to
the implementation of the Presidential Decree №1875 on “The measures of
strengthening the system of learning foreign languages” made on December 10,
2012 kids should begin being taught English at the first grade at schools. So that
children at preschool should be taught in order to adapt English lessons which
will take place at schools. Learning English at the early age supports
pronunciation and understanding on children in advance. However, teaching
English to children has some difficulties that educators try to find the best ways
of explanation almost every day. There is a range of effective methods and
materials such as using technical aids smartly and organizing useful activities
for the children at the early age which facilitate teaching respectively. Children at the age of 5-6 cannot be attentive and polite more than 10-15
minutes. For achieving even this grade educators should explain the lesson
clearcut and creatively. Teaching grammar is not suitable for preschool children. Instead, I think they are able to learn grammar by learning by heart short
sentences. Repeating after a teacher is easier than learning grammar rules which
is a cause of fatigue. However, this kind of ways is not enough to get the best results. Children want
to do physical activities and to be at somewhere interesting. Hence, educators
ought to utilize unusual ways of teaching. 1) Conducted with different activities,
as usual for the child: drawing a picture, dancing, listening to stories, drama,
movement and modeling—all, in fact, means of communication; 2) Organized in 126 Playing Competitive Games Games are useful device to make children pay attention spontaneously. s are useful device to make children pay attention spontaneously. Games are useful device to make children pay attention spontaneously. As Joseph Sparling mentioned (2019, 214): “Learning Games may be described
as As Joseph Sparling mentioned (2019, 214): “Learning Games may be described
as As Joseph Sparling mentioned (2019, 214): Learning Games may be described
as
‘simple, but deep.’ While the action of many of the games is simple, the
significance to the child’s development is profound.” Learning language through
games keeps children active and avoids exhaustion. As Arda Arikan (2011, 219)
stated “The fact that games are the most suitable instructional activities for
young learners is obvious because they are a natural part of their existence”. Especially, cooperation and competitive games contribute to children’s
activeness. At first, competition was considered as a destructive force for
children at any stage while cooperation was conceived as a good interaction. Later both of them were conceptualized partners (Sonja Sheridan and Pia
Williams, 2006, 5). Playing cooperative and competetive games during lessons
is effective if only it is utilized in moderation. Otherwise, constructive
competition can be changed into rival. Moreover, it should be vary every turn
according to the gender. Mostly, children are used to be divided into two groups
according to their own gender. Occasionally, a “mixed” group can be organized
that they adapt to cooperation no matter which group they are in. ‘simple, but deep.’ While the action of many of the games is simple, the
significance to the child’s development is profound.” Learning language through
games keeps children active and avoids exhaustion. As Arda Arikan (2011, 219)
stated “The fact that games are the most suitable instructional activities for
young learners is obvious because they are a natural part of their existence”. Especially, cooperation and competitive games contribute to children’s
activeness. At first, competition was considered as a destructive force for
children at any stage while cooperation was conceived as a good interaction. Later both of them were conceptualized partners (Sonja Sheridan and Pia
Williams, 2006, 5). Playing cooperative and competetive games during lessons
is effective if only it is utilized in moderation. Otherwise, constructive
competition can be changed into rival. Moreover, it should be vary every turn
according to the gender. Mostly, children are used to be divided into two groups
according to their own gender. ПРИКЛАДНЫЕ НАУКИ В СОВРЕМЕННОМ МИРЕ:
ТЕОРИЯ И ПРАКТИКА accordance with the children’s natural need for self-expression through music. We can infer from the passage above teaching children at the early childhood
should differ from teaching adolescents. For instance, teachers make their
lessons by utilizing visual aids and playing games properly. Effective Using Visual Aids As it was mentioned above, children can learn by heart new words easily and
effectively through pictures. However, children should repeat the words they
learned last lesson on each lesson in order to avoid forgetting. Teaching with the
help of pictures motivates children and they concentrate on the lesson. Teaching
English with singing songs is widely spread at preschool education. It is a vital
method of teaching which improves listening and pronunciation skills. Nicky
Dwiningrum (2016, 14) mentioned: “Someone who was learning second
language from the child, commonly their can pronounce like a native. Its differ
with person who does not begin learning second language until adult, they will
never have a native like accent”. In fact, learning English as a foreign language
requires pronunciation of native speakers. Through listening songs which are
authentic children will be subsidized with original pronunciation of English
language. Gestures and mimes play significant role while they are singing songs. Children memorize the song and terms of movement easily by doing variety
movements. Watching videos such as cartoons and clips in target language is
also beneficial. But children had better watch them after the lesson. Otherwise,
they can’t consentrate during explanation. Playing Competitive Games Playing Competitive Games Occasionally, a “mixed” group can be organized
that they adapt to cooperation no matter which group they are in. 127 ПРИКЛАДНЫЕ НАУКИ В СОВРЕМЕННОМ МИРЕ:
ТЕОРИЯ И ПРАКТИКА All in all, teaching children with variety of styles and strategies provides
efficient lessons. Not only styles and teaching materials, but also organizing the
lesson is vital for all teachers. Maybe teachers can achieve success during
lessons by traditional ways of teaching, but utilizing the other way of teaching as
well as using modern technologies helps children learn easily and memorize
long-time. References: •
Decree of the President of the Republic of Uzbekistan «On
measures to further improve foreign language learning system” №
1875 from December 10, 2012. •
Achkasova, N. The Best Ways of Teaching English to Children:
Using Children’s Operas in Teaching to 5- to 6- Year-Old Children
Natalya Achkasova High School of Economics, Moscow, Russia,
2013, p. 385. •
Arikan, A. Effectiveness of using games in teaching grammar to
young learners. İlköğretim Online, p. 219-229.2011 •
Dwiningrum, N. “ Teaching English Pronunciation to Young
Learners”, Jakarta, 2016, p. 14 •
. Maulana Rohman, I. The Effectiveness of Using Pictures in
Teaching Vocabulary”. Semarang, UIN Walisongo, 2016, p. 16 •
. Sheridan, S & Williams, P. Constructive Competition in
Preschool, University of Göteborg, Sweden. Journal of early
childhood research, 2006, p. 5 p
•
. Sparling, J. Abecedarian: An Early Childhood Education
Approach that has a Rich History and a Vibrant Present. International Journal of Early Childhood, 2019, p. 214. p
•
. Sparling, J. Abecedarian: An Early Childhood Education
Approach that has a Rich History and a Vibrant Present. International Journal of Early Childhood, 2019, p. 214. 128
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Cultural traits, infrastructure and feedback mechanisms as barriers to supply chain management in Brazil
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ORIGINAL
ARTICLE
Cultural traits, infrastructure and feedback
mechanisms as barriers to supply chain
management in Brazil
Traços culturais, infraestrutura e mecanismos de reforço como
barreiras para a gestão de cadeias de suprimento no Brasil
Leonardo Julianelli Ferreira1 , Leonardo Marques2
1
Instituto de Logistica e Supply Chain – ILOS, Rio de Janeiro, RJ, Brasil. E-mail: leonardo.julianelli@ilos.com.br
2
Audencia Business School, Nantes, France. E-mail: lmarques@audencia.com
How to cite: Ferreira, L. J., & Marques, L. (2022). Cultural traits, infrastructure and feedback
mechanisms as barriers to supply chain management in Brazil. Gestão & Produção, 29, e159.
http://doi.org/10.1590/1806-9649-2022v29e159
Abstract: The supply chain management (SCM) practices have been consolidated as important
tools for increasing productivity and, consequently, business competitiveness. This study shows
that peculiar aspects of culture and infrastructure in Brazil become barriers to collaboration and
integration, which helps to justify the country's difficulty in inserting its companies into global
supply chains. Based on the content analysis of interviews with prominent SCM executives in the
country, this work formulates a SCM model contextualized to the Brazilian reality covering three
cultural traits and three infrastructural traits. Fourteen propositions offer a fine-grained analysis
of feedback mechanisms between said traits that perpetuate the gap between SCM theory and
the Brazilian practice, hindering the advancement of SCM in Brazil. The model offers a guide for
companies that aim to unclog the bottlenecks to allow the country's participation in the complex
'dance' of the global SCM.
Keywords: Supply chain management; Culture; Infrastructure; Brazil.
Resumo: As práticas associadas à gestão integrada da cadeia de suprimentos, ou supply chain
management (SCM), têm se consolidado como importantes ferramentas para o aumento da
produtividade e, consequentemente, da competitividade empresarial. Este estudo mostra que
aspectos peculiares da cultura e da infraestrutura no Brasil aparecem como barreiras para
colaboração e integração entre empresas, o que ajuda a justificar a dificuldade do país de inserir
suas empresas nas cadeias de suprimentos globais. A partir da análise de conteúdo de
entrevistas com executivos destacados da área de SCM no país, este trabalho formula um
modelo de SCM contextualizado à realidade brasileira, com três traços culturais e três traços
infraestruturais. 14 proposições detalham os mecanismos de reforço entre estes traços que
perpetuam a distância entre a teoria SCM e a prática brasileira, impedindo o avanço do SCM no
Brasil. O modelo serve de guia para empresas que visem destravar amarras e permitir a
participação do país na complexa ‘dança’ do SCM global.
Palavras-chave: Gestão da cadeia de suprimentos; Cultura; Infraestrutura; Brasil.
Received July 18, 2021 - Accepted Oct.15, 2021
Financial support: None.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gestão & Produção, 29, e159, 2022 | https://doi.org/10.1590/1806-9649-2022v29e159
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1 Introduction
The supply chain management (SCM) paradigm arises at the end of the 1980s and gains
strength in the following decades with the understanding that companies compete not as
isolated entities, but as supply networks with suppliers and customers (Mentzer et al., 2001),
as globally organizations integrated in a non-linear fashion in successive adaptive cycles
(Wieland, 2021). Effective integration within this network is able to significantly improve
operational efficiency (Frohlich and Westbrook, 2001), becoming an important enabler for the
digitalization of the supply chain (Martins et al., 2020). Understanding the barriers that may
exist for SCM practices become appropriate and fundamental for countries such as Brazil at
a time when the low productivity is discussed, when growth is being stunted, and when there
is difficulty of being inserted into global trade and stop being merely a supporting agent and
supplier of basic inputs.
Despite studies showing that Brazilian companies began to use the term “integrated
logistics” in the early 1990s (Lavalle, 1995), we still have a long way ahead of us until reaching
the maturity of the model proposed by Bowersox et al. (1992) that proposed that an integrated
logistics system to provide a competitive advantage should have three dimensions:
formalization of the logistic functions, authority, and planning in order to obtain a orchestration
of the process; performance monitoring that allows the systematization of control and
continuous improvement; and adoption of information technology to ensure greater precision
and agility in the decision process (Tiwari, 2020).
In the 1990s, Brazilian companies advanced a great deal in structuring their logistic
activities (Figueiredo et al., 2003), but despite the intended benefits from collaborative
practices in the supply chain (Fawcett et al., 2008), adopting cooperation policies has
always presented a series of challenges for companies (Lockamy & McCormack, 2004;
Martins et al., 2020). One of the main difficulties in the business relationship is related
to the existing cultural barriers (Fawcett et al., 2015; Chu, 2020), which hinder building
relationships of trust (Canen & Canen, 2005). Furthermore, Fawcett et al. (2008, 2015)
also highlighted infrastructural issues as obstacles to the full adoption of SCM
initiatives. These barriers need to be studied in order to make full use of the benefits
intended to be reached when adopting SCM.
In Brazil, much is said about the “Brazil cost,” a generic term used to address the
structural, bureaucratic, and cultural difficulties that restrict economic growth. The most
recent evaluations available on the competitiveness of countries made by national and
international bodies illustrate the challenge and the need to seek solutions:
•
56th (/160 countries) in logistical competitiveness (Banco Mundial, 2018);
•
71st (/141 countries) in global competitiveness ranking (World Economic Forum, 2019);
•
17th (/18 countries) in competitiveness ranking of similar emerging countries (CNI, 2020).
These rankings consider factors such as availability, cost of capital, infrastructure,
logistics, tax burden, education, technology, and labor. Barriers in the country are noticeable
such as tax costs and infrastructure problems, as well as a legislation that hinders better
performance as in the case of port operations (ABTP, 2020). Illustrating the effects of these
inefficiencies, news about the difficulties in getting the grain crop to market, queues in ports,
food waste, and low productivity in the industry are commonplace in Brazilian news channels.
The adoption of integration and collaboration practices, as recommended by SCM,
plays a fundamental role in reducing Brazil’s low competitiveness, which makes it
important to understand the peculiar Brazilian aspects that may hinder and/or facilitate
adopting these practices. The purpose of this work is therefore to answer the following
research question:
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“How do cultural and infrastructural peculiarities in Brazil interact and create
barriers for supply chain integration?”
2 Theoretical Milestone
2.1 Collaboration across the supply chain
Even without a consensus on the definition of SCM, “integration,” “coordination,”
and “collaboration,” appear as key terms (Näslund and Williamson, 2010; Freitas et al.,
2019) for important authors and trade associations, according to Chart 1.
Chart 1. SCM Definitions.
Source
SCM Definition
SCM is a set of approaches utilized to efficiently integrate suppliers,
manufacturers, warehouses, and stores, so that merchandise is
Simchi-Levi et al.
produced and distributed in the right quantities, to the right locations and
(2003)
at the right time, in order to minimize system-wide costs while satisfying
service level requirements.
SCM is the management of a network of relationships within a firm and
between interdependent organizations and business units consisting of
material suppliers, purchasing, production facilities, logistics, marketing,
Stock et al. (2009) and related systems that facilitate the forward and reverse flow of
materials, services, finances and information from the original producer
to final customer with the benefits of adding value, maximizing
profitability through efficiencies, and achieving customer satisfaction.
SCM encompasses the planning and management of all activities
involved in sourcing and procurement, conversion, and all Logistics
Management activities. Importantly, it also includes coordination and
Council of SCM
collaboration with channel partners, which can be suppliers,
Professionals (2009)
intermediaries, third-party service providers, and customers. In essence,
Supply Chain Management integrates supply and demand management
within and across companies.
SCM has emerged to cope with a complex environment to plan,
Lakshmanasamy &
manage, coordinate, and integrate business activities, seeking to meet
Anil (2015)
customer requests while striving for better competitive advantages.
SCM should be rethought from its linear and fixed structure to a new
paradigm of successive adaptive cycles where organizations connect
Wieland (2021)
globally in a non-linear manner, as in a dance, allowing to deal with the
new complexities and risks,
Therefore, to understand the barriers to the effective adoption of SCM in Brazil and
how they are interrelated calls for understanding the barriers to “integration,”
“coordination,” and “collaboration” between companies in the Brazilian context.
2.2 Barriers to collaboration along the supply chain
Despite the numerous benefits sought with integrated supply chain management,
managers need to be aware of the challenges associated with implementing
collaborative practices with business partners (Freitas et al., 2019; Frohlich &
Westbrook, 2001). From the review by Leuschner et al. (2013), the barriers to SCM
were organized into two dimensions: culture and infrastructure.
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Cultural barriers. It is common for relationships between companies to be based on
the attempt to maximize the margin in transactions. Bargains by price, an “arm wrestle
match” at the end of the month, and the need to meet sales quotas/targets are an
impediment to collaboration. This type of relationship generates friction and wear over
time and creates an antagonistic and opportunistic behavior pattern in the actors
involved. And pressures for immediate results make it difficult to change the focus from
competition—short-term gains—to sharing information and joint decisions—long-term
gains (Chu, 2020; Freitas et al., 2019). Understanding how the traits of Brazilian culture
are able to contribute to deepening or mitigating antagonistic and competitive behaviors
may be decisive for SCM practices (Fawcett et al., 2008).
Infrastructure barriers. Adopting a policy of collaboration requires a great effort to
generate forecasts, reach a consensus, prepare orders, and mobilize operations
(Tiwari, 2020; Wanke & Ferreira, 2006). Furthermore, limitations in the external
environment such as inadequate infrastructure (Moavenzadeh et al., 2013), problems
in legislation (Power, 2005), and unskilled labor (Van Hoek et al., 2002) may make it
even more difficult to integrate supply chains. The reality of the infrastructure in Brazil
and its relations with the other peculiar Brazilian characteristics are fundamental to
understanding the barriers to SCM in the country.
The revised complete listing for this theoretical framework is found in Appendix 1
(Supplementary Material) and has been conducted with the support of the Atlas.TI
textual analysis software. Each term or expression with an idea associated with the
concept of SCM, its possible practices, tools, benefits, and barriers were registered in
the tool, indexing the source article and categorizing the relationships identified and
explained by each author. Based on these records, Atlas.TI was able to build “webs”
that represent these relationships that are grouped into two dimensions—culture and
infrastructure—as shown in Figure 1 and Figure 2.
Figure 1. Map of relations between cultural barriers with SCM. Source: the authors with support
from Atlas.TI software.
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Figure 2. Map of relations between infrastructure barriers with SCM. Source: the authors with
support from Atlas.TI software.
The initial revision of barriers to SCM in the cultural and infrastructural aspects was
based in the SCM literature. Since SCM literature is predominantly based on North
Global companies, this review was complemented by studies that characterize the
peculiarities of Brazilian culture and infrastructure. To achieve this objective, the review
of the theoretical framework combined references of Brazilian culture often consulting
seminal studies from the 1970s, 1980s, and 1990s with more recent studies that
promote the discussion of the impacts of Brazilian culture and infrastructure on
business management in general and on SCM specifically.
2.3 Brazilian supply chain context
2.3.1 Brazilian culture
Chu & Wood (2008) point out that understanding how the peculiarities of Brazilian culture
impact management is based on Brazil’s historical, cultural, social, and economic formation.
Brazilian national culture can be characterized by the large distance of power, by more
collective behaviors than individualistic, and the pressing need to avoid uncertainties (Chu,
2020; O’Keefe & O’Keefe, 2004). It could also be characterized by the central position in
relation to masculinity/femininity with a slight inclination to female values, which would lead to
a low orientation to results (Hofstede, 2001).
2.3.2 Brazilian infrastructure
In July 2018, the World Bank disclosed its new edition of the report that evaluated the
logistics and efficiency of the transportation infrastructure in 160 countries from the perception
of more than 1,000 entrepreneurs from around the world (Arvis et al., 2014). In it, Brazil
dropped down one position in relation to 2016, ranked 56th, lagging behind other Latin
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American countries such as Chile, Mexico, and Argentina. It should be pointed out that the
country has seen its performance fall in most of the questions evaluated by the research such
as the quality of transport infrastructure, its services, the efficiency of the customs clearance
process, cargo tracking, keeping to delivery deadlines, and the ease of finding freight at
competitive prices. Germany, Sweden, and Belgium held the top three positions, in that order.
In its 2020 report, the World Bank chose not to present a new ranking between countries.
The latest Global Competitiveness Report, 2019, corroborates these results and presents
an even more dramatic situation placing the Brazilian transport infrastructure in 85th position
among the 141 nations evaluated (World Economic Forum, 2019). Analyzing each transport
mode separately, Brazil was ranked in 116th position in the quality of its highways, 86th
position in the efficiency of its rail services, 104th position in the efficiency of its port services,
and 85th position in the efficiency of its air transport services.
The traits of Brazilian culture and infrastructure, which are not covered by traditional
SCM literature, are summarized in Chart 2. To counterbalance these peculiar elements
of the Brazilian context alongside the consensus in SCM makes it possible to identify
the barriers that prevent the development of SCM practices in Brazil.
Chart 2. Traits of Brazilian culture and infrastructure.
Brazilian trait
“Jeitinho”
Inequality of
power and
hierarchy
In Brazil it is common for people to judge themselves with special rights who exempt themselves from
following the same laws and generalizing norms made “for others” that led to the creation of the
popular expression: “Do you know who you are talking to?” (Chu & Wood, 2008). For Freire (1998),
the inequality that exists in the master-slave relations of the Brazilian colonial society profoundly
marked the country's social construct, resulting in this behavioral pattern. Inequality also found in the
organizational environment with a strong hierarchical structure and autocratic decision-making
processes (Freitas et al., 2019; Chu & Wood, 2008; Chu, 2020)
Flexibility
For Chu & Wood (2008, p. 4), “the flexibility that permeates behaviors in organizations in
the country translates into people’s adaptability and creativity.” This characteristic results
largely from the need to deal with the most diverse crises of a political, social, and/or
economic nature (Budde et al., 2019). This flexibility, which allows rapid adaptation to new
situations, discourages following recipes and manuals (Chu, 2020). Activities that require a
notion of order, constancy, and precision, so well executed by some nations, in Brazil are
often downplayed and considered of minor importance, suffering from adaptations,
“jeitinhos,” and shortcuts (Holanda & Cândido, 1978; Vieira et al., 2002; Nicodemo, 2014)
Plasticity
According to Holanda & Cândido (1978), the Portuguese colonizer is different from other colonizers
because of the absence of a feeling of racial superiority, which favored miscegenation and the
genuine interest in the new and exotic. This plasticity favors the easy adoption of foreign practices
and customs, as can be seen by the agricultural techniques used in the colony that were strongly
influenced by the Indian and African practices (Nicodemo, 2014). For Chu & Wood (2008, p. 4),
“historically and traditionally, adopting foreign concepts and references in management by the
organizations in Brazil is made without criticism, which reveals the high degree of permeability of the
nation to what is developed abroad. However, it is necessary to emphasize that such assimilation
may occur in some cases only superficially, deceiving a less attentive observer and indicating a
facade behavior.”
Personalism
Lomnitz (2009) highlights the importance of the networks of family members, friends, and
acquaintances for solving problems and/or obtaining some kind of advantage or privilege in
Latin American societies. Personal relationships and interests stand out in general and
community interests (Chu, 2020), using informal mechanisms to pass legal limits, reserved
for the “anonymous, isolated citizen without relations” (Chu & Wood, 2008, p. 5)
Formalism
Existence of a large number of laws, rules, norms, and procedures that seek to reduce risk,
ambiguity, and uncertainty, increasing control over people's behavior and action (Lee
Park et al., 2018; Barros & Prates, 1996). Apparently contrary to personalism, these
characteristics seem to be compatible through the “jeitinho,” as proposed by Da Matta
(1991), resulting in discrepancies between what is written and what is actually done (Chu &
Wood, 2008). This characteristic results in the creation of a number of activities and control
mechanisms in organizations (Barros & Prates, 1996).
Brazilian culture
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Description
The Brazilian style or “jeitinho” is the way found to weasel out of impersonal forms, especially laws
and norms that govern personal relations (Da Matta, 1991). It is an ambiguous mechanism that
allows reconciling personalist and formalistic characteristics found in the Brazilian people (Holanda &
Cândido, 1978) and according to the reading of Chu & Wood (2008, p. 4), it allows for a “double
reading, which may mean a conformist behavior of living with the unjust and unacceptable status quo
and can be seen as a way of surviving daily life, a recourse of cultural resistance,” which is
corroborated by Lee Park et al. (2018).
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Chart 2. Continued...
Brazilian infrastructure
Brazilian trait
Legal and tax
complexity
Description
The complexity of legislation and taxation in Brazil can be organized into five themes:
complexity, legal vulnerability, customs, tax wars, and regulation (Moraes & Souza, 2014)
Poor quality of
transport
infrastructure
Inefficiency of the customs clearance process, cargo tracking, delivery deadlines, and ease
of finding freight at competitive prices, aggravated by the imbalance between transport
modes with an excessive concentration on the road modal (Marchetti & Ferreira, 2012)
Inadequate
professional
training
Shortage of professionals with quantitative training and less experience than peers in other
countries (experience of 11 years in Brazil versus 20 years in the US - Ferreira, 2013)
3 Research method
3.1 Qualitative research
Although the literature on SCM is extensive, there is still little analysis about the
difficulties of implementing these initiatives in the Brazilian scenario, which justifies this
research being of an exploratory nature (Freitas et al., 2019; Gil, 2008). As for the
means, this research used a qualitative methodology to be able to achieve the
objectives it has proposed. Qualitative methods have been used when the study object
cannot be perfectly defined a priori, which is a characteristic of exploratory studies,
particularly in studies on SCM that aim to understand manager behavior, such as in
Silva et al. (2020) and Touboulic et al. (2018).
In this work, it was considered that a specific case could hardly be found where it
would be possible to observe the influence of all the peculiar Brazilian aspects on the
integration of the supply chain, so we then opted for a set of semi-structured interviews
with executives from the SCM area who were submitted to the hermeneutic method of
content analysis for building the propositions based on SCM in Brazil. With this we
expected to obtain as many propositions as possible, answering the question presented
in the best way possible.
The semi-structured interview is characterized by using basic questions supported
by theories and hypotheses related to the research theme, and that when answered by
the informant, allow the formulation of new hypotheses and consequently new
questions (Triviños, 1987). Triviños (1987) also states that this method of data
collection is useful not only in describing the phenomenon, “but also in explaining and
understanding its totality”. The research carried out the following steps: (a) validation
of the script of questions and assessment of the “Judge”, (b) pilot interview, and (c)
interview with the sample.
3.2 Sample collection and profile
Data collection was performed through face-to-face interviews in Rio de Janeiro and
in São Paulo or by a virtual conference platform (Skype) when holding face-to-face
meetings was impossible in the case of interviewees who were located in the US or
Europe. The interviews lasted between 38 and 75 minutes without any interruptions or
other complications with only the interviewee and the interviewer present.
Up until the data collection date, the respondents were responsible for the SCM area in
Brazil, though some were even responsible for all Latin America or the Americas, in
companies listed in the last ranking published by Gartner, Supply Chain Top 25, in the Food
& Beverage, Electronics, Hygiene & Cleaning, Clothing, Computing, and Automotive
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segments. A control interview was conducted with the SCM Vice-President of a large Brazilian
company with global operations and recognized with several national awards in SCM in order
to assess whether the company's origin could significantly influence the executive's view,
which was discarded. All executives interviewed for this work were Brazilian, but have at least
four years of professional experience working abroad, which allows us to compare business
practices in Brazil with other countries. It is also important to point out that their answers were
based on their entire business trajectory in SCM and not just the position and company at the
time of the interview. There were six men and two women with ages ranging from 38 to 65
years old, as summarized in Table 1.
Table 1. Summary of the study’s corpus.
Interview
Interviewee’s
location
Means used
Duration
(minutes)
Pages
transcribed
No. of
verbalizations
Interview 1
Brazil
Face-to-face
62
21
76
Interview 2
Peru
Video conference
40
14
34
Interview 3
Brazil
Face-to-face
38
12
43
Interview 4
USA
Video conference
75
32
71
Interview 5
Brazil
Video conference
50
19
41
Interview 6
Brazil
Video conference
53
14
39
Interview 7
Europe
Video conference
43
15
45
Interview 8
Brazil
Conference call
40
15
29
Using theoretical exhaustion as a way of deciding when to interrupt the data collection,
each interview was fully transcribed with cuts only of language errors and analyzed thoroughly
in search of new elements of analysis based on the elements mapped in Figures 1 and 2 and
in Chart 2. When new analysis elements were not found, the scheduling of new interviews
was closed, which took place after the eighth interview, according to Figure 3.
Figure 3. Theoretical exhaustion observed after eight interviews. Source: the authors.
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3.3 Content analysis
Content analysis has been widely used in qualitative research in the fields of social
sciences, health, psychology, education, and organizations over the past decades,
including in the area of SCM, as in Nascimento & Silva (2020). Bardin (2011, p. 38)
designated the term “content analysis” as “a set of techniques of communication
analysis in order to obtain by systematic procedures and description objectives, the
content of the indicator messages (quantitative or not), allowing the inference of
knowledge related to the conditions of production and reception (inferred variables) of
these messages”. For Godoy (1995), in this methodology of analysis, the researcher
seeks to understand the attributes and patterns that are behind the fragments of the
message analyzed, which obliges him to understand the direct meaning of
communication and to seek, through an effort to get “outside of oneself”, a new
meaning in what has been said, that is, a new message.
The content analysis was supported by Atlas.TI software, a tool widely used to
assist in examining textual material (Bandeira-de-Mello, 2006). According to abductive
reasoning, the study started from the deductive stage of literature review that generated
183 codes (main elements synthesized in Figures 1 and 2 and in Chart 2), which were
later complemented by the inductive process of analysis of the interviews that
generated 127 verbalizations. The contrast between the theoretical framework and the
content analysis generated a set of theoretical propositions that finalize this study.
Figure 4 presents a methodological guide that describes the abductive course
beginning in reviewing the theoretical framework, advancing to the data analysis, and
culminating in the conceptual model, besides also indicating that part of this process,
each Appendix (Supplementary Material) supports with detail and transparency of the
content analysis.
Figure 4. Methodological guide of the study. Source: the authors.
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4 Analysis and propositions
4.1 Cultural barriers
The peculiar aspects of Brazilian culture are summarized in Figure 5. The intricate
network of relationships built in the literature review was enriched with codes that
emerged from the interviews such as “invoice” and “indiscipline,” which made it possible
to illustrate and characterize some concepts, but also brought new elements such as
“regionalism” and the need to observe the “political/governmental context”. Within the
keys, the first algorithm represents the number of verbalizations in the interviews and
the second algorithm represents the number of aspects related to the term. Together
they help to understand the relative strength of each code.
Figure 5. Map of relations between cultural aspects with content analysis. Source: the authors
with support from Atlas.TI software.
4.2 Infrastructure barriers
In the case of infrastructure barriers, it was also possible to confirm the relevance
of some aspects such as the tax war and logistical tourism based on the joint analysis
of the network of relationships built on the literature review on peculiar aspects of
operations in Brazil and the codification carried out later, as presented in Figure 6.
Unlike the cultural aspects whose interviews brought relevant new aspects regarding
what had been presented in the literature review, the infrastructural issues seem well
mapped by literature, which suggests that studies on culture in SCM are still less
frequent or that infrastructural problems are more evident and/or relevant.
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Figure 6. Map of relations between infrastructure aspects with content analysis. Source: the
authors with support from Atlas.TI software.
4.3 Combined analysis of cultural and infrastructure aspects
Table 2 presents the verbalizations count for each analysis class: four cultural
dimensions and three infrastructural dimensions. In the discourse of the interviewees,
the results highlight the cultural aspects of flexibility and the adventurous character of
the Brazilian, as well as the country’s infrastructure problems.
Table 2. Analysis class and verbalizations count.
E1
E2
E3
E4
E5
E6
E7
E8
TT
Brazilian Culture
Flexible Adventure: traits related mainly to the flexibility
and plasticity of Brazilian culture and consequently to the
main characteristics of the “adventurous character”
6
1
7
17
0
7
6
6
50
Bureaucratic Formalization: recurrent characteristics
and situations in the discourses that are associated with
formalist aspects of Brazilian culture, almost always
mentioned negatively.
3
0
8
1
3
2
1
2
20
Unproductive “jeitinho”: aspects resulting from
personalism and the Brazilian way of doing things, which
appeared throughout the interviewee discourses, always
negatively.
8
6
0
6
1
0
4
2
27
Tangent Aspects: topics that emerged spontaneously
in the interviewee discourses and are complementary to
the analysis of Brazilian cultural peculiarities.
4
6
1
1
1
0
1
3
17
Brazilian Infrastructure
Limiting Infrastructure: an item that brings together the
main topics associated with aspects of the infrastructure
of operations in Brazil, almost always criticized in the
verbalizations.
11
7
10
4
3
2
7
5
49
Unfair Complexity: relevant aspects associated with the
results of the Brazilian legal and tax issues in SCM,
present in the interviewee statements.
9
4
6
5
3
1
4
4
36
Shortage of Professionals: presents highlighted traits
in the verbalizations about the level of education and
experience of SCM professionals in Brazil.
5
3
5
9
1
6
2
0
31
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Next, Table 3 of co-occurrence was put together, meaning that a new count was
performed, only now crossing the classes two by two and measuring the number of
verbalizations that were referenced to both classes concomitantly. This analysis makes
it possible to infer some non-explicit relationships in the content, formulating new
propositions from possible interpretations for these references.
Tangent Aspects
-
Flexible Adventure
1
-
Shortage of Professionals
3
1
-
Bureaucratic Formalization
1
3
1
-
Limiting Infrastructure
2
2
2
5
-
Unfair Complexity
1
4
1
6
16
-
Unproductive “jeitinho”
3
15
1
2
2
0
Unproductive
“ jeitinho”
Unfair
Complexity
Limiting
Infrastructure
Bureaucratic
Formalization
Shortage of
Professionals
Flexible
Adventure
Tangent
Aspects
Table 3. Co-occurrence of verbalizations between classes.
-
4.4 Contextualized SCM model
The inferred relationships between the analysis classes summarized in Tables 2 and 3
were condensed in Figure 7, which makes it possible to recognize more easily the three
cultural dimensions, the three infrastructural dimensions, and the recurrent mechanisms that
perpetuate practices, behaviors, rules, and resources recurrently criticized by the interviewees
and by literature as ultimately preventing the advancement of SCM in Brazil. The study also
points to an exception of a mitigating mechanism where cultural traits help to deal with
problems in the transport infrastructure and complex regulation.
Figure 7. Model (and Theoretical Propositions) of Brazilian Way SCM. Source: the authors.
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Even though there are some positive verbalizations about Brazilians’ capacity to
cope with adverse situations and the ability to adapt and adopt new practices, which in
the view of some executives interviewed is fundamental for SCM, much of the peculiar
characteristics of Brazil appear as barriers to the integrated management of the supply
chain, highlighting the cultural characteristics of formalism and flexibility, inadequate
vocational training, the precariousness of the transport infrastructure, and the complex
tax legislation.
Cultural contextualization of SCM in Brazil. The first set of aspects with an impact
on SCM in Brazil, identified in this work as “bureaucratic formalization,” is related to the
formalist characteristic of the national culture condensed into bureaucratic
requirements and inefficient use of time. The analysis reinforces how the bureaucratic
requirements existing in Brazil make SCM difficult in the country, resulting in
inefficiencies and making it difficult to coordinate actions across the supply chain.
Another expression of formalism is the reference made to using redundant control
mechanisms, considered necessary to deal with the lack of commitment and
indiscipline, but that contribute to the inefficient use of time, which can be considered
as a disadvantage to SCM insofar as a greater agility of chain response is required,
complementing the findings of Freitas et al. (2019) on cultural barriers to collaborative
initiatives. The importance of bureaucracy as a first barrier contrasts with studies on
collaboration and SCM focused on companies in the northern hemisphere
(Touboulic et al., 2018) or multi-country studies (Frohlich & Westbrook, 2001) that tend
to focus on the challenges of relationships between company agents or technology
adoption, respectively, and overlook the regulatory framework.
Proposition 1: The bureaucratic formalization expressed in excessive control and in
the inefficient use of time is a cultural barrier to SCM practices.
The second set of peculiar cultural traits refers to possible consequences from what
in Portuguese is called “jeitinho brasileiro”, meaning the Brazilian style of finding a way
through, a loophole, bending the rules, which is a behavior described by Da Matta
(1991) and Chu (2020) that could also be a quality of flexibility that would allow a
reconciliation of personalism and formalism present in Brazilian culture, but whose
analysis of the corpus content of this work was closer to the resignification proposed
by Chu & Wood (2008) who presented a predominantly negative view of this “jeitinho”.
The first consequence identified in the “jeitinho” is using shortcuts to circumvent
difficulties and challenges, which although being less intense in the interviewee
discourses than other barriers associated with cultural peculiarities, also seems to be
relevant. Despite a possible positive initial impression as an artifact to deal with the
challenges that arise in supply chain management, the tone used leaves no doubt that
it is a criticism of failing to face problems and the lack of a consistent and systematic
execution. Another consequence of the Brazilian “jeitinho” for SCM identified in this
study is the lack of commitment presented as a problem for corporate management in
general (Chu & Wood, 2008; Chu, 2020) and particularly for SCM practices, which
require a strong commitment from those involved.
The first reinforcement mechanism identified deals with the relation of the impact of
the unproductive “jeitinho” on strengthening the bureaucratic formalization that
demands management control mechanisms to deal with low productivity from the low
level of commitment. Confirming the literature (Moavenzadeh et al., 2013), the analysis
showed that the bureaucratic requirements and excessive rules pave the way for
corruption, which may represent an important barrier to SCM since distortions are
caused in the competition between companies and, due to the legal risk, discourage
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the exchange of information between companies. The reinforcement between the
unproductive “jeitinho” and bureaucracy has a parallel with studies that demonstrate
the negative effect that a short-term view can have on initiatives to implement lean
methodology, which is also based on integration (Erthal et al., 2021). The short-term
view dialogs with the relationship between “jeitinho” and bureaucracy.
Proposition 2: The unproductive “jeitinho,” characterized by using shortcuts and lack
of commitment, is a trait of the Brazilian culture that hinders SCM practices;
Proposition 3: The unproductive “jeitinho” requires additional control mechanisms,
amplifying bureaucratic obstacles to SCM practices.
The last set of peculiar cultural aspects is characterized here as “flexible adventure”
resulting from flexibility and plasticity, as described by Holanda & Cândido (1978) and
Chu (2020) and present in the interviews as individualism, immediacy, indiscipline,
adaptability, and impetus. Despite studies by Hofstede (2001) and O’Keefe & O’Keefe
(2004) situate the Brazilian culture as more collectivist than individualistic, the results
of the content analysis pointed out individualism as a characteristic present in the
country’s culture and an important barrier, confirming the analysis of the behavioral
barriers of Freitas et al. (2019). It was also possible to infer that individualism is partly
responsible for the preference for proprietary systems, which makes integration
between companies difficult, as pointed out by Fawcett et al. (2007). The trace of
immediacy again dialogs with the short-term orientation (Lacerda, 2011; O’Keefe &
O’Keefe, 2004; Chu, 2020) and appeared in the discourse of several interviewees as a
significant obstacle to adopting SCM practices in Brazil. Another cultural characteristic
perceived by Brazilian executives as restrictive to SCM practices is indiscipline, which
completes the adventurous character (Nicodemo, 2014) and the low orientation toward
long-term planning (O’Keefe & O’Keefe, 2004).
In addition to being a problem in itself, indiscipline present in “flexible adventure”
generates a second reinforcement mechanism. To deal with it, additional control
mechanisms are created, something characterized as undesirable for an effective
SCM, which corroborates the lack of orientation for the relationship (Freitas et al.,
2019). Together, propositions 3 and 5 perpetuate “bureaucratic formalization” as a
fundamental barrier to SCM practices in Brazil. This reinforcement mechanism exposes
the relationship between culture and regulation, a dimension little explored in SCM
studies (Marques et al., 2021).
In addition to its undesirable characteristic, the immediacy of “flexible adventure” is
related to the poor quality of transport infrastructure, whose long-term investments are
put off by adopting short-term policies, aggravating the infrastructure problems
mentioned by Marchetti & Ferreira (2012) and Assis et al. (2017). This third
reinforcement mechanism compromises the result of long-term oriented initiatives
(Erthal et al., 2021).
Proposition 4: The traits of individualism, immediacy, and indiscipline present in the
Brazilian culture form the negative side of the cultural aspect “flexible adventure”,
placing themselves as barriers to SCM practices;
Proposition 5: Indiscipline as a trait of the Brazilian “flexible adventure” leads to
creating additional control mechanisms, amplifying the bureaucracy as an obstacle
to SCM practices;
Proposition 6: The cultural trait of immediacy reinforces low investments in transport
infrastructure, amplifying barriers to SCM practices.
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In contrast to the reinforcement mechanisms, the cultural dimension of the “flexible
adventure” (Nicodemo, 2014) offers two traits that can contribute to SCM practices,
The first is the impetus, which has a direct relationship with insufficient and precarious
operational resources. In addition to being a virtue itself, one can assume that impetus
is a cultural characteristic that allows dealing with operational constraints posed by
limited infrastructure. The second positive trait is adaptability, which is directly related
to some infrastructural barriers in Brazil such as insufficient and precarious resources
and excessive rules. The executive discourse shows that adaptability is an important
artifact to deal with these restrictive aspects (Chu, 2020). The positive impacts of the
“flexible adventure” dimension are characterized as mitigating mechanisms,
highlighting favorable traits of the Brazilian culture for management (Tanure & Duarte,
2005) and offering a direction for SCM implementations.
Proposition 7(a-b): Impetus and adaptability are cultural traits of the Brazilian
“flexible adventure” that favor SCM practices by allowing to deal with insufficient
and precarious operational resources and excessive rules.
Infrastructure for SCM in Brazil. Most Brazilian infrastructure characteristics are
obstructive to the integrated management of the supply chain. The first set of peculiar
aspects of operations in Brazil is related to transport infrastructure, which was much
criticized by the executives interviewed. Reinforcing the study of Marchetti & Ferreira
(2012) and Assis et al. (2017), this work reinforces criticisms regarding the unbalanced
transport matrix and precarious and insufficient resources. The unbalanced transport
matrix refers to the fact that cargo handling in Brazil is concentrated in the road modal
with a relative low participation of other modes. The main impact of this distortion for
SCM would be a reduction in productivity and increase in costs across the chain. In
addition to the unbalanced transport matrix, it is possible to deduce the scarcity of
infrastructure resources in general, such as the inability of land port accesses to receive
the volume to be moved or the unavailability of railway lines in some regions of the
country, which results in a loss of efficiency and overload of the road modal, confirming
Assis et al. (2017).
A fourth reinforcement mechanism was identified: the limiting infrastructure feeding
the cultural aspect of indiscipline. The precarious infrastructure reinforces the
indiscipline as an antithesis of the execution of the actions planned since the
uncertainties resulting from the impoverished infrastructure generate disbelief in the
value of long-term planning activities, validating cultural and behavioral aspects raised
by Freitas et al. (2019). The identification of a mechanism where the infrastructure
reinforces the culture, after proposition 6 where culture prevents long-term
infrastructure improvements emphasizes culture and infrastructure as two key
components for SCM collaboration (Leuschner et al., 2013), but here completing the
design of a vicious cycle identified in this study:
Proposition 8: The unbalanced transport matrix and insufficient resources
characterize a “limiting infrastructure” for SCM practices;
Proposition 9: The “limiting infrastructure” reinforces the cultural aspect of
indiscipline, amplifying barriers to SCM practices.
The second set of infrastructural aspects refers to the combined characteristics in the term
“unjust complexity” depicting the legal and tax complexities existing in Brazil (Moraes & Souza,
2014): excessive rules, rules that generate inefficiencies and uncertainties in the rules. In
addition to the complexity and inefficiencies associated with legislation and taxation, frequent
changes in the rules creates uncertainties. The “unjust complexity” is a problem for
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understanding the rules, especially when many of them seem to be counterintuitive. This
nebulous environment favors a lack of transparency, the adoption of non-ethical practices
(Marques et al., 2021), and makes it difficult to take decisions and to build long-term
agreements, which are fundamental to the effective SCM.
A new reinforcement mechanism was identified in line with previous propositions
that dialog with the excessive focus on the short term. Excessive rules with their
contradictions discourage compliance with all legal obligations, reinforcing the cultural
aspect of indiscipline. The unjust complexity also favors individualistic and immediate
behaviors, thus emphasizing the flexible adventure in the Brazilian culture that is
obstructive to SCM practices and restricts the implementation of long-term oriented
projects (Erthal et al., 2021).
Beyond the complexity, current tax legislation causes enormous inefficiencies such
as “logistical tourism” and “fiscal war”. It is urgent to consider the complex tax legislation
and its impacts on any logistic network analysis in Brazil. In line with Moraes & Souza
(2014), this makes it difficult to manage the supply chain as companies often opt for
decisions that benefit them individually in the short term. Once again, new
reinforcement mechanisms are identified. The first mechanism comes from the legal
complexity of strengthening the infrastructure that has been precarized, since the
unnecessary displacement of loads contributes to the excessive use of the transport
infrastructure, which would already be deficient without this reinforcement and thus
contributing to its deterioration.
Proposition 10: The “unfair complexity” trait consists of having too many rules,
inefficient rules, and rule uncertainty, all of which hinder SCM practices;
Proposition 11: “Unfair complexity” reinforces the cultural aspects of “flexible
adventure” (individualism, immediacy, and indiscipline), amplifying obstacles to
SCM practices;
Proposition 12: “Unfair complexity” contributes to the deterioration and overuse of
transport infrastructure, amplifying barriers to SCM practices.
The last set of peculiar aspects of infrastructure in Brazil refers to the scarcity of
qualified professionals to work in SCM and point to nonspecific training, low technical
quality, and inexperience as possible causes, as previously pointed out by Freitas et al.
(2019). The main cause identified for the shortage of qualified professionals to work in
SCM in Brazil is the low technical quality in its education. The lack of knowledge of
concepts, models, and techniques seems to be an important barrier to SCM. This trait
characterized as “shortage of professionals” still feeds a last reinforcement mechanism,
strengthening cultural aspects of individualism and immediacy.
Proposition 13: Shortage of professionals is a barrier to SCM practices;
Proposition 14: Shortage of professionals reinforces the cultural aspects of
individualism and immediacy, amplifying obstacles to SCM practices.
5 Conclusion
5.1 Study’s contributions
This study shows that peculiar aspects of Brazilian culture and logistics
infrastructure can act as significant barriers in the implementation of supply chain
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management. Taking Brazil as an example, this study indicates that these aspects
show complex barriers to integration initiatives between companies, which helps justify
the country's difficulty in inserting its companies into global supply chains. The study
offers a model that organizes on the one hand three cultural dimensions—bureaucratic
formalization, unproductive jeitinho, and flexible adventure, and on the other three
infrastructural dimensions—transport-limiting infrastructure, unjust complexity, and
shortage of professionals that refine previous mappings of SCM barriers
(Leuschner et al., 2013). Next it presents propositions that expose the reinforcement
mechanisms that cause the culture to feed an inadequate infrastructure and vice-versa,
justifying why SCM initiatives in Brazil do not advance in line with the proposed theory
in the last 30 years (Mentzer et al., 2001; Frohlich & Westbrook, 2001).
On the one hand, undiscipline and the unproductive jeitinho reinforce the culture of
bureaucracy, the focus on the short term, and the consequent imprisonment in an
infrastructure that never modernizes and continues being excessively dependent on
road as it best suits short-term decisions (Figueiredo et al., 2003). On the other hand,
transport infrastructure, the perception of unjust regulatory complexity and the lack of
professional qualification reinforce the flexible adventure and immediate thinking.
These reinforcement mechanisms conflict with the definition of SCM from its origin, but
even more acutely, when the definition is updated to reflect the dynamism of global
supply chains that advance in a non-linear manner, in successive adaptive cycles, as
proposed by Wieland (2021). Effective network integration and collaboration is critical
to participating in global supply chains. Barriers in the sphere of a country such as those
identified in the case of Brazil can relegate companies to the periphery of the global
supply chains, or as Wieland’s analogy states, “leave them out of the dance”.
The exception in the map designed in this study is the mitigating mechanism
identified where the positive dimension of the flexible adventure as characterized by
the traces of impetus and adaptation helps managers and companies that need to
advance in a context of precarized infrastructure and excessive regulatory and legal
complexity. This mitigating mechanism offers two important inputs for SCM managers.
The first is the opportunity to explore positive aspects of local culture when
implementing new management practices (Erthal et al., 2021). Other mitigating
mechanisms not identified in this study may reinforce the advancement of SCM even
under non-favorable macro conditions. The second is to devise counter-mechanisms
capable of breaking the vicious cycle. The organizational culture adaptation planning
(Tanure & Duarte, 2005) can counter elements of the national culture (Chu, 2020),
reducing the resistance to implementing SCM while mitigating the reinforcement
mechanisms between culture and infrastructure.
This study advances in the discussion of the barriers to SCM for specific contexts
by exposing reinforcement mechanisms between culture and infrastructure while
mutually reinforcing themselves. In line with previous alerts made by Fawcett et al.
(2008, 2015), deficient infrastructure is a key barrier to unlocking SCM
implementations. But to this must be added the cultural context, which is in line with
Freitas et al. (2019) as it also creates barriers. The proposals in this study also serve
as a warning to other countries that identify themselves with the cultural and
infrastructural aspects reported in this study and probably face similar challenges in
adopting SCM. To unlock reinforcement mechanisms, countries exposed to the
problem must think of programs that act simultaneously in both dimensions in order to
ensure progress toward collaboration and favor participation in global supply chains.
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5.2 Study’s limitations
It is not possible to have the ambition of being complete, accurate, and profound
when dealing with the culture and infrastructure in Brazil at the same time and its
impacts on supply chain management. Thus, this is the first and largest limitation of
this study and there is no way to ensure that all relevant literature has been covered,
nor that the analyses have been comprehensive enough to point out all possible
propositions. Nor can it be denied that some of the proposals presented here,
especially those related to infrastructure in Brazil, have already been listed and that
academics and executives in the area of corporate logistics have already been aware
of, limiting the merit of their enunciation in this work to the fact that they receive new
meanings and impacts in the context of supply chain integration and not just as
operational challenges in themselves.
The propositions formulated, although significant and substantiated, were not
prioritized according to their importance or relevance to the supply chain integration,
and therefore it is not possible to prioritize the actions to be taken. Having said that, the
simple identification of barriers is not enough to overcome them, which can be
considered a limitation of this study. It should also be pointed out that despite the effort
to follow the method of content analysis with exemption, possible interpretation bias
related to preconceived concepts should not be disregarded, which together with a lack
of language training, may have resulted in the inadequate choice of words by
researchers.
Despite the limitations discussed here, it is believed that this work brings relevant
considerations to the evolution of SCM in Brazil, contributing to a better understanding
of the existing cultural barriers to adopting its practices in the country, deepening the
knowledge available.
5.3 Recommendations for future studies
This work should be seen as a new step in preparing an SCM theory that considers
the cultural and infrastructural characteristics of Brazil, enabling a full appropriation of
its benefits by companies and the country. However, there is still much to be studied
for developing management artifacts that allow the effective development and
application of collaborative practices between companies in the Brazilian context.
The natural continuation of this study seems to be preparing an objective
questionnaire that can be applied in stratified samples of SCM professionals in order
to validate or reject the propositions formulated here from statistical analyses, allowing
extrapolation or not of the findings in this work to different industries, companies of
distinct sizes and varied supply chain segments. A larger-scale study would make it
possible to control the effects of the industry’s maturity, for example.
It also seems necessary to carry out in-depth case studies with companies of
different sizes, industries, and segments along the chain in order to observe the
difficulties reported here in everyday situations so as to broaden the understanding of
the peculiarities identified, their impacts and mechanisms of functioning, thus allowing
to specify and deepen the results of this work.
Also in continuation to this study, a quantitative research would be very welcome
that would make it possible to prioritize the SCM barriers in Brazil pointed out in this
study. Ideally, this hierarchy should be conducted in such a way that an individualized
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diagnostic model could be reached, considering a set of predominant cultural and
infrastructural characteristics in a given company or chain.
Another desired development for this work, after the proper deepening of the
diagnosis carried out here, would be identifying and/or building mechanisms that would
enable the barriers to the integration of supply chains in Brazil to be overcome and with
this finally allow the insertion of the companies installed here in the global supply
chains.
Finally, it is considered important to deepen the knowledge about the peculiarities
of each region and their possible impacts on the integrated management of the supply
chain since regionalism emerged in the discourse several interviewees as an element
of fundamental importance for SCM in Brazil. There appears to be a great cultural
diversity and also of resources that would require a deep knowledge of these
peculiarities.
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Supplementary Material
Supplementary material accompanies this paper.
Appendix/Apêndice 1. Referências bibliográficas utilizadas na construção das
Figuras 1 e 2 e do Quadro 2.
Appendix/Apêndice 2. Verbalizações por classe.
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This material is available as part of the online article from https://www.scielo.br/j/gp
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An assessment of knowledge and utilisation of Mission Indradhanush at Qutub Vihar, New Delhi: A cross-sectional study
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ABSTRACT Background : The Mission Indradhanush, is a flagship programmestarted by the Government of India in 2014.The
objective is to achieve full immunization coverage for children up to 2 years of age and pregnant women. But the
urban area has faced a lot of implementation challenges to cover full immunization. Materials and Methods: The
community-based cross-sectional study was conducted in the peri-urban area of Qutub Vihar, Goyla Dairy, Southwest
District, New Delhi between the period of June 2022 and August 2022 to assess the immunization status of the
children along with the assessment of the knowledge, attitude, and practice among mothers having children below
two years of age. Results: Out of 155 participants, only 26 percent heard about Mission Indradhanush, and among
those 26 percent participants, only 45 percentknow about its ongoing immunization services. 14 percent of children
were partially vaccinated, 85percent of children were fully vaccinated, and one percent were not vaccinated. Conclusion: The age of the mother and literacy level of the father and mother is significantly related to the
vaccination status of the child. Almost all the child was vaccinated at a government facility and were registered at
a nearby facility. Most of the participants were unaware of Mission Indradhanushand its ongoing services at
government facilities. Community workers must strengthen the IEC activities related to Mission Indradhanushat
the community level for better utilization of services. Keywords: Mission Indradhanush, child immunization, immunization status, urban area, Qutub Vihar, peri-
urban area, and cross-sectional study. An assessment of knowledge and utilisation of Mission Indradhanush
at Qutub Vihar, New Delhi: A cross-sectional study
Akanksha, Nancy Modi, Nida Shaikh, Sumant Swain
International Institute of Health Management Research (IIHMR), New Delhi, India.
S Sheetal et al. JTVS (2022) 04:02
JTVS Online
Research Article
Open Access An assessment of knowledge and utilisation of Mission Indradhanush
at Qutub Vihar, New Delhi: A cross-sectional study
Akanksha, Nancy Modi, Nida Shaikh, Sumant Swain
International Institute of Health Management Research (IIHMR), New Delhi, India. S Sheetal et al. JTVS (2022) 04:02
JTVS Online
Research Article
Open Access INTRODUCTION next decade and set a target to save over 50 million
lives. IA2030 aims for s a world where everyone,
everywhere, at every age, fully benefits from vaccines next decade and set a target to save over 50 million
lives. IA2030 aims for s a world where everyone,
everywhere, at every age, fully benefits from vaccines Vaccines are provenone of the most affordable,
effective, and safe methods to provide protection to
infants and children from diseases, morbidity, and
mortality. The World Health Organization (WHO)
estimates that about two to three million deaths under
five years of age could be preventable through
immunization. Over time, many policies, programmes,
and targets have been adopted and implemented at the
international and national levels to improve immunization. Due to the COVID-19 pandemic and associated
disruptions, twenty-five million children unable to take
vaccination in 2021[1].Therefore, World Health
Organization has adopted the Immunization Agenda 2030
(IA2030) to address immunisation challenges over the Address for correspondence :
Dr Sumant Swain, Assistant Professor
International Institute of Health Management Research (IIHMR),
Plot No 3, Sector 18 A, Dwarka, New Delhi-110075, India. Email: sumanta.swain@gmail.com
ORCID ID: https://orcid.org/0000-0003-2513-1739 Address for correspondence :
Dr Sumant Swain, Assistant Professor
International Institute of Health Management Research (IIHMR),
Plot No 3, Sector 18 A, Dwarka, New Delhi-110075, India. Email: sumanta.swain@gmail.com
ORCID ID: https://orcid.org/0000-0003-2513-1739 This is an open access journal, and articles are distributed under
the terms of the Creative Commons Attribution Non Commercial
Share Alike 4.0 License, which allows others to remix, tweak,
and build upon the work non commercially, as long as
appropriate credit is given and the new creations are licensed
under the identical terms. How to cite this article: Akanksha, Modi N, Shaikh N,
Swain S. 2022.An assessment of knowledge and utilisation
of Mission Indradhanush at Qutub Vihar, New Delhi: A
cross-sectional study. THE THIRD VOICE REALITY AND
VISION. Vol No-4, Issue No-2, December; P: 28-33. Doi:
For reprints contact: voiceforvoiceless2013@gmail.com
Received
Reviewed
Accepted
Published
17-Aug-2022
18-Sept-2022
29-Oct.-2022
10-Nov-2022
Volume
Issue
Nov. ISSN
No. 4
No. 2
2022
2583-1852(P) Quick Response Code:
Available online at :
thirdvoice.voiceforvoiceless.in
DOI:
Article No - TVRV00020
ACCESS THIS ARTICLE ONLINE Quick Response Code: How to cite this article: Akanksha, Modi N, Shaikh N,
Swain S. 2022.An assessment of knowledge and utilisation
of Mission Indradhanush at Qutub Vihar, New Delhi: A
cross-sectional study. THE THIRD VOICE REALITY AND
VISION. Vol No-4, Issue No-2, December; P: 28-33. MATERIALS AND METHODS India has been implementing the largest immunization
programme in the country which annually covers more
than thirty million pregnant women and twenty-six million
children through the Universal Immunization Programme
(UIP)[3]. This programme was introduced in 1978 as
the ‘Expanded Programme of Immunization’ (EPI) by
the Ministry of Health and Family Welfare, Government
of India.This programme was renamed the ‘Universal
Immunization Programme,’ in 1985 and covered all
districts in the country by 1989-90. After many years of
implementation, UIP has only been able to fully immunize
65 percent of children in their first year of life[4]. Under
this programme, India provides free vaccination against
vaccine-preventable diseases such as diphtheria, polio,
pertussis,tetanus,measles, a severe form of childhood
tuberculosis, hepatitis B, meningitis, and pneumonia
(Hemophilus influenza type B infections), and Japanese
encephalitis (JE) in JE endemic districts with the
introduction of newer vaccines such as rotavirus
vaccine[5]. The Mission Indradhanush, a flagship
programme launched by the Government of India in 2014,
aims to achieve full immunization coverage for children
up to two years of age and pregnant women. India has
also launched Intensified Mission Indradhanush2.0 to
cover the unreachable with all accessible vaccinations
and speed up the coverage of children and pregnant
women in the designated districts and blocks from
December 2019 to March 2020. As of April 2021, during
the various phases of Mission Indradhanush, a total of
38.6 million children and 9.68 million pregnant women
have been vaccinated[6]. India launched Intensified
Mission Indradhanush(IMI) 4.0 on 7 February 2022 to
enhance the coverage of the Universal Immunization
Programme (UIP). The NFHS-5 report revealed that
full immunization coverage among children aged 12-23
months of age has increased from 62 percent (NFHS-
4) to 76.4 percent (NFHS-5) at the national level. According to this report, Delhi has 80 percent
immunization status with rural Delhi having 79 percent
immunization coverage for children. All the districts in
Delhi are listed under intensified routine immunization
in 2015. Southwest Delhi has 77 percent full immunization
coverage according to NFHS 5 data So the Southwest A community-based cross-sectional study was conducted
in the peri-urban area of Qutub Vihar, Goyla Dairy, South
West district, New Delhi between the period of June
2022 and August 2022. A small study was planned with
around twenty-five hundred of the population residing in
a part of this area. INTRODUCTION Doi: 28 8
© THE THIRD VOICE REALITY AND VISION : Vol No-4 | Issue No-2 | November,2022 | ISSN-2583-1852 (P) Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush to improve health and well-being. The objective of this
agenda is to maintain hard-won gains in immunization,
recover from the disruptions caused by COVID-19, and
achieve even more – by leaving no one behind, in any
situation or at any stage of life[2]. district of Delhi is lacking behind the immunisation target. With this cross-sectional study, we aimed to assess the
knowledge and utilisation of Mission Indradhanushat
Qutub Vihar, New Delhi, India. MATERIALS AND METHODS The study participants were mothers
of children of age less than 2 years from different age
groups, education levels, and diverse experience in the
immunization programme. Calculated using Stat Cal sample size calculator of Epi
Info for simple random sampling in a population survey
with the expected frequency of 14.85 percent, with a 10
percent acceptable margin of error, a design effect of
one, and a 95 percent of confidence level, the sample
size was estimated as 155. The snowball sampling technique was used for gaining
the desired numbers. The technique was followed in two
steps: (1) identification of mothers of all the children of
age 0-2 years from the ASHA registers in the Qutub
Vihar area. (2) These participants were asked to give
information about immunization-related information and
so on. This was continued till all 155 mothers were
identified and included in this study. The protocol was approved by the IIHMR Student
Research Review Board.After obtaining informed
consent, data on socioeconomic characteristics, parental
awareness regarding Mission Indradhanush, vaccination
status of the child, parental knowledge, attitude, and
practices regarding routine vaccination were collected
using a structured questionnaire designed through a
google form. Socio-economic status was assessed using
the Modified Kuppuswamy Scale. The collected data
were coded and analyzed with the help of STATA. Descriptive statistics and cross-tabulation were mainly
used for data analysis. RESULTS (Table:3)
Table 3: Source of information on Immunization
services
Source of Immunization services
Frequency
Percentage
ASHA
118
76%
AWW
17
11%
ANM
6
4%
INFORMAL SOURCES
14
9%
N
155
100%
Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush No-4 | Issue No-2 | November,2022 | ISSN-2583-1852 (P)
Table 2: Characteristics of studypopulation P Value
Variables
Awareness
P value
Awareness on P value
of Mission
Immunization
Indra Dhanush
Age of Mother
Less than 20 years
3
0.708
12
0.027
20-30 years
12
52
More than 30 years
2
5
Education Status of Mother
Uneducated
6
0.975
34
0.00
Up to High School
8
29
High School and above 3
6
Education Status of Father
Uneducated
8
0.002
26
0.002
Up to High School
4
31
High School and above 4
11
Occupation of Father
Unemployed
1
0.44
3
0.08
Wages
3
19
Regular Salaried
5
15
Self Employed
8
32
Place of Delivery
Institutional Delivery
14
0.105
61
0.108
At Home
3
8
Sources of information are very important to avail of
the services. 76 percent of the total participants got to
know about the immunization services from ASHA, 11
percent from AWW, 4 percent from ANM, and 9
percent from informal sources or self. (Table:3)
Table 3: Source of information on Immunization
services
Source of Immunization services
Frequency
Percentage
ASHA
118
76%
AWW
17
11%
ANM
6
4%
INFORMAL SOURCES
14
9%
N
155
100% were illiterate, 49 percent had an education up to high
school and only 18 percent hadan education above high
school. 86 percent of women were housewives and only
14 percent were employed. 92 percent of women had
undergone institutional delivery and 8 percent had home
delivery. This study found that 24percentof fathers of the child
were illiterate, 50 percent had an education up to high
school, and only 26percenthadan education above high
school. 5 percent of the fathers were unemployed,
35percentworked daily, 24percentwere regularly
salaried, and 36 percent were self-employed. RESULTS Out of a total of 155 participants, 11 percent of mothers
were in the age group (less than 20 years), 75 percent
in the age group (20-30 years), and 14 percent in the
age group (more than 30 years). 33 percent of mothers 29 © THE THIRD VOICE REALITY AND VISION : Vol No-4 | Issue No-2 | November,2022 | ISSN-2583-1852 (P) Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush rcent had an education up to high
ercent hadan education above high
women were housewives and only
ployed. 92 percent of women had
al delivery and 8 percent had home
at 24percentof fathers of the child
rcent had an education up to high
ercenthadan education above high
f the fathers were unemployed,
daily, 24percentwere regularly
cent were self-employed. (Table:1)
stics of the study population
ics
Frequency (N)
Percentage
18
11%
116
75%
21
14%
51
33%
75
49%
29
18%
134
86%
21
14%
143
92%
12
8%
her
Frequency
Percentage
38
24%
79
50%
38
26%
7
5%
55
35%
38
24%
Table 2: Characteristics of studypopulation P Value
Variables
Awareness
P value
Awareness on P value
of Mission
Immunization
Indra Dhanush
Age of Mother
Less than 20 years
3
0.708
12
0.027
20-30 years
12
52
More than 30 years
2
5
Education Status of Mother
Uneducated
6
0.975
34
0.00
Up to High School
8
29
High School and above 3
6
Education Status of Father
Uneducated
8
0.002
26
0.002
Up to High School
4
31
High School and above 4
11
Occupation of Father
Unemployed
1
0.44
3
0.08
Wages
3
19
Regular Salaried
5
15
Self Employed
8
32
Place of Delivery
Institutional Delivery
14
0.105
61
0.108
At Home
3
8
Sources of information are very important to avail of
the services. 76 percent of the total participants got to
know about the immunization services from ASHA, 11
percent from AWW, 4 percent from ANM, and 9
percent from informal sources or self. RESULTS (Table:1) Table 1: Characteristics of the study population
Maternal Characteristics
Characteristics
Frequency (N)
Percentage
Age of Mother
Less than 20 years
18
11%
20-30 years
116
75%
More than 30 years
21
14%
Education Status
Uneducated
51
33%
Up to High School
75
49%
High School and above
29
18%
Occupation
Housewife
134
86%
Employed
21
14%
Place of Delivery
Institutional
143
92%
At Home
12
8%
Characteristics of Father
Frequency
Percentage
Educational Status
Uneducated
38
24%
Up to High School
79
50%
High School and above
38
26%
Occupation
Unemployed
7
5%
Wages
55
35%
Regular Salaried
38
24%
Self Employed
55
36% Table 1: Characteristics of the study population
Maternal Characteristics Table 3: Source of information on Immunization
services 30 Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush Out of the total of twenty four participants whose The perception and attitude of the individual differ from
person to person on immunization. 28percentof
participants consider immunization as important, and 72
percent consider it very important. (Table:4) Table 4: Importance of Immunization for child Table 4: Importance of Immunization for child
Rate the Importance
of Immunization for child
Frequency
Percentage
Important
43
28%
Very important
112
72%
N
155
100% Out of 155 participants interviewed, 58 percent (n=90)
were the first child, 24 percent (n=37) was the second
child,14percent(n=22) were the third child and 4 percent
(n=6) wasthe fourth child or more. Out of those 90 first-
parity children, 83 children were fully vaccinated, 30
children out of 37-second parity were fully vaccinated,
14 children out of 22 third-parity children, and only 4 out
of 6 fourth-parity children were fully vaccinated. Out of
the total participants,91 percent got their child immunized
at a government facility, and only 9percentvisit a private
facility for their child vaccination. Out of which 94
percent got their child registered at the nearest health
facility. y
p
p
children were either partially vaccinated or not
vaccinated, fourteen were not aware of the vaccine or
time of vaccination, eight were not in the area at the
time of vaccination day, two children were sick after
past vaccination, one had a bad experience with
healthcare providers, and one had financial difficulty in
accessing the service. Only twopercent of participants
paid the cost of immunization. 17percent of children
experienced adverse effects after immunization. Figure 1: Birth Order of the children (n) Figure 1: Birth Order of the children (n) g
f
( ) RESULTS Out of
these 17 percent, only 8 percent (n=5) report to health
facilities the adverse effect after immunization. Out of
these 8 percent, 60 percent (n=3) visited UPHC and 40
percent (n=2) visited private clinics. The study instrument
also asked about personal experiences at the time of the
facility visits. 20percentof participants out of the
8percent(n=5) have given scale ratings. 60 percent have
given a four-scale rating and 20percent have given five
a scale rating for the level of satisfaction with facility
services. 90 percent of the total participants were aware
of the next immunization schedule. Acknowledgments: We would like to thank Director Prof. (Dr.) Sutapa B
Neogi for her support, and guidance and for facilitating
the visit to the Goyla diary area. We appreciate the
support and help of Dr.Abhaya Gupta, Dr. Riya Agarwal,
and Tarang Gupta from the International Institute of
Health Management Research (IIHMR), New Delhi,
India during the fieldwork of this study. CONCLUSION immunization. 94 percent of participants got their child
registered at a nearby facility. Most of the participants
had 2 children (42%) and 1st child was mostly immunized
(58%). The education of the father and mother has a
great impact on immunization and role of gender has a
greater role in immunization coverage. Mission Indradhanush is the most important flagship
program to immunize children. Over a period, this
programme has been modified to increase immunization
coverage including migrants, vulnerable populations,
children in pockets of less immunization coverage, and
high-risk, and hard-to-reach areas. Despite many
innovations and strategies, this programme faced lots of
problems in urban areas mainly peri-urban areas. IEC
activities and training of frontline health workers need
to be strengthened to succeed in this programme. The
collective and collaborative efforts of the healthcare
providers, communities, and beneficiaries would be
required to achieve the target of full immunization
coverage in the country, especially in the peri-urban
areas. This study showed that the IEC campaign is very poor
to reach out at the community level. Only 26 percent of
participants have heard about the mission of
Indradhanush. Most of the participants were unaware
of Mission Indradhanush. ASHA was the major source
of immunization services. Many of the study participants
consider immunization as very important (72%) for
children’s life. A similar study conducted by Summan et
al reported that children with mothers who had graduate
degrees had 75 percent coverage of full vaccination as
compared with 59 percent among children whose mothers
had no schooling. There was also a large urban–rural
divide in vaccination rates. Rural children had
80percentfull vaccination coverage as compared with
66 percent among urban (peri-urban areas) children [7]. Author Contributions: AK, NM & NS conceptualized the study, collected the
data, analysed the results, and drafted the manuscript
and SS analysed and drafted the final manuscript. All
authors read and approved the final manuscript. Financial Support and Sponsorship: Nil. This study found that almost all the children were fully
vaccinated (85%) and only 15 percent were not
vaccinated or partially vaccinated. Frances et al study
also indicated that parents found difficulties in accessing
routine immunization when traveling for work. This study
showed knowledge gaps regarding the benefits and risks
of vaccination, and fears surrounding certain vaccines
due to negative news reports and common side effects
[8]. Most of the participants were aware of the next
immunization schedule, indicating that people are aware
of the vaccination and its importance. The major reason
for partial or no vaccination was either not aware of the
vaccine or time of vaccination or not being in the area
at the time of vaccine day. Vaccination was free of cost
for almost all except for two percent who had to pay for
vaccination. Competing Interests: None declared. DISCUSSION The present study found that most (75%) of the mothers
were in the age group of 20-30 years, had high school
education (49%) and was a housewife (89%), and had
institutional delivery (92%). The majority of the fathers
had a high school education (50%) and were self-
employed (36%). Most of the participants we interviewed
had male children (53%) and were in the age group of
13 to 24 months (43%). Government facilities were
considered by 93 percent of participants for child Out of 155 participants, only 26 percent (n=17) heard
about Mission Indradhanush and among those
26percentparticipants, only 45 percent (n=69) know
about its ongoing immunization services. Out of 155 participants, only 26 percent (n=17) heard
about Mission Indradhanush and among those
26percentparticipants, only 45 percent (n=69) know
about its ongoing immunization services. Out of the total of 155 participants,14 percent of children
were partially vaccinated, 85 percent of children were
fully vaccinated, and 1 percent were not vaccinated. 31 © THE THIRD VOICE REALITY AND VISION : Vol No-4 | Issue No-2 | November,2022 | ISSN-2583-1852 (P) Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush REFERENCES 1. Vaccines and immunization. Accessed from https://
www.who.int/health-topics/vaccines-a nd-
immunization#tab=tab_1 [cited 2022 Dec 18]. 2. Immunization Agenda 2030: A Global Strategy to
Leave No One Behind. Accessedfrom https://
www.who.int/teams/immunization-vaccines-and-
biologicals/strategies/ia2030 [cited 2022 Dec 22]. ANMs were not provided sufficient information
regarding the name of the vaccine, diseases prevented,
normal or adverse effects of the vaccine, and their
management. Only post-immunization instructions were
given by ANM about the next schedule and benefits of
immunization cards. Few participants were not satisfied
with immunization services (11%) at the government
facility level. 3. Focus on Universal Immunization: Dr. Mansukh
Mandaviya launches Intensified Mission Indra
Dhanush (IMI) 4.0. Ministry of Health and Family
Welfare. Government of India. 07 FEB 2022. Accessed
from
https://pib.gov.in/ 32 © THE THIRD VOICE REALITY AND VISION : Vol No-4 | Issue No-2 | November,2022 | ISSN-2583 Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanus Akanksha et al.: An assessment of knowledge and utilisation of Mission Indradhanush PressReleasePage.aspx?PRID=1796099 [cited 2022
Dec 29]. 7. Summan A, Nandi A, Schueller E, Laxminarayan R. Public health facility quality and child immunization
outcomes in rural India: A decomposition analysis. Vaccine. 2022 Apr 6;40(16):2388-2398. doi: 10.1016/
j.vaccine.2022.03.017. Epub 2022 Mar 16. PMID:
35305825; PMCID: PMC8996686. 4. Immunization. Accessed from https://www.unicef.org/
india/what-we-do/immunization [cited 2022 Dec 18]. 4. Immunization. Accessed from https://www.unicef.org/
india/what-we-do/immunization [cited 2022 Dec 18]. 5. Immunization. National Health Mission. Accessed
from
https://nhm.gov.in/
index1.php?lang=1&level=2&sublinkid=824&lid=220
[cited 2022 Dec 29]. 8. Francis, M.R., Nuorti, J.P., Lumme-Sandt, K. et al. Vaccination coverage and the factors influencing
routine childhood vaccination uptake among
communities experiencing disadvantage in Vellore,
southern India: a mixed-methods study. BMC Public
Health 21, 1807 (2021). https://doi.org/10.1186/
s12889-021-11881-8 6. Focus on Universal Immunization: Dr. Mansukh
Mandaviya launches Intensified Mission Indra
Dhanush (IMI) 4.0. Ministry of Health and Family
Welfare. Government of India. 07 FEB 2022. Accessed from https://pib.gov.in/Press Release
Page.aspx?PRID=1796099 [cited 2022 Dec 29]. 6. Focus on Universal Immunization: Dr. Mansukh
Mandaviya launches Intensified Mission Indra
Dhanush (IMI) 4.0. Ministry of Health and Family
Welfare. Government of India. 07 FEB 2022. Accessed from https://pib.gov.in/Press Release
Page.aspx?PRID=1796099 [cited 2022 Dec 29]. 33 3
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CONTENTS OF THE NOTION AND ROLE OF PUBLIC ORDER AND OTHER THREATS IN THE BORDER CROSSING
|
Latgale National Economy Research/Latgales Tautsaimniecības Pētījumi
| 2,014
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cc-by
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SABIEDRISKĀS KĀRTĪBAS UN CITU APDRAUDĒJUMU
JĒDZIENU SATURS UN LOMA ROBEŽŠĶĒRSOŠANĀ
CONTENTS OF THE NOTION AND ROLE OF PUBLIC ORDER
AND OTHER THREATS IN THE BORDER CROSSING
Artūrs GAVEIKA
Mg. iur., viesdocents, Rēzeknes Augstskola
Rēzekne, Latvija
E–pasts: argavs@inbox.lv
Abstract. The concept of public policy as a legal concept is quite complicated, much
debated concept of jurisprudence, it is reflected in several laws of the Schengen acquis
and in the case law. Due to interpretation problems the attempts to proportionate
balance in public order interests ensuring free movement of persons in the European
Union space have become the subject of a number of judicial precedents in both
separate Schengen Member States as well as throughout the European Union. An
important step in conflict resolution is the concept and terminology analysis and
unification, which in the legal framework of the Schengen acquis must be initiated by
defining the basic concept s such as „public order”, „national security”, „threats to public
health” as well as to harmonize the terminology, the author offers a lecture. That is why
the author’s main suggestion in this study is to work out unitary and harmonized
terminology.
Keywords: border control, public order, public security, threats to public health.
Ievads
Tēma ir aktuāla sakarā ar Latvijas Republikas dalību Eiropas
Savienībā (ES) un Šengenas konvencijas darbības zonā, robežkontroles
normatīvā regulējuma pilnveidošanas nepieciešamību, Latgales reģiona
atrašanos ES ārējo sauszemes robežu tuvumā, uz kurām atrodas
vairākums Latvijas Republikas robežšķērsošanas vietu ar ļoti intensīvu
personu, mantu un transportlīdzekļu robežšķērsošanu (1.att.) un līdz ar
to – ievērojamu likumpārkāpumu un ieceļošanas liegumu skaitu.
Pētījums veikts 2012.-2013.g. periodā, pielietojot normatīvo aktu
un tiesu prakses analīzes, interpretācijas un salīdzināšanas metodes.
Raksta mērķis ir noskaidrot Latvijas robežkontroles normatīvā
regulējuma problemātiku sabiedriskās kārtības un citu apdraudējumu
jēdzienu izpratnē un piedāvāt tiesiskos risinājumus.
Pētījuma objekts ir pamatjēdzienu „sabiedriskā kārtība”, „valsts
drošība”, „sabiedrības veselības apdraudējums” piemērošanas normatīvā
regulējuma problemātika.
Pētījuma priekšmets – ES tiesību Šengenas acquis (Šengenas tiesību
kopums) un attiecīgais nacionālais normatīvais regulējums.
Sociālo zinātņu žurnāls Nr. 1(6)
61
6 000 000
1 614 090
4 418 561
1 650 695
4 511 600
2 400 000
1 400 615
3 600 000
4 137 724
4 800 000
1 200 000
0
2010. gads
2011.gads
2012.gads
personām par 2% (93 039) 2012.g. mazāk nekā 2011.g.
automašīnām par 2 % (36 605) 2012.g. mazāk nekā 2011.g.
1.attēls. Personu un automašīnu robežpārbaužu skaits Latvijā 2010. – 2012.g.
(36)
Pētījuma uzdevumi ir 1) izpētīt minēto jēdzienu tiesisko saturu un
pielietošanas prakses problemātiku un 2) piedāvāt šo jēdzienu
definējumus normatīvā regulējuma pilnveidošanai.
Pilnveidojot pamatjēdzienu „sabiedriskā kārtība”, „valsts drošība”,
„sabiedrības veselības apdraudējums” normatīvo regulējumu, būs
iespējams panākt efektīvāku publiskās kārtības institūciju darbību
robežpārbaudēs, kā robežkontroles būtiskā daļā.
Sabiedriskās kārtības un citu apdraudējumu jēdzieni Šengenas
acquis sistēmā
Šengenas konvencija nosaka, ka kontroli veic saskaņā ar vienotiem
principiem, ievērojot katrā valstī noteikto kompetenci un likumus un
ņemot vērā visu dalībvalstu intereses visās to teritorijās
(1.,6.p.,1.punkts). Attiecībā uz uzturēšanos, kas nav ilgāka par trim
mēnešiem sešu mēnešu laikā, uz trešo valstu valstspiederīgajiem attiecas
šādi ieceļošanas noteikumi (2.,5.p., 1.punkts):
viņam ir noteikts derīgs dokuments vai dokumenti, kas ļauj
šķērsot robežu;
viņam ir derīga vīza, ja tā ir vajadzīga (1.,6.p.,1.punkts) (derīga
vīza vajadzīga saskaņā ar Padomes Regulu (EK) Nr.539/2001
(3.), ar ko izveido to trešo valstu sarakstu, kuru pilsoņiem,
šķērsojot dalībvalstu ārējās robežas, ir jābūt vīzām, kā arī to
trešo valstu sarakstu, uz kuru pilsoņiem šī prasība neattiecas,
izņemot gadījumus, ja viņiem ir derīga uzturēšanas atļauja;
62
Latgales Tautsaimniecības pētījumi
vajadzības gadījumā viņš var uzrādīt dokumentus, kas pamato
paredzētās uzturēšanās mērķi un apstākļus, un viņa rīcībā ir
pietiekami iztikas līdzekļi paredzētās uzturēšanās laikam, kā
arī, lai atgrieztos valstī, no kuras ieceļo, vai tranzītam uz trešo
valsti, kurā ir garantēta viņa uzņemšana, vai arī viņš spēj
likumīgi iegūt šādus līdzekļus;
par viņu nav saņemti ziņojumi, t.sk., SIS (Šengenas
informācijas sistēmā) sakarā ar ko var liegt ieceļošanu;
viņš netiek uzskatīts par tādu, kas var apdraudēt kādas
Šengenas konvencijas dalībvalsts sabiedrisko kārtību, valsts
drošību vai starptautiskās attiecības; atbilstoši Šengenas
Robežu kodeksam– „viņus neuzskata par apdraudējumu kādas
dalībvalsts politikai, iekšējai drošībai, sabiedrības veselībai vai
starptautiskām attiecībām, un, jo īpaši, valstu datubāzēs par
viņiem nav izdots brīdinājums, lai minēto iemeslu dēļ atteiktu
ieceļošanas atļauju).
Salīdzinot uzskaitītās Šengenas konvencijas un Šengenas Robežu
kodeksa normas jāsecina, ka pēc būtības un pamatjēgas tās tiek
savstarpēji dublētas, kaut arī atšķirīgās redakcijās un terminoloģijā, kas
savukārt lielākā vai mazākā mērā deformē šo normu saturu. Tas savukārt
praksē var radīt un rada interpretācijas problēmas, piemēram, sakarā ar
tādu terminu lietošanu, kā derīgs dokuments (ceļošanas dokuments), nav
ziņots (SIS izdots brīdinājums), sabiedriskā kārtība (sabiedrības
veselība), valsts drošība (dalībvalsts politika, iekšējā drošība). Saskaņā ar
Šengenas Robežu kodeksu robežpārbaudes procesa ietvaros personām
var veikt minimālo vai pilno pārbaudi.
Saskaņā ar Šengenas Robežu kodeksa 7.panta 2.punktu minimālo
pārbaudi jāveic ātri un lietišķi, vajadzības gadījumā, izmantojot tehniskus
līdzekļus un pieejamās datubāzes. Minimālas pārbaudes mērķis ir
personas identificēšana, ceļošanas dokumenta derīguma kritēriju
pārbaude, kā arī viltojumu pazīmju neesamības pārbaude. Saskaņā ar
iepriekš minēto pantu, veicot minimālās pārbaudes personām, kas
izmanto Eiropas Kopienas tiesības brīvi pārvietoties, robežsargi tomēr
var izlases kārtībā pārbaudīt informāciju valstu un Eiropas attiecīgās
datubāzēs, lai pārliecinātos, ka attiecīgās personas nerada reālus,
konkrētus un pietiekami nopietnus draudus dalībvalstu iekšējai drošībai,
sabiedriskai kārtībai, starptautiskām attiecībām vai neapdraud
sabiedrības veselību (2.,7.p. 2.punkts). Trešo valstu valstspiederīgos
ieceļojot un izceļojot pārbauda pilnībā:
pārliecinās par to, vai trešās valsts valstspiederīgajam ir
dokuments (pase, vīza vai uzturēšanās atļauja) robežas
šķērsošanai un kura derīguma termiņš nav beidzies;
Sociālo zinātņu žurnāls Nr. 1(6)
63
rūpīgi pārbauda ceļošanas dokumenta derīgumu;
pārbauda ieceļošanas un izceļošanas spiedogu;
pārliecinās par attiecīgā trešās valsts valstspiederīgā ceļojuma
sākumpunktu un galapunktu, kā arī par iecerētās uzturēšanās
mērķi un, vajadzības gadījumā, atbilstīgajiem apstiprinājuma
dokumentiem;
pārliecinās par to, ka attiecīgajam trešās valsts piederīgajam ir
pietiekami iztikas līdzekļi gan iecerētās uzturēšanās laikam un
mērķim, gan lai atgrieztos izcelsmes valstī vai tranzītā dotos uz
trešo valsti;
pārliecinās par to, ka attiecīgais trešās valsts valstspiederīgais,
viņa transportlīdzeklis un vestie priekšmeti neapdraud kādas
dalībvalsts politiku, iekšējo drošību, sabiedrības veselību vai
starptautiskās attiecības; pārliecināšanās ietver tiešu datu
salīdzināšanu ar Šengenas informācijas sistēmā un attiecīgas
valsts datubāzēs esošiem datiem un brīdinājumiem par
personām un, vajadzības gadījumā, par priekšmetiem, kā arī
veicamās darbības, ko attiecīgā gadījumā veic, saņemot
brīdinājumu.
Pilna pārbaude izceļojot ir:
pārliecināšanās par to, ka trešo valstu valstspiederīgajam ir
robežas šķērsošanai derīgs dokuments;
rūpīga ceļošanas dokumenta pārbaude, vai tam nav viltojuma
pazīmju;
pārliecināšanās par to, ka trešās valsts valstspiederīgais nav
uzskatāms par draudu kādas dalībvalsts politikai, iekšējai
drošībai vai starptautiskām attiecībām;
Šengenas Robežu kodekss nosaka, ka robežkontroles atcelšana pie
iekšējām robežām neietekmē policijas pilnvaras, ko īsteno kompetentas
dalībvalstu iestādes saskaņā ar attiecīgās valsts tiesību aktiem, ja vien
pilnvaru īstenošana iedarbības ziņā nav līdzvērtīga robežpārbaudēm (tas
attiecas arī uz pierobežas teritorijām), jo:
par mērķi neizvirza robežkontroli;
balstās uz vispārēju tiesībsargājošos iestāžu informāciju un
pieredzi attiecībā uz iespējamiem sabiedriskās drošības
apdraudējumiem un konkrēti paredzēta pārrobežu
noziedzības apkarošanai;
veikta tā, ka tā noteikti atšķiras no sistemātiskajām personu
pārbaudēm pie ārējām robežām;
tiek veikta izlases kārtībā (2.,21p.).
64
Latgales Tautsaimniecības pētījumi
Robežkontroles atjaunošanas kontekstā būtisks ir Šengenas
Robežu
kodeksa
noteiktais
termins
„sabiedrības
veselības
apdraudējums” – slimība, kas potenciāli var izvērsties epidēmijā, kā
noteikts Pasaules Veselības organizācijas starptautiskajos veselības
aizsardzības noteikumos, kā arī citas infekcijas slimības vai lipīgas
parazītu slimības, ja uz tām attiecas aizsardzības noteikumi, kas attiecas
uz dalībvalstu valstspiederīgajiem (2.,2p.19.punkts). Šāds nosacījums ir
iekļauts uz ārējām robežām regulāri veicamo robežpārbaužu
(2.,7.p.2.punkts) drošības nosacījumos, taču, tāpat kā sabiedriskās
kārtības apdraudējums, nav minēts pie robežkontroles atjaunošanas
iemesliem uz iekšējām robežām, kaut gan tiek minēti tādi iemesli, kā
nopietns apdraudējums valsts politikai vai iekšējai drošībai. Turklāt
valsts politikas apdraudējuma saturs un jēga nav atklāta Šengenas acquis
ietvaros. 1994.g. Valsts robežas likuma (13.) (zaudējis spēku) 15.pants
noteica, ka, ja Latvijas Republikā vai kaimiņvalsts teritorijā draud
izplatīties infekcijas slimības, Valsts robežsardze, saskaņā ar MK lēmumu,
varēja apdraudētajos rajonos uz laiku ierobežot vai pārtraukt satiksmi
pāri valsts robežai. Taču 2009.g. Valsts robežas likumā (7.) šāda norma
vispār nav paredzēta, bet ir iekļauta norma par robežkontroles pagaidu
atjaunošanu uz iekšējās robežas tikai saskaņā ar Šengenas Robežu
kodeksa 23.panta 1.punkta noteikumu – “nopietns apdraudējums valsts
politikai vai iekšējai drošībai”.
Turpretī Krievijas Federācijas normatīvais regulējums valsts
drošības interesēs (arī pēc kaimiņvalstu lūguma) paredz iespēju slēgt
valsts robežu un uz noteiktu laiku pārtraukt personu kustību pāri robežai
(15,cт.9.), kā arī darbības sakarā ar sabiedrības veselības apdraudējumu,
ekoloģijas
apdraudējumu
un
citiem
apdraudējumiem,
t.sk.
kriminālpārkāpumu un administratīvo pārkāpumu apdraudējumiem (15,
cт.13.,14.). Līdzīga norma ir iekļauta arī Baltkrievijas Republikas
nacionālajā normatīvajā regulējumā (16, cт.5.) un līgumā par valsts
robežas režīmu starp Latvijas Republiku un Baltkrievijas Republiku, kurā
sabiedriskās kārtības un valsts drošības apdraudējumu regulējums ir
diezgan plašs un detalizēts: ekoloģiskais, sanitār–epidemioloģiskais,
dabas un tehnogeno katastrofu, starptautiskās noziedzības un nelegālās
migrācijas apdraudējumu diezgan konkrēts uzskaitījums (6, cт.26.–
29.,33.). Savukārt Šengenas Robežu kodeksa termins „sabiedrības
veselības apdraudējums” ir definēts pārāk sašaurināti, jo paredz
sabiedrības veselības apdraudējumus tikai no slimībām, taču tāds
apdraudējums var rasties arī dažādu ārkārtas situāciju (Gaveika, 2011)
rezultātā no dabas un tehnogēnajām katastrofām un avārijām.
Sociālo zinātņu žurnāls Nr. 1(6)
65
Apdraudējumu jēdzienu lietošanas judikatūra
Šengenas konvencijas 5.pants nosaka, ka tie ārvalstnieki, kas tiek
ielaisti kopējā brīvas pārvietošanās zonā, nedrīkst “tikt uzskatīti par
draudu jebkuras dalībvalsts sabiedriskajai kārtībai, nacionālajai drošībai
vai starptautiskajām attiecībām”. Šāda vienāda principa piemērošana uz
ārējām robežām nav vienkārša, jo personas tiek izvērtētas balstoties uz
nacionālo tiesību un tradīciju kritērijiem, kuri ir atšķirīgi dažādās
dalībvalstīs (11.,28.punkts). Sabiedriskās kārtības jēdziens ir diezgan
sarežģīts, kā arī plaši diskutēts juridisks jēdziens (Dubure, Fogels,
Fridrihsons u.c., 1998, 230.lpp.), tas ir sastopams vairākos Šengenas acquis
normatīvajos aktos un tiesu praksē, piemēram, lietā Ministerul
Administraţiei şi Internelor – Direcţia Generală de Paşapoarte Bucureşti
pret Gheorghe Jipa, kurā tiesa atzina, ka atbilstoši judikatūrai
“sabiedriskās kārtības jēdziens katrā ziņā nozīmē, ka papildus
sabiedriskiem traucējumiem, ko rada jebkurš likumpārkāpums, pastāv
faktisks, attiecīgajā brīdī esošs un pietiekami nopietns apdraudējums
sabiedrības pamata interesēm”. Turklāt, ņemot vērā šauro interpretāciju,
kāda ir izmantojama attiecībā uz visām atkāpēm no tiesībām uz
pārvietošanās brīvību, šīs brīvības ierobežojums ir pieļaujams vienīgi
tiktāl, ciktāl tas “ir pamatots tikai ar attiecīgā indivīda personisko
darbību, jo nav pieņemami apsvērumi, kas atdalīti no konkrētā gadījuma
iezīmēm vai pamatojas uz vispārējas profilakses apsvērumiem”, kā
rezultātā “pasākums, ar kuru tiek ierobežotas tiesības brīvi pārvietoties,
ir jāveic, ņemot vērā apsvērumus tikai par dalībvalsts, kura veic šo
pasākumu, sabiedriskās kārtības aizsardzību”. Tiesa arī atgādināja, ka “lai
arī dalībvalstīm saskaņā ar to vajadzībām, kuras katrā dalībvalstī un laika
periodā var atšķirties, principā ir tiesības noteikt sabiedriskās kārtības
un valsts drošības prasības, tomēr Kopienu kontekstā un it īpaši kā
pamatojums atkāpei no personu brīvas pārvietošanās pamatprincipa šīs
prasības ir jāinterpretē šauri un to saturu nevar noteikt katra dalībvalsts
vienpusēji bez [Savienības] iestāžu kontroles”(20., 22., 23.punkts).
Interpretācijas problēmu dēļ, mēģinājumos samērīgi sabalansēt
sabiedriskās kārtības nodrošināšanu ar personu brīvas pārvietošanās
nodrošināšanu, ir kļuvis par vairāku tiesu precedentu priekšmetu kā
atsevišķu Šengenas dalībvalstu (22.). Ar sabiedrisko kārtību var saprast
gan tādu kārtību sabiedriskās vietās, kas izpaužas realizējamās
subjektīvajās tiesībās un izpildāmos pilsoņu pienākumos, gan kā pilsoņu
tiesību un brīvību pastāvīgu aizsargāšanu, visu ar likumu amatpersonām
un pilsoņiem noteikto pienākumu obligātās izpildes uzraudzību, gan arī
citādākās interpretācijās. Sabiedriskās kārtības jēdziena skaidrojums nav
atrodams ne Šengenas acquis ietvaros, ne pat dažu valstu nacionālajā
normatīvajā regulējumā, kaut gan tieši no Rietumeiropas valstīm
66
Latgales Tautsaimniecības pētījumi
Francijas un Vācijas šis jēdziens 18.gs. guva izplatību Austrumeiropas un
citās valstīs (Бельский, 2004, c.231.).
Šengenas konvencijā noteikts, ka persona var ieceļot, ja tā netiek
uzskatīta par tādu personu, kura var apdraudēt kādas dalībvalsts
sabiedrisko kārtību, valsts drošību vai starptautiskās attiecības
(1.,5.p.1.punkts e) apakšpunkts). Savukārt Kodeksā noteikts, ka tai nav
jābūt personai, kuru uzskata par apdraudējumu kādas dalībvalsts
politikai, iekšējai drošībai, sabiedrības veselībai vai starptautiskām
attiecībām, un valstu datubāzēs par šo personu nav izdots brīdinājums,
lai minēto iemeslu dēļ atteiktu ieceļošanu (2.,5.p.,1.punkts
e) apakšpunkts).
Vairāku jēdzienu, piemēram, apdraudējumu definēšanas
daudzveidība („sabiedriskā kārtība vai valsts drošība” (2.,2.p.2.punkts),
„starptautisko attiecību apdraudējums” (2,5.p.e)punkts), „sabiedrības
veselības apdraudējums” (2.,2.p.19) punkts) „nopietns apdraudējums
valsts politikai vai iekšējai drošībai” (2.,23.p.1.punkts)) un citas
neprecizitātes ir novedušas pie Šengenas acquis vairāku pamatjēdzienu
dažādās interpretācijas un līdz ar to arī pie nekonsekvences Šengenas
konvencijas īstenošanā. Tā Lietas C–348/09 (18.) secinājumos tika atzīts,
ka seksuāla vardarbība pret četrpadsmitgadīgu nepilngadīgo, vardarbīga
dzimumtieksmes apmierināšana un izvarošana neietilpst nopietnu
(primāru) valsts drošības apsvērumu jēdzienā gadījumā, ja šīs darbības
tieši neapdraud iedzīvotāju mieru un fizisko drošību kopumā vai lielā tās
daļā, pat neskatoties uz to, ka vainīgais ir sodīts ar ilgstošu cietumsodu
un nav pat atzinis savu vainu, un tas palielina recidīva risku, tātad
apdraudējumu sabiedrībai (18.). Pretēji Šengenas konvencijas 96.pantā
noteiktajai sabiedriskās kārtības un valsts drošības interpretācijai, kas
paredz, ka šāds apdraudējums var izrietēt no ārvalstnieka, kas notiesāts
par noziedzīgu nodarījumu, par kuru paredzēta brīvības atņemšana
vismaz uz vienu gadu, vai ārvalstnieku, par kuru ir pamats uzskatīt, ka
viņš ir izdarījis smagus noziedzīgus nodarījumus, direktīvas 2004/38 (4.)
28.pantā sabiedriskās kārtības un valsts drošības jēdzieni jau ir nošķirti.
Direktīvas 2.punktā noteikts, ka dalībvalsts nedrīkst pieņemt lēmumu par
tādu Savienības pilsoņu vai viņu ģimenes locekļu izraidīšanu neatkarīgi
no valstiskās piederības, kuriem ir tiesības pastāvīgi uzturēties tās
teritorijā, izņemot nopietna sabiedriskās kārtības apdraudējuma vai
valsts drošības apsvērumu dēļ (4,28.p.). Savukārt direktīvas 2004/38
3. punktā noteikts, ka lēmumu par izraidīšanu nedrīkst pieņemt pret ES
pilsoņiem, izņemot, ja lēmums pamatojas uz nopietniem valsts drošības
apsvērumiem, ko definējušas dalībvalstis, t.i. ja pilsoņi:
a) ir uzturējušies uzņēmējā dalībvalstī iepriekšējos desmit gadus;
Sociālo zinātņu žurnāls Nr. 1(6)
67
b)
ir nepilngadīgi, izņemot, ja izraidīšana ir vajadzīga bērna
interesēs.
Iepriekšminēto jēdzienu salīdzinājums Direktīvas 2004/38
28. panta 2. un 3.punktā skaidri norāda uz atšķirību starp sabiedriskās
kārtības un valsts drošības jēdzieniem, no kuriem otrais norāda uz
augstāku svarīguma pakāpi nekā pirmais attiecībā uz to, kādos apstākļos
var nepiemērot ES pilsoņiem noteikto paplašināto aizsardzību. Abu šo
jēdzienu piemērošana krimināltiesību jomā atbilst divām atšķirīgām
krimināltiesiskām situācijām. Katra dalībvalsts ar tās nacionālajām
tiesībām nosaka savu sabiedrisko kārtību, jo tā definē, kāda veida rīcība
ir aizliegta, paredzot kriminālsodu. Šajā ziņā ir skaidrs, ka visas
krimināltiesību normas attiecas uz sabiedrisko kārtību tādējādi, ka šīs
tiesību normas pēc būtības ir obligātas un ar individuālu gribu nevar
izvēlēties tās neievērot. Tās ir radītas tieši tam, lai pakļautu individuālo
gribu, kuras sekas tiek uzskatītas kā sabiedrības vērtībām kaitējošas vai
bīstamas. Šo tiesību normu neievērošana rada dalībvalsts noteiktās
sabiedriskās kārtības traucējumu, kas ir lielāks vai mazāks atkarībā no
izdarītā noziedzīgā nodarījuma rakstura, jo sabiedriskās kārtības
traucējums parasti atspoguļojas valsts likumdevēja paredzētā soda
smagumā aizliegtās rīcības sodīšanai. Katrā konkrētajā lietā šī
izvērtēšana un attiecīgā gadījumā – izsvēršana, izpaužas faktiski
noteiktajā sodā, kas, ņemot vērā katras lietas raksturīgos apstākļus,
raksturo reāli nodarītā likumpārkāpuma pakāpi (4.). Policijas tiesību
zinātnieks Dr. A.Matvejevs norāda, ka sabiedriskā kārtība ir tāda kārtība
publiskās vietās, kura izpaužas cilvēku realizējamās subjektīvās tiesībās
un tiesību normās noteiktos izpildāmos pienākumos. Sabiedriskās
kārtības svarīga iezīme – regulēšana ar tiesību un morāles normām.
Turklāt galvenais uzsvars nosaukto sabiedrisko attiecību regulēšanā tiek
virzīts uz administratīvo un kriminālo tiesību normām. Mazāk bīstami
likumpārkāpumi, kas traucē sabiedrisko kārtību un sabiedrības drošību,
tiek kvalificēti kā administratīvie pārkāpumi, par kuriem Latvijas
Administratīvo pārkāpumu kodekss (turpmāk – LAPK) paredz
administratīvo atbildību (Matvejevs, 2009, 122.–123.lpp.).
Minēto
Šengenas
acquis
pamatjēdzienu
interpretācijas
problemātika izpaužas arī atsevišķos Eiropas Komisijas nekonsekventos
pārmetumos saistībā ar valstu iekšējās robežas šķērsojošo personu
sūdzībām par pārbaudēm pierobežā 2010.g. sakarā ar iespējamajām
regulārām pārbaudēm, kuras veic dažās iekšējās pierobežas zonās,
šķēršļiem netraucētām satiksmes plūsmām autoceļu robežšķērsošanas
vietās pie iekšējām robežām un kavētu paziņošanu par plānotu
robežkontroles atjaunošanu pie iekšējām robežām (27.). Taču nedaudz
vēlāk Eiropas Komisija, norūpējusies par Āfrikas politiskās krīzes
68
Latgales Tautsaimniecības pētījumi
izraisīto nelegālās imigrācijas risku Eiropā, ierosināja paredzēt stingrāku
Šengenas noteikumu piemērošanu un strukturētāku lēmumu
pieņemšanas mehānismu robežkontroles pagaidu atjaunošanā pie
iekšējām robežām, ja pastāv nopietns apdraudējums sabiedriskajai
kārtībai vai iekšējai drošībai tuvāk un konkrētāk neatklājot šo jēdzienu
juridisko saturu un apjomu (28.).
Ārkārtas apstākļos var uz laiku atjaunot robežkontroli pie iekšējām
robežām (2.,15. punkts), ja pastāv nopietns apdraudējums sabiedriskajai
kārtībai vai iekšējai drošībai. Iespēja atjaunot robežkontroli pie iekšējām
robežām ES mērogā tika izmantota ne mazāk kā 26 reizes. Vairumā
gadījumu robežkontroles atjaunošana notika saistībā ar liela mēroga
sporta pasākumiem, politiskajām demonstrācijām vai augsta līmeņa
politiskajām sanāksmēm (11.). Tā, piemēram, lai novērstu iespējamus
apdraudējumus NATO Parlamentārās Asamblejas pavasara sesijas norisei
Rīgā no 2010.g. 28.maija līdz 1.jūnijam, bija atjaunota pagaidu
robežkontrole uz iekšējām robežām (8.).
Personu kontroles atcelšana uz iekšējām robežām ļauj robežu
šķērsot ne tikai pilsoņiem, bet arī ārvalstniekiem, kuri var ieceļot un
uzturēties Šengenas valstu teritorijā līdz 3 mēnešiem, ja tiem ir derīgs
personu apliecinošs dokuments un vīza (ja tāda nepieciešama) (1.,5.p.).
Taču ārvalstniekiem var atteikt ieceļot Šengenas teritorijā, ja tie rada
draudus sabiedriskajai kārtībai un drošībai, informācija par kuriem tiek
iegūta no Šengenas informācijas sistēmas visos robežkontroles punktos
uz ārējām robežām visās Šengenas zonas valstīs. Ģenerāladvokāta
P.Mengoci (Paolo Mengozzi) secinājumos Lietā C–84/12 (17.) atzīts, ka ES
līmenī nav precīzi definēti Vīzu kodeksa 21.pantā un 32.panta 1.punktā
minētie ieceļošanas nosacījumi, kā arī riska izvērtējums un atteikuma
pamatojumi, kas var novest pie nepareiza lēmuma pieņemšanas vīzas
izsniegšanas procedūrā (14.). Līdztekus Šengenas robežu kodeksā un
Vīzu kodeksā noteiktajiem dalībvalsts politikas, iekšējās drošības,
sabiedrības veselības un starptautisko attiecību apdraudējumiem, kas
minēti pie ieceļošanas atteikuma iemesliem, Vīzu kodeksā tiek noteikti
papildus tādi vīzas izsniegšanas nosacījumi kā nelegālās imigrācijas
draudu neesamība, ieceļošanas mērķa pamatotība, iesniegto vīzas
pieteikuma dokumentu autentiskums, medicīniskā apdrošināšana un
uzturēšanās līdzekļu esamība (14.,21.,32.p.).
Tiesībsargājošo institūciju darbā ļoti svarīga ir Šengenas
konvencijas ceturtā sadaļa „Šengenas informācijas sistēma”, kas paredz
globālu informācijas sistēmu likumpārkāpumu apkarošanai un
dalībvalstu sadarbībai. Ar SIS izmantošanu ir saistītas nozīmīgas
izmaiņas imigrācijas procesa regulējumā katrā Šengenas dalībvalstī (27.),
lai stiprinātu sabiedrisko kārtību un drošību dalībvalstu teritorijā,
Sociālo zinātņu žurnāls Nr. 1(6)
69
nodrošinot ziņojumu pieejamību dalībvalstu kompetentajām institūcijām
un iestādēm (12.,1.p.), kaut arī šie ziņojumi dažkārt nesatur pietiekamu
sabiedriskās kārtības interešu pamatojumu, lai liegtu ieceļošanu
personām (21.). SIS ir kopīga tiesībaizsardzības iestāžu datu bāze, kurā
līdz 2012.g. bija ievadīti vairāk nekā 40 miljoni ziņojumu (skaits pieaug
par apmēram 3% mēnesī) (31.) no 28 valstīm (32.), ieskaitot Rumāniju
un Bulgāriju, kaut arī tās vēl joprojām nav Šengenas zonas dalībvalstis
(5.). No 2008.g. līdz 2013.g. SIS brīdinājumu kopskaits pieauga no 22,9
līdz 44 miljoniem (33.). SIS datubāzes ietilpība bija ierobežota tehnisko
iespēju dēļ. Bija plānots, ka līdz 2008.g. 31.dec. sāks darboties jauna
sistēma SIS II ar biometrijas datu izmantošanu un nacionālo informācijas
sistēmu integrēšanu, kas gala rezultātā sāka darboties tikai 2013.g. maijā
(31.).
Var arī piekrist H.J.Šreteram, ka harmonizācija ir ES tiesību un
pienākumu kopuma jēdziens un nozīmē atsevišķo ES dalībvalstu
atšķirīgo tiesisko un pārvaldes priekšrakstu saskaņošanu (35.,117.,
145.lpp.).
Konsolidētā Eiropas Kopienas dibināšanas līguma 307.pants
paredz, ka līgums neietekmē dalībvalstu starptautiski tiesiskās saistības,
kuras tās uzņēmušās pret vienu vai vairākām trešajām valstīm. Taču
saskaņā ar Kopienu Tiesas lēmumu lietā Commission v.Italy, Case [1962]
(23., 11.punkts) attiecībās ar citām dalībvalstīm piemērojami Eiropas
Kopienu tiesību akti. Turklāt, ja dalībvalsts konstatē savu starptautiski
tiesisko saistību nesaderību ar Eiropas Kopienu tiesību normām, tai
jāveic viss nepieciešamais, lai nesaderību novērstu.
Starptautiskās konvencijas par jūras satiksmes atvieglošanu (FAL)
5.panta otrajā daļā noteikts, ka „nekas šajā Konvencijā vai tās pielikumā
nevar tikt interpretēts kā aizliegums Līgumslēdzējvalsts valdībai
piemērot spēkā esošos pasākumus, ko šī valdība uzskata par vajadzīgiem,
lai saglabātu sabiedrisko morāli, kārtību un drošību vai lai novērstu
sabiedrības, dzīvnieku vai augu veselību apdraudošu slimību vai kaitēkļu
izplatību vai ievešanu „(9.).
Secinājumi un priekšlikumi
1.
Šengenas līgums un Šengenas konvencija ir viens no
ievērojamākajiem personu brīvas pārvietošanās sasniegumiem
starptautiskajā mērogā. Ņemot vērā šo līgumu ģeopolitisko nozīmi
un vistiešāko ietekmi uz konstitucionālajām tiesībām un
dalībvalstu suverenitāti, tie būtu pieskaitāmi pie ES dibināšanas
līgumiem, jo ar tiem tiek dibināta vienota personu brīvas
pārvietošanās telpa.
70
Latgales Tautsaimniecības pētījumi
2.
3.
4.
Salīdzinot sabiedriskās kārtības un citu apdraudējumu normas
Šengenas konvencijā un Kodeksā, jāsecina, ka pēc būtības un
pamatjēgas tās tiek savstarpēji dublētas, kaut arī atšķirīgās
redakcijās un terminoloģijā, kas savukārt lielākā vai mazākā mērā
deformē arī šo normu saturu. Tas savukārt praksē var radīt un rada
interpretācijas problēmas, piemēram, attiecībā uz tādu terminu
lietošanu kā: Šengenas konvencijā – nav ziņots, Kodeksā – SIS
izdots brīdinājums, tiesas spriedumā – „personas, par ko [Šengenas
informācijas sistēmā (SIS)] izdots brīdinājums, lai atteiktu
ieceļošanu”; Šengenas konvencijā – sabiedriskā kārtība, valsts
drošība vai starptautiskās attiecības, Kodeksā un tiesu praksē –
neuzskata par apdraudējumu kādas dalībvalsts politikai, iekšējai
drošībai, sabiedrības veselībai vai starptautiskām attiecībām, un, jo
īpaši, valstu datubāzēs par viņiem nav izdots brīdinājums, lai
minēto iemeslu dēļ atteiktu ieceļošanas atļauju.
Vairāki Rietumeiropas tiesību zinātnieki uzsver ES tiesību normu
harmonizācijas nepieciešamību, paredzot, ka ES tiesību kopuma
jēdziens nozīmē atsevišķo ES dalībvalstu atšķirīgo tiesisko un
pārvaldes priekšrakstu saskaņošanu. Taču autors uzsver arī ES
tiesību un starptautisko tiesību harmonizācijas nepieciešamību, kas
būtu īpaši svarīgs sabiedriskās kārtības un citu apdraudējumu
jēdzienu precizēšanā intensīvas pārrobežu komunikācijas
apstākļos, nosakot Šengenas acquis sistēmā, ka sabiedriskā kārtība
ir visu personām ar normatīvo regulējumu noteikto pienākumu
pastāvīga un precīza izpilde sabiedrībā, ja par sabiedriskās kārtības
likumpārkāpumiem saskaņā ar dalībvalsts nacionālo normatīvo
regulējumu ir paredzēta atbildība ne mazāka kā brīvības
atņemšana vismaz uz vienu gadu. Šāds regulējums būtu jāparedz
Šengenas robežu kodeksā 2.pantā (definīcijas).
ES līmenī nav unificēti un precīzi definēti arī dalībvalsts politikas,
iekšējās drošības, sabiedrības veselības, starptautisko attiecību
apdraudējumi, kas Šengenas robežu kodeksā minēti pie ieceļošanas
atteikuma iemesliem, bet Vīzu kodeksā papildus noteikti tādi vīzas
izsniegšanas nosacījumi, kā nelegālās imigrācijas draudu
neesamība, ieceļošanas mērķa pamatotība, iesniegto vīzas
pieteikuma dokumentu autentiskums, medicīniskā apdrošināšana
un uzturēšanās līdzekļu esamība. Harmonizējot Šengenas robežu
kodeksa jēdzienu „apdraudējums kādas dalībvalsts politikai,
iekšējai drošībai vai starptautiskām attiecībām, par Eiropas
Savienības un Šengenas konvencijas dalībvalstu drošības
apdraudējumu robežkontrolē jāuzskata starptautisko noziedzību,
masveida nelegālo migrāciju, terorismu, kontrabandu, narkotisko
Sociālo zinātņu žurnāls Nr. 1(6)
71
5.
vielu, radioaktīvo vielu, ieroču un sprāgstvielu nelikumīgu apriti un
citus ekstrēmisma apdraudējumus, kuru dēļ var izcelties
starpvalstu konflikti un starptautiskās situācijas saasināšanās. Šāds
regulējums papildus būtu jāparedz Šengenas robežu kodeksā
2.pantā (definīcijas).
Savukārt Šengenas robežu kodeksa jēdziena „„sabiedrības veselības
apdraudējums” – ir slimība, kas potenciāli var izvērsties epidēmijā,
kā noteikts Pasaules Veselības organizācijas starptautiskajos
veselības aizsardzības noteikumos, kā arī citas infekcijas slimības
vai lipīgas parazītu slimības, ja uz tām attiecas aizsardzības
noteikumi, kas attiecas uz dalībvalstu valstspiederīgajiem” ietvars
jāpapildina ar kaitēkļu masveida izplatības, ķīmiskā un cita
saindējuma apdraudējumiem, kas apdraud iedzīvotāju veselību un
apkārtējo vidi.
Izmantotā literatūra un avoti
Normatīvi akti:
1.
Konvencija, ar ko īsteno Šengenas nolīgumu (1985.gada 14.jūnijs) starp
Beniluksa Ekonomikas savienības valstu valdībām, Vācijas Federatīvās
Republikas valdību un Francijas Republikas valdību par pakāpenisku kontroles
atcelšanu pie kopīgām robežām. Ministru un valsts sekretāru Kopīgā
Deklarācija, tiekoties Šengenā 1990.gada 19.jūnijā. Oficiālais Vēstnesis. L 239,
22/09/2000 lpp. 0019 – 0062.
2.
Eiropas Parlamenta un Padomes Regula (EK) Nr. 562/2006 (2006.g. 15.marts),
ar kuru ievieš Kopienas Kodeksu par noteikumiem, kas reglamentē personu
pārvietošanos pār robežām (Šengenas robežu kodekss) [tiešsaiste]. Official
Juornal. L 105, 13.04.2006. [atsauce 2014.g. 20.jan.]. Pieejas veids:
http://europa.eu.int/eur-lex/lex/LexUriServ/site/lv/oj/2006/l_105/l_105
20060413lv00010032.pdf
3.
Eiropas Parlamenta un Padomes Regula (EK) Nr. 539/2001 (2001. gada 15.
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Summary
The present research was accomplished during the period of time from 2012 to 2013
using the laws, regulations and case law’s analysis, interpretation and methods of
comparison. The aim of the study is to find out the problems of Latvian border control
regulatory framework from the perspective of public order and other hazards and to
offer legal solutions. The object of the research is the adaptation problems of the basic
concepts of „public order”, „national security”, „threats to public health” in regulatory
framework. The subject of the research is EU Schengen acquis (Schengen set of
rights) and national regulatory framework. The tasks of the research are to explore
the concept of legal content and problems of applying it in practise, to propose
definitions of these concepts to improve the regulatory framework. By improving the
regulatory framework of basic concepts of „public order”, „national security”, „threats
to public health”, it will be possible to achieve more effective institution of public
policy in operations of border checks being a substantial part of border control.
The Schengen Agreement and the Schengen Convention are one of the remarkable
achievements to ensure free movement of persons on the international arena.
Considering geopolitical importance and the direct influence of these contracts on
constitutional rights and national sovereignty, they should be included in the EU's
establishing treaties, because with the help of these agreements a unitary free
movement space is founded.
Comparing public order and other threats of the rules in the Schengen Convention
and the Code, it can be concluded that essentially they are mutually duplicated,
although they are reflected having different versions and terminology, which in its
turn, more or less twists the content of the norms. Respectively, it can cause and
create problems of interpretation in practice, for instance, when using such terms as:
in the Schengen Convention - not reported, in the Code –SIS there is issued a warning,
in the judgment - „persons who were [the Schengen Information System (SIS)] issued
Sociālo zinātņu žurnāls Nr. 1(6)
75
a warning in order to refuse entry”; Schengen Convention - public policy, national
security or international relations, in the Code and in the case law – it was not
considered as a threat to any state policy, internal security, public health or
international relations, and in particular, in national data bases there is no issued
warning for them to refuse entry.
Several scholars in Western Europe underline the need for harmonizing the law in the
EU, provided that the EU law means different legal and administrative prescript
alignment of several individual EU Member States. Nevertheless, the author also
highlights the need for harmonizing the EU law and the international law which
would be particularly important in clarifying public policy and other hazards in the
intensive cross-border communication conditions, defining in the system of Schengen
acquis the fact that public policy comprises certain responsibilities which are
included in the law to ensure its permanent and accurate execution by members of
society being binding for all parties. As to the public order offenses, in accordance
with Member States' national regulatory framework, the liability foresees no less than
imprisonment for at least one year. This framework should be reflected in Article 2 of
the Schengen Borders Code (Definitions).
At the EU level there are no unified and clearly defined threats to State policy, internal
security, public health, international relations, which in the Schengen Borders Code
are mentioned as reasons to refuse entry, but in the Visa Code, in addition, there are
pointed out conditions for issuing visas such as, illegal immigration risks absence,
purpose of entry validity, submitted visa applications by the authenticity of
documents, medical insurance and residence resources existence. When harmonizing
the concepts “threat to public policy, internal security or international relations” of
the Schengen Borders Code, the threats to national security in the European Union
and in the Schengen Convention, it should be considered that international crime,
massive illegal migration, terrorism, smuggling, drugs, radioactive substances, illegal
movement of weapons and explosive and other extremist threats may become issues
that initiate interstate conflicts and aggravation in the international situation. That is
why, this framework should be also reflected in Article 2 of the Schengen Borders
Code (Definitions)
In its turn, the concept of the Schengen Borders Code, “threat to public health”- is
disease that potentially can expand to epidemic, as it is defined in the International
Health Regulations of the World Health Organization, and other infections or
contagious parasitic diseases, if they are the subject of protection provisions, related to
nationals of Member States”„ , framework should be complemented with a massive
spread of pests, chemical and other poisoning threats, that imperil public health and
the environment.
76
Latgales Tautsaimniecības pētījumi
|
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https://openalex.org/W2144548386
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https://europepmc.org/articles/pmc2778898?pdf=render
|
English
| null |
Cytokine signalling in human melanoma cells determines susceptability to statin-induced apoptosis
|
BMC pharmacology
| 2,009
|
cc-by
| 426
|
BioMed Central BioMed Central Background melanoma cells abrogated the pro-apoptotic effect of stat-
ins. g
Melanoma is one of the most aggressive and chemoresist-
ant cancer types in humans. Especially in late stages, effec-
tive therapeutic approaches are not available. Statins have
been investigated for their anti-proliferative and pro-
apoptotic effects in many tumor cells including
melanoma [1]. Beside paracrine signalling, melanoma
cells rely on a wide range of autocrine cytokine loops. Conclusion Taken together, theses data may open a possible new ther-
apeutic window for statins in late-stage melanoma ther-
apy which is based on IL-6 suppression by simvastatin in
the metastatic melanoma cell lines A375 and 518A2,
while early-stage melanoma cell lines, WM278 and
WM793B were virtually insensitive to statin treatment. Methods and results We have therefore screened the serum-free supernatant of
simvastatin-treated 518A2 melanoma cells for cytokines. While INF-γ, TNF-α, IL-1α, IL-1β, IL-10 and IL-12 were
not regulated by simvastatin, most strikingly, IL-6 levels
were significantly decreased. IL-6 is an important prog-
nostic marker in late stage melanoma. Due to this crucial
role in the autocrine regulation of the tumour growth this
cytokine was investigated in greater detail. A375 and
518A2 melanoma cells were transfected with a fluorescent
Stat-3 fusion protein and showed IL-6-mediated translo-
cation of Stat-3-YFP into the nucleus. This was followed
by a transient phosphorylation of Stat-3. Conversely, the
''IL-6-insensitive'' melanoma cell lines, WM278 and
WM793B, showed constitutively active Stat-3 phosphor-
ylation and virtually no regulation upon IL-6 addition. Interestingly, the latter cells were approximately 10-fold
less susceptible toward statin-induced caspase 3 activation
compared to A375 and 518A2 melanoma cells. Moreover,
addition of IL-6 to simvastatin-treated A375 and 518A2 Meeting abstract
Cytokine signalling in human melanoma cells determines
susceptability to statin-induced apoptosis
Christoph Minichsdorfer and Martin Hohenegger* Open Access from 15th Scientific Symposium of the Austrian Pharmacological Society (APHAR) Joint meeting with the Hungarian Society of Experimental and Clinical
Pharmacology (MFT) and the Slovenian Pharmacological Society (SDF)
Graz, Austria. 19-21 November 2009 Published: 12 November 2009
BMC Pharmacology 2009, 9(Suppl 2):A28
doi:10.1186/1471-2210-9-S2-A28 Published: 12 November 2009
BMC Pharmacology 2009, 9(Suppl 2):A28
doi:10.1186/1471-2210-9-S2-A28 This abstract is available from: http://www.biomedcentral.com/1471-2210/9/S2/A28
© 2009 Minichsdorfer and Hohenegger; licensee BioMed Central Ltd. References 1. Minichsdorfer C, Hohenegger M: Autocrine amplification loop in
statin-induced apoptosis of human melanoma cells. Br J Phar-
macol 2009, 157:1278-1290. 1. Minichsdorfer C, Hohenegger M: Autocrine amplification loop in
statin-induced apoptosis of human melanoma cells. Br J Phar-
macol 2009, 157:1278-1290. Page 1 of 1
(page number not for citation purposes) Page 1 of 1
(page number not for citation purposes)
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https://openalex.org/W4210637336
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https://ejournal.stai-tbh.ac.id/abdimasy/article/download/396/273
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Indonesian
| null |
Sosialisasi Pemilihan Karir di Sekolah Menengah Atas (SMA) Negeri 01 Reteh
|
ABDIMASY
| 2,021
|
cc-by-sa
| 4,591
|
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0
International License (CC-BY-SA) P-ISSN: 2745-7400│ E- ISSN: 2745-7419
Jurnal
Pengabdian dan Pemberdayaan Masyakat Sosialisasi Pemilihan Karir
di Sekolah Menengah Atas (SMA) Negeri 01 Reteh
Rika Devianti1), Mardiah2), Dina Liana3) Martina Napratilora4) Faridatul Munawaroh5)
Hendro Lisa6)
1,5Program Studi PIAUD, STAI Auliaurrasyidin Tembilahan, Indragiri Hilir, Riau, Indonesia
2,3,4Program Studi PGMI, STAI Auliaurrasyidin Tembilahan, Indragiri Hilir, Riau, Indonesia
6Program Studi ESY, STAI Auliaurrasyidin Tembilahan, Indragiri Hilir, Riau, Indonesia
Email: rika.devianti@stai-tbh.ac.id1; mardiah@stai-tbh.ac.id2; dina.liana@stai-tbh.ac.id3;
martina.napratilora@stai-tbh.ac.id4; faridatul.munawaroh@stai-tbh.ac.id5; hendro.lisa@stai-tbh.ac.id6
Cara Mensitasi Artikel ini:
Devianti, R., Mardiah, M., Liana, D., Napratilora, M., Munawaroh, F., & Lisa, H. (2021). Sosialisasi pemilihan
karir di sekolah menengah atas (SMA) Negeri 01 Reteh. Abdimasy: Jurnal Pengabdian dan Pemberdayaan
Masyarakat, 2(2), 92-103. https://doi.org/10.46963/ams.v2i2.396 Cara Mensitasi Artikel ini:
Devianti, R., Mardiah, M., Liana, D., Napratilora, M., Munawaroh, F., & Lisa, H. (2021). Sosialisasi pemilihan
karir di sekolah menengah atas (SMA) Negeri 01 Reteh. Abdimasy: Jurnal Pengabdian dan Pemberdayaan
Masyarakat, 2(2), 92-103. https://doi.org/10.46963/ams.v2i2.396 Abstract: Career selection is an important thing that needs to be
considered by students in adjusting their talents, interests or
abilities with the career to be chosen. However, in reality, there
are still cases of mismatch of talents, interests/ability with the
chosen career, causing individuals to become stressed, burdened
and even drop out of college. Thus, the need for socialization of
career selection in order to direct individuals to achieve success
in the future. This service is carried out at the State High School
(SMA) 01 Reteh. This type of research is community service with
the lecture method, and question and answer. The steps carried
out in this service are in the form of providing information about
education or position, student achievement, and evaluation. The
results of this service are students' understanding related to career
choice, it can be seen that they can adjust the choice of majors
according to their talents, interests and aspirations. DOI
https://doi.org/10.46963/ams.v2i2.396 PENDAHULUAN Jakarta ada pula yang masuk jurusan IPS
karena
nilai
mereka
yang
kurang
mencukupi untuk masuk jurusan IPA. Hal
ini
menyebabkan
kebimbangan
dan
konflik dalam diri siswa karena jurusan
yang ia tekuni saat ini tidak sejalan dengan
jurusan yang diinginkannya. Jakarta ada pula yang masuk jurusan IPS
karena
nilai
mereka
yang
kurang
mencukupi untuk masuk jurusan IPA. Hal
ini
menyebabkan
kebimbangan
dan
konflik dalam diri siswa karena jurusan
yang ia tekuni saat ini tidak sejalan dengan
jurusan yang diinginkannya. Pengangguran di Indonesia masih
tergolong tinggi. Hal ini memicu beberapa
dampak negative seperti kemiskinan dan
bunuh
diri
karena
tidak
sanggup
menghadapi kenyataan hidup. Salah satu
pemicu
pengangguran
adalah
ketidakkesesuaian karir dengan bakat atau
potensi
yang
dimiliki. Untuk
menyesuaikan pilihan karir dengan bakat
atau potensi yang dimiliki, tentunya
diawali dari peserta didik dibekali dengan
ilmu yang membantu mereka memahami
dirinya dan karir yang akan dipilihnya. Selanjutnya, menurut Melaty Ihsan
(2011) menyatakan di dalam jurnal
penelitiannya
menemukan
bahwa
sebanyak
50%
siswa
mengalami
kebingungan
dalam
pengambilan
keputusan. Salah satu faktorya adalah
begitu banyak pilihan jenjang pendidikan
dan
jenis
pekerjaan
yang
tersedia. Terbatasnya informasi berbagai pekerjaan
yang ada dalam masyarakat. Hal ini
membuat siswa menjadi berfikir atau
memilih sesuai apa yang diketahui,
sehingga terjadilah kesalahpahaman siswa
dalam memilih jurusan. Kesalahan yang sering dilakukan oleh
siswa
SMA
adalah
mereka
gagal
menentukan bakat apa yang mereka miliki
dan bidang apa yang ditekuni. Hal ini
menjadikan mereka salah masuk jurusan. Kemudian minimnya bekal pengetahuan
mengenai jurusan-jurusan di Perguruan
Tinggi
di
Indonesia. Inipula
yang
membuat mereka lebih memilih jurusan
yang nge-trend, ikutan teman atau
dorongan dari orangtua atau guru. Hal ini
berdampak pada siswa stress, putus
kuliah-menjadi pengangguran, dan pada
akhirnya penentuan karir di masa depan
yang tidak jelas. Selanjutnya, menurut Achour, Mohd
& Mohd Nor, Mohd R. (2014) Siswa
SMA pada umumnya masih menentukan
pilihan berdasarkan dukungan social
(teman sebaya, rekan kerja, orangtua,
guru). Hal penting dari dukungan social
adalah teman, pacar, guru, dan konselor. Teman adalah motivasi kuat, karena
dukungan yang diterima berbeda dari
dukungan yang diberikan oleh keluarga. Lebih lanjut, melihat hasil survey
Fatma Nuraqmarina & Erna Risnawati
(2018) yang dilaksanakan kepada siswa
kelas XII SMA di MAN Y Jakarta bahwa
kebimbangan siswa kelas XII SMA
diantaranya ragu dengan pilihannya,
merasa banyak saingan, untuk masuk
perguruan tinggi dan ketakutan nilai
akademik yang dimiliki tidak mencukupi
untuk pilihan jurusan yang diminati. DOI
https://doi.org/10.46963/ams.v2i2.396 Sejarah Artikel
Diterima
: 03/09/2021
Direvisi
: 23/12/2021
Diterbitkan : 31/12/2021 Editorial Address
Kampus Panam (Parit Enam)
STAI Auliaurrasyidin, Jl. Gerilya
No. 12 Tembilahan Barat, Riau,
Indonesia, 29213
abdimasy@stai-tbh.ac.id Editorial Address
Kampus Panam (Parit Enam)
STAI Auliaurrasyidin, Jl. Gerilya
No. 12 Tembilahan Barat, Riau,
Indonesia, 29213
abdimasy@stai-tbh.ac.id Abstrak: Pemilihan karir merupakan hal penting yang perlu
diperhatikan oleh siswa dalam rangka penyesuaian bakat, minat
atau kemampuan dengan karir yang akan dipilih. Namun, pada
kenyataannya, masih terdapat kasus ketidaksesuaian bakat,
minat/kemampuan
dengan
karir
yang
dipilih
sehingga
menyebabkan individu menjadi stress, terbebani bahkan putus
kuliah. Dengan demikian, perlunya sosialisasi pemilihan karir
guna mengarahkan individu pada ketercapaian kesuksesan
dikemudian hari. Pengabdian ini dilaksanakan di Sekolah
Menengah Atas (SMA) Negeri 01 Reteh. Jenis penelitian ini
adalah pengabdian pada masyarakat dengan metode ceramah,
dan tanya jawab. Langkah-langkah yang dilaksanakan dalam
pengabdian ini berupa pemberian informasi mengenai pendidikan
atau jabatan, pengungkapan karakteristik siswa, dan evaluasi. Hasil dari pengabdian ini berupa pemahaman siswa berkaitan
dengan pemilihan karir, hal ini terlihat bahwa mereka dapat
menyesuaikan pilihan jurusan dengan bakat, minat dan cita cita
mereka. Keywords:
Career Selection; Socialization;
Senior High School This work is licensed under a Creative Commons Attribution-ShareAlike 4.0
International License (CC-BY-SA) 92 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh PENDAHULUAN Kondisi tersebut sejalan dengan yang
dialami oleh siswa kelas XII MAN B Namun, hal ini berdampak pada
ketidak sesuaian pilihan karir dengan
kemampuan siswa. Padahal yang penting
untuk diperhatikan oleh siswa dalam
pemilihan karir harus didahului oleh
beberapa prosedur seperti melaksanakan
tes minat, bakat, dan tes psikologis
lainnya, menganalisis hasil belajar dan
mengisi angket untuk mengetahui cita-cita
siswa. Dari upaya tersebut diharapkan Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) 93 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh semua siswa dapat belajar atau bekerja
sesuai
dengan
kemampuan
mereka
masing-masing. perguruan tinggi, metode yang digunakan
adalah: 1. Ceramah,
tahapan
ceramah
dimaksudkan menyampaikan materi
kegiatan mengenai pilihan karir yang
berkaitan dengan program studi apa
saja yang ada di perguruan tinggi. Dengan
harapan
memberikan
pemahaman kepada siswa sehingga
dapat memberikan gambaran awal
mengenai pilihan program studi saat
akan melanjutkan ke perguruan tinggi
sesuai minat dan bakat siswa. Hal ini
berkaitan
dengan
pendidikan
masyarakat
yang
memberikan
penyuluhan terhadap siswa. Dari beberapa temuan di lapangan
tempat pengabdian akan dilaksanakan
masih ditemukan beberapa siswa kelas
XII belum mengetahui pilihan karir yang
akan diambil, sebagian yang lain merasa
mengikuti teman/pacar lebih utama, dan
bahkan ada sebagian lagi yang masih
bingung menentukan pilihan karir karna
tidak mengetahui beberapa jurusan atau
jenjang pendidikan yang ada, hal ini
berdampak pada pengambilan keputusan
oleh orang ketiga. Kemudian juga belum
ada TIM narasumber yang melaksanakan
sosialisasi terkait pemilihan karir setelah
menamatkan sekolah menengah atas. Lebih lanjut, beberapa siswa tidak
mengetahui bakat dan minat mereka
karena
tidak
semua
mengikuti
tes
psikologis, memilih salah satu jurusan
karena jurusan tersebut menarik ataupun
bergengsi, sebagian lagi mengungkapkan
bahwa ketidakpahaman mereka tentang
pentingnya kesesuaian bakat dan minat
dengan jurusan yang dipilih. Adapula
diantara siswa, pilihan jurusan sudah
ditentukan oleh orangtua. 2. Tanya
jawab,
tahapan
ini
memberikan
kesempatan
kepada
siswa
bertanya
tentang
materi
sosialisasi dan menyesuaikan bakat,
minat,
dan
cita-cita
maupun
harapannya di masa depan. 2. 3. Pembagian brosur terkait daftar
jurusan
yang
ada
dibeberapa
perguruan tinggi. 4. Advokasi
berkaitan
dengan
mendampingi
siswa
dalam
memahami keterkaitan antara bakat,
minat, cita-cita dengan pengambilan
keputusan karir kedepan. PENDAHULUAN Menelaah dari kasus yang telah
dipaparkan di atas maka penulis akan
melaksanakan
pengabdian
kepada
masyarakat
mengenai
sosialisasi
pemilihan karir di SMAN 01 Reteh Untuk
mengetahui bagaimana pemahaman siswa
pada pemilihan karir di SMAN 01 Reteh. Dengan
empat
metode
tersebut
diharapkan
mampu
memberikan
pemahaman yang baik kepada siswa,
kemudian untuk menunjang kelancaran
kegiatan, TIM juga memberikan brosur
atau poster atau tentang program studi
yang terdapat di beberapa Universitas. Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) HASIL DAN PEMBAHASAN Untuk turut membantu memberikan
informasi mengenai pemilihan karir di Pemilihan karir berkaitan dengan
suatu proses pengambilan keputusan yang 94 94 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Hanifan Akbar (2011) menyatakan
pemilihan
karier
merupakan
sebuah
proses yang dimulai sejak usia awal. Individu yang mampu menentukan pilihan
karier merupakan individu yang kompeten
memiliki kemampuan pengetahuan, skill,
talenta dan kemampuan untuk melangkah
maju. Mampu menyelesaikan masalah
dalam
pemilihan
karier
merupakan
individu yang kompeten. sangat penting dalam hidup setiap siswa
karena akan mempengaruhi terhadap
kehidupan yang akan dilaluinya, dan tidak
bisa dielakkan karena siswa tetap akan
menghadapinya setelah beberapa proses
tahap perkembangan terselesaikan. Pilihan karir merupakan suatu proses
ketika remaja mengarahkan diri kepada
suatu tahap baru dalam kehidupannya,
melihat posisi mereka dalam kehidupan
pembuatan keputusan karir mereka. Hal
ini diawali dari pemilihan jurusan di
universitas. Siswa yang lulus SMA,
SMEA, SMK, ALIYAH, dan jenjang
sederajat lainnya akan melanjutkan studi
ke perguruan tinggi baik PTN maupun
PTS (Lina Marliyah, dkk, 2004). Menurut Ginzberg dalam (Akbar,
2011 dalam Devi dkk, 2020) proses
pemilihan karir mencakup beberapa
tahapan, antara lain, pertama fantasi, yaitu
tahap seseorang memilih karirnya secara
sembarangan, tidak didasarkan pada
kemampuannya, sebaliknya didasarkan
pada rasa kagum dan terkesan terhadap
suatu profesi; kedua tentative, yaitu tahap
dimana seseorang mulai berkembang
dalam pilihan karir, seseorang mulai
menyadari bahwa minatnya berubah-ubah
dan mulai memikirkan karir apa yang
cocok untuk dirinya sesuai dengan
kemampuannya; ketiga realistic, yaitu
tahap ini seseorang memberikan penilaian
terhadap karir yang akan dipilihnya. Penilaian berasal dari pengalaman atau
pengetahuannya
tentang
karir
yang
dipilihnya
kemudian
dijadikan
pertimbangan
untuk memasuki
atau
menentukan jurusan yang dipilihny di
perguruan tinggi; keempat eksplorasi,
yaitu seseorang telah melakukan kegiatan-
kegiatan yang berkaitan dengan pilihan
karirnya akan mencapai keberhasilan atau
mengalami kegagalan. Keberhasilan atau
kegagalan yang dialami akan membentuk
pola
piker
dari
seseorang
mempertimbangkan kembali karir yang
telah dipilihnya; kelima kristalisasi, yaitu Memilih
Jurusan
kuliah
bukan
perkara
yang
mudah,
harus
dipertimbangkan baik baik, agar tidak
merasa terlambat bahwa pilihan jurusan
tidak sesuai dengan kepribadian sampai
pada Droup Out /DO atau dikeluarkannya
mahasiswa/I
karena
dikatakan
tidak
mampu mengikuti perkuliahan yang
diikutinya. Maka dari itu pemilihan
jurusan atau pemilihan karir harus sedini
mungkin harus dipertimbangkan. Salah
memilih jurusan merupakan kerugian
yang besar bagi karir dimasa depan. Cara memilih jurusan di universitas
yang baik: 1. Menyesuiakan Cita-cita, Minat dan
Bakat 2. Informasi yang Sempurna 3. Lokasi dan Biaya 3. Faktor-faktor Pemilihan Karir Pemilihan karir akan dipengaruhi
oleh dua factor, yaitu factor internal dan
factor eksternal. Dimana kedua factor
tersebut dapat memberikan pengaruh
positif terhadap arah pilihan karir siswa. Faktor internal merupakan factor yang
berasal dari dalam diri individu, seperti
nilai-nilai kehidupan, taraf inteligensi,
bakat khusus, sifat-sifat, pengetahuan, dan
keadaan jasmani. Sedangkan untuk factor
eksternal factor yang berasal dari luar diri
individu, seperti masyarakat, keadaan
social, status social ekonomi keluarga,
pengaruh keluarga, pendidikan sekolah,
pengaruh teman sebaya, dan tuntunan
jabatan (Winkel dan Sri Hastuti, 2004) HASIL DAN PEMBAHASAN Lokasi dan Biaya 4. Daya
Tampung
Jurusan/Peluang
Diterima 5. Masa Depan Karir dan Pekerjaan Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh seseorang berfikir lagi dan menyadari
bahwa untuk menentukan pilihan karir
harus mempertimbangkan factor-faktor
yang ada yang sangat mempengaruhi
dalam penentuan keputusan pilihan karir
yang sesuai; keenam spesifikasi, yaitu
pilihan
pekerjaan
atau
jurusan
dispesifikasikan lebih khusus. n Pemberdayaan Masyarakat
Rumpun
Ilmu
Jurusan Kuliah /
Program Studi
Kesehatan
Kedokteran
Kedokteran Gigi
Kedokteran Hewan
Kesehatan Masyarakat
Kesehatan Lingkungan
Ilmu Gizi
Keselamatan&
Kesehatan Kerja
Ilmu Keperawatan
Farmasi
Kedokteran Hewan
Nutrisi dan Teknologi
Pangan
Kebidanan
Fisioterafi
Ilmu Keolahragaan
Teknik Radiodiagnostik
dan Radioterapi
Manajemen Pelayanan
Rumah Sakit
Matematika
&
IPA
(MIPA)
Matematika
Fisika
Kimia
Biologi
Statistika
Astronomi
Bioteknologi
Geofisika
Meteorologi
Geografi
Biokimia
Metrologi
Aktuaria
Statistika Terapan
Mikrobiologi Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Daftar Jurusan Pemilihan jurusan atau karir yang
dapat menunjang kemampuan siswa
sangat berfariasi yang ada di beberapa
perguruan tinggi baik negeri maupun
swasta. Hal ini menunjukkan bahwa
pemerintah sangat berperan penting dalam
menunjang
tersalurnya
kemampuan-
kemampuan yang dimiliki setiap siswa
yang akan melanjutkan karir kedepan. Berikut
beberapa
prodi
yang
ada
dibeberapa perguruan tinggi yang perlu
diketahui dan menjadi acuan dalam
pemilihan karir di masa depan, yaitu: 96 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Bioentrepreneurship
Ilmu
Pangan
(Food
Science)
Matematika Bisnis
Fisika Medis
Kartografi
dan
Penginderaan Jauh
Pengelolaan
dan
Pemberdayaan SDA dan
Lingkungan
Sosial
dan
Humaniora
llmu Politik
Filsafat
Kriminologi
Psikologi
Ilmu Hukum
Sosiologi
Jurnalistik
Antropologi
Hubungan Internasional
(HI)
Ilmu
Kesejahteraan
Sosial
Ilmu Pemerintahan
Administrasi Publik
Administrasi Bisnis
Ilmu Komunikasi
Hubungan Masyarakat
Marketing
Communication
Penyiaran
(Broadcasting)
Periklanan (Advertiing)
Peradilan Agama
Politik Islam
Pembangunan
Sosial
dan Kesejahteraan
Business Law
Manajemen Komunikasi
Branding
Kearsipan
Sains Komunikasi dan
Pengembangan
Masyarakat
Ilmu
Keluarga
dan
Konsumen
Manajemen
Produksi
Media
Ekonomi dan
Bisnis
Akuntansi
Manajemen Keuangan
Menajemen
Sumber
Daya
Manusia
dan
Organisasi
Manajemen Operasi
Manajemen Pemasaran
Administrasi Fiskal
Ekonomi
Bisnis Internasional
Manajemen Informatika
Ekonomi Pembangunan
Techopreneurship
Green Economy
Manajemen Bisnis
Administrasi Niaga
Manajemen Keuangan
Syariah
Bisnis Islam
Business Creation
Kewirausahaan
Manajemen Bisnis dan
Pemasaran
Manajemen
Bisnis
Internasional
Ekonomi Syariah
Keuangan
Pemasaran Internasional Branding
Kearsipan
Sains Komunikasi dan
Pengembangan
Masyarakat
Ilmu
Keluarga
dan
Konsumen
Manajemen
Produksi
Media
Ekonomi dan
Bisnis
Akuntansi
Manajemen Keuangan
Menajemen
Sumber
Daya
Manusia
dan
Organisasi
Manajemen Operasi
Manajemen Pemasaran
Administrasi Fiskal
Ekonomi
Bisnis Internasional
Manajemen Informatika
Ekonomi Pembangunan
Techopreneurship
Green Economy
Manajemen Bisnis
Administrasi Niaga
Manajemen Keuangan
Syariah
Bisnis Islam
Business Creation
Kewirausahaan
Manajemen Bisnis dan
Pemasaran
Manajemen
Bisnis
Internasional
Ekonomi Syariah
Keuangan
Pemasaran Internasional 97
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. Daftar Jurusan 2 (2021) Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P ISSN: 2745 7400 │E ISSN: 2745 7419
Ekonomi Bisnis
Akuntansi Bisnis
Manajemen Pariwisata
Manajemen
Manajemen
Transportasi
Akuntansi Sektor Publik
Manajemen
Industri
Katering
Administrasi Keuangan
Manajemen
Bisnis
Telekomunikasi
Informatika
Sastra
dan
Budaya
Ilmu Sejarah
Sastra Inggris
Arkeologi
Sastra Jawa
Sastra Arab
Sastra Jepang
Sastra Indonesia
Sastra Rusia
Sastra Perancis
Sastra Korea
Sastra Jerman
Sastra Belanda
Sastra Cina
Sastra Sunda
Sastra Bali
Sastra Slavia
Sastra Minangkabau
Sastra Nusantara
Sejarah dan Kebudayaan
Islam
Komputer
dan
Teknologi
Teknik Informatika
Mobile Application &
Technology
Sistem
Informasi
(Manajemen
Informatika)
Teknologi Game
Ilmu Komputasi
Cyber Security
Bioinformatika
Sistem
Komputer
(Teknik Komputer)
Sistem Informasi Bisnis
Software Engineering
Sistem dan Teknologi
Informasi
Computerized
Accounting
Information
Systems
Audit
Accounting Information
Audio Engineering
Ilmu Komputer
Human
Computer
Interaction
Pendidikan
Pendidikan
Guru
Sekolah Dasar (PGSD)
Manajemen Pendidikan
Kurikulum
dan
Teknologi Pendidikan
Pendidikan
Luar
Sekolah
Pendidikan Luar Biasa
(PLB)
Pendidikan Anak Usia
Dini (PAUD)
Administrasi
Pendidikan
Pendidikan Bimbingan
Konseling
Ilmu Perpustakaan
Teologi Ekonomi Bisnis
Akuntansi Bisnis
Manajemen Pariwisata
Manajemen
Manajemen
Transportasi
Akuntansi Sektor Publik
Manajemen
Industri
Katering
Administrasi Keuangan
Manajemen
Bisnis
Telekomunikasi
Informatika
Sastra
dan
Budaya
Ilmu Sejarah
Sastra Inggris
Arkeologi
Sastra Jawa
Sastra Arab
Sastra Jepang
Sastra Indonesia
Sastra Rusia
Sastra Perancis
Sastra Korea
Sastra Jerman
Sastra Belanda
Sastra Cina
Sastra Sunda
Sastra Bali
Sastra Slavia
Sastra Minangkabau
Sastra Nusantara
Sejarah dan Kebudayaan
Islam
Komputer
dan
Teknologi
Teknik Informatika
Mobile Application &
Technology Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 99
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Daftar Jurusan 2 (2021) 98 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
│
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Kependudukan
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Pancasila
dan Kewarganegaraan
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Agama
Islam
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Pendidikan
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Pe
ngetahuan Alam
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Pertanian
Agronomi
dan
Hortikultura
Mikrobiologi Pertanian
Agribisnis
(Sosial
Ekonomi Pertanian)
Agroteknologi
Ilmu Kelautan
Peternakan
Agroeteknologi
Kehutanan
Budidaya
Perairan
(Akuakultur)
Teknologi Pangan
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Teknologi Pasca Panen
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Silvikulutur
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Tumbuhan
(Proteksi
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Lahan
(Ilmu Tanah)
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Hasil
Perikanan
Agrobisnis
Perikanan
(Sosial
Ekonomi
Perikanan)
Pengelolaan Hutan
Pemanfaatan
Sumberdaya Perikanan
Teknologi
Industri
Benih
Produksi Ternak
Teknologi Hasil Ternak
Rekayasa Pertanian
Budidaya Perikanan
Manajemen
Sumber
Daya Perairan
Manajemen Hutan
Penyuluhan
dan
Komunikasi Pertanian
Teknik Pertanian
Manajemen
Bisnis
Unggas
Profesi dan
Ilmu Terapan
Pariwisata
Pendidikan Kepolisian
Pendidikan Militer
Penerbang (Pendidikan
Pilot) Abdimasy: Jurnal Pengabdian d
Pendidikan
Kependudukan
Tafsir Hadits
Pendidikan
Pancasila
dan Kewarganegaraan
Pendidikan
Agama
Islam
Pendidikan Kepelatihan
Olahraga
Pendidikan
Jasmani
Kesehatan dan Rekreasi
Pendidikan
Ilmu
Pe
ngetahuan Alam
Pendidikan
Bahasa
Inggris
Pendidikan Bahasa dan
Sastra Indonesia
Pendidikan Sejarah
Pendidikan Matematika
Manajemen Pendidikan
Islam
Pendidikan Geografi
Pendidikan Bahasa Arab
Pertanian
Agronomi
dan
Hortikultura
Mikrobiologi Pertanian
Agribisnis
(Sosial
Ekonomi Pertanian)
Agroteknologi
Ilmu Kelautan
Peternakan
Agroeteknologi
Kehutanan
Budidaya
Perairan
(Akuakultur)
Teknologi Pangan
Rekayasa Pertanian
Teknologi Pasca Panen Teknologi Hasil Hutan
Silvikulutur
Konservasi Sumberdaya
Hutan dan Ekowisata
Ilmu Hama dan Penyakit
Tumbuhan
(Proteksi
Tanaman)
Teknologi
Industri
Pertanian (Agroindustri)
Manajemen
Sumberdaya
Lahan
(Ilmu Tanah)
Teknologi
Hasil
Perikanan
Agrobisnis
Perikanan
(Sosial
Ekonomi
Perikanan)
Pengelolaan Hutan
Pemanfaatan
Sumberdaya Perikanan
Teknologi
Industri
Benih
Produksi Ternak
Teknologi Hasil Ternak
Rekayasa Pertanian
Budidaya Perikanan
Manajemen
Sumber
Daya Perairan
Manajemen Hutan
Penyuluhan
dan
Komunikasi Pertanian
Teknik Pertanian
Manajemen
Bisnis
Unggas
Pariwisata
Pendidikan Kepolisian
Pendidikan Militer
Penerbang (Pendidikan
Pilot) 99
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Vol. 02. No. Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
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Vol. 02. No. 2 (2021) Daftar Jurusan 2 (2021) 99 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh
Pendidikan Intelijen
Komunikasi
Penerbangan
Lalu Lintas Udara
Manajemen Logistik
Seni
Desain Interior
Desain Produk
Furniture Design
Tata Boga
Desain Grafis
Animasi
DKV New Media
DKV
Creative
Advertising
Teknik
Teknik Pertambangan
Teknik Kelautan
Teknik Lingkungan
Teknik Metalurgi
Teknik Sipil
Arsitektur
Teknik Geodesi
Teknik Elektro
Teknik Mesin
Teknik Industri
Teknik Perkapalan
Teknik
Perencanaan
Wilayah
dan
Kota
(Planologi)
Teknik
Penerbangan
(Aeronautika
dan
Astronautika)
Oseanografi
Teknik Nuklir
Teknik Geologi
Teknik Otomotif
Teknobiomedik
Teknik
Perancangan
Jalan dan Jembatan
Teknik Refrigerasi dan
Tata Udara
Teknik Telekomunikasi
Teknologi Bioproses
Teknik Grafika
Transportasi Laut
Teknik
Otomasi
Manufaktur
dan
Mekatronika
Rekayasa hayati
Teknik Material
Automotive
and
Robotics Engineering
Teknik Tenaga Listrik
Teknik
Sistem
Komputer
Manajemen
Rekayasa
Industri
Teknik Bioenergi dan
Kemurgi
Industrial
RoboticsDesign
Teknik Kimia
Teknik Fisika
Teknik Geomatika
Teknik Perminyakan
Teknik Alat Berat
Rekayasa Infrastruktur
Lingkungan
Teknik Pesawat Udara
Teknik Telekomunikasi
dan Navigasi Udara
Teknik Bangunan dan
Landasan
Teknik Listrik Bandara Teknik
Perancangan
Jalan dan Jembatan
Teknik Refrigerasi dan
Tata Udara
Teknik Telekomunikasi
Teknologi Bioproses
Teknik Grafika
Transportasi Laut
Teknik
Otomasi
Manufaktur
dan
Mekatronika
Rekayasa hayati
Teknik Material
Automotive
and
Robotics Engineering
Teknik Tenaga Listrik
Teknik
Sistem
Komputer
Manajemen
Rekayasa
Industri
Teknik Bioenergi dan
Kemurgi
Industrial
RoboticsDesign
Teknik Kimia
Teknik Fisika
Teknik Geomatika
Teknik Perminyakan
Teknik Alat Berat
Rekayasa Infrastruktur
Lingkungan
Teknik Pesawat Udara
Teknik Telekomunikasi
dan Navigasi Udara
Teknik Bangunan dan
Landasan
Teknik Listrik Bandara Pendidikan Intelijen
Komunikasi
Penerbangan
Lalu Lintas Udara
Manajemen Logistik
Seni
Desain Interior
Desain Produk
Furniture Design
Tata Boga
Desain Grafis
Animasi
DKV New Media
DKV
Creative
Advertising
Teknik
Teknik Pertambangan
Teknik Kelautan
Teknik Lingkungan
Teknik Metalurgi
Teknik Sipil
Arsitektur
Teknik Geodesi
Teknik Elektro
Teknik Mesin
Teknik Industri
Teknik Perkapalan
Teknik
Perencanaan
Wilayah
dan
Kota
(Planologi)
Teknik
Penerbangan
(Aeronautika
dan
Astronautika)
Oseanografi
Teknik Nuklir
Teknik Geologi
Teknik Otomotif
Teknobiomedik Teknik
Perancangan
Jalan dan Jembatan
Teknik Refrigerasi dan
Tata Udara
Teknik Telekomunikasi
Teknologi Bioproses
Teknik Grafika
Transportasi Laut
Teknik
Otomasi
Manufaktur
dan
Mekatronika
Rekayasa hayati
Teknik Material
Automotive
and
Robotics Engineering
Teknik Tenaga Listrik
Teknik
Sistem
Komputer
Manajemen
Rekayasa
Industri
Teknik Bioenergi dan
Kemurgi
Industrial
RoboticsDesign
Teknik Kimia
Teknik Fisika
Teknik Geomatika
Teknik Perminyakan
Teknik Alat Berat
Rekayasa Infrastruktur
Lingkungan
Teknik Pesawat Udara
Teknik Telekomunikasi
dan Navigasi Udara
Teknik Bangunan dan
Landasan
Teknik Listrik Bandara 100 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Teknik
Ekonomi
Konstruksi
(Quantity
Surveyor)
Teknik
Sistem
Perkapalan
Teknik
Pengairan
(Sumber Daya Air)
Meteorologi Terapan
Arsitektur Lanskap
Teknik Konversi Energi
Teknik Perpipaan hasil belajar. Daftar Jurusan Karakteristik siswa
diungkap melalui tes standard dan
penilaian hasil belajar. Dalam hal ini,
pengungkapan karakteristik siswa,
sebelumnya telah dilaksanakan di
sekolah
tersebut
dengan
tes
psikologis (kecerdasan, bakat, dan
minat)
dan
angket,
wawancara
terkait dengan minat dan prestasi
belajar siswa. Hasil pengumpulan
data tersebut akan digunakan sebagai
pedoman
dan
penunjang
untuk
memberikan
siswa
pemahaman
terkait
pentingnya
penyetaraan
karakteristik siswa dengan pilihan
karir lanjutan. Teknik
Ekonomi
Konstruksi
(Quantity
Surveyor)
Teknik
Sistem
Perkapalan
Teknik
Pengairan
(Sumber Daya Air)
Meteorologi Terapan
Arsitektur Lanskap
Teknik Konversi Energi
Teknik Perpipaan Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Langkah-langkah dalam Pelaksanaan
Sosialisasi Pemilihan Karir Dari
beberapa
masalah
yang
ditemukan, ada beberapa langkah-langkah
yang akan dilakukan oleh TIM sosialisasi
secara sistematik, seperti: 3. Evaluasi. Evaluasi
dilaksanakan
untuk
menilai
sejauh
mana
pemahaman siswa dan kemampuan
siswa dalam menentukan pilihan
karir selanjutnya. Evaluasi dilakukan
dengan
Tanya
jawab
dengan
beberapa siswa, kemudian di sesi
terakhir pengevaluasian menyebar
kertas
yang
terdapat
beberapa
pertanyaan terkait dengan penentuan
karir. Selanjutnya meminta siswa
untuk menjawab pertanyaan tersebut,
kemudian meminta beberapa anak
untuk membacakan jawaban dari
pertanyaan yang sudah di berikan. kemudian memberikan motivasi. 3. Evaluasi. Evaluasi
dilaksanakan
untuk
menilai
sejauh
mana
pemahaman siswa dan kemampuan
siswa dalam menentukan pilihan
karir selanjutnya. Evaluasi dilakukan
dengan
Tanya
jawab
dengan
beberapa siswa, kemudian di sesi
terakhir pengevaluasian menyebar
kertas
yang
terdapat
beberapa
pertanyaan terkait dengan penentuan
karir. Selanjutnya meminta siswa
untuk menjawab pertanyaan tersebut,
kemudian meminta beberapa anak
untuk membacakan jawaban dari
pertanyaan yang sudah di berikan. kemudian memberikan motivasi. 3. 1. Pemberian
Informasi
Mengenai
Pendidikan atau Jabatan. informasi
yang diberikan oleh TIM sosialisasi
berupa
informasi
mengenai
pentingnya
siswa
menentukan
pilihan karir kedepan, informasi
tentang sekolah lanjutan atau tentang
perguruan tinggi dan jurusan yang
ada di perguruan tinggi tersebut. Informasi
tentang
pemanfaatan
dalam penggunaan berbagai fasilitas
atau kegiatan kegiatan yang ada di
sekolah yang disesuaikan dengan
kemampuan siswa tersebut dan
informasi lainnya terkait dengan
pemilihan karir. Dari kegiatan sosialisasi ini seluruh
siswa yang mengikuti sosialisasi terlihat
begitu bersemangat, setelah dipaparkan
materi mengenai pemilihan karir ini
terlihat bahwa adanya pemahaman siswa
berkaitan dengan pemilihan karir, hal ini
terlihat
bahwa
mereka
dapat
menyesuaikan pilihan jurusan dengan 2. Pengungkapan Karakteristik siswa. Karakteristik
siswa
yang
akan
diungkap
berkenaan
dengan
kemampuan
dasar
umum,
kecerdasan,
bakat,
minat,
dan
kecendrungan pribadi, serta prestasi 1
Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) 101 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh berkaitan dengan pemilihan karir, hal ini
terlihat
bahwa
mereka
dapat
menyesuaikan pilihan jurusan dengan
bakat, minat dan cita cita mereka, adapula
yang
mengungkapkan
bahwa
akan
mengganti jurusan yang sebelumnya
sudah ditentukan baik oleh peserta didik
itu sendiri maupun pilihan orang tuanya. bakat, minat dan cita cita mereka, adapula
yang
mengungkapkan
bahwa
akan
mengganti jurusan yang sebelumnya
sudah ditentukan baik oleh siswa itu
sendiri maupun pilihan orang tuanya. DAFTAR PUSTAKA Achour, M & Mohd Nor, Mohd R. (2014). “The Effects of Social
Support and Resilience on Life
Satisfaction of Secondary School
Students”. Journal of Academic and
Applied Studies, 4(1), 12-20. (http://
academians. org/ the -effects-of-
social-support-and-resilience-on-
life-satisfac-of-secondary-school-
students, diakses 10 Agustus 2020). Agus
Dariyo. (2004). Psikologi
Perkembangan
Dewasa
Muda. Jakarta: Grasindo. Brown, S. (2008). Career Development
and Counseling Putting Theory and
Research to Work. Canada: Wiley
John Wiley & Sons, Inc. Senada dengan pendapat Brown, Steven D
dan Lent, Robert W dalam (Brown, 2008)
mengemukakan
teori
Holland
menggambarkan
bagaimana
individu
berinteraksi dengan lingkungan mereka
dan bagaimana individu dan karakteristik
lingkungan
mengakibatkan
pilihan
kejuruan dan penyesuaian. Carson,
A. (2008). Aplication
Of
Holland’s Vovational Counseling
Practice
Related
to
Vocational
Education. McGill
Journal
Education. National Analysis, 3(2) Langkah-langkah dalam Pelaksanaan
Sosialisasi Pemilihan Karir Pemahanam siswa terkait dengan
kesesuaian atau kecocokan antara pilihan
karir dengan kemampuan yang dimiliki
memberikan dampak positif bagi mereka
dalam pengambilan keputusan yang lebih
baik dan terarah. Hal ini pula akan
berpengaruh
terhadap
kinerja
dan
produktifitas dalam menjalankan suatu
kegiatan
di
masa
hidupnya
demi
terwujudnya kesuksesan dan kebahagian
setiap siswa. Menurut Holland dalam
(Carson,
2008),
memandang
bahwa
kepuasan
kerja,
produktifitas,
dan
sebagainya bergantung pada tingkat
kecocokan antara karakteristik orang,
selanjutnya (kepribadian vokasional) dan
pekerjaan selanjutnya (lingkungan kerja). Senada dengan pendapat Brown, Steven D
dan Lent, Robert W dalam (Brown, 2008)
mengemukakan
teori
Holland
menggambarkan
bagaimana
individu
berinteraksi dengan lingkungan mereka
dan bagaimana individu dan karakteristik
lingkungan
mengakibatkan
pilihan
kejuruan dan penyesuaian. Abdimasy: Jurnal Pengabdian dan Pemberdayaan Masyarakat
P-ISSN: 2745-7400 │E- ISSN: 2745-7419
Vol. 02. No. 2 (2021) Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh Fatma Nuraqmarina & Erna Risnawati. (2018). Keputusan Pemilihan Karir:
Studi
Komparatif
pada
Siswa
Remaja Jurusan IPA dan IPS. Jurnal
Ilmiah
Psikologi,
5(2). DOI:
10.115575/psy.v5i2.3068. Hanifan Akbar. (2011). Kecendrungan
Pemilihan Karir Berdasarkan Gaya
Belajar Pada Siswa SMA Kelas XII. SIMPULAN Darito,
A. (2004). Psikologi
Perkembangan Dewasa Muda. Jakarta: Grasindo. Hasil
dari
pengabdian
yang
dilaksanakan TIM sosialisasi pemilihan
karir di SMAN 01 Reteh dengan
melakukan beberapa langkah, seperti
pemberian
informasi
mengenai
pendidikan atau jabatan, pengungkapan
karakteristik siswa, kemudian evaluasi. Dari tiga langkah tersebut diperoleh hasil
bahwa
adanya
pemahaman
siswa Devi Nurul Fikriyani, dkk. (2020). Pemilihan
Karir
Berdasarkan
Kepribadian Pada Siswa. Jurnal
Ilmiah Bimbingan dan Konseling. JIBK
Undiksha,
11(1). DOI:
http://dx.doi.org/10.23887/jibk.v10i
2. 102 Rika devianti, Mardiah, Dina liana, Martina napratilora, Faridatul munawaroh, Hendro lisa
Sosialisasi pemilihan karir di sekolah menengah atas (SMA) Negeri 01 Reteh https://salamadian.com/daftar-
jurusan-kuliah/ Online. Minggu 15
November 2020. https://salamadian.com/daftar-
jurusan-kuliah/ Online. Minggu 15
November 2020. Lina Marliyah, dkk. (2004). Persepsi
Terhadap Dukungan Orangtua dan
Pembuatan
Keputusan
Karir
Remaja. Jurnal
Provitae,
1(1)
Januari. Melaty Ihsan. (2011). Salah Memilih
Jurusan”. (Online),
(http:
//melatyihsan. blogspot. com /salah –
memilih - jurusan. html, diakses 18
September 2020). Winkel
dan
Sri
Hastuti. (2004). Bimbingan
dan
Konseling
di
Institusi Pendidikan. Yogyakarta:
Media Abadi. 103 103
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English
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Enhanced software and platform for the Town Energy Balance (TEB) model
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Journal of open source software
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cc-by
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To cite this version: D. Meyer, Robert Schoetter, Valéry Masson, S. Grimmond. Enhanced software and platform for
the Town Energy Balance (TEB) model. Journal of Open Source Software, 2020, 5 (50), pp.2008. 10.21105/joss.02008. meteo-03981081 Enhanced software and platform for the Town Energy
Balance (TEB) model
D. Meyer, Robert Schoetter, Valéry Masson, S. Grimmond D. Meyer, Robert Schoetter, Valéry Masson, S. Grimmond Distributed under a Creative Commons Attribution 4.0 International License HAL Id: meteo-03981081
https://meteofrance.hal.science/meteo-03981081v1
Submitted on 10 Feb 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Enhanced software and platform for the Town Energy
Balance (TEB) model D. Meyer1, 2, R. Schoetter3, V. Masson3, and S. Grimmond1
1 Department of Meteorology, University of Reading, Reading, UK 2 Department of Civil and
Environmental Engineering, Imperial College London, London, UK 3 CNRM UMR 3589, Université
Fédérale de Toulouse, Météo-France/CNRS, Toulouse, France D. Meyer1, 2, R. Schoetter3, V. Masson3, and S. Grimmond1
1 Department of Meteorology, University of Reading, Reading, UK 2 Department of Civil and
Environmental Engineering, Imperial College London, London, UK 3 CNRM UMR 3589, Université
Fédérale de Toulouse, Météo-France/CNRS, Toulouse, France DOI: 10.21105/joss.02008 DOI: 10.21105/joss.02008
Software
• Review
• Repository
• Archive
Editor: Matthew Sottile
Reviewers:
• @ethan-nelson
• @jayten
Submitted: 04 December 2019
Published: 09 June 2020
License
Authors of papers retain
copyright and release the work
under a Creative Commons
Attribution 4.0 International
License (CC BY 4.0). Software
• Review
• Repository
• Archive Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
1 nced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
1105/j
02008
1 Summary The Town Energy Balance (TEB) model (Masson, 2000) is a physically based single layer
Urban Canopy Model (UCM) to calculate the urban surface energy balance at neighborhood
scale assuming a simplified canyon geometry. It includes several capabilities (Table 1) that have
been extensively evaluated offline with flux observations (Lemonsu, Grimmond, & Masson,
2004; Leroyer, Mailhot, Bélair, Lemonsu, & Strachan, 2010; Masson, Grimmond, & Oke,
2002; Pigeon, Moscicki, Voogt, & Masson, 2008) and online coupled to atmospheric models
such as ALARO (Gerard, Piriou, Brožková, Geleyn, & Banciu, 2009) in ALARO-TEB (Hamdi,
Degrauwe, & Termonia, 2012), the Global Environmental Multiscale (GEM; Côté et al. (1998))
in GEM-TEB (Lemonsu, Belair, & Mailhot, 2009), Meso-NH (Lac et al., 2018; Lafore et al.,
1998) in TEB-MesoNH (Lemonsu & Masson, 2002), the Regional Atmospheric Modeling
System (RAMS; Pielke et al. (1992)) in RAMS-TEB (Freitas, Rozoff, Cotton, & Dias, 2007),
the Advanced Regional Prediction System (ARPS; Xue et al., (2000)) in ARPS-TEB (Rozoff,
Cotton, & Adegoke, 2003), and the Weather Research and Forecasting (WRF; Skamarock et
al. (2019)) in WRF-TEB (Meyer et al., 2020). Editor: Matthew Sottile
Reviewers:
• @ethan-nelson
• @jayten
Submitted: 04 December 2019
Published: 09 June 2020
License
Authors of papers retain
copyright and release the work
under a Creative Commons
Attribution 4.0 International
License (CC BY 4.0). Editor: Matthew Sottile
Reviewers:
• @ethan-nelson
• @jayten
Submitted: 04 December 2019
Published: 09 June 2020
License
Authors of papers retain
copyright and release the work
under a Creative Commons
Attribution 4.0 International
License (CC BY 4.0). Table 1: Main capabilities available in the Town Energy Balance (TEB) model (Masson, 2000). The number of features available in TEB has increased since its first version published in 2000. (*)
Capability not currently available in the TEB software. Modeling capability
Reference
Urban Surface Energy Balance and Snow
Masson (2000)
Building Energy Model (BEM)
Bueno et al. (2012), Pigeon et al. (2014)
In-canyon urban vegetation and variable
road orientation
Lemonsu et al. (2012)
Green roofs, irrigation of green roofs,
gardens and watering of roads
de Munck et al. (2013)
Solar panels for hot water and/or
photo-voltaic (PV)
Masson et al. (2014)
Human behavior related to building energy
consumption*
Schoetter et al. (2017)
Calculation of urban carbon dioxide
fluxes*
Goret et al. (2019)
Urban trees*
Redon et al. (2017; 2020) Acknowledgments We acknowledge the researchers who contributed to the scientific development of the TEB
code: from CNRM: Aude Lemonsu, Grégoire Pigeon, Cécile de Munck, Bruno Bueno, Marine
Goret, Emilie Redon; from IFSTTAR Katia Chancibaul, Xenia Stavropulos-Laffaille; and from
Environnement and Changement Climatique Canada (ECCC): Sylvie Leroyer. Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
2 License Here, we present an enhanced software and platform for the TEB model to help scientists
and practitioners wishing to use the TEB model in their research as a standalone software
application or as a library in their own software. This includes several features such as cross-
platform support for Windows, Linux, and macOS using CMake (Kitware Inc., 2020), static
and dynamic library generation for integration with other software/models, namelist-based
configuration, integration with MinimalDX (Meyer & Raustad, 2019) and PsychroLib (Meyer
& Thevenard, 2019) to improve the modelling of air conditioners (AC) and psychrometric
calculations respectively, a thin interface used in the coupling with WRF-CMake (Riechert &
Meyer, 2019), helper functions for Python for pre- and post-processing inputs and outputs
files, and a tutorial in Jupyter Notebook to allow users to quickly become familiar with the
general TEB modeling workflow. In the new platform we implement testing at every code
commit through continuous integration (CI) and automate the generation of documentation. The project is developed as a free, open source, community-driven project on GitHub (https:
//github.com/teb-model/teb) to support existing and new model applications with enhanced
functionality. We welcome contributions and encourage users to provide feedback, bug reports
and feature requests, via GitHub’s issue system at https://github.com/teb-model/teb/issues. References Bueno, B., Pigeon, G., Norford, L. K., Zibouche, K., & Marchadier, C. (2012). Development
and evaluation of a building energy model integrated in the TEB scheme. Geoscientific
Model Development, 5(2), 433–448. doi:10.5194/gmd-5-433-2012 Côté, J., Gravel, S., Méthot, A., Patoine, A., Roch, M., & Staniforth, A. (1998). The
Operational CMCMRB Global Environmental Multiscale (GEM) Model. Part I: Design
Considerations and Formulation. Monthly Weather Review. doi:10.1175/1520-0493(1998)
126<1373:TOCMGE>2.0.CO;2 de Munck, C., Pigeon, G., Masson, V., Meunier, F., Bousquet, P., Tréméac, B., Merchat, M.,
et al. (2013). How much can air conditioning increase air temperatures for a city like Paris,
France? International Journal of Climatology, 33(1), 210–227. doi:10.1002/joc.3415 Freitas, E. D., Rozoff, C. M., Cotton, W. R., & Dias, P. L. S. (2007). Interactions of
an urban heat island and sea-breeze circulations during winter over the metropolitan
area of São Paulo, Brazil. Boundary-Layer Meteorology, 122(1), 43–65. doi:10.1007/
s10546-006-9091-3 Gerard, L., Piriou, J.-M., Brožková, R., Geleyn, J.-F., & Banciu, D. (2009). Cloud and
Precipitation Parameterization in a Meso-Gamma-Scale Operational Weather Prediction
Model. Monthly Weather Review, 137(11), 3960–3977. doi:10.1175/2009MWR2750.1 Goret, M., Masson, V., Schoetter, R., & Moine, M.-P. (2019). Inclusion of CO2 flux modelling
in an urban canopy layer model and an evaluation over an old European city centre. Atmospheric Environment: X, 3, 100042. doi:10.1016/j.aeaoa.2019.100042 d software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
5/j
02008
2 Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
2 Hamdi, R., Degrauwe, D., & Termonia, P. (2012). Coupling the Town Energy Balance
(TEB) Scheme to an Operational Limited-Area NWP Model: Evaluation for a Highly
Urbanized Area in Belgium. Weather and Forecasting, 27(2), 323–344. doi:10.1175/
WAF-D-11-00064.1 Kitware Inc. (2020). CMake. Retrieved from https://cmake.org/ Lac, C., Chaboureau, J.-P., Masson, V., Pinty, J.-P., Tulet, P., Escobar, J., Leriche, M., et al. (2018). Overview of the Meso-NH model version 5.4 and its applications. Geoscientific
Model Development, 11(5), 1929–1969. doi:10.5194/gmd-11-1929-2018 Lafore, J. P., Stein, J., Asencio, N., Bougeault, P., Ducrocq, V., Duron, J., Fischer, C., et
al. (1998). The Meso-NH Atmospheric Simulation System. Part I: Adiabatic formulation
and control simulations. Annales Geophysicae, 20. doi:10.1007/s00585-997-0090-6 Lemonsu, A., Belair, S., & Mailhot, J. (2009). The New Canadian Urban Modelling Sys-
tem: Evaluation for Two Cases from the Joint Urban 2003 Oklahoma City Experiment. Boundary-Layer Meteorology, 133(1), 47–70. doi:10.1007/s10546-009-9414-2 Lemonsu, A., Grimmond, C. S. Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
3 Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
4 nced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
1105/joss 02008
4 References B., & Masson, V. (2004). Modeling the Surface Energy Bal-
ance of the Core of an Old Mediterranean City: Marseille. Journal of Applied Meteorology,
43, 16. doi:10.1175/1520-0450(2004)043<0312:MTSEBO>2.0.CO;2 Lemonsu, A., & Masson, V. (2002). Simulation of a Summer Urban Breeze Over Paris. Boundary-Layer Meteorology, 104(3), 463–490. doi:10.1023/A:1016509614936 Lemonsu, A., Masson, V., Shashua-Bar, L., Erell, E., & Pearlmutter, D. (2012). Inclusion of
vegetation in the Town Energy Balance model for modelling urban green areas. Geoscien-
tific Model Development, 5(6), 1377–1393. doi:10.5194/gmd-5-1377-2012 Leroyer, S., Mailhot, J., Bélair, S., Lemonsu, A., & Strachan, I. B. (2010). Modeling the
Surface Energy Budget during the Thawing Period of the 2006 Montreal Urban Snow
Experiment. Journal of Applied Meteorology and Climatology, 49(1), 68–84. doi:10. 1175/2009JAMC2153.1 Masson, V. (2000). A Physically-Based Scheme For The Urban Energy Budget In Atmospheric
Models. Boundary-Layer Meteorology, 94(3), 357–397. doi:10.1023/A:1002463829265 Masson, V., Bonhomme, M., Salagnac, J.-L., Briottet, X., & Lemonsu, A. (2014). Solar
panels reduce both global warming and urban heat island. Frontiers in Environmental
Science, 2. doi:10.3389/fenvs.2014.00014 Masson, V., Grimmond, C. S. B., & Oke, T. R. (2002). Evaluation of the town energy
balance (teb) scheme with direct measurements from dry districts in two cities. Journal
of Applied Meteorology, 41(10), 1011–1026. doi:10.1175/1520-0450(2002)041<1011:
EOTTEB>2.0.CO;2 Meyer, D., & Raustad, R. (2019). MinimalDX. Zenodo. doi:10.5281/zenodo.3562310 Meyer, D., Schoetter, R., Riechert, M., Verrelle, A., Tewari, M., Dudhia, J., Masson, V., et
al. (2020). WRF-TEB: Implementation and evaluation of the coupled Weather Research
and Forecasting (WRF) and Town Energy Balance (TEB) model. Journal of Advances in
Modeling Earth Systems. doi:10.1029/2019MS001961 Meyer, D., & Thevenard, D. (2019). PsychroLib: A library of psychrometric functions to
calculate thermodynamic properties of air. Journal of Open Source Software, 4(33), 1137. doi:10.21105/joss.01137 Pielke, R. A., Cotton, W. R., Walko, R. L., Tremback, C. J., Lyons, W. A., Grasso, L. D.,
Nicholls, M. E., et al. (1992). A comprehensive meteorological modeling system RAMS. Meteorology and Atmospheric Physics, 49(1-4), 69–91. doi:10.1007/BF01025401 d software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
5/j
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3 Meyer et al., (2020). Enhanced software and platform for the Town Energy Balance (TEB) model. Journal of Open Source Software, 5(50),
2008. https://doi.org/10.21105/joss.02008
3 Pigeon, G., Moscicki, M. A., Voogt, J. A., & Masson, V. (2008). Simulation of fall and winter
surface energy balance over a dense urban area using the TEB scheme. Meteorology and
Atmospheric Physics, 102(3-4), 159–171. doi:10.1007/s00703-008-0320-9 Pigeon, G., Zibouche, K., Bueno, B., Bras, J. L., & Masson, V. (2014). Improving the
capabilities of the town energy balance model with up-to-date building energy simula-
tion algorithms: An application to a set of representative buildings in paris. Energy and
Buildings, 76, 1–14. doi:10.1016/j.enbuild.2013.10.038 Redon, E. C., Lemonsu, A., Masson, V., Morille, B., & Musy, M. (2017). Implementation of
street trees within the solar radiative exchange parameterization of TEB in SURFEX v8.0. Geoscientific Model Development, 10(1), 385–411. doi:10.5194/gmd-10-385-2017 Redon, E., Lemonsu, A., & Masson, V. (2020). An urban trees parameterization for modeling
microclimatic variables and thermal comfort conditions at street level with the town energy
balance model (TEB-SURFEX v8.0). Geoscientific Model Development, 13(2), 385–399. doi:10.5194/gmd-13-385-2020 Riechert, M., & Meyer, D. (2019). WRF-CMake: Integrating CMake support into the Ad-
vanced Research WRF (ARW) modelling system. Journal of Open Source Software, 4(41),
1468. doi:10.21105/joss.01468 Rozoff, C. M., Cotton, W. R., & Adegoke, J. O. (2003). Simulation of St. Louis, Missouri,
land use impacts on thunderstorms. Journal of Applied Meteorology, 42(6), 716–738. doi:10.1175/1520-0450(2003)042<0716:SOSLML>2.0.CO;2 Schoetter, R., Masson, V., Bourgeois, A., Pellegrino, M., & Lévy, J.-P. (2017). Parametri-
sation of the variety of human behaviour related to building energy consumption in the
Town Energy Balance (SURFEX-TEB v. 8.2). Geoscientific Model Development, 10(7),
2801–2831. doi:10.5194/gmd-10-2801-2017 Skamarock, W. C., Klemp, J. B., Dudhia, J., Gill, D. O., Liu, Z., Berner, J., Wang, W., et al. (2019). A Description of the Advanced Research WRF Model Version 4. NCAR Technical
Note NCAR/TN-556+STR, 145. doi:10.5065/1dfh-6p97 Xue, M., Droegemeier, K. K., & Wong, V. (2000). The Advanced Regional Prediction System
(ARPS) – a multi-scale nonhydrostatic atmospheric simulation and prediction model. Part
I: Model dynamics and verification. Meteorology and Atmospheric Physics, 75(3), 161–
193. doi:10.1007/s007030070003
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Investigation of gene effects on fruit shape index and seed size in generations resulting from the crossing of Zucchini and hull-less seed Pumpkin
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Euphytica
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Investigation of gene effects on Fruit Shape Index
and Seed Size in Generations Resulting from The
Crossing of Zucchini and Hull-Less Seed Pumpkin
Sima Davoodi
Guilan University Faculty of Agriculture
Jamal-Ali Olfati ( jamalaliolfati@gmail.com )
Guilan University Faculty of Agriculture https://orcid.org/0000-0002-5485-8884
Babak Rabiei
Guilan University Faculty of Agriculture
Atefeh Sabouri
Guilan University Faculty of Agriculture
Research Article
Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
Epistasis, Generation mean analysis
Posted Date: June 22nd, 2021
DOI: https://doi.org/10.21203/rs.3.rs-590556/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Version of Record: A version of this preprint was published at Euphytica on August 18th, 2021. See the
published version at https://doi.org/10.1007/s10681-021-02911-y. Investigation of gene effects on Fruit Shape Index
and Seed Size in Generations Resulting from The
Crossing of Zucchini and Hull-Less Seed Pumpkin
Sima Davoodi
Guilan University Faculty of Agriculture
Jamal-Ali Olfati ( jamalaliolfati@gmail.com )
Guilan University Faculty of Agriculture https://orcid.org/0000-0002-5485-8884
Babak Rabiei
Guilan University Faculty of Agriculture
Atefeh Sabouri
Guilan University Faculty of Agriculture
Research Article
Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
Epistasis, Generation mean analysis
Posted Date: June 22nd, 2021
DOI: https://doi.org/10.21203/rs.3.rs-590556/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Version of Record: A version of this preprint was published at Euphytica on August 18th, 2021. See the
published version at https://doi.org/10.1007/s10681-021-02911-y. Investigation of gene effects on Fruit Shape Index
and Seed Size in Generations Resulting from The
Crossing of Zucchini and Hull-Less Seed Pumpkin Sima Davoodi
Guilan University Faculty of Agriculture
Jamal-Ali Olfati ( jamalaliolfati@gmail.com )
Guilan University Faculty of Agriculture https://orcid.org/0000-0002-5485-8
Babak Rabiei
Guilan University Faculty of Agriculture
Atefeh Sabouri
Guilan University Faculty of Agriculture * Corresponding Author: jamalaliolfati@gmail.com
16 * Corresponding Author: jamalaliolfati@gmail.com
16 Research Article Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
Epistasis, Generation mean analysis
Posted Date: June 22nd, 2021
DOI: https://doi.org/10.21203/rs.3.rs-590556/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Euphytica on August 18th, 2021. See the
published version at https://doi.org/10.1007/s10681-021-02911-y. Investigation of gene effects on fruit shape index and seed size in generations resulting from the
1
crossing of Zucchini and hull-less seed Pumpkin
2
Sima Davoodi 1, Jamalali Olfati 2*, Babak Rabiei 3, Atefeh Sabouri 4
3
1- Ph.D. student, Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of
4
Guilan
5
6
2- Associate Professor, Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of
7
Guilan
8
9
3- Professor, Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of
10
Guilan
11
12
4- Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences,
13
University of Guilan
14
15
* Corresponding Author: jamalaliolfati@gmail.com
16 Abstract
17 Fruit shape index (round shape) and seed size have important effects on pumpkin yield. To investigate
18
these traits and create the most desirable state, a cross was made between two pumpkin cultivars. The
19
objective of this study was to estimate the main gene effects (additive, dominant and di-genic epistasis)
20
and to determine the mode of inheritance for fruit shape and seed size by generation mean analysis. Six
21
generations, namely P1, P2, F1, F2, BC1, and BC2 from a cross between Zucchini and hull-less seed
22
Pumpkin, S10×P25, were constructed and evaluated for fruit length, fruit width, fruit shape index, and
23
some seed-related traits (seed length, seed width, seed thickness). The experiment was conducted in the
24
research field of the Faculty of Agricultural Science, University of Guilan, Rasht, Iran in 2019. Results
25
showed a significant difference between generations in terms of fruit and seed traits. Scale and joint scale
26
tests showed the presence of epistasis for some traits. According to the results of the average traits of
27
different generations, standard heterosis and hetrobeltiosis were observed. Concerning the fruit shape
28
index and seed width, there was an over-dominance effect. The broad-sense heritability of the traits was
29
relatively high for all traits and between 52 and 92%. Narrow sense heritability was between 26 and 86%
30
and relatively low for the fruit shape index and seed width. Therefore, selection of elite lines and
31
production of their hybrids are recommended as two methods suitable for breeding to achieve the round
32
shape index and larger seed size. 33 Investigation of gene effects on fruit shape index and seed size in generations resulting from the
1
crossing of Zucchini and hull-less seed Pumpkin
2 Sima Davoodi 1, Jamalali Olfati 2*, Babak Rabiei 3, Atefeh Sabouri 4 1- Ph.D. student, Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of
Guilan 2- Associate Professor, Department of Horticultural Sciences, Faculty of Agricultural Sciences,
Guilan 2- Associate Professor, Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of
Guilan 3- Professor, Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of
Guilan 3- Professor, Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of
Guilan 4- Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences,
University of Guilan * Corresponding Author: jamalaliolfati@gmail.com
16 Introduction Pumpkin is a plant of the Cucurbitaceae family, which according to the latest classification
38
statistics includes 118 genera and 825 species (Jeffrey et al. 1990). In Cucurbita, five cases of C. 39
argyrosperma, C. ficifolia, C. maxima, C. moschata, and C. pepo have been domesticated (Schaefer and
40
Renner 2011). Pumpkin has a rich and long history in terms of cultivation and domestication and unique
41
diversity in terms of morphological characteristics, particularly the shape of the fruit and has a wide range
42
of adaptation in cultivation (Savage et al. 2015). Pumpkins have a long history of consumption and are
43
used for many purposes, especially for seed. Despite the production of vegetables in Iran and its ranking
44
in terms of production in global markets and due to the area under cultivation and abundant production of
45
these products in the country, thus far, no significant activity has been performed in the field of seed
46
production, and most of the required seeds have been supplied from abroad (Hosseinzadeh and Amjadi
47
Souraki 2012). 48 Stewed squash (Cucurbita pepo L. var. pepo) is one of the species whose plants have limited
49
growth. The seeds of this squash have a uniform cream color; its leaves are multi-lobed with rough hairs
50
on the leaves. The tail of the fruit is hard, woody, and polygonal (angular). Its flowers are characterized
51
by short, thick, and conical flags (Decker-Walters and Walters 2000). 52
53
54 Hull-less seeded pumpkin (Cucurbita pepo L. var. pepo subsp. styriaca) is a species that has
55
found an important place in the medicine and food industries owing to fatty acids in its seed oil and high
56
protein content (Hosseinzadeh and Amjadi Souraki 2012). It has round fruit and more seeds per fruit. The
57
fruits of hull-less seeded pumpkins are yellowish-orange in color, and the seeds are olive green. One of
58
the most important characteristics of this plant is its skinless seeds (Mitra 2001). 59 One of the most diverse traits related to squash, which is also important to the consumer, is the
60
shape of the fruit. Fruit shape can vary from round to disc-shaped to very long (Paris 2008). Cucurbita
61
pepo L. is highly polymorphic for fruit shape and size. Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
34
Epistasis, Generation mean analysis
35 Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
34
Epistasis, Generation mean analysis
35 Keywords: Cucurbita pepo var. styriaca, Cucurbita pepo var. pepo, Additive effect, Dominant effect,
34
Epistasis, Generation mean analysis
35 1 1 36 Introduction Fruit shape and size are polygenically controlled,
62
and it has proven difficult to identify individual genes affecting these characteristics
63
Fruit shape is a highly important quantitative trait, which is closely associated with fruit
64
quality. There are no genetic models of fruit shapes (Hazra et al. 2007; Paris and Brown 2005; Wang et
65
al. 2012), and no reports are available on the genetic control of fruit shapes in pumpkins. The analysis of
66
fruit shape using the L/W ratio could be a useful approach to select other characteristics of pumpkin
67
shapes (ketsakul et al. 2020). 68
69 2 The hybrid production method can be used to achieve the desired traits, including fruits with a
72
round shape containing larger seeds. In addition, one of the methods to evaluate the results in hybrids is
73
to use the method of generation average analysis and calculation of genetic parameters. Generation means
74
analysis is an efficient technique to estimate important gene effects, such as additive effects, dominance,
75
and interaction in the development of quantitative traits (Amaefula et al. 2014). Generation means
76
analysis method was developed by Jinks and Jones (1958), Hayman (1958), and Mather and Jinks (1982). 77
In general, knowing nature genes involved in the development of traits act is the first step in advancing
78
breeding goals, so that if most of the gene effects are additive, it is recommended that the breeding
79
process be continued through direct selection for the trait. If the genetic effects are predominant, it is
80
recommended that the breeding route be continued to produce hybrid cultivars (Bernardo 2002; Azizi et
81
al. 2006; Rebolloza 2016). 82 According to researchers
83
84
85
Heritability for seed weight, seed length, and seed thickness was 76.4, 78.7, and
86
85.2%, respectively. A lower heritability for seed width (68.1%) can probably be due to the effect of
87
different degrees of expression of the hull-less trait on seed coat development along the seed margins. 88
Seed thickness and fruit weight did not correlate with each other. Seed weight and fruit weight as well as
89
seed length and fruit weight were positively correlated with each other. Therefore, it was possible to
90
obtain small fruits with large seeds by selections (Carle et al. 1994). Materials and methods:
108 Zucchini and hull-less seeded Pumpkin pure lines (P25 and S10) were selected for hybridization to
109
improve fruit shape (round) and seed size (large). Field experiments to generate populations and record
110
field data were performed during three consecutive planting seasons in the spring and summer of 2017-
111
2019 in the research field of the Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. In the
112
first year, two parental lines, Pumpkin (S10) and Zucchini (P25), which were previously purified by
113
Davoodi et al. (2015), were crossed to produce the seeds of the first generation (F1). At this stage, the
114
field cultivation system was considered in rows, and planting distance of two meters between rows and
115
one meter on the rows (Sajed et al. 2002; Elizabeth 2001; Latifi et al. 2012) and the steps were performed
116
by implementing the drip irrigation system, using cover mulch, and exercising necessary and appropriate
117
care in all stages of growth. In the second year, F1 seeds were planted in the field and were self-pollinated
118
to produce second-generation seeds (F2). Furthermore, many other F1-generation plants were backcrossed
119
with both parental lines to produce backcrossing generations (BC1 and BC2). In the third year, the seeds
120
of the six generations, including two parental lines (P1 and P2), along with F1, F2, BC1 and BC2 were
121
cultivated in a randomized complete block design with three replications in the research field of the
122
Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. The desired traits, such as fruit
123
length, fruit width, fruit shape index, seed length, seed width, and seed thickness were measured. Length
124
along the axis of each fruit from the place of the peduncle to the tail of the fruit and width at the
125
equatorial region of the fruit were measured. Measurements were performed on each seed using a digital
126
caliper based on millimeters. Seed length along the seed axis, seed width from the widest part of the seed,
127
and seed thickness from the thickest part of the seed were considered. 128 Random samples were selected from each generation, so that from each parent and the first
129
generation, 10 plants per replication and 30 plants, and from each of the backcrosses and the second
130
generation 30 plants per replication and 90 plants, were considered. Introduction 91 Al-Hamadany and Al-Lelah (2010) studied broad sense heritability (BSH), narrow-sense
92
heritability (NSH), and dominance degree for some fruit characteristics (fruit length, fruit diameter, and
93
average fruit weight) in summer squash. Broad sense heritability estimates were moderate to high for the
94
studied traits. Narrow sense heritability was low for the studied characteristics. Over dominance
95
controlled the inheritance of the studied traits. Narrow sense heritability was low and valued 13% (fruit
96
length), 21% (fruit diameter) and 27% (average fruit weight). 97 98
99
The
100
length-to-width ratio ranged from 1.5 to 2.5, the length-to-thickness ratio from 3.7 to 8.2, and the width-
101
to-thickness ratio from 2.0 to 4.9. Based on the results
102
103 3 3 Therefore, in this study, an attempt was made to create a combination through intersection of two
104
pumpkin species having a round shape in terms of fruit shape and a longer length, width and thickness in
105
terms of seed quality. Therefore, appropriate methods should be employed to improve the traits to be
106
determined by examining the effect of the desired genes. 107 Materials and methods:
108 Equations (2) to (5)
138
were used to test the presence of epistatic effects, and t-Student based on equations (6) to (9) was used to
139
test their significance (Mather and Jinks 1982):
140 (2) A = 2BC
̅̅̅̅1 −F̅1 −P̅1
141
(3) B = 2BC
̅̅̅̅2 −F̅1 −P̅2
142
(4) C = 4F̅2 −2F̅1 −P̅1 −P̅2
143
(5) D = 2F̅2 −BC
̅̅̅̅1 –BC
̅̅̅̅2
144
(6) tA=
A
√V𝑃̅1+ V𝐹̅1+4V 𝐵𝐶
̅̅̅̅1
145
(7) 𝑡𝐵=
B
√V 𝑃̅1+ V 𝐹̅1+4V 𝐵𝐶
̅̅̅̅2
146
(8) tC=
C
√𝑉𝑃̅1 +V𝑃̅2 +4V 𝐹̅1 +16V𝐹̅2
147
(9) tD=
D
√4VF̅2 + VBC
̅̅̅̅ +VBC
̅̅̅̅2
148 (2) A = 2BC
̅̅̅̅1 −F̅1 −P̅1
141
(3) B = 2BC
̅̅̅̅2 −F̅1 −P̅2
142
(4) C = 4F̅2 −2F̅1 −P̅1 −P̅2
143
(5) D = 2F̅2 −BC
̅̅̅̅1 –BC
̅̅̅̅2
144
(6) tA=
A
√V𝑃̅1+ V𝐹̅1+4V 𝐵𝐶
̅̅̅̅1
145
(7) 𝑡𝐵=
B
√V 𝑃̅1+ V 𝐹̅1+4V 𝐵𝐶
̅̅̅̅2
146
(8) tC=
C
√𝑉𝑃̅1 +V𝑃̅2 +4V 𝐹̅1 +16V𝐹̅2
147
(9) tD=
D
√4VF̅2 + VBC
̅̅̅̅ +VBC
̅̅̅̅2
148 To estimate the effects of genes controlling the studied traits, Equations (10) to (15) were used (Mather
149
and Jinks 1982):
150 To estimate the effects of genes controlling the studied traits, Equations (10) to (15) were used (Mather
149
and Jinks 1982):
150 151 (10) m=0.5P1
̅̅̅̅ + 0.5P2
̅̅̅̅ + 4F2
̅̅̅ − 2BC1
̅̅̅̅̅ − 2BC2
̅̅̅̅̅
152
(11) d = 0.5P1
̅̅̅̅ −0.5P2
̅̅̅̅
153 (10) m=0.5P1
̅̅̅̅ + 0.5P2
̅̅̅̅ + 4F2
̅̅̅ − 2BC1
̅̅̅̅̅ − 2BC2
̅̅̅̅̅
152
(11) d = 0.5P1
̅̅̅̅ −0.5P2
̅̅̅̅
153 (10) m=0.5P1
̅̅̅̅ + 0.5P2
̅̅̅̅ + 4F2
̅̅̅ − 2BC1
̅̅̅̅̅ − 2BC2
̅̅̅̅̅
152 (11) d = 0.5P1
̅̅̅̅ −0.5P2
̅̅̅̅
153 (12) h = 6BC1
̅̅̅̅̅ +6BC2
̅̅̅̅̅ −8F2
̅̅̅ −F1
̅̅̅ −1.5P1
̅̅̅̅ −1.5P2
̅̅̅̅
154
(13) i = 2(BC
̅̅̅̅1+BC
̅̅̅̅2 ) – 4𝐹2
̅̅̅̅
155
(14) j = 2(BC
̅̅̅̅1– BC
̅̅̅̅2 ) – P̅1+P̅2
or j=2𝐵𝐶1
̅̅̅̅̅̅– 𝑃1
̅̅̅̅– 2𝐵𝐶2
̅̅̅̅̅̅ + 𝑃2
̅̅̅̅
156
(15) l = P̅1 +P ̅2 + 2F1
̅̅̅̅ +4𝐹2
̅̅̅̅– 4BC
̅̅̅̅1– 4BC
̅̅̅̅2
157 (12) h = 6BC1
̅̅̅̅̅ +6BC2
̅̅̅̅̅ −8F2
̅̅̅ −F1
̅̅̅ −1.5P1
̅̅̅̅ −1.5P2
̅̅̅̅
154 (13) i = 2(BC
̅̅̅̅1+BC
̅̅̅̅2 ) – 4𝐹2
̅̅̅̅
155 (14) j = 2(BC
̅̅̅̅1– BC
̅̅̅̅2 ) – P̅1+P̅2
or j=2𝐵𝐶1
̅̅̅̅̅̅– 𝑃1
̅̅̅̅– 2𝐵𝐶2
̅̅̅̅̅̅ + 𝑃2
̅̅̅̅
156 To evaluate the best genetic model controlling each of the studied traits, joint scaling tests based on the
158
chi-squared test were used, and the degree of correspondence between the observed and expected means
159
(obtained from the significant values of genetic parameters) of the respective generations were
160
statistically tested. Materials and methods:
108 131 To analyze the data and evaluate the genetic effects of controlling the studied traits, the method
132
employed by Mather and Jinks (1982) according to Equation (1) was used:
133 (1) Y= m + α [d] +β [h] + α2[i] + 2αβ[j] + β2[l]
134 (1) Y= m + α [d] +β [h] + α2[i] + 2αβ[j] + β2[l]
134 Where Y is the mean of one generation, m is the mean of all generations in a cross, [d], [h], [i], [j] and [l]
135
are the effects of genetic parameters of Mather and Jinks’ model (1982) (additive, dominance, additive
136 4 4 ×additive epistasis, additive × dominance epistasis and dominance × dominance epistasis, respectively,
137
and α, β, α2, 2αβ and β2 are the coefficients of these genetic parameters, respectively). Equations (2) to (5)
138
were used to test the presence of epistatic effects, and t-Student based on equations (6) to (9) was used to
139
test their significance (Mather and Jinks 1982):
140 ×additive epistasis, additive × dominance epistasis and dominance × dominance epistasis, respectively,
137
and α, β, α2, 2αβ and β2 are the coefficients of these genetic parameters, respectively). Materials and methods:
108 161 The additive (VD), dominance (VH), and environmental (VE) variance components controlling the
162
phenotypic diversity of each of the studied traits were also calculated based on Equations (16) to (18)
163
(Kearsey and Pooni 1996):
164 The additive (VD), dominance (VH), and environmental (VE) variance components controlling the
162
phenotypic diversity of each of the studied traits were also calculated based on Equations (16) to (18)
163
(Kearsey and Pooni 1996):
164 5 5 )
BC2
+V
BC1
(V
–
F2
= 2 V
D
V
)
16
(
165
(17) VH = VF2 – VD – VE
166
(18) VE =
1
3 (VP1 + VP2 + VF1)
167 Where VD is the additive variance, VH is the dominance variance, VE is the environment variance, 𝑉𝑃1
̅̅̅̅ is
168
the first parent variance, 𝑉𝑃2
̅̅̅̅ is the first parent variance, 𝑉𝐹2
̅̅̅̅ is the second generation of variance, 𝑉𝐹1
̅̅̅̅ is
169
the first generation of variance, and 𝑉𝐵𝐶1
̅̅̅̅̅̅ and 𝑉𝐵𝐶2
̅̅̅̅̅̅ are the first and second back-crossing, respectively. 170
To determine the dominance deviations in different gene loci, the mean degree of dominance was
171
estimated based on Equation (19): (Mather and Jinks 1982):
172 (H/D)1/2= (
2V[H]
V[D]
1/2
)
173 (H/D)1/2= (
2V[H]
V[D]
1/2
)
173 Broad- and narrow-sense heritability were calculated using Equations 20 (Falconer and Mackay 1996)
174
and 21 (Mather and Jinks 1982):
175 Broad- and narrow-sense heritability were calculated using Equations 20 (Falconer and Mackay 1996)
174
and 21 (Mather and Jinks 1982):
175 hb
2 =
VF2−VE
VF2
=
V𝐷+V𝐻
V𝐷+V𝐻+V𝐸
176
hn2 = VD
VF2
=
𝑉𝐷
V𝐷+ V𝐻+ V𝐸
177 hb
2 =
VF2−VE
VF2
=
V𝐷+V𝐻
V𝐷+V𝐻+V𝐸
176
hn2 = VD
VF2
=
𝑉𝐷
V𝐷+ V𝐻+ V𝐸
177 The data normality test was performed using the SPSS software, and all other statistical and genetical
178
analyses were conducted using the SAS software (Kang 2003). To compare the means of the studied
179
generations, Tukey’s test was used at 5% and 1% probability levels. 180 The data normality test was performed using the SPSS software, and all other statistical and genetical
178
analyses were conducted using the SAS software (Kang 2003). To compare the means of the studied
179
generations, Tukey’s test was used at 5% and 1% probability levels. 180 Results and discussion:
181 The value of this trait in F2 and BC1
195
generations was higher than that of both parents, while in the BC2 generation (backcrossing with the
196
second parent), it was higher than the average of the parents but lower than the best parent value. 197
According to the results, it is possible that the back-cross method transfers the desired characteristics to
198
progenies. The average of the F2 generation compared to the F1 generation showed a decrease, which can
199
be attributed to the effects of self-pollination and the existence of the phenomenon of inbreeding
200
depression. Saad (2003) also confirmed these results by reporting the presence of heterosis in the fruit
201
and seed traits of squash. 202 the mean of the parents and even more valuable than the parents. Therefore, the phenomenon of
193
heterobeltiosis (heterosis compared to the best parent) was observed for this trait. These results are very
194
favorable considering the increase of fruit width and seed size. The value of this trait in F2 and BC1
195
generations was higher than that of both parents, while in the BC2 generation (backcrossing with the
196
second parent), it was higher than the average of the parents but lower than the best parent value. 197
According to the results, it is possible that the back-cross method transfers the desired characteristics to
198
progenies. The average of the F2 generation compared to the F1 generation showed a decrease, which can
199
be attributed to the effects of self-pollination and the existence of the phenomenon of inbreeding
200
depression. Saad (2003) also confirmed these results by reporting the presence of heterosis in the fruit
201
and seed traits of squash. 202 For the fruit shape index (fruit length to width ratio), considering that the optimal breeding goal
203
is the round shape (i.e. shape index near to one), the offspring of the new generation has values of the
204
shape index lower than those of the parents, which would be better due to higher yield (Ketsakul et al. 205
2020). Observation of the measurement results of this trait showed that the fruit shape index in the F1
206
generation (1.39) was decreased compared to the average of the parents (1.69), which was favorable
207
according to the intended purpose. Results and discussion:
181 Examination of the normal distribution in the measured data based on skewness and kurtosis tests
182
using the SPSS software indicated that the data related to all studied traits had a normal distribution
183
(Table 1). 184 Examination of the normal distribution in the measured data based on skewness and kurtosis test
182
using the SPSS software indicated that the data related to all studied traits had a normal distribution
183
(Table 1). 184
The results of the analysis of variance demonstrated a significant difference between the six
185
generations for all studied traits at the probability level 1% (Table 2); in other words, the genetic analysi
186
and review of their inheritance were possible. 187
Evaluation of the mean and standard error of the traits (Table 3) revealed a significant difference
188
between the means of parents and offspring for some traits, such as fruit length and width and seed length
189
and width. Furthermore, the phenomenon of standard heterosis was observed for these traits, meaning
190
that the average of the first generation (F1) for these traits was higher than the average parent. In the case
191
of seed length, the value of the F1 generation obtained from the crossing of two parents was higher than
192 The results of the analysis of variance demonstrated a significant difference between the six
185
generations for all studied traits at the probability level 1% (Table 2); in other words, the genetic analysis
186
and review of their inheritance were possible. 187 Evaluation of the mean and standard error of the traits (Table 3) revealed a significant difference
188
between the means of parents and offspring for some traits, such as fruit length and width and seed length
189
and width. Furthermore, the phenomenon of standard heterosis was observed for these traits, meaning
190
that the average of the first generation (F1) for these traits was higher than the average parent. In the case
191
of seed length, the value of the F1 generation obtained from the crossing of two parents was higher than
192 6 the mean of the parents and even more valuable than the parents. Therefore, the phenomenon of
193
heterobeltiosis (heterosis compared to the best parent) was observed for this trait. These results are very
194
favorable considering the increase of fruit width and seed size. Results and discussion:
181 On the contrary, the value of this trait did not considerably decrease in
208
the F2 generation compared to the F1 generation. In fact, in terms of achieving fruits with a round to oval
209
shape, a favorable population was obtained. This superiority of offspring over the average of parents can
210
indicate the existence of dominance or over-dominance effects in controlling this trait. The results of
211
backcross generations demonstrated that the fruit shape index in BC1 was less than that in mid-parents;
212
however, in BC2, it was more than that of mid-parents (Table 3). The fruit length of the first and second
213
generations corresponded to the average of the parents. The value of this trait in the offspring of BC1 and
214
BC2 was also less than that of mid-parents but more than that of parents with a lower value. Regarding
215
the fruit width trait, the average of F1 generation was higher than that of parents and higher than that of
216
the best parents. Therefore, the phenomenon of heterobeltiosis was observed for this trait in the current
217
experiment. Moreover, in the offspring of the F2 generation not only there was no decrease compared to
218
the F1 generation but also a higher value was obtained than the average of parents and even higher than
219
parents with a higher value. This result is highly desirable considering that we aimed to reduce the length
220
and increase the width of the fruit to reach the round shape of the fruit. This increase in value was also
221
observed about BC1, while the results of BC2 were decreased compared to both parents. 222 In the case of seed thickness, the mean F1 obtained from the crossing of two parents was lower
223
than the mean of the parents. Furthermore, the value of the F1 generation was less than the average of the
224
parents, but it was close to the average and higher than that of parents. The value of this trait in the results
225 7 7 of the F2 generation and the BC1 and BC2 was lower than the average of the parents and more than the
226
value of the parents with less seed thickness. Results and discussion:
181 227 The results of the scaling tests showed that none of the scaling tests A, B, C and D was
228
significant for the fruit shape index, seed length, seed width, and seed thickness traits, demonstrating that
229
the effects of epistasis (non-allelic effects) did not play a crucial role in controlling the diversity of the
230
mentioned traits. However, to ensure the results, it is better to calculate and test the effects of epistasis. 231
On the contrary, for fruit length and width, the B-scaling test was significant at the level of 5%
232
probability, which probably indicates the presence of an additive × dominance interaction (Table 4). 233 Table 5 presents the genetic parameters controlling each of the studied traits. The results
234
indicated that all genetic parameters were significant for fruit length, so that the dominance and
235
dominance × dominance interaction effects and the other components were significant at 5% and 1%
236
probability levels, respectively. In addition, the negative dominance effect and the positive dominance ×
237
dominance interaction effect for fruit length exhibited the presence of the duplicate epistasis effect of
238
reducing dominance alleles. On the contrary, for fruit width, except for the additive× dominance
239
interaction, which was significant at 1% probability level, the other effects were not significant. For the
240
fruit shape index, the additive and the additive × dominance interaction effects were significant at 1%
241
probability level, and the other components were not significant. 242 The results for seed traits indicated that the additive and additive × dominance interaction effects
243
were significant at 5% and 1% probability levels, respectively, while the other components were not
244
significant. Simple effects and interactions were significant except dominant × dominant interaction. 245
Conc Long & ambiguous. Please rewrite more clearly.erning seed thickness, only the additive effect was
246
significant at 1% probability level. The additive effect is investigated as a genetic effect in plant breeding,
247
which is significant according to the researcher and corresponds to the results of the experiment. In
248
addition, dominance genetic variations were significant for all studied mature fruit traits, except for
249
average mature fruit diameter, since it was not significant. 250 Table 6 presents the evaluation of the components of variance, along with heritability and degree
251
of dominance of each of the studied traits. Results and discussion:
181 In other words, it is better to use the hybrid production
277
method to improve these traits in the studied population. Other researchers, in studying the gene action of
278
different traits in squash, have reported similar results (Al-Ballat. 2008; Aruah et al. 2010; Najvot 2016). 279
Moreover, the average degree of dominance for fruit length, fruit width, seed length and seed thickness
280
varied from 0.27 to 0.94, demonstrating the relative or partial dominance gene action as well as the
281
importance of both additive and dominance effects in these traits. Accordingly, the selection can be made
282
in early generations (Al-Zabaee 2006; Al-Ballat 2008; Hussien 2015). 283
Table 1. Data normality test
284
Seed thickness
Seed width
Seed
length
Shape Index
Fruit
Width
Fruit
length
1.157
-0.183
-0.022
0.918
0.342
0.623
Skewness
1.148
0.294
-1.105
0.166
-1.193
-0.240
kurtosis
0.18
0.20
0.20
0.052
0.20
0.20
Sig (Kolmogorov-smirnov)
0.04
0.19
0.22
0.079
0.81
0.17
Sig (Shapiro-wilk) Concerning some traits, such as fruit shape index and seed width, relatively low narrow-sense
269
heritability indicates the effect of the dominance component in controlling these traits, and hybrid seed
270
production can be recommended to improve these traits. In other words, low narrow-sense heritability in
271
these traits confirms that there is less effect of additive variance and a positive effect of selection on these
272
traits (Rebolloza 2016). According to Al-Zabaee’s reports (2006), broad-sense heritability was high for
273
their studied traits, such as fruit length and fruit diameter, while narrow-sense heritability was moderate. 274 The average degree of dominance for the fruit shape index and seed width was more than one,
275
which probably indicates the presence of the over-dominance action of the genes controlling these traits;
276
hence, selection of these traits was ineffective. In other words, it is better to use the hybrid production
277
method to improve these traits in the studied population. Other researchers, in studying the gene action of
278
different traits in squash, have reported similar results (Al-Ballat. 2008; Aruah et al. 2010; Najvot 2016). 279
Moreover, the average degree of dominance for fruit length, fruit width, seed length and seed thickness
280
varied from 0.27 to 0.94, demonstrating the relative or partial dominance gene action as well as the
281
importance of both additive and dominance effects in these traits. Results and discussion:
181 The results indicated that concerning fruit length, fruit width,
252
seed length and seed thickness, the amount of additive variance more than the dominant variance
253
confirms the positive effect of selection on the breeding of these traits. However, in the case of the fruit
254
shape index, the additive variance was higher than the dominant variance, and regarding the seed width,
255
the two additives and dominance variance exhibited the same value. 256 8 8 The results showed that about fruit length, fruit width and seed length, the environmental
257
variance was less than the additive variance but greater than the dominance variance. The importance of
258
additive and dominance variance regarding the fruit shape index and the seed thickness has been
259
reported. This indicates the low effect of the environment on these traits in this experiment, but in the
260
case of seed width, environmental variance revealed a greater effect than both additive and dominance
261
variance did. 262 Regarding most of traits, broad-sense heritability was relatively high. Furthermore, high narrow-
263
sense heritability in some traits, such as fruit length and seed length, indicated that selection of the best
264
individuals based on these traits could be successful, since the phenotype expresses almost the same
265
genotype (Javanmard et al. 2018). These results were consistent with the findings obtained by Saad
266
(2003), showing that the values of broad- and narrow- sense heritability were high for fruit length; this
267
demonstrates the importance of additive genetic variance in the inheritance of this trait. 268 Concerning some traits, such as fruit shape index and seed width, relatively low narrow-sense
269
heritability indicates the effect of the dominance component in controlling these traits, and hybrid seed
270
production can be recommended to improve these traits. In other words, low narrow-sense heritability in
271
these traits confirms that there is less effect of additive variance and a positive effect of selection on these
272
traits (Rebolloza 2016). According to Al-Zabaee’s reports (2006), broad-sense heritability was high for
273
their studied traits, such as fruit length and fruit diameter, while narrow-sense heritability was moderate. 274
The average degree of dominance for the fruit shape index and seed width was more than one,
275
which probably indicates the presence of the over-dominance action of the genes controlling these traits;
276
hence, selection of these traits was ineffective. Results and discussion:
181 Accordingly, the selection can be made
282
in early generations (Al-Zabaee 2006; Al-Ballat 2008; Hussien 2015). 283 Table 1. Data normality test
284
Seed thickness
Seed width
Seed
length
Shape Index
Fruit
Width
Fruit
length
1.157
-0.183
-0.022
0.918
0.342
0.623
Skewness
1.148
0.294
-1.105
0.166
-1.193
-0.240
kurtosis
0.18
0.20
0.20
0.052
0.20
0.20
Sig (Kolmogorov-smirnov)
0.04
0.19
0.22
0.079
0.81
0.17
Sig (Shapiro-wilk)
285 9 Table 2. Analysis of variance for fruit and seed traits
Mean squares 287 Table 2. Analysis of variance for fruit and seed traits
287
Mean squares
Source of
Variation
df
Fruit length
Fruit width
Shape index
Seed length
Seed width
Seed
thickness
Replication
2
0.098ns
0.496ns
0.006ns
0.850*
0.198ns
0.102ns
Generation
5
19.163**
9.462**
0.297**
2.312* *
1.199**
0.515**
Error
10
1.746
1.074
0.014
0.175
0.089
0.034
C.V.%
6.180
7.356
7.889
2.619
3.280
8.005
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. 288
289 Table 2. Analysis of variance for fruit and seed traits
287
Mean squares
Source of
Variation
df
Fruit length
Fruit width
Shape index
Seed length
Seed width
Seed
thickness
Replication
2
0.098ns
0.496ns
0.006ns
0.850*
0.198ns
0.102ns
Generation
5
19.163**
9.462**
0.297**
2.312* *
1.199**
0.515**
Error
10
1.746
1.074
0.014
0.175
0.089
0.034
C.V.%
6.180
7.356
7.889
2.619
3.280
8.005
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. 288
289
Table 3. Comparison of means of the generations for all studied traits
290
Generation
Fruit length
(cm)
Mean±SE
Fruit width (cm)
Mean±SE
Shape index
Mean±SE
Seed length
(mm)
Mean±SE
Seed width
(mm)
Mean±SE
Seed thickness
(mm)
Mean±SE
P1
18.266±0.044
13.666±0.12
1.336±0.008
14.703±0.042
7.705±0.025
1.930±0.029
P2
25.576±0.053
12.433±0.06
2.056±0.014
15.709±0.043
8.954±0.046
3.105±0.028
F1
22.113±0.409
15.950±0.579
1.390±0.035
17.152±0.142
9.477±0.196
2.247±0.039
F2
21.856±1.139
14.653±0.833
1.496±0.095
16.123±0.512
8.500±0.078
2.134±0.189
BC1
19.445±0.827
16.050±0.697
1.216±0.104
16.706±0.395
9.111±0.078
2.132±0.150
BC2
21.023±0.886
11.803±0.193
1.783±0.073
15.503±0.363
8.815±0.148
2.450±0.174
HSD(0.01)
4.904
3.847
0.452
1.554
1.068
0.693
HSD(0.05)
3.747
2.94
0.346
1.187
0.816
0.529
291
Table 4. Scaling tests to assess the epistatic (non-allelic) effects
292
Scaling
test
Fruit length
Fruit width
Shape index
Seed length
Seed width
Seed thickness
A
ns
1.489
-2.484ns
0.294ns
-1.117ns
ns
0.907
-
ns
0.087
-
B
5.643*
4.777*
-0.12ns
1.444ns
0.846ns
0.452ns
C
0.678ns
-0.612ns
0.188ns
0.062ns
1.811ns
0.993ns
D
3.244ns
1.453ns
-0.007ns
0.018ns
-0.936ns
-0.314ns
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. Results and discussion:
181 293
Significance levels are shown based on the t-test compared to table t. 294
295
Table 5. Estimation of genetic effects for different generations resulting from the Zucchini and Hull-less seeded
296
Pumpkin for the studied traits
297
Genetic
parameter
Fruit length
Fruit width
Shape index
Seed
length
Seed width
Seed
thickness
m
28.411**
15.956**
1.683**
15.279**
6.460**
1.889**
d
-3.655**
0.616ns
-0.360**
-0.503*
-0.624**
-0.587**
h
-19.919*
-5.206ns
-0.453ns
1.503ns
5.045*
0.619ns
i
6 489ns
2 906ns
0 013ns
0 072ns
1 870**
0 628ns Replication
2
0.098
0.496
0.006
0.850*
0.198
0.102
Generation
5
19.163**
9.462**
0.297**
2.312* *
1.199**
0.515**
Error
10
1.746
1.074
0.014
0.175
0.089
0.034
C.V.%
6.180
7.356
7.889
2.619
3.280
8.005
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. 288
289
Table 3. Comparison of means of the generations for all studied traits
290
Generation
Fruit length
(cm)
Mean±SE
Fruit width (cm)
Mean±SE
Shape index
Mean±SE
Seed length
(mm)
Mean±SE
Seed width
(mm)
Mean±SE
Seed thickness
(mm)
Mean±SE
P1
18.266±0.044
13.666±0.12
1.336±0.008
14.703±0.042
7.705±0.025
1.930±0.029
P2
25.576±0.053
12.433±0.06
2.056±0.014
15.709±0.043
8.954±0.046
3.105±0.028
F1
22.113±0.409
15.950±0.579
1.390±0.035
17.152±0.142
9.477±0.196
2.247±0.039
F2
21.856±1.139
14.653±0.833
1.496±0.095
16.123±0.512
8.500±0.078
2.134±0.189
BC1
19.445±0.827
16.050±0.697
1.216±0.104
16.706±0.395
9.111±0.078
2.132±0.150
BC2
21.023±0.886
11.803±0.193
1.783±0.073
15.503±0.363
8.815±0.148
2.450±0.174
HSD(0.01)
4.904
3.847
0.452
1.554
1.068
0.693
HSD(0.05)
3.747
2.94
0.346
1.187
0.816
0.529
291
Table 4. Scaling tests to assess the epistatic (non-allelic) effects
292
Scaling
test
Fruit length
Fruit width
Shape index
Seed length
Seed width
Seed thickness
A
ns
1.489
-2.484ns
0.294ns
-1.117ns
ns
0.907
-
ns
0.087
-
B
5.643*
4.777*
-0.12ns
1.444ns
0.846ns
0.452ns
C
0.678ns
-0.612ns
0.188ns
0.062ns
1.811ns
0.993ns
D
3.244ns
1.453ns
-0.007ns
0.018ns
-0.936ns
-0.314ns
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. 293
Significance levels are shown based on the t-test compared to table t. 294
295 Table 3. Comparison of means of the generations for all studied traits 291
Table 4. Scaling tests to assess the epistatic (non-allelic) effects
292
Scaling
test
Fruit length
Fruit width
Shape index
Seed length
Seed width
Seed thickness
A
ns
1.489
-2.484ns
0.294ns
-1.117ns
ns
0.907
-
ns
0.087
-
B
5.643*
4.777*
-0.12ns
1.444ns
0.846ns
0.452ns
C
0.678ns
-0.612ns
0.188ns
0.062ns
1.811ns
0.993ns
D
3.244ns
1.453ns
-0.007ns
0.018ns
-0.936ns
-0.314ns
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively.
8 Results and discussion:
181 293
Significance levels are shown based on the t-test compared to table t. 294
295 Table 4. Scaling tests to assess the epistatic (non-allelic) effects
h
Fruit width
Shape index
Seed length
Seed w ns, ,
: Non significant and significant at 5 and 1% probability levels, respectively. 293
Significance levels are shown based on the t-test compared to table t. 294 Table 5. Estimation of genetic effects for different generations resulting from the Zucchini and Hull-less seeded
296
Pumpkin for the studied traits
297
Genetic
parameter
Fruit length
Fruit width
Shape index
Seed
length
Seed width
Seed
thickness
m
28.411**
15.956**
1.683**
15.279**
6.460**
1.889**
d
-3.655**
0.616ns
-0.360**
-0.503*
-0.624**
-0.587**
h
-19.919*
-5.206ns
-0.453ns
1.503ns
5.045*
0.619ns
i
-6.489ns
-2.906ns
0.013ns
-0.072ns
1.870**
0.628ns
j
4.154ns
7.260**
-0.413**
3.412**
1.752*
0.538ns
l
13.622*
5.200ns
0.160ns
0.370ns
-1.929ns
-0.261ns
𝑥2
0.160ns
0.035ns
0.0019ns
ns
0.0004
ns
0.0092
ns
0.035
ns,*,**: Non-significant and significant at 5 and 1% probability levels, respectively. 298
299 10 Table 6. Estimation of generation variance components for different generations resulting from the cross between
302
Zucchini and Hull-less seeded Pumpkin for the studied traits
303
Variance component
Fruit length
Fruit
width
Shape
index
Seed
length
Seed width
Seed
thickness
Additive variance
1.698
0.719
0.002
0.356
0.009
0.028
Dominance variance
0.086
0.072
0.004
0.013
0.009
0.012
Environmental variance
0.172
0.353
0.001
0.023
0.016
0.003
Narrow-sense heritability
0.86
0.62
0.28
0.90
0.26
0.64
Broad-sense heritability
0.91
0.69
0.85
0.94
0.52
0.92
The average degree of dominance
0.31
0.44
2
0.27
1.41
0.94
304
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380
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389 13
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0094649&type=printable
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English
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Prognosis and Risk Factors for Deterioration in Patients Admitted to a Medical Emergency Department
|
PloS one
| 2,014
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cc-by
| 5,506
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Abstract This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. nriksen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: DPH was supported by: University of Southern Denmark, The Research Foundation of Odense University Hospital, as well as an unrestricted grant from
the philanthropically private fund TrygFonden given to the University of Southern Denmark. ATL was supported by an unrestricted grant from the
philanthropically private fund TrygFonden given to University of Southern Denmark, and is employed as a consultant at the Department of Emergency Medicine,
Odense University Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dphenriksen@health.sdu.dk Introduction The aim of our study was to describe the risk factors and
prognosis of patients that arrive with normal vital signs but
clinically deteriorate within the first 24 hours after arrival. Most emergency departments (ED) are at constant risk of
overcrowding [1]. As a solution to this, specialised admission units
for acute medical patients have been implemented, which in turn
reduced waiting time, length of stay, and mortality [2]. Patients
who appear stable at arrival often receive low priority from the
nursing staff and their vital signs are insufficiently recorded[3–6]. However, clinical deterioration has been shown to be an
independent predictor of mortality and early identification could
improve prognosis [1,7,8]. Daniel Pilsgaard Henriksen1*, Mikkel Brabrand2, Annmarie Touborg Lassen1 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark, 2 Department of Medicine, Sydvestjysk Sygehus Abstract Objective: Patients that initially appear stable on arrival to the hospital often have less intensive monitoring of their vital
signs, possibly leading to excess mortality. The aim was to describe risk factors for deterioration in vital signs and the related
prognosis among patients with normal vital signs at arrival to a medical emergency department (MED). Design and setting: Single-centre, retrospective cohort study of all patients admitted to the MED from September 2010-
August 2011. Subjects: Patients were included when their vital signs (systolic blood pressure, pulse rate, respiratory rate, Glasgow Coma
Scale, oxygen saturation and temperature) were within the normal range at arrival. Deterioration was defined as a deviation
from the defined normal range 2–24 hours after arrival. Results: 4292 of the 6257 (68.6%) admitted to the MED had a full set of vital signs at first presentation, 1440/4292 (33.6%)
had all normal vital signs and were included in study, 44.0% were male, median age 64 years (5th/95th percentile: 21–90
years) and 446/1440 (31.0%) deteriorated within 24 hours. Independent risk factors for deterioration included age 65–84
years odds ratio (OR): 1.79 (95% confidence interval [CI]: 1.27–2.52), 85+ years OR 1.67 (95% CI: 1.10–2.55), Do-not-attempt-
to-resuscitate order OR 3.76 (95% CI: 1.37–10.31) and admission from the open general ED OR 1.35 (95% CI: 1.07–1.71). Thirty-day mortality was 7.9% (95% CI: 5.5–10.7%) among deteriorating patients and 1.9% (95% CI: 1.2–3.0%) among the
non-deteriorating, hazard ratio 4.11 (95% CI: 2.38–7.10). Conclusions: Among acutely admitted medical patients who arrive with normal vital signs, 31.0% showed signs of
deterioration within 24 hours. Risk factors included old age, Do-not-attempt-to-resuscitate order, admission from the open
general ED. Thirty-day mortality among patients with deterioration was four times higher than among non-deteriorating
patients. Further research is needed to determine whether intensified monitoring of vital signs can help to prevent
deterioration or mortality among medical emergency patients. Citation: Henriksen DP, Brabrand M, Lassen AT (2014) Prognosis and Risk Factors for Deterioration in Patients Admitted to a Medical Emergency
Department. PLoS ONE 9(4): e94649. doi:10.1371/journal.pone.0094649 Editor: Paul Robert Cleary, Health Protection Agency, United Kingdom Editor: Paul Robert Cleary, Health Protection Agency, United Kingdom Received January 10, 2014; Accepted March 19, 2014; Published April 9, 2014 Received January 10, 2014; Accepted March 19, 2014; Published April 9, 20 ed January 10, 2014; Accepted March 19, 2014; Published April 9 Copyright: 2014 Henriksen et al. Data Sources/Measurement Vital signs registered upon arrival to the department and in the
following 24 hours were extracted electronically from the patient
records. For each patient, we extracted data from the Danish
National
Patient
Register
[11]
(comorbidity
and
discharge
diagnoses), the Danish Civil Persons Register [12] (vital status
[dead or alive] and emigration status), Odense University
Pharmacoepidemiological Database [13], the Danish National
Cancer Register [14] and the Danish National Alcohol- and Drug
Treatment Register, the last three to identify immunosuppression
and prior alcoholism-related conditions. All patients were followed
up until death, emigration from the country or 30 days after
admission, whichever came first. Risk Factors for Deterioration Independent risk factors for clinical deterioration within the first
24 hours after arrival to the hospital included patients in the age
category 65–84 years (odds ratio [OR] 1.79 [95% CI: 1.27–2.52]);
patients in the age category 85+ years (OR 1.67 [95% CI: 1.10–
2.55]); patients admitted from the open general ED (OR 1.35
[95% CI: 1.07–1.71]) and patients with a DNAR recorded within
the first 24 hours after arrival to the hospital (OR 3.76 [95% CI:
1.37–10.31]) (Table 1). Ethics Committee Approval In compliance with Danish law, the study was notified to and
approved by the Danish Data Protection Agency (J No 2008-58-
0035), and the access to patient clinical records was approved by
the Danish National Board of Health (J No 3-3013-35). No further
ethical approval, or consent from participants, is needed for
register-based studies in Denmark. Data were anonymised and de-
identified prior data analysis. Initial vital signs were routinely collected upon arrival to the
hospital (blood pressure, heart rate, respiratory rate, rectal
temperature, peripheral oxygen saturation, recorded oxygen
therapy, and Glasgow Coma Scale). Any later vital signs were
collected as deemed necessary by the attending physician, but all
vital signs were routinely recorded at least every eight hours. There were no interventions related to the study, and all patients
received care following the standard operating procedures. We
excluded patients unidentified throughout the entire course of
admission, patients without a Danish civil registration number,
and patients without any recorded vital signs or with initial vital
signs recorded .2 hours after arrival to the hospital. We included
only the first admission of each patient, within the study period. The reporting of this study conforms to the STROBE statement
[15]. Deteriorating Patients in a Medical Emergency Department admitted medical patients from either primary care or from the
hospitals open mixed medical-surgical ED. Variables/Definitions We defined normal vital signs as systolic blood pressure . 100 mmHg, heart rate $50/min and #90/min, respiratory rate
$8/min and #20/min, rectal temperature $36.0u and #
38.0uCelsius, oxygen saturation $94% ($90% in patients with a
history of chronic obstructive pulmonary disease [COPD]) and no
need for oxygen therapy at arrival to the hospital and a Glasgow
Scale Score of 15 or registered as awake and alert. Clinical
deterioration was defined as vital signs beyond the definition of
normal. Immunosuppression was defined as one or more of the following
identified before the current admission: 1) A moderate to high intake of immunosuppressant medication
(corticosteroids or antineoplastic and immunomodulating agents)
2) Primary immunodeficiency diseases; 3) Acquired Immune
Deficiency Syndrome defining diseases; 4) A newly diagnosed
malignancy registered in the Danish Cancer Registry, up to a year
prior to admission; 5) Diagnosis of organ transplantation. Alcoholism-related condition was defined as at least one of the
following before the current admission: Statistical Methods Data were presented as medians with 5th and 95th percentiles
or proportions with 95% confidence intervals (CI), assuming a
binomial distribution. Univariate analysis of baseline characteris-
tics was performed using Chi-squared tests or Mann–Whitney U-
tests. A multivariable logistic regression analysis was computed
using
deterioration
as
the dependent
variable and
several
predefined potential risk factors for developing clinical deteriora-
tion within the first 24 hours after arrival to the hospital, as
independent variables. We included each independent variable in
the model without assessing their individual association with future
clinical deterioration. In each patient we included the first
recording of a deterioration among the individual vital signs
(systolic blood pressure, pulse rate, GCS, respiratory rate, oxygen
saturation and temperature). We computed Kaplan-Meier survival
plots of the individual vital signs, where the event was the time of
clinical deterioration. Thirty-day mortality was presented as
proportions as well as a Kaplan-Meier failure plot and log rank
test in a univariate analysis. If a vital sign was missing in the
reassessment of the patient, it was defined as if it was within
normal range. Statistical analyses were performed with Stata
version 13.0 (Stata Corporation LP, Texas, USA). Participants 1) A redeemed prescription of Disulfiram from 2007 and
onwards; 2) $2 admissions with a discharge diagnosis of an acute
alcohol episode; 3) At least one admission with a discharge
diagnosis of a chronic alcohol related diagnosis; 4) A registration in
The Danish National Register of Alcohol Abuse Treatment. A total of 6257 patients were admitted to the medical ED within
the study period.,4292/6257 (68.6%) had a full set of vital signs at
first presentation,1440/4292 (33.6%) had all normal vital signs
and were included (Figure 1). Median age among the included
patients was 64 years (21–90 years) and 44.0% were male. The
distribution of included patients with comorbidity in the Charlson
Comorbidity Index categories 0, 1–2 and .2 were 706 (49.0%),
311 (21.6%), 423 (29.4%), respectively. 446/1440 (31.0%) patients
had deteriorating vital signs within the first 24 hours after arrival
to the hospital. g
Comorbidity was reported by Charlson Comorbidity Index
based on discharge diagnoses in the last 10 years and up to seven
days prior the current admission. Patients were coded as Do-Not-Attempt-Resuscitation (DNAR)
if they had a DNAR order placed in the medical record within 24
hours of presentation. If a patient was characterised as having a
terminal illness or moribund, we defined them as having a DNAR
order as well. Study Design and Setting We conducted a retrospective cohort study of all consecutive
adult ($15 years) patients admitted to the medical ED at Odense
University Hospital over a one-year period (1 September 2010
through 31 August 2011). The medical ED serves a population of
235,000 adults and as a medical admission unit for the following
medical specialities: general internal medicine, infectious diseases,
gastroenterology, geriatric medicine, rheumatology, endocrinology
and respiratory medicine. The medical ED receives all acutely There is surprisingly little knowledge of how to monitor and
deal with deteriorating patients[3–7,9,10]. However, increased
knowledge could provide information to ‘‘post triage’’ follow-up
procedures in the emergency departments and admission units and
possibly lead to reduced morbidity and mortality. April 2014 | Volume 9 | Issue 4 | e94649 1 PLOS ONE | www.plosone.org Discussion Although several studies have focused on risk of clinical
deterioration among acutely admitted patients[5,7,16–18], studies
focusing on apparently low risk patients with normal vital signs on
arrival are few [3]. We show that 31% of all patients admitted to a
medical emergency department with normal vital signs on arrival Discharge Diagnosis Patients with a discharge diagnosis from the respiratory system
had the highest risk of deterioration, (98/153, 64.1%, 95% CI:
55.9–71.6%) followed by discharge diagnoses from the cardiac/
circulatory system 30 (35.3% [25.2%–46.4%]) (Table 2). April 2014 | Volume 9 | Issue 4 | e94649 PLOS ONE | www.plosone.org 2 Deteriorating Patients in a Medical Emergency Department Figure 1. Flow chart of recruitment of patients admitted to the medical emergency department. doi:10.1371/journal.pone.0094649.g001
Deteriorating Patients in a Me Figure 1. Flow chart of recruitment of patients admitted to the medical emergency department. doi:10.1371/journal.pone.0094649.g001 Mortality deterioration. The most common vital sign to deteriorate was
the pulse rate (194/533 deteriorations, 36.4%) followed by oxygen
saturation (187/533 deteriorations, 35.1%) (Figure 3). The overall 30-day mortality of the included patients was 3.8%
(95% CI: 2.8–4.9%). In the non-deteriorating group, 19/994
(1.9%, 95% CI: 1.2–3.0%) patients died compared to 35/446
(7.9%, 95% CI: 5.5–10.7%) patients in the deteriorating group
(Figure 2), unadjusted hazard ratio 4.11 (95% CI: 2.38–7.10). Of
all deteriorating patients, 18/314 (5.7%, 95% CI: 3.4–8.9%)
experiencing a single vital sign deteriorating within 24 hours after
arrival died within 30 days after admission, 12/99 (12.1%, 95%
CI: 6.4–20.2%) with two individual vital sign deteriorating and 5/
33 (15.2%, 95% CI: 5.1–31.9%) with three or more individual
vital sign deteriorating. Oxygen saturation expressed the shortest median time to
registered deterioration of 7.5 hours, (2.4–19.0 hours) whereas
temperature expressed the longest median time of 12.5 hours (3.3–
21.1 hours). For a detailed overview of the time of each individual
vital sign deterioration, see Figure 3. April 2014 | Volume 9 | Issue 4 | e94649 Vital Signs The six individual vital signs of interest contributed with 533
deteriorations among the 446 patients experiencing clinical April 2014 | Volume 9 | Issue 4 | e94649 PLOS ONE | www.plosone.org 3 Deteriorating Patients in a Medical Emergency Department Table 1. Risk factors for clinical deterioration within 24 hours in patients admitted to the medical emergency department with
normal vital signs registered at arrival. Non-deteriorating,
N = 994 (69.0%)
Deteriorating
N = 446 (31.0%)
Adjusted logistic
regression1
Gender (%)
Female
551 (68.4)
255 (31.6)
Ref
Male
443 (69.9)
191 (30.1)
0.90 (0.71–1.13)
Age in categories, years (%)
15–39
239 (75.6)
77 (24.4)
Ref
40–64
320 (74.6)
109 (25.4)
1.00 (0.70–1.41)
65–84
318 (62.4)
192 (37.6)
1.79 (1.27–2.52)
85+
117 (63.2)
68 (36.8)
1.67 (1.10–2.55)
Charlson Comorbidity index (%)
0
516 (73.1)
190 (26.9)
Ref
1–2
211 (67.8)
100 (32.2)
1.10 (0.81–1.49)
.2
267 (63.1)
156 (36.9)
1.19 (0.88–1.61)
Immunosuppression (%)
No
889 (69.3)
393 (30.7)
Ref
Yes
105 (66.5)
53 (33.5)
1.01 (0.70–1.47)
Alcoholism-related conditions (%)
No
901 (69.6)
394 (30.4)
Ref
Yes
93 (64.1)
52 (35.9)
1.44 (0.98–2.11)
Do-not-attempt-resuscitate order (%)
Resuscitate
988 (69.5)
434 (30.5)
Ref
Do-not attempt resuscitation
6 (33.3)
12 (66.7)
3.76 (1.37–10.31)
Admission at entry (%)
Primary care admitted
medical ED
642 (71.0)
262 (29.0)
Ref
Open general ED
352 (65.7)
184 (34.3)
1.35 (1.07–1.71)
1Adjusted for gender, age in categories, Charlson Comorbidity Index, immunosuppression, alcoholism-related conditions, Do-not-attempt-resuscitation order and
admission at entry. doi:10.1371/journal.pone.0094649.t001 Table 1. Risk factors for clinical deterioration within 24 hours in patients admitted to the medical emergency department with
normal vital signs registered at arrival. Table 1. Risk factors for clinical deterioration within 24 hours in patients admitted to the medical emergency department with
normal vital signs registered at arrival. Table 1. Risk factors for clinical deterioration within 24 hours in patients admitted to the medical emergency department with
normal vital signs registered at arrival. 1Adjusted for gender, age in categories, Charlson Comorbidity Index, immunosuppression, alcoholism-related conditions, Do-not-attempt-resuscitation order and
admission at entry. doi:10.1371/journal.pone.0094649.t001 deteriorated within the first 24 hours and that deteriorating
patients had four times higher 30-day all-cause mortality. (56.5%) [20]. Some have used admission to an ICU as endpoint
[7,17,21], others a relative comparison of risk score in each patient
[5], activation of a rapid response team [22,23] or a post hoc
definition of abnormal vital signs based on the subsequent
mortality [20]. Deteriorating Patients in a Medical Emergency Department Deteriorating Patients in a Medical Emergency Department Figure 2. Cumulative risk of death measured in days over a 30-
day period among patients with- (dashed line) and without
(solid line) recorded clinical deterioration within 24 hours after
admission to the medical emergency department. doi:10.1371/journal.pone.0094649.g002 There is little consensus on how frequently patients should be
re-evaluated after initial triage[4–6,9,10]. We found that the
deteriorating vital values were registered 4 to 13 hours after
arrival. The time to deterioration curves for the vital signs showed
almost linear trends the first 24 hours after arrival. However, this
observation is limited by the fact that the registration of vital signs
was not standardised, but to a great extent were recorded at
individual time frames. Few studies have previously investigated
the time to clinical deterioration from arrival to the hospital. Kennedy et al. found median time to ICU transfer to be 20 hours
in patients presenting at the emergency department with an
infection [17] and Bleyer et al. found that approximately 25% of
all deteriorations in hospitalised patients occurred within 24 hours
after admission [20]. The National Institute for Health and
Clinical Excellence recommends that physiological observations
should be monitored at least every 12 hours, depending on
decisions made at a senior level [25]. Figure 2. Cumulative risk of death measured in days over a 30-
day period among patients with- (dashed line) and without
(solid line) recorded clinical deterioration within 24 hours after
admission to the medical emergency department. doi:10.1371/journal.pone.0094649.g002 We found that 7.9% of the patients with registered clinical
deterioration died within 30 days after admission. This is a low
mortality rate compared to other studies [5,20]. We believe that
this is due to different definitions of deterioration, as an example
Kennedy et al used transfer to the ICU as a surrogate marker, a
threshold potentially more severe than single vital sign deteriora-
tion, and thereby indicating more severely ill patients [17]. Another possible explanation could be differences in case-mix
between studies. deterioration is a continuous process. Only recently, studies have
begun to emphasise the need for a consensus based framework to
define clinical deterioration similar to those used in ICUs [24]. We have chosen to use a less restrictive and more sensitive
definition of deterioration based on the normal ranges of the
individual vital signs likely to identify clinical deterioration earlier
than the aforementioned strategies. Vital Signs These inconsistencies in outcome make it difficult
to compare the results directly, and illustrate the fact that Previous studies have used varying definitions of clinical
deterioration. As a result, most of these studies show a lower
proportion of deterioration (1.7%–16.0%) than ours[5,7,16–19]
and only a single study found a higher proportion of deterioration Table 2. Distribution of primary discharge diagnosis from the medical emergency department among patients with- and without
registered clinical deterioration within 24 hours after admission. Table 2. Distribution of primary discharge diagnosis from the medical emergency department among patients with- and without
registered clinical deterioration within 24 hours after admission. Discharge Categories (ICD10), N = 1440
N, total
Deteriorating N, (%), [95% CI1]
Infections (A00–B99),
99
31 (31.3% [22.4%–41.4%])
Anemia and blood disease (D50–D89)
41
13 (31.7% [18.1%–48.1%])
Endocrine and metabolic (E00–E90),
139
41 (29.5% [22.1%–37.8%])
Mental and behavioral (F00–F99),
39
11 (28.2% [15.0%–44.9%])
Nervous system (G00–G99),
12
3 (25.0% [5.5%–57.2%])
Cardiac/Circulatory system (I00–I99)
85
30 (35.3% [25.2%–46.4%])
Respiratory system (J00–J99),
153
98 (64.1% [55.9%–71.6%])
Gastrointestinal system (K00–K93)
112
34 (30.4% [22.0%–39.8%])
Skin and subcutaneous tissue (L00–L99)
19
2 (10.5% [1.3%–33.1%])
Musculoskeletal and connective tissue (M00–M99)
129
22 (17.1% [11.0%–24.7%])
Genitourinary system (N00–N99)
68
23 (33.8% [22.8%–46.3%])
Toxicologic (T15–T98)
158
48 (30.4% [23.3%–38.2%])
General signs and symptoms and abnormal clinical and lab findings (R00–R94)
257
63 (24.5% [19.4%–30.2%])
Other
129
27 (20.9% [14.3%–29.0%])
195% CI: 95%. doi:10.1371/journal.pone.0094649.t002
PLOS ONE | www.plosone.org
4
April 2014 | Volume 9 | Issue 4 | e94649 f primary discharge diagnosis from the medical emergency department among patients with- and withou
ioration within 24 hours after admission. Table 2. Distribution of primary discharge diagnosis from the medical emergency department among p
registered clinical deterioration within 24 hours after admission. April 2014 | Volume 9 | Issue 4 | e94649 Deteriorating Patients in a Medical Emergency Department Deteriorating Patients in a Medical Emergency Department We did this to investigate if
these early changes in vital signs could be used to identify an at-risk
patient population and/or if the nursing staff and physicians
should pay extra attention to these ‘‘well appearing’’ patients. If we
had chosen admission to the ICU within the first 24 hours after
arrival
to
the
hospital
as
a
surrogate
marker
of
clinical
deterioration, only two patients would have been classified as
having deteriorated clinically and four patients if we had chosen a
cut-off of 48 hours as Kennedy et al did [17]. Increased 30-day mortality in deteriorating patients is probably
(to some degree) preventable if the deterioration is acted upon in
time, but one has to keep in mind that some fatalities are
inevitable. Hogan et al. showed that poor clinical monitoring was
considered the most dominant cause of preventable deaths in
acute hospitals [26] and Hands observed moderate to poor
adherence to monitoring procedures in patients with normal as
well as abnormal vital signs [4]. This highlights the need for
improved observation and follow-up. These procedures should be
able to identify clinical deterioration without compromising the
effectiveness of a busy emergency department. Figure 3. Kaplan Meier plot illustrating the different vital signs’ the probability of not yet having deteriorated among those who
eventually will deteriorate within 24 hours after admission to the medical emergency department. A patient could have more than one
of the five individual vital signs deteriorating at the same time, therefore the total number of deteriorating vital signs (at time 0) exceed the number
of deteriorating patients. Glasgow Coma Scale is not presented due to the small number of deteriorations (N = 2). doi:10.1371/journal.pone.0094649.g003 Figure 3. Kaplan Meier plot illustrating the different vital signs’ the probability of not yet having deteriorated among those who
eventually will deteriorate within 24 hours after admission to the medical emergency department. A patient could have more than one
of the five individual vital signs deteriorating at the same time, therefore the total number of deteriorating vital signs (at time 0) exceed the number
of deteriorating patients. Glasgow Coma Scale is not presented due to the small number of deteriorations (N = 2). doi:10.1371/journal.pone.0094649.g003 Figure 3. Kaplan Meier plot illustrating the different vital signs’ the probability of not yet having deteriorated among those who
eventually will deteriorate within 24 hours after admission to the medical emergency department. Strengths and Limitations of Study The main strength of our study is the large consecutive cohort of
acute medical patients with complete follow-up for 30-day all-
cause mortality. The main limitation is the absence of standardised
time intervals for re-evaluation of the patients. This introduces a
bias as the nurses might register vital signs more frequently in some
patients (e.g. presenting at increased risk). The current work was a
single-centre study from a medical ED at a university hospital,
which potentially could affect the generalisability of the study. However, the hospital is the only hospital in the area and is a
primary hospital for all residents in the hospital catchment area, in
parallel to highly specialised tertiary functions. Preliminary data from this study were presented as a poster presentation
at the International Conference on Emergency Medicine 27–30 June 2012,
Dublin, Ireland. The study was performed at the medical emergency department,
Odense University Hospital, Denmark from 1 September 2010–31 August
2011. References 9. Barfod C, Lauritzen MMP, Danker JK, So¨le´tormos G, Forberg JL, et al. (2012)
Abnormal vital signs are strong predictors for intensive care unit admission and
in-hospital mortality in adults triaged in the emergency department - a
prospective cohort study. Scand J Trauma Resusc Emerg Med 20: 28. doi:10.1186/1757-7241-20-28. 1. Guttmann A, Schull MJ, Vermeulen MJ, Stukel TA (2011) Association between
waiting times and short term mortality and hospital admission after departure
from emergency department: population based cohort study from Ontario,
Canada. Bmj 342: d2983-d2983. doi:10.1136/bmj.d2983. j
j
2. Scott I, Vaughan L, Bell D (2009) Effectiveness of acute medical units in
hospitals: a systematic review. Int J Qual Health Care 21: 397–407. doi:10.1093/intqhc/mzp045. 10. Wuerz R (2001) Emergency severity index triage category is associated with six-
month survival. ESI Triage Study Group. Acad Emerg Med 8: 61–64. doi:10.1111/j.1553-2712.2001.tb00554.x. 3. Barfod C, Lauritzen MMP, Danker JK, So¨le´tormos G, Berlac PA, et al. (2012)
The formation and design of the ‘‘Acute Admission Database’’- a database
including a prospective, observational cohort of 6279 patients triaged in the
emergency department in a larger Danish hospital. Scand J Trauma Resusc
Emerg Med 20: 29. doi:10.1186/1757-7241-20-29. j
11. Lynge E, Sandegaard JL, Rebolj M (2011) The Danish National Patient
Register. Scand J Public Health 39: 30–33. doi:10.1177/1403494811401482. 12. Pedersen CB (2011) The Danish Civil Registration System. Scand J Public
Health 39: 22–25. doi:10.1177/1403494810387965. 13. Hallas J (2001) Conducting pharmacoepidemiologic research in Denmark. Pharmacoepidemiol Drug Saf 10: 619–623. doi:10.1002/pds.638. 4. Hands C, Reid E, Meredith P, Smith GB, Prytherch DR, et al. (2013) Patterns in
the recording of vital signs and early warning scores: compliance with a clinical
escalation protocol. BMJ Qual Saf: 1–8. doi:10.1136/bmjqs-2013-001954. Pharmacoepidemiol Drug Saf 10: 619–623. doi:10.1002/pds.638 14. Gjerstorff ML (2011) The Danish Cancer Registry. Scand J Public Health 39:
42–45. doi:10.1177/1403494810393562. 5. Kellett J, Emmanuel A, Deane B (2011) Who will be sicker in the morning? Changes in the Simple Clinical Score the day after admission and the
subsequent outcomes of acutely ill unselected medical patients. Eur J Intern Med
22: 375–381. doi:10.1016/j.ejim.2011.03.005. 15. Von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, et al. (2007)
Strengthening the Reporting of Observational Studies in Epidemiology
(STROBE) statement: guidelines for reporting observational studies. BMJ 335:
806–808. doi:10.1136/bmj.39335.541782.AD. 6. Armstrong B, Walthall H, Clancy M, Mullee M, Simpson H (2008) Recording of
vital signs in a district general hospital emergency department. Conclusion Among acutely admitted medical patients who arrive with
normal vital signs, 31.0% deteriorated within 24 hours. Risk
factors included old age (85+ years), Do-not-attempt-to-resuscitate
order and if a patient was admitted from the open general ED. Thirty-day mortality among patients with deterioration was four
times higher than among patients without deterioration. This
pinpoints the need for an intensified follow up strategy on medical
emergency patients. We emphasise that the present study was not an attempt to
develop a clinical tool to predict and prevent morbidity and
mortality in initially ‘‘well appearing’’ patients, but an observa-
tional study where the primary aim was to describe this patient
population in the ED setting. Acknowledgments The authors would like to thank Professor Court Pedersen, Dept. of
Infectious Diseases, Odense University Hospital for insightful comments on
earlier drafts of this manuscript, and Thomas Henriksen for proofreading
of the manuscript. Author Contributions Conceived and designed the experiments: DPH MB ATL. Performed the
experiments: DPH MB ATL. Analyzed the data: DPH ATL. Contributed
reagents/materials/analysis tools: DPH MB ATL. Wrote the paper: DPH
MB ATL. Conceived and designed the experiments: DPH MB ATL. Performed the
experiments: DPH MB ATL. Analyzed the data: DPH ATL. Contributed
reagents/materials/analysis tools: DPH MB ATL. Wrote the paper: DPH
MB ATL. The study was conducted at a medical ED describing acutely
hospitalised medical patients so the risk factors’ strength of Deteriorating Patients in a Medical Emergency Department Deteriorating Patients in a Medical Emergency Department Although a single vital sign deterioration might appear as
unspecific we found large differences in the frequency of which
vital sign initially deteriorated and the following 30-day mortality. Deterioration in oxygen saturation was the most frequent cause of
deterioration and associated with the highest 30-day mortality. This could be because low oxygen saturation might identify a
more fragile patient group, namely patients suffering from chronic
respiratory diseases. Although a single vital sign deterioration might appear as
unspecific we found large differences in the frequency of which
vital sign initially deteriorated and the following 30-day mortality. association might be different among patients hospitalised to an
abdominal surgery department, cardiology patients or patients in
active chemotherapy. Deterioration in oxygen saturation was the most frequent cause of
deterioration and associated with the highest 30-day mortality. This could be because low oxygen saturation might identify a
more fragile patient group, namely patients suffering from chronic
respiratory diseases. There is always the possibility of residual confounding, which
could lead to different estimates when interpreting the indepen-
dent risk factors in the multivariate models. Due to the small number of patients died within 30 days after
admission we were not able to perform subgroup analysis of
mortality in the individual vital signs as well as in the different
discharge categories. In the present study, the most frequent discharge diagnosis
group among patients with normal vital signs admitted to the
medical ED was ‘‘General signs and symptoms and abnormal
clinical and lab findings (ICD-10: R00–R94)’’. This is in
concordance with a study by Schmidt et al. study regarding
patients admitted to the emergency department in a similar setting
as the present one [27]. We showed that the largest proportion of
patients deteriorating was patients’ with a discharge diagnosis of
the Respiratory system (ICD10: J00–J99). This might be because
the most common individual vital sign deteriorating was oxygen
saturation, and physicians assigning discharge diagnoses to the
patients course of admission. Deteriorating Patients in a Medical Emergency Department A patient could have more than one
of the five individual vital signs deteriorating at the same time, therefore the total number of deteriorating vital signs (at time 0) exceed the number
of deteriorating patients. Glasgow Coma Scale is not presented due to the small number of deteriorations (N = 2). doi:10.1371/journal.pone.0094649.g003 April 2014 | Volume 9 | Issue 4 | e94649 PLOS ONE | www.plosone.org 5 27. Schmidt M, Antonsen S, Hansen B, Møller J, Thordal C, et al. (2010) Mortality
following acute medical admission in Denmark: a feasibility study. Clin
Epidemiol 2: 195–203. doi:10.2147/CLEP.S12171. References Emerg Med J 25:
799–802. doi:10.1136/emj.2007.052951. 16. Buist M, Bernard S, Nguyen TV, Moore G, Anderson J (2004) Association
between clinically abnormal observations and subsequent in-hospital mortality: a
prospective study. Resuscitation 62: 137–141. doi:10.1016/j.resuscita-
tion.2004.03.005. j
7. Simchen E, Sprung CL, Galai N, Zitser-Gurevich Y, Bar-Lavi Y, et al. (2007)
Survival of critically ill patients hospitalized in and out of intensive care. Crit
Care Med 35: 449–457. doi:10.1097/01.CCM.0000253407.89594.15. 7. Simchen E, Sprung CL, Galai N, Zitser-Gurevich Y, Bar-Lavi Y, et al. (2007)
Survival of critically ill patients hospitalized in and out of intensive care. Crit
Care Med 35: 449–457. doi:10.1097/01.CCM.0000253407.89594.15. 17. Kennedy M, Joyce N, Howell MD, Lawrence Mottley J, Shapiro NI (2010)
Identifying infected emergency department patients admitted to the hospital
ward at risk of clinical deterioration and intensive care unit transfer. Acad Emerg
Med 17: 1080–1085. doi:10.1111/j.1553-2712.2010.00872.x. 8. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, et al. (2001) Early goal-
directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med
345: 1368–1377. doi:10.1056/NEJMoa010307. 8. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, et al. (2001) Early goal-
directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med
345: 1368–1377. doi:10.1056/NEJMoa010307. 6 April 2014 | Volume 9 | Issue 4 | e94649 PLOS ONE | www.plosone.org April 2014 | Volume 9 | Issue 4 | e94649 25. Centre for Clinical Practice at NICE (UK) (2007) Acutely Ill Patients in
Hospital: Recognition of and Response to Acute Illness in Adults in Hospital.
NICE Clin Guidel No50: 1–107. 23. Goldhill DR, Worthington L, Mulcahy A, Tarling M, Sumner A (1999) The
patient-at-risk team: identifying and managing seriously ill ward patients.
Anaesthesia 54: 853–860. 24. Jones D, Mitchell I, Hillman K, Story D (2013) Defining clinical deterioration.
Resuscitation 84: 1029–1034. doi:10.1016/j.resuscitation.2013.01.013. Deteriorating Patients in a Medical Emergency Department Deteriorating Patients in a Medical Emergency Department 18. Lighthall GK, Markar S, Hsiung R (2009) Abnormal vital signs are associated
with an increased risk for critical events in US veteran inpatients. Resuscitation
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patient-at-risk team: identifying and managing seriously ill ward patients. Anaesthesia 54: 853–860. 24. Jones D, Mitchell I, Hillman K, Story D (2013) Defining clinical deterioration. Resuscitation 84: 1029–1034. doi:10.1016/j.resuscitation.2013.01.013. 19. Fuhrmann L, Lippert A, Perner A, Østergaard D (2008) Incidence, staff
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20. Bleyer AJ, Vidya S, Russell GB, Jones CM, Sujata L, et al. (2011) Longitudinal
analysis of one million vital signs in patients in an academic medical center. Resuscitation 82: 1387–1392. doi:10.1016/j.resuscitation.2011.06.033. 26. Hogan H, Healey F, Neale G, Thomson R, Vincent C, et al. (2012) Preventable
deaths due to problems in care in English acute hospitals: a retrospective case
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001159. j
21. Subbe CP, Kruger M, Rutherford P, Gemmel L (2001) Validation of a modified
Early Warning Score in medical admissions. QJM 94: 521–526. j
21. Subbe CP, Kruger M, Rutherford P, Gemmel L (2001) Validation Early Warning Score in medical admissions. QJM 94: 521–526. 22. Farley H, Zubrow MT, Gies J, Kolm P, Mascioli S, et al. (2010) Emergency
department tachypnea predicts transfer to a higher level of care in the first 24
hours after ED admission. Acad Emerg Med 17: 718–722. doi:10.1111/j.1553-
2712.2010.00796.x. 27. Schmidt M, Antonsen S, Hansen B, Møller J, Thordal C, et al. (2010) Mortality
following acute medical admission in Denmark: a feasibility study. Clin
Epidemiol 2: 195–203. doi:10.2147/CLEP.S12171. PLOS ONE | www.plosone.org 7 PLOS ONE | www.plosone.org April 2014 | Volume 9 | Issue 4 | e94649 7
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https://openalex.org/W3094417216
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https://orca.cardiff.ac.uk/id/eprint/136904/1/1-s2.0-S2666379120301622-main.pdf
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English
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Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson’s Dementia in Humans
|
Cell reports medicine
| 2,020
|
cc-by
| 19,506
|
Correspondence
jeff.s.davies@swansea.ac.uk Correspondence
jeff.s.davies@swansea.ac.uk In Brief The gut-hormone acyl-ghrelin (AG) is
known to promote adult hippocampal
neurogenesis. Hornsby et al. combine
in vitro and in vivo rodent models,
alongside analysis of human plasma, to
show that unacylated-ghrelin (UAG)
impairs neurogenesis and that the
circulating AG:UAG ratio is reduced in
Parkinson’s dementia. Article Unacylated-Ghrelin Impairs Hippocampal
Neurogenesis and Memory in Mice and Is Altered in
Parkinson’s Dementia in Humans Graphical Abstract Authors
Amanda K.E. Hornsby, Luke Buntwal,
Maria Carla Carisi, ..., Zane B. Andrews,
David J. Burn, Jeffrey S. Davies Authors
Amanda K.E. Hornsby, Luke Buntwal,
Maria Carla Carisi, ..., Zane B. Andrews,
David J. Burn, Jeffrey S. Davies Graphical Abstract SUMMARY Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that
elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced
hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl
transferase-null (GOAT/) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by
acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF
signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is
important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating
AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients,
and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression
remained unchanged; however, GOAT+ cell number was reduced in PDD. We identify UAG as a regulator of
hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic
biomarker of dementia. Article
Unacylated-Ghrelin Impairs Hippocampal
Neurogenesis and Memory in Mice and Is
Altered in Parkinson’s Dementia in Humans Amanda K.E. Hornsby,1,6 Luke Buntwal,1,6 Maria Carla Carisi,1 Vanessa V. Santos,2 Fionnuala Johnston,3
Luke D. Roberts,1 Martina Sassi,1 Mathieu Mequinion,2 Romana Stark,2 Alex Reichenbach,2 Sarah H. Lockie,2
Mario Siervo,3,5 Owain Howell,1 Alwena H. Morgan,1 Timothy Wells,4 Zane B. Andrews,2 David J. Burn,3
and Jeffrey S. Davies1,7,* Amanda K.E. Hornsby,1,6 Luke Buntwal,1,6 Maria Carla Carisi,1 Vanessa V. Santos,2 Fionnuala Johnston,3
Luke D. Roberts,1 Martina Sassi,1 Mathieu Mequinion,2 Romana Stark,2 Alex Reichenbach,2 Sarah H. Lockie,2
Mario Siervo,3,5 Owain Howell,1 Alwena H. Morgan,1 Timothy Wells,4 Zane B. Andrews,2 David J. Burn,3
and Jeffrey S. Davies1,7,* y
1Molecular Neurobiology, Institute of Life Sciences, School of Medicine, Swansea University, Swansea, UK
2Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia
3Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
4School of Biosciences, Cardiff University, Cardiff, UK
5School of Life Sciences, Queen’s Medical Centre, The University of Nottingham Medical School, Nottingham NG7 2UH, UK
6These authors contributed equally
7Lead Contact
*C
d
j ff
d
i
@
k 7Lead Contact *Correspondence: jeff.s.davies@swansea.ac.uk
https://doi.org/10.1016/j.xcrm.2020.100120 *Correspondence: jeff.s.davies@swansea.ac.uk
https://doi.org/10.1016/j.xcrm.2020.100120 Article
Unacylated-Ghrelin Impairs Hippocampal
Neurogenesis and Memory in Mice and Is
Altered in Parkinson’s Dementia in Humans
Amanda K.E. Hornsby,1,6 Luke Buntwal,1,6 Maria Carla Carisi,1 Vanessa V. Santos,2 Fionnuala Johnston,3
Luke D. Roberts,1 Martina Sassi,1 Mathieu Mequinion,2 Romana Stark,2 Alex Reichenbach,2 Sarah H. Lockie,2
Mario Siervo,3,5 Owain Howell,1 Alwena H. Morgan,1 Timothy Wells,4 Zane B. Andrews,2 David J. Burn,3
and Jeffrey S. Davies1,7,*
1Molecular Neurobiology, Institute of Life Sciences, School of Medicine, Swansea University, Swansea, UK
2Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia
3Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
4School of Biosciences, Cardiff University, Cardiff, UK
5School of Life Sciences, Queen’s Medical Centre, The University of Nottingham Medical School, Nottingham NG7 2UH, UK
6These authors contributed equally
7Lead Contact
*Correspondence: jeff s davies@swansea ac uk
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OPEN ACCESS ll
OPEN ACCESS Cell Reports Medicine 1, 100120, October 20, 2020 ª 2020 The Author(s).
1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Cell Reports Medicine 1, 100120, October 20, 2020 ª 2020 The Author(s).
1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Highlights g
g
d Circulating unacylated-ghrelin (UAG) reduces hippocampal
neurogenesis d Circulating acyl-ghrelin (AG) rescues spatial memory deficit in
GOAT/ mice d UAG blocks the AG induced survival of newborn hippocampal
cells d Plasma AG:UAG and hippocampal GOAT+ cells are reduced
in Parkinson’s dementia Hornsby et al., 2020, Cell Reports Medicine 1, 100120
October 20, 2020 ª 2020 The Author(s). https://doi.org/10.1016/j.xcrm.2020.100120 ll ll INTRODUCTION tions.10 This process is impaired in neurodegeneration11 and de-
mentia12 but is enhanced by lifestyle factors, such as exercise13
and
calorie
restriction.14
Indeed,
neurogenic
impairments
observed in the familial Alzheimer disease (FAD) model,
5xFAD, are rescued by exercise. In addition, the suppression
of adult neurogenesis was associated with increased neuron
loss in 5xFAD, but not wild-type (WT) mice, suggesting a patho-
physiological link between impaired AHN and AD progression.15
Similarly, AHN is impaired in rodent models of Parkinson disease
(PD),16-18 and both NSPC and immature neuron number are
reduced in the DG of humans diagnosed with PD dementia
(PDD).11 However, therapeutic strategies that promote AHN
are limited. Circulating factors are known to both enhance1-4 and impair5-7
neuronal plasticity and learning in adult mammals. However,
the mechanisms underlying these effects are not completely un-
derstood. Systemic factors such as Growth Differentiation Fac-
tor 11 (GDF11)1 are reported to regulate the neural stem/progen-
itor cell (NSPC) niche in the adult hippocampus to promote new
neuron formation, termed adult hippocampal neurogenesis
(AHN), and cognition. Conversely, circulating Beta-2 microglo-
bulin (B2M)6 and eotaxin5 impair the same niche resulting in
reduced neurogenesis and impaired cognitive function. These
data demonstrate that the hippocampal neurogenic niche is
responsive to systemic factors, even in aged mammals, and sug-
gest that circulating factors act as important modulators of mne-
monic function. We recently showed that calorie restriction increased AHN
and hippocampal-dependent memory in a mechanism depen-
dent on signaling via the stomach hormone, acyl-ghrelin.14
Indeed, acyl-ghrelin, which is elevated during calorie restriction,
crosses the blood-brain barrier and binds to the growth hormone
secretagogue receptor (GHS-R) within the hippocampus and im-
proves spatial memory.19 Moreover, we showed that peripheral The birth and maturation of new neurons in the adult hippo-
campal dentate gyrus (DG) is essential for spatial pattern sepa-
ration memory,8,9 which is the ability to separate highly similar
components of memories into distinct memory representa- Figure 1. Unacylated-Ghrelin Inhibits Hippocampal Neurogenesis in Adult Mice
Peripheral administration of UAG or genetic ablation of GOAT reduces the number of dividing Ki67+ cells (A and B) and immature Dcx+ neurons (C and D) in the
mouse DG. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 200 mm (inset scale bar, 20 mm). *p <
0.05, **p < 0.01, ***p < 0.001 versus WT vehicle group. INTRODUCTION injection of acyl-ghrelin, at physiological doses, increases AHN
and enhances pattern separation memory in adult rats.20
Similarly, deletion of GHS-R in mice results in increased suscep-
tibility to chronic stress coupled with reduced neurogenesis
in the ventral DG.21 These studies clearly identify acyl-ghrelin
as an important pro-neurogenic circulating factor.22 In order
to generate acyl-ghrelin, ghrelin must undergo post-translational
acylation by the enzyme ghrelin-O-acyl transferase (GOAT),23,24
prior to binding and activating GHS-R signaling.25 Unacylated-
ghrelin (UAG) represents 80%–90% of circulating ghrelin and
is often considered an inactive precursor to acyl-ghrelin. Howev-
er, there is growing evidence that UAG functions as a hormone
distinct from acyl-ghrelin and GHS-R. For example, UAG in-
duces genome-wide changes in the expression of genes linked
to glucose and lipid metabolism in fat, muscle, and liver from
GHS-R/ mice,26 providing evidence for the existence of a
UAG receptor that is yet to be identified. UAG also inhibits
acyl-ghrelin actions that are mediated by GHS-R.27-29 For
example, UAG suppressed acyl-ghrelin-induced neuronal activ-
ity in the brainstem and prevented the acyl-ghrelin/GHS-R-medi-
ated increase in food intake.30 We therefore sought to determine INTRODUCTION Data shown are mean ± SEM n = 5–6 mice/group. Article
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OPEN ACCESS Figure 1. Unacylated-Ghrelin Inhibits Hippocampal Neurogenesis in Adult Mice
Articl
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OPEN ACCESS Figure 1. Unacylated-Ghrelin Inhibits Hippocampal Neurogenesis in Adult Mice g
y
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p
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Peripheral administration of UAG or genetic ablation of GOAT reduces the number of dividing Ki67+ cells (A and B) and immatu
mouse DG. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 200
0.05, **p < 0.01, ***p < 0.001 versus WT vehicle group. Data shown are mean ± SEM n = 5–6 mice/group. g
y
pp
p
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Peripheral administration of UAG or genetic ablation of GOAT reduces the number of dividing Ki67+ cells (A and B) and immature Dcx+ neurons (C and D) in the
mouse DG. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 200 mm (inset scale bar, 20 mm). *p <
0.05, **p < 0.01, ***p < 0.001 versus WT vehicle group. Data shown are mean ± SEM n = 5–6 mice/group. injection of acyl-ghrelin, at physiological doses, increases AHN
and enhances pattern separation memory in adult rats.20
Similarly, deletion of GHS-R in mice results in increased suscep-
tibility to chronic stress coupled with reduced neurogenesis
in the ventral DG.21 These studies clearly identify acyl-ghrelin
as an important pro-neurogenic circulating factor.22 In order
to generate acyl-ghrelin, ghrelin must undergo post-translational
acylation by the enzyme ghrelin-O-acyl transferase (GOAT),23,24
prior to binding and activating GHS-R signaling.25 Unacylated-
ghrelin (UAG) represents 80%–90% of circulating ghrelin and
is often considered an inactive precursor to acyl-ghrelin. Howev-
er, there is growing evidence that UAG functions as a hormone
distinct from acyl-ghrelin and GHS-R. For example, UAG in-
duces genome-wide changes in the expression of genes linked
to glucose and lipid metabolism in fat, muscle, and liver from
GHS-R/ mice,26 providing evidence for the existence of a
UAG receptor that is yet to be identified. UAG also inhibits
acyl-ghrelin actions that are mediated by GHS-R.27-29 For
example, UAG suppressed acyl-ghrelin-induced neuronal activ-
ity in the brainstem and prevented the acyl-ghrelin/GHS-R-medi-
ated increase in food intake.30 We therefore sought to determine whether UAG modulates hippocampal plasticity and memory
function and whether plasma levels of acyl-ghrelin and UAG
associate with dementia in humans. 2
Cell Reports Medicine 1, 100120, October 20, 2020 UAG Reduces the Number of New Adult-Born Immature
Neurons and Non-neuronal Cells in the Adult
Hippocampus Hippocampus
Next, as 70% of young adult-born neurons undergo Bax-medi-
ated programmed cell death during an early stage of differentia-
tion,35 we studied whether UAG reduced the number of new
adult-born DG neurons. To achieve this, mice in the above study
received an injection of BrdU to birth-date-proliferating cells on
day 2 of the infusion prior to brain dissection and immunochem-
ical analysis on day 7. The number of immature neurons (BrdU+/
Dcx+) was significantly reduced in GOAT/ mice36 (main effect
of genotype, p = 0.0171, F(1,18) = 6.896) (Figures 2A and 2D; Fig-
ure S1B). There was also a 30% reduction in the mean number
of BrdU+/Dcx+ cells in UAG-treated WT mice; however, this was
not statistically significant (p = 0.2757) (Figure 2A). Further anal-
ysis revealed a significant reduction in the number of new
adult-born cells that lack Dcx immunoreactivity (BrdU+/Dcx–) in
UAG-treated WT mice, relative to vehicle-treated WT mice (p =
0.0288) (interaction between genotype x treatment, p = 0.022,
F(1,18) = 6.282) (Figure 2B). Indeed, quantification of the relative
proportion of new adult-born cell types demonstrated that UAG
reduced the proportion of BrdU+/Dcx– cells in WT mice (p =
0.0029) (main effect on cell type, p = 0.0033, F(1, 36) = 9.924;
interaction between treatment x cell type, p = 0.0276, F(3, 36) =
3.413) (Figure 2C). These data suggest that UAG may play an
important role in regulating the number of new astro-glial cells
or new stem cells originating following asymmetric cell division. Analysis of a second mouse model with genetic deletion of
GOAT37 revealed a similar reduction in DG neurogenesis (Fig-
ures S3A–S3D). Importantly, genetic deletion of GOAT, in either
knockout model, did not affect the number of type II stem cells
(Sox2+) in the SGZ (p = 0.4433, Figures S1C and S1E and p =
0.4075 Figures S3E and S3F). To avoid counting a subset of as-
trocytes that co-express Sox2, we performed double immuno-
fluorescence, using anti-Sox2 and the astrocyte-specific anti-
S100b antibodies. Our findings demonstrate unaltered expres-
sion of Sox2+/S100b– cells in the SGZ of GOAT/ mice
(3,012 ± 114.0 cells) relative to WT mice (3,001 ± 133.8 cells) Next, as 70% of young adult-born neurons undergo Bax-medi-
ated programmed cell death during an early stage of differentia-
tion,35 we studied whether UAG reduced the number of new
adult-born DG neurons. UAG Reduces the Number of New Adult-Born Immature
Neurons and Non-neuronal Cells in the Adult
Hippocampus To achieve this, mice in the above study
received an injection of BrdU to birth-date-proliferating cells on
day 2 of the infusion prior to brain dissection and immunochem-
ical analysis on day 7. The number of immature neurons (BrdU+/
Dcx+) was significantly reduced in GOAT/ mice36 (main effect
of genotype, p = 0.0171, F(1,18) = 6.896) (Figures 2A and 2D; Fig-
ure S1B). There was also a 30% reduction in the mean number
of BrdU+/Dcx+ cells in UAG-treated WT mice; however, this was
not statistically significant (p = 0.2757) (Figure 2A). Further anal-
ysis revealed a significant reduction in the number of new
adult-born cells that lack Dcx immunoreactivity (BrdU+/Dcx–) in
UAG-treated WT mice, relative to vehicle-treated WT mice (p =
0.0288) (interaction between genotype x treatment, p = 0.022,
F(1,18) = 6.282) (Figure 2B). Indeed, quantification of the relative
proportion of new adult-born cell types demonstrated that UAG
reduced the proportion of BrdU+/Dcx– cells in WT mice (p =
0.0029) (main effect on cell type, p = 0.0033, F(1, 36) = 9.924;
interaction between treatment x cell type, p = 0.0276, F(3, 36) =
3.413) (Figure 2C). These data suggest that UAG may play an
important role in regulating the number of new astro-glial cells
or new stem cells originating following asymmetric cell division. UAG Inhibits Hippocampal Neurogenesis in Adult Mice g
To assess whether UAG regulates adult NSPC plasticity in the
sub-granular zone (SGZ) of the hippocampus, we analyzed the
effect of UAG administered peripherally for 7 days in WT and
GOAT/ mice.23 GOAT/ mice lack circulating acyl-ghrelin
but have elevated levels of UAG making them ideally suited to
assessing the loss of acyl-ghrelin coupled with increased plasma
UAG.31 Surprisingly, UAG-treated WT mice showed a significant
40% decrease in Ki67+-proliferating cells in the SGZ compared
to vehicle-treated WT mice (p = 0.0056) (Figures 1A and 1B). Similarly, genetic blockade of acyl-ghrelin signaling in GOAT/
mice reduced the number of dividing Ki67+ progenitor cells in the
SGZ (p = 0.0020) (main effect of genotype, p = 0.0175, F(1,19) =
6.766; interaction between genotype and treatment, p = 0.0074,
F(1,19) = 9.003) (Figures 1A and 1B). These findings were 2
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS (p = 0.9964) (Figures 2E and 2F), suggesting that the observed
reduction in dividing progenitors was not due to a reduced stem
cell pool but by modulation of the adult neurogenic niche. To
determine whether circulating immune factors, that are known
to inhibit AHN, were altered in UAG- and vehicle-treated
GOAT/ and WT mice, we quantified plasma levels of eotaxin,
fractalkine, interleukin-6 (IL-6), IL-10, RANTES, and tumor ne-
crosis factor-a (TNF-a). However, there were no significant al-
terations in levels of these circulating factors in GOAT/ mice
or in WT mice following UAG treatment (Figure S2). These
data identify GOAT-mediated acylation of ghrelin as an impor-
tant modulator of AHN, with UAG providing anti-neurogenic ac-
tivity opposing acyl-ghrelin’s pro-neurogenic effects.14,20 accompanied by a significant reduction in the number of double-
cortin positive (Dcx+) immature neurons within the SGZ of both
UAG-treated WT (p = 0.0106) and vehicle-treated GOAT/
mice (p = 0.0003) (main effect of genotype, p = 0.0096,
F(1,19) = 8.287; interaction between genotype x treatment, p =
0.0009, F(1,19) = 15.33) (Figures 1C and 1D). UAG did not further
exacerbate the reduction in proliferating cells and immature neu-
rons in GOAT/ mice, suggesting that UAG’s effect on neuro-
genesis may be via a saturable mechanism. The responsiveness
of the GOAT/ neurogenic niche to UAG treatment may be
attenuated by developmental compensation or exposure to
consistently high levels of UAG throughout development. UAG Inhibits Hippocampal Neurogenesis in Adult Mice While
UAG is often considered inactive, reports of UAG opposing
acyl-ghrelin function on hepatocyte gluconeogenesis,32 block-
ing acyl-ghrelin-induced food intake33 and suppressing activity
of the GH axis,34 provide support for our findings. Together,
these data suggest that the striking decrease in hippocampal
cell proliferation and immature neuron number is due to elevated
UAG, rather than simply loss of acyl-ghrelin. y
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To further test this finding, we quantified neurogenesis in mice
with genetic ablation of the ghrelin gene. These ghrelin/ mice38,
which lack both acyl-ghrelin and UAG, had no impairments in
adult neurogenesis. A previous study reported that the rate of
cell proliferation and neuronal differentiation were reduced in 8-
to 9-week-old male ghrelin/ mice compared to non-littermate
controls.39 However, our analyses of total NSPC number (Sox2;
p = 0.3674), cell proliferation (Ki67; p = 0.4797), immature neuron
number (Dcx; p = 0.1527), and new adult-born neuron number
(BrdU/NeuN; p = 0.6409) throughout the rostro-caudal extent of
the hippocampus in 6-month-old male and female ghrelin/
mice confirmed no change in any of these measures relative to
WT, sex-matched, littermate control mice (Figure S4). Our data
from ghrelin/ mice are consistent with the idea that the loss
of pro-neurogenic acyl-ghrelin and anti-neurogenic UAG resulted
in no net change in AHN. In contrast, GOAT/ mice lack acyl-
ghrelin but have high levels of UAG, suggesting that high circu-
lating UAG is at least partly responsible for the reduction in
AHN, rather than simply the loss of acyl-ghrelin. Indeed, this inter-
pretation is consistent with ourdata showing that elevated UAGin
WT mice results in reduced hippocampal plasticity. UAG and GOAT/ Reduce Markers of Hippocampal
Plasticity in Adult Mice Next, we assessed whether elevation of UAG disrupted other
molecular signatures of hippocampal impairment. The immedi-
ate early gene (IEG), c-Fos, which is associated with changes
in neuronal gene expression that promote learning and memory
function,40,41 was quantified in the DG of vehicle- or UAG-treated
WT and GOAT/ mice. In keeping with our findings of impaired
plasticity, we observed a significant reduction in immuno-label-
ing of c-Fos in both UAG-treated WT (58%, p = 0.0147) and
GOAT/ (44%, p = 0.0377) mice (main effect of treatment, p =
0.0392, F(1,16) = 5.046; interaction between genotype x treat-
ment, p = 0.0343, F(1,16) = 5.354) (Figures 3A and 3B). In addi-
tion, as acyl-ghrelin promotes F-actin expression in hippocam-
pal dendrites it is linked with the generation and re-modeling of
spines.42 Therefore, we analyzed dendritic spines on hippocam-
pal neurons from WT and GOAT/ mice. While there was no
change in total spine number between the two groups (p =
0.4695, Figure 3C), our analysis revealed a significant 53%
reduction in immature ‘‘stubby’’ spine number in GOAT/
mice (p = 0.0164) (main effect of spine type, p = 0.0001,
F(4,20) = 26.36) (Figures 3D and 3E). Dendritic spines form excit-
atory synapses with pre-synaptic axons and are essential for Analysis of a second mouse model with genetic deletion of
GOAT37 revealed a similar reduction in DG neurogenesis (Fig-
ures S3A–S3D). Importantly, genetic deletion of GOAT, in either
knockout model, did not affect the number of type II stem cells
(Sox2+) in the SGZ (p = 0.4433, Figures S1C and S1E and p =
0.4075 Figures S3E and S3F). To avoid counting a subset of as-
trocytes that co-express Sox2, we performed double immuno-
fluorescence, using anti-Sox2 and the astrocyte-specific anti-
S100b antibodies. Our findings demonstrate unaltered expres-
sion of Sox2+/S100b– cells in the SGZ of GOAT/ mice
(3,012 ± 114.0 cells) relative to WT mice (3,001 ± 133.8 cells) Cell Reports Medicine 1, 100120, October 20, 2020
3 Figure 2. Unacylated-Ghrelin Reduces the Number of New Adult-Born Non-neuronal Cells
(A–C) Genetic ablation of GOAT reduced the number of new neuroblasts (main effect of genotype) (A) and new non-neuronal cells (B). UAG treatment reduced
number of new non-neuronal cells (B, C). (D) Representative confocal image of DG from wild-type mouse showing new BrdU+/Dcx+ neuroblast (arrowhead) and a new BrdU+/Dcx– non-neuronal
(arrow), n = 5–6 mice/group. UAG and GOAT/ Reduce Markers of Hippocampal
Plasticity in Adult Mice Statistical analysis was
performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 200 mm (inset scale bar, 20 mm). *p < 0.05 versus WT vehicle group. Data
shown are mean ± SEM n = 5–6 mice/group. (C–E) Hippocampal dendritic spine number was unaltered in GOAT/ mice (C and D); however, spine sub-type analysis demonstrated a significant reduction in the
number of stubby spines (E). For spine analysis, statistical analysis was performed by Student’s t test (C) and two-way ANOVA followed by Fisher’s LSD test (E). Scale bar, 5 mm. n = 3 mice/group. All data shown are mean ± SEM. *p < 0.05 versus WT group. synaptic plasticity, with spine morphology linked to cognitive
function.43 Given the established role of hippocampal c-
Fos27-29 and dendritic spines44 in regulating plasticity and cogni-
tion, we reasoned that hippocampal-dependent learning and
memory may be impaired in GOAT/ mice. main effect of genotype, p = 0.0001, F(1,20) = 26.70. Figure S5A)
compared to WT mice. To assess whether this deficit could be
rescued by acyl-ghrelin, WT and GOAT/ mice were given daily
injections of either saline or acyl-ghrelin for one or seven days
prior to Y-maze testing. Acyl-ghrelin treatment 1 h before testing
did not alter performance in GOAT/ mice relative to vehicle-
treated GOAT/ mice (entries into novel arm, p = 0.1692 (Fig-
ure 4A); time in novel arm, p = 0.7972 (Figure S5A). However,
treatment with acyl-ghrelin for 7 days enhanced performance
relative to vehicle-treated GOAT/ mice (entries into novel
arm, p = 0.0114 (Figure 4B); time in novel arm, p = 0.0019 [Fig-
ure S5B]) and rescued the deficit in GOAT/ mice relative to
acyl-ghrelin-treated WT mice (entries into novel arm, p =
0.8263 [Figure 4B]; time in novel arm, p = 0.7254 [Figure S5B]);
main effect of arm choice, p % 0.0001, F(2, 60) = 145.1;
interaction between arm choice x genotype, p % 0.0001,
F(6, 60) = 7.426 [Figure 4B]). Interestingly, the rescue of spatial
memory performance in acyl-ghrelin-treated GOAT/ mice UAG and GOAT/ Reduce Markers of Hippocampal
Plasticity in Adult Mice (E) Type II NSPC number was unaltered in the DG of GOAT/ mice, n = 3 mice/group. (F) Representative confocal image of DG from wild-type mouse showing Sox2+/S100b– type II NSPCs (arrow) and Sox2+/S100b+ astrocytes (arrowhead
the DG. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 50 mm. *p < 0.05, **p < 0.01, versus WT veh
group. Data shown are mean ± SEM. Artic
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OPEN ACCESS Article Artic
OPEN ACCESS Figure 2. Unacylated-Ghrelin Reduces the Number of New Adult-Born Non-neuronal Cells the DG. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 50 mm. *p < 0.05, **p < 0.01, versus WT vehicle
group. Data shown are mean ± SEM. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 50 mm. *p < 0.05, **p < 0.01, versus WT vehicle
group. Data shown are mean ± SEM. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 50 mm. *p < 0.05, **p < 0.01, versus WT vehicle
group. Data shown are mean ± SEM. 4
Cell Reports Medicine 1, 100120, October 20, 2020 Figure 3. UAG and GOAT/ Reduce Hippocampal Plasticity in Adult Mice
A and B) Peripheral administration of UAG or genetic ablation of GOAT reduces the number of c-Fos+ neurons in the mouse DG. Statistical analysis was
performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons. Scale bar, 200 mm (inset scale bar, 20 mm). *p < 0.05 versus WT vehicle group. Data
hown are mean ± SEM n = 5–6 mice/group. C–E) Hippocampal dendritic spine number was unaltered in GOAT/ mice (C and D); however, spine sub-type analysis demonstrated a significant reduction in the
umber of stubby spines (E). For spine analysis, statistical analysis was performed by Student’s t test (C) and two-way ANOVA followed by Fisher’s LSD test (E). Scale bar, 5 mm. n = 3 mice/group. All data shown are mean ± SEM. *p < 0.05 versus WT group. Article
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OPEN ACCESS Figure 3. UAG and GOAT/ Reduce Hippocampal Plasticity in Adult Mice d GOAT/ Reduce Hippocampal Plasticity in Adult Mic Figure 3. UAG and GOAT/ Reduce Hippocampal Plasticity in Adult Mice
(A and B) Peripheral administration of UAG or genetic ablation of GOAT reduces the number of c-Fos+ neurons in the mouse DG. ghrelin Treatment Acyl-ghrelin treatment
for 7 days significantly increases the number of entries to the novel arm in GOAT/ mice on day 7 (B, p < 0.05) and day 28 (C, p < 0.05). (D) Schematic representations of Y-maze apparatus, indicating novel, old, and home arms. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons (n = 6 mice/group). *p < 0.05, **p < 0.01. All data shown are
mean ± SEM. ghrelin Treatment To determine whether the neurochemical and structural deficits
in hippocampal plasticity-related factors observed in GOAT/
mice resulted in memory impairments, adult WT and GOAT/
mice were tested using the hippocampal-dependent spatial
memory Y-maze task (Figure 4). GOAT/ mice displayed a
deficit in performance compared to WT mice, entering the novel
arm fewer times (p = 0.0023; interaction between arm choice x
genotype, p = 0.0007, F(6, 60) = 4.607. Figure 4A) and spending
significantly less time in the novel arm of the Y-maze (p = 0.0168; Cell Reports Medicine 1, 100120, October 20, 2020
5 Article Figure 4. Adult GOAT/ Mice Display Hippocampal-Dependent Spatial Memory Deficits that Are Rescued by Acyl-ghrelin Treatment
(A–C) Analysis of relative entries into each arm of the Y-maze shows that GOAT/ mice enter the novel arms less often (A and B; p < 0.01). Acyl-ghrelin treatment
for 7 days significantly increases the number of entries to the novel arm in GOAT/ mice on day 7 (B, p < 0.05) and day 28 (C, p < 0.05). (D) Schematic representations of Y-maze apparatus, indicating novel, old, and home arms. Statistical analysis was performed by 2-way ANOVA followed by Holm-Sidak post hoc comparisons (n = 6 mice/group). *p < 0.05, **p < 0.01. All data shown are
mean ± SEM
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S mory Deficits that Are Rescued by Acyl-ghrelin Treatmen Figure 4. Adult GOAT/ Mice Display Hippocampal-Dependent Spatial Memory Deficits that Are Rescued by Acyl-ghrelin Treatment
(A–C) Analysis of relative entries into each arm of the Y-maze shows that GOAT/ mice enter the novel arms less often (A and B; p < 0.01). Acyl-ghrelin treatmen
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GOAT/
i
d
7 (B
0 05)
d d
28 (C
0 05) Mice Display Hippocampal-Dependent Spatial Memory Deficits that Are Rescued by Acyl-ghrelin Treatment Figure 4. Adult GOAT/ Mice Display Hippocampal-Dependent Spatial Memory Deficits that Are Rescued g
p
y
pp
p
p
p
y
y
y g
(A–C) Analysis of relative entries into each arm of the Y-maze shows that GOAT/ mice enter the novel arms less often (A and B; p < 0.01). 6
Cell Reports Medicine 1, 100120, October 20, 2020 UAG Inhibits Acyl-Ghrelin-Mediated Hippocampal Cell
Proliferation and New Cell Survival In Vitro relative to vehicle-treated GOAT/ mice was observed on day
28, 21 days following the end of treatment (entries into novel
arm, p = 0.0357 [Figure 4C]). Similarly, acyl-ghrelin-treated WT
mice and GOAT/ mice performed comparably at this time
point (entries into novel arm, p = 0.7069 [Figure 4C]; time in novel
arm, p = 0.3413 [Figure S5C]; main effect of arm choice, p %
0.0001, F(2, 60) = 92.78; interaction between arm choice x geno-
type, p = 0.0006, F(3, 60) = 4.696 [Figure 4C]). Analysis of total
Y-maze arm entries suggest that GOAT/ mice do not have def-
icits in exploration (Figures S5E–S5G). These data are consistent
with our neurochemical findings and demonstrate that GOAT is
essential for intact hippocampal-dependent spatial memory. To determine whether the ghrelin peptides mediate direct effects
on the hippocampus, we used the thymidine analog, EdU, to
quantify cell proliferation and survival following treatment of hip-
pocampal cell cultures with acyl-ghrelin or UAG. The primary
hippocampal culture system, containing a mix of cell types (Fig-
ure S6), was used to show that 24 h acyl-ghrelin treatment signif-
icantly increased, in a dose dependent-manner, the number of
dividing EdU+ cells labeled in the final hour of treatment (p =
0.0186 [100 nM], p = 0.0205 [1,000 nM]). Similar treatment with
UAG had no effect (p = 0.9814 [100 nM], p = 0.8033 [1,000 nM] 6
Cell Reports Medicine 1, 100120, October 20, 2020 Figure 5. UAG Inhibits Acyl-ghrelin-Mediated Cell Proliferation and Survival in Primary Hippocampal Cultures
(A–C) Schematic representation of the cell proliferation assay (A). Acyl-ghrelin (AG) but not unacylated ghrelin (UAG), directly stimulated cell proliferation in a
GHSR-dependent manner (B and C). (D) Representative images of newly divided EdU+ cells in primary hippocampal cultures. (E) Schematic representation of the cell survival assay. (F) AG, but not UAG, increased new cell survival in a GHSR-dependent manner. Article
ll
OPEN ACCESS ll
OPEN ACCESS ll
OPEN ACCESS gure 5. UAG Inhibits Acyl-ghrelin-Mediated Cell Proliferation and Survival in Primary Hippocampal Cultures Figure 5. UAG Inhibits Acyl-ghrelin-Mediated Cell Proliferation and Survival in Primary Hippocampal Cultures
(A–C) Schematic representation of the cell proliferation assay (A). Acyl-ghrelin (AG) but not unacylated ghrelin (UAG), directly stimulated cell proliferation in a
GHSR-dependent manner (B and C). (D) Representative images of newly divided EdU+ cells in primary hippocampal cultures. (E) Schematic representation of the cell survival assay. Figure 5. Levels of Circulating Medium-Chain Fatty Acid
Substrates for GOAT Were Unchanged in PD and PDD To determine whether acyl-ghrelin induces the survival of
newborn cells via the action of soluble neurotrophic factors,
we treated primary hippocampal cells with acyl-ghrelin and
quantified the gene expression of BDNF, a known pro-neuro-
genic factor.45 BDNF mRNA was significantly increased in pri-
mary hippocampal cells following treatment with acyl-ghrelin
(Figure 6A). To determine whether this effect was relevant to
the in vivo hippocampus, we treated adult mice with a single
intra-peritoneal injection of acyl-ghrelin. Twenty-four hours later,
we collected brain tissue for RNAScope in situ hybridization (ISH)
analysis
to
show
that
acyl-ghrelin
treatment
significantly
increased BDNF IXa mRNA, specifically in the rostral granule
cell layer (GCL) of the DG, relative to the vehicle-treated mice
(p < 0.05, Figures 6B and 6C). These studies confirm that acyl-
ghrelin increases the expression of hippocampal BDNF in vitro
and in vivo. Subsequently, we collected the conditioned media
from treated primary hippocampal cultures and incubated
them with a separate culture of hippocampal NSPCs46 that do
not express GHS-R (Figure 6D; Figure S7). Conditioned media
collected from acyl-ghrelin treated primary hippocampal cells
significantly increased the number of surviving newborn cells
when incubated with hippocampal NSPCs. Notably, this pro-
survival effect was completely blocked by the addition of a
BDNF-neutralizing antibody to the hippocampal NSPC cultures
(Figures 6E and 6F), suggesting that acyl-ghrelin supports the
survival of newborn hippocampal via BDNF signaling. To determine whether there were changes in levels of circulating
GOAT substrate, we quantified medium-chain fatty acids
(MCFAs) C6, C8, and C10 in fasted plasma samples from healthy
control, PD, and PDD subjects. Levels of theses MCFAs, relative
to the standard, nonanoic acid (C9), were unaltered in each of the
three groups (Figures 7D–7F). Ghrelin Receptor Is Expressed in the Human DG and Is
Unchanged in PD and PDD In situ BaseScope analysis of human post-mortem hippocampal
brain tissue identified GHS-R1a mRNA expression within the
adult human DG (Figure 7G). Notably, quantification of GHS-
R1a in the hippocampal GCL from healthy control, PD, and
PDD subjects revealed no significant changes in receptor
expression in these groups (Figure 7H). GOAT Expression Is Reduced in the Hippocampal GCL of
PDD Brain To determine whether the reduced AG:UAG ratio may be
compensated by GOAT-mediated acylation of UAG at the hippo-
campus, we first performed western blot analysis of GOAT
expression in hippocampal lysates from control, PD, and PDD
brain. This assay demonstrated a significant decrease in GOAT
immunoblot density in both PD (p < 0.05) and PDD (p < 0.05) tis-
sue, relative to control (Figures 7I and 7J). These data are consis-
tent with impaired acyl-ghrelin signaling across all hippocampal
regions in both PD and PDD brain. For a specific assessment of
GOAT expression within the GCL, we quantified the number of
GOAT+ cells in this DG sub-region using immunohistochemistry
on hippocampal sections from control, PD, and PDD brain (Fig-
ure 7K). Our data reveal a significant reduction in the number of Article (p = 0.3202). Similarly, the pro-survival effect of acyl-ghrelin was
lost when the cells were cultured in the presence of [D-Lys3]-
GHRP-6 (p > 0.999) (Figure 5F). These data demonstrate that
acyl-ghrelin promotes cell proliferation and survival via a direct
hippocampal mechanism that is mediated by the ghrelin
receptor, GHS-R. In contrast, UAG did not directly affect cell
proliferation or survival, suggesting that its inhibitory effect on
neurogenesis may be induced indirectly or by opposing acyl-
ghrelin induced GHS-R signaling. To test whether UAG can
modulate acyl-ghrelin’s effect, we co-treated hippocampal
cell cultures with acyl-ghrelin and UAG at equimolar (1:1) and
non-equimolar doses (1:10 and 1:30), respectively. These non-
equimolar doses were used to reflect the elevated level of UAG
in GOAT/ mice and in our UAG-treated WT mice. Notably,
we report that UAG completely attenuated the pro-survival effect
of acyl-ghrelin at each of the ratios tested (Figures 5G and 5H). These findings do not rule out the presence of an alternate
UAG-specific receptor or steric effects that may inhibit GHS-R
signaling. However, the direct antagonistic effect of UAG on
acyl-ghrelin induced hippocampal cell survival, which is medi-
ated by GHS-R, provides compelling evidence for the ability of
UAG to block acyl-ghrelin-mediated GHS-R signaling in the
hippocampus. cell proliferation and survival in vitro, this may be reflected in
the plasma ratio of acyl-ghrelin to UAG (AG:UAG) in humans
diagnosed with dementia. We therefore hypothesized that circu-
lating AG:UAG ratios may be particularly affected in individuals
diagnosed with PDD compared to a cognitively unimpaired PD
group. To test this hypothesis, we recruited individuals with
PD, PDD, and age-matched healthy controls to determine fast-
ing and post-prandial levels of both acyl-ghrelin and UAG. In
keeping with our pre-clinical data, we found that the plasma ratio
of AG:UAG was significantly reduced in the PDD group
compared to the cognitively intact PD (p = 0.0033) and control
cohorts (p = 0.0145) (Figures 7A and 7B). Consistent with this
finding, cognitive impairment was correlated with a reduction
in plasma AG:UAG (Spearman r2 = 0.1164, p = 0.0145) (Fig-
ure 7C), suggesting this ratio may be valuable as a diagnostic
biomarker for human dementia. Interestingly, the cognitively
intact PD group did not have reduced acyl-ghrelin levels in either
the fasted state or 180 min after eating (Table S1), further
supporting a specific role for ghrelin in regulating mnemonic
function. UAG Inhibits Acyl-Ghrelin-Mediated Hippocampal Cell
Proliferation and New Cell Survival In Vitro UAG Inhibits Acyl-ghrelin-Mediated Cell Proliferation and Survival in Primary Hippocampal Cultures
(A–C) Schematic representation of the cell proliferation assay (A). Acyl-ghrelin (AG) but not unacylated ghrelin (UAG), directly stimulated cell proliferation in a
GHSR-dependent manner (B and C). Statistical analysis performed using two-way ANOVA followed by Dunnett’s post hoc test (B), one-way ANOVA followed by Dunnett’s post hoc test (C, F, and G). Scale bar, 400 mm. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle. Each independent experiment was performed three times, with each treatment condition
performed in triplicate. Data shown are mean ± SEM. Statistical analysis performed using two-way ANOVA followed by Dunnett’s post hoc test (B), one-way ANOVA followed by Dunnett’s post hoc test (C, F, and G). Scale bar, 400 mm. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle. Each independent experiment was performed three times, with each treatment condition
performed in triplicate. Data shown are mean ± SEM. (Figures 5A, 5B, and 5D). The proliferative effect of acyl-ghrelin
was lost when the cells were cultured in the presence of the
GHS-R-antagonist, [D-Lys3]-GHRP-6 (Figure 5C). In addition,
we used a modified protocol to quantify cell survival, whereby dividing cells were pulsed with EdU for the first 16 h of culture,
prior to acyl-ghrelin or UAG treatment (Figure 5E). Acyl-ghrelin
significantly increased the survival of newborn EdU+ cells after
4 days of treatment (p = 0.0003), while UAG had no effect Cell Reports Medicine 1, 100120, October 20, 2020
7 Cell Reports Medicine 1, 100120, October 20, 2020
7 Cell Reports Medicine 1, 100120, October 20, 2020
7 A
i l ll
OPEN ACCESS Acyl-ghrelin Increases Survival of Newborn
Hippocampal Cells via BDNF in a Non-cell-Autonomous
Manner Levels of Circulating Medium-Chain Fatty Acid
Substrates for GOAT Were Unchanged in PD and PDD Analysis
of
several
other
feeding-related
(PYY,
leptin), metabolism-related (insulin, GLP-1), and growth-related
(GH, IGF-1) hormones, as well as cytokines (IL-6, TNF-
alpha), revealed no significant differences between the groups
(Figure S8). cell proliferation and survival in vitro, this may be reflected in
the plasma ratio of acyl-ghrelin to UAG (AG:UAG) in humans
diagnosed with dementia. We therefore hypothesized that circu-
lating AG:UAG ratios may be particularly affected in individuals
diagnosed with PDD compared to a cognitively unimpaired PD
group. To test this hypothesis, we recruited individuals with
PD, PDD, and age-matched healthy controls to determine fast-
ing and post-prandial levels of both acyl-ghrelin and UAG. In
keeping with our pre-clinical data, we found that the plasma ratio
of AG:UAG was significantly reduced in the PDD group
compared to the cognitively intact PD (p = 0.0033) and control
cohorts (p = 0.0145) (Figures 7A and 7B). Consistent with this
finding, cognitive impairment was correlated with a reduction
in plasma AG:UAG (Spearman r2 = 0.1164, p = 0.0145) (Fig-
ure 7C), suggesting this ratio may be valuable as a diagnostic
biomarker for human dementia. Interestingly, the cognitively
intact PD group did not have reduced acyl-ghrelin levels in either
the fasted state or 180 min after eating (Table S1), further
supporting a specific role for ghrelin in regulating mnemonic
function. Analysis
of
several
other
feeding-related
(PYY,
leptin), metabolism-related (insulin, GLP-1), and growth-related
(GH, IGF-1) hormones, as well as cytokines (IL-6, TNF-
alpha), revealed no significant differences between the groups
(Figure S8). Acyl-ghrelin Increases Survival of Newborn
Hippocampal Cells via BDNF in a Non-cell-Autonomous
Manner Acyl-ghrelin Increases Survival of Newborn
Hippocampal Cells via BDNF in a Non-cell-Autonomous
Manner The Circulating Ratio of AG:UAG Is Reduced in PDD The Circulating Ratio of AG:UAG Is Reduced in PDD Mechanistically, we reasoned that as circulating levels of acyl-
ghrelin and UAG have opposing actions on neurogenesis and
cognition in mice and that UAG inhibits acyl-ghrelin-mediated 8
Cell Reports Medicine 1, 100120, October 20, 2020 ll
OPEN ACCESS igure 6. Acyl-Ghrelin Increases Survival of Newborn Hippocampal Cells via BDNF
A–C) 24 h acyl-ghrelin (1mM) increases the expression of BDNF mRNA in primary hippocampal cultures (A; *p < 0.05, Student’s t test, n = 3 mice/group) and in the
GCL of the adult mouse hippocampus (B; *p < 0.05, two-way ANOVA followed by Holm-Sidak post hoc test, n = 6 mice/group) (C; RNAScope BDNF IXa probes
enoted by white arrow heads. Scale bar, 100 mm). D) Schematic of in vitro experiment to determine the non-cell-autonomous effects of acyl-ghrelin on neurogenesis. Initial treatment of primary hippocampal
ultures with acyl-ghrelin, followed by transfer of conditioned media to hippocampal NSPCs and subsequent analysis of cell survival. E and F) Conditioned media from acyl-ghrelin (AG-CM)-treated primary hippocampal cultures increases survival of hippocampal NSPCs in a BDNF-
ependent manner (E). These data suggest that acyl-ghrelin stimulates the release of BDNF from primary hippocampal cells to promote the survival of newborn
ippocampal NSPCs in a non-cell-autonomous manner. Statistical analysis performed by one-way ANOVA followed by Dunnett’s post hoc test. **p < 0.01 . cale bar, 400 mm. Each independent in vitro experiment was performed three times, with each treatment condition performed in triplicate. Data shown are
mean ± SEM. Article
OPEN ACCESS Figure 6. Acyl-Ghrelin Increases Survival of Newborn Hippocampal Cells via BDNF gu e 6
cy G
e
c eases Su
a o
e
bo
ppoca
pa Ce s
a
(A–C) 24 h acyl-ghrelin (1mM) increases the expression of BDNF mRNA in primary hippocampal cultures (A; *p < 0.05, Student’s t test, n = 3 mice/group) and in the
GCL of the adult mouse hippocampus (B; *p < 0.05, two-way ANOVA followed by Holm-Sidak post hoc test, n = 6 mice/group) (C; RNAScope BDNF IXa probes
denoted by white arrow heads. Scale bar, 100 mm). (D) Schematic of in vitro experiment to determine the non-cell-autonomous effects of acyl-ghrelin on neurogenesis. Initial treatment of primary hippocampal
cultures with acyl-ghrelin, followed by transfer of conditioned media to hippocampal NSPCs and subsequent analysis of cell survival. GOAT+ granule layer cells specifically in PDD tissue (Figure 7L)
(p < 0.05). ghrelin on neurogenesis and learning, little was known about
the role of UAG, the most prevalent form of ghrelin, in this
context. Our results therefore identify a critical role for the
post-translational modification of ghrelin in regulating neurogen-
esis and learning and expand our understanding of ghrelin
biology in the adult hippocampus. The Circulating Ratio of AG:UAG Is Reduced in PDD GOAT+ granule layer cells specifically in PDD tissue (Figure 7L)
(p < 0.05). The Circulating Ratio of AG:UAG Is Reduced in PDD (E and F) Conditioned media from acyl-ghrelin (AG-CM)-treated primary hippocampal cultures increases survival of hippocampal NSPCs in a BDNF-
dependent manner (E). These data suggest that acyl-ghrelin stimulates the release of BDNF from primary hippocampal cells to promote the survival of newborn
hippocampal NSPCs in a non-cell-autonomous manner. Statistical analysis performed by one-way ANOVA followed by Dunnett’s post hoc test. **p < 0.01 . Scale bar, 400 mm. Each independent in vitro experiment was performed three times, with each treatment condition performed in triplicate. Data shown are
mean ± SEM. g
y
pp
p
(A–C) 24 h acyl-ghrelin (1mM) increases the expression of BDNF mRNA in primary hippocampal cultures (A; *p < 0.05, Student’s t test, n = 3 mice/group) and in the
GCL of the adult mouse hippocampus (B; *p < 0.05, two-way ANOVA followed by Holm-Sidak post hoc test, n = 6 mice/group) (C; RNAScope BDNF IXa probes
denoted by white arrow heads. Scale bar, 100 mm). (D) Schematic of in vitro experiment to determine the non-cell-autonomous effects of acyl-ghrelin on neurogenesis. Initial treatment of primary hippocampal
cultures with acyl-ghrelin, followed by transfer of conditioned media to hippocampal NSPCs and subsequent analysis of cell survival. (E
d F) C
diti
d
di
f
l
h
li
(AG CM) t
t d
i
hi
l
lt
i
i
l
f hi
l NSPC
i
BDNF reases the expression of BDNF mRNA in primary hippocampal cultures (A; *p < 0.05, Student’s t test, n = 3 mice/group) and in the
ampus (B; *p < 0.05, two-way ANOVA followed by Holm-Sidak post hoc test, n = 6 mice/group) (C; RNAScope BDNF IXa probes
Scale bar, 100 mm). t t
d t
i
th
ll
t
ff
t
f
l
h
li
i
I iti l t
t
t
f
i
hi
l (E and F) Conditioned media from acyl-ghrelin (AG-CM)-treated primary hippocampal cultures increases survival of hippocampal NSPCs in a BDNF-
dependent manner (E). These data suggest that acyl-ghrelin stimulates the release of BDNF from primary hippocampal cells to promote the survival of newborn
hippocampal NSPCs in a non-cell-autonomous manner. Statistical analysis performed by one-way ANOVA followed by Dunnett’s post hoc test. **p < 0.01 . Scale bar, 400 mm. Each independent in vitro experiment was performed three times, with each treatment condition performed in triplicate. Data shown are
mean ± SEM. Cell Reports Medicine 1, 100120, October 20, 2020
9 DISCUSSION The
long-term rescue of spatial memory, long after acyl-ghrelin has
cleared the circulation, is consistent with longer-term changes
in hippocampal plasticity such as AHN20 and increased c-Fos-
mediated transcriptional programs that support memory.14
However, further cellular analysis of new adult-born neurons
coupled with testing behavioral ‘‘pattern separation’’ perfor-
mance, that places emphasis on distinguishing similar but
distinct spatial contexts, is required to fully elucidate the behav-
ioral consequences of the acyl-ghrelin-mediated rescue of AHN
in GOAT/ mice. sion of plasticity related proteins, the NR2B subunit of the
NMDA receptor,47 and the GluA1 AMPA receptor subunit48 to
promote synaptic transmission and LTP. Our data suggest that
acute 1 h acyl-ghrelin treatment is not sufficient to trigger
changes that promote learning and memory. However, 7 days
of daily acyl-ghrelin treatment significantly improved perfor-
mance levels of GOAT/ mice to WT levels. In addition, the
rescue of spatial memory performance remained partially intact
when tested 21 days after the final acyl-ghrelin injection. The
long-term rescue of spatial memory, long after acyl-ghrelin has
cleared the circulation, is consistent with longer-term changes
in hippocampal plasticity such as AHN20 and increased c-Fos-
mediated transcriptional programs that support memory.14
However, further cellular analysis of new adult-born neurons
coupled with testing behavioral ‘‘pattern separation’’ perfor-
mance, that places emphasis on distinguishing similar but
distinct spatial contexts, is required to fully elucidate the behav-
ioral consequences of the acyl-ghrelin-mediated rescue of AHN
in GOAT/ mice. BrdU+/Dcx+ adult-born immature neurons in GOAT/ mice. Intriguingly, there was a reduction of new BrdU+/Dcx– adult-
born cells in UAG-treated WT mice and GOAT/ mice, suggest-
ing that UAG may play an important role in regulating new astro-
glial or new NSPCs in the hippocampus. Notably, the number of
type II NSPCs was similar in both WT and GOAT/ mice, sug-
gesting that the genetic ablation of GOAT during development
does not impair the generation of NSPCs in the hippocampal
niche. BrdU+/Dcx+ adult-born immature neurons in GOAT/ mice. Intriguingly, there was a reduction of new BrdU+/Dcx– adult-
born cells in UAG-treated WT mice and GOAT/ mice, suggest-
ing that UAG may play an important role in regulating new astro-
glial or new NSPCs in the hippocampus. Notably, the number of
type II NSPCs was similar in both WT and GOAT/ mice, sug-
gesting that the genetic ablation of GOAT during development
does not impair the generation of NSPCs in the hippocampal
niche. 10
Cell Reports Medicine 1, 100120, October 20, 2020 DISCUSSION (I and J) WB analysis of GOAT in hippocampal homogenates (I) identified a significant reduction in PD and PDD subjects (J) (n = 6/group). Figure 7. The Plasma Ratio of AG:UAG and Hippocampal GOAT Is Reduced in Humans with PD Dementia
(A) Pl
AG UAG
ti
i
h
lth
t
l
(
20) PD (
20)
d PDD (
8)
ti
t
d
f
ti
d
t
di l
diti
D tt d li Figure 7. The Plasma Ratio of AG:UAG and Hippocampal GOAT Is Reduced in Humans with PD Dementia breakfast consumption at time 0. (B) Area-under-the-curve (AUC) values demonstrate a significant reduction in AG:UAG ratio in control versus PDD and PD vers
(C) Correlation of cognition (MoCA score) with plasma AG:UAG (AUC). Statistical analysis was performed by Kruskal-Wallis test w
and Spearman correlation analysis (two-tailed). y
,
(
)
(G) BaseScope analysis of human hippocampal tissue identified ghrelin receptor (GHS-R1a1zz) mRNA within the dentate gyrus (red dots, top panel, white
arrowheads). Scale bar, 20 mm. Internal control mRNA probe PPIB1zz is also shown (red dots, bottom panel, white arrowheads). (H) GHS-R1a mRNA was not altered in PD or PDD brain. Statistical analysis was performed using a one-way ANOVA with the Tukey multiple-comparison test
(n = 5–7/group). hippocampal homogenates (I) identified a significant reduction in PD and PDD subjects (J) (n = 6/group). (I and J) WB analysis of GOAT in hippocampal homogenates (I) identified a significant reduction in PD and PDD subjects (J) (n
6/group). (K and L) IHC analysis of GOAT immunoreactivity (brown, white arrowheads) in hippocampal GCL (K) identified a significant reduction GOAT+ cell number in PDD
subjects (L) (n = 5–6/group). Scale bar, 25 mM. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparison. All data shown are mean ± SEM *p < 0 05 **p < 0 01 sion of plasticity related proteins, the NR2B subunit of the
NMDA receptor,47 and the GluA1 AMPA receptor subunit48 to
promote synaptic transmission and LTP. Our data suggest that
acute 1 h acyl-ghrelin treatment is not sufficient to trigger
changes that promote learning and memory. However, 7 days
of daily acyl-ghrelin treatment significantly improved perfor-
mance levels of GOAT/ mice to WT levels. In addition, the
rescue of spatial memory performance remained partially intact
when tested 21 days after the final acyl-ghrelin injection. DISCUSSION Previous findings support a role for acyl-ghrelin in improving hip-
pocampal neurogenesis, spine remodeling, LTP, and memory
function. The current study, to the best of our knowledge, dem-
onstrates a previously unknown function for UAG in reducing
hippocampal plasticity and spatial memory performance, and
opposing the cognitive enhancing effects of acyl-ghrelin. Despite several studies reporting the beneficial effects of acyl- We report a reduction in dividing Ki67+ hippocampal cells and
Dcx+ immature neurons following the genetic ablation of GOAT. These data are consistent with similar reductions in markers of
neurogenesis in WT mice treated with UAG. The impairment
in neurogenesis was further substantiated by a BrdU pulse-
chase study, which revealed a reduction in the number of new Cell Reports Medicine 1, 100120, October 20, 2020
9 Figure 7. The Plasma Ratio of AG:UAG and Hippocampal GOAT Is Reduced in Humans with PD Dementia
(A) Pl
AG UAG
ti
i
h
lth
t
l
(
20) PD (
20)
d PDD (
8)
ti
t
d
f
ti
d
t
di l
diti
D tt d li
i di
t
Article
ll
OPEN ACCESS ll
OPEN ACCESS t Figure 7. The Plasma Ratio of AG:UAG and Hippocampal GOAT Is Reduced in Humans with PD Dementia
(A) Plasma AG:UAG ratio in healthy controls (n = 20), PD (n = 20), and PDD (n = 8) patients under fasting and post-prandial conditions. Dotted line indicates
breakfast consumption at time 0. (B) Area-under-the-curve (AUC) values demonstrate a significant reduction in AG:UAG ratio in control versus PDD and PD versus PDD groups. (C) Correlation of cognition (MoCA score) with plasma AG:UAG (AUC). Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparison
and Spearman correlation analysis (two-tailed). (D–F) Analysis of plasma free fatty acid substrates; caproic acid (C6) (D), octanoic acid (C8) (E), and capric acid (C10) (F) revealed no differences between groups. The ratio of each analyte was normalized to the internal standard, nonanoic acid (C9). (G) BaseScope analysis of human hippocampal tissue identified ghrelin receptor (GHS-R1a1zz) mRNA within the dentate gyrus (red dots, top panel, white
arrowheads). Scale bar, 20 mm. Internal control mRNA probe PPIB1zz is also shown (red dots, bottom panel, white arrowheads). (H) GHS-R1a mRNA was not altered in PD or PDD brain. Statistical analysis was performed using a one-way ANOVA with the Tukey multiple-comparison test
(n = 5–7/group). DISCUSSION Analysis of other plasticity related markers also revealed
changes consistent with impaired hippocampal function. These
changes included a reduction in the number of c-Fos+ cells
within the DG in UAG-treated WT and GOAT/ mice. Moreover,
analysis of dendritic spines, which are essential for synaptic
plasticity, revealed a reduction in the number of ‘‘stubby’’ spines
present on hippocampal neurons from GOAT/ mice. As neuro-
genesis, IEGs, and dendritic spines are important for brain plas-
ticity and new memory formation, our data suggested impair-
ments
to
hippocampal-dependent
learning
and
memory. Indeed, hippocampal-dependent spatial memory testing re-
vealed a performance deficit in GOAT/ mice relative to WT
mice. Recent studies suggest that acyl-ghrelin induces expres- We recently showed in mice that the acyl-ghrelin receptor,
GHS-R, is expressed on mature DG neurons but not NSPCs.14
This finding was further substantiated by single-cell RNA 10
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS sequencing (RNA-seq) of distinct hippocampal cell populations
that report GHS-R expression in mature granule neurons but
not in NSPCs.49 We therefore propose that acyl-ghrelin pro-
motes hippocampal neurogenesis in a non-cell-autonomous
manner.22,50 To validate this, we show that acyl-ghrelin treat-
ment of hippocampal NSPCs in vitro had no effect on cell prolif-
eration—a result that was consistent with the absence of detect-
able GHS-R expression in these cells (Figure S7). To determine
whether acyl-ghrelin and UAG directly affect the hippocampus,
we treated primary hippocampal cultures, which contain a mixed
population of cells, including neurons and NSPCs (Figure S6). These studies demonstrated that acyl-ghrelin, but not UAG,
stimulated cell division and enhanced the survival of newly
divided cells in a GHS-R-dependent manner. More strikingly,
UAG was able to inhibit the neurogenic effect of acyl-ghrelin
in vitro, which is consistent with the inhibitory effect of UAG on
neurogenesis in WT mice. However, while UAG inhibited the
neurogenic effect of acyl-ghrelin in vitro, when given alone it
did not reduce basal hippocampal cell division, suggesting that
UAG may induce additional extra-hippocampal changes to
reduce neurogenesis in mice. Nonetheless, our in vitro studies
confirm that UAG inhibits the pro-neurogenic effect of acyl-ghre-
lin signaling in hippocampal cells and suggest that ghrelin, via
post-translational regulation, can fine-tune neurogenesis and
cognition in both positive and negative directions. lating factors that have been reported to modulate neurogenesis
and cognition, including leptin, GH, and IGF-1, were unchanged. DISCUSSION However, quantification of total ghrelin revealed a significant
reduction in the AG:UAG ratio in the PDD group compared to
both PD and control groups. This is in contrast to previous re-
ports of a reduction in circulating acyl-ghrelin in PD subjects.54,55
This difference may be explained by our stratification of PD
groups by cognitive performance. These findings are consistent
with our data from pre-clinical studies demonstrating that
elevated levels of UAG had detrimental effects on hippocampal
neurogenesis and spatial memory. Longitudinal studies are required to elucidate whether the
AG:UAG ratio may represent a prognostic PDD biomarker. In
addition, more extensive analysis of larger cohorts are needed
to understand the role of AG:UAG in clinically distinct popula-
tions of patients diagnosed with dementia. As biomarkers are
important for precision-medicine-based targeted therapies in
dementia, the need for blood-based biomarkers to complement
the high cost and invasive CSF and PET markers of amyloid-b
and Tau proteins are eagerly anticipated.56 Our findings identify
a possible blood-based biomarker associated with dementia
and emphasize the importance of assessing post-translational
modifications in patient cohorts. Interestingly, acyl-ghrelin treatment of GOAT/ mice, which
have high levels of UAG, restored learning and memory perfor-
mance to control levels. We therefore speculate that raising
the circulating AG:UAG ratio may ameliorate cognitive decline
in PDD patients via restoration of hippocampal plasticity and
pro-neurogenic signaling. Supporting this view, we show that
GHS-R1a mRNA is expressed in the DG of adult humans (Fig-
ure 7G). There were no statistically significant changes across
control, PD, and PDD cohorts; however, there was an almost
doubling of the mean GHS-R1a mRNA expression levels in
PDD versus control brain (Figure 7H). This change may reflect
a homeostatic response to the reduction in plasma AG:UAG
and therefore attenuated hippocampal ghrelin-signaling in
PDD. These data demonstrate that the hippocampal ghrelin-re-
ceptor is present in PDD brain and that elevation of the plasma
AG:UAG ratio should, in principle, result in activation of pro-
neurogenic hippocampal GHS-R1a signaling. Impairments in
hippocampal GHS-R1a signaling were recently identified in
5xFAD mice and in post-mortem human Alzheimer disease tis-
sue. Notably, co-activation of GHS-R1a/DRD1 rescued synaptic
and memory deficits in 5xFAD mice.57 We show that the levels of
octanoic acid, required for the post-translational formation of
acyl-ghrelin, were unaltered in PDD plasma (Figures 7D–7F). DISCUSSION Therefore, we speculate that enzyme activity for the acylation
and/or de-acylation of ghrelin may be impaired in PDD leading
to a reduction in the AG:UAG ratio. Of interest, recent studies
indicate that ghrelin undergoes tissue-dependent acylation,
including within the hippocampus, to support acyl-ghrelin
signaling.36,58 To understand the potential for UAG to undergo
tissue-dependent acylation, we quantified GOAT levels in the hu-
man hippocampus. Using crude hippocampal lysates, we show
a significant reduction in GOAT protein expression in both PD
and PDD brain (Figures 7I and 7J). Subsequently, we quantified
the number of GOAT+ cells within the GCL on brain tissue sec-
tions to reveal a reduction that was specific to the PDD As several soluble neurotrophic factors are known to promote
neurogenesis within the hippocampal niche, we reasoned that
acyl-ghrelin may stimulate the production and/or release of
such factors to promote neurogenesis. One such diffusible factor
that promotes neurogenesis51 and memory45 is BDNF. Indeed,
we show that BDNF mRNA was significantly increased in both
primary hippocampal cells (Figure 6A) and in granule cells of
the rostral, but not caudal, DG of adult mice (Figures 6B and
6C) following treatment with acyl-ghrelin. Notably, the rostral
pole of the DG is linked with regulation of spatial learning and
memory.52 To determine whether acyl-ghrelin regulates the sur-
vival of newborn hippocampal cells via BDNF, we collected the
conditioned media from treated primary hippocampal cultures
and incubated them with hippocampal NSPCs. This culture
method is well suited for testing this hypothesis as GHS-R is
not expressed in NSPCs. We demonstrate that conditioned me-
dia collected from acyl-ghrelin treated primary hippocampal
cells significantly increased the number of surviving newborn
cells when incubated with hippocampal NSPCs. Notably, the
pro-survival effect of conditioned media collected from acyl-
ghrelin treated primary cells was completely blocked by the
addition of a BDNF-neutralizing antibody to the hippocampal
NSPC cultures (Figures 6E and 6F). These data suggest
that acyl-ghrelin supports neurogenesis, at least in part, via
increased hippocampal BDNF signaling. Cell Reports Medicine 1, 100120, October 20, 2020
11 ACKNOWLEDGMENTS The authors would like to thank Dr. Jesus A. Gutierrez (Translational Science &
Technologies, Eli Lilly & Co., Indianapolis, IN, USA) for the supply of GOAT/
mice and Dr. Djordje Gveric (PUK Brain Bank at Imperial College London) for
the supply of post-mortem human brain tissue. We would also like to thank
Mrs. Sally James (Swansea University) for assistance with confocal micro-
scopy. The graphical abstract was created with BioRender.com. This work
was funded by grants from the Medical Research Council, UK (Grant no. G0902250 to J.S.D.), St. David’s Medical Foundation, UK (to J.S.D.), British
Society for Neuroendocrinology, UK (to Z.B.A. and J.S.D.), NHMRC Research
Fellowship, Australia (Grant no. APP1154974, to Z.B.A.), The Royal Society,
UK (to J.S.D.), and the National Institute for Health Research Newcastle
Biomedical Research Unit based at Newcastle Hospitals Foundation Trust
and Newcastle University, UK (to D.J.B. and J.S.D.).The study involving human
brain was funded by a Parkinson’s UK Innovation Award, UK (to J.S.D.). AUTHOR CONTRIBUTIONS A.K.E.H., L.B., M.C.C., V.V.S., F.J., L.D.R., M.S., M.M., R.S., A.R., S.H.L.,
M.S., O.H., A.H.M., T.W., Z.B.A., and J.S.D. performed the experiments and
generated the data. T.W., Z.B.A., D.J.B., and J.S.D. designed the study. J.S.D. prepared the figures and wrote the manuscript. All authors read and
approved the final manuscript. d KEY RESOURCES TABLE d KEY RESOURCES TABLE d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead Contact
B Materials Availability
B Data and Code Availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Study Approvals
B Animals
B Rat Primary Hippocampal Cell Culture
B Rat Hippocampal Stem Cells (HCN cells). B Human Brain
d METHOD DETAILS
B UAG Infusion
B Tissue Collection
B Immunohistochemistry
B Quantification of Labeled Cells
2
Cell Reports Medicine 1, 100120, October 20, 2020 d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead Contact
B Materials Availability
B Data and Code Availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Study Approvals
B Animals
B Rat Primary Hippocampal Cell Culture
B Rat Hippocampal Stem Cells (HCN cells). B Human Brain
d METHOD DETAILS
B UAG Infusion
B Tissue Collection
B Immunohistochemistry
B Quantification of Labeled Cells d RESOURCE AVAILABILITY
B Lead Contact
B Materials Availability d RESOURCE AVAILABILITY B Lead Contact Published: October 20, 2020 B Materials Availability B Data and Code Availability Article B RNAscope ISH on Mouse Brain Tissue
B Golgi-Cox Analysis of Dendritic Spines
B Milliplex Plasma Analysis
B Behavioral Testing
B Proliferation and Survival Assays
B Human Plasma Collection
B BaseScope ISH on Human Brain Tissue
B Immunohistochemistry of Human Brain Tissue
B Western Blot Assay of Human Brain Tissue
B Free Fatty Acid Analysis using Mass Spectrometry
QUANTIFICATION AND STATISTICAL ANALYSIS hippocampus (Figures 7K and 7L). Therefore, our studies sug-
gest that a reduction in plasma AG:UAG, coupled with the
inability of hippocampal cells to acylate UAG (via GOAT), leads
to reduced GHS-R1a signaling in the GCL and cognitive deficits
manifest in PDD. Of note, rodent toxin-based and genetic-based
models of PD16-18 have impaired hippocampal plasticity,
including neurogenesis. In future, these pre-clinical models
may be valuable tools in determining whether GHS-R1a agonists
or compounds that inhibit ghrelin de-acylation can rescue hippo-
campal plasticity and cognitive function. hippocampus (Figures 7K and 7L). Therefore, our studies sug-
gest that a reduction in plasma AG:UAG, coupled with the
inability of hippocampal cells to acylate UAG (via GOAT), leads
to reduced GHS-R1a signaling in the GCL and cognitive deficits
manifest in PDD. Of note, rodent toxin-based and genetic-based
models of PD16-18 have impaired hippocampal plasticity,
including neurogenesis. In future, these pre-clinical models
may be valuable tools in determining whether GHS-R1a agonists
or compounds that inhibit ghrelin de-acylation can rescue hippo-
campal plasticity and cognitive function. In summary, we describe how a post-translational modifica-
tion to a circulating factor modulates, in either direction, neuro-
genesis and cognition in mice. In addition, the reduction in circu-
lating
AG:UAG
ratio
correlated
with
dementia
in
human
neurodegenerative
disease. The
findings
extend
our
understanding of how adult brain plasticity is regulated by
circulating factors and suggest that manipulating the post-trans-
lational acylation of plasma ghrelin may offer therapeutic oppor-
tunities to ameliorate cognitive decline in human neurodegener-
ative disease. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. xcrm.2020.100120. Limitations of Study The plasma diagnostic biomarker data presented are from rela-
tively small cohorts. Further studies are needed, with increased
sample size, to validate our findings. Also, it is not known
whether the reduction in AG:UAG is specific to PDD or whether
it represents a broader measure of dementia in humans. Signif-
icant additional data are required to test these possibilities in
distinct dementia phenotypes. Similarly, it remains to be seen
whether the use of highly sensitive analytical chemistry tech-
niques (i.e., liquid chromatography-mass spectrometry [LC-
MS]) will provide greater insight into the biochemical composi-
tion of ghrelin species in plasma from individuals diagnosed
with dementia. Nonetheless, we provide a possible mechanistic
link between circulating ghrelin, hippocampal function, and PDD
in humans. DISCUSSION With the findings of our pre-clinical studies in mind, the reduc-
tion in NSPC and immature neuron number in the DG of humans
diagnosed with PDD11 and the impaired performance of PD pa-
tients in an Object Pattern Separation task, we reasoned that
ghrelin signaling may be impaired in PDD.53 Therefore, we quan-
tified plasma acyl-ghrelin in cognitively intact PD, PDD, and
healthy control subjects and reported no difference between
groups under fasted and fed conditions. Similarly, several circu- Cell Reports Medicine 1, 100120, October 20, 2020
11 ll
OPEN ACCESS DECLARATION OF INTERESTS Detailed methods are provided in the online version of this paper
and include the following: The authors declare no competing interests. The authors declare no competing interests. Received: November 5, 2019
Revised: May 20, 2020
Accepted: September 16, 2020
Published: October 20, 2020 Article ll
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232, 1843–1857. 62. Conrad, C.D., Galea, L.A.M., Kuroda, Y., and McEwen, B.S. (1996). Chronic stress impairs rat spatial memory on the Y maze, and this effect
is blocked by tianeptine pretreatment. Behav. Neurosci. 110, 1321–1334. 48. Ribeiro, L.F., Catarino, T., Santos, S.D., Benoist, M., van Leeuwen, J.F.,
and Esteban, J.A. (2014). Ghrelin triggers the synaptic incorporation of
AMPA receptors in the hippocampus. Proc. Natl. Acad. Sci. USA 111,
E149–D158. Article Kent, B.A., Beynon, A.L., Hornsby, A.K.E., Bekinschtein, P., Bussey, T.J.,
Davies, J.S., and Saksida, L.M. (2015). The orexigenic hormone acyl-ghre- Cell Reports Medicine 1, 100120, October 20, 2020
13 Article REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Rat anti-BrdU
BioRad
MCA6143: RRID: AB_2868611
Goat anti-Dcx (C-18)
Santa Cruz Biotechnology
sc-8066; RRID: AB_2088494
Rabbit anti-Sox2
Abcam
ab97959; RRID: AB_2341193
Mouse anti-S100b
Sigma
S2532; RRID: AB_477499
Rabbit anti-Ki67
Abcam
ab16667; RRID: AB_302459
Rabbit anti-GOAT antibody
Phoenix Pharmaceuticals
G-032-12; RRID: AB_2868614
Rabbit anti-GAPDH
Sigma
G9545; RRID: AB_796208
Rabbit anti-c-Fos
Santa Cruz
SC-52; RRID: AB_2106783
Rabbit anti-BDNF
Millipore
AB1779SP; RRID: AB_90994
Goat anti-GFAP
BioRad
AHP1468; RRID: AB_2294553
Rabbit anti-Sox2
Abcam
ab97959; RRID: AB_2341193
Mouse anti-NeuN
Millipore
MAB377; RRID: AB_2298772
Donkey anti-Goat AF-568
ThermoFisher
A11057; RRID: AB_2534104
Donkey anti-Mouse AF-568
ThermoFisher
A10037; RRID: AB_2534013
Donkey anti-rat AF-488
Life Technologies
A-21208; RRID: AB_2535794
Goat anti-rabbit AF-568
Life Technologies
A-11036; RRID: AB_10563566
Goat anti-mouse AF-488
Life Technologies
A-11001; RRID: AB_2534069
Biotinylated goat anti-rabbit
Vectorlabs
BA-1000; RRID: AB_2313606
Biotinylated donkey anti-goat
ThermoFisher
PA1-28663; RRID: AB_10980902
Biological Samples
Hippocampal brain tissue sections (for Basescope assay). PUK Brain Bank at Imperial
College London (ethical
approval: 07/MRE09/72)
N/A
Healthy controls (n = 5); Age (mean ± SD) 73.2 ± 24.27; Males 40%
PD (n = 7); Age (mean ± SD) 78.43 ± 5.798; Males 57.14%
PDD (n = 5); Age (mean ± SD) 80.6 ± 1.673; Males 40%
Hippocampal brain tissue sections (for IHC assay). PUK Brain Bank at Imperial
College London (ethical
approval: 07/MRE09/72)
N/A
Healthy controls (n = 5); Age (mean ± SD) 73.2 ± 24.27; Males 40%
PD (n = 6); Age (mean ± SD) 80 ± 5.292; Males 50%
PDD (n = 6); Age (mean ± SD) 77.33 ± 8.14; Males 50%
Hippocampal brain tissue (for WB assay). 63. Dubois, B., Burn, D., Goetz, C., Aarsland, D., Brown, R.G., Broe, G.A.,
Dickson, D., Duyckaerts, C., Cummings, J., Gauthier, S., et al. (2007). Diagnostic procedures for Parkinson’s disease dementia: recommenda-
tions from the movement disorder society task force. Mov. Disord. 22,
2314–2324. 49. Zeisel, A., Hochgerner, H., Lo¨ nnerberg, P., Johnsson, A., Memic, F., van
der Zwan, J., Ha¨ ring, M., Braun, E., Borm, L.E., La Manno, G., et al. (2018). Molecular architecture of the mouse nervous system. Cell 174,
999–1014. 64. Bankhead, P., Loughrey, M.B., Ferna´ ndez, J.A., Dombrowski, Y., McArt,
D.G., Dunne, P.D., McQuaid, S., Gray, R.T., Murray, L.J., Coleman,
H.G., et al. (2017). QuPath: Open source software for digital pathology im-
age analysis. Sci. Rep. 7, 16878. 50. Morgan, A.H., Andrews, Z.B., and Davies, J.S. (2017). Less is more:
Caloric
regulation
of
neurogenesis
and
adult
brain
function. J. Neuroendocrinol. 29, e12512. 65. Zhang, R., Brennan, M.L., Shen, Z., MacPherson, J.C., Schmitt, D., Mo-
lenda, C.E., and Hazen, S.L. (2002). Myeloperoxidase functions as a major
enzymatic catalyst for initiation of lipid peroxidation at sites of inflamma-
tion. J. Biol. Chem. 277, 46116–46122. 51. Bekinschtein, P., Kent, B.A., Oomen, C.A., Clemenson, G.D., Gage, F.H.,
Saksida, L.M., and Bussey, T.J. (2013). BDNF in the dentate gyrus is 14
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS Article PUK Brain Bank at Imperial
College London (ethical
approval: 07/MRE09/72)
N/A
Healthy controls (n = 6); Age (mean ± SD) 88 ± 5.865; Males 50%
PD (n = 6); Age (mean ± SD) 81.83 ± 6.432; Males 66.66%
PDD (n = 6); Age (mean ± SD) 81.83 ± 6.432; Males 66.66%
Human plasma samples;
Clinical Aging Research Unit,
Newcastle University (ethical
approval: 14/NE/0002)
N/A
Healthy controls (n = 20); Age (mean ± SD) 74 ± 6.28; Males 55%
PD (n = 20); Age (mean ± SD) 72.2 ± 5.51; Males 55%
PDD (n = 8); Age (mean ± SD) 74.75 ± 5.99; Males 87.5%
Chemicals, Peptides, and Recombinant Proteins
B27
ThermoFisher
17504
GlutaMax
ThermoFisher
35050
L-glutamine
ThermoFisher
25030081
Neurobasal media
ThermoFisher
21103
Penicillin-streptomycin-fungizone
ThermoFisher
15240062
Phosphate Buffered Saline
ThermoFisher
10010031
Poly-l-ornithine
Sigma
P4957
(Continued on next page) Cell Reports Medicine 1, 100120, October 20, 2020
e1 Article ll
OPEN ACCESS ll
OPEN ACCESS ll
OPEN ACCESS Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Laminin
AMS Bio
3400-010-02
DMEM F12
ThermoFisher
21331
Poly-d-lysine
Sigma
P6407
Accutase
Millipore
SCR005
N2
ThermoFisher
A1370701
Trypan Blue
ThermoFisher
15250061
[D-Lys3]-GHRP-6
Tocris
1922
Acyl-ghrelin
Tocris
1465
Ethylene glycol
Sigma
324558
Glycerol
Sigma
G5516
bFGF
Peprotech
100-18B
ImmPACT DAB
Vectorlabs
SK-4105
Sample loading buffer
BioRad
1610747
ECL Select
GE Healthcare
RPN2235
Prolong-gold anti-fade solution
Life Technologies
P36930
[D-Lys3]-GHRP-6
Tocris
1922
Acyl-ghrelin (rat)
Tocris
1465
Des-octanoyl-ghrelin
Tocris
2951
Ethylene glycol
Sigma
324558
Glycerol
Sigma
G5516
bFGF
Peprotech
100-18B
AEBSF
Sigma Aldrich
A8456
Ethanol > 99.5%
Sigma Aldrich
459836
Gill’s hematoxylin
Vectorlabs
H-3401-500
Vectamount mounting media
Vectorlabs
H-5000
Superfrost+ slides
VWR, France
631-0108
Bio-Plex Sheath Fluid
BioRad
171-000055
Vacutainer EDTA-plasma tubes
VWR, France
6450
Critical Commercial Assays
EdU detection kit
ThermoFisher
C10350
RNA scope 2.5 Red Assay
ACD Bio
322360
RNA scope BDNF probes
ACD Bio
461591
Milliplex-MAP 6-plex mouse cytokine magnetic bead panel kit
Millipore
SPR402
Milliplex MAP Kit - Human Metabolic Hormone Magnetic Bead Panel
Millipore
HMHEMAG-34K
EdU Click-IT assay
ThermoFisher
C10337
AllPrep DNA/RNA/Protein mini kit
QIAGEN
80204
Pierce BCA Protein Assay Kit
ThermoFisher
23227
VectaStain Elite ABC-HRP Kit
Vectorlabs
PK-6100
Human Ghrelin (Total) ELISA
Millipore
EZGRT-89K
Human Ghrelin (Active) ELISA
Millipore
EZGRA-88K
Human IGF-1 DuoSet ELISA
R&D Systems
DY291
Human GH DuoSet ELISA
R&D Systems
DY1067
BaseScope Reagent Kit - RED
Advanced Cell Diagnostics
322900
BaseScope Probe – BA-Hs-GHSR-tv1a-E1E2
Advanced Cell Diagnostics
709121
FD Rapid GolgiStain Kit
FD Neurotechnologies Inc. (Continued on next page) Article PK401A
Experimental Models: Cell Lines
Rat hippocampal primary cells
ThermoFisher
A1084101
Rat neural stem/progenitor cell-line
Hsieh Lab
Palmer et al.46
(Continued on next page)
Article
ll
OPEN ACCESS e2
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Experimental Models: Organisms/Strains
GOAT/ mice: 12-week old male mice on a C57BL/6 genetic
background were maintained at Monash University, Australia, with
approval from the Monash University Animal Ethics Committee. Regeneron Pharmaceuticals
(Tarrytown, NY, USA)
Bayliss et al.37
GOAT/ mice: 6-month old male mice on a C57BL/6 genetic
background were maintained at Cardiff University, UK, with approval
from the UK Animals (Scientific Procedures) Act 1986. Taconic Farms (Hudson,
NY, USA)
Hopkins et al.36
Ghrelin/ mice 5-month old female mice on a C57BL/6 genetic
background were maintained at Cardiff University, UK, with approval
from the UK Animals (Scientific Procedures) Act 1986. Dr Yuxiang Sun lab
(Texas A&M, TX, USA)
Sun et al.38
Oligonucleotides
Total BDNF mRNA oligos (NCBI: M61178)
PrimerDesign, UK
This paper
F: CGAGAGGTCTGACGACGACG
Total BDNF mRNA oligos (NCBI: M61178)
PrimerDesign, UK
This paper
R: GCGTCCTTATGGTTTTCTTCGTTG
Software and Algorithms
GraphPad Prism 8.0
https://www.graphpad.com/
N/A
ImageJ
https://www.imagej.nih.gov/
N/A
QuPath
https://qupath.github.io
N/A
Bio-Plex Manager v4.1
Bio-Rad
N/A
ImageLab Software v4.1 (ChemiDoc XRS)
BioRad
N/A
Other
In Cell Analyzer 2000
GE
N/A
Nikon Eclipse 50i microscope
Nikon
N/A
Fluorescent microscope (Axioscope)
Zeiss
N/A
Confocal microscope (LSM710 META). Zeiss
N/A
Axio Scan.Z1
Zeiss
N/A
LTQ Orbitrap XL
Thermo Scientific
N/A
CM1900 Cryostat
Leica
N/A
Freeezing-stage microtome (MicroM)
ThermoScientific
N/A
POLARstar Omega plate reader
BMG Labtech
N/A
Bioplex-200 System
Bio-Rad
N/A
Article
OPEN ACCESS Materials Availability This study did not generate datasets or code. Lead Contact Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jeff Da-
vies (jeff.s.davies@swansea.ac.uk). Human Brain The brain tissue was collected by the PUK Brain Bank at Imperial College London with ethical approval (07/MRE09/72) and was used
in this study with consent following peer review. A total of 17 subjects were included in the BaseScope analysis. Post-mortem hip-
pocampal tissue sections were prepared from formalin fixed frozen tissue, cryo-sectioned (6-8um thick) and mounted onto Super-
frost glass slides. Brain tissue was prepared from healthy controls (n = 5), with no evidence of degenerative disease or cognitive
decline, participants diagnosed with PD (n = 7) and participants diagnosed with PDD (n = 5). All cases had a post-mortem interval
of < 24h. Upon arrival, sections were stored at 70C and sections from across the anterior-posterior extent of the hippocampus
were visually determined for use in subsequent BaseScope assays. Animals Six-month old homozygous GOAT null (GOAT/) mice and their WT (C57BL/6) littermate controls23 were imported from Taconic
Farms (Hudson, NY) and housed in the JBIOS animal facility (Cardiff University) under standard laboratory conditions (12h L:D cycle)
with food and water available ad libitum. Six-month old Ghrelin null (Ghrelin/) mice and their WT littermates29 were bred from het-
erozygous x heterozygous mating in the animal facility at Cardiff University under standard conditions (as above). Founder stock were
a kind gift from Prof. Yuxiang Sun (Texas A&M University, College Station, USA). A second genetic model of GOAT ablation24 was
provided by Regeneron Pharmaceuticals. This GOAT/ line was generated using Velocigene technology. The GOAT gene sequence
(ATG-stop) was replaced with a lacZ reporter gene using the target vector, bacterial artificial chromosome (BAC). These mice orig-
inated from C57BL/6/129 targeted embryonic stem cells and mice were backcrossed onto a C57BL/6 mice background. Mice were
kept in standard laboratory conditions at Monash University with free access to food (chow diet, cat no. 8720610 Barastoc stock-
feeds, Victoria Australia), and water at 23C in a 12-hour light/dark cycle and were group-housed to prevent isolation stress, unless
otherwise stated. The allocation of mice into groups was performed in a randomized manner and data collection was performed by a
person blind to the treatment conditions. ARRIVE Guidelines on reporting in vivo experiments. The study involving humans was approved by Local Ethical Review (14/NE/
0002; IRAS project ID: 141456) at Newcastle University, consistent with the Mental Capacity Act 2005. ARRIVE Guidelines on reporting in vivo experiments. The study involving humans was approved by Local Ethical Review (14/NE/
0002; IRAS project ID: 141456) at Newcastle University, consistent with the Mental Capacity Act 2005. UAG Infusion Each mouse23 was fitted with an indwelling jugular vein catheter connected to a subcutaneous osmotic mini-pump (ALZET model
1007D) primed to deliver vehicle (sterile isotonic saline containing BSA (1mg/ml) and heparin (5U/ml) at 0.5ml/h) or UAG (48ug/
day) under isoflurane anesthesia. One day later all mice received an injection of thymidine analog, BrdU (50mg/kg i.p), to label dividing
cells. After seven days mice were re-anesthetized and killed by decapitation, the brains being excised whole and processed for
immunohistochemistry (IHC) as described below. Rat Hippocampal Stem Cells (HCN cells). These cells, herein referred to as Neural Stem/Progenitor Cells (NSPCs), initially isolated and cloned from Fisher 344 rats, were a kind
gift from Prof Jenny Hsieh’s lab (University of Texas, San Antonio, USA). These cells were cultured in DMEM F12 (ThermoFisher,
21331), supplemented with N2 supplement (ThermoFisher, A1370701), GlutaMax (ThermoFisher, 35050061), Penicillin-strepto-
mycin-fungizone (ThermoFisher, 15240062) and 20ng/ml bFGF/FGF2 (Peprotech, 100-18B). Tissue culture plastic was coated
with 10 mg/ml Poly-l-ornithine (PLO) (Sigma, P4957) and 5 mg/ml laminin (AMS Bio, 3400-010-02). Cells were maintained at 37C
in a 5% CO2 humidified incubator. Rat Primary Hippocampal Cell Culture Primary rat hippocampal cultures from the hippocampi of day-18 Fisher 344 rat embryos were grown in neurobasal media
(ThermoFisher, 21103), supplemented with B27 supplement (ThermoFisher, 17504), 200mM GlutaMax (ThermoFisher, 35050) and
25mM L-glutamine (ThermoFisher, 25030081). Tissue culture plastic was coated with poly-d-lysine (Sigma, P6407) at a concentration
of 4.5mg/cm2. Cells were maintained at 37C in a 5% CO2 humidified incubator. EXPERIMENTAL MODEL AND SUBJECT DETAILS Study Approvals
The animal procedures described, including those involving genetically modified animals, conformed to the UK Animals (Scientific
Procedures) Act 1986 and the Monash University Animal Ethics Committee guidelines. All procedures complied with the NC3Rs Study Approvals
The animal procedures described, including those involving genetically modified animals, conformed to the UK Animals (Scientific
Procedures) Act 1986 and the Monash University Animal Ethics Committee guidelines. All procedures complied with the NC3Rs Cell Reports Medicine 1, 100120, October 20, 2020
e3 Cell Reports Medicine 1, 100120, October 20, 2020
e3 A
i l ll
OPEN ACCESS Immunohistochemistry For DAB-immunohistochemical analysis of Ki67, Sox2 and DCX labeling, sections were washed in 0.1M PBS (2x10mins) and 0.1M
PBS-T (1x10 mins). Subsequently, endogenous peroxidases were quenched by washing in a PBS plus 1.5% H2O2 solution for 20 mi-
nutes. Sections were washed again (as above) and incubated in 5% NGS (NDS for DCX) in PBS-T for 1h. Sections were incubated
overnight at 4C with rabbit anti-Ki67 (1:500, ab16667, Abcam), rabbit anti-Sox2 (1:1000, ab97959, Abcam) or goat anti-DCX (1:200
Santa Cruz Biotechnology, USA), in PBS-T and 2% NGS (NDS for DCX) solution. Another wash step followed prior to incubation with
biotinylated goat anti-rabbit (1:400 Vectorlabs, USA) for Ki67 and Sox2 or biotinylated donkey anti-goat (1:400 Vectorlabs, USA) for
DCX, in PBS-T for 70 minutes. The sections were washed and incubated in ABC (Vectorlabs, USA) solution for 90 minutes in the dark
prior to another two washes in PBS, and incubation with 0.1M sodium acetate pH6 for 10 minutes. Immunoreactivity was developed
in nickel-enhanced DAB solution followed by two washes in PBS. Sections were mounted onto superfrost+ slides (VWR, France) and
allowed to dry overnight before being de-hydrated and de-lipified in increasing concentrations of ethanol. Finally, sections were incu-
bated in Histoclear (2x3 mins; National Diagnostics, USA) and coverslipped using Entellan mounting medium (Merck, USA). Slides
were allowed to dry overnight prior to imaging. For DAB-immunohistochemical analysis of c-Fos labeling, sections were washed and endogenous peroxidases quenched as
before. Sections were washed again (as above), before antigen retrieval in sodium citrate at 70C for 1h and subsequent blocking
in 5% NGS in PBS-T for 1h. Sections were incubated overnight at 4C with rabbit anti-c-Fos (1:4000, SC-52, Santa Cruz, USA) in
PBS-T and 2% NGS solution. Another wash step followed prior to incubation with biotinylated goat anti-rabbit (1:400 Vectorlabs,
USA) in PBS-T for 70 minutes. The sections were washed and incubated in ABC (Vectorlabs, USA) solution for 90 minutes in the
dark prior to another round of washing (as above) and subsequent tyramide signal amplification. Following incubation with bio-
tinylated tyramine (1:100) in PBS-T plus 0.1% H2O2 for 10 min, sections were washed in 0.1M PBS (1x10 mins) and 0.1M PBS-T
(2x10 mins), before a second 90 minutes ABC (Vectorlabs, USA) incubation, which was again performed in the dark. Sections
were then washed in 0.1M PBS (2x10 mins) and incubated with 0.1M sodium acetate pH6 for 10 minutes. Immunohistochemistry Immunoreactivity and tis-
sue processing was performed as described above. Tissue Collection Whole brain was removed and immediately fixed by immersion in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (pH 7.4) for
24h at 4C unless stated otherwise. Subsequently brains were cryoprotected in 30% sucrose solution (until sunk). Coronal sections
(30 mm) were cut into a 1:6 series along the entire rostro-caudal extent of the hippocampus using a freezing-stage microtome
(MicroM, ThermoScientific) and collected for IHC. All IHC was performed on free-floating sections at room temperature unless stated
otherwise. A sub-set of 12-week old GHSR/ mice (see Fig.S3) were terminally anesthetized and intra-cardially perfused with 4%
PFA prior to cryoprotection, as described above. e4
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS Immunohistochemistry For immunofluorescent analysis of BrdU+/Dcx+, sections were washed three times in PBS for 5 minutes, permeabilized in methanol at
20C for 2 minutes and washed (as before) prior to pre-treatment with 2N HCl for 30 minutes at 37C followed by washing in 0.1 M
borate buffer, pH8.5, for 10 minutes. Sections were washed as before and blocked with 5% normal donkey serum (NDS) in PBS plus
0.1% triton (PBS-T) for 60 minutes at room temperature. Sections were incubated overnight at 4C in rat anti-BrdU (1:400, AbD Se-
rotec) and goat anti-Dcx (1:200, Santa Cruz Biotechnology, USA) diluted in PBS-T. Tissue were washed as before and incubated in
donkey anti-rat AF-488 (1:500, Life Technologies, USA) and donkey anti-goat AF-568 (1:500, Life Technologies, USA) in PBS-T for
30 minutes in the dark. After another wash sections were mounted onto Superfrost+ slides (VWR, France) with prolong-gold anti-fade
solution (Life Technologies, USA). For immunofluorescent analysis of Sox2, sections were treated as above with the exception of antigen retrieval being performed in
sodium citrate at 70C for 1h (rather than 2N HCl or borate buffer) with subsequent blocking in 5% NGS. Immunoreactivity was de-
tected using rabbit anti-Sox2 (1:500, ab97959, Abcam) and goat anti-rabbit AF-568 (Life Technologies, USA). Nuclei were counter-
stained with Hoechst prior to mounting as described above. For immunofluorescent analysis of Sox2+/ S100b+, sections were washed three times in PBS for 5 minutes, permeabilised in meth-
anol for 2 minutes at 20C and washed again (as above). Antigen retrieval was performed with sodium citrate buffer for 1 hour at
70C, sections were washed as before and subsequently blocked with 5% normal goat serum (NGS) in PBS+0.1% Triton-X (PBS-T)
for 1 hour at room temperature. Sections were incubated overnight at 4C in rabbit anti-Sox2 (1:1000, ab97959, Abcam) diluted in
PBST. Sections were washed as before and incubated in goat anti-rabbit AF-568 (1:500, Life Technologies) in PBST for 30 minutes at
room temperature, in the dark. Following another wash step, sections were incubated for 1 hour at room temperature with mouse
anti-S100b (1:1000, S2532, Sigma) in PBST. Following another set of washes, sections were incubated in goat anti-mouse AF-
488 (1:500, Life Technologies) in PBST for 30 minutes at room temperature and protected from light. After a final wash sections
were mounted onto Superfrost+ Plus slides (VWR) with Prolong-gold anti-fade mounting solution (Invitrogen) and coverslipped. Quantification of Labeled Cells Immuno-stained brain tissue was imaged by light microscopy (Nikon 50i) (for DAB), fluorescent microscopy (Axioscope, Zeiss) or
confocal microscopy (LSM710 META, Zeiss). Immunofluorescent cells were manually counted bilaterally through the z axis using
a 3 40 objective and throughout the rostro-caudal extent of the granule cell layer (GCL). DAB-immunolabelled cells were quantified
using ImageJ software. Resulting numbers were divided by the number of coronal sections analyzed and multiplied by the distance
between each section to obtain an estimate of the number of cells per hippocampus (and divided by 2 to obtain the total per DG). For
quantification of immunoreactivity in GOAT/ mice24 where there were only rostral DG sections available, cell number was divided
by the DG area and expressed as counts per mm2. All analyses were performed blind to genotype and treatment. Article collected 24h later for RNAscope assay. Coronal sections (30 mm) were cut using a freezing-stage microtome (MicroM,
ThermoScientific) along the entire rostro-caudal extent of the hippocampus and collected in a 1:12 series, immersed in cryoprotec-
tant (0.1M PBS containing 30% ethylene glycol, 20% glycerol) and stored at 80C until use. Sections were first washed with TBS-
0.1%Tween20 (TBST) to remove residual cryoprotectant, followed by incubation in TBST-1.5% H2O2 for 20 minutes at RT. The
sections were subsequently rinsed in PBST and mounted onto SuperFrost Plus slides. Next, the slides were dehydrated in an ethanol
gradient (70%–100%) for 3 minutes each, then baked in a dry oven for 60 minutes at 60C. Target retrieval was performed by incu-
bation in target retrieval buffer (1X) in a steamer for 15 minutes, before washing with 100% ethanol. Pre-treatment protease digestion
was performed using the protease plus at 40C for 30 minutes in the HybEz oven rack. After this, slides were incubated with the
probes: BDNF IXa (461591), positive control PPIB (313911), or the negative control DapB (310043), for 2 hours at 40C. After this,
slides were washed with wash buffer (1X) and left in SSC buffer (5X) at RT overnight. Signal amplification was performed with
ACD bio amplification reagents (AMP1-6) in the HybEz oven. The stain was developed for 10 minutes using the Fast-RED A & B re-
agents (60:1). 50% Haematoxylin was used as a counter-stain to enhance visualization and contrast of the RNA puncta. VectaMount
was added per slide prior to coverslipping. Images were acquired using a light microscope (Nikon Eclipse 50i, 3 10 magnification)
and analyzed using the ImageJ software to quantify puncta per mm2 of GCL. Golgi-Cox Analysis of Dendritic Spines Impregnation of WT and GOAT/ mouse brains with Golgi-Cox solution was performed using the FD Rapid GolgiStainTM Kit
(PK401A, FD Neurotechnologies Inc.) according to the manufacturer’s instructions. Briefly, freshly dissected brains were rinsed
with Milli-Q water to remove residual blood. The tissue was immersed in impregnation solution (equal volumes solution A and solution
B) and stored, in the dark, at room temperature for 2 weeks. Impregnation solution was replaced with fresh solution after the first 24h. The tissue was transferred into solution C and stored, in the dark, at room temperature for 1 week. Solution C was replaced with fresh
solution after the first 24h of immersion. Brain tissue was subsequently frozen by slowly dipping the tissue into pre-cooled isopen-
tane, on dry-ice, for a few seconds. The tissue was placed on dry-ice for 1 minute, before being wrapped in foil and stored at 80C. Golgi-Cox impregnated brains were sectioned using a Leica CM1900 Cryostat (chamber temperature set to 15C; cutting stage
temperature set to 10C) at a thickness of 120mm and mounted onto glass slides that were rinsed in Solution C. Slides were dried
overnight at room temperature and protected from light. The slides were rinsed 2x with Milli-Q water, for 4 minutes each, before being
placed in a mixture of 1 part Solution D, 1 part Solution E and 2 parts Milli-Q water (DEQ solution) for 10 minutes. Slides were dehy-
drated in a series of 50%, 75% and 95% EtOH for 4 minutes each, followed by 4x 4 minute incubations in 100% EtOH. Subsequently,
the slides were delipified in Histoclear 3x for 4 minutes, and coverslipped using Entellan mounting media. Dendritic spines were analyzed using the Nikon Eclipse 50i microscope using a Nikon Plan 100x/1.25 oil objective. The images
were subsequently processed using ImageJ software in order to manually count dendritic spines, selected from secondary branches
of apical dendrites. Spines were categorised into the following groups; mushroom, thin spine, stubby spine, branched and filopo-
dium, as described59,60. For each genotype, 4-8 dendritic segments per mouse were analyzed. The spine density was presented
as the number of spines per mm dendritic length. RNAscope ISH on Mouse Brain Tissue RNAscope is a proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell. The assay was performed
according to the manufacturer’s instructions, including all buffers if not otherwise stated (ACD Bio, RNAscope 2.5 Red Assay,
322360). Briefly, male C57Bl6 mice (11-13 weeks old) were treated with acyl-ghrelin (300 mg/kg; i.p at 11.00h) before brains were Cell Reports Medicine 1, 100120, October 20, 2020
e5 A ti l ll
OPEN ACCESS Behavioral Testing To determine whether the neurochemical deficits observed in GOAT/ mice resulted in impaired hippocampal-dependent spatial
memory and whether this could be rescued by acyl-ghrelin, adult 12 week-old WT and GOAT/ mice (n = 6/group) were given daily
injections of either saline or acyl-ghrelin (300 mg/kg i.p) for 1 or 7 days prior to analysis of spatial memory using a Y-maze. This dose of
acyl-ghrelin was chosen as it has previously been shown to increase food intake. Injections were performed daily between 9-10am
when mice were in a fed state. A modified Y-maze task was used to assess spatial memory performance on day 1, 7 or 28 days after
the first acyl-ghrelin injection. All tests were performed in an experimental room with sound isolation and dim light. The animals were
carried to the test room for at least 1 hour of acclimation. Behavior was monitored using a video camera positioned above the ap-
paratuses and the videos were later analyzed by an experienced blinded researcher using video tracking software (CleverSys Inc,
Reston, VA, USA). The modified Y-maze measures spatial memory, as spatial orientation cues facilitate rodents to explore a novel
arm rather than returning to a previously visited arm61. This ethologically relevant test is based on the rodents’ innate curiosity to
explore novel areas and was chosen in these studies specifically because it does not require negative or positive reinforcers,
such as food rewards, as ghrelin is known to affect food intake and motivation. In addition, it has been validated as a hippocampal
relevant spatial task62, with impaired performance in different models of hippocampal damage. We used a Y-shaped gray Perspex
maze (30 cm x 10 cm x 16 cm) and each arm could be isolated by blocking entry with a sliding door. Sawdust from a mouse’s home
cage lined the maze during the trials and extra maze cues on the walls were placed 30-40 cm from the end of the arms to provide
spatial orientation cues. Behavior was tested across two trials, the first of which had one arm of the maze blocked off. Mice were
allowed to explore the reduced maze for 10 minutes and then returned to their home cage. The second trial was conducted 30 mi-
nutes after the first trial and both arms of the maze were opened. Mice were placed in the start (home) arm and allowed to explore the
full maze for 5 minutes. Proliferation and Survival Assays To determine the effect of ghrelin on proliferation, primary hippocampal cells were seeded at a density of 5x104 cells per well for 24h
with the appropriate treatments (acyl-ghrelin or UAG). To assess whether this proliferative affect was mediated by GHS-R, cells were
incubated with GHS-R antagonist D-Lys3 (1mM) 0.5h before acyl-ghrelin incubation. For the final 1h of treatments, half the media was
removed and incubated with EdU (well concentration 10mM), prior to fixation with 4% PFA. To determine the effect of acyl-ghrelin on
survival, primary hippocampal cells were seeded at a density of 5x104 cells per well for 16h in the presence of 10 mM EdU. The next
day cells were rinsed thoroughly with PBS and incubated with appropriate treatments for 4 days, before being fixed with 4% PFA. Proliferation/survival were assessed by EdU Click-IT assay (ThermoFisher C10337), counting the number of EdU-positive cells as a
proportion of Hoescht stained cells. Cells were imaged using a 3 20 objective (InCell Analyzer, GE Healthcare) with 9 fields of view per
well. Each independent experiment was performed three times, with each treatment condition performed in triplicate. To determine the effect of ghrelin and conditioned media on cell proliferation and survival the adult rat hippocampal stem cell line
(HCN) was used. These cells, herein referred to as Neural Stem/Progenitor Cells (NSPCs), initially isolated and cloned from Fisher 344
rats were a kind gift from Prof Jenny Hsieh’s lab (University of Texas, San Antonio, USA), were cultured in DMEM F12 (ThermoFisher,
21331), supplemented with N2 supplement (ThermoFisher, A1370701), GlutaMax (ThermoFisher, 35050061), Penicillin-strepto-
mycin-fungizone (ThermoFisher, 15240062) and 20ng/ml bFGF/FGF2 (Peprotech, 100-18B). Tissue culture plastic was coated
with 10 mg/ml Poly-l-ornithine (PLO) (Sigma, P4957) and 5 mg/ml laminin (AMS Bio, 3400-010-02). Cells were maintained at 37C
in a 5% CO2 humidified incubator. To examine the effect of conditioned media on the survival of newborn cells, NSPCs were seeded at a density of 5x104 cells per well
in a 96-well PLO/Laminin coated plate and incubated with EdU (10 mM) for 16h to label dividing cells. This time point coincided with
the end of primary hippocampal cells being treated with vehicle or acyl-ghrelin (1 mM) for 2 days. The NSPCs were washed three times
with PBS to remove residual media, and the vehicle (V-CM) or acyl-ghrelin (AG-CM) treated conditioned media was transferred from
the primary hippocampal cells to the NSPC cultures and incubated for 2 days. Milliplex Plasma Analysis Mouse plasma was analyzed using the Milliplex-MAP 6-plex mouse cytokine magnetic bead panel kit (Cat #SPR402), to analyze the
cytokines IL-6, eotaxin, fractakine, IL-10, RANTES and TNFa. The assay was performed according to the manufacturer’s guidelines. All reagents were brought to room temperature (RT) before use in the assay. Plasma samples were thawed at 4C and diluted 1:2 with
assay buffer. Antibody-immobilized beads were sonicated for 30 s and vortexed for 1 minute. 60ml from each antibody-bead vial was
added to the mixing bottle provided and the final volume made up to 3ml with assay buffer. Wash buffer (WB) was prepared by mixing
60ml 10X WB with 540ml deionized water. Serum matrix solution was prepared by adding 2ml assay buffer to the lyophilized serum
matrix. Subsequently, the mouse cytokine standard cocktail (Cat #MXM8070) was reconstituted with 0.25ml deionized water and a
1:5 serial dilution was made. WB (200ml) was added to each well of the 96-well plate before it was sealed and agitated for 10 minutes at room temperature. The
WB was removed and 25ml of each standard or control was added to the appropriate wells and 25ml of assay buffer to the sample
wells. Next, 25ml of serum matrix was added to the background, standards and control wells, and 25ml of diluted plasma sample was
added into the sample wells. Following this, the mixing bottle containing the antibody-bead mixture was vortexed and 25ml was
added to each well. The plate was sealed, wrapped in foil and incubated at 2-8C on a plate shaker for 16-18 hours. After incubation, the well contents were removed (using a handheld magnet) and the wells washed twice with 200ml WB. Subse-
quently, 25ml of detection antibodies were added to each well, the plate was then sealed, covered with foil and incubated for 1h at
room temperature, with agitation. Next, 25ml Streptavidin-Phycoerythrin was added to each well and incubated for 30 minutes as
before. Finally, with a hand-held magnet, well contents were removed and the plate was washed twice with 200ml WB. 150ml of sheath
fluid (BioRad Bio-Plex Sheath Fluid, Cat #171-000055) was added to each well for 5 minutes with agitation. The plate was assessed
using a Bio-Rad Bioplex-200 System with Bio-Plex Manager 4.1 software. e6
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS Proliferation and Survival Assays During this time, primary hippocampal cells were
treated with vehicle or acyl-ghrelin for a further 2 days. At this point the conditioned media was, once again, applied to the NSPCs
and incubated for a further 2 days. Following exposure to conditioned media for a total of 4 days, the NSPCs cells were fixed with 4%
PFA. The survival assay was performed in triplicate and assessed via the EdU Click-iT assay, as described above. Analysis was per-
formed using ImageJ software, with the number of EdU+ cells expressed as a proportion of DAPI+ cells. Behavioral Testing All behaviors were recorded and analyzed using tracking software. Novel arm exploration was recorded when
all four feet of each mouse entered the novel arm. The apparatus was cleaned with 80% ethanol between each trial and each animal. BaseScope ISH on Human Brain Tissue Sections were thawed at RT for five minutes, fixed with 10% NBF (Sigma Aldrich) for 1 h, then washed twice with PBS before dehy-
dration in increasing concentrations of ethanol. Sections were baked at 60C for 30min to improve tissue adhesion to the glass sur-
face. BaseScope assay (Advanced Cell Diagnostics) was performed according to supplier guidelines. Pre-treatment conditions were
optimized as follows; Hydrogen peroxide for 10 min at RT; target retrieval reagent for 15 min at 100C; dehydration in ethanol 100%
(Sigma Aldrich) for 3min. Sections were dried completely for 20 min at 60C, then protease IV was added to the dried slides for 30 min
at 40C. Custom BaseScope probes targeting human GHS-R1a were applied for 2 h at 40C as follows; for every case, sections were
incubated with negative (DABP) and positive (PPIB) probes as well as with GHS-R1a probes (Cat.no. 709121). After incubation, sec-
tions were stored overnight in freshly prepared 5x Saline Sodium Citrate (SSC) buffer pH 7.0 (from the 25x: 3M NaCl and 0.3M Sodium
Citrate dehydrate in ddH20). The following day, sections were incubated with the remaining reagents: AMP0 (30 min at 40C), AMP1
(15 min at 40C), AMP2 (30 min at 40C), AMP3 (30 min at 40C), AMP4 (15 min at 40C), AMP5 (30 min at RT) and AMP6 (15 min at
RT). Slides were rinsed twice with wash buffer between each incubation. A mixture of Fast Red A + B was prepared (1:60) and sec-
tions were incubated with the staining solution for 10 min at RT. Gill’s hematoxylin (Vector labs) counterstain solution (1:1 in ddH20)
was applied, then quickly rinsed and sections were left to dry for 30 min at 60C. Dried sections were mounted using VectaMount
mounting media (Vector labs) and imaged the following day. g
(
)
g
g
y
Tissue sections were digitised by whole slide-scanning using a Zeiss Axio Scan.Z1 at high resolution (0,11 to 0,15um/px), low to
none compression (15% to 0%) and 3 40 magnification. When necessary, the whole DG was reconstructed using Adobe Photoshop
(Adobe System Incorporated). Each file was then loaded into the open-source software QuPath64, the DG was manually drawn in
each file and the area measured by the software; subsequently the stain was manually counted using the ‘‘counter’’ tool - each
red puncta corresponding to a single mRNA molecule. BaseScope ISH on Human Brain Tissue The number of puncta and area were recorded separately, and the density
(number of dots per area) in the three GHS-R1a-stained sections was averaged and divided by the density of the positive control
stain – in order to account for any difference in tissue quality and RNA preservation. Finally, data were exported to GraphPad Prism
(GraphPad Software Inc.) for statistical analysis using one-way ANOVA. A ti l A ti l ll
OPEN ACCESS Article Whole blood was collected into Vacutainer EDTA-plasma tubes (Cat No: 6450) up to a volume of 7ml per tube and inverted gently
to prevent coagulation. Whole blood was transferred immediately to 3 cold centrifuge tubes; the first tube was used for quantification
of ghrelin and contained AEBSF (2mg/ml) to prevent proteinase activity. The second tube was used for analysis of insulin, leptin, PYY,
IL-6, GLP-1 (active) and TNFa. These 2 separate tubes were centrifuged at 2000xg for 15 minutes at 4C, resulting in 3 layers (top
plasma layer, mid white layer of leukocytes and a bottom layer of red blood cells). The top layer of plasma was carefully transferred
into sterile tubes (200ml aliquots), labeled with the date and identification number, and stored at 80C. The third tube retained
platelet-rich plasma for the analysis of IGF-1 and GH, this fraction of blood was spun at 1000xg for 15 minutes at 4C, aliquoted
and stored as stated above. Blood samples for assessing acyl-ghrelin were treated with 4-(2-Aminoethyl)-benzenesulfonyl fluoride (A8456, Sigma Aldrich) to
prevent de-acylation and analyzed using a multiplex assay (Milliplex MAP Kit - Human Metabolic Hormone Magnetic Bead Panel
HMHEMAG-34K, Millipore), with additional beads to quantify leptin, insulin, IL-6, TNFa, PYY and GLP-1 active. Total ghrelin was
analyzed by ELISA (EZGRT-89K Millipore). For IGF-1 and GH, platelet-rich plasma samples were analyzed using Human IGF-1
DuoSet ELISA (cat. No. DY291, R&D Systems), and Human GH DuoSet ELISA (Cat. No. DY1067, R&D Systems), using half-volume
Nunclon Microwell 96-well plates. Area under the curve (AUC) was calculated for each analyte, however, plasma samples that were
below the standard range for GH and IGF-1 were excluded. Outliers were identified using the ROUT test (Q = 1%) and removed prior
to analysis using a Kruskall-Wallis test with Dunn’s post hoc multiple comparison. Immunohistochemistry of Human Brain Tissue Fixed-frozen sections (controls n = 5, PD n = 6, PDD n = 6) stored at 80C were quickly thawed at RT and fixed for 1 h in 10% NBF
(Sigma-Aldrich, UK) to improve adherence to the glass slide. Sections were incubated for 8 min at RT with 1% H2O2 (Sigma-Aldrich) in
PBST, before incubation for 20 min at RT with 10% normal goat serum (Sigma-Aldrich). This was followed by overnight incubation at
4C with rabbit anti-GOAT antibody (1:500; Phoenix Pharmaceuticals, Germany). The following day, sections were incubated for 1 h
at RT with biotinylated anti-rabbit (1:500; Vector labs, UK) and subsequently for 1 h at RT with pre-mixed avidin-biotin complex (Vec-
tor labs, UK). ImmPactDAB (Vector labs, UK) was prepared according to manufacturer’s instructions and applied to the sections for
40 s before being washed away with tap water. Nuclei were counterstained for 60 s with Gill’s Haematoxylin 1:1 (Vector labs, UK)
before the sections were dehydrated and coverslipped using Entellan mounting media (Sigma-Aldrich, UK). Images were acquired
by whole slide scanning (as described above). Immunoreactive cells were manually counted within the GCL and the area measured
using QuPath software. All analysis was performed in a blinded manner. Human Plasma Collection All procedures involving human participants were performed at the Clinical Aging Research Unit, Newcastle University, with appro-
priate ethical approvals. 48 adults aged 60-85 were recruited; healthy controls (HC) (n = 20), PD (n = 20) and PDD according to level 1
Movement Disorder Task Force criteria for the diagnosis of PDD63 (n = 8). Montreal Cognitive Assessment (MoCA) was R 26/30 for
HC and PD, and % 25/30 for PDD. Participants with unexplained weight loss, obesity, BMI < 18 or > 30, diabetes, gastrointestinal
disease, smoking, deep brain stimulation or non-selective anticholinergic medication were excluded. Participants were tested fasted
and off PD medication. Blood was drawn in the fasted state (0) and at 5, 15, 30, 60, 120 and 180 minutes following a standard
breakfast. Cell Reports Medicine 1, 100120, October 20, 2020
e7 ll
OPEN ACCESS Free Fatty Acid Analysis using Mass Spectrometry y
y
g
p
y
Free fatty acids were extracted from control, PD and PDD human plasma collected at baseline following an overnight fast, using a
method as described by Zhang et al.65. Briefly 100ml of plasma was added to 250ml of a solvent mixture (1M acetic acid:2-propanol:-
hexane (2:20:30)) containing 2.5mg internal standard (nonanoic acid), this was mixed vigorously by vortex followed by the addition of
250ml hexane (ratio of 2.5:1 (solvent mixture:sample)). The samples were mixed vigorously by vortex and centrifuged for 10 minutes at
1100 x g, 4C. The upper organic layer was removed into clean glass vials. A second extraction step was performed by the addition of
250ml hexane (ratio of 2.5:1, solvent mixture:sample). The organic layers were pooled and dried under N2 flow or under vacuum, re-
suspended in Chloroform/Methanol (2:1, v/v) and stored at 70C. Samples were analyzed by direct infusion electrospray ionisation
mass spectrometry (ESI/MS) using the Thermo Scientific LTQ Orbitrap XL (in negative mode). Data was analyzed using Xcalibur
software. Cell Reports Medicine 1, 100120, October 20, 2020
e9 Western Blot Assay of Human Brain Tissue Frozen post-mortem hippocampal tissues were processed for western blot analysis. A total of 18 subjects were included in the
western blot analysis. Frozen hippocampal brain tissue (250mg) was prepared from controls (n = 6), PD (n = 6) and PDD (n = 6). All cases had a post-mortem interval of < 29h. Briefly, protein was extracted using the QIAGEN AllPrep DNA/RNA/Protein mini kit
according to manufacturer’s instructions (QIAGEN, 80204). Protein was quantified using the Pierce BCA Protein Assay Kit according e8
Cell Reports Medicine 1, 100120, October 20, 2020 Article ll
OPEN ACCESS to instructions (ThermoFisher, 23227) and the concentration of each sample standardized. Protein samples were combined with 4X
sample loading buffer (BioRad, 1610747) and made up to a final volume of 25 mL with water, boiled at 100C for 5 mins and loaded
onto 10% acrylamide gels and separated by SDS-PAGE. The separated proteins were transferred from the gel to the PVDF mem-
brane (BioRad 1620177). The blots were subsequently incubated overnight at 4C in TBST with 5% BSA with anti-GOAT (1:1000;
Phoenix Peptide) and anti-GAPDH (1:5000, Sigma) antibodies. Blots were visualized using the chemiluminescence method (ECL
Select, RPN2235; GE Healthcare) and levels were quantified using ImageLab Software v4.1 (ChemiDoc XRS, BioRad) and normal-
ized to GAPDH. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were carried out using GraphPad Prizm 6.0 for Mac. Data distribution were assessed using the Schapiro-Wilks
normality test. For normally-distributed data, comparison between two groups was assessed by two-tailed unpaired Student’s t test. For multiple groups with one variable factor, a one-way ANOVA was used, and where there were two variable factors a two-way
ANOVA was used. Appropriate post hoc tests were used as described in the Figure legends. Data displaying non-Gaussian distri-
bution were analyzed by non-parametric tests, as described in the text. Spearman correlation (two-tailed) and linear regression anal-
ysis were used to determine the goodness-of-fit between plasma AG:UAG (AUC) and cognition (MoCA). Data are presented as mean
± sem. *, p < 0.05; **, p < 0.01; ***, p < 0.001 were considered significant.
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Optimization of DNA Recovery and Amplification from Non-Carbonized Archaeobotanical Remains
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PloS one
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Optimization of DNA Recovery and Amplification from
Non-Carbonized Archaeobotanical Remains Nathan Wales1,2*, Kenneth Andersen , Enrico Cappellini , Marı
2
2
´a C. A´ vila-Arcos , M. Thomas P. Gilbert
2
2,3
1 Department of Anthropology, University of Connecticut, Storrs, Connecticut, United States of America, 2 Centre for GeoGenetics, University of Copenhagen,
Copenhagen, Denmark, 3 Department of Environment and Agriculture, Curtin University, Perth, Western Australia, Australia Abstract The authors confirm that his position does not alter adherence to the PLOS ONE policies on sharing data and materials. * E-mail: nathan.wales@snm.ku.dk of which originate from associated sediments. These darkly-
pigmented compounds are often inadvertently extracted together
with DNA and inhibit many DNA polymerases which are required
for genetic analyses [14]. Even when DNA eluates are visually
transparent, inhibitors may still be present, leading to PCR
failures. Abstract Ancient DNA (aDNA) recovered from archaeobotanical remains can provide key insights into many prominent
archaeological research questions, including processes of domestication, past subsistence strategies, and human
interactions with the environment. However, it is often difficult to isolate aDNA from ancient plant materials, and
furthermore, such DNA extracts frequently contain inhibitory substances that preclude successful PCR amplification. In the
age of high-throughput sequencing, this problem is even more significant because each additional endogenous aDNA
molecule improves analytical resolution. Therefore, in this paper, we compare a variety of DNA extraction techniques on
primarily desiccated archaeobotanical remains and identify which method consistently yields the greatest amount of
purified DNA. In addition, we test five DNA polymerases to determine how well they replicate DNA extracted from non-
charred ancient plant remains. Based upon the criteria of resistance to enzymatic inhibition, behavior in quantitative real-
time PCR, replication fidelity, and compatibility with aDNA damage, we conclude these polymerases have nuanced
properties, requiring researchers to make educated decisions as to which one to use for a given task. The experimental
findings should prove useful to the aDNA and archaeological communities by guiding future research methodologies and
ensuring precious archaeobotanical remains are studied in optimal ways, and may thereby yield important new
perspectives on the interactions between humans and past plant communities. ersen K, Cappellini E, A´vila-Arcos MC, Gilbert MTP (2014) Optimization of DNA Recovery and Amplification from Non-Carbonized
ns. PLoS ONE 9(1): e86827. doi:10.1371/journal.pone.0086827 Editor: Carles Lalueza-Fox, Institut de Biologia Evolutiva - Universitat Pompeu Fabra, Spain Received October 7, 2013; Accepted December 14, 2013; Published January 27, 2014 Copyright: 2014 Wales et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding is from: the Danish Council for Independent Research grant 10-081390 (NW, EC, and MTPG), the Danish National Research Foundation
(DNRF94) (MCAA and KA), and a dissertation fellowship from the American-Scandinavian Foundation (NW). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: M. Thomas P. Gilbert (Tom Gilbert) is an Editorial Board member for PLOS ONE. However, that in no way should influence the reception of
the manuscript. January 2014 | Volume 9 | Issue 1 | e86827 Goals for Polymerases in aDNA Amplification p
All extractions and PCR setups were performed in a dedicated
clean laboratory at the University of Copenhagen, which conforms
to the highest standards for the field [32]. Methodological
experiments on plant aDNA are fundamentally complicated by
limited numbers of suitable specimens and potentially variable
DNA preservation among samples, however extractions were
designed to minimize variability within a collection of samples. Over three rounds of experiments, sets of archaeobotanical
remains were extracted using three to five different methods,
and tested for DNA yield and purity. We refer to the methods
according to the leading author of the first publication to describe
the technique or the commercial name, as listed in Table 1. Appendix S1 provides detailed protocols for all methods, including
any modifications from the authors’ or manufacturers’ specifica-
tions. Ideally all traces of inhibitory substances would be removed in
the course of DNA extraction; however, in some instances these
substances remain, often leaving DNA eluates pigmented [20]. Such recalcitrant samples presumably contain humic acids and
DNA strands of the same molecular weight, and these molecules
consequently coprecipitate in purifications due to their shared
anionic properties [21]. Repeated purifications using silica and
other methods have been investigated [22–24], but since every
additional purification step can reduce DNA yield, and because
PCR inhibitors may not manifest themselves as obvious pigmen-
tation, it is advantageous to use polymerases that tolerate residual
inhibitors. Real-time quantitative PCR (qPCR) experiments have been
designed to study DNA from archaeobotanical remains [25], but
there has been little research into the compatibility of different
polymerases and PCR additives in qPCR. Exploratory experi-
mentation (N. Wales, unpublished data) suggested that some
polymerases do not exhibit normal amplification curves when
samples are pigmented or when certain PCR additives are
included in the reaction. As departures from ideal amplification
curves may lead to inaccurate DNA quantification, it is important
to know which polymerases yield consistent qPCR results under a
broad range of conditions. Archaeobotanical remains from a variety of contexts were
extracted, listed in Table 2. When deemed sufficiently intact, seeds
were cleaned in 0.5% bleach (NaClO) and rinsed in molecular
grade water before being extracted; seeds with small cracks or
other imperfections, indicated in Table 2, were instead wiped with
a towel. The cleaning of other types of archaeobotanical remains,
such as maize cobs and grape branches, was conducted by
removing exterior surfaces with sterile tools. Macrobotanical aDNA Extraction and Amplification Macrobotanical aDNA Extraction and Amplification Goals for aDNA Extractions Goals for aDNA Extractions the template molecule [28,29]. Depending on the research goals,
either of the available options may be preferable. For example, if a
polymerase does not copy damaged DNA molecules, bioinfor-
matic analyses are simplified as it can be assumed that damage is
not a factor in generating sequence variation. On the other hand,
if nearly all molecules are damaged, the polymerase may fail to
amplify anything, thus providing no data at all. Additionally, by
using a polymerase which pairs uracil with adenine, one may
argue for the authenticity of aDNA based upon damage patterns
[30,31]. It is therefore important to be fully aware of how a given
polymerase handles damage. The fundamental aim of DNA extractions of archaeobotanical
remains is to isolate as much endogenous DNA from a sample as
possible. Ancient samples characteristically have few copies of
endogenous DNA, and these molecules are usually fragmented
into segments less than a few hundred base pairs (bp) in length
[16]. Optimizing
aDNA recovery
has become even more
important in the era of HTS [17]. For conventional PCR-based
studies, it is only necessary for the locus of interest to be amplified,
and amplification can theoretically initiate from a single template
molecule. HTS, on the other hand, require a much larger
‘‘library’’ of DNA molecules (that is, DNA molecules from a
sample with special nucleotide adapters attached to each end). HTS platforms require libraries to be amplified to a specified
starting concentration, and if DNA extract concentrations are low,
more amplification cycles are required, leading to PCR drift and
clonality [18,19]. Materials and Methods The authors thank the following researchers for permission for
destructive analysis of archaeobotanical remains: Boris Gasparyan,
Institute of Archaeology and Ethnology, National Academy of
Sciences, Yerevan, Armenia; Giovanna Bosi and Anna Maria
Mercuri, Museo Di Paleobiologia e dell’Orto Botanico, Universita`
di Modena e Reggio Emilia, Modena, Italy; Girolamo Fiorentino,
Dipartimento di Beni Culturali, University of Salento, Lecce, Italy;
Mike Jacobs, Arizona State Museum; and Jose´ Luis Punzo-Dı´az,
Instituto Nacional de Antropologı´a e Historia, Centro INAH
Michoaca´n, Mexico. While it is important to extract as much DNA from an ancient
sample as possible, the DNA must also be relatively pure: clear of
other cellular components like proteins and lipids that might
otherwise hinder downstream analyses. For archaeobotanical
remains in particular, it is vital to remove substances which
impair enzymatic reactions, including humic acids and polyphe-
nols. January 2014 | Volume 9 | Issue 1 | e86827 Introduction Ancient DNA (aDNA) studies have become an integral part of
Quaternary research, providing invaluable anthropological and
biological insights, on issues as diverse as human evolution [1],
modern human migrations [2–4], plant and animal domestication
[5,6], and paleoecology [7]. Research on plant aDNA from
archaeological contexts is of particular interest because archae-
obotanical remains can provide important data on subsistence
patterns,
human
behavioral
variability,
domestication,
and
broader environmental issues [8–10]. Despite this rich potential,
relatively few researchers have studied aDNA from plant materials
[9,11]; the scarcity of this line of research can be partially
attributed to the many methodological challenges posed by ancient
plant materials. In their systematic review of aDNA techniques, Rohland and
Hofreiter [15] explore numerous protocols, the use of different
binding salts, incubation modifications, PCR additives, and DNA
polymerases. The results of the study have been influential in the
aDNA community and have been adopted by a number of
researchers, including for the prominent Neanderthal genome
project [1]. Nevertheless, Rohland and Hofreiter’s [15] investiga-
tion focused only upon aDNA from bones, and therefore the
findings may not be applicable to other aDNA source materials,
including ancient plant remains. In this article, we expand upon
Rohland and Hofreiter’s [15] work by examining the effectiveness
of various extraction techniques on non-charred archaeobotanical
remains and the relative capabilities of different polymerases to
amplify aDNA. Given the growing importance of high-throughput
sequencing (HTS) technologies in plant aDNA research [9], issues
and goals related to HTS are given special attention. In addition to the issues of contamination and biomolecular
degradation faced by all aDNA research [12], ancient plant
materials frequently contain compounds that impede DNA
extraction and enzymatic reactions, including the polymerase
chain reaction (PCR). In modern plant materials, polysaccharides
and polyphenols, such as tannins, pose significant problems for the
extraction of nucleic acids [13]; these compounds may still thwart
geneticists millennia after the death of a plant. In addition,
archaeological plant materials are often rich in humic acids, some January 2014 | Volume 9 | Issue 1 | e86827 1 PLOS ONE | www.plosone.org Goals for Polymerases in aDNA Amplification Most archaeobota-
nical remains were desiccated, although one set was waterlogged. No charred archaeobotanical remains were tested in these
experiments because burned remains often contain little or no
endogenous DNA that can be amplified by PCR [33–35]. This is
an important consideration because macrobotanical remains are
most frequently preserved at archaeological sites through charring
or carbonization [36]. Desiccation and waterlogging are compar-
atively less common processes by which plant remains become
preserved; nonetheless, desiccated and waterlogged macrobotani-
cals have been recovered from archaeological sites around the
world and are much more likely to contain endogenous aDNA
since they have not been exposed to high temperatures. Thus,
these experiments are most pertinent to non-charred remains,
although some findings may prove applicable to charred remains
in subsequent analyses. The fidelity of polymerases is an important concern, especially
when aDNA libraries are amplified for HTS. Ancient samples
frequently yield low levels of coverage for all loci, making it
challenging to identify which genetic motif is real and which is the
result of polymerase copy errors. The degraded and damaged nature of aDNA has a profound
effect on the performance of polymerases. In particular, research
has identified cytosine deamination, a hydrolysis reaction in which
cytosine is converted to uracil, as the main source of the problem
[26–28]. The presence of uracil in aDNA molecules has adverse
effects in PCR because DNA polymerases cannot add the
appropriate complementary nucleotide to the opposite DNA
strand. Instead, polymerases either 1) stop replicating the DNA
molecule, or 2) insert adenine which is complementary to uracil in
RNA. The latter scenario leads to an apparent C-to-T transition in January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 2 Macrobotanical aDNA Extraction and Amplification Table 1. Extraction techniques compared in this study. Experiment phase
Name
Method synopsis and relevant information
Reference
Phase 1
Epicentre
QuickExtract Plant DNA Extraction Solution. Designed to extract DNA from
modern plant remains in 10 minutes. Epicentre, Madison, WI
Finnzymes
Phire Plant Direct PCR kit. Sample incubated for 3 minutes in buffer and
immediately amplified. Thermo Fisher Scientific,
Waltham, MA
Gilbert
Digestion in SDS, DTT, and Proteinase K, followed by phenol and chloroform
extraction. Previously used to extract DNA from ancient grapes [58]. Gilbert et al. [37]
Japelaghi
Digestion in PVP, CTAB, and 2-mercaptoethanol followed by chloroform-
isoamylalcohol extraction. Goals for Polymerases in aDNA Amplification Method designed for modern plant remains rich in
tannins. Japelaghi et al. [13]
MO BIO
PowerLyzer PowerSoil DNA Isolation kit. Used to recover aDNA from humic-rich
soils [43,59]. MO BIO Laboratories, Carlsbad,
CA
Phase 21
Gilbert
See phase 1. See phase 1. Palmer
Digestion in CTAB, followed by chloroform-isoamyl alcohol extraction, and
purification in Qiagen MinElute column. Modified from Palmer et al. [39]
Rohland
Digestion in SDS, DTT, and Proteinase K, followed by DNA binding to silica
pellet. Silica extraction previously found to be optimal for extracting aDNA
from bones. Modified from Rohland and
Hofreiter [15]
Phase 3
Andersen
Digestion in 2-mercaptoethanol, DTT, and Proteinase K, followed by MOBIO
inhibitor removal, phenol and chloroform extraction, and Millipore filter
purification. Designed to recover aDNA from sediment. Experimental method
developed by Kenneth
Andersen
Gilbert
See phase 1. See phase 1. Palmer
See phase 2, but with purification in Millipore filter and Qiagen DNeasy silica
column. Exact method used to recover aDNA from ancient barley
remains [39]. Palmer et al. [39]
1Extraction methods in phase 2 were conducted in three ways: according to the specified directions, with MO BIO C2 and C3 solutions added before extraction, and with
MO BIO C2 and C3 solutions used after extraction. See the text and Appendix S1 for further details. doi:10.1371/journal.pone.0086827.t001 1Extraction methods in phase 2 were conducted in three ways: according to the specified directions, with MO BIO C2 and C3 solutions added before extraction, and with
MO BIO C2 and C3 solutions used after extraction. See the text and Appendix S1 for further details. doi:10.1371/journal.pone.0086827.t001 In extraction phase 1, seven sets of Vitis vinifera pips were
extracted. Grapes were tested because they contain a number of
PCR inhibitors and provide a challenge even for genetic studies of
modern material [13]. In extraction phase 1, a single seed was
extracted with a given method. Recognizing that DNA within
samples may be differentially preserved, phases 2 and 3 were
conducted on a homogenized collection of seeds from a given
context, thereby standardizing the amount of aDNA, contaminant
DNA, and inhibitory substances. In addition, a wider range of
species and contexts were tested in later extraction phases: four sets
of archaeobotanical remains were tested in phase 2 and eight sets
in phase 3. Goals for Polymerases in aDNA Amplification addition to conducting the extractions according to the specified
directions, the methods were modified with the addition of MO
BIO ‘C2’ and ‘C3’ solutions (MO BIO Laboratories, Carlsbad,
CA), reagents designed to precipitate humic acids and increase
sample purity. This modification was conducted either after
overnight incubation in digestion buffer or directly after DNA
extraction. The same criteria were used to compare the methods
as in phase 1, with the addition of sequencing rbcL products to
determine if endogenous DNA was recovered. g
For phase 3, the two top performing methods were further
compared, along with an experimental technique developed by
one of the authors of this paper (KA). This method, referred to as
the Andersen method, is part of an ongoing project to extract
aDNA from sediments, and therefore may not be fully optimized. Nonetheless, preliminary findings suggest the Andersen method
readily handles humic-rich sediments, and it was hypothesized the
technique may also effectively isolate DNA from archaeobotanical
remains. In addition to the above previously used testing criteria,
the three methods were compared using a qPCR assay for the rbcL
generic marker to more precisely determine the amount of plant
DNA recovered (for details, see ‘‘qPCR assay for quantifying DNA
in extraction phase 30 in Appendix S1). This approach was
deemed necessary because pigmentation in some extracts could
lead to erroneous DNA concentration readings in the Qubit
Fluorometer. In phase 1, we compared five extraction techniques which have
been designed for either ancient materials or modern plant
remains. Samples were tested in duplicate for the Gilbert et al. [37], Japelaghi et al. [13], and MO BIO methods; however, due to
a limited number of seeds from identical contexts, it was not
possible to perform duplicate extractions for the Epicentre and
Finnzymes techniques. Extraction methods were compared on the
basis of three criteria: DNA concentration measured on a Qubit
1.0 Fluorometer (Invitrogen, Carlsbad, CA), sample purity
measured on a NanoDrop 1000 spectrophotomer (Thermo
Scientific, Waltham, MA), and amplification success for the
ribulose-bisphosphate carboxylase (rbcL) gene, a universal plant
marker [38]. PCR conditions for the rbcL locus are listed in
Appendix S1. Comparison of DNA Polymerases The most promising method was advanced to phase 2, where it
was compared with Palmer et al.’s [39] extraction method (with
minor modifications as listed in Table 1) for ancient plants and a
silica pellet extraction, the top performing technique in Rohland
and Hofreiter’s [15] study on isolating aDNA from bones. In Enzymatic
inhibition
of
five
polymerases
was
tested
by
amplifying exogenous tiger (Panthera tigris) DNA ‘‘spiked’’ into
pigmented plant eluates. As indicated in Table 2, heavily
pigmented DNA extracts from two ancient plant samples were January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 3 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 Macrobotanical aDNA Extraction and Amplification y
Sample information
Archaeological context
Extraction phase
PCR tests
Name
Species
Tissue1
Site
Geographic location
Repository2
Provenience and age
1
2
3
ARE-A
Vitis vinifera
Pips
Areni-1
Areni, Armenia
IAE
Trench 1, square P30/31, locality 2,
spit 6. Medieval context. X
ARG
Vitis vinifera
Pips
Fossato
Argenta (FE), Italy
UMeRE
SU3 2.2. 1275–1325 A.D. X
CPR-A4
Vitis vinifera
Pips
Corso Porto Reno –
Via Vaspergolo
Ferrara, Italy
UMeRE
SU 1703. Medieval context. X
LUG
Vitis vinifera
Pips
Piazza Baracca
Lugo (RA), Italy
UMeRE
SU 144. 15th–16th c. A.D. X
Polymerase fidelity
PAR-A
Vitis vinifera
Pips
Piazza Municipale
Parma, Italy
UMeRE
SU 320. 4th–2nd c. B.C. X
SAM4
Vitis vinifera
Pips
Pozzo 1 Domagnano
San Marino, Republic of
San Marino
UMeRE
SU 565. Late Roman–Gothic
context. X
VAD-A
Vitis vinifera
Pips
Vasca Ducale Piazza
Municipale
Ferrara, Italy
UMeRE
SU 1050. 2nd half 15th c. A.D. X
Polymerase fidelity
CAS
Cornus mas
Seeds
Cassa di Risparmio
Modena, Italy
UMeRE
SU 31. Roman context. X
SAF
Vitis vinifera
Pips
Piazzale San
Francesco
Modena, Italy
UMeRE
SU 16. 10th–11th c. A.D. X
ARE-B5
Gossypium sp. Seeds
Areni-1
Areni, Armenia
IAE
Trench 1, square K35, spit 4. Medieval context. X
X
Inhibition
VAD-B
Olea europaea
Pits
Vasca Ducale Piazza
Municipale
Ferrara, Italy
UMeRE
SU 1050. 2nd half 15th c. A.D. X
X
Polymerase fidelity
ARE-C5
Vitis vinifera
Branch
Areni-1
Areni, Armenia
IAE
Trench 1, square N16, locality 29. Medieval context. X
CDM5
Zea mays
Cob
Cueva del Maguey 1
Pueblo Nuevo, Durango,
Mexico
INAH
Specimen ID: 10189. 1410625 14CYBP
X
CPR-B
Cornus mas
Seeds
Corso Porta Remo-Via
Vespergolo
Ferrara, Italy
UMeRE
SU 2597. Medieval context. Macrobotanical aDNA Extraction and Amplification used as inhibiting substances: medieval cotton (Gossypium sp.) seeds
from the Areni-1 site in Areni, Armenia, and medieval grape (Vitis
vinifera) pips from the Via San Pietro site in Modena, Italy. Varying
amounts of inhibiting solutions were added to PCR reactions, with
pigmented extracts representing up to 40% of the reaction volume. Polymerases were selected based upon either their ubiquity in
aDNA research, advertised fidelity, or purported ability to
overcome inhibition, as summarized in Table 3. PCR details for
each polymerase are located in Table S1. As bovine serum
albumin (BSA) has been shown to prevent inhibition and increase
the likelihood of amplification success in ancient samples [15,40],
reactions were conducted with and without 0.8 mg/mL BSA
additive. Therefore, the primary indication of success was taken to be the
rate of successful amplification of genetic plant markers. Based on
this criterion, the Gilbert method was the top performer, with
successful amplification of the rbcL marker in 10 out of 14
specimens, as listed in Table 4. Japelaghi’s method scored the
second most successes: 7 of 14. Therefore, the primary indication of success was taken to be the
rate of successful amplification of genetic plant markers. Based on
this criterion, the Gilbert method was the top performer, with
successful amplification of the rbcL marker in 10 out of 14
specimens, as listed in Table 4. Japelaghi’s method scored the
second most successes: 7 of 14. Amplification successes were compared using the generalized
estimating equations function in PASW Statistics 18.0 [45]. This
approach accommodates the presence of replicates for a given
method and controls for success rates within each set of samples,
even with limited numbers of samples. The Wald test found the
best performing technique, the Gilbert method, to have signifi-
cantly higher odds of amplifying the rbcL marker than the
Finnzymes and MO-BIO techniques (p = 0.001 and 0.018,
respectfully). The difference between the Gilbert method and the
other two methods was not statistically significant (Epicentre,
p = 0.061; Japelaghi, p = 0.273); however, qualitatively, it yielded
stronger, more distinct PCR bands than the others. AmpliTaq Gold, Omni Klentaq, and PfuTurbo Cx Hotstart
were further tested for potential use in qPCR assays by amplifying
spiked DNA in varying concentrations of inhibitors. Experimen-
tation suggested that BSA occasionally interfered with the
detection of fluorescence with AmpliTaq Gold; therefore, for
each polymerase, reactions were conducted with and without
0.8 mg/mL BSA. Macrobotanical aDNA Extraction and Amplification The effects of BSA and inhibition were observed
through changes in cycle threshold (Ct) and amplification curves. Experiment conditions are listed in Appendix S1 in the section
‘‘qPCR inhibition testing.’’ None of the methods yielded amplifiable DNA from ARE-A,
but this could be due to degradation of the sample (i.e. the
endogenous DNA was shorter than the 138 bp rbcL marker). Therefore, DNA concentrations and purity readings for this
sample are still considered germane. The mean amount of DNA
for the Gilbert method was 304.5 ng, nearly triple the second
highest value, 102.2 ng by Epicentre. After omitting the outliers
shown in the left side of Figure 1, values were compared using a
univariate generalized linear model (mixed model ANOVA) to
control for differences between specimens. The model determined
the
method
[F(4,
24) = 6.771,
p = 0.001],
specimen
[F(6,
24) = 5.566, p = 0.001], and interaction between method and
specimen [F(18, 24) = 9.607, p,0.001] to be statistically signifi-
cant. Tukey’s HSD post-hoc test finds the Epicentre and Gilbert
methods yield statistically significant greater amounts of DNA than
other methods (p,0.001), but the difference between the two is
not statistically significant (p = 0.885). Polymerase fidelity and compatibility with aDNA damage were
investigated through ‘‘deep sequencing’’ of an endogenous DNA
marker from ancient plant samples. This approach is commonly
used to characterize biodiversity in environmental samples [41],
including ancient ones [42]. In such studies, a universal genetic
marker for a group of organisms, such as plants or animals, is
amplified and sequenced on a HTS platform to identify all species
present in the sample and their relative proportions [43]. Here, the
aim of deep sequencing is to test thousands of copies of the plant
rbcL marker amplified from a sample to infer how often
polymerases make errors. PCR products from three ancient plant
samples, listed in Table 2, were sequenced on a Roche/454
Genome Sequencer FLX platform (for further information, see
‘‘Deep sequencing of rbcL products’’ in Appendix S1). Reads were
aligned to the expected sequence in Geneious Pro 5.5.7 [44] and
nucleotide
misincorporations, insertions, and
deletions
were
analyzed. The right side of Figure 1 depicts the ratio of light absorbance at
260 and 280 nm, where a ratio of 1.8 is commonly considered to
represent pure DNA [46]. Macrobotanical aDNA Extraction and Amplification None of the five methods consistently
reached a ratio of 1.8, perhaps due to the low amount of aDNA in
specimens, but the Gilbert method was the closest. After omitting
the five outliers, an ANOVA test found statistical differences in the
ratio of 260/280 between methods [F(4, 41) = 10.862, p,0.001],
and Tukey’s HSD post-hoc test found the Gilbert method to have
a statistically higher 260/280 ratio than the Finnzymes (p = 0.014)
and MO BIO (p,0.001), but not the Epicentre (p = 0.116) or
Japelaghi methods (p = 0.867). Comparison of DNA Polymerases X
PAR-B
Vitis vinifera
Pips
Piazza Municipale
Parma, Italy
UMeRE
SU 165. Medieval context. X
SUP4
Vitis vinifera
Waterlogged
pips
Loc. Scorpo
Supersano (LE), Italy
US
Excavated from well. 7th–8th c. A.D. X
THR
Zea mays
Kernels
Turkey House Ruin
Navajo County, Arizona
ASM
Specimen ID: 935. 723623
14CYBP
X
SPC
Vitis vinifera
Pips
Via San Pietro
Modena, Italy
UMeRE
Excavated from composting
feature. Medieval. Inhibition
1Tissues are desiccated except where noted. 2Samples provided by archaeologists and curators, as listed in acknowledgements. ASM: Arizona State Museum; IAE: Institute of Archaeology and Ethnology, National Academy of Sciences, Yerevan, Armenia; INAH: Instituto
Nacional de Antropologı´a e Historia, Centro INAH Michoaca´n, Mexico; UMeRE: Universita` di Modena e Reggio Emilia, Modena, Italy; US: University of Salento, Lecce, Italy. 3SU: Stratigraphic unit. 4Seeds cleaned by wiping with a dry paper towel. All other seeds cleaned by washing in 0.5% bleach. 5Samples cleaned by removing exterior (seed coat or bark) with sterile scalpel. doi:10.1371/journal.pone.0086827.t002 January 2014 | Volume 9 | Issue 1 | e86827 4 Extraction Comparisons Phase 1. The five extraction methods yielded highly variable
DNA concentrations, amplification success rates, and purity levels. The Epicentre and Finnzymes extraction methods frequently
yielded DNA eluates that were darkly pigmented. This is
significant because DNA concentrations, as measured on the
fluorometer, could produce anomalous readings if the pigmented
eluates
prevent
accurate
assessment
of
DNA-binding
dyes. Phase
2. As
the
Gilbert
method
performed
the
most
consistently in phase 1, with the highest rate of successful
amplification, the most DNA, and the purest eluates, it was
promoted to more testing in phase 2. In terms of amplification
success, all methods without C2/C3 solutions yielded PCR bands Table 3. Polymerases tested. Polymerase
Vendor
Notable features
AmpliTaq Gold
Applied Biosystems, Foster City, CA
Commonly used in aDNA research
Omni Klentaq
DNA Polymerase Technology, St. Louis, MO
Engineered to overcome multiple sources of inhibition, including blood and soil
PfuTurbo Cx Hotstart
Agilent Technologies, La Jolla, CA
Purportedly reads through uracil while maintaining high fidelity
Phire Hot Start II
Finnzymes (Thermo Fisher Scientific), Waltham, MA
Designed to overcome inhibition and features rapid processivity
Phusion Hot Start
Finnzymes (Thermo Fisher Scientific), Waltham, MA
Engineered for high fidelity and rapid processivity
doi:10.1371/journal.pone.0086827.t003 Table 3. Polymerases tested. Table 3. Polymerases tested. doi:10.1371/journal.pone.0086827.t003 January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 5 Macrobotanical aDNA Extraction and Amplification Table 4. Amplification success for extractions, phase 1. Generic rbcL plant marker amplified for given method1
Sample
Replicate2
Finnzymes
Epicentre
Gilbert
Japelaghi
MO BIO
ARE-A
A
2
2
2
2
2
B
2
2
2
2
2
ARG
A
2
2
(+)
2
2
B
2
2
+
2
2
CPR-A
A
+
(+)
+
2
+
B
2
2
+
(+)
2
LUG
A
2
+
+
+
+
B
2
+
+
+
+
PAR-A
A
2
2
+
+
2
B
2
(+)
+
2
2
SAM
A
2
2
2
(+)
2
B
2
2
2
2
2
VAD-A
A
(+)
+
(+)
+
(+)
B
2
(+)
+
+
2
Amplification successes
2/14
6/14
10/14
7/14
4/14
1+ indicates a distinct band on 2% agarose gel, (+) indicates a faint band, and 2 indicates no band. Macrobotanical aDNA Extraction and Amplification Macrobotanical aDNA Extraction and Amplification identifies the addition of C2/C3 solutions before (p = 0.010) or
after (p,0.001) an extraction to significantly reduce DNA
recovery. An ANOVA test on the extractions not modified with
C2/C3 solutions was significant for method [F(2, 6) = 10.109,
p = 0.012] and specimen [F(3, 6) = 42.802, p,0.001]. The Gilbert
method was found to recover significantly more DNA than the
Rohland
method
(p = 0.010),
but there was no
significant
difference
between
the
Gilbert
and
the
Palmer
methods
(p = 0.236). leading to the failure of nearly every reaction. The only samples
amplifiable without BSA were THR (successful in all three
methods) and VAD-B (a faint band in Palmer’s method). This
finding may have important implications for the use of BSA in
PCR on aDNA from non-charred archaeobotanical remains, as
discussed below. DNA purities were statistically identical, with mean 260/280
ratios
of
1.527
(sd = 0.188),
1.558
(sd = 0.157),
and
1.524
(sd = 0.245) for the Andersen, Gilbert, and Palmer methods,
respectively. The amount of DNA recovered by the methods was
more variable, as shown in top half of Figure 3. Mean DNA
recovery
was
highest
in
the
Gilbert
method
(1226.9 ng,
sd = 1909.1), followed by the Andersen (651.1 ng, sd = 722.2)
and Palmer (597.6 ng, sd = 968.6) methods. Log transformed
DNA yields were tested in a univariate generalized linear model
controlling for differences in specimens (mixed model ANOVA),
and were found to have significant effects for extraction method
[F(2, 14) = 6.539, p = 0.012] and specimen [F(3, 28) = 13.239,
p,0.001]. Post-hoc testing with Tukey’s HSD test found the
Gilbert method recovered a statistically significantly greater
amount of DNA than the Palmer method (p = 0.007), but not
the Andersen method (p = 0.184). The unmodified Gilbert and Palmer methods have statistically
identical mean 260/280 ratios: 1.465 and 1.515, respectively. The
Rohland method yielded ratios ranging from 1.10 to 4.87, likely
due to low DNA content or residual particles from the silica
extraction. When modified by C2/C3, the 260/280 ratios were
not consistently brought closer to the ideal value of 1.8, as can be
seen in Table S2. In all, there was no compelling evidence that the
C2/C3 additions improved DNA purity, however, they certainly
reduced DNA content. Phase 3. Extraction Comparisons 2Amplifications for Finnzymes and Epicentre were conducted at (A) full strength and (B) 10% dilutions to test for enzymatic inhibition rather than two separate
extractions of different seeds. doi:10.1371/journal.pone.0086827.t004 Generic rbcL plant marker amplified for given method1 1+ indicates a distinct band on 2% agarose gel, (+) indicates a faint band, and 2 indicates no band. 2Amplifications for Finnzymes and Epicentre were conducted at (A) full strength and (B) 10% dilutions to test for enzymatic inhibition rather than two separate
extractions of different seeds. doi:10.1371/journal.pone.0086827.t004 doi:10.1371/journal.pone.0086827.t004 for SAF and VAD-B samples, and the Rohland method also
produced a weak band for the CAS sample, as listed Table S2. Cloning and sequencing of the PCR bands showed that sequences
for the SAF and VAD-B samples were identical to the expected
sequence, or ,2 bp different from the sequence, an error rate
generally consistent with damaged DNA. None of the recovered
sequences of CAS sample from the Rohland method were closer
than 2 bp to the expected sequence and therefore likely represent
contamination. The unmodified Gilbert method yielded more DNA than the
other methods, and the addition of C2/C3 nearly always
decreased DNA yield, as seen in Figure 2. To control for major
differences in DNA recovery between specimens, DNA yield
values were compared after logarithmic transformation. Log
values were tested in a univariate generalized linear model
controlling for differences in specimens, and found to have
significant
effects
of
extraction
method
[F(2,
28) = 3.563,
p = 0.042], C2/C3 additives [F(2, 28) = 14.278, p,0.001], and
specimen [F(3, 28) = 13.239, p,0.001]. Tukey’s HSD post-hoc Figure 1. DNA yield and purity for extractions, phase 1. doi:10.1371/journal.pone.0086827.g001 Figure 1. DNA yield and purity for extractions, phase 1. doi:10.1371/journal.pone.0086827.g001 January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 Macrobotanical aDNA Extraction and Amplification The Gilbert and Palmer techniques were further
tested in the final extraction phase, along with the Andersen
sediment-style extraction. In terms of amplification success, the
methods performed similarly: the Andersen and Palmer methods
amplified six samples, while the Gilbert method amplified the
same six as well as PAR-B. PCR was also tested without BSA, The number of copies of the rbcL gene recovered by each
method varies dramatically between methods and samples. As seen Figure 2. DNA yield from extractions, phase 2. Maximum amount of DNA recovered in each specimen listed by corresponding symbol. doi:10.1371/journal.pone.0086827.g002 Figure 2. DNA yield from extractions, phase 2. Maximum amount of DNA recovered in each specimen listed by corresponding symbol. doi:10.1371/journal.pone.0086827.g002 January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 Macrobotanical aDNA Extraction and Amplification Figure 3. DNA yield and rbcL copies extracted during phase 3. DNA yield (top) calculated using a Qubit 1.0 Fluorometer and rbcL copies
(bottom) determined by qPCR. Values are scaled to the maximum value of each sample, with the highest value listed above the corresponding bar. Missing bars in lower portion of figure indicates that a sample did not amplify in qPCR. doi:10.1371/journal.pone.0086827.g003 Figure 3. DNA yield and rbcL copies extracted during phase 3. DNA yield (top) calculated using a Qubit 1.0 Fluorometer and rbcL copies
(bottom) determined by qPCR. Values are scaled to the maximum value of each sample, with the highest value listed above the corresponding bar. Missing bars in lower portion of figure indicates that a sample did not amplify in qPCR. doi:10.1371/journal.pone.0086827.g003 substances. With BSA, AmpliTaq Gold, Omni Klentaq, and Phire
overcame inhibition in at least one sample with 5% inhibitors. Omni Klentaq particularly exceled when BSA was added,
successfully
amplifying
reactions
containing
10%
inhibiting
solutions. substances. With BSA, AmpliTaq Gold, Omni Klentaq, and Phire
overcame inhibition in at least one sample with 5% inhibitors. Omni Klentaq particularly exceled when BSA was added,
successfully
amplifying
reactions
containing
10%
inhibiting
solutions. substances. With BSA, AmpliTaq Gold, Omni Klentaq, and Phire
overcame inhibition in at least one sample with 5% inhibitors. Omni Klentaq particularly exceled when BSA was added,
successfully
amplifying
reactions
containing
10%
inhibiting
solutions. Macrobotanical aDNA Extraction and Amplification in Figure 3, the number of rbcL copies as determined by qPCR
does not perfectly reflect the amount of DNA measured on the
Qubit fluorometer. This may indicate less pure eluates occasion-
ally yield errant values. It could also be possible the methods differ
in their ability to extract endogenous and exogenous DNA. To
control for the wide large range of values, a logarithmic
transformation was done, using log(x+1) to incorporate zero
values. A mixed model ANOVA found the method [F(2,
14) = 4.707,
p = 0.027]
and
specimen
[F(7,
14) = 5.646,
p = 0.003) to be significant factors in the number of recovered
rbcL copies. Tukey’s HSD post-hoc test determined the Andersen
method recovers significantly more rbcL copies than the Palmer
method (p = 0.043), but there is not statistical difference between
the Andersen and Gilbert methods (p = 0.995). Results provided
by the Gilbert method are also found to differ from those provided
by the Palmer method, but the differences are just beyond the
threshold of statistical significance (p = 0.051). Compatibility with qPCR. The three polymerases tested in
qPCR behaved very differently when amplifying spiked DNA in
the presence of BSA and inhibitors, as can be observed in Figure 4
and Table S3. The addition of BSA had a negative impact on the
amplification curve in AmpliTaq Gold, but not the other
polymerases. Increasing
concentrations
of
inhibitors
further
reduced the slope of the amplification phase of AmpliTaq Gold
reactions, and also affected PfuTurbo Cx Hotstart when inhibitors
reached 2.5%. Conversely, Omni Klentaq was remarkably
resilient to amplification inefficiencies due to inhibition. Fidelity. The PfuTurbo Cx Hotstart polymerase was unable
to amplify plant DNA in the LUG sample; therefore, 14 of the 15
possible
combinations
of
specimens
and
polymerases
were
analyzed. Deep sequencing of the rbcL plant marker showed the
vast majority of recovered sequences were consistent with the
expected endogenous sequence, listed in Table S4. The entire
dataset of sequencing reads is available online in Data S1–S14. All
reads differing from the expected sequence by more than 3 bp
were excluded from analyses, leaving 99.2%–99.9% of the original
data for each case. DNA Polymerases Ability
to
overcome
inhibition. The five polymerases
demonstrated great variability in overcoming inhibition from
substances found in ancient plant materials, as shown in Table 5. Without BSA additives, only Omni Klentaq and Phire Hot Start II
were successful amplifying spiked tiger DNA in the presence of
inhibitors, yielding PCR bands in reactions containing up to 1% of
the ARE-B eluate. The addition of BSA enabled all polymerases to
be functional in reactions containing at least 1% inhibiting Three polymerases yielded a small number of sequences that
could not be aligned to rbcL markers, shown in Table S4. Some of January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 8 Macrobotanical aDNA Extraction and Amplification Table 5. Amplification of spiked DNA in the presence of inhibiting substances. Polymerase1
BSA additive
Inhibiting solution
Amount of inhibitor
in reaction
AmpliTaq Gold
Omni Klentaq
PfuTurbo Cx
Phire
Phusion
No BSA
SPC
0%
+
+
+
+
+
0.1%
2
+
2
+
2
$1%
2
2
2
2
2
ARE-B
0%
+
+
+
+
+
0.1%
2
+
2
+
2
1%
2
(+)
2
(+)
2
$2.5%
2
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1+ indicates a distinct band on 2% agarose gel, (+) indicates a faint band, and 2 indicates no band. doi:10.1371/journal.pone.0086827.t005 1+ indicates a distinct band on 2% agarose gel, (+) indicates a faint band, and 2 indicates no band. doi:10.1371/journal.pone.0086827.t005 [47], listed in Table S5. Sequencing errors and DNA damage
undoubtedly contribute to the overall error rate, but they are
expected to be relatively constant across samples. As seen in
Figure 5, Phusion polymerase had a consistently lower error rate
than the other polymerases. A one-way ANOVA test found
statistical differences in the error rates between polymerases [F(4,
9) = 20.022, p,0.001] and Tukey’s HSD post-hoc test found these were determined to be chimeras of amplicons. Discussion These experiments provide a new perspective on how to extract
and amplify endogenous DNA from non-charred archaeobotani-
cal remains. Now that researchers are incorporating HTS
technologies into the study of aDNA from ancient plant remains
[19,35,48], these findings should prove especially useful, and may
aid future research on critical issues surrounding plant evolution,
domestication, and cultivation. Phusion was also found to have the lowest error rates for
nucleotide insertions and deletions, but several other polymerases
had similar rates, as seen in Table S5. A one-way ANOVA test
found statistically significant differences among the samples in
nucleotide deletion rates [F(4, 9) = 3.976, p = 0.040], but not
insertion rates [F(4, 9) = 2.031, p = 0.173]. Tukey’s HSD post-hoc
test found the deletion rate in Phusion to be statistically different
from that of AmpliTaq Gold (p = 0.025). In order to fully profit from HTS of ancient remains, steps should
be taken to optimize aDNA recovery. For archaeobotanical
remains, these concerns are not trivial, because samples are often
small and suboptimal approaches yield insufficient quantities of
DNA, potentially leading to the destruction of samples for little or no
gain. In the extraction experiments conducted here, the method that
consistently performed the best is that described by Gilbert et al. [37]. While this method was developed by one of the authors, it was
tested impartially, and found to recover more DNA with fewer co-
extracted inhibiting substances than other techniques, even across a
wide range of species and plant tissues. For previously untested
archaeobotanical remains, it logically follows the Gilbert method
provides the greatest chance for successful aDNA recovery. That
being said, in the final round of testing, an extraction method
developed for humic-rich sediments recovered more DNA from a
few specimens, suggesting that it may be necessary to test a couple of
methods for the most precious of samples. Of course, the insights
garnered during this testing are limited to the set of extraction
techniques used in the experiments. However, most methods
commonly employed on ancient plant remains combine elements
of the already tested approaches, so we do not anticipate such
techniques to perform drastically differently. Compatibility with damaged DNA. According to the
manufacturer, Phusion polymerase is incompatible with uracil,
causing DNA replication to stall. DNA Polymerases Notably,
Omni Klentaq had a relatively high percentage of non-aligning
reads. Additionally, Omni Klentaq was observed to occasionally
yield DNA smears on agarose gels, a characteristic consistent with
replication errors. Nucleotide substitution rates were calculated as the number of
incorrect nucleotides divided by the number of correct nucleotides Figure 4. Compatibility of polymerases with qPCR. Inhibitory substances extracted from the SPC sample prevented amplification of spiked
DNA in all reactions not including BSA, except for Omni Klentaq in 0.1% inhibitors (not shown). Unsuccessful amplifications, including PfuTurbo Cx
Hotstart in 5% inhibitors, are not included in figure. doi:10.1371/journal.pone.0086827.g004 Figure 4. Compatibility of polymerases with qPCR. Inhibitory substances extracted from the SPC sample prevented amplification of spiked
DNA in all reactions not including BSA, except for Omni Klentaq in 0.1% inhibitors (not shown). Unsuccessful amplifications, including PfuTurbo Cx
Hotstart in 5% inhibitors, are not included in figure. doi:10.1371/journal.pone.0086827.g004 January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 9 Macrobotanical aDNA Extraction and Amplification Figure 5. Overall substitution error rates on endogenous aDNA. Shorter bars represent fewer nucleotide misincorporations (higher
polymerase fidelity). Sequencing reads that differed from the expected rbcL sequence by .3 nucleotide substitutions were omitted prior to tallying
nucleotide calls and errors. As stated, the PfuTurbo Cx polymerase did not amplify the LUG sample. doi:10.1371/journal.pone.0086827.g005 Figure 5. Overall substitution error rates on endogenous aDNA. Shorter bars represent fewer nucleotide misincorporations (higher
polymerase fidelity). Sequencing reads that differed from the expected rbcL sequence by .3 nucleotide substitutions were omitted prior to tallying
nucleotide calls and errors. As stated, the PfuTurbo Cx polymerase did not amplify the LUG sample. doi:10.1371/journal.pone.0086827.g005 Phusion’s error rate to be significantly lower than the other
polymerases (versus AmpliTaq Gold: p = 0.006, PfuTurbo Cx:
p = 0.015, and Omni Klentaq and Phire: p,0.001). Differences
among the other polymerases were not statistically significant. January 2014 | Volume 9 | Issue 1 | e86827 Discussion Then, in a second
reaction, a high-fidelity polymerase, such as Phusion, is used to
copy DNA with minimal errors, and reach the required number of
DNA copies. Note that other strategies to deal with uracil in
aDNA exist [55], but they are not based on polymerases and are
therefore outside the realm of this article. [48,50]. CTAB is used to remove polysaccharides in modern
plants [51], but contrary to conventional wisdom, it may not be
necessary for non-charred archaeobotanical remains. Likewise,
silica pellet extractions have been shown to excel at isolating
aDNA from bones [15], but they did not perform as well on
ancient plant samples in our testing. Unsurprisingly, commercial
DNA extraction kits designed for use on freshly sampled modern
plants were found to perform very poorly on ancient samples. Therefore, we would generally discourage aDNA researchers from
using such kits on archaeobotanical remains, although similar kits
have successfully yielded plant aDNA in some instances [52,53]. One of the most striking findings of the polymerase tests was the
ability of Omni Klentaq to overcome inhibitory substances,
consistent with findings on archaeological fish bone samples [20]. Even in high levels of inhibitory substances derived from non-
charred ancient plant materials, like humic acids, Omni Klentaq
successfully amplified spiked DNA when used with BSA. Without
BSA, Omni Klentaq could still amplify DNA in the presence of
low levels of inhibitors, a feat not matched by AmpliTaq Gold or
Phusion. The significance of this property should not be
overlooked, because enzymatic inhibition is not always recognized
in the laboratory. For example, some DNA extracts in these
studies contain inhibiting substances even though they lacked
pigmentation. Omni Klentaq is also reliable in qPCR experiments
where enzymatic inhibition may be encountered. Unlike Ampli-
Taq Gold, Omni Klentaq exhibits an exemplar qPCR amplifica-
tion curve in the presence of BSA and inhibitors. Conversely,
Omni Klentaq may have slightly lower fidelity than AmpliTaq
Gold, and occasionally yields chimera amplicons, something not
observed in other polymerases. Therefore, it is not an ideal
polymerase to amplify libraries or other templates which will be
sequenced. Nevertheless, it is an excellent choice for amplifying
genetic markers in reticent samples and qPCR assays as it provides
a safeguard against undetected enzymatic inhibition. Comparative testing of polymerases also yielded a number of
important insights. Discussion Conversely, PfuTurbo Cx is
advertised as able to read uracil, resulting in an apparent C-to-T
transition on the template strand and G-to-A transition on the
complementary strand. Nucleotide substitutions rates in the other
three polymerases were compared to those of Phusion and
PfuTurbo Cx to determine if they follow similar patterns. As seen
in Figure 6, Phusion has lower error rates in C-to-T and G-to-A
transitions than the other polymerases. An ANOVA test on the
error rates for individual samples found statistically significant
differences in error rates for C-to-T [F(4, 9) = 30.846, p,0.001]
and G-to-A [F(4, 9) = 7.045, p = 0.007] transitions. Tukey’s HSD
post-hoc test on the C-to-T transitions found Phusion to have a
statistically different error rate than the other polymerases
(p#0.002 for each pairwise comparison). Tukey’s HSD post-hoc
test on the G-to-A transitions found Phusion to have a statistically
different error rate than AmpliTaq Gold (p = 0.028), Omni
Klentaq (p = 0.005), and PfuTurbo Cx (p = 0.034), but not Phire
(p = 0.103). Overall, none of the polymerases tested have a pattern
consistent with Phusion, indicating they pair uracil with adenine
rather than stalling. The expanded dataset with error rates for all
substitution types is available in Table S6. It is interesting to consider how the best extraction methods
compare to some others used in the field. For instance, the top two
performing methods do not include cetyltrimethylammonium
bromide (CTAB), a reagent used in many extraction methods on
ancient plant remains, both charred [35,49] and non-charred January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 10 Macrobotanical aDNA Extraction and Amplification Figure 6. Error rates of most frequent substitution types. High-low chart depicts the maximum and minimum error rates within the three
tested samples. Median values, represented by circles, are not included for PfuTurbo Cx because only two samples were amplified. doi:10.1371/journal.pone.0086827.g006 Figure 6. Error rates of most frequent substitution types. High-low chart depicts the maximum and minimum error rates within the three
tested samples. Median values, represented by circles, are not included for PfuTurbo Cx because only two samples were amplified. doi:10.1371/journal.pone.0086827.g006 nucleotides in a genetic marker or DNA library with a limited
number of PCR cycles (10 cycles, for example). January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org Appendix S1
Text with detailed extraction protocols,
PCR
information,
DNA
sequencing,
and
expanded
results.
(DOCX) Data S1
Deep-sequencing amplicon data of AmpliTaq
Gold on LUG sample in FASTQ format. (FASTQ) Some of the experimental methodology developed and refined
over the course of this study could also provide guidance for future
aDNA comparative experiments. For example, spectrophotomet-
ric detection of DNA in pigmented eluates was found to be
occasionally misleading, so quantification of endogenous aDNA
can be more reliably measured with qPCR and sequencing of
PCR products. Testing of newly engineered polymerases will
continue to be invaluable, and as demonstrated here, comparisons
of fidelity and compatibility with damaged nucleotides can be
successfully explored via HTS. Considering little is known about
the inhibitory effects on polymerases and other enzymes used in
the construction of DNA libraries, a similar set of experiments
could be undertaken to optimize this fundamental step of HTS
research. Data S2
Deep-sequencing amplicon data of AmpliTaq
Gold on VAD-A sample in FASTQ format. (FASTQ) Data S3
Deep-sequencing amplicon data of AmpliTaq
Gold on VAD-B sample in FASTQ format. (FASTQ) Data
S4
Deep-sequencing
amplicon
data
of
Omni
Klentaq on LUG sample in FASTQ format. (FASTQ) Data
S5
Deep-sequencing
amplicon
data
of
Omni
Klentaq on VAD-A sample in FASTQ format. (FASTQ) (DOCX) Table S6
Specific substitution frequencies and corre-
sponding error rate. Sequencing reads that differed from the
expected rbcL sequence by .3 nucleotide substitutions were
omitted prior to tallying nucleotide calls and errors. (DOCX) Conclusions As foreseen by Palmer et al. [9], the future of plant aDNA
research is very bright indeed. The introduction of high-
throughput sequencing technologies allows geneticists to delve
into ancient genomes in new and exciting ways. In fact, these
technologies have already been tested on aDNA extracted from
archaeobotanical remains [19,35,48]. However, in order for such
studies to become more widespread and for the discipline to reach
its full potential, it is critical the best available methods are used to
extract, amplify, and analyze DNA from ancient specimens. For
desiccated and waterlogged plant remains, this study is a step in
that direction, and to that end, we strongly encourage fellow
researchers to adopt the best performing extraction techniques, or
at a minimum, conduct head-to-head comparisons with more
familiar methods. Such experimentation will help advance plant
archaeogenetics into a more fruitful discipline, yielding unprece-
dented understandings of plant evolution, domestication, and
human-plant interactions. Data
S6
Deep-sequencing
amplicon
data
of
Omni
Klentaq on VAD-B sample in FASTQ format. (FASTQ) Data S7
Deep-sequencing amplicon data of PfuTurbo
Cx on VAD-A sample in FASTQ format. (FASTQ) Data S8
Deep-sequencing amplicon data of PfuTurbo
Cx on VAD-B sample in FASTQ format. (FASTQ) Data S9
Deep-sequencing amplicon data of Phire on
LUG sample in FASTQ format. (FASTQ) Data S10
Deep-sequencing amplicon data of Phire on
VAD-A sample in FASTQ format. (FASTQ) Data S11
Deep-sequencing amplicon data of Phire on
VAD-B sample in FASTQ format. (FASTQ) Table S4
Deep sequencing of rbcL markers to investi-
gate polymerase fidelity.
(DOCX) Table S4
Deep sequencing of rbcL markers to investi-
gate polymerase fidelity. (DOCX) method, AmpliTaq Gold nearly always failed to amplify endog-
enous plant markers unless BSA was added. While it might be
assumed that plant-specific extraction protocols, such as those
using CTAB, adequately purify DNA, they failed at virtually the
same rate as other methods. Thus, we encourage adding BSA in
PCR on non-charred archaeobotanical remains, contrary to the
approach in most plant aDNA studies [39,50,56]. method, AmpliTaq Gold nearly always failed to amplify endog-
enous plant markers unless BSA was added. While it might be
assumed that plant-specific extraction protocols, such as those
using CTAB, adequately purify DNA, they failed at virtually the
same rate as other methods. Thus, we encourage adding BSA in
PCR on non-charred archaeobotanical remains, contrary to the
approach in most plant aDNA studies [39,50,56]. Table S5
Polymerase error rates. Sequencing reads that
differed from the expected rbcL sequence by .3 nucleotide
substitutions were omitted prior to tallying nucleotide calls and
errors. As we have not extracted charred archaeobotanical remains in
these studies, we cannot directly test Giles and Brown’s [57]
argument that BSA has no benefit for PCR on charred
archaeobotanical remains and may reduce amplification success
because DNA molecules become bound to BSA along with
contaminants. However, it should be noted their study was based
on artificially charred seeds and may not reflect the complexity of
some archaeobotanical remains. For example, sediments adhering
to charred cereals may contain humic acids that could inhibit
PCR. Other things being equal, we suggest it is worth conducting
PCR with BSA to ensure enzymatic inhibition does not lead to
false negative results. Discussion One of the key findings is that no polymerase
excels in all categories; rather, they have nuanced properties and
should be selected with care, according to the goals and methods
in a given research project, as outlined below. Some of the findings
about particular polymerases have been reported [20,54], but the
results of these experiments can help select which polymerase to
use in different circumstances. One of the most commonly used polymerases in aDNA
research, AmpliTaq Gold, was found to perform well in many
categories, making it a good all-around polymerase. When used in
conjunction with BSA, it can overcome moderate amounts of
inhibition. Furthermore, it handles the most common form of
nucleotide damage, cytosine deamination. Therefore, AmpliTaq
Gold is well suited to amplify markers of interest in aDNA
libraries, albeit with some reservation due to its replication error
rate. Phusion, a polymerase designed to have very high fidelity, was
indeed found to have a much lower error rate than the other
polymerases. However, Phusion is incompatible with uracil and
stalls on damaged DNA templates. This is a critical concern for
amplification of genetic markers or aDNA libraries, because
Phusion will preferentially amplify non-damaged molecules,
precisely those originating from modern contaminants. Therefore,
some aDNA researchers, such as Green et al. [1], have devised a
two-step amplification approach to retain damaged DNA but keep
replication errors to minimum. First, a uracil-friendly polymerase,
such as AmpliTaq Gold, is used to amplify over damaged Another key discovery was that nearly all polymerases fail in the
presence of inhibiting substances from non-charred archaeobota-
nical remains, unless BSA is added. In reactions without BSA, only
Omni Klentaq and Phire could amplify spiked DNA, and even
then, only the smallest concentrations of inhibitors could be
overcome. When BSA was added to reactions containing small
amounts of inhibiting substances, all polymerases were successful. This finding is even more important given the amplification tests
from the third phase of extractions: irrespective of extraction January 2014 | Volume 9 | Issue 1 | e86827 January 2014 | Volume 9 | Issue 1 | e86827 11 PLOS ONE | www.plosone.org Macrobotanical aDNA Extraction and Amplification Table S4
Deep sequencing of rbcL markers to investi-
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pig domestication, migration, and turnover revealed by modern and ancient
DNA. Proc Natl Acad Sci USA 107(17): 7686–7691. Acknowledgments The authors thank the following researchers for permission for destructive
analysis of archaeobotanical remains: Boris Gasparyan, Institute of
Archaeology and Ethnology, National Academy of Sciences, Yerevan,
Armenia; Giovanna Bosi and Anna Maria Mercuri, Museo Di Paleobio-
logia e dell’Orto Botanico, Universita` di Modena e Reggio Emilia,
Modena, Italy; Girolamo Fiorentino, Dipartimento di Beni Culturali,
University of Salento, Lecce, Italy; Mike Jacobs, Arizona State Museum;
and Jose´ Luis Punzo-Dı´az, Instituto Nacional de Antropologı´a e Historia,
Centro INAH Michoaca´n, Mexico. From the Centre for GeoGenetics, we Supporting Information Table S1
PCR conditions for polymerases. (DOCX)
Table S2
Extraction phase 2 data. (DOCX)
Table S3
qPCR Ct values for polymerase inhibition
testing. (DOCX) Table S1
PCR conditions for polymerases. (DOCX) Data S12
Deep-sequencing amplicon data of Phusion on
LUG sample in FASTQ format. (FASTQ) Table S2
Extraction phase 2 data. (DOCX) Data S13
Deep-sequencing amplicon data of Phusion on
VAD-A sample in FASTQ format. (FASTQ) Table S3
qPCR Ct values for polymerase inhibition
testing. (DOCX) Table S3
qPCR Ct values for polymerase inhibition
testing. (DOCX) PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e86827 PLOS ONE | www.plosone.org 12 Macrobotanical aDNA Extraction and Amplification Data S14
Deep-sequencing amplicon data of Phusion on
VAD-B sample in FASTQ format. (FASTQ) Data S14
Deep-sequencing amplicon data of Phusion on
VAD-B sample in FASTQ format. (FASTQ) thank Aure´lien Ginolhac for additional programming advice and Ludovic
Orlando for recommendations on silica pellet extractions. We acknowledge
our appreciation to Morten Rasmussen, Kim Magnussen, and the Danish
National High-throughput Sequencing Centre for assistance in generating
the Roche/454 GS FLX data. The authors also thank two anonymous
reviewers who helped improve the manuscript. Special thanks goes to
Randi Garcia, University of Connecticut, for thoughtful considerations
about statistical tests. Author Contributions Conceived and designed the experiments: NW KA EC TG. Performed the
experiments: NW KA EC. Analyzed the data: NW MCAA. Contributed
reagents/materials/analysis tools: TG. Wrote the paper: NW KA EC
MCAA TG. References (2012)
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Application of the combined probabilisticstatistical methods of quantitative assessment of strength and durability of main pipelines with single and combined defects
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Ухтинский государственный технический университет, г. Ухта, Россия
*e-mail: ugtusovet@yandex.ru Ухтинский государственный технический университет, г. Ухта, Россия
*e-mail: ugtusovet@yandex.ru Аннотация. Магистральные газонефтепроводы являются важнейшими объ-
ектами топливно-энергетического комплекса государства. К ним предъявляют-
ся строгие требования по надежной и безопасной работе. Поэтому при экс-
плуатации магистральных трубопроводов необходимо производить оценку их
прочности (текущей прочности) и долговечности (прогнозной прочности). Стенка труб действующих магистральных трубопроводов испытывает
различные нагрузки и воздействия. Чтобы не допустить разрушения или от-
каза трубопровода, выполняются расчеты на прочность и долговечность, где
учитываются параметры воздействия (load) на трубопровод и параметры ме-
ханического сопротивления (resistance) труб. Поэтому применяется расчетная
модель «воздействие — сопротивление» для количественной оценки надеж-
ности магистрального трубопровода. В данной работе представлены основные теоретические положения мето-
дики оценки прочности и долговечности магистральных трубопроводов с
одиночными и комбинированными дефектами в рамках комбинированного
вероятностно-статистического подхода, а также рассмотрен пример исполь-
зования методики для участка магистрального трубопровода, обследованного
внутритрубным дефектоскопом. Ключевые слова: вероятность отказа; долговечность; надежность; пре-
дельное давление; проектное давление; прочность Alexander V. Salnikov*, Anatoly A. Ignatik Ukhta State Technical University, Ukhta, Russia
*e-mail: ugtusovet@yandex.ru 25.00.19 Строительство и эксплуатация нефтегазопроводов,
баз и хранилищ (технические науки) 25.00.19 Строительство и эксплуатация нефтегазопроводов,
баз и хранилищ (технические науки) 25.00.19 Строительство и эксплуатация нефтегазопроводов,
баз и хранилищ (технические науки) DOI: 10.31660/0445-0108-2019-5-115-124 DOI: 10.31660/0445-0108-2019-5-115-124 УДК 621.643.053
Применение комбинированной вероятностно-статистической методики
количественной оценки прочности и долговечности магистральных
трубопроводов с одиночными и комбинированными дефектами 71 РД-23.040.00-КТН-115-11. Нефтепроводы и нефтепродуктопроводы магистральные. Определение
прочности и долговечности труб и сварных соединений с дефектами. – М.: ОАО «АК “Транснефть”»,
2013. – 142 с.
82 СТО Газпром 2-2.3-112-2007. Методические указания по оценке работоспособности участков ма-
гистральных газопроводов с коррозионными дефектами. – Введ. 2007-08-28. – М.: ООО «Информаци-
онно-рекламный центр газовой промышленности», 2007. – 68 с. Введение Научно-обоснованная оценка степени опасности развивающихся в процессе
эксплуатации одиночных и особенно комбинированных дефектов труб маги-
стральных трубопроводов, прогноз их технического состояния — одна из ак-
туальных задач магистрального транспорта нефти и газа, напрямую влияющая
на планирование ремонтных работ линейной части. Адекватное и оперативное решение этой задачи зачастую осложняется от-
сутствием единообразия в нормативно-технических документах по оценке
технического состояния труб с дефектами, а также сложностью используемого
в методиках оценки прочности и долговечности труб с дефектами математиче-
ского аппарата. Проведенные ранее исследования [1–3] позволили сформулировать требо-
вания и разработать расчетную методику для количественной оценки прочно-
сти и долговечности магистральных трубопроводов, в которой используется
вероятностно-статистический подход, а в качестве базовой была принята мо-
дель «воздействие — сопротивление» [4]. В работе [3] параметром воздействия принято проектное давление pпроект, а
параметром сопротивления служит предельное давление pпред. Проектное и
предельное давления рассматриваются как случайные непрерывные величины,
имеющие распределение по некоторому закону, и их рассмотрение произво-
дится методами теории вероятности и математической статистики. Проектное
давление — это давление, действующее в рассматриваемой секции трубопро-
вода, а предельное давление — это максимальное избыточное давление, кото-
рое может выдержать труба с дефектом без разрушений и отказов. Предельное
давление pпред вычисляют при использовании расчетных схем и деформацион-
ных критериев предельных состояний трубопровода из документа 71 или при
использовании рекомендаций 82 из источников [5–9]. Исследование законов
распределения проектного и предельного давлений позволяет перейти к опре-
делению вероятности отказа магистрального трубопровода. Ukhta State Technical University, Ukhta, Russia
*e-mail: ugtusovet@yandex.ru Ukhta State Technical University, Ukhta, Russia
*e-mail: ugtusovet@yandex.ru Abstract. Main gas and oil pipelines are the most important objects of the fuel
and energy complex of the state. They are subjected to strict requirements for reli-
able and safe operation. Therefore, it is necessary to assess their strength (current
strength) and durability (prediction strength) when operating main pipelines. The wall of the pipes of the existing main pipelines is subjected to various
loads and influences. To prevent pipeline failure, strength and durability calcula-
tions are performed. The parameters of the load and mechanical resistance of the
pipes are taken into account when calculating. Therefore, the "load — resistance"
model is used to quantify the reliability of the main pipeline. № 5, 2019 115 № 5, 2019 № 5, 2019 Нефть и газ This paper presents the main theoretical provisions of the methodology for as-
sessing the strength and durability of trunk pipelines with single and combined de-
fects in the framework of a combined probabilistic-statistical approach, and also an
example of the use of the technique for a section of a trunk pipeline examined by
an in-line flaw detector. Key words: probability of failure; durability; reliability; maximum allowable
pressure; operating pressure; strength 71 РД-23.040.00-КТН-115-11. Нефтепроводы и нефтепродуктопроводы магистральные. Определение
прочности и долговечности труб и сварных соединений с дефектами. – М.: ОАО «АК “Транснефть”»,
2013. – 142 с. 82 СТО Газпром 2-2.3-112-2007. Методические указания по оценке работоспособности участков ма-
гистральных газопроводов с коррозионными дефектами. – Введ. 2007-08-28. – М.: ООО «Информаци-
онно-рекламный центр газовой промышленности», 2007. – 68 с. Объект и методы исследования Объект и методы исследования Величина S = pпред – pпроект называется запасом прочности. Она служит ко-
личественным показателем надежности трубопровода [10]. Если S ≥ 0 (или № 5, 2019 116 Нефть и газ pпред ≥ pпроект), то прочность конструкции выполняется. Если S < 0 (или
pпред < pпроект), то прочность конструкции не соблюдена. р
р
При расчете на прочность необходимо определить величины pпроект и pпред
в момент времени эксплуатации t = 0, то есть в момент выполнения диагно-
стики трубопровода. В ходе расчета на долговечность вычисляются величины
pпроект и pпред в моменты времени от t = 0 до момента перехода трубопровода
в предельное состояние t = tпред, где tпред — предельный срок эксплуатации
трубопровода, год. Величина времени t является переменной величиной при
расчете на долговечность. А при расчете на прочность переменной величиной
является давление p. Линейная часть магистрального трубопровода рассматривается как систе-
ма, состоящая из совокупности элементов. Элементом в рассматриваемой
методике является дефектная зона. Каждый элемент характеризуется одним
значением проектного давления pпроект и одним значением предельного
давления pпред. Как правило, для бездефектных зон труб pпроект < pпред и условие
прочности соблюдается, в том числе в будущие моменты времени. Бездефект-
ные зоны в разработанной методике не учитываются. Дефекты уменьшают
значение pпред. К тому же многие дефекты развиваются с течением времени, их
геометрические параметры увеличиваются по определенному механизму (кор-
розионному или циклическому), и еще больше уменьшается величина пре-
дельного давления pпред. р
Магистральный трубопровод с одиночными и комбинированными дефек-
тами, являющийся объектом исследования, разделяется на участки, на которых
расположены n соседних дефектных зон, где n ≥ 50 (это условие необходимо
для применения критерия Пирсона). Каждый участок трубопровода определяется совокупностью из n значений
pпроект и n значений pпред. Эти n значений проектного, а также предельного дав-
лений составляют выборки объемом n. С этими выборками производятся опе-
рации, известные из теории математической статистики. Определяются законы
распределения величин проектного и предельного давлений. На рисунке 1
схематично представлены графики плотностей законов распределения пре-
дельного и проектного давлений. Рис. 1. Модель «воздействие — сопротивление» для участка магистрального
трубопровода: fQ — кривая воздействия (плотности распределения проектного
давления); fR — кривая сопротивления (плотности распределения предельного давления);
Z — точка пересечения кривых; PZ — давление, соответствующее точке пересечения кривых Рис. 1. Объект и методы исследования Модель «воздействие — сопротивление» для участка магистрального
трубопровода: fQ — кривая воздействия (плотности распределения проектного
давления); fR — кривая сопротивления (плотности распределения предельного давления);
— точка пересечения кривых; PZ — давление, соответствующее точке пересечения кривых № 5, 2019 117 Нефть и газ Вероятность разрушения или перехода в другое предельное состояние, то
есть вероятность отказа, равна площади заштрихованной зоны (см. рис. 1). Для
этой зоны значения запаса прочности меньше нуля S < 0 (или pпред < pпроект). Вероятность отказа обозначается буквой V. Вероятность отказа следует срав-
нить
с
нормативной
величиной
Vнорм
[10],
являющейся
технико-
экономическим показателем. Если V ≤ Vнорм, то обеспечивается надежная рабо-
та рассматриваемого участка трубопровода. Если V > Vнорм, то надежная работа
не обеспечивается. С течением времени эксплуатации t прогнозируется
уменьшение значений pпред, а следовательно, значение V будет расти, что
ухудшает техническое состояние трубопровода. Для упрощения расчетов принимается, что проектное давление pпроект —
это постоянная величина, не являющаяся случайной и не зависящая от време-
ни эксплуатации. Величина же предельного давления pпред — это случайная
величина, изменяющаяся с течением времени эксплуатации трубопровода. То-
гда вероятность отказа V рассчитывается по следующей формуле: )
;
(
)
;
(
t
p
F
t
p
f
V
ПРОЕКТ
R
p
R
ПРОЕКТ
=
∫
=
∞
−
, (1) (1) где
)
;
(
t
p
F
ПРОЕКТ
R
— функция распределения случайной величины предель-
ного давления. где
)
;
(
t
p
F
ПРОЕКТ
R
— функция распределения случайной величины предель-
ного давления. В качестве одного детерминированного значения принято pпроект 5,9 МПа.
Следовательно, величина проектного давления не является случайной. р
р
р
p рое
,
Следовательно, величина проектного давления не является случайной. Если не учитывать высотные отметки трассы трубопровода, распределение
давления в МН выражается следующей формулой: )
(
0
0
0
к
x
x
p
p
L
L
p
p
−
⋅
−
=
, (2) (2) где px — проектное давление в точке с координатой x, МПа; x = L0x — дистан-
ция трубной секции от начала трубопровода по данным ВТД, км; p0 — давле-
ние в начальной точке трубопровода, принято p0 = 6,0 МПа; pк — давление в
конечной точке трубопровода, принято pк = 3,8 МПа; L — длина рассматри-
ваемого трубопровода, L = 142 км. ру
р
Высотные отметки трубопровода в формуле (2) не учитываются, поэтому
перепад высот принимаем Δz = 0. р
р
По формуле (2) для участка трубопровода 10–15 км имеем 9,5
)
8,3
0,6
(
0,
142
0,
10
0,6
10
=
−
⋅
−
=
км
p
МПа,
8,5
)
8,3
0,6
(
0,
142
0,
15
0,6
10
=
−
⋅
−
=
км
p
МПа. 9,5
)
8,3
0,6
(
0,
142
0,
10
0,6
10
=
−
⋅
−
=
км
p
МПа,
8,5
)
8,3
0,6
(
0,
142
0,
15
0,6
10
=
−
⋅
−
=
км
p
МПа. 9,5
)
8,3
0,6
(
0,
142
0,
10
0,6
10
=
−
⋅
−
=
км
p
МПа, По результатам расчетов за параметр воздействия принимаем проектное
давление pпроект = 5,9 МПа. p р
Величину предельного давления pпред считаем случайной величиной. Для
каждого из 63 дефектов потери металла по методике 93 вычисляется одно зна-
чение pпред. Рекомендации и расчетные схемы 104 были применены в среде Mi-
crosoft Excel. Полученная выборка значений pпред представлена в таблице 1. 9 3РД-23.040.00-КТН-115-11. – С. 58.
4 Результаты Рассмотрим частный случай расчета на прочность и долговечность магист-
рального трубопровода, когда учитываются только дефекты потери металла,
развивающиеся по коррозионному механизму. Для оценки прочности и долго-
вечности методами теории вероятности и математической статистики был вы-
бран один из участков магистрального нефтепровода (МН) Ухта — Ярославль
протяженностью 5 км. Исходные данные для выполнения вычислений приня-
ты по результатам внутритрубной диагностики (ВТД), проведенной дефекто-
скопом WM в 2005 году и из проектной документации. Для расчета предель-
ного давления pпред труб с дефектами потери металла из отчета ВТД использо-
вались следующие позиции: •
дистанция трубной секции от начала трубопровода; •
расположение дефекта от начала секции; •
расположение дефекта на трубе (внешнее или внутреннее); •
расположение дефекта на трубе (внешнее или вну •
толщина стенки трубной секции вне дефекта δ; •
длина L, ширина W и глубина H дефекта потери металла; •
угловые координаты границ дефекта Θнач и Θкон; •
категория участка МН. Расположение дефекта от начала секции, его геометрические параметры
(длина L, ширина W и глубина дефекта потери металла H) и угловые коорди-
наты границ дефекта (Θнач и Θкон) использовались для выявления комбиниро-
ванных коррозионных дефектов. Дефекты являются комбинированными (об-
разуют сочетание), если минимальное расстояние между их границами меньше
или равно 4·δ. На исследуемом участке МН установлено 63 дефекта потери
металла, среди них 7 являются комбинированными, состоящими из двух от-
дельных дефектов. Трубы рассматриваемого участка МН произведены из ста-
ли 17ГС (17Г1С). Величина проектного давления pпроект принята постоянной,
не зависящей от времени эксплуатации t. Как показали расчеты, проектное Нефть и газ
№ 5, 2019 118 № 5, 2019 Нефть и газ давление на исследуемом участке изменяется в пределах от 5,9 до 5,8 МПа. В качестве одного детерминированного значения принято pпроект = 5,9 МПа. Следовательно, величина проектного давления не является случайной. Д
10 4Там же. – С. 93. 9 3РД-23.040.00-КТН-115-11. – С. 58.
10 4Там же. – С. 93. В качестве одного детерминированного значения принято pпроект 5,9 МПа.
Следовательно, величина проектного давления не является случайной. Таблица 1 Таблица 1 Выборка значений предельных давлений pпред (в МПа), рассчитанных применительно
к дефектам потери металла, для участка трубопровода 10–15 км при t = 0 Выборка значений предельных давлений pпред (в МПа), рассчитанных применительно
к дефектам потери металла, для участка трубопровода 10–15 км при t = 0 Выборка значений предельных давлений pпред (в МПа), рассчитанных применительно
к дефектам потери металла, для участка трубопровода 10–15 км при t = 0
№
1
2
3
4
5
6
7
8
9
10
pпред
12,1
11,7
12,6
12,8
13,0
13,3
12,8
13,0
13,3
13,3
№
11
12
13
14
15
16
17
18
19
20
pпред
13,6
13,3
13,6
13,3
13,3
13,4
13,3
13,0
12,8
13,2
№
21
22
23
24
25
26
27
28
29
30
pпред
13,2
13,0
13,1
13,6
13,9
13,8
13,6
13,0
13,2
12,7
№
31
32
33
34
35
36
37
38
39
40
pпред
13,6
13,3
11,7
11,4
10,3
11,3
11,1
11,1
11,7
11,4
№
41
42
43
44
45
46
47
48
49
50
pпред
11,4
11,4
12,0
12,2
13,0
12,7
12,7
10,2
11,2
11,1
№
51
52
53
54
55
56
57
58
59
60
pпред
11,3
121,2
11,4
11,4
11,4
11,8
10,9
10,8
11,4
10,8
№
61
62
63
pпред
11,1
11,7
11,7 119 Нефть и газ № 5, 2019 Нефть и газ Для выборки pпред найдены среднее значение
ПРЕД
p
= 12,33 МПа (команда
«СРЗНАЧ» в Microsoft Excel) и стандартное отклонение σp пред = 1,0023 МПа
(команда «СТАНДОТКЛОН» в Microsoft Excel). Далее необходимо определить
закон распределения, которому подчиняется ряд значений pпред. р д
Рассматривались следующие законы распределения случайных величин:
й •
нормальный; •
Вейбулла; •
логнормальный; •
гамма-распределение. •
гамма-распределение. Проверка гипотез о законе распределения проводилась по критерию χ2
(Пирсона). Расчетные значения критерия для каждого закона распределения
приведены в таблице 2. Таблица 2 Расчетные значения критерия χ2 (Kрасч) для разных законов распределения
случайных величин при t = 0 Расчетные значения критерия χ2 (Kрасч) для разных законов распределения
случайных величин при t = 0
Закон
распределения
Нормальный
Вейбулла
Логнормальный
Гамма-
распределение
Kрасч
11,31
11,76
12,09
11,74 Выборка значении pпред была разделена на 7 интервалов с диапазоном
1 МПа от 8 до 15 МПа. Все четыре закона распределения являются двухпара-
метрическими. р
Критическое значение критерия при уровне значимости α = 0,01 и числе
степеней свободы m = 4 равно Kкрит =
2
4
;
01
,0
χ
= 13,3. В качестве одного детерминированного значения принято pпроект 5,9 МПа.
Следовательно, величина проектного давления не является случайной. Так как Kрасч < Kкрит для
всех законов распределения, то все эти законы можно применять для описания
случайной величины pпред. С течением времени эксплуатации магистрального трубопровода коррози-
онные дефекты увеличиваются в размерах, что приводит к снижению значений
предельного давления в исследуемой выборке. Откуда следует, что параметры
закона распределения случайной величины pпред изменяются во времени. Были рассмотрены выборки значений pпред через t = 1; 2; 3; 4; 5; 10 лет экс-
плуатации от даты проведения ВТД без проведения ремонтных работ на уча-
стках с коррозионными дефектами. Принимается, что с течением времени увеличиваются глубина дефекта H и
площадь продольного сечения дефекта A. Соответственно, необходимо знать
скорости развития глубины vH (мм/год) и площади продольного сече-
ния vA (мм2/год) дефекта. Величины скоростей vH и vA считаются постоянными
при эксплуатации трубопровода. Скорость роста дефекта потери металла в глу-
бину vH на внешней поверхности трубы определяется по формуле из работы [3] )
1(
3
2
1
00
0
K
K
K
t
H
H
Н
+
+
+
⋅
−
=
∆
ν
, (3) (3) где H0 — начальная глубина дефекта при t = 0, мм; H00 — глубина дефекта при
проведении предпоследней ВТД, мм; Δt — период времени между двумя по-
следними ВТД, год; K1 — коэффициент учета влияния удельного сопротивления
грунта; K2 — коэффициент учета влияния удельного сопротивления антикорро-
зионного покрытия; K3 — коэффициент учета влияния блуждающих токов. Нефть и газ
№ 5, 2019 120 № 5, 2019 Значения коэффициентов K1, K2, K3 приняты по рекомендациям 115:
K1 = 0,028; K2 = 0,077; K3 = 0,0. Также определено, что H00 = 0 и Δt = 5 лет. С учетом этого формула (3) примет следующий вид: Значения коэффициентов K1, K2, K3 приняты по рекомендациям 115:
K1 = 0,028; K2 = 0,077; K3 = 0,0. Также определено, что H00 = 0 и Δt = 5 лет. С учетом этого формула (3) примет следующий вид: 0
221
,0
H
Н
⋅
=
ν
. Скорость роста дефекта потери металла в глубину vH на внутренней по-
верхности трубы определяется по формуле (3), но без учета коэффициентов K1,
K2 и K3 (K1 = K2 = K3 = 0). Скорость роста площади продольного сечения дефекта потери металла vA
вычисляется по формуле из работы [4] 0
L
Н
A
⋅
=ν
ν
, Для времени эксплуатации t = 1; 2; 3; 4; 5 лет удовлетворительно описыва-
ют распределение случайной величины pпред нормальный закон и закон Вей-
булла. Для t = 10 лет пригоден только закон распределения Вейбулла. Наличие где L0 — начальная длина дефекта потери металла, мм. где L0 — начальная длина дефекта потери металла, мм. Площадь продольного сечения дефекта рассчитывается следующим образом:
A = (2/3)·L·H, причем используется параболическая аппроксимация площади. Через время эксплуатации с момента диагностики t = 1; 2; 3; 4; 5; 10 лет оп-
ределяются значения геометрических параметров дефектов: Ht, At и Lt. Вычис-
ления производятся в следующей последовательности: 1) Ht = H0 + vH · t;
2) At = A0 + vA · t; 3) Lt = (3 · At) / (2 · Ht). Отметим, что начальная площадь про-
дольного сечения дефекта A0 = (2/3) · L0 · H0. Величина ширины дефекта потери металла W считается неизменной с тече-
нием времени. В связи с тем что величины L и H дефектов возрастают при
эксплуатации трубопровода, а остальные исходные параметры, используемые
в расчетах на долговечность, не изменяются, то величина предельного давле-
ния pпред уменьшается. р
Для t = 1; 2; 3; 4; 5; 10 лет определены выборки из 63 значений pпред. Затем
были выполнены те же самые операции математической статистики, что и при
t = 0 (см. табл. 1 и 2). Расчетные значения критерия χ2 (Пирсона) Kрасч приве-
дены в таблице 3. Т б
3 Таблица 3 Расчетные значения критерия χ2 (Kрасч) для разных законов распределения
случайных величин при t = 0; 1; 2; 3; 4; 5; 10 лет Расчетные значения критерия χ2 (Kрасч) для разных законов распределения
случайных величин при t = 0; 1; 2; 3; 4; 5; 10 лет
Время
эксплуатации t,
год
Закон распределения
Нормальный
Вейбулла
Логнормальный
Гамма-
распределение
Расчетные значения критерия χ2 (Kрасч)
0
11,31
11,76
12,09
11,74
1
8,02
6,35
10,41
9,70
2
6,12
4,06
8,10
7,39
3
6,75
2,28
12,91
11,62
4
10,77
4,64
17,61
17,40
5
7,49
4,16
11,46
10,64
10
35,10
8,29
71,89
187,97 115 РД-23.040.00-КТН-115-11. – С. 115. Нефть и газ 121 № 5, 2019 № 5, 2019 121 Нефть и газ больших значений расчетного критерия Kрасч при t = 10 лет предположительно
связано с тем, что через 10 лет эксплуатации на исследуемом участке трубо-
провода будут находиться закритические (или недопустимые) дефекты потери
металла, для которых pпред < pпроект. Закритические дефекты необходимо ре-
монтировать для предотвращения аварий на магистральном трубопроводе. Однако закон распределения Вейбулла хорошо описывает распределение pпред
и при наличии на трубопроводе закритических дефектов. Графики плотностей распределения случайной величины pпред по закону
Вейбулла исследуемого участка МН представлены на рисунке 2. где L0 — начальная длина дефекта потери металла, мм. Эти графики
являются кривыми сопротивления модели «воздействие — сопротивление»
при разном значении времени эксплуатации трубопровода t от момента прове-
дения ВТД. Рис. 2. Кривые плотностей закона распределения Вейбулла для величин предельного
давления дефектных зон участка рассматриваемого трубопровода (10–15 км)
при времени эксплуатации от момента проведения ВТД t = 0; 1; 2; 3; 4; 5; 10 лет
без проведения ремонтных работ Рис. 2. Кривые плотностей закона распределения Вейбулла для величин предельного
давления дефектных зон участка рассматриваемого трубопровода (10–15 км)
при времени эксплуатации от момента проведения ВТД t = 0; 1; 2; 3; 4; 5; 10 лет
без проведения ремонтных работ Вероятности отказа V исследуемого участка трубопровода при разном зна-
чении t определены по формуле (1) с применением закона распределения Вей-
булла (табл. 4). Значения Vt в таблице 4, относящиеся к 5 км трубопровода и
переведенные к 1 000 км, должны быть сравнены с нормативным значе-
нием Vнорм, 1/1 000 км. Значение Vнорм назначается, в нашем случае принято
Vнорм = 0,050 1/1 000 км. Выводы В данной работе демонстрируются теоретические положения методики количе-
ственной оценки прочности и долговечности магистрального трубопровода с де-
фектами, где применен комбинированный вероятностно-статистический подход. Приведен пример использования методики по данным внутритрубной диагностики. Величину предельного давления дефектного участка трубопровода предла-
гается считать случайной величиной, описываемой двухпараметрическим за-
коном распределения Вейбулла и зависимой от времени эксплуатации. Вели-
чина проектного давления принимается постоянной величиной, не зависящей
от времени эксплуатации. Вероятность
отказа
магистрального
трубопровода
вычисляется
по
формуле (1) и сравнивается с нормативным значением величины отказа. По
итогам сравнения величин расчетной и нормативной вероятности отказа пла-
нируются объем и срок выполнения ремонтных работ для удаления дефектов. Обсуждение Как видно из таблицы 4, условие прочности через 5 лет (на конец пятого года)
эксплуатации не выполняется. При эксплуатации трубопровода недопустимо,
чтобы расчетная вероятность отказа превышала нормативную. Для уменьшения
расчетной вероятности отказа производятся ремонтные работы в дефектных зо-
нах, или снижается давление в трубопроводе. В нашем случае следует, например,
в начале пятого года эксплуатации (с момента проведения ВТД) или ранее про-
вести ремонтные работы по удалению дефектов. Необходимо отремонтировать
столько дефектов, чтобы вероятность отказа после ремонта была ниже норма-
тивной последующие несколько лет. В таблице 4 указана вероятность отказа че-
рез 5 лет эксплуатации, если произвести ремонт двух самых опасных дефекта 122 № 5, 2019 Нефть и газ потери металла, у которых наименьшие значения предельного давления. Отре-
монтированные дефекты не включаются в исследуемую выборку. Вероятности отказа Vt рассматриваемого участка трубопровода (10–15 км)
при t = 0; 1; 2; 3; 4; 5; 10 лет
Время
эксплуатации t,
год
Нормативная
вероятность
отказа Vнорм,
1/1 000 км
Расчетная
вероятность
отказа Vt,
1/5 км
Расчетная
вероятность
отказа Vt,
1/1 000 км
Выполнение
условия
прочности
V ≤ Vнорм
0
0,050
8,8·10-6
0,0018
да
1
1,7·10-5
0,0034
да
2
3,4·10-5
0,0068
да
3
6,3·10-5
0,0126
да
4
0,00013
0,026
да
5
0,00029
0,058
нет
10
0,00316
0,632
нет
5 (ремонт двух самых
опасных дефектов)
5,1·10-5
0,0102
да Вероятности отказа Vt рассматриваемого участка трубопровода (10–15 км)
при t = 0; 1; 2; 3; 4; 5; 10 лет Несмотря на проведение оценки прочности и долговечности участка трубо-
провода и ремонтных работ, внутритрубная диагностика должна производить-
ся в сроки, определенные действующим нормативно-техническим докумен-
том, для уточнения величины вероятности отказа, так как на участке трубо-
провода могут появиться новые дефекты потери металла. 1.
Сальников А. В., Шарыгин А. М., Игнатик А. А. Оценка прочности и долговечно-
сти труб с дефектами для эффективного планирования ремонтных работ на линейной части
магистральных трубопроводов // Территория Нефтегаз. – 2016. – № 9. – С. 114–121.
2.
Сальников А. В., Игнатик А. А. Совершенствование методики расчета труб на дол-
говечность с комбинированными дефектами типа «дефект геометрии с коррозионной поте-
рей металла» // Территория Нефтегаз. – 2018. – № 3. – С. 62–70.
3.
Игнатик А. А., Сальников А. В. Разработка методики расчета на прочность и дол-
говечность магистральных трубопроводов в рамках вероятностно-статистического подхода
// Трубопроводный транспорт: теория и практика. – 2017. – № 5 (63). – С. 42–45. 1.
Сальников А. В., Шарыгин А. М., Игнатик А. А. Оценка прочности и долговечно-
сти труб с дефектами для эффективного планирования ремонтных работ на линейной части
магистральных трубопроводов // Территория Нефтегаз. – 2016. – № 9. – С. 114–121. р
рр
р
ф
3.
Игнатик А. А., Сальников А. В. Разработка методики расчета на прочность и дол-
говечность магистральных трубопроводов в рамках вероятностно-статистического подхода
// Трубопроводный транспорт: теория и практика. – 2017. – № 5 (63). – С. 42–45. р
ру
р
д
рр
р
ф
2.
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Л. И. Быков [и др.]; под общ. ред. Л. И. Быкова – СПб.: Недра, 2006. – 824 с. 10. Типовые расчеты при сооружении и ремонте газонефтепроводов: учеб. пособие /
Л. И. Быков [и др.]; под общ. ред. Л. И. Быкова – СПб.: Недра, 2006. – 824 с. References 1. Salnikov, A. V., Sharygin, A. M. & Ignatik, A. A. (2016). Strength and durability evalu-
ation of pipes with defects for effective repair planning on the linear part of the main pipelines. Oil
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5. ASME B31G-2009 (Revision of ASME B31G-1991). (2009). Manual for Determining
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i upravlenie tekhnicheskim sostoyaniem. Moscow, Nedra Publ., 212 p. (In Russian). p
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7. Kharionovskiy, V. V. (2000). Nadezhnost' i resurs konstruktsiy gazoprovodov. Moscow,
Nedra Publ., 467 p. (In Russian). 8. Birillo, I. N., Yakovlev, A. Ya., Teplinskiy, Yu. A., Bykov I. Yu. & Voronin V. N. (2008). Otsenka prochnostnogo resursa gazoprovodnykh trub s korrozionnymi povrezhdeniyami. Moscow, CentrLitNefteGaz Publ., 168 p. (In Russian). p (
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9. Gasparyants, R. S. (2008). Raschet na prochnost' i dolgovechnost' truboprovodov s kor-
rozionnymi defektami poteri metalla. Oilfield Engineering, (1), pp. 34-39. (In Russian). 10. Bykov, L. I., Mustafin, F. M., Rafikov, S. K., Nechval, A. M. & Lavrentiev, A. E. (2006)
Pipeline construction and maintenance calculations. St. Petersburg, Nedra Publ., 824 p. (In Russian). Сведения об авторах Alexander V. Salnikov, Candidate of Engi-
neering, Associate Professor at the Department
of Design and Maintenance of Main Gas and
Oil Pipelines, Ukhta State Technical Universi-
ty, Ukhta, e-mail: ugtusovet@yandex.ru Сальников
Александр
Викторович,
к. т. н., доцент кафедры проектирования и
эксплуатации
магистральных
газо-
нефтепроводов, Ухтинский государственный
технический университет, г. Ухта, e-mail:
ugtusovet@yandex.ru Anatoly A. Ignatik, Postgraduate, Ukhta
State Technical University Anatoly A. Ignatik, Postgraduate, Ukhta
State Technical University Игнатик
Анатолий
Александрович,
аспирант,
Ухтинский
государственный
технический университет, г. Ухта № 5, 2019 № 5, 2019 124 Нефть и газ
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https://openalex.org/W2528260904
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https://www.nature.com/articles/srep34382.pdf
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English
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Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies
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Scientific reports
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Intravital imaging reveals
improved Kupffer cell-mediated
phagocytosis as a mode of action
of glycoengineered anti-CD20
antibodies received: 08 July 2016
accepted: 13 September 2016
Published: 04 October 2016 Capucine L. Grandjean1,2,*, Fabricio Montalvao1,2,*, Susanna Celli1,2, David Michonneau1,2,
Beatrice Breart1,2, Zacarias Garcia1,2, Mario Perro3, Olivier Freytag3, Christian A. Gerdes3 &
Philippe Bousso1,2 Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell
malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to
increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC)
and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab
may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse
liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after
anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we
provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell
depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered
potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis
were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab
and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an
important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. Anti-CD20 Ab is an effective therapy to treat B cell malignancies and a series of autoimmune diseases1–3. Dissecting its mode of action remains essential for the rational design of improved antibodies. Several in vitro
studies have contributed to the delineation of distinct possible mechanisms of action4 but few reports have
examined their respective contribution in vivo. We recently visualized the role of Kupffer cells in mediating
B cell phagocytosis within minutes of injection of a depleting mouse anti-mouse CD20 Ab5. In line with this,
Kupffer cells were also implicated in the clearance of circulating B16 tumor cells following administration of a
tumor-specific mAb6. Thus, B cell recirculation7 combined with depletion in the liver may contribute to the sys-
temic elimination of the B cell compartment. However, whether this mechanism is relevant for other anti-CD20
Abs and whether the liver is sufficient by itself to induce a broad B cell depletion remains to be established. Moreover, it remains unclear whether Kupffer-mediated B cell depletion can be tuned and improved by designing
anti-CD20 Abs with increased potency. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 08 July 2016
accepted: 13 September 2016
Published: 04 October 2016 1Dynamics of Immune Responses Unit, Equipe Labéllisée Ligue Contre le Cancer, Institut Pasteur, 75015 Paris,
France. 2INSERM U1223, 75015 Paris, France. 3Roche Innovation Center Zurich, Roche Pharma Research & Early
Development, Wagistrasse 18,8952 Schlieren, Switzerland. *These authors contributed equally to this work.
Correspondence and requests for materials should be addressed to P.B. (email: philippe.bousso@pasteur.fr) Intravital imaging reveals
improved Kupffer cell-mediated
phagocytosis as a mode of action
of glycoengineered anti-CD20
antibodies In particular, glycoengineering of anti-CD20 Fc portions has been shown
to improve antibody-dependent cellular cytotoxicity by NK cells and to increase phagocytosis by macrophages
in vitro8–11. To extend our previous findings, here we use both orthotopic liver transplants12 and intravital imaging13,14
to assess the importance of Kupffer cell-mediated B cell phagocytosis for the activity of mouse and human
anti-CD20 Abs. Specifically, we address the contribution of anti-CD20 Ab glycoengineering to the efficacy of B
cell depletion in the liver. 1Dynamics of Immune Responses Unit, Equipe Labéllisée Ligue Contre le Cancer, Institut Pasteur, 75015 Paris,
France. 2INSERM U1223, 75015 Paris, France. 3Roche Innovation Center Zurich, Roche Pharma Research & Early
Development, Wagistrasse 18,8952 Schlieren, Switzerland. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to P.B. (email: philippe.bousso@pasteur.fr) Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 1 www.nature.com/scientificreports/ Figure 1. The liver is sufficient to mediate systemic B cell depletion following anti-CD20 treatment. (A) Experimental set-up. A surgical procedure was undertaken to transplant WT livers into two FcRγ−/− and
one WT recipients. Mice were allowed to recover for 3 to 7 days before being injected with a single dose (50 μg)
of anti-CD20 Ab (5D2). Non-transplanted FcRγ−/− mice were used as negative controls. (B) B cell frequency
measured by flow cytometry in blood before, 2 h and 16 h following anti-CD20 treatment in the indicated mice. Values were normalized to the frequency measured prior to treatment. (C) B cell frequency in the spleen of WT
and FcRγ−/−recipients transplanted with a WT liver was measured by flow cytometry at 16 hr post-treatment. Numbers show the percentage of B cells after treatment. Figure 1. The liver is sufficient to mediate systemic B cell depletion following anti-CD20 treatment. ghfi
y
p
g
(A) Experimental set-up. A surgical procedure was undertaken to transplant WT livers into two FcRγ−/− and
one WT recipients. Mice were allowed to recover for 3 to 7 days before being injected with a single dose (50 μg)
of anti-CD20 Ab (5D2). Non-transplanted FcRγ−/− mice were used as negative controls. (B) B cell frequency
measured by flow cytometry in blood before, 2 h and 16 h following anti-CD20 treatment in the indicated mice. Values were normalized to the frequency measured prior to treatment. Intravital imaging reveals
improved Kupffer cell-mediated
phagocytosis as a mode of action
of glycoengineered anti-CD20
antibodies (C) B cell frequency in the spleen of WT
and FcRγ−/−recipients transplanted with a WT liver was measured by flow cytometry at 16 hr post-treatment. Numbers show the percentage of B cells after treatment. Methods Kupffer cells (green) and B cells (red) were labeled by i.v. injection of
fluorescently labeled anti-F4/80 Ab (2 μg) and anti-B220 Fab fragments, respectively. (D) Representative curve
showing the number of engulfed B cells (normalized per mm3) in the liver before and after rituximab (200 μg)
treatment. (E) Representative two-photon images obtained before and after rituximab treatment (200 μg),
highlighting rapid B cell phagocytosis by Kupffer cells (white arrows). Representative of 2–4 independent
experiments. Results are shown as mean ± SEM. Significance was assessed using an unpaired Student t-test. Figure 2. B cell depletion upon rituximab injection is mediated by Kupffer cells in human CD20 transgenic
mice. (A) Summary bar charts of the frequency of B cells in the blood of hCD20Tg mice 5 h following injection
of 50 μg rituximab or isotype control. (B) Experimental set-up. Splenocytes from WT (GFP+) or hCD20Tg
(mTomato+) mice were isolated and co-transferred into WT or FcRγ−/− recipient animals. After 24 h, mice were
treated i.v. with 50 μg rituximab or isotype control. (C) Summary bar charts of the ratio of hCD20Tg to WT B
cells in the blood 24 h following i.v. injection of 50 μg rituximab or isotype control. (D,E) hCD20Tg mice were
subjected to intravital imaging of the liver. Kupffer cells (green) and B cells (red) were labeled by i.v. injection of
fluorescently labeled anti-F4/80 Ab (2 μg) and anti-B220 Fab fragments, respectively. (D) Representative curve
showing the number of engulfed B cells (normalized per mm3) in the liver before and after rituximab (200 μg)
treatment. (E) Representative two-photon images obtained before and after rituximab treatment (200 μg),
highlighting rapid B cell phagocytosis by Kupffer cells (white arrows). Representative of 2–4 independent
experiments. Results are shown as mean ± SEM. Significance was assessed using an unpaired Student t-test. Figure 2. B cell depletion upon rituximab injection is mediated by Kupffer cells in human CD20 transgenic
mice. (A) Summary bar charts of the frequency of B cells in the blood of hCD20Tg mice 5 h following injection
of 50 μg rituximab or isotype control. (B) Experimental set-up. Splenocytes from WT (GFP+) or hCD20Tg
(mTomato+) mice were isolated and co-transferred into WT or FcRγ−/− recipient animals. After 24 h, mice were
treated i.v. with 50 μg rituximab or isotype control. (C) Summary bar charts of the ratio of hCD20Tg to WT B
cells in the blood 24 h following i.v. Methods Liver transplant. Orthotopic liver transplantation was adapted from Kamada et al.12 and performed under
a microdissecting microscope. To prepare mouse donor livers, ligaments, esophageal, pyloric, right adrenal veins
and the hepatic artery were cut. Liver were perfused and harvested after cutting the inferior vena cava, the portal
vein, and the superior vena cava. The inferior vena cava and the portal vein were prepared using the ‘cuff tech-
nique’. For mouse recipient liver removal, sutures were placed on the portal vein and the superior vena cava, the
portal vein, and the inferior vena cava were clumped before being cut. Anastomoses were performed by suture or
by sliding recipient vessels over cuffed donor vessels. Mice and antibodies. Wild type C57BL/6 mice were purchased from Charles River France. hCD20Tg mice7
(Genentech, USA), C57BL/6 GFP+ and FcRγ−/− mice were bred in our facility. All experiments were carried out
in agreement with relevant guidelines and regulations and approved by the Institut Pasteur committee on Animal
Welfare (CETEA) under the protocol code of CETEA2013–0077. Anti-CD20 mAbs included 5D2 (mIgG2a,
Genentech), WT and GE 18B12 (mIgG2a, Roche), rituximab and GA101 (Hôpital Necker), HY1.2 (mIgG2a) iso-
type control and mg053 (hIgG1) isotype control. 18B12 Ab glycoengineering was conducted using the GlycoMab
technology (Roche Glycart AG)15. Immunofluorescence. Perfused livers were cut, fixed overnight in paraformaldehyde and progressively
dehydrated in sucrose. Tissues were snap frozen in OCT compound (Tissue-Tek; Sakura). Sections were stained
with anti-F4/80 and anti-B220 Abs. Intravital two-photon imaging. Intravital imaging of the liver was performed using an upright DM
6000B/SP5 microscope (Leica Microsystems) and Chameleon Ultra Ti:Sapphire laser (Coherent) tuned at Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 2 www.nature.com/scientificreports/ Figure 2. B cell depletion upon rituximab injection is mediated by Kupffer cells in human CD20 transgenic
mice. (A) Summary bar charts of the frequency of B cells in the blood of hCD20Tg mice 5 h following injection
of 50 μg rituximab or isotype control. (B) Experimental set-up. Splenocytes from WT (GFP+) or hCD20Tg
(mTomato+) mice were isolated and co-transferred into WT or FcRγ−/− recipient animals. After 24 h, mice were
treated i.v. with 50 μg rituximab or isotype control. (C) Summary bar charts of the ratio of hCD20Tg to WT B
cells in the blood 24 h following i.v. injection of 50 μg rituximab or isotype control. (D,E) hCD20Tg mice were
subjected to intravital imaging of the liver. Methods injection of 50 μg rituximab or isotype control. (D,E) hCD20Tg mice were
subjected to intravital imaging of the liver. Kupffer cells (green) and B cells (red) were labeled by i.v. injection of
fluorescently labeled anti-F4/80 Ab (2 μg) and anti-B220 Fab fragments, respectively. (D) Representative curve
showing the number of engulfed B cells (normalized per mm3) in the liver before and after rituximab (200 μg)
treatment. (E) Representative two-photon images obtained before and after rituximab treatment (200 μg),
highlighting rapid B cell phagocytosis by Kupffer cells (white arrows). Representative of 2–4 independent
experiments. Results are shown as mean ± SEM. Significance was assessed using an unpaired Student t-test. 960 nm. The liver was exposed and a microscope slide was inserted underneath, immobilized using silicone paste
(Express 2 Putty Quick, 3M-Espe). A coversplit was placed on the top of the liver maintained by silicone paste. Results and Discussion (C) Summary bar charts showing percentages of engulfed B cells for the
indicated conditions. Representative of 2–4 independent experiments. Results are shown as mean ± SEM and were
compiled from mosaic images of liver sections (3 independent animals) containing > 1000 Kupffer cells. control following treatment, confirming that the graft procedure had no advert effects on the depletion pro-
cess. Most importantly, B cells were efficiently depleted in both grafted FcRγ−/− recipients carrying a WT liver,
as measured in blood (Fig. 1B) and spleens (Fig. 1C) indicating that the liver is indeed sufficient to mediate
broad B cell depletion of peripheral B cells. As expected5,16, no depletion was observed in non-transplanted
FcRγ−/− mice. We subsequently interrogated whether our previous findings using a murine anti-mouse CD20Ab
also pertain to clinically relevant anti-human CD20Abs such as rituximab. Treatment of BAC transgenic mice
expressing human CD20 (hCD20Tg)7 showed rapid B cell systemic depletion after a single injection of rituximab
(Fig. 2A and Fig. S2) consistent with a previous study17 and this effect was FcγR-dependent (Fig. 2B,C). Intravital
two-photon imaging of hCD20Tg liver revealed that circulating B cells arrested in the liver next to Kupffer cells
and were engulfed within minutes of rituximab injection. Moreover, within 20 min, most B cells had been phago-
cytized with only very few circulating B cells being detected in the liver at this time (Fig. 2D,E, Movie S1). Taken
together, these data further highlight the importance of the liver as a major B cell depletion site in response to
both anti-mouse or anti-human CD20 Abs.if Based on these results, we investigated whether modifications to anti-CD20 Abs could tune Kupffer-cell
mediated B cell phagocytosis efficacy. In this respect, glycoengineered anti-CD20 Abs have been shown to dis-
play higher potency for ADCC and APD in vitro11,18, but how these differences may translate in vivo remain
to be ascertained. First, a murine anti-mouse CD20 Ab (clone 18B12, referred to as WT anti-CD20) and its
glycoenginnered counterpart (GE anti-CD20) were compared for their B cell depletion efficacy in vivo at
30 min post-injection. Flow cytometric analyses revealed that early B cell depletion was more efficient with GE
anti-CD20 compared to WT anti-CD20 particularly at low doses (Fig. 3A). Quantification of engulfed B cells in
liver tissue sections identified a lower triggering threshold for GE anti-CD20 Ab (being active at doses as low as
0.3μg) compared to WT anti-CD20 Ab (Fig. 3B,C Fig. Results and Discussion We have previously shown using partial hepatectomy that the liver is necessary for optimal B cell depletion by
a mouse anti-CD20 Ab (5D2)5, a mechanism shown to be dependent on activating FcRs5,16. In addition, we
and others have shown that splenectomy has little effect on anti-CD20-mediated B cell depletion5,7. To assess
whether the liver is in fact sufficient to mediate systemic B cell depletion, we sought to create a system in which
Fc-dependent effector functions are restricted to the liver. For this purpose, we relied on orthotopic liver trans-
plantation, a particularly challenging surgical procedure in mice due to their small size (Fig. 1A). Nevertheless,
we successfully grafted two FcRγ−/− recipients with a WT liver and as a positive control, a WT recipient grafted
with a WT liver. Transplanted mice were treated with anti-CD20 Ab. PBL were collected prior to injection, at 2 h
and 16 h post-injection while spleens were harvested at 16 h. B cell depletion occurred in the WT transplanted Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 3 www.nature.com/scientificreports/ Figure 3. Mouse glycoengineered anti-CD20 Abs trigger enhanced Ab-dependent phagocytosis by Kupffer
cells in vivo. (A–C) WT or glycoengineered anti-CD20 mAbs (clone18B12) were used to treat WT mice at the
indicated doses. (A) B cell depletion efficacy was assessed in blood by flow cytometry at 30 min post-injection. (B) Frozen liver sections were stained using PE-labeled anti-F4/80 Ab and FITC-labeled anti-B220 Ab. B cell
phagocytosis by Kupffer cells is highlighted by white circles. Note that only the glycoengineered form of anti-CD20
trigger phagocytosis at low doses. (C) Summary bar charts showing percentages of engulfed B cells for the
indicated conditions. Representative of 2–4 independent experiments. Results are shown as mean ± SEM and were
compiled from mosaic images of liver sections (3 independent animals) containing > 1000 Kupffer cells. Figure 3. Mouse glycoengineered anti-CD20 Abs trigger enhanced Ab-dependent phagocytosis by Kupffer
cells in vivo. (A–C) WT or glycoengineered anti-CD20 mAbs (clone18B12) were used to treat WT mice at the
indicated doses. (A) B cell depletion efficacy was assessed in blood by flow cytometry at 30 min post-injection. (B) Frozen liver sections were stained using PE-labeled anti-F4/80 Ab and FITC-labeled anti-B220 Ab. B cell
phagocytosis by Kupffer cells is highlighted by white circles. Note that only the glycoengineered form of anti-CD20
trigger phagocytosis at low doses. Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 Results and Discussion S3), a finding that was also confirmed by intravital imaging
(Movie S2). Finally, using hCD20Tg mice, we compared two clinically relevant anti-human CD20 Abs, namely
rituximab and obinutuzumab (GA101), for their capacity to trigger Kupffer cell-mediated B cell phagocytosis
in vivo. GA101 is a glycoengineered type II anti-CD20 Ab shown to induce robust ADCC and APD in vitro11,18
due to the low fucose content of its Fc-portion, a feature that may contribute to its preclinical19 and clinical
activity20. Consistently, we found a more potent B cell depletion by GA101 at low doses (Fig. 4A). To investigate
how Kupffer cell activity may contribute to these differences, we relied on two-photon liver imaging of hCD20Tg
mice in response to rituximab and GA101. Interestingly, we visualized very efficient B cell arrest in the liver and
subsequent phagocytosis by Kupffer cells in response to low doses of GA101 whereas B cell continued to rapidly Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 4 www.nature.com/scientificreports/ Figure 4. Obinutuzumab triggers enhanced Ab-dependent phagocytosis by Kupffer cells in vivo
compared to rituximab. (A) Splenocytes from WT or hCD20Tg mice were isolated, labeled and co-
transferred into WT recipient mice. After 24 h, mice were treated i.v. with different doses of rituximab or
GA101 and blood was analyzed 1 hr later by flow cytometry. The summary bar charts show the ratio of
hCD20Tg to WT B cells (non depleted, used as an internal control) 1 h after injection of the indicated dose
of rituximab or GA101. (B–E) Intravital imaging of the liver of hCD20Tg mice during anti-CD20 treatment. Kupffer cells (green) and B cells (red) were labeled using anti-F4/80 Ab and anti-B220 Fab fragments,
respectively. (B) Representative curve showing the number of engulfed B cells (normalized per mm3) in
the liver following 0.4 μg GA101. (C) Figure shows representative two-photon images before and after
treatment with low doses of GA101 (0.4 μg), highlighting efficient B cell phagocytosis by Kupffer cells (white
squares and insets). Scale bar, 25 μm. (D) Representative two-photon images highlighting the absence of
B cell phagocytosis following 0.4 μg rituximab. Scale bar, 20 μm. (E) Each line represents the cell behavior
after anti-CD20 injection. Green squares represent cicrculating B cells, yellow squares represent contact
between a B and Kupffer cell and red squares represent engulfed B cells. Representative of 2–4 independent
experiments. Results are shown as mean ± SEM. Results and Discussion Significance was assessed using an unpaired Student t-test. Figure 4. Obinutuzumab triggers enhanced Ab-dependent phagocytosis by Kupffer cells in vivo
compared to rituximab. (A) Splenocytes from WT or hCD20Tg mice were isolated, labeled and co-
transferred into WT recipient mice. After 24 h, mice were treated i.v. with different doses of rituximab or
GA101 and blood was analyzed 1 hr later by flow cytometry. The summary bar charts show the ratio of
hCD20Tg to WT B cells (non depleted, used as an internal control) 1 h after injection of the indicated dose
of rituximab or GA101. (B–E) Intravital imaging of the liver of hCD20Tg mice during anti-CD20 treatment. Kupffer cells (green) and B cells (red) were labeled using anti-F4/80 Ab and anti-B220 Fab fragments,
respectively (B) Representative curve showing the number of engulfed B cells (normalized per mm3) in Figure 4. Obinutuzumab triggers enhanced Ab-dependent phagocytosis by Kupffer cells in vivo
compared to rituximab. (A) Splenocytes from WT or hCD20Tg mice were isolated, labeled and co- Figure 4. Obinutuzumab triggers enhanced Ab-dependent phagocytosis by Kupffer cells in vivo Figure 4. Obinutuzumab triggers enhanced Ab-dependent phagocytosis by Kupffer cells in vivo
compared to rituximab. (A) Splenocytes from WT or hCD20Tg mice were isolated, labeled and co-
transferred into WT recipient mice. After 24 h, mice were treated i.v. with different doses of rituximab or
GA101 and blood was analyzed 1 hr later by flow cytometry. The summary bar charts show the ratio of
hCD20Tg to WT B cells (non depleted, used as an internal control) 1 h after injection of the indicated dose
of rituximab or GA101. (B–E) Intravital imaging of the liver of hCD20Tg mice during anti-CD20 treatment. Kupffer cells (green) and B cells (red) were labeled using anti-F4/80 Ab and anti-B220 Fab fragments,
respectively. (B) Representative curve showing the number of engulfed B cells (normalized per mm3) in
the liver following 0.4 μg GA101. (C) Figure shows representative two-photon images before and after
treatment with low doses of GA101 (0.4 μg), highlighting efficient B cell phagocytosis by Kupffer cells (white
squares and insets). Scale bar, 25 μm. (D) Representative two-photon images highlighting the absence of
B cell phagocytosis following 0.4 μg rituximab. Scale bar, 20 μm. (E) Each line represents the cell behavior
after anti-CD20 injection. Green squares represent cicrculating B cells, yellow squares represent contact
between a B and Kupffer cell and red squares represent engulfed B cells. Results and Discussion Representative of 2–4 independent
experiments. Results are shown as mean ± SEM. Significance was assessed using an unpaired Student t-test. circulate in liver sinusoids of mice injected with the same dose of rituximab (Fig. 4B–E, Movie S3 and 4). Thus,
distinct features of anti-CD20 Ab such as glycoengineering, but also potentially epitope specificity, type I versus
type II or ability to induce antigenic modulation17,21 may confer variable Kupffer cell-mediated depletion activity
during therapy.if g
py
In summary, we have used five different mAbs directed against the murine or the human CD20 molecule to
show that antibody-dependent phagocytosis by Kupffer cells is a general mechanism for the systemic depletion
of circulating B cells. In addition, we provide evidence that the improved potency of glycoengineered anti-CD20
Abs in mediating B cell depletion in vivo is linked to their enhanced capacity to trigger Kupffer cell-mediated B
cell arrest and subsequent phagocytosis. Future work could address whether additional mechanisms contribute
to the elimination of non-circulating malignant B cells. Intravital imaging may help optimize mAbs therapy by
assessing how specific Ab modifications may finely tune their mode of action in vivo. 1. Lim, S. H. et al. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica 95, 135–143 (2010).
2. Chan, A. C. & Carter, P. J. Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol 10, 301–316 (2010).
3. Maloney, D. G. Anti-CD20 antibody therapy for B-cell lymphomas. The New England journal of medicine 366, 2008–2016 (2012). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Glennie, M. J. French, R. R. Cragg, M. S. & Taylor, R. P. Mechanisms of killing by anti-CD20 monoclonal antibodies. Mol Immuno
44, 3823–3837 (2007).h 5. Montalvao, F. et al. The mechanism of anti-CD20-mediated B cell depletion revealed by intravital imaging. J Clin Invest 123
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II. www.nature.com/scientificreports/ Biotechnology and bioengineering 93, 851–861 (2006).h gy
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7. Beers, S. A. et al. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood 115
5191–5201 (2010).fi 18. Mossner, E. et al. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 anti
with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood 115, 4393–4402 (2010). f
9. Herter, S. et al. Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab
in vitro and in xenograft models. Molecular cancer therapeutics 12, 2031–2042 (2013). in vitro and in xenograft models. Molecular cancer therapeutics 12 gt
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20. Goede, V. et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med 370, 1101–
(2014).f 1. Tipton, T. R. et al. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies
Blood 125, 1901–1909 (2015). 21. Tipton, T. R. et al. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies. Blood 125, 1901–1909 (2015). 21. Tipton, T. R. et al. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies. Blood 125, 1901–1909 (2015). Author Contributions C.L.G., F.M., C.A.G. and P.B. designed research, C.L.G., F.M., S.C., D.M., B.B. and Z.G. performed experiments,
C.L.G., F.M., S.C., D.M., B.B. and P.B. analyzed the data, D.M., M.P., O.F. and C.A.G. provided critical reagents,
C.L.G. and P.B. wrote the manuscript. Acknowledgements g
We wish to thank the members of the Bousso laboratory for critical review of the manuscript, This work was
supported by Institut Pasteur, Inserm, Fondation pour la Recherche Médicale, a Starting Grant from the European
Research Council (ERC) and a grant from the Institut ROCHE de Recherche et Médecine Translationnelle. We g
We wish to thank the members of the Bousso laboratory for critical review of the manuscript, This work was
supported by Institut Pasteur, Inserm, Fondation pour la Recherche Médicale, a Starting Grant from the European
Research Council (ERC) and a grant from the Institut ROCHE de Recherche et Médecine Translationnelle. We
thank Morgane Cheminant, Pierre Bruhns, Hugo Mouquet for providing reagents and Christian Klein, Oliver
Ast and Erwin van Puijenbroek for the generation and production of the glycoengineered muCD20 Ab 18B12. Research Council (ERC) and a grant from the Institut ROCHE de Recherche et Médecine Translationnelle. We
thank Morgane Cheminant, Pierre Bruhns, Hugo Mouquet for providing reagents and Christian Klein, Oliver
Ast and Erwin van Puijenbroek for the generation and production of the glycoengineered muCD20 Ab 18B12. thank Morgane Cheminant, Pierre Bruhns, Hugo Mouquet for providing reagents and Christian Klein, Oliver
Ast and Erwin van Puijenbroek for the generation and production of the glycoengineered muCD20 Ab 18B12. References 1. Lim, S. H. et al. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica 95, 135–143 (2010). 2. Chan, A. C. & Carter, P. J. Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol 10, 301–316 (2010). 3. Maloney, D. G. Anti-CD20 antibody therapy for B-cell lymphomas. The New England journal of medicine 366, 2008–2016 (2012). Scientific Reports | 6:34382 | DOI: 10.1038/srep34382 5 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: M.P., O.F., C.A.G are all employees of Roche Glycart AG. C.L.G., F.M., S.C.,
D.M., B.B., Z.G., P.B. received funding from Institut Roche de Recherche et Médecine Translationnelle. Competing financial interests: M.P., O.F., C.A.G are all employees of Roche Glycart AG. C.L.G., F.M., S.C.,
D.M., B.B., Z.G., P.B. received funding from Institut Roche de Recherche et Médecine Translationnelle. How to cite this article: Grandjean, C. L. et al. Intravital imaging reveals improved Kupffer cell-mediated
phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies. Sci. Rep. 6, 34382; doi: 10.1038/
srep34382 (2016). How to cite this article: Grandjean, C. L. et al. Intravital imaging reveals improved Kupffer cell-mediated
phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies. Sci. Rep. 6, 34382; doi: 10.1038/
srep34382 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 6 Scientific Reports | 6:34382 | DOI: 10.1038/srep34382
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Fatores associados à procura de serviços de saúde entre escolares brasileiros: uma análise da Pesquisa Nacional de Saúde do Escolar (PeNSE), 2012
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Correspondência
M. M. Oliveira
Secretaria de Vigilância em
Saúde, Ministério da Saúde.
SAF Sul, Trecho 2, Lotes
05/06, Bloco F, Torre I,
Brasília, DF
70070-600, Brasil.
maxomoura@gmail.com 1 Secretaria de Vigilância em
Saúde, Ministério da Saúde,
Brasília, Brasil.
2 Universidade Federal dos
Vales do Jequitinhonha e
Mucuri, Diamantina, Brasil. 1603
ARTIGO ARTICLE 1603
ARTIGO ARTICLE 1603
ARTIGO ARTICLE Factors associated with the demand for health
services by Brazilian adolescents: the National
School Health Survey (PeNSE), 2012 Factores asociados con el uso de los servicios de
salud por los escolares brasileños: un análisis de la
Encuesta Nacional de Salud Escolar (PeNSE), 2012 Max Moura de Oliveira 1
Silvânia Suely Caribé de Araújo Andrade 1
Maryane Oliveira Campos 2
Deborah Carvalho Malta 1 Max Moura de Oliveira 1
Silvânia Suely Caribé de Araújo Andrade 1
Maryane Oliveira Campos 2
Deborah Carvalho Malta 1 School Health; Adolescent Behavior; Adolescent;
Health Services Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 http://dx.doi.org/10.1590/0102-311X00165214 Introdução 2003, descreveu que pessoas do sexo feminino,
com 60 anos ou mais, de cor branca, com menor
nível socioeconômico, sem cobertura por plano
de saúde e com autopercepção de saúde ruim
tiveram maior probabilidade de utilizar a ESF. As
pessoas que tinham maior nível socioeconômico
e cobertura por plano de saúde utilizaram mais
outros serviços de saúde. Todavia, a implantação
da ESF foi uma ferramenta auxiliar na melhoria
das condições de saúde da população mais po
bre, minimizando as desigualdades em saúde 5. A utilização dos serviços de saúde é influenciada
por fatores individuais, como o perfil de necessi
dades de saúde, valores e preferências pessoais;
epidemiológicos e sociodemográficos, assim co
mo pelo acesso a estes serviços, associado tam
bém à sua oferta e qualidade do cuidado 1,2,3. O
uso dos serviços de saúde compreende tanto o
contato direto − consultas médicas e hospitali
zações, quanto o contato indireto − realização de
exames preventivos e diagnósticos. O processo
de utilização dos serviços de saúde é resultante
da interação do comportamento do indivíduo
que procura cuidados, e normalmente é respon
sável pelo primeiro contato; e do profissional que
conduz este cuidado e que na maioria das vezes
define o tipo e a intensidade de recursos para
resolver os problemas de saúde dos usuários 1. O estudo da utilização dos serviços de saúde é
um indicador importante para avaliação destes,
medindo a qualidade e a equidade da atenção à
saúde e possibilitando a orientação de políticas
públicas 4,5,6,7. Os estudos sobre utilização de serviços de
saúde entre adolescentes são pouco frequentes
no país, sendo na maioria locais 10,11. Destaca-se
que os adolescentes constituem um grupo priori
tário para promoção da saúde em razão dos com
portamentos que os expõem a diversas situações
de riscos para a saúde, uma vez que neste perío
do ocorrem intensas transformações cognitivas,
emocionais, sociais, físicas e hormonais. Nesta
fase da vida, ocorre experimentação de novos
comportamentos, que podem ser tanto fatores
de risco para a saúde, como exemplo, tabagismo
e sexo não protegido, quanto de proteção, como
alimentação saudável e escovação dentária 12. Logo, é importante estudar o comportamento
deste grupo etário frente à procura de serviços de
saúde. Segundo dados do censo demográfico, em
2000, os adolescentes de 10-14 anos e de 15-19
anos representavam 10,2% (17.348.067) e 10,6%
(17.939.815) em relação à população total, res
pectivamente. Abstract Os objetivos foram descrever a procura por ser
viços/profissionais de saúde por escolares bra
sileiros e identificar fatores associados. Foram
analisados dados da Pesquisa Nacional de Saú
de do Escolar 2012; estimadas as prevalências e
seus respectivos intervalos de 95% de confiança
(IC95%) do uso de serviços de saúde entre os es
colares. Foi feita regressão logística ajustada por
idade. Metade dos estudantes buscaram serviços
de saúde; a procura foi maior no sexo feminino;
os fatores associados foram: cor branca, escola
privada; escolaridade da mãe 12 anos ou mais;
ter tido relações sexuais; sofrido ferimento, dor
de dentes, tentativa de manter, perder ou ganhar
peso, chiado no peito nos últimos 12 meses, ter
hábitos de higiene adequados e conhecimento
dos pais sobre o que os filhos fazem no tempo li
vre. A busca por serviços de saúde foi maior no
sexo feminino e esteve associada com melhores
condições socioeconômicas, presença de sinto
mas e de comportamentos de risco/proteção. The objectives of this study were to describe the
demand for health services by adolescents and to
identify associated factors. The study analyzed
data from the National School Health Survey
2012 and calculated prevalence rates and 95%
confidence intervals (95%CI). Data analysis used
age-adjusted logistic regression. Half of all stu
dents required health services and the propor
tion was higher in girls. Factors associated with
demand for health services were white skin color,
enrollment in private school, maternal schooling
≥ 12 years, sexual activity, injuries, toothache, at
tempts to maintain, lose, or gain weight, wheez
ing, appropriate hygiene, and parents knowing
what their children did in their free time. The
highest demand for health services was mainly
by girls, and demand was associated with socio-
demographic characteristics, symptoms, and
health risk/protective behaviors. Correspondência
M. M. Oliveira
Secretaria de Vigilância em
Saúde, Ministério da Saúde. SAF Sul, Trecho 2, Lotes
05/06, Bloco F, Torre I,
Brasília, DF
70070-600, Brasil. maxomoura@gmail.com School Health; Adolescent Behavior; Adolescent;
Health Services School Health; Adolescent Behavior; Adolescent;
Health Services School Health; Adolescent Behavior; Adolescent;
Health Services Saúde Escolar; Comportamento do Adolescente;
Adolescente; Serviços de Saúde Saúde Escolar; Comportamento do Adolescente;
Adolescente; Serviços de Saúde Saúde Escolar; Comportamento do Adolescente;
Adolescente; Serviços de Saúde http://dx.doi.org/10.1590/0102-311X00165214 Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Oliveira MM et al. 1604 Introdução Em 2010, estes percentuais foram
reduzidos para 9% (17.348.067) para os adoles
centes de 10-14 anos e 8,9% (17.939.815) para os
adolescentes de 15-19 anos 13. Em ambos os cen
sos, a população de adolescentes corresponde a
cerca de 20% da população brasileira, ratificando
a importância de estudos neste grupo específico. Dados da Pesquisa Nacional por Amostra
de Domicílios (PNAD) de 2003 e 2008 no Brasil
mostraram que o Sistema Único de Saúde (SUS)
respondeu por 58,5% e 56,7% dos atendimentos,
respectivamente, realizando a maior parte das
internações, vacinação e consultas, mas somente
um terço das consultas odontológicas. No ano de
2008, observou-se que o relato de uso dos ser
viços de saúde ofertados pelo SUS reduzia con
forme o aumento na renda e na escolaridade;
houve decréscimo da proporção dos que procu
raram serviços de saúde para ações de prevenção
e aumento da busca por atendimento devido a
problemas odontológicos e acidentes e lesões, e
reabilitação 8. Em 2009, ocorreu a primeira edição da Pes
quisa Nacional de Saúde do Escolar (PeNSE) com
a finalidade de conhecer os fatores relacionados
ao risco e proteção à saúde dos adolescentes
brasileiros 14. Em 2012, foi realizada à segunda
edição, na qual foi inserido um módulo no ques
tionário sobre procura por serviços e profissio
nais de saúde 15. O presente estudo teve como
objetivo descrever a procura por serviços ou
profissionais de saúde por escolares brasileiros
e identificar os possíveis comportamentos de ris
co, condições de saúde e alguns fatores familiares
associados a essa procura. Ao utilizar dados da PNAD 2003 para analisar
diferenças entre usuários e não-usuários do SUS,
verificou-se predomínio de mulheres, crianças,
pretos e pardos, baixa escolaridade e renda entre
os que utilizaram serviços do SUS; associação en
tre estado de saúde regular/ruim e utilização dos
serviços do SUS, e entre o atendimento pelo SUS
e usuários de baixa escolaridade e renda 2. Um es
tudo com amostra representativa de dois distritos
de São Paulo, Brasil, em 2001, sobre a utilização
de serviços de saúde após implantação da Estra
tégia Saúde da Família (ESF) apontou que a renda
e a escolaridade não se constituíram fatores que
diferenciassem de forma significativa o perfil de
utilização de serviços de saúde e de procura por
assistência, indicando que a ESF pode contribuir
para maior equidade nessas situações 9. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Amostragem A amostra da PeNSE 2012 foi representativa para
o Brasil, as cinco grandes regiões, e as 26 capitais
dos estados brasileiros e do Distrito Federal. Para
o plano amostral, foram definidos 27 estratos ge
ográficos correspondendo a todas as capitais de
estados e Distrito Federal. Os demais municípios
foram agrupados dentro de cada uma das cinco
grandes regiões geográficas, formando cinco es
tratos. A amostra de cada estrato geográfico foi
alocada proporcionalmente ao número de esco
las segundo a dependência administrativa das
mesmas (pública e privada). Para cada um des
ses estratos, uma amostra de conglomerados em
dois estágios foi selecionada: 1o estágio - escolas;
2o estágio - turmas elegíveis nas escolas selecio
nadas (9o ano do Ensino Fundamental) 15. Para verificar os fatores associados à procura
por serviços ou profissionais de saúde pelos esco
lares, a variável desfecho foi oriunda da pergun
ta: “Nos últimos 12 meses você procurou algum
serviço ou profissional de saúde para atendimen
to relacionado à própria saúde?” (Respostas: sim;
não). Métodos Este estudo foi uma análise de dados provenien
tes da PeNSE 2012. A PeNSE é um estudo trans
versal trienal realizado pelo Instituto Brasileiro Outra pesquisa de base populacional rea
lizada em Porto Alegre, Rio Grande do Sul, em Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1605 médico particular; hospital; consultório odon
tológico; farmácia; laboratório ou clínica para
exames complementares; pronto-socorro ou
emergência; consultório de outro profissional de
saúde (fonoaudiólogo, psicólogo, etc.); serviço
de especialidades médicas ou policlínica; servi
ço de atendimento domiciliar; Outro); (c) “Nos
últimos 12 meses, quantas vezes você procurou
por algum posto de saúde (UBS)?” (respostas:
nenhuma vez; 1 ou 2 vezes nos últimos 12 meses;
3-5 vezes nos últimos 12 meses; 6-9 vezes nos úl
timos 12 meses; 10 ou mais vezes nos últimos 12
meses); (d) “Você foi atendido, na última vez que
procurou algum posto de saúde (UBS), nestes
últimos 12 meses?” (respostas: sim; não). de Geografia e Estatística (IBGE), em parceria
com o Ministério da Saúde e o Ministério da Edu
cação, cuja população de estudo foi composta
por escolares do 9o ano do Ensino Fundamental
(antiga 8a série) de escolas públicas e privadas
brasileiras 15. Amostragem Os fatores investigados potencialmente
associados a esta busca foram distribuídos por
grupos de variáveis (domínios): (a) característi
cas sociodemográficas dos escolares: sexo (femi
nino; masculino); idade (≤ 13 anos, 14 anos e > 15
anos); raça/cor: (branca, preta, amarela, parda e
indígena); dependência administrativa da escola
(pública: privada); região de residência (Sudeste;
Norte; Nordeste; Sul; Centro-oeste); (b) compor
tamentos de risco: tabagismo atual - nos últimos
30 dias (não; sim); tentativa de parar de fumar -
dentre os escolares que fumaram nos últimos 12
meses (nunca fumou, sim e não); uso de drogas
nos últimos 30 dias (não, sim); consumo abusivo
de álcool (não, sim); comportamento sexual (não
teve relação sexual; teve relação sexual com pre
servativo; teve relação sexual sem preservativo);
(c) situações de saúde: ter sofrido ferimento nos
últimos 12 meses (não; sim); atitude em relação
ao peso corporal (fazendo nada; tentando perder
peso; tentando ganhar peso; tentando manter
o mesmo peso); chiado no peito nos últimos 12
meses (não; sim); presença de dor de dente nos
últimos seis meses (não; sim) e atitudes relativas
a práticas de cuidado com o corpo como o hábito
de lavar as mãos antes de comer e após ida ao
banheiro (não; sim); (d) características familia
res: escolaridade da mãe em anos (0-8; 9-11; 12
e mais); moradia de pelo menos um dos pais na
mesma residência do adolescente (nenhum dos
pais; apenas com a mãe ou pai; ambos); conhe
cimento dos pais ou responsáveis sobre o tem
po livre dos filhos (nunca; raramente/às vezes;
maioria das vezes/sempre). No estrato formado pelos municípios não
capitais, as unidades primárias de amostragem
foram os agrupamentos de municípios, as uni
dades secundárias de amostragem foram as es
colas, e as turmas dessas escolas, as unidades
terciárias de amostragem. Todos os alunos das
turmas selecionadas, presentes no dia da cole
ta de dados, foram convidados a participar da
pesquisa. Outros detalhes do processo de amos
tragem podem ser consultados no relatório de
pesquisa publicado 15. Para que todos os alunos respondentes da
pesquisa representassem os escolares matricu
lados no 9o ano do Ensino Fundamental, que
frequentavam regularmente as aulas, foi calcu
lado peso amostral que considerou os estratos já
mencionados 15. Para a coleta de dados utilizou-
se um questionário estruturado autoaplicável,
respondidos em um smartphone, com cerca de
120 perguntas, organizadas em módulos temáti
cos, dentre eles, uso de serviços de saúde 15. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Variáveis estudadas Foram utilizadas para descrever o uso de ser
viços de saúde entre os escolares as seguintes
variáveis: (a) “Nos últimos 12 meses você pro
curou algum serviço ou profissional de saúde
para atendimento relacionado à própria saúde?”
(respostas: sim; não); (b) “Nos últimos 12 me
ses, qual serviço de saúde você procurou mais
frequentemente?” (respostas: unidade básica
de saúde (UBS) – posto de saúde; consultório Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Oliveira MM et al. 1606 Oliveira MM et al. Análise estatística consultório odontológico (8,1%), laboratório ou
exames complementares (2,9%) e consultório
de outros profissionais de saúde (1,5%). Entre os
alunos de escolas privadas a procura por consul
tório particular foi maior (42,9%). Inversamente,
entre os estudantes de escolas públicas, a pro
cura foi maior pela UBS (55,8%; IC95%: 55,0%-
56,6%) (Tabela 1). Por região de residência, as
prevalências foram estatisticamente diferentes
(p < 0,01), sendo maior no Sudeste (48,9%) e me
nor no Centro-oeste (43,8%). Inicialmente foram estimadas as prevalências e
seus respectivos intervalos de 95% de confian
ça (IC95%) do uso de serviços de saúde entre os
escolares (tipo de serviço, vezes que procurou o
posto de saúde e se foi atendido na última vez
que procurou atendimento), estratificadas por
sexo e dependência administrativa da escola. To
das as variáveis estudadas tiveram completitu
de maior que 99%, exceto escolaridade da mãe
(82,7%), dor de dente (91%) e comportamento
sexual (98,3%). As magnitudes das associações
entre o desfecho (procura por serviços ou pro
fissionais de saúde) e as variáveis independentes
foram medidas pelo odds ratio (OR) e seu IC95%,
obtidos por meio de modelagem múltipla por re
gressão logística, tendo a primeira categoria de
cada variável como referência. A maior frequência dos pesquisados procu
rou a UBS nos últimos 12 meses uma ou duas ve
zes (52,9%), todavia 4% dos estudantes buscaram
este serviço dez ou mais vezes. Houve diferença
significativa por sexo entre os que buscaram a
UBS de três a cinco vezes no último ano, sendo
maior entre estudantes do sexo feminino (17%)
e por dependência administrativa da escola en
tre todas as categorias de frequência por busca à
UBS (Tabela 1). Primeiramente, foi realizada uma análise uni
variada dentro de cada domínio. As variáveis que
se apresentaram associadas com nível de signifi
cância p ≤ 0,20 foram selecionadas para o modelo
múltiplo em cada domínio. Variáveis estudadas Posteriormente, foi
construído um modelo final considerando todas
estas variáveis. Os domínios foram incluídos de
forma sequencial: primeiro as características dos
escolares, seguidas por comportamentais, condi
ções de saúde e por último os fatores da família. O modelo final foi ajustado pela variável idade,
para controlar os efeitos dos comportamentos de
risco estudados. A proporção de escolares atendidos na últi
ma vez que procurou alguma UBS nos últimos 12
meses foi de 85,1%, sendo que escolares do sexo
feminino relataram mais atendimentos (86,1%). Não houve diferença quanto à dependência ad
ministrativa da escola (Tabela 1). Na análise de regressão logística múltipla, o
modelo final foi ajustado pela variável idade e
apontou que a procura pelos serviços de saúde
foi menor entre os estudantes do sexo masculino
(OR = 0,90; IC95%: 0,86-0,94); que referiram raça/
cor preta (OR = 0,90; IC95%: 0,84-0,97) e amarela
(OR = 0,85; IC95%: 0,76-0,96) quando compara
dos aos estudantes brancos. Segundo o tipo de
dependência administrativa da escola, alunos
das escolas privadas procuraram 1,29 vezes mais
(IC95%: 1,21-1,38) os serviços de saúde do que
aqueles das escolas públicas. Quando compara
dos aos residentes da Região Sudeste, escolares
das regiões Sul (OR = 0,86; IC95%: 0,81-0,92) e
Centro-oeste (OR = 0,79; IC95%: 0,74-0,84) pro
curaram menos os serviços de saúde. Observou-
se associação positiva entre a busca por serviços
de saúde e ter tido relação sexual com preserva
tivo (OR = 1,29; IC95%: 1,21-1,36) comparado aos
que nunca tiveram relação sexual (Tabela 2). As análises dos dados foram realizadas no
software Stata versão 11.0 (Stata Corp., College
Station, Estados Unidos), utilizando o coman
do survey para amostra complexa. A PeNSE foi
aprovada na Comissão Nacional de Ética em
Pesquisas do Ministério da Saúde, sob o pare
cer no 192/2012 referente ao Registro no 16805 do
CONEP/MS em 27 de março de 2012. As bases de
dados utilizadas para este estudo foram armaze
nadas de modo sigiloso e utilizadas apenas para
este fim. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Resultados A PeNSE em 2012 entrevistou 109.104 estudan
tes do 9o ano do Ensino Fundamental de escolas
públicas e privadas de todo o país. Entre os esco
lares entrevistados, 48% procuraram por algum
serviço ou profissional de saúde nos últimos 12
meses anteriores à pesquisa. A UBS foi o serviço
referido por 47,5% dos adolescentes. Destaca-
se que estudantes que se declararam brancos ti
veram o maior percentual de procura pela UBS
(49,6%) (dados não apresentados em tabela),
escolares do sexo feminino procuraram mais A procura por serviços de saúde foi maior en
tre os que referiram algum ferimento (OR = 1,29;
IC95%: 1,20-1,40), entre os estudantes que men
cionaram o hábito de lavar as mãos (OR = 1,31;
IC95%: 1,25-1,38), entre aqueles que tiveram al
guma tentativa de ação em relação ao peso cor
poral: perda (OR = 1,29; IC95%: 1,22-1,37), ga
nho (OR = 1,29; IC95%: 1,21-1,38) e manutenção
(OR = 1,4; IC95%: 1,32-1,49); entre os adolescen
tes que referiram chiado no peito (OR = 1,73;
IC95%: 1,64-1,83); e dor de dente (OR = 1,33; Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1607 1607 Tabela 1 Prevalência (e respectivos IC95%) de escolares do 9o ano do Ensino Fundamental que procuraram algum serviço ou profissional de saúde nos últimos 12
meses segundo sexo e dependência administrativa da escola. Pesquisa Nacional de Saúde do Escolar (PeNSE), Brasil, 2012. Prevalência (e respectivos IC95%) de escolares do 9o ano do Ensino Fundamental que procuraram algum serviço ou profissional de saúde nos últimos 12
meses segundo sexo e dependência administrativa da escola. Pesquisa Nacional de Saúde do Escolar (PeNSE), Brasil, 2012. Resultados Procura por serviço de saúde
Total
% (IC95%)
Sexo
Dependência administrativa
Masculino
Feminino
Privada
Pública
% (IC95%)
% (IC95%)
% (IC95%)
% (IC95%)
Nos últimos 12 meses (n = 108.647)
Sim
48,0 (47,6-48,5)
-
-
-
-
Não
51,7 (51,2-52,6)
-
-
-
-
Tipo de serviço (n = 53.317)
Unidade Básica de Saúde
47,5 (46,8-48,2)
47,4 (46,4-48,4)
47,6 (46,7-48,5)
14,1 (13,1-15,2)
55,8 (55,0-56,6)
Consultório particular
22,2 (21,7-22,8)
21,8 (21,0-22,6)
22,6 (21,9-23,4)
42,9 (41,5-44,4)
17,1 (16,5-17,7)
Hospital
10,2 (9,8-10,6)
10,9 (10,3-11,6)
9,5 (9,0-10,1)
12,9 (11,9-13,9)
9,5 (9,1-10,0)
Consultório odontológico
7,1 (6,7-7,4)
5,9 (5,5-6,4)
8,1 (7,6-8,6)
11,2 (10,3-12,2)
6,0 (5,7-6,4)
Farmácia
2,7 (2,5-3,0)
3,1 (2,7-3,4)
2,4 (2,2-2,7)
3,9 (3,4-4,5)
2,4 (2,2-2,7)
Laboratório ou exames complementares
2,6 (2,4-2,8)
2,2 (2,0-2,5)
2,9 (2,6-3,2)
4,4 (3,9-5,1)
2,1 (1,9-2,4)
Pronto socorro ou emergências
1,8 (1,6-1,9)
1,6 (1,4-1,9)
1,9 (1,7-2,2)
3,4 (2,9-4,0)
1,4 (1,2-1,5)
Consultório de profissionais de saúde
1,7 (1,6-1,9)
2,0 (1,7-2,3)
1,5 (1,3-1,8)
2,9 (2,4-3,4)
1,4 (1,3-1,6)
Serviço de especialidades médicas
0,1 (0,0-0,1)
0,9 (0,8-1,2)
0,1 (0,5-0,8)
0,9 (0,6-1,2)
0,7 (0,6-0,9)
Serviço de atendimento domiciliar
0,0 (0,0-0,0)
0,0 (0,0-0,0)
0,0 (0,0-0,0)
0,1 (0,0-0,2)
0,1 (0,0-0,2)
Outros
2,5 (2,3-2,7)
2,9 (2,6-3,2)
2,2 (1,9-2,5)
2,6 (2,2-3,1)
2,5 (2,3-2,7)
Vezes que procurou o posto de saúde nos
últimos 12 meses (n = 53.312)
Nenhuma
21,4 (20,8-21,9)
21,5 (20,7-22,4)
21,2 (20,5-22,0)
48,5 (47,0-50,0)
14,6 (14,1-15,2)
1 ou 2
52,9 (52,3-53,6)
53,2 (52,2-54,2)
52,7 (51,8-53,6)
36,7 (35,3-38,2)
57,0 (56,2-57,7)
3-5
16,3 (15,8-16,8)
15,4 (14,7-16,2)
17,0 (16,3-17,7)
9,9 (9,0-10,8)
17,9 (17,3-18,5)
6-9
4,6 (4,3-4,9)
4,5 (4,1-5,0)
4,7 (4,3-5,1)
2,5 (2,0-3,0)
5,2 (4,8-5,5)
10 ou mais
4,0 (3,8-4,3)
4,2 (3,8-4,6)
3,9 (3,5-4,2)
1,8 (1,4-2,2)
4,6 (4,3-4,9)
Atendido na última vez que procurou o
posto de saúde (n = 40.120)
Sim
85,0 (84,4-85,5)
83,7 (82,8-84,5)
86,1 (85,4-86,8)
85,5 (84,0-87,0)
84,9 (84,3-85,5)
Não
14,9 (14,3-15,4)
16,0 (15,2-16,9)
13,8 (13,1-14,5)
14,3 (12,9-15,9)
14,9 (14,4-15,5)
IC95%: intervalo de 95% de confiança. IC95%: 1,26-1,41). No domínio características fa
miliares, os fatores associados com a busca por
serviços de saúde foram: escolaridade materna
igual ou superior a 12 anos de estudo (OR = 1,31;
IC95%: 1,22-1,40), conhecimento dos pais sobre
o tempo livre dos filhos (OR = 1,32; IC95%: 1,24-
1,42) (Tabela 2). no semelhantemente à população adulta 16,17. A PNAD 1998 revelou que a procura regular por
serviços de foi maior em mulheres em todas as
faixas etárias estudadas, exceto em menores de
quatro anos 18. Resultados As mulheres tendem a procurar
mais os serviços de saúde, por serem mais pre
ocupadas com este assunto e isto aumenta su
as chances de diagnosticar doenças e contribui
para ampliar o acesso a práticas de promoção,
prevenção e tratamento 19,20. Os serviços mais
procurados também foram compatíveis com
dados da PNAD 2008 em adultos que utilizaram
mais as UBS 20. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Discussão Os dados deste trabalho apontam que, entre os
escolares entrevistados, cerca de metade procu
rou por algum serviço ou profissional de saúde
nos últimos 12 meses; e as UBS foram o serviço
mais acessado. A busca por serviços de saúde
entre os adolescentes foi maior no sexo femini Os escolares residentes na Região Sudeste fo
ram os que mais procuraram os serviços de saú
de. Dentre as cinco regiões geográficas, apesar da
Região Sudeste cobrir apenas 11% do território Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 1608 Oliveira MM et al. Tabela 2 Características e fatores associados dos escolares do 9º ano do Ensino Fundamental que procuraram algum serviço ou profissional de saúde nos últimos 12
meses e variáveis. Pesquisa Nacional de Saúde do Escolar (PeNSE), Brasil, 2012. Discussão Variáveis
Procura por serviços ou
profissionais de saúde *
Sim (IC95%)
OR bruta (IC95%)
Valor de p
OR ajustada **
(IC95%)
Valor de p
Características dos escolares
Sexo (n = 108.647)
Feminino
49,0 (48,3-49,6)
Referência
Referência
Masculino
47,0 (46,3-47,7)
0,92 (0,89-0,96)
0,01
0,90 (0,86-0,94)
0,01
Idade [anos] (n = 108.647)
≤ 13
49,3 (48,3-50,3)
Referência
-
-
14
47,9 (47,2-48,6)
0,95 (0,90-0,99)
0,03
-
-
≥ 15
47,2 (46,4-48,0)
0,92 (0,87-0,97)
0,01
-
-
Raça/cor (n = 108.587)
Branca
49,6 (48,8-50,4)
Referência
Referência
Preta
45,2 (43,9-46,5)
0,84 (0,79-0,89)
0,01
0,90 (0,84-0,97)
0,01
Amarela
46,6 (44,4-48,9)
0,89 (0,81-0,98)
0,02
0,85 (0,76-0,96)
0,01
Parda
47,8 (47,1-48,5)
0,93 (0,89-0,97)
0,01
0,96 (0,91-1,01)
0,12
Indígena
46,5 (44,0-49,0)
0,88 (0,80-0,98)
0,02
0,88 (0,78-1,00)
0,05
Dependência administrativa da
escola (n = 108.647)
Pública
46,4 (45,9-47,0)
Referência
Referência
Privada
55,7 (54,6-56,8)
1,45 (1,38-1,53)
0,01
1,29 (1,21-1,38)
0,01
Região de residência
(n = 108.647)
Sudeste
48,9 (48,0-49,8)
Referência
Referência
Norte
48,0 (47,2-48,8)
0,96 (0,92-1,01)
0,13
0,95 (0,89-1,00)
0,06
Nordeste
48,9 (48,0-49,7)
1,0 (0,95-1,05)
0,93
1,04 (0,98-1,10)
0,2
Sul
46,2 (45,2-47,1)
0,90 (0,85-0,94)
0,01
0,86 (0,81-0,92)
0,01
Centro-oeste
43,8 (43,0-44,6)
0,81 (0,78-0,85)
0,01
0,79 (0,74-0,84)
0,01
Comportamentos de risco
Tabagismo atual (n = 108.542)
Não
47,8 (47,3-48,3)
Referência
-
-
Sim
52,1 (50,0-54,3)
1,19 (1,09-1,30)
0,01
-
-
Tentativa de parar de fumar (n =
108.546)
Nunca fumou
47,8 (47,3-48,3)
Referência
Sim
53,2 (50,9-55,4)
1,24 (1,13-1,36)
0,01
-
-
Não
48,5 (45,3-51,7)
1,03 (0,90-1,17)
0,68
-
-
Uso de drogas (n = 108.503)
Não
47,9 (47,5-48,4)
Referência
-
-
Sim
52,5 (49,4-55,7)
1,20 (1,06-1,37)
0,01
-
-
Consumo abusivo de álcool
(n = 108.409)
Não
47,3 (46,7-47,8)
Referência
-
-
Sim
50,3 (49,3-51,2)
1,13 (1,08-1,18)
0,01
-
-
(
i
) Cad. Discussão Um
estudo que tratou da desigualdade de acesso em
consultas médicas, utilizando dados da PNAD
dos anos de 1998, 2003 e 2008, identificou que
na Região Sudeste a posse de plano particular de
saúde é o fator individual que auxilia na compre
ensão do acesso desigual nos serviços analisados
em favor dos mais ricos 21. Quanto mais elevada
a situação socioeconômica dos indivíduos ou das
regiões, melhor o estado de saúde e maior o aces
so aos serviços de saúde 23,24. tar não só a infecção por doenças sexualmente
transmissíveis (DST), como provocar gravidez
indesejada 30. Acredita-se que a maior procura
por serviços de saúde pelos adolescentes que já
tiveram relações sexuais pode estar associada à
prevenção de DST e aconselhamentos quanto a
métodos contraceptivos. No presente estudo, a busca por serviços de
saúde foi associada com o relato de ferimento,
tentativa de ação em relação ao peso corporal,
chiado no peito e dor de dente. As necessidades
de saúde – morbidade, gravidade e urgência da
doença – estão entre os determinantes de acesso
e utilização dos serviços de saúde apontados pela
literatura 30; assim como o comportamento do
indivíduo perante a doença 21,25 e a busca por
práticas de promoção à saúde e prevenção 30. Outra pesquisa apontou que as lesões graves
ocorreram em 10,3% dos adolescentes, em fun
ção de fatores diversos relacionados a atitudes
de risco como não usar cinto de segurança, não
usar capacetes ao andar de moto, uso de álcool,
dentre outras e que estas demandam a procura
de serviços de saúde frequentemente 31. O presente trabalho mostrou associação com
a busca por serviços de saúde: raça/cor branca,
estudar em escola privada e maior escolaridade
materna. Estes adolescentes buscaram mais o
consultório privado. Comparando a PeNSE e a
PNAD, as características socioeconômicas para
busca de serviços de saúde foram semelhantes
entre adultos e adolescentes: a população com
maior renda e escolaridade procura mais fre
quentemente os consultórios privados 15,20. Os
grupos de renda mais baixa procuram menos os
serviços de saúde independente da faixa etária
8,25,26. Em uma pesquisa realizada com 457 esco
lares de 12-17 anos de escolas públicas e privadas
no Município de Niterói, Rio de Janeiro, 47,7%
dos entrevistados procuraram algum serviço de
saúde nos últimos três meses. Discussão Houve uma maior
chance de procura por serviços de saúde entre os
estudantes de escolas privadas quando estão em
tratamento de doenças ou sentem necessidade
desta busca 11. Em um estudo qualitativo que buscou co
nhecer a percepção dos adolescentes quanto à
assistência à saúde e demanda dos serviços na
ESF, em uma escola pública no interior do Ceará,
Brasil, foi evidente que a busca dos adolescentes
pelo serviço de saúde foi motivada pela doen
ça e seus fatores associados; os autores ressal
tam que o modelo médico assistencialista ainda
se encontra como obstáculo à consolidação da
atenção integral proposta pela ESF no que diz
respeito à prevenção e promoção 32. A asma tem elevada prevalência nesta faixa
etária (23,2%) 33 e como doença crônica, justifi
ca o acompanhamento regular nos serviços de
saúde, tanto na atenção primária, quanto na ur
gência. Da mesma forma, eventos relacionados
à saúde bucal são frequentes em adolescentes,
sendo que 17% dos escolares apresentaram dor
de dentes nos últimos seis meses, fato que tam
bém aumenta a procura por serviços de saúde 34. Dentre as práticas de higiene, o hábito de la
var as mãos antes das refeições e após ir ao ba
nheiro foi associado à procura por serviços de
saúde. A promoção destas práticas é um com
ponente que traz implicações amplas no esta
do geral de saúde dos indivíduos 27, como por
exemplo, a prevenção de infecções respiratórias
e de doenças diarreicas. Boas práticas de auto
cuidado, como a higienização das mãos pode
contribuir no desenvolvimento da responsabili
dade perante o próprio bem-estar e na prática de
hábitos saudáveis 28. O conhecimento dos pais sobre o que os ado
lescentes faziam no tempo livre foi outro fator
relacionado à busca por serviços de saúde nes
ta pesquisa. O modelo ecológico de desenvolvi
mento humano, durante os primeiros anos de
vida, aborda a existência de uma complexa in
teração entre os fatores contextuais, incluindo a
influência da comunidade, colegas, escola, famí
lia, entre outros 35. Assim como neste trabalho,
outros estudos observaram a relação das caracte
rísticas familiares nos desfechos estudados entre
adolescentes, como na investigação relacionada
aos fatores de risco e proteção para doenças crô
nicas não transmissíveis 36; tabagismo entre ado Ter relação sexual foi o comportamento que
se manteve associado ao desfecho, comparado
aos que nunca tiveram relação. Discussão Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1609 1609 Tabela 2 (continuação)
Variáveis
Procura por serviços ou
profissionais de saúde *
Sim (IC95%)
OR bruta (IC95%)
Valor de p
OR ajustada **
(IC95%)
Valor de p
Comportamentos de risco
Comportamento sexual
(n = 107.299)
Não teve relação sexual
47,0 (46,4-47,5)
Referência
Referência
Teve relação sexual com
preservativo
51,7 (50,7-52,7)
1,21 (1,16-1,27)
0,01
1,29 (1,21-1,36)
0,01
Teve relação sexual sem
preservativo
48,1 (46,2-50,0)
1,05 (0,97-1,13)
0,27
1,14 (1,03-1,25)
0,01
Situações de saúde
Ter sofrido ferimento (n = 108.323)
Não
47,3 (46,8-47,8)
Referência
Referência
Sim
55,1 (53,7-56,6)
1,37 (1,29-1,46)
0,01
1,29 (1,20-1,40)
0,01
Hábito de lavar as mãos
(n = 108.139)
Não
44,2 (43,5-45,0)
Referência
Referência
Sim
50,4 (49,8-51,0)
1,28 (1,23-1,33)
0,01
1,31 (1,25-1,38)
0,01
Atitude em relação ao peso
corporal (n = 108.398)
Fazendo nada
42,2 (41,5-43,0)
Referência
Referência
Tentando perder peso
52,0 (51,1-52,9)
1,48 (1,41-1,55)
0,01
1,29 (1,22-1,37)
0,01
Tentando ganhar peso
49,4 (48,3-50,5)
1,34 (1,26-1,41)
0,01
1,29 (1,21-1,38)
0,01
Tentando manter o mesmo peso
52,5 (51,4-53,5)
1,51 (1,43-1,59)
0,01
1,40 (1,32-1,49)
0,01
Chiado no peito (n = 108.350)
Não
44,7 (44,2-45,2)
Referência
Referência
Sim
59,4 (58,4-60,4)
1,81 (1,73-1,90)
0,01
1,73 (1,64-1,83)
0,01
Dor de dente (n = 99.310)
Não
46,7 (46,1-47,2)
Referência
Referência
Sim
53,5 (52,4-54,5)
1,31 (1,25-1,38)
0,01
1,33 (1,26-1,41)
0,01
Característica familiar
Escolaridade da mãe [anos]
(n = 90.246)
0-8
47,2 (46,5-47,9)
Referência
Referência
09-11
49,5 (58,6-50,4)
1,10 (1,05-1,15)
0,01
1,05 (1,00-1,11)
0,05
12 e mais
54,4 (56,2-58,6)
1,51 (1,42-1,60)
0,01
1,31 (1,22-1,40)
0,00
Moradia de pelo menos um dos
pais na residência (n = 108.476)
Nenhum dos pais
48,6 (46,7-50,5)
Referência
-
-
Apenas com a mãe ou pai
46,8 (46,0-47,7)
0,93 (0,86-1,01)
0,09
-
-
Ambos
48,6 (48,0-49,2)
1,01 (0,92-1,08)
0,99
-
-
Conhecimento dos pais ou
responsáveis sobre o tempo livre
dos filhos (n = 108.359)
Nunca
42,7 (41,6-43,9)
Referência
Referência
Raramente/Às vezes
44,8 (43,9-45,7)
1,09 (1,02-1,16)
0,01
1,04 (0,97-1,12)
0,27
Maioria das vezes/Sempre
50,9 (50,3-51,5)
1,39 (1,32-1,47)
0,01
1,33 (1,24-1,42)
0,01
IC95%: intervalo de 95% de confiança. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Oliveira MM et al. 1610 brasileiro, representa 43% da população. Toda
via, destaca-se que o Sudeste concentra a maior
oferta de serviços de saúde do país 21, além de
concentrar 56% do produto interno bruto 22. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 Discussão Estudos realiza
dos no Brasil e no mundo mostram que a vida
sexual dos adolescentes tem início cada vez mais
cedo e que a precocidade está associada ao sexo
desprotegido e ao maior número de parceiros ao
longo da vida 29. A precocidade da primeira re
lação sexual pode trazer consequências graves
para a saúde dos adolescentes. O não uso do pre
servativo ou seu uso inadequado podem acarre Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1611 frequentavam a escola e estavam presentes no
dia da aplicação do questionário, entretanto, o
próprio absenteísmo escolar pode estar relacio
nado ao desfecho estudado. Ou até mesmo, outra
hipótese é que os alunos que estão fora da escola
podem ser aqueles que buscam menos os servi
ços de saúde. No país, o Ensino Fundamental é
universalizado, estima-se que o acesso à escola
entre adolescentes de 6-14 anos é de 97,4% e de
87,7% entre os de 15-19 anos, e não foram veri
ficadas diferenças nas proporções por regiões. Entretanto, houve diferenças mínimas entre os
quintis de rendimento familiar, padrão que se
repete para todas as Unidades da Federação 42. Por se tratar de um estudo transversal, não é pos
sível realizar inferências causais/temporais, pois
não foi possível avaliar se a procura dos serviços
de saúde é a causa ou consequência de algumas
variáveis associadas, como por exemplo, a asma. Não foram avaliados possíveis efeitos da coleta
de dados por meio de smartphones entre os ado
lescentes, principalmente em comportamentos
como sexual e de consumo de drogas. Estes efei
tos poderiam sugerir um viés de informação na
resposta a estes temas, por isso sugere-se a reali
zação de estudos específicos. lescentes e a realização de atividades sem con
sentimento dos pais 37; comportamento sexual
desprotegido entre adolescentes e viver em fa
mília monoparental e baixa supervisão dos pais
38. É importante o papel das famílias no cuidado
junto ao adolescente como fator de proteção que
influencia também no maior acesso aos serviços
de saúde. A participação da família na vida dos
escolares protege também de fatores de risco co
mo consumo de álcool, tabaco e outras drogas 39. Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 M. M. Oliveira, S. S. C. A. Andrade, M. O. Campos e
D. C. Malta contribuíram no delineamento do estudo,
na análise do banco de dados e na redação do manus
crito. Discussão Esta é a primeira pesquisa que aborda o aces
so de serviços de saúde entre adolescentes no
âmbito nacional, o que a torna relevante, uma
vez que há evidências que estudos sobre a procu
ra e a utilização de serviços de saúde contribuem
para a organização da assistência 8, e para o pla
nejamento de programas e políticas para este ci
clo de vida. Dentre elas, encontra-se o Programa
Saúde na Escola (PSE), que é uma política criada
em 2007, com o objetivo de promover saúde e
educação integral (promoção, prevenção, diag
nóstico e recuperação da saúde e formação), na
perspectiva da atenção integral à saúde de crian
ças, adolescentes e jovens do ensino público
básico, por meio da articulação intersetorial das
redes públicas de saúde e de educação e das de
mais redes sociais. A base do PSE é a articulação
entre a escola e a rede básica de saúde, a partir da
conformação de redes de corresponsabilidade 40. O estudo aponta que a procura pelos serviços
de saúde em escolares foi mediada por fatores
socioeconômicos e demográficos (maior busca
entre estudantes do sexo feminino, de cor bran
ca, estudantes de escolas privadas e cujos pais
têm maior escolaridade), pelas necessidades de
saúde; e pelo comportamento de maior cuidado
com a própria saúde. Destacamos a importância
do SUS no país, como ferramenta de redução das
desigualdades em saúde, garantindo o acesso
universal, bem como a expansão da atenção bá
sica, a porta de entrada para a maioria dos esco
lares. É fundamental a atenção integral à saúde
do adolescente com suas diversas dimensões,
uma vez que múltiplos contextos influenciam o
uso dos serviços de saúde. Outra iniciativa é as Diretrizes Nacionais para
a Atenção Integral à Saúde de Adolescentes e de
Jovens na Promoção, Proteção e Recuperação da
Saúde 41, de 2007, baseada na Política Nacional
de Atenção Integral à Saúde de Adolescentes e Jo
vens, e visa nortear as ações integradas às outras
políticas e programas já existentes no SUS, con
tribuindo na construção de estratégias interfede
rativas e intersetoriais, buscando a modificação
do quadro nacional de vulnerabilidade de ado
lescentes e de jovens 41. Uma limitação da pesquisa é que a entre
vista ocorre apenas entre os adolescentes que Oliveira MM et al. 1612 Resumen M. M. Oliveira, S. S. C. A. Andrade, M. O. Campos e
D. C. Malta contribuíram no delineamento do estudo,
na análise do banco de dados e na redação do manus
crito. El objetivo fue describir la demanda de servicios/profe
sionales de salud por parte de los estudiantes brasileños
e identificar posibles conductas de riesgo, condiciones
de salud y factores familiares asociados. Se realizó un
análisis de datos de la Encuesta Nacional de Salud Es
colar 2012; estimadas las tasas de prevalencia y sus in
tervalos de confianza (IC95%) para el uso de servicios
de salud. Asimismo, se efectuó una regresión logística
ajustada por edad. La mitad de los estudiantes buscó
servicios de salud; la demanda fue mayor en muje
res y factores asociados: color blanco, escuela privada;
educación de la madre (12 años o más); haber tenido
relaciones sexuales; sufrido daño, dolor de muelas, in
tentando mantener, perder o ganar peso, sibilancias los
últimos 12 meses, higiene adecuada y el conocimiento
de los padres sobre lo que sus hijos hacen en su tiempo
libre. La búsqueda de servicios de salud fue mayor en
mujeres y se asoció con mejores condiciones socioeco
nómicas, la presencia de síntomas y conductas de ries
go/protección. Salud Escolar; Conducta del Adolescent; Adolescente;
Servicios de Salud Salud Escolar; Conducta del Adolescent; Adolescente;
Servicios de Salud Cad. Saúde Pública, Rio de Janeiro, 31(8):1603-1614, ago, 2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1613 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
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Versão final reapresentada em 08/Jan/2015
Aprovado em 05/Fev/2015 FATORES ASSOCIADOS À PROCURA DE SERVIÇOS DE SAÚDE ENTRE ESCOLARES
1613 Sín
tese de indicadores sociais 2010: uma análise das
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Versão final reapresentada em 08/Jan/2015
Aprovado em 05/Fev/2015 Recebido em 04/Nov/2014
Versão final reapresentada em 08/Jan/2015
Aprovado em 05/Fev/2015
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https://europepmc.org/articles/pmc5543161?pdf=render
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English
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Vertical Paper Analytical Devices Fabricated Using the Principles of Quilling and Kirigami
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Scientific reports
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cc-by
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Bingbing Gao1, Junjie Chi1, Hong Liu 1,2 & Zhongze Gu1,2 Here we report the vertical paper analytical devices (vPADs) fabricated using the principles of quilling
and kirigami. What differentiates the vPADs from conventional paper microfluidic devices is that the
paper substrate used to fabricate the device is placed vertically to the device plane. The fabrication
of vPADs with high precision is instrument-free, requiring no photolithography, printing or heating. Two- and three-dimensional vPADs are fabricated for multiplex colorimetric assays of four biochemical
indicators and automated enzyme-linked immunosorbent assay of human myoglobin, respectively. Paper-based analytical devices such as dipsticks and lateral-flow test strips have long been used for point-of-care
diagnostics because they are fast, inexpensive and easy-to-use. In 2007, Whitesides et al. reported microfluidic
paper-based analytical devices (μPADs) based on two-dimensional (2D) and three-dimensional (3D) paper cap-
illary channels patterned using photolithography1. After that, many μPADs with interesting designs and func-
tions have been presented, such as origami-inspired μPADs (oPADs)2, 3 and slipPADs4. Paper-based analytical
devices have found a wide range of applications in multiplex, power-free, on-site chemical analysis, particularly
in underdeveloped areas. Various methods for patterning paper have been reported, including photolithography1,
plotting5, cutting6, inkjet printing7 and wax printing8. These methods selectively deliver energy, materials, or both
onto a sheet of paper plane to create capillary channels. Due to the inhomogeneity of cellulose paper, the lateral
resolution of the channel is larger than 500 μm even using sophisticated photolithography. Instrumental-free
fabrication of 2D and 3D devices with acceptable channel precision is still challenging. Quilling, which dates back to at least the 13th Century, is a form of art involving the use of paper strips
that are rolled and glued together to create decorative designs9, 10. As another type of paper art, kirigami is a
variation of origami that includes cutting of the paper rather than just paper folding11. The μPADs we report
here are inspired by these two types of paper arts. They are named vertical paper analytical devices (vPADs)
because the paper strips used to fabricate the device are placed vertically to the device plane. The 2D vPADs are
fabricated by rolling paper strips around a slotted stick. Double-sided adhesive tape is attached to both sides
of the paper strips for assembly of the device and also used as water-proof channel barriers. Aqueous solution
can then wick through the channel by capillary action. 3D vPADs are fabricated using the principles of both
kirigami and quilling. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 Vertical Paper Analytical Devices
Fabricated Using the Principles of
Quilling and Kirigami Received: 16 December 2016
Accepted: 23 June 2017
Published: xx xx xxxx Bingbing Gao1, Junjie Chi1, Hong Liu 1,2 & Zhongze Gu1,2 Bingbing Gao1, Junjie Chi1, Hong Liu 1,2 & Zhongze Gu1,2 Paper strips are first cut and then assembled into 3D devices. For the proof-of-concept,
multiplex colorimetric assays and automated enzyme-linked immunosorbent assay (ELISA) are carried out
on the vPADs.hl The vPADs have several interesting characteristics that are potentially useful. First, unlike most paper microflu-
idic device, the fabrication of vPADs is instrument-free. No photolithography, printing or heating is required6, 7, 12–16. This is useful for point-of-care diagnostics in developing countries which have infrastructure shortfalls such as
unreliable electric power and poor maintenance for sophisticated instruments17. Second, the channel width of
vPADs is determined by the thickness of the paper. So the fabrication of channels from 0.07 mm to 1 mm with
high precision is straightforward using commercially available paper, tape and hands18. Third, channels are rolled
into a small area on the chip. Using paper strips having a thickness of 80 μm, one can estimate that the maximum
length of channel per unit area is about 20 cm/cm2 for a 2D vPAD. This can be useful for high-throughput and/or
multiplex assays which require high channel density on a single chip19. Forth, because the paper strips are vertical
to the chip plane, the use of paper strips that are patterned will result in 3D devices with 3D channel network
for carrying out complicated bioassays1, 2. Fifth, the colour observed vertically to the paper plane is deeper due 1State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University,
Nanjing, 210096, China. 2Laboratory of Environment and Biosafety, Research Institute of Southeast University in
Suzhou, Suzhou, 215123, China. Correspondence and requests for materials should be addressed to H.L. (email:
liuh@seu.edu.cn) ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 1 www.nature.com/scientificreports/ Figure 1. (a) Photographs showing the quilling and kirigami processes. (b) Schematic illustration showing the
procedure for fabricating the 2D and 3D vPADs using the principles of quilling and kirigami. (c) Photographs
of the 2D vPAD before and after the introduction of 0.10 M PBS solution containing 0.010 M coomassie brilliant
blue. The inlet for introduction of the solution is indicated by the red arrow. Scale bar: 10 mm. (d) Photographs
of the 3D vPAD before and after the introduction of 0.10 M PBS solutions containing 0.010 M coomassie
brilliant blue, 0.010 M rhodamine B, 0.010 M methyl orange and 0.010 M bromocresol green, respectively. The
inlets for introduction of the solutions are indicated by the red arrows. Results and Discussion Paper-based analytical devices, including dipsticks, lateral-flow test strips and μPADs have been widely used in
biochemical analysis. In this work, we introduce another type of μPADs, which are intrinsically different from
conventional μPADs. The basic processes of kirigami and quilling processes are shown in Fig. 1a. For kirigami,
a sheet of paper was folded, cut, and finally unfolded to obtain a sheet of patterned paper, one can easily get
several duplicated patterns by single cutting. The cutting with scissors could also be replaced by automated cut-
ting machine such as a laser cutter to get more precise paper patterns. For quilling, double-sided adhesive tapes
were attached onto both sides of the paper strip, and the paper strip was rolled to assemble the device. Using the
principles of kirigami and quilling, we fabricated basic 2D and 3D vPADs as shown in Fig. 1b. For the 2D vPAD
(Fig. 1c), the double-sided tapes were attached on both sides of the paper strip not only for assembly of the device,
but also used as water-proof channel barriers to avoid the mixing of solutions in different channels. For the 3D
vPAD (Fig. 1d), the paper strip was first patterned using scissors and then assembled into the 3D device.hll ( g
)
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The 2D and 3D vPADs fabricated were used to demonstrate the ability of the devices to direct the flow of fluids
in two and three dimensions. Specifically, 0.10 M PBS solution (pH 7.4) containing 0.010 M coomassie brilliant
blue was dropcast onto the inlet of the 2D device (Fig. 1c), the blue-colored solution wicked evenly through
their designated channels without mixing with other channels. For 3D vPADs, 0.10 M PBS solutions (pH 7.4)
containing 0.010 M of the following dyes were dropcast onto the inlets of the device (indicated by the red arrows),
respectively: coomassie brilliant blue (blue), rhodamine B (red), methyl orange (orange) and bromocresol green
(green). The dye solutions flowed through the entire separated channels evenly without mixing or leaking. (g
)h
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In principle, the resolution of vPAD channels was determined by the thickness of paper strips. As the demonstra-
tion, six types of paper strips with different thicknesses ranging from 80–350 μm were obtained, and used to fabricate
the vPAD. As shown in Fig. 2a, a dye solution was used to test the liquid flow through each channel. Bingbing Gao1, Junjie Chi1, Hong Liu 1,2 & Zhongze Gu1,2 Scale bar: 10 mm. Figure 1. (a) Photographs showing the quilling and kirigami processes. (b) Schematic illustration showing the
procedure for fabricating the 2D and 3D vPADs using the principles of quilling and kirigami. (c) Photographs
of the 2D vPAD before and after the introduction of 0.10 M PBS solution containing 0.010 M coomassie brilliant
blue. The inlet for introduction of the solution is indicated by the red arrow. Scale bar: 10 mm. (d) Photographs
of the 3D vPAD before and after the introduction of 0.10 M PBS solutions containing 0.010 M coomassie
brilliant blue, 0.010 M rhodamine B, 0.010 M methyl orange and 0.010 M bromocresol green, respectively. The
inlets for introduction of the solutions are indicated by the red arrows. Scale bar: 10 mm. to increased optical path, which leads to a higher sensitivity for colorimetric detection. Finally, because only the
edge of the channel is exposed to the air, the problems for conventional devices such as water evaporation and
contamination can be easily solved3. Results and Discussion We measured the
flow length of an aqueous 0.010 M coomassie brilliant blue solution in three types of paper channels as a function of
time: (1) only paper; (2) paper having one side attached with tape; (3) paper channel having both sides attached with
tapes. The results showed that tapes slowed down the flow of solution through the capillary channel, which is reason-
able because the tape is less hydrophilic than paper. The channel width of the vPAD is determined by the thickness
of paper and tape. Therefore, by using really thin paper strips, large number of channels can be integrated in a device
with a small area. As shown in Fig. 2c, we can fabricate a vPAD with 50 parallel channels which were all connected to
one sample inlet. This device could be potentially useful for highthroughput and multiplex assays. ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 2 www.nature.com/scientificreports/ Figure 2. (a) Photographs of paper channels fabricated using paper stirps with different widths before and
after introduction of an aqueous solution containing 10 mM methyl orange. The width of the paper strips
are indicated. (b) Distance travelled by the solution as a function of time for different paper channels. (c)
Photographs of a vPAD device after introduction of an aqueous solution containing 10 mM coomassie brilliant
blue. Figure 2. (a) Photographs of paper channels fabricated using paper stirps with different widths before and
after introduction of an aqueous solution containing 10 mM methyl orange. The width of the paper strips
are indicated. (b) Distance travelled by the solution as a function of time for different paper channels. (c)
Photographs of a vPAD device after introduction of an aqueous solution containing 10 mM coomassie brilliant
blue. Figure 3. (a) Photographs showing the side and front views of three types of paper channels after introduction
of 0.010 M coomassie brilliant blue solution. The widths and thickness of the paper strips used to fabricate
the channel are indicated. Scale bar: 0.5 mm (b) Color intensity (Ic) of the channels measured from these
photographs. Figure 3. (a) Photographs showing the side and front views of three types of paper channels after introduction
of 0.010 M coomassie brilliant blue solution. The widths and thickness of the paper strips used to fabricate
the channel are indicated. Scale bar: 0.5 mm (b) Color intensity (Ic) of the channels measured from these
photographs. Results and Discussion When dye solution infiltrated through paper channels, uniformly dispersed dye molecules gradually concen-
trated at the edge of the paper as water evaporates, known as the coffee ring effect. More dye molecules aggregated
alone the edge compared with intermediate zone. Therefore the colour intensity of dyes observed from the side
view should be much higher than that from the front view, which could be used to increase the sensitivity for
colorimetric detection. We fabricated paper channels with different widths ranging from 500–2000 μm, as shown
in Fig. 3a. After introduction of solution containing 0.010 M coomassie brilliant blue, the optical photographs of ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 3 www.nature.com/scientificreports/ Figure 4. (a,b) Photographs of the paper strips attached with tapes for the device fabrication. (c) The resulted
2D vPAD for colorimetric detection of four analytes (GLU: glucose, UA: uric acid, CHO: cholesterol, TG:
triglycerides), scale bar: 2 mm. The colors of the paper strips are from the double-sided adhesive tapes used
to assemble the device. (d) The resulted fluidic channels and flow direction (indicated by arrows) of aqueous
sample in the device. (e) The vPAD for carrying out eight parallel assays. (f) The vPAD after introduction of
0.010 M coomassie brilliant blue solution. The assays results measured from the 2D vPAD for colorimetric
detection of four analytes: (g) GLU: glucose; (h) UA: uric acid; (i) CHO: cholesterol; and (j) TG: triglycerides. The arrow bars indicate the standard deviation of three replicated measurements. Figure 4. (a,b) Photographs of the paper strips attached with tapes for the device fabrication. (c) The resulted
2D vPAD for colorimetric detection of four analytes (GLU: glucose, UA: uric acid, CHO: cholesterol, TG:
triglycerides), scale bar: 2 mm. The colors of the paper strips are from the double-sided adhesive tapes used
to assemble the device. (d) The resulted fluidic channels and flow direction (indicated by arrows) of aqueous
sample in the device. (e) The vPAD for carrying out eight parallel assays. (f) The vPAD after introduction of
0.010 M coomassie brilliant blue solution. The assays results measured from the 2D vPAD for colorimetric
detection of four analytes: (g) GLU: glucose; (h) UA: uric acid; (i) CHO: cholesterol; and (j) TG: triglycerides. The arrow bars indicate the standard deviation of three replicated measurements. front and side views of the paper channels were collected. Results and Discussion The colour intensity of each channel was obtained from
a histogram of the channel after imported into Adobe Photoshop CS5. As shown in Fig. 3b, the colour intensity
of the channel measured from the side view was obviously higher than that measured from the front view. The
colour intensity of the front view increased with the thickness of the paper strips, which means longer optical path
resulted in higher colour intensity. The results indicated that vPAD channels can be used to increase the detection
signal of colorimetric assays. g
y
To demonstrate the applicability of vPADs to multiplex biochemical analysis, we first fabricated a 2D vPADs
for multiplex colorimetric detection of four analytes (glucose, uric acid, cholesterol and triglyceride) which are
important biochemical indicators of human health. The multiplex assays were carried out on the 2D vPADs
shown in Fig. 4. Two paper strips preloaded with assay reagents were used to fabricate the vPAD. Double-sided
adhesive tapes of four different colours were attached to the paper strips for assembly of the device, and the colour
of the tape indicated the target being analysed in each channel (red: glucose, blue: uric acid, yellow: cholesterol,
green: triglyceride). It is worth noting that, multiplex assays for eight analytes can also be simply carried out on
the vPADs by fabricating eight channels using the proposed method, as shown in Fig. 4e,f.h y
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The vPAD can be used for multiplex detection of analytes. Four important biochemical indicators (i.e glucose,
uric acid, cholesterol and triglyceride) were used as the model analytes because they are widely used for diag-
nostics. For example, blood glucose was used for diabetes diagnostics, uric acid is a metabolic product of purine
nucleotides which is associated with gout, diabetes and kidney stones, cholesterol is usually used for screening of
atherosclerotic risk and lipid metabolic disorders, and triglycerides are the main constituent of human body fat
and therefore utilized in the diagnosis and treatment of patients having diabetes mellitus, nephritis, liver obstruc-
tion, lipid metabolism disorders and numerous other endocrine diseases. For these biochemical species, the nor-
mal blood levels are 3.9–5.5 mM for glucose (tested while fasting)20, 0.20–0.430 mM (men) and 0.14–0.36 mM
(women) for uric acid21, 22, 2.9–6.0 mM for total cholesterol23, 24, and 0.56–1.7 mM for triglycerides25, 26.th As shown in Fig. Results and Discussion 4, the colour was developed in each detection reservoir after reaction of 20 min. The inten-
sity of the color change was correlated to the target concentration. To better quantify these results, the optical
photograph of each reservoir was obtained using an office scanner and imported into Adobe Photoshop CS5. The colour intensity was obtained from a histogram of the photograph. As shown in Fig. 4g, the intensity of the
brown colour was linearly correlated to the glucose concentration from 0.60 to 5.0 mM (Ic = 0.43 + 18 × CGLU). For uric acid, the intensity of the purple colour was linearly correlated to the concentration of uric acid from 50
to 400 µM (Ic = 0.68 + 0.14 × CUA) as showed in Fig. 4h. Figure 4i showed the intensity of the brown color was
linearly correlated to cholesterol concentration from 2.0 to 5.0 mM (Ic = 0.21 + 4.6 × CCHO). For triglycerides,
the intensity of the purple colour was linearly correlated to the triglyceride concentration from 0.60 to 2.4 mM
(Ic = 0.25 + 40 × CTG), as showed in Fig. 4j. The limit of detections, calculated as 3 times the standard deviation of ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 4 www.nature.com/scientificreports/ Figure 5. (a) Photographs of the paper strips (I) for the device fabrication
myoglobin ELISA. The colors of the paper strips are from the double-side
the device. III: the resulted fluidic channels and flow direction (indicated b
the device. Scale bars: 10 mm. (b) Schematic illustration showing the flow
3D vPAD during the ELISA of myoglobin. Assays reagents (i.e. I: anti-hum
labelled anti human myoglobin antibody, III: BCIP/NBT substrate) were p
intensity (Ic) of the detection reservoir measured after completion of the a
concentration (CMB) in the sample. The error bars represent standard dev
Inset: photographs of the detection reservoirs. The concentration of myog
each photograph. Figure 5. (a) Photographs of the paper strips (I) for the device fabrication and the resulted 3D vPAD (II) f
myoglobin ELISA. The colors of the paper strips are from the double-sided adhesive tapes used to assembl
the device. III: the resulted fluidic channels and flow direction (indicated by arrows) of aqueous sample in
the device. Scale bars: 10 mm. (b) Schematic illustration showing the flow direction of aqueous sample in t
3D vPAD during the ELISA of myoglobin. Assays reagents (i.e. Results and Discussion I: anti-human myoglobin antibody, II: ALP
labelled anti human myoglobin antibody, III: BCIP/NBT substrate) were preloaded on the test strips. (c) C
intensity (Ic) of the detection reservoir measured after completion of the assay as a function of myoglobin
concentration (CMB) in the sample. The error bars represent standard deviation of 3 replicate measuremen
Inset: photographs of the detection reservoirs. The concentration of myoglobin in the sample is indicated b
each photograph. Figure 5. (a) Photographs of the paper strips (I) for the device fabrication and the resulted 3D vPAD (II) for
myoglobin ELISA. The colors of the paper strips are from the double-sided adhesive tapes used to assemble
the device. III: the resulted fluidic channels and flow direction (indicated by arrows) of aqueous sample in
the device. Scale bars: 10 mm. (b) Schematic illustration showing the flow direction of aqueous sample in the
3D vPAD during the ELISA of myoglobin. Assays reagents (i.e. I: anti-human myoglobin antibody, II: ALP-
labelled anti human myoglobin antibody, III: BCIP/NBT substrate) were preloaded on the test strips. (c) Color
intensity (Ic) of the detection reservoir measured after completion of the assay as a function of myoglobin
concentration (CMB) in the sample. The error bars represent standard deviation of 3 replicate measurements. Inset: photographs of the detection reservoirs. The concentration of myoglobin in the sample is indicated below
each photograph. the sample containing no analyte divided by the slope of the calibration curve, were 0.42 mM for glucose, 35 µM
for uric acid, 0.55 mM for cholesterol and 0.32 mM for triglyceride, respectively. To demonstrate the applicability of the vPAD to more complex assays, a 3D vPAD was fabricated for auto-
mated ELISA27 of human myoglobin (Fig. 5a). Myoglobin is a protein found in the muscle tissue of human. It is
only found in the bloodstream after muscle injury, so the detection of blood myoglobin is used for diagnostics of
injury. The 3D vPAD for ELISA was fabricated using one NC strip and two paper strips, which were blocked with
the blocking solution. 1.0 µL of 0.50 mg/mL anti-human myoglobin antibody solution was transferred to location
I of the NC strip (Fig. 5b) and then the NC strip was blocked with the blocking solution. ALP-labelled anti-human
myoglobin antibody was dropcast into location II of one paper strip. Methods
Ch
i
l Chemicals and materials. Nitrocellulose (NC) was purchased from China Sinopham (Shanghai, China). Double-sided adhesive tape with different colours and tools for fabricating the vPADs were all bought from local
grocery store and supermarket. The 5-bromo-4-chloro-3‘-indolyphosphate p-toluidine salt/nitro-blue tetra-
zolium chloride (BCIP/NBT) substrate solution and substrate buffer solution, sodium dodecyl sulfate (SDS) were
obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Coomassie brilliant blue, rhodamine
B, methyl orange, bromocresol green, phosphate-buffered saline (PBS), polysorbate-20 (TWEEN-20), bovine
serum albumin (BSA), Whatman grade 1 chromatography paper (20 cm × 20 cm) and colorimetric glucose assay
kit was purchased from Sigma-Aldrich. The kits for colorimetric uric acid, cholesterol, triglyceride assays were
purchased from Biosino Bio-Technology & Science Inc. (Beijing, China). Mouse anti-human myoglobin, human
myoglobin, and alkaline phosphatase (ALP)-labelled goat anti-human myoglobin were purchased from Santa
Cruz Biotechnology Co., Ltd (Shanghai, China). All solutions were prepared with deionised water (18.0 MΩ
cm, Milli-Q Gradient System, Millipore) with ultraviolet sterilization. All reagents were used as received without
further purification. Experimental Procedures. For multiplex colorimetric assays, two paper strips (Whatman grade 1 chro-
matography paper) were used to fabricate the vPAD, as shown in Fig. 2. Double-sided adhesive tapes with four
different colours were attached to the paper for assembly of the device and also used as channel barrier. The color
of the tape indicated the target being analysed in each channel (red: glucose, blue: uric acid, yellow: cholesterol,
green: triglyceride). For the multiplex colorimetric assays, all of the reagents were from the assay kits and were
prepared according to the instructions of the kit provider. To preload the assay reagents onto the vPAD, 5.0 µL
aliquot of the reagent solution was dropcast into the detection reservoir at the end of each channel. The solution
was allowed to dry at 20 °C under nitrogen. To initiate the multiplex colorimetric assay, 20 µL sample was intro-
duced into the inlet at the centre of the device. After 20 min, an office scanner (HP C6180) was used to obtain
optical images of the detection reservoirs, the images were imported into Adobe Photoshop CS5 to measure
colour intensity. The detection of the four targets was based on a two-step reaction: (1) oxidase-catalyzed oxi-
dation of targets/hydrolytic product of targets to yield H2O2 and (2) horseradish peroxidase-catalyzed oxidation
of the substrate by H2O2 to yield a colored compound. Results and Discussion BCIP/NBT substrate was preloaded onto the ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 5 www.nature.com/scientificreports/ other paper strip at location III. The detection reservoir was indicated by red arrow in Fig. 5a. After introduction
of aqueous sample from the device inlet, the aqueous sample flowed through the 3D channel and initiate the
assay process, which included the capture of myoglobin by the antibody immobilized in location I, the binding
of ALP-labelled antibody with the captured myoglobin, the washing of the excess reagents and the ALP catalyzed
reaction involving BCIP/NBT for color generation. The four steps of the assay accomplished sequentially in the
channel. Finally, the color developed in the detection reservoir was measured and correlated to the myoglobin
concentration in the sample, as shown in Fig. 5c. For quantitative detection, the color intensity was plotted as a
function of the myoglobin concentration. As shown in Fig. 5c, the intensity of the color was linearly correlated
to the concentration from 0.050 to 2.0 µg/mL (Ic = 1.5 + 0.19 × CMB). The limit of detection, calculated as 3 times
the standard deviation of the sample containing no analyte divided by the slope of the calibration curve, was 32
ng/mL.h g
In conclusion, we have reported the vPADs for chemical analysis under resource-limited conditions. The fab-
rication of vPADs was based on the principles of quilling and kirigami, and thus the paper substrate used to fabri-
cate the device was vertical to the device plane. The fabrication of vPADs with high precision was instrument-free,
requiring no photolithography, printing or heating. To demonstrate the applicability of vPADs to chemical detec-
tion under resource-limited settings, 2D and 3D vPADs were fabricated and used for multiplex colorimetric
assays of four biochemical indicators and for automated ELISA of human myoglobin, respectively. However the
vPADs still have some limitations. For example, the time required for fabrication is still long compared with con-
ventional paper devices due to manual operation. Reproducible production on a large scale of the device will still
rely on instruments. The height of the channels is limited by the paper type that is commercially available. We will
solve these problems by further work and report them in due course. References Development of automated paper-based devices for sequentia
multistep sandwich enzyme-linked immunosorbent assays using inkjet printing. Lab Chip 13, 126–135 (2013). Acknowledgementsi g
We gratefully acknowledge financial support from Chinese Recruitment Program of Global Experts, Innovative
and Entrepreneurial Talent Recruitment Program of Jiangsu Province, the National Natural Science Foundation
of China (21405014, 21327902, 21635001), the Natural Science Foundation of Jiangsu (BK20140619), the
Science and Technology Development Program of Suzhou (ZXY201439), the Research Fund for the Doctoral
Program of Higher Education of China (20120092130006), State Key Project of Research and Development
(2016YFF0100802).The Project of Special Funds of Jiangsu Province for the Transformation of Scientific and
Technological Achievements (BA2015067). References Interfaces 1, 124–129 (2009) p g
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4. Li, X., Tian, J. F., Garnier, G. & Shen, W. Fabrication of paper-based microfluidic sensors by printing. Colloid Surf. B-Biointerfaces 76
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7. Apilux, A., Ukita, Y., Chikae, M., Chailapakul, O. & Takamura, Y. Methods
Ch
i
l To initiate the ELISA, aqueous sample containing myoglobin was intro-
duced from the inlet. After 30 min, an office scanner (HP C6180) was used to obtain optical image of the detection
reservoirs, the images were imported into Adobe Photoshop CS5 to measure colour intensity. Methods
Ch
i
l The detection of glucose was based on glucose oxidase
and o-dianisidine as the peroxidase substrate. The detection of uric acid was based on uricase and N-ethyl-N-
(2-hydroxy-3-sulfopropyl)-3-methylaniline as the peroxidase substrate. For cholesterol, the detection was based
on cholesterol esterase which catalyses the hydrolysis of cholesterol esters to yield free cholesterol. The peroxidase
substrates used for cholesterol detection was 4-aminoantipyrine and phenol. The determination of triglycerides
was based on hydrolysis of triglycerides by lipoprotein lipase to yield glycerol. The glycerol was then oxidised by
oxygen which was catalyzed by glycerol oxidase. The peroxidase substrates involved in the final color-generating
reaction were 4-aminophenazone and 4-chlorophenol. p
p
For ELISA, 1.0 µL aliquot of 50 mM PBS (pH 7.4) containing 1 mg/mL mouse anti-human myoglobin anti-
body was prepared and then mixed with a 1.0 µL aliquot of 50 mM Na2HPO4 solution (pH 7.5) containing 1.0%
(w/v) sucrose. 50 mM PBS (pH 7.5) solution containing 0.50% (w/v) BSA, and 50 mM phosphate buffer (pH 7.4)
containing 0.010% (w/v) SDS were used for blocking and wash, respectively. 50 mM PBS (pH 7.5) containing
25 µg/mL ALP-labelled anti-human myoglobin antibody and 0.10% (w/v) BSA were prepared. The BCIP/NBT
substrate solution was diluted 10 times with the substrate buffer solution. The 3D vPAD for ELISA was fabricated
using two paper strips which were blocked with the blocking solutions (green and blue) and one NC strips (red),
as shown in Fig. 3b. 1.0 µL aliquot of 0.50 mg/mL anti-human myoglobin solution was transferred to the NC
strip (at location I in Fig. 3b) using a pipette having a cotton thread on its tip. The NC strip was allowed to dry
for 1 hour, immersed in the blocking solution for 30 min, and finally washed by washing solution for 30 min at
25 °C. Double-sided adhesive tape (red) was attached to the NC strip for assembly of the device. 0.50 µL aliquot
of 0.50 mg/mL ALP-labelled anti-human myoglobin antibody was dropcast onto one paper strip (at location II in ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 6 www.nature.com/scientificreports/ Fig. 3b). 0.50 µL aliquot of BCIP/NBT substrate solution was dropcast onto the other paper strip (at location III
in Fig. 3b). Both paper strips were allowed to dry completely at 25 °C and then attached to double-sided adhesive
tape for assembly into the ELISA vPAD. References 1. Martinez, A. W., Phillips, S. T. & Whitesides, G. M. Three-dimensional microfluidic devices fabricated in layered paper and tape
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wax screen-printing. Analyst 136, 77–82 (2011). p
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3. Fenton, E. M., Mascarenas, M. R., Lopez, G. P. & Sibbett, S. S. Multiplex Lateral-Flow Test Strips Fabricated by Two-Dimensiona
Shaping. ACS Appl. Mater. Interfaces 1, 124–129 (2009).l Shaping. ACS Appl. Mater. Author Contributions Hong Liu and Zhongze Gu developed the idea. Bingbing Gao, and Hong Liu designed the experiments. Bingbing
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version for publication. ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 Additional Informationh Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
institutional affiliations. ScIEntIfIc REPOrTs | 7: 7255 | DOI:10.1038/s41598-017-07267-9 7 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Paternally Expressed, Imprinted Insulin-Like Growth Factor-2 in Chorionic Villi Correlates Significantly with Birth Weight
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PloS one
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cc-by
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Abstract Context: Fetal growth involves highly complex molecular pathways. IGF2 is a key paternally expressed growth hormone
that is critical for in utero growth in mice. Its role in human fetal growth has remained ambiguous, as it has only been
studied in term tissues. Conversely the maternally expressed growth suppressor, PHLDA2, has a significant negative
correlation between its term placental expression and birth weight. Objective: The aim of this study is to address the role in early gestation of expression of IGF1, IGF2, their receptors IGF1R and
IGF2R, and PHLDA2 on term birth weight. Design: Real-time quantitative PCR was used to investigate mRNA expression of IGF1, IGF2, IGF1R, IGF2R and PHLDA2 in
chorionic villus samples (CVS) (n = 260) collected at 11–13 weeks’ gestation. Expression was correlated with term birth
weight using statistical package R including correction for several confounding factors. Results: Transcript levels of IGF2 and IGF2R revealed a significant positive correlation with birth weight (0.009 and 0.04,
respectively). No effect was observed for IGF1, IGF1R or PHLDA2 and birth weight. Critically, small for gestational age (SGA)
neonates had significantly lower IGF2 levels than appropriate for gestational age neonates (p = 3?661027). Interpretation: Our findings show that IGF2 mRNA levels at 12 weeks gestation could provide a useful predictor of future
fetal growth to term, potentially predicting SGA babies. SGA babies are known to be at a higher risk for type 2 diabetes. This
research reveals an imprinted, parentally driven rheostat for in utero growth. Citation: Demetriou C, Abu-Amero S, Thomas AC, Ishida M, Aggarwal R, et al. (2014) Paternally Expressed, Imprinted Insulin-Like Gr
Villi Correlates Significantly with Birth Weight. PLoS ONE 9(1): e85454. doi:10.1371/journal.pone.0085454 Editor: Cees Oudejans, VU University Medical Center, Netherlands Received November 1, 2013; Accepted December 4, 2013; Published January 15, 2014 pyright: 2014 Demetriou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: CD is funded by Save the Baby Unit (http://www.savethebabyunit.org/). PS is funded by Great Ormond Street Hospital Children’s Charity (http://www. gosh.org/gen/). GEM Fetal growth and development research team is funded by the MRC (http://www.mrc.ac.uk/index.htm), Wellbeing of Women (http://www. wellbeingofwomen.org.uk/), Sparks (http://www.sparks.org.uk/) and the Wellcome Trust (http://www.wellcome.ac.uk/). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript. Paternally Expressed, Imprinted Insulin-Like Growth
Factor-2 in Chorionic Villi Correlates Significantly with
Birth Weight Charalambos Demetriou1,2, Sayeda Abu-Amero1, Anna C. Thomas1, Miho Ishida1, Reena Aggarwal3,
Lara Al-Olabi1, Lydia J. Leon1, Jaime L. Stafford1, Argyro Syngelaki4, Donald Peebles3,
Kypros H. Nicolaides4, Lesley Regan2, Philip Stanier1, Gudrun E. Moore1* 1 Fetal Development and Growth Research Group, Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, London, United Kingdom,
2 Department of Obstetrics and Gynaecology, St. Mary’s Campus, Imperial College London, London, United Kingdom, 3 Institute for Women’s Health, University College
London, London, United Kingdom, 4 Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, United Kingdom Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: gudrun.moore@ucl.ac.uk January 2014 | Volume 9 | Issue 1 | e85454 Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) Genomic DNA and cDNA from CVS were amplified by PCR
before sequencing. Primers (59 to 39 sequence) used for genomic
DNA
are
IGF2.F-
aacaccccacaaaagctcag;
IGF2.R-
tgcatg-
gattttggttttca;
IGF2R.F-
gaaacacaaaacctacgacc;
IGF2R.R-
agaacccaaaagagccaacc; PHLDA2.F- caaaccccgcacgccatgag and
PHLDA2.R- ctgtgcccattgcaaataaatc. The same primers were used
for cDNA with the exception of IGF2R.R- cctttggagtacgtgacaac. 20 ul reactions were set up and thermal cycling conditions were
94uC for 5 min, 94uC for 30 sec, 60uC for 30 sec, 72uC for 30 sec
for 35 cycles, and 72uC for 2 min. Genomic DNA and cDNA from CVS were amplified by PCR
before sequencing. Primers (59 to 39 sequence) used for genomic
DNA
are
IGF2.F-
aacaccccacaaaagctcag;
IGF2.R-
tgcatg-
gattttggttttca;
IGF2R.F-
gaaacacaaaacctacgacc;
IGF2R.R-
agaacccaaaagagccaacc; PHLDA2.F- caaaccccgcacgccatgag and
PHLDA2.R- ctgtgcccattgcaaataaatc. The same primers were used
for cDNA with the exception of IGF2R.R- cctttggagtacgtgacaac. 20 ul reactions were set up and thermal cycling conditions were
94uC for 5 min, 94uC for 30 sec, 60uC for 30 sec, 72uC for 30 sec
for 35 cycles, and 72uC for 2 min. Studies on IGF2 have reported conflicting results. Some
demonstrate no correlation between IGF2 expression and birth
weight [16] whereas others have variably shown that in SGA
pregnancies compared to controls, IGF2 expression is either
increased [22], decreased [15,23,24], or similar [25], including at
the protein level [26]. Moreover, studies have either shown no
significant relationship between IGF2 cord serum levels and size at
birth [27], or a positive effect on birth weight [28]. In others, IGF2
cord blood levels were significantly correlated with birth weight
only when its interaction with IGF2R was taken into account [29]. The profound role of the IGF1 and IGF2 pathways in the
regulation of fetal growth have been established largely based on
experiments using the mouse as a model [9,10,18]. Analysis in
humans has been hampered by the lack of available tissue to study
during the course of pregnancy and has therefore relied on the use
of term placenta, at a time when this tissue has become redundant
and therefore the levels of gene expression may no longer reliably
reflect the needs of the growing baby. To fully assess the role of
these growth factors and their receptors, we have focused on an
earlier developmental time point when these genes are much more
likely to measure functionally relevant expression levels. Studies on IGF2 have reported conflicting results. IGF2 Expression Correlates with Birth Weight imprinting [7]. It has been suggested that expression of the father’s
genes enhance fetal growth improving the success of the paternal
genome to be passed on. In contrast, the mother’s genome limits
fetal growth, distributing equal resources to each of her offspring,
whilst ensuring her own survival post birth allowing her to
reproduce again. Studies of imprinted genes generally support this
model, with one of the most striking examples being the reciprocal
imprinting effects and associated growth patterns for mouse
insulin-like growth factor 2 (Igf2) and its chelating receptor Igf2r
[8]. In transgenic mice, loss of function of the paternally-expressed
Igf2 results in a 40% reduction in birth weight which contrasts with
loss of the maternally-expressed Igf2r, resulting in a 30% increase
in birth weight [9,10]. In addition, IGF2 has been implicated in
two imprinted human growth disorders, the overgrowth, Beck-
with-Wiedemann syndrome (BWS) [11] and the growth restricting
Silver-Russell syndrome (SRS) [12]. Preparation of DNA and RNA from chorionic villus
samples DNA and RNA were extracted using the iPrep Purification
Instrument (Invitrogen), either by use of the iPrepTM ChargeS-
witchH gDNA Tissue Kit, or iPrepTM PureLinkTM Total RNA
and TrizolH Plus RNA kit including DNAse treatment, according
to the manufacturer’s instructions. Insulin-like growth factor 1 (IGF1) and its primary binding
receptor IGF1R are not imprinted. IGF1 exerts its growth
properties on almost every cell in the body. A homozygous partial
deletion of Igf1r in mice stunted height and weight, as well as
disrupted the pubertal growth spurt. A complete inactivation of
Igf1r is lethal in the neonatal period [18]. In contrast to Igf2
deficient mice, restriction of growth was seen to continue into the
postnatal period in the Igf1 mutants [19]. Polymerase Chain Reaction (PCR) Some
demonstrate no correlation between IGF2 expression and birth
weight [16] whereas others have variably shown that in SGA
pregnancies compared to controls, IGF2 expression is either
increased [22], decreased [15,23,24], or similar [25], including at
the protein level [26]. Moreover, studies have either shown no
significant relationship between IGF2 cord serum levels and size at
birth [27], or a positive effect on birth weight [28]. In others, IGF2
cord blood levels were significantly correlated with birth weight
only when its interaction with IGF2R was taken into account [29]. The profound role of the IGF1 and IGF2 pathways in the
regulation of fetal growth have been established largely based on
experiments using the mouse as a model [9,10,18]. Analysis in
humans has been hampered by the lack of available tissue to study
during the course of pregnancy and has therefore relied on the use
of term placenta, at a time when this tissue has become redundant
and therefore the levels of gene expression may no longer reliably
reflect the needs of the growing baby. To fully assess the role of
these growth factors and their receptors, we have focused on an
earlier developmental time point when these genes are much more
likely to measure functionally relevant expression levels. Study population Chorionic villus sampling (CVS) was performed at 11–13 weeks
of gestation in 260 singleton pregnancies that subsequently
resulted in normal live births at term. The samples were collected
from women undergoing CVS for prenatal diagnosis of chromo-
somal defects at King’s College Hospital London. The excess
tissue samples used for this study were obtained from women
agreeing to participate in research, which was approved by the
King’s College Hospital Ethics Committee. Demographic characteristics were recorded including maternal
age, racial origin, smoking status, parity and body mass index as
well as pregnancy outcomes such as gestational age at delivery, sex
and birth weight. Other birth parameters such as placental weight
were not available, nor were blood or tissue samples from the
newborn baby. The pregnancies were subdivided according to the
birth weight of neonates into small for gestational age (SGA) with
birth weight ,10th percentile, large (LGA) with birth weight
.90th percentile and appropriate (AGA). Pleckstrin homology-like domain family A member 2 (PHLDA2)
is maternally expressed, paternally imprinted, in both humans and
mice [13]. Involvement of Phlda2 in growth and development of
the placenta was demonstrated by knockout mouse models that
were associated with a significant increase in placental size during
mid to late gestation [14]. Studies that have investigated the
human placental expression of PHLDA2 report increased expres-
sion in SGA pregnancies and a negative association with birth
weight [15,16,17]. Preparation of DNA and RNA from chorionic villus
samples Reverse transcription Reverse transcriptase (RT) methodology was based on a
standard
protocol
using M-MLV
reverse transcriptase
and
random primer hexamers (Promega). Primers for the housekeep-
ing gene b-actin (ACTB) were used to check the integrity of the
cDNA and ensure no DNA contamination. The forward (F) and
reverse (R) primers spanned an intron and the sequences are: b-
actin.F- gtcttcccctccatcgtg and b-actin.R- ggtcatcttctcgcggttg. The analyses of human placental expression of IGF1, IGF2,
IG1R and IGF2R have previously been confined to samples
obtained at the time of birth. IGF1 under-expression was observed
in term placental samples from SGA pregnancies [20], while
mutations in the IGF1R gene led to abnormalities in the function
of IGF1 receptors that may also slow down intrauterine and
subsequent growth in humans [21]. Introduction and cardiovascular disease [4]. Hales et al., (1991) reported a
graded inverse association between birth weight and T2D risk with
the highest risks of T2D occurring at the lowest levels of birth
weight [5]. In a more recent meta-analysis, an increased risk for
T2D (OR = 1.47) is seen for SGA versus appropriate for
gestational age (AGA) neonates [6]. Fetal growth involves a complex interaction between genes and
the environment, with such significant complexity that many of the
molecular pathways remain to be elucidated. Normal fetal growth
depends on the successful nutrient exchange between the mother
and the fetus via the placenta. When this critical balance is
impaired it can result in a small for gestational age (SGA) baby [1]. SGA neonates have a reported incidence of ,6% of pregnancies
in developed countries and as high as 40% in some developing
countries [2,3]. SGA is also associated with an increased risk of
neonatal death. Although the survivors do exhibit catch up
growth, it is important to note that they remain at a higher risk for
common, late onset chronic diseases such as type 2 diabetes (T2D) One group of growth genes of particular interest are the
imprinted genes that are found almost exclusively in Eutherian
(placental) mammals. Genomic imprinting defines allele-specific,
differential expression of a gene, according to its parent of origin. If the paternal allele is expressed, the maternal allele is imprinted
(silenced) and vice versa. The ‘‘parental conflict hypothesis’’ is still
the most widely accepted explanation for the evolution of genomic January 2014 | Volume 9 | Issue 1 | e85454 January 2014 | Volume 9 | Issue 1 | e85454 1 PLOS ONE | www.plosone.org IGF2 Expression Correlates with Birth Weight IGF2 Expression Correlates with Birth Weight PLOS ONE | www.plosone.org Imprinting analysis Imprinting analysis of IGF2, IGF2R and PHLDA2 in CVS was
carried out by DNA sequence analysis of expressed single
nucleotide polymorphisms (SNP) in CVS gDNA after amplifica-
tion
using
specific
primers
(IGF2.F-
aacaccccacaaaagctcag;
IGF2R.R- cctttggagtacgtgacaac and PHLDA2.F- caaaccccgcacgc-
catgag). Corresponding cDNA samples were screened in patients
heterozygous for the IGF2 A/G (rs680), IGF2R A/G (rs1805075)
and PHLDA2 A/G (rs1056819) SNPs (http://www.ncbi.nlm.nih. gov/projects
/SNP/). Sequencing
reactions
were
prepared
according to the manufacturer’s instructions (Applied Biosystems)
using the ABI Prism Big Dye terminator cycle sequencing ready
reaction kit (BDT v1.1). Sequencing products were run on an ABI PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 2 IGF2 Expression Correlates with Birth Weight Figure 1. mRNA expression levels of IGF2, IGF2R, PHLDA2, IGF1 and IGF1R in chorionic villi. The expression levels of IGF2, IGF2R, PHLDA2,
IGF1 and IGF1R after standardization to the endogenous control gene L19, in relation to birth weight corrected for parity, sex, GA at term, maternal
BMI and smoking status. Significant associations were observed for CVS expression of A) IGF2 (p = 0.009) and B) IGF2R (p = 0.04). No significant
association was found for C) PHLDA2 (p = 0.73), for D) IGF1 (p = 0.48) or for E) IGF1R (p = 0.08). doi:10.1371/journal.pone.0085454.g001
IGF2 Expression Correlates with Birth Weight Figure 1. mRNA expression levels of IGF2, IGF2R, PHLDA2, IGF1 and IGF1R in chorionic villi. The expression levels of IGF2, IGF2R, PHLDA2,
IGF1 and IGF1R after standardization to the endogenous control gene L19, in relation to birth weight corrected for parity, sex, GA at term, maternal
BMI and smoking status. Significant associations were observed for CVS expression of A) IGF2 (p = 0.009) and B) IGF2R (p = 0.04). No significant
association was found for C) PHLDA2 (p = 0.73), for D) IGF1 (p = 0.48) or for E) IGF1R (p = 0.08). doi:10 1371/journal pone 0085454 g001 Figure 1. mRNA expression levels of IGF2, IGF2R, PHLDA2, IGF1 and IGF1R in chorionic villi. The expression levels of IGF2, IGF2R, PHLDA2,
IGF1 and IGF1R after standardization to the endogenous control gene L19, in relation to birth weight corrected for parity, sex, GA at term, maternal
BMI and smoking status. Significant associations were observed for CVS expression of A) IGF2 (p = 0.009) and B) IGF2R (p = 0.04). Imprinting analysis No significant
association was found for C) PHLDA2 (p = 0.73), for D) IGF1 (p = 0.48) or for E) IGF1R (p = 0.08). doi:10.1371/journal.pone.0085454.g001 Prism 3730 DNA analyzer, and the read-out was analysed with
SequencherTM v4.8 (Gene Codes Corporation). Prism 3730 DNA analyzer, and the read-out was analysed with
SequencherTM v4.8 (Gene Codes Corporation). gat; IGF1R.R- tccatgatgaccagtgttgg; PHLDA2.F- ccatccccgcagcc-
caaacc; PHLDA2.R- ccacgtcctagcctgggtcc; L19.F- gcggaagggta-
cagccaat and L19.R- caggctgtgatacatgtggcg. All primer sets were
free of primer-dimer products. The RTqPCR assays were run in
triplicate for each sample, with the gene of interest and
housekeeping gene run on the same 96-well plates. A control
pool of CVS cDNA was also included on each plate. Amplification
conditions include initial incubation at 95uC for 10 min and
repetitive denaturation at 95uC for 15 sec and annealing at 60uC
for 1 min for 40 cycles. Statistical analysis y
Standard multiple linear regression models were used to
examine the correlation of IGF1, IGF2, IGF2R, IGF1R and
PHLDA2 relative expression values to birth weight after adjust-
ment for gestational age at delivery, sex, parity, maternal age,
smoking status and maternal body mass index. Any outliers more
than 2 standard deviations from the mean were removed. The
analysis of variance (one-way ANOVA) was used to compare the
gene expression means between the three different categorical
birth weight groups (SGA, AGA, LGA) taking into consideration
any confounding factors. Statistical package ‘‘R’’ (version 2.13.0)
was used for the analyses and for calculating adjusted R-squared
(R2) values. Significance was defined as p,0.05. To investigate the whole growth spectrum we subdivided the
pregnancies into small for gestational age (SGA; n = 50) with birth
weight ,10th percentile, large (LGA; n = 65) with birth weight
.90th percentile and appropriate (AGA; n = 145). In the SGA
group compared to the AGA group, IGF2 expression was reduced
by statistically significant amounts (p = 3.661027), despite no
significant differences in the expression levels of individual
receptors (IGF2R, p = 0.76 or IGF1R, p = 0.15). In the LGA group
IGF2R expression was found to be higher than in the AGA group
(p = 0.02) and IGF1R expression was lower than in the SGA group
(p = 0.05) (figure 3). Given that IGF2 and PHLDA2 are both known to be imprinted
in mouse and human [8,13,30], we wanted to confirm that
monoallelic expression was maintained in CVS material, since
reversion to biallelic expression may impact on mRNA levels. Furthermore, IGF2R in the human population is reported to be
monoallelically expressed (i.e. imprinted) in only 10% of individ-
uals, which is in contrast to the mouse, where it is fully imprinted
[31]. We therefore wished to confirm its imprinting status in CVS
material and to investigate for any potential skewing. The
transcribed
SNPs
rs680
(IGF2),
rs1805075
(IGF2R)
and
rs1056819 (PHLDA2) were used to report on allele-specific
expression and thus determine imprinting status of IGF2, IGF2R
and PHLDA2. Genomic DNA extracted from the CVS from 200
patients were tested for heterozygosity at these SNPs, with 40
samples found to be informative at rs680 for IGF2, 24 samples at
rs1805075 for IGF2R and 21 samples at rs1056819 for PHLDA2. The 40 informative IGF2 individuals and the 21 informative
PHLDA2
individuals
were
all
found
to
be
monoallelically
expressed. Real-time quantitative PCR The quantitative expression analysis of the genes of interest,
IGF1, IGF2, IGF1R, IGF2R and PHLDA2 as well as the
endogenous control gene L19 (a housekeeping gene ubiquitously
expressed in the placenta) [16] was determined by real-time
quantitative PCR (RTqPCR) with SYBR Green (ABI) using the
StepOnePlus Real-Time PCR System (ABI). Primers (59 to 39
sequence) used for quantitative analysis are IGF1.F- ggaggctgga-
gatgtattgc; IGF1.R- acttgcttctgtcccctcct; IGF2.F- cgagagggacgtgtc-
gacc; IGF2.R- ggactgcttccaggtgtcata; IGF2R.F- ccggcgtgctctgga;
IGF2R.R- ccagagggtcacagtggaaga; IGF1R.F- ccaagggtgtggtgaaa- After amplification, quantitative expression levels were obtained
using the StepOne software (version 2.1). All triplicate cycle
threshold (CT) values were within 1 CT of each other. The
quantitative values for each triplicate were averaged and the PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 January 2014 | Volume 9 | Issue 1 | e85454 3 IGF2 Expression Correlates with Birth Weight Figure 2. mRNA expression levels of IGF1/IGF1R, IGF2/IGF2R and IGF2/IGF1R in chorionic villi. The expression levels of IGF1/IGF1R, IGF2/
IGF2R and IGF2/IGF1R after standardization to the endogenous control gene L19, in relation to birth weight corrected for parity, sex, GA at term,
maternal BMI and smoking status. No significant association was found for A) the ratio of IGF1 to IGF1R (p = 0.76), or for B) the ratio of IGF2 to IGF2R
(p = 0.93). Significant association was observed for CVS expression of C) the ratio of IGF2 to IGF1R (p = 0.005) and birth weight. doi:10.1371/journal.pone.0085454.g002 Figure 2. mRNA expression levels of IGF1/IGF1R, IGF2/IGF2R and IGF2/IGF1R in chorionic villi. The expression levels of IGF1/IGF1R, IGF2/
IGF2R and IGF2/IGF1R after standardization to the endogenous control gene L19, in relation to birth weight corrected for parity, sex, GA at term,
maternal BMI and smoking status. No significant association was found for A) the ratio of IGF1 to IGF1R (p = 0.76), or for B) the ratio of IGF2 to IGF2R
(p = 0.93). Significant association was observed for CVS expression of C) the ratio of IGF2 to IGF1R (p = 0.005) and birth weight. doi:10.1371/journal.pone.0085454.g002 As both IGF1 and IGF2 bind to the IGF1R and only IGF2
binds to the IGF2R we decided to look at this relationship between
the ligands and their receptors and any correlation they might
have with birth weight. Real-time quantitative PCR The ratio of IGF2 to IGF1R (p = 0.005) was
significantly associated with birth weight but no association was
found for the ratio of IGF1 to IGF1R (p = 0.76) or the ratio of IGF2
to IGF2R (p = 0.93) between CVS tissue and birth weight (figure 2). relative expression of the genes of interest was determined by a
ratio of their expression to that of the L19 housekeeping gene for
the same sample. Statistical analysis For the 24 samples informative for IGF2R, 21 (88%) Results In this study, we have investigated the relationship between
IGF1, IGF2, IGF2R, IGF1R and PHLDA2 mRNA levels in first-
trimester placental tissue with the birth weight of the resultant
term new born babies. We correlate this data taking into account
the carefully recorded birth parameters that may have a
confounding effect (gestational age, sex, parity, maternal BMI
and smoking status; figure S1). Regression analysis was performed
after standardization to the endogenous control gene L19, in
relation to birth weight corrected for confounding factors. Significant associations were observed for expression of IGF2
(p = 0.009) and IGF2R (p = 0.04) between CVS tissue and birth
weight (figure 1A–B). Importantly the range of IGF2 in relative
expression units was from 1.5–12 with one relative expression unit
being equivalent to a change in birth weight of 63 g. No
association was found for PHLDA2 (p = 0.73), IGF1 (p = 0.48) or
IGF1R (p = 0.08) between CVS tissue and birth weight (figure 1C–
E). PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 4 IGF2 Expression Correlates with Birth Weight Figure 3. mRNA expression levels of IGF2, IGF2R, and IGF1R in chorionic villi according to BW centile. Relative IGF2, IGF2R, and IGF1R
expression levels in the pregnancies with small (SGA), appropriate (AGA) and large (LGA) neonates. In the SGA group, compared to the AGA group, A)
IGF2 expression was lower (p = 3?661027), but there was no significant difference in the expression levels of B) IGF2R (p = 0?76) or C) IGF1R (p = 0?15). In the LGA group B) IGF2R expression was higher than in the AGA group (p = 0?02) and C) IGF1R expression was lower than in the SGA group
(p = 0?05). doi:10.1371/journal.pone.0085454.g003 Figure 3. mRNA expression levels of IGF2, IGF2R, and IGF1R in chorionic villi according to BW centile. Relative IGF2, IGF2R, and IGF1R
expression levels in the pregnancies with small (SGA), appropriate (AGA) and large (LGA) neonates. In the SGA group, compared to the AGA group, A)
IGF2 expression was lower (p = 3?661027), but there was no significant difference in the expression levels of B) IGF2R (p = 0?76) or C) IGF1R (p = 0?15). In the LGA group B) IGF2R expression was higher than in the AGA group (p = 0?02) and C) IGF1R expression was lower than in the SGA group
(p = 0?05). Results doi:10.1371/journal.pone.0085454.g003 doi:10.1371/journal.pone.0085454.g003 were biallellically expressed for IGF2R and 3 (12%) were
monoallelic, which is similar to the expected population frequency
(figure 4). Thus, skewed monoallelic/biallelic expression was
unlikely to be a factor reflected in the relative expression levels
associated with birth weight. promoter. However, in humans, IGF2R is polymorphic for its
imprinting status with 90% of individuals showing biallelic
expression [31]. This indicates a possible shift in the mechanism
of its regulation of expression away from that used in the mouse. Interestingly, here, the samples showing monallelic expression of
IGF2R had similar levels to those showing biallelic expression. promoter. However, in humans, IGF2R is polymorphic for its
imprinting status with 90% of individuals showing biallelic
expression [31]. This indicates a possible shift in the mechanism
of its regulation of expression away from that used in the mouse. Interestingly, here, the samples showing monallelic expression of
IGF2R had similar levels to those showing biallelic expression. As fetal size is subject to both positive and negative regulation,
IGF2 and IGF2R genes can exert opposite forces on the fetus,
achieving a fine degree of control over the growth process. As
levels of IGF2 increase in the fetus, the levels of IGF2R can also
increase, resulting in clearance of IGF2 from plasma and tissue
fluids, correcting and maintaining levels of IGF2 in the circulation. This acts as a fine-tuning rheostat designed to prevent babies from
overgrowth in order to maintain a normal growth trajectory. This
is also supported by the fact that no association was observed
between the IGF2/IGF2R expression levels and birth weight, as the
ligand and the receptor work tightly together to regulate growth. The same applies for IGF1 and its regulating receptor IGF1R. January 2014 | Volume 9 | Issue 1 | e85454 Discussion Biallelic expression, as shown in F), was found in the majority (88%) of informative cDNA samples
for IGF2R. doi:10.1371/journal.pone.0085454.g004 Figure 4. Imprinting analysis of IGF2, PHLDA2 and IGF2R. Representative sequencing chromat
CVS samples for the IGF2 A/G polymorphism (rs680) (A, B), one (from 21) for the PHLDA2 A/G polym
the IGF2R A/G polymorphism (rs1805075) (E, F, G, H). Heterozygous genomic CVS DNA sequence
expression from the corresponding cDNA samples are shown in B), D) and H). Monoallelic expressi
PHLDA2 samples as well as 12% of IGF2R samples. Biallelic expression, as shown in F), was found in
for IGF2R. doi:10.1371/journal.pone.0085454.g004 Figure 4. Imprinting analysis of IGF2, PHLDA2 and IGF2R. Representative sequencing chromatograms are shown from one of the 40 informative
CVS samples for the IGF2 A/G polymorphism (rs680) (A, B), one (from 21) for the PHLDA2 A/G polymorphism (rs1056819) (C, D) and two (from 24) for
the IGF2R A/G polymorphism (rs1805075) (E, F, G, H). Heterozygous genomic CVS DNA sequence is shown in A), C), E) and G). While monoallelic
expression from the corresponding cDNA samples are shown in B), D) and H). Monoallelic expression in cDNA was found in all informative IGF2 and
PHLDA2 samples as well as 12% of IGF2R samples. Biallelic expression, as shown in F), was found in the majority (88%) of informative cDNA samples
for IGF2R. doi:10 1371/journal pone 0085454 g004 doi:10.1371/journal.pone.0085454.g004 doi:10.1371/journal.pone.0085454.g004 contradict findings from studies involving mouse models which
highlighted IGF1 role in the regulation of both pre- and postnatal
growth [34]. There also remains a strong possibility that IGF1 is
indeed relevant to intrauterine growth in the later stages of
pregnancy as it is preparing the baby for a postnatal life. While
fetal chorionic fetal samples in the present cohort proved an
opportunity to assess correlation between gene expression and fetal
growth in early gestation (11–13 weeks), this narrow window might
not be the best to investigate IGF1 expression, as IGF1 does not
appear in the fetal circulation until later [35]. clusive results. However, in our study a statistically significant
association was observed between IGF2 expression levels in CVS
and birth weight, suggesting that the paternal genome is
promoting the baby’s growth earlier in pregnancy. Therefore,
the two parental genomes appear to be acting at different times
during pregnancy to control the fetal weight. Discussion As fetal size is subject to both positive and negative regulation,
IGF2 and IGF2R genes can exert opposite forces on the fetus,
achieving a fine degree of control over the growth process. As
levels of IGF2 increase in the fetus, the levels of IGF2R can also
increase, resulting in clearance of IGF2 from plasma and tissue
fluids, correcting and maintaining levels of IGF2 in the circulation. This acts as a fine-tuning rheostat designed to prevent babies from
overgrowth in order to maintain a normal growth trajectory. This
is also supported by the fact that no association was observed
between the IGF2/IGF2R expression levels and birth weight, as the
ligand and the receptor work tightly together to regulate growth. The same applies for IGF1 and its regulating receptor IGF1R. Previous studies have linked IGF2 expression to birth weight, by
showing that IGF2 in term placenta is decreased in SGA
pregnancies compared to controls [23,24]. However, the first
trimester IGF2 expression data reported in this study, provides the
first evidence for the role of this paternally expressed imprinted
gene as an in utero fetal growth enhancer this early in human
pregnancy. This is compatible with results previously described in
animal studies [9,32]. For example, in mouse experiments,
paternally expressed Igf2 was reported to control 40% of fetal
growth, with the maternally expressed gene Igf2r limiting fetal
growth [10,33]. In humans, IGF2 is consistently maternally
imprinted and therefore a paternal-expression driven growth In this study, expression of IGF1 in chorionic villi was not
associated with birth weight outcome. These results directly PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 January 2014 | Volume 9 | Issue 1 | e85454 5 IGF2 Expression Correlates with Birth Weight Figure 4. Imprinting analysis of IGF2, PHLDA2 and IGF2R. Representative sequencing chromatograms are shown from one of the 40 informative
CVS samples for the IGF2 A/G polymorphism (rs680) (A, B), one (from 21) for the PHLDA2 A/G polymorphism (rs1056819) (C, D) and two (from 24) for
the IGF2R A/G polymorphism (rs1805075) (E, F, G, H). Heterozygous genomic CVS DNA sequence is shown in A), C), E) and G). While monoallelic
expression from the corresponding cDNA samples are shown in B), D) and H). Monoallelic expression in cDNA was found in all informative IGF2 and
PHLDA2 samples as well as 12% of IGF2R samples. January 2014 | Volume 9 | Issue 1 | e85454 References 1. Pollack RN, Divon MY (1992) Intrauterine growth retardation: definition,
classification, and etiology. Clin Obstet Gynecol 35: 99–107. 15. McMinn J, Wei M, Schupf N, Cusmai J, Johnson EB, et al. (2006) Unbalanced
placental expression of imprinted genes in human intrauterine growth
restriction. Placenta 27: 540–549. 1. Pollack RN, Divon MY (1992) Intrauterine growth retardation: definition,
classification, and etiology. Clin Obstet Gynecol 35: 99–107. 2. Brodsky D, Christou H (2004) Current concepts in intrauterine growth
restriction. J Intensive Care Med 19: 307–319. 16. Apostolidou S, Abu-Amero S, O’Donoghue K, Frost J, Olafsdottir O, et al. (2007) Elevated placental expression of the imprinted PHLDA2 gene is
associated with low birth weight. J Mol Med 85: 379–387. 3. Albertsson-Wikland K, Wennergren G, Wennergren M, Vilbergsson G, Rosberg
S (1993) Longitudinal follow-up of growth in children born small for gestational
age. Acta Paediatr 82: 438–443. 17. Ishida M, Monk D, Duncan AJ, Abu-Amero S, Chong J, et al. (2012) Maternal
inheritance of a promoter variant in the imprinted PHLDA2 gene significantly
increases birth weight. Am J Hum Genet 90: 715–719. g
4. Barker DJ (1992) Fetal growth and adult disease. Br J Obstet Gynaecol 99: 275–
276. 18. Klammt J, Pfaffle R, Werner H, Kiess W (2008) IGF signaling defects as causes
of growth failure and IUGR. Trends Endocrinol Metab 19; 197–205. 5. Hales CN, Barker DJ, Clark PM, Cox LJ, Fall C, et al. (1991) Fetal and infant
growth and impaired glucose tolerance at age 64. BMJ 303: 1019–1022. 19. Le Roith D, Scavo L, Butler A (2001) What is the role of circulating IGF-I? Trends Endocrinol Metab 12: 48–52. 6. Harder T, Rodekamp E, Schellong K, Dudenhausen JM, Plagemann A (2007)
Birth weight and subsequent risk of type 2 diabetes: A meta-analysis. Am J Epidemiol 165: 849–857. 20. Koutsaki M, Sifakis S, Zaravinos A, Koutroulakis D, Koukoura O, et al. (2011)
Decreased placental expression of hPGH, IGF-I and IGFBP-1 in pregnancies
complicated by fetal growth restriction. Growth Horm IGF Res 21: 31–36. J
p
7. Moore T, Haig D (1991) Genomic imprinting in mammalian development: a
parental tug-of-war. Trends Genet 7: 45–49. 21. Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, et al. (2003)
IGF-I receptor mutations resulting in intrauterine and postnatal growth
retardation. N Engl J Med 349: 2211–22. 8. Willison K (1991) Opposite imprinting of the mouse Igf2 and Igf2r genes. Trends Genet 7: 107–109. 9. IGF2 Expression Correlates with Birth Weight To better understand the causes and consequences of fetal
growth restriction as a human pregnancy complication, we focused
on available tissue from the first trimester, as this time window is
more likely to accurately reflect the in utero growth potential. We
used a larger sample size and we investigated the whole growth
spectrum including not only SGA but also LGA neonates. We
reasoned that extending the investigation of these genes to samples
displaying macrosomic birth weight would also reveal further
correlations between growth parameters and gene expression. In
this study, LGA neonates had higher IGF2R expression levels
compared to AGA neonates and lower IGF1R levels compared to
SGA babies. Similar results have been reported in placenta tissue
for IGF1R mRNA levels alone [36], and this suggests that fetuses
with
high
birth
weight
are
producing
more
IGF2R
and
correspondingly less IGF1R, which could remove IGF2. This
balance may represent another important compensatory mecha-
nism in response to fetal overgrowth. This is also supported by the
fact that a significant association was observed between the ratio of
IGF2 to IGF1R and birth weight, suggesting that the levels of
IGF1R in LGA neonates decrease as their IGF2 levels increase, to
avoid any further increase in size. An important aim for antenatal care is the prediction, detection
and treatment of anomalous fetal growth. We know that variation
in size at birth results from interaction between fetal genetic factors
and the maternal genetic and uterine environment. In this study
we also implicate the important role of the father’s genes in fetal
growth in utero. Understanding the developmental role, function
and parent-of-origin effect of critical imprinted genes during
human fetal growth will make an important contribution to
effective
clinical
evaluation
of
growth
disorders
and
their
association with longer term, metabolically related, health risks
such as T2D. Determination of paternal IGF2 expression in the
first trimester, potentially in maternal blood, may act as a
predictor of fetal growth trajectory for later in the pregnancy. An
early
growth
biomarker
could
be
invaluable
to
alert
Obstetricians and Neonatologists towards closer ‘at risk’ pregnan-
cy surveillance. References DeChiara TM, Efstratiadis A, Robertson EJ (1990) A growth-deficiency
phenotype in heterozygous mice carrying an insulin-like growth factor II gene
disrupted by targeting. Nature 345: 78–80. 22. Abu-Amero SN, Ali Z, Bennett P, Vaughan JI, Moore GE (1998) Expression of
the insulin-like growth factors and their receptors in term placentas: a
comparison between normal and IUGR births. Mol Reprod Dev 49: 229–235. 10. Wang ZQ, Fung MR, Barlow DP, Wagner EF (1994) Regulation of embryonic
growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372:
464–467. p
p
23. Koukoura O, Sifakis S, Soufla G, Zaravinos A, Apostolidou S, et al. (2011) Loss
of imprinting and aberrant methylation of IGF2 in placentas from pregnancies
complicated with fetal growth restriction. Int J Mol Med 28: 481–487. 11. Weksberg R, Shen DR, Fei YL, Song QL, Squire J (1993) Disruption of insulin-
like growth factor-2 in Beckwith Wiedemann syndrome. Nat Genet 5:143–149. 24. Guo L, Choufani S, Ferreire J, Smith A, Chitayat D, et al. (2008) Altered gene
expression and methylation of the human chromosome 11 imprinted region in
small for gestational age (SGA) placentae. Developmental Biology 320: 79–91. 12. Eggermann T, Meyer E, Obermann C, Heil I, Schuler H, et al. (2005) Is
maternal duplication of 11p15 associated with Silver-Russell syndrome? J Med
Genet 42: e26. 25. Street ME, Seghini P, Fieni S, Ziveri MA, Volta C, et al. (2006) Changes in
interleukin-6 and IGF system and their relationships in placenta and cord blood
in newborns with fetal growth restriction compared with controls. Eur J Endocrinol 155: 567–574. 13. Qian N, Frank D, O’Keefe D, Dao D, Zhao L, et al. (1997) The IPL gene on
chromosome 11p15.5 is imprinted in humans and mice and is similar to
TDAG51, implicated in Fas expression and apoptosis. Hum Mol Genet 6: 2021–
2029. 26. Laviola L, Perrini S, Belsanti G, Natalicchio A, Montrone C, et al. (2005)
Intrauterine growth restriction in humans is associated with abnormalities in
placental insulin-like growth factor signaling. Endocrinology 146: 1498–1505. 14. Frank D, Fortino W, Clark L, Musalo R, Wang W, et al. (2002) Placental
overgrowth in mice lacking the imprinted gene Ipl. Proc Natl Acad Sci USA 99:
7490–7495. p
g
g
g
gy
27. Klauwer D, Blum WF, Hanitsch S, Rascher W, Lee PD, et al. Supporting Information Figure S1
Confounding factors. Correlation of birth weight
with gestational age at term (p = 4.8610215; Fig. S1A), with
maternal BMI (p = 0.0012; Fig. S1B), with gender (p = 1.461027;
Fig. S1C), with parity (p = 7.561025; Fig. S1D), and maternal
smoking status (p = 0.33; Fig S1E). (TIF) The ‘‘small baby syndrome hypothesis’’ proposed by Barker et
al., (1993) suggests that there is an inverse linear relation between
birth weight and T2D [37]. This is also supported by a large meta-
analysis [38]. The mechanisms by which birth weight is related to
T2D is still under debate, Barker et al. claim that this relationship
reflects long-term consequences of under-nutrition in utero [37],
whereas others do not see under-nutrition as playing a significant
role [39]. Interestingly, specific fetal IGF2 paternal haplotypes are
linked to higher maternal glucose levels that increase the risk of
gestational diabetes [40]. Nevertheless the amount of IGF2
available from the placenta early in the first trimester is likely to
be a major factor in promoting growth. Author Contributions Conceived and designed the experiments: CD SAA ACT PS GEM. Performed the experiments: CD SAA ACT MI RA LA LJL JLS. Analyzed
the data: CD MI RA LA LJL JLS. Contributed reagents/materials/
analysis tools: AS DP KHN LR PS GEM. Wrote the paper: CD SAA ACT
MI RA LA LJL JLS AS DP KHN LR PS GEM. Provided chorionic villus
samples: KHN. Gathered the sample’s clinical data: AS. Provided obstetric
advice and discussion: DP LR. Discussion This supports the
idea that whilst increased fetal growth is important early on, it
must still require careful regulation by the mother to ensure a
successful birth. In our study, SGA neonates had significantly lower IGF2
expression levels compared to AGA neonates, but no differences
were observed between the levels of the receptors IGF2R and
IGF1R. This highlights the importance of the IGF2 ligand rather
than the receptor level in determining size of the neonate in SGA
pregnancies. This is in agreement with previous studies investi-
gating IGF2 mRNA levels in the placentas from growth-restricted
pregnancies [23,24]. Although previous studies have shown that placental PHLDA2
expression is negatively associated with birth weight [16,17], in our
study no statistically significant association was observed between
PHLDA2 expression levels in CVS and birth weight. This suggests
that maternally expressed PHLDA2 is suppressing the baby’s
growth later in pregnancy rather than early on. Previous studies
investigating term placental IGF2 expression have shown incon- January 2014 | Volume 9 | Issue 1 | e85454 PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 6 IGF2 Expression Correlates with Birth Weight relationship to birthweight, length and gestational age in healthy newborns. Acta
Paediatr 86: 826–833. 34. Le Bouc Y, Gircquel C, Holzenberger M (2003) Physiology of somatotropic axis:
interest of gene inactivation experiments. Bull Acad Natl Med 187: 1225–43. 28. Smerieri A, Petraroli M, Ziveri MA, Volta C, Bernasconi S, et al. (2011) Effects
of cord serum insulin, IGF-II, IGFBP-2, IL-6 and cortisol concentrations on
human birth weight and length: pilot study. PLoS One 6: e29562. 35. Demendi C, Bo¨rzso¨nyi B, Nagy ZB, Rigo´ J Jr, Pajor A, et al. (2011) Gene
expression patterns of insulin-like growth factor 1, 2 (IGF-1, IGF-2) and insulin-
like growth factor binding protein 3 (IGFBP-3) in human placenta from preterm
deliveries: influence of additional factors. Eur J Obstet Gynecol 160: 40–44. 29. Ong K, Kratzsch J, Kiess W, Costello M, Scott C, et al. (2000) Size at birth and
cord blood levels of insulin, insulin-like growth factor I (IGF-I), IGF-II, IGF-
binding protein-1 (IGFBP-1), IGFBP-3, and the soluble IGF-II/mannose-6-
phosphate receptor in term human infants. The ALSPAC Study Team. Avon
Longitudinal Study of Pregnancy and Childhood. J Clin Endocrinol Metab 85:
4266–4269. 36. Iniguez G, Gonzalez CA, Argandona F, Kakarieka E, Johnson MC, et al. (2010)
Expression and protein content of IGF-I and IGF-I receptor in placentas from
small, adequate and large for gestational age newborns. Horm Res Paediatr 73:
320–327. 37. Barker DJ, Hales CN, Fall CH, Osmond C, Phipps K, et al. (1993) Type 2 (non-
insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syn-
drome X): relation to reduced fetal growth. Diabetologia 36: 62–67. 30. Giannoukakis N, Deal C, Paquette J, Goodyer CG, Polychronakos C (1993)
Parental genomic imprinting of the human IGF2 gene. Nat Genet 4: 98–101. 31. Monk D, Arnaud P, Apostolidou S, Hills FA, Kelsey G, et al. (2006) Limited
evolutionary conservation of imprinting in the human placenta. Proc Natl Acad
Sci USA 103: 6623–6628. g
g
38. Whincup PH, Kaye SJ, Owen CG, Huxley R, Cook DG, et al. (2008) Birth
weight and risk of type 2 diabetes A systematic review. JAMA 300: 2886–2897. 39. Hofman PL, Regan F, Jackson WE, Jefferies C, Knight DB, et al. (2004)
Premature birth and later insulin resistance. N Engl J Med 351: 2179–2186. 32. Constaˆncia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, et al. (2002) Placental-specific IGF-II is a major modulator of placental and fetal
growth. Nature 417: 945–948. 40. References (1997) IGF-I,
IGF-II, free IGF-I and IGFBP-1, -2 and -3 levels in venous cord blood: 7 PLOS ONE | www.plosone.org January 2014 | Volume 9 | Issue 1 | e85454 January 2014 | Volume 9 | Issue 1 | e85454 37. Barker DJ, Hales CN, Fall CH, Osmond C, Phipps K, et al. (1993) Type 2 (non-
insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syn-
drome X): relation to reduced fetal growth. Diabetologia 36: 62–67. IGF2 Expression Correlates with Birth Weight Petry CJ, Seear RV, Wingate DL, Manico L, Acerini CL, et al. (2011)
Associations between paternally transmitted fetal IGF2 variants and maternal
circulating glucose concentartions in pregancy. Diabetes 60: 3090–3096. g
33. Lau MM, Stewart CE, Liu Z, Bhatt H, Rotwein P, et al. (1994) Loss of the
imprinted IGF2/cation-independent mannose 6-phosphate receptor results in
fetal overgrowth and perinatal lethality. Genes Dev 8: 2953–2963. January 2014 | Volume 9 | Issue 1 | e85454 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
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English
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Rubella sero-prevalence among children in Kilimanjaro region: a community based study prior to the introduction of rubella vaccine in Tanzania
|
The Italian Journal of Pediatrics/Italian journal of pediatrics
| 2,017
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cc-by
| 5,841
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Rubella sero-prevalence among children in
Kilimanjaro region: a community based
study prior to the introduction of rubella
vaccine in Tanzania Nikolas A. S. Chotta1,7*, Melina Mgongo1,2, Jacqueline G. Uriyo1,2, Sia E. Msuya2,5,6, Babill Stray-Pedersen1,2,4
and Arne Stray-Pedersen3 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Childhood rubella infection is a mild, self-limiting illness. Rubella infection among pregnant women
however, is a major public health concern. Depending on gestation age, it may result in fetal death, stillbirth or a
new-born with congenital rubella syndrome (CRS). Maternal antibodies protect young infants from rubella infection
and lifelong immunity is acquired by vaccination or post-rubella infection. This study aims at characterizing rubella
infection and its epidemiology in the Kilimanjaro region, prior to the introduction of the rubella vaccine in Tanzania. Methods: This was a population based cross-sectional study, covering all the seven districts in Kilimanjaro region,
North-eastern Tanzania. The study population included children of 0 to 36 months of age and their mothers/primary
caretakers. A multistage sampling method was used to obtain a representative sample of the children. Interviews were
conducted using a structured questionnaire. Dried blood spot (DBS) samples were taken from eligible children. Rubella
specific IgG antibodies were detected from eluted serum by enzyme-linked immunosorbent assay (ELISA). Descriptive
statistics were used to summarize the data, the difference between groups was tested by Fishers exact test or
chi square test as appropriate. Univariate and multivariate analysis was used, with rubella sero-positive groups
as dependent variables and the socio-demographic, children, paediatric and parental factors as independent
variables, the Odds ratio and their 95% confidence intervals were calculated to assess the strength of association between
the dependent and independent variables. A p value less than 0.05 was considered significant. Results: The overall rubella sero-prevalence was 1.8%. Rural residence was associated with greater risk for rubella infection. Other family characteristic did not predict rubella infection. Conclusions: This study highlights the low natural immunity to rubella among children prior to the introduction of rubella
vaccine in Tanzania. Our research underscores the need for an effective rubella vaccination program to prevent CRS. More
epidemiologic and immunologic studies are needed to guide the vaccination deployment and administration
strategy in Tanzania. Keywords: Childhood, ELISA, Immunity, Prevalence, Rubella, Vaccination * Correspondence: sagumochotta@gmail.com
1Institute of Clinical Medicine, University of Oslo, Oslo, Norway
7P.O. Box 8418, Moshi, Tanzania
Full list of author information is available at the end of the article © The Author(s). * Correspondence: sagumochotta@gmail.com
1Institute of Clinical Medicine, University of Oslo, Oslo, Norway
7P.O. Box 8418, Moshi, Tanzania
Full list of author information is available at the end of the article Chotta et al. Italian Journal of Pediatrics (2017) 43:63
DOI 10.1186/s13052-017-0379-3 Chotta et al. Italian Journal of Pediatrics (2017) 43:63
DOI 10.1186/s13052-017-0379-3 Open Access Open Access Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chotta et al. Italian Journal of Pediatrics (2017) 43:63 Page 2 of 7 Page 2 of 7 Background cases, rubella infection and associated CRS is oftentimes
missed and/or under-reported. Rubella is a mild and self-limiting illness [1]. The causative
agent of rubella is the rubella virus, a single stranded RNA
virus of the genus Rubivirus in the Togaviridae family. Ru-
bella virus maintains only one serotype exclusively able to
affect humans [2]. Transmission can occur through respira-
tory droplets or direct contact. Vertical transmission of ru-
bella virus is also possible [1]. In Tanzania, though vaccination against rubella was
started in 2014 (Additional file 1), there are few studies
describing the epidemiology of rubella or the efficacy of
interventions [22–24]. More epidemiologic studies are
needed to estimate gross disease burden, and explore
disease distribution along with associated risk factors for
rubella infection. This information could provide the
evidence base to better inform the planning, monitoring
and evaluation of rubella prevention program [22, 25]. Congenital rubella infection has been shown to cause the
most severe form of rubella disease. This route of transmis-
sion results from maternal rubella infection before concep-
tion or in early months of pregnancy. Depending upon the
timing of fetal infection, infection may result in loss of preg-
nancy or congenital rubella syndrome (CRS) [1, 3]. Our study aims to determine the pre-vaccination ru-
bella sero-prevalence among children in the Kilimanjaro
region of Tanzania [26, 27]. Predictors of rubella infec-
tion including: age, maternal age, parity, urban versus
rural settings and other socio-economic factors were ex-
plored [28]. Our findings will provide pilot data for fu-
ture large-scale rubella studies, and aid in the planning
for immediate public health campaigns as well as long-
term rubella elimination policy [29]. Before the development of the rubella vaccine, rubella
infection was prevalent worldwide. Outbreaks occurred
in cycles every 5–9 years [1]. In countries where rubella
vaccination campaigns have not yet begun or where ad-
ministration is currently sub-optimal, rubella outbreaks
continue to expose susceptible women to an increased
risk for miscarriages, still births or CRS in their new-
borns [4–8]. Population susceptibility to infection is pre-
dicted by herd immunity, population density, place of
residence, socio-economic factors, as well as other epi-
demiologic indicators [9–12]. Study design and area The study was a community based cross-sectional study,
covering all the seven districts of the Kilimanjaro region
in North-eastern Tanzania. Kilimanjaro is among 30 ad-
ministrative regions of Tanzania, and covers an area of
1831.32 km2. Kilimanjaro maintains an estimated popu-
lation of 1,640,087, of those, 451,911 of which are
women of the reproductive age. The fertility rate (per
woman) is 4.8, with an annual growth rate of 1.6%. In
Kilimanjaro,
there
are
152,198
children
aged
0–
36 months. Most residents (up to 80%) engage in sub-
sistence farming and live in rural areas [30]. g
Prevention of rubella is aimed at reducing the risk of
congenital infection and consequent CRS [1, 3, 12]. Re-
cent research has recommended that inclusion of rubella
containing vaccines (RCVs) in universal childhood vac-
cination programs, could be targeted as a major preven-
tion strategy [10]. Other available prevention strategies
include: routine childhood vaccination, mass vaccination
of adolescents and adults, or a combined strategy [13–
15]. In order to select an optimal, evidence-based vac-
cination strategy for a particular geographic region, fur-
ther information regarding the epidemiology of rubella
infection is needed, along with a better understanding of
desired outcomes (control or elimination) and availabil-
ity of resources [16]. Study population
h
d The
study
population
included
children
of
0
to
36 months of age and their mothers/primary caretakers,
in seven districts of the Kilimanjaro region. The other
inclusion criteria was presence of blood sample for ru-
bella testing. The specific exclusion criteria was; children
with proven vaccination with a rubella containing vac-
cine, were not eligible for this study.. Rubella vaccines are available in monovalent or in
combined forms like; Measles and Rubella (MR), Mea-
sles, Mumps and Rubella (MMR), Measles, Mumps, Ru-
bella and Varicella (MMRV), the vaccines are safe and
effective, with rubella sero-conversion rates of >95%
after a single dose [16–18]. To be effective, rubella vac-
cination coverage must be high enough to achieve herd
immunity. Vaccination coverage of less than 80% may
result in a shift of infection to young adults, with result-
ant increase in CRS [5, 19]. Routine and high vaccin-
ation coverage has successfully eliminated rubella in
most North American and European countries [20, 21]. Interviews Interviews were conducted using a structured question-
naire. Information collected included: social, economic
and demographic characteristics, such as age, sex, mater-
nal education level, marital status, occupation, income,
household facilities, reproductive history, pediatric and
child health history. Sampling method Sampling method
From June 2010 to May 2011, a multistage sampling
method was used to obtain a representative sample of
children 0 to 36 months [31]. Sampling was first con-
ducted by a random cluster sampling method, and fifty
clusters were selected from 7 districts proportionate to
the population size. This was achieved from a sampling
frame, which included the number of all children 0 to
36 months in each village, with a column for cumulative
population. The total population was divided by the Most epidemiologic data on rubella infection are de-
rived from ongoing Measles surveillance. Given that ru-
bella is a mild disease and asymptomatic in up to 50% of Chotta et al. Italian Journal of Pediatrics (2017) 43:63 Page 3 of 7 Page 3 of 7 number of clusters to get the sampling interval of
166,808/50. The first cluster was determined by multi-
plying a computer generated random number between 0
and 1 and the sampling interval. By adding the previous
number to the sampling interval, the subsequent clusters
were located, to the total of fifty clusters. A compact
segment sampling method was applied to select fifty
children 0 to 36 months in each cluster. The selected
clusters were mapped into segments of fifty children,
each segment was allocated a number on a piece of
paper, and one was selected randomly. In each selected
segment, a quota sampling method was used for the
children to be recruited. Recruitment was done by visit-
ing all households in the segment until fifty children
were enrolled. In the case that fewer than fifty eligible
children were enrolled in a segment, enrollment went to
a second randomly selected segment. To maximize re-
cruitment, prior information was given to mothers/care-
takers and their eligible children to be available on the
day of interview. titer ≤10 UI/ml. Equivocal results were defined as a titer
of 10–15 UI/ml, indeterminate specimens were retested
until test results were conclusive. Data management Data obtained from questionnaires and laboratory test
results were entered and verified for consistency. Statis-
tical analysis was performed using the Statistical Package
for Social Sciences (SPSS) for windows version 16. Ru-
bella infection status was assigned as the dependent vari-
able,
and
all
other
epidemiologic
indicators
were
assigned as the independent variables. Descriptive statis-
tics were used to summarize the data. Results are
expressed
as
real
numbers
and
percentages
were
assigned to rubella sero-prevalence results. Differences
between groups were compared using Fishers exact test
or chi square test as appropriate. Univariate and multi-
variate analysis were used, with rubella sero-positive
groups assigned as dependent variables and epidemio-
logic factors assigned as independent variables. Odds ra-
tios, along with their 95% confidence intervals, were
calculated to assess the strength of association between
the dependent and independent variables. A p-value of
<0.05 was considered statistically significant. Results A total of 1870 children (0 to 36 months) were enrolled
and provided blood samples tested for rubella. The ma-
jority (93.6%) of patient enrolled were 7–36 months. The 1751 (93.6%) of the children in the 7–36 months
group, were included in this analysis. The mean age of
the participants was 15.9 months (SD = 6.5), and the
male enrollment rate was 902/1751 (51.5%). Children
were enrolled from all districts of Kilimanjaro region,
and their residence statuses were classified as 25.8%
urban and 74.2% rural. The children’s mean hemoglobin
level was 10.2 (SD = 1.5), and 97% of them had complete
vaccination status for their age group. Blood sample collection and laboratory methods Blood sample collection and laboratory methods
Dried blood spots (DBS) were taken from eligible chil-
dren by finger or heel prick. Blood was dropped onto
standardized filter papers, and a corresponding partici-
pant identification number was written on the sample. The filter papers were air dried and sealed in airtight
plastic bags. The samples were sent to the Kilimanjaro
Christian Medical Centre (KCMC) Clinical laboratory,
where they were stored in a minus 20 degree Celsius
freezer prior to analysis. Rubella specific IgG antibodies
were detected from eluted serum by enzyme-linked im-
munosorbent assay (ELISA), using commercial test kits
from Human Diagnostics worldwide, HUMAN Gesell-
schaft für Biochemica und Diagnostica mbH, Germany
(ref. 51,208). The presence of rubella-specific IgG was
determined by comparing the optical density (OD) to
the cut off ranges (index). A positive test was deter-
mined when the absorbance test result at 450 nm was
≥0.8. For quantitative estimation of rubella IgG in posi-
tive test specimens, the OD was converted to Inter-
national Units per milliliter by plotting a standard curve
for each assay according to the test kit instructions. The
estimated levels were read off the graph using their indi-
vidual A450. A positive test was defined as a titer equal
or more than 15UI/ml, a negative result was defined as a The interviewed mother’s mean age was 28.7 (SD = 7)
years. Many of the participant mothers (48.8%) were
peasant farmers. Antenatal attendance was 98.2%, deliv-
ery in medical facility was 86.8% and 95.2% of mothers
practiced breast feeding on demand. Rubella sero-prevalence among participant children
was 1.8% (32/1751). Rubella sero-prevalence increased
with age from 1.1% in the 7 to12 month age group, to
2.3% in 13 to 24 month and 25 to 36 month age groups. We
observed
that,
rubella
sero-prevalence
varied
among the districts. Same district demonstrated the
highest sero-prevalence (5.1%) and Moshi rural district
maintained
the
lowest
sero-prevalence
(1.2%)
(p = 0.001). Children in Rombo and Moshi districts had
lower sero-prevalence compared to those in Same dis-
trict (OR 0.3, 95% CI: 0.1, 0.8) for Rombo, and (OR 0.2, Rubella sero-prevalence among participant children
was 1.8% (32/1751). Rubella sero-prevalence increased
with age from 1.1% in the 7 to12 month age group, to
2.3% in 13 to 24 month and 25 to 36 month age groups. We
observed
that,
rubella
sero-prevalence
varied
among the districts. Blood sample collection and laboratory methods Same district demonstrated the
highest sero-prevalence (5.1%) and Moshi rural district
maintained
the
lowest
sero-prevalence
(1.2%)
(p = 0.001). Children in Rombo and Moshi districts had
lower sero-prevalence compared to those in Same dis-
trict (OR 0.3, 95% CI: 0.1, 0.8) for Rombo, and (OR 0.2, Chotta et al. Italian Journal of Pediatrics (2017) 43:63 Page 4 of 7 Page 4 of 7 95% CI: 0.1, 06) for Moshi district. Children of Mothers
doing small business/unskilled jobs had lower rubella
sero-prevalence as compared to those of skilled/profes-
sional’s (OR 0.1, 95% CI: 0.02, 0.7). We observed no
positive rubella test results from individuals sampled
from Hai, Siha and Moshi urban districts (Table 1). had increased odds as compared to those breastfed on de-
mand (OR 5.6, 95% CI: 1.5, 20.6) (Table 2). We did not
observe statistically significant variation in rubella sero-
prevalence depending upon maternal age, maternal educa-
tion, parity and the number of live children. In Multivariate analysis Moshi district had significantly
lower odds of rubella sero-prevalence (OR 0.3, 95% CI:
0.1, 0.8) (Table 2). Rubella sero-prevalence was statistically different de-
pending upon urban versus rural residences (p = 0.018). Mother’s occupation (p = 0.038), place of delivery
(p = 0.025), and breast feeding type (p = 0.027) were also
statistically significant epidemiologic factors. Children de-
livered at home had increased odds of rubella as com-
pared to those delivered at health facilities (OR 2.6 95%
CI: 1.2,5.8), Children who were breastfed on time table avariables with missing information Discussion This is the first community based study, characterizing
rubella epidemiology in Kilimanjaro region, Tanzania. The study found a low overall rubella sero-prevalence
in the region (1.8%), signifying low natural immunity to Table 1 Background characteristics and rubella seropositivity, N = 1751
Variable name
N
%
Rubella sero-positive % (n)
Chi- square
p-value
Total
1751
100
1.8 (32)
Child’s gender
Male
902
51.5
1.9 (17)
0.5
Female
849
48.5
1.8 (15)
Child’s age categories
7–12 months
647
37
1.1 (7)
0.182
13–24 months
932
53.2
2.3 (21)
25–36 months
172
9.8
2.3 (4)
Child’s District of residence
Same
312
17.8
5.4 (17)
< 0.001
Mwanga
132
7.5
2.3 (3)
Rombo
359
20.5
1.7 (6)
Moshi District
448
25.6
1.3 (6)
Moshi municipal
180
10.3
-
Hai
172
9.8
-
Siha
148
8.5
-
Child’s Residence (N = 1727)
Rural
1282
74.2
2.2 (28)
0.018
Urban
445
25.8
0.7 (3)
Mother’s occupationa (N = 1690)
Unemployed
299
17.7
4 (1.3)
0.038
Peasant farmer/casual
934
55.3
20 (2.1)
Small scale business
367
21.7
2 (0.5)
Skilled/professional
90
5.3
4 (4.4)
Place of deliverya (N = 1724)
Medical facility
1505
87.3
1.5 (22)
0.025
Home
219
12.7
4.1 (9)
Breast feeding typea (N = 1219)
On demand
1160
95.2
0.9 (11)
0.027
Time table
59
4.8
5.1 (3)
avariables with missing information Chotta et al. Italian Journal of Pediatrics (2017) 43:63 Page 5 of 7 Table 2 Factors associated with rubella sero-prevalence among children aged 7–36 months
Exposure variables
Crude
OR
P-
value
95% C. I. Adjusted
OR
P-
value
95% C. I. Discussion Lower
Upper
Lower
Upper
District
Same
1
-
-
-
1
-
-
-
Mwanga
0.404
0.153
0.116
1.401
0.525
0.337
0.141
1.956
Rombo
0.295
0.011
0.115
0.758
0.393
0.072
0.142
1.088
Moshi District
0.236
0.003
0.092
0.604
0.268
0.015
0.093
0.776
Residence
Rural
1
-
-
-
1
-
-
-
Urban
0.304
0.051
0.092
1.005
2.497
0.203
0.611
10.199
Mother’s occupation
Skilled
1
-
-
-
1
-
-
-
Unemployed
0.292
0.086
0.071
1.190
0.613
0.512
0.142
2.647
Peasant farmer
0.470
0.178
0.157
1.408
0.412
0.345
0.065
2.601
Small business & unskilled
0.118
0.014
0.021
0.654
0.855
0.896
0.081
9.014
Place of delivery
Medical facility
1
-
-
-
1
-
-
-
Home
2.648
0.015
1.210
5.797
2.037
0.378
0.419
9.901
Breast feeding
On demand
1
-
-
-
1
-
-
-
Time table
5.596
0.010
1.518
20.625
5.179
0.052
0.989
27.136
OR Odds ratio, CI Confidence interval Table 2 Factors associated with rubella sero-prevalence among children aged 7–36 months OR Odds ratio, CI Confidence interval 7–12 months. The observed waning of passive anti-
bodies at 7 months, may explain the increasing suscepti-
bility, leading to increased rubella infection for infants
above 6 months in our study [33]. rubella, rendering children to a high susceptibility (98.2%)
for infection in the event of an outbreak of wild rubella
virus. This low level of natural immunity warrants the inclu-
sion
of
the
rubella
vaccine
in
routine
childhood
immunization in this region. This finding is similar to other
pre-vaccine rubella epidemiologic studies, documenting the
peak age of infection among children of 5–9 years [22]. Ru-
bella epidemics tend to repeat every 5–9 years, which can
partly explain the low sero-prevalence in young children
born prior to an epidemic period [24]. The results are simi-
lar with findings from Bangladesh where Sultana et al. re-
ported a lack of protective antibodies against rubella among
children 3 months to 5 years, however demonstrated in-
creasing rubella sero-prevalence with age to 71% at
>10–15 years [26]. Our findings provide important information in choos-
ing the appropriate vaccination age. In Tanzania, rubella
vaccine is combined with the measles vaccine (MR), and
since rubella is a mild disease, measles immunology and
epidemiology determines the optimal timing for MR vac-
cination. Currently, MR is administered in two doses,
the first dose at 9–12 months, and the second dose at
15–18 months [34]. Competing interests
h
h
d
l
h Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ contributions Authors’ contributions
NSAC, JGU, SEM, BSP and ASP designed the study. NSAC, MM and JGU
participated in data collection. NSAC, MM, SEM, BSP and ASP analyzed
the data and interpreted the results. NSAC wrote the initial manuscript. All authors read and approved the final manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Abbreviations CRS: Congenital rubella syndrome; DBS: Dried blood spot; ELISA: Enzyme
linked immunosorbent assay; HIV: Human immunodeficiency virus;
IgG: Immunoglobulin G; KCMC: Kilimanjaro Christian Medical Centre;
MMRV: Measles Mumps Rubella Varicella; OD: Optical density;
PCR: Polymerase chain reaction; RCV: Rubella containing vaccines;
REK: Regional Committee for Medical & Health Research Ethics;
WHO: World Health Organization 3. Quintana E, Solórzano C, Torna N, et al. Congenital rubella syndrome: a
matter of concern. Rev Panam Salud Publica. 2015;37(3):179–86. 4. Mirambo M, Majigo M, Aboud S, et al. Serological makers of rubella infection in
Africa in the pre vaccination era: a systematic review. BMC Res Notes. 2015;8:716. ;
( )
4. Mirambo M, Majigo M, Aboud S, et al. Serological makers of rubella infection in
Africa in the pre vaccination era: a systematic review. BMC Res Notes. 2015;8:716. 5. Lanzieri T, Segatto T, Sequiera M, et al. Burden of congenital rubella
syndrome after a community-wide rubella outbreak, Rio Branco, Acre, Brazil,
2000-2001. Pediatr Infect Dis J. 2003;22(4):323–9. 6. Banatvala JE, Brown DW. Rubella. Lancent. 2004;363(9415):1127 37. 7. World Health Organization. Global measles and rubella strategic plan: 2012–
2020. Geneva: World Health Organization; 2012. whqlibdoc.who.int/
publications/2012/9789241503396_eng.pdf. Accessed 2 Mar 2016. References Additional file 1: Past and current Childhood immunization schedule in
Tanzania. (DOCX 12 kb) 1. World Health Organization. Rubella vaccines. WHO Position Paper. Wkly
Epidemiol Rec. 2011;86:301–316. http://www.who.int/wer/2011/wer8629.pdf. Accessed 20 Mar 2016. 2. Kolawole O, Anjorin E, Adokanle D, et al. Serology of rubella IgG antibody in
pregnant women in Osogbo, Nigeria. Int J Prev Med. 2014;5(3):287–92. 2. Kolawole O, Anjorin E, Adokanle D, et al. Serology of rubella IgG antibody in
pregnant women in Osogbo, Nigeria. Int J Prev Med. 2014;5(3):287–92. 3. Quintana E, Solórzano C, Torna N, et al. Congenital rubella syndrome: a
matter of concern. Rev Panam Salud Publica. 2015;37(3):179–86. 4. Mirambo M, Majigo M, Aboud S, et al. Serological makers of rubella infection in
Africa in the pre vaccination era: a systematic review. BMC Res Notes. 2015;8:716. 5. Lanzieri T, Segatto T, Sequiera M, et al. Burden of congenital rubella
syndrome after a community-wide rubella outbreak, Rio Branco, Acre, Brazil,
2000-2001. Pediatr Infect Dis J. 2003;22(4):323–9. 6. Banatvala JE, Brown DW. Rubella. Lancent. 2004;363(9415):1127–37. 7. World Health Organization. Global measles and rubella strategic plan: 2012–
2020. Geneva: World Health Organization; 2012. whqlibdoc.who.int/
publications/2012/9789241503396_eng.pdf. Accessed 2 Mar 2016. 8. Seetoo K, Carlos MP, Blythe D, Trivedi L, Myers R, England T, Agee C, Arnold
B, Dobbs C, McIntyre M,Ramirez E. Three cases of congenital rubella
syndrome in the postelimination era-Maryland, Alabama, andIllinois, 2012. Morbidity and Mortality Weekly Report. 2013;62(12):226-9. http://www.cdc. gov/.../mm6212.pdf. 9
Strebel P Dabbagh A Gacic A Reef S et al Progress towards Control of pregnant women in Osogbo, Nigeria. Int J Prev Med. 2014;5(3):287–92. 3. Quintana E, Solórzano C, Torna N, et al. Congenital rubella syndrome: a
matter of concern. Rev Panam Salud Publica. 2015;37(3):179–86. 4. Mirambo M, Majigo M, Aboud S, et al. Serological makers of rubella infection in
Africa in the pre vaccination era: a systematic review. BMC Res Notes. 2015;8:716. Discussion Our findings on rubella Sero-
epidemiology, confirms that, the schedule for MR vac-
cine at this age will be an effective timing, as 98% of the
children have no immunity against rubella infection. This study found an association between rubella sero-
prevalence and district of residence. Other test variables
including: gender, age, rural versus urban residence,
mother’s occupation, place of delivery and breast feeding
type were determined to be statistically insignificant fac-
tors in our analysis [24, 27]; however, as reported else-
where, these factors were found to be significant factors
for pre-vaccine era rubella epidemiology [26]. Manirakiza
and colleagues in a Central African study reported no gen-
der differences in rubella sero-prevalence; however, they
did report an increase in rubella sero-prevalence with in-
creasing age [27]. Similarly, Ki and colleagues reported an Infants below 7 months were not included in the ana-
lysis as all had low antibodies levels below the test cut-
off threshold. The low antibody titers can be attributed
to
non–immune
mothers,
a
finding
reported
by
Mwambe and colleagues in a study of pregnant women
in Mwanza, Tanzania [32]. Early waning of passive anti-
bodies, is another possible explanation for low level of
antibodies in children below 7 months, a finding re-
ported by Manirakiza and colleagues in Central African
Republic [27], where maternal rubella antibodies sero-
prevalence rates were >45% among infants 0–3 months,
decreasing to >10% at 4–6 months and finally to zero at Chotta et al. Italian Journal of Pediatrics (2017) 43:63 Page 6 of 7 increase in sero-prevalence with increasing child age in a
study among Korean children [35]. A study in Mwanza,
Tanzania conducted by Mirambo et al., found that, rural
residence and child age were statistically significant associ-
ated factors for rubella infection [24]. Higher risk of ru-
bella infection in rural areas was also reported in a recent
Kenyan study, by Kombich and colleagues, where lower
social economic status and age (above 7 years) were other
associated risks for rubella infection [36]. The differences
in sero-prevalence between urban and rural settings
points to special considerations for targeted vaccination
campaigns to specific high risk communities. Ethics approval and consent to participate The study protocol was approved by the Tanzanian National institute of
Medical Research, certificate number 938. Ethical clearance to use the data
and blood samples for analysis was obtained from Kilimanjaro Christian
Medical College Research and ethics review committee; certificate number
917 and approval for the project was given by the Regional Ethics
Committee (REK) in South East D Norway. Before data collection,
informed consent was obtained from each participant. Identity of the
study participants was masked from the research team, and only
participant identification numbers were used in the data analysis portion
of this study. Availability of data and materials The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Acknowledgements
W
f l
h We are grateful to the families for participating in this study. We would also
like to thank the management and staff of KCMC clinical laboratory for their
valuable contribution. We are grateful to the Regional and District medical
officers for the permission to conduct the study. This study was financially
supported by Letten foundation, Norway. 8. Seetoo K, Carlos MP, Blythe D, Trivedi L, Myers R, England T, Agee C, Arnold
B, Dobbs C, McIntyre M,Ramirez E. Three cases of congenital rubella
syndrome in the postelimination era-Maryland, Alabama, andIllinois, 2012. Morbidity and Mortality Weekly Report. 2013;62(12):226-9. http://www.cdc. gov/.../mm6212.pdf. 9. Strebel P, Dabbagh A, Gacic A, Reef S, et al. Progress towards. Control of
rubella and prevention of CRS-worldwide morbidity & mortality weekly
report. 2010;59(40):1307–10. 9.
Strebel P, Dabbagh A, Gacic A, Reef S, et al. Progress towards. Control of
rubella and prevention of CRS-worldwide morbidity & mortality weekly
report. 2010;59(40):1307–10. ccessed 0
a
0 6.
2.
Kolawole O, Anjorin E, Adokanle D, et al. Serology of rubella IgG antibody in
pregnant women in Osogbo, Nigeria. Int J Prev Med. 2014;5(3):287–92.
3.
Quintana E, Solórzano C, Torna N, et al. Congenital rubella syndrome: a
matter of concern. Rev Panam Salud Publica. 2015;37(3):179–86.
4.
Mirambo M, Majigo M, Aboud S, et al. Serological makers of rubella infection in
Africa in the pre vaccination era: a systematic review. BMC Res Notes. 2015;8:716.
5.
Lanzieri T, Segatto T, Sequiera M, et al. Burden of congenital rubella
syndrome after a community-wide rubella outbreak, Rio Branco, Acre, Brazil,
2000-2001. Pediatr Infect Dis J. 2003;22(4):323–9.
6.
Banatvala JE, Brown DW. Rubella. Lancent. 2004;363(9415):1127–37.
7.
World Health Organization. Global measles and rubella strategic plan: 2012–
2020. Geneva: World Health Organization; 2012. whqlibdoc.who.int/
publications/2012/9789241503396_eng.pdf. Accessed 2 Mar 2016.
8.
Seetoo K, Carlos MP, Blythe D, Trivedi L, Myers R, England T, Agee C, Arnold
B, Dobbs C, McIntyre M,Ramirez E. Three cases of congenital rubella
syndrome in the postelimination era-Maryland, Alabama, andIllinois, 2012.
Morbidity and Mortality Weekly Report. 2013;62(12):226-9. http://www.cdc.
gov/.../mm6212.pdf.
9
S
b l P D bb
h A G
i A R
f S
l P
d
C
l
f Author details
1 1Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 2Better Health
for African Mother and Child, Moshi, Tanzania. 3Institute of Public Health,
Department of forensic medicine, University of Oslo, Oslo, Norway. 4Division
of Gynaecology and Obstetrics, Oslo University Hospital, Rikshospitalet, Oslo,
Norway. 5Institute of Public Health, Department of Community Health,
Kilimanjaro Christian Medical University College, Moshi, Tanzania. 6Institute of
Public Health, Department of Epidemiology and Biostatistics, Kilimanjaro
Christian Medical University College, Moshi, Tanzania. 7P.O. Box 8418, Moshi,
Tanzania. We recommended further epidemiologic studies to es-
tablish the burden and risk factors for rubella infection
and CRS, as well as to establish geographical immunity
gaps. Post-rubella vaccine introduction studies are neces-
sary to characterize current vaccine coverage, potential
barriers to intervention, safety and efficacy of RCVs, with
special focus on disadvantaged rural populations. Christian Medical University College, Moshi, Tanzania. 7P.O. Box 8418, Moshi,
Tanzania. Received: 1 May 2017 Accepted: 13 July 2017 Conclusions
h d The data presented in this study were obtained before intro-
duction of rubella vaccine in Kilimanjaro region, Tanzania. The study findings of low natural immunity to rubella
among children in the age group of 7 to 36 months is signifi-
cant, as well as the increase of rubella prevalence with age,
which poses a risk for rubella epidemics and increase in
CRS. These findings justify the need for introduction of ru-
bella immunization to eliminate rubella transmission. A suc-
cessful immunization strategy in Tanzania demands further
evidence-based guidance from additional epidemiologic and
immunologic studies performed across this region. Funding
N
f
di No funding was granted for this research. Page 7 of 7 Chotta et al. Italian Journal of Pediatrics (2017) 43:63 10. Robertson S, Cutts F, Samuel R, Diaz-Ortega J. Control of congenital rubella
and congenital rubella syndrome (CRS) in developing countries, part 2:
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position paper. Paediatr Child Health. 2007;12:9. 13. Grant B. Rubella and congenital rubella syndrome control and elimination –
Global progress 2002-2012. Mortal Morb Wkly Rep. 2013;62(48):983–6. 14. Lanzieri T, Pinto D, Prevots D. Impact of rubella vaccination strategy on the
occurrence of congenital rubella syndrome. J Pediatr. 2007;83(5):415–21. 15. Taneja D, Sharma P. Targeting rubella for elimination. Indian J Public Health. 2012;56(4):269. 16. Strebel P, Gacic-Dobo M, Reef S, et al. Global use of rubella vaccines. J Infect
Dis. 2011;204(12):579. 17. Centers for Disease Control and Prevention, Recommended immunizations
for children from birth through 6 years old. 2015. http://www.cdc.gov/
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mumps, 2013: summary recommendations of the advisory committee on
immunization practices (ACIP). MMWR. 2013;62(RR04):1–34. 19. Lessler J, Jessica C, Metcalf E. Balancing evidence and uncertainty when
considering rubella vaccine introduction. PLoS One. 8(7):e67639. doi:10. 1371/journal.pone.0067639. 20. Kinoshita R, Nishiura H. Assessing herd immunity against rubella in Japan: a
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Analysis of public opinion evolution of COVID-19 based on LDA-ARMA hybrid model
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Complex & intelligent systems
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Complex & Intelligent Systems (2021) 7:3165–3178
https://doi.org/10.1007/s40747-021-00514-7 Complex & Intelligent Systems (2021) 7:3165–3178
https://doi.org/10.1007/s40747-021-00514-7 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract The aim of this study was to explore a method for developing an emotional evolution classification model for large-scale
online public opinion of events such as Coronavirus Disease 2019 (COVID-19), in order to guide government departments
to adopt differentiated forms of emergency management and to correctly guide online public opinion for severely afflicted
areas such as Wuhan and those afflicted elsewhere in China. We propose the LDA-ARMA deep neural network for dynamic
presentation and fine-grained categorization of a public opinion events. This was applied to a huge quantity of online public
opinion texts in a complicated setting and integrated the proposed sentiment measurement algorithm. To begin, the Latent
Dirichlet Allocation (LDA) was employed to extract information about the topic of comments. The autoregressive moving
average model (ARMA) was then utilized to perform multidimensional sentiment analysis and evolution prediction on large-
scale textual data related to COVID-19 published by netizens from Wuhan and other countries on Sina Weibo. The results
show that Wuhan netizens paid more attention to the development of the situation, treatment measures, and policies related
to COVID-19 than other issues, and were under greater emotional pressure, whereas netizens in the rest of the country paid
more attention to the overall COVID-19 prevention and control, and were more positive and optimistic with the assistance
of the government and NGOs. The average error in predicting public opinion sentiment was less than 5.64%, demonstrating
that this approach may be effectively applied to the analysis of large-scale online public sentiment evolution. Keywords Coronavirus disease 2019 (COVID-19) · LDA-ARMA hybrid model · Large-scale online public opinion ·
Emotional evolution Dynamic prediction Keywords Coronavirus disease 2019 (COVID-19) · LDA-ARMA hybrid model · Large-scale online public opinion ·
Emotional evolution · Dynamic prediction Analysis of public opinion evolution of COVID‑19 based on LDA‑ARMA
hybrid model Muni Zhuang1,2 · Yong Li3 · Xu Tan1 · Lining Xing4 · Xin Lu4 Received: 2 March 2021 / Accepted: 22 August 2021 / Published online: 4 September 2021
© The Author(s) 2021 * Xin Lu
xin.lu@flowminder.org
1
School of Software Engineering, Shenzhen Institute
of Information Technology, Shenzhen 518172, China
2
National Institute for Data Science in Health and Medicine,
Xiamen University, Xiamen 361005, China
3
School of Economics and Management, Changsha
University, Changsha 410022, China
4
College of Systems Engineering, National University
of Defense Technology, Changsha 410022, China * Xin Lu
xin.lu@flowminder.org Introduction prevention and control since the founding of the People's
Republic of China [1]. It was also listed by the World
Health Organisation as the sixth most significant global
public health emergency, after the Ebola epidemic in
Africa in 2019 [2, 3]. Due to the large number of casualties
and high economic and property losses, the physical and
mental health of much of the population was threatened to
a considerable extent, resulting in different degrees of ten-
sion, panic and other negative emotions [4]. In his work on
the prevention of COVID-19, General Secretary Xi Jinping
emphasised that “we must mobilise all kinds of forces to
comprehensively strengthen psychological counselling”
and has specifically pointed out that “we must strengthen
the guidance of public opinion.” COVID-19 has produced
massive amounts of public opinion data in the form of text
on the Internet. In this context, it is quite necessary to ana-
lyse this valuable public sentiment information, clarify the
development context, the characteristics and laws of public
emotion and opinion evolution on COVID-19, explore the At the moment, of public health incidents are unavoid-
able for our human society, most notably an outbreak of
a novel coronavirus pneumonia (COVID-19) in Wuhan,
China, in early 2020. This was a major public health emer-
gency, with the highest speed of transmission, the broadest
range of infection, and the highest level of difficulty of 1
School of Software Engineering, Shenzhen Institute
of Information Technology, Shenzhen 518172, China 2
National Institute for Data Science in Health and Medicine,
Xiamen University, Xiamen 361005, China Vol.:(0123456789)
1 3 3166 Complex & Intelligent Systems (2021) 7:3165–3178 level, down to phrases and aspects. However, sentiment
information is not used in these works during modelling.i differences in emotional evolution between Wuhan and rest
of China and provide specific measures for public opin-
ion prevention and response to government departments. These content fall into the research category of big data
text mining and public sentiment analysis. In the field of sentiment prediction, the Box-Jenkins
methodology, which employs autoregressive moving average
model (ARMA) linear models, has dominated several fields
of time series forecasting. Box and Jenkins [19] popular-
ized ARMA models in 1970 by developing a model-building
methodology that included an iterative three-stage process of
model selection, parameter estimation, and model checking. In a real-world scenario, text may be expressed in a com-
plex context that includes English, slang, emoticons, etc. Introduction To
obtain better results, we propose an improved LDA-ARMA
theme analysis model that can effectively solve the problem
of complex public opinion analysis. Traditional sentiment analysis methods are mainly based
on dictionaries, machine learning, Dictionary Plus Machine
learning, weak labelling and deep learning [5]. For example,
Rao et al. proposed a social sentiment monitoring dictionary
based on a sentiment dictionary [6]; Tripathy et al. com-
bined the naive Bayes (NB) approach, maximum entropy
(ME), stochastic gradient descent (SGD) and support vector
machine (SVM) machine learning algorithms with n-gram
models for sentiment analysis [7]; Jie et al. proposed a classi-
fication algorithm that combined a dictionary with machine
learning [8]; and Ma et al. proposed the enhancement of the
long short-term memory (LSTM) artificial neural network
by integrating both explicit and implicit knowledge, in an
approach known as perceptual LSTM [9]. Experimental
results have shown that these methods improved the accu-
racy of sentiment analysis to a certain extent. First, we construct a Python crawler framework to cap-
ture content relating to COVID-19 from microblogs on Sina
Weibo published by netizens of Wuhan and others across
China. In this complex context, which includes the mixed
use of Chinese and English and emoticons, we aimed to
model the dynamic evolution and obtain a fine-grained
understanding of the emotional evolution of public opinion
of an event from a multi-dimensional perspective, and to
predict and analyse the laws and emotional trends of this
evolution. We consider that the weights of feature words in
the original LDA “bag-of-words” models is uniform, which
leads to the problem that the topic model tends to high-fre-
quency words. We introduced a Gaussian function to set
different weights for feature words in order to improve the
rationality and independence of the distribution of topics. Then, based on the four mainstream sentiment dictionar-
ies, we manually labelled specific sentiment words for the
event and emoticons after sinicisation, and a sentiment value
measurement algorithm based on machine learning was pro-
posed that was combined with the ARMA model to give
reasonable predictions of the emotional evolution of this
epidemic event. We therefore explore the rules of evolution
and the differences between netizens of Wuhan and others in
terms of public opinion on COVID-19, and forms the whole
process public opinion analysis framework from collection
and processing, mining and analysis to strategy support. 1 3 1 3 Introduction It provides certain theoretical basis support and scientific
decision support for optimizing the public opinion collec-
tion function of microblog platform and assisting relevant
management departments to effectively guide and control
network public opinion when dealing with similar events. However, these traditional methods of sentiment analysis
are limited to judging the overall sentiment of the entire text,
and rely on the manual labelling of text features, meaning
that the processing efficiency is low when handling large
amounts of unstructured text information. Topic extraction
technology [5] is an emerging technology for the automatic
annotation and extraction of words, phrases and sentences in
the field of text mining, and can effectively solve this prob-
lem. At present, a great deal of research on topic extraction
technology is applied in the fields of emotional informa-
tion mining, emotional tendency analysis, emotional evolu-
tion analysis, and performance evaluation, such as the term
frequency-inverse document frequency (TF-IDF) algorithm
(based on word frequency statistics [10]), probabilistic latent
semantic analysis (PLSA) [11] and latent Dirichlet alloca-
tion (LDA) [12] (based on the topic probabilistic model),
automatic keyword extraction systems such as GenEx [13],
KEA [14] (based on machine learning) and high-level text
feature extraction and sentiment classification models (based
on deep learning) [15]. It is worth noting that in recent years, LDA has been
successfully utilized to classify the sentiments. He and Lin
propose a novel probabilistic modelling approach for senti-
ment analysis based on LDA termed the joint sentiment/
topic model (JSP) [16]. This model is completely unsuper-
vised. Li et al. present a Sentiment-LDA model for sentiment
analysis with global topics and local dependence [17]. Jo and
Oh describe two models, Sentence-LDA (SLDA) and Aspect
and Sentiment Unification Model (ASUM), to address the
challenge of automatically determining which aspects are
evaluated in reviews and how sentiments for different aspects
are expressed [18]. Sentiment classification is done at a finer The main contributions of our proposed work are high-
lighted as follows: (a) We introduced a Gaussian function to
improve the LDA model. This method can achieve a more
rational and fine-grained division of text topics; (b) we pro-
pose an emotional value measurement algorithm suitable
for complex contexts; (c) we propose a large-scale network
sentiment evolution model that combines improved LDA
and ARMA models. Improved LDA topic model First, the Gaussian function shown in (1) is used to dif-
ferentiate the words. LDA is a document topic generation model that was pro-
posed by Blei [12] in 2003. The model analyses the topics
of text documents in the form of a probability distribution,
and performs topic clustering or text classification accord-
ing to this distribution. The modelling process is illustrated
in Fig. 1. D = {di|i ∈{1, 2, … , M}} is defined as a set
containing M documents; di = {dis|s ∈{1, 2, … , S}
} is a
document composed of S sentences (i.e. a set of sentences);
wi = {wij|j ∈{1, 2, … , Ni}} is the set of words resulting
from word segmentation of document di ; Ni is the num-
ber of words in document di ; N=∑M
i=1 Ni is the number of
words in the set of documents D; zi = {zij|j ∈{1, 2, … , Ni}}
is the set of topics corresponding to the set of words wi ; and
K =|⋃M
i=1 zi| is the total number of topics in the document
set D. The LDA modelling process can be parsed as follows: (1)
F(wij) =
1
σ
√
2π
exp
−
(Frwij −Mnwi)2
2휎2 (1) In this equation, Frwij is the word frequency of the word
wij in the document di , Mnwi is the average frequency of
all words in the set wi corresponding to the document di ,
and 흈2 represents the variance. Next, in order to ensure that the total number of words
in the improved document di remains unchanged, the
weight of word wij is calculated using (2): (2)
Weightwij =
Ni × F(wij)
∑Ni
j=1 Frwij × F(wij) (2) Step 1. Set the initial probability of each document di to
P(di); In order to better analyse the improved word weight
퐖퐞퐢퐠퐡퐭wij calculation method, in the process of Gibbs sam-
pling algorithm derivation of parameters θ and φ, the joint
distribution of all variables in the LDA model is calculated
as shown in (3) as follows: Fig. Introduction Complex & Intelligent Systems (2021) 7:3165–3178 3167 The remainder of the paper is organized as follows: Sec-
tion “Sentiment analysis and prediction algorithm based on
the hybrid LDA-ARMA model” improved the LDA topic
model and ARMA time series model, and describes our
proposed model and algorithm in detail; empirical analysis
of public opinion and emotions in regard to COVID-19 are
presented in Sect. “Empirical analysis of public opinion and
emotions in regard to COVID-19”; Sect. “Analysis of the
differences in the evolution of public opinion and sentiment
between Netizens of Wuhan and other locations in China”
analysis of the differences in the evolution of public opinion
and sentiment between netizens of Wuhan and other loca-
tions in China; finally, Sect. “Conclusion” concludes this
paper. Step 2. Given the parameter 훂 , which obeys the Dir-
ichlet distribution, sample from the distribution to gener-
ate the topic distribution 휽i for document di; Step 3. Sample from the topic distribution 휽i to generate
the theme zij of the jth word in the document di; j
Step 4. Given the parameter β which obeys the Dirichlet
prior distribution, sample from this distribution to generate
the word distribution 흋zij of topic zij; j
Step 4. Given the parameter β which obeys the Dirichlet
prior distribution, sample from this distribution to generate
the word distribution 흋zij of topic zij; j
Step 5. Generate word wij from the word distribution 흋zij j
Step 5. Generate word wij from the word distribution 흋zij In the original LDA topic model, the text is expressed
in the form of “bag-of-words”, which disregard the differ-
ences in weight. The distribution of topics is often inclined
towards high-frequency words, which affects the accuracy. Therefore, inspired by Wang Rui's research [20], we use a
Gaussian function-based word weight distribution to iden-
tify potential topic information from within a large-scale
text. The ideas on which this improvement are based are
as follows: 1 3 Improved LDA topic model 1 LDA topic analysis model (3)
P(wi, zi, 휃i, Φ|훼, 훽) =
Ni
∏
j=1
P(wij|||휑Zij )P(zij||휃i)⋅P(휃i|훼)⋅P(Φ|훽) (3) In this equation, ∏Ni
j=1 P(wij 흋Zij )P(zij 휽i) is the probabil-
ity of all words in the ith document produced by 휽i;P(휽i|휶)
is the “text topic” distribution probability of the document
di generated by the Dirichlet parameter α defined above;
and P(횽|휷) is the “topic word” distribution matrix gener-
ated by the Dirichlet parameter β defined above. Fig. 1 LDA topic analysis model 3168 Complex & Intelligent Systems (2021) 7:3165–3178 mathematical expression for the ARMA model is obtained
as follows: Then according to (3), using Bayes’ rule and the Dirichlet
distribution, a Dirichlet-multinomial conjugation is used to
obtain Gibbs samples. The estimated values of parameters
흋c
zij and 휽di
zij in the Bayesian framework are expressed as in (4)
and (5): (8)
Xt =
p
∑
m=1
휂mXt−m +
q
∑
n=1
휇n휀t−n + 휀t (8) The steps of our prediction algorithm, which is based on
the ARMA model, can be described as follows: The steps of our prediction algorithm, which is based on
the ARMA model, can be described as follows: (4)
̂휑c
zij =
Numc
zij + 훽c
∑N
c=1 Numc
zij + 훽c
(5)
̂휃di
zij =
Numdi
zij + 훼zij
∑K
zij=1 Numdi
zij + 훼zij (4)
̂휑c
zij =
Numc
zij + 훽c
∑N
c=1 Numc
zij + 훽c
(5)
̂휃di
zij =
Numdi
zij + 훼zij
∑K
zij=1 Numdi
zij + 훼zij the ARMA model, can be described as follows: (4) Step 1. Zero-average processing is performed on the time
series value Xt , and a stationarity test is then applied to Xt
[22]. If the result is not stable, the difference method (DM)
is applied until the data after the difference are stable. (5) f
Step 2. A white noise test is carried out on the stationary
data. If the test result is a stationary white noise sequence,
the orders p and q of the ARMA model are obtained by
calculating the autocorrelation function (ACF) and partial
autocorrelation function (PACF), and a fit to the ARMA(p,
q) model is calculated using the StatsModels package. The
value of the Akaike information criterion (AIC) is calculated
for different combinations of (p, q), and the minimum value
of AIC (p, q) is taken as the estimate of (p, q). ARMA time series model The autoregressive moving average (ARMA) time series
model is composed of an autoregressive model (AR)
and a moving average model (MA) [21]. In a time series
X = {X1, X2, … , XT} , the value at a certain moment in the
preset time series of the ARMA model is related to the value
of the first p time series and the first q random disturbances
entering the system, and from this we can predict the value
at the next moment. Let Xt be an autoregressive process that
is affected by the values of the first p time series, as shown
in (6): Improved LDA topic model In (4) and (5), c is the tag word and ̂흋c
zij is the distribution
probability that c belongs to the topic zij ; ̂훉
퐝퐢
퐳퐢퐣 is the distribu-
tion probability that document di contains the topic zij . Obvi-
ously, when the marked word c is a high-frequency word,
the probability that it belongs to the topic zij and the docu-
ment di is higher, resulting in the topic distribution tending
to tilt toward high-frequency words. After the improvements
introduced via (1) and (2), when the tag word c is assigned
to the topic zij , it is given a corresponding weight. In (4) and
(5), the values of 퐍퐮퐦c
zij and 퐍퐮퐦di
zij are given differentiated
word weights 퐖퐞퐢퐠퐡퐭wij . The improved LDA topic model
can therefore achieve a finer-grained and more reasonable
division of text data by topic. Step 3. The least squares estimation method is used to
calculate the remaining unknown parameters η and μ in the
model. The result of dynamic prediction at instant t + 1 is
expressed as follows: (9)
X
t+1 = 휂1X
t + ⋯+ 휂pX
t+1−p + et −휇1휀t −⋯−휇q휀t+1−, (9) where X
′
t is a sequence of zero mean value. In this way, the
time series ARMA model is obtained to realise the predic-
tive analysis of text data and the evolution of public opinion. 1 3 1 3 Improved LDA‑ARMA model and algorithm
for sentiment analysis and prediction In order to analyse the topics in microblog text data repre-
senting public opinion of an epidemic event in a complex
context, including calculation, classification and prediction
of sentiment values, we propose an improved LDA-ARMA
model and algorithm. A flow chart for our model is shown
in Fig. 2. The proposed model algorithm can be summarised by the
following steps: (6)
Xt = 휂1Xt−1 + 휂2Xt−2 + ⋯+ 휂pXt−p + et, (6) Step 1. Pre-processing of text in a complex context. The
document set D of the public opinion corpus is pre-pro-
cessed by applying emoticon sinicization, English–Chinese
translation, sentence segmentation, word segmentation, stop
words, and punctuation marks. For each document, the sen-
tence set di and the word set wi are obtained. Step 1. Pre-processing of text in a complex context. The
document set D of the public opinion corpus is pre-pro-
cessed by applying emoticon sinicization, English–Chinese
translation, sentence segmentation, word segmentation, stop
words, and punctuation marks. For each document, the sen-
tence set di and the word set wi are obtained. where 휼1, 휼2, … , 휼p are autoregressive coefficients, and et is
the error term. The error term et has a dependency on the
different values of the time series, and its moving average
process can be expressed in (7) as follows: (7)
et = 휇1휀t−1 + 휇2휀t−2 + ⋯+ 휇q휀t−q + 휀t, (7) Step 2. Calculate the number of topics with the improved
LDA model. The topic similarity is set to SK , and the Gibbs
sampling algorithm is used to obtain the distribution param-
eters for each "text topic" and "topic-word". A Gaussian where μ1, μ2, … , μq are moving average coefficients and εt
is a white noise sequence. By substituting (7) into (6), the Complex & Intelligent Systems (2021) 7:3165–3178 3169 Fig. 2 Flow chart of improved
LDA-ARMA model for senti-
ment analysis and prediction Fig. 2 Flow chart of improved
LDA-ARMA model for senti-
ment analysis and prediction function is used to adjust the weights of the high- and low-
frequency words for use in (4) and (5). Finally, the similarity
SK[23] corresponding to the number of different topics K is
calculated. When SK is a minimum, the number of topics K
is optimal, and the improved LDA "topic word" distribution
is obtained. value is calculated as 흀2E(wij) . Improved LDA‑ARMA model and algorithm
for sentiment analysis and prediction Following this, we traverse the
negative words before the emotion word; when the number of
negative words is odd, the sentiment value of the sentiment
word 흀2E(wij) takes a negative value, and when the number
of negative words is even or zero, a positive value is taken. We
then traverse the punctuation marks behind the emotion word
and match the corresponding emotion degree. If an exclama-
tion mark appears behind the emotion word, the value of 흀1
is two; otherwise, it is one. In addition, the index coordinates
of sentence dis are labelled, and the sentiment degree E′(dis
)
is calculated based on the position of the index coordinates. Step 3. Construction of the sentiment dictionary and
labelling of the sentiment words. Let E(wij) be the senti-
ment value of the word wij . The words are matched with
the constructed sentiment dictionary for the mixed-language
context. The emotion polarity is automatically labelled for
those words that can be automatically matched, labelling
positive emotion words as E(wij) = 1, negative emotion
words as E(wij) = −1 and neutral emotion words as E(wij) =
0. After sinicization, the emoticons are regarded as special
emotional words wij , which are manually labelled according
to the labelling rules for emotional words and are added into
the emotion dictionary. E dis
=
⎧
⎪
⎨
⎪⎩
1, Ifdis is the first sentence of a paragraph
0.5, Ifdisis the last sentence of a paragraph
0, otherwise (10) (10) Finally, the equation for the sentiment value E(dis) of each
sentence dis is as follows: Step 4. Calculation of the emotional value of sentences. We
set the sentiment degree of a punctuation mark behind a senti-
ment word to 흀1 , and the degree adverb in front of the emo-
tional word as 흀2 . Based on the word sentiment value E(wij)
obtained in Step 3, we traverse the degree adverb in front of
the sentiment word. For each sentiment word wij , the sentiment (11)
E(dis
) = 휆1
Ni
∑
j=1
휆2E(wij) + E(dis
) (11) 3170 Complex & Intelligent Systems (2021) 7:3165–3178 locations in China during the COVID-19 epidemic, we took
COVID-19 as the research background and used “Wuhan”
as the search keyword in the “Advanced Search” on Sina
Weibo to crawl texts relating to the public opinion of Wuhan
netizens. Improved LDA‑ARMA model and algorithm
for sentiment analysis and prediction The position retrieval was terminated in the second
stage, and the regularization method was used to exclude the
microblog located in "Wuhan," and this section of the text
was taken as the public opinion text of other netizens across
the country. For the above conditions, we built a crawler
framework using Python. From 0:00 on January 18th to
24:00 on February 10th, 2020, we collected comments from
Wuhan and other Chinese netizens on Sina Weibo. In order
to ensure that the data samples were as evenly distributed
as possible, we collected the first 10% of the data per hour
and finally got 59,579 pieces of blog data of Wuhan netizens
and 67,877 pieces of data from other Internet users in China,
giving a total of 127,456 items. Thus, the sentiment value for document di is
E
di
= ∑S
s=1 E(dis) . We then find the best threshold for
positive and negative sentiment classification using the F1
score [24] and conditionally judge E
(
di
) using a Boolean
expression. When E(di) is higher than the threshold, it is
judged to be a positive emotion; otherwise, it is a negative
emotion. Based on the results of this conditional judgment,
we calculate the number of documents with positive senti-
ment E ′ pos(di ), the number with negative sentiment E ′
neg(di ), the mean value of documents with the positive sen-
timent MnEpos(di ) and the mean value of documents with
the negative sentiment MnEneg(di ). The result of the docu-
ment sentiment analysis is then RT = (E ′ pos(di ), E ′ neg(di ),
MnEpos(di ), MnEneg(di)). p
g
Step 5. Carry out sentiment analysis of a multi-dimen-
sional "text topic". The results of the text sentiment analysis
RT calculated in Step 4 are integrated into the time series set
of documents: D_time = {(d1, X1
), (d2, X2
), … , (dM, XT
)} ,
based on the division into time slices, to give the result
time_RT = {time_RT1, time_RT2, … , time_RTT} for the
emotional time series in the "text time" dimension. We then
visually calculate the results for the emotional distribution
under different "text-topics", and combine RT with the
"topic word" distribution to obtain the emotional distribution
results [25, 26]. Next, we divide time_RT into K different
topic names to obtain the emotional time series distribution
result time_RE = {time_RE1,time_RE2,…,time_RET } under
different "text topic". Improved LDA‑ARMA model and algorithm
for sentiment analysis and prediction The text data were pre-processed following Step 1 in part
C of Section II. First, the regular Python functions Findall
and Subn were used to extract English words from the com-
ment text. After machine translation, the English words were
backfilled, and the emoticons in the blog posts were regu-
larly matched and sinicised. Next, we carried out batch seg-
mentation of the text based on Chinese words by calling the
Segmentor function in the LTP word segmentation tool from
the Harbin Institute of Technology. Finally, we removed
invalid data and meaningless URLs. Through this iterative
process, 49,187 items of data from Weibo generated by neti-
zens of Wuhan were finally obtained, and 56,447 items from
other netizens nationwide, giving a total of 105,634. The
datasets were classified into a training set and a test set with
a ratio of 7:3. The training and test sets for Wuhan netizens
contained 34,431 and 14,756 items, respectively, and those
for the other locations in China were 39,513 and 16,934,
respectively. In this way, the sentence set di and word set wi
were constructed for netizens of Wuhan and other locations
in China. f
Step 6. Prediction of public sentiment trend. Using time_
RT as the training set of prediction model. Based on the
training set, the improved LDA-ARMA model is trained
iteratively until the loss function reaches a minimum and
the optimal robust model is obtained [27, 28]. We use 10%
of the data in the training set as the verification set and
repeatedly verify the model using these data to obtain the
optimal combination of hyperparameters. We then combine
the training set and the verification set, and use a fivefold
cross-validation method to select the optimal model to pre-
dict the public sentiment trend for time slice t + 1, and use
the prediction result X
t+1 as the test data. Finally, the predic-
tion performance of the model is evaluated by calculating
the mean absolute percentage error (MAPE) [29] for the
LDA-ARMA model. In order to analyse the evolution of sentiment on this
topic for the two groups of residents, we used our improved
LDA topic model. First, we iteratively calculated the aver-
age similarity SK of the number of topics using Step 2 of the
algorithm, as described in part C of Sect. 2, to obtain the
optimal number of topics. Improved LDA‑ARMA model and algorithm
for sentiment analysis and prediction We carried out numerous experi-
mental tests, setting α = 50/K, β = 0.01, and using fivefold
cross-validation; the training data before and after optimisa-
tion using the Gaussian function were iterated 2000 times
each, and the test data were iterated 1000 times each. When
SK is the minimum, the number of topics K is the optimal
value. For K = 4, a comparison of the experimental results
shows that the topic similarity before and after applica-
tion of the improved algorithm was SK = 0.737 and SK =
0.641, respectively. The LDA model improved by the use of
the Gaussian function gave better performance in terms of 1 3 Table 2 Distribution of topics discussed by other netizens of China in regard to the COVID-19 epidemic after application of the improved LDA
algorithm
Topic
Keywords
Topic 1
Virus (0.0592), Pneumonia (0.0523), Quarantine (0.0495), Epidemic Situation (0.0462), Mask (0.0387), Responsible (0.0321), Gov-
ernment (0.0216), Normal (0.0178), One (0.0151)
Topic 2
Hospital (0.0643), Medical Staff (0.0435), Protect (0.0331), Infect (0.0297), Front Line (0.0285), Patient (0.0217), Angel (0.0114),
Salute (0.0102), Rumor (0.0095)
Topic 3
Come on (0.0906), Safety (0.0524), Thank (0.0473), Precious (0.0364), Together (0.0143), Spring Festival (0.0127), People (0.0116),
Unite (0.0104), Journalism (0.065)
Topic 4
Contribution (0.0461), Materials (0.0369), Overcome (0.0355), Red Cross (0.0314), do (0.0306), Support (0.0272), Protect (0.0224),
Japan (0.0168), PUMC (0.0082) Table 1 Distribution of topics discussed by Wuhan netizens in regard to the COVID-19 epidemic and public opinion after application of the
improved LDA algorithm
Topic
Keywords
Topic 1
Pneumonia (0.0381), Virus (0.0303), Epidemic Situation (0.0216), Mask (0.0178), Zhong Nanshan (0.0072), Diagnosis (0.0054),
SARS (0.0024), Seafood (0.0013), People (0.0009)
Topic 2
Lockdown (0.0545), Wuhan (0.0351), Government (0.0314), Hospital (0.0238), Thunder God Mountain (0.0119), disinfection
(0.0104), compatriots (0.0097), awesome (0.0093), no (0.0076)
Topic 3
Come on (0.0803), China (0.0674), Safety (0.0557), Zhu Yilong (0.0431), Warm Spring (0.0225), Contribution (0.0204), Relay
(0.0186), Direct Broadcast (0.0176), Help (0.0164)
Topic 4
Hospital (0.0407), Help (0.0351), Worried (0.0334), Ill (0.0278), Cry (0.0195), Bed (0.0143), Quarantine (0.0142), Government
(0.0096), Stay (0.0087) Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion Until
28th January, the number of negative texts by Wuhan’s neti-
zens was always higher than the number of positive texts,
reaching a peak on the 23rd January. The number of posi-
tive texts by other netizens in the country showed a steady We processed these 36,496 sentiment words using Step
4 in part C of Section II, to obtain the text sentiment clas-
sification and sentiment analysis results RT(Wuhan) and
RT(Nationwide). The best value for the classification thresh-
old for positive and negative sentiments was 0.61. In order
to further explore the trend in sentiment evolution from
the perspective of multi-dimensional topics, according to
Step 5 of the algorithm in part C of Section II, sentiment
analysis was carried out on the full dataset using multiple
dimensions of "text topic", and the time_RT(Wuhan) and
time_RT(Nationwide) results of the "text-time" emotional
time series evolution of Wuhan netizens and other netizens
across the country were obtained, as shown in Figs. 3 and 4. In order to deepen our understanding of the trend and
evolution of public opinion of the COVID-19 epidemic,
based on a multi-dimensional analysis of the topic, the posi-
tive and negative words of four mainstream emotion dic-
tionaries were used to analyse microblogs. These were the
HowNet Chinese vocabulary for sentiment analysis, Tsing-
hua University’s dictionary of praising and derogatory words
in Chinese, Dalian University of Technology’s sentiment
vocabulary ontology database in Chinese, and Taiwan Uni-
versity’s NTUSD. After filtering repeated sentiment words Figures 3a and 4a show that compared with other neti-
zens in China, the numbers of positive and negative texts by
Wuhan netizens fluctuated greatly during this period. Until
28th January, the number of negative texts by Wuhan’s neti-
zens was always higher than the number of positive texts,
reaching a peak on the 23rd January. 1 3 Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion To carry out an in-depth analysis of the evolution of the
opinions and emotions of residents of Wuhan and other 1 3 1 3 3171 Complex & Intelligent Systems (2021) 7:3165–3178 topic clustering. The distributions of "topic words" reflecting
public opinion on the COVID-19 epidemic by netizens of
Wuhan and other locations in China are shown in Tables 1
and 2, respectively. from the four major dictionaries, we obtained 16,639 words
relating to praising words and 18,084 derogatory words. Fol-
lowing Step 3 of the algorithm in Sect. 1.3, emotion words
relating to specific events were manually marked, and finally,
1773 new emotion words (including 647 praising words and
1126 derogatory words) were extracted. The total number of
words in the emotion dictionary was 36,496. According to the probability distribution in Table 1, the
top two keywords with the highest probability in Topic 1
were P(Pneumonia) = 0.0381 and P(Virus) = 0.0303. Topic
1 is, therefore, referred to here as “Pneumonia virus”. Simi-
larly, Topic 2 is referred to as “Lockdown of Wuhan city”,
Topic 3 “Go China”, and Topic 4 “Hospital”. The topics
in Table 2 are referred to as “Pneumonia virus”, “Hospi-
tal staff”, “Encourage” and “Contribution”. From the core
theme words, it appears that Wuhan netizens were more
concerned with the development of the epidemic situation,
treatment measures and related policies, while netizens of
other areas paid more attention to the overall prevention and
control of COVID-19. words in the emotion dictionary was 36,496. We processed these 36,496 sentiment words using Step
4 in part C of Section II, to obtain the text sentiment clas-
sification and sentiment analysis results RT(Wuhan) and
RT(Nationwide). The best value for the classification thresh-
old for positive and negative sentiments was 0.61. In order
to further explore the trend in sentiment evolution from
the perspective of multi-dimensional topics, according to
Step 5 of the algorithm in part C of Section II, sentiment
analysis was carried out on the full dataset using multiple
dimensions of "text topic", and the time_RT(Wuhan) and
time_RT(Nationwide) results of the "text-time" emotional
time series evolution of Wuhan netizens and other netizens
across the country were obtained, as shown in Figs. 3 and 4. Figures 3a and 4a show that compared with other neti-
zens in China, the numbers of positive and negative texts by
Wuhan netizens fluctuated greatly during this period. Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion The number of posi-
tive texts by other netizens in the country showed a steady Table 1 Distribution of topics discussed by Wuhan netizens in regard to the COVID-19 epidemic and public opinion after application of the
improved LDA algorithm
Topic
Keywords
Topic 1
Pneumonia (0.0381), Virus (0.0303), Epidemic Situation (0.0216), Mask (0.0178), Zhong Nanshan (0.0072), Diagnosis (0.0054),
SARS (0.0024), Seafood (0.0013), People (0.0009)
Topic 2
Lockdown (0.0545), Wuhan (0.0351), Government (0.0314), Hospital (0.0238), Thunder God Mountain (0.0119), disinfection
(0.0104), compatriots (0.0097), awesome (0.0093), no (0.0076)
Topic 3
Come on (0.0803), China (0.0674), Safety (0.0557), Zhu Yilong (0.0431), Warm Spring (0.0225), Contribution (0.0204), Relay
(0.0186), Direct Broadcast (0.0176), Help (0.0164)
Topic 4
Hospital (0.0407), Help (0.0351), Worried (0.0334), Ill (0.0278), Cry (0.0195), Bed (0.0143), Quarantine (0.0142), Government
(0.0096), Stay (0.0087) ics discussed by Wuhan netizens in regard to the COVID-19 epidemic and public opinion after application of the Table 2 Distribution of topics discussed by other netizens of China in regard to the COVID-19 epidemic after application of the improved LDA
algorithm
Topic
Keywords
Topic 1
Virus (0.0592), Pneumonia (0.0523), Quarantine (0.0495), Epidemic Situation (0.0462), Mask (0.0387), Responsible (0.0321), Gov-
ernment (0.0216), Normal (0.0178), One (0.0151)
Topic 2
Hospital (0.0643), Medical Staff (0.0435), Protect (0.0331), Infect (0.0297), Front Line (0.0285), Patient (0.0217), Angel (0.0114),
Salute (0.0102), Rumor (0.0095)
Topic 3
Come on (0.0906), Safety (0.0524), Thank (0.0473), Precious (0.0364), Together (0.0143), Spring Festival (0.0127), People (0.0116),
Unite (0.0104), Journalism (0.065)
Topic 4
Contribution (0.0461), Materials (0.0369), Overcome (0.0355), Red Cross (0.0314), do (0.0306), Support (0.0272), Protect (0.0224),
Japan (0.0168), PUMC (0.0082) 1 3 Complex & Intelligent Systems (2021) 7:3165–3178 3172 Fig. 3 Emotional evolution and distribution of opinions of Wuhan
netizens with regard to COVID-19
Fig. 4 Emotional evolution and distribution of opinions of other neti-
zens in China with regard to COVID-19 Fig. 4 Emotional evolution and distribution of opinions of other neti-
zens in China with regard to COVID-19 Fig. 3 Emotional evolution and distribution of opinions of Wuhan
netizens with regard to COVID-19 Fig. 3 Emotional evolution and distribution of opinions of Wuhan
netizens with regard to COVID-19 attitudes of Wuhan netizens were more negative than those
of other netizens in China during this period. 1 3 1 3 Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion Wuhan netizens had more negative Figures 3b and 4b show that the mean values for positive
emotions of Wuhan netizens and the mean values for the
negative emotions of other netizens in China tended towards
the optimal threshold line, indicating that the emotional Complex & Intelligent Systems (2021) 7:3165–3178 3173 positive text evolution paths in Figs. 3a and 7c are highly
consistent. It can be inferred that the number of positive
texts shown in Fig. 3a underwent rapid growth from 23rd to
28th January, mainly due to the positive emotional growth
of the theme “Go China”. Although the “pneumonia virus”
and “Go China” showed a slow downward trend, negative
emotions related to “Hospital” continued to rise. Through
tracing, it was found that most of these netizens were diag-
nosed patients or their families, and a small number of
medical staff. It is preliminarily inferred that the medical
conditions of some diagnosed patients and the issue of sup-
plies for medical staff during this period were not adequately
addressed. However, the negative emotion related to “lock-
down of Wuhan City” reached its highest value in the initial
stages of the crisis, and then decreased rapidly, indicating
that when the news of lockdown was released, Wuhan neti-
zens generally showed negative and panic emotions. How-
ever, with the active involvement of officials and disclosure
of information, the anxiety of Wuhan netizens was alleviated
and their rationality was gradually restored. Fig. 5 Distribution of public opinion and sentiment tendency of
Wuhan netizens Fig. 5 Distribution of public opinion and sentiment tendency of
Wuhan netizens Fig. 6 Distribution of public opinion and sentiment tendency of other
netizens in China Figure 8 shows that the positive emotions for all the four
topics showed an upward trend. The positive emotion related
to “Hospital staff” was the largest, and the negative emotion
was the smallest, indicating that netizens of other regions of
China had positive emotion towards the efforts of medical
staff during the COVID-19 epidemic. The highest fluctua-
tions were seen for the negative emotions towards “dona-
tions”. Through source tracing, it was found that the negative
sentiment of these netizens mainly arose from the “mask
gate” incident, which stimulated negative emotions such as
dissatisfaction and anger. The donation by Japan and other
friendly countries to China then aroused the gratitude and
praise of these residents. Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion It is worth
noting that with the evolution of public opinion, the aver-
age negative sentiment of Wuhan netizens showed a down-
ward trend overall, reflecting the fact that the outlook for the
COVID-19 epidemic was not optimistic during this period. increase during this period, and the difference between
the number of positive and negative text increased. It can
be inferred that residents of Wuhan, as the centre of the
COVID-19 outbreak, were extremely nervous and panicked
in the initial stages, due to uncertainty about the source,
transmission routes, mortality rate, and treatability of the
virus. Meanwhile, the lockdown of Wuhan on the 23rd
January intensified the social panic and psychological pres-
sure on Wuhan netizens. Although other netizens across the
country showed panic at the beginning of the outbreak, the
duration of this phase of public opinion was not long; they
quickly overcame their negative emotions and turned them
into positive and optimistic attitudes. p
p
g
p
In order to further analyse the sentiment distribution
regarding the core topics in microblogs by the two groups,
the "topic word" sentiment classification was combined
with the coarse-grained sentiment classification result RT
to obtain the "topic word" sentiment classification. The the-
matic sentiment distributions of netizens of Wuhan and other
locations in China were obtained as shown in Figs. 5 and 6. According to Fig. 5, except for “Go China”, Wuhan neti-
zens had mainly negative emotions for the other three core
topic words, with negative comment rates of 65%, 72%,
and 82%, respectively. Wuhan netizens had more negative In order to further analyse the sentiment distribution
regarding the core topics in microblogs by the two groups,
the "topic word" sentiment classification was combined
with the coarse-grained sentiment classification result RT
to obtain the "topic word" sentiment classification. The the-
matic sentiment distributions of netizens of Wuhan and other
locations in China were obtained as shown in Figs. 5 and 6. According to Fig. 5, except for “Go China”, Wuhan neti-
zens had mainly negative emotions for the other three core
topic words, with negative comment rates of 65%, 72%,
and 82%, respectively. 1 3 Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion prediction results for the positive and negative emotions
of both groups of residents are shown in Figs. 9 and 10.i Figures 9 and 10 show that there was no significant
change in the positive and negative emotional trends of
Wuhan’s netizens in the forecast data. In general, the
positive emotional trend of Wuhan netizens fluctuated,
while the negative emotional trend decreased slowly. The
sentiment trends of the other netizens across the country
are more obvious. Compared with Wuhan netizens, the
positive sentiments of these residents increased signifi-
cantly, while negative sentiments decreased considerably. In fact, with the gradual decline in the diagnosis and sus-
pected rates of new coronary penumonia, and the gradual
increase in the cure rate, the negative emotions of ten-
sion and extreme panic experienced by Wuhan netizens
and other netizens across the country were alleviated to
varying degrees, and the themes of public opinion also
became more diverse. The topics discussed by netizens
began to turn to problems such as the brutal requisition of
college dormitories, live broadcast chaos of school online Multi‑dimensional visual empirical analysis
of covid‑19 public opinion and emotion Judging from the evolution of the COVID-19 public
opinion of Wuhan netizens and other netizens across the
country, most Wuhan netizens were actively fighting the
epidemic. However, Wuhan as a disaster area, the medi-
cal resources and supplies of confirmed patients, patients'
families and medical staff in Wuhan still need to be attached
great importance. Other netizens across the country acted
as “supervisors” for prevention and control of COVID-19
during the epidemic; they actively responded to the call for
anti-epidemic measures at home and effectively reduced the
probability of virus transmission through practical actions. They also actively participated in prevention and control
work online, such as transmitting their experience and skills
in relation to prevention and control of the disease and social
circles and activity track of patients diagnosed, collecting
materials or providing channels for donations to frontline
workers, which provided a broad basis for the development
and popularisation of epidemic prevention and control. At
the same time, in view of the mistakes and anomie related
to such work, the public opinion of other netizens across the Fig. 6 Distribution of public opinion and sentiment tendency of other
netizens in China emotions about “Hospital” and “lockdown of Wuhan City”. Figure 6 shows that other netizens across the country had a
positive and optimistic attitude towards COVID-19, espe-
cially on the themes of "Hospital staff" and “Encourage”, but
the emotional attitude towards the theme of “Contribution”
was mixed, and the reason for this is worth investigating
further. In order to grasp the trends in the emotional evolution of
public opinion on COVID-19 of the two groups over time,
the distribution results for the emotional tendency of the
different topics were divided into time slices (T = 24). We
obtained the "text topic" emotional time-series distributions
time_RE (Wuhan) and time_RE (Nationwide) for the two
groups as shown in Figs. 7 and 8. Figure 7 shows that for these four topics, positive emo-
tion generally increased over time. It is worth noting that the 1 3 Complex & Intelligent Systems (2021) 7:3165–3178 3174 Fig. 7 Evolution of public opinion and sentiment of Wuhan netizens on four topics of the COVID-19 Fig. 7 Evolution of public opinion and sentiment of Wuhan netizens on four topics of the COVID-19 country played an important role in monitoring and promot-
ing efficient prevention and control. 1 3 An empirical analysis of the dynamic prediction
of COVID‑19 public opinion and emotion We aimed to obtain further insight into the develop-
ment trends and evolution of public opinion on COVID-
19, to make accurate predictions for the development
of public opinion on this issue, and to provide a rea-
sonable theoretical basis for government departments
to formulate differentiated public opinion prevention
measures. Following Step 6 of the algorithm in part C
of Section II, the "text-time" sentiment analysis result
time_RT = {time_RT1, time_RT2, … , time_RT24} was sub-
stituted into the improved LDA-ARMA hybrid model
for iterative training. After cross-validating the trained
optimal model on the training and verification sets, we
predicted comment data and the emotional evolution pro-
cess for the period from 11 to 21th February 2020. The Complex & Intelligent Systems (2021) 7:3165–3178 3175 p
g
y
Fig. 8 Evolution of public opinion and sentiment of other netizens in China on four topics of the COVID-19 Fig. 8 Evolution of public opinion and sentiment of other netizens in China on four topics of the COVID-19 Fig. 8 Evolution of public opinion and sentiment of other netizens in China on four topics of the COVID-19 Fig. 8 Evolution of public opinion and sentiment of other netizens in China on four topics of the COVID-19 teaching, and the emergence of COVID-19 in Shandong
prison. measures and psychological counselling provided by the
government departments. In order to further verify the prediction results, we
calculated the average absolute percentage error MAPE
values and found that the values for the positive and nega-
tive emotions of Wuhan netizens were 4.74% and 5.87%,
respectively, while those of other netizens in China were
2.59% and 9.37%, respectively. It can be seen that despite
the large scale of the experimental data and the presence
of a mixture of languages, the MAPE values for the test
set still achieved good results, with an average error rate
of less than 5.64%. This proves that the improved LDA-
ARMA model can effectively simulate the evolution of
public opinion on COVID-19 and can predict the trends
of this opinion. These results indicate that netizens of
other locations in China were able to overcome negative
emotions about COVID-19 more quickly, while Wuhan
netizens needed more time, more powerful anti-epidemic 1 3 Analysis of the differences in the evolution
of public opinion and sentiment
between Netizens of Wuhan and other
locations in China In order to more intuitively reflect the differences of public
opinion on this topic between residents of Wuhan and other
areas of China, the topics discussed by the two groups are
presented in the form of word clouds based on the prob-
ability distribution of their occurrence, as shown in Fig. 11. From the analysis in Fig. 11 and the previous two sec-
tions, we can see that the emotional attitudes of Wuhan
netizens were more negative and pessimistic than those
expressed by netizens of other parts of the country. As the 1 3 1 3 1 3 Complex & Intelligent Systems (2021) 7:3165–3178 3176 Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic
Fig. 10 Positive and negative emotion prediction results for other net-
izens in China during the COVID-19 epidemic Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic Fig. 10 Positive and negative emotion prediction results for other net- Fig. 10 Positive and negative emotion prediction results for other net-
i
i Chi
d
i
th COVID 19
id
i Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic Fig. 10 Positive and negative emotion prediction results for other net-
izens in China during the COVID-19 epidemic Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic Fig. 9 Positive and negative emotion prediction results for Wuhan
netizens during the COVID-19 epidemic Fig. 11 Word clouds for subjects addressed by netizens of Wuhan
(left) and other locations in China (right) during the COVID-19 epi-
demic centre of the COVID-19 outbreak, Wuhan’s netizens lacked
a deep knowledge of COVID-19 in the early stages, and this
was evident from public opinion; in addition, internet media
amplified the negative effects of information dissemination,
and the negative emotions of Wuhan netizens spread rapidly
and widely, giving rise to the threat of large-scale group
psychological crises. In the early stages of the epidemic,
the government of Wuhan should have taken the initiative
to voice and answer questions promptly, so that information
was open and transparent, thereby alleviating the negative
emotions of Wuhan netizens. 1 3 1 3 Analysis of the differences in the evolution
of public opinion and sentiment
between Netizens of Wuhan and other
locations in China Second, in the middle and late
stages of the epidemic, it should have provided effective psy-
chological assistance to the masses to help them overcome
the temptation to panic, which would have been conducive
to social stability and development. Fig. 11 Word clouds for subjects addressed by netizens of Wuhan
(left) and other locations in China (right) during the COVID-19 epi-
demic frontline of COVID-19. Especially when news of the heroic
deeds of medical staff was widely spread via microblogs,
the empathy of other netizens in China was aroused; these
people were acutely aware of the difficulty of anti-epidemic
work, and this resolved a certain amount of anxiety and dis-
satisfaction. The COVID-19 epidemic deepened the mutual
understanding between doctors and patients and provided
an opportunity to improve the current close relationships
between doctors and patients. In addition, the central gov-
ernment quickly coordinated medical resources across the
country, built Huoshenshan and Leishenshan hospitals, and
opened numerous shelter hospitals within a short time; this The emotional evolution of other netizens across the
country has generally been positive and optimistic, and
positive emotions have far outweighed negative ones. These
netizens were relatively far away from the epidemic-stricken
area, and when responding to the call for anti-epidemic dis-
ease at home, they paid a great deal of attention to the medi-
cal staff and grassroots workers who were fighting on the Complex & Intelligent Systems (2021) 7:3165–3178 3177 should be biased towards enterprises and individuals to help
them recover to pre-epidemic levels. provided solid reassurance in terms of the prevention and
control of COVID-19, demonstrated efficient response and
governance capabilities and made the public opinion of
other netizens in the country develop positively spontane-
ously. It is undeniable that COVID-19 has greatly affected
the daily life and level of production of small and medium-
sized enterprises and ordinary people. In the middle and
late stages of the COVID-19 epidemic, the allocation of
resources by the state should be adjusted to help enterprises
and individuals return to a normal (i.e. pre-epidemic) level
as soon as possible. The limitation of this study is that it is based on single
case, making it impossible to derive a general causal rela-
tionship between the level of public pleasure and govern-
ment performance. Conclusion The article examines public opinion of COVID-19 as
expressed online and builds an improved LDA-ARMA
hybrid model for a complex context, for example involving
a mixture of Chinese and English text, slang and emoticons. We analyse and predict the evolution of sentiment within
public opinion data in an attempt to find the laws underlying
the evolution of emotion around large-scale public opinion
events and propose an algorithm to measure the emotional
value of text based on machine learning. Using an evolution-
ary analysis of topics, this article analyses the thematic fea-
tures of different research subjects in multiple dimensions,
and by applying "text topic" sentiment analysis, we analyse
the public opinion of netizens of Wuhan and other loca-
tions across China on different topics. We use our proposed
LDA-ARMA hybrid model to predict the evolution of public
opinion and emotional trends in complex contexts and show
that the average error rate does not exceed 5.64%. Acknowledgements This work was supported by the National Sci-
ence Foundation of China (Nos. 71771213, 91846301, 71790615,
82041020, 72025405), the Program of Guangdong Innovative Research
Team (Nos. 2020KCXTD040), the Basic Research Project of Science
and Technology Plan of Shenzhen (Nos. SZIITWDZC2021A02,
JCYJ20200109141218676) and the Hunan Science and Technology
Plan Project (Nos. 2020TP1013, 2020JJ4673, 2019GK2131). Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
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permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Based on the evolution of the emotions of Wuhan neti-
zens, this empirical research shows that these people are
under a lot of emotional pressure; there is, therefore, a need
for the relevant departments to offer timely psychological
assistance and guidance of public opinion to avoid a large-
scale psychological crisis. Analysis of the differences in the evolution
of public opinion and sentiment
between Netizens of Wuhan and other
locations in China This study, on the other hand, presents a
preliminary framework for the government to respond rap-
idly to public emergencies, especially during times of public
emergency. In future research, we will continue to explore
the application of deep neural networks in the tracking and
governance of large-scale emergencies, as well as attempt to
integrate more modal data, such as video, voice and bullet
screen, so as to more accurately capture the emotional evolu-
tion of Internet users, in order to provide targeted guidance
and governance for Internet users in different regions or dif-
ferent psychological states. Conclusion The evolution trends in the emo-
tions of other netizens in China show that their emotional
states are more positive. This positive and optimistic mood
can help Wuhan netizens relieve their emotional pressure,
arouse the national unity and cohesion of other netizens in
the country and call on the masses to actively fight COVID-
19. Based on the differences in emotional evolution between
netizens of Wuhan and other locations in China, the targeted
allocation of the resources of the country can be achieved
during the COVID-19 epidemic. In the early stages of an
epidemic such as COVID-19, medical, financial and infor-
mation resources should have been quickly concentrated, the
disease should be stabilised quickly, and information should
be open and transparent, in order to resolve the negative
emotions expressed in public opinion. In the middle and
later stages of the epidemic, financial and human resources Association for Computational Linguistics, pp 417–424. https://
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objective optimization. IEEE Trans Evolut Comput IEEE 22:3–18 g
6. Rao YH et al (2014) Building emotional dictionary for senti-
ment analysis of online news. World Wide Web Int Web Inf Syst
17(4):723–742. https://doi.org/10.1007/s11280-013-0221-9 j
p
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21. Ohtsuka Y et al (2010) Forecasting electricity demand in Japan:
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Data Anal 54(11):2721–2735. https://doi.org/10.1016/j.csda.2009.
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7. Tripathy A (2016) Classification of sentiment reviews using
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and control. Wiley, Hoboken 8. Jie J and Rui X (2017) Microblog sentiment classification via
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tion algorithm for mixed-variable newsvendor problems. IEEE
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TEVC.2019.2932624 11. Hofmann T (2001) Unsupervised learning by probabilistic latent
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Mach Learn Res 3:993–1022 27. Wang R, Purshouse RC, Fleming PJ (2013) Preference-inspired
coevolutionary algorithms for many-objective optimization. IEEE
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TEVC.2012.2204264 13. Turney PD (2000) Learning algorithms for keyphrase extraction. Inf Retrieval 2(4):303–336i 14. Frank E et al (1999) Domain-specific keyphrase extraction. In:
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1145/1099554.1099628 28. Wang R, Zhang Q, Zhang T (2016) Decomposition-based algo-
rithms using pareto adaptive scalarizing methods. References 1. Xi JP (2020) Speech at the meeting to coordinate the promotion of
the prevention and control of the new crown pneumonia epidemic
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People's Daily, 24th Feb. http://www.gov.cn/xinwen/2020-02/24/
content_5482502.htm. 1. Xi JP (2020) Speech at the meeting to coordinate the promotion of
the prevention and control of the new crown pneumonia epidemic
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People's Daily, 24th Feb. http://www.gov.cn/xinwen/2020-02/24/
content_5482502.htm. 2. Wang C (2020) A novel coronavirus outbreak of global health
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S0140-6736(20)30185-9 2. Wang C (2020) A novel coronavirus outbreak of global health
concern. The Lancet 10223:470–473. https://doi.org/10.1016/
S0140-6736(20)30185-9 (
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3. Yu T et al (2020) Timely mental health care for the 2019 novel
coronavirus outbreak is urgently needed. Lancet Psychiatry
3:228–229. https://doi.org/10.1016/S2215-0366(20)30046-8 3. Yu T et al (2020) Timely mental health care for the 2019 novel
coronavirus outbreak is urgently needed. Lancet Psychiatry
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4. Li S (2020) The impact of COVID-19 epidemic declaration on
psychological consequences: a study on active weibo users. Int
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h17062032 4. Li S (2020) The impact of COVID-19 epidemic declaration on
psychological consequences: a study on active weibo users. Int
J Environ Res Public Health 6:1–9. https://doi.org/10.3390/ijerp
h17062032 5. Turney PD (2002) Thumbs up or thumbs down? Semantic orienta-
tion applied to unsupervised classification of reviews. Meeting on 1 3 3178 Complex & Intelligent Systems (2021) 7:3165–3178 Association for Computational Linguistics, pp 417–424. https://
doi.org/10.3115/1073083.1073153 IEEE Trans
Evol Comput 20(6):821–837. https://doi.org/10.1109/TEVC.2016.
2521175 15. Ruangkanokmas P et al (2016) Deep belief networks with feature
selection for sentiment classification. In: International Conference
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Current Data Gaps in Modeling Essential Worker Absenteeism Due to COVID-19
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LETTER TO THE EDITOR LETTER TO THE EDITOR OTHER IMPACTS ON ABSENTEEISM Absenteeism due to an exposure requiring quarantine,
and/or in order to care for a family or other household
member, also has an unspecified impact on absenteeism
rates. This is potentially exacerbated by an overbur-
dened testing system and many households being
unable to accommodate at-home self-isolation in the
United States.4 In our review, we did not find any
data on absenteeism related to illness of a family or
other household member. Additionally, the prolonged
nature of the pandemic has taken an emotional toll:
53% of US adults say that COVID-19-related stress
has had a negative impact on their mental health,
up from 39% in May.5 This could potentially result
in increased absenteeism or an extended or permanent
leave of absence. Current Data Gaps in Modeling Essential Worker
Absenteeism Due to COVID-19 Zackery White, MPH; Jeff Schlegelmilch, MPH, MBA; Jackie Ratner; Gunjan Saxena;
Kevin Wongsodirdjo; Susanna Aguilar; Daniel Kushner; Jim Ortega; Aleksi Paaso, PhD;
Shay Bahramirad, PhD © Society for Disaster Medicine and Public Health, Inc. 2020. This is an Open Access article, distributed under the terms of the Creative Commons
Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any
medium, provided the original work is properly cited. DOI: 10.1017/dmp.2020.353
//doi.org/10.1017/dmp.2020.353 Published online by Cambridge University Press ABSTRACT With the uncertain physical and mental health implications of COVID-19 infection, companies have taken a
myriad of actions that aim to reduce the risk of employees contracting the virus, with most grounded in
reducing or eliminating in-person interactions. Our preliminary analysis indicates that while there is some
data to support modelling absenteeism, there are gaps in the available evidence, requiring the use of
assumptions that limit precision and efficacy for decision support. Improved data on time-to-recovery after
hospitalization, absenteeism due to family or other household member illness, and mental health’s impact
on returning to work will support the development of more robust absenteeism models and analytical
approaches. Key Words: absenteeism, COVID-19, critical Infrastructure, modelling, pandemics Key Words: absenteeism, COVID-19, critical Infrastructure, modelling, pandemic W
i
h W
ith the uncertain physical and mental
health implications of the coronavirus
disease (COVID-19) infection, compa-
nies have taken a myriad of actions that aim to reduce
the risk of employees contracting the virus, with
most grounded in reducing or eliminating in-person
interactions. However, utilities and other essential
industries may be unable to fully eliminate the need
for in-person attendance. The National Center for
Disaster Preparedness (NCDP) at Columbia University’s
Earth Institute has been supporting the electric utility
company Commonwealth Edison (ComEd) to help
identify parameters for modeling workforce absenteeism. As part of this effort, NCDP has developed a research
dashboard to track publications that are non-, pre-,
and post- peer review to support analytical parameter
assumptions. While more than 80 publications have
been analyzed, there are many data gaps that pose a chal-
lenge for the accurate forecasting of employee absentee-
ism (Figure 1). non-hospitalized patients revealed that, on average,
symptoms in outpatients resolved after 3 weeks with
a standard deviation of 1.25 weeks, not accounting
for the possibility that an employee’s symptoms could
worsen, requiring hospitalization and extending his
or her absence.2,3 We have found no data nor proxy
for estimating the recovery time after discharge, though
we would expect that those hospitalized, especially
those requiring intensive care, would need extensive
time to recuperate prior to re-entering the workforce. DISEASE COURSE OF COVID-19 In the simplest scenario, employees who contract
COVID-19 would miss work and return 24 hours after
their fever resolves and their other symptoms improve,
and at least 10 days have passed since symptom onset.1
However, the disease course is not well studied in
moderate cases, as most studies use hospital records,
which reflect severe cases and do not capture the time
frame post-discharge until returning to work. Two pre-
peer-reviewed studies with longitudinal follow-up for Our preliminary analysis indicates that, while there are
some data to support modeling absenteeism, there are
gaps in the available evidence, requiring the use of REFERENCES 1. CDC. Discontinuation of isolation for persons with COVID-19 not in
healthcare settings. United States Centers for Disease Control and
Prevention. Updated July 20, 2020. https://www.cdc.gov/coronavirus/
2019-ncov/hcp/disposition-in-home-patients.html. Accessed August 19,
2020. 1. CDC. Discontinuation of isolation for persons with COVID-19 not in
healthcare settings. United States Centers for Disease Control and
Prevention. Updated July 20, 2020. https://www.cdc.gov/coronavirus/
2019-ncov/hcp/disposition-in-home-patients.html. Accessed August 19,
2020. 2. O’Keefe JB, Tong EJ, Datoo O’Keefe GA, Tong DC. Predictors of disease
duration and symptom course of outpatients with acute COVID-19: a retro-
spective cohort study. medRxiv. 2020. The authors have no conflicts of interest to declare. The authors have no conflicts of interest to declare. Disaster Medicine and Public Health Preparedness e10 VOL. 15/NO. 4 Disaster Medicine and Public Health Preparedness Employee Absenteeism Due to COVID-19 Employee Absenteeism Due to COVID-19 Swim Lane Diagram of the Conceptual Inner Workings of Employee Absenteeism and Events That Alter the Timeline for the
Return to Work. Swim Lane Diagram of the Conceptual Inner Workings of Employee Absenteeism and Events Th
Return to Work. Return to Work. (The shaded boxes indicate parameter values that have evidence to support the development of more robust absenteeism models. This diagram does not represent all events that an individual may experience.) (The shaded boxes indicate parameter values that have evidence to support the development of more robust absenteeism models. This diagram does not represent all events that an individual may experience.) (The shaded boxes indicate parameter values that have evidence to support the development of more robust absenteeism models. This diagram does not represent all events that an individual may experience.) Conflict of Interest Statement
The authors have no conflicts of interest to declare. assumptions that limit precision and efficacy for decision sup-
port. Improved data on time-to-recovery after hospitalization,
absenteeism due to family or other household member illness,
and mental health’s impact on returning to work will support
the development of more robust absenteeism models and ana-
lytical approaches. We also acknowledge that this analysis and
approach are not exhaustive, and that additional factors for
absenteeism should be identified and analyzed as the evi-
dence-base is further developed. Correspondence and reprint requests to Jeff Schlegelmilch, Columbia University,
National Center for Disaster Preparedness, 215 W 125th Street, Suite 303,
New York, New York 10027 (e-mail: js4645@columbia.edu) About the Authors 3. Mizrahi B, Shilo S, Rossman H, et al. Longitudinal symptom dynamics of
COVID-19 infection in primary care. medRxiv. 2020. The National Center for Disaster Preparedness at The Earth Institute,
Columbia University, New York, NY (Mr White, Mr Schlegelmilch, Ms Ratner,
Ms Saxena, Mr Wongsodirdjo) and Commonwealth Edison, Chicago, IL
(Ms Aguilar, Mr Kushner, Mr Ortega, Dr Paaso, Dr Bahramirad). 4. Sehgal AR, Himmelstein DU, Woolhandler S. Feasibility of separate rooms
for home isolation and quarantine for COVID-19 in the United States. Ann
Intern Med. 2020. 5. KFF.org. Hamel L, Kearney A, Kirzinger A, et al. KFF Health Tracking
Poll – July 2020. Henry Kaiser Family Foundation. July 27, 2020. https://
www.kff.org/coronavirus-covid-19/report/kff-health-tracking-poll-july-2020/. Accessed September 1, 2020. Correspondence and reprint requests to Jeff Schlegelmilch, Columbia University,
National Center for Disaster Preparedness, 215 W 125th Street, Suite 303,
New York, New York 10027 (e-mail: js4645@columbia.edu) e11 https://doi.org/10.1017/dmp.2020.353 Published online by Cambridge University Press
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Attribute driven inverse materials design using deep learning Bayesian framework
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npj computational materials
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INTRODUCTION ANNs were used to predict organic reactions,8 properties of
inorganic materials.9 Recently, deep variants of the ANN are also
used for molecular property predictions.10 These ANNs based
methods are essentially solving the forward problem in which
material properties are computed for an input molecular structure. However, actual materials design is naturally an inverse problem,
that is, specific technology-enabling properties requirements are
known, but often we do not have the materials that have these
properties. For example, in the case of multi-junction solar cells,
the band-gap, exciton binding energy and carrier mobility
requirements
of
each
layer
for
achieving
target
quantum
efficiency are known, and we need to design materials that meet
these requirements. Thus to advance computational materials
design significantly, one needs to ask the question, can we train
machines to learn designing materials? Given that the atomic
composition and structure determines the material property, can
machines
provide
suggestions
on
materials/structures
to
fabricate? Imagine having to design a new material for a target application
from scratch. Conventional materials design practice relies on trial-
and-error approach. One typically starts with a known material by
comparing its properties against the target property, and then
incorporating a compositional/structural change to update the
design. This process is performed iteratively, primarily driven by
intuition/experience of the materials scientist. In the recent past,
with the advent of efficient physics-based modelling that span a
wide length scale from atomistic to continuum regime, properties
are determined in-silico to minimise efforts involved in experi-
mental realisation of candidate materials and measurement of
their properties and thus helping speed-up the design iteration
process.1–3 This approach was further augmented by optimisation
schemes that attempted at designing materials with optimum
properties by using physics-based simulations informed optimisa-
tion. The latter approach is exemplified by evolutionary algorithms
or other gradient based optimisation techniques when appro-
priate.4 It takes many, often times hundreds of simulations before
a
reasonable
material/structure
can
be
found. Since
each
simulation is computationally expensive, these methods become
prohibitively slow as molecular/material/device size and complex-
ity grow. In contrast to the optimisation approach, data-driven
approaches based on materials informatics have been employed
in the last decade, with particular emphasis on fingerprinting and
determining previously unknown underlying correlations between
structures and properties to derive “surrogate models”.5,6 More
recently, machine learning approaches are rapidly emerging,
where shallow learning methods like artificial neural networks
(ANNs) are trained to assist the materials design process. www.nature.com/npjcompumats 1Next Gen Research, Samsung Advanced Institute of Technology, Samsung R & D Institute, Bangalore 560037, India. 2Autonomous Material Development Lab, Samsung
Electronics, 130 Samsung-ro, Suwon, Gyeonggi-do 443-803, Republic of Korea. *email: p.tagade@samsung.com; shashi.adiga@samsung.com ARTICLE
OPEN
Attribute driven inverse materials design using deep
learning Bayesian framework sh M. Tagade1*, Shashishekar P. Adiga1*, Shanthi Pandian1, Min Sik Park2, Krishnan S. Hariharan1 and Su Much of computational materials science has focused on fast and accurate forward predictions of materials properties, for example,
given a molecular structure predict its electronic properties. This is achieved with first principles calculations and more recently
through machine learning approaches, since the former is computation-intensive and not practical for high-throughput screening. Searching for the right material for any given application, though follows an inverse path—the desired properties are given and the
task is to find the right materials. Here we present a deep learning inverse prediction framework, Structure Learning for Attribute-
driven Materials Design Using Novel Conditional Sampling (SLAMDUNCS), for efficient and accurate prediction of molecules
exhibiting target properties. We apply this framework to the computational design of organic molecules for three applications,
organic semiconductors for thin-film transistors, small organic acceptors for solar cells and electrolyte additives with high redox
stability. Our method is general enough to be extended to inorganic compounds and represents an important step in deep learning
based completely automated materials discovery. npj Computational Materials (2019) 5:127 ; https://doi.org/10.1038/s41524-019-0263-3 Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences INTRODUCTION ANNs are
used to replace/minimise the computationally expensive first
principles calculations in the optimisation loop, greatly reducing
overall design time. For example, ANNs were used for high-
throughput screening of organic molecules for use in solar cells
based on ANN predicted frontier orbital energy levels.7 Further, In this paper, we posit that the current state of the art materials
design approaches use machine learning primarily as an enabler
for implementing traditional high-throughput screening, that is,
essentially solving the forward problem in a computationally
efficient manner. Recent advancements in machine learning
algorithms has a potential to solve the inverse materials design
problem in a fully automated framework. However, development
of such a framework is impeded by some key challenges. First, a
compact and accurate numerical representation of molecular
structures is required to digitise the known chemical space. Second,
a
computationally
efficient
structure
to
properties
mapping is required to ensure practicality of the framework. Although ANNs partially resolve this challenge, training the ANNs
require a large properties database, introducing a new challenge. Finally, an appropriate search algorithm is required that is capable
of scanning a chemical space of known and hitherto unknown ublished in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. ules, while ensuring that the candidate structures form a
molecule. h
dd
d ll th
h ll
i
thi
T
l
Bayes theorem. In its most widely used form, the Bayes the
specified as
Posterior
p(molecule | property)
Chemical space of molecular
structures with
desired properties
Likelihood
p(property|molecule)
Semi-supervised deep learning
of
structure-property correlation
Prior
p(molecule)
Unsupervised learning
of
molecular structures
Structure - Property
Correlation
(a)
(b)
(c)
0100010....00111001
0101110....10110100
0001000....01101011
0111010....10111001
0100100....10101000
0100011....11100001
1101010....10101001
0101101....11001101
v
h
p(molecule)
Binary representation of
molecular structures
database
Restricted
Boltzmann machine
p(properties | molecule)
CH3
O
O
OH
COC1=C(C=CC(=C1)C=O)O
010001.......11000110
Molecular
Structure
SMILES
Representation
Binary
Representation
RBM
Properties
GB-RBM
+
=
010001.......11000
Properties
Deep Network
010001.......11000
Molecular Structure
Properties
(d)
3
H C
N
H2C
NH
N
N
H2N
H3C
N
H3C
N
N
NH2
CH3
HN
O
CH3
N
N
H2N
CH3
HN
N
H
CH3
N
N
H2N
H3C
N
N
N
N
NH2
H3C
N
H3C
N
H
H3C
N
N
H2N
MCMC Sampling
(e)
The four stages of deep learning based inverse materials design in SLAMDUNCS framework. Bayesian inference shown in
one of SLAMDUNCS. INTRODUCTION The framework first digitises the molecular structures using a fingerprinting approach shown in b. The
cal structure encoded in the molecular fingerprints are embedded in the RBM using unsupervised learning of molecular st
ase, as shown in c. The RBM quantifies prior probability on the chemical space. Semi-supervised deep learning approach show
o obtain a molecular structure-property correlation, quantifying the likelihood of a molecular structure exhibiting the target pro
osterior distribution combines prior in c with the likelihood in d to encode a chemical space of molecules possessing target pro
ic Markov chain is simulated to sample from this posterior distribution, as shown in e. P.M. Tagade et al. 2 Posterior
p(molecule | property)
Chemical space of molecular
structures with
desired properties
Likelihood
p(property|molecule)
Semi-supervised deep learning
of
structure-property correlation
Prior
p(molecule)
Unsupervised learning
of
molecular structures
(a) (a) (c)
0100010....00111001
0101110....10110100
0001000....01101011
0111010....10111001
0100100....10101000
0100011....11100001
1101010....10101001
0101101....11001101
v
h
p(molecule)
Binary representation of
ole
la
t
t
e
Restricted (b)
CH3
O
O
OH
COC1=C(C=CC(=C1)C=O)O
010001.......11000110
Molecular
Structure
SMILES
Representation
Binary
Representation (c) (b) p(molecule) Structure - Property
Correlation
p(properties | molecule)
RBM
Properties
GB-RBM
+
=
010001.......11000
Properties
Deep Network
010001.......11000
Molecular Structure
Properties
(d)
3
H C
N
H2C
NH
N
N
H2N
H3C
N
H3C
N
N
NH2
CH3
HN
O
CH3
N
N
H2N
CH3
HN
N
H
CH3
N
N
H2N
H3C
N
N
N
N
NH2
H3C
N
H3C
N
H
H3C
N
N
H2N
MCMC Sampling
(e) Structure - Property
Correlation
p(properties | molecule)
RBM
Properties
GB-RBM
+
=
010001.......11000
Properties
Deep Network
010001.......11000
Molecular Structure
Properties
(d) (d) 3
H C
N
H2C
NH
N
N
H2N
H3C
N
H3C
N
N
NH2
CH3
HN
O
CH3
N
N
H2N
CH3
HN
N
H
CH3
N
N
H2N
H3C
N
N
N
N
NH2
H3C
N
H3C
N
H
H3C
N
N
H2N
MCMC Sampling (e) MCMC Sampling MCMC Sampling Fig. 1
The four stages of deep learning based inverse materials design in SLAMDUNCS framework. Bayesian inference shown in a is the
backbone of SLAMDUNCS. The framework first digitises the molecular structures using a fingerprinting approach shown in b. The rules of
chemical structure encoded in the molecular fingerprints are embedded in the RBM using unsupervised learning of molecular structures
database, as shown in c. The RBM quantifies prior probability on the chemical space. npj Computational Materials (2019) 127 Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences RESULTS Our use of semi-supervised deep
learning allows us to implement our proposed approach with a
relatively small properties database. y
p
p
We will illustrate the practicability of our approach with three
exemplar organic materials design problems: n- and p-type
organic semiconductors for organic thin film transistors (OTFTs),21
small organic acceptor molecules for use in organic solar cells22
and high stability electrolytes for lithium-ion batteries.23 These
problems are chosen to demonstrate that in the continuous
evolution of the use of computers in materials science, deep
learning based inverse design presented here takes us one step
forward in going beyond subject matter experts’ knowledge
based intuition. In the current practice, using chemical classifica-
tion of substituents and developing quantitative rules, experts can
predict how electron donors and acceptors in conjugated
molecules alter the reduction/oxidation potentials in specific
classes or narrow range of molecules. However, as the molecule
becomes more complex, properties’ predictions become increas-
ingly hard. We first show that because our forward method is able
to learn “chemical rules”, in the form of how the relative
arrangement of elements and bonds in a molecule together
decide its properties with high accuracy, we can predict properties
of complex molecules that chemical intuition alone cannot. Further, the unsupervised learning of our approach enables us
to take advantage of Bayesian inference to predict previously
unknown (not in the database) molecules that have target
properties. For example, to design stable molecules for electro-
lytes, target reduction and oxidation stability as dictated by
reduction and oxidation potentials is used as an input to predict
new molecules. In the case of n- and p-type semiconductors and
organic solar cell acceptors target values/windows for energies of
the frontier orbitals are used to design new molecules. In all cases,
SLAMDUNCS is able to predict valid molecules with target
properties and we then validate the properties of a small set of
these new molecules using first principles simulations. While we
have demonstrated inverse design here for designing organic
molecules, our approach is general enough to be applied to a
wide class of materials systems. Unsupervised learning of molecular structures Unsupervised learning of molecular structures
We use unsupervised learning of molecular structures to quantify
prior probability distribution in our Bayesian approach. Our choice
of an unsupervised learning algorithm is constrained by two
considerations. RESULTS Depending on the application,
different molecular descriptors such as Coulomb matrices,24 bag
of bonds,25 overlap matrix,26 deep tensor neural networks,27 count
of functional groups present in the structure28 etc. have been used
for molecular representation at various level of granularities. Aim
of this paper is to develop an inverse molecular design framework,
as such, our choice of fingerprinting is primarily motivated by ease
and
uniqueness
with
which
molecular
descriptor
can
be
converted to the molecular structure. We use canonical SMILES29
representation, which is a widely used text based line notation for
organic molecules, as a molecular descriptor, as it allows for
capturing most important chemical features of organic molecules,
is particularly amenable for digital representation and it can be
easily and uniquely converted to the molecular structure. Note
that the SMILES representation is widely used in the literature as a
molecular descriptor, however, one-hot encoding is often used to
digitise the SMILES representation. In our approach, we use binary
representation to digitize the SMILES. We digitise the SMILES
representation by first obtaining ASCII equivalent of each SMILES
character, and subsequently converting the ASCII to an eight-bit
binary number (see Fig. 1b). The resultant binary vector is used as
a molecular fingerprint. This approach allows molecular finger-
printing
with
complete
positional
and
atomic
connectivity
information without using any additional chemical information. This approach has the inherent advantage of compact representa-
tion over one-hot encoding. For instance, the one-to-one mapping
of SMILES representation would require 67 one-hot encoding
characters. In this work, since we consider molecular structures of
SMILES length upto 100 characters, the length of one-hot
encoding would be 6700. Such high dimensionality substantially
increases the number of parameters of the underlying deep
network requiring large training dataset. Our use of 8-bit binary
representation for SMILES digitisation results in a binary vector of
length 800, while preserving uniqueness of the representation. As
the resultant deep network has smaller number of parameters, the
network can be efficiently trained using smaller training dataset. We claim several innovations of our proposed approach. Previous approaches primarily comprised intrinsic screening with
hand-crafted features,19 whereas, unsupervised learning in our
approach enables us to perform intrinsic as well as extrinsic
screening. Moreover, previous approaches are limited to human
specified design spaces with heuristic or design intent search
rules,20 whereas, the proposed approach is a fully automated
artificial intelligence based materials design framework with a
minimal human intervention. RESULTS attributes. Our choice of this Bayesian approach is motivated by
two considerations. First, optimisation methods used in the state
of the art inverse materials design algorithms require specification
of heuristic rules to ensure generation of valid molecular
structures. In the Bayesian approach, appropriate choice of the
prior distribution ensures that the search space is constrained to
valid molecular structures. We use unsupervised learning16–18 on a
large dataset of known molecular structures to quantify this prior. This unsupervised learning enables us to encode the chemical
rules of molecule formation as a prior probability distribution of
the Bayes theorem. Thus, the proposed approach does not require
specification of any heuristic rules. Second, the optimisation
methods predict a single molecular structure by maximising/
minimising a desired molecular attribute. As against the single
point prediction of optimisation methods, the Bayesian approach
predicts
posterior
probability
distribution
that
encode
the
chemical space of molecules with desired attributes. Sampling
from this distribution allows us to generate a large set of valid
molecular structures for a given application. This helps us in
further accelerating the inverse materials design. attributes. Our choice of this Bayesian approach is motivated by
two considerations. First, optimisation methods used in the state
of the art inverse materials design algorithms require specification
of heuristic rules to ensure generation of valid molecular
structures. In the Bayesian approach, appropriate choice of the
prior distribution ensures that the search space is constrained to
valid molecular structures. We use unsupervised learning16–18 on a
large dataset of known molecular structures to quantify this prior. This unsupervised learning enables us to encode the chemical
rules of molecule formation as a prior probability distribution of
the Bayes theorem. Thus, the proposed approach does not require
specification of any heuristic rules. Second, the optimisation
methods predict a single molecular structure by maximising/
minimising a desired molecular attribute. As against the single
point prediction of optimisation methods, the Bayesian approach
predicts
posterior
probability
distribution
that
encode
the
chemical space of molecules with desired attributes. Sampling
from this distribution allows us to generate a large set of valid
molecular structures for a given application. This helps us in
further accelerating the inverse materials design. RESULTS
Numerical representation of molecular structures Numerical representation of molecular structures Numerical representation of molecular structures
Numerical representation of molecular structures using the
molecular fingerprinting forms a critical step of a data-driven
inverse materials design approach. INTRODUCTION Semi-supervised deep learning approach shown in d is
used to obtain a molecular structure-property correlation, quantifying the likelihood of a molecular structure exhibiting the target properties. The posterior distribution combines prior in c with the likelihood in d to encode a chemical space of molecules possessing target properties. Ergodic Markov chain is simulated to sample from this posterior distribution, as shown in e. Bayes theorem. In its most widely used form, the Bayes theorem is
specified as molecules, while ensuring that the candidate structures form a
valid molecule. We have addressed all these challenges in this paper. To resolve
the first challenge, we have developed a binary representation to
digitise the molecular structure. We resolve the second challenge
by using semi-supervised learning approach to obtain structure to
properties mapping. This semi-supervised algorithm can be
trained using a smaller dataset compared to the supervised
training of ANNs. We address the final challenge by approaching
the inverse materials design from a Bayesian perspective11–13 (see
Fig. 1a). At the core of this Bayesian approach is a solution of the posterior / likelihood ´ prior
(1) (1) posterior / likelihood ´ prior
(1) where the prior probabilistically encompasses available knowl-
edge of the uncertain system,14 the likelihood is defined using
new measurement or evidence,15 while the posterior denotes
updated probability after assimilating the new evidence. For
inverse materials design, prior probabilistically quantifies validity
of a given molecular structure, whereas, the likelihood15 specifies
probability of a molecular structure exhibiting our desired where the prior probabilistically encompasses available knowl-
edge of the uncertain system,14 the likelihood is defined using
new measurement or evidence,15 while the posterior denotes
updated probability after assimilating the new evidence. For
inverse materials design, prior probabilistically quantifies validity
of a given molecular structure, whereas, the likelihood15 specifies
probability of a molecular structure exhibiting our desired npj Computational Materials (2019) 127 P.M. Tagade et al. 3 Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 RESULTS Flexibility of this semi-supervised learning
approach allow us to create a variety of deep networks like Deep
Boltzmann Machine43 and Deep Neural Network,44 and train the
network using same training dataset. p
We have trained the RBM using molecular structures dataset
consisting of 306153 randomly chosen molecules in the com-
pound
identifier
range
of
1–1200000
from
the
PubChem
database.36 We evaluate training accuracy of the RBM using its
reconstruction ability. For instance, given any input vector v and a
set of RBM weights and biases, the RBM can be used to obtain a
hidden layer by sampling from p hjv
ð
Þ and subsequently obtaining
a reconstructed vector by sampling from p vjh
ð
Þ. We define the
reconstruction error as the sum of Euclidean distance between
input vectors from the training dataset and the corresponding
reconstructed vectors. Supplementary Fig. 1a shows the recon-
struction error reduction with number of training iterations (also
known as epochs). Supplementary Fig. 1b shows reconstructed
SMILES with increasing number of epochs. Reconstruction ability
of the RBM increases as training progresses, as can be seen from
Supplementary Fig. 1b, where reconstructed visible layer output
approaches towards valid SMILES. The RBM also allows us to
construct new molecular structures using Gibbs sampling.37 For a
given input vector v, we implement Gibbs sampling as follows. We
first use the trained RBM to calculate p hjv
ð
Þ and obtain the hidden
layer output h by sampling from this distribution. Subsequently,
we calculate p vjh
ð
Þ and sample v from this distribution. We repeat
these steps a pre-determined number of times to generate
samples from the RBM. Finally, we convert the sampled binary
vectors
to
SMILES
representation
for
construction
of
new
molecular structures. Supplementary Fig. 1c shows a set of
randomly created molecular structures using Gibbs sampling. g
g
We have trained our network using a database of molecular
orbital energies and redox potentials predicted using first
principles calculation performed by density functional theory
(DFT). Detailed
analysis
of
this
database
is
presented
in
Supplementary Fig. 2. Supplementary Fig. 2a shows distribution
of the number of compounds against the SMILES length. Total
85:7% of the compounds in the database have SMILES length ≤50
while the SMILES length ≤100 covers 99:85% of the molecules in
the database. Thus for all the results presented in this paper, we
have only considered molecules with SMILES length of upto 100
characters. RESULTS Moreover, since the DBN is constructed using RBMs, it
has a capability to efficiently encode the binary vectors that are
used as molecular descriptors. However, we cannot use the RBM
for unsupervised learning of properties that are real numbers. This
issue is resolved by a recently developed variant of the RBM,
known as Gaussian-Bernoulli RBM42 (GB-RBM). Contrary to the
RBM, visible layer of the GB-RBM is defined using a vector of real
numbers with probability of each node specified using indepen-
dent Gaussian distributions. We use a small property database to
train this GB-RBM in an unsupervised manner. y
y
y
g
The probability density function of RBM is defined using a
Boltzmann distribution given by p v; h
ð
Þ ¼ exp E
ð
Þ
Z
;
(2) p v; h
ð
Þ ¼ exp E
ð
Þ
Z
; (2) where Z is a partition function and E is the energy defined as
E ¼ vTWh bv ch;
(3) E ¼ vTWh bv ch; (3) E ¼ vTWh bv ch;
(3) where v is visible layer, h is hidden layer, W is a weight matrix,
while b and c are biases in visible and hidden layer, respectively. We use maximum likelihood estimation for training the RBM on
molecular structures database.35 After training, we marginalise
over h to obtain p v
ð Þ. pð Þ
The marginal distribution p v
ð Þ facilitates understanding of the
SMILES
representation. Training
the
RBM
entails
maximum
likelihood estimation of p v
ð Þ that encodes both localised and
neighbourhood information present in the SMILES representation. For instance, if a specific character like “A” is not present in the
SMILES dataset, the trained RBM assigns it a probability zero, and
as such, this character is never sampled by the RBM. Similarly, a
triple bond between carbon and oxygen never exists in nature,
thus, combination of characters “C # O” is not present in the
SMILES dataset. Hence, the trained RBM assigns zero probability to
such sequence of characters. As such the RBM essentially learns,
through probability assignments, the rules of valid molecule
formation that are embedded in the SMILES representation. We invert the trained GB-RBM and stack together with the DBN
to construct a deep network (see Fig. 1d). We use a small
structure-property database to train the resultant deep network in
a supervised manner. RESULTS As we use binary vectors as molecular descriptors,
we require a machine learning architecture that can efficiently
encode the binary numbers. Furthermore, the architecture is
required to have capability to quantify probability distributions. Although the unsupervised learning algorithms like Gaussian
mixture models30 are capable of quantifying probability distribu-
tions, they are primarily designed for real numbers, and as such,
fail to efficiently encode the binary vectors. Several autoenco-
ders31 are capable of learning to construct valid molecular
structures by efficiently encoding the binary vectors, however,
they fail to quantify probability distributions which is key to our
proposed Bayesian approach. In our proposed approach, we use
restricted Boltzmann machine (RBM)32 for unsupervised learning,
as it is specifically designed for efficient encoding of binary vectors
and is capable of quantifying probability distributions. Note that
though the RBMs were initially developed for applications like face
recognition,33 recently, RBMs are successfully used of applications
in chemical and physical sciences.34 However, to the best of our
knowledge, RBMs are never used for quantification of prior
distribution in the Bayesian inference framework. The RBM network consists of a visible and a hidden layer, where
each node of the visible layer is connected to every node of the shed in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. 4 hidden layer (see Fig. 1c). Every node of the visible and hidden
layer is a binary number that takes value of 0 or 1 with a
probability. This probability is learned by training the RBM. available only for a very small subset of molecules. Supervised
learning algorithms fail to fully utilise information from the
molecular structures database, whereas a small size of the
property database often results in the poor accuracy of the
machine learning predictions. We propose a semi-supervised deep
learning approach39 for structure-property correlation that can
provide accurate predictions even with a comparatively small
property dataset. In this approach, we first use unsupervised
learning to train a Deep Belief Network (DBN)40 using the
molecular structure database. DBN contains more than one
hidden layers, where a pair of subsequent layers is a RBM. Thus,
we construct this DBN using a stack of pre-trained RBMs.41 We
have used the DBN for this unsupervised learning, as it allows us
to reuse already pre-trained RBM for quantifying prior probability
distribution. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences RESULTS Dependence of properties on the molecular structures
enforces correlation amongst properties for a given structure, as
can be observed for highest occupied orbital (HOMO) energies
and oxidation potential from Supplementary Fig. 2b and between
lowest
unoccupied
molecular
orbital
(LUMO)
energies
and
reduction potential in Supplementary Fig. 2c. SLAMDUNCS is designed to exploit the information content in
the correlations amongst properties and between the molecular
structure and properties for efficient training with minimal
properties dataset. We evaluate this information content using
the Pearsons correlation coefficient. For any two random variables
x and y, the Pearsons correlation coefficient is given by ρ x; y
ð
Þ ¼ covðx; yÞ
σxσy
;
(4) (4) where covðx; yÞ is a covariance between x and y, while σx and σy
denotes standard deviation of x and y, respectively. Semi-supervised learning of structure-property correlation We need a computationally efficient approach to forward predict
the properties given a molecular structure. Due to high computa-
tional cost of first principles calculations, we use machine learning
for efficient forward property predictions. Previously, different
supervised learning algorithms are used in the literature38 for
property prediction of a given molecule. However, supervised
learning requires a very large dataset for accurate training, again
incurring high computational cost. In practice, we have a very
large molecular structures database, however, property values are Figure 2a shows the Pearsons correlation coefficient between
the molecular properties and output of every layer of the DBN,
which is trained in unsupervised
manner using molecular
structures dataset. Even though the properties data is not used
for training, every added layer of DBN increasingly extracts
properties information that is embedded in the molecular
structure, as is evident from the increasing correlation between
properties and the DBN layer output. The highest correlation
between the DBN output and properties is greater than 0.3, with npj Computational Materials (2019) 127 P.M. Tagade et al. 5 (a)
HOMO
LUMO
Oxid. Red. HOMO
LUMO
Oxid. Red. 1
-0.045
1
-0.85
-0.038
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. 1
-0.05
1
-0.88
-0.039
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. RESULTS 0.96
-0.048
0.97
-0.84
-0.035
0.95
-0.14
-0.78
0.21
1
1.0
0.5
0.0
-0.5
-1.0
(b)
Data-Data
Prediction - Prediction
Data - Prediction
(c)
HOMO
LUMO
Oxidation
Reduction
Train
Train
Train
Train
Test
Test
Test
Test
7.5
5.0
2.5
0.0
2.5
nergy, eV/ Potential, V
Data
Prediction
(d) (a) (a) HOMO
LUMO
Oxid. Red. HOMO
LUMO
Oxid. Red. 1
-0.045
1
-0.85
-0.038
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. 1
-0.05
1
-0.88
-0.039
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. 0.96
-0.048
0.97
-0.84
-0.035
0.95
-0.14
-0.78
0.21
1
1.0
0.5
0.0
-0.5
-1.0
(b)
Data-Data
Prediction - Prediction
Data - Prediction
(c)
HOMO
LUMO
Oxidation
Reduction
Train
Train
Train
Train
Test
Test
Test
Test
HOMO
LUMO
Oxidation
Reduction
7.5
5.0
2.5
0.0
2.5
Energy, eV/ Potential, V
Data
Prediction
(d)
Fig. 2
Forward property prediction using semi-supervised deep learning. Properties information embedded in molecular structures is
extracted using unsupervised learning of DBN, as shown in a. The Pearson’s correlation coefficient between activation probability of each
node and the properties is shown in the figure. The DBN network used in this paper is shown in the rightmost part of the figure. Highest
correlation for each layer output is shown in the leftmost part of the figure. Similarly, correlation between the properties is shown in b. The
quantile-quantile plot for deep learning predicted properties is shown in c. Violin plot in d shows comparison of the PDF for predicted
properties and dataset. Kulback–Liebler (KL) divergence between the PDFs is also shown in the figure. HOMO
LUMO
Oxid. Red. HOMO
LUMO
Oxid. Red. 1
-0.045
1
-0.85
-0.038
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. 1
-0.05
1
-0.88
-0.039
1
-0.14
-0.78
0.21
1
HOMO
LUMO
Oxid. Red. 0.96
-0.048
0.97
-0.84
-0.035
0.95
-0.14
-0.78
0.21
1
1.0
0.5
0.0
-0.5
-1.0
(b)
Data Data
P ediction
P ediction
Data
Prediction (b) HO
LU
O
R
HO
LU
O
R
HO
LU
O
R
Data-Data
Prediction - Prediction
Data - Prediction
(c)
HOMO
LUMO
Oxidation
Reduction
Train
Train
Train
Train
Test
Test
Test
Test H
Prediction - Prediction Data - Prediction (c) HOMO
LUMO
Oxidation
Reduction
7.5
5.0
2.5
0.0
2.5
Energy, eV/ Potential, V
Data
Prediction
(d) (d) Reduction Fig. 2
Forward property prediction using semi-supervised deep learning. RESULTS We first
evaluate the prediction accuracy of the trained deep network by
comparing the Pearsons correlation coefficient for the predicted
properties against the corresponding correlation coefficient for
the properties from the dataset (left subfigure of Fig. 2b). Middle
subfigure of the Fig. 2b shows Pearsons correlation coefficient
obtained for predicted properties. Comparison of left and middle
subfigures of Fig. 2b shows that the Pearsons correlation
coefficient obtained from data and predictions are close to each
other, demonstrating that the semi-supervised training approach
has enabled accurate learning of the correlations amongst the
properties. This learning capability is further investigated in the
rightmost subfigure of the Fig. 2b, where the Pearsons correlation
coefficient between the properties from the dataset and the
predictions is shown. As can be observed from the figure, the
correlation coefficient between properties from the dataset and
the corresponding properties predicted by the deep network are
close to 1. the correlation reaching ±0.46 for the LUMO. This ability to extract
the properties information only from the molecular structures
database is a key strength of our proposed approach. Similarly,
Pearsons correlation coefficient amongst properties is shown in
Fig. 2b. For the DFT predicted properties in the database (left
subfigure
of
Fig. 2b),
HOMO
energy
show
high
negative
correlation (−0.77) with oxidation potential, while, the LUMO
energy show high negative correlation (−0.85) with the reduction
potential. Moreover, HOMO energies have noticeable correlation
with all the other properties. SLAMDUNCS extract this correlation
by unsupervised training of GB-RBM using the properties database
W
i
d
f
i
d
i i
f pp
y
The error statistics is further explored in Supplementary Fig. 3. Supplementary Fig. 3a shows the distribution of mean absolute
error as a function of smiles length, while, the cumulative
distribution function (CDF) of absolute error in the property
prediction is shown in the Supplementary Fig. 3b. Absolute error
corresponding to CDF = 0.2 −0.9 is also tabulated in the figure. Absolute error less than 0.065 is achieved for 20% of the dataset,
whereas for 90% of the dataset, absolute error ≤0.427 eV/V is
achieved for all the properties. We investigate failed predictions of
our deep network in the Supplementary Fig. 3c, where we list five
molecules with the highest error for each property. For HOMO
prediction, highest error is observed for tetrapyradine. HOMO
prediction error is also high for molecules consisting nitro
functional group. RESULTS Highest error for LUMO prediction is observed
for diazene, whereas, error is also high for molecules containing
azide functional group. For oxidation potential prediction, higher
error is observed for molecules containing Si or F. For reduction
potential prediction, highest error is observed for cumene
hydroperoxide, however, a specific trend in prediction error with
respect to functional groups is not observed for reduction
potential prediction. y
p
g
g
p
p
We use a structure-properties dataset for supervised training of
the deep network. A total of 60000 data points are used for
training while remaining data points are used for testing. We first
evaluate the prediction accuracy of the trained deep network by
comparing the Pearsons correlation coefficient for the predicted
properties against the corresponding correlation coefficient for
the properties from the dataset (left subfigure of Fig. 2b). Middle
subfigure of the Fig. 2b shows Pearsons correlation coefficient
obtained for predicted properties. Comparison of left and middle
subfigures of Fig. 2b shows that the Pearsons correlation
coefficient obtained from data and predictions are close to each
other, demonstrating that the semi-supervised training approach
has enabled accurate learning of the correlations amongst the
properties. This learning capability is further investigated in the
rightmost subfigure of the Fig. 2b, where the Pearsons correlation
coefficient between the properties from the dataset and the
predictions is shown. As can be observed from the figure, the
correlation coefficient between properties from the dataset and
the corresponding properties predicted by the deep network are
close to 1. Distribution of the DFT calculated and the deep learning
predicted properties is shown in Fig. 2d using violin plots. We use
Kullback–Liebler (KL) divergence45 to quantify distance between
these distributions. For any two probability distributions p x
ð Þ and
q x
ð Þ, the KL divergence between the distributions p and q is given
by KL pjjq
½
¼
Z 1
1
p x
ð Þlog p x
ð Þ
q x
ð Þ
dx:
(5) (5) Figure 2c shows quantile-quantile plots for the predictions
using the trained network. Predictions are close to 45° line for all
the properties, demonstrating high prediction accuracy of the
trained network. The KL divergence is always non-negative, with KL pjjq
½
¼ 0 only if
the distributions p x
ð Þ and q x
ð Þ are same everywhere. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 RESULTS Properties information embedded in molecular structures is
extracted using unsupervised learning of DBN, as shown in a. The Pearson’s correlation coefficient between activation probability of each
node and the properties is shown in the figure. The DBN network used in this paper is shown in the rightmost part of the figure. Highest
correlation for each layer output is shown in the leftmost part of the figure. Similarly, correlation between the properties is shown in b. The
quantile-quantile plot for deep learning predicted properties is shown in c. Violin plot in d shows comparison of the PDF for predicted
properties and dataset. Kulback–Liebler (KL) divergence between the PDFs is also shown in the figure. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 P.M. Tagade et al. 6 Table 1. Error statistics for forward prediction. HOMO
LUMO
Oxidation
Reduction
Train
Test
Train
Test
Train
Test
Train
Test
R2
0.9184
0.9164
0.9506
0.9497
0.9109
0.9070
0.9203
0.9160
MAE (eV/V)
0.1407
0.1435
0.1464
0.1476
0.1496
0.1510
0.1782
0.1808
<0:05 (eV/V)
27.27
20.78
26.4
21.07
25.78
26.05
23.00
22.80
>0:5 (eV/V)
2.07
3.62
2.64
4.64
2.96
3.05
5.71
6.07
This table lists coefficient of determination (R2) and mean absolute error (MAE) of deep learning predictions. Percentage of molecules with absolute error
<0:05 eV/V and >0:5 eV/V are also reported in the table Table 1. Error statistics for forward prediction. reported in Pereira et al.38 This comparison is also shown in the
Supplementary Table 1. the correlation reaching ±0.46 for the LUMO. This ability to extract
the properties information only from the molecular structures
database is a key strength of our proposed approach. Similarly,
Pearsons correlation coefficient amongst properties is shown in
Fig. 2b. For the DFT predicted properties in the database (left
subfigure
of
Fig. 2b),
HOMO
energy
show
high
negative
correlation (−0.77) with oxidation potential, while, the LUMO
energy show high negative correlation (−0.85) with the reduction
potential. Moreover, HOMO energies have noticeable correlation
with all the other properties. SLAMDUNCS extract this correlation
by unsupervised training of GB-RBM using the properties database
We use a structure-properties dataset for supervised training of
the deep network. A total of 60000 data points are used for
training while remaining data points are used for testing. RESULTS During prediction, we predict the
DNN output by randomly dropping out each layer according to
the dropout probability. A large number of samples are collected
by repeating this procedure for pre-specified number of times. The mean, variance and other higher order moments can be
calculated from these samples to efficiently quantify uncertainty in
the deep learning predictions. If desired, SLAMDUNCS can admit
this quantified uncertainty as a model structural uncertainty in the dropout while training.46 The use of dropout in the DNN training
also allows us to quantify uncertainty in the model predictions.47
For instance, the forward network can be trained with a pre-
specified dropout probability. During prediction, we predict the
DNN output by randomly dropping out each layer according to
the dropout probability. A large number of samples are collected
by repeating this procedure for pre-specified number of times. The mean, variance and other higher order moments can be
calculated from these samples to efficiently quantify uncertainty in
the deep learning predictions. If desired, SLAMDUNCS can admit
this quantified uncertainty as a model structural uncertainty in the g
g
We then solve the inverse materials design problem from a
Bayesian inference perspective. A typical Bayesian inference solves
a Bayes theorem given by p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ;
(6) (6) RESULTS These
properties of KL divergence allows us to compare the closeness
and the difference between two probability distributions. For all
the properties, KL divergence is lower than 0.03, which shows that
our trained network has an ability to predict the distribution of
properties with high accuracy. Non-Gaussian nature of the
distributions, as is evident from the violin plot, exemplifies non-
linear nature of the structure-properties relationship. Low KL
divergence demonstrates that this non-linearity is also accurately
captured by the SLAMDUNCS. Error statistics for the properties prediction is summarised in
Table 1. As can be observed from Table 1, coefficient of
determination (R2) greater than 0.9 is achieved for all the
properties. For more than 20% of the molecules, deep learning
predicts all the properties with absolute error ≤0.05 eV/V, whereas,
the properties are predicted with absolute error more than
0.5 eV/V for ≤6:1% of the molecules. We have also compared
performance of our approach against against kernel ridge and
support vector regression. For comparison, we use same training
and testing data for all the three aproaches. In Supplementary
Table 1, we have summarised mean absolute error in prediction
for these three models. As can be observed from the table, our
approach performs better than both kernel ridge and support
vector regression. In Pereira et al.,38 authors have compared
accuracy of different sub-Angstrom level molecular descriptors
and machine learning algorithms for prediction of orbital energies. We have compared our results against the best performing model y
The deep network trained in this paper essentially learns the
mapping between the molecular structure and the corresponding
properties. Traditionally, the deep networks are treated as
deterministic models, however, the deep architectures proposed
in this paper have a capability to quantify uncertainty in the model
predictions. For instance when DBM is used as the deep network,
we use Gibbs sampling for property prediction. The Gibbs
sampling estimates complete probability distribution of the DBM
output, enabling uncertainty quantification in the property
predictions. When DNN is used as the deep network, we use P.M. Tagade et al. 7 definition of the likelihood that ensures low posterior probability
for the region with high prediction error. dropout while training.46 The use of dropout in the DNN training
also allows us to quantify uncertainty in the model predictions.47
For instance, the forward network can be trained with a pre-
specified dropout probability. Design of n- and p-type semiconductors for organic thin film
transistors Design of n- and p-type semiconductors for organic thin film
transistors First, we will use SLAMDUNCS to design n-type and p-type organic
semiconductor molecules. In organic thin film transistor (OTFT)
applications, the energy required to add an electron or remove an
electron determines whether a molecule is a suitable n-type or p-
type semiconductor, respectively.51 This necessitates that the
energy of the lowest unoccupied molecular orbital (LUMO) is low
enough for an n-type carrier so that it can accommodate an extra
electron easily or that the energy of the highest occupied
molecular orbital (HOMO) for p-type molecule is high enough that
an electron can be removed easily. Additionally, the device
performance is strongly dependent on the electronic structure of
the metal-semiconductor interface. It is important to choose a
metal contact with a work function that matches the highest
occupied
molecular
orbital
(HOMO)
or
lowest
unoccupied
molecular orbital (LUMO) of the organic semiconductor within a
few tenths of an electronvolt, such that desirable charge injection
efficiency and low operational voltage are achieved. While
previous computational design studies have focused mainly on
high throughput screening, here we demonstrate the applicability
of SLAMDUNCS to design new molecules with target orbital
energy values driven by the design intent. gy
y
g
In Fig. 3, we illustrate the design of n-type and p-type organic
semiconductors using SLAMDUNCS. Finding high performance n-
type organic semiconductors has been a challenge because, if the
LUMO of the n-type molecules is not low enough, the injected
electrons can be lost to species that can readily accept an electron
such as the silanol groups in the semiconductor-dielectric
interface or to O2/H2O molecules in ambient air.21 This leads to
poor performance of the electron transport layer as carriers are
annihilated. Therefore, the key to obtain stable n-type transport is
to find materials with low LUMO energies. Additionally, lowering
the LUMO level also helps in achieving better match with work
function of the electrode which can help make an Ohmic contact. As such, we seek as low an energy value as possible for the LUMO
to design an n-type OTFT device. Similarly, we desire as high an
energy value as possible for the HOMO to design a p-type OTFT
device. A typical OTFT device is shown is shown in Fig. 3a. The
figure also shows desirable energy levels for LUMO of a n-type
device (bottom left subfigure of Fig. p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ; However, the resultant posterior distribu-
tion is analytically intractable. Thus, we use a sampling based
approach to approximate the posterior distribution. In particular,
we use the Markov Chain Monte Carlo algorithm to sample from
the posterior distribution. We initiate the Markov Chain Monte
Carlo sampling from a random molecular structure, and use the
Metropolis-Hastings48,49 acceptance criterion to move the chain
forward. We discard pre-defined number of initial steps, known as
the equilibration period, and collect the desired number of
samples afterwards. The resultant Markov chain is known as the
ergodic Markov chain. Structures sampled after the equilibration
period provide a set of molecules with desired attributes. Key
advantage of Metropolis-Hastings algorithm is its ability to sample
from the posterior distribution without having to evaluate the
normalising constant of the probability distribution.50 This enables
conditional sampling, where we constrain the prior chemical
space to molecular structures with pre-defined attributes, like
minimum length of conjugated bonds or number of cyclic rings
present in the molecular structures, and sample from the resultant
posterior distribution. The SLAMDUNCS predicted LUMO energies for randomly
chosen 50 candidate molecular structures, that are extrinsic to
the training dataset, are validated using DFT. The comparison is
shown in Fig. 3d. As can be observed from the figure, SLAMDUNCS
predictions matches closely with the DFT predictions. For this
validation set, SLAMDUNCS predict the LUMO energies with mean
absolute error of 0.1534 eV against the DFT calculations, with the
maximum absolute error of 0.581 eV. It is important to note that,
SLAMDUNCS automatically discovered molecules from two well-
known n-type material classes: Rylene diimides and n-type
polyacene materials. For example, molecules 61, 72 and 80
reported in the Supplementary Table 2 are derivatives of perylene
diimide. Similarly, polyacene molecules modified by electron
withdrawing groups, diethylene thiol pentacene (molecule 45 in
Supplementary Table 2), dimethoxy anthracene dione (molecule
16
in
Supplementary
Table
2),
are
also
predicted. Unlike
conventional design approaches, our algorithm discovers these
molecules automatically without the need for searching in a
particular n-type material class. In addition to that, new molecules
previously not reported for n-type applications, have been
predicted. For example, molecules based on benzodithiophene
(BDT) have been previously reported as p-type materials.53 Our
inverse prediction has suggested adding electron withdrawing
aldehyde group to BDT to bring the LUMO level to −3.02 eV and
make it n-type. p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ; where p v
ð Þ is prior, p yjv
ð
Þ is likelihood and p vjy
ð
Þ is a posterior
probability distribution. For inverse materials design, we specify
the prior as probability that a given molecular structure is valid,
whereas, the likelihood is specified as probability of molecular e mean, variance and other higher order moments can be
culated from these samples to efficiently quantify uncertainty in
e deep learning predictions. If desired, SLAMDUNCS can admit
s quantified uncertainty as a model structural uncertainty in the
where p v
ð Þ is prior, p yjv
ð
Þ is likelihood and p vjy
ð
Þ is a posterior
probability distribution. For inverse materials design, we specify
the prior as probability that a given molecular structure is valid,
whereas, the likelihood is specified as probability of molecular
(a)
(d)
(e)
(c)
(b)
. 3
SLAMDUNCS predicted organic thin film transistors (OTFTs). A typical OTFT and desirable levels of orbital energies with respect to
ork function of the contact are shown in a. Orbital energies of the inverse predicted molecular structures for n-type and p-type OTFTs are
own in b, c, respectively. The figure also shows joint probability contours of the orbital energies in the background. d SLAMDUNCS
edicted LUMO energies are compared against the DFT calculations for 50 candidate molecular structures for n-type OTFT. Similarly, e
mpares SLAMDUNCS predicted and DFT calculated HOMO energies of 50 candidate p-type OTFT molecular structures. blished in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences
npj Computational Materials (2019) 127 (a)
(c)
(b) (b) (a)
(c)
(b) (b) (a) (a)
(d)
(c)
(b) (d)
(c) (c) (d) (e) (e) (e) Fig. 3
SLAMDUNCS predicted organic thin film transistors (OTFTs). A typical OTFT and desirable levels of orbital energies with respect to
work function of the contact are shown in a. Orbital energies of the inverse predicted molecular structures for n-type and p-type OTFTs are
shown in b, c, respectively. The figure also shows joint probability contours of the orbital energies in the background. d SLAMDUNCS
predicted LUMO energies are compared against the DFT calculations for 50 candidate molecular structures for n-type OTFT. Similarly, e
compares SLAMDUNCS predicted and DFT calculated HOMO energies of 50 candidate p-type OTFT molecular structures. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 P.M. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ; Tetrazole bonded to the nitrogen of Isoindole
(molecule 37) are also another n-type molecule discovered by our
algorithm. The predictions of n-type molecules in this work are
based on the LUMO energy, which is only a necessary condition
for easy electron injection and stable operation. However, in
practice, high electron mobility constitutes the other important
criterion for a molecule to be an attractive n-channel material. We
have not trained SLAMDUNCS for correlation between molecular
structure and mobility since mobility is determined by both
molecular structure and the crystal structure of the organic
molecule and thus it is computationally complex. In the Marcus
theorem
formalism,
widely
adopted
for
calculating
charge
transport in organic semiconductor crystals, the mobility is related
to molecular structure through reorganisation energy and to the
crystal structure through transfer integral as well as number of
nearest neighbours and the hopping distance.54 We foresee that
with adequate data relating molecular structure to reorganisation
energy and transfer integral to train the forward model in
SLAMDUNCS, inverse prediction of molecules with target mobility
values is possible. Even with the capability to predict molecules
with target LUMO values is very useful in considering alternatives
to existing options for n-type materials. d
k
l
l
h p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ; Tagade et al. 8 candidate molecule can accommodate an extra electron extracted
from the source. To generate a set of valid molecular structures,
we simulated 1000 parallel ergodic Markov chains initialised from
randomly chosen structures. Figure 3b shows orbital energies of
the predicted structures. Joint probability contours are also shown
in the background. As can be observed from the figure, a vast
majority of candidate n-type organic semiconductors are expected
to have LUMO energies in the −3.1 to −3.4 eV range with the
corresponding HOMO energies in −5.5 to −7.0 eV range. A list of
predicted molecular structures is provided in Supplementary
Table 2. Although a large number of molecular structures are
generated by simulating a number of parallel ergodic Markov
chains, only 100 structures are reported in the table for brevity. Validity of the listed structures is confirmed by optimising the
structures in Avogadro52 from SMILES representation. We also
performed an automated search to check if the molecule is
present in the PubChem database. properties satisfying the desired attributes. We learn the prior on
molecular structures database using RBM, while, we use the
structures-property correlation learned using the deep network to
specify the likelihood. However, the resultant posterior distribu-
tion is analytically intractable. Thus, we use a sampling based
approach to approximate the posterior distribution. In particular,
we use the Markov Chain Monte Carlo algorithm to sample from
the posterior distribution. We initiate the Markov Chain Monte
Carlo sampling from a random molecular structure, and use the
Metropolis-Hastings48,49 acceptance criterion to move the chain
forward. We discard pre-defined number of initial steps, known as
the equilibration period, and collect the desired number of
samples afterwards. The resultant Markov chain is known as the
ergodic Markov chain. Structures sampled after the equilibration
period provide a set of molecules with desired attributes. Key
advantage of Metropolis-Hastings algorithm is its ability to sample
from the posterior distribution without having to evaluate the
normalising constant of the probability distribution.50 This enables
conditional sampling, where we constrain the prior chemical
space to molecular structures with pre-defined attributes, like
minimum length of conjugated bonds or number of cyclic rings
present in the molecular structures, and sample from the resultant
posterior distribution. properties satisfying the desired attributes. We learn the prior on
molecular structures database using RBM, while, we use the
structures-property correlation learned using the deep network to
specify the likelihood. npj Computational Materials (2019) 127 Design of n- and p-type semiconductors for organic thin film
transistors We can
seek ambipolar organic semiconductors in this region, if applica-
tion demands. All the predicted candidate molecular structures for
p-type organic semiconductors are listed in Supplementary Table
3. We validate the SLAMDUNCS prediction for randomly selected
50 structures that are extrinsic to the training dataset. Figure 3e
compare SLAMDUNCS predictions with the DFT calculations for
these 50 molecular structures. As can be observed from the figure,
SLAMDUNCS predicted HOMO energies match closely with the
DFT calculations. For this validation set, SLAMDUNCS predicts
HOMO energies with mean absolute error of 0.2021 eV against the
DFT calculations, with maximum absolute error of 0.455 eV. One strategy used to overcome this challenge is to increase the
open-circuit
voltage,
by
increasing
the
difference
between
ionisation potential of the donor and the electron affinity of the
acceptor. While this leads to decrease in driving force for exciton
separation at the donor:acceptor interface due to decreasing
energy difference between LUMO of donor and LUMO of the
acceptor, it has been demonstrated that as low as 0.1 eV LUMO
offset between donor and acceptor is capable of efficient exciton
separation.57 As new donor molecules are considered for BHJ, the
acceptor molecules have to be energy level matched so that open
circuit voltage is maximised, while still providing a driving force for
the exciton separation. One strategy used to overcome this challenge is to increase the
open-circuit
voltage,
by
increasing
the
difference
between
ionisation potential of the donor and the electron affinity of the
acceptor. While this leads to decrease in driving force for exciton
separation at the donor:acceptor interface due to decreasing
energy difference between LUMO of donor and LUMO of the
acceptor, it has been demonstrated that as low as 0.1 eV LUMO
offset between donor and acceptor is capable of efficient exciton
separation.57 As new donor molecules are considered for BHJ, the
acceptor molecules have to be energy level matched so that open
circuit voltage is maximised, while still providing a driving force for
the exciton separation. p
Here, we demonstrate the inverse prediction capability of
SLAMDUNCS to predict non-fullerene organic acceptors for use
with P3HT (Poly(3-hexylthiophene-2,5-diyl)) donor, as shown in
Fig. 4a. To determine the desired target attributes of the acceptor,
we first calculated orbital energies of P3HT using DFT. Design of n- and p-type semiconductors for organic thin film
transistors 3a), and HOMO of a p-type
device (bottom right subfigure of Fig. 3a). For n-type OTFT, we
seek organic molecules with LUMO < 3:0 eV, such that the For p-type semiconductors, we seek organic molecules with
HOMO energies > 5:6 eV that can allow efficient release of
electron to the source, as shown in bottom right subfigure of the
Fig. 3a. The
candidate
p-type
organic
semiconductors,
as
predicted by SLAMDUNCS, are expected to have HOMO energies npj Computational Materials (2019) 127 P.M. Tagade et al. 9 MDUNCS predicted organic solar cells. A typical solar cell and the desired trend in orbital energies is shown in a. Orbital energies
rse predicted molecular structures are shown in b. The figure also shows joint probability contours of the orbital energies in the
d. In c, SLAMDUNCS predicted LUMO energies are compared against the DFT calculations for 50 candidate molecular structures. Fig. 4
SLAMDUNCS predicted organic solar cells. A typical solar cell and the desired trend in orbital energies is shown in a. Orbital energies
of the inverse predicted molecular structures are shown in b. The figure also shows joint probability contours of the orbital energies in the
b
k
d I
SLAMDUNCS
di
d LUMO
i
d
i
h
DFT
l
l
i
f
50
did
l
l Fig. 4
SLAMDUNCS predicted organic solar cells. A typical solar cell and the desired trend in orbital energies is shown in a. Orbital energies
of the inverse predicted molecular structures are shown in b. The figure also shows joint probability contours of the orbital energies in the
background. In c, SLAMDUNCS predicted LUMO energies are compared against the DFT calculations for 50 candidate molecular structures. Fig. 4
SLAMDUNCS predicted organic solar cells. A typical solar cell and the desired trend in orbital energies is shown in a. Orbital energies
of the inverse predicted molecular structures are shown in b. The figure also shows joint probability contours of the orbital energies in the
background. In c, SLAMDUNCS predicted LUMO energies are compared against the DFT calculations for 50 candidate molecular structures. in a narrow band of −5.6 to −5.2 eV, whereas, the LUMO energies
have a comparatively wider band of 0 V to 3 eV (see Fig. 3c). Note that there is a small region of overlap between the candidate
n-type and p-type organic semiconductors (see Fig. 3b, c). Design of n- and p-type semiconductors for organic thin film
transistors DFT
predicted LUMO energy of −2.52 eV for P3HT, thus we seek
acceptors with LUMO energies in the range −2.62 to −2.72 eV
that corresponds to the LUMO energies lower by 0.1–0.2 eV as
compared to P3HT. We predicted a large number of candidate
molecules by simulating 1000 ergodic Markov chains with
equilibration period of 30000 samples. Distribution of orbital
energies of the predicted molecules is shown in the Fig. 4b. This
desired range of LUMO energies also bound the HOMO energies
to a narrow band of −5.5 to −7.0 eV (see Fig. 4b), ensuring the
hole transfer from acceptor to the donor. A list of predicted
candidate organic molecules for OSC is provided in Supplemen-
tary Table 4. For brevity, we have only reported randomly selected
100 molecular structures. We performed first principle DFT
calculations for the first 50 molecular structures listed in the
Supplementary Table 4. In Fig. 4c, the DFT calculated LUMO
energies are compared against the SLAMDUNCS predictions. The
comparison shows high prediction accuracy of the SLAMDUNCS,
with mean absolute error of 0.2140 eV against the DFT calcula-
tions, whereas, the maximum absolute error is 0.6877 eV. Unlike Examples of p-type molecules SLAMDUNCS predicted outside
of the training database include amino modified acenaphthalene
(molecule 26 in Supplementary Table 3) and bromine modified
biphenyl dicarbonitrile (molecule 87 in Supplementary Table 3). It
is interesting to point out that, SLAMDUNCS has correctly learned
that even though the parent biphenyl dicarbonitrile has a HOMO
level (6.49 eV) lower than the cut-off value for p-type molecules,
bromine modification brings it −5.35 eV that is within the
target range. npj Computational Materials (2019) 127 Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences Design of non-fullerene acceptor molecules for bulk-
heterojunction organic solar cells Here, we demonstrate the inverse prediction capability of
SLAMDUNCS to predict organic structures that have reduction
potential lower than the anode and an electrochemical window of
4.8 V or better. These desired attributes ensure stability of the
electrolyte across full operating range of the lithium-ion cell, as
shown in Fig. 5a. Thus, SLAMDUNCS is used to predict molecules
with the reduction potential <−3.35 V against standard hydrogen
electrode. Predicted candidate molecular structures are listed in
Supplementary Table 5. Figure 5b shows the distribution of redox
potentials for the predicted molecular structures. Although the
marginal distributions of the redox potentials is unimodal, the
joint distribution shows two distinct modes, first mode near
reduction potential of −3.7 V and another mode near the
reduction potential of −4.15 V. We have validated the deep
learning prediction of redox potential for 50 randomly selected
candidate molecules. The DFT calculation and deep learning
prediction of the reduction potential is compared in Fig. 5c. For
the candidate structures considered for validation, SLAMDUNCS
predicts the reduction potential with maximum error of 0.5918 V
and mean absolute error of 0.2004 V. High redox stable electrolyte design
Finally, we consider the practical problem of designing redox-
stable electrolytes for lithium-ion batteries. The goal is to design
new organic solvents that have both reduction and oxidation
stability against the reducing potential at the anode and the
oxidising potential at the cathode, respectively.23,58,59 The redox
stability is determined by redox potential, which is determined by
how easy it is to inject/remove an electron from the molecule and
is an important parameter for the design of new electrolytes. Previously, electrolyte design relied on making minor tweaks, such
as replacing an ethylene based core with a propylene based core,
since electrochemical properties are not widely reported for all
molecules in chemical databases. Design of non-fullerene acceptor molecules for bulk-
heterojunction organic solar cells As a second demonstration, we use SLAMDUNCS to design non-
fullerene acceptors for bulk-heterojunction (BHJ) organic solar
cells (OSC). The discovery of BHJ by Heeger et al.55 thrust OSCs to
the forefront of photovoltaic materials research since this concept
provided efficient route for exciton dissociation in many donor-
acceptor systems by drastically reducing the distance travelled by
exciton for separation.56 This has led to devices with high
photovoltaic external quantum efficiencies and high photocurrent. However, this came at the cost of significant photovoltage losses. npj Computational Materials (2019) 127 shed in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. 10 Fig. 5
SLAMDUNCS predicted organic molecules for lithium-ion battery electrolytes. A typical lithium-ion battery and desired trend in
electrolyte redox potentials is shown in a. Redox potentials of the inverse predicted molecular structures are shown in b. Joint probability
contours of the redox potentials is also shown in the background. Fifty molecules are randomly selected for validation. Redox potential for the
selected molecules is obtained using DFT. In c, the DFT calculated reduction potential are compared against the deep learning predictions. Fig. 5
SLAMDUNCS predicted organic molecules for lithium-ion battery electrolytes. A typical lithium-ion battery and desired trend in
electrolyte redox potentials is shown in a. Redox potentials of the inverse predicted molecular structures are shown in b. Joint probability
contours of the redox potentials is also shown in the background. Fifty molecules are randomly selected for validation. Redox potential for the
selected molecules is obtained using DFT. In c, the DFT calculated reduction potential are compared against the deep learning predictions. the previous example, where organic semiconductor molecules of
LUMO values below a cut-off or HOMO values above a cut-off
were sought, here organic acceptors were sought that have LUMO
values in a very narrow range and yet SLAMDUNCS was able to
predict several candidate acceptor molecules is noteworthy. For
example, SLAMDUNCS has interestingly suggested an adaman-
tane structure modified by nitrosol and Cl (molecule 27 in
Supplementary Table 4) as one of the candidates. It is important to
note
that
adamantane,
which
is
the
smallest
diamondoid
molecule, is not a very good electron acceptor. However, the
addition of nitroso group and Cl brings the LUMO level down just
enough to be in the target range. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. 11 molecular structures extrinsic to the database without using any
heuristic or combinatorial rules, enabling fully automated design
of molecules for target applications. The proposed approach is
efficient and robust, we expect that it can be extended to directly
design other complex materials as well, as long as a reasonably
diverse dataset for learning structure-property relationship is
available and that the property-relevant structural information can
be digitised. This dataset can be purely experimental, purely based
on physics-based calculations or a combination of the two. Currently, our forward model is trained on organic molecular
database, thus, our inverse model is applicable to only problems in
the organic space. Within the organic space also, it is trained to
inverse predict molecules for a small set of properties such as
HOMO, LUMO and Redox potentials. However, same approach can
be used to train the model for other properties of the organic
molecules. Additionally, the same inverse design architecture can
be extended to train for predictions in the inorganic space, by
properly choosing the fingerprints. Note that an alternative to the
SMILES representation is required for structure encoding in the
inorganic space. Table 2. The table lists number of predicted molecules that are not
present in training dataset or not listed in the PubChem. n-type
OTFT
p-type
OTFT
Solar
cell
Stable
electrolyte
Not in PubChem
25
31
34
27
Not in training dataset
42
36
31
42
% of new molecules
67
65
70
69 that are outside the training dataset or not listed in the PubChem. If the molecule is not present in the PubChem database, distance
between the molecule and training dataset is reported using
Levenshtein (λ) and Ratcliff-Obershelp (ρ) distance. Minimum
distance between molecular structure and the training dataset is
reported in the table. As can be observed from the table, out of
the 400 predicted molecules, 67:75% of the molecules are new. p
,
We have validated the properties prediction by SLAMDUNCS for
50 candidate molecular structures for each application. Although
highly accurate, the prediction error is found to be high for some
of the candidate structures. In the Supplementary Fig. 4 of
Supplementary Information, we investigate the candidate struc-
tures with highest error for each application. Top row of the figure
shows initial structure for each application, while the bottom row
shows the corresponding structure optimised using DFT. Validation To summarise, we have developed a deep learning based inverse
prediction framework, SLAMDUNCS, for design of materials with
target properties. In particular, we apply Bayesian inference for
design of organic molecules with desired properties in three
representative applications: high stability electrolytes for lithium-
ion batteries, n- and p-type organic semiconductors for OTFTs and
small organic acceptor molecules for use in organic solar cells. We
successfully generated molecules not in the database with target
properties, a small subset of which were validated with first
principles simulations. This is primarily enabled by our use of
Bayesian framework that allowed us to obtain a set of molecular
structure as against single structures predicted by the state of the
art optimisation methods. Moreover, our use of RBM to encode
the fundamental chemical rules allowed us to generate the A small subset of SLAMDUNCS predicted molecular structures that are not
present in the reference dataset were validated using the first principles
calculations. The methodology outlined for reference dataset creation was
used for validating the predicted molecular structures using the first
principles calculations. Reference dataset creation The molecular properties database was generated using density functional
theory (DFT) calculations on 77,547 molecules randomly selected from the
PubChem database36 with compound identifier numbers between 1 and
136200. The resultant database is a well representative mixture of small
and large molecules, as well as long chains, as can be seen from the
distribution of number of molecules against the SMILES length (see
Supplementary Fig. 2a). All the first principles calculations were performed
using the Gaussian 09 software package62 with B3LYP functional and 6-311
+G (d,p) basis sets with the polarised continuum model63 as a solvation
environment with an effective dielectric constant of 28.86. Unconstrained
optimisation was used to optimise all the geometries in this solvation
environment. The oxidation and reduction reactions were respectively
simulated by removing or adding an electron from the neutral molecule. The oxidation potential was obtained from the difference in the total free
energies of the oxidised and neutral molecules. The reduction potential
was similarly calculated from the difference in the total free energies of the
reduced and neutral molecules. Each of these applications have additional secondary criteria,
such as transport properties, solvation energy, and parameters
that affect processing that need to be considered while designing. The candidate structures can be down-selected considering these
additional properties before experimentally realising them. Alter-
natively, SLAMDUNCS can be used to design molecules by
simultaneously
considering
multiple
target
properties
in
a
coherent and holistic approach. For p-
type OTFT, initial and optimised structures are similar (see middle
column of the Supplementary Fig. 4), however, the structure
contains chlorine atom attached to a Benzene ring. This presence
of chlorine atom results in a higher error in HOMO prediction. For
n-type OTFT, initial structure obtained from the SMILES represen-
tation contains a ring of alternate carbon and oxygen atoms
attached to nepthanol, however when optimised using DFT,
consists of two side chains formed by breaking of the ring. Right
column of the Supplementary Fig. 4 shows the candidate structure
for redox-stable organic electrolyte that has highest error in
reduction potential prediction. As can be observed from the
figure, the candidate structure consists of two rings of four atoms. When optimized for anion using DFT (bottom structure of the
right column in Supplementary Fig. 4), both the four atom rings
break due to high strain. As the deep learning algorithms of
SLAMDUNCS are not trained for capturing this behaviour, error in
reduction potential prediction is high for this candidate structure. Each of these applications have additional secondary criteria,
such as transport properties, solvation energy, and parameters
that affect processing that need to be considered while designing. The candidate structures can be down-selected considering these
additional properties before experimentally realising them. Alter-
natively, SLAMDUNCS can be used to design molecules by
simultaneously
considering
multiple
target
properties
in
a
coherent and holistic approach. While we have applied SLAMDUNCS for designing organic
molecules, it can be readily adapted to design inorganic materials
such as semiconductors, piezoelectric and thermoelectric materi-
als where one would need to learn on relationship between crystal
structure and properties. Our work clearly demonstrates that a fast
prediction of molecules/materials with target properties is indeed
possible. We strongly believe that it can be easily extended to
other types of materials and a diverse set of properties, including
thermal, electrical and optical performance. Moreover, SLAM-
DUNCS can be used for inverse prediction of molecules with
multiple target properties simply by using multivariate probability
distribution to quantify the likelihood function. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 Design of non-fullerene acceptor molecules for bulk-
heterojunction organic solar cells To address this issue, first
principles calculation of redox potentials was used to screen
molecules with the goal of populating the database with redox
potentials to accelerate design of organic electrolytes with high
voltage stability window for energy storage applications in
consumer electronics and electric vehicles.60 Further to this,
artificial neural network based forward prediction tools for
predicting the oxidation and reduction potentials of organic
compounds existing in the massive organic compound database,
PubChem was developed by one of the co-authors.61 Examples of new electrolyte additive molecules predicted by
SLAMDUNCS include phosphoryl attached to two methyl pentane
groups (molecule 55 in Supplementary Table 4) and dipropyl
iodomethylcarbonate (molecule 51 in Supplementary Table 5),
both of which are not in the pubchem database. Additionally,
SLAMDUNCS has suggested many other molecules that are in the
pubchem database but have not been considered for the
electrolyte application. For example, dimethyl silyl carbonate
(molecule 26 in Supplementary Table 5) and 2,4,6 triisopropyl-
1,3,5-trioxane are two interesting suggestions made by the inverse
prediction algorithm. For the representative problems considered above, SLAM-
DUNCS predicted a large number of molecular structures extrinsic
to the database that meet the primary essential criterion as
required by the target application. For each application, 100
predicted molecules are listed in the Supplementary Tables 2–5 of
the Supplementary Information. Table 2 lists number of molecules npj Computational Materials (2019) 127 P.M. Tagade et al. P.M. Tagade et al. 12 The posterior distribution p vjy
ð
Þ probabilistically encompasses a chemical
space of molecular structures with the desired target properties. We
sample from the posterior distribution by simulating an ergodic Markov
chain with p vjy
ð
Þ as a stationary distribution. We initiate the Markov chain
from a random structure v0 and use following steps to simulate the
Markov chain: structure, while, the likelihood is specified in terms of the expert opinion
on the desired attributes of a given application. Subsequently, we simulate
an ergodic Markov chain with the posterior as a stationary distribution, this
provides us a set of valid molecules with desired attributes. In the
following, we provide a detailed step-by-step description of the proposed
deep learning Bayesian framework. (a) Sample a candidate structure v from a proposal distribution
q vjvi
ð
Þ. Molecular fingerprinting. We obtain a canonical SMILES representation of
known molecules from the PubChem database. In this paper, we have only
considered the molecules with SMILES length less than or equal to 100
characters. For the molecules with a SMILES representation of less than 100
characters, we concatenate the SMILES string with null characters to obtain
a resultant 100 character SMILES string. Each SMILES character is
subsequently converted to an equivalent 8 bit binary representation, and
the resultant binary vector of length 800 is used as a molecular fingerprint. q
j
ð
Þ
(b) Compute the prior probability p v
ð
Þ using Eq. 8. y
ð
Þ
g
(c) Use deep learning to predict the properties of the candidate
structure v. Obtain the likelhood p yjv
ð
Þ by probabilistically comparing
predicted properties with the target properties y. p
p
p
g
p
p
y
(d) Compute the Metropolis-Hastings acceptance criterion α ¼ p yjv
ð
Þp v
ð
Þq vijv
ð
Þ
p yjvi
ð
Þp vi
ð Þq vjvi
ð
Þ :
(10) (10) Specification of prior using restricted Boltzmann machine. Molecular
fingerprinting using the binary variables allow us to represent the
molecules as a Bernoulli random vector, v. The molecular structures
database, represented using Bernoulli random vectors, is treated as
samples from a multivariate probability distribution p v
ð Þ. We quantify p v
ð Þ
using restricted Boltzmann machine (RBM). RBM is a two layer network
with a probability distribution given by The candidate structure v is accepted with probability α. REFERENCES Likelihood estimation using deep learning. SLAMDUNCS admit expert
opinion on desired molecular attributes to define the likelihood function
p yjv
ð
Þ, where y is a vector of target properties. For a given molecular
structure v, the likelihood function quantifies probability of the molecule
possessing target properties. Thus, complete specification of the likelihood
function require prediction of molecular properties of the structure v. We
have used semi-supervised deep learning for molecular properties
prediction. We have constructed the deep network using a stack of pre-
trained RBMs. 1. Jain, A., Shin, Y. & Persson, K. A. Computational predictions of energy materials
using density functional theory. Nat. Rev. Mater. 1, 15004 (2016). 1. Jain, A., Shin, Y. & Persson, K. A. Computational predictions of energy materials
using density functional theory. Nat. Rev. Mater. 1, 15004 (2016). 2. Ceder, G. Opportunities and challenges for first-principles materials design and
applications to li battery materials. MRS Bull. 35, 693–701 (2010). 3. Dingreville, R., Karnesky, R. A., Puel, G. & Schmitt, J.-H. Review of the synergies
between computational modeling and experimental characterization of materials
across length scales. J. Mater. Sci. 51, 1178–1203 (2016). 4. Le, T. C. & Winkler, D. A. Discovery and optimization of materials using evolu-
tionary approaches. Chem. Rev. 116, 6107–6132 (2016). We have first defined a DBN created by stacking three RBMs. The visible
layer of the DBN consists of 800 nodes, whereas, the hidden layers are
defined using 800, 500 and 200 nodes. We have used a greedy algorithm
to train the DBN, where we use the molecular structures database to train
the first RBM and train the subsequent RBMs using prediction of the
previous RBM as the training data. All the RBMs are trained for 5000 epochs
using momentum algorithm with initial momentum of 0.5 for first 5 epochs
and the momentum of 0.9 for the remaining epochs. We start the training
with learning rate 1e02 and uniformly reduce the learning rate to 1e05. Similarly, we have pre-trained the top GB-RBM using the properties
database. We have used 20 Gibbs sampling steps and fixed learning rate of
1e05 for pre-training the GB-RBM. 5. Ramprasad, R., Batra, R., Pilania, G., Mannodi-Kanakkithodi, A. & Kim, C. Machine
learning in materials informatics: recent applications and prospects. NPJ Comput. Mater. 3, 54 (2017). 6. Tagade, P. M. et al. CODE AVAILABILITY The codes are publicly available at https://github.com/piyushtagade/SLAMDUNCS. Received: 14 January 2019; Accepted: 25 November 2019; p vjy
ð
Þ / p yjv
ð
Þp v
ð Þ: DATA AVAILABILITY The
dataset
used
in
this
work
is
publicly
available
at
https://github.com/
piyushtagade/SLAMDUNCS. The
dataset
used
in
this
work
is
publicly
available
at
https://github.com/
piyushtagade/SLAMDUNCS. (8) For all the experiments presented in this paper, the RBM is constructed
using 800 visible nodes and 500 hidden nodes. This RBM is trained using a
contrastive-divergence algorithm with a single Gibbs sampling step (CD1
algorithm). We have used momentum algorithm64 to implement the
stochastic gradient descent for minimisation of loss in the CD1 algorithm. We use 80% of the molecular structures database and train the RBM for
5000 epochs. We start the training with learning rate 1e02 and uniformly
reduce the learning rate to 1e05. Momentum is fixed at 0.5 for initial 5
epochs, which is subsequently increased to 0.9 for the remaining epochs. P.M. Tagade et al. To define the proposal distribution, we multiply the weights and biases
of the RBM trained on the molecular structures database by a random
variable β 2 ½0; 1. We use this heated RBM as a proposal distribution. We
use alternate Gibbs sampling to obtain a candidate molecular structure v
from this proposal distribution. We start sampling after equilibration period
of 30000 steps to ensure appropriate ergodicity of the Markov chain. p v; h
ð
Þ ¼ exp vTWh þ bv þ ch
ð
Þ
Z
;
(7)
where h denotes hidden layer. We marginalise over h to obtain
p v
ð Þ ¼ expðbvÞ 1 þ exp vTW þ c
ð
Þ
ð
Þ
Z
:
(8) (7) (7) Details of the deep learning Bayesian framework Details of the deep learning Bayesian framework The molecular structures with desired attributes are predicted using a
deep learning Bayesian framework. At the core of the proposed framework
is a Bayesian inference methodology that provide a completely rational
data assimilation framework using the Bayes theorem. Unlike traditional
data assimilation applications, SLAMDUNCS uses Bayesian inference for
predicting valid molecular structures with desired attributes. In this
framework, the prior is defined as a probability of validity of a molecular partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences npj Computational Materials (2019) 127 P.M. Tagade et al. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. 13 14. D’Agostini, G. Bayesian reasoning in high-energy physics: principles and applica-
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implemented Bayesian inference and deep learning algorithms. M.S.P. created the
molecular properties database. P.M.T. carried out the calculations. S.P., S.P.A., and
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material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/. Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences © The Author(s) 2019 ADDITIONAL INFORMATION 44. Silver, D. et al. Mastering the game of go with deep neural networks and tree
search. Nature 529, 484 (2016). Supplementary information is available for this paper at https://doi.org/10.1038/
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a simple way to prevent neural networks from overfitting. J. Mach. Learning Res. 15, 1929–1958 (2014). Reprints and permission information is available at http://www.nature.com/
reprints npj Computational Materials (2019) 127 Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences P.M. Tagade et al. P.M. Tagade et al. P.M. Tagade et al. 14 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
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Unsteady Blade Element-Momentum Method Including Returning Wake Effects
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Journal of Aerospace Technology and Management
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cc-by
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Unsteady Blade Element-Momentum Method
Including Returning Wake Effects Cláudio Tavares Silva1,2, Maurício Vicente Donadon1 ABSTRACT: The wind energy research has grown substantially
in the past few years, considerably fostered by the pursuit
for a clean and sustainable energy source. Improvements on
the design methods are increasingly needed. The purpose
of this research is to investigate the use of the Loewy´s lift
deficiency function (LDF), also named Returning Wake Model,
coupled with a non-stationary Blade Element-Momentum
Method (BEM). The LDF simulates the influence of the wake
behind the wind turbine on its capacity to generate power. It is
expected that this model reduce the dependency of the several
empirical parameters necessary in other wake models which
are currently used. Aiming to validate the results obtained in
this new approach they are compared with those provided by
commercial computational software and they have proven to
be very consistent. It is concluded that the method is feasible
to be used as an efficient design and optimization tool of upwind
horizontal axis wind turbine blades. doi: 105028/jatm.v5i1.163 doi: 105028/jatm.v5i1.163 1.Instituto Tecnológico de Aeronáutica – São José dos Campos/SP – Brazil 2.Universidade Tecnológica Federal do Paraná – Curitiba/PR – Brazil
Author for correspondence: Cláudio Tavares Silva | Praça Marechal Eduardo Gomes, 50 – Vila das Acácias | CEP 12228-900 São José dos Campos/SP – Brazil |
E-mail: ctavares@ita.br
Received: 19/09/12 | Accepted: 07/02/13 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 don, M.V. using different settings on wind speed, rotational speed
and pitch angle. The method couples the momentum
theory with local events taking place at the actual blades. The blade is analyzed as a number of independent stream
tubes. In each one, the induced velocity is calculated by
performing the conservation of momentum, and the
aerodynamic forces are found with the 2D aerodynamic
theory and airfoil data. The stream tubes are discretized
into N annular elements. The lateral boundary of the
elements does not admit any flow across them. Some
assumptions are made for the annular elements: no radial
dependence, that is, one element cannot be affected by the
others; the forces from the blade on the flow are constant
in each annular element, corresponding to a rotor with a
number of blades. these semi-empirical engineering models. Furthermore, an
analytical resolution results in a more elegant mathematical
solution, computationally feasible and sufficiently fast to
provide blade design optimization. The induced effects generated by the shed wake have
been modeled using two general approaches: dynamic inflow
methods and vortex wake methods. The principles of the dynamic inflow approach are
attributed to Carpenter and Freidovich (1953). The idea
is to consider the unsteady aerodynamic lag of the inflow
development over the rotor disk in response to changes in
blade pitch inputs of changes in rotor thrust. They are written
in the form of ordinary differential equations, with a time
constant (or constants) representing the dynamic lag in the
build-up of the inflow. One of its less satisfying aspects is that
time constants must be obtained by experimental calibrations. A
correction
known
as
Prandtl´s
tip
loss
factor (Glauert, 1935) is introduced to correct this latter
assumption in order to compute a rotor with a finite
number or blades. Vortex wake models are based on the assumption of
an incompressible potential flow, with all vorticity being
assumed concentrated within vortex filaments (which in the
case of rotors require a coupling to the blade lift distribution). The induced velocity field can be determined through the
application of the Biot-Savart low. Different approaches
are encompassed ranging from prescribed to free vortex
techniques. The prescribed wake models are strictly applicable
when the operating conditions are nominally steady-state, i.e.,
in a steady wind. The free vortex methods have fewer potential
limitations. They have been widely developed to be used in
helicopters rotor analyses. don, M.V. Free vortex methods are based
on discretized, finite-difference representation of the
governing equations for the wake, and when solved, they
track the evolution of discrete vortex elements through the
flow. The number of discrete elements per vortex filaments
can be very large, making the tracking process memory
intensive and computationally demanding. A relative velocity Vrel seen by a blade section is a
combination of axial velocity V0(1–a), in which a is the
axial induction factor, and the tangential velocity (1–a')
ϖr, where a' is the radial induction factor at the rotor
plane (Fig. 1). The angle θ is the local pitch angle of the
blade element, i.e., the local angle between the chord
and the plane of rotation. It is a combination of the pitch
angle, measured between the tip chord, the rotor plane
and the twist of the blade, relative to the tip chord. ϕ is
the flow angle, measured between the plane of rotation
and the relative velocity. The local angle of attack α is
obviously found. Figure 1. Velocities at rotor plane. rel
V
(
)
0 1
V
a
(
)
1
r
a
+
Rotor plane Leishman (2002) presents more details about dynamic
inflow models and vortex wake models. INTRODUCTION Wind turbines operate in a hostile environment in which
strong flow fluctuations, mainly due to the nature of the wind,
can produce high and variable loads on its components. These
loads, combined with the elastic behavior of the turbine
structural components, compromise the overall energy
generation efficiency and cannot be neglected during the
design phase. The need for experimental and computational
approaches to investigate the behavior of unsteady loads
produced on a wind turbine blade has grown in proportion to
the growing nominal power and size of the actual horizontal
axis wind turbines. The objective of this work is to evaluate the mathematical
model of the Loewy´s lift deficiency function (LDF) to be
included in a computational package enabling the optimal
design of horizontal axis wind turbine blades. This model
allows the inclusion of non-stationarities as well as an
approximation of the effects of the downwind wake dynamics
on turbine aerodynamic performance. This inclusion
of non-stationarities and the effects of the downwind wake
allow more realistic results for the aerodynamic loads than
the simple Blade Element-Momentum (BEM) method, and
consequently leads to better designs of structural components. KEYWORDS: Blade element method, Loewy´s lift deficiency
function, Non stationary BEM, Wind turbine, Wind turbine
blade design, Returning wake model. The overwhelming majority of computer packages and
procedures actually use semi-empirical models to represent
the influence of the wake in the overall aerodynamic
performance of a wind turbine blade. The LDF (Loewy, 1957)
is an analytical solution based on the classical Theodorsen
theory (Theodorsen, 1935). The use of an analytical model
reduces the dependence on experimental tests for the
adjustment of empirical parameters needed to calibrate J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. 28 y
g
g P
rb
r
x2
y2
y3
3
y4
z
z
z
2
x3
x4
4
tilt
cone
rt
rs
Figure 2. Coordinate systems. The algorithm for the BEM model can be summarized
as the sequence of steps that follows. Since different
control volumes are assumed to be independent, each
blade element can be treated separately and the solution
at one radius can be computed before solving another
radius. The following algorithm is applied for each
control volume. Step (1): Initialize a' (axial induction factor) and á (radial
induction factor), typically a = a' = 0. Step (2): Compute the flow angle ϕ. Step (2): Compute the flow angle ϕ. Step (3): Compute the local angle of attack α.ft Step (4): Read off the lift Cl(α) and drag Cd(α) coefficients
from a table. Step (4): Read off the lift Cl(α) and drag Cd(α) coefficients
from a table. Step (5): Compute Cn and Ct, respectively normal and
tangential aerodynamic force coefficients. Step (6): Recalculate a and a'. Step (7): If a and a' have changed more than a tolerable
amount, repeat step (2), or else finish. Step (8): Compute local loads on the blade element. All equations necessary to perform the above algorithm
can are described by Burton (2001) and Hansen (2007). All equations necessary to perform the above algorithm
can are described by Burton (2001) and Hansen (2007). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 THE CLASSICAL BLADE ELEMENT MOMENTUM
METHOD The BEM method presented by Glauert (1935) enables
to calculate the steady loads and also the thrust and power Figure 1. Velocities at rotor plane. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Unsteady Blade Element-Momentum Method Including Returning Wake Effects 29 UNSTEADY BEM MODEL In order to obtain good estimates of the annual energy
production of a wind turbine, a steady BEM method is
adequate to compute the steady power curve. But in reality
the rotor of a wind turbine feels the inherent unsteadiness
of the wind caused by atmospheric turbulence, wind shear
and the presence of the tower. It is necessary to use an
unsteady BEM method to compute realistically this variable
behavior of the wind. Figure 2. Coordinate systems. One simple model and additional coordinate systems can
by placed at the wind turbine and its blades so it is possible to
know the relative position of any blade element at any time. This simple model is depicted at Fig. 2. The essence of the BEM method is to determine the
induced velocity and thus the local angle of attack. This is
achieved by a summation of vectors, Vrel = V0+ Vrot+W, all
written at the element blade coordinate system (System 4,
Fig. 3), in which the induction velocity is the term W. An inertial system of coordinates is placed at tower
base and named System 1. System 2 is non-rotating and
fixed in the nacelle. System 3 is solidary to the shaft
turbine and rotates with it, and system 4 is aligned with
one of the blades. The tilt and cone angles are shown and
the azimuthal position of the blade is set by de wing angle
θwing, not depicted. With the induced velocity known, the flow angle and
angle of attack are found. (
)
,
,
tan
rel z
p
rel y
V
V
ϕ
α
ϕ
β
θ
=
=
−
+
-
(1) (1) g
The undisturbed wind velocity seen by the blade is found
by a simple coordinate transformation clearly detailed by
Hansen (2007). Bramwell et al. (1976) states that Glauert’s relation
between thrust and this induced velocity for a gyrocopter in Bramwell et al. (1976) states that Glauert’s relation
between thrust and this induced velocity for a gyrocopter in Silva, C.T. and Donadon, M.V. 0
Silva, C.T. and Donadon, M.V. 30 Figure 3. Relative and induced wind speeds in System 4. Rotor plane
rel
V
W
+
0
V
rot
V blade pointing downstream is deeper into the wake than
a blade pointing upstream. UNSTEADY BEM MODEL This means that an upstream
blade sees a higher wind speed and thus produces higher
loads than the downstream blade, which produces a
beneficial yawing moment that will try to turn the rotor
more into the wind, thus enhancing yaw stability. The yaw
model describes the distribution of the induced velocity. If a yaw model is not included, the BEM method will not
be able to predict the restoring yaw moment, according
to Hansen (2007): rel
V Figure 3. Relative and induced wind speeds in System 4. (
)
0
0 1
tan
cos
2
wing
r
R
χ
θ
θ
⎞
⎛
⎛
⎞
=
+
−
⎜
⎟
⎟
⎜
⎝
⎠
⎠
⎝
W
W
(5) (5) forward flight (similar to the result of the lifting line for an
elliptically loaded circular wing) is: in which the wake skew angle, X, is defined as the angle
between the wind velocity in the wake and the rotational axis
of the rotor. θ0 is the angle in which the blade is deepest into
the wake. The skew angle can be found as: (
)
0
2
n
T
W
A
ρ
=
⋅
=
+
⋅
n W
V
n n W
(2) (2) (2) (
)
0
0
cosX
⋅
+
=
+
n
V
W
n V
W
(6) (6) in which n is the unit vector in the direction of the thrust,
which in System 3 has the coordinates n=[0,0,1]T. The skew angle is assumed to be constant with the radius
and can be computed at a radial position close to r/R = 0.7. It is assumed that only the lift contributes to the
induced velocity, and that the induced velocity acts in
the opposite direction to the lift. The force from this blade
at radial position is assumed to affect the air in the area
dA = 2πrdr/B, so that all B blades cover the entire annulus of
the rotor disc at radius r. The induced velocity is now known at the new
azimuthal position at time t+∆t, θwing(t+ ∆t)= θwing(t)+
ω∆t. The angle of attack can thus be evaluated from the
equation and the lift and drag coefficients can be looked
up from a table. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 LOEWY´S LIFT DEFICIENCY FUNCTION Step (5): Compute relative velocity to the blade element
using old values for induced velocity; The problem of calculating the aerodynamic loading
on an oscillating profile was first approached by Glauert
(1929), but it was only properly solved by Theodorsen
(1935). Theodorsen’s approach gives the solution for
unsteady aerodynamic loading on a 2D oscillating airfoil
in an inviscid and incompressible flow, and subject to the
assumption of small disturbances. Theodorsen’s problem
is to obtain the solution for loading on the surface of
the airfoil under the condition of forced harmonic
oscillations. Step (6): Calculate flow angle and thus the angle of attack
(Eq. 1); Step (6): Calculate flow angle and thus the angle of attack
(Eq. 1); Step (7): Determine static drag and lift coefficients from
tables; Step (7): Determine static drag and lift coefficients from
tables; Step (7): Determine static drag and lift coefficients from
tables; Step (8): Compute lift and drag for each blade element
(Eq. 8); Step (9): Compute loads (Eq. 7); Step (9): Compute loads (Eq. 7); Step (9): Compute loads (Eq. 7); Step (10): Compute new equilibrium values for induced
velocities (Eqs. 3 and 4); Step (11) Calculate the azimuthal variation from Eq. 5
and compute the induced velocity for each blade; Step (11) Calculate the azimuthal variation from Eq. 5
and compute the induced velocity for each blade; For a simple harmonic motion of the airfoil the solution
given by Theodorsen in a way that represents a transfer
function relating the forcing input (angle of attack) and the
aerodynamic response (pressure distribution, lift, and pitching
moment). The approach is summarized by Bisplinghoff et al. (1955). See also Bramwell et al. (1976) and Johnson (1980) for
a detailed exposition of the theory. Step (12): Compute momentum, thrust and power; Step (13): Increment time step and repeat from step (5). The equations of the BEM method must be solved
iteratively. The flow angle and thus the angle of attack depend
on induced velocity. But the described algorithm is unsteady,
therefore time is used as relaxation. After blades have moved
in one time step an azimuthal angle of ∆θwing=ω∆t (for
small ∆t´s), values from the previous time step are used on
the right hand side of equations for W when updating new
values for induced velocity. This can be taken into account
since induced velocity changes relatively slowly in time. UNSTEADY BEM MODEL The normal, pz, and tangential, py, loads
can be determined from: The following expression can be derived for one blade,
according to Hansen (2007), (
)
(
)
0
0
cos
cos
2
4
2
n
z
L
dr
BL
W
W
rdr
rF
F
B
ϕ
ϕ
π
πρ
ρ
−
−
=
=
+
⋅
+
⋅
V
n n W
V
n n W
(3) cos
sin
sin
cos
y
zp
L
D
p
L
D
φ
φ
φ
φ
=
+
=
−
(7) (7) in which in which For the tangential component a similar expression is
postulated, 2
2
1
1
2
2
d
ler
l
ler
L
V
cC
D
V
cC
ρ
ρ
=
=
(8) (8) (
)
0
sin
4
t
y
BL
W
W
rF
ϕ
πρ
−
=
=
+
⋅
V
n n W
(4) (4) The algorithm can be resumed like the following: Step (1): Initialize all necessary data (geometry and run
parameters); in which F is Prandtl’s tip loss factor. Step (2): Initialize the position and velocity of bla
Step (3): Discretize the blades into N elements; in which F is Prandtl’s tip loss factor. Step (2): Initialize the position and velocity of bla
Step (3): Discretize the blades into N elements; Step (3): Discretize the blades into N elements; If the rotor is yawed (and/or tilted), there will be an
azimuthal variation of the induced velocity, so that it is
lower when the blade is pointing upstream in relation to
when the same blade, half a revolution later, is pointing
downstream. The physical explanation for it is that a Step (4): Initialize the induced velocity; - for n=1 to max time step (t=n∆t) - for each blade - for each element 1 to N J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Unsteady Blade Element-Momentum Method Including Returning Wake Effects 31 LOEWY´S LIFT DEFICIENCY FUNCTION This eliminates the need of calculating the induction factors
and use of tolerance. Theodorsen’s theory is not suitable for studies involving
rotors. In these types of problems sections of blades can
find wake vorticity due to other rotor blades, as well as the
returning wake from the blade in question. This fact was
recognized by Loewy (1957) and Jones (1958). They built
a two-dimensional model of a 2-D blade section with a
returning shed wake, as shown in Fig. 4. As in Theodorsen’s model, the shed wake is modeled with
2-D flat surfaces of vortices, but now with a series of surfaces
below the airfoil section with a vertical separation h, which
depends on the speed induced by the rotor disc V and the
number of blades Nb. Loewy shows that, in this case, the lift
on the blade can be expressed by replacing the function
Theodorsen by the named Loewy’s function. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 DETERMINISTIC WIND MODEL If an average induced velocity vi=λΩR is assumed, then
during a single rotor revolution the shed wake generated by a
single blade will be at a distance h=(2π/Ω)vi below the rotor. For a multiple blade rotor the spacing is (2π)vi/ ΩNb, i.e. dynamic inflow, yaw and tower shadow, turbulence, wake
dynamics and interactions blade/wake, and the dynamic
stall. The adoption of a non-stationary BEM method
using a dynamic inflow model is a solution that tends
to enhance the results, proving to be a good option to
introduce the study of non-stationarity in the design of a
wind turbine blade. However the same problems related to
the physics of the method remain. If an average induced velocity vi=λΩR is assumed, then
during a single rotor revolution the shed wake generated by a
single blade will be at a distance h=(2π/Ω)vi below the rotor. For a multiple blade rotor the spacing is (2π)vi/ ΩNb, i.e. 2
4
b
h
R
b
N b
λΩ
π
λ
Ω
σ
=
=
(11) (11) Unsatisfactory
aspects
of
the
inflow
theory
are the so-called dynamic time constants employed in
the methods. They are developed using the concept
of apparent mass and inertia of the fluid surrounding
the rotor (noncirculatory effect) as opposed to the delay of
the dynamic evolution of wake vortices (circulatory
effects). The concept of apparent mass applied to the rotor
also assumes equivalence between the apparent force of
the rotor disk accelerating in a stopped fluid and the force
in a fluid accelerating through a permeable actuator disc,
which certainly is not an accurate analogy. in which σ is the rotor solidity. in which σ is the rotor solidity. Representative results from Loewy’s theory show
that the main consequence of including shed vorticity
below the blade is that it serves to amplify or attenuate
the unsteady lift response, depending on the reduced
frequency, wake spacing and wake phase. The most
important effects are for lower reduced frequencies,
with oscillations at the harmonics of the rotor rotational
frequency (Leishman, 2000). Loewy proposes a solution to the problem of
aerodynamics of rotors affected by non-stationarity
generated by shed wake. It is based on Theodorsen’s
solution applying a suitable physical model for Several aspects related to the non-stationarity of
the operating environment of a wind turbine need to
be addressed during its project. DETERMINISTIC WIND MODEL The exponential model used to simulate wind shear. It controls the wind speed according to the altitude, and the
shear parameter used is 0.2. Detailed information about this
wind shear model was described by Hansen (2007). The wind is also influenced by the presence of the
tower. The simple model used to simulate the tower
shadow assumes potential flow. All details about
this simple model were also described by Hansen (2007). This model is a bad approximation for a downwind
machine, in which each blade passes the tower wake
once every revolution. However, for an upwind
machine, the object of this study, the model provides
good estimation. Also, the turbulent part of the real
atmospheric wind should be added for a realistic time
simulation for a wind turbine. For this initial investigation
no atmospheric turbulence is added to the simulation. (
)
( ) ( )
( )
( )
( ) ( )
( )
( )
(
)
2
1
1
2
2
1
0
1
0
2
,
,
2
H
k
J
k W
C k
h
H
k
iH
k
J
k
iJ
k
W
ω Ω
+
ʹ
=
+
+
+
(9) (9) in which it is known for Loewy’s function of Loewy’s lift
deficiency function. For a rotor with Nb blades the complex function W is
written as: Δψ ω Ω
(
)
(
)
1
2
1
,
,
,
i
b
i
e
kh
N
b
b
kh
W
N
e
e
b
ω
π
Ω
ω
Δψ
Ω
−
(10) (10) J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. 32 2
Silva, C.T. and Donadon, M.V. ,
, Figure 4. Loewy’s returning wake problem (Leishman, 2000). C
C
C
C
C
C
C
C
C
C
C
C
∆ψ
γb
γb
γw
= 0
n=0, m=0
n=1, m=0
h
h
4λ
m=1
m=2
m=1
m=2
m=1
m=2
n=2, m=0
x,ζ
∞
∞
α=
θ
=
V
i
ω
t
b
σ ∆ψ Figure 4. Loewy’s returning wake problem (Leishman, 2000). The wake spacing ratio h/b can be determined from the
spacing of vortex sheets that are laid down below the rotor. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 DETERMINISTIC WIND MODEL Among them there are
the main variations in wind speed (gust and wind shear), J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 33
Unsteady Blade Element-Momentum Method Including Returning Wake Effects 33 rotorcraft aerodynamics. The results obtained in the
solution of problems related to helicopter hovers, and
duly validated with experimental results, confirm the
efficiency of the method. Table 1. General characteristics of rotor and turbine. Rotor diameter
80
m
Number of blades
3
Hub height
61.5
m
Tower height
60
m
Tilt angle of rotor to horizontal
4
deg
Cone angle of rotor
0
deg
Blade set angle
0
deg
Rotor overhang
3.7
m
Rotational sense of rotor, viewed from
upwind
Clockwise
Position of rotor relative to tower
Upwind
Aerodynamic control surfaces
Pitch
Radial position of root station
1.25
m
Cut in windspeed
4
m/s
Cut out windspeed
25
m/s The aerodynamics of a helicopter hover resembles in
many aspects the aerodynamics of the blades of a wind
generator. This similarity, coupled with the need for a
mathematical model for the wind generator blade design
that considers the conditions of non-stationarity of the
phenomena involved in its aerodynamics, are the main
motivators for this work. The corrections using Loewy’s model are applied for
the Equation 2.8 in order to provide the lift deficiency
described above. The corrected equations can be rewritten. No corrections are applied to the drag force. 2
12
rel
l
L
V
cC C
ρ
=
(12) (12) J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. vertical shear model with wind shear coefficient 0.2 and
the potential flow model for the tower shadow. Details
about the models for wind shear end tower shadow were
described by Hansen (2007). Figures 6 and 7 illustrate the wind condition for one blade
element. Figure 6 is an illustration of the magnitude of the wind
for a blade element. Figure 7 shows the wind speed distribution
influenced by the presence of the tower (tower shadow). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 7. Wind speed components. Z component for θyaw=0 degress
γ component for θyaw=0 degress
Wind speed (component Z) [m/s]
Wind speed (component Y) [m/s]
Blade azimuth,
[rad]
wing
θ
Blade azimuth,
[rad]
wing
θ
0
1
2
3
4
5
6
0
1
2
3
4
5
6
13
0.4
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4
12.5
12
11.5
10.5
10
11
Figure 6. Vectors representing wind. Wind spedd vectors for θyaw=0 degress
ground height
lateral distance from tower axis
wind speed
85
80
75
70
65
60
55
50
45
40
35
-20
-25
-15
-5
5
15
25
5
10
-10
0
0
10
20
-5
x[m]
Wind turbine 3D view - yaw=0º, tilt=-4, wing=0º, cone=0º
Radial position (r=30 m)
Blade element (theta=0 degree)
Tower (height=60 m)
Nacele (overhang=3.7 m)
Pás (lenght=40 m)
z[m]
y[m]
100
90
80
70
60
50
40
30
20
10
0
-30
-20
-10
0
10
20
30
-5
0
Figure 5. 3D view of the wind turbine. described by Hansen (2007). influenced by the presence of the tower (tower shadow). x[m]
Wind turbine 3D view - yaw=0º, tilt=-4, wing=0º, cone=0º
Radial position (r=30 m)
Blade element (theta=0 degree)
Tower (height=60 m)
Nacele (overhang=3.7 m)
Pás (lenght=40 m)
z[m]
y[m]
100
90
80
70
60
50
40
30
20
10
0
-30
-20
-10
0
10
20
30
-5
0
Figure 5. 3D view of the wind turbine. x[m]
Wind turbine 3D view - yaw=0º, tilt=-4, wing=0º, cone=0º
Radial position (r=30 m)
Blade element (theta=0 degree)
Tower (height=60 m)
Nacele (overhang=3.7 m)
Pás (lenght=40 m)
z[m]
y[m]
100
90
80
70
60
50
40
30
20
10
0
-30
-20
-10
0
10
20
30
-5
0
Figure 5. 3D view of the wind turbine. Figure 6. Vectors representing wind. VERIFICATION AGAINST AN INDUSTRY-
STANDARD SOFTWARE The commercial package used for result validation is
the GH Bladed V4.1, distributed by GL Garrad Hassan
(2012), an independent renewable energy consultancy. GH Bladed is an industry-standard integrated software
package for the design and certification of onshore and
offshore turbines. It provides user with a design tool that
has been extensively validated against measured data
from a wide range of turbines and enables the conduction
of the full range of performance and loading calculations
(Garrad Hassan & Partners, 2011). Table 2. Blade geometry. Distance along
blade (m)
Chord (m)
Aerodynamic
twist (deg)
Aerofoil
section
0
2.07
0
cylinder
1.15
2.07
0
cylinder
3.44
2.76
9
cylinder
5.74
3.44
13
NASA
LS(1)-0421
9.19
3.44
11
NASA
LS(1)-0421
16.07
2.76
7.8
NASA
LS(1)-0421
26.41
1.84
3.3
NASA
LS(1)-0417
35.59
1.15
0.3
NASA
LS(1)-0413
38.23
0.69
2.75
NASA
LS(1)-0413
38.75
0.03
4
NASA
LS(1)-0413 Its manual postulates that GH Bladed uses the same
methods employed at the present work. That is, combined
blade element and momentum theory, wake rotation with
radial induction, tip and hub loss models, which is suppressed
for the present comparison, dynamic wake model and
dynamic stall, also suppressed. An educational version of de GH Bladed GH Bladed 4.1,
with a limitation of 10 blade elements, is used. The object of study is a 2MW wind turbine. The main
used parameters and data are shown in Tables 1 and 2. All necessary data, like angle of attack, lift, drag and moment
coefficients are described by McGee and Beasley (1976). The developed computational routine uses these same
data to be compared with the results of both packages. It
also uses de same model for wind shear, i.e., the exponential Just for visualization, BEM NE has a graphical interface
that shows a simplified illustration of the wind turbine and his
main geometrical parameter. This is shown at Fig. 5. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. Silva, C.T. and Donadon, M.V. 34 RESULTS AND DISCUSSION blade element. For the BEM NE results for the lift coefficient,
Loewy’s lift deficiency function is applied to correct its value. The most important aerodynamic and performance
results are shown and compared here. It is important to
mention that the GH Bladed package has a pitch control
module that changes the pitch angle of the blades in each
time step according to the wind speed, rotor speed and
output power. The developed computational routine, named
here BEM NE, uses this same pitch angle control map in
order to achieve the correct level of the shaft power, once it
does not have a control module. This control map is depicted
in Fig. 8b as also the shaft power (Fig. 8a) against different
wind speeds. Again, the mean relative errors are small: respectively
+0.28% e +7.68% for relative wind speed and lift coefficient. tfi
The most important result, the output shaft power, is
shown in Fig. 13. The relative mean error is now -0.66%. Results obtained until this point show a good correlation
between the GH Bladed and the BEM NE for the wind
speed 12 m/s. Now the same comparison of results for
the wind speed 18 m/s is shown in the middle of the operation
wind speed range. Figures 14 until 18 show these comparison. For wind speed 18 m/s the correspondent pitch angle is 14.9
degrees, as shown in Fig. 8. The blades of the wind turbine used in this study are
designed to produce 2MW power between wind speeds
of 12 and 25 m/s, at the rotor speed 18 rpm. Results at 12
and 18 m/s wind speed are compared. Relative mean errors are compatible with the ones obtained
for wind speed 12 m/s. The most important parameter, the
shaft power, has a relative error of -4.1%. It is observed that the power curve obtained from BEM
NE has more spikes then the one from GH Bladed. The
discontinuities coincide with the passage of the blade through
the tower shadow. Even though both packages use the same
model for the tower shadow, these discrepancies can be
justified by the absence of any kind of pitch control on the
BEM NE. At the current stage of the present study, the model
is simplified because its primary objective is to investigate the
viability of LDF as a simulation for the influence of the wake
shed behind the rotor. 3
Unsteady Blade Element-Momentum Method Including Returning Wake Effects 35 Silva, C.T. and Donadon, M.V. Wind spedd vectors for θyaw=0 degress
ground height
lateral distance from tower axis
wind speed
85
80
75
70
65
60
55
50
45
40
35
-20
-25
-15
-5
5
15
25
5
10
-10
0
0
10
20
-5 Figure 6. Vectors representing wind. Wind spedd vectors for θyaw=0 degress
ground height
lateral distance from tower axis
wind speed
85
80
75
70
65
60
55
50
45
40
35
-20
-25
-15
-5
5
15
25
5
10
-10
0
0
10
20
-5 wind speed Figure 6. Vectors representing wind. Figure 6. Vectors representing wind. Figure 5. 3D view of the wind turbine. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 7. Wind speed components. Z component for θyaw=0 degress
γ component for θyaw=0 degress
Wind speed (component Z) [m/s]
Wind speed (component Y) [m/s]
Blade azimuth,
[rad]
wing
θ
Blade azimuth,
[rad]
wing
θ
0
1
2
3
4
5
6
0
1
2
3
4
5
6
13
0.4
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4
12.5
12
11.5
10.5
10
11 γ component for θyaw=0 degress
Wind speed (component Y) [m/s]
Blade azimuth,
[rad]
wing
θ
0
1
2
3
4
5
6
0.4
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4 Z component for θyaw=0 degress
Wind speed (component Z) [m/s]
Blade azimuth,
[rad]
wing
θ
0
1
2
3
4
5
6
13
12.5
12
11.5
10.5
10
11 Z component for θyaw=0 degress Blade azimuth,
[rad]
wing
θ Figure 7. Wind speed components. Figure 7. Wind speed components. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 RESULTS AND DISCUSSION Therefore the pitch angle is maintained
constant during all time steps simulation. On the contrary,
Bladed has a variation of the pitch angle during the simulation,
as shown in Fig. 19. At first the results obtained for 12 m/s wind speed is
shown. Figures 9 and 10 compare the geometrical results of
flow angle and angle of attack, respectively. The results shown
are obtained from the blade element located 26.407 m from
the rotor center. This blade element is the most representative
one. It is located in 70% length position of the blade. During the 12 seconds of simulation, deviations on
the results are minimal, as demonstrated in Figs. 9 and 10. Mean relative error at inflow angle is -1.47% and mean relative
error at angle of attack is -2.71%. Figures 11 and 12 show the comparison of the relative wind
speed and the corresponding lift coefficient of the investigated Figure 8. Shaft Power (a) and Pitch angle (b) control map. (b)
Hub wind speed [m/s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
4
6
8
10
12
14
16
18
20
22
24
26
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Shaf power [MW]
Hub wind speed [m/s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
4
6
8
10
12
14
16
18
20
22
24
26
2.2
20
15
10
5
0
-5
Pitch angle [deg]
(a) Hub wind speed [m/s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
4
6
8
10
12
14
16
18
20
22
24
26
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Shaf power [MW]
(a) (b)
Hub wind speed [m/s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
4
6
8
10
12
14
16
18
20
22
24
26
2.2
20
15
10
5
0
-5
Pitch angle [deg] (b) (a) Figure 8. Shaft Power (a) and Pitch angle (b) control map. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. 36 6
Silva, C.T. and Donadon, M.V. Silva, C.T. and Donadon, M.V. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 10. Angle of attack (wind speed 12 m/s). RESULTS AND DISCUSSION Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 9. Inflow angle (wind speed 12 m/s). Time [s]
Infow angle [deg]
Infow angle for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
11
10.5
10
9.5
9
8.5
8
7.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 9. Inflow angle (wind speed 12 m/s). Time [s]
Infow angle [deg]
Infow angle for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
11
10.5
10
9.5
9
8.5
8
7.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 9. Inflow angle (wind speed 12 m/s). Time [s]
Infow angle [deg]
Infow angle for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
11
10.5
10
9.5
9
8.5
8
7.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Infow angle for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m Infow angle [deg] Figure 9. Inflow angle (wind speed 12 m/s). Figure 10. Angle of attack (wind speed 12 m/s). Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m Angle of attack [deg]
5 Figure 10. Angle of attack (wind speed 12 m/s). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Unsteady Blade Element-Momentum Method Including Returning Wake Effects 37 3 J. Aerosp. Technol. RESULTS AND DISCUSSION Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 12. Lift coefficient (wind speed 12 m/s). Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 11. Relative wind speed (12 m/s wind speed). Time [s]
Relative wind speed [m/s]
Relative wind speed for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
55
54.5
54
53.5
53
52.5
52
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 11. Relative wind speed (12 m/s wind speed). Time [s]
Relative wind speed [m/s]
Relative wind speed for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
55
54.5
54
53.5
53
52.5
52
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 11. Relative wind speed (12 m/s wind speed). Figure 12. Lift coefficient (wind speed 12 m/s). Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 12. Lift coefficient (wind speed 12 m/s). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. 38 8
Silva, C.T. and Donadon, M.V. Silva, C.T. and Donadon, M.V. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 14. Inflow angle (wind speed 18 m/s). Time [s]
Infow angle [deg]
Infow angle for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
22
21
20
19
18
17
16
15
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 13. Measured shaft power (wind speed 12 m/s). RESULTS AND DISCUSSION Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 13. Measured shaft power (wind speed 12 m/s). Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
7
6.5
6
5.5
5
4.5
4
3.5
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Angle of attack for blade 1 at wind speed 12 m/s - Distance along blade 26.407 m Figure 13. Measured shaft power (wind speed 12 m/s). Figure 14. Inflow angle (wind speed 18 m/s). Time [s]
Infow angle [deg]
Infow angle for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
22
21
20
19
18
17
16
15
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Infow angle [deg]
Infow angle for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
22
21
20
19
18
17
16
15
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 14. Inflow angle (wind speed 18 m/s). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Unsteady Blade Element-Momentum Method Including Returning Wake Effects 39 39 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
39
Figure 16. Relative wind speed (wind speed 18 m/s). RESULTS AND DISCUSSION Time [s]
Realative wind speed [m/s]
Relative wind speed for blade 1 at nominal wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
58
57
56
55
54
53
52
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
2
1
0
-1
-2
-3
-4
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 15. Angle of attack (wind speed 18 m/s). Time [s]
Angle of attack [deg]
Angle of attack for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
2
1
0
-1
-2
-3
-4
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 15. Angle of attack (wind speed 18 m/s). Figure 15. Angle of attack (wind speed 18 m/s). Figure 16. Relative wind speed (wind speed 18 m/s). Time [s]
Realative wind speed [m/s]
Relative wind speed for blade 1 at nominal wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
58
57
56
55
54
53
52
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Figure 16. Relative wind speed (wind speed 18 m/s). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Silva, C.T. and Donadon, M.V. Silva, C.T. and Donadon, M.V. 0
Silva, C.T. and Donadon, M.V. 40 Silva, C.T. and Donadon, M.V. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013
Figure 18. Measured shaft power (wind speed 18 m/s). RESULTS AND DISCUSSION Time [s]
Measured shaf power [MW]
Measured shaf power at wind speed 18 m/s
0
1
2
3
4
5
6
7
8
9
10
11
12
2.2
2.15
2.1
2.05
2
1.95
1.9
1.85
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Time [s]
Lif coefcient
Lif coefcient for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 17. Lift coefficient (wind speed 18 m/s). Time [s]
Lif coefcient
Lif coefcient for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 17. Lift coefficient (wind speed 18 m/s). Time [s]
Lif coefcient
Lif coefcient for blade 1 at wind speed 18 m/s - Distance along blade 26.407 m
0
1
2
3
4
5
6
7
8
9
10
11
12
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE
Figure 17. Lift coefficient (wind speed 18 m/s). Figure 17. Lift coefficient (wind speed 18 m/s). Figure 18. Measured shaft power (wind speed 18 m/s). Time [s]
Measured shaf power [MW]
Measured shaf power at wind speed 18 m/s
0
1
2
3
4
5
6
7
8
9
10
11
12
2.2
2.15
2.1
2.05
2
1.95
1.9
1.85
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Measured shaf power [MW]
Measured shaf power at wind speed 18 m/s
0
1
2
3
4
5
6
7
8
9
10
11
12
2.2
2.15
2.1
2.05
2
1.95
1.9
1.85
GH Bladed
mean (GH Bladed)
mean (BEM NE)
BEM NE Measured shaf power at wind speed 18 m/s Figure 18. Measured shaft power (wind speed 18 m/s). J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Unsteady Blade Element-Momentum Method Including Returning Wake Effects 41 4 Figure 19. CONCLUSION The
comparisons
show
that
the
model
has
good performance in terms of computational speed and
the differences between its results and those provided by the
commercial software used as validation parameter are very
small, being compatible with the optimization design method. The
comparisons
show
that
the
model
has
good performance in terms of computational speed and
the differences between its results and those provided by the
commercial software used as validation parameter are very
small, being compatible with the optimization design method. The work presented an alternative approach to
predict the performance of upwind horizontal-axis wind
turbine design using the unsteady BEM theory and an
analytical model for the wake shed behind the rotor. This
alternative approach is employed in order to verify the
viability of using an analytical model for the returning
wake effects, which does not have any empirical parameters
or the need for experimental calibration. The numerical
approach is very stable and fast, even being written in an
interactive computation environment. The computational
processing time necessary for any case is less than 10
seconds, and there were not numerical crashes. Based on the
good approximation of results, when compared with others
provided by commercial and established computational
package, the mathematical model presented in this paper
introduces an alternative tool for the wind turbine design,
especially for upwind rotors. RESULTS AND DISCUSSION Pitch angle for wind speed (a) 12 m/s and (b) 18 m/s from Bladed. Time [s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
0
2
4
6
8
10
12
0.80
0.78
0.76
0.74
0.72
0.70
0.68
0.66
0.64
Blade 1 pitch angle [deg]
Time [s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
0
2
4
6
8
10
12
15.02
15.00
14.98
14.96
14.94
14.92
14.90
14.88
14.86
14.84
14.82
Nominal pitch angle [deg]
(b)
(a) Time [s]
Bladed Educational - Licensed to: Instituto
Tecnológico de Aeronáutica
0
2
4
6
8
10
12
15.02
15.00
14.98
14.96
14.94
14.92
14.90
14.88
14.86
14.84
14.82
Nominal pitch angle [deg]
(b) Figure 19. Pitch angle for wind speed (a) 12 m/s and (b) 18 m/s from Bladed. Burton,T., 2001, “Wind Energy Handbook”, Ed. John Wiley and Sons
Ltd., Chichester, New York, 624 p.
Carpenter, P.J. and Fridovich, B., 1953, “Effect of A Rapid Blade-Pitch
Increase on the Thrust and Induced-Velocity Response of a Full-Scale
Helicopter Rotor”, NACA TN 3044. Carpenter, P.J. and Fridovich, B., 1953, “Effect of A Rapid Blade-Pitch
Increase on the Thrust and Induced-Velocity Response of a Full-Scale
Helicopter Rotor”, NACA TN 3044. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 Burton,T., 2001, “Wind Energy Handbook”, Ed. John Wiley and Sons
Ltd., Chichester, New York, 624 p. Burton,T., 2001, “Wind Energy Handbook”, Ed. John Wiley and Sons
Ltd., Chichester, New York, 624 p. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 ACKNOWLEDGEMENTS The authors acknowledge the financial support from
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), by Programa Institucional de Qualificação Docente para
a Rede Federal de Educação Profissional e Tecnológica (PIQDTec),
of Universidade Tecnológica Federal do Paraná (UTFPR). The authors acknowledge the financial support received
for this work from Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), contract number
303287/2009-8. J. Aerosp. Technol. Manag., São José dos Campos, Vol.5, No 1, pp.27-42, Jan.-Mar., 2013 2
Silva, C.T. and Donadon, M.V. 42 42
Silva, C.T. and Donadon, M.V. Garrad Hassan & Partners, 2011, “Bladed Theory Manual Version
4.1 Multibody Dynamics”, St. Vincent’s Works, Silverthome Lane,
Bristol BS2 0QD, England. Leishman, J.G., 2002, “Challenges in Modeling the Unsteady
Aerodynamics of Wind Turbines”, 21st ASME Wind Energy Symposium
and 40th AIAA Aerospace Sciences Meeting Reno, NV. GL Garrad Hassan, 2012, http://www.gl-garradhassan.com. Leishman, J.G., 2000, “Principles of Helicopter Aerodynamics”,
Cambridge University Press, The Edinburgh Building, Cambridge CB2
2RU, UK. Glauert, H., 1935, “Airplane Propellers”, Aerodynamic Theory (W.F. Durand, ed.), Div. L, Chapter XI. Berlin:Springer Verlag. Loewy, R.G., 1957, “A Two-dimensional Approximation to the Unsteady
Aerodynamics of Rotary Wings”, J. Aeronaut. Sci. Vol. 24, No 2, pp
81-92. Glauert, H., 1929, “The Force and Moment on an Oscillating Airfoil”,
Rep. Mem. Aeronaut., Res. Comm., Great Britain, No. 1561. Hansen, M.O.L., 2007, “Aerodynamics of wind turbines”, Earthscan,
Camden High Street London, NW1 0JH, UK, 2nd ed, pp 8-12. McGee, R.J. and Beasley, W.D., 1976, “The Aerodynamic
Characteristics of An Initial Low-speed Family of Airfoils for General
Aviation Applications”, NASA TM X-72843, NASA Langley Research
Center, Hampton, VA, USA. Johnson,W., 1980, “Helicopter Theory”, Princeton University Press. Jones, J.P., 1958, “The Influence of the Wake on the Flutter and
Vibration of Rotor Blades”, the Aeronaut. Quart., Vol. 9, No 3, pp
258-286. Theodorsen, T., 1935, “General Theory of Aerodynamic Instability and
the Mechanism of Flutter”, NACA report 496, pp 413-33.
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https://openalex.org/W4226073973
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https://www.researchsquare.com/article/rs-185959/latest.pdf
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English
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Glucose-Induced Biofilm Formation in Bacillus thuringiensis KPWP1 is Associated with Increased Cell Surface Hydrophobicity and Increased Production of Exopolymeric Substances
|
Current microbiology
| 2,021
|
cc-by
| 7,491
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Glucose induced biofilm formation in Bacillus
thuringiensis KPWP1 is associated with increased
cell surface hydrophobicity and increased
production of exopolymeric substances
Sushmita Jha
Indian Institute of Science Education and Research Kolkata
Nirbhay Bhadani
Indian Institute of Science Education and Research Kolkata
Abhinash Kumar
Indian Institute of Science Education and Research Kolkata
Tapas Kumar Sengupta
(
senguptk@iiserkol.ac.in
)
Indian Institute of Science Education and Research-Kolkata
https://orcid.org/0000-0001-6482-914X
Original Article
Keywords: Bacillus thuringiensis, Biofilm, Glucose, Cell-surface hydrophobicity
Posted Date: February 4th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-185959/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License
Version of Record: A version of this preprint was published at Current Microbiology on December 14th,
2021. See the published version at https://doi.org/10.1007/s00284-021-02699-z. Glucose induced biofilm formation in Bacillus
thuringiensis KPWP1 is associated with increased
cell surface hydrophobicity and increased
production of exopolymeric substances
Sushmita Jha
Indian Institute of Science Education and Research Kolkata
Nirbhay Bhadani
Indian Institute of Science Education and Research Kolkata
Abhinash Kumar
Indian Institute of Science Education and Research Kolkata
Tapas Kumar Sengupta
(
senguptk@iiserkol.ac.in
)
Indian Institute of Science Education and Research-Kolkata
https://orcid.org/0000-0001-6482-914X
Original Article
Keywords: Bacillus thuringiensis, Biofilm, Glucose, Cell-surface hydrophobicity
Posted Date: February 4th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-185959/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at Current Microbiology on December 14th,
2021. See the published version at https://doi.org/10.1007/s00284-021-02699-z. Abstract Bacillus thuringiensis are agriculturally and medically important bacteria as they produce insecticidal Cry
proteins and can form biofilm on different plant surfaces. Previous studies reported that the ubiquitous
carbon source glucose could induce restricted motility and fractal pattern formation in the growing
colonies of pH, salt and arsenate tolerant Bacillus thuringiensis KPWP1. As bacteria are evolved with the
ability to exhibit multicellular behaviour and biofilm formation under limiting conditions for survival, the
present study was focused on exploring the effect of glucose in biofilm formation by Bacillus
thuringiensis KPWP1. Significant rise in biofilm loads were observed with increased glucose
concentrations in growth media. Compared to control, six times more biofilm load was marked in
presence of 2% of glucose. Interestingly, it was observed that the effect was glucose specific and also not
due to any change in sugar induced physicochemical property of the growth media as addition of
galactose or arabinose could not induce any significant increase in KPWP1 biofilm load. Scanning
electron-, confocal laser scanning-microscopic studies and biochemical tests revealed that increased
concentrations of glucose could induce increased production of exopolymeric substances, increased
number of densely-packed micro-colonies in KPWP1 biofilm and increased hydrophobicity and adherence
properties in KPWP1cells. Original Article Version of Record: A version of this preprint was published at Current Microbiology on December 14th,
2021. See the published version at https://doi.org/10.1007/s00284-021-02699-z. Page 1/18 1. Introduction Thousands of years ago, human beings adapted a survival strategy to stay in communities, especially
communities inclusive of people with one-of-a-kind abilities. They realized that a community is far more
likely to continue to exist through the department of hard work– one makes food, other gathers source,
nonetheless any other protects the community towards invaders (Johnson LR et al.2008; Verplaetse et
al.2015; Verplaetse et al.2017). Microbes exhibit two kinds of growth modes i.e. planktonic cells and
sessile aggregate that is called the biofilm. Biofilms are a critical component of the natural surroundings
(Jamal et al.2015) and they are found in multiple habitats from soil (Maeder et al.2002) to space
(Horneck et al.2006), roots of the tree to the leaves, outer dead skin layer (Grice et al.2009) to the
intestines. Majority of microorganisms have the potency to construct biofilm on a wide range of surfaces including
biotic and abiotic surfaces (Verplaetse et al.2015) by producing the extracellular polymeric substance
(EPS) (Jamal et al.2015; Verplaetse et al.2015). For the most part in biofilm, microorganisms involve
about 10% of the dry mass, whereas the remaining 90% are possessed by EPS (Kavitaet al. 2013). It
mainly consists of different macromolecules like proteins, polysaccharides, lipids and extracellular DNA
(Kavita et al.2014). The EPS fill in between the shape of the biofilm constituents. Numerous factors, including source and abundance of nutrients, osmolality, temperature, and
anaerobiosis were suggested to affect biofilm formation and EPS production (Lim et al. 2004). Although
glucose is a preferred carbon source for most of the living organisms, there are conflicting reports on the
effect of glucose in bacterial biofilm formation. Although it was observed that glucose can inhibit biofilm Page 2/18 Page 2/18 formation by multiple species of Enterobacteriaceae, it was reported that glucose supplementation in
growth media enhance biofilm formation in E. faecalis. Such divergent results indicate a bimodal fashion
in occurrence of glucose mediated biofilm formation in enterococci (Pillai et al.2004). formation by multiple species of Enterobacteriaceae, it was reported that glucose supplementation in
growth media enhance biofilm formation in E. faecalis. Such divergent results indicate a bimodal fashion
in occurrence of glucose mediated biofilm formation in enterococci (Pillai et al.2004). Bacteria belonging to Bacillus cereus group display a broad range of existence and ecological niches and
consist of useful as well as pathogenic lines. 1. Introduction Bacillus thuringiensis, a Gram-positive, spore-forming
microbe, belong to Bacillus cereus group which is used as a biological pesticide as this microbe produces
insecticidal toxins (cryI, cryIV, cryIII etc) that target selected insect hosts ( Gill et al.1992;Bravo et al.2007;
Gill et al.1992; Guan et al. 2014;Qi JiaHeLing et al. 2016).Initially, spores and crystalline insecticidal
proteins produced by B. thuringiensis have been used to control insect pests since the 1950s and are
often applied as liquid sprays (Saiyad et al.2017). But the problems arise when they get washed off from
the plant surface due to heavy rainfall and irrigation. If biofilm formation of B. thuringiensis is
encouraged onto the plant surface, the cells can adhere to the plant surface by secreting an extracellular
matrix and ensure the insecticidal property without being engineered the crop. Therefore, studies on
biofilm formation by Bacillus thuringiensis are increasingly recognized as an important area of research. Previous studies from our laboratory revealed that increased concentration of glucose in growth media
resulted in decreased number of flagella on KPWP1 cell surface (Roy et al.2010). There are contrasting
reports on the relation between swarming and biofilm formation in bacterial world. In one hand, it is
generally observed that swarming motility and biofilm formation are inversely correlated (Caiazza et
al.2007;Verstraeten et al.2008; Guttenplan et al.2013) whereas, recent findings indicate that for some
bacteria, there may be a positive correlation between bacterial swarming and biofilm formation (Fuente-
Núñez et al.2012; Park et al.2018). It is, therefore, important to know whether swarming and biofilm
formation are inversely or positively correlated in Bacillus thuringiensis KPWP1 and also to know whether
glucose can influence the biofilm formation by Bacillus thuringiensis KPWP1. The aim of the present study was to investigate the effect of glucose in biofilm formation of Bacillus
thuringiensis KPWP1 and also to characterize the changes in EPS composition, cell morphology and
surface hydrophobicity in KPWP1 biofilms due to the presence of differential concentrations of glucose in
growth media. 2.1 Bacterial strains and growth conditions Bacillus thuringiensis KPWP1, (Roy et al.2010) isolated from Kolkata port water, were grown in 24-well
plates in nutrient broth (peptone − 0.5%, NaCl-0.5% and beef extract-0.3%) in presence of different
concentration of glucose, arabinose and galactose as required for experiments. Planktonic growth of
bacteria was monitored by measuring the turbidity/O. D of bacterial suspension at 600 nm by using a UV-
vis spectrophotometer (Schimadzu UV 2600) 2.2 Biofilm assay 2.4 Scanning Electron Microscopy In order to visualize the biofilm formed by KPWP1 cells, SEM was performed by using the protocol
described by Chakraborty et al.2016 with minor modification. Biofilm was first formed onto the coverslip placed vertically in 24 well plates. The coverslips were washed
with PBS and fixed with 200 µl of glutaraldehyde (2.5%) solution for 1 hour at room temperature in dark. The coverslips were then re-suspended in 200 µl of 0.1% OsO4 and incubated at room temperature for 30
minutes. After repeated (three times) wash with PBS, cells were again washed in 30%, 50%, 70%, 90%, and
100% alcohol respectively. Finally, 20 µl of 100% ethanol was added onto the coverslips. The samples
were then dried using desiccators and then were coated with a thin layer of conducting metal, (gold-
palladium) in sputter coater (Quorum technologies ltd.) The samples were observed under field emission
scanning electron microscope (Carl Zeiss) using smart SEM software 2.3 Confocal laser scanning microscopy of biofilm Confocal laser scanning microscopy (CLSM) was performed to measure three-dimensional structure and
related topological parameters of KPWP1 biofilms. For that, KPWP1 biofilms were formed on coverslip by
using the same method as described above (method Sect. 2.3). The coverslips, containing the biofilms,
were washed with PBS two times gently and then stained with 0.001% acridine orange and incubated in
dark for 10 mins. The stained coverslips were washed with PBS to remove the excess stain and observed
under confocal microscope The bio-volume (µm3 /µm2), mean thickness (µm), volume (µm3), skewness and kurtosis of KPWP1 were
quantified from the confocal stacks using the Zen software. The bio-volume is defined as the volume of
the biomass (µm3) divided by the surface area of the substratum (µm2). The skewness determines the
porosity in the biofilm which helps the cells to get access to the nutrient. The kurtosis value determines
the adherence property of the bacteria (Dasgupta et al.2013). 2.2 Biofilm assay Page 3/18 Page 3/18 Page 3/18 Biofilm formation was assayed was performed by the method described by (Pui et al.2017) with minor
modification. For assay of biofilm formation by Bacillus thuringiensis, 106 cells number of KPWP1 cells
in 1 ml were grown for 48 hours in nutrient broth in 24 well plates n presence of 1 % and 2% of sugars at
37 ˚C under shaking condition (150 rpm). The planktonic cells were removed and the growth of
planktonic cells was measured spectrophotometrically. Wells (contained biofilm) of plates were gently
washed with phosphate buffered saline (PBS) twice and incubated with 0.1% crystal violet (CV) for 15
mins at room temperature. After incubation, excess stain was removed by washing the wells with PBS. CV attached to the biofilm was extracted with 1 ml of 30% acetic acid. The biofilm load was calculated by
measuring the optical density (O.D) of the extracted crystal violet at 600 nm by using microplate. 2.6 Microbial adhesion to the hydrocarbons (MATH) assay In order to measure the hydrophobicity of cell surface of bacteria grown under different conditions, MATH
assay was performed by using hydrophobic hydrocarbon n-hexadecane (Sigma, purity > 99%) (Tyfa et al. 2015). For that, KPWP1 cells were grown in Nutrient Broth in presence of different concentrations of
glucose. The optical densities of cell suspensions were adjusted to 0.6 by diluting the suspensions with
PBS (A0). Next, 1.5 ml of adjusted cell suspensions were thoroughly mixed with 0.5 ml of n-hexadecane
for 30 min at room temperature. The mixtures were then allowed to settle for 30 min and the aqueous
layers were collected and OD of the collected aqueous layers was measured at 600 nm (A1). The
percentage hydrophobicity was calculated by the formula; images of live and dead cells were stained with SYTO 9 dye and Propidium iodide (PI), respectively and
the images were captured by using fluorescence microscope (Olympus provis ax70) images of live and dead cells were stained with SYTO 9 dye and Propidium iodide (PI), respectively and
the images were captured by using fluorescence microscope (Olympus provis ax70) images of live and dead cells were stained with SYTO 9 dye and Propidium iodide (PI), respectively and
the images were captured by using fluorescence microscope (Olympus provis ax70) 2.5 Live-dead staining of biofilm The presence of live and dead cells in KPWP1 biofilm was measured by staining the biofilm (formed on
coverslips) with the live/dead BACLIGHT bacterial viability kit (Invitrogen, paisley, UK) (He et al.2017). The Page 4/18 Page 4/18 Page 4/18 Percentage hydrophobicity = [1-A1/A0]*100
2.7 Staining of Exopolymeric substances (EPS)
components with concanavalin A and Nile red The EPS is consisting of embedded carbohydrate, proteins and lipids which gives a structural integrity for
the three-dimensional biofilm lattice. The dispersion of extracellular polysaccharides was examined
utilizing fluorochromes that interact with particular oligosaccharide subunits. Concanavalin A (Con A)
was used to stain glycoconjugates (mannose specific) part of biofilm flocs. The biofilm onto the
coverslips was stained with 500 mg/l of the dye followed by incubation for 15 mins in dark [35]. The
samples were washed with PBS and observed under confocal laser scanning microscopy with an
excitation wavelength of 485 nm and emission at 530 nm. Similarly, Nile red (2.5mg/l) localized lipids
such as triglycerides phospholipids, and neutral lipid droplets within cells. The staining procedure was the
same as above about the incubation time, in this case, is 10 mins followed by PBS wash (Chen et
al.2009). 2.8 Quantification of carbohydrate, protein and eDNA in EPS
of KPWP1 biofilms The anthrone method was used for quantification of the carbohydrate content of EPS (Khan et al.2014). In brief, 160 µl of anthrone reagent (0.125% anthrone [wt./vol] in 94.5% [vol/vol] H2SO4) was mixed with
80 µl of the sample and incubated at 100°c for 14 min and after that cooled at 4°C for 5 min. The
absorbance at 625 nm was measured utilizing a microplate reader. The Bradford method was used to
measure the protein concentration in EPS. In brief, 400 µl of the filtered sample was blended tenderly with
100 µl of Bradford reagent 96 well plate. After 5 min of blending, 200 µl of the blend was utilized to
measure the absorbance at 595 nm with a microplate reader ( Kawaguchi et al.2000).For eDNA
quantification, KPWP1 biofilms were grown in 24 well plates. The planktonic cells were discarded after 48
hours of incubation and the wells were washed with 0.9% NaCl solution for two times. 1 ml of the 0.9%
NaCl was added to the wells and dispensed properly. The mixture was vortexed vigorously for 1 min and Page 5/18 Page 5/18 centrifuged at 5,500 x g at 4°C for 10 mins (Panariello et al.2017). The supernatant was collected and the
eDNA content was measured by using Nanodrop 1000 spectrophotometer (Thermo scientific Asheville,
NC, USA). centrifuged at 5,500 x g at 4°C for 10 mins (Panariello et al.2017). The supernatant was collected and the
eDNA content was measured by using Nanodrop 1000 spectrophotometer (Thermo scientific Asheville,
NC, USA). 2.9 Statistical analysis Graph Prism 6 was used to analyse the data. At least three independent data of anindividual experiment
were taken and mean ± SE was used to express all the results. 3. Results
3.1 Effect of glucose on growth and biofilm formation of
Bacillus thuringiensis Planktonic cell growth of KPWP1 in presence of glucose was monitored by measuring the turbidity (A600)
by using UV- visible spectrometer. 1.33- and 1.35-fold increase in the growth of the planktonic cells was
observed in case of glucose supplemented media with 1 % and 2 % of glucose with respect to control,
although, no significant difference in planktonic growth was observed between media containing
different glucose percentage (Fig. 1A). Interestingly, significant rise in biofilm load was observed with the increase in glucose concentrations in
growth media. An increment of 11.45 and 18.36-fold in biofilm load was marked in 1% and 2 % of
glucose supplemented nutrient broth respectively, with respect to control (Nutrient broth without added
glucose) (Fig. 1B). The results indicate that KPWP1 was able to form better biofilm in presence of
glucose. Moreover, the observed increase in biofilm formation was found to be glucose specific and also
not due to any change in physicochemical property (e.g., osmolarity) of growth media as addition of
increased concentrations of galactose or arabinose could not induce significant biofilm formation as
compared to glucose (Fig. 1C). 3.3 Scanning Electron Microscopy of KPWP1 biofilm 3.3 Scanning Electron Microscopy of KPWP1 biofilm We further explored the effect of glucose on Bacillus thuringeinsis KPWP1 biofilm at the levels of cell
morphology and presence of EPS by scanning electron microscopy (SEM). Biofilms were formed on glass
coverslips in 24-well microtiter plates and visualised by SEM. The scanning electron microscopy images
of KPWP1 cells in biofilms grown in absence of any added glucose revealed that monolayer of a few
spores adhering to the coverslips (Fig. 3A.i and 3A.ii). Interestingly, the outcome due to the addition of 1%
glucose in growth media presents a contrasting picture. At the lower magnification, it was detected that
heterogenous cell population of KPWP1 adhering to the substratum forming multiple layers with void
spaces (Fig. 3B.i. and 3B.ii.) which most probably help in nutrient circulation and excretion of the waste
products. The higher magnification of the images revealed that the heterogenous cell population consist
of densely packed region of elongated cells, normal healthy cells and spores embedded in EPS of KPWP1
biofilm. The effect of 2% glucose on KPWP1 biofilm is also captivating. The images revealed the
presence of densely packed 3-D structure of KPWP1 cells along with the void spaces with more EPS
compare to the 1% glucose and the higher magnification gives an exact view of the cell morphology
where it is observed that the heterogeneously cells population is present trapped in EPS (Fig. 3C.i and
3C.ii.) 3.2 Topological parameters of KPWP1 biofilm In order to characterize the topological parameters of KPWP1 biofilm, confocal laser scanning
microscopy was done on KPWP1 biofilm formed on glass coverslips. The bio-volume, mean thickness,
biomass, kurtosis and skewness of the 48-hour grown biofilms were analysed to evaluate changes in the
biofilm structure with increased glucose concentrations in growth media. As shown in Fig. 2A, 2B and 2C
the mean thickness, biovolume and biomass of the bacterial micro-colonies of the 48-h biofilms
increased gradually as the glucose concentration increased. The thickness of the biofilm in 1% and 2%
glucose supplemented media was found to be 1.43 and 1.86 times more than the control while the
volume increases by 1.48 and 1.82-folds, respectively (Fig. 2A). The biomass of the KPWP1 biofilm was
also observed to be increased by 2.06-fold in 2% glucose concentrations (Fig. 2C). Page 6/18
The skewness and kurtosis value are the two statistical parameters that measures porosity in biofilm and
adherence of the bacteria to the surface respectively. Lesser the skewness value more is the porosity that enables the bacteria to access more nutrient. From the figure (Fig. 2D), the lowest skewness value was
observed in the case of 2% glucose containing media which implies the presence of more porosity in the
formed biofilm. The highest kurtosis value (Fig. 2E) was again noticed in 2% glucose supplemented
media states that the adherence property of bacteria increases as glucose is added in the media. Figure 2F shows representative CLSM images of KPWP1 biofilms, in which the bacterial micro-colonies at
in 1% and 2% of glucose-containing media were denser, and more aggregated than that of control. 3.6 Identification and estimation of EPS in K 3.6 Identification and estimation of EPS in KPWP1 Biofilm Exopolymeric substances in bacterial biofilm are generally made of polysaccharides, proteins, glycolipids
and eDNA. Total polysaccharide was determined biochemically by utilizing anthrone reagent. The carbohydrate
content is 8.45 and 31.70-fold more in 1% and 2% glucose (Fig. 6A). Bradford reagent is used to quantify
the protein content in the EPS matrix. The results suggest that the total protein content is 9.8 and 36.24-
fold more in more in 1% and 2% glucose with respect to control (Fig. 6B). eDNA was quantified by
spectrophotometric analysis. Notably, eDNA contents in KPWP1 biofilm was found to be 1.9 and 5.01
folds more in 1% and 2% glucose with respect to the control condition with no added glucose in growth
medium (Fig. 6C). In order to check the relative presence and quantify the carbohydrate residue the EPS matrix was stained
with Con A Fig. (6D). The observed intensity for Con A which binds with the mannose residue is 2.8 and
3.1 folds (Fig. 6E) more in the matrix 1% and 2% glucose with respect to control. Nile red was used for the assessment of lipids residue present in the EPS matrix of KPWP1. The lipophilic
stain Nile red shows the presence of lipid or exceedingly hydrophobic areas within biofilms (Fig. 6F). The
intensity for Nile red was 1.74 and 4.60 times more in the EPS matrix of 1% and 2% glucose with respect
to control (Fig. 6G). 3.4 Glucose induced change in cell surface hydrophobicity
in KPWP1 cells Cell surface hydrophobicity (CSH) was assessed using the microbial adhesion assay for hydrocarbons
(MATH). High CSH enables microorganisms to attach to the hydrophobic surface and form a biofilm. The
results show that the least percentage hydrophobicity in control. The maximum percentage
hydrophobicity is observed in case 2% glucose (Fig. 4). The CSH changes as the cell surface
macromolecules changes with response to environmental stimulus and nutrient variation. The results
clearly depict that addition of 1% and 2% glucose in nutrient media increases the percentage
hydrophobicity by 3.77 and 4.85 folds respectively. Cell surface hydrophobicity has been distinguished as
a measurable trademark for microscopic organisms. This property has been usually investigated in the
adherence of microscopic organisms to surfaces. Nutrients play an important role in altering the cell
surface hydrophobicity which eventually assists the bacteria to form biofilms. Thus, glucose treatment
applied in the present study caused significant variation in biofilm organization.fi Page 7/18 3.5 Live dead staining of bacteria in KPWP1 biofilms Both live and dead bacteria embrace the biofilm of Bacillus thuringiensis were observed when they were
allowed to develop on 18mm coverslips in 24 well plates. By using the BACLIGHT live/dead viability
probe (molecular probes), we observed that cell death is manifested, in the micro colonies (Fig. 5). As it is
reported that dead cells play important role in the adherence of the bacteria while forming biofilm (Desai
et al.2019), imaging of live/dead cells in KPWP1 biofilm, grown in absence and presence of added
glucose was performed. The overlapped images of SYTO 9 and PI of the live dead staining depicts that
the dead cells (indicate by red) are present at the bottom while the live cells (indicate by green) are
present at the top of the biofilm. It is also observed that with the increase of the glucose percentages, the
intensity of the dead cells also increases in the KPWP1 biofilm which indicates that the dead cells might
helped the live cells to adhere to the substratum during biofilm formation. Cell death inside microcolonies is an imperative physiological occasion that plays a part in subsequent
differentiation and dispersal of a subpopulation of surviving biofilm cells (Webb et al.2003). 4. Discussion Biofilm development is a multiplex procedure that is influenced by many factors, which include growth
condition, stress and surface attributes (Kostakioti et al.2013). Microbial biofilms are captivating areas to
study because of their wide varieties in nature, importance in infection and use in bioremediation and Page 8/18 industries. Advances in molecular and biochemical methods have helped in improving our
comprehension of biofilm structure and functions (Velmourougane et al.2017). Exploiting naturally
occurring- and in-situ biofilm devolvement are promising ways for future advances in agricultural field as
they can possibly give different advantages like increase crop production, protection of the economically
important plants from insects and improvement of the crop variety (Franklin et al.2015).Investigations
uncovered the intricate conditions experienced in biofilms and during infection create extraordinary
heterogeneity inside the populace (Bisht et.al 2019). Regarding the regulation of biofilm formation, it was reported that in case of Bacillus subtilis, glucose
repressed biofilm development through the catabolic control protein CcpA (Stanley et al.2003). On the
other hand, interesting and contrasting reports showed that addition of glucose with NaCl in Tryptic Soy
broth (TSB) altogether favoured more biofilm formation in Bacillus cereus cells compared to the addition
of glucose or glycerol or NaCl in growth media (Kwon et al.2017). Many studies suggest that the biofilm
develops in restricted supplement of nutrients or in the presence of any sort of stress i.e. antibiotic, high
salt or a corrosive substance like acid (Khatoon et al.2018).It is also reported that the nutrient
accessibility affects adherence, biofilm arrangement and synthesis of EPS (Xiao et al.2017). Recent studies revealed the biofilm forming abilities the Bacillus thuringiensis isolates invitro and on
plant surfaces (Verplaetse et al.2015). B. thuringiensis biofilms on plant surfaces thus can help the plant
by protecting it from the pathogens like agricultural pests. It is also reported that Bacillus thuringiensis
spores have a higher hydrophobicity, conferring them a higher adhesive potential to diverse materials
(Wiencek et al.1990 ). Interesting findings in recent times revealed that Bacillus thuringiensis cells
undergoes differentiations and represent a mixed population of heterogenous cells under stressed
conditions (Verplaetse et al.2015). Previous study from our laboratory revealed that addition of higher concentrations of glucose in growth
media could induce restricted swarming motility due to impaired flagellation in Bacillus thuringiensis
KPWP1 on nutrient agar plates (Roy et al.2010). 4. Discussion This finding had raised the question whether the
restricted motility in presence of glucose can trigger the biofilm forming ability in Bacillus thuringiensis
KPWP1 cells? The present study was therefore to explore whether restricted motility induced by glucose
can regulate the adherence property, production of EPS components, thus biofilm formation ability in
Bacillus thuringiensis KPWP1. The results from this present study clearly indicate that the presence of
added glucose in nutrient-rich media induces biofilm formation (Fig. 1B) by KPWP1 as a result of
increase in EPS production (Fig. 6) and cell surface hydrophobicity (Fig. 4) and such induced biofilm
formation is glucose specific as addition of galactose or arabinose in growth media could not induce any
significant biofilm formation by KPWP1(Fig. 1D). The increased biofilm formation, in presence of glucose, was manifested by the increased thickness
(Fig. 2A), bio-volume (Fig. 2B), and biomass (Fig. 2C). Moreover, it was observed that the skewness
(Fig. 2D) which reflects the intracellular void spaces and the kurtosis values (Fig. 2E) which expresses the D) which reflects the intracellular void spaces and the kurtosis values (Fig. 2 Page 9/18 Page 9/18 adherence of microscopic organisms in KPWP1 biofilm also increased with the increased glucose
concentrations in growth media. It is reported that EPSs add to the mass and 3-D structure of the biofilm framework [46]. The present
study revealed that the EPS components i.e. polysaccharides, proteins, lipids and eDNA concentrations
increased (Fig. 6A, 6B and 6C) in KPWP1 biofilms with the increase in glucose concentration in growth
media. In the 48-h grown biofilms, EPS contents - sugar, proteins lipid and eDNA were altogether higher in
the presence of higher concentrations of glucose than that of control condition and supports the
observed increase in physical appearance of EPS in SEM images of KPWP1 biofilms grown in different
glucose concentrations (Fig. 3). Furthermore, it is also indicative that the EPSs formed in presence of 1
and 2% glucose helps in the adherence of the bacteria to the surface (Fig. 6). Collectively, the present
study uncovered the effect of glucose on biofilm arrangement of KPWP1 cells. Glucose prompts the
adherence property of the Bacillus thuringiensis KPWP1 cells and induces more EPS production resulting
in more biofilm formation. 4. Discussion The results of the present study signify that restricted motility in Bacillus thuringiensis KPWP1 induced by
glucose helps the bacterial cells to form biofilm, thus give a major premise to a more elaborate
investigation of the in-vivo biofilm formation by this insecticidal bacterium Bacillus thuringiensis on plant
surfaces, particularly in response to glucose. Not required Not required Consent for publication: All authors have consent for publication of this study Funding: The study was funded by IISER Kolkata. Conflicts of interest/Competing interests The authors of the paper declare that there is no conflict of interest. Authors' contributions: Page 10/18 SJ and TKS conceived the study and designed the experiments. SJ, NB and AB performed the
experiments and analysed the data. SJ and TKS have written the manuscript. SJ and TKS conceived the study and designed the experiments. SJ, NB and AB performed the
experiments and analysed the data. SJ and TKS have written the manuscript. Acknowledgements The authors gratefully acknowledge Mr Ritabrata Ghosh for help with confocal imaging and Mr
Kashinath Sahu for help with SEM imaging at IISER-K. SJ, NB and AB are recipients of fellowship from
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pattern on the human enamel surface. Int J Oral Sci 9:74–79 51. Yaron S, Römling U et al (2014) Biofilm formation by enteric pathogens and its role in plant
colonization and persistence. Microbial biotechnology 7:6: 496–516 51. Yaron S, Römling U et al (2014) Biofilm formation by enteric pathogens and its role in plant
colonization and persistence. Microbial biotechnology 7:6: 496–516 Figures Figure 1
Growth and biofilm formation by Bacillus thuringiensis KPWP1 in absence and presence of glucose. Planktonic growth of KPWP1 in NB after 48 hours of incubation in absence and presence glucose (A) and
other sugars (C). Biofilm formation of KPWP1 in NB after 48 hours of incubation in absence and presence
of glucose (B) and other sugars (D). Data represent mean values ±SE of 3 biological replicates. The
experiment was repeated three times. Asterisks indicate statistically significant differences when
compared to control (*P<0.05; **P<0.01; ***P<0.001; two-tailed Student’s-test). Figure 1 Figure 1 Figure 1 Growth and biofilm formation by Bacillus thuringiensis KPWP1 in absence and presence of glucose. Planktonic growth of KPWP1 in NB after 48 hours of incubation in absence and presence glucose (A) and
other sugars (C). Biofilm formation of KPWP1 in NB after 48 hours of incubation in absence and presence
of glucose (B) and other sugars (D). Data represent mean values ±SE of 3 biological replicates. The
experiment was repeated three times. Asterisks indicate statistically significant differences when
compared to control (*P<0.05; **P<0.01; ***P<0.001; two-tailed Student’s-test). Page 14/18 Figure 2
Topological feature of the biofilm formed by Bacillus thuringiensis KPWP1 in 1% Glucose and 2%
Glucose after 48 hours of incubation,thickness (A) volume, (B) biomass (C), kurtosis (D), skewness (E)
and representative CLSM images of biofilm grown in presence of different percentages of glucose of
KPWP1 (F). Data represent mean values ±SE of 3 biological replicates. The experiment was repeated
three times. Asterisks indicate statistically significant differences when compared to control (*P<0.05;
**P<0.01; ***P<0.001; two-tailed Student’s-test). Figure 2 Figure 2 Topological feature of the biofilm formed by Bacillus thuringiensis KPWP1 in 1% Glucose and 2%
Glucose after 48 hours of incubation,thickness (A) volume, (B) biomass (C), kurtosis (D), skewness (E)
and representative CLSM images of biofilm grown in presence of different percentages of glucose of
KPWP1 (F). Data represent mean values ±SE of 3 biological replicates. The experiment was repeated
three times. Asterisks indicate statistically significant differences when compared to control (*P<0.05;
**P<0.01; ***P<0.001; two-tailed Student’s-test). Page 15/18 Figure 3
Scanning Electron Micrograph (SEM) of biofilm of KPWP1 that developed on a glass coverslip after 48
hours period in nutrient broth with different percentage of glucose, Control (no Glucose added) at 3.00K X
and 10.00K X magnification (A.i, A.ii), 1% Glucose at 3.00K X and 10.00K X magnification (B.i, B.ii), 2%
Glucose at 3.00K X and 10.00K X magnification (C.i, C.ii) respectively. Figure 3 Scanning Electron Micrograph (SEM) of biofilm of KPWP1 that developed on a glass coverslip after 48
hours period in nutrient broth with different percentage of glucose, Control (no Glucose added) at 3.00K X
and 10.00K X magnification (A.i, A.ii), 1% Glucose at 3.00K X and 10.00K X magnification (B.i, B.ii), 2%
Glucose at 3.00K X and 10.00K X magnification (C.i, C.ii) respectively. Figure 4
Estimation of adhesion property of KPWP1 cells grown in absence and presence of glucose. Figure 4 Estimation of adhesion property of KPWP1 cells grown in absence and presence of glucose. Estimation of adhesion property of KPWP1 cells grown in absence and presence of glucose. Page 16/18 Page 16/18 Page 16/18 Figure 5
Analysis of Live /dead cells in the biofilm of KPWP1by using PI and Syto 9. Figure 5 Figure 5 Analysis of Live /dead cells in the biofilm of KPWP1by using PI and Syto 9. Page 17/18 Figure 6
Quantification of Exo polymeric substance in KPWP1 biofilm in absence and presence of Glucose. Carbohydrate residue (A), Protein (B), eDNA (C), Staining and quantification of mannose respectively by
Con A (D, E) and Staining of lipid by quantification of respectively by Nile Red. Data represent mean
values ±SE of 3 biological replicates. The experiment was repeated three times. Asterisks indicate
statistically significant differences when compared to control (*P<0.05; **P<0.01; ***P<0.001; two-tailed
Student’s-test). Figure 6 Quantification of Exo polymeric substance in KPWP1 biofilm in absence and presence of Glucose. Figure 2 Carbohydrate residue (A), Protein (B), eDNA (C), Staining and quantification of mannose respectively by
Con A (D, E) and Staining of lipid by quantification of respectively by Nile Red. Data represent mean
values ±SE of 3 biological replicates. The experiment was repeated three times. Asterisks indicate
statistically significant differences when compared to control (*P<0.05; **P<0.01; ***P<0.001; two-tailed
Student’s-test). Page 18/18
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Additive Manufacturing as A New Technique of Fabrication
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Corresponding author: Amir Dehghanghadikolaei, Oregon State University, USA.
Email: amir.dehghanghadikolaei@rockets.utoledo.edu
Received: July 05, 2018
Accepted: July 18, 2018
Published: July 19, 2018
Editor: Alessandra Caggiano, University of Naples Federico II
Citation:Amir Dehghanghadikolaei (2018) Additive Manufacturing as A New Technique of Fab-
rication. Journal of 3D Printing and Applications - 1(1):3-4. https://doi.org/10.14302/issn.2831
-8846.j3dpa-18-2207 Freely Available Online Freely Available Online Additive Manufacturing as A New Technique of
Fabrication Amir Dehghanghadikolaei 1,*
1Oregon State University, USA from tough alloys that cause difficulties during
conventional fabrication methods [2]. One of these
materials is an alloy of nickel and titanium, known as
NiTi, which offers excellent biocompatibility properties
and shape memory and superelastic behaviors. Using
AM techniques offers significantly easier fabricating
procedure for this specific alloy [3]. However, similar to
any other new techniques, AM has to undergo
numerous investigations regarding its effects on change
of mechanical properties, corrosion behavior and overall
performance. From other perspective, the effect of post
treatment processes such as heat treatment, coating,
machining and finishing should be investigated as the
parts are fabricated in a layer-by-layer fabricated
process [4, 5]. In the next step, one has to compare
the change of all the mentioned parameters and reveal
the effect of fabrication process. Additive Manufacturing (AM) is the most recent
method to fabricate 3D components. The general idea
of this process is to use 3D CAD files, slice the file into
desired values depending on accuracy, and send them
to a computer processing unit [1]. Several AM
techniques are used for fabricating metallic alloys that
powder-based methods are the most common ones. Selective laser sintering (SLS), self-propagation high
temperature synthesis (SHS), selective laser melting
(SLM), direct metal sintering (DMLS) and electron beam
melting (EMB) are the most common ones. Other
uncommon additive manufacturing techniques are laser
engineered net shaping (LENS), direct light fabrication
(DLF), laser consolidation, laser cladding and shape
deposition manufacturing (SDM) which are flow-based. Most of these processes are executed under controlled
atmosphere or with support of inert gases to purge the
chamber and keep oxygen level below a standard value. One of the most important applications of AM processes
could be named as fabricating patient specified implants To date, many AM processes are being
investigated on fabricating 3D parts out of different
materials such as plastics, polymers, metals, and www.openaccesspub.org J3DPA CC-license DOI : 10.14302/issn.2831-8846.j3dpa-18-2207
Vol-1 Issue 1 Pg. no.– 3
Corresponding author: Amir Dehghanghadikolaei, Oregon State University, USA. Email: amir.dehghanghadikolaei@rockets.utoledo.edu
Received: July 05, 2018
Accepted: July 18, 2018
Published: July 19, 2018
Editor: Alessandra Caggiano, University of Naples Federico II
Citation:Amir Dehghanghadikolaei (2018) Additive Manufacturing as A New Technique of Fab-
rication. Journal of 3D Printing and Applications - 1(1):3-4. https://doi.org/10.14302/issn.2831
-8846.j3dpa-18-2207 Freely Available Online Freely Available Online ceramics. Additive Manufacturing as A New Technique of
Fabrication Thus, there is always an opportunity to have
a scientific look at defects of each of these studies and
find a solution. “Journal of 3D printing and applications”
encourages the submission of papers focusing on AM of
engineering materials, post processing of AM parts and
investigation of new possibilities in this field. All forms
of papers are welcomed, including review, original
research article, perspective, short communications, etc. CC-license DOI : 10.14302/issn.2831-8846.j3dpa-18-2207
Vol-1 Issue 1 Pg. no.– 4 References 1. Elahinia, M., et al., Additive manufacturing of NiTiHf
high temperature shape memory alloy. Scripta
Materialia, 2018. 145: p. 90-94. 2. Moghaddam, N.S., et al., Anisotropic tensile and
actuation properties of NiTi fabricated with selective
laser melting. Materials Science and Engineering: A,
2018. 724: p. 220-230. 2. Moghaddam, N.S., et al., Anisotropic tensile and
actuation properties of NiTi fabricated with selective
laser melting. Materials Science and Engineering: A,
2018. 724: p. 220-230. 3. Elahinia, M.H., et al., Manufacturing and processing
of NiTi implants: A review. Progress in materials
science, 2012. 57(5): p. 911-946. 4. Dehghan
Ghadikolaei,
A. and
M. Vahdati,
Experimental study on the effect of finishing
parameters on surface roughness in magneto-
rheological
abrasive
flow
finishing
process. Proceedings
of
the
Institution
of
Mechanical
Engineers,
Part
B:
Journal
of
Engineering
Manufacture, 2015. 229(9): p. 1517-1524. 5. Dehghanghadikolaei, A., J. Ansary, and R. Ghoreishi,
Sol-gel process applications: A mini-review. Proc. Nat. Res. Soc, 2018. 2: p. 02008. www.openaccesspub.org J3DPA CC-license DOI : 10.14302/issn.2831-8846.j3dpa-18-2207
Vol-1 Issue 1 Pg. no.– 4 DOI : 10.14302/issn.2831-8846.j3dpa-18-2207
Vol-1 Issue 1 Pg. no.– 4 Vol-1 Issue 1 Pg. no.– 4 www.openaccesspub.org J3DPA CC-license DOI : 10.14302/issn.2831-8846.j3dpa-18-2207
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English
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Trends and innovations in the formulation of plant-based foods
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Food production, processing and nutrition
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cc-by
| 11,891
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Abstract Globally, the production, distribution, sale and consumption of plant-based foods (PBFs) are on the increase due to
heightened consumer awareness, a growing demand for clean label products, widespread efforts to promote and
embrace sustainable practices, and ethical concerns over animal-derived counterparts. This has led to the exploration
of several strategies by researchers and the food industry to develop alternative milk, cheese, meat, and egg products
from various plant-based sources using technologies such as precision fermentation (PF), scaffolding, extrusion, and
muscle fibre simulation. This work explores current alternative protein sources and PBFs, production trends, innova-
tions in formulation, nutritional quality, as well as challenges restricting full utilization and other limitations. However,
PBFs have several limitations which constrain their acceptance, including the beany flavour of legumes, concerns
about genetically modified foods, cost, nutritional inadequacies associated micronutrient deficiencies, absence of
safety regulations, and the addition of ingredients that are contrary to their intended health-promoting purpose. The
review concludes that investing in the development of PBFs now, has the potential to facilitate a rapid shift to large
scale consumption of sustainable and healthy diets in the near future. Christabel Tachie1, Ifeanyi D. Nwachukwu2 and Alberta N. A. Aryee1* Christabel Tachie1, Ifeanyi D. Nwachukwu2 and Alberta N. A. Aryee1* Food Production, Processing
and Nutrition Food Production, Processing
and Nutrition Tachie et al. Food Production, Processing and Nutrition (2023) 5:16
https://doi.org/10.1186/s43014-023-00129-0 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16
https://doi.org/10.1186/s43014-023-00129-0 Open Access Highlights Highlights
• Various novel food products have been developed from plants
• Animal-derived foods have a higher risk of diet-related metabolic disorders
• Improving PBF characteristics and nutrient composition will increase patronage
• PBFs production is more environmentally friendly than animal-derived foods
• PBF industry still in its infancy requiring adequate safety regulations and quality standards
Keywords Plant-based foods, Vegetarian diet, Sustainability, Healthy diets, Precision fermentation *Correspondence:
Alberta N. A. Aryee
aaryee@desu.edu Correspondence:
Alberta N. A. Aryee
aaryee@desu.edu
Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Page 2 of 14 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition Graphical Abstract Graphical Abstract Graphical Abstract Legumes g
Pulse is coined from the Latin word “puls” and belongs
to the legume family grown for its edible seeds (Semba
et al. 2021; Tidåker et al. 2021). All pulses are legumes
but not vice versa. Legumes are distinct from pulses as
“pods or fruits” containing seeds or dry grains (S. Kumar
& Pandey 2020). Legumes are affordable, nutrient-dense,
and excellent sources of protein, dietary fibre and minor
nutrients such as iron and vitamins, and include len-
tils, chickpeas, pinto beans, red kidney beans, soybean,
peanuts, and fresh pod beans (Keshavarz et al. 2020). Recently, an Israeli company developed a new protein
isolate from their cross-bred pea with about 65—72%
protein to be used as a base in plant-based products
with improved nutritional benefits and functional prop-
erties including high solubility, emulsification and gela-
tion (Southey 2022a). Due to the lower glycaemic index
of legumes and their high dietary fiber content, regular
consumption can help maintain normoglycemia and
lower the risk of cardiovascular diseases (Bresciani &
Marti 2019; Mullins & Arjmandi 2021). Legumes are a
generally healthier replacement for conventional meat
and for improved nutritional value, they can be com-
plemented with other plant sources such as cereals
(Proveg 2019). It has also been reported that incorporat-
ing pulses into meat products reduces lipid oxidation,
microbial spoilage and also enhances the functional
properties in the samples such as swelling, emulsification
and water/oil holding capacity (Mulla et al. 2022; Puro-
hit et al. 2016). For instance, adding red lentils, green
peas and grass pea flour to pasta improved its nutritional
characteristics and served as a natural colour additive
(Teterycz et al. 2020). The color effect was contributed
by carotenoids and chlorophyll in the seed coat and coty-
ledons of bean samples with the red lentil showing the
highest colour intensity (Teterycz et al. 2020). Conventional protein Conventional sources of proteins such as red meat, poul-
try, dairy products, eggs and seafood typically represent
the source of the majority of proteins used as food, with
protein content ranging from the lowest in eggs (51%) to
the highest in the milk protein, calcium caseinate (86–
93%) (Atallah et al. 2021; Gorissen et al. 2018). Animal-
based protein sources are regarded as high-quality since
they contain essential amino acids that are needed for
the various metabolic functions in the body. Bovine milk
contributes up to 85% of milk consumed globally with a
protein content of about 3.4% and contains all the essen-
tial amino acids in large amounts (Małecki et al. 2021). In red meat, total protein content ranges from ~ 19—31%
(Boler & Woerner 2017). The average protein content of
whole and freshly-laid egg is approximately 12.6% with
the egg white contributing about 10% of the total proteins
(Boler & Woerner 2017; Réhault-Godbert et al. 2019;
Salter 2019). In addition to their considerable protein
content, eggs also have the added advantage of being low
in calories and more affordable compared to red meat
(Małecki et al. 2021). Although animal-based proteins are
very nutritious, highly digestible and bioavailable (Asche-
mann-Witzel et al. 2020), frequent consumption of red
and processed meats (including bacon, ham and sausage)
has been linked to diseases such as cancer, type-2 diabe-
tes and obesity (Salter 2019; Zheng et al. 2019). Given the
world’s burgeoning population, longer average lifespan
and higher average purchasing power, the correspond-
ing increase food demands and nutritional requirements
underlines the urgent need for exploring novel sources of
food proteins (Salter 2019). Introduction Aside from macro-nutrients such as
proteins, PBFs also contain wide variety of carbohydrate Page 3 of 14 Page 3 of 14 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 The Agri-food system is simultaneously a significant
contributor to and at risk of adverse climate change
events due to the emission of GHGs in the normal course
of agricultural activities such as livestock production. Lifestyle changes and specifically changes in eating habits
have been reported to promote environmental sustain-
ability (Semba et al. 2021). For instance, eating according
to the Eatwell plate will minimize GHG emissions and
land use by 45% and 49%, respectively (Askew 2022b). Replacing meat-based diets with PBFs has the potential
to enhance soil enrichment through nitrogen fixation, as
well as decrease water use and reduce the carbon foot-
print (Keshavarz et al. 2020; Semba et al. 2021). Seeds and nuts Seeds and nuts such as flaxseed, peanuts and almonds
are notably high in proteins, but are generally lacking in
certain amino acids, making them insufficient in meet-
ing amino acid needs e.g., peanuts lack valine and lysine. Others including cottonseed, sunflower seed, sesame
seed, pumpkin seed, hazelnut, grape seed, walnut, can-
ola seed, hemp seed and canola are important oilseed
crops with relatively high protein meal content (Access-
wire 2022a; Langyan et al. 2022). Some seeds such as
pumpkin, sunflower and hemp have also been used to
produce alternative eggs, cheese and flour products
respectively (Accesswire 2022b; Watson 2021). Proteins
from canola seeds used in sports beverages and fro-
zen desserts exhibit good emulsification properties and
improve colour and consistency (Watson 2022b). The Introduction types with varying role in the development of a diverse
intestinal flora and a substantial amount of micronu-
trients such as vitamins, minerals, and bioactive com-
pounds (Hever & Cronise 2017; Samtiya et al. 2021). For instance, soy products also represent a good source
of calcium and isoflavones which may help maintain
healthy bones and lower the incidence of osteoporosis
(Qin et al. 2022). Consumers’ concern about their choice of food, and their
possible health and environmental implications has led
to noticeable changes in dietary patterns and a growing
shift to the consumption of PBFs, mostly for the purpose
of promoting healthful living, conserving animal life
and enhancing environmental sustainability (Bresciani
& Marti 2019; Estell et al. 2021; Małecki et al. 2021;
Nychas et al. 2021). There now exists an increased con-
sciousness among many consumers to adopt PBFs due to
ethical concerns, campaigns to reduce livestock use and
meat consumption by animal rights/welfare organiza-
tions, and the heightened emission of environmentally
harmful greenhouse gases (GHG) as a result of animal-
based food production (M. Kumar et al. 2022). Other
drivers include excessive use of resources, urbanization,
improper distribution of protein intake (overconsump-
tion) in developed countries often with negative health
implications, as well as population growth (Aschemann-
Witzel et al. 2020; World Economic Forum 2019). However, PBFs are generally lacking in certain essen-
tial amino acids and are consequently regarded as
incomplete proteins (M. Kumar et al. 2022). It has been
reported that the consumption of PBFs reduced the
risk of cardiovascular disease, enhanced cardiovascular
health, controlled glycaemic levels, as well as lowered
blood cholesterol level, obesity, and blood pressure
(Kahleova et al. 2017; Kim et al. 2019). PBFs in the
form of soy products partially substituted for meat have
been shown to improve insulin sensitivity (Van Nielen
et al. 2014) and enhance the gastrointestinal response
to regulate glycaemic index (Kahleova et al. 2017). In
addition, plant-based products such as meatless burg-
ers from Beyond Meat® and Impossible Foods® contain
no cholesterol and are designed to have significantly
lower saturated fat contents than traditional beef burg-
ers (Zheng et al. 2019). Several studies have shown that PBFs help to delay
the onset, reduce the risk of and even prevent certain
disease conditions (Bowman 2020; Kahleova et al. 2017;
Kim et al. 2019). Food waste materials Every year, billions of dollars are lost in countries all over
the world as a result of food waste. For instance, about
3.5 billion tons of peel waste is generated from bananas
annually. Banana peels contain a considerable amount
of carbon compounds that decompose to generate vola-
tile odoriferous compounds, GHGs and which therefore
contribute to adverse climate change events (Ewing-
Chow 2022). The peels also contain proteins, dietary
fibre, polyunsaturated fatty acids, calcium and vitamin
A (Ewing-Chow 2022), as well as antioxidants such as
carotenoids and polyphenolic compounds. Banana peels
have also been used for ethanol production and as a pri-
mary material for pectin extraction (Shalini 2015). Whole
banana peel and flour can be incorporated into bread,
tea, dried snacks (with peels) and bacon alternatives
to increase their nutritional value (Martins et al. 2019;
Wells 2021). Due to its ability to foam, gelate, thicken, and emulsify
foods, aquafaba (spent liquid from cooked chickpea) has
caught the interest of vegans and those who design culi-
nary products. It is stable over a wide range of pH and
temperature conditions and is very process tolerant. In
addition, aquafaba contains no fat and starch, thus mak-
ing it different from protein isolate and chickpea flour. Instead, it is a diluted solution that contains soluble poly-
saccharides, phenolic compounds, saponins, low molecu-
lar weight, and water-soluble proteins (mainly albumin)
(Mustafa & Reaney 2020). The main organic waste prod-
ucts from food production include seeds, peels, bracts,
leaves, roots, bark, and midribs. Numerous bioactive sub-
stances including phytochemicals with nutritional and
functional value can potentially be found in these waste
products. For instance, the peels of pineapples contain
about 222—428 mg GAE/100 g DW phenolics and can
be used for several functionalities in processed foods
including substrates for single-cell proteins, prebiotics,
anti-browning agents, texture enhancers and as additives
in new products (Pattnaik et al. 2021). Barley and wheat
are present in spent grain, a by-product that is typically
discarded following the mashing process in beer-mak-
ing (Garcia-Garcia et al. 2019). Other alternative and emerging protein sourcesh The cell, clean or lab-grown meat concept involves the
cultivation of animal cells to produce meat, outside the
body of real animals, with similar sensory characteris-
tics and nutritional profiles as the conventional meat
(Innova Communcations 2021; Specht 2018; Swartz &
Bomkamp 2020). Lab cultured meat and fish products
have been developed to prevent the extinction of certain
species, and produce meat with fewer antibiotics and
microbial contamination (Rodriguez Fernandez 2022). To
produce lab-cultured meat, the cells needed to cultivate
the meat are obtained from healthy animals, grown in
bioreactors or cultivators on nutrient-rich media includ-
ing amino acids, vitamins, inorganic salts and other
supplements (Swartz & Bomkamp 2020). The nutri-
ent medium fed to the system is varied and this differ-
entiates the cells into various muscles, fat and tissues as
seen in meat. This process is usually completed within
2–8 weeks, depending on the meat of interest (Swartz
& Bomkamp 2020). In fact, foods such as ground beef,
steak, pork and poultry, sausage, fish and egg white have
been produced by the food industry using cell culture
(Rodriguez Fernandez 2019). Results from Szejda et al. (2021) in their study to determine the acceptance rate of
cell-cultured meat in the US and UK showed that about
40%, 10% and 25—30% of consumers are eager to try cul-
tured meat, willing to spend more on cultured meat, and
make regular purchases, respectively. Consumers also
revealed that cultured meat with Food and Drug Author-
ity (FDA) or United States Department of Food and Agri-
culture (USDA) approval, and which are non-genetically
modified will be preferred and should be labelled culti-
vated or cultured meat instead of cell-based meat. The
cultivated meat concept is still in its infant stages and has
a huge potential for growth depending on full consumer
acceptability and the establishment of adequate regula-
tory standards for their production. Plant‑based sources Plant-based protein sources include legumes, seeds and
nuts, and by-products/waste from food production have
been studied and utilized to simulate meat and other ana-
logues. Their nutritional and functional properties, health
benefits and food applications are described below. Tachie et al. Food Production, Processing and Nutrition Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Page 4 of 14 wheat and rice which are obtained after polishing rice are
also extracted due to their amino acid content for use in
food products. oil extracted from rapeseed, sunflower and palm is used
by industries to produce plant-based butter and marga-
rine. Alternative or vegan cheese has also been produced
from cashew and macadamia nuts oil while starch is
often added to make hard cheese (gouda) when needed
(Southey 2022c). Plant‑based meat (PBMs) PBM is produced to mimic meat not only for vegetarians
but for ‘meat lovers’ or flexitarians (Bellone et al. 2022;
Clayton & Specht 2021). The production of PBM involves
analysing and selecting appropriate plants and other raw
materials (Allen 2018). Modified legume protein isolates
for example, processed using hydrostatic pressure tech-
nology when included in PBMs improve functional prop-
erties during the product formulation process (Clayton
& Specht 2021; Mulla et al. 2022; Semba et al. 2021). A
combination of legumes e.g., pea and oat protein will
not only add to the required protein content (60–70%)
for good texture in extruded meat products but will also
improve the amino acid profile, wettability, and stabil-
ity over a wide temperature range (Lantmännen 2022). The hardness and dryness of alternate meat products,
however, increase as the percentage of oat protein in the
formulation increases (Lantmännen 2022). Other nutri-
ents such as starch, fats, and additives such as lecithin are
added to improve the sensory characteristics of PBMs. Burger formulations which include lecithin are juicier
and tastier, uniformly brown and sizzle when baked
compared to those produced without lecithin (Crok-
laan 2022), while the commercial citrus fibre, Herbacel,
has been reported to improve final product texture and
freeze–thaw stability, and also minimizes cooking and
frying loss (Herbafood 2022). In order to obtain the desirable fibrous nature of meat,
plant proteins are texturized through shearing, extrusion
and spinning. The temperature involved in the extru-
sion process accounts for the structural and chemical
changes in plant protein to improve their functionality in
the PBM products (Krintiras et al. 2016). The character-
istics and cost of PBM can be improved by substituting
cell-cultured fat into pure cultured PBM products (Rubio
et al. 2020). For instance, cell-cultured pork fat from Mis-
sion Barns is incorporated into the hybrid pork alter-
native from Herotein to improve the taste as well as to
subsidise production cost (Neo 2021). Other producers
such as Better meat and Tyson foods in order not to lose
the market to PBM industries blend meat products with
plant-based ingredients to produce hybrid (50/50) prod-
ucts which taste better and have reduced environmental
impact than 100% meat (Hill 2021). Plant‑based foods and alternative protein products
A sustainable diet, as defined by the Food and Agriculture
Organization (FAO) must be safe, healthy, have sufficient
nutrients and low environmental impact (Askew 2022b). Food waste materials Fish
food, beverages, protein bars and pharmaceuticals can
also be formulated using insect proteins (Grand View
Research 2022). Protein isolate made from Gryllodes sig-
illatus was reported to have high foaming capacity and
stability of 99% and 92%, while Schistocerca gregaria and
Tenebrio molitor, had an emulsion stability of 51.31 and
50.40 respectively, making them suitable to be incorpo-
rated into food (Zielińska et al. 2018). Food waste materials Spent grains with about
11% protein and 12.7% carbohydrates and beer yeast can
be used as feed for rearing insects such as mealworms
to serve as an alternative protein source in human food
(Mussato 2014; Varelas 2019), while cereal bran such as Given the continuing rise in global population, demand
for seafood could double by 2050, thus highlighting the
necessity for research into alternative seafood produc-
tion using methods such as fermentation technology. However, seafood has varied and unique flavour profiles
with respect to each fish type hence the difficulty in mim-
icking them. Thus, to create desirable seafood products,
culinary and sensory needs must be aligned with scien-
tific and technological realities in order to develop ‘whole
cut’ products (Southey, 2022b). Insect protein is also growing in popularity. Insects
contain proteins, mono- and polyunsaturated fats, Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition Page 5 of 14 minerals (about 17% of zinc and 25% of iron) and some
vitamins (Van Huis 2015). The profit from insect pro-
tein was $249.9 million in 2020 and is projected to con-
tinue to rise annually by a compound rate of 27.4% from
2021 to 2028 (Grand View Research 2022). The interest
in insects as a novel protein source especially in the US,
U.K, France, Germany, Thailand, India, South Africa, and
Kenya is due to the high demand for alternative proteins
(De Castro et al. 2018; Grand view research 2022; Leb-
lanc 2019). Insects contain peptides with antihyperten-
sive, antimicrobial and antioxidant properties, and can be
used in food applications (De Castro et al. 2018). Insects
are consumed as food and used as livestock feed in most
parts of the world where they are obtained from the wild
or forest population (De Castro et al. 2018; Salter 2019;
Van Huis 2015). Insects are efficient feed converters with
minimal dependence on water for their survival (Grio-
pro 2016; Van Huis 2015). They are poikilothermic, with
no body fat and are able to conserve water due to their
tough exoskeleton (Griopro 2016). Their production
requires less land and pesticide use. For bulk produc-
tion, insects are reared in automated systems as mini-
livestock (Van Huis 2015). Plant‑based meat (PBMs) Meat, egg, dairy products, bakery products (snacks), bev-
erages, and dietary supplements have been developed
from plant and alternative protein sources (Bunge 2022). The traditional source of these products, for instance
cheese, has unique characteristics which confer distinct
taste and flavour. For alternative cheese, producers must
observe and take note of the interactions between ingre-
dients, processing methods effects, proportion and vari-
ous formulations, for easy replication and optimization
(Edlong 2022). In plant-based meat products, the com-
mon characteristics consumers expect are taste/flavour Food waste materials The insect order Coleoptera
(beetles, aquatic beetles, wood-boring larvae, and dung
beetles), Orthoptera (locusts, grasshoppers, crickets)
and Lepidoptera (caterpillars, butterflies, and moths)
are widely used in insect-based oil, protein powder, and
flour because they contain high amounts of protein
and are easy to breed (Grand view research 2022). Fish
food, beverages, protein bars and pharmaceuticals can
also be formulated using insect proteins (Grand View
Research 2022). Protein isolate made from Gryllodes sig-
illatus was reported to have high foaming capacity and
stability of 99% and 92%, while Schistocerca gregaria and
Tenebrio molitor, had an emulsion stability of 51.31 and
50.40 respectively, making them suitable to be incorpo-
rated into food (Zielińska et al. 2018). intensity, texture (firmness and hardness), and nutritional
value (Griffith foods 2020). minerals (about 17% of zinc and 25% of iron) and some
vitamins (Van Huis 2015). The profit from insect pro-
tein was $249.9 million in 2020 and is projected to con-
tinue to rise annually by a compound rate of 27.4% from
2021 to 2028 (Grand View Research 2022). The interest
in insects as a novel protein source especially in the US,
U.K, France, Germany, Thailand, India, South Africa, and
Kenya is due to the high demand for alternative proteins
(De Castro et al. 2018; Grand view research 2022; Leb-
lanc 2019). Insects contain peptides with antihyperten-
sive, antimicrobial and antioxidant properties, and can be
used in food applications (De Castro et al. 2018). Insects
are consumed as food and used as livestock feed in most
parts of the world where they are obtained from the wild
or forest population (De Castro et al. 2018; Salter 2019;
Van Huis 2015). Insects are efficient feed converters with
minimal dependence on water for their survival (Grio-
pro 2016; Van Huis 2015). They are poikilothermic, with
no body fat and are able to conserve water due to their
tough exoskeleton (Griopro 2016). Their production
requires less land and pesticide use. For bulk produc-
tion, insects are reared in automated systems as mini-
livestock (Van Huis 2015). The insect order Coleoptera
(beetles, aquatic beetles, wood-boring larvae, and dung
beetles), Orthoptera (locusts, grasshoppers, crickets)
and Lepidoptera (caterpillars, butterflies, and moths)
are widely used in insect-based oil, protein powder, and
flour because they contain high amounts of protein
and are easy to breed (Grand view research 2022). Plant‑based dairy products About 7.4% of the market for milk is occupied by plant-
based dairy alternatives (Laila et al. 2021; Schiano
et al. 2020) and this is likely to rise by 8.8% between 2021
and 2031 (Southey 2022d). Plant-based dairy products Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition Page 6 of 14 Page 6 of 14 products with improved organoleptic properties (Teng
et al. 2021). Chai et al. (2020), described the use of fer-
mentation to transform substrates into value-added
products such as enzymes, peptides, probiotics and other
biotechnological products using endogenous microbes,
starter culture or a portion of a previously fermented
product. PF is one of the newest and major technologies
used to produce PBFs where microbes are redesigned to
produce specific, customised and recombinant molecules
to yield new food ingredients. The goal of PF is to pro-
duce newer protein sources with desirable textural and
taste characteristics for increased consumer acceptance
(Vanhercke & Colgrave 2022). It targets the microbial
genome where genetic information of specific proteins is
modified. contain milk sourced from legumes (soy, lupine chick-
pea), nuts (coconut, hazel), seeds (sunflower, hemp),
pseudo cereals (quinoa), and cereals (oat), and have dis-
tinct flavours (Tangyu et al. 2019). It has been argued
that milk from plant sources should be diversified in
order to obtain maximum nutritional benefits and to pre-
vent exploitation of the market for a particular product
(Marinova & Bogueva 2020). The lactose-free property of plant-based dairy products
is of interest to many lactose-intolerant consumers (Laila
et al. 2021). A recent survey conducted by Cargill revealed
that 20% of consumers opted for plant-based dairy prod-
ucts to support animal life conservation (Loria 2018). Another study in Canada revealed that most parents rec-
ommended the use of plant-based dairy because they do
not contain antibiotics and hormones which can affect
the health of their children (Laila et al. 2021). Other rea-
sons for the increase in the non-dairy industry are scar-
city of bovine-derived milk in some areas, as well as low
cholesterol levels and high bioactive phytochemicals
of plant-based milk (Paul et al. 2019). The plant-based
dairy industry seeks to produce milk with organoleptic
and nutritional properties similar to conventional milk. Barriers/limitations to adoption of plant‑based
foodsh Though PBFs patterns were associated with better con-
sumer and environmental health, and people are being
encouraged to consume more PBFs, their impact vary
greatly. For instance, the higher scores for unhealthy
plant-based diet index were associated with higher con-
sumption of unhealthy plant-based diet such as refined
grains, sugary drinks, fruit juice, potatoes, and sweets/
desserts (Musicus et al. 2022). Some plant-based ingredients such as legumes and
cereals contain varying amounts of anti-nutrients such as
phytates, saponins, tannins, protease and amylase inhibi-
tors, and goitrogens that limit the amount of the ingredi-
ent that can be used in formulation due to their ability
to form complexes with proteins and minerals reduc-
ing protein digestibility and overall nutritional quality,
inhibit mineral absorption, cause stomach discomfort,
and toxic when accumulated (Acquah et al. 2021; Sam-
tiya et al. 2020). To minimize anti-nutrient content and
cooking time, pulses are mostly soaked for several hours,
however this may be inconvenient for some consumers
(Szczebyło et al. 2020). The poor bioavailability of certain
minerals (e.g., calcium, zinc, iron and iodine) and low
content of vitamins (A, B2, B12, and D) has necessitated
critical examination of PBFs and the inclusion of supple-
mentary/alternative sources of these nutrients (Protudjer
& Mikkelsen 2020). Plant‑based dairy products In the manufacturing of non-dairy milk, milk extract
from the desired plant source is combined with other
ingredients such as oil, additives (enzyme deactivators),
emulsifiers, thickeners (locust bean gum), flavours, pre-
servatives and nutrients (tricalcium phosphate), before
being filtered, sterilized and properly homogenised to
prevent separation over time (Aydar et al. 2020; Mccle-
ments 2020). Some examples of commercial plant-based
dairy products currently available to consumers include
cheese, milk and yoghurt (Ganeshram 2021; Veganuary
2022). A commercial plant-based powdered milk which
was produced with the intention of reducing the weight
of the final product for convenience and which consum-
ers only need to dissolve in water as needed, is also cur-
rently available to consumers (Southey 2022d). i
Other technologies used in the production of PBM
through cell culture include tissue engineering, and cell-
based therapeutics (Specht 2018). Extrusion is another
processing technique used for commercial production
of PBFs where all ingredients are mixed, preconditioned,
cooked, and extruded through dies (Moses 2022). Based
on the company’s product specifications, the extruded
product is subjected to further processes including trim-
ming, marinating and grinding. Production methods and innovations
in formulation Several methods and innovations including blending,
cell culture, precision fermentation (PF), and genetic
engineering have been explored in the manufacture of
PBFs. Most PBF producers mimic the sensory properties
and nutritional characteristics of conventional protein
sources to attract consumers and obtain a wider mar-
ket beyond vegetarians (Nwachukwu & Aluko 2021). In
Table 1, some PBF producers, products and target for
sustainability are shown. Fermentation is a convenient, adaptable, technology
that preserves food, increases shelf life, enhances nutri-
tional quality, and has been used for developing new Page 7 of 14 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Table 1 PBF industries, products and sustainability goals
Company
Location
Product/name
Ingredients
Production
Target
References
Impact food
Finless food
Emeryville, California, U.S
Seafood (tuna)
Pea protein isolate, starch,
algae
Blending and Cell culturing
Prevent overfishing and
blue fin species extinction
Zero- food waste
Crawford 2022; Watson 2022c
Merit food
Manitoba, Canada
Puratein Canola protein and
pea protein
Canola seeds
Proprietary extraction and
membrane filtration
Reduced environmental
impact, uses less water
Merit Functional foods 2022a,
2022b
NotCo
Santiago, Chile
Not burger
Not Milk
Notblood, Notmatrix, pea
protein, pineapple cabbage
and seeds
Artificial intelligence
(Giuseppe) generates plant
combinations followed by
blending ingredients
Low carbon and water foot-
print. Production methods and innovations
in formulation Minimizes pressure on
land and energy usage
Watson 2022b
Perfeggt Egg
Germany
Alternative Egg
Fava beans
Not available
Uses 5% fresh water, emits
25.7% GHG and less land
use
Southey 2021
Helaina
New York, U.S
Infant formula (breast milk
alternative)
Yeast
PF
Production method with a
low environmental footprint
Marston 2022
Nepra food
Chile
Propasta, Hemp seed flour
(N-50), Cheese
Hemp seeds
Cold press, mill and blend
hemp meal with other
ingredients
Uses less land, water and
energy resources to pro-
duce hemp seed
Accesswire, 2022a; Nepra
Foods 2022a, 2022b
Ynsects
Paris, France
Protein powder for feed
(Ynmeal) and food (Adalba
Pro)
Molitor larvae (feed) and
buffalo meal worm (food)
Proprietary technology
Low carbon emission and
land use
Grand view research, 2022;
Ynsect 2022
Veg of Lund
Sweden
Dug potato milk
Pea protein, rapeseed oil,
emulsifiers and chicory fibre
Blending
About 75% lower climate
footprint, minimized water
and land usage
Synergy foods 2022;
Anay 2021
Current food
San Francisco, U.S
Kuleana
Radish, bamboo, algae,
potato
Proprietary (scaffolding)
technology
Restore the ocean, no
planet strain
Buxton 2022; Shieber 2020
Hooray foods
San Francisco, U.S
Bacon alternative
Rice flour and cassava
Encapsulation of fats and
other ingredient
Combat climate change,
prevent animal mishandling
and slaughtering
Lyra 2021; Wilder 2021
Herotein
China
Hybrid beef and pork
products
Soy, pea and wheat and cell
cultured fats
Muscle fibre simulation
technique and cell culture
Low environmental impact
Neo 2021
Spero
San Jose, U.S
Egg and cream cheese
alternative
Sunflower (cheese) seeds,
pumpkin seeds (egg) mush-
room extract
Not available
Uses 96% and 99% less land
and CO2 emission for pro-
duction compared to nuts
and dairy, respectively
Watson 2021; Spero
Foods 2022a, 2022b
Impossible food
California, U.S
Burger, sausage and
chicken-like nuggets
Soybean and potato
Genetic engineering using
yeast
Conserve water, save
wildlife and reduce GHG
emission footprint
Morrison 2021
Roslin Technologies
UK
Cultivated meat
Animal stem cells
Advanced cell technology
Conserve animal life and
prevent negative envi-
ronmental impact due to
animal production
Roslin Technologies 2022 Page 8 of 14 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 A poorly formulated PBFs using legumes with low
non-heme iron content and absorption compared to
meat requires that consumers eat more to meet the iron
requirement (Semba et al. 2021). Production methods and innovations
in formulation Consuming high lev-
els of heme raises body iron content and increases the
risk of type-2 diabetes (Zheng et al. 2019). For instance,
commercial meatless burgers contain high amounts
of sodium (16—17%) and heme (20—25%) per serving
(Beyond Meat 2022; Zheng et al. 2019). 2021). The FDA also recommends the consumption
of < 10% (239 kilo calories) SF per day at age 2 or older,
18 mg iron and < 2300 mg of sodium (Na) (U.S Depart-
ment of Agriculture 2020). In Table 2, the nutrient com-
position of some plant-based products is presented. In addition, the media used for culturing meat are
mostly sourced from the fetal blood of a slaughtered
pregnant cow which makes it expensive and at odds with
the stance of animal welfare/rights groups (Rodriguez
Fernandez 2022). y
g
For people with food allergies, navigating diets that
also minimizes nutritional deficiencies can be challeng-
ing. With about 467 allergens from certain PBF sources
and about 436 being allocated to specific food protein
families including 2S albumin, non-specific lipid trans-
fer proteins, legumins, cereal prolamins, and profilins
(Costa et al. 2022), greater attention is warranted by con-
sumers with food allergies. Among the 170 foods that
have been found to trigger allergenic reactions in the US,
peanut, tree nut, wheat and soy are predominant, as well
as some cross-reactivity between plant-based sources. Sesame seeds, lupines, mustard, buckwheat, gluten
from wheat and soy protein have also been associated
with allergenic reactions in other parts of the world
(Bresciani & Marti 2019; Hertzler et al. 2020). Aller-
gic responses happen when the immune system targets
and attacks typically safe dietary proteins, causing tem-
porary to severe and life-threatening symptoms (Hert-
zler et al. 2020). Consumers may consider other nuts,
cereals and legumes as alternatives (Protudjer & Mik-
kelsen 2020). The use of precautionary allergen labelling
is strongly encouraged. PBMs have organoleptic properties which are very dif-
ferent from conventional meat, are costly, and often come
with unfamiliar ingredients on product labels (Morri-
son 2022). Additionally, alternative cheese is costlier with
less nutritional value (Southey 2022c), while some plant-
based milk produced from nuts such as almonds requires
a lot of water during production (Southey 2022d). Production methods and innovations
in formulation Most consumers view PF as an unnatural and synthetic
process that is directly linked to genetically engineered/
modified (GM) foods which are seen by some consum-
ers as a threat to human health (Teng et al. 2021). Peo-
ple seeking PBFs expect foods made from real plants due
to cultural or personal reasons or otherwise considered
“modified/unnatural”. Food engineered through PF also
has “ill-disposed” labels such as ‘nature identical’, and
‘precision fermentation’ with no explicit information on
the content of the food (Bellingham 2022) which raises
doubts about their consumption. Aside from the substan-
tial increase in yield and final product purity, there is no
distinction between naturally produced foods and those
synthesized through GM technology (Teng et al. 2021). Another potential PBFs pitfall is the susceptibility to
aflatoxin invasion, favism and high alkaloid contents
in peanuts, fava beans and lupins, strong beany flavour,
extended processing time and lack of standardized tech-
niques minimise the consumption of legumes (Acquah
et al. 2021; Semba et al. 2021). Many consumers are uncomfortable and largely unen-
thusiastic about consuming insects (Leblanc 2019). Lastly, lack of convenience for meatless meals, limited
options to choose from, and negative reactions by other
consumers also serve as a barrier to the consumption of
PBFs (Graça et al. 2019). The processing of many traditional vegetarian dishes
(tofu) requires less oil and salt. However, some plant-
based burgers and sausages available in the market
contain more salt and saturated fats (SF), leading to
increased calories and salt content when consumed (Tso
& Forde 2021). Some consumers have also expressed
concerns that plant-based dairy products are costly, not
readily available in the supermarkets and that some have
high sugar content which can affect oral health (Aydar
et al. 2020; Laila et al. 2021). Excessive use of sugar and
salt to mask undesirable characteristics in plant-based
products might limit its intake since the main reason
for the switch to these products is for health benefits
(Pratt 2020). It is recommended that the diet consumed
per day should contribute 200 cal or less (5—6%) SF out
of the 2000 cal needed (American Heart Association, Regulations and safety concerns PBFs are mostly displayed next to traditional products
in most supermarkets. Product label captures consum-
ers’ attention and must be informative to avoid mislead-
ing them. It is expected that the FDA will present a draft
label guidance for PBF products based on consumers’
understanding of the distinction between PBF terms
and nutritional composition from conventional products
(Glick 2022). The type and major ingredient/composition
must be clearly identified on the label with health claims
thoroughly reviewed by FDA (Glick 2022). Producers
should also include the score on animal welfare, water
and carbon footprint on their labels (Southey 2022d). Recently, France banned the use of terms used to
describe traditional meat such as meat, steak and bacon Page 9 of 14 Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Table 2 Nutrient composition of PBFs and FDA recommended daily intake of nutrients
Based on a 2000-cal daily diet
DV*
PBF
Amount of Nutrient (%) per
serving
References
Total fat (TF) (78 g)
SF (20 g)
Total carbohydrate (275 g)
Dietary fiber (28 g)
Sodium (2300 mg)
Potassium (4700 mg)
Impossible beef burger
Na – 16
SF – 16
Fe – 25
TF – 18
Total Carbohydrates – 3
Potassium – 15
Link 2022
Sweet Earth Burritos
Na – 24
SF – 35
Fe – 10
TF – 23
Total carbohydrate – 17
Potassium – 6
Sweet Earth 2022
Morning Star Vegan Cheese Burger
Na -27
SF – 20
Fe – 10
TF – 23
Total Carbohydrates – 4
Potassium – 4
MorningStar Farms 2022
Violife parmesan cheese
Na – 17
SF – 23
TF – 8
Total Carbohydrates – 3
Potassium- 0
MyFoodDiary 2022
Pepita Eggs
Na – 69
SF – 24
Fe – 20
TF – 17
Total Carbohydrates – 1
Potassium – 4
MyFitnessPal 2022 Table 2 Nutrient composition of PBFs and FDA recommended daily intake of nutrients Based on a 2000-cal daily diet for PBF product to prevent confusion among consumers
(Askew 2022a). been unfolded. While cell-based meat is currently being
regulated in Europe under Novel Food Regulation, the
US has not finalised the decision on which regulatory
body (FDA or the USDA) should handle cell-cultured
products (Robertson 2022). Producers are thus seeking
directions from consultants for cell-cultured products to
meet both FDA and USDA standards on good manufac-
turing practices, hygiene and hazard analysis critical con-
trol point (HACCP). Regulations and safety concerns Conventional protein sources have been widely stud-
ied in contrast to plant-based protein sources and their
alternatives (Boler & Woerner 2017; Małecki et al. 2021;
Salter 2019). PBF are highly susceptible to microbial load
than animal-based products due to the varied ingredients
and macronutrients which are combined in formulating
them (Engstrom 2021). PBFs must be carefully handled
and monitored during production and safety regulations
should be set to govern their use to satisfy consumers’
quality demands. Rules regarding storage for PBFs should
be properly established and the right cooking tem-
peratures necessary to eliminate microbes must be set. Luchansky et al. (2020) reported that the 62.8 °C—73.9 °C
minimum cooking temperature set by USDA for animal-
based meat also reduced salmonella, and Listeria mono-
cytogens load in both beef and plant-based burger to the
same level. In addition, insects reared on organic by-
products are prone to contamination which might com-
promise their safety (Van Huis 2015). Conclusion and future perspectivehi The benefits associated with the consumption of PBF
and increasing demand have attracted a lot of consumer
interest and incentivized entrepreneurs to venture into
PBF businesses. Knowing the barriers associated with
adopting PBFs and improving the characteristics based
on these limitations will help increase the patronage of
PBFs. A change in the current dietary pattern for PBFs
is encouraged to minimize the contribution of animal-
based products to climate change. The nascent nature
of entomophagy and the novelty of the plant-based food
industry require constant research and development
(R&D) to improve product characteristics and consumer
acceptance. The fat component of plant-based meat products is
usually sourced from coconut oil which has a low melt-
ing point (25%) as compared to beef fat (42—45%) (Wat-
son 2022a). Coconut fat tends to leak out or melt quickly
while cooking (decreasing juiciness), and it lacks the
distinct flavour found in either pork, beef or mutton fat,
therefore, requiring other food additives during process-
ing to make it desirable. This challenge can be overcome
by using microbes for example oleaginous fungi through
genetic engineering to produce fats with characteris-
tics similar to animal fats in plant-based meat products
(Watson 2022a). The structure and fibrous nature of
PBMs can be improved by incorporating fungi-based
products and fermented foods to mimic whole-cut meat
and seafood (Morrison 2022). Instead of producing PBFs
to only mimic conventional sources with respect to sen-
sory characteristics, producers should focus on increas-
ing the nutrient composition of these products (Tso &
Forde 2021) and providing a list of familiar ingredients
on labels (Morrison 2022). In addition, further research
should be done to develop products with reduced fat,
sodium and sugar content which are major nutrition
concerns. Plant-based ingredients must be diversified to increase
repeat purchases and patronage and improve function-
ality in developing products. The growth conditions of
these ingredients influence the sustainability of the final
product. Growing and supplying these ingredients at
the local or grassroots level will minimize the emission
associated with transportation while bringing consum-
ers closer to ingredient sources hence satisfying their
need for transparency (Askew 2022b). For this reason,
some companies have introduced blockchain technol-
ogy, where all the company’s information is kept and
made accessible to consumers to promote transparency
(Askew 2022b). Improving characteristics of plant‑based foods
to increase patronageh The texture, taste and nutrition of PBFs should be equiva-
lent to those of their animal-based counterparts in order
to favorably compete and be fully accepted by consumers
(Merit Functional ). Other factors such as price and famil-
iarity can affect the patronage of PBFs (Watson 2022c). f
High antinutrient composition in PBFs may be mini-
mized through pre-treatment procedures such as roast-
ing, soaking, fermentation, sprouting, milling and
removal of bran (Samtiya et al. 2020). Germinating
legumes for example Bambara groundnut usually for
three days minimizes antinutrients, improves protein Most manufacturers aim to upscale their products but
the regulation for cell-based meat for instance has not Tachie et al. Food Production, Processing and Nutrition (2023) 5:16 Page 10 of 14 ingredients such as oils and emulsifiers should be added
to create that colloidal effect which is a reason for the
desirable attributes of dairy milk (Mcclements 2020). and starch digestibility, enhances functional proper-
ties (pasting, water absorption capacity) and is rec-
ommended as a good pre-treatment method (Chinma
et al. 2021). Genetic mutation can reduce the antinutri-
ents to mineral acid ratio in legumes but such products
must be processed with caution to minimize the leach-
ing of nutrients for example when soaked or cooked
(Hummel et al. 2020). Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or
analysed during the current study. Funding The acceptance of plant-based milk will increase if
they are formulated to have similar functional proper-
ties which include foaming and stability in beverages and
structure, as conventional milk with no beany flavour. The beany flavour in soy milk may be eliminated through
processing. Substituting non-dairy milk for conventional
milk in products will be easier. To improve non-dairy
milk, the natural structure of the plant sources which will
be used should be disintegrated and other plant-based This work is supported by the U.S. Department of Agriculture, National Insti-
tute of Food and Agriculture [Grant no. 2021–67022-34148]. Acknowledgements
None Acknowledgements
None Authors’ contributions ANAA: Conceptualization, funding acquisition, project administration, supervi-
sion, writing—review & editing; CT: Writing—original draft; IDN: Writing—
review & editing. The authors read and approved the final manuscript. Conclusion and future perspectivehi The prolamin technology developed by Motif Food
Works in partnership with scientists from the Univer-
sity of Guelph uses corn to make plant-based cheese with
increased springiness (Cumbers 2021). In the extrud-
able fat technology, fat is passed through an extruder
and mixed with plant protein to develop a final product
with a marbling effect as seen in meat (Cumbers 2021). Beyond Meat has recently introduced a new burger with
less fat (35%) (Synergy foods 2022). Approaches such as
improving existing products based on consumers’ feed-
back when adopted by PBF manufacturers will help to
maintain the industry. Overall, current evidence suggests that PBFs are a bet-
ter and more viable alternative to animal proteins as they
confer health benefits, use fewer production resources,
and produce comparably more sustainable processing
methods and techniques. Ethics approval and consent to participate
Not applicable. y
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English
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Independent influence of negative blood cultures and bloodstream infections on in-hospital mortality
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BMC infectious diseases
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* Correspondence: carlv@ohri.ca
1Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario,
Canada
3Ottawa Hospital Research Institute, Clinical Epidemiology Program, ASB-1,
1053 Carling Ave, Ottawa, Ontario K1Y 4E9, Canada
Full list of author information is available at the end of the article © 2014 van Walraven and Wong; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Open Access Independent influence of negative blood cultures
and bloodstream infections on in-hospital
mortality Carl van Walraven1,3,4* and Jenna Wong2 Carl van Walraven1,3,4* and Jenna Wong2 Abstract Background: The independent influence of blood culture testing and bloodstream infection (BSI) on hospital
mortality is unclear. y
Methods: We included all adults treated in non-psychiatric services at our hospital between 2004 and 2011. We
identified all blood cultures and their results to determine the independent association of blood culture testing and
BSI on death in hospital using proportional hazards modeling that adjusted for important covariates. Results: Of 297 070 hospitalizations, 48 423 had negative blood cultures and 5274 had BSI. 12 529 (4.2%) died in
hospital. Compared to those without blood cultures, culture-negative patients and those with BSI were sicker. Culture-negative patients had a significantly increased risk of death in hospital (adjusted hazard ratio [HR] ranging
between 3.1 and 4.4 depending on admission urgency, extent of comorbidities, and whether the blood culture was
taken in the intensive care unit). Patients with BSI had a significantly increased risk of death (adj-HR ranging between
3.8 and 24.3] that was significantly higher when BSI was: diagnosed within the first hospital day; polymicrobial; in
patients who were exposed to immunosuppressants or were neutropenic; or due to Clostridial and Candidal
organisms. Death risk in culture negative and bloodstream infection patients decreased significantly with time. Methods: We included all adults treated in non-psychiatric services at our hospital between 2004 and 2011. We
identified all blood cultures and their results to determine the independent association of blood culture testing and
BSI on death in hospital using proportional hazards modeling that adjusted for important covariates. g
p
g
y
p
j
g g
between 3.1 and 4.4 depending on admission urgency, extent of comorbidities, and whether the blood culture was
taken in the intensive care unit). Patients with BSI had a significantly increased risk of death (adj-HR ranging between
3.8 and 24.3] that was significantly higher when BSI was: diagnosed within the first hospital day; polymicrobial; in
patients who were exposed to immunosuppressants or were neutropenic; or due to Clostridial and Candidal
organisms. Death risk in culture negative and bloodstream infection patients decreased significantly with time. Conclusions: Risk of death in hospital is independently increased both in patients with negative blood
cultures and further in those with bloodstream infection. Death risk associated with bloodstream infections
varied by the patient’s immune status and the causative microorganism. Keywords: Blood stream infections, Hospital mortality, Outcomes, Proportional hazards modelin Blood culture utilization and results Other time-dependent covariates were also examined. We linked to the hospital’s laboratory dataset to determine
each patient’s neutrophil count throughout the admission;
counts <500 × 109 neutrophils/mL were classified as neu-
tropenic. We also linked to the pharmacy dataset to deter-
mine if patients received immunosuppressive medications
including enteral or parenteral steroids, methotrexate, aza-
thioprine, mycophenolate mofetil, cyclosporine, or anti-
thymocyte globulin. We linked to the laboratory database to determine if and
when each hospitalized patient had a blood culture mea-
sured. Cultures collected in the emergency department
prior to admission were also captured and attributed to the
hospitalization. Each day of each person’s hospitalization
was divided into six-hour sections starting at the time the
patient was admitted to hospital. For each of these quarter-
day segments, we determined whether or not the person
had at least one blood culture measured. Results of all blood cultures were then retrieved. Likely
contaminants were identified using criteria modified
from Richter [17] in which coagulase-negative staphylo-
cocci, aerobic and anaerobic diphtheroids, Micrococcus
spp., Bacillus spp., and viridans group streptococci were
classified as contaminants in the absence of another blood
culture within 48 hours growing the same organism. Mi-
croorganisms identified on the final report were clustered
by their genus into microorganism groups that were based
on the chapter (in a commonly used textbook of infectious van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 2 of 11 diseases [18]) which contained most information about
the organism. Blood cultures growing more than one or-
ganism were classified as polymicrobial. have determined the influence of simply having a blood
culture measured during the hospital – independent of
its results – on hospital mortality in a broad patient
population. Study setting This study took place at The Ottawa Hospital (TOH), a
tertiary-care teaching facility with three in-patient sites
that averaged 20 000 admissions annually during the
study period. TOH functions within a publicly funded
health care system, is the sole trauma centre for the re-
gion, and provides most of the region’s oncological, thor-
acic surgery, and neurosurgical care. In the present study, we used a validated extension of
the Escobar model to calculate each person’s daily risk
of death in hospital [22]. The “Daily Escobar” model in-
cluded all of the covariates in the Escobar model but
expressed LAPS as a time-dependent covariate (i.e. its
value was allowed to change with time) in which we
used the most extreme value of each laboratory test in
each 6-hour segment to calculate the LAPS. It also in-
cluded three additional time-dependent covariates: ad-
mission to intensive care unit [determined by linking to
the hospital’s patient location table]; performance of
significant operative procedures [23] [determined by link-
ing to the hospital’s procedure table]; and awaiting long-
term care status. The “Daily Escobar” model also had
excellent discrimination (concordance probability of 0.895,
95% CI 0.889-0.902) and calibration. Patients We
included
all
non-psychiatric
hospitalizations
of
people over 15 years of age between 1 April 2004 and 31
March 2011 including same-day surgeries. This study
period was chosen to maximize the study sample size
given the data available at the time we conducted the
study. Psychiatry admissions were not included since
physically ill patients potentially requiring blood cultures
are rarely admitted to – or are readily transferred from –
the psychiatry service. We excluded patients who were
transferred from or to other hospitals since we could not
get admission data or mortality status, respectively. The
unit of analysis in this study was the hospitalization. Outcome and covariates Knowing the effect of bloodstream infections on pa-
tient outcomes is needed to determine their importance
in patient care. In this study, we measured the independ-
ent influence of blood cultures and bloodstream infec-
tions on the risk of death in hospital. The primary outcome of the study was all-cause death
in hospital. The primary covariate was the risk of death
in hospital calculated using an extension of a model by
Escobar et al. [19]. The “Escobar model” estimates the
probability of death in hospital based on covariates avail-
able at the time of admission including: patient age and
sex; admission urgency (i.e. elective or emergent) and
service (i.e. medical or surgical); admission diagnosis;
severity of acute illness as measured by the Laboratory-
based Acute Physiology Score (LAPS); chronic comor-
bidities as measured by the Elixhauser score [20]; and
admission diagnosis [19]. The Escobar model was highly
discriminative, well calibrated, and was externally vali-
dated in our center with a c-statistic of 0.901 [21]. Background manifestations of the infection, and may indicate a sig-
nificant turning point in a patient’s health. Blood cultures are commonly ordered in clinical practice
with bloodstream infections found in almost all medical
specialties. Blood cultures are typically ordered when
physicians suspect the possibility of a bloodstream infec-
tion. Both the test and its result signal important events
in a patient’s care: blood cultures are often performed
when patients are ill or when their conditions signifi-
cantly worsen; bloodstream infections frequently change
treatment, often invoke a search for both causes and Despite the prevalence of blood cultures and the im-
portance of bloodstream infections, their impact on hos-
pitalized patient outcomes is unclear. Previous studies of
blood cultures and hospital mortality have focused on
specific patient populations [1-6], specific microorgan-
isms [7-10], or both [11]. Other studies focused their
mortality analysis on patients with documented blood-
stream infections to the exclusion of those with negative
blood cultures [12-16]. A few studies have compared
hospital mortality in patients with or without a blood-
stream infection but studied a restricted population in-
cluding those admitted with acute on chronic hepatic
failure [1] and critically ill patients with catheter-related
blood stream infections [2]. Finally, no previous studies van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 3 of 11 Other time-dependent covariates offered to the model
included those capturing each patient’s neutropenic status
and exposure to immunosuppressant medications. To de-
termine if death risk varied by microorganism, we subclas-
sified bloodstream infection patients by the microorganism
group of the isolate(s). Also, we repeated the model using
only one admission per patient (i.e. a patient-level analysis
rather than an admission level analysis) to determine if our
conclusions were sensitive to this modification. Finally, we
determined the possible influence of censoring patients at
hospital discharge by conducting a competing risks model
using the methods described by Wolkewitz et al. {13665}. This was accomplished by assessing the size of parameter
estimates in a replicate of our final model that had time to
discharge from hospital as the outcome while censoring
patients who died during their admission. The study was
approved by The Ottawa Hospital Research Ethics Board. We described how blood culture utilization and blood-
stream infections changed hospital mortality by calculat-
ing the daily expected number of deaths in each patient
group. For all patients in each group, we calculated the
estimated risk of death using the ‘Daily Escobar’ model
and then summed these daily risks to determine the ex-
pected number of deaths in each group on each day. In
this analysis, patients with a blood culture had their ref-
erence time (i.e. time 0) set at the date of their first
blood culture. Patients with no blood cultures had their
reference time set at the middle of their admission. We then determined the association of blood culture
results with time to death in hospital using a survival
model. Observation started at hospital admission and
was censored at discharge with observation time broken
into six hour segments. Blood culture procurement and
bloodstream infection status was expressed as time-
dependent binomial covariates. We added to the model
a term expressing the number of quarters since the
blood culture had been done to quantify changes over
time between the association of blood culture measure-
ment and death in hospital. The best fitting polynomial
transformation that captured this change was determined
using a modification of a SAS macro from Sauerbrei [24]. We included terms capturing polymicrobial status and
timing of bloodstream infection (categorized as admis-
sion [positive blood culture procured within first day
of admission] vs. hospital [blood culture procured more
than 24 hours after admission]). van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 We also determined if
significant interactions (p < 0.001) existed between each
covariate and blood culture measurement or bloodstream
infections. Results During the study period, a total of 317 194 adult in-
patient encounters (in which patients were neither trans-
ferred from or to another facility) occurred at our hospital. 20 124 of these hospitalizations were excluded because pa-
tients were admitted and discharged from the psychiatry
service. This left a total of 297 070 hospitalizations consisting
of 186 182 patients (Table 1). Most hospitalizations had
no blood cultures (n = 243 373, 81.9%), 16.3% of admis-
sions (N = 48 423) had all negative cultures, and 1.8%
(N = 5274) had at least one documented bloodstream in-
fection. Regardless of the result, patients having one or
more blood cultures during the hospitalization were not-
ably sicker than those without (Table 1): they were older;
they had higher Elixhauser [20] scores (indicating more
extensive chronic illnesses); they were more likely to be
admitted urgently and had a much higher predicted risk
of death in hospital; they had more extensive perturba-
tions of their laboratory tests (as indicated by their LAP
Scores); they were more likely to be treated in the inten-
sive care unit or to be exposed to immunosuppressants
at any time during the admission; and they had a longer
median length of stay. The fully adjusted model determining the association
of blood cultures and bloodstream infections with death
in hospital controlled for each patient’s daily expected
risk of death in hospital. This was quantified using the
“Daily Escobar” model described above to calculate the
Xβ from the model for each patient on each day. This
daily death risk was expressed as a time-dependent covari-
ate but was kept constant after patients were diagnosed
with a bloodstream infection. This was done because the
inclusion of the daily death risk in the model – including
that after bloodstream infection was treated – would mute
the influence of bloodstream infection on outcomes. This
is because the treatment of bloodstream infection should
decrease the daily death risk score (see Figure 1) and
lower daily death risk scores are strongly associated with
a decreased risk of death. Therefore, including in the
model post-treatment daily death risk scores of blood-
stream infection patients would, because of confounding,
bias the association between bloodstream infection and
hospital death towards the null. Fisher highlighted this
potential problem with survival models using time-
dependent covariates [25]. Analysis For baseline descriptive purposes, we separated patients
into three exclusive groups: no blood cultures done dur-
ing hospitalization; at least one blood culture but no
bloodstream infection; or one or more bloodstream infec-
tions. The prevalence - or median value - of each covariate
within each group was described. Blood culture utilization
was expressed as an incidence density reported as the
number of tests per 100 patient-days. van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Blood culture utilization and results The incident rate for blood culture utilization was 3.7
culture sets per 100 patient days; bloodstream infection
incident rate was 0.25 per 100 patient days. Blood cul-
ture utilization was heavily weighted to the start of the
hospital stay; blood culture utilization rates were highest
on the first hospital day (4.5 cultures per 100 patient-days)
while utilization was subsequently significantly lower (an
average of 0.68 cultures per 100 patient-days [range 0.55-
0.79]). Bloodstream infection rates were also highest on
the first hospitalization day (0.41 bloodstream infections van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 4 of 11 Figure 1 Daily expected risk of death by blood culture status. The daily hazard of death was calculated for each person using a validated
predictive model that captured daily values of important, patient-level covariates [22]. Within each group (no blood culture [red], negative blood
culture [grey], positive blood culture [blue]), these were summed and standardized to 1000 population (vertical axis). The horizontal axis displays
the hospital day relative to the first blood culture; for patients with no blood culture, the hospitalization midpoint was used as the reference. The
dip in the ‘no blood culture’ group is due to increased prevalence at zero time of short stay admissions (which have the lowest expected risk
of death). Figure 1 Daily expected risk of death by blood culture status. The daily hazard of death was calculated for each person using a validated
predictive model that captured daily values of important, patient-level covariates [22]. Within each group (no blood culture [red], negative blood
culture [grey], positive blood culture [blue]), these were summed and standardized to 1000 population (vertical axis). The horizontal axis displays
the hospital day relative to the first blood culture; for patients with no blood culture, the hospitalization midpoint was used as the reference. The
dip in the ‘no blood culture’ group is due to increased prevalence at zero time of short stay admissions (which have the lowest expected risk
of death). Expected death risk varied by patient group. Blood culture utilization and results In pa-
tients without blood cultures, the daily expected risk of
death averaged 3.5 deaths per 1000 patient-days (range
1.8-5.0); the lowest expected death rates for this group
occurred at Time 0 (Figure 1) likely due to an increased
prevalence at this point of short stay admissions which
have the lowest expected risk of death. In contrast, the
daily expected risk of death in patients with blood cul-
tures - regardless of their result - averaged 7.5 deaths
per 1000 (range 5.6-8.7) prior to the blood culture test-
ing (Figure 1). On the day of the blood culture, however,
expected death rates increased significantly to 14.3 deaths
per 1000 patient-days in patients with negative cultures
and 24.6 deaths per 1000 patient-days in patients with
bloodstream infections. Expected death rates returned to
pre-testing values within 4 days of blood culture procure-
ment (Figure 1). per 100 patient-days) and decreased significantly for the
rest of the hospitalization (mean rate of 0.03 bloodstream
infections per 100 patient-days, range 0.02-0.05). Table 2 shows that the majority of people undergoing
testing had only one set of blood cultures (37 243 people,
69.4% of those having any blood culture). As well, 4721 of
the 5274 people (89.5%) with at least one positive test had
only one bloodstream infection. The ten most common
microorganisms identified in the positive blood cultures
are shown in Table 3 with Enterobacteriacae, S. Aureus,
and Streptococci being the most common. 637 of a total of
7549 bloodstream infections (8.4%) were polymicrobial
with one or more polymicrobial bloodstream infections
occurring in 502 (0.2%) hospitalizations. Blood cultures, bloodstream infections and unadjusted
risk of hospital mortality Table 1 Description of study hospitalizations Unless stated, numbers in right column are percentages. single-microbial bloodstream infections (Figure 2A). The
increased risk of death associated with negative blood cul-
tures and bloodstream infection both decreased signifi-
cantly over time. ratio for death in patients with bloodstream infection
ranged between 6.2 and 15.8 depending on both the tim-
ing of the bloodstream infection (i.e. at admission vs. in-
hospital) and the number of organisms in the positive
culture: patients with bloodstream infection detected after
the first hospitalization day had a significantly higher
unadjusted risk of death compared to bloodstream in-
fections detected at admission; unadjusted death risk was
higher in polymicrobial bloodstream infections than in ratio for death in patients with bloodstream infection
ranged between 6.2 and 15.8 depending on both the tim-
ing of the bloodstream infection (i.e. at admission vs. in-
hospital) and the number of organisms in the positive
culture: patients with bloodstream infection detected after
the first hospitalization day had a significantly higher
unadjusted risk of death compared to bloodstream in-
fections detected at admission; unadjusted death risk was
higher in polymicrobial bloodstream infections than in The unadjusted relative hazard of death in hospital with
negative blood cultures and bloodstream infection varied
significantly with several covariates. Compared to a patient
without blood culture testing, the unadjusted relative risk
of death associated with negative blood cultures was sig-
nificantly higher in patient groups having a lower risk of
death including: females (Figure 2B); younger patients
(Figure 2C); those admitted electively to hospital (Figure 2D);
patients with fewer comorbidities (Figure 2E); those with-
out immunosuppressants (Figure 2G); and patients not in
the intensive care unit (Figure 2H). The increased risk of
death associated with bloodstream infection interacted
significantly with neutropenia (Figure 2I) and immuno-
suppressant exposure (Figures 2F and 2G); compared to
patients without blood culture testing, the risk of death
with bloodstream infection was extensively and signifi-
cantly higher in patients who were neutropenic and those
exposed to immunosuppressants. Blood cultures, bloodstream infections and unadjusted
risk of hospital mortality Both negative blood cultures and bloodstream infec-
tion were associated with an increased risk of death in
hospital (Figure 2A). The unadjusted death risk in pa-
tients with negative blood cultures was five times that of
people without blood cultures. The unadjusted hazard Patients without blood cultures were significantly less
likely to die in hospital (2.1%) than those with all nega-
tive blood cultures (13.3%) or those with at least one
bloodstream infection (20.7%) (Table 1; χ2 value 16172,
p < 0.0001). van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 5 of 11 Page 5 of 11 Table 1 Description of study hospitalizations
No blood culture
(N=243 373, 81.9%)
1+ Blood culture, all negative
(N=48 423, 16.3%)
1+ Blood culture, 1+ positive
(N=5274, 1.8%)
All patients
(N=297, 7%)
Patient
Mean age (SD)
53.2
20.2
62.3
18.8
63.4
17.9
54.8
20.2
Female
145 772
59.9
23 414
48.4
2403
45.6
171 589
57.8
Elixhauser score*:
<0
11206
4.6
1719
3.6
212
4.0
13 137
4.4
0
140 027
57.5
12 461
25.7
1177
22.3
153 665
51.7
>0
91 140
37.9
34 243
70.7
3885
73.7
130 268
43.8
Admission
Emergent admission
135 962
55.9
43 914
90.7
4856
92.1
184 732
62.2
Admitted to surgical service
82 721
34.0
11 116
23.0
1012
19.2
94 849
31.9
Median death risk (IQR)**
0.4%
0.1-2.1
5.1%
1.2-15.6
8.0%
2.1-22.6
0.6%
0.17-4.0
LAP score****:
0
152 338
62.6
9742
20.1
804
15.2
162 884
54.8
>0
91 035
37.4
38 681
79.9
4470
84.8
134 186
45.2
Hospitalization
Intensive care unit***
4137
1.7
7845
16.2
1297
24.5
13 369
4.5
Surgical procedure***
66 928
27.5
8813
18.2
964
18.2
76 649
25.8
Neutrophils < 500 × 109/L***
730
0.3
3147
6.5
609
11.5
4456
1.5
Immunosupressant***
6814
2.8
14 140
29.2
1763
33.3
22 579
7.6
Awaiting placement anytime
97
0.04
3680
7.6
434
8.2
4159
1.4
Median LOS (IQR)
4
2-6
9
4-19
12
6-28
4
2-8
Died in hospital
5013
2.1
6423
13.3
1093
20.7
12 529
4.2
Unless stated, numbers in right column are percentages. SD standard deviation, IQR interquartile range. *Measures number and severity of comorbidities [21]. **Measured using Escobar model [20] using covariate values at hospital admission. ***At any time during the hospitalization. ****Laboratory-based Acute Physiology Score; quantifies deviations of important laboratory tests from normal [20]. Blood cultures, bloodstream infections and unadjusted
risk of hospital mortality Table 2 Summary of blood culture utilization and results
Number of blood culture sets
0
1
2
3+
Total
NUMBER OF
POSITIVE
CULTURES
0
243 373
34 854
8023
5546
291 796
(98.2%)
1
-
2389
1101
1231
4721 (1.6%)
2
-
-
107
317
424 (0.1%)
3+
-
-
-
129
129 (0.04%)
TOTAL
243 373
37 243
9231
7223
297 070
(81.9%)
(12.5%)
(3.1%) (2.4%)
A maximum of 1 culture set per person per six hour period was counted. Table 2 Summary of blood culture utilization and results Table 2 Summary of blood culture utilization and results Page 6 of 11 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Table 3 Description of 8334 microoganisms identified in 7549 positive cultures
Microorganism group
Isolates
Frequency
% Total (% of grou
Enterbacteriacae
-
3024
36.3
Escherichia coli
1659
(54.9)
Klebsiella pneumoniae
534
(17.7)
Enterobacter cloacae
221
(7.3)
Other
610
(20.2)
Staphylococcus Aureus
-
1065
12.8
Streptococci
-
979
11.7
Streptococcus pneumoniae
321
(32.8)
Group B Streptococcus (s. Blood cultures, bloodstream infections and unadjusted
risk of hospital mortality agalactiae)
131
(13.4)
Viridans group Streptococcus
105
(10.7)
Other
422
(43.1)
Enterococcus (including Streptococcus bovis)
-
689
8.3
Enterococcus faecalis
412
(59.8)
Enterococcus faecium
211
(30.6)
Enterococcus species
31
(4.5)
Other
35
(5.1)
Candida
-
574
6.9
Candida albicans
292
(50.9)
Candida (torulopsis) glabrata
119
(20.7)
Candida parapsilosis
65
(11.3)
Other
98
(17.1)
Other gram negative and gram-variable bacilli
-
538
6.5
Pseudomonas aeruginosa
381
(70.8)
Gram-negative bacilli
69
(12.8)
Achromobacter (alcaligenes) xylosoxidans
17
(3.2)
Other
71
(13.2)
Anaerobic gram-positive nonsporulating bacilli
-
239
2.9
Propionibacterium acnes
114
(47.7)
Propionibacterium species
102
(42.7)
Eubacterium lentum
16
(6.7)
Other
7
(2.9)
Other anaerobes
-
160
1.9
Bacteroides fragilis
99
(61.9)
Bacteroides thetaiotaomicron (fragilis gr.)
13
(8.1)
Fusobacterium nucleatum
10
(6.3)
Other
38
(23.8)
Methicillin resistant staphylococcus aureus
-
153
1.8
Clostridium
-
116
1.4
Clostridium perfringens
49
(42.2)
Clostridium septicum
23
(19.8)
Clostridium species
18
(15.5)
Other
26
(22.4)
Other
-
797
9.6
Coagulase-negative Staphylococcus
158
(19.8) Table 3 Description of 8334 microoganisms identified in 7549 positive cultures Staphylococcus Aureus
Streptococci Candida Other Page 7 of 11 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Table 3 Description of 8334 microoganisms identified in 7549 positive cultures (Continued)
Stenotrophomonas maltophilia
70
(8.8)
Haemophilus influenzae
52
(6.5)
Other
517
(64.9)
Blood cultures growing two organisms in the same class were counted once. Microorganisms were classified using Mandell [19]. Table 3 Description of 8334 microoganisms identified in 7549 positive cultures (Continued) Figure 2 Stratified and unadjusted influence of negative blood cultures and bloodstream infection on risk of death in hospital. These
figures plot the association between day from blood culture (horizontal axis) with relative hazard of all-cause death in hospital (vertical axis) for
patients with negative blood culture (red lines) and bloodstream infection (blue lines). These estimates were generated from models in which
blood cultures were expressed as time-dependent covariates and are stratified by the characteristic in each title; therefore, the displayed hazard
of death is relative to patients in that strata who did not have blood culture testing. Plot A shows the unadjusted association. In the remaining
plots (Plot B through I), the stratifying variable for the analysis is presented atop the plot. The p-value for all of the interactions presented here
(i.e. plots B to F) is ≤0.0001. Adjusted association of blood cultures and, bloodstream
infections with hospital mortality vs. 3E). In addition, the adjusted risk of death associated
with bloodstream infections increased significantly in pa-
tients exposed to immunosuppressives, those who were
neutropenic, or both. On the first day of bloodstream in-
fection, the adjusted relative hazard of death in hospital
varied from a low of 3.8 [95% CI 3.1-4.6] (in: emergently
admitted patients; with low comorbidity; not in the ICU;
without neutropenia; not on immunosuppresives; with a
bloodstream infection from a single organism during the
hospitalization) to 24.3 [95% CI 16.6-35.4] (in: electively
admitted patients; with high comorbidity; in the ICU; with
neutropenia; on immunosuppresives; with a polymicrobial
bloodstream infection identified in the first 24 hours of
the hospitalization). As with negative blood cultures, the
increased risk of death from bloodstream infections de-
creased significantly over time (Figure 3B-3E). Adjusted association of blood cultures and, bloodstream
infections with hospital mortality After adjusting for important covariates, the risk of
death in hospital was significantly higher in patients with
negative blood cultures (Figure 3A, Additional file 1). This
risk ranged between 3.1 and 4.4 times that of people
without blood culture testing, varying significantly by
admission urgency (risk tended to be higher in elective ad-
missions), patient comorbidity (risk tended to be higher in
sicker patients), and ICU status (risk tended to be higher
in ICU patients). The increased risk of death associated
with negative blood cultures decreased significantly over
time. Bloodstream infections conferred a significant, additional
adjusted risk of death in hospital beyond that from blood
culture testing (Figure 3B-E, Additional file 1). This risk
was higher in bloodstream infections identified in the
first 24 hours of the admission and in those due to mul-
tiple microorganisms (Figure 3B vs. 3D and Figure 3C The influence of bloodstream infection on the inde-
pendent risk of death in hospital varied significantly
by the microorganism isolated in the culture (Figure 4). A
B
C
D
E A
B
C
D
E
Figure 3 Adjusted influence of negative blood cultures/bloodstream infection on hospital mortality by significant effect modifiers. These plots present the adjusted hazard ratio (vertical axes) for the association of negative blood cultures (“No BSI”) and bloodstream infection
with death in hospital in the first 2 weeks following blood culture testing (horizontal axes). Adjusted hazard ratios for negative blood cultures are
presented for all combinations of admission urgency (elective vs. Blood cultures, bloodstream infections and unadjusted
risk of hospital mortality re 2 Stratified and unadjusted influence of negative blood cultures and bloodstream infection on risk of death in h
l
h
i i
b
d
f
bl
d
l
(h
i
l
i )
i h
l i
h
d
f ll
d
h i
h
i l ( Figure 2 Stratified and unadjusted influence of negative blood cultures and bloodstream infection on risk of death in hospital. These
figures plot the association between day from blood culture (horizontal axis) with relative hazard of all-cause death in hospital (vertical axis) for
patients with negative blood culture (red lines) and bloodstream infection (blue lines). These estimates were generated from models in which
blood cultures were expressed as time-dependent covariates and are stratified by the characteristic in each title; therefore, the displayed hazard
of death is relative to patients in that strata who did not have blood culture testing. Plot A shows the unadjusted association. In the remaining
plots (Plot B through I), the stratifying variable for the analysis is presented atop the plot. The p-value for all of the interactions presented here
(i.e. plots B to F) is ≤0.0001. Figure 2 Stratified and unadjusted influence of negative blood cultures and bloodstream infection on risk of death in hospital. These
figures plot the association between day from blood culture (horizontal axis) with relative hazard of all-cause death in hospital (vertical axis) for
patients with negative blood culture (red lines) and bloodstream infection (blue lines). These estimates were generated from models in which
blood cultures were expressed as time-dependent covariates and are stratified by the characteristic in each title; therefore, the displayed hazard
of death is relative to patients in that strata who did not have blood culture testing. Plot A shows the unadjusted association. In the remaining
plots (Plot B through I), the stratifying variable for the analysis is presented atop the plot. The p-value for all of the interactions presented here
(i.e. plots B to F) is ≤0.0001. Page 8 of 11 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Adjusted association of blood cultures and, bloodstream
infections with hospital mortality emergent), patient comorbidity status (low comorbidity [Elixhauser score of 1]
vs. high comorbidity [Elixhauser score of 12]), and intensive care unit (ICU) status. Adjusted hazard ratios for bloodstream infection (calculated for
patients with low comorbidity, low comorbidity, and not in the ICU) are presented for all combinations of immunosuppressant exposure and
neutropenia. All hazard ratios adjust for: patient age; patient sex; admission service (i.e. medical or surgical) and diagnosis; severity of acute illness
as measured by the Laboratory-based Acute Physiology Score (LAPS); chronic comorbidities as measured by the Elixhauser score [20]; treatment
in the intensive care unit; performance of significant operative procedures [23]; awaiting long-term care status; [19] and exposure to
immunosuppressant medications. All adjusted hazard ratios use as a comparator a person an electively admitted person with low comorbidity not in
the ICU who has no blood culture measured. Figure 3 Adjusted influence of negative blood cultures/bloodstream infection on hospital mortality by significa ed influence of negative blood cultures/bloodstream infection on hospital mortality by significant effect modifiers. h
d
d h
d
(
l
) f
h
f
bl
d
l
(“N
BSI”)
d bl
d
f Figure 3 Adjusted influence of negative blood cultures/bloodstream infection on hospital mortality by significant effect modifiers. These plots present the adjusted hazard ratio (vertical axes) for the association of negative blood cultures (“No BSI”) and bloodstream infection
with death in hospital in the first 2 weeks following blood culture testing (horizontal axes). Adjusted hazard ratios for negative blood cultures are
presented for all combinations of admission urgency (elective vs. emergent), patient comorbidity status (low comorbidity [Elixhauser score of 1]
vs. high comorbidity [Elixhauser score of 12]), and intensive care unit (ICU) status. Adjusted hazard ratios for bloodstream infection (calculated for
patients with low comorbidity, low comorbidity, and not in the ICU) are presented for all combinations of immunosuppressant exposure and
neutropenia. All hazard ratios adjust for: patient age; patient sex; admission service (i.e. medical or surgical) and diagnosis; severity of acute illness
as measured by the Laboratory-based Acute Physiology Score (LAPS); chronic comorbidities as measured by the Elixhauser score [20]; treatment
in the intensive care unit; performance of significant operative procedures [23]; awaiting long-term care status; [19] and exposure to
immunosuppressant medications. All adjusted hazard ratios use as a comparator a person an electively admitted person with low comorbidity not in
the ICU who has no blood culture measured. Adjusted association of blood cultures and, bloodstream
infections with hospital mortality Bloodstream infections with Clostridial, Candida, and
other gram negative/variable bacilli had adjusted death
risks that were notably higher than the other micro-
organisms. Figure 4 also highlights the striking increased
risk of death in hospital from bloodstream infection when
patients were neutropenic and exposed to immuno-
supressives. addition, consideration of competing risks indicated little
chance of biased results in our original model with minis-
cule parameter estimates in the competing risks model for
all study covariates (Additional file 3). Adjusted association of blood cultures and, bloodstream
infections with hospital mortality van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 9 of 11 Figure 4 Independent association of specific microorganism classes with hospital death risk. These plots present the adjusted hazard ratio
of death in hospital (horizontal axis) for patients without blood culture testing, those with negative blood cultures, and those with bloodstream
infections caused by different microorganisms (vertical axis). Estimates are provided with 95% confidence intervals and are presented for patients
without immunosuppressive or neutropenia (Plot A) and for patients with immunosuppressive or neutropenia (Plot B). In both plots, adjusted
hazard ratios are relative to people without blood culture testing. Microorganisms whose adjusted relative hazard is statistically distinct from
patients with culture negative blood cultures are indicated in red. Figure 4 Independent association of specific microorganism classes with hospital death risk. These plots present the adjusted hazard ratio
of death in hospital (horizontal axis) for patients without blood culture testing, those with negative blood cultures, and those with bloodstream
infections caused by different microorganisms (vertical axis). Estimates are provided with 95% confidence intervals and are presented for patients
without immunosuppressive or neutropenia (Plot A) and for patients with immunosuppressive or neutropenia (Plot B). In both plots, adjusted
hazard ratios are relative to people without blood culture testing. Microorganisms whose adjusted relative hazard is statistically distinct from
patients with culture negative blood cultures are indicated in red. Figure 4 Independent association of specific microorganism classes with hospital death risk. These plots present the adjusted hazard ratio
of death in hospital (horizontal axis) for patients without blood culture testing, those with negative blood cultures, and those with bloodstream
infections caused by different microorganisms (vertical axis). Estimates are provided with 95% confidence intervals and are presented for patients
without immunosuppressive or neutropenia (Plot A) and for patients with immunosuppressive or neutropenia (Plot B). In both plots, adjusted
hazard ratios are relative to people without blood culture testing. Microorganisms whose adjusted relative hazard is statistically distinct from
patients with culture negative blood cultures are indicated in red. Bloodstream infections with Clostridial, Candida, and
other gram negative/variable bacilli had adjusted death
risks that were notably higher than the other micro-
organisms. Figure 4 also highlights the striking increased
risk of death in hospital from bloodstream infection when
patients were neutropenic and exposed to immuno-
supressives. Discussion
k This indicates that patients undergoing
blood culture testing are sicker than others, even after
adjusting for measured covariates that distinguished
these patient groups (Table 1). Obviously, we do not be-
lieve that the actual act of measuring blood cultures in-
creases the risk of death in hospital. Instead, we suspect
that an increased risk of subsequent bad outcomes re-
gardless of the blood cultures result is due to the test in-
dicating a sicker population than is indicated by the
measured covariates. We believe that this phenomenon
is likely true for other tests (such as electrocardiogram,
portable chest radiograph, cardiac enzymes, and arterial
blood gases) that are done when patients deteriorate
acutely. This observation should be considered when ana-
lyzing the influence of abnormal test results on hospital
outcomes. Second, we found that the increased independ-
ent risk of death in hospital associated with negative blood
cultures and bloodstream infections was maximal at the
time that the blood culture was procured and decreased
significantly over time. This likely reflects the benefit of
treatments given to surviving patients. Finally, the harmful
effect of bloodstream infections was highest with particu-
lar microorganisms (notably Clostridial and Candidal)
and in immunocompromised hosts (those exposed to im-
munosuppressive agents and those with neutropenia). These results confirm how such patients with bloodstream
infection must be treated aggressively. Our study has several notable attributes. We captured
all hospitalizations, all blood cultures, and all bloodstream
infections at our hospital during the study period. Our
statistical model recognized the time-dependent nature of
blood culture testing, bloodstream infections, and their as-
sociation with death in hospital. However, several poten-
tial limitations of our study should be kept in mind. First,
although we found that bloodstream infection was associ-
ated with an increased risk of death in hospital, we have
no way of determining whether the infection actually
caused the death. Primary data review would be needed to
determine – if possible – whether or not the bloodstream
infection caused a particular patient’s death. Such analyses
are necessary to determine if and how we might intervene
to improve outcomes with hospital-associated blood-
stream infections. Second, our data did not account for
treatment of bloodstream infections. Outcomes in patients
admitted for community acquired pneumonia are im-
proved significantly in those who receive antibiotics more
quickly [27]. Berjohn et al. Discussion
k To our knowledge, this is the most extensive examin-
ation of the association between blood culture testing
and bloodstream infections on hospital mortality. We
found that blood culture testing was common and that
bloodstream infections were detected in almost 2% of
hospitalizations. Patients undergoing blood culture test-
ing were much sicker and had a notably higher risk of
death in hospital that peaked around the time of their
test. Even after adjusting for important confounders,
patients with negative blood cultures still had a signifi-
cantly increased risk of death in hospital. The risk of
death was higher still in those with bloodstream infec-
tions with the risk being highest in bloodstream infec-
tions that: were detected in the first hospital day; were
polymicrobial; occurred during a neutropenic episode or The study’s conclusions did not change significantly
when we repeated the analysis with the patient – rather
than the admission – as the unit of analysis (Additional
file 2). All but one of the parameter estimates in the pa-
tient model - that for polymicrobial status - remained
within the 95% confidence intervals of the hospitalization
model (Additional file 1). Changes in the influence of
polymicrobial status on mortality might be due to a
change in statistical power (i.e. a reduction in the number
of polymicrobial cases when the unit of analysis changed
to the patient) or might indicate that repeated polymicro-
bial bloodstream infections (which would be in the model
having the hospitalization – but not the patient - as the
unit of analysis) have a particularly poor outcome. In van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 10 of 11 van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 10 of 11 while exposed to immunosuppressants; or those due to
Clostridial and Candidal organisms. hospital longer tend to be sicker, such analyses could
falsely attribute mortality risk from confounders in these
patients to nosocomial bloodstream infection. Analyz-
ing bloodstream infection as a time-dependent covariate
within a survival model – as we did in this analysis –
avoids this potential bias [25]. Our study has several important findings. First, we
found that the independent risk of death in hospital in-
creased significantly whenever blood cultures were or-
dered, even when those blood cultures did not grow a
microorganism. Discussion
k [11] found that patients with
pneumococcal bacteremia receiving least 1 active anti-
biotic within 4 hours of blood cultures were significantly
less likely to die in hospital (odds ratio [OR], 0.47; 95%
confidence interval [CI], 0.2-1.0). It is possible that timeli-
ness of appropriate antibiotics - and other interventions to
control infection - could have an independent influence
on death risk in all patients with bloodstream infections. Third, we did not have access to information about poten-
tial sources or causes of bloodstream infections, such as
catheters. It is possible that mortality risk associated with
bloodstream infections may change significantly based on
the presence of foreign bodies. Fourth, the utilization of
blood cultures requires a physician’s response to clinical
data input and are not – by themselves – a pathophysio-
logical marker. Physicians will vary in their response to
various clinical data and, as such, will have different
thresholds or likelihoods for ordering blood cultures. Therefore, the external validity of our findings to other
centres could be questioned. However, supporting the
generalizability of our findings is the large size and long
duration of the study as well as its complete capture of all
blood cultures at our large institution, all of which will en-
sure a large number of different physicians who were cap-
tured by the analysis. Our study has several interesting comparisons with
previous analyses of bloodstream infections and hospital
mortality. The distribution of microorganisms that we
identified in our cohort was very similar to that identi-
fied in previous analyses [2,5,12,13,15]. Similar to our re-
sults, several other analyses have found particularly high
mortality rates in patients with Candidal bloodstream
infection [5,6,14-16]. To our knowledge, ours is the most
extensive analysis that included patients without blood
cultures and those with negative blood cultures; this
characteristic is necessary to precisely gauge the influ-
ence of bloodstream infection on mortality risk relative
to other patients and independent of confounders associ-
ated with the actual measurement of blood cultures. Finally, several studies had previously found – in contrast
to our study – that mortality risk was higher in those with
nosocomial bloodstream infections [2,12,14,16]. We be-
lieve that these analyses are susceptible to time-dependent
bias [26] since patients must remain alive in hospital for a
specified period of time to be classified with nosocomial
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hospital is independently increased in patients with van Walraven and Wong BMC Infectious Diseases 2014, 14:36
http://www.biomedcentral.com/1471-2334/14/36 Page 11 of 11 either negative blood cultures or bloodstream infections. In addition, death risk associated with bloodstream in-
fections varied significantly by the patient’s immune sta-
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inpatient risk adjustment methodology was valid in an external patient
population. J Clin Epidemiol 2010, 63(7):798–803. Additional files Additional file 1: Final model for the adjusted association of blood
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(Additional file 1) having the admission or the patient as the unit of
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Correction: Shallow-Water Northern Hemisphere Jaera (Crustacea, Isopoda, Janiridae) Found on Whale Bones in the Southern Ocean Deep Sea: Ecology and Description of Jaera tyleri sp. nov
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PloS one
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Correction
Correction: Shallow-Water Northern Hemisphere Jaera
(Crustacea, Isopoda, Janiridae) Found on Whale Bones in
the Southern Ocean Deep Sea: Ecology and Description
of Jaera tyleri sp. nov Correction
Correction: Shallow-Water Northern Hemisphere Jaera
(Crustacea, Isopoda, Janiridae) Found on Whale Bones in
the Southern Ocean Deep Sea: Ecology and Description
of Jaera tyleri sp. nov Correction Correction: Shallow-Water Northern Hemisphere Jaera
(Crustacea, Isopoda, Janiridae) Found on Whale Bones in
the Southern Ocean Deep Sea: Ecology and Description
of Jaera tyleri sp. nov The PLOS ONE Staff Citation: The PLOS ONE Staff (2014) Correction: Shallow-Water Northern
Hemisphere Jaera (Crustacea, Isopoda, Janiridae) Found on Whale Bones in the
Southern Ocean Deep Sea: Ecology and Description of Jaera tyleri sp. nov. PLOS
ONE 9(6): e101092. doi:10.1371/journal.pone.0101092 Citation: The PLOS ONE Staff (2014) Correction: Shallow-Water Northern
Hemisphere Jaera (Crustacea, Isopoda, Janiridae) Found on Whale Bones in the
Southern Ocean Deep Sea: Ecology and Description of Jaera tyleri sp. nov. PLOS
ONE 9(6): e101092. doi:10.1371/journal.pone.0101092
Published June 17, 2014
Copyright: 2014 The PLOS ONE Staff. This is an open-accessarticle distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. Published June 17, 2014 Copyright: 2014 The PLOS ONE Staff. This is an open-accessarticle distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. The PLOS ONE Staff Figure 3 is illegible in the XML and PDF of the article. Please
see the correct version of Figure 3 below. Published June 17, 2014 Copyright: 2014 The PLOS ONE Staff. This is an open-accessarticle distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org June 2014 | Volume 9 | Issue 6 | e101092 1 June 2014 | Volume 9 | Issue 6 | e101092 ference
ure 3. Depth distribution in Janiridae. A) Species numbers per depth. The abscissa divides the total number of speci
imum richness is just under 155 species; B) Bathymetric ranges of janirid genera; in bracketsare the numbers of species pe
0.1371/journal.pone.0093018.g003 Figure 3. Depth distribution in Janiridae. A) Species numbers per depth. The abscissa divides the total number of spec
maximum richness is just under 155 species; B) Bathymetric ranges of janirid genera; in bracketsare the numbers of species p
doi:10.1371/journal.pone.0093018.g003 Figure 3. Depth distribution in Janiridae. A) Species numbers per depth. The abscissa divides the total number of species in half, thus the
maximum richness is just under 155 species; B) Bathymetric ranges of janirid genera; in bracketsare the numbers of species per family. doi:10.1371/journal.pone.0093018.g003 i
3 D
th di t ib ti
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f Figure 3. Depth distribution in Janiridae. A) Species numbers per depth. The abscissa divides the total number of species in half, thus the
maximum richness is just under 155 species; B) Bathymetric ranges of janirid genera; in bracketsare the numbers of species per family. doi:10.1371/journal.pone.0093018.g003 1. Linse K, Jackson JA, Malyutina MV, Brandt A (2014) Shallow-Water Northern
Hemisphere Jaera (Crustacea, Isopoda,Janiridae) Found on Whale Bones in the
Southern Ocean Deep Sea: Ecology and Description of Jaera tyleri sp. nov. PLoS
ONE 9(3): e93018. doi:10.1371/journal.pone.0093018 Reference June 2014 | Volume 9 | Issue 6 | e101092 PLOS ONE | www.plosone.org 2
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A RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA Based 5G Wireless Networks
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Received February 4, 2020, accepted February 25, 2020, date of publication March 2, 2020, date of current version March 16, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2977773 A RAN Resource Slicing Mechanism for
Multiplexing of eMBB and URLLC Services
in OFDMA Based 5G Wireless Networks This work was supported by the Luxembourg National Research Fund (FNR), under the AFR research grant entitled ‘‘Learning-Assisted
Cross-Layer Optimization of Cognitive Communnication networks’’ and ‘‘5G-Sky: Interconnecting the Sky in 5G and Beyond – A Joint
Communication and Control Approach.’’ ABSTRACT
Enhanced mobile broadband (eMBB) and ultra-reliable and low-latency communica-
tions (URLLC) are the two main expected services in the next generation of wireless networks. Accom-
modation of these two services on the same wireless infrastructure leads to a challenging resource allocation
problem due to their heterogeneous specifications. To address this problem, slicing has emerged as an
architecture that enables a logical network with specific radio access functionality to each of the supported
services on the same network infrastructure. The allocation of radio resources to each slice according to their
requirements is a fundamental part of the network slicing that is usually executed at the radio access network
(RAN). In this work, we formulate the RAN resource allocation problem as a sum-rate maximization problem
subject to the orthogonality constraint (i.e., service isolation), latency-related constraint and minimum rate
constraint while maintaining the reliability constraint with the incorporation of adaptive modulation and
coding (AMC). However, the formulated problem is not mathematically tractable due to the presence
of a step-wise function associated with the AMC and a binary assignment variable. Therefore, to solve
the proposed optimization problem, first, we relax the mathematical intractability of AMC by using an
approximation of the non-linear AMC achievable throughput, and next, the binary constraint is relaxed
to a box constraint by using the penalized reformulation of the problem. The result of the above two-step
procedure provides a close-to-optimal solution to the original optimization problem. Furthermore, to ease the
complexity of the optimization-based scheduling algorithm, a low-complexity heuristic scheduling scheme
is proposed for the efficient multiplexing of URLLC and eMBB services. Finally, the effectiveness of the
proposed optimization and heuristic schemes is illustrated through extensive numerical simulations. INDEX TERMS
Network slicing, RAN radio resource allocation, sum-rate maximization, scheduling,
eMBB and URLLC INDEX TERMS
Network slicing, RAN radio resource allocation, sum-rate maximization, scheduling,
eMBB and URLLC. I. INTRODUCTION wireless networks requires to support a large variety of ser-
vices and applications with different requirements. Towards
this achievement, the fifth-generation (5G) of wireless net-
works is expected to support the three major usage scenarios,
which, according to ITU-R, are classified as enhanced mobile
broadband (eMBB), ultra-reliable and low latency commu-
nications (URLLC) and massive machine-type communi-
cations [3]–[6]. A brief characterization of theses services
is provided as follows: eMBB service requires higher data The third and fourth generations (3G and 4G) of wireless net-
works have already revolutionized social behaviors through
empowering the generalization of social networking on wire-
less mobile devices [2]. In order to further improve our cities,
living environment, and industries, the next generation of The associate editor coordinating the review of this manuscript and
approving it for publication was Giuseppe Araniti
. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 45674 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA has been introduced in [12] for wireless virtual networks
(WVNs), which dynamically assign a specific number of RBs
to each VN to assure services to its users. The authors of [13]
have studied the problems of admission control and resource
provisioning in Orthogonal Frequency Division Multiple
Access (OFDMA) based WVNs. In [14], the energy-efficient
sub-carrier and power allocation strategy with WVN has
been proposed for a single-cell OFDMA system. Though the
above-mentioned works have considered some constraints
to maintain isolation between the slices, they may not be
applicable for the next generation networks due to lack of
considerations for the end-to-end QoS requirements of dif-
ferent services. rates to further improve the current mobile services such as
high definition (HD) video and virtual reality (VR); URLLC
service concentrates on supporting low-latency transmissions
of small packets with high reliability and it covers appli-
cations such as autonomous vehicles, industrial automation,
and vehicular communications; mMTC supports the services
that connect a massive number of devices where each device
transmits small data packets intermittently and it covers the
applications like smart cities. pp
Out of the aforementioned three services, the main two
services to be supported in the next release of 5G wireless
networks are eMBB and URLLC [7], and thus are considered
in this paper. I. INTRODUCTION However, the current one-size-fits-all network
model is not suitable to accommodate these services [8]. Fur-
thermore, accommodating these different wireless services
in the same physical network while assuring their potential
co-existence is also a major challenge. To address this prob-
lem, the next generation of wireless networks is expected to
exploit the highly flexible and scalable network architectures
to support the diverse applications from the aforementioned
different services. In this context, recently, network slicing
has emerged as a promising network architecture for allo-
cating resources to different wireless services with diverse
quality-of-service (QoS) needs [9]. In this approach, the com-
mon physical network infrastructure is sliced into multiple
end-to-end logical networks, where each logical network acts
as a dedicated network for a specific service. Specifically,
each logical network or a network slice consists of a col-
lection of particular radio access mechanisms and network
functions and needs to be isolated from other slices that can
be acquired through logical partitioning and radio resource
virtualization. As aforementioned, the 5G NR has to support multiple
number of services and a huge variety of applications. Using
the simplified queuing analysis, in [15], the authors have
shown that the dynamic multiplexing of URLLC and eMBB
traffic significantly improves the total resource efficiency of
a wireless system. The authors of [16] have investigated the
radio-channel and QoS aware packet scheduling technique
for the multiplexing of radio eMBB and URLLC services on
the same radio resources in accordance with their demanding
QoS requirements. In this work [16], the proposed scheduling
algorithm dynamically adjusts the BLEP of URLLC trans-
missions according to the available traffic load and also a new
channel quality indicator (CQI) estimation mechanism was
introduced to improve the accuracy of the URLLC link adap-
tion process. Furthermore, to advance the scheduler given
in [15], a packet-size and control channel aware resource
allocation method has been investigated in [17]. However,
the proposed heuristic scheduling algorithms in [16], [17]
satisfy the QoS requirements of URLLC service through
prioritizing the service (i.e., allocation of large number of
resources), but cannot guarantee the isolation between the
service slices under the high URLLC loads (i.e., eMBB users
may not get enough RBs under the high URLLC traffic). Network slicing is executed both on the Radio Access
Network (RAN) and the Core Network (CN). I. INTRODUCTION Thus, both parts
of the network require to be sliced into multiple overlaid
instances to serve the different types of services. In this
paper, we focus on RAN slicing, whose challenges reside in
the management of heterogeneous traffic demands coming
from a variety of multiple users and services, and the limited
available radio resources to satisfy these needs. To this end,
the dynamic allocation of radio resources aligned with the
instantaneous user traffic demands represents a major chal-
lenge. In this paper, we address the RAN slicing problem by
dynamically assigning radio resource blocks (RBs) to each
user according to its traffic demand in the network. In the
following sub-sections, we review the related works from the
literature and highlight the contributions of this paper. y
g
g
g
On the another hand, puncturing based schemes have
been recently proposed in the literature to eliminate the
queuing delay of randomly occurred URLLC traffic through
placing the URLLC traffic on the ongoing eMBB traf-
fic [6], [18]–[20]. In this regard, [6] uses information the-
oretic results to achieve expressions for the average eMBB
rates under URLLC puncturing for different decoding meth-
ods for uplink eMBB traffic superposed or punctured by the
URLLC users. However, the authors of [6] have not consid-
ered the design of joint scheduling schemes for eMBB and
URLLC data traffic. In [18], a punctured scheduling mech-
anism has been introduced for the transmission of latency
critical traffic on a shared channel with the eMBB traffic,
wherein the authors have utilized recovery techniques for
eMBB transmissions, a service-specific heuristic scheduling
algorithm and link adaption to increase the efficiency of the
proposed scheme. The authors of [19] have proposed an
online joint scheduling algorithm to improve the network
utility of eMBB while assuring the stringent requirements
of URLLC and studied optimal online joint URLLC/eMBB A. REVIEW OF RELATED WORKS Dynamic RAN radio resource allocation mechanisms have
received recently increasing attention in the literature. Some
of the relevant research studies are summarized in the fol-
lowing. In order to provide services to different service
providers, a resource sharing approach for an efficient slicing
of LTE network into multiple virtual networks (VNs) has
been studied in [11]. A new slicing and scheduling technique 45675 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA schedulers within the broad class of channel state dependent
but mini-slot-homogeneous policies by using a more general
class of convex and threshold loss models. threshold-based approach is considered to relax the binary
constraint in the optimization problem while a penalized
formulation is considered in this work for the mathematical
tractability. Besides, we propose a heuristic algorithm to solve
the formulated optimization problem, which was missing
in [1]. The main contributions of this paper are summarized
in the following. Furthermore, a risk-sensitive approach for the efficient
allocation of radio resources to the eMBB and URLLC trans-
missions has been investigated in [20]. This work formulated
the resource allocation problem as an optimization problem
to maximize the overall eMBB data rate while assuming the
risk of eMBB using the conditional value at risk (CVaR) func-
tion as a risk measure. In general, the punctured scheduling
algorithms proposed in [6], [18]–[20] prioritize the URLLC
service when the URLLC traffic arrives sporadically and
places the URLLC traffic on ongoing eMBB traffic that
leads to the isolation problems and also greatly reduces the
eMBB data rate and reliability at the higher URLLC traffic. Also, the aforementioned puncturing mechanisms decrease
the decoding ability due to the potential inter-user interfer-
ence and also increase the control channel (CCH) overhead
due to the utilization of extra dedicated CCHs for indicating
the URLLC overlapping positions to the eMBB receiver. Moreover, most of the aforementioned works [15]–[20] have
proposed the heuristic scheduling algorithms for dynamic
multiplexing of eMBB and URLLC users and also consid-
ered the mini-slot (i.e., 2 OFDM symbols) based schedul-
ing process. A. REVIEW OF RELATED WORKS Nevertheless, none of works have targeted a
design of the RAN resource slicing mechanism that effi-
ciently optimize the currently available LTE standard radio
resources (i.e., 0.5ms each transmission time interval (TTI),
1ms sub-frame and 10ms frame) between eMBB and URLLC
services according to their isolation constraints and QoS
requirements such as latency, reliability and minimum data
rate. Moreover, most of the works e.g. [15], [19], [20] did not
consider the AMC based link adaption process. • Firstly, an AMC based resource allocation scheme is
proposed for the dynamic multiplexing of eMBB and
URLLC users on the same RAN infrastructure of a
wireless network. In this model, the data traffic of each
eMBB user is considered to be full-buffered with an
infinite packet size and the data traffic of each URLLC
user is generated using the 3GPP’s FTP3 model [3] with
B bytes of packet size • Secondly, we formulate the above resource allocation
problem as an optimization problem to maximize the
overall sum-rate of the network while satisfying the
latency-related constraint of URLLC users and the mini-
mum rate constraint of the eMBB users. However, due to
the presence of the AMC scheme and binary assignment
variable, the formulated optimization problem becomes
analytically intractable. To address this issue, as a first
step, by considering the two approximation functions,
the step-wise optimization problem is transformed into
a continuous linear problem. In the next step, a penalized
formulation is considered to relax the binary constraint
(i.e., assignment variable). Using the above two steps,
we provide the solution to the proposed optimization
problem for RAN slicing. • Thirdly, a heuristic scheduling algorithm is proposed
to overcome the complexity, time-consumption and
in-feasibility problems of the proposed optimization
problem. B. CONTRIBUTIONS • Finally, the performances of the proposed schemes are
evaluated and compared through extensive simulations
to illustrate their capability to perform a dynamic allo-
cation of resources for different services while satisfying
their reliability, latency and minimum rate requirements. In the above context, different from the existing works, in this
work, we propose a RAN resource slicing technique for
the efficient multiplexing of eMBB and URLLC services
in wireless networks by considering an AMC scheme. Sub-
sequently, this resource slicing problem is formulated as
an optimization problem to maximize the sum rate of all
users, while satisfying the isolation constraint and stringent
QoS constraints of the users such as latency and reliability. In this AMC based resource optimization problem, each RB’s
achievable data rate is measured by the chosen modulation
and coding scheme (MCS) instead of Shannon rate formula. Furthermore, different signal-to-noise ratio (SNR) levels are
modeled based on the specific user channel conditions to
choose the MCS in accordance to their target block error
rate (BLER) which depends on the reliability constraint of
each service. This work builds on the author’s previous pub-
lication [1]. In [1], a simple network model (without consid-
eration of queues and traffic models) is considered for the
analysis of slicing based resource allocation while this paper
considers a queue based system model and different traffic
models for eMBB and URLLC. Furthermore, in [1], a hard The remainder of the paper is organized as follows. Next
section provides the system model and a detailed description
of the radio resources considered in the scheduling process. In Section III, the proposed RAN resource optimization prob-
lem and its solution are presented. A low-complexity heuristic
algorithm for the dynamic resource allocation is presented
in Section IV. Section V provides the performance analysis
of the proposed scheduling algorithms using the extensive
numerical evaluations. Finally, the conclusions are drawn in
Section VI. II. SYSTEM MODEL We consider the downlink (DL) OFDMA scenario of a
single-cell cellular network, where a base station (BS) is
located at the center of the cell, and M user equipment (UEs)
are distributed randomly across the network area as shown 45676 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA TABLE 1. Summary of notations. FIGURE 1. Illustration of a DL single-cell cellular network serving
heterogeneous services (URLLC and eMBB). TABLE 1. Summary of notations. TABLE 1. Summary of notations. FIGURE 1. Illustration of a DL single-cell cellular network serving
heterogeneous services (URLLC and eMBB). as shown in Fig. 1. At each TTI, the proposed technique
analyses the queues’ status and provides the allocation of RBs
according to the formulated mathematical optimization prob-
lem. However, the buffer congestion may show a significant
effect on the QoS requirements. To avoid these disadvantages,
we make the following assumptions: (1) Congestion control
mechanisms are applied at higher layers that detect potential
congestion and temporally reduce the transmission data rate,
(2) infinite buffer size that avoids the packet loss due to buffer
overflow. Note that the URLLC packet latency measurements
provided in the manuscript correspond to the gap (measured
in TTIs) between the time that the particular URLLC packet
has entered the queue and the time that the packet has been
scheduled and left the queue. Furthermore, these available
packets in the buffer (i.e., queue) of each UE are served by
the BS on the First-In and First-Out (FIFO) basis. in Fig. 1. The distributed UEs are associated with different
types of services such as eMBB and URLLC (i.e., different
services exist in the network). In the network, the UE associ-
ated with the URLLC service generates bursts of small pack-
ets of B bytes following the Poisson Point Process (PPP) with
the arrival rate of λ [packets/sec]. This traffic model is termed
as FTP3 in 3GPP [4]. Furthermore, the UE associated with the
eMBB service generates continuous traffic (i.e., full-buffer
traffic) with the infinite packet size. Traffic requested by the
users located within the considered RAN undergo the spe-
cific admission and congestion control schemes implemented
in the L3 and above OSI layers. II. SYSTEM MODEL In this paper, we consider the
4G LTE standard radio-frame numerology1 for radio resource
allocation to DL transmissions, which is also considered as a
candidate numerology for upcoming 5G systems. First, each
user computes the channel quality indicators (CQIs) for all
the available RBs and feeds back its CQIs to the BS. If the
RB is allocated to the specific UE, the AMC method allows
the wireless system to choose the appropriate MCS according
to the received CQI. Based on the BLER or probability of
error, and the received CQI feedback from the user, the min-
imum SNR threshold is set to obtain the appropriate MCS. For instance, if MCS14 is chosen, the SE of the MCS14 is are available, where each RE comprises of a sub-carrier and
an OFDM symbol [23], [24]. In this paper, we consider the
4G LTE standard radio-frame numerology1 for radio resource
allocation to DL transmissions, which is also considered as a
candidate numerology for upcoming 5G systems. First, each
user computes the channel quality indicators (CQIs) for all
the available RBs and feeds back its CQIs to the BS. If the
RB is allocated to the specific UE, the AMC method allows
the wireless system to choose the appropriate MCS according
to the received CQI. Based on the BLER or probability of
error, and the received CQI feedback from the user, the min-
imum SNR threshold is set to obtain the appropriate MCS. For instance, if MCS14 is chosen, the SE of the MCS14 is 5.12 bits/symbol and each RE carries 5.12 bits (using MCS
Table. 2). As a result, each RB carries 60×5.12 = 307.2 bits
on average. 1A mixture of different numerology defined in [2] is out of scope and left
for the future work. II. SYSTEM MODEL These schemes allow the
base station to make independent decisions about what set
of packets to accept and/or what bit rates to offer to elastic
traffic users competing for the same bandwidth. In general,
if traffic loads are manageable, these are queued and sched-
uled in due time according to each slice QoS and priority. In our case, and as the general assumption in the literature,
e.g. [15], we assume that the data from higher layers are
received at the serving BS and stored in their respective
user-specific transmission buffer until they get to be served The BS serves all the UEs in the cell, indexed by U =
{1, 2, . . . .M}, through the available radio resources. In our
analysis, the available radio resources are two-dimensional
(2D), i.e., including both time and frequency domains.The
DL frequency bandwidth is partitioned into F sub-bands
indexed by f
= {1, 2, . . . F} and the time dimension is
divided into transmission time intervals (TTIs) indexed by
t
= {1, 2, . . . N} with the duration of 0.5 ms as shown
in Fig. 2. Thus, a total F number of RBs are available for
DL transmission in a one-time slot. As defined in 3GPP
5G-NR [10], an RB is the minimum time-frequency resource
that can be allocated to a specific user, which consists of 7
OFDM symbols and 12 consecutive sub-carriers (for a com-
plete bandwidth of 180 KHz) [21], [22]. Therefore, each
RB (i.e., 12 sub-carriers, 7 OFDM symbols) includes 84
Resource Elements (REs), after accounting for the refer-
ence signals overhead, approximately ρ = 60 REs per RB 45677 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA FIGURE 2. Illustration of time and frequency radio resource grid in the considered system
scenario. FIGURE 2. Illustration of time and frequency radio resource grid in the considered system
scenario. ABLE 2. Modulation and Coding Schemes (MCS) for eMBB and URLLC services with different BLERs. TABLE 2. Modulation and Coding Schemes (MCS) for eMBB and URLLC services with different BLERs. TABLE 2. Modulation and Coding Schemes (MCS) for eMBB and URLLC services with different BLERs. are available, where each RE comprises of a sub-carrier and
an OFDM symbol [23], [24]. III. PROBLEM FORMULATION AND
PROPOSED SOLUTION Now, wu is expressed as where Ru
mbb, Ru
llc are the bit rates of the uth eMBB and
URLLC users, and wu is the weight factor of the uth URLLC
user. In order to prioritize the URLLC user that accumulates
more packets in its queue, wu is included in the URLLC
individual user rates. Now, wu is expressed as F
X
f =1
kp+p
X
t=kp+1
xu
t,f ≥1; k = 0, 1, 2, 3.., kmax;
∀u ∈U2
(C3) (C3) wu =
Q(l)
u
P
u∈U2
Q(l)
u
(2) More precisely, when a URLLC user is scheduled
(i.e., u ∈U2), constraint (C3) enforces that at least one RB
for every p TTIs is scheduled for each URLLC users until
the required number of TTIs to vacate the queue. When the
number of available URLLC packets in the queue becomes
very high, the scheduler needs to allocate RBs to the user
by following the given constraint (C3) in order to vacate as
much as possible number of packets. Otherwise, when the
available packets in the queue are fewer, then the scheduler
needs to assign RBs until the queue becomes empty. To do
so, the variable kmax is defined as (2) where Q(l)
u represents the queue length of uth user on the lth
frame measured in bits. A high wu value indicates a high
priority URLLC user. The bit rate of each user that belongs
to either eMBB or URLLC service, Ru
s, s ∈{mbb, llc} is
computed as Ru
s =
N
X
t=1
F
X
f =1
xu
t,f Ru
t,f ,
∀u ∈U1 : s = mbb;
∀u ∈U2 : s = llc;
(3) (3) kmax = min
N
p
−1,
Q(l)
u
ρ · ϕ
−1
(6) (6) (6) where the bit rate of user u operating in sub-band f at TTI t
can be expressed as where ϕ represents the SE of the MCS that ensures that the
whole packet can be sent. For example, assume that the queue
length of the uth URLLC user is 1024, and MCS13 is selected
for the scheduled RB. Using the formula in (6), the variable
kmax is computed as ⌈
1024
60×4.52⌉−1 = 3, and the required
number of RBs for the assumed queue length is estimated as
ψRB = kmax + 1, i.e., 3 + 1 = 4. III. PROBLEM FORMULATION AND
PROPOSED SOLUTION The fifth-generation (5G) wireless networks are expected
to support users from multiple services. These services are
mainly classified as latency-critical and rate-based services. The latency-critical service needs to satisfy delay constraint,
while the rate-based service requires a minimum rate to
support continuous traffic demand. Therefore, an efficient
scheduling mechanism is essential to allocate the resources
for these two type of services by satisfying their require-
ments (i.e., aforementioned constraints). In other words,
in this work, we develop a slice-aware RAN radio resource 45678 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA different BLER targets are provided in Table 2 [25]. Note that
in Table 2, we have provided two different MCS depending
on the BLER target. In particular, BLER = 10−3 is used for
to the URLLC service, while BLER = 10−1 is used for the
eMBB service. Also, perfect channel information is assumed
at the BS for all users, and the total available power Pmax is
considered to be equally distributed over all available RBs
for a TTI (i.e., allocated power to each RB is P = Pmax/F). Then, the received SNR (γ u
t,f ) of the uth user in sub-band f at
TTI t can be expressed as different BLER targets are provided in Table 2 [25]. Note that
in Table 2, we have provided two different MCS depending
on the BLER target. In particular, BLER = 10−3 is used for
to the URLLC service, while BLER = 10−1 is used for the
eMBB service. Also, perfect channel information is assumed
at the BS for all users, and the total available power Pmax is
considered to be equally distributed over all available RBs
for a TTI (i.e., allocated power to each RB is P = Pmax/F). Then, the received SNR (γ u
t,f ) of the uth user in sub-band f at
TTI t can be expressed as allocation mechanism [30], which shares the available radio
resources by considering the specific constraints for eMBB
and URLLC users to make sure that the performance guar-
antees of slices are satisfied and the slices do not adversely
affect the each other’s performance. Particularly, in this
section, we address the AMC based sum-rate maximization
problem for the dynamic allocation of radio resources to the
wireless system consisting of multiple services. III. PROBLEM FORMULATION AND
PROPOSED SOLUTION A total T number of frames are considered for the schedul-
ing process, wherein each frame consists of F number of RBs
and N number of TTIs. The problem consists in assigning the
total number of N × F RBs to the active users. During the
scheduling round (i.e., for every TTI), each RB is assigned
to a single user. Denoting xu
t,f a binary assignment variable,
which is 1 if the RB (t, f ) is allocated to the user u, otherwise,
it is 0. Then, the binary constraint is mathematically written
as γ u
t,f =
P|hu
t,f |2d−α
BS,u
σ 2
,
(5) (5) where hu
t,f represents the channel gain of user u on a sub-band
f at the TTI t, dBS,u is the distance between the BS and the
UE, α is the path loss exponent and σ 2 is the noise power. Note that the inter-cell interference is assumed to be mitigated
using suitable interference avoidance mechanisms such as
in [34], [35]. as xu
t,f =
1; If RB (t, f ) is allocated to user ’u’
0;
Otherwise
(C1) (C1) We maximize the above objective function given in (1) sub-
ject to the constraints as follows. The aforementioned binary
constraint (C1) or decision variable maintains the allocation
of RBs to users and the constraint (C2) assures that an RB is
only allocated to a single user (i.e., called as the orthogonality
constraint). In this work, we assume the presence of both the eMBB and
URLLC users in the network. The sets U1 = {1, 2, . . . , L}
and U2 = {1, 2, . . . , K}, represent the sets of users associated
with eMBB and URLLC services, respectively. The objective
function, i.e., the total sum-rate of the network, is then given
by X
u∈U1
X
u∈U2
xu
t,f ≤1;
∀t, f
(C2) (C2) Rtot =
X
u∈U1
Ru
mbb +
X
u∈U2
wuRu
llc
(1) (1) The next constraint (C3) introduces to control the transmis-
sion latency requirement of URLLC users. where Ru
mbb, Ru
llc are the bit rates of the uth eMBB and
URLLC users, and wu is the weight factor of the uth URLLC
user. In order to prioritize the URLLC user that accumulates
more packets in its queue, wu is included in the URLLC
individual user rates. III. PROBLEM FORMULATION AND
PROPOSED SOLUTION Assuming p = 2, a total
(p × ψRB) TTIs, i.e., 2 × 4 = 8 are required to vacate the Ru
t,f = BRB · T · F(γ u
t,f )
[bits]
(4) (4) where BRB is the bandwidth of an RB, T is the transmis-
sion time length of each slot and F(.) is the spectral effi-
ciency (SE) of the selected MCS from Table. 2 according to
the achievable SNR. In our study, Mmcs distinct MCSs are
considered and the corresponding SNR levels of MCS for 45679 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA FIGURE 3. Rate of eMBB, URLLC services using true MCS given
in Table 2 and approximated rate functions. queue of this particular URLLC user. Note that the number
of scheduling TTIs should not exceed the available TTIs in
a frame such that by setting the minimum condition as given
in (6), we can assure the feasible scheduling process. Furthermore, each of the assigned RB to the URLLC user
should at least transmits a complete data packet. In
this
regard, constraint (C4) enforces the condition that every
scheduled RB for the URLLC user transmits more than the
minimum number of bits (i.e., one packet size denotes by Rmin
measured in bits). It means that when an RB is scheduled to
a uth URLLC user (i.e., xu
t,f = 1), the resulting rate from that
assigned RB should be greater than or equal to Rmin number of
bits. Moreover, we assume that the packet size of all URLLC
users is same and the packet segmentation is not allowed. Thus, this constraint helps to transmit at least a packet through
the assigned RB. xu
t,f · Ru
t,f ≥xu
t,f · Rmin; u ∈U2
(C4) (C4) FIGURE 3. Rate of eMBB, URLLC services using true MCS given
in Table 2 and approximated rate functions. For every URLLC user, the constraints (C3) and (C4)
together ensures the transmission of at least one packet of data
for every p time slots till to reach the kmax number of TTIs. Note that the backlogged packets in the queue are transmitted
in the early next frame. III. PROBLEM FORMULATION AND
PROPOSED SOLUTION F
X
f =1
N
X
t=1
xu
t,f Ru
t,f ≥Rth; u ∈U1
(C5) (C5) However, the presented function F(.) in (7), given by (4),
is a step-wise function that makes the optimization problem
mathematically intractable and complex to solve. Inspired
by [26], to simplify the problem, using the received SNR
and target BLER, we make use of two SE approximation
functions (i.e., differentiable continuous functions) for eMBB
and URLLC services that can be expressed as Finally, to ensure a minimum throughput for the eMBB
service, the constraint (C5) confirms that every scheduled
eMBB user at least transmit Rth number of bits for every
frame. F
X
f =1
N
X
t=1
xu
t,f Ru
t,f ≥Rth; u ∈U1
(C5) (C5) Fmbb(γ u
t,f , βmbb) = log2(1 +
γ u
t,f
0mbb
),
(8)
Fllc(γ u
t,f , βllc) = log2(1 +
γ u
t,f
0llc
)
(9) The major objective of the proposed optimization problem
is to maximize the total sum-rate of users associated with
eMBB and URLLC services through performing dynamic
RBs allocation subject to a set of constraints. After the for-
mulation of max sum-rate, the term of sum-data rate for
URLLC service is required in conjunction with the latency
constraint in order to adhere to the URLLC requirements,
which has to be implemented by taking into account the avail-
able queue lengths of URLLC users (and updating the weight
at each scheduling time).Therefore, the optimization problem
is formulated as a scheduling of the time-frequency radio
resources to the different users according to their available
traffics (i.e., queue lengths) and respective QoS requirements. Mathematically, the optimization problem is expressed as (8) (9) where 0mbb =
−ln(5βmmb)
0.45
and 0llc =
−ln(5βllc)
1.25
represent
the SNR gaps, and βmbb and βllc represent the target BLER
for eMBB and URLLC users, respectively. The proposed
approximate functions are compared to the values achieved
with AMC Table. 2 in Fig. 3, where we can observe that (8)
and (9) provide a good approximation to the AMC step-wise
functions. P1 :
max
{xu
t,f } Rtot A. HEURISTIC ALGORITHM FOR SCHEDULING
OF EMBB AND URLLC USERS In this section, we propose a low-complexity greedy heuris-
tic algorithm for scheduling the eMBB and URLLC users
efficiently. This algorithm is proposed to maximize the over-
all sum-rate of the users in the network while prioritizing
the URLLC users. A total T number of frames are con-
sidered for the complete scheduling process, wherein each
frame consists of F number of RBs and N number of TTIs
(i.e., N × F number of RBs for the complete frame). The
proposed heuristic algorithm is executed for the given number
frames T. In particular, at each frame l, l
=
1, . . . T,
the URLLC users are firstly scheduled based on the best
RB according to their channel conditions, followed by the
scheduling on the remaining RBs for the eMBB, again based
on their channel conditions. After completion of the schedul-
ing of a particular frame l, the queues of the URLLC UEs are
updated with the new arrived packets and the unscheduled
packets of the previous frame. Clearly, the proposed heuristic
provide the priority for URLLC users in order to satisfy the
latency-related requirement. Summarizing, the scheduling
process at each frame ’l’ is executed in the steps as follows: P3 :
max
{xu
t,f }
ˆRtot +
X
u∈U1
X
u∈U2
F
X
f =1
N
X
t=1
ζ1P(xu
t,f )
(15)
subject to (C1 :) 0 ≤xu
t,f ≤1
(16)
(C2), (C3), (C4) and (C5) in (P1)
(17) (15) (16)
(17) By assuming X = xu
t,f , the penalty function is defined
as P(X) = (X2 −X), which is a convex function in the
region of [0, 1]. The function P(X) produces no penalty at
X = 0 or 1 and increases the penalty as X moves away
from 0 or 1 with the maximum penalty at X = 0.5. For
example, when X = 0.5, the total incurred penalty is (0.5)2 −
(0.5) = −0.25. Further, by selecting the penalty parame-
ter ζ1 appropriately, the binary nature of X can be accom-
plished. Note that the objective function in P3 i.e., ˆRtot −
F
N
p
j
−P
u∈U1
P
u∈U2
FP
f =1
NP
t=1
ζ1P(xu
t,f )
, is a difference of convex and concave functions. Thus, the problem P3 belongs to
the class of difference of convex (DC) programming [27]. A. HEURISTIC ALGORITHM FOR SCHEDULING
OF EMBB AND URLLC USERS In this regard, we utilize an iterative algorithm based on
convex-concave procedure (CCP) to solve the DC problem
in (14). CCP is a dynamic tool to estimate the stationary
point of the DC problems. In this algorithm, the following two
steps are performed iteratively till its converges: (i) Assume
Xk−1 is the estimate of X in the (k −1)th iteration. In the
kth iteration, the affine approximation around the estimate of
Xk−1 is utilized to replace the convex part of the objective
(i.e., the penalty summation part in (15)). (ii) The update
Xk+1 is acquired by solving the following convex problem: Step 1. SNRs Computation: Compute the received SNRs
of all users on all the available RBs using (5) in the first
TTI of every frame. Note that within 1 frame, assume that
the channel remains temporal invariant, while it may change
from carrier to carrier (i.e., RB to RB). Therefore, the compu-
tation of SNRs on the first TTI is sufficient for the complete
scheduling process of a frame. Step 2. RBs Assignment to Users: The URLLC service is
prioritized than the eMBB service, hence, the active URLLC
users schedule first according to the process as follows:
first, identify the highest SNR received RB for every active
URLLC user using Step 1 and next, compute the MCS and
SE of the selected RBs by comparing the SNR values with the
ones in Table 2. Note that, in this work the packet segmenta-
tion is not allowed for the URLLC users, so that the user can
select the MCS according to the packet size (i.e., the user can
select the lower MCS instead of achieved higher MCS, which
is sufficient to transmit one packet). Then, using the updated
queue and the SE of the selected RB, the number of required
RBs to transmit the available traffic is computed as P4 :
max
{X} [ ˆRtot + ζ1
X
u∈U1
X
u∈U2
F
X
f =1
N
X
t=1
(X −Xk−1)∇P(X)]
(18)
subject to(C1) 0 ≤X ≤1; ∀u, t, f
(19)
(C2), (C3), (C4) and (C5) in (P1)
(20) The above algorithm is based on the CCP framework,
thus, a feasible initial point is enough to converge the algo-
rithm to a stationary point [28]. Note that the initial fea-
sible point need not to be binary, but it should satisfy all
the other constraints in the optimization problem. The optimization problem (P1) is now reformulated as The optimization problem (P1) is now reformulated as (i.e., without penalty function), wherein the constraint (C1) is
relaxed between 0 and 1. Now, the problem P4 is a convex
problem that can be solved by the standard optimization
software tools such as CVX [29]. The proposed solution
optimizes the resource allocation across both frequency and
time simultaneously, covering the complete frame. The final
output of the problem shows in each scheduling round which
users should be served on which RB. P2 :
max
{xu
t,f }
ˆRtot
(13)
subject to
(C1), (C2), (C3), (C4) and (C5) in (P1)
(14) (13) Due to the binary constraint (C1), the problem P2 is com-
binatorial. Therefore, to avoid the combinatorial nature of P1,
the assignment variable xu
t,f is relaxed to a box constraint
between 0 and 1, and the relaxation penalty is added using
the function P(xu
t,f ) so that the relaxed problem produces
the output favorable to either 0 or 1. The problem P2 is
reformulated with the penalty parameter ζ1 as VOLUME 8, 2020 A. HEURISTIC ALGORITHM FOR SCHEDULING
OF EMBB AND URLLC USERS Therefore,
we set the initial feasible point by solving the problem P1 ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉),
∀u ∈U2
(21) (21) III. PROBLEM FORMULATION AND
PROPOSED SOLUTION The achievable data on each RB using the aforementioned
approximation functions can be written as Mathematically, the optimization problem is expressed as ru,s
t,f = B · T · Fs(γ, βs), s ∈{mbb, llc}[bits]
(10) (10) P1 :
max
{xu
t,f } Rtot
(7) (7) Using (10), the bit-rate of each user that belongs to either
eMBB or URLLC service can be reformulated as Using (10), the bit-rate of each user that belongs to either
eMBB or URLLC service can be reformulated as ct to xt,f ∈{0, 1};
∀u, t, f
(C1)
X
u∈U1
X
u∈U2
xu
t,f ≤1;
∀t, f
(C2)
F
X
f =1
kp+p
X
t=kp+1
xu
t,f ≥1;
k = 0, 1, 2, 3.., kmax; u ∈U2
(C3)
xu
t,f · Ru
t,f ≥xu
t,f · Rmin; u ∈U2
(C4)
eMBB or URLLC service can be reformulated as
ˆ
Rus =
N
X
t=1
F
X
f =1
xu
t,f ru,s
t,f
(11)
Now, using (11), the objective function sum-rate of all
users can be reformulated as
ˆRtot =
X
u∈U1
ˆRu
mbb +
X
u∈U2
wu ˆRu
llc
(12) to xt,f ∈{0, 1};
∀u, t, f
(C1)
X
u∈U1
X
u∈U2
xu
t,f ≤1;
∀t, f
(C2)
F
X
f =1
kp+p
X
t=kp+1
xu
t,f ≥1;
k = 0, 1, 2, 3.., kmax; u ∈U2
(C3)
xu
t,f · Ru
t,f ≥xu
t,f · Rmin; u ∈U2
(C4)
eMBB
Now
users ˆ
Rus =
N
X
t=1
F
X
f =1
xu
t,f ru,s
t,f
(11) (11) Now, using (11), the objective function sum-rate of all
users can be reformulated as t
kp+1
k = 0, 1, 2, 3.., kmax; u ∈U2
(C3)
Ru
t,f ≥xu
t,f · Rmin; u ∈U2
(C4)
ˆRtot =
X
u∈U1
ˆRu
mbb +
X
u∈U2
wu ˆRu
llc
(12) (12) 45680 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉),
∀u ∈U2
(21) data for uth URLLC user on lth frame : G(l)
u
2: Initialization: l = 1 and queue: ζ (0)
u
= 0;
3: while l ≤T do
4:
for u = 1 : Num do
5:
for f = 1 : F do
6:
Estimate the received SNRs of the user on all
RBs using (5)
7:
end for
8:
if u ∈U2 then
9:
Find the highest SNR received RB:
f ∗= arg maxu∈U2 γ u
t,f
10:
Get the MCS and SE of the highest SNR
received RB using Table 2;
11:
Get the queue length: Q(l)
u = G(l)
u + ζ (l−1)
u
12:
Compute the required number of RBs:
ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉)
13:
Assign the selected RBs to the user for every
p TTIs until to reach the ku RBs;
14:
Remove the selected RBs from the scheduling
process in that TTI;
15:
end if
16:
if u ∈U1 then
17:
Find the highest SNR received RBs:
f ∗= arg maxu∈U1 γ u
t,f
18:
Get the MCS and SE of the RB;
19:
Assign the selected RBs to the user in every
TTI;
20:
end if
21:
end for
22:
Compute the delivered data of URLLC users:
R(l)
u = ku · ρ · ϕ; ∀u ∈U2
23:
Queue status update of of URLLC user:
ζ (l)
u
= Q(l)
u −R(l)
u ;∀u ∈U2
24:
Compute the delivered data of eMBB users:
R(l)
u = Pηu
i=1 ρ · ϕu, ∀u ∈U1
25:
l = l + 1;
26: end while Step 3: Computation of Delivered Data and Queue Update:
After completion of the scheduling process for the complete
frame (i.e., up to N number of TTIs), compute the sum-rate
(i.e., delivered data rate) of every user using the assigned
number of RBs and SE of the selected RB. Next, estimate the
undelivered data traffic by subtracting the delivered data traf-
fic from the available data traffic (i.e., generated or original
data traffic ). Finally, update the queues with the undelivered
data traffic, that is scheduled in the early next frame. The
same procedure continues until the end of the frames. The
complete procedure of heuristic scheduling scheme is sum-
marized in Algorithm 1. B. COMPLEXITY ANALYSIS The computational complexity of the optimization problem
depends on the complexities of the following two procedures
provided in Section III: (i) Convex-Concave procedure (CCP)
involved in the convex problem P4, and (ii) Initial feasi-
ble point selection procedure using the convex problem P1. The convex problem P4 has MNF decision variables and
2MNF +NF +K(kmax +1)+M linear constraints. Therefore,
the computational complexity of P4 is O((MNF)3(2MNF +
NF+K(kmax+1)+M)). Similarly, the convex problem P1 has
MNF decision variables and 2MNF + NF + Kkmax + 2K + L
linear constraints. Hence, the computational complexity of P1
is O((MNF)3(2MNF + NF + Kkmax + 2K + L)) [32]. The complexity of heuristic algorithm is majorly due to the
one while iterations and executing two argmaxs. The com-
plexity for executing argmax is proportional to the number of
elements being sorted. Therefore, the complexity of heuristic
algorithm is O(L+F +K) for each RB and the total complex-
ity is O(FL + F2 + FK) [33]. From this complexity analysis,
it is easy to observe that the computational complexity of the
optimization method is significantly larger compared to the
heuristic method as expected. IV. NUMERICAL EVALUATIONS In this section, we simulate and compare the performance
of the proposed optimization based and heuristic schedul-
ing algorithms for the allocation of resources to the exist-
ing eMBB and URLLC users in the downlink single-cell
OFDMA scenario. ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉),
∀u ∈U2
(21) Later, assign the selected RB for every assuming p TTIs
until to meet the ku number of RBs. After scheduling the
URLLC users in every TTI, remove the assigned RBs from 45681 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA Algorithm 1 Heuristic Algorithm for Scheduling of eMBB
and URLLC Users the scheduling and update the available RBs for eMBB users
scheduling. Now, the eMBB users first identify the highest
SNR received RBs using Step 1 and next, estimate the MCS
and SEs of the selected RBs by comparing the SNR values
with the provided Table 2, and finally the selected RBs are
assigned to every user in every scheduling TTI until the end
of frame. Algorithm 1 Heuristic Algorithm for Scheduling of eMBB
and URLLC Users
1: Inputs:
• Number of TTIs: N
• Number of RBs: F
• Class of eMBB and URLLC users: U1 and U2
• Total number of users: Num = U1 ∪U2
• Number of frames: T
• Gen. ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉),
∀u ∈U2
(21) data for uth URLLC user on lth frame : G(l)
u
2: Initialization: l = 1 and queue: ζ (0)
u
= 0;
3: while l ≤T do
4:
for u = 1 : Num do
5:
for f = 1 : F do
6:
Estimate the received SNRs of the user on all
RBs using (5)
7:
end for
8:
if u ∈U2 then
9:
Find the highest SNR received RB:
f ∗= arg maxu∈U2 γ u
t,f
10:
Get the MCS and SE of the highest SNR
received RB using Table 2;
11:
Get the queue length: Q(l)
u = G(l)
u + ζ (l−1)
u
12:
Compute the required number of RBs:
ku = max(⌈N/p⌉, ⌈Q(l)
u /ρ · ϕ⌉)
13:
Assign the selected RBs to the user for every
p TTIs until to reach the ku RBs;
14:
Remove the selected RBs from the scheduling
process in that TTI;
15:
end if
16:
if u ∈U1 then
17:
Find the highest SNR received RBs:
f ∗= arg maxu∈U1 γ u
t,f
18:
Get the MCS and SE of the RB;
19:
Assign the selected RBs to the user in every
TTI;
20:
end if
21:
end for
22:
Compute the delivered data of URLLC users:
R(l)
u = ku · ρ · ϕ; ∀u ∈U2
23:
Queue status update of of URLLC user:
ζ (l)
u
= Q(l)
u −R(l)
u ;∀u ∈U2
24:
Compute the delivered data of eMBB users:
R(l)
u = Pηu
i=1 ρ · ϕu, ∀u ∈U1
25:
l = l + 1;
26: end while Algorithm 1 Heuristic Algorithm for Scheduling of eMBB
and URLLC Users
1: Inputs:
• Number of TTIs: N
• Number of RBs: F
• Class of eMBB and URLLC users: U1 and U2
• Total number of users: Num = U1 ∪U2
• Number of frames: T
• Gen. A. SIMULATION ENVIRONMENT the channel between the BS and the each user is considered
as a Nakagami-m fading channel. Also, the path loss expo-
nent (α) is set to 3 for all the communication links. Our
simulations are executed for 10 frames, where each frame
consists of 20 TTIs (i.e., a total time of 10ms) and 100
RBs for each TTI. Each RB comprises of 12 sub-carriers, We mainly concentrate on the resource scheduling for a
downlink wireless network, where a single BS is deployed
at the center of the cell coverage area with the radius
of 250m, and L eMBB users and K URLLC users are dis-
tributed randomly within the cell coverage area. In this model, 45682 45682 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA TABLE 3. Simulation parameters. TABLE 3. Simulation parameters. FIGURE 4. Cumulative distribution of achieved eMBB rates (per frame)
for different URLLC loads using the proposed scheduling algorithms and
baseline methods. FIGURE 4
Cumulative distribution of achieved eMBB rates (per frame) TABLE 3. Simulation parameters. FIGURE 4. Cumulative distribution of achieved eMBB rates (per frame)
for different URLLC loads using the proposed scheduling algorithms and
baseline methods. 7 OFDM symbols, and a total of 60 REs after accounting
for the reference signals. Moreover, each sub-carrier has a
carrier-spacing of 15 KHz. Hence, the bandwidth of each RB
is 180 KHz and the available complete bandwidth for the BS
is 20 MHz. Also, we assume that the additive white Gaussian
noise power on each sub-band is 10−10W. Importantly, to sat-
isfy the reliability of each service, we assume a high BLER
target (i.e., βmbb = 10−1) for eMBB users as compared
to URLLC user’s BLER target (i.e., βllc = 10−3). Further,
we consider that each UE has a buffer to store the generated
packets prior to serve. In this network model, we assume
that each URLLC UE generates the small bursts of data
following the FTP3 model with the mean arrival rate of λ
payloads per frame (i.e., for example λ = 2.12 packets/10ms,
equal to 212 packets/sec), and each eMBB user generates the
packet with an infinite size. The complete set of simulation
parameters is provided in Table 3. (i.e., the minimum rate requirement, Rth = 30 Kbits per
frame). A. SIMULATION ENVIRONMENT The heuristic algorithm first greedily assigns RBs
to URLLC users by giving them priority, and subsequently
assigns the remaining RBs (i.e., left after scheduling of the
URLLC users) to the eMBB users, which are not enough
sometimes to achieve the minimum rate. Also, in the eMBB
users scheduling process, the heuristic algorithm greedily
assigns the RBs to the users which receive the highest SNRs,
so that the users with lowest SNRs cannot get the required
number of RBs to achieve the minimum rate. In contrast,
the optimization based scheduling algorithm provides the
RBs to users by respecting the isolation between service
slices, so that it satisfies the minimum rate condition of
eMBB users regardless of the channel conditions. The results
confirm that the heuristic algorithm, although lighter in com-
plexity compared to the proposed optimization procedure,
it fails in satisfying isolation and minimum rate requirements. y g
q
In Fig. 5, we evaluate the ECDF of the latency in the deliv-
ered URLLC packets measured as the gap between the TTI
that the packets have entered the queue and the TTI the packet
has been scheduled and left the queue. Therefore, the total
packet latency time is computed as the sum of packet waiting
time in the queue, the required time to assign the RB and
data transmission (i.e., scheduling time). From the results
in Fig. 5, it is observed that adjusting the value of p in (C3)
(i.e., difference between the scheduling time intervals is less)
the total latency can be reduced. For instance, by assigning
an RB to URLLC user for every 2 TTIs, 40% of packets
experience approximately 3 TTIs less latency as compared
to the assignment of RB to URLLC users for every 4 TTIs. As expected, the heuristic scheduling algorithm shows a
slightly better performance compared to the optimization
based scheduling algorithm in terms of latency. The heuristic
based algorithm schedules RBs to the URLLC users before B. RESULTS AND DISCUSSIONS Further,
as can be seen from the results, the sum of unscheduled
packets is increased with p = 4 compared to p = 2 as
expected. By considering the assignment of RBs to URLLC
users in the large TTI gaps (i.e., high values of p), obviously
reduce the number of resources for URLLC users and leads to
huge number of unscheduled packets in the queues of URLLC
users. In Fig. 7, we show the average sum-rate of all users
bt i
d b
i
th
d
ti i
ti
b
d
h d l
FIGURE 6. Sum of URLLC users queues on every frame for different TTI
assignment strategies (p) at the URLLC packet arrival rate of λ = 6.36
packets/frame and L = 10 and K = 10. FIGURE 7. Average sum rate of eMBB, URLLC and total users with varying
BS powers at 2 scheduling TTIs gap (i.e., p = 2) and L = 10 and K = 5. and URLLC users. From results, it is noticed that the per-
formance of the heuristic scheme is almost as same as the
performance of the optimization-based scheduling scheme
(i.e., the performance gap of the two algorithms is negligible). The resulting average sum-rate using the heuristic method is
a tight upper bound when all the active users in the network
b l
t th URLLC
i
O th
t
th
lti FIGURE 6. Sum of URLLC users queues on every frame for different TTI
assignment strategies (p) at the URLLC packet arrival rate of λ = 6.36
packets/frame and L = 10 and K = 10. FIGURE 7. Average sum rate of eMBB, URLLC and total users with varying
BS powers at 2 scheduling TTIs gap (i.e., p = 2) and L = 10 and K = 5. and URLLC users. From results, it is noticed that the per-
f
f h
h
i i
h
i
l
h FIGURE 5. Cumulative distribution of URLLC latency for different TTI
assignment strategies at the URLLC packet arrival rate of λ = 2.12
packets/frame and L = 10 and K = 5. FIGURE 6. Sum of URLLC users queues on every frame for different TTI
assignment strategies (p) at the URLLC packet arrival rate of λ = 6.36
packets/frame and L = 10 and K = 10. FIGURE 5. B. RESULTS AND DISCUSSIONS Cumulative distribution of URLLC latency for different TTI
assignment strategies at the URLLC packet arrival rate of λ = 2.12
packets/frame and L = 10 and K = 5. FIGURE 6. Sum of URLLC users queues on every frame for different TTI
assignment strategies (p) at the URLLC packet arrival rate of λ = 6.36
packets/frame and L = 10 and K = 10. FIGURE 6. Sum of URLLC users queues on every frame for different TTI
assignment strategies (p) at the URLLC packet arrival rate of λ = 6.36
packets/frame and L = 10 and K = 10. eMBB users by giving them priority, but, the optimization
method schedules RBs to users in any TTI of the considered
interval range by following the constraints. This is the reason
for heuristic algorithm to achieve the better performance in
terms of latency compared to the optimization method. The results in Fig. 4 and Fig. 5 show the trade off between
the minimum data rate of eMBB users and the latency of
URLLC users. Using the optimization based scheduling pro-
cess, the minimum data rate requirement for eMBB users
is achieved, while providing a good latency-related perfor-
mance for URLLC users. In contrast, the heuristic scheduling
scheme improves the performance of URLLC users in terms
of latency, but it fails to achieve the minimum data rate for
eMBB users. Fig. 6 shows the sum of the queue status (i.e., unscheduled
packets (ζ (l)
u )) of URLLC users on every frame after exe-
cuting the scheduling process using the proposed algorithms
for different p values. It can be observed that both optimal
and heuristic are vacating the URLLC packets for low values
of ‘‘p’’, while this is not the case when ‘‘p’’ increases. Further,
as can be seen from the results, the sum of unscheduled
packets is increased with p = 4 compared to p = 2 as
expected. By considering the assignment of RBs to URLLC
users in the large TTI gaps (i.e., high values of p), obviously
reduce the number of resources for URLLC users and leads to
huge number of unscheduled packets in the queues of URLLC
users. FIGURE 7. Average sum rate of eMBB, URLLC and total users with varying
BS powers at 2 scheduling TTIs gap (i.e., p = 2) and L = 10 and K = 5. and URLLC users. B. RESULTS AND DISCUSSIONS We compare the performance of the proposed methods in
Section III with the performance of baseline methods random
scheduler (RS) (i.e., distributes RBs randomly to URLLC and
eMBB users), equally distributed scheduler (EDS) (i.e., dis-
tributes RBs equally to URLLC and eMBB users) [31] and
proportional fair (PF) scheduler [15] in terms of achieved
delivered data rate for eMBB users. In Fig. 4, we illustrate
the empirical cumulative distribution function (ECDF) of
achieved delivered data rates of eMBB users on every frame
using the proposed methods, RS, EDS, and PF. From the
results in Fig. 4, it can be observed that the baseline methods
RS, EDS, and PF fail to achieve the minimum delivered rate
(per frame) for many of eMBB users. Also, it is evident
from the results that using the heuristic scheduling algorithm,
some of the eMBB users fail to satisfy the constraint (C5) 45683 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA FIGURE 5. Cumulative distribution of URLLC latency for different TTI
assignment strategies at the URLLC packet arrival rate of λ = 2.12
packets/frame and L = 10 and K = 5. eMBB users by giving them priority, but, the optimization
method schedules RBs to users in any TTI of the considered
interval range by following the constraints. This is the reason
for heuristic algorithm to achieve the better performance in
terms of latency compared to the optimization method. The results in Fig. 4 and Fig. 5 show the trade off between
the minimum data rate of eMBB users and the latency of
URLLC users. Using the optimization based scheduling pro-
cess, the minimum data rate requirement for eMBB users
is achieved, while providing a good latency-related perfor-
mance for URLLC users. In contrast, the heuristic scheduling
scheme improves the performance of URLLC users in terms
of latency, but it fails to achieve the minimum data rate for
eMBB users. Fig. 6 shows the sum of the queue status (i.e., unscheduled
packets (ζ (l)
u )) of URLLC users on every frame after exe-
cuting the scheduling process using the proposed algorithms
for different p values. It can be observed that both optimal
and heuristic are vacating the URLLC packets for low values
of ‘‘p’’, while this is not the case when ‘‘p’’ increases. B. RESULTS AND DISCUSSIONS Fur-
ther, the following outcomes are observed for the three dif-
ferent existence scenarios: (i) when all the active users in the
cell belong to eMBB service, the resulting average sum-rate
performance of users is the tight upper bound, (ii) if all the
active users in cell are associated with the URLLC service,
then the resulting average sum-rate performance of users is
the tight lower bound, and (iii) The resulting average sum-rate
performance of users is lower than the performance of all
eMBB users and higher than the performance of all URLLC
users when the active users in cell belong to both types of
services. The reliability constraint is more strict for URLLC
service, therefore, in order to ensure the transmission with
high success probability, the URLLC users select the lower
MCS compared to the eMBB users. This is the main reason
for the aforementioned results, shown in Fig. 7. g
In Fig. 8 and Fig. 9, we illustrate the average sum-rate of
the eMBB users and URLLC users, respectively, achieved by
the proposed scheduling algorithm for different scheduling
TTI gaps (i.e., different p values) and packets arrival rate of
URLLC users. As can be seen from results, it is clear that the
average sum-rate of URLLC users decreases by increasing
the value of p. In contrast, the average sum-rate of the eMBB
users increases by increasing the value of p. Specifically,
this effect is very dominant at the higher packets arrival
rate. This is happened due to the constraint presented in the
problem that allocates only an RB to the URLLC users for
every p TTIs. Obviously, this condition favors improving the
sum-rate of eMBB users by assigning a higher number of
RBs. Due to the allocation of less number of RBs instead of
the required number of RBs, the unscheduled data packets
are stacked in the queues for the longer period. For example,
assume that a URLLC user is generating the data packets FIGURE 10. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with a new constraint (C3a)). with the arrival rate of 8 (packets/frame). B. RESULTS AND DISCUSSIONS This is the main reason
for the aforementioned results, shown in Fig. 7. In Fig. 8 and Fig. 9, we illustrate the average sum-rate of
the eMBB users and URLLC users, respectively, achieved by
the proposed scheduling algorithm for different scheduling
TTI gaps (i.e., different p values) and packets arrival rate of
URLLC users. As can be seen from results, it is clear that the
average sum-rate of URLLC users decreases by increasing
the value of p. In contrast, the average sum-rate of the eMBB
users increases by increasing the value of p. Specifically,
this effect is very dominant at the higher packets arrival
rate. This is happened due to the constraint presented in the
problem that allocates only an RB to the URLLC users for
TTI
Ob i
l
thi
diti
f
i
i
th
FIGURE 9. Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 10. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with a new constraint (C3a)). with the arrival rate of 8 (packets/frame). According to the
arrival rate, a total 8 number of RBs are required to vacate
the complete packets, which means that for every 10 TTIs,
at least 4 RBs should be assigned to the user in the case of
5ms transmission latency. But, the constraint (C3) allocates
only 1 RB for every 10 TTIs (i.e., in total 2 RBs), which
l
d t
6
h d l d
k t i
th
th
k t FIGURE 9. Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 10. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with a new constraint (C3a)). FIGURE 8. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 9. B. RESULTS AND DISCUSSIONS From results, it is noticed that the per-
formance of the heuristic scheme is almost as same as the
performance of the optimization-based scheduling scheme
(i.e., the performance gap of the two algorithms is negligible). The resulting average sum-rate using the heuristic method is
a tight upper bound when all the active users in the network
belong to the URLLC service. On the contrary, the resulting
average sum rate using the optimization method is a tight
upper bound when all the active users in the network are
from the eMBB service. Also, we observe that the average
sum-rate of all users increases with the BS power as expected. As the BS power increases, the received SNR at the user In Fig. 7, we show the average sum-rate of all users
obtained by using the proposed optimization-based schedul-
ing scheme and the heuristic scheme. Specifically, we show
the results by considering the three different scenarios of
users existence in the cell: (i) all the distributed users belong-
ing to eMBB service, (ii) all the distributed users belonging
to URLLC service, and (iii) the presence of both the eMBB 45684 VOLUME 8, 2020 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA FIGURE 8. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). increases so that the respective user chooses the higher MCS
which subsequently increases the sum-rate of the user. Fur-
ther, the following outcomes are observed for the three dif-
ferent existence scenarios: (i) when all the active users in the
cell belong to eMBB service, the resulting average sum-rate
performance of users is the tight upper bound, (ii) if all the
active users in cell are associated with the URLLC service,
then the resulting average sum-rate performance of users is
the tight lower bound, and (iii) The resulting average sum-rate
performance of users is lower than the performance of all
eMBB users and higher than the performance of all URLLC
users when the active users in cell belong to both types of
services. The reliability constraint is more strict for URLLC
service, therefore, in order to ensure the transmission with
high success probability, the URLLC users select the lower
MCS compared to the eMBB users. B. RESULTS AND DISCUSSIONS Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 8. Average sum rate of URLLC with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 9. Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 9. Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). FIGURE 9. Average sum rate of eMBB with different URLLC users
scheduling TTI gaps and packet arrival rates at 40 dBm of BS power
(using the scheduling algorithm with constraint (C3)). increases so that the respective user chooses the higher MCS
which subsequently increases the sum-rate of the user. Fur-
ther, the following outcomes are observed for the three dif-
ferent existence scenarios: (i) when all the active users in the
cell belong to eMBB service, the resulting average sum-rate
performance of users is the tight upper bound, (ii) if all the
active users in cell are associated with the URLLC service,
then the resulting average sum-rate performance of users is
the tight lower bound, and (iii) The resulting average sum-rate
performance of users is lower than the performance of all
eMBB users and higher than the performance of all URLLC
users when the active users in cell belong to both types of
services. The reliability constraint is more strict for URLLC
service, therefore, in order to ensure the transmission with
high success probability, the URLLC users select the lower
MCS compared to the eMBB users. This is the main reason
for the aforementioned results, shown in Fig. 7. increases so that the respective user chooses the higher MCS
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pp. 2840–2843. 45687 VOLUME 8, 2020 P. Korrai et al.: RAN Resource Slicing Mechanism for Multiplexing of eMBB and URLLC Services in OFDMA BJÖRN OTTERSTEN (Fellow, IEEE) was born
in Stockholm, Sweden, in 1961. He received the
M.S. degree in electrical engineering and applied
physics from Linkoping University, Linkoping,
Sweden, in 1986, and the Ph.D. degree in electrical
engineering from Stanford University, Stanford,
CA, USA, in 1990. V. CONCLUSION He has held research positions
with the Department of Electrical Engineering,
Linkoping University; the Information Systems
Laboratory, Stanford University; the Katholieke
Universiteit Leuven, Leuven, Belgium; and the University of Luxembourg,
Luxembourg. From 1996 to 1997, he was the Director of research with
ArrayComm, Inc., a start-up in San Jose, CA, based on his patented tech-
nology. In 1991, he was appointed as a Professor of signal processing with
the Royal Institute of Technology (KTH), Stockholm, Sweden. From 1992 to
2004, he was the Head of the Department for Signals, Sensors, and Systems,
KTH. From 2004 to 2008, he was the Dean of the School of Electrical
Engineering, KTH. He is currently the Director for the Interdisciplinary
Centre for Security, Reliability and Trust, University of Luxembourg. ASHOK BANDI (Student Member, IEEE) was
born in Kunkalagunta, India, in 1988. He received
the M.Tech. degree in electronics and communi-
cation engineering from the National Institute of
Technology (NIT), Tiruchirappalli, India, in 2012. He is currently pursuing the Ph.D. degree in elec-
trical engineering with the University of Luxem-
bourg. He has worked on physical layer design and
development for WLAN 802.11a/n/ac at Imgaina-
tion Technologies, Hyderabad, India, from 2012 to
2015, and at National Instruments, Bengaluru, India, from 2015 to 2016. He was worked as a Project Associate with the Department of ECE, IISc
Bengaluru, from 2016 to May 2017. He joined the Interdisciplinary Centre
for Security, Reliability, and Trust, University of Luxembourg, Luxembourg,
in June 2017. He is working on sparse signal recovery and joint update
of integer and non-linear variables in MINLP problems that appear in for
wireless communications within the Project PROSAT(on-board PROcessing
techniques for high throughput SATellites), funded under FNR CORE-PPP
Framework. He is a Fellow of EURASIP. He was a recipient of the IEEE Signal Pro-
cessing Society Technical Achievement Award, in 2011, and the European
Research Council advanced research grant twice, from 2009 to 2013 and
from 2017 to 2022. He has coauthored journal articles that received the
IEEE Signal Processing Society Best Paper Award, in 1993, 2001, 2006, and
2013, and seven IEEE conference papers best paper awards. He is currently
a member of the editorial boards of EURASIP Signal Processing Journal,
EURASIP Journal of Advances Signal Processing, and Foundations and
Trends of Signal Processing. V. CONCLUSION He has served as an Associate Editor for the
IEEE TRANSACTIONS ON SIGNAL PROCESSING and the Editorial Board of the IEEE
Signal Processing Magazine. SYMEON
CHATZINOTAS
(Senior
Member,
IEEE) received the M.Eng. degree in telecommu-
nications from the Aristotle University of Thes-
saloniki, Thessaloniki, Greece, in 2003, and the
M.Sc. and Ph.D. degrees in electronic engineer-
ing from the University of Surrey, Surrey, U.K.,
in 2006 and 2009, respectively. He was involved in
numerous research and development projects for
the Institute of Informatics Telecommunications,
National Center for Scientific Research Demokri-
tos; the Institute of Telematics and Informatics, Center of Research and Tech-
nology Hellas; and the Mobile Communications Research Group, Center
of Communication Systems Research, University of Surrey. He is currently
the Co-Head of the SIGCOM Research Group, Interdisciplinary Centre for
Security, Reliability, and Trust, University of Luxembourg, Luxembourg, and
also a Visiting Professor with the University of Parma, Italy. He has more than
300 publications, 3000 citations, and an H-Index of 30 according to Google
Scholar. He was a co-recipient of the 2014 IEEE Distinguished Contributions
to Satellite Communications Award, the CROWNCOM 2015 Best Paper
Award, and the 2018 EURASIC JWCN Best Paper Award. 45688 VOLUME 8, 2020
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Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
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Revista Brasileira de Enfermagem
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REVIEW REVIEW RESUMO
Obj ti Objetivos: analisar o conceito de alteração da condição de pele em recém-nascidos internados
na Unidade de Terapia Intensiva Neonatal. Métodos: trata-se de uma análise de conceito
operacionalizada mediante scoping review. A busca foi realizada em três partes: a primeira,
nas fontes Scopus e Web of Science; a segunda, no Google Acadêmico®; e a terceira, mediante
lista paralela de referências. Resultados: de acordo com os tipos de alterações de pele, as
mais frequentes foram eritema/vermelhidão e lesões por pressão. Para a análise de conceito,
o atributo “lesões ou alterações na pele” apresentou maior evidência. Os antecedentes mais
frequentes foram idade gestacional, peso ao nascer e fatores relacionados à internação
hospitalar. Dentre os consequentes, infecção/sepse apresentou destaque. Conclusões:
este estudo permite o aprimoramento da visão dos profissionais de saúde em relação às
alterações na condição de pele dos neonatos e, portanto, pode contribuir para uma prática
de enfermagem segura e sistematizada. Quenia Camille Soares MartinsI
ORCID: 0000-0002-4036-2423 IUniversidade Federal do Rio Grande do Norte. Santa Cruz,
Rio Grande do Norte, Brazil. IIUniversidade Federal do Rio Grande do Norte. Natal,
Rio Grande do Norte, Brazil. How to cite this article: Descritores: Pele; Recém-Nascido; Segurança do Paciente; Cuidados de Enfermagem;
Unidade de Terapia Intensiva Neonatal. Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC,
Martins QCS. Alteration of skin condition in newborns
admitted to neonatal intensive care: a concept analysis. Rev Bras Enferm. 2022;75(4):e20210473. https://doi.org/10.1590/0034-7167-2021-0473 Dayara Ainne de Sousa AraújoI
ORCID: 0000-0002-0593-2443 Jéssica Naiara de Medeiros AraújoI
ORCID: 0000-0002-9115-3285 Amanda Barbosa da SilvaII
ORCID: 0000-0002-5410-7060
Josanyelem Vidal LopesI
ORCID: 0000-0002-4210-2991 y
g
Descriptors: Skin; Newborn; Patient Safety; Nursing Care; Intensive Care Units, Neonatal. Alteração da condição de pele em recém-nascidos internados em terapia intensiva neonatal: análise de conceito
Alteración de la condición de piel en recién nacidos internados en cuidado intensivo neonatal: análisis de concepto Alteração da condição de pele em recém-nascidos internados em terapia intensiva neonatal: análise de conceito
Alteración de la condición de piel en recién nacidos internados en cuidado intensivo neonatal: análisis de concepto 1
Rev Bras Enferm. 2022;75(4): e20210473
9
of RESUMEN Objetivos: analizar concepto de alteración de la condición de piel en neonatos internados
en Unidades de Cuidado Intensivo Neonatal. Métodos: análisis de operacionalización de
concepto mediante scoping review. Búsqueda realizada en tres partes: la primera, en las
fuentes Scopus y Web of Science; la segunda, en el Google Académico®; y la tercera, mediante
lista paralela de referencias. Resultados: conforme los tipos de alteraciones de piel, las
más frecuentes fueron eritema/enrojecimiento y lesiones por presión. Para el análisis de
concepto, el atributo “lesiones o alteraciones en la piel” presentó mayor evidencia. Los
antecedentes más frecuentes fueron edad gestacional, peso al nacer y factores relacionados
a la internación. Entre los consecuentes, infección/sepsis presentó destaque. Conclusiones:
este estudio permite el perfeccionamiento de la visión de profesionales de salud en relación
a las alteraciones en la condición de piel de los neonatos y, así, puede contribuir para una
práctica de enfermería segura y sistematizada. ABSTRACT Dayara Ainne de Sousa AraújoI
ORCID: 0000-0002-0593-2443
Jéssica Naiara de Medeiros AraújoI
ORCID: 0000-0002-9115-3285
Amanda Barbosa da SilvaII
ORCID: 0000-0002-5410-7060
Josanyelem Vidal LopesI
ORCID: 0000-0002-4210-2991
Ana Clara DantasII
ORCID: 0000-0002-5634-7498
Quenia Camille Soares MartinsI Objectives: to analyze the concept of alteration of skin condition in newborns admitted
to the Neonatal Intensive Care Unit. Methods: this is a concept analysis operationalized by
scoping review. The search was conducted in three parts: the first, in sources like Scopus
and Web of Science; the second, in Google Scholar®; and the third, through a parallel list
of references. Results: according to the types of skin, the most frequent alterations were
erythema/redness and pressure injuries. The concept analysis was more evident in the attribute
“skin lesions or alterations” than the others. The most frequent antecedents were gestational
age, birth weight, and factors related to hospitalization. Among the consequences stood out
infection/sepsis. Conclusions: this study allows improving the vision of health professionals
regarding alterations in skin condition of neonates and, therefore, may contribute to a safe
and systematized nursing practice. INTRODUCTION The daily life at NICUs subjects the NB to several moments
of risk for alterations in skin condition. According to a survey
that identified the frequency of adherence of the nursing staff
to patient safety actions at the NICU, by using a previously vali-
dated instrument, the technologies that increase the survival of
NBs requiring intensive care can cause cutaneous lesions when
misused. To avoid complications, adverse events, and worsening
of clinical status, the multi-professional team must be oriented
about the conditions of the NB, emphasizing that adverse events
can prolong hospitalization and even lead to death(9). The skin is vitally important and is responsible for developing
several functions, such as thermoregulation, infection control,
immune vigilance, hydro electrolytic homeostasis maintenance,
endocrine secretion, and tactile sensation. Therefore, it directly
interferes with the metabolism, especially in the newborn (NB). It is composed of the dermis, formed essentially by collagen and
elastin; and the epidermis, composed of four sub-layers, including
the stratum corneum, which is relevant because it is the outermost
portion of the skin(1). Another study analyzed notifications of adverse events on the
Health Surveillance Notification System (Sistema de Notificação
de Vigilância Sanitária) and found out that 65.6% were related to
medications and that skin lesions, phlebitis, and hematomas were
frequent in NICUs(10). To reduce damage and adverse events related
to health care, the Brazilian Network of Nursing and Patient Safety
(Rede Brasileira de Enfermagem e Segurança do Paciente) seeks to
promote and protect human health and maintain the permanent
improvement of services with quality. In 2013, patient safety protocols
were instituted to prevent pressure injuries, which aims to prevent
the occurrence of skin lesions(7,11). The integrity of the stratum corneum, which is the most su-
perficial layer of the skin, is related to gestational age at birth. Up
to 23 weeks, the skin may be translucent, gelatinous, and highly
fragile, presenting a significant compromised skin barrier. Preterm
newborn’s skin (PTNS) with ≤ 37 weeks has structural differences
compared to the pediatric and adult population; consequently,
the case of a lesion is potentially high(2-3). Given these structural specificities of the newborn’s skin, the
lower the gestational age, the higher the risk. So, the chances of
infection may increase, which is the leading cause of neonatal
morbidity and mortality, besides causing definitive scars and
functional alterations(4). Lesions tend to prolong hospitalization
time and increase treatment costs. Corresponding author: Corresponding author:
Dayara Ainne de Sousa Araújo
E-mail: dayara-ainne@hotmail.com EDITOR IN CHIEF: Antonio José de Almeida Filho
ASSOCIATE EDITOR: Maria Itayra Padilha Descriptores: Piel; Recién Nacido; Seguridad del Paciente; Cuidados de Enfermería; Unidades
de Cuidado Intensivo Neonatal. Submissão: 06-24-2021 Approval: 12-04-2021 VERSÃO ON-LINE ISSN: 1984-0446 1
Rev Bras Enferm. 2022;75(4): e20210473
9
of https://doi.org/10.1590/0034-7167-2021-0473 https://doi.org/10.1590/0034-7167-2021-0473 Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Ethical aspects In the Neonatal Intensive Care Units (NICU), several types of
caring and practices on the NBs involve the skin, such as bathing,
using skin solutions for antisepsis, and caring with transepider-
mal loss, which may predispose or enhance the appearance of
lesions. Several procedures are also carried out, like dressings and
venous and arterial punctures. The use of adhesives on catheters
for oxygen therapy also irritates the skin of those NBs(7). Since this is a review study, no human beings were involved,
which exempts approval by the Research Ethics Committee. OBJECTIVES The maintenance of skin integrity is essential, especially in the
critical period, since factors such as dermatitis, burns, ulcers, trauma,
injuries due pressure, and shear can harm the protective function. Thus, integrity maintenance becomes an essential factor in building
preventing strategies and then directing the continuation of interven-
tions by the multi-professional team. For that, caring for neonates’
skin has become a concern, especially in Neonatal Admission Units(6). To analyze the concept of alteration of skin condition in new-
borns admitted to the Neonatal Intensive Care Unit. INTRODUCTION Thus, more than dealing with
the principal diagnosis, managing the skin of the preterm newborn
is fundamental while providing care(4). It is known that excessive NB handling can cause physical and
physiological stress, such as changes in respiratory pattern and
heart rate, pain, and alterations in skin integrity. For this reason,
the professional needs to identify such patterns to prevent and
reduce the damages to health resulting from the assistance(12). NANDA-International (NANDA-I) presents risk factors that act
directly on skin integrity, such as radiation, excretions, hydration,
hyperthermia, hypothermia, pressure on bony prominence, and
humidity. It also presents internal factors, such as alteration in the
volume of liquids and inadequate nutrition. The damage to the in-
tegrity may also be associated with pharmaceutical agents, altered
sensitivity, altered skin turgor, the use of adhesives, and arterial
puncture. Moreover, one of its risk populations is the age extremes(5). However, to define the alterations of skin conditions that affect
NBs admitted to NICUs, it is necessary to know the concept that
defines them. Also, the elements that make up this concept so that
the assistance provided by the multidisciplinary team can be targeted
and have quality. It is noteworthy that the concept is an idea about a
phenomenon, essential for developing scientific evidence and con-
tributing to clinical practice and the construction of nursing science(13). Organization of data/Data analysis The initial screening was done by dynamically reading the
studies’ titles and abstracts, followed by a complete reading of
the selected studies. Repeated studies were counted only once,
and those that did not fit the eligibility criteria and were not
available for access were excluded. Design of study The second phase of the search occurred in Google Scholar®,
using the following keywords, identified based on the first part of
the search: “Newborn Skin Condition Scale”; “Neonatal Intensive
Care Unit”; “Nursing Care”; “Newborn Skin Condition”; “Neonatal Skin
Condition Score”; “Intensive Care Units, Neonatal”; “Nursing Care” with
the following combinations: “Newborn Skin Condition Score” AND
“Neonatal Intensive Care Unit “; “Newborn Skin Condition Score” AND
“Nursing Care”; “Newborn Skin Condition” and “Neonatal Intensive
Care Unit”; “Neonatal Skin Condition Score” AND “Intensive Care
Unit, Neonatal”; “Neonatal Skin Condition Score” AND “Nursing Care.”
For the selection of studies, the following inclusion criteria were
adopted: complete studies available in the data sources addressing
newborn skin condition, in Portuguese, Spanish, and English. Ab-
stracts, editorials, correspondence, and expert opinion were excluded. The second phase of the search occurred in Google Scholar®,
using the following keywords, identified based on the first part of
the search: “Newborn Skin Condition Scale”; “Neonatal Intensive
Care Unit”; “Nursing Care”; “Newborn Skin Condition”; “Neonatal Skin
Condition Score”; “Intensive Care Units, Neonatal”; “Nursing Care” with
the following combinations: “Newborn Skin Condition Score” AND
“Neonatal Intensive Care Unit “; “Newborn Skin Condition Score” AND
“Nursing Care”; “Newborn Skin Condition” and “Neonatal Intensive
Care Unit”; “Neonatal Skin Condition Score” AND “Intensive Care
Unit, Neonatal”; “Neonatal Skin Condition Score” AND “Nursing Care.” Given this, the concept “ alteration of skin condition in new-
borns “ was chosen to be analyzed from the perspective of NBs
admitted to the NICU regarding the handling of NBs, invasive
procedures, devices, among others. In the literature, diffuse
definitions appear for “skin alterations in newborns; therefore,
defining a concept for that ensures more patient safety, quality
of service, and nursing care directed to the user’s needs. For the selection of studies, the following inclusion criteria were
adopted: complete studies available in the data sources addressing
newborn skin condition, in Portuguese, Spanish, and English. Ab-
stracts, editorials, correspondence, and expert opinion were excluded. In this line of thought, to support the conceptual analysis, a
scoping review was used according to the recommendations of
the Joana Briggs Institute(15), based on the PRISMA extension for
scoping reviews (PRISMA-ScR)(17). The study was registered in the
Open Science Framework study platform and assigned the follow-
ing URL: https://osf.io/hvcn6/(18). Study protocol; criteria of inclusion and exclusion The second part was carried
out in Google Scholar® using keywords identified in the first part of
the search, and the third part was performed through a parallel list
of references(15). Study protocol; criteria of inclusion and exclusion The scoping review was selected because it allows the inclu-
sion of studies of various natures. To carry it out, we initially used
a research protocol composed of the following steps: objectives;
research question; identification of relevant studies by searching
the literature through electronic databases; selection of studies,
with the establishment of eligibility criteria; mapping and extrac-
tion of data; and presentation of the results(15). A protocol was prepared to extract the data with the study’s
methodological information (study title, indexed data source, au-
thors, language, continent and year of publication, methodology
used, type of approach, and level of evidence) and items related
to the concept analysis (types of skin alterations, possible defini-
tions for the concept, attributes of the alteration of skin condition,
antecedents, and consequents of the alteration of skin condition,
and empirical references). This study structured the guiding question using the PCC
strategy - P (population), C (concept), and C (context)(15). So, the
population was the newborns, the concept was Alteration of skin
condition, and the context involved the Neonatal Intensive Care
Unit. Thus, we formulated the following central question: What is
the concept of alteration of skin condition in newborns admitted
to the Neonatal Intensive Care Unit? The subsequent questions of
the study were: What is the alteration of skin condition in newborns
at the Neonatal Intensive Care Unit? What are the attributes, ante-
cedents, and consequents of the concept “Alteration of skin condi-
tion in newborns” admitted to the Neonatal Intensive Care Unit? As for the level of evidence was adopted the Joanna Briggs
Collaborating Center classification(18). The studies were evaluated
as follows: Level I - Evidence obtained from a systematic review
of randomized controlled trials; Level II - Evidence achieved in the
randomized controlled trial; Level III.1 - Evidence obtained from well-
designed controlled trials without randomization; Level III.2 - Evidence
acquired from well-designed cohort or case-control studies; Level
III.3 - Level IV - Opinions of respected authorities based on clinical
criteria and experience, descriptive studies, or expert committee
reports. Finally, tables and charts were used to present the results(18). As recommended by the method, the search was performed in
three parts: in the first part, the data sources Scopus (Elsevier) and
Web of Science (Elsevier) were used. Design of study This study is a concept analysis based on Walker and Avant’s
model(14), operationalized through a scoping review(15) and carried
out between September and December 2020. Another issue is the monitoring technologies in the NICUs, like
cardiorespiratory (pulse oximetry using photoplethysmography,
electrocardiography [ECG], and impedance pneumography) based
on electrical potential differences through skin patches. They have
disadvantages over the use of adhesive sensors because they
have potential to cause lesions in neonates’ skin. There is also the
management of daily assessment of the NB by professionals, which
may increase the risk of hypothermia and circulatory disturbances
during the examination(8). Concept analysis is a method capable of synthesizing and
understanding a concept already introduced in the literature. It
aims to standardize the description of a phenomenon and allow
effective communication about it, reducing vague, ambiguous,
and incoherent terminology, to make it more functional in theory,
research, and practice. In that sense, the concept analysis is in
the literature in various methods and strategies. We used in this
study the model of Walker and Avant(13-14). 2
Rev Bras Enferm. 2022;75(4): e20210473
9
of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. The search conducted in each data source occurred through
the content accessed by the Federal University of Rio Grande do
Norte, via Federated Academic Community (CAFe), through the
Coordination for the Improvement of Higher Education Personnel
(CAPES) gateway. The method proposed by Walker and Avant (2019) is traditional
and easy to apply, is based on Wilson’s proposal, and includes the
execution of eight steps: (1) Select a concept; (2) Determine the
objectives or purposes of analysis; (3) Identify all possible uses of
the concept; (4) Determine the defining attributes; (5) Construct a
model case; (6) Identify other cases: borderline, related, and con-
trary; (7) Identify antecedents and consequents of the concept; (8)
Define empirical referents. In this study, we carried out the eight
recommended steps(14-16). RESULTS After searching the databases, 2,828 studies were found in Scopus
(Elsevier) and 33 studies in the Web of Science. In Google Scholar®,
1,000 studies were found, totaling 3,861. From these, 173 were un-
available electronically. After analysis of titles and abstracts, 3,582
were excluded for not meeting the eligibility criteria, and 40 counted
only once since they were duplicates, leaving a total of 66 studies. After reading those complete texts, all were eligible and in-
cluded in the final sample. Then, one study was also included in
the reverse search of the parallel references list, totaling a final After searching the databases, 2,828 studies were found in Scopus
(Elsevier) and 33 studies in the Web of Science. In Google Scholar®,
1,000 studies were found, totaling 3,861. From these, 173 were un-
available electronically. After analysis of titles and abstracts, 3,582
were excluded for not meeting the eligibility criteria, and 40 counted
only once since they were duplicates, leaving a total of 66 studies. For the data sources, an advanced search was made using
the indexed descriptors (Medical Subject Headings - MeSH): Skin;
Newborn; Patient Safety; Intensive Care Units, Neonatal; Nursing
Care. We used the Boolean operator AND for the following
cross-references: 1# “Skin” AND “Patient Safety” AND “Newborn”;
2# “Skin” AND “Newborn” AND “Intensive Care Units, Neonatal”; 3#
“Skin” AND “Newborn” AND “Nursing Care”; 4# “Skin” AND “Intensive
Care Units, Neonatal” AND “Nursing Care.” y
y
After reading those complete texts, all were eligible and in-
cluded in the final sample. Then, one study was also included in
the reverse search of the parallel references list, totaling a final 3
Rev Bras Enferm. 2022;75(4): e20210473
9
of 3
9
of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. sample of 67 studies. Figure 1 shows the flowchart of studies
selected from the data sources and Google Scholar®. Identifying the use of the concept The concept “Alteration of skin condition in newborns” was
selected to perform the concept analysis. Inclusion
Eligibility
Screening
Identification
Selected Articles
(n = 106)
Full articles analyzed
(n = 66)
Articles considered eligible
(n = 66)
Articles included in the review
(n = 67)
Studies identified by searching
the data sources
(N = 3,861)
Duplicate articles
(n = 40)
Articles included through
parallel reference list (n = 1)
Articles excluded for not
meeting eligibility criteria
(n = 3,582)
Articles unavailable
electronically (n = 173)
Figure 1 – Flowchart of literature search and inclusion of articles, according
to PRISMA-ScR guidelines(17) (adapted), Santa Cruz, Rio Grande do Norte,
Brazil, 2020 Articles excluded for not
meeting eligibility criteria
(n = 3,582)
Articles unavailable
electronically (n = 173) According to the studies, the possible usages of the concept identified
in the sample were: the altered epidermis and/or dermis(5); change in
skin color(19); partial loss of dermal thickness, evidenced as a superficial
wound(20); and lesion, which is suggestive of an end or advanced point
of damage to the skin by pressure (a mechanical force)(3). Identifying a model case and a contrary case In addition, we built a model case and a contrary case for
alteration of skin condition in newborns admitted to the NICU. The prevailing skin alteration was erythema, with 38.80%,
also described as “redness” in some studies. Pressure injury was
also evidenced in 20.90% of the studies, followed by abrasion,
stripping, and dermatitis, with 19.40%. Essential Attributes The attributes are associated with the concept because they
describe characteristics, allowing a comprehensive analysis,
which can improve the understanding of this concept. After
reading the studies, five essential attributes were identified and
appear in Table 2. Articles included through
parallel reference list (n = 1) Table 2 – Essential attributes of the concept “Alteration of skin condition
in newborns” in Neonatal Intensive Care Unit, Santa Cruz, Rio Grande do
Norte, Brazil, 2020 Table 2 – Essential attributes of the concept “Alteration of skin condition
in newborns” in Neonatal Intensive Care Unit, Santa Cruz, Rio Grande do
Norte, Brazil, 2020 Table 2 – Essential attributes of the concept “Alteration of skin condition
in newborns” in Neonatal Intensive Care Unit, Santa Cruz, Rio Grande do
Norte, Brazil, 2020
Attributes*
n
%
Lesions or skin alterations
Skin lesions
35
15
52.23%
22.38%
Skin alteration
6
8.95%
Skin Inflammation
1
1.49%
Lesions to the skin, nerves, or tendons
1
1.49%
*Variable that accepts more than one option. Articles included in the review
(n = 67) Figure 1 – Flowchart of literature search and inclusion of articles, according
to PRISMA-ScR guidelines(17) (adapted), Santa Cruz, Rio Grande do Norte,
Brazil, 2020 In terms of the selected studies characterization, the years
of publication date from 1999 to 2020, most of them published
in the last five years (55.22%). According to the location, South
America appeared most frequently (47.76%), and the language
that prevailed was English (62.69%). Regarding the method used,
literature reviews prevailed with 17.91%, followed by descriptive
studies in 16.42% of the sample. Thus, the most frequent attributes were injuries or skin altera-
tions (52.23% of the studies). It is worth noting that not all studies
in the sample presented requirements for concept attributes. The most used approach was quantitative (76.13%), present-
ing only one study with mixed methods (1.49%). The level IV
of scientific evidence was presented in 47.76% of the studies,
followed by level III.3 in 25.37%. In Table 1 are listed the types of
lesions presented in the studies. *Variable that accepts more than one option. Model case for “Alteration of skin condition in a newborn” Full-term newborn admitted to the NICU due to perinatal
asphyxia during delivery. Severe general condition, hypocoric,
anicteric, acyanotic, afebrile, normotensive lambdoid and breg-
matic fontanelles. Under residual sedation from fentanyl and
dormonid, she presents isochoric and mydriatic pupils, without
pupillary photoreaction, absence of muscle tone, and primitive
reflexes. Hemodynamically unstable, under the use of dopamine
through infusion pump, presents decreased tissue perfusion. On
invasive mechanical ventilation, presents Ronchi sounds during
pulmonary auscultation, but with little secretion in the airways. The newborn presents a flaccid abdomen with fluid-air noises, an
umbilical stump in mummification process, and without phlogistic
signs. With an orogastric tube, she accepted breast milk. Diuresis
by indwelling urinary catheter, urine with a light-yellow aspect
and without sediments. No edema in the limbs, but they are stiff. Skin evaluation: dry and broken skin all over the body, mainly in
the lower limbs, evaluated by the Newborn Skin Condition Scale
(NSCS). In addition, in the areas of alterations/lesions, she presents
phlogistic signs and possible tendon impairment. Table 1 – Types of skin alterations in newborns admitted to the Neonatal
Intensive Care Unit, Santa Cruz, Rio Grande do Norte, Brazil, 2020
Types of alterations*
n
%
Erythema/Redness
Pressure Injuries
26
14
38.80%
20.90%
Abrasion
13
19.40%
Stripping
13
19.40%
Dermatitis
13
19.40%
Skin Rupture
Edema
10
9
14.92%
13.43%
Necrosis
9
13.43%
Dryness
9
13.43%
Burns
9
13.43%
Hematoma/Eczema
Blister
Eruptions
Folliculitis
Maceration
9
5
3
2
1
13.43%
7.46%
4.47%
2.98%
1.49%
*Variable that accepts more than one option. Table 1 – Types of skin alterations in newborns admitted to the Neonatal
Intensive Care Unit, Santa Cruz, Rio Grande do Norte, Brazil, 2020 4
Rev Bras Enferm. 2022;75(4): e20210473
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of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Model case for “Alteration of skin condition in a newborn” This case model, adapted from the researcher’s clinical practices, was
developed in a university hospital in Rio Grande do Norte in 2019. In
this case, it is noteworthy that the author identified the most frequent
attributes highlighted by the studies: skin lesions, skin changes, skin
inflammation, nerve, or tendon lesions. The antecedents are the events or incidents that anticipate
the skin alterations and are directly related to the characteristics
of prematurity. In the category “Related to gestational age,” the
most frequent antecedents were immature skin barrier (13.43%)
and thermoregulation (10.44%). In the category “Related to birth
weight,” low weight was the most evident in 32.83% of the studies. As for the antecedents related to hospitalization, medical devices
were highlighted in 31.34%. Contrary case for “Alteration of skin condition in a newborn” Preterm newborn admitted to the NICU. A general stable state,
normal color, anicteric, acyanotic, afebrile. Under residual seda-
tion, she shows isochoric and photo reagent pupils, presence of
muscle tone, and primitive reflexes. Hemodynamically stable, with
good perfusion. In ambient air, presents a wheezing sound during
pulmonary auscultation. The newborn has a flaccid abdomen with
fluid-air sounds, umbilical stump in mummification process, and no
phlogistic signs, accepting breast milk. Spontaneous diuresis, urine
with a light-yellow aspect. No edema in the limbs. Skin evaluation:
intact skin, using skin hydration, and obeying safe care protocols. The newborn did not present alterations when evaluated by the
Newborn Skin Condition Scale (NSCS). The consequences most prevalent in the studies were: infec-
tions/sepsis (28.35%), pain (2.98%), and dehydration. Those are
the factors that can occur due to the alteration of skin condition
in newborns. It is noteworthy that not all studies in the sample
presented requirements for consequences of the concept. Identifying the empirical references Furthermore, to support the concept analysis, some empirical
references were found in the studies that allow measuring the
“alteration of skin condition in newborns” (Table 4). Based on
them, we can classify the risk of having the lesion. They also help
standardize and direct care to the patients’ needs, decreasing the
risk of complications. The contrary case is fictitious, contradicting the attributes of this
study. For this purpose, according to the possible usages of the con-
cept, with the critical attributes, and the elaboration of the cases, it was
built a definition to be addressed for the concept “Alteration of skin
condition in newborns,” namely: alteration in color, thickness, and skin
hydration in newborns, associated or not with the presence of lesions. Table 4 – Empirical references for “Alteration of skin condition in newborns”
identified in the studies, Santa Cruz, Rio Grande do Norte, Brazil, 2020 Table 4 – Empirical references for “Alteration of skin condition in newborns”
identified in the studies, Santa Cruz, Rio Grande do Norte, Brazil, 2020 Table 4 – Empirical references for “Alteration of skin condition in newborns”
identified in the studies, Santa Cruz, Rio Grande do Norte, Brazil, 2020
Empirical references*
n
%
Newborn Skin Condition Scale
4
5.97%
Braden Q scale
4
5.97%
Braden QD scale
2
2.98%
Neonatal Skin Risk Assessment Scale
1
1.49%
Dubowitz Neonatal Maturity Rating Scale
1
1.49%
Skin Condition Score (Lane e Drost)
1
1.49%
Glamorgan Scale
1
1.49%
*Variable that accepts more than one option. Antecedents e consequents The antecedents and consequents for “Alteration of skin condi-
tion in newborns” are shown in Table 3. Table 3 – Categorization of the antecedents and consequences for “Altera-
tion of skin condition in newborns” in a Neonatal Intensive Care Unit, Santa
Cruz, Rio Grande do Norte, Brazil, 2020 Antecedents*
n
%
Gestational age-related
Immature skin barrier
9
13.43%
Thermoregulation
7
10.44%
Transepidermal water loss
4
5.97%
Impaired immunity
3
4.47%
Increased percutaneous absorption
1
1.49%
Capillarity fragility
1
1.49%
Birth weight related
Low weight at birth
22
32.83%
Malnutrition
2
2.98%
Lack of subcutaneous tissue
1
1.49%
Dehydration
1
1.49%
Nutrition Source
1
1.49%
Hospitalization-related
Medical Devices
21
31.34%
Skincare
9
13.43%
Length of hospitalization
5
7.46%
Invasive procedures
4
5.97%
Chemical Agents
3
4.47%
Consequents*
N
%
Infections/sepsis
19
28.35%
Pain
2
2.98%
Dehydration
2
2.98%
Inflammation
1
1.49%
Skin reactions
1
1.49%
*Variable that accepts more than one option. According to Table 4, the most frequent empirical references
in the sample were the Newborn Skin Condition Scale and the
Braden Q Scale (5.97% of the studies), followed by the Braden
QD Scale (2.98%). We reiterate that not all studies in the sample
presented elements for for the concept’s empirical references. DISCUSSION In the
neonate, these alterations are common due to the complexity of
this period. The lesion is aggressive to the skin, which threatens
its functionality; thus, noticing the existence of alterations, even
before the development of the lesion, is one of the fundamental
roles for health prevention(27-28). So, a literature review demonstrates that the topical application
of 2% chlorhexidine gluconate is related to infection reduction(34). Another study describes economic and clinical benefits with its
use, reducing the incidence of morbidity and mortality(36). How-
ever, the use of chlorhexidine is not recommended for neonatal
patients due to concerns about dermatitis and systemic absorption,
indicated only for those with gestational age above 27 weeks(34). Still, on the subject under discussion, another review study
shows recent reports that the bath with chlorhexidine reduces the
incidence of central catheter-associated bloodstream infections,
but there is still no evidence related to neonates and premature
babies(37). In PTNBs less than 32 weeks, the Association of Women’s
Health Obstetric and Neonatal Nurses (AWHONN) recommends
using only warm water with a cotton swab(1). Skin inflammation may be related to atopic dermatitis, which
is chronic and genetic, multifactorial, relapsing, and varying
severity. It presents intense pruritus, erythematous or vesicular
maculopapular lesions, with stripping, accompanied by dryness,
crusts, and liquefaction, with higher prevalence in children(29-30). The empirical references address scales that assess the skin
condition and the risk of developing lesions to establish daily,
safe, and systematic care. Thus, it is recommended the use of
these scales to standardize the assessment performed by profes-
sionals and assist in nursing interventions, avoiding the occur-
rence of discrepancies in assessments due to the subjectivity of
each professional(7). The lesions denote distinct complexities and consequences,
so full attention becomes essential. The dermis is a thick layer
of connective tissue that extends itself to the subcutaneous tis-
sue. The presence of nerves and tendons indicates the need for
special treatment for the lesion since the damage has reached
other tissues, affecting homeostasis and causing pain(31). It was observed in the study that antecedents contribute
to skin alterations and lesions in NBs, characterized mainly by
gestational age, birth weight, and factors related to hospitaliza-
tion. The antecedents refer to the possible etiological factors that
will result in a gradual and intense response depending on the
degree of exposure(14). DISCUSSION The partial loss of dermal thick-
ness, evidenced as a superficial wound, is associated with stage
2 pressure injury, abrasion, and even minor lesions caused by
adhesive removal — those lesions result from skin exposure to
aggressive elements, whether physical, mechanical, or chemical(3). According to the lesions or skin alterations — the most fre-
quent critical or essential attributes — other authors report that
the neonate’s skin continues to develop after birth, undergoing
a process of extrauterine adaptation. In this sense, neonates’
safety depends on damage-free care to maintain and restore
physiological stability. A condition associated with immature
skin in neonates is transepidermal water loss, which contributes
to dehydration, thermal instability, electrolyte imbalances, and
the possible presence of alterations and other lesions in the
newborn’s skin(2,25-26). For the antecedents related to hospitalization, medical devices
stood out. In neonatology, these devices are the main etiological
and extrinsic factors for developing injuries. Some research reports
that these injuries can be avoided with preventive measures, such
as choosing the appropriate size of the device, giving preference
to less harmful materials, regularly assessing the skin area under
the device, protecting, and moisturizing the site in contact, and
performing repositioning. Thus, it is recommended the removal
of these devices as soon as possible(24,32,34). According to the lesions or skin alterations — the most fre-
quent critical or essential attributes — other authors report that
the neonate’s skin continues to develop after birth, undergoing
a process of extrauterine adaptation. In this sense, neonates’
safety depends on damage-free care to maintain and restore
physiological stability. A condition associated with immature
skin in neonates is transepidermal water loss, which contributes
to dehydration, thermal instability, electrolyte imbalances, and
the possible presence of alterations and other lesions in the
newborn’s skin(2,25-26). According to the most frequent consequences of the concept,
infections of multiple etiologies occur because of colonization
of microorganisms, adherence of medical devices developing
biofilm, and maternal separation, which generates stress in early
life(34). An observational study conducted in Canada showed a
reduction in central catheter-associated bloodstream infections
using 2% chlorhexidine gluconate(35). A skin lesion is considered any unusual finding on its surface
and can be classified as primary when it represents an initial sign
of a pathological process or secondary when it corresponds to the
result of a late formation or trauma of the primary lesion. DISCUSSION While examining the studies used to back up the concept
analysis on the alterations of the skin condition in NBs admitted
to the NICU, it was observed that most of them were published
in the last five years, showing a recent production on the subject. The neonate’s skin is complex and fragile, making it more
susceptible to the appearance of lesions of various etiologies. According to the most frequent alterations, the use of topical
antiseptics is the leading cause of erythema and may be the
primary cause for the development of other types of lesions,
such as dermatitis(11,21-22). Pressure injuries may be related to immobility; however, the
use of medical devices in neonates stood out as a significant factor
in triggering this type of lesion since their usage for therapy and
recovery of newborns is essential. Thus, it is crucial to protect the
skin area where each device is applied. Other factors contributing 5
9
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of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. to the risk of pressure injury are friction, shear, ineffective nutri-
tion, insufficient tissue perfusion and oxygenation(23-24). hospitalization conditions. Breast milk is often supplemented
with formula, and this dietary deficiency considerably increases
the possibility of developing skin lesions as well as significantly
influences the healing process(33). When identified the possible uses of the concept, it was verified
that the change in skin color could be associated with erythema,
hematomas, and cyanosis - the latter indicating poor perfusion,
which can lead to tissue necrosis. The partial loss of dermal thick-
ness, evidenced as a superficial wound, is associated with stage
2 pressure injury, abrasion, and even minor lesions caused by
adhesive removal — those lesions result from skin exposure to
aggressive elements, whether physical, mechanical, or chemical(3). When identified the possible uses of the concept, it was verified
that the change in skin color could be associated with erythema,
hematomas, and cyanosis - the latter indicating poor perfusion,
which can lead to tissue necrosis. DISCUSSION This study stands out the Newborn Skin Condition Scale, which
evaluates the skin condition of neonates in three variables: dry-
ness, erythema, and rupture. On this scale, 3 is the worst score;
and 9, the best. In addition, it is an easy-to-understand instru-
ment that can be inserted into the nursing care practice at the
NICU, contributing to standardizing the assessment of lesions
and improving the quality of care provided(4,7). As for gestational age, a study conducted in the United States
showed that the lower the gestational age at birth, the greater the
probability of developing lesions. It is known that transepidermal
water loss is also associated with gestational age because, due
to more significant immaturity, the skin tends to be drier in the
first week of life. Thus, along with impaired skin integrity, there
are risks of thermoregulation failure(32). The Braden Q Scale was adapted from the Braden Scale used
in adults. Its variables consist of seven risk factors: mobility;
activity; sensory perception, humidity; nutrition; friction and
shear; tissue perfusion, and oxygenation. The score ranges from
7 to 28 points, with the lowest score referring to a higher risk of
developing pressure injuries. The Braden QD Scale is an adapta-
tion of the Braden Q Scale; it assesses the same variables with
the addition of two others: the number of medical devices and
skin repositioning/protection. The scale score is allowed to range A study developed in Hungary reports that NBs with low birth
weight have an increased risk of adverse events(33). Also associ-
ated with low birth weight is malnutrition since the NB admitted
to the NICU often do not have adequate nutrition due to their 6
Rev Bras Enferm. 2022;75(4): e20210473
9
of 6
9
of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. CONCLUSIONS Given the results obtained and considering the objective of
this research, the concept analysis of alteration of skin condition
in newborns hospitalized in the Neonatal Intensive Care Unit,
operationalized through scoping review, obtained a sample
of 67 studies. The concept obtained as essential attributes the
skin lesions or alterations, presenting as antecedents the factors
related to gestational age, birth weight, and hospitalization. As
consequents were highlighted the infections of various etiologies. As a result, the definition constructed in this research to address
the concept was as follows: alteration in color, thickness, and
hydration of the skin in newborns, associated or not with the
presence of lesions. Furthermore, it should be noted that the NICU is an environment
that welcomes NBs and their families; therefore, it depends on
the support of the entire multi-professional team. Nursing profes-
sionals play an essential role in maintaining the living conditions
of highly complex NBs, by aligning care practices with scientific
evidence, acting in the management of the environment and the
nursing team. In addition, with the execution and planning of
individual and priority care linked to the prevention of alterations
of skin condition of the NB, they promote safe and quality care(39). Study limitations The limitations of this study may be related to the data sources
chosen for the methodological approach and the limit of three
languages, which may have contributed to obscuring the inclu-
sion of other important surveys on the topic. Therefore, we recommend new studies with other designs
about that subject to deepen the knowledge in this field since
neonate skincare may not be standardized due to its complexity,
and there are still many disagreements in the literature. Contributions to the Fields of Nursing, Health or Public Policy from 0 to 20 points. When the risk of lesions increases, the score
increases too, and a score ≥ 13 indicates that the patient is at risk
of developing the lesion. It is noteworthy that the scales can be
inserted in the assistance provided by the nursing staff(25,38). Since
the Braden QD scale predicts the risk of immobility and medical
device-related pressure injuries in children, it can include from
NBPTs to 21-year-old patients(25). This study can contribute to professionals’ broader and im-
proved view of the concept “altered skin condition in newborns”
since skincare is a predictor of service quality, health care and
ensures patient safety. Furthermore, when it comes to nursing, it
provides systematized care with human responses, contributing
to developing new clinical indicators for nursing diagnoses and
supporting the development of preventive intervention strategies
against skin condition alterations in newborns admitted to NICUs. Huffines and Logsdon developed the Neonatal Skin Risk As-
sessment Scale, based on the Braden scale specific to the neonatal
population, with six subscales: general physical condition; mental
status; mobility; activity; nutrition; and humidity. Each subscale
receives 1 point for a total of 6 to 24. The higher the score, the
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newborns: observational study. J Clin Nurs. 2019;28(21-22):3807-16. https://doi.org/10.1111/jocn.14998 33. Meszes A, Tálosi G, Máder K, Orvos H, Kemény L, Csoma ZR. Lesions requiring wound management in a central terti
care unit. World J Pediatric. 2017;13(2):165-72. https://doi.org/10.1007/s12519-016-0070-6 34. Paplawski S. REFERENCES Prevention of central line-associated blood stream infections in the neonatal intensive care unit: a liter
Nurs. 2020;26(3):142-8. https://doi.org/10.1016/j.jnn.2020.01.013 35. Westlin T, Cowden C, Mwananyanda L, Kapasa ML, Machona S, Pierre C, et al. Impact of chlorhexidine baths on suspected sepsis and blood
stream infections in hospitalized neonates in Zambia. Int J Infect Dis. 2020;96:54-60. https://doi.org/10.1016/j.ijid.2020.03.043 35. Westlin T, Cowden C, Mwananyanda L, Kapasa ML, Machona S, Pierre C, et al. Impact of chlorhexidine baths on suspected sepsis and blood
stream infections in hospitalized neonates in Zambia. Int J Infect Dis. 2020;96:54-60. https://doi.org/10.1016/j.ijid.2020.03.043 36. Oliveira AC, Gama CS. What to use in preoperative skin preparation: povidone-iodine or chlorhexidine?. Rev SOBECC. 2018;23(3):155-9. https://doi.org/10.5327/Z1414-4425201800030007 36. Oliveira AC, Gama CS. What to use in preoperative skin preparation: povidone-iodine or chlorhexidine?. Rev SOBECC. 2018;23(3):155-9. https://doi.org/10.5327/Z1414-4425201800030007 8
Rev Bras Enferm. 2022;75(4): e20210473
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of Alteration of skin condition in newborns admitted to neonatal intensive care: a concept analysis
Araújo DAS, Araújo JNM, Silva AB, Lopes JV, Dantas AC, Martins QCS. 39. Silva ACL, Santos GN, Aoyama EA. A importância da assistência de enfermagem na unidade de terapia intensiva neonatal. ReBIS [Internet]. 2020[cited 2021 Jan 15];2(1):49-54. Available from: https://revistarebis.rebis.com.br/index.php/rebis/article/view/69 39. Silva ACL, Santos GN, Aoyama EA. A importância da assistência de enfermagem na unidade de terapia intensiva neonatal. ReBIS [Internet]. 2020[cited 2021 Jan 15];2(1):49-54. Available from: https://revistarebis.rebis.com.br/index.php/rebis/article/view/69 9
Rev Bras Enferm. 2022;75(4): e20210473
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Completeness and accuracy of national cancer and death registration for outcome ascertainment in trials - an ovarian cancer exemplar
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Completeness and accuracy of natio
death registration for outcome ascer
trials - an ovarian cancer exemplar
Jatinderpal K Kalsi
University College London Institute for Women's Health
Andy Ryan
University College London Institute of Clinical Trials and Methodology
Aleksandra Gentry-Maharaj
University College London Institute of Clinical Trials and Methodology
Danielle Crump
University College London Institute for Women's Health
Naveena Singh
Barts Health NHS Trust
Matthew Burnell
University College London Institute of Clinical Trials and Methodology
Elizabeth Benjamin
HCA International Pathology Laboratories, London
Sophia Apostolidou
University College London Institute of Clinical Trials and Methodology
Mariam Habib
Imperial College London
Susan Massingham
University College London Institute of Clinical Trials and Methodology
Karpinskyj Chloe
University College London Institute of Clinical Trials and Methodology
Robert Woolas
University College London Institute of Clinical Trials and Methodology
Martin Widschwendter
University College London Institute for Women's Health
Lesley Fallowfield
Brighton and Sussex Medical School
Stuart Campbell
Create Health London
Steve Skates Completeness and accuracy of national cancer and
death registration for outcome ascertainment in
trials - an ovarian cancer exemplar Page 1/22 Page 1/22 Massachusetts General Hospital
Alistair J McGuire
London School of Economics
Max Parmar
University College London Institute of Clinical Trials and Methodology
Ian Jacobs
University of New South Wales
Usha Menon
(
u.menon@ucl.ac.uk
)
University College London Institute of Clinical Trials and Methodology
https://orcid.org/0000-0003-
3708-1732
Research
Keywords: outcomes review, adjudication, randomised controlled trial, ovarian cancer, screening,
UKCTOCS, registry
Posted Date: December 8th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-31549/v2
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published on January 25th, 2021. See the published
version at https://doi.org/10.1186/s13063-020-04968-x. Massachusetts General Hospital
Alistair J McGuire
London School of Economics
Max Parmar
University College London Institute of Clinical Trials and Methodology
Ian Jacobs
University of New South Wales
Usha Menon
(
u.menon@ucl.ac.uk
)
University College London Institute of Clinical Trials and Methodology
https://orcid.org/0000-0003-
3708-1732
Research
Keywords: outcomes review, adjudication, randomised controlled trial, ovarian cancer, screening,
UKCTOCS, registry
Posted Date: December 8th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-31549/v2
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published on January 25th, 2021. See the published
version at https://doi.org/10.1186/s13063-020-04968-x. Research Version of Record: A version of this preprint was published on January 25th, 2021. See the published
version at https://doi.org/10.1186/s13063-020-04968-x. Page 2/22 Page 2/22 Abstract Background: There is a trend to increasing use of routinely collected health data to ascertain outcome
measures in trials. We report on completeness and accuracy of national ovarian cancer and death
registration in the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Methods: Of the 202638 participants, 202632 were successfully linked and followed through national
cancer and death registries of Northern Ireland, Wales and England. Women with registrations of any of
19 pre-defined ICD-10 codes suggestive of tubo-ovarian cancer or notification of ovarian/tubal/peritoneal
cancer from hospital episode statistics or trial sites were identified. Copies of hospital and primary care
notes were retrieved and reviewed by an independent outcomes review committee. National registration
of site and cause of death as ovarian/tubal/peritoneal cancer (C56/C57/C48) obtained up to three
months after trial censorship was compared to that assigned by outcomes review (reference standard). Results: Outcome review was undertaken in 3110 women on whom notification was received between
2001 and 2014. Ovarian cancer was confirmed in 1324 of whom 1125 had a relevant cancer registration. Sensitivity and specificity of ovarian/tubal/peritoneal cancer registration was 85.0%(1125/1324; 95%CI
83.7%-86.2%) and 94.0%(1679/1786; 95%CI93.2%-94.8%), respectively. Of 2041 death registrations
reviewed, 681 were confirmed to have a tubo-ovarian cancer of whom 605 had relevant death registration. Sensitivity and specificity was 88.8%(605/681; 95%CI86.4%-91.2%) and 96.7%(1482/1533;
95%CI95.8%-97.6%%) respectively. When multiple electronic health record sources were considered,
sensitivity for cancer site increased to 91.1%(1206/1324; 95%CI89.4%-92.5%) and for cause of death
94.0%(640/681; 95%CI91.9%-95.5%). 95%CI95.8%-97.6%%) respectively. When multiple electronic health record sources were considered,
sensitivity for cancer site increased to 91.1%(1206/1324; 95%CI89.4%-92.5%) and for cause of death
94.0%(640/681; 95%CI91.9%-95.5%). Of 1232 with cancer registration, 8.7%(107/1232) were wrongly designated as ovarian/tubal/peritoneal
cancers by the registry and 4.0%(47/1172) of confirmed tubo-ovarian cancers were mis-registered. In 656
with death registrations, 7.8%(51/656) were wrongly assigned as ovarian/tubal/peritoneal cancers whilst
6.2%(40/645) of confirmed tubo-ovarian cancer deaths were mis-registered. Conclusion: Follow-up of trial participants for tubo-ovarian cancer using national registry data will result
in incomplete ascertainment particularly of site due in part to the latency of registration. This can be
reduced by using other routinely collected data such as hospital episode statistics. Central adjudication
by experts though resource intensive adds value by improving accuracy of diagnoses. Trial registration: ISRCTN22488978, date of trial registration: 6/4/2000 Introduction Currently most trials use multiple sources for outcome ascertainment such as case record forms, patient
questionnaires and electronic national health datasets. The latter includes cancer and death registrations,
hospital administrative records (e.g. hospital episode statistics in the UK), national audit data and
specialised health datasets. This is often followed by central adjudication to ensure consistency and Page 3/22 Page 3/22 minimize bias.1 Central review requires medical records to be retrieved, checked for completeness,
redacted with regards to any reference to randomisation group and patient identifiers and the collated
information reviewed by a specialist committee. Given the expensive and time-consuming nature of this
process, there is a clear need to evaluate alternative strategies. There has been an explosion in the use of electronic national datasets for clinical research. As the data
quality of such resources improve and access processes for research become more streamlined, this is
likely to be a less resource intensive and less costly option for outcome ascertainment in trials. Use of
cancer and death registration alone is especially relevant to large randomised controlled trials of cancer
screening where cancer site and disease specific cause of death (CoD) are primary outcome measures. Data from the U.S. National Lung Screening Trial (NLST) suggests that using death certificate CoD alone
(18%; 95% CI: 4.2-25.0) would not have impacted on the published (20%; 95% CI: 6.7-26.7) lung cancer
mortality reduction where the process included central adjudication.2 This was also noted in the Finnish
prostate cancer screening trial.3 However, other trials have raised concerns about completeness and
accuracy of such data.4 In the Health Insurance Plan of New York breast screening trial4, there was no
screening benefit based on death certificates alone whereas analysis performed using adjudicated data
showed a significant effect. This raises the need for further investigation before universal adoption. In the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS)5.6 follow-up relies on
multiple data sources including national electronic health related datasets with expert adjudication
(outcomes review committee) being used for confirmation of ovarian cancer diagnosis and CoD. This
was felt necessary as ovarian cancer often presents late with widely disseminated disease, heightening
the possibility of disease misclassification. In addition, decreased investigation in older women, accident
and emergency presentation and mortality within the first month of presentation could contribute to
ovarian cancer being reported as a malignant neoplasm of unknown origin (ICD-10 code C80)7. Introduction The trial
data provides an opportunity to determine the completeness and accuracy of national ovarian cancer and
death registration in England, Wales and Northern Ireland using outcomes review as the reference
standard. Methods Between April 2001 and September 2005, 202638 postmenopausal women aged 50-74 from the general
population were recruited. They were randomised to annual screening by transvaginal ultrasound (50639)
or a multimodal strategy using serum CA125 interpreted by the Risk of Ovarian Cancer Algorithm (ROCA),
followed by ultrasound as a second-line test (50640) or control (no intervention; 101359).5,6 The full trial
protocol is accessible at https://www.ctu.mrc.ac.uk/studies/all-studies/u/ukctocs/. Trial registration is
ISRCTN22488978. This analysis is based on data collected prospectively up to the end of the initial
follow-up phase of UKCTOCS (2001-2014). Page 4/22
Multiple sources were used for follow-up of trial participants (Table 1).6 Women who provided written
consent for follow-up through national registries were flagged using their NHS number, date of birth and address for cancer and/or death registrations with NHS Digital (England and Wales) and the Northern
Ireland Cancer Registry and Business Services Organisation Health and Social Care Northern Ireland (BSO
Northern Ireland), respectively. Censorship data for the initial analysis of trial data was 31st December
2014.6 The last notification prior to data freeze for trial outcome analysis was received for both cancer
and deaths for England and Wales on March 25th, 2015 (NHS Digital); deaths for Northern Ireland on April
9th, 2015 (BSO, Northern Ireland), cancers for Northern Ireland on April 15th, 2015 (Northern Ireland Cancer
Registry). For women resident in England, data was also available for 2001-2012 from Hospital Episodes
Statistics (HES) and up to 2014 from the National Cancer Intelligence Network (NCIN). Additionally, all
women were sent two follow-up postal questionnaires, 3-5 years after randomisation and in 2014. The
UKCTOCS coordinating centre also received direct notification from the 13 trial centres, and trial
participants or their relatives. Outcomes Review Committee An outcomes review committee (ORC) was set up to confirm ovarian cancer diagnosis and death. The
committee consisted of two pathologists with specialist interest in gynaecological cancer and two
gynaecological oncologists. The ORC members were independent of trial conduct and blinded to the
randomisation group. Identification of cases and collation of evidence Identification of cases and collation of evidence Cases were identified for OR based on a cancer/death registration with any one of 19 preselected
International Classifications of Disease (10th Revision, ICD-10) codes (WHO 2003)7 (Supplementary Table
1, see Additional file 1) or a notification of possible ovarian cancer through the other sources listed above. The preselected ICD-10 codes were defined at trial initiation as those potentially associated with an
underlying ovarian cancer.6 Cases with an ICD-10 C80 (n=936) death registration and a cancer
registration of a non-ovarian malignancy were reviewed by a designated gynaecological oncologist and
excluded from the below process. Dedicated Coordinating Centre personnel initiated collection of pertinent documents from National Health
Service (NHS) Trusts, general practices, local health authorities, private hospitals, and hospices. These
included copies of medical records, histology, cytology reports, operative notes, diagnostic imaging
reports, hospital letters and discharge summaries, multidisciplinary meeting summaries,
chemotherapy/radiotherapy notes and autopsy reports. Procurement of the histology report was
obligatory in cases where pathological examination had been undertaken. The documents were
organized in chronological order and any direct or indirect mention of randomization group redacted, prior
to submission to ORC. The minimum dataset required for review comprised three separate documents
which could include in addition to those listed above, death certificates and cancer registrations. Outcome review Outcome review Page 5/22 Figure 1 shows a schematic for outcomes review. For all cases, a cancer review form (Supplementary
Figure 1, see Additional file 3) and where applicable, a death review form (Supplementary Figure 2, see
Additional file 4) was completed. If the reviewer deemed the documentation adequate, then a cancer
diagnosis or CoD was assigned. An algorithm with detailed rules for site allocation (Supplementary
Figure 3, see Additional file 5) ensured robust, reproducible and transparent assignment in various clinical
scenarios. In all cases where there was a discrepancy regarding ovarian cancer classification between the
reviewer and cancer or death registry, the case was forwarded to a second ORC member for an
independent review. If both reviewers were in agreement, the assigned diagnosis/CoD was accepted. If
there was disagreement, a third ORC member independently reviewed the case and a consensus decision
was made based on the majority view. If a reviewer was unable to make a definitive diagnosis, the
reviewer could request either further information or review of pathology slides, or a case discussion at an
ORC meeting. Outcomes review initially used the WHO 20037 rules for classifying cases into tubo-ovarian or primary
peritoneal. More recently all the peritoneal cancers were reviewed and reclassified as tubo-ovarian
cancers based on WHO revised 2014 criteria8. This states that peritoneal cancers can only be diagnosed
when there is no demonstrable gross or microscopic ovarian or tubal involvement. 9 Analysis All trial participants were included in this analysis (Figure 2). Sensitivities and specificities of
cancer/death registration of ovarian cancer obtained up to three months post censorship date were
calculated, using central adjudication as the reference standard. We also calculated the sensitivity if all
available national electronic health related datasets sources (Table 1) were used. The definitions for
sensitivity and specificity were as defined by the STARD 2015 guidelines (http://www.equator-
network.org/reporting-guidelines/stard) In addition, in all cases with a cancer registration and/or death certification, overall agreement of disease
site and CoD between cancer/death registries and OR was assessed using the Cohen’s kappa (κ)
statistic10, with confidence intervals calculated using 1000 bootstrap samples. Zero weighting was
applied to all off-diagonal cell entries. We also considered the false negative rate (FNR=1-sensitivity) of cancer registration and death
certification over time, with ORC confirmed diagnosis and CoD as the gold standard and summarising
with a test for proportional trend (Stata package ptrend). This test takes a standard χ2 test of cancer
registration (or death certification) versus year, on k=(row-1)×(column-1) degrees of freedom (df) and
partitions the χ2 value into a linear component (trend test) on 1 df and a ‘departure’ from linearity
component on k-1 df. We excluded the year 2014 from the cancer registration analysis, as during
exploratory analysis there was a large number of missing registrations. Funding Performance characteristics of cancer and death registration for tubo-ovarian cancer Our algorithm identified 3110 women requiring outcomes review for tubo-ovarian cancer diagnosis. The
ORC confirmed 1324 as having tubo-ovarian cancer. Of them, 1125 women had an ovarian, tubal or
primary peritoneal cancer registration. Overall sensitivity and specificity of ovarian, tubal or primary
peritoneal cancer registration within 3 months of censorship date was 85.0% (1125/1324; 95% CI
83.7%-86.2%) and 94.0% (1679/1786; 95% CI 93.2%-94.8%), respectively (Figure 2). In an additional 81 of
the 1324 women, we received an ovarian, tubal or primary peritoneal cancer notification from other
national datasets (death registration 51, HES 27, NCIN 3). If all available national electronic health related
datasets (Table 1) were considered, sensitivity would be 91.1% (1206/1324; 95% CI 89.4%-92.5%). Figure
3a and Supplementary Table 2 (see Additional file 2) show proportion of missing cancer registrations
over time. Excluding the last year (2014), there was no statistical evidence of a trend in the proportion of
missing cancer registrations (p=0.261) or any other form of temporal variability (p=0.549). Our algorithm identified 2214 women who died and required outcomes review for CoD. Only one of these
women did not have a death registration as she had died outside the UK. The ORC confirmed 681 as
having died due to tubo-ovarian cancer. Of them, 605 women had an ovarian, tubal or primary peritoneal
cancer CoD registration (Table 2). Overall sensitivity and specificity of ovarian, tubal or primary peritoneal
cancer death registration within 3 months of the censorship date was 88.8% (605/681; 95% CI Our algorithm identified 2214 women who died and required outcomes review for CoD. Only one of these
women did not have a death registration as she had died outside the UK. The ORC confirmed 681 as
having died due to tubo-ovarian cancer. Of them, 605 women had an ovarian, tubal or primary peritoneal
cancer CoD registration (Table 2). Overall sensitivity and specificity of ovarian, tubal or primary peritoneal
cancer death registration within 3 months of the censorship date was 88.8% (605/681; 95% CI
86.4%-91.2%) and 96.7% (1482/1533; 95% CI 95.8%-97.6%%) respectively (Figure 2). In the remaining 76
women, 29 women had an ovarian, tubal or primary peritoneal cancer registration, six had an ovarian
cancer HES record. If all available national electronic health related datasets (Table 1) were considered,
sensitivity would be 94.0% (640/681; 95% CI 91.9%-95.5%). The false negative rates by year are presented
in Figure 3b (Supplementary Table 2, see Additional file 2). Performance characteristics of cancer and death registration for tubo-ovarian cancer We found strong evidence of a negative linear
trend in proportion of missing death certificates (p=0.00002) but no further departure from that linearity
(p=0.474). Results Baseline characteristics of the trial population have been previously described.11 Of the 202638
postmenopausal women, 202632 (99.9%) women were electronically flagged for cancer and death
registrations in the relevant national registries of England, Wales and Northern Ireland (Figure 2). Funding Page 6/22 The current analysis was funded by a peer reviewed NIHR HTA grant (16/46/01) and The Eve Appeal. The
funders were not involved in the analyses nor in the writing of this report. Agreement of registry and outcome review assigned tubo-ovarian cancer diagnosis There was a relevant ICD-10 cancer registration in 1474 of the 3110 women who underwent outcomes
review for potential tubo-ovarian cancer (Table 2). 107 (8.7%; 107/1232) of 1232 cases registered as
ovarian, tubal or primary peritoneal cancer were reclassified on outcome review as malignant neoplasm
of unknown origin (2.7%; 33/1232), no cancer (2.1%; 26/1232) and other primary cancer (3.9%; 48/1232). Page 7/22 Page 7/22 Over one third were endometrium (41.6%; 20/48) About one fourth (27.1%; 13/48) of the other primary
cancers were metastatic to the ovary. In addition, 47 (4.0%; 47/1172) of the 1172 women confirmed as having tubo-ovarian cancer by
outcomes review would have been missed (Table 2). Almost three-fourths of these women were
registered as neoplasms of the ovary of uncertain behaviour (ICD-10 D39.1). The majority of these were
reclassified on outcomes review as borderline epithelial or non-epithelial ovarian cancer (ICD-10 C56.0). Majority (80.3%; 10/12) of the missed invasive epithelial cancers were at advanced stage (Table 3). The
overall agreement (85.4%; 1259/1474) between disease site ascribed by the cancer registries and those
designated by the ORC was moderate (Cohen’s kappa (κ) 0.55; 95% CI 0.5-0.6). Agreement of registry and outcome review assigned ovarian cancer deaths At censorship, 2041 cases had undergone outcomes review and had a relevant ICD-10 death registration. Of the 656 ovarian cancer death registrations, 92.2% (605/656) were confirmed on outcomes review. The
CoD in 51 (7.8%; 51/656) of 656 women registered as having died of ovarian, tubal or primary peritoneal
cancer were reclassified on outcome review as non-ovarian. This include malignant neoplasm of
unknown origin (51.0%; 26/51), other malignancy (37.3%; 19/51) or non-cancer causes (11.8%; 6/51). Over half of the other cancer deaths were due to endometrial cancer (52.6%; 10/19) (Table 2). In addition,
40 (6.2%; 40/645) of 645 women confirmed by outcomes review as having died of tubo-ovarian cancer
were registered as having died of non-tubo-ovarian cancer causes (Table 3). The agreement (86.3%;
1763/2041) between cancer registry and OR was substantial (Kappa 0.78; 95% CI 0.76-0.80). Main Findings Our findings demonstrate that in trials using follow-up through national cancer registration alone in
England, Wales and Northern Ireland, 15% of tubo-ovarian cancer cases may be missed. Similarly, use of
national death registration alone would result in 11% of tubo-ovarian cancer deaths being missed. The
proportions of missing cancer registrations showed no decline over time unlike the deficit in disease
specific death registrations. These rates could be halved if additional national electronic datasets such as
hospital episode statistics are used. When extrapolating these results, two facts must be taken into
consideration. We included cancer and death registrations of primary peritoneal cancer in ovarian cancer
incidence and mortality statistics. This is not the norm adopted by UK Office of National Statistics or US
SEER Registries. We obtained final follow-up data from the registries three months after the censorship
date in order to fulfil reporting guidelines for the trial. Longer intervals from censorship to obtaining
registry data are likely to reduce the rate in the last year. Adjudication by an independent review process can improve the accuracy of cancer and death
registrations. In those where a cancer registration was available, 9% of registry reported tubo-ovarian
cancers would have inaccurate cancer site assignment. Additionally, 4% of the final outcomes confirmed Page 8/22 Page 8/22 cancers would have been missed as they were registered as non-ovarian cancers. Reliance on death
registration alone, would have resulted in a similar proportion (7.8%) of deaths being wrongly assigned to
tubo-ovarian cancers and 6.2% of ovarian cancer deaths being missed. cancers would have been missed as they were registered as non-ovarian cancers. Reliance on death
registration alone, would have resulted in a similar proportion (7.8%) of deaths being wrongly assigned to
tubo-ovarian cancers and 6.2% of ovarian cancer deaths being missed. Strengths and Limitations A key strength of our analysis is that we compared cancer diagnoses and causes of death reported by the
national registries of England, Wales and Northern Ireland with those assigned through independent
central adjudication in a population cohort of over 3000 women. Of the 202638 women, 202632 (99.9%)
women were electronically flagged using their NHS number to the relevant national cancer and death
registries. The multiple additional data sources (Table 1) we used to identify potential ovarian cancer
cases further ensured that we had a complete population dataset of cases. We did not restrict the study to women with the tubo-ovarian cancer specific ICD-10 codes (C56, C57.0,
C48.1 and C48.2) but used 19 possible ICD-10 codes, in particular malignant neoplasm primary site
unknown (ICD-10 C80) that might have potentially included tubo-ovarian cases. The peritoneal cancers reported by cancer registries were included as tubo-ovarian in this analysis. While
this is the norm among clinicians and researchers, these cancers are not included in national tubo-
ovarian statistics. We did this to ensure that registry completeness rates were not underestimated due to
the 2014 WHO revision of the primary site definition.8 As the latter is adopted by pathology departments
the world over, it is likely that cancers previously denoted as primary peritoneal will be registered as tubo-
ovarian. A limitation is that the cases were diagnosed over a prolonged period between 2001 and 2014. To
address this we explored time trends. While there were no time trends in missing cancer registration data,
the completeness of death registration data improved over the years. Interpretation Use of UK cancer registry data alone would have allowed us to detect 85% of the tubo-ovarian cancer
cases. This is an improvement from the 78% sensitivity that we reported in our previous trial where
women were diagnosed with these cancers between 1986 and 1993.12 It is likely that the rates have
improved further in the more recent years. Use of multiple national electronic data sets especially hospital
administration records can augment these rates. A recent report on tubo-ovarian cancers diagnosed
between 2004 and 2012 in Switzerland also found that the hospital registry data provided
complementary information to that from the cancer registries.13 Sourcing data directly from live
healthcare systems allows trials to overcome the latency in the national cancer registration systems. A
median latency of 18 (range: 4–60) months to completion of incidence ascertainment was reported in a
recent survey of European Network of Cancer Registries.14 Page 9/22 Page 9/22 The overall agreement regarding ovarian cancer site between OR and cancer registries was moderate. A
key area of discrepancy was related to tumours defined by the ICD-10 code D39.1; neoplasms of the
ovary of unknown or uncertain behaviour. On outcome review, the majority were reclassified as malignant
neoplasm of the ovary (ICD-10 codes C56), either borderline epithelial ovarian or granulosa cell tumours. Most of these discrepancies likely reflect historical coding practices and classification systems used by
regional registries prior to centralization of services in 2013. Furthermore, coding for ovarian cancers
since 2011 uses the ICD-O-3 system,15 where the topographical code (site of cancer) is clearly separated
from the morphological code which includes the behaviour (malignant/ benign). In the current system, as
borderline ovarian tumours are classified as C56 (topographical code) with a morphology code of 1, there
is less likelihood of the cases not being registered as ovarian cancer. However our data for 2012 to 2014
are not sufficiently large to confirm this. The 9% of tubo-ovarian cancers reported by the registries that were classed as ‘not ovarian’ cancers on
review were equally distributed between three main groups: (1) miscoding of a benign mass as a cancer,
(2) other primary cancer, and (3) malignant neoplasm, primary site unknown (C80). The other primary
group included just under a third of cancers that metastasized to the ovary, which might have led to the
confusion. Interpretation The ovaries are a frequent site for metastases, with 5-30% of ‘ovarian masses’ being
secondary metastasis reported from non-ovarian cancers.16 The cause of death on death certificates is not always accurate17 due to a variety of reasons
(inexperience of certifying physician, lack of sufficient time/information, coding errors). However, in our
trial there was substantial agreement regarding primary site between death registry reported ovarian
cancer deaths and those assigned by the review committee. This has also been reported in screening
trials of other cancers (e.g. prostate) which used adjudication panels.18,19 Nonetheless 6.2% additional
tubo-ovarian cancer deaths were identified through the adjudication process. A high proportion were
registered as malignancies with an unknown primary site (C80) and all were advanced stage. In
advanced cancer, where multiple sites are involved, it may be difficult to assign the primary site.20 The
lack of consistency in the approach adopted by pathologists to assignment of primary site during our
trial led to a proposal for unified criteria for tubo-ovarian site assignment21 which has since been adopted
in international pathology reporting22 and clinical guidelines.23 The adjudication process used in the UKCTOCS trial is not dissimilar to that used in the Prostate Lung
Colorectal and Ovarian Cancer screening trial.24 In contrast to the Cluster randomised trial of PSA testing
for prostate cancer25 where data was abstracted into vignettes for expert review, we provided copies of
original clinical notes to the ORC members. To minimise ascertainment bias any reference to the
trial/randomisation arms was redacted in these documents. Review only occurred if the minimal required
number of documents was available. ORC members who were highly experienced gynaecological
oncology surgeons or pathologists used an algorithm developed specifically during the trial to assign
cancer site. To ensure accuracy and reproducibility over the years and across assessors, we audited the
reviews and introduced a classification algorithm (Supplementary Figure 3, see Additional File 5). The Page 10/22 Page 10/22 resource implications of central adjudication can be significant, as it requires many hours of highly
trained senior staff time. Often as in our trial, it is challenging to estimate costs as reviewers usually
volunteer their time and work outside normal office hours. Conclusion Our data suggests that follow-up of trial participants for tubo-ovarian cancer using national registry data
will result in incomplete ascertainment particularly of site due in part to the latency of registration. This
can be reduced by using hospital administrative data, which has shorter latency. Central adjudication by
experts though resource intensive adds value by improving accuracy of diagnoses. Revised classification
systems for ovarian cancer8 will improve accurate identification and aid reproducibility of site
assignment but this will require pathologists and clinicians to apply this new system uniformly across the
globe. Interpretation To save the effort and cost of adjudication, we
used individual as opposed to group adjudication, a work flow that involved more than one assessment
only when there was discrepancy between the primary reviewer and registered ICD-10 code with group
review limited to those where there was discrepancy between two reviewers. Adjudication also provides information on other key variables such as staging and morphology which in
the past have been poorly documented by registries26 but now improving.27 In our trial we observed a
stage shift in the multimodal screening arm.6 This would not have been quantifiable using data from the
registries alone without the detailed review of all available clinical documents as undertaken in
UKCTOCS. Kahan et al28 using a simulation model concluded that outcome misclassification can lead to biased
treatment effect estimates and reduced power, potentially resulting in an erroneous conclusion regarding
efficacy. The implementation of strategies to reduce misclassification is therefore of critical importance
in clinical trials. In UKCTOCS, the main impact of adjudication was in improving accuracy of tubo-ovarian
cancer. On death outcomes, there was no significant difference on the mortality impact of screening
between the analyses which used ORC adjudicated disease specific deaths and the sensitivity analysis
limited to deaths with the disease specific ICD-10 codes (ICD-10 C56, C57 and C48) for tubo-ovarian
cancer.10 Outcomes review committee (ORC) Outcomes review committee (ORC) Abbreviations United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author
on reasonable request. Ethics approval and consent to participate The study was approved by the UK North West Multicentre Research Ethics Committees (North West
MREC 00/8/34) on 21st June 2000 with site-specific approval from the local regional ethics committees
and the Caldicott guardians (data controllers) of the primary care trusts. All trial participants provided
written informed consent. The study was conducted in accordance to the Declaration of Helsinki and
Good Clinical Practice guidelines. Cause of death (CoD) Cause of death (CoD) Risk of Ovarian Cancer Alogorithm (ROCA) Page 11/22 Office of National Statistics (ONS) Business Services Organisation, Health and Social Care Northern Ireland (BSO Northern Ireland)
International Classifications of Disease, (Revision 10)(ICD10) National Health Service (NHS) General practitioners (GP) Funding The current analysis is supported by National Institute for Health Research (NIHR) HTA grant (16/46/01)
and The Eve Appeal. UKCTOCS was funded by Medical Research Council (G9901012 and G0801228),
Cancer Research UK (C1479/A2884), and the Department of Health, with additional support from The Eve
Appeal. Researchers at UCL are supported by the NIHR University College London Hospitals (UCLH)
Biomedical Research Centre and MRC CTU at UCL core funding (MR_UU_12023). The current analysis is supported by National Institute for Health Research (NIHR) HTA grant (16/46/01)
and The Eve Appeal. UKCTOCS was funded by Medical Research Council (G9901012 and G0801228),
Cancer Research UK (C1479/A2884), and the Department of Health, with additional support from The Eve
Appeal. Researchers at UCL are supported by the NIHR University College London Hospitals (UCLH)
Biomedical Research Centre and MRC CTU at UCL core funding (MR_UU_12023). Disclaimer: Views expressed in this publication are those of the authors and not necessarily those of the
NHS, the NIHR or the Department of Health and Social Care. Author Contributions This analysis was conceived by UM, JKK and AR. SM, SA, MH, DMC, CK collated clinical notes for review
by Outcomes Review Committee. NS, MW, EB and RW performed the Outcome Review. JKK, UM and AGM
performed the literature search. AR prepared dataset and MB performed the statistical analysis. JKK, AR,
MB, AGM and UM interpreted the data. JKK, AR, AGM and UM prepared the figures and tables. AR, JKK,
AGM, MB and UM contributed to writing of manuscript. AGM, SA, MH, DMC, JKK, SM, CK and UM were
involved with trial implementation and data collection. All authors (JKK, AR, DMC, NS, MB, EB, SA, MH,
SM, CK, AGM, RW, MW, LF, SC, SS, AM, MP, IJ, UM) critically reviewed and approved the report before
submission. Authors LF, SC, SS, AM and MP contributed to the trial design initiated by IJ and UM. Acknowledgments We thank the volunteers without whom the trial would not have been possible and everyone involved in
conduct and oversight. We are also very grateful to the current members of the UKCTOCS Trial Steering
Committee - Prof Henry Kitchener (Chair), Prof Julietta Patnick, Prof Jack Cuzick and Ms Annwen Jones. Competing Interests UM has stock ownership in Abcodia awarded to her by UCL. IJ is a co-inventor of the Risk of Ovarian
Cancer Algorithm (ROCA). The ROCA has been licensed to Abcodia Ltd by the owners of the ROCA,
Massachusetts General Hospital (MGH) and Queen Mary University of London (QMUL). IJ has a financial
interest in Abcodia Ltd as a shareholder and director and is entitled to a royalty payments via MGH and
QMUL from any commercial use of the ROCA. He is a trustee (2012–14) and Emeritus Trustee (2015 to
present) for The Eve Appeal. SJS co-developed the risk of ovarian cancer algorithm with all rights
assigned to MGH and QMUL. MGH has co-licensed software to Abcodia implementing the
algorithm. SJS receives personal fees from the LUNGevity Foundation and SISCAPA Assay Technologies
as a member of their Scientific Advisory Boards and from Abcodia as a consultant. SA is funded by a
research grant from Abcodia. All other authors declare no competing interests. Page 12/22 References 1. Dechartres A, Boutron I, Roy C, Ravaud P. Inadequate planning and reporting of adjudication
committees in clinical trials: recommendation proposal. Clin. Epidemiol. 62, 695-702 (2009). 2. Marcus PM, Doria-Rose VP, Gareen IF, Brewer B, Clingan K, Keating K, et al Did death certificates and
a death review process agree on lung cancer cause of death in the National Lung Screening Trial? Clin Trials. 2016 13:434-8. 3. Mäkinen T, Karhunen P, Aro J, Lahtela J, Määttänen L, Auvinen A. Assessment of causes of death in
a prostate cancer screening trial. Int J Cancer. 122, 413-417 (2008). 4. Doria-Rose VP, Marcus PM, Miller AB, Bergstralh EJ, Mandel JS, Tockman MS, et al. Does the source
of death information affect cancer screening efficacy results? A study of the use of mortality review
versus death certificates in four randomized trials. Clin Trials. 7, 69-77 (2010). Page 13/22 Page 13/22 5. Menon U, Gentry-Maharaj A, Ryan A, Sharma A, Burnell M, Hallett R, et al. Recruitment to multicentre
trials--lessons from UKCTOCS: descriptive study. 337, a2079 (2008). 6. Jacobs I, Menon U, Ryan A, Gentry-Maharaj A, Burnell M, Kalsi JK et al Ovarian cancer screening and
mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised
controlled trial. Lancet 387, 945–956 (2016). 7. World Health Organisation: International Statistical Classification of Disease and Related Health
Problems Codes, (tenth revision) 2nd (2003) 8. World Health Organisation: International Statistical Classification of Disease and Related Health
Problems Codes, (tenth revision) (2014) 9. Prat J; FIGO Committee on Gynecologic Oncology. Staging classification for cancer of the ovary,
fallopian tube, and peritoneum. Int J Gynaecol Obstet. 124(1):1-5. (2014) 10. Cohen, J. "A coefficient of agreement for nominal scales". Educational and Psychological
Measurement. 20, 37–46 (1960). 11. Menon U, Gentry-Maharaj A, Hallett R, Ryan A, Burnell M, Sharma A, et al. Sensitivity and specificity
of multimodal and ultrasound screening for ovarian cancer, and stage distribution of detected
cancers: results of the prevalence screen of the UK Collaborative Trial of Ovarian Cancer Screening
(UKCTOCS). Lancet Oncol. 10:327-40 (2009) 12. MacDonald N, Sibley K, Rosenthal A, Menon U, Jayarajah A, Oram D et al A comparison of national
cancer registry and direct follow-up in the ascertainment of ovarian cancer. Br J Cancer. ;80:1826-7. (1999) 13. Wieser S, Schmidt M, Kind AB, Heinzelmann-Schwarz VA. Ovarian cancer in Switzerland: incidence
and treatment according to hospital registry data. Swiss Med Wkly. 2018 Jul 29;148:w14647. References doi:
10.4414/smw.2018.14647. eCollection 2018 Jul 16. 14. Zanetti R, Schmidtmann I, Sacchetto L, Binder-Foucard F, Bordoni A, Coza D, et al. Completeness and
timeliness: Cancer registries could/should improve their performance. Eur J Cancer. 51, 1091-1098
(2015). 15. Internationational Classification of Diseases for Oncology http://codes.iarc.fr/topography Accessed
1st Aug 2018 16. Sung-Jong Lee, Jeong-Hoon Bae, A-Won Lee, Seo-Yun Tong, Yong-Gyu Park, and Jong-Sup Park. Clinical Characteristics of Metastatic Tumors to the Ovaries. J Korean Med Sci 24, 114–119 (2009). 16. Sung-Jong Lee, Jeong-Hoon Bae, A-Won Lee, Seo-Yun Tong, Yong-Gyu Park, and Jong-Sup Park. Clinical Characteristics of Metastatic Tumors to the Ovaries. J Korean Med Sci 24, 114–119 (2009). 17. Mieno MN, Tanaka N, Arai T, Kawahara T, Kuchiba A, Ishikawa S, et al Accuracy of Death Certificates
and Assessment of Factors for Misclassification of Underlying Cause of Death. J Epidemiol. 26, 191-
198 (2016). 17. Mieno MN, Tanaka N, Arai T, Kawahara T, Kuchiba A, Ishikawa S, et al Accuracy of Death Certificates
and Assessment of Factors for Misclassification of Underlying Cause of Death. J Epidemiol. 26, 191-
198 (2016). 18. Godtman R, Holmberg E, Stranne J, Hugosson J. High accuracy of Swedish death certificates in men
participating in screening for prostate cancer: a comparative study of official death certificates with a
cause of death committee using a standardized algorithm. Scand J Urol Nephrol. 45, 226-232 (2011). 18. Godtman R, Holmberg E, Stranne J, Hugosson J. High accuracy of Swedish death certificates in men
participating in screening for prostate cancer: a comparative study of official death certificates with a
cause of death committee using a standardized algorithm. Scand J Urol Nephrol. 45, 226-232 (2011). Page 14/22 Page 14/22 19. Turner EL, Metcalfe C, Donovan JL, Noble S, Sterne JA, Lane JA, et al. Contemporary accuracy of
death certificates for coding prostate cancer as a cause of death: Is reliance on death certification
good enough? A comparison with blinded review by an independent cause of death evaluation
committee. Br J Cancer. 28;115, 90-94 (2016). 20. Singh N, Gilks CB, Wilkinson N, McCluggage WG. Assessment of a new system for primary site
assignment in high-grade serous carcinoma of the fallopian tube, ovary, and peritoneum. Histopathology. 67, 331-337 (2015). 21. Singh N, Gilks CB, Hirschowitz L, Kehoe S, McNeish IA, Miller D, et al. Primary site assignment in tubo-
ovarian high-grade serous carcinoma: Consensus statement on unifying practice worldwide. Gynecol
Oncol 141, 195-198. (2016) 22. References Kahan BC, Feagan B, Jairath V. (2017). A comparison of approaches for adjudicating outcomes in
clinical trials. Trials.18, 266. DOI 10.1186/s13063-017-1995-3 28. Kahan BC, Feagan B, Jairath V. (2017). A comparison of approaches for adjudicating outcomes in
clinical trials. Trials.18, 266. DOI 10.1186/s13063-017-1995-3 Tables Due to technical limitations, Tables 1-3 are only available as downloads in the supplemental files
section. References McCluggage WG, Judge MJ, Clarke BA, et al. Data set for reporting of ovary, fallopian tube and
primary peritoneal carcinoma: recommendations from the International Collaboration on Cancer
Reporting (ICCR). Mod Pathol 28,1101-1122. (2015) 22. McCluggage WG, Judge MJ, Clarke BA, et al. Data set for reporting of ovary, fallopian tube and
primary peritoneal carcinoma: recommendations from the International Collaboration on Cancer
Reporting (ICCR). Mod Pathol 28,1101-1122. (2015) 23. Colombo N,Sessa C,du Bois A,Ledermann J,McCluggage WG,McNeish I, et al ESMO-ESGO Ovarian
Cancer Consensus Conference Working Group. ESMO-ESGO consensus conference
recommendations on ovarian cancer: pathology and molecular biology, early and advanced stages,
borderline tumours and recurrent disease Ann Oncol.30:672-705. (2019) 24. Miller AB, Yurgalevitch S, Weissfeld JL; Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial
Project Team.Death review process in the Prostate, Lung,Colorectal and Ovarian (PLCO) Cancer
Screening Trial. Control Clin Trials 21(6 Suppl):400S-406S (2000). 24. Miller AB, Yurgalevitch S, Weissfeld JL; Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial
Project Team.Death review process in the Prostate, Lung,Colorectal and Ovarian (PLCO) Cancer
Screening Trial. Control Clin Trials 21(6 Suppl):400S-406S (2000). 25. Williams NJ, Hill EM, Ng SY, Martin RM, Metcalfe C, Donovan JL, et al. CAP Cause of Death
Committee (2015). Standardisation of information submitted to an end-point committee for cause of
death assignment in a cancer screening trial. BMC Med Res Methodol 15, 6. 25. Williams NJ, Hill EM, Ng SY, Martin RM, Metcalfe C, Donovan JL, et al. CAP Cause of Death
Committee (2015). Standardisation of information submitted to an end-point committee for cause of
death assignment in a cancer screening trial. BMC Med Res Methodol 15, 6. 26. Walters S, Maringe C, Butler J, Brierley JD, Rachet B, Coleman MP. Comparability of stage data in
cancer registries in six countries: lessons from the International Cancer Benchmarking Partnership. Int J Cancer. 132, 676-685. (2013). 26. Walters S, Maringe C, Butler J, Brierley JD, Rachet B, Coleman MP. Comparability of stage data in
cancer registries in six countries: lessons from the International Cancer Benchmarking Partnership. Int J Cancer. 132, 676-685. (2013). 27. McPhail S, Johnson S, Greenberg D, Peake M, Rous B. Stage at diagnosis and early mortality from
cancer in England. Br J Cancer. 112, Suppl 1:S108-15 27. McPhail S, Johnson S, Greenberg D, Peake M, Rous B. Stage at diagnosis and early mortality from
cancer in England. Br J Cancer. 112, Suppl 1:S108-15 28. Figures Page 15/22 Page 15/22 Figure 1 Outcomes Review process Page 16/22 Figure 1 Outcomes Review process Page 17/22 Figure 2 Evaluation of national registrations of tubo-ovarian* cancer/death compared to the reference standard
(identification through multiple sources, hospital notes retrieval and independent outcomes review) Page 18/22 Page 18/22 Figure 2 Evaluation of national registrations of tubo-ovarian* cancer/death compared to the reference standard
(identification through multiple sources, hospital notes retrieval and independent outcomes review) Page 19/22 Page 19/22 gure 3
roportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Page 20/22
Figure 3
Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Figure 3 Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Figure 3 Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Proportion of missing registrations* of tubo-ovarian cancers by year of diagnosis Page 20/22 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 21/22
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The influence of transformed Reynolds number suppression on gas transfer parameterizations and global DMS and CO&lt;sub&gt;2&lt;/sub&gt; fluxes
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Atmospheric chemistry and physics
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The influence of transformed Reynolds number suppression on
gas transfer parameterizations and global DMS and CO2 fluxes
Alexander Zavarsky1 and Christa A. Marandino2
1independent researcher, Kiel, Germany
2GEOMAR H l h l
C
f
O
R
h Ki l G Parameteri-
zations of k are another source of uncertainty in calculating
fluxes. The flux F can be directly measured, e.g., with the
eddy covariance technique, together with 1C in order to de-
rive k and estimate a k parameterization (Eq. 2). ktotal = F
1C =
F
cwater −cair · H
(2) (2) It is very common that ktotal is parameterized with wind
speed and all wind speed parameterizations have in com-
mon that ktotal increases monotonically with increasing wind
speed. This assumption is sensible, as higher wind speed in-
creases turbulence both on the air side and the water side and
hence the flux. Additional processes like bubble generation
can additionally enhance gas transfer. The total gas transfer
velocity ktotal, which is measured by eddy covariance or other
direct flux methods, can split into the water-side gas trans-
fer velocity kwater and the air-side gas transfer velocity kair
(Eq. 3). It is very common that ktotal is parameterized with wind
speed and all wind speed parameterizations have in com-
mon that ktotal increases monotonically with increasing wind
speed. This assumption is sensible, as higher wind speed in-
creases turbulence both on the air side and the water side and
hence the flux. Additional processes like bubble generation
can additionally enhance gas transfer. The total gas transfer
velocity ktotal, which is measured by eddy covariance or other
direct flux methods, can split into the water-side gas trans-
fer velocity kwater and the air-side gas transfer velocity kair
(Eq. 3). The influence of transformed Reynolds number suppression on
gas transfer parameterizations and global DMS and CO2 fluxes
Alexander Zavarsky1 and Christa A. Marandino2
1independent researcher, Kiel, Germany
2GEOMAR H l h l
C
f
O
R
h Ki l G Correspondence: Alexander Zavarsky (alexz@mailbox.org) Correspondence: Alexander Zavarsky (alexz@mailbox.org) Received: 12 January 2018 – Discussion started: 12 February 2018
Revised: 13 December 2018 – Accepted: 29 January 2019 – Published: 11 February 2019 Received: 12 January 2018 – Discussion started: 12 February 2018
Revised: 13 December 2018 – Accepted: 29 January 2019 – Published: 11 February 2019 Abstract. Eddy covariance measurements show gas transfer
velocity suppression at medium to high wind speed. A wind–
wave interaction described by the transformed Reynolds
number is used to characterize environmental conditions fa-
voring this suppression. We take the transformed Reynolds
number parameterization to review the two most cited wind
speed gas transfer velocity parameterizations: Nightingale
et al. (2000) and Wanninkhof (1992, 2014). We propose
an algorithm to adjust k values for the effect of gas trans-
fer suppression and validate it with two directly measured
dimethyl sulfide (DMS) gas transfer velocity data sets that
experienced gas transfer suppression. We also show that the
data set used in the Nightingale 2000 parameterization ex-
perienced gas transfer suppression. A compensation of the
suppression effect leads to an average increase of 22 % in the
k vs. u relationship. Performing the same correction for Wan-
ninkhof 2014 leads to an increase of 9.85 %. Additionally, we
applied our gas transfer suppression algorithm to global air–
sea flux climatologies of CO2 and DMS. The global applica-
tion of gas transfer suppression leads to a decrease of 11 %
in DMS outgassing. We expect the magnitude of Reynolds
suppression on any global air–sea gas exchange to be about
10 %. ductance, which includes all processes promoting and sup-
pressing gas transfer. cair and cwater are the respective air-side
and water-side concentrations. H is the dimensionless form
of the Henry’s law constant. F = ktotal · 1C = ktotal · (cwater −cair · H)
(1) (1) 1C is typically measured with established techniques, al-
though the distance of the measurements from the interface
introduces uncertainties in the flux calculation. Parameteri-
zations of k are another source of uncertainty in calculating
fluxes. The flux F can be directly measured, e.g., with the
eddy covariance technique, together with 1C in order to de-
rive k and estimate a k parameterization (Eq. 2). 1C is typically measured with established techniques, al-
though the distance of the measurements from the interface
introduces uncertainties in the flux calculation. 1
Introduction 1
ktotal
=
1
kwater
+ H
kair
(3) (3) Gas flux F between the ocean and the atmosphere is com-
monly described as the product of the concentration differ-
ence 1C between the liquid phase (seawater) and the gas
phase (atmosphere) and the total gas transfer velocity ktotal. 1C acts as the forcing potential difference and k as the con- We focus, in this work, on kwater, which is the sum of the
interfacial gas transfer ko and the bubble-mediated gas trans-
fer kb (Eq. 4). We focus, in this work, on kwater, which is the sum of the
interfacial gas transfer ko and the bubble-mediated gas trans-
fer kb (Eq. 4). Atmos. Chem. Phys., 19, 1819–1834, 2019
https://doi.org/10.5194/acp-19-1819-2019
© Author(s) 2019. This work is distributed under
the Creative Commons Attribution 4.0 License. A. Zavarsky and C. A. Marandino: Gas transfer suppression model There are two main goals of this study: (1) develop and
use a simplistic algorithm to adjust for gas transfer suppres-
sion; (2) illustrate that gas transfer suppression is ubiqui-
tous, showing up in our most used gas transfer parameter-
izations. To address goal 1, we develop a gas transfer sup-
pression model and apply it to two DMS eddy covariance
data sets. To address goal 2, we investigate the two most
commonly used gas parameterizations (both cited more than
1000 times each) for the occurrence of gas transfer suppres-
sion. The Nightingale et al. (2000) parameterization (N00)
contains data from the North Sea, Florida Strait and Georges
Bank between 1989 and 1996. The N00 parameterization
is derived from changes in the ratio of SF6 and 3He (dual-
tracer method). We also investigate the Wanninkhof (2014)
gas transfer parameterization (W14), which is an update to
Wanninkhof (1992). They calculate the amount of CO2 ex-
changed between the ocean and atmosphere using a global
ocean 14C inventory. This 14C inventory is already influenced
by gas transfer suppression as it is globally averaged. They
deduce a quadratic k vs. wind speed parameterization using
a wind speed climatology. Both k parameterizations (N00,
W14) are monotonically increasing with wind speed. Retr = utr · Hs
νair
· cos(θ)
(7) (7) Retr is the Reynolds number transformed into the reference
system of the moving wave. utr is the wind speed trans-
formed into the wave’s reference system, Hs the significant
wave height, νair the kinematic viscosity of air and θ the an-
gle between the wave direction and direction of utr in the
wave’s reference system. This parameterization is based on
the model of air flowing around a sphere (Singh and Mittal,
2004). The flow is laminar and attached all around the sphere
at low Re (Retr < 10). However, this condition does not oc-
cur in the oceanic environment as utr would have to be around
3×10−5 m s−1 (using Hs = 3 m and νair = 10−5 m2 s−1). At
101 < Retr < 105, vortexes form at the lee side of the sphere
and the flow separates. This is the state of gas transfer sup-
pression and occurs approximately at utr from 3 × 10−5 to
3 m s−1. A. Zavarsky and C. A. Marandino: Gas transfer suppression model of this model. The Retr parameterization shows that the sup-
pression is primarily dependent on wind speed, wave speed,
wave height and a directional component. kwater = ko + kb
(4) (4) kwater = ko + kb It is noteworthy that, so far, only gas transfer velocities
deduced by eddy covariance have shown a gas transfer sup-
pression. This may be due to the spatial (1 km) and tempo-
ral (30 min) resolution of eddy covariance measurements, or
to the types of gases measured (e.g., CO2; dimethyl sulfide,
DMS; organic VOCs). The use of rather soluble gases (DMS,
acetone, methanol) means that the gas transfer velocity will
not be greatly influenced by bubble-mediated gas transfer. Gas transfer suppression only affects ko (Zavarsky et al.,
2018). Another direct flux measurement technique, the dual-
tracer method, utilizes sulfur hexafluoride (SF6) or 3He. The
dual-tracer measurement usually lasts over a few days but
could have a similar spatial resolution as eddy covariance. SF6 and 3He are both very insoluble and heavily influenced
by the bubble effect. Hence, if the gas transfer suppression
only affects ko, kb could be the dominant process, masking
the gas transfer suppression. Additionally, the long measure-
ment period could decrease the likelihood of detection of gas
transfer suppression as the conditions for suppression might
not be persistent over a few days. Schmidt number (Sc) scaling (Eq. 5) is used to compare gas
transfer velocities of different gases. Sc scaling only applies
to ko and kair. Sc is the ratio of the viscosity ν to the diffusiv-
ity D of the respective gas in seawater. Sc = ν
D
(5)
ko,Sc
ko,660
=
Sc
660
n
(6) (5) (6) The exponent n is chosen depending on the surface proper-
ties. For smooth surfaces n = −2
3 and for rough wavy sur-
faces n = −1
2 (Komori et al., 2011). In this study n = −1
2 is
used. In contrast to commonly accepted gas transfer velocity
parameterizations, parameterizations based on direct flux
measurements by eddy covariance systems have shown a
decrease or flattening of k with increasing wind speed at
medium to high wind speeds (Bell et al., 2013, 2015; Yang
et al., 2016; Blomquist et al., 2017). We use the transformed
Reynolds number Retr (Zavarsky et al., 2018) to identify in-
stances of gas transfer suppression. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1820 2.4
Transformed Reynolds number The Reynolds number describes the balance of inertial forces
and viscous forces. It is the ratio of the typical length and
velocity scale over the kinematic viscosity. The transformed
Reynolds number, in Eq. (11), uses the wind speed utr trans-
formed into the wave’s reference system. The significant
wave height Hs is used as the typical length scale. The dif-
ference between wind direction and wave direction is given
by the angle θ. Between θ = 0 and θ = 90◦the air flowing
over the wave experiences, due to the angle of attack, a dif-
ferently shaped and streamlined wave. The factor cos(θ) is
multiplied by Hs to account for directional dependencies and
shape influences (Fig. A1). cp = g · Tp
2π
(8) cp = g · Tp
2π (8) 2
Methods The kinematic viscosity ν of air is dependent on air density ρ
and the dynamic viscosity µ of air, Eq. (9). 2.1
WAVEWATCH III® (WWIII) ν(T,p) =
µ(T )
ρ(T,p)
(9) (9) We use wave data from the WWIII model hindcast run by
the Marine Modeling and Analysis Branch of the Environ-
mental Modeling Center of the National Centers for Envi-
ronmental Prediction (NCEP; Tolman, 1997, 1999, 2009). The model is calculated for the global ocean surface exclud-
ing ice-covered areas with a temporal resolution of 3 h and
a spatial resolution of 0.5◦× 0.5◦. The data for the specific
analysis of the N00, W14 parameterizations and the Knorr11
cruise (Sect. 4.1–4.3) were obtained from the model for the
specific locations and times of the measurements. The data
for the global analysis, Sect. 4.4, were obtained for the total
year 2014. The model also provides the u (meridional) and
v (zonal) wind vectors, assimilated from the Global Forecast
System, used in the model. We retrieved wind speed, wind
direction, bathymetry, wave direction, wave period and sig-
nificant wave height. We converted the wave period Tp to
phase speed cp, assuming deep water waves, using Eq. (8)
(Hanley et al., 2010). The dynamic viscosity is dependent on temperature T and
can be calculated using Sutherland’s law (White, 1991)
(Eq. 10). µ = µ0 ·
T
T0
2
3
(10) (10) µ0 = 1.716×10−5 N s m−2 at T0 = 273 K (White, 1991). Air
density is dependent on temperature T and air pressure p and
was calculated using the ideal gas law. A. Zavarsky and C. A. Marandino: Gas transfer suppression model We linearly interpolated all data sets to the grid and times
of the WWIII model. transfer suppression. There, we calculate an estimate for the
magnitude of gas transfer suppression on a monthly local ba-
sis. 2.2
Auxiliary variables Surface air temperature T , air pressure p, sea surface temper-
ature SST and sea ice concentration were retrieved from the
ERA-Interim reanalysis of the European Centre for Medium-
Range Weather Forecasts (Dee et al., 2011). It provides a 6-
hourly time resolution and a global 0.125◦× 0.125◦spatial
resolution. Sea surface salinity (SSS) was extracted from the
Takahashi climatology (Takahashi et al., 2009). Retr = utr · Hs
ν
· cos(θ)
(11) (11) Air–sea partial pressure difference (1pCO2) was obtained
from the Takahashi climatology. 1pCO2, in the Takahashi
climatology, is calculated for the year 2000 CO2 air concen-
trations. Assuming an increase in both the air concentration
and the partial pressure in the water side, the partial pres-
sure difference remains constant. The data set has a monthly
temporal resolution, a 4◦latitudinal resolution and a 5◦lon-
gitudinal resolution. 3
Gas transfer suppression model Below |Retr| ≤6.96×105 flow separation between the wind
flowing above the wave and the flow entering the trough sup-
presses gas transfer (Zavarsky et al., 2018). As a result, com-
mon wind speed parameterizations of k are not applicable
(Eq. 1). To provide a magnitude for this suppression, we pro-
pose an alternative wind speed ualt, which is lower than u10. This decrease accounts for the effect of gas transfer suppres-
sion. ualt represents the wind speed with the maximum pos-
sible k in these conditions, hence an increase in u beyond ualt
does not result in an increase in k. Thus, ualt can then be used
with k parameterizations to calculate the gas flux. DMS water concentrations were taken from the Lana DMS
climatology (Lana et al., 2011). These are provided with a
monthly resolution and a 1◦× 1◦spatial resolution. The air
mixing ratio of DMS was set to zero (cair,DMS = 0). Taking
air mixing ratios into account, the global air–sea flux of DMS
reduces by 17 % (Lennartz et al., 2015). We think this ap-
proach is reasonable as we look at the relative flux change
due to gas transfer suppression only. Given a set wave field (constant Hs, wave direction and
speed), if the relative wind speed in the reference system
of the wave utr is high enough that |Retr| > 6.96 × 105, no Atmos. Chem. Phys., 19, 1819–1834, 2019 A. Zavarsky and C. A. Marandino: Gas transfer suppression model When utr, and as a consequence Retr, is further in-
creased (Retr > 105), turbulence in the boundary layer be-
tween the air and the sphere counteracts the flow separation
and reduces the surface area on which the separation acts. This means that an increased relative wind speed utr favors
unsuppressed conditions. 5 In addition, we use wind and wave data for the year 2014,
calculate Retr and perform an analysis of the impact of gas
transfer suppression on the yearly global air–sea exchange of
DMS and CO2. So far global estimates of air–sea exchange
of DMS have been based on k parameterizations, which have
not included a mechanism for gas transfer suppression. We
provide an iterative calculation of the effect of gas transfer
suppression on existing DMS climatologies. For global CO2
budgets, the widely used W14 and Tak09 (Takahashi et al.,
2009) parameterizations already include a global average gas A flux measurement at values of |Retr| ≤6.96×105 is gas
transfer suppressed (Zavarsky et al., 2018). The threshold
presents a binary treatment of the problem. We adopt this
treatment since stall conditions, flow detachment and reat-
tachment in aerodynamics are also binary. Describing transi-
tion conditions is beyond the scope of the first introduction Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ 1821 Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ 1822 A. Zavarsky and C. A. Marandino: Gas transfer suppression model Figure 1. Work flow of the gas transfer suppression model. In the case of suppressed gas transfer, the output is the adjusted wind speed ualt,
which can then be used in gas transfer parameterizations. The step size 1s can be adapted freely, but considerations of resolution and
computing power have to be made. We set 1s = 0.3 m s−1 for this paper. Figure 1. Work flow of the gas transfer suppression model. In the case of suppressed gas transfer, the output is the adjusted wind speed ualt,
which can then be used in gas transfer parameterizations. The step size 1s can be adapted freely, but considerations of resolution and
computing power have to be made. We set 1s = 0.3 m s−1 for this paper. fer and measurement. Therefore, we change the wind speed
only. suppression occurs. In the “unsuppressed” case, k can be
estimated by common gas transfer parameterizations. If the
wind speed u10, in the earth’s reference system, is getting
close to the wave’s phase speed, utr in the wave’s refer-
ence system gets smaller and |Retr| drops below the thresh-
old; thus, flow separation happens and suppression occurs. We propose a stepwise (1s) reduction of u10 to calculate
when the wind–wave system changes from the flow sepa-
ration regime (|Retr| < 6.96 × 105) to a normal flow regime
(|Retr| > 6.96 × 105). This can be used to estimate the mag-
nitude of the suppression. We recalculate Retr with a lower
ualt = u10−i·1s and iterate i = 0, 1, 2, 3 ... as long as Retr is
below the threshold (flow separation). If Retr crosses to the
non-suppressing regime, the iteration is stopped and the ac-
tual ualt can be used as an alternative wind speed. The itera-
tion steps are (1) calculate Retr using ualt = u10 −i · 1s and
(2) determine if |Retr| ≤6.96 × 105. (3) If yes, i = i + 1 and
continue with step (1). If no, break the loop. The step size
in this model was 0.3 m s−1. We think this step size allows
for a good balance between computing time and velocity res-
olution. The minimum velocity for ualt is 0 m s−1. Figure 1
shows a flowchart of the algorithm. This algorithm is applied
to every box at every time step. 3.1
Gas transfer The difference between ualt and u10 directly relates to the
magnitude of gas transfer suppression. ualt can be used in
two ways: (1) u10 can be directly replaced by ualt. This is
only possible for parameterizations with a negligible bubble
contribution (like DMS), as we assume that the gas transfer
suppression only affects ko. As a result, one gets a k estima-
tion using the lower wind speed ualt. This is an estimate of
the reduction of k by gas transfer suppression. (2) For param-
eterizations of rather insoluble gases, like CO2, SF6 and 3He,
one needs to subtract 1k from the unsuppressed k parameter-
ization. This adjustment is done by inserting u10 −ualt into
a ko parameterization (Eq. 12) and subtracting 1k. In this
paper, ZA18 from Zavarsky et al. (2018) is used as the pa-
rameterization of ko. The magnitude of the gas transfer sup-
pression is given by Eq. (12). 1k = ko (u10) −ko (ualt) = (3.1 · u10 −5.7)
−(3.1 · ualt −5.7) = 3.1 · (u10 −ualt)
(12) (12) A change in the parameters of the wave field is, in our
opinion, not feasible as the wave field is influenced to a cer-
tain extent by swell that is externally prescribed. Swell trav-
els long distances and does not necessarily have a direct re-
lation to the wind conditions at the location of the gas trans- For the global flux of DMS we use the bulk gas transfer
formula (Eq. 1). The global DMS gas flux calculations are
based on the following k parameterizations: ZA18 and the
quadratic parameterization N00. For every grid box and ev-
ery time step we calculate ualt according to the description in
Sect. 3. If ualt is lower than u10 from the global reanalysis, For the global flux of DMS we use the bulk gas transfer
formula (Eq. 1). The global DMS gas flux calculations are
based on the following k parameterizations: ZA18 and the
quadratic parameterization N00. For every grid box and ev-
ery time step we calculate ualt according to the description in
Sect. 3. If ualt is lower than u10 from the global reanalysis, Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ 1823 A. Zavarsky and C. A. Marandino: Gas transfer suppression model Figure 2. Adjustments to the SO234-2/235 DMS k vs. u relationship. 3.1
Gas transfer The datapoints with |Retr| ≺6.96 × 105 were adjusted using the gas
transfer suppression model. Black circles denote k values at the original wind speed u10. Colored filled circles denote the k value at wind
speed equals to ualt. The color shows the significant wave height. If a datapoint has a concentric black and filled circle, it was not adjusted,
as it was not subject to gas transfer suppression. The black solid line is the ZA18 parameterization. The dotted line is the linear fit to the
datapoints before the adjustment; the dashed line is the linear fit after the adjustment. Figure 2. Adjustments to the SO234-2/235 DMS k vs. u relationship. The datapoints with |Retr| ≺6.96 × 105 were adjusted using the gas
transfer suppression model. Black circles denote k values at the original wind speed u10. Colored filled circles denote the k value at wind
speed equals to ualt. The color shows the significant wave height. If a datapoint has a concentric black and filled circle, it was not adjusted,
as it was not subject to gas transfer suppression. The black solid line is the ZA18 parameterization. The dotted line is the linear fit to the
datapoints before the adjustment; the dashed line is the linear fit after the adjustment. proof of concept, we quantify the influence of gas transfer
suppression on N00 and W14 and provide unsuppressed esti-
mates. Finally, we apply the wind speed adjustment to global
flux estimates of DMS. For CO2, we estimate the magnitude
of gas transfer suppression. then gas transfer suppression occurs. Subsequently, ualt to-
gether with Eq. (12) is used in the specific bulk gas trans-
fer formulas (Eqs. 13–14). For ZA18, ualt can be directly
inserted into the ZA18 parameterization (Eq. 13). How-
ever, other parameterizations, e.g., N00, which are based on
measurements with rather insoluble gases, have a signifi-
cant bubble-mediated gas transfer contribution. As a con-
sequence, we subtract the linearly dependent 1k using the
ZA18 parameterization, to account for the gas transfer sup-
pression in ko (Eq. 14). 4.1
Adjustment of the interfacial gas transfer Figures 2 and 3 show the unsuppressed DMS gas transfer ve-
locities for the SO234-2/235 and the Knorr11 cruises. We
shift the measured datapoints, which are gas transfer sup-
pressed, along the x axis by replacing u10 with ualt. The shift
along the x axis is equivalent to an addition of 1k, for a
given k vs. u relationship, to balance gas transfer suppres-
sion (see Appendix). The black circles indicate the original
data set at u10. The colored circles are k values plotted at
the adjusted wind speed ualt. If a black circle and a colored
circle are concentric, the datapoint was not suppressed and
therefore no adjustment was applied. For comparison, the pa-
rameterization ZA18 is plotted in both figures. Both figures
show the significant wave height with the color bar. Flim,ZA18 = [kZA18 (u10) −1k] · 1C
= (3.1 · ualt −5.37) · 1C
(13) (13) Flim,N00&other=
kN00&other (u10) −1k
· 1C
=
kN00&other (u10) −3.1 · (u10 −ualt)
· 1C (14) Sea ice concentration from the ERA-Interim reanalysis was
included as a linear factor in the calculation. A sea ice con-
centration of 90 %, for example, results in a 90 % reduction
of the flux. Each time step (3 h) of the WWIII model provided
a global grid of air–sea fluxes with and without gas transfer
suppression. These single time steps were summed up to get
a yearly flux result. Figure 2 illustrates the linear fits to the data set before (dot-
ted) and after (dashed) the adjustment. The suppressed data-
points from 14 to 16 m s−1 moved closer to the linear fit after
an adjustment with ualt. The high gas transfer velocity val-
ues at around 13 m s−1 and above 35 cm h−1 were moved to
11 m−1. This means a worsening of the k estimate by the lin-
ear fit. These datapoints have very low 1C values (Zavarsky
et al., 2018), therefore we expect a large scatter as a result
from Eq. (2). 4
Results We test the adjustment of u10 →ualt with two data sets of
DMS gas transfer velocities, Knorr11 (Bell et al., 2017) and
SO234-2/235 (Zavarsky et al., 2018). Both data sets experi-
enced gas transfer suppression at high wind speed. Using this Figure 3 also shows an improvement of the linear fit es-
timates. The gas transfer suppressed datapoints were as- Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ The mean of the absolute value is presented in the last two columns. Table 1. Mean differences between the reference fits (column one) and the adjusted and unadjusted k data sets. A negative value describes
that the fit, on average, overestimates the actual measured data. The mean of the absolute value is presented in the last two columns. Table 1. Mean differences between the reference fits (column one) and the adjusted and unadjusted k data sets. A negative value describes
that the fit, on average, overestimates the actual measured data. The mean of the absolute value is presented in the last two columns. Table 2. Linear fits to the adjusted and unadjusted data sets of Knorr11 and SO234-2/235. The error estimates correspond to a 95 % confidence
interval. Knorr11
SO234-2/235
Unadjusted
k660 = 0.52 ± 0.4 · u + 5.79 ± 4.82
k660 = 2 ± 0.42 · u + 0.94 ± 2.48
Adjusted
k660 = 2.27 ± 0.5 · u −3.29 ± 4.08
k660 = 2.28 ± 0.45 · u −0.63 ± 4.14 ed and unadjusted data sets of Knorr11 and SO234-2/235. The error estimates correspond to a 95 % confidence signed the new wind speed ualt, resulting in better agreement
to ZA18. The change of the linear fit to the unsuppressed and
suppressed data set can be seen in the dotted (before) and
dashed (after) line. The adjusted datapoints at 12–16 m s−1
are still, relative to the linear estimates, heavily gas trans-
fer suppressed. A reason could be that the significant wave
height of these points is larger than 3.5 m and they expe-
rienced high wind speed. A shielding of wind by the large
wave or an influence of water droplets on the momentum
transfer is suggested as another reason (Yang et al., 2016;
Bell et al., 2013). In principle, we agree that these processes
may be occurring, but only during exceptional cases of high
winds and wave heights. The Reynolds gas transfer suppres-
sion (Zavarsky et al., 2018) occurs over a wider range of wind
speeds and wave heights, but obviously does not capture all the flux suppression. Therefore, it appears that several pro-
cesses, including shielding and influence of droplets, may be
responsible for gas transfer suppression and they are not all
considered in our model. This marks the upper boundary for
environmental conditions for our model. www.atmos-chem-phys.net/19/1819/2019/ 1824 A. Zavarsky and C. A. Marandino: Gas transfer suppression mod A. Zavarsky and C. A. Marandino: Gas transfer suppression model 1824
A. Zavarsky and C. A. Marandino: Gas transfer suppression model
Figure 3. Adjustments to the Knorr11 DMS k vs. u relationship. The datapoints with |Retr| ≺6.96×105 were adjusted using the gas transfer
suppression model. Black circles denote k values at the original wind speed u10. Colored filled circles denote the k value at wind speed equal
to ualt. The color shows the significant wave height. If a datapoint has a concentric black and filled circle, it was not adjusted, as it was not
subject to gas transfer suppression. The solid black line is the ZA18 parameterization. The dotted line is the linear fit to the datapoints before
the adjustment; the dashed line is the linear fit after the adjustment. Figure 3. Adjustments to the Knorr11 DMS k vs. u relationship. The datapoints with |Retr| ≺6.96×105 were adjusted using the gas transfer
suppression model. Black circles denote k values at the original wind speed u10. Colored filled circles denote the k value at wind speed equal
to ualt. The color shows the significant wave height. If a datapoint has a concentric black and filled circle, it was not adjusted, as it was not
subject to gas transfer suppression. The solid black line is the ZA18 parameterization. The dotted line is the linear fit to the datapoints before
the adjustment; the dashed line is the linear fit after the adjustment. Figure 3. Adjustments to the Knorr11 DMS k vs. u relationship. The datapoints with |Retr| ≺6.96×105 were adjusted using the gas transfer
suppression model. Black circles denote k values at the original wind speed u10. Colored filled circles denote the k value at wind speed equal
to ualt. The color shows the significant wave height. If a datapoint has a concentric black and filled circle, it was not adjusted, as it was not
subject to gas transfer suppression. The solid black line is the ZA18 parameterization. The dotted line is the linear fit to the datapoints before
the adjustment; the dashed line is the linear fit after the adjustment. Table 1. Mean differences between the reference fits (column one) and the adjusted and unadjusted k data sets. A negative value describes
that the fit, on average, overestimates the actual measured data. 4.2
Nightingale parameterization FS14 datapoint showed an average wave height of 0.6 m and
wind speed of 4.7 m s−1. It is questionable if a flow separa-
tion and a substantial wind–wave interaction can be estab-
lished at this small wave height. This could mark the lower
boundary for the Reynolds gas transfer suppression model
(Zavarsky et al., 2018). Taking out either one or both of
these measurements (GB11 or FS14) changes the correlation
(Spearman’s rank) to −0.62 p = 0.0233 (excluding GB11),
−0.59 p = 0.033 (excluding FS14) and −0.79 p = 0.0025
(excluding GB11 and FS14). All three are significant. The
solid black line in Fig. 4b is a fit to all points except GB11
and FS14, and based on Eq. (15). We expect a negative correlation between the suppression
index and the relation of the individual measurement vs. the
N00 parameterization. The higher the suppression index, the
higher the gas transfer suppression and the lower the gas
transfer velocity k with respect to the average parameteri-
zation. The correlation (Spearman’s rank) is −0.43 with a
significance level (p value) of 0.11. This is not significant. However, we must take a closer look at two specific points:
(1) point 11, GB11 that shows low measurement percentage
despite a low suppression index, and (2) point 14, FS14 that
shows high measurement percentage despite a high suppres-
sion index. GB11 at the Georges Bank showed an average
significant wave height of 3.5 m, with a maximum of 6 m
and wind speed between 9 and 13 m s−1. Transformed wind
speeds utr are between 4 and 20 m s−1. As already discussed
in Sect. 4.1 using the Knorr11 data set, wave heights above
3.5 m could lead to gas transfer suppression without being
captured by the Reynolds gas transfer suppression model
(Zavarsky et al., 2018). High waves together with the strong
winds could mark an upper limit of the gas transfer suppres-
sion model (Zavarsky et al., 2018). On the other hand, the y(x) = a1 + a2 ·
1
x −a3
(15) (15) We choose this functional form and hypothesize that gas
transfer suppression is not linear, but rather has a threshold
(Zavarsky et al., 2018). This means that the influence of sup-
pression on gas transfer is relatively low with a small sup-
pression ratio, but increases strongly. 4.2
Nightingale parameterization The N00 parameterization is a quadratic wind-speed-
dependent parameterization of k. It is widely used, especially
for regional bulk CO2 gas flux calculations as well as for
DMS flux calculations in Lana et al. (2011). The parame-
terization is based on dual-tracer measurements in the water
performed in the North Sea (Watson et al., 1991; Nightingale
et al., 2000) as well as data from the Florida Strait (FS; Wan-
ninkhof et al., 1997) and Georges Bank (GB; Wanninkhof,
1992). )
We analyzed each individual measurement that was used
in the parameterization to assess the amount of gas transfer
suppressing instances that are within the N00 parameteriza-
tion. The single measurements, which are used for fitting the
quadratic function of the N00 parametrization, are shown to-
gether with N00 in Fig. 4a. As the measurement time of the
dual-tracer technique is on the order of days, we interpolated
the wind and wave data, obtained from the WWIII model for
the specific time and location, to 1 h time steps and calculated
the number of gas transfer suppressing and gas transfer non-
suppressing instances. Fig. 4b shows the suppression index,
which is the ratio of gas suppressing instances to the number
of datapoints (x axis). The value 1 indicates that all of the in-
terpolated 1 h steps were gas transfer suppressed. The y axis
of Fig. 4 depicts the relation of the individual measurements
to the N00 parameterization. A ratio (y axis) of 1 indicates
that the measurement point is exactly the same as the N00 pa-
rameterization. A value of 1.1 would indicate that the value
was 10 % higher than predicted by the N00 parameterization. Figure 4. Individual dual-tracer measurements that contribute to
the N00 (solid line) parameterization (a). The relationship of the
gas suppression ratio to the measurement and N00 ratio (b). The
solid line in (b) is a fit to the suppression to measurement and N00
relationship. A higher suppression ratio indicates a longer influence
of gas transfer suppression on the datapoint. The two red circles de-
note the outlier points that are discussed in the text. The solid black
line is a fit using the function y(x) = a1 + a2 ·
1
x−a3 . The fit coeffi-
cients are a1 = 1.52, a2 = 0.14 and a3 = 1.18. A. Zavarsky and C. A. Marandino: Gas transfer suppression model 1825 Figure 4. Individual dual-tracer measurements that contribute to
the N00 (solid line) parameterization (a). The relationship of the
gas suppression ratio to the measurement and N00 ratio (b). The
solid line in (b) is a fit to the suppression to measurement and N00
relationship. A higher suppression ratio indicates a longer influence
of gas transfer suppression on the datapoint. The two red circles de-
note the outlier points that are discussed in the text. The solid black
line is a fit using the function y(x) = a1 + a2 ·
1
x−a3 . The fit coeffi-
cients are a1 = 1.52, a2 = 0.14 and a3 = 1.18. function ZA18 k660 = 3.1±0.37·u10−5.37±2.35 (Zavarsky
et al., 2018), but the slopes barely overlap within the 95 %
confidence interval. www.atmos-chem-phys.net/19/1819/2019/ Table 1 shows the average offset between every datapoint
and the linear fit ZA18. A reduction of the average offset can
be seen for all data combinations. The last two columns of
Table 1 show the mean absolute error. The absolute error also
decreases with the application of our adjustments. The linear
fits to the two data sets, before and after the adjustments, are
given in Table 2. The slopes for the two altered data sets show a good agree-
ment. However, we do not account for the suppression en-
tirely. The adjusted slopes are both in the range of the linear www.atmos-chem-phys.net/19/1819/2019/ Atmos. Chem. Phys., 19, 1819–1834, 2019 Atmos. Chem. Phys., 19, 1819–1834, 2019 4.2
Nightingale parameterization It utilizes a global, annual
averaged, gas transfer velocity of 14C and relates it to re-
motely sensed wind speed. This means that the average gas
transfer velocity has experienced the average global occur-
rence of gas transfer suppression and therefore is incorpo-
rated into the k vs. u parameterization. The interesting point about this parameterization is that it
should already include a global average gas transfer sup-
pressing factor. The parametrization is independent of local
gas transfer suppression events. It utilizes a global, annual
averaged, gas transfer velocity of 14C and relates it to re-
motely sensed wind speed. This means that the average gas
transfer velocity has experienced the average global occur-
rence of gas transfer suppression and therefore is incorpo-
rated into the k vs. u parameterization. On average, the new parameterization is 22 % higher than
the original N00 parameterization. This increase is caused
by the heavy gas transfer suppression of the individual mea-
surements. As we believe that this suppression only affects
the interfacial ko gas exchange, it might not be easily visible
(decreasing k vs. u relationship) in parameterizations based
on dual-tracer gas transfer measurements, because of the po-
tential of a large bubble influence. The calculation of the unsuppressed N00 parameteriza-
tion is an example application for this adjustment algo-
rithm. We advise using the unsuppressed parameterization
(N00 + 22 %) for flux calculations with very insoluble gases
like SF6or3He. We hypothesize that the original N00 con-
tains a large bubble component, as it is based on SF6 and
3He measurements, which is compensated by the gas trans-
fer suppression. Therefore, the original N00 has been widely
used for regional CO2 gas flux calculations. The quadratic coefficient, a, is calculated by dividing the
averaged gas transfer velocity kglob by u2 and the wind dis-
tribution, distu, of u. a =
kglob
Pu2 · distu
(18) (18) The quadratic coefficient then defines the wind-speed-
dependent gas transfer velocity k (Eq. 19). k = a · u2
(19) k = a · u2 (19) 4.2
Nightingale parameterization The fit coefficients are
a1 = 1.52, a2 = 0.14 and a3 = 1.18. We choose this functional form and hypothesize that gas
transfer suppression is not linear, but rather has a threshold
(Zavarsky et al., 2018). This means that the influence of sup-
pression on gas transfer is relatively low with a small sup-
pression ratio, but increases strongly. The fit coefficients are
a1 = 1.52, a2 = 0.14 and a3 = 1.18. Figure 5 shows the unsuppressed datapoints, according to
the gas transfer suppression model (Sect. 3). We do not adjust
the individual datapoints along the wind speed axis (x axis),
as the parameterization has a significant bubble contribution,
but add 1k (Eq. 12) to make up for the suppressed part of
total k. Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ 1826 A. Zavarsky and C. A. Marandino: Gas transfer suppression mod A. Zavarsky and C. A. Marandino: Gas transfer suppression model 1826
A. Zavarsky and C. A. Marandino: Gas transfer suppression model
Figure 5. Adjusted individual measurements, comprising the N00 parameterization, resulting from the algorithm described in Sect. 3. The
difference between ualt and the original u10 was added to k using the linear parameterization ZA18, which accounts for the suppression of ko
due to wind–wave interaction. The solid black line is the original N00 parametrization. The red line is a new quadratic fit to the adjusted
datapoints k = 0.359 · u2. Figure 5. Adjusted individual measurements, comprising the N00 parameterization, resulting from the algorithm described in Sect. 3. The
difference between ualt and the original u10 was added to k using the linear parameterization ZA18, which accounts for the suppression of ko
due to wind–wave interaction. The solid black line is the original N00 parametrization. The red line is a new quadratic fit to the adjusted
datapoints k = 0.359 · u2. A new quadratic fit was applied to the adjusted datapoints
(Eq. 16, Fig. 5). fore in the proposed range (Wanninkhof, 2014). The W14 pa-
rameterization is given in Eq. (17). k660,W14 = 0.251 · (u10)2
(17) k660 = 0.359 · u2
(16) (17) k660 = 0.359 · u2 (16) The interesting point about this parameterization is that it
should already include a global average gas transfer sup-
pressing factor. The parametrization is independent of local
gas transfer suppression events. 4.3
Wanninkhof parameterization Additionally, we plotted the
occurrences split into ocean basins and northern and south-
ern hemispheres. Two trends are visible. There is a higher
percentage of gas transfer suppression in the Northern Hemi-
sphere and, on the time axis, the peak is in the respective (bo-
real and austral) summer season. The Southern Hemisphere
has a water-to-landmass ratio of 81 %, the Northern Hemi-
sphere’s ratio is 61 %. The area of free open water is there-
fore greater in the Southern Hemisphere. Gas transfer sup-
pression is favored by fully developed seas without remote
swell influence. In the Southern Hemisphere, the large open
ocean areas, where swell can travel longer distances, provide
an environment with less gas transfer suppression. The peak
in summer and minimum in winter can be associated with the
respective sea ice extent on the Northern Hemisphere and
Southern Hemisphere. Figure 7 shows that seas, which are
usually ice-covered in winter, have a high ratio of gas trans-
fer suppression. speed parametrization. A global average gas transfer veloc-
ity of kglob = 16.5 cm h−1 (Naegler, 2009) results in a coef-
ficient a = 0.2269, using the NCEP wind speed distribution. The value for a becomes 0.2439 with the ualt distribution. This is a 9.85 % increase. Our calculated value of a = 0.2269
differs from the W14 value of a = 0.251 because we use a
different wind speed distribution. The W14 uses a Rayleigh
distribution with σ = 5.83, our NCEP-derived σ = 6.04 and
the adjusted NCEP σ = 5.78. This means that the W14 uses
a wind speed distribution with a lower global average speed. However, for the estimation of a suppression effect we calcu-
late the difference between using the NCEP wind speed and
the adjusted wind speed distribution. For the calculation of
a, we did not use a fitted Rayleigh function but the adjusted
wind speed distribution from Fig. 6. A comparison of W14, N00 and the unsuppressed param-
eterizations is shown in Fig. 6b. N00 shows the lowest re-
lationship between u and k. W14 shows a parameterization
with a global-averaged gas transfer suppression influence and
is therefore slightly higher than N00. It appears that the gas
transfer suppression is overcompensating the smaller bubble-
mediated gas transfer of CO2 (W14). The unsuppressed N00
is significantly higher than the W14 + 9.85 %. 4.3
Wanninkhof parameterization The Fig. 6a shows the global wind speed distribution of the
year 2014 taken from the WWIII model, which is based on
the NCEP reanalysis. Additionally, we added the distribu-
tion taking our wind speed adjustment into account. At the
occurrence of gas transfer suppression, we calculated ualt as
the representative wind speed for the unsuppressed transfer,
as described in Sect. 3. The distribution of ualt shifts higher
wind speed (10–17 m s−1) to lower wind speed regimes (0–
7 m s−1). This alters the coefficient for the quadratic wind The W14 parameterization estimates the gas transfer veloc-
ity using the natural disequilibrium between ocean and at-
mosphere of 14C and the bomb 14C inventories. The total
global gas transfer over several years is estimated by the in-
flux of 14C in the ocean (Naegler, 2009) and the global wind
speed distribution over several years. The parameterization
from W14 is for winds averaged over several hours. The
WWIII model wind data, used here, are 3 hourly and there- Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ A. Zavarsky and C. A. Marandino: Gas transfer suppression model 1827 Figure 6. Wind speed distributions for the year 2014 (a). The solid line is NCEP-derived wind speed distribution, the dashed line the wind
speed distribution of the adjusted wind speed ualt. Comparison of original and adjusted k vs. wind speed parameterizations (b). Figure 6. Wind speed distributions for the year 2014 (a). The solid line is NCEP-derived wind speed distribution, the dashed line the wind
speed distribution of the adjusted wind speed ualt. Comparison of original and adjusted k vs. wind speed parameterizations (b). month in the year 2014. The average yearly global percent-
age is 18.6 %. The minimum is 15 % in March and April
and the maximum is 22 % in June–August. Coastal areas and
marginal seas seem to be more influenced than open oceans. The reason could be that gas transfer suppression is likely
to occur at developed wind seas when the wind speed is in
the same direction and magnitude as the wave’s phase speed. At coastal areas and marginal seas, the sea state is less in-
fluenced by swell and waves that were generated at a re-
mote location. Landmasses block swell from the open ocean
to marginal seas. The intra-annual variability of gas transfer
suppression is shown in Fig. 8. 4.3
Wanninkhof parameterization We hypoth-
esize that this difference is based on the different bubble-
mediated gas transfer of He, SF6, and CO2. 4.4
Global analysis We used the native global grid (0.5◦× 0.5◦) from the WWIII
for the global analysis. The datapoints from the DMS and
CO2 climatologies as well as all auxiliary variables were in-
terpolated to this grid. The global reduction of the CO2 and DMS flux is cal-
culated using Eqs. (13)–(14) and shown for every month in
Figs. 9 and 10. These magnitudes represent the reduction of
interfacial gas transfer due to gas transfer suppression. Most
areas with a reduced influx of CO2 into the ocean are in the Figure 7 shows the percentage of gas-transfer-suppressed
datapoints with respect to the total datapoints for every www.atmos-chem-phys.net/19/1819/2019/ www.atmos-chem-phys.net/19/1819/2019/ Atmos. Chem. Phys., 19, 1819–1834, 2019 1828 A. Zavarsky and C. A. Marandino: Gas transfer suppression model A. Zavarsky and C. A. Marandino: Gas transfer suppression model Figure 7. The global probability of experiencing gas transfer suppression during the respective month (2014). The percentage is the number
of gas transfer suppressed occurrences with respect to the total datapoints with a 3 h resolution. Figure 7. The global probability of experiencing gas transfer suppression during the respective month (2014). The percentage is the number
of gas transfer suppressed occurrences with respect to the total datapoints with a 3 h resolution. Table 3. 2014 DMS flux in teragrams. Retr indicates an applica-
tion of the gas transfer suppression model. The last two rows are
estimated from global climatologies. Northern Hemisphere. The only reduced CO2 influx areas
of the Southern Hemisphere are in the South Atlantic and
west of Australia and New Zealand. Significantly reduced
CO2 efflux areas are found in the northern tropical Atlantic,
especially in the boreal summer months, the northern Indian
Ocean and the Southern Ocean. The maximum monthly re-
duction of influx (oceanic uptake) is 18.7 mmol m−2 day−1. The maximum monthly reduction of efflux (oceanic out-
gassing) is 12.9 mmol m−2 day−1. Parameterization
Flux (Tg DMS yr−1)
N00
50.72
N00 Retr
45.47
ZA18
56.22
ZA18 Retr
51.07
Lana et al. (2011)
54.39
Lennartz et al. (2015)
45.5 The absolute values of DMS flux reduction (Fig. 9), due
to gas transfer suppression, coincide with the summer max-
imum of DMS concentration and therefore large air–sea
fluxes (Lana et al., 2011; Simó and Pedrós-Alió, 1999). The
northern Indian Ocean during boreal winter also shows a
high level (10 µmol m2 day−1) of reduction. The highest wa-
ter concentrations and fluxes in the Indian Ocean are found
in boreal summer (Lana et al., 2011), which is less influenced
by gas transfer suppression. for the year 2014. We use our estimations of ualt and
Eq. (14) to subtract gas transfer suppression from the orig-
inal N00 parameterization. The resulting reduced total emis-
sion is 45.47 Tg DMS yr−1, which is a reduction of 11 %. The linear parameterization ZA18 estimates an emission
of 56.22 Tg DMS yr−1. Using the gas transfer suppression
algorithm and Eq. (13), the global amount is reduced to
51.07 Tg DMS yr−1, which is a reduction of 11 %. Global for the year 2014. www.atmos-chem-phys.net/19/1819/2019/ We use our estimations of ualt and
Eq. (14) to subtract gas transfer suppression from the orig-
inal N00 parameterization. The resulting reduced total emis-
sion is 45.47 Tg DMS yr−1, which is a reduction of 11 %. The linear parameterization ZA18 estimates an emission
of 56.22 Tg DMS yr−1. Using the gas transfer suppression
algorithm and Eq. (13), the global amount is reduced to
51.07 Tg DMS yr−1, which is a reduction of 11 %. Global The DMS emissions from the ocean to the atmosphere
are shown in Table 3. The calculated total emission from
the original N00 parameterization is 50.72 Tg DMS yr−1 Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ Zavarsky and C. A. Marandino: Gas transfer suppression model 1829 A. Zavarsky and C. A. Marandino: Gas transfer suppression model Figure 8. The probability of experiencing gas transfer suppression during the respective month (2014) divided into ocean basins and hemi-
spheres. The Southern Ocean was added to the southern part of the respective ocean basin. The percentage is the number of gas transfer
suppressed instances with respect to the total datapoints with a 3 h resolution. Figure 8. The probability of experiencing gas transfer suppression during the respective month (2014) divided into ocean basins and hemi-
spheres. The Southern Ocean was added to the southern part of the respective ocean basin. The percentage is the number of gas transfer
suppressed instances with respect to the total datapoints with a 3 h resolution. Figure 9. The absolute change of CO2 gas transfer due to suppression for each month of 2014. Negative values (blue) denote areas where a
flux into the ocean is reduced by the shown value. Positive values denote areas where flux out of the ocean is reduced by the shown value. The change is calculated using the bulk flux formula (Eq. 1) and 1k (Eq. 12). Figure 9. The absolute change of CO2 gas transfer due to suppression for each month of 2014. Negative values (blue) denote areas where a
flux into the ocean is reduced by the shown value. Positive values denote areas where flux out of the ocean is reduced by the shown value. The change is calculated using the bulk flux formula (Eq. 1) and 1k (Eq. 12). Atmos. Chem. Phys., 19, 1819–1834, 2019 Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ 1830 A. Zavarsky and C. A. Marandino: Gas transfer suppression model A. Zavarsky and C. A. Marandino: Gas transfer suppression model Figure 10. The absolute change of DMS gas transfer due to suppression for each month of 2014. The shown magnitudes denote the reduction
by gas transfer suppression. The change is calculated using the bulk flux formula (Eq. 1) and 1k (Eq. 12). Figure 10. The absolute change of DMS gas transfer due to suppression for each month of 2014. The shown magnitudes denote the reduction
by gas transfer suppression. The change is calculated using the bulk flux formula (Eq. 1) and 1k (Eq. 12). estimates are 54.39 Tg DMS yr−1 (Lana et al., 2011) and
45.5 Tg DMS yr−1 (Lennartz et al., 2015). As stated above,
a difference in wind speed or sea ice coverage could be the
reason for the difference in the global emission estimated be-
tween the Lana climatology and our calculations with the
N00 parameterization. Lennartz et al. (2015) use the water
concentrations from the Lana climatology, but include air-
side DMS concentrations, which reduces the flux by 17 %. We do not include air-side DMS concentrations but gas trans-
fer suppression, which reduces the flux by 11 %. We can
expect a reduction of 20 %–30 % when including both pro-
cesses. direction) of the ocean waves have to be known or retrieved
from wave models. The calculation is iterative and can be
easily implemented. The effect of this adjustment is shown
with two data sets from the Knorr11 (Bell et al., 2017) and
the SO234-2/235 cruises (Zavarsky et al., 2018). Both data
sets show, after the adjustment, a better agreement with the
linear ZA18 parameterizations (Tables 1 and 2), which only
contains unsuppressed gas transfer velocity measurements
from the SO 234-2/235 cruise. Generally, the adjustments
may be only applied to the interfacial gas transfer velocity ko. direction) of the ocean waves have to be known or retrieved
from wave models. The calculation is iterative and can be
easily implemented. The effect of this adjustment is shown
with two data sets from the Knorr11 (Bell et al., 2017) and
the SO234-2/235 cruises (Zavarsky et al., 2018). Both data
sets show, after the adjustment, a better agreement with the
linear ZA18 parameterizations (Tables 1 and 2), which only
contains unsuppressed gas transfer velocity measurements
from the SO 234-2/235 cruise. www.atmos-chem-phys.net/19/1819/2019/ Generally, the adjustments
may be only applied to the interfacial gas transfer velocity ko. We investigated the individual measurements leading to
the N00 gas transfer parameterization for the influence of
gas transfer suppression. We think that the overall parame-
terization is heavily influenced by gas transfer suppression,
but the suppression is likely masked by bubble-mediated gas
transfer, due to the solubility of the dual-tracer measurement
gases. We show a significant negative correlation between
the occurrence of gas transfer suppression and the ratio of
the individual measurements to the N00 parameterization. We applied an adjustment due to gas transfer suppression
and fitted a new quadratic function to the adjusted data set. www.atmos-chem-phys.net/19/1819/2019/ A. Zavarsky and C. A. Marandino: Gas transfer suppression model A. Zavarsky and C. A. Marandino: Gas transfer suppression model Using the Retr parameter, one can evaluate if a flux mea-
surement or flux calculation is influenced by gas transfer
suppression. For unsuppressed conditions and rather solu-
ble gases, such as DMS, we recommend the use of a lin-
ear parameterization (e.g., ZA18). For gases with a simi-
lar solubility as CO2, we recommend the use of the ad-
justed W14 + 9.85 % parameterization. The adjusted N00
(N00 + 22 %) parameterization is recommended for very in-
soluble gases. In case of gas transfer suppression, we rec-
ommend the previous parameterizations together with our it-
erative approach to adjust u to ualt (Fig. 1) with the use of
Eqs. (13)–(14). For global calculations, we recommend the
use of the Wanninkhof parameterizations W14 (Wanninkhof,
2014), as it already has an average global gas transfer sup-
pression included. The new parameterization is on average 22 % higher than the
original N00 parameterization. This leads to the conclusion
that gas transfer suppression influences gas transfer param-
eterizations, even if it is not directly visible, via a smaller
slope. Asher and Wanninkhof (1998) state that SF6/3He gas
transfer measurements could lead to a 23 % overestimation
of CO2 gas transfer velocities. After adjusting of N00 for gas
transfer suppression, the difference between gas transfer ve-
locities of the original N00 and the adjusted version closely
matches this estimation. For the W14 parameterization we used a global wind
speed climatology for the year 2014 and applied the gas
transfer suppression model u10 →ualt. Using the distribution
function of ualt we calculated an unsuppressed gas transfer
parameterization. The coefficient of the unsuppressed param-
eterization is 9.85 % higher than the original one. W14 al-
ready includes the global average of gas transfer suppres-
sion. Therefore the increase, due to the adjustment, is ex-
pected to be less than the one for N00, which is strongly
suppressed. The original N00 is lower than W14, but after
adjustment N00 is larger than the unsuppressed W14, which
is expected due to the larger bubble-mediated gas transfer
of He and SF6 over CO2. Data availability. The wave data are available at the website of the
NOAA Environmental Modeling Center. The ERA-Interim data are
available at the website of the ECMWF. The data are stored at the
data portal of GEOMAR Kiel. 5
Conclusions We provide a first approach to adjust k values for the gas
transfer suppression due to wind–wave interaction (Zavarsky
et al., 2018) and therefore to account for the effect of this sup-
pression. Retr and the resulting alternative wind speed ualt
can be calculated from standard meteorological and oceano-
graphic variables. Additionally, the condition (period, height, www.atmos-chem-phys.net/19/1819/2019/ Atmos. Chem. Phys., 19, 1819–1834, 2019 1831 A. Zavarsky and C. A. Marandino: Gas transfer suppression model We think that gas transfer suppression has a global influ-
ence on air–sea gas exchange of 10 %–11 %. These numbers
are supported by the adjustment of the W14 parametriza-
tion as well as a global DMS gas transfer calculation. Local
conditions may lead to much higher influences. Gas trans-
fer velocity parameterizations from regional data sets might
be heavily influenced by gas transfer suppression. We have
shown this for the N00 parameterization. This should be con-
sidered with their use. Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ Data availability. The wave data are available at the website of the
NOAA Environmental Modeling Center. The ERA-Interim data are
available at the website of the ECMWF. The data are stored at the
data portal of GEOMAR Kiel. www.atmos-chem-phys.net/19/1819/2019/ A. Zavarsky and C. A. Marandino: Gas transfer suppression model A. Zavarsky and C. A. Marandino: Gas transfer suppression model 1832 Figure B1. Illustration of the gas transfer suppression adjustments
either along the wind speed or gas transfer velocity axis. Figure A1. The streamlined shape of a wave (cylindrical half
sphere) that experiences wind flowing over it from various angles
θ. Figure A1. The streamlined shape of a wave (cylindrical half
sphere) that experiences wind flowing over it from various angles
θ. Figure A1. The streamlined shape of a wave (cylindrical half
sphere) that experiences wind flowing over it from various angles
θ. Figure B1. Illustration of the gas transfer suppression adjustments
either along the wind speed or gas transfer velocity axis. Appendix A: Directional dependencies Figure A1 shows the shape of the wave (half sphere) as ex-
perienced by the wind flowing over it with a certain angle θ. The larger θ, the more streamlined the wave (half sphere). The more streamlined the wave, the more difficult it is to gen-
erate turbulence; this counteracts the flow detachment and as
a consequence gas transfer suppression occurs. Appendix B: Adjustment of wind speed or adjustment
of k A shift on the x axis from u10 to ualt is equivalent to an in-
crease in k by 1k, when related to a linear relationship. We
use the ZA18 parameterization as a reference (Eq. 12), which
is a linear relationship describing ko, as gas transfer sup-
pression only affects interfacial gas transfer. Figure B1 illus-
trates the two different possibilities of adjusting suppressed
gas transfer values. Wind at an angle of θ = 90◦does not experience a wave
crest or trough, but rather an along-wind corrugated sur-
face. In this case there should be no gas transfer suppres-
sion. Zavarsky et al. (2018) predict a unsuppressed condition
around Retr = 0, which coincides with θ ≈90◦or utr →0. Both conditions rarely occur and must be investigated in the
future. The adjustments of the two DMS data sets (SO234-2/235
and Knorr11) are done by shifting u10 along the x axis
to ualt. We want to test whether u10 can be directly replaced
by ualt for ko parameterizations. Gas transfer suppression ad-
justments for bubble-influenced parameterizations are done
by adding 1k, which is directly related to the difference
1u = u10 −ualt. Atmos. Chem. Phys., 19, 1819–1834, 2019 www.atmos-chem-phys.net/19/1819/2019/ Atmos. Chem. Phys., 19, 1819–1834, 2019 References Naegler, T.: Reconciliation of excess
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vided and collected the data. AZ prepared the manuscript with con-
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Surfaces, 2010, Kyoto University, available at: http://hdl.handle. net/2433/156156 (last access: 5 January 2018), 2011. Acknowledgements. The authors thank Kirstin Krüger, the chief
scientist of the R/V Sonne cruise (SO234-2/235), as well as
the captain and crew. We thank the Environmental Modeling
Center at the NOAA/National Weather Service for providing the
WAVEWATCH III® data. We thank the European Centre for
Medium-Range Weather Forecasts for providing the ERA-Interim
data. This work was carried out under the Helmholtz Young
Investigator Group of Christa A. Marandino, TRASE-EC (VH-NG-
819), from the Helmholtz Association. The cruise 234-2/235 was
financed by the BMBF, 03G0235A. Lana, A., Bell, T. G., Simo, R., Vallina, S. M., Ballabrera-Poy,
J., Kettle, A. J., Dachs, J., Bopp, L., Saltzman, E. S., Ste-
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Partial trisomy 21 map: Ten cases further supporting the highly restricted Down syndrome critical region (HR‐DSCR) on human chromosome 21
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European Journal of Human Genetics (2019) 27:1–688
https://doi.org/10.1038/s41431-019-0404-7 European Journal of Human Genetics (2019) 27:1–688
https://doi.org/10.1038/s41431-019-0404-7 MEETING ABSTRACTS June 16–19, 2018, Fiera Milano Congressi, Milan Italy Sponsorship: Publication of this supplement was sponsored by the European Society of Human Genetics. All content was
reviewed and approved by the ESHG Scientific Programme Committee, which held full responsibility for the abstract
selections. Disclosure Information In order to help readers form their own judgments of potential bias in published abstracts, authors
are asked to declare any competing financial interests. Contributions of up to EUR 10 000.- (Ten thousand Euros, or equivalent value in kind) per year per company are considered
“Modest”. Contributions above EUR 10 000.- per year are considered “Significant”. Abstracts from the 51st European Society of Human Genetics
Conference: Posters © European Society of Human Genetics 2019 P01.01A Results: Analytical sensitivity was 96.00%(95%CI:80.46-
99.28) (24/25) and specificity was 99.76%(95%CI:98.67-
99.96) (422/423) not statistically different from that reported
in a previous clinical validation/verification (OR 0.12;95%
CI:0.01-0.97 and OR 0.54;95%CI:0.07-4.31). The specifi-
city must be considered a lower-bound estimate as the
sample classified as “false positives” may be a true positive
from an undiagnosed mother or affected fetus. P01 Reproductive Genetics/Prenatal Genetics 22q11.2DS were tested. This study was approved by the
laboratory IRB. All samples were de-identified before study. 22q11.2DS were tested. This study was approved by the
laboratory IRB. All samples were de-identified before study. Internal analytical verification of a targeted microarray-
based cell-free DNA test for 22q11.2 deletion Internal analytical verification of a targeted microarray-
based cell-free DNA test for 22q11.2 deletion F. R. Grati, L. Marcato, B. Malvestiti, S. Crippa, L. Martinoni,
V. Zanatta, S. Saragozza, B. Grimi, F. Maggi, G. Simoni F. R. Grati, L. Marcato, B. Malvestiti, S. Crippa, L. Martinoni,
V. Zanatta, S. Saragozza, B. Grimi, F. Maggi, G. Simoni TOMA, Advanced Biomedical Assays S.p.A., Busto Arsizio,
Italy Conclusions: We have verified the internal analytical
performances of a targeted microarray-based cfDNA test for
22q11.2 deletions inside the typical 3Mb region in the
decentralized laboratory match the performance specifica-
tion of the source laboratory. The low FPR, below 0.5%, for
this cfDNA test expansion is critical when testing low-risk
population as it highly impacts PPV. Objectives: Laboratories are required to verify their assays
determining that the test is being performed correctly, even
if kit/software are CE-IVD marked with suitable perfor-
mance specifications or a new test is implemented using a
technology that is already well established in a source/
reference laboratory. This study presents the internal ana-
lytical verification after the implementation of 22q11.2DS
cfDNA test by a targeted microarray-based technology in an
independent decentralized European laboratory. F.R. Grati: D. Speakers Bureau/Honoraria (speakers
bureau, symposia, and expert witness); Modest; Roche. F. Consultant/Advisory Board; Modest; Roche. L. Marcato:
None. B. Malvestiti:
None. S. Crippa:
None. L. Martinoni: None. V. Zanatta: None. S. Saragozza: None. B. Grimi: None. F. Maggi: E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; TOMA Advanced Biomedical Assays S.p.A. G. Simoni: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Significant; TOMA
Advanced Biomedical Assays S.p.A. F.R. Grati: D. Speakers Bureau/Honoraria (speakers
bureau, symposia, and expert witness); Modest; Roche. F. Consultant/Advisory Board; Modest; Roche. L. Marcato: Methods:
Analytical
sensitivity:
25
samples
with
22q11.2DS were tested (2 maternal plasma and 23 simulated
pregnancy samples). Deletions spanned through the A-D
22q11.2 LCRs, sizes ranged from 2.37 and 2.89Mb and
simulated fetal fractions ranged from 7 to 39%. Analytical
specificity: 423 prospectively ascertained maternal plasma
samples
with
no
known
diagnosis
of
fetal/maternal Methods:
Analytical
sensitivity:
25
samples
with
22q11.2DS were tested (2 maternal plasma and 23 simulated
pregnancy samples). Deletions spanned through the A-D
22q11.2 LCRs, sizes ranged from 2.37 and 2.89Mb and
simulated fetal fractions ranged from 7 to 39%. Analytical
specificity: 423 prospectively ascertained maternal plasma
samples
with
no
known
diagnosis
of
fetal/maternal J. del Picchia 2 Washington Univ School of Medicine in St. Louis, St. Louis,
MO, United States Washington Univ School of Medicine in St. Louis, St. Louis,
MO, United States Materials and Methods: A total of 570 spontaneous
miscarriage cases were collected between 2002 and 2017,
referred to our laboratory from all over the country. DNA
was extracted from placental or fetal tissue. Samples that
did not contain tissue, appropriate for DNA analysis (7%)
were excluded. QF-PCR focused on chromosomes 13, 18,
21, X and Y was performed on 530 samples. One hundred
of them were additionally analyzed for aneuploidies
involving chromosomes 15, 16 and 22. Introduction: The atypical chemokine receptor 3 (ACKR3)
is highly expressed in vascularized structures including the
placenta
and
umbilical
cord. Aberrant
expression
of
ACKR3/ligands resulting in dysregulation of trophoblast-
endometrial interaction has been implicated in recurrent
pregnancy loss, intrauterine growth restriction, preterm
labor, and preeclampsia. Copy number variations (CNV’s)
overlapping with ACKR3 have been documented in patients
with
variable
findings
including
neurodevelopmental
defects (NDD’s), and in 0.01% of the healthy population
(DGV). No CNV’s involving ACKR3 have been reported in
pregnancy loss. Here we present our novel data from our
retrospective
chromosome
microarray
analysis
(CMA)
associating
duplications
of
ACKR3
with
spontaneous
abortions (SAB’s) and intrauterine fetal demise (IUFD). Introduction: The atypical chemokine receptor 3 (ACKR3)
is highly expressed in vascularized structures including the
placenta
and
umbilical
cord. Aberrant
expression
of
ACKR3/ligands resulting in dysregulation of trophoblast-
endometrial interaction has been implicated in recurrent
pregnancy loss, intrauterine growth restriction, preterm
labor, and preeclampsia. Copy number variations (CNV’s)
overlapping with ACKR3 have been documented in patients
with
variable
findings
including
neurodevelopmental
defects (NDD’s), and in 0.01% of the healthy population
(DGV). No CNV’s involving ACKR3 have been reported in
pregnancy loss. Here we present our novel data from our
retrospective
chromosome
microarray
analysis
(CMA)
associating
duplications
of
ACKR3
with
spontaneous
abortions (SAB’s) and intrauterine fetal demise (IUFD). Results: Aneuploidy was found in 117 (22% of the
cases). Thirty-five of them (29.9%) were with triploidy, 27
(23%) - with trisomy 21, 24 (20%) - with monosomy X, 17
(14%) - with trisomy 13 and 14(11.9%) - with trisomy 18. Additionally, trisomy 15 was found in 2 cases, trisomy 16 -
in 4 and trisomy 22 in 3 cases out from 100. Conclusions: QF-PCR analysis, being rapid and cost-
efficient proved to be useful for genetic analysis of
miscarriage
samples. 1qGenomics Laboratory, Esplugues de Llobregat, Spain,
2Laboratori Citogenètica Molecular. Servei de Medicina
Genètica i Molecular. Instituto Pediátrico de Enfermedades
Raras (IPER). Hospital Sant Joan de Déu, Barcelona, Spain,
3Universitat Pompeu Fabra, Hospital del Mar - IMIM, and
CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain,
4Women’s and Children’s Health Network and University of
Adelaide, Adelaide, Australia Washington Univ School of Medicine in St. Louis, St. Louis,
MO, United States Although
the
vast
majority
of
chromosomal aneuploidies in miscarriages are de novo,
such results provide valuable information for the clinicians
and genetic counselors and for the couple. Materials
and
Methods:
Retrospective
CMA
was
performed from ~4,700 samples to identify CNV’s over-
lapping with ACKR3 (aka CXCR7; 2q37.3), of which ~350
were products of conception (POC). Results: This study revealed 11 CNV’s overlapping with
ACKR3. Nine gains were detected from six SAB’s and three
IUFD (7 males, 2 females) ranging from small intervals
(n = 4, 36.8-65.5 kb) to trisomy 2 (n = 4) and tetrasomy 2
(n = 1). These gains account for 0.19% of all CMA cases and
2.6% of all POC cases. A 610 kb gain and a 6.1 Mb loss were
observed from blood samples of two patients with NDD’s. R. Raynova: None. S. Andonova: None. S. Bichev:
None. I. Bradinova: None. V. Dimitrova: None. M. Tzankova: None. S. Savova: None. A. Savov: None. P01.06B Clinical implementation of a custom oligonucleotide array-
CGH. Experience in the largest cohort of Spanish prenatal
samples (>3400 samples) Clinical implementation of a
custom oligonucleotide array-CGH. Experience in the
largest cohort of Spanish prenatal samples
(>3400 samples) Conclusions: This study is the first to report rare
duplications of ACKR3 in SAB’s and IUFD, providing
additional information to existing genomics data. Findings
from this study may aid in enhanced understanding of
ACKR3’s
role
in
pregnancy,
genetic
counseling
and
management of pregnancy loss. O. Villa1, M. Viñas1, P. Muñoz1, L. Vila1, C. Hernando1,2,
N. Fornés1, S. Cano1, A. Zurano1, M. García-Aragonés1,
L. Pérez-Jurado1,3,4, L. Armengol1 I. Amarillo: None. V. Wesevich: None. M. Smith:
None. D. Gray: None. 1University Hospital of Obstetrics and Gynecology, National
Genetic Laboratory, Sofia, Bulgaria, 2University Hospital of
Obstetrics and Gynecology, Sofia, Bulgaria P01.013C
Potential role of ACKR3/CXCR7 duplication in pregnancy
loss Introduction: Spontaneous loss of pregnancy before the
fetus reaches viability is the most frequent pregnancy
complication. The term miscarriage includes all pregnancy
losses from the time of conception until 20 weeks of
gestation. According to published data about 50% of first-
trimester pregnancy losses are the consequence of fetal
chromosomal abnormalities. Most of these abnormalities
are numerical (86%). I. Amarillo, V. Wesevich, M. Smith, D. Gray L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1,
T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4,
M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1 L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1,
T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4,
M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1 L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1,
T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4,
M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1 Introduction: The implementation of genomic array in
prenatal routines, when accompanied by pre- and post-test
genetic counselling, has demonstrated its utility by fulfilling
the longstanding need for a diagnostic test with a higher
resolution and higher diagnostic yield than its predecessor,
the conventional karyotype. 1Service of Genetic Medicine, University Hospitals of Geneva,
Geneva, Switzerland, 2Department of Genetic Medicine and
Development, University of Geneva, Geneva, Switzerland,
3Gynecology and obstetric private clinic, Geneva, Switzerland,
4Department of gynecology and obstetrics, University
Hospitals of Geneva, Geneva, Switzerland Materials and methods: Array CGH was performed in
3.438 prenatal samples (2.604 amniotic fluids, 728 corions
and
106
fetal
samples),
using
a
custom
60K
oligonucleotide-based microarray (qChip® CM) designed
to maximize the detection of clinically relevant copy-
number alterations, and minimize the detection of variants
of unknown significance (VOUS). As a general rule, VOUS
with unclear phenotypic effect according to current knowl-
edge, and some susceptibility variants are not reported. Materials and methods: Array CGH was performed in
3.438 prenatal samples (2.604 amniotic fluids, 728 corions
and
106
fetal
samples),
using
a
custom
60K
oligonucleotide-based microarray (qChip® CM) designed
to maximize the detection of clinically relevant copy-
number alterations, and minimize the detection of variants
of unknown significance (VOUS). As a general rule, VOUS
with unclear phenotypic effect according to current knowl-
edge, and some susceptibility variants are not reported. Introduction: Prenatal ultrasound allows the detection of
fetal malformation syndromes which often remain without
conclusive diagnosis. In case of a recurrent fetal phenotype
an autosomal recessive disorder is suspected. Introduction: Prenatal ultrasound allows the detection of
fetal malformation syndromes which often remain without
conclusive diagnosis. In case of a recurrent fetal phenotype
an autosomal recessive disorder is suspected. Results: We identified a total of 247 pathogenic or
probably pathogenic alterations (detection rate: 7.18%) and
60 VOUS (1.74%). As expected, the greatest pathogenic
detection rate (7.66%, 202/2637) was among fetuses with
ultrasound anomalies, while detection rate was 5.61% (45/
801) in ecografically normal gestations (2.24%, 18/801 only
with altered maternal serum screning). The vast majority of
the VOUS were inherited from a non-affected parent
(89.65%) and could be reclassified as most likely benign. L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1,
T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4,
M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1 Conclusions: Targeted WES is an effective tool for
mutation detection in rare autosomal recessive disorders
causing recurrent undiagnosed fetal phenotypes, allowing
accurate recurrence risk counseling and early prenatal
diagnosis for future pregnancies. L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1,
T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4,
M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1 Materials and Methods: We report on 2 families with
recurrent fetal anomalies: -Family A: Consanguineous couple from Afghanistan,
who experienced 2 terminated pregnancies due to severe
brain malformation with hydrocephalus and absence of
cerebellar vermis. -Family B: Non-consanguineous couple from Kosovo
who experienced 2 perinatal deaths after pregnancies
marked by third trimester polyhydramnios and fetal
akinesia. Both
newborns
presented
at
post-mortem
examination distal joints flexion contractures. Conclusions: Our series reinforces the clinical utility of
prenatal microarray testing: it nearly doubles the diagnostic
yield of conventional karyotype (110/247 with variants
<10Mb), with no significant increase in the frequency of
VOUS that could interfere in decision making. In our
experience, we highlight the importance of implementing
aCGH in prenatal routines (for all gestations with an
indication of invasive fetal sampling). We performed on stored fetal DNA whole exome
sequencing (WES) with targeted bioinformatic analysis. In
family A, we analyzed genes associated with brain
malformation and in family B genes known to cause
arthrogryposis and fetal akinesia. O. Villa: A. Employment (full or part-time); Significant;
qGenomics Laboratory. M. Viñas: None. P. Muñoz: A. Employment (full or part-time); Significant; qGenomics
Laboratory. L. Vila: A. Employment (full or part-time);
Significant; qGenomics Laboratory. C. Hernando: A. Employment (full or part-time); Significant; qGenomics
Laboratory. N. Fornés: A. Employment (full or part-time);
Significant; qGenomics Laboratory. S. Cano: A. Employ-
ment (full or part-time); Significant; qGenomics Laboratory. A. Zurano: A. Employment (full or part-time); Significant;
qGenomics Laboratory. M. García-Aragonés: A. Employ-
ment (full or part-time); Significant; qGenomics Laboratory. L. Pérez-Jurado: A. Employment (full or part-time);
Significant; qGenomics Laboratory. L. Armengol: A. Employment (full or part-time); Significant; qGenomics
Laboratory. Results: In family A, we found a novel nonsense
homozygous mutation in LARGE1 causing a rare form of
congenital muscular dystrophy-dystroglycanopathy with
brain and eye anomalies (OMIM: 613154) and in family
B a novel nonsense homozygous mutation in GLDN
causing lethal congenital contracture syndrome 11 (OMIM:
617194). In both families, Sanger sequencing confirmed
homozygosity in the proband and in the second affected
fetus, and also confirmed carrier status in both parents. Based on these results, we were able to offer invasive
prenatal testing for both families. Conclusions: Targeted WES is an effective tool for
mutation detection in rare autosomal recessive disorders
causing recurrent undiagnosed fetal phenotypes, allowing
accurate recurrence risk counseling and early prenatal
diagnosis for future pregnancies. P01.05A 1qGenomics Laboratory, Esplugues de Llobregat, Spain,
2Laboratori Citogenètica Molecular. Servei de Medicina
Genètica i Molecular. Instituto Pediátrico de Enfermedades
Raras (IPER). Hospital Sant Joan de Déu, Barcelona, Spain,
3Universitat Pompeu Fabra, Hospital del Mar - IMIM, and
CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain,
4Women’s and Children’s Health Network and University of
Adelaide, Adelaide, Australia Aneuploidy rate in 530 miscarriage cases R. Raynova1, S. Andonova1, S. Bichev1, I. Bradinova1,
V. Dimitrova2, M. Tzankova2, S. Savova2, A. Savov1 R. Raynova1, S. Andonova1, S. Bichev1, I. Bradinova1,
V. Dimitrova2, M. Tzankova2, S. Savova2, A. Savov1 R. Raynova1, S. Andonova1, S. Bichev1, I. Bradinova1,
V. Dimitrova2, M. Tzankova2, S. Savova2, A. Savov1 1University Hospital of Obstetrics and Gynecology, National
Genetic Laboratory, Sofia, Bulgaria, 2University Hospital of
Obstetrics and Gynecology, Sofia, Bulgaria 3 Abstracts from the 51st European Society of Human Genetics Conference: Posters Antenatal presentation of Bardet-Biedl syndrome: the
question of phenotype-genotype correlations Muller1,2 1Department of Experimental and Clinical Biomedical
Sciences “Mario Serio”, Centre of Excellence DeNo, Florence,
Italy, 2Andrology Department, Fundació Puigvert, Universitat
Autònoma de Barcelona, Instituto de Investigaciones
Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Spain,
3Genetics Department and Biomedical Reseach Institute,
Hospital de Sant Pau, Center for Biomedical Research on Rare
Diseases (CIBERER), and Department of Genetics and
Microbiology, Universitat Autònoma de Barcelona, Barcelona,
Spain, 4Hospital Germans Trias i Pujol, Badalona, Spain 1Laboratoire de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France,
2Laboratoire de Génétique médicale, UMR_S INSERM U1112,
IGMA, Faculté de Médecine FMTS, Université de Strasbourg,
Strasbourg, France, 3Complex Systems and Translational
Bioinformatics, ICube, University of Strasbourg, CNRS,
Illkirch, France, 4: Gynécologie-obstétrique, centre de
dépistage anténatal, hôpital Maison-Blanche, CHU de Reims,
Reims, France, 5Institut d'Histologie, Icube, UMR7357,
Université de Strasbourg, Strasbourg, France, 6Service de
Pathologie, UF6349 Fœtopathologie, Hôpitaux Universitaires
de Strasbourg, Strasbourg, France, 7INSERM U1163, Institut
IMAGINE, Paris, Paris, France, 8Service d'Histologie-
Embryologie-Cytogénétique, Hôpital Necker-Enfants Malades,
Assistance Publique-Hôpitaux de Paris, Paris, France,
9Laboratoire de Cardiogénétique, Malformations cardiaques
congénitales, Hôpitaux Civils de Lyon, Lyon, France,
10Anatomie et Cytologie Pathologiques, Hôpital Edouard
Herriot, Hôpitaux Civils de Lyon, Lyon, France,
11Département de pathologie, centre hospitalier Est, Hôpitaux
Civils de Lyon, Lyon, France, 12Service d'Anatomie-Cytologie
Pathologique, Centre Hospitalier Universitaire de Bordeaux,
Bordeaux, France, 13Service de Biologie du Développement,
Hôpital Robert Debré, Assistance Publique-Hôpitaux de Paris,
Paris, France, 14Service de Pathologie, Hôpital Jeanne de
Flandres, Centre Hospitalier Régional Universitaire de Lille,
Lille, France, 15Service de Génétique Médicale, Centre
Hospitalier Universitaire de Poitiers, Poitiers, France, 16Unité
de Génétique Médicale et Cytogénétique, Centre Hospitalier
Universitaire de Nîmes, Nîmes, France, 17Service d'Anatomie
Pathologique, Hôpital Pontchaillou, Université Rennes 1,
Rennes, France, 18Unité de Fœtopathologie, Hôpital Antoine
Béclère, Assistance Publique-Hôpitaux de Paris, Clamart,
France, 19Unité de fœtopathologie, Service de Génétique
Médicale, Centre Hospitalier Universitaire de Montpellier,
Montpellier, France, 20Clinique de Génétique Guy Fontaine, 1Laboratoire de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France,
2Laboratoire de Génétique médicale, UMR_S INSERM U1112,
IGMA, Faculté de Médecine FMTS, Université de Strasbourg,
Strasbourg, France, 3Complex Systems and Translational
Bioinformatics, ICube, University of Strasbourg, CNRS,
Illkirch, France, 4: Gynécologie-obstétrique, centre de
dépistage anténatal, hôpital Maison-Blanche, CHU de Reims,
Reims, France, 5Institut d'Histologie, Icube, UMR7357,
Université de Strasbourg, Strasbourg, France, 6Service de
Pathologie, UF6349 Fœtopathologie, Hôpitaux Universitaires
de Strasbourg, Strasbourg, France, 7INSERM U1163, Institut
IMAGINE, Paris, Paris, France, 8Service d'Histologie-
Embryologie-Cytogénétique, Hôpital Necker-Enfants Malades,
Assistance Publique-Hôpitaux de Paris, Paris, France,
9Laboratoire de Cardiogénétique, Malformations cardiaques
congénitales, Hôpitaux Civils de Lyon, Lyon, France,
10Anatomie et Cytologie Pathologiques, Hôpital Edouard Background: The etiology of non-obstructive azoospermia
(NOA) remains unknown in about 40% of cases and a
genetic origin is likely to be involved in idiopathic NOA. P01.09A C. Krausz1, A. Riera-Escamilla2, C. Chianese2, D. Moreno-
Mendoza2, O. Rajmil2, M. Bogliolo3, I. Blanco4, E. Ars2, E. Ruiz-
Castañé2, J. Surrallés3 Antenatal presentation of Bardet-Biedl syndrome: the
question of phenotype-genotype correlations Genes implicated in stem cell proliferation and DNA repair
may cause isolated NOA or be responsible for syndromic
diseases, such as Fanconi Anemia (FA). In about 10% of
FA cases the diagnosis is delayed until adulthood when a
malignant tumor is diagnosed. Methods:
Whole-Exome
Sequencing
(WES)
in
an
idiopathic NOA patient (index case) with consanguineous
parents. Sanger sequencing of the FANCA gene in the
brother of the index case and in 27 selected NOA patients. DEB-induced chromosome breakage test. Results: a rare pathogenic homozygous FANCA variant
(c.2639G>A) was identified in the index case, affected by
Sertoli Cell only syndrome. The patient’s brother (also with
NOA) carried the same genotype. The two brothers did not
manifest overt anemia, though chromosomal breakage test
confirmed FA. In 27 selected NOA patients with similar
testicular phenotype and borderline/mild hematological altera-
tions revealed one additional SCOS patient with compound
heterozygosis in FANCA (c.3788_3790delTCT;c.3913C>T). Herriot, Hôpitaux Civils de Lyon, Lyon, France,
11Département de pathologie, centre hospitalier Est, Hôpitaux
Civils de Lyon, Lyon, France, 12Service d'Anatomie-Cytologie
Pathologique, Centre Hospitalier Universitaire de Bordeaux,
Bordeaux, France, 13Service de Biologie du Développement,
Hôpital Robert Debré, Assistance Publique-Hôpitaux de Paris,
Paris, France, 14Service de Pathologie, Hôpital Jeanne de
Flandres, Centre Hospitalier Régional Universitaire de Lille,
Lille, France, 15Service de Génétique Médicale, Centre
Hospitalier Universitaire de Poitiers, Poitiers, France, 16Unité
de Génétique Médicale et Cytogénétique, Centre Hospitalier
Universitaire de Nîmes, Nîmes, France, 17Service d'Anatomie
Pathologique, Hôpital Pontchaillou, Université Rennes 1,
Rennes, France, 18Unité de Fœtopathologie, Hôpital Antoine
Béclère, Assistance Publique-Hôpitaux de Paris, Clamart,
France, 19Unité de fœtopathologie, Service de Génétique
Médicale, Centre Hospitalier Universitaire de Montpellier,
Montpellier, France, 20Clinique de Génétique Guy Fontaine, Conclusions: we identified a specific subgroup of NOA
patients with mild or borderline hematological alterations
presenting high frequency of occult FA (7.1%). This
discovery have important clinical implications: the screen-
ing for FANCA mutations in such patients may allow the
identification of undiagnosed FA; it corroborates previous
epidemiological observations reporting a higher risk of
morbidity (including cancer) and a lower life expectancy in
infertile men in respect to fertile, normozoospermic men. Conclusions: we identified a specific subgroup of NOA
patients with mild or borderline hematological alterations
presenting high frequency of occult FA (7.1%). P01.08D C. Krausz: None. A. Riera-Escamilla: None. C. Chianese: None. D. Moreno-Mendoza: None. O. Rajmil:
None. M. Bogliolo: None. I. Blanco: None. E. Ars: None. E. Ruiz-Castañé: None. J. Surrallés: None. Whole exome sequencing in non-obstructive azoospermia
allows the identification of a high-risk subgroup of infertile
men for undiagnosed Fanconi Anemia, a cancer-prone
disease P01.07C L. Quteineh: None. M. Guipponi: None. A. Godhino:
None. E. Hammar: None. T. Nouspikel: None. L. Lemmens: None. J.M. Pellegrinelli: None. M. Abramo-
wicz: None. J.L. Blouin: None. S. Fokstuen: None. Targeted exome sequencing for mutation detection in rare
autosomal recessive disorders in families with recurrent
undiagnosed fetal anomalies J. del Picchia 4 Antenatal presentation of Bardet-Biedl syndrome: the
question of phenotype-genotype correlations L. Mary1, K. Chennen2,3, C. Stoetzel2, E. Alanio-Detton4,
C. Antal5,6, T. Attie-Bitach7,8, P. Bouvagnet9, R. Bouvier10,
A. Buenerd11, D. Carles12, A. Delezoide13, L. Devisme14,
B. Gilbert-Dussardier15, F. Guimiot13, P. Khau Van Kien16,
P. Loget17, J. Martinovic18, M. Perez19, F. Petit20, L. Pinson21,
C. Rooryck-Thambo22, O. Poch3, H. Dollfus2,23,24,
E. Schaefer2,23, J. Y. Jung1, S. Lee1, S. Oh2, C. Park1, J. Park1, J. Jun1 1Seoul National University College of Medicine, Seoul, Korea,
Republic of, 2Seoul Metropolitan Government Seoul National
University Boramae Medical Center, Seoul, Korea, Republic of Introduction: Bardet-Biedl syndrome (BBS) is an emble-
matic ciliopathy associating retinal dystrophy, obesity,
postaxial polydactyly, learning disabilities and renal dys-
function. Before birth, enlarged/cystic kidneys as well as
polydactyly revealed by ultrasound (US) are the usual hints
to consider this diagnosis in absence of familial history. However, these symptoms are not specific of BBS, raising
the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely
challenge for this heterogeneous disease (21 known BBS
genes). We report a large cohort of BBS fetuses to better
characterize antenatal phenotype-genotype correlations. Objective: The pathogenesis of birth defects is multi-
factorial, and comparing the concordance rate of birth
defects according to zygosity in the twin can help to
understand the genetic and environmental impacts of the
occurrence of birth defects. The objective of this study was
to determine the concordance rate of birth defects in central
nervous system (CNS) and cardiovascular system (CV),
according to the zygosity. Method: Twins born at Seoul National University
Hospital were examined. Zygosity was confirmed by sex,
chorionicity, and DNA analysis of umbilical cord blood. PCR amplified short tandem repeat (STR analysis) was
conducted with the DNA of cord blood. Materials and Methods: Prenatal US and/or autopsic
data from 74 interrupted fetuses with putative BBS
diagnosis were collected. Materials and Methods: Prenatal US and/or autopsic
data from 74 interrupted fetuses with putative BBS
diagnosis were collected. Results: Using targeted Next Generation Sequencing, we
established a molecular diagnostic in 52 cases mainly in BBS
genes (47 cases) following the classical gene distribution, but
also in other ciliopathy genes (5 cases). Polydactyly (81%, of
postaxial localization only) and renal cysts (72%) were the
most prevalent symptoms in BBS-mutated fetuses. However,
autopsy revealed polydactyly missed by US in 44% of cases. Hydrometrocolpos, evocative of BBS, was found in 3 cases. Ductal plate anomalies, hepatic portal fibrosis, cardiovascular
or central nervous system anomalies were rare (6, 4 and 6
cases respectively). Results: Using targeted Next Generation Sequencing, we
established a molecular diagnostic in 52 cases mainly in BBS
genes (47 cases) following the classical gene distribution, but
also in other ciliopathy genes (5 cases). Polydactyly (81%, of
postaxial localization only) and renal cysts (72%) were the
most prevalent symptoms in BBS-mutated fetuses. Y. Jung: None. S. Lee: None. S. Oh: None. C. Park:
None. J. Park: None. J. Jun: None. Antenatal presentation of Bardet-Biedl syndrome: the
question of phenotype-genotype correlations This
discovery have important clinical implications: the screen-
ing for FANCA mutations in such patients may allow the
identification of undiagnosed FA; it corroborates previous
epidemiological observations reporting a higher risk of
morbidity (including cancer) and a lower life expectancy in
infertile men in respect to fertile, normozoospermic men. Funding: Instituto Carlos III (FIS/FEDER-PI14/01250) Abstracts from the 51st European Society of Human Genetics Conference: Posters 5 Centre Hospitalier Régional Universitaire de Lille, Lille,
France, 21Département de Génétique Médicale, Centre
Hospitalier Régional Universitaire de Montpellier,
Montpellier, France, 22Laboratoire Maladies rares, génétique
et métabolisme, Centre Hospitalier Universitaire de Bordeaux,
Bordeaux, France, 23Service de Génétique Médicale, IGMA,
Hôpitaux Universitaires de Strasbourg, Strasbourg, France,
24Centre des Affections Rares en Génétique Ophtalmologique,
FSMR SENSGENE, Hôpitaux Universitaires de Strasbourg,
Strasbourg, France Poch: None. H. Dollfus: None. E. Schaefer: None. J. Muller: None. Poch: None. H. Dollfus: None. E. Schaefer: None. J. Muller: None. P01.10B
Different pattern of concordance rate in birth defects
according to the zygosity in twins Y. Jung1, S. Lee1, S. Oh2, C. Park1, J. Park1, J. Jun1 Y. Jung1, S. Lee1, S. Oh2, C. Park1, J. Park1, J. Jun1 However,
autopsy revealed polydactyly missed by US in 44% of cases. Hydrometrocolpos, evocative of BBS, was found in 3 cases. Ductal plate anomalies, hepatic portal fibrosis, cardiovascular
or central nervous system anomalies were rare (6, 4 and 6
cases respectively). Result: The risk of birth defects in CNS and CV of the
second twin (F2) was increased when birth defects was present
in the first twin (F1) (table). However, higher concordance
rates of CNS birth defects were observed in MZ than in DZ
[probandwise concordance rate (%, 95% CI): 50.00 (15.70-
84.30) in MZ vs. 0.00 (0.00-23.16) in DZ, p < 0.01], whereas
the concordance rate of CV birth defects was not different
[probandwise concordance rate (%, 95% CI): 26.67 (14.60-
41.94) in MZ vs. 20.29 (11.56-31.69) in DZ, p = NS] Conclusion: The concordance rate according to the
zygosity was different between CNS and CV birth defects. It may be speculated that CNS birth defects are highly
genetically affected, whereas CV birth defects are affected
by the environment. Conclusion: Polydactyly and renal anomalies are con-
firmed as major prenatal manifestations for BBS. Poly-
dactyly must be carefully controlled in case of apparent
isolated renal anomalies. The use of prenatal “fast track”
NGS in case of enlarged/cystic kidneys and/or polydactyly
has a high utility for diagnosis and prognosis for improved
parental information. Birth defects in the 2nd twin
Birth defects in
the 1st twin (-)
Birth defects in
the 1st twin (+)
p-value
Central nervous system
0.5% (12/2375)
25% (2/8)
<0.05
- Monozygotic
0.4% (3/681)
66.7% (2/3)
<0.001
- Dizygotic
0.5% (9/1694)
0% (0/5)
NS
Cardiovascular system
1.9% (45/2327)
23.2% (13/56)
<0.001
- Monozygotic
2.4% (16/661)
26.1% (6/23)
<0.001
- Dizygotic
1.7% (29/1666)
21.2% (7/33)
<0.001 L. Mary: None. K. Chennen: None. C. Stoetzel: None. E. Alanio-Detton: None. C. Antal: None. T. Attie-Bitach:
None. P. Bouvagnet: None. R. Bouvier: None. A. Buenerd: None. D. Carles: None. A. Delezoide: None. L. Devisme: None. B. Gilbert-Dussardier: None. F. Guimiot: None. P. Khau Van Kien: None. P. Loget:
None. J. Martinovic: None. M. Perez: None. F. Petit:
None. L. Pinson: None. C. Rooryck-Thambo: None. O. Y. Jung: None. S. Lee: None. S. Oh: None. C. Park:
None. J. Park: None. J. Jun: None. 6 J. del Picchia P01.11C F. Vogel, K. Brüsehafer, S. Kishore, K. Kandaswamy, M. Weiss,
G. Oprea, A. Rolfs, P. Bauer Centogene AG, Rostock, Germany Staneva: None. M. Mihova: None. B. Zaharova: None. A. Savov: None. Comprehensive Carrier Screening using a combination of
NGS panel, ddPCR and RPA Centogene AG, Rostock, Germany DNA was subsequently
extracted from buccal cells and from suspensions of
cultivated lymphocytes used for karyotyping. DNA profil-
ing was performed with a special attention on markers
located on X and Y-chromosomes. Materials and Methods: Cytogenetic analysis of whole
blood lymphocytes was performed twice - after delivery and
at age of 6 months of the children. DNA was subsequently
extracted from buccal cells and from suspensions of
cultivated lymphocytes used for karyotyping. DNA profil-
ing was performed with a special attention on markers
located on X and Y-chromosomes. Results: Routine NGS processing covers ≥99% of
targeted bases at ≥20 reads. For a comprehensive carrier
evaluation, ”pathogenic”, ”likely pathogenic” and strong
VUS (class 3.1) are reported. Couples are offered complete
screening for partner one and check for partner two genes
with actionable variants. Results: A demonstrable blood chimerism was detected
by karioyping and DNA analysis after birth in both twins. Karyotypes for twin 1 (healthy female) and twin 2 (healthy
male) at time of birth were chi46,XY[78]/46,XX[22] and
chi46,XY[69]/46,XX[31], respectively. After 6 months the
karyotypes detected were chi46,XY[87]/46,XX[13] and
chi46,XY[86]/46,XX[14], respectively. DNA data from
cultivated
lymphocytes
was
in
accordance
with
the
cytogenetic results and confined the blood chimerism. This
phenomenon was not detected by DNA analysis of buccal
cells of both twins. Conclusions:
CentoScreen®
offers
a
comprehensive
carrier screening to accurately determine genetic risks. It
can be especially used in couples from regions with high
consanguinity as well as ethnicities with high incidence of
certain genetic diseases even without any family history of
genetic disease to understand their genetic risks. F. Vogel: A. Employment (full or part-time); Significant;
Centogene AG. K. Brüsehafer: A. Employment (full or
part-time); Significant; Centogene AG. S. Kishore: A. Employment (full or part-time); Significant; Centogene AG. K. Kandaswamy: A. Employment (full or part-time);
Significant; Centogene AG. M. Weiss: A. Employment
(full or part-time); Significant; Centogene AG. G. Oprea:
A. Employment (full or part-time); Significant; Centogene
AG. A. Rolfs:
A. Employment
(full
or
part-time);
Significant; Centogene AG. P. Bauer: A. Employment
(full or part-time); Significant; Centogene AG. Conclusions: An efficient use of both cytogenetic and
molecular analysis techniques in this case of blood but not
buccal cells chimerism was demonstrated. When XX/XY
chimerism is detected in blood cells, a careful monitoring of
reproductive organs of the twins is recommended. In this
case no genital anomalies are detected up to now. S. Andonova:
None. S. Hadjidekova:
None. R. Centogene AG, Rostock, Germany Centogene AG, Rostock, Germany S. Andonova1, S. Hadjidekova2,3, R. Staneva2,3, M. Mihova3,
B. Zaharova1, A. Savov1 Introduction: Carrier screening is a genetic test used to
determine if a healthy person is a carrier of a recessive
genetic disease. The goal of carrier screening is to help
individuals understand their risks of having a child with a
genetic disorder and review the range of options available to
guide pregnancy and family planning. 1National Genetic Laboratory, UHOG "Maichin dom", Sofia,
Bulgaria, 2Department of Medical Genetics, Medical
University of Sofia, Sofia, Bulgaria, 3Woman Health Hospital
"Nadezhda", Sofia, Bulgaria Method & test design rationale: Autosomal and X-
linked recessive disorders were selected based on the
following criteria: Early onset and high-severity disorders,
high carrier frequency, availability of treatment and effect
on quality of life. Based on this, a NGS panel of 331 genes
was designed assessing CCDS +/-20 bases and relevant
deep intronic mutations from HGMD® and Centogene’s
proprietary
variant
database
CentoMD®. An
in-house
developed pipeline provides CNV calling on NGS data. Technically challenging but relevant risk genes (FMR1,
SMN1, CYP21A2) are analyzed by additional assays based
on ddPCR, RPA and Sanger. Adult-onset conditions and X-
linked genes in males are not analyzed. Method & test design rationale: Autosomal and X-
linked recessive disorders were selected based on the
following criteria: Early onset and high-severity disorders,
high carrier frequency, availability of treatment and effect
on quality of life. Based on this, a NGS panel of 331 genes
was designed assessing CCDS +/-20 bases and relevant
deep intronic mutations from HGMD® and Centogene’s
proprietary
variant
database
CentoMD®. An
in-house
developed pipeline provides CNV calling on NGS data. Technically challenging but relevant risk genes (FMR1,
SMN1, CYP21A2) are analyzed by additional assays based
on ddPCR, RPA and Sanger. Adult-onset conditions and X-
linked genes in males are not analyzed. Introduction: Blood-chimerism in dizigotic twins is very
rare condition with presence of two genetically distinct cell
lines in one individual that are derived from two separate
zygotes. It can occur through intrauterine transfer of
hematopoietic cells between the fetuses via vascular ana-
stomoses. Here we present a case of IVF/ICSI pregnancy
initially defined as monozygotic; during second trimester a
sex-discordance between twins was noted. Materials and Methods: Cytogenetic analysis of whole
blood lymphocytes was performed twice - after delivery and
at age of 6 months of the children. Centre for Medical Genetics and Reproductive Medicine
GENNET, Prague, Czech Republic We have designed a NGS (next-generation sequencing)
amplicon-based panel testing 835 key mutations of 77 genes
which can influence reproductive health of prospective
parents or can cause recessive disorders in offspring. We
have developed a bioinformatic pipeline using local instal-
lation of Ensembl genomic database for annotation and
SQL server variant database for data handling and clinical
reporting. To replace MLPA and fragmentation analysis
methods we developed coverage analysis-based CNV
detection of frequent large deletions of SMN1 and CFTR
genes. A software tool developed for the application gen-
erates
report
semi-automatically. Results
are
grouped
according to clinical impact: (1) mutations in genes asso-
ciated with severe recessive disorders in offspring (e.g. SMN1, CFTR, GJB2 genes), (2) mutations in set of genes
predisposing to blood hypercoagulation- trombophilic pro-
file (F2, F5, MTHFR, ANXA5- M2 haplotype), (3) ovarian
response to FSH (FSHR polymorphism). We analysed 3238 samples In the year 2017: 1296
couples in IVF programme (2592 individuals), 354 gamete
donors (F 339, M 15) and 141 individuals with a
reproductive disorder without compatibility testing (F 88,
M 53). The most frequent occurrence of carriers was
recorded in the commonly investigated genes (SMN1,
CFTR, GJB2), but also in other genes (e.g. BTD, MEFV,
ABCA4, SERPINA1, ACADS, DHCR7, PAH, AR). 28
couples with reproductive risks were identified, who were
offered
preimplantation
genetic
testing
of
monogenic
diseases (PGT-M). So far we have done IVF with PGT-M
in four cases (AR, ABCA4, CFTR genes) and invasive
prenatal diagnosis in two cases after a spontaneous
conception (SERPINA1 and PAH genes). A. Röpke: None. C. Brenker: None. M. Hoffmann:
None. C. Krallmann: None. S. Kliesch: None. T. Strünker: None. F. Tüttelmann: None. 1TOMA, Advanced Biomedical Assays S.p.A, Busto Arsizio,
Italy, 2Department of Obstetrics & Gynecology, NYC Health +
Hospitals/Jacobi, Bronx, NY, NY, United States, 3Icahn School
of Medicine at Mount Sinai, New York, NY, United States,
4Genomed SA, Warsaw, Poland, 5Hospital Central de Maputo,
Maputo, Mozambique, 6Faculdade de Medicina da
Universidade Eduardo Mondlane, Maputo, Mozambique P01.13A Comprehensive Carrier Screening using a combination of
NGS panel, ddPCR and RPA CarrierTest - one year experience with expanded
preconception carrier screening Abstracts from the 51st European Society of Human Genetics Conference: Posters 7 L. Dohnalová, F. Lhota, Z. Vilímová, F. Zembol, I. Soldatova,
M. Bittóová, M. Koudová, D. Stejskal We report on three brothers and one sister born to non-
consanguineous parents. The sister and two of the brothers
suffer from hearing loss. In addition, the affected brothers,
but not the sister, are infertile. Although semen analyses
yielded normal sperm concentrations, motility, and mor-
phology (= normozoospermia), the sperm failed to fertilize
the oocytes in vitro. However, both brothers conceived a
child after ICSI. The sister and the unaffected brother each
conceived children spontaneously. Conventional chromo-
somal analysis of the affected brothers demonstrated
apparently normal karyotypes, whereas array-CGH revealed
a homozygous loss of approximately 45 kb on chromosome
15q15.3. Homozygous microdeletions in 15q15.3 lead to
the deafness-infertility syndrome (OMIM 611102) that is
characterized by prelingual hearing loss and infertility in
males but not in females. This homozygous loss was also
demonstrated in the sister. Both parents were heterozygous
for this deletion. The deletion encompasses the genes
CKMT1B, STRC and CATSPER2. CKMT1B is translated
to a mitochondrial creatine kinase. STRC encodes stereo-
cilin, a protein required for the function ofthe outer hair
cells in the inner ear. CATSPER2 encodes a subunit of the
sperm-specific CatSper Ca2+-channel complex. CatSper
controls the intracellular Ca2+ concentration and, thereby,
the swimming behavior of sperm. Functional analysis of
sperm from one of the affected brothers demonstrated a lack
of functional CatSper channels - this deficit explains the
infertility and IVF failure (Brenker et al., 2018 https://doi. org/10.1073/pnas.1717929115). This work was carried out
within the frame of the DFG Clinical Research Unit ‘Male
Germ Cells: from Genes to Function‘ (CRU 326). P01.15C Positive predictive value (PPV) estimates for cell-free DNA
based screening and choice of confirmatory invasive
procedure: experience of a large Italian referral prenatal
diagnostic laboratory L. Dohnalová: None. F. Lhota: None. Z. Vilímová:
None. F. Zembol: None. I. Soldatova: None. M. Bittóová:
None. M. Koudová: None. D. Stejskal: None. L. Dohnalová: None. F. Lhota: None. Z. Vilímová:
None. F. Zembol: None. I. Soldatova: None. M. Bittóová:
None. M. Koudová: None. D. Stejskal: None. F. R. Grati1, B. Grimi1, L. Branca1, L. Marcato1, B. Malvestiti1,
K. Bajaj2, S. J. Gross3, J. C. P. B. Ferreira4,5,6, G. Simoni1,
F. Maggi1 F. R. Grati1, B. Grimi1, L. Branca1, L. Marcato1, B. Malvestiti1,
K. Bajaj2, S. J. Gross3, J. C. P. B. Ferreira4,5,6, G. Simoni1,
F. Maggi1 1Institute of Human Genetics, University of Münster, Münster,
Germany, 2Centre of Reproductive Medicine and Andrology
(CeRA), University Hospital Münster, Münster, Germany A. Röpke1, C. Brenker2, M. Hoffmann1, C. Krallmann2,
S. Kliesch2, T. Strünker2, F. Tüttelmann1 Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 In women <35y and ≥35y the PPV for T21, T18 and
T13 was, 92%(81,97)*, 57%(25,84)*, 33.3%(12,65)*, and
90(60,98)*, 100%(34,100)*, 0%(0,49)*, respectively. Most
of prenatal confirmations were performed on amniocytes
(81/116;69.8%); this trend was more evident when the high-
risk result involved a SCA (28/33;84.8%) versus a trisomy
(54/84;64.3%) (OR: 3.1111;95%CI: 1.1-8.9). However,
CVS shows a higher PPV (28/30,93.3%) for non-mosaic
trisomies than AF (37/54,68.5%) (OR: 6.43;95%CI: 1.4-
30.2). Results: PPV for T13, T18, T21 was 23.1%, 66.7%,
91.9%, respectively. PPV for SCAs was 7.7% for 45,X,
50% for 47,XXX, 30.8% for 47,XXY and 50% for 47,
XYY. In women <35y and ≥35y the PPV for T21, T18 and
T13 was, 92%(81,97)*, 57%(25,84)*, 33.3%(12,65)*, and
90(60,98)*, 100%(34,100)*, 0%(0,49)*, respectively. Most
of prenatal confirmations were performed on amniocytes
(81/116;69.8%); this trend was more evident when the high-
risk result involved a SCA (28/33;84.8%) versus a trisomy
(54/84;64.3%) (OR: 3.1111;95%CI: 1.1-8.9). However,
CVS shows a higher PPV (28/30,93.3%) for non-mosaic
trisomies than AF (37/54,68.5%) (OR: 6.43;95%CI: 1.4-
30.2). Method:38.095 pregnant women undergoing genome-
wide cfDNA-based NIPT were enrolled in the study. Sequencing data were analyzed using algorithms for
common fetal aneuploidies, aneuploidies and subchromo-
somal aberrations. Clinical outcomes were obtained in
37.804 pregnancies. Method:38.095 pregnant women undergoing genome-
wide cfDNA-based NIPT were enrolled in the study. Sequencing data were analyzed using algorithms for
common fetal aneuploidies, aneuploidies and subchromo-
somal aberrations. Clinical outcomes were obtained in
37.804 pregnancies. Results:Clinically relevant chromosomal abnormalities
were detected in 630 (1.7%) pregnancies, 560 (1.6%) of
which were confirmed by invasive prenatal diagnosis. In
500/560 cases common aneuploidies were involved, 30/560
were rare autosomal trisomies (RAT) and 30/560 were
segmental imbalances. 60 fetal conditions would have
otherwise been overlooked if only a conventional NIPT had
been performed. The specificity for common aneuploidy,
RAT and segmental aneuploidies was 99.92%, 99.98%, and
99.98%, respectively; the sensitivity was 100%. Results:Clinically relevant chromosomal abnormalities
were detected in 630 (1.7%) pregnancies, 560 (1.6%) of
which were confirmed by invasive prenatal diagnosis. In
500/560 cases common aneuploidies were involved, 30/560
were rare autosomal trisomies (RAT) and 30/560 were
segmental imbalances. 60 fetal conditions would have
otherwise been overlooked if only a conventional NIPT had
been performed. The specificity for common aneuploidy,
RAT and segmental aneuploidies was 99.92%, 99.98%, and
99.98%, respectively; the sensitivity was 100%. F. Fiorentino, S. Bono, F. Pizzuti, A. Polverari, S. Duca,
M. Faieta, M. Baldi, M. Sessa, L. Diano, F. Spinella Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 1TOMA, Advanced Biomedical Assays S.p.A, Busto Arsizio,
Italy, 2Department of Obstetrics & Gynecology, NYC Health +
Hospitals/Jacobi, Bronx, NY, NY, United States, 3Icahn School
of Medicine at Mount Sinai, New York, NY, United States,
4Genomed SA, Warsaw, Poland, 5Hospital Central de Maputo,
Maputo, Mozambique, 6Faculdade de Medicina da
Universidade Eduardo Mondlane, Maputo, Mozambique A. Röpke1, C. Brenker2, M. Hoffmann1, C. Krallmann2,
S. Kliesch2, T. Strünker2, F. Tüttelmann1 1Institute of Human Genetics, University of Münster, Münster,
Germany, 2Centre of Reproductive Medicine and Andrology
(CeRA), University Hospital Münster, Münster, Germany J. del Picchia 8 Introduction: Conventional cell-free fetal DNA (cfDNA)-
based non-invasive prenatal testing (NIPT) focuses on
detection of common aneuploidies, leaving a gap of ~17%
of clinically relevant chromosomal abnormalities that would
go undetected. Genome-wide NIPT would greatly expand
the range of chromosomal rearrangements detectable. In this
study, we expanded conventional cfDNA-based NIPT to
cover the entire genome in a large general population of
pregnant women, in order to assess the incidence of chro-
mosomal abnormalities not detectable by traditional NIPT. Introduction: Conventional cell-free fetal DNA (cfDNA)-
based non-invasive prenatal testing (NIPT) focuses on
detection of common aneuploidies, leaving a gap of ~17%
of clinically relevant chromosomal abnormalities that would
go undetected. Genome-wide NIPT would greatly expand
the range of chromosomal rearrangements detectable. In this
study, we expanded conventional cfDNA-based NIPT to
cover the entire genome in a large general population of
pregnant women, in order to assess the incidence of chro-
mosomal abnormalities not detectable by traditional NIPT. Introduction: The aim of this study is to determine the PPV
for common trisomies and SCAs using cfDNA screening,
based on karyotype results obtained following a high-risk
cfDNA testing result. Materials and Methods: Initial CfDNA screening was
ordered by the referring physicians and performed by a
variety of technologies. One-hundreds and eighteen con-
firmatory samples were sent to our lab following a high risk
cfDNA test result. PPV stratified by maternal age was
calculated for maternal age-dependent aneuploidies. Results: PPV for T13, T18, T21 was 23.1%, 66.7%,
91.9%, respectively. PPV for SCAs was 7.7% for 45,X,
50% for 47,XXX, 30.8% for 47,XXY and 50% for 47,
XYY. 1Clinical Genetic Unit, Fondazione IRCCS Ca' Granda,
Ospedale Maggiore Policlinico, Milan, Italy, 2Department of
Obstetrics and Gynaecology 'L. Mangiagalli', Fondazione
IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 F. Spinella: None. Male infertility and deafness caused by homozygous
deletion of CATSPER2 and STRC on 15q15.3 Conclusions: The higher rate of confirmatory AF after a
SCA high-risk result probably indicates that referring
clinicians are aware of the proneness of sex chromosomes
to generate confined placental mosaicism, therefore causing
a decreased PPV when using CVS for SCAs. A possible
hypothesis for the higher PPV of CVS for non-mosaic
trisomies might be the performance of an ultrasound-scan
before the choice of confirmatory invasive procedure,
whereby amniocentesis is preferred when no ultrasound
anomalies are identified. * 95th% CI Conclusion:The results of this study demonstrate that
genome-wide cfDNA analysis represents an enhanced
screening tool for prenatal detection of chromosomal
abnormalities, allowing identification of clinically relevant
imbalances that are not detectable by conventional cfDNA
testing. This screening provides improved detection rate as
compared to conventional NIPT, with no appreciable
decrease in specificity. These findings provide substantial
evidence for the feasibility of introducing genome-wide
NIPT into routine prenatal diagnosis practice. Conclusion:The results of this study demonstrate that
genome-wide cfDNA analysis represents an enhanced
screening tool for prenatal detection of chromosomal
abnormalities, allowing identification of clinically relevant
imbalances that are not detectable by conventional cfDNA
testing. This screening provides improved detection rate as
compared to conventional NIPT, with no appreciable
decrease in specificity. These findings provide substantial
evidence for the feasibility of introducing genome-wide
NIPT into routine prenatal diagnosis practice. F.R. Grati: None. B. Grimi: None. L. Branca: None. L. Marcato: None. B. Malvestiti: None. K. Bajaj: None. S.J. Gross: None. J.C.P.B. Ferreira: None. G. Simoni: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; TOMA Advanced Bio-
medical Assays S.p.A. F. Maggi: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; TOMA, Avanced Biomedical Assays, S.p.A. F.R. Grati: None. B. Grimi: None. L. Branca: None. L. Marcato: None. B. Malvestiti: None. K. Bajaj: None. S.J. Gross: None. J.C.P.B. Ferreira: None. G. Simoni: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; TOMA Advanced Bio-
medical Assays S.p.A. F. Maggi: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; TOMA, Avanced Biomedical Assays, S.p.A. F. Fiorentino: None. S. Bono: None. F. Pizzuti: None. A. Polverari: None. S. Duca: None. M. Faieta: None. M. Baldi: None. M. Sessa: None. L. Diano: None. F. Spinella: None. F. Fiorentino: None. S. Bono: None. F. Pizzuti: None. A. Polverari: None. S. Duca: None. M. Faieta: None. M. Baldi: None. M. Sessa: None. L. Diano: None. L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3,
D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1 L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3,
D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1
1Clinical Genetic Unit, Fondazione IRCCS Ca' Granda,
Ospedale Maggiore Policlinico, Milan, Italy, 2Department of
Obstetrics and Gynaecology 'L. Mangiagalli', Fondazione
IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, test If the woman refused the CGH-array, a
karyotype analysis was carried out using the long-term
culture method. Results: chromosomal abnormalities were detected in 58
fetuses (23%), particularly in pregnancies complicated by
ultrasound
abnormalities. In
192
fetuses
(77%),
the
karyotype, after short-term analysis, was normal. All these
women were counselled: 74% agreed to proceed with CGH-
array analysis while26% refused. In these cases, karyotyp-
ing
was
completed
with
long-term
culture
methods
confirming the chromosomal normality. Only 19% of the
women with a fetus with an increased NT (> 3.5 mm) or
ultrasound abnormalities, but with a normal karyotype,
refused CGH-array compared to 28% of the women with a
high risk after combined test principally due to altered
biochemistry. Submicroscopic chromosomal abnormalities
were detected only in two cases (1.5%). One was an
incidental finding with the detection of a microdeletion
causative of dystrophinopathy in a female fetus. In the other
case, it was a pathogenic 22q11.21 microduplication in a
fetus with a NT < 3.5 mm. Results: Meta analysis was conducted in a pooled cohort
of 10,614 fetuses based on the 10 largest studies (N > 300)
of a total of 19 relevant studies. In 0.84%, 95%CI [0.55%,
1.30%] of fetuses a submicroscopic pathogenic aberration
was detected prenatally. The onset/penetrance of the
submicroscopic findings was studied in 10,314 fetuses out
of 8 papers that presented aberrant cases with all necessary
details. The prevalence of early onset syndromic disorders
due to a submicroscopic aberration was calculated to be
1:270, based on 0.37%, 95%CI [0.27%, 0.52%] cases where
aberrations were specified. Conclusions: This systematic review shows that a
significant proportion of fetuses in a general pregnant
population carry a submicroscopic pathogenic CNV. Based
on these figures all women should be informed on their
individual risk for all pathogenic chromosome aberrations
and not only for common trisomies. M.I. Srebniak: None. M. Joosten: None. M.F.C.M. Knapen: None. L.R. Arends: None. M. Polak: None. S. van
Veen:
None. A.T.J.I. Go:
None. D. Van
Opstal: None. Conclusions: CGH-array analysis, performed only after a
multidisciplinary counselling, should also be part of the
investigation in fetuses with biochemical high risk after a
combined test. test Genome-wide cell-free fetal DNA screening in routine non-
invasive prenatal testing practice: a prospective study on
over 38.000 clinical cases L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3,
D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1
1Clinical Genetic Unit, Fondazione IRCCS Ca' Granda,
Ospedale Maggiore Policlinico, Milan, Italy, 2Department of
Obstetrics and Gynaecology 'L. Mangiagalli', Fondazione
IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3,
D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1 L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3,
D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1 Genoma Group, Rome, Italy Abstracts from the 51st European Society of Human Genetics Conference: Posters 9 Italy, 3Laboratory of Medical Genetics, Fondazione IRCCS Ca'
Granda, Ospedale Maggiore Policlinico, Milan, Italy Italy, 3Laboratory of Medical Genetics, Fondazione IRCCS Ca'
Granda, Ospedale Maggiore Policlinico, Milan, Italy Objectives: To establish the frequency of pathogenic sub-
microscopic chromosome aberrations in fetuses that are not
at increased risk for unbalanced structural chromosome
aberrations, a systematic literature search was performed. The aim was to determine whether high resolution testing
for submicroscopic aberrations is beneficial in a general
pregnant population. Introduction: to investigate the incremental yield of
detecting submicroscopic chromosomal abnormalities by
genomic microarray compared to karyotyping in high risk
fetuses after combined testing. Introduction: to investigate the incremental yield of
detecting submicroscopic chromosomal abnormalities by
genomic microarray compared to karyotyping in high risk
fetuses after combined testing. Methods: On 3rd June 2016 Embase and PubMed
databases were systematically searched for all relevant
articles on prevalence of pathogenic submicroscopic CNVs
in fetuses tested due to advanced maternal age or parental
anxiety. Relevant full text articles were analyzed and based
on the extracted data the prevalence of submicroscopic
CNVs was calculated. Materials and Methods: A total of 250 fetuses, with a
high risk after combined test, were tested by conventional
CVS karyotyping. If the short-term cytogenetic analysis
appeared to be normal, women were informed about CGH-
array analysis. If the woman refused the CGH-array, a
karyotype analysis was carried out using the long-term
culture method. Materials and Methods: A total of 250 fetuses, with a
high risk after combined test, were tested by conventional
CVS karyotyping. If the short-term cytogenetic analysis
appeared to be normal, women were informed about CGH-
array analysis. Chromothriptic events in healthy people: pay attention to
''innocent'' insertional translocations Chromothriptic events in healthy people: pay attention to
''innocent'' insertional translocations R. Silipigni: None. S. Guerneri: None. D. Alberico: None. S. Boito: None. I. Fabietti: None. M. Disegni: None. F. Lalatta: None. N. E. KURTAS1, L. Zumerle2, L. Leonardelli2, U. Giussani3,
A. Pansa3, L. Cardarelli4, V. Bertini5, E. Errichiello1,
M. Delledonne2, O. Zuffardi1 1Erasmus MC, Rotterdam, Netherlands, 2Erasmus University,
Rotterdam, Netherlands P01.20D L. Ronzoni: None. N. Persico: None. A. Sajeva: None. L. Ronzoni: None. N. Persico: None. A. Sajeva: None. P01.21A The influence of chromosomal microarray and NIPT on the
diagnostic yield in 6811 high risk pregnancies without The
influence of chromosomal microarray and NIPT on the
diagnostic yield in 6811 high risk pregnancies without
ultrasound anomalies M.I. Srebniak: None. M.F.C.M. Knapen: None. M. Polak: None. M. Joosten: None. K.E.M. Diderich: None. W.F.J. van IJcken: None. R. Heydanus: None. A. Dijkman: None. T. Toolenaar: None. F.A.T. de Vries:
None. J. Knijnenburg: None. A.T.J.I. Go: None. R.H. Galjaard: None. D. Van Opstal: None. P01.18B Chromosomal SNP microarray
analysis replaced karyotyping in in 2012 and since 2014 a
choice between NIPT and diagnostic testing with micro-
array was offered to women with an increased risk for
common aneuploidy (as a part of a national TRIDENT
study). Material and Methods: The frequency of pathogenic
fetal unbalanced chromosome aberrations was studied in
6811 high risk pregnancies without ultrasound anomalies
referred for prenatal testing in 2009-2015 due to advanced
maternal age, abnormal first trimester screening results
(with nuchal translucency < 3.5mm) or recurrence risk for
chromosome aberration. Chromosomal SNP microarray
analysis replaced karyotyping in in 2012 and since 2014 a
choice between NIPT and diagnostic testing with micro-
array was offered to women with an increased risk for
common aneuploidy (as a part of a national TRIDENT
study). Results: The introduction of microarray led to an
additional yield of submicroscopic pathogenic chromosome
aberrations in 1.9% in fetuses without ultrasound anomalies. The introduction of NIPT led to a decrease of invasive tests,
but also of the diagnostic yield. Conclusions: Since 33% of pathogenic fetal chromosome
aberrations were different from the common aneuploidies
and triploidy, whole genome analysis should be offered
after invasive sampling. Because NIPT (as a second
screening) has resulted in a decreased diagnostic yield it
should be accompanied by an appropriate pre-test counsel-
ing in high risk pregnancies. Conclusions: Since 33% of pathogenic fetal chromosome
aberrations were different from the common aneuploidies
and triploidy, whole genome analysis should be offered
after invasive sampling. Because NIPT (as a second
screening) has resulted in a decreased diagnostic yield it
should be accompanied by an appropriate pre-test counsel-
ing in high risk pregnancies. N.E. Kurtas: None. L. Zumerle: None. L. Leonardelli:
None. U. Giussani: None. A. Pansa: None. L. Cardarelli:
None. V. Bertini: None. E. Errichiello: None. M. Delledonne: None. O. Zuffardi: None. N.E. Kurtas: None. L. Zumerle: None. L. Leonardelli:
None. U. Giussani: None. A. Pansa: None. L. Cardarelli:
None. V. Bertini: None. E. Errichiello: None. M. Delledonne: None. O. Zuffardi: None. 1Community Genetics, Public Health Services, Ministry of
Health, Jerusalem, Israel, 2Recanati Genetics Institute,
Beilinson Hospital, Rabin Medical Center, Petach Tikva, 1Erasmus MC, Clinical Genetics, Rotterdam, Netherlands,
2Erasmus MC, Department of Obstetrics and Gynecology,
Rotterdam, Netherlands, 3Institute of Psychology, Erasmus
University Rotterdam, Rotterdam, Netherlands, 4Erasmus MC,
Rotterdam, Netherlands, 5Department of Obstetrics and
Gynecology, Amphia Hospital, Breda, Netherlands, P01.18B Prevalence of submicroscopic chromosome aberrations in
pregnancies without increased risk for structural
chromosome aberrations - a systematic review of the
literature 1Department of Molecular Medicine, University of Pavia,
Pavia, Italy, 2Department of Biotechnologies, University of
Verona, Verona, Italy, 3Laboratorio di Genetica, Ospedali
Riuniti di Bergamo, Bergamo, Italy, 4Laboratorio Analisi
CITOTEST, Padova, Italy, 5Laboratory of Medical Genetics,
Azienda Ospedaliero-Universitaria Pisana, S. Chiara
Hospital, Pisa, Italy M. I. Srebniak1, M. Joosten1, M. F. C. M. Knapen1, L. R. Arends1, M. Polak2, S. van Veen1, A. T. J. I. Go1, D. Van Opstal1 M. I. Srebniak1, M. Joosten1, M. F. C. M. Knapen1, L. R. Arends1, M. Polak2, S. van Veen1, A. T. J. I. Go1, D. Van Opstal1
1Erasmus MC, Rotterdam, Netherlands, 2Erasmus University,
Rotterdam, Netherlands 1Erasmus MC, Rotterdam, Netherlands, 2Erasmus University,
Rotterdam, Netherlands 10 J. del Picchia 6Department of Obstetrics and Gynecology, Reinier de Graaf
Gasthuis, Delft, Netherlands, 7Department of Gynecology,
Albert Schweitzer Hospital Dordrecht, Dordrecht,
Netherlands, 8Department of Obstetrics and Gynecology,
Erasmus MC, Rotterdam, Netherlands Chromothripsis is characterized by extensive genomic
rearrangements consisting in multiple deletions and dis-
ordered orientation of the rearranged portions of one or few
chromosomes. By whole genome sequencing (WGS) in
three unrelated families, we demonstrated that in one parent
of each family a balanced chromothripsis was present
causing a genomic imbalance in the index case consisting in
a deletion and a non-contiguous duplication within 3q22.1-
q26.31 in case 1, a simple two-way reciprocal translocation
t(6;14) in case 2, and a complex rearrangement involving
chromosomes 6, 7 and 15 in case 3. It is noteworthy that a
parental chromothripsis at the origin of the proband’s
imbalance was far from predictable in two of them, in which
the proband’s rearrangement was at first interpreted as de
novo in case 1 and consisting in a simple translocation in
case 2. In all parents-of-origin a small size fragment of the
shattered chromosome was inserted into an additional
chromosome. Our findings strongly indicate that (i) both
simple and complex unbalanced rearrangements, can in fact
be recombinant chromosomes derived by a balanced chro-
mothripsis present in one healthy parent; (ii) WGS inves-
tigations in the parent may reveal unexpected genomic
complexity that are impossible to foresee by conventional
and molecular cytogenetic approaches; (iii) insertional
translocations cannot anymore be considered three break-
points events but rather are the spy of more complex rear-
rangements. P01.22B Chromosomal microarray analysis in fetuses with double
renal collecting system A. Singer1, I. Maya2, A. Frumkin3, S. Zeligson4, R. Berger5,
S. Ben Shachar6, C. Vinkler7, L. Sagi-Dain8
1Community Genetics, Public Health Services, Ministry of
Health, Jerusalem, Israel, 2Recanati Genetics Institute,
Beilinson Hospital, Rabin Medical Center, Petach Tikva, P01.18B Our partially novel and partially confirmatory
data call for WGS as first tier genomic analysis in order to
properly
evaluate
any possible risk
for
chromosome
imbalances at following pregnancies. Chromothripsis is characterized by extensive genomic
rearrangements consisting in multiple deletions and dis-
ordered orientation of the rearranged portions of one or few
chromosomes. By whole genome sequencing (WGS) in
three unrelated families, we demonstrated that in one parent
of each family a balanced chromothripsis was present
causing a genomic imbalance in the index case consisting in
a deletion and a non-contiguous duplication within 3q22.1-
q26.31 in case 1, a simple two-way reciprocal translocation
t(6;14) in case 2, and a complex rearrangement involving
chromosomes 6, 7 and 15 in case 3. It is noteworthy that a
parental chromothripsis at the origin of the proband’s
imbalance was far from predictable in two of them, in which
the proband’s rearrangement was at first interpreted as de
novo in case 1 and consisting in a simple translocation in
case 2. In all parents-of-origin a small size fragment of the
shattered chromosome was inserted into an additional
chromosome. Our findings strongly indicate that (i) both
simple and complex unbalanced rearrangements, can in fact
be recombinant chromosomes derived by a balanced chro-
mothripsis present in one healthy parent; (ii) WGS inves-
tigations in the parent may reveal unexpected genomic
complexity that are impossible to foresee by conventional
and molecular cytogenetic approaches; (iii) insertional
translocations cannot anymore be considered three break-
points events but rather are the spy of more complex rear-
rangements. Our partially novel and partially confirmatory
data call for WGS as first tier genomic analysis in order to
properly
evaluate
any possible risk
for
chromosome
imbalances at following pregnancies. Introduction: Prenatal diagnostics has been impacted by
technological changes in the past decade, which have
affected the diagnostic yield. The aim of this study was to
evaluate the impact of SNP array and non-invasive prenatal
testing (NIPT) on the diagnostic yield and the number of
invasive tests in our center. Material and Methods: The frequency of pathogenic
fetal unbalanced chromosome aberrations was studied in
6811 high risk pregnancies without ultrasound anomalies
referred for prenatal testing in 2009-2015 due to advanced
maternal age, abnormal first trimester screening results
(with nuchal translucency < 3.5mm) or recurrence risk for
chromosome aberration. A. Singer1, I. Maya2, A. Frumkin3, S. Zeligson4, R. Berger5,
S. Ben Shachar6, C. Vinkler7, L. Sagi-Dain8 Abstracts from the 51st European Society of Human Genetics Conference: Posters 11 The fibrous sheath is a unique cytoskeletal structure located
in the principle piece of the sperm flagellum with more than
13 protein components. AKAP4 is the most abundant pro-
tein in the fibrous sheath, which interacts with at least 3
other proteins. The molecular structure and functionality of
the fibrous sheath are largely unknown. We collected a
clinic sample of sperms featured by dysplasia of fibrous
sheath (DFS), leading to a failure of natural conception. The
sperm donor is an offspring of consanguineous family, and
we identified an inherited homozygous truncating mutation
in his genome by whole-exome sequencing. The affected
protein is one of the components of fibrous sheath, and this
mutation caused a shortened protein which lost part of the
original functions. The mice model with this mutation
introduced by CRISPR-Cas9 technique showed similar
phenotype to the human, with sperms of reduced number
and lower motility due to flagellum malfunction. Strikingly,
we observed that, unlike AKAP4 knock-out mice model,
the knock-out mice model we constructed for the novel
gene exerted major effect on testis, manifested by sig-
nificant size/weight reduction and azoospermia. Micro-
scopic observation of testis slices and in-situ hybridization
showed abnormal cross-section of the seminal vesicles and
disorganized progression from spermatogonia to spermatid,
when comparing the knock-out mice model with the control
ones. Therefore, we concluded that this gene is of critical
importance
to
normal
spermatogenesis
and
testis
development. Israel, 3. Department of Genetic and Metabolic Diseases,
Hadassah, Hebrew University Medical Center, Jerusalem,
Israel, 4Medical Genetics Institute, Shaare Zedek Medical
Center, Jerusalem, Israel, 5Maccabi Health Services, Rehovot,
Israel, 6. Genetics Institute, Sorasky Medical Center, Tel Aviv,
Israel, 7Genetic Institute, "Wolfson", Holon, Israel, 8Genetics
Institute, Carmel Medical Center, Haifa, Israel Israel, 3. Department of Genetic and Metabolic Diseases,
Hadassah, Hebrew University Medical Center, Jerusalem,
Israel, 4Medical Genetics Institute, Shaare Zedek Medical
Center, Jerusalem, Israel, 5Maccabi Health Services, Rehovot,
Israel, 6. Genetics Institute, Sorasky Medical Center, Tel Aviv,
Israel, 7Genetic Institute, "Wolfson", Holon, Israel, 8Genetics
Institute, Carmel Medical Center, Haifa, Israel Duplication of the renal collecting system is one of the most
common variants of urinary tract anatomy with an estimated
incidence of about 1% . This condition is characterized by
two pelvicalyceal units draining a single kidney. It can be
either complete or partial. The objective of our study was to
examine the rate for chromosomal aberrations in isolated
prenatal sonographic finding. Data from all chromosomal
microarray analyses (CMA) reported to the Ministry of
Health between January 2013 and December 2016 were
retrospectively obtained from a computerized database. All
pregnancies with sonographic diagnosis of isolated duplex
renal collecting system and documentation of CMA result
were included. Rate of abnormal CMA findings in these
cases was compared to that of the general population risk,
based on a systematic review encompassing 9272 cases
with normal ultrasound, and a local data of 5541 pregnan-
cies undergoing CMA due to maternal request. One
pathogenic CMA finding was found amongst 98 pregnan-
cies with double collecting system (1.02%), not sig-
nificantly different from the risk for abnormal CMA results
in the general population. In addition, two variants of
unknown significance were demonstrated (2.04%). This is
the first report describing the rate of chromosomal anoma-
lies in pregnancies with isolated duplex renal collecting
system. It is suggested that routine invasive prenatal testing
with CMA analysis in such cases is no more useful than in
the general population. Prospective larger studies are nee-
ded to guide the optimal management of pregnancies with
isolated duplex renal collecting system. L. Sun: None. L. Huang: None. Z. Chen: None. N. Li: None. P01.24D Use of prenatal exome sequencing in fetuses with
ultrasound anomalies M. Segura-Puimedon, B. Rodríguez-Santiago, A. Vallmajó,
M. Codina-Solà, B. Campos, D. Datta, I. Banchs, H. Mattlin,
Y. Sarria, O. Abad, J. Rodríguez, L. Pérez-Jurado, L. Armengol
Quantitative Genomic Medicine Laboratories, qGenomics,
Esplugues de Llobregat, Spain M. Segura-Puimedon, B. Rodríguez-Santiago, A. Vallmajó,
M. Codina-Solà, B. Campos, D. Datta, I. Banchs, H. Mattlin,
Y. Sarria, O. Abad, J. Rodríguez, L. Pérez-Jurado, L. Armengol
Quantitative Genomic Medicine Laboratories, qGenomics,
Esplugues de Llobregat, Spain A. Singer: None. I. Maya: None. A. Frumkin: None. S. Zeligson: None. R. Berger: None. S. Ben Shachar: None. C. Vinkler: None. L. Sagi-Dain: None. A component of sperm fibrous sheath has major effect on
testis morphology and functionality Introduction: Whole exome sequencing is a diagnostic tool
in postnatal settings for individuals with a suspected genetic
condition. Recently, its application in prenatal settings has
increased and is sporadically used as a diagnostic tool. We
present here our experience using this approach in a pre-
natal setting. A component of sperm fibrous sheath has major effect on
testis morphology and functionality L. Sun1, L. Huang2, Z. Chen3, N. Li2 NOVAGEN, Buenos Aires, Argentina Introduction: In the past decade, NGS-based technologies
have been disruptive in many areas of clinical genetics,
mainly related to the diagnosis of known entities. Repro-
ductive medicine has not been excluded from these
advances, and expanded carrier screening (ECS) has
become increasingly used, both for couples at risk and
general
population.Here
we
describe
challenges
and
opportunities of an NGS-based ECS panel for recessive
disorders.Material and Methods: After variant calling of
120 cases using an in-house developed pipeline for pro-
cessing data of 483 genes sequenced on a Nextseq 550
platform, variants were classified according to ACMG
guidelines and our clinical geneticists’ and molecular biol-
ogists’ criteria.Results: 39.2% of the patients were found to
be carriers for at least one pathogenic/likely pathogenic
variant for 48 different diseases. A pathogenic variant por
G6PD (X-linked haemolytic anemia) was found in an
apparently healthy man. 91 unique variants of uncertain
significance (39 classified as “Conflict of interpretation” in
ClinVar) were found in a homozygous/hemizygous state,
which when present in genes related to recessive full-
penetrance and early-onset disease were deemed likely
benign.Conclusions: NGS-based ECS represents an unique
opportunity to identify couples at risk of having children
with recessive conditions but also to calculate variant fre-
quencies from countries underrepresented in global con-
sortiums,
diagnose
individuals
with
low-penetrance
disorders and classify homozygous/hemizygous variants of
uncertain significance as benign. ACMG guidelines are
mainly focused on diagnosis of affected individuals, but
variant classification for ECS must be based on data not
related to the phenotype. Conclusion: Exome sequencing is a valuable diagnostic
tool in fetuses with ultrasound anomalies, especially when
skeletal anomalies are present or Noonan syndrome is
suspected. M. Segura-Puimedon: A. Employment (full or part-
time); Significant; Quantitative Genomic Medicine Labora-
tories, qGenomics, Barcelona, Spain. B. Rodríguez-San-
tiago: A. Employment (full or part-time); Significant;
Quantitative Genomic Medicine Laboratories, qGenomics. A. Vallmajó: A. Employment (full or part-time); Signifi-
cant; Quantitative Genomic Medicine Laboratories, qGe-
nomics. M. Codina-Solà: A. Employment (full or part-
time); Significant; Quantitative Genomic Medicine Labora-
tories, qGenomics. B. Campos: A. Employment (full or
part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. D. Datta: A. Employment (full
or part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. I. Banchs: A. Employment (full
or part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. H. Mattlin: A. Employment (full
or part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. Y. Sarria: A. Employment (full
or part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. O. P01.26B
Challenges and opportunities in variant interpretation in
NGS-based expanded carrier screening. First results from
an Argentinian cohort S. Menazzi, M. Fabbro, D. Lorenzi, M. Bilinski, C. Fernandez,
M. Galain, P. Nicotra, V. Chekherdemian, F. Nodar, S. Papier
NOVAGEN, Buenos Aires, Argentina S. Menazzi, M. Fabbro, D. Lorenzi, M. Bilinski, C. Fernandez,
M. Galain, P. Nicotra, V. Chekherdemian, F. Nodar, S. Papier S. Menazzi, M. Fabbro, D. Lorenzi, M. Bilinski, C. Fernandez,
M. Galain, P. Nicotra, V. Chekherdemian, F. Nodar, S. Papier L. Sun1, L. Huang2, Z. Chen3, N. Li2 L. Sun1, L. Huang2, Z. Chen3, N. Li2 1Department of Reproductive Medicine,Guangzhou Women
and Children's Medical Center, Guangzhou, China,
2Guangzhou Institute of Pediatrics,Guangzhou Women and
Children's Medical Center, Guangzhou, China, 3Department of
Reproductive Medicine,Guangzhou Women and Children's
Medical Center, Guangzhou, China, Guangzhou, China Material and Methods: Exome sequencing was per-
formed in 42 fetal samples carrying different ultrasound
anomalies. 30 samples were from evolutive pregnancies and
12 were from legal pregnancy interruptions. In 10 of the
samples previous prenatal CGH-array was performed with J. del Picchia 12 Significant; Quantitative Genomic Medicine Laboratories,
qGenomics. negative result. Segregation studies were performed in cases
with a candidate variant when possible. Results: Common reasons for referral were skeletal
anomalies, polymalformated fetuses, cerebral anomalies or
monogenic disorder suspicion (Noonan syndrome). Patho-
genic variants were identified in n = 8 (19%) of samples,
being previously described or de novo in the index case. In
n = 9 (21%) cases, variants of unknown significance were
identified, and in two of them inheritance was consistent
with expected pattern. In more than half of the cases (52%)
we were not able to identify any candidate variant. Diagnostic yield was highest in fetuses with skeletal
anomalies, where pathogenic variants were identified in
(n = 6) 60% of cases, and in fetuses with clinical suspicion
of Noonan syndrome, where a pathogenic variant was found
in 75% of the samples. No pathogenic variants were found
in
polymalformated
fetuses
or
fetuses
with
cerebral
anomalies. Results: Common reasons for referral were skeletal
anomalies, polymalformated fetuses, cerebral anomalies or
monogenic disorder suspicion (Noonan syndrome). Patho-
genic variants were identified in n = 8 (19%) of samples,
being previously described or de novo in the index case. In
n = 9 (21%) cases, variants of unknown significance were
identified, and in two of them inheritance was consistent
with expected pattern. In more than half of the cases (52%)
we were not able to identify any candidate variant. Diagnostic yield was highest in fetuses with skeletal
anomalies, where pathogenic variants were identified in
(n = 6) 60% of cases, and in fetuses with clinical suspicion
of Noonan syndrome, where a pathogenic variant was found
in 75% of the samples. No pathogenic variants were found
in
polymalformated
fetuses
or
fetuses
with
cerebral
anomalies. P01.28D
Molecular autopsy is an important tool in the diagnosis of
lethal fetal disorders and structural anomalies Molecular autopsy is an important tool in the diagnosis of
lethal fetal disorders and structural anomalies A. Yeung1,2,3, M. Regan1,4, M. Hunter1,3 1Monash Genetics, Monash Health, Melbourne, Australia,
2Victorian Clinical Genetics Services, Murdoch Children's
Research Institute, Parkville, Australia, 3Department of
Paediatrics, Monash University, Melbourne, Australia,
4Family Cancer and Genetics, Melbourne Health, Parkville,
Australia University Medical Center Groningen, Groningen,
Netherlands A significant impact on clinical
management was noted in the families who received a
molecular diagnosis: 2/9 families were informed that a
causative variant was de novo, restoring reproductive
confidence;
3/9
patients
were
counselled
regarding
increased
recurrence
risk
and
proceeded
to
prenatal
diagnosis in subsequent pregnancies, and 3/9 couples were
referred to an IVF service for preimplantation genetic
diagnosis. Results: A diagnosis was established in 9/19 cases (47%)
on the basis of likely pathogenic or pathogenic variants
detected in genes with a concordant Mendelian phenotype:
RIT1, RAF1, L1CAM, AGRN, MTM1, CHRNB1, CEP290,
COL1A1, and PKHD1. Variants of unknown significance
were detected in 2/19. A significant impact on clinical
management was noted in the families who received a
molecular diagnosis: 2/9 families were informed that a
causative variant was de novo, restoring reproductive
confidence;
3/9
patients
were
counselled
regarding
increased
recurrence
risk
and
proceeded
to
prenatal
diagnosis in subsequent pregnancies, and 3/9 couples were
referred to an IVF service for preimplantation genetic
diagnosis. Results: A total of 169 couples were tested, 52 potential
high-risk couples and 117 general public couples as part of
an population-based implementation study. Five couples,
referred for diagnostic reasons, shared carriership of one of
the diseases tested. All remaining couples tested normal. Reporting times averaged at 38 days, and in some cases
even within 2 weeks. Conclusions: Our combined approach for ECS testing
allows for a fast, simplified procedure to report a combined
risk to couples, forestalling the burden of individual
findings. Broader implementation (e.g. general public via
their GP) seems warranted, and is supported by these first
results. Future international discussions will guide further
development of such important screening tests. Conclusion: Genomic sequencing enhances the diagnos-
tic yield of standard fetal imaging and autopsy and
improves patient care. A. Yeung: None. M. Regan: None. M. Hunter: None. NOVAGEN, Buenos Aires, Argentina Abad: A. Employment (full
or part-time); Significant; Quantitative Genomic Medicine
Laboratories, qGenomics. J. Rodríguez: A. Employment
(full
or
part-time);
Significant;
Quantitative
Genomic
Medicine Laboratories, qGenomics. L. Pérez-Jurado: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Quantitative Genomic
Medicine Laboratories, qGenomics. F. Consultant/Advisory
Board; Significant; Quantitative Genomic Medicine Labora-
tories, qGenomics. L. Armengol: E. Ownership Interest
(stock, stock options, patent or other intellectual property); S. Menazzi: A. Employment (full or part-time); Sig-
nificant; NOVAGEN. M. Fabbro: A. Employment (full or
part-time);
Significant;
NOVAGEN. D. Lorenzi:
A. Employment (full or part-time); Significant; NOVAGEN. M. Bilinski: A. Employment (full or part-time); Significant;
NOVAGEN. C. Fernandez: A. Employment (full or part-
time); Significant; NOVAGEN. M. Galain: A. Employ-
ment (full or part-time); Significant; NOVAGEN. P. Abstracts from the 51st European Society of Human Genetics Conference: Posters 13 Nicotra: A. Employment (full or part-time); Significant;
NOVAGEN. V. Chekherdemian: A. Employment (full or
part-time); Significant; NOVAGEN. F. Nodar: A. Employ-
ment (full or part-time); Significant; NOVAGEN. S. Papier: A. Employment (full or part-time); Significant;
NOVAGEN. P01.28D
Molecular autopsy is an important tool in the diagnosis of
lethal fetal disorders and structural anomalies P01.27C K. M. Abbott, T. Dijkhuizen, M. Veldhuis, J. Schuurmans, I. van
Langen, R. J. Sinke K. M. Abbott, T. Dijkhuizen, M. Veldhuis, J. Schuurmans, I. van
Langen, R. J. Sinke Introduction: Genomic sequencing is emerging as an
important tool in the diagnosis of lethal fetal disorders and
structural malformations. We sought to determine the clin-
ical utility of genomic sequencing as an adjunct to standard
antenatal imaging and fetal autopsy, as well as the impact of
molecular diagnosis on clinical care. University Medical Center Groningen, Groningen,
Netherlands University Medical Center Groningen, Groningen,
Netherlands University Medical Center Groningen, Groningen,
Netherlands Introduction:
Expanded
carrier
screening
(ECS)
has
broadened in recent years from high risk population-
targeted testing to general public screening. and the main
challenge now is choosing the most applicable test design
for the intended population. Here we describe the ECS test
developed at our department of Genetics and our initial
results. Materials and Methods: We performed a retrospective
review of perinatal cases referred to the Monash Genetics
Unit between 2015 and 2017 in the setting of structural
malformations or fetal-death-in-utero. 19 fetuses were
identified in whom genomic testing had been performed
following a normal microarray and findings on antenatal
imaging and autopsy suggestive of an underlying mono-
genic disorder. Testing comprised either a targeted panel or
whole
exome
sequencing
in
a
clinically
accredited
laboratory. Materials and Methods: Based on focus group discus-
sions, we designed and implemented a couple-based ECS
multi-gene test for 70 rare, early onset and serious recessive
Mendelian conditions using NGS technologies. Concentrat-
ing on couple-based screening, emphasis was on the
combined risk for having affected children. The a priori
risk of being a carrier couple is approximately 1 in 150 and
increases for those referred for medical reasons (e.g. consanguinity). Only results with high predictive value
regarding affected offspring were reported in the combined
result. Materials and Methods: Based on focus group discus-
sions, we designed and implemented a couple-based ECS
multi-gene test for 70 rare, early onset and serious recessive
Mendelian conditions using NGS technologies. Concentrat-
ing on couple-based screening, emphasis was on the
combined risk for having affected children. The a priori
risk of being a carrier couple is approximately 1 in 150 and
increases for those referred for medical reasons (e.g. consanguinity). Only results with high predictive value
regarding affected offspring were reported in the combined
result. Results: A diagnosis was established in 9/19 cases (47%)
on the basis of likely pathogenic or pathogenic variants
detected in genes with a concordant Mendelian phenotype:
RIT1, RAF1, L1CAM, AGRN, MTM1, CHRNB1, CEP290,
COL1A1, and PKHD1. Variants of unknown significance
were detected in 2/19. NGS studies in structurally abnormal fetuses with a normal
chromosomal microarray analysis. Clinical experience P01.29A NGS studies in structurally abnormal fetuses with a normal
chromosomal microarray analysis. Clinical experience K.M. Abbott: None. T. Dijkhuizen: None. M. Veld-
huis: None. J. Schuurmans: None. I. van Langen: None. R.J. Sinke: None. 14 J. del Picchia A. Borrell1, M. Pauta1, V. Borobio1, B. Marquès1, C. Badenas2,
M. Milà2 Methods: 77 unrelated fetal samples underwent exome
sequencing between 2012-2017. Indications, turnaround
time, diagnostic rates, and pregnancy outcomes were
analyzed. 1BCNatal.Hospital Clínic Barcelona, Barcelona, Catalonia,
Spain, 2CDB.Hospital Clínic Barcelona, Barcelona, Catalonia,
Spain Results: The most common indication for fetal exome
sequencing was multiple malformations (21/77, 27%),
followed by isolated brain malformations (15/77, 19%). Twelve fetuses (15%) were referred for isolated increased
nuchal translucency (IINT). Exome analysis was diagnostic
for 16 fetuses (21%); when sub-classified to fetal mal-
formations vs. IINT it became clear that exome analysis did
not reveal any known or probable pathogenic variants in
IINT whereas among the remaining fetuses, a molecular
diagnosis was reached in 16/65 (25%). Proband-only cases
received a diagnosis more often than trio exomes. Results: The most common indication for fetal exome
sequencing was multiple malformations (21/77, 27%),
followed by isolated brain malformations (15/77, 19%). Twelve fetuses (15%) were referred for isolated increased
nuchal translucency (IINT). Exome analysis was diagnostic
for 16 fetuses (21%); when sub-classified to fetal mal-
formations vs. IINT it became clear that exome analysis did
not reveal any known or probable pathogenic variants in
IINT whereas among the remaining fetuses, a molecular
diagnosis was reached in 16/65 (25%). Proband-only cases
received a diagnosis more often than trio exomes. Objective: The elective genetic testing for structurally
abnormal fetuses is chromosomal microarray analysis
(CMA). We investigated the value of next generation
sequencing (NGS) studies in fetuses with selected structural
anomalies and normal CMA. Methods: During a 30-month period, NGS studies were
performed on fetal DNA extracted from amniocytes or
chorionic villi in 25 structurally abnormal fetuses with a
normal CMA. NGS studies included a single gene analysis
(n = 6), gene panel (n = 14), or clinical exome sequencing
(n = 5). Single gene analysis was performed in suspected
syndromes caused by a single candidate gene (thanatopho-
ric dysplasia, CHARGE, Mowat-Wilson...) and when a
panel or exome was not available in our center (Noonan,
lissencephaly). A gene panel was used when for a specific
fetal syndrome or sign have multiple candidate genes (large
echogenic kidneys, Noonan syndrome, craneosynostosis⋯). P01.29A Finally, clinical exome sequencing was performed in
recurrent
or
multisystem
anomalies
with
no
specific
syndrome suspicion. Conclusion: Exome sequencing has the potential to
provide molecular diagnoses in cases where conventional
prenatal cytogenetic testing is negative. A referral bias of
consanguineous cases could account for the high diagnostic
rate for proband-only sequencing. Syndrome-specific prog-
nostic information enables parents to make informed
decisions, whereas challenges include time limitations and
variant interpretation in the setting of non-specific fetal
findings. As we report only established disease-gene
associations, further segregation and functional studies in
a research setting are expected to significantly increase the
diagnostic yield. H. Daum: None. V. Meiner: None. O. Elpeleg: None. T. Harel: None. Results: In 40% (10/25) of the cases, NGS provided
definitive diagnoses. It was provided in 1/6 (17%) of single
gene analyses (thanatophoric dysplasia), in 8/14 (57%) of
gene panels (4 by CAKUT panel, 2 by Noonan, one by a
craniosynostosis and one by skeletal dysplasia panels), and
in 2/5 (40%) of clinical exomes (primary microcephaly and
Bohring-Opitz syndrome). Natera, Inc., San Carlos, CA, United States Introduction: Pregnancies receiving a ‘no result’ on non-
invasive prenatal testing (NIPT) due to low fetal fraction
(‘no result’ LFF) are at increased risk for trisomies 18 and
13 (T18, T13) and digynic triploidy (DT). The Fetal-
Fraction–Based Risk (FFBR) method has recently been
validated to assess SNP-based NIPT ‘no result’ LFF preg-
nancies given fetal fraction, maternal weight, and gesta-
tional age. This method demonstrated high sensitivity for
pregnancies affected by T18, T13, and DT (high risk, ≥1%). The objective of this study was to retrospectively apply the
FFBR model to SNP-based NIPT ‘no result’ LFF cases that
later had products-of-conception (POC) testing. A. Borrell: None. M. Pauta: None. V. Borobio: None. B. Marquès: None. C. Badenas: None. M. Milà: None. P01.30B Fetal exome sequencing: yield and limitations in a single
tertiary center Fetal exome sequencing: yield and limitations in a single
tertiary center P01.31C Low fetal fraction and digynic triploidy: products-of-
conception testing supports Fetal-Fraction-Based Risk
model for non-invasive prenatal testing Conclusion: NGS provides a definitive diagnosis in 40%
of fetuses with selected structural anomalies and normal
CMA results. The most frequent diagnoses were specific
skeletal dysplasias, Noonan syndrome in persistent nuchal
fold +/- hydrops, and autosomal recessive renal diseases in
large echogenic kidneys. S. Krinshpun, W. DiNonno, T. McKanna, A. Ryan, K. Martin S. Krinshpun, W. DiNonno, T. McKanna, A. Ryan, K. Martin Natera, Inc., San Carlos, CA, United States H. Daum, V. Meiner, O. Elpeleg, T. Harel H. Daum, V. Meiner, O. Elpeleg, T. Harel Hadassah, Jerusalem, Israel Objective: To explore the indications and diagnostic out-
comes of fetal exomes in a single referral center. Materials and Methods: Over 30,000 consecutive POC
samples submitted January 2014-December 2017 were Abstracts from the 51st European Society of Human Genetics Conference: Posters 15 5.88% for groups 1, 2 and 3, respectively (P= 0.0042). The
buccal smear also exhibited physiological pattern of a low-
level mosaicism, however, the level of mosaicism was
statistically insignificant in different age groups and, on
average, was 4.01% (P = 0.530). It is shown that mosaicism
in buccal smear is represented by two cell lines: one is with
disomy
and
another
one
includes
monosomy
on
chromosome X. reviewed. Pregnancies with both SNP-based NIPT ‘no
result’ LFF and SNP-based POC results, which included
parent of origin, were selected for application of the
FFBR model. 5.88% for groups 1, 2 and 3, respectively (P= 0.0042). The
buccal smear also exhibited physiological pattern of a low-
level mosaicism, however, the level of mosaicism was
statistically insignificant in different age groups and, on
average, was 4.01% (P = 0.530). It is shown that mosaicism
in buccal smear is represented by two cell lines: one is with
disomy
and
another
one
includes
monosomy
on
chromosome X. Results: A total of 338 POC cases also had NIPT
performed. Of these cases, 48 (14.2%) had ‘no result’ LFF
(expected given its association with pregnancy loss). The
prevalence of chromosome abnormalities in the ‘no result’
LFF cases was 66.7% (32/48); DT was the most common
(43.8% [14/32]). All DT cases (100%) received a high
FFBR score. Conclusion: The obtained data can be a reference for the
evaluation of low-level mosaicism in fertile women. Conclusion: The obtained data can be a reference for the
evaluation of low-level mosaicism in fertile women. A. Tarlycheva: None. Z. Markova: None. T. Volkova:
None. N. Shilova: None. Conclusion: DT was a significant source of chromosome
abnormalities among pregnancy losses that previously had a
SNP-based NIPT ‘no result’ LFF. Retrospective application
of FFBR demonstrated that these cases could have been
identified as at-risk at time of SNP-based NIPT, allowing
for a more informed genetic counseling and prenatal
management. Exome sequencing reveals novel IGSF10 variation in
patients with hypergonadotropic hypogonadism Exome sequencing reveals novel IGSF10 variation in
patients with hypergonadotropic hypogonadism R. Colombo1,2, A. Jolly3, Y. Bayram3, E. Karaca3, N. Di Simone4,
G. Scambia4, T. Tos5, S. Jhangiani6, Z. C. Akdemir6, J. E. Posey3, J. R. Lupski3,6,7 S. Krinshpun: A. Employment (full or part-time);
Significant; Natera, Inc. W. DiNonno: A. Employment
(full or part-time); Significant; Natera, Inc. T. McKanna:
A. Employment (full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant;
Natera, Inc. K. Martin: A. Employment (full or part-time);
Significant; Natera, Inc. 1Institute of Clinical Biochemistry, Faculty of Medicine,
Catholic University and Policlinico Agostino Gemelli, Rome,
Italy, 2Center for the Study of Rare Hereditary Diseases,
Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy,
3Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX, United States, 4Department
of Obstetrics and Gynecology, Faculty of Medicine, Catholic
University and Policlinico Agostino Gemelli, Rome, Italy,
5Department of Medical Genetics, Sami Ulus Children’s
Hospital, Ankara, Turkey, 6Human Genome Sequencing
Center, Baylor College of Medicine, Houston, TX, United
States, 7Department of Pediatrics, Baylor College of Medicine,
Houston, TX, United States A case of Down syndrome with isodicentric chromosome 21
misdiagnosed by noninvasive prenatal testing (NIPT) A case of Down syndrome with isodicentric chromosome 21
misdiagnosed by noninvasive prenatal testing (NIPT) J. Pietrzak1, C. Wleczyk1, K. Bernatowicz2, A. Kashyap2,
E. Studniak1, W. Bonda1, M. Kossowski1, M. Ryłów1,
M. Bryśkiewicz1, S. Zajączek2 1Clinical Institute of Medical Genetics, University Medical
Centre Ljubljana, Ljubljana, Slovenia, 2Andrology Unit,
Reproductive Unit, Department of Obstetrics and Gynecology,
University Medical Centre Ljubljana, Ljubljana, Slovenia,
3Research Centre for Genetic Engineering and Biotechnology,
Skopje, Macedonia, The Former Yugoslav Republic of,
4Institute of Human Genetics, Medical Faculty, University of
Belgrade, Belgrade, Serbia 1Cytogenetic Unit, Department of Laboratory Diagnostics,
Pomeranian Medical University, Szczecin, Poland,
2Department of Genetics and Pathology, Pomeranian Medical
University, Szczecin, Poland Isodicentric chromosome 21 is an extremely rare chromo-
somal aberration. Only several cases have been reported
worldwide. The phenotype of patients with 46,idic(21)
(q22.3) karyotype is generally concordant with patients who
have simple trisomy 21. Isodicentric chromosome 21 is an extremely rare chromo-
somal aberration. Only several cases have been reported
worldwide. The phenotype of patients with 46,idic(21)
(q22.3) karyotype is generally concordant with patients who
have simple trisomy 21. Introduction: Infertility affects about 5% of adult human
males and despite efforts in understanding genetic basis of
male infertility, a large number of cases still remain to be
explained. Previous methods of detecting disease-related
genes included large family studies. In disorders, such as
infertility, natural selection prevents transmission of muta-
tions, and therefore many genes whose mutations cause
infertility are not yet known. We report a girl born in 38-th week from first pregnancy;
prenatal screening (USG and a double test) performed in
13-th week of gestation predicted a 6-fold increased risk of
Down
syndrome. Noninvasive
prenatal
testing
using
cffDNA from mother's blood did not show increased risk
of trisomy 21 in fetus. Invasive prenatal testing was not
performed. After birth, phenotypic features of trisomy 21
were observed in the child. Materials and Methods: In order to investigate potential
roles of de novo mutations in male infertility we performed
trio whole exome re-sequencing in 13 infertile males and
their parents. All infertile males were diagnosed with
idiopathic azoospermia. For all subjects library preparation
was performed with Nextera Coding Exome Capture Kit
(Illumina, San Diego, CA), with subsequent sequencing on
Illumina HiSeq 2500 platform. Cytogenetic testing performed from the periferal blood
revealed an unbalanced karyotype 46,XX,idic(21)(q22.3). The result were confirmed using Methods: FISH, and
MLPA. A. Hodić1, A. Maver1, B. Zorn2, D. Plaseska-Karanfilska3,
M. Ristanović4, I. Novaković4, B. Peterlin1 A. Hodić1, A. Maver1, B. Zorn2, D. Plaseska-Karanfilska3,
M. Ristanović4, I. Novaković4, B. Peterlin1 P01.33A
Age-dependent gonosomal mosaicism in fertile women IGSF10
encodes a 2623-amino-acid member of the immunoglobulin
superfamily that is likely involved in controlling early
migration of neurons expressing gonadotropin-releasing
hormone. All
three missense IGSF10 variants affect
conserved amino acid residues, were predicted to be
deleterious by several SNV scoring algorithms, and are
present in genomic databases with a frequency <2x10-5. informed about phenomena of germinal mosaicism in
gonadal DNA. these families, each with one apparently sporadic case,
biallelic variation in IGSF10 was found. Two of the three
families had reported parental consanguinity. IGSF10
encodes a 2623-amino-acid member of the immunoglobulin
superfamily that is likely involved in controlling early
migration of neurons expressing gonadotropin-releasing
hormone. All
three missense IGSF10 variants affect
conserved amino acid residues, were predicted to be
deleterious by several SNV scoring algorithms, and are
present in genomic databases with a frequency <2x10-5. In
this
case,
NIFTY
test
could
not
identify
the
abnormality. Further studies are needed to assess sensitivity
of NIPT in the cases of Down syndrome, not caused by
simple trisomy 21. We conclude that invasive prenatal diagnosis should be
proposed in all pregnancies with increased trisomy risk,
even
if
NIPT
results
are
negative. High
resolution
microarray testing can be helpful in identification of
microdeletions in patients with idic 21 and could delineate
genotype -phenotype correlations. Conclusions: A WES approach enabled the identification
of novel IGSF10 mutations in females with HH, thus
expanding the spectrum of genes involved in impaired
ovarian response to Gn. J. Pietrzak: None. C. Wleczyk: None. K. Bernatowicz:
None. A. Kashyap: None. E. Studniak: None. W. Bonda:
None. M. Kossowski: None. M. Ryłów: None. M. Bryśkiewicz: None. S. Zajączek: None. R. Colombo: None. A. Jolly: None. Y. Bayram: None. E. Karaca: None. N. Di Simone: None. G. Scambia:
None. T. Tos: None. S. Jhangiani: None. Z.C. Akdemir:
None. J.E. Posey: None. J.R. Lupski: None. De novo mutations in idiopathic male infertility De novo mutations in idiopathic male infertility P01.33A
Age-dependent gonosomal mosaicism in fertile women Age-dependent gonosomal mosaicism in fertile women A. Tarlycheva1, Z. Markova1, T. Volkova2, N. Shilova1 A. Tarlycheva1, Z. Markova1, T. Volkova2, N. Shilova1 A. Tarlycheva1, Z. Markova1, T. Volkova2, N. Shilova1 1Federal Budgetary State Institution "Research Centre for
Medical Genetics", Moscow, Russian Federation, 2Clinic
"Moja sem'ja", Moscow, Russian Federation Introduction: One of the most significant features of the
human genome is high variability. Genomic variations
occur during ontogenesis in various tissues and organs
leading to the tissue-specific mosaicism. However, phe-
nomenon and mechanisms of a low-level gonosomal
mosaicism in women of reproductive age have not been
properly described and clarified. Introduction: Hypergonadotropic hypogonadism (HH) is
characterized
by
hypogonadism
due
to
an
impaired
response of the gonads to gonadotropins (Gn) and a sec-
ondary lack of sex steroid production and elevated Gn
levels. HH can be caused by environmental factors and
congenital disorders that affect ovarian development and
function, as well as syndromic and non-syndromic single
gene disorders. However, in most cases of gonadal dys-
function the molecular etiology remains an enigma. Materials and Methods: preparations from buccal smear
(34) and peripheral venous blood (32) each woman had at
least one healthy child. Three groups were formed that
included woman of different ages: 20-29 years (1), 30-39
years
(2)
and
40-49
years
(3). FISH-analysis
with
centromeric
probes
on
chromosomes
X
and
18
in
accordance with the standard protocol. Subjects and Methods: To identify novel molecular
causes in HH we applied whole exome sequencing (WES)
to 33 affected female individuals from 30 unrelated families
including 22 with parental consanguinity. Subjects and Methods: To identify novel molecular
causes in HH we applied whole exome sequencing (WES)
to 33 affected female individuals from 30 unrelated families
including 22 with parental consanguinity. Results: WES revealed likely pathogenic variants in
known
HH-associated
genes
in
7/30
families
(23%)
including AR, NOBOX, MCM8, PADI6, PSMC3IP, and
TG. In seven unrelated families, we identified likely
pathogenic variants in candidate disease genes. In three of Results: The study found that in the blood of healthy
women there is a physiological low-level mosaicism with a
clear trend in increased proportion of abnormal cells
associated with the increasing age to 1.83%, 2.23% and 16 J. del Picchia these families, each with one apparently sporadic case,
biallelic variation in IGSF10 was found. Two of the three
families had reported parental consanguinity. Dr. Lal PathLabs Ltd., New Delhi, India The chance of finding a pathogenic aberration in in IUFD
with congenital anomalies was higher than other women. Homozygosity analysis had no advantage over CNV
analysis. Conclusions: CMA resolves more IUFD cases and
therefore should be implicated in such cases. O. Lobel: None. S. Zeligson: None. M. Bar Meir: None. R. Michaelson- cohen: None. S. Koka: None. P. Schwed:
None. A. Samueloff: None. O. Weiss: None. M. Ben
Uziyahu: None. E. Levy-Lahad: None. R. Segel: None. A case of Down syndrome with isodicentric chromosome 21
misdiagnosed by noninvasive prenatal testing (NIPT) Microarray additionally revealed a terminal micro-
deletion sized 11.2 kbp on chromosome 21. Both parents
were tested and confirmed negative for any chromosomal
aberration from blood and fibroblasts and they have been Results: We identified de novo mutations in three
infertile males. Among genes with de novo mutations,
two (NEURL4, BRD2) were previously implicated in
reproduction in animal models. Previous studies have
shown that NEURL4 contributes to germ cell formation in 17 Abstracts from the 51st European Society of Human Genetics Conference: Posters Drosophila, while the BRD2 is essential for chromatin
remodeling during spermatogenesis in mice. The third gene
with de novo mutation (SEMA5A) has not yet been
implicated in reproduction, but it shows expression in testis. O. Lobel, S. Zeligson, M. Bar Meir, R. Michaelson- cohen,
S. Koka, P. Schwed, A. Samueloff, O. Weiss, M. Ben Uziyahu,
E. Levy-Lahad, R. Segel P01.39C Extended genetic analyses in infertile men with non-
obstructive azoospermia S.K. Bhattacharya: A. Employment (full or part-time);
Significant; Dr. Lal PathLabs Ltd. V. Lal: A. Employment
(full or part-time); Significant; Dr. Lal PathLabs Ltd.. Dr. Lal PathLabs Ltd., New Delhi, India Karyotyping has an important role in the genetic work-up of
POC specimens, since approximately one half of mis-
carriages are due to chromosomal imbalances. The three
primary methods used to obtain karyotype are 1) Classical
cytogenetics 2) Targeted FISH and 3) aCGH. Each of these
methods has its advantages and disadvantages. Addition-
ally, the TAT is significantly long. The targeted FISH, only
covers 5-7 chromosomes and therefore provides incomplete
information. It cannot detect any structural abnormalities. Prenatal diagnosis by karyotype determination is done
mostly to provide assurance, since majority of the preg-
nancies would have a normal karyotype. Therefore, fast and
accurate information is highly critical for management of
the pregnancy. However, the same limitations mentioned
for POC also apply to prenatal diagnosis. To overcome
these challenges, we recently validated and adapted a novel
cytogenetic technology ‘Interphase Chromosome Profiling
(ICP) (InteGen LLC, USA) to assess the molecular kar-
yotype of 200 miscarriage material and 80 amniotic fluid
samples using interphase nuclei. For POC and AF samples,
using ICP probes, all numerical, most balanced and unba-
lanced structural aberrations, and all Robertsonian translo-
cations can be detected. Using ICP, we obtained results
from all (100%) samples and the TAT was significantly
reduced to less than 1 day. However, with a proper work-
flow, results can be delivered in less than 2 hours. Karyotyping has an important role in the genetic work-up of
POC specimens, since approximately one half of mis-
carriages are due to chromosomal imbalances. The three
primary methods used to obtain karyotype are 1) Classical
cytogenetics 2) Targeted FISH and 3) aCGH. Each of these
methods has its advantages and disadvantages. Addition-
ally, the TAT is significantly long. The targeted FISH, only
covers 5-7 chromosomes and therefore provides incomplete
information. It cannot detect any structural abnormalities. Methods: We performed CMA on placental tissue of
IUFD, gathered information regarding mothers and fetuses
charachteristics, and compared with information of women
who underwent prenatal diagnosis during the same period. Results: CMA finding was an independent predictor with
higher prevalence in IUFD than in live-born pregnancies. The chance of finding a pathogenic aberration in in IUFD
with congenital anomalies was higher than other women. Homozygosity analysis had no advantage over CNV
analysis. Results: CMA finding was an independent predictor with
higher prevalence in IUFD than in live-born pregnancies. Shaare Zedek Medical Center, Jerusalem, Israel Conclusions: We identified potentially new genes and
mechanisms involved in the pathogenesis of male infertility. Background: 30-50% of clinically recognized pregnancies
end in intra-uterine fetal death (IUFD), 20% within the
second and the third trimester, 25-60% without determined
cause. Genetic
work-up
may
lower
the
uncertainty,
acknowledge the recurrence risks and enable wiser man-
agement of future pregnancies. Microarray technology
(CMA) plays a major role in identifying genetic etiology of
prenatal and postnatal pathologies, raising detection rate
compared with karyotyping. An advantage of CMA in
IUFD workup, is usage of directly extracted DNA without a
culture, therefore without viability issues. The CMA chip
includes copy number and singe nucleotide polymorphism
probes, enabling identification of small copy number
changes, mosaicism and homozygous regions throughout
the genome. We charachterized CMA findings in IUFD
cases in the Israeli population to find: - the characteristics
and frequency of the chromosomal aberrations in IUFD
compared to livebirths. - Do maternal and fetal determinants
have impact on chromosomal aberration prevalence in
IUFD cases? - Can finding of higher percentage of homo-
zygous regions in IUFD explain the etiology? A. Hodić: None. A. Maver: None. B. Zorn: None. D. Plaseska-Karanfilska: None. M. Ristanović: None. I. Novaković: None. B. Peterlin: None. P01.37A Interphase Chromosome Profling (ICP) as a rapid, sensitive
and cost-effective diagnostic tool for Prenatal and Postnatal
settings S. K. Bhattacharya, V. Lal S. K. Bhattacharya, V. Lal F. Tüttelmann1, C. Krallmann2, Y. Stratis1, M. Hoffmann1,
L. Hankamp1, S. Burkhardt1, C. Dreier1, C. Ruckert1,
J. Gromoll2, P. F. Wieacker1, S. Kliesch2, A. Röpke1 J. Gromoll2, P. F. Wieacker1, S. Kliesch2, A. Röpke1 F. Tüttelmann1, C. Krallmann2, Y. Stratis1, M. Hoffmann1, P01.38B The impact of chromosomal microarray analysis on
resolving intra-utrine fetal death cases in Israeli population 18 J. del Picchia 1Institute of Biomedicine and Translational Medicine,
University of Tartu, Tartu, Estonia, 2Andrology Centre, Tartu
University Hospital, Tartu, Estonia 1Institute of Human Genetics, University of Münster, Münster,
Germany, 2Centre of Reproductive Medicine and Andrology
(CeRA), University Hospital Münster, Münster, Germany Introduction: Infertility affects 5-7% of men. The current
pipeline at the andrology clinic is able to assign a definite
cause for 40% of patients, including genetic errors in 8% of
cases, whereas 60% of them remain idiopathic (Punab M
et al. 2017 Hum Reprod). Considering the complexity of
spermatogenesis, it is likely that a substantial proportion of
this
uncharacterised
aetiology
may
be
explained
by
unknown genetic factors. Currently, the role of genomic
copy number variants (CNVs) in male infertility is not well
defined. We aimed to characterise genome-wide profile of
CNVs among Estonian men with idiopathic infertility. Male infertility is a clinically and genetically highly het-
erogeneous disease, mostly caused by spermatogenetic
failure. The most severe form is non-obstructive azoos-
permia (NOA). In the majority of NOA cases, a genetic
origin is suspected but current genetic testing, comprising
cytogenetic analysis and AZF deletion screening, only
discovers the cause in about 17%. Introduction: Infertility affects 5-7% of men. The current
pipeline at the andrology clinic is able to assign a definite
cause for 40% of patients, including genetic errors in 8% of
cases, whereas 60% of them remain idiopathic (Punab M
et al. 2017 Hum Reprod). Considering the complexity of
spermatogenesis, it is likely that a substantial proportion of
this
uncharacterised
aetiology
may
be
explained
by
unknown genetic factors. Currently, the role of genomic
copy number variants (CNVs) in male infertility is not well
defined. We aimed to characterise genome-wide profile of
CNVs among Estonian men with idiopathic infertility. Recently, we expanded our analyses of NOA patients that
attended
the
Centre
of
Reproductive
Medicine
and
Andrology (CeRA). First, the routine chromosomal and
AZF analyses were performed. In a second step, sequence
analysis of three genes was carried out. Material and Methods: All patients (n = 211) and
controls (n = 100) were recruited and clinically character-
ized at the Andrology Centre, Tartu University Hospital. Cases comprised of idiopathic non-obstructive azoospermia
or oligozoospermia patients. Controls represented partners
of pregnant women. Two novel CEP290 pathological variants prenatally
identified by targeted next-generation sequencing using a
custom Meckel-Gruber gene panel Two novel CEP290 pathological variants prenatally
identified by targeted next-generation sequencing using a
custom Meckel-Gruber gene panel P01.38B Hoffmann: None. L. Hankamp: None. S. Burkhardt: None. C. Dreier: None. C. Ruckert: None. J. Gromoll: None. P.F. Wieacker: None. S. Kliesch: None. A. Röpke: None. A. Punab: None. L. Kasak: None. M. Punab: None. E. Laasik: None. A. Valdner: None. M. Laan: None. P01.38B CNV calling utilized genome-wide
SNP data (HumanOmniExpress-24 BeadChip) and was
performed using alternative CNV prediction algorithms in
parallel. CNVs were validated with TaqMan (specific loci)
or aCGH (microdeletions/duplications). Chromosomal analyses were performed in 399 patients of
whom 60 (15.0%) were identified with numerical (47,XXY;
47,XYY) or structural aberrations (46,XX; aberrant Y
chromosomes; translocations; inversions). AZF deletions
were found in 1.8% (6 of 335). The coding sequence of TEX11, NR5A1 and DMRT1
was analysed in 123 patients. Potentially pathogenic
variants were identified in 6 patients (4.9%). One mutation
in TEX11 (c.450C>T) and one in NR5A1 (c.712G>A) were
already published, whereas novel mutations were detected
in NR5A1 (1x c.1079C>T) and in DMRT1 (2x c.308A>G,
1x c.436C>G). The coding sequence of TEX11, NR5A1 and DMRT1
was analysed in 123 patients. Potentially pathogenic
variants were identified in 6 patients (4.9%). One mutation
in TEX11 (c.450C>T) and one in NR5A1 (c.712G>A) were
already published, whereas novel mutations were detected
in NR5A1 (1x c.1079C>T) and in DMRT1 (2x c.308A>G,
1x c.436C>G). Results: Infertility patients and fertile men did not differ
in their overall CNV load. However, an enrichment of
asymptomatic carriers of known microdeletions and micro-
duplications was observed among patients. Additionally, a
novel recurrent CNV overlapping an uncharacterized testis-
specific gene, was detected solely in seven infertility cases. Replication analysis for its association with male infertility
is ongoing in a larger Estonian cohort. In conclusion, the basic genetic analyses in men with
NOA by conventional cytogenetic analysis and AZF
screening revealed the expected number of aberrations. Through sequencing of three genes, which have been
confirmed as responsible for spermatogenetic failure, an
additional 5% of men carrying possibly pathogenic variants
were identified. Conclusions: Diagnostic yield for the patients with
impaired spermatogenesis may be increased via introducing
profiling of genome-wide genomic rearrangements into
clinical routine. This work was carried out within the frame of the DFG
Clinical Research Unit ‘Male Germ Cells: from Genes to
Function‘ (CRU 326). Grants: IUT34-12, Happy Pregnancy project. Grants: IUT34-12, Happy Pregnancy project. Grants: IUT34-12, Happy Pregnancy project. F. Tüttelmann: None. C. Krallmann: None. Y. Stratis:
None. M. Hoffmann: None. L. Hankamp: None. S. Burkhardt: None. C. Dreier: None. C. Ruckert: None. J. Gromoll: None. P.F. Wieacker: None. S. Kliesch: None. A. Röpke: None. A. Punab: None. L. Kasak: None. M. Punab: None. E. Laasik: None. A. Valdner: None. M. Laan: None. F. Tüttelmann: None. C. Krallmann: None. Y. Stratis:
None. M. A. Punab1, L. Kasak1, M. Punab2, E. Laasik1, A. Valdner1,
M. Laan1 A. Punab1, L. Kasak1, M. Punab2, E. Laasik1, A. Valdner1,
1 A. Punab1, L. Kasak1, M. Punab2, E. Laasik1, A. Valdner1,
M. Laan1 1LabGenetics, San Sebastian de los Reyes, Spain, 2Hospital
Universitario Miguel Servet, Zaragoza, Spain Profile of copy number variants in Estonian men with
impaired spermatogenesis Profile of copy number variants in Estonian men with
impaired spermatogenesis M. Alameda Garcia1, E. Del Nuevo Martinez1, S. Garcia
Gomez1, M. Labrador Ranz1, S. Izquierdo Alvarez2, A. Rodríguez
Valle2, M. Miramar Gallart2, A. Sesto Yague1, J. Puente-Prieto1 M. Alameda Garcia1, E. Del Nuevo Martinez1, S. Garcia
Gomez1, M. Labrador Ranz1, S. Izquierdo Alvarez2, A. Rodríguez
Valle2, M. Miramar Gallart2, A. Sesto Yague1, J. Puente-Prieto1 1LabGenetics, San Sebastian de los Reyes, Spain, 2Hospital
Universitario Miguel Servet, Zaragoza, Spain 19 Abstracts from the 51st European Society of Human Genetics Conference: Posters Meckel-Gruber syndrome (MKS) is a lethal autosomal
recessive disorder characterized by a classic ultrasound triad
of occipital encephalocele, polycystic kidneys and postaxial
polydactyly. anomalies of the central nervous system, dys-
plasias and malformations. The mortality is 100%.. In this
case report, a prenatal sample from a fetus with MKS
clinical features was screened for 21 genes using a targeted
next-generation sequencing panel using a Ion PGM System
(Thermo Fisher Scientific). one year was included in the study. Plasma samples were
taken from newborns. All newborn’s physical examinations
were performed and enrolled. The total microRNA isolated
from plasma samples was reverse transcribed into the
cDNA. Quantitative real time -PCR was performed with
specific primers for miR-16 reference gene and miR-17, 21,
23, 92, 141, 145, 191, 483 target genes. REST software was
used for the normalization of relative expression values. The plasma levels of the target miRNA molecules were
showed comparable difference between control and ART
groups. All three target miRNA molecule were displayed
significantly higher levels of expression in ART babies than
controls. In our preliminary results signed that infertility-
causing miRNAs in parents might be cause of congenital
anomalies in newborns. Project code:TTU-2016-1709 Two novel pathological variants, both resulting in stop
codons,
p.Ser1198*
(c.3593C>A)
and
p.Ser1648*
(c.4943C>G), were found in the CEP290 gene which codes
for a centrosomal protein of 290 kDa involved in early and
late steps of cilia formation. Sanger sequencing confirmed
the carrier status of the parents. B. Gozum: None. A. Toylu: None. B. Nur: None. M. Sakinci: None. M. Özekinci: None. O.A. Clark: None. E. Mihci: B. Research Grant (principal investigator, colla-
borator or consultant and pending grants as well as grants
already received); Significant; Akdeniz University Scientific
Research Projects Foundation. Feasibility and concerns of preimplantation genetic
diagnosis for mitochondrial DNA disorders Feasibility and concerns of preimplantation genetic
diagnosis for mitochondrial DNA disorders J. Steffann1, S. Monnot1, N. Gigarel1, A. Rotig2, M. Rio1,
N. Frydman3, L. Hesters3, A. Munnich1, J. Bonnefont1 Profile of copy number variants in Estonian men with
impaired spermatogenesis Our results show that our Meckel-Gruber targeted next-
generation sequencing 21-gene panel is an effective tool for
the identification of pathological variants involved in this
syndrome and confirms the possibility of obtaining a faster
and accurate prenatal genetic diagnosis. M. Alameda Garcia: Other; Significant; LabGenetics. E. Del Nuevo Martinez: Other; Significant; LabGenetics. S. Garcia
Gomez:
Other;
Significant;
LabGenetics. M. Labrador
Ranz:
Other;
Significant;
LabGenetics. S. Izquierdo Alvarez: None. A. Rodríguez Valle: None. M. Miramar Gallart: None. A. Sesto Yague: Other;
Significant; LabGenetics. J. Puente-Prieto: Other; Signifi-
cant; LabGenetics. P01.43C 1Paris Descartes University and Necker Hospital, Paris,
France, 2INSERM UMR1163 Imagine Institute, Paris, France,
3Paris-Sud and Paris-Saclay University, Antoine-Béclère
Hospital, Clamart, France Comparison of microRNA profiles in infants born with and
without assisted reproduction techniques Comparison of microRNA profiles in infants born with and
without assisted reproduction techniques B. Gozum, A. Toylu, B. Nur, M. Sakinci, M. Özekinci, O. A. Clark, E. Mihci B. Gozum, A. Toylu, B. Nur, M. Sakinci, M. Özekinci, O. A. Clark, E. Mihci Preimplantation genetic diagnosis (PGD) is an alternative
procedure to prenatal diagnosis for couples at-risk to have
children affected with a severe genetic disease, such as a
mitochondrial DNA (mtDNA) disorder. PGD relies on the
genetic analysis of one or a few cells sampled from in-vitro
fertilized embryos, between day 3 and day 5 of develop-
ment. In the case of mtDNA disorders, quantification of the
mutant load on these cells is performed in order to assess
the risk for the embryo to develop a severe mitochondrial
disease, either in utero or in childhood. Our 15-year
experience supports the reliability of such procedure. Overall 15 heteroplasmic patients were included in our PGD
program. A total of 26 cycles were started, 25 oocytes
retrievals and 16 embryo transfers were performed, result-
ing in 3 pregnancies and birth of 3 children. The mutant
load assessed on a single blastomere sampled from 94
embryos was very close to the mutant load of the remaining
cell/embryo,
for
all
mutations
tested:
m.8344A>G,
m.3243A>G,
m.8993T>G,
m.8993T>C,
m.9185T>C, A homozygous donor splice-site mutation in the meiotic
gene MSH4 causes primary ovarian insufficiency A homozygous donor splice-site mutation in the meiotic
gene MSH4 causes primary ovarian insufficiency I. Lund1,2,3, E. Vestergaard1,2,3, N. Becher1,2,3, D. Lildballe1,2,
P. Schelde4, L. Hatt4, R. Singh4, O. Petersen5,2, N. Uldbjerg5,
I. Vogel1,2,3 C. Carlosama1, M. Elzaiat2, L. C. Patiño1, H. E. Mateus1, R. A. Veitia2, P. Laissue1 C. Carlosama1, M. Elzaiat2, L. C. Patiño1, H. E. Mateus1, R. A. Veitia2, P. Laissue1 1Aarhus University Hospital, Department of Clinical Genetics,
Aarhus N., Denmark, 2Center for Fetal Diagnostics, Aarhus
University, Aarhus University Hospital, Aarhus N., Denmark,
3Department of Biomedicine, Aarhus University, Aarhus N.,
Denmark, 4Arcedi Biotech Aps, Vejle, Denmark, 5Aarhus
University Hospital, Department of Gynecology and
Obstetrics, Aarhus N., Denmark 1Center For Research in Genetics and Genomics-CIGGUR,
GENIUROS Research Group, School of Medicine and Health
Sciences, Universidad del Rosario, Bogota, Colombia, 2Institut
Jacques Monod, Université Paris Diderot, Paris, France Premature ovarian insufficiency (POI) is a pathology
affecting women under 40 years of age characterized by an
early cessation of menses and high FSH levels. Despite
recent progresses in molecular diagnosis, the etiology of
POI remains idiopathic in most cases. Whole-exome
sequencing of members of a Colombian family affected
by POI allowed us to identify a novel homozygous donor
splice-site mutation in the meiotic gene MSH4 (MutS
Homolog 4). The variant followed a strict mendelian seg-
regation within the family and was absent in a cohort of 135
women over 50 years of age without history of infertility,
from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site
mutation induced skipping of exon 17. At the protein level,
the mutation p.Ile743_Lys785del is predicted to lead to the
ablation of the highly conserved Walker B motif of the
ATP-binding domain, thus inactivating MSH4. Our study
describes the first MSH4 mutation associated with POI and
increases the number of meiotic/DNA mismatch repair
genes formally implicated as being responsible for this
condition. Introduction: Mosaicism is characterized by a normal and
an abnormal cell-line. Test results on cell-free DNA
(cfDNA) in maternal plasma can be compromised as the
fraction of abnormal cells may be too low for detection. We
wanted to explore the detection rate of both confined pla-
cental and fetal mosaicism using cfDNA sequencing on
maternal plasma. Methods and Material: We retrieved data on invasive
samples from mosaic pregnancies obtained from 2014 to
2017. P01.45A
Detecting confined placental and fetal mosaicism using cell-
free DNA sequencing on maternal plasma Detecting confined placental and fetal mosaicism using cell-
free DNA sequencing on maternal plasma Akdeniz University, Antalya, Turkey Akdeniz University, Antalya, Turkey The incidence of congenital anomalies in ART babies is
higher than that of babies born with spontaneous pregnancy. The cause of this situation is unknown. Three mechanisms
are known to cause congenital anomalies in ART preg-
nancies: point mutation, chromosomal disorders, epigenetic
abnormalities. An important factor in epigenetics is micro-
RNA. Studies have shown microRNAs are associated with
fertility and development.The aim of this study to demon-
strate whether infertility-related miRNAs are different in
children born with spontaneous pregnancy compared to
those born with ART. The other aim of the study to show
whether miRNAs are associated with anomalies and dys-
morphic findings in patients. A total of 38 term newborns
included the study. In Akdeniz University Hospital, a baby
born with 21 ART and 17 spontaneous pregnancies within J. del Picchia 20 m.10197G>A. Most of the transferred embryos (17/25)
were heteroplasmic, and 2/3 neonates carried the maternal
mtDNA mutation, questioning the long-term prognosis of
these patients. PGD remains a cumbersome procedure with
a low success rate, which cannot be applied to homoplasmic
or critically homoplasmic patients. There is therefore a
strong need to develop alternative procedures such as
nuclear transfer. m.10197G>A. Most of the transferred embryos (17/25)
were heteroplasmic, and 2/3 neonates carried the maternal
mtDNA mutation, questioning the long-term prognosis of
these patients. PGD remains a cumbersome procedure with
a low success rate, which cannot be applied to homoplasmic
or critically homoplasmic patients. There is therefore a
strong need to develop alternative procedures such as
nuclear transfer. however a high-level mosaicism of trisomy 21 was missed. In the future, we need to learn more about placental
mosaicism in general and in particular the comparability
between the detection rates of the new non-invasive
methods. I. Lund: None. E. Vestergaard: None. N. Becher:
None. D. Lildballe: None. P. Schelde: A. Employment
(full or part-time); Significant; Arcedi Biotech Aps. L. Hatt: A. Employment (full or part-time); Significant;
Arcedi Biotech Aps. R. Singh: A. Employment (full or
part-time); Significant; Arcedi Biotech Aps. O. Petersen:
None. N. Uldbjerg: None. I. Vogel: None. I. Lund: None. E. Vestergaard: None. N. Becher:
None. D. Lildballe: None. P. Schelde: A. Employment
(full or part-time); Significant; Arcedi Biotech Aps. L. Hatt: A. Employment (full or part-time); Significant;
Arcedi Biotech Aps. R. Singh: A. Employment (full or
part-time); Significant; Arcedi Biotech Aps. O. Petersen:
None. N. Uldbjerg: None. I. Vogel: None. J. Steffann: None. Akdeniz University, Antalya, Turkey S. Monnot: None. N. Gigarel: None. A. Rotig: None. M. Rio: None. N. Frydman: None. L. Hesters: None. A. Munnich: None. J. Bonnefont: None. J. Steffann: None. S. Monnot: None. N. Gigarel: None. A. Rotig: None. M. Rio: None. N. Frydman: None. L. Hesters: None. A. Munnich: None. J. Bonnefont: None. C. Carlosama: None. M. Elzaiat: None. L.C. Patiño:
None.
H.E.
Mateus:
None.
R.A.
Veitia:
None.
P.
Laissue: None.
A. M
C. H
N. Si A. Marichal, B. Grisart, J. Billard, S. Brohée, D. Feret,
C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier,
N. Simonis, K. Dahan A homozygous donor splice-site mutation in the meiotic
gene MSH4 causes primary ovarian insufficiency On maternal plasma, we retrospectively performed
cfDNA testing by genome-wide massive parallel sequen-
cing and VeriSeq-NIPT analysis software. Results: CfDNA detected placental mosaicism in 59%
(n = 16). The false negative rate of placental mosaicism
using cfDNA testing was 41% (n = 11). The mean level of
mosaicism in the invasive samples was 72.0% in the
detected cases and 20% in the false negative cases
(p < 0.05). Fetal mosaicism, confirmed by amniocentesis,
was detected by cfDNA sequencing in 63% (5/8 cases). A
mosaic trisomy 21 case which was confirmed both by CVS
(84% T21 cells) and AC was missed by cfDNA. This work was supported by the Universidad del
Rosario grant: [CS/ABN062/GENIUROS 017] and by
the
Fondation
pour
la
Recherche
Médicale
grant:
[DEQ20150331757]. C.C. and M.E. on the one hand, and
R.A.V. and P.L. on the other hand contributed equally to
this work. Conclusion: CfDNA sequencing is capable of detecting
placental mosaicism in 59% of the cases. It seems that the
level of mosaicism in the invasive samples predicts whether
or not cfDNA testing is able to detect the abnormal cell-line; Abstracts from the 51st European Society of Human Genetics Conference: Posters 21 C. Carlosama: None. M. Elzaiat: None. L.C. Patiño:
None. H.E. Mateus:
None. R.A. Veitia:
None. P. Laissue: None. A. Marichal, B. Grisart, J. Billard, S. Brohée, D. Feret,
C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier,
N. Simonis, K. Dahan Synlab, Esplugues de Llobregat, Spain Paired-end MPSS allows digital counting of plasma cfDNA
while also measuring fragments length. cfDNA size dif-
ferences can be used to determine fetal fraction (FF) and to
improve sensitivity by additionally applying counting sta-
tistics on short (fetal) fragments. NeoBona is the first test using such approach, we
evaluated its performance by screening a large cohort of
consecutive average risk gestations. Prospective study of 19151 pregnancies (575 twins)
screened for common trisomies, including XY aneuploidies
in 57% of cases. NeoBona test was used to determine the likelihood of
aneuploidy (Tscore) based on FF, counting statistics and
cfDNA size distribution where cut-offs were applied to
classify normal and aneuploid cases. Test results were provided in 99.2% gestations, 288 T21,
63 T18 and 27 T13, in 23 cases detected with FF between
0.8 and 3%. Invasive procedures were performed in 99%
risk pregnancies, 5 false positives were observed for T21, 2
T18 and 3 for T13 (FPR 0.03%, 0.01% and 0.02%); 1 T21
was missed (DR 99.7%). XY aneuploidies were reported in
39 cases, follow-up available for 14 with 4 FP results (FPR
0.13%). Vanishing twins of discrepant sex were suspected
in 5 cases and 4 maternal X aneuploidies were identified. A. Marichal: None. B. Grisart: None. J. Billard: None. S. Brohée: None. D. Feret: None. C. Hougardy: None. S. Mary: None. S. Rombout: None. P. Hilbert: None. C. Meunier: None. N. Simonis: None. K. Dahan: None. Paired-end MPSS and the bioinformatics approach of
NeoBona allowed detecting aneuploidies even at fetal
fractions below 1% while reducing FPR. Removing the
need of a lower FF limit allowed cfDNA analysis to be
successful on a high proportion of clinical cases extending
the benefits of cfDNA screening to a larger population of
pregnancies. P01.49A External assessment of the quality of cell free fetal DNA
non-invasive prenatal testing for aneuploidies Z. C. Deans1, F. Khawaja1, R. Hastings2, K. Rack2, S. Patton3,
W. Gutowska-Ding3, L. Jenkins4, S. Allen5, L. S. Chitty6,
E. Sistermans7 V. Cirigliano: None. E. Ordoñez: None. L. Rueda:
None. S. Nicolas: None. M. Grau: None. I. Castilla:
None. C. Puertollano: None. M. Lechuga: None. M. Cañadas: None. 1UK NEQAS for Molecular Genetics, Edinburgh, United
Kingdom, 2CEQAS, Oxford, United Kingdom, 3EMQN,
Manchester, United Kingdom, 4Great Ormond Street NHS
Foundation Trust, London, United Kingdom, 5Birmingham
Women’s and Childrens NHS Foundation Trust, Birmingham, Map of maternal copy number variation highlighted by
NIPT analysis V. Cirigliano, E. Ordoñez, L. Rueda, S. Nicolas, M. Grau,
I. Castilla, C. Puertollano, M. Lechuga, M. Cañadas V. Cirigliano, E. Ordoñez, L. Rueda, S. Nicolas, M. Grau,
I. Castilla, C. Puertollano, M. Lechuga, M. Cañadas Z. C. Deans1, F. Khawaja1, R. Hastings2, K. Rack2, S. Patton3,
W. Gutowska-Ding3, L. Jenkins4, S. Allen5, L. S. Chitty6,
E. Sistermans7 P01.47C
Clinical application of paired-end MPSS for cfDNA
screening of common aneuploidies Since July 2017 reimbursement of NIPT is entirely covered
during pregnancy for all pregnant women in Belgium. In
our institute we processed more than 12000 samples over a
6 month period. Our in house NIPT workflow allows the
identification of trisomies involving chromosome 13, 18
and 21 but also trisomies affecting other autosomes as well
as sex chromosomes aneuploidies. Intrachromosomal rear-
rangements are also investigated using a 1 Mb sliding
window approach. This allowed us to identify fetal rear-
rangements in a number of cases but also maternal rear-
rangements with an unpreceded power. Indeed given the
prevalence of maternal cfDNA in purified cfDNA prepared
from the blood of pregnant mothers, some rather small
rearrangements can be pin-pointed quite reliably. Interest-
ingly, some unusually large maternal rearrangements (>1
Mb) have also been observed. Maternal CNVs can usually
be distinguished from fetal ones by the level of significance
of the intrachromosomal Z-score. Some of these CNVs
were futher confirmed by CGH arrays using maternal con-
stitutional DNA. The majority of these CNVs are duplica-
tions (68 duplications and 43 deletions) some of which
overlap syndromic genes. Some correspond to large known
CNVs. However, some large CNVs ranging from 1 to 5 Mb
seem to be rare familial deletions or duplications probably
not associated with any pathogenic phenotype. Indirect
screening of the whole maternal population using NIPT
offers a unique opportunity to identify large probably
benign CNVs. A map of these rare familial CNVs char-
acterized by CGH will presented. M. Hýblová1, J. Budiš2, F. Ďuri3, M. Kucharík2, G. Minarik1,
T. Szemes2 1Medirex, Bratislava, Slovakia, 2Geneton, Bratislava, Slovakia,
3CVTI, Bratislava, Slovakia P01.48D Results: Among 76 twin pregnancies, 70 were identified
correctly and 6 cases were found as uninformative. All of
6 samples fell to the group with fetal fraction lower
than 10%. Results: Ninety-five laboratories from 30 countries
participated. The use of maternal plasma allowed any
testing methods to be applied. Two critical errors were
reported; one false positive and an incorrect high-risk
trisomy 18 result. Reports lacked details of methods,
limitations, and many formats made it difficult to identify
key clinical recommendations. Conclusion: Novel algorithm SCAR predicted correctly
92% cases however fetal fraction under 10% critically
affected reliable sex determination in boy-girl and boy-boy
pregnancies. M. Hýblová: None. J. Budiš: None. F. Ďuri: None. M. Kucharík: None. G. Minarik: None. T. Szemes: None. Conclusions: Growing international demand for partici-
pation demonstrates the clinical need for an independent
evaluation of NIPT practice. This pilot EQA has demon-
strated that the use of real maternal samples distributed at
ambient temperature has enabled global participation with
very low sample failure rate (2%). The genotyping accuracy
was good but review of the large number of reports
submitted highlighted the need for further standardisation
and guidance on NIPT reporting. P01.48D 22 J. del Picchia United Kingdom, 6UCL Great Ormond Street Institute of Child
Health, London, United Kingdom, 7VUmc medical Center
Amsterdam, Amsterdam, Netherlands Introduction: Currently used approaches for determination
of fetal sex in noninvasive prenatal testing (NIPT) have
shown limitations in correct prediction of fetal sex in cases
of twin pregnancies. According to recent information only
SNP based tests of NIPT category are able to determine
fetal sex for each of the twin. Introduction: To deliver a high standard of laboratory
testing, external quality assessment (EQA) is required to
provide important information for clinicians, laboratories
and patients, demonstrating that accurate testing is being
performed and reported. Providing an EQA for cell free
fetal DNA (cffDNA) testing is challenging as sample
acquisition (availability and scalability) is a limiting factor. The delivery and results of a large international pilot EQA
for laboratory NIPT for aneuploidy using maternal plasma
samples is described. Introduction: To deliver a high standard of laboratory
testing, external quality assessment (EQA) is required to
provide important information for clinicians, laboratories
and patients, demonstrating that accurate testing is being
performed and reported. Providing an EQA for cell free
fetal DNA (cffDNA) testing is challenging as sample
acquisition (availability and scalability) is a limiting factor. The delivery and results of a large international pilot EQA
for laboratory NIPT for aneuploidy using maternal plasma
samples is described. Aim: To test the feasibility and to validate the new
bioinformatic algorithm called SCAR to predict sex of both
fetuses in twin pregnancies with utilisation of whole
genome coverage genomic scan of circulating DNA from
pregnant plasma. Materials and Methods: Low coverage whole genome
sequencing analysis was performed on MiSeq and NextSeq
platforms on circulating DNA of 76 pregnant women with
twins according to previously published protocol. For each
of the fetuses sex determination algorithm SCAR predicted
the most probable combination of twin sexes: girl-girl; girl-
boy or boy-boy according to fetal fraction counted from
fragment lengths and reads mapped on Y chromosome. All
predictions were verified after delivery. Materials and Methods: Eighty-six maternal plasma
samples from pregnancies with known outcomes (low/high-
risk for common aneuploidy) were obtained from the
RAPID sample bank. Three EQA providers (CEQAS,
EMQN, and UKNEQAS for Molecular Genetics) delivered
the pilot assessing NIPT and reporting The submitted
reports were assessed and feedback provided for genotyp-
ing, interpretation and clerical accuracy. Chromosomal microarray coupled to genome-wide non-
invasive prenatal testing (NIPT) to minimize the number of
unnecessary invasive procedures Chromosomal microarray coupled to genome-wide non-
invasive prenatal testing (NIPT) to minimize the number of
unnecessary invasive procedures B. Oneda, P. Sirleto, R. Baldinger, M. Taralczak, P. Joset,
M. Zweier, D. Niedrist, S. Azzarello-Burri, K. Steindl, A. Rauch
Institute of Medical Genetics, University of Zurich,
Schwerzenbach, Switzerland, Zurich, Switzerland B. Oneda, P. Sirleto, R. Baldinger, M. Taralczak, P. Joset, B. Oneda, P. Sirleto, R. Baldinger, M. Taralczak, P. Joset, M. Zweier, D. Niedrist, S. Azzarello-Burri, K. Steindl, A. Rauch Z.C. Deans: None. F. Khawaja: None. R. Hastings:
None. K. Rack: None. S. Patton: None. W. Gutowska-
Ding: None. L. Jenkins: None. S. Allen: None. L.S. Chitty: None. E. Sistermans: None. Z.C. Deans: None. F. Khawaja: None. R. Hastings:
None. K. Rack: None. S. Patton: None. W. Gutowska-
Ding: None. L. Jenkins: None. S. Allen: None. L.S. Chitty: None. E. Sistermans: None. Z.C. Deans: None. F. Khawaja: None. R. Hastings:
None. K. Rack: None. S. Patton: None. W. Gutowska-
Ding: None. L. Jenkins: None. S. Allen: None. L.S. Chitty: None. E. Sistermans: None. Institute of Medical Genetics, University of Zurich,
Schwerzenbach, Switzerland, Zurich, Switzerland Institute of Medical Genetics, University of Zurich,
Schwerzenbach, Switzerland, Zurich, Switzerland Prospective clinical results on genome-wide NIPT are still
few and there is little follow up and little data available on
test accuracy. We received 1921 samples including 108
twin pregnancies for NIPT. 93.6% of the cases were ana-
lyzed genome-wide. Additionally, in order to assess test
limitations, we performed NIPT retrospectively in 90 cases
with a variety of segmental aberrations. In the prospective
cohort, 176 samples showed chromosomal abnormalities,
144 of small size. In the latter, we performed chromosomal
microarray analysis on maternal DNA obtained from the
NIPT
tube
and
suggested
invasive
testing
only
for P01.54B K. Tsangaras, P. Mina, M. Ioannides, C. Loizides, A. Achilleos,
E. Kypri, G. Koumbaris, P. C. Patsalis Validation of novel bioinformatic algorithm SCAR for fetal
sex determination in twin pregnancies Validation of novel bioinformatic algorithm SCAR for fetal
sex determination in twin pregnancies Abstracts from the 51st European Society of Human Genetics Conference: Posters 23 aberrations not of maternal origin. We found maternal
CNVs in 7.8% of the total cases which were surprisingly
large in several instances. We detected and confirmed
pathologic chromosomal abnormalities in 32 samples, six of
which would not have been detected if NIPT had been
restricted to common trisomies. The positive predictive
value for the common trisomies was 100% and a retro-
spective questionnaire for quality control showed no evi-
dence for a false negative result. The positive predictive
value for non-maternal segmental anomalies was 50%. Thus
the number of ‘’unnecessary’’ invasive procedures pro-
voked by genome wide NIPT was as low as 0.3%. We
correctly detected all chromosomal aberrations bigger than
6.3 Mb in size in the retrospective cohort. Altogether, we
demonstrate that genome wide NIPT does not lead to a
significant loss of specificity, if in cases with segmental
abnormalities, maternal chromosomal microarray testing is
performed prior to invasive testing. analyzed using a proprietary statistical analysis pipeline
developed to test for deletions in each of the syndromes. Results: The assay was able to correctly classify all
abnormal and normal samples resulting in 100% specificity
and specificity. Conclusions: Using a proprietary target capture enrich-
ment technology and novel multi-engine copy number
detection pipeline we accurately detected all normal and
abnormal samples. This novel microdeletion NIPT method
overcomes the limitations of other methodologies and
increases the number of diseases that can be reliably
detected by NIPT, thus offering more choices to couples
towards an informed management of their pregnancy. K. Tsangaras: A. Employment (full or part-time);
Significant; NIPD Genetics. P. Mina: A. Employment (full
or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant;
NIPD Genetics. A. Achilleos: A. Employment (full or part-
time); Significant; NIPD Genetics. E. Kypri: A. Employ-
ment (full or part-time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Signifi-
cant; NIPD Genetics. P.C. Patsalis: A. Employment (full or
part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time);
Significant; NIPD Genetics. P. Mina: A. Employment (full
or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant;
NIPD Genetics. A. Achilleos: A. Non-invasive prenatal testing of microdeletion syndromes Non-invasive prenatal testing of microdeletion syndromes Validation of novel bioinformatic algorithm SCAR for fetal
sex determination in twin pregnancies Employment (full or part-
time); Significant; NIPD Genetics. E. Kypri: A. Employ-
ment (full or part-time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Signifi-
cant; NIPD Genetics. P.C. Patsalis: A. Employment (full or
part-time); Significant; NIPD Genetics. B. Oneda: None. P. Sirleto: None. R. Baldinger: None. M. Taralczak: None. P. Joset: None. M. Zweier: None. D. Niedrist: None. S. Azzarello-Burri: None. K. Steindl:
None. A. Rauch: None. B. Oneda: None. P. Sirleto: None. R. Baldinger: None. M. Taralczak: None. P. Joset: None. M. Zweier: None. D. Niedrist: None. S. Azzarello-Burri: None. K. Steindl:
None. A. Rauch: None. M. Nicolaou, C. Loizides, M. Ioannides, K. Tsangaras, P. Mina,
A. Achilleos, E. Kypri, G. Koumbaris, P. C. Patsalis Introduction: The discovery of cffDNA in maternal plasma
has greatly facilitated the development of NIPT of fetal
aneuploidies. However, sub-chromosomal copy number
change detection still remains a challenge. Towards this
goal, we employed a proprietary hybrid capture-based
technology and novel bioinformatics pipeline for the
detection of microdeletion syndromes. By leveraging the
inherent high enrichment uniformity and high read depth of
this in-solution hybridization NIPT method we achieved
accurate non-invasive detection of fetal microdeletion syn-
dromes. The assay combines multiple depth of coverage-
based and fragment size-based ploidy detection engines to
detect 1p36, DiGeorge, Wolf-Hirschhorn, and Smith-
Magenis microdeletion syndromes with high sensitivity
and specificity. NIPD Genetics, Nicosia, Cyprus M. Nicolaou, C. Loizides, M. Ioannides, K. Tsangaras, P. Mina,
A. Achilleos, E. Kypri, G. Koumbaris, P. C. Patsalis NIPD Genetics, Nicosia, Cyprus Introduction: We hereby present a novel NIPT of major
aneuploidies, microdeletions and 50 monogenic diseases
with moderate and severe phenotypes, including Hemato-
logical, Kidney, Opthalmological, Neurological, Inherited
Metabolic Diseases, such as Thalassaemia, Cystic Fibrosis,
Phenylketonuria and Tay-Sachs. Methods: cfDNA was obtained from 300 pregnancies
referred for NIPT at 10th-15th week of gestation for
identification of 651 causative mutations in 50 disease
associated genes. A study including another 1000 pregnan-
cies using cfDNA and paternal DNA is ongoing for NIPT of
major aneuploidies, microdeletions and 50 monogenic
diseases. An enriched sequencing library was prepared
using custom TArget Capture Sequences (TACS) as
previously described. TACS were designed based on
genomic locations of known causative mutations for
monogenetic
diseases
under
investigation. Enriched Methods: cfDNA was obtained from 300 pregnancies
referred for NIPT at 10th-15th week of gestation for
identification of 651 causative mutations in 50 disease
associated genes. A study including another 1000 pregnan-
cies using cfDNA and paternal DNA is ongoing for NIPT of
major aneuploidies, microdeletions and 50 monogenic
diseases. An enriched sequencing library was prepared
using custom TArget Capture Sequences (TACS) as
previously described. TACS were designed based on
genomic locations of known causative mutations for
monogenetic
diseases
under
investigation. Enriched Materials and Methods: cfDNA was extracted from 752
unaffected first trimester pregnancy plasma samples and 29
affected prenatal and synthetic samples. Enrichment probes
were designed to span the syndromes’ critical regions
avoiding low copy repeats and repetitive elements. All
samples were enriched using hybrid capture technology as
previously described. Enriched sequencing libraries were Materials and Methods: cfDNA was extracted from 752
unaffected first trimester pregnancy plasma samples and 29
affected prenatal and synthetic samples. Enrichment probes
were designed to span the syndromes’ critical regions
avoiding low copy repeats and repetitive elements. All
samples were enriched using hybrid capture technology as
previously described. Enriched sequencing libraries were 24 J. del Picchia We recently identified a possible malignancy in a 25-year
old women. The chromosome profile resembles aberrations
previously seen in patients with colon carcinoma (loss of
8p, gain of 8q and 20). During subsequent colonoscopy, a
possible precursor adenomatous polyps was removed. Follow-up is ongoing. Also, some well-known (maternal)
CNVs have been identified, such as Hereditary Neuropathy
with
liability
to
Pressure
Palsies
(HNPP)
deletions
(OMIM162500). Furthermore, several deletions and dupli-
cations with unknown clinical significance have been
detected that affected the chromosomal Z-scores of the
NIPT analysis. NIPD Genetics, Nicosia, Cyprus In five cases thus far, the presence of an
extra X-chromosome in the mother was present, these were
communicated. products were sequenced using NGS and the data was
processed using a custom bioinformatics pipeline. Results: For the initial 300 samples, a high number of
causative mutations was identified and a selection of those
was confirmed using Sanger sequencing. For the ongoing
1000 samples, causative mutations were identified and the
fetal risk for aneuploidies, microdeletions and monogenic
disorders was determined. Conclusions: This is the first time that NIPT is made
available for a high number of single gene diseases together
with aneuploidies and microdeletions, opening a new
chapter in prenatal screening. The cumulative risk for the
fetus is estimated to be as high as 1/125. This novel NIPT is
expandable to hundreds of single gene diseases and can be
taken potentially by all pregnant women as early as the 10th
week of gestation Conclusions: This is the first time that NIPT is made
available for a high number of single gene diseases together
with aneuploidies and microdeletions, opening a new
chapter in prenatal screening. The cumulative risk for the
fetus is estimated to be as high as 1/125. This novel NIPT is
expandable to hundreds of single gene diseases and can be
taken potentially by all pregnant women as early as the 10th
week of gestation With the plummeting costs of NGS on the one hand and
the advent of better bio-informatic tools for analysis on the
other hand, guidelines are clearly needed to guide us in this
new genetic, healthcare landscape of secondary findings. M. Nicolaou:
A. Employment
(full
or
part-time);
Significant; NIPD Genetics. C. Loizides: A. Employment
(full or part-time); Significant; NIPD Genetics. M. Ioan-
nides: A. Employment (full or part-time); Significant; NIPD
Genetics. K. Tsangaras: A. Employment (full or part-
time); Significant; NIPD Genetics. P. Mina: A. Employ-
ment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant;
NIPD Genetics. E. Kypri: A. Employment (full or part-
time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Sig-
nificant; NIPD Genetics. M. Baetens: None. T. Sante: None. S. Vergult: None. M. De Smet: None. S. Janssens: None. O. Vanakker:
None. B. Callewaert:
None. B. Poppe:
None. A. Dheedene: None. B. Menten: None. P01.55C Non-invasive prenatal testing (NIPT): how to handle
secondary findings of maternal chromosomal abnormalities Objective:
Using
a
whole-genome
sequencing
NIPT
approach, our laboratory began offering screening for rare
autosomal trisomies (RATs) in 2017. This study presents
our initial clinical experience. M. Baetens, T. Sante, S. Vergult, M. De Smet, S. Janssens,
O. Vanakker, B. Callewaert, B. Poppe, A. Dheedene, B. Menten
Center for Medical Genetics Ghent, Ghent, Belgium M. Baetens, T. Sante, S. Vergult, M. De Smet, S. Janssens,
O. Vanakker, B. Callewaert, B. Poppe, A. Dheedene, B. Menten Clinical experience with noninvasive prenatal testing
(NIPT) for rare autosomal trisomies F. M. Liao1, D. Huynh1, S. Kim2, V. Corey2, K. Curnow2,
W. Seltzer1, S. Beruti2, S. Bhatt2 1Illumina, Inc, Redwood City, CA, United States, 2Illumina,
Inc, San Diego, CA, United States Outcomes, n (%) F.M. Liao: A. Employment (full or part-time); Signifi-
cant; Illumina, Inc. D. Huynh: None. S. Kim: A. Employ-
ment (full or part-time); Significant; Illumina, Inc. V. Corey: A. Employment (full or part-time); Significant;
Illumina, Inc. K. Curnow: A. Employment (full or part-
time); Significant; Illumina, Inc. W. Seltzer: F. Consultant/
Advisory Board; Significant; Illumina, Inc. S. Beruti: A. Employment (full or part-time); Significant; Illumina, Inc. S. Bhatt:
F. Consultant/Advisory
Board;
Significant;
Illumina, Inc. a 4 lymphoma, 1 colon cancer, 1 Stage IV cholangiocarcinoma
diagnosed a year after delivery, 1 ovarian teratoma, and 1 unspecified. Outcome of high risk for digynic triploidy results on SNP-
based non-invasive prenatal testing T. McKanna1, J. Chaperon1, A. Ryan1, S. Leonard1, K. Martin1,
H. Hedriana2 1Natera, Inc., San Carlos, CA, United States, 2University of
California, Davis, CA, United States 1Natera, Inc., San Carlos, CA, United States, 2University of
California, Davis, CA, United States P01.57A T. McKanna: A. Employment (full or part-time);
Significant; Natera, Inc. J. Chaperon: A. Employment
(full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant; Natera, Inc. S. Leonard: None. K. Martin: None. H. Hedriana: F. Consultant/Advisory Board; Modest; Natera, Inc. Outcome of high risk for digynic triploidy results on SNP-
based non-invasive prenatal testing Center for Medical Genetics Ghent, Ghent, Belgium Center for Medical Genetics Ghent, Ghent, Belgium Method: Maternal blood samples from over 10,000 sin-
gleton pregnancies were analyzed in the CLIA-certified
Illumina Laboratory (Redwood City, CA) by the Verifi™
Plus Prenatal Test. Sequencing data was computationally
processed with chromosome-specific quantitative scores
determined using sequence coverage and fetal fraction. Classification thresholds for each chromosome were derived
to maximize specificity while accounting for differences in
prevalence for each RAT. Method: Maternal blood samples from over 10,000 sin-
gleton pregnancies were analyzed in the CLIA-certified
Illumina Laboratory (Redwood City, CA) by the Verifi™
Plus Prenatal Test. Sequencing data was computationally
processed with chromosome-specific quantitative scores
determined using sequence coverage and fetal fraction. Classification thresholds for each chromosome were derived
to maximize specificity while accounting for differences in
prevalence for each RAT. NIPT has become a widely implemented screening test for
the detection of fetal trisomy 13, 18 and 21. Over 8000
NIPT analyses have been performed thus far at the Center
for Medical Genetics Ghent, using shallow whole genome
sequencing
(sWGS)
protocol. Around
0,6%
samples
showed an increased risk for trisomy 13, 18 or 21. Also in
0,6% analyses, we reported another chromosomal abnorm-
ality, including other fetal aneuploidies but also several
clinically relevant maternal CNVs. The detection and dis-
closure of these (secondary) maternal aberrations poses
ethical dilemmas. Aberrations such as the unfortunate
detection of a (predisposition to) malignancy or other
incidental findings are not always straightforward to
disclose. Results: 43 cases (0.4%) were reported as RAT screen
positive. The most common RAT identified was trisomy 22,
followed by trisomies 7 and 9. The average maternal age
(35.0 years) and gestational age (12.4 weeks) of the screen
positive cohort were similar to the whole study cohort. However, some high-risk indications, abnormal ultrasound
(1.8x)
and
history
suggestive
of
increased
risk
for Abstracts from the 51st European Society of Human Genetics Conference: Posters 25 triploidy was 23.3% (7/30). Maternal neoplasm was found
in 26.7% (8/30) cases. aneuploidy (5.5x), were more frequently listed in the screen
positive cohort than in the whole study cohort. Clinical
outcome was available in 8 cases (18.6%): 2 confirmed
positives (1 full trisomy 9; 1 segmental 9p duplication), 2
false positives, 3 miscarriages, and 1 elective termination
without confirmatory testing; >99% of pregnancies are
ongoing. aneuploidy (5.5x), were more frequently listed in the screen
positive cohort than in the whole study cohort. Center for Medical Genetics Ghent, Ghent, Belgium Clinical
outcome was available in 8 cases (18.6%): 2 confirmed
positives (1 full trisomy 9; 1 segmental 9p duplication), 2
false positives, 3 miscarriages, and 1 elective termination
without confirmatory testing; >99% of pregnancies are
ongoing. Conclusions: This post hoc analysis of SNP-based NIPT
data revealed a PPV of 23.3% for pregnancies determined to
be at high risk for DT. An additional and unexpected
finding was the similar number of maternal neoplasm cases. While a small cohort, these results suggest that maternal
neoplasm should be included in the differential diagnosis of
high risk DT results on SNP-based NIPT. Conclusions: Our 0.4% screen-positive frequency is
consistent with previous studies, though some differences
were noted in the relative RAT prevalence.1,2 Results
obtained through NIPT early in pregnancy can be valuable
for clinical management. Ongoing outcome collection will
provide more insight into the biological aspects of RATs. Table. Outcomes
from
suspected
DT
pregnancies
determined via SNP-based NIPT Outcomes, n (%)
Cases (N=30)
Normal fetal and maternal outcome
12 (40.0)
Maternal neoplasma
8 (26.7)
Triploidy suspected by ultrasound
4 (13.3)
Confirmed triploidy
3 (10.0)
Complete molar pregnancy
1 (3.3)
Early fetal demise
1 (3.3)
Ongoing early gestation
1 (3.3) Widespread use of Non Invasive Prenatal Testing(NIPT) :
experience of a Belgian genetic Center Widespread use of Non Invasive Prenatal Testing(NIPT) :
experience of a Belgian genetic Center Introduction: Single nucleotide polymorphism (SNP)-
based non-invasive prenatal testing (NIPT) is uniquely able
to identify the extra haplotype and parental origin in triploid
pregnancies. The objective of this study was to establish a
positive predictive value (PPV) for pregnancies suspected to
be at high risk for digynic (maternal) triploidy (DT) via
SNP-based NIPT. For apparent false positive cases, possible
maternally-derived causes were investigated. B. Grisart, J. Billard, S. Brohée, A. Marichal, D. Feret,
C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier,
N. Simonis, K. Dahan
Centre de Génétique Humaine, Institut de Pathologie et de
Génétique, Charleroi (Gosselies), Belgium B. Grisart, J. Billard, S. Brohée, A. Marichal, D. Feret,
C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier,
N. Simonis, K. Dahan Centre de Génétique Humaine, Institut de Pathologie et de
Génétique, Charleroi (Gosselies), Belgium Centre de Génétique Humaine, Institut de Pathologie et de
Génétique, Charleroi (Gosselies), Belgium Non Invasive Prenatal Testing was developed in our insti-
tute in 2014 using an in house whole genome approach. For
more than 3 years, this test was open for all pregnant
women at their own expense. During this period we tested
7041 maternal blood with a positive screening rate for
chromosome 13, 18 and 21 of 0,10%, 0,20% and 1,3%
respectively. Since July 2017 NIPT has been reimbursed in
Belgium for all pregnant women. This resulted in a sharp
increase in activity as in 6 months more than 12000 samples
were processed. The rates of trisomy 13, 18 and 21 over this
period were 0.05%, 0.06% and 0.4% respectively. Beside Materials and Methods: Retrospective outcome data
were collected for SNP-based NIPTs performed between
January 1, 2015 and December 31, 2017 and coded as high
risk for DT. IRB-approved outcomes comprised: number of
fetuses, ultrasound findings, results of cytogenetic testing
including parental origin of triploidy, and maternal medical
findings. Results: A total of 39 cases with suspected DT were
identified and outcome data were obtained for 30 (77%)
cases (see Table). The PPV for confirmed or suspected J. del Picchia 26 Vanadis Diagnostics – a PerkinElmer company, Sollentuna,
Sweden Vanadis Diagnostics – a PerkinElmer company, Sollentuna,
Sweden Vanadis Diagnostics – a PerkinElmer company, Sollentuna,
Sweden Non-Invasive Prenatal Testing (NIPT) is increasing in
interest for detection of aneuploidies due to these tests
giving a more reliable result than obtained from traditional
first trimester screening. NIPT should not only be available
to high-risk pregnancies but for all women. With Vanadis
NIPT, the aim was to fulfill this criterium, making NIPT
available for all women by creating a fully automated
method with simple preparation need and minimal hands-on
time and thereby reducing both complexity and cost. Vanadis NIPT is a sequencing and PCR free, probe-based
technology used to label targets on chromosome 13, 18, 21
and Y, thereby allowing for trisomy screening (13, 18 and
21) as well as sex determination. The assay consists of four
enzymatic steps resulting in Rolling Circle Amplification
Products (RCPs) for each of these four chromosomes. The
RCPs are labeled with four different dyes, one for each
chromosome, and deposited onto a nano-pore filter from
where the labeled objects are counted by imaging. We will
present performance characteristics for the Vanadis NIPT
assay, including data for real clinical samples, to show the
analytical precision and clinical feasibility to correctly
identify trisomy 13, 18 and 21 as well as the sex of
the fetus. Results: To date, we have performed NIPD for 48
referrals (UK and international) and reported 16 normal, 18
unaffected carrier and 10 affected pregnancies. For 4 cases,
a complete result could not be issued due to persistent low
fetal fraction, a recombination event or lack of informative
SNPs. Of the 48 diagnostic tests, we have so far received
postnatal confirmation of 15 results, with no discrepancies. Conclusions: NIPD by RHDO is a robust assay, which is
feasible to provide in a clinical setting for both X-linked and
autosomal recessive disorders. The assay could be extended
to increase the availability of NIPD for many monogenic
disorders. E.C. Young: None. B. Bowns: None. A. Gerrish: None. M. Parks: None. S. Court: None. S. Clokie: None. C. Mashayamombe-Wolfgarten: None. J. Hewitt: None. D. Williams: None. T. Cole: None. M. Griffiths: None. S. Allen: None. P01.59C P01.59C
Making NIPT available to all pregnant women Å. Janfalk Carlsson Å. Janfalk Carlsson P01.60D these common trisomies other trisomies involving chro-
mosomes 4, 6, 7, 8, 14, 15, 16, 20 and 22 were identified,
most of them (>94%) were not confirmed on invasive
samples. In case of trisomy for chromosome 6, 7, 11, 14, 15
and 20, uniparental disomy was evaluated in the normal
fetus. Intrachromosome analysis using a 1 Mb sliding
window approach, allowed to identify some micro rear-
rangements affecting the fetus. These ones ranged from a
few megabases to several tenth of megabases and were
confirmed by CGH on amniocytes. Sex chromosome
aneuploidies could be technically identified but a Belgian
prenatal consortium (www.BeSHG.be) decided not to report
these sex chromosomes aneuploidies. Indeed generalization
of NIPT would screen almost the whole population for these
sex aneuploidies as well as for susceptibility loci. This
raised ethical questions which have to be addressed. Non-invasive prenatal diagnosis (NIPD) of single gene
disorders by relative haplotype dosage (RHDO): review of
18 months of clinical service E. C. Young1, B. Bowns1, A. Gerrish1, M. Parks2, S. Court1,
S. Clokie1, C. Mashayamombe-Wolfgarten1, J. Hewitt1,
D. Williams1, T. Cole1, M. Griffiths1, S. Allen1 1West Midlands Regional Genetics Service, Birmingham,
United Kingdom, 2Nonacus Ltd., Birmingham, United
Kingdom P01.59C
Making NIPT available to all pregnant women Materials and Methods: The test involves targeted
enrichment of thousands of SNPs across multiple genomic
regions and massively parallel sequencing (Illumina MiSeq)
of cfDNA followed by RHDO analysis. Maternal, paternal
and proband genomic DNA samples are tested alongside
cfDNA for haplotype phasing and to measure fetal fraction. Our method can test 2-3 pregnancies on a single MiSeq run,
thus centralising testing and decreasing costs. The devel-
opment of an automated analysis pipeline has increased
capacity further. There is no requirement to confirm positive
results. Materials and Methods: The test involves targeted
enrichment of thousands of SNPs across multiple genomic
regions and massively parallel sequencing (Illumina MiSeq)
of cfDNA followed by RHDO analysis. Maternal, paternal
and proband genomic DNA samples are tested alongside
cfDNA for haplotype phasing and to measure fetal fraction. Our method can test 2-3 pregnancies on a single MiSeq run,
thus centralising testing and decreasing costs. The devel-
opment of an automated analysis pipeline has increased
capacity further. There is no requirement to confirm positive
results. 1West Midlands Regional Genetics Service, Birmingham,
United Kingdom, 2Nonacus Ltd., Birmingham, United
Kingdom Introduction: We have developed and implemented a
relative haplotype dosage (RHDO)- based method for NIPD
of multiple single gene disorders (SGD), including spinal
muscular atrophy (SMA), Duchenne and Becker muscular
dystrophies (DMD/BMD), cystic fibrosis (CF) and con-
genital adrenal hyperplasia (CAH). Diagnostic services for
SMA and DMD/BMD were launched in September 2016,
followed by the launch of a CF service in December 2017. B. Grisart: None. J. Billard: None. S. Brohée: None. A. Marichal: None. D. Feret: None. C. Hougardy: None. S. Mary: None. S. Rombout: None. P. Hilbert: None. C. Meunier: None. N. Simonis: None. K. Dahan: None. Predicting fetoplacental chromosomal mosaicism during
non-invasive prenatal testing P01.62B Predicting fetoplacental chromosomal mosaicism during
non-invasive prenatal testing Å. Janfalk Carlsson: None. Å. Janfalk Carlsson: None. Å. Janfalk Carlsson: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 27 translucency but normal karyotype and CMA: impact on
the counselling and need for further genetic investigations R. Ficarella1, M. Mucciolo2, C. Votino1, F. R. Lepri2, M. F. Antonucci1, A. L. Buonadonna1, V. Lanari2, P. Volpe1, 1Centre for Human Genetics - UZ Leuven, Leuven, Belgium,
2Department of Human Genetics, Faculty of Medicine “Ibn Al
Jazzar”, Sousse, Tunisia 1ASL Bari, Bari, Italy, 2Bambino Gesù Paediatric Hospital,
Roma, Italy Objective: Non-invasive prenatal detection of trisomies 21,
18 and 13 can be achieved with high accuracy through
sequencing of cell-free DNA (cfDNA) found in maternal
blood. Using a genome-wide approach, fetal aneuploidies
other than the common trisomies can also be detected. Fetoplacental mosaicism is the main cause for false posi-
tive/negative NIPT results. We further improved the ana-
lytical
power
of
genome-wide
cfDNA
screening
by
enabling the detection of fetoplacental mosaicism. Objectives: To investigate the outcome for fetuses with
nuchal translucency (NT) ≥3.5 mm but normal karyotype/
CMA. Objectives: To investigate the outcome for fetuses with
nuchal translucency (NT) ≥3.5 mm but normal karyotype/
CMA. Methods: All patients were referred to our institution for
NT≥3.5 mm from 2012 to 2016. We followed prenatally all
patients
until
delivery
and
pregnancy
outcome
was
recorded. Targeted Resequencing was performed using a
panel including 15 RASopathies genes. p
g
p
g
Results: We identified 74 fetuses. An adverse perinatal
outcome was observed in 27% of cases. US follow-up
showed 10 cases with cardiac malformations (major in 6/
10); diaphragmatic hernia (1); Dandy Walker Malformation
plus skeletal dysplasia (1); pyelectasis (1), and IUGR (1
monochorionic twins). 54% of these cases (40/74) were
analyzed using our customized panel of RASopathies
genes. Four
variants
were
identified,
the
aminoacid
substitutions Val1432Phe and Arg2452Cys, in the NF1
gene, and a Thr7Arg and Thr159Pro, in the LZTR1 gene. Four additional intronic variants were also identified, but no
one altered the splice site according to the prediction tools. Thirty additional fetuses with NT ≥3.5 mm had been
previously analyzed, leading to the identification of two
variants, the Gln506Pro in the PTPN11 gene, and the
Glu63Lys in the KRAS gene. Method: Aneuploidy detection was combined with fetal
fraction estimation to enable the detection of placental
chromosomal mosaicism. This pipeline was applied to
whole genome sequencing data derived from ~20.000
maternal plasma samples. Following an abnormal NIPT,
test results were validated by conventional invasive prenatal
or postnatal genetic testing. Method: Aneuploidy detection was combined with fetal
fraction estimation to enable the detection of placental
chromosomal mosaicism. translucency but normal karyotype and CMA: impact on
the counselling and need for further genetic investigations Novelli: None. N. Brison: None. M. Neofytou: None. L. Dehaspe:
None. B. Bayindir: None. K. Van Den Bogaert: None. L. Dardour: None. H. Peeters: None. H. Van Esch: None. G. Van Buggenhout: None. A. Vogels: None. J. Breck-
pot: None. T. de Ravel: None. E. Legius: None. K. Devriendt: None. J.R. Vermeesch: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; Cartagenia. Other; Modest; Collaboration with
Cartagenia. N. Brison: None. M. Neofytou: None. L. Dehaspe:
None. B. Bayindir: None. K. Van Den Bogaert: None. L. Dardour: None. H. Peeters: None. H. Van Esch: None. G. Van Buggenhout: None. A. Vogels: None. J. Breck-
pot: None. T. de Ravel: None. E. Legius: None. K. Devriendt: None. J.R. Vermeesch: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; Cartagenia. Other; Modest; Collaboration with
Cartagenia. translucency but normal karyotype and CMA: impact on
the counselling and need for further genetic investigations This pipeline was applied to
whole genome sequencing data derived from ~20.000
maternal plasma samples. Following an abnormal NIPT,
test results were validated by conventional invasive prenatal
or postnatal genetic testing. p
g
g
Results: The new analysis pipeline identified 134, 24 and
7 non-mosaic trisomies 21, 18 and 13 respectively. All for
whom follow-up information was available were confirmed
upon
invasive
testing. The
incidence
of
other,
rare
autosomal trisomies (RATs) was ~0.3%, with trisomy 7
and 16 being the most prevalent. Three of these RATs,
predicted as full trisomies in the placenta, were found to be
mosaic in the fetus; 25 other RATs were predicted to be
mosaic, 8 of which have been confirmed in placental tissue. The new pipeline also correctly predicted twin pregnancies
with discordant fetal sex. Results: The new analysis pipeline identified 134, 24 and
7 non-mosaic trisomies 21, 18 and 13 respectively. All for
whom follow-up information was available were confirmed
upon
invasive
testing. The
incidence
of
other,
rare
autosomal trisomies (RATs) was ~0.3%, with trisomy 7
and 16 being the most prevalent. Three of these RATs,
predicted as full trisomies in the placenta, were found to be
mosaic in the fetus; 25 other RATs were predicted to be
mosaic, 8 of which have been confirmed in placental tissue. The new pipeline also correctly predicted twin pregnancies
with discordant fetal sex. Conclusion:
Even
with
normal
karyotype/CMA,
a
NT>99th centile is associated with an adverse pregnancy
outcome, as in one third of cases a congenital malformation
and/or a miscarriage was observed. More interestingly our
study demonstrate a RASopathy gene variant in 4/10 (10%)
fetuses, in the absence of ultrasound markers that could
address the diagnostic suspicion. Conclusion:
Even
with
normal
karyotype/CMA,
a
NT>99th centile is associated with an adverse pregnancy
outcome, as in one third of cases a congenital malformation
and/or a miscarriage was observed. More interestingly our
study demonstrate a RASopathy gene variant in 4/10 (10%)
fetuses, in the absence of ultrasound markers that could
address the diagnostic suspicion. Conclusions: This improved analysis pipeline permits the
detection of autosomal aneuploidies and pinpoints preg-
nancies at risk of fetoplacental mosaicism. This knowledge
can influence estimation of the risk for miscarriage, aid in
genetic counselling and improve prenatal management. R. Ficarella: None. M. Mucciolo: None. C. Votino:
None. F.R. Lepri: None. M.F. Antonucci: None. A.L. Buonadonna: None. V. Lanari: None. P. Volpe: None. M. Gentile: None. A. P01.64D Novel pathogenic splice variant in PALB2 gene causing
anemia Fanconi identified by transcriptomic analysis N. Brison1, M. Neofytou1, L. Dehaspe1, B. Bayindir1, K. Van
Den Bogaert1, L. Dardour2, H. Peeters1, H. Van Esch1, G. Van
Buggenhout1, A. Vogels1, J. Breckpot1, T. de Ravel1, E. Legius1,
K. Devriendt1, J. R. Vermeesch1 translucency but normal karyotype and CMA: impact on
the counselling and need for further genetic investigations I. Viakhireva1, E. Musatova1,2, E. Pomerantseva3,
Y. Shcherbatyuk4, S. Korobkov4, S. Zhikriveckaya2
5
1 6 I. Viakhireva1, E. Musatova1,2, E. Pomerantseva3, Y. Shcherbatyuk4, S. Korobkov4, S. Zhikriveckaya2, F. Konovalov5, M. Skoblov1,6 I. Viakhireva1, E. Musatova1,2, E. Pomerantseva3,
Y. Shcherbatyuk4, S. Korobkov4, S. Zhikriveckaya2, P01.63C Pregnancy outcome for fetuses with increased nuchal Pregnancy outcome for fetuses with increased nuchal 28 J. del Picchia 1Center for Medical Genetics Ghent, Ghent University
Hospital, Ghent, Belgium, 2Department for Reproductive
Medicine, Ghent University Hospital, Ghent, Belgium 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Center of Genetics and Reproductive Medicine
"Genetico", LLC, Moscow, Russian Federation, 3Center of
Genetics and Reproductive Medicine, Moscow, Russian
Federation, 4Hospital Lapino, MD Medical Group, Moscow,
Russian Federation, 5Genomed, Ltd, Moscow, Russian
Federation, 6Moscow Institute of Physics and Technology,
Dolgoprudny, Russian Federation 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Center of Genetics and Reproductive Medicine
"Genetico", LLC, Moscow, Russian Federation, 3Center of
Genetics and Reproductive Medicine, Moscow, Russian
Federation, 4Hospital Lapino, MD Medical Group, Moscow,
Russian Federation, 5Genomed, Ltd, Moscow, Russian
Federation, 6Moscow Institute of Physics and Technology,
Dolgoprudny, Russian Federation Preimplantation genetic diagnosis (PGD) for chromosomal
rearrangements is used to avoid the transfer of embryos with
genomic aberrations to the uterus and hence to improve
implantation rate and avoid miscarriage or the birth of
children with congenital anomalies. Currently, genomic
microarrays are predominantly used for the detection of
unbalanced structural abnormalities and aneuploidies in
embryos from parents at risk. There are however several
limitations to the use of microarrays such as constraints on
resolution and throughput. With the advent of massive
parallel sequencing (MPS), we investigated the use of
shallow whole genome sequencing for PGD (CNVseq) on
trophectoderm biopsies in our clinical diagnostic workflow. PGD was performed on embryos of translocation carriers in
combination with vitrification and frozen embryo transfer in
non-stimulated cycles. Data were collected from January
2016 onwards. Fanconi anemia is rare congenital disease caused by muta-
tions in genes responsible for DNA repair and expressing in
chromosomal instability. Although the main symptom is
anemia, clinical pattern differs in parents with mutations in
genes according to different complementation group. The
most severe clinical pattern is described in cases with
PALB2 gene mutations. These children develop severe
anemia and early onset of different oncological diseases
such as medulloblastoma, Wilms tumor, different leuke-
mias. We report clinical case of a child with Fanconi anemia
died because of medulloblastoma at the age 4 years
10 months. The disease was caused by frameshifting
mutation in PALB2 gene c. 172_175del inherited from
mother
and
novel
intronic
deletion
NC_000016.9:g. 23625423delAAAAATA inherited from father. Frameshift
mutation was identified by exome sequencing of the
affected child. A. Dheedene1, I. De Croo2, E. Van den Abbeel2, P. De Sutter2,
K. Tilleman2, B. Menten1 P01.67C I. Viakhireva: None. E. Musatova: None. E. Pomer-
antseva: None. Y. Shcherbatyuk: None. S. Korobkov:
None. S. Zhikriveckaya: None. F. Konovalov: None. M. Skoblov: None. Whole-exome sequencing identifies novel causative variants
and expands the phenotypic spectrum of PLK4-related
primary microcephaly P. Boonsawat1, R. Asadollahi1, D. Niedrist1, P. Joset1, J. Wisser2,
H. Budka3, P. K. Bode4, H. Sticht5, K. Steindl1, A. Rauch1 P01.63C The deletion was identified in father’s blood
by transcriptomic analysis. Functional analysis of mutation
in minigene system confirmed its pathogenicity. As the
family was interested in having a healthy child, under-
standing of molecular causes of disease in this family
allowed to perform preimplantation genetic testing for
monogenic disease (PGT-M). One IVF cycle was per-
formed and 10 embryos were biopsied for PGT-M. According to the PGT-M results, it was determined that 3
embryos had both variants in heterozygose stage, 1 embryo
inherited only c. 172_175del variant, 4 embryos inherited
only NC_000016.9:g. 23625423delAAAAATA variant and
2 embryos did not inherit either of two variants. Pre-
implantation testing for aneuploidies was performed for
these two embryos and they defined as euploid and were
recommended for transfer. In total 45 PGD cycles have been performed for
reciprocal (n = 39) and Robertsonian (n = 5) translocation
and inversion (n = 1) carriers (total number of embryos =-
185). Almost 60% of the analysed embryos showed
chromosomal aberrations, which is in line with our earlier
results with microarrays (Christodoulou et al., Fertilty&S-
terility, 2017). The CNVseq protocol shows especially for
small chromosomal segments better results than micro-
arrays, and a resolution of ~5 Mb is achieved. Furthermore,
many samples can be processed in batch leading to higher
throughput. Besides abnormalities due to the parental
rearrangement, also other chromosome abnormalities were
detected in our cohort. We describe the successful implementation of CNVseq
on blastocysts in patients with a chromosomal rearrange-
ment to identify euploid embryos for transfer. A. Dheedene: None. I. De Croo: None. E. Van den
Abbeel: None. P. De Sutter: None. K. Tilleman: None. B. Menten: None. 1Institute of Medical Genetics, Schlieren-Zurich, Switzerland,
2Department of Obstetrics, Zurich, Switzerland, 3Institute of
Neuropathology, Zurich, Switzerland, 4Institute of Pathology P01.66B Methods: We sequenced transcriptomes and miRNomes
from term placental samples from normal (n = 8) and PE
(n = 8) pregnancies and 1st trimester samples from 2 RPL
cases and electively terminated pregnancies (ETP; n = 8). Differential
expression
was
tested
using
DESeq
and
DESeq2. g:Profiler was used for enrichment analysis. Methods: We sequenced transcriptomes and miRNomes
from term placental samples from normal (n = 8) and PE
(n = 8) pregnancies and 1st trimester samples from 2 RPL
cases and electively terminated pregnancies (ETP; n = 8). Differential
expression
was
tested
using
DESeq
and
DESeq2. g:Profiler was used for enrichment analysis. Results: In placentas of RPL cases, we detected 195
transcripts with altered expression in RPL compared to ETP
(1). Over 60% of genes with altered expression in RPL
possess binding sites for E2F transcription factors. E2F
regulates the cell cycle and coordinates the mammalian
endocycle and placental development. Results: In placentas of RPL cases, we detected 195
transcripts with altered expression in RPL compared to ETP
(1). Over 60% of genes with altered expression in RPL
possess binding sites for E2F transcription factors. E2F
regulates the cell cycle and coordinates the mammalian
endocycle and placental development. Expression of 215 genes was altered in PE (2). Promoters
of down-regulated genes (n = 173) exhibited strong enrich-
ment for binding sites for transcription factors AP2, SP1
and LRF. Promoters of 77 genes (44.5%) contain potential
response elements for all three transcription factors. Correlation
analysis
between
microRNA
and
gene
expression identified an extensive network of coordinated
expression involving multiple transcripts and microRNAs. Conclusions: The E2F family of transcription factors
represents a potential central coordinator of the shut-down
of nuclear and cellular functions leading to fetal demise. Inadequate AP2, SP1 and LRF activity along with altered
microRNA levels may drive gene expression changes in
pre-eclampsia. (1)Sõber et al. SciRep (2016): 38439. (1)Sõber et al. SciRep (2016): 38439. (1)Sõber et al. SciRep (2016): 38439. (2)Sõber et al. SciRep (2015): 13336. Grants: IUT34-12 (Estonian Research Agency), Happy
Pregnancy (SA Archimedes). Grants: IUT34-12 (Estonian Research Agency), Happy
Pregnancy (SA Archimedes). P. Boonsawat: None. R. Asadollahi: None. D. Niedrist:
None. P. Joset: None. J. Wisser: None. H. Budka: None. P.K. Bode: None. H. Sticht: None. K. Steindl: None. A. Rauch: None. S. Sõber: None. M. Reiman: None. K. Rull: None. M. Laan: None. Preimplantation genetic testing for cystic fibrosis and
aneuploidy in clinical practice Preimplantation genetic testing for cystic fibrosis and
aneuploidy in clinical practice P01.66B Preimplantation genetic diagnosis for chromosomal
rearrangements using shallow whole genome sequencing at
the blastocyst stage 1Institute of Medical Genetics, Schlieren-Zurich, Switzerland,
2Department of Obstetrics, Zurich, Switzerland, 3Institute of
Neuropathology, Zurich, Switzerland, 4Institute of Pathology A. Dheedene1, I. De Croo2, E. Van den Abbeel2, P. De Sutter2,
K. Tilleman2, B. Menten1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 29 and Molecular Pathology, Zurich, Switzerland, 5Institute of
Biochemistry, Erlangen, Germany Introduction: Approximately 5% of pregnancies suffer
from pre-eclampsia (PE) and 3% of couples are affected by
recurrent pregnancy loss (RPL). Loss-of-function variants in PLK4, encoding a key regulator
of centriole duplication, cause autosomal recessive micro-
cephaly and chorioretinopathy 2 (MCCRP2). Currently,
only 13 cases from six families have been reported har-
boring four recessive variants. Following whole-exome
sequencing analysis in 61 microcephalic cases, we identi-
fied novel causative PLK4 variants in two aborted sib
fetuses and an additional unrelated child. In the two fetuses,
we found a nonsense variant and a serine substitution in
compound heterozygous (CH) state, which the latter likely
creates an additional phosphorylation site in the phospho-
degron element of PLK4, leading to reduced protein level
via accelerated autodestruction. Autopsy examination of the
fetuses revealed white matter neuronal heterotopia and
cerebellar vermis hypoplasia in one, and absence of corpus
callosum in the other, apart from facial dysmorphism and
microcephaly. Additional physical anomalies included 2-
lobed right lung and accessory spleen, which have not been
previously reported in MCCRP2. Furthermore, we identi-
fied (likely) pathogenic CH variants in the unrelated child,
presenting with primary microcephaly, facial dysmorphism
and moderate speech delay. Brain MRI showed simplified
cortical gyri, dysplastic corpus callosum, and novel finding
of large cerebellum-brain stem relative to the supratentorial
region. Considering our cases and those previously repor-
ted, we consistently observed simplified gyri, abnormal
corpus callosum and neuronal heterotopia, suggesting the
importance of PLK4 in the regulation of neuronal migra-
tion. Moreover, we report a novel MRI finding as well as
additional organ anomalies in MCCRP2, and describe the
first deleterious missense variant located in the phospho-
degron element outside the main PLK4 domains. Aims: To identify mechanisms affecting gene expression
in common pregnancy complications PE and RPL. 1Institute of Biomedicine and Translational Medicine,
University of Tartu, Tartu, Estonia, 2Department of Obstetrics
and Gynaecology, University of Tartu, Tartu, Estonia,
3Women’s Clinic of Tartu University Hospital, Tartu, Estonia P01.68D Evidence for key roles of transcription factors and
microRNAs in orchestrating placental gene expression
patterns in common pregnancy complications GENNET s.r.o., Praha, Czech Republic S. Sõber1, M. Reiman1, K. Rull1,2,3, M. Laan1 S. Sõber1, M. Reiman1, K. Rull1,2,3, M. Laan1 Introduction: Cystic fibrosis (CF) due to mutations in the
CFTR gene is the most frequent reason for preimplantation
genetic testing of monogenic disorders (PGT-M). Many
women requiring PGD for CF are at advanced age that is a
limiting factor of IVF success. 1Institute of Biomedicine and Translational Medicine,
University of Tartu, Tartu, Estonia, 2Department of Obstetrics
and Gynaecology, University of Tartu, Tartu, Estonia,
3Women’s Clinic of Tartu University Hospital, Tartu, Estonia Materials and methods: We have performed PGT for
CF in 81 IVF cycles for 51 couples between the years 2007 30 J. del Picchia and 2017. The total number of examined embryos is 438. We have examined 317 samples of blastomeres from
cleavage stage embryos and 121 samples of trophectoderm
from blastocysts. The PGT-M for was performed by
haplotyping using whole genome amplification (WGA)
and multiplex fluorescence PCR analysis of short tandem
repeat polymorphisms (STR markers) linked to the CFTR
gene. We have recently added the aneuploidy detection
(PGT-A) by NGS using Ion Torrent Proton as a second step
in the evaluation of trophectoderm samples. Results: All samples were correctly classified and all
abnormalities
were
detected
including
numerical
and
structural rearrangements. Results obtained were in agree-
ment with array CGH. Conclusions:
Targeted
sequencing
is
the
preferred
method for applications requiring high read depth. This
assay in combination with a novel bioinformatics pipeline
can be used for the genome-wide screening of fertilized
embryos (PGS/PGD). It can also be used in cases where
limited number of cells from affected tissues/individuals are
available. Results: We have amplified the DNA from 295 out of
317 single blastomeres (93%) and 104 out of 121
trophectoderm samples (86%). We have found 246 embryos
not affected by CF (62%) or other abnormalities of
chromosome 7 (monosomy, trisomy) using the haplotype
analysis. We have further analysed the amplified DNA from
72 trophectoderm samples and detected aneuploidy in 31
out of them (43 %). M. Ioannides: A. Employment (full or part-time);
Significant; NIPD Genetics. A. Achilleos: A. Employment
(full or part-time); Significant; NIPD Genetics. K. Tsan-
garas: A. Employment (full or part-time); Significant;
NIPD Genetics. C. Loizides: A. Employment (full or part-
time); Significant; NIPD Genetics. P. Mina: A. Employ-
ment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant;
NIPD Genetics. C. Sismani: None. G. P01.70B Implementation of target capture enrichment on single and
few cells for the robust detection of embryo abnormalities R. Staneva1,2, S. Hadjidekova1,2, S. Andonova3, A. Savov3,
S. Bitchev3, S. Yaneva2, M. Pancheva4, M. Serafimova2,
T. Chaushev2, K. Nikolova2, D. Toncheva1, G. Stamenov2 M. Ioannides1, A. Achilleos1, K. Tsangaras1, C. Loizides1,
P. Mina1, E. Kypri1, C. Sismani2, G. Koumbaris1, P. C. Patsalis1
1NIPD Genetics, Nicosia, Cyprus, 2The Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus M. Ioannides1, A. Achilleos1, K. Tsangaras1, C. Loizides1,
P. Mina1, E. Kypri1, C. Sismani2, G. Koumbaris1, P. C. Patsalis1 1Department of Medical Genetics, Medical University of Sofia,
Sofia, Bulgaria, 2Women's Health Hospital "Nadezhda", Sofia,
Bulgaria, 3National Genetic Laboratory, UHOG “Maichin
dom”, Sofia, Bulgaria, 4Women's Health Hospital, Sofia,
Bulgaria 1NIPD Genetics, Nicosia, Cyprus, 2The Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus 1NIPD Genetics, Nicosia, Cyprus, 2The Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus Introduction: High throughput non-invasive prenatal test-
ing (NIPT) technologies have demonstrated safe, accurate
and reliable results for the detection of fetal abnormalities,
relying on the detection and analysis of cell free fetal DNA
(cffDNA) in maternal plasma. However, such analysis is
often limited by the low abundance of DNA, as in the case
of fertilized embryos. Therefore, the development of novel,
sensitive approaches which can provide reliable results from
single/few cells is necessary. Background: In the last decade preimplantation genetic
testing (PGT) for severe genetic diseases emerged as a
viable alternative to prenatal testing and termination of
pregnancy for couples where one or both partners is a
carrier or suffering from a debilitating genetic condition. Background: In the last decade preimplantation genetic
testing (PGT) for severe genetic diseases emerged as a
viable alternative to prenatal testing and termination of
pregnancy for couples where one or both partners is a
carrier or suffering from a debilitating genetic condition. Materials & Methods: 7 couples opted for IVF-PGT
after extensive genetic counseling. Indications were beta-
thalassemia, epidermolysis bullosa, myotonic dystrophy
type 1, Huntingtion disease, Fragile-X syndrome, hemo-
philia A and Duchenne muscular dystrophy. Trophectoderm
biopsy
of
32
5-day
embryos
was
performed. DNA
amplification was achieved by Repli-g (Qiagen). For all
trinucleotide repeat disorders allele size was determined by
fragment length analysis (TNR Diagnostics). RT-PCR was
applied for epidermolysis bullosa and beta-thalassemia Materials and Methods: Amplified DNA isolated from
seven and 17 embryos was obtained from 3-day and 5-day
biopsy cases. GENNET s.r.o., Praha, Czech Republic Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Sig-
nificant; NIPD Genetics. Conclusions: We have successfully used trophectoderm
biopsy and whole genome amplification to combine the
preimplantation genetic testing of a monogenic disorder
(PGT-M) with aneuploidy detection (PGT-A) in clinical
practice. Conclusions: We have successfully used trophectoderm
biopsy and whole genome amplification to combine the
preimplantation genetic testing of a monogenic disorder
(PGT-M) with aneuploidy detection (PGT-A) in clinical
practice. J. Diblík:
None. I. Soldátova:
None. M. Sekowská: None. P01.71C Various approaches to preimplantation genetic testing -
experience from seven monogenic disorders Various approaches to preimplantation genetic testing -
experience from seven monogenic disorders Preimplantation genetic testing for polycystic kidney
disease is an option for affected families Preimplantation genetic testing for polycystic kidney
disease is an option for affected families 1Institute of Molecular and Cell Biology, University of Tartu,
Tartu, Estonia, 2Competence Center on Health Technologies,
Tartu, Estonia, 3Department of Medical Biotechnology and
Translational Medicine Università degli Studi di Milano,
Milan, Italy, 4Medical Cytogenetics and Molecular Genetics
Lab, IRCCS Istituto Auxologico Italiano, Milan, Italy,
5Genetics of Common Disorders, Division of Genetics and Cell
Biology, San Raffaele Scientific Institute, Milan, Italy, 6Chair
of Obstetrics and Gynaecology, University of Tartu, Tartu,
Estonia V. Berckmoes1, P. Verdyck1, P. De Becker1, A. De Vos2,
G. Verheyen2, P. Van der Niepen3, W. Verpoest2, I. Liebaers1,
M. Bonduelle1, M. De Rycke1 V. Berckmoes1, P. Verdyck1, P. De Becker1, A. De Vos2,
G. Verheyen2, P. Van der Niepen3, W. Verpoest2, I. Liebaers1,
M. Bonduelle1, M. De Rycke1 1Centre for Medical Genetics, Brussel, Belgium, 2Centre for
Reproductive Medicine, Brussel, Belgium, 3Nephrology &
Hypertension Department, Brussel, Belgium Introduction: In this study, preimplantation genetic testing
(PGT) data for polycystic kidney disease (PKD) from 2005
until 2016 are reported. As males affected with autosomal
dominant PKD (ADPKD) may present with reproductive
system abnormalities and infertility, the clinical outcome
was compared between couples with the female partner
affected with ADPKD and couples with the male partner
affected with ADPKD. Premature ovarian failure (POF) is considered as a multi-
factorial and heterogeneous condition affecting approxi-
mately 1% women of reproductive age. Despite the
extensive research, the considerably complex pathogenesis
of POF is still not well understood. POF can develop as
result of a broad spectrum of pathogenic mechanisms
including genetic, autoimmune and iatrogenic causes that
leads to follicular dysfunction or depletion. In recent years,
many research studies are trying to find out the genetic
component of the disease by using different high-resolution
methods. We have performed a case-control genetic asso-
ciation study, using high-resolution SNP microarrays to
investigate DNA copy number variations (CNVs) of 216
Italian women presenting POF phenotype and 240 women
from the Italian general population as a control. All patient
and control samples were collected at the Division of
Genetics and Cell Biology, San Raffaele Scientific Institute
and University of Milan, Italy and genotyped by Illumina Materials and Methods: Sixteen single-cell clinical tests
for PKD based on multiplex PCR of STR markers were
applied for 91 PGT cycles for 43 couples. P01.73A
DNA copy number variations in a cohort of 216 Italian
women with premature ovarian failure P01.73A
DNA copy number variations in a cohort of 216 Italian
women with premature ovarian failure K. Teearu1, O. ilina1, O. Tšuiko1,2, A. Marozzi3, P. Finelli3,4,
I. Bestetti3,4, D. Toniolo5, A. Salumets2,6, A. Kurg1 P01.70B Conclusion: IVF-PGT is a valid option for couples at risk
to have a child with severe genetic condition. R. Staneva: None. S. Hadjidekova: None. S. Ando-
nova: None. A. Savov: None. S. Bitchev: None. S. Yaneva: None. M. Pancheva: None. M. Serafimova:
None. T. Chaushev: None. K. Nikolova: None. D. Toncheva: None. G. Stamenov: None. P01.70B TArget Capture Sequences (TACS) were
designed at a median resolution of 1Mb spanning all
chromosomes
and
were
used
to
perform
in-solution
hybridization capture enrichment as previously described1. Novel bioinformatics algorithms were also developed to
determine the ploidy status of the samples. Materials and Methods: Amplified DNA isolated from
seven and 17 embryos was obtained from 3-day and 5-day
biopsy cases. TArget Capture Sequences (TACS) were
designed at a median resolution of 1Mb spanning all
chromosomes
and
were
used
to
perform
in-solution
hybridization capture enrichment as previously described1. Novel bioinformatics algorithms were also developed to
determine the ploidy status of the samples. 31 Abstracts from the 51st European Society of Human Genetics Conference: Posters (Microsynth), Sanger sequencing for haemophilia A and sex
determination by aCGH for DMD. All protocols were tested
in advance on donated unviable embryos. after five treatment cycles. The clinical pregnancy rate and
live birth delivery rate was significantly lower for couples
with the male partner affected with ADPKD compared with
couples with the female partner affected with ADPKD. However, female age was the only variable significantly
associated with live birth delivery rate. Results: DNA from 31 embryos was available for testing
after amplification (96,9%). DNA analysis was successful
for all 31 embryos (100%). There were 21 unaffected
embryos and 3 females (for the DMD case). Four of the
couples (57,1%) achieved pregnancy after the first transfer,
2 couples (28,6%) after the second and only one (14,3%)
did not get pregnant after three transfers, giving a 85,7%
success rate of the IVF-PGT procedure. Except one
pregnancy that has not yet reached time for prenatal
confirmation, all PGT results were confirmed by DNA
testing of CVS samples, 6 healthy babies were delivered. Conclusions: This study shows that PGT for PKD
performed in our centre offers good reproductive outcomes
from both fresh and frozen embryo transfers. Males affected
with ADPKD who suffer from infertility should be advised
to seek treatment on time to improve their chances of
conceiving a child. V. Berckmoes: None. P. Verdyck: None. P. De Becker:
None. A. De Vos: None. G. Verheyen: None. P. Van der
Niepen: None. W. Verpoest: None. I. Liebaers: None. M. Bonduelle: None. M. De Rycke: None. Conclusion: IVF-PGT is a valid option for couples at risk
to have a child with severe genetic condition. GeneDx, Gaithersburg, MD, United States Guy’s and St Thomas’ NHS Foundation Trust, London, United
Kingdom Guy’s and St Thomas’ NHS Foundation Trust, London, United
Kingdom Introduction: Fetal ultrasound abnormalities pose a unique
diagnostic challenge. In order to help increase the diag-
nostic
yield
of
underlying
genetic
etiologies
exome
sequencing (ES) is gaining use in the prenatal setting. There is currently no clear consensus on the cost-benefit of
detailed genome-wide copy-number analysis for pregnan-
cies with ultrasound anomalies. Decisions regarding test
resolution should consider evidence concerning diagnostic
yield, cost (technical and analytical), reporting times and the
number of uncertain and incidental findings. From 2012 to
2017 we investigated pregnancies with ultrasound anoma-
lies using a prenatal array testing strategy designed to
minimise uncertain results and incidental findings whilst
identifying clinically significant abnormalities; a 3Mb
backbone resolution was supplemented with high resolution
analysis of 23 regions known to be associated with severe,
fully penetrant syndromes. Review of 480 of these cases
(after anonymization) at an average of 120kb resolution
found no severe, fully penetrant imbalance had been mis-
sed. European guidelines now recommend a genome-wide
resolution of at least 400kb (the evidence base for this
guideline is unclear). To comply with this, in 2017 we
introduced a 400kb analysis strategy, with high resolution
analysis of 25 syndrome regions; only findings associated
with the ultrasound anomalies and actionable or severe,
early-onset incidental findings were reported. Of 201 pre-
natal samples tested, 22 were reported as abnormal,
including one incidental finding, 178 as normal and one Methods: We retrospectively analyzed ES results on 233
deceased fetuses and 33 ongoing pregnancies with ultra-
sound anomalies. Methods: We retrospectively analyzed ES results on 233
deceased fetuses and 33 ongoing pregnancies with ultra-
sound anomalies. Results: Of the 233 deceased fetuses, 76% of testing was
performed as proband-parent trios. Fifty-six percent were
male and 44% female. Most cases had multiple congenital
anomalies (MCA) (78%). The most common ultrasound
findings were central nervous system (CNS) anomalies
(51%),
hydrops
(33%),
skeletal
abnormalities
(30%),
cardiovascular
defects
(28%),
neuromuscular
findings
(26%), and genitourinary anomalies (24%). A definitive
molecular diagnosis was identified in 28% of cases, a
possible diagnosis in 36%, only a candidate gene was
reported in 10%, and 26% of cases had no reportable
variants. We also analyzed 33 prenatal specimens from
ongoing pregnancies. All cases were trios and had non-
diagnostic standard genetic testing prior to ES. The diagnostic yield of exome sequencing in the prenatal
setting: A clinical laboratory experience The diagnostic yield of exome sequencing in the prenatal
setting: A clinical laboratory experience Preimplantation genetic testing for polycystic kidney
disease is an option for affected families Employ-
ment (full or part-time); Significant; GeneDx. R. Chikar-
mane: A. Employment (full or part-time); Significant;
GeneDx. J. Juusola: A. Employment (full or part-time);
Significant; GeneDx. A. Telegrafi:
A. Employment
(full
or
part-time);
Significant; GeneDx. C. Yates: A. Employment (full or
part-time); Significant; GeneDx. K.G. Monaghan: A. Employment (full or part-time); Significant; GeneDx. E. Ryan: A. Employment (full or part-time); Significant;
GeneDx. B. Friedman: A. Employment (full or part-time);
Significant; GeneDx. H. Sroka: A. Employment (full or
part-time); Significant; GeneDx. R. Willaert: A. Employ-
ment (full or part-time); Significant; GeneDx. R. Chikar-
mane: A. Employment (full or part-time); Significant;
GeneDx. J. Juusola: A. Employment (full or part-time);
Significant; GeneDx. K. Teearu: None. O. ilina: None. O. Tšuiko: None. A. Marozzi: None. P. Finelli: None. I. Bestetti: None. D. Toniolo: None. A. Salumets: None. A. Kurg: None. K. Teearu: None. O. ilina: None. O. Tšuiko: None. A. Marozzi: None. P. Finelli: None. I. Bestetti: None. D. Toniolo: None. A. Salumets: None. A. Kurg: None. Prenatal array CGH: comparison of 400kb and 3Mb
resolution A. Telegrafi, C. Yates, K. G. Monaghan, E. Ryan, B. Friedman,
H. Sroka, R. Willaert, R. Chikarmane, J. Juusola K. Mann, J. Ahn, S. Bint, C. Brown, C. Mackie Ogilvie K. Mann, J. Ahn, S. Bint, C. Brown, C. Mackie Ogilvie K. Mann, J. Ahn, S. Bint, C. Brown, C. Mackie Ogilvie Preimplantation genetic testing for polycystic kidney
disease is an option for affected families Results: A diagnosis was obtained for 93.3% of the
analysed
embryos
of
which
36.8%
were
genetically
transferable. Transfer of 74 embryos in 53 fresh cycles
and transfer of 34 cryopreserved embryos in 33 frozen-
warmed embryo transfer cycles resulted in a live birth
delivery rate of 38.4% per transfer with 31 singleton live
births, 2 twin live births and 1 ongoing pregnancy. The
observed cumulative delivery rate was 57.8% per couple 32 J. del Picchia PsychArray BeadChips at the Estonian Genome Center
University of Tartu Genotyping core in Tartu, Estonia. Microarray data,
analyzed using
different algorithms,
revealed both, genomic regions containing genes previously
associated with POF (e.g. 26.8Mb 1q41-q44 duplication
affecting FMN2 gene) and novel potentially clinically sig-
nificant CNVs (e.g. 11p15.2 microdeletion). In addition to
autosomal CNVs, we identified several POF critical regions
on the X chromosome. The currently known POF genes
only account for a small proportion of patients, while the
majority remain without a genetic diagnosis. Using whole-
genome DNA microarrays, the present study provides novel
insight into the implications of CNVs in genetic aetiology
of POF. PsychArray BeadChips at the Estonian Genome Center
University of Tartu Genotyping core in Tartu, Estonia. Microarray data,
analyzed using
different algorithms,
revealed both, genomic regions containing genes previously
associated with POF (e.g. 26.8Mb 1q41-q44 duplication
affecting FMN2 gene) and novel potentially clinically sig-
nificant CNVs (e.g. 11p15.2 microdeletion). In addition to
autosomal CNVs, we identified several POF critical regions
on the X chromosome. The currently known POF genes
only account for a small proportion of patients, while the
majority remain without a genetic diagnosis. Using whole-
genome DNA microarrays, the present study provides novel
insight into the implications of CNVs in genetic aetiology
of POF. identified in 30.3% of cases, a possible diagnosis in 33.3%,
only a candidate gene was reported in 6.1%, and 30.3% had
no reportable variants. Conclusion: Although interpretive and ethical issues
remain a concern in the prenatal setting, ES may identify
genetic variants responsible for fetal anomalies and impact
prognosis, medical management, and recurrence risks. A. Telegrafi:
A. Employment
(full
or
part-time);
Significant; GeneDx. C. Yates: A. Employment (full or
part-time); Significant; GeneDx. K.G. Monaghan: A. Employment (full or part-time); Significant; GeneDx. E. Ryan: A. Employment (full or part-time); Significant;
GeneDx. B. Friedman: A. Employment (full or part-time);
Significant; GeneDx. H. Sroka: A. Employment (full or
part-time); Significant; GeneDx. R. Willaert: A. Case report: Gonosomal placental mosaicism leads to a
false-positive NIPT result Case report: Gonosomal placental mosaicism leads to a
false-positive NIPT result T. Harasim1, A. Wagner1, U. Heinrich1, E. Krimmel1,
M. Delius2, I. Rost1, H. Klein1 The introduction of whole exome sequencing (WES) in
genome diagnostics has dramatically changed the current
practice in clinical genetics. It is expected that WES and
ultimately whole genome sequencing will replace current
routine clinical practice (array based technologies), not only
after birth but also during pregnancy. The implementation
of WES in a prenatal setting for genetic analysis of fetuses
with multiple congenital abnormalities has the potential to
increase the diagnostic yield and thereby improving prog-
nostic information for professionals and expectant parents. However, there are a number of factors that need to be taken
into account before WES could be part of the prenatal
diagnostic workup. These include technical and practical
aspects like long turn-around-times (TATs), costs, difficul-
ties in interpreting variants and limited possibilities to
determine the fetal phenotype. Intensive collaboration
within a multi-disciplinary team consisting of a molecular
laboratory specialist, a clinical geneticist and a fetal medi-
cine specialist is required. Furthermore, the possibility of
detecting variants of unknown significance (VOUS) or
incidental findings (IF) may lead to ethical dilemmas and
demands careful pre- and post-test counselling. 1Center for Human Genetics and Laboratory Diagnostics, Dr. Klein, Dr. Rost and Colleagues, Martinsried, Germany,
2Department of Gynecology and Obstetrics of the hospital of
the Ludwig-Maximilians-University, Munich, Germany Introduction: A 37 years old pregnant woman with no fetal
ultrasound abnormalities received a NIPT result at early
gestational age indicating a monosomy X. For confirmatory
purpose, amniocentesis including aCGH was performed: the
fetal karyotype was reported to be normal, male (46,XY)
instead of the expected 45,X. Perinatally, another NIPT
with high sequencing depth was requested (Prenatalis®,
MVZ Martinsried) in concert with postpartal FISH of pla-
cental villi to shed light into these discordant results. Method: Prenatalis® was performed with ~ 21 million
reads used for detection of aneuploidy 13, 18, 21, X and Y. Postpartal FISH analysis was done on nuclei of placental
villi by using the centromere specific DXZ1-, DYZ3- and
D18Z- probes (Cytocell, Cambridge, UK). Results: Prenatalis® confirmed the initial monosomy X
result at a fetal fraction of 39% (GA:36+6). FISH-analysis
revealed placental mosaicism with a dominant X0-cell line
(86% of all nuclei) in combination with a XY cell line
(10%). 4% of nuclei showed two X-chromosomes, probably
a contamination of maternal cells. P01.77A 1Radboud university medical centre, Nijmegen, Netherlands,
2Maastricht University Medical Centre, Maastricht,
Netherlands P01.78B
Rapid whole exome sequencing; implementation in the
prenatal setting I. Feenstra1, Y. Arens2, S. de Munnik1, A. C. Deden1, A. C. J. Gijsbers1, V. van der Schoot2, W. van Zelst-Stams1, E. Sikkel1,
D. Smeets1, K. Neveling1, M. Nelen1, H. Yntema1 K. Mann: None. J. Ahn: None. S. Bint: None. C. Brown: None. C. Mackie Ogilvie: None. GeneDx, Gaithersburg, MD, United States Seventy-
three percent were male and 27% female. Most cases had
MCA
(79%). Common
ultrasound
findings
included
cardiovascular anomalies (33%), genitourinary abnormal-
ities (33%), CNS defects (30%), and hydrops (30%). For all
ongoing pregnancies, a definitive molecular diagnosis was Abstracts from the 51st European Society of Human Genetics Conference: Posters 33 failed. Only one case (0.5% of samples), a de novo 1.6 Mb
15q25 deletion (OMIM 614294) would not have been
identified using our previous analysis strategy. Fourteen
unique imbalances (7% of samples) were not reported fol-
lowing detailed variant classification; in addition, four
susceptibility loci (class 4 variants) were not disclosed. A
cost-benefit
comparison
of
these
strategies
will
be
discussed. failed. Only one case (0.5% of samples), a de novo 1.6 Mb
15q25 deletion (OMIM 614294) would not have been
identified using our previous analysis strategy. Fourteen
unique imbalances (7% of samples) were not reported fol-
lowing detailed variant classification; in addition, four
susceptibility loci (class 4 variants) were not disclosed. A
cost-benefit
comparison
of
these
strategies
will
be
discussed. failed. Only one case (0.5% of samples), a de novo 1.6 Mb
15q25 deletion (OMIM 614294) would not have been
identified using our previous analysis strategy. Fourteen
unique imbalances (7% of samples) were not reported fol-
lowing detailed variant classification; in addition, four
susceptibility loci (class 4 variants) were not disclosed. A
cost-benefit
comparison
of
these
strategies
will
be
discussed. T. Harasim: None. A. Wagner: None. U. Heinrich:
None. E. Krimmel: None. M. Delius: None. I. Rost: None. H. Klein: None. I. Bestetti1,2, C. Barbieri3, A. Sironi1,2, C. Castronovo1,
C. Caslini2, R. Rossetti4, A. Pistocchi2, A. Rajkovic5,6,7, C. Sala3,
D. Toniolo3, L. Persani4,8, A. Marozzi2, P. Finelli1,2 I. Bestetti1,2, C. Barbieri3, A. Sironi1,2, C. Castronovo1,
C. Caslini2, R. Rossetti4, A. Pistocchi2, A. Rajkovic5,6,7, C. Sala3,
D. Toniolo3, L. Persani4,8, A. Marozzi2, P. Finelli1,2 1Lab. of Medical Cytogenetics and Molecular Genetics, IRCCS
Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy,
2Dep. of Medical Biotechnology and Translational Medicine,
University of Milan, Milan, Italy, 3Division of Genetics and
Cell Biology, San Raffaele Research Institute and Vita Salute
University, Milan, Italy, 4Lab. of Endocrine and Metabolic
Research and Division of Endocrine and Metabolic Diseases,
IRCCS Istituto Auxologico Italiano, Cusano Milanino, Milan,
Italy, 5Dep. of Obstetrics, Gynecology, and Reproductive
Sciences, Magee-Womens Research Institute, University of
Pittsburgh, Pittsburgh, PA, United States, 6Dep. of Pathology,
University of Pittsburgh, Pittsburgh, PA, United States, 7Dep. of Human Genetics, University of Pittsburgh, Pittsburgh, PA,
United States, 8Dep. of Clinical Sciences and Community
Health, University of Milan, Milan, Italy P. Noveski, M. Terzic, M. Vujovic, M. Kuzmanovska,
E. Sukarova Stefanovska, D. Plaseska-Karanfilska Research Center for Genetic Engineering and Biotechnology
“Georgi D. Efremov”, Macedonian Academy of Sciences and
Arts, Skopje, Macedonia, The Former Yugoslav Republic of The quantitative fluorescent polymerase chain reaction (QF-
PCR) has proven to be a reliable method for detection of
common fetal chromosomal aneuploidies, with advantages
over conventional karyotyping such as cost-effectiveness,
reliability and requirement of only small amount of mate-
rial. However, there are some technical shortcomings,
involving the necessity to perform two or more multiplex
PCR reactions simultaneously for a given sample or the
uncertainty of aneuploidy determination when the STR
(short tandem repeats) height ratio is unusual due to large
size difference between alleles. Here, we present an in-
house one-tube multiplex QF-PCR method including 20
PCR markers (14 STR markers and 6 fixed size) for rapid
prenatal diagnosis of chromosome 13, 18, 21, X and Y
aneuploidies. In order to improve the aneuploidy classifi-
cation of a given diallelic STR marker, we used a total of
7630 diallelic genotypes (88 trisomic and 7542 normal)
from 871 samples to employ multilevel logistic regression
analysis using "height ratio" and "allele size difference" as
fixed effects and "marker" as random effect. We employed
two regression models, one for the 2:1 height ratio (n = 47)
and second for the 1:2 height ratio (n = 41) of the trisomic
diallelic markers. Case report: Gonosomal placental mosaicism leads to a
false-positive NIPT result The patient delivered a
phenotypically normal, male baby, who was not karyotyped
further. Until now, rapid WES with short TATs has been
performed on a case-by-case basis in our centre in a small
number of pregnancies in the second or third trimester. We
will provide an overview of the workflow, the challenges
and the difficulties encountered. To warrant an accurate,
more widespread implementation of prenatal WES, there is
a strong need for clear criteria, data-sharing and an (inter)
national guideline. Conclusion: The results presented above describe con-
fined placental mosaicism of a dominant monosomy X cell
line in concert with a low level XY-cell line. It can be
deduced from the NIPT results, that the placenta released
cfDNA exclusively from 45,X loci, since y-chromosomal
cfDNA could not be detected during NIPT. Since placental
cfDNA only serves as a proxy for the fetus, confirmation of
positive NIPT results are highly recommended. I. Feenstra: None. Y. Arens: None. S. de Munnik:
None. A.C. Deden: None. A.C.J. Gijsbers: None. V. van
der Schoot: None. W. van Zelst-Stams: None. E. Sikkel:
None. D. Smeets: None. K. Neveling: None. M. Nelen:
None. H. Yntema: None. J. del Picchia 34 I. Bestetti1,2, C. Barbieri3, A. Sironi1,2, C. Castronovo1,
C. Caslini2, R. Rossetti4, A. Pistocchi2, A. Rajkovic5,6,7, C. Sala3,
D. Toniolo3, L. Persani4,8, A. Marozzi2, P. Finelli1,2 Both models achieved 100% specificity
for marker aneuploidy classification on training data as
compared to 98.3% (2:1 ratio) and 97.9% (1:2 ratio) spe-
cificity when using only height ratio for classification. In
conclusion, adjusting for the allele size difference and
marker type improves the STR classification, eliminates
sample re-testing and reinforces the robustness of the QF-
PCR method for prenatal testing. POI is a heterogeneous group of disorders that affect
women fertility whose genetic origin has been clarified in
less than 30% of cases. To unveil new causative genes
essential for ovarian function we searched for rare high-
penetrance Copy Number Variants (CNVs) in a cohort of 67
46,XX patients affected by the most severe phenotype
(primary amenorrhea). High-resolution array-CGH analysis
detected 72 rare CNVs according to the Database of
Genomic Variants in 49 patients. CNVs gene content ana-
lysis and disease prioritization selected 37 CNVs involving
2 POI-associated genes and 42 putative candidate genes
(e.g. TP63, VLDLR). The research of this CNVs in an ad-
hoc cohort of 134 control women supported their actual
rarity. Despite the presence of ovary genes also in the ad-
hoc cohort, chi-q and Wilcoxon tests showed in patients a
significant
enrichment
of
ovary-related
CNVs/genes
(P=0.0132/P=0.0126) supporting array-CGH as a valuable
tool in identifying novel POI molecular defects. Array-CGH
genes identified together with their predicted interactors,
and other known ovary/POI-related genes (n = 226) were
then screened in a targeted-WES analysis of 102 secondary
amenorrhea patients. After filtering variants (MAF<0.005;
LoF SNVs inclusion) a total of 375 possibly pathogenic
SNVs were found in 31 array-genes, 12 interactor-genes,
and 83 ovary/POI-related genes. Burden test analysis versus
1000G_EUR control women confirmed a statistical sig-
nificance for 1 interactor-TP63 gene (P=1.65E-07) and 2
ovary/POI-related
genes
(FSHR,
P=1.66E-05;FOXO3,
P=7.31E-05). This combined approach allowed to increase
the knowledge about POI pathogenesis and will probably
provide the basis for a more accurate genetic diagnosis of
POI patients. P. Noveski: None. M. Terzic: None. M. Vujovic: None. M. Kuzmanovska: None. E. Sukarova Stefanovska:
None. D. Plaseska-Karanfilska: None. S. Saini1, R. Foley1, C. Kingsley1, E. Wang1, M. Schmid1,
P. Bogard1, A. Ramos2 I. Tkach1, N. Huleyuk1, D. Zastavna1,2, M. Tyrka2 Spontaneous abortions with aneuploid anomalies
(monosomy X, trisomy 21, trisomy 16 and triploidy) were
characterized by shorter telomeres, compared to sponta-
neous abortions, subgroup with euploid (46,XN) karyotype. Results: Relative telomere length was measured from
total genomic DNA using a real-time polymerase chain
reaction approach. In this study, we examined relative
telomere length in 80 spontaneously eliminated embryos
and in 25 embryos eliminated due to induced abortions. Relative telomere length in spontaneous abortions was
significantly lower (P = 0.000001) compared to the induced
abortions. Spontaneous abortions with aneuploid anomalies
(monosomy X, trisomy 21, trisomy 16 and triploidy) were
characterized by shorter telomeres, compared to sponta-
neous abortions, subgroup with euploid (46,XN) karyotype. Materials and Methods: This retrospective study was
conducted on 73 first-trimester POCs (September 2014-
February 2017). The POCs were collected from 73 women
with at least one previous miscarriage and analysed for
chromosomal anomalies using QF-PCR and aCGH as part
of the routine clinical evaluation. Results: Chromosome aberrations were detected in 52/73
POCs (71.2%), of which 41 (56.2%) were identified by QF-
PCR and an additional 11 (15.1%) by aCGH. Numerical
aberrations constituted the majority (92.3%) of abnormal-
ities, with trisomies as the most common subtype (72.9%). Causative structural aberrations were found in three samples
(5.8%) and variant of unknown significance in one sample. The
frequency
of
chromosome
aberrations
was
not
dependent on the number of previous miscarriages, whereas
it significantly increased with advanced maternal age. Conclusion: Spontaneously lost pregnancies are char-
acterized by shortened telomeres, especially in embryos
with aneuploidies. We hypothesize that the shortening of
telomeres is involved in the processes leading to sponta-
neous abortions. I. Tkach: None. N. Huleyuk: None. D. Zastavna: None. M. Tyrka: None. Conclusions: The results of our comprehensive genetic
analyses of POCs of RM couples confirm the QF-PCR and
aCGH combination as an effective diagnostic strategy. Considering the high frequency of chromosome aberrations,
a routine genetic analysis of POCs should be considered,
which could improve the clinical approach to couples with
miscarriage. Conclusions: The results of our comprehensive genetic
analyses of POCs of RM couples confirm the QF-PCR and
aCGH combination as an effective diagnostic strategy. Considering the high frequency of chromosome aberrations,
a routine genetic analysis of POCs should be considered,
which could improve the clinical approach to couples with
miscarriage. I. Tkach1, N. Huleyuk1, D. Zastavna1,2, M. Tyrka2 1Institute of Hereditary Pathology, NAMS of Ukraine, Lviv,
Ukraine, 2Department of Biotechnology and Bioinformatics,
Faculty of Chemistry, Rzeszow University of Technology,
Rzeszow, Poland 1Institute of Hereditary Pathology, NAMS of Ukraine, Lviv,
Ukraine, 2Department of Biotechnology and Bioinformatics,
Faculty of Chemistry, Rzeszow University of Technology,
Rzeszow, Poland L. Lovrečić1, N. Pereza2, H. Jaklič1, S. Ostojić2, B. Peterlin1 L. Lovrečić1, N. Pereza2, H. Jaklič1, S. Ostojić2, B. Peterlin1 1Clinical Institute of Medical Genetics, University Medical
Center Ljubljana, Department of Gynaecology and Obstetrics,
Ljubljana, Slovenia, 2Faculty of medicine, University of Rijeka,
Department of biology and medical genetics, Rijeka, Croatia Background:
Telomeres
are
transcriptionally
inactive
genomic areas, which, if shortened, are associated with
pathological processes, unsuccessful fertilization, aging,
and death. Telomere dysfunction has also been linked to
chromosomal rearrangements and genomic instability. The
role of telomeres in postnatal life has been extensively
studied and discussed both in physiological as well as in
pathological processes. However, the role of telomere
length in prenatal development is still poorly understood,
and mainly concerns the preimplantation stage. The aim of
this study was to estimate relative telomere length in
spontaneously eliminated human embryos between 5th and
12th week of gestation. Introduction: Although previous studies have shown that
embryonic chromosome aberrations are the most common
cause of recurrent miscarriage (RM), the comprehensive
genetic evaluation using the combination of quantitative
fluorescence-polymerase chain reaction (QF-PCR) and
array-comparative genomic hybridisation (aCGH) was not
used systematically for their detection in the clinical setting. We aimed to investigate the frequency and type of chro-
mosome aberrations in POCs of couples with at least one
previous miscarriage using the QF-PCR and a-CGH
strategy. Introduction: Although previous studies have shown that
embryonic chromosome aberrations are the most common
cause of recurrent miscarriage (RM), the comprehensive
genetic evaluation using the combination of quantitative
fluorescence-polymerase chain reaction (QF-PCR) and
array-comparative genomic hybridisation (aCGH) was not
used systematically for their detection in the clinical setting. We aimed to investigate the frequency and type of chro-
mosome aberrations in POCs of couples with at least one
previous miscarriage using the QF-PCR and a-CGH
strategy. Results: Relative telomere length was measured from
total genomic DNA using a real-time polymerase chain
reaction approach. In this study, we examined relative
telomere length in 80 spontaneously eliminated embryos
and in 25 embryos eliminated due to induced abortions. Relative telomere length in spontaneous abortions was
significantly lower (P = 0.000001) compared to the induced
abortions. P01.80D High-resolution array-CGH analysis and Targeted Whole
Exome Sequencing on patients affected by Primary
Ovarian Insufficiency (POI) identified new genes involved
in oocyte grow and differentiation Abstracts from the 51st European Society of Human Genetics Conference: Posters 35 L. Lovrečić: None. N. Pereza: None. H. Jaklič: None.
S. Ostojić: None. B. Peterlin: None.
P01.82B
Telomere shortening as the main indicator of non-viable
fetus elimination L. Lovrečić: None. N. Pereza: None. H. Jaklič: None. S. Ostojić: None. B. Peterlin: None. I. Bestetti: None. C. Barbieri: None. A. Sironi: None. C. Castronovo: None. C. Caslini: None. R. Rossetti:
None. A. Pistocchi: None. A. Rajkovic: None. C. Sala:
None. D. Toniolo: None. L. Persani: None. A. Marozzi:
None. P. Finelli: None. P01.83C The RHD signal in each case
correlated with fetal fraction and was therefore consistent
with an RHD-positive fetal source on the background of an
RHD-negative maternal source. As expected, no RHD
sequences were detected for samples with 0% fetal fraction. Conclusion: Targeted cfDNA testing using DANSR
assays has the potential to determine fetal RHD status and
be used as a noninvasive screening method to identify
pregnancies at increased risk for RhD immunization. The lack of a precise definition of genetic content of the r
(16) and its mosaic form leads to uncertain prognosis of
clinical outcome. S. Saini: A. Employment (full or part-time); Significant;
Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. R. Foley: A. Employment (full or part-time); Significant;
Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. C. Kingsley: A. Employment (full or part-time); Signifi-
cant; Ariosa Diagnostics Inc., Roche Sequencing Solutions
Inc. E. Wang:
A. Employment
(full
or
part-time);
Significant; Ariosa Diagnostics Inc., Roche Sequencing
Solutions Inc. M. Schmid: A. Employment (full or part-
time); Significant; Ariosa Diagnostics Inc., Roche Sequen-
cing Solutions Inc. P. Bogard: A. Employment (full or
part-time); Significant; Ariosa Diagnostics Inc., Roche
Sequencing Solutions Inc. A. Ramos: None. S. Saini: A. Employment (full or part-time); Significant;
Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. R. Foley: A. Employment (full or part-time); Significant;
Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. C. Kingsley: A. Employment (full or part-time); Signifi-
cant; Ariosa Diagnostics Inc., Roche Sequencing Solutions
Inc. E. Wang:
A. Employment
(full
or
part-time);
Significant; Ariosa Diagnostics Inc., Roche Sequencing
Solutions Inc. M. Schmid: A. Employment (full or part-
time); Significant; Ariosa Diagnostics Inc., Roche Sequen-
cing Solutions Inc. P. Bogard: A. Employment (full or
part-time); Significant; Ariosa Diagnostics Inc., Roche
Sequencing Solutions Inc. A. Ramos: None. After genetic counseling the couple opted to continue the
pregnancy. At birth no major malformations were observed
and a lower level of mosaic r(16) was observed in
peripheral blood. C. Kingsley: A. Employment (full or part-time); Signifi-
cant; Ariosa Diagnostics Inc., Roche Sequencing Solutions
Inc. E. Wang:
A. Employment
(full
or
part-time);
Significant; Ariosa Diagnostics Inc., Roche Sequencing
Solutions Inc. M. Schmid: A. Employment (full or part-
time); Significant; Ariosa Diagnostics Inc., Roche Sequen-
cing Solutions Inc. P. Bogard: A. Employment (full or
part-time); Significant; Ariosa Diagnostics Inc., Roche
Sequencing Solutions Inc. A. Ramos: None. O. Nagy1, J. Kárteszi2, M. Hartwig2, M. Tihanyi2, É. Erhardt3,
A. Patócs4, A. Ujfalusi1 F. Brito1, M. Silva1, C. Alves1, C. Ferreira1, S. Serafim1,
L. Simão1, B. Marques1, S. Pedro1, A. Tarelho1, J. Furtado1,
P. Lopes1, N. Silva1, M. Viegas1, A. Fernandes2, F. Teixeira2,
S. Gomes3, H. Correia1 P01.83C The mosaicism, as well as limitations of CMA in those
cases, prevent a refined characterization of these genomic
imbalances and pose a challenge in genetic counseling. F. Brito: None. M. Silva: None. C. Alves: None. C. Ferreira: None. S. Serafim: None. L. Simão: None. B. Marques: None. S. Pedro: None. A. Tarelho: None. J. Furtado: None. P. Lopes: None. N. Silva: None. M. Viegas: None. A. Fernandes: None. F. Teixeira: None. S. Gomes: None. H. Correia: None. F. Brito: None. M. Silva: None. C. Alves: None. C. Ferreira: None. S. Serafim: None. L. Simão: None. B. Marques: None. S. Pedro: None. A. Tarelho: None. J. Furtado: None. P. Lopes: None. N. Silva: None. M. Viegas: None. A. Fernandes: None. F. Teixeira: None. S. Gomes: None. H. Correia: None. Gomes: None. H. Correia: None. P01.83C Targeted cfDNA Analysis Using DANSR assays for
Determination of Fetal RHD Status 36 J. del Picchia 1Instituto Nacional de Saúde Dr. Ricardo Jorge, Unidade de
Citogenética, Lisboa, Portugal, 2Hospital Central do Funchal,
Serviço de Pediatria, Funchal, Portugal, 3Hospital Central do
Funchal, Serviço de Obstetrícia, Funchal, Portugal 1Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc., San
Jose, CA, United States, 2Roche Sequencing Solutions, San
Jose, CA, United States Objectives: To develop a targeted cell-free (cfDNA) test,
the Harmony® prenatal test, enhancement that allows
determination
of
fetal
RhD
status
in
RhD-negative
pregnant women. Objectives: To develop a targeted cell-free (cfDNA) test,
the Harmony® prenatal test, enhancement that allows
determination
of
fetal
RhD
status
in
RhD-negative
pregnant women. Ring chromosomes are rare cytogenetic findings (prenatal
frequency ~ 0.0075%) often associated with an abnormal
phenotype, depending of the chromosomal origin, genetic
content and the presence of a mosaic. Supernumerary ring
chromosome 16 [r(16)] is rarely observed and mosaicism
makes the genotype/phenotype correlation difficult. Method: 12 simulated pregnancy plasma samples with
known RHD genotype were prepared by titrating non-
pregnant, RHD-positive cfDNA (fetal source) into non-
pregnant, female RHD-negative cfDNA (maternal source)
to simulate fetal fractions of 5%, 10% and 15%. A 0%
sample served as a negative control. Digital Analysis of
Selected Regions (DANSR) assays targeting exons 2, 3, 4, 5
and 7 of the RHD gene were added to existing DANSR
assays and the generated DANSR products were hybridized
onto a custom DNA microarray for analysis of fetal fraction
and determination of fetal RHD status using the fetal
fraction optimized algorithm FORTE. We report a de novo mosaic r(16) detected after prenatal
diagnosis in a woman referred for advanced maternal age. Multiplex ligation-dependent probe amplification (MLPA)
for aneuploidy testing of chromosomes 13, 18, 21 and X
was normal. Karyotype was 47,XX,+r[10]/46,XX[15]. Chromosomal
microarray
analysis
(CMA)
on
DNA
obtained from long-term cultured amniocytes did not detect
any alterations. MLPA with a pericentromeric probe kit on
an uncultured sample showed a chromosome 16 gain,
encompassing 16p11.2 and 16q11.2 regions, including
TGFB1I1, AHSP, VPS35 and ORC6 genes, leading to
partial characterization of the r(16). Although no phenotype
has been correlated with overexpression of these genes, the
16p11.2 region is associated with neurodevelopmental
disorders. Nevertheless individuals with microduplication
of 16p11.2 and normal development have been described. Results: In all 12 simulated pregnancy samples, RHD
sequences were detected. 1Division of Clinical Genetics, Department of Laboratory
Medicine, Faculty of Medicine, University of Debrecen, P01.85A A novel partial deletion of the NR5A1 gene in a female
patient with 46, XY disorder of sex development Prenatal diagnosis of mosaic ring chromosome 16 - a rare
event with uncertain prognosis Prenatal diagnosis of mosaic ring chromosome 16 - a rare
event with uncertain prognosis A novel partial deletion of the NR5A1 gene in a female
patient with 46, XY disorder of sex development F. Brito1, M. Silva1, C. Alves1, C. Ferreira1, S. Serafim1,
L. Simão1, B. Marques1, S. Pedro1, A. Tarelho1, J. Furtado1,
P. Lopes1, N. Silva1, M. Viegas1, A. Fernandes2, F. Teixeira2,
S. Gomes3, H. Correia1 1Division of Clinical Genetics, Department of Laboratory
Medicine, Faculty of Medicine, University of Debrecen, Abstracts from the 51st European Society of Human Genetics Conference: Posters 37 Debrecen, Hungary, 2Hospital of Zala County, Zalaegerszeg,
Hungary, 3Department of Pediatrics, University of Pécs, Pécs,
Hungary, 4Department of Laboratory Medicine, Endocrin
Genetics Laboratory, Semmelweis University, Budapest,
Hungary processing. The disease affects 1 out of 6000 newborns,
while about 1 out of 50 individuals are carriers. processing. The disease affects 1 out of 6000 newborns,
while about 1 out of 50 individuals are carriers. SMN1 has a paralog SMN2, which differs in coding
sequence by just 1 base. SMN2 produces a functional
protein though less efficiently than SMN1. These genes are
located 500 kb apart on chromosome 5, which permits
frequent recombination events that result in deletions,
duplications or chimeras. Evolution has selected for multi-
ple copies of SMN2 since having several copies can
partially compensate for a non-functional SMN1 gene. Because of the severity of SMA, carrier and newborn
screening is important. To be utilized in carrier screening,
an assay has to detect one copy of SMN1 with 100%
sensitivity, which necessitates distinguishing every SMN1
copy from SMN2. NR5A1 (Steroidogenic factor 1, SF-1) is a transcriptional
regulator of genes required for normal adrenal and gonadal
development and function. Mutations in NR5A1 have been
identified in patients with various forms of disorders of sex
development (DSD), including gonadal dysgenesis with or
without adrenal insufficiency. To date microdeletion or
partial deletion involving the NR5A1 gene have been
reported in only a few of cases with DSD.We present a
patient with female external genitalia, clitoromegaly, bilat-
eral ingiunal hernia containing testicles, mixed internal
genitalia (uterus, Fallopian tube, epididymis), minor facial
dysmorphism, normal adrenal function, low testosterone,
high FSH levels. A new candidate biomarker in Spermatogonial Stem Cell
maturation : Dynamin 2 A new candidate biomarker in Spermatogonial Stem Cell
maturation : Dynamin 2 Y. YUKSELTEN1, O. S. AYDOS1, A. SUNGUROGLU1,
K. AYDOS2 P01.85A Family history is negative for disorders of
sex development or premature ovarian failure. Chromosome
analysis revealed a 46,XY karyotype. Array CGH did not
detect pathogenic copy number variations. Next generation
sequencing of the most common DSD genes did not iden-
tify pathogenic variants. Genomic DNA was screened for
small deletion/duplication of genes commonly affected in
DSD using the SALSA Intersex MLPA kit (MRC-Holland)
according to manufacturer’s protocol. We identified a novel
partial deletion encompassing the exons 5 and 6 of the
NR5A1 gene leading to haploinsufficiency of the gene, that
is alone sufficient to cause the patient’s abnormal sexual
development.This report expands upon the range of muta-
tions associated with NR5A1 gene, further confirms the role
of NR5A1 deletions in 46,XY DSD and emphasises the
utility of MLPA as a genomic screening tool in the workup
of DSDs of unclear etiology.This study was supported by
Ministry of National Economy, Hungary GINOP-2.3.2-15-
2016-00039 An NGS assay was developed to detect one copy of
SMN1 and the number of SMN2 copies in SMA carriers in
an easy and scalable protocol suitable for automatization. The assay is performed in one tube, using a ligation-free
method. Testing of clinical research samples with known
numbers of SMN1 and SMN2 has demonstrated the
precision of the assay to detect the number of SMN copies. The protocol can also be easily expanded to include all
SMN1 mutations and/or be combined with assays to detect
variants in other known carrier-screening genes for future
clinical research. Research use only. Not for diagnostic use. R. Hrdlickova: None. J. Nehyba: None. C. Clear:
None. D. Fox: None. A. Kothandaraman: None. P01.87C Dynamin 2 (DNM2) belongs to the GTPase superfamily. Beside having important roles in the regulation of mem-
brane fission and fusion events dynamins induce differ-
entiation of germ cells in spermatogenesis. P01.87C
A novel next generation sequencing assay for the detection
of SMA carrier status and the number of copies of SMN2
R. Hrdlickova, J. Nehyba, C. Clear, D. Fox, A. Kothandaraman
Bioo Scientific, Austin, TX, United States P01.87C
A novel next generation sequencing assay for the detection
of SMA carrier status and the number of copies of SMN2 Y. YUKSELTEN1, O. S. AYDOS1, A. SUNGUROGLU1,
K. AYDOS2 1Ankara University, School of Medicine, Department of
Medical Biology, Ankara, Turkey, 2Ankara University, School
of Medicine, Department of Urology, Ankara, Turkey O. Nagy: None. J. Kárteszi: None. M. Hartwig: None. M. Tihanyi: None. É. Erhardt: None. A. Patócs: None. A. Ujfalusi: None. R. Hrdlickova, J. Nehyba, C. Clear, D. Fox, A. Kothandaraman
Bioo Scientific, Austin, TX, United States S. O. AYDOS1, Y. YUKSELTEN1, A. SUNGUROGLU1,
K. AYDOS2 S. O. AYDOS1, Y. YUKSELTEN1, A. SUNGUROGLU1,
K. AYDOS2 Background: Preimplantation genetic diagnosis (PGD) of
spinal muscular atrophy (SMA) is vulnerable to mis-
diagnosis arising from allele drop-out or exogenous DNA
contamination. We describe a PGD strategy that detects
SMA with high diagnostic confidence and accuracy. 1Ankara University, School of Medicine, Department of
Medical Biology, Ankara, Turkey, 2Ankara University, School
of Medicine, Department of Urology, Ankara, Turkey 1Ankara University, School of Medicine, Department of
Medical Biology, Ankara, Turkey, 2Ankara University, School
of Medicine, Department of Urology, Ankara, Turkey Methods: A triplex PCR-minisequencing assay was
developed for reliable detection of SMN1 and SMN2. A
single-tube assay was developed to simultaneously amplify
13 highly polymorphic microsatellite markers located
within 0.5 Mb of the 1.7 Mb duplicated SMN region on
chromosome 15q13.2. Single cells were subjected to whole
genome amplification, and separate aliquots were used for
triplex SMN1/2 detection and for tridecaplex microsatellite-
based haplotyping. The strategy was validated on 24 single
cells isolated from cell lines of an SMA case-parent trio. Spermatogonial stem cells (SSCs) have major roles on
spermatogenesis and male fertility. Identification of SSCs
and determination of their effects on impaired spermato-
genesis are very important for elucidation of the etiology of
male infertility. In this study, our aim was to investigate
CD90, CD29, CD49f and POU5F1 genes expressions and
the relationship between these biomarkers with impaired
spermatogenesis in non-obstructive azoospermia (NOA)
and obstructive azoospermia (OA) as control group. NOA
group consisted of 20 hypospermatogenesis (HS), 20
maturation arrest (MA), 20 Sertoli Cell Only syndrome
(SCO) patients. The biomarkers were analyzed by RT-PCR
array. All groups were compared with the control group. CD29 and CD49f gene expressions showed 0.08 ± 0.01 and
0.65 ± 0.15 fold decreases (p < 0.05), in the HS group, while
CD90 and POU5F1 genes showed 1.20 ± 0.21 and 0.83
± 0.24 fold changes, respectively (p > 0.05). In the MA
group, CD90, CD49f and POU5F1 gene expressions
showed
0.40 ± 0.05,
0.22 ± 0.03
and
0.16 ± 0.02
fold
decrease (p < 0.05) respectively, while CD29 gene showed
0.85 ± 0.10 fold change (p > 0.05). In the SCO group,
CD90, CD29 and POU5F1 gene expressions showed 4.73
± 1.03, 2.10 ± 0.47 and 3.56 ± 0.68 fold increases, (p <0.05)
respectively, while CD49f gene expression showed 1.67
± 0.37 fold change (p > 0.05). Robust preimplantation genetic diagnosis of spinal
muscular atrophy combining triplex SMN1 detection with
multi-microsatellite haplotyping S. S. Chong1,2,3, M. Zhao1, M. Lian2, F. S. H. Cheah2 S. S. Chong1,2,3, M. Zhao1, M. Lian2, F. S. H. Cheah2 Y. Yukselten: None. O.S. Aydos: None. A. Sungur-
oglu: None. K. Aydos: None. 1Department of Paediatrics, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore, Singapore,
2Khoo Teck Puat - National University Children's Medical
Institute, National University Health System, Singapore,
Singapore, 3Department of Laboratory Medicine, National
University Hospital, Singapore, Singapore P01.87C
A novel next generation sequencing assay for the detection
of SMA carrier status and the number of copies of SMN2 R. Hrdlickova, J. Nehyba, C. Clear, D. Fox, A. Kothandaraman
Bioo Scientific, Austin, TX, United States In this study, our aim was to investigate dynamin 2 gene
expression and the relationship between this biomarker with
impaired spermatogenesis in non-obstructive azoospermia
(NOA) and obstructive azoospermia (OA) as control group. NOA group consisted of 20 hypospermatogenesis (HS), 20
maturation arrest (MA), 20 Sertoli Cell Only syndrome
(SCO) patients. The biomarker was analyzed by RT-
PCR array. Bioo Scientific, Austin, TX, United States Spinal muscular dystrophy (SMA) is an inherited autosomal
recessive neuromuscular disease caused by a defective
SMN1 gene. SMN1 codes for a protein involved in RNA 38 J. del Picchia patients. We believe that the curative protocols which will
be developed against the effects of these markers in sper-
matogenic defects will be very valuable in terms of human
reproductive health. patients. We believe that the curative protocols which will
be developed against the effects of these markers in sper-
matogenic defects will be very valuable in terms of human
reproductive health. patients. We believe that the curative protocols which will
be developed against the effects of these markers in sper-
matogenic defects will be very valuable in terms of human
reproductive health. When compared with the control group, DNM2 gene
expression in HP, MA and SCO groups showed 1.60 ± 0.30
(p > 0.05), 0.28 ± 0.05 (p < 0.001) and 0.86 ± 0.18 (p > 0.05)
fold changes, respectively. S.O. Aydos: None. Y. Yukselten: None. A. Sungur-
oglu: None. K. Aydos: None. The dynamin family of proteins has important regulatory
roles in membrane remodelling and endocytosis. By this
way they also affects the cell viability. Thus, the decrease in
DNM2 gene expression in MA group suggests that
clinically DNM2 expression deficiency may cause matura-
tion arrest. DNM2 is thought to be an important marker for
the understanding the etiology of male infertility and in the
development
of
treatment
protocols
for
azoospermic
patients with MA. P01.89A Expression profiles of Spermatogonial Stem Cells’ marker
genes in azoospermic patients with different pathologies Expression profiles of Spermatogonial Stem Cells’ marker
genes in azoospermic patients with different pathologies S.S. Chong: None. M. Zhao: None. M. Lian: None. F.S.
H. Cheah: None. P01.92D P01.92D
Phenotype variability of NR5A1 (SF1) gene mutations in
DSD patients S. O. AYDOS1, Y. YUKSELTEN1, A. SUNGUROGLU1,
K. AYDOS2 As a conclusion, it was found
that SSCs biomarkers exhibit different profiles in NOA Results: Triplex SMN1/2 PCR-minisequencing reliably
detected single-copy SMN1 even in the presence of five
SMN2 copies. Only SMN2 was detected in all SMA-affected
samples. Observed heterozygosities of the 13 flanking
microsatellite markers ranged from 0.56 to 0.8, and 98.4%
of genotyped individuals were heterozygous for 2 or more
markers on either side of SMN1. Triplex SMN1/2 PCR-
minisequencing results were completely correlated with
observed marker diplotypes in all tested samples. Conclusion: Triplex detection of SMN1 and SMN2,
combined with linked multi-marker diplotyping, improves
diagnostic confidence for SMA PGD. The highly poly-
morphic tridecaplex microsatellite panel can potentially be
informative in most if not all at-risk couples. S.S. Chong: None. M. Zhao: None. M. Lian: None. F.S. H. Cheah: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 39 P01.91C
Numerical and structural genomic aberrations in
spontaneous abortions, detected by array CGH analysis preimplantation genetic diagnosis with transfer of unaf-
fected embryos or the use of donor gametes might be
considered for therapy. preimplantation genetic diagnosis with transfer of unaf-
fected embryos or the use of donor gametes might be
considered for therapy. Acknowledgment: SNSF grant No IZ73Z0_152454. Acknowledgment: SNSF grant No IZ73Z0_152454. K. Belemezova1,2, M. Rizov1, M. Hristova-Savova1, E. Nikolova1,
T. Timeva3,4, T. Milachich3, M. Yunakova3, P. Andreeva3,5,
P. Chaveeva3, A. Shterev3, V. Djonov6, I. Dimova1,7 K. Belemezova: None. M. Rizov: None. M. Hristova-
Savova: None. E. Nikolova: None. T. Timeva: None. T. Milachich: None. M. Yunakova: None. P. Andreeva:
None. P. Chaveeva: None. A. Shterev: None. V. Djonov:
None. I. Dimova: None. 1Genetic Laboratory, SAGBAL „Dr Shterev“, Sofia, Bulgaria,
2Clinical Immunology Laboratory, St Ivan Rilski Hospital,
Medical University Sofia, Sofia, Bulgaria, 3SAGBAL „Dr
Shterev“, Sofia, Bulgaria, 4"Angel Kanchev" University of
Ruse, Ruse, Bulgaria, 5South-West University "Neofit Rilski",
Blagoevgrad, Bulgaria, 6Institute of Anatomy, University of
Bern, Bern, Switzerland, 7Laboratory of Genomic diagnostics,
Medical University Sofia, Sofia, Bulgaria Phenotype variability of NR5A1 (SF1) gene mutations in
DSD patients V. B. Chernykh1, N. Y. Raygorodskaya2, N. V. Bolotova2, J. V. Paltseva3 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Saratov State Medical University, Saratov,
Russian Federation, 3Clinical Hospital of Medical University
named SR Mirotvortsev, Saratov, Russian Federation 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Saratov State Medical University, Saratov, Background: Chromosomal abnormality in the product of
conception is the reason for 50-70% of abortions. Chro-
mosomal microarray analysis of fetal tissue has been pro-
posed as a technique to evaluate the cause of isolated and
recurrent early pregnancy loss (miscarriages) and later
pregnancy loss (intrauterine fetal demise). Russian Federation, 3Clinical Hospital of Medical University
named SR Mirotvortsev, Saratov, Russian Federation Background: NR5A1(Steroidogenic Factor 1, SF1) gene
mutations result in various forms of disorders of sex
development (DSD), adrenal insufficiency, primary hypo-
gonadism, male and female infertility. Materials and Methods: In this study, we applied array
CGH method for analysis of chromosomal imbalances at a
high resolution in 24 samples of spontaneous abortions. Higher levels of circulating mRNA for Tenascin X (TNXB)
gene in maternal plasma at the second trimester of
pregnancy in isolated congenital ventricular septal defects Higher levels of circulating mRNA for Tenascin X (TNXB)
gene in maternal plasma at the second trimester of
pregnancy in isolated congenital ventricular septal defects M. Grigorova1, M. Punab2, O. Poolamets2, M. Adler1,2,
V. Vihljajev2, M. Laan1 1Institute of Biomedicine and Translational Medicine,
University of Tartu, Tartu, Estonia, 2Andrology Unit, Tartu
University Hospital, Tartu, Estonia C. Lapucci1, D. Morano2, S. Berto1, L. Walczer Baldinazzo1,
D. Prandstraller3, F. Cro'1, A. Farina4 1Genetic department, Synlab Italy, Castenedolo, Brescia, Italy,
2Department of Morphology, Surgery and Experimental
Medicine, Section of Obstetrics and Gynecology, University of
Ferrara, Azienda Ospedaliero-Universitaria S.Anna, Cona,
Ferrara, Italy, 3Pediatric Cardiology and Adult Congenital
Unit, Sant'Orsola Malpighi Hospital, Bologna, Italy, 4Division
of Obstetrics and Prenatal Medicine, DIMEC Sant'Orsola
Malpighi Hospital, University of Bologna, Bologna, Italy Introduction: Testosterone (T) is a central androgenic
hormone. Sex hormone-binding globulin (SHBG) mod-
ulates T bioactivity. The current understanding of genetic
variation contributing to male reproductive hormone levels
is moderate. We aimed to confirm or reject genetic asso-
ciations of top-loci (SHBG, GCKR, SLCO1B1, JMJD1C)
from GWA-studies for SHBG and T, and uncover addi-
tional genetic effects on male fertility-related parameters (J
Endocr Soc. 2017 1(6): 560-576). Introduction: Maternal plasma is a source of circulating
placental nucleic acids; therefore it is a powerful tool for
prenatal screening for congenital heart diseases (CHD). Previous studies showed that the identification of circulating
mRNAs of MAPK1, IQGAP1 and Visfatin in maternal
plasma had different gene expressions between CHD foe-
tuses and control foetuses. This method allowed us to fur-
ther investigate the expression of Tenascin X gene (TNXB)
published as involved in CHD. Levels of circulating mRNA
for the TNXB were investigated in pregnancies with ven-
tricular septal defects during 2nd trimester of pregnancy. Material and Methods: Study groups: young men
(n = 540; 19.3 ± 1.8 years), severe idiopathic male inferti-
lity patients (n = 641; 31.6 ± 6.0 years), male partners of
pregnant women (n = 324; 31.9 ± 6.6 years). Recruitment:
Andrology
Unit,
Tartu
University
Hospital,
Estonia. Analysis: genetic associations with reproductive parameters
(linear regression, meta-analysis). Results. Robust associations with SHBG for SHBG
rs1799941 (meta-analysis: P=3.7x10-14; beta = 4.67(0.62)
nmol/L), SHBG rs727428 (P=7.3x10-11; -3.74(0.57), SHBG
Pro185Leu (rs6258; P=1.2x10-4, -12.2(3.17) and GCKR
Pro446Leu (rs1260326; P=1.5x10-4; -2.2(0.59). Total T
correlates
with
genetically
modulated
SHBG
levels
(r=0.48-0.74, P < 0.0001), guaranteeing stable availability
of free T. Results: The
Aim:
To
evaluate
the
clinical
variability
of
ambiguous phenotypes and the gender assignment in DSD
patients with SF1 mutations. Results:
Genomic aberration
%
Trisomy 22
8
Trisomy 16
8
Monosomy Х
8
Trisomy 18
4
Trisomy 19
4
Trisomy 20
4
Trisomy 21
4
Trisomy 15
4
Double trisomy 18 and 19
4
Duplication 7р
4
Tetrasomy 9р
4
Chromotripsis
8
Euploid
36 Materials and Methods: Clinical examination, hormonal
tests, ultrasound, laparoscopy, standard cytogenetic exam-
ination, and DNA analyses, including Sanger and next-
generation sequencing. Results: Case 1. A 46,XY female patient, aged 18 months
with clitorophallus. A small testis was detected in the right
labioscrotal fold. Hormonal tests showed LH - 0.25 IU/l,
FSH - 12.9 IU/l, and testosterone after hCG stimulation -
0.3 nmol/l. Pelvic ultrasonography and laparoscopy showed
the uterus, fallopian tubes and dysgenetic abdominal gonad
on the left side. DNA analysis detected the heterozygous
nonsense mutation c.256delA of the NR5A1 gene. Case 2. A 46,XY boy, aged 5 months, with undermasculinized
Prader III genitalia, perineal hypospadias and small testes in
bifid scrotum. Uterus was not found. Hormonal tests
showed testosterone - 5.6 nmol/l, LH - 2.2 IU/l, FSH -
5.0 IU/l, and AMH - 43.8 ng/ml. DNA analysis found
heterozygous p.R313C mutation of the NR5A1 gene. 36 Conclusion: In 15 of 24 spontaneous abortions (64%),
genomic anomalies were discovered by array CGH analysis. Two thirds of them could be detected by the rapid DNA
analysis (offered in our country as a rapid QF-PCR analysis
for aneuploidies 13, 15, 16, 18, 21, 22, X and Y). Numerical
anomalies were detected in 73% of aberrant cases, and in
27% - structural aberrations/chromothripsis were revealed. For couples with recurrent pregnancy loss and evidence of a
structural genetic abnormality in one of the parents, Conclusions: The 46,XY patients with heterozygous
NR5A1 mutations presented various phenotypes of gonadal
development. The patient with nonsense mutation presented
female phenotype with gonadal dysgenesis and Müllerian
derivates. The male patient with NR5A1 missense mutation
developed
genitalia
ambiguous
with
moderated Conclusions: The 46,XY patients with heterozygous
NR5A1 mutations presented various phenotypes of gonadal
development. The patient with nonsense mutation presented
female phenotype with gonadal dysgenesis and Müllerian
derivates. The male patient with NR5A1 missense mutation
developed
genitalia
ambiguous
with
moderated 40 J. del Picchia undermasculinization and no Müllerian structures, that is
characteristic
for
partial
46,XY
gonadal
(testicular)
dysgenesis. C. Lapucci: None. D. Morano: None. S. Results: Berto: None. L. Walczer Baldinazzo: None. D. Prandstraller: None. F. Cro': None. A. Farina: None. V.B. Chernykh: None. N.Y. Raygorodskaya: None. N. V. Bolotova: None. J.V. Paltseva: None. V.B. Chernykh: None. N.Y. Raygorodskaya: None. N. V. Bolotova: None. J.V. Paltseva: None. Laiko Hospital, Athens, Greece Introduction: Thalassaemias are the most common mono-
genic disorders worldwide. In the majority of cases prenatal
diagnosis of embryos at risk is performed through an
invasive procedure that has 1-2% probability of miscarriage. We have previously developed a non-invasive prenatal
testing (NIPT) approach, based on analyzing free fetal
DNA. Here, we present the results of 32 cases where the
parental beta globin gene mutations are inherited by the
fetus. Our method is based on High Resolution Melting
(HRM) analysis and covers all possible combinations of
fetal inheritance. Results: At a sequencing depth of 8M reads on a
NextSeq™500 or 550, the limit of detection was below 3%
FF for each chromosome-of-interest. Test performance was
determined using 3107 samples, of which 21 (0.7%) were
not reported because of QC failure on the only plasma
aliquot available. Sensitivity was 98.9% (90/91), 90.0%
(18/20), and 100% (8/8) for trisomy 21, trisomy 18, and
trisomy 13, respectively. Specificities were ≥99.9% for all
three trisomies; there were 8 false-positive results (one
trisomy 21, three trisomy 18, and four trisomy 13). Concordance for sex chromosome aneuploidies ranged
from 80.0-100%. Materials and Methods: ffDNA is isolated from 2ml
maternal plasma and screened for the presence of β globin
gene parental mutations based on an in-house developed
HRM approach. Previously described SNPs in the vicinity
of β globin gene, for which parents presented distinct
haplotypes, were also determined and used for identification
of the fetal fraction. Sex determination was also helpful in
case of male embryos. Materials and Methods: ffDNA is isolated from 2ml
maternal plasma and screened for the presence of β globin
gene parental mutations based on an in-house developed
HRM approach. Previously described SNPs in the vicinity
of β globin gene, for which parents presented distinct
haplotypes, were also determined and used for identification
of the fetal fraction. Sex determination was also helpful in
case of male embryos. Results: Our hitherto results (32 cases) cover all possible
combinations like male or female embryos that inherited
either the paternal or the maternal mutation and cases where
the embryo has inherited both parental mutations. Our
results succeeded in obtaining the same diagnosis with the
preceded analysis of corresponding chorionic villus sam-
pling, showing up to now 100% diagnostic capacity. Conclusions: VeriSeq NIPT Solution showed good test
performance
for
the
detection
of
fetal
chromosomal
aneuploidies, even at low fetal fractions. P01.95C Objectives: To develop and evaluate performance of a
novel and highly automated, paired-end sequencing-based
noninvasive prenatal test (NIPT) for fetal chromosomal
aneuploidies on chromosomes 21, 18, 13, X, and Y. Laiko Hospital, Athens, Greece This paired-end
sequencing-based NIPT assay detects fetal chromosomal
aneuploidies with high sensitivities and specificities and a
low assay failure rate. Conclusions: The described approach, after further
evaluation, may allow its application at the diagnostic level
as a primary or secondary method for NIPT for beta
thalassaemia and if accordingly adopted it may be used for
other monogenic diseases as well. Conclusions: The described approach, after further
evaluation, may allow its application at the diagnostic level
as a primary or secondary method for NIPT for beta
thalassaemia and if accordingly adopted it may be used for
other monogenic diseases as well. S. Duenwald:
A. Employment
(full
or part-time);
Significant; Illumina, Inc. E. Ownership Interest (stock,
stock options, patent or other intellectual property); Modest;
Illumina, Inc. D. Vavrek: A. Employment (full or part-
time); Significant; Illumina, Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; Illumina, Inc. K. Meier: A. Employment (full or
part-time); Significant; Illumina, Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; Illumina, Inc. C. Deciu: A. Employment (full or
part-time); Significant; Illumina, Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property); M. Karapanou: None. M. Vlahou: None. A. Floratos:
None. A. Balassopoulou: None. E. Boutou: None. M. Karapanou: None. M. Vlahou: None. A. Floratos:
None. A. Balassopoulou: None. E. Boutou: None. S. Duenwald1, D. Vavrek2, K. Meier2, C. Deciu2, M. Garcia-
Ransom3, M. Halks-Miller4 S. Duenwald1, D. Vavrek2, K. Meier2, C. Deciu2, M. Garcia-
Ransom3, M. Halks-Miller4 M. Grigorova: None. M. Punab: None. O. Poolamets:
None. M. Adler:
None. V. Vihljajev:
None. M. Laan: None. 1Illumina, Inc., San Francisco, CA, United States, 2Illumina,
Inc., San Diego, CA, United States, 3N/A, San Francisco, CA,
United States, 4Grail, San Francisco, CA, United States Higher levels of circulating mRNA for Tenascin X (TNXB)
gene in maternal plasma at the second trimester of
pregnancy in isolated congenital ventricular septal defects Among infertile men, SHBG Pro185Leu shows
downstream effect on LH (P=5.1x10-5; -1.66(0.57) IU/L)
and FSH (P=3.4x10-3; -2.48(1.23) IU/L). No associations
for SHBG Asp327Asn (rs6259), SLCO1B1 Val174Ala
(rs4149056), JMJD1C rs7910927. Materials and Methods: The circulating mRNA assay
was isolated from blood samples collected in several
Institutes in Italy from March 2016 to July 2017. TNXB
gene expression was investigated by RT-PCR in 10 women
carrying foetus with ventricular septal defects and 31
controls at 19-24 weeks of gestation. Results: RT-PCR showed that TNXB gene expression
was higher in foetuses with ventricular septal defects with a
2-ΔΔCT value of 4.38 ± 3.01 vs 1.00 ± 0.80 in controls. CSH2 was used as housekeeping gene which expression
value was normalised based on several gestational ages. Conclusions. We replicated previously reported associa-
tions and detected additional effects for four of seven
analysed
GWAS
hits. These
variants
are
promising
candidates for the future studies of hypotestosteronemia in
aging men, as well as promising pharmacogenetic targets
for hormone replacement therapy in males with fertility
problems. Conclusions:
The
data
confirmed
a
link
between
circulating mRNA and CHD; ventricular septal defects
could be associated with abnormal level of TNXB mRNA
during the 2nd trimester of pregnancy. In future, the
variation of TNXB gene expression will be used to identify
the foetuses with CHD, however this molecular marker will
be assessed throughout prospective studies in a wider
population. Funding: European Union through the European Regio-
nal
Development
Fund
(project
Happy
Pregnancy, Funding: European Union through the European Regio-
nal
Development
Fund
(project
Happy
Pregnancy, 41 Abstracts from the 51st European Society of Human Genetics Conference: Posters 41 3.2.0701.12-0047) and Estonian Research Council (IUT34-
12, ETF9030, PUT181). M. Grigorova: None. M. Punab: None. O. Poolamets:
None. M. Adler:
None. V. Vihljajev:
None. M. Laan: None. Evaluation of an in-house-developed HRM based approach
for Non-invasive prenatal diagnosis of beta thalassaemia M. Karapanou, M. Vlahou, A. Floratos, A. Balassopoulou,
E. Boutou Methods: We developed a PCR-free, paired-end sequen-
cing-based NIPT, the VeriSeq™NIPT Solution, that
estimates fetal fraction (FF) and screens for fetal chromo-
somal aneuploidies in maternal plasma samples. Perfor-
mance of VeriSeq NIPT Solution was determined by
analyzing 3057 frozen maternal plasma samples that had
been tested previously using a single-end sequencing-based
NIPT (VeriުPrenatal Test, Illumina, Inc.); samples were
blinded prior to reanalysis. Clinical outcomes (cytogenetic
analysis or newborn physical examination) were available
for all cases used to determine assay sensitivity and
specificity. Laiko Hospital, Athens, Greece FEDERAL STATE BUDGETARY INSTITUTION «RESEARCH
CENTRE FOR MEDICAL GENETICS», Moscow, Russian
Federation Introduction: Trophoblast cells in blood of pregnant
women are a potential target for non-invasive prenatal
testing, but the number of trophoblasts is very low. Obtaining the quantity and quality of DNA sufficient for
subsequent analysis is key point in the possibility of
studying the genetic material of single cells. To compare
three different methods of whole genome amplification in
model experiment was performed. Materials and Methods: We present a case of a 37-year-
old TS women who had normal fertility and three
documented pregnancies. Cytogenetic analysis were based
on the analysis of 200 metaphases. According to standard
procedures, metaphase chromosomes were obtained from
phytohaemagglutinin stimulated lymphocyte cultures from
peripheral blood. The chromosomes were analyzed by
Giemsa banding and karyotype according to the Use of the
International System for Human Cytogenetic Nomenclature. Materials and Methods: There were used 10 samples of
artificially created mixtures. The artificial mixtures were
prepared by mixing samples of peripheral venous blood of
adult with cells’ samples of chorionic villus with known
karyotype. There was used filtration through polycarbonate
filters to isolate trophoblasts from artificial mixtures. Isolated cells were fixed with paraformaldehyde during
sample preparation. Detection of trophoblasts on the filters
was carried out by immunocytochemical staining with
monoclonal antibodies to cytokeratin 7. Isolation of single
cytokeratin7-positive cells was performed by laser micro-
dissection. WGA performed by three different Methods:
linker adapter PCR (LA-PCR), degenerate oligonucleotide
primed
PCR
(DOP-PCR)
and
multiple
displacement
amplification (MDA). Analysis of molecular karyotype of
WGA products was performed by comparative genomic
hybridization. Lengths and concentrations of WGA pro-
ducts and results of comparative genomic hybridization
results were compared. Materials and Methods: There were used 10 samples of
artificially created mixtures. The artificial mixtures were
prepared by mixing samples of peripheral venous blood of
adult with cells’ samples of chorionic villus with known
karyotype. There was used filtration through polycarbonate
filters to isolate trophoblasts from artificial mixtures. Isolated cells were fixed with paraformaldehyde during
sample preparation. Detection of trophoblasts on the filters
was carried out by immunocytochemical staining with
monoclonal antibodies to cytokeratin 7. Isolation of single
cytokeratin7-positive cells was performed by laser micro-
dissection. WGA performed by three different Methods:
linker adapter PCR (LA-PCR), degenerate oligonucleotide
primed
PCR
(DOP-PCR)
and
multiple
displacement
amplification (MDA). Analysis of molecular karyotype of
WGA products was performed by comparative genomic
hybridization. FEDERAL STATE BUDGETARY INSTITUTION «RESEARCH
CENTRE FOR MEDICAL GENETICS», Moscow, Russian
Federation Lengths and concentrations of WGA pro-
ducts and results of comparative genomic hybridization
results were compared. Results: Female patient with a phenotype of TS contacted
a genetic counselor after two lost pregnancy. The result of
cytogenetic analysis: 45,X[188]/46,X,r(X)(p22.1q28)[12]. After a few months, the patient came pregnant and prenatal
diagnosis
was
conducted. The
third
pregnancy
was
successfully completed by the birth of a healthy boy. Conclusions:TS women typically experience gonadal
dysfunction that results in amenorrhea and sterility. Most
likely, an ovum with the ring X chromosome can be fertile
and can produce a viable zygote.TS associated with an X
ring chromosome, r (X), is rare and this view is our country
first case report on normal pregnancy in TS with 45,X/46,X,
r(X) mosaicism. S.D. Teofilov: None. T.P. Ostojic: None. M.R. Bula-
tovic:
None. J.D. Jovanovic:
None. O.V. Miljanovic: None. S.D. Teofilov: None. T.P. Ostojic: None. M.R. Bula-
tovic:
None. J.D. Jovanovic:
None. O.V. Miljanovic: None. Results: LA-PCR showed the highest sensitivity, speci-
ficity and uniformity of amplification in comparison with
other methods. Conclusion: The results of model experiment show that
not all the WGA methods used are applicable for the
analysis of single trophoblast cells, isolated by filtration and
fixed with paraformaldehyde in the process of sample
preparation. P02 Sensory disorders (eye, ear, pain) P01.96D Performance of the VeriSeq NIPT Solution - a novel PCR-
free, paired-end sequencing-based noninvasive prenatal
screening test 42 J. del Picchia E. Musatova: None. A. Tveleneva: None. Z. Markova:
None. N. Shilova: None. Modest; Illumina, Inc. M. Garcia-Ransom: E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Modest; Illumina, Inc. M. Halks-Miller: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; Illumina, Inc. E. Musatova: None. A. Tveleneva: None. Z. Markova:
None. N. Shilova: None. Modest; Illumina, Inc. M. Garcia-Ransom: E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Modest; Illumina, Inc. M. Halks-Miller: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; Illumina, Inc. Clinical Center of Montenegro, Podgorica, Montenegro E. Musatova, A. Tveleneva, Z. Markova, N. Shilova Introduction: Turner syndrome (TS) is a chromosomal
condition that affects development in females, caused by the
loss of an X-chromosome or X-structural abnormalities in
the X-chromosome.This condition occurs in about 1 in
2,500 newborn girls. The most frequent constitutional kar-
yotype of TS patients is 45,X. Small percentage individuals
with TS also have another cell line with 46 chromosomes
due to presence of a ring chromosome X instead of normal
X chromosome FEDERAL STATE BUDGETARY INSTITUTION «RESEARCH
CENTRE FOR MEDICAL GENETICS», Moscow, Russian
Federation The compare results of different whole genome
amplification methods for purposes of cell-based
noninvasive prenatal diagnosis S. D. Teofilov, T. P. Ostojic, M. R. Bulatovic, J. D. Jovanovic,
O. V. Miljanovic S. D. Teofilov, T. P. Ostojic, M. R. Bulatovic, J. D. Jovanovic,
O. V. Miljanovic Clinical Center of Montenegro, Podgorica, Montenegro P01.98B Rare mosaicism with ring X chromosome in female patient
with Turner syndrome and normal fertility: a case report Two novel homozygous splicing mutations in ARL2BP
cause autosomal recessive Retinitis Pigmentosa Two novel homozygous splicing mutations in ARL2BP
cause autosomal recessive Retinitis Pigmentosa M. Bauwens1, S. Naessens1, C. Van Cauwenbergh1,2, T. Van
Laethem1, S. De Jaegere1, I. Balikova2, Y. Sznajer3, J. De
Zaeytijd2, B. P. Leroy2,4, E. De Baere1 A. Fiorentino1, J. Yu2, G. Arno1,3, N. Pontikos1, S. Halford2,
S. Broadgate2, M. Michaelides3,1, K. J. Carss4,5, F. L. Raymond5,6, M. E. Cheetham1, A. R. Webster1,3, S. M. Downes7,
A. J. Hardcastle1, NIHR-BioResource Rare Diseases
Consortium, UK Inherited Retinal Dystrophy Consortium 1Center for Medical Genetics Ghent, Ghent University, Ghent,
Belgium, 2Department of Ophthalmology, Ghent University
and Ghent University Hospital, Ghent, Belgium, 3Centre de
Génétique Humaine, Cliniques universitaires St. Luc,
Université catholique de Louvain, Brussels, Belgium, 4Division
of Ophthalmology and Center for Cellular & Molecular
Therapeutics, The Children's Hospital of Philadelphia,
Philadelphia, PA, United States 1UCL Institute of Ophthalmology, London, United Kingdom,
2University of Oxford, Oxford, United Kingdom, 3Moorfields
Eye Hospital, London, United Kingdom, 4Department of
Haematology, University of Cambridge, Cambridge, United
Kingdom, 5Cambridge University Hospitals NHS Foundation
Trust, Cambridge, United Kingdom, 6Department of Medical
Genetics, University of Cambridge, Cambridge, United
Kingdom, 7Oxford Eye Hospital, John Radcliffe Hospital,
Oxford, United Kingdom Stargardt disease (STGD1) is a prevalent autosomal reces-
sive inherited retinal disease (IRD), hallmarked by a large
proportion of mono-allelic cases with one coding ABCA4
mutation, representing an interesting cohort to elucidate
missing heritability. We aimed to find the second patho-
genic allele in eleven mono-allelic STGD1 patients without
pathogenic coding or non-coding sequence variant after
ABCA4 locus resequencing. Targeted copy number variant
(CNV) analysis using a customized platform (arrEYE)
interrogating coding and non-coding regions of IRD genes,
revealed four novel ABCA4 CNVs in unrelated STGD1
patients. A 10 kb deletion spanning ABCA4 exons 10-11
was identified in one patient. An out-of-frame deletion of
exon 40-50 (19.112 bp) was identified in another patient
and eliminates the 3’UTR of the gene, most likely rendering
the resulting transcript unstable and prone to nonsense-
mediated decay. An in-frame tandem duplication (exons 2-
6) of 26 kb was identified in another patient. A non-coding
intronic tandem duplication of 7 kb was identified in intron
1, occurring in trans with a mild mutation. We hypothesize
a regulatory or splicing effect as a consequence of the
duplication. All CNVs were delineated and investigated via
bioinformatics analyses, pointing to a replicative-based
mechanism for three out of the four CNVs and to non-
homologous end joining for one CNV. P02.02A P02.02A
Coding and non-coding structural variants of ABCA4 Abstracts from the 51st European Society of Human Genetics Conference: Posters 43 contribute to the missing heritability in Stargardt disease, a
prevalent inherited retinal disease Two novel homozygous splicing mutations in ARL2BP
cause autosomal recessive Retinitis Pigmentosa These findings add
to the ABCA4 mutational spectrum, characterized by a
paucity of CNVs, with eight deletions reported so far. Finally, we highlight the importance of investigating non-
coding regions of ABCA4 in mono-allelic STGD1 patients. Introduction: Retinitis pigmentosa is the most common
inherited retinal dystrophy, affecting approximately 1 in
4,000 individuals. Mutations in ARL2BP, encoding ADP-
ribosylation factor-like 2 binding protein, have recently
been implicated as a cause of autosomal recessive retinitis
pigmentosa (arRP) with 3 homozygous variants identified to
date (c.101-1G>C, c.134T>G [p.Met45Arg], c.207+1G>T)
in cases of Arab-Muslim, European and Moroccan origin. Materials and Methods: Whole-genome sequencing
(WGS) or whole-exome sequencing (WES) was performed
in 1051 unrelated individuals recruited to the UK Inherited
Retinal Disease Consortium and NIHR-BioResource Rare
Diseases research studies. Sanger sequencing was used to
validate the next generation sequencing data, and RT-PCR
analysis was performed on RNA extracted from blood from
affected individuals to test for altered splicing of ARL2BP. Materials and Methods: Whole-genome sequencing
(WGS) or whole-exome sequencing (WES) was performed
in 1051 unrelated individuals recruited to the UK Inherited
Retinal Disease Consortium and NIHR-BioResource Rare
Diseases research studies. Sanger sequencing was used to
validate the next generation sequencing data, and RT-PCR
analysis was performed on RNA extracted from blood from
affected individuals to test for altered splicing of ARL2BP. Results:
Homozygous
variants
in
ARL2BP
(NM_012106.3) were identified in two unrelated indivi-
duals with RP. The variants, c.207+1G>A and c.390
+5G>A, at conserved splice donor sites for intron 3 and
intron 5 respectively, were predicted to alter the pre-mRNA
splicing of ARL2BP. RT-PCR spanning the affected exon/
intron boundaries showed both variants caused abnormal
splicing of ARL2BP in samples from affected individuals
compared to controls. M. Bauwens: None. S. Naessens: None. C. Van
Cauwenbergh: None. T. Van Laethem: None. S. De
Jaegere: None. I. Balikova: None. Y. Sznajer: None. J. De
Zaeytijd:
None. B.P. Leroy:
None. E. De
Baere: None. Conclusions: Our study identified 2 homozygous variants
in ARL2BP as a rare cause of arRP. Further studies are
required to define the underlying disease mechanism
causing retinal degeneration as a result of mutations in
ARL2BP. A. Fiorentino: None. J. Yu: None. G. Arno: None. N. Pontikos: None. S. Halford: None. S. Broadgate: None. M. Michaelides: None. K.J. Carss: None. F.L. Raymond: A. Fiorentino: None. J. Yu: None. G. Arno: None. N. Pontikos: None. S. Halford: None. S. Broadgate: None. M. Michaelides: None. K.J. P02.03B P02.03B
Two novel homozygous splicing mutations in ARL2BP
cause autosomal recessive Retinitis Pigmentosa V. Toulis1, M. Costa2, G. Marfany1,3,4 A cell model of ATXN3 knockdown was
generated using RNAi in human ARPE-19 cells, which was
analyzed
by
western-blot
and
immunofluorescence
detection. Methods: Functional analysis in Atxn3 murine models
was performed and the retinal phenotype was studied by
immunofluorescence
detection,
transmission
electron
microscopy and photoreceptor isolation for a more detailed
description. A cell model of ATXN3 knockdown was
generated using RNAi in human ARPE-19 cells, which was
analyzed
by
western-blot
and
immunofluorescence
detection. Material and methods: Five-generation Polish family
participated in the study. Clinical exome sequencing on the
proband’s DNA and family segregation analysis of the
identified variants were performed. Audiological assess-
ment included pure tone audiometry (PTA), impedance
audiometry,
transient
evoked
otoacoustic
emissions
(TEOAE)
and
auditory
brainstem
responses
(ABRs). Vestibular system function was evaluated using ocular
and
cervical
vestibular
evoked
myogenic
potentials
(oVEMP, cVEMP). Temporal bone computed tomography
was also performed. Material and methods: Five-generation Polish family
participated in the study. Clinical exome sequencing on the
proband’s DNA and family segregation analysis of the
identified variants were performed. Audiological assess-
ment included pure tone audiometry (PTA), impedance
audiometry,
transient
evoked
otoacoustic
emissions
(TEOAE)
and
auditory
brainstem
responses
(ABRs). Vestibular system function was evaluated using ocular
and
cervical
vestibular
evoked
myogenic
potentials
(oVEMP, cVEMP). Temporal bone computed tomography
was also performed. Results: The Atxn3 knockout mouse retinas showed a
significant elongation of the photoreceptors, mislocalization
of cone-specific phototransduction proteins, with concomi-
tant elongation of the connecting cilium -the gate through
which the proteins are transported to the photoreceptor outer
segment-, and diminished phagocytosis of the photoreceptor
outer segments by the retinal pigmented epithelium (RPE). These results were also confirmed in vitro in ARPE-
19 cells. Results: Genetic testing revealed a novel probably
pathogenic c.553G>A (p.Asp185Asn) TBC1D24 variant,
which fully segregated with HL in the studied family. Clinically, progressive HL involving mainly high frequen-
cies was observed. No TEOAE were recorded in the study
subjects and no or increased threshold of the stapedial
muscle reflex was found. Function of the vestibulocochlear
nerve measured by ABR was normal. No vestibular
dysfunction and anatomical abnormalities of cochleoves-
tibular system were detected. Conclusions: Our work supports the key role of ATXN3
in retinal development and maintenance, particularly in the
ciliogenesis and protein trafficking in photoreceptors,
thereby highlighting ATXN3 as a good candidate gene for
inherited retinal dystrophies. Grants: V.T. is in receipt of a FPI fellowship (BES-2014-
068639, MINECO). V. Toulis1, M. Costa2, G. Marfany1,3,4 D. Oziębło1,2, G. Tacikowska3, K. Kochanek4, H. Skarżyński5,
M. Ołdak1 1Departament de Genètica, Microbiologia i Estadística,
Facultat de Biologia, Universitat de Barcelona, Barcelona,
Spain, 2Department of Neurology, Medical School, University
of Michigan, Ann Arbor, MI, United States, 3Institut de
Biomedicina de la Universitat de Barcelona (IBUB),
Barcelona, Spain, 4Centro de Investigación Biomédica en Red
de Enfermedades Raras (CIBERER), Instituto de Salud Carlos
III, Barcelona, Spain 1Departament de Genètica, Microbiologia i Estadística,
Facultat de Biologia, Universitat de Barcelona, Barcelona,
Spain, 2Department of Neurology, Medical School, University
of Michigan, Ann Arbor, MI, United States, 3Institut de
Biomedicina de la Universitat de Barcelona (IBUB),
Barcelona, Spain, 4Centro de Investigación Biomédica en Red
de Enfermedades Raras (CIBERER), Instituto de Salud Carlos
III, Barcelona, Spain 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Postgraduate School
of Molecular Medicine, Medical University of Warsaw,
Warsaw, Poland, 3Department of Otoneurology, Institute of
Physiology and Pathology of Hearing, Warsaw, Poland,
4Department of Experimental Audiology, Institute of
Physiology and Pathology of Hearing, Warsaw, Poland, 5Oto-
Rhino-Laryngology Surgery Clinic, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Postgraduate School
of Molecular Medicine, Medical University of Warsaw,
Warsaw, Poland, 3Department of Otoneurology, Institute of
Physiology and Pathology of Hearing, Warsaw, Poland,
4Department of Experimental Audiology, Institute of
Physiology and Pathology of Hearing, Warsaw, Poland, 5Oto-
Rhino-Laryngology Surgery Clinic, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland Introduction: An expanded CAG repeat on the ATXN3
gene, encoding the deubiquitinating enzyme ATXN3, cau-
ses Spinocerebellar Ataxia Type 3 (SCA3), a late onset
autosomal dominant neurodegenerative disorder. The phy-
siological role of the wild-type ATXN3 protein, however, is
not completely understood. Since deubiquitination seems to
play a crucial role in photoreceptor development and dif-
ferentiation, we aimed to define the function of ATXN3 in
retinal formation combining animal and cell models. Background:
To
date
different
genetic
variants
in
TBC1D24 gene were causally involved in the development
of neurological syndromes and profound prelingual hearing
loss (HL) inherited in a recessive manner (DFNB86). In
2014 the first and so far only TBC1D24 pathogenic variant
has been linked with postlingual autosomal dominant HL
(DFNA65). Methods: Functional analysis in Atxn3 murine models
was performed and the retinal phenotype was studied by
immunofluorescence
detection,
transmission
electron
microscopy and photoreceptor isolation for a more detailed
description. Two novel homozygous splicing mutations in ARL2BP
cause autosomal recessive Retinitis Pigmentosa Carss: None. F.L. Raymond: 44 J. del Picchia Spain), La Marató TV3 (project 201417.30), SGR2014-
0932 (Generalitat de Catalunya). V. Toulis: None. M. Costa: None. G. Marfany: None. None. M.E. Cheetham: None. A.R. Webster: None. S.M. Downes: None. A.J. Hardcastle: None. P02.04C
ATXN3 in retina: from ataxia to candidate gene for retinal
dystrophies None. M.E. Cheetham: None. A.R. Webster: None. S.M. Downes: None. A.J. Hardcastle: None. A study of the genetics of cholesteatoma through systematic
review and whole exome sequencing I. Jovanovic1, T. Djuric1, M. Zivkovic1, S. Jesic2,3, A. Stankovic1 B. A. Jennings1, C. Philpott1, M. F. Bhutta2, D. Swan3,
G. Willis4, J. Woods5, P. Prinsley5 1Laboratory for Radiobiology and Molecular Genetics, Vinca
Institute of Nuclear Sciences, University of Belgrade, Belgrade,
Serbia, 2Medical Faculty, University of Belgrade, Belgrade,
Serbia, 3Clinic for Otorhinolaryngology and Maxillofacial
Surgery, Clinical Centre of Serbia, Belgrade, Serbia 1UEA, Norwich, United Kingdom, 2Brighton and Sussex
University NHS Trust, Brighton, United Kingdom, 3NCIMB,
Aberdeen, United Kingdom, 4Norfolk and Norwich University
Hospital, Norwich, United Kingdom, 5James Paget University
Hospital, Great Yarmouth, United Kingdom Aberdeen, United Kingdom, 4Norfolk and Norwich University
Hospital, Norwich, United Kingdom, 5James Paget University
Hospital, Great Yarmouth, United Kingdom Introduction: Chronic otitis media is followed by irrever-
sible tissue damage and destruction of the middle ear
structures. Cholesteatoma is an expanding, destructive epi-
thelial lesion within the middle ear, commonly associated
with chronic otitis media. Molecular mechanisms of chronic
otitis media with cholesteatoma (COMch) and without
cholesteatoma (COM) are challenging subject in research. The aim of this study was to perform transcriptome profil-
ing of tissue from COMch and COM, and subsequent net-
work analysis, to identify candidate miRNA regulatory hubs
that can potentially serve as molecular discriminators of
these two pathologies. Introduction: Chronic otitis media is followed by irrever-
sible tissue damage and destruction of the middle ear
structures. Cholesteatoma is an expanding, destructive epi-
thelial lesion within the middle ear, commonly associated
with chronic otitis media. Molecular mechanisms of chronic
otitis media with cholesteatoma (COMch) and without
cholesteatoma (COM) are challenging subject in research. The aim of this study was to perform transcriptome profil-
ing of tissue from COMch and COM, and subsequent net-
work analysis, to identify candidate miRNA regulatory hubs
that can potentially serve as molecular discriminators of
these two pathologies. Introduction A cholesteatoma is a mass of keratinising
epithelium in the middle ear. It is a rare disorder, associated
with significant morbidity. Its OMIM entry (#604183) cites
minimal evidence for Mendelian inheritance, but we have
observed 31 multiply affected families in Norfolk; including
individuals with bilateral disease, suggesting a genetic
component for its aetiology. Methods We conducted a systematic literature review
(SR) to identify any published studies about the genetics of
cholesteatoma and established a national biobank for
subsequent whole exome sequencing (WES) studies of
familial disease. P02.07B Integrative identification of miRNA regulatory hubs as
candidate molecular discriminators of chronic otitis media
pathologies V. Toulis1, M. Costa2, G. Marfany1,3,4 Work is supported by SAF2013-
49069-C2-1-R (Ministerio de Economía y Competitividad, Grants: V.T. is in receipt of a FPI fellowship (BES-2014-
068639, MINECO). Work is supported by SAF2013-
49069-C2-1-R (Ministerio de Economía y Competitividad, Conclusions: Our results represent the first independent
confirmation of TBC1D24 involvement in the development
of autosomal dominant HL and the first thorough clinical
characteristics of TBC1D24-induced autosomal dominant Abstracts from the 51st European Society of Human Genetics Conference: Posters 45 HL. The identified TBC1D24 variant affects the cochlear
component of the auditory system and results in a high
frequency HL usually observed in the third decade of life. HL. The identified TBC1D24 variant affects the cochlear
component of the auditory system and results in a high
frequency HL usually observed in the third decade of life. miRNAs to become clinically applicable biomarkers and
therapeutic targets. miRNAs to become clinically applicable biomarkers and
therapeutic targets. Acknowledgements: This work was funded by Serbian
Ministry of Education, Science and Technological devel-
opment Grant OI175085. Supported by: 2016/22/E/NZ5/00470 Supported by: 2016/22/E/NZ5/00470 D. Oziębło: None. G. Tacikowska: None. K. Kocha-
nek: None. H. Skarżyński: None. M. Ołdak: None. I. Jovanovic: None. T. Djuric: None. M. Zivkovic:
None. S. Jesic: None. A. Stankovic: None. A study of the genetics of cholesteatoma through systematic
review and whole exome sequencing We have also completed a pilot sequencing
study to identify candidate variants that segregate with the
disease phenotype (using NimbleGen exome capture; and
the Illumina HiSeq4000 platform). Materials
and
Methods:
Transcriptome
data
were
obtained from COMch (n = 2 patients) and COM tissue
(n = 4 patients), by employing Illumina iScan microarray
technology. Differentially expressed genes (DEGs) were
identified using R/Bioconductor limma package. Functional
analysis of DEGs and determination of network miRNAs by
reverse mapping of target DEGs was done using miRNet
software. Nonspecific miRNAs were excluded. Results In our SR, we identified 8 case-series with
multiply-affected families and associations with congenital
malformation syndromes. DNA and clinical data have been
collected from 42 participants (from 9 multiply affected
Norfolk families) to date. In 2018, participants will also be
recruited from 10 additional UK centres. Our pilot WES
study of 16 participants from 4 families identified 95,437
variants. Variant filtering, using pedigree analysis, has
identified 430 candidate genes for further filtering using the
Ensembl Variant Effect Predictor. Results: Transcriptome analysis identified 169 DEGs. Significant biological processes involving DEGs covered
epithelial cell and keratinocyte differentiation and extra-
cellular structure organization. miRNet determined hsa-
miR-8485, hsa-miR-24-3p, hsa-miR-34a-5p, hsa-miR-9-5p,
hsa-miR-375, hsa-miR-145-5p, hsa-miR-21-5p as top hub
miRNas with centrality degree ≥8. Conclusions: The identified hub miRNAs should be the
guide mark for future studies on mechanisms of miRNA
activity in chronic otitis media pathologies. Also, hub
miRNAs should be evaluated for their utilization in
prevention and therapy, based on the growing potential of Results: Transcriptome analysis identified 169 DEGs. Significant biological processes involving DEGs covered
epithelial cell and keratinocyte differentiation and extra-
cellular structure organization. miRNet determined hsa-
miR-8485, hsa-miR-24-3p, hsa-miR-34a-5p, hsa-miR-9-5p,
hsa-miR-375, hsa-miR-145-5p, hsa-miR-21-5p as top hub
miRNas with centrality degree ≥8. Conclusion We have completed our SR (see PROSPERO
register
CRD42015023579)
and
established
the
first
biobank to explore the genetics-of-cholesteatoma. A WES
strategy and bioinformatics pipeline have been developed in
the pilot study; and preliminary filtering has identified
candidate variants that could have an impact on TGF β
signalling and inflammatory processes. Conclusion We have completed our SR (see PROSPERO
register
CRD42015023579)
and
established
the
first
biobank to explore the genetics-of-cholesteatoma. A WES
strategy and bioinformatics pipeline have been developed in
the pilot study; and preliminary filtering has identified
candidate variants that could have an impact on TGF β
signalling and inflammatory processes. P02.08C 1Institute of Physiology and Pathology of Hearing, Warsaw,
Poland., Warsaw, Poland, 2Department of Medical Genetics,
Warsaw Medical University, Warsaw, Poland, Warsaw,
Poland, 3Postgraduate School of Molecular Medicine, Warsaw
Medical University, Warsaw, Poland, Warsaw, Poland The relationship between defects at the middle ear bone
chain and NLRP3 and OPG gene polymorphisms at
chronic otitis media patients S. Keskin1, P. Ata2, B. Erkal2, T. Dogan2, A. Eren3,
A. Tatlipinar4 S. Keskin1, P. Ata2, B. Erkal2, T. Dogan2, A. Eren3,
A. Tatlipinar4 Hearing loss (HI) is a highly heterogenic and frequent dis-
ability that affects human senses. Calcium- and integrin-
binding protein 2 (CIB2) is one of the genes involved in HI
pathogenesis. The CIB2 protein is responsible for main-
taining Ca2+ homeostasis in cells and interacting with
integrins. The aim of presented study was to establish the
frequency of CIB2-related hearing impairment based on
pooled DNA sequencing. DNA samples derived from 900
patients with HI were pooled and 180 pools were obtained
(5 DNA samples/pool). The whole CIB2 coding region was
amplified in the DNA pools and sequenced on MiSeq
apparatus (Illumina). Ten novel, potentially pathogenic
variants were identified within the pools. DNA samples
derived from pools with detected variants were separately
resequenced via direct sequencing to confirm the presence
of the variants as well as to assign the variants to a parti-
cular sample. Five of the primarily detected variants were
confirmed in the heterozygous form, which was in line with
the NGS results, the following five variants are currently
being verified. Thus, our results confirmed that pool-seq is a
cost and time effective approach that can be used for
screening of causative HI variants in a large group of
patients. This study was supported by National Science
Centre grant 2011/03/D/NZ5/05592. 1Fatih Sultan Mehmet Research and Training Hospital,
Istanbul, Turkey, 2Marmara University School of Medicine,
Istanbul, Turkey, 3Koc University, Istanbul, Turkey, 4Okan
University School of Medicine, Istanbul, Turkey Introduction: The purpose of this research is to investigate
the relation between osteoprotogerin, NLRP3 inflamma-
some gene polymorphisms and ossicular chain defects. Materials and Methods: There were 96 participants,
composed of 30 patients with type 1 tympanoplasty due to
chronic otitis media between 01/06//2013 - 01/06/2016, had
intact ossicular chain; 20 patients who had ossiculoplasty
because of ossicular chain erosion, and 46 healthy controls. DNA was isolated from peripheral blood using Puregene
Qiagen kit. P02.09D P02.09D
CIB2 gene in the pathogenesis of hearing loss - results of
pooled DNA high throughput sequencing P02.09D
CIB2 gene in the pathogenesis of hearing loss - results of
pooled DNA high throughput sequencing A. Pollak1, J. Gzik1, A. Jacoszek2,3, U. Lechowicz1, P. Stawiński1,
R. Ploski2, H. Skarżynski1, M. Oldak1 PAX6 sequence variants affecting splicing cause сongenital
aniridia A study of the genetics of cholesteatoma through systematic
review and whole exome sequencing Conclusions: The identified hub miRNAs should be the
guide mark for future studies on mechanisms of miRNA
activity in chronic otitis media pathologies. Also, hub
miRNAs should be evaluated for their utilization in
prevention and therapy, based on the growing potential of Conclusions: The identified hub miRNAs should be the
guide mark for future studies on mechanisms of miRNA
activity in chronic otitis media pathologies. Also, hub
miRNAs should be evaluated for their utilization in
prevention and therapy, based on the growing potential of 46 J. del Picchia polymorphism was higher at tympanoplasty and ossiculo-
plasty patients as a whole compared to controls. The level of
OPG at inner ear swabs taken from tuba Eustachi opening at
nasopharynx was increased at tympanoplasty group. B.A. Jennings: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; The Bernice Bibby
Research Trust. C. Philpott: B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Modest; Rosetrees Trust
(A1136), The Bernice Bibby Research Trust. M.F. Bhutta:
None. D. Swan: None. G. Willis: None. J. Woods: None. P. Prinsley: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants
already
received);
Modest;
Royal
College
of
Surgeons, The Bernice Bibby Research Trust. S. Keskin: None. P. Ata: None. B. Erkal: None. T. Dogan: None. A. Eren: None. A. Tatlipinar: None. P02.08C OPG and NLRP3 were analyzed at real-time
PCR reaction with melting curve analysis. Also inner ear
swabs were used for RNA isolation and OPG and NLRP3
expression were analyzed with Real-time quantitativePCR. Materials and Methods: There were 96 participants,
composed of 30 patients with type 1 tympanoplasty due to
chronic otitis media between 01/06//2013 - 01/06/2016, had
intact ossicular chain; 20 patients who had ossiculoplasty
because of ossicular chain erosion, and 46 healthy controls. Results: There were 37 males (38.5%) and 59 females
(61.5%) with an age range of 15 - 70 years, The average age
was
35.05 ± 14.43
years. There
was
no
statistically
significant difference between three groups according to
OPG c.226A>C (p.Thr76Pro) and NLRP3 c.592G>A (p. Val198Met) polymorphisms. But when analyzed between
the controls and patient group as a whole there was a
significant difference of OPG c.226A>C (p.Thr76Pro)
polymorphism. The expression level of OPG was higher
at
the
tympanoplasty
group
compared
to
ossiculoplasty group A. Pollak: None. J. Gzik: None. A. Jacoszek: None. U. Lechowicz: None. P. Stawiński: None. R. Ploski: None. H. Skarżynski: None. M. Oldak: None. A. Pollak: None. J. Gzik: None. A. Jacoszek: None. U. Lechowicz: None. P. Stawiński: None. R. Ploski: None. H. Skarżynski: None. M. Oldak: None. Evaluation of genetic variants associated with congenital
stationary night blindness 2 (CSNB2) Evaluation of genetic variants associated with congenital
stationary night blindness 2 (CSNB2) A. Filatova: None. T. Vasilyeva: None. M. Skoblov:
None. A. Marakhonov: None. A. Voskresenskaya: None. R. Zinchenko: None. A. Mihalich1, G. Cammarata2, E. Ponti1, G. Tremolada2,
S. Bianchi Marzoli2, A. Di Blasio1 O. M. Messina-Baas1, C. Leon-Oviedo2, J. M. Valdes-Miranda3,
M. R. Rivera-Vega2, R. Vega-Gama2, N. Xilotl-DeJesus2, P02.10A Introduction: Aniridia is a rare autosomal dominant
panocular disorder caused by mutations in the PAX6 gene or
chromosome 11p13 rearrangements. Materials and Methods: DNA samples for analysis were
obtained from patients with congenital aniridia (110 patients
from 84 unrelated families). The search for mutations in the
PAX6 gene was carried out by Sanger sequencing, MLPA
and analysis of heterozygosity loss (LOH) in proband. To
determine the effects of identified SNVs on PAX6 pre-
mRNA splicing we used in vitro minigene assay. Results: Molecular analysis of a large cohort of aniridia
patients from Russia conducted earlier revealed a significant
proportion of PAX6 mutations affecting splicing (14 from
81 mutations). We focused on 8 SNVs affected slicing: 6
deep-intronic and 2 exonic. These variants were classified
as variant of unknown significance (VUS), benign or likely
pathogenic according to ACMG recommendations. Human
Splicing Finder and IntSplice on-line tools analysis predict
them to disrupt PAX6 pre-mRNA splicing. To validate this
hypothesis we used a minigene system and showed that all
investigated sequence variants except one affect splicing. These variants result in open reading frame shifting,
premature termination codon formation following by RNA
degradation by nonsense-mediated decay. Thus, investi-
gated SNVs produce a null allele and haploinsufficiency of
the PAX6-function. So putative mutations were reclassified
as loss of function. O.M. Messina-Baas: None. C. Leon-Oviedo: None. J. M. Valdes-Miranda: None. M.R. Rivera-Vega: None. R. Vega-Gama: None. N. Xilotl-DeJesus: None. V. Martí-
nez-Montoya: None. M.G. Tovar-Ayala: None. L.M. Gonzalez-Huerta:
None. S.A. Cuevas-
Covarrubias: None. Conclusions: Using functional in vitro analysis we
confirmed the pathogenicity of 7 PAX6 mutations affecting
splicing. Our results emphasized the necessity of such
analysis and advanced search for PAX6 mutations. This
work is supported by RFBR grant 17-04-00475. 1Istituto Auxologico Italiano, Lab. of Medical Genetics,
Milano, Italy, 2Istituto Auxologico Italiano, Dept. of
Neurophthalmology and Electrophysiology, Milano, Italy A. Mihalich1, G. Cammarata2, E. Ponti1, G. Tremolada2,
S. Bianchi Marzoli2, A. Di Blasio1 P02.10A PAX6 sequence variants affecting splicing cause сongenital
aniridia Conclusion: There was no statistically significant differ-
ence of OPG and NLRP3 gene polymorphisms among three
groups. But TNFRSF11B (OPG) c.226A>C (p.Thr76Pro) Abstracts from the 51st European Society of Human Genetics Conference: Posters 47 V. Martínez-Montoya4, M. G. Tovar-Ayala2, L. M. Gonzalez-
Huerta2, S. A. Cuevas-Covarrubias5 V. Martínez-Montoya4, M. G. Tovar-Ayala2, L. M. Gonzalez-
Huerta2, S. A. Cuevas-Covarrubias5 V. Martínez-Montoya4, M. G. Tovar-Ayala2, L. M. Gonzalez-
Huerta2, S. A. Cuevas-Covarrubias5 A. Filatova1, T. Vasilyeva1, M. Skoblov1,2, A. Marakhonov1,2,
A. Voskresenskaya3, R. Zinchenko1,4 A. Filatova1, T. Vasilyeva1, M. Skoblov1,2, A. Marakhonov1,2,
A. Voskresenskaya3, R. Zinchenko1,4 1Oftalmologia, Hospital General de Mexico, Mexico DF,
Mexico, 2Genetica, Hospital General de Mexico, Mexico DF,
Mexico, 3Gentica, Hospital General de Mexico, Mexico DF,
Mexico, 4Genética, Hospital General de Mexico, Mexico DF,
Mexico, 5Genetica, Hospital General de Mexico, Facultad de
Medicina, Universidad Nacional Autonoma de Mexico, Mexico
DF, Mexico 1Research Centre of Medical Genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology
(State University), Dolgoprudny, Russian Federation,
3Cheboksary branch of S. Fyodorov Eye Microsurgery Federal
State Institution, Cheboksary, Russian Federation, 4Pirogov
Russian National Research Medical University, Moscow,
Russian Federation Cataracts are one important cause of blindness around the
world. Primary congenital cataract has several patterns of
inheritance and can be syndromic and non-syndromic. More
than 100 loci have been associated with primary congenital
cataract; autosomal dominant is the principal form of
inheritance. Cataract is due to opacities of the lens resulting
in defects of the refractive index. This produces alterations
in the lens structure resulting in light scattering with a
significant concentration of high-molecular-weight protein
aggregates. The aim of the present study was to analyze,
through exome sequencing, a sample of Mexican patients
with non-familial primary congenital cataract. Genomic
DNA was extracted through conventional methods and
analyzed
through
exome
sequencing;
Sanger
direct
sequencing was performed to confirm the exome results. Molecular analysis identified defects in the HSF4, BFSP1,
GCNT2, GJA8, BFSP1, NHS and MAF genes. These
alterations were not found in healthy members of the
families and 100 normal controls. This study allowed to
describe the genes associated with primary congenital cat-
aract in a sample of Mexican patients and enriched the
spectrum of these molecular defects associated with her-
editary cataract. Introduction: Aniridia is a rare autosomal dominant
panocular disorder caused by mutations in the PAX6 gene or
chromosome 11p13 rearrangements. P02.11B The
results of these tests indicate that vision impairment in
CSNB2 patients may derive from a decreased synaptic
transmission between photoreceptor and second-order neu-
rons. The Cav1.4 channel is involved in this process and
CACNA1F gene encodes the pore-forming subunit α1. Since CACNA1F is located on the X chromosome, Cav1.4
channelopathies are typically affecting male patients. Materials and Methods: We performed NGS of 32
known retinal disease genes on DNA of 3 male patients
with CSNB2 and different symptoms of the disease. In two
patients CACNA1F mRNA was also studied in peripheral
lymphocytes. Results: We have identified 3 unreported CACNA1F
variants : in exon 4 c.425dupC, in exon 43 c.G5123C and in
exon 48 c. 5800delG. The first variant causes insertion of a
stop codon that could determine mRNA degradation by
Nonsense Mediated Decay (NMD). However, mRNA
evaluation revealed that the transcript was present. The
second variant is located in a splicing site and skipping of
exon 43 was demonstrated by mRNA sequencing. The third
variant determinates a frameshift. In this patient mRNA was
not available. Conclusions: These results confirm that, in CSNB2
patients, variants in the same gene may be associated with
different phenotypic characteristics. Moreover, they high-
light the importance of studying gene expression and
protein function to assess the biological significance of the
variants detected. P.I. Buonfiglio: None. C.D. Bruque: None. V. Lotersz-
tein: None. E. Goldschmidt: None. A.B. Elgoyhen: None. V.K. Dalamón: None. P.I. Buonfiglio: None. C.D. Bruque: None. V. Lotersz-
tein: None. E. Goldschmidt: None. A.B. Elgoyhen: None. V.K. Dalamón: None. P02.14A A. Mihalich: None. G. Cammarata: None. E. Ponti:
None. G. Tremolada: None. S. Bianchi Marzoli: None. A. Di Blasio: None. Personalized stem cell therapy to correct corneal defects
due to a unique homozygous-heterozygous mosaicism of
Ectrodactyly-Ectodermal dysplasia-Clefting syndrome Di Blasio: None. P02.13D P. Nespeca1, V. Barbaro2, A. Nasti1, P. Raffa1, A. Migliorati1,
S. Ferrari2, E. Palumbo3, M. Bertolin2, C. Breda2, F. Miceli4,
A. Russo3, L. Caenazzo1, D. Ponzin2, G. Palù1, C. Parolin1, E. Di
Iorio1 Large-scale molecular analysis of Hereditary Hearing Loss
genes in Argentinean deaf patients: lookingfora needle in a
haystack Large-scale molecular analysis of Hereditary Hearing Loss
genes in Argentinean deaf patients: lookingfora needle in a
haystack P02.11B Analysis through exome sequencing in patients with non
familial primary congenital cataract J. del Picchia 48 Hereditary Hearing Loss (HHL) is a common trait affecting
1 in 2000 new born children. The presence of over 100
different genes involved in HHL, lead us to go on board
with Whole Exome Sequencing (WES) in order to search
for the causative mutations. The main objective of this
project was to diagnose Argentinean deaf families and
discover novel mutations or new genes involved in
pathology. We designed a flowchart to exclude all the
spurious variations obtained and target for few candidates. To approach this, we filtered results from WES, and can-
didate variations were segregated throughout family mem-
bers. Variations positively selected, were analyzed using
bioinformatic predictors and tracked in public databases. Additionally, conservation studies, structure and functional
domain analysis in proteins, and in-vivo studies were per-
formed. Using this strategy we analysed 15 WES results. We identified 16 causative mutations in 12 families with
syndromic and non-syndromic hearing loss (11 missense, 4
frameshift and 1 splicing site mutations). Six were novel
and functional studies of some of the identified mutations,
using Zebra fish models, are under way. In the remaining 3
families, variables of uncertain significance were detected
(Vous). To our knowledge this is the first study using WES
to diagnose deaf patients in Argentina. We show in the
present study that our flowchart is advantageous and note-
worthy for large-scale molecular analysis in deaf patients. These findings clearly highlight the importance of genetic
studies followed by in-sílico and in-vivo validation to better
understand the genetic basis of Hereditary Hearing loss. Introduction: CSNB2 is a non-progressive retinal disorder
with clinical features that include reduced visual acuity,
nystagmus and variable myopia or hypermetropia. Char-
acteristic
abnormalities
are
detected
upon
electro-
retinography, autofluorescence and infrared imaging. The
results of these tests indicate that vision impairment in
CSNB2 patients may derive from a decreased synaptic
transmission between photoreceptor and second-order neu-
rons. The Cav1.4 channel is involved in this process and
CACNA1F gene encodes the pore-forming subunit α1. Since CACNA1F is located on the X chromosome, Cav1.4
channelopathies are typically affecting male patients. Introduction: CSNB2 is a non-progressive retinal disorder
with clinical features that include reduced visual acuity,
nystagmus and variable myopia or hypermetropia. Char-
acteristic
abnormalities
are
detected
upon
electro-
retinography, autofluorescence and infrared imaging. Large-scale molecular analysis of Hereditary Hearing Loss
genes in Argentinean deaf patients: lookingfora needle in a
haystack P. Nespeca1, V. Barbaro2, A. Nasti1, P. Raffa1, A. Migliorati1, P. Nespeca1, V. Barbaro2, A. Nasti1, P. Raffa1, A. Migliorati1, S. Ferrari2, E. Palumbo3, M. Bertolin2, C. Breda2, F. Miceli4, S. Ferrari2, E. Palumbo3, M. Bertolin2, C. Breda2, F. Miceli4, A. Russo3, L. Caenazzo1, D. Ponzin2, G. Palù1, C. Parolin1, E. Di
Iorio1 P. I. Buonfiglio1, C. D. Bruque2, V. Lotersztein3,
E. Goldschmidt4, A. B. Elgoyhen1, V. K. Dalamón1 1Departments of Molecular Medicine, Padova, Italy,
2Fondazione Banca degli Occhi, Venezia, Italy, 3Departments
of Biology, Padova, Italy, 4Departments of Neuroscience,
Napoli, Italy E. Goldschmidt4, A. B. Elgoyhen1, V. K. Dalamón1 1Instituto de Investigaciones en Ingeniería Genética y Biología
Molecular “Dr. Héctor Torres” - INGEBI/CONICET, CABA,
Argentina, 2Centro Nacional de Genética Médica A.N.L.I.S. Dr. Carlos G. Malbrán, CABA, Argentina, 3Servicio de
Genética del Hospital Militar Central Cirujano Mayor Dr. Cosme Argerich, CABA, Argentina, 4Servicio de Genética del
Hospital General de Agudos Dr. Juan A. Fernández, CABA,
Argentina Introduction: Ectrodactyly-Ectodermal dysplasia-Clefting
syndrome is a rare autosomal dominant disease caused by
mutations in the p63 gene. To date, approximately 40 dif-
ferent p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and Abstracts from the 51st European Society of Human Genetics Conference: Posters 49 Toulouse, France, 7West Midlands Regional Clinical Genetics
Service and Birmingham Health Partners, Birmingham
Women’s and Children’s NHS Foundation Trust, Birmingham,
United Kingdom resolve the progressive corneal degeneration due to a pre-
mature aging of limbal epithelial stem cells. Here, we
describe a unique case of a young EEC patient, who was,
surprisingly, homozygous for a novel and de novo R311K
missense mutation in the p63 gene. resolve the progressive corneal degeneration due to a pre-
mature aging of limbal epithelial stem cells. Here, we
describe a unique case of a young EEC patient, who was,
surprisingly, homozygous for a novel and de novo R311K
missense mutation in the p63 gene. Defects in eye morphogenesis can lead to a spectrum of
structural ocular disorders, including anophthalmia, micro-
phthalmia and coloboma (AMC). These conditions, affect-
ing approximately 6-13 per 100,000 individuals, are
clinically and genetically heterogeneous. The number of
genes known to be involved in AMC is rapidly growing, yet
only approximately 25% of patients receive a genetic
diagnosis. Large-scale molecular analysis of Hereditary Hearing Loss
genes in Argentinean deaf patients: lookingfora needle in a
haystack Following whole-exome sequencing of 53 indi-
viduals with developmental eye anomalies, we identified
one patient with bilateral microphthalmia and polydactyly
carrying a de novo nonsynonymous variant (NM_012300.2:
c.1087C>T, p.[Arg363Trp]) in FBXW11. This gene codes
for an F-box protein involved in the regulation of cyto-
plasmic levels of β-catenin and therefore plays an important
role in the Wnt signalling, a fundamental growth-control
pathway also implicated in eye and limb development. Screening of FBXW11 in a further 252 patients with
developmental eye anomalies identified three previously
unreported 3’ untranslated region (UTR) variants in three
unrelated individuals. In situ hybridisation experiments
showed FBXW11 expression in developing human eye and
brain, including the retinal neuroepithelium, telencephalon
and the cerebellum. We also demonstrated that zebrafish
models with reduced expression of the FBXW11 ortholo-
gues fbxw11a and fbxw11b display smaller, misshapen and
underdeveloped eyes, and abnormal pectoral fin and jaw
development. Material and Methods: Genetic analysis was performed
in p63 gene to identify the disease-causing mutation. FISH,
qRT-PCR and SNPs assay were executed to discover an
additional
alteration
in
p63. From
oral
biopsy,
the
heterozygous mutant cells were expanded performing clonal
analysis. Results: The analysis of the degree of somatic mosaicism
in leukocytes and oral mucosal epithelial stem cells
(OMESCs) showed that the 80% were homozygous mutant
cells
and
20%
were
heterozygous. Cytogenetic
and
molecular analyses excluded genomic alterations, thus
suggesting a de novo mutation followed by an allelic gene
conversion of the wild-type allele by de novo mutant allele
as a possible mechanism to explain the homozygous
condition. The R311K-p63 OMESCs were able to generate
well-organized and stratified epithelia in vitro, resembling
the features of healthy tissues. Results: The analysis of the degree of somatic mosaicism
in leukocytes and oral mucosal epithelial stem cells
(OMESCs) showed that the 80% were homozygous mutant
cells
and
20%
were
heterozygous. Cytogenetic
and
molecular analyses excluded genomic alterations, thus
suggesting a de novo mutation followed by an allelic gene
conversion of the wild-type allele by de novo mutant allele
as a possible mechanism to explain the homozygous
condition. The R311K-p63 OMESCs were able to generate
well-organized and stratified epithelia in vitro, resembling
the features of healthy tissues. P02.16C
Molecular diagnosis in paediatric retinal dystrophies Molecular diagnosis in paediatric retinal dystrophies 1Faculty of Health and Life Sciences, Oxford Brookes
University, Oxford, United Kingdom, 2Department of Cell and
Developmental Biology, University College London, London,
United Kingdom, 3Great Ormond Street Institute of Child
Health, University College London, London, United Kingdom,
4Service de Génétique Médicale, Hôpital Purpan, CHU
Toulouse, Toulouse, France, 5UMR 1056 Inserm - Université
de Toulouse, Toulouse, France, 6UDEAR, Université de
Toulouse, UMRS 1056 INSERM-Université Paul Sabatier, P02.15B Mutations in F-Box and WD40 Domain Protein 11
(FBXW11) are associated with developmental eye and digit
anomalies F. Ceroni: None. R.M. Young: None. R.J. Holt: None. B. Crespo: None. N. Chassaing: None. D.A. Bax: None. C. Zazo Seco: None. P. Calvas: None. D. Gerrelli: None. S.W. Wilson: None. N.K. Ragge: None. F. Ceroni: None. R.M. Young: None. R.J. Holt: None. B. Crespo: None. N. Chassaing: None. D.A. Bax: None. C. Zazo Seco: None. P. Calvas: None. D. Gerrelli: None. S.W. Wilson: None. N.K. Ragge: None. F. Ceroni: None. R.M. Young: None. R.J. Holt: None. B. Crespo: None. N. Chassaing: None. D.A. Bax: None. F. Ceroni: None. R.M. Young: None. R.J. Holt: None. B. Crespo: None. N. Chassaing: None. D.A. Bax: None. C. Zazo Seco: None. P. Calvas: None. D. Gerrelli: None. S.W. Wilson: None. N.K. Ragge: None. C. Zazo Seco: None. P. Calvas: None. D. Gerrelli: None. S.W. Wilson: None. N.K. Ragge: None. F. Ceroni1, R. M. Young2, R. J. Holt1, B. Crespo3,
N. Chassaing4,5, D. A. Bax1, C. Zazo Seco6, P. Calvas4,5,
D. Gerrelli3, S. W. Wilson2, N. K. Ragge1,7 F. Ceroni1, R. M. Young2, R. J. Holt1, B. Crespo3,
N. Chassaing4,5, D. A. Bax1, C. Zazo Seco6, P. Calvas4,5,
D. Gerrelli3, S. W. Wilson2, N. K. Ragge1,7 P02.16C P02.16C
Molecular diagnosis in paediatric retinal dystrophies Large-scale molecular analysis of Hereditary Hearing Loss
genes in Argentinean deaf patients: lookingfora needle in a
haystack Conclusion: This study supports the rationale for the
development of cultured autologous oral mucosal epithelial
stem cell sheets obtained by selected heterozygous R311K-
p63 stem cells, as an effective and personalized therapy for
this unique case of EEC syndrome, thus bypassing gene
therapy approaches. P. Nespeca: None. V. Barbaro: None. A. Nasti: None. P. Raffa: None. A. Migliorati: None. S. Ferrari: None. E. Palumbo: None. M. Bertolin: None. C. Breda: None. F. Miceli: None. A. Russo: None. L. Caenazzo: None. D. Ponzin: None. G. Palù: None. C. Parolin: None. E. Di
Iorio: None. Our results support the role of FBXW11 in eye and limb
development, likely via modulation of the Wnt pathway,
and therefore this gene represents an important candidate
for human developmental disorders. M. Borregán Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Introduction: Hereditary retinal dystrophies (HRD) are a
group of conditions with a great clinical and genetic het-
erogeneity; more than 300 responsible genes have been
identified. The most prevalent HRD is retinitis pigmentosa
(RP) (1: 4000). 50 J. del Picchia genes were included). The causal variants on both alleles
were found in 35% of patients (32 of 90). The most frequent
causes of HL detected in the Czech population were bial-
lelic probably pathogenic variants in following genes
STRC, MYO15A, CDH23 and OTOG. Detection rate for
each panel testing was different, namely 34%, 40% and
12.5%. In the first two panels most patients with at least one
affected sibling were included. On the contrary only
patients with sporadic HL were included in the last panel. Possible explanation for lower detection rate is that patients
with acquired or autosomal dominant inherited HL were
included in the last panel. Supported- by MH CR AZV 16-
31173A Materials and Methods: Prospective study that includes
98 patients were evaluated in a tertiary hospital from 2010
to 2017. The clinical diagnosis was made through clinical
exploration: visual acuity, biomicroscopy, fundus explora-
tion and electrophysiological tests. According to the
patient's collaboration, computerized campimetry, wide-
field retinography, optical coherence tomography and
microperimetry were performed. For the genetic diagnosis,
a customized panel of 155 genes (IRD150) was designed by
next generation sequencing and applied to children with
clinical diagnosis of HRD. Results: Identification of the causative variant was
achieved in 70% of cases. In our cohort, mutations in
ABCA4 gene were the most frequent, followed by the
BEST1 gene (Best's disease) and RPGR (X-linked RP). 33%
of molecular findings have not been previously described. Variant segregation within families was performed in order
to increase evidence of pathogenicity. Adequate genetic
counselling was offered in all cases. Regarding therapeutic
options, 5 families with a diagnosis of ACL and mutations
in RPE65 that could be candidates for gene therapy have
been identified. In 97% of cases molecular diagnosis was
concordant with clinical diagnostic orientation. Results: Identification of the causative variant was
achieved in 70% of cases. In our cohort, mutations in
ABCA4 gene were the most frequent, followed by the
BEST1 gene (Best's disease) and RPGR (X-linked RP). 33%
of molecular findings have not been previously described. Variant segregation within families was performed in order
to increase evidence of pathogenicity. Adequate genetic
counselling was offered in all cases. 31173A D. Safka Brozkova: None. S. Poisson Markova: None. P. Seeman: None. HARS2 sequence variants identified in young individuals
with severe sensorineural hearing impairment HARS2 sequence variants identified in young individuals
with severe sensorineural hearing impairment H. Karstensen1, N. Rendtorff1, L. Hindhede1, A. Stein2,
R. Hartmann-Petersen2, K. Lindorff-Larsen2, A. Højland3,4,
M. Petersen3,4, L. Tranebjærg1,5 H. Karstensen1, N. Rendtorff1, L. Hindhede1, A. Stein2,
R. Hartmann-Petersen2, K. Lindorff-Larsen2, A. Højland3,4,
M. Petersen3,4, L. Tranebjærg1,5 Conclusion: The identification of the molecular cause in
HRD is essential for diagnosis and genetic counselling to
the patient and the family. In some cases, a visual prognosis
and potential treatment could be provided. 1Kennedy Centre, Clinical Genetics Clinic, Copenhagen
University hospital, Glostrup, Denmark, 2The Linderstrøm-
Lang Centre for Protein Science, Department of Biology,
University of Copenhagen, Copenhagen, Denmark,
3Department of Clinical Genetics, Aalborg University hospital,
Aalborg, Denmark, 4Department of Clinical Medicine, Aalborg
University hospital, Aalborg, Denmark, 5Department of
Clinical Medicine, Panum Institute, University of Copenhagen,
Copenhagen, Denmark M. Borregán: None. Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Regarding therapeutic
options, 5 families with a diagnosis of ACL and mutations
in RPE65 that could be candidates for gene therapy have
been identified. In 97% of cases molecular diagnosis was
concordant with clinical diagnostic orientation. The improved DNA diagnostics and genetic causes of early
onset hereditary hearing loss in the Czech Republic The improved DNA diagnostics and genetic causes of early
onset hereditary hearing loss in the Czech Republic D. Safka Brozkova, S. Poisson Markova, P. Seeman D. Safka Brozkova, S. Poisson Markova, P. Seeman
2nd Medical School and University Hospital Motol, Prague,
Czech Republic;, Prague 5, Czech Republic The genetics of hearing impairment (HI) is complex and
highly heterogeneous, and variation in the same gene may
be associated with different types of both syndromic and
non-syndromic HI. Here we focus on HI, in association with
the HARS2 gene, where biallelic missense variants pre-
viously have been associated with Perrault syndrome in two
studies. The published HARS2 variants were all identified in
patients, ascertained clinically because of the combination
of HI and gonadal dysgenesis, the clinical hallmarks of
Perrault syndrome. We have investigated the presence of
HARS2 sequence variants in 79 cases (42 females; mean age
23
±
21.5 years) with HI. Using Next Generation
Sequencing we have identified three potential pathogenic
missense variants. In a family with three affected siblings
(two girls age 12 and 15, and one boy age 19), we identified
compound heterozygosity for the variants: p.Lys58Glu and
p.Arg150Cys, and in a 6 year-old girl we identified com-
pound heterozygosity for the variants: p.Arg150Cys and p. Arg327Gln. Supporting evidence for the functional defects 2nd Medical School and University Hospital Motol, Prague,
Czech Republic;, Prague 5, Czech Republic Hereditary hearing loss (HL) is the most common sensory
deficit. About 60% of cases have genetic origin. The largest
group of HL patients shows autosomal recessive inheri-
tance. Up to 40% of HL in the Czech Republic is caused by
biallelic mutations in the GJB2 gene. We have enlarged the
diagnostics for the second most frequent type of HL -
DFNB16. We use a simple and fast quantitative compara-
tive fluorescent PCR and MLPA for detection of deletions
or CNVs of the STRC gene. For patients who are not
homozygous for the STRC deletion we use masivelly par-
allel sequencing (MPS) of panel of all genes already asso-
ciated with autosomal recessive and X -linked types of
NSHL. P02.20C Evidence against TMPRSS3/GJB2 digenic inheritance of
hearing loss - practical lesson learned in the era of high
throughput sequencing The improved DNA diagnostics and genetic causes of early
onset hereditary hearing loss in the Czech Republic HaloPlex or SureSelect technology was used for
library preparation.Overall 90 patients were tested by 3
consecutively updated gene panel versions (62, 67 and 70 Abstracts from the 51st European Society of Human Genetics Conference: Posters 51 of the variants were obtained using in silico modeling of
HARS2 variants identified in this and previous studies, by
predicting both the change in protein stability (ΔΔG) and of
potential loss of function using a co-variation-based ana-
lysis of multiple HARS2 sequence alignments. The three
female patients with HARS2 variants, compatible with
autosomal recessive inheritance, are young for determining
signs of ovarian dysgenesis, but will be followed carefully. The present study raises the possibility for non-syndromic
HI being the only phenotypic effect of biallelic HARS2
variation. negative patients, a diagnostic yield comparable to other
reports. In remaining patients, molecular diagnosis could
not be achieved, where five patients were carrying one
likely causative variant in genes associated with autosomal
recessive HL. Identification of genetic cause delivers an
important information for the genetic counseling and to
some extend enables HL clinical prediction and verbal
communication improvement with cochlear implants. 1Battellino Int Adv Otol 2011;7:372-378 2Battelino J Laryngol Otol 2012;126:763-9 K. Trebušak Podkrajšek: None. T. Obreza: None. J. Kovač: None. S. Bertok: None. T. Battelino: None. S. Battelino: None. K. Trebušak Podkrajšek: None. T. Obreza: None. J. Kovač: None. S. Bertok: None. T. Battelino: None. S. Battelino: None. H. Karstensen: None. N. Rendtorff: None. L. Hind-
hede: None. A. Stein: None. R. Hartmann-Petersen:
None. K. Lindorff-Larsen: None. A. Højland: None. M. Petersen: None. L. Tranebjærg: None. M. Oldak1, U. Lechowicz1, A. Pollak1, D. Oziębło1,2,
H. Skarżyński3 K. Trebušak Podkrajšek1,2, T. Obreza1, J. Kovač1, S. Bertok1,
T. Battelino1,2, S. Battelino3,2 K. Trebušak Podkrajšek1,2, T. Obreza1, J. Kovač1, S. Bertok1,
T. Battelino1,2, S. Battelino3,2 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Postgraduate School
of Molecular Medicine, Medical University of Warsaw,
Warsaw, Poland, 3Oto-Rhino-Laryngology Surgery Clinic,
Institute of Physiology and Pathology of Hearing, Warsaw,
Poland 1University Medical Centre Ljubljana, University Children´s
Hospital, Ljubljana, Slovenia, 2University of Ljubljana,
Faculty of Medicine, Ljubljana, Slovenia, 3University Medical
Centre Ljubljana, Department of Otorhinolaryngology and
Cervicofacial Surgery, Ljubljana, Slovenia Introduction: Hereditary hearing loss (HL) is characterized
by a vast phenotypic and genetic heterogeneity. Up to date
it was associated to 119 genes; 73 with non-syndromic and
46 with syndromic HL (Hereditary Hearing Loss Home-
page, http://hereditaryhearingloss.org, February 2018). In
Slovenians, 26.6% of congenitally deaf patients1 and 11%
of progressive HL patients2 had biallelic GJB2 mutations,
where other genetic causes were limitedly exploited so far. Background: Hearing loss (HL) is the most common dis-
ability of human senses and genetic factors significantly
contribute to its development. For some HL genes digenic
inheritance has been documented. Recently it has been
proposed for TMPRSS3 and GJB2 recessive pathogenic
variants. As the data were not convincing, we aimed to
verify the hypothesis. Material and methods: From our genetic database of HL
patients with at least one TMPRSS3 pathogenic variants
(n = 42) we have selected individuals with additional
pathogenic variants in the GJB2 gene (n = 3) and recruited
for the study all of the available family members. Segregation analysis of the respective TMPRSS3 and
GJB2 pathogenic variants within the families was per-
formed on genomic DNA by Sanger sequencing. Material and Methods: 58 HL patients (12 congenital,
46 progressive HL) with negative GJB2 and GJB6 testing
were subjected to targeted NGS with TruSightOne Sequen-
cing Panel on the MiSeq platform followed by interpretation
of variants in selected 111 HL associated genes and
subsequent Sanger sequencing confirmation. Novel variants
were evaluated with in silico prediction tools (Mutation
Taster, CADD). Results: The strategy has allowed to identify four
individuals who were double heterozygous for pathogenic
TMPRSS3 and GJB2 variants. Two individuals from two
different families had GJB2 c.35delG and TMPRSS3
c.208delC and in two other individuals from one family
GJB2
c.35delG
together
with
TMPRSS3
c.1343T>C
variants were found. None of these subjects has ever
reported hearing problems and their hearing status was
normal. Genetic testing of hearing loss related genes in Slovene
patients with next generation sequencing Genetic testing of hearing loss related genes in Slovene
patients with next generation sequencing M. Oldak1, U. Lechowicz1, A. Pollak1, D. Oziębło1,2,
H. Skarżyński3 M. Oldak1, U. Lechowicz1, A. Pollak1, D. Oziębło1,2,
H. Skarżyński3 Molecular Diagnostics of Hereditary Hearing Loss using
Next-generation sequencing (NGS) in Estonian patients Molecular Diagnostics of Hereditary Hearing Loss using
Next-generation sequencing (NGS) in Estonian patients R. Teek1, H. Roomere1, S. Pajusalu1,2, K. Õunap1,2 R. Teek1, H. Roomere1, S. Pajusalu1,2, K. Õunap1,2 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia Introduction: HHL is characterized by a high genetic
heterogeneity hampering accurate molecular diagnosis that
is essential for a proper genetic counselling and therapeutic
options. Introduction: Hearing loss (HL) is the most common
sensory disorder worldwide. Despite this, DNA diagnostics
of HL is complicated due to the heterogeneity of the dis-
ease. The most common cause for autosomal-recessive non-
syndromic HL in the Caucasian population is mutations in
the GJB2 gene. We know from our previous study that the
most frequent mutations found among Estonian children
with early onset HL were c.35delG and p.M34T (36%) in
GJB2 gene. Still, other genes causing HL are rarely inves-
tigated in Estonian patients with HL. Study group consist of
60 probands with HL as main complaint or one of the the
clinical features and who referred to geneticist during 2015-
2017. In patients with isolated HL mutations in the GJB2
gene were excluded by GJB2 gene sequencing. Among 60
cases in 46 patients HL was the main complaint and in rest
of 14 cases HL was one of the symptoms in addition to
other clinical symptoms. Methods: 166 Italian HHL cases (46 familial and
120 sporadic) were screened with a TRS panel of 96
deafness genes, using Ion Torrent PGM™. Annotated
variants were filtered according to the pattern of inheritance,
frequency, and pathogenicity. In negative cases, HD-SNPs
arrays were used to identify CNVs using Genome Studio
software for allele detection and genotype calling followed
by PennCNV analysis. Methods: 166 Italian HHL cases (46 familial and
120 sporadic) were screened with a TRS panel of 96
deafness genes, using Ion Torrent PGM™. Annotated
variants were filtered according to the pattern of inheritance,
frequency, and pathogenicity. In negative cases, HD-SNPs
arrays were used to identify CNVs using Genome Studio
software for allele detection and genotype calling followed
by PennCNV analysis. Results: Approximately 40% of cases (66/166) were
positive for GJB2 mutations, In the remaining 100 cases,
TRS and SNPs array led to the characterization of
approximately 30% of cases (30/100). P02.22A
Targeted Re-Sequencing (TRS) and high density SNP array
for the molecular characterisation of Hereditary Hearing
Loss (HHL) S. Lenarduzzi1, A. Morgan2, S. Cappellani1, V. Pecile1,
M. Morgutti1, E. Orzan1, U. Ambrosetti3, M. La Bianca1,
F. Faletra1, E. Grosso4, F. Sirchia1, A. Sensi5, C. Graziano6,
M. Seri6, P. Gasparini1,2, G. Girotto1,2 Supported by: 2011/03/D/NZ5/05592 Supported by: 2011/03/D/NZ5/05592 M. Oldak: None. U. Lechowicz: None. A. Pollak: None. D. Oziębło: None. H. Skarżyński: None. P02.21D 1IRCSS Burlo Garofolo, Trieste, Italy, 2University of Trieste,
Trieste, Italy, 3Univ. Milano U.O.C. Audiologia/Fondazione
IRCCS Cà Granda Ospedale Maggiore Policlinico, Milano,
Italy, 4Città della Salute e della Scienza University Hospital,
Medical Genetics Unit, 10126, Torino, Italy, 5Department of
Clinical Pathology, Medical Genetics Unit, Pievesestina,
47522, Cesena, Italy, 6Unit of Medical Genetics, S. Orsola-
Malpighi Hospital, Bologna, Italy M. Oldak1, U. Lechowicz1, A. Pollak1, D. Oziębło1,2,
H. Skarżyński3 Results: The strategy has allowed to identify four
individuals who were double heterozygous for pathogenic
TMPRSS3 and GJB2 variants. Two individuals from two
different families had GJB2 c.35delG and TMPRSS3
c.208delC and in two other individuals from one family
GJB2
c.35delG
together
with
TMPRSS3
c.1343T>C
variants were found. None of these subjects has ever
reported hearing problems and their hearing status was
normal. Results: Causative variants were detected in 33 patients
in 16 genes (COL2A1, DIAPH3, ILDR, MYH14, MYO15A,
MYO6, MYO7A, PDZD7, POU4F3, SLC17A8, TBC1D24,
TCOF1, TECTA, USH2A, WFS1, TMPRSS3), most fre-
quently in USH2A (19%), TMPRSS3 (16%) and SLC17A8
(9,7%). Six variants were not reported so far. Conclusions:
Causative
variants
explaining
clinical
presentation were detected in 28(48,3%) of GJB2/GJB6 52 J. del Picchia R. Teek: None. H. Roomere: None. S. Pajusalu: None. K. Õunap: None. R. Teek: None. H. Roomere: None. S. Pajusalu: None. K. Õunap: None. Conclusions: Access to a large genetic database of HL
patients allowed us to identify as many as four individuals
with concomitant pathogenic variants in TMPRSS3 and
GJB2 genes and without HL. Our data provide evidence
against TMPRSS3/GJB2 digenic inheritance of HL. As high
throughput sequencing is increasingly used for genetic
testing, particular caution should be taken to not over-
interpret the findings. Z. Ravesh1,2, B. Wissinger2, M. Ansar1 Z. Ravesh1,2, B. Wissinger2, M. Ansar1 1Institute of Human Genetics, Polish Academy of Sciences,
Poznan, Poland, 2Poznan University of Medical Sciences,
Poznan, Poland, 3Institute of Computer Science, Warsaw
University of Technology, Warsaw, Poland, 4Baylor College of
Medicine, Department of Molecular & Human Genetics,
Houston, TX, United States, 5Medical University of Warsaw,
Warsaw, Poland, 6Linköping University, Linkoping, Sweden,
7Medical University of Bialystok, Bialystok, Poland, 8Medical
Centre Vigor Med, Leszno, Poland, 9Baylor College of
Medicine, Houston, TX, United States, 10Baylor College of
Medicine, Department of Molecular & Human Genetics and
Department of Pediatrics, Houston, TX, United States,
11Human Genome Sequencing Center, Baylor College of
Medicine, Houston, TX, United States, 12Texas Children's
Hospital, Houston, TX, United States 1Department of Biochemistry, Lab of Genomics, Quaid-i-Azam
University, Islamabad, Pakistan, 2Institute for Ophthalmic
Research, Tübingen, Germany Introduction: Hereditary retinal dystrophies (RD) are a
group of heterogeneous disorders caused by mutations in
over 200 genes. RDs can be subdivided into different
groups based on the primary degeneration of rod or cone
photoreceptor cells. This study was conducted to investigate
the underlying RD genes and mutation in consanguineous
families from Pakistan. Material and Methods: Families were recruited after
informed consent. Peripheral blood was collected and
genomic
DNA
was
extracted
according
to
standard
procedures. Homozygosity mapping was performed using
Affymetrix Gene Chip Human Mapping 250 K-NspI arrays. The data were analyzed using Homozygosity Mapper
software. Primers for amplifying all exons and intron
boundaries
were
designed
with
Primer3plus
software
followed by PCR and Sanger sequencing. Minigene
splicing assay and DNA walking were performed on
respective samples. Objectives: High myopia is an eye disorder with a refrac-
tive error greater than -6 diopters (D) with both environ-
mental and genetic factors involved. Although a number of
high myopia loci have been identified, the pathogenic genes
in the general population have not been determined. The
aim of the study was to identify sequence variants causative
for high myopia in families from Central Europe. Objectives: High myopia is an eye disorder with a refrac-
tive error greater than -6 diopters (D) with both environ-
mental and genetic factors involved. Although a number of
high myopia loci have been identified, the pathogenic genes
in the general population have not been determined. The
aim of the study was to identify sequence variants causative
for high myopia in families from Central Europe. P02.23B J. Swierkowska1, J. A. Karolak1,2, T. Gambin3,4, M. Rydzanicz5,
A. Frajdenberg6, M. Mrugacz7, M. Podfigurna-Musielak8,
P. Stankiewicz9, J. R. Lupski10,11,12, M. Gajecka1,2 Molecular Diagnostics of Hereditary Hearing Loss using
Next-generation sequencing (NGS) in Estonian patients In particular, we
identified: 1) the first case of UPD involving LOXHD1, with
the presence of both a small isodisomy segment spanning
LOXHD1 and a heterodisomy on the remaining parts of
Chr18, suggesting that the UPD is the result of a non-
disjunction in meiosis I, followed by trisomy rescue, 2) two
large deletions in OTOA and STRC, 3) four patients (13%)
with mutations in TECTA which is thus the second major
player in the Italian population (in both autosomal recessive
and
dominant
families),
followed
by
TMPRSS3
(3/
100,10%), MYO6, MYO7A, MYO15A, PDZD7, and ACTG1
(2/100,7% each). Methods: the NGS subpanel of genes associated with HL
was performed (107 genes, Illumina TruSightOne panel) in
46 cases. For the other 14 cases with non-isolated HL only
selected gene analysis or different subpanels were done. Results: we found that the mutated genes were most
commonly associated with Usher, Waardenburg and Alport
syndrome. There were some mutations in genes related to
non-syndromic hearing loss: MYO15A, TECTA, MAR-
VELD2, MYO6 and STRC gene. Conclusion: The using of NGS gene subpanel analysis
for HL is valuable and increased the detection of genetic
etiology of HL for 33% (20 patients). Conclusions: Thanks to this approach approximately
58% (96/166) of cases were characterized confirming the
large mutation’s spectrum of HHL genes, as well as the Conclusions: Thanks to this approach approximately
58% (96/166) of cases were characterized confirming the
large mutation’s spectrum of HHL genes, as well as the Abstracts from the 51st European Society of Human Genetics Conference: Posters 53 efficacy of TRS and HD-SNPs arrays in helping an accurate
molecular diagnosis. S. Lenarduzzi: None. A. Morgan: None. S. Cappellani:
None. V. Pecile: None. M. Morgutti: None. E. Orzan:
None. U. Ambrosetti: None. M. La Bianca: None. F. Faletra: None. E. Grosso: None. F. Sirchia: None. A. Sensi: None. C. Graziano: None. M. Seri: None. P. Gasparini: None. G. Girotto: None. genetic testing in Pakistani families to minimize the risk of
recessive disorders. genetic testing in Pakistani families to minimize the risk of
recessive disorders. efficacy of TRS and HD-SNPs arrays in helping an accurate
molecular diagnosis. S. Lenarduzzi: None. A. Morgan: None. S. Cappellani:
None. V. Pecile: None. M. Morgutti: None. E. Orzan:
None. U. Ambrosetti: None. M. La Bianca: None. F. Faletra: None. E. Grosso: None. F. Sirchia: None. A. Sensi: None. C. Graziano: None. M. Seri: None. P. Gasparini: None. G. Girotto: None. Molecular Diagnostics of Hereditary Hearing Loss using
Next-generation sequencing (NGS) in Estonian patients Z. Ravesh: None. B. Wissinger: None. M. Ansar: None. P02.24C
Variants in the FLRT3 and SLC35E2B identified using
whole-exome sequencing in seven high myopia families
from Central Europe P02.24C
Variants in the FLRT3 and SLC35E2B identified using
whole-exome sequencing in seven high myopia families
from Central Europe Diagnostics of inherited retinal disorders: Slovenian
experience using clinical exome sequencing S. Van de Sompele1, K. Van Schil1, C. Van Cauwenbergh1,2,
T. Rosseel1, S. De Jaegere1, T. Van Laethem1, I. Balikova2, B. P. Leroy1,2,3, F. Coppieters1, E. De Baere1 Diagnostics of inherited retinal disorders: Slovenian
experience using clinical exome sequencing M. Volk1, A. Maver1, A. Fakin2, H. Jaklic1, M. Hawlina2,
B. Peterlin1 1Center for Medical Genetics, Ghent University and Ghent
University Hospital, Ghent, Belgium, 2Department of
Ophthalmology, Ghent University and Ghent University
Hospital, Ghent, Belgium, 3Division of Ophthalmology, The
Children’s Hospital of Philadelphia, Philadelphia, PA, United
States 1Clinical Institute of Medical Genetics, UMC Ljubljana,
Ljubljana, Slovenia, 2Eye Hospital, UMC Ljubljana, Ljubljana,
Slovenia Introduction: Inherited retinal disorders are diagnostically
challenging group of clinically and genetically hetero-
geneous diseases that may affect the entire retina or may be
restricted to the macula. In addition, they may also be an
ophthalmic manifestation of a systemic condition. There are
approximately 261 genes associated with inherited retinal
degeneration (https://sph.uth.edu/retnet/). We present our
experiences using clinical exome sequencing for diagnostics
of inherited retinal disorders. Inherited retinal diseases (IRD) are a major cause of early-
onset blindness, caused by mutations in >250 disease genes. We investigated the currently known IRD genes (RetNet
panel, https://sph.uth.edu/retnet) in 253 IRD probands using
whole-exome sequencing (WES). We identified (likely) causative variants in 55% of IRD
probands, in frequently as well as rarely mutated RetNet
genes. Specifically, two unrelated probands with simplex
retinitis pigmentosa (RP) were homozygous for a novel
frameshift variant in RAX2, in which so far only mono-
allelic mutations have been described in dominant cone-rod
dystrophy and macular dystrophy, suggesting a novel
disease association to a known IRD gene. Secondly,
ARL3 was recently proposed as a novel disease gene for
autosomal dominant RP (ADRP). Interestingly, we identi-
fied the same ARL3 variant in an ADRP family, as well as a
novel missense variant in a simplex RP patient, corroborat-
ing its involvement in ADRP. Lastly, in a proband
diagnosed with non-syndromic IRD, bi-allelic truncating
variants in CEP164 were identified. Previous observations
suggest
a
strong
genotype-phenotype
correlation
of
CEP164-disease, with truncating mutations causing early-
onset phenotypes of dysplasia and malformation in different
organs, and hypomorphic alleles causing late-onset degen-
erative
phenotypes
including
IRD. Our
observations Materials and Methods: 40 Slovenian patients with
referral
of
suspected
inherited
retinal
disorder
were
submitted to our institution. We performed targeted analysis
of retinopathy-associated genes in the exome sequencing
data. P02.25D Exome-based RetNet panel analysis in a Belgian cohort
with inherited retinal disease (IRD) expands the molecular
and phenotypic spectrum of recently identified IRD genes Z. Ravesh1,2, B. Wissinger2, M. Ansar1 Overall, RetNet-WES analysis revealed an underlying
genetic defect in 55% of a Belgian IRD cohort, expanding
their disease associations (RAX2), supporting their role in
IRD (ARL3), and providing new insights into phenotypic
consequences
of
bi-allelic
loss-of-function
alleles
(CEP164). J. Swierkowska:
None. J.A. Karolak:
None. T. Gambin: None. M. Rydzanicz: None. A. Frajdenberg:
None. M. Mrugacz: None. M. Podfigurna-Musielak:
None. P. Stankiewicz: None. J.R. Lupski: None. M. Gajecka: None. Funding:
BOF15/GOA/011;
01D04716;
AUGE/13/
023; FWO. S. Van de Sompele: None. K. Van Schil: None. C. Van
Cauwenbergh: None. T. Rosseel: None. S. De Jaegere:
None. T. Van Laethem: None. I. Balikova: None. B.P. Leroy: None. F. Coppieters: None. E. De Baere: None. Z. Ravesh1,2, B. Wissinger2, M. Ansar1 Methods: Seventeen individuals from seven unrelated
Central European families with hereditary high myopia
were assessed using whole-exome sequencing analyses. Selected variants were further evaluated using Sanger
sequencing and the segregation analyses in other family
members. Results: Homozygosity mapping identified a novel locus
in family A, one novel gene (C8ORF37) in family B and a
large novel genomic deletion of the (LCA5) gene in
family C. Conclusion: Our study indicates the heterogeneous
nature of retinal dystrophies in Pakistan. Although earlier
studies explored number of RD families, but underlying
genes are still unknown for significant proportion of
Pakistani families. Theoretically the traditional screening
methods were successful, however genome wide scan were
further implemented to improve the detection of underlying
variations in Retinal Dystrophies. Therefore the amalgama-
tion of traditional and modern molecular techniques is
required for accurate identification of mutations. It is
anticipated that these findings will contribute to future Results: Out of a total of 72 variants identified, two novel
missense variants c.1642G>C in FLRT3 and c.938C>T in
SLC35E2B segregate with high myopia in the HM-78
family. Results: Out of a total of 72 variants identified, two novel
missense variants c.1642G>C in FLRT3 and c.938C>T in
SLC35E2B segregate with high myopia in the HM-78
family. Conclusions: FLRT3 and/or SLC35E2B could represent
disease candidate genes and may be potentially responsible
for high myopia in the HM-78 family. Our data suggest that
there are different genetic backgrounds of high myopia in
each multiplex family, indicating the complex genetic
causes of high myopia. This study was supported by
National Science Centre in Poland, (Doctoral Scholarship 54 J. del Picchia therefore challenge the hypothesis that null mutations
underlie the most severe end of the phenotypic spectrum. Overall, RetNet-WES analysis revealed an underlying
genetic defect in 55% of a Belgian IRD cohort, expanding
their disease associations (RAX2), supporting their role in
IRD (ARL3), and providing new insights into phenotypic
consequences
of
bi-allelic
loss-of-function
alleles
(CEP164). Etiuda 2013/08/T/NZ5/00754), and Ministry of Science and therefore challenge the hypothesis that null mutations
underlie the most severe end of the phenotypic spectrum. Etiuda 2013/08/T/NZ5/00754), and Ministry of Science and
Higher Education of Poland (27/GM/2017) and United
States National Institutes of Health, National Human
Genome Research Institute/National Heart Lung and Blood
Institute (UM1HG0067542). therefore challenge the hypothesis that null mutations
underlie the most severe end of the phenotypic spectrum. P02.28C Clinical exome sequencing as a genomic approach for the
diagnosis of unsolved cases of inherited retinal dystrophies M. Martín-Sánchez1, M. González-del Pozo1,2, N. Bravo-Gil1,2,
C. Méndez-Vidal1, E. Rodríguez-de la Rúa3,4, S. Borrego1,2,
G. Antiñolo1,2 M. Martín-Sánchez1, M. González-del Pozo1,2, N. Bravo-Gil1,2,
C. Méndez-Vidal1, E. Rodríguez-de la Rúa3,4, S. Borrego1,2,
G. Antiñolo1,2 C. Méndez-Vidal1, E. Rodríguez-de la Rúa3,4, S. Borrego1,2,
G. Antiñolo1,2 C. Méndez-Vidal1, E. Rodríguez-de la Rúa3,4, S. Borrego1,2,
G. Antiñolo1,2 N. Bravo-Gil1,2, M. Martín-Sánchez1, M. González-del Pozo1,2,
C. Méndez-Vidal1,2, S. Borrego1,2, G. Antiñolo1,2 C. Méndez-Vidal1,2, S. Borrego1,2, G. Antiñolo1,2 1Department of Maternofetal Medicine, Genetics and
Reproduction, Institute of Biomedicine of Seville, University
Hospital Virgen del Rocio/CSIC/University of Seville, Seville,
Spain, 2Centro de Investigación Biomédica en Red de
Enfermedades Raras (CIBERER), Seville, Spain, 3Department
of Ophthalmology, University Hospital Virgen Macarena,
Seville, Spain, 4Retics Patologia Ocular. OFTARED. Instituto
de Salud Carlos III (ISCIII), Madrid, Spain 1Department of Maternofetal Medicine, Genetics and
Reproduction, Institute of Biomedicine of Seville, University
Hospital Virgen del Rocio/CSIC/University of Seville, Seville,
Spain, 2Centro de Investigación Biomédica en Red de
Enfermedades Raras (CIBERER), Seville, Spain, 3Department
of Ophthalmology, University Hospital Virgen Macarena,
Seville, Spain, 4Retics Patologia Ocular. OFTARED. Instituto
de Salud Carlos III (ISCIII), Madrid, Spain 1Department of Maternofetal Medicine, Genetics and
Reproduction, Institute of Biomedicine of Seville, University
Hospital Virgen del Rocio/CSIC/University of Seville, Seville,
Spain, 2Centro de Investigación Biomédica en Red de
Enfermedades Raras (CIBERER), Seville, Spain Introduction: Genomic approaches based on the analysis
of previously disease-associated genes have shown a high
diagnostic efficiency in Inherited Retinal Dystrophies
(IRD). However, around 40-50% of cases remain unsolved
after their application. Introduction: Inherited Retinal Dystrophies (IRDs) are a
group of rare disorders characterized by photoreceptors
degeneration. Phenotypic and genetic heterogeneity hamper
the diagnosis of IRDs. Although NGS technologies have
shown to be helpful in this endeavor, some cases remain
unsolved even after analyzing the whole exome. Remark-
ably, a number of patients carry only one mutated allele in a
recessive
gene,
suggesting
that
screening
non-coding
regions could improve the diagnosis. Materials and Methods: Targeted enrichment of around
5000 clinically relevant genes (SureSelect Focused Exome,
Agilent) and Illumina HiSeq 3000 sequencing, were used
for the molecular diagnosis of 12 unsolved IRD cases
previously analyzed by a retinal gene panel. Results:
Application of our data
analysis
pipeline
allowed the identification of causal mutations in 4 out of
the 12 cases. Whole-gene panel sequencing in patients with inherited
retinal dystrophies and mono-allelic variants: contribution
of deep-intronic mutations and CNVs Whole-gene panel sequencing in patients with inherited
retinal dystrophies and mono-allelic variants: contribution
of deep-intronic mutations and CNVs Diagnostics of inherited retinal disorders: Slovenian
experience using clinical exome sequencing Mitochondrial sequence was also analysed based on
the off-target exome reads. Filtered variants were analysed
according to population frequency, characterization in
ClinVar database, putative impact of the variant and
predicted pathogenicity. Results: Causative pathogenic variants were detected in
25 patients (63%). Of these, we confirmed the referral
diagnosis in 23 cases, and reclassified initial diagnosis in
two cases, one to mitochondriopathy and one to 16q11.2
microdeletion syndrome. In addition, we found variants of
unknown clinical significance (VUS) in 6 cases and 9 cases
with negative results. Altogether, we identified 19 known
pathogenic and 12 novel pathogenic or likely pathogenic
variants, 11 VUS and a microdeletion. Results: Causative pathogenic variants were detected in
25 patients (63%). Of these, we confirmed the referral
diagnosis in 23 cases, and reclassified initial diagnosis in
two cases, one to mitochondriopathy and one to 16q11.2
microdeletion syndrome. In addition, we found variants of
unknown clinical significance (VUS) in 6 cases and 9 cases
with negative results. Altogether, we identified 19 known
pathogenic and 12 novel pathogenic or likely pathogenic
variants, 11 VUS and a microdeletion. 55 Abstracts from the 51st European Society of Human Genetics Conference: Posters Conclusion: Our results demonstrate high diagnostic
yield using clinical exome sequencing in diagnostics of
inherited retinal disorders. Clinical exome sequencing
should be a first-tier investigation not only because of
genetic heterogeneity but also because of the potential to
identify novel findings. mutations. Our approach to discover the second mutant
allele in patients with mono-allelic mutations, together with
our reliable CNVs detection algorithm, marks a step
forward to increase diagnosis rate of IRDs. mutations. Our approach to discover the second mutant
allele in patients with mono-allelic mutations, together with
our reliable CNVs detection algorithm, marks a step
forward to increase diagnosis rate of IRDs. g
Funding: PI15-01648 (ISCIII and European Union
ERDF/ESF, “Investing in your future”), ER17P1AC702/
2018 (CIBERER), CTS-1664 (Government of Andalusia). M. Martín-Sánchez: None. M. González-del Pozo:
None. N. Bravo-Gil: None. C. Méndez-Vidal: None. E. Rodríguez-de la Rúa: None. S. Borrego: None. G. Antiñolo: None. Funding: PI15-01648 (ISCIII and European Union
ERDF/ESF, “Investing in your future”), ER17P1AC702/
2018 (CIBERER), CTS-1664 (Government of Andalusia). M. Volk: None. A. Maver: None. A. Fakin: None. H. Jaklic: None. M. Hawlina: None. B. Peterlin: None. M. Volk: None. A. Maver: None. A. Fakin: None. H. Jaklic: None. M. Hawlina: None. B. Peterlin: None. M. Martín-Sánchez: None. M. González-del Pozo:
None. N. Diagnostics of inherited retinal disorders: Slovenian
experience using clinical exome sequencing Bravo-Gil: None. C. Méndez-Vidal: None. E. Rodríguez-de la Rúa: None. S. Borrego: None. G. Antiñolo: None. Multiple differentially methylated regions specific to
keratoconus overlap known keratoconus linkage loci Multiple differentially methylated regions specific to
keratoconus overlap known keratoconus linkage loci U. Lechowicz1, T. Gambin2,3, A. Pollak1, A. Podgorska1,
P. Stawinski1, A. Franke4, B. Petersen4, M. Firczuk5, M. Oldak1,
H. Skarzynski6, R. Ploski7 M. Gajecka1,2, J. A. Karolak1,2, M. Rydzanicz3, P. Gasperowicz3,
R. Ploski3, J. P. Szaflik4, M. Kabza1 M. Gajecka1,2, J. A. Karolak1,2, M. Rydzanicz3, P. Gasperowicz3,
R. Ploski3, J. P. Szaflik4, M. Kabza1 H. Skarzynski6, R. Ploski7 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Institute of Computer
Science, Warsaw University of Technology,, Warsaw, Poland,
3Department of Medical Genetics, Institute of Mother and
Child at Warsaw, Warsaw, Poland, 4Institute of Clinical
Molecular Biology, Kiel University, Kiel, Germany,
5Department of Immunology, Center of Biostructure Research,
Medical University of Warsaw, Warsaw, Poland, 6Oto-Rhino-
Laryngology Surgery Clinic, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 7Department of
Medical Genetics, Centre of Biostructure, Medical University
of Warsaw, Warsaw, Poland 1Poznan University of Medical Sciences, Poznan, Poland,
2Institute of Human Genetics, Polish Academy of Sciences,
Poznan, Poland, 3Medical University of Warsaw, Warsaw,
Poland, 4Medical University of Warsaw, Warsawa, Poland Keratoconus (KTCN) is a complex degenerative eye dis-
order in which development both genetic and environmental
or behavioral components are involved. In order to verify if
DNA methylation may also play a role in KTCN etiology,
reduced representation bisulfite sequencing (RRBS) of
human corneas obtained from five KTCN and five non-
KTCN individuals was performed. Multiple differentially
methylated regions (DMRs) specific to KTCN were detec-
ted and many of them overlap previously identified KTCN
linkage loci (3p14.3, 15q24.1, 20p13, 5q35.2, 13q32.3) and
chromosome arms (2q, 4q, 5p, 9p, 14q, and 17q). For many
of these regions candidate genes and variants were never
identified and our results may allow for significant nar-
rowing of the genomic regions of interest and reduce the list
of putative KTCN genes. We also reanalyzed the previously
described RNA-Seq dataset of 25 KTCN and 25 non-KTCN
human corneas and found that 12 genes downregulated in
KTCN (IQGAP2, SYNJ2, CYP1B1, MYO1G, WNT5A,
PARVB, MGLL, CDC25B, PSG3, FHL2, CAMK1D, and
THEMIS)
and
six
upregulated
genes
(WNT3,
RB1,
AC098617.1, RPS6KA2, PELI2, and PLXNA4) overlapped
or were located in the near vicinity of the identified DMRs. Particularly interesting were the DNA methylation changes
in two genes encoding Wnt ligands (Wnt3 and Wnt5A), as
they provide a potential explanation for the Wnt signaling
pathway deregulation observed in KTCN. P02.28C Research was supported by the grants 2011/03/D/NZ5/
05592 and 2014/13/B/NZ2/01248. N. Bravo-Gil: None. M. Martín-Sánchez: None. M. González-del Pozo: None. C. Méndez-Vidal: None. S. Borrego: None. G. Antiñolo: None. U. Lechowicz: None. T. Gambin: None. A. Pollak:
None. A. Podgorska: None. P. Stawinski: None. A. Franke: None. B. Petersen: None. M. Firczuk: None. M. Oldak: None. H. Skarzynski: None. R. Ploski: None. P02.28C Three of these cases carried variants in IRD-
associated
genes
not
initially
included
in
the
panel
(FAM161A, MFRP and RP1L1) and 1 harbored a mutation
in the orf15 of RPGR that, although it was included in the
panel, represents a highly repetitive region technically
difficult to capture. Furthermore, in 2 other families, we
found candidate mutations in genes not previously asso-
ciated with IRD for which segregation and/or additional
studies are currently pending. Materials and Methods: We performed a targeted
sequencing study of 29 patients, 25 harboring mono-
allelic mutations. The design comprised the entire genomic
sequence of three genes (USH2A, ABCA4 and CEP290), the
coding exons and flanking intronic bases of 76 IRDs related
genes and two disease-associated intronic regions. Materials and Methods: We performed a targeted
sequencing study of 29 patients, 25 harboring mono-
allelic mutations. The design comprised the entire genomic
sequence of three genes (USH2A, ABCA4 and CEP290), the
coding exons and flanking intronic bases of 76 IRDs related
genes and two disease-associated intronic regions. Results: Thirty-two mutations (8 novel) were identified in
18 probands (diagnostic rate: 62.07%). Among the variants,
two were CNVs in USH2A, comprising one deletion (exons
22-55) and one duplication (exons 46-47) in two different
patients. No deep-intronic mutations were detected. Conclusions: These results allowed us to improve the
efficiency of our retinal panel and suggest that most of
unsolved cases could carry understudied mutations in IRD
genes (deep intronic or large rearrangements) or variants in
genes not yet associated with any phenotype. In this regard,
it will be increasingly difficult to identify new genotype-
phenotype correlations of already known genes. Therefore, Conclusions: Sequencing entire genes represents an
intermediate strategy between exon-targeted and whole-
genome sequencing, while reducing costs, time and effort. In our cohort, deep-intronic mutations may not play a
significant role in the etiopathogenesis of IRDs in contrast
to CNVs, which account for about 10% of the total 56 J. del Picchia in our experience, genome sequencing would be the most
appropriate approach for these cases. in our experience, genome sequencing would be the most
appropriate approach for these cases. demonstrated only for two variants and it was relatively
weak (P < 0.05). Summarizing, ISVS strategy and ISVS
Simulator are useful for detection of genetic variants caus-
ing AR diseases. Funding: PI15-01648 (ISCIII and European Union
ERDF/ESF, “Investing in your future”), CIBERER, CTS-
1664 (Andalusian Government). Multiple differentially methylated regions specific to
keratoconus overlap known keratoconus linkage loci Supported by
National Science Centre in Poland, 2012/05/E/NZ5/02127. M. Gajecka: None. J.A. Karolak: None. M. Rydzanicz:
None
P
Gasperowicz: None
R
Ploski: None
J P Autosomal recessive diseases (ARD) are typically caused
by a limited number of mutations whose identification is
challenged by their low prevalence. Our purpose was to
develop a novel approach allowing an efficient search for
mutations causing ARD and evaluation of their pathogeni-
city without a control group. We developed Iterative
Sequencing and Variant Screening (ISVS) approach based
on iterative cycles of gene sequencing and mutation
screening, and ISVS Simulator software (http://zsibio.ii.pw. edu.pl/shiny/isvs/) for assessment of detected variants’
significance. As shown by simulations, ISVS efficiently
identifies and correctly classifies pathogenic mutations
except for cases where the gene of interest has extremely
high number of low frequency nonpathogenic variants. By
applying ISVS, we found 4 known and 9 novel (p.C73Y, p. S124L, p.C194Mfs*17, c.782 + 2 T > A, c.953-5 A > G, p. L325Q, p.D334Mfs*24, p.R436G, p.M448T) TMPRSS3
variants among deaf patients. For 3 known and 5 novel
variants the disease association was supported by ISVS
Simulator odds >90:1. Pathogenicity of 6 novel mutations
has been supported by in-silico predictions of variants’
deleteriousness. By directly comparing variant prevalence
in
patients
and
controls,
disease
association
was M. Gajecka: None. J.A. Karolak: None. M. Rydzanicz:
None. P. Gasperowicz: None. R. Ploski: None. J.P. Szaflik: None. M. Kabza: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 57 P02.31B
Mutations in TUBB4B cause a distinctive sensorineural
disease Pôle d'Imagerie et Explorations
Fonctionnelles, CHRU de Lille, Hôpital Roger Salengro, Lille,
France, 5Pediatric ENT Department, Hôpital Necker-Enfants
Malades, APHP and Paris Descartes University, Paris,
France, 6Centre Hospitalier National d'Ophtalmologie des
Quinze-Vingts, Paris, France, 7Ophthalmology Department,
Hôpital Lariboisière, APHP and Paris Diderot University,
Paris, France, 8Visual Exploration Department, Hôpital
Lariboisière, APHP, Paris, Diderot University, Paris, France,
9Cell Division and Reproduction. Institut Jacques Monod,
CNRS, University Paris Diderot, Paris, France, 10Cell Imaging
Core Facility of the Structure Fédérative de Recherche Necker
INSERM US24/CNRS UMS3633 Imagine and Paris Descartes
University, Paris, France, 11Bioinformatics Platform, Imagine
and Paris Descartes University, Paris, France, 12Genomics
Platform, Imagine and Paris Descartes University, Paris,
France, 13Unité d'Embryo-foetopathologie, Hôpital Necker-
Enfants Malades, APHP and Paris Descartes University,
Paris, France, 14Department of Pediatric Radiology, Hôpital
Necker-Enfants Malades, APHP, Paris, Descartes University,
Paris, France P02.31B
Mutations in TUBB4B cause a distinctive sensorineural
disease Institut Jacques Monod,
CNRS, University Paris Diderot, Paris, France, 10Cell Imaging
Core Facility of the Structure Fédérative de Recherche Necker
INSERM US24/CNRS UMS3633 Imagine and Paris Descartes
University, Paris, France, 11Bioinformatics Platform, Imagine
and Paris Descartes University, Paris, France, 12Genomics
Platform, Imagine and Paris Descartes University, Paris,
France, 13Unité d'Embryo-foetopathologie, Hôpital Necker-
Enfants Malades, APHP and Paris Descartes University,
Paris, France, 14Department of Pediatric Radiology, Hôpital
Necker-Enfants Malades, APHP, Paris, Descartes University,
Paris, France Acknowledgements: We are grateful to the families for
their participation in the study. This work was supported by
“S’entendre”, by grants from the Retina France to IP,
UNADEV-
AVIESAN
ITMO
MNP
to
JMR,
FRM
(DEQ20160334869) to JD, and by a grant (GM097376)
from the NIH to NJC. Acknowledgements: We are grateful to the families for
their participation in the study. This work was supported by
“S’entendre”, by grants from the Retina France to IP,
UNADEV-
AVIESAN
ITMO
MNP
to
JMR,
FRM
(DEQ20160334869) to JD, and by a grant (GM097376)
from the NIH to NJC. 3Department of Biochemistry & Molecular Pharmacology,
NYU Langone Medical Center., New York NY 10016, NY, United States, 4Service d'Exploration de la Vision et Neuro-
ophtalmologie. Pôle d'Imagerie et Explorations S. Méchaussier: None. R. Luscan: None. A. Paul:
None. G. Tian: None. X. Gérard: None. S. Defoort-
Dhellemmes: None. N. Loudon: None. I. Audo: None. S. Bonin: None. J. LeGargasson: None. J. Dumont: None. N. Goudin: None. M. Garfa-Traore: None. M. Bras:
None. A. Pouliet: None. B. Bessières: None. N. Boddaert:
None. S. Lyonnet: None. N.J. Cowan: None. J. Rozet:
None. S. Marlin: None. I. Perrault: None. United States, 4Service d'Exploration de la Vision et Neuro-
ophtalmologie. Pôle d'Imagerie et Explorations S. Méchaussier: None. R. Luscan: None. A. Paul:
None. G. Tian: None. X. Gérard: None. S. Defoort-
Dhellemmes: None. N. Loudon: None. I. Audo: None. S. Bonin: None. J. LeGargasson: None. J. Dumont: None. N. Goudin: None. M. Garfa-Traore: None. M. Bras:
None. A. Pouliet: None. B. Bessières: None. N. Boddaert:
None. S. Lyonnet: None. N.J. Cowan: None. J. Rozet:
None. S. Marlin: None. I. Perrault: None. United States, Service d Exploration de la Vision et Neuro
ophtalmologie. P02.31B
Mutations in TUBB4B cause a distinctive sensorineural
disease stability. Functional
analysis
in
cultured
cells
over-
expressing FLAG-tagged wild-type or mutant TUBB4B and
in patient skin-derived fibroblasts showed that the mutant
TUBB4Bs were able to fold, form αβ-heterodimers and co-
assemble into the endogenous MT lattice. However, the
dynamics of growing MTs were consistently altered,
showing that the mutations have a significant dampening
impact on normal MT growth. Our findings provide a link
between sensorineural disease and anomalies in MT beha-
vior, and describe a syndromic LCA unrelated to ciliary
dysfunction. S. Méchaussier1, R. Luscan2, A. Paul2, G. Tian3, X. Gérard1,
S. Defoort-Dhellemmes4, N. Loudon5, I. Audo6, S. Bonin7,
J. LeGargasson8, J. Dumont9, N. Goudin10, M. Garfa-Traore10,
M. Bras11, A. Pouliet12, B. Bessières13, N. Boddaert14,
S. Lyonnet2, N. J. Cowan3, J. Rozet1, S. Marlin2, I. Perrault1 1Laboratory of Genetics in Ophthalmology (LGO),
INSERMU1163 INSTITUT IMAGINE, Paris Descartes
University, Paris, France, 2Laboratory of Embryology and
genetics of human malformation, INSERMU1163 INSTITUT
IMAGINE, Paris Descartes University, Paris, France,
3Department of Biochemistry & Molecular Pharmacology,
NYU Langone Medical Center., New York NY 10016, NY, 1Laboratory of Genetics in Ophthalmology (LGO),
INSERMU1163 INSTITUT IMAGINE, Paris Descartes
University, Paris, France, 2Laboratory of Embryology and
genetics of human malformation, INSERMU1163 INSTITUT
IMAGINE, Paris Descartes University, Paris, France,
3Department of Biochemistry & Molecular Pharmacology,
NYU Langone Medical Center., New York NY 10016, NY,
4 1Laboratory of Genetics in Ophthalmology (LGO),
INSERMU1163 INSTITUT IMAGINE, Paris Descartes
University, Paris, France, 2Laboratory of Embryology and
genetics of human malformation, INSERMU1163 INSTITUT
IMAGINE, Paris Descartes University, Paris, France,
3Department of Biochemistry & Molecular Pharmacology,
NYU Langone Medical Center., New York NY 10016, NY,
United States, 4Service d'Exploration de la Vision et Neuro-
ophtalmologie. Pôle d'Imagerie et Explorations
Fonctionnelles, CHRU de Lille, Hôpital Roger Salengro, Lille,
France, 5Pediatric ENT Department, Hôpital Necker-Enfants
Malades, APHP and Paris Descartes University, Paris,
France, 6Centre Hospitalier National d'Ophtalmologie des
Quinze-Vingts, Paris, France, 7Ophthalmology Department,
Hôpital Lariboisière, APHP and Paris Diderot University,
Paris, France, 8Visual Exploration Department, Hôpital
Lariboisière, APHP, Paris, Diderot University, Paris, France,
9Cell Division and Reproduction. P02.32C Introduction: The genetic architecture in Meniere’s disease
(MD), an inner ear disorder defined by episodic vertigo,
sensorineural hearing loss (SNHL) and tinnitus, is not
known. A panel of 69 hearing loss genes have been
sequenced targeting rare variants in MD. Introduction: The genetic architecture in Meniere’s disease
(MD), an inner ear disorder defined by episodic vertigo,
sensorineural hearing loss (SNHL) and tinnitus, is not
known. A panel of 69 hearing loss genes have been
sequenced targeting rare variants in MD. Materials and Methods: Nine hundred thirty DNA
samples (890 cases and 40 controls) were pooled (each pool
= 10 samples) and libraries were generated by HaloPlex
PCR target enrichment system. Paired-end sequencing was
performed in a Nextseq500 instrument. BWA and GATK
were used for alignment and quality control. Variant calling
was made through VarScan2. The estimated minor allelic
frequencies (MAF) were compared with public references
values in multiple populations, including Spanish variant
server database. Materials and Methods: Nine hundred thirty DNA
samples (890 cases and 40 controls) were pooled (each pool
= 10 samples) and libraries were generated by HaloPlex
PCR target enrichment system. Paired-end sequencing was
performed in a Nextseq500 instrument. BWA and GATK
were used for alignment and quality control. Variant calling
was made through VarScan2. The estimated minor allelic
frequencies (MAF) were compared with public references
values in multiple populations, including Spanish variant
server database. Results: An enrichment of certain rare variants in cases
was observed in genes such as MARVELD2. Some intronic
variants with unknown significance showed a higher MAF
compared with available data from Spanish population,
including SLC12A2, TRIOBP, KCNQ1 and KCNE3 genes. Prioritizing pathogenicity prediction tools suggest that some
of them should be consider as MD candidate variants. So, a
novel synonymous variant in MARVELD2 gene in 3
unrelated individuals was found and validated by Sanger
sequencing. Conclusions: Spanish population has a specific enrich-
ment of rare variants in some hearing loss genes. The
involvement of MARVELD2 variant in MD has to be
investigated. The functional role of the rest of the variants in
SNHL and MD remains to be established. Acknowledgments: Funded by 2016-Target sequencing
from the Meniere Society, UK and Luxembourg National
Research Fund (INTER/Mobility/17/11772209). E. Ranza: None. H. Chung: None. M. Ansar: None. Y. M. Waryah: None. P. Makrythanasis: None. E. Falcon-
net: None. M. Guipponi: None. A.K. Narsani: None. F.A. Santoni: None. A.M. Waryah: None. H. Bellen: None. S. E. P02.33D Spanish rare variants with unknown significance in
candidate hearing loss genes for Meniere disease in Spain P02.32C Antonarakis: None. A. Gallego-Martinez: None. T. Requena: None. P. Román-Naranjo: None. D. Bobbili: None. P. May: None. J. Dopazo: None. J. Lopez-Escamez: None. P. Roman-Naranjo1, A. Gallego-Martinez1, M. C. Moleon-
Gonzalez2, D. R. Bobbili3, T. Requena-Navarro1, P. May3, J. A.
Lopez-Escamez1,2,3 1Centro Pfizer-Universidad de Granada-Junta de Andalucía de
Genómica e Investigación Oncológica (GENYO), Granada,
Spain, 2Luxembourg Centre for Systems Biomedicine (LCSB),
Université du Luxembourg, Esch-sur-Alzette, Luxembourg,
3Clinical Bioinformatics Research Area, Fundación Progreso y
Salud, Hospital Virgen del Rocío, Sevilla, Spain, 4Department
of Otolaryngology, Instituto de Investigación Biosanitaria ibs.
GRANADA, Hospital Universitario Virgen de las Nieves,
Granada, Spain 1Otology & Neurotology Group CTS495, Department of
Genomic Medicine, Centre for Genomics and Oncological
Research - Pfizer/University of Granada/Andalusian Regional
Government (GENYO), Granada, Spain, 2Department of
Otolaryngology, Instituto de Investigación Biosanitaria ibs.
GRANADA, Hospital Universitario Virgen de las Nieves,
Granada, Spain, 3Luxembourg Centre for Systems Biomedicine P02.32C Overexpression of the wild type Par1
protein in the developing eye caused a mild defect in eye
morphology and a mild reduction in ERG amplitude. Expression of the Par1 Arg792Gly mutation, the corre-
sponding mutation observed in the patients, induced
severely reduced eye size that are rough, and cause a near
complete loss of the amplitude of the ERG. Our data in flies
and human indicate that the MARK3 variants correspond to
a loss of function variant in human and flies, although the
precise nature of the allele remains to be determined. GeneMatcher
search did
not detect
additional
cases. MARK3 is also known to interact with Mitf, which causes
the COMMAD syndrome (MIM 617306) that includes
severe microphthalmia. We conclude that MARK3 is a novel
candidate for visual impairment with affected ocular
anatomy. Developmental eye birth defects often severely reduce
vision. We studied a cohort of more than 150 Pakistani
consanguineous families with eye birth defects with at least
two affected individuals. Families were analyzed by a
combination of exome sequencing and homozygosity
mapping. In one family (F105) three affected individuals
were reported with progressive eye phthisis and visual
impairment, and we identified a non-synonymous homo-
zygous variant (NM_001128918.2:c.1708C>G:p.Arg570-
Gly) in MARK3. Given that MARK3 is highly conserved in
flies (I: 55%; S: 67%) the fly homologue, par1, was
knocked down in the eye during development. This resulted
to a significant reduction in eye size and reduced amplitude
of the electroretinograms, suggesting an impairment in
visual transduction. Overexpression of the wild type Par1
protein in the developing eye caused a mild defect in eye
morphology and a mild reduction in ERG amplitude. Expression of the Par1 Arg792Gly mutation, the corre-
sponding mutation observed in the patients, induced
severely reduced eye size that are rough, and cause a near
complete loss of the amplitude of the ERG. Our data in flies
and human indicate that the MARK3 variants correspond to
a loss of function variant in human and flies, although the
precise nature of the allele remains to be determined. GeneMatcher
search did
not detect
additional
cases. MARK3 is also known to interact with Mitf, which causes
the COMMAD syndrome (MIM 617306) that includes
severe microphthalmia. We conclude that MARK3 is a novel
candidate for visual impairment with affected ocular
anatomy. P02.32C Progressive shrinking of the eye and visual impairment
caused by biallelic variants in the MARK3 gene E. Ranza1, H. Chung2,3, M. Ansar4, Y. M. Waryah5, Narsani7, F. A. Santoni4,8, A. M. Waryah5, H. Bellen2,3,9, S. E. Antonarakis4 Narsani7, F. A. Santoni4,8, A. M. Waryah5, H. Bellen2,3,9, S. E. Antonarakis4 1Service of Genetic Medicine, University Hospitals of Geneva,
Geneva, Switzerland, 2Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston, TX, United
States, 3Jan and Dan Duncan Neurological Research Institute,
Texas Children's Hospital, Houston, TX, United States,
4Department of Genetic Medicine and Development, University
of Geneva, Geneva, Switzerland, 5Molecular Biology and
Genetics Department, Medical Research Center, Liaquat
University of Medical and Health Sciences, Jamshoro,
Pakistan, 6Biomedical Research Foundation of the Academy of
Athens, Athens, Greece, 7Institute of Ophthalmology, Liaquat
University of Medical and Health Sciences, Jamshoro,
Pakistan, 8Department of Endocrinology Diabetes and
Metabolism, University Hospital of Lausanne, Lausanne,
Switzerland, 9Howard Hughes Medical Institute, Houston, TX,
United States Leber congenital amaurosis (LCA) is a neurodegenerative
disease of photoreceptor cells that causes blindness within
the first year of life. It occasionally occurs in syndromic
metabolic diseases and plurisystemic ciliopathies. Using
exome sequencing in a multiplex family and three sporadic
cases with an atypical association of LCA with early-onset
hearing loss, we identified two heterozygous mutations
affecting Arg391 in the β-tubulin 4B isotype-encoding gene
(TUBB4B). Inspection of the atomic structure of the
microtubule (MT) protofilament reveals that the β-tubulin
Arg391 residue contributes to a binding pocket that inter-
acts with α-tubulin contained in the longitudinally adjacent
αβ-heterodimer, consistent with a role in maintaining MT J. del Picchia 58 Developmental eye birth defects often severely reduce
vision. We studied a cohort of more than 150 Pakistani
consanguineous families with eye birth defects with at least
two affected individuals. Families were analyzed by a
combination of exome sequencing and homozygosity
mapping. In one family (F105) three affected individuals
were reported with progressive eye phthisis and visual
impairment, and we identified a non-synonymous homo-
zygous variant (NM_001128918.2:c.1708C>G:p.Arg570-
Gly) in MARK3. Given that MARK3 is highly conserved in
flies (I: 55%; S: 67%) the fly homologue, par1, was
knocked down in the eye during development. This resulted
to a significant reduction in eye size and reduced amplitude
of the electroretinograms, suggesting an impairment in
visual transduction. (LCSB), University of Luxembourg, Esch-sur-Alzette,
Luxembourg (LCSB), University of Luxembourg, Esch-sur-Alzette,
Luxembourg recently proposed hypothesis suggests the involvement of
the gastrointestinal (gut) microbiome in (obesity-related)
knee Osteoarthritis pain, through a low grade systemic
inflammation mediated by bacterial endotoxins from the gut
microbiome. Introduction: Meniere’s disease (MD), an inner ear dis-
order characterized by vertigo, sensorineural hearing loss
and tinnitus, involves 7.5 cases in 100.000 people. MD
shows familial aggregation and we have found rare variants
in FAM136A, DTNA, PRKCB and SEMA3D genes in single
families, showing genetic heterogeneity. We present a new
family with autosomal dominant MD segregating novel
variants for this condition. Materials and Methods: Gut microbial composition was
determined by 16S ribosomal RNA-sequencing (n = 1,427,
Rotterdam Study population). Association analysis were
done in MaAs. We used the relative abundancy of gut
microbiome taxonomies, adjusted for age, sex, technical
covariates, and BMI. Knee joint pain measures are based on
the standardised pain questionnaires(WOMAC) pain score. Materials and Methods: A Spanish family including 3
affected women with MD, suggestive of an autosomal-
dominant pattern of inheritance, was diagnosed. DNA was
isolated from blood samples to perform whole-exome
sequencing in the 3 cases. Single nucleotide variants
(SNVs) and insertions/deletions (indels) were identified
and annotated by GATK and Scalpel after quality controls. These variants were filtered by exome data from 1579
Spanish controls and the cut-off for minor allele frequency
(MAF) was 0.001. Variants were prioritized according to
pathogenicity by multiple bioinformatics tools. Results: We find four highly significant associations
(FDR<0.05) with knee joint pain on different taxonomic
levels (Class-Order-Family-Genus) leading to the bacterial
genus of Streptococcus (FDR= 1.96E-05,). Additional
adjustment for BMI did not affect the identified association
Also, the relative abundancy of Streptococcus in the gut is
significantly associated with amount of effusion (assessed
by
MRI),
a
measure
for
knee
joint
inflammation
(FDR=1.1E-2, n = 373). Conclusions: We identified a significant positive associa-
tion between Streptococcus abundance and knee joint pain
and inflammation in the knee. This association seems
independent of BMI. Streptococcus species are known to
potentially cause osteomyelitis and rheumatic fever, an
inflammatory joint disease affecting the heart and articular
joints, indicating that Streptococcus itself or released
components (such as membrane vescicles), can directly
target the joint. Results: A total of 2822 rare SNPs and 779 rare indels
segregated the phenotype. We identified 7 candidate
variants with a MAF lower than 0.001. (LCSB), University of Luxembourg, Esch-sur-Alzette,
Luxembourg A novel non-
synonymous SNV in DIABLO gene (c.C353G;p.T118R)
and
a
non-synonymous
SNV
in
SLC7A8
gene
(rs146946494) were the two best rated variants. Conclusions: Familial MD shows genetic heterogeneity. This study is the basis for a forthcoming functional study to
evaluate how these variants are involved in MD. Conclusions: Familial MD shows genetic heterogeneity. This study is the basis for a forthcoming functional study to
evaluate how these variants are involved in MD. C.G. Boer: None. D. Radjabzeh: None. C. Medina-
Gomez: None. D. Schiphof: None. P. Arp: None. F. Rivadeneira: None. A.G. Uitterlinden: None. J.P. Hays:
None. R. Kraaij: None. J.B.J. van Meurs: None. Acknowledgments: Funded by 2016-Target sequencing
from the Meniere Society, UK and Luxembourg National
Research Fund (INTER/Mobility/17/11772209). Acknowledgments: Funded by 2016-Target sequencing
from the Meniere Society, UK and Luxembourg National
Research Fund (INTER/Mobility/17/11772209). P. Roman-Naranjo: None. A. Gallego-Martinez: None. M.C. Moleon-Gonzalez: None. D.R. Bobbili: None. T. Requena-Navarro: None. P. May: None. J.A. Lopez-
Escamez: None. P. Roman-Naranjo: None. A. Gallego-Martinez: None. M.C. Moleon-Gonzalez: None. D.R. Bobbili: None. T. Requena-Navarro: None. P. May: None. J.A. Lopez-
Escamez: None. P02.36C Mutation screening and tissue expression patterns
implicate SRY-box 14 (SOX14) in human eye and brain
developmental anomalies Mutation screening and tissue expression patterns
implicate SRY-box 14 (SOX14) in human eye and brain
developmental anomalies P02.34A
Novel and ultrarare allelic variants in DIABLO and
SLC7A8 genes in familial Meniere’s disease Novel and ultrarare allelic variants in DIABLO and
SLC7A8 genes in familial Meniere’s disease A. Gallego-Martinez1, T. Requena1, P. Román-Naranjo1,
D. Bobbili2, P. May2, J. Dopazo3, J. Lopez-Escamez1,2,4 A. Gallego-Martinez1, T. Requena1, P. Román-Naranjo1,
D. Bobbili2, P. May2, J. Dopazo3, J. Lopez-Escamez1,2,4 1Otology & Neurotology Group CTS495, Department of
Genomic Medicine, Centre for Genomics and Oncological
Research - Pfizer/University of Granada/Andalusian Regional
Government (GENYO), Granada, Spain, 2Department of
Otolaryngology, Instituto de Investigación Biosanitaria ibs. GRANADA, Hospital Universitario Virgen de las Nieves,
Granada, Spain, 3Luxembourg Centre for Systems Biomedicine Abstracts from the 51st European Society of Human Genetics Conference: Posters 59 1Oxford Brookes University, Oxford, United Kingdom,
2University College London, London, United Kingdom,
3Université de Toulouse, Toulouse, France, 4Hôpital Purpan,
Toulouse, France, 5Universidad Mayor, Santiago, Chile,
6University of British Columbia, Vancouver, BC, Canada,
7Hôpital Jeanne de Flandre, Lille, France, 8Wellcome Trust
Sanger Institute, Cambridge, United Kingdom, 9University of P02.35B
The role of the gut microbiome in osteoarthritis and
joint pain Conclusions: Here, we show that LVAVA haplotype of
the
OPN1LW
gene
and
MVAVA
haplotype
of the
OPN1MW gene cause apparently nonsyndromic high
myopia in young patients but lead to progressive cone
dystrophy with deuteranopia and protanopia in middle-aged
patients corresponding to a previously unknown disease
course. To the best of our knowledge, this is the first report
on the joint effect of these toxic haplotypes in the two opsin
genes on chromosome X. Supported by Ministry of
National Economy, Hungary GINOP-2.3.2-15-2016-00039. O. Orosz: None. I. Balogh: None. G. Losonczy: None. R.J. Holt: None. D. Gold Diaz: None. N. Chassaing:
None. L.E. Valdivia: None. A.W. Wyatt: None. J. Plaisancié: None. D. Bourgeois: None. C. Vincent-
Delorme: None. R. Osborne: None. D.A. Bax: None. C. Santos: None. S. Broadgate: None. L. Cooper-Charles:
None. L.E. Allen: None. D. McMullan: None. S.W. Wilson: None. D. Gerrelli: None. P. Calvas: None. N.K. Ragge: None. O. Orosz: None. I. Balogh: None. G. Losonczy: None. P02.35B
The role of the gut microbiome in osteoarthritis and
joint pain Here, we describe a new genotype-phenotype correlation
attributed to rare exon 3 interchange haplotypes simulta-
neously present in the long- and middle-wavelength sensi-
tive opsin genes (L- and M-opsin genes). Anophthalmia, microphthalmia and coloboma (AMC) are
developmental eye anomalies which occur in approximately
3 in 10,000 births. They are a genetically heterogeneous
group of conditions, with over 300 genes having been
identified as underlying them. However, only approxi-
mately 25% of patients receive a genetic diagnosis,
depending on phenotype. The most frequent genetic cause
of severe AMC are alterations in SOX2, a member of the
SOXB family of transcription factors which have important
functions in early central nervous system development. Both SOX2 and SOX14 bind the same transcription factor
binding site, acting as enhancers and repressors, respec-
tively. Therefore, SOX14 may mediate SOX2 targeted gene
transcription and so be a candidate for AMC. We screened
SOX14 in 306 individuals with developmental eye anoma-
lies and identified four families carrying variants: a de novo
heterozygous c.242G>T (p.Arg81Leu), a maternally inher-
ited frameshift c.722delA, a de novo deletion of 7.78Mb
including SOX14, and a paternally inherited SOX14 dupli-
cation. However, the link between the identified variations
and the ocular phenotype still remain to be demonstrated. Furthermore, in situ hybridisation experiments using human
embryonic tissue demonstrated that SOX14 is expressed in
the eye and regions of the brain, including the hindbrain and
diencephalon. Although, we developed a zebrafish model
carrying CRISPR-induced mutations of sox14, these fish
showed no alterations in eye development or gross anato-
mical abnormalities. We consider SOX14 to be a likely
important candidate in mammalian nervous system devel-
opment and should be considered a candidate for AMC
disorders. Materials and Methods: A multigenerational family
with X-linked high myopia and cone dystrophy was
investigated by clinical exome sequencing. Materials and Methods: A multigenerational family
with X-linked high myopia and cone dystrophy was
investigated by clinical exome sequencing. Results: Affected male patients had infantile onset
myopia with normal visual acuity and color vision until
their forties. Visual acuity decreased thereafter, along with
the development of severe protan and deutan color vision
defects. A mild decrease in electroretinography response of
cone photoreceptors was detected in childhood, which
further deteriorated in middle-aged patients. Rods were also
affected, however, to a lesser extent than cones. Clinical
exome sequencing revealed the LVAVA and MVAVA
toxic haplotypes in the OPN1LW and OPN1MW opsin
genes, respectively. P02.38A A recurrent intergenic variant upstream of PRDM13 causes
autosomal dominant progressive bifocal chorioretinal
atrophy in two unrelated pedigrees O. Orosz1, I. Balogh1, G. Losonczy2 G. Arno1,2, R. S. Silva1,2, N. Pontikos1,2, V. Cipriani1,2,
S. Defoort-Dhellemmes3, A. Kalhoro1,2, K. J. Carss4,5, F. L.
Raymond4,6, V. van Heyningen1, A. T. Moore1,2,7, B. Puech3,
A. R. Webster1,2 P02.35B
The role of the gut microbiome in osteoarthritis and
joint pain R. J. Holt1, D. Gold Diaz2, N. Chassaing3,4, L. E. Valdivia2,5,
A. W. Wyatt6, J. Plaisancié2, D. Bourgeois4, C. Vincent-
Delorme7, R. Osborne8, D. A. Bax1, C. Santos2, S. Broadgate9,1,
L. Cooper-Charles10, L. E. Allen10, D. McMullan10, S. W. Wilson2, D. Gerrelli2, P. Calvas4,3, N. K. Ragge1,11 C. G. Boer, D. Radjabzeh, C. Medina-Gomez, D. Schiphof,
P. Arp, F. Rivadeneira, A. G. Uitterlinden, J. P. Hays, R. Kraaij,
J. B. J. van Meurs C. G. Boer, D. Radjabzeh, C. Medina-Gomez, D. Schiphof,
P. Arp, F. Rivadeneira, A. G. Uitterlinden, J. P. Hays, R. Kraaij,
J. B. J. van Meurs C. G. Boer, D. Radjabzeh, C. Medina-Gomez, D. Schiphof,
P. Arp, F. Rivadeneira, A. G. Uitterlinden, J. P. Hays, R. Kraaij,
J. B. J. van Meurs
Erasmus MC, Rotterdam, Netherlands 1Oxford Brookes University, Oxford, United Kingdom,
2University College London, London, United Kingdom,
3Université de Toulouse, Toulouse, France, 4Hôpital Purpan,
Toulouse, France, 5Universidad Mayor, Santiago, Chile,
6University of British Columbia, Vancouver, BC, Canada,
7Hôpital Jeanne de Flandre, Lille, France, 8Wellcome Trust
Sanger Institute, Cambridge, United Kingdom, 9University of Erasmus MC, Rotterdam, Netherlands Introduction: Osteoarthritis a degenerative joint disease is
predominantly thought to be due to mechanical and genetic
factors. However, also chronic inflammation plays a causal
role in Osteoarthritis and Osteoarthritis related joint pain. A 60 J. del Picchia Oxford, Oxford, United Kingdom, 10Birmingham Women’s
Hospital, Birmingham, United Kingdom, 11West Midlands
Regional Clinical Genetics Service and Birmingham Health
Partners Birmingham Women’s and Children’s Hospital NHS
Foundation Trust, Birmingham, United Kingdom 1Divison of Clinical Genetics, Department of Laboratory
Medicine, Debrecen, Hungary, 2Department of
Ophthalmology, Zuyderland-Eyescan, Sittard-Geleen,
Netherlands Introduction: Rare interchange haplotypes in exon 3 of the
OPN1LW and OPN1MW opsin genes cause X-linked
myopia, color vision defect, and cone dysfunction. The
severity of the disease varies on a broad scale from non-
syndromic high myopia to blue cone monochromatism. Here, we describe a new genotype-phenotype correlation
attributed to rare exon 3 interchange haplotypes simulta-
neously present in the long- and middle-wavelength sensi-
tive opsin genes (L- and M-opsin genes). Introduction: Rare interchange haplotypes in exon 3 of the
OPN1LW and OPN1MW opsin genes cause X-linked
myopia, color vision defect, and cone dysfunction. The
severity of the disease varies on a broad scale from non-
syndromic high myopia to blue cone monochromatism. 1UCL Institute of Ophthalmology, London, United Kingdom,
2Moorfields Eye Hospital, London, United Kingdom, 1UCL Institute of Ophthalmology, London, United Kingdom,
2Moorfields Eye Hospital, London, United Kingdom,
3Exploration de la Vision et Neuro-Ophtalmologie, Centre P02.39B C. Chiereghin1, M. Robusto2, L. Mauri3, P. Primignani3,
P. Castorina4, U. Ambrosetti4, S. Duga1,2, R. Asselta1,2,
G. Soldà1,2 1Humanitas Clinical and Research Center, Rozzano, Italy,
2Humanitas University, Department of Biomedical Sciences,
Pieve Emanuele, Italy, 3S.S. Genetica Medica, ASST Grande
Ospedale Metropolitano Niguarda, Milano, Italy,
4Dipartimento di Scienze Cliniche e di Comunità, Università
degli Studi di Milano and Fondazione IRCCS Cà Granda
Ospedale Maggiore Policlinico, UO Audiologia, Milano, Italy Background: Progressive bifocal chorioretinal atrophy
(PBCRA) is characterised by macular (and subsequent
nasal) chorioretinal atrophic lesions evident during infancy. Prior genetic linkage pinpointed the disease locus to chro-
mosome 6q14-16.2 overlapping the North Carolina Macular
Dystrophy (NCMD) locus (MCDR1), a non-progressive
developmental macular dystrophy in which mutations
upstream of PRDM13 have been implicated. Introduction: Nonsyndromic sensorineural hearing loss
(NSHL) is one of the most common congenital disorders in
humans and is characterized by a high genetic hetero-
geneity. Recently,
an
homozygous
missense
variant
(NM_003059.2:c.338G>A:p.C113Y) in the SLC22A4 gene
(DNFB60 locus on chromosome 5), which encodes the
organic cation transporter OCTN1, has been described in
two
Tunisian
families
affected
by
autosomal
recessive NSHL. Methods: Whole genome sequencing was performed to
interrogate structural and single nucleotide variants in 5
PBCRA affected individuals from 2 unrelated families,
including the 6q14-16.2 linked family (family 1) to gain
insight into the cause of PBCRA. Methods: Whole genome sequencing was performed to
interrogate structural and single nucleotide variants in 5
PBCRA affected individuals from 2 unrelated families,
including the 6q14-16.2 linked family (family 1) to gain
insight into the cause of PBCRA. Results: Seven novel variants were identified on the
disease haplotype (chr6:98117898-103695199) in 3 indivi-
duals from family 1. Eleven novel variants were shared
between the 2 affected individuals of family 2 at the same
locus. A
single
variant
(chr6:100046804T>C),
7.8kb
upstream of the PRDM13 gene, was identified in both
families, haplotype analysis confirmed that the variant arose
independently. Materials and Methods: Genome-wide linkage analysis
with OmniExpressExome-8 v1.4 BeadChip array (Illumina)
was performed on a large consanguineous NSHL family of
Moroccan origin to highlight the genomic loci most likely
to be involved in the disease. Whole-exome sequencing
(WES) was then carried out on two affected siblings. Results: Genome-wide linkage analysis on the pedigree
pointed to a unique strong linkage signal peak (LOD > 3.5)
in an interval of about 3Mb on chromosome 5q23.3-q31.1,
encompassing the SLC22A4 gene, delimited by markers
rs11241999 and rs2237060. P02.39B Moreover, WES analysis
identified the presence, at the homozygous state, of the
previously described p.C113Y mutation in both affected
siblings. Finally, Sanger sequencing confirmed the presence
of the p.C113Y variant in all 6 affected relatives, and one
unaffected sibling. The entire SLC22A4 gene was screened
in additional 7 NSHL patients coming from North African
countries, but no likely pathogenic variants were found. Discussion: We report the likely pathogenic variant in
two unrelated PBCRA families and expand the non-coding
variant spectrum upstream of PRDM13; the PBCRA variant
lies 5.7kb closer to PRDM13 than the 3 variants previously
implicated in NCMD. Duplications encompassing PRDM13
have also been implicated in NCMD. Discussion: We report the likely pathogenic variant in
two unrelated PBCRA families and expand the non-coding
variant spectrum upstream of PRDM13; the PBCRA variant
lies 5.7kb closer to PRDM13 than the 3 variants previously
implicated in NCMD. Duplications encompassing PRDM13
have also been implicated in NCMD. Discussion: We report the likely pathogenic variant in
two unrelated PBCRA families and expand the non-coding
variant spectrum upstream of PRDM13; the PBCRA variant
lies 5.7kb closer to PRDM13 than the 3 variants previously
implicated in NCMD. Duplications encompassing PRDM13
have also been implicated in NCMD. Taken together this suggests altered spatio-temporal
expression of PRDM13 is a candidate disease mechanism
in the phenotypically distinct NCMD and PBCRA. Since
both disorders affect the macula at birth, exploring the
functional distinctions between these variants will be key to
understanding the disease mechanisms and the importance
of PRDM13 in the context of normal retinal development. Taken together this suggests altered spatio-temporal
expression of PRDM13 is a candidate disease mechanism
in the phenotypically distinct NCMD and PBCRA. Since
both disorders affect the macula at birth, exploring the
functional distinctions between these variants will be key to
understanding the disease mechanisms and the importance
of PRDM13 in the context of normal retinal development. Taken together this suggests altered spatio-temporal
expression of PRDM13 is a candidate disease mechanism
in the phenotypically distinct NCMD and PBCRA. Since
both disorders affect the macula at birth, exploring the
functional distinctions between these variants will be key to
understanding the disease mechanisms and the importance
of PRDM13 in the context of normal retinal development. P02.37 Dmyopia and late-onset progressive cone dystrophy
associate to LVAVA/MVAVA exon 3 interchange
haplotypes of opsin genes on chromosome O. Orosz1, I. Balogh1, G. Losonczy2 Abstracts from the 51st European Society of Human Genetics Conference: Posters 61 Hospitalier Universitaire, Lille, France, 4NIHR Bioresource –
Rare Diseases, Cambridge University Hospitals NHS
Foundation Trust, Cambridge Biomedical Campus,
Cambridge, United Kingdom, 5Department of Haematology,
University of Cambridge, NHS Blood and Transplant Centre,
Cambridge, United Kingdom, 6Department of Medical
Genetics, Cambridge Institute for Medical Research,
University of Cambridge, Cambridge, United Kingdom,
7Ophthalmology Department, UCSF School of Medicine, San
Francisco, CA, United States Hospitalier Universitaire, Lille, France, 4NIHR Bioresource –
Rare Diseases, Cambridge University Hospitals NHS
Foundation Trust, Cambridge Biomedical Campus,
Cambridge, United Kingdom, 5Department of Haematology,
University of Cambridge, NHS Blood and Transplant Centre,
Cambridge, United Kingdom, 6Department of Medical
Genetics, Cambridge Institute for Medical Research,
University of Cambridge, Cambridge, United Kingdom,
7Ophthalmology Department, UCSF School of Medicine, San
Francisco, CA, United States 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan 1Hospital General de Mexico, Mexico DF, Mexico, 2Genetica,
Hospital General de Mexico, Mexico DF, Mexico, 3Genetica,
Hospital General de Mexico, Facultad de Medicina,
Universidad Nacional Autonoma de Mexico, Mexico DF,
Mexico 1Hospital General de Mexico, Mexico DF, Mexico, 2Genetica,
Hospital General de Mexico, Mexico DF, Mexico, 3Genetica,
Hospital General de Mexico, Facultad de Medicina,
Universidad Nacional Autonoma de Mexico, Mexico DF,
Mexico Introduction: Retinitis Pigmentosa (RP) is a group of
genetically heterogeneous conditions of retinal dystrophies. PAX6 is a highly conserved transcription factor expressing
in the eyes and controls the development of eyes. Null
mouse mutation of Pax6 exhibit Small eye (Sey) phenotype,
similar to the heterozygous mutations of Cad, a gene
involved in de novo pyrimidine biosynthesis and tightly
regulated during the cell cycle. In our previous study
demonstrated that PAX6 regulated transcription of Cad. Functional
studies
of
PAX6
mutations
were
largely
unknown, our study is to investigate whether the PAX6 non-
stop mutation affects the gene expression of CAD as a way
of functional analysis. Introduction: Congenital nystagmus is the most common
eye movement disorder, with bilateral and involuntary
oscillations of the eye, visual alteration and erroneous
postures of the head. Nystagmus has been related to
alterations in different genes with several patterns of
inheritance. Apparently, X-linked inheritance pattern is the
most common. Nystagmus can be a syndromic or non-
syndromic entity. The PAX6 gene has been associated with
different eye diseases such as optic nerve/eye colobomas,
aniridia, anterior dysgenesis, cataract with corneal dystro-
phy, foveal hypoplasia, keratitis and optic nerve hypoplasia. The PAX6 gene is also involved in congenital nystagmus
with
photophobia,
posterior
embryoxoton
and
foveal
hypoplasia. Materials and Methods: Structural modeling was used
to predict the influence of the non-stop mutation. In vitro
luciferase promoter assay and in vivo zebrafish knockdown
and rescue experiments were performed to test the function
of PAX6 mutation. Materials and Methods: Structural modeling was used
to predict the influence of the non-stop mutation. In vitro
luciferase promoter assay and in vivo zebrafish knockdown
and rescue experiments were performed to test the function
of PAX6 mutation. Objective: In this study, we described a large Mexican
family of four generations with idiopathic congenital
nystagmus and no other alterations of the structures of the
eye and a novel PAX6 gene mutation. Results: The non-stop mutation we identified from a
human RP family were predicted to have additional 36 a.a. forming an α-helical structure. 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan We hypothesized that the
mutation would lead to improper binding of PAX6 to the
CAD
promoter
and
lose
transactivation
ability. The
luciferase promoter assay, PAX6 non-stop mutation abol-
ished CAD promoter activity. The in vivo zebrafish
morpholino knockdown with overexpressing the non-stop
mutation PAX6 developed retinal abnormalities. Material and Methods: Genomic DNA was extracted
from peripheral blood of 15 members of a Mexican family
and 100 normal controls. They were analyzed through
exome sequencing. Results: It was detected in PAX6 gene the mutation
c.382C>T. This mutation was not found in healthy
members
of
the
family,
normal
controls
and
world
databases. Conclusions: Our studies suggest that the non-stop
mutation in PAX6 may reduce the expression of CAD
leading to an insufficient development of the retina. Conclusions: Our studies suggest that the non-stop
mutation in PAX6 may reduce the expression of CAD
leading to an insufficient development of the retina. Conclusion: The result include a mutation not previously
reported in the literature. This mutation involves the PAX6
gene associated to nystagmus congenital with an autosomal
dominant pattern and no other clinical manifestations. This
is of great relevance for the genetic diagnosis of idiopathic
congenital nystagmus associated to PAX6 gene. W. Lin: None. C. Liu: None. L. Hu: None. C. Chung:
None. J. Chien: None. M. Lin: None. S. Huang: None. Y. Ching: None. W. Lin: None. C. Liu: None. L. Hu: None. C. Chung:
None. J. Chien: None. M. Lin: None. S. Huang: None. Y. Ching: None. L. A. M. Demain1,2, D. Antunes3, A. Heiberg4, J. O’Sullivan1,2,
S. S. Bhaskhar1,2, R. T. O’Keefe5, W. G. Newman1,2 P02.39B Conclusion: This represents the first independent replica-
tion of the involvement of SLC22A4 in autosomal recessive
NSHL, highlighting the importance of this gene, and of the
p.C113Y variant, at least in the North African population. This
study
was
supported
by
Fondazione
Cariplo
(grant#2013-0825). G. Arno: None. R.S. Silva: None. N. Pontikos: None. V. Cipriani:
None. S. Defoort-Dhellemmes:
None. A. Kalhoro: None. K.J. Carss: None. F.L. Raymond: None. V. van Heyningen: None. A.T. Moore: None. B. Puech:
None. A.R. Webster: None. G. Arno: None. R.S. Silva: None. N. Pontikos: None. V. Cipriani:
None. S. Defoort-Dhellemmes:
None. A. Kalhoro: None. K.J. Carss: None. F.L. Raymond: None. V. van Heyningen: None. A.T. Moore: None. B. Puech:
None. A.R. Webster: None. C. Chiereghin: None. M. Robusto: None. L. Mauri:
None. P. Primignani: None. P. Castorina: None. U. Ambrosetti: None. S. Duga: None. R. Asselta: None. G. Soldà: None. J. del Picchia 62 P02.42A
Functional study of a PAX6 non-stop mutation causing
autosomal dominant Retinitis Pigmentosa
W. Lin1, C. Liu1, L. Hu1, C. Chung1, J. Chien2, M. Lin1,
S. Huang1, Y. Ching1 Autosomal dominant nystagmus in a large family
associated to a novel mutation in the PAX6 gene Autosomal dominant nystagmus in a large family
associated to a novel mutation in the PAX6 gene Autosomal dominant nystagmus in a large family
associated to a novel mutation in the PAX6 gene R. Vega-Gama1, L. M. Gonzalez-Huerta2, M. R. Rivera-Vega2,
J. M. Valdes-Miranda2, N. Xilotl-DeJesus2, V. Martínez-
Montoya2, M. Tovar-Ayala2, S. A. Cuevas-Covarrubias3 W. Lin1, C. Liu1, L. Hu1, C. Chung1, J. Chien2, M. Lin1,
S. Huang1, Y. Ching1 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan J. Lenk1, J. Porrmann2, A. Kahlert2, M. Smitka3, I. Eger4,
E. Schröck2, K. Hackmann2, R. Herber1, F. Raiskup1,
A. Tzschach2 1Department of Ophthalmology, Universitätsklinikum Carl
Gustav Carus, Dresden, Germany, 2Technische Universität
Dresden, Institute of Clinical Genetics, Dresden, Germany,
3Children´s hospital, Universitätsklinikum Carl Gustav Carus,
Dresden, Germany, 4Department of Neuropediatrics,
Städtisches Klinikum Görlitz, Görlitz, Germany Posterior amorphous corneal dystrophy (PACD) (OMIM
612868) is a rare autosomal dominant disorder character-
ized by partial or complete posterior lamellar corneal opa-
cification, decreased corneal thickness and flattening of the
corneal curvature. Onset of the disease is in the first years of
life. PACD is associated with heterozygous deletions in
chromosome band 12q21.33-q22 harbouring the genes
DCN (Decorin, OMIM 125255), KERA (Keratocan, OMIM
603288), LUM (Lumican, OMIM 600616) and EPYC
(Epiphycan, OMIM 601657) which encode small leucine-
rich proteoglycans. Only four families with deletions of this
region have been published to date. We report on a 7-year-
old male patient with PACD in whom an interstitial deletion
in 12q21.33 was detected by array CGH. Subsequent FISH
analyses in the parents revealed a balanced insertional
translocation of this 12q21.33 segment into the long arm of
one chromosome 13 in the mother. This family corroborates
the association of 12q21.33 deletions with PACD and
constitutes the first example of the involvement of a
balanced chromosome aberration that predisposes to this
rare disorder. Perrault syndrome is a rare recessive condition characterised
by sensorineural hearing loss (SNHL) and primary ovarian
insufficiency (POI). Additional phenotypes, most com-
monly neurological, may also present. Perrault syndrome is
clinically and genetically heterogeneous. Six causative
genes have been identified to date, five of which function in
mitochondrial homeostasis. In a number of cases no cau-
sative variants have been identified in known Perrault
syndrome genes. Perrault syndrome may be difficult to
clinically distinguish from overlapping phenotypes such as
mitochondrial DNA depletion syndrome 7, which causes
SNHL, POI and severe neurological dysfunction. We
identified two cases of apparent Perrault syndrome due to
variants in genes associated with other conditions. We
identified a homozygous known pathogenic variant RMND1
c.713A>G, p.(Asn238Ser) in a proband with SNHL, POI
and renal acidosis. RMND1 is essential for mitochondrial
translation and recessive variants in RMND1 have been
associated with renal defects, neurological phenotypes and
SNHL. In a proband with SNHL, POI and mild intellectual
disability we identified compound heterozygous rare var-
iants
in
XPNPEP3;
c.263A>G,
p.Gln88Arg
and
c.1261C>G, p.His421Asp as the likely cause of the phe-
notype. XPNENP3 is involved in mitochondrial protein
processing. P02.44C
12q21.33 deletion in a patient with posterior amorphous
corneal dystrophy J. Lenk1, J. Porrmann2, A. Kahlert2, M. Smitka3, I. Eger4,
E. Schröck2, K. Hackmann2, R. Herber1, F. Raiskup1,
A. Tzschach2 Homozygous loss of function variants in
XPNENP3 have been linked to nephronophthisis with some
individuals also affected by SNHL. POI has not been
reported secondary to variants in RMND1 or XPNENP3 and
reflects the later onset and sex-limited nature of this phe-
notype. Overlapping phenotypes such as those reported here
may account for some of the genotypic and phenotypic
variation seen in Perrault syndrome. J. Lenk: None. J. Porrmann: None. A. Kahlert: None. M. Smitka: None. I. Eger: None. E. Schröck: None. K. Hackmann: None. R. Herber: None. F. Raiskup: None. A. Tzschach: None. P02.43B Perrault-like phenotypes may account for some of the
genetic and phenotypic heterogeneity within Perrault
syndrome R. Vega-Gama: None. L.M. Gonzalez-Huerta: None. M.R. Rivera-Vega: None. J.M. Valdes-Miranda: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. M. Tovar-Ayala: None. S.A. Cuevas-Covarrubias: None. M. Tovar-Ayala: None. S.A. Cuevas-Covarrubias: None. M. Tovar-Ayala: None. S.A. Cuevas-Covarrubias: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 63 1Division of Evolution and Genomic Sciences, Faculty of
Biology, Medicine and Health, School of Biological Sciences,
University of Manchester, Manchester, United Kingdom,
2Manchester Centre for Genomic Medicine, Manchester
University NHS Foundation Trust, Manchester, United
Kingdom, 3Medical Genetics Department, Hospital de Dona
Estefânia, Centro Hospitalar Lisboa Central, Lisbon,
Portugal, 4Department of Medical Genetics, Oslo University
Hospital, Oslo, Norway, 5Division of Cellular & Molecular
Function, Faculty of Biology, Medicine and Health, University
of Manchester, Manchester, United Kingdom J. Lenk1, J. Porrmann2, A. Kahlert2, M. Smitka3, I. Eger4,
E. Schröck2, K. Hackmann2, R. Herber1, F. Raiskup1,
A. Tzschach2 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Postgraduate School
of Molecular Medicine, Medical University of Warsaw,
Warsaw, Poland, 3Oto-Rhino-Laryngology Surgery Clinic,
Institute of Physiology and Pathology of Hearing, Warsaw,
Poland D. Oziębło1,2, A. Adamiok1, A. Sarosiak1,2, H. Skarżyński3,
M. Ołdak1 First independent confirmation of the PTPRQ gene
involvement in autosomal dominant hearing loss First independent confirmation of the PTPRQ gene
involvement in autosomal dominant hearing loss D. Oziębło1,2, A. Adamiok1, A. Sarosiak1,2, H. Skarżyński3,
M. Ołdak1 D. Oziębło1,2, A. Adamiok1, A. Sarosiak1,2, H. Skarżyński3,
M. Ołdak1 D. Oziębło1,2, A. Adamiok1, A. Sarosiak1,2, H. Skarżyński3,
M. Ołdak1 1Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland, 2Postgraduate School
of Molecular Medicine, Medical University of Warsaw,
Warsaw, Poland, 3Oto-Rhino-Laryngology Surgery Clinic,
Institute of Physiology and Pathology of Hearing, Warsaw,
Poland L.A.M. Demain: None. D. Antunes: None. A. Heiberg:
None. J. O’Sullivan: None. S.S. Bhaskhar: None. R.T. O’Keefe: None. W.G. Newman: None. L.A.M. Demain: None. D. Antunes: None. A. Heiberg:
None. J. O’Sullivan: None. S.S. Bhaskhar: None. R.T. O’Keefe: None. W.G. Newman: None. P02.44C Background: Hearing loss (HL) is the most common birth
defect affecting about 1-6/1000 births and the most 64 J. del Picchia Material and Methods: This research has been con-
ducted using the UK Biobank (UKBB) resources and two
sets of clinically ascertained RRD cases (close to 1000
genotyped cases). A discovery genome-wide association
study was carried out for retinal detachment using the
largest dataset, UKBB, using BOLT-lmm which allows
rapid analysis of large (N> 5000) datasets. Associated
conditions, high myopia and cataract, were analysed in
UKBB, to evaluate the amount of common genetic
underpinning. RRD associations were replicated using the
datasets of clinically ascertained cases and controls. common disability of human senses. Genetic factors play an
important role in the development of HL. The PTPRQ gene
has been previously reported in the context of autosomal
recessive HL and in 2017 for the first time in the devel-
opment of autosomal dominant HL. common disability of human senses. Genetic factors play an
important role in the development of HL. The PTPRQ gene
has been previously reported in the context of autosomal
recessive HL and in 2017 for the first time in the devel-
opment of autosomal dominant HL. Material and methods: A five-generation Polish family
with progressive, high frequency autosomal dominant HL
was recruited for the study. Genomic DNA was isolated
from peripheral blood samples or buccal swabs of available
family members. Clinical exome sequencing was conducted
in the proband’s DNA sample. Family segregation analysis
of the identified variants was performed using Sanger
sequencing. Results:
Discovery
GWAS
was
performed
using
N=3977 self reported or hospital record linked retinal
detachment cases and revealed three genome-wide sig-
nificant signals as well as a low but significant heritability,
24% on the liability scale. High myopia showed substan-
tially higher heritability, with, potentially mechanistically
interesting, some but not all the top signals also influencing
retinal
detachment. Two
of
the
three
genome-wide
significant retinal detachment associations seem specific to
this phenotype with no association with high myopia,
cataract, nor any phenotypes from publicly available
PheWAS databases. One of those hits, in the FAT3 gene,
was replicated in the clinically ascertained datasets. Results: Molecular genetic testing showed the presence
of probably pathogenic c.6881G>A (p.Trp2294*) variant in
the PTPRQ gene, which fully segregated with HL observed
in the family. P02.44C The identified variant is located in the last
coding exon of the PTPRQ gene and introduces a premature
stop codon. The c.6881G>A transition has not been
reported in population databases. To date, the PTPRQ
variant has been described in one family worldwide and is
the only PTPRQ genetic variant causally involved in
autosomal dominant HL. Conclusions: Identification of the c.6881G>A variant
provides an independent confirmation of the PTPRQ
involvement in autosomal dominant HL, which is progres-
sive, affects high frequencies and is usually diagnosed in the
first decade of life. Conclusions: Identification of the c.6881G>A variant
provides an independent confirmation of the PTPRQ
involvement in autosomal dominant HL, which is progres-
sive, affects high frequencies and is usually diagnosed in the
first decade of life. Conclusions. Biobank resources especially linkage to
health records are a very promising complement to studies
of well ascertained cases. Conclusions. Biobank resources especially linkage to
health records are a very promising complement to studies
of well ascertained cases. T.S. Boutin: None. D.G. Charteris: None. A. Chandra:
None. D. Mitry: None. V. Vitart: None. T.S. Boutin: None. D.G. Charteris: None. A. Chandra:
None. D. Mitry: None. V. Vitart: None. Supported by: 2016/22/E/NZ5/00470 Supported by: 2016/22/E/NZ5/00470 D. Oziębło: None. A. Adamiok: None. A. Sarosiak:
None. H. Skarżyński: None. M. Ołdak: None. D. Oziębło: None. A. Adamiok: None. A. Sarosiak:
None. H. Skarżyński: None. M. Ołdak: None. P02.47B Whole exome sequencing of a cohort of Polish patients with
retinal disorders - NeuStemGen project P02.46A P02.46A
Using UK Biobank for common conditions of poorly
characterised genetic aetiology: the example of retinal
detachment R. Szymańczak1, P. Łyszkiewicz1, A. Wąsowska1,
E. Matczyńska1, W. Krysa1, K. Kamińska1, E. Ewa Suchecka1,
J. Kosakowski1, M. Jurkowska1, A. Pałucha1, M. Jędrzejowska1,
S. Teper2, E. Wylęgała2, E. Pius-Sadowska3, A. Machalińska3,
A. M. Boguszewska-Chachulska1 R. Szymańczak1, P. Łyszkiewicz1, A. Wąsowska1,
1
1
1 T. S. Boutin1, D. G. Charteris2, A. Chandra3, D. Mitry4,
V. Vitart1 S. Teper2, E. Wylęgała2, E. Pius-Sadowska3, A. Machalińska3,
A. M. Boguszewska-Chachulska1 1MRC HGU, MRC Institute of Genetics and Molecular
Medicine, Edinburgh, United Kingdom, 2Moorfields Eye
Hospital, London, United Kingdom, 3Southend University
Hospital, Westcliff-on-Sea, United Kingdom, 4Department of
Ophthalmology, Royal Free NHS Foundation Trust, London,
United Kingdom 1MRC HGU, MRC Institute of Genetics and Molecular
Medicine, Edinburgh, United Kingdom, 2Moorfields Eye
Hospital, London, United Kingdom, 3Southend University
Hospital, Westcliff-on-Sea, United Kingdom, 4Department of
Ophthalmology, Royal Free NHS Foundation Trust, London,
United Kingdom 1Genomed SA, Warsaw, Poland, 2Chair and Ophthalmology
Department, II School of Medicine with the Division of
Dentistry in Zabrze, Medical University of Silesia, Katowice,
Poland, 3Pomeranian Medical University, Szczecin, Poland 1Genomed SA, Warsaw, Poland, 2Chair and Ophthalmology
Department, II School of Medicine with the Division of
Dentistry in Zabrze, Medical University of Silesia, Katowice,
Poland, 3Pomeranian Medical University, Szczecin, Poland The goal of the NeuStemGen project is to identify novel
biomarkers, which may serve as diagnostic and prevention
targets in degenerative disorders. Identification of recurrent
pathogenic variants will allow to develop new therapies,
such as genome editing and gene therapy, for patients with
progressive,
untreatable
retinal
dystrophies
and
degenerations. Introduction: Rhegmatogenous retinal detachment (RRD)
is a common cause of emergency ophthalmic intervention. There is some evidence for a genetic contribution to idio-
pathic RRD but studies have been limited. Here we eval-
uated the use of the UK Biobank Resource to increase
insight into RRD genetic aetiology. 65 Abstracts from the 51st European Society of Human Genetics Conference: Posters The specific approach adopted, based on WES, served to
identify not only pathogenic variants in genes associated
retinal disorders but also in novel genes. P02.46A Physiology, National Institute on Alcohol Abuse and
Alcoholism, NIH, Bethesda, MD, United States, 4Unit on
Neural Circuits, National Institute of Neurological Disorders
and Stroke, NIH, Rockville, MD, United States, 5Laboratory of
Immunology, National Eye Institute, NIH, Bethesda, MD,
United States, 6Section of Molecular Mechanisms of
Glaucoma, Laboratory of Molecular and Developmental
Biology National Eye Institute, NIH, Bethesda, MD, United
States, 7School of Life Sciences, University of Science and
Technology of China, Hafei, China, 8Institute of Biomedical
and Genetic Engineering (IBGE), Islamabad, Pakistan,
9University College London, Institute of Ophthalmology,
London, United Kingdom, 10Faculty of Science, COMSATS
Institute of Information Technology, Islamabad, Pakistan,
11Department of Molecular and Human Genetics, Baylor
College of Medicine, Huston, TX, United States, 12Department
of Human Genetics, Donders Institute for Brain, Cognition and
Behaviour, Radboud University Medical Center, Nijmegen,
Netherlands, 13Faculty of Medicine, University of
Southampton, Southampton, United Kingdom, 14National
Centre of Excellence in Molecular Biology, University of the
Punjab, Lahore, Pakistan, 15Allama Iqbal Medical College,
University of Health Sciences, Lahore, Pakistan, 16National
Eye Institute, NIH, Bethesda, MD, United States WES was performed for the cohort of 94 patients with
clinical symptoms of retinal dystrophies while the POL-
GENOM genomic database of long-lived Poles was used as
the control group. An in-house bioinformatic pipeline was
applied to analyse SNVs and InDels. Further analysis
involved Gemini, enabling an analysis of the full set of
samples in search for rare pathogenic variants in the whole
exome. CNVs were identified using XHMM, these results
were validated
using aCGH arrays (180K), showing
conformity for larger structural variants. Pathogenic variants were identified predominantly in
genes already known to cause retinal degenerations (such as
ABCA4, USH2A, EYS, RHO) although variants in novel
genes, previously reported as single cases, were also
uncovered. A gain of chr 8 was identified as a possible
cause of retinal dystrophy in one patient. The results of this analysis will support further develop-
ment of the targeted retinal panel covering most pathogenic
variants occurring in the Polish population and allowing for
a fast, low-cost genetic analysis, preceding selection of a
personalised therapy. Supported by NCBiR, Project No.STRATEGMED1/
234261/2/NCBR/2014. Supported by NCBiR, Project No.STRATEGMED1/
234261/2/NCBR/2014. Retinitis pigmentosa (RP) is an inherited eye disease char-
acterised by photoreceptor death and retinal degeneration,
resulting in vision loss. This condition affects ~1:4000
individuals worldwide and is highly clinically and geneti-
cally heterogeneous, presenting with variable symptoms
and inheritance patterns. P02.46A We identified a homozygous
missense alteration (c.75C>A, p.D25E) in the CLCC1 gene,
which encodes a presumptive intracellular chloride channel
highly expressed in the retina, associated with autosomal
recessive RP in eight consanguineous families from Pakis-
tani and the UK. The p.D25E alteration decreased CLCC1
channel function accompanied by accumulation of mutant
protein in granules within the ER lumen. In keeping with
these findings, Clcc1+/- KO mice displayed depressed
electroretinogram and photoreceptor number. Together
these findings define a single founder gene mutation as a
cause of RP in families of Pakistani descent, and strongly
suggest that CLCC1 function is crucial for maintaining
retinal integrity and function. This work was supported by
National Eye Institute Grant R01EY021237-01 (SAR),
National Human Genome Research Institute (NHGRI)/
National Heart Lung and Blood Institute (NHLBI) to the
Baylor Hopkins Center for Mendelian Genomics (UM1
HG006542,
JRL),
Medical
Research
Council
UK
(G1002279), the Newlife Foundation for Disabled Children
(SG/15-16/12), Fight For Sight (Ref 2027), Wellcome Trust
209083/Z/17/Z. R. Szymańczak: None. P. Łyszkiewicz: None. A. Wąsowska: None. E. Matczyńska: None. W. Krysa:
None. K. Kamińska: None. E. Ewa Suchecka: None. J. Kosakowski: None. M. Jurkowska: None. A. Pałucha:
None. M. Jędrzejowska: None. S. Teper: None. E. Wylęgała: None. E. Pius-Sadowska: None. A. Macha-
lińska: None. A.M. Boguszewska-Chachulska: None. 1RILD Wellcome Wolfson Centre, Royal Devon & Exeter NHS
Foundation Trust, University of Exeter, Exeter, United
Kingdom, 2Ophthalmic Genetics and Visual Function Branch,
National Eye Institute, NIH, Bethesda, MD, United States,
3Section on Model Synaptic Systems, Laboratory of Molecular P02.49D Haplotypes constructed by Whole exome sequencing to
map and identify a novel disease-causing RP2 gene variant
from a recessive X linked Retinities Pigmentosa family Genetic screening for Italians patients affected with
Retinitis Pigmentosa V. Errichiello1, S. Carboni2, G. Pagliaroli2, V. Caputo1,
C. Strafella1,3, G. Campoli2, C. Peconi2, F. Sangiuolo1,
G. Novelli1, R. Cascella1,4, E. Giardina1,2 Y. Ching1, W. Fan2, W. Lin1, W. Tsai1, L. Hu1, S. Huang1,
R. Chung3 1Molecular Biology and Human Genetics, Hualien, Taiwan,
2Whole-Genome Research Core Laboratory of Human
Diseases, Chang Gung Memorial Hospital, Keelung, Taiwan,
3National Health Research Institutes, Zhunan, Taiwan 1Department of Biomedicine and Prevention, “Tor Vergata”
University, Roma, Italy, 2Laboratory of Genomic Medicine
UILDM, IRCCS Santa Lucia Foundation, Roma, Italy,
3Emotest Laboratory, Pozzuoli, Italy, 4Department of Chemical
Pharmaceutical and Biomolecular Technologies, Catholic
University “Our Lady of Good Counsel” Laprakë, Rruga
Dritan Hoxha, Tirana, Albania 1Department of Biomedicine and Prevention, “Tor Vergata”
University, Roma, Italy, 2Laboratory of Genomic Medicine
UILDM, IRCCS Santa Lucia Foundation, Roma, Italy,
3Emotest Laboratory, Pozzuoli, Italy, 4Department of Chemical
Pharmaceutical and Biomolecular Technologies, Catholic
University “Our Lady of Good Counsel” Laprakë, Rruga
Dritan Hoxha, Tirana, Albania Introduction: Retinitis pigmentosa (RP) is a genetically
heterogeneous with more than 70 RP loci currently known. A three-generation RP family exhibiting X linked recessive
pattern were studied. Introduction: Retinitis Pigmentosa (RP, OMIM #268000)
is a degenerative disorder affecting peripheral retina which
is caused by a progressive loss of photoreceptors. The
genetics of RP is highly heterogeneous, with several asso-
ciated genes mainly implicated in the phototransduction
cascade. RP shows autosomal dominant, autosomal reces-
sive, X-linked and mitochondrial inheritance patterns. One
of the most investigated gene associated with autosomal RP
is RHO (3q21-q24), which encodes for Rodopsin and is
essential for vision in low-light conditions. In this context,
the genetics of RP was studied considering the screening of
RHO as a first-level analysis and a panel of 24 putative
genes as second-level step. Materials and Methods: Linkage analysis was per-
formed using 18 microsatellite markers. Whole exome
sequencing was used for mutational analysis. Results: Polymorphic microsatellite markers were used to
map the disease interval to a 48 Mb region on the X
chromosome between the markers DXS1068 and DXS1196. Whole exome sequencing (WES) was performed on a
selected sib-pair within the family. Total 1973 SNPs were
found to be located within the critical interval. Among these
SNPs, 139 were shared between the obligated carrier and
the affected male offspring but not the phenotypically
normal male offspring. We also utilized the WES identified
polymorphic SNPs mapped within the critical disease
interval to construct detailed haplotype for the sib-pair. P02.48C Hardy: None. G. Arno: None. S. Hull:
None. M. Khan: None. J. Fasham: None. G.V. Harlalka:
None. M. Michaelides: None. A.T. Moore: None. Z. Akdemir: None. S. Jhangiani: None. J.R. Lupski: None. F.P.M. Cremers: None. R. Qamar: None. A. Salman:
None. J.K. Chilton: None. J. Self: None. F. Kabir: None. M. Naeem: None. M. Ali: None. J. Akram: None. P.A. Sieving: None. S. Riazuddin: None. S. Riazuddin: None. J. Hejtmancik:
None. E.L. Baple:
None. A.H. Crosby: None. therefore subjected to the second-step analysis, which
reported a number of variants. At the moment, the resulting
variants are under investigation and validation. Conclusion: the present study highlights a major burden
of genes other than RHO associated with the disease in the
Italian population. Given the genetic heterogeneity of RP, it
would be highly helpful and faster to perform a large-scale
screening of genes compared to the slower and labor-
intensive traditional approaches. F.P.M. Cremers: None. R. Qamar: None. A. Salman:
None. J.K. Chilton: None. J. Self: None. F. Kabir: None. V. Errichiello: None. S. Carboni: None. G. Pagliaroli:
None. V. Caputo: None. C. Strafella: None. G. Campoli:
None. C. Peconi: None. F. Sangiuolo: None. G. Novelli:
None. R. Cascella: None. E. Giardina: None. M. Naeem: None. M. Ali: None. J. Akram: None. P.A. Sieving: None. S. Riazuddin: None. S. Riazuddin: None. J. Hejtmancik:
None. E.L. Baple:
None. A.H. Crosby: None. P02.48C Mutation in the intracellular chloride channel CLCC1
associated with autosomal recessive retinitis pigmentosa I. D'Atri1, L. Li2, X. Jiao2, F. Ono3, R. Nelson4, C. Chan5,
N. Nakaya6, Z. Ma2, Y. Ma2, X. Cai7, L. Zhang7, S. Lin1,
A. Hameed8, B. A. Chioza1, H. Hardy1, G. Arno9, S. Hull9,
M. Khan10, J. Fasham1, G. V. Harlalka1, M. Michaelides9, A. T. Moore9, Z. Akdemir11, S. Jhangiani12, J. R. Lupski11, F. P. M. Cremers12, R. Qamar10, A. Salman13, J. K. Chilton1, J. Self13,
F. Kabir14, M. Naeem14, M. Ali14, J. Akram15, P. A. Sieving16,
S. Riazuddin14, S. Riazuddin14, J. Hejtmancik2, E. L. Baple1,
A. H. Crosby1 I. D'Atri1, L. Li2, X. Jiao2, F. Ono3, R. Nelson4, C. Chan5,
N. Nakaya6, Z. Ma2, Y. Ma2, X. Cai7, L. Zhang7, S. Lin1,
A. Hameed8, B. A. Chioza1, H. Hardy1, G. Arno9, S. Hull9,
M. Khan10, J. Fasham1, G. V. Harlalka1, M. Michaelides9, A. T. Moore9, Z. Akdemir11, S. Jhangiani12, J. R. Lupski11, F. P. M. Cremers12, R. Qamar10, A. Salman13, J. K. Chilton1, J. Self13,
F. Kabir14, M. Naeem14, M. Ali14, J. Akram15, P. A. Sieving16,
S. Riazuddin14, S. Riazuddin14, J. Hejtmancik2, E. L. Baple1,
A. H. Crosby1 1RILD Wellcome Wolfson Centre, Royal Devon & Exeter NHS
Foundation Trust, University of Exeter, Exeter, United
Kingdom, 2Ophthalmic Genetics and Visual Function Branch,
National Eye Institute, NIH, Bethesda, MD, United States,
3Section on Model Synaptic Systems, Laboratory of Molecular 66 J. del Picchia I. D'Atri: None. L. Li: None. X. Jiao: None. F. Ono:
None. R. Nelson: None. C. Chan: None. N. Nakaya:
None. Z. Ma: None. Y. Ma: None. X. Cai: None. L. Zhang: None. S. Lin: None. A. Hameed: None. B.A. Chioza: None. H. Hardy: None. G. Arno: None. S. Hull:
None. M. Khan: None. J. Fasham: None. G.V. Harlalka:
None. M. Michaelides: None. A.T. Moore: None. Z. Akdemir: None. S. Jhangiani: None. J.R. Lupski: None. F.P.M. Cremers: None. R. Qamar: None. A. Salman:
None. J.K. Chilton: None. J. Self: None. F. Kabir: None. M. Naeem: None. M. Ali: None. J. Akram: None. P.A. Sieving: None. S. Riazuddin: None. S. Riazuddin: None. J. Hejtmancik:
None. E.L. Baple:
None. A.H. Crosby: None. I. D'Atri: None. L. Li: None. X. Jiao: None. F. Ono:
None. R. Nelson: None. C. Chan: None. N. Nakaya:
None. Z. Ma: None. Y. Ma: None. X. Cai: None. L. Zhang: None. S. Lin: None. A. Hameed: None. B.A. Chioza: None. H. Genetic screening for Italians patients affected with
Retinitis Pigmentosa Additional crossing-over evens were revealed on and the
disease interval were mapped into two intervals: chrx:
38,911,177 -68890047; and chr X: 69,155,432-69,261,818). The causative mutation (RP2 c.102G>A; Lys34Lys, a RP2
splicing donor site mutation) was then identified. Results: Polymorphic microsatellite markers were used to
map the disease interval to a 48 Mb region on the X
chromosome between the markers DXS1068 and DXS1196. Whole exome sequencing (WES) was performed on a
selected sib-pair within the family. Total 1973 SNPs were
found to be located within the critical interval. Among these
SNPs, 139 were shared between the obligated carrier and
the affected male offspring but not the phenotypically
normal male offspring. We also utilized the WES identified
polymorphic SNPs mapped within the critical disease
interval to construct detailed haplotype for the sib-pair. Additional crossing-over evens were revealed on and the
disease interval were mapped into two intervals: chrx:
38,911,177 -68890047; and chr X: 69,155,432-69,261,818). The causative mutation (RP2 c.102G>A; Lys34Lys, a RP2
splicing donor site mutation) was then identified. Matherial and Methods: 100 Italian RP patients were
analyzed by this two steps-analysis, through direct sequen-
cing
and
NGS
on
IonTorrent
S5
(Thermo
Fisher). Concerning NGS analysis, a 20X coverage was fixed as
minimum depth of coverage. Results: the first-step analysis identified a variant in the
fourth exon of RHO gene, namely c.G760T. The variant
was found in 1 patient, while the remaining 99 patients were
negative for this level of analysis. These patients were Conclusion: We have combined the positional informa-
tion obtained from the linkage and haplotype analysis to
identify the genetic intervals harboring the disease-causing 67 Abstracts from the 51st European Society of Human Genetics Conference: Posters gene, and also utilized DNA polymorphisms identified from
WES as additional genetic markers to further mapped the
crossing-over evens as a creative strategy for positional
cloning. gene, and also utilized DNA polymorphisms identified from
WES as additional genetic markers to further mapped the
crossing-over evens as a creative strategy for positional
cloning. J. Känsäkoski: None. S. Tuupanen: A. Employment
(full or part-time); Significant; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant;
Blueprint Genetics. P. Siivonen: A. Employment (full or
part-time); Significant; Blueprint Genetics. K. Kämpjärvi:
A. Employment (full or part-time); Significant; Blueprint
Genetics. M. Mehine: A. Employment (full or part-time);
Significant; Blueprint Genetics. M. Valori: A. Employment
(full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Genetic screening for Italians patients affected with
Retinitis Pigmentosa Employment (full or part-time); Signifi-
cant; Blueprint Genetics. E. Sankila: F. Consultant/
Advisory Board; Modest; Blueprint Genetics. E. Salminen:
A. Employment (full or part-time); Significant; Blueprint
Genetics. S. Myllykangas: E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Blueprint Genetics. T. Alastalo: E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Significant; Blueprint Genetics. J.W. Kosken-
vuo: E. Ownership Interest (stock, stock options, patent or
other intellectual property); Significant; Blueprint Genetics. MOST: 104-2311-B-320 -001 - MOST: 104-2311-B-320 -001 - Y. Ching: None. W. Fan: None. W. Lin: None. W. Tsai:
None. L. Hu:
None. S. Huang:
None. R. Chung: None. 1Blueprint Genetics, Helsinki, Finland, 2Helsinki University
Eye Hospital, Helsinki, Finland Retinitis pigmentosa (RP) is the most common form of
inherited retinal degeneration affecting around 1:3,000
individuals worldwide. Classical RP is characterized by
progressive rod-cone dysfunction. Patients initially present
with night blindness and tunnel vision, followed by
decreased visual acuity and macular affectation. RP is
clinically and genetically heterogeneous, and it can be
inherited in an autosomal dominant, autosomal recessive,
and X-linked manner. The majority of the X-linked RP,
associated with a severe phenotype, is caused by mutations
in the RPGR gene. All known mutations causing RPGR-
related
retinal
dystrophies
are
found
to
affect
the
RPGRORF15 isoform, which contains a unique C-terminal
567-aa exon called ORF15. ORF15 is a mutational hotspot
for RPGR-associated RP, accounting for two-thirds of all
disease-causing mutations. The exon ORF15, however,
includes a highly repetitive, purine-rich sequence, which
generally performs poorly in next-generation sequencing
(NGS)-based assays. To address the clinical importance of
the RPGR ORF15 and the lack of high quality NGS-based
diagnostics, we have developed a novel test to detect var-
iants in the ORF15 region. This test combines NGS analysis
with Illumina NovaSeq 6000 platform and Sanger sequen-
cing, specifically optimised for this region. We have vali-
dated the test using samples with known RPGR ORF15
variants, and additionally tested patients with a clinical
suspicion of X-linked RP, who have remained negative in
previous genetic testing. We show that the test has high
clinical sensitivity and specificity for detecting RPGR
ORF15 variants, and that it improves the genetic diagnostics
of inherited retinal dystrophies. Improved genetic diagnostics of RPGR ORF15-associated
retinal dystrophy J. Känsäkoski1, S. Tuupanen1, J. Sistonen1, P. Siivonen1,
K. Kämpjärvi1, M. Mehine1, M. Valori1, P. Salmenperä1,
E. Sankila2, E. Salminen1, S. Myllykangas1, T. Alastalo1, J. W. Koskenvuo1 1Blueprint Genetics, Helsinki, Finland, 2Helsinki University
Eye Hospital, Helsinki, Finland 1Blueprint Genetics, Helsinki, Finland, 2Helsinki University
Eye Hospital, Helsinki, Finland An integrated molecular approach to characterize the
genetic bases of hearing loss in an Italian cohort Conclusions: Disease-causing TE insertions can be
missed by traditional genetic testing methods. However,
they can be detected by using WGS data with specific
methods, not systematically implemented. Large -scale
implementation of such algorithms will allow the discovery
of more disease-causing TE insertions and better assessment
of the frequency of this category of mutational events. F. Cesca1, E. Bettella1, R. Polli1, E. Leonardi1, M. C. Aspromonte1, S. Bigoni2, R. Santarelli3, A. Murgia1 1Laboratory of Molecular Genetics of Neurodevelopment,
Department of Women’s and Children’s Health, University of
Padova, Padova, Italy, 2Medical Genetics Unit, Ferrara
University Hospital, Ferrara, Italy, 3Audiology and Phoniatric
Service, Treviso Regional Hospital, Treviso, Italy E. Tavares: None. A. Vig: None. S. Li: None. G. Billingsley: None. W. Sung: None. A. Vincent: None. B. Thiruvahindrapuram: None. E. Héon: None. E. Tavares: None. A. Vig: None. S. Li: None. G. Billingsley: None. W. Sung: None. A. Vincent: None. B. Thiruvahindrapuram: None. E. Héon: None. P02.53D Non-syndromic hearing loss is characterized by a vast
genetic heterogeneity with more than 160 loci described in
humans and 100 genes so far identified. With the aim of
targeting genes strongly associated, in Caucasians, with
NSHL or with SHL which onset is usually characterized by
isolated deafness (i.e. Pendred and Usher syndrome), we
developed an NGS targeted panel of 59 genes, to obtain an
advanced efficient diagnostic tool. The Ion Torrent PGMTM
platform combined with a customized bioinformatics pipe-
line was used for the analysis of 87 DNA samples collected
from clinically highly selected Italian subjects negative for
GJB2 mutations/GJB6 deletions. An etiological diagnosis
was established in 41 of these subjects, with an overall
diagnostic yield of 47%. An early molecular diagnosis of
Usher syndrome was achieved in 3 unrelated children car-
rying mutations in ADGRV1, CDH23 and USH2A genes. NGS allowed the identification of a homozygous as well as
a heterozygous deletion in the STRC gene; the latter dele-
tion was found in-trans with a known STRC pathogenic
variant. The deletions were confirmed by q-PCR with pri-
mers that excluded the highly homologous STRC pseudo-
gene. The novel likely causative variants identified were
located in the following genes: ACTG1, ADGRV1, CDH23,
CEACAM16, COCH, COL11A2, EYA4, GJB3, KCNQ4,
MYO7A, PCDH15, PTPRQ, SLC17A8, TMPRSS3. Our
targeted panel coupled with a solid bioinformatics pipeline
has proved a sensitive molecular tool with a high diagnostic
yield. We demonstrate the importance and efficacy of
integrating the powerful NGS technology with a compre-
hensive data processing and a careful clinical evaluation. P02.52C P02.52C
SVAF retrotransposon insertion in BBS1 gene, leading to
Bardet- Biedl Syndrome P02.52C
SVAF retrotransposon insertion in BBS1 gene, leading to
Bardet- Biedl Syndrome E. Tavares1, A. Vig1,2, S. Li1, G. Billingsley1, W. Sung3,
A. Vincent1,2,4, B. Thiruvahindrapuram3, E. Héon1,2,4 E. Tavares1, A. Vig1,2, S. Li1, G. Billingsley1, W. Sung3,
A. Vincent1,2,4, B. Thiruvahindrapuram3, E. Héon1,2,4 1Genetics and Genome Biology, Toronto, ON, Canada,
2Institute of Medical Science, University of Toronto, Toronto,
ON, Canada, 3The Centre for Applied Genomics, Toronto, ON,
Canada, 4Ophthalmology and Vision Sciences. The Hospital
for Sick Children, Toronto, ON, Canada Introduction: Active transposable elements (TE) account
for over 0.02% of the human genome. One de novo inser-
tion is believed to happen with each 10-100 live births. TEs
can cause disease by inserting themselves into coding or
regulatory portions of genes, facilitating duplications and
deletions, among other mechanisms. Due to their repetitive
nature, traditional sequencing methods often fail to detect
TEs. Here we report a Bardet-Biedl syndrome (BBS) patient
who is compound heterozygous for the most frequent
disease-causing mutation in BBS1 and an inserted SVAF
retrotransposon. Methods
and
Results:
Whole-genome
sequencing
(WGS) was performed for a female BBS patient with an
identified maternally inherited mutation M390R. No other
mutation was identified in BBS1 using dense microarray
analysis, qPCR assays, and whole exome sequencing. An
exonic TE insertion was detected in exon 13 of BBS1 in by
visual inspection of the WGS read files. Following Sanger
sequencing, we determined the insertion to be a paternally Methods
and
Results:
Whole-genome
sequencing
(WGS) was performed for a female BBS patient with an
identified maternally inherited mutation M390R. No other
mutation was identified in BBS1 using dense microarray
analysis, qPCR assays, and whole exome sequencing. An
exonic TE insertion was detected in exon 13 of BBS1 in by
visual inspection of the WGS read files. Following Sanger
sequencing, we determined the insertion to be a paternally 68 J. del Picchia Y. Kao: None. W. Fan: None. R. Chung: None. W. Lin: None. W. Tsai: None. L. Hu: None. S. Huang: None. Y. Ching: None. inherited SVAF element of ~2 kb. We have not identified
the same SVAF insertion upon screening twenty-four BBS
negative patients, 2 with disease-causing mutations in
BBS1. However, screening for other TE insertions are yet
to be completed. In vitro functional analysis of a novel RP2 alleles In vitro functional analysis of a novel RP2 alleles Y. KAO1, W. Fan2, R. Chung3, W. Lin1, W. Tsai1, L. Hu1,
S. Huang1, Y. Ching1 1Tzu Chi University, Hualien, Taiwan, 2Chang Gung Memorial
Hospital, Keelung, Taiwan, 3National Health Research
Institutes, Zhunan, Taiwan Introduction: Retinitis pigmentosa (RP), a hereditary het-
erogeneous disease with a prevalence of 1/4000, char-
acterized by the degeneration of photoreceptors leading to
progressive loss of vision in patients. We have identified a
three-generation X-linked RP family. Materials and Methods:
Eighteen X chromosome
microsatellite markers were used to locate disease locus,
and one of the Sibling-pairs within the family were used to
performed Whole-exome sequencing. Results: The disease locus was mapped between markers
DXS1068 and DXS1196. Whole-exome sequencing identi-
fied 139 possible SNPs located within the critical diseases-
causing
intervals. Considering
that
co-segregation
of
mutation and genes expression patterns, the RP2_c.102G>
A point mutation might be the responsible mutation of this
family's disease. It is a novel allele of RP2 gene with allele
frequency <1%, and no known functional impact. At protein
level the mutation appears to be no functional impact
(Lys34> Lys), however, c.102G> A located at the last
nucleotide of the splicing donor site of exon1, suggesting
that this mutation might cause intron retention at the
transcriptional level. A MiniGene assay was designed to
validate the functional consequences of this mutation. Results: The disease locus was mapped between markers
DXS1068 and DXS1196. Whole-exome sequencing identi-
fied 139 possible SNPs located within the critical diseases-
causing
intervals. Considering
that
co-segregation
of
mutation and genes expression patterns, the RP2_c.102G>
A point mutation might be the responsible mutation of this
family's disease. It is a novel allele of RP2 gene with allele
frequency <1%, and no known functional impact. At protein
level the mutation appears to be no functional impact
(Lys34> Lys), however, c.102G> A located at the last
nucleotide of the splicing donor site of exon1, suggesting
that this mutation might cause intron retention at the
transcriptional level. A MiniGene assay was designed to
validate the functional consequences of this mutation. F. Cesca: None. E. Bettella: None. R. Polli: None. E. Leonardi: None. M.C. Aspromonte: None. S. Bigoni:
None. R. Santarelli: None. A. Murgia: None. F. Cesca: None. E. Bettella: None. R. Polli: None. E. Leonardi: None. M.C. Aspromonte: None. S. Bigoni:
None. R. Santarelli: None. A. Murgia: None. P02.55B 1Department of Biomedicine and Prevention, “Tor Vergata”
University, Rome, Italy, 2Emotest Laboratory, Pozzuoli, Italy,
3UOSD Retinal Pathology PTV Foundation “Policlinico Tor
Vergata”, Rome, Italy, 4Laboratory of Genomic Medicine
UILDM, IRCCS Santa Lucia Foundation, Rome, Italy,
5Department of Chemical Pharmaceutical and Biomolecular
Technologies, Catholic University “Our Lady of Good
Counsel”, Tirana, Albania P02.55B
Stereocilin gene mutations associated with vertigo:
Expansion of the DFNB16 phenotype STRC, encoding stereocilin, is
expressed in the cochlea and in the vestibular organ where it
ensheats the kinocilium suggesting a role for the protein in
sensing balance and spatial orientation. Our findings
support such a function for stereocilin in the vestibular
organ and expand the phenotype associated with DFNB16. Results and Conclusions: Clinical investigations con-
firmed pathological vestibular responses in two siblings and
hearing loss in all three affected individuals. The cousin had
a history compatible with a vestibular disorder. DNA
analysis revealed that the siblings were homozygous for a
STRC stop variant (c.4027C>T) and their cousin was
compound heterozygous for the stop variant and a 90kb
deletion spanning the STRC gene. These results are
consistent with DFNB16. STRC, encoding stereocilin, is
expressed in the cochlea and in the vestibular organ where it
ensheats the kinocilium suggesting a role for the protein in
sensing balance and spatial orientation. Our findings
support such a function for stereocilin in the vestibular
organ and expand the phenotype associated with DFNB16. Results: The SNPs rs11671784 (miRNA-27, G/A) and
rs2910164 (miRNA-146a, C/G), advanced age, smoking
and dietary habits were significantly associated with AMD
risk (p<0.05). Genetic/epigenetic variants appeared to
contribute to AMD susceptibility for 23% while non-
genetic variants accounted for 10% of disease. Concern-
ing gene-environment interactions, we found that AMD-
associated genes may be involved in the alteration of
Bruch's membrane and angiogenesis, contributing to the
exacerbation of aging and environmental damages. Grants: Supported by the Swedish Research Council
(2015-02424). Grants: Supported by the Swedish Research Council
(2015-02424). Conclusions: Our study provides an overview of genetic/
epigenetic and non-genetic factors characterizing AMD
susceptibility in Italian population. These data may be
applied to develop a “population-specific precision medi-
cine” approach able to prevent AMD or improve patients’
quality of life. C.A. Frykholm: None. J. Klar: None. T. Tomanovic:
None. A. Ameur: None. N. Dahl: None. C.A. Frykholm: None. J. Klar: None. T. Tomanovic:
None. A. Ameur: None. N. Dahl: None. C.A. Frykholm: None. J. Klar: None. T. Tomanovic:
None. A. Ameur: None. N. Dahl: None. P02.55B
Stereocilin gene mutations associated with vertigo:
Expansion of the DFNB16 phenotype 1Department of Biomedicine and Prevention, “Tor Vergata”
University, Rome, Italy, 2Emotest Laboratory, Pozzuoli, Italy,
3UOSD Retinal Pathology PTV Foundation “Policlinico Tor
Vergata”, Rome, Italy, 4Laboratory of Genomic Medicine
UILDM, IRCCS Santa Lucia Foundation, Rome, Italy,
5Department of Chemical Pharmaceutical and Biomolecular
Technologies, Catholic University “Our Lady of Good
Counsel”, Tirana, Albania C. A. Frykholm1, J. Klar1,2, T. Tomanovic3, A. Ameur2,
N. Dahl1,2 C. A. Frykholm1, J. Klar1,2, T. Tomanovic3, A. Ameur2,
N. Dahl1,2 1Department of Immunology, Genetics and Pathology,
Uppsala, Sweden, 2Science for Life Laboratory, Biomedical
Centre, Uppsala University, Uppsala, Sweden, 3Department of
Hearing and Balance Disorders, Karolinska University
Hospital, Stockholm, Sweden Introduction:
Susceptibility
to
Age-related
Macular
Degeneration (AMD) strictly depends on genetic, epige-
netic and environmental factors. Previous results high-
lighted
prominent
differences
concerning
genetic
contributors to AMD in Italian population compared to
worldwide groups. Among genetic variables, SNPs of CFH,
ARMS2, IL-8, TIMP3, SLC16A8, RAD51B, VEGFA and
COL8A1 were significantly associated with the risk of
AMD in our cohort. Given these data, this study aimed to
evaluate the contribution of genetic, epigenetic (SNPs of
miRNA-146a, miRNA-31, miRNA-23a, miRNA-27, miRNA-
20a and miRNA-150 genes) and environment factors (age,
sex, smoking, diet) to exudative AMD. Introduction: Vestibular disorders comprise a group of
diseases with transient or permanent loss of vestibular
function characterized by vertigo and imbalance. Isolated
vestibulopathy is rare and more often associated with
migraine,
Ménière
disease,
ataxia
or
sensorineural
hearing loss. Materials and Methods: We examined two siblings and
their first cousin with childhood onset of episodic vertigo
and sensorineural hearing loss. Hearing loss and vestibular
dysfunction was investigated by audiometry, SVH, cVEMP
and oVEMP. DNA was analyzed using exome sequencing
and SNP-array. Materials and Methods: 976 exudative AMD patients
and 1000 controls were subjected to an epigenotyping
analysis through Real-Time PCR and direct sequencing. Biostatistical analysis was performed to calculate associa-
tion and estimate the contribution of genetics, epigenetics
and
environment
to
AMD
susceptibility. Gene-
environment
interactions
were
evaluated
by
bioinformatic tools. y
Results and Conclusions: Clinical investigations con-
firmed pathological vestibular responses in two siblings and
hearing loss in all three affected individuals. The cousin had
a history compatible with a vestibular disorder. DNA
analysis revealed that the siblings were homozygous for a
STRC stop variant (c.4027C>T) and their cousin was
compound heterozygous for the stop variant and a 90kb
deletion spanning the STRC gene. These results are
consistent with DFNB16. C. Strafella1,2, V. Errichiello1, V. Caputo1, F. Sangiuolo1,
F. Ricci3, G. Novelli1, A. Cusumano3, R. Cascella4,5, C. Strafella1,2, V. Errichiello1, V. Caputo1, F. Sangiuolo1,
3
1
3
4 5 In vitro functional analysis of a novel RP2 alleles Conclusion: We have identified a novel splicing-site
mutation of RP2 gene from an X-Link RP family. In vitro
functional
analysis
using
MiniGene
assay
has
been
performed to verify the functional impact of the mutation. Conclusion: We have identified a novel splicing-site
mutation of RP2 gene from an X-Link RP family. In vitro
functional
analysis
using
MiniGene
assay
has
been
performed to verify the functional impact of the mutation. Abstracts from the 51st European Society of Human Genetics Conference: Posters 69 E. Giardina1,4 Genetic, epigenetic and environmental contributors to Age-
related Macular Degeneration susceptibility F. Ricci3, G. Novelli1, A. Cusumano3, R. Cascella4,5, E. Giardina1,4 C. Strafella1,2, V. Errichiello1, V. Caputo1, F. Sangiuolo1
F. Ricci3, G. Novelli1, A. Cusumano3, R. Cascella4,5, 1University of Trieste, Trieste, Italy, 2IRCCS-Burlo Garofolo,
Trieste, Italy Trieste, Italy Introduction: Dominant phenotypes related to WFS1
mutations were known to be less severe than the Wolfram
syndrome (WS) recessive phenotype, characterized by dia-
betes mellitus, optic atrophy, diabetes insipidus and deaf-
ness. Dominant
phenotypes
included
isolated
low-
frequency sensorineural hearing loss, optic atrophy and
hearing impairment, isolated adult-onset diabetes and iso-
lated congenital nuclear cataracts. More recently, De Franco
et al. (Diabetes 2017) described a severe congenital
Wolfram-like syndrome (CWLS), characterized by con-
genital progressive hearing loss, neonatal diabetes mellitus
and cataract, due to de novo dominant mutations in WFS1. Introduction: HHL is genetically heterogeneous and at
least 40% of cases are not characterized by mutations in
known genes. Thus, we applied WES followed by “in vitro”
and “in vivo” functional studies for the discovery of
new genes. Methods: 14 Italian HHL families, negative for muta-
tions in 96 deafness-genes, were analyzed by WES (Ion
Proton™). Variants were filtered according to: a) pattern of
inheritance, b) frequency, c) pathogenicity. Functional
studies on new candidates were carried out by in vitro/
in vivo experiments. Results: WES allowed the discovery of five new HHL-
genes: PSIP1 (https://doi.org/10.1038/srep18568), TBL1Y,
SPATC1L, PLS1 and ATP2B2. As regards PSIP1, a
nonsense variant in a 3-generation family was identified;
RNAseq and immunolabeling confirmed gene expression in
mouse inner ear. For TBL1Y, a missense variant was
detected in a large Y-linked family; functional experiments
demonstrated TBL1Y expression in human cochlea and an
early degradation of the mutated protein. For SPATC1L, a
nonsense variant in a 3-generation family was identified;
protein modeling revealed a reduced structural stability
(loss of part of the C-terminus) confirmed by western blot
(presence of a shorter protein isoform). Finally, for PLS1
and ATP2B2 (known as a CDH23 modifier) Zebrafish KI
models of the identified variants (a missense and a nonsense
variant, respectively, in two dominant HHL families) are at
the final stages of validation. WES data of the remaining 9
families are now under investigation. Materials and Methods: We reported 3 unrelated cases
with this new dominant phenotype, among our French
cohort of 116 patients carrying at least 1 WFS1mutation. Results: The dominant known p.Glu809Lys mutation
was found de novo for 2 unrelated girls, respectively 12 and
3 years-old, who presented CWLS during the first year of
life, associated with psychomotor retardation, failure to
thrive, amblyopia, dysmorphic features and cerebellar
hypoplasia. Dominant WFS1 mutations in a new congenital phenotype 1University of Trieste, Trieste, Italy, 2IRCCS-Burlo Garofolo,
Trieste, Italy We also described a 20 years-old patient, who
had developed with diabetes and deafness before 1 year of
age and bilateral cataract diagnosed at 18 months, asso-
ciated with glaucoma, amblyopia, cerebellar ataxia, short
stature, hypothyroidism and hypogonadism. The clinical
presentation was very suggestive of CWLS. We found a
heterozygous
p.His860Asp
WFS1
mutation,
that
was
previously described at compound heterozygous state in a
case of WS, but with congenital deafness and occuring De
Novo, asking the question of its pathogenicity. Conclusions: Our approach, based on WES followed by
functional studies, already proved to be effective for the
discovery of new HHL-genes. Conclusions:
We
highlight
the
expanding
clinical
spectrum of WFS1-related disorders with 3 case reports of
severe congenital dominant phenotype. G. Girotto: None. A. Morgan: None. M. Brumat:
None. M. Di Stazio: None. S. Cappellani: None. E. Campana: None. U. Ambrosetti: None. M. La Bianca:
None. E. Orzan: None. P. Gasparini: None. A. Chaussenot: None. C. Rouzier: None. C. Vincent-
Delorme: None. M. Bonnet-Dupeyron: None. G. Auge:
None. V. Paquis-Flucklinger: None. A. Chaussenot: None. C. Rouzier: None. C. Vincent-
Delorme: None. M. Bonnet-Dupeyron: None. G. Auge:
None. V. Paquis-Flucklinger: None. P02.58A 1Department of Medical Genetics, National Centre for
Mitochondrial Diseases, Archet 2 Hospital, Nice, France,
2Nice Sophia-Antipolis University, CNRS UMR 7284, INSERM
U1081, Institute for Research on Cancer and Aging, Nice,
France, 3Jeanne de Flandre hospital, Lille, France,
4Laboratoire de Biologie Médicale, Centre hospitalier de
Valence, Valence, France G. Girotto1,2, A. Morgan1, M. Brumat1, M. Di Stazio1,
S. Cappellani2, E. Campana1, U. Ambrosetti1, M. La Bianca2,
E. Orzan2, P. Gasparini1,2 1University of Trieste, Trieste, Italy, 2IRCCS-Burlo Garofolo,
Trieste, Italy Multidisciplinary team work to get differential diagnosis of
a reverse phenotype in retinal disease P02.56C Genetic, epigenetic and environmental contributors to Age-
related Macular Degeneration susceptibility C. Strafella: None. V. Errichiello: None. V. Caputo:
None. F. Sangiuolo: None. F. Ricci: None. G. Novelli:
None. A. Cusumano: None. R. Cascella: None. E. Giardina: None. E. Giardina1,4 J. del Picchia 70 P02.58A
Discovery of new Hereditary Hearing Loss (HHL) genes by
Whole Exome Sequencing (WES) and in vitro/in vivo
functional studies: five years of experience
G. Girotto1,2, A. Morgan1, M. Brumat1, M. Di Stazio1,
S. Cappellani2, E. Campana1, U. Ambrosetti1, M. La Bianca2,
E. Orzan2, P. Gasparini1,2 A. Chaussenot1,2, C. Rouzier1,2, C. Vincent-Delorme3,
M. Bonnet-Dupeyron4, G. Auge1, V. Paquis-Flucklinger1,2
1Department of Medical Genetics, National Centre for
Mitochondrial Diseases, Archet 2 Hospital, Nice, France,
2Nice Sophia-Antipolis University, CNRS UMR 7284, INSERM
U1081, Institute for Research on Cancer and Aging, Nice,
France, 3Jeanne de Flandre hospital, Lille, France,
4Laboratoire de Biologie Médicale, Centre hospitalier de
Valence, Valence, France A. Chaussenot1,2, C. Rouzier1,2, C. Vincent-Delorme3,
M. Bonnet-Dupeyron4, G. Auge1, V. Paquis-Flucklinger1,2
1Department of Medical Genetics, National Centre for
Mitochondrial Diseases, Archet 2 Hospital, Nice, France,
2Nice Sophia-Antipolis University, CNRS UMR 7284, INSERM
U1081, Institute for Research on Cancer and Aging, Nice,
France, 3Jeanne de Flandre hospital, Lille, France,
4Laboratoire de Biologie Médicale, Centre hospitalier de
Valence, Valence, France P02.59B Multidisciplinary team work to get differential diagnosis of
a reverse phenotype in retinal disease Dominant WFS1 mutations in a new congenital phenotype Dominant WFS1 mutations in a new congenital phenotype Abstracts from the 51st European Society of Human Genetics Conference: Posters 71 L. Candita1, B. Boschi1, E. Contini1, I. Passerini1, A. Sodi2, A. L. Nutini1, O. Colavecchio1, T. Morgani1, E. Ronconi1, E. Pelo1
1SOD Diagnostica genetica, AOU Careggi, Firenze, Italy,
2Dipartimento di Chirurgia e Medicina Traslazionale, Clinica
Oculistica, AOU Careggi, Firenze, Italy L. Candita1, B. Boschi1, E. Contini1, I. Passerini1, A. Sodi2, A. L. Nutini1, O. Colavecchio1, T. Morgani1, E. Ronconi1, E. Pelo1 1Dept. of Clinical Genetics, Leiden University Medical Center,
Leiden, Netherlands, 2Dept. Nephrology, University Medical
Center Groningen, Groningen, Netherlands, 3Dept. Internal
Medicine, Ziekenhuisgroep Twente, Almelo, Netherlands,
4Dept. Gastroenterology and Hepatology, Radboud University
Medical Center, Nijmegen, Netherlands, 5Dept. Nephrology,
Leiden University Medical Center, Leiden, Netherlands, 6Dept. Nephrology, Radboud University Medical Center, Nijmegen,
Netherlands, 7Dept. Nephrology, Erasmus Medical Center
Rotterdam, Rotterdam, Netherlands, 8Dept. of Human
Genetics, Leiden University Medical Center, Leiden,
Netherlands 1SOD Diagnostica genetica, AOU Careggi, Firenze, Italy,
2Dipartimento di Chirurgia e Medicina Traslazionale, Clinica
Oculistica, AOU Careggi, Firenze, Italy We report a case of a 23 year old woman referred to our
center with a diagnosis of Leber congenital amaurosis
(LCA). An accurate clinical re-evaluation detected dystro-
phy of retinal pigment epithelium, Franceschetti sign, exo-
tropia and nistagmus. Clinical signs and symptoms were
therefore not suggestive for LCA. A genetic counseling was
then offered to this patient. We performed NGS analysis of
137 retinal dystrophy associated genes. Mutational and
CNV
analysis
were
performed. Pathogenic
variants
c.1666del in heterozygosis in CEP290 gene and c.484G>A
in heterozygosis in CRB1 gene and probably pathogenic
variant c.1054C>T in heterozygosis in CACNA2D4 gene
were detected. Variant c.1666del was reported in associa-
tion with LCA (Hui Wang et al., 2015) in compound het-
erozygosis with a pathogenic variant in CEP290 gene;
variant c.484G>A in CRB1 gene was reported in one family
with pigmented paravenous chorioretinal atrophy (PPCRA)
(McKay et al., 2005) dominantly inherited. The cose-
gregation analysis showed that proband’s father carried all
the three familial variants. No defined clinical symptoms of
impairment visual loss were reported. Clinical and mole-
cular findings are not always clear: therefore we recommend
a multidisciplinary patient management in rare pathology. P02.59B NGS data should lead to clinical revaluation, early diag-
nosis in mild symptomatic relatives and sometimes lead to
achieve reverse phenotype. In conclusion, we requested
clinical revaluation of the proband and her parents by our
physicians, to gain also a clinical PPCRA diagnosis, which
should justify a CRB1 dominant disease with variable
expression or incomplete penetrance. Autosomal dominant polycystic kidney disease is an
inherited disease characterized by progressive cyst forma-
tion in both kidneys and renal function loss, which ulti-
mately leads to end-stage renal failure. A well-defined
clinical cohort of 339 ADPKD patients that gave informed
consent and were screened for participation in a clinical trial
was analysed for the presence of PKD1 and PKD2 muta-
tions. The molecular analysis is relevant for the trial since
disease progression is partly determined by mutation type. Mutation analysis was performed by Sanger sequencing and
MLPA which resulted in a mutation detection ratio of 94%. Mutation negative patients were analysed for mutations in
GANAB, HNF1B or PKHD1 but no mutation was detected. Four mutation negative patients were sequenced with a
NGS approach (Illumina HiSeq4000 platform, targets cap-
tured using custom-designed gene panel specific Agilent
SureSelectXTClearseq enrichment kit). Data analysis was
performed using an in house developed pipeline (stringent
post-sequencing annotation pipeline based on BWA, GATK
and VEP and various filtering steps in LOVD+). In 3 out of
4 patients a pathogenic mutation was detected in PKD1 or
PKD2. Mutations were missed by Sanger sequencing
because of allelic drop-out, or a gap in the overlapping
Sanger sequencing fragments. The remaining 16 patients
are currently being analyzed with the same NGS approach. In conclusion, mutation analysis in a well-defined ADPKD
cohort has an extremely high mutation detection rate (94%). The NGS approach is a very useful addition to standard
mutation analysis techniques especially in very poly-
morphic regions of the genome like the PKD1 genomic
region. L. Candita: None. B. Boschi: None. E. Contini: None. I. Passerini: None. A. Sodi: None. A.L. Nutini: None. O. Colavecchio: None. T. Morgani: None. E. Ronconi:
None. E. Pelo: None. P03 Internal organs & endocrinology (lung, kidney,
liver, gastrointestinal)
P03.01D
High diagnostic yield in well-defined cohort of ADPKD
patients by conventional sequencing and NGS
M. Losekoot1, A. Tholens1, M. Phylipsen1, E. Meijer2, F. W. Visser2,3, J. P. H. Drenth4, J. W. de Fijter5, J. F. Wetzels6,
R. Zietse7, R. T. Gansevoort2, D. J. M. Peters8 P03 Internal organs & endocrinology (lung, kidney,
liver, gastrointestinal) M. Losekoot: None. A. Tholens: None. M. Phylipsen:
None. E. Meijer: None. F.W. Visser: None. J.P.H. Drenth: None. J.W. de Fijter: None. J.F. Wetzels: None. R. Zietse:
None. R.T. Gansevoort:
None. D.J.M. Peters: None. P03.02
ANGS allele drop out in a family with NPHP4 related
nephronophthisis M. Losekoot1, A. Tholens1, M. Phylipsen1, E. Meijer2, F. W.
Visser2,3, J. P. H. Drenth4, J. W. de Fijter5, J. F. Wetzels6,
R. Zietse7, R. T. Gansevoort2, D. J. M. Peters8 High diagnostic yield in well-defined cohort of ADPKD
patients by conventional sequencing and NGS High diagnostic yield in well-defined cohort of ADPKD
patients by conventional sequencing and NGS M. Losekoot1, A. Tholens1, M. Phylipsen1, E. Meijer2, F. W. Visser2,3, J. P. H. Drenth4, J. W. de Fijter5, J. F. Wetzels6,
R. Zietse7, R. T. Gansevoort2, D. J. M. Peters8 P03.02
ANGS allele drop out in a family with NPHP4 related
nephronophthisis A. V. Kirov1, L. Grozdanova2, T. Todorov3,1, A. Todorova1,3 So far, some founder mutations have been identified with
many of them being region- or ethnicity-specific. COL4A5
c.1871G>A, p.(Gly624Asp) pathogenic variant is known to
be prevalent in AS patients from Slovenia (6 out of 17),
Hungary (3 out of 10) and Greece and lead to late age at
onset of end stage renal disease. In contrast to these
populations, the mutation is considered to be rare in the US,
Northern and Western Europe or Japan. Here we show that
the mutation was detected in 7 AS patients out of 49 with
genetically confirmed diagnosis from Russia. 1IMDL Genome Centre Bulgaria, Sofia, Bulgaria, 2Department
of Medical Genetics, Medical University Hospital “St. George”, Plovdiv, Bulgaria, 3GMDL Genica, Sofia, Bulgaria Here we report a family with one child died soon after birth
due to bilateral renal agenesis and one terminated early
pregnancy due to the same reason. The family was referred
for genetic counseling and molecular-genetic testing to our
lab. DNA from the affected children was not available so
we perform whole exome sequencing of both parents. We
analyzed the data searching for heterozygous genetic variant
in the known nephronophthisis and polycystic kidney dis-
ease associated genes. However, only one heterozygous
variant in exon 23 of NPHP4 gene was detected in the
mother: NM_015102.4: c.3292G>A (p.Ala1098Thr). Some
authors reported few patients with only one NPHP4 muta-
tion so we could not exclude the possibility of incomplete
penetrance of the genetic variant. We confirm the genetic
finding with standard Sanger sequencing of both parents. Surprisingly, the father was also a heterozygous carrier of
the same variant. We double check the finding and the NGS
coverage (above 100x in both patients) and again it was
missing from the NGS data of the father. It is well known
that both cytosine methylation and DNA structures known
as G-quadruplexes (G4s) in some region contributed to
allelic dropout (ADO) in PCR based reactions and NGS
library preparation. However the genetic region in proxi-
mity to our genetic variant is not GC-rich and as far as we
know this region is also not methylated. Obviously ADO in
NGS era is something that we still need to keep in mind and
continue to investigate in our routine laboratory work. A. V. Kirov1, L. Grozdanova2, T. Todorov3,1, A. Todorova1,3 Materials and Methods: The population sample con-
tained 76 apparently unrelated pediatric patients (1 to 17
years old) from diverse range of regions in the European
part of Russian Federation with confirmed or suspected
diagnosis of AS according to current guidelines. NGS
sequencing was performed using Ion PGM (AmpliSeq
panel). Results: We confirmed the diagnosis in 49 patients
including 43 with X-linked AS (harboring COL4A5 gene
mutations) and 1 with digenic COL4A5 and COL4A3
inheritance. Seven of them were bearing the COL4A5
c.1871G>A, p.(Gly624Asp) mutation which corresponds to
14% frequency in the sample. We demonstrate that the
mutation is characterized by late age at onset of hematuria
(>48 months) and absence of proteinuria in childhood. The
research was supported by RFBR grant 18-34-00708 to L.I. S. and Minzdrav government grant №115022070016. L.I. Shagam: None. V.S. Sukhorukov: None. T.A. Kuznetsova:
None. M.E. Aksenova:
None. V.V. Dlin: None. L.I. Shagam: None. V.S. Sukhorukov: None. T.A. Kuznetsova:
None. M.E. Aksenova:
None. V.V. Dlin: None. P03.03B P. Halat-Wolska1, E. Ciara1, J. Antoniewicz2, K. Gadomska-
Prokop2, L. Obrycki2, M. Rydzanicz3, J. Kosińska3,
D. Siestrzykowska1, B. Chałupczyńska1, P. Stawiński3,4,
D. Jurkiewicz1, D. Piekutowska-Abramczuk1, M. Pelc1,
P. Kowalski1, D. Wicher1, A. Cieślikowska1, P. Iwanowski1,
M. Gierla2, A. Łuba2, A. Niemirska2, D. Runowski2,
W. Jarmużek2, J. Lesiak2, M. Gorzkowska-Paczwa2,
A. Borowski2, J. Latoszyńska2, M. Podymniak-Grzeszykowska2,
R. Grenda2, K. Chrzanowska1, R. Płoski3, M. Krajewska-
Walasek1, M. Litwin2 P. Halat-Wolska1, E. Ciara1, J. Antoniewicz2, K. Gadomska-
Prokop2, L. Obrycki2, M. Rydzanicz3, J. Kosińska3,
D. Siestrzykowska1, B. Chałupczyńska1, P. Stawiński3,4,
D. Jurkiewicz1, D. Piekutowska-Abramczuk1, M. Pelc1,
P. Kowalski1, D. Wicher1, A. Cieślikowska1, P. Iwanowski1,
M. Gierla2, A. Łuba2, A. Niemirska2, D. Runowski2,
W. Jarmużek2, J. Lesiak2, M. Gorzkowska-Paczwa2,
A. Borowski2, J. Latoszyńska2, M. Podymniak-Grzeszykowska2,
R. Grenda2, K. Chrzanowska1, R. Płoski3, M. Krajewska-
Walasek1, M. Litwin2 P. Halat-Wolska1, E. Ciara1, J. Antoniewicz2, K. Gadomska-
Prokop2, L. Obrycki2, M. Rydzanicz3, J. Kosińska3,
D. Siestrzykowska1, B. Chałupczyńska1, P. Stawiński3,4,
D. Jurkiewicz1, D. Piekutowska-Abramczuk1, M. Pelc1,
P. Kowalski1, D. Wicher1, A. Cieślikowska1, P. Iwanowski1,
M. Gierla2, A. Łuba2, A. Niemirska2, D. Runowski2,
W. Jarmużek2, J. Lesiak2, M. Gorzkowska-Paczwa2,
A. Borowski2, J. Latoszyńska2, M. Podymniak-Grzeszykowska2,
R. Grenda2, K. Chrzanowska1, R. Płoski3, M. Krajewska-
Walasek1, M. Litwin2 COL4A5 G624D: abundance of the Alport syndrome
mutation in Russia along with Greek, Hungarian and
Slovenian populations suggests it is a frequent mutation in
Eastern Europe with mild phenotype L. I. Shagam1, V. S. Sukhorukov1,2, T. A. Kuznetsova1, M. E. Aksenova1, V. V. Dlin1 P03.04C Identification of the genetic background of Polish patients
with suspected Alport Syndrome using next-generation
sequencing Identification of the genetic background of Polish patients
with suspected Alport Syndrome using next-generation
sequencing A.V. Kirov: None. L. Grozdanova: None. T. Todorov:
None. A. Todorova: None. L. I. Shagam1, V. S. Sukhorukov1,2, T. A. Kuznetsova1, M. E.
Aksenova1, V. V. Dlin1 1Veltishev Pediatric Clinical Research Institute of Pirogov
Russian National Research Medical University, Moscow,
Russian Federation, 2Sechenov University, Moscow, Russian
Federation 1Department of Medical Genetics, The Children's Memorial
Health Institute, Warsaw, Poland, 2Department of Nephrology,
The Children's Memorial Health Institute, Warsaw, Poland,
3Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland, 4Department of Genetics, Institute of
Physiology and Pathology of Hearing, Warsaw, Poland Introduction: Alport syndrome (AS) is a familial hematuria
caused by mutations in COL4A3, COL4A4 and/or COL4A5
genes which lead to defects in glomerular filtration barrier. P03.02 J. del Picchia 72 A. V. Kirov1, L. Grozdanova2, T. Todorov3,1, A. Todorova1,3 73 Abstracts from the 51st European Society of Human Genetics Conference: Posters B. Guillen-Guio1, J. M. Lorenzo-Salazar2, A. Corrales1,3,
E. Espinosa4, A. Muriel5, L. Lorente6, M. M. Martín7,
C. Rodríguez-Gallego8, J. Solé-Violán9, A. Ambrós10,
D. Carriedo11, J. Blanco5, J. M. Añón12, J. M. Añón12,
J. Villar3,13, C. Flores1,2,3, the Genetics of Sepsis (GEN-SEP)
Network B. Guillen-Guio1, J. M. Lorenzo-Salazar2, A. Corrales1,3,
E. Espinosa4, A. Muriel5, L. Lorente6, M. M. Martín7,
C. Rodríguez-Gallego8, J. Solé-Violán9, A. Ambrós10,
D. Carriedo11, J. Blanco5, J. M. Añón12, J. M. Añón12,
J. Villar3,13, C. Flores1,2,3, the Genetics of Sepsis (GEN-SEP)
Network Introduction: Alport syndrome (AS) is a clinically and
genetically heterogeneous nephropathy associated with
sensorineural hearing loss and ocular anomalies, with thin
basement membrane nephropathy being at the mildest end
of the disorder spectrum. In most AS cases pathogenic
variants can be found in COL4A5 (XL~80%) whereas
COL4A3 and COL4A4 are associated with autosomal
recessive (AR~15%) and dominant (AD~5%) forms. Introduction: Alport syndrome (AS) is a clinically and
genetically heterogeneous nephropathy associated with
sensorineural hearing loss and ocular anomalies, with thin
basement membrane nephropathy being at the mildest end
of the disorder spectrum. In most AS cases pathogenic
variants can be found in COL4A5 (XL~80%) whereas
COL4A3 and COL4A4 are associated with autosomal
recessive (AR~15%) and dominant (AD~5%) forms. 1Research Unit, Hospital Universitario N.S. de Candelaria,
Universidad de La Laguna, Santa Cruz de Tenerife, Spain,
2Genomics Division, Instituto Tecnológico y de Energías
Renovables (ITER), Santa Cruz de Tenerife, Spain, 3CIBER de
Enfermedades Respiratorias, Instituto de Salud Carlos III,
Madrid, Spain, 4Department of Anesthesiology, Hospital
Universitario N.S. de Candelaria, Santa Cruz de Tenerife,
Spain, 5Intensive Care Unit, Hospital Universitario Rio
Hortega, Valladolid, Spain, 6Intensive Care Unit, Hospital
Universitario de Canarias, La Laguna, Santa Cruz de Tenerife,
Spain, 7Intensive Care Unit, Hospital Universitario N.S. de
Candelaria, Santa Cruz de Tenerife, Spain, 8Department of
Immunology, Hospital Universitario Dr Negrín, Las Palmas de
Gran Canaria, Spain, 9Intensive Care Unit, Hospital
Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain,
10Intensive Care Unit, Hospital General de Ciudad Real,
Ciudad Real, Spain, 11Intensive Care Unit, Complejo
Hospitalario Universitario de León, León, Spain, 12Intensive
Care Unit, Hospital Universitario La Paz, Madrid, Spain,
13Research Unit, Hospital Universitario Dr. Negrin, Las
Palmas de Gran Canaria, Spain Materials and Methods: In our study we examined a
group of 36 unrelated Polish patients with suspected AS. To
identify the molecular basis of the disease, we conducted
next-generation sequencing (NGS) using Illumina TruSight
One
Sequencing
Panel,
which
allowed
simultaneous
analysis of all genes encoding the subunits of type IV
collagen. Results: A genetic etiology was established in 32
patients. Overall, 31 pathogenic or likely pathogenic
variants
in
COL4A3,
COL4A4
(both
AR~12%
and
AD~12%) and COL4A5 (XL~75%) were identified, includ-
ing 10 known mutations and 21 novel variants expanding
the list of COL4A3-5 alterations. Changes were randomly
distributed across all coding regions of COL4A3-5, with no
indices of a genetic “hot spot”, however we revealed
variants repeated four times (COL4A5: c.1871G>A) or
twice
(COL4A3:
c.2083G>A;
COL4A5:
c.2414G>T,
c.3399delA) in our study group. Conclusions: Diagnosis of patients with suspected AS
was confirmed at the molecular level in ~89% cases. Our
findings correspond to the data reported worldwide. NGS is
efficient, reduces screening time and cost, and provides an
expanding diagnostic tool to investigate the genetic back-
grounds of AS, which will improve personalized diagnos-
tics, genetic and prognostic counseling for the patients and
their relatives. Introduction: The acute respiratory distress syndrome
(ARDS) is a complex syndrome of severe acute hypoxemic
respiratory failure. Here, we describe the results of the
discovery stage for the first genome-wide association study
(GWAS) of sepsis-induced ARDS. Introduction: The acute respiratory distress syndrome
(ARDS) is a complex syndrome of severe acute hypoxemic
respiratory failure. Here, we describe the results of the
discovery stage for the first genome-wide association study
(GWAS) of sepsis-induced ARDS. P. Halat-Wolska: None. E. Ciara: None. J. Antonie-
wicz: None. K. Gadomska-Prokop: None. L. Obrycki:
None. M. Rydzanicz: None. J. Kosińska: None. D. Siestrzykowska: None. B. Chałupczyńska: None. P. Stawiński: None. D. Jurkiewicz: None. D. Piekutowska-
Abramczuk: None. M. Pelc: None. P. Kowalski: None. D. Wicher: None. A. Cieślikowska: None. P. Iwanowski:
None. M. Gierla: None. A. Łuba: None. A. Niemirska:
None. D. Runowski: None. W. Jarmużek: None. J. Lesiak: None. M. Gorzkowska-Paczwa: None. A. Bor-
owski: None. J. Latoszyńska: None. M. Podymniak-
Grzeszykowska: None. R. Grenda: None. K. Chrza-
nowska: None. R. Płoski: None. M. Krajewska-Walasek:
None. M. Litwin: None. Materials and Methods: We performed a GWAS on
672 sepsis patients admitted into intensive care units. After
quality
control
steps
and
variant
imputation
in
the
Haplotype Reference Consortium data, 7.8 million variants
with a minor allele frequency ≥1% were analyzed. Logistic
regressions were carried out based on the Wald test,
considering sex, age and the APACHE II score as
covariates. GCTA-COJO was used to identify the indepen-
dent loci. Gene-set enrichment analysis was assessed with
EnrichR. P. Halat-Wolska: None. E. Ciara: None. J. Antonie-
wicz: None. K. Gadomska-Prokop: None. L. Obrycki:
None. M. Rydzanicz: None. J. Kosińska: None. D. Siestrzykowska: None. B. Chałupczyńska: None. P. Stawiński: None. D. Jurkiewicz: None. D. Piekutowska-
Abramczuk: None. M. Pelc: None. P. Kowalski: None. D. Wicher: None. A. Cieślikowska: None. P. Iwanowski:
None. M. Gierla: None. A. Łuba: None. A. Niemirska:
None. D. Runowski: None. W. Jarmużek: None. J. Lesiak: None. M. Gorzkowska-Paczwa: None. A. Bor-
owski: None. J. Latoszyńska: None. M. Podymniak-
Grzeszykowska: None. R. Grenda: None. K. Chrza-
nowska: None. R. Płoski: None. M. Krajewska-Walasek:
None. M. Litwin: None. Results: A suggestive association (p < 5.0e-5) with ARDS
was found for 53 independent loci (lowest p = 2.6e-7). Top
hits were significantly enriched in genes linked to VEGF
ligand-receptor interactions (p = 3.5e-4), and located near
genes previously associated with other respiratory traits
including lung function, chronic obstructive pulmonary
disease, asthma, and idiopathic pulmonary fibrosis. P03.05D Genome-wide association study of sepsis-induced acute
respiratory distress syndrome: discovery stage Conclusions: We have identified putative novel genetic
variants associated with sepsis-induced ARDS. Replication 74 J. del Picchia that increased autophagy may be strictly associated to renal
cystogenesis in OFD type I syndrome. analyses are currently underway. These results will advance
our understanding of ARDS pathogenesis and will likely
allow identifying new therapeutic targets. Conclusions: Altogether our data suggest that autophagy
alterations may be a common pathogenic mechanism in CK. Dissection of the molecular mechanisms underlying cyst
formation in OFD type I will allow elucidating the role of
autophagy in CK and could disclose new therapeutic
avenues
for
renal
cystic
disease. Supported
by
the
Polycystic Kidney Disease Foundation and the Telethon
Foundation Funding: ISCIII (PI11/00623, PI16/00049) and co-
financed by the European Regional Development Funds,
“A way of making Europe” from the European Union;
Agreement OA17/008 with ITER to strengthen scientific
and technological education, training, research, develop-
ment and innovation in Genomics, Personalized Medicine
and
Biotechnology;
Fellowship
from
the
ACIISI
(TESIS2015010057) co-funded by European Social Fund
to BGG. M. Morleo: None. U. Formisano: None. S. Brillante:
None. D. Iaconis: None. S. Maione: None. E. Damiano:
None. R. Tammaro: None. C. Settembre: None. B. Franco: None. B. Guillen-Guio: None. J.M. Lorenzo-Salazar: None. B. Guillen-Guio: None. J.M. Lorenzo-Salazar: None. A. Corrales: None. E. Espinosa: None. A. Muriel: None. L. Lorente: None. M.M. Martín: None. C. Rodríguez-
Gallego: None. J. Solé-Violán: None. A. Ambrós: None. D. Carriedo: None. J. Blanco: None. J.M. Añón: None. J. M. Añón: None. J. Villar: None. C. Flores: None. P03.07B
Genotype/phenotype correlations in ADPKD M. Audrezet1, E. Cornec-Legall1, Y. Le Meur1, C. FEREC1,2 M. Audrezet1, E. Cornec-Legall1, Y. Le Meur1, C. FEREC1,2 M. Audrezet: None. E. Cornec-Legall: None. Y. Le
Meur: None. C. Ferec: None. P03.06A
Autophagy inhibition ameliorates renal cystic disease in
OFD type I syndrome 1University of Brest, BREST, France, 2INSERM UMR1078,
Brest, France 1University of Brest, BREST, France, 2INSERM UMR1078,
Brest, France M. Morleo1,2, U. Formisano1, S. Brillante1, D. Iaconis1,
S. Maione1, E. Damiano1, R. Tammaro1, C. Settembre1,
B. Franco1,2 Autosomal dominant polycystic kidney disease (ADPKD) is
one of the most common gene kidney disorder with a
worldwide prevalence of 1/400 to 1/1000. ADPKD is a
genetically heterogeneous disease with two main genes
PKD1 and PKD2 responsible of the disorder .Recently a
new gene,GANAB has been reported to be involved in
polycystic diseases(Porath et al., AJHG 2016) . We have
completely analyzed the coding sequence of a large cohort
of more than 4500 ADPKD patients (The genkyst cohort
from the western part of France (2500 patients) and patients
from different centers of France,(2000 patients).The analy-
sis were performed first by Sanger and more recently by
New Generation Sequencing (NGS). We have found an
overall detection rate of 93% in our cohort,75% being
mutated in PKD1 and 18% in PKD2 .We have performed a
genotype/phenotype correlation (Audrezet et al., Hum
Mut,2012 ;Cornec-Le Gall et al., JASN,2013) and showed
that the type of mutation in PKD1, truncating mutations was
55.6 years versus non truncating mutations were associated
with a 12 years delay in ESRD. We have only found 6
families with a mutation in the GANAB gene .To investi-
gate the molecular basis of prenatal form of ADPKD we
screened 42 patients with early ADPKD and we showed
that additional PKD variation inherited from the unaffected
parent were identified in 37.2% of patients ..These results
suggest that hypomorphic mutations could explain at least a
part
of the
clinical
variability
observed in
ADPKD
(Audrezet et al., JASN,2015) M. Morleo1,2, U. Formisano1, S. Brillante1, D. Iaconis1,
S. Maione1, E. Damiano1, R. Tammaro1, C. Settembre1,
B. Franco1,2 L. Rocha1,2, S. Fernandes1, J. P. Oliveira1,2 1Genetics, Department of Pathology, Faculty of Medicine &
Instituto de Investigação e Inovação em Saúde, Universidade
do Porto, Porto, Portugal, 2Medical Genetics – São João
Hospital Centre, Porto, Portugal Introduction: Autosomal dominant polycystic kidney dis-
ease (ADPKD) is a multi-organic hereditary disorder,
responsible for 7-10% incident cases of end-stage renal
failure (ESRF). In most populations, pathogenic variants in
PKD1 (OMIM#601313) and in PKD2 (OMIM#173910)
account respectively for 85% and 15% of ADPKD cases. Although both forms of ADPKD have similar pathogenesis,
the onset of clinical manifestations and progression to ESRF
occurs at young ages in patients with PKD1 mutations, who
reach ESRF on average 10-15 years earlier than those with
PKD2 mutations. The allelic heterogeneity is high for both
genes, making NGS an appropriate first-tier approach to
genetic diagnosis. Introduction: Autosomal dominant polycystic kidney dis-
ease (ADPKD) is a multi-organic hereditary disorder,
responsible for 7-10% incident cases of end-stage renal
failure (ESRF). In most populations, pathogenic variants in
PKD1 (OMIM#601313) and in PKD2 (OMIM#173910)
account respectively for 85% and 15% of ADPKD cases. Although both forms of ADPKD have similar pathogenesis,
the onset of clinical manifestations and progression to ESRF
occurs at young ages in patients with PKD1 mutations, who
reach ESRF on average 10-15 years earlier than those with
PKD2 mutations. The allelic heterogeneity is high for both
genes, making NGS an appropriate first-tier approach to
genetic diagnosis. Introduction: Autosomal dominant polycystic kidney dis-
ease (ADPKD) is a multi-organic hereditary disorder,
responsible for 7-10% incident cases of end-stage renal
failure (ESRF). In most populations, pathogenic variants in
PKD1 (OMIM#601313) and in PKD2 (OMIM#173910)
account respectively for 85% and 15% of ADPKD cases. Materials and Methods: Six patients clinically diag-
nosed with BSCL from four families were enrolled. Exons
and exon-intron boundaries of AGPAT2 and CAVIN1 were
studied by Sanger sequencing method. Results: Homozygous two known mutations (c.685G>T;
c.514G>A) and a novel mutation (c.316+1G>T) were
detected in AGPAT2 in five patients from three families. They were admitted between 6 months and 11 years of age. The patients had reduced subcutaneous fat (5/5), muscular
hypertrophy (5/5), enlarged hands and feet (3/5), acanthosis
nigricans (2/5), hepatomegaly (3/5), hypertriglyceridemia
(5/5), hyperinsulinemia (3/5) and low serum leptin level (1/
5). During their follow-up period 4 to 7 years, hypertrophic
cardiomyopathy and hyperinsulinemia have developed in
only one patient. 1Telethon Institute of Genetics and Medicine-TIGEM, Pozzuoli,
Italy, 2Federico II University, Naples, Italy 1Telethon Institute of Genetics and Medicine-TIGEM, Pozzuoli,
Italy, 2Federico II University, Naples, Italy Introduction: Oral-facial-digital type I syndrome is ascri-
bed to cilia dysfunction and characterized by abnormalities
of face, oral cavity and digits and by renal cystic disease
(CK). The causative gene codifies for OFD1, a centrosomal/
basal body protein, necessary for primary cilia formation. Interestingly, recent data established a link between CK,
primary
cilia,
cilioproteins
and
autophagy,
a
self-
degradative process. Results:
Mass
spectrometry
analysis
identified
autophagy-related proteins among putative OFD1 interac-
tors. In addition, we demonstrated that OFD1-depleted renal
cells show increased autophagic flux and that OFD1 exerts a
direct functional role on autophagosome biogenesis in an
mTOR and cilia-independent manner. To test the physio-
logical relevance of these findings we moved to in vivo
studies and demonstrated enhanced autophagic flux both at
precystic and cystic stages in two Ofd1 mutants (Ofd1-IND
and Ofd1;creKsp). Moreover, we achieved renal specific
inactivation in kidneys of both Ofd1 and Atg7, a key player
of autophagy. Histological analysis showed a significant
reduction in the number and size of cysts in creKsp;Ofd1y/fl;
Atg7fl/flmutants compared to creKsp;Ofd1y/fl;Atg7+/+ mice,
as revealed by quantification of the cystic index, suggesting M. Audrezet: None. E. Cornec-Legall: None. Y. Le
Meur: None. C. Ferec: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 75 L. Rocha1,2, S. Fernandes1, J. P. Oliveira1,2 Last patient, a 2-year-old boy with pyloric
stenosis operation history, had reduced subcutaneous fat,
muscular hypertrophy, hypertrophic cardiomyopathy, and
elevated CK value. Homozygous mutation in CAVIN1
(c.259C>T) was detected. Hypertriglyceridemia and hyper-
insulinemia were observed at 5 years of age. Results: Homozygous two known mutations (c.685G>T;
c.514G>A) and a novel mutation (c.316+1G>T) were
detected in AGPAT2 in five patients from three families. They were admitted between 6 months and 11 years of age. Methods: DNA was extracted from 40 unrelated ADPKD
patients. PKD1 and PKD2 genes were analyzed by NGS
sequencing (Ion Torrent PGM). Heterozygosity for clini-
cally
relevant
variants
was
confirmed
by
Sanger
sequencing. Results: Ten of the 40 patients (25%) were heterozygous
for a single-nucleotide c.181C>T transition in PKD2 exon 1
- predicting the nonsense variant p.(Gln61Ter) - which co-
segregated with a relatively mild form of ADPKD in the
affected families, most of which are from a circumscribed
region in the river Douro valley. Conclusions: As distinct from other BSCL types, pyloric
stenosis and high CK values in an infant should suggest
BSCL type-4. Interestingly, a previously reported patient
with the same mutation of our BSCL type-4 patient had
similar features including achalasia/pyloric stenosis, devel-
opmental hip dysplasia and high CK levels. Discussion: The PKD2 p.(Gln61Ter) variant is reported
in the Mayo Clinic ADPKD Mutation Database and is not
recognized as polymorphic human variation. Its high
prevalence in a limited region of the north of Portugal
suggests a “founder” effect. Accordingly, we have changed
our genotyping approach to mildly affected ADPKD
families originating from the critical geographic region,
and screen first for that PKD2 variant. N. Gunes: None. T. Erkan: None. T. Kutlu: None. H. Onay: None. T. Atik: None. B. Tüysüz: None. N. Gunes: None. T. Erkan: None. T. Kutlu: None. H. Onay: None. T. Atik: None. B. Tüysüz: None. N. Gunes1, T. Erkan1, T. Kutlu1, H. Onay2, T. Atik2, B. Tüysüz1 P03.10A A chronic obstructive pulmonary disease and interaction
between single nucleotide polymorphisms of FAM13A gene
and smoking A chronic obstructive pulmonary disease and interaction
between single nucleotide polymorphisms of FAM13A gene
and smoking L. Rocha:
None. S. Fernandes:
None. J.P. Oliveira: None. L. Rocha:
None. S. Fernandes:
None. J.P. Oliveira: None. P03.08C 1İstanbul University, Cerrahpasa Medical Faculty, İstanbul,
Turkey, 2Ege University, Faculty of Medicine, İzmir, Turkey 1İstanbul University, Cerrahpasa Medical Faculty, İstanbul,
Turkey, 2Ege University, Faculty of Medicine, İzmir, Turkey Introduction:
Berardinelli-Seip
congenital
generalized
lipodystrophy (BSCL) is a rare disorder due to homozygous
mutations of AGPAT2 (type-1), BSCL2 (type-2), CAV1
(type-3) and CAVIN1 (type-4) genes and characterized by
reduced adipose tissue, muscular hypertrophy, hepatome-
galy, insulin resistance and hypertriglyceridemia. Here, we
report the longitudinal observation and comparison of the
patients with BSCL type-1 and 4. P03.09D Clinical and molecular aspects of the patients with
Berardinelli-Seip congenital lipodystrophy types 1 and 4 1Institute for Human Genomic Study, Seoul, Korea, Republic
of, 2Department of Radiology, Korea University Ansan
Hospital, Ansan, Korea, Republic of, 3Division of Pulmonary,
Sleep and Critical Care Medicine, Department of Internal
Medicine, Korea University Ansan Hospital, Ansan, Korea,
Republic of N. Gunes1, T. Erkan1, T. Kutlu1, H. Onay2, T. Atik2, B. Tüysüz1 J. del Picchia 76 Background and aims: Although smoking is a primary
risk factor for chronic obstructive pulmonary disease
(COPD), genetic polymorphisms within the FAM13A gene
have been consistently reported to association with pul-
monary function and/or COPD in genome-wide association
studies. We aimed to investigate the effect of FAM13A gene
variants that interact with ever smoking on COPD and
emphysema risk. Background and aims: Although smoking is a primary
risk factor for chronic obstructive pulmonary disease
(COPD), genetic polymorphisms within the FAM13A gene
have been consistently reported to association with pul-
monary function and/or COPD in genome-wide association
studies. We aimed to investigate the effect of FAM13A gene
variants that interact with ever smoking on COPD and
emphysema risk. progress in a discovery of new CP genes, the genetic cause
of the disease, in the majority of the patients, remains
unknown. Aim: To identify novel susceptibility genes in early onset
CP patients using whole exome sequencing (WES). Patients and Methods: Six patients (mean age at
diagnosis 10 years) with idiopathic or hereditary/familial
CP with undetermined cause of the disease and their
relatives were included for WES. Before, Sanger sequen-
cing was performed to exclude the genetic causes of CP. WES data (HiSeq 2500, Illumina) were compared between
index patient and affected or/and unaffected relatives. The
variants were selected taking into account: in silico
prediction
(SIFT,
MutTaster),
minor
allele
frequency
(MAF <0.01), gene function and expression in the pancreas,
and the variant co-segregation with CP. Methods: Using a community-based cohort, we analyzed
the association between genetic variants of FAM13A gene
and COPD (GOLD stage >1) / emphysema (e.g., total lung
volume, emphysema volume and emphysema ratio on
computed tomography) risks using multivariate logistic and
linear regression models (total n = 3,400). Furthermore,
similar analyses were conducted after stratification by
smoking status Results:. We detected 3 variants in known CP genes: two
recurrent CFTR variants (p.L997F and p.R75Q) and one
novel p.L100V*21 variant (introducing a STOP codon) in
CTRC. P03.09D We identified potentially pathogenic variants in 5
novel genes: PNLIP (p.Q323L), GCK (p.V101M), TRPV6
(p.V239Sfs*53 and p.L576R), SCNN1G (p.I224V) and
SERPINA12 (p.G327D). The TRPV6 and SERPINA12
variants showed autosomal dominant inheritance. In 5/6
of the index patients, the trans-heterozygous variants in two
different genes were observed. Results: Five common variants (rs1458551, rs2609264,
rs2609261, rs2609260 and rs7671167) annotated to the
FAM13A gene were shown to have an additive effect on
COPD and emphysema risk, whereas rs3756050 was only
associated with a significantly higher risk for COPD (risky
homozygote odds ratio (OR) = 1.49 (95% CI 1.13-1.96)). Finally, we identified significant interaction between the
rs3756050 and ever smoking (P for interaction = 0.01). Conclusions: We confirmed the previously reported
association of FAM13A with COPD as well as emphysema
risk. The genetic variant of FAM13A gene also interacted
with ever smoking to affect the risk of higher COPD risk. This study was supported by the Korea Centers for Disease
Control and Prevention grant (2011-E71004-00, 2012-
E71005-00,
2013-E71005-00,
2014-E71003-00),
the
National Research Foundation of Korea grant funded by
the Korea government (NRF-2016R1A2B4012155 and
NRF-2017R1A6A3A11034663),
and
the
Korea
University Grant. Conclusions: Using WES approach, we identified novel
variants and susceptibility genes candidates in CP. Their
clinical significance needs to be elucidated by the functional
and the case-control studies. Financed: National Science Center Poland:2015/19/B/
NZ5/02224 A.M. Rygiel: None. A. Kujko: None. G. Oracz: None. T. Gambin: None. J. Kosińska: None. K. Wejnarska:
None. K. Wertheim-Tysarowska: None. E. Kołodziejc-
zyk: None. R. Płoski: None. J. Bal: None. A.M. Rygiel: None. A. Kujko: None. G. Oracz: None. T. Gambin: None. J. Kosińska: None. K. Wejnarska: None. K. Wertheim-Tysarowska: None. E. Kołodziejc-
zyk: None. R. Płoski: None. J. Bal: None. S. Kim: None. K.Y. Lee: None. R.E. Kim: None. C. Shin: None. S. Kim: None. K.Y. Lee: None. R.E. Kim: None. C. Shin: None. P03.12C P03.12C
The association between CEL-HYB1 allele and idiopathic/
familial chronic pancreatitis in Polish pediatric patients 1Institute of Mother and Child, Warsaw, Poland, 2The
Children’s Memorial Health Institute, Warsaw, Poland,
3Haukeland University Hospital,, Bergen, Norway, 4Jan
Kochanowski University of Kielce, Kielce, Poland, 5Oncology
Center of the Holy Cross, Kielce, Poland, 6University of
Bergen, Bergen, Norway P03.11B The analysis of > 1200 cases by
this approach showed that: a) in the 6% of alleles with
multiple mutations they are distributed on different genes
(duplications); b) in the 33% of cases with the Q318X
mutation there is a concomitant duplicated normal gene,
resulting in a non-pathological allele; c) in 37% of cases
with a whole gene deletion, this includes part of the con-
tiguous TNXB gene, resulting in 21OHD potentially asso-
ciated with EDS signs. In very rare cases (<1%) with
particularly complex arrangements, interpretative doubts
may persist. The complexity of 21OHD genetic analysis
therefore requires the integration of multiple techniques and
an excellent knowledge of the peculiarities of the locus. An
incomplete investigation can lead to erroneous interpreta-
tions of the result with serious consequences in clinical
management and genetic counseling. An accurate clinical
evaluation of patients with 21OHD should always include
the search for EDS signs. The approach described here has
proved to be fundamental for a correct interpretation of
complex arrangements and, especially in cases of pre-
conceptional and prenatal tests, to improve the genetic
counseling, as well as the clinical classification/manage-
ment of the patients. Introduction: The replacement of a part of the carboxyl
ester lipase gene (CEL) and its pseudogene (CELP) can
create CEL-HYB1 allele which has been recently shown to
elevate susceptibility to chronic pancreatitis (CP) in adults
patients of European but not Asian ancestry, suggesting that
it is an European CP risk factor. However, the replication
studies are lacking. Aim: To evaluate the risk associated with the CEL-HYB1
allele in the pediatric Polish CP patients as compared to
control group. Patients and Method: We enrolled a single-center cohort
of 147 unrelated CP children (mean age at diagnosis 12
years) including 64 idiopathic (ICP) or familial idiopathic
(FCP) patients and 83 CP patients with various ethological
risk factors (anatomical/physiological dysfunctions and/or
genetics risk factors). The control group consisted of 331
ethnically matched individuals (mean age 45).The CEL-
HYB1 allele was detected using PCR, confirmed by Sanger
sequencing. Results: The CEL-HYB1 allele is significantly over-
represented in ICP/FCP patients compared to controls
(9.4% vs 2.1%, P=0.0098) with OR of 4.8 (95%Cl 1.5-
13.3) but not in a CP group with known ethological factors
(1.2% vs 2.1%, P>0.05, OR=0.57, 95% CI (0.05-3.4). We
identified two CP families with CEL-HYB1 allele and
confirmed the co-segregation of the allele with the disease. CYP21A2 mutations in congenital adrenal hyperplasia due
to 21 hydroxylase deficiency in Turkish population CYP21A2 mutations in congenital adrenal hyperplasia due
to 21 hydroxylase deficiency in Turkish population O. Cilingir1, E. Simsek2, B. Durak Aras1, E. Erzurumluoglu1,
C. Binay3, M. A. Temena1, S. Kocagil1, S. Artan1 O. Cilingir1, E. Simsek2, B. Durak Aras1, E. Erzurumluoglu1,
C. Binay3, M. A. Temena1, S. Kocagil1, S. Artan1 M. Rygiel: None. 1Department of Medical and Surgical Sciences, University of
Bologna, Bologna, Italy, 2Department of Women, Children and
Urological Diseases, Medical Genetics Unit, "S.Orsola-
Malpighi" University-Hospital, Bologna, Italy P03.11B The association between CEL-HYB1 allele and idiopathic/
familial chronic pancreatitis in Polish pediatric patients Novel susceptibility genes candidates of chronic
pancreatitis identified by whole exome sequencing A. Kujko1, G. Oracz2, K. Fjeld3, K. Wejnarska2, K. Wertheim-
Tysarowska1, E. Kołodziejczyk2, J. Bal1, D. Koziel4, A. Kowalik5,
S. Gluszek4, A. Molven6, A. M. Rygiel1 A. M. Rygiel1, A. Kujko1, G. Oracz2, T. Gambin1, J. Kosińska3,
K. Wejnarska4, K. Wertheim-Tysarowska1, E. Kołodziejczyk2,
R. Płoski3, J. Bal1 A. M. Rygiel1, A. Kujko1, G. Oracz2, T. Gambin1, J. Kosińska3,
K. Wejnarska4, K. Wertheim-Tysarowska1, E. Kołodziejczyk2,
R. Płoski3, J. Bal1 A. M. Rygiel1, A. Kujko1, G. Oracz2, T. Gambin1, J. Kosińska3, A. M. Rygiel1, A. Kujko1, G. Oracz2, T. Gambin1, J. Kosińska3, K. Wejnarska4, K. Wertheim-Tysarowska1, E. Kołodziejczyk2,
R. Płoski3, J. Bal1 1Institute of Mother and Child, Warsaw, Poland, 2The
Children’s Memorial Health Institute, Warsaw, Poland,
3Medical University of Warsaw, Warsaw, Poland, 4The
Children’s Memorial Health Institute, Warsaw, Poland The early onset chronic pancreatitis (CP) is often associated
with mutations in a subset of CP genes. Despite of a 77 Abstracts from the 51st European Society of Human Genetics Conference: Posters Introduction: The replacement of a part of the carboxyl
ester lipase gene (CEL) and its pseudogene (CELP) can
create CEL-HYB1 allele which has been recently shown to
elevate susceptibility to chronic pancreatitis (CP) in adults
patients of European but not Asian ancestry, suggesting that
it is an European CP risk factor. However, the replication
studies are lacking. Aim: To evaluate the risk associated with the CEL-HYB1
allele in the pediatric Polish CP patients as compared to
control group. The 21-OHD is caused by mutations in the CYP21A2 gene,
that maps within a repeated module (RCCX) in a region
(6p21) with a high recombination frequency. Some RCCX
arrangements can complicate and significantly modify the
interpretation of the genetic tests. Our goal was to obtain the
correct diagnosis of complex cases by integrating: complete
gene sequencing, segregation analysis of variants/SNPs,
CNVs detection by MLPA. P03.11B Conclusions: Our replication study show that the CEL-
HYB1 allele is a significant CP risk factor in idiopathic and
familial CP pediatric patients. The screening for CEL-HYB1
allele should be considered in CP diagnostics in European
ancestry patients. S. Menabò: None. A. Balsamo: None. A. Cassio: None. L. Mazzanti: None. M. Seri: None. L. Baldazzi: None. S. Menabò: None. A. Balsamo: None. A. Cassio: None. L. Mazzanti: None. M. Seri: None. L. Baldazzi: None. Financed by National Science Center, Poland:2015/19/B/
NZ5/02224 P03.14A A. Kujko: None. G. Oracz: None. K. Fjeld: None. K. Wejnarska: None. K. Wertheim-Tysarowska: None. E. Kołodziejczyk: None. J. Bal: None. D. Koziel: None. A. Kowalik: None. S. Gluszek: None. A. Molven: None. A. M. Rygiel: None. A. Kujko: None. G. Oracz: None. K. Fjeld: None. K. Wejnarska: None. K. Wertheim-Tysarowska: None. E. Kołodziejczyk: None. J. Bal: None. D. Koziel: None. A. Kowalik: None. S. Gluszek: None. A. Molven: None. A. M. Rygiel: None. S. Menabò1, A. Balsamo1, A. Cassio1, L. Mazzanti1, M. Seri1,
L. Baldazzi2 P03.13D 1Eskisehir Osmangazi University, Faculty of Medicine,
Department of Medical Genetics, Eskisehir, Turkey, 2Eskisehir
Osmangazi University, Faculty of Medicine, Department of
Pediatric Endocrinology, Eskisehir, Turkey, 3Tekirdag Corlu
State Hospital, Tekirdag, Turkey Congenital adrenal hyperplasia, due to 21-hydroxylase
deficiency (21-OHD), isolated or associated with Ehlers
Danlos syndrome (EDS): technical and counseling
problems related to molecular diagnosis S. Menabò1, A. Balsamo1, A. Cassio1, L. Mazzanti1, M. Seri1,
L. Baldazzi2
1Department of Medical and Surgical Sciences, University of
Bologna, Bologna, Italy, 2Department of Women, Children and
Urological Diseases, Medical Genetics Unit, "S.Orsola-
Malpighi" University-Hospital, Bologna, Italy S. Menabò1, A. Balsamo1, A. Cassio1, L. Mazzanti1, M. Seri1,
L. Baldazzi2 Congenital adrenal hyperplasia (CAH) caused by 21-
hydroxylase deficiency is a common autosomal recessive
disorder due to mutations in the CYP21A2 gene. Defects in
this gene lead to adrenal insufficiency, ambiguous genitalia,
salt-wasting in classic form of CAH, and hyperandrogenism
during childhood or early adulthood in milder form of
CAH, known as nonclassic CAH (NCAH). Mutations are J. del Picchia 78 mostly
caused
by a rearrangement
during intergenic
recombination between CYP21A2 and its non-functional
pseudogene CYP21AP with very high percentage of
sequence homology. This study aimed at analyzing the
frequency of 11 prevalent mutations in 183 patients. Mutations, including P30L, 8bp deletion, exon 6 cluster,
L307 frameshift, R356W, R483P, I2 splice, I712N, V281L,
Q318 and, P453S were analyzed by reverse-hybridization
strip-based assay (CAH Strip Assay). While 62,3% of 183
patients (114/183) have wild type alleles, the revealed
CAH-related variants are given in the table. Interestingly,
homozygous Del8bpE3 and I2 splice mutations was found
in two siblings. Segregation analysis showed that the
mother was heterozygous for these compound mutations. After confirmation of paternity, we are now focussing on
the possibility of uniparental disomy (UPD). Further studies
for this case were planned to clarify the genetic mechanisms
underlying this situation. 1University Children's Hospital Skopje, Skopje, Macedonia,
The Former Yugoslav Republic of, 2University of Cambridge,
Metabolic Research Laboratories, Cambridge, United
Kingdom Introduction: Congenital hypothyroidism (CH), defined as
lack of thyroid hormones at birth, is the most common
neonatal endocrine disorder affecting 1 in 2000-4000
newborns worldwide. While up to 20% of CH cases are
hereditary, the majority of cases are sporadic with unknown
etiology. Mutations in at least 15 different genes have been
associated with CH. The genetics of CH has not been stu-
died in Macedonia previously. P03.17D
Development of a biochip array for the rapid detection of
32 common cystic fibrosis mutations Development of a biochip array for the rapid detection of
32 common cystic fibrosis mutations Development of a biochip array for the rapid detection of
32 common cystic fibrosis mutations O. Cilingir: None. E. Simsek: None. B. Durak Aras:
None. E. Erzurumluoglu: None. C. Binay: None. M.A. Temena: None. S. Kocagil: None. S. Artan: None. P03.16C
Molecular characterization of congenital hypothyroidism
with “gland in situ” in Macedonia
N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 O. Cilingir: None. E. Simsek: None. B. Durak Aras:
None. E. Erzurumluoglu: None. C. Binay: None. M.A. Temena: None. S. Kocagil: None. S. Artan: None. M. J. Latten1, D. A. Bailie2, C. A. Graham1, M. A. Crockard1,
J. V. Lamont1, S. P. FitzGerald1 M. J. Latten1, D. A. Bailie2, C. A. Graham1, M. A. Crockard1,
J. V. Lamont1, S. P. FitzGerald1 P03.13D Material and Methods: A multigenic sequencing of CH
candidate genes including TG, TPO, DUOX2, DUOXA2,
SLC5A5, SLC26A4, IYD and TSHR was performed in a
cohort of 22 CH patients with “gland in situ” (GIS), both
familial and sporadic cases, associated with permanent,
transient or subclinical phenotype. Table. Percentage of mutation frequencies
MUTATIONS
HETERO-
ZYGOUS (%)
HOMOZ-
YGOUS (%)
COMPOUND MUTA Table. Percentage of mutation frequencies Table. Percentage of mutation frequencies
MUTATIONS
HETERO-
ZYGOUS (%)
HOMOZ-
YGOUS (%)
COMPOUND MUTATIONS
HETERO-
ZYGOUS (%)
HOMOZ-
YGOUS (%)
P30L
1,47
–
I2 Splice
Del 8bp E3
1,47
Del 8bp E3
I2 Splice
4,41
Del 8bp E3
2,94
–
I2 Splice
V281L
1,47
E6 Cluster
2,94
2,94
I2 Splice
Q318X
2,94
L307fs
–
–
I712N
Q318X
1,47
R356W
–
–
V281L
I2 Splice
1,47
R483P
–
–
V281L
Q318X
1,47
I2 Splice
–
–
V281L
P453S
2,94
Del 8bp E3
I2 Splice
P30L
1,47
I712N
2,94
–
P30L
I2 Splice
Del 8bp E3
5,88
V281L
20,59
8,84
L307fs
Q318X
R356W
1,47
Q318X
25,00
–
P30L
I2 Splice
Del 8bp E3
V281L
1,47
P453S
2,94
1,47
TOTAL
58,82
13,25
22,05
5,88 Results: Mutations were identified in 27% of patients in
the TPO, TSHR and DUOX2 genes. In two siblings with
severe persistent CH, monoallelic c.1187_1188insGCCG
mutation in TPO gene was detected. A child with prenatally
diagnosed goiter was compound heterozygous for two
mutations in TPO gene (c.31_50dup/ c.1313G>A). Two
novel mutations were detected in TSHR gene in children
with subclinical hypothyroidism c.1516G>A and c.692
+1_692+4delGTGA. A
previously
known
mutation
c.4637A>G in the DUOX2 gene was detected in hetero-
zygous state in one child with transient hypothyroidism. Conclusion: Genetic variants were not frequently found
in Macedonian CH patients, thus the etiology of CH with
GIS
remains
elusive. Factors
other
than
known
dyshormonogenesis-associated genes or the TSHR have to
be considered, as well as future studies with whole exome
sequencing for elucidating the cause of hypothyroidism. N. Zdraveska: None. N. Schoenmakers: None. V. Anastasovska: None. M. Kocova: None. N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 1Randox Laboratories Ltd, Crumlin, United Kingdom,
2Northern Ireland Regional Genetics Centre, Belfast Health
and Social Care Trust, City Hospital, Belfast, United Kingdom
Introduction: Cystic Fibrosis (CF) is an autosomal reces-
sive genetic condition which affects vital organs, mostly the
lungs, by clogging them with thick, sticky mucus. Persistent P03.16C Molecular characterization of congenital hypothyroidism
with “gland in situ” in Macedonia Molecular characterization of congenital hypothyroidism
with “gland in situ” in Macedonia Molecular characterization of congenital hypothyroidism
with “gland in situ” in Macedonia
N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 Introduction: Cystic Fibrosis (CF) is an autosomal reces-
sive genetic condition which affects vital organs, mostly the
lungs, by clogging them with thick, sticky mucus. Persistent N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1,
M. Kocova1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 79 To investigate the pathophysiology of cystic fibrosis (CF)
several animal models have been developed including
mouse, pig, and ferret; however, none of them perfectly
recapitulates all human patient symptoms. On the contrary,
zebrafish (Danio rerio) recently emerged as a powerful
genetic model system to better understand CF onset and to
develop new pharmacological treatments. Indeed, zebrafish
embryos present only innate immune system and the zeb-
rafish cftr gene is highly conserved with the human ortho-
logue. cftr-loss-of-function zebrafish embryos mimic CF
human defects in response to infection of P. aeruginosa,
presenting a dampened respiratory burst response, a reduced
neutrophil migration and defects in endocrine organs
function. infections can lead to chronic lung problems. The disorder
is caused by mutations in the Cystic Fibrosis Transmem-
brane Conductance Regulator gene (CFTR) and to date,
over 2,000 activating mutations have been reported. In most
populations, however, a panel of 30-35 mutations will
detect over 90% of disease causing variants. Materials and Methods: An assay was designed for
detection of 32 common CF mutations and the intron 8
polypyrimidine tract variant 5,7,9 T, using genomic DNA. Following target-specific multiplex-PCR, amplicons were
hybridised to a 7 x 7 array of Discrete Test Regions (DTR)
on a biochip (Randox Laboratories Ltd, UK). Using the
Evidence Investigator analyser. Biochips were imaged and
analysed automatically. Assay run time was <3 hours. Pre-
characterised
samples
(n = 50)
containing
known
CF
mutations were assessed and results compared against the
predicate genotyping. In our previous work, we demonstrated that P. aerugi-
nosa infection in mice and Galleria mellonella larvae could
be cured by administration of phages, the natural enemies of
bacteria. Phage therapy, used for decades in Eastern Europe,
is gathering interest as a therapeutic alternative or a
complementary treatment to antibiotics. P03.16C The goal of this
project is to in vivo validate the efficacy of phage therapy
against P. aeruginosa infections using the CF-zebrafish
animal model. Both wild-type and cftr-loss-of-function
zebrafish embryos, were infected with P. aeruginosa by
microinjection, followed by phage administration. The
therapeutic effects of phages was evaluated, following
embryo mortality, bacterial burden, neutrophil migration
and immune response. In addition, we plan to combine the
phage treatment with antibiotics to verify if combination of
the two treatments has a positive outcome against P. aeruginosa infections. Results:
The
array
was
verified
using
50
pre-
characterised samples containing known CF mutations, as
well as the intron 8 polypyrimidine tract, in order to confirm
specificity of the biochip for detection of mutations in the
CFTR gene. Each of the 32 targets was assessed at least
once. 100% concordance was achieved. Conclusions: This rapid screening technique enables the
simultaneous analysis of 32 common mutations associated
with cystic fibrosis. This will aid in the confirmation of
suspected cases of CF and also in the identification of those
who are carriers of a mutation. M.J. Latten:
A. Employment
(full
or
part-time);
Significant; Randox Laboratories Ltd. D.A. Bailie: C. Other Research Support (supplies, equipment, receipt of
drugs or other in-kind support); Significant; Randox
Laboratories Ltd. C.A. Graham: A. Employment (full or
part-time); Significant; Randox Laboratories Ltd. M.A. Crockard: A. Employment (full or part-time); Significant;
Randox Laboratories Ltd. J.V. Lamont: A. Employment
(full or part-time); Significant; Randox Laboratories Ltd. S. P. FitzGerald:
A. Employment
(full
or
part-time);
Significant; Randox Laboratories Ltd. Funding Grant: Italian Cystic Fibrosis Research Foun-
dation FFC#22.2017 Funding Grant: Italian Cystic Fibrosis Research Foun-
dation FFC#22.2017 M. Cafora: None. F. Forti: None. G. Deflorian: None. L. Ferrari: None. D. Ghisotti: None. F. Briani: None. A. Pistocchi: None. 1Dip. Biotecnologie Mediche e Medicina Traslazionale,
Università degli Studi di Milano, Milan, Italy, 2Dip.
Bioscienze, Università degli Studi di Milano, Milan, Italy,
3Istituto Fondazione FIRC di Oncologia Molecolare IFOM,
Milan, Italy M. Cafora1, F. Forti2, G. Deflorian3, L. Ferrari3, D. Ghisotti2,
F. Briani2, A. Pistocchi1 M. O. Woods, A. Pirzada, G. Zhai, B. Fernandez M. O. Woods, A. Pirzada, G. Zhai, B. Fernandez M. O. Woods, A. Pirzada, G. Zhai, B. Fernandez P03.18A Conclusions: The minor T allele of rs35705950 is
associated with FPF and likely contributes to the significant
proportion of FPF families without a known genetic
predisposition. Results: Seven previously described and 15 possible
DSD associated rare variants were identified in 10 different
genes within a total of 19 cases, lead our diagnostic rate to
43%. The structural alteration in one novel coding region
was simulated in three dimensional protein modeling
program. Results: Seven previously described and 15 possible
DSD associated rare variants were identified in 10 different
genes within a total of 19 cases, lead our diagnostic rate to
43%. The structural alteration in one novel coding region
was simulated in three dimensional protein modeling
program. Supported by the Regional Partnership Program of the
CIHR; the NL Lung Association; and the generous
donations from the family of Craig L. Dobbin. M.O. Woods: None. A. Pirzada: None. G. Zhai: None. B. Fernandez: None. Conclusions: This study revealed new data for known
and novel associated variants and acknowledged mutation
frequencies in cases with DSD from Turkey. Our results
underscored the critical role of early diagnosis which is
valuable for proper management
of gonadal tumors,
expedient treatments and definitive genetic counseling for
families. Improving of CRISPR/Cas9 efficacy for F508del mutation
correction in cystic fibrosis S. A. Smirnikhina1, A. Anuchina1, K. Kochergin-Nikitsky1,
E. Adilgereeva1, A. Lavrov1,2 A. Aghayev: None. G. Toksoy: None. S. Poyrazoglu:
None. B. Karaman: None. S. Avcı: None. Z. Yavas Abalı:
None. U. Altunoglu: None. F. Bas: None. F. Darendeliler:
None. S. Basaran: None. Z. Uyguner: None. 1Federal State Budgetary Institution “Research Centre for
Medical Genetics” of the Russian Academy of, Moscow,
Russian Federation, 2The Russian National Research Medical
University Named after N.I. Pirogov, Moscow, Russian
Federation 1Federal State Budgetary Institution “Research Centre for
Medical Genetics” of the Russian Academy of, Moscow,
Russian Federation, 2The Russian National Research Medical
University Named after N.I. Pirogov, Moscow, Russian
Federation P03.18A In vivo validation of phage therapy against Pseudomonas
aeruginosa infections using zebrafish as a new model for
cystic fibrosis A. Aghayev1, G. Toksoy1, S. Poyrazoglu2, B. Karaman1, S. Avcı1,
Z. Yavas Abalı2, U. Altunoglu1, F. Bas2, F. Darendeliler2,
S. Basaran1, Z. Uyguner1 M. Cafora1, F. Forti2, G. Deflorian3, L. Ferrari3, D. Ghisotti2,
F. Briani2, A. Pistocchi1 1Dept. Med. Genet, Istanbul Med. Faculty, Ist. Uni., Istanbul,
Turkey, 2Dept. Ped. Endocrinology, Istanbul Med. Faculty, Ist. Uni., Istanbul, Turkey 1Dip. Biotecnologie Mediche e Medicina Traslazionale,
Università degli Studi di Milano, Milan, Italy, 2Dip. Bioscienze, Università degli Studi di Milano, Milan, Italy,
3Istituto Fondazione FIRC di Oncologia Molecolare IFOM,
Milan, Italy Introduction: Disorders of sexual development (DSD) are
defined as congenital conditions covering a wide spectrum
of clinical expressions from mild hypospadias to abnormal J. del Picchia 80 gonadal abnormalities causing complete sex reversal. Early
diagnosis is valuable as it will impact the individual’s
mental well-being and overall life quality, including the
advisability for genetic counseling. cohort. A case-control analysis was carried out using 110
affected individuals and 277 healthy controls from the
Newfoundland population. Results: There was a significant association between
rs35705950 genotypes and IPF. The odds ratios for all
individuals affected with IPF who were heterozygous and
homozygous
for
the
variant
allele
of
this
promoter
polymorphism respectively were 5.4 (95% CI, 3.3 to 9.6,
P < .001) and 12.2 (95% CI, 3.3 to 44.7, P < .001). Furthermore, two families displayed segregation of the
variant allele with the phenotype. Using SISA, we showed
that, in one of these families, the likelihood that the minor
allele segregates with PF by chance is 1.56%. Materials and Methods: We evaluated the results of 44
DSD cases for gross deletion/duplication with MLPA and
for sequence alteration via in-house-designed next genera-
tion sequencing (NGS) targeted gene panel, using an Ion
Torrent
platform,
covering
all
exon
and
exon-intron
boundaries of 31 DSD associated genes. Cases with
chromosomal abnormalities except one case with 47,XXY
were excluded from the study group. Screening of targeted
regions that were missed by AmpliSeq primer design is
covered by Sanger sequencing. Segregation analysis was
performed for very rare and novel missense alterations. Conclusions: The minor T allele of rs35705950 is
associated with FPF and likely contributes to the significant
proportion of FPF families without a known genetic
predisposition. P03.20C Association between a promoter SNP in MUC5B and
idiopathic pulmonary fibrosis in the Newfoundland
population Association between a promoter SNP in MUC5B and
idiopathic pulmonary fibrosis in the Newfoundland
population Association between a promoter SNP in MUC5B and
idiopathic pulmonary fibrosis in the Newfoundland
population Genome editing using CRISPR/Cas9 seems to be the most
promising way for gene therapy to correct F508del mutation
in CFTR gene in cystic fibrosis. Design of sgRNA to DNA
sequence near mutation is determined by the presence of
PAM for Cas9. It is possible to design only one sgRNA for
F508del mutation for spCas9 - sgCFTR#1, but in HEK293T
cell culture this sgRNA demonstrated low efficiency in
indels formation in combination with different spCas9
(13.8%), compared to other sgRNAs to CFTR gene (13-
18%), as well as to GFP gene (34.2%). qPCR data showed
that sgCFTR#1 and sgGFP_1 expression levels were low
and did not differ in 0 and 7 hours after transfection; but
expression level of sgGFP_1 became almost 15-fold higher
than sgCFTR#1 in 24 hours after transfection; 30 hours
after transfection - 22-fold higher. Attempts to increase
sgCFTR#1 expression by adding addition expression cas-
sette to the plasmid, fusing sgCFTR#1 with active sgGFP_1 Memorial University of Newfoundland, St. John's, NL, Canada Memorial University of Newfoundland, St. John's, NL, Canada Background: Idiopathic pulmonary fibrosis (IPF) is a late-
onset, complex genetic disease characterized by inflamma-
tion and scarring of the lung parenchyma. To date, hetero-
zygous causal variations in TERT, TERC, SFTPC, SFTPA2,
that account for 2-20% of IPF, have been documented. More recently, a promoter variant (rs35705950) upstream of
MUC5B has been shown to be associated with IPF. Methods: All probands in this study were previously
screened for variants in the four abovementioned PF genes. A TaqMan SNP Genotyping assay and a 7900HT Real-time
PCR analyzer were used to genotype rs35705950 in our Abstracts from the 51st European Society of Human Genetics Conference: Posters 81 and using hybrid promoter were made, however, increasing
of sgCFTR#1 efficacy was not received. Attempts to sta-
bilize sgCFTR#1 by including G-quadruplexes to its
sequence, shortening and addition of GG to the 5'-region
also failed. Cultivation of the edited cells at lower tem-
perature also did not lead to improved results. Further
attempts to enhance sgCFTR#1 expression and its stabili-
zation should be performed, or other Cas9 enzymes, which
expand the ability to select sgRNA direct to F508del
mutation can be used. The work was partially supported by
Russian Science Foundation (agreement 17-75-20095 from
25/07/2017) and Russian Academy of Sciences. Introduction: As neonatal cholestasis is a common infant
disease with more than 50 possible etiologies, fast and com-
prehensive diagnosis is desirable to initiate early therapy. Also
for other hepatopathies, especially those with acute liver
failure, rapid diagnosis is important, as they could constitute
contraindication to liver transplantation or can be treated with
specific therapies. We used four different whole-exome-
sequencing-(WES)-based in-silico panels to analyze genes
associated with different liver-related signs and symptoms. Introduction: As neonatal cholestasis is a common infant
disease with more than 50 possible etiologies, fast and com-
prehensive diagnosis is desirable to initiate early therapy. Also
for other hepatopathies, especially those with acute liver
failure, rapid diagnosis is important, as they could constitute
contraindication to liver transplantation or can be treated with
specific therapies. We used four different whole-exome-
sequencing-(WES)-based in-silico panels to analyze genes
associated with different liver-related signs and symptoms. Methods: DNA was extracted from blood samples of 66
patients (age: 0-46, median 2.50 years, 37 males). Library
preparation was performed with TruSeq Nano DNA Library
Preparation Kit (Illumina, San Diego, USA) or xGen
Exome Research Panel (IDT, Leuven, Belgium). P03.22A Diagnosis of monogenetic metabolic hepatopathies by
whole-exome sequencing Memorial University of Newfoundland, St. John's, NL, Canada The
Table shows details: Panel
signs and
symptoms
genes
Number
of
requests
Number of patients with
(likely)
pathogenic
variants*
VUS+
pathogenic
variant°
VUS* heterozygous
pathogenic
variants or
VUS°
no
significant
findings
1
Cholestasis
with low
gamma-
glutamyltran-
sferase
ATP8B1,
ABCB11,
TJP2,
AKR1D1,
CYP7B1,
AMACR,
ABCD3,
BAAT,
CLDN1,
HSD3B7,
NR1H4,
SLC25A13
19
4
1
1
1
12
2
Cholestasis
with high
gamma-
glutamyltran-
sferase
ABCB4,
JAG1,
NOTCH2
13
–
–
3
–
10
3
Acute liver
failure with
suspected
lysosomal
storage
disease,
mitochondri-
al DNA
depletion
syndrome or
Wilson
disease
MPV17,
POLG,
DGUOK,
C10ORF2,
TRMU,
GFM1,
BCS1L,
NPC1,
NPC2,
ATP7B
12
4
–
1
2
5
4
Hepatopathi-
es/increased
transaminas-
es without
primary
cholestasis
ATP7B,
FBP1,
G6PC,
GBE1,
GYS2,
PHKA2,
PHKB,
PHKG2,
PYGL,
SLC37A4,
SLC2A2
22
2
–
–
7
13
66
10
1
5
10
40
*Two variants for recessive, one for dominant traits
°for recessive traits Panel
signs and
symptoms
genes
Number
of
requests
Number of patients with
(likely)
pathogenic
variants*
VUS+
pathogenic
variant°
VUS* heterozygous
pathogenic
variants or
VUS°
no
significant
findings
1
Cholestasis
with low
gamma-
glutamyltran-
sferase
ATP8B1,
ABCB11,
TJP2,
AKR1D1,
CYP7B1,
AMACR,
ABCD3,
BAAT,
CLDN1,
HSD3B7,
NR1H4,
SLC25A13
19
4
1
1
1
12
2
Cholestasis
with high
gamma-
glutamyltran-
sferase
ABCB4,
JAG1,
NOTCH2
13
–
–
3
–
10
3
Acute liver
failure with
suspected
lysosomal
storage
disease,
mitochondri-
al DNA
depletion
syndrome or
Wilson
disease
MPV17,
POLG,
DGUOK,
C10ORF2,
TRMU,
GFM1,
BCS1L,
NPC1,
NPC2,
ATP7B
12
4
–
1
2
5
4
Hepatopathi-
es/increased
transaminas-
es without
primary
cholestasis
ATP7B,
FBP1,
G6PC,
GBE1,
GYS2,
PHKA2,
PHKB,
PHKG2,
PYGL,
SLC37A4,
SLC2A2
22
2
–
–
7
13
66
10
1
5
10
40
*Two variants for recessive, one for dominant traits
°for recessive traits 1Hannover Medical School, Institute of Human Genetics,
Hannover, Germany, 2Hannover Medical School, Paediatric
Gastroenterology and Hepatology, Hannover, Germany,
3Hannover Medical School, Hannover Unified Biobank (HUB),
Hannover, Germany Memorial University of Newfoundland, St. John's, NL, Canada Libraries
were sequenced on a NextSeq 500 (Illumina). Data analysis
was done with the NGS pipeline megSAP (https://github. com/imgag/megSAP). Variants were filtered using GSvar
(University Hospital Tübingen, Germany). Variant classifi-
cation was done using Alamut Visual (Interactive Biosoft-
ware, Rouen, France) according to standards and guidelines
of American College of Medical Genetics and Genomics. S.A. Smirnikhina: A. Employment (full or part-time);
Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Russian Science Foundation, Russian
Academy of Sciences. A. Anuchina: A. Employment (full
or part-time); Significant; Research Centre for Medical
Genetics. B. Research Grant (principal investigator, colla-
borator or consultant and pending grants as well as grants
already received); Significant; Russian Science Foundation,
Russian Academy of Sciences. K. Kochergin-Nikitsky: A. Employment (full or part-time); Significant; Research
Centre for Medical Genetics. B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Significant; Russian
Science Foundation, Russian Academy of Sciences. E. Adilgereeva: A. Employment (full or part-time); Signifi-
cant; Research Centre for Medical Genetics. B. Research
Grant (principal investigator, collaborator or consultant and
pending
grants
as
well
as
grants
already
received);
Significant; Russian Science Foundation, Russian Academy
of Sciences. A. Lavrov: A. Employment (full or part-time);
Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Russian Science Foundation, Russian
Academy of Sciences. Results: In 25/66 patients we detected (likely) pathogenic
variants or variants of unknown significance (VUS). A. Stalke1, A. Schöner-Heinisch1, B. Auber1, U. Baumann2,
T. Illig1,3, B. Skawran1, B. Schlegelberger1, G. Schmidt1,
E. Pfister1 Search for genetic risk factors in Hirschsprung's disease
associated enterocolitis by Whole Exome Sequencing Search for genetic risk factors in Hirschsprung's disease
associated enterocolitis by Whole Exome Sequencing 1Biochemistry and Molecular Genetics Department, Hospital
Clinic of Barcelona and IDIBAPS, Barcelona, Spain, 2Centre
for Biomedical Research on Rare Diseases (CIBERER), ISCIII,
Barcelona, Spain, 3Universitat Pompeu Fabra, Barcelona,
Spain, 4Pneumology Department, Hospital Clinic of Barcelona
and IDIBAPS, Barcelona, Spain, 5Centro de Investigación
Biomédica en Red de Enfermedades Respiratorias (CIBERES),
ISCIII, Madrid, Spain, 6Technion Israel Institute of
Technology, Haifa, Israel T. Bachetti1, G. Santamaria1, M. Mosconi2, S. Sartori3, M. De
Filippo3, A. Pini Prato4, I. Ceccherini1, F. Lantieri5 1Molecular Genetics Laboratory, G. Gaslini Inst., Genoa,
Italy, 2UOC Pediatric Surgery, G. Gaslini Inst., Genoa, Italy,
3Center for Translational Genomics and BioInformatics,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 4Children
Hospital, AON SS Antonio e Biagio e Cesare Arrigo,
Alessandria, Italy, 5Health Science Department (DISSAL) –
Biostatistics Unit, University of Genoa, Genoa, Italy 1Molecular Genetics Laboratory, G. Gaslini Inst., Genoa,
Italy, 2UOC Pediatric Surgery, G. Gaslini Inst., Genoa, Italy,
3Center for Translational Genomics and BioInformatics,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 4Children
Hospital, AON SS Antonio e Biagio e Cesare Arrigo,
Alessandria, Italy, 5Health Science Department (DISSAL) –
Biostatistics Unit, University of Genoa, Genoa, Italy Introduction: Heritable pulmonary arterial hypertension is
a rare disease inherited as an autosomal dominant disease. Introduction: Heritable pulmonary arterial hypertension is
a rare disease inherited as an autosomal dominant disease. Mutations in the BMPR2 gene have been detected in 75-
80% of the cases. However, only about 20% of all mutation
carriers
develop
the
disease,
indicating
a
reduced
penetrance. Introduction: Hirschsprung's disease (HSCR) is a con-
genital gut malformation. The most serious and life-
threatening complication is enterocolitis (HAEC), which
occurs in one third of the patients. The evident susceptibility
to HAEC in HSCR patients suggests a genetic background
that can lead to abnormal inflammatory response. Mutations in the BMPR2 gene have been detected in 75-
80% of the cases. However, only about 20% of all mutation
carriers
develop
the
disease,
indicating
a
reduced
penetrance. Material and Methods: We have studied a large family
affected by pulmonary arterial hypertension and segregating
with the pathogenic missense mutation p.Arg491Gln with a
penetrance of ~30%. In order to identify genetic variants
that may affect the penetrance in this family, we genotyped
20 mutation carriers (6 affected and 14 asymptomatic) and
12
healthy
individuals
with
the
Illumina
Infinium
CoreExome-24 BeadChip. P03.23B I. Madrigal: None. P. Puigdevall: None. L. Piccari:
None. I. Blanco: None. J. Barberà: None. D. Geiger:
None. M. Milà:
None. R. Castelo:
None. C. Badenas: None. Genetic linkage analysis identifies candidate modulators of
reduced penetrance in heritable pulmonary arterial
hypertension I. Madrigal1,2, P. Puigdevall3, L. Piccari4, I. Blanco4,5,
J. Barberà4,5, D. Geiger6, M. Milà1,2, R. Castelo3, C. Badenas1
1Biochemistry and Molecular Genetics Department, Hospital
Clinic of Barcelona and IDIBAPS, Barcelona, Spain, 2Centre
for Biomedical Research on Rare Diseases (CIBERER), ISCIII,
Barcelona, Spain, 3Universitat Pompeu Fabra, Barcelona,
Spain, 4Pneumology Department, Hospital Clinic of Barcelona
and IDIBAPS, Barcelona, Spain, 5Centro de Investigación
Biomédica en Red de Enfermedades Respiratorias (CIBERES),
ISCIII, Madrid, Spain, 6Technion Israel Institute of
Technology, Haifa, Israel I. Madrigal1,2, P. Puigdevall3, L. Piccari4, I. Blanco4,5,
J. Barberà4,5, D. Geiger6, M. Milà1,2, R. Castelo3, C. Badenas1 I. Madrigal1,2, P. Puigdevall3, L. Piccari4, I. Blanco4,5,
J. Barberà4,5, D. Geiger6, M. Milà1,2, R. Castelo3, C. Badenas1 Diagnosis of monogenetic metabolic hepatopathies by
whole-exome sequencing A. Stalke1, A. Schöner-Heinisch1, B. Auber1, U. Baumann2,
T. Illig1,3, B. Skawran1, B. Schlegelberger1, G. Schmidt1,
E. Pfister1 1Hannover Medical School, Institute of Human Genetics,
Hannover, Germany, 2Hannover Medical School, Paediatric
Gastroenterology and Hepatology, Hannover, Germany,
3Hannover Medical School, Hannover Unified Biobank (HUB),
Hannover, Germany 82 J. del Picchia Conclusions: We found a significant enrichment of
HPAH-related traits by a regulatory region within q24.2-
q31.1, located upstream from the FIGN gene. Full genome
sequencing and functional assays will be required to
validate the linkage regions, identify the actual variant
modulating the penetrance and unravel the molecular
mechanism
that
triggers
the
disease
in
the
family
under study. Conclusion: WES-based panel analysis represents a fast
and comprehensive tool to diagnose hereditary hepatopa-
thies in order to improve therapy and thus patient’s
prognosis. The WES-based approach further offers the
possibility to analyze the remaining exome data in patients
without clear diagnosis after panel analysis. A. Stalke: None. A. Schöner-Heinisch: None. B. Auber: None. U. Baumann: None. T. Illig: None. B. Skawran: None. B. Schlegelberger: None. G. Schmidt:
None. E. Pfister: None. Acknowledgments: Instituto de Salud Carlos III (PI15/
00483) and ‘fondos FEDER’, AGAUR (2017SGR1134),
“CERCA Programme / Generalitat de Catalunya” Acknowledgments: Instituto de Salud Carlos III (PI15/
00483) and ‘fondos FEDER’, AGAUR (2017SGR1134),
“CERCA Programme / Generalitat de Catalunya” Search for genetic risk factors in Hirschsprung's disease
associated enterocolitis by Whole Exome Sequencing Materials and Methods: To search for genetic factors
predisposing to HAEC, we have performed an exome next
generation sequencing (NGS) on 24 HSCR patients, 12 with
enterocolitis episodes (HAEC) and 12 without (HSCR-
only). Patients were selected based on Italian ancestry and
absence
of
additional
anomalies. The
exomes
were
sequenced with Illumina at a 50X coverage and variants
were filtered based on depth >= 10 and quality >=
10,
obtaining 77396 variants. These were further selected based
on allele frequency in databases and impact on the protein,
and on higher frequency in HAEC than in HSCR-only
patients. Results: Genetic linkage analysis showed two candidate
regions in chromosome 2 (q24.2-q31.1 and q33.1-q33.3) to
host the modifier and confer susceptibility to the disease
among BMPR2 mutation carriers. The modifier is predicted
to be common in the population and to be inherited in an
autosomal recessive mode. The first region is located 30 Mb
upstream from BMPR2, while the second region overlaps
the BMPR2 locus itself. Results: We have thus identified 86 variants in 90 genes,
which were ranked based on their frequency in the general
population,
predicted
effect,
association
p-value,
and
recurrence in the samples. We also explored the gene Results: We have thus identified 86 variants in 90 genes,
which were ranked based on their frequency in the general
population,
predicted
effect,
association
p-value,
and
recurrence in the samples. We also explored the gene Abstracts from the 51st European Society of Human Genetics Conference: Posters 83 pathways, biological role, and PubMed citations, particu-
larly in regard to immune and inflammation processes. We
are currently validating the five top variants and replicating
the results on a larger panel of 25 HAEC and 45 HSCR-
only patients. monoallelic EHHADH mutation, inherited only from the
mother that presented bilaterally kidney stones and urinary
calculi history, associated with Fanconi renotubular syn-
drome 3. The same genetic background was identified also
in fetal DNA extracted from amniotic fluid. Conclusions: We have identified a few very promising
candidate genes. The most promising variant/s will undergo
functional
tests
to
confirm
its/their
role
in
HAEC
development. Treatment of IIH in early age is problematic as vitamin D
therapy often exacerbates hypercalcemia. P03.26A The application of targeted sequencing and whole exome
analysis to identify disease-causing variants in familial
pulmonary fibrosis 1Medical Genetics Unit, Department of Biomedical
Experimental and Clinical Sciences, Firenze, Italy, 2Medical
Genetics Unit, Meyer Children's University Hospital, Florence,
Italy, Firenze, Italy, 3Nephrology and dialysis Unit, Meyer
Children's Hospital, Florence, Firenze, Italy, 4Prenatal
Diagnosis Unit, Meyer Children's University Hospital, Firenze,
Italy, 5Department of Diagnostic Imaging, Meyer Children's
University Hospital, Firenze, Italy, 6Medical Genetics Unit,
Department of Biomedical Experimental and Clinical Sciences
"Mario Serio", University of Florence, Florence, Italy S. Wilkinson1,2, U. Hodgson3, F. Honti1, H. Beckwith1,
P. Molyneaux4, W. O. Cookson2, T. Maher4, M. Moffatt2,
T. Laitinen3, D. J. Morris-Rosendahl1 S. Wilkinson1,2, U. Hodgson3, F. Honti1, H. Beckwith1,
P. Molyneaux4, W. O. Cookson2, T. Maher4, M. Moffatt2,
T. Laitinen3, D. J. Morris-Rosendahl1 1Clinical Genetics and Genomics Laboratory, Royal Brompton
and Harefield NHS Foundation Trust, London, United
Kingdom, 2Genomic Medicine, National Heart and Lung
Institute, Imperial College London, London, United Kingdom,
3Department of Pulmonology, University of Helsinki, Helsinki,
Finland, 4Fibrosis Research Group, National Heart and Lung
Institute, Imperial College London, London, United Kingdom Idiopathic infantile hypercalcemia (IIH) is characterized by
severe hypercalcemia with failure to thrive, vomiting,
dehydration, and nephrocalcinosis (NC). NC has not a well
understood genesis. Monogenic causes were restricted to
rare genetic syndromes/tubulopathies. Introduction: Idiopathic Pulmonary Fibrosis (IPF) is one of
the most common forms of interstitial pneumonia and is
irreversible. It has a late age of onset, mostly between 55-75
years (median 66 years) and average survival rate of 3 years
post diagnosis. Using next-generation sequencing (NGS) of
whole exome (WES) and a targeted respiratory gene panel
(Respigene) on Finnish and UK families with familial PF
(FPF), we identified many new potentially pathogenic var-
iants causative of FPF. We examined a 24 month old child for advanced bilateral
medullary
nephrocalcinosis
(accidentally
detected
at
15 months), failure to thrive, mild hypercalcemia/hypercal-
ciuria,
moderate
hypophosphatemia
without
proximal
tubulopathy, renal failure and skeletal dysplasia. He’s the
firstborn of healthy consanguineous parents and the mother
was pregnant; a maternal uncle has had recurrent nephro-
litiasis since the age of 22 years old. Whole exome
sequencing (WES) was performed in order to clarify the
molecular diagnosis and to offer a prenatal test. P03.25D F. Peluso: None. V. Palazzo: None. L. Dosa: None. G. Traficante: None. S. Landini: None. R. Artuso: None. A. Provenzano: None. P. Reho: None. E. Bosi: None. G. Carignani: None. A. La Barbera: None. A. Pagliazzi:
None. G. Forzano: None. F. Becherucci: None. M. Materassi: None. R. Biagiotti: None. C. Defilippi: None. P. Romagnani: None. S. Giglio: None. Search for genetic risk factors in Hirschsprung's disease
associated enterocolitis by Whole Exome Sequencing Considering the
possibility to check an affected subject from the first month
of life since the mother decided to continue the pregnancy,
our data could give new insights about the temporal
expression profile of SLC34A1 in the kidney and an
explanation to the development of hypercalcemia in child-
hood, where intestinal absorption and renal handling of
calcium and phosphate likely differ from older patients with
other forms of IIH. Acknowledgments: Funded by the Italian Ministry of
Health (grant GR-2011-02347381) T. Bachetti: None. G. Santamaria: None. M. Mosconi:
None. S. Sartori: None. M. De Filippo: None. A. Pini
Prato: None. I. Ceccherini: None. F. Lantieri: None. Whole exome sequencing to detect the cause of early onset
nephrocalcinosis Whole exome sequencing to detect the cause of early onset
nephrocalcinosis F. PELUSO1, V. Palazzo2, L. Dosa2, G. Traficante2, S. Landini1,
R. Artuso2, A. Provenzano1, P. Reho1, E. Bosi1, G. Carignani1,
A. La Barbera1, A. Pagliazzi1, G. Forzano1, F. Becherucci3,
M. Materassi3, R. Biagiotti4, C. Defilippi5, P. Romagnani3,6,
S. Giglio1,2 M. Materassi3, R. Biagiotti4, C. Defilippi5, P. Romagnani3,6,
S. Giglio1,2 P03.26A Materials and Methods: WES and targeted analysis of
42 genes previously associated with interstitial lung disease
(ILD) was performed on 25 individuals (21 affected and 4
unaffected) from families with severe, early-onset FPF. WES revealed a novel homozygous SLC34A1 mutation
linked to IIH and Fanconi renotubular syndrome 2, and a J. del Picchia 84 decreased
IVC
levels
were
strongly
associated
with
increased risk of IPF (p< 2.9x10-10). The non-coding allele
that decreased IVC levels increased IVD expression (p<
3.4x10-12). Thus, we hypothesized that low IVC levels are
associated with higher risk of IPF. To test our hypothesis,
we ran a case-control pilot study and found that IVC levels
are decreased in IPF cases relative to controls (P = 2x10-3). Therefore, these data strongly suggest the IVC metabolic
pathway could play a role in IPF etiology. W
tl
fi
i
lt i
l decreased
IVC
levels
were
strongly
associated
with
increased risk of IPF (p< 2.9x10-10). The non-coding allele
that decreased IVC levels increased IVD expression (p<
3.4x10-12). Thus, we hypothesized that low IVC levels are
associated with higher risk of IPF. To test our hypothesis,
we ran a case-control pilot study and found that IVC levels
are decreased in IPF cases relative to controls (P = 2x10-3). Therefore, these data strongly suggest the IVC metabolic
pathway could play a role in IPF etiology. We are currently confirming our results in a larger case Variant assessment was performed according to ACMG
guidelines, using an in-house bioinformatics pipeline for
classification of SNVs and CNVs, configured to rare
respiratory conditions. Results: Twelve of 25 individuals were found to have
variants in ACMG classes 3-5 (VUS to pathogenic), in
genes previously associated with FPF and which segregated
in families where available. Seven patients were found to
have potentially pathogenic variants in TERT, TERC and
RTEL1, genes all previously associated with telomerase and
telomere integrity. 13/21 (61.9%) patients were positive for
the
MUC5B
SNP,
rs35705950,
as
opposed
to
1/4
unaffected individuals. No CNVs were found in any genes
related to ILD. We are currently confirming our results in a larger case-
control study. We are currently confirming our results in a larger case-
control study. Conclusion: IPF presents seriously unmet clinical needs. Building on strong preliminary data, we propose that IVC
levels are associated with higher risk of IPF. P03.28C S. Wilkinson: None. U. Hodgson: None. F. Honti:
None. H. Beckwith: None. P. Molyneaux: None. W.O. Cookson: None. T. Maher: None. M. Moffatt: None. T. Laitinen: None. D.J. Morris-Rosendahl: None. P03.26A IVC represents
a clinically relevant biomarker and its enzyme, IVD, could
represent an entirely novel IPF drug target. Conclusion: These findings confirm that genes involved
in telomere function play a major role in familial pulmonary
fibrosis. These findings have potential implications for
family members and raise the possibility of predictive
testing and early therapeutic intervention in FPF. A. Cerani: None. S. Ross: None. D.A. Schwartz: None. P. Wolters: None. B. Richards: None. A. Cerani: None. S. Ross: None. D.A. Schwartz: None. P. Wolters: None. B. Richards: None. Interallelic interactions mediated by the oligomerization of
podocin E. Balogh1,2, P. Stráner3, G. Schay4, C. Arrondel5, Á. Mikó6,2,
G. L’Auné1,2, A. Benmerah5, A. Perczel3, D. K. Menyhárd3,
C. Antignac5,7,8, G. Mollet9, K. Tory1,2 P03.27B A metabolic biomarker for idiopathic pulmonary fibrosis: a
two sample Mendelian randomization study and a case-
control study A metabolic biomarker for idiopathic pulmonary fibrosis: a
two sample Mendelian randomization study and a case-
control study 1MTA-SE Lendulet Nephrogenetic Laboratory, Budapest,
Hungary, 2Semmelweis University, Ist Department of
Pediatrics, Budapest, Hungary, 3MTA-ELTE Protein Modeling
Research Group and Laboratory of Structural Chemistry and
Biology, Eötvös Loránd University, Budapest, Hungary,
4Semmelweis University, Department of Biophysics and
Radiation Biology, Budapest, Hungary, 5Laboratory of
Hereditary Kidney Diseases, INSERM, UMR 1163, Imagine
Institute, Paris, France, 6MTA-SE Lendulet Nephrogenetic
Laboratory, Hungarian Academy of Sciences, Budapest,
Hungary, 7Université Paris Descartes-Sorbonne Paris Cité,
Imagine Institute, Paris, France, 8Assistance Publique –
Hôpitaux de Paris, Hôpital Necker-Enfants Malades,
Département de Génétique, Paris, France, 9Laboratory of
Hereditary Kidney Diseases, INSERM, UMR 1163, Paris,
France A. Cerani1,2, S. Ross1,2, D. A. Schwartz3, P. Wolters4,
B. Richards1,2 A. Cerani1,2, S. Ross1,2, D. A. Schwartz3, P. Wolters4,
B. Richards1,2 1McGill University, Montreal, QC, Canada, 2Lady Davis
Institute for Medical Research, Montreal, QC, Canada,
3University of Colorado, Aurora, CO, United States,
4University of California - San Francisco, San Francisco, CA,
Canada Introduction: Idiopathic pulmonary fibrosis (IPF) is a
lethal disease without current effective treatments. There is,
therefore, an urgent need to find and validate biomarkers for
diagnosis, prognosis, as well as potential drug targets. We
propose to combine genomics and metabolomics to meet
these objectives. NPHS2, the major gene of steroid-resistant nephrotic syn-
drome, encodes podocin, a membrane-anchored component
of the slit diaphragm. We formerly showed that its R229Q
variant is pathogenic only when trans-associated to specific
3’ missense mutations secondary to an altered C-terminal
dimerization. We aimed to determine the membrane tar-
geting and the oligomerization of podocin in function of its
C-terminal integrity. Podocin localization was studied in
podocytes
co-transfected
with
GFP-
and
HA-tagged Methods and Results: To identify potential metabolites
associated with IPF, we used an IPF GWAS that identified
novel single nucleotide polymorphisms (SNP) associated
with isovaleryl dehydrogenase (IVD), whose inhibition is
known to increase the metabolite isovalerylcarnitine (IVC). Concurrently, we collaborated in a metabolite GWAS in
healthy subjects that found the same IPF-associated SNP to
be associated with IVC blood levels. Through 2-sample
Mendelian randomization, we observed that genetically Abstracts from the 51st European Society of Human Genetics Conference: Posters 85 podocin variants. Oligomerization was assessed by mea-
suring the FRET efficiency between maleimide-stained
podocin variants and by size exclusion chromatography. We found the oligomerization to occur exclusively through
the C-terminal tail (residues 283-382): principally through
the 283-313 (H1) and the 332-348 residues. A metabolic biomarker for idiopathic pulmonary fibrosis: a
two sample Mendelian randomization study and a case-
control study Schay: None. C. Arrondel: None. Á. Mikó: None. G. L’Auné: None. A. Benmerah: None. A. Perczel: None. D. K. Menyhárd:
None. C. Antignac:
None. G. Mollet:
None. K. Tory: None. E.G. Sheridan:
None. S. Bonnefoy:
None. C.M. Watson: None. K.D. Kernohan: None. M. Lemos: None. S. Hutchinson1: None. J. Poulter: None. C. O'Call-
aghan: None. R.A. Hirst: None. A. Rutman: None. L. Huang: None. T. Hartley: None. D. Grynspan: None. C. Li: None. I.M. Carr: None. D.B. Bonthron: None. M. Leroux: None. K.B. Boycott: None. P. Bastin: None. A metabolic biomarker for idiopathic pulmonary fibrosis: a
two sample Mendelian randomization study and a case-
control study The interallelic
interactions are mediated by oligomerization: while the
monomer-forming R286Tfs*17 podocin remained mem-
branous irrespective of the coexpressed podocin variant,
podocin variants with an intact H1 significantly influenced
each other’s localization (r2=0.68, P = 9.2x10-32). The
dominant negative effect resulting in intracellular retention
of
the
pathogenic
F344Lfs*4-R229Q
heterooligomer
occured in parallel with a reduction in the FRET efficiency,
suggesting a conformational rearrangement. In contrast,
oligomerization with membranous podocin variants pre-
vented the endocytosis of F344Lfs*4 or F344* podocin
mutants induced by C-terminal truncation. In conclusion,
oligomerization of podocin can mediate both a dominant
negative effect and interallelic complementation. Interallelic
interactions of 3’ NPHS2 mutations are not restricted to the
R229Q variant and have to be considered in compound
heterozygous individuals. Supported by MTA-SE Lendulet
Research
Grant
(LP2015-11/2015),
NKFIA/OTKA
K109718, K116305, KH125566, MedinProt Synergy grant,
(ANR-10-IAHU-01), EURenOmics (2012-305608), ANR
(GenPod project ANR-12-BSV1-0033.01). Defects in motile cilia result in conditions characterised by
impaired pulmonary mucus clearance, susceptibility to
chronic recurrent respiratory infections, male infertility and
laterality defects. Here we report that biallelic variants in
LRRC56 (also known as ODA8), result in a phenotype
comprising laterality defects and chronic pulmonary infec-
tions. Investigation of cultured patient epithelial cells
revealed
severely
dyskinetic
cilia,
but
no
structural
abnormalities on transmission electron microscopy. In
human cells, we show that LRRC56 interacts with the
intraflagellar transport (IFT) protein IFT88. In the model organism Trypanosoma brucei, we show
LRRC56 is recruited during later phases of flagellar
assembly before being removed during flagellum matura-
tion, after cell division. We disrupted anterograde or
retrograde IFT in T Brucei by use of RNAi. This showed
that LRRC56 acts as an IFT cargo, but only at later stages of
flagellar construction. LRRC56 localisation was unchanged in DNAI1RNAi and
ODA7RNAi mutants that are defective in their dynein arm
constitution or cytoplasmic preassembly, respectively. Thus
LRRC56 does not require the presence of dynein arms to be
associated with flagella. In T. brucei carrying LRRC56 null mutations, or a
mutation (p.Leu259Pro) corresponding p.Leu140Pro variant
seen in one of the affected families, we observed abnormal
ciliary beat patterns and an absence of outer dynein arms
restricted to the distal portion of the axoneme. These
findings confirm that deleterious variants in LRRC56 result
in human disease, and suggest this protein has a likely role
in dynein transport during cilia assembly that is evolutio-
narily important for cilia motility. E. Balogh: None. P. Stráner: None. G. P03.29D Biallelic Mutations in LRRC56, encoding a protein
associated with intraflagellar transport, causes defects in
mucociliary clearance and laterality E. G. Sheridan1, S. Bonnefoy2, C. M. Watson1, K. D. Kernohan3,
M. Lemos4, S. Hutchinson12, J. Poulter1, C. O'Callaghan5, R. A. Hirst6, A. Rutman6, L. Huang3, T. Hartley3, D. Grynspan7,
C. Li8, I. M. Carr1, D. B. Bonthron1, M. Leroux8, Care4Rare
Canada Consortium, K. B. Boycott3, P. Bastin2 1University of Leeds, Leeds, United Kingdom, 2Institut Pasteur,
Paris, France, 3University of Ottawa, Ottawa, ON, Canada,
4Institut Pateur, Paris, France, 5University College London,
London, United Kingdom, 6University of Leicester, Leicester,
United Kingdom, 7Children’s Hospital of Eastern Ontario,
Ottawa, ON, Canada, 8Simon Fraser University, Burnaby, BC,
Canada 1Latvian Biomedical Research and Study Centre, Riga, Latvia,
2Riga East Clinical University Hospital, Riga, Latvia, 3Pauls
Stradins Clinical University Hospital, Riga, Latvia I. Elbere1, I. Kalnina1, I. Silamikelis1, I. I. Dindune1,
L. Gulbinska1, L. Zaharenko1, I. Radovica-Spalvina1, D. Gudra1,
D. Fridmanis1, I. Konrade2, V. Pirags1,3, J. Klovins1 P03.30A Metformin induced changes in gut microbiome composition
in healthy individuals and newly-diagnosed type 2 diabetes
patients 1University of Leeds, Leeds, United Kingdom, 2Institut Pasteur,
Paris, France, 3University of Ottawa, Ottawa, ON, Canada,
4Institut Pateur, Paris, France, 5University College London,
London, United Kingdom, 6University of Leicester, Leicester,
United Kingdom, 7Children’s Hospital of Eastern Ontario,
Ottawa, ON, Canada, 8Simon Fraser University, Burnaby, BC,
Canada 1University of Leeds, Leeds, United Kingdom, 2Institut Pasteur,
Paris, France, 3University of Ottawa, Ottawa, ON, Canada,
4Institut Pateur, Paris, France, 5University College London,
London, United Kingdom, 6University of Leicester, Leicester,
United Kingdom, 7Children’s Hospital of Eastern Ontario,
Ottawa, ON, Canada, 8Simon Fraser University, Burnaby, BC,
Canada I. Elbere1, I. Kalnina1, I. Silamikelis1, I. I. Dindune1,
L. Gulbinska1, L. Zaharenko1, I. Radovica-Spalvina1, D. Gudra1,
D. Fridmanis1, I. Konrade2, V. Pirags1,3, J. Klovins1
1Latvian Biomedical Research and Study Centre, Riga, Latvia,
2Riga East Clinical University Hospital, Riga, Latvia, 3Pauls
Stradins Clinical University Hospital, Riga, Latvia J. del Picchia 86 Metformin is a biguanide class agent widely used as a first-
line treatment for type 2 diabetes (T2D). Despite its
advantages, metformin has variable therapeutic effects,
contraindications, and side effects. Previous findings have
led to the hypothesis that both the beneficial and adverse
effects of metformin are partially explained by its interac-
tion with the gut microbiome, yet details of these mechan-
isms remain obscure. Introduction: Kidney diseases can be caused by a wide
spectrum of underlying conditions and, in practice, the
exact cause of a patient’s renal disease often remains
unknown. Knowing the precise genetic defect is important
in terms of its diagnostic, prognostic value and appropriate
genetic counselling and is likely to become more crucial for
directing specific therapy. NGS has opened up a new field
allowing the identification of previously undiagnosed her-
editary nephropathies. This study aims to investigate the
etiology of genetic kidney diseases in the clinical routine of
our hospital´s patient population. Metformin is a biguanide class agent widely used as a first-
line treatment for type 2 diabetes (T2D). Despite its
advantages, metformin has variable therapeutic effects,
contraindications, and side effects. Previous findings have
led to the hypothesis that both the beneficial and adverse
effects of metformin are partially explained by its interac-
tion with the gut microbiome, yet details of these mechan-
isms remain obscure. The study was conducted to investigate the effects of
metformin treatment on the composition of human gut
microbiome. P03.30A Results: Regarding the analysis, it is done differently
depending on the patient: restricted to the proband and
known genes in patients with a specific phenotype, or open
to a trio (including parents) when clinicians suggest an
unspecific phenotype. Diagnostic yields are around 51-54%. Probable changes in the gut microbiome induced by
metformin intake were assessed employing massive parallel
sequencing of the 16S rRNA gene V3 region. A week-long metformin treatment rapidly and signifi-
cantly decreased alpha diversity of gut microbiome among
both groups. Concurrently, significant changes in several
taxonomic groups were observed, including an increase in
abundance
of
opportunistic
pathogens
representing
Escherichia-Shigella
genus,
and
decrease
in
possibly
harming family Peptostreptococcaceae. Together these
findings support the role of metformin in the modulation
of the gut microbiome composition and the hypothesis that
there may be some specific interactions further linked with
efficacy and side effects of metformin. Remarking is the genetic analysis of the PKD1 (responsible
for most ADPKD cases), a diagnostic challenge: the 5′-
region of the gene overlapped with a pseudogene. Our
approach “in a tube” validated by Long-Rage PCR and
Sanger sequencing yielded around 80% and it is cost-
effective. Conclusions: We share our management of the inherited
nephropathies cases under a model pointed out to precision
medicine, which includes a multidisciplinary committee for
discussing the cases included in this customized NGS panel. J. Nevado: None. R. Mena: None. V. Gómez del Pozo:
None. R. Peces: None. M. Melgosa: None. L. Espinosa:
None. R. Selgas: None. P.D. Lapunzina: None. This project was supported by ERDF No.:1.1.1.1/16/A/
091. I. Elbere: None. I. Kalnina: None. I. Silamikelis: None. I.I. Dindune: None. L. Gulbinska: None. L. Zaharenko:
None. I. Radovica-Spalvina: None. D. Gudra: None. D. Fridmanis: None. I. Konrade: None. V. Pirags: None. J. Klovins: None. Genetic causes of early onset obesity are frequently
identified in a tertiary pediatric obesity cohort Genetic causes of early onset obesity are frequently
identified in a tertiary pediatric obesity cohort L. Kleinendorst1, O. Abawi2, A. E. Brandsma3, M. H. Jongejan4,
E. F. C. Van Rossum2, B. Van der Zwaag5, E. L. T. Van den
Akker2, M. M. Van Haelst6,1 L. Kleinendorst1, O. Abawi2, A. E. Brandsma3, M. H. Jongejan4,
E. F. C. Van Rossum2, B. Van der Zwaag5, E. L. T. Van den
Akker2, M. M. Van Haelst6,1 P03.30A The microbial DNA was extracted from: Materials and Methods: The Nefroseq® panel V2.0 is a
targeted NGS panel containing 350 genes involved in
inherited nephropathies, optimizing libraries from Kappa,
Target enrichment by SeqCap EZ system (RocheNimble-
gen), and sequencing on a NextSeq500 platform (Illumina). Results: Regarding the analysis, it is done differently
depending on the patient: restricted to the proband and
known genes in patients with a specific phenotype, or open
to a trio (including parents) when clinicians suggest an
unspecific phenotype. Diagnostic yields are around 51-54%. Remarking is the genetic analysis of the PKD1 (responsible
for most ADPKD cases), a diagnostic challenge: the 5′-
region of the gene overlapped with a pseudogene. Our
approach “in a tube” validated by Long-Rage PCR and
Sanger sequencing yielded around 80% and it is cost-
effective. (1.) Stool samples obtained from 18 healthy nondiabetic
individuals at three time points during metformin treatment:
M0 - before metformin treatment, M24h - 24 hours after the
first metformin dose, and M7d - after a week-long
metformin administration; Materials and Methods: The Nefroseq® panel V2.0 is a
targeted NGS panel containing 350 genes involved in
inherited nephropathies, optimizing libraries from Kappa,
Target enrichment by SeqCap EZ system (RocheNimble-
gen), and sequencing on a NextSeq500 platform (Illumina). Materials and Methods: The Nefroseq® panel V2.0 is a
targeted NGS panel containing 350 genes involved in
inherited nephropathies, optimizing libraries from Kappa,
Target enrichment by SeqCap EZ system (RocheNimble-
gen), and sequencing on a NextSeq500 platform (Illumina). g
q
g
q
p
Results: Regarding the analysis, it is done differently
depending on the patient: restricted to the proband and
known genes in patients with a specific phenotype, or open
to a trio (including parents) when clinicians suggest an
unspecific phenotype. Diagnostic yields are around 51-54%. Remarking is the genetic analysis of the PKD1 (responsible
for most ADPKD cases), a diagnostic challenge: the 5′-
region of the gene overlapped with a pseudogene. Our
approach “in a tube” validated by Long-Rage PCR and
Sanger sequencing yielded around 80% and it is cost-
effective. (2.) Stool samples acquired from 11 newly-diagnosed
T2D patients at two concordant time points - M0 and M7d. (2.) Stool samples acquired from 11 newly-diagnosed
T2D patients at two concordant time points - M0 and M7d. J. Klar1, H. Engstrand Lilja2, J. Mattisson1, N. Dahl1 1Department of Immunology, Genetics and Pathology, Uppsala
University, Uppsala, Sweden, 2Department of Women's and
Children's Health, Section of Paediatric Surgery, Uppsala
University Hospital, Uppsala, Sweden Table 1. Underlying genetic causes identified in our
pediatric obesity patient cohort Genetic disorder
No. of
patients
Characteristics
Melanocortin-4 receptor,
dominantly inherited obesity
5
Heterozygous MC4R
mutation
Melanocortin-4 receptor
deficiency
2
Homozygous or compound
heterozygous MC4R
mutation
Leptin receptor deficiency
5
Homozygous or compound
heterozygous LEPR
mutation
16p11.2 deletion syndrome
3
Typical 16p11.2 deletion
Melanocortin-2 receptor
accessory protein 2 associated
obesity
2
Heterozygous MRAP2
mutation in 2 sibs
Pseudohypoparathyroidism
type 1A
2
Heterozygous GNAS
mutation
Central hypothyroidism
1
Heterozygous IGSF1
mutation
Cohen syndrome
1
Homozygous VPS13B
mutation
Mental retardation,
autosomal dominant 39
1
Heterozygous MYT1L
mutation
Maternal UPD 14
1
mUPD 14
Proprotein convertase-1
associated obesity
1
Heterozygous PCSK1
mutation Introduction: Oesophageal atresia (OA) is the most com-
mon congenital anomaly of the oesophagus with an inci-
dence
of
around
1/3500
births. OA
with
a
low
tracheoesophageal fistula constitute 85% of all cases and
syndromic OA at least 50% of cases. Genetic variants have
been identified in a proportion of patients with syndromic
OA but the genetics behind isolated forms remains elusive. Introduction: Oesophageal atresia (OA) is the most com-
mon congenital anomaly of the oesophagus with an inci-
dence
of
around
1/3500
births. OA
with
a
low
tracheoesophageal fistula constitute 85% of all cases and
syndromic OA at least 50% of cases. Genetic variants have
been identified in a proportion of patients with syndromic
OA but the genetics behind isolated forms remains elusive. Materials and Methods: We identified three families
with recurrent and isolated OA suggesting inherited factors
behind
the
malformation. Whole
genome
sequencing
(WGS; Illumina) was performed on DNA from affected
individuals (n = 6) and presumed obligate and healthy
carriers (n = 4). Materials and Methods: We identified three families
with recurrent and isolated OA suggesting inherited factors
behind
the
malformation. Whole
genome
sequencing
(WGS; Illumina) was performed on DNA from affected
individuals (n = 6) and presumed obligate and healthy
carriers (n = 4). Results and Conclusions: We identified single nucleo-
tide variants (SNVs) and small insertions/deletions (indels)
using GATK, and structural variants (SVs) using Manta. After stringent filtering (frequency = 0) against the Swedish
1000 genome project (SweGen), only one gene (DOCK4)
remained that contains rare SNVs in all 10 individuals. P03.32C Targeted-NGS panel for inherited nephropathies: an
example of clinical utility in a tube Targeted-NGS panel for inherited nephropathies: an
example of clinical utility in a tube 1Academic Medical Center, Amsterdam, Netherlands,
2Erasmus MC, University Medical Center Rotterdam,
Rotterdam, Netherlands, 3Maasstad Hospital, Rotterdam,
Netherlands, 4Franciscus Gasthuis, Rotterdam, Netherlands,
5University Medical Center Utrecht, Utrecht, Netherlands, 6VU
university medical center, Amsterdam, Netherlands 1Academic Medical Center, Amsterdam, Netherlands,
2Erasmus MC, University Medical Center Rotterdam,
Rotterdam, Netherlands, 3Maasstad Hospital, Rotterdam,
Netherlands, 4Franciscus Gasthuis, Rotterdam, Netherlands,
5University Medical Center Utrecht, Utrecht, Netherlands, 6VU
university medical center, Amsterdam, Netherlands J. Nevado1,2, R. Mena1, V. Gómez del Pozo1, R. Peces3,
M. Melgosa4, L. Espinosa4, R. Selgas3, P. D. Lapunzina1,2
1INGEMM-IdiPaz, Madrid, Spain, 2CIBERER, Madrid, Spain,
3Adult Nephrology Department, HULP-IdiPaz, Madrid, Spain,
4Pediatric Nephrology Department, HULP-IdiPaz, Madrid,
Spain J. Nevado1,2, R. Mena1, V. Gómez del Pozo1, R. Peces3,
M. Melgosa4, L. Espinosa4, R. Selgas3, P. D. Lapunzina1,2 1INGEMM-IdiPaz, Madrid, Spain, 2CIBERER, Madrid, Spain,
3Adult Nephrology Department, HULP-IdiPaz, Madrid, Spain,
4Pediatric Nephrology Department, HULP-IdiPaz, Madrid,
Spain Obesity is predominantly considered a multifactorial dis-
order. In unselected patient cohorts, an underlying genetic
diagnosis can be established in only a minority of cases. Abstracts from the 51st European Society of Human Genetics Conference: Posters 87 Table (continued)
Genetic disorder
No. of
patients
Characteristics
Proopiomelanocortin
associated obesity
1
Deletion 2p, including
POMC gene
Pseudohypoparathyroidism
type 1B
1
Heterozygous STX16
mutation
Single-minded 1 associated
obesity
1
Deletion 6q16.3, including
SIM1 gene
Spastic paraplegia type 11
1
Compound heterozygous
SPG11 mutation They typically present with early-onset, severe obesity. Establishing a genetic diagnosis can lead to personalized
treatment, reduce stigma and support reproductive decision-
making. This study provides an overview of obesity-
associated mutations and copy number variations (CNV’s)
identified in this selected population. In 174 obese children, referred to the pediatric obesity
center Centrum Gezond Gewicht between 2012 and 2017,
diagnostic sequencing of 52 obesity-associated genes, CNV
detection by SNP-microarray analysis and, on clinical
suspicion, specific additional diagnostics were performed to
identify genetic causes of obesity. In 28 patients (16.1%), an underlying genetic cause was
identified (table 1). In an additional 22 patients (12.6%), a
novel CNV or sequence variant of unknown clinical
significance (VUS) was shown in obesity-associated genes,
for which the role in the phenotype has yet to be confirmed. L. Kleinendorst:
None. O. Abawi:
None. A.E. Brandsma: None. M.H. Jongejan: None. E.F.C. Van
Rossum: None. B. Van der Zwaag: None. E.L.T. P03.32C Van
den Akker: None. M.M. Van Haelst: None. A definitive obesity-associated genetic diagnosis was
made in 16.1% of our patients with early-onset obesity. This
may increase, if follow-up studies in patients with VUS
confirm a causal role for their variants. This diagnostic yield
is relatively high compared to similar studies and shows that
genetic testing can be highly relevant in selected obese
patients, especially when personalized treatment becomes
available. Isolated oesophageal atresia associated with rare structural
variants identified by whole genome sequencing Isolated oesophageal atresia associated with rare structural
variants identified by whole genome sequencing J. Klar1, H. Engstrand Lilja2, J. Mattisson1, N. Dahl1 J. Klar1, H. Engstrand Lilja2, J. Mattisson1, N. Dahl1 Next Generation Sequencing (NGS) differential genetic
analysis in polycystic kidney diseases: work in progress T. Szabó1, Z. Maróti2, T. Kalmár2, L. Madar3, É. Gombos3,
O. Orosz3, K. Tory4,5, I. Balogh3 T. Szabó1, Z. Maróti2, T. Kalmár2, L. Madar3, É. Gombos3,
O. Orosz3, K. Tory4,5, I. Balogh3 G. Pipitone1, S. Calzavara1, R. Magistroni2,3, S. Merella1,
A. Boletta2, M. Ferrari1,4,5, P. Carrera1,4 G. Pipitone1, S. Calzavara1, R. Magistroni2,3, S. Merella1,
A. Boletta2, M. Ferrari1,4,5, P. Carrera1,4 1Department of Pediatrics, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary, 2Department of Pediatrics,
Faculty of Medicine, University of Szeged, Szeged, Hungary,
3Division of Clinical Genetics, Department of Laboratory
Medicine, Faculty of Medicine, University of Debrecen,
Debrecen, Hungary, 4Ist Department of Pediatrics,
Semmelweis University, Budapest, Hungary, 5MTA-SE
Lendulet Nephrogenetic Laboratory, Budapest, Hungary 1Clinical molecular biology laboratory, IRCCS San Raffaele,
Milano, Italy, 2Molecular basis of polycystic kidney disease
unit, Division of genetics and cell biology, IRCCS San
Raffaele, Milano, Italy, 3Division of nephrology and dialysis A. O.U. Policlinico, University of Modena and Reggio Emilia,
Modena, Italy, 4Unit of genomics for human disease diagnosis,
Division of genetics and cell biology, IRCCS San Raffaele,
Milano, Italy, 5University Vita-Salute San Raffaele, Milano,
Italy 1Clinical molecular biology laboratory, IRCCS San Raffaele,
Milano, Italy, 2Molecular basis of polycystic kidney disease
unit, Division of genetics and cell biology, IRCCS San
Raffaele, Milano, Italy, 3Division of nephrology and dialysis A. O.U. Policlinico, University of Modena and Reggio Emilia,
Modena, Italy, 4Unit of genomics for human disease diagnosis,
Division of genetics and cell biology, IRCCS San Raffaele,
Milano, Italy, 5University Vita-Salute San Raffaele, Milano,
Italy Introduction: Autosomal recessive polycystic kidney dis-
ease (ARPKD) is genetically one of the least heterogeneous
ciliopathies, resulting primarily from mutations of PKHD1. However, 15-40% of patients diagnosed with ARPKD are
found not to carry PKHD1 mutations. Aim of this study was
to identify whether mutations in other genes might explain
these cases. Polycystic Kidney Diseases are clinically and genetically
heterogeneous conditions mainly caused by mutations in
PKD1/2. In the absence of a family history and/or in the
presence of atypical presentations, other cystic diseases or
other clinical conditions should be considered for a differ-
ential diagnosis. Thanks to NGS is now possible to mod-
ulate
genetic
test
ranging
from
a
few
genes
to
a
comprehensive clinical-exome testing, allowing differential
genetic analysis based on the clinical suspicion. To evaluate
this approach, we applied the illumina Clinical-Exome NGS
protocol in 10 patients negative for ADPKD. J. Klar1, H. Engstrand Lilja2, J. Mattisson1, N. Dahl1 Furthermore, we identified a total of 186 SVs in the affected
individuals. Interestingly, we observe a significant enrich-
ment of rare SVs on chromosome 21, suggesting a role for
this chromosome in the aetiology of isolated OA in our
cohort. 88 J. del Picchia Grants: This work was supported by grants from the
Swedish Research Council (2015-02424). Grants: This work was supported by grants from the
Swedish Research Council (2015-02424). G. Pipitone: None. S. Calzavara: None. R. Magistroni:
None. S. Merella: None. A. Boletta: None. M. Ferrari:
None. P. Carrera: None. J. Klar: None. H. Engstrand Lilja: None. J. Mattisson:
None. N. Dahl: None. J. Klar: None. H. Engstrand Lilja: None. J. Mattisson:
None. N. Dahl: None. P03.38A
Prader-Willi syndrome: clinical particularities found in 15
patients Follow-up revealed: skeletal deformities that con-
traindicated GH therapy in one case, epilepsy and multiple
allergies in another and central fever that led to death in
another. In conclusion, we present a clinical study on 15
PWS patients to illustrate particular features and long-time
evolution. Prader-Willi syndrome (PWS) is a genetic disorder char-
acterized by severe hypotonia and feeding difficulties in
early infancy, followed by excessive eating and morbid
obesity in childhood, intellectual disability, small hands and
feet and hypogonadism. The genetic defect (microdeletion,
uniparental disomy or imprinting defect) involves region
15q11.2-q13. We performed a clinical and genetic study of
15 patients diagnosed with PWS in Iaşi Medical Genetics
Center to evaluate the relevance of clinical features for the
diagnosis, identify particularities and possible complica-
tions. All cases were confirmed using MS-MLPA or FISH
testing. We have identified neonatal hypotonia in 11/15
cases, feeding difficulties in infancy in 12/15, excessive
weight gain in 9/15 (6/15 being under GH therapy), char-
acteristic facial features in 13/15 and acromicria in 8/15. Micropenis (2/6), cryptorchidism (4/6) and hypoplasia of
labia minora (1/9) indicated the presence of hypogenitalism. High pain threshold was seen in 5/15 cases, sleep apnea as
well as skin picking in 4/15, ADHD, strabismus and
incontinence were associated in 2/15. One case associated
central fever. Particular features identified included: skeletal
anomalies (5/15), congenital heart defects (4/15), cerebral
malformations (3/15), ADHD (2/15), epilepsy (1/15) and
multiple allergies (1/15). The correlation of the clinical
features with the type of genetic defect will be illustrated in
detail. Follow-up revealed: skeletal deformities that con-
traindicated GH therapy in one case, epilepsy and multiple
allergies in another and central fever that led to death in
another. In conclusion, we present a clinical study on 15
PWS patients to illustrate particular features and long-time
evolution. Materials
and
Methods:
Using
Next
Generation
Sequencing (NGS) approach and Clinical-Exome Sequen-
cing Panel with 4813 genes, we analysed 74 genes related to
PCD and other pediatric lung diseases in a cohort of 21
Serbian patients with clinically suspected PCD. Results: After variant filtering and prioritization, the
molecular diagnosis of PCD was achieved in 14/21
(66.67%) patients. The most frequently mutated gene was
DNAH5 (23.33%). We have also genetically diagnosed
asthma, NRDS, and bronchiectasis without CF, leading to
detection rate of 95.24% (20/21). Identified variants were in
homozygous,
compound
heterozygous
and
trans-
heterozygous state. P03.39B The importance of comprehensive genomic profiling in
differential diagnosis and discovery of novel disease causing
genetic variants in patients with pediatric lung diseases T. Szabó: None. Z. Maróti: None. T. Kalmár: None. L. É T. Szabó: None. Z. Maróti: None. T. Kalmár: None. L. Madar: None. É. Gombos: None. O. Orosz: None. K. Tory: None. I. Balogh: None. M. Z. Andjelkovic1, P. Minic2,3, M. Vreca1, M. Stojiljkovic1,
A. Skakic1, A. Sovtic2, M. Rodic2, V. Skodric-Trifunovic4,3,
N. Maric5, J. Visekruna2, V. Spasovski1, S. Pavlovic1 M. Z. Andjelkovic1, P. Minic2,3, M. Vreca1, M. Stojiljkovic1,
A. Skakic1, A. Sovtic2, M. Rodic2, V. Skodric-Trifunovic4,3,
N. Maric5, J. Visekruna2, V. Spasovski1, S. Pavlovic1 Next Generation Sequencing (NGS) differential genetic
analysis in polycystic kidney diseases: work in progress I. Balogh: None. of Sciences and NKFIA/OTKA K109718, KH125566 (to
Kálmán Tory). T. Szabó: None. Z. Maróti: None. T. Kalmár: None. L. Madar: None. É. Gombos: None. O. Orosz: None. K. Tory: None. I. Balogh: None. of Sciences and NKFIA/OTKA K109718, KH125566 (to
Kálmán Tory). P03.38A
Prader-Willi syndrome: clinical particularities found in 15
patients 1Institute of Molecular Genetics and Genetic Engineering,
Belgrade, Serbia, 2Mother and Child Health Care Institute of
Serbia, Belgrade, Serbia, 3School of Medicine, University of
Belgrade, Belgrade, Serbia, 4Clinic for Pulmonology, Clinical
Center of Serbia, Belgrade, Serbia, 5Clinic for children
diseases, Banja Luka, Belgrade, Serbia S. Calapod1, A. Hrisca2, R. Popescu1, E. Gorduza3,
M. Gramescu3, M. Panzaru1, L. Butnariu1, C. Rusu1 1"St.Mary” Children's Hospital - Medical Genetics Center,
Iasi, Romania, 2"St.Spiridon” Emergency Clinical Hospital -
Department of Endocrinology, Iasi, Romania, 3”Gr. T. Popa”
University of Medicine and Pharmacy - Department of Medical
Genetics, Iasi, Romania Introduction: Primary ciliary dyskinesia (PCD) is a rare
inherited autosomal recessive or X-linked disorder that
mainly affects lungs. PCD diagnosis requires well-described
clinical phenotype combined with the identification of
underlying genetic causes since clinical symptoms of PCD
overlaps with symptoms of other pediatric lung diseases. The aim of the study was to point out the significance of
comprehensive genomic profiling in differential diagnosis
of presumed PCD patients and detection of novel disease-
causing genetic variants. Prader-Willi syndrome (PWS) is a genetic disorder char-
acterized by severe hypotonia and feeding difficulties in
early infancy, followed by excessive eating and morbid
obesity in childhood, intellectual disability, small hands and
feet and hypogonadism. The genetic defect (microdeletion,
uniparental disomy or imprinting defect) involves region
15q11.2-q13. We performed a clinical and genetic study of
15 patients diagnosed with PWS in Iaşi Medical Genetics
Center to evaluate the relevance of clinical features for the
diagnosis, identify particularities and possible complica-
tions. All cases were confirmed using MS-MLPA or FISH
testing. We have identified neonatal hypotonia in 11/15
cases, feeding difficulties in infancy in 12/15, excessive
weight gain in 9/15 (6/15 being under GH therapy), char-
acteristic facial features in 13/15 and acromicria in 8/15. Micropenis (2/6), cryptorchidism (4/6) and hypoplasia of
labia minora (1/9) indicated the presence of hypogenitalism. High pain threshold was seen in 5/15 cases, sleep apnea as
well as skin picking in 4/15, ADHD, strabismus and
incontinence were associated in 2/15. One case associated
central fever. Particular features identified included: skeletal
anomalies (5/15), congenital heart defects (4/15), cerebral
malformations (3/15), ADHD (2/15), epilepsy (1/15) and
multiple allergies (1/15). The correlation of the clinical
features with the type of genetic defect will be illustrated in
detail. Next Generation Sequencing (NGS) differential genetic
analysis in polycystic kidney diseases: work in progress Analysis of
106 genes, selected due to their relation with atypical form
of PKD, was performed using an in-house validated pipe-
line (100% sensitivity, >94% specificity). Considering a
population frequency <1% in the 1000KG and Exac Data-
bases and basing on the consistency with the clinical phe-
notypes we focused on 16 candidate variants in total. Eight
of these variants were private and novel. We found: 4 novel
variants in HNF1B, BBS9, REN, GLIS3 and 5 already
described variants in BBS9, PKHD1, GLIS3, NPHP3 and
WDPCP. Moreover, one novel stop-gained and one start-
lost variant were found in INVS and GLIS3 and three
intronic and one splice donor variants were found in
TMEM138, TMEM67, IFT43 and COL4A1. Interestingly,
one frameshift variant was found in PKD1, previously
misdiagnosed by Sanger sequencing, indicating a good
performance of NGS. All variants, except one (start-lost
variant in GLIS3), were confirmed by Sanger sequencing as
well as the re-evaluation of clinical phenotypes and the
potential for family and functional studies. Materials and Methods: Thirty-six unrelated patients
with the clinical diagnosis of ARPKD were tested by
PKHD1 sequencing and MLPA. Patients without biallelic
mutations were reevaluated and tested for second locus
mutations in function of the phenotype, followed, if
negative, by clinical exome sequencing. Results: Twenty-eight patients (78%) carried PKHD1
point mutations, three of whom were heterozygotes for
small-scale alterations. Two of the three patients possessed
either a duplication of exons 33-35 or a large deletion
involving exons 1-55 in trans. All eight patients without
PKHD1 mutations had mutations in other genes (PKD1
(n = 2), HNF1B (n = 3), NPHP1, TMEM67, PKD1/TSC2). Perinatal respiratory failure, a kidney length >+4SD and
early-onset hypertension were specific features of PKHD1-
associated ARPKD. Conclusions: We found all ARPKD cases without
PKHD1 point mutations to be phenocopies, and none to
be explained by biallelic PKHD1 copy number variations. Based on this non-consanguineous cohort, screening for
copy number variations is indicated only in patients with a
heterozygous point mutation. This work was supported by
OTKA K109076 and Ministry of National Economy,
Hungary, GINOP-2.3.2-15-2016-00039 (to István Balogh,
Zoltán Maróti and Tibor Kalmár), MTA-SE Lendulet
Research Grant (LP2015-11/2015) of Hungarian Academy Abstracts from the 51st European Society of Human Genetics Conference: Posters 89 of Sciences and NKFIA/OTKA K109718, KH125566 (to
Kálmán Tory). T. Szabó: None. Z. Maróti: None. T. Kalmár: None. L. Madar: None. É. Gombos: None. O. Orosz: None. K. Tory: None. Genes with mutational enrichment in metastatic clear cell
kidney cancer associate with patient mortality Genes with mutational enrichment in metastatic clear cell
kidney cancer associate with patient mortality Background: The integrity of the intestinal lymphatics is
essential for proper nutrient absorption and tissue home-
ostasis. Damage to the lymphatic endothelial cells (LECs)
leads to an enteric loss of protein-rich lymphatic fluid, i.e. protein-losing enteropathy (PLE). We investigated the
genetic cause of PLE in two patients, first-degree cousins
once removed, who presented at 22 and 2.5 years. A. Mendoza-Alvarez1,2, B. Guillen-Guio2, C. Hernandez-Perez3,
A. Baez-Ortega4, J. M. Lorenzo-Salazar1, S. Lakhwani-
Lakhwani2, M. D. C. Maeso5, M. Morales3, C. Flores1,2,6 1Genomics Division, Instituto Tecnológico y de Energías
Renovables (ITER), Santa Cruz de Tenerife, Spain, 2Research
Unit, Hospital Universitario Nuestra Señora de Candelaria,
Universidad de La Laguna, Santa Cruz de Tenerife, Spain,
3Service of Medical Oncology, Hospital Universitario Nuestra
Señora de Candelaria, Santa Cruz de Tenerife, Spain,
4Transmissible Cancer Group, Department of Veterinary
Medicine, University of Cambridge, Cambridge, United
Kingdom, 5Department of Pathology, Hospital Universitario
Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain,
6CIBER de Enfermedades Respiratorias, Instituto de Salud
Carlos III, Madrid, Spain Methods: Whole exome sequencing was performed for
two affected and five healthy family members. Variants
were filtered based on autosomal recessive inheritance
model, population frequencies, and predicted functional
effect. Results: We identified a rare homozygous variant
(NM_031310.2:c.101T>C; p.Leu34Pro) in PLVAP, which
co-segregated with the disease. The variant is predicted
deleterious by bioinformatics algorithms, affecting the
protein’s transmembrane domain. Electron microscopy
(EM) of the younger patient's duodenal biopsy revealed
preserved endothelial fenestral diaphragms. Introduction: Kidney cancer only represents 2-3% of all
cancers. However, more than 60% of the patients die after
2-3 years from diagnosis. Here we aimed to evaluate the
association of somatic genetic variation with mortality by
metastatic kidney cancer. Discussion: The plasmalemma vesicle-associated protein
(PLVAP) is the building block of LEC fenestral dia-
phragms, which serve as a filtration system that blocks
passage of large molecules and pathogens through the
intestines. Mouse models of Plvap-deficiency demonstrate
PLVAP’s important contribution to LEC barrier function. Recently, a homozygous nonsense variant in PLVAP was
reported in a single patient with severe congenital PLE and
hypertriglyceridemia. We now show that bi-allelic missense
variants in PLVAP can cause an attenuated form of the
syndrome, albeit apparently intact endothelial diaphragms
on EM. P03.40C
PLVAP in protein-losing enteropathy: a homozygous
missense variant leads to an attenuated phenotype A. Kurolap1,2, O. Eshach-Adiv1, C. Gonzaga-Jauregui3,
K. Dolnikov1, A. Mory1, T. Paperna1, T. Hershkovitz1, J. D. Overton3, M. Kaplan1, Y. Zohar1, A. R. Shuldiner3, G. Berger1,
H. N. Baris1 A. Kurolap1,2, O. Eshach-Adiv1, C. Gonzaga-Jauregui3,
K. Dolnikov1, A. Mory1, T. Paperna1, T. Hershkovitz1, J. D. Overton3, M. Kaplan1, Y. Zohar1, A. R. Shuldiner3, G. Berger1,
H. N. Baris1 A. Kurolap1,2, O. Eshach-Adiv1, C. Gonzaga-Jauregui3,
K. Dolnikov1, A. Mory1, T. Paperna1, T. Hershkovitz1, J. D. Overton3, M. Kaplan1, Y. Zohar1, A. R. Shuldiner3, G. Berger1,
H. N. Baris1 1Rambam Health Care Campus, Haifa, Israel, Haifa, Israel,
2Technion – Israel Institute of Technology, Haifa, Israel,
3Regeneron Genetics Center, Tarrytown, NY, United States 1Rambam Health Care Campus, Haifa, Israel, Haifa, Israel,
2Technion – Israel Institute of Technology, Haifa, Israel,
3Regeneron Genetics Center, Tarrytown, NY, United States P03.38A
Prader-Willi syndrome: clinical particularities found in 15
patients We also detected a novel homozygous
frameshift mutation that leads to premature stop codon in
DNAI1 gene (c. 947_948insG, p. Thr318TyrfsTer11), in
two affected PCD siblings. For detailed characterization of
novel genetic variant we performed RT-qPCR, in silico
prediction of variant impact at protein level and protein
analysis by Western Blot. Conclusions: Analysis of the genomic profile of PCD
suspected patients improves differential diagnosis and
enables prenatal and carrier testing. It also leads to better
understanding of the molecular basis of pediatric lung
diseases. S. Calapod: None. A. Hrisca: None. R. Popescu: None. E. Gorduza: None. M. Gramescu: None. M. Panzaru:
None. L. Butnariu: None. C. Rusu: None. S. Calapod: None. A. Hrisca: None. R. Popescu: None. E. Gorduza: None. M. Gramescu: None. M. Panzaru:
None. L. Butnariu: None. C. Rusu: None. M.Z. Andjelkovic: None. P. Minic: None. M. Vreca:
None. M. Stojiljkovic: None. A. Skakic: None. A. Sovtic:
None. M. Rodic: None. V. Skodric-Trifunovic: None. N. 90 J. del Picchia Interest (stock, stock options, patent or other intellectual
property); Significant; Regeneron Pharmaceuticals Inc. K. Dolnikov: None. A. Mory: None. T. Paperna: None. T. Hershkovitz: None. J.D. Overton: A. Employment (full or
part-time); Significant; Regeneron Pharmaceuticals Inc. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Regeneron Pharmaceu-
ticals Inc. M. Kaplan: None. Y. Zohar: None. A.R. Shuldiner: A. Employment (full or part-time); Significant;
Regeneron Pharmaceuticals Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Regeneron Pharmaceuticals Inc. G. Berger:
None. H.N. Baris: None. Maric: None. J. Visekruna: None. V. Spasovski: None. S. Pavlovic: None. Genes with mutational enrichment in metastatic clear cell
kidney cancer associate with patient mortality Thus, our report establishes the role of PLVAP in
the pathophysiology of PLE, and expands the phenotypic
and mutation spectrums of the disorder, underscoring the
importance of PLVAP in LEC barrier function in the gut. Materials and Methods: Whole-exome sequencing from
paired tumor/normal FFPE tissue obtained from ten patients
with
metastatic
clear-cell
renal
cell
carcinoma
was
performed with AmpliSeq Exome RDY kit with the Ion
Proton System (Thermo Fisher Scientific). Somatypus and
VEP were used to identify and annotate single nucleotide
variants and small indels. A gene-based burden of putative
somatic mutations was calculated, and mutation enrichment
assessed by the Fisher’s exact test. Gene-set enrichment
analysis (GSEA) was evaluated with EnrichR. Results: Mean exome-wide target coverage was 127-fold
for tumor and 130-fold for matched normal samples. A total
of 9,220 putative somatic variants mapping to 5,256 genes A. Kurolap: None. O. Eshach-Adiv: None. C. Gon-
zaga-Jauregui: A. Employment (full or part-time); Sig-
nificant; Regeneron Pharmaceuticals Inc. E. Ownership Results: Mean exome-wide target coverage was 127-fold
for tumor and 130-fold for matched normal samples. A total
of 9,220 putative somatic variants mapping to 5,256 genes Abstracts from the 51st European Society of Human Genetics Conference: Posters 91 microbiome perturbations associate with ICU mortality in
septic patients. were detected across samples. Mutation enrichment was
associated with mortality at nominal significance in 138
genes (lowest p = 2.0e-6), and GSEA on this subset
evidenced a significant link with renal cancer (p = 2.3e-9),
specifically for 49 of the genes. Materials and Methods: We analyzed DNA from 41
bronchoalveolar aspirates from non-pulmonary septic ICU
patients at sepsis diagnosis (8h), and after 24h and 72h. Bacterial abundance was obtained by DNA sequencing of
the 16S rRNA V4. QIIME was used for operative
taxonomic units (OTUs) assignment. Conclusions: Mutational burden in 138 genes associate
with mortality among metastatic kidney cancer patients,
which represents a concise gene set for conducting focused
validation and functional studies. Results: The core microbiome (taxa in >50% of patients)
was composed by 46 OTUs and its number diminished in
deceased compared to surviving patients at all collection
times. Diversity differences were evident very early (17 vs
45 OTUs in deceased and survivors at 8h of sepsis
diagnosis, respectively; p < 0.01). The four most abundant
taxa
in
non-survivors
comprised
>89%
of
the
core
microbiome, while these only represented 15% of the core
microbiome in survivors. Lung metagenomics to predict patient mortality by non-
pulmonary sepsis B. Guillen-Guio1, D. Domínguez2, A. Baez-Ortega3, F. Lorenzo-
Diaz4, A. Díaz-de Usera5, R. González-Montelongo5,
R. Hernández-Bisshopp2, J. Arias2, L. Soto2, J. Belda6,
E. Espinosa2, J. Alcoba-Florez7, J. Villar8,9, C. Flores1,5,9 B. Guillen-Guio1, D. Domínguez2, A. Baez-Ortega3, F. Lorenzo-
Diaz4, A. Díaz-de Usera5, R. González-Montelongo5, R. Hernández-Bisshopp2, J. Arias2, L. Soto2, J. Belda6,
E. Espinosa2, J. Alcoba-Florez7, J. Villar8,9, C. Flores1,5,9 1Research Unit, Hospital Universitario N.S. de Candelaria,
Universidad de La Laguna, Santa Cruz de Tenerife, Spain,
2Department of Anesthesiology, Hospital Universitario N.S. de
Candelaria, Santa Cruz de Tenerife, Spain, 3Transmissible
Cancer Group, Department of Veterinary Medicine, University
of Cambridge, Cambridge, United Kingdom, 4Department of
Biochemistry, Microbiology, Cell Biology and Genetics,
Universidad de La Laguna, Santa Cruz de Tenerife, Spain,
5Genomics Division, Instituto Tecnológico y de Energías
Renovables (ITER), Santa Cruz de Tenerife, Spain,
6Department of Anesthesiology, Hospital Clínico Universitario
de Valencia, Valencia, Spain, 7Department of Microbiology,
Hospital Universitario N.S. de Candelaria, Santa Cruz de
Tenerife, Spain, 8Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain, 9CIBER de
Enfermedades Respiratorias, Instituto de Salud Carlos III,
Madrid, Spain B. Guillen-Guio: None. D. Domínguez: None. A. Baez-
Ortega: None. F. Lorenzo-Diaz: None. A. Díaz-de Usera:
None. R. González-Montelongo: None. R. Hernández-
Bisshopp: None. J. Arias: None. L. Soto: None. J. Belda:
None. E. Espinosa: None. J. Alcoba-Florez: None. J. Villar: None. C. Flores: None. 1Department of Pediatrics, Oncology, Hematology and
Diabetology, Medical University of Lodz, Lodz, Poland,
2Department of of Clinical Genetics, Medical University of
Lodz, Lodz, Poland, 3Department of of Dermatology, Pediatric
and Oncology Dermatology, Medical University of Lodz, Lodz,
Poland Genes with mutational enrichment in metastatic clear cell
kidney cancer associate with patient mortality Funding:
Fundación
CajaCanarias
(SALUCAN11);
Agreement OA17/008 with ITER to strengthen scientific
and technological education, training, research, develop-
ment and innovation in Genomics, Personalized Medicine
and
Biotechnology;
ACIISI
fellowship
to
BGG
(TESIS2015010057) co-funded by European Social Fund;
CEDeI fellowship (Cabildo de Tenerife) to AMA. A. Mendoza-Alvarez: None. B. Guillen-Guio: None. C. A. Mendoza Alvarez: None. B. Guillen Guio: None. C. Hernandez-Perez: None. A. Baez-Ortega: None. J.M. Lorenzo-Salazar: None. S. Lakhwani-Lakhwani: None. M.D.C. Maeso:
None. M. Morales:
None. C. Flores: None. Conclusions: A reduction in bacterial lung diversity
within 8h of sepsis diagnosis associates with ICU mortality,
providing a novel prognostic biomarker at an early stage of
disease. Hernandez-Perez: None. A. Baez-Ortega: None. J.M. Lorenzo-Salazar: None. S. Lakhwani-Lakhwani: None. M.D.C. Maeso:
None. M. Morales:
None. C. Flores: None. M.D.C. Maeso:
None. M. Morales:
None. C. Flores: None. Funding: ISCIII (PI11/00623, PI16/00049) and co-
financed by the European Regional Development Funds,
“A way of making Europe” from the European Union;
Agreement OA17/008 with ITER to strengthen scientific
and technological education, training, research, develop-
ment and innovation in Genomics, Personalized Medicine
and
Biotechnology;
Fellowships
from
the
ACIISI
(TESIS2015010057) co-funded by European Social Fund
to BGG and the Spanish Ministry of Education, Culture and
Sports (FPU16/01435) to ADU. P03.43B Lung metagenomics to predict patient mortality by non-
pulmonary sepsis Searching for causal treatment in patients with Wolfram
syndrome Searching for causal treatment in patients with Wolfram
syndrome A. Zmyslowska1, M. Borowiec2, E. Polakowska1, A. Lesiak3,
W. Mlynarski1 Introduction: Death by sepsis remains high in patients
admitted into intensive care units (ICUs) worldwide. Iden-
tifying early biomarkers of disease prognosis remains an
urgent
necessity. Here
we
hypothesized
that
lung J. del Picchia 92 involves the highest number of affected members docu-
mented. In all patients the non-syndromic long segment
disease (HSCR type 2) is present. At the age of 8 months
the proposita was admitted to Cincinnati Children´s Hos-
pital for ileostomy under the care of one of the authors (EP),
then a resident in pediatrics. During the following 15 years
she required 41 surgical procedures of the GI tract or the
rectum. In spite of the complicated course she is well
adjusted to her disorder. This family was re-investigated in
November 2017, including two daughters of the proposita
also affected with long segment Hirschsprung disease. Both
daughters also required several additional surgical proce-
dures. Hirschsprung disease, a genetically heterogeneous
and clinically variable disorder, results from the absence or
malfunction of intestinal ganglion cells. At least three signal
effector pathways are required for normal migration and
development of intramural intestinal ganglion cells, (i) the
RET tyrosine-kinase receptor and its ligand GDNF (glial-
cell-derived neurotrophic factor,
OMIM 600837), (ii)
endothelin type B receptor (EDNRB, OMIM 600837) and
its ligand EDN3 (endothelin 3, OMIM 131244), (iii)
SOX10 transcription factor (OMIM 602229). Introduction: Wolfram syndrome (WFS) is an example of
neurodegenerative disease due to increased ER stress
coexisting with diabetes mellitus with no causal treatment. WFS is caused by recessive mutations in the WFS1 gene
among which are the mutations of premature termination
codons (PTCs). Some prospects for the causal treatment of
WFS patients can give a readthrough of PTCs. The use of a
chemical compound e.g. PTC124 can result in bypassing
the PTCs and a continuation of translation. The aim of the
study was to evaluate a repairing of a genetic defect by
using PTC124 in WFS patients with confirmed mutations
of PTCs. Materials
and
Methods:
Diagnosis
of
WFS
was
confirmed by direct sequencing of the WFS1 gene. On
fibroblasts from skin biopsies of WFS patients and healthy
individuals the in vitro studies were performed involving
the induction of ER stress (Tunicamycin) with a subsequent
using a compound PTC124 (Ataluren). Searching for causal treatment in patients with Wolfram
syndrome Evaluation of ER
stress induction was performed by mRNA expression
analysis of wolframin and markers of the ER stress
(7900HT Real Time PCR; Applied Biosystems, USA). Materials
and
Methods:
Diagnosis
of
WFS
was
confirmed by direct sequencing of the WFS1 gene. On
fibroblasts from skin biopsies of WFS patients and healthy
individuals the in vitro studies were performed involving
the induction of ER stress (Tunicamycin) with a subsequent
using a compound PTC124 (Ataluren). Evaluation of ER
stress induction was performed by mRNA expression
analysis of wolframin and markers of the ER stress
(7900HT Real Time PCR; Applied Biosystems, USA). Results: In 12/15 of WFS patients the mutations of PTCs
were identified. The most specific markers of ER stress in
patients
with
WFS
should
be
considered:
GRP78,
GADD153
(CHOP)
and
ATF4. Thus,
a
compound
PTC124 may unfortunately increase the ER stress. E. Passarge: None. A.N. Ziegler: None. R.J. Hopkin:
None. H.M. Saal: None. P04 Skeletal, connective tissue, ectodermal and skin
disorders Conclusions: It seems that PTC124 by ER stress
increasing can not be used as a potential causal treatment
for the WFS patients. Conclusions: It seems that PTC124 by ER stress
increasing can not be used as a potential causal treatment
for the WFS patients. P03.46A
Afifty-year follow-up of familial Hirschsprung disease Afifty-year follow-up of familial Hirschsprung disease Children's Memorial Health Institute, Warsaw, Poland P04.01A Supported by the National Science Centre grants No
2014/15/B/NZ5/01579 and 2015/19/B/NZ5/02243. Supported by the National Science Centre grants No
2014/15/B/NZ5/01579 and 2015/19/B/NZ5/02243. Microduplication of 13q31.3 region: clinical and molecular
analysis based on a new case A. Zmyslowska:
None. M. Borowiec:
None. E. Polakowska:
None. A. Lesiak:
None. W. Mlynarski: None. M. Młynek, M. Kucharczyk, D. Wicher, A. Cieślikowska,
A. Gutkowska, M. Krajewska-Walasek M. Młynek, M. Kucharczyk, D. Wicher, A. Cieślikowska,
A. Gutkowska, M. Krajewska-Walasek Children's Memorial Health Institute, Warsaw, Poland E. Passarge1, A. N. Ziegler2, R. J. Hopkin3, H. M. Saal3
1Institut für Humangenetik, Universitätsklinikum Essen, Essen,
Germany, 2., Harrison, OH, United States, 3Human Genetics
Division, Cincinnati Children´s Hospital Medical Center,
Cincinnati, OH, United States E. Passarge1, A. N. Ziegler2, R. J. Hopkin3, H. M. Saal3 Microduplication of 13q31.3 is a rare genomic disorder
associated with abnormal growth and skeletal development. To date, less than ten comparable submicroscopic duplica-
tions involving this region have been described. The most
important phenotypic features include developmental delay,
digital malformations, growth abnormalities, macrocephaly
and facial dysmorphism. MIR17HG (encoding the miR-
17~92 polycistronic miRNa cluster) and GPC5 are the
major
candidate
genes
for
the
genotype-phenotype
correlation. E. Passarge1, A. N. Ziegler2, R. J. Hopkin3, H. M. Saal3 1Institut für Humangenetik, Universitätsklinikum Essen, Essen,
Germany, 2., Harrison, OH, United States, 3Human Genetics
Division, Cincinnati Children´s Hospital Medical Center,
Cincinnati, OH, United States We describe familial Hirschsprung disease (OMIM 142623)
with histologically proven congenital intestinal aganglio-
nosis in the proposita, her two affected brothers, her two
affected daughters and in the extended family three first
cousins (two males and one female). Originally this family
was described in 1967 as part of systematic study of the
genetics of Hirschsprung disease (Family 21 in E. Passarge,
New Eng J Med 1967; 276:138-143). It presumably Here, we report on the case of a 3 4/12 year-old girl
referred for genetic counseling because of excessive height
and weight (above the 97th centile) and dysmorphic
features (macrocephaly, frontal bossing, hyperthelorism,
long palpebral fissures, long philtrum, tilted upper lip, pug Abstracts from the 51st European Society of Human Genetics Conference: Posters 93 nose, widely spaced teeth). In the neonatal period umbilical
hernia was observed. At the clinical examination, indistinct
speech, laxity of hand joints, long and overlapping toes,
café au lait spot on the thigh and hypoplastic nipples
were noted. Results: In all models we noted similar extensive and
progressive vertebral hypermineralization (Table 1), starting
during embryonic development, with spinal deformities and
scoliosis in adults. Contrary to previously reported data,
abcc6a was essential for neither embryonic survival nor
morphological development. Conventional G-banding karyotyping revealed a de novo
translocation
46,XX,t(1;13)(q21;q13.31). Whole-genome
oligonucleotide microarray analysis revealed a 11.47-Mb
cryptic interstitial duplication of the 13q31.1q32.1 region
associated
with
the
translocation
breakpoints
(chr13:85583670-97054435; GRCh37). Microarray studies
in both parents gave normal results, proving de novo
occurrence of this aberration in the child. P04.03C Functional characterization of a predicted regulatory
element associated with alopecia areata (AA) Children's Memorial Health Institute, Warsaw, Poland Conclusions: Observing an identical ossification pheno-
type in 3 independent models confirms its specificity and
indicates for the first time a direct relation between abcc6a
deficiency and dysregulated osteogenesis. The phenotype is
readily quantifiable and an excellent readout for compound
screening. Table 1: Mineralization semi-quantified at 10 days post
fertilization (Mean ± SD; *P<0.05) In conclusion, this study contributes additional informa-
tion for the 13q31.3 microduplication and strongly support
the involvement of miR-17~92 cluster of miRNA and
GPC5 gene in normal growth and skeletal development. +/+ or Control
-/- or Morpholino
CRISPR (c.180delTCGG)
100% ± 9.6
121% ± 8.8 *
Sa963 (c.2250+1G>A)
100% ± 15.8
135% ± 40.2 *
Morpholino abcc6a
100% ± 19.2
155% ± 29.6 * This study was supported by the NCN Grant No. UMO-
2016/21/B/NZ5/02541. M. Młynek: None. M. Kucharczyk: None. D. Wicher:
None. A. Cieślikowska: None. A. Gutkowska: None. M. Krajewska-Walasek: None. M. Van Gils: None. A. Willaert: None. E. De Vilder:
None. P. Coucke: None. O. Vanakker: None. A homozygous missense change in canine ALPL, an animal
model for hypophosphatasia A homozygous missense change in canine ALPL, an animal
model for hypophosphatasia K. Kyöstilä1,2,3, P. Syrjä1, S. Hundi1,2,3, A. Lappalainen4,
R. Viitmaa4, V. Karkamo5, H. Lohi1,2,3 Functional characterization of a predicted regulatory
element associated with alopecia areata (AA) To our knowledge, this is the first time naturally-
occurring inherited hypophosphatasia has been described in
animals, and we hope our findings contribute to under-
standing of the disease. Taken together, with our experiments we could prove
allele-dependent silencing properties of the examined
regulatory region. Regulation of the IL2RA gene might
play a relevant role in the development of regulatory and
effector T-cells in AA. Grants: This study was supported by the Academy of
Finland, Jane and Aatos Erkko Foundation and Wisdom
Health. Funded by the EKFS-Promotionskolleg, Bonn. Funded by the EKFS-Promotionskolleg, Bonn. M.M. Mattern: None. A. Tafazzoli: None. S. Sivalin-
gam: None. J. Schultze: None. K. Ludwig: None. M.M. Nöthen: None. P. Kokordelis: None. R.C. Betz: None. K. Kyöstilä: None. P. Syrjä: None. S. Hundi: None. A. Lappalainen: None. R. Viitmaa: None. V. Karkamo:
None. H. Lohi: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Wisdom Health. F. Consultant/Advisory
Board;
Modest;
Genoscoper
Laboratories Oy. Functional characterization of a predicted regulatory
element associated with alopecia areata (AA) del Picchia We identified a region mapping into a predicted T-cell
enhancer, which might influence the expression of the
nearby AA-susceptibility gene IL2RA. Analysis of CTCF
sites revealed co-localization of IL2RA and the RE in the
same CTCF flanked domain. DLAs of the cloned RE
showed significant decrease of gene expression compared to
controls. Furthermore, gene expression was significantly
suppressed
after
integration
of
the
validated
IL2RA
promoter. However, introduction of the identified SNP led
to significantly less suppression. Taken together, with our experiments we could prove
allele-dependent silencing properties of the examined
regulatory region. Regulation of the IL2RA gene might
play a relevant role in the development of regulatory and
effector T-cells in AA. We identified a region mapping into a predicted T-cell
enhancer, which might influence the expression of the
nearby AA-susceptibility gene IL2RA. Analysis of CTCF
sites revealed co-localization of IL2RA and the RE in the
same CTCF flanked domain. DLAs of the cloned RE
showed significant decrease of gene expression compared to
controls. Furthermore, gene expression was significantly
suppressed
after
integration
of
the
validated
IL2RA
promoter. However, introduction of the identified SNP led
to significantly less suppression. Taken together, with our experiments we could prove
allele-dependent silencing properties of the examined
regulatory region. Regulation of the IL2RA gene might
play a relevant role in the development of regulatory and
effector T-cells in AA. We identified a region mapping into a predicted T-cell
enhancer, which might influence the expression of the
nearby AA-susceptibility gene IL2RA. Analysis of CTCF
sites revealed co-localization of IL2RA and the RE in the
same CTCF flanked domain. DLAs of the cloned RE
showed significant decrease of gene expression compared to
controls. Furthermore, gene expression was significantly
suppressed
after
integration
of
the
validated
IL2RA
promoter. However, introduction of the identified SNP led
to significantly less suppression. missense variant in a conserved domain of the tissue-
nonspecific alkaline phosphatase gene, ALPL. The ALPL
variant showed full segregation with the disease in a cohort
of 489 KBDs, and was absent from 303 dogs from control
breeds. Conclusions: In humans, near 350 ALPL mutations are
known to cause hypophosphatasia, a metabolic bone disease
with
heterogeneous
clinical
manifestations. We
have
identified a recessive ALPL missense change in dogs with
clinical and pathological findings compatible with hypopho-
sphatasia. Loss of GPNMB causes autosomal recessive amyloidosis
cutis dyschromica in humans 1Department of Veterinary Biosciences, University of Helsinki,
Helsinki, Finland, 2Department of Molecular Genetics,
Folkhälsan Institute of Genetics, Helsinki, Finland, 3Research
Programs Unit, Molecular Neurology, University of Helsinki,
Helsinki, Finland, 4Department of Equine and Small Animal
Medicine, University of Helsinki, Helsinki, Finland, 5Section of
Companion Animal Pathology, Finnish Food Safety Authority
Evira, Helsinki, Finland C. Yang1, S. Lin2,3, C. Chiang4,5, Y. Wu2,3, W. H'ng1, C. Chang1,
Y. Chen1, J. Wu1 1Academia Sinica, Taipei, Taiwan, 2Mackay Medical College,
New Taipei City, Taiwan, 3Mackay Memorial Hospital, Taipei,
Taiwan, 4Tri-Service General Hospital, Taipei, Taiwan,
5National Defense Medical Center, Taipei, Taiwan Introduction: The purebred dogs are affected with similar
skeletal disease as humans. In the present study, we have
performed clinical, pathological and genetic examinations
to characterize a previously unknown, autosomal recessive
skeletal disease in the Karelian Bear Dog (KBD) breed. Amyloidosis cutis dyschromica (ACD) is a distinct form of
primary cutaneous amyloidosis characterized by generalized
hyperpigmentation
mottled
with
small
hypopigmented
macules on the trunks and limbs. Families and sporadic
cases have been reported predominantly in East and
Southeast Asian ethnicities; however, the genetic cause has
not been elucidated. Homozygous premature nonsense
mutations leading to loss of function of GPNMB contribute
to the iris pigment dispersion phenotype in mouse pig-
mentary glaucoma. However, no mutations in GPNMB have
been identified in human pigmentary glaucoma and pigment
dispersion syndrome. We establish that the compound het-
erozygosity or homozygosity of GPNMB truncating alleles
is the cause of autosomal recessive ACD. Six nonsense or
frameshift mutations were identified in nine individuals
diagnosed with ACD. Immunofluorescence analysis of skin Materials and Methods: Clinical examinations were
performed on six affected dogs and pathological examina-
tion on seven dogs. Exome sequencing was carried out on
one affected dog. Results: Altogether seven affected puppies were recog-
nized in the KBDs. The clinical phenotype varied from a
failure to thrive and seizures at 2 weeks of age to a growth
defect and severe ambulatory difficulties at 12 weeks of age. Serum analysis indicated low alkaline phosphatase activity
and hypercalcemia. Radiographic and pathological exam-
inations revealed defective skeletal mineralization and
ossification. Exome sequencing identified a homozygous Results: Altogether seven affected puppies were recog-
nized in the KBDs. The clinical phenotype varied from a
failure to thrive and seizures at 2 weeks of age to a growth
defect and severe ambulatory difficulties at 12 weeks of age. Functional characterization of a predicted regulatory
element associated with alopecia areata (AA) M. Van Gils1, A. Willaert1, E. De Vilder1,2, P. Coucke1,2,
O. Vanakker1,2 M. M. Mattern1,2, A. Tafazzoli1,2, S. Sivalingam1,2, J. Schultze3,
K. Ludwig2, M. M. Nöthen1,2, P. Kokordelis1,2, R. C. Betz1,2 1Center for Medical Genetics, Ghent, Belgium, 2Ghent
University Hospital, Ghent, Belgium 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
University of Bonn, Bonn, Germany, 3Life & Medical Sciences-
Institute (LIMES), University of Bonn, Bonn, Germany 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
University of Bonn, Bonn, Germany, 3Life & Medical Sciences-
Institute (LIMES), University of Bonn, Bonn, Germany Introduction: Pseudoxanthoma elasticum is an ectopic
mineralization disease due to biallelic ABCC6 mutations. As no curative therapy is available, development of reliable
disease models for compound screening is necessary. Zeb-
rafish, which have a functional abcc6a orthologue, are an
excellent candidate model organism, if it has a reliable
phenotype. For a morpholino-induced knockdown and a
missense mutant model of abcc6a phenotypic discrepancies
are reported, questioning their validity. Therefore, we
developed a complete CRISPR/Cas9 knockout model and
compared its phenotype to a novel mutant (Sa963) and our
morpholino model. Alopecia areata (AA) is a genetic complex hair loss disorder
characterized by patchy hair loss. Genetic studies have
supported the hypothesis that AA is autoimmune in nature. The identified loci only explain a limited proportion of
disease heritability. The goal of the current project is to
functionally characterize a regulatory element (RE) and its
effect on IL2RA and other genes in close vicinity. Based on
data of a recent meta-analysis on AA, we performed a
genome-wide in-silico analysis of REs. We cloned a T-cell
specific RE, which contains the identified lead SNP, into a
pGL4.23 vector to perform dual-luciferase-assay (DLA) in
Jurkat cells. Further, we analyzed the effect of the reference
and alternative allele on gene transcription in association
with a minimal promoter or with a promoter of a predicted
target gene. Materials and Methods: Both carriers of the CRISPR-
induced
(c.180delTCGG)
and
Sa963
(c.2250+1G>A)
heterozygotes were out- and incrossed to F4 generations. Morpholino dosage was optimized to 3ng and co-injected
with 4.5ng p53-morpholino. Development was monitored
during embryonic development into adulthood. Mineraliza-
tion was assessed via alizarin red S staining and ImageJ
analysis. 94 J. P04.06B L. M. Hochfeld1,2, D. Broadley3, N. V. Botchkareva3, M. P. Philpott4, S. Schoch5, R. C. Betz1, M. M. Nöthen1,2, S. Heilmann-
Heimbach1,2 MetaXcan gene-based association analysis yields novel
insights into male-pattern baldness biology S. Heilmann-Heimbach1,2, L. M. Hochfeld1,2, V. Schüller3, the
MAAN Consortium, M. M. Nöthen1,2 1Institute of Human Genetics, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
2Department of Genomics, Life&Brain Center, University of
Bonn, Bonn, Germany, 3School of Chemistry and Bioscience,
Centre for Skin Sciences, University of Bradford, Bradford,
United Kingdom, 4Centre for Cell Biology and Cutaneous
Research, Blizard Institute, Barts and The London School of
Medicine and Dentistry, Queen Mary University of London,
London, United Kingdom, 5Department of Neuropathology,
University of Bonn Medical Center, Bonn, Germany 1Institute of Human Genetics, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
2Department of Genomics, Life&Brain Center, University of
Bonn, Bonn, Germany, 3Institute for Medical Biometry,
Informatics, and Epidemiology, University of Bonn, Bonn,
Germany Male-pattern baldness (MPB) is a heritable trait and GWAS
have implicated more than 100 genomic regions. The
majority of associated variants however are located in
noncoding regions and their functional relevance remains
unclear. Integrative analyses that link genetic variation with
biological function are needed to identify relevant genes and
mechanisms. Here, we used MetaXcan (Barbeira et al.,
bioRxiv) on summary statistics from two published large-
scale genetic studies for MPB (Heilmann-Heimbach et al.,
2017; Hagenaars et al., 2017) and gene-expression data
from human hair follicle (own data) and the GTEx-project
(skin, adipose tissue, blood) to test for an association
between MPB and the genetically determined regulation of
gene-expression in these tissues. The analysis identified
associations between MPB and expression levels of 30 (hair
follicle) to 637 genes (skin). Twenty-seven genes showed
significant association in one or more tissues after Bonfer-
roni correction (P<0.05/27300 gene-tissue pairs). These
included previously implicated genes (e.g. WNT10A, IRF4, The WNT10A-locus on 2q35 is a known risk locus for AGA. A reduced WNT10A expression in hair follicle (HF) of risk
allele carriers for rs7349332 supports the role of WNT10A
as the functionally relevant gene (Heilmann et al., 2013). This lead variant is in high LD (r2=0.96, D’=0.99) with
rs3856551, located within a binding site for the transcrip-
tion factor EBF1. Loss of GPNMB causes autosomal recessive amyloidosis
cutis dyschromica in humans Serum analysis indicated low alkaline phosphatase activity
and hypercalcemia. Radiographic and pathological exam-
inations revealed defective skeletal mineralization and
ossification. Exome sequencing identified a homozygous Abstracts from the 51st European Society of Human Genetics Conference: Posters 95 biopsies showed that GPNMB is expressed in all epidermal
cells, with the highest staining observed in melanocytes. GPNMB staining is significantly reduced in the lesional
skin
of
affected
individuals. Hyperpigmented
lesions
exhibited significantly increased amounts of DNA/keratin-
positive amyloid deposits in the papillary dermis and infil-
trating macrophages compared with hypo-/depigemented
macules. Depigmentation of the lesions was attributable to
loss of melanocytes. Intracytoplamic fibrillary aggregates
were observed in keratinocytes scattered in the lesional
epidermis. Thus,
our
analysis
indicates
that
loss
of
GPNMB, which has been implicated in melanosome for-
mation, autophagy, phagocytosis, tissue repair, and negative
regulation of inflammation, underlies autosomal recessive
ACD, and provides insights into the etiology of amyloidosis
and pigment dyschromia. TWIST1) and novel candidate genes such as SPRR1B and
TRADD, which have a reported role in keratinocyte biology
and apoptosis, thereby rendering an involvement of these
processes in early MPB-pathogenesis likely. The fact that
associations with previously implicated genes were detected
in
subcutaneous
adipose
tissue
(TWIST1)
and
blood
(WNT10A) lends further support to the hypothesis, that cells
in the perifollicular environment play a role in MPB. In
summary, our analyses provide evidence for an MPB-
associated genetic regulation of plausible candidate genes
(SPRR1B,TRADD,WNT10A)
and
biological
processes
(apoptosis,adipogenesis) and yield novel insights into the
functional effects of current GWAS findings. TWIST1) and novel candidate genes such as SPRR1B and
TRADD, which have a reported role in keratinocyte biology
and apoptosis, thereby rendering an involvement of these
processes in early MPB-pathogenesis likely. The fact that
associations with previously implicated genes were detected
in
subcutaneous
adipose
tissue
(TWIST1)
and
blood
(WNT10A) lends further support to the hypothesis, that cells
in the perifollicular environment play a role in MPB. In
summary, our analyses provide evidence for an MPB-
associated genetic regulation of plausible candidate genes
(SPRR1B,TRADD,WNT10A)
and
biological
processes
(apoptosis,adipogenesis) and yield novel insights into the
functional effects of current GWAS findings. S. Heilmann-Heimbach: None. L.M. Hochfeld: None. V. Schüller: None. M.M. Nöthen: None. S. Heilmann-Heimbach: None. L.M. Hochfeld: None. V. Schüller: None. M.M. Nöthen: None. P04.07C C. Yang: None. S. Lin: None. C. Chiang: None. Y. Wu:
None. W. H'ng: None. C. Chang: None. Y. Chen: None. J. Wu: None. Evidence for a functional interaction between WNT10A and
EBF1 in the development of androgenetic alopecia (AGA) Evidence for a functional interaction between WNT10A and
EBF1 in the development of androgenetic alopecia (AGA) Division of Human Genetics, Bern, Switzerland Introduction: Distal arthrogryposis type 5 (DA5) is char-
acterized by multiple congenital contractures of distal
extremities associated with ocular abnormalities, facial
dysmorphism and restrictive lung disease and caused by
heterozygous PIEZO2 gain of function mutations. 1Laboratory of cytogenetics, molecular genetics and
reproductive biology, Farhat HACHED Hospital, Sousse,
Tunisia, 2Departement. of Restorative Dentistry and
Endodontology, Ghent University Hospital, Ghent, Belgium,
3Department of Medical Genetics, MRB, Ghent University
Hospital, Ghent, Belgium, 4Private Practice, Sousse, Tunisia 1Laboratory of cytogenetics, molecular genetics and
reproductive biology, Farhat HACHED Hospital, Sousse,
Tunisia, 2Departement. of Restorative Dentistry and
Endodontology, Ghent University Hospital, Ghent, Belgium,
3Department of Medical Genetics, MRB, Ghent University
Hospital, Ghent, Belgium, 4Private Practice, Sousse, Tunisia Case report: A 4 year old boy presented with severe club
foot, camptodactyly, short stature, ophtalmoplegia, deep-set
eyes and micrognathia. The family history revealed that his
father had congenital arthrogryposis with short stature,
keratoconus and astigmatism. The clinical presentation
being very suggestive for DA5. Sanger sequencing of
PIEZO2 was performed initially and revealed no mutation. NGS panel for distal arthrogryposis did also reveal no
mutation in the analyzed genes. As no MLPA is available
for PIEZO2, in silico CNV analysis was performed and
detected a deletion of exons 32 and 33 in PIEZO2 (c.4634-? _4855+?del). The deletion was confirmed by RT-qPCR. Introduction: Andontia refers to the total absence of den-
tition. It can affect permanent and/or temporary dentition. In
most cases, anodontia is associated with different clinical
manifestations as a part of syndrome but also non-
syndromic forms have been described although their etiol-
ogy is often unclear. Material and Methods: In this study we explored a
Tunisian family with most likely a recessive form of
anodontia of permanent dentition in the proband whereas
the
parents
and
some
other
family
members
show
hypodontia. We performed DNA targeted sequencing of
candidate genes, PAX9, MSX1, WNT10A and AXIN2 and
consequently whole exome sequencing (WES). Material and Methods: In this study we explored a
Tunisian family with most likely a recessive form of
anodontia of permanent dentition in the proband whereas
the
parents
and
some
other
family
members
show
hypodontia. We performed DNA targeted sequencing of
candidate genes, PAX9, MSX1, WNT10A and AXIN2 and
consequently whole exome sequencing (WES). Discussion: Heterozygous gain of function mutations in
PIEZO2 cause DA5, Gordon and Marden-Walker syndrome
whereas homozygous loss of function mutations cause
muscular atrophy, perinatal respiratory distress, progressive
arthrogryposis, scoliosis and loss of proprioception. P04.06B This mutation,
predicted to be disease causing, is located at the NFRKB-
WHL domain which belongs to a group of helical domains
involved in protein-protein interactions. This might suggest
that the interaction activity is probably altered in the first
stages of development. Conclusions: This study revealed for the first time the
involvement of the NFRKB gene in tooth agenesis. NFRKB
might be involved in transcriptional regulation. Further
qPCR studies and functional studies will be performed. M. Haddaji Mastouri: None. P. De Coster: None. N. Ben Salah: None. A. Touati: None. I. Veerecke: None. A. Zaghabani: None. A. Saad: None. D. H’mida-Ben
Brahim: None. P. Coucke: None. L.M. Hochfeld:
None. D. Broadley:
None. N.V. Botchkareva: None. M.P. Philpott: None. S. Schoch:
None. R.C. Betz:
None. M.M. Nöthen:
None. S. Heilmann-Heimbach: None. Novel heterozygous deletion in PIEZO2 identified by NGS
in a familial case with distal arthrogryposis type 5 Novel heterozygous deletion in PIEZO2 identified by NGS
in a familial case with distal arthrogryposis type 5 C. Rieubland, S. Gallati, A. Schaller C. Rieubland, S. Gallati, A. Schaller M. Haddaji Mastouri1, P. De Coster2, N. Ben Salah1, A. Touati1,
I. Veerecke3, A. Zaghabani4, A. SAAD1, D. H’mida-Ben
Brahim1, P. Coucke3 P04.06B Interestingly, the gene encoding EBF1 is
located at the 5q33.3-AGA risk locus, suggesting that
changes in EBF1 mediated regulation of WNT10A expres-
sion may contribute to AGA. To investigate this potential
interaction,
we
performed
luciferase
assays
by
co-
transfecting (i) luciferase vectors containing the WNT10A
promoter and the 2q35-EBF1 binding site with either the
risk or the alternate allele and (ii) an EBF1 expression
vector. Our experiments in HEK cells showed that EBF1
activates the WNT10A promoter and that the WNT10A/
EBF1 interaction was increased with the risk allele, which
together with the previous mRNA expression data suggests J. del Picchia 96 that EBF1 acts as a negative regulator of WNT10A. To
confirm this interaction in AGA relevant tissue, we tested
for co-expression of WNT10A and EBF1 in a published
RNA-Seq dataset of mouse HF (Joost et al., 2016) and
performed immunofluorescence co-staining in human HF
and skin. The analyses revealed the strongest co-expression
in HF keratinocytes. Therefore, the initial luciferase assays
are repeated in human keratinocytes. In parallel, EBF1
knockout experiments in human HF are under way. Taken
together, our data provide the first evidence for a functional
interaction
between
WNT10A
and
EBF1
in
AGA
pathobiology. that EBF1 acts as a negative regulator of WNT10A. To
confirm this interaction in AGA relevant tissue, we tested
for co-expression of WNT10A and EBF1 in a published
RNA-Seq dataset of mouse HF (Joost et al., 2016) and
performed immunofluorescence co-staining in human HF
and skin. The analyses revealed the strongest co-expression
in HF keratinocytes. Therefore, the initial luciferase assays
are repeated in human keratinocytes. In parallel, EBF1
knockout experiments in human HF are under way. Taken
together, our data provide the first evidence for a functional
interaction
between
WNT10A
and
EBF1
in
AGA
pathobiology. of the NFRKB gene on the other allele. This mutation,
predicted to be disease causing, is located at the NFRKB-
WHL domain which belongs to a group of helical domains
involved in protein-protein interactions. This might suggest
that the interaction activity is probably altered in the first
stages of development. of the NFRKB gene on the other allele. This mutation,
predicted to be disease causing, is located at the NFRKB-
WHL domain which belongs to a group of helical domains
involved in protein-protein interactions. This might suggest
that the interaction activity is probably altered in the first
stages of development. of the NFRKB gene on the other allele. Genetic investigation of a rare form of severe tooth
agenesis: Anodontia Genetic investigation of a rare form of severe tooth
agenesis: Anodontia Camptodactyly-arthropathy-coxa vara-pericarditis
syndrome in a large family: A clinical condition with a
diagnostic challange Years later, and after the discovery of PRG4 mutations
in CACP, another patient from another family with a
diagnosis of JIA who was not responsive to anti-rheumatic
treatment was referred with a suspicion of a genetic disorder
of the skeleton. With the help of the pedigree analysis, it
was revealed that these two families were actually related,
and a recent contact with the first family could be
established years after. Conclusion: The CaSR kodon 990 AA allele seemed to
be a risk factor for bone health in our patient group. Among
CRF patients with increased hemodialysis duration osteo-
porosis and fracture risks were increased. Risk of develop-
ing bone disease for female patients was higher than that
of males. Results: Molecular analysis in all patients revealed a
homozygous
deletion
of
two
nucleotide
(c.1910_1911delCT) in the highly repetitive region of exon
6 of PRG4 in all patients. Results: Molecular analysis in all patients revealed a
homozygous
deletion
of
two
nucleotide
(c.1910_1911delCT) in the highly repetitive region of exon
6 of PRG4 in all patients. P. Ata: None. B. Erkal: None. D. Gultekin: None. B. Altas: None. B. Celik: None. D. Kayır: None. A. Low:
None. A. Eren: None. S. Tuglular: None. Conclusions: Patients with CACP syndrome can easily
be misdiagnosed as JIA, leading to unnecessary treatment
and a delay for the actual diagnosis. In patients with
arthropathic conditions, CACP should be kept in mind,
especially in the presence of progressive symptoms and
parental consanguinity. Conclusions: Patients with CACP syndrome can easily
be misdiagnosed as JIA, leading to unnecessary treatment
and a delay for the actual diagnosis. In patients with
arthropathic conditions, CACP should be kept in mind,
especially in the presence of progressive symptoms and
parental consanguinity. Division of Human Genetics, Bern, Switzerland We
hypothesize that the in frame deletion of exons 32 and 33
causes a dominant negative effect resulting in a gain of
function of the channel activity. Results: Sequencing of the 4 genes did not reveal any
causative mutation. Therefore, we performed WES on the
anodontia patient. We selected six homozygous mutations
and segregation analysis on the remaining family members. One of these is a missense mutation located in NFRKB gene
(Nuclear Factor Related to Kappa-B-binding protein) in the
proband. Interestingly, in the rest of the family this mutation
is compound heterogeneous with most likely a duplication Conclusion: This is the first report of an intragenic
deletion in PIEZO2 in a family with autosomal dominant
DA5 diagnosed by NGS. Inframe deletions of PIEZO2
might be a frequent cause of DA5. As no MLPA is available Abstracts from the 51st European Society of Human Genetics Conference: Posters 97 for PIEZO2, in silico CNV analysis using NGS may be a
useful tool to diagnose intragenic deletions. S. Oguz: None. P.O. Simsek Kiper: None. G.E. Utine:
None. Y. Alanay: None. S. Ozen: None. K. Boduroglu:
None. M. Alikasifoglu: None. S. Oguz: None. P.O. Simsek Kiper: None. G.E. Utine:
None. Y. Alanay: None. S. Ozen: None. K. Boduroglu:
None. M. Alikasifoglu: None. C. Rieubland:
None. S. Gallati:
None. A. Schaller: None. C. Rieubland:
None. S. Gallati:
None. A. Schaller: None. Camptodactyly-arthropathy-coxa vara-pericarditis
syndrome in a large family: A clinical condition with a
diagnostic challange Materials and Methods: Fortyfıve CRF patients on
hemodialysis
treatment
aged
between
15-80yrs
who
admitted between 1994-2017 were included. Serum PTH,
Ca2+ and P values were examined. The control group
included 50 healthy patients without CRF, bone disease and
osteoporotic risk factors. Genomic DNA isolation was
performed using the Gentra Qiagen kit. CaSR allele-specific
PCR analysis was performed.Findings: Patients’ average
age was 60.5 ± 17.5 years, (male/ female 53.3% / 46.7%)
Patients with hemodialysis treatment for less than 3 years
had normal CaxP value while this value was increased for
patients with hemodialysis over 3 years. The pathological
CaxP value was found in 8,3% (n = 2/24) of the male
patients and 38,09% (n = 8/21) of the female patients (p =
0,029). In the
patient
group,
the
990th
codon
AA
polymorphism in the CaSR gene was found to be 32.4%
(n = 12) while in the control group it was 23.1% (n = 6). Introduction:
Camptodactyly-arthropathy-coxa
vara-
pericarditis syndrome (CACP; OMIM 208250) is a rare
autosomal recessive disorder caused by PRG4 mutations. PRG4 product is a glycoprotein called lubricin. We report
on the clinical and molecular findings of two related
families who were initially diagnosed with JIA, and PRG4
mutations were identified in the follow-up. Materials and Methods: After the referral of the first
family with three affected individuals with CACP, a linkage
analysis was done and a high LOD score on the long arm of
chromosome 1 was found. However, no further studies
could be performed, and the family was lost in the follow-
up. Years later, and after the discovery of PRG4 mutations
in CACP, another patient from another family with a
diagnosis of JIA who was not responsive to anti-rheumatic
treatment was referred with a suspicion of a genetic disorder
of the skeleton. With the help of the pedigree analysis, it
was revealed that these two families were actually related,
and a recent contact with the first family could be
established years after. Materials and Methods: After the referral of the first
family with three affected individuals with CACP, a linkage
analysis was done and a high LOD score on the long arm of
chromosome 1 was found. However, no further studies
could be performed, and the family was lost in the follow-
up. Camptodactyly-arthropathy-coxa vara-pericarditis
syndrome in a large family: A clinical condition with a
diagnostic challange P. Ata1, B. Erkal1, D. Gultekin2, B. Altas2, B. Celik2, D. Kayır2,
A. Low2, A. Eren3, S. Tuglular4 S. Oguz1, P. O. Simsek Kiper2, G. E. Utine2, Y. Alanay3,
S. Ozen4, K. Boduroglu2, M. Alikasifoglu1 1Marmara University, School of Medicine Medical Genetics,
Istanbul, Turkey, 2Marmara University, School of Medicine,
Istanbul, Turkey, 3Koc University, Istanbul, Turkey, 4Marmara
University, School of Medicine Nephrology, Istanbul, Turkey 1Hacettepe University Faculty of Medicine, Department of
Medical Genetics, Ankara, Turkey, 2Hacettepe University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Genetics, Ankara, Turkey, 3Acıbadem University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Genetics, Istanbul, Turkey, 4Hacettepe University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Rheumatology, Ankara, Turkey 1Hacettepe University Faculty of Medicine, Department of
Medical Genetics, Ankara, Turkey, 2Hacettepe University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Genetics, Ankara, Turkey, 3Acıbadem University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Genetics, Istanbul, Turkey, 4Hacettepe University
Faculty of Medicine, Department of Pediatrics, Department of
Pediatric Rheumatology, Ankara, Turkey Introduction: The aim of this study is to examine the effect
of the CaSR genotype, which significantly affects Ca, P,
PTH values on the disease progression in chronic renal
failure (CRF) patients. Introduction: The aim of this study is to examine the effect
of the CaSR genotype, which significantly affects Ca, P,
PTH values on the disease progression in chronic renal
failure (CRF) patients. Materials and Methods: Fortyfıve CRF patients on
hemodialysis
treatment
aged
between
15-80yrs
who
admitted between 1994-2017 were included. Serum PTH,
Ca2+ and P values were examined. The control group
included 50 healthy patients without CRF, bone disease and
osteoporotic risk factors. Genomic DNA isolation was
performed using the Gentra Qiagen kit. CaSR allele-specific
PCR analysis was performed.Findings: Patients’ average
age was 60.5 ± 17.5 years, (male/ female 53.3% / 46.7%)
Patients with hemodialysis treatment for less than 3 years
had normal CaxP value while this value was increased for
patients with hemodialysis over 3 years. The pathological
CaxP value was found in 8,3% (n = 2/24) of the male
patients and 38,09% (n = 8/21) of the female patients (p =
0,029). In the
patient
group,
the
990th
codon
AA
polymorphism in the CaSR gene was found to be 32.4%
(n = 12) while in the control group it was 23.1% (n = 6). 1Istituto Giannina Gaslini, Genova, Italy, 2Università di Pisa,
Pisa, Italy 1Istituto Giannina Gaslini, Genova, Italy, 2Università di Pisa,
Pisa, Italy Chiari malformation type I (CMI) is a congenital abnorm-
ality of the cranio-cerebral junction with an incidence of 1
in 1280. CMI is characterized by underdevelopment of the
occipital bone and posterior fossa (PF) and consequent
cerebellar tonsil herniation. The presence for a genetic basis
to CMI is supported by many lines of evidence. The cellular
and molecular mechanisms leading to CM1 are poorly
understood. The occipital bone formation is dependent on
complex interactions between genes and molecules with
pathologies resulting from disruption of this delicate pro-
cess. Whole-exome sequencing of affected and not affected
individuals from two Italian families with non isolated CMI
was undertaken. Single nucleotide and short insertion-
deletion
variants
were
prioritized
using
KGGSeq-
knowledge-based platform. We identified three hetero-
zygous missense variants: DKK1 c121G>A (p.(A41T)) in
the first family, and the LRP4 c.2552C>G (p.(T851R)) and
BMP1 c.941G>A (p.(R314H)) in the second family. The
variants were located at highly conserved residues, segre-
gated with the disease, but they were not observed in more
than 100 unaffected in-house controls. DKK1 encodes for a
potent soluble WNT inhibitor that binds to LRP5 and LRP6,
and is itself regulated by BMPs. DKK1 is required for
embryonic head development and patterning. LRP4 is a
novel osteoblast expressed receptor for DKK1 and a WNT
and BMP signaling pathways integrator. Screening of
DKK1 in a cohort of 65 CMI sporadic patients identified
another missense variant, the c.359G>T (p.(R120L)), in two
unrelated patients. These findings implicated the WNT
signaling
in
the
correct
development
of
the
cranial
mesenchyme originating the PF. Chiari malformation type 1 (CM1) is a congenital anom-
aly of cranio-cerebral junctions characterized by under-
development of the occipital bone and posterior fossa (PF)
and consequent cerebellar tonsil herniation across the
foramen magnum. This condition can impair the normal
flow of cerebral spinal fluid (CSF) leading to syr-
ingomyelia. Patients display a high degree of clinical
variability depending on the compression of the tissue,
nerves and on the buildup of CSF pressure. CM1 is also
associated with known syndromes, (e.g. craniosynos-
tosis), and is often reported as clinical sign in more
complex phenotypes, but the molecular mechanisms of
isolated CM1 are not yet known. To understand the
molecular basis and which factors could contribute to the
high heterogeneity, we performed WES of 42 trios with
sporadic and syndromic, without craniosynostosis, CM1. 1Istituto Giannina Gaslini, Genova, Italy, 2Università di Pisa,
Pisa, Italy WES of syndromic cases provided a diagnosis of distinct
genetic disorders, in which pathogenic variants were
associated to bone dysplasia, growth retardation and
resistance to gonadotropins. In sporadic cases, variants in
genes involved in common molecular pathways have been
identified; these are all associated with modeling and
deposition of bone matrix and with regulatory processes,
all requested in the same pathway. In 75% of trios we
found variants in the same recurrent genes, shared both by
isolated and syndromic CM1 cases; functional tests on
bone biopsies of these patients are underway to demon-
strate the pathogenicity of these genes. Our data remark
complex interactions among several genes involved in
different steps of the same pathways, underlying the high
clinical-genetic heterogeneity of CM1. P. De Marco: None. L. Tattini: None. A. Accogli:
None. G. Piatelli: None. M. Pavanello: None. D. Tortora:
None. A. Cama: None. V. Capra: None. A. La Barbera: None. A. Provenzano: None. L. Tiberi:
None. R. Artuso: None. V. Palazzo: None. P. Reho:
None. E. Bosi: None. A. Pagliazzi: None. F. Peluso: None. S. Landini: None. I. Sani: None. L. Giunti: None. S. Guarducci: None. M. Pantaleo: None. B. Lucherini:
None. M. Scagnet:
None. L. Genitori:
None. S. Giglio: None. P04.12D P04.12D
WES in 42 trios of syndromic and isolated Chiari
Malformation type 1: how to define the genetic cause in a
high clinical heterogeneous condition 98 J. del Picchia A. La Barbera1, A. Provenzano1, L. Tiberi1, R. Artuso2,
V. Palazzo2, P. Reho1, E. Bosi1, A. Pagliazzi1, F. Peluso1,
S. Landini1, I. Sani2, L. Giunti2, S. Guarducci2, M. Pantaleo2,
B. Lucherini2, M. Scagnet3, L. Genitori3, S. Giglio1 P. De Marco1, L. Tattini2, A. Accogli1, G. Piatelli1,
M. Pavanello1, D. Tortora1, A. Cama1, V. Capra1 1Medical Genetics Unit, Department of Clinical and
Experimental Biomedical Sciences 'Mario Serio', University of
Florence, Florence, Italy, 2Medical Genetics Unit, Meyer
Children’s University Hospital, Florence, Italy, 3Neurosurgery
Unit, Anna Meyer Children's Hospital, University of Florence,
Florence, Italy 1Medical Genetics Unit, Department of Clinical and
Experimental Biomedical Sciences 'Mario Serio', University of
Florence, Florence, Italy, 2Medical Genetics Unit, Meyer
Children’s University Hospital, Florence, Italy, 3Neurosurgery
Unit, Anna Meyer Children's Hospital, University of Florence,
Florence, Italy P04.13A P. De Marco1, L. Tattini2, A. Accogli1, G. Piatelli1,
M. Pavanello1, D. Tortora1, A. Cama1, V. Capra1 P. De Marco1, L. Tattini2, A. Accogli1, G. Piatelli1,
M. Pavanello1, D. Tortora1, A. Cama1, V. Capra1 Cell Laboratory, Centre of Excellence for Advanced Sciences,
National Research Centre, Cairo, Egypt Introduction: Cleft palate is one of the most common
congenital craniofacial defects that may present alone or in
association with various genetic disorders. Repairing such
defect is very important to restore oral functions and normal
facial features. Regenerative medicine and stem cell therapy
are emerging fields that have shown great potentials in
treating various diseases. Here, we follow a regenerative
approach by introducing a method in which we use auto-
logous bone marrow mononuclear cells (BMMNCs) com-
bined
with
platelet-rich
fibrin
(PRF)
and
nano-
hydroxyapatite for bone regeneration in patients with uni-
lateral alveolar clefts. Introduction: CHST11 is a membrane protein of Golgi that
catalyses the transfer of sulphate to position 4 of the N-
acetylgalactosamine residue of chondroitin. Chondroitin
sulphate is the predominant proteoglycan in cartilage, and
its sulphation is important in the developing growth plate of
cartilage. A homozygous deletion encompassing part of the
gene and the embedded microRNA MIR3922 had been
detected in a woman with hand/foot malformation and
malignant lymphoproliferative disease. Chst11 deficient
mouse has severe chondrodysplasia, congenital arthritis and
neonatal lethality. We searched for the causative variant for
the unusual combination of limb malformations with vari-
able expressivity accompanied by skeletal defects in a
consanguineous Pakistani kindred. Materials and Methods: The study included 10 patients
with unilateral alveolar cleft defects and of age range 8 to 15
years. Autologous BMMNCs were isolated using the
density gradient separation method and seeded on a
collagen sponge. The seeded collagen sponge was then
used in combination with nano-hydroxyapatite and auto-
logous PRF to repair alveolar cleft defects via the
regenerative approach. The effectiveness of the technique
was evaluated after 12 months of follow up via clinical and
radiographic assessments. Materials and Methods: We performed detailed clinical
investigations in family members. Homozygosity mapping
using SNP genotype data was performed to map the disease
locus and exome sequencing to identify the underlying
molecular defect. Materials and Methods: We performed detailed clinical
investigations in family members. Homozygosity mapping
using SNP genotype data was performed to map the disease
locus and exome sequencing to identify the underlying
molecular defect. Results: The limb malformations include brachydactyly,
overriding digits and clino-symphalangism in hands and
feet, and syndactyly and hexadactyly in feet. Skeletal
defects include scoliosis, dislocated patellae and fibulae,
and pectus excavatum. The disease locus is mapped to a
1.6-Mb region at 12q23, harbouring a homozygous in-frame
deletion of 15 nucleotides in CHST11. H. H. El-Ahmady1, A. F. Abd Elazeem2,3,1, N. E. B. Ahmed2,3,
M. A. Abdelrahman2, M. A. Abderazik1 Cell Laboratory, Centre of Excellence for Advanced Sciences,
National Research Centre, Cairo, Egypt Novel variant
c.467_481del (p.L156_N160del) is deduced to lead to the
deletion of five evolutionarily highly conserved amino acids
and predicted as damaging to protein by in silico analysis. Results: All patients healed probably without any
complications. During the 12-month follow-up, no donor
site morbidities have been reported. Postoperative pain,
bleeding, and swelling were within the normal for same
surgical
procedures. Cone
beam
radiographs
showed
successful complete bone regeneration in all cases. Conclusion: Results of this study suggest that a mixture
of autologous BMMNCs, nano-hydroxyapatite, and PRF
greatly promote bone regeneration providing a novel
therapeutic strategy for alveolar cleft defects. Conclusions: Our findings confirm the crucial role of
CHST11 in skeletal morphogenesis and show that CHST11
defects have variable manifestations that include a variety
of limb malformations and skeletal defects. Conclusions: Our findings confirm the crucial role of
CHST11 in skeletal morphogenesis and show that CHST11
defects have variable manifestations that include a variety
of limb malformations and skeletal defects. H.H. El-Ahmady: None. A.F. Abd Elazeem: None. N. E.B. Ahmed: None. M.A. Abdelrahman: None. M.A. Abderazik: None. (Grant: TUBITAK114Z829) R. Shabbir: None. G. Nalbant: None. N. Ahmad: None. S. Malik: None. A. Tolun: None. R. Shabbir: None. G. Nalbant: None. N. Ahmad: None. S. Malik: None. A. Tolun: None. 1Al-Azhar Cleft Lip and Palate Treatment Center, Faculty of
Dental Medicine for Girls, Al-Azhar university, Cairo, Egypt,
2Department of Oro-dental Genetics, Center of Medical
Excellence, National Research Centre, Cairo, Egypt, 3Stem P04.16D
Expanded phenotypic spectrum of type I collagenopathy Expanded phenotypic spectrum of type I collagenopathy P04.14B A small homozygous CHST11 deletion in chondrodysplasia,
brachydactyly, overriding digits, clino-symphalangism and
synpolydactyly A small homozygous CHST11 deletion in chondrodysplasia,
brachydactyly, overriding digits, clino-symphalangism and
synpolydactyly R. Shabbir1, G. Nalbant2, N. Ahmad3, S. Malik1, A. Tolun2 Abstracts from the 51st European Society of Human Genetics Conference: Posters 99 1Quaid-i-Azam University, Islamabad, Pakistan, 2Bogazici
University, Istanbul, Turkey, 3Institute of Biomedical and
Genetic Engineering, Islamabad, Pakistan Cell Laboratory, Centre of Excellence for Advanced Sciences,
National Research Centre, Cairo, Egypt Cell Laboratory, Centre of Excellence for Advanced Sciences,
National Research Centre, Cairo, Egypt P04.15C
Regenerative approach for treating maxillofacial defects
associated with some genetic disorders P04.15C
Regenerative approach for treating maxillofacial defects
associated with some genetic disorders J. Lee1, B. Lim2, M. Choi3, T. Cho2, J. Chae2 1Gachon University Gil Medical Center, Incheon, Korea,
Republic of, 2Seoul National University Children’s Hospital,
Seoul, Korea, Republic of, 3Seoul National University College
of Medicine, Seoul, Korea, Republic of H. H. El-Ahmady1, A. F. Abd Elazeem2,3,1, N. E. B. Ahmed2,3,
M. A. Abdelrahman2, M. A. Abderazik1 It is known that type I collagenopathy has a broad-spectrum
phenotypic variability. Here, we report a case of a Korean
girl with a heterozygous COL1A1 mutation who had an
atypical presentation. A 26-month-old girl presented with It is known that type I collagenopathy has a broad-spectrum
phenotypic variability. Here, we report a case of a Korean
girl with a heterozygous COL1A1 mutation who had an
atypical presentation. A 26-month-old girl presented with J. del Picchia 100 delayed motor development and failure to thrive. She had
severe growth retardation. She exhibited right-sided plagi-
ocephaly, blue sclerae, and facial dysmorphism, including a
small pointed chin, frontal bossing, and a triangular face,
but had microcephaly. Whole-exome sequencing revealed a
novel de novo heterozygous sequence variant in COL1A1
(p.Gly1127Asp), which was validated by Sanger sequen-
cing. Radiological finding showed generalized osteoporosis
with progressive scoliosis of the spine without evidence of
platyspondyly related to fractures and bowing of the long
bones, and markedly delayed carpal bone age. Muscle
pathology showed a marked size variation of myofibers and
selective type 1 atrophy. This study expanded the clinical
and genetic spectrum of type I collagenopathy with a
COL1A1 variant. Therefore, we suggest that type I col-
lagenopathy should be considered in the patients who have
some features of osteogenesis imperfecta simultaneously
with atypical features such as facial dysmorphism. depending on whether the CL/P is associated with another
anomaly or not. In general, patients with syCL/P follow
Mendelian inheritance, while those with nsCL/P, have a
complex etiology and as such, do not adhere to Mendelian
inheritance. Genome-wide association studies (GWAS)
have identified approximately 30 risk loci for nsCL/P,
which could explain a small fraction of heritability. Materials and Methods: To identify variants causing
nsCL/P, we conducted Whole Exome Sequencing (WES)
on 84 individuals with nsCL/P, drawn from multiplex
families (n = 46). P04.15C
Regenerative approach for treating maxillofacial defects
associated with some genetic disorders Results: We identified rare damaging variants in four
genes known to be mutated in syCL/P: TP63 (1 family),
TBX1 (1 family), LRP6 (1 family) and GRHL3 (2 families),
and clinical reassessment confirmed the isolated nature of
their CL/P. Conclusion: These data demonstrate that CL/P patients
without cardinal signs of a syndrome may still carry a
mutation in a gene linked to syCL/P. Rare coding and non-
coding variants in syCL/P genes could in part explain the
controversial question of “missing heritability” for nsCL/P. Therefore, gene panels designed for diagnostic testing of
syCL/P should be used for nsCL/P patients, especially when
there is at least 3rd degree family history. This would allow
a
more
precise
management,
follow-up
and
genetic
counseling. Moreover,
stratified
cohorts
would
allow
hunting for genetic modifiers. Conclusion: These data demonstrate that CL/P patients
without cardinal signs of a syndrome may still carry a
mutation in a gene linked to syCL/P. Rare coding and non-
coding variants in syCL/P genes could in part explain the
controversial question of “missing heritability” for nsCL/P. Therefore, gene panels designed for diagnostic testing of
syCL/P should be used for nsCL/P patients, especially when
there is at least 3rd degree family history. This would allow
a
more
precise
management,
follow-up
and
genetic
counseling. Moreover,
stratified
cohorts
would
allow
hunting for genetic modifiers. J. Lee: None. B. Lim: None. M. Choi: None. T. Cho:
None. J. Chae: None. P04.17A Whole exome sequencing identifies mutations in 10% of
familial non-syndromic cleft lip and/or palate patients in
genes mutated in well-known syndromes M. Basha1, B. Demeer2, N. Revencu3, S. Theys4, S. Bou Saba5,
O. Boute6, B. Devauchelle7, G. François8, B. Bayet9,
M. Vikkula10 M. Basha1, B. Demeer2, N. Revencu3, S. Theys4, S. Bou Saba5,
O. Boute6, B. Devauchelle7, G. François8, B. Bayet9,
M. Vikkula10 M. Basha: None. B. Demeer: None. N. Revencu: None. S. Theys: None. S. Bou Saba: None. O. Boute: None. B. Devauchelle: None. G. François: None. B. Bayet: None. M. Vikkula: None. 1de Duve Institute, Brussels, Belgium, 2Center for Human
Genetics, CLAD nord de France, CHU Amiens, Amiens,
France, 3Center for Human Genetics, Cliniques universitaires
Saint-Luc, University of Louvain, Brussels, Belgium, 4Pediatric
Dentistry and Oral Care for Special Needs, Cliniques
universitaires Saint-Luc, University of Louvain, Brussels,
Belgium, 5Department of Orthodontics and dentofacial
orthopedics, Cliniques universitaires Saint-Luc, University of
Louvain, Brussels, Belgium, 6Center for Human Genetics,
Lille, France, 7Service of Maxillofacial Surgery and
Stomatology, CHU Amiens-Picardie, Amiens, France,
8Department of Pediatrics, Cliniques universitaires Saint-Luc,
University of Louvain, Brussels, Belgium, 9Centre
Labiopalatin, Division of Plastic Surgery, Cliniques
universitaires Saint-Luc, University of Louvain, Brussels,
Belgium, 10Human Molecular Genetics, de Duve Institute,
University of Louvain, Brussels, Belgium M. Barba1, L. Di Pietro1, C. Bernardini1, L. Massimi1,
G. Tamburrini1, S. Della Longa2, A. Arcovito1, O. Parolini1,
S. A. Boyadjiev3, W. Lattanzi1 BBS9 as potential tissue-specific key protein in BBSome
binding and ciliary trafficking in NCS patients BBS9 as potential tissue-specific key protein in BBSome
binding and ciliary trafficking in NCS patients M. Barba1, L. Di Pietro1, C. Bernardini1, L. Massimi1,
G. Tamburrini1, S. Della Longa2, A. Arcovito1, O. Parolini1,
S. A. Boyadjiev3, W. Lattanzi1 M. Barba1, L. Di Pietro1, C. Bernardini1, L. Massimi1,
G. Tamburrini1, S. Della Longa2, A. Arcovito1, O. Parolini1,
S. A. Boyadjiev3, W. Lattanzi1 1Università Cattolica del Sacro Cuore, Rome, Italy,
2University of L'Aquila, L'Aquila, Italy, 3University of
California Davis, Sacramento, CA, United States Nonsyndromic craniosynostosis (NSC) is a congenital
malformation due to the premature ossification of calvarial
sutures, with an unclear molecular etiopathogenesis. The
Bardet-Biedl Syndrome-associated gene 9 (BBS9), already
associated to NCS by GWAS, encodes a member of the
well-characterized class of BBS proteins that interact
through their C-term to form an octameric complex named
BBSome, necessary for ciliogenesis and ciliary function. Preliminary data identified a suture specific signature, Introduction: Oral clefts i.e. clefts of the lip and/or cleft
palate (CL/P) are the most common craniofacial birth
defects with an approximate incidence of ~1/700. To date
physicians stratify patients with oral clefts into either syn-
dromic CL/P (syCL/P) or non-syndromic CL/P (nsCL/P) Abstracts from the 51st European Society of Human Genetics Conference: Posters 101 including BBS9 and several genes involved in primary
cilium signaling and assembly. In particular, we showed the
overexpression of 5 spliced isoforms of BBS9 in fused
suture specimens of single-suture midline NCS patients. The aim of this study is to clarify the mechanism through
which BBS9 exerts its regulatory function during the
osteogenic commitment and differentiation of somatic stem
cells inside skull bone. We confirmed through qPCR the
overexpression of the selected BBS9 splice isoforms in
mesenchymal stromal cells (CMSC) isolated from fused (p)
and unfused (n) sutures of NCS patients. Then, we assessed
by immunofluorescence that p-CMSC showed a reduced
number of primary cilia compared with same patient-n-
CMSC. Computational modeling of the upregulated iso-
forms showed structural differences in the C-term of the
proteins, predicting that their binding affinity within the
BBSome may be affected. Taken together, these data sug-
gest that selected BBS9 protein isoforms show a tissue-
specific increased expression in osteogenic precursors
residing in NCS fused sutures, owing to a less effective
assembly of the BBSome, which impairs ciliogenesis. Targeted exome sequencing analysis in Turkish non-
syndromic craniosynostosis patients Targeted exome sequencing analysis in Turkish non-
syndromic craniosynostosis patients M. Barba: None. L. Di Pietro: None. C. Bernardini:
None. L. Massimi: None. G. Tamburrini: None. S. Della
Longa: None. A. Arcovito: None. O. Parolini: None. S.A. Boyadjiev: None. W. Lattanzi: None. E. Yilmaz, B. Nur, E. Mihci, O. M. Alper BBS9 as potential tissue-specific key protein in BBSome
binding and ciliary trafficking in NCS patients [Funding support: NIH-NIDCR grant R01DE16886, Fed-
erazione GENE and Università Cattolica] By describing 10 new patients recruited from a population
attending centres for Human Genetics, we further delineate
the clinical spectrum of a new syndromic craniosynostosis
resembling Crouzon syndrome. Singularly, it is inherited
according to an autosomal recessive mode of inheritance. Missense mutations in IL11RA, a gene encoding alpha
subunit of interleukin 11 receptor, were at cause in all five
families, four of them not reported, including two in the Ig-
like C2-type domain. A subset of patients had an associated
connective tissue disorder with joint hypermobility and
intervertebral discs fragility. Two of our patients had syr-
ingomyelia. A lower figure of teeth anomalies than pre-
viously reported in the two large series of patients evaluated
in dental institutes points towards an ascertainment bias. E. Brischoux-Boucher: None. A. Trimouille: None. G. Baujat: None. A. Goldenberg: None. E. Schaefer: None. B. Guichard: None. P. Hannequin: None. S. Baer: None. C. Cabrol: None. E. Weber: None. G. Godfrin: None. M. Lenoir: None. D. Lacombe: None. C. Collet: None. L. Van Maldergem: None. Characterization of the calvarial suture skeletogenic stem
cell niche in nonsyndromic craniosynostosis Characterization of the calvarial suture skeletogenic stem
cell niche in nonsyndromic craniosynostosis 1Department of Pathology and Genetics, University of
Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden,
2Department of Clinical Pathology and Genetics, Sahlgrenska
University Hospital, Gothenburg, Sweden, 3Department of
Plastic Surgery, University of Gothenburg, The Sahlgrenska
Academy, Gothenburg, Sweden 1Department of Pathology and Genetics, University of
Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden,
2Department of Clinical Pathology and Genetics, Sahlgrenska
University Hospital, Gothenburg, Sweden, 3Department of
Plastic Surgery, University of Gothenburg, The Sahlgrenska
Academy, Gothenburg, Sweden M. Barba1, L. Di Pietro2, C. Prampolini2, V. Orticelli2,
L. Massimi2, P. Frassanito2, S. A. Boyadjiev3, M. Caldarelli2,
G. Tamburrini2, O. Parolini2, W. Lattanzi2 1Università Cattolica del Sacro Cuore, Roma, Italy,
2Università Cattolica del Sacro Cuore, Rome, Italy,
3University of California Davis, Sacramento, CA, United States 1Università Cattolica del Sacro Cuore, Roma, Italy, 1Università Cattolica del Sacro Cuore, Roma, Italy, 2Università Cattolica del Sacro Cuore, Rome, Italy, Università Cattolica del Sacro Cuore, Rome, Italy, Approximately 1 in 2000 children are affected by cranio-
synostosis. During the last few years, mutations in several
genes have been identified as cause of early closing of the
cranial sutures. The list increases constantly as new genes
with relation to craniosynostosis are detected. Craniosy-
nostosis occurs isolated or associated with other symptoms
including malformations as part of several syndromic dis-
orders - the most known are represented by Pfeiffer,
Crouzon, Jackson-Weiss, Antley-Bixler, Apert, Saethre-
Chotzen and Muenke syndromes. The aim of our project
was to study the prevalence and spectrum of the genetic
disorders which are associated with early suture closing in a
unique patient cohort including 144 individuals with mostly
uni- or bicoronal synostosis. The mutational screening of
our patient cohort has been performed by using a custom-
designed
NGS-enrichment
panel
including
63
craniosynostosis-related genes selected from OMIM. In 91
out of 144 screened patients (approximately 63%), either a
known previously reported pathogenic variant or a likely
pathogenic variant has been detected. As expected, the
majority of variants have occurred in the craniosynostosis
“core genes”: FGFR2, TWIST1, FGFR3, TCF12, EFNB1
and POR. However, novel likely pathogenic variants have
been observed also in IL11RA, KMT2D and SKI-genes. Our
study shows that a broad genetic screening using a targeted
NGS assay have a high diagnostic yield in a large cohort of
patients with craniosynostosis. P04.19C Our data seem to suggest that in NCS the
in vivo tissue microenvironment may cause the enhanced
osteogenic differentiation of suture MSCs leading to pre-
mature suture closure. Funding support: Federazione GENE
and Università Cattolica del Sacro Cuore Conclusion: Our data highlights the importance of
increased diagnostic rate (35.3%) with the use of this
panel, and help to solve the genotype-phenotype correla-
tions in nsCRN. M. Barba: None. L. Di Pietro: None. C. Prampolini:
None. V. Orticelli: None. L. Massimi: None. P. Frassa-
nito: None. S.A. Boyadjiev: None. M. Caldarelli: None. G. Tamburrini:
None. O. Parolini:
None. W. Lattanzi: None. Acknowledgement: This research was supported by
grant from Scientific Research Project Council of Akdeniz
University (#TDK-2015-933). The outcome of broad genetic screening in a cohort of 144
patients with craniosynostosis The outcome of broad genetic screening in a cohort of 144
patients with craniosynostosis P04.22B E. Yilmaz: None. B. Nur: None. E. Mihci: None. O.M. Alper: None. P04.21A A. Topa1,2, A. Rohlin2, L. Lovmar2, G. Stenman1,2, L. Kölby3 A. Topa: None. A. Rohlin: None. L. Lovmar: None. G.
Stenman: None. L. Kölby: None. P04.19C IL11RA-related Crouzon-like autosomal recessive
craniosynostosis in ten new patients: which are the
differences? Introduction: Craniosynostosis is described as an early
fusion of one or more calvarial sutures. Early closure of
these sutures results with new head shape that inhibits the
normal growth and development of the brain. Worldwide,
the estimated prevalence of the syndrome is 1 in 2500. Craniosynostosis is divided into two sub-goups: syndromic
and non-syndromic (nsCRN). More than 70% of the
patients have nsCRN. Although more than 50 genes were
identified, the genetic cause is mostly unknown. Approxi-
mately 24% of cases can be genetically identified. E. Brischoux-Boucher1, A. Trimouille2, G. Baujat3,
A. Goldenberg4, E. Schaefer5, B. Guichard6, P. Hannequin7,
S. Baer5, C. Cabrol1, E. Weber8, G. Godfrin9, M. Lenoir10,
D. Lacombe2, C. Collet11, L. Van Maldergem1 1Centre de genetique humaine CHU Besançon, Besancon,
France, 2Service de génétique médicale CHU Bordeaux,
Bordeaux, France, 3Institut Imagine CHU Necker-Enfants-
Malades, Paris, France, 4Service de genetique CHU Rouen,
Rouen, France, 5Service de genetique médicale CHU
Strasbourg, Strasbourg, France, 6Service de chirurgie maxillo-
faciale CHU Rouen, Rouen, France, 7Service de
nrurochirurgie CHU Rouen, Rouen, France, 8Service de
chirurgie maxillo-faciale CHU Besançon, Besancon, France,
9Service de neurochirurgie CHU Besançon, Besancon, France,
10Service de radiologie CHU Besançon, Besancon, France,
11Service de biochimie et biologie moléculaire CHU
Lariboisière, Paris, France Method: Unrelated seventeen nsCRN Turkish cases have
been sequenced by using Dysmorphia and Dsyplasia
Research Panel v2, includes 519 genes, of Ion AmpliSeq. IonS5 sequencing technology was used and data were
analyzed with Ingenuity software. Genetic variants in
several genes known to cause craniosynostosis were
filtered. Clinically pathogenic variants were checked and
confirmed by Sanger sequencing. Results: In six of the cases (35.3%), we identified six
different mutations (including three novel ones) in ERF,
FREM1 and TCF12 genes. The novel missense mutations
are p.P1802L(c.5405C>T) and p.G1493R(c.4477G>A) in
FREM1, and frameshift mutation is c.1106_111delCTCT-
CAC(p.P369fs*26)
in
TCF12
gene. POLYPHEN, 102 J. del Picchia calvarial niche. Our data seem to suggest that in NCS the
in vivo tissue microenvironment may cause the enhanced
osteogenic differentiation of suture MSCs leading to pre-
mature suture closure. Funding support: Federazione GENE
and Università Cattolica del Sacro Cuore PROVEAN and SIFT analysis revealed that p.P1802L and
p.G1493R are predicted to be deleterious and damaging
with scores of 0.997; -9.07; 0.003 and 1.000; -3.99; 0.019,
respectively. calvarial niche. Characterization of the calvarial suture skeletogenic stem
cell niche in nonsyndromic craniosynostosis 3University of California Davis, Sacramento, CA, United States 3University of California Davis, Sacramento, CA, United States Nonsyndromic craniosynostosis (NCS) is the congenital
premature fusion of skull sutures. The suture mesenchyme
houses a skull-specific stem cell niche, plausibly impaired
in NCS fused suture sites. To test this hypothesis, we
characterized the stem cell niche of open and fused sutures
of NCS patients. Lineage-specific markers were analyzed,
by qPCR and immunofluorescence, in suture tissues and in
calvarial mesenchymal stem cells (CMSC). MSC from
alternative tissues served as controls. We analyzed the
localization of THY1 (skeletal stemness-marker), GLI1
(putative
calvarial
stemness-marker)
and
AXIN2
(mesenchymal cell fate determinant) in suture tissue sec-
tions: AXIN2 resulted mainly expressed at the endosteal
ossified side, while THY1 and GLI1 were primarily
expressed within the trabeculae, enriched with proliferating
cells. Both NCS suture tissues and CMSC isolated thereof
expressed reduced levels of TEK and ENPEP (bone marrow
stem cells differentiation markers) compared with controls. AXIN2 levels were higher in open suture-derived CMSC
than in fused suture cells and in controls. Upon in vitro
osteogenic induction, the expression of THY1 and GLI1
decreased, whereas AXIN2 levels increased, in both open-
and fused- suture derived CMSC. CMSCs isolated from
both fused and unfused sutures shared the same marker
expression profile, indicating that explant cultures allowed
selecting
comparable
cell
populations,
THY1+/GLI1+
representing the stem cell population within the human A. Topa: None. A. Rohlin: None. L. Lovmar: None. G. Stenman: None. L. Kölby: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 103 Ultrastructural elastic fiber morphology in cutis laxa
reflects the underlying pathogenesis and supports a novel
clinical classification A. Beyens1,2, R. De Rycke3, H. Syryn1, B. Fischer-Zirnsak4,
T. Van Damme1, I. Hausser5, M. De Bruyne3, M. Morroni6,
S. Nampoothiri7, K. Mahesh8, U. Kornak4, Z. Urban9, S. Hadj-
Rabia10, C. Bodemer11, S. De Schepper2, E. C. Davis12,
B. Callewaert1 Conclusion: Our novel nosology of the CL syndromes
provides a practical approach to the broad differential
diagnosis of CL syndromes. The classification forms a basis
to integrate the clinical presentation with the pathogenesis
and ultrastructural EF defects and might bode for new
management guidelines and therapeutic approaches. 1Center for Medical Genetics, Ghent University Hospital,
Ghent, Belgium, 2Department of Dermatology, Ghent
University Hospital, Ghent, Belgium, 3Department of
Biomedical Molecular Biology, Ghent University, Ghent,
Belgium, 4Institute of Medical Genetics and Human Genetics,
Charité-Universitätsmedizin Berlin, Berlin, Germany,
5Institute of Pathology, Universitätsklinikum Heidelberg,
Heidelberg, Germany, 6Department of Experimental and
Clinical Medicine, Section of Neuroscience and Cell Biology,
School of Medicine, Università Politecnica delle Marche and
Electron Microscopy Unit, United Hospitals, Ancona, Italy,
7Department of Pediatric Genetics, Amrita Institute of Medical
Sciences and Research Center, Kochi, India, 8Department of
Pediatric Cardiology, School of Medicine, Kochi, India,
9Department of Human Genetics, Graduate School of Public
Health, University of Pittsburgh, Pittsburgh, PA, United States,
10Service de Dermatologie, CHU Paris - Hôpital Necker-
Enfants Malades, Paris, France, 11Centre MAGEC (Maladies
rares Génétiques a Expression Cutanée), Service de
Dermatologie, CHU Paris - Hôpital Necker-Enfants Malades,
Paris, France, 12Department of Anatomy and Cell Biology,
McGill University, Montréal, QC, Canada (Grant Reference BOF01N04516) A. Beyens: None. R. De Rycke: None. H. Syryn: None. B. Fischer-Zirnsak: None. T. Van Damme: None. I. Hausser: None. M. De Bruyne: None. M. Morroni: None. S. Nampoothiri: None. K. Mahesh: None. U. Kornak:
None. Z. Urban: None. S. Hadj-Rabia: None. C. Bodemer: None. S. De Schepper: None. E.C. Davis:
None. B. Callewaert: None. P04.24D microscopy in skin biopsies of all CL subtypes and found
discriminative and specific findings that correlate with the
main presenting symptoms (emphysema, arterial tortuosity,
skeletal defects/mental disability with or without intrauter-
ine growth retardation/cataract). Moreover, EF ultrastuc-
tural
morphology
reflects
the
involved
molecular
pathogenesis and provides new insights in elastic fiber
biogenesis. Ultrastructural elastic fiber morphology in cutis laxa
reflects the underlying pathogenesis and supports a novel
clinical classification Microbial signatures in TIF1γ autoantibody positive
dermatomyositis patients S. Megremis1, T. Walker1, X. He1, J. O’Sullivan1, A. Payton1,
N. Pendleton1, L. Hampson1, R. Cooper2, W. Ollier1, H. Chinoy1,
I. Hampson1, J. Lamb1 1Center for Life Course Epidemiology and Systems Medicine,
Faculty of Medicine, University of Oulu, Oulu, Finland,
2Center for Cell – Matrix Research, Faculty of Biochemistry
and Molecular Medicine, University of Oulu, Oulu, Finland,
3Medical Research Center Oulu, Oulu University Hospital and
University of Oulu, Oulu, Finland, 4Biocenter Oulu, University
of Oulu, Oulu, Finland, 5Research Unit of Medical Imaging,
Physics and Technology, Faculty of Medicine, University of
Oulu, Oulu, Finland, 6Finnish Institute of Occupational
Health, Health and Work Ability, and the Disability Prevention
Center, Oulu, Finland 1University of Manchester, Manchester, United Kingdom,
2University of Liverpool, Liverpool, United Kingdom Introduction: Tripartite motif-containing (TRIM) proteins
are involved in innate immunity. Reports support a role for
microbial infections in dermatomyositis (DM); TIF1γ
(TRIM33) autoantibody positive patients may have reduced
ability to restrict pathogen infection. Lumbar disc degeneration (LDD) is one of the contributing
factors behind low back pain (LBP). Modic change (MC) is
a phenotype of LDD and is visualized as bone marrow
signal intensity changes on magnetic resonance imaging. MC is a heritable trait with heritability estimation around
30%. It is strongly associated with LBP. We studied two
families to identify predisposing variants for MC. Nine
individuals were chosen for whole exome sequencing. We
focused on rare (MAF < 0.01) and private variants with
harmful in silico predictions and variants located in reg-
ulatory regions. The identified variants were genotyped
from additional family members. One rare variant co-
segregated with MC in each family. In the Family I, the
observed variant was an insertion and deletion mutation in
the HSPG2 gene, resulting in a premature stop codon. HSPG2 encodes a heparin sulfate proteoglycan, which is a
structural protein expressed in mammalian cartilage and
basement membranes. Rare autosomal recessive disorders
with osteochondrodysplasia are caused by mutations in the
HSPG2 gene. In the Family II, a single nucleotide poly-
morphism in the MAML1 gene was identified. MAML1 is Material and Methods: Serum total Ig from 20 DM
patients and 20 age-matched healthy controls were pooled
for competitive panning and clonal expansion of the
FliTrxTM bacterial random-peptide surface display system. DNA
libraries
were
sequenced
using
pair-end
high-
throughput sequencing. Translated peptide sequences were
searched for maximum exact matches against the NCBI
microbial database and assigned to taxa. P04.25A
Diverse mechanisms of germline and somatic mosaicism in
CYLD cutaneous syndrome M. Arefi1, V. Wilson2, D. Bajwa1, S. Zwolinski2, N. Sinclair1,
P. Brennan2, N. Bown2, D. Bourn2, M. Santibanez-Koref1,
J. Burn1, N. Rajan1 1Institute of Genetic Medicine, Newcastle upon Tyne, United
Kingdom, 2Northern Genetics Service, Newcastle upon Tyne,
United Kingdom Background: Cutis laxa (CL) syndromes are a hetero-
geneous group of multisystem disorders that share a loose,
redundant skin as a common feature reflecting elastic fiber
(EF) deficiency. The pathogenesis of each of the CL sub-
types is different but affects elastogenesis. However, light
microscopy of the dermis is non-discriminative and the
recently observed vast molecular heterogeneity mitigates
the clinical validity and practicality of the current classifi-
cation, based on the mode of inheritance and systemic
involvement. Six unsolved cases that met clinical diagnostic criteria for
CYLD cutaneous syndrome, deemed to be mutation nega-
tive following Sanger sequencing, were investigated for
clinical and genetic features of mosaicism. One case
demonstrated 8% blood mosaicism for a pathogenic muta-
tion using a NGS assay targeting CYLD. We found no
causative lesions in the blood of five of the six remaining
cases. In two cases, we investigated tumour tissue. In the
first case, two tumour samples demonstrated a recurrent
19bp deletion in the skin only. In the second, five distinct
mutations resulting in a stop codon in CYLD were detected
in five tumours. SNP array analysis of three tumours
demonstrated a recurrent 5.5Mb deletion encompassing
CYLD and 23 other genes. This patient had a daughter who
had developmental delay and unilateral renal agenesis, but
no cylindromas. This phenotype has been reported in other
patients with large germline deletions including CYLD,
suggesting transmission of the deletion. The remaining Aims: We aim to classify the CL subtypes by means of a
simple flowchart and to evaluate correlations between EF
ultrastructural morphology (as evaluated by transmission
electron microscopy) and clinical presentation. Results: Following literature review, we developed a 7-
step flowchart to classify the CL subtypes. As a proof of
principle, we systematically evaluated 68 CL patients from
our in-house database and could allocate 95% of patients to
the
right
gene. We
performed
transmission
electron J. del Picchia 104 autoantibodies and dermatomyositis. The increased pre-
sence of viral peptides in DM patients, particularly ssRNA
and retro-transcribing viruses, agrees with pathways known
to be regulated by TRIMs and points towards increased
susceptibility for certain viral families. A whole exome study identifies novel candidate genes for
vertebral bone marrow signal changes (Modic changes) A whole exome study identifies novel candidate genes for
vertebral bone marrow signal changes (Modic changes) P04.25A
Diverse mechanisms of germline and somatic mosaicism in
CYLD cutaneous syndrome The increased
detection of peptides against potentially ‘pathogenic’ but
not ‘non-pathogenic’ bacteria from the same families might
play a key role in the development of the disease. three cases had clinical features suggesting cutaneous
mosaicism. We conclude that, firstly, mosaicism may be
detectable in the blood and account for the presence of
cylindromas in the skin. Secondly, some patients may only
have a unilateral linear arrangement or cluster of tumours,
with absence of mosaic mutation in the blood suggesting
cutaneous mosaicism alone. Thirdly, mosaic mutations
detectable in the blood may affect the germline and be
transmissible to offspring; where this is due to a contiguous
deletion involving CYLD, the phenotype may involve
multiple organ systems. S. Megremis: None. T. Walker: None. X. He: None. J. O’Sullivan: None. A. Payton: None. N. Pendleton: None. L. Hampson: None. R. Cooper: None. W. Ollier: None. H. Chinoy: None. I. Hampson: None. J. Lamb: None. M. Arefi: None. V. Wilson: None. D. Bajwa: None. S. Zwolinski: None. N. Sinclair: None. P. Brennan: None. N. Bown: None. D. Bourn: None. M. Santibanez-Koref:
None. J. Burn: None. N. Rajan: None. M. Arefi: None. V. Wilson: None. D. Bajwa: None. S. Zwolinski: None. N. Sinclair: None. P. Brennan: None. N. Bown: None. D. Bourn: None. M. Santibanez-Koref:
None. J. Burn: None. N. Rajan: None. P04.26B M. Kraatari1,2,3, S. Skarp1,2,4, J. Niinimäki3,5, J. Karppinen1,3,6,
M. Männikkö1,2,4 M. Kraatari1,2,3, S. Skarp1,2,4, J. Niinimäki3,5, J. Karppinen1,3,6,
M. Männikkö1,2,4 Microbial signatures in TIF1γ autoantibody positive
dermatomyositis patients Microbial signatures in TIF1γ autoantibody positive
dermatomyositis patients C.D. Durmaz:
None. J.A. McGrath: None. P. Ertop: None. A. Okçu
Heper: None. A. Boyvat: None. H. Ilgın Ruhi: None. E. Gökpınar İli: None. S. Vural: None. C.D. Durmaz:
None. J.A. McGrath: None. P. Ertop: None. A. Okçu
Heper: None. A. Boyvat: None. H. Ilgın Ruhi: None. M. Kraatari: None. S. Skarp: None. J. Niinimäki:
None. J. Karppinen: None. M. Männikkö: None. R. Solc1, K. HIrschfeldova2 E. Gökpınar İli1, S. Vural2, C. D. Durmaz1, J. A. McGrath3,
P. Ertop2, A. Okçu Heper4, A. Boyvat2, H. Ilgın Ruhi1 1Charles University in Prague, Faculty of Science, Department
of Anthropology and Human Genetics, Prague, Czech
Republic, 2Charles University in Prague, First Faculty of
Medicine, Institute of Biology and Medical Genetics, Prague,
Czech Republic 1Charles University in Prague, Faculty of Science, Department
of Anthropology and Human Genetics, Prague, Czech
Republic, 2Charles University in Prague, First Faculty of
Medicine, Institute of Biology and Medical Genetics, Prague,
Czech Republic 1Department of Medical Genetics, Ankara University School of
Medicine, Ankara, Turkey, 2Department of Dermatology,
Ankara University School of Medicine, Ankara, Turkey, 3St
John's Institute of Dermatology, King's College London, Guy's
Hospital, London, United Kingdom, 4Department of
Pathology, Ankara University School of Medicine, Ankara,
Turkey 1Department of Medical Genetics, Ankara University School of
Medicine, Ankara, Turkey, 2Department of Dermatology,
Ankara University School of Medicine, Ankara, Turkey, 3St
John's Institute of Dermatology, King's College London, Guy's
Hospital, London, United Kingdom, 4Department of
Pathology, Ankara University School of Medicine, Ankara,
Turkey Introduction: The human SHOX gene encodes an impor-
tant growth regulating transcription factor. Heterozygous
deletions of the gene or deletions of one of its numerous
enhancers are responsible for Lėri-Weill dyschondrosteosis
(LWD) and small portion of idiopathic short stature (ISS). Effect of reciprocal duplications is less distinct. The aim of
our study was to compare frequency, extent and distribution
of SHOX
gene
and associated elements duplications
between the LWD/ISS patients and population sample. A
preliminary analysis indicated that rather than the difference
in frequency it is the difference in distribution of duplicated
areas. A meta-analysis of published cases was performed to
confirm the consistency of these data. Introduction: Dystrophic epidermolysis bullosa (DEB) is a
rare form of genodermatoses characterized by blistering
condition, caused by COL7A1 gene mutations with domi-
nant or recessive inheritance. Autosomal dominant DEB
may remain throughout life in a mild form. However,
phenotypic aggravation may be seen between patients. Here, we present a family with seven affected members
from three-generations with phenotypic heterogeneity. Materials and Methods: Genomic DNA was isolated
from peripheral blood samples of the patients and COL7A1
gene was sequenced. Material and Methods: For the purpose of meta-
analysis, merged groups of published cases were created:
LWD patients (31 individuals), ISS patients (29 individuals)
and population sample (36 individuals). P04.28D Comparison of the distribution of duplicated regions
associated with SHOX gene between LWD/ISS patients and
population sample - conclusions of the meta-analysis Intrafamilial phenotypic heterogeneity in dominant
dystrophic epidermolysis bullosa associated with G2043R
mutation in COL7A1 Microbial signatures in TIF1γ autoantibody positive
dermatomyositis patients Material and Methods: Serum total Ig from 20 DM
patients and 20 age-matched healthy controls were pooled
for competitive panning and clonal expansion of the
FliTrxTM bacterial random-peptide surface display system. DNA
libraries
were
sequenced
using
pair-end
high-
throughput sequencing. Translated peptide sequences were
searched for maximum exact matches against the NCBI
microbial database and assigned to taxa. Results:
DM
patients
exhibited
higher
number
of
microbial peptide reads (22x106 vs 14x106) and unique
microbial taxa (32x105 vs 28x105). Viral peptides were
higher in DM patients (4x106 vs 2x106) and occupied larger
space in the complete microbial IgOme (12% vs 9%). Peptides of cellular microbial origin also were increased in
DM patients but with no change in the relative abundance
(63% vs 64%). Specific differences were observed for
dsDNA (Herpesvirales), ssRNA (Orthomyxoviridae), and
retro-transcribing viruses (HIV) and for potentially patho-
genic bacteria (Streptococcaceae). Conclusions: This is the first systematic high-throughput
investigation of a link between microbial exposures, TIF1γ Conclusions: This is the first systematic high-throughput
investigation of a link between microbial exposures, TIF1γ Abstracts from the 51st European Society of Human Genetics Conference: Posters 105 to the Index patient; which decreased her generalized
lesions and severe pruritus very effectively. to the Index patient; which decreased her generalized
lesions and severe pruritus very effectively. considered to be a transcriptional coactivator in the Notch
signaling pathway which regulates many biological func-
tions including cartilage development and homeostasis. MAML1 has been reported to affect the activity of RUNX2,
a transcription factor essential in the osteoblast differentia-
tion. RUNX2 has been reported to be highly expressed in
degenerated discs. We identified two promising candidate
genes for MC, HSPG2 and MAML1. Our findings are novel
in lumbar spine degenerative phenotypes. considered to be a transcriptional coactivator in the Notch
signaling pathway which regulates many biological func-
tions including cartilage development and homeostasis. MAML1 has been reported to affect the activity of RUNX2,
a transcription factor essential in the osteoblast differentia-
tion. RUNX2 has been reported to be highly expressed in
degenerated discs. We identified two promising candidate
genes for MC, HSPG2 and MAML1. Our findings are novel
in lumbar spine degenerative phenotypes. Conclusions: Genetic and environmental factors as well
as hormonal status may be responsible for the intrafamilial
phenotypic heterogeneity. Also, we would like to empha-
size IVIG as a treatment option in DEB patients with more
severe phenotype. E. Gökpınar İli: None. S. Vural: None. Degradation routes of trafficking-defective VLDLR
mutants associated with dysequilibrium syndrome Dyssegmental dysplasia (DD) is a rare autosomal recessive
skeletal disorder characterized by congenital short limbed
dwarfism. Based on clinical features, it is classified into two
types;
the
severe,
lethal
Silverman-Handmaker
type
(DDSH)
and
milder
form
Rolland-Desbuquois
type
(DDRD). Among the molecularly confirmed DD patients,
most are DDSD type. There have been no reports in the
patients with molecularly confirmed DDRD. Here, we
report a case with homozygous biallelic mutation in
HSPG2, diagnosed as DDRD based on clinical features in
neonatal period. The proband was 15-years-old boy. He was
the first child of non-consanguineous and healthy parents. Physical examination revealed cleft lip palate, shortening of
limbs, clubfoot, flexion contractures of bilateral knees and
elbows, right inguinal hernia, and narrow chest. X-rays
showed small thorax and vertebral body size difference,
dumbbell-shaped tubular bones. Based on the radiological
investigation, he was diagnosed as DDRD. Now, he is 15
years old, with the height 120 cm (-7.5 SD), weight 27.6 kg
(-2.8 SD), and had short distance walkable. To confirm the
diagnosis molecularly, we performed Mendelian exome
using the TruSight One Sequencing Panel (Illumina, Inc.,
San Diego, CA, USA). Captured DNA was sequenced on a
MiSeq platform (Illumina) with 151 bp paired-end reads. Targeted resequencing and Sanger sequencing identified a
novel biallelic homozygous variant c.9970G>A, p.G3324R
in HSPG2. This is a first case report with molecularly
confirmed
DDRD. These
results
suggest
genotype-
phenotype correlation in the HSPG2 related disorders. United Arab Emirates University, Al-Ain, United Arab
Emirates United Arab Emirates University, Al-Ain, United Arab
Emirates Introduction: Endoplasmic reticulum associated degrada-
tion (ERAD) of misfolded proteins by the ubiquitin-
proteasome system is a recurrent theme in rare genetic
disorders and the process crucially involve ER-membrane
complexes such as HRD1-SEL1L. Previously, we have
reported that missense mutations in the Very Low Density
Lipoprotein Receptor gene (VLDLR), causing Dysequili-
brium syndrome (DES), disrupt ligand-binding, due to
retention of the mutants in ER. This study explores in detail
the degradation routes of these ER-retained VLDLR
mutants. Materials and Methods: The missense pathogenic
VLDLR variants have been generated by QuikChange site-
directed mutagenesis. The constructs were expressed in
HEK293 cells and analyzed by immuno-pull down assays
and Western blotting. Protein turn-over studies were
conducted by translation shut-off assays and inhibition of
proteasomal/lysosomal
degradation. The
HRD1-SEL1L
knockout HEK293 cell lines have been generated by
CRISPR/Cas9. Materials and Methods: The missense pathogenic
VLDLR variants have been generated by QuikChange site-
directed mutagenesis. The constructs were expressed in
HEK293 cells and analyzed by immuno-pull down assays
and Western blotting. Protein turn-over studies were
conducted by translation shut-off assays and inhibition of
proteasomal/lysosomal
degradation. The
HRD1-SEL1L
knockout HEK293 cell lines have been generated by
CRISPR/Cas9. Results: We show that VLDLR mutants are retained in
the ER for prolonged periods which could be facilitated by
association with the ER resident chaperone calnexin. The
mutants were found to be aggregation prone and capable of
eliciting ER stress. Inhibition studies suggested that these
mutants are degraded partially by the proteasomal pathway. Further, the degradation of VLDLR wild type and a mutant
were delayed in CRISPR/Cas9 edited SEL1L knock-out
cells which could be reversed by exogenous expression
of SEL1L. K. Kurosawa: None. K. Shono: None. T. Yokoi: None. N. Harada:
None. M. Akahira-Azuma:
None. Y. Enomoto: None. Y. Tsurusaki: None. N. Aida: None. K. Kurosawa: None. K. Shono: None. T. Yokoi: None. N. Harada:
None. M. Akahira-Azuma:
None. Y. Enomoto: None. Y. Tsurusaki: None. N. Aida: None. K. Kurosawa, K. Shono, T. Yokoi, N. Harada, M. Akahira-
Azuma, Y. Enomoto, Y. Tsurusaki, N. Aida R. Solc: None. K. HIrschfeldova: None. P04.30B Kanagawa Children's Medical Center, Yokohama, Japan 1Department of Genomics, Life&Brain, Bonn, Germany,
2Institute of Human Genetics, University of Bonn, Bonn, R. Solc1, K. HIrschfeldova2 Only carriers of the
duplication with one brakepoint within the chromosomal
region chrX:398,000-980,000 (hg19) were included. Extent,
distribution and relative frequency of duplications were
compared among merged groups. Material and Methods: For the purpose of meta-
analysis, merged groups of published cases were created:
LWD patients (31 individuals), ISS patients (29 individuals)
and population sample (36 individuals). Only carriers of the
duplication with one brakepoint within the chromosomal
region chrX:398,000-980,000 (hg19) were included. Extent,
distribution and relative frequency of duplications were
compared among merged groups. Results: p.G2043R (c.6127G>A) mutation was found in
the index patient and verified in the other affected family
members. One patient in the family was diagnosed with
DEB while others had severe pruritus and lichenified linear
plaques located more prominently on the extensor surface of
the arms and shins. Disease onset was early infancy with a
significant increase in complaints after puberty. In female
patients, who had a more pronounced phenotype, pregnancy
was associated with exacerbation of the disease. Nail
dystrophy of hands and feet was present in all patients. Index patient had moderately high serum IgE levels. Skin
biopsy revealed eosinophilic infiltrate, and the previous
diagnosis was in favor of acquired epidermolysis bullosa. Intravenous immunoglobulin (IVIG) treatment was initiated Results: p.G2043R (c.6127G>A) mutation was found in
the index patient and verified in the other affected family
members. One patient in the family was diagnosed with
DEB while others had severe pruritus and lichenified linear
plaques located more prominently on the extensor surface of
the arms and shins. Disease onset was early infancy with a
significant increase in complaints after puberty. In female
patients, who had a more pronounced phenotype, pregnancy
was associated with exacerbation of the disease. Nail
dystrophy of hands and feet was present in all patients. Index patient had moderately high serum IgE levels. Skin
biopsy revealed eosinophilic infiltrate, and the previous
diagnosis was in favor of acquired epidermolysis bullosa. Intravenous immunoglobulin (IVIG) treatment was initiated Results: There was a significant difference in the relative
frequency of CNE-9 enhancer duplications (11 vs. 3) and
complete SHOX (exon1-6b) duplications (4 vs. 24) (p-value
0.0139 and p-value 0.000014, respectively) between the
merged LWD sample and the merged population sample. Conclusion: We propose, partial duplications of the
SHOX gene coding sequences and small duplications
encompassing
the
CNE-9
enhancer
are
the
highly 106 J. del Picchia P04.31C penetrating alleles associated with the increased risk of
LWD/ISS development. Acknowledgement: The study was
supported by Charles University (UNCE204022) and its
Grant Agency (GAUK202615). 0 .3 C
Biallelic homozygous mutation of HSPG2 in a patient with
dysssegmental dysplasia, Rolland-Desbuquois type Biallelic homozygous mutation of HSPG2 in a patient with
dysssegmental dysplasia, Rolland-Desbuquois type K. Kurosawa, K. Shono, T. Yokoi, N. Harada, M. Akahira-
Azuma, Y. Enomoto, Y. Tsurusaki, N. Aida P04.32D Congenital anonychia and uncombable hair syndrome: co-
inheritance of homozygous mutations in RSPO4 and PADI3 Conclusions: ER retention of VLDLR mutants involves
binding to calnexin, elevated ER stress, and delayed
degradation which is dependent on SEL1L. Since LDLR
family members share common structural domains, com-
mon mechanisms may be involved in their processing and
trafficking (31M254). M. T. Romano1,2, C. K. Hsu3,4,5, A. Nanda6, E. Rashidghamat3,
J. Y. W. Lee3, H. Y. Huang4, C. Songsantiphap3,7, J. Y. Y. Lee4,
H. Al-Ajmi6, M. A. Simpson8, C. Tziotzios3, R. C. Betz1,2, J. A. McGrath3 1Department of Genomics, Life&Brain, Bonn, Germany,
2Institute of Human Genetics, University of Bonn, Bonn, B.R. Ali: None. P. Kizhakkedath: None. A. John:
None. L. Al-Gazali: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 107 1Queens University Medical School, Kingston, ON, Canada,
2IWK Health Centre, Halifax, NS, Canada, 3University of
Washington, Seattle, WA, United States Germany, 3St John’s Institute of Dermatology, King's College
London, London, United Kingdom, 4Department of
Dermatology, National Cheng Kung University Hospital,
Tainan, Taiwan, 5Institute of Clinical Medicine, National
Cheng Kung University, Tainan, Taiwan, 6As’ ad Al-Hamad
Dermatology Center, Al Sabah Hospital, Kuwait city, Kuwait,
7Department of Dermatology, Chulalongkorn University,
Bangkok, Thailand, 8Division of Genetics and Molecular
Medicine, King's College London, London, United Kingdom We present an adult woman with cEDS, bilateral hip dis-
location and obstetric anesthetic complications. The patient
had generalized joint hypermobility, childhood skin fragi-
lity and shoulder dislocations. Her bilateral congenital hip
dislocations were treated with Plavik harness. She was
diagnosed with cEDS at age 15 following evaluation with
multiple affected family members. There was an extensive
family history of cEDS features in 7 individuals over three
generations spanning in age from 7 to 80 years, none were
known to have had bone fragility or vascular complications. The patient had a normal echocardiogram during preg-
nancy. The patient presented with severe post-partum
orthostatic headache after failed epidural anesthesia, dur-
ing which there was unexpected dural puncture. She
recovered after epidural blood patch treatment, following
unsuccessful sphenopalatine ganglion block. Sequencing of
a large panel of EDS genes, identified the COL1A1 variant
c. 934C>T, p.Arg312Cys. This variant has been reported
with a classical EDS like phenotype. The child of patient 1
reported in Malfait et al. (2007), had congenital hip dis-
location (1). The variant is mentioned in the 2017 EDS
nosology for both cEDS and vascular type EDS (2). P04.32D Whilst
three patients with this same pathogenic variant have been
reported to have had a vascular complication, a recent report
of a large family was more reassuring (3). We present
further cumulative phenotypic data on a large family with
this genotype. Ectodermal dysplasia comprises a heterogeneous group of
genetic disorders defined by developmental defects in
ectoderm-derived tissues. The great heterogeneity of the
symptoms is often an obstacle for the identification of the
causative gene, although the use of next generation
sequencing has brought new insights. Here, we investigated
a 4-year-old Kuwaiti boy showing both congenital ano-
nychia and uncombable hair syndrome. Through whole
exome sequencing (WES) we identified mutations in two
separate genes, demonstrating that the patient’s phenotype
comes from the overlap of two autosomal recessive dis-
orders. With regards to anonychia, we identified a pre-
viously known homozygous splice-site mutation in RSPO4. The encoded protein, R-spondin, is expressed in nail
mesenchyme and is an activator of the Wnt/β-catenin
pathway. For the hair abnormality, we found a novel
homozygous missense mutation in PADI3. This gene
encodes for peptidylarginine deiminases 3 (PADI3), which
is involved in deiminating trichohyalin in the hair follicle,
contributing to its structure.All mutations were verified by
Sanger sequencing. Furthermore, the PADI3 mutation was
investigated
through
immunoblotting
and
immuno-
fluorescence in a keratinocyte cell line, showing both lower
expression and formation of aggregates for the mutant
protein compared to wild-type. In conclusion, our case
highlights the value of WES in identifying co-inheritance of
two distinct conditions in consanguineous pedigrees, giving
rise to an ectodermal dysplasia phenotype. J. Duong: None. A. Rideout: None. J. Beis: None. S. Parkash:
None. U. Schwarze:
None. A. Vandersteen: None. J. Duong: None. A. Rideout: None. J. Beis: None. S. Parkash:
None. U. Schwarze:
None. A. Vandersteen: None. P04.34B Molecular genetic analysis of Ehlers-Danlos Syndrome in
Northwestern region of Russia M.T. Romano: None. C.K. Hsu: None. A. Nanda:
None. E. Rashidghamat: None. J.Y.W. Lee: None. H.Y. Huang: None. C. Songsantiphap: None. J.Y.Y. Lee:
None. H. Al-Ajmi: None. M.A. Simpson: None. C. Tziotzios: None. R.C. Betz: None. J.A. McGrath: None. M.T. Romano: None. C.K. Hsu: None. A. Nanda:
None. E. Rashidghamat: None. J.Y.W. Lee: None. H.Y. Huang: None. C. Songsantiphap: None. J.Y.Y. Lee:
None. H. Al-Ajmi: None. M.A. Simpson: None. C. Tziotzios: None. R.C. Betz: None. J.A. McGrath: None. T. I. Kadurina1, E. A. Serebriakova1,2, L. N. Abbakumova3, Y. A. Barbitoff4,2,5, D. E. Polev2, A. S. Glotov2 1North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, 2Biobank of
the Research park, Saint-Petersburg State University, Saint-
Petersburg, Russian Federation, 3Saint-Petersburg State
Pediatric Medical University, Saint-Petersburg, Russian
Federation, 4Bioinformatics Institute, Saint-Petersburg,
Russian Federation, 5Department of Genetics and
Biotechnology, Saint-Petersburg State University, Saint-
Petersburg, Russian Federation J. Duong1, A. Rideout2, J. Beis2, S. Parkash2, U. Schwarze3,
A. Vandersteen2 A. Vandersteen2 J. Duong1, A. Rideout2, J. Beis2, S. Parkash2, U. Schwarze3,
A Vandersteen2 P04.33A A second patient with Classical Ehlers-Danlos Syndrome
(cEDS) and congenital hip dislocation caused by the
pathogenic variant COL1A1 c. 934C>T, p.Arg312Cys J. del Picchia 108 Introduction: Ehlers-Danlos Syndrome (EDS) is a hetero-
geneous group of connective tissue disorder, caused by
metabolic imbalance of collagen, the structure and function
of myomatrix also the synthesis of proteoglycans. Accord-
ing to a new international classification of EDS (2017), now
it is classified in 13 different types and it is associated with
mutations in 19 genes. The exception is the hypermobility
type EDS (hEDS) the diagnosis of which is based only on
clinical criteria. progressive,
moderate
hearing
loss
in
addition
to
a
disproportionate short-limb dwarfism with distalward short-
ening of extremities and postaxial hexadactyly. Her brother
as well as other members of the family also presented
disproportionate short stature and anomalies of the limbs. Next generation sequencing with a customized skeletal
dysplasia panel containing over 370 genes and subsequent
bioinformatics analysis disclosed two homozygous muta-
tions in EVC2 (c.2653C>T; p.Arg885*) and COL11A2
(c.966dup; p.Thr323Hisfs*19), respectively. Sanger sequen-
cing showed that both parents were heterozygous carriers
for both EVC2 and COL11A2 mutations and the sister was a
heterozygous carrier for the COL11A2 mutation but wild
type at the EVC2 mutation position. Materials and Methods: 12 patients with hEDS were
examined. NGS was applied to 10 patients with hEDS and
19 genes associated with EDS were analyzed (ADAMTS2,
B3GALT6, B4GALT7, C1R, C1S, CHST14, COL1A1,
COL1A2, COL12A1, COL3A1, COL5A1, DSE, FLNA,
FKBP14, PLOD1, PRDM5, SLC39A13, TNXB, ZNF469). Additionally, direct PCR method was performed to identify
del30kb in the TNXB for two patients. Materials and Methods: 12 patients with hEDS were
examined. NGS was applied to 10 patients with hEDS and
19 genes associated with EDS were analyzed (ADAMTS2, B3GALT6, B4GALT7, C1R, C1S, CHST14, COL1A1,
COL1A2, COL12A1, COL3A1, COL5A1, DSE, FLNA,
FKBP14, PLOD1, PRDM5, SLC39A13, TNXB, ZNF469). Additionally, direct PCR method was performed to identify
del30kb in the TNXB for two patients. This study highlights a dual molecular diagnosis in a
patient with a blending of two distinct phenotypes and
illustrates the advantage and importance of this staple
technology to facilitate rapid and comprehensive genetic
dissection of a heterogeneous phenotype. The differentia-
tion between phenotypic expansion of a genetic disorder
and a blended phenotype that is due to more than one
distinct genetic aberration is essential in order to reduce the
diagnostic odyssey endured by patients. P04.33A Results:
We
identified
a
heterozygous
mutation
c.2818G>A in one patient in the ADAMTS2 gene with
uncertain clinical significance. Mutations in the ADAMTS2
are associated with autosomal recessive, dEDS. In addition,
we detected one heterozygous mutation c.3023C>T with
uncertain clinical significance in another patient in the
COL5A1 gene which is associated with cEDS. A hetero-
zygous del30kb in the TNXB was identified by direct PCR
method in two patients. P.L. Bahena Carbajal: None. B. Vona: None. R. Maroofian: None. G. Mendirattac: None. M. Croken:
None. S. Peng: None. S. Peng: None. X. Ye: None. J. Rezazadeh: None. C. Lekszas: None. T. Haaf: None. L. Edelmannc: None. L. Shic: None. Conclusions: Our results indicate that heterozygous
mutations in genes associated with different types of EDS
can be the cause of hEDS, which indicates genetic
heterogeneity of this pathology and needs further research. P04.37A Application of a machine learning approach to identify
miRNA fingerprint signatures in recessive dystrophic
epidermolysis bullosa Application of a machine learning approach to identify
miRNA fingerprint signatures in recessive dystrophic
epidermolysis bullosa T.I. Kadurina: None. E.A. Serebriakova: None. L.N. Abbakumova: None. Y.A. Barbitoff: None. D.E. Polev:
None. A.S. Glotov: None. T.I. Kadurina: None. E.A. Serebriakova: None. L.N. Abbakumova: None. Y.A. Barbitoff: None. D.E. Polev:
None. A.S. Glotov: None. P04.35C R. Zauner1, M. Wimmer1, T. Lettner1, S. Atzmüller2, J. Pröll2,
C. Guttmann-Gruber1, E. M. Murauer1, J. Reichelt1, D. Strunk3,
J. W. Bauer4, V. Wally1 P. L. Bahena Carbajal1, B. Vona1, R. Maroofian2, Dual diagnosis of Ellis-van Creveld syndrome and hearing
loss in a consanguineous family Dual diagnosis of Ellis-van Creveld syndrome and hearing
loss in a consanguineous family P. L. Bahena Carbajal1, B. Vona1, R. Maroofian2,
G. Mendirattac3, M. Croken3, S. Peng3, S. Peng3, X. Ye3,
J. Rezazadeh4, C. Lekszas1, T. Haaf1, L. Edelmannc3, L. Shic3 P. L. Bahena Carbajal1, B. Vona1, R. Maroofian2, 1EB House Austria, Research Program for Molecular Therapy
of Genodermatoses Department of Dermatology, University
Hospital of the Paracelsus Medical University, Salzburg,
Austria, 2Red Cross Transfusion Service for Upper Austria,
Linz, Austria, 3Institute for Experimental and Clinical Cell
Therapy, Paracelsus Medical University, Salzburg, Austria,
4Department of Dermatology, University Hospital of the
Paracelsus Medical University, Salzburg, Austria P. L. Bahena Carbajal1, B. Vona1, R. Maroofian2, G. Mendirattac3, M. Croken3, S. Peng3, S. Peng3, X. Ye3, G. Mendirattac3, M. Croken3, S. Peng3, S. Peng3, X. Ye3,
4
1
1
3
3 J. Rezazadeh4, C. Lekszas1, T. Haaf1, L. Edelmannc3, L. Shic3 J. Rezazadeh4, C. Lekszas1, T. Haaf1, L. Edelmannc3, L. Shic3 1Institut of Humangenetics, Würzburg, Germany, 2University
of Exeter Medical School, Exeter, United Kingdom, 3Icahn
School of Medicine at Mount Sinai, New York, NY, United
States, 4Genetic counselling and Rehabilitation Unit, Welfare
organization, South Khorasan, Iran, Islamic Republic of 1Institut of Humangenetics, Würzburg, Germany, 2University
of Exeter Medical School, Exeter, United Kingdom, 3Icahn
School of Medicine at Mount Sinai, New York, NY, United
States, 4Genetic counselling and Rehabilitation Unit, Welfare
organization, South Khorasan, Iran, Islamic Republic of Recessive dystrophic epidermolysis bullosa (RDEB) repre-
sents one of the most severe subforms of a rare geno-
dermatosis and is caused by mutations within the COL7A1
gene. Absent or dysfunctional type VII collagen impedes
the structural integrity of the molecular link between epi-
dermis and underlying dermis, which manifests in severe Multilocus analysis of rare or genetically heterogeneous
diseases is a distinct advantage of next generation sequen-
cing over conventional single-gene investigations. Multilocus analysis of rare or genetically heterogeneous
diseases is a distinct advantage of next generation sequen-
cing over conventional single-gene investigations. We described a female proband from a large consangui-
neous
Iranian
family
who
manifests
postlingual, Abstracts from the 51st European Society of Human Genetics Conference: Posters 109 blister formation sublamina densa and erosions of skin and
mucous membranes. RDEB patients suffer from chronically
impaired wound healing and an extraordinary high risk of
developing a particularly aggressive form of squamous cell
carcinoma (SCC). Dual diagnosis of Ellis-van Creveld syndrome and hearing
loss in a consanguineous family So far there is only very limited data
available on non-coding RNA events in RDEB cancer
progression. epidermal juction. Clinically, patients suffer from extra-
ordinary sensitivity of the skin to mechanic friction or
trauma and present with an increased risk of infections. One
major concern in this patient population relates to the high
incidence of developing a particularly aggressive form of
squamous cell carcinomas (SCCs), resulting in a highly
increased mortality due to frequent metastatic spreading. Recent studies have demonstrated a significantly deregu-
lated miRNome in RDEB-SCC in vitro. In order to inves-
tigate the cause of the observed altered miRNA abundance,
we analyzed expression levels of major components of the
miRNA biogenesis pathway via microarray, sqRT-PCR and
Western blot. Sanger sequencing of mutational hotspots was
performed for major components of the miRNA processing
machinery. Cultured primary skin cells derived from patient
SCCs and keratinocytes and healthy controls served as
experimental groups. In order to widen the therapeutic spectrum for RDEB
cancer patients we aim to improve our understanding on the
impact of differentially expressed small non-coding miR-
NAs on disease progression and to explore their potential as
biomarkers. Therefore, Illumina miRNA-seq was performed
on RDEB-SCC, UV-SCC, RDEB and nonEB keratinocytes
derived from skin biopsies. Fingerprint miRNAs able to
discriminate RDEB-SCCs from the other experimental
groups were derived applying a neural network machine
learning approach. Specific self-organizing maps were
trained on our miRNA-seq data sets and co-expression
modules of miRNAs with differential expression profiles
extracted. In a guilt-by-association assessment based on
microarray expression data, we could demonstrate an
enrichment of target mRNAs with strong correlation to
signature miRNAs, in hallmark signatures like epithelial-to-
mesenchymal transition, which is related to the observed
aggressive phenotype in RDEB-SCCs. We found significantly increased RNA and protein
expression levels of DROSHA in SCC cells as compared
to healthy controls, without indication of mutagenic events
in reported hotspots. Karyotyping of RDEB SCC cells
confirmed chromosomal abberations in regions of interest
concerning DROSHA. A dysregulation of canonical miRNA biogenesis is
associated with many diseases, particularly cancer, and
could prove instrumental in further deepening our under-
standing of triggers and promoters of SCC in RDEB. Eventually, our set of identified fingerprint miRNAs may
provide the foundation for the identification of biomarkers
and drugable targets. M. Wimmer: None. R. Zauner: None. A. Waldmann:
None. H. Bodocian: None. M. Ablinger: None. S. Atzmueller: None. J. Proell: None. D. Strunk: None. Dual diagnosis of Ellis-van Creveld syndrome and hearing
loss in a consanguineous family J. W. Bauer: None. J. Reichelt: None. V. Wally: None. R. Zauner: None. M. Wimmer: None. T. Lettner:
None. S. Atzmüller: None. J. Pröll: None. C. Guttmann-
Gruber: None. E.M. Murauer: None. J. Reichelt: None. D. Strunk: None. J.W. Bauer: None. V. Wally: None. R. Zauner: None. M. Wimmer: None. T. Lettner:
None. S. Atzmüller: None. J. Pröll: None. C. Guttmann- Gruber: None. E.M. Murauer: None. J. Reichelt: None. D. Strunk: None. J.W. Bauer: None. V. Wally: None. Gruber: None. E.M. Murauer: None. J. Reichelt: None. D. Strunk: None. J.W. Bauer: None. V. Wally: None. Correction of type XVII collagen using spliceosome-
mediated RNA trans-splicing Correction of type XVII collagen using spliceosome-
mediated RNA trans-splicing Major player in miRNA processing appear altered in
recessive dystrophic epidermolysis bullosa squamous cell
carcinoma M. Reisenberger, P. Schlager, J. Reichelt, J. W. Bauer,
U. Koller, S. Hainzl, M. Ablinger, T. Lettner, V. Wally M. Reisenberger, P. Schlager, J. Reichelt, J. W. Bauer,
U. Koller, S. Hainzl, M. Ablinger, T. Lettner, V. Wally M. Wimmer1, R. Zauner1, A. Waldmann1, H. Bodocian1,
M. Ablinger1, S. Atzmueller2, J. Proell2, D. Strunk3, J. W. Bauer1, J. Reichelt1, V. Wally1 EB House Austria, Research Program for Molecular Therapy
of Genodermatoses, salzburg, Austria EB House Austria, Research Program for Molecular Therapy
of Genodermatoses, salzburg, Austria Ret-
roviral transduction of a JEB keratinocyte line harbouring a
compound heterozygous mutation (exon 52 and 53) with
the most functional RTM (RTM135mSU), coding for the
COL17A1 wild type exons 34-56, led to restoration of type
XVII collagen expression at mRNA level and protein level
assessed by immunofluorescence. We found that a binding
domain covering the intron/exon junction and the removal
of splice sites in the RTMs coding region resulted in an
increased trans-splicing efficiency. Our results indicate that
the SMaRT technology is a promising tool for the devel-
opment of an RNA therapy for JEB patients. COL17A1 pre-mRNA, thereby inducing a trans-splicing
reaction that results in the generation of a hybrid mRNA,
containing RTM-derived and endogenous gene portions. In
a minigene-based approach we first studied the impact of
various RTM sequence modifications on trans-splicing
efficiency and specificity. The modifications included
binding domain optimization, removal of the COL17A1
3’UTR and removal of potential cryptic splice sites. Ret-
roviral transduction of a JEB keratinocyte line harbouring a
compound heterozygous mutation (exon 52 and 53) with
the most functional RTM (RTM135mSU), coding for the
COL17A1 wild type exons 34-56, led to restoration of type
XVII collagen expression at mRNA level and protein level
assessed by immunofluorescence. We found that a binding
domain covering the intron/exon junction and the removal
of splice sites in the RTMs coding region resulted in an
increased trans-splicing efficiency. Our results indicate that
the SMaRT technology is a promising tool for the devel-
opment of an RNA therapy for JEB patients. Material and Methods: As FLNB is a large gene with 46
exons, we preferred exome sequencing index patients from
all the seven families as this is cost-efficient at our center
followed by Sanger validation and segregation analysis. Material and Methods: As FLNB is a large gene with 46
exons, we preferred exome sequencing index patients from
all the seven families as this is cost-efficient at our center
followed by Sanger validation and segregation analysis. Results: Clinical features of ten patients (six females and
four males) included short stature (9/9), short neck (6/10),
pectus carinatum (5/10), facial dysmorphism (2/10) and
cleft lip and palate (1/10). Radiological features were fused
vertebrae (9/10), carpal fusion (10/10), tarsal fusion (5/5),
scoliosis (9/10), lumbar lordosis (2/10) and crowding of ribs
(9/10). EB House Austria, Research Program for Molecular Therapy
of Genodermatoses, salzburg, Austria Seven novel homozygous variants were identified in
FLNB: c.6317del [p.(Pro2106ArgfsX12)] in patient 1,
c.1493del [p.(Glu498GlyfsX4)] in patient 2, c.1243C>T
[p.(Arg415X)]
in
patient
3,
c.1204del
[p.(Val402-
TryfsX88)] in patient 4, c.28G>T [p.(Glu10X)] in patient
5, c.1429delinsCT [p.(Val477Leufs*2)] in patients 6 and 7,
and c.1592dup [p.(His532ThrfsX9)] in patients 8, 9 and 10. Conclusion: Our work demonstrates that spondylocarpo-
tarsal
synostosis
syndrome
has
a
unique
pattern
of
anomalous vertebral segmentation and all the reported
patients have truncating variants in FLNB. The study is
funded by Indian Council of Medical Research. Conclusion: Our work demonstrates that spondylocarpo-
tarsal
synostosis
syndrome
has
a
unique
pattern
of
anomalous vertebral segmentation and all the reported
patients have truncating variants in FLNB. The study is
funded by Indian Council of Medical Research. M. Reisenberger: None. P. Schlager: None. J. Reichelt:
None. J.W. Bauer: None. U. Koller: None. S. Hainzl:
None. M. Ablinger:
None. T. Lettner:
None. V. Wally: None. S. Salian: None. A. Shukla: None. H. Shah: None. S.N. Bhat: None. V.R. Bhat: None. S. Nampoothiri: None. R. Shenoy: None. S.R. Phadke: None. S.V. Hariharan:
None. K.M. Girisha: None. P04.40D Seven additional families with spondylocarpotarsal
synostosis syndrome with novel biallelic deleterious
variants in FLNB EB House Austria, Research Program for Molecular Therapy
of Genodermatoses, salzburg, Austria Distinct subtypes of junctional EB (JEB) are caused by
mutations within the COL17A1 gene, encoding type XVII
collagen, which leads to fragility of the skin and blister
formation within the lamina lucida upon minor friction. So
far, there is no causal therapy available for this EB subtype. SMaRT is a post-transcriptional therapeutic approach,
which uses the cellular splicing machinery to replace
mutation-bearing exons. The aim of this study was to apply
SMaRT therapy for the correction of any mutation down-
stream from COL17A1 exon 33. We engineered a set of
RNA
trans-splicing
molecules
(RTMs),
which
bind 1EB House Austria, Research Program for Molecular Therapy
of Genodermatoses, Department of Dermatology, University
Hospital of the Paracelsus Medical University, Salzburg,
Austria, 2Red Cross Transfusion Service for Upper Austria,
Linz, Austria, 3Institute for Experimental and Clinical Cell
Therapy, Paracelsus Medical University, Salzburg, Austria Recessive dystrophic epidermolysis bullosa (RDEB) is
characterized by loss-of-function mutations in the COL7A1
gene, resulting in impaired or absent type VII collagen
protein, a major anchoring molecule at the dermal- 110 J. del Picchia COL17A1 pre-mRNA, thereby inducing a trans-splicing
reaction that results in the generation of a hybrid mRNA,
containing RTM-derived and endogenous gene portions. In
a minigene-based approach we first studied the impact of
various RTM sequence modifications on trans-splicing
efficiency and specificity. The modifications included
binding domain optimization, removal of the COL17A1
3’UTR and removal of potential cryptic splice sites. Ret-
roviral transduction of a JEB keratinocyte line harbouring a
compound heterozygous mutation (exon 52 and 53) with
the most functional RTM (RTM135mSU), coding for the
COL17A1 wild type exons 34-56, led to restoration of type
XVII collagen expression at mRNA level and protein level
assessed by immunofluorescence. We found that a binding
domain covering the intron/exon junction and the removal
of splice sites in the RTMs coding region resulted in an
increased trans-splicing efficiency. Our results indicate that
the SMaRT technology is a promising tool for the devel-
opment of an RNA therapy for JEB patients. COL17A1 pre-mRNA, thereby inducing a trans-splicing
reaction that results in the generation of a hybrid mRNA,
containing RTM-derived and endogenous gene portions. In
a minigene-based approach we first studied the impact of
various RTM sequence modifications on trans-splicing
efficiency and specificity. The modifications included
binding domain optimization, removal of the COL17A1
3’UTR and removal of potential cryptic splice sites. Novel clinical features in frontometaphyseal dysplasia 2
caused by a recurrent mutation in MAP3K7 Novel clinical features in frontometaphyseal dysplasia 2
caused by a recurrent mutation in MAP3K7 S. Salian1, A. Shukla1, H. Shah1, S. N. Bhat1, V. R. Bhat1,
S. Nampoothiri2, R. Shenoy3, S. R. Phadke4, S. V. Hariharan5,
K. M. Girisha1 S. Salian1, A. Shukla1, H. Shah1, S. N. Bhat1, V. R. Bhat1,
S. Nampoothiri2, R. Shenoy3, S. R. Phadke4, S. V. Hariharan5,
K. M. Girisha1 P04.45A
Genetic analysis of genodermatoses in domestic animals P04.45A
Genetic analysis of genodermatoses in domestic animals L. G. Stuessel1,2, L. M. Hochfeld1,2, J. Schroeder1,2, F. Thieme1,2,
T. Hess1,2, J. Gehlen1,2, S. Heilmann-Heimbach1,2, M. Knapp3,
E. Mangold2, A. Rada-Iglesias4,5, K. U. Ludwig1,2 A. Bauer1,2, P. Balmer1,3, M. A. T. Brunner1,4, M. Caduff1,2,
M. De Lucia5, C. Drögemüller1,2, M. Drögemüller1,2,
V. Jagannathan1,2, J. Nimmo6, L. Murgiano1,2,7, B. S. Sayar1,8,
N. Tarasova9, K. Timm10, R. E. Towers11,12, G. Zur13,
E. Müller1,8,14, P. Roosje1,3, M. M. Welle1,4, T. Leeb1,2 1Department of Genomics, Life&Brain Center, Bonn,
Germany, 2Institute of Human Genetics, University of Bonn,
Bonn, Germany, 3Institute of Medical Biometry Informatics
and Epidemiology, University of Bonn, Bonn, Germany,
4Center for Molecular Medicine Cologne (CMMC), University
of Cologne, Cologne, Germany, 5Cologne Excellence Cluster
for Cellular Stress Responses in Aging-Associated Diseases
(CECAD), University of Cologne, Cologne, Germany 1Dermfocus, University of Bern, Bern, Switzerland, 2Institute of
Genetics, Vetsuisse Faculty, University of Bern, Bern,
Switzerland, 3Division of Clinical Dermatology, Department of
Clinical Veterinary Medicine, Vetsuisse Faculty, University of
Bern, Bern, Switzerland, 4Institute of Animal Pathology,
Vetsuisse Faculty, University of Bern, Bern, Switzerland, 5San
Marco Veterinary Clinic and Laboratory, Padova, Italy,
6ASAP Laboratory, Mulgrave, Victoria, Australia, 7Section of
Ophthalmology, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, PA, United States, 8Department of
Biomedical Research, Molecular Dermatology and Stem Cell
Research, University of Bern, Bern, Switzerland, 9Russian
Akhal-Teke Association, Moscow, Russian Federation,
10Vetderm, Hünenberg, Switzerland, 11Institute of Medical
Genetics, Cardiff University, Cardiff, United Kingdom, 12Tees,
Esk and Wear Valleys NHS Foundation Trust, United
Kingdom, 13Veterinary Teaching Hospital, The Koret School of
Veterinary Medicine, The Hebrew University of Jerusalem,
Rehovot, Israel, 14Clinic for Dermatology, Inselspital, Bern
University Hospital, Bern, Switzerland GWAS for nonsyndromic cleft lip with/without cleft palate
(nsCL/P) have identified 40 risk loci for this human cra-
niofacial malformation. The vast majority of these asso-
ciated loci map to non-coding genomic regions. One
possible mechanism by which variants at these loci might
exert their regulatory effect is the control of microRNA
(miRNA) expression and miRNA-mediated gene regula-
tion, in disease-relevant tissue. In the present study, we
sought to identify candidate miRNAs in human neural crest
cells (hNCCs), an early precursor cell population of facial
tissue, and integrate GWAS data to identify potential sus-
ceptibility miRNA candidates that might be involved in
nsCL/P. MiRNA profiling was performed in four independent
hNCC samples, previously generated from induced plur-
ipotent stem cells, using the Affymetrix miRNA 4.0 array
platform. A. Costantini1, C. Wallgren-Pettersson2, O. Mäkitie1,3,4,5 The 17-year-old boy has the characteristic
skeletal and facial features of FMD2. However, several
novel features were also observed, including multiple
hemangiomas, hand and foot abnormalities, growth retar-
dation, spina bifida, Sprengel deformity, Chiari malforma-
tion and ocular manifestations. He also showed keloid scars
but, in contrast to other patients harboring the same muta-
tion, he does not have intellectual disability. This report
expands the clinical spectrum of FMD2 caused by the
recurrent c.1454C>T, p.(Pro485Leu) mutation in MAP3K7. identified as expressed in hNCCs, none of which mapped
to the known nsCL/P loci. However, positional integration
of GWAS summary statistics revealed 25 variants (at P-
value <0.01) that were located within 1kb of a candidate
miRNA. Association of one variant, a low-frequency
variant at chr. 2q37.3 close to miRNA-149, was confirmed
in a replication analysis of an independent nsCL/P cohort. Target gene prediction with miRWalk2.0 and literature
research revealed that miRNA149 likely binds known
nsCL/P candidate genes such as FGFR1, BMP9 and
RUNX2, and has been shown to differentially interact with
MTHFR upon folate deficiency. Although further functional
characterization is required and currently ongoing, our
study suggests that miRNA-149 might be involved as
regulatory mechanism in facial development. L.G. Stuessel: None. L.M. Hochfeld: None. J. Schroe-
der: None. F. Thieme: None. T. Hess: None. J. Gehlen:
None. S. Heilmann-Heimbach: None. M. Knapp: None. E. Mangold:
None. A. Rada-Iglesias:
None. K.U. Ludwig: None. A. Costantini: None. C. Wallgren-Pettersson: None. O. Mäkitie: None. L.G. Stuessel: None. L.M. Hochfeld: None. J. Schroe-
der: None. F. Thieme: None. T. Hess: None. J. Gehlen:
None. S. Heilmann-Heimbach: None. M. Knapp: None. E. Mangold:
None. A. Rada-Iglesias:
None. K.U. Ludwig: None. A. Costantini1, C. Wallgren-Pettersson2, O. Mäkitie1,3,4,5 1Kasturba Medical College, Manipal, Udupi, India,
2Department of Pediatric Genetics, Amrita Institute of Medical
Sciences and Research Centre, Cochin, India, 3Department of
Pediatrics, KS Hegde Medical Academy, Mangalore, India,
4Department of Medical Genetics, Sanjay Gandhi
Postgraduate Institute of Medical Sciences, Lucknow, India,
5Department of Pediatrics, Sree Avittom Thirunal Hospital,
Government Medical College, Thiruvananthapuram, India 1Department of Molecular Medicine and Surgery and Center
for Molecular Medicine, Karolinska Institutet, Stockholm,
Sweden, 2Folkhälsan Institute of Genetics and Department of
Medical and Clinical Genetics, University of Helsinki,
Helsinki, Finland, Helsinki, Finland, 3Folkhälsan Institute of
Genetics and University of Helsinki, Helsinki, Finland,
4Children's Hospital, University of Helsinki and Helsinki
University Hospital, Helsinki, Finland, 5Department of
Clinical Genetics, Karolinska University Hospital, Stockholm,
Sweden Introduction: Location and/or type of variants in FLNB
result in a spectrum of osteochondrodysplasias: spondylo-
carpotarsal synostosis syndrome and Larsen syndrome that
are milder and atelosteogenesis I and III, and Boomerang
dysplasia that are perinatal lethal. So far, nine bi-allelic loss-
of-function variants in FLNB are reported to cause spon-
dylocarpotarsal synostosis syndrome in nine families. We
aimed to identify pathogenic variants in FLNB in ten
patients from seven families with spondylocarpotarsal
synostosis syndrome. Frontometaphyseal dysplasia 2 (FMD2) is a skeletal dys-
plasia
with
supraorbital
hyperostosis
combined
with
undermodeling of the bones, joint contractures and some
extraskeletal features. It is caused by heterozygous muta-
tions in MAP3K7, encoding the Mitogen-Activated Protein
Kinase 7. MAP3K7 is activated by TGF-β and plays an
important role in osteogenesis. Less than 20 patients with
FMD2 and MAP3K7 mutations have been described thus
far. Three out of four patients harbor a recurrent missense Frontometaphyseal dysplasia 2 (FMD2) is a skeletal dys-
plasia
with
supraorbital
hyperostosis
combined
with
undermodeling of the bones, joint contractures and some
extraskeletal features. It is caused by heterozygous muta-
tions in MAP3K7, encoding the Mitogen-Activated Protein
Kinase 7. MAP3K7 is activated by TGF-β and plays an
important role in osteogenesis. Less than 20 patients with
FMD2 and MAP3K7 mutations have been described thus
far. Three out of four patients harbor a recurrent missense Abstracts from the 51st European Society of Human Genetics Conference: Posters 111 mutation,
NM_003188.3:
c.1454C>T,
p.(Pro485Leu),
which leads to a more severe phenotype than mutations in
other domains. Here we describe an additional patient with
FMD2 caused by the recurrent c.1454C>T MAP3K7
mutation, identified as a de novo variant by whole-genome
sequencing. Novel case with a double “apparently” balanced
rearrangement disrupting EXT1 in a patient with
hereditary multiple exostoses Novel case with a double “apparently” balanced
rearrangement disrupting EXT1 in a patient with
hereditary multiple exostoses Novel case with a double “apparently” balanced
rearrangement disrupting EXT1 in a patient with
hereditary multiple exostoses A. Alexandrou1, N. Salameh1, I. Papaevripidou1, N. Nicolaou2,
P. Myriathopoulos1, A. Ketoni1, P. Evangelidou1, G. A. Tanteles2, C. Sismani1,3 A. Alexandrou1, N. Salameh1, I. Papaevripidou1, N. Nicolaou2,
P. Myriathopoulos1, A. Ketoni1, P. Evangelidou1, G. A. Tanteles2, C. Sismani1,3 A. Alexandrou1, N. Salameh1, I. Papaevripidou1, N. Nicolaou2,
P. Myriathopoulos1, A. Ketoni1, P. Evangelidou1, G. A. Tanteles2, C. Sismani1,3 Materials and Methods: Genetic mapping and whole
genome sequencing approaches were used. Results: We identified candidate causative variants for
several genodermatoses including variants in genes that had
not previously been associated with disease phenotypes in
humans. As an example, we discovered genetic variants in
SUV39H2 in dogs with hereditary nasal parakeratosis,
which revealed a role for SUV39H2 in keratinocyte
differentiation. 1Cytogenetics and Genomics Department, The Cyprus Institute
of Neurology and Genetics, Nicosia, Cyprus, 2Clinical
Genetics Clinic, The Cyprus Institute of Neurology and
Genetics, Nicosia, Cyprus, 3The Cyprus School of Molecular
Medicine, The Cyprus Institute of Neurology and Genetics,
Nicosia, Cyprus 1Cytogenetics and Genomics Department, The Cyprus Institute
of Neurology and Genetics, Nicosia, Cyprus, 2Clinical
Genetics Clinic, The Cyprus Institute of Neurology and
Genetics, Nicosia, Cyprus, 3The Cyprus School of Molecular
Medicine, The Cyprus Institute of Neurology and Genetics,
Nicosia, Cyprus Selected domestic animal models for human genodermatoses
Gene
Phenotype
Species
Human disorder (MIM#)
ASPRV1
ichthyosis
dog
? EDA
X-linked
hypohidrotic
ectodermal dysplasia
dog, cattle 305100
FAM83G hereditary footpad
hyperkeratosis
dog
palmoplantar
keratoderma and
exuberant scalp hair
IKBKG
incontinentia
pigmenti
horse
300291
MBTPS2
brindle 1
horse
300918,
308205, 308800
NSDHL
congenital
cornification disorder
dog
308050, 300831
OCA2
oculocutaneous
albinism, type 2
dog
203200, 227220
ST14
naked foal syndrome horse
602400
SUV39H2 hereditary nasal
parakeratosis
dog
? TSR2
streaked hairlessness cattle
? Hereditary multiple exostoses (HME) is an autosomal
dominant skeletal disorder characterized by the develop-
ment of multiple, circumscript, occasionally painful and
usually symmetric bony protuberances called osteochon-
dromas. HME is caused by EXT1 and EXT2 loss of function
mutations. Most pathogenic mutations are nonsense fol-
lowed by missense mutations and deletions. We report on a
patient with a rare and complex genotype resulting in a
classical HME phenotype. P04.45A
Genetic analysis of genodermatoses in domestic animals After stringent filtering, 152 miRNAs were Introduction: Spontaneous mutants in domestic animal
species are valuable models to study heritable human J. del Picchia 112 P04.46B disorders. Purebred animals are kept in closed populations
necessitating a certain degree of inbreeding, which favors
the expression of recessive alleles. Due to the unique
population structure of purebred animals, identification of
disease causing genetic variants is often more straightfor-
ward than in humans. disorders. Purebred animals are kept in closed populations
necessitating a certain degree of inbreeding, which favors
the expression of recessive alleles. Due to the unique
population structure of purebred animals, identification of
disease causing genetic variants is often more straightfor-
ward than in humans. P04.48D
Italian validation of the functional difficulties questionnaire
(FDQ-9) and its correlation with major determinants of
quality of life in adults with hypermobile Ehlers-Danlos
syndrome/hypermobility spectrum disorders P04.48D
Italian validation of the functional difficulties questionnaire
(FDQ-9) and its correlation with major determinants of
quality of life in adults with hypermobile Ehlers-Danlos
syndrome/hypermobility spectrum disorders S. Morlino1, C. Dordoni2, I. Sperduti3, C. Piedimonte4,
M. Ritelli2, M. Colombi2, P. Grammatico1, M. Castori5 S. Morlino1, C. Dordoni2, I. Sperduti3, C. Piedimonte4,
M. Ritelli2, M. Colombi2, P. Grammatico1, M. Castori5 1Lab. Medical Genetics, Dep. Molecular Medicine, Sapienza
University, S.Camillo-Forlanini Hospital, Rome, Italy, 2Div. Biology and Genetics, Dep. Molecular and Translational
Medicine, University of Brescia, Brescia, Italy, 3Biostatistics,
IRCCS San Gallicano Dermatologic Institute, Rome, Italy,
4Department of Experimental Medicine, Sapienza University,
"Umberto I" Hospital, Rome, Italy, 5Div. Medical Genetics,
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo
(FG), Italy Ectodermal dysplasias (EDs) encompass a large group of
clinically and genetically heterogeneous hereditary dis-
orders that are defined by abnormal development of at least
two ectodermal structures, which include but are not limited
to hair, nails, teeth and sweat glands. A rare form of auto-
somal recessive ED that is usually characterized by hypo-
trichosis and nail dystrophy only is known as pure hair and
nail ectodermal dysplasia (PHNED). To date, mutations in
KRT85, KRT74, and HOXC13 have been reported in
patients and families of different ethnic origin with PHNED. Here, we studied two sisters from a consanguineous Iranian
marriage who were not only affected by hypotrichosis and
nail dysplasia but also lacrimal duct obstruction (LDO). Homozygosity mapping and GeneDistiller analysis sug-
gested linkage of the disease to chromosome 12q13.13. In
this region, KRT85, KRT74, and HOXC13 were the most
plausible candidate genes. Sanger sequencing of HOXC13
revealed a hitherto undescribed homozygous 28 bp insertion
mutation
(c.837_838insACTTGCGGCTAGCAAGTT-
CATCACCAAA; p. A280Tfs*4) in exon 2 in both affected
children that was present in the heterozygous state in the
parents. The 2017 EDS International Classification defined the new
criteria for hypermobile Ehlers-Danlos syndrome (hEDS),
which is now considered one end of a continuous spectrum
originating from isolated, non-syndromic joint hypermobi-
lity (JH) and passing through hypermobility spectrum dis-
orders (HSD). Preliminary data indicate a link between JH
and neurodevelopmental disorders, and the strongest evi-
dence is the non-causal association with developmental
coordination disorder (DCD) in children. Assessing DCD in
adults is difficult and the recently described functional dif-
ficulties questionnaire 9 (FDQ-9) is one of the few available
tools. Novel case with a double “apparently” balanced
rearrangement disrupting EXT1 in a patient with
hereditary multiple exostoses Selected domestic animal models for human genodermatoses
Gene
Phenotype
Species
Human disorder (MIM#) Mutations in EXT1 and EXT2 were excluded by Sanger
sequencing. The patient was subsequently referred for
karyotype and array-CGH analyses. Results obtained were
validated with FISH and qRT-PCR and parental studies
determined the mode of inheritance. Chromosomal analysis revealed a de novo “apparently”
double balanced rearrangement: a balanced translocation
between chromosomes 2 and 3 at breakpoints 2q22 and
3q13.2 and a pericentric 8p23.1q24.1 inversion both of
which were confirmed by FISH analysis. Subsequently,
array-CGH analysis revealed a novel heterozygous deletion
within the EXT1 gene at the inversion breakpoints,
rendering the inversion as unbalanced. The inheritance
mode as well as the size of the deletion was further
investigated by qRT-PCR and the deletion was character-
ized as a de novo 3.1kb deletion removing exon 10. The
inversion in combination with the 8p23.1 deletion most
likely abolishes the transcription of EXT1 downstream of
exon 10 hence resulting in a truncated protein. Conclusions: The study provides new candidate genes
for genodermatoses in human and veterinary medicine and a
better understanding of the genotype-phenotype correlation. Grant:
Swiss
National
Science
Foundation
CRSII3_160738/1 To conclude, a rare and novel pathogenic cause of HME
is presented in this study, highlighting the importance of
additional comprehensive cytogenetic investigation when
EXT1 and EXT2 mutation analysis is negative. A. Bauer: None. P. Balmer: None. M.A.T. Brunner:
None. M. Caduff: None. M. De Lucia: None. C. Drögemüller: None. M. Drögemüller: None. V. Jagan-
nathan: None. J. Nimmo: None. L. Murgiano: None. B.S. Sayar: None. N. Tarasova: None. K. Timm: None. R.E. Towers: None. G. Zur: None. E. Müller: None. P. Roosje: None. M.M. Welle: None. T. Leeb: None. A. Bauer: None. P. Balmer: None. M.A.T. Brunner:
None. M. Caduff: None. M. De Lucia: None. C. Drögemüller: None. M. Drögemüller: None. V. Jagan-
nathan: None. J. Nimmo: None. L. Murgiano: None. B.S. Sayar: None. N. Tarasova: None. K. Timm: None. R.E. Towers: None. G. Zur: None. E. Müller: None. P. Roosje: None. M.M. Welle: None. T. Leeb: None. A. Alexandrou: None. N. Salameh: None. I. Papaevri-
pidou: None. N. Nicolaou: None. P. Myriathopoulos:
None. A. Ketoni: None. P. Evangelidou: None. G.A. Tanteles: None. C. Sismani: None. 113 Abstracts from the 51st European Society of Human Genetics Conference: Posters An insertion mutation in HOXC13 underlies pure hair and
nail ectodermal dysplasia with lacrimal duct obstruction An insertion mutation in HOXC13 underlies pure hair and
nail ectodermal dysplasia with lacrimal duct obstruction A. Humbatova:
None. R. Maroofian:
None. M. Romano: None. A. Tafazzoli: None. M. Behnam: None. N. Dilaver: None. N. Nouri: None. M. Salehi: None. S. Wolf: None. J. Frank: None. P. Kokordelis: None. R. Betz: None. A. Humbatova1,2,3, R. Maroofian4, M. Romano1,2, A. Tafazzoli1,2,
M. Behnam5, N. Dilaver6, N. Nouri5, M. Salehi5,7, S. Wolf1,2,
J. Frank8, P. Kokordelis1,2, R. Betz1,2 P04.47C when clinically evaluating individuals and families with this
rare variant of ED. An insertion mutation in HOXC13 underlies pure hair and
nail ectodermal dysplasia with lacrimal duct obstruction P04.48D 1Institute of Human Genetics, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
2Department of Genomics, Life&Brain Center, Bonn,
Germany, 3Institute of Genetic Resources, Azerbaijan National
Academy of Sciences, Baku, Azerbaijan, 4Molecular & Clinical
Sciences Research Institute, St. George's University of London,
Cranmer Terrace, London, United Kingdom, 5Medical
Genetics Laboratory of Genome, Isfahan, Iran, Islamic
Republic of, 6Swansea University Medical School, Swansea
University, Wales, United Kingdom, 7Division of Genetics and
Molecular Biology, School of Medicine, Isfahan University of
Medical Sciences, Isfahan, Iran, Islamic Republic of,
8Department of Dermatology, Venereology and Allergology,
University Medical Center Göttingen, Göttingen, Germany P04.48D
Italian validation of the functional difficulties questionnaire
(FDQ-9) and its correlation with major determinants of
quality of life in adults with hypermobile Ehlers-Danlos
syndrome/hypermobility spectrum disorders The aims of this study were: (i) to validate FDQ-9 in
Italian and to normalize its values in 230 Italian non-clinical
patients; and (i) to explore the relationship of FDQ-9 with
the brief pain inventory, composite autonomic symptom
score 31, multidimensional fatigue inventory, ADHD self-
report version 1.1, and the SF-36 for quality of life in 105
Italian adults with hEDS/HSD. Validity of FDQ-9 was
assessed by the Pearson test in 10 bilingual individuals and
5 bilingual hEDS/HSD patients who completed the ques-
tionnaire in English and Italian. In the hEDS/HSD group,
67% patients had a value of FDQ-9 above the cut-off and,
therefore, a high probability of DCD in their developmental
age. Multivariate analysis was carried out comparing FDQ-
9 values with features of pain, fatigue, autonomic dys-
function, ADHD and quality of life, and demonstrated an LDO has not previously been described in association
with PHNED although this symptom has been frequently
observed in other types of ED. Therefore, LDO might have
been neglected or underdiagnosed in earlier reports of
PHNED. The clinical and molecular genetic findings in the
family expand the phenotypic and mutation spectrum of
PHNED and suggest that LDO should be examined for 114 J. del Picchia influence of a past history of coordination troubles on
chronic symptoms in adults with hEDS/HSD. Our pre-
liminary data open wider management and therapeutic
perspectives for coordination troubles in hypermobile
individuals. France, 16Centre National de Recherche en Génomique
Humaine, Evry, France, 17Département de Génétique et Centre
de Référence Déficiences Intellectuelles de Causes Rares,
Hôpital de la Pitié-Salpétrière, AP–HP, Paris, France,
18Department of Human Genetics, Faculty of Medicine, McGill
University, Montreal, QC, Canada, 19Service de Dermatologie,
CHU Dijon Bourgogne, Dijon, France S. Morlino: None. C. Dordoni: None. I. Sperduti:
None. C. Piedimonte: None. M. Ritelli: None. M. Colombi:
None. P. Grammatico:
None. M. Castori: None. Whole exome sequencing (WES) is as a powerful tool for
deciphering the genetic basis of developmental disorder,
either single nucleotides variants (SNV), indels, or recently
copy number variants (CNV). In mosaic development dis-
orders involving the skin, it has allowed detection of post-
zygotic mutations (mSNV) in various genes, but detection
of mosaic CNV (mCNV) still relies on conventional cyto-
genetic studies, such as array-CGH. We sought to develop
an all-in-one strategy for patients with mosaic disorders,
using trio-WES (lesional skin versus parent’s blood). P04.49A P04.49A
Detection of mosaic Copy-Number Variations from Whole-
Exome Sequencing in mosaic pigmentation disorders using
XHMM and a custom SNP approach A. SORLIN1,2,3, É. Tisserant2,3, J. Thevenon1,3,2, Y. Duffourd2,3,
P. Kuentz2,3,4, V. Carmignac2,3, V. Cormier-Daire5, C. Michot5,
V. Malan6, M. Beaujard6, F. Morice-Picard7, C. Rooryck-
Thambo8, C. Vincent-Delorme9, T. Smol10, É. Boudry-Labis10,
S. Hadj Rabia11, A. Phan12, M. Cordier13, M. Till13,
D. Sanlaville13, J. St-Onge3,2,14, C. Thauvin-Robinet1,2,3,
A. Mosca Boidron15,2,3, R. Olaso16, A. Boland16, J. Deleuze16,
B. Keren17, L. Faivre1,2,3, J. Rivière2,3,14,18, P. Callier2,3,15,
P. Vabres2,3,19 We performed WES in 74 patients with cutaneous mosaic
disorders. We detected 6 mCNV, all in a subgroup of 21
patients with hypomelanosis of Ito without known patho-
genic SNV: 3 mosaic trisomy (chromosomes 7, 12, 15) and
3 smaller
mCNV. Blood and skin karyotypes were
previously negative, due either to the absence of mosaic
cells in blood or to their elimination from cultured
fibroblasts. For the 3 mosaic trisomies, SNP inheritance
and b-allele frequency provided information on the parental
origin of extra chromosomes and clues to understand the
underlying mechanism. We have confirmed that chromo-
somal mosaicism is associated with mosaic pigmentation
disorders (6/21, 29% in our cohort). An appropriate fresh
tissue sample is essential. Our combined approach showed a
good efficiency to detect both (m)SNV and (m)CNV in a
single one-step assay, doubling our diagnostic rate, and
offering new perspectives in the study of mosaic develop-
mental disorder. P04.48D
Italian validation of the functional difficulties questionnaire
(FDQ-9) and its correlation with major determinants of
quality of life in adults with hypermobile Ehlers-Danlos
syndrome/hypermobility spectrum disorders We
combined a SNV detection pipeline with a CNV detection
approach, based on both a read-depth approach and a cus-
tom SNP-based approach. P04.50B self-designed panel of 86 genes (Roche NimbleGen SeqCap
EZ System) for library preparation and MiSeq sequencer
(Illumina) were used, followed by Sanger sequencing/
MLPA for mutations verification. self-designed panel of 86 genes (Roche NimbleGen SeqCap
EZ System) for library preparation and MiSeq sequencer
(Illumina) were used, followed by Sanger sequencing/
MLPA for mutations verification. Results of diagnostics of ichthyoses and epidermolysis
bullosa using dedicated next generation sequencing panel Results of diagnostics of ichthyoses and epidermolysis
bullosa using dedicated next generation sequencing panel K. Wertheim-Tysarowska1, D. Śniegórska1, A. Grabarczyk1,
S. Radomska1, A. Kutkowska-Kaźmierczak1, J. Sawicka1,
M. Jackiewicz1, P. Bialik1, A. Kujko1, A. M. Rygiel1,
K. Niepokoj1, L. Ruszkowska2, K. Osipowicz3, R. Smigiel4,
B. Wawrzycki5, K. Wozniak3, A. Jazela-Stanek6, A. Jakubiuk-
Tomaszuk7, A. Eckersdorf-Mastalerz8, A. Barczyk1,
D. Marańska9, I. Dąbrowska-Wójciak10, K. Ebner11,
J. Castaneda1, N. Bezniakow1, M. Firek-Pędras12, E. Obersztyn1,
M. Pasinska13, A. Pietrzyk14, K. Szczałuba15, J. Wierzba16,
P. Wlasienko1, C. Kowalewski3, J. Bal1 Results: We identified full genotype in 86/103 (83%) of I
and 7/8 (87%) of EB patients. In 12/103 (12%) of I patients
we didn’t detect any mutation, while in 6 (5 I and 1 EB) we
found mutation in one allele only. Overall, we detected
mutations in 19 distinct genes. Furthermore, in 10 patients,
in addition to their primary disease-causing mutations,
harbored also another possibly pathogenic mutation in one
allele of the other gene, including semi-dominant mutations
in FLG. Results: We identified full genotype in 86/103 (83%) of I
and 7/8 (87%) of EB patients. In 12/103 (12%) of I patients
we didn’t detect any mutation, while in 6 (5 I and 1 EB) we
found mutation in one allele only. Overall, we detected
mutations in 19 distinct genes. Furthermore, in 10 patients,
in addition to their primary disease-causing mutations,
harbored also another possibly pathogenic mutation in one
allele of the other gene, including semi-dominant mutations
in FLG. K. Wertheim-Tysarowska1, D. Śniegórska1, A. Grabarczyk1,
S. Radomska1, A. Kutkowska-Kaźmierczak1, J. Sawicka1,
M. Jackiewicz1, P. Bialik1, A. Kujko1, A. M. Rygiel1,
K. Niepokoj1, L. Ruszkowska2, K. Osipowicz3, R. Smigiel4,
B. Wawrzycki5, K. Wozniak3, A. Jazela-Stanek6, A. Jakubiuk-
Tomaszuk7, A. Eckersdorf-Mastalerz8, A. Barczyk1,
D. Marańska9, I. Dąbrowska-Wójciak10, K. Ebner11,
J. Castaneda1, N. Bezniakow1, M. Firek-Pędras12, E. Obersztyn1,
M. Pasinska13, A. Pietrzyk14, K. Szczałuba15, J. Wierzba16,
P. Wlasienko1, C. Kowalewski3, J. Bal1 Conclusions: In conclusion, our panel proved to be an
efficient and sensitive first-line diagnostic tool. P04.50B Our data
provide further information regarding molecular epidemiol-
ogy of I and EB and also focus on the presence of secondary
mutations,
which
have
important
impact
on
genetic
counseling and, presumably, may affect therapy. Supported
by grant NCN 2014/13/D/NZ5/03304 1Medical Genetic Department, Institute of Mother and Child,
Warsaw, Poland, 2Department of Paediatric Dermatology,
Specialist Hospital Miedzyleski, Warsaw, Poland, 3Department
of Dermatology and Immunodermatology, Warsaw Medical
University, Warsaw, Poland, 4Department of Paediatrics and
Rare Disorders,Wroclaw Medical University, Warsaw, Poland,
5Department of Dermatology, Venereology and Pediatric
Dermatology, Medical University of Lublin, Lublin, Poland,
6The Children’s Memorial Health Institute, Warsaw, Poland,
7Podlaskie Medical Center "Genetics", Bialystok, Poland,
8Department of Clinical Genetics, Medical University of Lodz,
Lodz, Poland, 9Department of Adult Dermatology, Specialist
Hospital Miedzyleski, Warsaw, Poland, 10Department of
Neonatology, SALVE ZOZ, Lodz, Poland, 11Department of
Intensive Care and Anaesthesiology for Children, Medical
University of Lodz,, Lodz, Poland, 12Clinical Hospital No. 6 of
the Silesian Medical University in Katowice, Katowice,
Poland, 13Department of Clinical Genetics - Collegium
Medicum Nicolaus Copernicus University in Bydgoszcz,
Bydgoszcz, Poland, 14Department of Genetics and Pathology,
Pomeranian Medical University, Szczecin, Poland, 15MedGen
Medical Center, Warsaw, Poland., Warsaw, Poland,
16Departments of Pediatrics, Hematology, Oncology and
Department of General Nursery, Medical University of
Gdansk, Gdansk, Poland K. Wertheim-Tysarowska:
None. D. Śniegórska:
None. A. Grabarczyk: None. S. Radomska: None. A. Kutkowska-Kaźmierczak: None. J. Sawicka: None. M. Jackiewicz: None. P. Bialik: None. A. Kujko: None. A.M. Rygiel: None. K. Niepokoj: None. L. Ruszkowska: None. K. Osipowicz: None. R. Smigiel: None. B. Wawrzycki:
None. K. Wozniak: None. A. Jazela-Stanek: None. A. Jakubiuk- Tomaszuk: None. A. Eckersdorf-Mastalerz:
None. A. Barczyk: None. D. Marańska: None. I. Dąbrowska-Wójciak: None. K. Ebner: None. J. Casta-
neda: None. N. Bezniakow: None. M. Firek-Pędras:
None. E. Obersztyn: None. M. Pasinska: None. A. Pietrzyk: None. K. Szczałuba: None. J. Wierzba: None. P. Wlasienko: None. C. Kowalewski: None. J. Bal: None. P04.49A 1Centre de Génétique, CHU Dijon Bourgogne, Dijon, France,
2INSERM 1231, Génétique des Anomalies du Développement,
Université Bourgogne Franche-Comté, Dijon, France,
3Fédération Hospitalo-Universitaire Médecine
Translationnelle et Anomalies du Développement, CHU Dijon
Bourgogne, Dijon, France, 4Génétique Biologique Histologie,
CHRU de Besançon, Besançon, France, 5AP-HP, Hôpital
Necker-Enfants malades, Genetics Departement, Centre of
Reference for Skeletal Dysplasia, INSERM UMR 1163, Institut
Imagine, University Paris Descartes-Sorbonne Paris Cité,
Paris, France, 6Service d'Histologie-Embryologie-
Cytogénétique, Hôpital Universitaire Necker-Enfants Malades,
Paris, France, 7Service de génétique médicale, CHU de
Bordeaux-GH Pellegrin, Bordeaux, France, 8Laboratoire de
génétique moléculaire, CHU de Bordeaux-GH Pellegrin,
Bordeaux, France, 9Service de génétique clinique, CHRU de
Lille - Hôpital Jeanne de Flandre, Lille, France, 10Laboratoire
de Génétique médicale, CHRU de Lille - Hôpital Jeanne de
Flandre, Lille, France, 11Service de dermatologie, Hôpital
Universitaire Necker-Enfants Malades, Paris, France,
12Pediatric Dermatology Department, Hôpital Femme Mère
Enfant, Hospices Civils de Lyon, Lyon, France, 13Service de
génétique, GH Est-Hôpital Femme Mère Enfant, Hospices
Civils de Lyon, Lyon, France, 14Child Health and Human
Development Program, Research Institute of the McGill
University Health Centre, Montreal, QC, Canada,
15Laboratoire de génétique moléculaire et de Cytogénétique,
Plateau Technique de Biologie, CHU Dijon Bourgogne, Dijon, A. Sorlin: None. É. Tisserant: None. J. Thevenon:
None. Y. Duffourd: None. P. Kuentz: None. V. Car-
mignac: None. V. Cormier-Daire: None. C. Michot:
None. V. Malan: None. M. Beaujard: None. F. Morice-
Picard: None. C. Rooryck-Thambo: None. C. Vincent-
Delorme: None. T. Smol: None. É. Boudry-Labis: None. S. Hadj Rabia: None. A. Phan: None. M. Cordier: None. M. Till: None. D. Sanlaville: None. J. St-Onge: None. C. Thauvin-Robinet: None. A. Mosca Boidron: None. R. Olaso: None. A. Boland: None. J. Deleuze: None. B. Keren: None. L. Faivre: None. J. Rivière: None. P. Callier: None. P. Vabres: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 115 P04.50B
Results of diagnostics of ichthyoses and epidermolysis
bullosa using dedicated next generation sequencing panel Novel mutations and clinical variability in Van der Woude
Syndrome Novel mutations and clinical variability in Van der Woude
Syndrome B. Cavdarli, V. Topcu, A. Bakir B. Cavdarli, V. Topcu, A. Bakir B. Cavdarli, V. Topcu, A. Bakir Department of Medical Genetics, Ankara Numune Education
and Research Hospital, Ankara, Turkey Grant references: Fundación Ramón Areces U. Esperón Moldes: None. M. Ginarte Val: None. M. Santamariña Pena: None. B. Rordríguez Lage: None. L. Rodríguez Pazos: None. A. Vega Gliemmo: None. U. Esperón Moldes: None. M. Ginarte Val: None. M. Santamariña Pena: None. B. Rordríguez Lage: None. L. Rodríguez Pazos: None. A. Vega Gliemmo: None. Introduction: Ectrodactyly, also known as split hand/foot
malformation, is seen in 1 of 8,500-25,000 newborn as
isolated or part of a syndrome. There are various types of
ectrodactyly that are inherited with autosomal recessive,
dominant and X linked manner. Ectrodactyly is also a
component of Hartsfield syndrome resulting in homozygous
or heterozygous mutations in Ig-like C2-type 2 domain
(conserved) of FGFR1 gene. Our purpose for presenting this
work is to discuss homozygous mutations in the upstream
region of FGFR1 gene conserved region may be a cause of
isolated ectrodactyly. P04.52D Materials and Methods: Two 26 and 27 years-old
Spanish siblings who presented with congenital ichthyosis
clinical manifestations, and tested negative for autosomal
recessive congenital ichthyosis (ARCI) related genes, were
sent to our service. Clinical features were carefully assessed
and genetic analysis was performed in both patients and
their parents. In silico predictions of mutational effects and
further characterization by RNA study was performed for
one putative splicing variant identified. Materials and Methods: Two 26 and 27 years-old
Spanish siblings who presented with congenital ichthyosis
clinical manifestations, and tested negative for autosomal
recessive congenital ichthyosis (ARCI) related genes, were
sent to our service. Clinical features were carefully assessed
and genetic analysis was performed in both patients and
their parents. In silico predictions of mutational effects and
further characterization by RNA study was performed for
one putative splicing variant identified. Results: Two novel FATP4 mutations were found in both
patients:
one
frameshift
variant,
c.1322dup,
p. Gly442Argfs*2
and
one
intronic
substitution,
c.988-
19A>G. Parents were heterozygous for each of the variants. Molecular characterization of c.988-19A>G showed that
this variant creates one aberrant transcript that lacks the first
45bp of exon 8, which encodes the end of the protein ATP/
AMP motif, and it also leads to a deregulation of naturally
occurring isoforms. In silico analyses predicted the variant
to alter the recognition sites for splicing regulatory proteins. A.C. Ceylan: None. I. Vargel: None. A.C. Ceylan: None. I. Vargel: None. P04.52D Ichthyosis prematurity syndrome identified for the first
time in the Spanish population. Unprecedented molecular
characterization of a FATP4 splicing variant in humans U. Esperón Moldes1, M. Ginarte Val2, M. Santamariña Pena1,
B. Rordríguez Lage1, L. Rodríguez Pazos3, A. Vega Gliemmo1 Introduction: Ichthyoses (I) and epidermolysis bullosa
(EB) are rare, monogenic skin comprising several dozens of
clinical entities, caused by mutations in over 36 and 21
genes, in I and EB, respectively. Low incidence, genetic
diversity, overlapping phenotypes and age-dependent dis-
ease course negatively influence the detection rate in tra-
ditional diagnostic procedure based on phenotype to
genotype approach. The aim of the study was to elaborate
the cost effective genodermatoses-dedicated next generation
sequencing (NGS) panel. 1Fundación pública galega de medicina xenómica, Santiago de
compostela, Spain, 2Servicio de dermatología del complexo
hospitalario universitario de santiago de compostela, Santiago
de compostela, Spain, 3Servicio de dermatología del complexo
hospitalario universitario de vigo, Vigo, Spain Introduction: Ichthyosis prematurity syndrome (IPS) is a
rare syndromic form of autosomic recessive ichthyosis
caused by mutations in FATP4. To date, three different
FATP4 splice site mutations have been associated with IPS, Materials and Methods: We enrolled 103 and 8 patients
presenting clinical symptoms of I and EB, respectively. A J. del Picchia 116 as the cause of vWS syndrome. Mutations are mostly
clustered in exons encoding functional domains. Mutations
in the IRF6 gene are also associated with the Popliteal
Pterygium Syndrome(OMIM 119500), which has similar
orofacial features with vWS; however, it also presents with
popliteal webs, syndactyly, and genital anomalies. Here we
report, 43 patients with vWS at 6 different families and two
of them have novel mutations, which have not reported
before. A novel c.841-2A>C mutation was identified in
family A and a novel c.881T>A mutation was identified in
family B. These mutations will provide a better under-
standing of the phenotypic effect of exon 7 mutations. Due
to variable expression, the same mutation can lead to dif-
ferent clinical manifestations. However, different mutations
in the same genes can also be presenting with several
phenotypes. For this reason, the clinical effect of new
mutations help us for better understanding the causes of the
disease. It contributes to the genetic counselling. but none of them has been yet characterized at RNA level in
humans. but none of them has been yet characterized at RNA level in
humans. P04.55C Conclusions: This study describes, for the first time, two
cases of genetically diagnosed IPS in the Spanish popula-
tion, adding new mutations to the current list of FATP4
pathogenic variants. It also characterizes the non canonical
splice-site c.988-19A>G mutation, being the first splicing
study in FATP4 in humans. Do homozygous mutations in the upstream of Ig-like C2-
type 2 domain of FGFR1 result in isolated ectrodactyly? Higher diagnostic yield can be achieved by using multigene
NGS panel in testing patients with Marfan-related
disorders Higher diagnostic yield can be achieved by using multigene
NGS panel in testing patients with Marfan-related
disorders Higher diagnostic yield can be achieved by using multigene
NGS panel in testing patients with Marfan-related
disorders M. Ritelli1, C. Dordoni1, E. Giacopuzzi1, N. Chiarelli1,
V. Cinquina1, M. Venturini2, M. Colombi1 M. Ritelli1, C. Dordoni1, E. Giacopuzzi1, N. Chiarelli1,
V. Cinquina1, M. Venturini2, M. Colombi1 A. karamzade1, Z. Golchehre1, M. Keramatipour1, M. Saberi1,
A. Nasrollahzadeh1, M. Arabpour1, P. Nourmohammadi2,
R. Behdad3 1Division of Biology and Genetics, Department of Molecular
and Translational Medicine, University of Brescia, Brescia,
Italy, 2Division of Dermatology, Department of Clinical and
Experimental Sciences, Spedali Civili University Hospital,
Brescia, Italy 1Division of Biology and Genetics, Department of Molecular
and Translational Medicine, University of Brescia, Brescia,
Italy, 2Division of Dermatology, Department of Clinical and
Experimental Sciences, Spedali Civili University Hospital,
Brescia, Italy 1Tehran university of medical sciences, Tehran, Iran, Islamic
Republic of, 2Pishgam Biotech Company, NGS Department,
North Kargar Street, Tehran, Iran, Islamic Republic of,
3Watson Genetic Laboratory, North Kargar Street, Tehran,
Iran, Islamic Republic of 1Tehran university of medical sciences, Tehran, Iran, Islamic
Republic of, 2Pishgam Biotech Company, NGS Department,
North Kargar Street, Tehran, Iran, Islamic Republic of,
3Watson Genetic Laboratory, North Kargar Street, Tehran,
Iran, Islamic Republic of Linkeropathies are a group of heterogeneous syndromes
along a spectrum of skeletal and connective tissue disorders
(CTDs) with the full disease range yet to be defined. Lin-
keropathy genes encode for enzymes branching glycosa-
minoglycan chains onto proteoglycans via a common
tetrasaccharide linker; XYLT1 and XYLT2 encode for xylo-
syltransferases, B4GALT7 and B3GALT6 for galactosyl-
transferases, and B3GAT3 for a glucuronyltransferase. XYLT1 and XYLT2 are associated respectively with Des-
buquois dysplasia type 2 and with spondylo-ocular syn-
drome, B4GALT7 and B3GALT6 with spondylodysplastic
Ehlers-Danlos
syndrome
(spEDS). For
B3GAT3,
23
patients, all but one from 9 consanguineous families with
homozygous missense variants, have been described ran-
ging in phenotypic severity from mild to severe and
resembling Larsen-, Antley-Bixler-, Shprintzen-Goldberg-,
and Geroderma osteodysplastica-like syndromes. Here, we
report on a 16-year-old girl with a clinical suspicion of
spEDS. She was born to non-consanguineous parents and
presented with facial dysmorphism (dolichocephaly, pro-
minent
forehead,
enophthalmos,
midface
hypoplasia,
micrognathia, low-set ears), short stature, muscle hypotonia,
severe kyphoscoliosis, joint laxity with recurrent disloca-
tions, pectus carinatum, bilateral radio-ulnar synostosis,
atlanto-occipital instability, and bilateral pes planovalgus. A. C. CEYLAN1, I. VARGEL2 1Ankara Yildirim Beyazit Universi̇ty, Department of Medical
Genetics, Ankara, Turkey, 2Hacetepe University, Faculty of
Medicine, Department of Plastic Reconstructive and Aesthetic
Surgery, Ankara, Turkey Materials and Methods: We performed next-generation
sequencing (NGS) panel (Trusight one sequencing panel)
for a 2 years old female with left-hand ectrodactyly. Neuromotor and growth development of the patient were
normal and no abnormality was observed in cranial MRI
and
echocardiography. All
laboratory
tests
including
biochemical and metabolic screenings were also normal. Materials and Methods: We performed next-generation
sequencing (NGS) panel (Trusight one sequencing panel)
for a 2 years old female with left-hand ectrodactyly. Neuromotor and growth development of the patient were
normal and no abnormality was observed in cranial MRI
and
echocardiography. All
laboratory
tests
including
biochemical and metabolic screenings were also normal. Van der Woude Syndrome (OMIM 119300) is an autosomal
dominant clinical condition characterized by cleft palate,
cleft lip, and lower lip pits. vWS is the most common cause
of syndromic cleft lip-palate, and also 2% of all cleft lip and
palate cases. vWS presents with variable phenotypic fea-
tures and high penetrance. Interferon Regulatory Factor 6
(IRF6) gene mutations have been reported as the cause of
vWS. The 3th and 4th exons of the gene encode DNA
binding domain; 7,8, and 9th exons encode protein binding
domain. More than 300 IRF6 mutations have been reported Results:
A
homozygous
mutation
(c.386A>C;
p. Asp129Ala) was detected with NGS panel in the upstream
of Ig-like C2-type 2 domain of FGFR1 gene. p.Asp129Ala
hasn’t been reported before and insilico tools predicted the
mutation as deleterious. The mutation is detected as 117 Abstracts from the 51st European Society of Human Genetics Conference: Posters congenital hip dislocation, atrial septal defect, and anterior
ectopic anus. Trio analysis by WES revealed compound
heterozygosity
for
two
novel
missense
mutations
in
B3GAT3, thus expanding its allelic repertoire. We provide a
comparative overview of the phenotypic features of lin-
keropathies headlining the extended phenotypic range of
B3GAT3 mutations that overlaps with skeletal dysplasias
and other CTDs including EDS, hence offering future per-
spectives for EDS nosology and clinical research in
this field. heterozygous in unaffected parents and expression analysis
currently continues. congenital hip dislocation, atrial septal defect, and anterior
ectopic anus. Trio analysis by WES revealed compound
heterozygosity
for
two
novel
missense
mutations
in
B3GAT3, thus expanding its allelic repertoire. A. C. CEYLAN1, I. VARGEL2 We provide a
comparative overview of the phenotypic features of lin-
keropathies headlining the extended phenotypic range of
B3GAT3 mutations that overlaps with skeletal dysplasias
and other CTDs including EDS, hence offering future per-
spectives for EDS nosology and clinical research in
this field. Conclusion:
Hartsfield
syndrome,
characterized
by
ectrodactyly and holoprosencephaly, is the result of FGFR1
gene conserved domain mutations. Up to now, FGFR1
mutations have not been reported in patients with isolated
ectrodactyly. This report is unique in terms of showing that
homozygous mutations in upstream of Ig-like C2-type 2
domain
of
FGFR1
gene
are
related
with
isolated
ectrodactyly. B. Cavdarli: None. V. Topcu: None. A. Bakir: None. B. Cavdarli: None. V. Topcu: None. A. Bakir: None. M. Ritelli: None. C. Dordoni: None. E. Giacopuzzi:
None. N. Chiarelli: None. V. Cinquina: None. M. Venturini: None. M. Colombi: None. P04.57A Further delineation of the linkeropathy syndrome due to
glucuronyltransferase I-deficiency in a family with B3GAT3
compound heterozygosity for two novel mutations revealed
by whole exome sequencing P04.62B CALMPlex: a multi-gene panel for variant detection in
patients with Phakomatoses and overlapping disorders 1Center for Medical Genetics, Ghent University Hospital,
Ghent, Belgium, 2Department of Pediatrics, Ghent University
Hospital, Ghent, Belgium, 3Department of Biomedical
Molecular Biology, Ghent University, Ghent, Belgium,
4Department of Head, Neck and Maxillofacial Surgery, Ghent
University Hospital, Ghent, Belgium, 5Department of
Cardiology, Ghent University Hospital, Ghent, Belgium,
6Department of Pediatric Cardiology, Ghent University
Hospital, Ghent, Belgium, 7Department of Orthopedic Surgery,
Ghent University Hospital, Ghent, Belgium T. Giugliano1, C. Santoro2, A. Torella1,3, G. Esposito1,3,
S. Perrotta2, V. Nigro1,3, G. Piluso1 1Dipartimento di Medicina di Precisione, Università degli
Studi della Campania “Luigi Vanvitelli”, Napoli, Italy,
2Dipartimento della Donna, del Bambino e di Chirurgia
Generale e Specialistica, Università degli Studi della
Campania “Luigi Vanvitelli”, Napoli, Italy, 3TIGEM (Telethon
Institute of Genetics and Medicine), Pozzuoli, Italy 1Dipartimento di Medicina di Precisione, Università degli
Studi della Campania “Luigi Vanvitelli”, Napoli, Italy,
2Dipartimento della Donna, del Bambino e di Chirurgia
Generale e Specialistica, Università degli Studi della
Campania “Luigi Vanvitelli”, Napoli, Italy, 3TIGEM (Telethon
Institute of Genetics and Medicine), Pozzuoli, Italy Introduction: Myhre syndrome (MYHRS) (MIM 139210)
is a rare autosomal dominant multisystem connective tissue
disorder, characterized by mental retardation, cardiopathy,
skeletal abnormalities, short stature, scleroderma and lar-
yngeal stenosis. So far, all reported cases were due to de
novo gain-of-function missense mutations in SMAD4,
encoding the SMAD protein commonly required for both
transforming growth factor-beta and bone morphogenic
proteins signal transduction. Phakomatoses, characterized by pigmentary manifestations,
are phenotypically overlapping disorders often difficult to
distinguish only on the basis of clinic in early childhood. Nevertheless, a proper diagnosis is essential for an appro-
priate clinical management. We developed CALMPlex, a
targeted NGS platform for molecular diagnosis of Phako-
matoses. It comprises 70 disease-causing genes, including
genes responsible for Phakomatoses, RNF135 and SUZ12
as NF1 flanking genes involved in 17q11 microdeletion,
SPRED2 and SPRED3 as SPRED1 homologous genes and
all the genes of RASopathies. It also includes 25 candidate
genes identified as direct interactors of the selected disease
genes with different bioinformatics tools (i.e. STRING,
GeneMANIA). We enrolled 149 children (0-18 years) with
clinical diagnosis of Phakomatoses. Additional 13 samples
with already known mutations were used as training set to
validate CALMPlex. 10/149 patients were clinically diag-
nosed as NF1 not confirmed by Sanger sequencing at RNA
analysis. P04.61A I. Meerschaut: None. A. Beyens: None. W. Steyaert:
None. R. De Rycke: None. B. Menten: None. K. Bonte:
None. T. De Backer: None. S. Janssens: None. F. Malfait: None. J. Panzer: None. F. Plasschaert: None. P. Coucke:
None. D. De
Wolf:
None. B. Callewaert: None. Higher diagnostic yield can be achieved by using multigene
NGS panel in testing patients with Marfan-related
disorders Nearly 30% found
in this study were novel variants. gene (64%), while 5 samples had possible causative variants
in five other genes. Missense variants were the most
common causative variants and in total 7 novel variants
were also identified. gene (64%), while 5 samples had possible causative variants
in five other genes. Missense variants were the most
common causative variants and in total 7 novel variants
were also identified. Conclusion: This study showed using NGS panel of
genes associated with Marfan-related disorders, gives a
higher diagnostic yield for such patients. Nearly 30% found
in this study were novel variants. Conclusion: This study showed using NGS panel of
genes associated with Marfan-related disorders, gives a
higher diagnostic yield for such patients. Nearly 30% found
in this study were novel variants. Conclusions: We report on two novel probands with
MYHRS. To our knowledge our data represent the first
familial case of MYHRS and further widens the clinical
spectrum of the disorder. TEM analysis implicates both
collagen and elastic fiber anomalies and may shed novel
insights on the relation between growth factor signaling and
extracellular matrix homeostasis. A. karamzade: None. Z. Golchehre: None. M. Kera-
matipour: None. M. Saberi: None. A. Nasrollahzadeh:
None. M. Arabpour: None. P. Nourmohammadi: None. R. Behdad: None. Grants: FWO: G028415N to PC and BC Grants: FWO: G028415N to PC and BC Two novel probands with Myhre syndrome identified
through whole exome sequencing Two novel probands with Myhre syndrome identified
through whole exome sequencing I. Meerschaut1,2, A. Beyens1, W. Steyaert1, R. De Rycke3,
B. Menten1, K. Bonte4, T. De Backer5, S. Janssens1, F. Malfait1,
J. Panzer6, F. Plasschaert7, P. Coucke1, D. De Wolf6,
B. Callewaert1 I. Meerschaut1,2, A. Beyens1, W. Steyaert1, R. De Rycke3,
B. Menten1, K. Bonte4, T. De Backer5, S. Janssens1, F. Malfait1,
J. Panzer6, F. Plasschaert7, P. Coucke1, D. De Wolf6,
B. Callewaert1 Higher diagnostic yield can be achieved by using multigene
NGS panel in testing patients with Marfan-related
disorders Medical
history
included
severe
low
bone
density, Introduction: Marfan syndrome is a life threatening con-
dition with estimated prevalence of 1:5,000-1:10,000. All
cases appear to be due to heterozygous mutation in FBN1
gene. However, there are other heritable conditions with
partially overlapping phenotypes caused by other genetic
defects. Here, we compare the diagnostic yield of using
NGS multigene panel with FBN1-only testing, in 34
patients. Methods:
Targeted
NGS
was
applied
to
analyze
34 samples from individuals with clinical presentation of
Marfan-related disorders. Twenty samples were analyzed
for FBN1 gene only, and 14 samples were analyzed for a
panel of 14 genes named as “Marfan, Aneurysm and
Related Disorders”. Target regions captured with Nimble-
gen chip, followed by NGS on Illumina platform. Candidate
variants were interpreted according to ACMG-guideline for
variant interpretation 2015. Methods:
Targeted
NGS
was
applied
to
analyze
34 samples from individuals with clinical presentation of
Marfan-related disorders. Twenty samples were analyzed
for FBN1 gene only, and 14 samples were analyzed for a
panel of 14 genes named as “Marfan, Aneurysm and
Related Disorders”. Target regions captured with Nimble-
gen chip, followed by NGS on Illumina platform. Candidate
variants were interpreted according to ACMG-guideline for
variant interpretation 2015. Results: In 10 samples, out of 20 samples, that were
analyzed for FBN1 gene only, a strong candidate causative
variant with pathogenic, likely pathogenic or VUS classi-
fication were detected. Other 10 samples end up with a
negative result. On the other hand, all 14 samples tested for
the panel of 14 genes, resulted strong candidate causative
variants. 9 out of 14 carried a causative variant in FBN1 118 J. del Picchia patients harbor the recurrent heterozygous c.1486C>T (p. Arg496Cys) mutation in SMAD4 (NM_005359). TEM of
the dermis of the second proband shows dens collagen and
irregular elastin cores with globular deposits and almost
absent surrounding microfibrils. patients harbor the recurrent heterozygous c.1486C>T (p. Arg496Cys) mutation in SMAD4 (NM_005359). TEM of
the dermis of the second proband shows dens collagen and
irregular elastin cores with globular deposits and almost
absent surrounding microfibrils. gene (64%), while 5 samples had possible causative variants
in five other genes. Missense variants were the most
common causative variants and in total 7 novel variants
were also identified. Conclusion: This study showed using NGS panel of
genes associated with Marfan-related disorders, gives a
higher diagnostic yield for such patients. Trakya University, Medical Faculty, Department of Medical
Genetics, Edirne, Turkey Introduction: Bone mineralisation disorders are a very
heterogenious group of bone disorders both in terms of
clinical manifestations and genetic background. Next Gen-
eration Sequencing (NGS) is a powerfull technology
allowing analysis of a lot of genes of a number of patients
simultaneusly with a low cost in a short time. We aimed to
report six novel variations of bone mineralisation disorders-
related genes in 6 different patients directed to our depart-
ment
with
clinical
diagnosis
of
abnormal
bone
mineralisation. Non-syndromic cleft lip with or without cleft palate (nsCL/
P) is one of the most common congenital malformations and
has a multifactorial etiology. To date, a number of common
risk variants have been identified for nsCL/P, explaining
about 25% of the genetic heritability. We hypothesize that
some of the remaining genetic liability is explained by rare
dominant de novo mutations. In order to identify such rare
de novo events, we performed whole exome sequencing
(WES) in 50 nsCL/P patients and their unaffected parents. The analysis resulted in 33 rare de novo events in 33 genes. To find further support for these candidate genes and to
strengthen our hypothesis of dominant de novo events
adding to nsCL/P etiology, the candidate genes were sub-
jected to a multiplex resequencing study with single mole-
cule molecular inversion probes (smMIPs) in a multiethnic
case/control sample of Arabian, Mexican and Central Eur-
opean ancestry (ncases=1,061, ncontrols=1,591). The assay Materials and Methods: Genomic DNA samples were
isolated from EDTA-blood samples of 11 patients. Libraries
were prepared with Osteo-GeneSGKit DensidadOsea, IVD
-CE kit according to instructions of manufacturer’s. Fastq
generation was performed by MiSeq Reporter (v2.5.1;
Illumina Inc.) Genomize Seq. Platform was used for variant
calling and filtering. Varsome was used for in silico analysis
of variants and ClinVar and HGMD databases were
accepted as refference for known variant interpretation, Materials and Methods: Genomic DNA samples were
isolated from EDTA-blood samples of 11 patients. Libraries
were prepared with Osteo-GeneSGKit DensidadOsea, IVD
-CE kit according to instructions of manufacturer’s. Fastq
generation was performed by MiSeq Reporter (v2.5.1;
Illumina Inc.) Genomize Seq. Platform was used for variant
calling and filtering. P04.62B CALMPlex detected all disease-causing mutations Materials and Methods: We report on two adult
probands with MYHRS identified through whole exome
sequencing and performed transmission electron micro-
scopy (TEM) on a skin biopsy. Results: The first proband presented with a congenital
heart defect and vertebral anomalies, but normal skin,
stature and intelligence. Later-on, she developed severe
laryngeal stenosis. She has two similarly affected children. The second proband presented with visual impairment
following lensectomy in childhood, short stature, brachy-
dactyly, stiff skin and decreased peripheral sensitivity. Both Abstracts from the 51st European Society of Human Genetics Conference: Posters 119 define genetic background of this type of heterogenious
diseases. define genetic background of this type of heterogenious
diseases. define genetic background of this type of heterogenious
diseases. in the training set and a NF1 mutation in 100% (10/10) of
molecularly unresolved NF1 cases. In the other 139 cases,
CALMPlex identified the molecular defect in 57%(80/139)
of them: 77 had mutations in genes fitting the clinical
diagnosis (NF1, NF2, SPRED1, PTPN11, KIT, TSC1,
TSC2, LZTR1 and PPP1CB), while 3 had mutations in an
unexpected gene respect to initial clinical suspicion. Pathogenic variants in candidate genes have also been
detected in some of patients remained undiagnosed but a
further characterization is necessary. CALMPlex is a useful
first-tier test for genetic evaluation of these phenotypically
overlapping conditions especially when only pigmentary
manifestations occur. H. Tozkır: None. S. Demir: None. H. Gürkan: None. D. Eker: None. E. Atli: None. H. Tozkır: None. S. Demir: None. H. Gürkan: None. D. Eker: None. E. Atli: None. Large-scale resequencing study of nsCL/P candidate genes
in 1061 nsCL/P cases and 1591 controls N. Ishorst1,2, L. Henschel1,2, F. Thieme1,2, D. Drichel3,
S. Sivalingam1,2, S. L. Mehrem1,2, A. C. Fechtner1,2,
A. Heimbach1,2, M. Alblas1,2, K. Keppler1,2, A. Hoischen4,5,6,
K. Aldhorae7, B. Braumann8, M. Martini9, L. Gölz10,
H. Reutter1,11, S. Nowak1, M. Knapp12, M. M. Nöthen1,2,
M. Nothnagel3, T. Becker13, K. U. Ludwig1,2, E. Mangold1 T. Giugliano: None. C. Santoro: None. A. Torella:
None. G. Esposito: None. S. Perrotta: None. V. Nigro:
None. G. Piluso: None. T. Giugliano: None. C. Santoro: None. A. Torella:
None. G. Esposito: None. S. Perrotta: None. V. Nigro:
None. G. Piluso: None. 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life&Brain Center,
University of Bonn, Bonn, Germany, 3Cologne Center for
Genomics, University of Cologne, Cologne, Germany,
4Department of Human Genetics, Radboud University Medical
Center, Nijmegen, Netherlands, 5Department of Internal
Medicine, Radboud University Medical Center, Nijmegen,
Netherlands, 6Radboud Institute for Molecular Life Sciences,
Radboud University Medical Center, Nijmegen, Netherlands,
7Orthodontic Department, College of Dentistry, Thamar
University, Thamar, Yemen, 8Department of Orthodontics,
University of Cologne, Cologne, Germany, 9Department of
Oral and Maxillo-Facial-Plastic Surgery, University of Bonn,
Bonn, Germany, 10Department of Orthodontics, University of
Bonn, Bonn, Germany, 11Department of Neonatology,
Children's Hospital, University of Bonn, Bonn, Germany,
12Institute of Medical Biometry, Informatics and Epidemiology,
University of Bonn, Bonn, Germany, 13Institute for Community
Medicine, University of Greifswald, Greifswald, Germany Genetic diagnosis of bone mineralisation disorders with
Next Generation Sequencing and definition of five novel
pathogenic variations H. Tozkır, S. Demir, H. Gürkan, D. Eker, E. Atli Trakya University, Medical Faculty, Department of Medical
Genetics, Edirne, Turkey P04.66B Opsismodysplasia - report on long-term follow-up of a
previously described and two new Portuguese cases P. Maia Almeida1,2, H. G. Santos3, J. M. Saraiva1,4, J. Seabra5,
C. Reis1, M. Venâncio1,6, K. Heath7, V. Cormier-Daire8, S. B
Sousa1,6 P. Maia Almeida1,2, H. G. Santos3, J. M. Saraiva1,4, J. Seabra5,
C. Reis1, M. Venâncio1,6, K. Heath7, V. Cormier-Daire8, S. B
Sousa1,6 Discussion: Once thought to be a lethal condition, OPSM
turned out to have a wider phenotypic variability, well-
illustrated in the description of these 3 patients. Despite the
growing number of reported surviving cases, information is
lacking on natural history and long-term follow-up. 1Medical Genetics Unit, Hospital Pediátrico, Centro
Hospitalar e Universitário de Coimbra, Coimbra, Portugal,
2Faculty of Health Sciences, University of Beira Interior,
Covilhã, Portugal, 3Serviço de Genética Médica,
Departamento de Pediatria, Centro Hospitalar Lisboa Norte,
Hospital de Santa Maria, Centro Académico de Medicina de
Lisboa, Lisboa, Portugal, 4University Clinic of Pediatrics,
Faculty of Medicine, University of Coimbra, Coimbra,
Portugal, 5Serviço de Ortopedia Pediátrica, Hospital
Pediátrico, Centro Hospitalar e Universitário de Coimbra,
Coimbra, Portugal, 6University Clinic of Genetics, Faculty of
Medicine, University of Coimbra, Coimbra, Portugal,
7Institute of Medical and Molecular Genetics (INGEMM),
IdiPAZ and Skeletal dysplasia multidisciplinary Unit (UMDE),
Hospital Universitário La Paz, Madrid, Spain and CIBERER,
ISCIII, Madrid, Madrid, Spain, 8Department of Medical
Genetics, INSERM U1163, Université Paris-Descartes, Institut
Imagine, Hôpital Necker-Enfants Malades, Paris, France,
Paris, France P. Maia Almeida: None. H. G. Santos: None. J. M. Saraiva: None. J. Seabra: None. C. Reis: None. M. Venâncio: None. K. Heath: None. V. Cormier-Daire:
None. S. B Sousa: None. Trakya University, Medical Faculty, Department of Medical
Genetics, Edirne, Turkey Alblas: None. K. Keppler: None. A. Hoischen: None. K. Aldhorae: None. B. Braumann: None. M. Martini: None. L. Gölz: None. H. Reutter: None. S. Nowak: None. M. Knapp: None. M.M. Nöthen: None. M. Nothnagel: None. T. Becker:
None. K.U. Ludwig:
None. E. Mangold: None. N. Ishorst: None. L. Henschel: None. F. Thieme: None. D. Drichel: None. S. Sivalingam: None. S.L. Mehrem:
None. A.C. Fechtner: None. A. Heimbach: None. M. Alblas: None. K. Keppler: None. A. Hoischen: None. K. Aldhorae: None. B. Braumann: None. M. Martini: None. L. Gölz: None. H. Reutter: None. S. Nowak: None. M. Knapp: None. M.M. Nöthen: None. M. Nothnagel: None. T. Becker:
None. K.U. Ludwig:
None. E. Mangold: None. Trakya University, Medical Faculty, Department of Medical
Genetics, Edirne, Turkey Varsome was used for in silico analysis
of variants and ClinVar and HGMD databases were
accepted as refference for known variant interpretation, Results: We defined 5 novel and 4 known mutations in 9
(4 variants in COL1A1, 2 variants in COL1A2, 1 variant in
PHEX, 1 variant in TGFB1 and 1 variant in SLC34A1) out
of 11 patients (81.81 %). Conclusion: We suggest that sequencing of the genes
associated with bone mineralisation simultaneously with
Next Generation Sequencing offers a practical approach to 120 J. del Picchia SHIP2, one of the phosphatases that catalyse depho-
sphorylation
at
the
5-position
of
phosphoinositides. OPSMD is characterised by severe pre and post-natal
micromelia with extremely short hands and feet, large
fontanel, craniofacial dysmorphisms and typical radio-
graphic features such as major delay in bone maturation,
platyspondyly,
squared
metacarpals
and
metaphyseal
cupping. was designed using the standard MIPgen pipeline and was
successful for 32 genes. Libraries were sequenced on an
Illumina HiSeq2500 2x125bp using Illumina v4 paired-end
chemistry. Raw reads were aligned with BWA and variants
were called with UnifiedGenotyper. Downstream analysis
included filtering for CADD ≥15 and MAF ≤0.1% fol-
lowed by a manual inspection of reads and a validation step
including segregation analysis. Our preliminary results of
the resequencing approach show the presence of further rare
variants/rare de novo events in our candidate genes in nsCL/
P patients. Further results will be presented at the
conference. Case Reports: We report on three unrelated Portuguese
patients, two male and one female, with the diagnosis of
OPSM based on their clinical, radiographic and molecular
findings. Patient 1 was the 6th case described in the literature
and is now 25 years, likely one of the oldest known patients
alive. From his follow-up we highlight: adult height of
1.05m; spinal surgery for severe scoliosis (4 years); severe
atlantoaxial
instability;
bilateral
cryptorchidism;
mitral
valve prolapse; noninvasive ventilation during sleep (since
15 years); without other significant problems. Patient 2 (5
years) has also typical features while Patient 3 (8 years) has
a milder phenotype, in accordance with his genotype:
compound heterozygous for c.1497+5G>C and c.1649T>C
(p.Phe550Ser) in INPPL1 gene. Patients 1 and 2 are
homozygous for null mutations: c.2719C>T(p.Arg907*)
and c.768-769del(p.Glu258Alafs*45), respectively. Neuro-
developmental assessments showed normal cognitive func-
tion and marked motor delay. N. Ishorst: None. L. Henschel: None. F. Thieme: None. D. Drichel: None. S. Sivalingam: None. S.L. Mehrem:
None. A.C. Fechtner: None. A. Heimbach: None. M. P04.67C Whole exome sequencing in Finnish families identifies new
candidate genes for osteoarthritis S. Skarp1,2, O. Kämäräinen2, G. Wei2, E. Jakkula2,
I. Kiviranta3,4, H. Kröger5, J. Auvinen1, P. Lehenkari6, L. Ala-
Kokko7, M. Männikkö1,2 1Center for Life Course Health Research, Faculty of Medicine,
University of Oulu, Oulu, Finland, 2Faculty of Biochemistry
and Molecular Medicine, University of Oulu, Oulu, Finland,
3Department of Orthopaedics and Traumatology, University of
Helsinki and Helsinki University Hospital, Helsinki, Finland,
4Department of Orthopaedics and Traumatology, Jyväskylä
Central Hospital, Jyväskylä, Finland, 5Department of
Orthopaedics and Traumatology, Kuopio University Hospital
and Kuopio Musculoskeletal Research Unit, University of Introduction: Opsismodysplasia (OPSM) is a rare auto-
somal recessive spondyloepimetaphyseal dysplasia caused
by biallelic mutations in INPPL1 gene, which encodes S. Skarp1,2, O. Kämäräinen2, G. Wei2, E. Jakkula2,
I. Kiviranta3,4, H. Kröger5, J. Auvinen1, P. Lehenkari6, L. Ala-
Kokko7, M. Männikkö1,2 In one family we found evidence for a
digenic inheritance pattern due to heterozygous variants in
COL1A1 and COL1A2.Conclusions: The findings in our
large cohort demonstrate the clinical utility of gene panel
testing for OI. Results: In all but two patients, pathogenic variants in
known disease genes were detected. We observed a total of
24 novel mutations and 24 known OI mutations, of which
several were recurrent. In one patient no relevant mutation
was found, and another patient harboured a class III
COL1A1 intronic variant. The percentage of autosomal
recessive forms due to mutations in BMP1, FKBP10,
LEPRE1, SERPINF1, and WNT1 was unusually high
(48%). Cases with FKBP10, IFITM5, and WNT1 mutations
could best be distinguished clinically. Most severe forms
were due to IFITM5 and LEPRE1 mutations, followed by
qualitative COL1A1, SERPINF1, and WNT1 mutations. Quantitative COL1A1 mutations and COL1A2 mutations
had milder effects. In one family we found evidence for a
digenic inheritance pattern due to heterozygous variants in
COL1A1 and COL1A2.Conclusions: The findings in our
large cohort demonstrate the clinical utility of gene panel
testing for OI. Materials and Methods: Eight subjects from three
families with hip and knee OA were studied using whole
exome sequencing. We focused on rare exonic variants with
predicted pathogenicity and variants located in active
promoter or strong enhancer regions. The software tool
ANNOVAR was used for functional annotation and minor
allele frequencies were obtained from the Exome Aggrega-
tion Consortium (ExAC) and Sequencing Initiative Suomi
(SISu) database. Expression of identified genes in human
bone and cartilage tissue was studied using PCR. Materials and Methods: Eight subjects from three
families with hip and knee OA were studied using whole
exome sequencing. We focused on rare exonic variants with
predicted pathogenicity and variants located in active
promoter or strong enhancer regions. The software tool
ANNOVAR was used for functional annotation and minor
allele frequencies were obtained from the Exome Aggrega-
tion Consortium (ExAC) and Sequencing Initiative Suomi
(SISu) database. Expression of identified genes in human
bone and cartilage tissue was studied using PCR. Results: Two rare variants co-segregated with OA in two
families. In Family 8 a missense variant was observed in the
OLIG3 gene that encodes a transcription factor known to be
associated with rheumatoid arthritis and inflammatory
polyarthritis. In Family 12 the observed variant was located
in the transcription start site of the FIP1L1 gene. FIP1L1
participates in the regulation of polyadenylation. Both
FIP1L1 and OLIG3 were observed to be expressed in
human bone and cartilage tissues. Grant: DBT-BMBF Cooperative Science Program (BT/
IN/Germany-BMBF/05/GK/2015-16) Grant: DBT-BMBF Cooperative Science Program (BT/
IN/Germany-BMBF/05/GK/2015-16) G.S. Bhavani: None. J. Mrosk: None. H. Shah: None. J. Hecht: None. U. Krüger: None. A. Shukla: None. U. Kornak: None. K.M. Girisha: None. Conclusion: The exome sequencing revealed novel
candidate
genes
for
OA. OLIG3
and
FIP1L1
may
participate in the regulatory events leading to OA. S. Skarp: None. O. Kämäräinen: None. G. Wei: None. E. Jakkula: None. I. Kiviranta: None. H. Kröger: None. Sclerostin and bone: The role of the SOST gene in
osteoporosis and fragility fractures in Malta J. Auvinen: None. P. Lehenkari: None. L. Ala-Kokko:
None. M. Männikkö: None. Sclerostin and bone: The role of the SOST gene in
osteoporosis and fragility fractures in Malta 1Kasturba Medical College, Manipal, India, 2Institute of
Medical Genetics and Human Genetics, Berlin, Germany,
3Berlin-Brandenburg Center for Regenerative Therapies,
Berlin, Germany, 4Max Planck Institute for Molecular
Genetics, Berlin, Germany P04.68D Mutation spectrum, genotype-phenotype correlation &
digenic inheritance in a large Indian cohort of osteogenesis
imperfecta Mutation spectrum, genotype-phenotype correlation &
digenic inheritance in a large Indian cohort of osteogenesis
imperfecta 121 Abstracts from the 51st European Society of Human Genetics Conference: Posters Eastern Finland, Kuopio, Finland, 6Department of Anatomy
and Cell biology and Surgery Clinic, Medical Research
Center, University of Oulu and Oulu University Hospital,
Oulu, Finland, 7Connective Tissue Gene Tests, Allentown, PA,
United States Eastern Finland, Kuopio, Finland, 6Department of Anatomy
and Cell biology and Surgery Clinic, Medical Research
Center, University of Oulu and Oulu University Hospital, Introduction: Osteogenesis Imperfecta (OI) is a clinically
and genetically heterogeneous disorder. Till date, patho-
genic variants in approximately twenty genes are known to
cause OI with mutations in the type 1 collagen genes
(COL1A1 and COL1A2) being the most common cause. Materials and Methods: In our study, we evaluated the
clinical features of OI in 50 Indian index patients. Grouping
according to phenotypic and radiographic features revealed
four individuals with Bruck syndrome, three patients with
hypertrophic callus and twenty with extreme bone bowing. The molecular evaluation was done by a small custom
designed gene panel or exome sequencing. Materials and Methods: In our study, we evaluated the
clinical features of OI in 50 Indian index patients. Grouping
according to phenotypic and radiographic features revealed
four individuals with Bruck syndrome, three patients with
hypertrophic callus and twenty with extreme bone bowing. The molecular evaluation was done by a small custom
designed gene panel or exome sequencing. Introduction: Osteoarthritis (OA) is the most common
degenerative joint disease characterized by progressive
degradation of the joint cartilage. OA is a complex disease
that has a strong genetic background with heritability esti-
mations of 39% and 60% for knee and hip OA, respectively. The objective of this study was to identify rare variants
predisposing subjects to OA in three Finnish families. Results: In all but two patients, pathogenic variants in
known disease genes were detected. We observed a total of
24 novel mutations and 24 known OI mutations, of which
several were recurrent. In one patient no relevant mutation
was found, and another patient harboured a class III
COL1A1 intronic variant. The percentage of autosomal
recessive forms due to mutations in BMP1, FKBP10,
LEPRE1, SERPINF1, and WNT1 was unusually high
(48%). Cases with FKBP10, IFITM5, and WNT1 mutations
could best be distinguished clinically. Most severe forms
were due to IFITM5 and LEPRE1 mutations, followed by
qualitative COL1A1, SERPINF1, and WNT1 mutations. Quantitative COL1A1 mutations and COL1A2 mutations
had milder effects. G. S. Bhavani1, J. Mrosk2, H. Shah1, J. Hecht3, U. Krüger3,
A. Shukla1, U. Kornak2,3,4, K. M. Girisha1 Department of Applied Biomedical Science, Faculty of Health
Sciences, Msida, Malta Department of Applied Biomedical Science, Faculty of Health
Sciences, Msida, Malta G. S. Bhavani1, J. Mrosk2, H. Shah1, J. Hecht3, U. Krüger3,
A. Shukla1, U. Kornak2,3,4, K. M. Girisha1
1Kasturba Medical College, Manipal, India, 2Institute of
Medical Genetics and Human Genetics, Berlin, Germany,
3Berlin-Brandenburg Center for Regenerative Therapies,
Berlin, Germany, 4Max Planck Institute for Molecular
Genetics, Berlin, Germany G. S. Bhavani1, J. Mrosk2, H. Shah1, J. Hecht3, U. Krüger3,
A. Shukla1, U. Kornak2,3,4, K. M. Girisha1 G. S. Bhavani1, J. Mrosk2, H. Shah1, J. Hecht3, U. Krüger3,
A. Shukla1, U. Kornak2,3,4, K. M. Girisha1 Introduction: Sclerostin is an important regulator of the
bone remodelling cycle acting as an inhibitor of the cano-
nical Wnt signalling pathway, resulting in reduced bone
mass. The aim of the study was to identify known or novel
SOST variants associated with osteoporosis and fracture
susceptibility in the Malta Osteoporotic Fracture Study
(MOFS). 122 J. del Picchia clubbing, prominent sweating of hands and feet and joints
pain. In older patient pachydermia and excessive sweating
was noticeable, however in younger patient the skin is not
yet affected. HPGD gene analysis was performed and in
both of them the same two truncating mutations were found
– c.175_176delCT and c.373delC. Biparental origin of
these
mutations
was
confirmed. First
mutation
(c.175_176delCT) is the most common mutation of HPGD
gene in Caucasian population, whereas second mutation
(c.373delC) has not been reported previously. We assume that the c.373delC truncating mutation of
HPGD gene, together with the c.175_176delCT mutation,
might be the founder mutations in Polish population,
responsible
for
more
cases
of
Pachydermoperiostosis
syndrome. clubbing, prominent sweating of hands and feet and joints
pain. In older patient pachydermia and excessive sweating
was noticeable, however in younger patient the skin is not
yet affected. HPGD gene analysis was performed and in
both of them the same two truncating mutations were found
– c.175_176delCT and c.373delC. Biparental origin of
these
mutations
was
confirmed. First
mutation
(c.175_176delCT) is the most common mutation of HPGD
gene in Caucasian population, whereas second mutation
(c.373delC) has not been reported previously. Materials and Methods: Sanger sequencing of the SOST
exons and their intronic flanking regions, together with the
promoter and 3’untraslated region (UTR) was performed in
200 individuals having normal and low bone mineral
density (BMD). Department of Applied Biomedical Science, Faculty of Health
Sciences, Msida, Malta Results: A total of 10 known and 3 novel variants were
identified including: rs851055, rs140960915, rs117857467,
rs59613373,
rs17882143,
rs768384322,
rs199560099,
rs17883310, rs17881550, rs17886183, c.220+134(T>C),
c*331(A>G) and c*390(C>A). Preliminary logistic regres-
sion with regards to the rs851055 promoter variant (G>A),
indicate that homozygosity for the A allele was associated
with a protective effect on BMD at the lumbar spine, LS
(Age adjusted Odds ratio: 0.1 [95% confidence interval
0.03-0.8]) and total hip, TH (OR: 0.3 [0.1-1.0]). The
rs17881550 3’ UTR insertion (-/G) also showed a protective
effect on LS BMD (OR 0.1 [0.03-0.7]), TH (OR 0.2 [0.04-
0.9]), and also with fracture risk (OR 0.2 [0.05-0.9]) in
women with the homozygous mutant genotype compared to
women with the homozygous wild-type genotype. We assume that the c.373delC truncating mutation of
HPGD gene, together with the c.175_176delCT mutation,
might be the founder mutations in Polish population,
responsible
for
more
cases
of
Pachydermoperiostosis
syndrome. K. Bernatowicz: None. A. Kashyap: None. C. Wlec-
zyk: None. S. Zajączek: None. Polymorphisms in genes involved in the base excision
repair (BER) pathway are associated with susceptibility to
Paget´s disease of bone Polymorphisms in genes involved in the base excision
repair (BER) pathway are associated with susceptibility to
Paget´s disease of bone Conclusion: Observations suggest that the rs851055 and
rs17881550 variants might be affecting transcriptional
activation or epigenetic mechanisms resulting in altered
Sclerostin function. All variants will be replicated in the
entire MOFS collection to determine association with BMD
and fracture susceptibility at different anatomical sites. Paget´s disease of bone C. Gutiérrez-Cerrajero1,2, R. Usategui-Martín1,2, S. Jiménez-
Vázquez2, I. Calero-Paniagua1,3,4, J. García-Aparicio1,5,
L. Corral-Gudino1,6, J. del Pino-Montes1,4, R. González-
Sarmiento1,2,7 C. Gutiérrez-Cerrajero1,2, R. Usategui-Martín1,2, S. Jiménez-
Vázquez2, I. Calero-Paniagua1,3,4, J. García-Aparicio1,5, L. Corral-Gudino1,6, J. del Pino-Montes1,4, R. González-
Sarmiento1,2,7 Sarmiento1,2,7 D. Scerri: None. A. Xuereb Anastasi: None. M.M. Formosa: None. 1Instituto De Investigación Biómedica De Salamanca (IBSAL),
Salamanca, Spain, 2Unidad de Medicina Molecular, Facultad
de Medicina, Universidad de Salamanca, Salamanca, Spain,
3Servicio de Reumatología, Hospital Universitario,
Salamanca, Spain, 4Servicio de Medicina Interna, Hospital
Virgen de la Luz, Cuenca, Spain, 5Servicio de Medicina
Interna Hospital Universitario, Salamanca, Spain, 6Servicio de
Medicina Interna, Hospital del Bierzo, Ponferrada, Spain,
7Laboratorio 14, Instituto de Biología Molecular Y Celular del
Cáncer, Salamanca, Spain P04.70B A novel recurrent mutation (c.373delC) in HPGD gene is
responsible for Pachydermoperiostosis K. Bernatowicz1, A. Kashyap1, C. Wleczyk2, S. Zajączek1 K. Bernatowicz1, A. Kashyap1, C. Wleczyk2, S. Zajączek1 1Department of Genetics and Pathology, Pomeranian Medical
University, Szczecin, Poland, 2Cytogenetic Unit, Department of
Laboratory Diagnostics, Pomeranian Medical University,
Szczecin, Poland Introduction: Paget´s disease of bone (PDB) is a chronic
bone metabolic disorder. Currently, PDB is the second most
frequent bone disorder. PDB is a focal disorder affecting the
skeleton segmentally. Its cause is unknown, but it has been
hypothesised that somatic mutations could be responsible
for the mosaicism described in PDB patients. Therefore, our
hypothesis is that defective response to DNA damage may
lead to somatic mutations, which in turn increase the risk
of PDB. Pachydermoperiostosis (primary hypertrophic osteoarthro-
pathy, Touraine-Solente-Gole syndrome, MIM 167100) is a
rare genetic disorder characterized by digital clubbing,
pachydermia and periostosis. Genetic background of this
disease has been recently revealed. Homozygous and
compound heterozygous mutations in HPGD gene cause
insufficiency of 15-hydroxyprostaglandin dehydrogenase,
an enzyme responsible for PGE2 catabolism. Elevated
levels of PGE2 were reported in all homozygous patients,
thus leading to chronic inflammation process, most strik-
ingly affecting joints. Materials and Methods: We analysed polymorphisms in
DNA repair genes involved in the BER, NER and DSBR
pathways in order to evaluate the role of these variants in
modulating PDB risk. We recently diagnosed two unrelated patients from non-
consanguineous families, age 7 and 23, both with digital 123 Abstracts from the 51st European Society of Human Genetics Conference: Posters Results: We found statistically significant differences in
genotypic and allelic distribution for polymorphisms in
genes involved in the BER pathway. Our results showed
that carrying the allele T of the XRCC1 rs1799782
polymorphism and the allele G of the APEX rs1130409
polymorphism increased the risk of developing PDB. Reference Centre for vEDS (Georges Pompidou European
Hospital, Paris, France). Results : We found 3 unrelated cases harboring C1R or
C1S missense variants: p.Tyr302Cys (previously reported)
and p.Cys309Phe in C1R, p.Cys321Ser in C1S. The two last
variants affect cysteines in CCP1 domains, like most
described pathogenic variants. These affected cases had
three characteristic features: early-onset periodontitis with
complete tooth loss, easy bruising and pretibial hyperpig-
mentation. Two were sporadic cases and one had a family
history of periodontitis. The negative cases had unusual
periodontal manifestations and severe joint hypermobility. P04.70B We are currently building a database of confirmed pEDS
patients to study the role of pathogenic variants in the
immune response because of recurrent infections (40% of
cases). Conclusions: These polymorphisms could cause a lower
DNA repair efficiency and this might lead to local somatic
mutations favouring the bone metabolic alterations char-
acteristic of PDB. This is the first report showing an
association between polymorphism in genes involved in the
BER pathway and PDB. This work was supported by a grant from Instituto de
Salud Carlos III (Ministry of Economy and Competitive-
ness) (ISC IIII-FEDER: PI16/01920) C. Gutiérrez-Cerrajero: None. R. Usategui-Martín:
None. S. Jiménez-Vázquez: None. I. Calero-Paniagua: C. Gutiérrez-Cerrajero: None. R. Usategui-Martín:
None. S. Jiménez-Vázquez: None. I. Calero-Paniagua: Conclusions : We confirmed pEDS is a rare condition
with a well-defined phenotype including the three features
abovementioned. The clinical diagnosis should lead to
genotype C1R and C1S. The pathophysiology remains to be
discovered, including the impact in the immune response
system. None. J. García-Aparicio: None. L. Corral-Gudino:
None. J. del
Pino-Montes:
None. R. González-
Sarmiento: None. P04.72D Screening a French series of Ehlers-Danlos syndrome
patients with periodontal manifestations reveals new
variants in C1R and C1S and refines the clinical features of
periodontal Ehlers-Danlos syndrome A. Legrand: None. M. Devriese: None. S. Adham:
None. K. Benistan:
None. E. Schaefer:
None. R. Jaussaud: None. M. Frank: None. X. Jeunemaitre:
None. J. Albuisson: None. A. Legrand: None. M. Devriese: None. S. Adham:
None. K. Benistan:
None. E. Schaefer:
None. R. Jaussaud: None. M. Frank: None. X. Jeunemaitre:
None. J. Albuisson: None. A. Legrand1,2, M. Devriese1, S. Adham1, K. Benistan3,
E. Schaefer4, R. Jaussaud5, M. Frank1, X. Jeunemaitre1,2,
J. Albuisson1,2 A. Legrand1,2, M. Devriese1, S. Adham1, K. Benistan3, A. Legrand1,2, M. Devriese1, S. Adham1, K. Benistan3, P04.73A
PLS3 deletions lead to severe spinal osteoporosis and
disturbed bone matrix mineralization PLS3 deletions lead to severe spinal osteoporosis and
disturbed bone matrix mineralization E. Schaefer4, R. Jaussaud5, M. Frank1, X. Jeunemaitre1,2,
J. Albuisson1,2 1Département de génétique et Centre de Référence des
Maladies Vasculaires, Hôpital Européen Georges Pompidou,
Assistance Publique-Hôpitaux de Paris, Paris, France,
2INSERM, U970, Paris Centre de Recherche Cardiovasculaire,
Paris, France, 3Département de Génétique, Hôpital Raymond
Poincaré, Assistance Publique-Hôpitaux de Paris, Garches,
France, 4Service de Génétique Médicale, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France,
5Département de Médecine Interne et Immunologie Clinique,
CHU de Nancy-Hôpitaux de Brabois, Vandoeuvre-Lès-Nancy,
France A. J. Kämpe1, A. Costantini1, Y. Levy-shraga2,3, L. Zeitlin4,
P. Roschger5, F. Taylan1, A. Lindstrand1,6, E. P. Paschalis5,
S. Gamsjaeger5, A. Raas-Rothschild7, M. Hövel8, H. Jiao9,
K. Klaushofer5, C. Grasemann10, O. Mäkitie1,6,11 1Department of Molecular Medicine and Surgery and Center
for Molecular Medicine, Stockholm, Sweden, 2Pediatric
Endocrinology Unit, Edmond and Lily Safra Children's
Hospital, Sheba Medical Center, Tel-Hashomer, Israel,
3Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv,
Israel, 4Pediatric Orthopedic Department, Dana-Dwek
Children's Hospital, Sourasly Medical Center, Tel-Aviv, Israel,
5Ludwig Boltzmann Institute of Osteology at the Hanusch
Hospital of WGKK and AUVA Trauma Centre Meidling, 1st
Medical Department Hanusch Hospital, Vienna, Austria,
6Department of Clinical Genetics, Karolinska University
Hospital, Stockholm, Sweden, 7Institute for Rare Diseases, The
Danek Gertner Institute of Human Genetics, Sheba Medical
Center, Tel-Hashomer, Israel, 8Department of Orthopedics
and Trauma Surgery, University Hospital Essen and the
University of Duisburg-Essen, Essen, Germany, 9Department
of Biosciences and Nutrition, and Science for Life Laboratory, Background : Pathogenic variants in C1R and C1S were
recently discovered as a cause for periodontal Ehlers-
Danlos syndrome (pEDS, previously EDS VIII). EDS are
connective tissue disorders defined by major criteria: joint
laxity and skin alterations. pEDS share several features with
vascular EDS (vEDS), like acrogeria, gum fragility and
arterial or gastrointestinal ruptures. Methods : C1R and C1S were screened by Sanger
Sequencing in a series of 20 cases with periodontal
manifestations and addressed to the French National Methods : C1R and C1S were screened by Sanger
Sequencing in a series of 20 cases with periodontal
manifestations and addressed to the French National 124 J. del Picchia Karolinska Institutet, Stockholm, Sweden, 10Klinik für
Kinderheilkunde II, University Hospital Essen and the
University of Duisburg-Essen, Essen, Germany, 11Children's
Hospital, University of Helsinki and Helsinki University
Hospital, Helsinki, Finland Background. Polydactyly can be isolated or encountered in
more than 300 syndromes, mostly ciliopathies. Methods. P04.73A
PLS3 deletions lead to severe spinal osteoporosis and
disturbed bone matrix mineralization We describe a girl with a BBS8 with two
novel molecular anomalies: a new splice site mutation in
intron 13 of TTC8 and a duplication including GPC6, a
candidate gene in the pathogenesis of PAP-A2. The
patient’s
polydactyly
is
a
feature
of
BBS,
but
the
duplication of GPC6 has probably influenced the phenotype
of the patient, with polydactyly of all four limbs. Conclusion. We describe a girl with a BBS8 with two
novel molecular anomalies: a new splice site mutation in
intron 13 of TTC8 and a duplication including GPC6, a
candidate gene in the pathogenesis of PAP-A2. The
patient’s
polydactyly
is
a
feature
of
BBS,
but
the
duplication of GPC6 has probably influenced the phenotype
of the patient, with polydactyly of all four limbs. Conclusion. We describe a girl with a BBS8 with two
novel molecular anomalies: a new splice site mutation in
intron 13 of TTC8 and a duplication including GPC6, a
candidate gene in the pathogenesis of PAP-A2. The
patient’s
polydactyly
is
a
feature
of
BBS,
but
the
duplication of GPC6 has probably influenced the phenotype
of the patient, with polydactyly of all four limbs. J. Harvengt: None. U. Schierloh: None. S. Bulk: None. G. Pierquin: None. J. Harvengt: None. U. Schierloh: None. S. Bulk: None. G. Pierquin: None. P04.74B Post axial polydactyly: isolated symptom or part of a
syndrome. A case of BBS8 with two novel molecular
anomalies P04.76D Conclusion: Our results indicate that PLS3 deletions lead
to severe spinal osteoporosis and defective bone matrix
mineralization, and suggest that PLS3 is directly involved in
the mineralization process. Conclusion: Our results indicate that PLS3 deletions lead
to severe spinal osteoporosis and defective bone matrix
mineralization, and suggest that PLS3 is directly involved in
the mineralization process. The inflammasome pathway is involved in PXE through
IL1B upregulation in patients with a severe cardiovascular
PXE phenotype The inflammasome pathway is involved in PXE through
IL1B upregulation in patients with a severe cardiovascular
PXE phenotype A.J. Kämpe: None. A. Costantini: None. Y. Levy-
shraga: None. L. Zeitlin: None. P. Roschger: None. F. Taylan: None. A. Lindstrand: None. E.P. Paschalis:
None. S. Gamsjaeger: None. A. Raas-Rothschild: None. M. Hövel: None. H. Jiao: None. K. Klaushofer: None. C. Grasemann: None. O. Mäkitie: None. E. Y. G. De Vilder1,2,3, L. Martin4, G. Lefthériotis5, P. Coucke1,
A. De Paepe1, F. Van Nieuwerburgh6, O. M. Vanakker1 1Center for Medical Genetics, Ghent University Hospital,
Ghent, Belgium, 2Department of Ophthalmology, Ghent
University Hospital, Ghent, Belgium, 3PhD Fellow of the
Research Foundation - Flanders, Brussels, Belgium,
4Department of Dermatology, Angers University Hospital,
Angers, France, 5Department of Vascular Investigations, Nice
University Hospital, Nice, France, 6Department of
Pharmaceutics, Laboratory of Pharmaceutical Biotechnology,
Ghent University, Ghent, Belgium P04.73A
PLS3 deletions lead to severe spinal osteoporosis and
disturbed bone matrix mineralization A young girl aged 5 was investigated for
dysmorphic features, speech delay, obesity, strabismus and
post axial polydactyly of the four limbs. Results. Array CGH revealed a de novo heterozygous
duplication of uncertain significance in the 13q31.3 region
including the gene GPC6. A panel analysis for Bardet Biedl
Syndrome (BBS) revealed a new maternally inherited
pathogenic splice site mutation in intron 13 of the TTC8
gene (c.1317+1G>A) and a likely pathogenic splice site
variant in the exon 5 of the TTC8 gene inherited from the
father (c.459G>A (p.Thr153Thr)). Introduction: PLS3 is one of the genes associated with X-
linked osteoporosis in children and pathogenetic variants
have thus far been reported in 16 families. However, our
understanding of PLS3’s specific role in bone metabolism
and how it exerts its effects is still poor. To study this
further we investigated 3 children from 2 different families
with PLS3 deletions. Discussion. A previous publication of a case of PAP-A2
with a similar duplication has postulated GPC6 as a
candidate gene for PAPA-A2. For our patient, the additional
symptoms have led to test for BBS. This analysis showed a
never described mutation: a new pathogenic splice site
mutation in intron 13. According to the algorithms, the
variant c.1317+1G>A leads to a loss of the natural splice
site
leading
to
exon
skipping,
inclusion
of
intronic
sequences or usage of a cryptic splice site. Material and Methods: Family 1 consisted of 2 brothers,
11 and 7 years old, and their healthy parents. For the older
brother, a transiliac bone biopsy was taken and extensively
analyzed using histomorphometry, Quantitate Backscattered
Electron Imaging and Raman microspectroscopy. Family 2
consisted of a 12-year-old boy, his osteoporotic mother and
healthy father. Massive parallel sequencing and array-CGH
was used to genetically evaluate all subjects. Results:
In
both
families
the
affected
boys
had
childhood-onset osteoporosis with severe spinal involve-
ment and multiple compression fractures. Both brothers in
Family 1 had a deletion of exon 4-16 in PLS3, which was
inherited from their mother. The index boy in Family 2 had
a larger deletion, also inherited from his mother, which
spanned the entire PLS3 gene. All children had a normal
biochemical profile. Results from the bone tissue analyses
showed a strong hypomineralization of the bone and a
striking increase in osteoid volume, osteoid thickness and
mineralizing lag time. Conclusion. J. HARVENGT1, U. SCHIERLOH2, S. BULK1, G. PIERQUIN1 1CHU Liège, Liège, Belgium, 2CHL KannerKlinik,
Luxembourg, Luxembourg Introduction: Pseudoxanthoma elasticum (PXE), an auto-
somal recessive ectopic mineralization disorder, causes
skin, eye and cardiovascular symptoms. The disease shows Abstracts from the 51st European Society of Human Genetics Conference: Posters 125 Universität Erlangen-Nürnberg and Universitätsklinikum
Erlangen, Erlangen, Germany, 5Department of Dermatology,
Georg-August-University Göttingen, Göttingen, Germany,
6Department of Rheumatology, Fachklinik Bad Bentheim, Bad
Bentheim, Germany, 7Department of Internal Medicine I, José-
Carreras Centrum for Immuno- and Gene Therapy, University
of Saarland Medical School, Homburg/Saar, Germany,
8Department of Dermatology, University of Münster, Münster,
Germany, 9Division of Medical Inflammation Research,
Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden striking
phenotypic
variability
without
underlying
genotype-phenotype correlations. Therefore, we evaluated
the collective modifying effect of rare variants on the PXE
cardiovascular disease severity. striking
phenotypic
variability
without
underlying
genotype-phenotype correlations. Therefore, we evaluated
the collective modifying effect of rare variants on the PXE
cardiovascular disease severity. Methods: Whole Exome Sequencing and burden tests
(SKAT-O and C-alpha) were performed in 12 PXE patients
with an extreme cardiovascular phenotype (severe/mild). Functional validation included inflammasome stimulation
with LPS/ATP in dermal fibroblasts of PXE patients with
extreme cardiovascular phenotypes (4 severe/6 mild) and 2
healthy controls. Readout comprised qPCR analysis for
NLRP1 and inflammasome outcome parameter IL1B. Psoriatic arthritis (PsA) and psoriasis vulgaris (PsV) are
common chronic inflammatory disorders of complex etiol-
ogy. In a mouse-model, psoriasis-like skin and joint
symptoms were aggravated in a NADPH oxidase deficient
strain, indicating regulation by reactive oxygen species
(ROS). A subunit of the human NADPH oxidase is encoded
by NCF1, a gene located in a structurally complex genomic
region on chromosome 7q11.23 including two homologous
pseudogene copies. The considerable homology of the
genomic region comprising NCF1, its pseudogenes and
further
genes
predisposes
to
genomic
rearrangements
resulting in variability of copy number (CN). A reduced
NCF1 CN and a functional missense variant (c.269G>A/p. Arg90His) in NCF1, causing a reduced ROS production,
have recently been identified as strong genetic risk factors in
other autoimmune diseases. Those associations combined
with the animal model prompted us to analyze both variants
in 1,248 PsA, 1,157 PsV patients and 932 controls. NCF1
and pseudogene CNs were determined by qPCR, the
functional variant was genotyped with a nested PCR strat-
egy. J. HARVENGT1, U. SCHIERLOH2, S. BULK1, G. PIERQUIN1 We did not observe evidence for association with the
NCF1 CN nor with the functional variant in carriers of the
most frequent CN ratio (4 pseudogenes : 2 gene copies)
despite 97% power to detect nominally significant asso-
ciation with PsA or PsV with c.269G>A/p.Arg90His. The
negative findings make a role of these functional NCF1
variants in psoriasis unlikely, but since oxidative burst
seems to play a role in autoimmune disorders, other path-
ways resulting in deviant ROS production might play a role
in the pathogenesis of the disease. Results: Sixteen (SKAT-O) and 74 (C-alpha) genes were
identified as significant modifiers of the PXE cardiovascular
disease and were enriched for 3 pathways: calcium
homeostasis, vascular disease and apoptosis. One modifier,
NLRP1, is linked to vascular disease and apoptosis, and was
withheld for functional validation. IL1B was upregulated in
dermal fibroblasts of PXE patients with a severe versus mild
cardiovascular phenotype (fold change (FC) +/- 8; Mann-
Whitney (MW) test: p < 0.001) and a severe phenotype
versus healthy controls (FC +/- 51; MW test: p < 0.001). In
addition, baseline IL1B expression was significantly higher
in PXE patients versus healthy controls (FC +/- 10; MW
test: p < 0.001). Conclusion: This study provides functional validation for
a predicted modifier gene in the PXE cardiovascular
phenotype, and for the first time implicates inflammasome
involvement in the PXE pathophysiology. This will help to
direct future research on the mechanisms underlying PXE
and related soft tissue calcification disorders (FWO14/ASP/
084). E.Y.G. De
Vilder:
None. L. Martin:
None. G. Lefthériotis: None. P. Coucke: None. A. De Paepe:
None. F. Van
Nieuwerburgh:
None. O.M. Vanakker: None. Association analyses of functional NCF1 variants in
psoriatic arthritis and psoriasis vulgaris Association analyses of functional NCF1 variants in
psoriatic arthritis and psoriasis vulgaris U. D. Hüffmeier1, S. Löhr1, A. B. Ekici1, S. Uebe1, M. Köhm2,
F. Behrens2, B. Böhm2, M. Sticherling3, G. Schett4, R. Mössner5,
A. Nimeh6, G. Assmann7, J. Rech4, V. Oji8, R. Holmdahl9,
H. Burkhardt2, A. Reis1 Funding: BMBF-Metarthros-01EC1407A Funding: BMBF-Metarthros-01EC1407A U.D. Hüffmeier: None. S. Löhr: None. A.B. Ekici:
None. S. Uebe: None. M. Köhm: None. F. Behrens: None. B. Böhm: None. M. Sticherling: None. G. Schett: None. R. Mössner: None. A. Nimeh: None. G. Assmann: None. J. Rech: None. V. Oji: None. R. Holmdahl: None. H. Burkhardt: None. A. Reis: None. 1Human Genetics, University of Erlangen, Erlangen, Germany,
2Division of Rheumatology and IME, Fraunhofer Project
Group Translational Medicine and Pharmacology, Johann
Wolfgang Goethe University, Frankfurt am Main, Germany,
3Department of Dermatology, University Hospital Erlangen,
Erlangen, Germany, 4Department of Internal Medicine 3 -
Rheumatology and Immunology, Friedrich-Alexander- Analysis of the genetic background of psoriasis in a
Hungarian cohort A. Penyige1, E. A. Janka2, I. Sawhney2, A. Csordás2, Z. Kovács2,
A. Szegedi2, P. Holló3, S. Fiatal4, R. Ádány4, D. Törőcsik2, É. Remenyik2 1Univ. of Debrecen, Faculty of Medicine, Department of
Human Genetics, Debrecen, Hungary, 2Univ. of Debrecen,
Faculty of Medicine, Department of Dermatology, Debrecen,
Hungary, 3Semmelweis University, Department of
Dermatovenereology and Dermatooncology, Budapest,
Hungary, 4Univ. of Debrecen, Faculty of Public Health,
Department of Preventive Medicine, Debrecen, Hungary Introduction: Collagens provide stability and resilience to
extracellular matrix of connective tissues, including dermis, Introduction: Collagens provide stability and resilience to
extracellular matrix of connective tissues, including dermis,
blood vessels and bone. Therefore, collagen genes could be
involved
in
the
etiopathogenesis
of
Psoriasis
(Ps,
OMIM#177900)
and
Psoriatic
Arthritis
(PsA,
OMIM#607507), which display dysfunction and instability
of the connective tissues. COL10A1 (rs3812111, A/T),
COL6A5 (rs12488457, A/C), COL8A1 (rs13081855, G/T)
and the miR-146a (rs2910164, G/C) were selected as
potential biomarkers for Ps and PsA susceptibility. Introduction: Psoriasis is chronic, inflammatory disorder of
the skin, often associated with arthritis and systemic co-
morbidities. Previous genetic studies established that psor-
iasis susceptibility has strong genetic components beside
environmental risk factors. Materials and Methods: 393 Ps, 424 PsA and 600
controls were genotyped by Real Time-PCR and subjected
to biostatistic analysis using chi-square test and evaluation
of ORs. The potential pathogenetic impact of the associated
genes was then investigated by bioinformatic tools. Materials and Methods: 19 SNPs in 15 candidate genes
were genotyped in 782 psoriasis patients and 2000 controls
and their association with psoriasis was assessed in a case-
control study. Departures from HWE were tested using
Fischer exact test. Allelic and genotypic association tests
were performed by using either χ2 test or Fisher’s exact test,
then allelic and genotypic odds ratios with 95% confidence
intervals were calculated, the model was adjusted to
confounding factors, too. Association of inferred haplotypes
with psoriasis was assessed by a log-additive model. Pathway and network analysis was also carried out. Materials and Methods: 19 SNPs in 15 candidate genes
were genotyped in 782 psoriasis patients and 2000 controls
and their association with psoriasis was assessed in a case-
control study. Departures from HWE were tested using
Fischer exact test. Allelic and genotypic association tests
were performed by using either χ2 test or Fisher’s exact test,
then allelic and genotypic odds ratios with 95% confidence
intervals were calculated, the model was adjusted to
confounding factors, too. V. Caputo1, V. Errichiello1, C. Strafella1,2, A. Mazzotta3,
G. Novelli1, F. Sangiuolo1, E. Campione4, R. Cascella1,5,
E. Giardina1,6 V. Caputo1, V. Errichiello1, C. Strafella1,2, A. Mazzotta3,
G. Novelli1, F. Sangiuolo1, E. Campione4, R. Cascella1,5,
E. Giardina1,6 V. Caputo1, V. Errichiello1, C. Strafella1,2, A. Mazzotta3,
G. Novelli1, F. Sangiuolo1, E. Campione4, R. Cascella1,5,
E. Giardina1,6 V. Caputo: None. V. Errichiello: None. C. Strafella:
None. A. Mazzotta:
None. G. Novelli:
None. F. Sangiuolo: None. E. Campione: None. R. Cascella:
None. E. Giardina: None. 1Department of Biomedicine and Prevention, “Tor Vergata”
University, Rome, Italy, 2Emotest Laboratory, Pozzuoli, Italy,
3UOSD Dermatology, San Camillo Forlanini Hospital, Rome,
Italy, 4Department of Systems Medicine, Division of
Dermatology, University of Rome “Tor Vergata”, Rome, Italy,
5Department of Chemical Pharmaceutical and Biomolecular
Technologies, Catholic University “Our Lady of Good
Counsel” Laprakë, Rruga Dritan Hoxha, Tirana, Albania,
6Laboratory of Genomic Medicine UILDM, IRCCS Santa
Lucia Foundation, Rome, Italy P04.79C P04.79C
Evidence of common and differential genetic biomarkers
for Ps and PsA Evidence of common and differential genetic biomarkers
for Ps and PsA 126 J. del Picchia Skeletal abnormalities in SATB2-associated syndrome M. Rio1, M. Mouille1, S. Breton2, J. Souberbielle3, G. Maruani3,
A. Afenjar4, J. Amiel1,5, Y. Capri6, M. Fouillet7, A. Goldenberg8,
C. Michot1,9, C. Mignot4, L. Perrin6, A. Putoux10, C. Quelin11,
J. Van Gils12, V. Cormier Daire1,5,9 N. Guleray1, P. O. Simsek Kiper2, G. E. Utine2, K. Boduroglu2,
M. Alikasifoglu1 1Department of Medical Genetics, Hacettepe University
Faculty of Medicine, Ankara, Turkey, 2Department of Pediatric
Genetics, Department of Pediatrics, Hacettepe University
Faculty of Medicine, Ankara, Turkey 1Département de génétique, Hôpital Necker-Enfants Malades,
APHP, paris, France, 2Centre de référence maladies osseuses
constitutionnelles, Hôpital Necker-Enfants Malades, paris,
France, 3Laboratoire d'explorations fonctionnelles, Hôpital
Necker-Enfants Malades, APHP, paris, France, 4Département
de génétique, Hôpital Trousseau, APHP, paris, France,
5INSERM UMR1163, Institut Imagine, Université Paris
Descartes Sorbonne Paris Cité, Paris, France, 6Département
de génétique, Hôpital Robert debré, APHP, paris, France,
7Service de pédiatrie, CHU Lyon, Lyon, France, 8Département
de génétique, CHU Rouen, Rouen, France, 9Centre de
référence maladies osseuses constitutionnelles, Hôpital
Necker-Enfants Malades, Paris, France, 10Service de
génétique, CHU Lyon, Lyon, France, 11Service de génétique,
CHU Rennes, Rennes, France, 12service de génétique, CHU
Bordeaux, Bordeaux, France Introduction: Roberts/SC Phocomelia syndrome (MIM
268300,269000) is a rare autosomal recessive disorder
characterized by limb anomalies, craniofacial dysmorphism,
pre- and postnatal growth failure, developmental delay and
a variety of visceral anomalies. Roberts syndrome is part of
a spectrum named ‘’cohesinopathies’’ with varying phe-
notypic expression, and is caused by mutations in ESCO2. ESCO2 encodes a protein that establishes sister chromatid
cohesion during S phase. In this study, we report on 4 new
patients with previously unreported endocrine findings. Introduction: Roberts/SC Phocomelia syndrome (MIM
268300,269000) is a rare autosomal recessive disorder
characterized by limb anomalies, craniofacial dysmorphism,
pre- and postnatal growth failure, developmental delay and
a variety of visceral anomalies. Roberts syndrome is part of
a spectrum named ‘’cohesinopathies’’ with varying phe-
notypic expression, and is caused by mutations in ESCO2. ESCO2 encodes a protein that establishes sister chromatid
cohesion during S phase. In this study, we report on 4 new
patients with previously unreported endocrine findings. Material and Methods: Three patients clinically and
radiologically diagnosed with Roberts syndrome, were
screened for ESCO2 variations using Sanger sequencing
analysis
while
one
patient
diagnosed
with
FATCO
syndrome underwent whole exome sequencing. Material and Methods: Three patients clinically and
radiologically diagnosed with Roberts syndrome, were
screened for ESCO2 variations using Sanger sequencing
analysis
while
one
patient
diagnosed
with
FATCO
syndrome underwent whole exome sequencing. Analysis of the genetic background of psoriasis in a
Hungarian cohort Association of inferred haplotypes
with psoriasis was assessed by a log-additive model. Pathway and network analysis was also carried out. Results: rs12488457 (A/C, COL6A5), rs13081855 (G/T,
COL8A1) and rs2910164 (G/C, miR-146a) were associated
with both Ps [rs12488457: p = 2.97*10-9;, OR (C): 1.75,
CI95%: 1.44-2.13; rs13081855: p = 0.001, OR (T): 1.79,
CI95%:1.24-2.59; rs2910164: p=0.01, OR (G): 1.29,
CI95%:1.04-1.61] and PsA [rs12488457: p = 1.24*10-5,
OR (C): 2.46, CI95%: 2.03-2.97; rs13081855: p=9.06*10-
6, OR (T): 2.17, CI95%:1.53-3.06; rs2910164: p=0.04, OR
(G):
1.23
CI95%:1.0-1.51],
while
rs3812111
(A/T,
COL10A1) showed significant association with PsA only
[p = 0.008, OR (T):1.29, CI95%:1.07-1.57]. The bioinfor-
matic analysis reported that COL6A5, COL8A1 and miR-
146a may be potentially involved in alteration of the
proliferation, neovascularization and inflammation path-
ways leading to Ps and PsA. On the other hand, COL10A1
was implicated in bone metabolism mechanisms which are
dysregulated in PsA. Results: rs12488457 (A/C, COL6A5), rs13081855 (G/T,
COL8A1) and rs2910164 (G/C, miR-146a) were associated
with both Ps [rs12488457: p = 2.97*10-9;, OR (C): 1.75,
CI95%: 1.44-2.13; rs13081855: p = 0.001, OR (T): 1.79, Results: 12 SNPs in 11 candidate genes showed
significant association with psoriasis susceptibility. Six
SNPs have protective effect and six confer increased risk for
psoriasis revealing modest effect on phenotype. The same
variants were found to be significantly associated with the
early onset of psoriasis. Protecting and sensitizing haplo-
types were identified on chromosomes 1, 5, 6 and 7. Conclusion: Network analysis of disease associated
genes
-
LCE3D,
PLCL2,
IL12B,
TNF,
TRAF3IP2,
TNFAIP3, IL6, PON1, FTO and SPATA2 - identified six
functional
modules
centered
around
TNF,
TNFAIP3,
IL12B/IL6, SPATA2, PON1 and PLCL2. These results
underlie the importance of TNF and NFκB signaling
pathways, cytokine production and inflammatory response
in psoriasis patients and hints connections to obesity,
asthma, atherosclerosis, and IBD among others. Conclusion:
COL6A5,
COL8A1,
miR-146a
and
COL10A1 represent new susceptibility biomarkers for Ps
and PsA, reflecting thereby the existence of differential
mechanisms
underlying
the
etiopathogenesis of these
pathologies. Abstracts from the 51st European Society of Human Genetics Conference: Posters 127 A. Penyige: None. E.A. Janka: None. I. Sawhney:
None. A. Csordás: None. Z. Kovács: None. A. Szegedi:
None. P. Holló: None. S. Fiatal: None. R. Ádány: None. D. Törőcsik: None. É. Remenyik: None. patients
with
Roberts
syndrome
might
be
evaluated
endocrinologically during their follow-up. N. Guleray: None. P.O. Simsek Kiper: None. G.E. Utine:
None. K. Boduroglu:
None. M. Alikasifoglu: None. Skeletal abnormalities in SATB2-associated syndrome The SATB2-associated syndrome (SAS) has been recently
proposed as a new clinically recognizable syndrome that
results from deleterious alterations of the SATB2 gene in
humans. The characteristic features are intellectual dis-
ability with absent or limited speech development, beha-
vioral problems, craniofacial abnormalities with cleft or
high palate, and dental abnormalities. About fifty patients
have been reported in the literature. Skeletal abnormalities
such as tibial bowing, bone fragility or osteoporosis have
been reported in patients, suggesting a higher frequency of
skeletal complications in SAS. The role of SATB2 in the
regulation of skeletal development has recently been
demonstrated. Indeed, SATB2 is a regulator of the Osx
promoter, itself responsible for the differentiation of
mesenchymal cells into osteoblasts. In this context, we
decided
to
perform
a
non-interventional,
multicentric
research to understand mechanisms that lead to osteopenia
in SAS patients. For all patients, we carried out a complete
phosphocalcic assessment including markers of bone for-
mation and bone resorption. We also reviewed skeletal
radiographies and bone densitometry when available. We
report here our data of 23 patients with SAS (21 with Results:
Sanger
sequencing
revealed
3
pathogenic
mutations, including a novel mutation identified in a
conserved region of ESCO2. While the patient with novel
c.1433_1437delCTTTT mutation had severe intellectual
disability, the other two patients with previously reported
c.1131+1G>A
splice
donor
mutation
presented
with
generalized decreased bone mineral density and hypothyr-
oidism, respectively. Finally in the patient with clinical
findings resembling FATCO syndrome, WES analysis
revealed c.879_880delAG. In this individual premature
adrenarche without significant hormonal imbalance was
found and this finding was attributed to the Roberts
syndrome. Conclusion: This study demonstrates that premature
adrenarche and hypothyroidism may accompany Roberts
syndrome. ESCO2 expression in thyroid and adrenal glands
as well as the previous description of thyroid agenesis,
hypothyroidism and osteoporosis in another ‘’cohesino-
pathy’’ Cornelia de Lange syndrome further support this
observation. This study provides further evidence that J. del Picchia 128 disease causing mutations in known short stature genes in
19% of patients. We now performed exome sequencing in
211 clinically characterized families without mutations in
known short stature genes to identify potential candidate
genes. Variants were assessed for a potential effect on the
gene and its product using multiple lines of evidence
including expression in chondrocytes. We found 21 genes
mutated in at least two patients. Skeletal abnormalities in SATB2-associated syndrome Six were especially strong
candidates (CPZ, EDEM3, FBRS, RASA3, SLC7A8 and
USP45) with mutations in at least two patients. The
resulting proteins participate in protein degradation, tran-
scriptional regulation and protein transport. One out-
standing candidate gene, RASA3, is a member of the RAS-
MAPK pathway. Mutations in other members of this
pathway are known to cause diseases of the RASopathy
complex, with short stature as a main symptom. The 2
patients carrying de novo mutations in RASA3 share some
clinical characteristics with other RASopathy patients. We
recently identified a third patient with a de novo mutation in
RASA3. As GH treatment is discussed for some RASopathy
patients, successful application in one of our patients sug-
gested that GH might be indicated in the treatment of these
patients. In conclusion, using exome analyses in patients
with idiopathic short stature, we found 21 strong candidate
genes in 40 patients. Thus, Exome sequencing can therefore
be of great value to identify the underlying genetic cause in
these individuals. intragenic pathogenic variant and 2 with a microdeletion)
aged from 4 to 33 years. A clinically significant fracture
history was present in 10 patients. Osteopenia was present
in skeletal radiographies of 10/10 patients. Low mineral
density ascertained by osteodensitometry was present in 4/
10 patients. We hope that ungoing results will allow better
clinical management of SAS patients. M. Rio: None. M. Mouille: None. S. Breton: None. J. Souberbielle: None. G. Maruani: None. A. Afenjar:
None. J. Amiel: None. Y. Capri: None. M. Fouillet: None. A. Goldenberg: None. C. Michot: None. C. Mignot:
None. L. Perrin: None. A. Putoux: None. C. Quelin:
None. J. Van Gils: None. V. Cormier Daire: None. P04.84D
SJOGREN-LARSSON SYNDROME IN TWO SISTERS
WITH AN INTRONIC VARIANT OF ALDH3A2 GENE:
CLINICAL FINDINGS P04.84D
SJOGREN-LARSSON SYNDROME IN TWO SISTERS
WITH AN INTRONIC VARIANT OF ALDH3A2 GENE:
CLINICAL FINDINGS F. Fernandez1, G. Pi2, M. Carmona1, L. Pedrola1, M. Evole3,
A. Zuñiga1, J. Cervera1 F. Fernandez1, G. Pi2, M. Carmona1, L. Pedrola1, M. Evole3,
A. Zuñiga1, J. Cervera1 1Unidad de Genética. HUP La Fe., Valencia, Spain, 2Serv. Pediatria. Hospital de la Ribera., Alzira, Spain, 3Serv. Dermatología. HUP La Fe., Valencia, Spain 1Unidad de Genética. HUP La Fe., Valencia, Spain, 2Serv. Pediatria. Hospital de la Ribera., Alzira, Spain, 3Serv. Dermatología. HUP La Fe., Valencia, Spain Short stature affects 3% of the population. We recently
demonstrated that exome sequencing is able to identify Introduction: Sjögren-Larsson syndrome is a neurocuta-
neous disease due to an inborn anomaly of lipid metabolism Identification of novel candidate genes for idiopathic short
stature using whole exome sequencing Identification of novel candidate genes for idiopathic short
stature using whole exome sequencing C. T. Thiel1, N. N. Hauer1, C. Vogl1, R. Ahmadian2, P. S. Dhandapany3, B. Popp1, C. Büttner1, S. Ube1, H. Sticht4,
F. Ferrazzi1, A. B. Ekici1, A. de Luca5, E. Schöller1,
S. Schuhmann1, K. E. Heath6, A. Hisado-Oliva6, P. Klinger7,
S. Boppudi8, J. Kelkel9, A. M. Jung9, C. Kraus1, U. Trautmann1,
A. Wiesener1, K. Kutsche10, A. Rauch11, D. Wieczorek12,
T. Rohrer9, M. Zenker8, H. Dörr13, A. Reis1 1Institute of Human Genetics, FAU Erlangen-Nürnberg,
Erlangen, Germany, 2Institute of Biochemistry and Molecular
Biology II, Medical Faculty, Heinrich-Heine University,
Düsseldorf, Germany, 3The Mindich Child Health and
Development Institute Icahn School of Medicine at Mount
Sinai, New York, NY, United States, 4Institute of Biochemistry
FAU Erlangen-Nürnberg, Erlangen, Germany, 5Mendel
Laboratory Casa Sollievo della Sofferenza Hospital IRCCS
San Giovanni Rotondo, Rome, Italy, 6Institute of Medical and
Molecular Genetics and Skeletal dysplasia Multidisciplinary
Unit Hospital Universitario La Paz Universidad Autónoma de
Madrid IdiPAZ and CIBERER, Madrid, Spain, 7Department of
Orthopaedic Rheumatology, FAU Erlangen-Nürnberg,
Erlangen, Germany, 8Institute of Human Genetics Otto‐von‐
Guericke University Magdeburg, Magdeburg, Germany,
9Division of Pediatric Endocrinology Department of Pediatrics
and Neonatology Saarland University Hospital, Homburg/
Saar, Germany, 10Institute of Human Genetics University
Medical Center Hamburg-Eppendorf, Hamburg, Germany,
11Institute of Medical Genetics University of Zurich, Zürich,
Switzerland, 12Institute of Human-Genetics University
Duesseldorf, Duesseldorf, Germany, 13Department of
Pediatrics and Adolescent Medicine FAU Erlangen-Nürnberg,
Erlangen, Germany C.T. Thiel: None. N.N. Hauer: None. C. Vogl: None. R. Ahmadian: None. P.S. Dhandapany: None. B. Popp:
None. C. Büttner: None. S. Ube: None. H. Sticht: None. F. Ferrazzi: None. A.B. Ekici: None. A. de Luca: None. E. Schöller: None. S. Schuhmann: None. K.E. Heath:
None. A. Hisado-Oliva: None. P. Klinger: None. S. Boppudi: None. J. Kelkel: None. A.M. Jung: None. C. Kraus: None. U. Trautmann: None. A. Wiesener: None. K. Kutsche: None. A. Rauch: None. D. Wieczorek: None. T. Rohrer: None. M. Zenker: None. H. Dörr: None. A. Reis: None. P04.84D
SJOGREN-LARSSON SYNDROME IN TWO SISTERS
WITH AN INTRONIC VARIANT OF ALDH3A2 GENE:
CLINICAL FINDINGS
F. Fernandez1, G. Pi2, M. Carmona1, L. Pedrola1, M. Evole3,
A. Zuñiga1, J. Cervera1
1Unidad de Genética. HUP La Fe., Valencia, Spain, 2Serv. Pediatria. Hospital de la Ribera., Alzira, Spain, 3Serv. Dermatología. HUP La Fe., Valencia, Spain Abstracts from the 51st European Society of Human Genetics Conference: Posters 129 Haploinsufficiency of the transcription factor Short Stature
Homeobox (SHOX) manifests as a spectrum of clinical
phenotypes, ranging from disproportionate short stature and
Madelung deformity to isolated short stature. Here, we
describe five infants with molecularly confirmed diagnoses
of SHOX haploinsufficiency who presented in-utero with
short long bones during routine antenatal scanning from as
early as 19 weeks gestation. Other fetal growth parameters
were normal. The molecular basis of SHOX haploinsuffi-
ciency was distinct in each case. In four cases SHOX hap-
loinsufficiency
was
inherited
from
a
previously
undiagnosed parent. In our de novo case, SHOX hap-
loinsufficiency reflected the formation of a derivative sex
chromosome during paternal meiosis. Final adult height in
the SHOX deficient parents ranged from -1.9 to -1.2 SDS. All affected parents had disproportionately short limbs and
two affected mothers had bilateral Madelung deformity. To
our knowledge, SHOX haploinsufficiency has not pre-
viously been reported to present in-utero. Our experience
illustrates that SHOX deficiency should form part of the
differential diagnosis of fetal short long bones and suggests
a low threshold for genetic testing. This should be parti-
cularly targeted at, but not limited to, families with a history
of features suggestive of SHOX deficiency. Data on the
postnatal growth of our index cases is presented which
demonstrates that antenatal presentation of SHOX hap-
loinsufficiency is not indicative of severe postnatal growth
restriction. Early identification of SHOX deficiency will
enable accurate genetic counselling reflecting a good post-
natal outcome and facilitate optimal initiation of growth
hormone therapy. and characterized by congenital ichthyosis, intellectual
deficit and spasticity. Half of the patients approximately are
not able to walk. It is due to mutations of the ALDH3A2
gene (17p11.2), which encodes the enzyme required in the
oxidation of fatty alcohols in fatty acids. Transmission is
autosomal recessive. They usually
live to
adulthood
requiring care. Neurological symptoms and intellectual
deficit do not evolve after puberty. Early symptomatology
suggests more serious involvement. GEN: ALDH3A2 gg
Case report: Our index case was a 20-year-old female
patient with neurological problems (seizures and spasticity)
and congenital bone malformations. She presented a dry,
rough, and scaly with a brownish or yellowish tone skin in
the trunk and extremities, hyperpigmented hyperkeratotic
plaques predominantly in flexures and abundant scalp
scaling. P04.85A SHOX haploinsufficiency presenting with isolated short
long bones in the second and third trimester She had a 15-year-old sister with the same
symptomatology, but who also has a spastic, scissor gait,
mental retardation and most severe skin lesions. A genetic
study was carried out, detecting the variant c.471 + 1delG
in the ALDH3A2 gene in homozygous state. This variant is
described as pathological with an autosomal recessive
pattern of inheritance, which confirms at the genetic level
the clinical suspicion of SJOGREN-LARSSON SYN-
DROME. The variant detected is also present in sister of
the patient with the same clinical diagnosis but with a most
severe phenotype. Genetic confirmation is of great impor-
tance in these patients, due to the clinical follow-up they
require, and offers the possibility of assessing genetic
counseling. F. Fernandez: None. G. Pi: None. M. Carmona: None. L. Pedrola: None. M. Evole: None. A. Zuñiga: None. J. Cervera: None. S. Ramachandrappa: None. A. Kulkarni: None. H. Gandhi: None. C. Ellis: None. R. Hutt: None. L. Roberts:
None. R. Hamid: None. A. Papageorghiou: None. S. Mansour: None. P04.86B S. Ramachandrappa1, A. Kulkarni1, H. Gandhi2, C. Ellis3,
R. Hutt4, L. Roberts4, R. Hamid5, A. Papageorghiou6,
S. Mansour1 S. Ramachandrappa1, A. Kulkarni1, H. Gandhi2, C. Ellis3,
R. Hutt4, L. Roberts4, R. Hamid5, A. Papageorghiou6,
S. Mansour1 Functional missense and splicing variants in the retinoic
acid catabolizing enzyme CYP26C1 in idiopathic short
stature S. Mansour1 G. A. Rappold1, A. Montalbano1, L. Juergensen2, M. Fukami3,
C. T. Thiel4, N. H. Hauer4, R. Roeth1, B. Weiss1, Y. Naiki5,
T. Ogata6, D. Hassel2 1South West Thames Regional Genetics Unit, London, United
Kingdom, 2Department of Obstetrics and Gynaecology, Surrey
and Sussex Healthcare NHS Trust, Surrey, United Kingdom,
3Department of Obstetrics and Gynaecology, Epsom and St
Helier University Hospitals NHS Trust, Epsom, United
Kingdom, 4Department of Obstetrics and Gynaecology, Royal
Surrey County Hospital NHS Foundation Trust, Guildford,
United Kingdom, 5Department of Obstetrics and Gynaecology,
Croydon Health Services NHS Trust, Croydon, United
Kingdom, 6Fetal Medicine Unit, St George’s University of
London, London, United Kingdom G. A. Rappold1, A. Montalbano1, L. Juergensen2, M. Fukami3,
C. T. Thiel4, N. H. Hauer4, R. Roeth1, B. Weiss1, Y. Naiki5,
T. Ogata6, D. Hassel2 1Department of Human Molecular Genetics, Institute of
Human Genetics, Heidelberg University, Heidelberg,
Germany, 2Department of Internal Medicine III, Cardiology,
Heidelberg University, Heidelberg, Germany, 3Department of
Molecular Endocrinology, National Research Institute for
Child Health and Development, Tokyo, Japan, 4Institute of
Human Genetics, Friedrich-Alexander-Universität Erlangen-
Nürnberg, Erlangen, Germany, 5Division of Endocrinology J. del Picchia 130 for all pediatric subspecialties. The registry mainly included
patients with the genetics disorders of the skeleton. Age at
diagnosis, geographical region of referral, family history,
parental consanguinity, clinical diagnosis, molecular diag-
nosis
and
inheritance
patterns
when
available
were
reviewed
respectively. The
study
was
approved
by
Hacettepe University Noninterventional Clinical Research
Ethics Board (Ref No: GO 17/321). and Metabolism, National Center for Child Health and
Development, Tokyo, Japan, 6Department of Pediatrics,
Hamamatsu University School of Medicine, Hamamatsu,
Japan for all pediatric subspecialties. The registry mainly included
patients with the genetics disorders of the skeleton. Age at
diagnosis, geographical region of referral, family history,
parental consanguinity, clinical diagnosis, molecular diag-
nosis
and
inheritance
patterns
when
available
were
reviewed
respectively. The
study
was
approved
by
Hacettepe University Noninterventional Clinical Research
Ethics Board (Ref No: GO 17/321). Height is a complex quantitative trait with a high herit-
ability. Short stature is diagnosed when height is sig-
nificantly below the average of the general population for
that person´s age and sex. We have recently found that the
retinoic acid degrading enzyme CYP26C1 is a modifier for
SHOX deficiency phenotypes towards more severe clinical
manifestations. Here, we asked whether damaging variants
in CYP26C1 alone could lead to short stature. Clinical, demographic and nosologic characterisation of the
genetic disorders of the skeleton in Turkey: The skeletal
dysplasia registry Clinical, demographic and nosologic characterisation of the
genetic disorders of the skeleton in Turkey: The skeletal
dysplasia registry S. Mansour1 We per-
formed exome and Sanger sequencing to analyze 856
individuals with short stature where SHOX deficiency was
previously excluded. Three different damaging missense
variants and one splicing variant were identified in six
independent individuals; the functional significance of the
identified variants was tested in vitro or in vivo using
Zebrafish as a model. The genetic and functional data
reported here indicate that CYP26C1 represents a novel
gene underlying growth disorders and that damaging var-
iants in the absence of SHOX mutations can lead to short
stature. Results: The findings of a total of 884 patients were
reviewed. The overall consanguinity rate in our study is
52% with some regional differences. A comparison is made
in two time periods, 2005-2009 and 2009-2018. The
registry has enabled scientific contributions through clinical,
radiographic and molecular delineation of different clinical
diagnosis including identification of founder mutations in
FKBP10 in autosomal recessive OI and SFRP4 mutations in
Pyle disease. Additional patients with recently identified
genes including RSPRY1, EXTL3, XYLT2, EXOC6 and
PGAP3 were also diagnosed and registered during this time
period. Conclusions: The present study provides further informa-
tion about the clinical and nosological characteristics of the
genetic disorders of the skeleton in Turkey. Despite
limitations this registry still represents the first of its own
kind from Turkey and around, and is a useful tool not only
for the physicians but also for the families. G.A. Rappold:
None. A. Montalbano:
None. L. Juergensen: None. M. Fukami: None. C.T. Thiel: None. G.A. Rappold:
None. A. Montalbano:
None. L. Juergensen: None. M. Fukami: None. C.T. Thiel: None. G.A. Rappold:
None. A. Montalbano:
None. L. Juergensen: None. M. Fukami: None. C.T. Thiel: None. N.H. Hauer: None. R. Roeth: None. B. Weiss: None. Y. Naiki: None. T. Ogata: None. D. Hassel: None. N.H. Hauer: None. R. Roeth: None. B. Weiss: None. Y. Naiki: None. T. Ogata: None. D. Hassel: None. N.H. Hauer: None. R. Roeth: None. B. Weiss: None. Y. Naiki: None. T. Ogata: None. D. Hassel: None. P.O. Simsek-Kiper: None. G.E. Utine: None. E.Z. Taskiran: None. C. Kosukcu: None. U. Arslan: None. Y. Alanay:
None. M. Alikasifoglu:
None. K. Boduroglu: None. A Syrian patient with Steel syndrome due to compound
heterozygous COL27A1 mutations with hitherto
undescribed colobomas extending the clinical spectrum L. Pölsler1, B. Simma2, U. Schatz1, J. Zschocke1, S. Rudnik1 L. Pölsler1, B. Simma2, U. Schatz1, J. Zschocke1, S. Rudnik1 1Division of Human Genetics; Medical University Innsbruck,
Innsbruck, Austria, 2Department of Pediatrics and Adolescent
Medicine; Academic Teaching Hospital LKH Feldkirch,
Feldkirch, Austria 1Division of Human Genetics; Medical University Innsbruck,
Innsbruck, Austria, 2Department of Pediatrics and Adolescent
Medicine; Academic Teaching Hospital LKH Feldkirch,
Feldkirch, Austria Split hand-split foot malformation (SHFM) is a rare con-
dition that occurs in 1 in 8500-25000 newborns and
accounts for 15% of all limb reduction defects. SHFM is
heterogeneous and can be isolated, associated with other
malformations or syndromic. The mode of inheritance is
mostly autosomal dominant with incomplete penetrance,
but can be X-linked or autosomal recessive. Seven loci are
currently known: SHFM1 at 7q21.2q22.1 (DLX5 gene),
SHFM2 at Xq26, SHFM3 at 10q24q25, SHFM4 at 3q27
(TP63 gene), SHFM5 at 2q31 and SHFM6 as a result of
mutations in WNT10B (chromosome 12q13). Duplications
at 17p13.3 are seen in SHFM when isolated or associated
with long bone deficiency. Tandem genomic duplications at
chromosome 10q24 involving at least the DACTYLIN gene
are associated with SHFM3. No point mutation in any of the
genes residing within the region has been identified so far,
but duplication of exon 1 of the BTRC gene may explain the
phenotype, with likely complex alterations of gene regula-
tion mechanisms that would impair limb morphogenesis. We report on 32 new index cases identified by array-CGH
and/or by qPCR, including some prenatal ones, leading to
termination for the most severe. Twenty-three cases were
presenting with SHFM and 7 with monodactyly only. Two
had an overlapping phenotype. Additional findings were
identified in 5 (renal dysplasia, cutis aplasia, hypogonadism
and agenesis of corpus callosum with hydrocephalus). We
present their clinical and radiological findings and review
the literature on this rearrangement that seems to be one of
the most frequent cause of SHFM. Introduction: The combination of short stature, bilateral
congenital dislocation of the hip, carpal coalition, disloca-
tion of the radial head, cavus deformity, scoliosis, and
vertebral anomalies was first described in 1993 by Steel
et al. (OMIM #615155) in twenty-three children from
Puerto Rico. It is caused by deficient matrix protein col-
lagen 27 alpha 1 expressed in cartilage, skin, and tendons
and is inherited as an autosomal recessive trait. P04.88D Materials
and
Methods:
“The
Skeletal
Dysplasia
Registry” was established in 2005 at Hacettepe University
Pediatric Genetics Department, a tertiary reference center Materials
and
Methods:
“The
Skeletal
Dysplasia
Registry” was established in 2005 at Hacettepe University
Pediatric Genetics Department, a tertiary reference center Abstracts from the 51st European Society of Human Genetics Conference: Posters 131 11Service de Genetique, Nancy, French Guiana, 12Service de
Genetique, Hopital Robert Debre, Paris, France, 13Service de
Genetique, Nantes, France, 14American University, Beirut,
Lebanon, 15Service de Genetique, Angers, France,
16Department of Paediatrics, Montreal, QC, Canada,
17Laboratoire de Cytogenetique, Bois-Guillaume, France,
18Clinical Genetics Department, Saint George's Hospital,
London, United Kingdom, 19North West Genetics Service,
Harrow, United Kingdom, 20Laboratoire de genetique
chromosomique, Marseille, France, 21Clinical Genetics
Department, Saint George’s Hospital, London, United
Kingdom, 22Clinical Genetics Department, Great Ormond
Street Hospital, London, United Kingdom, 23Pathology
Department, Queen Elizabeth Hospital, Woolwich, United
Kingdom, 24CPDPN, CHU Lille, Lille, French Guiana,
25Service de chirurgie orthopedique, CHU Lille, Lille, France,
26CPDPN, CHU Lille, Lille, France 11Service de Genetique, Nancy, French Guiana, 12Service de
Genetique, Hopital Robert Debre, Paris, France, 13Service de
Genetique, Nantes, France, 14American University, Beirut,
Lebanon, 15Service de Genetique, Angers, France,
16Department of Paediatrics, Montreal, QC, Canada,
17Laboratoire de Cytogenetique, Bois-Guillaume, France,
18Clinical Genetics Department, Saint George's Hospital,
London, United Kingdom, 19North West Genetics Service,
Harrow, United Kingdom, 20Laboratoire de genetique
chromosomique, Marseille, France, 21Clinical Genetics
Department, Saint George’s Hospital, London, United
Kingdom, 22Clinical Genetics Department, Great Ormond
Street Hospital, London, United Kingdom, 23Pathology
Department, Queen Elizabeth Hospital, Woolwich, United
Kingdom, 24CPDPN, CHU Lille, Lille, French Guiana,
25Service de chirurgie orthopedique, CHU Lille, Lille, France,
26CPDPN, CHU Lille, Lille, France None. C. Baumann: None. A. David: None. C. Farra:
None. E. Colin: None. S. Jacquemont: None. A. Rossi:
None. S. Mansour: None. N. Ghali: None. A. Moncla:
None. N. Lahiri: None. J. Hurst: None. E. Pollina: None. C. Patch: None. A. Valat: None. A. Mezel: None. P. Bourgeot: None. S. Manouvrier-Hanu: None. None. C. Baumann: None. A. David: None. C. Farra: P04.88D Duplication of 10q24 locus: broadening the clinical and
radiological spectrum P. O. Simsek-Kiper1, G. E. Utine1, E. Z. Taskiran2, C. Kosukcu3,
U. Arslan4, Y. Alanay5, M. Alikasifoglu2, K. Boduroglu1 M. Holder-Espinasse1, A. Jamsheer2, F. Escande3, J. Andrieux3,
F. Petit4, A. Sowinska-Seidler2, M. Socha2, A. Jakubiuk-
Tomaszuk5, M. Gerard6, M. Matthieu-Dramard7, V. Cormier-
Daire8, A. Verloes9, A. Toutain10, G. Plessis6, P. Jonveaux11,
C. Baumann12, A. David13, C. Farra14, E. Colin15,
S. Jacquemont16, A. Rossi17, S. Mansour18, N. Ghali19,
A. Moncla20, N. Lahiri21, J. Hurst22, E. Pollina23, C. Patch1,
A. Valat24, A. Mezel25, P. Bourgeot26, S. Manouvrier-Hanu4 1Hacettepe University School of Medicine Pediatric Genetics
Unit, Ankara, Turkey, 2Hacettepe University School of
Medicine Medical Genetics Department, Ankara, Turkey,
3Hacettepe University Institute of Health Sciences,Department
of Bioinformatics, Ankara, Turkey, 4Hacettepe University
Institute of Public Health, Ankara, Turkey, 5Acıbadem
University Faculty of Medicine Department of Pediatric
Genetics, İstanbul, Turkey 1Clinical Genetics Department, Guy’s Hospital, London,
United Kingdom, 2Department of Medical Genetics, Poznan,
Poland, 3CHU Lille, Institut de Biochimie et Genetique
Moleculaire, Lille, France, 4CHU Lille, Clinique de Genetique
Guy Fontaine, Lille, France, 5Medical Genetics Unit,
Bialystok, Poland, 6Service de Genetique, Caen, France,
7Service de Genetique, Amiens, France, 8Institut Imagine,
Paris, France, 9Service de Genetique, Hopital Robert-Debre,
Paris, France, 10Service de Genetique, Tours, France, Introduction: We aimed to determine the clinical, demo-
graphic and nosologic characteristics of the genetic dis-
orders of the skeleton in Turkey over the past 13 years
(February 2005-February 2018). Transcriptome analysis of skin fibroblasts with dominant
negative COL3A1 mutations provides insights into the
molecular pathology of vascular Ehlers-Danlos syndrome B. Tüysüz1, N. Güneş1, G. Yeşil2, E. Özer1, D. Uludağ-Alkaya1,
D. Pehlivan3, J. Lupski3 B. Tüysüz1, N. Güneş1, G. Yeşil2, E. Özer1, D. Uludağ-Alkaya1,
D. Pehlivan3, J. Lupski3 N. Chiarelli, G. Carini, N. Zoppi, M. Ritelli, M. Colombi N. Chiarelli, G. Carini, N. Zoppi, M. Ritelli, M. Colombi 1İstanbul University, Cerrahpasa Medical Faculty, İstanbul,
Turkey, 2Bezmialem Vakif University, Department of Medical
Genetics,, İstanbul, Turkey, 3Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX,
United States 1İstanbul University, Cerrahpasa Medical Faculty, İstanbul,
Turkey, 2Bezmialem Vakif University, Department of Medical
Genetics,, İstanbul, Turkey, 3Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX,
United States Division of Biology and Genetics, Department of Molecular
and Translational Medicine, University of Brescia, Brescia,
Italy Division of Biology and Genetics, Department of Molecular
and Translational Medicine, University of Brescia, Brescia,
Italy Vascular Ehlers-Danlos syndrome (vEDS) is a dominant
inherited connective tissue disorder caused by mutations in
the COL3A1 gene encoding type III collagen (COLLIII), the
major expressed collagen in blood vessels and hollow
organs. The majority of COL3A1 causative variants are
glycine substitutions and in-frame splice mutations in the
triple helix domain that through a dominant negative effect
are associated with the severe clinical spectrum character-
ized by fragility of soft connective tissues with arterial and
organ ruptures. Herein, we performed gene expression
profiling in cultured skin fibroblasts from three patients with
different
structural
COL3A1
mutations. Transcriptome
analysis revealed significant expression changes of several
genes involved in maintenance of endoplasmic reticulum
(ER) homeostasis, COLLs folding, extracellular matrix
(ECM) organization, proteasome complex, and cell cycle
regulation. Protein analyses showed that aberrant COLLIII
expression causes the disassembly of many structural ECM
constituents, such as fibrillins, EMILINs, elastin, perlecan,
decorin, and versican, all playing a crucial role in the vas-
cular system. Furthermore, the altered distribution of the ER
marker protein disulfide isomerase PDI and the strong
reduction of the COLLs-modifying enzyme FKBP22 are
consistent with the disturbance of ER-related homeostasis
and COLLs post-translational modifications, indicated by
microarray. Our findings provide a picture of the gene
expression changes in vEDS skin fibroblasts and highlight
that
dominant
negative
mutations
in
COL3A1
affect
maturation and deposition into the ECM of several struc-
tural proteins crucial to the integrity of soft connective tis-
sues, and that ER dysfunction might play an important role
in the etiology of this severe vascular disorder. A Syrian patient with Steel syndrome due to compound
heterozygous COL27A1 mutations with hitherto
undescribed colobomas extending the clinical spectrum Causative
mutations in the gene COL27A1 have been identified pri-
marily as a possible founder effect in Puerto Ricans and in
only two more families, in one of which the patient also
presented hearing loss. Here we report a girl aged 9 years born to non-
consanguineous Syrian parents with characteristic features
of Steel syndrome (short stature, massive malalignment of
large joints, kyphoskoliosis, hearing loss) and matching
facial dysmorphism (large, laterally extended palpebral
fissures, arched eyebrows, flat midface, long philtrum, and
short nose with low hanging columella) who also showed
bilateral colobomas of the irides, retinae, and uveae with
unilateral affection of the macula, which have not been
described before. Her intelligence seemed normal, as was an
MRI of the brain. Results: Exome sequencing identified two novel com-
pound heterozygous variants in COL27A1: c.93del, p. (Phe32Leufs*71)
in
exon
2
and
c.3075del,
p. (Lys1026Argfs*33) in exon 26 in this child. Her parents
were confirmed heterozygous carriers. Conclusions: Our findings extend the clinical spectrum
of this exceptionally rare disorder and provide evidence of
developmental defects of the eye caused by COL27A1
mutations. M. Holder-Espinasse: None. A. Jamsheer: None. F. Escande: None. J. Andrieux: None. F. Petit: None. A. Sowinska-Seidler: None. M. Socha: None. A. Jakubiuk-
Tomaszuk: None. M. Gerard: None. M. Matthieu-
Dramard: None. V. Cormier-Daire: None. A. Verloes:
None. A. Toutain: None. G. Plessis: None. P. Jonveaux: Abstract ESHG 2018 Milano. Version 1.2 02.02.2018 Abstract ESHG 2018 Milano. Version 1.2 02.02.2018 Abstract ESHG 2018 Milano. Version 1.2 02.02.2018 132 J. del Picchia L. Pölsler: None. B. Simma: None. U. Schatz: None. J. Zschocke: None. S. Rudnik: None. L. Pölsler: None. B. Simma: None. U. Schatz: None. J. Zschocke: None. S. Rudnik: None. B. Tüysüz: None. N. Güneş: None. G. Yeşil: None. E. Özer: None. D. Uludağ-Alkaya: None. D. Pehlivan:
None. J. Lupski: None. N. Chiarelli: None. G. Carini: None. N. Zoppi: None.
M. Ritelli: None. M. Colombi: None. A. IJpma1,2, D. Heijsman1,3, H. T. Bruggenwirth1,3, D. Majoor-
Krakauer1,3 A. IJpma1,2, D. Heijsman1,3, H. T. Bruggenwirth1,3, D. Majoor-
Krakauer1,3 1Erasmus MC, Rotterdam, Netherlands, 2Clinical
Bioinformatics, Rotterdam, Netherlands, 3Department of
Clinical Genetics, Rotterdam, Netherlands Introduction: Aortopathies represent heterogeneous group
of rare inherited disorders with a variable phenotype ran-
ging form
of aortic aneurysm/dissection
with/without
associated cardiac valvular disease. We studied the dis-
tribution of variants within selected candidate genes in a
representative cohort of Czech paediatric-/adult patients. Introduction: Abdominal Aortic Aneurysm (AAA) has a
prevalence of 5% in the elderly population. An AAA occurs
when the aorta below the renal arteries expands to a dia-
meter of 3cm or more. In the Netherlands, each year
approximately 5000 AAA patients are hospitalized and
around 750 people die due to AAA rupture. Materials and Methods: Massively parallel sequencing
was performed in 120 unrelated individuals (average age
42,5 years) using a custom-made panel comprising either
136 or 229 cardiac/aortic conditions-related genes (Nimble-
Gen/Illumina). Detected variants were validated by Sanger
DNA sequencing and segregation analysis. In 40 “sequen-
cing-negative” cases CNVs in the FBN1, TGFBR1 and
TGFBR2 genes were examined by MLPA (MRC-Holland). Materials and Methods: Massively parallel sequencing
was performed in 120 unrelated individuals (average age
42,5 years) using a custom-made panel comprising either
136 or 229 cardiac/aortic conditions-related genes (Nimble-
Gen/Illumina). Detected variants were validated by Sanger
DNA sequencing and segregation analysis. In 40 “sequen-
cing-negative” cases CNVs in the FBN1, TGFBR1 and
TGFBR2 genes were examined by MLPA (MRC-Holland). Hypothesis: Approximately 20% of AAA patients are
familial and our hypothesis is that genetic predisposition is
a significant cause for abdominal aneurysm pathology. Our
goal is to identify the genes that play a role in the formation
of AAA. Hypothesis: Approximately 20% of AAA patients are
familial and our hypothesis is that genetic predisposition is
a significant cause for abdominal aneurysm pathology. Our
goal is to identify the genes that play a role in the formation
of AAA. Methods: Our study population consists of approxi-
mately 1250 AAA patients. So far we sequenced 548 of
these AAA patients. Complete Genomics whole genome
sequencing (WGS) was performed in 3 families (15
individuals)and whole exome sequencing (WES) on the
illumina
platform
using
Agilent
Haloplex
and
CRE
sureselect exome capturing technology was performed in
71 families (175 individuals) and 358 single familial AAA
patients. Burden analysis was used to identify genes
enriched in our AAA population. A. IJpma1,2, D. Heijsman1,3, H. T. Bruggenwirth1,3, D. Majoor-
Krakauer1,3 Results:
Pathogenic/likely
pathogenic
DNA
variant
(Class ≥4) were found in 10/120 (8.3%) cases, while
VUS (Class 3) were detected in 40/120 (33.3%) patients
comprising genes FBN1, NOTCH1, FBN2, MYH11 and
others. Majority of pathogenic variants were observed in
FBN1 (20.0%), while CNVs were not identified. Interest-
ingly, pathogenic variants were also observed in 7/120
(5,8%) patients within aortopathy-unrelated genes confer-
ring other cardiovascular risks. Conclusions: As expected majority of variants were
identified in connective tissue-related genes. The overall
lower variant detection rate corresponds to published data. The detection of pathogenic or potentially pathogenic
variants for other cardiac conditions (e.g. arrhythmias)
demonstrates the diagnostic usefulness of broader gene
panels. Segregation analysis together with clinical examina-
tion of positive cases increases the utility of DNA
sequencing,
thereby
underscores
the
multidisciplinary
character of our approach and usefulness of cooperating
with compliant at risk families. Supported by 00064203,
CZ.2.16/3.1.00/24022,
LD14073,
IGA
NT13770.15-
27682A and IKEM: 00023001; 9039. Results: We present the detailed workflow of the analysis
of the genomics data. We will discuss several candidate
genes identified such as the enrichment for variants we
identified in the COL4A2 gene. Results: We present the detailed workflow of the analysis
of the genomics data. We will discuss several candidate
genes identified such as the enrichment for variants we
identified in the COL4A2 gene. Conclusions: In 118 out of 512 families a variant in a
diagnostic AAA gene was found. Analysis of all genes in
the exome dataset led to the identification of several
candidate genes that show variants in more than one AAA
family and that have not been linked to AAA before. Conclusions: In 118 out of 512 families a variant in a
diagnostic AAA gene was found. Analysis of all genes in
the exome dataset led to the identification of several
candidate genes that show variants in more than one AAA
family and that have not been linked to AAA before. A. IJpma: None. D. Heijsman: None. H.T. Bruggen-
wirth: None. D. Majoor-Krakauer: None. A. IJpma: None. D. Heijsman: None. H.T. Bruggen-
wirth: None. D. Majoor-Krakauer: None. Transcriptome analysis of skin fibroblasts with dominant
negative COL3A1 mutations provides insights into the
molecular pathology of vascular Ehlers-Danlos syndrome Introduction: TRPV4 is a calcium permeable non-selective
cation channel expresses in different tissues. TRPV4 muta-
tions have been implicated in autosomal dominant diseases
of skeletal and peripheral nervous system. Here, we report
TRPV4 mutations in three patients with spondylometaphy-
seal dysplasia Kozlowski type (SMDK), metatropic dys-
plasia (MD) and scapuloperoneal spinal muscular atrophy
(SPSMA). Metod and Result: Patient-1 was a 3-year-old girl. She
had normal height, short trunk, lumbar scoliosis, general-
ized severe platyspondyly, left proximal femoral metaphysis
irregularity,
short
femoral
necks
and
delayed
carpal
ossification which were compatible with SMDK. Progres-
sive scoliosis, waiddling gait and metaphyseal irregularity
of right distal ulna and radial bones were developed with
aging. At 8.5 years of age her height was -2.5 SDS. Patient-
2, a 2-year-old boy had torticollis (noticed in 3rd month),
narrow chest with normal stature (-1.7SD), was diagnosed
as MD especially shortening of long tubular bones, tail-like
sacral
appendage
and
radiological
features
including
defective ossification of servical bodies, platyspondyly,
metaphyseal flaring, and delayed epiphyseal ossification. His height is -1.7 SD at 4.5 years of age. Heterozygous
TRPV4
mutations
c.1781G>A
and
c.2396C>G
were
detected by Sanger sequencing in Patient 1 and 2,
respectively. Patient-3, a 1-year-old boy had laryngomala-
cia, torticollis, hip dysplasia, muscle weakness, bilateral pes
equinovarus, and scoliosis. Mild platyspondyly and acet-
abular irregularity were present radiologically. Using exome
sequencing, we identified heterozygous novel mutation in
TRPV4 (c.806G>A). The patient was diagnosed SPSMA
phenotype. N. Chiarelli: None. G. Carini: None. N. Zoppi: None. M. Ritelli: None. M. Colombi: None. N. Chiarelli: None. G. Carini: None. N. Zoppi: None. M. Ritelli: None. M. Colombi: None. Conclusion: This study showed both neuromuscular
diseases and skeletal dysplasia due to TRPV4 mutation in
infantile period should be kept in mind. 133 Abstracts from the 51st European Society of Human Genetics Conference: Posters P05 Cardiovascular disorders P. Votypka1, A. Krebsova2, P. Norambuena1, V. Zoubkova1,
M. Vlckova1, M. Nemcikova1, M. Havlovicova1,
A. Puchmajerova3, M. Balascakova1, M. Macek, Jr.1 P05.01A
Genetic variants in familial abdominal aortic aneurysms 1Department of Biology and Medical Genetics, 2nd Faculty of
Medicine, Charles University and Motol, Prague, Czech
Republic, 2Department of Cardiology, Institute for Clinical and
Experimental Medicine, Prague, Czech Republic, 3GENNET,
Prague, Czech Republic MicroRNAs in Arrhythmogenic Cardiomyopathy: from
tissue-profile to circulating-signature 1Department of Cardiac, Thoracic and Vascular Sciences,
University of Padua, Padua, Italy, 2Department of Clinical and
Experimental Cardiology, University of Amsterdam,
Amsterdam, Netherlands M. Bueno Marinas1, R. Celeghin1, M. Cason1, E. Lazzarini1,
D. Paladin2, G. Thiene1, C. Basso1, K. Pilichou1 1Department of Cardiac, Thoracic and Vascular Sciences. University of Padua, Padua, Italy, 2Ditta Dr. Dino Paladin,
Padua, Italy Background. Arrhythmogenic cardiomyopathy (AC) is an
inherited myocardial disease characterized by fibro-fatty
replacement
of
the
myocardium
and
life-threatening
arrhythmias caused mainly by low penetrant mutations in
desmosome-encoding genes. The molecular mechanism
underlying disease pathogenesis is still unclear. To deter-
mine molecular pathways underlying disease onset, gene
expression profiling was performed on AC patients and
transgenic mice with a desmoglein-2 (dsg2) mutation. Background: Arrhythmogenic cardiomyopathy (AC) is a
clinically and genetically heterogeneous disease, char-
acterized by progressive myocardial fibro-fatty replacement
and high risk of sudden cardiac death. Half of AC patients
harbor private desmosomal gene mutations. MicroRNAs
(miRNA) have been associated with numerous pathophy-
siological conditions, as gene-expression regulatory mole-
cules. Their role in AC is largely unknown. Background: Arrhythmogenic cardiomyopathy (AC) is a
clinically and genetically heterogeneous disease, char-
acterized by progressive myocardial fibro-fatty replacement
and high risk of sudden cardiac death. Half of AC patients
harbor private desmosomal gene mutations. MicroRNAs
(miRNA) have been associated with numerous pathophy-
siological conditions, as gene-expression regulatory mole-
cules. Their role in AC is largely unknown. Methods. RNA-Seq was carried out, separately, on the
right (RV) and the left (LV) ventricle of 9 AC heart-
transplanted
patients
carrying
pathogenic
desmosomal
mutations paired with 6 age-matched nondiseased donors. RNA-Seq was carried out also on 8 transgenic mice over-
expressing NS-dsg2 mutation (TgNS) at 2 different age-
groups (<2weeks and >3weeks), to deduct genetic/epige-
netic interference-factors and on a paired age-matched
control group comprised 6 over-expressing wild type dsg2
(TgWt) and 2Wt mice. Methods: The study cohort comprised 59 genotype-
positive AC-subjects and 24 healthy controls (Ctrl). 84-
miRNA array analysis was carried out on frozen right-
ventricle myocardial tissue samples derived from 8 AC
heart-transplanted patients; 9 AC-whole blood samples, and
6 Ctrls. miRNA validation was performed by qPCR (ΔΔCt
method) on 42-AC and 18-Ctrl. Results: miRNA profiling on AC-tissue samples dis-
played a genotype-related profile showing 19 differentially
expressed miRNAs in PKP2 carriers, 15 in DSP carriers and
14 in DSG2 carriers. Differential gene expression analysis in Arrhythmogenic
Cardiomyopathy P05.02B P. Votypka: None. A. Krebsova: None. P. Noram-
buena: None. V. Zoubkova: None. M. Vlckova: None. M. Nemcikova: None. M. Havlovicova: None. A. Puchma-
jerova:
None. M. Balascakova:
None. M. Macek,
Jr.: None. Targeted massively parallel sequencing of a representative
cohort of Czech patients with various rare aortopathies
demonstrates the clinical utility of genetic testing and the
need for a multidisciplinary approach to at risk families P. Votypka: None. A. Krebsova: None. P. Noram-
buena: None. V. Zoubkova: None. M. Vlckova: None. M. Nemcikova: None. M. Havlovicova: None. A. Puchma-
jerova:
None. M. Balascakova:
None. M. Macek,
Jr.: None. 134 J. del Picchia M. Cason1, M. Bueno Marinas1, R. Celeghin1, E. Lazzarini1,
S. Rizzo1, K. Ludwig1, C. R. Bezzina2, C. A. Remme2, B. Bauce1,
G. Thiene1, C. Basso1, K. Pilichou1 MicroRNAs in Arrhythmogenic Cardiomyopathy: from
tissue-profile to circulating-signature A common signature was identified
between PKP2 and DSP carriers (PKP2/DSP profile),
different from DSG2 profile. In silico target prediction of
both profiles marked Hippo Signaling Pathway (p-value
1.6e-9 and 6.4e-6). Analysis of AC-tissue sample-data as a
unique group confirmed 26 miRNAs (AC-tissue profile)
with predicted targets in the AC pathway (p-value 0.01). AC-blood miRNA profiling showed a 14-miRNA signature,
of which 10 miRNAs were found also in AC-tissue profile. Results. 1136 and 822 differentially expressed genes
(DEGs) were respectively identified in the RV and LV of
AC patients. Further, 143 DEGs were identified comparing
TgNS<2weeks and TgNS>3weeks gene expression profil-
ing. Finally, 82 DEGs were shared comparing human and
murine (TgNS>3weeks) expression-profiling among which
genes most linked to the suppression of the canonical WNT/
β-catenin and the activation of TGF-β signaling pathways. However, 29 DEGs were identified in TgNS<2weeks
comparing them to age-matched controls (WT<2weeks
and TgWt < 2weeks). Conclusions: A genotype-related miRNA profile was
observed on AC-tissue samples, as to reflect clinical
variability. In addition, 10 miRNAs in common were
identified
between
AC-tissue
and
AC-blood
profiles,
demonstrating a specific AC-miRNA signature. In silico
analysis highlighted pathways involved in AC pathogenesis
demonstrating a key role of miRNAs in AC. Conclusions. Transcriptome profiling enabled the identifica-
tion of the culprit molecule aiding cell-cell contact detachment
under stress conditions and wound healing repair through
suppression of the WNT/β-catenin signalling pathway. Conclusions. Transcriptome profiling enabled the identifica-
tion of the culprit molecule aiding cell-cell contact detachment
under stress conditions and wound healing repair through
suppression of the WNT/β-catenin signalling pathway. M. Cason: None. M. Bueno Marinas: None. R. Celeghin: None. E. Lazzarini: None. S. Rizzo: None. K. Ludwig: None. C.R. Bezzina: None. C.A. Remme: None. B. Bauce: None. G. Thiene: None. C. Basso: None. K. Pilichou: None. M. Bueno Marinas: None. R. Celeghin: None. M. Cason: None. E. Lazzarini: None. D. Paladin: None. G. Thiene: None. C. Basso: None. K. Pilichou: None. M. Bueno Marinas: None. R. Celeghin: None. M. Cason: None. E. Lazzarini: None. D. Paladin: None. G. Thiene: None. C. Basso: None. K. Pilichou: None. J. Ramírez1, S. van Duijvenboden2, I. Ntalla1, B. Mifsud1, H. R.
Warren1, E. Tzanis1, M. Orini3, A. Tinker1, P. D. Lambiase2,
P. B. Munroe1 P05.06B The effect of the expression level of tissue inhibitor of
metalloproteinase-3 on the development of atherosclerosis
in patients with myocardial infarction Differential gene expression analysis in Arrhythmogenic
Cardiomyopathy Differential gene expression analysis in Arrhythmogenic
Cardiomyopathy Abstracts from the 51st European Society of Human Genetics Conference: Posters 135 G. Celebi1, F. Guclu-Geyik1, D. Ozsoy2, C. Yildiz2, M. Yildiz3,
D. Oksen3, E. Komurcu-Bayrak1 1William Harvey Research Institute, London, United Kingdom,
2Institute of Cardiovascular Science, University College
London, London, United Kingdom, 3Barts Heart Centre, St
Bartholomews Hospital, London, United Kingdom 1Aziz Sancar Institute of Experimental Medicine,Istanbul
University, Istanbul, Turkey, 2Institute of Cardiology,Istanbul
University, Istanbul, Turkey, 3Anesthesiology and
Reanimation,Istanbul University Cardiology Institute, Istanbul,
Turkey Introduction: Reduced heart rate (HR) responses to exer-
cise (ΔHRex) and to recovery (ΔHRrec) are associated with
higher cardiovascular mortality rates, possibly due to
abnormalities in autonomic balance. We aimed to discover
single-nucleotide
polymorphisms
associated
with
both
indices and to identify associated biological pathways. Introduction: Reduced heart rate (HR) responses to exer-
cise (ΔHRex) and to recovery (ΔHRrec) are associated with
higher cardiovascular mortality rates, possibly due to
abnormalities in autonomic balance. We aimed to discover
single-nucleotide
polymorphisms
associated
with
both
indices and to identify associated biological pathways. TIMP3, a member of the Tissue Inhibitors of Metallopro-
teinases (TIMPs) binds to components of the extracellular
matrix and forms insoluble complex. TIMP3 has a crucial
regulatory role in adipose tissue. Our aim is to reveal the
relationship between TIMP3 expression and atherosclerosis
pathogenicity in peri-coronary epicardial adipose tissue
(EAT) and circulating leukocytes of patients with myo-
cardial infarction (MI). Methodologically, the expression
levels of TIMP3 were investigated in patients with MI (n
= 46) who had atherosclerosis severity determined by
SYNTAX and GENSINI scores that compared with the
control group (n = 24). The expression levels of TIMP3 in
the leukocytes (n = 69) and peri-coronary EAT (n = 23)
obtained from coronary artery bypass graft surgery using
qRT-PCR. The expression results were analyzed using the
comparative CT method, and results were statistically
evaluated. Previously unpublished findings showed that
TIMP3 expression levels were significantly lower in post-
mortem advanced atherosclerotic plaques than in normal
arteries (p < 0.05) and that TIMP-3 protein was present in
plaque enriched with macrophages of the coronary artery
sections. P05.06B Bioinformatics analyses highlighted neural
development and adrenergic modulation by the autonomic
nervous system pathways. Conclusion: Our results demonstrate that ΔHRex and
ΔHRrec are genetically modulated. Our biological findings
support the potential link between genetics and autonomic
modulation and highlight several plausible candidate genes
for both traits. Future studies will confirm the contributions
of our identified genetic variants to cardiovascular risk. J. Ramírez: None. S. van Duijvenboden: None. I. Ntalla: None. B. Mifsud: None. H.R. Warren: None. E. Tzanis: None. M. Orini: None. A. Tinker: None. P.D. Lambiase: None. P.B. Munroe: None. G. Celebi: None. F. Guclu-Geyik: None. D. Ozsoy:
None. C. Yildiz: None. M. Yildiz: None. D. Oksen: None. E. Komurcu-Bayrak: None. M. Sinitsky1,2, E. Velikanova1, D. Shishkova1, M. Asanov1,
A. Ponasenko1, A. Kutikhin1 P05.08D Gene expression signature in human immortalized venous
endothelial cells, human coronary artery and human
internal thoracic artery endothelial cells exposed to
different types of mineral-organic nanoparticles P05.06B In this study, it was determined that expressions of
TIMP3 increased 1.25 fold in peri-coronary EAT and
decreased 4.5 fold in circulating leukocytes as compared to
control samples (p > 0.05). The data were evaluated in detail
according to conventional risk factors. In conclusion,
TIMP3 expression levels in leukocytes and fat tissue
affecting the development of atherosclerosis were evaluated
in patients with MI. These analyses are still ongoing in more
patient samples. This study was supported by Scientific
Research Projects Coordination Unit of Istanbul University
(Project numbers:28473 and 21496) Methods: A total of 67,257 participants in an exercise
test from the UK Biobank study were included. We
calculated differences in HR at peak exercise (ΔHRex)
and at 1-minute post-peak exercise (ΔHRrec) with respect
to resting HR. Next, we randomly divided participants
into discovery (N = 40,000) and replication (N = 27,257)
cohorts and we performed a genome-wide association
study (GWAS) for each trait in the discovery dataset and
validated the findings in the replication cohort. We finally
conducted a combined meta-analysis of GWAS using the
full cohort for both traits. Methods: A total of 67,257 participants in an exercise
test from the UK Biobank study were included. We
calculated differences in HR at peak exercise (ΔHRex)
and at 1-minute post-peak exercise (ΔHRrec) with respect
to resting HR. Next, we randomly divided participants
into discovery (N = 40,000) and replication (N = 27,257)
cohorts and we performed a genome-wide association
study (GWAS) for each trait in the discovery dataset and
validated the findings in the replication cohort. We finally
conducted a combined meta-analysis of GWAS using the
full cohort for both traits. Results: We robustly validated six and eight independent
SNPs for ΔHRex and ΔHRrec, respectively. The combined
analysis revealed a further eight and seven SNPs for each
respective trait that were genome-wide significant (P <
5x10-8). In total, 14 and 15 SNPs were identified for ΔHRex
and ΔHRrec, respectively, with eight SNPs being common
across traits. Bioinformatics analyses highlighted neural
development and adrenergic modulation by the autonomic
nervous system pathways. Results: We robustly validated six and eight independent
SNPs for ΔHRex and ΔHRrec, respectively. The combined
analysis revealed a further eight and seven SNPs for each
respective trait that were genome-wide significant (P <
5x10-8). In total, 14 and 15 SNPs were identified for ΔHRex
and ΔHRrec, respectively, with eight SNPs being common
across traits. 1Research Institute for Complex Issues of Cardiovascular
Diseases, Kemerovo, Russian Federation, 2Federal Research P05.07C
Identification of novel loci for heart rate response to
exercise and recovery P05.07C
Identification of novel loci for heart rate response to
exercise and recovery M. Sinitsky1,2, E. Velikanova1, D. Shishkova1, M. Asanov1,
A. Ponasenko1, A. Kutikhin1 1Research Institute for Complex Issues of Cardiovascular
Diseases, Kemerovo, Russian Federation, 2Federal Research 136 J. del Picchia Genetic
testing
for
inherited
cardiomyopathies
has
improved in the last decade using next generation sequen-
cing (NGS). Although CNV of large segments of DNA may
significantly affect transcription and translation of cardio-
myopathic genes, these abnormalities remain often unrec-
ognized even with the use of NGS. Center of Coal and Coal Chemistry, Kemerovo, Russian
Federation Center of Coal and Coal Chemistry, Kemerovo, Russian Introduction: Mineral-organic nanoparticles (bions) are a
result of increasing concentration of precipitating ions or
failure in the mechanism of their excretion from human
body. It was found that the atherosclerosis and heart valves
calcification risk factors are similar to ones of bions for-
mation. This indicates that bions play role in the patho-
genesis of cardiovascular calcification. We have performed whole exome sequencing in a cohort
of 323 patients with dilated cardiomyopathy. We used a
read-depth coverage strategy to call CNVs from the short-
read sequence data. Detected CNVs were then validated by
q-PCR, RT-PCR and western blot analysis. The prevalence
of major CNVs in the cohort was 2%. We have found large
deletions in DMD, LAMP2, FLNC and MYH7 genes, which
were predicted to cause major structural and functional
abnormalities of the affected genes. The corresponding
pedigrees and clinical phenotypes will be presented. Assessment of CNVs with whole exome sequencing
elucidates genetic architecture in a substantial proportion
of patients with dilated cardiomyopathy. It should be a
routine part of NGS bioinformatics. Supported by the
research grant: AZV-MZ 15-27682A and IKEM 00023001. L. Piherova: None. F. Majer: None. A. Krebsova: Materials and Methods: Confluent cultures of human
immortalized venous endothelial cells (EA.hy 926), human
coronary artery (HCAEC) and human internal thoracic
artery endothelial cells (HITAEC) exposed to different
types of bions (magnesium phosphate bions [MFB],
spherical calcium phosphate bions [SCFB], and needle-
like calcium phosphate bions [ICPB]) were used in this
study. Expression of LDLR, VLDLR, SCARF1, NOS3,
PXDN genes was evaluated by RT-qPCR. Results were
normalized by three housekeeping genes (ACTB, GAPDH,
B2M). Expression level was calculated by Pfafflmethod. Results: 2-fold decreasing of SCARF1 expression was
detected in EA.hy 926 culture exposed to SCFB. In
HCAEC culture we found no significant differences in
gene expression signature. Rare variants in cardiomyopathy related genes - a
Portuguese cohort population Rare variants in cardiomyopathy related genes - a
Portuguese cohort population A. M. Coutinho1, J. Tavares1, M. Carmo-Fonseca2,
D. Antunes1,3 A. M. Coutinho1, J. Tavares1, M. Carmo-Fonseca2,
D. Antunes1,3 Conclusions: Exposure by mineral-organic nanoparticles
can lead to changing in gene expression signature in
different types of endothelial cells depending on type of
bions. HITAEC are more sensitive to such exposure. The
reported study was funded by Russian Foundation for Basic
Research according to the research project №17-04-00570. M. Sinitsky: None. E. Velikanova: None. D. Shish-
kova: None. M. Asanov: None. A. Ponasenko: None. A. Kutikhin: None. Conclusions: Exposure by mineral-organic nanoparticles
can lead to changing in gene expression signature in
different types of endothelial cells depending on type of
bions. HITAEC are more sensitive to such exposure. The
reported study was funded by Russian Foundation for Basic
Research according to the research project №17-04-00570. 1GenoMed - Diagnósticos de Medicina Molecular SA, Lisbon,
Portugal, 2Instituto de Medicina Molecular, Faculdade de
Medicina, Universidade de Lisboa, Lisbon, Portugal, 3Medical
Genetics Department, Hospital de Dona Estefânia, Centro
Hospitalar Lisboa Central, Lisbon, Portugal Introduction: With the latter advances in high throughput
sequencing technologies there was an increasing demand
for broader comprehensive Next-Generation Sequencing
(NGS) gene panels to screen for mutations in genetic dis-
eases with heterogeneous etiology. In cardiology, genetic
testing has been routinely offered to patients to improve
prognosis through appropriate lifestyle and medical inter-
ventions. There are now 340 gene entries retrieved under
“cardiomyopathy” search in OMIM and 375 genes under
the HPO superclass “Abnormal myocardium morphology”
(HP:0001637). P05.07C
Identification of novel loci for heart rate response to
exercise and recovery In HITAEC culture the exposure
by all types of bions caused an increase in VLDLR gene
expression. HITAEC cultures exposed by all types of
mineral-organic
nanoparticles
are
characterized
by
enhanced
expression
of
all
studied
genes
compared
to HCAEC. L. Piherova: None. F. Majer: None. A. Krebsova:
None. P. Melenovska: None. V. Stranecky: None. T. Palecek: None. M. Kubanek: None. S. Kmoch: None. 1Research Unit for Rare Disease, Charles University, Prague,
Czech Republic, 2Department of Cardiology, Institute of
Clinical and Experimental Medicine, Prague, Czech Republic,
32nd Internal Clinic, Charles University, Prague, Czech
Republic P05.09A Assessment of CNVs in dilated cardiomyopathies by whole
exome sequencing P05.11C
Identification of gene mutations in pediatric
cardiomyopathy by whole exome sequencing P05.11C
Identification of gene mutations in pediatric
cardiomyopathy by whole exome sequencing 1Center of Genomic Medicine, University of Medicine and
Pharmacy “Victor Babes” Timisoara, Timisoara, Romania,
2Institute of Emergency for Cardiovascular Diseases "Prof.dr. C.C.Iliescu", University of Medicine and Pharmacy "Carol
Davila" Bucharest, Bucharest, Romania, 3Clinical Emergency
Hospital for Children “Louis Turcanu” Timisoara, University
of Medicine and Pharmacy “Victor Babes” Timisoara,
Timisoara, Romania, 4Emergency Clinical County Hospital,
Timisoara. Department of Pediatrics, University of Medicine
and Pharmacy “Victor Babes” Timisoara, Timisoara,
Romania, 5Department of Genetics, University of Medicine and
Pharmacy of Craiova, Craiova, Romania 1Center of Genomic Medicine, University of Medicine and
Pharmacy “Victor Babes” Timisoara, Timisoara, Romania,
2Institute of Emergency for Cardiovascular Diseases "Prof.dr. C.C.Iliescu", University of Medicine and Pharmacy "Carol
Davila" Bucharest, Bucharest, Romania, 3Clinical Emergency
Hospital for Children “Louis Turcanu” Timisoara, University
of Medicine and Pharmacy “Victor Babes” Timisoara,
Timisoara, Romania, 4Emergency Clinical County Hospital,
Timisoara. Department of Pediatrics, University of Medicine
and Pharmacy “Victor Babes” Timisoara, Timisoara,
Romania, 5Department of Genetics, University of Medicine and
Pharmacy of Craiova Craiova Romania K. Rojnueangnit1, B. Sirichongkolthong1, R. Wongwandee1,
T. Khetkham1, S. Noojarern2, A. Khongkraparn2,
D. Wattanasirichaigoon2 K. Rojnueangnit1, B. Sirichongkolthong1, R. Wongwandee1,
T. Khetkham1, S. Noojarern2, A. Khongkraparn2,
D. Wattanasirichaigoon2 1Thammasat University, Klong-Luang, Thailand, 1Thammasat University, Klong-Luang, Thailand,
2Ramathibodi Hospital, Mahidol University, Bangkok,
Thailand 2Ramathibodi Hospital, Mahidol University, Bangkok,
Thailand Introduction: Primary cardiomyopathy in children is rare
but serious condition with a high mortality rate. Hyper-
trophic and dilated cardiomyopathies are the most common
presentations. Etiology has been mainly idiopathic; how-
ever, with the use of next generation sequencing techniques,
it has been noted that up to nearly half of idiopathic
pediatric cases arose from a specific genetic mutation. Therefore, this study aimed to identify genetic causes of
primary unexplained cardiomyopathy. Romania, 5Department of Genetics, University of Medicine and
Pharmacy of Craiova, Craiova, Romania Romania, 5Department of Genetics, University of Medicine and
Pharmacy of Craiova, Craiova, Romania Congenital heart diseases are genetically heterogeneous. Targeted next-generation sequencing (NGS) can identify
the genetic causes in a significant proportion of the
population. Aim: We tested a targeted NGS specific gene panel in
patients, adults and children, with syndromic and non-
syndromic cardiac involvement. Materials and Methods: Children with primary unex-
plained cardiomyopathy, ranging from newborns to 20-year
olds, were recruited during March 2016 to May 2017 at
Thammasat University Hospital. Assessment of CNVs in dilated cardiomyopathies by whole
exome sequencing Conclusions: Although genetic analysis through com-
prehensive multigene NGS approach potentially increases
diagnostic rate, it also raises new challenges. Rare benign
specific population polymorphisms add extra difficulty
regarding pathogenicity classification of variants. Further
characterization
studies
of
specific
populations
could
improve classification variants algorithm, preferably within
public databases with the possibility to identify variants by
nationality. K. Rojnueangnit: None. B. Sirichongkolthong: None. R. Wongwandee: None. T. Khetkham: None. S. Noojar-
ern:
None. A. Khongkraparn:
None. D. Wattanasirichaigoon: None. Assessment of CNVs in dilated cardiomyopathies by whole
exome sequencing L. Piherova1, F. Majer1, A. Krebsova2, P. Melenovska1,
V. Stranecky1, T. Palecek3, M. Kubanek2, S. Kmoch1 L. Piherova1, F. Majer1, A. Krebsova2, P. Melenovska1,
V. Stranecky1, T. Palecek3, M. Kubanek2, S. Kmoch1 1Research Unit for Rare Disease, Charles University, Prague,
Czech Republic, 2Department of Cardiology, Institute of
Clinical and Experimental Medicine, Prague, Czech Republic,
32nd Internal Clinic, Charles University, Prague, Czech
Republic Methods: The study population comprised 150 unrelated
patients diagnosed with cardiomyopathies, namely Hyper-
trophic Cardiomyopathy (HCM), Dilated Cardiomyopathy
(DCM)
and
Arrhythmogenic
Right
Ventricular 137 Abstracts from the 51st European Society of Human Genetics Conference: Posters Cardiomyopathy (ARVC). Up to 57 genes associated with
cardiomyopathies were sequenced, mostly by NGS. Cardiomyopathy (ARVC). Up to 57 genes associated with
cardiomyopathies were sequenced, mostly by NGS. follow-up (hypocalcemia and pace-maker induced dilated
cardiomyopathy). 118 gene lists for cardiomyopathy were
analyzed in 12 included cases. In cases of syndromic
cardiomyopathy, specific genes were added to aid the
analysis, but none was detected. Pathogenic and likely
pathogenic mutations were identified in 5 patients: SOS1,
HRAS, TTN, FLNC and TXNRD2. follow-up (hypocalcemia and pace-maker induced dilated
cardiomyopathy). 118 gene lists for cardiomyopathy were
analyzed in 12 included cases. In cases of syndromic
cardiomyopathy, specific genes were added to aid the
analysis, but none was detected. Pathogenic and likely
pathogenic mutations were identified in 5 patients: SOS1,
HRAS, TTN, FLNC and TXNRD2. Results: Pathogenic variants were detected in 39 patients
and in further 50 patients it was found an uncertain
significance/likely pathogenic variant, namely novel var-
iants in 17 patients (11.3%). One novel variant was detected
in two unrelated DCM patients in the LMNA gene -
c.490G>A, p.(Asp164Asn), suggesting an increased fre-
quency in the Portuguese population. Conclusion: Our cohort demonstrated that 41% of our
cases were not actually idiopathic. In light of this revelation
and despite its high cost, genetic testing is also useful in
determining genetic risk in the family as well as helping to
predict the prognosis of the cardiomyopathy. Conclusions: Although genetic analysis through com-
prehensive multigene NGS approach potentially increases
diagnostic rate, it also raises new challenges. Rare benign
specific population polymorphisms add extra difficulty
regarding pathogenicity classification of variants. Further
characterization
studies
of
specific
populations
could
improve classification variants algorithm, preferably within
public databases with the possibility to identify variants by
nationality. Targeted Next-Generation Sequencing in Patients with
Non-syndromic and syndromic Congenital Heart Disease A.M. Coutinho: None. J. Tavares: None. M. Carmo-
Fonseca: None. D. Antunes: None. Targeted Next-Generation Sequencing in Patients with
Non-syndromic and syndromic Congenital Heart Disease A. Chirita-Emandi1, N. Andreescu1, R. Jurcut2, G. Doros3,
A. Popoiu3, A. Lacatusu4, A. Dobrescu5, M. Puiu1 P05.13A Targeted Next-Generation Sequencing in Patients with
Non-syndromic and syndromic Congenital Heart Disease P05.11C
Identification of gene mutations in pediatric
cardiomyopathy by whole exome sequencing The study was conducted with the
support of the RFBR (№16-04-00840\16). NA1C;FBN1;PTPN1;SOS1;BRAF;LPL;ABCC9
and
APOE genes. Twenty-five variants present in public
databases, with very rare allele frequency, have been
previously linked to cardiomyopathy or relevant syndromic
phenotypes. Twenty-four were novel mutations, currently
not found in public databases, of which 9 were classified as
VUS. Two patients carried pathogenic variants for two
diseases (heterozygotes in different genes), with possibly
synergistic deleterious effects. Conclusion: First-line targeted genetic NGS testing
identified
a
significant
variant
(pathogenic
or
likely
pathogenic) for the phenotype, in 51,5%(17/33) of the
cases with syndromic cardiac involvement, and in 40.9%
(18/44) of the cases with non-syndromic cardiomyopathies,
providing the opportunity for diagnostic, risk stratification
and prevention, along with genetic counselling. Funding: Development of Existing Infrastructure and
Creation of New Infrastructure POSCCE-A2-O2.2.1-2013-
1, Center of Genomic Medicine of the University of
Medicine and Pharmacy ‘Victor Babes’ Timisoara. A. Chirita-Emandi: None. N. Andreescu: None. R. Jurcut: None. G. Doros: None. A. Popoiu: None. A. Lacatusu: None. A. Dobrescu: None. M. Puiu: None. A. Chirita-Emandi: None. N. Andreescu: None. R. Jurcut: None. G. Doros: None. A. Popoiu: None. A. Lacatusu: None. A. Dobrescu: None. M. Puiu: None. I.A. Goncharova: None. M.S. Nazarenko: None. T.B. Pecherina: None. V.V. Kashtalap: None. N.B. Tara-
senko:
None. O.L. Barbarash:
None. V.P. Puzyrev: None. P05.11C
Identification of gene mutations in pediatric
cardiomyopathy by whole exome sequencing Complete patient history
and
physical
examination
data
were
collected
by
a
geneticist; cardiac examination and echocardiogram was
performed by pediatric cardiologists. Whole exome sequen-
cing was performed in all cases. Results: The patient cohort included 77 patients(52
males;28.4 ± 22.6years), 41 people with hypertrophic/
dilated cardiomyopathy, 3 with arrhythmogenic cardiomyo-
pathy, and 33 with syndromic cardiac involvement (includ-
ing RASopathies and fibrillinopathies). Amplicon libraries
for 174 related genes were generated using the TruSight-
Cardio®kit(Illumina,CA) and sequenced using the Illumina
MiSeq platform. Sequence data were processed using
ANNOVAR software. Thirty-six variants (30 missense, 4 Results: Fourteen patients were enrolled in our study: 8
cases were dilated type, and 6 were hypertrophic. Two were
excluded given identification causes during period of J. del Picchia 138 environment R. Isolate MI showed the association with
genes of various biological pathways: immune response
CD79A (rs3810153), IFNGR1 (rs17181457), endothelial
dysfunction
KIAA1462(rs3739998),
reparations
LIG1
(rs20579),
fibrogenesis
(ADAMDEC1(rs3765124). In
groups with risk factors, it was found that: MI and HT
associations with genes involved in fibrogenesis ITGA4
(rs1143674), ITGB5(rs6778643, rs1007856), ADAMDEC1
(rs3765124),
CDKN2BAS1(rs1333049)
and
immune
response IFNGR1 (rs17181457); MI, HT and DLE asso-
ciations with genes involved in fibrogenesis (ITGA4
(rs1143674), ITGB5(rs6778643), ADAMDEC1(rs3765124),
CDKN2BAS1(rs1333049)),
immune
response
IFNGR1
(rs17181457), homeostasis of glucose and low-density
lipoproteins (TAS 2R38(rs1726866), LDLR(rs2738446)). “Syntropy of cardiovascular continuum“ associations with
genes involved in fibrogenesis (CDKN2BAS1(rs1333049),
(MTAP(rs7023329)), endothelial dysfunction KIAA1462
(rs3739998), lipid metabolism APOA2(rs5082). In conclu-
sion, isolate MI and MI with risk factors had different
genetic susceptibility profiles. Cardiovascular continuum
comorbidity was characterized by genes involved in various
biological processes. The study was conducted with the
support of the RFBR (№16-04-00840\16). environment R. Isolate MI showed the association with
genes of various biological pathways: immune response
CD79A (rs3810153), IFNGR1 (rs17181457), endothelial
dysfunction
KIAA1462(rs3739998),
reparations
LIG1
(rs20579),
fibrogenesis
(ADAMDEC1(rs3765124). In
groups with risk factors, it was found that: MI and HT
associations with genes involved in fibrogenesis ITGA4
(rs1143674), ITGB5(rs6778643, rs1007856), ADAMDEC1
(rs3765124),
CDKN2BAS1(rs1333049)
and
immune
response IFNGR1 (rs17181457); MI, HT and DLE asso-
ciations with genes involved in fibrogenesis (ITGA4
(rs1143674), ITGB5(rs6778643), ADAMDEC1(rs3765124),
CDKN2BAS1(rs1333049)),
immune
response
IFNGR1
(rs17181457), homeostasis of glucose and low-density
lipoproteins (TAS 2R38(rs1726866), LDLR(rs2738446)). “Syntropy of cardiovascular continuum“ associations with
genes involved in fibrogenesis (CDKN2BAS1(rs1333049),
(MTAP(rs7023329)), endothelial dysfunction KIAA1462
(rs3739998), lipid metabolism APOA2(rs5082). In conclu-
sion, isolate MI and MI with risk factors had different
genetic susceptibility profiles. Cardiovascular continuum
comorbidity was characterized by genes involved in various
biological processes. P05.15C The association of genes of fibrogenesis to the development
of cardiovascular continuum comorbidity KRIT1 loss of function induces a chronic Nrf2-mediated
adaptive homeostasis that sensitizes cells to oxidative stress:
implication for Cerebral Cavernous Malformation disease I. A. Goncharova1,2, M. S. Nazarenko1,2, T. B. Pecherina2, V. V. Kashtalap2, N. B. Tarasenko1, O. L. Barbarash2, V. P. Puzyrev1 E. Trapani1, C. Antognelli2, S. Delle Monache3, A. Perrelli1,
C. Fornelli1, V. Benedetti1, S. Sarri1, G. Costantino1, F. Geddo1,
A. Zotta1, L. Goitre1, S. F. Retta1 1Scientific Research Institute of Medical Genetics, Tomsk,
Russian Federation, 2Research Institute for Complex Problems
of Cardiovascular Diseases, Kemerovo, Russian Federation 1Scientific Research Institute of Medical Genetics, Tomsk,
Russian Federation, 2Research Institute for Complex Problems
of Cardiovascular Diseases, Kemerovo, Russian Federation 1Department of Clinical and Biological Sciences, Orbassano,
TO, Italy, 2Department of Experimental Medicine, Perugia,
Italy, 3Department of Biotechnological and Applied Clinical
Sciences, L'Aquila, Italy The aim of this study was to assess the genetic structure of
cardiovascular continuum comorbidity. The study included
531 patients with myocardial infarction (MI) and 285
Russian inhabitants of Siberia. Рatients were divided into
groups: 113 MI patients without risk factors (аrterial
hypertension (HT), dyslipidemia (DLE), type 2 diabetes
mellitus (T2D)); 146 MI patients with HT; 96 MI patients
with HT and HDL; 96 MI patients with HT, HDL and T2D
designated as “syntropy of cardiovascular continuum”. Genotyping of 58 SNPs was performed on Sequenom
MassARRAY (USA). These SNPs are localized in the
genes involved in fibrogenesis and/or associated with car-
diovascular diseases and atherosclerotic plaque stability. Statistical data analysis was performed in the software KRIT1 (CCM1) is a disease gene responsible for Cerebral
Cavernous Malformations (CCM), a major cerebrovascular
disease of proven genetic origin affecting 0.3-0.5% of the
population. Previously, we demonstrated that KRIT1 loss-
of-function is associated with altered redox homeostasis,
suggesting a novel pathogenic mechanism for CCM disease
and raising the possibility that KRIT1 loss exerts pleiotropic
effects on multiple redox-sensitive mechanisms. To address
this possibility, we investigated major redox-sensitive
pathways and enzymatic systems that play critical roles in Abstracts from the 51st European Society of Human Genetics Conference: Posters 139 fundamental
cytoprotective
mechanisms
of
adaptive
responses to oxidative stress, including the master Nrf2
antioxidant defense pathway and its downstream target
Glyoxalase 1 (Glo1), a pivotal stress-responsive defense
enzyme involved in cellular protection against glycative and
oxidative stress through the metabolism of methylglyoxal
(MG). P05.16D Introduction: The clinical presentation of congenital heart
disease (CHD) is often accompanied by diverse comorbid-
ities within and outside the cardiovascular system. There is
significant heterogeneity in the presence of comorbidities
and this can make genetic diagnosis and identification of
new CHD-associated genes challenging, especially for rare
forms of CHD, where cohorts are usually small. This is
evident in low diagnostic yields of standard criteria for
suspicion of 22q11-deletion syndrome (22q11DS), and
limited identification of genes associated with Ebstein
Anomaly (EA). Quantification of DNA copy number variations in patients
with coronary artery disease by digital droplet PCR Quantification of DNA copy number variations in patients
with coronary artery disease by digital droplet PCR A. A. Sleptsov1, M. S. Nazarenko1,2,3, N. R. Valiakhmetov1, A. N. Kazantsev2, O. L. Barbarash2, V. P. Puzyrev1 A. A. Sleptsov1, M. S. Nazarenko1,2,3, N. R. Valiakhmetov1, A. N. Kazantsev2, O. L. Barbarash2, V. P. Puzyrev1 1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk,
Russian Federation, 2Institute for complex issues of 1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk,
Russian Federation, 2Institute for complex issues of
cardiovascular diseases, Kemerovo, Russian Federation,
3Siberian State Medical University, Tomsk, Russian Federation 3Siberian State Medical University, Tomsk, Russian Federation 3Siberian State Medical University, Tomsk, Russian Federation Materials
and
Methods:
Taking
advantage
of
a
concentration of cases of rare forms of CHD in a single
center in Colombia, unusually large cohorts of patients with
suspicion
of
22q11DS
and
EA
were
assembled. A
comorbidity database was constructed and multivariate
statistical analysis was carried out to identify correlations
between comorbidities in different subgroups in each
cohort. Materials
and
Methods:
Taking
advantage
of
a
concentration of cases of rare forms of CHD in a single
center in Colombia, unusually large cohorts of patients with
suspicion
of
22q11DS
and
EA
were
assembled. A
comorbidity database was constructed and multivariate
statistical analysis was carried out to identify correlations
between comorbidities in different subgroups in each
cohort. Introduction: Recently we performed the genome-wide
analysis of CNVs in patients with coronary artery disease
(CAD) by using array-CGH. We found 90 CNVs, and 13%
of them were novel. The aim of this study was to determine
the frequencies of the several candidate CNVs (SFMBT1,
PRKRA, and SIRPB1). Materials and Methods: We have extracted DNA
specimens from white blood cells of 100 patients with
CAD, 100 patients with both CAD and diabetes mellitus 2
type (DM2), and 130 persons without any clinical and
laboratory features of atherosclerosis, at the same ages. Digital droplet PCR (QX200) along with TaqMan Assays
was used to identify CNVs. Materials and Methods: We have extracted DNA
specimens from white blood cells of 100 patients with
CAD, 100 patients with both CAD and diabetes mellitus 2
type (DM2), and 130 persons without any clinical and
laboratory features of atherosclerosis, at the same ages. Digital droplet PCR (QX200) along with TaqMan Assays
was used to identify CNVs. P05.15C Experimental outcomes showed that KRIT1 loss-of-
function induces a redox-sensitive sustained upregulation of
Nrf2 and Glo1, and a drop in intracellular levels of major
apoptosis-protective proteins, including MG-modified heat
shock protein 70 (Hsp70) and 27 (Hsp27), leading to a
chronic
adaptive
redox
homeostasis
that
counteracts
intrinsic oxidative stress but increases susceptibility to
oxidative DNA damage and apoptosis. While supporting
and extending the pleiotropic functions of KRIT1, these
findings shed new light on the mechanistic relationship
between KRIT1 loss-of-function and enhanced cell sensi-
tivity to oxidative stress, thus providing valuable new
insights into CCM pathogenesis and novel options for the
development of preventive and therapeutic strategies. However, patients with CAD and DM2 had more frequently
loss of SFMBT1 (16%) than patients with CAD (8%). The
frequency of gain 2q31.2 (PRKRA) was 6% in patients with
CAD whereas in control group we identified it in one
person only. The frequency of loss 20p13 (SIRPB1) was
67% in both groups. Conclusion: It is likely that the identified CNV loss in the
3p21.1 region (SFMBT1) plays a certain role in the risk of
developing CAD and DM2 (p = 0.006). At the same time,
we detected a trend towards increased frequency of the gain
in the 2q31.2 region (PRKRA) in patients with CAD. A.A. Sleptsov: None. M.S. Nazarenko: None. N.R. Valiakhmetov:
None. A.N. Kazantsev:
None. O.L. Barbarash: None. V.P. Puzyrev: None. Multivariate analysis of comorbidities in congenital heart
disease: Making sense of phenotypic heterogeneity Multivariate analysis of comorbidities in congenital heart
disease: Making sense of phenotypic heterogeneity R. Cabrera1, M. Miranda-Fernández1, V. Huertas-Quiñones1,
G. Alberto1, N. Sandoval1, C. M. Restrepo2, P. Laissue2,
C. Silva2, K. Moreno Medina1, R. Dennis Verano1 E. Trapani: None. C. Antognelli: None. S. Delle
Monache: None. A. Perrelli: None. C. Fornelli: None. V. Benedetti: None. S. Sarri: None. G. Costantino: None. F. Geddo: None. A. Zotta: None. L. Goitre: None. S.F. Retta: None. 1Fundacion Cardioinfantil - Instituto de Cardiología, Bogota,
Colombia, 2Universidad de la Rosario, Bogota, Colombia 1Fundacion Cardioinfantil - Instituto de Cardiología, Bogota,
Colombia, 2Universidad de la Rosario, Bogota, Colombia Quantification of DNA copy number variations in patients
with coronary artery disease by digital droplet PCR Results: The data show phenotypic heterogeneity in CHD
can mask the existence of identifiable subgroups which can
be used to improve diagnosis and identify genetic variants
in CHD. In patients meeting criteria for 22q11.2DS,
multivariate analysis of comorbidities can improve the
specificity
of
clinical
evaluations
without
sacrificing
sensitivity. In patients with EA, multivariate analysis
revealed a distinct homogenous subgroup with a likely Results: We detected the loss in the 3p21.1 region
(SFMBT1) in 11.48% patients and 8.5% in control group. 140 J. del Picchia there is evidence that knockout mouse has ventricular septal
defects and hypoplastic aortic valve. there is evidence that knockout mouse has ventricular septal
defects and hypoplastic aortic valve. distinct genetic etiology, presenting with pre-excitation
arrhythmias with reduced outflow tract obstruction and
improved survival. Grant reference: British Heart Foundation Conclusions: Multivariate analysis can reveal clinically
relevant patterns in comorbidity datasets of phenotypically
heterogeneously disease cohorts. This approach can be used
in the identification of novel genetic causes and the
improvement of clinical diagnostic criteria. E. Fotiou: None. S. Williams: None. D. Page: None. K. Hentges: None. B. Keavney: None. Molecular autopsy reveals clues for genetic basis of
congenital valve defect R. Cabrera: None. M. Miranda-Fernández: None. V. Huertas-Quiñones:
None. G. Alberto:
None. N. Sandoval: None. C.M. Restrepo: None. P. Laissue: None. C. Silva: None. K. Moreno Medina: None. R. Dennis
Verano: None. R. Cabrera: None. M. Miranda-Fernández: None. V. Huertas-Quiñones:
None. G. Alberto:
None. N. Sandoval: None. C.M. Restrepo: None. P. Laissue: None. C. Silva: None. K. Moreno Medina: None. R. Dennis
Verano: None. F. A. R. Madia1, A. T. Dias2, É. A. Zanardo2, J. G. Damasceno2,
A. M. Nascimento1, T. V. M. M. Costa2, S. N. Chehimi1, G. M. Novo-Filho2, M. M. Montenegro2, Y. G. Oliveira2, A. B. Freitas2,
L. L. Vieira2, R. Schultz3, F. T. Gonçalves4, C. Fridman4, C. A. Kim5, L. D. Kulikowski1,2 Integration of large-scale genomic data sources to identify
novel genetic loci for congenital heart disease Integration of large-scale genomic data sources to identify
novel genetic loci for congenital heart disease 1Laboratorio de Citogenomica, Departamento de Pediatria,
Faculdade de Medicina FMUSP, Universidade de Sao Paulo,
Sao Paulo, Brazil, 2Laboratorio de Citogenomica,
Departamento de Patologia, Faculdade de Medicina FMUSP,
Universidade de Sao Paulo, Sao Paulo, Brazil, 3Servico de
Anatomia Patologica, Departamento de Patologia, Faculdade
de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo,
Brazil, 4Departamento de Medicina Legal, Etica Medica e
Medicina Social e do Trabalho, Faculdade de Medicina
FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil,
5Unidade de Genetica, Departamento de Pediatria, Instituto da
Crianca, Hospital das Clinicas HCFMUSP, Universidade de
Sao Paulo, Sao Paulo, Brazil 1Laboratorio de Citogenomica, Departamento de Pediatria,
Faculdade de Medicina FMUSP, Universidade de Sao Paulo,
Sao Paulo, Brazil, 2Laboratorio de Citogenomica,
Departamento de Patologia, Faculdade de Medicina FMUSP,
Universidade de Sao Paulo, Sao Paulo, Brazil, 3Servico de
Anatomia Patologica, Departamento de Patologia, Faculdade
de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo,
Brazil, 4Departamento de Medicina Legal, Etica Medica e
Medicina Social e do Trabalho, Faculdade de Medicina
FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil,
5Unidade de Genetica, Departamento de Pediatria, Instituto da
Crianca, Hospital das Clinicas HCFMUSP, Universidade de
Sao Paulo, Sao Paulo, Brazil E. Fotiou, S. Williams, D. Page, K. Hentges, B. Keavney E. Fotiou, S. Williams, D. Page, K. Hentges, B. Keavney Division of Cardiovascular Science, Manchester, United
Kingdom Background: Small nucleotide variants and copy number
variants (CNV) have been found to affect congenital heart
disease (CHD) risk. Yet, the identification of the genetic
causes of CHD remains quite challenging. Purpose: To integrate data on both classes of variation
associated with non-syndromic CHD cases as a better
means for identification of candidate genes predisposing
to CHD. Introduction: Congenital heart defect (CHD) consists in a
large set of functional and structural anomalies that arise
during the cardiac embryogenesis including septal defects,
valve defects, and outflow tract anomalies. Valve defect is
an important cause of mortality and morbidity. However,
the genetic basis of congenital valve defect is unclear. Methods: Here, we have updated a previously published
CHD case CNV list and generated a control CNV list using:
a) DECIPHER database, b) ISCA database, c) ECARUCA
database, d) 1000 Genome phase 3 dataset, e) DGV
database and f) published literature. Results: Analysing deleted (del) and duplicated (dup)
CNVs independently resulted in unique case CNV regions
not present in the controls. Somatic DNA methylation and copy number landscape of
coronary artery disease patients Somatic DNA methylation and copy number landscape of
coronary artery disease patients 1FIMM, Institute for Molecular Medicine, University of
Helsinki, Helsinki, Finland, 2Department of Internal Medicine,
University of Michigan, Ann Arbor, MI, United States,
3Department of Public Health, Clinicum, Faculty of Medicine,
University of Helsinki, Helsinki, Finland M. S. Nazarenko1,2,3, A. V. Markov1, A. A. Sleptcov1, A. N. Kazancev3, O. L. Barbarash3, V. P. Puzyrev1 1Research Institute of Medical Genetics, Tomsk, Russian
Federation, 2Siberian State Medical University, Tomsk,
Russian Federation, 3Research Institute for Complex Problems
of Cardiovascular Diseases, Kemerovo, Russian Federation Severe hypercholesterolemia is one of the most significant
risk factors for coronary artery disease (CAD), and it has a
strong and complex genetic predisposition. Currently, not
even patients with inherited forms of severe hypercholes-
terolemia are adequately identified in Finland, and only a
part of their mutation load is known. DNA methylation and copy number variations (CNVs) of
affected and healthy vascular tissues of patients with
atherosclerosis had not been investigated in detail. The
Illumina
HumanMethylation27
BeadChip
and
Agilent
SurePrintG3 HumanCGH+SNP 2×400K microarrays were
used for DNA testing from right coronary arteries in the
area
of
advanced
atherosclerotic
plaques
and
atherosclerotic-resistant internal mammary arteries of six
patients with coronary artery disease. The atherosclerotic
plaques to compare with the healthy arteries were char-
acterized by predominate DNA hypermethylation changes. These genes were annotated with muscle system process
and positive regulation of cytosolic calcium ion con-
centration in Gene Ontology terms. In contrast, hypo-
methylated genes encode molecules belonging to different
biological processes such as development, immune/inflam-
mation responses, lipid storage, and programmed cell death. In atherosclerotic plaques the most pronounced hypo-
methylation was registered in 2q31.1 (HOXD4/HOXD3/
MIR10B), 7p15.2 (HOXA7) and 11p11.2 (ALX4). More-
over, methylation changes at 2q31.1 in blood cells were
consistently associated with smoke and ischemic stroke. We
identified 90 high-confidence CNVs that were present in
matched arteries studied. Gene Ontology analysis revealed
enrichments in the immune/inflammation responses and
olfactory transduction. Furthermore, two patients contained
the gain in 10q24.31 (ERLIN1) that affected only the blood
DNA but not arteries. There was not overlap between both To unravel the underlying genetic architecture, we
screened 206 individuals with severe hypercholesterolemia
(LDL-cholesterol >= 5mmol/l) in the prospective GeneR-
ISK study, encompassing 7,328 subjects, aged 45-64 years,
from Southern Finland. Integration of large-scale genomic data sources to identify
novel genetic loci for congenital heart disease In
conclusion, although DNA methylation differences do not
appear to be linked to the copy number changes in arteries
of patients with coronary heart disease both mechanisms
may be important in the disease. Grants: FAPESP: 09/53105-9 and FINEP-CT INFRA
0160/12 SP8. Grants: FAPESP: 09/53105-9 and FINEP-CT INFRA
0160/12 SP8. M.S. Nazarenko: None. A.V. Markov: None. A.A. Sleptcov: None. A.N. Kazancev: None. O.L. Barbarash:
None. V.P. Puzyrev: None. F.A.R. Madia: None. A.T. Dias: None. É.A. Zanardo:
None. J.G. Damasceno: None. A.M. Nascimento: None. T.V.M.M. Costa: None. S.N. Chehimi: None. G.M. Novo-Filho:
None. M.M. Montenegro:
None. Y.G. Oliveira: None. A.B. Freitas: None. L.L. Vieira: None. R. Schultz: None. F.T. Gonçalves: None. C. Fridman:
None. C.A. Kim: None. L.D. Kulikowski: None. P05.21A N. Junna1, P. Ripatti1, I. Surakka1,2, S. Ripatti1,3, E. Widén1 Integration of large-scale genomic data sources to identify
novel genetic loci for congenital heart disease Further filtering led to the
identification of 54 novel candidate protein-coding genes in
del CNVs present only in non-syndromic CHD cases and
with high/medium impact variants in exome data from our
cohort of Tetralogy of Fallot patients. Moreover, we have
identified 50 genes in those unique case CNV regions that
were previously shown to be associated with CHD such as
GATA4 for atria septal defects and NKX2-6 for conotruncal
heart malformations. Materials and Methods: We investigate the contribution
of genomic alterations in the valve defect`s pathogenesis
using molecular methods in 18 cases postmortem of
stillbirth and infant from Serviço de Verificação de Óbitos,
HC-FMUSP. DNA samples from skin, diaphragm, and
heart tissues were evaluated using AmpFℓSTR® MiniFi-
ler™PCR Amplification Kit (Life Technologies™, Cali-
fornia, USA) and Multiplex Ligation-dependent Probe
Amplification (MLPA) with different kits (MCR-Holland,
Amsterdam, the Netherlands). Results: In 8 out of 18 stillbirth and infant (44,4%) show
alterations in the genome, including trisomy 18 (5 cases),
trisomy 21 (2 cases) and duplication of 4p16 (1 case). The
tricuspid valve defect was reported in all syndromes
describe above. Besides that, the mitral valve defect was
associated with deletion of 4p16, and abnormalities of aortic
and pulmonary valve was associated with trisomy 18. Conclusions: We demonstrate a promising new strategy
with the integration of large-scale genomic data sources to
identify novel candidate genes for CHD and their contribu-
tion in heart development. We are currently performing
functional work with our strongest candidate gene for which Abstracts from the 51st European Society of Human Genetics Conference: Posters 141 Conclusion: Molecular autopsy is a significant tool for
the characterization of the basis of cardiac valve defect and
also become vital for an accurate genetic counseling. DNA methylation and copy number changes in arteries. In
conclusion, although DNA methylation differences do not
appear to be linked to the copy number changes in arteries
of patients with coronary heart disease both mechanisms
may be important in the disease. DNA methylation and copy number changes in arteries. In
conclusion, although DNA methylation differences do not
appear to be linked to the copy number changes in arteries
of patients with coronary heart disease both mechanisms
may be important in the disease. DNA methylation and copy number changes in arteries. P05.22B Monogenic and complex risk factors for ischemic heart
disease in Finland - Elucidating the role of severe
hypercholesterolemia P05.24D
Refinement of coronary artery disease risk assessment by
genomic information Refinement of coronary artery disease risk assessment by
genomic information Refinement of coronary artery disease risk assessment by
genomic information J. Partanen1, P. Ripatti1, N. Mars1, P. Pöllänen2,
K. Hotakainen3,4, J. Partanen5, E. Widén1, S. Ripatti1,6 J. Partanen1, P. Ripatti1, N. Mars1, P. Pöllänen2,
K. Hotakainen3,4, J. Partanen5, E. Widén1, S. Ripatti1,6 1Institute for Molecular Medicine Finland, University of
Helsinki, Helsinki, Finland, 2Carea – Kymenlaakso Social and
Health Services, Kotka, Finland, 3Department of Clinical
Chemistry and Hematology, Medicum, Faculty of Medicine,
University of Helsinki, Helsinki, Finland, 4Mehiläinen Oy,
Helsinki, Finland, 5Finnish Red Cross Blood Service, Helsinki,
Finland, 6Department of Public Health, Clinicum, Faculty of
Medicine, University of Helsinki, Helsinki, Finland Hyperlipidemia,
particularly
increased
LDL-cholesterol
(LDL-C) or triglycerides (TGs), is a treatable risk factor
for coronary artery disease (CAD). In addition to high-
penetrant mutations in genes like LDLR, APOB and PCSK9,
hyperlipidemias can be a consequence of polygenic burden. Whereas monogenic familial hypercholesterolemia (FH)
increases CAD risk considerably due to high lifelong
exposure to LDL-C, it is unclear whether a high polygenic
load
of
LDL-C
or
TG-increasing
variants
increases
CAD risk. Introduction: Current cardiovascular disease (CVD) risk
assessment relies on clinical risk scores, which fail to
identify a large proportion of individuals who develop
CVD. Novel biomarkers may complement such scores. Genomic information has improved risk estimation in sev-
eral prospective cohort studies and is therefore the most
promising candidate to enhance clinical risk assessment. In this prospective cohort study, we constructed incident
CAD events (n=970) from the genotyped FINRISK
population
cohort
(n=20499)
linked
with
healthcare
registries. We tested if high (>90th percentile) polygenic
scores for LDL-C or TG increased CAD risk. Finnish LDLR FH mutations increased LDL-C 3.5 mmol/l
on average, and CAD risk considerably (HR 3.67 [1.18-
11.43]). In contrast, high LDL-C score increased LDL-C
0.8 mmol/l and CAD risk only marginally (HR 1.22 [1.00-
1.49]). Materials and Methods: We established a novel cohort
of 7439 coronary artery disease (CAD)-free 45-65-year-old
individuals from southern Finland. We estimated their CAD
risk using 1) an established national clinical risk score, and
2) the clinical score combined with a genetic risk score
(GRS) for CAD. We returned both estimates to the
participants. We aimed to 1) evaluate the CAD risk
spectrum of the cohort, 2) assess how combining the GRS No monogenic large-effect TG-increasing variants were
present. Polygenic hyperlipidemias and coronary artery disease risk The CAD risk associated with a high polygenic load of
LDL-C or TG-increasing variants depends directly on their
effect on lipid levels. The clinical utility of polygenic scores
needs further study. P. Ripatti1, J. T. Rämö1, S. Söderlund2,3, I. Surakka1,4, A. S. Havulinna1,5, N. B. Freimer6, V. Salomaa5, A. Palotie1,7,8,
M. Taskinen2,9, S. Ripatti1,10 P. Ripatti: None. J.T. Rämö: None. S. Söderlund:
None. I. Surakka: None. A.S. Havulinna: None. N.B. Freimer: None. V. Salomaa: None. A. Palotie: F. Consultant/Advisory Board; Modest; Pfizer Genetics Scien-
tific Advisory Panel. M. Taskinen: B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending grants as well as grants already received); Modest;
Amgen, Novo Nordisk, Merck, Sharpe & Dohme. Other;
Modest; Amgen, Novo Nordisk, SanofiAventis, Chiesi,
Astra Zeneca, Pfizer. S. Ripatti: None. 1Institute for Molecular Medicine Finland, Helsinki, Finland,
2Research Programs Unit, Diabetes & Obesity, University of
Helsinki, Helsinki, Finland, 3Department of Internal Medicine,
Helsinki University Hospital, Helsinki, Finland, 4Department
of Internal Medicine, University of Michigan, Ann Arbor, MI,
United States, 5Department of Public Health Solutions,
National Institute for Health and Welfare, Helsinki, Finland,
6Center for Neurobehavioral Genetics, Semel Institute for
Neuroscience and Human Behavior, University of California,
Los Angeles, CA, United States, 7The Stanley Center for
Psychiatric Research, The Broad Institute of MIT and Harvard,
Cambridge, MA, United States, 8Psychiatric &
Neurodevelopmental Genetics Unit, Massachusetts General
Hospital, Boston, MA, United States, 9Clinical Research
Institute HUCH, Ltd., Helsinki, Finland, 10Department of
Public Health, Clinicum, Faculty of Medicine, University of
Helsinki, Helsinki, Finland Somatic DNA methylation and copy number landscape of
coronary artery disease patients Exome-sequencing identified a
novel, likely causal mutation in LDLR (R574L), but
surprisingly
we
didn’t
identify
any
of
the
hypercholesterolemia-associated
LDLR-mutations
pre-
viously shown to be enriched in Finland. Neither did we
identify potentially causal mutations in APOB or PCSK9. Polygenic
modeling
(106
LDL-cholesterol-associated
SNPs) explained 17% of the LDL-cholesterol variation in
the cohort but suggested only slight clustering of polygenes
with severe hypercholesterolemia. Evaluating the use of lipid-lowering medication in the
GeneRISK-cohort, we found that only 4.3% of individuals
with severe hypercholesterolemia were on lipid-lowering
treatment at baseline. This is considerably less than the
10.1% that were treated in the full cohort. Our preliminary
data further show that at follow-up 1.5 years later, lipid-
lowering medication had been initiated only to an additional
3.3% of the individuals with severe hypercholesterolemia. Given that patients with hypercholesterolemia are poorly
identified and treated, there is an unmet need not only to
further
characterize
causal
variants
underlying
severe 142 J. del Picchia hypercholesterolemia but also to raise awareness among
caretakers to take action to reduce the disease burden. hypercholesterolemia but also to raise awareness among
caretakers to take action to reduce the disease burden. 1.54]). Comparing hypertriglyceridemic individuals (TG
>2.5 mmol/l) to individuals with TG <1.7 mmol/l, those
with high TG score had higher CAD risk (HR 2.11 [1.58-
2.81]) than those without high TG score (HR 1.61 [1.33-
1.94]) despite comparable TG levels (p=0.022). Only 5.2%
of the hypertriglyceridemic individuals received fibrate
treatment. 1.54]). Comparing hypertriglyceridemic individuals (TG
>2.5 mmol/l) to individuals with TG <1.7 mmol/l, those
with high TG score had higher CAD risk (HR 2.11 [1.58-
2.81]) than those without high TG score (HR 1.61 [1.33-
1.94]) despite comparable TG levels (p=0.022). Only 5.2%
of the hypertriglyceridemic individuals received fibrate
treatment. N. Junna: None. P. Ripatti: None. I. Surakka: None. S. Ripatti: None. E. Widén: None. 1University of Cambridge, Cambridge, United Kingdom,
2University of Oxford, Oxford, United Kingdom J. Partanen: None. P. Ripatti: None. N. Mars: None. P. Pöllänen: None. K. Hotakainen: A. Employment (full or
part-time); Significant; Mehiläinen Oy. J. Partanen: None. E. Widén: None. S. Ripatti: None. J. Partanen: None. P. Ripatti: None. N. Mars: None. P. Pöllänen: None. K. Hotakainen: A. Employment (full or
part-time); Significant; Mehiläinen Oy. J. Partanen: None. E. Widén: None. S. Ripatti: None. Background: Several pharmacological enhancers of lipo-
protein lipase (LPL) are in preclinical/early-clinical devel-
opment for dyslipidemia, but it is unknown if they will
reduce cardio-metabolic disease risk when added to existing
lipid-lowering drugs. Human genetics can be used to study
if genetic-differences in LPL-mediated lipolysis contribute
to these disease independent of pathways targeted by
existing drugs. P05.25A A novel candidate frameshift mutation for F.B. Isik: None. M.D. Sozuguzel: None. N. Genc: None.
E.F. Caralan: None. Z. Dogru: None. C. Akdeniz: None.
V. Tuzcu: None. H. Cangul: None. Results: Genotyped participants numbered 5125. Those
with high CAD risk (10-year risk ≥10%) increased from 406
(7.9%) to 452 (8.8%) with the combined score; 113 were
reclassified to a lower risk category and 159 to the high-risk
category. The combined score refined CAD risk clinically
meaningfully in 950 (18.5%) participants. Of the 452 high-
risk participants based on the combined score, 197 (44%)
smoked, 165 (37%) were obese, 80 (18%) had diabetes, 310
(69%) had LDL-C >3 mmol/l, 98 (22%) used statins and
205 (45%) used antihypertensives. Results: Genotyped participants numbered 5125. Those
with high CAD risk (10-year risk ≥10%) increased from 406
(7.9%) to 452 (8.8%) with the combined score; 113 were
reclassified to a lower risk category and 159 to the high-risk
category. The combined score refined CAD risk clinically
meaningfully in 950 (18.5%) participants. Of the 452 high-
risk participants based on the combined score, 197 (44%)
smoked, 165 (37%) were obese, 80 (18%) had diabetes, 310
(69%) had LDL-C >3 mmol/l, 98 (22%) used statins and
205 (45%) used antihypertensives. Catecholaminergic Polymorphic Ventricular Tachycardia F. B. Isik, M. D. Sozuguzel, N. Genc, E. F. Caralan, Z. Dogru,
C. Akdeniz, V. Tuzcu, H. Cangul Methods:
Using individual-level
genetic data from
390,470 individuals and a “factorial” design, we investi-
gated the independent and combined consequences on
coronary disease and type 2 diabetes risk of triglyceride-
lowering
LPL-alleles
and
LDL-C-lowering
alleles
at
different genes, including those encoding targets of current
LDL-C-lowering therapy (HMGCR, NPC1L1 and PCSK9). A novel candidate frameshift mutation for Catecholaminergic Polymorphic Ventricular Tachycardia P05.24D
Refinement of coronary artery disease risk assessment by
genomic information High TG score, however, increased TG 0.6 mmol/l
on average, and CAD risk significantly (HR 1.26 [1.04- 143 Abstracts from the 51st European Society of Human Genetics Conference: Posters to the clinical score refines risk estimation, and 3) identify
high-risk individuals with actionable clinical characteristics. to the clinical score refines risk estimation, and 3) identify
high-risk individuals with actionable clinical characteristics. to the clinical score refines risk estimation, and 3) identify
high-risk individuals with actionable clinical characteristics. F.B. Isik: None. M.D. Sozuguzel: None. N. Genc: None. E.F. Caralan: None. Z. Dogru: None. C. Akdeniz: None. V. Tuzcu: None. H. Cangul: None. P05.27C
Variation in the LPL gene, low density lipoprotein-
cholesterol lowering alleles and risk of coronary disease
and type 2 diabetes L. A. Lotta1, I. Stewart1, S. Sharp1, F. Day1, S. Burgess1,
J. Luan1, L. Cai1, L. Wittemans1, N. Kerrison1, K. Khaw1,
M. McCarthy2, S. O'Rahilly1, R. Scott1, D. Savage1, J. Perry1,
C. Langenberg1, N. Wareham1 Conclusions: Incorporating the CAD GRS to the clinical
risk score considerably refined CAD risk estimation. High-
risk individuals presented multiple risk factors and inter-
vention opportunities. 1University of Cambridge, Cambridge, United Kingdom,
2University of Oxford, Oxford, United Kingdom Medipol University, istanbul, Turkey Catecholaminergic Polymorphic Ventricular Tachycardia
(CPVT) is a cardiac disorder which characterized by
arrhythmias, sudden cardiac arrest after physical activity or
emotional stress which could result from CASQ2 gene
mutations
which
follow
autosomal
recessive
pattern. CASQ2 encodes the protein Calsequestrin-2 which is a
high-capacity calcium-binding protein acting as an internal
calcium store in muscle. Therefore, it plays a pivotal role in
excitation-contraction coupling which regulates the rate of
heart beats. In this study, we identified a novel mutation on
CASQ2 (NM_001232.3) gene in two, non-related patients
with overlapping symptoms such as repetitive syncope and
polymorphic ventricular tachycardia. Molecular analysis of
the 2 patients’ by next generation sequencing showed a
homozygous splice variant p.L79X (c.237delT) which is not
currently present in clinical databases. We evaluated this
novel variant as a likely pathogenic variant because of its
potential nonsense effect. In vivo functional studies at
transcriptional and translational level are underway to
demonstrate the causative role of this mutation in patho-
genesis of CPVT. Results: People carrying a higher than median load of
both triglyceride-lowering LPL-alleles and LDL-C lowering
alleles had an odds ratio (OR) for coronary disease of 0.73
compared to people below the median of both exposures
(95% confidence interval [CI], 0.70 to 0.76; p = 2.8×10-52),
which was a greater effect than that observed in people with
higher than median load of either exposure. Triglyceride-
lowering LPL-alleles were strongly associated with lower
diabetes risk (odds ratio per standard deviation genetically-
lower triglycerides, 0.69; 95% CI, 0.62 to 0.76; p = 2.6×10-
13). In
factorial
analyses,
this
protective
association
neutralized the association of LDL-C lowering alleles with
a
higher
diabetes
risk
(pheterogeneity
in
effect
estimates = 0.0094). Conclusions:
Triglyceride-lowering
LPL-alleles
and
LDL-C-lowering genetic mechanisms have independent
contributions to a lower risk of coronary disease. These
findings provide human genetics evidence to support the
development of agents that enhance LPL-mediated lipolysis
for use in the context of LDL-C-lowering therapy. 144 J. del Picchia L.A. Lotta: None. I. Stewart: None. S. Sharp: None. F. Day: None. S. Burgess: None. J. Luan: None. L. Cai:
None. L. Wittemans: None. N. Kerrison: None. K. Khaw: None. M. McCarthy: Other; Significant; Grants
from Eli Lilly, Roche, AstraZeneca, Merck, Janssen,
Servier, Novo Nordisk, Sanofi-Aventis, Boehringer Ingel-
heim, Pfizer, and Takeda; and honoraria from Novo Nordisk
and Pfzier. S. Digenic mutations of MYH7 and RYR2 in siblings
manifesting with severe cardiac dysfunction Digenic mutations of MYH7 and RYR2 in siblings
manifesting with severe cardiac dysfunction P05.29A
Increasing the number of genes in the genetic screening of
dilated cardiomyopathy: is more actually more? Increasing the number of genes in the genetic screening of
dilated cardiomyopathy: is more actually more? E. Vanhoutte, J. Verdonschot, G. Claes, A. van den Wijngaard,
A. Helderman-van den Enden, M. Hazebroek, S. Heymans,
H. Brunner, I. Krapels E. Vanhoutte, J. Verdonschot, G. Claes, A. van den Wijngaard,
A. Helderman-van den Enden, M. Hazebroek, S. Heymans,
H. Brunner, I. Krapels Medipol University, istanbul, Turkey O'Rahilly: Other; Significant; Personal fees
from Pfizer, AstraZeneca, iMed, and ERX Pharmaceuticals
for serving on advisory boards and scientific panels. R. Scott: A. Employment (full or part-time); Significant; Full-
time employment with GSK.. D. Savage: None. J. Perry:
None. C. Langenberg: None. N. Wareham: None. L.A. Lotta: None. I. Stewart: None. S. Sharp: None. F. Day: None. S. Burgess: None. J. Luan: None. L. Cai:
None. L. Wittemans: None. N. Kerrison: None. K. Khaw: None. M. McCarthy: Other; Significant; Grants
from Eli Lilly, Roche, AstraZeneca, Merck, Janssen,
Servier, Novo Nordisk, Sanofi-Aventis, Boehringer Ingel-
heim, Pfizer, and Takeda; and honoraria from Novo Nordisk
and Pfzier. S. O'Rahilly: Other; Significant; Personal fees
from Pfizer, AstraZeneca, iMed, and ERX Pharmaceuticals
for serving on advisory boards and scientific panels. R. Scott: A. Employment (full or part-time); Significant; Full-
time employment with GSK.. D. Savage: None. J. Perry:
None. C. Langenberg: None. N. Wareham: None. Conclusions: We report sibling cases manifesting with
severe cardiac dysfunction possibly due to digenic MYH7
and RYR2 mutations. Digenic mutations may cause severer
clinical manifestation than that caused by each mutation. M. Nagasaka: None. M. Taniguchi-Ikeda: None. H. Inagaki: None. I. Morioka: None. H. Kurahashi: None. K. Iijima: None. M. Nagasaka1,2, M. Taniguchi-Ikeda1,3,4, H. Inagaki4,
I. Morioka1, H. Kurahashi4, K. Iijima1 Whole-exome sequencing identified
two variants in the siblings, a heterozygous missense variant
in MYH7 (c.728G>A) inherited from the father, and in
RYR2 (c.5428G>C), inherited from the mother. The variant
in MYH7 was reported as a pathogenic allele. The variant in
RYR2 was considered likely to be pathogenic since PHRD-
like scaled Combined Annotation-Dependent Depletion
(CADD) score suggested deleteriousness (25.9). Case Report: The proband was born at 34 weeks of
gestation with a birth weight of 2452g. He presented with
severe cardiac dysfunction at birth and needed strict
intensive care. At 3 years-old, he had intractable epilepsy
and severe neurodevelopmental impairment. An elder
brother who was born at 39 weeks of gestation died shortly
after birth because of cardiac failure. There were strong
family history of sudden cardiac death in the father’s trait. The mother manifested with heart failure after the delivery
of the proband and presented with tachycardia during
treadmill stress test. Whole-exome sequencing identified
two variants in the siblings, a heterozygous missense variant
in MYH7 (c.728G>A) inherited from the father, and in
RYR2 (c.5428G>C), inherited from the mother. The variant
in MYH7 was reported as a pathogenic allele. The variant in
RYR2 was considered likely to be pathogenic since PHRD-
like scaled Combined Annotation-Dependent Depletion
(CADD) score suggested deleteriousness (25.9). Case Report: The proband was born at 34 weeks of
gestation with a birth weight of 2452g. He presented with
severe cardiac dysfunction at birth and needed strict
intensive care. At 3 years-old, he had intractable epilepsy
and severe neurodevelopmental impairment. An elder
brother who was born at 39 weeks of gestation died shortly
after birth because of cardiac failure. There were strong
family history of sudden cardiac death in the father’s trait. Results: In 51 patients, genetic analysis of the 347-heart
gene panel has been completed. Variants of unknown
significance, likely pathogenic or pathogenic variants were
detected in 15 (29%), 3 (6%) and 0 (0%) patients
respectively. So
far,
likely
pathogenic
variants
were
identified in ANK2, KCNQ1 and FLNC. The gene with
the highest yield is FLNC. Mutations in FLNC are
associated with myopathy and cardiomyopathies. ANK2
and
KCNQ1
are
associated
with
inherited
primary
arrhythmias. Results: In 51 patients, genetic analysis of the 347-heart
gene panel has been completed. Variants of unknown
significance, likely pathogenic or pathogenic variants were
detected in 15 (29%), 3 (6%) and 0 (0%) patients
respectively. M. Nagasaka1,2, M. Taniguchi-Ikeda1,3,4, H. Inagaki4,
I. Morioka1, H. Kurahashi4, K. Iijima1 Background: Dilated cardiomyopathy (DCM) is char-
acterized by systolic dysfunction with dilatation of the left
ventricle and/or right ventricle. The Dutch Society for
Clinical Genetic Laboratory Diagnostics (VKGL) pre-
viously recommended genetic testing in DCM patients
using a core panel of 47 cardiomyopathy-associated genes
(including TTN, MYH7, PLN and LMNA). However, DCM
is a complex multifactorial disorder involving multiple
genes. Other genes are probably involved in the pathogen-
esis. Therefore, a panel containing 347 genes involved in
cardiac disorders was composed. 1Department of Pediatrics, Kobe University Graduate School
of Medicine, Kobe, Japan, 2Department of Pediatrics and
Neonatology,Takatsuki General Hospital, Takatsuki, Japan,
3Division of Genetic Counseling, Kobe University Hospital,
Kobe, Japan, 4Division of Molecular Genetics, Institute for
Comprehensive Medical Science, Fujita Health University,
Toyoake, Japan 1Department of Pediatrics, Kobe University Graduate School
of Medicine, Kobe, Japan, 2Department of Pediatrics and
Neonatology,Takatsuki General Hospital, Takatsuki, Japan,
3Division of Genetic Counseling, Kobe University Hospital,
Kobe, Japan, 4Division of Molecular Genetics, Institute for
Comprehensive Medical Science, Fujita Health University,
Toyoake, Japan Introduction: Mutations in the Myosin heavy chain 7
(MYH7) are associated with inherited cardiomyopathies. On
the other hand, mutations in the Ryanodine receptor 2
(RYR2) are associated with fatal arrhythmias such as cate-
cholaminergic polymorphic ventricular tachycardia. Both of
them exhibit autosomal dominant patterns of inheritance. Aims: To determine the yield of genetic variants in gene
panel testing involving 347 heart related genes in 100
genetically unsolved DCM patients in a Dutch population. Aims: To determine the yield of genetic variants in gene
panel testing involving 347 heart related genes in 100
genetically unsolved DCM patients in a Dutch population. Methods: 100 DCM patients without (likely) pathogenic
mutations in the 47 core cardiomyopathy genes were
offered genetic analysis through whole exome sequencing
using a targeted filter consisting of 347 heart related genes. p
Case Report: The proband was born at 34 weeks of
gestation with a birth weight of 2452g. He presented with
severe cardiac dysfunction at birth and needed strict
intensive care. At 3 years-old, he had intractable epilepsy
and severe neurodevelopmental impairment. An elder
brother who was born at 39 weeks of gestation died shortly
after birth because of cardiac failure. There were strong
family history of sudden cardiac death in the father’s trait. The mother manifested with heart failure after the delivery
of the proband and presented with tachycardia during
treadmill stress test. Maastricht University Medical Center, Maastricht,
Netherlands M. Nagasaka1,2, M. Taniguchi-Ikeda1,3,4, H. Inagaki4,
I. Morioka1, H. Kurahashi4, K. Iijima1 J. Dean1, E. Cleary2, C. McWilliam3, R. McGowan4, W. Lam2,
D. O'Sullivan1, S. Tennant1 Similarly, SMR was
calculated in parents of 128 present-day DCM patients
with a TTNtv using the reverse parent-offspring method. Subgroups were compared with Poisson regression. Results: In 20,522 person-years, overall mortality was
not significantly increased in three historical pedigrees
compared to the general population (SMR 1.06, 95% CI
0.95-1.18, p = 0.162). However, mortality was significantly
increased in subjects living after 1965 (SMR 1.27, 95% CI
1.04-1.53, p = 0.009), and subjects aged ≥60 (SMR 1.17,
95% CI 1.01-1.35, p = 0.02). Mutation-specific differences
in
mortality
were
observed. Reverse
parent-offspring
analysis showed excess mortality (SMR 1.26, 95% CI
1.07-1.48, p = 0.003), driven by subjects aged ≥60. J. Dean: None. E. Cleary: None. C. McWilliam: None. R. McGowan: None. W. Lam: None. D. O'Sullivan:
None. S. Tennant: None. Conclusion: The natural history of TTNtv showed a
relatively mild phenotype with significant excess mortality
after 1965 and in subjects aged ≥60 years. Increased
mortality above 60 years was also seen in parents of
present-day patients. With increasing life expectancy,
TTNtv-associated
mortality
will
likely
become
more
prevalent. J. Dean1, E. Cleary2, C. McWilliam3, R. McGowan4, W. Lam2,
D. O'Sullivan1, S. Tennant1 J. Dean1, E. Cleary2, C. McWilliam3, R. McGowan4, W. Lam2,
D. O'Sullivan1, S. Tennant1 1NHS Grampian, Aberdeen, United Kingdom, 2NHS Lothian,
Edinburgh, United Kingdom, 3NHS Tayside, Dundee, United
Kingdom, 4NHS Greater Glasgow, Glasgow, United Kingdom Dilated cardiomyopathy (DCM) is aetiologically hetero-
geneous, and up to 35% of cases are found to be familial
after investigation of first degree relatives. Amongst those
with genetic DCM, truncating mutations in TTN are thought
to be the most common cause, being found in around 30%
of familial cases in most series. In Scotland, patients
referred for genetic investigation of dilated cardiomyopathy
are tested using 2 gene panels, one with 13 genes encoding
components of the sarcomere including TTN, and the other
including 5 genes also associated with cardiac disorders
with an arrhythmia phenotype (SCN5A, ABCC9, PLN,
DES and LMNA). The 5 gene arrhythmia panel came into
use in 2017. We report an audit of the outcome of this
approach. In 2017, 106 patients with DCM were referred for
genetic testing in Scotland (population ~5.4 million). Only
25% had a genetic variant likely to cause DCM, 8% being
associated with truncating mutations in TTN. The majority
(17%) had variants in other cardiac genes (including 5% in
TNNT2, 4% in ABCC9 and 2% in MYH7, other genes
contributed a smaller number to the total). This real world
data from the Scottish Laboratory Consortium Molecular
Diagnostic service suggests that in Scotland, TTN accounts
for fewer cases than might be expected from the literature. A 25% overall detection rate nevertheless suggests that
targeted gene panel testing using genes for which good
genotype-phenotype data is available remains a useful way
to investigate for inherited DCM. Background: Truncating Titin variants (TTNtv) are a major
cause of dilated cardiomyopathy (DCM), a major cause of
heart failure. TTNtv are also frequently present in the
general population and associated with a variable pheno-
type. The prognosis of asymptomatic TTNtv carriers is
poorly understood. Objectives: To assess the clinical rele-
vance of TTNtv by analysing TTNtv associated all-cause
mortality in historical pedigrees and in present-day patients. Methods: Haplotype analysis identified five recurrent A-
band TTNtv as founder mutations. Three multigenerational
pedigrees were traced back to 18th century ancestors. Using
the Family Tree Mortality Ratio method, standardized
mortality ratios (SMR, standardized for sex, age and
calendar-period) were calculated. M. Jansen1, A. F. Baas1, K. Y. van Spaendonck-Zwarts2, A. S.
Ummels1, A. van den Wijngaard3, J. D. H. Jongbloed4, M. A. van A targeted 18 gene panel approach detects a likely genetic
cause in 25% of Scottish Dilated Cardiomyopathy patients 1University Medical Center Utrecht, University Utrecht,
Utrecht, Netherlands, 2Academic Medical Centre, Amsterdam,
Netherlands, 3Maastricht University Medical Centre,
Maastricht, Netherlands, 4University Medical Centre
Groningen, Groningen, Netherlands, 5Erasmus Medical
Center, Rotterdam, Netherlands, 6Academic Medical Center,
Amsterdam, Netherlands, 7Erasmus Medical Centre,
Rotterdam, Netherlands, 8VU University Medical Centre,
Amsterdam, Netherlands, 9University Medical Centre
Groningen, University of Groningen, Groningen, Netherlands,
10Leiden University Medical Centre, Leiden, Netherlands M. Nagasaka1,2, M. Taniguchi-Ikeda1,3,4, H. Inagaki4,
I. Morioka1, H. Kurahashi4, K. Iijima1 So
far,
likely
pathogenic
variants
were
identified in ANK2, KCNQ1 and FLNC. The gene with
the highest yield is FLNC. Mutations in FLNC are
associated with myopathy and cardiomyopathies. ANK2
and
KCNQ1
are
associated
with
inherited
primary
arrhythmias. The mother manifested with heart failure after the delivery
of the proband and presented with tachycardia during
treadmill stress test. Whole-exome sequencing identified
two variants in the siblings, a heterozygous missense variant
in MYH7 (c.728G>A) inherited from the father, and in
RYR2 (c.5428G>C), inherited from the mother. The variant
in MYH7 was reported as a pathogenic allele. The variant in
RYR2 was considered likely to be pathogenic since PHRD-
like scaled Combined Annotation-Dependent Depletion
(CADD) score suggested deleteriousness (25.9). Conclusions: It is likely that FLNC has a major role in
the pathogenesis of DCM. Therefore, FLNC should be part
of the core panel of cardiomyopathy genes. Furthermore,
variants in primary arrhythmia genes might explain a small
number of patients. 145 Abstracts from the 51st European Society of Human Genetics Conference: Posters E. Vanhoutte: None. J. Verdonschot: None. G. Claes:
None. A. van den Wijngaard: None. A. Helderman-van
den Enden: None. M. Hazebroek: None. S. Heymans:
None. H. Brunner: None. I. Krapels: None. Slegtenhorst5, R. H. Lekanne Deprez6, M. W. Wessels5,
M. Michels7, A. C. Houweling8, E. T. Hoorntje9, P. J. T. M. Helderman-van den Enden3, D. Q. M. C. Barge-Schaapveld10,
J. van Tintelen6, M. P. van den Berg4, A. A. M. Wilde6, H. Ploos
van Amstel1, E. A. M. Hennekam1, F. W. Asselbergs1, E. J. G. Sijbrands5, D. Dooijes1 Mortality Risk associated with Truncating Founder
Mutations in Titin P05.32D P05.33A
FABP4 gene expression in epicardial adipose tissue is
related to obesity associated coronary heart disease P05.33A
FABP4 gene expression in epicardial adipose tissue is
related to obesity associated coronary heart disease P05.31C Mortality Risk associated with Truncating Founder
Mutations in Titin Mortality Risk associated with Truncating Founder
Mutations in Titin M. Jansen1, A. F. Baas1, K. Y. van Spaendonck-Zwarts2, A. S. Ummels1, A. van den Wijngaard3, J. D. H. Jongbloed4, M. A. van 146 J. del Picchia M. Jansen:
None. A.F. Baas:
None. K.Y. van
Spaendonck-Zwarts: None. A.S. Ummels: None. A. van
den Wijngaard: None. J.D.H. Jongbloed: None. M.A. van Slegtenhorst: None. R.H. Lekanne Deprez: None. M. W. Wessels: None. M. Michels: None. A.C. Houweling:
None. E.T. Hoorntje: None. P.J.T.M. Helderman-van
den Enden: None. D.Q.M.C. Barge-Schaapveld: None. J. van Tintelen: None. M.P. van den Berg: None. A.A.M. Wilde: None. H. Ploos van Amstel: None. E.A.M. Hennekam:
None. F.W. Asselbergs:
None. E.J.G. Sijbrands: None. D. Dooijes: None. and also with heart failure severity and ACE inhibitors
intake. Conclusions: DNA methylation changes in unstable
CAPs are associated with activation of immune response
and impaired remodeling of artery wall, which can reflect
the underlying processes of plaque destabilization on
epigenetic level. The study is supported by a grant of
Russian Scientific Foundation (16-15-10150). A.V. Markov: None. M.S. Nazarenko: None. A. Zarubin: None. D.V. Sharysh: None. A.N. Kazantsev:
None. O.L. Barbarash: None. V.P. Puzyrev: None. DNA methylation changes in destabilization of carotid
atherosclerotic plaque A. V. Markov1, M. S. Nazarenko1,2,3, A. Zarubin1,2, D. V. Sharysh2, A. N. Kazantsev3, O. L. Barbarash3, V. P. Puzyrev1,2 V. Miroshnikova1, A. Panteleeva2, E. Polyakova3, I. Pobozheva4,
N. Razgildina4, O. Belyaeva3, E. Baranova3, O. Berkovich3,
S. Pchelina4 1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk,
Russian Federation, 2Siberian State Medical University,
Tomsk, Russian Federation, 3Research Institute for Complex
Issues of Cardiovascular Diseases, Kemerovo, Russian
Federation 1Petersburg nuclear physics institute, Gatchina, Russian
Federation, 2Petersburg nuclear physics institute, NRC,
Gatchina, Russian Federation, 3First Pavlov State Medical
University, St. Petersburg, Russian Federation, 4Petersburg
nuclear physics institute, NRC "Kurchatov Institute",
Gatchina, Russian Federation Introduction: Destabilization of carotid atherosclerotic
plaque (CAP) is known to be the cause of cerebrovascular
ischemia and stroke, but epigenetic framework for this
complex process is poorly investigated. Our study aimed to
compare genome-wide DNA methylation profiles in cells of
stable and unstable atherosclerotic plaques of carotid
arteries. Objective: The fatty acid-binding protein 4 (FABP4) has
been described as a biomarker for adiposity. Being
expressed by both adipocytes and macrophages it takes part
in intracellular lipid traffic and acts as an important adipo-
cytokine. Increased circulating FABP4 level is associated
with obesity, insulin resistance and atherosclerosis. How-
ever, little is known about FABP4 gene expression in epi-
cardial adipose tissue (EAT) during obesity and its potential
influence on coronary atherosclerosis development. Materials and Methods: CAPs were obtained from 16
patients (10 males, 6 females, aged 65 ± 6 years) and
divided into histologically stable (n = 8) and unstable
(n = 8) samples. CpG methylation in specimens was
estimated
using
Infinium
MethylationEPIC
BeadChip
(Illumina) and analyzed in R/Bioconductor. The aim of this study was to evaluate FABP4 mRNA
expression in EAT in patients with obesity and coronary
heart disease (CHD). Results: After discard of sex chromosome-mapped,
polymorphic, cross-hybridized and poorly detected micro-
array probes, of remained 775836 sites, 1829 CpG-sites
showed differential methylation (|Δβ|>0.15) according to
CAP instability (p < 0.05) and located commonly beyond
CpG-islands. Hypomethylated 1194 sites were overrepre-
sented genes functioning in immune cells and participating
in different biological processes associated with immune
response (i.e. leukocyte activation, T-cell differentiation,
etc.). Other 635 sites with elevated methylation level in
unstable plaques resided within genes largely involved in
morphogenesis and developmental processes (i.e. cytoske-
leton
organization,
cell
adhesion,
etc.). University of Nottingham, Nottingham, United Kingdom Introduction: Familial Hypercholesterolaemia (FH) is a
common inherited cause of raised cholesterol. However,
over 80% of people with FH are still not identified, leading
to many avoidable heart attacks and early deaths. This study
assessed the validity of a new FH case-finding algorithm
(FAMCAT), intended for application to patients’ primary
health care records, in a large population cohort. Material and methods: Using routine primary care data
from the UK Clinical Practice Research Datalink (CPRD),
we used primary care disease codes to select subjects with
clinical FH diagnoses who were free from CVD at baseline,
and matched them with population controls according to
age, sex and general practice. Cox proportional hazards
regression models stratified on the matched pairs, were used
to determine hazard ratios (HR) for CVD (up to 10 years
follow-up) between FH patients and their matched controls. Material and methods: Using routine primary care data
from the UK Clinical Practice Research Datalink (CPRD),
we used primary care disease codes to select subjects with
clinical FH diagnoses who were free from CVD at baseline,
and matched them with population controls according to
age, sex and general practice. Cox proportional hazards
regression models stratified on the matched pairs, were used
to determine hazard ratios (HR) for CVD (up to 10 years
follow-up) between FH patients and their matched controls. Material and Methods: Analysis of 747,194 patients’
data from QRESEARCH, a UK primary care database, was
conducted by applying FAMCAT regression equations to
predict probability of FH. There were 1,397 patients
diagnosed
with
definite
FH. Prediction
accuracy
of
FAMCAT, determined by the area under the receiver
operating characteristics curve (AUC), was compared to use
of established Simon-Broome and Dutch Lipid Clinic
criteria for FH. Material and Methods: Analysis of 747,194 patients’
data from QRESEARCH, a UK primary care database, was
conducted by applying FAMCAT regression equations to
predict probability of FH. There were 1,397 patients
diagnosed
with
definite
FH. Prediction
accuracy
of
FAMCAT, determined by the area under the receiver
operating characteristics curve (AUC), was compared to use
of established Simon-Broome and Dutch Lipid Clinic
criteria for FH. Results: The incidence rates of CVD in patients with FH
(n = 14073; 4,481 CVD events) and in the controls
(n = 42,130; 1,767 CVD events) were 25.8 and 3.1 per
1000 person-years respectively. DNA methylation changes in destabilization of carotid
atherosclerotic plaque FABP4
mRNA levels within obese subgroups were highly corre-
lated with proinflammatory macrophage marker CD68
mRNA levels (r = 0.602, p < 0.01). Conclusions: Our results indicate that FABP4 gene
expression in EAT is associated with coronary heart disease
developed on obesity background. Conclusions: Our results indicate that FABP4 gene
expression in EAT is associated with coronary heart disease
developed on obesity background. Conclusion: Individuals with clinical FH have signifi-
cantly higher risk of coronary heart disease, stroke/transient
ischaemic attack and peripheral vascular disease over time. Funder: NIHR HTA Programme Grant V. Miroshnikova: None. A. Panteleeva: None. E. Polyakova: None. I. Pobozheva: None. N. Razgildina:
None. O. Belyaeva: None. E. Baranova: None. O. Berkovich: None. S. Pchelina: None. B. Iyen: None. S. Weng: None. R. Akyea: None. J. Kai:
None. J. Leonardi-Bee: None. S.E. Humphries: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Steve E Humphries was funded by the
British Heart Foundation (BHF PG08/008) and by the
NIHR UCLH BRC. F. Consultant/Advisory Board; Modest;
Steve E Humphries was a member of the NICE Familial
Hypercholesterolaemia
Guideline
Development
group
(2016–2017). Other; Modest; Medical Director and minor-
ity
shareholder
of
a
UCL
spin-out
company
called
StoreGene, which uses a 20 SNP genetic test in combina-
tion with the classical risk profile, to estimate individual
CVD risk. P. Roderick: None. N. Qureshi: F. Consultant/
Advisory Board; Modest; Nadeem Qureshi was a member
of the NICE Familial Hypercholesterolaemia Guideline
Development group (2016–2017). B. Iyen: None. S. Weng: None. R. Akyea: None. J. Kai:
None. J. Leonardi-Bee: None. S.E. Humphries: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Steve E Humphries was funded by the
British Heart Foundation (BHF PG08/008) and by the
NIHR UCLH BRC. F. Consultant/Advisory Board; Modest;
Steve E Humphries was a member of the NICE Familial
Hypercholesterolaemia
Guideline
Development
group
(2016–2017). Other; Modest; Medical Director and minor-
ity
shareholder
of
a
UCL
spin-out
company
called
StoreGene, which uses a 20 SNP genetic test in combina-
tion with the classical risk profile, to estimate individual
CVD risk. P. Roderick: None. N. Qureshi: F. Consultant/
Advisory Board; Modest; Nadeem Qureshi was a member
of the NICE Familial Hypercholesterolaemia Guideline
Development group (2016–2017). The incidence and risk of cardiovascular disease outcomes
in patients with Familial Hypercholesterolaemia: a
matched survival analysis using a large UK primary care
prospective cohort The incidence and risk of cardiovascular disease outcomes
in patients with Familial Hypercholesterolaemia: a
matched survival analysis using a large UK primary care
prospective cohort B. Iyen1, S. Weng1, R. Akyea1, J. Kai1, J. Leonardi-Bee2, S. E. Humphries3, P. Roderick4, N. Qureshi1 B. Iyen1, S. Weng1, R. Akyea1, J. Kai1, J. Leonardi-Bee2, S. E. Humphries3, P. Roderick4, N. Qureshi1 B. Iyen1, S. Weng1, R. Akyea1, J. Kai1, J. Leonardi-Bee2, S. E. Humphries3, P. Roderick4, N. Qureshi1 1Division of Primary Care, University of Nottingham,
Nottingham, United Kingdom, 2Division of Epidemiology and
Public Health, University of Nottingham, Nottingham, United
Kingdom, 3Cardiovascular Genetics, Institute of
Cardiovascular Science, University College London, London,
United Kingdom, 4Faculty of Medicine, Primary care and
Population sciences, University of Southampton, Southampton,
United Kingdom 1Division of Primary Care, University of Nottingham,
Nottingham, United Kingdom, 2Division of Epidemiology and
Public Health, University of Nottingham, Nottingham, United
Kingdom, 3Cardiovascular Genetics, Institute of
Cardiovascular Science, University College London, London,
United Kingdom, 4Faculty of Medicine, Primary care and
Population sciences, University of Southampton, Southampton,
United Kingdom Improving identification of familial hypercholesterolaemia
in primary care using FAMCAT (Familial
Hypercholesterolaemia Case Ascertainment Tool):
validation in a large population database Improving identification of familial hypercholesterolaemia
in primary care using FAMCAT (Familial
Hypercholesterolaemia Case Ascertainment Tool):
validation in a large population database Introduction: Heterozygous familial hypercholesterolemia
(FH) is the most common inherited cause of raised LDL-
cholesterol and is associated with an increased risk of cor-
onary heart disease (CHD). This study aimed to determine if
FH is associated with other cardiovascular diseases (CVD)
including transient ischaemic attack (TIA), stroke and per-
ipheral vascular disease (PVD). W. Stephen, J. Kai, R. Akyea, N. Qureshi DNA methylation changes in destabilization of carotid
atherosclerotic plaque Unsupervised
analysis revealed moderate correlation of the first principal
components with measures of lipid core and cap of CAPs, Results: After discard of sex chromosome-mapped,
polymorphic, cross-hybridized and poorly detected micro-
array probes, of remained 775836 sites, 1829 CpG-sites
showed differential methylation (|Δβ|>0.15) according to
CAP instability (p < 0.05) and located commonly beyond
CpG-islands. Hypomethylated 1194 sites were overrepre-
sented genes functioning in immune cells and participating
in different biological processes associated with immune
response (i.e. leukocyte activation, T-cell differentiation,
etc.). Other 635 sites with elevated methylation level in
unstable plaques resided within genes largely involved in
morphogenesis and developmental processes (i.e. cytoske-
leton
organization,
cell
adhesion,
etc.). Unsupervised
analysis revealed moderate correlation of the first principal
components with measures of lipid core and cap of CAPs, Methods: We analyzed: 1) group of 50 patients with
CHD which consisted of 26 obese persons and 24 normal
weight persons; 2) control group of 12 individuals without
CHD which consisted of 6 obese persons and 6 normal
weight persons. EAT samples were obtained from CHD
patients during coronary bypass surgery and from control
persons during heart valve surgery. Atherosclerosis severity
was evaluated by coronary angiography. FABP4 and
macrophage marker CD68 mRNA levels in EAT were
determined by real-time RT-PCR. Results: FABP4 mRNA levels were significantly reduced
in obese CHD patients when compared with obese controls
(p < 0.01). Among normal weight persons there was no
difference in FABP4 expression between patients with 147 Abstracts from the 51st European Society of Human Genetics Conference: Posters 10.34 (95% CI: 9.56-11.17; p < 0.0001), 6.51 (95% CI:
5.65-7.51; p < 0.0001) and 6.93 (95% CI: 5.89-8.16;
p < 0.0001) respectively. These hazard ratios remained
significant after adjusting for known CVD risk factors. 10.34 (95% CI: 9.56-11.17; p < 0.0001), 6.51 (95% CI:
5.65-7.51; p < 0.0001) and 6.93 (95% CI: 5.89-8.16;
p < 0.0001) respectively. These hazard ratios remained
significant after adjusting for known CVD risk factors. CHD and corresponding controls without CHD. FABP4
mRNA levels within obese subgroups were highly corre-
lated with proinflammatory macrophage marker CD68
mRNA levels (r = 0.602, p < 0.01). CHD and corresponding controls without CHD. FABP4
mRNA levels within obese subgroups were highly corre-
lated with proinflammatory macrophage marker CD68
mRNA levels (r = 0.602, p < 0.01). CHD and corresponding controls without CHD. P05.37A Fibromuscular dysplasia: Identifying potential genetic
causal variants by whole-exome sequencing 1Department of Pediatrics, Section Molecular Genetics,
University Medical Center Groningen, University of
Groningen, Groningen, Netherlands, 2Department of Vascular
Medicine, University Medical Center Groningen, University of
Groningen, Groningen, Netherlands, 3Department of Vascular
Medicine, Academic Medical Center, Amsterdam, Netherlands,
4Department of Genetics, University Medical Center
Groningen, University of Groningen, Groningen, Netherlands E. Vanhoutte, G. Claes, I. Krapels, P. de Leeuw, H. Brunner,
A. Kroon E. Vanhoutte, G. Claes, I. Krapels, P. de Leeuw, H. Brunner,
A. Kroon University of Nottingham, Nottingham, United Kingdom Compared with controls,
the hazard ratio for CVD among those with clinical FH was
8.87 (95% CI: 8.31-9.47, p < 0.0001). The hazard ratios for
coronary heart disease (CHD), TIA/stroke and PVD were 148 J. del Picchia without genetic predisposition. 16,8% of women with high
LDL-c carried mutations causing familial hypercholester-
olemia, whereas 21% were predisposed to high LDL-c
based on polygenic risk-scores. Women without such
genetic predisposition exhibited a significantly unfavorable
lifestyle. Results: FAMCAT resulted in an overall AUC of 0.805
(95% confidence interval [CI] 0.793 - 0.817), performing
significantly better in identifying FH than Simon-Broome
(SB) criteria (0.692, 95% CI 0.680 - 0.705) or Dutch Lipid
Clinic (DLC) criteria (0.716, 95% CI 0.703 - 0.729). Prediction of FH by minority ethnic group resulted in AUCs
ranging from 0.755 (95% CI 0.626 - 0.885) for Asian/Asian
British to 0.871 (95% CI 0.808 - 0.934) for Black/Black
British/African. Conclusions: Our study demonstrates the need for early
assessment of cardiovascular risk profiles in apparently
healthy
young
women
to
identify
those
with
LDL
cholesterol levels above the 99th percentile for their age:
1) 17% of the cases appeared molecularly diagnosed with
familial hypercholesterolemia; 2) our data indicate that an
unfavorable lifestyle is significantly associated with severe
hypercholesterolemia in genetically unaffected women,
which may need further evaluation and advice to prevent
future cardiovascular complications. Conclusions: The FAMCAT algorithm offers significant
improvement for identifying FH compared to DLC or SB
criteria in UK primary care practice. FAMCAT may require
recalibration in, and for use other international and
ethnically diverse population groups. Further research
assessing the clinical utility of FAMCAT with genetic test
diagnosis is recommended. Grants:
CVON2011-2016;
CVON2017-2020;
FP7-
603091; NHS 2015T068 Grants:
CVON2011-2016;
CVON2017-2020;
FP7-
603091; NHS 2015T068 W. Stephen: F. Consultant/Advisory Board; Modest;
roadtohealth ltd. J. Kai: None. R. Akyea: None. N. Qureshi: None. W. Stephen: F. Consultant/Advisory Board; Modest;
roadtohealth ltd. J. Kai: None. R. Akyea: None. N. Qureshi: None. A. Rimbert: None. J. Balder: None. X. Zhang: None. M. Viel: None. R. Kanninga: None. F. van Dijk: None. P. Lansberg:
None. R.J. Sinke:
None. J. Kuivenhoven: None. A. Rimbert: None. J. Balder: None. X. Zhang: None. M. Viel: None. R. Kanninga: None. F. van Dijk: None. P. Lansberg:
None. R.J. Sinke:
None. J. Kuivenhoven: None. Genetics, lifestyle and cholesterol in young women Genetics, lifestyle and cholesterol in young women A. Rimbert1, J. Balder1,2, X. Zhang3, M. Viel4, R. Kanninga4,
F. van Dijk4, P. Lansberg1, R. J. Sinke4, J. Kuivenhoven1 Maastricht University Medical Center, Maastricht,
Netherlands Background: Fibromuscular dysplasia (FMD) is a nonin-
flammatory, nonartherosclerotic disorder of medium-sized
arteries. FMD leads to arterial stenosis, occlusion, aneur-
ysms and dissection, most commonly involving renal and
internal carotid arteries. Clinical manifestations commonly
include hypertension, dizziness and pulsatile tinnitus. Introduction: Low-density lipoprotein cholesterol (LDL-
c), a causal risk factor for atherosclerosis, is mostly studied
upon clinical events. Women are usually affected later in
life than men and are underdiagnosed and undertreated in
cardiovascular investigations. This study addresses genetic
and lifestyle factors affecting LDL-c in young women. The etiology of FMD remains unknown. Since FMD
occurs in families in approximately 10%, a genetic origin
seems plausible. Methods: Premenopausal women with LDL-c ≤1st
percentile (≤50 mg/dl; n = 119) and ≥99th percentile (≥
186
mg/dl;
n = 121)
were
selected
from
a
Dutch
population-based cohort (Lifelines). We applied a one-step,
novel NGS-based gene-panel to establish genetic origins of
hypo- and hypercholesterolemia. A “healthy lifestyle score”
was extracted from questionnaires. Aims: To identify genetic variants involved in FMD. Methods: So far, genetic analysis through whole exome
sequencing was performed in five patients with FMD. Only
rare and novel truncating, missense and deletion variants
predicted to be conserved and deleterious are considered to
be of interest. Results: 15,7% of women with low LDL-c carried
mutations causing monogenic hypocholesterolemia and
49,6% were genetically predisposed to low LDL-c based
on extremely low weighted genetic risk-scores. A healthier
lifestyle was not associated with low LDL-c in women Results: In one patient, we identified a missense variant
in COL4A1, a nonsense variant in COL4A2 and an 85kb
deletion in ACTA2. In two other patients, a missense variant
in JAG1 and a missense variant in COL4A2 were identified
respectively. 149 Abstracts from the 51st European Society of Human Genetics Conference: Posters The collagen IV network seems to be critical for structural
integrity in the basement membrane. COL4A1 and COL4A2
mutations are associated with multisystem disorders with
abnormalities in the vasculature and other organs. strong evidence of heterogeneity in allelic effects on T2D
(pHET<1.5x10-4, Bonferroni
correction) correlated
with
ancestry at 16% of signals, including LEP (rs7778167,
pHET=4x10-25, East Asian specific) and multiple associa-
tions at/near KCNQ1 and TCF7L2 (representing ethnic-
specific/-differentiated
effects). T2D-associated
variants
showed significant enrichment (odds-ratio range 1.90-6.63;
p<0.05) in coding exons, pancreatic islet enhancers and
promoters, adipose enhancers, and binding sites for tran-
scription factors, including NKX2.2 and FOXA2. Discovery and fine-mapping of type 2 diabetes susceptibility
loci in diverse populations using more than a million
individuals A. Mahajan1,2, H. Kitajima1, X. Sim3, M. C. Y. Ng4, W. Zhang5,
J. E. Below6, K. J. Gaulton7, A. P. Morris1,8, on behalf of the
DIAMANTE Consortium A. Mahajan: None. H. Kitajima: None. X. Sim: None. M.C.Y. Ng: None. W. Zhang: None. J.E. Below: None. K.J. Gaulton: None. A.P. Morris: None. A. Mahajan: None. H. Kitajima: None. X. Sim: None. M.C.Y. Ng: None. W. Zhang: None. J.E. Below: None. K.J. Gaulton: None. A.P. Morris: None. 1Wellcome Centre for Human Genetics, Oxford, United
Kingdom, 2Oxford Centre for Diabetes, Endocrinology and
Metabolism, Radcliffe Department of Medicine, University of
Oxford, Oxford, United Kingdom, 3Saw Swee Hock School of
Public Health, National University of Singapore, Singapore,
Singapore, 4Center for Genomics and Personalized Medicine
Research, Wake Forest School of Medicine, Winston-Salem,
Winston-Salem, NC, United States, 5School of Public Health,
Imperial College London, London, United Kingdom, 6Human
Genetics Center, University of Texas Health Science Center at
Houston, Houston, TX, United States, 7Department of
Pediatrics, University of California San Diego, La Jolla, CA,
United States, 8Department of Biostatistics, University of
Liverpool, Liverpool, United Kingdom Maastricht University Medical Center, Maastricht,
Netherlands Increased
sample size, population diversity, and annotation-informed
fine-mapping substantially improved localisation of poten-
tial causal variants compared with previous efforts, and
highlighted 76 signals with a single variant accounting for
>80% of the posterior probability of association (PPA); of
these 35 signals had PPA of >99%. Clustering of signal-
specific annotation enrichment highlighted distinct clades of
T2D associations driven by different underlying molecular
processes. These analyses represent the most comprehen-
sive view of the genetic contribution to T2D to date and,
through integration with expression quantitative trait loci in
disease-relevant tissues, point to previously unreported
effector genes and mediating molecular mechanisms at
several loci. Mutations in JAG1 causes Alagille syndrome in which
vascular anomalies including bilateral renal artery stenosis
leading to hypertension and internal carotid artery aneur-
ysms have been reported. Literature reports one mutation in ACTA2 causing
dilatation of proximal internal carotid artery, occlusive
disease of terminal internal carotid artery, and abnormally
straight course of intracranial arteries. Conclusions: FMD is likely to be a multifactorial
disorder, however vascular and connective tissue genes
might be involved in the pathogenesis of FMD. E. Vanhoutte: None. G. Claes: None. I. Krapels: None. P. de Leeuw: None. H. Brunner: None. A. Kroon: None. P05.38B Discovery and fine-mapping of type 2 diabetes susceptibility
loci in diverse populations using more than a million
individuals P05.39C P05.39C
Copy number variation analysis in cardiac congenital
septal defects P05.39C
Copy number variation analysis in cardiac congenital
septal defects G. Crauciuc1, F. Tripon1, L. Gozar2, R. Togănel2, C. Bănescu3 G. Crauciuc1, F. Tripon1, L. Gozar2, R. Togănel2, C. Bănescu3 1University of Medicine and Pharmacy, Tîrgu Mureş,
Romania, 2Emergency Institute for Cardiovascular Diseases
and Transplantation, Tîrgu Mureş, Romania, 3Genetics
Laboratory, Center for Advanced Medical and Pharmaceutical
Research, University of Medicine and Pharmacy, Tîrgu Mureş,
Romania Congenital cardiac septal defects (CCSD) are the most
frequently type of congenital heart malformation, often
occurs sporadically, without an obvious cause in most
cases. Recent studies demonstrated the involvement of
several genes variation in cardiac development process,
factors that have been underestimated in the past. The aim
of our study was to evaluate if the CCSD patients present
copy number variations (CNVs) and establish whether the
CNVs investigation should be performed in management of
this patients. A number of 26 pediatric patients were
enrolled. The CNVs were determined using the multiplex
ligation-dependent probe amplification (MLPA) technique To discover type 2 diabetes (T2D) loci and enhance fine-
mapping resolution, we conducted the largest meta-analysis
of genome-wide association studies of the disease to date by
aggregating 171,262 cases and 1,075,072 controls from
diverse populations (45% non-European ancestry). We
identified 208 loci at genome-wide significance (p<5x10-8),
including 41 mapping outside regions previously implicated
in T2D (accounting for those discovered in European-
specific component of this study). Across these loci, con-
ditional analyses revealed a total of 342 distinct signals of
association (locus-wide significance, p<10-5). We observed 150 J. del Picchia and P234-A3 SALSA MLPA probemix which contain
probe for GATA 3 and GATA 4 gene analysis. The MLPA
analysis revealed CNVs in two patients. Both of them
present a duplication located in 10p14 (GATA 3 gene,
exons 4 and 5). Several patients present a high ratio for
8p23.1 (GATA 4 gene) and a low ratio for 10p14 (GATA 3
gene) but the signals were statistically insignificant. Six
patients present low ratio for flanking probe (CELF2 probe,
according to the manufacturer CNVs of flanking probes are
unlikely to be related to the condition tested). Twenty
patients received recommendation for target sequencing of
different exons. Based on our results, we may consider the
mentioned MLPA kit as a first step in CCSD patient’s
management. and P234-A3 SALSA MLPA probemix which contain
probe for GATA 3 and GATA 4 gene analysis. The MLPA
analysis revealed CNVs in two patients. 1Molecular and Clinical Sciences Institute, St George's
University of London, London, United Kingdom, 2Department
of Genetics, Faculty of Science, Shahid Chamran University of
Ahvaz, Ahvaz, Iran, Islamic Republic of, 3Narges Medical
Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran,
Islamic Republic of P05.39C
Copy number variation analysis in cardiac congenital
septal defects Results: Analyses of 25 HCM patients revealed a total of
54 variants in cardiac genes of which 9 (16,7%) were
pathogenic, 5 (%9,2) were potentially pathogenic, 34 (63%)
were variant of unknown significance (VUS) and 6 (11,1%)
were likely benign. Only one of the patient (4%) had no
suspicious variant in examined genes. 10 patients (40%)
carried
at
least
one pathogenic/potentially pathogenic
mutations in MYH6, MYH7, MYBPC3, TNNI3, TNNT2,
DSP, DPP6, JUP, KCNQ1 and SCN5A genes, while other
14 patients (56%) had at least one VUS variant. A total of
12 novel variants were identified, which consist of 1
frameshift insertion in JUP gene, 1 frameshift deletion in
DPP6 gene and 9 missense variants in DPP6, MYH7,
MYH6, GJA5, NKX2-5, TMPO and TRDN genes. An
interesting finding is co-occurrence of dominant pathogenic/
likely pathogenic variants in different genes, as in the three
cases explains the complexity and severity of the observed
phenotypes. G. Crauciuc: None. F. Tripon: None. L. Gozar: None. R. Togănel: None. C. Bănescu: None. G. Crauciuc: None. F. Tripon: None. L. Gozar: None. R. Togănel: None. C. Bănescu: None. G. Crauciuc: None. F. Tripon: None. L. Gozar: None. R. Togănel: None. C. Bănescu: None. P05.39C
Copy number variation analysis in cardiac congenital
septal defects Both of them
present a duplication located in 10p14 (GATA 3 gene,
exons 4 and 5). Several patients present a high ratio for
8p23.1 (GATA 4 gene) and a low ratio for 10p14 (GATA 3
gene) but the signals were statistically insignificant. Six
patients present low ratio for flanking probe (CELF2 probe,
according to the manufacturer CNVs of flanking probes are
unlikely to be related to the condition tested). Twenty
patients received recommendation for target sequencing of
different exons. Based on our results, we may consider the
mentioned MLPA kit as a first step in CCSD patient’s
management. Introduction:
Familial
Hypertrophic
cardiomyopathy
(HCM) is a multigenic heart condition characterized by
hypertrophy of the cardiac muscle. The prevalence of HCM
is estimated at 1:500 in the general population.The aim of
our study was evaluation of complex genetic profile of next
generation sequencing (NGS) results in Turkish patients
with HCM. Introduction:
Familial
Hypertrophic
cardiomyopathy
(HCM) is a multigenic heart condition characterized by
hypertrophy of the cardiac muscle. The prevalence of HCM
is estimated at 1:500 in the general population.The aim of
our study was evaluation of complex genetic profile of next
generation sequencing (NGS) results in Turkish patients
with HCM. Methods: We designed a targeted cardiac panel of 68
genes using NGS technology on Iontorrent PGM. Results: Analyses of 25 HCM patients revealed a total of
54 variants in cardiac genes of which 9 (16,7%) were
pathogenic, 5 (%9,2) were potentially pathogenic, 34 (63%)
were variant of unknown significance (VUS) and 6 (11,1%)
were likely benign. Only one of the patient (4%) had no
suspicious variant in examined genes. 10 patients (40%)
carried
at
least
one pathogenic/potentially pathogenic
mutations in MYH6, MYH7, MYBPC3, TNNI3, TNNT2,
DSP, DPP6, JUP, KCNQ1 and SCN5A genes, while other
14 patients (56%) had at least one VUS variant. A total of
12 novel variants were identified, which consist of 1
frameshift insertion in JUP gene, 1 frameshift deletion in
DPP6 gene and 9 missense variants in DPP6, MYH7,
MYH6, GJA5, NKX2-5, TMPO and TRDN genes. An
interesting finding is co-occurrence of dominant pathogenic/
likely pathogenic variants in different genes, as in the three
cases explains the complexity and severity of the observed
phenotypes. Next Generation Sequencing (NGS) panel revealed new
candidate genes and variants in 25 Hypertrophic
Cardiomyopathy patients Next Generation Sequencing (NGS) panel revealed new
candidate genes and variants in 25 Hypertrophic
Cardiomyopathy patients B. Turkgenc1, S. G. Temel2,3, F. Uysal4, S. Ugan Atik5,
F. Oztunc5, A. Sulu6, F. Ekici7, C. Ayabakan8, E. Odemis9,
A. Saygili10, A. Koka5, I. Ozkan Akinci11, Y. Alanay12,13,
A. Celiker14, A. Ozer15, M. C. Yakicier16 1Acibadem Genetic Diagnostic Center, Istanbul, Turkey,
2Department of Medical Genetic, Faculty of Medicine, Uludag
University, Bursa, Turkey, 3Department of Histology and
Embryology, Faculty of Medicine, Uludag University, Bursa,
Turkey, 4Department of Pediatric Cardiology, Faculty of
Medicine, Uludag University, Bursa, Turkey, 5Department of
Pediatric Cardiology, Cerrahpasa Medical Faculty, Istanbul,
Turkey, 6Department of Pediatric Cardiology, Faculty of
Medicine, Gaziantep University, Gaziantep, Turkey,
7Department of Pediatric Cardiology, Faculty of Medicine,
Akdeniz University, Antalya, Turkey, 8Department of Pediatric
Cardiology, Baskent University Istanbul Hospital, Istanbul,
Turkey, 9Department of Pediatric Cardiology, Acibadem
University, Acibadem Atakent Hospital, Istanbul, Turkey,
10Department of Pediatric Cardiology, Medical Faculty of
Acibadem University, Istanbul, Turkey, 11Department of
Anesthesiology and Reanimation, Istanbul Medical Faculty,
Istanbul, Turkey, 12Department of Medical Genetics, Acibadem
University, Istanbul, Turkey, 13Department of Pediatric
Genetic, Acibadem Maslak Hospital, Istanbul, Turkey,
14Department of Pediatric Cardiology, Faculty of Medicine,
Koç University, Istanbul, Turkey, 15Department of Medical
Biology and Genetics, Marmara University, Istanbul, Turkey,
16Department of Molecular Biology and Genetic, Acibadem
University, Faculty of Science, Istanbul, Turkey Conclusion:
Identification
of
pathogenic/potentially
pathogenic variants and novel candidate variants/genes
has led to new opportunities for prevention and therapy of
lethal HCM. Acknowledgements: The study was supported
by SANTEZ Grant (0253. STZ.2013-2), Turkey. Conclusion:
Identification
of
pathogenic/potentially
pathogenic variants and novel candidate variants/genes
has led to new opportunities for prevention and therapy of
lethal HCM. Acknowledgements: The study was supported
by SANTEZ Grant (0253. STZ.2013-2), Turkey. B. Turkgenc: None. S.G. Temel: None. F. Uysal: None. S. Ugan Atik: None. F. Oztunc: None. A. Sulu: None. F. Ekici: None. C. Ayabakan: None. E. Odemis: None. A. Saygili: None. A. Koka: None. I. Ozkan Akinci: None. Y. Alanay: None. A. Celiker: None. A. Ozer: None. M.C. Yakicier: None. B. Turkgenc: None. S.G. Temel: None. F. Uysal: None. S. Ugan Atik: None. F. Oztunc: None. A. Sulu: None. F. Exome sequencing in Russian families with noncompaction
cardiomyopathy Exome sequencing in Russian families with noncompaction
cardiomyopathy P05.43C Six of
them are located in the coding regions of the gene and lead
to an amino acid substitution. To assess the pathogenicity of
each SNV, we performed a functional investigation of the
corresponding variant of the receptor proteins. Materials and Methods: Binding, uptake, and degrada-
tion of 125-I-labeled LDL by cultured CHO cells expres-
sing wild-type and mutant LDLR proteins were compared
in the presence and absence of an excess of unlabelled LDL. Levels of LDLR mRNA expression were determined by
qRT-PCR 48 h after transfection with plasmids. Results: Four out of the six LDLR mutants showed a
markedly reduced in vitro receptor activity, suggesting their
role in the pathogenesis of FH. Conclusions: According to in silico predictions and
conservation analysis in multiple species, two of the six
identified LDLR variants are likely benign SNVs. The
remaining four variants can be categorized as “pathogenic
variants” that expand the spectrum of FH-linked LDLR
mutations in the Argentinian population. A. Gómez: None. G. Giunta: None. L. Helman: None. A. Pontoglio: None. L. Kaeser: None. U. Toscanini:
None. R. Colombo: None. L. Cuniberti: None. R. Maroofian: None. N. Mazaheri: None. M. Zamani:
None. G. Shariati:
None. A. Sedaghat:
None. Y. Jamshidi: None. H. Galehdari: None. R. Maroofian: None. N. Mazaheri: None. M. Zamani:
None. G. Shariati:
None. A. Sedaghat:
None. Y. Jamshidi: None. H. Galehdari: None. 1PRICAI-FUNDACIÓN FAVALORO, Buenos Aires, Argentina,
2Unidad Metabólica, Hospital Universitario Fundación
Favaloro (HUFF), Buenos Aires, Argentina, 3Laboratorio de
Lípidos y Aterosclerosis, IMETTYB‒Universidad Favaloro‒
CONICET, Buenos Aires, Argentina, 4Center for the Study of
Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan
Hospital, Milan, Italy, 5Institute of Clinical Biochemistry,
Faculty of Medicine, Catholic University and Policlinico
Agostino Gemelli, Rome, Italy P05.43C The
variant occurs in the protein C-terminal region of JPH2, just
upstream of a 22-amino acid transmembrane
anchor
responsible for binding of the protein to the sarcoplasmic/
endoplasmic
reticulum,
thereby
potentially
disrupting
binding. Our findings add to the growing evidence that
mutations in JPH2 play a role in HCM; and suggest that this
novel biallelic truncating mutation can give rise to severe,
early-onset pediatric cardiomyopathy. Pediatric
cardiomyopathies
represent
a
clinically
and
genetically heterogeneous group of disorders affecting the
ventricular myocardium, with an annual incidence of ~ 1.5
per 100,000 children. They are associated with substantial
morbidity and mortality: up to 40% die or undergo trans-
plantation. Here we studied two unrelated consanguineous
families from the same geographic region of Iran, with 4
children who died due to infantile cardiomyopathy. Affec-
ted offspring presented with severe hypertrophic cardio-
myopathy and right atrium enlargement in utero, at birth, or
in early childhood. Whole exome sequencing (WES) of
DNA from the proband of the first family led to identifi-
cation of a novel homozygous frameshift variant (p. Glu641*) in JPH2. The parents of the proband and a
healthy
sibling
were
heterozygous. Echocardiography
revealed no abnormalities in the carriers. Due to unavail-
ability of samples WES could only be carried out for the
parents of the second family, however the same hetero-
zygous frameshift variant was identified in both parents. The variant is novel and absent from population databases
including gnomAD, the ethnically-matched GME variome,
Iranome, and in ~1000 WES in-house control subjects from
the same geographic region as the families investigated. The
variant occurs in the protein C-terminal region of JPH2, just
upstream of a 22-amino acid transmembrane
anchor
responsible for binding of the protein to the sarcoplasmic/
endoplasmic
reticulum,
thereby
potentially
disrupting
binding. Our findings add to the growing evidence that
mutations in JPH2 play a role in HCM; and suggest that this
novel biallelic truncating mutation can give rise to severe,
early-onset pediatric cardiomyopathy. Pediatric
cardiomyopathies
represent
a
clinically
and
genetically heterogeneous group of disorders affecting the
ventricular myocardium, with an annual incidence of ~ 1.5
per 100,000 children. They are associated with substantial
morbidity and mortality: up to 40% die or undergo trans-
plantation. Here we studied two unrelated consanguineous
families from the same geographic region of Iran, with 4
children who died due to infantile cardiomyopathy. P05.43C Affec-
ted offspring presented with severe hypertrophic cardio-
myopathy and right atrium enlargement in utero, at birth, or
in early childhood. Whole exome sequencing (WES) of
DNA from the proband of the first family led to identifi-
cation of a novel homozygous frameshift variant (p. Glu641*) in JPH2. The parents of the proband and a
healthy
sibling
were
heterozygous. Echocardiography
revealed no abnormalities in the carriers. Due to unavail-
ability of samples WES could only be carried out for the
parents of the second family, however the same hetero-
zygous frameshift variant was identified in both parents. The variant is novel and absent from population databases
including gnomAD, the ethnically-matched GME variome,
Iranome, and in ~1000 WES in-house control subjects from
the same geographic region as the families investigated. The
variant occurs in the protein C-terminal region of JPH2, just
upstream of a 22-amino acid transmembrane
anchor
responsible for binding of the protein to the sarcoplasmic/
endoplasmic
reticulum,
thereby
potentially
disrupting
binding. Our findings add to the growing evidence that
mutations in JPH2 play a role in HCM; and suggest that this
novel biallelic truncating mutation can give rise to severe,
early-onset pediatric cardiomyopathy. Introduction: Mutations in the gene encoding the low-
density lipoprotein receptor (LDLR) are involved in the
molecular
etiology
of
autosomal
dominant
familial
hypercholesterolemia (FH), an hereditary condition asso-
ciated with coronary heart disease and the risk of premature
death. Based on FH prevalence among Caucasians, it is
expected that >80,000 cases are present in Argentina, less
than 1% of which have been identified so far. Previously, in
a cohort of Argentinian FH patients we detected eight
unreported LDLR single nucleotide variants (SNVs). Six of
them are located in the coding regions of the gene and lead
to an amino acid substitution. To assess the pathogenicity of
each SNV, we performed a functional investigation of the
corresponding variant of the receptor proteins. Introduction: Mutations in the gene encoding the low-
density lipoprotein receptor (LDLR) are involved in the
molecular
etiology
of
autosomal
dominant
familial
hypercholesterolemia (FH), an hereditary condition asso-
ciated with coronary heart disease and the risk of premature
death. Based on FH prevalence among Caucasians, it is
expected that >80,000 cases are present in Argentina, less
than 1% of which have been identified so far. Previously, in
a cohort of Argentinian FH patients we detected eight
unreported LDLR single nucleotide variants (SNVs). A. Gómez1, G. Giunta2,3, L. Helman2, A. Pontoglio4, L. Kaeser1,
U. Toscanini1, R. Colombo4,5, L. Cuniberti3 P05.43C P05.43C
Identification of a novel homozygous loss-of-function
variant in JPH2 in two unrelated families affected by lethal
Neonatal hypertrophic cardiomyopathy Identification of a novel homozygous loss-of-function
variant in JPH2 in two unrelated families affected by lethal
Neonatal hypertrophic cardiomyopathy
R. Maroofian1, N. Mazaheri2,3, M. Zamani2,3, G. Shariati2,3,
A. Sedaghat2,3, Y. Jamshidi1, H. Galehdari2,3
1Molecular and Clinical Sciences Institute, St George's
University of London, London, United Kingdom, 2Department
of Genetics, Faculty of Science, Shahid Chamran University of
Ahvaz, Ahvaz, Iran, Islamic Republic of, 3Narges Medical
Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran,
Islamic Republic of R. Maroofian1, N. Mazaheri2,3, M. Zamani2,3, G. Shariati2,3,
A. Sedaghat2,3, Y. Jamshidi1, H. Galehdari2,3 R. Maroofian1, N. Mazaheri2,3, M. Zamani2,3, G. Shariati2,3,
A. Sedaghat2,3, Y. Jamshidi1, H. Galehdari2,3 1Molecular and Clinical Sciences Institute, St George's
University of London, London, United Kingdom, 2Department
of Genetics, Faculty of Science, Shahid Chamran University of
Ahvaz, Ahvaz, Iran, Islamic Republic of, 3Narges Medical
Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran,
Islamic Republic of 151 Abstracts from the 51st European Society of Human Genetics Conference: Posters Pediatric
cardiomyopathies
represent
a
clinically
and
genetically heterogeneous group of disorders affecting the
ventricular myocardium, with an annual incidence of ~ 1.5
per 100,000 children. They are associated with substantial
morbidity and mortality: up to 40% die or undergo trans-
plantation. Here we studied two unrelated consanguineous
families from the same geographic region of Iran, with 4
children who died due to infantile cardiomyopathy. Affec-
ted offspring presented with severe hypertrophic cardio-
myopathy and right atrium enlargement in utero, at birth, or
in early childhood. Whole exome sequencing (WES) of
DNA from the proband of the first family led to identifi-
cation of a novel homozygous frameshift variant (p. Glu641*) in JPH2. The parents of the proband and a
healthy
sibling
were
heterozygous. Echocardiography
revealed no abnormalities in the carriers. Due to unavail-
ability of samples WES could only be carried out for the
parents of the second family, however the same hetero-
zygous frameshift variant was identified in both parents. The variant is novel and absent from population databases
including gnomAD, the ethnically-matched GME variome,
Iranome, and in ~1000 WES in-house control subjects from
the same geographic region as the families investigated. Functional analysis of six novel LDLR mutations in the
Argentinian population Functional analysis of six novel LDLR mutations in the
Argentinian population A. N. Meshkov1, O. V. Kulikova1, R. P. Myasnikov1, N. V. Shcherbakova1, A. A. Zharikova1, S. N. Koretsky1, A. V. Kiseleva1, A. I. Ershova1, M. S. Kharlap1, E. N. Basargina2,
N. A. Sdvigova2, E. A. Mershina3, V. E. Sinitsyn3, O. M. Drapkina1, S. A. Boytsov1 A. Gómez1, G. Giunta2,3, L. Helman2, A. Pontoglio4, L. Kaeser1,
U. Toscanini1, R. Colombo4,5, L. Cuniberti3 1PRICAI-FUNDACIÓN FAVALORO, Buenos Aires, Argentina, 1National Medical Research Center for Preventive Medicine,
Moscow, Russian Federation, 2National Medical Research
Center of Children's Health, Moscow, Russian Federation,
3Federal Center of Treatment and Rehabilitation, Moscow,
Russian Federation 1National Medical Research Center for Preventive Medicine,
Moscow, Russian Federation, 2National Medical Research
Center of Children's Health, Moscow, Russian Federation,
3Federal Center of Treatment and Rehabilitation, Moscow,
Russian Federation Introduction: Left ventricular noncompaction cardiomyo-
pathy (LVNC), a relatively rare cardiomyopathy, is char-
acterized by a high incidence of serious complications and
early death. Over 60 genes associated with the development J. del Picchia 152 variants from 73 patients with LQT. For all patients we did
exome sequencing using Agilent Focused Exome enrich-
ment panel and Illumina HiSeq2500. For variant calling and
pathogenicity scoring we followed ACMG guidelines. We
also confirmed all mutations by Sanger sequencing. No
disease-causing mutations were identified in 14 cases sug-
gesting new genes might be involved in pathogenesis. 59
patients had 91 disease-causing variants in LQT and cardiac
arrhythmias associated genes. 27 patients had more than one
variant in those genes. We discovered 22 new variants, that
were not reported previously in dbNSFP, Clinvar, OMIM,
HGMD, 1000Genomes and ExAC . In almost half cases
(46)
we
discovered
pathogenic
variants
in
KCNE1,
KCNH2, KCNQ1 or SCN5A genes. In remaining cases (45)
we found pathogenic variants in 18 other genes, including 7
variants in ANK2, which are rarely reported in LQT
patients.This work was funded by Fundamental Scientific
Research Program of the Russian Academy of Sciences for
2013-2020. of family cases of LVNC are described, in most cases the
inheritance type is autosomal dominant. However, causal
variants in these genes are detected in less than 50% of
families. The search for new genes is actual. Materials and Methods: We formed the cohort of
patients with LVNC and their relatives of 1 and 2 degrees of
kinship. The enrollment of participants was done by cascade
and reverse-cascade methods in the heart failure center. Probands should have met echocardiographic and cardiac
MRI criteria for noncompaction. Molecular testing was
performed using exome sequencing for members of 14
families (43 participants). We searched for pathogenic and
probably pathogenic variants in 66 genes associated with
LVNC and additionally in 122 genes associated with other
cardiomyopathies. Results: In 8 out of 14 families pathogenic or probably
pathogenic variants of the nucleotide sequence were
identified. D. Tavian1, S. Missaglia1, P. E. Maltese2, S. Michelini3,
M. Bertelli2 1University, Milan, Italy, 2Magi-onlus, Rovereto, Italy, 3San
Giovanni Battista Hospital, Rome, Italy Introduction: Forkhead transcription factor FOXC2 is
essential for the correct development and maintenance of
lymphatic system. Mutations in the FOXC2 gene are asso-
ciated with primary lymphedema-distichiasis syndrome
(LD), a congenital autosomal dominant disorder that causes
a dysfunction of the lymphatic vessels. It has been
demonstrated that FOXC2 variations can either reduce or
increase
protein
function. To
clarify
the
molecular
mechanism through which FOXC2 mutations can affect
transcriptional activity, we analyzed two missense muta-
tions (p.L80F and p.R121H), two frameshift variations (p. H199Pfs264* and p.M276Dfs186*) and one nonsense
mutation (p.Y109*), previously identified in LD patients. 1PRICAI-FUNDACIÓN FAVALORO, Buenos Aires, Argentina, Only 4 variants were found in genes associated
with LVNC - 2 pathogenic variants in TTN and 2 probably
pathogenic in MYBPC3 and TPM1. One pathogenic variant
was found in VCL and 3 probably pathogenic variants were
found in DES, TBX1 and FHOD3 associated with other
cardiomyopathies. E.G. Okuneva: None. A.A. Kozina: None. K. Tsuka-
nov: None. A. Krasnenko: None. P. Shatalov: None. T.A. Trofimova:
None. V.M. Solovyev:
None. V.V. Ilinsky: None. E.G. Okuneva: None. A.A. Kozina: None. K. Tsuka-
nov: None. A. Krasnenko: None. P. Shatalov: None. T.A. Trofimova:
None. V.M. Solovyev:
None. V.V. Ilinsky: None. Conclusions:
The
data
obtained
may
indicate
the
detection of 4 new genes (DES, VCL, TBX1, FHOD3)
associated with LVNC. The reported study was funded by
RFBR according to the research project #17-04-00521. Long QT syndrome mutation spectrum in Russians E. G. Okuneva1, A. A. Kozina1,2, K. Tsukanov1, A. Krasnenko1,
P. Shatalov3, T. A. Trofimova3, V. M. Solovyev3, V. V. Ilinsky1 1Genotek Ltd.(Genotek IT.), Moscow, Russian Federation,
2Institute of Biomedical Chemistry, Moscow, Russian
Federation, 3The Research and Clinical Institute for Pediatrics
named after Academician Yuri Veltischev of the Pirogov
Russian National Research Medical University of the Russian
Ministry of Health, Moscow, Russian Federation 1Genotek Ltd.(Genotek IT.), Moscow, Russian Federation,
2Institute of Biomedical Chemistry, Moscow, Russian
Federation, 3The Research and Clinical Institute for Pediatrics
named after Academician Yuri Veltischev of the Pirogov
Russian National Research Medical University of the Russian
Ministry of Health, Moscow, Russian Federation Mutations in more than 16 genes can cause long QT syn-
drome (LQT). Detection of variants in these genes is
necessary for diagnostics, targeted gene therapy, behavioral
management, family screening and prophylaxis of sudden
cardiac death. We report frequencies of disease-causing Material
and
Methods:
To
investigate
subcellular
localization, HeLa cells have been transfected with FOXC2
mutant plasmids, expressing FOXC2 with GFP at the N-
terminus,
and
immunofluorescence
analysis
has
been
performed. Subsequently, transactivation activity has been Material
and
Methods:
To
investigate
subcellular
localization, HeLa cells have been transfected with FOXC2
mutant plasmids, expressing FOXC2 with GFP at the N-
terminus,
and
immunofluorescence
analysis
has
been
performed. Subsequently, transactivation activity has been Material
and
Methods:
To
investigate
subcellular
localization, HeLa cells have been transfected with FOXC2
mutant plasmids, expressing FOXC2 with GFP at the N-
terminus,
and
immunofluorescence
analysis
has
been
performed. Subsequently, transactivation activity has been Lymphedema distichiasis syndrome: dominant FOXC2
mutations causing protein aggregation and loss of
transcriptional activity Lymphedema distichiasis syndrome: dominant FOXC2
mutations causing protein aggregation and loss of
transcriptional activity A.N. Meshkov: None. O.V. Kulikova: None. R.P. Myasnikov: None. N.V. Shcherbakova: None. A.A. Zharikova: None. S.N. Koretsky: None. A.V. Kiseleva:
None. A.I. Ershova: None. M.S. Kharlap: None. E.N. Basargina: None. N.A. Sdvigova: None. E.A. Mershina:
None. V.E. Sinitsyn: None. O.M. Drapkina: None. S.A. Boytsov: None. D. Tavian1, S. Missaglia1, P. E. Maltese2, S. Michelini3,
M. Bertelli2 D. Tavian1, S. Missaglia1, P. E. Maltese2, S. Michelini3,
M. Bertelli2 153 Abstracts from the 51st European Society of Human Genetics Conference: Posters associated with a markedly increase on cell proliferation
and migration. M13 phage-RGD characterized a strong
increase in MMP-9, eNOS and VEGF-A mRNAs as well as
leading to higher expression level of VEGF-A protein,
MMP-9 activity and NO release in HUVECs in comparison
to other surfaces. evaluated by a Luciferase assay. Finally, the effects of
missense and nonsense mutations on FOXC2 protein
structure have been evaluate using bioinformatics tools. Results:
p.L80F,
p.R121H,
p.H199Pfs264*
and
p. M276Dfs186* were able to correctly localize in the nucleus,
but they produced FOXC2 protein aggregates characterized
by partial or total loss of transcriptional activity. Moreover,
p.H199Pfs264* and p.M276Dfs186 protein aggregation
involved also chromatin structure. The p.Y109* also caused
the formation of protein aggregates, even though it was
mainly localized in the cytoplasm. Finally, the analysis of
FOXC2 protein structure revealed that missense and
nonsense
mutations
dramatically
modified
the
DNA
binding-site conformation. Conclusion:
Beneficial
effect
of
M13
phage-RGD
propose these nanostructures as an advantageous and
potential biomaterial in order to promote angiogenesis,
which are becoming feasible therapeutic applications for
variety of ailments. Conclusion:
Beneficial
effect
of
M13
phage-RGD
propose these nanostructures as an advantageous and
potential biomaterial in order to promote angiogenesis,
which are becoming feasible therapeutic applications for
variety of ailments. Z. Safari: None. M. Sadeghizadeh: None. Z. Safari: None. M. Sadeghizadeh: None. Z. Safari: None. M. Sadeghizadeh: None. The role of genetic variation in phenotype variability and
response to treatment in Marfan syndrome The role of genetic variation in phenotype variability and
response to treatment in Marfan syndrome Conclusions: In Lymphedema-distichiasis, FOXC2 mis-
localization, structure modification and nuclear or cytoplas-
mic aggregates formation can be considered the main cause
of loss of protein function B. Loeys1, J. Meester1, E. Franssen1, G. Vandeweyer1,
A. Verstraeten1, H. C. Dietz2, L. Van Laer1, Marfan trial -
Pediatric Heart Network investigators D. Tavian: None. S. Missaglia: None. P.E. Maltese:
None. S. Michelini: None. M. Bertelli: None. 1Center for Medical Genetics, University of Antwerp/Antwerp
University Hospital, Antwerp, Belgium, 2McKusick Nathans
Institute for Genetic Medicine, Johns Hopkins University,
Baltimore, MD, United States Z. Safari, M. Sadeghizadeh Z. Safari, M. Sadeghizadeh Induction of angiogenesis by M13 bacteriophage-RGD
nanofibrous surfaces on human umbilical vein endothelial
cells Induction of angiogenesis by M13 bacteriophage-RGD
nanofibrous surfaces on human umbilical vein endothelial
cells In a large cohort of 300 clinically diagnosed MFS patients,
we addressed the following questions: Which proportion of
classic MFS patients has an FBN1 mutation? Are there
genotype-phenotype correlations? Does the nature of the
FBN1 mutation predict cardiovascular treatment outcome? Next-generation targeted resequencing of FBN1 and related
genes was followed by deletion/duplication testing. Patho-
genic FBN1 mutation was identified in 94% of patients
(including 15 del/dups). Three percent of MFS patients had
non-FBN1 mutations. A similar small fraction had no causal
variant but was clinically indistinguishable from the FBN1
mutation positives. We confirm prior literature that muta-
tions creating or deleting cysteines are significantly asso-
ciated
with
lens
dislocation
(p = 10-7). Premature
termination codons are more often associated with skeletal
findings, but not with cardiovascular severity. We did not
observe a correlation between mutations in the middle
region of FBN1 and phenotypical severity. Mutations at the
C- and N-terminal (exons 1-16/60-66) end tend to lead to a
milder cardiovascular phenotype (p = 0.047). We observed
no difference in aortic root size or progression nor in
treatment outcome comparing dominant negative with
haploinsufficient mutations. No effect of mutation location
on treatment outcome could be detected. Patients that star-
ted treatment before age 10 had less aortic growth than
older patients, irrespective of treatment type. A compre-
hensive molecular analysis identifies an FBN1 mutation in Next generation sequencing in the diagnosis of Marfan
syndrome and related disorders: an efficient global
approach in the SNV/CNV detection Bio-informatical analysis was performed
on CLC Genomics Workbench software; Single Nucleotide
Variants (SNV) annotation was completed with a Python
script. Copy Number Variation (CNV) were sought through
comparison of coverage depths, standardized for each
amplicon to those of a group of controls. Material and Methods: Sequencing was carried out on
MiSeq (Illumina). Bio-informatical analysis was performed
on CLC Genomics Workbench software; Single Nucleotide
Variants (SNV) annotation was completed with a Python
script. Copy Number Variation (CNV) were sought through
comparison of coverage depths, standardized for each
amplicon to those of a group of controls. Results: Pathogenic mutations were identified in 20 cases
(57%; DN-type: 9 missense and 11 HI-type: 5 nonsense, 2
frameshift, 3 splice mutations and 1 large deletion affecting
exons 2-4). There was no significant difference between the
occurrence of major CV symptoms (aortic dilatation and/or
dissection) in the patient cohorts with or without FBN1
mutations
(60%
vs. 85%,
p = 0.13). There
was
no
significant difference between the effect of DN and HI
mutations on the major CV traits (66% vs. 100%, p = 0.07). Among patients with non-cysteine missense mutations, CV
symptoms occurred with significantly lower probability
compared to DN mutations affecting cysteine residues or HI
mutations (25% vs. 100%, p < 0.01). Results: Pathogenic mutations were identified in 20 cases
(57%; DN-type: 9 missense and 11 HI-type: 5 nonsense, 2
frameshift, 3 splice mutations and 1 large deletion affecting
exons 2-4). There was no significant difference between the
occurrence of major CV symptoms (aortic dilatation and/or
dissection) in the patient cohorts with or without FBN1
mutations
(60%
vs. 85%,
p = 0.13). There
was
no
significant difference between the effect of DN and HI
mutations on the major CV traits (66% vs. 100%, p = 0.07). Among patients with non-cysteine missense mutations, CV
symptoms occurred with significantly lower probability
compared to DN mutations affecting cysteine residues or HI
mutations (25% vs. 100%, p < 0.01). Results: To date, more than 600 probands have been
sequenced on this capture panel; a potentially pathogenic
variant was found in approximatively 200 patients. This led
us to identify the disease causing variation in new genes in
families in which the most frequent genes had already been
excluded. This global approach enabled us to expand the
phenotypic spectrum of pathogenic variants in some genes. TMU, Tehran, Iran, Islamic Republic of Introduction: The ability to create, remodeling and regulate
the human blood system holds wide range of medical uti-
lization and carries possible therapeutic effects on patients
with variety of cardiovascular diseases. Commonly, Clinical
use of autologous vessels has been reported; However,
transplant rejection unfortunately occurs in most patients. Likewise,
biomaterial
tissue
engineering
on
vascular
replacement devices has been recently improved. Materials and Method: In the following study, compar-
ing the ability of M13 Bacteriophage, M13 bacteriophage-
RGD and Gelatin surfaces to enhance the angiogenic
potential
of
human
umbilical
vein
endothelial
cells
(HUVECs) was investigated, The response of HUVECs
cells to surfaces were studied through cell apoptosis, cell
proliferation,
growth
factors
secretion
(VEGF),
and
angiogenic-endogenic-associated genes (Matrix metallopep-
tidase 9 (MMP9), Endothelial nitric oxide synthase eNOS). Real Time-PCR, ELISA, Nitric Oxide assay and Zymo-
graphy was operated on samples. After 2 days of cell
seeding, Gene and Protein expression data were analyzed
and compared by two-way analysis of variance. Result: Our results show that M13 phage-RGD appeared
Non-cytotoxic to the cells. Furthermore, these result was 154 J. del Picchia The chosen technology is reliable for SNV and CNV
detection and flexible since the targeted genes can easily be
adapted as scientific knowledge develops. The chosen technology is reliable for SNV and CNV
detection and flexible since the targeted genes can easily be
adapted as scientific knowledge develops. the overwhelming majority of MFS patients. With one
exception, no major genotype-phenotype correlations can
be identified. Importantly, early start of treatment has better
outcome with regards to aortic root growth. the overwhelming majority of MFS patients. With one
exception, no major genotype-phenotype correlations can
be identified. Importantly, early start of treatment has better
outcome with regards to aortic root growth. P. Arnaud: None. N. Hanna: None. L. Benarroch:
None. M. Reocreux: None. K. Diallo: None. S. Gazal:
None. M. Langeois: None. L. Gouya: None. G. Jondeau:
None. C. Boileau: None. B. Loeys: None. J. Meester: None. E. Franssen: None. G. Vandeweyer: None. A. Verstraeten: None. H.C. Dietz: None. L. Van Laer: None. Next generation sequencing in the diagnosis of Marfan
syndrome and related disorders: an efficient global
approach in the SNV/CNV detection Genotype-phenotype correlations in Marfan-syndrome for
the prediction of severe cardiovascular manifestations A. Bors1, P. Kövy1, R. Stengl2, K. Benke2,3, B. Ágg2,3, M. Pólos2,3,
N. Daradics2, G. Mátyás4, Z. Szabolcs2,3, H. Andrikovics1 P. Arnaud1,2, N. Hanna1,2, L. Benarroch2, M. Reocreux3,
K. Diallo3,4, S. Gazal5,4, M. Langeois6, L. Gouya6, G. Jondeau6,2,
C. Boileau1,2 P. Arnaud1,2, N. Hanna1,2, L. Benarroch2, M. Reocreux3,
K. Diallo3,4, S. Gazal5,4, M. Langeois6, L. Gouya6, G. Jondeau6,2,
C. Boileau1,2 1Central Hospital of Southern Pest - National Institute of
Hematology and Infectious Diseases, Budapest, Hungary,
2Heart and Vascular Center, Semmelweis University,
Budapest, Hungary, 3Hungarian Marfan Foundation,
Budapest, Hungary, 4Center for Cardiovascular Genetics and
Gene Diagnostics, Schlieren-Zurich, Switzerland 1Département de Génétique, AP-HP, Hôpital Bichat, PARIS,
France, 2INSERM U1148, Paris, France, 3AP-HP, Hôpital
Bichat, PARIS, France, 4INSERM U1137, Paris, France,
5Harvard University, Department of Epidemiology, Boston,
MA, United States, 6Centre National Maladies Rares,
Syndrome de Marfan et pathologies apparentées, AP-HP,
Hôpital Bichat, PARIS, France Introduction: Marfan syndrome (MFS) is an autosomal-
dominant, systemic connective tissue disorder, with a pre-
valence of ~1:5000. The most important, life-threatening
complication is aortic dissection, therefore the identification
and the follow-up of aortic dilatation as well as prophylactic
surgery is essential. In most cases, heterozygous FBN1 gene
mutations are responsible for the disease, leading to the
reduction (haploinsufficiency=HI) or to the abnormal
structure (dominant negative type mutation=DN) of the
fibrillin-1 protein. Introduction: The Genetic Laboratory in Bichat-Hospital
(Paris) receives each year samples for approximatively 600
probands suspected for Marfan Syndrome (MFS) or Related
Disorders (RD) nationwide. Genetic heterogeneity is high
and new genes are regularly discovered. Initially, the
strategy for molecular diagnosis was based on successive
Sanger sequencing of the main genes, associated with
MLPA analysis. The objective was to evaluate on these
probands a NGS capture panel strategy, targeting 25 known
disease causing genes. Aims: We examined the association of FBN1 sequence
variations with cardiovascular (CV) involvement. Methods: Phenotypic evaluation was carried out accord-
ing to the revised Ghent nosology, while molecular genetic
analysis of the FBN1 gene was performed using next-
generation sequencing and Sanger-sequencing techniques in
35 MFS patients. Methods: Phenotypic evaluation was carried out accord-
ing to the revised Ghent nosology, while molecular genetic
analysis of the FBN1 gene was performed using next-
generation sequencing and Sanger-sequencing techniques in
35 MFS patients. Material and Methods: Sequencing was carried out on
MiSeq (Illumina). Next generation sequencing in the diagnosis of Marfan
syndrome and related disorders: an efficient global
approach in the SNV/CNV detection Moreover, several pathogenic CNV have been pinpointed,
either in some genes that were not screened for CNV before. Conclusions: NGS technologies enable us to have a
unique strategy in the molecular diagnosis of MFS and RD. Conclusions: NGS technologies enable us to have a
unique strategy in the molecular diagnosis of MFS and RD. 155 Abstracts from the 51st European Society of Human Genetics Conference: Posters Conclusion: The identification of disease-causing FBN1
mutations in MFS patients can improve the accuracy of the
risk estimation of aortic disorders and planning prophylactic
aortic root reconstruction surgeries. Conclusion: The identification of disease-causing FBN1
mutations in MFS patients can improve the accuracy of the
risk estimation of aortic disorders and planning prophylactic
aortic root reconstruction surgeries. (miR-101-3p, miR-199a-5p, miR-125a-5p) were hypoex-
pressed
in
unstable
plaques. These
results
are
well
explained by microRNA-associated pathological processes
described in atherosclerosis. Principal component and
cluster analyses showed similar results. Two first principal
components accounted for 99% of data variability and
showed a fine division of samples by stability, which can
indirectly indicate that the selected microRNAs characterize
the development of the pathological process. A. Bors: None. P. Kövy: None. R. Stengl: None. K. Benke: None. B. Ágg: B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Modest; Hungarian
National Research, Development and Innovation Office;
(grant number NVKP_16-1- 2016-0017), New National
Excellence Program of the Ministry of Human Capacities of
Hungary”; (ÚNKP-17- 3-I- SE-31; BÁ). M. Pólos: None. N. Daradics: None. G. Mátyás: None. Z. Szabolcs: None. H. Andrikovics: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; Janos Bolyai Research
Scholarship of the Hungarian Academy of Sciences. A. Bors: None. P. Kövy: None. R. Stengl: None. K. Benke: None. B. Ágg: B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Modest; Hungarian
National Research, Development and Innovation Office;
(grant number NVKP_16-1- 2016-0017), New National
Excellence Program of the Ministry of Human Capacities of
Hungary”; (ÚNKP-17- 3-I- SE-31; BÁ). M. Pólos: None. N. Daradics: None. G. Mátyás: None. Z. Szabolcs: None. H. Andrikovics: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; Janos Bolyai Research
Scholarship of the Hungarian Academy of Sciences. Genotype-phenoype correlation in probands with three
recurrent (founder) mutations in the MYH7 gene (V964L,
M982T and G1057S) Genotype-phenoype correlation in probands with three
recurrent (founder) mutations in the MYH7 gene (V964L,
M982T and G1057S) A. A. Zarubin1,2, A. V. Markov1, M. S. Nazarenko1,2,3, D. V. Sharysh2, A. N. Kazantsev3, O. L. Barbarash3, V. P. Puzyrev1,2 A. A. Zarubin1,2, A. V. Markov1, M. S. Nazarenko1,2,3, D. V. Sharysh2, A. N. Kazantsev3, O. L. Barbarash3, V. P. Puzyrev1,2 K. J. A. F. van Kaam1, M. B. Hoos1, C. M. Marcelis2, J. G. Post3,
R. M. Oldenburg4, Y. M. Hoedemaekers5, F. A. M. Duijkers6,
F. H. J. van Tienen1, A. van den Wijngaard1, I. P. C. Krapels1 1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk,
Russian Federation, 2Siberian State Medical University,
Tomsk, Russian Federation, 3Research Institute for Complex
Issues of Cardiovascular Diseases, Kemerovo, Russian
Federation 1MUMC+, Maastricht, Netherlands, 2Radboud UMC,
Nijmegen, Netherlands, 3UMCU, Utrecht, Netherlands,
4Erasmus MC, Rotterdam, Netherlands, 5UMCG, Groningen,
Netherlands, 6AMC, Amsterdam, Netherlands Background: MicroRNAs have been reported to participate
in atherogenesis and to serve as markers of atherosclerosis,
but the involvement of microRNAs in destabilization of
atherosclerotic plaque is under-investigated. Objective:
Identification of microRNAs differentially expressed in the
cells of carotid atherosclerotic plaques depending on the
degree of its stability. Aims: MYH7 is one of the most frequently mutated genes
in different types of cardiomyopathies. We aimed to gain
more insight in the genotype-phenotype correlation of three
recurrent mutations (two founder mutations and one hotspot
mutation) in the S2 structural subdomain of the MYH7 gene
(V964L, M982T and G1057S). Moreover, we aimed to
reach national consensus on the interpretation and classifi-
cation of these three mutations. Material and Methods: The samples of carotid athero-
sclerotic plaque were obtained from 14 patients. After
histological analysis, all samples were divided into stable
(n = 8)
and
unstable
(n = 6)
atherosclerotic
plaques. MicroRNA expression was analyzed by massive parallel
sequencing (miRNA-seq). Material and Methods: The samples of carotid athero-
sclerotic plaque were obtained from 14 patients. After
histological analysis, all samples were divided into stable
(n = 8)
and
unstable
(n = 6)
atherosclerotic
plaques. MicroRNA expression was analyzed by massive parallel
sequencing (miRNA-seq). Methods: We retrospectively collected information on
clinical and cardiologic characteristics as well as additional
risk factors possibly influencing the phenotype of 55
probands with one of the three recurrent mutations V964L
(n = 30), M982T (n = 16) and G1057S (n = 9) in MYH7. Next generation sequencing in the diagnosis of Marfan
syndrome and related disorders: an efficient global
approach in the SNV/CNV detection Conclusions:
Of
161 microRNAs
identified to
be
differentially expressed between stable and unstable carotid
plaques, nine microRNAs can reliably be associated with
destabilization of atherosclerotic lesion. The study is
supported by RSF №16-15-10150. A.A. Zarubin: None. A.V. Markov: None. M.S. Nazarenko: None. D.V. Sharysh: None. A.N. Kazantsev:
None. O.L. Barbarash: None. V.P. Puzyrev: None. Genotype-phenoype correlation in probands with three
recurrent (founder) mutations in the MYH7 gene (V964L,
M982T and G1057S) If
available, results of segregation analysis in (first degree)
family members were retrieved. Results and Discussion: We found 161 microRNAs to
be differentially expressed between stable and unstable
atherosclerotic plaques. After disposal of microRNAs with
low expression level (<10 CPM) in all samples and having a
strong correlation with clinical features (including drug
intake and other diseases), only 9 microRNAs showed
significant differences. Six of them (let-7b-5p, miR-7704,
miR-328-3p,
miR-1291,
miR-370-3p,
miR-148a-5p)
showed increased level of expression, and three microRNAs Results: Probands with one of the mutations V964L and
M982T displayed varying types of CMP whereas probands
with the G1057S mutation all had a diagnosis of HCM. In
the majority of these probands additional risk factors (most
often
hypertension)
and/or
a
mutation
in
a
second Results: Probands with one of the mutations V964L and
M982T displayed varying types of CMP whereas probands
with the G1057S mutation all had a diagnosis of HCM. In
the majority of these probands additional risk factors (most
often
hypertension)
and/or
a
mutation
in
a
second J. del Picchia 156 sarcomeric gene were identified. The limited amount of
segregation data are inclonclusive at this point in time. sarcomeric gene were identified. The limited amount of
segregation data are inclonclusive at this point in time. Methods: Whole-exome sequencing (WES) and genome-
wide genotyping data, derived from an Italian cohort of
~1,600 early-onset myocardial infarction (MI) cases and
1,600 controls, were analyzed to test whether rare/common
variants affecting the expression levels of the F10 gene,
encoding FX, are associated with MI risk. MYH7 mutation
V964L (n = 30) M982T (n = 16) G1057S (n = 9)
Second
sarcomeric gene
mutation
7 (23%)
5 (31%)
1 (11%)
Hypertension
5 (17%)
4 (25%)
5 (56%)
Second mutation
AND
hypertension
4 (13%)
–
–
Other risk factors
1 (3%)
2 (13%)
–
No risk factors
13 (43%)
5 (31%)
3 (33%) MYH7 mutation
V964L (n = 30) M982T (n = 16) G1057S (n = 9) Results:
Bioinformatics
analyses,
measurements
of
plasma FX levels, and data mining in publicly-available
databases were used to assess the pathogenicity of the rare
variants identified by WES in the F10 gene. An enrichment
in the burden of potentially-deleterious variants was
observed among controls (OR=0.47, P=0.02), thus high-
lighting a reduced MI risk in subjects carrying one F10
inactivating mutation. P05.55C E. Paraboschi: None. S. Kathiresan: B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Bayer. F. Consultant/Advisory Board; Modest;
Catabasis. L. Gigante: None. F. Peyvandi: F. Consultant/
Advisory Board; Modest; Ablynx, F. Hoffmann-La Roche. D. Ardissino: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Astra Zeneca, Bayer,
Glaxosmithkline, Eli Lilly & Company, Pfizer, Novartis. R. Asselta: None. S. Duga: None. Genotype-phenoype correlation in probands with three
recurrent (founder) mutations in the MYH7 gene (V964L,
M982T and G1057S) Moreover, the case-control associa-
tion analysis performed on common polymorphisms known
to influence F10 levels (GTEx data) evidenced a significant
protective effect of the rs4907485T>G variant. In particular,
the GG genotype associated with lowered F10 expression in
the GTEx database was enriched among controls (37.7% vs
33.2%; OR=0.92, CI=0.84-1.02, P=0.0093), again sug-
gesting that lowered F10 levels are protective against the
disease. Conclusion: The high number of additional risk factors
(mainly second sarcomeric gene mutations and/or hyperten-
sion) in probands with the mutations V964L, M982T and
G1057S in MYH7 supports the notion that these variants
may be not be causal by themselves, but are possibly
modifiers of disease. K.J.A.F. van Kaam: None. M.B. Hoos: None. C.M. Marcelis: None. J.G. Post: None. R.M. Oldenburg: K.J.A.F. van Kaam: None. M.B. Hoos: None. C.M. Marcelis: None. J.G. Post: None. R.M. Oldenburg:
None. Y.M. Hoedemaekers: None. F.A.M. Duijkers:
None. F.H.J. van Tienen: None. A. van den Wijngaard:
None. I.P.C. Krapels: None. Conclusion: We showed for the first time that variants
lowering FX levels are associated with reduced MI risk,
thus supporting, on a genetic basis, clinical trials’ results
showing that FX inhibition is beneficial for the treatment/
prevention of atherothrombotic events. None. Y.M. Hoedemaekers: None. F.A.M. Duijkers:
None. F.H.J. van Tienen: None. A. van den Wijngaard:
None. I.P.C. Krapels: None. None. Y.M. Hoedemaekers: None. F.A.M. Duijkers:
None. F.H.J. van Tienen: None. A. van den Wijngaard: Genetic variants lowering the levels of coagulation factor X
are protective against myocardial infarction Genetic variants lowering the levels of coagulation factor X
are protective against myocardial infarction E. Paraboschi1, S. Kathiresan2, L. Gigante3, F. Peyvandi4,5,
D. Ardissino3, R. Asselta1,6, S. Duga1,6 1Humanitas University, Pieve Emanuele, Italy, 2Center for
Genomic Medicine, Massachusetts General Hospital, Boston,
MA, United States, 3Division of Cardiology, Azienda
Ospedaliero-Universitaria di Parma, Parma, Italy, 4Angelo
Bianchi Bonomi Haemophilia and Thrombosis Centre,
Fondazione IRCCS Ca' Granda Ospedale Maggiore
Policlinico, and Luigi Villa Foundation, Milan, Italy,
5Department of Pathophysiology and Transplantation,
Università degli Studi di Milano, Milan, Italy, 6Humanitas
Clinical and Research Center, Rozzano, Italy P05.56D
Family study shows the phenotypic and genetic spectrum of
familial noncompaction cardiomyopathy J. I. van Waning1, K. Caliskan1, M. Michels1, A. F. L. Schinkel1,
M. Dalinghuis1, Y. M. Hoedemaekers2, M. W. Wessels1,
I. Kardys1, A. IJpma1, R. M. W. Hofstra1, M. A. van
Slegtenhorst1, D. Majoor-Krakauer1 Background: Coagulation factor X (FX) plays a pivotal
role in the clotting process, being responsible for thrombin
generation; emerging evidence suggests its involvement
also in non-hemostatic processes, including inflammation. Recent data demonstrated that FX inhibition reduces the
risk of recurrent atherothrombotic events in patients with
acute coronary syndrome. 1Erasmus MC, Rotterdam, Netherlands, 2UMCG, Groningen,
Netherlands Introduction: Noncompaction cardiomyopathy (NCCM) is
a heterogeneous cardiomyopathy characterized by excessive 157 Abstracts from the 51st European Society of Human Genetics Conference: Posters Spain, 7Servicio de Neumología, Complejo Hospitalario de
Pontevedra, Pontevedra, Spain Spain, 7Servicio de Neumología, Complejo Hospitalario de
Pontevedra, Pontevedra, Spain trabeculation of the left ventricle (LV). Patients diagnosed
with NCCM, may also have a dilated left ventricle or a
hypertrophic septum. Pulmonary Arterial Hypertension (PAH) is a rare and pro-
gressive disease characterized by vascular remodeling and
the increase of vascular resistance that leads to right heart
failure and, ultimately, death. PAH genetic basis has been
slowly uncovered during the last decades. After screening
with a sequencing panel for PAH related genes, several
mutations were detected in the ATP-Binding Cassette
transporter subfamily C member 8 (ABCC8) (Exon 3 c. G298A p.E100K, Exon3 c.2694+1G>A, Exon 11 c. C1643T p.T548M, Exon 26 c.3288_3289del pL1096fs,
Exon 27 c.G3384A p.D1132N), a gene widely related to
congenital hyperinsulinism. DNA fragments, wild type and
mutated sequence, were cloned into the pSPL3 vector. After
PCR and Sanger sequencing to confirm the fragment
insertion into the plasmid, pSPL3 was transfected into the
COS-7 cell line by triplicate. 48 hours’ post-transfection,
RNA was isolated and cDNA was generated using RT-
PCR. Lastly, a high fidelity polymerase was used to perform
a PCR using primers surrounding vector’s exon trap, the
product was analyzed via electrophoresis and the bands of
interest were sequenced. In silico analysis predicted a
moderate ANNOVAR effect in 3/5 mutations, high in 1/5
and no effect in the last one. Our experimental results
showed an altered splicing pattern in 1/5 mutations checked. Sequencing is in progress to confirm the differential pattern. In conclusion, minigene assay is a simple and effective
method to check for splicing alterations and pathogenicity
of the variants detected. Nonetheless, analysis of patient´s
RNA will be definitive to confirm our results. P05.58B Landscape of mutations found by gene panel routine
sequencing for pulmonary hypertension Landscape of mutations found by gene panel routine
sequencing for pulmonary hypertension P05.56D
Family study shows the phenotypic and genetic spectrum of
familial noncompaction cardiomyopathy Purpose: Indentify if index cases and affected relatives
had similar NCCM phenotypes, and if cardiac phenotypes
were linked to genotypes. Methods: A retrospective family study assessed the
familial cardiac phenotype and genotype in 114 families of
consecutively diagnosed cases with NCCM. Results: Family screening identified 109 relatives with a
cardiomyopathy from 58 (51%) families. More affected
relatives were identified in families with a mutation (29% vs
15%; p < 0.001). 33% of the mutation carriers did not have
a cardiomyopathy. Clinical features in relatives were less
severe than in index cases, 51% of the relatives diagnosed
with NCCM were asymptomatic (p < 0.001). The non-
dilated phenotype of NCCM segregated in families of index
NCCM cases with normal LV dimensions, and the dilated
NCCM phenotype predicted risk of having a dilated LV for
relatives (p = 0.002). Dilated NCCM were older (p =-
0.017), had more often LV systolic dysfunction (p < 0.001),
RV systolic dysfunction (p = 0.012), MACE (p = 0.007)
and was associated with mutations in the tail of MYH7
(p < 0.001). Hypertrophic NCCM was associated with
MYBPC3 mutations and HCM in the family (p = 0.006). HCM or DCM without trabeculation in the family increased
risk for MACE in NCCM patients (p = 0.05). Conclusion: From the phenotype of the index case, the
familial mutation and the phenotypes of relatives, predic-
tions can be made on risk for phenotype and outcome in
relatives. J.I. van Waning: None. K. Caliskan: None. M. Michels: None. A.F.L. Schinkel: None. M. Dalinghuis:
None. Y.M. Hoedemaekers: None. M.W. Wessels: None. I. Kardys: None. A. IJpma: None. R.M.W. Hofstra:
None. M.A. van
Slegtenhorst:
None. D. Majoor-
Krakauer: None. M. Lago-Docampo:
None. J. Tenorio:
None. P. Escribano: None. A. Baloira: None. P. Lapunzina: None. D. Valverde: None. P05.57A Splicing mechanism evaluation of NGS detected variants
using hybrid minigenes 1Département de Génétique, Hôpital Pitié-Salpêtrière, Paris,
France, 2Centre de Référence de l’Hypertension Pulmonaire
Sévère, Service de Pneumologie, Hôpital de Bicêtre, DHU
thorax Innovation, Le Kremlin Bicêtre, France, 3M3C-
Cardiologie pédiatrique, Hôpital Necker-enfants malades,
Paris, France, 4Département de Pneumologie et Addictologie;
CHU Arnaud de Villeneuve, Montpellier, France, 5Service de
cardiologie maladies vasculaires, CHU Gabriel Montpied, 1University of Vigo, Vigo, Spain, 2Instituto de Investigación
Sanitaria Galicia Sur (IIS-Galicia Sur), Vigo, Spain, 3Centro
de Investigación Biomédica (CINBIO), Vigo, Spain, 4Instituto
de Genética Médica y Molecular (INGEMM), Hospital
Universitario La Paz-IdiPaz, Universidad Autónoma de
Madrid, Madrid, Spain, 5CIBER de enfermedades Raras
(CIBERER), Insitituto de Salud Carlos III, Madrid, Spain,
6Servicio de Cardiología, Hospital 12 de Octubre, Madrid, I. Ntalla1,2, L. Weng3,4, H. R. Warren1,5, Y. Jamshidi6, P. B.
Munroe1,5, S. A. Lubitz3,4,7, the CHARGE EKG consortium I. Ntalla1,2, L. Weng3,4, H. R. Warren1,5, Y. Jamshidi6, P. B. Munroe1,5, S. A. Lubitz3,4,7, the CHARGE EKG consortium Clermont-Ferrand, France, 6Département de Pneumologie-
CHRU Nancy-Université de Lorraine, Vandoeuvre-les-nancy,
France, 7Service de pneumologie, CHU de Lyon HCL - GH
Est-Hôpital Louis Pradel, Bron, France, 8Service de
pneumologie, Hôpital Pasteur-CHU Nice, Nice, France,
9Service de pneumologie, Hôpital Larrey, Toulouse, France,
10Service de Pneumologie, CHU Nord de Marseille, Marseille,
France, 11- Service de pneumologie, CHU de Bordeaux
Hôpital Haut-lévêque, Pessac, France, 12Service de
Cardiologie Infantile et Congénitale, CHRU Lille-Hôpital
Cardiologique, Lille, France 1William Harvey Research Institute, Barts and The London
School of Medicine and Dentistry, Queen Mary University of
London, London, United Kingdom, 2Centre for Genomic
Health, Queen Mary University of London, London, United
Kingdom, 3Program in Medical and Population Genetics,
Broad Institute of MIT and Harvard, Cambridge, MA, United
States, 4Cardiovascular Research Center, Massachusetts
General Hospital, Boston, MA, United States, 5National
Institute for Health Research Barts Cardiovascular Biomedical
Research Unit, Queen Mary University of London, London,
United Kingdom, 6Genetics Unit, Cardiovascular and Cell
Sciences Institute, St George's University of London, London,
United Kingdom, 7Cardiac Arrhythmia Service, Massachusetts
General Hospital, Boston, MA, United States Introduction: Since the discovery of BMPR2 mutations in
2000, mutations in several other genes, related or not to
BMPR2 signaling, have been discovered, making pulmon-
ary hypertension (PH) a heterogenous genetic disease. Here,
we sought to determine the genetic architecture of PH gene
mutations in the french PH cohort. Introduction: The electrocardiographic PR interval repre-
sents cardiac atrioventricular conduction, a critical physio-
logical process that is associated with arrhythmias and all-
cause mortality. Yet the biological determinants of the PR
interval remain incompletely understood. We conducted the
largest genetic association study of the PR interval to date. Introduction: The electrocardiographic PR interval repre-
sents cardiac atrioventricular conduction, a critical physio-
logical process that is associated with arrhythmias and all-
cause mortality. Yet the biological determinants of the PR
interval remain incompletely understood. We conducted the
largest genetic association study of the PR interval to date. Methods: We analyzed 227 patients with a clinical
diagnosis of pulmonary arterial hypertension (PAH) or
pulmonary veno-occlusive disease (PVOD), by a targeted
capture panel including known PH genes. Methods: We analyzed 227 patients with a clinical
diagnosis of pulmonary arterial hypertension (PAH) or
pulmonary veno-occlusive disease (PVOD), by a targeted
capture panel including known PH genes. I. Ntalla1,2, L. Weng3,4, H. R. Warren1,5, Y. Jamshidi6, P. B.
Munroe1,5, S. A. Lubitz3,4,7, the CHARGE EKG consortium Expression quantitative trait locus
(eQTL) analysis using the GTEx portal revealed 5 variants
associated with gene expression levels in right atrial
appendage (TRAK1, SMARCB1, SYNE2, DEK, DNAH11). Gene Ontology enrichment analysis including only the
nearest genes to both known and novel variants indicated
further enrichment of biological processes involving heart
and cardiac muscle tissue development with the addition of
the newly identified genes. Conclusions: Gene panel NGS sequencing is an efficient
tool to improve the knowledge of PAH genetic architecture. In our cohort, the major gene remains BMPR2, however
mutations in other PH genes account for a small number of
cases. Identification of these mutations allows the involve-
ment of these genes in PH to be confirmed since only few
cases were previously reported in the literature. Conclusions: Gene panel NGS sequencing is an efficient
tool to improve the knowledge of PAH genetic architecture. In our cohort, the major gene remains BMPR2, however
mutations in other PH genes account for a small number of
cases. Identification of these mutations allows the involve-
ment of these genes in PH to be confirmed since only few
cases were previously reported in the literature. F. Soubrier: None. M. Eyries: None. B. Girerd: None. D. Montani: None. M. Levy: None. D. Bonnet: None. A. Bourdin: None. R. Trésorier: None. A. Chaouat: None. V. Cottin: None. C. Sanfiorenzo: None. G. Prévot: None. M. Reynaud-Gaubert10: None. C. Dromer: None. A. Houeijeh: None. M. Humbert: None. Conclusions: Our results implicate specific genes which
determine PR interval and highlight the complex polygenic
nature of atrioventricular conduction. Future analyses will
assess the relations between genetic determinants of the PR
interval and cardiac arrhythmias. Conclusions: Our results implicate specific genes which
determine PR interval and highlight the complex polygenic
nature of atrioventricular conduction. Future analyses will
assess the relations between genetic determinants of the PR
interval and cardiac arrhythmias. I. Ntalla1,2, L. Weng3,4, H. R. Warren1,5, Y. Jamshidi6, P. B.
Munroe1,5, S. A. Lubitz3,4,7, the CHARGE EKG consortium Results: Following clinical examination, 159 subjects
were classified as idiopathic PAH (iPAH), 11 as familial
PAH (fPAH), 7 as drugs or toxin induced PAH and 50 as
PVOD. A mutation was identified in 42 of the 227 analyzed
subjects: 27 in iPAH, 7 in fPAH and 8 in PVOD. No
mutation was identified in patients with drugs or toxin
induced PAH. Methods: We combined genome-wide association results
for the PR interval from 55 studies encompassing 293,051
individuals (271,570 European, 8,173 African, 12,823
Hispanic, and 763 Asian ancestry) using fixed-effect
meta-analysis. Analyses included ~12 million variants
(minor allele frequency, MAF>0.1%) imputed using the
1000 Genomes Project reference panel. We identified 23 mutations in BMPR2, 2 in ACVRL1, 5 in
TBX4, 1 in GDF2, 1 in SMAD9 and 9 biallelic mutations in
EIF2AK4. No pathogenic mutation was identified in
KCNK3
or
CAV1. However
1
variant
of
unknown
significance was identified in each of these genes. We identified 23 mutations in BMPR2, 2 in ACVRL1, 5 in
TBX4, 1 in GDF2, 1 in SMAD9 and 9 biallelic mutations in
EIF2AK4. No pathogenic mutation was identified in
KCNK3
or
CAV1. However
1
variant
of
unknown
significance was identified in each of these genes. Results: We identified 217 regions (152 novel) associated
with PR interval exceeding genome-wide significance
(p < 5x10-8). Among
novel
regions,
we
identified
3
missense variants annotated as deleterious and/or possibly
damaging in KIAA1755, ARHGEF40, and SPSB3; and 7
variants in high LD (r2>0.8) with missense variants in
DERL3, DUSP13, DNAH11, C10orf71, ACCN4, CHPF,
OBSL1 and DALRD3. Expression quantitative trait locus
(eQTL) analysis using the GTEx portal revealed 5 variants
associated with gene expression levels in right atrial
appendage (TRAK1, SMARCB1, SYNE2, DEK, DNAH11). Gene Ontology enrichment analysis including only the
nearest genes to both known and novel variants indicated
further enrichment of biological processes involving heart
and cardiac muscle tissue development with the addition of
the newly identified genes. Results: We identified 217 regions (152 novel) associated
with PR interval exceeding genome-wide significance
(p < 5x10-8). Among
novel
regions,
we
identified
3
missense variants annotated as deleterious and/or possibly
damaging in KIAA1755, ARHGEF40, and SPSB3; and 7
variants in high LD (r2>0.8) with missense variants in
DERL3, DUSP13, DNAH11, C10orf71, ACCN4, CHPF,
OBSL1 and DALRD3. Multi-ancestry genome-wide association meta-analysis of
293,000 individuals identifies 217 regions for the
electrocardiographic PR interval Splicing mechanism evaluation of NGS detected variants
using hybrid minigenes F. Soubrier1, M. Eyries1, B. Girerd2, D. Montani2, M. Levy3,
D. Bonnet3, A. Bourdin4, R. Trésorier5, A. Chaouat6, V. Cottin7,
C. Sanfiorenzo8, G. Prévot9, M. Reynaud-Gaubert1010,
C. Dromer11, A. Houeijeh12, M. Humbert2 M. Lago-Docampo1,2,3, J. Tenorio4,5, P. Escribano6, A. Baloira7,
P. Lapunzina4,5, D. Valverde1,2,3 M. Lago-Docampo1,2,3, J. Tenorio4,5, P. Escribano6, A. Baloira7,
P. Lapunzina4,5, D. Valverde1,2,3 1University of Vigo, Vigo, Spain, 2Instituto de Investigación
Sanitaria Galicia Sur (IIS-Galicia Sur), Vigo, Spain, 3Centro
de Investigación Biomédica (CINBIO), Vigo, Spain, 4Instituto
de Genética Médica y Molecular (INGEMM), Hospital
Universitario La Paz-IdiPaz, Universidad Autónoma de
Madrid, Madrid, Spain, 5CIBER de enfermedades Raras
(CIBERER), Insitituto de Salud Carlos III, Madrid, Spain,
6Servicio de Cardiología, Hospital 12 de Octubre, Madrid, 1Département de Génétique, Hôpital Pitié-Salpêtrière, Paris,
France, 2Centre de Référence de l’Hypertension Pulmonaire
Sévère, Service de Pneumologie, Hôpital de Bicêtre, DHU
thorax Innovation, Le Kremlin Bicêtre, France, 3M3C-
Cardiologie pédiatrique, Hôpital Necker-enfants malades,
Paris, France, 4Département de Pneumologie et Addictologie;
CHU Arnaud de Villeneuve, Montpellier, France, 5Service de
cardiologie maladies vasculaires, CHU Gabriel Montpied, J. del Picchia 158 Genome-wide association analysis of recurrent myocardial
infarction in UK Biobank identifies suggestive evidence for
association to twenty seven loci J. Tenorio1, M. Orcholsky2, I. Hernández3, P. Navas4, P. Arias5,
E. Shamskhou2, K. Yuan2, E. Granda1, V. De Jesús-Pérez2,
P. Escribano-Subías3, P. Lapunzina1 O. Giannakopoulou1,2, S. Kanoni1,2, P. Giardoglou3,
K. Kelaidoni3, UKBiobank CardioMetabolic Consortium CHD
working group, G. Dedoussis3, P. Deloukas1,2,4 1INGEMM, Madrid, Spain, 2Stanford University, Stanford, CA,
United States, 3Hospital Universitario 12 de Octubre, Madrid,
Spain, 4Hospital Universitario Gregorio Marañón, Madrid,
Spain, 5Institute of Medical and Molecular Geneticist, Madrid,
Spain 1INGEMM, Madrid, Spain, 2Stanford University, Stanford, CA,
United States, 3Hospital Universitario 12 de Octubre, Madrid,
Spain, 4Hospital Universitario Gregorio Marañón, Madrid,
Spain 5Institute of Medical and Molecular Geneticist Madrid K. Kelaidoni3, UKBiobank CardioMetabolic Consortium CHD
working group, G. Dedoussis3, P. Deloukas1,2,4 1William Harvey Research Institute, Barts &the London
Medical School, Queen Mary University of London, London,
United Kingdom, 2Centre for Genomic Health, Queen Mary
University of London, London, United Kingdom, 3Department
of Nutrition-Dietetics, Harokopio University, Athens, Greece,
4Princess Al-Jawhara Al-Brahim Centre of Excellence in
Research of Hereditary Disorders (PACER-HD), King
Abdulaziz University, Jeddah, Saudi Arabia Background: Pulmonary Arterial Hypertension (PAH) is a
rare disease of unclear etiology that is associated with
abnormally increased pulmonary pressures and chronic
right heart failure. Use of whole exome sequencing (WES)
has led to the discovery of gene variants linked to PAH
pathobiology. The main aim of this project was to perform
WES analysis of 56 unrelated PAH patients to identify gene
variants potentially involved in PAH. Background: Despite the decline in their incidence rates,
the recurrent myocardial infarction (MI) events are asso-
ciated with significant morbidity, short- and long- term
mortality. Relative to our understanding of risk for first
events, the aetiology of recurrent MI is poorly understood. Material and Methods: Bioinformatic and in silico
analysis was applied to WES data from patients with
idiopathic, heritable and secondary induced PAH. Expres-
sion of WES candidate genes was assessed in pulmonary
microvascular endothelial cells (PMVECs) from healthy
donors and PAH patients. Functional gene analysis was
carried via tube formation and scratch assays. Material and Methods: Bioinformatic and in silico
analysis was applied to WES data from patients with
idiopathic, heritable and secondary induced PAH. Expres-
sion of WES candidate genes was assessed in pulmonary
microvascular endothelial cells (PMVECs) from healthy
donors and PAH patients. Functional gene analysis was
carried via tube formation and scratch assays. Methods: We used UK-Biobank, a large prospective
cohort of 500,000 individuals, to investigate the genetic
predisposition of recurrent MI. P05.59C Multi-ancestry genome-wide association meta-analysis of
293,000 individuals identifies 217 regions for the
electrocardiographic PR interval Multi-ancestry genome-wide association meta-analysis of
293,000 individuals identifies 217 regions for the
electrocardiographic PR interval 159 Abstracts from the 51st European Society of Human Genetics Conference: Posters I. Ntalla: None. L. Weng: None. H.R. Warren: None.
Y.
Jamshidi:
None.
P.B.
Munroe:
None.
S.A.
Lubitz: None. J. Tenorio: None. M. Orcholsky: None. I. Hernández:
None. P. Navas: None. P. Arias: None. E. Shamskhou:
None. K. Yuan: None. E. Granda: None. V. De Jesús-
Pérez:
None. P. Escribano-Subías:
None. P. Lapunzina: None. P05.60D P05.60D
Identification of causative genes or phenotype modifiers
variants associated with Pulmonary Arterial Hypertension Division of Primary Care, Nottingham, United Kingdom Division of Primary Care, Nottingham, United Kingdom Introduction: Statins are recommended to prevent cardio-
vascular disease (CVD). In many patients, statins do not
achieve optimal cholesterol lowering effects due to clinical
and genetic factors. This large population-based cohort
study assessed LDL cholesterol response to statins and its
association with future incidence of CVD. Biological mechanisms underlying stress cardiomyopathy
(SCM, also known as Takotsubo cardiomyopathy), are
poorly understood. SCM can occur sporadically, often in
association with a stressful event, or in clusters after major
natural disasters. Our primary study cohort consisted of 28
women who suffered SCM as a result of two devastating
earthquakes that struck the city of Christchurch, New
Zealand, in 2010 and 2011. Since then, a further 5 cases
arising from the Kaikoura 2016 event have also been added
to the CNV analysis cohort, bringing the total case number
to 33. To seek possible underlying genetic factors, array
comparative genomic hybridisation was carried out on these
subjects. The most striking finding from these analyses was
the observation of a high rate of rare, heterogeneous copy
number variants (CNV) of uncertain clinical significance (in
13/33 subjects). Several of these CNVs clearly impacted on
genes of cardiac relevance including RBFOX1, GPC5,
KCNRG, CHODL, and GPBP1L1. There is no physical
overlap between the CNVs, and the genes they impact do
not fall into a clear pathophysiological pathway. However,
the recognition that SCM cases display a high rate of
unusual CNV, and that SCM predisposition may therefore
be associated with these CNVs, offers a novel perspective
and a new approach by which to understand this proble-
matic and enigmatic condition. Material and Methods: 200,225 patients (mean age of
62.8 years; 47.5% females), with at least 2 cholesterol
measurements and without prior CVD at baseline, were
followed from their first statin prescription date, in a
population-based cohort using electronic health records
from the UK Clinical Practice Research Datalink. A greater
than 40% reduction in baseline cholesterol level within
24 months was classified optimal statin response in line
with national guidelines. Cox regression models were used
to determine hazard ratios for incident CVD events between
optimal and non-optimal statin responders. Results: A total of 97,377 (48.6%) of patients were
optimal statin responders. During the follow-up, 21,973
incident CVD events occurred (10,310 in optimal respon-
ders and 11,663 in non-optimal responders). Division of Primary Care, Nottingham, United Kingdom The rate of
CVD was 18.8 and 16.5 per 1000 person-life years for non-
optimal and optimal responders respectively. In optimal
responders, compared with non-optimal responders, the
hazard ratio (95% CI) for incident CVD was 0.87 (0.84 –
0.89; p < 0.0001). The association was not confounded by
family history and other CVD risk factors. K. Doudney: None. C.J. Lacey: None. P.G. Bridgman:
None. R.T. Mulder: None. J.J. Zarifeh: None. B. Kimber: None. M. Cadzow: None. M.A. Black: None. T.R. Merriman: None. K. Lehnert: None. V. Bickley:
None. J.F. Pearson: None. V.A. Cameron: None. M.A. Kennedy: None. K. Doudney: None. C.J. Lacey: None. P.G. Bridgman:
None. R.T. Mulder: None. J.J. Zarifeh: None. B. Kimber: None. M. Cadzow: None. M.A. Black: None. T.R. Merriman: None. K. Lehnert: None. V. Bickley:
None. J.F. Pearson: None. V.A. Cameron: None. M.A. Kennedy: None. Conclusions:
Optimal
response
to
statins
within
24 months after initiation was associated with a decreased
risk of CVD in UK general population. Further pharmaco-
genetic studies may indicate reasons for non-optimal
response. Conclusions:
Optimal
response
to
statins
within
24 months after initiation was associated with a decreased
risk of CVD in UK general population. Further pharmaco-
genetic studies may indicate reasons for non-optimal
response. Funder: AMGEN Independent Research Grant Funder: AMGEN Independent Research Grant Rare copy number variants involving cardiac function and
development genes detected in earthquake induced
takotsubo cardiomyopathy patients O. Giannakopoulou: None. S. Kanoni: None. P. Giardoglou: None. K. Kelaidoni: None. G. Dedoussis:
None. P. Deloukas: None. K. Doudney1, C. J. Lacey2, P. G. Bridgman1, R. T. Mulder2, J. J. Zarifeh1, B. Kimber2, M. Cadzow3, M. A. Black3, T. R. Merriman3, K. Lehnert4, V. Bickley1, J. F. Pearson2, V. A. Cameron2, M. A. Kennedy2 Genome-wide association analysis of recurrent myocardial
infarction in UK Biobank identifies suggestive evidence for
association to twenty seven loci We performed a GWAS in
3386 UK-Biobank participants admitted to hospital due to
MI at least twice within a period of 28 days - 1.5 years and
8567 controls with one unique hospital record with MI
diagnosis or MI hospital admissions, which occurred
outside the aforementioned period. Results: A mutation in CRIPAK was found in a high
significant percentage in PAH patients compare to controls. CRIPAK is the key protein responsible for regulation of
PAK1, a major signaling mediator of VEGF responsible for
promoting
proliferation,
migration
and
survival
of
PMVECs. PAH PMVECs lysates demonstrated significant
reduction in CRIPAK protein levels along with concomi-
tantly increased phosphoPAK1 levels. PAH PMVECs
exhibit impaired tube formation and motogenic responses
in culture. Thus, transfection of CRIPAK siRNA into
healthy PMVECs significantly reduced angiogenic response
to VEGF-A, as evidenced by reduced tube network
formation and gap closure in matrigel and scratch assays,
respectively. Results: In total, 215 variants representing 27 loci
reached a suggestive significance level of 10-5. Among
these, 17 loci have been implicated in coronary artery
disease (CAD) and other cardiovascular phenotypes (eg. KCNN2, KLF4, CACNB2, ADIPOR2, KLF5, PKD1L3),
known CAD risk factors (blood pressure, CACNB2; lipid
levels, ABHD4), cardiac remodelling (MAP3K5, SEMA3A),
and abnormalities in platelets and coagulation (GRM7,
KALRN, P2RY1). Five of the identified genes (CHD7, IST1,
KIAA1958, MAP3K5, UBFD1) were also found to be
differentially expressed six months after a MI in 39 MI
survivors (Greek Recurrent Myocardial Infarction Cohort)
that had not experienced any recurrent event during that
period (p-adj ≤10-5). Conclusion: WES analysis has identified CRIPAK as a
potential modifier gene in PAH. Reduced CRIPAK could
contribute to PAH by reducing endothelial viability,
promoting small vessel loss and accelerating vascular
remodeling. Conclusions: We identified 27 loci associated with
increased risk of recurrent MI. We aim to identify 160 J. del Picchia independent datasets to replicate our findings, aiming to a
greater understanding of recurrent MI determinants. Sup-
porting British Heart Foundation grant to O.G (FS/14/66/
31293) as well as grants already received); Significant; Independent
Research Grant from AMGEN. Should the minor genes be included in NGS panels for
inherited cardiac diseases? Patient variation in cholesterol response and the risk of
cardiovascular disease: A UK population-based cohort
study R. K. Akyea, B. Iyen, N. Qureshi, J. Kai, S. Weng R. K. Akyea, B. Iyen, N. Qureshi, J. Kai, S. Weng 1Canterbury District Health Board, Christchurch, New
Zealand, 2University of Otago Christchurch, Christchurch,
New Zealand, 3University of Otago Dunedin, Dunedin, New
Zealand, 4University of Auckland, Auckland, New Zealand Missense variants in genes connected with cardiovascular
diseases in supercentenarians Missense variants in genes connected with cardiovascular
diseases in supercentenarians D. N. Nikolova1, D. Serbezov1, L. Balabanski1, M. Mihaylova1,
V. Damyanova1, Z. Hammoudeh1, D. Nesheva1, S. Karachanak-
Yankova1, R. Staneva1, O. Antonova1, R. Vazharova2,
S. Hadjidekova1, D. Toncheva1 Brompton and Harefield NHS Foundation Trust, London,
United Kingdom 1Fondazione Policlinico A. Gemelli, Rome, Italy, 2Catholic
University of the Sacred Heart, Rome, Italy Sudden cardiac death (SCD) is one of the commonest
causes of mortality in infants and older individuals. Many
cases are a result of pathogenic variants in cardiac ion-
channel
and
cardiomyopathy-related
genes;
however
numerous cases remain unexplained. Kennedy et al. (2016)
and Guimier et al. (2016) (both Am J Hum Genet) reported
cases of both infantile and alcohol-induced SCD caused by
bi-allelic variants in the PPA2 gene, which encodes a
mitochondrial pyrophosphatase essential for cell phosphate
metabolism. Here, we report further cases of infantile and
possible alcohol-induced SCD caused by compound het-
erozygosity for PPA2 variants. Family 1 involved the sud-
den unexplained death of two infants aged less than 1 year. Post-mortem revealed a structurally normal heart in both. Exome sequencing detected two likely pathogenic PPA2
variants in both: p.(Ser61Phe) (previously reported by
Guimier et al.) and p.(Arg127Leu) (previously reported by
Kennedy et al.). Bi-parental inheritance was confirmed, and
we were able to provide predictive genetic testing for a third
healthy infant.Family 2 involved the SCD of a 14-year old
whose heart showed fibrosis at post-mortem. Two variants
were detected in PPA2 by Sanger sequencing: a likely
pathogenic variant, p.(Glu172Lys) (previously detected in a
family where acute sensitivity to alcohol manifested as
myocardial fibrosis (Kennedy et al.) and a VUS, p. (Thr159Met) (not previously reported). Functional studies
of p.(Thr159Met) are on-going. These findings add to the
previous reports of autosomal recessive SCD caused by
pathogenic variants in PPA2, and expand the diagnostic
pathways available to investigate SCD. Introduction: Genetic testing in inherited cardiac diseases
has been revolutionized by NGS technology: the NGS-
based panel approach, indeed, has enabled to test a large
number of genes simultaneously, differently from the tra-
ditional Sanger, limited to the most prevalent and char-
acterized genes. Therefore, recently, so called “minor
genes” have been included in diagnostic NGS panels,
causing interpretative issue, related to the high prevalence
of variants of unknown significance (VUS), not clearly
associated to the disease. Here, we report our experience
with expanded gene panels for genetic testing. Brompton and Harefield NHS Foundation Trust, London,
United Kingdom Materials
and
Methods:
82
consecutive
patients,
19 suspected Hypertrophic Cardiomyopathy (HCM), 29
Arrhythmogenic
Right
Ventricular
Cardiomyopathy
(ARVC), 12 Long QT syndrome (LQTS), 16 Brugada
syndrome (BrS) and 6 Idiopathic Ventricular Fibrillation
(IVF) patients have been analyzed using 3 different custom
sequencing panels, including 11 genes for HCM, 9 for
ARVC, 12 for LQTS and IVF and 4 for BrS. Results: 28 probands carried at least one potentially
pathogenic variant (34%). According to the ACMG guide-
lines, the majority of the identified variants (70%) were
VUS, while 18% were Likely Pathogenic (LP) and 12%
Pathogenic (P). 30% of the VUS have been identified in
minor genes, 8 in the LQTS/IVS and 1 in the HCM panel,
respectively. On the other hand, no LP or P variant was
identified in minor genes. M. Edwards: None. S. Wilkinson: None. F. van den
Broek: None. L. Brett: None. J. Till: None. J.A. Mayr:
None. T. Homfray: None. D. Morris-Rosendahl: None. Conclusions: These data showed that the inclusion of
minor genes in the genetic screening does not significantly
increase the detection rate, while it is associated with an
increase in the rate of VUS. P05.66B V. Novelli: None. D.F. Tiziano: None. C.M. Russo:
None. F. Perella: None. F. Crea: None. P. Zeppilli: None. M. Genuardi: None. 1Clinical Genetics & Genomics Laboratory, Royal Brompton
and Harefield NHS Trust, London, United Kingdom,
2Department of Pediatrics, Paracelsus Medical University
Salzburg, Salzburg, Austria, 3Paediatric Cardiology, Royal P05.64D R.K. Akyea: None. B. Iyen: None. N. Qureshi: None. J. Kai: None. S. Weng: B. Research Grant (principal
investigator, collaborator or consultant and pending grants Should the minor genes be included in NGS panels for
inherited cardiac diseases? Abstracts from the 51st European Society of Human Genetics Conference: Posters 161 V. Novelli1,2, D. F. Tiziano2, C. M. Russo2, F. Perella2,
F. Crea2,1, P. Zeppilli2,1, M. Genuardi2,1
1Fondazione Policlinico A. Gemelli, Rome, Italy, 2Catholic
University of the Sacred Heart, Rome, Italy V. Novelli1,2, D. F. Tiziano2, C. M. Russo2, F. Perella2,
F. Crea2,1, P. Zeppilli2,1, M. Genuardi2,1 P05.65A
Sudden cardiac death caused by bi-allelic variants in the
PPA2 gene M. Edwards1, S. Wilkinson1, F. van den Broek2, L. Brett1,
J. Till3, J. A. Mayr2, T. Homfray1, D. Morris-Rosendahl1 M. Edwards1, S. Wilkinson1, F. van den Broek2, L. Brett1,
J. Till3, J. A. Mayr2, T. Homfray1, D. Morris-Rosendahl1 1Medical University Sofia, Sofia, Bulgaria, 2Sofia University”
St.Kliment Ohridski”,Faculty of Medicine,Department of
Biology, Medical Genetics and Microbiology, Sofia, Bulgaria
Introduction: During the last decades, mankind suffers
unprecedented
demographic
changes
as
a
result
of
decreased/controlled
birthrates. Cardiovascular, 1Medical University Sofia, Sofia, Bulgaria, 2Sofia University”
St.Kliment Ohridski”,Faculty of Medicine,Department of
Biology, Medical Genetics and Microbiology, Sofia, Bulgaria
Introduction: During the last decades, mankind suffers
unprecedented
demographic
changes
as
a
result
of
decreased/controlled
birthrates. Cardiovascular, 1Medical University Sofia, Sofia, Bulgaria, 2Sofia University”
St.Kliment Ohridski”,Faculty of Medicine,Department of
Biology, Medical Genetics and Microbiology, Sofia, Bulgaria 1Medical University Sofia, Sofia, Bulgaria, 2Sofia University”
St.Kliment Ohridski”,Faculty of Medicine,Department of
Biology, Medical Genetics and Microbiology, Sofia, Bulgaria 1Clinical Genetics & Genomics Laboratory, Royal Brompton
and Harefield NHS Trust, London, United Kingdom,
2Department of Pediatrics, Paracelsus Medical University
Salzburg, Salzburg, Austria, 3Paediatric Cardiology, Royal Introduction: During the last decades, mankind suffers
unprecedented
demographic
changes
as
a
result
of
decreased/controlled
birthrates. Cardiovascular, 162 J. del Picchia Introduction: Heart transplantation is the best therapeutic
option for selected patients with end-stage heart failure. However, the immunological barrier between the donor and
recipient still limits long-term survival. Immunosuppressive
drugs are needed to avoid rejection, but cause an increased
incidence of cancer and infections. Besides HLA, also other
genetic factors play a role in graft rejection. We aim to
identify genetic variants in the patient and in the donor that
are
involved
in
rejection
and
survival
after
heart
transplantation. neurodegenerative
and
malignant
diseases
are
mostly
responsible for populations’ mortality. Twin studies show
genetic factors play crucial role in longevity. Super-
centenarians are individuals who have reached 110 or more
years. The analysis of their genomes could help to clarify
the role of genetic variants or reclassify others. Materials and Methods: In publicly available database
of supercentenarians (Gierman HJ et al., 2014) are listed
altogether
110,000
variants. We
selected
216
genes
connected with atherosclerotic vessel changes, lipoprotein
signaling or cholesterol metabolism and analyzed the
nononsynonymous variants according to their clinical
significance (ClinVar). Methods: The iGeneTRAiN consortium consists of over
30,000 solid organ transplant recipients and donors. We
included 1,043 heart transplant recipients and 783 donors
from five different hospitals. P05.65A
Sudden cardiac death caused by bi-allelic variants in the
PPA2 gene We tested over 8 million high
quality variants for association with time to rejection, and
time to death. We used a mixed models approach to take
relatedness and ancestry into account. Results: 151 nonsynonymous variants in 216 cardiovas-
cular genes are found in the list of supercentenarians. After
applying filtering criteria, we listed 35 variants in 21 genes
divided into three categories (,,Pathogenic,,,Conflicting
interpretation of pathogenicity“, and,,Protective“). 8 var-
iants are in APOB gene, followed by 3 variants in APOE
gene. 16 of 35 variants have MAF<0,01%. Of them only
rs769452 in APOE gene is absent in supercentenarians,
while seven others are absent in control group. Results: We identified nine loci that were significantly
associated with rejection (P < 5x10-8); two in donors and
seven in recipients. In addition, we identified one locus in
recipients that was associated with survival. Results: We identified nine loci that were significantly
associated with rejection (P < 5x10-8); two in donors and
seven in recipients. In addition, we identified one locus in
recipients that was associated with survival. Conclusion: We identified a total of ten loci that are
associated with rejection and survival. We aim to increase
our sample of heart transplant donors and recipients in the
near future. In addition, we will conduct cross-organ meta-
analyses including lung, liver, and kidney transplants,
maximizing statistical power to identify novel variants. We ultimately aim to translate genetic data into clinical
applications such as more optimal genomic compatibility
matching of donor-recipient pairs and immune suppression
therapy dosing. Conclusion: We particularly focus on variants with
unknown significance in supercentenarians. On the basis of
our results we could speculate the pathogenic nature of
rs769452 and possible protective role of the other seven
variants in this study. At the moment we are undertaking “in
silico” modeling in attempt to reclassify those as “patho-
genic” or “benign”. Thus it would be easier to make clinical
decisions in the future. D.N. Nikolova: None. D. Serbezov: None. L. Bala-
banski: None. M. Mihaylova: None. V. Damyanova:
None. Z. Hammoudeh: None. D. Nesheva: None. S. Karachanak-Yankova: None. R. Staneva: None. O. Antonova: None. R. Vazharova: None. S. Hadjidekova:
None. D. Toncheva: None. J. van Setten: None. B. Chang: None. N. de Jonge:
None. M.V. Holmes: None. C.C. Baan: None. O.C. Manintveld: None. A.M.A. Peeters: None. F. Domin-
guez: None. K.K. Khush: None. P. Garcia-Pavia: None. J.W. Rossano: None. R.A. de Weger: None. J.H. P05.65A
Sudden cardiac death caused by bi-allelic variants in the
PPA2 gene Moore:
None. B. Keating: None. F.W. Asselbergs: None. J. van Setten1, B. Chang2, N. de Jonge1, M. V. Holmes2, C. C.
Baan3, O. C. Manintveld3, A. M. A. Peeters3, F. Dominguez4,
K. K. Khush5, P. Garcia-Pavia4, J. W. Rossano2, R. A. de
Weger1, J. H. Moore2, B. Keating2, F. W. Asselbergs1
1University Medical Center Utrecht, Utrecht, Netherlands,
2University of Pennsylvania, Philadelphia, PA, United States,
3Erasmus MC, Rotterdam, Netherlands, 4Puerta de Hierro
University Hospital, Madrid, Spain, 5Stanford University,
Stanford, CA, United States P05.68D Whole exome sequencing in 186 sporadic transposition of
the great arteries cases reveals complex genetic etiology W. CHEN, X. Liu, Y. Fu, Z. Zhou
Fuwai Hospital Chinese Academy of Medical Sciences
&Peking Union Medical College, Beijing, China J. van Setten1, B. Chang2, N. de Jonge1, M. V. Holmes2, C. C. Baan3, O. C. Manintveld3, A. M. A. Peeters3, F. Dominguez4,
K. K. Khush5, P. Garcia-Pavia4, J. W. Rossano2, R. A. de
Weger1, J. H. Moore2, B. Keating2, F. W. Asselbergs1
1University Medical Center Utrecht, Utrecht, Netherlands,
2University of Pennsylvania, Philadelphia, PA, United States,
3Erasmus MC, Rotterdam, Netherlands, 4Puerta de Hierro
University Hospital, Madrid, Spain, 5Stanford University,
Stanford, CA, United States J. van Setten1, B. Chang2, N. de Jonge1, M. V. Holmes2, C. C. Baan3, O. C. Manintveld3, A. M. A. Peeters3, F. Dominguez4,
K. K. Khush5, P. Garcia-Pavia4, J. W. Rossano2, R. A. de
Weger1, J. H. Moore2, B. Keating2, F. W. Asselbergs1
1University Medical Center Utrecht, Utrecht, Netherlands,
2University of Pennsylvania, Philadelphia, PA, United States,
3Erasmus MC, Rotterdam, Netherlands, 4Puerta de Hierro
University Hospital, Madrid, Spain, 5Stanford University,
Stanford, CA, United States J. van Setten1, B. Chang2, N. de Jonge1, M. V. Holmes2, C. C. Baan3, O. C. Manintveld3, A. M. A. Peeters3, F. Dominguez4,
K. K. Khush5, P. Garcia-Pavia4, J. W. Rossano2, R. A. de
Weger1, J. H. Moore2, B. Keating2, F. W. Asselbergs1 W. CHEN, X. Liu, Y. Fu, Z. Zhou Fuwai Hospital Chinese Academy of Medical Sciences
&Peking Union Medical College, Beijing, China Fuwai Hospital Chinese Academy of Medical Sciences
&Peking Union Medical College, Beijing, China Introduction: Transposition of the great arteries (TGA) is a
rare life-threatening congenital heart disease with little
known etiology. Some family-based genetic variants are
seldom observed in sporadic TGA subjects, indicating a
distinct genetic etiology in sporadic TGA. We sought to 163 Abstracts from the 51st European Society of Human Genetics Conference: Posters National Yang-Ming University, Taipei, Taiwan, 8Division of
Endocrine and Metabolism, Tri-Service General Hospital,
Taipei, Taiwan, 9Graduate Institute of Medical Sciences,
National Defense Medical Center, Taipei, Taiwan, 10Graduate
Institute of Medical Genomics and Proteomics, National
Taiwan University, Taipei, Taiwan, 11Department of Internal
Medicine, National Taiwan University Hospital, Taipei,
Taiwan, 12Institute of Biomedical Science, Academia Sinica,
Taipei, Taiwan, 13Division of Cardiovascular Medicine, Falk
Cardiovascular Research Building, Stanford University School
of Medicine, Stanford, CA, United States, 14Los Angeles
Biomedical Research Institute, Harbor-UCLA Medical Center,
Torrance, CA, United States explore the genetic etiology of sporadic TGA at different
levels including mutations, genes and pathways. Materials and Methods: 186 sporadic TGA cases and
182 obesity patients without cardiac disease phenotype
were submitted to whole exome sequencing. Variants
calling and filtering were performed according to GATK
best practice pipeline. Single-variant association tests and
gene-based burden testing were performed to identify rare,
disruptive mutations. We also aggregated mutations in 4434
gene sets and performed gene-set based burden tests. Materials and Methods: 186 sporadic TGA cases and
182 obesity patients without cardiac disease phenotype
were submitted to whole exome sequencing. Variants
calling and filtering were performed according to GATK
best practice pipeline. Single-variant association tests and
gene-based burden testing were performed to identify rare,
disruptive mutations. We also aggregated mutations in 4434
gene sets and performed gene-set based burden tests. Results: Few single mutations and genes could achieve
exome-wide significance after multiple testing corrections. However, 45 gene sets were found statistically significant
by gene-set based burden tests (adjust P-value <0.05). These gene sets were mainly involved in embryonic organ
development, cilium organization and cellular response to
stimulus, which is consistent with our current understanding
about the genetic basis underlying CHD. For example,
there was a significant enrichment of rare disruptive
mutations in TGA (5.95 mutations per individual) compared
to control cohort (4.88 mutations per individual) in the
gene set GO_CILIUM_ORGANIZATION (adjust P-value-
= 4.36E-06). W. CHEN, X. Liu, Y. Fu, Z. Zhou Introduction: Chromosome 12q23-q24 has been linked to
triglyceride levels by linkage studies, and it contains the
Insulin-like growth factor 1 (IGF1) gene. However, asso-
ciation between IGF1 and triglyceride levels was not well
investigated and remained unclear. Materials and Methods: We investigated the association
between IGF1 and triglyceride levels by using two
independent samples collected in Taiwan: the first sample
consists of 954 siblings in 397 families from the Stanford
Asian
Pacific
Program
in
Hypertension
and
Insulin
Resistance (SAPPHIRe); the second sample consists of
13,193 unrelated subjects from the Taiwan biobank (TWB)
project. Five tag single-nucleotide polymorphisms (tag-
SNPs) within IGF1 were analyzed. Conclusions: Our study revealed a polygenic burden of
rare disruptive mutations in sporadic TGA, implying an
extensive and complex genetic etiology underlying TGA. The findings of this study may help stratify patients for
guiding the therapeutic management of their clinical care. W. Chen: None. X. Liu: None. Y. Fu: None. Z. Zhou: None. Results: First, based on the SAPPHIRe sample, we found
that one IGF1 tag-SNP was associated with triglyceride
levels (β = -0.049, p = 0.0043). Then, subset analyses in
the TWB sample showed that this association appeared in
subjects with a family history (FH) of hypertension (β =
-0.045, p = 0.0000034), but not in subjects without an FH
(p = 0.61). A re-examination of the SAPPHIRe sample
confirmed that this association appeared in subjects with an
FH of hypertension (β = -0.068, p = 0.0025), but not in
subjects without an FH (p = 0.32). Exome sequencing in disclosing causes of unexpected death
in child - single genetic center experience M. Mijovic1, A. Miletic1, B. Dimitrijevic1, B. Peterlin2, A. Maver2,
J. Ruml Stojanovic1, M. Zivanovic1, G. Cuturilo1,3 1University Children's Hospital, Department of Medical
Genetics, Belgrade, Serbia, 2Clinical Institute of Medical
Genetics, University Medical Centre Ljubljana, Ljubljana,
Slovenia, 3Faculty of Medicine, University of Belgrade,
Belgrade, Serbia Introduction: In the period of last two years in genetic
outpatient clinic in Serbia we had six families with a family
history of unexpected death of a child. Methods: We performed exome sequencing for all. Results: In two cases, there was polylethality with sudden
cardiovascular death in a previously healthy child and other
family members. We found causative RYR2 gene variant in
first and two variants of unknown significance in KCNA5
and FBN1 gene in second family. Pathogenic variant in
ABCD1 gene (X-linked adrenoleucodystrophy) explains
fulminant and unexpected death of boy during gastrointest-
inal infection. MECP2 gene duplication (associated with
infection susceptibility) was discovered by NGS in the boy
with
intellectual
disabilities
and
epilepsy
who
died
unexpectedly from pneumonia. The girl with undiagnosed
metabolic disease without signs of respiratory failure died in
sleep; in her sibling we found the pathogenic variants in the
SURF1 gene for COX IV deficiency. The boy with
dysmorphic features, epilepsy and sudden death was
diagnosed with hemizigot variant in the HUWE1 gene. Introduction: Venous malformations (VMs) are caused by
activating, somatic TIE2 or PIK3CA mutations, activating
AKT and mTOR. In a pilot study, we demonstrated efficacy
of sirolimus as a personalised therapy. We subsequently set
up a trial to assess the efficacy and safety of sirolimus in a
larger cohort of patients (Vascular Anomaly - Sirolimus -
Europe, VASE). Methods: VASE is a prospective, multicentric European
phase III cross over clinical trial. 250 patients with various
vascular malformations refractory to standard treatments are
enrolled. Evaluation includes clinical history and examina-
tion, QOL questionnaire, coagulation analysis, and MRI
before and after one year of treatment. Genotyping of tissue
biopsies is performed. Conclusion: Many genetic diseases, not only cardiovas-
cular, increase risk for unexpected death during childhood. Early genetic referral, genomic testing and genetic counse-
lig are crucial. M. Mijovic: None. A. Miletic: None. B. Dimitrijevic:
None. B. Peterlin: None. A. Maver: None. J. Ruml
Stojanovic:
None. M. Zivanovic:
None. G. Cuturilo: None. M. Mijovic: None. A. Miletic: None. B. Dimitrijevic:
None. B. Peterlin: None. A. Maver: None. J. Ruml
Stojanovic:
None. M. Zivanovic:
None. G. Cuturilo: None. L. Boon1, J. Hammer1, D. Steven1, A. Van Damme2,
A. Dompmartin3, M. Sevestre4, J. Rössler5, A. Bisdorff6,
I. Quere7, S. Schmitz8, P. Clapuyt9, F. Hammer10, C. Legrand11,
M. Vikkula12, E. Seront13 IGF1 gene is associated with triglyceride levels in subjects
with family history of hypertension from the SAPPHIRe
and TWB projects IGF1 gene is associated with triglyceride levels in subjects
with family history of hypertension from the SAPPHIRe
and TWB projects W. Wang1,2, Y. Chiu2, R. Chung2, C. Hwu3,4, I. Lee5,6,7, C. Lee8,9,
Y. Chang10,11,12, K. Hung2, T. Quertermous13, Y. I. Chen14, C. A. Hsiung2 W. Wang1,2, Y. Chiu2, R. Chung2, C. Hwu3,4, I. Lee5,6,7, C. Lee8,9,
Y. Chang10,11,12, K. Hung2, T. Quertermous13, Y. I. Chen14, C. A. Hsiung2 Conclusions: The successful replication in two indepen-
dent samples indicated that IGF1 is associated with
triglyceride levels in subjects with an FH of hypertension
in Taiwan. 1The Ph.D. Program for Translational Medicine, College of
Medical Science and Technology, Taipei Medical University,
Taipei, Taiwan, 2Division of Biostatistics and Bioinformatics,
Institute of Population Health Sciences, National Health
Research Institutes, Zhunan, Taiwan, 3Section of
Endocrinology and Metabolism, Department of Medicine,
Taipei Veterans General Hospita, Taipei, Taiwan, 4Faculty of
Medicine, National Yang-Ming University School of medicin,
Taipei, Taiwan, 5Division of Endocrinology and Metabolism,
Department of Internal Medicine, Taichung Veterans General
Hospital, Taichung, Taiwan, 6School of Medicine, Chung Shan
Medical University, Taichung, Taiwan, 7School of Medicine, This study was supported by the National Health
Research Institutes (PH-104-PP03, PH-105-PP03, PH-106-
PP03), the Taipei Medical University (TMU101-AE1-B67),
and the Ministry of Science and Technology (MOST 106-
2314-B-038-052-MY3) in Taiwan. W. Wang: None. Y. Chiu: None. R. Chung: None. C. Hwu: None. I. Lee: None. C. Lee: None. Y. Chang: None. K. Hung: None. T. Quertermous: None. Y.I. Chen:
None. C.A. Hsiung: None. 164 J. del Picchia Exome sequencing in disclosing causes of unexpected death
in child - single genetic center experience Results: During 2016-17, 44 patients (median age 44y;
2y-71y), including 31 VMs, 4 lymphatic malformations, 5
capillary-venous malformations, and 4 syndromic patients,
were enrolled. Sirolimus was well tolerated with mostly
mild and easily manageable side effects. There was no
complete response, but 89% of patients (n = 39) presented a
rapid clinical improvement with reduction of pain and/or
coagulation abnormalities, decrease in size of lesions, and/
or improvement in quality of life (QOL). Currently, 29
patients have been treated with sirolimus for ≥12 months
and 8 for ≥6 months. The 1-year radiological evaluation
demonstrated VM reduction ≥10% in 45% of 21 evaluable
patients. P05.70B 1Center for Vascular Anomalies, Division of Plastic Surgery,
Cliniques universitaires Saint Luc, University of Louvain,
Brussels, Belgium, 2Department of Pediatric Hemato-
oncology, Cliniques universitaires Saint Luc, University of
Louvain, Brussels, Belgium, 3Dermatology Department, CHU
Caen, University Caen Normandie, Caen, France,
4Department of Vascular Medicine, Amiens university
Hospital, Amiens, France, 5Pediatric Hematology/Oncology,
Medical Centre - University Freiburg, Freiburg, Germany,
6Department of Neuroradiology, Lariboisière Hospital, Paris,
France, 7Centre Hospitalier Universitaire, Montpellier,
Montpellier, France, 8Department of Head and Neck Surgery,
Cliniques universitaires Saint-Luc, University of Louvain,
Brussels, Belgium, 9Division of Pediatric Radiology, Cliniques
universitaires Saint Luc, University of Louvain, Brussels,
Belgium, 10Division of Interventional Radiology, Cliniques
universitaires Saint-Luc, University of Louvain, Brussels,
Belgium, 11Institut of Statistics, Biostatistics, and Actuarial
Sciences, University of Louvain, Louvain-la-Neuve, Belgium,
12Human Molecular Genetics, de Duve Institute, University of
Louvain, Brussels, Belgium, 13Institut Roi Albert II,
Department of Medical Oncology, Cliniques universitaires
Saint Luc, University of Louvain, Brussels, Belgium Exome sequencing in disclosing causes of unexpected death
in child - single genetic center experience Exome sequencing in disclosing causes of unexpected death
in child - single genetic center experience M. Vikkula12, E. Seront13 Mutation spectrum in a Kazakhstani cohort with
ventricular tachycardia: targeted sequencing study Mutation spectrum in a Kazakhstani cohort with
ventricular tachycardia: targeted sequencing study A. R. Akilzhanova1, C. Guelly2, Z. Abilova1, S. Rakhimova1,
A. Akhmetova1, U. Kairov1, O. Nuralinov3, G. Rashbayeva3,
S. Trajanoski2, Z. Zhumadilov1, M. Bekbossynova3 W. Bonda1, K. Bernatowicz2, A. Kashyap2, M. Kossowski1,
E. Studniak1, M. Ryłów1, C. Wleczyk1, M. Bryśkiewicz1,
J. Pietrzak1, S. Zajączek2 W. Bonda1, K. Bernatowicz2, A. Kashyap2, M. Kossowski1,
E. Studniak1, M. Ryłów1, C. Wleczyk1, M. Bryśkiewicz1,
J. Pietrzak1, S. Zajączek2 A. Akhmetova1, U. Kairov1, O. Nuralinov3, G. Rashbayeva3,
S. Trajanoski2, Z. Zhumadilov1, M. Bekbossynova3
1Center for life sciences, National Laboratory Astana,
Nazarbayev University, Astana, Kazakhstan, 2Center of
Medical Research, Medical University of Graz, Graz, Austria,
3National Scientific Cardiac Surgery Center, Astana,
Kazakhstan 1Center for life sciences, National Laboratory Astana,
Nazarbayev University, Astana, Kazakhstan, 2Center of
Medical Research, Medical University of Graz, Graz, Austria,
3National Scientific Cardiac Surgery Center, Astana,
Kazakhstan 1Center for life sciences, National Laboratory Astana,
Nazarbayev University, Astana, Kazakhstan, 2Center of 1Cytogenetic Unit, Department of Laboratory Diagnostics,
Pomeranian Medical University, Szczecin, Poland,
2Department of Genetics and Pathology, Pomeranian Medical
University, Szczecin, Poland Medical Research, Medical University of Graz, Graz, Austria,
3National Scientific Cardiac Surgery Center Astana Medical Research, Medical University of Graz, Graz, Austria,
3 Medical Research, Medical University of Graz, Graz, Austria,
3National Scientific Cardiac Surgery Center, Astana, 3National Scientific Cardiac Surgery Center, Astana, Kazakhstan Kazakhstan Introduction: Ventricular tachycardia (VT) is a common
symptom in cardiac disorders of different etiology. Aim: to
investigate genetic basis of VT in patients with cardio-
myopathy in Kazakhstan using targeted NGS (design of
new HaloPlex gene panel). Williams syndrome (Williams Beuren syndrome, MIM
194050) is a multisystem disorder with variable phenotypic
expression, caused by microdeletions of chromosomal
region 7q11.23. Cardinal features of the syndrome are:
supravalvular aortic stenosis (SVAS), mental retardation,
and distinctive facial features, commonly known as ‘elfin
facies’. Size of the deletion may vary, mostly between 1.5 to
1.8 Mbp. Vast majority of cases contains deletion of mul-
tiple genes (>25) along with the ELN gene. Material and Methods:
We enrolled
92 patients,
diagnosed with either coronary heart disease (CHD), dilated
cardiomyopathy (DCM) or idiopathic ventricular tachycar-
dia (iVT) in a study to evaluate the genetic profile and
variants in known 96 cardiac risk genes by targeted next
generation sequencing (NGS). We report a 12 month old boy with congenital SVAS and
pulmonary stenosis. P05.71C Precision therapy for venous malformations: efficacy and
safety of sirolimus in vascular malformations, preliminary
results of a phase III clinical trial VASE Conclusion: Sirolimus showed impressive efficacy in
slow-flow vascular malformations with activation of the Abstracts from the 51st European Society of Human Genetics Conference: Posters 165 PI3K-AKT-mTOR pathway due to somatic mutations. It
reduced pain and improved functional restraint in the
majority of patients. This underscores the results of our
earlier pilot study on 20 VMs. cardiac risk genes in a similar pattern and at a comparable
frequency. This study was supported by a grant from the
Ministry Education and Science, Republic of Kazakhstan
(AP05134683). cardiac risk genes in a similar pattern and at a comparable
frequency. This study was supported by a grant from the
Ministry Education and Science, Republic of Kazakhstan
(AP05134683). cardiac risk genes in a similar pattern and at a comparable
frequency. This study was supported by a grant from the
Ministry Education and Science, Republic of Kazakhstan
(AP05134683). L. Boon: None. J. Hammer: None. D. Steven: None. A. Van Damme: None. A. Dompmartin: None. M. Sevestre: A.R. Akilzhanova: None. C. Guelly: None. Z. Abilova:
None. S. Rakhimova: None. A. Akhmetova: None. U. Kairov: None. O. Nuralinov: None. G. Rashbayeva:
None. S. Trajanoski: None. Z. Zhumadilov: None. M. Bekbossynova: None. L. Boon: None. J. Hammer: None. D. Steven: None. A. Van Damme: None. A. Dompmartin: None. M. Sevestre: None. J. Rössler: None. A. Bisdorff: None. I. Quere:
None. S. Schmitz: None. P. Clapuyt: None. F. Hammer:
None. C. Legrand:
None. M. Vikkula:
None. E. Seront: None. None. J. Rössler: None. A. Bisdorff: None. I. Quere:
None. S. Schmitz: None. P. Clapuyt: None. F. Hammer:
None. C. Legrand:
None. M. Vikkula:
None. E. Seront: None. P05.72D Deletion of a small fragment of chromosome 7q11.23
containing ELN gene may be responsible for supravalvular
aortic stenosis with pulmonary stenosis and no other
features of Williams syndrome ZeClinics, Barcelona, Spain ZeClinics, Barcelona, Spain Cardiovascular disease represents a heavy burden for
societies and was responsible of the premature death of 17
million people worldwide in 2015. Dilated cardiomyopathy
(DCM) is a common form of heart disease with high mor-
tality. It affects 1 in 250 people, principally young adults
and children. DCM is characterized by ventricular dilatation
and impaired cardiac function, due to a dramatic decrease in
ejection fraction. DCM is also associated with increased
risks for cardiac arrhythmia and sudden cardiac death. To
achieve better patient stratification and identify new drug-
gable targets, which might allow finding novel therapies, it
is crucial to identify genes associated to DCM progression. Different NGS studies have identified genetic variants with
a possible association to DCM. However, the use of animal
models is required to validate phenotypically their real
impact in DCM progression. The genetic and physiologic
homology between humans and zebrafish allows perform-
ing functional genomic studies with this model organism. Hereby, we have developed an experimental platform that
combines fast gene inactivation and robust cardiovascular
phenotyping in zebrafish: ZeCardio. ZeCardio combines the
advantages of CRISPR/Cas9 for generating F0 mutant lar-
vae with a high mutagenesis efficiency rate (CRISPANTS)
and a high-throughput imaging system allowing morpho-
logical and functional analysis of cardiovascular pheno-
types. Such platform represents a fast and reliable screening
method for validating novel DCM associated genes/ther-
apeutic targets and, eventually, for discovering novel
therapies to treat this disease. This project has received
funding from the European Union’s Horizon 2020 research
and innovation programme under grant agreement No
755988. 1Universidad Católica de Murcia (UCAM), Murcia, Spain,
2Servicio de Neurología. Hospital Comarcal del Noroeste,
Caravaca, Spain, 3Grupo Applied Statistical Methods in
Medical Research. Universidad Católica de Murcia (UCAM),
Murcia, Spain, 4Centro de Bioquímica y Genética Clínica. Hospital Clínico Universitario Virgen de la Arrixaca, Murcia,
Spain, 5IMIB- Arrixaca, Murcia, Spain, 6CIBERER-ISCIII,
Madrid, Spain, 7Sección Genética Médica. Servicio de
Pediatría. Hospital Clínico Universitario Virgen de la
Arrixaca., Murcia, Spain Introduction: Acute intermittent porphyria (AIP) is a haem
biosynthesis disorder, leading to an overproduction of δ-
aminolevulinic acid (ALA) and porphobilinogen (PBG). AIP is characterized by acute neurovisceral attacks. Chronic
kidney disease (CKD) is a long-term complication, corre-
lated with the frequency of attacks. Urinary ALA and PBG
seem to promote renal tubular toxicity. Variants of human
peptide transporter 2 (PEPT2), which mediates reabsorption
of ALA in renal tubular cells, predict CKD in AIP. Mutation spectrum in a Kazakhstani cohort with
ventricular tachycardia: targeted sequencing study No dysmorphic features were observed
during several hospitalizations and visits in the genetic
clinics. Genitourinary, skeletal, skin and endocrine anoma-
lies were excluded, except of subclinical hypothyroidism. Growth parameters are normal. It is too early to determine
neurological development in this patient, however no
psychomotor retardation was diagnosed so far. Results: By sequencing 92 clinically well diagnosed
patients we observed a total of 168 mutations (61 distinct)
listed in the Human Genome Mutation Database (HGMD)
and
another
256
rare/unique
variants
with
elevated
pathogenic potential. The majority of CHD patients carried
known mutations and rare variants with high pathogenetic
potential in the same genes and at a comparable frequency
as observed for DCM and iVT patients. The most abundant
mutations observed for the CHD group locate to MYBPC3,
DMD, LAMA2, MYH6 and GAA. Mutations in the
PRKAG2 gene were overrepresented in the CHD subgroup,
correlating to the prominent role of disturbed energy
metabolism in CHD development and progression. MLPA testing for 7q11.23 region revealed a deletion in
the exon 33 of ELN gene. Microarray (aCGH) analysis
showed deletion of approximately 68kbp containing part of
ELN gene and LIMK1 gene, that is adjacent to ELN. Isolated SVAS is described as a separate nosological
entity in OMIM (#185500). Part of the patients with this
diagnosis carry a heterozygous point mutation, or intragenic
deletions in ELN gene. Conclusions:
Our
study
indicates
that
individuals
presenting with VT secondary to CHD, DCM or of
idiopathic etiology carry multiple rare mutations and
potentially pathogenetic sequence variants in classical 68kb deletion in the 7q11.23 is much smaller than all the
other deletions reported in ISCA and Decipher database. This report support the hypothesis that deletion in the 166 J. del Picchia Williams syndrome region limited to ELN gene may cause
nonsyndromic SVAS and pulmonary stenosis. consultant and pending grants as well as grants already
received); Significant; Zeclinics. Williams syndrome region limited to ELN gene may cause
nonsyndromic SVAS and pulmonary stenosis. W. Bonda: None. K. Bernatowicz: None. A. Kashyap:
None. M. Kossowski: None. E. Studniak: None. M. Ryłów: None. C. Wleczyk: None. M. Bryśkiewicz: None. J. Pietrzak: None. S. Zajączek: None. W. Bonda: None. K. Bernatowicz: None. A. Kashyap:
None. M. Kossowski: None. E. Studniak: None. M. Ryłów: None. C. Wleczyk: None. M. Bryśkiewicz: None. J. Pietrzak: None. S. Zajączek: None. V. Di Donato, S. Dyballa, J. Terriente V. Di Donato, S. Dyballa, J. Terriente V. Di Donato, S. Dyballa, J. Terriente P05.74B
ZECARDIO: A zebrafish genetic screening platform for
cardiovascular disease association studies M. Barreda-Sanchez1, J. Buendía2, C. Carazo-Díaz3,
G. Glover4,5,6, M. C. Martínez-Romero4,5,1,6, M. J. Ballesta-
Martínez7,1,5,6, V. López-González7,5,6, M. J. Sánchez-Soler7,5,1,
L. Rodriguez7, R. Gil-Ferrer7, E. Guillén-Navarro7,5,6 M. Barreda-Sanchez1, J. Buendía2, C. Carazo-Díaz3,
G. Glover4,5,6, M. C. Martínez-Romero4,5,1,6, M. J. Ballesta-
Martínez7,1,5,6, V. López-González7,5,6, M. J. Sánchez-Soler7,5,1,
L. Rodriguez7, R. Gil-Ferrer7, E. Guillén-Navarro7,5,6 M. Barreda-Sanchez1, J. Buendía2, C. Carazo-Díaz3,
G. Glover4,5,6, M. C. Martínez-Romero4,5,1,6, M. J. Ballesta-
Martínez7,1,5,6, V. López-González7,5,6, M. J. Sánchez-Soler7,5,1,
L. Rodriguez7, R. Gil-Ferrer7, E. Guillén-Navarro7,5,6 V. Di Donato, S. Dyballa, J. Terriente IDIBELL, Barcelona, Spain NRF2, encoded by NFE2L2 gene, is the master regulator of
endogenous antioxidant responses. Oxidative damage is a
shared and early-appearing feature in X-linked adrenoleu-
kodystrophy (X-ALD) patients and the corresponding
mouse model (Abcd1- mouse). X-ALD is a rare neurome-
tabolic disease caused by the loss of function of the per-
oxisomal transporter ABCD1. In mice, Abcd1 loss results in
a phenotype similar to adrenomyeloneuropathy (AMN), the
most common X-ALD phenotype. Here, we identify an
impaired NRF2 antioxidant response caused by aberrant
activity of GSK-3β. We find that GSK-3β inhibitors can
significantly reactivate the blunted NRF2 response in
patients’ fibroblasts. In the mouse models (Abcd1- and
Abcd1-/Abcd2-/- mice), oral administration of dimethyl
fumarate (DMF/ BG12/Tecfidera), an FDA-approved NRF2
activator, normalized i) mitochondrial depletion, ii) bioe-
nergetic failure, iii) oxidative damage and iv) inflammation,
highlighting an intricate cross-talk governing energetic and
redox homeostasis in X-ALD. Strikingly, DMF halted
axonal degeneration and locomotor disability. Our results
indicate that therapies activating NRF2 hold therapeutic
potential for X-ALD and other axonopathies with impaired
GSK-3β/NRF2 axis. Supported by grants from the Spanish
Institute for Health Carlos III and ‘Fondo Europeo de
Desarrollo Regional (FEDER), Union Europea, una manera
de hacer Europa’ [PFIS FI12/00457] to P.R-R., FIS PI14/
00410 to A.P., FIS PI15/00857 to S.F. We present the systematic analysis of the largest cohort of
patients with the rare metabolic disorder alkaptonuria
(AKU). In 166 patients, including those from the SONIA2
(Suitability of Nitisinone in Alkaptonuria 2) and SOFIA
(Subclinical Ochronotic Features In Alkaptonuria) studies,
we identified 22 novel homogentisate 1,2-dioxygenase
(HGD) gene variants by DNA sequencing, and four novel
larger genomic deletions within HGD gene by MLPA
(Multiplex Ligation-dependent Probe Amplification). In
addition, we analyzed by minigene reporter assay seven
novel or previously reported HGD variants predicted to
affect splicing. Two of them were shown to cause exon
skipping or cryptic splice-site activation. Thus, using DNA
sequencing, MLPA and in vitro splicing analysis we were
able to identify AKU-causing mutation in 328 of 332 AKU
chromosomes (98.8%). However, in two patients, only one
HGD mutation was found, and in one case, no HGD variant
has been identified. No patient’s cDNA is available in order
to verify whether some deep intronic HGD variants affect-
ing correct exon splicing represent an alternative mutation
mechanism in these cases. ZeClinics, Barcelona, Spain Our aim
was to analyse PEPT2 genotype and the onset of CKD in
Spanish AIP carriers. Materials and Methods: PEPT2 alleles were determined
by sequencing and haplotype analysis of two tag SNPs in
exons 13 and 15. Urinary PBG and ALA were measured by
chromatography and spectrophotometry. Glomerular filtra-
tion rate was estimated with CKD-EPI Creatinine Equation
(eGFR). Results: 50 AIP carriers, 52% symptomatic, were
included. Multiple lineal regression analysis showed no
association between levels of urinary ALA and PBG and
PEPT2 genotype. Carriers of at least one PEPT2*2 allele
showed higher eGFR (μ: 92,08 ml/min per 1.73 m2;
SE:5.22) compared with PEPT2*1/*1 carriers (μ: 78.37 ml/
min per 1.73 m2; SE: 5.54), but with moderate statistical
evidence (p = 0.07). 28.57% of PEPT2*1/*1 carriers,
16.67% of *1/*2 carriers and 0% of *2/*2 carriers presented V. Di Donato: A. Employment (full or part-time);
Significant; ZeClinics. S. Dyballa: A. Employment (full
or part-time); Significant; ZeClinics. J. Terriente: A. Employment (full or part-time); Significant; ZeClinics. B. Research Grant (principal investigator, collaborator or Abstracts from the 51st European Society of Human Genetics Conference: Posters 167 eGFR<60 (p = 0.21). The occurrence of acute attacks was
associated with lower eGFR and higher prevalence of CKD
(p < 0.01). P06.03C 26 novel homogentisate 1,2, dioxygenase (HGD) gene
variants, in vitro splicing analysis and genotype-phenotype
correlations in the largest cohort of patients with
alkaptonuria (AKU) Conclusions: In our population, PEPT2 genotyping may
be a useful tool to predict risk to CKD although the
occurrence of acute attacks seems to be a more powerful
predictor. M. Sekelska1, J. Kralovicova2, I. Sevcikova2, A. Soltysova1,
B. Olsson3, D. B. Asher4,5,6, D. E. V. Pires5,6, L. Ranganath7,
A. Zatkova1 M. Sekelska1, J. Kralovicova2, I. Sevcikova2, A. Soltysova1,
B. Olsson3, D. B. Asher4,5,6, D. E. V. Pires5,6, L. Ranganath7,
A. Zatkova1 M. Barreda-Sanchez: None. J. Buendía: None. C. Carazo-Díaz: None. G. Glover: None. M.C. Martínez-
Romero: None. M.J. Ballesta-Martínez: None. V. López-
González: None. M.J. Sánchez-Soler: None. L. Rodri-
guez:
None. R. Gil-Ferrer:
None. E. Guillén-
Navarro: None. 1Biomedical Research Center, Slovak Academy of Sciences,
Bratislava, Slovakia, 2Center of Biosciences, Institute of
Molecular Physiology and Genetics, Slovak Academy of
Science, Bratislava, Slovakia, 3Clinical Development, Swedish
Orphan Biovitrum AB, Stockholm, Sweden, 4Department of
Biochemistry and Molecular Biology, Bio21 Institute,
University of Melbourne, Victoria, Australia, 5Department of
Biochemistry, Cambridge University, Cambridge, United
Kingdom, 6Instituto René Rachou, FIOCRUZ, Belo Horizonte,
Mg, Brazil, 7Department of Clinical Biochemistry and
Metabolism, Royal Liverpool University Hospital, Liverpool,
United Kingdom P06.02B Aberrant regulation of the GSK-3β/NRF2 axis unveils a
novel therapy for adrenoleukodystrophy A. Pujol, P. Ranea-Robles, N. Launay, M. Ruiz, S. Fourcade A. Pujol, P. Ranea-Robles, N. Launay, M. Ruiz, S. Fourcade IDIBELL, Barcelona, Spain M. Rhee1, A. Solyom2 1Enzyvant, Boston, MA, United States, 2Enzyvant, Basel,
Switzerland 1Enzyvant, Boston, MA, United States, 2Enzyvant, Basel,
Switzerland Introduction: Farber disease is an ultra-rare autosomal
recessive lysosomal storage disease characterized by defi-
ciency of the enzyme acid ceramidase, caused by mutations
in the ASAH1 gene. Resulting accumulation of the pro-
inflammatory and pro-apoptotic sphingolipid
ceramide
causes typical symptoms with a broad spectrum of severity. Materials and Methods: A review of 96 case studies of
Farber patients revealed 64 patient cases where time to
diagnosis could be estimated. Introduction: Barth syndrome (BTHS) is an X-linked
recessive disease caused by mutations in tafazzin gene
(TAZ) which lead to cardiolipin deficiency and mitochon-
drial dysfunction. Male patients have variable clinical
findings - cardiomyopathy, skeletal myopathy, prepubertal
short stature, neutropenia, 3-methylglutaconic aciduria. Female carriers are usually asymptomatic. Results: Diagnosis is generally based on the triad of (1)
hoarse
or
weak
voice,
(2)
joint
inflammation
and
contractures, and (3) subcutaneous nodules. Patients with
a more severe, rapidly progressive, phenotype often
manifest with the triad of symptoms in early infancy,
and also exhibit signs of central nervous system (CNS)
and respiratory involvement. Patients with more moder-
ately progressive phenotypes often present with one of the
classic symptoms during early childhood and develop the
full triad over months to years. The average and median
times to diagnosis (TTD) for severe phenotype patients
was significantly shorter than the average and median
TTDs for moderate phenotype patients. Generally, the
emergence of all three typical symptoms led to the
suspicion of Farber disease and genetic and/or enzyme
activity testing. Materials and Methods: We report male and female
siblings with left ventricular noncompaction and hypotonia. At age of three months, female was found to have mild
aortic valve stenosis with increased trabeculations of the left
ventricle. At age 9 years she had noted a mild persistent
muscle weakness and easy fatigability. The patient had a
normal female karyotype. No biochemical abnormalities. Materials and Methods: We report male and female
siblings with left ventricular noncompaction and hypotonia. At age of three months, female was found to have mild
aortic valve stenosis with increased trabeculations of the left
ventricle. At age 9 years she had noted a mild persistent
muscle weakness and easy fatigability. The patient had a
normal female karyotype. No biochemical abnormalities. Farber disease (acid ceramidase deficiency): time to
diagnosis is influenced by disease severity, indicating that
more slowly progressive disease may be underdiagnosed 1National Genetic Laboratory, Department of Obstetrics and
Gynecology, Medical University of Sofia and Department of
Analytical Chemistry, Faculty of Chemistry and Pharmacy,
Sofia University “St. Kl. Ohridski”, Sofia, Bulgaria,
2Department of Clinical Genetics, University Pediatric
Hospital, Medical University, Sofia, Bulgaria, 3Department of
Medical Chemistry and Biochemistry, Medical University and
Genetic Medico-Diagnostic Laboratory “Genica”, Sofia,
Bulgaria, 4Medical University and Genetic Medico-Diagnostic
Laboratory “Genica”, Sofia, Bulgaria, 5Department of
Neurology, University Pediatric Hospital, Medical University,
Sofia, Bulgaria, 6Department of Pediatric Cardiology,
National Heart Hospital, Sofia, Bulgaria M. Rhee1, A. Solyom2 P06.05A M. Sekelska: None. J. Kralovicova: None. I. Sevci-
kova: None. A. Soltysova: None. B. Olsson: None. D.B. Asher: None. D.E.V. Pires: None. L. Ranganath: None. A. Zatkova: None. The first manifesting case of Barth syndrome in a
heterozygous female patient with normal karyotype M. B. Ivanova1, D. M. Avdjieva-Tzavella2, A. P. Todorova3,
H. M. H. M. Kathom2, I. T. Yordanova3, T. P. Todorov4, I. O. Litvinenko5, A. T. Dasheva-Dimitrova6, R. T. Tincheva2 IDIBELL, Barcelona, Spain For the first time in AKU, we
performed a genotype-phenotype correlation study for the
four most frequent HGD mutations (G161R, M368V,
A122V, ivs1-1G>A), identified in 139 patients participating
in the SONIA2 study. We found no correlation of either
urine excretion or serum HGA concentrations to a specific
HGD genotype. Most probably, these parameters are not
suitable for this kind of analysis, since they are affected by
external factors, such as renal function and dietary intake of
tyrosine. (7FP- DevelopAKUre 304985) A. Pujol: None. P. Ranea-Robles: None. N. Launay:
None. M. Ruiz: None. S. Fourcade: None. A. Pujol: None. P. Ranea-Robles: None. N. Launay:
None. M. Ruiz: None. S. Fourcade: None. J. del Picchia 168 P06.06B
Clinical, biochemical and genetic spectrum of 70 patients
with ACAD9 deficiency: is riboflavin supplementation
effective? E. Mastantuono1,2, B. Repp1, C. L. Alston3, M. Schiff4, T. B. Haack1,2, A. Rötig5, A. Ardissone6, A. Lombes7, C. B. Catarino8,
D. Diodato9, G. Schottmann10, J. Poulton11, A. Burlina12,
A. Jonckheere13, A. Munnich5, D. Ghezzi6, D. Rokicki14,
D. Wellesley15, D. Martinelli16, E. Lamantea6, E. Ostergaard17,
E. Pronicka14, G. Pierre18, H. J. Smeets19, I. Scurr20, I. F. De
Coo21, I. Moroni6, J. Smet22, J. A. Mayr23, L. De Meirleir24,
M. Schuelke10, M. Zeviani25, R. McFarland3, S. Seneca26,
T. Klopstock8, T. Meitinger2,1, T. M. Strom1,2, U. Herberg27,
W. Sperl23, M. Nassogne28, H. Ling29, F. Fang29, P. Freisinger30,
R. Van Coster31, R. W. Taylor3, J. Häberle32, J. Vockley33,
H. Prokisch2, S. Wortmann1,23,2 1IHG, Helmholtz Zentrum, Munich, Germany, 2IHG, TUM,
Munich, Germany, 3Wellcome Trust Centre, Newcastle, United
Kingdom, 4INSERM, Uni. Diderot, Paris, France, 5UMR1163,
Uni. Descartes, Paris, France, 6Istituto Neurologico Besta,
Milan, Italy, 7INSERM, Inst. Cochin, Paris, France, 8Neurol. Dept., Friedrich-Baur-Inst., Munich, Germany, 9Dept. Neurogen., Bambino Gesù, Rome, Italy, 10NCRC, Charité,
Berlin, Germany, 11Nuffield Repr. Health, Oxford Uni., Oxford,
United Kingdom, 12Inher. Met., Padova Uni., Padova, Italy,
13Ped. Dept., Antwerp Uni., Antwerp, Belgium, 14Med. Genet. Dept., Children's Memorial Health Instit., Warsaw, Poland,
15Wessex Clin. Genet., Princ. Anne Hosp., Southampton,
United Kingdom, 16Genet. Div., Bambino Gesù, Rome, Italy,
17Clin. Genet., Copenhagen Uni., Copenaghen, Denmark,
18Metab. Dept., Bristol Royal, Bristol, United Kingdom, 19Clin. Genet. Dept., Maastricht Uni., Maastricht, Netherlands, 20Clin. Genet. Dept., St Michael's Hosp., Bristol, United Kingdom,
21Neur. Dept, Erasmus MC, Rotterdam, Netherlands, 22Ped. Neurol., Ghent Uni., De Pintelaan, Belgium, 23Ped. Dept.,
SALK+PMU, Salzburg, Austria, 24Repr. and Genet., Vrije
Uni., Brussel, Belgium, 25MRC, Cambridge, United Kingdom,
26Med. Genet., Vrije Univ., Brussel, Belgium, 27Ped. Cardiol. Dept., Uni. of Bonn, Bonn, Germany, 28Univ. Catholique de
Louvain, Brussels, Belgium, 29Beijing Hosp., Bejiing, China,
30Ped. Dept, Reutlingen Hosp., Reutlingen, Germany, 31Ped. Neurol., Ghent Univ., De Pintelaan, Belgium, 32Metab. Div.,
Children' Hosp., Zurich, Switzerland, 33Ped. Dept., Pittsburgh
Uni., Pittsburgh, PA, United States E. Mastantuono: None. B. Repp: None. C.L. Alston:
None. M. Schiff: None. T.B. Haack: None. A. Rötig:
None. A. Ardissone: None. A. Lombes: None. C.B. Catarino: None. D. Diodato: None. G. Schottmann:
None. J. Poulton: None. A. Burlina: None. A. Jonc-
kheere: None. A. Munnich: None. D. Ghezzi: None. D. Rokicki: None. D. Wellesley: None. D. Martinelli: None. E. Lamantea: None. E. Ostergaard: None. E. Pronicka:
None. G. Pierre: None. H.J. Smeets: None. I. Scurr:
None. I.F. De Coo: None. I. Moroni: None. J. Smet:
None. J.A. M. Rhee1, A. Solyom2 Her younger brother showed a severe phenotype of BTHS
- dilated cardiomyopathy with noncompaction of the left
ventricle and endocardial fibroelastosis, cyclic neutropenia,
muscle weakness. Metabolic testing showed elevated levels
of urine 3-methylgultaconic acid and 3-methylglutaric acid. Conclusions: The analysis indicates that TTD is longer in
those patients with more moderate and attenuated pheno-
types, potentially due to the low index of suspicion in
patients who may have not yet developed all three typical
symptoms, and points to the need for additional education
on the broad phenotype spectrum and indications for testing
of acid ceramidase deficiency. Results: The molecular genetic testing showed that both
siblings
carry
a
novel
mutation:
c.253insC,
p. (Arg85Profs*54) in exon 3 of the TAZ gene. The
heterozygous female patient is manifesting carrier while the
severely affected male proband was hemizygous for the X-
linked TAZ gene mutation. M. Rhee: A. Employment (full or part-time); Significant;
Enzyvant. A. Solyom: A. Employment (full or part-time);
Significant; Enzyvant. Conclusions: We identified a novel TAZ gene insertion
that is associated with a classical phenotype of BTHS in the
male sibling and a milder clinical presentation in his sister. This is the first report of a manifesting female carrier
of BTHS. Abstracts from the 51st European Society of Human Genetics Conference: Posters 169 M.B. Ivanova: None. D.M. Avdjieva-Tzavella: None. A.P. Todorova: None. H.M. H. M. Kathom: None. I.T. Yordanova: None. T.P. Todorov: None. I.O. Litvinenko:
None. A.T. Dasheva-Dimitrova:
None. R.T. Tincheva: None. Mitochondrial acyl-CoA dehydrogenase family member 9
(ACAD9) is an essential assembly factor of mitochondrial
respiratory chain complex I. The clinical presentation of
ACAD9 deficiency is dominated by cardiomyopathy. Other features are lactic acidosis, myopathy and devel-
opmental delay. Here we describe the genetic, clinical and
biochemical findings in a cohort of 70 patients, of whom
29 previously unpublished. Among the disease-causing
biallelic ACAD9 variants identified, 34 were known and
18 previously unreported variants. No patients harbored
biallelic loss of function mutations, indicating that this
combination is unlikely to be compatible with life. For the
causal pathogenic variants distributed across the gene, no
obvious genotype-phenotype correlation was observed. The majority of the patients presented in the first year of
life. For this subgroup the survival was poor (50% not
surviving the first two years) comparing to patients with a
later presentation (more than 90% surviving 10 years). 1IHG, Helmholtz Zentrum, Munich, Germany, 2IHG, TUM,
Munich, Germany, 3Wellcome Trust Centre, Newcastle, United
Kingdom, 4INSERM, Uni. Diderot, Paris, France, 5UMR1163,
Uni. Descartes, Paris, France, 6Istituto Neurologico Besta,
Milan, Italy, 7INSERM, Inst. Cochin, Paris, France, 8Neurol.
Dept., Friedrich-Baur-Inst., Munich, Germany, 9Dept.
Neurogen., Bambino Gesù, Rome, Italy, 10NCRC, Charité,
Berlin, Germany, 11Nuffield Repr. Health, Oxford Uni., Oxford,
United Kingdom, 12Inher. Met., Padova Uni., Padova, Italy,
13Ped. Dept., Antwerp Uni., Antwerp, Belgium, 14Med. Genet.
Dept., Children's Memorial Health Instit., Warsaw, Poland,
15Wessex Clin. Genet., Princ. Anne Hosp., Southampton,
United Kingdom, 16Genet. Div., Bambino Gesù, Rome, Italy,
17Clin. Genet., Copenhagen Uni., Copenaghen, Denmark,
18Metab. Dept., Bristol Royal, Bristol, United Kingdom, 19Clin.
Genet. Dept., Maastricht Uni., Maastricht, Netherlands, 20Clin.
Genet. Dept., St Michael's Hosp., Bristol, United Kingdom,
21Neur. Dept, Erasmus MC, Rotterdam, Netherlands, 22Ped.
Neurol., Ghent Uni., De Pintelaan, Belgium, 23Ped. Dept.,
SALK+PMU, Salzburg, Austria, 24Repr. and Genet., Vrije
Uni., Brussel, Belgium, 25MRC, Cambridge, United Kingdom,
26Med. Genet., Vrije Univ., Brussel, Belgium, 27Ped. Cardiol.
Dept., Uni. of Bonn, Bonn, Germany, 28Univ. Catholique de
Louvain, Brussels, Belgium, 29Beijing Hosp., Bejiing, China,
30Ped. Dept, Reutlingen Hosp., Reutlingen, Germany, 31Ped.
Neurol., Ghent Univ., De Pintelaan, Belgium, 32Metab. Div.,
Children' Hosp., Zurich, Switzerland, 33Ped. Dept., Pittsburgh
Uni., Pittsburgh, PA, United States P06.06B P06.06B
Clinical, biochemical and genetic spectrum of 70 patients
with ACAD9 deficiency: is riboflavin supplementation
effective? M. Rhee1, A. Solyom2 The most common clinical findings were cardiomyopathy
(85%), muscular weakness (75%) and exercise intolerance
(72%). Interestingly, severe intellectual deficits were only
reported in one patient and severe developmental delays
in just four patients. The majority of the patients (70%)
were
able
to
perform
normal
daily-life
activities. Remarkably, our results show that riboflavin treatment
improves complex I activity for most of patient-derived
fibroblasts tested. This effect was also reported for the
majority of the treated patients and is mirrored in the
survival data. In the patient group with disease-onset
below one year of age, a statistically-significant better
survival for patients treated with riboflavin was observed. P06.06B
Clinical, biochemical and genetic spectrum of 70 patients
with ACAD9 deficiency: is riboflavin supplementation
effective? 1Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 2Faculty of Medicine, Vilnius University, Vilnius,
Lithuania Introduction: Setting population specific cut-off levels is
essential in cost-effective newborn screening programs. This study describes the results obtained in the first year of a
public congenital adrenal hyperplasia (CAH) screening
program in Lithuania. Wilson disease (WD) is a rare inherited disorder associated
with the copper accumulation in different organs. WD is
caused by mutations in the ATP7B gene. The clinical phe-
notype of WD is modified by variants in other genes
including COMMD1 and XIAP. Protein COMMD1 takes
part in the transport of copper. XIAP is antiapoptotic pro-
tein, whose activity depends on the cytoplasmic copper
concentration. Hereby the interactions of these proteins can
be involved in a clinical polymorphism of the WD. Materials and Methods: 17-alpha-hydroxyprogesterone
(17-OHP) concentrations in dry blood spots from 27175
neonates were measured using a neonatal screening test. 17-
OHP concentrations were compared in accordance to
gender, gestation status, 5 gestational age (GA) categories,
6 gestational weight (GW) categories and the age at blood
sampling (ABS). 95th percentile cut-off values were
calculated for GA and GW categories and evaluated using
data of 4 confirmed CAH cases. Analysis was significant at
P < 0.05. Materials and Methods: 17-alpha-hydroxyprogesterone
(17-OHP) concentrations in dry blood spots from 27175
neonates were measured using a neonatal screening test. 17-
OHP concentrations were compared in accordance to
gender, gestation status, 5 gestational age (GA) categories,
6 gestational weight (GW) categories and the age at blood
sampling (ABS). 95th percentile cut-off values were
calculated for GA and GW categories and evaluated using
data of 4 confirmed CAH cases. Analysis was significant at
P < 0.05. Materials and Methods: We examined 89 WD patients
by NGS method. We designed targeted panel NimbleGen
SeqCap
EZ
Choice:
151012_HG38_CysFib_EZ_HX3
(ROCHE) for analysis ATP7B, COMMD1 and XIAP genes. Different algorithms were used to predict the effects of the
mutations. Results: A total of 27175 infants (13142 females, 13911
males, 122 unknown) were screened. 1469 (5,4%) new-
borns were born at pre-term (PT) and 25706 (94,6%) at full-
term (FT). Mean 17-OHP concentrations were significantly
higher in PT vs FT group (28.99 ± 31,68 mmol/l vs 11,9
SD ± 9.0 mmol/l, P < 0.05). Mean 17-OHP was higher in
males (13.46 ± 12.63 mmol/l vs 12.13 mmol/l, P < 0.05). Lower GW and GA categories had higher mean 17-OHP
concentrations (P < 0.05). No significant correlation was
found between ABS and 17-OHP concentration. P06.06B
Clinical, biochemical and genetic spectrum of 70 patients
with ACAD9 deficiency: is riboflavin supplementation
effective? Mayr: None. L. De Meirleir: None. M. Schuelke: None. M. Zeviani: None. R. McFarland: None. S. Seneca: None. T. Klopstock: None. T. Meitinger:
None. T.M. Strom: None. U. Herberg: None. W. Sperl:
None. M. Nassogne: None. H. Ling: None. F. Fang:
None. P. Freisinger: None. R. Van Coster: None. R.W. Taylor: None. J. Häberle: None. J. Vockley: None. H. Prokisch: None. S. Wortmann: None. 18Metab. Dept., Bristol Royal, Bristol, United Kingdom, 19Clin. Genet. Dept., Maastricht Uni., Maastricht, Netherlands, 20Clin. Genet. Dept., St Michael's Hosp., Bristol, United Kingdom, 170 J. del Picchia M. S. Balashova1,2, I. G. Tuluzanovskaya1, M. I. Filimonov1,
N. A. Zhuchenko1, O. V. Solov'eva1, O. S. Glotov3,4,5, A. S. Glotov3,4,5, M. A. Fedyakov5,3, I. V. Polyakova3,5, A. M. Sarana3,5, S. G. Shcherbak3,5, T. E. Ivashchenko6, Y. A. Barbitov3, O. V. Romanova5, T. M. Ignatova1, A. Y. Asanov1 1Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 2Faculty of Medicine, Vilnius University, Vilnius,
Lithuania Finally, all
4 cases would be detected by proposed cut-off values. Results: 34 mutations in ATP7B gene were detected. Two
most frequent mutations were c.3207C>A (48% of alleles)
and c.3190G>A (7% of alleles). Single rare mutations were
found in 29% of cases. All our patients had mutations of
both copies of the ATP7B. Two patients were heterozygous
carriers of variants in COMMD1 (c.180+4C>T, c.340C>T). Two patients had variants c.925G>C, c.355A>G in XIAP
gene; benign variant c.1268A>C were detected in our group
very often (63% of cases). Conclusions: Birth at preterm, lower gestational age,
lower gestational weight and male gender were associated
with higher 17-OHP concentration. The sensitivity and
specificity of the cut-off values needs to be evaluated by
further studies. Conclusions: Birth at preterm, lower gestational age,
lower gestational weight and male gender were associated
with higher 17-OHP concentration. The sensitivity and
specificity of the cut-off values needs to be evaluated by
further studies. Conclusion: We discovered no significant pathogenic
mutations in the COMMD1 gene in patients but found two
polymorphisms. We have found 3 polymorphisms in the
XIAP. The meaning of these results remains unclear. The
study
was
implemented
under
the
Russian
Science
Foundation grant №14-50-00069. M. Petkeviciene: None. K. Sablauskas: None. R. Marcinkute: None. M. Smirnova: None. J. Songailiene:
None. A. Utkus: None. M. Petkeviciene: None. K. Sablauskas: None. R. Marcinkute: None. M. Smirnova: None. J. Songailiene:
None. A. Utkus: None. None. A. Utkus: None. The frequency of mutations and polymorphisms in the
genes involved in the metabolism of copper (ATP7B,
COMMD1 and XIAP) M. Petkeviciene1, K. Sablauskas2, R. Marcinkute2,
M. Smirnova1, J. Songailiene1, A. Utkus1 1Sechenov First Moscow State Medical University, Moscow,
Russian Federation, 2Center of Genetics and Reproductive
Medicine «Genetico», Moscow, Russian Federation, 3Saint
Petersburg State University, Saint Petersburg, Russian
Federation, 4D.O.Ott Research Institute of Obstetrics,
Gynecology and Reproductology, Saint Petersburg, Russian
Federation, 5City Hospital №40, Saint Petersburg, Russian
Federation, 6Scientific Research Institute of Obstetrics and
Gynecology by D.O.Otta of the Russian academy of medical
sciences, Saint Petersburg, Russian Federation 1Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 2Faculty of Medicine, Vilnius University, Vilnius,
Lithuania 1Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 2Faculty of Medicine, Vilnius University, Vilnius,
Lithuania P06.11C
Plasma and urinary metabolomic profiles of Down
syndrome correlate with alteration of mitochondrial
metabolism M. Caracausi1, V. Ghini2,3, C. Locatelli4, M. Mericio5,
A. Piovesan1, F. Antonaros1, M. C. Pelleri1, L. Vitale1, R. A. Vacca6, F. Bedetti5, M. C. Mimmi7, C. Luchinat2,8, P. Turano2,8,
P. Strippoli1, G. Cocchi5 J. L. Santos1, R. Cataldo1, F. Rosso1, C. Bravo1, F. Allende2,
S. Solari2, M. I. Hodgson1 J. L. Santos1, R. Cataldo1, F. Rosso1, C. Bravo1, F. Allende2,
S. Solari2, M. I. Hodgson1 1Department of Nutrition, Diabetes and Metabolism. School of
Medicine. Pontificia Universidad Católica de Chile, Santiago,
Chile, 2Departmento de Laboratorios Clínicos. School of
Medicine. Pontificia Universidad Católica de Chile, Santiago,
Chile 1Department of Experimental, Diagnostic and Specialty
Medicine, University of Bologna, Bologna, Italy, 2Center of
Magnetic Resonance, University of Florence, Sesto Fiorentino,
Florence, Italy, 3Consorzio Interuniversitario Risonanze
Magnetiche Metallo Proteine, Sesto Fiorentino, Florence,
Italy, 4Neonatology Unit, St. Orsola-Malpighi Polyclinic,
Bologna, Italy, 5Neonatology Unit, St. Orsola-Malpighi
Polyclinic, Department of Medical and Surgical Sciences,
University of Bologna, Bologna, Italy, 6Institute of
Biomembranes, Bioenergetics and Molecular Biotechnologies,
National Council of Research, Bari, Italy, 7Department of
Medical and Biological Sciences, University of Udine, Udine,
Italy, 8Department of Chemistry, University of Florence, Sesto
Fiorentino, Florence, Italy Introduction: A Chilean proband diagnosed with perma-
nent neonatal diabetes and severe malabsorptive diarrhea
was found to carry two loss-of-function mutations in the
NEUROG3 gene (c.82G>T and 404T> C). Introduction: A Chilean proband diagnosed with perma-
nent neonatal diabetes and severe malabsorptive diarrhea
was found to carry two loss-of-function mutations in the
NEUROG3 gene (c.82G>T and 404T> C). Aim: to measure gut-derived hormones during a meal test
to gain insights on pathophysiological processes that may
explain the impaired intestinal function of the proband
carrying NEUROG3 mutations. Subjects and Methods: Proband, parents and controls
were submitted to a standardized Oral Liquid Meal Test
(237 ml; 220 Kcal; 29 g CHO) with measures of plasma
levels of Gastric Inhibitory Polypeptide (GIP), Glucagon‐
Like Peptide‐1 (GLP-1), Pancreatic Polypeptide (PP),
Fibroblast-Growth-Factor-19
(FGF-19),
C-peptide,
7α-
hydroxy-4-cholesten-one (C4) and platelet serotonin. Introduction: Down syndrome (DS) is caused by the pre-
sence of a supernumerary copy of the human chromosome
21 (Hsa21) and is the most frequent genetic cause of
intellectual disability (ID). Key traits of DS are the dis-
tinctive facies and cognitive impairment. Results and Discussion: Near-absence of GIP, GLP1, PP
and C-peptide were found during the meal test in the
proband, in contrast to controls. Increased bile acid synthesis and near-absence of gut-
derived hormones in a patient with neonatal diabetes and
severe diarrhea caused by mutations in the neurogenin-3
gene Increased bile acid synthesis and near-absence of gut-
derived hormones in a patient with neonatal diabetes and
severe diarrhea caused by mutations in the neurogenin-3
gene None. Y.A. Barbitov: None. O.V. Romanova: None. T.
M. Ignatova: None. A.Y. Asanov: None. J.L. Santos: None. R. Cataldo: None. F. Rosso: None. C. Bravo: None. F. Allende: None. S. Solari: None. M.I. Hodgson: None. J.L. Santos: None. R. Cataldo: None. F. Rosso: None. C. Bravo: None. F. Allende: None. S. Solari: None. M.I. Hodgson: None. P06.08D M.S. Balashova: None. I.G. Tuluzanovskaya: None. M.I. Filimonov: None. N.A. Zhuchenko: None. O.V. Solov'eva: None. O.S. Glotov: None. A.S. Glotov: None. M.A. Fedyakov: None. I.V. Polyakova: None. A.M. Sarana: None. S.G. Shcherbak: None. T.E. Ivashchenko: The frequency of mutations and polymorphisms in the
genes involved in the metabolism of copper (ATP7B,
COMMD1 and XIAP) M.A. Fedyakov: None. I.V. Polyakova: None. A.M. Sarana: None. S.G. Shcherbak: None. T.E. Ivashchenko: Abstracts from the 51st European Society of Human Genetics Conference: Posters 171 None. Y.A. Barbitov: None. O.V. Romanova: None. T. M. Ignatova: None. A.Y. Asanov: None. P06.11C
Plasma and urinary metabolomic profiles of Down
syndrome correlate with alteration of mitochondrial
metabolism FGF19 plasma levels were
extremely low in the proband (26.3 pg/ml versus 118 ± 63
pg/ml in n = 7 controls), while C4 levels were very high
(83.4 ng/ml vs 16.3 ± 12 ng/ml in n = 47 controls). Interestingly, platelet serotonin levels, which reflects its
gut-derived production, were extremely low in the proband
(27 ng/109 platelets vs 772 ± 239 in n = 64 controls). Values of plasma C4 and platelet 5HT of the patient felt
outside
the
prediction
limits
for
univariate
normal
distribution. Materials and Methods: We conducted for the first time
an analysis of the Nuclear Magnetic Resonance (NMR)-
detectable part of the metabolome in plasma and urine
samples, studying 67 subjects with DS and 29 normal
subjects as controls selected among DS siblings. Results: Multivariate analysis of the NMR metabolomic
profiles showed a clear discrimination (up to of 80%
accuracy) between the DS and the control groups. The
univariate
analysis
of
plasma
and
urine
revealed
a
significant alteration for some interesting metabolites. Remarkably, most of the altered concentrations were
consistent with the 3:2 gene dosage model, suggesting
effects caused by the presence of three copies of Hsa21
rather than two: DS/normal ratio in plasma was 1.23
(pyruvate), 1.39 (fumarate), 1.47 (succinate), 1.33 (lactate),
1.4 (formate). Conclusion: The proband with loss-of-function muta-
tions in NEUROG3 gene displays extremely-low platelet
serotonin levels and plasma concentrations of gut-derived
incretins. The combination of low plasma FGF19 and very
high C4 plasma levels suggests an increased hepatic
synthesis of bile acids as a contributor of the severe
malabsorptive diarrhea. FONDECYT 1150416. Conclusions: Several significantly altered metabolites are
produced at the beginning or during the Krebs cycle. Accounting
for
sex,
age
and
fasting
state
did
not 172 J. del Picchia significantly affect the main result of both multivariate and
univariate analysis. P06.11C
Plasma and urinary metabolomic profiles of Down
syndrome correlate with alteration of mitochondrial
metabolism Human Genetics, Technische Universität München, Munich,
Germany, 4Institut für Neurogenomik, Helmholtz Zentrum
München, Neuherberg, Germany, 5Institute of Medical
Genetics and Applied Genomics, University of Tübingen,
Tübingen, Germany, 6Department of Neurology and Institute
of Neuropathology, University Hospital RWTH Aachen,
Aachen, Germany, 7Department of Neurology, University
Hospitals Leuven and KU Leuven, Leuven, Belgium, 8Klinik für
Kinder- und Jugendmedizin, Klinikum Frankfurt Hoechst,
Frankfurt, Germany, 9Department of Genetics, La Pitié-
Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris,
Paris, France, 10Center for Rare Childhood Disorders,
Translational Genomics Research Institute, Phoenix, AZ,
United States, 11Department of Medical Genetics, Warsaw
Medical University, Warsaw, Poland, 12Department of
Pathology, Children’s Memorial Health Institute, Warsaw,
Poland, 13Department of Pediatrics, Nutrition and Metabolic
Diseases, The Children's Memorial Health Institute, Warsaw,
Poland significantly affect the main result of both multivariate and
univariate analysis. y
M. Caracausi: None. V. Ghini: None. C. Locatelli:
None. M. Mericio:
None. A. Piovesan:
None. F. Antonaros: None. M.C. Pelleri: None. L. Vitale: None. R.A. Vacca: None. F. Bedetti: None. M.C. Mimmi: None. C. Luchinat: None. P. Turano: None. P. Strippoli: None. G. Cocchi: None. M. Caracausi: None. V. Ghini: None. C. Locatelli:
None. M. Mericio:
None. A. Piovesan:
None. F. Antonaros: None. M.C. Pelleri: None. L. Vitale: None. R.A. Vacca: None. F. Bedetti: None. M.C. Mimmi: None. C. Luchinat: None. P. Turano: None. P. Strippoli: None. G. Cocchi: None. Hematopoietic stem cell transplant does not prevent
neurological deterioration in infants with Farber disease Visual and hearing impairment, in keeping with
the previously described phenotype in FDXR defects, is
reported in four of five investigated families. Functional tests performed in two available fibroblast cell
lines confirm reduction in the FDXR protein level on
Western blot. A. Alayoubi: None. C. Goudie: None. P. Tibout: None. A. Alayoubi: None. C. Goudie: None. P. Tibout: None. M. Duval: None. B. Maranda: None. D. Mitchell: None. J. Mitchell: None. J. Mitchell: None. 1Department of Medical Genetics, Children’s Memorial Health
Institute, Warsaw, Poland, 2Institute of Human Genetics,
Helmholtz Zentrum München, Munich, Germany, 3Institute of Hematopoietic stem cell transplant does not prevent
neurological deterioration in infants with Farber disease Hematopoietic stem cell transplant does not prevent
neurological deterioration in infants with Farber disease A. Alayoubi1,2, C. Goudie1, P. Tibout3, M. Duval4, B. Maranda5,
D. Mitchell1, J. Mitchell1 A. Alayoubi1,2, C. Goudie1, P. Tibout3, M. Duval4, B. Maranda5,
D. Mitchell1, J. Mitchell1 1McGill University, Montreal, QC, Canada, 2Taibah
University, Madinah, Saudi Arabia, 3Laval University, Quebec
City, QC, Canada, 4University of Montreal, Montreal, QC,
Canada, 5Université de Sherbrooke, Sherbrooke, QC, Canada Introduction: FDRX gene encodes the mitochondrial
membrane-associated ferredoxin reductase, the sole enzyme
essential for the biosynthesis of iron-sulfur (Fe-S) clusters
and heme formation. Fe-S clusters proteins are involved in
enzymatic catalysis and gene expression, mitochondrial
respiration, DNA replication, DNA repair, and iron home-
ostasis. Recently, FDXR defects have been shown to cause a
novel mitochondrial disease with auditory neuropathy and
optic atrophy. Introduction: FDRX gene encodes the mitochondrial
membrane-associated ferredoxin reductase, the sole enzyme
essential for the biosynthesis of iron-sulfur (Fe-S) clusters
and heme formation. Fe-S clusters proteins are involved in
enzymatic catalysis and gene expression, mitochondrial
respiration, DNA replication, DNA repair, and iron home-
ostasis. Recently, FDXR defects have been shown to cause a
novel mitochondrial disease with auditory neuropathy and
optic atrophy. Farber disease (FD) is an inherited autosomal recessive
disorder of lipid metabolism. The hallmark of the disease is
systemic accumulation of ceramide due to lysosomal acid
ceramidase (ACDase) deficiency. The involvement of the
central nervous system is critical in this disorder leading to
rapid deterioration and death within a few years after birth. Efforts to treat patients by hematopoietic stem cell trans-
plant (HSCT) have resulted in favorable results in the
absence of neurological manifestations. We report the out-
comes of HSCT in two patients with FD who received early
HSCT and had neurological deterioration post-transplant. We also present a new understanding of the limitations of
HSCT in FD management based on our observations of the
clinical course of the two patients after therapy. <!--End-
Fragment--> Patients and Results: Here, we describe nine novel
FDXR pathogenic variants revealed by whole exome
sequencing of 8 affected individuals from 6 families. The
clinical presentation in these individuals is varied, and
includes
families
with
more
severe
phenotypes
then
previously described. Early-onset progressive leukoence-
phalopathy
and brain atrophy, Leigh
syndrome,
and
medullary and cerebellar atrophy are reported in three
families. D. Piekutowska-Abramczuk1, S. L. Stenton2,3, M. Gusic2,3,
E. Ciara1, M. Wagner3,2,4, T. Haack3,2,5, K. G. Claeys6,7,
L. Schrod8, C. Nava9, V. Narayanan10, D. Jurkiewicz1, P. Halat-
Wolska1, M. Pelc1, K. Chrzanowska1, R. Płoski11, T. Meitinger3,2,
M. Pronicki12, E. Pronicka1,13, H. Prokisch2,3 1Department of Medical Genetics, Children’s Memorial Health
Institute, Warsaw, Poland, 2Institute of Human Genetics, 1University of California, San Francisco, San Francisco, CA,
United States, 2California State University, Stanislaus,
Turlock, CA, United States 1University of California, San Francisco, San Francisco, CA,
United States, 2California State University, Stanislaus,
Turlock, CA, United States S. Beck-Woedl1, K. Grundmann-Hauser1, R. Buchert-Lo1,
A. Dufke1, M. Sturm1, O. Riess1, M. Hoopmann2, T. Haack1,
M. Stampfer1 Gaucher disease [GD] is an autosomal recessive lysosomal
storage disease caused by mutations in the GBA gene
resulting in deficient glucocerebrosidase activity and accu-
mulation of glucosylceramide [GL1] in reticuloendothelial
cells. The pathophysiologic mechanism of GD is multi-
factorial including cell engorgement, as well as a lipid-
specific immune response to accumulating GL1 and lyso-
GL1. The non-neuronopathic form, type 1, is more common
in the Ashkenazi Jewish [AJ] population with an estimated
prevalence of ~1:850. We describe a 16 year old male with
paternal AJ ancestry who presented at age 3 with spleno-
megaly. Reduced glucocerebrosidase activity and com-
pound heterozygosity (N370S/L444P) in the GBA gene
were consistent with a diagnosis of type 1 GD [GD1]. At
age 12 the patient was noted to have anemia, hematochezia,
weight loss and diarrhea; he was diagnosed with inflam-
matory bowel disease (IBD). His father and paternal aunt
also have IBD. This umbrella term refers to a state of
chronic inflammation of the gastrointestinal tract with an
autoimmune etiology. The prevalence of IBD in the Wes-
tern world is >0.3% with a higher prevalence in the AJ
population. Surprisingly, despite the higher prevalence of
both GD and IBD in the AJ population, to our knowledge
no case of co-occurrence of these two diagnoses has been
reported. This suggests the possibility that the GD1
inflammatory state may have an effect on the expression of
IBD. Relevant registry data will be reviewed and described
and treatment implications will be discussed. 1Medical Genetics and Applied Genomics, Tuebingen,
Germany, 2Department of Gynecology, Tuebingen, Germany 1Medical Genetics and Applied Genomics, Tuebingen,
Germany, 2Department of Gynecology, Tuebingen, Germany Introduction: The sensitivity and specificity of prenatal
ultrasound diagnostics significantly improved over the last
years and fetal anomalies are currently being detected in up
to 3% of pregnancies. Exome sequencing is underway to
become a routine assay in the downstream diagnostic
algorithm. However, the clinical interpretation of identified
variants remains challenging due to limited and mostly non-
standardized prenatal phenotypic features for most disease-
associated genes We report on a fetus with multiple ultrasound abnorm-
alities including increased nuchal translucency, ascites,
hydrops fetalis, and left-sided hydrothorax. P06.14B Novel FDXR pathogenic variants expand the clinical
spectrum related to human ferredoxin reductase defects Conclusions:
Our
study
contribute
to
the
further
delineation of molecular and clinical spectrum related to
FDRX defects. Conclusions:
Our
study
contribute
to
the
further
delineation of molecular and clinical spectrum related to
FDRX defects. D. Piekutowska-Abramczuk1, S. L. Stenton2,3, M. Gusic2,3,
E. Ciara1, M. Wagner3,2,4, T. Haack3,2,5, K. G. Claeys6,7,
L. Schrod8, C. Nava9, V. Narayanan10, D. Jurkiewicz1, P. Halat-
Wolska1, M. Pelc1, K. Chrzanowska1, R. Płoski11, T. Meitinger3,2,
M. Pronicki12, E. Pronicka1,13, H. Prokisch2,3 This study was partially supported by CMHI grants:
S148/16, S145/16, 238/16, mitoNET (German Network for
Mitochondrial Diseases) and GENOMIT (European Net-
work for Mitochondrial Diseases). This study was partially supported by CMHI grants:
S148/16, S145/16, 238/16, mitoNET (German Network for
Mitochondrial Diseases) and GENOMIT (European Net-
work for Mitochondrial Diseases). 1Department of Medical Genetics, Children’s Memorial Health
Institute, Warsaw, Poland, 2Institute of Human Genetics, D. Piekutowska-Abramczuk:
None. S.L. Stenton:
None. M. Gusic: None. E. Ciara: None. M. Wagner:
None. T. Haack: None. K.G. Claeys: None. L. Schrod: Abstracts from the 51st European Society of Human Genetics Conference: Posters 173 None. C. Nava:
None. V. Narayanan:
None. D. Jurkiewicz: None. P. Halat-Wolska: None. M. Pelc:
None. K. Chrzanowska: None. R. Płoski: None. T. Meitinger: None. M. Pronicki: None. E. Pronicka: None. H. Prokisch: None. O. Riess: None. M. Hoopmann: None. T. Haack: None. M. Stampfer: None. P06.19C
Clinical and Genetic Analysis in a Rare Case of Hereditary
FructoseIntolerance P06.20D Clinical Manifestations and Molecular Aspects of
Phosphoribosylpyrophosphate Synthetase Superactivity in
Females E. Ponzi: None. A. Maiorana: None. V. Alesi: None. F. Lepri: None. S. Genovese: None. S. Loddo: None. M. Mucciolo: None. A. Novelli: None. C. Dionisi-Vici: None. Glycogen storage disease type III: identification of the first
case of uniparental disomy and two novel deletions Results: Patient 1 showed the homozygous variant
c.3904insA, patient 2 resulted in apparent homozygosity
for W1401X mutation, patient 3 carried a homozygous
deletion of the first 4 exons of AGL. Since discordant results
from segregation studies showed the carrier status in only
one parent of patients 1 and 2, further investigations
revealed a paternal disomy of chromosome 1 (UPD1) in
patient 1, and a paternal inherited 349 kb deletion of
chromosome 1 including AGL gene in patient 2. Results: Patient 1 showed the homozygous variant
c.3904insA, patient 2 resulted in apparent homozygosity
for W1401X mutation, patient 3 carried a homozygous
deletion of the first 4 exons of AGL. Since discordant results
from segregation studies showed the carrier status in only
one parent of patients 1 and 2, further investigations
revealed a paternal disomy of chromosome 1 (UPD1) in
patient 1, and a paternal inherited 349 kb deletion of
chromosome 1 including AGL gene in patient 2. Conclusions:
The
genetic
characterization
of
three
GSDIII patients allowed to describe the first case of GSDIII
resulting from UPD1 and two new deletions of the AGL
gene. Moreover, UPD can play an important role even in
case of imprinted genes. Particularly, ARHI is a maternally
imprinted tumor suppressor gene, implicated in growth and
oncogenesis. It could be speculated that ARHI overexpres-
sion could have a role in the severe short stature of patient 1
with paternal UPD1. The study emphasizes the importance
of parental segregation studies especially in patients with
recessive conditions to look for specific genetic causes of
disease and to estimate properly the risk of family
recurrence. M. Militaru: None. A. Maris: None. M. Militaru:
None. M. tefănu: None. M. Pop: None. I. Blănaru:
None. D. Militaru: None. E. Dronca: None. 1University of California, San Francisco, San Francisco, CA,
United States, 2California State University, Stanislaus,
Turlock, CA, United States Thoracentesis of
the fetus was performed to relieve the fetal lung and pleural
effusion was used for genetic testing. Cytogenetic analysis
showed a normal female karyotype. In addition, a fetal
blood sample was used to exclude spherocytosis. The fetus
had a highly increased number of reticulocytes. Results: Prenatal exome sequencing identified compound
heterozygous mutations in GLB1, a previously reported
pathogenic missense variant on the paternal allele and a
novel nonsense variant on the maternal allele. Recessive-
type
GLB1
variants
have
been
associated
with
the
lysosomal storage disorder GM1 gangliosidosis which is
characterized by progressive neurodegeneration. M. Sabbadini: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; SanofiGenzyme. D. Speakers Bureau/Honoraria (speakers bureau, symposia,
and expert witness); Modest; SanofiGenzyme, Medscape. F. Consultant/Advisory Board; Modest; Invitae. R. Galla-
gher: B. Research Grant (principal investigator, collabora-
tor or consultant and pending grants as well as grants
already received); Modest; SanofiGenzyme. F. Consultant/
Advisory Board; Modest; Horizon Pharma, Alexion. P. Bawa: None. Conclusion: After the exclusion of aneuploidy, exome-
based trio analysis is a fast and cost-efficient diagnostic tool
in cases with multiple unspecific fetal anomalies. On the
one hand establishing a diagnosis enables an informed
choice about future pregnancies and provides information
on the prognosis and the recurrence risk, on the other hand
unclear results may cause uncertainty. Thus the challenge
remains to offer trio-exome analysis in a responsible
manner in prenatal diagnostic settings. S. Beck-Woedl: None. K. Grundmann-Hauser: None. R. Buchert-Lo: None. A. Dufke: None. M. Sturm: None. S. Beck-Woedl: None. K. Grundmann-Hauser: None. R. Buchert-Lo: None. A. Dufke: None. M. Sturm: None. 174 J. del Picchia M. Militaru1,2, A. Maris3, M. Militaru3, M. tefănu2, M. Pop2,
I. Blănaru2, D. Militaru1, E. Dronca1
1Iuliu Haieganu University of Medicine and Pharmacy, Cluj-
Napoca, Romania, 2Genetic Center, Cluj-Napoca, Romania,
32nd Paediatrics Hospital, Cluj-Napoca, Romania M. Militaru1,2, A. Maris3, M. Militaru3, M. tefănu2, M. Pop2,
I. Blănaru2, D. Militaru1, E. Dronca1 K. Hodaňová Research Unit for Rare Diseases, Department of Pediatrics
and Adolescent Medicine, Praha 2, Czech Republic Research Unit for Rare Diseases, Department of Pediatrics
and Adolescent Medicine, Praha 2, Czech Republic Glycogen storage disease type III: identification of the first
case of uniparental disomy and two novel deletions 1Iuliu Haieganu University of Medicine and Pharmacy, Cluj-
Napoca, Romania, 2Genetic Center, Cluj-Napoca, Romania,
32nd Paediatrics Hospital, Cluj-Napoca, Romania E. Ponzi1, A. Maiorana1, V. Alesi2, F. Lepri2, S. Genovese2,
S. Loddo2, M. Mucciolo2, A. Novelli2, C. Dionisi-Vici1 Hereditary fructose intolerance is a very rare autosomal
recessive, metabolic disorder with unknown global pre-
valence. Carrier frequency is estimated at 1 in 70 indivi-
duals, especially in those of Caucasian origin. The key
enzyme in fructose metabolism is aldolase B produced by
the liver at low constant levels; when dietary fructose is
ingested, the enzyme becomes active and metabolizes the
fructose-1-phosphate and 1,6-biphosphate to 3-phosphate-
glyceraldehyde. Due to mutations in the ALDOB gene
(located on chromosome 9q31.1), the enzyme activity can
be drastically reduced (85-100%). In Europe, more than
80% of mutations are A150P, A175D and N335K. Here, we
present a case of a 15 months year old patient with a history
of liver insufficiency, currently with hepatomegaly, failure
to thrive, fever and diarrhea. In evolution, the patient
became comatose (Glasgow score 8) with severe hypogly-
cemia but without ketonuria. Clinical and paraclinical test-
ing raised the question of hereditary fructose intolerance vs. hydroxyglutaric aciduria. Genetic testing revealed that the
patient
was
a
compound
heterozygote
with
A174D
(c.524C>A) mutation in exon 5 and 4 bp deletion in exon 3
(c.113-1_115) of ALDOB gene. Subsequent genetic testing
of parents showed that they were both heterozygotes: the
mother with A174D (c.524C>A) mutation and the father
with 4bp deletion in exon 3. The particularities of this case
are the presence of liver insufficiency and coma in a child,
without
ketonuria
and
neurological
long-term
con-
sequences, that was eventually diagnosed as a rare case
hereditary fructose intolerance. 1Metabolic Unit, Department of Specialist Pediatrics, Bambino
Gesù Children's Hospital, Rome, Italy, 2Medical genetics
laboratory department, Bambino Gesù Children’s Hospital,
Rome, Italy Introduction: Glycogen storage disease type III (GSDIII) is
caused by mutations of AGL gene with debranching enzyme
deficiency. Introduction: Glycogen storage disease type III (GSDIII) is
caused by mutations of AGL gene with debranching enzyme
deficiency. Materials and Methods: Molecular analysis of three
GSDIII patients was performed through Sanger and Next
Generation Sequencing; CGHarray e SNParray were used
for further genetic investigations. Besides typical clinical
features of GSDIII, patient 1 showed a severe growth
retardation (<3 SD), whereby endocrinological studies
resulted negative. Glycogen storage disease type III: identification of the first
case of uniparental disomy and two novel deletions Glycogen storage disease type III: identification of the first
case of uniparental disomy and two novel deletions P06.19C Objectives:
Phosphoribosylpyrophosphate
synthetase
(PRPS) superactivity is an X-linked disorder characterized
by urate overproduction (OMIM 300661). This condition is Abstracts from the 51st European Society of Human Genetics Conference: Posters 175 hemochromatosis and as a determinant in cases with pri-
mary iron overload. hemochromatosis and as a determinant in cases with pri-
mary iron overload. thought to rarely affect women, and when it does, the
clinical presentation is mild. We describe a 16 year old
African-American female who developed progressive tophi,
nephrolithiasis, and acute kidney failure due to urate over-
production. Family history included a mother with tophac-
eous gout who developed end-stage kidney disease due to
nephrolithiasis
and
an
affected
sister
with
polyarticular gout. Material and methods: 24 patients, belonging to 8
distinct genealogies with type 4 hemochromatosis and
showing both high interfamily and intrafamily variability in
iron overload degree were investigated for PCBP2 variants. At the same time, PCBP2 was studied in 30 unrelated
individuals with hemochromatosis but not carrying muta-
tions in the HFE, TFR2, HAMP, HJV and SLC40A1genes. The entire coding region and the intron-exon boundaries of
PCBP2 were sequenced with Sanger method in these
patients. Methods: Whole exome sequencing was performed in
affected females and their fathers. Results: Mutational analysis revealed a new c.520G>A
(p.G174R) mutation in the PRPS1 gene. The mutation
resulted in decreased PRPS inhibition by ADP. Results: We did not identify any PCBP2 point variant in
the examined sample. Results: We did not identify any PCBP2 point variant in
the examined sample. Conclusions: Clinical findings in previously reported
females with PRPS superactivity showed a high clinical
penetrance of this disorder with a mean serum urate level of
8.5 ± 4.1 mg/dl (506 ± 247 umol/L) and a high prevalence
of gout. These findings indicate that all women in families
with PRPS superactivity should be genetically screened for
a mutation (for clinical management and genetic counsel-
ing). In addition, women with tophaceous gout, gout
presenting in childhood, or a strong family history of
severe gout should be considered for PRPS1 mutational
analysis. Conclusions: Clinical findings in previously reported
females with PRPS superactivity showed a high clinical
penetrance of this disorder with a mean serum urate level of
8.5 ± 4.1 mg/dl (506 ± 247 umol/L) and a high prevalence
of gout. P06.19C These findings indicate that all women in families
with PRPS superactivity should be genetically screened for
a mutation (for clinical management and genetic counsel-
ing). In addition, women with tophaceous gout, gout
presenting in childhood, or a strong family history of
severe gout should be considered for PRPS1 mutational
analysis. Conclusion: Our study was not able to confirm the
hypothesis of PCBP2 as a modifier gene in type 4
hemocromatosis. Also, PCBP2 did not appear to be
causative of our cases with non-molecularly characterized
hemochromatosis. We believe that the analysis should be
expanded to more patients to get conclusive considerations
on PCBP2 as a gene possibly involved in iron overload
disorders. Conclusion: Our study was not able to confirm the
hypothesis of PCBP2 as a modifier gene in type 4
hemocromatosis. Also, PCBP2 did not appear to be
causative of our cases with non-molecularly characterized
hemochromatosis. We believe that the analysis should be
expanded to more patients to get conclusive considerations
on PCBP2 as a gene possibly involved in iron overload
disorders. M. Valiante: None. S. Majore: None. G. Musci: None. M. Lipari: None. M.C. Bonaccorsi di Patti: None. I. Bottillo: None. F. Polticelli: None. C. De Bernardo:
None. A. Baiocchini: None. P. Grammatico: None. K. Hodaňová: None. National Research and Applied Medicine Centre “Mother and
Child”, Minsk, Belarus Introduction: Mucopolysaccharidosis type II (MPS II;
Hunter syndrome; OMIM 309900) is a life –limiting,
multisystemic
disease
with
varying
presentation
and
severity. This X-linked lysosomal storage disorder is caused
by mutations in IDS gene, encoding the lysosomal enzyme
iduronate-2-sulfatase (EC 3.1.6.13). For clinical purposes,
patients are generally considered to be in one of two cate-
gories according to the presence or absence of cognitive
impairment. Here we present the results of IDS gene
mutations analysis in Hunter patients from Belarus. Introduction: Disorders of lipid metabolism are very
common. Hypercholesterolemia plays an important role in
the pathogenesis of atherosclerosis and can be effectively
treated by lifestyle changes and drugs. Predominantly, the
clinical phenotype is caused by mutations in the LDLR or
APOB genes, but also mutations in other genes rarely result
in monogenic hypercholesterolemia. Materials and Methods: Next generation sequencing of
the LDLR, PCSK9 and APOE genes and part of APOB gene
(exon 26) using ADH Master kit (Multiplicom, Belgium);
Sanger sequencing of the ABCG5, ABCG8 and LDLRAP1
genes. DNA samples have been collected within the
framework of the MedPed project. Materials and Methods: From early 1980-th 44 MPS II
patients were diagnosed in Belarus giving an estimated
incidence of 1.2 per 100,000 live births. Patients belong to
several generations of 30 families with female-carriers. The
genomic DNA from blood leukocytes of the patients and
carriers from 17 families was isolated and the entire coding
region and flanking intronic sequences of IDS gene were
amplified by PCR. Amplified PCR products were purified
and sequenced directly by an ABI 3500 Genetic Analyzer. Results: We assessed a spectrum of mutations in more
than 3900 unrelated patients with clinical diagnosis of
hypercholesterolemia. Predominantly,
we
have
found
mutations in the LDLR gene (22.3%) and in the APOB
gene (11.0%). 208 unique allelic variants in the LDLR gene
have been detected. We have found also pathological
variants in the PCSK9, ABCG5 and LDLRAP1 genes. ACMG criteria were used for pathogenicity evaluation of
detected sequence variants. Results: 10 different mutations were identified: exon 3 -
c.236G>A (p.R88H), exon 5 - c.511T>G (p.C171G), exon
6 - c.788delC (p.Y264Tfs*17), exon 7 - c.1004A>G (p. H335R), exon 8 - c.1007-2delA, c.1035G>T (p.W345C),
c.1085_1086delTA (p.Y362Cfx*249), exon 9 - c.1402C>T
(p.R468W),
c.1403G>A
(p.R468Q),
c.1425G>A
(p. W475X). Only the mutations of exon 9 were revealed in
more than one family, all others were unique. In vivo effect of pyridoxine administration on CBS mutants del Picchia Conclusions: The mutation of exon 5 - c.511T>G is
novel, never reported before in patients with Hunter
syndrome and predicted to be pathogenic. Results: Plasma CBS activity ranged 0-0.7% of controls
in non-responders even on combined therapy with methio-
nine restriction, pyridoxine and/or betaine. In contrast,
plasma CBS activity in responders not taking pyridoxine
was 5.1% (range 0-20.3%) and significantly increased on
pyridoxine to 26.2% (range 7.3-72.1%) indicating in vivo
rescue of the activity of mutant CBS in liver. N. Gusina: None. S. Miasnikov: None. E. Budzejka:
None. A. Gusina: None. The spectrum of pathological sequence variants in patients
with lipid metabolism disorder in the Czech Republic Conclusion: Pyridoxine administration partially rescued
in vivo enzyme activity of selected CBS-mutant. On the
other hand large doses of pyridoxine did not increase the
activity of CBS in non-responsive patients. This study
supports
the
recent
recommendation,
that
pyridoxine
therapy is not necessary in non-responsive patients with
homocystinuria. Supported from institutional projects RVO-
VFN64165 and ProgresQ26. L. Tichý1, V. Soška2, M. Vrablík3, T. Honzík4, Z. Urbanová4,
H. Vaverková5, T. Freiberger6 1Centre of molecular biology and gene therapy, University
Hospital Brno, Brno, Czech Republic, 2Department of
Biochemistry, Masaryk University, Brno, Czech Republic,
3Third Medical Department, First Faculty of Medicine,
Charles University and General Faculty Hospital, Prague,
Czech Republic, 4Department of Paediatrics and Adolescent
Medicine, First Faculty of Medicine, Charles University and
General University Hospital, Prague, Czech Republic,
5Department of Internal Medicine III – Nephrology,
Rheumatology and Endocrinology, University Hospital
Olomouc and Faculty of Medicine and Dentistry, Palacky
University, Olomouc, Czech Republic, 6Molecular Genetics
Laboratory, Centre for Cardiovascular Surgery and
Transplantation, Brno, Czech Republic P. Jesina: None. V. Kozich: None. J. Sokolova: None. J. Krijt: None. T. Honzik: None. J. Zeman: None. In vivo effect of pyridoxine administration on CBS mutants P. Jesina, V. Kozich, J. Sokolova, J. Krijt, T. Honzik, J. Zeman
Dept. of Pediatrics, Prague, Czech Republic M. Valiante1, S. Majore1, G. Musci2, M. Lipari1, M. C. Bonaccorsi di Patti3, I. Bottillo1, F. Polticelli4, C. De Bernardo1,
A. Baiocchini5, P. Grammatico1 P. Jesina, V. Kozich, J. Sokolova, J. Krijt, T. Honzik, J. Zeman Dept. of Pediatrics, Prague, Czech Republic Introduction: Pathophysiology of pyridoxine responsive-
ness in patients with homocystinuria is elusive with a pos-
sible
chaperoning
activity
of
pyridoxal
5´-phosphate. However, direct demonstration of in vivo effect on mutant
enzyme activity is lacking. We described previously that
cystathionine-beta-synthase (CBS) is released into circula-
tion from several organs, mostly from liver. Here we used
this method to assess in vivo effect of pyridoxine adminis-
tration on the rescue of CBS-mutant in 15 patients with
homocystinuria. 1Medical Genetics, Sapienza University of Rome, S. Camillo-
Forlanini Hospital, Rome, Italy, 2Department of Biosciences
and Territory, University of Molise, Pesche, Italy, 3Department
of Biochemical Sciences, Sapienza University of Rome, Rome,
Italy, 4Department of Sciences, University Roma Tre, Rome,
Italy, 5Laboratory of Pathology, INMI "L.Spallanzani", Rome,
Italy 1Medical Genetics, Sapienza University of Rome, S. Camillo-
Forlanini Hospital, Rome, Italy, 2Department of Biosciences
and Territory, University of Molise, Pesche, Italy, 3Department
of Biochemical Sciences, Sapienza University of Rome, Rome,
Italy, 4Department of Sciences, University Roma Tre, Rome,
Italy, 5Laboratory of Pathology, INMI "L.Spallanzani", Rome,
Italy Introduction: Over the last decade, many mechanisms
orchestrating iron metabolism have been highlighted. A
number of molecules involved in iron homeostasis have
been progressively identified, shedding light into hereditary
conditions leading to iron excess or deficiency. Among
them, the recently recognized iron chaperone poly(rC)-
binding protein 2 (PCBP2) seems to have a crucial role in
transferring intracellular iron to the iron exporter ferro-
portin1. The aim of this study was to investigate PCBP2 as
a candidate gene in disorders of iron metabolism. We ana-
lysed
PCBP2
as
a
possible
modifier
in
type
4 Methods: We analyzed plasma CBS activity in 8
pyridoxine non-responders (homozygotes/compound het-
erozygotes
for
mutations
p.A69fs*94;
p.C165Y;
p. A71Pfs*24; p.V10Wfs*71; p.K211*; p.G246Dfs*52; p. W409_G453del; p.A155T; p.E144K) and 7 partial/full
responders (homozygotes/compound heterozygotes carry-
ing mutations p.P145L; p.I278T; p.P49L; p.R336H; p. R336C on at least one allele). CBS activity was measured
by LC-MS/MS. 176 J. N. Gusina, S. Miasnikov, E. Budzejka, A. Gusina N. Gusina, S. Miasnikov, E. Budzejka, A. Gusina National Research and Applied Medicine Centre “Mother and
Child”, Minsk, Belarus The genetic origin of Hunter syndrome in Belarus The genetic origin of Hunter syndrome in Belarus Two novel mutations in ALPL gene associated with mild
phenotype of hypophosphatasia in Russia cohort study Two novel mutations in ALPL gene associated with mild
phenotype of hypophosphatasia in Russia cohort study M. A. Fedyakov1,2, Y. A. Eismont1, T. E. Ivaschenko3, I. B. Sosnina4, E. V. Snegova4, N. Y. Shved1,3, A. M. Sarana1,2, S. G. Scherbak1,2, N. Y. Kalinchenko5, T. M. Pervunina6, E. Y. Gurkina6, O. S. Glotov1,2,3 National Research and Applied Medicine Centre “Mother and
Child”, Minsk, Belarus Conclusions: Our study presents a large spectrum of
causative mutations, including mutations in rarely involved
genes, in Czech patients with lipid metabolism disorders. 177 Abstracts from the 51st European Society of Human Genetics Conference: Posters We would like to thank the physicians from the national
and regional centres of the Czech MedPed project. This
work was funded by the grants numbers AZV 16-29084A
and AZV 15-28277A. Conclusions: We presented the first data of ALPL gene
mutation spectrum in North-Western region of Russia. Most
of identified mutations were heterozygous that confirm
dominant-negative effect in late forms of HPP. Two novel
missense mutations were detected and associated with mild
phenotype of disease. This study was supported by Russian
Science Foundation №14-50-00069. L. Tichý: None. V. Soška: None. M. Vrablík: None. T. Honzík: None. Z. Urbanová: None. H. Vaverková: None. T. Freiberger: None. M.A. Fedyakov: None. Y.A. Eismont: None. T.E. Ivaschenko: None. I.B. Sosnina: None. E.V. Snegova:
None. N.Y. Shved: None. A.M. Sarana: None. S.G. Scherbak: None. N.Y. Kalinchenko: None. T.M. Pervu-
nina: None. E.Y. Gurkina: None. O.S. Glotov: None. P06.26B Mutation of IARS2 causes cataracts, growth hormone
deficiency, sensorimotor polyneuropathy, sensorineural
hearing loss, short stature, and type II esophageal
achalasia: expanding the clinical phenotype 1City Hospital 40, Saint-Petersburg, Russian Federation,
2Saint-Petersburg State University, Saint-Petersburg, Russian
Federation, 3D.O.Ott Research Institute of Obstetrics,
Gynecology and Reproductology, Saint-Petersburg, Russian
Federation, 4Consultative and diagnostic center for children,
Saint-Petersburg, Russian Federation, 5Endocrinology
Research Centre, Moscow, Russian Federation, 6Almazov
National Medical Research Centre, Saint-Petersburg, Russian
Federation E. Ghayoor Karimiani1,2, B. Vona3, T. Haaf4, R. Maroofian2,
S. Shahrokhzadeh1, N. Ahangari1, B. Vona4, A. Alahmad5,
L. He6, K. Thompson7, R. W. Taylor7, J. Movaffagh8, N. Amiri8,
M. Doosti1, R. Boostani9 E. Ghayoor Karimiani1,2, B. Vona3, T. Haaf4, R. Maroofian2,
S. Shahrokhzadeh1, N. Ahangari1, B. Vona4, A. Alahmad5,
L. He6, K. Thompson7, R. W. Taylor7, J. Movaffagh8, N. Amiri8,
M. Doosti1, R. Boostani9 E. Ghayoor Karimiani1,2, B. Vona3, T. Haaf4, R. Maroofian2,
S. Shahrokhzadeh1, N. Ahangari1, B. Vona4, A. Alahmad5,
L. He6, K. Thompson7, R. W. Taylor7, J. Movaffagh8, N. Amiri8,
M. Doosti1, R. Boostani9 1Next Generation Genetic Clinic, Mashhad, Iran, Islamic
Republic of, 2Genetics and Molecular Cell Sciences Research
Centre, St George’s University of London,, London, United
Kingdom, 3Institute of Human Genetics, Julius Maximilians
University, Würzburg,, Germany, 4Institute of Human
Genetics, Julius Maximilians University, Würzburg, Germany,
52Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience,, Newcastle University, Newcastle upon Tyne,
United Kingdom, 6Wellcome Centre for Mitochondrial
Research, Institute of Neuroscience, Newcastle University,
ewcastle upon Tyne, United Kingdom, 7Wellcome Centre for
Mitochondrial Research, Institute of Neuroscience, Newcastle
University, Newcastle University, Newcastle upon Tyne, United
Kingdom, 8Targeted Drug Delivery Research Center,
Pharmaceutical Technology Institute, Mashhad University of
Medical Sciences, Mashhad, Iran, Islamic Republic of,
9Department of Neurology, Faculty of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran, Islamic
Republic of Introduction: Hypophosphatasia (HPP) is a rare heritable
metabolic disorder characterized by defective mineraliza-
tion of bone and/or teeth in the presence of reduced activity
of unfractionated serum alkaline phosphatase (ALP). Late
forms of HPP (childhood, adult, odontohypophosphatasia)
are predominantly caused by heterozygous and compound-
heterozygous missense-mutations in ALPL gene and often
remain undiagnosed. Genetic analysis provides determining
of diagnosis in cases with suspected HPP. Furthermore it
expands our knowledge of mutation spectrum in mild and
latent forms of the disease. Materials and Methods: We analyzed genomic DNA
samples from 55 unrelated individuals with signs of HPP. Minimal criteria’s to include in study were recurrent low
levels of ALP and low growth. P06.25A Two novel mutations in ALPL gene associated with mild
phenotype of hypophosphatasia in Russia cohort study P06.26B A compara-
tive in silico protein structural analysis provides a possible
explanation for the comparatively mild phenotype com-
pared to the other reported patients with IARS2 pathogenic
variants. Our findings provide further support that biallelic
mutations in IARS2 result in an extremely rare but dis-
tinctive and clinically recognisable phenotype in human. Iranian proband with distinctly overlapping features to
CAGSSS was subjected to whole exome sequencing and
bioinformatics analysis. This revealed a novel homozygous
missense variant in exon 21 (c.2625C>T, p.Pro909Ser) that
was included in a 14.3 Mb run of homozygosity affecting
the same proline residue described in the original French-
Canadian kindred. This study reveals an expansion of the
CAGSSS phenotypic spectrum to include type II esopha-
geal achalasia and proposes the bone phenotypes of this
syndrome may also appear as a mild manifestation. Fur-
thermore,
patient-derived
fibroblasts
showed
normal
respiratory chain enzyme activity, as well as unchanged
OXPHOS protein subunits and IARS2 levels. A compara-
tive in silico protein structural analysis provides a possible
explanation for the comparatively mild phenotype com-
pared to the other reported patients with IARS2 pathogenic
variants. Our findings provide further support that biallelic
mutations in IARS2 result in an extremely rare but dis-
tinctive and clinically recognisable phenotype in human. MDs are severe, progressive and often because of their
multi-systemic character lead to early death. Lack of time is
the biggest problem for physicians and parents, that’s why
Rapid WES is a unique diagnostic tool and should be
performed in cases where a MD is suspected in critical ill
children hospitalized in the intensive care units with rapid
progression of severe, life-threatening symptoms, to definite
diagnosis on the genetic level. Onset of
symptoms (age)
1 day
3 month
5 month
1 day
Death (age)
1,5 month
10 month
–
6 week
Suspected
disorder
Mitochondrial
disorder, Van der
Knapp or Canavan
disease
Leigh-like
syndrome
Leigh-
like
syndrome
Mitochondrial
disorder
Performed
genetic tests
before WES
–
Panel of genes (selected
exons) responsible for
Leigh and Leigh-like
syndromes
–
Time for WES
results
14 days
7 days 7 days
5 days
Gene
PC
POLG1
SCO2
TRMT10C
Diagnosis
Pyruvate
carboxylase
deficiency
Alpers
syndrome
Leigh
syndrome
Mitochondrial
disorder E. Ghayoor Karimiani: None. B. Vona: None. T. Haaf:
None. R. Maroofian: None. S. Shahrokhzadeh: None. N. Ahangari: None. B. Vona: None. A. Alahmad: None. L. He: None. K. Thompson: None. R. W. Taylor: None. J. Movaffagh: None. N. P06.27C P06.27C
Rapid whole-exome sequencing (WES) in the critically ill
infants hospitalized in the intensive care units M. Biela1, K. Szmyd2, M. Bloch1, E. Szmida3, A. Walczak4,
G. Kostrzewa4, J. Kosinska4, M. Rydzanicz4, P. Stawinski4,
A. Biernacka4, M. M. Sasiadek3, R. Ploski4, R. Smigiel1 M. Biela1, K. Szmyd2, M. Bloch1, E. Szmida3, A. Walczak4,
G. Kostrzewa4, J. Kosinska4, M. Rydzanicz4, P. Stawinski4,
A. Biernacka4, M. M. Sasiadek3, R. Ploski4, R. Smigiel1 1Department of Paediatrics and Rare Disorders, Wroclaw
Medical University, Wrocław, Poland, 2Lower Silesia
Children's Hospice, Wrocław, Poland, 3Department of
Genetics, Wroclaw Medical University, Wrocław, Poland,
4Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland 1Department of Paediatrics and Rare Disorders, Wroclaw
Medical University, Wrocław, Poland, 2Lower Silesia
Children's Hospice, Wrocław, Poland, 3Department of
Genetics, Wroclaw Medical University, Wrocław, Poland,
4Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland 1Department of Paediatrics and Rare Disorders, Wroclaw
Medical University, Wrocław, Poland, 2Lower Silesia
Children's Hospice, Wrocław, Poland, 3Department of
Genetics, Wroclaw Medical University, Wrocław, Poland,
4Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland M. Biela: None. K. Szmyd: None. M. Bloch: None. E. Szmida: None. A. Walczak: None. G. Kostrzewa: None. J. Kosinska: None. M. Rydzanicz: None. P. Stawinski:
None. A. Biernacka: None. M.M. Sasiadek: None. R. Ploski: None. R. Smigiel: None. M. Biela: None. K. Szmyd: None. M. Bloch: None. E. Szmida: None. A. Walczak: None. G. Kostrzewa: None. J. Kosinska: None. M. Rydzanicz: None. P. Stawinski:
None. A. Biernacka: None. M.M. Sasiadek: None. R. Ploski: None. R. Smigiel: None. Among congenital metabolic diseases mitochodrial dis-
orders (MD) are the most common. This highly hetero-
geneous (genetically and clinically) group of disorders is
caused by mutations in the mitchondrial or the nuclear
genome. Every organ or tissue could be affected, althought
the symptoms from the CNS and skeletal muscles are the
most common. P06.26B Primers’ system for Sanger
sequencing was designed and validated for 2-12 exons of
ALPL gene. First exon of this gene was excluded because
it’s non-coding and GC-rich region. Materials and Methods: We analyzed genomic DNA
samples from 55 unrelated individuals with signs of HPP. Minimal criteria’s to include in study were recurrent low
levels of ALP and low growth. Primers’ system for Sanger
sequencing was designed and validated for 2-12 exons of
ALPL gene. First exon of this gene was excluded because
it’s non-coding and GC-rich region. The gene IARS2 encodes a mitochondrial isoleucyl-tRNA
synthetase, a highly conserved nuclear encoded enzyme
required for the synthesis of charged tRNA for translation. Recently, an extended French-Canadian family and a
Danish proband have associated biallelic pathogenic var-
iants in IARS2 with a rare autosomal recessive syndrome
abbreviated CAGSSS characterized by cataracts, growth
hormone
deficiency,
sensorineuropathy,
sensorineural
hearing loss, and skeletal dysplasia. Genomic DNA from an Results:
Among
cohort
of
patient
we
detected
9
pathogenic mutations (16,4% detection rate): seven in
heterozygous and two in compound-heterozygous. Most of
identified mutations were p.E191K (6 times). Furthermore 2
novel missense mutation were detected - p.N323I in exon 9
(heterozygous) and p. Y101C in exon 5 (compound
heterozygous with p.E191K in patient with childhood form
of HPP). 178 J. del Picchia WES was performed using SureSelect V5 kit and
HiSeq1500 sequencing. Results were avaible after 5-14
days and a genetically confirmed diagnose was made for
every patient (3 in life,1 postmortom). The following
disorders were diagnosed: Leigh syndrome, Alpers syn-
drome, pyruvate carboxylase deficiency and in one patinet
with suspicion of MD two heterozygous mutations in
TRMT10C were identified. The results allowed to estabilish
prognosis, therapical decissions (in 3 of them hospis care
was taken) and familial councelling. Iranian proband with distinctly overlapping features to
CAGSSS was subjected to whole exome sequencing and
bioinformatics analysis. This revealed a novel homozygous
missense variant in exon 21 (c.2625C>T, p.Pro909Ser) that
was included in a 14.3 Mb run of homozygosity affecting
the same proline residue described in the original French-
Canadian kindred. This study reveals an expansion of the
CAGSSS phenotypic spectrum to include type II esopha-
geal achalasia and proposes the bone phenotypes of this
syndrome may also appear as a mild manifestation. Fur-
thermore,
patient-derived
fibroblasts
showed
normal
respiratory chain enzyme activity, as well as unchanged
OXPHOS protein subunits and IARS2 levels. P06.26B Amiri: None. M. Doosti: None. R. Boostani: None. 1Regeneron Genetics Center, Regeneron Pharmaceuticals Inc.,
Tarrytown, NY, United States, 2Regeneron Pharmaceuticals
Inc., Tarrytown, NY, United States JUMC, Krakow, Poland JUMC, Krakow, Poland Introduction: PPARG encodes peroxisome proliferator-
activated receptor gamma, a nuclear hormone receptor
which plays a crucial role in both glucose and lipid
metabolism. Materials and Methods: Genetic testing in a 44-year old
female patient with familial partial lipodystrophy was
performed by targeted NGS sequencing using a panel of
28 monogenic diabetes genes. Confirmation of the p. [(Asp116Gly)]; c.[347A>G] PPARG gene variant as well as
verification
of
potential
carriers
in
the
family
were
subsequently
performed
using
Sanger
sequencing
in
3130xl Genetic Analyzer. Clinical characteristics was
subsequently assessed for the mutation carriers. Conclusions: Our data show that partial lipodystrophy is
an underdiagnosed condition and that its prevalence might
be
higher
than
previously
reported.Carriers
of
lipodystrophy-associated variants show metabolic abnorm-
alities in the spectrum of lipodystrophy disease, however
they are often diagnosed with type 2 diabetes and
unspecified metabolic syndrome. Results: Two programs were utilized in order to assess
the impact of the detected missense variant on protein
function: SIFT (Sorting Intolerant From Tolerant)[http://sift. bii.a-star.edu.sg/www/SIFT_intersect_coding_submit.html] Results: Two programs were utilized in order to assess
the impact of the detected missense variant on protein
function: SIFT (Sorting Intolerant From Tolerant)[http://sift. bii.a-star.edu.sg/www/SIFT_intersect_coding_submit.html]
and PolyPhen-2 (Polymorphism Phenotyping v2) [http://
genetics.bwh.harvard.edu/pph2/]. These analyses supported
the deleterious character of the mutation by revealing a
PROVEAN score of -4,982. The amino acid position of
116Asp was also demonstrated to have a high degree of
conservation between species. We confirmed this variant in
7 (out of 20) family members. They were diagnosed with
diabetes, dyslipidemia, ischemic heart disease and suffered
from myocardial infarctions before the age of forty. Additional family members have been recruited to further
explore the impact of the mutation on the phenotype. C. Gonzaga-Jauregui: A. Employment (full or part-
time); Significant; Regeneron Pharmaceuticals. E. Owner-
ship
Interest
(stock,
stock
options,
patent
or
other
intellectual property); Significant; Regeneron Pharmaceu-
ticals. J. Altarejos: A. Employment (full or part-time);
Significant; Regeneron Pharmaceuticals. E. Ownership
Interest (stock, stock options, patent or other intellectual
property);
Significant;
Regeneron
Pharmaceuticals. J. Staples: A. Employment (full or part-time); Significant;
Regeneron Pharmaceuticals. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Regeneron Pharmaceuticals. T.M. Teslovich:
A. Employment (full or part-time); Significant; Regeneron
Pharmaceuticals. E. Ownership
Interest
(stock,
stock
options, patent or other intellectual property); Significant;
Regeneron Pharmaceuticals. A. Baras: A. Employment
(full or part-time); Significant; Regeneron Pharmaceuticals. Molecular assessment of variants in inherited
lipodystrophy genes: prevalence and clinical impact in a
large clinical care cohort C. Gonzaga-Jauregui1, J. Altarejos2, J. Staples1, T. M. Teslovich1, Geisinger-Regeneron DiscovEHR Collaboration,
A. Baras1, J. Gromada2, O. Gottesman1, F. Dewey1 We present four unrelated infants with MD manifested by
multi-systemic failures and where ealier metabolic and
genetic examinations haven’t confirmed the diagnosis, for
which Rapid WES was perfomed. 179 Abstracts from the 51st European Society of Human Genetics Conference: Posters E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Regeneron Pharmaceu-
ticals. J. Gromada: A. Employment (full or part-time);
Significant; Regeneron Pharmaceuticals. E. Ownership
Interest (stock, stock options, patent or other intellectual
property);
Significant;
Regeneron
Pharmaceuticals. O. Gottesman: A. Employment (full or part-time); Significant;
Regeneron Pharmaceuticals. F. Dewey: A. Employment
(full or part-time); Significant; Regeneron Pharmaceuticals. E. Ownership Interest (stock, stock options, patent or other
intellectual
property);
Significant;
Regeneron
Pharmaceuticals. Introduction:
Inherited
Lipodystrophies
are
disorders
characterized by loss of adipose tissue accompanied by
metabolic dysregulation. They are divided into Congenital
Generalized Lipodystrophies (CGLs) and Familial Partial
Lipodystrophies (FPLs). Lipodystrophies are considered
rare genetic disorders with reported prevalences ranging
from 1 in 10 million for CGLs, to 1 in 1 million for FPLs. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Regeneron Pharmaceu-
ticals. J. Gromada: A. Employment (full or part-time);
Significant; Regeneron Pharmaceuticals. E. Ownership
Interest (stock, stock options, patent or other intellectual
property);
Significant;
Regeneron
Pharmaceuticals. O. Gottesman: A. Employment (full or part-time); Significant;
Regeneron Pharmaceuticals. F. Dewey: A. Employment
(full or part-time); Significant; Regeneron Pharmaceuticals. E. Ownership Interest (stock, stock options, patent or other
intellectual
property);
Significant;
Regeneron
Pharmaceuticals. Introduction:
Inherited
Lipodystrophies
are
disorders
characterized by loss of adipose tissue accompanied by
metabolic dysregulation. They are divided into Congenital
Generalized Lipodystrophies (CGLs) and Familial Partial
Lipodystrophies (FPLs). Lipodystrophies are considered
rare genetic disorders with reported prevalences ranging
from 1 in 10 million for CGLs, to 1 in 1 million for FPLs. Methods: We interrogated the electronic health record
(EHR) information for ~1.6 million individuals in the
Geisinger Health System for lipodystrophy/lipoatrophy
diagnosis
codes. We
performed
genetic
analyses
of
individuals
with
available
genetic
data
from
our
Geisinger-Regeneron DiscovEHR collaboration to identify
likely causative variants of lipodystrophy in these patients. Clinical characterization of a novel (Asp116Gly) mutation
in PPARgamma gene identified in a large Polish family Results: Of 18 lipodystrophy-diagnosed patients with
available genetic data, we identified potentially pathogenic
variants in known genes in 8 individuals. Of these, 4
individuals carry the pathogenic variant in LMNA (p. R482Q) associated with Dunnigan FPL. There were
additional 12 individuals harboring a molecular finding
for
the
condition
but
no
documented
diagnosis
of
lipodystrophy.EHR review of these individuals showed
that they have metabolic abnormalities consistent with
lipodystrophy including diabetes, dyslipidemia, and pan-
creatitis. We surveyed the DiscovEHR cohort for patho-
genic and likely pathogenic variants in lipodystrophy genes
and evaluated the clinical and metabolic profiles of variant
carriers. We observed a burden of metabolic dysregulation
in these patients. Clinical characterization of a novel (Asp116Gly) mutation
in PPARgamma gene identified in a large Polish family Results: Of 18 lipodystrophy-diagnosed patients with
available genetic data, we identified potentially pathogenic
variants in known genes in 8 individuals. Of these, 4
individuals carry the pathogenic variant in LMNA (p. R482Q) associated with Dunnigan FPL. There were
additional 12 individuals harboring a molecular finding
for
the
condition
but
no
documented
diagnosis
of
lipodystrophy.EHR review of these individuals showed
that they have metabolic abnormalities consistent with
lipodystrophy including diabetes, dyslipidemia, and pan-
creatitis. We surveyed the DiscovEHR cohort for patho-
genic and likely pathogenic variants in lipodystrophy genes
and evaluated the clinical and metabolic profiles of variant
carriers. We observed a burden of metabolic dysregulation
in these patients. M. Szopa, B. Zapala, A. Jamsheer, A. Ludwig-Galezowska,
M. Malecki M. Szopa, B. Zapala, A. Jamsheer, A. Ludwig-Galezowska,
M. Malecki P06.29A Clinical characterization of a novel (Asp116Gly) mutation
in PPARgamma gene identified in a large Polish family M. Szopa, B. Zapala, A. Jamsheer, A. Ludwig-Galezowska,
M. Malecki P06.31C
Development of an antisense-mediated exon skipping
therapeutic strategy for Mucolipidosis II Newcastle University, Newcastle upon Tyne, United Kingdom L. Matos, R. Vilela, F. Coutinho, P. Gaspar, S. Alves Introduction: m.3243A>G, the most common pathogenic
mitochondrial DNA (mtDNA) mutation, is associated with
a range of clinical features, which progress at variable rates,
making prognosis difficult to predict. We aimed to describe
and understand the cause of this heterogeneity. National Health Institute Doutor Ricardo Jorge, Porto,
Portugal Lysosomal storage diseases (LSDs) are a class of inherited
metabolic diseases caused by mutations in proteins critical
for lysosomal function. Among them is ML II, which is
caused by the deficiency of the GlcNAc-phosphotransfer-
ase, a key enzyme for the trafficking of lysosomal hydro-
lases
to
the
lysosome. GlcNAc-phosphotransferase
is
encoded by two genes: GNPTAB and GNPTG. One of the
most frequent mutations is a deletion on exon 19 of the
GNPTAB gene that disrupts the reading frame impairing the
production of an active enzyme and the targeting of lyso-
somal enzymes. Despite broad understanding of the mole-
cular causes behind this and other LSDs, the same progress
has not been observed in the development of therapies, with
current treatments still mostly symptomatic. Therefore,
alternative options should be investigated. One possibility is
the modulation of splicing by antisense oligonucleotides
(AOs) with the purpose of altering the mature mRNA and
the final protein. This work intends to develop a RNA-based
therapeutic agent through the use of AOs capable of indu-
cing the skipping of exon 19 of the GNPTAB gene and
circumvent the effects of this ML II mutation. Different
2’O-Methyl AOs were designed and tested. We have
already succeeded in inducing the skipping of exon 19 in
control and ML II patient fibroblasts. At biochemical level,
48 hours following transfection, enzyme activity suffered a
small increase in patients fibroblasts for all enzymes tested,
even if the results are still much lower than the observed for
controls. Methods: We examined the phenotypic profile of 238
m.3243A>G carriers from the UK MRC Mitochondrial
Disease Patient Cohort using the Newcastle Mitochondrial
Disease Adult Scale and evaluated which commonly
assayed tissue (blood, urine, skeletal muscle) represents
the m.3243A>G mutation load and mtDNA copy number
most strongly associated with disease burden. We modelled
the role of risk factors and additive nuclear genetic factors
in the development of specific phenotypes within 46
pedigrees from the cohort. P06.31C
Development of an antisense-mediated exon skipping
therapeutic strategy for Mucolipidosis II Results: Age and m.3243A>G heteroplasmy level are
associated with disease burden in all three tissues (R2
range = 0.18-0.27, P<0.001); a greater proportion of the
variation in disease burden is explained if mtDNA copy in
skeletal muscle is included (R2=0.40, P<0.001). Common
phenotypic features include hearing impairment, psychiatric
involvement and ataxia; age and heteroplasmy levels are
poor predictors of phenotypic severity. We found high to
moderate heritability estimates for psychiatric involvement,
cognition, ataxia, migraine and hearing impairment (h2
range = 0.40-0.76, P<0.05). Conclusion:
Our
results
indicate
that
m.3243A>G
heteroplasmy, skeletal muscle mtDNA copy number and
age explain some of the variation in m.3243A>G-related
disease burden suggesting that nuclear genetic factors
influence clinical outcomes, paving the way for future
work identifying these. Conclusion:
Our
results
indicate
that
m.3243A>G
heteroplasmy, skeletal muscle mtDNA copy number and
age explain some of the variation in m.3243A>G-related
disease burden suggesting that nuclear genetic factors
influence clinical outcomes, paving the way for future
work identifying these. This work is supported by a Wellcome Trust Fellowship
(204709/Z/16/Z) to SJP and the Wellcome Centre for
Mitochondrial Research (203105/Z/16/Z). Conclusion:
Our
results
indicate
that
m.3243A>G
heteroplasmy, skeletal muscle mtDNA copy number and
age explain some of the variation in m.3243A>G-related
disease burden suggesting that nuclear genetic factors
influence clinical outcomes, paving the way for future
work identifying these. Financing:
FCT/
PTDC
/BBB-BMD/6301/2014
and
Asociación Nour de Mucolipidosis This work is supported by a Wellcome Trust Fellowship
(204709/Z/16/Z) to SJP and the Wellcome Centre for
Mitochondrial Research (203105/Z/16/Z). L. Matos: None. R. Vilela: None. F. Coutinho: None. P. Gaspar: None. S. Alves: None. S.J. Pickett: None. J.P. Grady: None. Y. Shiau Ng:
None. C.L. Alston: None. E. Blakely: None. S.A. Hardy:
None. C.L. Feeney: None. A.A. Bright: None. A.M. Schaefer: None. R.J.Q. McNally: None. I.J. Wilson:
None. H.J. Cordell: None. G.S. Gorman: None. R.W. Taylor:
None. D.M. Turnbull:
None. R. McFarland: None. S. J. Pickett1, J. P. Grady1, Y. Shiau Ng1, C. L. Alston1,
E. Blakely1, S. A. Hardy1, C. L. Feeney1, A. A. Bright1, A. M.
Schaefer1, R. J. Q. McNally2, I. J. Wilson3, H. J. Cordell3, G. S.
Gorman1, R. W. Taylor1, D. M. Turnbull1, R. McFarland1 identification of families with rare forms of lipodystrophy,
with the potential for consequently tailoring early treatment
and prevention of IHD to prolong life expectancy. M. Szopa: None. B. Zapala: None. A. Jamsheer: None. A. Ludwig-Galezowska: None. M. Malecki: None. 1Wellcome Centre for Mitochondrial Research, Newcastle
University, Newcastle upon Tyne, United Kingdom, 2Institute
of Health and Society, Newcastle University, Newcastle upon
Tyne, United Kingdom, 3Institute of Genetic Medicine,
Newcastle University, Newcastle upon Tyne, United Kingdom JUMC, Krakow, Poland Conclusion: We assessed a family with autosomal
dominant lipodystrophy related to a new PPARG mutation. The potential utility of NGS was confirmed for the J. del Picchia 180 S. J. Pickett1, J. P. Grady1, Y. Shiau Ng1, C. L. Alston1,
E. Blakely1, S. A. Hardy1, C. L. Feeney1, A. A. Bright1, A. M. Schaefer1, R. J. Q. McNally2, I. J. Wilson3, H. J. Cordell3, G. S. Gorman1, R. W. Taylor1, D. M. Turnbull1, R. McFarland1 Heterogeneity in m.3243A>G-related mitochondrial
disease: The role of mtDNA heteroplasmy, copy number,
age and nuclear factors P06.35C Biallelic mutations in MRPS34 lead to instability of the
small mitoribosomal subunit and Leigh syndrome P. Dawod1,2, B. Rovcanin2, M. Brankovic2,3, A. Marjanovic2,3,
M. Jankovic3,2, I. Novakovic2,3, I. Dujmovic3,2, J. Jancic4,2,
V. Kostic3,2 P. Dawod1,2, B. Rovcanin2, M. Brankovic2,3, A. Marjanovic2,3,
M. Jankovic3,2, I. Novakovic2,3, I. Dujmovic3,2, J. Jancic4,2,
V. Kostic3,2 P. Dawod1,2, B. Rovcanin2, M. Brankovic2,3, A. Marjanovic2,3,
M. Jankovic3,2, I. Novakovic2,3, I. Dujmovic3,2, J. Jancic4,2,
V. Kostic3,2 N. J. Lake1,2, B. D. Webb3,4, D. A. Stroud5, T. R. Richman6,
B. Ruzzenente7, A. G. Compton8,2, H. S. Mountford1,2,9,
J. Pulman7, C. Zangarelli7, M. Rio10, N. Bodaert11,
Z. Assouline10, M. D. Sherpa3,12, E. E. Schadt3,12, S. M. Houten3,12, J. Byrnes13, E. M. McCormick13, Z. Zolkipli-
Cunningham13,14, K. Haude15, Z. Zhang15, K. Retterer15,
R. Bai15, S. E. Calvo16,17,18, V. K. Mootha19,17,18,
J. Christodoulou1,2, A. Rotig20, A. Filipovska6,21, I. Cristian22,23,
M. J. Falk13,24, M. D. Metodiev25, D. R. Thorburn1,2,26 N. J. Lake1,2, B. D. Webb3,4, D. A. Stroud5, T. R. Richman6,
B. Ruzzenente7, A. G. Compton8,2, H. S. Mountford1,2,9,
J. Pulman7, C. Zangarelli7, M. Rio10, N. Bodaert11,
Z. Assouline10, M. D. Sherpa3,12, E. E. Schadt3,12, S. M. Houten3,12, J. Byrnes13, E. M. McCormick13, Z. Zolkipli-
Cunningham13,14, K. Haude15, Z. Zhang15, K. Retterer15,
R. Bai15, S. E. Calvo16,17,18, V. K. Mootha19,17,18,
J. Christodoulou1,2, A. Rotig20, A. Filipovska6,21, I. Cristian22,23,
M. J. Falk13,24, M. D. Metodiev25, D. R. Thorburn1,2,26 1Faculty of Medicine, Cairo, Egypt, 2Faculty of Medicine,
Belgrade, Serbia, 3Neurology Clinic, Belgrade, Serbia, 4Child
and Adolescent Neurology and Psychiatry Clinic, Belgrade,
Serbia 1Faculty of Medicine, Cairo, Egypt, 2Faculty of Medicine,
Belgrade, Serbia, 3Neurology Clinic, Belgrade, Serbia, 4Child
and Adolescent Neurology and Psychiatry Clinic, Belgrade,
Serbia Introduction: Leber hereditary optic neuropathy (LHON)
is now regarded as the most frequent neurological disorder
caused by mutations in a mitochondrial DNA (mtDNA),
characterized by selective degeneration of retinal ganglion
cells, optic atrophy, and central vision loss. Besides primary
mutations number of secundary DNA changes affect phe-
notype. Aim of this study was to investigate the presence of
mutations in mtDNA among Serbian patients affected
with LHON. P06.32D Heterogeneity in m.3243A>G-related mitochondrial
disease: The role of mtDNA heteroplasmy, copy number,
age and nuclear factors Abstracts from the 51st European Society of Human Genetics Conference: Posters 181 P06.35C 1Murdoch Children’s Research Institute, Royal Children’s
Hospital, Melbourne, Australia, 2Department of Paediatrics,
University of Melbourne, Melbourne, Australia, 3Department
of Genetics and Genomic Sciences, Icahn School of Medicine
at Mount Sinai, New York, NY, United States, 4Department of
Pediatrics, Icahn School of Medicine at Mount Sinai, New
York, NY, United States, 5Department of Biochemistry and
Molecular Biology, Monash Biomedicine Discovery Institute,
Monash University, Clayton Campus, Melbourne, Australia,
6Harry Perkins Institute of Medical Research and Centre for
Medical Research, University of Western Australia, Perth,
Australia, 7Institute Imagine, Paris, France, 8Murdoch
Children’s Research Institute, Royal Children’s Hospital,
Melbourne, Austria, 9Department of Biological and Medical
Sciences, Faculty of Health Sciences, Oxford Brookes
University, Oxford, Oxford, United Kingdom, 10Departments of
Pediatric, Neurology and Genetics, Hospital Necker-Enfants-
Malades, Paris, France, 11Pediatric Radiology Department,
Hospital Necker Enfants Malades, AP-HP, University Rene
Descartes, PRES Sorbonne Paris Cite, INSERM U1000 and
UMR 1163, Institute Imagine,, Paris, France, 12Icahn Institute
for Genomics and Multiscale Biology, Icahn School of
Medicine at Mount Sinai, New York, NY, United States,
13Division of Human Genetics, Department of Pediatrics, The
Children’s Hospital of Philadelphia, Philadelphia, PA, United
States, 14Division of Neurology, Department of Pediatrics, The
Children’s Hospital of Philadelphia, Philadelphia, PA, United
States, 15GeneDx, Gaithersburg, MD, United States, 16Howard
Hughes Medical Institute, Department of Molecular Biology,
Massachusetts General Hospital,, Boston, MA, United States,
17Department of Systems Biology, Harvard Medical School,
Boston, MA, United States, 18Broad Institute of MIT and
Harvard, Cambridge, MA, United States, 19Howard Hughes
Medical Institute, Department of Molecular Biology,
Massachusetts General Hospital, Boston, MA, United States,
20NSERM U1163, Paris Descartes University - Sorbonne Paris
Cite, Institute Imagine, Paris, France, 21School of Molecular
Sciences, University of Western Australia,, Crawley, Australia,
22Nemours Children’s Hospital,, Orlando, FL, United States, Material and Methods: Individuals included in this
study were recruited from the Child and Adolescent
Neurology and Psychiatry Clinic and from Neurology
Clinic CCS, Belgrade, Serbia. All examined individuals for
17 unrelated familes had characteristic clinical presentation
suggesting the presence of LHON. Multiple segmental PCR
amplicons of mtDNA have been sequenced by Sanger’s
method and the obtained results were compared with the
referent mtDNA sequence. Material and Methods: Individuals included in this
study were recruited from the Child and Adolescent
Neurology and Psychiatry Clinic and from Neurology
Clinic CCS, Belgrade, Serbia. All examined individuals for
17 unrelated familes had characteristic clinical presentation
suggesting the presence of LHON. P06.36D
Mutations in NDUFAF8 cause Leigh syndrome with an
isolated complex I deficiency The synthesis of all 13 mitochondrial DNA (mtDNA)-
encoded protein subunits of the human oxidative phos-
phorylation (OXPHOS) system is carried out by mito-
chondrial
ribosomes
(mitoribosomes). Defects
in
the
stability
of
mitoribosomal
proteins
or
mitoribosome
assembly impair mitochondrial protein translation, causing
combined OXPHOS enzyme deficiency and clinical dis-
ease. Here we report four autosomal-recessive pathogenic
mutations in the gene encoding the small mitoribosomal
subunit protein, MRPS34, in six subjects from four unre-
lated
families
with
Leigh
syndrome
and
combined
OXPHOS defects. Whole-exome sequencing was used to
independently identify all variants. Two splice-site muta-
tions were identified, including homozygous c.321+1G>T
in a subject of Italian ancestry and homozygous c.322-
10G>A in affected sibling pairs from two unrelated families
of Puerto Rican descent. In addition, compound hetero-
zygous MRPS34 mutations were identified in a proband of
French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a
nonsense (c.94C>T [p.Gln32*]) variant. We demonstrated
that these mutations reduce MRPS34 protein levels and the
synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative
proteomics showed that the mitochondrial translation defect
was caused by destabilization of the small mitoribosomal
subunit and impaired monosome assembly. Lentiviral-
mediated expression of wild-type MRPS34 rescued the
defect in mitochondrial translation observed in skin fibro-
blasts from affected subjects, confirming the pathogenicity
of MRPS34 mutations. Our data establish that MRPS34 is
required for normal function of the mitoribosome in humans
and furthermore demonstrate the power of quantitative
proteomic analysis to identify signatures of defects in spe-
cific cellular pathways in fibroblasts from subjects with
inherited disease. C. L. Alston1,2, M. T. Veling3,4, J. Heidler5, L. S. Taylor6,
L. He6,2, A. Broomfield7, J. Pavaine8, H. Prokisch9,10,
S. Wortmann9,10,11, P. E. Bonnen12, R. McFarland1,2,
I. Wittig5,13,14, D. J. Pagliarini3,4, R. W. P06.35C Multiple segmental PCR
amplicons of mtDNA have been sequenced by Sanger’s
method and the obtained results were compared with the
referent mtDNA sequence. Results: The most frequent primary mutations mt.3460
G>A in ND1 and mt. 11778 G>A in ND4 was found in 6
out of 15 and in 10 out of 15 families, respectively. LOHN
primary mutation 11778 G>A was associated with ND1
mt.3394 T>C secondary mutation caused the evolutionary
Conserved tyrosine to histidine (Y30H). The presence of
both 11778 G>A and 3394 T>C mutations appear to
contribute to higher penetrance of LHON. Many other
mutations were detected in various parts of mitochiondrial
genome. Some of haplotype variants were linked prefer-
ential to primary mutations. Results: The most frequent primary mutations mt.3460
G>A in ND1 and mt. 11778 G>A in ND4 was found in 6
out of 15 and in 10 out of 15 families, respectively. LOHN
primary mutation 11778 G>A was associated with ND1
mt.3394 T>C secondary mutation caused the evolutionary
Conserved tyrosine to histidine (Y30H). The presence of
both 11778 G>A and 3394 T>C mutations appear to
contribute to higher penetrance of LHON. Many other
mutations were detected in various parts of mitochiondrial
genome. Some of haplotype variants were linked prefer-
ential to primary mutations. Conclusion: Both primary and secondary mutations in
mtDNA need to be better characterized in light of their
association with clinical manifestations and progression of
Leber hereditary optic neuropathy. Leber hereditary optic neuropathy. P. Dawod: None. B. Rovcanin: None. M. Brankovic:
None. A. Marjanovic: None. M. Jankovic: None. I. Novakovic: None. I. Dujmovic: None. J. Jancic: None. V. Kostic: None. 182 J. del Picchia 23Division of Genetics, Arnold Palmer Hospital for Children,
Orlando, FL, United States, 24University of Pennsylvania
Perelman School of Medicine, Philadelphia, PA, United States,
25INSERM U1163, Paris Descartes University - Sorbonne
Paris Cite, Institute Imagine, Paris, France, 26Victorian
Clinical Genetic Services, Royal Children’s Hospital,
Melbourne, Australia part-time); Significant; GeneDx. R. Bai: A. Employment
(full or part-time); Significant; GeneDx. S.E. Calvo: None. V.K. Mootha: None. J. Christodoulou: None. A. Rotig:
None. A. Filipovska: None. I. Cristian: None. M.J. Falk:
None. M.D. Metodiev: None. D.R. Thorburn: None. P06.36D
Mutations in NDUFAF8 cause Leigh syndrome with an
isolated complex I deficiency Introduction: Mitochondrial diseases are a clinically het-
erogeneous group of disorders caused by dysfunction of the
mitochondrial respiratory chain. Some mitochondrial dis-
orders affect a single organ, while many involve multiple
systems such as skeletal muscle, brain, heart and liver,
leading to diagnostic difficulties. Here we present three
patients who were originally suspected to have a primary
disease of skeletal muscle, leukodystrophy and brain
malformation. Results: We describe three unrelated individuals who
harbour biallelic variants in NDUFAF8, a recently identified
ancillary factor required for assembly of the complex I
holoenzyme. We provide functional evidence to support the
pathogenicity of these NDUFAF8 variants and unequivo-
cally establish this gene as a cause of complex I deficiency
in association with an exclusively Leigh-like clinical
presentation. Materials and methods: Patients were recruited from
three paediatric neurology clinics in Turkey: Izmir, Malatya
and Diyarbakir. Whole exome sequencing (WES) was
performed using Illumina exome capture (38 Mb target). Data analysis was carried out on the RD-Connect Genome-
Phenome Analysis Platform. Standard filtering criteria with
MAF<1% and high/moderate VEP were used, as well as a
list consisting of >5,000 medically interpretable genes. Conclusions: Functional experimentation including com-
plementation studies and complexome profiling of subject
cell lines establishes NDUFAF8 as the twelfth complex I
assembly
factor
associated
with
human
disease
and
validates the importance of orphan gene characterisation. Results: We identified a homozygous frameshift variant
(p.Glu41GlyfsTer10) in NDUFA12 and a homozygous
missense variant (p.Gln85His) in NDUFS3, both associated
with Leigh syndrome due to mitochondrial complex I
deficiency (OMIM# 256000), and a homozygous nonsense
variant (p.His158ProfsTer8) in TACO1 associated with
mitochondrial complex IV deficiency (OMIM# 220110). All the variants were highly pathogenic and were absent in
the control population, suggesting they were disease-
causing. Critical clinical review and metabolic analysis
confirmed the mitochondrial deficiency. Acknowledgements: NIHR doctoral fellowship (NIHR-
HCS-D12-03-04); Wellcome Centre for Mitochondrial
Research (203105/Z/16/Z); MRC Centre for Neuromuscular
Diseases (G0601943); NHS Highly Specialised Service for
Rare
Mitochondrial
Disorders;
The
Lily
Foundation;
Deutsche Forschungsgemeinschaft: SFB 815/Z1; BMBF
mitoNET: 01GM1113B. The views expressed are those of
the author(s) and not necessarily the NHS, NIHR or DoH. C.L. Alston: None. M.T. Veling: None. J. Heidler:
None. L.S. Taylor: None. L. He: None. A. Broomfield:
None. J. Pavaine: None. H. Prokisch: None. S. Wort-
mann: None. P.E. Bonnen: None. R. McFarland: None. I. Wittig:
None. D.J. Pagliarini:
None. R.W. Taylor: None. P06.36D
Mutations in NDUFAF8 cause Leigh syndrome with an
isolated complex I deficiency Conclusions:
Next
generation
sequencing
has
the
advantage of allowing an unbiased genetic diagnosis. We
described three cases that had been initially diagnosed as
myopathy, brain malformation and leukodystrophy, and
WES resulted in the diagnoses of mitochondrial disorders. Importantly, this will allow for appropriate clinical manage-
ment of these patients. P06.36D
Mutations in NDUFAF8 cause Leigh syndrome with an
isolated complex I deficiency Taylor1,2 1Wellcome Centre for Mitochondrial Research, Newcastle
University, Newcastle upon Tyne, United Kingdom, 2NHS
Highly Specialised Service for Rare Mitochondrial Disorders,
Newcastle upon Tyne, United Kingdom, 3Morgridge Institute
for Research, Madison, WI, United States, 4Department of
Biochemistry, University of Wisconsin–Madison, Madison, WI,
United States, 5Functional Proteomics, Goethe-Universität,
Frankfurt am Main, Germany, 6Wellcome Centre for
Mitochondrial Research, Newcastle upon Tyne, United
Kingdom, 7Manchester Centre for Genomic Medicine, St
Mary's Hospital, Manchester Academic Health Science Centre
(MAHSC), Manchester, United Kingdom, 8Department of
Paediatric Neuroradiology, Royal Manchester Children's
Hospital, Central Manchester Foundation Trust, Manchester,
United Kingdom, 9Institute of Human Genetics, Technische
Universität München, München, Germany, 10Institute of
Human Genetics, Helmholtz Zentrum München, Neuherberg,
Germany, 11Department of Pediatrics, Salzburger
Landeskliniken (SALK), Paracelsus Medical University
(PMU), Salzburg, Austria, 12Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX,
United States, 13Cluster of Excellence “Macromolecular
Complexes”, Goethe-Universität, Frankfurt am Main,
Germany, 14German Center for Cardiovascular Research
(DZHK), Partner site RheinMain, Frankfurt, Germany Introduction: Mitochondrial diseases are clinically and
genetically heterogeneous metabolic conditions. Complex I
deficiency is the most common biochemical diagnosis,
particularly prevalent in paediatric patients. Mitochondrial
disease is caused by mutations affecting the mitochon-
drion’s own genome (mtDNA) or one of the ~1150 nuclear-
encoded mitoproteome components. The limited genotype:
phenotype correlation in mitochondrial disease means next-
generation sequencing methodologies are critical for the
rapid genetic diagnosis of patients. N.J. Lake: None. B.D. Webb: None. D.A. Stroud:
None. T.R. Richman: None. B. Ruzzenente: None. A.G. Compton: None. H.S. Mountford: None. J. Pulman:
None. C. Zangarelli: None. M. Rio: None. N. Bodaert:
None. Z. Assouline: None. M.D. Sherpa: None. E.E. Schadt: None. S.M. Houten: None. J. Byrnes: None. E. M. McCormick: None. Z. Zolkipli-Cunningham: None. K. Haude: A. Employment (full or part-time); Significant;
GeneDx. Z. Zhang: A. Employment (full or part-time);
Significant; GeneDx. K. Retterer: A. Employment (full or Materials and Methods: Whole exome and targeted
next-generation
sequencing
was
performed
for
three Abstracts from the 51st European Society of Human Genetics Conference: Posters 183 subjects with suspected mitochondrial disease. Functional
evaluation of identified variants was undertaken using
subject fibroblasts and/or muscle biopsy, including cDNA
studies, complexome profiling, complementation studies
and assessment of steady-state levels. subjects with suspected mitochondrial disease. Functional
evaluation of identified variants was undertaken using
subject fibroblasts and/or muscle biopsy, including cDNA
studies, complexome profiling, complementation studies
and assessment of steady-state levels. Unexpected genetic diagnosis of mitochondrial disease in
three consanguineous Turkish families A. Topf: None. Y. Oktay: None. S. Balaraju: None. E. Yılmaz: None. E. Sönmezler: None. A. Yaramis: None. S. Güngör: None. S. Laurie: None. S. Beltran: None. I. Gut: None. H. Lochmüller: None. S. Hiz: None. R. Horvath: None. A. Topf1, Y. Oktay2,3, S. Balaraju1, E. Yılmaz2, E. Sönmezler2,
A. Yaramis4, S. Güngör5, S. Laurie6,7, S. Beltran6,7, I. Gut6,7,
H. Lochmüller1, S. Hiz8, R. Horvath1 1Newcastle University, Newcastle, United Kingdom, 2Izmir
Biomedicine and Genome Center, Dokuz Eylul University
Health Campus, Izmir, Turkey, 3Dokuz Eylul University,
School of Medicine, Department of Medical Biology, Izmir,
Turkey, 4Pediatric Neurology Clinic, Diyarbakir Memorial
Hospital, Diyarbakir, Turkey, 5Inonu University, Faculty of
Medicine, Turgut Ozal Research Center, Department of
Paediatric Neurology, Malatya, Turkey, 6CNAG-CRG, Centre
for Genomic Regulation, Barcelona Institute of Science and
Technology, Barcelona, Spain, 7Universitat Pompeu Fabra,
Barcelona, Spain, 8Dokuz Eylul University, School of
Medicine, Department of Paediatric Neurology, Izmir, Turkey P06.37A Unexpected genetic diagnosis of mitochondrial disease in
three consanguineous Turkish families Innovative Next Generation Sequencing strategy for
screening mitochondrial DNA mutations as a robust
alternative of conventional methods Mitochondria harbor multiple copies of a maternally
inherited non-nuclear genome (mtDNA) and defects in its
replication or nucleotide metabolism cause point mutations,
deletions, or depletion of mtDNA. These genetic alterations
can lead to multi-system syndromes, including neuromus-
cular and neurodegenerative diseases. To date, approaches
based on Sanger technology and Southern blot have formed
the basis of mtDNA screening but these technologies are
inherently hampered by limitations in speed, throughput,
resolution, and associated costs. Mitochondria harbor multiple copies of a maternally
inherited non-nuclear genome (mtDNA) and defects in its
replication or nucleotide metabolism cause point mutations,
deletions, or depletion of mtDNA. These genetic alterations
can lead to multi-system syndromes, including neuromus-
cular and neurodegenerative diseases. To date, approaches
based on Sanger technology and Southern blot have formed
the basis of mtDNA screening but these technologies are
inherently hampered by limitations in speed, throughput,
resolution, and associated costs. Materials and Methods: The Maltese cohort included 13
probands (7 children and 6 adults) and 2 unaffected
relatives. Whole exome sequencing and bioinformatics
analysis were carried out at the Centro Nacional de Análisis
Genómico (CNAG-CRG) in Barcelona. Phenotypic data of
each participant was recorded on PhenoTips. Exome data
was
analysed
on
the
RD-Connect
Genome-Phenome
Analysis Platform. In this study, we describe a robust strategy for screening
mtDNA alterations through a PCR free NGS approach in
order to assess with high accuracy point mutations and
single or multiple large deletions in both homoplasmic or
heteroplasmic state. Mitochondrial DNA is extracted from
biological samples with the Mitochondrial DNA Isolation
Kit (Abcam), processed according to Nextera XT DNA
library Prep Kit (Illumina), and sequenced on MiSeq
benchtop sequencer (Illumina). The output obtained for
each sample analyzed is represented by the full sequence of
mtDNA at an high depth of coverage. We developed an
innovative bioinformatic analysis of deep NGS data to
detect mitochondrial haplogroups, heteroplasmic mutations
and large mtDNA deletions with higher accuracy and
sensitiveness respect to standard techniques. In addition,
this approach allows us to precisely map and quantify large
heteroplasmic mtDNA deletions from the analysis of NGS
coverage data, avoiding the bias introduced by polymerase
amplification for shorter mtDNA molecules that character-
ize the NGS approaches published in the literature so far. Results: A comparative analysis of rare autosomal
recessive mutations shows that some patients share the
same variants. Diagnosis of mitochondrial disorders by whole exome
sequencing Diagnosis of mitochondrial disorders by whole exome
sequencing Innovative Next Generation Sequencing strategy for
screening mitochondrial DNA mutations as a robust
alternative of conventional methods Rare missense mutations in the mitochond-
rially encoded cytochrome B gene (MT-CYB) at positions
14766 and 15326 were present in 6 and 11 of the probands
respectively. The mtDNA mutation at position 15326
(rs2853508) was not present in the reference Maltese
Exome
database,
whereas
that
at
position
14766
(rs57236041) had a frequency of 59%. Conclusion: The exome sequence data generated through
this collaborative research will aid in establishing a genetic
diagnosis for these rare disease patients. The research leading to these results has received funding
from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 2012-305444, the 2016 BBMRI-LPC WES call and the
Malta Government Scholarship Scheme. A. Legati: None. N. Zanetti: None. C. Péron: None. E. Lamantea: None. D. Ghezzi: None. J. Vella: None. J. Borg: None. D. Soler: None. N. Vella:
None. J. Aquilina: None. E. Said: None. I. Borg: None. A.E. Felice: None. Innovative Next Generation Sequencing strategy for
screening mitochondrial DNA mutations as a robust
alternative of conventional methods Innovative Next Generation Sequencing strategy for
screening mitochondrial DNA mutations as a robust
alternative of conventional methods A. Legati, N. Zanetti, C. Péron, E. Lamantea, D. Ghezzi
Fondazione IRCCS Istituto Neurologico ‘Carlo Besta’, Milan,
Italy A. Legati, N. Zanetti, C. Péron, E. Lamantea, D. Ghezzi A. Legati, N. Zanetti, C. Péron, E. Lamantea, D. Ghezzi Fondazione IRCCS Istituto Neurologico ‘Carlo Besta’, Milan,
Italy Fondazione IRCCS Istituto Neurologico ‘Carlo Besta’, Milan,
Italy Human mitochondria produce ATP and metabolites to
support development and maintain cellular homeostasis. J. del Picchia 184 Mitochondria harbor multiple copies of a maternally
inherited non-nuclear genome (mtDNA) and defects in its
replication or nucleotide metabolism cause point mutations,
deletions, or depletion of mtDNA. These genetic alterations
can lead to multi-system syndromes, including neuromus-
cular and neurodegenerative diseases. To date, approaches
based on Sanger technology and Southern blot have formed
the basis of mtDNA screening but these technologies are
inherently hampered by limitations in speed, throughput,
resolution, and associated costs. In this study, we describe a robust strategy for screening
mtDNA alterations through a PCR free NGS approach in
order to assess with high accuracy point mutations and
single or multiple large deletions in both homoplasmic or
heteroplasmic state. Mitochondrial DNA is extracted from
biological samples with the Mitochondrial DNA Isolation
Kit (Abcam), processed according to Nextera XT DNA
library Prep Kit (Illumina), and sequenced on MiSeq
benchtop sequencer (Illumina). The output obtained for
each sample analyzed is represented by the full sequence of
mtDNA at an high depth of coverage. We developed an
innovative bioinformatic analysis of deep NGS data to
detect mitochondrial haplogroups, heteroplasmic mutations
and large mtDNA deletions with higher accuracy and
sensitiveness respect to standard techniques. In addition,
this approach allows us to precisely map and quantify large
heteroplasmic mtDNA deletions from the analysis of NGS
coverage data, avoiding the bias introduced by polymerase
amplification for shorter mtDNA molecules that character-
ize the NGS approaches published in the literature so far. A. Legati: None. N. Zanetti: None. C. Péron: None. E. Lamantea: None. D. Ghezzi: None. P06.39C
Diagnosis of mitochondrial disorders by whole exome Introduction: The Malta BioBank (BBMRI.mt) partici-
pated in the BBMRI-Large Prospective Cohort (BBMRI-
LPC) whole exome sequencing (WES) call with a colla-
borative research project with Hacettepe University, Turkey
to sequence a total of 50 exomes from patients with
genetically undiagnosed mitochondrial disorders. High-throughput sequencing of the whole mitochondrial
genome in 974 patients with mitochondrial disease: New
insights and challenges for the interpretation of
mitochondrial DNA variants High-throughput sequencing of the whole mitochondrial
genome in 974 patients with mitochondrial disease: New
insights and challenges for the interpretation of
mitochondrial DNA variants J. Vella1,2, J. Borg1,3, D. Soler4, N. Vella5, J. Aquilina5, E. Said6,
I. Borg6,7, A. E. Felice1,2,6 1Malta BioBank (BBMRI.mt), Centre of Molecular Medicine
and Biobanking, University of Malta, Msida, Malta,
2Department of Physiology and Biochemistry, Faculty of
Medicine and Surgery, University of Malta, Msida, Malta,
3Department of Applied Biomedical Science, Faculty of Health
Sciences, University of Malta, Msida, Malta, 4Department of
Paediatrics, Mater Dei Hospital, Msida, Malta, 5Department
of Neuroscience, Mater Dei Hospital, Msida, Malta,
6Department of Pathology, Mater Dei Hospital, Msida, Malta,
7Department of Pathology, Faculty of Medicine and Surgery,
University of Malta, Msida, Malta C. Bris1,2, D. Goudenège1,2, V. Desquiret-Dumas1,2, M. Charif2,
N. Gueguen1,2, S. Belal2, C. Verny3,2, G. Lenaers2,
D. Bonneau1,2, P. Reynier1,2, P. Amati-Bonneau1,2,
V. Procaccio1,2 C. Bris1,2, D. Goudenège1,2, V. Desquiret-Dumas1,2, M. Charif2,
N. Gueguen1,2, S. Belal2, C. Verny3,2, G. Lenaers2,
D. Bonneau1,2, P. Reynier1,2, P. Amati-Bonneau1,2,
V. Procaccio1,2 1Biochemistry and Genetics Department, Angers University
Hospital, Angers, France, 2UMR CNRS 6015-INSERM U1083,
University of Angers, Angers, France, 3Neurology department,
Angers University Hospital, Angers, France 1Biochemistry and Genetics Department, Angers University
Hospital, Angers, France, 2UMR CNRS 6015-INSERM U1083,
University of Angers, Angers, France, 3Neurology department,
Angers University Hospital, Angers, France Mitochondrial diseases owe their clinical heterogeneity to
the dual origin of mitochondrial proteins: nuclear and Abstracts from the 51st European Society of Human Genetics Conference: Posters 185 mitochondrial genomes (mtDNA). We used whole mtDNA
analysis by Next Generation Sequencing (NGS) in a cohort
of 974 patients with suspected mitochondrial disease. Our
study demonstrated the power of our strategy, especially
using uroepithelial cells as source of mtDNA, with the
identification of a pathogenic variant in 130 patients
(13.3%). However, massive parallel sequencing used raised
several issues such as: 1. Low pathogenic mtDNA variant
loads. The identification of low mutation rates contributes
to improved diagnosis, especially among relatives. How-
ever, evaluating the clinical relevance of these low mutation
rates in probands is complex, as for the m.3243A> G,
detected at low levels for 16 patients.2. Pathogenic var-
iants unrelated to patient phenotype. Nevertheless, some
of them could be regarded as "actionable mutations” calling
for specific recommendations in patient management.3. Variants of unknown significance. High-throughput sequencing of the whole mitochondrial
genome in 974 patients with mitochondrial disease: New
insights and challenges for the interpretation of
mitochondrial DNA variants Bonneau: None. P. Reynier: None. P. Amati-Bonneau:
None. V. Procaccio: None. P06.43C Mutations in MRPS14 cause intellectual disability, neonatal
lactic acidosis, cachexia and hypertrophic cardiomyopathy
with distinct dysmorphic features Mutations in MRPS14 cause intellectual disability, neonatal
lactic acidosis, cachexia and hypertrophic cardiomyopathy
with distinct dysmorphic features High-throughput sequencing of the whole mitochondrial
genome in 974 patients with mitochondrial disease: New
insights and challenges for the interpretation of
mitochondrial DNA variants A novel variant was
identified in 3.7% of the patients highlighting the difficulty
in prioritizing them, because of the weakness of in silico
tools and databases, and the lack of guidelines.4. Inte-
grative analysis. Currently, diagnostic laboratory do not
use information such as mitochondrial haplogroups or rare
co-occurrences of mtDNA variants, for explaining pheno-
typic differences within patients carrying the same muta-
tion. Our study shows that mtDNA screening by NGS
significantly improves the diagnosis of mitochondrial dis-
orders. However, the technique increases the complexity of
appreciating the variants identified, thereby posing new
challenges in the molecular diagnosis of mitochondrial
diseases. Here, we report an abnormal processing of precursor
proteins in human mitochondria by the mitochondrial
intermediate peptidase (MIPEP) leading to mitochondrial
disease. Most nucleus-encoded mitochondrial proteins are
synthesized in the cytosol as precursor proteins carrying an
N-terminal targeting signal that is cleaved upon import into
mitochondria by the matrix processing peptidase (MPP) and
in some cases by other processing peptidases, like MIPEP
to ensure protein stability and functionality. Using exome
sequencing, we identified mutations in the MIPEP gene in a
patient
presenting
psychomotor
retardation,
cerebellar
syndrome and axonal neuropathy. MRI shows hyperinten-
sities in the bilateral atrophic putamen. Western immunoblotting of patient fibroblast extracts
revealed strong decease of the mutant MIPEP protein and
an accumulation of the unprocessed precursor of MRPL12 –
a bona fide MIPEP substrate. Analysis of the assembly and
function of OXPHOS using blue-native (BN) PAGE and
respirometry, respectively, showed defects in the biogenesis
and activity of complexes I, IV and V in subject’s
fibroblasts. We carried out functional complementation
experiments in patient fibroblasts stably expressing wild
type MIPEP protein, which showed restoration of MRPL12
processing and OXPHOS assembly and function thereby
confirming that MIPEP is the disease-causing gene. Our
studies further expand the genotypic and phenotypic
heterogeneity of MIPEP-linked mitochondrial disease and
provide important insights into its pathophysiology. J. Pulman: None. B. Ruzzenente: None. L. Bianchi:
None. C. Bole-Feysot: None. P. Nitschké: None. A. Munnich: None. A. Rötig: None. M.D. Metodiev: None. C. Bris: None. D. Goudenège: None. V. Desquiret-
Dumas: None. M. Charif: None. N. Gueguen: None. S. Belal: None. C. Verny: None. G. Lenaers: None. D. Bonneau: None. P. Reynier: None. P. Amati-Bonneau:
None. V. Procaccio: None. C. Bris: None. D. Goudenège: None. V. Desquiret-
Dumas: None. M. Charif: None. N. Gueguen: None. S. Belal: None. C. Verny: None. G. Lenaers: None. D. P06.42B C. B. Jackson1, M. Huemer2,3, R. Bolognini4, F. Martin5,
B. Donner6, G. Szinnai7, J. Nuoffer8, A. Wartiovaara9,
A. Schaller4 C. B. Jackson1, M. Huemer2,3, R. Bolognini4, F. Martin5,
B. Donner6, G. Szinnai7, J. Nuoffer8, A. Wartiovaara9,
A. Schaller4 Mutations in the mitochondrial intermediate peptidase
(MIPEP) cause multiple OXPHOS deficiency, psychomotor
retardation, cerebellar syndrome and axonal neuropathy 1Biomedicum Helsinki, Helsinki, Finland, 2University
Children's Hospital Basel, Basel, Switzerland, 3Division of
Metabolism and Children’s Research Center, Zürich,
Switzerland, 4Division of Human Genetics, Bern, Switzerland,
5Université de Strasbourg, Strasbourg, France, 6Division of
Cardiology, Basel, Switzerland, 7Division of Pediatric
Endocrinology, Basel, Switzerland, 8Institute of Clinical
Chemistry, Bern, Switzerland, 9Biomedicum Helsinki, Bern,
Finland 1Biomedicum Helsinki, Helsinki, Finland, 2University
Children's Hospital Basel, Basel, Switzerland, 3Division of
Metabolism and Children’s Research Center, Zürich,
Switzerland, 4Division of Human Genetics, Bern, Switzerland,
5Université de Strasbourg, Strasbourg, France, 6Division of
Cardiology, Basel, Switzerland, 7Division of Pediatric
Endocrinology, Basel, Switzerland, 8Institute of Clinical
Chemistry, Bern, Switzerland, 9Biomedicum Helsinki, Bern,
Finland J. Pulman , B. Ruzzenente , L. Bianchi , C. Bole-Feysot ,
P. Nitschké1, A. Munnich2,1, A. Rötig1, M. D. Metodiev1 1UMR1163, Université Paris Descartes, Sorbonne Paris Cité,
Institut IMAGINE, Paris, France, 2Departments of Pediatrics,
Radiology and Genetics, Hôpital Necker-Enfants Malades,
Paris, France 1UMR1163, Université Paris Descartes, Sorbonne Paris Cité,
Institut IMAGINE, Paris, France, 2Departments of Pediatrics,
Radiology and Genetics, Hôpital Necker-Enfants Malades,
Paris, France Mitochondrial diseases represent a large group of rare and
heterogeneous genetic disorders that are associated with
different disease mechanisms. Introduction: Multiple respiratory dysfunction is asso-
ciated
with
defects
in
mitochondrial
replication
and J. del Picchia 186 Introduction: Complex III (CIII) presents the center of the
mitochondrial respiratory chain. Its deficiency is one of the
least common oxidative phosphorylation defects associated
with mitochondrial disease, with pathogenic variants in 10
genes encoding complex III subunits or assembly factors
identified
and
associated
with
a
broad
phenotypical
spectrum. translation. Possibly, due to their detrimental effect, defects
of the mitochondrial ribosome are rare. Here we report that
a mutated essential mitochondrial small ribosome subunit
causes mental retardation, cachexia, muscle hypotonia,
hypertrophic cardiomyopathy with elevated lactate in a
Turkish girl born to consanguineous parents. Introduction: Complex III (CIII) presents the center of the
mitochondrial respiratory chain. Its deficiency is one of the
least common oxidative phosphorylation defects associated
with mitochondrial disease, with pathogenic variants in 10
genes encoding complex III subunits or assembly factors
identified
and
associated
with
a
broad
phenotypical
spectrum. P06.42B Materials and Methods: DNA from blood was used for
Next Generation Sequencing via MitoExome panel. Mito-
chondrial respiratory chain function was assessed in skeletal
muscle via enzymatic activity and in primary fibroblasts via
oxymetric measurements and native mitochondrial complex
assembly. Ribosomal RNA stability was analysed by
Northern-blotting and qPCR in fibroblasts. Mitochondrial
translation capacity was determined in fibroblasts by 35S-
methionine incorporation into newly synthesized proteins
and separated on SDS-PAGE. For complementatio, wild-
type MRPS14 cDNA was cloned into a lentiviral vector and
stable lines generated via antibiotic selection. Material and methods: Two unrelated individuals with
early-onset hypertrophic cardiomyopathy and lactic acidosis
underwent genetic analysis (whole exome sequencing, RT-
PCR, Sanger sequencing). Functional studies were per-
formed in proband derived fibroblasts. Results: We identified biallelic mutations in UQCRFS1,
encoding the ubiquinol-cytochrome c reductase, Rieske
iron-sulfur polypeptide 1, a catalytic subunit of the CIII. One
proband
carried
homozygous
intronic
splice-site
variant. Splicing analysis revealed deletion of 30 nucleo-
tides. The other proband had two compound heterozygous
missense variants. Proband derived fibroblasts showed
deficient oxygen consumption rate. Western blot analysis
of isolated mitochondria showed a reduction of UQCRFS1
in affected probands, as well as a decrease of CIII assembly
factor UQCC2. We also observed reduction of complex I
subunit NDUFS4. Furthermore, Blue-Native gel electro-
phoresis of digitonin-solubilized mitochondria showed
decrease of both complex III and complex I. Results: We identified biallelic mutations in UQCRFS1,
encoding the ubiquinol-cytochrome c reductase, Rieske
iron-sulfur polypeptide 1, a catalytic subunit of the CIII. One
proband
carried
homozygous
intronic
splice-site
variant. Splicing analysis revealed deletion of 30 nucleo-
tides. The other proband had two compound heterozygous
missense variants. Proband derived fibroblasts showed
deficient oxygen consumption rate. Western blot analysis
of isolated mitochondria showed a reduction of UQCRFS1
in affected probands, as well as a decrease of CIII assembly
factor UQCC2. We also observed reduction of complex I
subunit NDUFS4. Furthermore, Blue-Native gel electro-
phoresis of digitonin-solubilized mitochondria showed
decrease of both complex III and complex I. Results: Targeted next generation sequencing revealed a
homozygous variant in mitochondrial small ribosomal
protein
14
(MRPS14). Biochemical
characterisation
revealed an enzymatic complex IV deficiency in skeletal
muscle
with
total
mitochondrial
translation
greatly
decreased in fibroblasts
alongside all mitochondrially
encoded respiratory subunits. Lentiviral complementation
confirmed the pathogenicity of the novel variant. Results: Targeted next generation sequencing revealed a
homozygous variant in mitochondrial small ribosomal
protein
14
(MRPS14). P06.42B Biochemical
characterisation
revealed an enzymatic complex IV deficiency in skeletal
muscle
with
total
mitochondrial
translation
greatly
decreased in fibroblasts
alongside all mitochondrially
encoded respiratory subunits. Lentiviral complementation
confirmed the pathogenicity of the novel variant. Conclusions: This is the first mutation in any mitochon-
drial ribosomal protein so far reported not affecting
ribosomal stability, but drastically decreasing mitochondrial
translation capacity. Conclusions: Until now, variants in UQCRFS1 and
impaired complex III activity have only been reported in
relation with gastric and breast cancer. Here we present
evidence that biallelic variants in UQCRFS1 can cause
mitochondrial disease with early onset cardiomyopathy and
lactic acidosis. Combined complex III/I deficiency seems to
be the common pattern of complex III subunit/assembly
factor defects. Conclusions: Until now, variants in UQCRFS1 and
impaired complex III activity have only been reported in
relation with gastric and breast cancer. Here we present
evidence that biallelic variants in UQCRFS1 can cause
mitochondrial disease with early onset cardiomyopathy and
lactic acidosis. Combined complex III/I deficiency seems to
be the common pattern of complex III subunit/assembly
factor defects. C.B. Jackson: None. M. Huemer: None. R. Bolognini:
None. F. Martin: None. B. Donner: None. G. Szinnai:
None. J. Nuoffer: None. A. Wartiovaara: None. A. Schaller: None. M. Gusic: None. G. Schottmann: None. R.G. Feich-
tinger: None. M. Wagner: None. J.A. Mayr: None. C. Du: None. C. Lee: None. N. Lorenz: None. E. Gill: None. S. Morales-Gonzales: None. D.M. Panneman: None. A. Rötig: None. R.J.T. Rodenburg: None. S.B. Wortmann:
None. H. Prokisch: None. M. Schuelke: None. M. Gusic: None. G. Schottmann: None. R.G. Feich-
tinger: None. M. Wagner: None. J.A. Mayr: None. C. Du: None. C. Lee: None. N. Lorenz: None. E. Gill: None. S. Morales-Gonzales: None. D.M. Panneman: None. A. Rötig: None. R.J.T. Rodenburg: None. S.B. Wortmann:
None. H. Prokisch: None. M. Schuelke: None. H. Saei Ahan1, M. Alaei2, S. Dabagh Bagheri3, H. Bagherian3,
A. Setoodeh4, M. Abiri1,3, S. Zeinali2,3,5 1HelmholtzZentrum München, Neuherberg, Germany,
2Technical University Munich, Munich, Germany,
3Charité–Universitätsmedizin Berlin, Berlin, Germany,
4Salzburger Landeskliniken (SALK) and Paracelsus Medical
University (PMU), Salzburg, Austria, 5Medizinische
Hochschule Hannover, Hannover, Germany, 6Municipal
Hospital Dresden, Dresden, Germany, 7RadboudUMC,
Nijmegen, Netherlands, 8Institut Imagine, Paris, France P06.45A P06.45A
Homozygosity mapping in maple syrup urine disease
patients from Iran: Identification of novel, recurrent
mutations and in silico analysis of novel mutations H. Saei Ahan1, M. Alaei2, S. Dabagh Bagheri3, H. Bagherian3,
A. Setoodeh4, M. Abiri1,3, S. Zeinali2,3,5 P06.44D Recessive mutations in UQCRFS1, encoding the Rieske
iron-sulfur protein, are associated with mitochondrial
complex III deficiency, lactic acidosis and cardiomyopathy M. Gusic1,2, G. Schottmann3, R. G. Feichtinger4, M. Wagner1,2,
J. A. Mayr4, C. Du5, C. Lee3, N. Lorenz6, E. Gill3, S. Morales-
Gonzales3, D. M. Panneman7, A. Rötig8, R. J. T. Rodenburg7,
S. B. Wortmann1,2,4, H. Prokisch1,2, M. Schuelke3 M. Gusic1,2, G. Schottmann3, R. G. Feichtinger4, M. Wagner1,2,
J. A. Mayr4, C. Du5, C. Lee3, N. Lorenz6, E. Gill3, S. Morales-
Gonzales3, D. M. Panneman7, A. Rötig8, R. J. T. Rodenburg7,
S. B. Wortmann1,2,4, H. Prokisch1,2, M. Schuelke3 M. Gusic1,2, G. Schottmann3, R. G. Feichtinger4, M. Wagner1,2,
J. A. Mayr4, C. Du5, C. Lee3, N. Lorenz6, E. Gill3, S. Morales-
Gonzales3, D. M. Panneman7, A. Rötig8, R. J. T. Rodenburg7,
S. B. Wortmann1,2,4, H. Prokisch1,2, M. Schuelke3 A. Setoodeh , M. Abiri , , S. Zeinali , ,
1Medical Genetic and Molecular Biology Department, Faculty
of Medicine, Iran University of Medical Science,, Tehran, Iran,
Islamic Republic of, 2Pediatric Endocrinology and
Metabolism, Mofid Children’s Hospital, Shahid Beheshti M. Gusic1,2, G. Schottmann3, R. G. Feichtinger4, M. Wagner1,2,
J. A. Mayr4, C. Du5, C. Lee3, N. Lorenz6, E. Gill3, S. Morales-
Gonzales3, D. M. Panneman7, A. Rötig8, R. J. T. Rodenburg7,
S. B. Wortmann1,2,4, H. Prokisch1,2, M. Schuelke3 M. Wagner, B. Alhaddad, R. Berutti, H. Prokisch, T. Strom,
S. B. Wortmann R285X)) and two novel point mutations
[(c.599 C>T (p. P200L), c.484 A>G (p. N162D)]. In DBT
gene we found novel homozygote deletion of exon 5,6 and
7 in one patient as well as a point mutation and deletion
(c.363delCT/ c.1238T>C). M. Wagner: D. Speakers Bureau/Honoraria (speakers
bureau, symposia, and expert witness); Modest; Bayer
Vital. B. Alhaddad: None. R. Berutti: None. H. Prokisch:
None. T. Strom: None. S.B. Wortmann: None. Rothschild1,2 1Sheba Medical Center, Ramat-Gan, Israel, 2Sackler Faculty
of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 3FDNA Inc.,
Boston, MD, United States The most commonly used techniques to detect
single nucleotide variants in the mtDNA are Sanger
sequencing
of
certain
mutations
or
next
generation
sequencing of the whole mtDNA. With the introduction of
exome and genome sequencing, it is now possible to detect
variants on both the nDNA and the mtDNA with one
comprehensive approach. We performed an analysis of the
mtDNA on exome sequencing data from leucocyte derived
DNA of 1,692 individuals for which exome sequencing was
performed in a diagnostic setting with various indications. In total, 26 (likely) clinical relevant variants in the mtDNA
were detected. This resulted in 40 conclusive diagnoses
equaling an overall diagnostic yield of ~2.4%. Looking only
at the solved cases, 5.7% had disease due to mutations in
the mtDNA. Of the 26 different variants, three were listed as
“reported” in MitoMap and we confirmed pathogenicity by
functional tests and/or segregation analysis. An additional
eight novel, to date unpublished variants were identified. Of
note, we did not only identify causative mutations in
patients with suspected mitochondrial disease. However,
read-depth of the mtDNA was not enough to reliably detect
deletions in the mtDNA and exome sequencing should be
complemented by e.g. long-range PCR in certain cases. This
study shows that mutations in the mtDNA can be found in a
significant proportion of patients with suspected monogenic
disorders. Exome sequencing can reliably detect those
variants resulting in a high diagnostic yield. Introduction: Maple syrup urine disease (MSUD) is a rare
inborn error of metabolism of branched-chain amino acid
metabolism. The disease prevalence is higher in populations
with the higher rate of consanguineous marriage like Iran
(38.6%). Different mutations have been previously reported
in BCKDHA, BCKDHB, DBT, and DLD is known to be
responsible for MSUD phenotype. Materials and Methods: In this study, two sets of
multiplex polymorphic STR (short tandem repeat) markers
linked
to
the
above-mentioned
genes
were
used
in
homozygosity mapping in order to find probable pathogenic
changes in 40 studied families. The families who showed
homozygous haplotypes for BCKDHA, BCKDHB and
DBT genes were subsequently sequenced. Results: Our findings revealed that exon 2, 4 and 6 of
BCKDHA gene contained most of the mutations which
were novel. The changes include one reported point
mutation (c.890G>A (p. R297H)), 7 nucleotide insertion
(c.355-356 Ins 7nt (p. D355D fs)) and a splice site mutation
(c.288G>A). In BCKDHB gene we identified one reported
(c.853 C>T (p. B. Pode-Shakked1,2, Y. Levi1, N. Fleischer3, L. Wolf3,
Y. Finezilber1,2, L. Greenbaum1,2, S. Putter1,2, A. Raas-
Rothschild1,2 187 Abstracts from the 51st European Society of Human Genetics Conference: Posters University of Medical Sciences,, Tehran, Iran, Islamic
Republic of, 3Dr. Zeinali’s Medical Genetics Laboratory,
Kawsar Human Genetics Research Center,, Tehran, Iran,
Islamic Republic of, 4Department of Pediatrics, Tehran
University of Medical Sciences,, Tehran, Iran, Islamic
Republic of, 5Department of Molecular Medicine,
Biotechnology Research Center, Pasteur Institute of Iran,,
Tehran, Iran, Islamic Republic of University of Medical Sciences,, Tehran, Iran, Islamic
Republic of, 3Dr. Zeinali’s Medical Genetics Laboratory,
Kawsar Human Genetics Research Center,, Tehran, Iran,
Islamic Republic of, 4Department of Pediatrics, Tehran
University of Medical Sciences,, Tehran, Iran, Islamic
Republic of, 5Department of Molecular Medicine,
Biotechnology Research Center, Pasteur Institute of Iran,,
Tehran, Iran, Islamic Republic of Mitochondrial diseases can result from mutations in either
the mitochondrial DNA (mtDNA) or the nuclear DNA
(nDNA). The most commonly used techniques to detect
single nucleotide variants in the mtDNA are Sanger
sequencing
of
certain
mutations
or
next
generation
sequencing of the whole mtDNA. With the introduction of
exome and genome sequencing, it is now possible to detect
variants on both the nDNA and the mtDNA with one
comprehensive approach. We performed an analysis of the
mtDNA on exome sequencing data from leucocyte derived
DNA of 1,692 individuals for which exome sequencing was
performed in a diagnostic setting with various indications. In total, 26 (likely) clinical relevant variants in the mtDNA
were detected. This resulted in 40 conclusive diagnoses
equaling an overall diagnostic yield of ~2.4%. Looking only
at the solved cases, 5.7% had disease due to mutations in
the mtDNA. Of the 26 different variants, three were listed as
“reported” in MitoMap and we confirmed pathogenicity by
functional tests and/or segregation analysis. An additional
eight novel, to date unpublished variants were identified. Of
note, we did not only identify causative mutations in
patients with suspected mitochondrial disease. However,
read-depth of the mtDNA was not enough to reliably detect
deletions in the mtDNA and exome sequencing should be
complemented by e.g. long-range PCR in certain cases. This
study shows that mutations in the mtDNA can be found in a
significant proportion of patients with suspected monogenic
disorders. Exome sequencing can reliably detect those
variants resulting in a high diagnostic yield. Mitochondrial diseases can result from mutations in either
the mitochondrial DNA (mtDNA) or the nuclear DNA
(nDNA). P06.47C Conclusion: Computational approaches were used to
analyze these novel mutations in terms of their impact on
protein structure. Computational structural modeling indi-
cated that these mutations might affect structural stability
and multimeric assembly of branched-chain keto acid
dehydrogenase complex (BCKDC). Common facial phenotype of patients with Mucolipidosis
type IV: a clinical observation reaffirmed by facial
dysmorphology novel analysis technology Common facial phenotype of patients with Mucolipidosis
type IV: a clinical observation reaffirmed by facial
dysmorphology novel analysis technology B. Pode-Shakked1,2, Y. Levi1, N. Fleischer3, L. Wolf3,
Y. Finezilber1,2, L. Greenbaum1,2, S. Putter1,2, A. Raas-
Rothschild1,2 B. Pode-Shakked1,2, Y. Levi1, N. Fleischer3, L. Wolf3,
Y. Finezilber1,2, L. Greenbaum1,2, S. Putter1,2, A. Raas-
Rothschild1,2 H. Saei Ahan: None. M. Alaei: None. S. Dabagh
Bagheri: None. H. Bagherian: None. A. Setoodeh: None. M. Abiri: None. S. Zeinali: None. H. Saei Ahan: None. M. Alaei: None. S. Dabagh
Bagheri: None. H. Bagherian: None. A. Setoodeh: None. M. Abiri: None. S. Zeinali: None. Institute of Human Genetics, Munich, Germany P06.46B Exome sequencing can reliably identify mtDNA variants
increasing the diagnostic yield in a diagnostic context Background: Mucolipidosis type IV (ML-IV) is a rare
autosomal recessive lysosomal storage disease, caused by
mutations in the MCOLN1 gene. It manifests with non-
specific symptoms of developmental delay, esotropia and
even corneal clouding. While the clinical phenotype,
molecular basis and underlying pathomechanism have been Institute of Human Genetics, Munich, Germany J. del Picchia 188 described, the diagnosis of ML-IV remains elusive and
patients are often misdiagnosed. Our clinical observation
was that ML IV patients share common and identifiable
facial features, which have yet to be included in the clinical
phenotype as described in the literature to date. Objective
and methods: In order to validate these findings using an
objective and digital tool, two-dimensional facial images of
ten patients with ML-IV, obtained at various ages, were
analyzed
using
facial
dysmorphology
novel
analysis
(FDNA). This technology utilizes various measurements
extracted from automatically-detected facial points from
facial photographs, to recognize distinct dysmorphic fea-
tures and analyze their similarities to known facial patterns,
termed gestalts. described, the diagnosis of ML-IV remains elusive and
patients are often misdiagnosed. Our clinical observation
was that ML IV patients share common and identifiable
facial features, which have yet to be included in the clinical
phenotype as described in the literature to date. Objective
and methods: In order to validate these findings using an
objective and digital tool, two-dimensional facial images of
ten patients with ML-IV, obtained at various ages, were
analyzed
using
facial
dysmorphology
novel
analysis
(FDNA). This technology utilizes various measurements
extracted from automatically-detected facial points from
facial photographs, to recognize distinct dysmorphic fea-
tures and analyze their similarities to known facial patterns,
termed gestalts. variants were reported. Bioinformatics analysis was done by
SWISS-MODEL, the mutant proteins were generated by
homology from the wild-type GALNS 4FDI template
obtained
from
PDB
database
and
visualization
was
performed using Swiss-PdbViewer. The predictive analysis
was run in PolyPhen-2 software (Polymorphism Phenotyp-
ing v2) and SIFTS human protein v1.03 software. Results: 79% of the cohort was homozygous and 21%
were compound heterozygous. The mutation c.901G>T was
the most frequent mutation with 74% of the alleles 10,5%
followed by mutation c.1156C>T. In addition, 1 novel
mutation was described in c.214T>A predictive analysis
identify it as pathogenic variant. Conclusion: This study reveals the mutation spectrum of
MPS IVA in the Colombian population. P06.49A P06.49A
Biallelic missense and deep intronic NDUFAF6 variants,
unraveled by exome sequencing and mRNA analysis, in
patients with Leigh syndrome A. Catania1, A. Ardissone1, D. Verrigni2, A. Legati1, A. Reyes3,
E. Lamantea1, D. Diodato2, I. Moroni1, E. Bertini2,
A. Robinson3, R. Carrozzo2, M. Zeviani3, D. Ghezzi1,4 A. Catania1, A. Ardissone1, D. Verrigni2, A. Legati1, A. Reyes3, A. Catania1, A. Ardissone1, D. Verrigni2, A. Legati1, A. Reyes3, E. Lamantea1, D. Diodato2, I. Moroni1, E. Bertini2, P06.46B The mutation
spectrum data for MPS IVA disorder in the Colombian
population are not yet completely characterized. The high
prevalence of the c.901G>T mutation suggest it is a founder
effect cause diseases in this particular region, and In
addition as a migration process in the Andean region. This
spectrum data will be useful in the provision of better
genetic counseling, and prenatal diagnosis. Results: When analyzed in comparison to a control
cohort of unaffected cases (n = 100) and a cohort of cases
diagnosed with syndromes other than ML-IV (n = 100), the
ML-IV cohort showed a mean area-under-the-curve (AUC)
of 0.77 (SD, 0.19) and 0.87 (SD, 0.05), respectively. Conclusions: We describe for the first time recognizable
facial features typical in patients with ML-IV. Reaffirmed
by the objective FDNA technology, the described common
facial gestalt adds to the tools currently available for
clinicians and may thus assist in reaching an earlier
diagnosis of this rare and underdiagnosed disorder. H. Pachajoa: None. E. Candelo: None. G. Caicedo:
None. G. Porras:
None. D. Ramirez:
None. L. Diaz: None. B. Pode-Shakked: None. Y. Levi: None. N. Fleischer:
A. Employment (full or part-time); Significant; FDNA Inc. L. Wolf: A. Employment (full or part-time); Significant;
FDNA Inc. E. Ownership Interest (stock, stock options,
patent or other intellectual property); Significant; FDNA
Inc. Y. Finezilber: None. L. Greenbaum: None. S. Putter:
None. A. Raas-Rothschild: None. Molecular Characterisationof MPS IVA patients in Andean
region of Colombia Molecular Characterisationof MPS IVA patients in Andean
region of Colombia 1Foundation IRCCS Neurological Institute Besta, Milan, Italy,
2“Bambino Gesù” Children’s Hospital, IRCCS, Rome, Italy,
3Medical Research Council - Mitochondrial Biology Unit,
University of Cambridge, Cambridge, United Kingdom,
4University of Milan, Milan, Italy H. Pachajoa1, E. Candelo1, G. Caicedo1, G. Porras2,
D. Ramirez1, L. Diaz1 1Universidad Icesi, Cali, Colombia, 2COMFAMILIAR Clinic,
Pereira, Colombia P06.50B 1Laboratory of Genomic Diagnostics, Molecular Medicine
centre, Department of Medical Chemistry and biochemistry,
Medical Faculty, MU- Sofia, Sofia, Bulgaria, 2Laboratory of
Genomic Diagnostics, Molecular Medicine centre, Department
of Medical chemistry and biochemistry, Department of Medical
Genetics, Medical Faculty, MU- Sofia, Sofia, Bulgaria,
3Pediatric clinic „Alexandrovska University Hospital“,
Medical University of Sofia, Sofia, Bulgaria, 4Department of
Clinical genetics, Specialized Hospital for active treatment of
pediatric diseases “Prof. Ivan Mitev”, Sofia, Sofia, Bulgaria P06.51C Targeted next generation sequencing utility in diagnosis of
complex metabolic and neurological disorders D. L. Kachakova1, K. Mihova1, I. Popov1, I. Dimova2,
E. Simeonov3, A. Kadum4, I. Kremensky1, V. Mitev1, R. P. Kaneva1 1Universidad Icesi, Cali, Colombia, 2COMFAMILIAR Clinic,
Pereira, Colombia Introduction: NADH dehydrogenase complex I assembly
factor 6 (NDUFAF6) gene encodes a mitochondrial protein
which is essential for early assembly stages of mitochon-
drial respiratory complex I. Biallelic mutations in NDU-
FAF6 have been identified as responsible for cases of
autosomal
recessive
Leigh
syndrome
associated
with
mitochondrial complex I deficiency. Patients and Methods:
we studied two siblings and two unrelated subjects with
Leigh syndrome. Whole exome sequencing (WES), quan-
titative PCR and sequencing on NDUFAF6 transcript were
performed on patients’ biological specimens Introduction: Mucopolysaccharidoses (MPS) are a group
of inherited metabolic lysosomal storage disorders. A sub-
group of this is Morquio disease, an autosomal recessive
condition which overall incidence is 0.68 per 100,000 live
births. In Colombia, studies suggest that MPS IVA is likely
the highest prevalence worldwide. Materials and Methods: Sixteen families and nineteen
patients from a different region of the country were tested
for mutation identification, the sequence was compared to
the GALNS reference sequence NM_000512.4, and gene Abstracts from the 51st European Society of Human Genetics Conference: Posters 189 had a genetic diagnosis identified, resulting in improved
treatment for 620 patients. Results: By WES, we found a variant in NDUFAF6
(c.532G>C:p.A178P) in two siblings and a singleton
unrelated subject, all affected by Leigh syndrome. The
same missense mutation was recently described in a patient
presenting Leigh syndrome and complex I deficiency,
associated with an almost monoallelic expression of the
mutated allele at transcriptional level; nevertheless, the
second pathogenic mutation remained unidentified. Here we
provide evidence that the second allelic mutation consists of
a deep intronic variant present in all affected individuals,
including the previously published case. Through mRNA
analysis we demonstrated that the identified intronic
mutation is responsible for the formation of an alternative
splice site, leading to production of an aberrant transcript. Results: By WES, we found a variant in NDUFAF6
(c.532G>C:p.A178P) in two siblings and a singleton
unrelated subject, all affected by Leigh syndrome. The
same missense mutation was recently described in a patient
presenting Leigh syndrome and complex I deficiency,
associated with an almost monoallelic expression of the
mutated allele at transcriptional level; nevertheless, the
second pathogenic mutation remained unidentified. Here we
provide evidence that the second allelic mutation consists of
a deep intronic variant present in all affected individuals,
including the previously published case. 1Universidad Icesi, Cali, Colombia, 2COMFAMILIAR Clinic,
Pereira, Colombia Through mRNA
analysis we demonstrated that the identified intronic
mutation is responsible for the formation of an alternative
splice site, leading to production of an aberrant transcript. Further testing by whole-genome (N=69) or whole-
exome (N=24) sequencing defined the genetic diagnosis in
34 individuals and identified 14 novel causes of NDM. Eleven patients had pathogenic variants in disease genes for
which neonatal hyperglycaemia had not been previously
reported as part of the phenotype (COQ2, COQ9, LPL,
OXCT1, NARS2, TARS2). Pathogenic variants in four genes
thought to cause diseases unrelated to NDM were found in
69 patients in our cohort (GATA6, STAT3, LRBA, WFS1). Four novel disease genes were identified in 14 patients. Our integrated diagnostic and research approach defined
the genetic cause of NDM in 88.7% of patients (with
improved treatment for 19%) and identified 14 novel
genetic causes of NDM. Conclusions: A detailed analysis of whole exome
sequencing data together with functional validation based
on mRNA analysis may reveal pathogenic variants even in
non-exonic regions. Acknowledgments: Telethon Grant
GGP15041; Mariani Foundation; MRC-QQR (2015-2020)
grant; the ERC advanced grant FP7-322424, NRJ-Institut
de France grant; Italian Ministry of Health. The Telethon
Biobank (grant GTB12001J) supplied biological specimens. Conclusions: A detailed analysis of whole exome
sequencing data together with functional validation based
on mRNA analysis may reveal pathogenic variants even in
non-exonic regions. Acknowledgments: Telethon Grant
GGP15041; Mariani Foundation; MRC-QQR (2015-2020)
grant; the ERC advanced grant FP7-322424, NRJ-Institut
de France grant; Italian Ministry of Health. The Telethon
Biobank (grant GTB12001J) supplied biological specimens. Conclusions: A detailed analysis of whole exome
sequencing data together with functional validation based
on mRNA analysis may reveal pathogenic variants even in
non-exonic regions. Acknowledgments: Telethon Grant
GGP15041; Mariani Foundation; MRC-QQR (2015-2020)
grant; the ERC advanced grant FP7-322424, NRJ-Institut
de France grant; Italian Ministry of Health. The Telethon
Biobank (grant GTB12001J) supplied biological specimens. A. Catania: None. A. Ardissone: None. D. Verrigni:
None. A. Legati: None. A. Reyes: None. E. Lamantea:
None. D. Diodato: None. I. Moroni: None. E. Bertini:
None. A. Robinson: None. R. Carrozzo: None. M. Zeviani: None. D. Ghezzi: None. E. De Franco: None. M.N. Wakeling: None. M.B. Johnson: None. S.E. Flanagan: None. S. Ellard: None. A. T. Hattersley: None. Integration of research within clinical care identifies 14
novel genetic causes of neonatal diabetes Integration of research within clinical care identifies 14
novel genetic causes of neonatal diabetes E. De Franco, M. N. Wakeling, M. B. Johnson, S. E. Flanagan,
S. Ellard, A. T. Hattersley University of Exeter Medical School, Exeter, United Kingdom P06.52D M. M. Peić1, N. Maksimović1, V. Vidović2, S. Vidovic2, B. Jekić1,
T. Damnjanović1, M. Grk1, M. Duanović Pjević1, I. Novaković1 University of Exeter Medical School, Exeter, United Kingdom In the second patient with epilepsy
and progressive muscular hypotonia a new pathogenic
frameshift variant in SLC16A2 gene mutation was found
leading to Allan-Herndon-Dudley syndrome. Conclusions: This approach provided a diagnosis in
about 50% of patients in whom clinical and laboratory
evaluation did not allowed to identify a single candidate
gene, overtaking genetic heterogeneity and clinical varia-
bility. Remarkably, a diagnosis was established in 33% of
patients belonging to the no candidate gene class. Our study
shows that NGS technique is cost-effective compared to
Sanger sequencing of multiple genes, and represents a
powerful tool for the diagnosis of inborn errors of
metabolism presenting with persistent hypoglycemia Conclusions: Our results prove that tNGS is cost
effective and efficient method, which allows a more precise
diagnosis to be made for many complex disorders and could
be considered earlier in the diagnostic practice. Acknowl-
edgements: The research was supported by grant DUNK01-
2/2009 by NSF, Ministry of Education and Science. E. Ponzi: None. A. Maiorana: None. F. Lepri: None. M. Mucciolo: None. A. Novelli: None. C. Dionisi-
Vici: None. D.L. Kachakova: None. K. Mihova: None. I. Popov:
None. I. Dimova: None. E. Simeonov: None. A. Kadum:
None. I. Kremensky: None. V. Mitev: None. R.P. Kaneva: None. P06.53A Polymorphisms in PPARG gene: association with obesity-
related metabolic traits in a Serbian adolescent population University of Exeter Medical School, Exeter, United Kingdom Neonatal diabetes (NDM) is diagnosed before 6 months of
age and has >25 known genetic causes. Since 2000 the
Exeter laboratory has offered free genetic testing to anyone
diagnosed with NDM. Testing of all known genetic causes
of NDM using Sanger sequencing, methylation-specific-
MLPA and targeted next-generation-sequencing is per-
formed by the Exeter Genetics laboratory using the same
pipeline and quality parameters of diagnostic tests. Patients
without a genetic diagnosis are tested by whole-exome/
genome sequencing as part of the novel genetic aetiologies
research study. Introduction: Metabolic and neurological conditions are a
huge group of disorders and syndromes, characterized by
clinical variability and extreme genetic heterogeneity. Tar-
geted next generation sequencing (tNGS) provides possi-
bility for precise genetic diagnosis of metabolic and
neurological disorders. Materials and methods: Six patients with metabolic and/
or neurological symptoms were referred in 2016/2017 to the
Laboratory of Genome Diagnostics for performing tNGS. The analysis was implemented on MiSeq Illumina with
TruSight One kit. Resutls: In one of the patients with
neurodegenerative disease and congenital defect in glyco-
sylation we did not find clinically relevant variants leading
to the observed phenotype. In two of the patients with We assessed the success rate of this approach in terms of
1) the proportion of patients with a genetic diagnosis
identified; 2) how many novel genetic causes were found. 1673 NDM patients referred from 93 countries had
comprehensive genetic testing. 1484 individuals (88.7%) We assessed the success rate of this approach in terms of
1) the proportion of patients with a genetic diagnosis
identified; 2) how many novel genetic causes were found. 1673 NDM patients referred from 93 countries had
comprehensive genetic testing. 1484 individuals (88.7%) 190 J. del Picchia Results: A proven diagnosis was obtained in 78% of
patients in SCG, in 49% in MCG and in 33% in NCG. The
diagnostic yield was 48% for HI, 66% per FAOD and
ketogenesis defects, 59% for GSDs and other carbohydrate
disorders, and 67% for mitochondrial disorders. clinical diagnosis glycogenosis type IXa and methylmalonic
acidemia genetic testing confirmed the diagnosis and we
found mutations in PHKA2 and ММАА respectively. The
mutation in PHKA2 was new frameshift variant. In two
patients with epilepsy the genetic testing clarified the
diagnosis. In one of these patients homozygous pathogenic
mutation leading to severe reduction of PRODH activity
was found. The test changed the diagnosis from epilepsy to
hyperpolinemia, type I. Persistent hypoglycemia in children: contribution of NGS
in diagnosis of inborn errors of metabolism 1Institute of Human Genetics, Belgrade, Serbia, 2Faculty of
Medicine, University of Banja Luka, Banja Luka, Bosnia and
Herzegovina 1Institute of Human Genetics, Belgrade, Serbia, 2Faculty of
Medicine, University of Banja Luka, Banja Luka, Bosnia and
Herzegovina E. Ponzi1, A. Maiorana1, F. Lepri2, M. Mucciolo2, A. Novelli2,
C. Dionisi-Vici1 E. Ponzi1, A. Maiorana1, F. Lepri2, M. Mucciolo2, A. Novelli2,
C. Dionisi-Vici1 1Metabolic Unit, Department of Specialist Pediatrics, Bambino
Gesù Children's Hospital, Rome, Italy, 2Medical genetics
laboratory department, Bambino Gesù Children’s Hospital,
Rome, Italy 1Metabolic Unit, Department of Specialist Pediatrics, Bambino
Gesù Children's Hospital, Rome, Italy, 2Medical genetics
laboratory department, Bambino Gesù Children’s Hospital,
Rome, Italy Introduction:
The
peroxisome
proliferator-activated
receptor γ (PPAR-γ) is a candidate gene for obesity and
type 2 diabetes mellitus. Protein product PPAR-γ is a lipid-
activated transcription factor that has a main role in the
expression of genes involved in adipocyte differentiation
and function. Although the association of a common single
nucleotide polymorphism (SNP) of the PPAR-γ gene,
Pro12Ala (34C>G, rs1801282), with obesity has been
reported in various populations, these data are not con-
clusive. Similarly,
conflicting
results,
were
obtained
regarding the association between metabolic phenotypes
and another frequent variant, a silent mutation of the PPAR-
γ gene, His477= (1431C>T, rs3856806). Introduction: Hypoglycemia is an important cause of
pediatric morbidity and is often due to inborn errors of
metabolism (IEM) that present with clinical, biochemical
and genetic heterogeneity. We have developed a rapid and
accurate strategy for the molecular diagnosis of hypogly-
cemia- associated IEMs. Materials and Methods: 64 patients were tested through
a custom gene panel of 65 genes, which included five
disease categories: hyperinsulinemic hypoglycemia (HI),
fatty acid-oxidation (FAOD) and ketogenesis defects,
ketolysis defects, glycogen storage diseases (GSDs) and
other disorders of carbohydrate metabolism, and mitochon-
drial disorders. Molecular data were compared with clinical
and biochemical data. Patients, investigated through an
extensive workup, were divided in 3 diagnostic classes: a)
single candidate gene (SCG-9/64), b) multiple candidate
genes (MCG-43/64) and c) no candidate gene (NCG-12/
64). This study aimed to estimate whether the PPAR-γ
rs1801282 and PPAR-γ rs3856806 SNPs are linked with
obesity-related metabolic traits in a Serbian adolescent
population. This study aimed to estimate whether the PPAR-γ
rs1801282 and PPAR-γ rs3856806 SNPs are linked with
obesity-related metabolic traits in a Serbian adolescent
population. Functional genomics' studies are mandatory for clarifying
pathogenicity of novel genetic variants detected by NGS in
OXPHOS disorders In all cases, the functional genomics’ approach was
essential for clarifying pathogenicity, with implications for
genetic counselling. M. Grazina1,2, M. Simões1, M. Bacalhau1,2, C. Ribeiro1, M. J. Santos1,2, M. C. Macário3, J. Durães3, L. Diogo3, P. Garcia3,
M. Rocha4, A. Vincent4, S. Hardy4, A. C. Rego1,2, H. Girão5,
L. Wong6, R. W. Taylor4 Financial Support: Feder funds through the Operational
Competitiveness Program – COMPETE2020. Financial Support: Feder funds through the Operational
Competitiveness Program – COMPETE2020. M. Grazina: None. M. Simões: None. M. Bacalhau:
None. C. Ribeiro: None. M.J. Santos: None. M.C. Macário: None. J. Durães: None. L. Diogo: None. P. Garcia: None. M. Rocha: None. A. Vincent: None. S. Hardy: None. A.C. Rego: None. H. Girão: None. L. Wong: None. R.W. Taylor: None. 1Center for Neuroscience and Cell Biology, Coimbra,
Portugal, 2Faculty of Medicine, University of Coimbra,
Coimbra, Portugal, 3Reference Centre of Inherited Metabolic
Diseases, MetabERN - Coimbra Hospital and Universitary
Centre, Coimbra, Portugal, 4Wellcome Centre for
Mitochondrial Research, Institute of Neuroscience, The
Medical School, Newcastle University, Newcastle Upon Tyne,
United Kingdom, 5iCBR - Institute for Biomedicine and
Research, University of Coimbra, Coimbra, Portugal,
6Mitochondrial Diagnostic Laboratory, Baylor College of
Medicine, Houston, TX, United States Hardy: None. A.C. Rego: None. H. Girão: None. L. Wong: None. R.W. Taylor: None. P06.54B Functional genomics' studies are mandatory for clarifying
pathogenicity of novel genetic variants detected by NGS in
OXPHOS disorders Persistent hypoglycemia in children: contribution of NGS
in diagnosis of inborn errors of metabolism Materials and Methods: Anthropometric and biochem-
ical parameters were measured in 84 adolescent patients at
the age of 15, whose BMI was over 85th percentile. Body
mass index (BMI), fasting glucose, triglyceride, systolic and
diastolic blood pressure and total cholesterol were mea-
sured. Subjects were genotyped for Pro12Ala and 1431C>T Materials and Methods: Anthropometric and biochem-
ical parameters were measured in 84 adolescent patients at
the age of 15, whose BMI was over 85th percentile. Body
mass index (BMI), fasting glucose, triglyceride, systolic and
diastolic blood pressure and total cholesterol were mea-
sured. Subjects were genotyped for Pro12Ala and 1431C>T 191 Abstracts from the 51st European Society of Human Genetics Conference: Posters SNPs by PCR-restriction fragment length polymorphism
analysis. Patient 2. Epileptic encephalopathy (8y, male); moderate
OXPHOS decrease and novel detected homozygous muta-
tion (FASTKD2). Functional analyses revealed decreased
protein expression and respiratory rate/ATP production,
with increased glycolytic capacity (fibroblasts). Results: The Pro12Ala variant was associated with
higher diastolic blood pressure in male subjects carrying
G allele (p = 0,041). The 1431C>T variant was associated
with higher systolic blood pressure in male subjects
carrying T allele (p = 0,002). Patient
3. Case of CPEO
(62y,
female);
multiple
OXPHOS deficiencies (muscle) and two mtDNA alterations
(m.7486G>A, MT-TS1; mt-tRNASer(UCN), 4,977bp deletion)
[Bacalhau et al. Neuromuscular Disorders, in press]. Diverse functional studies unveiled the energy failure
cellular causes. Conclusions: Results of our preliminary study indicate
that a rare variant Pro12Ala (allele G), and 1431C>T rare
variant (allele T) appear as risk factors for higher diastolic
and systolic blood pressure, respectively, in overweight and
obese male adolescents. Conclusion:
We
present
three
cases
of
OXPHOS
diseases due to different genetic causes: 1 novel nuclear
mutation in a gene affecting a known protein (surfeit 1,
assembly factor of complex IV); 1 novel nuclear mutation
in a gene affecting a recently known protein; 2 mtDNA
alterations (1 known, 1 unclassified). Limitations of NGS in
mutation detection and evaluation of OXPHOS activity as
the only functional parameter are addressed. M.M. Peić: None. N. Maksimović: None. V. Vidović:
None. S. Vidovic: None. B. Jekić: None. T. Damnjanović:
None. M. Grk: None. M. Duanović Pjević: None. I. Novaković: None. Sequential approach to identify disease causing variants in
patients with mitochondrial dysfunction of a Hungarian
cohort For interpretation, the Exomiser algorithm was
applied at first, using variant and HPO descriptors, to rank
variants based on predicted pathogenicity and semantic
similarity to known phenotypes. Secondly, multi-gene-
panel analysis was focused to disease-related genes that are
presumptive to the pathogenesis (e.g. neurodegeneration). The ranked variants were further filtered using in-house data
warehouse. After screening known causal variants of can-
didate genes a large number of annotated features were
considered to fine-tune the hypothesis about causality of a
given variant to reach cc. 30% diagnostic rate. To assess
candidates, item-level similarity of phenotypic terms and
reverse-phenotyping of hallmark symptoms were often
needed to complement clinical data with the typical, but
routinely not tested manifestations of the possible inherited
disorder. phenylalanine (Phe) levels: classic, moderate and mild
PKU, and mild hyperphenylalaninemia (MHP). For each
genotypic combination, the predicted phenylalanine hydro-
xylase (PAH) residual activity (PRA) and the sum of
arbitrary assigned values (AV) were determined. Genotype-
based predictions of responsiveness to BH4 were also
analyzed. Results: A strong relationship between genotypic sever-
ity, according to the level of PRA, and the inverse of pre-
treatment Phe levels was observed (t = 4.79, P<0.0001). The
observed
phenotype
matched
the
AV
predicted
phenotype in 48% of the cases. A BH4-responsiveness rate
of 37.0% was estimated. Results: A strong relationship between genotypic sever-
ity, according to the level of PRA, and the inverse of pre-
treatment Phe levels was observed (t = 4.79, P<0.0001). The
observed
phenotype
matched
the
AV
predicted
phenotype in 48% of the cases. A BH4-responsiveness rate
of 37.0% was estimated. Conclusions: The high degree of discordance found
when the AV sum prediction system was employed fell to
33% once the phenotypes reported at the BIOPKU database
were also considered. We estimated that 81% of the patients
were potential candidates for a BH4 loading test. Analyzing
the functionally hemizygous patients, we found that the
moderate common mutations, p.R261Q, p.V388M, and p. I65T contributed decisively to the high genotype-phenotype
discordance. Despite these discrepancies, genotype con-
tinues to be the main determinant of metabolic outcome in
most patients with PKU, anticipating their dietary needs and
BH4 responsiveness. V. Molnár: None. A. Gézsi: None. A. Illés: None. P. Balicza: None. B. Berta: None. D. Csabán: None. I.J. Jimoh: None. A. Gál: None. M.J. Molnár: None. E. Vieira Neto: B. P06.56D Genotype-phenotype correlations and BH4 predicted
responsiveness in patients with phenylketonuria from Rio
de Janeiro, Southeast Brazil E. Vieira Neto1,2, F. Laranjeira3, D. Quelhas3, I. Ribeiro3,
A. Seabra3, N. Mineiro3, L. M. Carvalho4, L. Lacerda3, M. G. Ribeiro2 E. Vieira Neto1,2, F. Laranjeira3, D. Quelhas3, I. Ribeiro3,
A. Seabra3, N. Mineiro3, L. M. Carvalho4, L. Lacerda3, M. G. Ribeiro2 Sequential approach to identify disease causing variants in
patients with mitochondrial dysfunction of a Hungarian
cohort Sequential approach to identify disease causing variants in
patients with mitochondrial dysfunction of a Hungarian
cohort V. Molnár, A. Gézsi, A. Illés, P. Balicza, B. Berta, D. Csabán,
I. J. Jimoh, A. Gál, M. J. Molnár
Institute of Genomic Medicine and Rare Disorders,
Semmelweis University, Budapest, Hungary V. Molnár, A. Gézsi, A. Illés, P. Balicza, B. Berta, D. Csabán,
I. J. Jimoh, A. Gál, M. J. Molnár V. Molnár, A. Gézsi, A. Illés, P. Balicza, B. Berta, D. Csabán,
I. J. Jimoh, A. Gál, M. J. Molnár Institute of Genomic Medicine and Rare Disorders,
Semmelweis University, Budapest, Hungary Institute of Genomic Medicine and Rare Disorders,
Semmelweis University, Budapest, Hungary Introduction:
Genetic
causes
of
OXPHOS
disorders
include mutations in mitochondrial and/or nuclear genomes. Introduction:
Genetic
causes
of
OXPHOS
disorders
include mutations in mitochondrial and/or nuclear genomes. With the significant increase of genetic diagnoses following
NGS techniques, demonstrating the pathogenicity of novel
variants became a challenging issue. With the significant increase of genetic diagnoses following
NGS techniques, demonstrating the pathogenicity of novel
variants became a challenging issue. Genetic diagnosis of disorders with mitochondrial dys-
function is often challenging due to the phenotypic varia-
bility and considerable genetic heterogeneity. Here we
would share our experiences on analysis of 192 individual
WES data using phenotype-driven prioritization or candi-
date genes-based filtering. Patients with positive family
history were enrolled if targeted genetic tests failed to yield
conclusive results. Multisystemic and neurodegenerative
diseases were preferred with high-quality health records. Phenotype profiles were reconstructed from clinical doc-
umentation using HPO (#14.5+/-10.7terms/patient). The We present 3 patients with novel genetic variants in
whom functional studies were mandatory for illuminating
pathogenicity. Case reports: Patient 1. Leigh syndrome (40y, male);
complex IV deficiency (29.7% activity) and novel homo-
zygous deletion (c.-11_13del, SURF1 gene) [Ribeiro et al. Mitochondrion31(2016)84–88], detected by Sanger sequen-
cing and not by NGS, demonstration of complex IV
assembly (fibroblasts). J. del Picchia 192 selected cases were classified into disease-groups based on
the main presenting symptoms: 27%mitochondrial/multi-
systemic disease, 12%early-onset Parkinson, 7%dementia
and 11%NBIA/dystonia syndromes. Besides these groups,
24% unclassified and 19% were used as unaffected relative
or healthy control; 128 as singletons, 64 individuals as
members of 26 examined families were analyzed, respec-
tively. WES was performed on Illumina HiSeq2500 plat-
form. Sequential approach to identify disease causing variants in
patients with mitochondrial dysfunction of a Hungarian
cohort Research Grant (principal investiga-
tor, collaborator or consultant and pending grants as well as
grants already received); Modest; Coordination for the
Improvement of Higher Level Personnel (Capes) of the
Ministry of Education, Brazil. F. Laranjeira: None. D. Quelhas: None. I. Ribeiro: None. A. Seabra: None. N. Mineiro: None. L.M. Carvalho: None. L. Lacerda: None. M.G. Ribeiro: None. Mutation analysis of the PAH gene in phenylketonuria
patients from Rio de Janeiro, Southeast Brazil Mutation analysis of the PAH gene in phenylketonuria
patients from Rio de Janeiro, Southeast Brazil 1Agência Nacional de Saúde Suplementar, Rio de Janeiro,
Brazil, 2Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil, 3Centro de Genética Médica Doutor Jacinto
Magalhães, Porto, Portugal, 4Instituto Estadual de Diabetes e
Endocrinologia Luiz Capriglione, Rio de Janeiro, Brazil E. V. Neto1,2, F. E. R. Laranjeira3, D. Quelhas3,4, A. Seabra3,5,
N. Mineiro3, I. Ribeiro3,4, L. M. Carvalho6, L. Lacerda3, M. G. Ribeiro2 1Agência Nacional de Saúde Suplementar, Gerência de
Monitoramento Assistencial, Rio de Janeiro, Brazil,
2Universidade Federal do Rio de Janeiro, Instituto de
Puericultura e Pediatria Martagão Gesteira, Serviço de
Genética Médica, Rio de Janeiro, Brazil, 3Unidade de
Bioquímica Genética, Centro Genética Médica Jacinto
Magalhães, Centro Hospitalar Porto, Porto, Portugal,
4Unidade Multidisciplinar de Investigação Biomédica, Porto,
Portugal, 5Faculdade de Ciências / Instituto de Ciências
Biomédicas Abel Salazar, Universidade do Porto, Porto, Introduction: The clinical phenotypes of phenylketonuria
(PKU) are highly variable. This has been attributed to
genetic heterogeneity and frequent compound hetero-
zygosis. The correlations between phenotypic character-
istics, BH4 predicted responsiveness, and the causative
mutations found in PKU patients from Rio de Janeiro,
Brazil, were evaluated. Materials and Methods: A total of 102 completely
genotyped patients were included. They were assigned to
one of the following phenotypes according to pre-treatment 193 Abstracts from the 51st European Society of Human Genetics Conference: Posters Portugal, 6Instituto de Diabetes e Endocrinologia Luiz
Capriglione, Serviço de Metabologia, Rio de Janeiro, Brazil Portugal, 6Instituto de Diabetes e Endocrinologia Luiz
Capriglione, Serviço de Metabologia, Rio de Janeiro, Brazil 4Kawsar Human Genetics research Center, Tehran, Iran,
Islamic Republic of, 5Department of Medical Genetics and
Molecular Biology, School of Medicine, Iran University of
Medical Sciences, Tehran, Iran, Islamic Republic of Phenylketonuria (PKU) is an autosomal recessive disease
resulting from mutations in the PAH gene. Most of the
patients are compound heterozygotes, and the combination
of mutations is a major factor in determining the phenotypic
differences found among them. The mutational spectrum of
PKU in the state of Rio de Janeiro, Southeast Brazil, was
analyzed by sequencing the PAH gene from genomic DNA
of 102 patients. Deletions and duplications were also
screened using MLPA analysis. Both mutated alleles were
identified in all patients. Nine (8.8%) homozygous and 93
(91.2%) compound heterozygous patients were found. The
spectrum included 37 causative mutations, including a new
mutation - p.G312C. PMM2-CDG patients gestalt: is recognizable enough? PMM2-CDG patients gestalt: is recognizable enough? A. Martinez-Monseny, M. Bolasell, C. Arjona, N. Fleischer,
F. Palau, M. Serrano E.V. Neto: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; FBM Indústria Farm-
acêutica Lda., Anápolis, Goiás, Brasil, Danone Lda., São
Paulo, Brasil. F.E.R. Laranjeira: None. D. Quelhas:
None. A. Seabra: None. N. Mineiro: None. I. Ribeiro:
None. L.M. Carvalho: None. L. Lacerda: None. M.G. Ribeiro: None. E.V. Neto: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; FBM Indústria Farm-
acêutica Lda., Anápolis, Goiás, Brasil, Danone Lda., São
Paulo, Brasil. F.E.R. Laranjeira: None. D. Quelhas:
None. A. Seabra: None. N. Mineiro: None. I. Ribeiro:
None. L.M. Carvalho: None. L. Lacerda: None. M.G. Ribeiro: None. Sant Joan de Deu Hospital, Barcelona, Spain P06.58B P06.58B
Molecualr genetics of a cohort of more than 770 cases of
PKU in a consanguineous population 1Pasteur Institute of Iran, Tehran, Iran, Islamic Republic of,
2Kawsar Human Genetics Research Center, Tehran, Iran,
Islamic Republic of, 3Department of Dermatology and
Cutaneous Biology, Sidney Kimmel Medical College, Thomas
Jefferson University,, Philadelphia, PA, United States, Mutation analysis of the PAH gene in phenylketonuria
patients from Rio de Janeiro, Southeast Brazil Missense, nonsense, and splicing
variants corresponded to 63.7%, 2.9% and 22.6% of the
mutant alleles, respectively. Large (1.5%), and small dele-
tions, inframe (5.4%) and with frameshift (3.9%), com-
prised the remainder. The most frequent mutations were: p. V388M
(12.7%),
p.R261Q
(11.8%),
IVS10-11G>A
(10.3%), IVS2+5G>C (6.4%), p.S349P (6.4%), p.R252W
(5.4%), p.I65T (4.4%), and p.T323del (4.4%). The Iberian
Peninsula, especially Portugal, is the major source of PAH
mutations in Rio de Janeiro. Mutations that have other
geographical origins, such as IVS2+5G>C, p.G352Vfs*48,
and IVS12+1G>A were also detected. Genetic drift and
founder effect may be responsible for the high frequencies
of the mutations p.S349P and p.T323del in this population. Grant from "Coordination for the Improvement of Higher
Level Personnel (Capes) of the Ministry of Education,
Brazil’ Phenylketonuria (PKU) is an inborn error of amino acid
metabolism caused by mutations in phenylalanine hydro-
xylase (PAH). Herein, we reported that among the 772
patients diagnosed with PKU, there are 635 classic (82%)
and 137 previously reported non-classic subtypes (18%)
from all ethnicities of 31 provinces in Iran. The disease
causing mutations were found in 611 out of 635 classic
(with a diagnostic detection rate of 96%) and in 97 out of
137 non-classic patients (with a diagnostic detection rate of
71%). To the best of our knowledge, this report is the most
comprehensive study of the molecular genetics of PKU in
Iran, identifying 100 distinct mutations in the PAH gene,
including 15 previously unreported mutations. Interestingly,
we found unique cases of PKU with uniparental disomy,
germline mosaicism and co-inheritance with another Men-
delian single-gene disorder that provides new insights for
improving the genetic counseling, prenatal diagnosis (PND)
and/or pre-implantation genetic diagnosis (PGD) for inborn
error of metabolism group of disorders. S. Zeinali: None. T. Shirzad: None. A.H. Saeidian:
None. H. Bagherian: None. M. Abiri: None. Sant Joan de Deu Hospital, Barcelona, Spain Background: Phosphomanomutase deficiency (PMM2-
CDG, MIM#212065) is the most frequent congenital dis-
order of glycosylation. Initially PMM2-CDG was defined as
a combination of systemic involvement, developmental
delay due to cerebellar atrophy, peculiar fat pads and
inverted nipples. It seems to be mild and not specific dys-
morphic facial features that may change with age. We aim
to describe the dysmorphic facial traits and draw a recog-
nizable facial pattern. S. Zeinali1, T. Shirzad2, A. H. Saeidian3, H. Bagherian4,
M. Abiri5 A. Martinez-Monseny, M. Bolasell, C. Arjona, N. Fleischer,
F. Palau, M. Serrano P06.60D RNASeq profiling of Pompe disease patients reveals a
compensatory transcriptional response centered around
mTOR inhibition A. Eliyahu1,2, M. Christine V.Malicdan3,4, T. Vilboux4,5, B. Ben-
Zeev6,2, B. Pode-Shakked1,2,7, A. Dori7,2,8, N. Shelestovich2,9,
D. Marek-Yagel1,2, H. Pri-Chen4,10, I. Blatt11, L. He12,
Y. Anikster1,2,13 A. Eliyahu1,2, M. Christine V.Malicdan3,4, T. Vilboux4,5, B. Ben-
Zeev6,2, B. Pode-Shakked1,2,7, A. Dori7,2,8, N. Shelestovich2,9,
D. Marek-Yagel1,2, H. Pri-Chen4,10, I. Blatt11, L. He12,
Y. Anikster1,2,13 A. Gheldof1, S. Seneca1, K. Stouffs1, L. Vo Ngoc1, A. Jansen2,
M. De Rademaeker1, H. Laeremans3, A. Jonckheere4, L. De
Meirleir5 A. Gheldof1, S. Seneca1, K. Stouffs1, L. Vo Ngoc1, A. Jansen2,
M. De Rademaeker1, H. Laeremans3, A. Jonckheere4, L. De
Meirleir5 1Metabolic Disease Unit, Edmond and Lily Safra Children's
Hospital, Sheba Medical Center, Tel-Hashomer, Israel,
2Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv,
Israel, 3NIH Undiagnosed Diseases Program, Common Fund,
Office of the Director, NIH and National Human Genome
Research Institute, NIH, Bethesda, MD, United States,
4Medical Genetics Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD, United
States, 5Inova Translational Medicine Institute, Falls Church,
VA, United States, 6PediatricNeurology Unit, Edmond and Lily
Safra Children's Hospital, Sheba Medical Center, Tel-
Hashomer, Israel, 7The Dr. Pinchas Borenstein Talpiot
Medical Leadership Program, Sheba Medical Center, Tel-
Hashomer, Israel, 8Joseph Sagol Neuroscience Center, Sackler
Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel,
9Center for Mitochondrial and Epigenomic Medicine,
Children's Hospital of Philadelphia Research Institute,,
Philadelphia, PA, United States, 10Graduate Partnership
Program(GPP), National Institute of Health (NIH), Bethesda,
MD, United States, 11Department of Neurology, Sheba Medical
Center, Tel-Hashomer, Israel, 12Wellcome Centre for
Mitochondrial Research, Institute of Neuroscience, The
Medical School, Newcastle University, Newcastle upon Tyne,
United Kingdom, 13The Wohl Institute for Translational
Medicine, Sheba Medical Center, Tel-Hashomer, Israel 1Centre of Medical Genetics, UZBrussel, Brussels, Belgium,
2Centre Paediatric Neurology, UZBrussel, Brussels, Belgium,
3Flemish Center for Detection of Metabolic Diseases, Hôpital
Erasme, Brussels, Belgium, 4Centre of Paediatric Neurology,
UZAntwerpen, Antwerp, Belgium, 5Centre of Paediatric
Neurology, UZBrussel, Brussels, Belgium Pompe disease is a lysosomal storage disorder which is
caused by alpha-glucosidase deficiency and leads to accu-
mulation of glycogen in the lysosomes. The disease phe-
notype is mainly characterized by muscle weakness and
hypotonia, finally resulting in respiratory difficulties. While
the clinical manifestations of the disease have been well
documented, it remains unclear what the consequences of
the excessive glycogen storage are at the transcriptional
level. P06.58B
Molecualr genetics of a cohort of more than 770 cases of
PKU in a consanguineous population As far as
this application is open access and easily available, it is
considered a useful tool for daily clinical practice. Further
studies will study these dysmorphic features in early ages
and its correlation with neurological involvement and
genotype. A. Gheldof: None. S. Seneca: None. K. Stouffs: None. L. Vo Ngoc: None. A. Jansen: None. M. De Rademaeker:
None. H. Laeremans: None. A. Jonckheere: None. L. De
Meirleir: None. A. Martinez-Monseny: None. M. Bolasell: None. C. Arjona: None. N. Fleischer: None. F. Palau: None. M. Serrano: None. P06.58B
Molecualr genetics of a cohort of more than 770 cases of
PKU in a consanguineous population Methods: We evaluated the frequency of occurrence of
clinical symptoms and analysed the performance of facial
gestalt computer-assisted image analysis in PMM2-CDG
patients. We
used
the
Research
application
of
the
Face2Gene tool (FDNA Inc. Boston, MA, USA). It
generated a classification model on three groups of frontal
photos:
the
group-PMM2,
unaffected
and
Angelman
Syndrome, because it was the most frequent syndrome-
match offered by the automated tool. Methods: We evaluated the frequency of occurrence of
clinical symptoms and analysed the performance of facial
gestalt computer-assisted image analysis in PMM2-CDG
patients. We
used
the
Research
application
of
the
Face2Gene tool (FDNA Inc. Boston, MA, USA). It
generated a classification model on three groups of frontal
photos:
the
group-PMM2,
unaffected
and
Angelman
Syndrome, because it was the most frequent syndrome-
match offered by the automated tool. S. Zeinali1, T. Shirzad2, A. H. Saeidian3, H. Bagherian4,
M. Abiri5 S. Zeinali1, T. Shirzad2, A. H. Saeidian3, H. Bagherian4,
M. Abiri5 1Pasteur Institute of Iran, Tehran, Iran, Islamic Republic of,
2Kawsar Human Genetics Research Center, Tehran, Iran,
Islamic Republic of, 3Department of Dermatology and
Cutaneous Biology, Sidney Kimmel Medical College, Thomas
Jefferson University,, Philadelphia, PA, United States, 194 J. del Picchia and Regulation) gene network. In all tested Pompe patients
we observe a 6-10 fold transcriptional upregulation of the
DEPTOR mRNA, which is an inhibitor of the mTOR
pathway. Additionally, an mRNA downregulation of sev-
eral mTOR activating genes including GAS6, Rhes and
MC4R was detected as well, further hinting to an overall
inhibition of the mTOR pathway. These results suggest that
cells of Pompe patients initiate a compensatory response to
try to restore the disturbed lysosomal functionality. Whether
this cell intrinsic restoration mechanism is specific for
Pompe patients or is common for lysosomal storage dis-
eases in general remains to be investigated. Results: We included 34 patients, 20 boys and 14 girls,
aged 6-18, of Caucasian ethnicity. The confusion matrix
describing a multiclass comparison of actual class as
compared to a predicted class showed a true positive rate
for each group with values significantly better than a
random assignment of photos. The area under the receiver
operating characteristics curve (AUC of ROC) demon-
strated
values
p > 0,85
(p-value < 0,05)
in
all
binary
comparisons. Conclusions: Facial features of PMM2-CDG patients
present a recognizable and differentiated gestalt. P06.61A A biallelic novel mutation in the COQ5 C-
methyltransferase gene gives a diagnosis of a new sub-type
of primary CoQ10 deficiency A biallelic novel mutation in the COQ5 C-
methyltransferase gene gives a diagnosis of a new sub-type
of primary CoQ10 deficiency P06.63C P06.63C
RNA-seq of whole-blood in Asian extreme childhood obese
subjects indicates defects in oxidative phosphorylation and
mitochondrial pathways RNA-seq of whole-blood in Asian extreme childhood obese
subjects indicates defects in oxidative phosphorylation and
mitochondrial pathways RNA-seq of whole-blood in Asian extreme childhood obese
subjects indicates defects in oxidative phosphorylation and
mitochondrial pathways P06.60D For this purpose, we performed RNAseq on fibro-
blasts of four Pompe patients and compared this to four
controls. Transcriptional profiling revealed the presence of a
compensatory effect of the cells to increase lysosomal
functionality which seems to be centered around inhibition
of the mTOR (mammalian Target of Rapamycin) pathway. It has been shown that inhibition of the mTOR pathway
leads to nuclear localization of TFEB and subsequent acti-
vation of the CLEAR (Coordinated Lysosomal Expression 195 Abstracts from the 51st European Society of Human Genetics Conference: Posters Primary Coenzyme Q10 (CoQ10; MIM# 607426) defi-
ciencies are an emerging group of inherited Mitochondrial
disorders with heterogeneous clinical phenotype. Over a
dozen genes are involved in the biosynthesis of CoQ10, and
mutations in several of these are associated with human
disease. However, mutations in COQ5 (MIM# 616359),
catalyzing the only C-methylation in the CoQ10 Synthetic
pathway, have not been implicated in human disease. Here,
we report three female siblings of Iraqi-Jewish descent, who
had varying degrees of cerebellar ataxia, encephalopathy,
seizures, and cognitive disability. We describe the remark-
able mechanism of this molecular mutation and outline
some of the major challenges in diagnosis via molecular
analysis. In the cases described, a duplication in a non-
coding region of the CoQ5 gene was not diagnosable by
WES, and only through WGS with targeted focus of a
suspicious area, was it possible to reach a diagnosis of a
new disease with treatment potential. Duplications in the
COQ5 gene, lead to reduced levels of CoQ10 in peripheral
white blood cells of all affected individuals and reduced
CoQ10 levels in the only muscle tissue available from one
affected proband. CoQ10 supplementation led to clinical
improvement and increased the concentrations of CoQ10 in
blood. This is the first report of primary CoQ10 deficiency
caused by loss of function of COQ5, with delineation of the
clinical, laboratory, histological, and molecular features,
and
insights
regarding
targeted
treatment
with
CoQ10 supplementation. Early diagnosis of this disease
may be of great value for for successful treatment and
intervention strategies. an estimated total birth frequency of at least 1:500. In 2017,
the European Commission established the non-profit net-
work MetabERN, which connects 69 health care providers
in 18 EU countries and delivers care to approximately
43,000 patients with IMDs. An important instrument to
improve diagnosis, treatment and wellbeing of patients is a
systematic collection of data in a registry. P06.60D an estimated total birth frequency of at least 1:500. In 2017,
the European Commission established the non-profit net-
work MetabERN, which connects 69 health care providers
in 18 EU countries and delivers care to approximately
43,000 patients with IMDs. An important instrument to
improve diagnosis, treatment and wellbeing of patients is a
systematic collection of data in a registry. Project description: The Unified European Registry for
Inherited Metabolic Disorders (U-IMD) project started in
February 2018. The project has three major activities: a/to
establish a patient registry for the MetabERN based on the
common data elements of the European Platform on Rare
Disease Registration; U-IMD will be the first unified
European registry that encompasses all IMDs, b/to upgrade
already existing IMD registries to the standard of U-IMD,
starting with the registry of the International Working
Group on Neurotransmitter Related Disorders (iNTD) and
c/to develop a standard for minimal core data sets shared by
the MetabERN and the European Rare Kidney Disease
Reference Network (ERKNET). The diverse nature of the
heterogeneous etiological and clinical spectrum of the IMDs
necessitates collection of a minimal set of common data
elements and the usage of controlled and standardized
vocabularies such as Human Phenome Ontology or WHO
ATC classifications for the description of the clinical
phenotype and treatment strategies, respectively. Project description: The Unified European Registry for
Inherited Metabolic Disorders (U-IMD) project started in
February 2018. The project has three major activities: a/to
establish a patient registry for the MetabERN based on the
common data elements of the European Platform on Rare
Disease Registration; U-IMD will be the first unified
European registry that encompasses all IMDs, b/to upgrade
already existing IMD registries to the standard of U-IMD,
starting with the registry of the International Working
Group on Neurotransmitter Related Disorders (iNTD) and
c/to develop a standard for minimal core data sets shared by
the MetabERN and the European Rare Kidney Disease
Reference Network (ERKNET). The diverse nature of the
heterogeneous etiological and clinical spectrum of the IMDs
necessitates collection of a minimal set of common data
elements and the usage of controlled and standardized
vocabularies such as Human Phenome Ontology or WHO
ATC classifications for the description of the clinical
phenotype and treatment strategies, respectively. Acknowledgement: The U-IMD is supported by EU
project 777259 from CHAFEA. S. Kölker: None. F. Gleich: None. T. Opladen: None. M. Scarpa: None. C. Dionisi Vici: None. A. Garcia-
Cazorla: None. P06.60D V. Kozich: None. S. Kölker: None. F. Gleich: None. T. Opladen: None. M. Scarpa: None. C. Dionisi Vici: None. A. Garcia-
Cazorla: None. V. Kozich: None. S. Kölker: None. F. Gleich: None. T. Opladen: None. M. Scarpa: None. C. Dionisi Vici: None. A. Garcia-
Cazorla: None. V. Kozich: None. A. Eliyahu: None. M. Christine V.Malicdan: None. T. Vilboux: None. B. Ben-Zeev: None. B. Pode-Shakked:
None. A. Dori: None. N. Shelestovich: None. D. Marek-
Yagel: None. H. Pri-Chen: None. I. Blatt: None. L. He:
None. Y. Anikster: None. U-IMD: Unified European Registry for Inherited Metabolic
Disorders as a patient database for MetabERN U-IMD: Unified European Registry for Inherited Metabolic
Disorders as a patient database for MetabERN U-IMD: Unified European Registry for Inherited Metabolic
Disorders as a patient database for MetabERN R. Dorajoo1, S. Ooi2, W. Liu1, L. Wang1, J. Kang1, J. Liu1,3,4,
Y. Lee2,5,6 1Genome Institute of Singapore, Agency for Science,
Technology and Research, Singapore, Singapore, 2Department
of Paediatrics, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore, 3Saw Swee
Hock School of Public Health, National University of
Singapore, Singapore, Singapore, 4Department of Medicine,
Yong Loo Lin School of Medicine, National University of
Singapore, Singapore, Singapore, 5Singapore Institute for
Clinical Sciences, Agency for Science, Technology, and
Research, Singapore, Singapore, 6Division of Paediatric
Endocrinology, Khoo Teck Puat-National University
Children's Medical Institute, National University Hospital,
National University Health System, Singapore, Singapore S. Kölker1, F. Gleich1, T. Opladen1, M. Scarpa2, C. Dionisi Vici3,
A. Garcia-Cazorla4, V. Kozich5 S. Kölker1, F. Gleich1, T. Opladen1, M. Scarpa2, C. Dionisi Vici3,
A. Garcia-Cazorla4, V. Kozich5 1Universitätsklinikum Heidelberg, Heidelberg, Germany,
2Helios Dr. Horst Schmidt Kliniken Wiesbaden, Wiesbaden,
Germany, 3Ospedale Pediatrico Bambino Gesu, Rome, Italy,
4Hospital Sant Joan de Deu, Pediatric Research Institute and
CIBERER, Barcelona, Spain, 5Department of Pediatrics and
Adolescent Medicine, General Faculty Hospital in Prague,
Prague, Czech Republic Introduction: Inherited metabolic disorders (IMD) are a
prominent group of over 700 monogenic rare diseases with 196 J. del Picchia Sanfilippo C syndrome is a rare lysosomal storage disorder
caused by mutations in the HGSNAT gene, which encodes
an enzyme involved in heparan sulphate (HS) degradation. It is characterized by a severe and progressive neurode-
generation for which no effective treatment exists. Introduction: Whole-blood transcriptomics have high-
lighted novel disease pathways. We aimed to evaluate for
differentially expressed genes (DEGs) in whole-blood
between 34 extreme obese and 30 normal weight children. We further evaluated the role of identified DEGs with
diabetes related phenotypes in 138 independent childhood
samples. Introduction: Whole-blood transcriptomics have high-
lighted novel disease pathways. We aimed to evaluate for
differentially expressed genes (DEGs) in whole-blood
between 34 extreme obese and 30 normal weight children. We further evaluated the role of identified DEGs with
diabetes related phenotypes in 138 independent childhood
samples. Previously, we demonstrated the usefulness of siRNAs
targeting EXTL2 genes (involved in HS synthesis) as an
effective short-term substrate reduction therapy (SRT), on
Sanfilippo C patients’ fibroblasts. Now, we use different
lentiviral vectors encoding shRNAs targeting EXTL2 to
analyse their long-term effect. P06.65A R. Dorajoo: None. S. Ooi: None. W. Liu: None. L. Wang: None. J. Kang: None. J. Liu: None. Y. Lee: None. R. Dorajoo: None. S. Ooi: None. W. Liu: None. L. Wang: None. J. Kang: None. J. Liu: None. Y. Lee: None. U-IMD: Unified European Registry for Inherited Metabolic
Disorders as a patient database for MetabERN García-Morant: None. D. Grinberg: None. L. Vilageliu:
None. I. Canals: None. Characterization of genetic variants in centenarians
associated with obesity, metabolic syndrome and
hypertriglyceridemia P06.64D
Substrate reduction therapy approach for Sanfilippo C
syndrome: use of iPSC and iPSC-derived neurons from
patients as cellular models U-IMD: Unified European Registry for Inherited Metabolic
Disorders as a patient database for MetabERN We observe a clear reduction
in EXTL2 mRNA levels sixty days after transduction and an
evident decrease of the HS amounts. Methods: Extreme childhood obesity was defined as
onset <10 years of age and BMI ≥98th percentile. Normal
weight subjects were defined as a BMI between 18.5 kg/m2
and 24.5 kg/m2. Whole-blood was collected in Paxgene
tubes. RNA were library-prepped using Illumina Truseq v2
kits and sequenced on the Hiseq4000 in 15-plexes. Sequenced reads were QCed using Illumina chastity filters
and FastQC. QCed reads were mapped to HG19 using
tophat and transformed to CPM and normalized between-
samples using log2 transformation. All association tests
were performed in Deseq2, adjusted for age, sex, ethnicity
and sequencing batches. Due to the good results obtained, now we are using
neurons derived from patients’ induced pluripotent stem
cells (iPSC) as a cellular model. We are using an established
protocol to differentiate those iPSC into neurons within a
week. Neurons show mature signatures and functional
properties after one month. This technique will provide new
insights in the usefulness of this treatment in the main
affected cell type. To evaluate this therapeutic option, we
are analysing the neurons, focusing on aspects such as the
inhibition of the EXTL2 gene at the mRNA level or the
accumulation of HS over time by immunocytochemistry. Results: Mean reads of samples were 20.5 million reads. Analysis for DEGs between obese and normal weight
children identified 34 DEGs (Bonferroni adjusted-p < 0.05
and
fold-change
>1.4
or
<-1.4). DEGs
were
over-
represented in oxidative phosphorylation and mitochondrial
pathways (COX7C, NDUFS5, NDUFS4 and UQCRB). We
further evaluated the role of DEGs with multiple diabetes
related traits in an additional 138 samples and identified
nominally significant associations with fasting glucose,
fasting insulin and HOMA-IR levels (p between 0.044 and
0.0006, EMILIN2, HMGB2, NDUSF4, NDUSF5, PVRL2,
STAB1 and TXN). Our preliminary results in patients’ fibroblasts indicate
that shRNAs could be a long-term SRT and open a door for
the development of a promising therapeutic approach for
Sanfilippo C syndrome. Fundings: Catalan Government (2014SGR 932), Spanish
Government (SAF201456562R), Asoc. Stop Sanfilippo
(Spain), MPS España. Conclusion: Whole-blood RNA-seq analyses identified
genes in oxidative phosphorylation and mitochondrial
pathways associated with extreme childhood obesity that
may also play a role in diabetes-related pathways. N. Benetó:
None. E. Creus-Bachiller:
None. M. García-Morant: None. D. Grinberg: None. L. Vilageliu:
None. I. Canals: None. N. Benetó:
None. E. Creus-Bachiller:
None. M. 1Department of Genetics, Microbiology and Statistics, Faculty
of Biology, University of Barcelona, CIBERER, IBUB, IRSJD,
Barcelona, Spain, 2Stem Cells, Aging and Neurodegeneration
Group, Lund Stem Cell Center, University Hospital, Lund,
Sweden P06.64D The aim of this study is to
characterize genetic variations associated with obesity,
metabolic syndrome and hypertriglyceridemia. Materials and Methods: Our analysis was performed on
a group of 17 supercentenarians and 34 controls. We used
the publicly available whole-genome sequence database
(Gierman HJ et al., 2014) and focused our attention on
genetic variations at 25 genes associated with obesity and
metabolic syndrome. Results: We identified 5 variations in genes TAS1R2,
CLOCK, FABP2, ADRB2, TCF7L2 with pathogenic or
protective effect, distributed unequally between the two
studied groups. Two of the variations, in genes CLOCK and
FABP2, can be discussed as polymorphisms of probably
protective significance because their frequency is higher in
the group of the supercentenarians. The variations in genes
ADRB2, TCF7L2 are with pathogenic significance and their
frequency is again higher in the group of supercentenarians,
which means that it is possible to refer to gene interactions
that determine different penetrance or to impacts of other
genetic mechanisms. According to some data, the TAS1R2
gene polymorphism is risky for diabetes and dyslipidemia. Discussion: The data for the higher frequencies of the
possible protective variants in the CLOCK and FABP2 and
low frequencies of pathogenic variants in the TAS1R2 and
ADRB2 genes in the group of supercentenarian compared to
the control group is in line with our hypothesis but they
need further research. G. Singh: None. P. Raina: None. H.S. Sandhu: None. I. Sharma: None. V. Sharma: None. I. Sethi: None. R. Kapoor: None. V. Vig: None. E. Rai: None. S. Sharma:
None. A. Bhanwer: None. M. Mihaylova:
None. V. Damyanova:
None. L. Balabanski: None. D. Serbezov: None. D. Nikolova:
None. S. Karachanak-Yankova: None. D. Nesheva:
None. Z. Hammudeh: None. S. Hadjidekova: None. O. Antonova: None. R. Staneva: None. R. Vazharova:
None. D. Toncheva: None. Association study of candidate genes with diabetic
nephropathy in North Indian Population Association study of candidate genes with diabetic
nephropathy in North Indian Population P06.64D P06.64D
Substrate reduction therapy approach for Sanfilippo C
syndrome: use of iPSC and iPSC-derived neurons from
patients as cellular models M. Mihaylova1, V. Damyanova1, L. Balabanski1, D. Serbezov1,
D. Nikolova1, S. Karachanak-Yankova1, D. Nesheva1,
Z. Hammudeh1, S. Hadjidekova1, O. Antonova1, R. Staneva1,
R. Vazharova2, D. Toncheva1 M. Mihaylova1, V. Damyanova1, L. Balabanski1, D. Serbezov1, M. Mihaylova1, V. Damyanova1, L. Balabanski1, D. Serbezov1,
D. Nikolova1, S. Karachanak-Yankova1, D. Nesheva1, Z. Hammudeh1, S. Hadjidekova1, O. Antonova1, R. Staneva1,
R. Vazharova2, D. Toncheva1 N. Benetó1, E. Creus-Bachiller1, M. García-Morant1,
D. Grinberg1, L. Vilageliu1, I. Canals2,1 1Department of Medical Genetics, Medical Faculty, Medical
University of Sofia, Bulgaria, 2 “Zdrave” s, Sofia, Bulgaria,
2Department of Biology, Medical Genetics and Microbiology,
Faculty of Medicine, Sofia University” St.Kliment Ohridski”,
Sofia, Bulgaria 1Department of Genetics, Microbiology and Statistics, Faculty
of Biology, University of Barcelona, CIBERER, IBUB, IRSJD,
Barcelona, Spain, 2Stem Cells, Aging and Neurodegeneration
Group, Lund Stem Cell Center, University Hospital, Lund,
Sweden Introduction: It is likely that the supercentenarian’s gen-
ome will not contain pathogenic variations or it’s possible Abstracts from the 51st European Society of Human Genetics Conference: Posters 197 20-30% of type 2 diabetes (T2D) patients are more likely to
develop DN. Genetic vulnerability has been anticipated as
an important factor for the development and progression of
diabetic nephropathy and various research efforts have been
executed worldwide to identify the susceptible genes for
diabetic nephropathy. Several
single nucleotide poly-
morphisms (SNPs) have been observed in different genes
which have been found to play a major role in the genetic
susceptibility to DN. In this study five SNPs, each from five
candidate genes like ELMO1, CD2AP, VEGFA, HLA-G and
TERF1 have been evaluated to study the susceptibility of
these genes in the development of DN. 1260 subjects (374
diabetic nephropathy samples and 886 healthy controls)
have been included in this study. Genotyping was per-
formed, using High throughput AGENA MassArray tech-
nique. Four
SNPs,
ELMO1
(rs741301),
VEGFA
(rs2010963), HLA-G (rs1063320) and TERF1 (rs2010441)
showed strong association with the development of DN,
whereas one marker rs1485780 (CD2AP) did not show any
association. The study highlights that risk of diabetes-
associated renal ailment may be enhanced by risk alleles at
different susceptibility loci, in the presence of hyperglyce-
mia. The grant to Gurvinder Singh by UGC-UPA Scheme
and the grant No. F.8-2/2008(NS/PE),UGC,India to AJS
Bhanwer through CPEPA has been acknowledged. to have some protective alleles. P06.67C Homozygous mutation p.Arg192Cys of the TALDO1 gene
causes primary amenorrhea and hepatosplenomegaly
without liver cirrhosis in an adult woman D. Ilencikova1, F. Laccone2, R. Braun1, H. C. Duba1 1Institute of Medical Genetics, Linz, Austria, 2Institute of
Medical Genetics, Wien, Austria 1Institute of Medical Genetics, Linz, Austria, 2Institute of
Medical Genetics, Wien, Austria G. Singh1,2, P. Raina1, H. S. Sandhu1, I. Sharma2, V. Sharma2,
I. Sethi2, R. Kapoor3, V. Vig4, E. Rai2, S. Sharma2, A. Bhanwer1 G. Singh1,2, P. Raina1, H. S. Sandhu1, I. Sharma2, V. Sharma2,
I. Sethi2, R. Kapoor3, V. Vig4, E. Rai2, S. Sharma2, A. Bhanwer1 Introduction: Transladolase deficiency (TALDO, OMIM
606003) represents a recently recognized error of the pen-
tose phosphate pathway. Until now, it has been reported in
only 11 children, but there are no data about adult patients
with this disorder 1Department of Human Genetics, Guru Nanak Dev University,
Amritsar, India, 2Human Genetics Research Group, School of
Biotechnology, Shri Mata Vaishno Devi University, Katra,
India, 3Heart Station and Diabetes Clinic, Amritsar, India,
4Dr. S.B. Sohan Singh Eye Hospital, Amritsar, India Material and Methods: We report about an 32-year-old
Austrian woman with congenital hepatosplenomegaly, liver
dysfunction, gonadal streaks and mild facial dysmorphia. She was born with neonatal oedema and atrial septum
defect. In the neonatal period, she experienced sepsis with
leucopenia and thrombocytopenia. At the age of one year,
an inhomogenity of liver parenchyma was seen without the
typical signs of cirrhosis. As the parents declined liver Diabetic Nephropathy (DN) or Diabetic Kidney Disease
(DKD) is characterized by functional and structural changes
with the predominant changes in mesangial expansion,
glomerular sclerosis and Glomerular Basement Membrane
(GBM) thickening. It has been observed that approximately J. del Picchia 198 biopsy at that time, it was performed in adulthood because
of bleeding from oesophageal varices. Histology didn´t
show features of cirrhosis and no portal hypertensia was
diagnosed, however pulmonal-arterial hypertension was
present. Cholelithiasis and pancytopenia persist until now,
without any serious clinical problems. At the age of 32
years her liver is 6 cm, her spleen 12 cm large and the sex
hormone
status
is
comparable
to
that
of
a
climacteric woman. reference genome (GRGh37/hg19) and variants were called
using Freebayes. biopsy at that time, it was performed in adulthood because
of bleeding from oesophageal varices. Histology didn´t
show features of cirrhosis and no portal hypertensia was
diagnosed, however pulmonal-arterial hypertension was
present. Cholelithiasis and pancytopenia persist until now,
without any serious clinical problems. At the age of 32
years her liver is 6 cm, her spleen 12 cm large and the sex
hormone
status
is
comparable
to
that
of
a
climacteric woman. 1Institute of Medical Genetics, Linz, Austria, 2Institute of
Medical Genetics, Wien, Austria Results: The c.95A>G variant was sequenced with a
mean-coverage of 83 and 23 in the WB and DBS samples,
respectively. Father and child were heterozygous (A/G) for
the variant, while the mother was homozygous (A/A),
which was consistent for DBS and WB. Conclusion: DBS can be used for mutation identification
when WB is unavailable. Linked-reads may be essential for
detection of specific diplotypes when compound hetero-
zygosity plays a role in the pathogenesis.Funding: The
project is funded by the Danish and Faroese Governments. The
diagnosis
of
TALDO
was
set
using
EXOM
sequencing focusing primarily on hepatospenomegaly in
the search for potential candidate genes. L. Lydersen: None. Ó. Mortensen: None. B. á Steig:
None. G. Andorsdóttir: None. N.O. Gregersen: None. Results: The homozygous mutation p.Arg192Cys of the
TALDO1 Gene, as found in our patient, was already
reported in a 2 year old child with liver fibrosis and speech
delay (Wamelink et al., 2008). Results: The homozygous mutation p.Arg192Cys of the
TALDO1 Gene, as found in our patient, was already
reported in a 2 year old child with liver fibrosis and speech
delay (Wamelink et al., 2008). Next generation sequencing (NGS) for mitochondrial
respiratory chain disorders (MRCD): new genes,
cautionary tales and lessons learnt Next generation sequencing (NGS) for mitochondrial
respiratory chain disorders (MRCD): new genes,
cautionary tales and lessons learnt D. Ilencikova: None. F. Laccone: None. R. Braun:
None. H.C. Duba: None. L. G. Riley1,2, M. J. Cowley3, V. Gayevskiy3, T. Roscioli3,
S. Balasubramaniam4, D. R. Thorburn5,6, M. Bahlo7,6, C. M. Sue8,3,2, D. Coman9,10, M. Kava11, K. Bhattacharya1, C. J. Ellaway1,2, J. Christodoulou5,6,2,1 P06.71C Conclusions: In summary, this is the first report ever
about an adult woman with Transladolase deficiency
describing her specific clinical course. Whole-exome sequencing of a case-unaffected parent’s trio
using barcoded DNA from dried blood spots Whole-exome sequencing of a case-unaffected parent’s trio
using barcoded DNA from dried blood spots 1Children's Hospital at Westmead, Sydney, Australia,
2University of Sydney, Sydney, Australia, 3Garvan Institute of
Medical Research, Sydney, Australia, 4The Children’s Hospital
at Westmead, Sydney, Australia, 5Murdoch Childrens Research
Institute, Melbourne, Australia, 6University of Melbourne,
Melbourne, Australia, 7The Walter and Eliza Hall Institute of
Medical Research, Melbourne, Australia, 8Kolling Institute of
Medical Research, Sydney, Australia, 9Lady Cilento Children’s
Hospital, Brisbane, Australia, 10Wesley Hospital, Brisbane,
Australia, 11Perth Children’s Hospital, Perth, Australia 1Children's Hospital at Westmead, Sydney, Australia,
2University of Sydney, Sydney, Australia, 3Garvan Institute of
Medical Research, Sydney, Australia, 4The Children’s Hospital
at Westmead, Sydney, Australia, 5Murdoch Childrens Research
Institute, Melbourne, Australia, 6University of Melbourne,
Melbourne, Australia, 7The Walter and Eliza Hall Institute of
Medical Research, Melbourne, Australia, 8Kolling Institute of
Medical Research, Sydney, Australia, 9Lady Cilento Children’s
Hospital, Brisbane, Australia, 10Wesley Hospital, Brisbane,
Australia, 11Perth Children’s Hospital, Perth, Australia L. Lydersen1, Ó. Mortensen1, B. á Steig2, G. Andorsdóttir1, N. O. Gregersen1 1Genetic Biobank of the Faroe Islands, Tórshavn, Faroe
Islands, 2General Medical Department, National Hospital of
the Faroe Islands,, Tórshavn, Faroe Islands Introduction: The National Hospital of the Faroe Islands
has screened new-borns for heritable traits since 1986 using
blood samples collected on filter paper. To use the current
22.000 filter cards for genetic research has retrospect
advantages, however, challenging due to degraded samples
and the limited quantities of DNA obtained from each card,
which may be inadequate for next-generation sequencing
(NGS) analyses. As NGS introduces it´s own limitations
with short reads, we investigate the possibility to use dried
blood spot (DBS) samples together with linked-reads for
detection of a disease causing mutation (c.95A>G) in
carnitine-transporter deficiency disease, and whether this
approach may replace the trio information for true haplo-
type detection. Introduction: MRCD diagnosis is an arduous journey, with
early NGS being a logical diagnostic approach. We report
our extensive analysis of trio whole genome sequencing
(WGS) in 40 children with suspected MRCD, focusing on
genes not previously associated with MRC function. Introduction: MRCD diagnosis is an arduous journey, with
early NGS being a logical diagnostic approach. We report
our extensive analysis of trio whole genome sequencing
(WGS) in 40 children with suspected MRCD, focusing on
genes not previously associated with MRC function. Introduction: MRCD diagnosis is an arduous journey, with
early NGS being a logical diagnostic approach. P06.72D P06.72D
Methylmalonic Aciduria cblB type cellular model:
Hepatocyte differentiation from iPSC and pharmacological
chaperones evaluation P06.72D
Methylmalonic Aciduria cblB type cellular model:
Hepatocyte differentiation from iPSC and pharmacological
chaperones evaluation Á. Briso-Montiano1, A. Gámez1, S. Brasil1, L. R. Desviat1,
M. Ugarte1, J. García-Fernández2, C. Pérez-Cerdá1, B. Pérez3 E. Richard, Á. Briso-Montiano, S. Brasil, L. R. Desviat,
M. Ugarte, B. Pérez E. Richard, Á. Briso-Montiano, S. Brasil, L. R. Desviat,
M. Ugarte, B. Pérez 1Centro de Biología Molecular (CBM) "Severo Ochoa", Centro
de Diagnóstico de Enfermedades Moleculares, Universidad
Autónoma de Madrid, Ciberer, Madrid, Spain, 2Instituto de
Investigaciones Químicas, CSIC, Sevilla, Spain, 3Centro de
Biología Molecular (CBM), Madrid, Spain Centro de Biología Molecular (CBM) "Severo Ochoa", Centro
de Diagnóstico de Enfermedades Moleculares, Universidad
Autónoma de Madrid, Ciberer, Madrid, Spain The functional characterization of Phosphomannomutase 2
(PMM2) disease-causing mutations has suggested that
PMM2-CDG could be a conformational disease. Therapies
to
ameliorate
clinical
symptoms
could
be
addressed
improving the protein folding to restore total or partially its
native state. In this sense, from a 10,000 compound library
screening the compound 1-(3-chlorophenyl)-3-3-bis(pyr-
idine-2-yl)urea (compound VIII) stood out, based on its
pharmacochemical properties, enhancing the enzymatic
activity and stability of a number of destabilizing PMM2
mutations. These results provided a promising chemical
structure as a starting lead for new therapeutic agents
against this severe orphan disease. The aim of this work was
setting-up an optimization process by methodological
sequential rounds from a battery of chemical analogs of
compound VIII in order to improve the physicochemical
properties and cytotoxicity: Up to 795 analogs were eval-
uated and results showed 165 analogs that passed every
reactivity filter by in silico SmartsFilter analysis. In a first
selection of 25 analogs for in vitro analysis, 4 of these The understanding of the cellular and molecular mechan-
isms underlying inherited metabolic disorders (IMDs) is
essential for developing new strategies for their prevention
and treatment. Due to the genotype variability of IMDs and
the upcoming of personalized medicine has prompted the
emergence of developing new models. The aim of this work
was the generation of a hepatic model of methylmalonic
aciduria cblB type by hepatocyte differentiation of induced
pluripotent stem cells (IPSCs) generated by reprogramming
of patient-derived fibroblasts. This organic aciduria is
caused by the deficiency of ATP: cob(I)alamin adenosyl-
transferase (ATR) encoded by the MMAB gene. Whole-exome sequencing of a case-unaffected parent’s trio
using barcoded DNA from dried blood spots We report
our extensive analysis of trio whole genome sequencing
(WGS) in 40 children with suspected MRCD, focusing on
genes not previously associated with MRC function. Introduction: MRCD diagnosis is an arduous journey, with
early NGS being a logical diagnostic approach. We report
our extensive analysis of trio whole genome sequencing
(WGS) in 40 children with suspected MRCD, focusing on
genes not previously associated with MRC function. Methods: Trio WGS was performed at the Kinghorn
Centre for Clinical Genomics using the Illumina HiSeq X
sequencing platform: 95% of the nuclear genome covered to
> 15× depth; mitochondrial genome covered to >3,000×
depth. We identified SNVs and INDELS using GATK,
copy number and structural variation using ClinSV and
mitochondrial DNA variants using mity. Materials and Methods: The case-unaffected parent’s
trio, was whole-exome sequenced using DNA obtained
from DBS and whole blood (WB). DNA was barcoded
using the ChromiumTM Genome Kit. Exomes were captured
using the SureSelectXT Human All Exon kit and sequenced
on the NextSeq 500. The linked-reads were aligned to the Materials and Methods: The case-unaffected parent’s
trio, was whole-exome sequenced using DNA obtained
from DBS and whole blood (WB). DNA was barcoded
using the ChromiumTM Genome Kit. Exomes were captured
using the SureSelectXT Human All Exon kit and sequenced
on the NextSeq 500. The linked-reads were aligned to the Results: Overall diagnostic yield was > 60% (most
involving nuclear genes), including three putative new
disease genes. Six cases with clinical, biochemical and/or
enzymatic features consistent with a MCRD disorder had
mutations in “non-MRCD” genes, including ARX (two Abstracts from the 51st European Society of Human Genetics Conference: Posters 199 expressed relevant hepatic markers analyzed by immuno-
fluorescence. Finally, the hepatocytes generated were used
for evaluation of potential pharmacological chaperones
previously described (N-{[(4-chlorophenyl)carbamothioyl]
amino]-2-phenylacetamide
and
4-(4-(4-fluorophenyl)-5-
methyl-1H-pyrazol-3-yl)benzene-1,3-diol)) in combination
with hydroxocobalamin, providing evidences of its positive
effect on the activity of the mutant ATR hepatocytes. Hence, our findings provide an experimental suitable model
for the investigation of the hepatotoxicity of new drugs and
the pathogenesis of this severe disease serving also as
ex vivo platform for organoids generation and therapeutic
applications. affected half-brothers), SLC39A8 (two sibs with Leigh
disease), and single cases with mutations in HRAS (Costello
syndrome), EPG5 (Vici syndrome), SKIV2L (Trichohepa-
toenteric syndrome) and G6PC (Glycogen Storage Disorder
type 1a). Whole-exome sequencing of a case-unaffected parent’s trio
using barcoded DNA from dried blood spots Conclusions: Suspected MRCD might be associated with
“non-MRCD” genes, with potential explanations including:
clinical picture is a phenocopy of MRCD (MRC abnorm-
alities are a red herring); functional MRC abnormalities are
real, indicative of a previously unrecognised contribution to
the phenotype; the phenotype may be “blended” reflecting
contributions from two disease genes. These findings
highlight the importance of vigilance for “non-MRCD”
genes as the genetic aetiology in certain cases, and the
benefit of comprehensive, unbiased WGS. Research funded
by a NSW OHMR Sydney Genomics Collaborative grant. L.G. Riley: None. M.J. Cowley: None. V. Gayevskiy:
None. T. Roscioli: None. S. Balasubramaniam: None. D. R. Thorburn: None. M. Bahlo: None. C.M. Sue: None. D. Coman: None. M. Kava: None. K. Bhattacharya: None. C.J. Ellaway: None. J. Christodoulou: None. Conclusions: Suspected MRCD might be associated with
“non-MRCD” genes, with potential explanations including:
clinical picture is a phenocopy of MRCD (MRC abnorm-
alities are a red herring); functional MRC abnormalities are
real, indicative of a previously unrecognised contribution to
the phenotype; the phenotype may be “blended” reflecting
contributions from two disease genes. These findings
highlight the importance of vigilance for “non-MRCD”
genes as the genetic aetiology in certain cases, and the
benefit of comprehensive, unbiased WGS. Research funded
by a NSW OHMR Sydney Genomics Collaborative grant. PI13/01239 ISCIII; Fundación Isabel Gemio; LCF/PR/
PR16/11110018 Fundación la Caixa PI13/01239 ISCIII; Fundación Isabel Gemio; LCF/PR/
PR16/11110018 Fundación la Caixa E. Richard: None. Á. Briso-Montiano: None. S. Brasil:
None. L. R. Desviat: None. M. Ugarte: None. B. Pérez: None. E. Richard: None. Á. Briso-Montiano: None. S. Brasil:
None. L. R. Desviat: None. M. Ugarte: None. B. Pérez: None. L.G. Riley: None. M.J. Cowley: None. V. Gayevskiy:
None. T. Roscioli: None. S. Balasubramaniam: None. D. R. Thorburn: None. M. Bahlo: None. C.M. Sue: None. D. Coman: None. M. Kava: None. K. Bhattacharya: None. C.J. Ellaway: None. J. Christodoulou: None. P07.01A
Association study of TNFAIP3 gene polymorphisms in
three different autoimmune diseases P07.01A
Association study of TNFAIP3 gene polymorphisms in
three different autoimmune diseases L. Brick1, P. Bross2, D. Ang3, C. Cömert2, J. Palmfeldt2, B. F. Meaney4, M. Kozenko1, C. Georgopoulos3 A. Latini1, C. Perricone2, P. Conigliaro3, S. Colafrancesco2,
G. Novelli1, P. Borgiani1, C. Ciccacci1 1Department of Genetics, McMaster Children's Hospital,
Hamilton, ON, Canada, 2Research Unit for Molecular
Medicine, Aarhus University and University Hospital, Aarhus,
Denmark, 3Department of Biochemistry, University of Utah
School of Medicine, Salt Lake City, UT, United States,
4Division of Pediatric Neurology, McMaster Children's
Hospital, Hamilton, ON, Canada 1Department of Biomedicine and Prevention, Genetics Section,
University of Rome Tor Vergata, Rome, Italy, 2UOC
Reumatologia, Dipartimento di Medicina Interna e Specialità
Mediche, Sapienza Università di Roma, Rome, Italy, 3Clinic of
Rheumatology, Allergology and Clinical Immunology,
Department of Medicina dei Sistemi, University of Rome Tor
Vergata, Rome, Italy 1Department of Biomedicine and Prevention, Genetics Section,
University of Rome Tor Vergata, Rome, Italy, 2UOC
Reumatologia, Dipartimento di Medicina Interna e Specialità
Mediche, Sapienza Università di Roma, Rome, Italy, 3Clinic of
Rheumatology, Allergology and Clinical Immunology,
Department of Medicina dei Sistemi, University of Rome Tor
Vergata, Rome, Italy Hypomyelinating leukodystrophy type 4 (HLD4) (OMIM
612233) is a progressive neurodegenerative disorder char-
acterized
by
hypotonia,
psychomotor
delay,
acquired
microcephaly, mental retardation, and seizures. Typically,
the condition onsets in infancy and is fatal within the first
two decades. Brain MRI is consistent with diffuse hypo-
myelination. Some patients have increased urinary ethyl-
malonic acid. HLD4 is attributed to autosomal recessive
mutations in the HSPD1 gene, encoding the mitochondrial
Hsp60 chaperonin. Introduction: Autoimmune diseases (AIDs) are complex
diseases that share several susceptibility genetic loci. Tumor
necrosis factor alpha inducible protein 3 (TNFAIP3)
encodes the ubiquitin-modifying enzyme A20, that down-
regulates inflammation by restricting NF-kB, a transcription
factor that regulates expression of various pro-inflammatory
genes. Variants in TNFAIP3 gene have been described as
associated with susceptibility to several AIDs. Here, we
analyzed two TNFAIP3 polymorphisms in Italian patients
with systemic lupus erythematosus (SLE), rheumatoid
arthritis (RA), and Sjögren’s syndrome (pSS), to verify if
TNFAIP3 is involved in genetic predisposition to AIDs also
in Italian population. We present a 4 year-old boy with speech and motor
delays. Brain MRI showed diffuse hypomyelination of the
cerebral and cerebellar white matter. Examination revealed
a normal head circumference, brisk reflexes, mild ataxia and
bilateral
postural
tremor. Urine
metabolic
screening
detected
increased
ethylmalonic
acid. P06.74B De novo heterozygous HSPD1 variants: A novel mechanism
in hypomyelinating leukodystrophy type 4? P06.72D Fibroblasts
from a patient bearing a hypomorphic destabilizing muta-
tion in this gene (p.Ile96Thr) were reprogrammed using a
commercial kit based on Sendai virus vectors. After the
molecular and functional characterization of the iPSC line,
these cells were differentiated in vitro into definitive
endoderm and then incubated with specific factors, aimed at
hepatocyte
differentiation. IPSC-derived
hepatocytes 200 J. del Picchia We used an Escherichia coli genetic assay system to assess
the function of the mutant Hsp60(L47V) protein. Pre-
viously, we showed by complementation that E. coli cells
expressing the wild-type human Hsp60 protein and its co-
chaperone Hsp10 protein can survive deletion of the
otherwise essential and homologous groESgroEL genes of
E. coli. Here, we found that co-expression of the wild-type
human Hsp10 and mutant Hsp60(L47V) does not support
growth of these E. coli cells. Our results demonstrate that
the function of the mutant Hsp60(L47V) protein is
compromised, at least when expressed in E. coli. This case
highlights a possible novel autosomal dominant mechanism
for HLD4. 25 structures have shown no concentration-dependent
inhibitory effect on enzymatic activity, a mild stability
improvement and higher PMM2 activity in a mutant cellular
model bearing a destabilizing mutation (p.T237M). This
workflow for developing a potential therapy for PMM2-
CDG has shown a dynamic progression from a promising
pharmacological chaperone to 4 new compounds that pas-
sed critical selection points, allowing the process to move
forward to the next screening levels in another cellular
models which will provide new potential structures with
pharmacological effects. PI16/00573 MINECO-FEDER; Fundación Isabel Gemio;
LCF/PR/PR16/11110018 Fundación la Caixa
Á PI16/00573 MINECO-FEDER; Fundación Isabel Gemio;
LCF/PR/PR16/11110018 Fundación la Caixa
Á L. Brick: None. P. Bross: None. D. Ang: None. C. Cömert: None. J. Palmfeldt: None. B.F. Meaney: None. M. Kozenko: None. C. Georgopoulos: None. L. Brick: None. P. Bross: None. D. Ang: None. C. Cömert: None. J. Palmfeldt: None. B.F. Meaney: None. M. Kozenko: None. C. Georgopoulos: None. Á. Briso-Montiano: None. A. Gámez: None. S. Brasil:
None. L. R. Desviat: None. M. Ugarte: None. J. García-
Fernández:
None. C. Pérez-Cerdá:
None. B. Pérez: None. P07.01A
Association study of TNFAIP3 gene polymorphisms in
three different autoimmune diseases Whole
exome
sequencing was completed, and detected a de novo
heterozygous HSPD1 variant (c.139T>G, p.L47V). Methods: We recruited 315 SLE patients, 196 pSS
patients, 187 RA patients, and 236 healthy controls. Genotyping of rs2230926 and rs6920220 in TNFAIP3 gene
was performed by allelic discrimination assay. We carried Mass spectrometry analyses indicated similar amounts of
mutant and wild type proteins in our patient’s fibroblasts. Abstracts from the 51st European Society of Human Genetics Conference: Posters 201 out a case/control association study and a genotype/
phenotype correlation analysis. (38.48%). This suggests a strong relationship between the
genetic alteration and the observed phenotype. However,
further studies of the segregation pattern of the variant are
needed, as well as functional validation. Results: Higher risk to develop SLE was observed for
rs2230926
(P=0.02,
OR=1.92). No
association
was
observed with the pSS susceptibility, but the variant allele
seems to confer a higher risk to develop lymphoma in pSS
patients. In RA patients, the presence of RF and ACPA
resulted significantly associated with rs2230926 variant
allele. We observed a significant association between the
variant allele of rs6920220 and SLE (P=0.03, OR=1.53),
pSS (P=0.02, OR=1.69) and RA (P=0.00001, OR=2.58)
susceptibility. Furthermore, SLE patients carrying the
rs6920220 variant allele showed a higher risk to develop
pericarditis, pleurisy and kidney complications. Conclusion: Target sequencing is a suitable approach to
detect likely causative genetic variants in autoinflammatory
disease patients. The use of NGS techniques in clinical
immunology will lead to several benefits in basic science
and clinical practice. This study was funded by grants SAF2012-35025 and
SAF2015-68472-C2-2-R from the Ministerio de Economía
y Competitividad (Spain) and FEDER to FC. L. Batlle-Masó: None. A. Mensa-Vilaró: None. M. Solís-Moruno: None. M. Tormo: None. T. Marquès-
Bonet: None. J.I. Aróstegui: None. F. Casals: None. Conclusion: Our results support the importance of the
TNFAIP3 gene variants role in the development of different
autoimmune diseases. 1ITU, Istanbul, Turkey, 2Cerrahpasa Medical School, Istanbul,
Turkey 1ITU, Istanbul, Turkey, 2Cerrahpasa Medical School, Istanbul,
Turkey 1ITU, Istanbul, Turkey, 2Cerrahpasa Medical School, Istanbul,
Turkey L. Batlle-Masó1, A. Mensa-Vilaró2,3, M. Solís-Moruno1,
M. Tormo4, T. Marquès-Bonet1, J. I. Aróstegui2,3, F. Casals4 L. Batlle-Masó1, A. Mensa-Vilaró2,3, M. Solís-Moruno1, L. Batlle-Masó1, A. Mensa-Vilaró2,3, M. Solís-Moruno1, Normal 0 false false false EN-US JA X-NONE /* Style
Definitions */ table.MsoNormalTable {mso-style-name:"-
Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-col-
band-size:0; mso-style-noshow:yes; mso-style-priority:99;
mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
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theme-font:minor-latin;
mso-hansi-font-family:Cambria;
mso-hansi-theme-font:minor-latin;} M. Tormo4, T. Marquès-Bonet1, J. I. Aróstegui2,3, F. Casals4 M. Tormo4, T. Marquès-Bonet1, J. I. Aróstegui2,3, F. Casals4 1Institut de Biologia Evolutiva (UPF-CSIC), Barcelona, Spain,
2Hospital Clínic-IDIBAPS, Barcelona, Spain, 3Hospital Sant
Joan de Déu, Barcelona, Spain, 4Servei de Genòmica (UPF),
Barcelona, Spain Introduction: Autoinflammatory diseases are usually dif-
ficult to diagnose due to their high phenotypic heterogeneity
and variable expression. In some cases, diagnosis difficul-
ties can be resolved using genetic testing. Next-generation
sequencing (NGS) is a cost-effective approach to identify
likely causative genetic variants. This identification can lead
to a better understanding of the disease and increase the
number of diagnosed patients. According to this, the main
goal of this study was to use NGS to detect genetic variants
likely to be causative of the disease in pediatric patients
with autoinflammatory symptoms. Introduction: Behçet’s disease (BD) is a systemic
inflammatory disorder. Investigation of the proteome profile
will facilitate our understanding of disease processes. We
aimed to identify proteins specific to BD and related
pathways through proteomic analyses performed on PBMC
samples. Introduction: Behçet’s disease (BD) is a systemic
inflammatory disorder. Investigation of the proteome profile
will facilitate our understanding of disease processes. We
aimed to identify proteins specific to BD and related
pathways through proteomic analyses performed on PBMC
samples. Methods: Study groups were composed of active BD
(N=33), inactive BD(N=26), and healthy controls(N=28). PBMC protein samples from each group were pooled and
then separated using 2D-DIGE. Protein spots with at least 2
times
differentially-expressed
were
compared
among
groups, and identified by MALDI-TOF-MS. Bioinformatic
pathway
analyses
were
carried
out
through
KEGG,
PANTHER and STRING databases. Materials and Methods: We performed target sequen-
cing (TruSight™One panel) in 26 samples from patients
with clinical suspicion of autoinflammatory disease. Next,
we performed bioinformatics analysis to detect likely
causative genetic variants. For the most relevant candidates
we corroborate our findings using Sanger sequencing. Investigation of Peripheral Blood Mononuclear Cells
(PBMC) Proteome Profile in Behcet’s Disease Investigation of Peripheral Blood Mononuclear Cells
(PBMC) Proteome Profile in Behcet’s Disease A. Kirectepe Aydın1, Y. Özgüler2, D. Ucar2, E. Seyahi2,
H. Yazici2, E. Tahir Turanli1 Application of clinical exome sequencing panel in early
onset autoinflammatory disease patients Application of clinical exome sequencing panel in early
onset autoinflammatory disease patients P07.03C A. Latini: None. C. Perricone: None. P. Conigliaro:
None. S. Colafrancesco: None. G. Novelli: None. P. Borgiani: None. C. Ciccacci: None. The human-restricted duplicated form of the α7 nicotinic
receptor, CHRFAM7A: expression and transcriptional
regulation in inflammatory cells Z. Litwinska1, A. Pietrzyk2, K. Luczkowska1, A. Sobus1,
E. Paczkowska1, G. Helbig3, B. Machalinski1 A. Maroli1, S. Di Lascio1, S. Cardani1, L. Drufuca1,2,
M. Locati1,2, D. Fornasari1,3, R. Benfante3,1 1Department of General Pathology, Pomeranian Medical
University, Szczecin, Poland, 2Cytogenetic Unit, Department of
Laboratory Diagnostics, Pomeranian Medical University,
Szczecin, Poland, 3Department of Hematology and Bone
Marrow Transplantation, Medical University of Silesia,
Katowice, Poland 1Department of General Pathology, Pomeranian Medical
University, Szczecin, Poland, 2Cytogenetic Unit, Department of
Laboratory Diagnostics, Pomeranian Medical University,
Szczecin, Poland, 3Department of Hematology and Bone
Marrow Transplantation, Medical University of Silesia,
Katowice, Poland 1Dept. of Medical Biotechnology and Translational Medicine,
Università degli Studi di Milano, Milan, Italy, 2Humanitas
Clinical and Research Center, Rozzano, Italy, 3CNR -
Neuroscience Institute, Milan, Italy Introduction: The α7 nicotinic acetylcholine receptor
(CHRNA7) plays a role in the modulation of the inflam-
matory response through the activation of the “cholinergic
anti-inflammatory pathway”. In humans, a recombination
event involving the exon 5 to 10 of CHRNA7 gene, fused to
four novel exons A, B, C and D (FAM7A), gave rise to the
CHRFAM7A gene. This hybrid gene, located on chromo-
some 15q13-q14, 1.6 Mb apart from CHRNA7, is highly
expressed in inflammatory cells, where it can regulate the
anti-inflammatory effects of α7 activation. Acute treatment
of macrophages with LPS down-regulates CHRFAM7A by a
mechanism driven by NF-κB, paralleled by CHRNA7 up-
regulation. As
studies
are
emerging,
which
identify
CHRFAM7A expression alteration in inflammatory or
infective pathologies, the regulation of its expression may
become a key step in the modulation of inflammation. However, the region driving the transcriptional regulation
of CHRFAM7A gene in human immune tissues is largely
unknown. Introduction: Tyrosine kinase inhibitors (TKIs) are the
first-line therapy for most chronic myeloid leukemia (CML)
patients, however some are unresponsive to it or develop
resistance. Recently, microRNAs (miRNAs) have been
implicated in the progression of CML and the development
of TKI resistance. miRNA-146a and miRNA-155-5p are
involved in MAPK signaling pathway, cell proliferation and
apoptosis. In this study we aimed to investigate expression
of miR-146a and miR-155-5p in CML patients and, where
possible, to identify how TKI treatment affects this
expression. Materials and Methods: Bone marrow (BM) samples
were obtained from newly diagnosed CML patients (n =-
16) and healthy controls (n = 18). From one patient we
took 3 samples: one before TKI therapy initiation and two
samples after (6, 12 months). 1ITU, Istanbul, Turkey, 2Cerrahpasa Medical School, Istanbul,
Turkey Di Lascio: None. S. Cardani:
None. L. Drufuca: None. M. Locati: None. D. Fornasari:
None. R. Benfante: None. A. Maroli: None. S. Di Lascio: None. S. Cardani:
None. L. Drufuca: None. M. Locati: None. D. Fornasari:
None. R. Benfante: None. 1ITU, Istanbul, Turkey, 2Cerrahpasa Medical School, Istanbul,
Turkey Results: A total of 369 protein spots were detected by
2D-DIGE. 115 for active vs inactive BD, 118 for active BD
vs healthy controls, 129 spots for inactive BD vs healthy Results: We evaluated the performance of the kit to
capture autoinflammatory candidate genes. We found likely
causative genetic variants of the disease in 10 patients 202 J. del Picchia Materials and Methods: human macrophages and THP-
1 cell line have been used to characterized the CHRFAM7A
regulatory region. controls were identified. The strongest 45 spots were further
analyzed through MALDI-TOF-MS. Fructose-bisphosphate
aldolase-C, calreticulin, ficolin-1, fibrinogen alpha chain,
fibrinogen beta chain, filamin-A, FUSE-binding protein-1,
phosphoglycerate kinase-1, stathmin, vinculin, hnRNP-M,
WD repeat-containing protein-1, HSPA8, myosin light
polypeptide-6, talin-1 and tropomyosin alpha-3 chain were
differentially expressed between groups. Materials and Methods: human macrophages and THP-
1 cell line have been used to characterized the CHRFAM7A
regulatory region. Results and Conclusions: we provide a detailed analysis
of the CHRFAM7A gene regulatory region and its pro-
inflammatory stimuli responsiveness. Furthermore, given
the anti-inflammatory potential of the acetylcholinesterase
inhibitor
donepezil,
we
investigated
the
CHRFAM7A
expression profile in macrophages treated with donepezil,
showing an unexpected up-regulation of both CHRFAM7A
and CHRNA7 gene, thus highlighting a possible role for
CHRFAM7A gene product in the control and modulation of
the cholinergic anti-inflammatory pathway, and/or in the
modulation of CHRNA7 function. Results and Conclusions: we provide a detailed analysis
of the CHRFAM7A gene regulatory region and its pro-
inflammatory stimuli responsiveness. Furthermore, given
the anti-inflammatory potential of the acetylcholinesterase
inhibitor
donepezil,
we
investigated
the
CHRFAM7A
expression profile in macrophages treated with donepezil,
showing an unexpected up-regulation of both CHRFAM7A
and CHRNA7 gene, thus highlighting a possible role for
CHRFAM7A gene product in the control and modulation of
the cholinergic anti-inflammatory pathway, and/or in the
modulation of CHRNA7 function. Conclusion: We identified proteins that involve in
glycolysis, complement/coagulation and coagulation activa-
tion pathways. The expression of proteins involved in ER
protein processing (calreticulin, HSPA8, GRP78-BiP) were
down-regulated
in
BD
patients
compared
to
healthy
controls, which raised the importance of ER stress in
Behcet disease pathogenesis. <!--EndFragment--> Funding: CNR Research project on aging A. Kirectepe Aydın: None. Y. Özgüler: None. D. Ucar:
None. E. Seyahi: None. H. Yazici: None. E. Tahir
Turanli: None. A. Maroli: None. S. Di Lascio: None. S. Cardani:
None. L. Drufuca: None. M. Locati: None. D. Fornasari:
None. R. Benfante: None. A. Maroli: None. S. The human-restricted duplicated form of the α7 nicotinic
receptor, CHRFAM7A: expression and transcriptional
regulation in inflammatory cells Quantitative assessment of the
expression
of
miRNA-146a
and
miRNA-155-5p
was
performed by qRT-PCR using qScript™microRNA cDNA
Synthesis Kit, specific primers, iQ™
SYBR® Green 203 Abstracts from the 51st European Society of Human Genetics Conference: Posters Supermix and Bio-Rad CFX96 Real-Time PCR Detection
System. identified several previously undescribed candidate variants
that
could
explain
the
hyperosinophilia
driven
auto-
immunity phenotype. We have also performed immunolo-
gical assays to validate these variants. We conclude that
WGS is the best investigation in rare cases of hyper-
eosinophilia driven autoimmunity. This test improves time
to diagnosis, helps rule out known chromosomal/single
gene causes and identifies novel intronic variants that are
overlooked by exome testing alone. Results:
miRNA-146a
expression
was
significantly
elevated in CML patients (p =
0,006), expression of
miRNA-155-5p did not differ between groups. Both tested
miRNAs showed constant decrease in expression in 6 and
12 month after TKI therapy initiation. Conclusions:
miRNAs,
including
miRNA-146a,
miRNA-155-5p, are promising biomarker candidates for
CML diagnosis and prognosis. The evident drop in
expression levels of these miRNAs after TKI therapy
suggests that they might also be viable targets for
monitoring drug response. However, the precise contribu-
tion of miRNA-146a and miRNA-155-5p to CML patho-
genesis remains to be further elucidated. A. Marwaha: None. G. Latino: None. R. Laxer: None. E. Grunebaum: None. V. Kim: None. E. Pope: None. M. Kirby-Allen: None. R. Schneider: None. M. Weinstein:
None. A. Naqkvi: None. R. Berard: None. L. Dupuis:
None. R. Jobling: None. J. Stavropoulos: None. A. Muise: None. T. Eiwegger: None. R. Mendoza: None. A. Marwaha: None. G. Latino: None. R. Laxer: None. E. Grunebaum: None. V. Kim: None. E. Pope: None. M. Kirby-Allen: None. R. Schneider: None. M. Weinstein:
None. A. Naqkvi: None. R. Berard: None. L. Dupuis:
None. R. Jobling: None. J. Stavropoulos: None. A. Muise: None. T. Eiwegger: None. R. Mendoza: None. Z. Litwinska: None. A. Pietrzyk: None. K. Lucz-
kowska: None. A. Sobus: None. E. Paczkowska: None. G. Helbig: None. B. Machalinski: None. Z. Litwinska: None. A. Pietrzyk: None. K. Lucz-
kowska: None. A. Sobus: None. E. Paczkowska: None. G. Helbig: None. B. Machalinski: None. P07.06B C. Marconi1, M. Faleschini2, F. Palombo3,4, R. Bottega5,
V. Bozzi6, P. Noris6, A. Balduini7, T. Pippucci1, A. Savoia5,2,
C. L. Balduini6, N. Katsanis8, A. Pecci6, M. Seri1 Loss of the phosphatase PTPRJ causes migration defects of
megakaryocytes and thrombocytopenia Loss of the phosphatase PTPRJ causes migration defects of
megakaryocytes and thrombocytopenia Whole Genome Sequencing in a cohort of children with
hypereosinophilia driven autoimmunity Whole Genome Sequencing in a cohort of children with
hypereosinophilia driven autoimmunity A. Marwaha, G. Latino, R. Laxer, E. Grunebaum, V. Kim,
E. Pope, M. Kirby-Allen, R. Schneider, M. Weinstein, A. Naqkvi,
R. Berard, L. Dupuis, R. Jobling, J. Stavropoulos, A. Muise,
T. Eiwegger, R. Mendoza 1University of Bologna -Department of Medical and Surgical
Sciences, Bologna, Italy, 2Department of Medical Sciences,
University of Trieste, Trieste, Italy, 3Unit of Neurology,
Department of Biomedical and NeuroMotor Sciences
(DIBINEM), University of Bologna, Bologna, Italy, 4IRCCS
Institute of Neurological Sciences, Bologna, Italy, 5IRCCS
Burlo Garofolo Mother and Child Institute, Trieste, Italy,
6Internal Medicine Department, University of Pavia – IRCCS
Policlinico San Matteo Foundation, Pavia, Italy, 7Department
of Molecular Medicine, University of Pavia, Pavia, Italy,
8Center for Human Disease Modeling, Duke University
Medical Center, Durham, NC, United States Hospital for Sick Children, Toronto, ON, Canada Hypereosinophillia is a common, often transient, phenotype
with a broad differential. However, there exists a small
subset of patients that have a constitutively increased
eosinophil count, resulting in severe end organ auto-
immunity and requiring ongoing immunosuppressive ther-
apy. Previously described genetic causes for these rarer
forms of hypereosinophilia include; chromosomal rearran-
gement promoting bone marrow eosinophil production,
TCR clonality or recently described single gene disorders
(e.g. STAT3 and MALT1). We present a cohort of three
children who presented with dysmorphic features and
hypereosinophilia driven autoimmunity of the skin and
gastrointestinal system. These children all had extensive
genetic testing including; microarray, TCR clonality stu-
dies, chromosomal rearrangement FISH studies and exome
sequencing, however, only variants of uncertain sig-
nificance were identified. We therefore performed whole
genome sequencing (WGS) with the hypothesis that an
intronic regulatory variant is responsible for the hyper-
eosinophilia driven autoimmunity observed. We present
here a detailed phenotyping of this cohort, the variants of
uncertain significance that were identified by microarray
and exome sequencing and the results of the WGS. We have Inherited thrombocytopenias (IT) are characterized by
decreased circulating platelets that can be associated with
other phenotypes (bone marrow aplasia, haematological
tumors, renal failure). Through exome sequencing we identified a new recessive
IT due to loss of PTPRJ (Protein Tyr-Phosphatase, Receptor
type J). In two affected siblings we observed two heterozygous
variants causing a premature stop insertion on both alleles
and the loss of mRNA and protein, as we observed in
patients’ platelets. PTPRJ is a transmembrane tyrosine-
phosphatase highly expressed in megakaryocytes (Mks) and
platelets. A mouse model of PTPRJ inactivation shows
defects in Mk migration, platelet production, platelet
activation and aggregation. The two probands with PTPRJ
mutations presented with thrombocytopenia and moderate J. del Picchia 204 Multiple myeloma (MM) is the second most common
hematologic
malignancy
that
forms
in
plasma
cell. Recently, we identified ELL2 as a susceptibility gene for
MM. To understand its mechanism of action, we performed
expression quantitative trait locus (eQTL) analysis in
CD138+ plasma cells from 1,630 MM patients from four
populations. We show that the MM risk allele lowers ELL2
expression in these cells (Pcombined=2.5×10-27; βcombined=-
0.24 s.d.), but not in peripheral blood or other tissues. A
total of 67 single-nucleotide polymorphisms and 5 small
insertions/deletions are highly correlated with the best-
supported sentinel MM risk variant (rs1423269) and the
strongest ELL2 expression variant (rs9314162) (r2 > 0.8). Hospital for Sick Children, Toronto, ON, Canada Using bioinformatic approaches we identified 8 variants that
might alter the efficiency of ELL2 transcription. We made
luciferase vectors for each of these variants and transfected
them into three MM plasma cell lines (L363, OPM2, and
RPMI-8226) and two cell lines representing other hemato-
logic lineages (K562 and MOLM-13). Among those, three
risk variants (rs3777189-C, rs3777185-C and rs4563648-G)
yielded decreased luciferase activity relative to their corre-
sponding protective variants in plasma cell lines, but not in
non-plasma cell lines. Further analysis reveals that the MM
risk allele associates with upregulation of gene sets related
to ribosome biogenesis, and knockout/knockdown and
rescue experiments in plasmocytoma cell lines support a
cause-effect relationship. Our results provide mechanistic
insight into MM predisposition. spontaneous bleeding. Patients’ platelets showed defective
activation and aggregation and a global decrease in tyrosine
phosphorylation after stimulation with a GPVI agonist
associated with reduced activation of the tyrosine kinase
Src. Mks differentiated in vitro from patients’ blood
progenitors showed impaired maturation and defective
migration. Exploiting a zebrafish line with fluorescently
labeled
thrombocytes,
we
demonstrated
a
significant
decrease of the circulating thrombocytes in the PTRPJ
KO with morpholino and the phenotype rescue through
injection with human wt mRNA. Moreover, silencing of
PTPRJ in the human Mk cell line Dami cells induced the
migration and maturation defects observed in patients’ Mks. In summary, we discovered a novel form of IT. Pathogenetic mechanisms include impaired Mk migration
and maturation. These abnormalities may be mediated by
reduced activation of Src, which is therefore recognized as a
target of PTPRJ in humans. C. Marconi: None. M. Faleschini: None. F. Palombo:
None. R. Bottega: None. V. Bozzi: None. P. Noris: None. A. Balduini: None. T. Pippucci: None. A. Savoia: None. C.L. Balduini: None. N. Katsanis: None. A. Pecci: None. M. Seri: None. P07.09A The multiple myeloma risk allele at 5q15 lowers ELL2
expression and increases ribosomal gene expression in
malignant plasma cells This work was supported by the Swedish Foundation for
Strategic Research (KF10-0009), the Knut and Alice
Wallenberg Foundation (2012.0193), the Swedish Research
Council (2012-1753), Cancerfonden (2017/265). M. Ali1, R. Ajore1, A. Wihlborg1, A. Niroula1, B. Swaminathan1,
E. Johnsson1, O. Stephens2, G. Morgan2, T. Meissner3,
I. Turesson1, H. Goldschmidt4,5, U. Mellqvist6, U. Gullberg1,
M. Hansson1,7, K. Hemminki8,9, H. Nahi10, A. Waage11,
N. Weinhold2, B. Nilsson1,12 M. Ali: None. R. Ajore: None. A. Wihlborg: None. A. Niroula: None. B. Swaminathan: None. E. Johnsson:
None. O. Stephens:
None. G. Morgan:
None. T. Meissner: None. I. Turesson: None. H. Goldschmidt:
None. U. Mellqvist: None. U. Gullberg: None. M. Hansson: None. K. Hemminki: None. H. Nahi: None. A. Waage: None. N. Weinhold: None. B. Nilsson: None. 1Lund University, Hematology and Transfusion Medicine,
Department of Laboratory Medicine, Lund, Sweden, 2Myeloma
Institute for Research and Therapy, University of Arkansas for
Medical Sciences, Little Rock, AR, United States, 3Avera
Cancer Institute, Department of Molecular and Experimental
Medicine, Sioux Falls, SD, United States, 4Department of
Internal Medicine V, University of Heidelberg, Heidelberg,
Germany, 5National Center for Tumor Diseases, Ulm,
Germany, 6Section of Hematology, South Elvsborg Hospital,
Borås, Sweden, 7Hematology Clinic, Skåne University
Hospital, Lund, Sweden, 8German Cancer Research Center,
Heidelberg, Germany, 9Center for Primary Health Care
Research, Lund University, Malmö, Sweden, 10Center for
Hematology and Regenerative Medicine, Karolinska Institutet,
Stockholm, Sweden, 11Department of Cancer Research and
Molecular Medicine, Norwegian University of Science and
Technology, Trondheim, Norway, 12Broad Institute, 7
Cambridge Center, Cambridge, MA, United States Genomic and functional evaluation of the role of TNFSF14
gene in susceptibility to multiple sclerosis Genomic and functional evaluation of the role of TNFSF14
gene in susceptibility to multiple sclerosis M. Zuccalà*1, N. Barizzone*1, C. Basagni*1, E. Boggio*1,
L. Gigliotti*1, M. Sorosina*2, R. Bordoni3, F. Clarelli2,
S. Anand3, E. Mangano3, D. Vecchio4, F. Esposito2, E. Corsetti1,
G. Predebon1, R. Cantello4, V. Martinelli2, G. Comi2, M. Leone5,
G. De Bellis3, U. Dianzani*1, F. Martinelli-Boneschi*2,6,
S. D'Alfonso*1
1University of Eastern Piedmont-Department of Health
Sciences, Novara, Italy, 2Laboratory of Human Genetics of M. Zuccalà*1, N. Barizzone*1, C. Basagni*1, E. Boggio*1,
L. Gigliotti*1, M. Sorosina*2, R. Bordoni3, F. Clarelli2,
S. Anand3, E. Mangano3, D. Vecchio4, F. Esposito2, E. Corsetti1,
G. Predebon1, R. Cantello4, V. Martinelli2, G. Comi2, M. Leone5,
G. De Bellis3, U. Dianzani*1, F. Martinelli-Boneschi*2,6,
S. D'Alfonso*1 M. Zuccalà*1, N. Barizzone*1, C. Basagni*1, E. Boggio*1,
L. Gigliotti*1, M. Sorosina*2, R. Bordoni3, F. Clarelli2,
S. Anand3, E. Mangano3, D. Vecchio4, F. Esposito2, E. Corsetti1,
G. Predebon1, R. Cantello4, V. Martinelli2, G. Comi2, M. Leone5,
G. De Bellis3, U. Dianzani*1, F. Martinelli-Boneschi*2,6,
S. D'Alfonso*1 1University of Eastern Piedmont-Department of Health
Sciences, Novara, Italy, 2Laboratory of Human Genetics of 1University of Eastern Piedmont-Department of Health
Sciences, Novara, Italy, 2Laboratory of Human Genetics of 1University of Eastern Piedmont-Department of Health
Sciences, Novara, Italy, 2Laboratory of Human Genetics of P07.11C
Single-cell transcriptomics uncovers cellular and molecular
determinants of tissue myeloid cell heterogeneity in
homeostasis and cancer P07.11C
Single-cell transcriptomics uncovers cellular and molecular
determinants of tissue myeloid cell heterogeneity in
homeostasis and cancer Neurological Diseases, San Raffaele Scientific Institute,
Milano, Italy, 3National Research Council of Italy, Institute for
Biomedical Technologies, Milano, Italy, 4MS Centre, SCDU
Neurology, AOU Maggiore della Carità, Novara, Italy, 5SC
Neurologia, Dipartimento di Scienze Mediche, IRCCS Casa
Sollievo della Sofferenza, San Giovanni Rotondo (Foggia),
Italy, 6Department of Biomedical Sciences for Health,
University of Milan, Milano, Italy Neurological Diseases, San Raffaele Scientific Institute,
Milano, Italy, 3National Research Council of Italy, Institute for
Biomedical Technologies, Milano, Italy, 4MS Centre, SCDU
Neurology, AOU Maggiore della Carità, Novara, Italy, 5SC
Neurologia, Dipartimento di Scienze Mediche, IRCCS Casa
Sollievo della Sofferenza, San Giovanni Rotondo (Foggia),
Italy, 6Department of Biomedical Sciences for Health,
University of Milan, Milano, Italy Abstracts from the 51st European Society of Human Genetics Conference: Posters 205 P07.11C G. Barbiera1, M. Genua1, F. Cilenti1, D. Iodice1, E. Dugnani2,
A. Citro2, A. Capotondo3, M. Milani1, A. Cantore1, N. Coltella1,
L. Barbarossa1, G. Martino3, L. Piemonti2, L. Naldini1,
R. Ostuni1 Over 200 multiple sclerosis (MS) susceptibility genes
were identified. Among these, the strongest non-HLA
signal in the Italian population maps in the Tumor
Necrosis
Factor
(ligand)
superfamily
member
14
(TNFSF14) gene encoding for LIGHT, a transmembrane
glycoprotein expressed on various immune cells and
involved in dendritic cells (DC) maturation. We demon-
strated through a fine-mapping approach that an intronic
variant is the primarily associated one. Cis-eQTL analysis
from different databases showed that carriers of MS risk
allele have a lower TNFSF14 RNA expression in EBV-
transformed lymphoblastoid cell lines (Geuvadis, Bio-
portal, Gtex) and in PBMCs (Gtex). These data are con-
sistent with the imbalance against the risk allele observed
in heterozygous individuals (p < 0.0001, RNAseq on 97
lymphoblastoid cells, Geuvadis). Consistently, in PBMC
of 84 Italian MS and 80 healthy controls (HC), individuals
with
MS
risk
genotype
produced
lower
levels
of
TNFSF14 transcript (p = 1.1e-4) and MS patients were
the minor producers (p = 0.031). Analysis on peripheral
blood of HC(N=37) with flow cytometry showed that in
myeloid DC (CD11c+) the homozygous individuals for
the risk allele had a higher percentage of LIGHT positive
cells (p-value = 0.04). In conclusion, we propose that an
altered TNFSF14 expression in immune cells driven by an
intronic variant can contribute to MS pathogenesis. Par-
ticularly, this variant seems to be associated with a low
TNFSF14 RNA expression in a mixed population of
PBMCs and with a higher percentage of LIGHT positive
cells in myeloid dendritic cells, suggesting a cell specific
influence of this variant on LIGHT expression at the
protein level. 1San Raffaele Telethon Institute for Gene Therapy, Milan,
Italy, 2Diabetes Research Institute (DRI), San Raffaele
Hospital, Milan, Italy, 3Institute for Experimental Neurology
(INSPE), IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: The innate immune system is highly com-
plex and comprises cell populations with organ-specific
properties. Key open questions include how the tissue of
origin shapes the phenotype of myeloid cells and how this
heterogeneity is altered in pathological conditions. Here, we
combine single-cell (sc)RNA-Seq and immunophenotypic
analyses to build a comprehensive view of the tissue mouse
myeloid cell landscape, at steady state and in models of
pancreatic adenocarcinoma (PDAC). A new workflow for classification of genetic variants
pathogenicity applied to hereditary recurrent fevers by the
International Study Group for Systemic AutoInflammatory
Diseases (INSAID) M. E. van Gijn1,2, I. Ceccherini3, Y. Shinar4, E. C. Carbo1,
M. Slofstra5, J. I. Arostgui6, G. Sarrabay7, D. Rowczenio8,
E. Omoyımnı9, B. Peynircioglu10, F. Milhavet11, M. A. Swertz5,
I. Touitou11 M. E. van Gijn1,2, I. Ceccherini3, Y. Shinar4, E. C. Carbo1,
M. Slofstra5, J. I. Arostgui6, G. Sarrabay7, D. Rowczenio8, E. Omoyımnı9, B. Peynircioglu10, F. Milhavet11, M. A. Swertz5,
I. Touitou11 1Department of Genetics, University Medical Center Utrecht,
Utrecht, Netherlands, 2Laboratory of Translational
Immunology, University Medical Center Utrecht, Utrecht,
Netherlands, 3DUOC Medical Genetics, Giannina Gaslini
Institute, Genova, Italy, 4Laboratory of FMF and
autoinflammatory diseases, Sheba Medical center, Tel
Hashomer, Israel, 5Department of Genetics, University
Medical Center Groningen, Groningen, Netherlands,
6Department of Immunology, Hospital Clinic-IDIBAPS,
Barcelona, Spain, 7Laboratory of rare and autoinflammatory
diseases, CHU Montpellier, Montpellier, France, 8National
Amyloidosis Centre, Division of Medicine, UCL, Royal Free
Hospital, London, United Kingdom, 9UCL Great Ormond
Street Institute of Child Health (ICH), London, United
Kingdom, 10Department of Medical Biology and Genetics,
Hacettepe University Faculty of Medicine, Ankara, Turkey,
11Laboratory of rare and autoinflammatory diseases, CHU
Montpellier, Monpellier, France M.E. van Gijn: None. I. Ceccherini: None. Y. Shinar:
None. E.C. Carbo: None. M. Slofstra:
None. J.I. Arostgui: None. G. Sarrabay: None. D. Rowczenio:
None. E. Omoyımnı: None. B. Peynircioglu: None. F. Milhavet: None. M.A. Swertz: None. I. Touitou: None. P07.13A Atypical paroxysmal nocturnal hemoglobinuria presenting
with autoinflammatory symptoms is caused by germline
and somatic mutations involving PIGT Y. Murakami1, T. Hirata1, S. Murata1, T. Kinoshita1,
M. Kawamoto2, S. Murase2, H. Yoshimura2, N. Kohara2,
N. Inoue3, M. Osato4, J. Nishimura4, Y. Ueda4, Y. Kanakuru4,
P. M. Krawitz5, A. Knaus5, M. Jäger6, R. Flöttmann6,
T. Eggermann7, B. Hoechsmann8, M. Anliker8,
H. Schrezenmeier8 Y. Murakami1, T. Hirata1, S. Murata1, T. Kinoshita1,
M. Kawamoto2, S. Murase2, H. Yoshimura2, N. Kohara2,
N. Inoue3, M. Osato4, J. Nishimura4, Y. Ueda4, Y. Kanakuru4,
P. M. Krawitz5, A. Knaus5, M. Jäger6, R. Flöttmann6,
T. Eggermann7, B. Hoechsmann8, M. Anliker8,
H. Schrezenmeier8 Y. Murakami1, T. Hirata1, S. Murata1, T. Kinoshita1,
M. Kawamoto2, S. Murase2, H. Yoshimura2, N. Kohara2,
N. Inoue3, M. Osato4, J. Nishimura4, Y. Ueda4, Y. Kanakuru4,
P. M. Krawitz5, A. Knaus5, M. Jäger6, R. Flöttmann6,
T. Eggermann7, B. Hoechsmann8, M. Anliker8,
H. Schrezenmeier8 Background: Hereditary recurrent fevers (HRF) are rare
inflammatory diseases sharing similar clinical symptoms,
and effectively treated with anti-inflammatory biological
drugs. Accurate diagnosis of HRF relies heavily on genetic
testing, yet in the big data era the clinical significance of
most gene variants remains unsolved or controversial. 1Research Institute for Microbial Diseases, Osaka University,
Osaka, Japan, 2Department of Neurology, Kobe City Medical
Center General Hospital, Kobe, Japan, 3Osaka Medical Center
for Cancer, Osaka, Japan, 4Department of Hematology and
Oncology, Graduate School of Medicine, Osaka University,
Osaka, Japan, 5Institute for Genomic Statistics and
Bioinformatics - University Hospital Bonn, Bonn, Germany,
6Institute for Medical Genetics and Human Genetics, Charité
University Medicine Berlin, Berlin, Germany, 7Institute for
Human Genetics, RWTH Aachen, Aachen, Germany, 8Institute
of Transfusion Medicine, University of Ulm, Ulm, Germany Methods: We configured a MOLGENIS web-platform to
share and analyze pathogenicity classifications of the
variants, and to manage a consensus-based classification
process. Four experts in HRF genetics submitted indepen-
dent classifications of 858 variants of 4 well known HRF
genes: MEFV, TNFRSF1A, NLRP3 and MVK. Classifica-
tions were driven to consensus by recruiting 4 more expert
opinions and by targeting discordant classifications in 5
iterative rounds. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired
disorder of the blood-forming system. Typically, affected
hematopoetic stem cells (HSCs) in PNH harbour a single
somatic loss-of-function mutation in the X-linked PIGA
gene. P07.11C We noted a lower pathogenic variant load and a
higher fraction of variants with unknown or unsolved
clinical significance in the MEFV gene. all genes. We noted a lower pathogenic variant load and a
higher fraction of variants with unknown or unsolved
clinical significance in the MEFV gene. Conclusion: Applying a consensus driven process on the
pathogenicity assessment of experts yielded rapid classifi-
cation of almost all variants of four HRF genes. The high-
throughput database will profoundly assist clinicians and
geneticists in the diagnosis of HRFs. The configured
MOLGENIS platform and consensus evolution protocol
are usable for assembly of other variant-pathogenicity
databases. The MOLGENIS software is available for reuse
at http://github.com/molgenis/molgenis, and the specific
HRF configuration is available at http://molgenis.org/said/. The HRF pathogenicity-classifications will be publically on
the INFEVERS database at http://fmf.igh.cnrs.fr/ISSAID/
infevers/. P07.11C Introduction: The innate immune system is highly com-
plex and comprises cell populations with organ-specific
properties. Key open questions include how the tissue of
origin shapes the phenotype of myeloid cells and how this
heterogeneity is altered in pathological conditions. Here, we
combine single-cell (sc)RNA-Seq and immunophenotypic
analyses to build a comprehensive view of the tissue mouse
myeloid cell landscape, at steady state and in models of
pancreatic adenocarcinoma (PDAC). Methods: Tissue-resident myeloid cells (CD45+CD11b+)
were isolated from 9 tissues in healthy mice (blood, bone
marrow (BM), spleen, lung, liver, pancreas, brain, colon
and intestine) and subjected to scRNA-Seq. Ortho- or
hetero-topic PDAC models were established by intrapan-
creatic or subcute injection of DT6606 cells in C57/BL6
mice. Tumor-infiltrating, circulating and BM myeloid cells
were analysed throughout disease progression by scRNA-
Seq, immunophenotypic and histological analyses. Results:
scRNA-Seq
analysis
shows
tissue-specific
heterogeneity in monocytes, neutrophils, dendritic cells
and macrophages populations at the steady state and
identified putative gene networks underlying this organ
specialization. Pancreatic cancer had a dramatic impact on
the composition and transcriptional heterogeneity of tumor-
infiltrating
and
circulating
pools
of
monocytes
and
neutrophils, and also affected BM myelopoiesis at the
single-cell level. Differential gene expression analysis in
tumor-associated vs. steady-state myeloid cells revealed
transcriptional programs aberrantly activated in the PDAC
immune microenvironment. M. Zuccalà*:
None. N. Barizzone*:
None. C. Basagni*: None. E. Boggio*: None. L. Gigliotti*: None. M. Sorosina*: None. R. Bordoni: None. F. Clarelli:
None. S. Anand: None. E. Mangano: None. D. Vecchio:
None. F. Esposito: None. E. Corsetti: None. G. Pre-
debon: None. R. Cantello: None. V. Martinelli: None. G. Comi: None. M. Leone: None. G. De Bellis: None. U. Dianzani*: None. F. Martinelli-Boneschi*: None. S. D'Alfonso*: None. Conclusions: Our analyses link cellular and molecular
alterations
in
the
immune
microenvironment
to
the
progression of PDAC. These results have implications for
the design of cell and gene therapy strategies aiming at
stimulating anti-tumor immunity in pancreatic cancer. G. Barbiera: None. M. Genua: None. F. Cilenti: None. D. Iodice: None. E. Dugnani: None. A. Citro: None. A. Capotondo: None. M. Milani: None. A. Cantore: None. 206 J. del Picchia N. Coltella: None. L. Barbarossa: None. G. Martino:
None. L. Piemonti:
None. L. Naldini:
None. R. Ostuni: None. all genes. We noted a lower pathogenic variant load and a
higher fraction of variants with unknown or unsolved
clinical significance in the MEFV gene. all genes. P07.13A Herein we report four cases of this new subgroup: A
predisposing germline mutation in PIGT, which is an
autosomal gene of the glycosylphosphatidylinositol (GPI)- Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired
disorder of the blood-forming system. Typically, affected
hematopoetic stem cells (HSCs) in PNH harbour a single
somatic loss-of-function mutation in the X-linked PIGA
gene. Herein we report four cases of this new subgroup: A
predisposing germline mutation in PIGT, which is an
autosomal gene of the glycosylphosphatidylinositol (GPI)- Results: A consensus classification was reached for 804/
858 variants (94%). None of the unsolved variants (6%)
remained with opposite classifications (e.g. pathogenic
versus benign). New mutational hotspots were found in Abstracts from the 51st European Society of Human Genetics Conference: Posters 207 real-time TaqMan assay. Cytokine concentration in sera
was measured by enzyme-linked immunosorbent assay. Localization of PAD4 and PAD2 protein was indicated by
immunohistochemistry. We also generate Padi2−/−mice
and performed experimental arthritis. We demonstrated that
the clinical disease score was significantly decreased in
Padi4−/−mice and Padi4 expression was induced by CII
immunization. In Padi4−/−mice sera, serum anti-type II
collagen (CII) IgM, IgG, and inflammatory cytokine levels
were also significantly decreased compared with those in
wild-type mice sera. Interestingly, Padi2 expression was
compensationally induced in CD11b+ cells of Padi4-/-
mice. We also demonstrated that the clinical disease score
was significantly decreased in Padi2−/−CIA mice. anchor synthesis pathway, is followed by a second somatic
hit. Deep sequencing and array-CGH identified acquired
deletions on chromosome 20q in PNH cells that include
PIGT and a commonly deleted region in myelodysplastic
syndromes, that is known to be differentially methylated. This results in a complete loss of expression of certain
genes at this locus which is also thought to contribute to the
clonal expansion. The deficiency of GPI-anchored proteins
on PNH cells results in a lack of complement regulation.In
contrast to classical PNH, PIGT mutations impair loading of
substrate to the anchor and thus result in an accumulation of
unbound GPI molecules.This difference in the pathophy-
siology can also be visualised by FACS analysis of blood:
While CD55 and CD59 expression is reduced in all PNH
cells, the atypical PNH cells can be discriminated by a
specific antibody that binds free GPI anchors. Besides
classical PNH symptoms of anemia, thrombosis, and
hemolysis, patients with PIGT-mutations also manifest with
additional autoinflammatory symptoms. P07.15C Y. Murakami: None. T. Hirata: None. S. Murata:
None. T. Kinoshita: None. M. Kawamoto: None. S. Murase: None. H. Yoshimura: None. N. Kohara: None. N. Inoue: None. M. Osato: None. J. Nishimura: None. Y. Ueda: None. Y. Kanakuru: None. P.M. Krawitz: None. A. Knaus: None. M. Jäger: None. R. Flöttmann: None. T. Eggermann:
None. B. Hoechsmann:
None. M. Anliker: None. H. Schrezenmeier: None. Rare regulatory variant in the MEF2D gene is associated
with SLE in Swedish patients and contributes to the gene
regulation and splicing S. V. Kozyrev1, F. H. G. Farias1,2, J. Dahlqvist1, D. Leonard3,
M. Wilbe4,5, S. N. Abramov1,6, A. Alexsson3, G. R. Pielberg1,
H. Hansson-Hamlin7, G. Andersson4, K. Tandre3, M. L. Eloranta3, L. Rönnblom3, K. Lindblad-Toh1,8 P07.13A It is hypothesized
that the free GPI-anchor that accumulates in affected cells is
causally related to autoinflammation. Based on these find-
ings, we propose the new entity of atypical PNH. It appears that Padi4 and Padi2 enhance collagen-initiated
inflammatory responses. Our results revealed that PAD4
affected on expression of various cytokines and also
controlled Padi genes. A. Suzuki:
None. Y. Kochi:
None. K. Yamamoto: None. A. Suzuki:
None. Y. Kochi:
None. K. Yamamoto: None. A. Suzuki:
None. Y. Kochi:
None. K. Yamamoto: None. P07.14B 1Department of Medical Biochemistry and Microbiology,
Uppsala University, Uppsala, Sweden, 2The Genome Institute,
Washington University School of Medicine, St. Louis, MO,
United States, 3Department of Medical Sciences, Uppsala
University, Uppsala, Sweden, 4Department of Animal Breeding
and Genetics, Swedish University of Agricultural Sciences
(SLU), Uppsala, Sweden, 5Department of Immunology,
Genetics and Pathology, Uppsala University, Uppsala,
Sweden, 6Institute of Fundamental Medicine and Biology,
Kazan Federal University, Kazan, Russian Federation,
7Department of Clinical Sciences, Swedish University of
Agricultural Sciences (SLU), Uppsala, Sweden, 8Broad
Institute, Cambridge, MA, United States Functional study of Peptidylarginine Deiminase genes in
arthritis model mice A. Suzuki, Y. Kochi, K. Yamamoto RIKEN, Yokohama, Japan This is until recently undescribed variant. In this study, we
also analysed structural effect of found sequence variant in
silico. In particular, we used crystal structure of N-terminal
domain of human platelet receptor GPIb-alpha. Replace-
ment of aliphatic amino-acid Leu 59 with charged, polar
and larger arginine most probably disrupts the protein
structure. Results: Whole-exome sequencing identified a hetero-
zygous
single-nucleotide
change
in
GP1BA
(exone2:
c.176T>G), encoding a p.Leu59Arg substitution in the N-
terminal domain, segregating with macrothrombocytopenia. This is until recently undescribed variant. In this study, we
also analysed structural effect of found sequence variant in
silico. In particular, we used crystal structure of N-terminal
domain of human platelet receptor GPIb-alpha. Replace-
ment of aliphatic amino-acid Leu 59 with charged, polar
and larger arginine most probably disrupts the protein
structure. Results: We identified a novel rare regulatory variant
rs200395694 located in the MEF2D gene encoding for the
myocyte-specific enhancer factor 2D transcription factor
associated with SLE in Swedish patients (total 504 SLE
patients and 839 healthy controls, p = 0.013, CI=1.1-10). The risk allele was strongly associated with the triad of
disease manifestations including Raynaud’s phenomenon,
anti-RNP and anti-Sm antibodies (p = 0.00046, CI 5.05-∞). The
region
has
properties
of
an
active
cell-specific
enhancer,
differentially
affected
by
the
alleles
of
rs200395694. In addition, the risk allele exerts inhibitory
effect on the splicing of the alternative tissue-specific
isoform, and thus may modify the target gene set regulated
by this isoform. Conclusions: A germinal GP1BA mutation (exone2:
c.176T>G) disrupts the molecular structure of the protein
and is the reason of hereditary thrombocytopenia in this
family. Conclusions: We present evidence of genetic association
of a novel rare regulatory variant rs200395694 with SLE in
Swedish patients. Supported by Ministry of Health of the Czech Republic,
grant nr. 16-29447A. All rights reserved. Supported by Ministry of Health of the Czech Republic,
grant nr. 16-29447A. All rights reserved. K. Staňo Kozubik: None. J. Trizuljak: None. M. Peová: None. K. Pál: None. K. Réblová: None. L. Radová: None. Š. Pospíšilová: None. M. Doubek: None. S.V. Kozyrev: None. F.H.G. Farias: None. J. Dahlq-
vist: None. D. Leonard: None. M. Wilbe: None. S.N. Abramov: None. A. Alexsson: None. G.R. Pielberg:
None. H. Hansson-Hamlin: None. G. Andersson: None. K. Tandre: None. M.L. Eloranta: None. L. Rönnblom:
None. K. Lindblad-Toh: None. P07.16D K. Tokunaga1, Y. Omae1, L. Toyo-oka1, H. Yanai1, P07.17A A novel susceptibility locus CD53 in tuberculosis identified
by pathogen lineage-based genome-wide association study A novel germline mutation in GP1BA gene in family with
hereditary macrothrombocytopenia A novel germline mutation in GP1BA gene in family with
hereditary macrothrombocytopenia S. Wattanapokayakit2, N. Smittipat3, P. Paliittapongarnpim
2
4
2 S. Wattanapokayakit2, N. Smittipat3, P. Paliittapongarnpim3,
N. Wichukchinda2, T. Mushiroda4, S. Mahasirimongkol2 N. Wichukchinda2, T. Mushiroda4, S. Mahasirimongkol2 K. Staňo Kozubik1,2, J. Trizuljak1,2, M. Peová1, K. Pál1,
K. Réblová1, L. Radová1, . Pospíilová1,2, M. Doubek1,2 K. Staňo Kozubik1,2, J. Trizuljak1,2, M. Peová1, K. Pál1,
K. Réblová1, L. Radová1, . Pospíilová1,2, M. Doubek1,2 1Dept Human Genetics, Grad Sch Medicine, Univ Tokyo,
Tokyo, Japan, 2Medical Life Sciences Institute, Department of
Medical Sciences, Ministry of Public Health, Nonthaburi,
Thailand, 3National Center for Genetic Engineering and
Biotechnology, Pathum Thani, Thailand, 4RIKEN Center for
Integrative Medical Sciences, Yokohama, Japan RIKEN, Yokohama, Japan Previously, peptidylarginine deiminase type 4 (PADI4) was
identified as a susceptibility gene for Rheumatoid arthritis
(RA) by genome-wide association studies. Peptidyl citrul-
line is a target antigen of anti-citrullinated peptide anti-
bodies (ACPAs), and only PADs (translated protein from
PADI genes) can provide peptidyl citrulline via modifica-
tion of protein substrates. Also the distribution of PADI4
and PADI2 has overlap in immune cells. The aim of this
study was to investigate the relationship between PADI4
gene and PADI2 gene in the progression of RA. Introduction: Systemic lupus erythematosus (SLE) is an
autoimmune disorder with heterogeneous clinical manifes-
tations and complex etiology. The common associated
SNPs explain only a small part of disease heritability sug-
gesting the contribution from rare genetic variants, unde-
tectable in GWAS. We searched for novel rare variants
associated with SLE. Padi4−/−DBA1J and wild-type mice were immunized
with bovine type II collagen (CII) to develop collagen-
induced arthritis (CIA). Expression of various inflammatory
cytokines and Padi genes in immune cells was detected by J. del Picchia 208 members: four of them showing signs of thrombocytopenia,
two healthy family members and one family member with
borderline platelet count. Exome libraries were prepared
according to the protocol for Nimblegen SeqCap EZ Exome
v3 and sequencing was performed on NextSeq 500 for all of
them. Found variants of individuals with thrombocytopenia
phenotype were compared to variants of healthy family
members. Materials and Methods: 144 SLE patients and 17
controls were used for targeted re-sequencing of coding and
conserved regulatory regions within and around 215
candidate genes. The variant enriched in cases was
validated by genotyping in additional 360 patients and
822 healthy controls. Fisher’s exact test and logistic
regression were used for genetic association analysis. The
regulatory effect of the novel variant was studied by EMSA,
luciferase reporter assays and minigenes. Materials and Methods: 144 SLE patients and 17
controls were used for targeted re-sequencing of coding and
conserved regulatory regions within and around 215
candidate genes. The variant enriched in cases was
validated by genotyping in additional 360 patients and
822 healthy controls. Fisher’s exact test and logistic
regression were used for genetic association analysis. The
regulatory effect of the novel variant was studied by EMSA,
luciferase reporter assays and minigenes. Results: Whole-exome sequencing identified a hetero-
zygous
single-nucleotide
change
in
GP1BA
(exone2:
c.176T>G), encoding a p.Leu59Arg substitution in the N-
terminal domain, segregating with macrothrombocytopenia. 1Central European Institute of Technology, Brno, Czech
Republic, 2University Hospital and Faculty of Medicine, Brno,
Czech Republic 1Central European Institute of Technology, Brno, Czech
Republic, 2University Hospital and Faculty of Medicine, Brno,
Czech Republic 1Central European Institute of Technology, Brno, Czech
Republic, 2University Hospital and Faculty of Medicine, Brno,
Czech Republic Tuberculosis (TB) is a major global infectious disease that is
caused by Mycobacterium tuberculosis (M. tb) and TB
onset is known to be affected by host genetic factors. In this
study, we focused on the heterogeneity of M. tb lineages
and assessed its possible interaction with host genetic fac-
tors. Genome-wide
association
analyses
stratified
by
pathogen lineage information and age at onset revealed that
two SNPs on chromosome 1p13 were specifically asso-
ciated with non-Beijing lineage infected old age onset cases Introduction: Hereditary thrombocytopenias are a rare and
heterogeneous
group
of
disorders,
associated
with
approximately 30 causal genes involved in the process of
megakaryopoesis and thrombopoesis. Pathological muta-
tions lead to disruption of these processes and origin of
thrombocytopenia. Materials and Methods: We identified a family with
autosomal
dominant
thrombocytopenia
and
increased
platelet volume. We performed analysis of eight family P08 Intellectual Disability 1Medical Genetics, Department of Molecular Medicine,
University of Pavia, Pavia, Italy, 2Department of Pediatrics,
Fondazione IRCCS Policlinico San Matteo, University of
Pavia, Pavia, Italy, 3Department of Pediatric Hematology-
Oncology, IRCCS Bambino Gesù Children's Hospital, Rome,
Italy, 4B Cell Physiopathology Unit, IRCCS Bambino Gesù
Children's Hospital, Rome, Italy, 5Cell Factory, Pediatric
Haematology/Oncology, Fondazione IRCCS Policlinico San
Matteo, Pavia, Italy P07.18B E. Errichiello: None. A. Licari: None. P. Merli: None. R. Carsetti: None. P. Comoli: None. G. Marseglia: None. O. Zuffardi: None. E. Errichiello: None. A. Licari: None. P. Merli: None. R. Carsetti: None. P. Comoli: None. G. Marseglia: None. O. Zuffardi: None. Non-response to vaccines: still an enigma? B-cell
transcription factor POU2F2/OCT2 is a potential candidate E. Errichiello1, A. Licari2, P. Merli3, R. Carsetti4, P. Comoli5,
G. Marseglia2, O. Zuffardi1 E. Errichiello1, A. Licari2, P. Merli3, R. Carsetti4, P. Comoli5,
G. Marseglia2, O. Zuffardi1 E. Errichiello1, A. Licari2, P. Merli3, R. Carsetti4, P. Comoli5,
G. Marseglia2, O. Zuffardi1 Whole-exome sequencing revealed a shared
heterozygous frameshift variant (c.1285dupC;p.Leu429-
ProfsTer73) affecting POU2F2 (19q13.2), a non-OMIM
gene with low tolerance to loss-of-function variations
(pLI=0.97), encoding the transcription factor OCT2 (octa-
mer-binding protein 2) that regulates immunoglobulin
expression in germinal center B-cells. The variant was
unreported in gnomAD and was shown to segregate in the
family and to have occured de novo in the mother. Impor-
tantly, heterozygous knock-out mice show a pathological
phenotype restricted to immune/hematopoietic system with
reduction of B-cells and IgM, thus recapitulating our
patients’ phenotype. Analysis of mRNA from B-LCLs and
fibroblasts of both carriers revealed a stable mutant trascript
and excluded mRNA decay, suggesting a dominant-
negative effect; in contrast, somatic POU2F2 amplifica-
tions, leading to demonstrated overexpression, have been
described in diffuse large B-cell lymphomas. Further
functional assays showed severe deficiency of switched
memory B-cells and reduced surface and intracellular
immunoglobulin expression in both patients. In conclusion,
our preliminary findings identified a novel gene likely
involved in B-cell anergy and highlighted POU2F2 as an
attractive target for enhancing humoral immune response to
vaccination. K. Tokunaga: None. Y. Omae: None. L. Toyo-oka:
None. H. Yanai: None. S. Wattanapokayakit: None. N. Smittipat:
None. P. Paliittapongarnpim:
None. N. Wichukchinda:
None. T. Mushiroda:
None. S. Mahasirimongkol: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 209 impaired, with selective deficit of B-memory cells and IgM
production. Whole-exome sequencing revealed a shared
heterozygous frameshift variant (c.1285dupC;p.Leu429-
ProfsTer73) affecting POU2F2 (19q13.2), a non-OMIM
gene with low tolerance to loss-of-function variations
(pLI=0.97), encoding the transcription factor OCT2 (octa-
mer-binding protein 2) that regulates immunoglobulin
expression in germinal center B-cells. The variant was
unreported in gnomAD and was shown to segregate in the
family and to have occured de novo in the mother. Impor-
tantly, heterozygous knock-out mice show a pathological
phenotype restricted to immune/hematopoietic system with
reduction of B-cells and IgM, thus recapitulating our
patients’ phenotype. Analysis of mRNA from B-LCLs and
fibroblasts of both carriers revealed a stable mutant trascript
and excluded mRNA decay, suggesting a dominant-
negative effect; in contrast, somatic POU2F2 amplifica-
tions, leading to demonstrated overexpression, have been
described in diffuse large B-cell lymphomas. Further
functional assays showed severe deficiency of switched
memory B-cells and reduced surface and intracellular
immunoglobulin expression in both patients. In conclusion,
our preliminary findings identified a novel gene likely
involved in B-cell anergy and highlighted POU2F2 as an
attractive target for enhancing humoral immune response to
vaccination. (p = 4.86E-08, OR=1.72 [95%CI=1.41-2.09], n = 314),
but were not associated with Beijing lineage infected old
age onset cases (p = 0.0870, OR=1.26 [95%CI=0.97-
1.64], n = 155), when we compared them to the population
matched 782 healthy controls. These SNPs were associated
with both East-African Indian (EAI) and Euro-American
lineages in the non-Beijing lineage group. These SNPs were
located near CD53, which encodes a leukocyte surface
glycoprotein and has not been reported to be associated with
TB onset. However, interestingly, one of the significant
SNPs was previously reported as a cis-expression quanti-
tative trait locus (eQTL) of CD53 expression level in den-
dritic cells infected by M. tb. This is the first report of TB
pathogen lineage-based genome-wide association study and
successfully identified a TB-associated locus at a genome-
wide significance level. The present results indicated that
host genetic risk in TB is affected by pathogen genetic
background and demonstrate the importance of analyzing
the
interaction
between
host
and
pathogen
genomic
variations. impaired, with selective deficit of B-memory cells and IgM
production. P08.01A
Targeted NGS of the TSC1/TSC2 genes R. Polli1, E. Bettella1, E. Leonardi1, M. Aspromonte1, F. Cesca1,
S. Rossato2, I. Toldo3, A. Murgia1 1Laboratory of Molecular Genetics of Neurodevelopment,
Padua, Italy, 2Pediatrics Unit,San Bortolo Hospital, Vicenza,
Italy, 3Neuropediatrics Unit,Department of Women’s and
Children’s Health, Padua, Italy Unresponsiveness to vaccines affects 2-10% of individuals,
representing an extraordinary limitation for infection pre-
vention worldwide. Genetic determinants are still mainly
unknown, although in recent years GWAS identified
potential susceptibility loci (HLA-DQ, HLA-DR, CXCR5). We investigated a patient and her daughter with unrespon-
siveness to vaccines (tetanus, diphtheria, hepatitis B,
poliovirus) and intermittent infectious episodes, but other-
wise unremarkable clinical history. Lymphocyte prolifera-
tion assay to tetanus and diphtheria toxoids was highly Tuberous sclerosis TSC (MIN:191100,613254) is an auto-
somal dominant disorder characterized by benign tumor
growths in multiple organ systems. In 75-90% of cases TSC
is due to mutations in the TSC1 (OMIM # 605284) or TSC2
(OMIM# 191092) genes. Somatic mosaicism potentially
account for up to 26% of TSC cases. We report the results
of NGS analysis in 3 familial and 8 sporadic unrelated cases
referred with clinically diagnosed (9) or highly suspected
TSC (2). DNA samples from peripheral blood leukocytes 210 J. del Picchia and oral mucosa were analyzed by AmpliSeq custom panel
covering the coding sequence of the two genes. NGS was
performed on the Ion Torrent PGM platform. Data analysis
was performed with ION Torrent Suite v.5; median read
depth was ≥500x. Variants were annotated with wAN-
NOVAR and filtered based on MAF (<1%), phylogenetic
conservation and CADD score; pathogenicity was evaluated
by 12 in silico tools and Sanger sequencing validation
performed for all the filtered likely pathogenic variants. We
identified 6 heterozygous seemingly germ-line mutations,
two of which novel, in cases with a definite clinical diag-
nosis:
2
TSC2
frameshift
deletions
(NM_000548:
c.5076delG;c.935delT)
and1
TSC2
splice-mutation
(NM_000548:c.2221-2A>C), 2 TSC1 rare single nucleo-
tide variants (NM_000368:c.569 C>G;c.647 T>C) and 1
frameshift deletion (NM_000368; c.709_716 del). The latter
was present in the DNA of the proband's father as mosaic
mutation at about 10% of mutant allele frequency. Five
subjects remained without a molecular diagnosis; we plan to
extend our NGS analysis in negative samples with the
inclusion of TSC1 and TSC2 promoter, UTRs and intronic
regions These latter two cases and published features of individuals
with even larger 13q31.3 overlapping duplications sug-
gested that gene expression imbalance of GPC5 could be
causative. P08.03C
15q13.3 microdeletion and microduplication in patients
with neurodevelopment disorders 15q13.3 microdeletion and microduplication in patients
with neurodevelopment disorders R. Polli: None. E. Bettella: None. E. Leonardi: None. M. Aspromonte: None. F. Cesca: None. S. Rossato:
None. I. Toldo: None. A. Murgia: None. E. Dagytė1,2, A. Matulevičienė1,2, L. Ambrozaitytė1,2,
R. Laimutė2, B. Aleksiūnienė1,2, B. Burnytė1,2, A. Utkus1,2 E. Dagytė1,2, A. Matulevičienė1,2, L. Ambrozaitytė1,2, E. Dagytė1,2, A. Matulevičienė1,2, L. Ambrozaitytė1,2, P08.01A
Targeted NGS of the TSC1/TSC2 genes The limited extent of the rearrangement and the
normal expression level of GPC5 in cells of our proband,
however, provides evidence against this hypothesis. Our results suggest that duplication of the miR-17~92
cluster is linked to a new syndrome characterized by
features mirroring those of Feingold syndrome type 2,
which is associated with haploinsufficiency of the region. Whereas deletion of the region is linked with short stature
and microcephaly, duplication is on the contrary associated
with overgrowth and macrocephaly. Similar dosage depen-
dent mirror phenotypes on BMI and head circumference
have been previously reported for deletion and duplications
of the 1q21.1, 2p15, 16p11.2 BP4-BP5, 16p11.2 BP2-BP3
and 17p11.2 CNVs. E. Siavriene: None. E. Preiksaitienė: None. Z. Mal-
dzienė: None. L. Ambrozaitytė: None. L. Gueneau:
None. A. Reymond: None. V. Kučinskas: None. P08.05A Array-CGH cohort of 1500 patients with
Neurodevelopmental Disorders: Copy Number Variation in
16p13.11 Novel ADAT3 variants associated with RNA modification
defects and autosomal recessive neurodevelopmental
disorders M. Val1, I. M. Carreira1,2,3, L. M. Pires1, J. Ribeiro1,2,3,
N. Lavoura1, S. I. Ferreira1,2, M. Venancio4, F. Ramos4, J. B. Melo1,2,3 A. Thuresson1, J. Ramos2, J. Halvardson1, E. Kuchinskaya3,
R. Maroofian4, E. Ghayoor Karimiani4, N. Mazaheri5,
H. Galehdari5, G. Shariati6, D. Fu2, L. Feuk1 1Laboratório de Citogenética e Genómica, Faculdade de
Medicina da Universidade Coimbra, Coimbra, Portugal,
2iCBR-CIMAGO- Centro de Investigação em Meio Ambiente,
Genética e Oncobiologia, Faculdade de Medicina da
Universidade Coimbra, Coimbra, Portugal, 3CNC- IBILI,
Universidade de Coimbra, Coimbra, Portugal, 4Serviço de
Genética Médica, Hospital Pediátrico, Centro Hospitalar e
Universitário de Coimbra, Coimbra, Portugal 1Uppsala University, Uppsala, Sweden, 2University of
Rochester, Rochester, NY, United States, 3Linköping
University, Linköping, Sweden, 4University of London,
London, United Kingdom, 5Shahid Chamran University of
Ahvaz, Ahvaz, Iran, Islamic Republic of, 6Ahvaz Jundishapur
University of Medical Sciences, Ahvaz, Iran, Islamic Republic
of 1Uppsala University, Uppsala, Sweden, 2University of
Rochester, Rochester, NY, United States, 3Linköping
University, Linköping, Sweden, 4University of London,
London, United Kingdom, 5Shahid Chamran University of
Ahvaz, Ahvaz, Iran, Islamic Republic of, 6Ahvaz Jundishapur
University of Medical Sciences, Ahvaz, Iran, Islamic Republic
of Chromosomal region 16p13.11 is structurally complex,
subdivided into three single-copy sequence blocks called
intervals I, II and III. Each block is flanked by low-copy
repeats (LCRs) with highly homologous DNA sequences
making it prone to non-allelic homologous recombination
(NAHR) being a major source of de novo genomic rear-
rangements. 16p13.11 copy number variants (CNVs) have
variable sizes (0,8 to 3,3Mb), encompassing one or more of
the
three
intervals. Interval
II
(chr16:15.48-16.32Mb,
GRCh37/hg19) CNVs, have the higher number of patients
reported so far, involving a set of eight genes, including
NDE1, referred as a strong candidate gene for neurodeve-
lopmental disorders. Clinical features of patients with
microdeletions
or
microduplications
at
chromosome
16p13.11, have been associated with a range of neurode-
velopmental disorders including autism spectrum disorders Post-transcriptional modifications of tRNAs are important
for the regulation and efficiency of translation as well as for
fidelity and stability of the tRNA structure. Defects in these
modifications have been implicated in human diseases such
as neurological and mitochondrial disorders, diabetes and
cancer. P08.02B R. Laimutė2, B. Aleksiūnienė1,2, B. Burnytė1,2, A. Utkus1,2 R. Laimutė2, B. Aleksiūnienė1,2, B. Burnytė1,2, A. Utkus1,2 Microduplication of the 13q31.3 miR17-92 cluster results in
a syndrome with features opposite to those associated with
Feingold syndrome 2 1Institute of Biomedical Sciences, Faculty of Medicine, Vilnius
University, Vilnius, Lithuania, 2Centre for Medical Genetics,
Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania E. Siavriene1, E. Preiksaitienė1, Z. Maldzienė1, L. Ambrozaitytė1,
L. Gueneau2, A. Reymond2, V. Kučinskas1 The proximal long arm of chromosome 15 contains a cluster
of low copy repeats (LCRs), located at breakpoints BP1-
BP5. These mediate various deletions and duplications via
non-allelic homologous recombination. BP4-BP5 micro-
deletion/duplication syndrome may include features of
ASD, a variety of neuropsychiatric disorders, and cognitive
impairment. We report two patients with a deletion within
BP3-BP5 and two patients with smaller duplication within
BP4-BP5. A 15q13.2-13.3 microdeletion (chr15:30955149-
32515681) which encompasses seven protein-coding genes
(ARHGAP11B,
FAN1,
MTMR10,
TRPM1,
KLF13,
OTUD7A, CHRNA7) was detected for the 1st patient 1 y boy
with hypotonia, psychomotor retardation and dysmorphic
features. 2nd patient 8 y boy has a 15q13.1-13.3 deletion
(chr15:29247469-32515681)
which
encompasses
17
protein-coding genes (APBA2, FAM189A1, NDNL2, TJP1,
GOLGA8J, GOLGA8T, CHRFAM7A, GOLGA8R, GOL-
GA8Q,
GOLGA8H,
ARHGAP11B,
FAN1,
MTMR10,
TRPM1, KLF13, OTUD7A, CHRNA7). The patient was
referred
to
clinical
geneticist
due
to
psychomotor 1Department of Human and Medical Genetics, Institute of
Biomedical Sciences, Faculty of Medicine, Vilnius University,
Vilnius, Lithuania, 2Center for Integrative Genomics,
University of Lausanne, Lausanne, Switzerland Deletion of the miR17~92 cluster is associated with Fein-
gold syndrome type 2 (OMIM: #614326) characterized by
short stature, microcephaly, skeletal abnormalities, and
intellectual disability. Deletion of the miR17~92 cluster is associated with Fein-
gold syndrome type 2 (OMIM: #614326) characterized by
short stature, microcephaly, skeletal abnormalities, and
intellectual disability. In the present study, we report a female individual
presenting with opposite features such as tall stature and
macrocephaly, besides developmental delay, skeletal and
digital abnormalities and a de novo ~840kb duplication of
13q31.3
(91166748-92010901)
encompassing
only
miR17~92 cluster. Two individuals carrying overlapping
duplications
of
the
miR17~92
cluster
including
the
proximal GPC5 were previously described. They similarly
present with macrocephaly, developmental delay, skeletal
and digital abnormalities as well as growth abnormalities. Abstracts from the 51st European Society of Human Genetics Conference: Posters 211 retardation, ASD and dysmorphic features. 3rd patient with
expressive language impairment and attention problems has
307 kb size duplication at 15q13.3 (chr15:32018731-
32325676). P08.02B Duplication
region
encompasses
part
of
OTUD7A gene and part of CHRNA7 gene. 4th patient with
psychomotor delay and short stature has 194 kb duplication
(chr15:32114055-323208134) involving a distal part of
OTUD7A gene. The differential diagnosis of the 15q13.3
microdeletion/microduplication
comprises
an
extensive
spectrum of diseases. There is no consistent or recognizable
phenotype. The BP4-BP5 microdeletion/microduplication
events span CHRNA7, a candidate gene for seizures. However, none of these patients reported here have epi-
lepsy. Both deletion and duplication encompasses OTUD7A
gene which is critical gene for brain function. (ASD), attention-deficit hyperactivity disorder (ADHD),
intellectual disability (ID) and schizophrenia. In our cohort
of 1500 patients, with ID, ASD and congenital anomalies,
studied by Agilent 180K oligonucleotide array-CGH, we
have identified 17 patients with 16p13.11 CNVs (9 dele-
tions and 8 duplications). The majority of the patients
showed high clinical variability with a wide range of phe-
notypic
manifestations:
developmental
delay,
autism,
speech delay, learning difficulties, behavioural problems,
epilepsy, microcephaly and physical dysmorphisms. In our
data, an higher male:female 16p13.11 CNV ratio has not
been detected. In 66% of the cases, the alteration was
inherited from unaffected parents confirming that duplica-
tions and deletions at 16p13.11 represent incomplete
penetration but that predispose to a range of neurodeve-
lopmental disorders. E. Dagytė: None. A. Matulevičienė: None. L. Ambro-
zaitytė: None. R. Laimutė: None. B. Aleksiūnienė: None. B. Burnytė: None. A. Utkus: None. M. Val: None. I.M. Carreira: None. L.M. Pires: None. J. Ribeiro: None. N. Lavoura: None. S.I. Ferreira: None. M. Venancio: None. F. Ramos: None. J.B. Melo: None. M. Val: None. I.M. Carreira: None. L.M. Pires: None. J. Ribeiro: None. N. Lavoura: None. S.I. Ferreira: None. M. Venancio: None. F. Ramos: None. J.B. Melo: None. P08.04D P08.04D
Array-CGH cohort of 1500 patients with
Neurodevelopmental Disorders: Copy Number Variation in
16p13.11 P08.06B Whole-exome sequencing of patients with Angelman-like
phenotypes and no aberration detected in the UBE3A gene M. Williams1,2,3, J. Harraway3, J. McGaughran4,2, C. Patel4,
S. Mehta4,5, A. Harris6,7, M. Lipke8, J. Pinner9,10, H. Goel11,12,
B. Hanna11, C. Stutterd13,14,15, T. Y. Tan13,15, S. White13,15,
A. Yeung13, H. Heussler1,16,6 Results:
Using
ACMG-AMP
variant-interpretation
guidelines, a pathogenic or likely pathogenic variant was
identified in the 14/37 patients (37.8%). Variants were
detected in the ARID1B, BCL11A (2 patients), CDKL5,
DEAF1, GABRA1, HDAC8, HIVEP2, KAT6A, NEXMIF
(KIAA2022), PIGN, POGZ, PURA and TRAPPC9 genes. 1Mater Research Institute – The University of Queensland,
Woolloongabba, Queensland, Australia, 2Faculty of Medicine,
The University of Queensland, St Lucia, Queensland,
Australia, 3Genetic Pathology, Mater Pathology, South
Brisbane, Queensland, Australia, 4Genetic Health Queensland,
Royal Brisbane and Women’s Hospital, Herston, Queensland,
Australia, 5Clinical Genetics Department, Addenbrookes
Hospital, Cambridge, United Kingdom, 6Child Development
Program, Lady Cilento Children’s Hospital, South Brisbane,
Queensland, Australia, 7School of Clinical Medicine -
Children's Health Queensland, The University of Queensland,
South Brisbane, Queensland, Australia, 8Queensland
Metabolic Medicine Lifespan Service, Lady Cilento Children’s
Hospital, South Brisbane, Queensland, Australia, 9Department
of Medical Genomics, Royal Prince Alfred Hospital,
Camperdown, New South Wales, Australia, 10Sydney Centre
for Clinical Genetics, Sydney Children’s Hospital, Randwick,
New South Wales, Australia, 11Hunter Genetics, Waratah, New
South Wales, Australia, 12University of Newcastle, Callaghan,
New South Wales, Australia, 13Victorian Clinical Genetics
Services, Murdoch Children’s Research Institute, Parkville,
Victoria, Australia, 14Department of Neurology, Royal
Children’s Hospital, Parkville, Victoria, Australia,
15Department of Paediatrics University of Melbourne Conclusion: Results from this cohort advocate the utility
of WES, following microarray and UBE3A testing, for
patients with Angelman-like phenotypes or neurodevelop-
mental
disorders
with
unclear
or
atypical
clinical
phenotypes. Conclusion: Results from this cohort advocate the utility
of WES, following microarray and UBE3A testing, for
patients with Angelman-like phenotypes or neurodevelop-
mental
disorders
with
unclear
or
atypical
clinical
phenotypes. M. Williams: None. J. Harraway: None. J. McGaugh-
ran: None. C. Patel: None. S. Mehta: None. A. Harris:
None. M. Lipke: None. J. Pinner: None. H. Goel: None. B. Hanna: None. C. Stutterd: None. T.Y. Tan: None. S. White: None. A. Yeung: None. H. Heussler: None. P08.05A Shariati: None. D. Fu: None. L. Feuk: None. Methods: Singleton WES was performed on a retro-
spective cohort of 37 patients with a clinical diagnosis of
AS or a syndromic intellectual disability with clinical
feature(s) in common with AS. None of the patients had an
identified UBE3A aberration, and all had normal microarray
test results. Variant analysis was restricted to a curated list
of
812
genes
associated
with
Angelman
syndrome,
intellectual
disability
and
related
neurodevelopmental
disorders. P08.05A The conversion of adenosine (A) to inosine (I) of the first
base in the anticodon of tRNA enables alternative pairing
with U, C or A at the wobble position of mRNA codons. The A to I conversion is catalysed by a heterodimeric
adenosine deaminase complex consisting of the ADAT2
and ADAT3 subunits. To date, only a single homozygous
founder mutation, c.430G>A; p.Val144Met, has been
detected in ADAT3. The main features of these patients
are
cognitive
impairment,
strabismus,
hypotonia
and
spasticity or epilepsy. 212 J. del Picchia Parkville, Victoria, Australia, 16Child Health Research Centre,
The University of Queensland, South Brisbane, Queensland,
Australia Using exome sequencing, we identified novel compound
heterozygous variants in ADAT3 in two different non-
consanguineous families, and the homozygous founder
mutation in one consanguineous family. The variants c. [587C>T];[820C>T]
(p.[Ala196Val];[Gln274*]
were
detected in three affected siblings, and c.[928_936del];
[946C>G] (p.[Cys310_Met312del];[His316Asp] in a single
case. All four cases share many of the features presented by
the previously detected founder mutation. The mutated
amino acids are highly conserved, and Sanger sequencing
of cDNA from the three affected siblings shows a
significantly lowered expression of the allele harbouring
the nonsense variant. Further functional studies are on-
going to investigate the pathogenicity of these variants. Introduction: Angelman syndrome (AS) is a neurodeve-
lopmental disorder characterised by moderate to severe
developmental delay, absent or near absent speech, gait
ataxia, microcephaly and seizures. Deficient expression or
function of the maternally inherited UBE3A allele results in
AS. Approximately 5-10% of patients with a presumed
diagnosis of AS do not have an identifiable molecular
cause. A proportion of these patients may have a defect of
maternal UBE3A expression which is not detectable by
current test methods. However, there are other rare syn-
dromes which have clinical features that overlap with AS. Whole-exome sequencing (WES) may have diagnostic uti-
lity in these patients. A. Thuresson: None. J. Ramos: None. J. Halvardson:
None. E. Kuchinskaya: None. R. Maroofian: None. E. Ghayoor Karimiani: None. N. Mazaheri: None. H. Galehdari: None. G. Shariati: None. D. Fu: None. L. Feuk: None. A. Thuresson: None. J. Ramos: None. J. Halvardson:
None. E. Kuchinskaya: None. R. Maroofian: None. E. Ghayoor Karimiani: None. N. Mazaheri: None. H. Galehdari: None. G. Shariati: None. D. Fu: None. L. Feuk: None. A. Thuresson: None. J. Ramos: None. J. Halvardson:
None. E. Kuchinskaya: None. R. Maroofian: None. E. Ghayoor Karimiani: None. N. Mazaheri: None. H. Galehdari: None. G. P08.07C Whole exome sequencing identifies new genes responsible
for Angelman-like syndrome C. Aguilera1, A. Ruiz1, E. Gabau2, N. Baena1, N. Capdevila2,
A. Ramírez2, V. Delgadillo2, S. Ourani2, C. Brun2, S. Derdak3,
S. Laurie3, M. Guitart1 A. Ramírez2, V. Delgadillo2, S. Ourani2, C. Brun2, S. Derdak3,
S. Laurie3, M. Guitart1 1Genetics Laboratory, UDIAT-Centre Diagnòstic.Parc Taulí
Hospital Universitari. Institut d’Investigació i Innovació Parc C. Aguilera1, A. Ruiz1, E. Gabau2, N. Baena1, N. Capdevila2,
A. Ramírez2, V. Delgadillo2, S. Ourani2, C. Brun2, S. Derdak3,
S. Laurie3, M. Guitart1 C. Aguilera1, A. Ruiz1, E. Gabau2, N. Baena1, N. Capdevila2, 213 Abstracts from the 51st European Society of Human Genetics Conference: Posters Taulí I3PT. Universitat Autònoma de Barcelona., Sabadell,
Spain, 2Paediatric Unit. ParcTaulí Hospital Universitari. Institut d’Investigació i Innovació Parc Taulí I3PT. Universitat
Autònoma de Barcelona., Sabadell, Spain, 3CNAG-CRG,
Centre for Genomic Regulation (CRG), Barcelona Institute of
Science and Technology (BIST), Barcelona, Spain Taulí I3PT. Universitat Autònoma de Barcelona., Sabadell,
Spain, 2Paediatric Unit. ParcTaulí Hospital Universitari. Institut d’Investigació i Innovació Parc Taulí I3PT. Universitat
Autònoma de Barcelona., Sabadell, Spain, 3CNAG-CRG,
Centre for Genomic Regulation (CRG), Barcelona Institute of
Science and Technology (BIST), Barcelona, Spain Cowley6,7, M. E. Dinger6,7, J. A. Rosenfeld8, R. Xiao8, M. T. Cho9, L. B. Henderson9, M. J. Guillen Sacoto9, A. Begtrup9,
M. Hamad10, M. Shinawi11, M. Andrews11, M. C. Jones12,
K. Lindstrom13, S. Kayani14, M. Snyder15, M. Villanueva16,
A. Schteinschnaider16, T. Roscioli2,17, E. P. Kirk2,18, A. Bye2,18,
J. Merzaban4, L. Jaremko4, M. Jaremko4, R. K. Sachdev2,18,
F. S. Alkuraya5,19,20, S. T. Arold4 Introduction: Approximately 10% of patients with an
Angelman syndrome (AS) phenotype remain without a
molecular diagnosis. Some of these AS-like syndrome
patients may harbor alternative genetic defects that present
overlapping
clinical
features
with
AS. Whole-exome
sequencing (WES) has been successfully applied to iden-
tify the genetic bases of intellectual disability and autism. 1Genetics of Learning Disability (GoLD) Service, Waratah,
Australia, 2Sydney Children's Hospital, Randwick, Australia,
3School of Women's and Children's Health, University of New
South Wales, Randwick, Australia, 4King Abdullah University
of Science and Technology, Thuwal, Saudi Arabia,
5Department of Genetics, King Faisal Specialist Hospital and
Research Center, Riyadh, Saudi Arabia, 6The Kinghorn
Cancer Centre, Garvan Institute of Medical Research,
Darlinghurst, Australia, 7St Vincent’s Clinical School,
University of New South Wales, Darlinghurst, Australia,
8Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX, United States, 9GeneDx,
Gaithersburg, MD, United States, 10King Khalid University
Hospital, King Saud University, Riyadh, Saudi Arabia,
11Division of Genetics and Genomic Medicine, Washington
University School of Medicine, St. Louis, MO, United States,
12Division of Genetics, Department of Pediatrics, University of
California, San Diego, CA, United States, 13Division of
Genetics and Metabolism, Phoenix Children's Hospital,
Phoenix, AZ, United States, 14University of Texas Southwestern
Medical Center, Dallas, TX, United States, 15Department of
Neurology, Children's Health, Dallas, TX, United States,
16Fundación para la Lucha contra las Enfermedades
Neurológicas de la Infancia, Buenos Aires, Argentina,
17Neuroscience Research Australia, Randwick, Australia,
18School of Women’s and Children’s Health, University of New
South Wales, Randwick, Australia, 19Department of Anatomy
and Cell Biology, College of Medicine, Alfaisal University,
Riyadh, Saudi Arabia, 20Saudi Human Genome Program, King
Abdulaziz City for Science and Technology, Riyadh, Saudi
Arabia Materials and Methods: 17 patients who met the
consistent clinical features of AS and lack a molecular
diagnosis were selected. WES was performed in 17 parents-
patient trios in a Hiseq2000 platform (Illumina) using the
SureSelectXT
Human
All
Exon
V5+UTR
(Agilent). Variants were filtered according to their allele frequency
in the ExAC database and their effect on the protein. Pathogenicity of missense variants was evaluated using
bioinformatics tools. Results: Candidate variants were identified in 14 of 17
patients. Ten of those variants were de novo. According to
the recommendation of the ACMG/AMP, six pathogenic
variants were identified in SATB2, SYNGAP1, ASXL3,
SLC6A1, SPTAN1 and SMARCE1 genes whereas four likely
pathogenic missense variants were identified in other
neurodevelopmental genes. Clinical reevaluation is being
performed in those patients with pathogenic and likely
pathogenic variants. Conclusion: Exome sequencing has proved a valuable
tool to identify the genetic defect in AS-like patients. Newly
identified genes should be added to the expanding list of
differential diagnoses for patients presenting with AS-like
features. We thank Instituto de Salud Carlos III (PI16/
01411), Asociación Española de Síndrome de Angelman
and Fundació Parc Taulí-Institut d’Investigació i Innovació
ParcTaulí I3PT for their financial support. Polyglutamine
expansions
in
the
transcriptional
co-
repressor ATN1, at 12p13.31, have been linked to the
neurodegenerative condition dentatorubral-pallidoluysian
atrophy (DRPLA) via a proposed toxic gain of function. We present detailed phenotypic information on seven
unrelated individuals with de novo missense and in-frame
insertion variants within an evolutionarily conserved 16
amino acid ‘poly HX repeat’ motif of ATN1. The subjects
have severe cognitive impairment, hypotonia, a recogni-
sable facial gestalt and variable congenital anomalies but
lack the progressive symptoms typical of DRPLA. E. E. Palmer1,2,3, S. Hong4, F. Al Zahrani5, M. Omar Hashem5,
F. A. Aleisa5, H. M. Jalal Ahmed4, T. Kandula2,3, R. Macintosh2,
A. Minoche6, C. Puttick6, V. Gayevskiy6, A. P. Drew6, M. J. We
show that a variant in ATN1’s HX repeat is sufficient to
perturb the structural features of the repeat with several Polyglutamine
expansions
in
the
transcriptional
co-
repressor ATN1, at 12p13.31, have been linked to the
neurodegenerative condition dentatorubral-pallidoluysian
atrophy (DRPLA) via a proposed toxic gain of function. We present detailed phenotypic information on seven
unrelated individuals with de novo missense and in-frame
insertion variants within an evolutionarily conserved 16
amino acid ‘poly HX repeat’ motif of ATN1. The subjects
have severe cognitive impairment, hypotonia, a recogni-
sable facial gestalt and variable congenital anomalies but
lack the progressive symptoms typical of DRPLA. We
show that a variant in ATN1’s HX repeat is sufficient to
perturb the structural features of the repeat with several C. Aguilera: None. A. Ruiz: None. E. Gabau: None. N. Baena: None. N. Capdevila: None. A. Ramírez: None. V. Delgadillo: None. S. Ourani: None. C. Brun: None. S. Derdak: None. S. Laurie: None. M. Guitart: None. C. Aguilera: None. A. Ruiz: None. E. Gabau: None. N. Baena: None. N. Capdevila: None. A. Ramírez: None. V. P08.08D De novo variants disruting the HX repeat motif of ATN1
cause a non-progressive neurocognitive disorder with
recognisable facial features and congenital malformations J. del Picchia 214 effects. It alters ligand binding, including the binding to
several proteins which are important in ribosomal function
and regulation of gene expression. In addition, the mutation
affects the nuclear localization of the HX repeat motif. These data suggest that the variant affects the transcriptional
repression activity of ATN1, leading to an apparent gain of
function effect. Our study provides valuable insights into
the function of the HX repeat motifs and ATN1’s primary
roles regulating neuronal and other organ system develop-
ment. This research provides an example of phenotypically
distinct allelic disorders in humans, revealing the power of
unbiased genomic technologies and international colla-
borations in providing diagnoses for individuals with
complex congenital disorders. 1Department of Pediatrics, Division of Medical Genetics,
University of Utah, Salt Lake City, UT, United States, 2Child
Neuropsychiatric Unit - Epilepsy Center, S. Paolo Hospital,
Department of Health Sciences, Università degli Studi di
Milano, Milano, Italy, 3Department of Pathology, University of
Utah, Salt Lake City, UT, United States, 4ARUP Laboratories,
Salt Lake City, UT, United States, 5Division of Medical
Genetics, Alberta Children's Hospital, Calgary, AB, Canada,
6Department of Pediatrics and Neurology &
Neurotherapeutics, UT Southwestern Medical Center, Dallas,
TX, United States, 7Department of Pediatrics, University of
Montreal, Montreal, QC, Canada, 8Clinical Genetics/
Dysmorphology, University of California San Diego/Rady
Children’s Hospital, San Diego, CA, United States,
9Ambulantes Gesundheitszentrum Humangenetik, Charité
Universitätsmedizin Berlin, Berlin, Germany Grants: National Health and Medical Research; King
Abdulaziz City for Science and Technology; King Salman
Center for Disability Research; Saudi Human Genome
Program and the King Abdullah University of Science and
Technology. Introduction: Dias-Logan syndrome is a recently described
condition characterized by intellectual disability (ID) and
persistence of fetal hemoglobin (HbF). It is caused by
haploinsufficiency of BCL11A (2p16.1), encoding a tran-
scription factor of the SWI/SNF chromatin remodeling
complex. E.E. Palmer: None. S. Hong: None. F. Al Zahrani:
None. M. Omar Hashem: None. F.A. Aleisa: None. H.M. E.E. Palmer: None. S. Hong: None. F. Al Zahrani:
None. M. Omar Hashem: None. F.A. Aleisa: None. H.M. Jalal Ahmed: None. T. Kandula: None. R. Macintosh:
None. A. Minoche: None. C. Puttick: None. V. Gayevs-
kiy: None. A.P. Drew: None. M.J. Cowley: None. M.E. Dinger:
None. J.A. Rosenfeld:
B. P08.08D Research
Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Baylor Genetics Laboratory. R. Xiao: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Baylor Genetics Laboratory. M.T. Cho: A. Employment (full or part-time); Significant;
GeneDx. L.B. Henderson: A. Employment (full or part-
time); Significant; GeneDx. M.J. Guillen Sacoto: A. Employment (full or part-time); Significant; GeneDx. A. Begtrup: A. Employment (full or part-time); Significant;
GeneDx. M. Hamad: None. M. Shinawi: None. M. Andrews: None. M.C. Jones: None. K. Lindstrom: None. S. Kayani: None. M. Snyder: None. M. Villanueva:
None. A. Schteinschnaider: None. T. Roscioli: None. E.P. Kirk: None. A. Bye: None. J. Merzaban: None. L. Jaremko: None. M. Jaremko: None. R.K. Sachdev:
None. F.S. Alkuraya: None. S.T. Arold: None. E.E. Palmer: None. S. Hong: None. F. Al Zahrani:
None. M. Omar Hashem: None. F.A. Aleisa: None. H.M. Jalal Ahmed: None. T. Kandula: None. R. Macintosh:
None. A. Minoche: None. C. Puttick: None. V. Gayevs-
kiy: None. A.P. Drew: None. M.J. Cowley: None. M.E. Dinger:
None. J.A. Rosenfeld:
B. Research
Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Baylor Genetics Laboratory. R. Xiao: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Baylor Genetics Laboratory. M.T. Cho: A. Employment (full or part-time); Significant;
GeneDx. L.B. Henderson: A. Employment (full or part-
time); Significant; GeneDx. M.J. Guillen Sacoto: A. Employment (full or part-time); Significant; GeneDx. A. Begtrup: A. Employment (full or part-time); Significant;
GeneDx. M. Hamad: None. M. Shinawi: None. M. Andrews: None. M.C. Jones: None. K. Lindstrom: None. S. Kayani: None. M. Snyder: None. M. Villanueva:
None. A. Schteinschnaider: None. T. Roscioli: None. E.P. Kirk: None. A. Bye: None. J. Merzaban: None. L. Jaremko: None. M. Jaremko: None. R.K. Sachdev:
None. F.S. Alkuraya: None. S.T. Arold: None. Methods: We reviewed the medical records of our
patients with changes in BCL11A and those in the literature,
and assessed the frequency of the main manifestations. Methods: We reviewed the medical records of our
patients with changes in BCL11A and those in the literature,
and assessed the frequency of the main manifestations. Results: Our patients (2-11 y) presented with hypotonia
(6/6), ID (6/6), persistence of HbF (3/3), brain abnormalities
(4/6), strabismus (4/6), and seizures (2/6). No birth defects
were observed. P08.08D Two had de novo deletions and four had a
de novo pathogenic variant in BCL11A. By review of the literature, we found 16 additional
individuals with point mutations and 25 with 2p15p16.1
microdeletions. Including our patients, the most frequent
manifestations were ID (100%), varying from mild to
profound, persistent HbF (100%), distinctive facial features
(95%), hypotonia (87%), microcephaly (67%), abnormal
brain MRI (67%), consisting of cortical dysplasia, corpus
callosum hypoplasia or cerebellar hypoplasia, and growth
delay (41%). Epilepsy was present in 16%: age at onset
ranged 2 m - 3 y, and seizures were mostly drug-resistant. Interestingly, in most of the patients the facial gestalt
resembled Alfa Thalassemia Intellectual Disability (ATRX),
caused by mutations in a gene encoding another SWI/SNF-
like protein. By review of the literature, we found 16 additional
individuals with point mutations and 25 with 2p15p16.1
microdeletions. Including our patients, the most frequent
manifestations were ID (100%), varying from mild to
profound, persistent HbF (100%), distinctive facial features
(95%), hypotonia (87%), microcephaly (67%), abnormal
brain MRI (67%), consisting of cortical dysplasia, corpus
callosum hypoplasia or cerebellar hypoplasia, and growth
delay (41%). Epilepsy was present in 16%: age at onset
ranged 2 m - 3 y, and seizures were mostly drug-resistant. Interestingly, in most of the patients the facial gestalt
resembled Alfa Thalassemia Intellectual Disability (ATRX),
caused by mutations in a gene encoding another SWI/SNF-
like protein. A. Peron1,2, C. Carlston3,4, J. Palumbos1, T. Tvrdik4,
P. Ferreira5, D. Haffner6, P. Campeau7, L. Bird8, L. Graul-
Neumann9, A. Openshaw4, A. Lamb3,4, P. Paulraj3,4,
E. Andersen3,4, M. Rong3,4, J. C. Carey1, D. H. Viskochil1 Telomere shortening in Down syndrome and cerebral palsy The main sub-group is Kabuki syndrome patients for which
we have analysed 83 patients finding 26 (16% of the total)
of them carrying KMT2D and KDM6A pathogenic point
pathogenic
variants. Moreover
we
found
pathogenic
variants in 9/37 Rubinstein-Taybi syndrome patients, 3/11
Cornelia de Lange,syndrome 2/10 in Floating-Harbor
syndrome, 2/3 Sotos syndrome, and 1/1in Wiedemann-
Steiner syndrome. Then we extended our analysis searching
for additional causative genes alteration in those patients
with overlapping phenotypes, finding unexpected results
supporting the need of using NGS for this group of diseases
with molecular and clinical overlapping. Results: To date we have NGS-sequenced 165 patients
and we found pathogenic variants in 31% of the all patients. The main sub-group is Kabuki syndrome patients for which
we have analysed 83 patients finding 26 (16% of the total)
of them carrying KMT2D and KDM6A pathogenic point
pathogenic
variants. Moreover
we
found
pathogenic
variants in 9/37 Rubinstein-Taybi syndrome patients, 3/11
Cornelia de Lange,syndrome 2/10 in Floating-Harbor
syndrome, 2/3 Sotos syndrome, and 1/1in Wiedemann-
Steiner syndrome. Then we extended our analysis searching
for additional causative genes alteration in those patients
with overlapping phenotypes, finding unexpected results
supporting the need of using NGS for this group of diseases
with molecular and clinical overlapping. Conclusions: The study of chromatinopathies may offer a
unique opportunity to learn about the role of epigenetics in
health and disease. Since the pathogenic sequences are
unknown for most of the cases, we highlight the importance
to analyse them with NGS. B. Augello: None. C. Gervasini: None. V. Massa: None. G.M. Squeo: None. E.A. Colombo: None. D. Milani:
None. M.C. Gandini: None. M. Castori: None. E. Di
Fede: None. I. Adipietro: None. N. Malerba: None. G. Merla: None. E. Bueno-Martínez: None. J.J. Tellería: None. C. Cieza-Borrella: None. J.A. Mirón-Canelo: None. R. González-Sarmiento: None. E. Bueno-Martínez: None. J.J. Tellería: None. C. Cieza-Borrella: None. J.A. Mirón-Canelo: None. R. González-Sarmiento: None. E. Bueno-Martínez: None. J.J. Tellería: None. C. Cieza-Borrella: None. J.A. Mirón-Canelo: None. R. González-Sarmiento: None. P08.12D Ending the diagnostic odyssey by clinical whole exome /
genome sequencing (CWEW/CWGS) P08.09A Dias-Logan syndrome: delineating a newly recognized
disorder of transcriptional regulation Dias-Logan syndrome: delineating a newly recognized
disorder of transcriptional regulation Conclusions: Our study expands the phenotype of
BCL11A mutations to include early onset seizures and
brain abnormalities, and the overlapping manifestations
between Dias-Logan syndrome and ATRX suggest con-
vergence on a common pathway of transcription regulation
of hemoglobin genes. A. Peron1,2, C. Carlston3,4, J. Palumbos1, T. Tvrdik4,
P. Ferreira5, D. Haffner6, P. Campeau7, L. Bird8, L. Graul-
Neumann9, A. Openshaw4, A. Lamb3,4, P. Paulraj3,4,
E. Andersen3,4, M. Rong3,4, J. C. Carey1, D. H. Viskochil1 A. Peron: None. C. Carlston: None. J. Palumbos:
None. T. Tvrdik: None. P. Ferreira: None. D. Haffner: A. Peron: None. C. Carlston: None. J. Palumbos:
None. T. Tvrdik: None. P. Ferreira: None. D. Haffner: 215 Abstracts from the 51st European Society of Human Genetics Conference: Posters None. P. Campeau: None. L. Bird: None. L. Graul-
Neumann: None. A. Openshaw: None. A. Lamb: None. P. Paulraj: None. E. Andersen: None. M. Rong: None. J. C. Carey: None. D.H. Viskochil: None. B. Augello1, C. Gervasini2, V. Massa2, G. M. Squeo1, E. A.
Colombo2, D. Milani3, M. C. Gandini2, M. Castori1, E. Di Fede2,
I. Adipietro1, N. Malerba1, G. Merla1 1Division of Medical Genetics, IRCCS Casa Sollievo della
Sofferenza, San Giovanni Rotondo, Italy, 2Medical Genetics,
Dept. Health sciences, Università degli Studi di Milano,
Milano, Italy, 3UOSD Pediatria ad alta intensità di cura,
Fondazione IRCCS Cà Granda Ospedale Maggiore
Policlinico, Milano, Italy Telomere shortening in Down syndrome and cerebral palsy E. Bueno-Martínez1, J. J. Tellería2,3, C. Cieza-Borrella1, J. A. Mirón-Canelo4, R. González-Sarmiento5 1Unidad de Medicina Molecular. Departamento de Medicina,
Salamanca, Spain, 2IBSAL, Salamanca, Spain, 3Hospital
Clinico de Valladolid, Valladolid, Austria, 4Deppartamento de
Medicina Preventiva, Salud Pública y Microbiología Médica,
Salamanca, Spain, 5IBSAL y IBMCC, Salamanca, Spain Introduction: Aberrant structure and function of chromatin,
by altering various components of the epigenetic machin-
ery, causes a number of human diseases. Intellectual dis-
ability appears to be a common phenotype feature, although
these disorders affect multiple organs. There is emerging
importance recognition of this spectrum of disorders, that
we termed ‘chromatinopathies’. Intellectual disability has a global prevalence of 1-3%. Down syndrome and Cerebral Palsy are two entities asso-
ciated to intellectual disability with high impact in society
and easily diagnosed. In recent decades, life expectancy in
people with intellectual disability has increased and has
been accompanied by premature aging whose genetic cause
remains unknown. Telomere shortening is involved in the
cellular and body aging. We have studied by quantitative
real time PCR the telomere length in subjects with Down
syndrome(66 males, 47 females, age 11-69y, mean 37.2)
and Cerebral Palsy (34 males, 20 females, age 11-80y,
mean 32.7), and compared with a control group in order to
identify differences that could explain the accelerated aging
of both pathological groups. We found differences in telo-
mere length between subjects with Down syndrome and
Cerebral Palsy for ages over 35y and when compared both
groups to healthy subjects matched by age for all ages (p
< 0.001) in all cases. The analysis of the genotype dis-
tribution of two polymorphisms associated with a low tel-
omerase activity: TERT -1327C>T (rs2735940) and TERC
-63G>A (rs2793607) did not show any difference between
groups. Analysis of telomere length in siblings with a
similar age to those of individuals with cerebral palsy as
well as those of their parents corrected for age showed
shorter telomeres length than control subjects with similar
age. These data suggest that early telomere shortening could
increase susceptibility to cerebral palsy or that both entities
could share genetic predisposition factors. Materials and Methods: We generated a targeted NGS
custom-made gene panel to sequence 66 genes that are
causative of 54 chromatinopathies, including Kabuki, Au-
Kline,
Charge,
Wiedemann
Steiner,
Rubinstein-Taybi,
Floating Harbor, and Cornelia de Lange syndromes. Results: To date we have NGS-sequenced 165 patients
and we found pathogenic variants in 31% of the all patients. NGS panel for chromatinopathies, implications for
diagnosis and research P08.13A The search of biological processes affected by CNTN6
microdeletion in neurons, derived from induced pluripotent
stem cells of a patient with intellectual disability and 3p26.3
microdeletion C. F. Wells1, S. Heide2, P. Charles3, I. Marey2, D. Héron2,4,
C. Nava2,4, T. Courtin2, B. Isidor5, A. Piton6,7, B. Gérard7,
J. Buratti8, M. Fradin9, C. Dubourg10, L. Pasquier11,
L. Faivre12,13, N. Philip14, M. Milh15, G. Lesca16,17, P. Edery16,17,
D. Sanlaville16,17, A. Liquier18, A. Dieux19, T. Attié-Bitach20,21,
E. Colin22, D. Bonneau22,23, B. Keren2, D. Geneviève1 P08.11C The enrichment
analysis for identification of functional groups of genes was
performed. Materials and Methods: Clinical Whole Exome and
Genome Sequencing (CWES and CWGS) were performed
with bioinformatics analysis done using in-house algorithm. The overall interpretation was based on the clinical,
laboratory and imaging findings, and pathomechanism. Materials and Methods: Clinical Whole Exome and
Genome Sequencing (CWES and CWGS) were performed
with bioinformatics analysis done using in-house algorithm. Materials and Methods: Clinical Whole Exome and
Genome Sequencing (CWES and CWGS) were performed
with bioinformatics analysis done using in-house algorithm. The overall interpretation was based on the clinical,
laboratory and imaging findings, and pathomechanism. Results: Over 100 cases had been referred to us and the
disease entities were heterogeneous. Some cases were
potentially
actionable,
these
include
Allan-Herndon-
Dudley syndrome, benign recurrent intrahepatic cholestasis
(BRIC), coenzyme Q6 deficiency-related nephrotic syn-
drome, coenzyme Q10 deficiency, osteogenesis imperfecta
type VII, steroid-resistant nephrotic syndrome, X-linked
adrenoleukodystrophy, etc. A new treatment for GNAO1-
related epilepsy was found by this group. Novel disease-
causing genes were discovered, for example, AK9 in
congenital myasthenic syndrome (CMS) and EBF3 in
Moebius syndrome. Results: The comparison of gene expression in WT and
CNTN6 microdeletion neurons revealed 756 DEG (142
downregulated and 614 upregulated genes). The enrichment
analysis
of
the
underexpressed
genes
revealed
their
involvement in responses to glucocorticoid and corticoster-
oid, astrocyte development, basic amino acid and trans-
membrane transport. Overexpressed genes were involved in
regulation of synaptic signalling, trans-synaptic signalling,
anterograde trans-synaptic signalling, chemical synaptic
transmission, cell-cell
signalling, nervous system and
neuron development, regulation of postsynaptic membrane
potential. Conclusions: The transcriptome analysis of iPSC-derived
neurons with the CNTN6 microdeletion revealed the
alteration of the expression of other genes significant for
CNS functioning. This study was supported by Russian
Science Foundation, grant 14-15-00772. Conclusions:
Patients
with
undiagnosed
disease
>3 months should undergo CWES/CWGS. The clinical
interpretation of CWES/CWGS is not straightforward, and
requires in-depth knowledge in both advanced laboratory
and clinical medicine. Therefore, it should be handled by
specialists with experience in clinical genomics. M.E. Lopatkina: None. V.S. Fishman: None. M.M. Gridina: None. N.A. Skryabin: None. T.V. Nikitina:
None. A.A. Kashevarova: None. L.P. Nazarenko: None. O.L. Serov: None. I.N. Lebedev: None. M.E. Lopatkina: None. V.S. Fishman: None. M.M. Gridina: None. N.A. Skryabin: None. T.V. Nikitina:
None. A.A. Kashevarova: None. L.P. Nazarenko: None. O.L. Serov: None. I.N. Lebedev: None. C. Law: None. C. Lam: None. P08.11C Ending the diagnostic odyssey by clinical whole exome /
genome sequencing (CWEW/CWGS) NGS panel for chromatinopathies, implications for
diagnosis and research C. Law, C. Lam 216 J. del Picchia Introduction:
Microdeletions
and
microduplications
affecting CNTN6 have been described in patients with
neurodevelopmental disorders. We aimed to find out the
basic biological processes involved in the realization of
pathogenic microdeletion effects in the central nervous
system (CNS) via analysis of differentially expressed genes
(DEG) in neurons, derived from induced pluripotent stem
(iPS) cells of a patient with intellectual disability and
CNTN6 microdeletion. Introduction:
Microdeletions
and
microduplications
affecting CNTN6 have been described in patients with
neurodevelopmental disorders. We aimed to find out the
basic biological processes involved in the realization of
pathogenic microdeletion effects in the central nervous
system (CNS) via analysis of differentially expressed genes
(DEG) in neurons, derived from induced pluripotent stem
(iPS) cells of a patient with intellectual disability and
CNTN6 microdeletion. Department of Pathology, The University of Hong Kong, Hong Kong, China Kong, China Introduction: There are more than 7,000 rare diseases
affecting 300 million populations worldwide. Unfortu-
nately, the diagnosis of rare diseases is challenging and very
often, there is a delay between disease onset and the time of
the correct diagnosis. This is also known as “diagnostic
odyssey”. Over the past years, we had closed a number of
“diagnostic odysseys” in this locality and we realized there
is a strong need to provide rare diseases diagnostic service. For this reason, we developed the first Undiagnosed Dis-
eases Program (UDP) in Hong Kong which was supported
by the S. K. Yee Medical Foundation. Materials and Methods: Two iPS cell clones with
CNTN6 microdeletion and three wild-type (WT) cell clones
with different genotypes were differentiated into cortical
neurons. The neuronal RNA was examined using SurePrint
G3 Human Gene Expression 8×60K Microarray Kit
(Agilent Technologies, USA). Empirical Bayes statistical
test was applied for bioinformatic analysis (p-value < 0.05;
expression levels differ more than 2 times). The enrichment
analysis for identification of functional groups of genes was
performed. Materials and Methods: Two iPS cell clones with
CNTN6 microdeletion and three wild-type (WT) cell clones
with different genotypes were differentiated into cortical
neurons. The neuronal RNA was examined using SurePrint
G3 Human Gene Expression 8×60K Microarray Kit
(Agilent Technologies, USA). Empirical Bayes statistical
test was applied for bioinformatic analysis (p-value < 0.05;
expression levels differ more than 2 times). 1Research Institute of Medical Genetics, Tomsk National
Research Medical Center, Tomsk, Russian Federation,
2Institute of Cytology and Genetics SB RAS, Novosibirsk,
Russian Federation Russian Federation M. E. Lopatkina1, V. S. Fishman2, M. M. Gridina2, N. A.
Skryabin1, T. V. Nikitina1, A. A. Kashevarova1, L. P.
Nazarenko1, O. L. Serov2, I. N. Lebedev1 Expanding the phenotype of CTNNB1-mutated individuals
and description of the fetal phenotype Lebedev1 1Research Institute of Medical Genetics, Tomsk National
Research Medical Center, Tomsk, Russian Federation,
2Institute of Cytology and Genetics SB RAS, Novosibirsk,
Russian Federation Abstracts from the 51st European Society of Human Genetics Conference: Posters 217 1Département de Génétique Médicale, Maladie Rares et
Médecine Personalisée, Centre de référence anomalies du
développement et syndrome Malformaifs, CHU de Montpellier,
Montpellier, France, 2APHP, GH Pitié-Salpêtrière,
Département de Génétique, Paris, France, 3APHP, GH Pitié-
Salpêtrière, Centre de Référence de Neurogénétique, Paris,
France, 4Centre de Référence `Déficiences Intellectuelles de
Causes Rares', Paris, France, 5Service de Génétique Médicale,
CHU de Nantes, Nantes, France, 6Department of Translational
Medicine and Neurogenetics, Institut Génétique Biologie
Moléculaire Cellulaire, Illkirch, France, 7Laboratoire de
diagnostic génétique, Hôpitaux Universitaires de Strasbourg,
Strasbourg, France, 8CNRS UMR 3571: Genes, Synapses and
Cognition, Institut Pasteur, Paris, France, 9Unité de Génétique
clinique, CHU de Rennes, Rennes, France, 10Unité de
Génétique clinique, CHU de Rennes,, Paris, France, 11CHU
Rennes, Service de Génétique Clinique, CNRS UMR6290,
Université Rennes1, Rennes, France, 12Equipe GAD, INSERM
LNC UMR 1231, Faculté de Médecine, Université de
Bourgogne Franche-Comté, Dijon, France, 13Centre de
Génétique et Centre de Référence Anomalies du
Développement et Syndromes Malformatifs de l'Interrégion
Est, Centre Hospitalier Universitaire Dijon, Dijon, France,
14Département de génétique médicale, Hôpital de la Timone,
APHM, Marseille, France, 15Service de neurologie
pédiatrique, Hôpital de la Timone, APHM, Marseille, France,
16Hospices Civils de Lyon, Service de Génétique, Centre de
Référence Anomalies du Développement, Bron, France,
17INSERM U1028, CNRS UMR5292, UCB Lyon 1, Centre de
Recherche en Neurosciences de Lyon, GENDEV Team, Lyon,
France, 18Laboratoire de Cytogénétique, Biomnis, Lyon,
France, 19Service de génétique clinique Guy Fontaine CHRU
de Lille - Hôpital Jeanne de Flandre, Lille, France, 20APHP,
Hôpital Necker-Enfants Malades, Service d'Histologie-
Embryologie-Cytogénétique, Paris, France, 21INSERM U1163,
Hôpital Necker-Enfants Malades, Institut Imagine, Paris,
France, 22Département de biochimie et génétique, CHU
d’Angers, Angers, France, 23Equipe MitoLab, CNRS UMR
6015, Inserm U1083, Institut MitoVasc of Angers, CHU
d’Angers, Angers, France 1Département de Génétique Médicale, Maladie Rares et
Médecine Personalisée, Centre de référence anomalies du
développement et syndrome Malformaifs, CHU de Montpellier,
Montpellier, France, 2APHP, GH Pitié-Salpêtrière,
Département de Génétique, Paris, France, 3APHP, GH Pitié-
Salpêtrière, Centre de Référence de Neurogénétique, Paris,
France, 4Centre de Référence `Déficiences Intellectuelles de
Causes Rares', Paris, France, 5Service de Génétique Médicale,
CHU de Nantes, Nantes, France, 6Department of Translational
Medicine and Neurogenetics, Institut Génétique Biologie
Moléculaire Cellulaire, Illkirch, France, 7Laboratoire de
diagnostic génétique, Hôpitaux Universitaires de Strasbourg,
Strasbourg, France, 8CNRS UMR 3571: Genes, Synapses and
Cognition, Institut Pasteur, Paris, France, 9Unité de Génétique
clinique, CHU de Rennes, Rennes, France, 10Unité de
Génétique clinique, CHU de Rennes,, Paris, France, 11CHU
Rennes, Service de Génétique Clinique, CNRS UMR6290,
Université Rennes1, Rennes, France, 12Equipe GAD, INSERM
LNC UMR 1231, Faculté de Médecine, Université de
Bourgogne Franche-Comté, Dijon, France, 13Centre de
Génétique et Centre de Référence Anomalies du
Développement et Syndromes Malformatifs de l'Interrégion
Est, Centre Hospitalier Universitaire Dijon, Dijon, France,
14Département de génétique médicale, Hôpital de la Timone,
APHM, Marseille, France, 15Service de neurologie
pédiatrique, Hôpital de la Timone, APHM, Marseille, France,
16Hospices Civils de Lyon, Service de Génétique, Centre de
Référence Anomalies du Développement, Bron, France,
17INSERM U1028, CNRS UMR5292, UCB Lyon 1, Centre de
Recherche en Neurosciences de Lyon, GENDEV Team, Lyon,
France, 18Laboratoire de Cytogénétique, Biomnis, Lyon,
France, 19Service de génétique clinique Guy Fontaine CHRU
de Lille - Hôpital Jeanne de Flandre, Lille, France, 20APHP,
Hôpital Necker-Enfants Malades, Service d'Histologie-
Embryologie-Cytogénétique, Paris, France, 21INSERM U1163,
Hôpital Necker-Enfants Malades, Institut Imagine, Paris,
France, 22Département de biochimie et génétique, CHU
d’Angers, Angers, France, 23Equipe MitoLab, CNRS UMR
6015, Inserm U1083, Institut MitoVasc of Angers, CHU
d’Angers, Angers, France exome sequencing. Expanding the phenotype of CTNNB1-mutated individuals
and description of the fetal phenotype We decribe new signs in CTNNB1-
mutated individuals and the fetal phenotype. Thirteen have
SNVs and two have deletions. New signs include conden-
sing bone anomalies, severe acne and reproductive organ
malformation. One fetus was diagnosed after termination of
pregnancy for agenesis of corpus callosum and gene panel
analysis. The other fetus had microcephaly (-3DS), array-
CGH was performed and revealed a deletion encompassing
CTNNB1. Conclusion: We expand the phenotype of CTNNB1-
mutated individuals and describe the fetal phenotype and
new mutations. More patients are needed to confirm the
frequency of rarer signs. X-rays in both males and females
and abdominal ultrasound in females should be carried out
to respectively assess bone anomalies and reproductive
organ malformations. Conclusion: We expand the phenotype of CTNNB1-
mutated individuals and describe the fetal phenotype and
new mutations. More patients are needed to confirm the
frequency of rarer signs. X-rays in both males and females
and abdominal ultrasound in females should be carried out
to respectively assess bone anomalies and reproductive
organ malformations. C.F. Wells: None. S. Heide: None. P. Charles: None. I. Marey: None. D. Héron: None. C. Nava: None. T. Courtin: None. B. Isidor: None. A. Piton: None. B. Gérard: None. J. Buratti: None. M. Fradin: None. C. Dubourg: None. L. Pasquier: None. L. Faivre: None. N. Philip: None. M. Milh: None. G. Lesca: None. P. Edery:
None. D. Sanlaville: None. A. Liquier: A. Employment
(full or part-time); Significant; Biomnis. A. Dieux: None. T. Attié-Bitach: None. E. Colin: None. D. Bonneau: None. B. Keren: None. D. Geneviève: None. Expanding the phenotype of CTNNB1-mutated individuals
and description of the fetal phenotype Expanding the phenotype of CTNNB1-mutated individuals
and description of the fetal phenotype M. E. Lopatkina1, V. S. Fishman2, M. M. Gridina2, N. A. Skryabin1, T. V. Nikitina1, A. A. Kashevarova1, L. P. Nazarenko1, O. L. Serov2, I. N. DDX3X mutations in 11 french patients with intellectual
disability : new phenotypic features DDX3X mutations in 11 french patients with intellectual
disability : new phenotypic features DDX3X mutations in 11 french patients with intellectual
disability : new phenotypic features V. Ruault1,2, C. Coubes2, P. Charles3, M. Vincent4, M. Nizon4,
C. Mignot3, A. Delahaye-Duriez5, C. Thauvin6, N. Jean-
Marçais6, A. Garde6, L. Faivre6, Y. Alembik7, A. Gouronc7,
B. Durand7, C. Nava3, B. Keren3, C. Depienne3, F. Tran Mau
Them6, M. Willems2, B. Gérard7, D. Geneviève2,1 1Université de Montpellier, Montpellier, France, 2Service de
génétique clinique, CHU de Montpellier, Montpellier, France,
3Service de génétique de la Pitié Salpetrière, Paris, France,
4Service de génétique clinique, CHU de Nantes, Nantes,
France, 5Service de génétique clinique Robert Debré, Paris,
France, 6Centre de génétique CHU Dijon, Dijon, France,
7Service de génétique CHU de Strasbourg, Strasbourg, France Introduction: CTNNB1 constitutional mutations were first
described in intellectual disability by De Ligt et al. in 2012. Thirty-one individuals have been reported so far and a
further 16 non-published mutated individuals are reported
by the Deciphering Developmental Disorders study. The
CTNNB1 gene encodes for the highly-conserved beta-cate-
nine which has an major role in the Wnt-signalling
pathway. Intellectual
disability
affects
approximately
2.5%
of
humans. The prevalence of DDX3X (X-linked, MIM
*300160) mutations in intellectual disability (ID) is not yet
known, but have been reported in 45 patients with ID. Other
symptoms
included
hypotonia,
movement
disorders,
microcephaly, behavior problems and epilepsy. Several
additional features were noted, including hyperlaxity, skin
abnormalities, cleft lip, cleft palate, hearing loss, visual Results: Using French National networks, we collected
clinical data of 13 individuals (including one previously
published patient) from 11 centers and 2 fetuses. Individuals
were diagnosed using array-CGH, gene panels, solo or trio J. P08.16D Different mutations in DEAF1 lead to clinically distinct
dominant and recessive forms of intellectual disability Mutations in DEAF1, encoding a crucial transcription factor
in
central
nervous
system
development
during
early
embryogenesis, were reported to lead to autosomal domi-
nant mental retardation 24 (MRD24; MIM 615828) and
autosomal recessive dyskinesia, seizures, and intellectual
developmental disorder (DYSEIDD; MIM 617171). We
aimed at understanding genotype-phenotype correlations of
15 novel patients with likely pathogenic DEAF1 variants
identified by exome sequencing. In 13 cases, de novo
DEAF1 variants resulted in single amino acid changes in the
SAND domain. Two unrelated patients had inherited com-
pound heterozygous DEAF1 variants, which were located
outside the SAND domain and both consisted of a combi-
nation of a loss-of-function mutation with a milder mutation
on the other allele. Analysis of transcriptional repression
activity at the DEAF1 promoter showed that the tested de
novo variants impaired DEAF1 function, while this effect
could not be observed for recessive variants. De novo
DEAF1 variants caused moderate/severe intellectual dis-
ability with limited or absent speech and behavioral pro-
blems (mood swings, autism, self-aggression and sleep
disturbance). Recessive DEAF1 variants resulted addition-
ally in more severe psychomotor delay, movement disorder
and MRI abnormalities. Epilepsy, occurring in recessive
patients and most of dominant patients, was frequently
difficult
to
treat. Some
patients
used
non-verbal M. J. Nabais Sá1, P. J. Jensik2, M. J. Parker3, N. Lahiri4, E. P. McNeil5, K. Hibbs6, H. Y. Kroes7, C. T. R. M. Stumpel8, A. P. A. Stegmann8, R. J. Hagerman9, R. E. Harrison10, M. Splitt11,
T. Montgomery11, E. E. Palmer12, R. K. Sachdev12, H. C. Mefford13, A. A. Scott14, J. A. Martinez-Agosto15, R. Lorenz16,
N. Orenstein17, J. N. Berg18, J. Cobben19, E. J. Marco20, B. B. A. de Vries1, A. T. DDX3X mutations in 11 french patients with intellectual
disability : new phenotypic features Coubes: None. P. Charles: None. M. Vincent: None. M. Nizon: None. C. Mignot: None. A. Delahaye-Duriez: None. C. Thauvin: None. N. Jean-
Marçais: None. A. Garde: None. L. Faivre: None. Y. Alembik: None. A. Gouronc: None. B. Durand: None. C. Nava: None. B. Keren: None. C. Depienne: None. F. Tran Mau Them: None. M. Willems: None. B. Gérard:
None. D. Geneviève: None. DDX3X mutations in 11 french patients with intellectual
disability : new phenotypic features del Picchia 218 Dept Pediatrics, Division of Child Development and Behavior,
University of California Davis Health, Davis, CA, United
States, 10Dept Clinical Genetics, Nottingham University
Hospitals NHS Trust, Nottingham, United Kingdom,
11Northern Genetics Service, Newcastle upon Tyne Hospitals
NHS Foundation Trust; Institute of Human Genetics,
International Centre for LifeCentral Parkway, Newcastle-
upon-Tyne, United Kingdom, 12Sydney Children’s Hospital,
Randwick; School of Women’s and Children’s Health, UNSW
Medicine, The University of New South Wales, Sydney, NSW,
Australia, 13Division of Genetic Medicine, Dept Pediatrics,
University of Washington, Seattle, WA, United States,
14Division of Genetic Medicine, Seattle Children’s Hospital,
Seattle, WA, United States, 15Dept Human Genetics, David
Geffen School of Medicine at UCLA; Division of Medical
Genetics, Dept Pediatrics, David Geffen School of Medicine at
UCLA, Los Angeles, CA, United States, 16Neuropediatrics,
Pediatrics, Bad Wildungen, Germany, 17Pediatric Genetics
Clinic, Schneider Children’s Medical Center of Israel, Petach
Tikva; Sackler Faculty of Medicine, Tel Aviv University, Tel
Aviv, Israel, 18Dept Clinical Genetics, Ninewells Hospital and
Medical School; Clinical Genetics, University of Dundee,
Dundee, Angus, United Kingdom, 19Dept Pediatric Neurology,
Academic Medical Center, Amsterdam, Netherlands, 20Dept
Neurology, Psychiatry&Pediatrics, University of California,
San Francisco, CA, United States impairment, and precocious puberty. So far, all amino acid
substitutions in females were de novo and localized in one
of the two protein subdomains : the helicase ATP-binding
domain or the helicase C-terminal. We report 11 French
females patients with DDX3X mutations, carrying 8 new
mutations, including one substitution outside those two
domains : c.113A>G. We describe the first case of mother-
daughter transmission of DDX3X mutation : c.543+2_543
+3del. Those two patients are schizophrenics. All patients
present ID, with speech and walk delay. Six present feeding
difficulties. Two patients are obese, two others have thyroid
issues. One has ASD, another one had neuroblastoma in
young childhood. None of them present hearing loss.This
report leads to 56 patients with DDX3X mutations report in
the
litterature
and
expand
the
phenotypic
spectrum
of DDX3X. V. Ruault: None. C. Coubes: None. P. Charles: None. V. Ruault: None. C. Coubes: None. P. Charles: None. M. Vincent: None. M. Nizon: None. C. Mignot: None. A. Delahaye-Duriez: None. C. Thauvin: None. N. Jean-
Marçais: None. A. Garde: None. L. Faivre: None. Y. Alembik: None. A. Gouronc: None. B. Durand: None. C. Nava: None. B. Keren: None. C. Depienne: None. F. Tran Mau Them: None. M. Willems: None. B. Gérard:
None. D. Geneviève: None. V. Ruault: None. C. P08.16D Vulto-vanSilfhout1 1Dept Human Genetics, Radboud University Medical Center,
Nijmegen, Netherlands, 2Dept Physiology, Southern Illinois
University School of Medicine, Carbondale, IL, United States,
3Sheffield Clinical Genetics Service, OPD2 Northern General
Hospital, Sheffield, United Kingdom, 4Dept Clinical Genetics,
St George’s University Hospitals NHS Foundation Trust & St
George’s, University of London, London, United Kingdom,
5University of Minnesota, Minneapolis, MN, United States,
6Dept Pediatrics, Division of Genetics and Metabolism,
University of Minnesota, Minneapolis, MN, United States,
7Dept Genetics, University Medical Center Utrecht, Utrecht,
Netherlands, 8Dept Clinical Genetics and GROW-School for
Oncology and Developmental Biology, Maastricht University
Medical Center, Maastricht, Netherlands, 9Medical
Investigation of Neurodevelopmental Disorders (MIND)
Institute, University of California Davis School of Medicine; De novo variants in SNAP25 cause a spectrum of
developmental and epileptic encephalopathy De novo variants in SNAP25 cause a spectrum of
developmental and epileptic encephalopathy K. Platzer: None. W.K. Chung: None. Z. Powis: None. E. Brilstra: None. M. McDonald: None. M. Mikati:
None. M.T. Cho: None. A.C. Taylor: None. A. Wadley:
None. J.A. Sullivan: None. V. Shashi: None. J.R. Lemke: None. K. Platzer: None. W.K. Chung: None. Z. Powis: None. E. Brilstra: None. M. McDonald: None. M. Mikati:
None. M.T. Cho: None. A.C. Taylor: None. A. Wadley:
None. J.A. Sullivan: None. V. Shashi: None. J.R. Lemke: None. K. Platzer1, W. K. Chung2, Z. Powis3, E. Brilstra4,
M. McDonald5, M. Mikati6, M. T. Cho7, A. C. Taylor8,
A. Wadley8, J. A. Sullivan5, V. Shashi5, J. R. Lemke1 K. Platzer1, W. K. Chung2, Z. Powis3, E. Brilstra4,
M. McDonald5, M. Mikati6, M. T. Cho7, A. C. Taylor8,
A. Wadley8, J. A. Sullivan5, V. Shashi5, J. R. Lemke1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 219 spasms, focal and generalized seizures. Four remained
refractory to therapy. Three individuals did not attain
walking skills by age eleven years or later. Movement
disorders of dystonia or choreoathetosis were seen in two
individuals. Brain imaging revealed two individuals show-
ing generalized volume loss. In addition, one case presented
with signs of a leukoencephalopathy. Further symptoms
include microcephaly, ataxia, cortical visual impairment,
congenital myasthenia and congenital hip dysplasia and
contractures. All causative variants constitute de novo
missense variants located in the t-SNARE coiled-coil
homology domain 1 & 2, both showing a significantly
reduced number of missense variation in controls, indicating
a selective constraint. communication methods, suggesting better receptive than
expressive language. We provide further insight in the
genotype
and
phenotype
spectrum
of
DEAF1-related
dominant and recessive intellectual disability. Detailed
phenotype information, segregation and functional analysis
are fundamental to determine the pathogenicity of novel
variants and to improve the care of these patients. M.J. Nabais Sá: None. P.J. Jensik: None. M.J. Parker:
None. N. Lahiri: None. E.P. McNeil: None. K. Hibbs:
None. H.Y. Kroes: None. C.T.R.M. Stumpel: None. A.P. A. Stegmann:
None. R.J. Hagerman:
None. R.E. Harrison: None. M. Splitt: None. T. Montgomery: None. E.E. Palmer: None. R.K. Sachdev: None. H.C. Mefford:
None. A.A. Scott: None. J.A. Martinez-Agosto: None. R. Lorenz: None. N. Orenstein: None. J.N. Berg: None. J. Cobben: None. E.J. Marco: None. B.B.A. de Vries:
None. A.T. Vulto-vanSilfhout: None. M.J. Nabais Sá: None. P.J. Jensik: None. M.J. Parker:
None. N. Lahiri: None. E.P. McNeil: None. K. Hibbs:
None. H.Y. Kroes: None. C.T.R.M. Stumpel: None. A.P. A. Stegmann:
None. R.J. Hagerman:
None. R.E. Harrison: None. M. Splitt: None. T. Montgomery: None. E.E. Palmer: None. R.K. Sachdev: None. H.C. Mefford:
None. A.A. Scott: None. J.A. Martinez-Agosto: None. R. Lorenz: None. N. Orenstein: None. J.N. Berg: None. J. Cobben: None. E.J. Marco: None. B.B.A. de Vries:
None. A.T. Vulto-vanSilfhout: None. Conclusion: De novo variants in SNAP25 cause a
spectrum of developmental and epileptic encephalopathy. Further patients and studies are needed to improve our
understanding of the phenotypic spectrum and elucidate the
effects of the variants on protein function. P08.18B The role of recessive inheritance in early-onset epileptic
encephalopathies: a combined whole-exome sequencing and
high-resolution copy number study 1Institute of Human Genetics, University of Leipzig Hospitals
and Clinics, Leipzig, Germany, 2Department of Pediatrics,
Columbia University Medical Center, New York, NY, United
States, 3Ambry Genetics, Aliso Viejo, CA, United States,
4Department of Genetics, Utrecht University Medical Center,
Utrecht, Netherlands, 5Division of Medical Genetics,
Department of Pediatrics, Duke University, Durham, NC,
United States, 6Division of Pediatric Neurology, Department of
Pediatrics, Duke University, Durham, NC, United States,
7GeneDX, Gaithersburg, MD, United States, 8Section of
Genetics, Department of Pediatrics, University of Oklahoma,
Oklahoma City, OK, United States The role of recessive inheritance in early-onset epileptic
encephalopathies: a combined whole-exome sequencing and
high-resolution copy number study S. Papuc1,2, L. Abela3,4,5, K. Steindl1, A. Begemann1,
T. Simmons3, B. Schmitt3,4, M. Zweier1, B. Oneda1, E. Socher6,
L. Crowther3, G. Wohlrab3, L. Gogoll1, M. Poms3, M. Seiler7,
M. Papik1, R. Baldinger1, A. Baumer1, R. Asadollahi1, J. Kroell-
Seger8, R. Schmid9, T. Iff10, T. Schmitt-Mechelke11, K. Otten8,
A. Hackenberg3, M. Addor12, A. Klein13, S. Azzarello-Burri1,
H. Sticht6, P. Joset1, B. Plecko3,4,5, A. Rauch1,5,14 S. Papuc1,2, L. Abela3,4,5, K. Steindl1, A. Begemann1,
T. Simmons3, B. Schmitt3,4, M. Zweier1, B. Oneda1, E. Socher6,
L. Crowther3, G. Wohlrab3, L. Gogoll1, M. Poms3, M. Seiler7,
M. Papik1, R. Baldinger1, A. Baumer1, R. Asadollahi1, J. Kroell-
Seger8, R. Schmid9, T. Iff10, T. Schmitt-Mechelke11, K. Otten8,
A. Hackenberg3, M. Addor12, A. Klein13, S. Azzarello-Burri1,
H. Sticht6, P. Joset1, B. Plecko3,4,5, A. Rauch1,5,14 1Institute of Medical Genetics, University of Zurich, Schlieren-
Zurich, Switzerland, 2Victor Babes National Institute of
Pathology, Bucharest, Romania, 3Division of Child Neurology,
University Children's Hospital Zurich, Zurich, Switzerland,
4CRC Clinical Research Center University, Children’s
Hospital Zurich, Zurich, Switzerland, 5radiz—Rare Disease
Initiative Zürich, Clinical Research Priority Program for Rare
Diseases University of Zurich, Zurich, Switzerland, 6Institute of
Biochemistry, Friedrich-Alexander-Universität Erlangen-
Nürnberg (FAU), Erlangen, Germany, 7Emergency Clinic,
University Children's Hospital Zurich, Zurich, Switzerland,
8Children’s department, Swiss Epilepsy Centre, Clinic Lengg,
Zurich, Switzerland, 9Division of Child Neurology,
Kantonsspital Winterthur, Winterthur, Switzerland, 10County
Hospital of Triemli, Zurich, Switzerland, 11Division of Child De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy F. Tran Mau-Them1,2, A. Vitobello1,2, L. Guibaud3,4,
L. Duplomb2, B. Keren5, K. Lindstrom6, I. Marey5,
F. Mochel5,7,8, M. van den Boogaard9, R. Oegema9, C. Nava5,
A. Masurel10, T. Jouan1,2, F. Jansen11, M. Au12, A. Chen13,
M. Cho14, Y. Duffourd2, P. Dickson15, V. Moin15,
A. Begemann16, M. Zweier16, B. Zieba17, T. Schmitt-Mechelke18,
K. van Gassen9, S. Nelson19, J. Graham12, J. Friedman20,
L. Faivre2,10, F. Ebstein17, H. Lin15, C. Thauvin Robinet1,2 F. Tran Mau-Them1,2, A. Vitobello1,2, L. Guibaud3,4,
L. Duplomb2, B. Keren5, K. Lindstrom6, I. Marey5,
F. Mochel5,7,8, M. van den Boogaard9, R. Oegema9, C. Nava5,
A. Masurel10, T. Jouan1,2, F. Jansen11, M. Au12, A. Chen13,
M. Cho14, Y. Duffourd2, P. Dickson15, V. Moin15,
A. Begemann16, M. Zweier16, B. Zieba17, T. Schmitt-Mechelke18,
K. van Gassen9, S. Nelson19, J. Graham12, J. Friedman20,
L. Faivre2,10, F. Ebstein17, H. Lin15, C. Thauvin Robinet1,2 Intoduction: Early-onset epileptic (EE) and combined
developmental
and
epileptic
encephalopathies
(DEE)
represent a group of epilepsies characterized by generally
poor outcome. A few whole exome or genome sequencing
studies have emphasized the causative role of de novo
mutations in a growing number of EE/DEE disease genes,
yet leaving the majority of patients without etiological
diagnosis. The aim of this study was to further elucidate the
genetic etiology of EE/DEE. P08.19C
De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy P08.19C
De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy Neurology, Children’s Hospital, Lucerne, Switzerland,
12Service de Génétique Médicale, Centre Hospitalier
Universitaire Vaudois CHUV, Lausanne, Switzerland,
13Division of Child Neurology, University Children’s Hospital
Basel and Berne, Berne, Switzerland, 14Neuroscience Center
Zurich, University of Zurich and ETH Zurich, Zurich,
Switzerland De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy 1UF Innovation en diagnostic génomique des maladies rares,
CHU Dijon, Dijon, France, 2INSERM UMR1231 GAD, F-
21000, Dijon, France, 3Université Claude Bernard Lyon I,
CHU de Lyon, Lyon, France, 4Service de Radiologie, Hôpital-
Femme-Mère-Enfant, Hospices Civils de Lyon, Lyon, France,
5Département de Génétique, Hôpital Pitié-Salpêtrière 47-83
Boulevard de l'Hôpital 75013, Paris, France, 6Division of
Genetics and Metabolic Phoenix Children's Hospital, Phoenix,
AZ, United States, 7Inserm U 1127, CNRS UMR 7225,
Sorbonne Universités, UPMC Univ Paris 06, Paris, France,
8UMR S 1127, Institut du Cerveau et de la Moelle épinière,
ICM, Paris, France, 9Department of Genetics, University
Medical Center, Utrecht, Netherlands, 10Centre de Référence
maladies rares « Anomalies du Développement et syndrome
malformatifs » de l’Est, Centre de Génétique, Hôpital
d’Enfants, FHU TRANSLAD, CHU Dijon Bourgogne, Dijon,
France, 11Department of Child Neurology, Brain Center
Rudolf Magnus, University Medical Center, Utrecht,
Netherlands, 12Department of Pediatrics, Division of Medical
Genetics, Cedars-Sinai Medical Center and Harbor-UCLA
Medical Center, Los Angeles, CA, United States, 13Division of
Pediatric Neurology, Department of Pediatrics, Harbor-UCLA
Medical Center, Los Angeles, CA, United States, 14GeneDx,
Gaithersburg, MD, United States, 15Division of Medical
Genetics, Department of Pediatrics, Harbor-UCLA Medical
Center,, Torrance, CA, United States, 16Institute of Medical
Genetics, University of Zurich, Zurich, Switzerland, 17Institut
für Medizinische Biochemie und Molekularbiologie,
Universitätsmedizin Greifswald, Greifswald, Germany,
18Division of Pediatric Neurology, Children's Hospital,
Lucerne, Switzerland, 1918. Departments of Human Genetics
and Psychiatry, David Geffen School of Medicine at UCLAHU
Dijon, Los Angeles, CA, United States, 2019. Department of
Neurosciences and Pediatrics UCSD/Rady Children's Hospital
San Diego, Rady Children's Institute for Genomic Medicine,
San Diego, CA, United States Materials and Methods: We performed high-resolution
chromosomal
microarray
analysis
and
whole-exome
sequencing in 63 independent patients with EE/DEE. Assessment of pathogenicity included molecular modelling
of missense variants and untargeted plasma-metabolomics
in selected patients. Results: We yielded a diagnosis in ~42% of cases with
causative copy number variants in 6 patients (~10%),
(likely) pathogenic sequence variants in 14 known and two
newly confirmed disease genes in 20 patients (~32%),
including compound heterozygosity for causative sequence
and copy number variants in one patient. 38% of diagnosed
cases were caused by recessive genes, albeit with one allele
occurring de novo in two instances. Notably, the recessive
gene SPATA5 was found causative in 3% of our cohort but
was difficult to detect and therefore may have been
underdiagnosed in previous studies. Introduction: Introduction:
Synaptosomal-associated
Protein-25
(SNAP25), predominantly expressed in the brain, is part of
the SNARE complex (soluble N-ethylmaleimide-sensitive
factor attachment protein receptor) required for proper
presynaptic vesicle docking and fusion. Heterozygous de
novo variants in SNAP25 have previously been separately
reported in three individuals with intellectual disability (ID),
epileptic encephalopathy, ataxia and congenital myasthenia. Results: We have collected detailed phenotypic data on at
least four additional cases with de novo variants in SNAP25. Combined with the three publishes cases, all seven
individuals presented with ID with three of them classified
as severe, three as moderate and one as mild. Five
individuals developed seizures with a spectrum of epileptic J. del Picchia 220 De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy We further support
candidacy of four previously described genes, three of
which also followed a recessive inheritance pattern. Results: We yielded a diagnosis in ~42% of cases with
causative copy number variants in 6 patients (~10%),
(likely) pathogenic sequence variants in 14 known and two
newly confirmed disease genes in 20 patients (~32%),
including compound heterozygosity for causative sequence
and copy number variants in one patient. 38% of diagnosed
cases were caused by recessive genes, albeit with one allele
occurring de novo in two instances. Notably, the recessive
gene SPATA5 was found causative in 3% of our cohort but
was difficult to detect and therefore may have been
underdiagnosed in previous studies. We further support
candidacy of four previously described genes, three of
which also followed a recessive inheritance pattern. Conclusion: Our results confirm the importance of de
novo causative gene variants in EE/DEE, but additionally
illustrate the major role of mostly compound heterozygous
or hemizygous recessive inheritance and consequently high
recurrence risk. S. Papuc: None. L. Abela: None. K. Steindl: None. A. Begemann: None. T. Simmons: None. B. Schmitt: None. M. Zweier: None. B. Oneda: None. E. Socher: None. L. Crowther: None. G. Wohlrab: None. L. Gogoll: None. M. Poms: None. M. Seiler: None. M. Papik: None. R. Baldinger: None. A. Baumer: None. R. Asadollahi:
None. J. Kroell-Seger: None. R. Schmid: None. T. Iff:
None. T. Schmitt-Mechelke: None. K. Otten: None. A. Hackenberg: None. M. Addor: None. A. Klein: None. S. Azzarello-Burri: None. H. Sticht: None. P. Joset: None. B. Plecko: None. A. Rauch: None. 18Division of Pediatric Neurology, Children's Hospital,
Lucerne, Switzerland, 1918. Departments of Human Genetics
and Psychiatry, David Geffen School of Medicine at UCLAHU
Dijon, Los Angeles, CA, United States, 2019. Department of
Neurosciences and Pediatrics UCSD/Rady Children's Hospital
San Diego, Rady Children's Institute for Genomic Medicine,
San Diego, CA, United States Developmental and epileptic encephalopathies (DEEs) are
severe clinical conditions characterized by stagnation or Abstracts from the 51st European Society of Human Genetics Conference: Posters 221 decline of cognitive and behavioural abilities preceed,
accompanied or followed by seizures. Because DEEs are
clinically and genetically heterogeneous, next-generation
sequencing - especially Whole Exome Sequencing (WES) -
is becoming a first-tier strategy to identify the molecular
etiologies of these disorders. P08.20D Background: Exome trio analysis is an effective strategy to
identify
potentially
causal
variants,
along
with
their
inheritance pattern, on rare genetic disorders. This approach
has entered the medical practice as an effective diagnostic
test transforming the molecular diagnosis and clinical
management of undiagnosed genetic diseases. De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy By combining WES analysis
and international data sharing we identified 10 unrelated
individuals with DEE and de novo heterozygous truncating
variants in the interferon regulatory factor 2-binding pro-
tein-like gene (IRF2BPL). The 10 individuals allowed
delineation of a consistent neurodevelopmental disorder
characterized
by
disturbed/normal
initial
psychomotor
development
followed
by
severe
global
neurological
regression, usually starting in childhood, and epilepsy with
non-specific EEG abnormalities and variable CNS anoma-
lies including cerebral or cerebellar atrophy. IRF2BPL, also
known as enhanced at puberty protein 1 (EAP1), encodes a
transcriptional regulator containing a C-terminal RING-
finger domain common to E3 ubiquitin ligases. This domain
is required for its repressive and transactivating transcrip-
tional properties. The variants identified are expected to
encode for protein lacking the C-terminal RING-finger
domain. Taken together these data support the causative
role of truncating IRF2BPL variants in paediatric neurode-
generation and expands the spectrum of transcriptional
regulators identified as molecular factors implicated in
genetic developmental and epileptic encephalopathies. Geneva, Geneva, Switzerland, 7Khyber Medical University,
Kohat, Pakistan The study of consanguineous families has proven valuable
in the identification of novel causative genes for monogenic
disorders. In a large study of consanguineous families
focusing in intellectual disability, we combined exome
sequencing and homozygosity mapping and identified two
consanguineous families with homozygous loss-of-function
variants in FBXL3. In the first family, from Lebanon,
FBXL3 was the only gene among five candidates with a loss
of function variant (NM_012158.2:c.445C>T:p.(Arg149*));
and in the second family, from Pakistan, the FBXL3 variant
(NM_012158.2:c.884del:p.(Leu295Tyrfs*25)) was the only
one segregating in five affected individuals in two family
loops. In both families, the patients presented with intel-
lectual disability, short stature and mild facial dysmorph-
ism, a relatively large nose with bulbous tip. Fbxl3 variants
have been engineered in mice and are associated with dis-
turbances of the circadian rhythm and behavioral problems. Consanguinity provides the opportunity to identify novel
candidate genes for various phenotypes and enhance the
diagnostic yield of mendelian disorders. P. Makrythanasis: None. S.A. Paracha: None. M. Ansar: None. A. Megarbane: None. F.A. Santoni: None. M. Guipponi: None. E. Ranza: None. S.F. Shah: None. E. Falconnet: None. M.T. Sarwar: None. J. Ahmed: None. S.E. Antonarakis: None. P. Makrythanasis: None. S.A. Paracha: None. M. Ansar: None. A. Megarbane: None. F.A. Santoni: None. M. Guipponi: None. E. Ranza: None. S.F. Shah: None. E. Falconnet: None. M.T. Sarwar: None. J. Ahmed: None. S.E. Antonarakis: None. F. Tran Mau-Them: None. A. De novo truncating variants in the intronless IRF2BPL
gene are responsible for developmental epileptic
encephalopathy Vitobello: None. L. Guibaud: None. L. Duplomb: None. B. Keren: None. K. Lindstrom: None. I. Marey: None. F. Mochel: None. M. van den Boogaard: None. R. Oegema: None. C. Nava:
None. A. Masurel: None. T. Jouan: None. F. Jansen:
None. M. Au: None. A. Chen: None. M. Cho: None. Y. Duffourd: None. P. Dickson: None. V. Moin: None. A. Begemann: None. M. Zweier: None. B. Zieba: None. T. Schmitt-Mechelke: None. K. van Gassen: None. S. Nelson: None. J. Graham: None. J. Friedman: None. L. Faivre: None. F. Ebstein: None. H. Lin: None. C. Thauvin Robinet: None. P08.21A Diagnostic yield of exome trio analysis to identify the
genetic etiology in 404 undiagnosed cases I. Diez, M. Martinez-Garcia, R. Sanchez-Alcudia, C. Rodriguez,
R. Perez-Carro, I. Sanchez-Navarro, E. Mata, E. Fernandez-
Tabanera, M. Carcajona, L. De la Vega, D. Rodriguez,
G. Benito, N. Sánchez-Bolivar, P. Maietta, J. Botet, S. Alvarez NIMGenetics, Madrid, Spain NIMGenetics, Madrid, Spain 1University of Geneva, Geneva, Switzerland, 2Biomedical
Research Insitution of the Academy of Athens, Athens, Greece,
3Khyber Medical University, Peshawar, Pakistan, 4Institut
Jérôme Lejeune, Paris, France, 5University Hospital of
Lausanne, Lausanne, Switzerland, 6University Hospitals of FBXL3, novel candidate for autosomal recessive
intellectual disability P. Makrythanasis1,2, S. A. Paracha3, M. Ansar1, A. Megarbane4,
F. A. Santoni5,1, M. Guipponi6, E. Ranza6, S. F. Shah7,
E. Falconnet6, M. T. Sarwar1,3, J. Ahmed3, S. E. Antonarakis1,6
1University of Geneva, Geneva, Switzerland, 2Biomedical
Research Insitution of the Academy of Athens, Athens, Greece,
3Khyber Medical University, Peshawar, Pakistan, 4Institut
Jérôme Lejeune, Paris, France, 5University Hospital of
Lausanne, Lausanne, Switzerland, 6University Hospitals of Material And Methods: We performed exome sequen-
cing using Ion AmpliSeqTM Exome RDY technology (Life
Technologies) and SureSelectXT Human All Exon V6
technology (Agilent Technologies). Sequencing reads were
analyzed using Torrent Suite software and an in-house
pipeline, respectively. Trio annotated variants using ION J. del Picchia 222 Reporter were prioritized with an in-house analytical
pipeline. non-consanguineous Japanese parents. She was hypotonic
and her development was delayed. She showed moderate
ID. Physical examination revealed no dysmorphic features. At 6 years of age, she showed accelerated growth. She was
diagnosed with precocious puberty. (Patient 2) The 5-year-
old female was the younger sister of patient 1. Her devel-
opment was delayed. She stated to walk alone after 3 years
old. She spoke no meaningful words. She showed severe
ID. Physical examination revealed no dysmorphic features. Reporter were prioritized with an in-house analytical
pipeline. Results: We present the analysis of 404 trios referred to a
single institution. Patients were mainly children with
syndromic intellectual disability (46%). The genetic etiol-
ogy was potentially elucidated in 129 probands harboring
82 causal variants and 47 likely causative variants,
achieving a 32% genetic diagnostic rate. Among these
patients, 80 harbored de novo variants, 12 hemizygous
maternally inherited variants, 10 in compound heterozygous
variants, 21 newly homozygous variants and 6 variants
inherited from parents. Patients with syndromic intellectual
disability (42%, 78/187) and specific neurological disorders
(40%, 19/48) showed higher molecular diagnostics rates
than patients with non-neurologic disorders (26%, 8/31) and
non-syndromic intellectual disability (17%, 24/138). Methods: With the approval of our institutional ethics
committee, the samples were analyzed using WES. Results: Biallelic loss-of-function mutations of EZH1
were found in the sisters. Their parents and unaffected sister
was heterozygous for the mutation. Conclusions: Probability of being LoF intolerant for
EZH1 is very low. Heterozygous deletion of EZH1 was
non-candidate for developmental disorder. Haploinsuffi-
ciency of EZH1 may not cause abnormal methylation of
H3K27. Our results show that biallelic loss-of-function
mutations of EZH1 may cause developmental disorder with
precocious puberty. FBXL3, novel candidate for autosomal recessive
intellectual disability Conclusions: In our cohort exome trio analysis provide a
diagnostic yield of 32% in patients whom traditional
molecular diagnostics strategies were uninformative. The
implementation of exome trio analysis as a first-tier
diagnostic approach will provide a higher diagnostic yield
and a cost-efficient option particularly in rare syndromic
intellectual disabled patients. N. Okamoto: None. H. Sakamoto: None. K. Yanagi:
None. T. Kaname: None. I. Diez: None. M. Martinez-Garcia: None. R. Sanchez-
Alcudia: None. C. Rodriguez: None. R. Perez-Carro:
None. I. Sanchez-Navarro: None. E. Mata: None. E. Fernandez-Tabanera: None. M. Carcajona: None. L. De
la Vega: None. D. Rodriguez: None. G. Benito: None. N. Sánchez-Bolivar: None. P. Maietta: None. J. Botet:
None. S. Alvarez: None. Clinical and molecular characterizationof three cases of
Floating-Harbor syndrome A. M. Travessa1, P. Dias1, L. Sampaio2, I. Cordeiro1, S. Martins3,
A. Sousa3, A. B. Sousa1 P08.22B 1Serviço de Genética Médica, Departamento de Pediatria,
Hospital de Santa Maria, Centro Hospitalar Lisboa Norte,
Centro Académico de Medicina de Lisboa, Lisbon, Portugal,
2Unidade de Endocrinologia Pediátrica, Serviço de Pediatria
Médica, Departamento de Pediatria, Hospital de Santa Maria,
Centro Hospitalar Lisboa Norte, Centro Académico de
Medicina de Lisboa, Lisbon, Portugal, 3Centro de
Neurodesenvolvimento, Serviço de Pediatria Médica,
Departamento de Pediatria, Hospital de Santa Maria, Centro
Hospitalar Lisboa Norte, Centro Académico de Medicina de
Lisboa, Lisbon, Portugal Biallelic loss-of-function mutations of EZH1 may cause
novel developmental disorder Biallelic loss-of-function mutations of EZH1 may cause
novel developmental disorder N. Okamoto1, H. Sakamoto2, K. Yanagi2, T. Kaname2 N. Okamoto1, H. Sakamoto2, K. Yanagi2, T. Kaname2 N. Okamoto1, H. Sakamoto2, K. Yanagi2, T. Kaname2 1Department of Medical Genetics, Osaka Women's and
Children's Hospital, Osaka, Japan, 2Department of Genome
Medicine, National Center for Child Health and Development,
Tokyo, Japan 1Department of Medical Genetics, Osaka Women's and
Children's Hospital, Osaka, Japan, 2Department of Genome
Medicine, National Center for Child Health and Development,
Tokyo, Japan Introduction: EZH1 is a component of a Polycomb repres-
sive complex-2 (PRC2) that mediates methylation of
H3K27. It has important roles in the maintenance of
embryonic stem cell pluripotency and plasticity. Mutations
of PRC2 subunits often cause over growth and neurological
diseases. Weaver syndrome is characterized by overgrowth,
intellectual disability (ID), and characteristic facial features. Mutations in EZH2 have been identified as a cause of
Weaver syndrome. We report sisters with biallelic loss-of-
function mutations of EZH1. Clinical report: (Patient 1)
The 7-year-old female was the first child of healthy and Introduction: Floating-Harbor syndrome (FHS) (MIM#
136140) is a very rare condition characterized by short
stature with delayed bone age, expressive language delay
and typical facial dysmorphism. FHS is due to heterozygous
pathogenic variants in the SRCAP gene. We present three
cases of FHS. Our aim is to highlight the clinical and
molecular features of this syndrome. Cases description: We present three patients, aged 5, 10
and 11 years old, all with proportionate short stature with
delayed bone age, relative macrocephaly, and mild to Abstracts from the 51st European Society of Human Genetics Conference: Posters 223 moderate intellectual disability/ developmental delay with
language impairment. Other common features included
recurrent respiratory infections, hypotonia in the neonatal
period, brachydactyly, 5th finger clinodactyly, high-pitched
voice and dental problems. One of the patients presented
also deafness, constipation, Arnold-Chiari malformation,
and absence seizures. Two cases were medicated with
growth hormone (GH). In all cases, the diagnosis was
suggested by the facial gestalt and confirmed by the
identification
of
the
c.7303C>T,
p.(Arg2435*)
or
c.7330C>T,
p.(Arg2444*)
pathogenic
variants
in
the
SRCAP gene. moderate intellectual disability/ developmental delay with
language impairment. Other common features included
recurrent respiratory infections, hypotonia in the neonatal
period, brachydactyly, 5th finger clinodactyly, high-pitched
voice and dental problems. One of the patients presented
also deafness, constipation, Arnold-Chiari malformation,
and absence seizures. Two cases were medicated with
growth hormone (GH). In all cases, the diagnosis was
suggested by the facial gestalt and confirmed by the
identification
of
the
c.7303C>T,
p.(Arg2435*)
or
c.7330C>T,
p.(Arg2444*)
pathogenic
variants
in
the
SRCAP gene. P08.26B diagnostic approach with genetic tests of global
developmental delay and/or intellectual disability: single
tertiary centre experience tertiary centre experience Daejeon St. Mary's hospital, Daejeon, Korea, Republic of Daejeon St. Mary's hospital, Daejeon, Korea, Republic of A.M. Travessa: None. P. Dias: None. L. Sampaio:
None. I. Cordeiro: None. S. Martins: None. A. Sousa:
None. A.B. Sousa: None. Diagnostic approach with genetic tests of global develop-
mental delay and/or intellectual disability P08.25A Background: Global developmental delay (GDD) and
intellectual disability (ID) are common cause for referral to
pediatric department. Recent advance in genetic tests have
resulted that multiple diagnostic approach including brain
imaging, metabolic tests, or genetic tests is possible for
children with GDD or ID. N. Okamoto1, H. Sakamoto2, K. Yanagi2, T. Kaname2 expected, targeting both the mGluR5 and the GABAergic
pathways simultaneously did not result in a synergistic
effect, but in a slight worsening of the social behavior
phenotype. This does implicate that both pathways are
interconnected and important for social behavior. Our
results underline the tremendous fine-tuning that is needed
to reach the excitatory-inhibitory balance in the synapse in
relation to social behavior. We believe that alternative
strategies focused on combination therapy should be further
explored, including targeting pathways in different cellular
compartments or cell-types. S. Zeidler: None. H. De Boer: None. R. Hukema: None. R. Willemsen: None. Conclusions: FHS should be considered in the differ-
ential diagnosis of short stature and language developmental
delay, and can be recognized by the typical facial gestalt. We report the second case of FHS with Arnold-Chiari
malformation and a case with toenails hypoplasia. The use
of GH is controversial, and should be evaluated in these
cases. Long term follow-up is needed to understand the
evolution of the phenotype of patients with this very rare
entity. J. Han Daejeon St. Mary's hospital, Daejeon, Korea, Republic of S. Zeidler, H. De Boer, R. Hukema, R. Willemsen S. Zeidler, H. De Boer, R. Hukema, R. Willemsen Methods: We retrospectively reviewed the medical
records of children with GDD/ID attending the pediatric
neurology department of Daejeon St. Mary’s Hospital
during the period from January 2016 through March 2017. Combination therapy in fragileX syndrome; possibilities
and pitfalls illustrated by targeting themGluR5 and GABA
pathway simultaneously Combination therapy in fragileX syndrome; possibilities
and pitfalls illustrated by targeting themGluR5 and GABA
pathway simultaneously Abstract G. Pascolini1, E. Agolini2, S. Majore1, A. Novelli2,
P. Grammatico1, M. Digilio3 G. Pascolini1, E. Agolini2, S. Majore1, A. Novelli2,
P. Grammatico1, M. Digilio3 Background: HNRNPU (OMIM *6022869) encodes a
member of the heterogeneous nuclear ribonucleoprotein
(hnRNP) family which mediates different aspects of RNA
transport and metabolism by forming ribonucleoprotein
complexes in the nucleus. Heterozygous HNRNPU variants
have been associated with global developmental delay,
seizures and autistic features; additional symptoms such as
cardiac and renal abnormalities as well as dysmorphic
features have been reported. 1Medical Genetics Laboratory, Department of Molecular
Medicine, Sapienza University, San Camillo-Forlanini
Hospital, Rome, Rome, Italy, 2Medical Genetics Laboratory,
Bambino Gesù Paediatric Hospital, IRCCS, Rome, Italy,
Rome, Italy, 3Medical Genetics Unit, Bambino Gesù Paediatric
Hospital, IRCCS, Rome, Italy, Rome, Italy Case Presentation: Here we report the identification of
previously unreported de novo variants in two index
patients presenting with intellectual disability, speech
impairment
and
epilepsy. Additional
clinical
features
included cardiac defects, recurrent infections, hypermobility
of the joints, muscular hypotonia and brain atrophy. Case Presentation: Here we report the identification of
previously unreported de novo variants in two index
patients presenting with intellectual disability, speech
impairment
and
epilepsy. Additional
clinical
features
included cardiac defects, recurrent infections, hypermobility
of the joints, muscular hypotonia and brain atrophy. Introduction: A recent syndromic condition with cranio-
facial dysmorphisms, comprising congenital ocular defect
and neurodevelopmental delay named Helsmoortel-Van der
Aa
Syndrome
(HVDAS)(OMIM#615873),
has
been
described and molecularly defined, identifying pathogenic
mutations
in
the
ADNP
gene
(OMIM#611386)
as
biological cause. Introduction: A recent syndromic condition with cranio-
facial dysmorphisms, comprising congenital ocular defect
and neurodevelopmental delay named Helsmoortel-Van der
Aa
Syndrome
(HVDAS)(OMIM#615873),
has
been
described and molecularly defined, identifying pathogenic
mutations
in
the
ADNP
gene
(OMIM#611386)
as
biological cause. Methods: We performed exome sequencing on the index
patients. Variant confirmation and carrier testing was done
by Sanger sequencing. Materials and Methods: We report on two children,
displaying intellectual disability (ID) and peculiar congeni-
tal eyes anomalies, referred to a clinical genetics evaluation
to better define their condition. Results: Exome sequencing and downstream variant
prioritization led us to identify predictively truncating de
novo Variants in HNRNPU including a heterozygous
stop_variant (c.1801C>T, p.Arg601*) and a frameshift
variant (c.974del, p.Ala325Alafs*14). Results: Both patients resulted to carry a de novo
nonsense mutation in the ADNP gene, identified by Next
Generation Sequencing analysis (NGS). P08.27C Helsmoortel-Van der Aa Syndrome as emerging clinical
diagnosis in intellectually disabled children with autistic
traits and ocular involvement Abstract Conclusion: Here we report on the phenotypic features
associated with novel truncating de novo mutations in
HNRNPU. Global
developmental
delay
and
epilepsy
present as common features observed in all HNRNPU cases
reported to date. Apart from the central nervous system
additional organ systems appear to be variable affected. Our
findings support the role and implication of HNRNPU in the
development and functions of different body organs in
addition to the central nervous system. Conclusions: The review of present and literature reports,
suggests that the diagnosis of HVDAS should be suspected
in patients with ID accompanied by behavioral features in
the Autism Spectrum Disorder and distinctive craniofacial
phenotype. Among dysmorphisms due to malformation of
the periorbital region, ptosis appears to be particularly
recurrent in HVDAS. Furthermore, the present patients
could support the inclusion of the HVDAS associated with
specific mutations clustering within a small ADNP genomic
region
among
clinical
conditions
reminiscent
of
the
blepharophimosis/mental retardation syndromes (BMRS). W. Habhab: None. R. Buchert: None. L. Laugwitz:
None. M. Grimmel: None. M. Sturm: None. B. Oehl-
Jaschkowitz: None. A. Kuster: None. T. Haack: None. G. Pascolini: None. E. Agolini: None. S. Majore: None. A. Novelli:
None. P. Grammatico:
None. M. Digilio: None. P08.29A A novel HS6ST2 variant reduces the enzyme activity in two
brothers with severe myopia and syndromic intellectual
disability Identification of novel variants in HNRNPU associated with
global developmental delay, epilepsy and multiple organ
system defects Erasmus MC, Rotterdam, Netherlands Fragile X syndrome (FXS) is the most common mono-
genetic cause of intellectual disability and autism. The
disorder is characterized by altered synaptic plasticity in the
brain. Synaptic plasticity is tightly regulated by a complex
balance of different synaptic pathways. In FXS, various
synaptic pathways are disrupted, including the excitatory
metabotropic glutamate receptor 5 (mGluR5) and the inhi-
bitory γ-aminobutyric acid (GABA) pathways. Targeting
each of these pathways individually, has demonstrated
beneficial effects in animal models, but not in patients with
FXS. This lack of translation might be due to over-
simplification of the disease mechanisms when targeting
only one affected pathway, in spite of the complexity of the
many pathways implicated in FXS. In this report we outline
the hypothesis that targeting more than one pathway
simultaneously, a combination therapy, might improve
treatment effects in FXS. In addition, we present a glance of
the first results of chronic combination therapy on social
behavior in Fmr1 KO mice. In contrast to what we Results: A total of 75 children were investigated for
GDD/ID in the pediatric neurology departments. Ten
patients (13%) were diagnosed with specific disease such
as Rett syndrome, Prader Willi syndrome. Chromosomal
microarray was performed as 1st tier test and 25 patients
(33%) diagnosed. Brain structural abnormalities were
showed 8 patients (11%) and two patients diagnosed
Fragile X syndrome. Thirty patients who did not revealed
etiology of GDD/ID received gene panel by next generation
sequencing. Eight patients were found out underlying
genetic etiology: CHD8, ZDHCC9, CACNA1H, SMARCB1,
FOXP1, and KCNK18. However, twenty two patients
(29%) were remained uncertain cause of GDD/ID. Conclusion: This study provides information on pedia-
tricians to diagnostic approach of children with GDD/ID. Early detection of detection of GDD/ID can help treatment
planning and assignment of the recurrence risk for siblings,
and emotional relief for the family. Practicality, precision, J. del Picchia 224 A novel HS6ST2 variant reduces the enzyme activity in two
brothers with severe myopia and syndromic intellectual
disability W. Habhab1, R. Buchert1, L. Laugwitz1, M. Grimmel1,
M. Sturm1, B. Oehl-Jaschkowitz2, A. Kuster3, T. Haack1 yield, and genome-based diagnosis in medical care must be
defined in future researches and will need prospective study
designs. 1Institute of Medical Genetics and Applied Genomics,
Tuebingen, Germany, 2Gemeinschaftspraxis für
Humangenetik, Biomedizinisches Zentrum, 66424 Homburg
Saar, Germany, 3Neurometabolism department, Nantes
Hospital and University, Nantes, France 1Institute of Medical Genetics and Applied Genomics,
Tuebingen, Germany, 2Gemeinschaftspraxis für
Humangenetik, Biomedizinisches Zentrum, 66424 Homburg
Saar, Germany, 3Neurometabolism department, Nantes
Hospital and University, Nantes, France J. Han: None. P08.30B 1Division of Pathology, Fondazione IRCCS Ca' Granda
Ospedale Maggiore Policlinico, Milano, Italy, 2Department of
Pathophysiology & Transplantation, Università degli Studi di
Milano, Milano, Italy, 3Medical Genetics, Department of
Health Sciences, Università degli Studi di Milano, Milano,
Italy, 4Pediatric Highly Intensive Care Unit, Fondazione
IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano,
Italy, 5Department of Medical Biotechnology and
Translational Medicine, Università degli Studi di Milano,
Milano, Italy, 6GenomiX4Life, Università degli Studi di
Salerno, Salerno, Italy M. Vlckova1, K. Sterbova2, J. Neupauerova2, D. Stanek2,
H. Zunova1, V. Moslerova1, L. Sedlackova2, J. Paderova1,
M. Havlovicova1, P. Seeman2, Z. Sedlacek1, P. Lassuthova2 M. Vlckova1, K. Sterbova2, J. Neupauerova2, D. Stanek2,
H. Zunova1, V. Moslerova1, L. Sedlackova2, J. Paderova1,
M. Havlovicova1, P. Seeman2, Z. Sedlacek1, P. Lassuthova2 1Department of Biology and Medical Genetics, Charles
University 2nd Faculty of Medicine and University Hospital
Motol, Prague, Czech Republic, 2Department of Paediatric
Neurology, Charles University 2nd Faculty of Medicine and
University Hospital Motol, Prague, Czech Republic Pathogenic variants in HUWE1 have been described as the
cause of a broad spectrum of phenotypes ranging from
isolated craniosynostosis to non-syndromic or syndromic Pathogenic variants in HUWE1 have been described as the
cause of a broad spectrum of phenotypes ranging from
isolated craniosynostosis to non-syndromic or syndromic Introduction: An Italian family composed of healthy non-
consanguineous parents, two affected male children and
one healthy son came to our attention. The affected sibs
presented with febrile seizures, EEG irregular diffuse
spike-wave anomalies, severe myopia, mild facial dys-
morphisms and developmental delay. Their phenotypic
features resembled those associated with Brooks Wis-
niewski Brown (BWB) syndrome even though probands
were wild type for HUWE1, described in literature as
causative of BWB. isolated craniosynostosis to non syndromic or syndromic
X-linked
intellectual
disability
(ID). Some
of
the
syndromic cases were clinically diagnosed as the Brooks,
Juberg-Marsidi or Kabuki-like syndromes. To our knowl-
edge, 16 families with HUWE1 mutations have been
reported, and all variants were missense. No genotype-
phenotype correlation has been identified yet. We present a
novel
family
with
a
missense
HUWE1
variant
NM_031407.6:c.12195G>C p.(Trp4065Cys) in two male
cousins. Both boys suffered from ID, epilepsy and autism,
and showed similar facial phenotypes with epicanthus,
strabism, short palpebral fissures, low-set dysplastic ears,
prominent nose and broad columella. One of the boys had a
short stature and failure to thrive. P08.28D Identification of novel variants in HNRNPU associated with
global developmental delay, epilepsy and multiple organ
system defects Abstracts from the 51st European Society of Human Genetics Conference: Posters 225 L. Paganini1,2, L. Fontana1,2, E. Bonaparte1,2, D. Rovina3,
D. Milani4, S. Esposito4, L. Hadi5, M. Chetta6, L. Riboni5,
S. Sirchia3, S. Tabano1,2, M. Miozzo1,2 L. Paganini1,2, L. Fontana1,2, E. Bonaparte1,2, D. Rovina3,
D. Milani4, S. Esposito4, L. Hadi5, M. Chetta6, L. Riboni5,
S. Sirchia3, S. Tabano1,2, M. Miozzo1,2 P08.30B
HUWE1 gene variants as the cause of snydromic or
nonsyndromic intellectual disability: spectrum of the
HUWE1 associated phenotypes Vilnius university, Vilnius, Lithuania Due to extensive locus heterogeneity the causes of intel-
lectual disability are numerous and unknow in many cases. For such spectrum of disorders the variation of de novo
mutations (DNMs) are less frequent and potentially more
deleterious, could offer insights into risk-determining genes
or integrated deleterious effect of DNMs. Accordingly, the
main interest of this study was to evaluate the observed rate
and consequence of de novo point and indels mutations in
the exomes of ID subjects and their sibs. The data set
consisted of the Lithuanian patients with ID and relevant
family members samples. Sequencing data of 9 parent-
offspring trios exomes generated by sequencer SOLiD
5500™and 33 more parent-offspring and 16 parent-sibs
trios exomes were generated by Illumina™. Considering
platform of sequencing primary analysis performed by
Lifescope™and GATK™respectively. DNMs called by
VarScan. Called potentially DNMs were filtered, manually
reviewed by the IGV and validated by Sanger sequencing. Functional annotation of all identified DNMs were per-
formed using ANNOVAR. The findings indicate that the
number of DNMs varies between 1 and 12. Preliminary
results of single nucleotide DNM rate before validation step
is 3.1×10-6, 95% CI [2.2x10-6; 4.2x10-6], and de novo rate
for indels - 1.1×10-6, 95% CI [5.4x10-8; 5.7x10-6]. Func-
tional
analysis
revealed
rs398123009
(CM1211547),
rs121909121
(CM072075),
rs28934906
(CM992178)
DNMs
that
determine
Schuurs-Hoeijmakers
(OMIM:
615009), Pitt-Hopkin (OMIM: 602272) and Rett syndrome
(OMIM: 312750). Functional analysis of remaining DNMs,
their clusters and hot spots are ongoing. This study sup-
ported by the Lithuanian-Swiss cooperation program under
UNIGENE project agreement no. CH-3-ŠMM-01/04. Introduction: Most of the over 200 phenotypes associated
with X-linked intellectual disability (XLID; 10% of cases of
ID in boys) are not specific and next-generation sequencing
techniques are crucial for their accurate diagnosis. HUWE1
missense variants have been described as causative in a
limited number of XLID families with wide phenotypic
variability. Methods: Clinical and molecular characterization of all
cases with HUWE1 point mutations identified in our
department
through
retrospective
analysis
of
medical
records and literature comparison. Results: We report 9 affected males from 6 unrelated
families, each with a distinct HUWE1 missense variant
identified. One variant had already been reported and was
considered likely pathogenic. Five were novel: 1 was
considered
as
likely
pathogenic,
3
as
of
uncertain
significance and 1 as benign due to its presence on the
unaffected brother (case excluded). One was de novo and 5
were inherited. P08.31C X-linked intellectual disability: report on 6 families with
HUWE1 missense mutations and literature review S.M. Ribeiro: None. J. Sá: None. A. Beleza: None. C. Reis: None. F. Ramos: None. P. Louro: None. S. Maia:
None. H. Williams: None. M. Harakalova: None. G. Van
Haaften: None. M. Simões: None. C. Barroso: None. H. Froufe: None. C. Egas: None. J.M. Saraiva: None. S.B. Sousa: None. M. Venâncio: None. S. M. Ribeiro1, J. Sá1, A. Beleza1, C. Reis1, F. Ramos1,
P. Louro1, S. Maia1, H. Williams2, M. Harakalova3, G. Van
Haaften3, M. Simões4, C. Barroso4,5, H. Froufe4, C. Egas4,5,
J. M. Saraiva1,6, S. B. Sousa1,6, M. Venâncio1,6 1Medical Genetics Unit, Hospital Pediatrico, Centro
Hospitalar Universitário de Coimbra, Coimbra, Portugal,
2GOSgene, Genetics and Genomic Medicine Programme, UCL
GOS Institute of Child Health, London, United Kingdom,
3Department of Medical Genetics, Centre for Molecular
Medicine, University Medical Centre Utrecht, Utrecht,
Netherlands, 4Biocant, Transfer Technology Association,
Cantanhede, Portugal, 5Center for Neuroscience and Cell
Biology, University of Coimbra, Coimbra, Portugal,
6University Clinic of Genetics, Faculty of Medicine, University
of Coimbra, Coimbra, Portugal P08.30B M. Harakalova: None. G. Van
Haaften: None. M. Simões: None. C. Barroso: None. H. Froufe: None. C. Egas: None. J.M. Saraiva: None. S.B. Sousa: None. M. Venâncio: None. 2020 program, Portugal 2020, European Union, through the
European Regional Development Fund. L. Pranckeniene, A. Jakaitiene, L. Ambrozaityte, V. Kucinskas L. Pranckeniene, A. Jakaitiene, L. Ambrozaityte, V. Kucinskas P08.30B Their facial phenotype
was also very similar to patients with a neighboring variant
(p.(R4063Q), Friez et al., 2016)), but not to the other
previously published cases whose variants were more
distant. To our knowledge, autism has not been associated
with HUWE1 yet, in contrast to craniosynostosis, which has
been reported repeatedly but was not present in our patients. Interestingly, craniosynostosis has been described only in
patients carrying different amino acid substitutions in
position 110. Our report contributes to the delineation of
the HUWE1 phenotypic spectrum which is apparently broad
and seems not to be strongly influenced by the localization
of the variant, with a possible exception of craniosynostosis. The phenotypic heterogeneity must be considered in
interpretation of exome sequencing data. Supported by
MH CR AZV 15-33041A, 17-29423A and 00064203. Material and Methods: WES of all the family members
was set up. To evaluate the effect of the identified variant,
site-directed mutagenesis was performed on a commercial
expression vector containing HS6ST2 cDNA. Transient
expression of both wild type and mutant transcripts was
carried out in HEK293 cells and HS6ST2 enzymatic
activity was assayed. Results and Conclusion: WES highlighted the novel
maternally-inherited mutation c.916 G>C (G306R) at
HS6ST2 gene, present in hemizygous state in the two
probands and absent in their healthy brother. HS6ST2 maps
at Xq26.2, a locus associated with X-linked mental
retardation and recessive myopia, and encodes a member
of the heparan sulfate (HS) sulfotransferase (ST) family
expressed in brain and eye during development. c.916 G>C
variant affects HS6ST2 substrate binding site and its effect
was considered “deleterious” by many in-silico tools. In-
vitro enzymatic assay revealed that HS6ST2 mutant isoform
maintained 36% of transferase activity, supporting our
hypothesis of its involvement in the pathological phenotype
establishment. M. Vlckova: None. K. Sterbova: None. J. Neupauer-
ova: None. D. Stanek: None. H. Zunova: None. V. Moslerova: None. L. Sedlackova: None. J. Paderova:
None. M. Havlovicova: None. P. Seeman: None. Z. Sedlacek: None. P. Lassuthova: None. L. Paganini: None. L. Fontana: None. E. Bonaparte:
None. D. Rovina: None. D. Milani: None. S. Esposito:
None. L. Hadi: None. M. Chetta: None. L. Riboni: None. S. Sirchia: None. S. Tabano: None. M. Miozzo: None. 226 J. del Picchia 2020 program, Portugal 2020, European Union, through the
European Regional Development Fund. S.M. Ribeiro: None. J. Sá: None. A. Beleza: None. C. Reis: None. F. Ramos: None. P. Louro: None. S. Maia:
None. H. Williams: None. M. Tessarech1, A. Guichet1, M. Barth1,2, S. Vuillaumier3,4,
J. Durigneux1, I. Pellier1, T. Dupré3,4, A. Bruneel3, P. Van
Bogaert1, N. Seta3,5, D. Bonneau1,2, E. Colin1,2 M. Moreno-Igoa1, B. Hernández-Charro1, M. Artigas-López1,
A. Bengoa-Alonso1, M. M. Mehrjouy2, N. Tommerup2, M. A. Ramos-Arroyo1 1CHU ANGERS, ANGERS, France, 2UMR CNRS 6015,
INSERM U1083, Angers, France, 3AP-HP, Hôpital Bichat-
Claude Bernard, PARIS, France, 4INSERM U1149 CRB3,
Université Denis Diderot, Paris 7, Paris, France, 5Université
Paris Descartes, Paris, France 1S.Genética, Complejo Hospitalario de Navarra, Pamplona,
Spain, 2ICMM, University of Copenhagen, Copenhagen,
Denmark 1S.Genética, Complejo Hospitalario de Navarra, Pamplona,
Spain, 2ICMM, University of Copenhagen, Copenhagen,
Denmark Introduction: FAAH2 encodes a fatty acid amide hydrolase
that plays a role in the complex neural endocannabinoid
signaling system. FAAH2 gene maps to chromosome Xp11
and has been suggested as a possible candidate gene for X-
linked intellectual disability (XLID). We present a female
patient with intellectual disability (ID) and epilepsy, bearing
a de novo translocation spanning FAAH2 gene. Introduction: FAAH2 encodes a fatty acid amide hydrolase
that plays a role in the complex neural endocannabinoid
signaling system. FAAH2 gene maps to chromosome Xp11
and has been suggested as a possible candidate gene for X-
linked intellectual disability (XLID). We present a female
patient with intellectual disability (ID) and epilepsy, bearing
a de novo translocation spanning FAAH2 gene. Congenital glycosylation deficits (CDG syndromes) are a
group of pathologies caused by a defect of synthesis of
glycoproteins. COG7 is a gene encoding one of the subunits
of the Golgi oligomeric complex (COG), involved in
intracellular trafficking and modification of glycoproteins. The pathogenic variants of COG7 cause CDG type -IIe,
resulting in abnormalities of the N- and 0-glycosylation
pathway. So far, only eight cases have been reported in the
literature. We describe two sisters from a Moroccan con-
sanguineous couple and review the literature. These two
sisters present a phenotype associating moderate to severe
intellectual impairment with abnormalities in cerebral MRI,
unexplained episodes of fever with macrophagic activation
syndrome-like, hepatomegaly and growth retardation. They
are also insensitive to pain in the extremities, which has
never been described before. Through the association of
homozygosity mapping and exome sequencing, we have
highlighted a homozygous intronic variant c. 170-7A>G in
the COG7 gene, previously reported in a consanguineous
family evoking a founder effect of this variant. Biochemi-
cally, the electrophoresis of transferrin on a Guthrie blood
spot was normal and only the 2D isoform analysis of
transferrin and apoC3 were able to confirm the diagnosis of
CDG type- IIe. Vilnius university, Vilnius, Lithuania The X-inactivation patterns were normal in
3/5 heterozygous mothers. Conclusions: Our clinical findings are in accordance with
the literature. All patients had significant global develop-
ment
delay
/
ID
with
limited
speech
and
variable
dysmorphisms. One had pre- and postnatal severe short
stature and postnatal microcephaly. Features not previously
reported include a multicentric ganglioneurocytoma and a
linear hypopigmented nevus. Our results reinforce the likely
pathogenic role of HUWE1-missense variants in XLID and
the difficulty in interpreting novel variants in this gene. Conclusions: Our clinical findings are in accordance with
the literature. All patients had significant global develop-
ment
delay
/
ID
with
limited
speech
and
variable
dysmorphisms. One had pre- and postnatal severe short
stature and postnatal microcephaly. Features not previously
reported include a multicentric ganglioneurocytoma and a
linear hypopigmented nevus. Our results reinforce the likely
pathogenic role of HUWE1-missense variants in XLID and
the difficulty in interpreting novel variants in this gene. L. Pranckeniene: None. A. Jakaitiene: None. L. Ambrozaityte: None. V. Kucinskas: None. This work was partly funded by the In2Genome, ref
CENTRO-01- 0247- FEDER-017800, of the CENTRO This work was partly funded by the In2Genome, ref
CENTRO-01- 0247- FEDER-017800, of the CENTRO 227 Abstracts from the 51st European Society of Human Genetics Conference: Posters M. Tessarech1, A. Guichet1, M. Barth1,2, S. Vuillaumier3,4,
J. Durigneux1, I. Pellier1, T. Dupré3,4, A. Bruneel3, P. Van
Bogaert1, N. Seta3,5, D. Bonneau1,2, E. Colin1,2 This observation of a consanguineous
family allows the identification of two new cases of CDG
type -IIe with the variant c. 170-7A>G in the COG7 gene. We extend the phenotype of CDG syndromes caused by
COG7 and show the importance of transferrin and ApoC3
analysis in 2D electrophoresis for the diagnostic orientation
of CDG syndromes. Patient and Methods: 34-years old female proband with
mild-moderate ID and absence and generalized seizures,
since the age of 7 years. Conventional and molecular
karyotypes
were
performed. FISH
experiments
were
completed using BAC clones for breakpoint’s approach. Rearrangement breakpoints were characterized by whole
genome sequencing and validated by Sanger sequencing. X-
inactivation studies were also performed. Patient and Methods: 34-years old female proband with
mild-moderate ID and absence and generalized seizures,
since the age of 7 years. Conventional and molecular
karyotypes
were
performed. FISH
experiments
were
completed using BAC clones for breakpoint’s approach. Rearrangement breakpoints were characterized by whole
genome sequencing and validated by Sanger sequencing. X-
inactivation studies were also performed. Results:
Cytogenetic
analysis
showed
an
apparent
balanced translocation [46,XX,t(X;10)(p11.2;q25)dn] and
absence of cryptic genomic imbalances. Translocation
breakpoints were narrowed by FISH between RP11-
466I19 and RP11-613D18 probes at 10q25, and RP11-
141B06 and RP11-497J09 at Xp11.2. Sequencing analysis
revealed two disrupted genes at translocation breakpoints:
FAAH2 (Xp11.2) and NHLRC2 (10q25). The normal X-
chromosome was > 90% inactive. Conclusions: Our patient displayed a skewed pattern of
X-inactivation of the normal X-chromosome, silencing the
normal FAAH2 gene, while the disrupted allele is predicted
to render a non-functional protein. No mutations in the
NHLRC2 gene or associated phenotypes have been reported
in humans and animal models suggest embryonic lethality
in recessive inheritance. We, therefore, conclude that the
FAAH2 gene is likely to play an important role in
neurodevelopment, causing XLID and epilepsy. M. Tessarech: None. A. Guichet: None. M. Barth:
None. S. Vuillaumier: None. J. Durigneux: None. I. Pellier: None. T. Dupré: None. A. Bruneel: None. P. Van
Bogaert: None. N. Seta: None. D. Bonneau: None. E. Colin: F. Consultant/Advisory Board; Modest; Novartis
Pharma SAS. M. Tessarech: None. A. Guichet: None. M. Barth:
None. S. Vuillaumier: None. J. Durigneux: None. I. Pellier: None. T. Dupré: None. A. Bruneel: None. P. Van
Bogaert: None. N. Seta: None. D. Bonneau: None. E. Colin: F. Consultant/Advisory Board; Modest; Novartis
Pharma SAS. M. Moreno-Igoa: None. B. Hernández-Charro: None. M. Artigas-López: None. A. Bengoa-Alonso: None. M.M. Mehrjouy: None. N. De novo FBXO11 mutations are associated with intellectual
disability, microcephaly and behavioural anomalies De novo FBXO11 mutations are associated with intellectual
disability, microcephaly and behavioural anomalies A. Kuechler1, D. Fritzen2, J. Becker2, S. Peters2, M. Sturm3,
H. Hundertmark2, A. Schmidt2, M. Kreiß2, T. Strom4,
D. Wieczorek5, T. Haack3,6, S. Beck-Wödl7, K. Cremer2,
H. Engels2 1Institut für Humangenetik, Universitätsklinikum Essen,
Universität Duisburg-Essen, Essen, Germany, 2Institute of
Human Genetics, University of Bonn, School of Medicine &
University Hospital Bonn, Bonn, Germany, 3IInstitute of
Medical Genetics and Applied Genomics, University of
Tübingen, Tübingen, Germany, 4Institute of Human Genetics,
Helmholtz Zentrum München, Neuherberg, Germany,
5Heinrich-Heine-University, Medical Faculty, Institute of
Human Genetics, Düsseldorf, Germany, 6Institute of Human
Genetics, Technische Universität München, München,
Germany, 7Institute of Medical Genetics and Applied
Genomics, University of Tübingen, Tübingen, Germany More than 1000 genes are known in intellectual disability
(ID) and new genes are still discovered monthly. Because of
this genetic heterogeneity, Next Generation Sequencing
(NGS) has allowed great progress. We included 676 probands with ID and their parents after
clinical evaluation and negative testing, i.e array CGH,
Fragile X and, for some cases, targeted sequencing or small
NGS panels. We performed medical exome sequencing
(MES) with the Illumina TruSight One (including 4813
genes known in pathology) for 262 patients. And then we
performed whole exome sequencing (WES) with the Roche
Medexome for 464 patients, including 50 patients with
negative MES. Intellectual disability (ID) has an estimated prevalence of
1.5-2% and in most affected persons its genetic basis
remains unclear. Whole exome sequencing (WES) has
proven to be a valuable tool to identify causative gene
defects and has shown that a large proportion of sporadic ID
cases results from de novo mutations. We made 55/262 (21%) diagnoses with MES and 202/
464 (44%) with WES. 38% of the diagnostic variants
identified with WES were in genes absent from MES
because they were too recently discovered. Besides, we
found 17/50 (34%) diagnoses with WES in patients with no
diagnosis with MES. Moreover, we identified 25 additional
likely pathogenic variants with WES in yet unpublished
genes (ongoing international collaborations). All diagnoses
were
validated
through
strong
collaboration
between
clinical and biological geneticists. Here, we present two unrelated patients with common
clinical features and deleterious de novo variants in
FBXO11 detected by WES. Patient 1 has mild ID, mild
microcephaly, hyperkinetic disorder, corrected cleft lip and
alveolus, mild brain atrophy and mild dysmorphism. P08.37A years, which makes a panel of genes rapidly obsolete
whereas WES remains always up to date and sees its
diagnostic rate increase with scientific advances. Medical exome sequencing vs whole exome sequencing in
the diagnosis of intellectual disability C. Nava: None. B. Keren: None. C. Mignot: None. B. Julien: None. C. Estrade: None. S. Karagic: None. A. Lafitte: None. E. Lejeune: None. C. Mach: None. V. Olin: None. T. Courtin: None. A. Afenjar: None. D. Doummar: None. M. Moutard: None. T. Billette de
Villemeur: None. M. Nougues: None. S. Valence: None. B. Héron: None. D. Rodriguez-Levi: None. L. Burglen:
None. S. Whalen: None. D. Haye: None. S. Heide: None. P. Charles: None. I. Marey: None. C. Depienne: None. D. Héron: None. C. Nava: None. B. Keren: None. C. Mignot: None. B. Julien: None. C. Estrade: None. S. Karagic: None. A. Lafitte: None. E. Lejeune: None. C. Mach: None. V. Olin: None. T. Courtin: None. A. Afenjar: None. D. Doummar: None. M. Moutard: None. T. Billette de
Villemeur: None. M. Nougues: None. S. Valence: None. B. Héron: None. D. Rodriguez-Levi: None. L. Burglen:
None. S. Whalen: None. D. Haye: None. S. Heide: None. P. Charles: None. I. Marey: None. C. Depienne: None. D. Héron: None. C. Nava1,2,3, B. Keren1,2, C. Mignot1,2, B. Julien1, C. Estrade1,
S. Karagic1, A. Lafitte1, E. Lejeune1, C. Mach1, V. Olin1,
T. Courtin1, A. Afenjar4,5, D. Doummar6, M. Moutard6,
T. Billette de Villemeur6, M. Nougues6, S. Valence6, B. Héron6,
D. Rodriguez-Levi6, L. Burglen4,7, S. Whalen4,7, D. Haye1,5,
S. Heide1,2,5, P. Charles1,2,5, I. Marey1,2,5, C. Depienne1,3,
D. Héron1,2,5 C. Nava1,2,3, B. Keren1,2, C. Mignot1,2, B. Julien1, C. Estrade1,
S. Karagic1, A. Lafitte1, E. Lejeune1, C. Mach1, V. Olin1,
T. Courtin1, A. Afenjar4,5, D. Doummar6, M. Moutard6,
T. Billette de Villemeur6, M. Nougues6, S. Valence6, B. Héron6,
D. Rodriguez-Levi6, L. Burglen4,7, S. Whalen4,7, D. Haye1,5,
S. Heide1,2,5, P. Charles1,2,5, I. Marey1,2,5, C. Depienne1,3,
D. M. Tessarech1, A. Guichet1, M. Barth1,2, S. Vuillaumier3,4,
J. Durigneux1, I. Pellier1, T. Dupré3,4, A. Bruneel3, P. Van
Bogaert1, N. Seta3,5, D. Bonneau1,2, E. Colin1,2 Tommerup: None. M.A. Ramos-
Arroyo: None. 228 J. del Picchia P08.37A Héron1,2,5 1Département de génétique, Hôpital Pitié-Salpêtrière, APHP,
Paris, France, 2Groupe de Recherche Clinique (GRC)
"déficience intellectuelle et autisme", UPMC, Paris, France,
3INSERM U 1127, CNRS UMR 7225, Sorbonne Universités,
UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et de la
Moelle épinière, ICM, Paris, France, Paris, France,
4Département de génétique, Hôpital Armand-Trousseau,
APHP, Paris, France, 5Centre de Référence Déficiences
Intellectuelles de Causes Rares, Hôpital de la Pitié-Salpêtrière,
Paris, France, 6Service de Neuropédiatrie, Hôpital Armand-
Trousseau, APHP, Paris, France, 7Centre de Référence
Malformations et maladies congénitales du cervelet, Hôpital
Armand-Trousseau, Paris, France New KIAA1033 mutations in 3 patients with syndromic
intellectual disability 1Carmel Medical Center, Haifa, Israel, 2Bnai Zion Medical
Center, Haifa, Israel Background: Numerous genes have been implicated in
non-syndromic mental retardation. The association of
ZNF674 gene mutations in isolated X-linked intellectual
disability is controversial. While some studies have shown a
clear relationship between mutations and deletions in
ZNF674 gene with mental retardation, other authors have
contradicted these findings. Our report presents a novel
mutation in ZNF674 gene. The cases: We describe two 48-
old and 39-old brothers with non-syndromic mental retar-
dation. Fragile X syndrome genetic testing and chromoso-
mal microarray analysis were normal. Maternal examination
was positive for skewed X-inactivation. Results: In KS iPSC and hESCs, we observed signifi-
cantly fewer cells (~10%) in the S-phase of the cell cycle
when compared to control cells. Accordingly, in these cells
we found a decrease in the expression of CCDN1 and E2F3,
two genes important for G1-S transition in the cell cycle. Additionally, we observed a delay in neuronal differentia-
tion of KS iPSC and hESCs compared to control lines. In
particular, at day 20 of the differentiation protocol, we
observed a 4 fold decrease in formation of CD44-CD184-
CD24+ neuronal cells in KS mutant cells compared to
control cells. Results: Whole exome sequencing of the older brother
revealed a novel hemizygous 46360713 A>G (Leu-104-
Pro) mutation in ZNF674 gene. The variant was not found
in large exome databases, and in-silico prediction programs
classified the mutation as "pathogenic". According to
sequence alignment of the ZNF674 protein from bacteria
to human, Leu104 is a highly conserved residue throughout
evolution. Family segregation analysis demonstrated the
mutation in the younger brother with mental retardation,
while two healthy males (a brother and a maternal uncle) Conclusions: Our results suggest that correct dosage of
KMT2D is essential for normal progression of cell cycle in
pluripotent stem cells and that altered cell cycle may
underlie delay in neuronal differentiation and ID in KS. S. Cuvertino: None. S. Kimber: None. S. Banka: None. S. Cuvertino, HipSci Consortium, S. Kimber, S. Banka A. Kuechler: None. D. Fritzen: None. J. Becker: None. S. Peters: None. M. Sturm: None. H. Hundertmark:
None. A. Schmidt: None. M. Kreiß: None. T. Strom:
None. D. Wieczorek: None. T. Haack: None. S. Beck-
Wödl: None. K. Cremer: None. H. Engels: None. A. Kuechler: None. D. Fritzen: None. J. Becker: None. S. Peters: None. M. Sturm: None. H. Hundertmark:
None. A. Schmidt: None. M. Kreiß: None. T. Strom:
None. D. Wieczorek: None. T. Haack: None. S. Beck-
Wödl: None. K. Cremer: None. H. Engels: None. Faculty of Biology, Medicine, and Health, Manchester, United
Kingdom Faculty of Biology, Medicine, and Health, Manchester, United
Kingdom Introduction: Kabuki syndrome (KS) is a multi-systemic
intellectual disability (ID) disorder. Loss-of-function (LoF)
KMT2D mutations are responsible for a vast majority of
cases of KS. KMT2D encodes a Histone3 Lysine4 methyl-
transferase but the underlying mechanism of ID in KS is
unknown. De novo FBXO11 mutations are associated with intellectual
disability, microcephaly and behavioural anomalies Trio
WES detected a heterozygous de novo 1bp insertion in the
splice donor site of exon 3 which is predicted unequivocally
to result in aberrant splicing. Patient 2 showed ID, growth
retardation, mild microcephaly, hyperkinetic and restless
behaviour, as well as mild dysmorphism. WES detected a
heterozygous de novo nonsense mutation. Thus nearly 40% of our WES diagnoses would not have
been found with MES, although MES theoretically included
all known ID genes when it was designed. This is explained
by the discovery of many new genes in ID over the past 3 229 Abstracts from the 51st European Society of Human Genetics Conference: Posters FBXO11 (Homo sapiens F-box protein 11) encodes a
member of the F-box protein family which form part of the
SCF ubiquitin ligases. Two ID patients with de novo
variants in FBXO11 are mentioned without additional
clinical
data
by
Lelieveld
et
al. (Nat
Neurosci. 2016;19:1194-6). Only one patient with clinical information
and a de novo FBXO11 mutation has been reported by
Martínez et al. (JMG 2017;54:87-92). Interestingly, this
patient carries the identical mutation as our patient 2 and
also displays ID, growth retardation, microcephaly, beha-
vioural anomalies, and dysmorphisms. Thus, we propose
deleterious de novo mutations in FBXO11 as a novel cause
of
ID
and
possibly
microcephaly
and
behavioural
anomalies. were negative. The mother was found to be a heterozygous
carrier. Conclusion: Our research sheds light on the few
controversial reports describing the relationship between
ZNF674 gene mutations and intellectual disability, support-
ing this association. L. Sagi-Dain: None. V. Adir: None. G. Larom-Khan:
None. A. Harari-Shaham: None. O. Sadeh: None. S. Sagi: None. A. Peleg: None. J. Haddad-Halloun: None. P08.44D
Cell cycle and neuronal differentiation defects in Kabuki-
KMT2D mutant hESC and iPSC lines Cell cycle and neuronal differentiation defects in Kabuki-
KMT2D mutant hESC and iPSC lines S. Cuvertino, HipSci Consortium, S. Kimber, S. Banka P08.41A Whole exome sequencing reveals a novel missense mutation
in the ZNF674 gene, underpinning its association with X-
linked mental retardation L. Sagi-Dain1, V. Adir1, G. Larom-Khan1, A. Harari-Shaham1,
O. Sadeh1, S. Sagi2, A. Peleg1, J. Haddad-Halloun1 L. Sagi-Dain1, V. Adir1, G. Larom-Khan1, A. Harari-Shaham1,
O. Sadeh1, S. Sagi2, A. Peleg1, J. Haddad-Halloun1 Materials and Methods: We generated (1) induced
pluripotent stem cell (iPSC) lines from fibroblasts of
patients with heterozygous LoF KMT2D mutations; and
(2) a LoF heterozygous KMT2D in a wildtype human
embryonic stem cell (hESC) line using the CRISPR-Cas9
approach. We differentiated the KS iPSC and hESC lines to
form cortical neurons. 1Carmel Medical Center, Haifa, Israel, 2Bnai Zion Medical
Center, Haifa, Israel P08.47C RNA sequencing and pathway analysis identify important
pathways involved in hypertrichosis and intellectual
disability in patients with Wiedemann-Steiner syndrome 1Centre de Génétique et Centre de Référence Anomalies du
Développement et Syndromes Malformatifs de l’Interrégion
Est, Centre Hospitalier Universitaire Dijon, Dijon, France,
2Génétique des Anomalies du Développement, UMR1231,
Université de Bourgogne, Dijon, France, 3Fédération
Hospitalo-Universitaire Médecine Translationnelle et
Anomalies du Développement (TRANSLAD), Centre
Hospitalier Universitaire Dijon, Dijon, France, 4Division of
Genetics, Johns Hopkins All Children's Hospital, Johns
Hopkins University School of Medicine, St Petersburg, FL,
United States, 5GeneDx, 207 Perry Pkwy, Gaithersburg, MD,
United States N. Lebrun1, L. Mietton1, I. Giurgea2, A. Goldenberg3,
B. Saintpierre1, J. Hamroune1, A. Afenjar4, P. Billuart1,
T. Bienvenu1 N. Lebrun1, L. Mietton1, I. Giurgea2, A. Goldenberg3,
B. Saintpierre1, J. Hamroune1, A. Afenjar4, P. Billuart1,
T. Bienvenu1 1Université Paris Descartes, Institut Cochin, CNRS
(UMR8103), Paris, France, 2U.F. de Génétique moléculaire,
Hôpital Armand Trousseau, Assistance Publique - Hôpitaux de
Paris; INSERM UMR S933, Faculté de médecine Sorbonne
Universités, Paris, France, 3Service de génétique, CHU de
Rouen et Inserm U1079, Université de Rouen, Centre Nor-
mand de Génomique Médicale et Médecine Personnalisée,
Rouen, France, 4Service de génétique et embryologie
médicales, Centre de référence Maladie du cervelet, CHU
Paris Est - Hôpital d'Enfants Armand-Trousseau, Paris,
France In 2011, KIAA1033/SWIP has been associated with auto-
somal recessive intellectual disability (AR-ID) in a large
consanguineous family comprising seven affected indivi-
duals with moderate ID and short stature. Since that, no
other case of KIAA1033 mutation has been reported. Here,
we report 3 patients from two unrelated couples with syn-
dromic ID due to compound heterozygous KIAA1033
mutations ascertained by whole exome sequencing (WES). Two sisters, aged 5.5 and 4 years, had a nonsense and a
missense mutation inherited from their parents (p.Gln442*
and p.Asp1048Gly), and presented with learning dis-
abilities, macrocephaly, dysmorphic features, and skeletal
features, associated with congenital absence of the right
internal carotid with bilateral sensorineural hearing loss in
the youngest. Our third patient was aged 32 years, had 2
missense
mutations
inherited
from
each
parent
(p. Lys1079Arg and p.His503Arg), and presented with mild
ID, short stature and microcephaly. KIAA1033 encodes a
large protein named WASH4/SWIP which is part of the
WASH complex. WASH complex is involved in the reg-
ulation of the fission of tubules that serve as transport
intermediates during endosome sorting. N. Lebrun: None. L. Mietton: None. I. Giurgea: None.
A. Goldenberg: None. B. Saintpierre: None. J. Ham-
roune: None. A. Afenjar: None. P. Billuart: None. T.
Bienvenu: None. P08.45A 230 J. del Picchia J. Delanne1,2,3, M. Assoum2, M. L. Crenshaw4, I. M. Wentzensen5, K. McWalter5, M. T. Cho5, P. Kuentz1,2,3,
J. Thevenon1,2,3, Y. Duffourd1,2,3, C. Thauvin-Robinet1,2,3,
L. Faivre1,2,3 P08.48D
GENIDA, an international participative cohort study on
genetic forms of intellectual disability and autism spectrum
disorders: analyses of Koolen-deVries, Kleefstra, KBG and
MECP2duplications syndromes Table (continued)
#
GENIDA
cohorts - top11
Registered
families
Active
participants
Family access
to analyses
Professionals
of reference
3. KBG syndrome
55
42
Overview /
MCQ
C. Ockeloen
4. MECP2
duplication
syndrome
47
30
Overview /
MCQ
H. van Esch
5. Valproate
Neurodev. project
46
31
Under
preparation
F. Francis &
M. Nosten
6. RASopathies
34
23
Overview only A. Verloes &
B. Kerr
7. Cockayne
syndrome
30
24
Overview only N. Calmels
8. 22q11.2
duplication
19
13
Overview only none yet
9. MED13L
12
9
Overview only none yet
10. DYRK1A
9
6
Under
preparation
A. Piton
11. PCDH19
6
6
Overview only none yet
... ... ... ... ... ... Total
on GENIDA
860
535 P08.48D
GENIDA, an international participative cohort study on
genetic forms of intellectual disability and autism spectrum
disorders: analyses of Koolen-deVries, Kleefstra, KBG and
MECP2duplications syndromes F. P. Colin1, T. Mazzucotelli1, D. A. Koolen2, T. Kleefstra2,
H. van Esch3, C. Ockeloen2, P. Parrend4, J. Mandel1 1Department of Neurogenetics and Translational Medicine,
Institut de génétique et de biologie moléculaire et cellulaire
(IGBMC) – INSERM U964 – CNRS UMR7104 – University of
Strasbourg, Illkirch-Graffenstaden, France, 2Department of
Human Genetics, Radboud University Medical Centre,
Nijmegen, Netherlands, 3Department of Human Genetics,
University of Leuven, Leuven, Belgium, 4iCube laboratory,
CNRS UMR 7357, University of Strasbourg, ECAM
Strasbourg-Europe, Strasbourg, France Many recurrent CNVs and more than 700 genes are impli-
cated in genetic forms of intellectual disability (ID) or
autism spectrum disorders (ASD), but often with limited
information on the clinical spectrum and natural history. We
initiated cohorts study for genetic causes of ID/ASD, called
GENIDA (https://genida.unistra.fr), whereby clinical infor-
mation is entered by the family of the affected individual
based on a clinical questionnaire (41 MCQ and 5 text
qualitative questions, including adverse effect of drugs) that
is currently available in 5 languages. We are internationally
recruiting and have constituted a collection of cohorts
(table1-top11). We have a questionnaire completion level
above 85% for our best 300 participants. We have used
Koolen-deVries syndrome to confirm that in general the
data is in line with the literature. P08.47C Previous members
of WASH complex KIAA0196/WASHC5 have already been
implicated in AR-ID with brain and cardiac malformations,
under the designation of the Ritscher-Schinzel syndrome. WES has proved its efficiency to find replications of genes
with insufficient data in the literature to be defined as a new
OMIM gene. We conclude that KIAA1033 is responsible of
a
non-recognizable
AR-ID
phenotype,
and
additional
descriptions will be needed to refine the clinical phenotype. Growing number of histone modifiers are involved in
human neurodevelopmental disorders, suggesting that
proper regulation of chromatin state is essential for the
development of the central nervous system. Among them,
heterozygous de novo variants in KMT2A, a gene coding
for histone methyltransferase, have been associated with
Wiedemann-Steiner Syndrome (WSS), a rare develop-
mental disorder mainly characterized by intellectual dis-
ability (ID) and hypertrichosis. As KMT2A is known to
regulate the expression of multiple target genes through
methylation of lysine 4 of histone 3 (H3K4me), we sought
to investigate the transcriptomic consequences of KMT2A
variants involved in WSS. Using fibroblasts from four
WSS patients harboring loss-of-function KMT2A variants,
we performed RNA sequencing and identified a number of
genes for which transcription was altered in KMT2A-
mutated cells compared to control ones. Strikingly, ana-
lysis of the pathways and biological functions sig-
nificantly deregulated between patients with WSS and
healthy individuals revealed a number of processes pre-
dicted to be altered that are relevant for hypertrichosis and
intellectual disability, the cardinal signs of this disease. Network analysis of the present RNA-Seq data suggest
that eNOS, WNT and BMP signaling pathways alterations
may be linked to cognitive dysfunction and hypertrichosis
in WSS. N. Lebrun: None. L. Mietton: None. I. Giurgea: None. A. Goldenberg: None. B. Saintpierre: None. J. Ham-
roune: None. A. Afenjar: None. P. Billuart: None. T. Bienvenu: None. J. Delanne: None. M. Assoum: None. M.L. Crenshaw:
None. I.M. Wentzensen: None. K. McWalter: None. M. T. Cho: None. P. Kuentz: None. J. Thevenon: None. Y. Duffourd:
None. C. Thauvin-Robinet:
None. L. Faivre: None. J. Delanne: None. M. Assoum: None. M.L. Crenshaw:
None. I.M. Wentzensen: None. K. McWalter: None. M. T. Cho: None. P. Kuentz: None. J. Thevenon: None. Y. Duffourd:
None. C. Thauvin-Robinet:
None. L. Faivre: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 231 P08.48D
GENIDA, an international participative cohort study on
genetic forms of intellectual disability and autism spectrum
disorders: analyses of Koolen-deVries, Kleefstra, KBG and
MECP2duplications syndromes Importantly, we reported in
detail growth parameters over time and the expected timing
of delayed developmental milestones, including speech and
language development, showed that social skills are rela-
tively more preserved compared to other communication
skills, and calculated - for the first time - the mean age of
seizure onset (5.8 yo - n = 56). We are now extending our
effort to other syndromes and will also present these results. This project shows the willingness and effectiveness of
parents in participative studies. Direct comparison with
published data allows us to search for novel and/or sig-
nificant comorbidities and should promote better healthcare. F.P. Colin: None. T. Mazzucotelli: None. D.A. Koolen:
None. T. Kleefstra: None. H. van Esch: None. C. Ockeloen: None. P. Parrend: None. J. Mandel: None. F.P. Colin: None. T. Mazzucotelli: None. D.A. Koolen:
None. T. Kleefstra: None. H. van Esch: None. C. Ockeloen: None. P. Parrend: None. J. Mandel: None. A pseudogene increasing LRFN5 expression in a patient
with 14q21.2 deletion and autism A pseudogene increasing LRFN5 expression in a patient
with 14q21.2 deletion and autism G. Cappuccio1, M. Alagia1, R. Borzone2, S. Attanasio2,
R. Genesio1, A. Mormile1, L. Litsch1, B. Granese1, G. Terrone1,
S. Banfi2, E. Del Giudice1, N. Brunetti-Pierri1
1Federico II Univeristy Naples Italy
2Tigem Pozzuoli Italy G. Cappuccio1, M. Alagia1, R. Borzone2, S. Attanasio2,
R. Genesio1, A. Mormile1, L. Litsch1, B. Granese1, G. Terrone1,
S. Banfi2, E. Del Giudice1, N. Brunetti-Pierri1 1Federico II Univeristy, Naples, Italy, 2Tigem, Pozzuoli, Italy P08.51C P08.51C
KCNT2-related developmental and epileptic
encephalopathy - a novel disease entity with potential for
targeted treatment 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia, 3Department of Clinical Genetics, United
Laboratories, Tartu University Hospital, Tallinn, Estonia,
4Children’s Clinic, Tartu University Hospital, Tartu, Estonia,
5Department of Paediatrics, Institute of Clinical Medicine,
University of Tartu, Tartu, Estonia, 6Division of Genetics and
Genomics, Department of Medicine, Boston Children’s
Hospital, Harvard Medical School, Boston, MA, United States,
7Broad Institute of MIT and Harvard, Cambridge, MA, United
States KCNT2-related developmental and epileptic
encephalopathy - a novel disease entity with potential for
targeted treatment P. Ambrosino1, M. Soldovieri1, T. Bast2, P. Turnpenny3,
S. Uhrig4, S. Biskup4, M. Döcker4, T. Fleck5, I. Mosca1,
L. Manocchio1, N. Iraci6, M. Taglialatela7, J. Lemke8 1University of Molise, Department of Medicine and Health
Sciences "Vincenzo Tiberio", Campobasso, Italy, 2Epilepsy
Center Kork, Kehl, Germany, 3Clinical Genetics, Royal Devon
& Exeter NHS Foundation Trust, Exeter, United Kingdom,
4CeGaT GmbH and Praxis für Humangenetik Tübingen,
Tübingen, Germany, 5University Heart Center Freiburg, Bad
Krotzingen, Germany, 6University of Salerno, Department of
Pharmacy, Salerno, Italy, 7University of Naples “Federico II”,
Department of Neuroscience, Naples, Italy, 8University of
Leipzig, Institute of Human Genetics, Leipzig, Germany Introduction: The MED13L gene was first associated with
transposition of the great arteries and intellectual disability
(ID). Later, it was recognised as distinctive syndromic ID
phenotype
presenting
moderate
to
severe
ID,
facial
anomalies, severe speech delay and muscular hypotonia in
majority cases. At least 26 cases have been previously
described. Variants in several potassium channel genes have been
found in developmental and epileptic encephalopathies
(DEE). KCNT1 and KCNT2 belong to the SLO2 family of
Na+-dependent (K+) channel genes, of which the latter has
not been reliably associated with human disorders. We
report two independent individuals with de novo variants in
KCNT2 at position R190. One of the two had West syn-
drome evolving to Lennox-Gastaut syndrome, the second
individual had DEE with malignant migrating partial sei-
zures of infancy. In vitro analysis suggested a gain-of- Methods: We analysed retrospectively the results of
4813-gene panel and exome sequencing analyses (1495
cases) performed during 2014-2017 at Tartu University
Hospital. In 25 unsolved exome sequencing trios, whole
genome sequencing (WGS) was performed at Broad
Institute. Copy number variant (CNV) calling from WGS
data was performed using Manta software. 1Federico II Univeristy, Naples, Italy, 2Tigem, Pozzuoli, Italy In agreement with this
hypothesis, LRFN5 expression was increased following
transfection of the chr14.232.a pseudogene in the patient’s
fibroblasts. regulates LRFN5 expression. In agreement with this
hypothesis, LRFN5 expression was increased following
transfection of the chr14.232.a pseudogene in the patient’s
fibroblasts. Conclusion: The chr14.232.a pseudogene is predicted to
bind miRNAs and based on the data generated so far, we
speculate that the chr14.232.a pseudogene functions as a
miRNA decoy to regulate LRFN5 expression through
sequestration of miRNAs targeting LRFN5. In conclusion,
this study may unravel a novel mechanism of gene
regulation involved in neurodevelopmental disorders. Conclusion: The chr14.232.a pseudogene is predicted to
bind miRNAs and based on the data generated so far, we
speculate that the chr14.232.a pseudogene functions as a
miRNA decoy to regulate LRFN5 expression through
sequestration of miRNAs targeting LRFN5. In conclusion,
this study may unravel a novel mechanism of gene
regulation involved in neurodevelopmental disorders. G. Cappuccio: None. M. Alagia: None. R. Borzone:
None. S. Attanasio:
None. R. Genesio:
None. A. Mormile: None. L. Litsch: None. B. Granese: None. G. Terrone: None. S. Banfi: None. E. Del Giudice: None. N. Brunetti-Pierri: None. Conclusion: We identified a pathogenic variant in
MED13L gene in 0.2% of cases in our patient cohort. It
makes MED13L one of the most common ID-associated
genes among our diagnostic cohort. Funding: Estonian
Research Council PUT355. P08.50B Three new cases of MED13L defect caused by a de novo
novel frameshift mutations and a complex rearrangement K. Õunap: None. K. Reinson: None. E. Õiglane-Shlik:
None. M.H. Wojcik: None. M. Lek: None. J.L. Marshall:
None. Ü. Murumets: None. T. Reimand: None. K. Õunap: None. K. Reinson: None. E. Õiglane-Shlik:
None. M.H. Wojcik: None. M. Lek: None. J.L. Marshall:
None. Ü. Murumets: None. T. Reimand: None. K. Õunap1,2, K. Reinson3,2, E. Õiglane-Shlik4,5, M. H. Wojcik6,7,
M. Lek7, J. L. Marshall7, Ü. Murumets1, T. Reimand1,2 1Federico II Univeristy, Naples, Italy, 2Tigem, Pozzuoli, Italy Introduction: Autism spectrum disorder (ASD) is a dis-
order with impaired social relationships, language and
communication, and is frequently associated with intellec-
tual disability. Underlying genetic defects can be identified
in 30-40% of ASD patients by chromosomal microarray
analysis and whole exome sequencing. Matherials and Methods: We report a 16 year-old boy
with ASD bearing a microdeletion at chromosome 14q21.2
inherited from the father who has borderline cognitive
impairment. The deletion affects a ‘gene desert’ and LRFN5
is the closest gene in the non-deleted interval. LRFN5
encodes a protein involved in synaptic plasticity that has
been implicated in neurodevelopmental phenotypes. #
GENIDA
cohorts - top11
Registered
families
Active
participants
Family access
to analyses
Professionals
of reference
1. Koolen-deVries
syndrome
223
188
All -
except text
D. Koolen
2. Kleefstra
sydrnome
124
74
Overview /
MCQ
T. Kleefstra Results: We found decreased mRNA expression of both
LRFN5 gene and chr14.232.a pseudogene included within
the deleted interval in the proband’s fibroblasts compared to
controls. We hypothesized the pseudogene chr14.232.a Results: We found decreased mRNA expression of both
LRFN5 gene and chr14.232.a pseudogene included within
the deleted interval in the proband’s fibroblasts compared to
controls. We hypothesized the pseudogene chr14.232.a J. del Picchia 232 and 5.5-year-old male). Two patients had frameshift
mutations
-
c.4245_4246del
p.(His1415Glnfs*2)
and
c.5488_5495dup p.(Ser1833Ilefs*22). In the third case,
CNV calling from WGS data revealed complex rearrange-
ment with four breakpoints in MED13L gene. This patient
has tandem duplication: chr12:116,661,676-116,668,880
and a deletion: chr12:116,662,161-116,675,475. The dele-
tion disrupts exon 2 of MED13L gene presumably causing
loss of gene function. All patients presented with mild to
moderate ID, muscular hypotonia, ataxia or coordination
problems
and
facial
dysmorphism. Epilepsy,
skeletal
anomalies and strabismus were noticed once. No congenital
heart anomalies were detected. One patient with complex
CNV had severe speech defect. and 5.5-year-old male). Two patients had frameshift
mutations
-
c.4245_4246del
p.(His1415Glnfs*2)
and
c.5488_5495dup p.(Ser1833Ilefs*22). In the third case,
CNV calling from WGS data revealed complex rearrange-
ment with four breakpoints in MED13L gene. This patient
has tandem duplication: chr12:116,661,676-116,668,880
and a deletion: chr12:116,662,161-116,675,475. The dele-
tion disrupts exon 2 of MED13L gene presumably causing
loss of gene function. All patients presented with mild to
moderate ID, muscular hypotonia, ataxia or coordination
problems
and
facial
dysmorphism. Epilepsy,
skeletal
anomalies and strabismus were noticed once. No congenital
heart anomalies were detected. One patient with complex
CNV had severe speech defect. regulates LRFN5 expression. P08.51C Results: We identified a novel de novo variant in
MED13L gene in three cases (6 and 6.5-year-old females Abstracts from the 51st European Society of Human Genetics Conference: Posters 233 function responsive to quinidine. A precision medicine
approach in one of the two patients with add-on therapy of
quinidine resulted in increase of alertness and vigilance,
mild developmental progression, improved EEG and a
temporary decrease of seizure frequency. We suggest that
KCNT2-related disorders share similar phenotypic and
in vitro functional and pharmacological features with
KCNT1-related disorders and thus may represent a further
example for disorders potentially responsive to targeted
treatment opportunities. TRAP1 genes in 176 unrelated ADS patients revealed no
relevant variants in RUVBL1, whereas in TRAP1 eight rare
variants were identified, including the p.Gln639Ter. TRAP1 genes in 176 unrelated ADS patients revealed no
relevant variants in RUVBL1, whereas in TRAP1 eight rare
variants were identified, including the p.Gln639Ter. Conclusions: Mutations in TRAP1, especially the p. Gln639Ter, may be associated with ADS. DNA purified
from blood of disease discordant MZTs should not be used
to perform molecular tests due to possible blood chimerism
that may mask genetic differences. Support: National Science Centre (NCN) Poland, Grant
2014/13/B/NZ5/00287 P. Ambrosino: None. M. Soldovieri: None. T. Bast:
None. P. Turnpenny: None. S. Uhrig: None. S. Biskup:
None. M. Döcker: None. T. Fleck: None. I. Mosca: None. L. Manocchio: None. N. Iraci: None. M. Taglialatela:
None. J. Lemke: None. P. Ambrosino: None. M. Soldovieri: None. T. Bast:
None. P. Turnpenny: None. S. Uhrig: None. S. Biskup:
None. M. Döcker: None. T. Fleck: None. I. Mosca: None. L. Manocchio: None. N. Iraci: None. M. Taglialatela:
None. J. Lemke: None. M. Rydzanicz: None. A. Walczak: None. I. Chojnicka:
None. G. Kostrzewa: None. J. Kosińska: None. P. Gasperowicz: None. N. Chojnacka: None. P. Stawiński:
None. R. Płoski: None. P. Ambrosino: None. M. Soldovieri: None. T. Bast:
None. P. Turnpenny: None. S. Uhrig: None. S. Biskup:
None. M. Döcker: None. T. Fleck: None. I. Mosca: None. L. Manocchio: None. N. Iraci: None. M. Taglialatela:
None. J. Lemke: None. Confirmation of a recurrent mutation in NACC1 causing a
severe neurodevelopmental disorder Confirmation of a recurrent mutation in NACC1 causing a
severe neurodevelopmental disorder Molecular study of autism spectrum disorder discordant
monozygotic twins indicates the role of TRAP1 defects in
disease susceptibility K. Steindl1, A. Bahr1, A. Hackenberg2, M. Papik1, P. Joset1,
A. Rauch1 M. Rydzanicz1, A. Walczak1, I. Chojnicka2, G. Kostrzewa3,
J. Kosińska1, P. Gasperowicz1, N. Chojnacka1, P. Stawiński1,
R. Płoski1 M. Rydzanicz1, A. Walczak1, I. Chojnicka2, G. Kostrzewa3,
J. Kosińska1, P. Gasperowicz1, N. Chojnacka1, P. Stawiński1,
R. Płoski1 M. Rydzanicz1, A. Walczak1, I. Chojnicka2, G. Kostrzewa3, M. Rydzanicz1, A. Walczak1, I. Chojnicka2, G. Kostrzewa3, 1Institute of Medical Genetics, University of Zurich, 8902
Schlieren, Switzerland, 2Division of Child Neurology,
Children's Hospital Zurich, 8032 Zurich, Switzerland J. Kosińska1, P. Gasperowicz1, N. Chojnacka1, P. Stawiński1,
R. Płoski1 J. Kosińska1, P. Gasperowicz1, N. Chojnacka1, P. Stawiński1,
R. Płoski1 1Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland, 2Department of Health and Rehabilitation
Psychology, Faculty of Psychology, University of Warsaw,
Warsaw, Poland, 3Department of Forensic Medicine, Medical
University of Warsaw, Warsaw, Poland, Warsaw, Poland Only recently, the NACC1 gene was shown to harbor a
recurrent mutation leading to a complex congenital neuro-
developmental disorder in 7 affected children (Schauch
et al. 2017). All patients presented with a highly similar
phenotype including severe intellectual disability (ID),
developmental delay, cataract, microcephaly, severe infan-
tile epilepsy, failure to thrive, irritability and stereotypic
hand
movements. Whole
exome
sequencing
(WES)
revealed the same de novo missense mutation in the
NACC1 gene in all 7 children: c.892C>T p.(Arg298Trp). The NACC1 gene (nucleus accumbens associated protein 1)
encodes a transcriptional repressor and has, until then, only
been considered a candidate gene for ID due to its func-
tional role and a missense mutation found by Gilissen et al.,
2014 in a single patient with ID. Here, we describe a 5-year
old girl with ID, microcephaly, cataract and movement
abnormalities, leading to an initial diagnosis of a Rett-like
phenotype in 2016. Panel sequencing of genes associated
with Rett syndrome and an Array-CGH have been negative
in our patient. Subsequently, using Trio-WES in the girl and
her parents, the recurrent mutation c.892C>T in the NACC1
was found as a de novo mosaic and was the most likely
disease-causing variant in this family. Comparison of the
girls’ clinical symptoms with those described by Schauch
et al. showed a highly congruent phenotype. Our findings
support the hypothesis that this NACC1 mutation causes a
very specific phenotype. Confirmation of a recurrent mutation in NACC1 causing a
severe neurodevelopmental disorder Introduction: Monozygotic twins (MZTs) have been con-
sidered to be physically and genetically identical. However,
a significant number of disease-discordant MZTs, including
pairs discordant for autism spectrum disorder (ASD), have
been observed. Introduction: Monozygotic twins (MZTs) have been con-
sidered to be physically and genetically identical. However,
a significant number of disease-discordant MZTs, including
pairs discordant for autism spectrum disorder (ASD), have
been observed. Materials
and
Methods:
Three
male
MZT
pairs
discordant for ASD was recruited. DNA purified from hair
follicles was used for whole exome sequencing (WES). For
replication amplicon deep sequencing (ADS) was per-
formed using DNA form MZTs’ blood and hair follicles. Further validation was performed using 176 unrelated ADS
patients. Materials
and
Methods:
Three
male
MZT
pairs
discordant for ASD was recruited. DNA purified from hair
follicles was used for whole exome sequencing (WES). For
replication amplicon deep sequencing (ADS) was per-
formed using DNA form MZTs’ blood and hair follicles. Further validation was performed using 176 unrelated ADS
patients. Results: In one MZTs pair WES analysis revealed
discordant missense variant in RUVBL1 (p.Phe329Leu)
and a nonsense variant in TRAP1 (p.Gln639Ter). In autistic
twin’s hair follicles ADS confirmed the presence of
RUVBL1 variant with the mutant allele frequency (MAF)
42% and the TRAP1 variant (MAF 8%); both variants were
absent from hair follicles DNA of the healthy brother. However, in blood DNA both variants had the same MAF
(RUVBL1 22%, TRAP1 2%) in both twins. Subsequent
analysis of the whole coding sequences of RUVBL1 and 234 J. del Picchia K. Steindl: None. A. Bahr: None. A. Hackenberg:
None. M. Papik: None. P. Joset: None. A. Rauch: None. 1Department of Genetic Medicine and Development, University
of Geneva, Geneva, Switzerland, 2Institute of Basic Medical
Sciences, Khyber Medical University, Peshawar, Pakistan,
3Department of Genetics, University of Karachi, Karachi,
Pakistan, 4Molecular Biology and Genetics Department,
Medical Research Center, Liaquat University of Medical and
Health Sciences, Jamshoro, Pakistan, 5Biomedical Research
Foundation of the Academy of Athens, Athens, Greece,
6Service of Genetic Medicine, University Hospitals of Geneva,
Geneva, Switzerland, 7Department of Medical Genetics, Oslo
University Hospital and University of Oslo, Oslo, Norway,
8Department of Endocrinology Diabetes and Metabolism,
University Hospital of Lausanne, Lausanne, Switzerland,
9Department of Bio Sciences, Faculty of Life Sciences,
Muhammad Ali Jinnah University, Karachi, Pakistan Radboud University Medical Center Nijmegen, Nijmegen,
Netherlands Noonan syndrome (NS) is a relatively common genetic
disorder, characterized by distinctive facial features, short
stature, congenital heart defect and developmental delay of
variable degree. Known causative genes account for 70-
80% of clinically diagnosed NS patients, but the genetic
basis for the remaining cases is not known. The majority of
the mutations identified in NS and other RASopathies are
gain-of-function which result in increased RAS/MAPK
signaling. However, previous studies have demonstrated
that copy number variations, containing critical genes
related to the RAS/MAPK signaling pathway, play a minor
role in RASopathies. We describe a 7 year old girl that was
clinically diagnosed with Noonan syndrome at the age of
10 months. Using exome sequencing, we recently identified
a novel de novo ~3,2 Mbp deletion of multiple genes on
chromosome
10p12.1p11.22,
including
MAP3K8. MAP3K8 is an oncogene, encoding a MAP kinase kinase
kinase, that can phosphorylate and activate MAP2K1,
which is associated with NS and CFC syndrome. We
hypothesize that the 10p12.1p11.22 deletion leads to a
dysregulation of the RAS/MAPK signaling pathway and
therefore has an association with the phenotype of our
patient. Functional analysis will be the next step to provide
additional
evidence
that
(micro)deletions
containing
MAP3K8 could result in NS. Consanguinity, practiced in a substantial fraction of human
populations, reveals numerous rare recessive phenotypes
because of the extensive regions of homozygosity by
decent. The average total size of homozygosity is 253Mb in
the offspring of consanguineous parents; this is 10fold
higher than that of 25Mb in outbred individuals. Similarly,
the rare homozygous variants (<2% frequency) in the cod-
ing regions are 57 in the offspring of first-cousins compared
to 18 in outbred individuals. In Pakistan, the frequency of
consanguineous marriages approaches 70%. We have
initiated a Swiss-Pakistani project to identify novel reces-
sive gene candidates for two phenotypes: intellectual dis-
ability (ID) and visual impairment (VI). We have collected
samples from 145 ID and 205 VI families of first cousin
marriages with at least 2 affecteds. Exome sequence of one
affected and genotyping of the whole family (parents, all
affected and unaffected siblings) has been completed in 114
ID and 146 VI families to date. The likely causative gene/
variant in known genes was found in 67% of the VI and
34% in the ID families. Thus, there are more unknown
recessive genes for ID. P08.55C
Identification of a novel de novo deletion of
MAP3K8 suggestive of causing Noonan syndrome E. K. S. M. Leenders, C. Marcelis, A. P. A. Stegmann, T. Rinne,
I. van de Burgt Radboud University Medical Center Nijmegen, Nijmegen,
Netherlands We have identified 21 novel can-
didate genes in VI and 14 in ID (to be presented in the
conference). International matchmaking identified addi-
tional families in 20% of the candidate genes. Careful
evaluation of the phenotypes is mandatory to assess the
possibility of 2 or more causative genes, to eliminate false
negative
results. International
databases
from
con-
sanguineous
individuals
are
needed
to
facilitate
the
assignment of pathogenicity to homozygous variants. E.K.S.M. Leenders: None. C. Marcelis: None. A.P.A. Stegmann: None. T. Rinne: None. I. van de Burgt: None. S. E. Antonarakis1, S. A. Paracha2, S. Imtiaz3, A. Nazir2, Y. M.
Waryah4, P. Makrythanasis1,5, S. Qureshi2, S. Saeed3,
E. Falconnet1, M. Guipponi6, C. Borel1, M. A. Ansari3,
E. Frengen7, E. Ranza6, F. A. Santoni1,8, I. Shah2, K. Gul9,
M. T. Sarwar2,1, J. Ahmed2, A. M. Waryah4, M. Ansar1 P08.57A
Two novel splicing mutations in the OTUD6B gene
associated with intellectual disability and seizures 1All Wales Medical Genetics Service, Cardiff, United Kingdom,
2Division of Cancer and Genetics, Cardiff University, Cardiff,
United Kingdom, 3Department of Medical Genetics, Our
Lady’s Children’s Hospital, Dublin, Ireland, 4Division of
Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON,
Canada, 5Department of Medical Genetics, Liverpool Women's
Hospital, Liverpool, United Kingdom, 6North West Thames
Regional Genetic Service, Kennedy Galton Centre, Northwick
Park Hospital, Harrow, United Kingdom, 7Department of
Clinical Genetics, Northern General Hospital, Sheffield,
United Kingdom, 8Wellcome Trust Sanger Institute,
Cambridge, United Kingdom L. Straniero1, V. Rimoldi1, M. Bellini2, S. Duga1,3, G. Soldà1,3,
R. Asselta1,3 1Humanitas University, Pieve Emanuele (Milano), Italy, 2Dept
of Paediatrics and Neonatology, Guglielmo da Saliceto
Hospital, Piacenza, Italy, 3Humanitas Clinical and Research
Center, Rozzano, Italy 1Humanitas University, Pieve Emanuele (Milano), Italy, 2Dept
of Paediatrics and Neonatology, Guglielmo da Saliceto
Hospital, Piacenza, Italy, 3Humanitas Clinical and Research
Center, Rozzano, Italy Introduction Biallelic mutations in the ovarian tumor
domain-containing 6B (OTUD6B) gene, coding for a deu-
biquitinating enzyme, were recently described to cause an
intellectual disability syndrome characterized by seizures
and dysmorphic features in 6 families worldwide. Börjeson-Forssman-Lehmann
syndrome
(BFLS,
OMIM
301900) is a rare X-linked condition associated with intel-
lectual
disability,
dysmorphic
features
and
endocrine
abnormalities. In recent years, a distinct phenotype, similar
to Coffin-Siris syndrome, has been described in females
with de novo mutations in PHF6. We ascertained 13
patients (8 males and 5 females) with PHF6 mutations who
were identified through clinical or research testing. Two
families with 5 affected males had the same novel inframe
deletion (p.E338del). Another male patient had a novel
missense (p.D353H) which is the most distal pathogenic
mutation yet described in PHF6. Female patients tend to
have large de novo deletions, duplications or truncating
mutations. Consistent features in the subjects included
intellectual disability and the typical facial gestalt: broad
nasal bridge, deep-set eyes, prominent or arched eyebrows,
synophrys, brachydactyly, syndactyly, and large ears with
fleshy lobes. Obesity, hormonal deficiencies and delayed
puberty were common in male patients, whereas females
often had linear skin hyperpigmentation, abnormal teeth and
autistic traits. Unusual features in our series included
umbilical hernias, keloid scarring, peri-ungal fibromas,
absent vaginal orifice, lower limb motor neuropathy and
talipes. The majority of our patients had squints or refrac-
tive errors. One had dysplastic optic discs while another had
chorioretinal pigmentation and atrophy. One female patient
with a novel missense mutation (p.G248V) had cortical
abnormalities and poorly-controlled nocturnal frontal epi-
lepsy. P08.58B A series of patients with PHF6-related disease: novel
mutations and an expansion of the phenotype P08.57A
Two novel splicing mutations in the OTUD6B gene
associated with intellectual disability and seizures This is further evidence that females with BFLS are
at risk of malformations of cortical development. Materials and Methods We report on a 5-year-old
Italian girl, presenting mild intellectual disability, speech
and motor delay, and recurrent seizures. Whole-exome
sequencing was performed on the proband DNA using the
SureSelect Human All Exon V6 kit (Agilent) and the
NextSeq500 instrument (Illumina). The identified mutations
were confirmed by Sanger sequencing and their functional
role was assessed by performing RT-PCR assays on the
RNA extracted from peripheral blood mononuclear cells of
the patient and her parents. Results and Conclusions We identified two candidate
heterozygous splicing mutations in the OTUD6B gene. These variants (c.324+1G>C and c.405+1G>A), both
reported in the ExAC database with a frequency lower
than the 1%, affect the donor splicing site of exon 2 and 3,
respectively. Sanger sequencing confirmed the segregation
of the variants in the family, showing that both parents are
carriers of one mutation. RT-PCR experiments demon-
strated that both variants affect OTUD6B splicing and lead
to the production of aberrant transcripts, the major ones
being, in both cases, the skipping of the upstream exon. Quantitative analysis performed by competitive-fluorescent
RT-PCR on the patient RNA showed that the proband
presents less than 1% of wild-type transcripts, further
strengthening the causative role of these variants. Results and Conclusions We identified two candidate
heterozygous splicing mutations in the OTUD6B gene. These variants (c.324+1G>C and c.405+1G>A), both
reported in the ExAC database with a frequency lower
than the 1%, affect the donor splicing site of exon 2 and 3,
respectively. Sanger sequencing confirmed the segregation
of the variants in the family, showing that both parents are
carriers of one mutation. RT-PCR experiments demon-
strated that both variants affect OTUD6B splicing and lead
to the production of aberrant transcripts, the major ones
being, in both cases, the skipping of the upstream exon. Quantitative analysis performed by competitive-fluorescent
RT-PCR on the patient RNA showed that the proband
presents less than 1% of wild-type transcripts, further
strengthening the causative role of these variants. L. Straniero: None. V. Rimoldi: None. M. Bellini:
None. S. Duga: None. G. Soldà: None. R. Asselta: None. V. Jain: None. A. Clarke: None. S. Davies: None. S. Doyle: None. G. Graham: None. L. Greenhalgh: None. S. Holder: None. D. Johnson: None. S.A. Lynch: None. E. McCann: None. C. Pottinger: None. D.D.D. study: None. A.E. Fry: None. Asymmetrical and Chemical-modified Donor-DNA Leads
to Efficient Knock-ins of Pathogenic Mutations at CRISPR-
Cas9-induced DNA Double Strand Breaks V. Jain1, A. Clarke1,2, S. Davies1, S. Doyle3, G. Graham4,
L. Greenhalgh5, S. Holder6, D. Johnson7, S. A. Lynch3,
E. McCann5, C. Pottinger1, D. D. D. study8, A. E. Fry1,2 P08.56D 35 novel recessive candidate genes for intellectual disability
and visual impairment by using 260 consanguineous
families S.E. Antonarakis: None. S.A. Paracha: None. S. Imtiaz: None. A. Nazir: None. Y.M. Waryah: None. P. Makrythanasis: None. S. Qureshi: None. S. Saeed: None. E. Falconnet: None. M. Guipponi: None. C. Borel: None. M.A. Ansari: None. E. Frengen: None. E. Ranza: None. F.A. Santoni: None. I. Shah: None. K. Gul: None. M.T. Sarwar: None. J. Ahmed: None. A.M. Waryah: None. M. Ansar: None. S. E. Antonarakis1, S. A. Paracha2, S. Imtiaz3, A. Nazir2, Y. M. Waryah4, P. Makrythanasis1,5, S. Qureshi2, S. Saeed3,
E. Falconnet1, M. Guipponi6, C. Borel1, M. A. Ansari3,
E. Frengen7, E. Ranza6, F. A. Santoni1,8, I. Shah2, K. Gul9,
M. T. Sarwar2,1, J. Ahmed2, A. M. Waryah4, M. Ansar1 S. E. Antonarakis1, S. A. Paracha2, S. Imtiaz3, A. Nazir2, Y. M. Waryah4, P. Makrythanasis1,5, S. Qureshi2, S. Saeed3,
E. Falconnet1, M. Guipponi6, C. Borel1, M. A. Ansari3,
E. Frengen7, E. Ranza6, F. A. Santoni1,8, I. Shah2, K. Gul9,
M. T. Sarwar2,1, J. Ahmed2, A. M. Waryah4, M. Ansar1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 235 1All Wales Medical Genetics Service, Cardiff, United Kingdom,
2Division of Cancer and Genetics, Cardiff University, Cardiff,
United Kingdom, 3Department of Medical Genetics, Our
Lady’s Children’s Hospital, Dublin, Ireland, 4Division of
Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON,
Canada, 5Department of Medical Genetics, Liverpool Women's
Hospital, Liverpool, United Kingdom, 6North West Thames
Regional Genetic Service, Kennedy Galton Centre, Northwick
Park Hospital, Harrow, United Kingdom, 7Department of
Clinical Genetics, Northern General Hospital, Sheffield,
United Kingdom, 8Wellcome Trust Sanger Institute,
Cambridge, United Kingdom 1All Wales Medical Genetics Service, Cardiff, United Kingdom,
2Division of Cancer and Genetics, Cardiff University, Cardiff,
United Kingdom, 3Department of Medical Genetics, Our
Lady’s Children’s Hospital, Dublin, Ireland, 4Division of
Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON,
Canada, 5Department of Medical Genetics, Liverpool Women's
Hospital, Liverpool, United Kingdom, 6North West Thames
Regional Genetic Service, Kennedy Galton Centre, Northwick
Park Hospital, Harrow, United Kingdom, 7Department of
Clinical Genetics, Northern General Hospital, Sheffield,
United Kingdom, 8Wellcome Trust Sanger Institute,
Cambridge, United Kingdom E. McCann5, C. Pottinger1, D. D. D. study8, A. E. Fry1,2 V. Jain1, A. Clarke1,2, S. Davies1, S. Doyle3, G. Graham4,
5
6
7
3 M. Rodriguez de los Santos1,2, A. Knaus3, B. Fischer-Zirnsak1,
L. Wittler4, S. Mundlos1,2, U. Kornak1, P. Krawitz3 M. Rodriguez de los Santos1,2, A. Knaus3, B. Fischer-Zirnsak1,
L. Wittler4, S. Mundlos1,2, U. Kornak1, P. Krawitz3 1Institute for Medical Genetics and Human Genetics, Charité
Universitätsmedizin, Berlin, Germany, 2Max-Planck-Institute
for Molecular Genetics, FG Development & Disease, Berlin,
Germany, 3Institute for Genomic Statistics and Bioinformatics,
University of Bonn, Bonn, Germany, 4Max-Planck-Institute for
Molecular Genetics, Department Developmental Genetics,
Berlin, Germany 1Institute for Medical Genetics and Human Genetics, Charité
Universitätsmedizin, Berlin, Germany, 2Max-Planck-Institute
for Molecular Genetics, FG Development & Disease, Berlin,
Germany, 3Institute for Genomic Statistics and Bioinformatics,
University of Bonn, Bonn, Germany, 4Max-Planck-Institute for
Molecular Genetics, Department Developmental Genetics,
Berlin, Germany 1Institute of Genomic Medicine, Università Cattolica del Sacro
Cuore, Roma, Italy, 2Unit of Pediatrics and Medical Genetics,
I.R.C.C.S. Associazione Oasi Maria Santissima, Troina, Italy,
3Epilepsy and Clinical Neurophysiology Unit, Scientific
Institute IRCCS "Eugenio Medea", Conegliano, Italy,
4Maternal and Child Department, University of Parma,
Parma, Italy, 5Department of Pediatric Neurosciences, Carlo
Besta Neurological Institute, IRCCS Foundation,, Milan, Italy,
6Department Experimental and Clinical Medicine, University
of Sassari, Sassari, Italy, 7Department of Medical Genetics,
Gaetano Rummo Hospital, Benevento, Italy, 8Department of
Pediatric Neurosciences, Carlo Besta Neurological Institute,
IRCCS Foundation,, Roma, Italy, 9Department of
Neurosciences and Neurorehabilitation, IRCCS Bambino Gesù
Children Hospital, Roma, Italy, 10Medical Genetics, University
of Siena, Siena, Italy, 11Department of Advanced Diagnostic
and Clinical Trials, Institute for Maternal and Child Health-
IRCCS “Burlo Garofolo”, Trieste, Italy, 12Genetics and
Molecular Medicine Unit, Anna Meyer Children’s University
Hospital,, Florence, Italy, 13Child Neurology Unit, Istituto di
Ricovero e Cura a Carattere Scientifico, Institute of
Neurological Sciences, Bologna, Italy, 14Center of Myology
and Neurodegenerative Disorders, Istituto Giannina Gaslini,
Genova, Italy, 15Department of Sciences for Health Promotion
and Mother and Child Care "Giuseppe D'Alessandro",
University of Palermo, Palermo, Italy, 16Department of
Pediatrics, Regional Hospital of Bolzano, Bolzano, Italy,
17Medical Genetics Unit, Meyer Children's Hospital, Florence,
Italy, 18U.O.C Anatomia Patologica, AOR Ospedale "San
Carlo", Potenza, Italy, 19Stella Maris Clinical Research
Institute for Child and Adolescent Neuropsychiatry, Pisa, Italy,
20Unit of Medical Genetics, S. Orsola-Malpighi Hospital,
Bologna, Italy, 21Clinical Genetics Unit, Department of
Obstetrics and Pediatrics, AUSL-IRCCS of Reggio Emilia,
Reggio Emilia, Italy Introduction: Glycosylphosphatidylinositol (GPI) anchors
attach a broad range of proteins to the cell membrane that
have diverse roles in cell adhesion, signaling and comple-
ment regulation. Patients with a biosynthesis deficiency of
the GPI anchor reveal global developmental delay, seizures
and multiple malformations. M. Rodriguez de los Santos1,2, A. Knaus3, B. Fischer-Zirnsak1,
L. Wittler4, S. Mundlos1,2, U. Kornak1, P. Krawitz3 S. Mundlos:
None. U. Kornak: None. P. Krawitz: None. M. Rodriguez de los Santos1,2, A. Knaus3, B. Fischer-Zirnsak1,
L. Wittler4, S. Mundlos1,2, U. Kornak1, P. Krawitz3 Around 30 genes are involved
in the molecular pathway and most pathogenic mutations
have shown to be hypomorphic. However, the patho-
mechanisms on a cellular, tissue or organ level remain
unclear and suitable animal models are lacking. We there-
fore chose to establish a mouse line for one of the most
prevalent
pathogenic
missense
mutations observed in
humans so far PIGV c.1022C>A, p.Ala341Glu. Methods: We co-transfected mouse embryonic stem
(mES) cells with PX459-pSpCas9(BB)-2A-Puro (Addgene)
vector and single-stranded oligodeoxynucleotides (ssODN)
including the disease-causing mutation PIGV c.1022C>A. Positive clones were expanded and characterized by flow
cytometry. Results: We successfully integrated the pathogenic
mutation PIGV c.1022C>A in mES cells using ssODNs
with asymmetrical homology arms which were modified at
the ends with phosphoro-thioat bonds. With this strategy,
we were able to obtain up to 30% of knock-in (KI) clones. Furthermore, we observed that sgRNAs located directly at
the missense mutation generated only homozygous clones. In contrast, we obtained heterozygous clones using sgRNA
binding 13 bp upstream or downstream from the missense
mutation. The generated PIGV-deficient mES cells were
analyzed by flow cytometry and revealed a reduced surface
expression of GPI-linked markers. In contrast, we obtained heterozygous clones using sgRNA
binding 13 bp upstream or downstream from the missense
mutation. The generated PIGV-deficient mES cells were
analyzed by flow cytometry and revealed a reduced surface
expression of GPI-linked markers. Conclusion: We here present a valid gene-editing
strategy to generate efficiently KIs of missense mutations
in mES cells via the CRISPR-Cas9 system. Pitt-Hopkins syndrome (PTHS, OMIM #610954) is a rare
neurodevelopmental
disorder
caused
by
TCF4
hap-
loinsufficiency. Clinical features include severe intellectual
disability,
distinctive
facial
characteristics,
breathing
anomalies, postnatal microcephaly, and recurrent behavioral
abnormalities. We studied 320 subjects referred to our
Institute with a clinical suspicion of PTHS, who, in most
cases, had already undergone several genetic tests with M. Rodriguez de los Santos: None. A. Knaus: None. B. Fischer-Zirnsak: None. L. Wittler: None. S. Mundlos:
None. U. Kornak: None. P. Krawitz: None. Pitt-Hopkins syndrome (PTHS, OMIM #610954) is a rare
neurodevelopmental
disorder
caused
by
TCF4
hap-
loinsufficiency. Clinical features include severe intellectual
disability,
distinctive
facial
characteristics,
breathing
anomalies, postnatal microcephaly, and recurrent behavioral
abnormalities. We studied 320 subjects referred to our
Institute with a clinical suspicion of PTHS, who, in most
cases, had already undergone several genetic tests with M. Rodriguez de los Santos: None. A. Knaus: None. B. Fischer-Zirnsak: None. L. Wittler: None. P08.59C Asymmetrical and Chemical-modified Donor-DNA Leads
to Efficient Knock-ins of Pathogenic Mutations at CRISPR-
Cas9-induced DNA Double Strand Breaks J. del Picchia 236 G. Marangi1, S. Frangella1, S. Ricciardi1, P. Chiurazzi1,
D. Orteschi1, S. Lattante1, R. Pettinato2, F. Martinez3,
C. Magnani4, C. Pantaleoni5, C. Perria6, G. Scarano7, V. Saletti8,
P. Bonanni3, G. Vasco9, C. Lo Rizzo10, A. Volzone3, E. Alfei5,
F. Faletra11, S. Romano12, A. Renieri10, M. Giannotta13,
C. Minetti14, C. Bruno14, M. Piccione15, F. Stanzial16, M. Della
Monica17, A. Ardissone5, M. Di Giacomo18, A. Battaglia19,
C. Graziano20, L. Garavelli21, M. Zollino1 G. Marangi1, S. Frangella1, S. Ricciardi1, P. Chiurazzi1,
D. Orteschi1, S. Lattante1, R. Pettinato2, F. Martinez3,
C. Magnani4, C. Pantaleoni5, C. Perria6, G. Scarano7, V. Saletti8,
P. Bonanni3, G. Vasco9, C. Lo Rizzo10, A. Volzone3, E. Alfei5,
F. Faletra11, S. Romano12, A. Renieri10, M. Giannotta13,
C. Minetti14, C. Bruno14, M. Piccione15, F. Stanzial16, M. Della
Monica17, A. Ardissone5, M. Di Giacomo18, A. Battaglia19,
C. Graziano20, L. Garavelli21, M. Zollino1 G. Marangi1, S. Frangella1, S. Ricciardi1, P. Chiurazzi1,
D. Orteschi1, S. Lattante1, R. Pettinato2, F. Martinez3,
C. Magnani4, C. Pantaleoni5, C. Perria6, G. Scarano7, V. Saletti8,
P. Bonanni3, G. Vasco9, C. Lo Rizzo10, A. Volzone3, E. Alfei5,
F. Faletra11, S. Romano12, A. Renieri10, M. Giannotta13,
C. Minetti14, C. Bruno14, M. Piccione15, F. Stanzial16, M. Della
Monica17, A. Ardissone5, M. Di Giacomo18, A. Battaglia19,
C. Graziano20, L. Garavelli21, M. Zollino1 P08.60D Pitt-Hopkins syndrome: dissecting the clinical and genetic
heterogeneity of conditions in the phenotypic spectrum Abstracts from the 51st European Society of Human Genetics Conference: Posters 237 normal results. We performed clinical evaluation and
genetic analyses according to the following procedure: 1)
detailed phenotype characterization by means of direct
observation or the use of a specific questionnaire; 2) direct
sequencing and MLPA of the TCF4 gene; 3) NGS analysis
of a panel of genes; 4) standard karyotype/ FISH analysis. Whole exome sequencing (WES) was performed in 16
patients. By clinical evaluation, a subset of 158/320 subjects
was included in the PTHS spectrum. Among the 146 sub-
jects analyzed by techniques 1) to 4), pathogenic variants
were identified in 58 (40%), involving the following genes:
TCF4 (tot:42), UBE3A (tot:4), MECP2 (tot:6), FOXG1
(tot:3), ZEB2 (tot:2), ATRX (tot:1). Further causative var-
iants were identified by WES in 4/12 analyzed patients, in
EHMT1, SZT2, ASXL3 and GABRB2. Through an in-depth
molecular characterization of a further TCF4 variant non
associated with a classical PTHS phenotype, and a critical
review of other similar cases in scientific literature, we
suggest an intragenic phenotypical map of TCF4, reflecting
the selective disruption of different functional domains of
the protein. resulted in identification of a de novo p.Glu198Lys
mutation in the PPP2R5D-gene. This mutation has been
previously described and is associated with the most severe
phenotype. Interestingly, in our patient profound myopia,
strabismus convergens fixus and retinal punched out lesions
were identified. To our knowledge, no retinal abnormalities
were associated with mutations in the PPP2R5D-gene
until now. Punched out lesions are associated with inflammation of
the retina and choroidea, described in posterior uveitis as
well as after congenital infection. There was no history of
uveitis or congenital infection in our patient. Interestingly,
such lesions have been described in Aicardi syndrome. Aicardi syndrome is a neurodevelopmental disorder char-
acterized by infantile spasms, agenesis of the corpus
callosum, and retinal abnormalities. The cause of the
disease remains unclear. We see some overlap in the phenotype of PPP2R5D-
mutations and Aicardi syndrome. Mutations in PPP2R5D-
gene have not been previously identified in Aicardi cohort. This case illustrates the clinical phenotype and natural
history of a PPP2R5D-mutation in an adult patient. We
recommend to perform ophthalmological examination in
patients with PPP2R5D-mutation to identify visual impair-
ment and to find out whether the retinal abnormalities are
part of the PPP2R5D-phenotype. G. Marangi: None. S. P08.60D Frangella: None. S. Ricciardi:
None. P. Chiurazzi:
None. D. Orteschi:
None. S. Lattante: None. R. Pettinato: None. F. Martinez: None. C. Magnani: None. C. Pantaleoni: None. C. Perria:
None. G. Scarano: None. V. Saletti: None. P. Bonanni:
None. G. Vasco: None. C. Lo Rizzo: None. A. Volzone:
None. E. Alfei: None. F. Faletra: None. S. Romano:
None. A. Renieri: None. M. Giannotta: None. C. Minetti:
None. C. Bruno: None. M. Piccione: None. F. Stanzial:
None. M. Della Monica: None. A. Ardissone: None. M. Di Giacomo: None. A. Battaglia: None. C. Graziano:
None. L. Garavelli: None. M. Zollino: None. A. Kattentidt: None. J.T.H.N. de Faber: None. R. Pfundt: None. R. Kersseboom: None. Dysmorphic phenotype in patients with RAB39B mutation Dysmorphic phenotype in patients with RAB39B mutation D. Casas-Alba1,2, A. Martínez-Monseny1, L. Martorell1,
C. Arjona1, F. Palau1, M. Serrano1,3 D. Casas-Alba1,2, A. Martínez-Monseny1, L. Martorell1,
C. Arjona1, F. Palau1, M. Serrano1,3 D. Casas-Alba1,2, A. Martínez-Monseny1, L. Martorell1,
C. Arjona1, F. Palau1, M. Serrano1,3 P08.61A 1Genetic Medine and Pediatric Institute for Rare Diseases,
Hospital Sant Joan de Deu (University of Barcelona),
Esplugues de Llobregat, Spain, 2Pediatrics Department,
Hospital Sant Joan de Deu (University of Barcelona),
Esplugues de Llobregat, Spain, 3Pediatric Neurology
Department, Hospital Sant Joan de Deu (University of
Barcelona),, Esplugues de Llobregat, Spain P08.63C Mutations in several genes encoding components or
regulators of the Rho signaling pathway (e.g. CDC42,
ARHGAP31, TRIO, HACE1, ELMO2, DOCK6 and SMPX)
have been identified in human disorders recently. We
propose that this emerging sub-category of rare develop-
mental disorders to be designated as Rhopathies with RAC1
as its central player. Our and others’ work indicate that
some Rhopathies may be amenable to treatment. C. Soussi Zander1, A. Thuresson1, J. Zhao1, J. Halvardson1,
E. Månsson2, E. Stenninger2, U. Holmlund3, Y. Öhrner3,
L. Feuk1 RAC1 missense mutations cause diverse phenotypes and
define Rhopathies as a new group of developmental
disorders M. R. F. Reijnders1, N. M. Ansor2,3, M. Kousi4, W. W. Yue5, P. L. Tan4, K. Clarkson6, J. Clayton-Smith2,7, K. Corning6, J. R. Jones6, W. W. K. Lam8, G. M. S. Mancini9, C. Marcelis1,
S. Mohammed10, R. Pfundt1, M. Roifman11,12, R. Cohn11,
D. Chitayat11,12, Deciphering Developmental Disorders Study,
N. Katsanis4, T. H. Millard2, H. G. Brunner1,13, S. Banka2,7 M. R. F. Reijnders1, N. M. Ansor2,3, M. Kousi4, W. W. Yue5, P. L. Tan4, K. Clarkson6, J. Clayton-Smith2,7, K. Corning6, J. R. Jones6, W. W. K. Lam8, G. M. S. Mancini9, C. Marcelis1,
S. Mohammed10, R. Pfundt1, M. Roifman11,12, R. Cohn11,
D. Chitayat11,12, Deciphering Developmental Disorders Study,
N. Katsanis4, T. H. Millard2, H. G. Brunner1,13, S. Banka2,7 M.R.F. Reijnders: None. N.M. Ansor: None. M. Kousi:
None. W.W. Yue: None. P.L. Tan: None. K. Clarkson:
None. J. Clayton-Smith: None. K. Corning: None. J.R. Jones: None. W.W.K. Lam: None. G.M.S. Mancini:
None. C. Marcelis: None. S. Mohammed: None. R. Pfundt: None. M. Roifman: None. R. Cohn: None. D. Chitayat: None. N. Katsanis: None. T.H. Millard: None. H.G. Brunner: None. S. Banka: None. 1Radboud University Medical Center, Nijmegen, Netherlands,
2University of Manchester, Manchester, United Kingdom,
3Universiti Sains Malaysia, Penang, Malaysia, 4Duke
University, Durham, NC, United States, 5University of Oxford,
Oxford, United Kingdom, 6Greenwood Genetic Center,
Greenwood, SC, United States, 7Manchester Centre for
Genomic Medicine, St. Mary's Hospital, Manchester, United
Kingdom, 8Western General Hospital, Edinburgh, United
Kingdom, 9Erasmus Medical Center, Rotterdam, Netherlands,
10Guy’s and St Thomas’ Hospital, London, United Kingdom,
11University of Toronto, Toronto, ON, Canada, 12Mount Sinai
Hospital, Toronto, ON, Canada, 13Maastricht University
Medical Center, Maastricht, Netherlands 1Radboud University Medical Center, Nijmegen, Netherlands,
2University of Manchester, Manchester, United Kingdom,
3Universiti Sains Malaysia, Penang, Malaysia, 4Duke
University, Durham, NC, United States, 5University of Oxford,
Oxford, United Kingdom, 6Greenwood Genetic Center,
Greenwood, SC, United States, 7Manchester Centre for
Genomic Medicine, St. Mary's Hospital, Manchester, United
Kingdom, 8Western General Hospital, Edinburgh, United
Kingdom, 9Erasmus Medical Center, Rotterdam, Netherlands,
10Guy’s and St Thomas’ Hospital, London, United Kingdom,
11University of Toronto, Toronto, ON, Canada, 12Mount Sinai
Hospital, Toronto, ON, Canada, 13Maastricht University
Medical Center, Maastricht, Netherlands Intellectual disability due to a PPP2R5D-mutation; what do
we know about natural history and adult phenotype? The maternal uncle was a 57-year-old male with severe ID. Both showed long and hypomimic face, bilateral ptosis,
lower lid ectropion, thick and everted lower lip, protruding
ears and drooling and swallowing difficulties, unlike other
family members. Two novel variants were identified in
RAB39B and HCFC1 in the proband, the mother and the
maternal uncle, but not in the brother. The first one was a
hemizygous mutation in RAB39B (NM_171998.3:c.137dup,
p.Ser47Leufs*44) with a likely pathogenic significance. The second one was a hemizygous nonsense mutation in
HCFC1 (NM_005334.2:c.2984C>G, p.Thr995Ser), pre-
dicted to be probably tolerated. These genes are not known
to interact with each other (dSysMap). X-chromosome
inactivation in the mother was not skewed. In silico modeling, mouse fibroblasts spreading assays
and in vivo overexpression assays using zebrafish as a
surrogate model demonstrated that (a) the p.Cys18Tyr and
p.Asn39Ser variants are dominant-negative alleles and
result in reduced neuronal proliferation, microcephaly and
cerebellar abnormalities; (b) the p.Tyr64Asp variant is
constitutively active; and (c) the effect of other variants is
likely context dependent. The second one was a hemizygous nonsense mutation in
HCFC1 (NM_005334.2:c.2984C>G, p.Thr995Ser), pre-
dicted to be probably tolerated. These genes are not known
to interact with each other (dSysMap). X-chromosome
inactivation in the mother was not skewed. RNAi-mediated knockdown of CYFIP homologue, sara1,
in drosophila neurons with constitutively active p.Tyr64Asp
rac1 variant resulted increased the rate of embryonic
lethality. Conclusions: In addition to ID and ASD, RAB39B
mutations
could
be
associated
with
a
characteristic
dysmorphic phenotype. Conclusions:
RAC1
missense
mutations
orchestrate
diverse human phenotypes with an extraordinary spread of
~10 SD of head circumferences. These findings highlight
the importance of RAC1 in neuronal development and
demonstrate the complexity of defining novel rare diseases
with extreme phenotypic variability. D. Casas-Alba: None. A. Martínez-Monseny: None. L. Martorell: None. C. Arjona: None. F. Palau: None. M. Serrano: None. Intellectual disability due to a PPP2R5D-mutation; what do
we know about natural history and adult phenotype? Intellectual disability due to a PPP2R5D-mutation; what do
we know about natural history and adult phenotype? A. Kattentidt1, J. T. H. N. de Faber2, R. Pfundt3, R. Kersseboom1
1Zuidwester, Spijkenisse, Netherlands, 2The Rotterdam Eye
Hospital, Rotterdam, Netherlands, 3Department of Clinical
Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands A. Kattentidt1, J. T. H. N. de Faber2, R. Pfundt3, R. Kersseboom1 A. Kattentidt1, J. T. H. N. de Faber2, R. Pfundt3, R. Kersseboom1 A. Kattentidt1, J. T. H. N. de Faber2, R. Pfundt3, R. Kersseboom1
1Zuidwester, Spijkenisse, Netherlands, 2The Rotterdam Eye
Hospital, Rotterdam, Netherlands, 3Department of Clinical
Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands 1Zuidwester, Spijkenisse, Netherlands, 2The Rotterdam Eye
Hospital, Rotterdam, Netherlands, 3Department of Clinical
Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands 1Zuidwester, Spijkenisse, Netherlands, 2The Rotterdam Eye
Hospital, Rotterdam, Netherlands, 3Department of Clinical
Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands Background: Mutations in the gene RAB39B (Xq28) have
been
associated
with
X-linked
mental
retardation-72
(#MIM300271, including intellectual disability (ID) and
autism spectrum disorders (ASD)), and Waisman syndrome
(#MIM311510), characterized by ID and early-onset Par-
kinsonism. RAB39B regulates GluA2 trafficking to deter-
mine synaptic AMPAR composition. In this report we
describe a family with two affected males with severe ID
and characteristic dysmorphic features not previously
reported in patients with RAB39B mutation. PPP2R5D is a gene associated with neurodevelopmental
disorders and intellectual disability. The phenotype includes
large ventricles, macrocephaly, hydrocephalus and (partial)
corpus callosum agenesis. Data on the natural history in
adulthood is limited. We describe a female, 42 years, with a severe intellectual
disability, macrocephaly, corpus callosum agenesis, open
vertebral bow and no seizures. Trio exome sequencing 238 J. del Picchia Introduction: RAC1 is a highly conserved Rho GTPase
under strict mutational constraint. Introduction: RAC1 is a highly conserved Rho GTPase
under strict mutational constraint. Methods: Clinical phenotyping, MLPA and whole exome
sequencing (WES) combined with targeted analysis focused
on 119 genes related to X-linked ID was performed on four
members of this family. Methods and Results: We report seven individuals with
intellectual disability and distinct de novo missense RAC1
mutations. These included four patients with microcephaly
(OFC between -2.5 to -5 SD; p.Cys18Tyr, p.Asn39Ser, p. Pro73Leu and p.Cys157Tyr), two with macrocephaly (OFC
+4.16 and +4.5 SD; p.Val51Met and p.Val51Leu) and one
individual with normal OFC (p.Tyr64Asp). Results: The proband case, a 18-year-old male, presented
with ID, ASD, epilepsy and behavioral and sleep disorders. C. Soussi Zander1, A. Thuresson1, J. Zhao1, J. Halvardson1,
E. Månsson2, E. Stenninger2, U. Holmlund3, Y. Öhrner3, P08.64D Whole genome sequencing of consanguineous families
reveals novel pathogenic variants in intellectual disability Abstracts from the 51st European Society of Human Genetics Conference: Posters 239 1Department of Immunology, Genetics and Pathology, Science
for Life Laboratory, Uppsala, Sweden, 2Department of
pediatrics, Örebro university hospital, Örebro, Sweden,
3Department of pediatrics, Västmanlands hospital Västerås,
Västerås, Sweden Sheffield, United Kingdom, 4Department of Genetics, Aberdeen
Royal Infirmary, Aberdeen, United Kingdom, 5Wessex Clinical
Genetics Service, G Level, Princess Anne Hospital,
Southampton, United Kingdom, 6Cheshire and Merseyside
Clinical Genetic Service, Liverpool Women's NHS Foundation
Trust, Liverpool, United Kingdom, 7Department of Clinical
Genetics, City Hospital Campus, Nottingham, United
Kingdom, 8Institute of Cancer and Genetics, University
Hospital of Wales, Cardiff, United Kingdom, 9Temple Street
Children’s Hospital, Dublin, Ireland, 10Department of Clinical
Genetics, Cambridge University Hospitals NHS Foundation
Trust, Cambridge, United Kingdom, 11Clinical Genetics Unit,
Birmingham Women's Hospital, Edgbaston, Birmingham,
United Kingdom, 12Sheffield Clinical Genetics Service,
Sheffield Children's Hospital, Sheffield, United Kingdom,
13Northern Genetics Service, Newcastle upon Tyne Hospitals,
Newcastle upon Tyne, United Kingdom, 14Clinical Genetics,
Royal Devon & Exeter NHS Foundation Trust, Exeter, United
Kingdom, 15Department of Clinical Genetics, University
Hospitals of Leicester NHS Trust, Leicester Royal Infirmary,
Leicester, United Kingdom, 16University of Exeter Medical
School, Institute of Biomedical and Clinical Science, RILD,
Royal Devon & Exeter Hospital, Barrack Road, Exeter, United
Kingdom, 17MRC Human Genetics Unit, MRC IGMM,
University of Edinburgh, Western General Hospital,
Edinburgh, United Kingdom Intellectual disability (ID) is a common disorder affecting
more than 2% of the population, but the majority of patients
receive no molecular diagnosis. The disorder is highly
genetically heterogeneous, with estimates of more than
2,500 autosomal ID genes. Autosomal recessive ID genes
have primarily been identified by studies of patients from
consanguineous families. Here we used whole genome
sequencing to study six consanguineous families that had
previously been tested using whole exome sequencing
without obtaining a molecular diagnosis. For four of the six
families (66%) we identified pathogenic variants in genes
previously reported in patients with ID (PIGN, SMC1A,
FRRS1L, and RTTN). Additionally, in one patient we
identified variants in both FRMD4A1 and COL27A1. The
COL27A1 variant explains the skeletal malformations in the
patient, but also expands the phenotype of this gene to
involve hearing impairment and ID. Our study highlights
the benefits of whole genome sequencing in families where
diagnostic exome sequencing was unsuccessful. P08.64D Our results
provide new pathogenic variants in several autosomal
recessive ID genes and describe a second finding of
pathogenic variation in COL27A1 in a patient with both
skeletal malformations and ID. Large scale sequencing is illuminating the genetic archi-
tecture of rare diseases. We analyzed 7,447 exome-
sequenced families from the Deciphering Developmental
Disorders study, and estimated the genome-wide contribu-
tion of recessive coding variation in both known and as-yet-
undiscovered genes. Our approach is the first to allow a
properly calibrated estimate of recessive burden. We found
that the proportion of cases attributable to recessive coding
variants was only 3.6% in patients of European ancestry,
compared to 50% explained by de novo coding mutations. It
was higher (31%) in patients with Pakistani ancestry, due to
elevated autozygosity. Half of this recessive burden is
attributable to known genes. Three genes were significantly
enriched for biallelic variants after stringent Bonferroni
correction. One is a new disease gene (EIF3F), and another,
KDM5B, is already reported in association with dominant
disease. The signal in EIF3F (p = 1.2×10-10) is driven by a
single missense variant (frequency ~0.1%) that was homo-
zygous in 9 DDD probands, and we are currently testing its
effect in cell lines. KDM5B (p = 1.1×10-7) appears to follow
a complex mode of inheritance, in which heterozygous loss-
of-function (LoF) variants show incomplete penetrance and
homozygous LoFs are fully penetrant. Through simulations,
we estimate the number of as-yet-undiscovered genes that
act by a recessive coding mechanism. Our results suggest
that recessive coding variants only account for a small C. Soussi Zander: None. A. Thuresson: None. J. Zhao:
None. J. Halvardson: None. E. Månsson: None. E. Stenninger: None. U. Holmlund: None. Y. Öhrner: None. L. Feuk: None. 1Wellcome Sanger Institute, Hinxton, United Kingdom,
2Division of Cardiovascular Medicine, Radcliffe Department of
Medicine, University of Oxford, Oxford, United Kingdom,
3Sheffield Clinical Genetics Service, Sheffield Children's NHS
Foundation Trust, OPD2, Northern General Hospital, P08.66B
The functional consequences of SCN2A mutations
determine the phenotype A. Begemann1, M. Acuña2, M. Zweier1, H. Sticht3, K. Steindl1,
M. Besnard1, A. Hackenberg4, L. Abela4, B. Plecko4,
K. Yamakawa5, Y. Inoue6, A. Baumer1, P. Joset1, R. Asadollahi1,
H. Zeilhofer2,7,8, A. Rauch1,7,8,9 A. Begemann1, M. Acuña2, M. Zweier1, H. Sticht3, K. Steindl1,
M. Besnard1, A. Hackenberg4, L. Abela4, B. Plecko4,
K. Yamakawa5, Y. Inoue6, A. Baumer1, P. Joset1, R. Asadollahi1,
H. Zeilhofer2,7,8, A. Rauch1,7,8,9 A. Begemann1, M. Acuña2, M. Zweier1, H. Sticht3, K. Steindl1,
M. Besnard1, A. Hackenberg4, L. Abela4, B. Plecko4, A. Begemann1, M. Acuña2, M. Zweier1, H. Sticht3, K. Steindl1,
M. Besnard1, A. Hackenberg4, L. Abela4, B. Plecko4, K. Yamakawa5, Y. Inoue6, A. Baumer1, P. Joset1, R. Asadollahi1,
H. Zeilhofer2,7,8, A. Rauch1,7,8,9 K. Yamakawa5, Y. Inoue6, A. Baumer1, P. Joset1, R. Asadollahi1,
H. Zeilhofer2,7,8, A. Rauch1,7,8,9 1University of Zurich, Institute of Medical Genetics, Schlieren-
Zurich, Switzerland, 2University of Zurich, Institute of
Pharmacology and Toxicology, Zurich, Switzerland,
3Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute
of Biochemistry, Erlangen, Germany, 4University Children's
Hospital, Division of Child Neurology, Zurich, Switzerland,
5RIKEN Brain Science Institute, Laboratory for Neurogenetics,
Wako-shi, Saitama, Japan, 6Shizuoka Institute of Epilepsy and
Neurological Disorders, National Epilepsy Center, Shizuoka,
Japan, 7radiz - Rare Disease Initiative Zürich, Clinical
Research Priority Program for Rare Diseases, Zurich,
Switzerland, 8University of Zurich and ETH Zurich,
Neuroscience Center Zurich, Zurich, Switzerland, 9University
of Zurich, Zurich Center for Integrative Human Physiology,
Zurich, Switzerland A. Begemann: None. M. Acuña: None. M. Zweier:
None. H. Sticht: None. K. Steindl: None. M. Besnard:
None. A. Hackenberg: None. L. Abela: None. B. Plecko:
None. K. Yamakawa: None. Y. Inoue: None. A. Baumer:
None. P. Joset: None. R. Asadollahi: None. H. Zeilhofer:
None. A. Rauch: None. A. Begemann: None. M. Acuña: None. M. Zweier:
None. H. Sticht: None. K. Steindl: None. M. Besnard:
None. A. Hackenberg: None. L. Abela: None. B. Plecko:
None. K. Yamakawa: None. Y. Inoue: None. A. Baumer:
None. P. Joset: None. R. Asadollahi: None. H. Zeilhofer:
None. A. Rauch: None. 1Department of Human Genetics, Radboud university medical
Center, Nijmegen, Netherlands, 2Department of Medical
Genetics, Telemark Hospital, Ulefossveien, Skien, Norway,
3Greenwood Genetic Center, Greenwood, SC, United States,
4Department of Clinical Genetics, Erasmus medical center,
Rotterdam, Netherlands, 5Department of Pathology, Oslo
University Hospital, the Norwegian Radium Hospital, Oslo, P08.65A Quantifying the contribution of recessive coding variation
to developmental disorders H. C. Martin1, W. D. Jones1, J. Stephenson1, J. Handsaker1,
R. McIntyre1, M. Bruntraeger1, G. Gallone1, J. McRae1,
E. Prigmore1, P. Short1, M. Niema1, J. Kaplanis1, E. Radford1,
N. Akawi2, M. Balasubramanian3, J. Dean4, R. Horton5,
A. Hulbert6, D. S. Johnson3, K. Johnson7, D. Kumar8, S. Lynch9,
S. G. Mehta10, J. Morton11, M. Parker12, M. Splitt13, P. D. Turnpenny14, P. C. Vasudevan15, M. Wright13, A. Bassett1, C. F. Wright16, D. R. FitzPatrick17, H. V. Firth10, M. E. Hurles1, J. C. Barrett1 240 J. del Picchia fraction of currently undiagnosed individuals, and that
future studies should focus on noncoding variants and
polygenic mechanisms. therefore investigate the functional effects of six mutations
to elucidate the different pathomechanisms leading to
EE or ID. H.C. Martin: None. W.D. Jones: None. J. Stephenson:
None. J. Handsaker: None. R. McIntyre: None. M. Bruntraeger: None. G. Gallone: None. J. McRae: None. Methods: Six pathogenic SCN2A mutations underlying
either EE, ID or BFNIE were selected for functional studies. Biophysical properties of recombinant wildtype and mutant
Nav1.2 channels were measured using voltage-clamp of
transiently transfected HEK293T cells co-expressing aux-
iliary β-subunits and EGFP. In-silico protein modeling was
used to gain insight into the structural effects of the
mutations. E. Prigmore: None. P. Short: None. M. Niema: None. J. Kaplanis: None. E. Radford: None. N. Akawi: None. M. Balasubramanian: None. J. Dean: None. R. Horton:
None. A. Hulbert: None. D.S. Johnson: None. K. Johnson: None. D. Kumar: None. S. Lynch: None. S.G. Mehta: None. J. Morton: None. M. Parker: None. M. Splitt: None. P.D. Turnpenny: None. P.C. Vasudevan:
None. M. Wright: None. A. Bassett: None. C.F. Wright:
None. D.R. FitzPatrick: None. H.V. Firth: None. M.E. Hurles: E. Ownership Interest (stock, stock options, patent
or other intellectual property); Significant; Congenica Ltd. F. Consultant/Advisory Board; Significant; Congenica Ltd. J C
Barrett: F
Consultant/Advisory Board; Modest; Results: Both SCN2A missense mutations causing EE
showed profound gating changes in patch-clamp experi-
ments, whereas all ID mutations, nonsense and missense,
exhibited no relevant current. The BFNIE mutation showed
a small change of channel inactivation resulting in a small
gain of function. The protein modeling suggested structural
aberrations for all studied missense mutations consistent
with the electrophysiological findings. Splitt: None. P.D. Turnpenny: None. P.C. Vasudevan:
None. M. Wright: None. A. Bassett: None. C.F. Wright:
None. D.R. FitzPatrick: None. H.V. Firth: None. M.E. Hurles: E. P08.65A Ownership Interest (stock, stock options, patent
or other intellectual property); Significant; Congenica Ltd. F. Consultant/Advisory Board; Significant; Congenica Ltd. J.C. Barrett: F. Consultant/Advisory Board; Modest;
Genomics plc. Discussion: By examining the functional consequences
of SCN2A mutations causing epileptic encephalopathy, self-
limited familial neonatal-infantile epilepsy and intellectual
disability this study contributes to the elucidation of
mechanisms leading to the broad phenotype variability
reported for SCN2A mutations. Our findings support the
hypothesis that complete loss-of-function mutations lead to
intellectual
disability
without
seizures,
small
gain-of-
function mutations cause BFNIE and epileptic encephalo-
pathy mutations exhibit variable but profound Nav1.2 gating
changes. P08.67C P08.67C
A recurrent de novo missense mutation in SMARCB1
causes severe intellectual disability and choroid plexus
hyperplasia with resultant hydrocephalus A recurrent de novo missense mutation in SMARCB1
causes severe intellectual disability and choroid plexus
hyperplasia with resultant hydrocephalus I. J. Diets1, T. Prescott2, N. L. Champaigne3, G. M. S. Mancini4,
B. Krossnes5, R. Frič6, K. Kocsis7, M. C. J. Jongmans1,
T. Kleefstra1 I. J. Diets1, T. Prescott2, N. L. Champaigne3, G. M. S. Mancini4,
B. Krossnes5, R. Frič6, K. Kocsis7, M. C. J. Jongmans1,
T. Kleefstra1 I. J. Diets1, T. Prescott2, N. L. Champaigne3, G. M. S. Mancini4,
B. Krossnes5, R. Frič6, K. Kocsis7, M. C. J. Jongmans1,
T. Kleefstra1 1Department of Human Genetics, Radboud university medical
Center, Nijmegen, Netherlands, 2Department of Medical
Genetics, Telemark Hospital, Ulefossveien, Skien, Norway,
3Greenwood Genetic Center, Greenwood, SC, United States,
4Department of Clinical Genetics, Erasmus medical center,
Rotterdam, Netherlands, 5Department of Pathology, Oslo
University Hospital, the Norwegian Radium Hospital, Oslo, Objective: Mutations in the voltage-gated sodium channel
type 2 (Nav1.2) lead to a broad spectrum of phenotypes
ranging from self-limited familial neonatal-infantile epi-
lepsy (BFNIE) to very severe epileptic encephalopathy (EE)
to intellectual disability without seizures (ID), yet the
underlying mechanisms determining this phenotypic varia-
bility are incompletely understood. In this study, we 241 Abstracts from the 51st European Society of Human Genetics Conference: Posters Norway, 6Department of Neurosurgery, Oslo University
Hospital - Rikshospitalet, and Faculty of Medicine, Oslo,
Norway, 7Children's Hospital Colorado, University of
Colorado, Department of Clinical Genetics and Metabolism,
Denver-Aurora, CO, United States Division, IRCCS Fondazione Istituto Neurologico C. Besta,
Milan, Italy Smith-Magenis
Syndrome
(SMS) [MIM:182290] is
a
genomic disorder caused by RAI1 gene haploinsufficiency
and characterized by intellectual disability, craniofacial
dysmorphisms, behavioral and sleep disturbances, speech
and motor delay. Here we describe a 17 years old girl with a
clinical suspicion of SMS. Upon previous exclusion of
17p11.2 SMS locus deletion, RAI1 mutational screening
identified the yet unreported heterozygous variant p. A1091D predicted as likely benign and inherited from the
healthy mother. Moreover, MLPA analysis revealed a de
novo heterozygous deletion encompassing RAI1 exon 5,
that encodes PHD functional domain, consistent with the
initial clinical suspicion.In order to verify that the de novo
deletion results in RAI1 haploinsufficiency, RT-qPCR stu-
dies were carried out but showed an unexpected significant
increase in blood transcript levels of both patient and
mother compared to those of 10 controls. P08.67C Moreover, a
specific allelic dosage analysis revealed that the deleted
allele is overexpressed in the patient and concerns the
inherited maternal allele. This result confirms SMS diag-
nosis, as an overexpression of an aberrant transcript lacking
the functional domain was detected.Our finding supports the
hypothesis that RAI1 overexpression, shared with the
mother, can be mediated by a cis element. Promoter and
regulatory regions are under study aiming at identifying
variants that could be related with RAI1 overexpression
finding. Despite RAI1 overexpression causes Potocki-
Lupski Syndrome (PTLS) [MIM:610883], the mother
does not resemble a PTLS clinical phenotype. This might be
explained by the PTLS high phenotype variability or by
penetrance
defect
as
previously
reported
in
other
familiar cases. Introduction: SMARCB1 encodes a subunit of the SWI/
SNF-complex involved in chromatin remodeling. Mutations
in this gene can give rise to three conditions. Heterozygous
loss of function germline mutations cause the rhabdoid
tumor
predisposition
syndrome
and
schwannomatosis. Presumed gain of function missense mutations in exon 8
and 9 result in Coffin Siris syndrome, which is characterized
by intellectual disability (ID), coarse facial features and fifth
digit anomalies. Methods: By a gene matching approach, individuals with
a similar SMARCB1 mutation were identified. Informed
consent was obtained and patient data were collected to
further establish genotype-phenotype relationship. Results:
A
recurrent
de
novo
missense
mutation
(c.110G>A;p.Arg37His) in exon 2 of SMARCB1, encoding
the DNA-binding domain, was identified in four individuals
from different genetic centers. They shared a distinct
phenotype consisting of profound ID and hydrocephalus
due to choroid plexus hyperplasia. Other shared features
include severe neonatal feeding difficulties, congenital
heart-, kidney-, and eye anomalies, obstructive sleep apnea
and anemia. Conclusion: The p.Arg37His mutation in the DNA
binding domain of SMARCB1 causes a distinctive syn-
drome, probably through a gain of function, which is
characterized by severe ID and hydrocephalus resulting
from choroid plexus hyperplasia. This report broadens the
phenotypic
spectrum
associated
with
mutations
in
SMARCB1. I.J. Diets: None. T. Prescott: None. N.L. Champaigne:
None. G.M.S. Mancini: None. B. Krossnes: None. R. I.J. Diets: None. T. Prescott: None. N.L. Champaigne:
None. G.M.S. Mancini: None. B. Krossnes: None. R. Frič: None. K. Kocsis: None. M.C.J. Jongmans: None. T. Kleefstra: None. I.J. Diets: None. T. Prescott: None. N.L. Champaigne:
None. G.M.S. Mancini: None. B. Krossnes: None. R. Frič: None. K. Kocsis: None. M.C.J. Jongmans: None. T. Kleefstra: None. A. Sironi: None. I. Bestetti: None. C. Boninsegna:
None. M. 1Lab. of Medical Cytogenetics and Molecular Genetics, IRCCS
Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy,
2Dep. of Medical Biotechnology and Translational Medicine,
University of Milan, Milan, Italy, 3Developmental Neurology P08.69A P08.68D
RAI1 intragenic deletion and concomitant overexpression
in a syndromic patient: Smith-Magenis or Potocki-Lupski
syndrome? P08.68D
RAI1 intragenic deletion and concomitant overexpression
in a syndromic patient: Smith-Magenis or Potocki-Lupski
syndrome? Maternal transmission of mild Coffin-Siris syndrome
phenotype due to a SOX11 missense mutation B. Hoffmann1, G. Gillessen-Kaesbach1, H. Lüdecke2,
I. Hüning1, D. Wieczorek2
1Institut für Humangenetik, Universität zu Lübeck, Lübeck,
Germany, 2Institut für Humangenetik, Universitätsklinikum,
Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
Since 2014, mutations in SOX11 are known to cause mild
Coffin-Siris syndrome (CSS) phenotype. SOX11 is a B. Hoffmann1, G. Gillessen-Kaesbach1, H. Lüdecke2,
I. Hüning1, D. Wieczorek2 P08.67C Masciadri: None. S. Russo: None. C. Panta-
leoni: None. S. D'Arrigo: None. L. Larizza: None. P. Finelli: None. Frič: None. K. Kocsis: None. M.C.J. Jongmans: None. T. Kleefstra: None. B. Hoffmann1, G. Gillessen-Kaesbach1, H. Lüdecke2,
I. Hüning1, D. Wieczorek2 A. Sironi1,2, I. Bestetti1,2, C. Boninsegna1, M. Masciadri1,
S. Russo1, C. Pantaleoni3, S. D'Arrigo3, L. Larizza1, P. Finelli1,2
1Lab. of Medical Cytogenetics and Molecular Genetics, IRCCS
Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy,
2Dep. of Medical Biotechnology and Translational Medicine,
University of Milan, Milan, Italy, 3Developmental Neurology A. Sironi1,2, I. Bestetti1,2, C. Boninsegna1, M. Masciadri1,
S. Russo1, C. Pantaleoni3, S. D'Arrigo3, L. Larizza1, P. Finelli1,2 1Institut für Humangenetik, Universität zu Lübeck, Lübeck,
Germany, 2Institut für Humangenetik, Universitätsklinikum,
Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany 1Lab. of Medical Cytogenetics and Molecular Genetics, IRCCS
Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy,
2Dep. of Medical Biotechnology and Translational Medicine,
University of Milan, Milan, Italy, 3Developmental Neurology Since 2014, mutations in SOX11 are known to cause mild
Coffin-Siris syndrome (CSS) phenotype. SOX11 is a J. del Picchia 242 The ST3GAL3 gene located on chr1p34.1 encodes for the
β-galactosidase-α2,3 sialyltransferase-III (ST3Gal-III), a
glycoprotein/ganglioside sialylytransferase present in Golgi
membrane and playing a role in interactions of brain cells
with their environment thought glycocalyx. Pathogenic
variants in ST3GAL3 were reported in three consanguineous
families only in patients with nonsydromic intellectual
disability or with epileptic encephalopathy (West syn-
drome) and severe developmental delay. We report three
unrelated patients (two brothers and a sporadic case) to
further delineate the phenotype associated with ST3GAL3
mutations and describe with more details the phenotype of
previously reported patients. All of our three novel patients
had early epilepsy and severe to profound intellectual dis-
ability. The epilepsy was transient in one and pharmacore-
sistant
in
the
siblings
with
a
more
severe
neurodevelopmental phenotype. We identifed by whole
exome sequencing and with a sequencing gene panel two
novel homozygous variants in ST3GAL3: the c.765-1G>A
intronic variant in one patient and the c.1015C>T (p. Arg339*) nonsense mutation in the brothers. Our three
patients and those of the literature share severe neurode-
velopmental delay without language acquisition. Epilepsy is
constant and variable ranging from few seizures to phar-
macoresistant epileptic encephalopathy. The siblings had
the most severe phenotype reported to date and micro-
cephaly, a feature not previously reported in the condition. MRI, EEG and the patients’ morphology are non specific. We report the first truncating mutation and the first splice
site mutation predicted to product exon skipping, which
may explain that the patient has a less severe phenotype. transcription factor of the PAX6-BAF complex, which is
proposed to play a role in brain development. Novel ST3GAL3 mutations in three patients with
intellectual disability and epilepsy Novel ST3GAL3 mutations in three patients with
intellectual disability and epilepsy B. Hoffmann1, G. Gillessen-Kaesbach1, H. Lüdecke2,
I. Hüning1, D. Wieczorek2 Here
we
report
for
the
first
time
of
a
maternal
transmission of Coffin-Siris syndrome (CSS) due to a
SOX11 missense mutation. We present two daughters (14
and 10 years of age) of non-consanguineous parents with
intellectual disability and muscular hypotonia. Both sisters
showed mild dysmorphic facial features such as short
philtrum, thick lips, low-set ears and strabism. Cogan ocular
motor apraxia was present in both sisters. Mother and both
daughters showed hypoplastic nails of the fifth toes as sign
of mild CSS. The mother also had a history of learning
difficulties. A coloboma of the iris was present. Karyotyping and array-CGH gave normal results. NGS
panel diagnostics for CSS revealed a missense variant in
SOX11 [c.139G>A; p.(Gly47Ser)] in both sisters and their
mother. Four in silico-tools (PROVEAN, SIFT, Polyphen-2
and MutationTaster) predicted the mutation as probably
pathogenic. A review of the literature showed that until now only six
patients with de novo mutations in SOX11 have been
described. All of them showed intellectual disability and
hypoplastic nails of the fifth toes. Some of these patients
had Cogan ocular
motor apraxia. Facial dysmorphic
features seem not to be specific. We suggest that the
combination of Cogan ocular motor apraxia, developmental
delay and hypoplastic nails of fifth toes are important
diagnostic criteria for recognizing patients for mutations in
SOX11. B. Hoffmann: None. G. Gillessen-Kaesbach: None. H. Lüdecke: None. I. Hüning: None. D. Wieczorek: None. L. Ruaud: None. D. Héron: None. H. Maurey: None. C. Mehler-Jacob: None. D. Doummar: None. S. Heide:
None. C. Nava: None. B. Keren: None. C. Mignot: None. M. A. Zelenova1,2, S. G. Vorsanova1,2, Y. B. Yurov1,2, S. A.
Korostelev3, O. S. Kurinnaia1,2, I. Y. Iourov1,2,4 P08.71C The use of genetic databases
of patients with intellectual disability and other described
clinical traits is evidencing correlations with specific
molecular alterations or combination of them. Materials and Methods. To reveal candidate processes
for neurobehavioral problems, we used high-resolution
genome-wide CNV scan in 191 children with congenital
abnormalities and idiopathic ID (Affymetrix CytoScan HD)
and in 11 children with Rett syndrome-like phenotype
without MECP2 mutations (Nimblegen 12×135K). All
children were not exhibiting cytogenetically visible chro-
mosomal abnormalities and detectable genomic rearrange-
ments and epigenetic changes. A bioinformatic algorithm
(Iourov et al., 2014) was applied for candidate pathway
prioritization using KEGG, Reactome, Gene Ontology,
NCBI biosystems databases. Materials and Methods. To reveal candidate processes
for neurobehavioral problems, we used high-resolution
genome-wide CNV scan in 191 children with congenital
abnormalities and idiopathic ID (Affymetrix CytoScan HD)
and in 11 children with Rett syndrome-like phenotype
without MECP2 mutations (Nimblegen 12×135K). All
children were not exhibiting cytogenetically visible chro-
mosomal abnormalities and detectable genomic rearrange-
ments and epigenetic changes. A bioinformatic algorithm
(Iourov et al., 2014) was applied for candidate pathway
prioritization using KEGG, Reactome, Gene Ontology,
NCBI biosystems databases. Patients and Methods: We present an exhaustive
description of Copy Number Variants (CNVs) identified
in chromosome 16p from 191 CytoScan HD Affymetrix
arrays performed in syndromic patients and their relation-
ship with severe alterations in corporal and body weight
composition. Patients and Methods: We present an exhaustive
description of Copy Number Variants (CNVs) identified
in chromosome 16p from 191 CytoScan HD Affymetrix
arrays performed in syndromic patients and their relation-
ship with severe alterations in corporal and body weight
composition. Results. Pathway prioritization yielded enrichment of 4
pathway clusters in both cohorts: vesicles functioning,
Notch signaling pathway, actin functioning, transcription
regulation. Among these clusters, the SNAP receptor
activity pathway (GO:0005484) was found to be the most
significantly
prioritized. Synaptic
vesicles
are
mostly
located at presynaptic terminal and carry neuromediators. Alterations to vesicles docking and exocytosis can affect
neurotransmitter
release
leading
to
neurobehavioral
diseases. Results: Variants were localized in chromosome 16p and
were classified as: (I) previously non-described 10 Mb
duplication in the 16p13.2p12.3 region considered causal of
the developed phenotype which consisted on intellectual
disability and obesity, and (II) CNVs localized in the
16p11.2 region characterized by their low prevalence but
with recurrence in syndromic patients with severe altera-
tions in the corporal weight. P08.72D Effect of modifier genes within Copy Number Variations in
chromosome 16p on the body weight of intellectual
disability patients P08.71C L. Ruaud1,2, D. Héron2,3,4, H. Maurey5, C. Mehler-Jacob5,
D. Doummar6,7, S. Heide2,3,4, C. Nava2,3,4, B. Keren2,3,4,
C. Mignot2,3,4 L. Ruaud1,2, D. Héron2,3,4, H. Maurey5, C. Mehler-Jacob5,
D. Doummar6,7, S. Heide2,3,4, C. Nava2,3,4, B. Keren2,3,4,
C. Mignot2,3,4 Alterations to synaptic vesicles pathways are likely to be
involved in non-syndromic intellectual disability M. A. Zelenova1,2, S. G. Vorsanova1,2, Y. B. Yurov1,2, S. A. Korostelev3, O. S. Kurinnaia1,2, I. Y. Iourov1,2,4 1Centre de Génétique Humaine, CHU Besançon, Besançon,
France, 2Département de Génétique, APHP, GH Pitié-
Salpêtrière, Paris, France, 3Centre de Référence Déficiences
Intellectuelles de Causes Rares, Paris, France, 4Groupe de
Recherche Clinique (GRC) 'déficience intellectuelle et autisme'
UPMC, Paris, France, 5Service de neurologie pédiatrique,
APHP, HU Paris Sud Site Kremlin Bicêtre, Le Kremlin-
Bicêtre, France, 6Service de Neuropédiatrie - Unité de
neuropédiatrie et pathologie du développement, APHP,
Hôpital Armand Trousseau, Paris, France, 7Centre de
Référence Cervelet, Paris, France 1FSBSI «Mental Health Research Center», Moscow, Russian
Federation, 2Academician Yu.E. Veltishchev Research Clinical
Institute of Pediatrics, N.I. Pirogov Russian National Research
Medical University, Ministry of Health of the Russian
Federation, Moscow, Russian Federation, 3The State
Educational Institution of Professional Training under the
Federal Agency of Health Care and Social Development,
Moscow, Russian Federation., Moscow, Russian Federation,
4FSBEI FPE «Russian Medical Academy of Postgraduate
Education» of the Ministry of Healthcare of the Russian
Federation, Moscow, Russian Federation 243 Abstracts from the 51st European Society of Human Genetics Conference: Posters Badajoz, Spain., Badajoz, Spain, 33. Laboratorio de
Citogenética, Complejo Hospital Universitario de Vigo, Vigo,
Spain., Vigo, Spain Introduction. Pathway prioritization is a challenging yet
promising tool for uncovering mechanisms and therapy
targets for intellectual disability (ID). Using big genomic
data and advanced in silico techniques, it is possible to
unmask pathways and processes, alterations to which can be
causative to neurobehavioral diseases. Introduction. Pathway prioritization is a challenging yet
promising tool for uncovering mechanisms and therapy
targets for intellectual disability (ID). Using big genomic
data and advanced in silico techniques, it is possible to
unmask pathways and processes, alterations to which can be
causative to neurobehavioral diseases. Introduction: Syndromic patients could manifest intellec-
tual disability associated to early-onset weight alteration
(underweight and/or obesity). The diverse heterogeneity in
its etiology has improved the development of techniques for
genetic diagnosis, allowing the molecular characterization
of new syndromic phenotypes. P08.71C Proximal 16p11.2 CNVs had a
dose-dependent effect: underweight in case of duplication
and obesity in case of deletion. Our analysis has allowed
suggesting KCTD13 gene as candidate to produce the
physiopathology of the 16p11.2 proximal syndromes and
SH2B1 gene for the 16p11.2 distal phenotypes. Conclusions. Since synaptic vesicles fusion with pre-
synaptic plasma membrane is essential for nerve impulse
transmission, these data do not only hallmark alterations to
synaptic vesicles pathways involved in non-syndromic
intellectual disability, but also indicates possible therapeutic
targets for molecular interventions. Finally, it seems that
synaptic vesicles pathways represent an intriguing target for
further studies of ID pathogenesis. Supported by RSF (14-
35-00060). Conclusion: CNVs of chromosome 16 postulate it as
genomic hotspot of alterations in the body mass index in
syndromic patients, allowing the primary prevention of
comorbidities with its detection. Studies in syndromic
individuals could constitute a reliable model to evaluate
hypothalamic satiety disorders. M.A. Zelenova: None. S.G. Vorsanova: None. Y.B. Yurov: None. S.A. Korostelev: None. O.S. Kurinnaia:
None. I.Y. Iourov: None. F. Gimeno-Ferrer: None. D. Albuquerque: None. C. Guzmán Luján: None. G. Marcaida Benito: None. M. Aleu Pérez-Gramunt: None. V. Ballesteros Cogollos:
None. E. Galán Gómez: None. C. Torreira: None. R. Rodríguez-López: None. F. Gimeno-Ferrer1, D. Albuquerque1, C. Guzmán Luján1,
G. Marcaida Benito1, M. Aleu Pérez-Gramunt1, V. Ballesteros
Cogollos1, E. Galán Gómez2, C. Torreira3, R. Rodríguez-López1
11. Genomics Group for the Study of Obesity, Research
Foundation of the General University Hospital, Valencia,
Spain, 22.Servicio de Pediatría del Hospital Materno Infantil, 11. Genomics Group for the Study of Obesity, Research
Foundation of the General University Hospital, Valencia,
Spain, 22.Servicio de Pediatría del Hospital Materno Infantil, F. Gimeno-Ferrer1, D. Albuquerque1, C. Guzmán Luján1,
G. Marcaida Benito1, M. Aleu Pérez-Gramunt1, V. Ballesteros
Cogollos1, E. Galán Gómez2, C. Torreira3, R. Rodríguez-López1
11. Genomics Group for the Study of Obesity, Research 1Cytogenetics and Molecular Genetics Laboratory, Istituto
Auxologico Italiano, Milano, Italy, 2Department of S. Guzzetti1, L. Calzari1, L. Buccarello1, V. Cesari2, I. Toschi3,
S. Cattaldo4, F. Pregnolato5, S. Mazzola6, S. Russo1 S. Guzzetti1, L. Calzari1, L. Buccarello1, V. Cesari2, I. Toschi3,
S. Cattaldo4, F. Pregnolato5, S. Mazzola6, S. Russo1
1Cytogenetics and Molecular Genetics Laboratory, Istituto
Auxologico Italiano, Milano, Italy, 2Department of P08.73A Taurine administration recovers motor and learning
deficits in an Angelman syndrome mouse model Taurine administration recovers motor and learning
deficits in an Angelman syndrome mouse model 1Cytogenetics and Molecular Genetics Laboratory, Istituto
Auxologico Italiano, Milano, Italy, 2Department of 244 J. del Picchia B. Liesfeld15, T. Polster17, D. Mitter1, K. Platzer1, J. Hentschel1,
J. Lemke1, R. Jamra1 Agricultural and Environmental Sciences University of Milan,
Milano, Italy, 3of Agricultural and Environmental Sciences,
Universityof Milano, Milano, Italy, 4HPLC laboratory,
Neurobiology, Istituto Auxologico Italiano, Piancavallo (NO),
Italy, 5Experimental Laboratory of Immunological and
Rheumatologic Researches, Istituto Auxologico Italiano,
Milano, Italy, 6Department of Veterinary Medicine, University
of Milano, Milano, Italy 1Institute of Human Genetics, Leipzig, Germany, 2Praxis für
Humangenetik Leipzig, Leipzig, Germany, 3Hospital for
Children and Adolescents, University Medical Center Leipzig,
Leipzig, Germany, 4Division of Neuropediatrics, Hospital for
Children and Adolescents, University Medical Center Leipzig,
Leipzig, Germany, 5Institute of Human Genetics and
Anthropology, Heinrich Heine University Düsseldorf,
Düsseldorf, Germany, 6Department of Human Genetics, Ruhr-
University, Bochum, Germany, 7Institute of Human Genetics,
University Lübeck, Lübeck, Germany, 8Institute of Human
Genetics, University Hospital Essen, Essen, Germany,
9Division for Neuropaediatrics and Metabolic Medicine,
Center for Paediatric and Adolescent Medicine, Heidelberg
University Hospital, Heidelberg, Germany, 10EKO Children‘s
Hospital, Witten/Herdecke University, Oberhausen, Germany,
11Institute of Human Genetics, University of Bonn School of
Medicine and University Hospital of Bonn, Bonn, Germany,
12Centogene AG, Rostock, Germany, 13CeGaT GmbH, Center
for Genomics and Transcriptomics, Tübingen, Germany,
14Institute of Clinical Genetics, Technische Universität
Dresden, Dresden, Germany, 15Limbus Medical Technologies
GmbH, Rostock, Germany, 16Institute of Human Genetics,
University Hospital Magdeburg, Magdeburg, Germany,
17Bethel Epilepsy Center, Hospital Mara GmbH, Bielefeld,
Germany Angelman syndrome (AS, MIM 105830) is a rare neuro-
developmental
disorder
affecting
1:10-20000
children. Patients show moderate to severe intellectual disability,
ataxia and absence of speech. Currently no treatment is
available. Studies on both post-mortem AS human brains
and mouse models revealed dysfunctions in the extra
synaptic GABA receptors implicated in the pathogenesis. Our study focused on taurine, a free intracellular aminoacid,
abundant in brain and considered an inhibitor neuro-
transmitter with neuroprotective properties. As taurine acts
as agonist of GABA-A receptors, expressing various
GABA-A subunits, we aimed to investigate if it might
ameliorate AS symptoms. Since the mice weaning, we
orally and chronically administered 1 g/kg taurine in water
to Ube3a deficient mice. P08.73A Cattaldo:
None. F. Pregnolato: None. S. Mazzola: None. S. Russo: None. P08.73A In order to test the improvement of
motor and cognitive skills, rotarod, NORT and Open Field
tests were assayed at 7, 14, 21 and 30 weeks, while bio-
chemical tests and aminoacid dosages were carried on
respectively by western blot and HPLC on frozen brains. An increased level of GFAP and an activation of ERK1/2
pathway were observed in the total brain of transgenic mice. Taurine treatment significantly rescued motor and learning
skills and restored the level of the glial marker GFAP and of
pERK1-2/ERK1-2 ratio. Our study indicates oral taurine
administration as a potential therapy to ameliorate motor
deficits and learning difficulties in AS. Introduction: We present our results of scientific evalua-
tion of clinical trio exome sequencing of neurodevelop-
mental disorders (NDD). Introduction: We present our results of scientific evalua-
tion of clinical trio exome sequencing of neurodevelop-
mental disorders (NDD). Materials and Methods: We performed trio exome
sequencing of 203 undiagnosed NDD cases. Of these, 59%
were preceded by a large, but negative diagnostic panel. Sequencing was performed by Centogene or CeGaT using
well-established enrichment kits and sequencing platforms. Bioinformatic analyses were performed by Limbus. If
exome sequencing did not reveal a clear causative variant
in an established NDD gene, we searched for candidate
genes on scientific basis. To estimate the relevance of
candidate genes, we established a scoring system using 13
parameters, based on inheritance, gene and variant attri-
butes, and published literature. Materials and Methods: We performed trio exome
sequencing of 203 undiagnosed NDD cases. Of these, 59%
were preceded by a large, but negative diagnostic panel. Sequencing was performed by Centogene or CeGaT using
well-established enrichment kits and sequencing platforms. Bioinformatic analyses were performed by Limbus. If
exome sequencing did not reveal a clear causative variant
in an established NDD gene, we searched for candidate
genes on scientific basis. To estimate the relevance of
candidate genes, we established a scoring system using 13
parameters, based on inheritance, gene and variant attri-
butes, and published literature. Funded by ORSA Angelman parents’ association grant
08A401 and Ministery of Health 08C002_2010 Funded by ORSA Angelman parents’ association grant
08A401 and Ministery of Health 08C002_2010 S. Guzzetti: None. L. Calzari: None. L. Buccarello:
None. V. Cesari: None. I. Toschi: None. S. Cattaldo:
None. F. Pregnolato: None. S. Mazzola: None. S. Russo: None. S. Guzzetti: None. L. Calzari: None. L. Buccarello:
None. V. Cesari: None. I. Toschi: None. S. Scientific yield of clinical exome sequencing of
neurodevelopmental disorders Muschke16, Abstracts from the 51st European Society of Human Genetics Conference: Posters Abstracts from the 51st European Society of Human Genetics Conference: Posters 245 Université de Montréal, Montreal, QC, Canada, 4Laboratory
of embryology and genetics of congenital malformations,
INSERM UMR 1163, Institut Imagine, Paris, France, 5Institut
Imagine, Paris Descartes-Sorbonne Paris Cité University,
Paris, France, 6Departement of Medical Genetics, Trondheim
University Hospital, Trondheim, Norway, 7Division of
Genetics, Children's Hospital of Philadelphia, Philadelphia,
PA, United States, 8Department of Pediatrics, Perelman School
of Medicine, University of Pennsylvania, Philadelphia, PA,
United States, 9Medical Genomics and Metabolic Genetics
Branch, National Human Genome Research Institute, National
Institutes of Health, Bethesda, MD, United States,
10Department of Medical Genetics, University of British
Columbia, Vancouver, BC, Canada, 11CHRU Brest, Génétique
médicale, Brest, France, 12Service de Génétique, CHU
Poitiers, Poitiers, France, 13EA3808 CiMoTheMA Université
Poitiers, Poitiers, France, 14GeneDx, Gaithersburg, Nantes,
MD, United States, 15GeneDx, Gaithersburg, MD, United
States, 16Department of Clinical Genetics, Oxford University
Hospitals NHS Trust, Oxford, United Kingdom, 17Department
of Molecular and Human Genetics, Baylor College of
Medicine, Houston, TX, United States, 18Human Genome
Sequencing Center, Baylor College of Medicine, Houston, TX,
United States, 19Department of Molecular Physiology and
Biophysics, Baylor College of Medicine, Houston, TX, United
States, 20Texas Children's Hospital, Houston, TX, United
States, 21Norwegian National Unit for Newborn Screening,
Oslo University Hospital, Oslo, Norway, 22Institute of Clinical
Medicine, University of Oslo, Oslo, Norway, 23Center for
Individualized Medicine, Mayo Clinic, Rochester, MN, United
States, 24Department of Laboratory Medicine and Pathology,
Mayo Clinic, Rochester, MN, United States, 25Division of
Neurogenetics and Hugo W. Moser Research Institute,
Kennedy Krieger Institute, Baltimore, MD, United States,
26Department of Genome Sciences, University of Washington
School of Medicine, Seattle, WA, United States, 27Department
of Pharmacology, Creighton University Medical School,
Omaha, NE, United States, 28Yorkshire Regional Genetics
Service, Chapel Allerton Hospital, Leeds Teaching Hospitals
NHS Trust, Leeds, United Kingdom, 29Department of Genetics,
University of North Carolina School of Medicine, Chapel Hill,
NC, United States, 30Department of Genetics, University of
Groningen, University Medical Center Groningen, Groningen,
Netherlands, 31Division of Medical Genetics, Department of
Pediatrics, Duke University Medical Center, Durham, NC,
United States, 32Institute for Genomic Medicine, Columbia
University Medical Center, New York, NY, United States,
33Department of Medicine, Royal College of Surgeons in
Ireland, St Stephen's Green, Dublin, Ireland, 34Division of
Pediatric Neurology, University of Alberta, Edmonton, AB, UNC13A, GRIA4, PUM2, TOP1, WDFY3, NPTX1 and
SPEN. Scientific yield of clinical exome sequencing of
neurodevelopmental disorders Scientific yield of clinical exome sequencing of
neurodevelopmental disorders Scientific yield of clinical exome sequencing of
neurodevelopmental disorders Results: A reliable clinical genetic diagnosis was made in
60 (29.5%) trios, corresponding to 20% in cases preceded
by panel diagnostics and 43% in cases without prescreen-
ing. In the remaining 143 (70.5%) trios, re-evaluation
revealed potentially causative variants in 146 candidate
genes. We applied our scoring system to prioritize the
relevance of these variants. The scores varied between 2 and
11.9. The top 10% of the scores (score > 9) are ASIC1,
TANC2, FBXL19, KMT2E, GLS, ACTL6B, GRIN3B, CUX1, B. Büttner1, S. Martin1, I. Krey1, D. Le Duc1, T. Bartolomaeus1,
H. Constanze1, D. Huhle2, W. Kiess3, A. Merkenschlager4,
M. Bernhard3, R. Pfäffle3, F. Hornemann3, D. Wieczorek5,
S. Hoffjan6, Y. Hellenbroich7, A. Kuechler8, M. Elgizouli8,
S. Syrbe9, J. U. Schlump10, J. Schumacher11, A. Rolfs12,
S. Biskup13, N. Di Donato14, A. Tzschach14, Y. Schmitz15,
S. Leye15, R. Ewald15, I. Schanze16, M. Zenker16, P. Muschke16, B. Büttner1, S. Martin1, I. Krey1, D. Le Duc1, T. Bartolomaeus1,
H. Constanze1, D. Huhle2, W. Kiess3, A. Merkenschlager4,
M. Bernhard3, R. Pfäffle3, F. Hornemann3, D. Wieczorek5,
S. Hoffjan6, Y. Hellenbroich7, A. Kuechler8, M. Elgizouli8,
S. Syrbe9, J. U. Schlump10, J. Schumacher11, A. Rolfs12,
S. Biskup13, N. Di Donato14, A. Tzschach14, Y. Schmitz15,
S. Leye15, R. Ewald15, I. Schanze16, M. Zenker16, P. Scientific yield of clinical exome sequencing of
neurodevelopmental disorders Conclusion: For the majority of the candidates with
highest
scoring
variants
additional
cases
have
been
identified through our network of collaborators. This
confirms disease associations and enables spin-off studies. Our results illustrate the enormous scientific value of the re-
evaluation of the substantial amount of negative exome
sequencing samples. B. Büttner: None. S. Martin: None. I. Krey: None. D. Le Duc: None. T. Bartolomaeus: None. H. Constanze:
None. D. Huhle: None. W. Kiess: None. A. Merkens-
chlager: None. M. Bernhard: None. R. Pfäffle: None. F. Hornemann: None. D. Wieczorek: None. S. Hoffjan:
None. Y. Hellenbroich: None. A. Kuechler: None. M. Elgizouli: None. S. Syrbe: None. J.U. Schlump: None. J. Schumacher: None. A. Rolfs: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Centogene, Rostock, Germany. S. Biskup: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; CeGaT. N. Di Donato:
None. A. Tzschach: None. Y. Schmitz: A. Employment
(full or part-time); Significant; Limbus. S. Leye: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Limbus. R. Ewald: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Limbus. I. Schanze:
None. M. Zenker: None. P. Muschke: None. B. Liesfeld:
E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Limbus. T. Polster:
None. D. Mitter: None. K. Platzer: None. J. Hentschel:
None. J. Lemke: None. R. Jamra: None. 1CHU Nantes, Service de Génétique Médicale, Nantes, France,
2l’institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes,
France, 3Centre de Recherche du CHU Sainte-Justine et B. Cogne1,2, E. Beauregard-Lacroix3, J. Rousseau3,
S. Ehresmann3, T. Garcia3, C. Gordon4,5, C. von der Lippe6,
C. Skraban7,8, J. Johnston9, A. Lehman10, P. Parent11,
B. Gilbert-Dussardier12,13, K. McWalter14, M. T. Cho15,
U. Kini16, Z. Coban Akdemir17, J. Punetha17, S. Jhangiani18,
X. Song17, D. A. Scott19,17,20, A. Stray-Pedersen21,22,
P. Blackburn23,24, J. S. Cohen25, H. Stessman26,27, M. Blyth28,
J. Berg29, E. Gerkes30, V. Shashi31, J. Sullivan31, D. B.
Goldstein32,33, R. Redon2, J. R. Lupski17,20,18, F. Bolduc34,35,36,
Deciphering Developmental Disorder study, TRRAP consortium,
S. Bezieau1,2, S. Kury1,2, P. M. Campeau3 Center for Medical Genetics Ghent (CMGG), Ghent, Belgium Introduction:
The
implementation
of
whole
exome
sequencing (WES) in the clinic has rapidly increased the
diagnostic yield in patients with (syndromic) intellectual
disability (ID) and/or epilepsy. Here we present an over-
view of the first 109 patients that were analyzed with our
accredited ID & Epilepsy panel, a WES based targeted
panel analysis. Introduction:
The
implementation
of
whole
exome
sequencing (WES) in the clinic has rapidly increased the
diagnostic yield in patients with (syndromic) intellectual
disability (ID) and/or epilepsy. Here we present an over-
view of the first 109 patients that were analyzed with our
accredited ID & Epilepsy panel, a WES based targeted
panel analysis. Material
and
Methods:
gDNA
was
enriched
for
(coding) exons with the Sureselect All Exon v6 kit (Agilent
Technologies) followed by paired-end 2x150bp sequencing
on a HiSeq3000 platform (Illumina). Raw sequence reads
were processed using an in-house developed pipeline
(Seqplorer) and data analysis was limited to a panel of
1109 selected Intellectual Disability & Epilepsy genes. Results: We’ve found a possible molecular explanation
for the aberrant phenotype of the patient in 23% of the
cases. The majority of the (likely) pathogenic variants arose
de novo (71%), few cases could be explained by variants in
genes related to respectively X-linked recessive disorders
(17%) or autosomal recessive disorders (8%). Several
interesting findings were observed, for example, we’ve
identified de novo SATB2 missense variants in two
unrelated patients affecting the same codon but different
nucleotide. B. Cogne: None. E. Beauregard-Lacroix: None. J. Rousseau: None. S. Ehresmann: None. T. Garcia: None. B. Cogne: None. E. Beauregard-Lacroix: None. J. Rousseau: None. S. Ehresmann: None. T. Garcia: None. C. Gordon: None. C. von der Lippe: None. C. Skraban:
None. J. Johnston: None. A. Lehman: None. P. Parent:
None. B. Gilbert-Dussardier: None. K. McWalter: A. Employment (full or part-time); Significant; GeneDx. M.T. Cho: A. Employment (full or part-time); Significant;
GeneDx. U. Kini: None. Z. Coban Akdemir: None. J. Punetha: None. S. Jhangiani: None. X. Song: None. D.A. Scott: None. A. Stray-Pedersen: None. P. Blackburn:
None. J.S. Cohen: None. H. Stessman: None. M. Blyth:
None. J. Berg: None. E. Gerkes: None. V. Shashi: None. J. Sullivan: None. D.B. Goldstein: None. R. Redon:
None. J.R. Lupski: E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest;
23andMe,
Lasergen. F. Modest; Regeneron. F. Bolduc: None. S. Bezieau: None.
S. Kury: None. P.M. Campeau: None. Modest; Regeneron. F. Bolduc: None. S. Bezieau: None. S. Kury: None. P.M. Campeau: None. of Medical Genetics, University of Alberta, Edmonton, AB,
Canada P08.75C Genotype-phenotype correlation associated with de novo
missense variants in TRRAP: from autism spectrum
disorder to syndromic intellectual disability B. Cogne1,2, E. Beauregard-Lacroix3, J. Rousseau3,
S. Ehresmann3, T. Garcia3, C. Gordon4,5, C. von der Lippe6,
C. Skraban7,8, J. Johnston9, A. Lehman10, P. Parent11,
B. Gilbert-Dussardier12,13, K. McWalter14, M. T. Cho15,
U. Kini16, Z. Coban Akdemir17, J. Punetha17, S. Jhangiani18,
X. Song17, D. A. Scott19,17,20, A. Stray-Pedersen21,22,
P. Blackburn23,24, J. S. Cohen25, H. Stessman26,27, M. Blyth28,
J. Berg29, E. Gerkes30, V. Shashi31, J. Sullivan31, D. B. Goldstein32,33, R. Redon2, J. R. Lupski17,20,18, F. Bolduc34,35,36,
Deciphering Developmental Disorder study, TRRAP consortium,
S. Bezieau1,2, S. Kury1,2, P. M. Campeau3 246 J. del Picchia Center for Medical Genetics Ghent (CMGG), Ghent, Belgium Consultant/Advisory
Board; Conclusion: To conclude, since the introduction of whole
exome sequencing in the Center for Medical Genetics
Ghent, we were able to provide a possible molecular
diagnosis (that could explain the clinical features) for a high
percentage (23%) of patients. This is in concordance with
previous reports in literature with a diagnostic yield ranging
from 16-29.4% (de Ligt, 2012 and Monroe GR, 2016). B. Cogne: None. E. Beauregard-Lacroix: None. J. Rousseau: None. S. Ehresmann: None. T. Garcia: None. C. Gordon: None. C. von der Lippe: None. C. Skraban:
None. J. Johnston: None. A. Lehman: None. P. Parent:
None. B. Gilbert-Dussardier: None. K. McWalter: A. Employment (full or part-time); Significant; GeneDx. M.T. Cho: A. Employment (full or part-time); Significant;
GeneDx. U. Kini: None. Z. Coban Akdemir: None. J. Punetha: None. S. Jhangiani: None. X. Song: None. D.A. Scott: None. A. Stray-Pedersen: None. P. Blackburn:
None. J.S. Cohen: None. H. Stessman: None. M. Blyth:
None. J. Berg: None. E. Gerkes: None. V. Shashi: None. J. Sullivan: None. D.B. Goldstein: None. R. Redon:
None. J.R. Lupski: E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest;
23andMe,
Lasergen. F. Consultant/Advisory
Board; S. Vergult: None. E. D'haenens: None. M. Baetens:
None. F. Coppieters: None. M. De Smet: None. A. Dheedene: None. T. Rosseel: None. T. Sante: None. B. Menten: None. P08.76D Acetylation of histone lysine residues is a major component
of transcriptional regulation. This tightly regulated reaction
is performed by histone acetyltransferases (HATs). The
recruitment and activity of HATs depend on a multiprotein
complex, that includes cofactors and a large scaffolding
protein of 3859 amino acids called TRansformation/tRan-
scription domain-Associated Protein (TRRAP). Through an
international collaboration and by use of GeneMatcher, 15
de novo variants in TRRAP (NM_001244580.1) were
identified from research and clinical exome sequencing
cohorts, in 21 patients from 20 families (1 germinal
mosaicism). All variants were absent from gnomAD. Remarkably, a strong genotype to phenotype correlation
was observed with two clinical spectra. The first is a com-
plex, multi-systemic syndrome characterized by severe
intellectual disability (ID) in addition to various mal-
formations of the brain, heart, kidneys and genitourinary
system. Patients with this phenotype carried missense var-
iants
(I1031M, R1035Q, S1037R, E1104G, E1106K,
G1111W, G1159R) that clustered around a substitution
identified in 5 individuals (A1043T). The second pre-
sentation includes individuals with autism spectrum dis-
order and/or ID and epilepsy, with another cluster of
variants including R1859C, W1866C, W1866R, G1883R
and P1932L as well as non-clustering variants (L805F and
F860L). TRRAP is highly conserved evolutionarily and is
among the top five genes intolerant to missense variations. RNAseq on patient cell lines identified several hundred
misregulated genes; with an enrichment in genes involved
in neuronal development and axonal guidance, develop-
mental processes, cell-cell adhesion and motility. qRT-
PCR, ATACseq and Immuno-histochemistry experiments
are ongoing to confirm the results and gain further insight
into pathophysiology. P08.76D
Analysis of whole exome sequencing data with a panel of
genes associated with intellectual disability and epilepsy in
a diagnostic lab P08.76D
Analysis of whole exome sequencing data with a panel of
genes associated with intellectual disability and epilepsy in
a diagnostic lab S. Vergult, E. D'haenens, M. Baetens, F. Coppieters, M. De
Smet, A. Dheedene, T. Rosseel, T. Sante, B. Menten Center for Medical Genetics Ghent (CMGG), Ghent, Belgium Center for Medical Genetics Ghent (CMGG), Ghent, Belgium The phenotypic spectrum of WWOX-related Epileptic
Encephalopathy: 20 additional cases and review of the
literature P08.77A The phenotypic spectrum of WWOX-related Epileptic
Encephalopathy: 20 additional cases and review of the
literature Abstracts from the 51st European Society of Human Genetics Conference: Posters 247 J. Piard1, L. Hawkes2, M. Milh3, L. Villard3, R. Borgatti4,
M. Fradin5, Y. Capri6, D. Héron7, M. Nougues8, C. Nava9,
O. Tarta Arsene10, D. Shears11, Y. Sogawa12, D. Johnson13,
H. Firth14, P. Vasudevan15, G. Jones15, M. Nguyen-Morel16,
T. Busa17, A. Roubertie18, M. van den Born19, M. Koenig20,
E. Brischoux-Boucher21, C. Mignot7, U. Kini2, C. Philippe22 syndrome). We report on 20 additional patients with
WOREE syndrome. syndrome). We report on 20 additional patients with
WOREE syndrome. SNVs and CNVs were identified by means of pange-
nomic approaches (WES and aCGH) and/or WWOX
targeted molecular screening. All missenses identified
(included eight novel mutations) are classified as pathogenic
or likely pathogenic according to the ACMG recommenda-
tions. The phenotype of our patients was consistent with
previously reported cases. All individuals had severe
developmental delay (inability to walk and no speech
development) and early onset epilepsy. In contrast to
previous reports, growth retardation (6%) and microcephaly
(20%) were not prominent signs in our series. The most
striking dysmorphic features were a round hypotonic face
with full cheeks and a short neck. Additional medical
problems were visual impairment (75%), spine deformity
(65%), feeding (70%) and respiratory (40%) problems. Brain MRI was abnormal in 80% of patients showing
corpus callosum hypoplasia (75%) and progressive cerebral
atrophy (55%). By aggregating our patients with all cases
reported so far, there are currently 37 patients with a
WOREE syndrome. No clear genotype-phenotype correla-
tion is emerging. It was initially claimed that homozygous
or compound heterozygous missense(s) genotypes could
lead to a SCAR12 phenotype. In our cohort, we describe 5
patients
with
missense(s)
genotypes
and
a
WOREE
syndrome. The most severe clinical presentation seems to
be associated with genotypes corresponding to virtual
WWOX full knock-down. P08.77A Shears: None. Y. Sogawa: None. D. Johnson: None. H. Firth: None. P. Vasudevan: None. G. Jones: None. M. Nguyen-Morel:
None. T. Busa: None. A. Roubertie: None. M. van den
Born: None. M. Koenig: None. E. Brischoux-Boucher:
None. C. Mignot:
None. U. Kini:
None. C. Philippe: None. J. Piard: None. L. Hawkes: None. M. Milh: None. L. Villard: None. R. Borgatti: None. M. Fradin: None. Y. Capri: None. D. Héron: None. M. Nougues: None. C. Nava: None. O. Tarta Arsene: None. D. Shears: None. Y. Sogawa: None. D. Johnson: None. H. Firth: None. P. Vasudevan: None. G. Jones: None. M. Nguyen-Morel:
None. T. Busa: None. A. Roubertie: None. M. van den
Born: None. M. Koenig: None. E. Brischoux-Boucher:
None. C. Mignot:
None. U. Kini:
None. C. Philippe: None. T. Besnard1,2, F. Ebstein3, L. Fuqua4, M. Lefèbvre5,6,
B. Cogné1,2, X. Latypova1,2, W. Deb1, D. Quinquis1, F. Bolduc7,8,
S. Nambot5,6, M. Hempel9, D. Li10, F. Tran Mau-Them5,6, K. W.
Gripp11, E. Infante12, M. McGuire12,13, T. Smol14,15,
J. Ghoumid14,16, T. Maarup17, E. Calonico17, L. L. Immken18,
D. Martin-Coignard19, S. Yang20, M. Cho20, D. Lessel9,
C. Thauvin-Robinet5,6, L. Faivre5,6, B. Isidor1,2, S. Küry1,2,
S. Bézieau1,2 P08.77A 1Centre de Génétique Humaine, Université de Franche-Comté,
Besançon, France, 2Oxford Centre for Genomic Medicine,
Oxford University Hospitals NHS Foundation Trust, Oxford,
United Kingdom, 3UMR_S 910 - Equipe de Neurogénétique
Humaine, Marseille, France, 4Unità Operativa Complessa di
Neuropsichiatria Età Evolutiva Neuroriabilitazione, Bosisio
Parini, Italy, 5Service de Pédiatrie, St Brieuc, France, 6Service
de Génétique, Hôpital Robert Debré, Paris, France, 7Departement de Genetique et Centre de Reference «
Deficiences intellectuelles de causes rares », APHP, Groupe
Hospitalier Pitie-Salpêtriere, Paris, France, 8Neuropédiatrie et
Unité d'électrophysiologie clinique, Centre de Référence des
Maladies Neuromusculaires de l'EST parisien et DHU I2B,
Hôpital d'Enfants Armand Trousseau, Paris, France,
9INSERM, U 1127, CNRS UMR 7225, Sorbonne Universités,
UPMC Univ Paris, Institut du Cerveau et de la Moelle
épinière, Paris, France, 10Pediatric Neurology Clinic,
"Alexandru Obregia" Clinical Psychiatric Hospital, "Carol
Davila" University of Medicine, Bucharest, Romania, 112. Oxford Centre for Genomic Medicine, Oxford University
Hospitals NHS Foundation Trust, Oxford, United Kingdom,
12Division of Pediatric Neurology, Children's Hospital of
Pittsburgh, Pittsburgh, PA, United States, 13Clinical Genetics,
Sheffield Children’s Hospital, Sheffield, United Kingdom,
14Department of Clinical Genetics, Cambridge University
Hospitals, NHS Foundation Trust, Addenbrooke’s Hospital,
Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, United Kingdom, 15Clinical
Genetics Department, University Hospitals Leicester NHS
Trust, Leicester, United Kingdom, 16Pediatric department,
Grenoble -Alp university hospital, Grenoble, France,
17Département de Génétique, Hôpital Timone, Marseille,
France, 18Service de Neuropédiatrie, Centre Hospitalier
Universitaire de Montpellier, Montpellier, France,
19Department for Clinical Genetics, Erasmus MC, 3000,
Rotterdam, Netherlands, 20Laboratoire de Génétique
Moléculaire, EA7402 Institut Universitaire de Recherche
Clinique, Montpellier, France, 21Centre de génétique
Humaine, Besançon, France, 2221. Laboratoire de génétique
moléculaire et de Cytogénétique, Plateau Technique de
Biologie, CHU de Dijon et Université de Bourgogne, Dijon,
France 7Departement de Genetique et Centre de Reference «
Deficiences intellectuelles de causes rares », APHP, Groupe
Hospitalier Pitie-Salpêtriere, Paris, France, 8Neuropédiatrie et
Unité d'électrophysiologie clinique, Centre de Référence des
Maladies Neuromusculaires de l'EST parisien et DHU I2B,
Hôpital d'Enfants Armand Trousseau, Paris, France, 7Departement de Genetique et Centre de Reference «
Deficiences intellectuelles de causes rares », APHP, Groupe
Hospitalier Pitie-Salpêtriere, Paris, France, 8Neuropédiatrie et
Unité d'électrophysiologie clinique, Centre de Référence des
Maladies Neuromusculaires de l'EST parisien et DHU I2B,
Hôpital d'Enfants Armand Trousseau, Paris, France, J. Piard: None. L. Hawkes: None. M. Milh: None. L. Villard: None. R. Borgatti: None. M. Fradin: None. Y. Capri: None. D. Héron: None. M. Nougues: None. C. Nava: None. O. Tarta Arsene: None. D. P08.78B Ubiquitin-proteasome system impairment and intellectual
disability: the CUL4B example P09.001A
Dissecting tissue-specific functional networks associated
with 16p11.2 reciprocal genomic disorder using CRISPR
engineered human iPS and mouse models P. Razaz1,2,3, D. J. Tai1,2,3, S. Erdin1,2,3, T. Aneichyk1,2,3,
T. Arbogast4, A. Ragavendran1, A. Stortchevoi1,2, B. B. Currall1,2,3, C. E. F. Esch1,2,3, E. Morini1,2, W. Ma1,2, R. J. Kelleher1,2, C. Golzio4,5, N. Katsanis4, J. F. Gusella1,2,3,6,
M. Talkowski1,2,3,6 Introduction: Pathogenic variants in the CUL4B gene are
responsible for the clinical phenotype of CUL4B-related X-
linked intellectual disability (ID) or Cabezas syndrome
(MIM #300354). CUL4B encodes a scaffold protein, the
cullin-4B which is enrolled in the Cullin4B-RING ubiquitin
ligase (E3) complex. This complex plays an essential role in
the recognition and ubiquitination of proteins which occurs
prior to their cellular degradation by the 26S proteasome. 1Center for Genomic Medicine and Department of Neurology,
Massachusetts General Hospital, BOSTON, MA, United States,
2Department of Neurology, Harvard Medical School, Boston,
MA, United States, 3Program in Medical and Population
Genetics, Broad Institute of MIT and Harvard, Cambridge,
MA, United States, 4Center for Human Disease Modeling,
Duke University Medical Center, Durham, NC, United States,
5Institut de Génétique et de Biologie Moléculaire et Cellulaire,
Illkirch, France, 6Department of Genetics, Harvard Medical
School, Boston, MA, United States Materials and Methods: Through an international multi-
centric investigation based on exome sequencing, we
identified 15 male individuals with unpublished CUL4B
pathogenic variants. In order to determine if the ubiquitina-
tion/protein system (UPS) was affected by these CUL4B
alterations, we performed protein analysis by native SDS-
PAGE and western-blot on peripheral blood mononuclear
cells (PBMCs) from subjects and healthy controls. Materials and Methods: Through an international multi-
centric investigation based on exome sequencing, we
identified 15 male individuals with unpublished CUL4B
pathogenic variants. In order to determine if the ubiquitina-
tion/protein system (UPS) was affected by these CUL4B
alterations, we performed protein analysis by native SDS-
PAGE and western-blot on peripheral blood mononuclear
cells (PBMCs) from subjects and healthy controls. Results: We identified 9 frameshift indels, 1 in-frame
indel; 4 nonsense and 1 missense variants. All affected
individuals presented with mild to severe ID with speech
and motor delay, along with more variable neurological,
skeletal and dysmorphic features. Interestingly, the 20S and
26S proteasome complexes of the tested
individuals
exhibited a higher chymotrypsin-like activity than those of
the controls. This finding was confirmed by measuring the Reciprocal genomic disorders (RGDs) represent a recurrent
class of copy number variants (CNVs) that collectively
comprise a major contributor to neurodevelopmental dis-
orders (NDD) and altered anthropometric traits. Ubiquitin-proteasome system impairment and intellectual
disability: the CUL4B example Ubiquitin-proteasome system impairment and intellectual
disability: the CUL4B example T. Besnard1,2, F. Ebstein3, L. Fuqua4, M. Lefèbvre5,6,
B. Cogné1,2, X. Latypova1,2, W. Deb1, D. Quinquis1, F. Bolduc7,8,
S. Nambot5,6, M. Hempel9, D. Li10, F. Tran Mau-Them5,6, K. W. Gripp11, E. Infante12, M. McGuire12,13, T. Smol14,15,
J. Ghoumid14,16, T. Maarup17, E. Calonico17, L. L. Immken18,
D. Martin-Coignard19, S. Yang20, M. Cho20, D. Lessel9,
C. Thauvin-Robinet5,6, L. Faivre5,6, B. Isidor1,2, S. Küry1,2,
S. Bézieau1,2 Germline bi-allelic mutations in WWOX have been asso-
ciated with spinocerebellar ataxia type 12 (SCAR12) and
WWOX-Related-Epileptic-Encephalopathy
(WOREE 248 J. del Picchia 1CHU de Nantes, Service de Génétique Médicale, Nantes,
France, 2L'institut du thorax, INSERM, CNRS, UNIV Nantes,
Nantes, France, 3Institute of Medical Biochemistry and
Molecular Biology, University Medicine Greifswald,
Greifswald, Germany, 4Lineagen, Salt Lake City, UT, United
States, 5Inserm UMR 1231 GAD, Génétique des Anomalies du
Développement, Dijon, France, 6Centre de référence
«Anomalies du Développement et Syndromes Malformatifs»,
Centre de Génétique, Hôpital d’Enfants, CHU Dijon,
Université de Bourgogne, Dijon, France, 7Neuroscience and
Mental Health Institute, University of Alberta, Edmonton, AB,
Canada, 8Department of Medical Genetics, University of
Alberta, Edmonton, AB, Canada, 9Institute of Human Genetics,
University Medical Center Hamburg-Eppendorf, Hamburg,
Germany, 10Center for Applied Genomics, The Children’s
Hospital of Philadelphia, Philadelphia, PA, United States,
11Division of Medical Genetics, Nemours/Alfred I. duPont
Hospital for Children, Wilmington, DE, United States,
12Children's Hospital of Pittsburgh of UPMC, University of
Pittsburgh, Pittsburgh, PA, United States, 13Department of
Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX, United States, 14Université de Lille, EA7364
RADEME, Lille, France, 15Institut de génétique médicale,
CHU Lille, Lille, France, 16Service de génétique clinique, CHU
Lille, Lille, France, 17Kaiser Permanente, Los Angeles, CA,
United States, 18Specially For Children Genetics, Austin, TX,
United States, 19Unité de Génétique, CHU Le Mans, Le Mans,
France, 20GeneDX, Gaithersburg, MD, United States degradation rate of the Suc-LLVY-AMC substrate in
whole-cell extracts. degradation rate of the Suc-LLVY-AMC substrate in
whole-cell extracts. Conclusions: Our initial results suggest a direct implica-
tion of CUL4B in the regulating function of proteasome
26S. CUL4B could indirectly contribute to the maintenance
of cellular protein homeostasis. However, the mechanisms
through which these interactions are exercised and their
consequences, particularly in the pathological context of
intellectual impairment, still remain to be clarified. T. Besnard: None. F. Ebstein: None. L. Fuqua: None. M. Lefèbvre: None. B. Cogné: None. X. Latypova: None. W. Deb: None. D. Quinquis: None. F. Bolduc: None. S. Ubiquitin-proteasome system impairment and intellectual
disability: the CUL4B example Nambot: None. M. Hempel: None. D. Li: None. F. Tran
Mau-Them: None. K.W. Gripp: None. E. Infante: None. M. McGuire: None. T. Smol: None. J. Ghoumid: None. T. Maarup: None. E. Calonico: None. L.L. Immken:
None. D. Martin-Coignard: None. S. Yang: None. M. Cho: None. D. Lessel: None. C. Thauvin-Robinet: None. L. Faivre: None. B. Isidor: None. S. Küry: None. S. Bézieau: None. P09 Neurogenetic and psychiatric disorders P09.001A
Dissecting tissue-specific functional networks associated
with 16p11.2 reciprocal genomic disorder using CRISPR
engineered human iPS and mouse models P09.001A
Dissecting tissue-specific functional networks associated
with 16p11.2 reciprocal genomic disorder using CRISPR
engineered human iPS and mouse models Here, we
systematically dissected the functional networks associated
with 16p11.2 RGD from transcriptome analyses of 70 mice
with reciprocal CNV of the syntenic 7qF3 region across Abstracts from the 51st European Society of Human Genetics Conference: Posters 249 cortex, striatum, and cerebellum, as well as liver, white and
brown adipose tissues in a subset of 16 mice (n =
250 samples). We integrated these data with brain tissues
from a Kctd13 mouse model (a putative driver of 16p11.2
neuroanatomical phenotypes, n = 50), and CRISPR-engi-
neered, isogenic 16p11.2 iPSC-derived NSCs (n = 25) and
induced neurons (n = 27). The strongest magnitude of effect
sizes from 7qF3 were observed across brain regions by
comparison to non-brain tissues (cortex 7qF3 region aver-
age p-value = 8.80E-35; non-brain p = 0.0013), reflecting
the ~3x higher basal expression changes. Coexpression
network analyses isolated a consistent module of 16p11.2
genes, as well as a module that was highly enriched for
constrained
genes
(ExAC
pLI≥0.9),
autism-associated
genes, early fetal development coexpression networks
derived from BrainSpan, and neurological phenotypes and
processes. Differentially expressed genes (DEGs) were
enriched in this ‘constrained’ module network; moreover,
DEGs from the Kctd13 mouse coalesced into this same
module
(cortex
enrichment
p = 7.82E-41),
suggesting
overlap in altered transcriptional networks between full
length CNV and deletion of KCTD13 alone. These analyses
identify a tissue-specific impact of 16p11.2 RGD that
converges on a module of co-expressed genes that are
intolerant to genetic perturbation and associated with critical
processes in human neurodevelopment. Sciences, Faculty of Biology, Medicine and Health, University
of Manchester, Manchester Academic Health Science Centre,
Manchester, United Kingdom, 8INSERM UMR-1163,
Laboratory of Neurogenetics and Neuroinflammation, Paris,
France, 9Paris Descartes University, Sorbonne-Paris-Cité,
Imagine Institute, Paris, France Aicardi-Goutières syndrome (AGS) is a genetically deter-
mined inflammatory encephalopathy characterized by a
resemblance to congenital infection. Early onset in child-
hood is often observed, but variability in presentation and
course is recognized, encompassing peripheral spasticity to
severe psychomotor disability. To date, mutations in seven
genes have been described to cause AGS. RNASEH2B gene
(MIM# 610326), encoding the RibonucleaseH2 beta sub-
unit, is most frequently mutated, seen in association with
~40% of diagnosed patients. Here we report a new case of
AGS in a 4 year old girl who, after an infectious gastro-
enteritis, developed a left spastic hemiparesis with pre-
dominant dystonia of the upper limb, in association with
white matter abnormalities on brain MRI. P09.001A
Dissecting tissue-specific functional networks associated
with 16p11.2 reciprocal genomic disorder using CRISPR
engineered human iPS and mouse models Biochemical
investigation revealed high neopterin and slightly elevated
interferon (IFN)-α levels in the cerebrospinal fluid, with a
variable elevation of IFN-regulated-gene transcripts in the
peripheral blood. By targeted next generation sequencing
performed on genomic DNA, we identified a hitherto
unreported RNASEH2B intronic variant, c.699-9C>G, in the
compound heterozygous state with the recurrent mutation
c.529G>A (p.Ala177Thr). Sanger sequencing confirmed
biallelic segregation consistent with autosomal recessive
inheritance. The study of RNASEH2B transcript expression
in leucocytes by RT-PCR in the proband, and her healthy
heterozygous carrier mother, demonstrated that the novel
variant creates a new 3’ acceptor splice site upstream of the
boundary of exon 9, inducing the retention of an intronic
fragment in the mature mRNA, with predicted loss of the
STOP-codon. An investigation of the impact of this variant
on RNASEH2B transcript stability and translated protein is
in progress. P. Razaz: None. D.J. Tai: None. S. Erdin: None. T. Aneichyk: None. T. Arbogast: None. A. Ragavendran:
None. A. Stortchevoi: None. B.B. Currall: None. C.E.F. Esch: None. E. Morini: None. W. Ma: None. R.J. Kelleher: None. C. Golzio: None. N. Katsanis: None. J. F. Gusella: None. M. Talkowski: None. Molecular characterization of a novel RNASEH2B splice
site mutation responsible for Aicardi-Goutieres syndrome Molecular characterization of a novel RNASEH2B splice
site mutation responsible for Aicardi-Goutieres syndrome S. Samaan1,2, S. Valence3,4, F. Renaldo5, C. Maalouf1,
I. Dorboz2, K. Boussaid5, M. Elmaleh6, G. Rice7, O. Boespflug-
Tanguy2,5, Y. Crow8,9, D. Rodriguez3,4 S. Samaan1,2, S. Valence3,4, F. Renaldo5, C. Maalouf1,
I. Dorboz2, K. Boussaid5, M. Elmaleh6, G. Rice7, O. Boespflug-
Tanguy2,5, Y. Crow8,9, D. Rodriguez3,4 S. Samaan1,2, S. Valence3,4, F. Renaldo5, C. Maalouf1,
I. Dorboz2, K. Boussaid5, M. Elmaleh6, G. Rice7, O. Boespflug-
Tanguy2,5, Y. Crow8,9, D. Rodriguez3,4 S. Samaan: None. S. Valence: None. F. Renaldo: None. C. Maalouf: None. I. Dorboz: None. K. Boussaid: None. M. Elmaleh: None. G. Rice: None. O. Boespflug-Tanguy:
None. Y. Crow: None. D. Rodriguez: None. 1Department of Medical Genetics, UF Molecular Genetics,
Robert Debre University Hospital APHP, 75019, Paris,
France, 2INSERM UMR-S1141, DHU PROTECT, Robert
Debre University Hospital APHP, 75019, Paris, France,
3Department of Neuropaediatrics, Armand Trousseau
University Hospital, APHP, 75012, Paris, France, 4GRC
ConCer-LD, Sorbonne Universités, UPMC-Paris 6 University,
Paris, France, 5Department of Neuropaediatric and metabolic
diseases, Robert Debre University Hospital, APHP, 75019,
Paris, France, 6Department of Radiology, Robert Debre
University Hospital, APHP, 75019, Paris, France, 7Division of
Evolution and Genomic Sciences, School of Biological M. Elmaleh: None. G. Rice: None. O. Boespflug-Tanguy:
None. Y. Crow: None. D. Rodriguez: None. P09.003C Molecular characterization of a novel RNASEH2B splice
site mutation responsible for Aicardi-Goutieres syndrome P09.004D A whole exome study of Alzheimer’s Diseases which is
augmented by population data found the noble AD risk
genes A whole exome study of Alzheimer’s Diseases which is
augmented by population data found the noble AD risk
genes J. Kim Ilsan Hospital, Goyang-shi, Korea, Republic of 250 J. del Picchia Alzheimer disease (AD) has high heritability. The AD
Sequencing Project (ADSP) which includes 10,909 parti-
cipants, aimed to find additional AD risk genes harboring
low frequency and rare coding variants. However, ADSP
may be still underpowered for very rare alleles. In this
study, we augmented statistical power by using the ExAC
database as comparison group for the ADSP cases and tried
the noble AD risk and protective genes. In order to avoid
the population bias, we included only Non-Finnish Eur-
opean population in ExAC. In ADSP, we used European
ancestry and excluded outliers by the plot of the first two
principal components. We selected variants with minor
allele frequencies (MAF) between that of the ADSP cases
and ADSP controls. The MAF differences were tested by
Pearson’s chi-square tests. The MAF of gene-based analysis
were obtained by collapsing MAFs of variants within gene
boundaries. In the variant level, the well-known AD genes-
TOMM40, TREM2, and MS4A6A-are replicated. The
noble genes with genome wide significant level (p = 5x10-
8) were 38 genes including C10orf2, CHRD, and CROCC. In the gene-based level using burden test, we selected genes
with
p < 1x10-6. The
well-known
AD
genes-ABCA7,
SORL1, and TREM2-are replicated. The significant noble
genes were 69 genes including AGAP1, ALYREF, and
TYRO3. We not only replicate known AD risk genes-
ABCA7, MS4A6A, SORL1, and TREM2, but also found
the noble candidate AD risk genes. Our augmentation
methods can be applied to the whole exome sequencing
studies on other diseases. P09.004D University Hospital, Department of Neuropathology, F 76000,
Normandy Center for Genomic and Personalized Medicine,
Rouen, France, 6Institute of Genetic Medicine, Newcastle
University, Newcastle Upon Tyne, United Kingdom, 7National
CJD Research & Surveillance Unit, Centre for Clinical Brain
Sciences, University of Edinburgh, Western General Hospital,
Edinburgh, United Kingdom, 8Department of Neuropathology,
John Radcliffe Hospital, Oxford, United Kingdom,
9Department of Basic and Clinical Neuroscience, Institute of
Psychiatry, Psychology and Neuroscience, King’s College
London, London, United Kingdom, 10Department of research,
Rouvray Psychiatric Hospital, Sotteville-lès-Rouen, France,
11Institute of Genetic Medicine, International Centre for Life,
Newcastle University, Newcastle Upon Tyne, United Kingdom,
12Department of Internal Medicine and Radboud Center for
Infectious Diseases (RCI), Radboud University Medical
Center, Nijmegen, Netherlands Introduction: In sporadic Alzheimer disease (sAD), highly
penetrant pathogenic variants have been reported in the
autosomal-dominant genes APP, PSEN1 and PSEN2 in a
minority of early-onset cases (onset before 66 years), some
of them occurring as de novo germline events. Given the
recent knowledge on seeding and spreading of neuro-
pathological lesions in AD, we hypothesized that somatic
variants in these genes may contribute to sAD etiology in a
proportion of patients with no germline pathogenic variant. Methods: We applied an ultra-sensitive technique of
single-molecule molecular inversion probes (smMIP)-based
deep NGS to 100 brain and 355 blood samples from
445 sAD patients from France, the Netherlands and the UK,
including 83.5% early-onset cases. The panel included APP,
PSEN1, PSEN2, genes with a de novo germline mutation in
a trio study (VPS35, MARK4), the risk factor gene SORL1
and 5 genes involved in APP processing. J. Kim: None. P09.005A Steehouwer: None. S. Lelieveld: None. S. Rousseau: None. A. Richard:
None. M. Oud: None. F. Marguet: None. A. Laquerriere:
None. C.M. Morris: None. J. Attems: None. C. Smith: G. Nicolas: None. R. Acuña-Hidalgo: None. M.J. Keogh: None. O. Quenez: None. M. Steehouwer: None. S. Lelieveld: None. S. Rousseau: None. A. Richard:
None. M. Oud: None. F. Marguet: None. A. Laquerriere:
None. C.M. Morris: None. J. Attems: None. C. Smith: 5Normandie Univ, UNIROUEN, Inserm U1245 and Rouen P09.005A Assessing and challenging the somatic variant hypothesis in
sporadic Alzheimer disease G. Nicolas1,2, R. Acuña-Hidalgo2,3, M. J. Keogh4, O. Quenez1,
M. Steehouwer2, S. Lelieveld2, S. Rousseau1, A. Richard1,
M. Oud2, F. Marguet5, A. Laquerriere5, C. M. Morris6,
J. Attems6, C. Smith7, O. Ansorge8, S. Al Sarraj9, T. Frebourg1,
D. Campion1,10, D. Hannequin1, D. Wallon1, C. Gilissen2, P. F. Chinnery4, J. A. Veltman11,2, A. Hoischen2,12 Results: We identified 9 somatic variants in 2 brain and 7
blood samples, with variant allele fractions ranging from
0.2% to 10.8%; 6/9 had a ratio below 0.5% (0.22-0.48%). All nine were confirmed by independent amplicon-based
deep sequencing with similar fractions. Two somatic
variants mapped to APP, although they were interpreted
as likely benign. The other somatic variants were located in
SORL1 (n = 5), NCSTN (n = 1) and MARK4 (n = 1). Two
of the SORL1 variants might have contributed to the disease
while the other variants remain of unknown significance. Results: We identified 9 somatic variants in 2 brain and 7
blood samples, with variant allele fractions ranging from
0.2% to 10.8%; 6/9 had a ratio below 0.5% (0.22-0.48%). All nine were confirmed by independent amplicon-based
deep sequencing with similar fractions. Two somatic
variants mapped to APP, although they were interpreted
as likely benign. The other somatic variants were located in
SORL1 (n = 5), NCSTN (n = 1) and MARK4 (n = 1). Two
of the SORL1 variants might have contributed to the disease
while the other variants remain of unknown significance. 1Normandie Univ, UNIROUEN, Inserm U1245 and Rouen
University Hospital, Department of Genetics and CNR-MAJ, F
76000, Normandy Center for Genomic and Personalized
Medicine, Rouen, France, 2Department of Human Genetics,
Radboud University Medical Center, Nijmegen, Netherlands,
3Max Planck Institute for Molecular Genetics, RG
Development & Disease, Berlin, Germany, 4Department of
Clinical Neurosciences, University of Cambridge, Cambridge
Biomedical Campus & MRC Mitochondrial Biology Unit,
University of Cambridge, Cambridge, United Kingdom,
5Normandie Univ UNIROUEN Inserm U1245 and Rouen Conclusion: Somatic variants in the autosomal dominant
AD genes may not be a common cause of sAD, including
early-onset cases. G. Nicolas: None. R. Acuña-Hidalgo: None. M.J. Keogh: None. O. Quenez: None. M. Steehouwer: None. S. Lelieveld: None. S. Rousseau: None. A. Richard:
None. M. Oud: None. F. Marguet: None. A. Laquerriere:
None. C.M. Morris: None. J. Attems: None. C. Smith: G. Nicolas: None. R. Acuña-Hidalgo: None. M.J. Keogh: None. O. Quenez: None. M. 1Normandie Univ, UNIROUEN, Inserm U1245 and Rouen
University Hospital, Department of Genetics and CNR-MAJ, F
76000, Normandy Center for Genomic and Personalized
Medicine, Rouen, France, 2Department of Human Genetics,
Radboud University Medical Center, Nijmegen, Netherlands,
3Max Planck Institute for Molecular Genetics, RG
Development & Disease, Berlin, Germany, 4Department of
Clinical Neurosciences, University of Cambridge, Cambridge
Biomedical Campus & MRC Mitochondrial Biology Unit,
University of Cambridge, Cambridge, United Kingdom,
5N
di U i
UNIROUEN I
U1245
d R Leukocyte telomere shortening is associated with
progressive cognitive decline in amnestic mild cognitive
impairment and Alzheimer’s disease 1Department of Genetics and CNR-MAJ, Normandie Univ,
UNIROUEN, Inserm U1245 and Rouen University Hospital,
Normandy Centre for Genomic and Personalized Medicine,
Rouen, France, 2Inserm, U1167, RID-AGE - Risk factors and
molecular determinants of aging-related diseases, Lille,
France, 3Institut Pasteur de Lille, Lille, France, 4University
Lille, U1167 - Excellence Laboratory LabEx DISTALZ, Lille,
France, 5University of Bordeaux, Inserm, Bordeaux
Population Health Research Center, UMR1219, Bordeaux,
France, 6Department of Neurology and CNR-MAJ, Normandie
Univ, UNIROUEN, Inserm U1245 and Rouen University
Hospital, Normandy Centre for Genomic and Personalized
Medicine, Rouen, France, Rouen, France, 7Centre National de
Recherche en Génomique Humaine (CNRGH), Institut de
Biologie François Jacob, CEA, Evry, France, 8McGill
University and Génome Québec Innovation Centre, Montréal,
QC, Canada, 9CNR-MAJ, and Department of Neurology,
Université de Lille, CHU, Inserm UMR-S 1171, Lille, France,
10Inserm UMR-1087/CNRS UMR 6291, l’institut du thorax,
Univ. Nantes, Nantes, France, 11Department of Genetics,
Normandie Univ, UNIROUEN, Inserm U1245 and Rouen
University Hospital, Normandy Centre for Genomic and
Personalized Medicine, Rouen, France, 12Department of
Genetics, Neurology and CNR-MAJ, Normandie Univ,
UNIROUEN, Inserm U1245 and Rouen University Hospital,
Normandy Centre for Genomic and Personalized Medicine,
Rouen, France, 13Inserm UMR-1078, CHRU Brest, Univ. Brest, Brest, France, 14Centre Hospitalier Universitaire de
Lille, Epidemiology and Public Health Department, Lille,
France, 15Department of Research, Centre hospitalier du
Rouvray, Sotteville-les-Rouen, France D. Scarabino1, M. Peconi2, E. Broggio3, S. Magliulo2,
G. Gambina3, R. M. Corbo2 D. Scarabino1, M. Peconi2, E. Broggio3, S. Magliulo2,
G. Gambina3, R. M. Corbo2 1CNR Institute of Molecular Biology and Pathology, Rome,
Italy, 2Sapienza University, Rome, Italy, 3Department of
Neuroscience, University and Hospital of Verona, Verona,
Italy Introduction: Numerous studies have reported an associa-
tion between shortened leukocyte telomere length (LTL)
and increased risk of Alzheimer’s disease (AD). In this
study we investigated the relationship between LTL and AD
development, including in the analysis patients with
amnestic mild cognitive impairment (aMCI), a clinical
entity considered prodromal of AD. Materials and Methods: LTL (T/S ratio) was measured
in patients with AD (n = 61) or aMCI (n = 46), and
compared with LTL of age-matched controls (n = 56). Results: significant LTL differences were observed
between controls, aMCI and AD patients (p < 0.0001), with
mean LTL values ( ± s.d) in the order: AD patients (0.70 ±
0.15) < aMCI patients (0.80 ± 0.14) < controls (0.88 ±
0.15). 251 Abstracts from the 51st European Society of Human Genetics Conference: Posters Leukocyte telomere shortening is associated with
progressive cognitive decline in amnestic mild cognitive
impairment and Alzheimer’s disease A positive relationship (linear regression p = 0.004)
was observed between LTL and cognitive performance
(measured by Mini Mental State Examination score). LTL
did not differ by apolipoprotein E (APOE) genotype. Conclusions: The shortened LTL observed in AD
patients appears to stem from progressive telomere erosion
possibly correlated with the cognitive decline characterizing
conversion from aMCI to AD. LTL reduction, indicating
active
cell proliferation, may reflect immune system
involvement in AD pathogenesis. Conclusions: The shortened LTL observed in AD
patients appears to stem from progressive telomere erosion
possibly correlated with the cognitive decline characterizing
conversion from aMCI to AD. LTL reduction, indicating
active
cell proliferation, may reflect immune system
involvement in AD pathogenesis. Introduction: Alzheimer disease (AD) is a complex dis-
order with high genetic component. Associations of rare
variants with AD risk have recently been reported, however
the limited panels of variants under scrutiny or the sample
sizes restricted the possibility to assess their effect fully. Grant 2016 by La Sapienza University of Rome D. Scarabino: None. M. Peconi: None. E. Broggio:
None. S. Magliulo: None. G. Gambina: None. R.M. Corbo: None. D. Scarabino: None. M. Peconi: None. E. Broggio:
None. S. Magliulo: None. G. Gambina: None. R.M. Corbo: None. Materials and Methods: We took advantage from the
ADES-FR dataset to focus on TREM2, SORL1, ABCA7
genes. We generated whole-genome (n-955) or whole-
exome (n = 2,106) sequencing data and performed gene-
based burden association analyses on 927 late-onset AD
(LOAD, onset > 65 years) cases, 852 early-onset (EOAD,
onset ≤65 years) cases and 1,273 controls. Contribution to Alzheimer's disease risk of rare variants in
TREM2, SORL1 and ABCA7 in 1,779 cases and 1,273
controls None. O. Ansorge: None. S. Al Sarraj: None. T.
Frebourg: None. D. Campion: None. D. Hannequin:
None.
D.
Wallon:
None.
C.
Gilissen:
None.
P.F.
Chinnery:
None.
J.A.
Veltman:
None.
A.
Hoischen: None. C. Charbonnier1, C. Bellenguez2,3,4, G. Nicolas1, B. Grenier-
Boley2,3,4, O. Quenez1, K. Le Gennec1, G. Chauhan5, D. Wallon6,
S. Rousseau1, A. Richard1, A. Boland7, G. Bourque8, H. M. Munter8, R. Olaso7, V. Meyer7, A. Rollin-Sillaire9, F. Pasquier9,
L. Letenneur5, R. Redon10, J. Dartigues5, C. Tzourio5,
T. Frebourg11, M. Lathrop8, J. Deleuze7, D. Hannequin6,12,
E. Genin13, P. Amouyel2,3,4,14, S. Debette5, J. Lambert2,3,4,
D. Campion1,15 C. Charbonnier1, C. Bellenguez2,3,4, G. Nicolas1, B. Grenier-
Boley2,3,4, O. Quenez1, K. Le Gennec1, G. Chauhan5, D. Wallon6,
S. Rousseau1, A. Richard1, A. Boland7, G. Bourque8, H. M. Munter8, R. Olaso7, V. Meyer7, A. Rollin-Sillaire9, F. Pasquier9,
L. Letenneur5, R. Redon10, J. Dartigues5, C. Tzourio5,
T. Frebourg11, M. Lathrop8, J. Deleuze7, D. Hannequin6,12,
E. Genin13, P. Amouyel2,3,4,14, S. Debette5, J. Lambert2,3,4,
D. Campion1,15 C. Charbonnier1, C. Bellenguez2,3,4, G. Nicolas1, B. Grenier-
Boley2,3,4, O. Quenez1, K. Le Gennec1, G. Chauhan5, D. Wallon6,
S. Rousseau1, A. Richard1, A. Boland7, G. Bourque8, H. M. Munter8, R. Olaso7, V. Meyer7, A. Rollin-Sillaire9, F. Pasquier9,
L. Letenneur5, R. Redon10, J. Dartigues5, C. Tzourio5,
T. Frebourg11, M. Lathrop8, J. Deleuze7, D. Hannequin6,12,
E. Genin13, P. Amouyel2,3,4,14, S. Debette5, J. Lambert2,3,4,
D. Campion1,15 miR-146a and miR-181a are potential biomarkers for the
progression of mild cognitive impairment to Alzheimer’s
disease miR-146a and miR-181a are potential biomarkers for the
progression of mild cognitive impairment to Alzheimer’s
disease 1University of Athens, School of Medicine, Athens, Greece,
2University of Patras School of Health Sciences, Department of
Pharmacy, Patras, Greece, 3University of Thessaly School of
Medicine, Larissa, Greece, 4University of Thessaly, School of
Medicine, Larissa, Greece, 5Papageorgiou hospital,
Thessaloniki, Greece, 6United Arab Emirates University P09.007C Despite different
effect sizes and varying cumulative MAF, TREM2, SORL1
and ABCA7 rare PT and SD variants contribute similarly to
the heritability of EOAD and explain between 1.1 and 1.5%
of EOAD heritability each, compared with 9.12% for
APOEε4. Conclusion: Beyond a confirmation of the association of
TREM2, SORL1 and ABCA7 rare variants with AD risk,
our study provides a clearer insight into the classes of rare
variants involded, namely SD variants sharing a common
loss of function mechanism with PT variants, and sheds
light on the genetic heterogeneity of AD. Despite different
effect sizes and varying cumulative MAF, TREM2, SORL1
and ABCA7 rare PT and SD variants contribute similarly to
the heritability of EOAD and explain between 1.1 and 1.5%
of EOAD heritability each, compared with 9.12% for
APOEε4. C. Charbonnier: None. C. Bellenguez: None. G. Nicolas: None. B. Grenier-Boley: None. O. Quenez:
None. K. Le Gennec: None. G. Chauhan: None. D. Wallon: None. S. Rousseau: None. A. Richard: None. A. Boland: None. G. Bourque: None. H.M. Munter: None. R. Olaso: None. V. Meyer: None. A. Rollin-Sillaire:
None. F. Pasquier: None. L. Letenneur: None. R. Redon:
None. J. Dartigues:
None. C. Tzourio:
None. T. Frebourg: None. M. Lathrop: None. J. Deleuze: None. D. Hannequin: None. E. Genin: None. P. Amouyel:
None. S. Debette:
None. J. Lambert:
None. D. Campion: None. A. Ansari: None. E. Maffioletti: None. M. Marizzoni:
None. O. Blin: None. G. Frisoni: None. J. Richardson:
None. R. Bordet: None. M. Gennarelli: None. L.B. Chiavetto: None. A. Ansari: None. E. Maffioletti: None. M. Marizzoni:
None. O. Blin: None. G. Frisoni: None. J. Richardson:
None. R. Bordet: None. M. Gennarelli: None. L.B. Chiavetto: None. K. Mitropoulos1, E. Merkouri Papadima2, G. Xiromerisiou3,
A. Balasopoulou2, K. Charalambidou2, V. Galani2, K. Zafeiri2,
E. Dardiotis4, S. Ralli4, G. Deretzi5, A. John6, K. Kydonopoulou7,
E. Papadopoulou7, A. di Pardo8, F. Akcimen9, A. Loizedda10,
V. Dobričić11, I. Novaković11, V. S. Kostić11, C. Mizzi12, B. A.
Peters13, A. N. Basak9, S. Orrù10, E. Kiskinis14, D. N. Cooper15,
S. Gerou7, R. Drmanac13, M. Bartsakoulia12, E. Tsermpini12,
G. M. Hadjigeorgiou4, B. R. Ali16, T. Katsila12, G. P. Patrinos12 1Università degli Studi di Brescia, Brescia, Italy, 2IRCCS
Centro S. Giovanni di Dio Fatebenefratelli, Brescia, Italy, 3Aix
Marseille University, UMR-CNRS 7289, Service de
Pharmacologie Clinique, AP-HM, Marseille, France, 4Memory
Clinic and LANVIE - Laboratory of Neuroimaging of Aging,
University Hospitals and University of Geneva, Geneva,
Swaziland, 5Neurosciences Therapeutic Area,
GlaxoSmithKline R&D, Stevenage, United Kingdom,
6University of Lille, Inserm, CHU Lille, U1171 - Degenerative
and vascular cognitive disorders, F-59000, Lille, France,
73Faculty of Psychology, eCampus University, Novedrate
(Como), Italy P09.007C 252 J. del Picchia Mild cognitive impairment (MCI) is a transitional stage
between normal aging and Alzheimer's disease (AD). Not
all MCI subjects convert to AD but some remain MCI, and
the identification of biomarkers that could give alarm for
dementia development would be of great usefulness in the
clinical practice. MicroRNAs (miRNAs) are small non-
coding RNAs that play a pivotal role in gene expression and
in many neuronal mechanisms going to synaptic plasticity,
neurogenesis, neurodegeneration and apoptosis. In this
study we investigated if baseline blood levels of a set of
candidate miRNAs (miR-22, miR-24-3p, miR-101, miR-
146a, miR-181a, miR-181b, miR-186, miR-339, and miR-
590) may be associated to AD conversion after 2 years in a
group of 45 MCI patients (of whom 19 were converted). Expression level of miR-146a (p = 0. 0.036) and miR-181a
(p = 0. 0.026) showed a significant upregulation in MCI
subjects that converted to AD. A significant negative cor-
relation was found between levels of miR-146a (p = 0.006)
and miR-181a (p = 0.001) in blood and Aβ-42 concentra-
tion in CSF, while no association was evidenced for P-Tau,
and T-Tau. Moreover the increase in miR-146a was asso-
ciated with volume reduction in hippocampus and its sub-
fields (CA1 p = 0.013, subiculum p = 0.027) and increased
levels of miR-146a (p = 0.031) and miR-181a (p = 0.002)
were correlated with diffusivity alterations in the cingulum,
a white matter tract connecting temporal with frontal and
parietal lobes. In conclusion, the data obtained support a
possible usefulness of blood miR-146a and miR-181a levels
as biomarkers for illness progression in MCI patients. Results: When aggregating rare (MAF<1%) protein
truncating (PT) and missense variants predicted damaging
by three bioinformatics tools (strictly damaging, SD),
association with EOAD risk reached exome-wide signifi-
cance (p < 2.5x10-6). Missense variants predicted damaging
by less than three bioinformatics tools were not associated
with EOAD risk. No exome-wide significant signal was
detected in the LOAD sample. Conclusion: Beyond a confirmation of the association of
TREM2, SORL1 and ABCA7 rare variants with AD risk,
our study provides a clearer insight into the classes of rare
variants involded, namely SD variants sharing a common
loss of function mechanism with PT variants, and sheds
light on the genetic heterogeneity of AD. p
f
E. Dardiotis4, S. Ralli4, G. Deretzi5, A. John6, K. Kydonopoulou7,
E. Papadopoulou7, A. di Pardo8, F. Akcimen9, A. Loizedda10,
V. Dobričić11, I. Novaković11, V. S. Kostić11, C. Mizzi12, B. A.
Peters13, A. N. Basak9, S. Orrù10, E. Kiskinis14, D. N. Cooper15,
S. Gerou7, R. Drmanac13, M. Bartsakoulia12, E. Tsermpini12,
G. M. Hadjigeorgiou4, B. R. Ali16, T. Katsila12, G. P. Patrinos12
1University of Athens, School of Medicine, Athens, Greece,
2University of Patras School of Health Sciences, Department of
Pharmacy, Patras, Greece, 3University of Thessaly School of
Medicine, Larissa, Greece, 4University of Thessaly, School of
Medicine, Larissa, Greece, 5Papageorgiou hospital,
Thessaloniki, Greece, 6United Arab Emirates University P09.009A Genomic variants in the FTO gene are associated with
sporadic amyotrophic lateral sclerosis in Greek patients A. Ansari1, E. Maffioletti1, M. Marizzoni2, O. Blin3, G. Frisoni2,4,
J. Richardson5, R. Bordet6, M. Gennarelli1,2, L. B. Chiavetto2,7 1Università degli Studi di Brescia, Brescia, Italy, 2IRCCS
Centro S. Giovanni di Dio Fatebenefratelli, Brescia, Italy, 3Aix
Marseille University, UMR-CNRS 7289, Service de
Pharmacologie Clinique, AP-HM, Marseille, France, 4Memory
Clinic and LANVIE - Laboratory of Neuroimaging of Aging,
University Hospitals and University of Geneva, Geneva,
Swaziland, 5Neurosciences Therapeutic Area,
GlaxoSmithKline R&D, Stevenage, United Kingdom,
6University of Lille, Inserm, CHU Lille, U1171 - Degenerative
and vascular cognitive disorders, F-59000, Lille, France,
73Faculty of Psychology, eCampus University, Novedrate
(Como), Italy 253 Abstracts from the 51st European Society of Human Genetics Conference: Posters None. A. John: None. K. Kydonopoulou: None. E. Papadopoulou: None. A. di Pardo: None. F. Akcimen:
None. A. Loizedda:
None. V. Dobričić:
None. I. Novaković: None. V.S. Kostić: None. C. Mizzi: None. B.A. Peters: A. Employment (full or part-time); Significant;
Complete Genomics Inc. A.N. Basak: None. S. Orrù:
None. E. Kiskinis: None. D.N. Cooper: None. S. Gerou:
A. Employment (full or part-time); Significant; ANALYSI
Biomedical Laboraotories. R. Drmanac: A. Employment
(full or part-time); Significant; Complete Genomics. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Complete Genomics Inc. M. Bartsakoulia: None. E. Tsermpini: None. G.M. Hadjigeorgiou: None. B.R. Ali: None. T. Katsila: None. G.P. Patrinos: None. College of Medicine and Health Sciences, Department of
Pathology, Al-Ain, United Arab Emirates, 7ANALYSI
Diagnostic Laboratories SA, Thessaloniki, Greece,
8Northwestern University Departments of Neurology and
Physiology, Chcago, IL, IL, United States, 9Bogazici
University, Suna and Inan Kirac Foundation, Istanbul, Turkey,
10University of Cagliari, Department of Medical Sciences and
Public Health, Cagliari, Italy, 11Institute of Neurology CCS,
School of Medicine, University of Belgrade, Belgrade, Serbia,
12Department of Pharmacy, University of Patras School of
Health Sciences, Patras, Greece, 13Complete Genomics Inc,
Mountain View, CA, United States, 14Departments of
Neurology and Physiology, Northwestern University Feinberg
School of Medicine, Chicago, IL, United States, 15Cardiff
University, Institute of Medical Genetics, Cardiff, United
Kingdom, 16United Arab Emirates University, College of
Medicine and Health Sciences, Department of Pathology, Al-
Ain, United Arab Emirates D. Gieruszczak-Białek, A. Skorka D. Gieruszczak-Białek, A. Skorka P09.010B Polymicrogyria in patient with Angelman Syndrome-
coincidence or a new feature ? Amyotrophic lateral sclerosis (ALS) is a devastating disease
whose complex pathology has been associated with a strong
genetic component in the context of both familial and
sporadic disease. Herein, we adopted a stepwise approach,
including whole genome and conventional Sanger sequen-
cing, in order to explore further the genetic basis of sporadic
ALS (sALS) in 3 patient cohorts of Greek (n = 150),
Turkish (n = 148) and Sardinian (n = 114) origin. Whole
genome sequencing yielded a total of 174 variants that were
found in 6 Greek sALS patients and in none of the 5
ethnically-matched controls, mostly intergenic (n = 144)
and intronic (n = 23) variants. Next, genes were clustered
by metabolic or disease network and the most prominent
ones were further analyzed in the entire 3 patient cohorts. Our analysis revealed a positive association between FTO
gene variants and sALS in the Greek patient cohort, while
linkage disequilibrium analyses were suggestive of a spe-
cific disease-associated haplotype for FTO gene variants. In
addition, qRT-PCR analysis of fibroblasts, undifferentiated
embryonic stem cells (ESCs), astrocytes, neural PAX6+
progenitors, as well as stem cell-derived spinal cord motor
neurons (MNs) and cortical neurons (CNs) indicated that
FTO gene expression is relatively neuron-specific and
notably, is most highly expressed in motor neurons. To our
knowledge, this is the first study to present a possible
association between FTO gene variants and the genetic
etiology of sALS, while the lack of association between
FTO variants and sALS in patients of Sardinian and Turkish
descent may suggest a founder effect in the Greek popula-
tion. <!--EndFragment--> D. Bartholdi1, S. Meier1, F. Joncourt1, S. Gallati1, M. Steinlin2 1University of Helsinki, Helsinki, Finland, 2The Folkhälsan
Institute of Genetics, Helsinki, Finland, 3University of Oulu,
Oulu, Finland 1Division of Human Genetics, University Children’s Hospital,
Bern, Switzerland, 2Department of Neuropediatrics, University
Children’s Hospital, Bern, Switzerland Introduction: Anxiety disorders include a large spectrum
of heterogeneous conditions and rank among the most
common health concerns in human medicine. Anxiety dis-
orders are known to be heritable, but genetically complex. Dogs suffer from various naturally occurring breed-specific
compulsions and phobias such as noise sensitivity and fear,
which respond to human anxiolytics and can be measured. This study aimed to find new anxiety loci in dogs. Hereditary cerebellar ataxias (CAs) are a genetically hetero-
geneous group of disorders with different inheritance patterns
and a variety of neurological symptoms. To date, more than
40 loci have been identified for the autosomal dominant
forms. Here, we describe a 10 year-old girl who, at the age of
2 years, presented with mild motor delay, gait instability,
dysarthria and oculomotor symptoms. Brain MRI showed
mild cerebellar atrophy. The family history is negative. We
performed targeted exome analysis and identified a hetero-
zygous de novo missense variant in the CACNA1G gene
which has not been described previously and is classified as
likely pathogenic, according to the actual ACMG-guidelines. CACNA1G encodes Cav3.1, a T-type calcium channel
belonging to the family of voltage-gated calcium channels. Cav3.1 is highly expressed in cerebellar neurons as well as in
thalamic
relay
neurons. The
variant
described
here
(c.5152C>G; p.(Arg1718Gly)) induces an amino acid change
in the voltage sensor S4 segment of Cav3.1. It is located in
close proximity to the recurrent CACNA1G missense variant
c.5144G>A, reported in French and Japanese families with
CA (Coutelier et al. and Morino et al., both 2015). The pre-
viously reported patients with the recurrent CACNA1G variant
all presented with symptom onset in adolescence or adult-
hood. The present case highlights that CACNA1G has also to
be considered in children with CA with very early onset. In
analogy to episodic ataxia type 2, which is caused by variants
in CACNA1A, we started a treatment with acetazolamide and
will report on the efficacy thereof. Materials and Methods: A total of 330 German
Shepherd dogs were phenotyped for two anxiety traits,
noise sensitivity (NS) and fear towards novel humans and
situations (fear) using a behavioral survey. D. Bartholdi1, S. Meier1, F. Joncourt1, S. Gallati1, M. Steinlin2 Each dog was
given a score describing the severity of the phenotype,
ranging from 0 for controls to 1-60 for NS and 0.5-13.5 for
fear. The dogs were genotyped using Illumina’s canine HD
SNP arrays and analysed for phenotype-genotype associa-
tions using single-locus method (PLINK) and single-locus
mixed model approach (GenABEL). Results: Genomic regions on chromosome 20 and
chromosome 7 were significantly associated with NS and
fear, respectively. The NS locus includes known anxiety
and hearing related genes, such as the glutamate receptor 7
gene. The fear locus was syntenic to a locus in human
18p11 that has been linked to psychiatric illnesses. Conclusions: The findings revealed new anxiety loci in
dogs overlapping several genes associated with human
neuropsychiatric disorders. Further investigation of the
causative variants within the loci has a potential to shed
light on the biological basis of the disorders in both species. R. Sarviaho: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Doctoral Programme
in Integrative Life Science, University of Helsinki. O. Hakosalo:
B. Research
Grant
(principal
investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; The Finnish Cultural
Foundation. K. Tiira: None. S. Sulkama: None. E. D. Bartholdi: None. S. Meier: None. F. Joncourt:
None. S. Gallati: None. M. Steinlin: None. P09.013A
A novel variant in CACNA1G is associated with early onset
cerebellar ataxia R. Sarviaho1,2, O. Hakosalo1,2, K. Tiira1,2, S. Sulkama1,2,
E. Salmela1,2, M. Hytönen1,2, M. J. Sillanpää3, H. Lohi1,2
1University of Helsinki, Helsinki, Finland, 2The Folkhälsan
Institute of Genetics, Helsinki, Finland, 3University of Oulu,
Oulu, Finland R. Sarviaho1,2, O. Hakosalo1,2, K. Tiira1,2, S. Sulkama1,2,
E. Salmela1,2, M. Hytönen1,2, M. J. Sillanpää3, H. Lohi1,2 D. Bartholdi1, S. Meier1, F. Joncourt1, S. Gallati1, M. Steinlin2
1Division of Human Genetics, University Children’s Hospital,
Bern, Switzerland, 2Department of Neuropediatrics, University
Children’s Hospital, Bern, Switzerland D. Bartholdi1, S. Meier1, F. Joncourt1, S. Gallati1, M. Steinlin2 Medical University of Warsaw, Warsaw, Poland Medical University of Warsaw, Warsaw, Poland Angelman syndrome (AS) is a neurogenetic imprinting
disorder attributable to the reduced expression of maternally
inherited UBE3A gene on chromosome 15. Angelman
syndrome is characterized by a combination of severe
intelactual impairement with limited speech, epilepsy,
ataxic gait, unique behavior and psychiatric comorbidities. MRI usually reveals only a small sized central nervous
system or minor abnormalities, such as mild cortical atro-
phy,
dysmyelination
and
focal
white
matter
signal
abnormalities. To our knowledge there is no patient with
polymicorgyria
in
previously
reported
patients
with
Angelman syndrome. Polymicrogyria is genetically het-
erogeneous, and only in a small minority of patients, a
definite genetic cause has been identified. Here we report
the patient with Angelman syndrome and polymicrogyria. The patient was born at term, birth measurements were
normal. At the neonatal period he was presented with
hypotonia. Brain MRI reavealed polymicrogyria. At the age
of eight months, because of developmental delay and slight
dysmorphic features he was referred to geneticist. Whole-
genome oligonucleotide microarray analysis revealed a 5.75
Mb deletion of chromosome 15q11.2q13.1 (15:22765628-
28520313; GRCh37) encompassing the critical region for
AS/PWS. Further MS-MLPA test identified hypomethyla-
tion of SNRPN and MAGEL2 locus as well as confirmed
the maternal Class I deletion extending from BP1 to BP3. FISH studies in both parents gave normal results, proving
the de novo occurrence of this aberration in the child. Further studies (NGS) are needed to determine the possible K. Mitropoulos: None. E. Merkouri Papadima: None. G. Xiromerisiou: None. A. Balasopoulou: None. K. Charalambidou: None. V. Galani: None. K. Zafeiri:
None. E. Dardiotis: None. S. Ralli: None. G. Deretzi: 254 J. del Picchia Salmela: None. M. Hytönen: None. M.J. Sillanpää:
None. H. Lohi: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; ERCStG, Jane ja
Aatos Erkko Foundation, ERANET-NEURON CBGC. F. Consultant/Advisory Board; Significant; Mars Petcare from
1.1.2018. genetic causes of polymicrogyria in our patient.aCHG and
MLPA studies were performed in Department of Medical
Genetics, Chidren' s Memorial Health Institute. D. Gieruszczak-Białek: None. A. Skorka: None. P09.014B An unstable ATTTC repeat mutation within the Disabled 1
gene causes cerebellar Purkinje cell alterations, DAB1 RNA Abstracts from the 51st European Society of Human Genetics Conference: Posters 255 switch, and Reelin-DAB1 signalling dysregulation in
Spinocerebellar ataxia type 37 A novel intronic ATM gene mutation affecting splicing in a
patient with Ataxia-Telangiectasia E. Arslan Ates1,2, A. Turkyilmaz3, M. A. Soylemez3, B. B. Geckinli3, P. Ata3, A. Arman3, A. I. Guney3 1Marmara University Pendik Training and Research Hospital,
Pendik, Turkey, 2Istanbul University Institute of Graduate
Studies in Science and Engineering, Istanbul, Turkey,
3Marmara University School of Medicine, Pendik, Turkey The spinocerebellar ataxias (SCAs) are characterised by
progressive cerebellar ataxia variably associated with oph-
thalmoplegia, pyramidal and extrapyramidal signs, demen-
tia, pigmentary retinopathy, seizures, lower motor neuron
signs, or peripheral neuropathy. We previously reported a
novel spinocerebellar ataxia subtype, SCA37, linked to an
11-Mb genomic region on 1p32 in a large Spanish ataxia
pedigree, characterised by a pure cerebellar syndrome dis-
tinctively presenting with early-altered vertical eye move-
ments. Here we demonstrate the segregation of an unstable
intronic ATTTC pentanucleotide repeat mutation within the
5’-noncoding regulatory region of the gene encoding the
reelin adaptor protein implicated in neuronal migration
DAB1, as the causative genetic defect of the disease in four
Spanish SCA37 families. Neuropathology revealed severe
loss of Purkinje cells (PCs) with abundant astrogliosis,
empty baskets, occasional axonal spheroids, and hyper-
trophic fibers by phosphorylated neurofilament immunos-
taining in the cerebellar cortex. The remaining PCs showed
loss
of
calbindin
immunoreactivity,
aberrant
dendrite
arborisation, nuclear pathology, and multiple ubiquitinated
perisomatic granules immunostained for DAB1. A sub-
population of Purkinje cells was found ectopically mis-
positioned within the cerebellar cortex. Importantly, we
demonstrate that the ATTTC repeat mutation dysregulated
DAB1 expression and induced a DAB1 RNA switch
resulting in the up-regulation of Reelin-DAB1 and PI3K/
AKT signalling in the SCA37 cerebellum. This study
reveals the unstable ATTTC pentanucleotide repeat muta-
tion within the DAB1 gene as the underlying genetic cause Introduction: Ataxia-telangiectasia (AT) is a rare auto-
somal recessive disorder characterized by progressive
ataxia, chorea, myoclonus beginning in early childhood. The other characteristic feature of the disease is tel-
angiectases in the eyes and also on the skin. ATM gene
mutations which encodes a protein involved in cell division
and DNA repair, are resposible for AT. Introduction: Ataxia-telangiectasia (AT) is a rare auto-
somal recessive disorder characterized by progressive
ataxia, chorea, myoclonus beginning in early childhood. The other characteristic feature of the disease is tel-
angiectases in the eyes and also on the skin. ATM gene
mutations which encodes a protein involved in cell division
and DNA repair, are resposible for AT. switch, and Reelin-DAB1 signalling dysregulation in
Spinocerebellar ataxia type 37 and provides evidence of cerebellar Reelin-DAB1 signal-
ling dysregulation in the spinocerebellar ataxia type 37. This work was funded by the Spanish Health Institute
Carlos III (CP14/00029; FIS PI14/00136; PI14/01159; FIS
PI17/00534). M. Corral-Juan1, C. Serrano-Munuera2, A. Rábano3, D. Cota-
González1, A. Segarra Roca1, L. Ispierto4, A. T. Cano Orgaz5,
A. D. Adarmes6, C. Méndez-del-Barrio6, S. Jesús6, P. Mir6,7,
V. Volpini8, R. Alvarez-Ramo4, I. Sánchez1, A. Matilla-Dueñas1 M. Corral-Juan: None. C. Serrano-Munuera: None. A. Rábano: None. D. Cota-González: None. A. Segarra
Roca: None. L. Ispierto: None. A.T. Cano Orgaz: None. A.D. Adarmes: None. C. Méndez-del-Barrio: None. S. Jesús: None. P. Mir: None. V. Volpini: None. R. Alvarez-
Ramo:
None. I. Sánchez:
None. A. Matilla-
Dueñas: None. 1Functional and Translational Neurogenetics Unit,
Department of Neuroscience, Health Sciences Research
Institute Germans Trias i Pujol (IGTP), Can Ruti Campus,
Badalona, Spain, 2Neurology Section, Hospital Sant Joan de
Deu de Martorell, Martorell, Spain, 3Fundación CIEN,
Madrid, Spain, 4Neurodegeneration Unit, Neurology Service,
Department of Neuroscience, University Hospital Germans
Trias i Pujol (HUGTiP), Can Ruti Campus, Badalona, Spain,
5Neurology Service, Hospital de Mataró, Mataró, Spain,
6Unidad de Trastornos del Movimiento, Servicio de
Neurología. Instituto de Biomedicina de Sevilla (IBiS),
Hospital Universitario Virgen del Rocío, CSIC, Universidad de
Sevilla, Sevilla, Spain, 7CIBERNED, Sevilla, Spain, 8IDIBELL,
L'Hospitalet de Llobregat, Spain A. Persico3 The aim of this
study was to compare the array-CGH diagnostic yield in 91
ADHD subjects divided into two groups according to the
clinical diagnosis: (1) 48 subjects diagnosed with ADHD as
primary diagnosis, co-morbid with learning disabilities,
conduct disorders, motor coordination disorders, opposi-
tional defiant disorders and mood disorders (2) 43 subjects
in which ADHD was co-morbid with autism and/or intel-
lectual disability. Aim of the study: Identification of prospective candidate
genes that could play a role in the aetiology of autism by
microarray. Aim of the study: Identification of prospective candidate
genes that could play a role in the aetiology of autism by
microarray. Patients and Methods: CNV analysis (Cytoscan HD
Affymetrix, CytoSNP-12 Illumina) was performed in 93
patients of Caucasian ethnicity - 63 males and 30 females -
with autism, PDD-NOS and Asperger’s syndrome, pre-
dominantly from simplex families. Systematic analysis of
the genes involved in CNVs was performed using databases
Decipher, OMIM, DGV and SFARI databases. Results: We detected 188 OMIM genes affected by
CNV. 28 of them are associated with neurodevelopment
disorders in OMIM database. Genes ARX (SFARI score S),
EIF2S3, GAL, GNAS, GRN, KLHL15, MSX2, NRXN1
(SFARI score 2), ORC6, PRODH, PTCHD1 (SFARI score
2), POLA1, SOX3, TSPAN7 and ZIC1 are associated with
autosomal dominant diseases with mental retardation,
intellectual disability, schizophrenia, agenesis of the corpus
callosum, frontotemporal lobar degeneration, craniosynos-
tosis, and lissencephaly, epilepsy and seizure. Results: We detected 188 OMIM genes affected by
CNV. 28 of them are associated with neurodevelopment
disorders in OMIM database. Genes ARX (SFARI score S), EIF2S3, GAL, GNAS, GRN, KLHL15, MSX2, NRXN1
(SFARI score 2), ORC6, PRODH, PTCHD1 (SFARI score
2), POLA1, SOX3, TSPAN7 and ZIC1 are associated with
autosomal dominant diseases with mental retardation,
intellectual disability, schizophrenia, agenesis of the corpus
callosum, frontotemporal lobar degeneration, craniosynos-
tosis, and lissencephaly, epilepsy and seizure. Materials and Methods: Array-CGH technology was
performed using the Human Genome CGH SurePrint G3
Microarray 4x180K Kit (Agilent). Results: We detected pathogenic and likely pathogenic
CNVs in 37% (16/43) subjects in which ADHD was co-
morbid with autism and/or intellectual disability and in 21%
(10/48)
subjects
diagnosed
with
ADHD
as
primary
diagnosis (exact P=0.105, n.s.). Detection of CNVs of
unknown clinical significance was similar in the two groups
being 10% and 8% in group (2) and in group (1)
respectively. A. Persico3 1University Campus Bio-medico of Rome, Roma, Italy,
2Mafalda Luce Center for Pervasive Neurodevelopmental
Disorders, Milan, Italy, 3Interdepartmental Program "Autism
0-90" "G. Martino" University Hospital, University of Messina,
Messina, Italy 1Institute of Medical Genetics, Olomouc, Czech Republic,
2Institute of Molecular and Translation Medicine, Olomouc,
Czech Republic, 3Gennet, Prague, Czech Republic Introduction: Copy number variants (CNVs) play an
important role in susceptibility to autism. Clinical sig-
nificance, however, is still unclear in many of them. Gene
content of CNVs seems to be crucial for determination of
the significance. Aim of the study: Identification of prospective candidate
genes that could play a role in the aetiology of autism by
microarray Introduction: Copy number variants (CNVs) play an
important role in susceptibility to autism. Clinical sig-
nificance, however, is still unclear in many of them. Gene
content of CNVs seems to be crucial for determination of
the significance. Introduction: Copy number variants (CNVs) play an
important role in susceptibility to autism. Clinical sig-
nificance, however, is still unclear in many of them. Gene
content of CNVs seems to be crucial for determination of
the significance. Introduction and objective: Attention Deficit Hyper-
activity Disorder (ADHD) is a common and heritable neu-
rodevelopmental
disorder
characterized
by
persistent
inattention, hyperactivity and impulsivity. ADHD is fre-
quently comorbid with other neuropsychiatric disorders. Array-CGH is the first-tier genetic test for patients with
idiopathic autism and intellectual disability with a reported
diagnostic yield ranging from 4% to 30%. Yet, its utility in
the ADHD clinics is more controversial. The aim of this
study was to compare the array-CGH diagnostic yield in 91
ADHD subjects divided into two groups according to the
clinical diagnosis: (1) 48 subjects diagnosed with ADHD as
primary diagnosis, co-morbid with learning disabilities,
conduct disorders, motor coordination disorders, opposi-
tional defiant disorders and mood disorders (2) 43 subjects
in which ADHD was co-morbid with autism and/or intel-
lectual disability. Introduction and objective: Attention Deficit Hyper-
activity Disorder (ADHD) is a common and heritable neu-
rodevelopmental
disorder
characterized
by
persistent
inattention, hyperactivity and impulsivity. ADHD is fre-
quently comorbid with other neuropsychiatric disorders. Array-CGH is the first-tier genetic test for patients with
idiopathic autism and intellectual disability with a reported
diagnostic yield ranging from 4% to 30%. Yet, its utility in
the ADHD clinics is more controversial. A novel intronic ATM gene mutation affecting splicing in a
patient with Ataxia-Telangiectasia Method: After the clinical evalution of the case next
generation sequencing was performed for ATM gene
analysis. Detected intronic mutation was investigated for
splicing affect. From peripheral blood lymphocytes mRNA
was isolated and via revers transcription cDNA was
obtained. ATM gene exon 48-50 was sequenced using
spesific primers targeting related region. Result: A 6 year-old girl was referred us with the
diagnosis of AT. She was born at term to consanguineous
parents. She had gait disturbance and dysartria for 3 years. Multiple cutaneous telangiectases were observed on her
face, trunk and limbs. Next-generation sequencing analysis
of ATM gene revealed homozygous c.7308-15A>G varia-
tion in IVS49. Parents were carrying the variation in
heterozygous site. Human Splicing Finder predicted that the
mutation could activate an intronic cryptic acceptor site. We
designed primers for amplification of related exons (49-50)
from cDNA for evaluating splicing pattern. A fourteen
nucleotide insertion from intron 49 were detected between
exon 49 and 50, resulting premature termination of
translation at codon 2439. 256 J. del Picchia be associated with significantly greater diagnostic yield with
a larger sample size. be associated with significantly greater diagnostic yield with
a larger sample size. Conclusion: In this study we investigated the affect of an
intronic mutation on splicing and revealed the molecular
diagnosis of the AT case. <!--EndFragment--> C. Lintas: None. A. Costa: None. L. Gorrieri: None. M. Baccarin: None. C. Picinelli: None. P. Tomaiuolo:
None. C. Cannizzaro: None. M. Canali: None. R. Sacco:
None. A. Persico: None. C. Lintas: None. A. Costa: None. L. Gorrieri: None. M. Baccarin: None. C. Picinelli: None. P. Tomaiuolo:
None. C. Cannizzaro: None. M. Canali: None. R. Sacco:
None. A. Persico: None. E. Arslan Ates: None. A. Turkyilmaz: None. M.A. Soylemez: None. B.B. Geckinli: None. P. Ata: None. A. Arman: None. A.I. Guney: None. Appropriateness of genetic testing in the ADHD clinics: a
comparative study C. Lintas1, A. Costa1, L. Gorrieri1, M. Baccarin2, C. Picinelli2,
P. Tomaiuolo2, C. Cannizzaro1, M. Canali1, R. Sacco1,
A. Persico3 Z. Capkova1, P. Capkova1, J. Srovnal2, K. Staffova2,
V. Becvarova3, M. Trkova3, V. Curtisova1, M. Hajduch2,
M. Prochazka1 V. Becvarova3, M. Trkova3, V. Curtisova1, M. Hajduch2,
M. Prochazka1 An unusual high frequency of natural fetal loss in a
Colombian cohort with Autism Spectrum Disorder A. Lopez1, D. Nuñez2, C. Velasco2, R. Chaskel1, E. Ferro3,
C. Lattig2 A. Lopez1, D. Nuñez2, C. Velasco2, R. Chaskel1, E. Ferro3,
C. Lattig2 We applied genome-wide oligonucleotide microarrays
(OGT) with average resolution 30 kpz for identification and
characterization of CNVs in patients with ASDs. The
analyses of the patients’ genomes were performed using
arrays contained approximately 180,000 oligonucleotide
probes that covered the entire human genome and allow to
accurate detection of copy number variation at the exon
level. For the study 95 patients were qualified. 1Fundacion Santa Fe de Bogota, Bogota, Colombia,
2Universidad de los Andes, Bogota, Colombia, 3Clinica
Montserrat, Bogota, Colombia Autism spectrum disorders (ASD) are neurodevelopmental
disorders that share difficulties in communication, social
interactions and stereotyped behaviors. ASD has a herit-
ability of 64 - 91% indicating a high genetic component. Clinically recognized pregnancy loss is relatively common
in the population with an estimate between 12-20%. How-
ever, this risk increases substantially, frequencies between
58-65%, for genetic diseases such as Edwards and Patau
syndromes. Here we describe an unusual high rate of pre-
vious natural fetal losses in a cohort clinically diagnosed
with ASD. We have clinically ascertained 45 family trios
composed of mother, father and child with autism, of which
44% (20 mothers) had previous natural fetal losses; 14 of
them had one previous natural fetal loss, and 6 mothers had
two or more previous natural fetal losses. Age was not a
critical factor in our cohort suggesting that clinically
recognized pregnancy loss might be associated with
increases risk of autism. Chromosomal microarray analysis revealed 18 nonpoly-
morphic CNVs, ranging in size from 15 kb to 3.1 Mb, in 17
(17.9%) patients. We identified pathogenic or potentially
pathogenic CNVs in 9 individuals with ASDs (9.5%),
whereas CNVs with unknown clinical significance were
identified in 9.5% of cases. All of the identified CNVs were
submicroscopic in size and therefore could not have been
detected by standard karyotype analysis. Our results
confirmed the importance of array CGH in detection of
CNVs in patients with ASDs. B. Wiśniowiecka-Kowalnik: None. M. Kędzior: None. E. Obersztyn: None. A. Kutkowska-Kaźmierczak: None. B. Wiśniowiecka-Kowalnik: None. M. Kędzior: None. E. Obersztyn: None. A. Kutkowska-Kaźmierczak: None. N. Bezniakow: None. J. Castañeda: None. A. Barczyk:
None. A. Sobczyńska-Tomaszewska: None. K. Czerska:
None. B. Nowakowska: None. N. Bezniakow: None. J. Castañeda: None. A. Barczyk:
None. A. Sobczyńska-Tomaszewska: None. K. Czerska:
None. B. Nowakowska: None. B. Wiśniowiecka-Kowalnik1, M. Kędzior1, E. Obersztyn1, Supported by MH CZ – DRO (FNOL, 00098892), IGA
UP
LF_2018_005,
TACR
TE02000058,
NCMG
LM201591 a NPU LO1304. Supported by MH CZ – DRO (FNOL, 00098892), IGA
UP
LF_2018_005,
TACR
TE02000058,
NCMG
LM201591 a NPU LO1304. 1Institute of Mother and Child, Warsaw, Poland, 2MEDGEN,
Warsaw, Poland Autism Spectrum Disorders (ASDs) are one of the most
prevalent groups of neurodevelopmental disorder that
affects around 1-2% of the population with the average
male to female ratio 4-5:1. A strong genetic contribution to
the etiology of ASDs has been recognized in ~ 25-40% of
patients. In ~ 7-14% of individuals with ASDs, submicro-
scopic chromosomal copy-number variants (CNVs) are one
such contributing factor. Because of the large genetic het-
erogeneity of ASDs, high-resolution whole-genome ana-
lyses such as aCGH are useful tools to study the
etiopathogenesis of these disorders. Z. Capkova: None. P. Capkova: None. J. Srovnal:
None. K. Staffova: None. V. Becvarova: None. M. Trkova: None. V. Curtisova: None. M. Hajduch: None. M. Prochazka: None. A. Kutkowska-Kaźmierczak1, N. Bezniakow1, J. Castañeda1, Facial dysmorphisms as biomarkers for autism spectrum
disorder Facial dysmorphisms as biomarkers for autism spectrum
disorder A. Lopez: None. D. Nuñez: None. C. Velasco: None. R. Chaskel: None. E. Ferro: None. C. Lattig: None. A. Lopez: None. D. Nuñez: None. C. Velasco: None. R. Chaskel: None. E. Ferro: None. C. Lattig: None. I. Menashe1, M. Ragoler1, A. Algrabili2, O. Bar2, I. Dinstein1,
G. Meiri3 I. Menashe1, M. Ragoler1, A. Algrabili2, O. Bar2, I. Dinstein1,
G. Meiri3 I. Menashe1, M. Ragoler1, A. Algrabili2, O. Bar2, I. Dinstein1,
G. Meiri3 An unusual high frequency of natural fetal loss in a
Colombian cohort with Autism Spectrum Disorder This work was supported by Colciencias Grant 744-2016
and Vicerrectoria de Investigaciones, Universidad de los
Andes, Bogota - Colombia A. Barczyk1, A. Sobczyńska-Tomaszewska2, K. Czerska2, B. Nowakowska1 B. Nowakowska1 1Ben-Gurion University of the Negev, Beer Sheva, Israel,
2FDNA, Herzelia, Israel, 3Soroka University Medical Center,
Beer Sheva, Israel B. Wiśniowiecka-Kowalnik1, M. Kędzior1, E. Obersztyn1,
A. Kutkowska-Kaźmierczak1, N. Bezniakow1, J. Castañeda1,
A. Barczyk1, A. Sobczyńska-Tomaszewska2, K. Czerska2,
B. Nowakowska1 A. Persico3 Conclusions: In our cohort we identified several new
possible candidate genes associated with autism (EIF2S3,
GAL, GRN, KLHL15, MSX2, ORC6, PRODH, POLA1,
SOX3, ZIC1), in addition to previously reported ones
(ARX, GNAS, NRXN1, PTCHD1, TSPAN7). However,
further
study
is
essential
for
determination
of
their
significance in aetiology of ASD. Conclusions: Array CGH is a valuable diagnostic tool to
detect pathogenic and likely pathogenic CNVs in ADHD-
affected subjects, even in the absence of comorbidity with
autism and/or intellectual disability, although the latter may Abstracts from the 51st European Society of Human Genetics Conference: Posters 257 Supported by MH CZ – DRO (FNOL, 00098892), IGA
UP
LF_2018_005,
TACR
TE02000058,
NCMG
LM201591 a NPU LO1304. Z. Capkova: None. P. Capkova: None. J. Srovnal:
None. K. Staffova: None. V. Becvarova: None. M. Trkova: None. V. Curtisova: None. M. Hajduch: None. M. Prochazka: None. P09.019C We thus examined whether CNVs and SNVs in
genes involved in regulation of toxicant exposure, namely
in detoxification processes and physiological permeability
barriers, occur more frequently in individuals with ASD
than in control subjects. For this purpose, publicly available
genomic datasets (AGP, SSC, ARRA, DGV) and the
Comparative Toxicogenomics Database were analyzed. CNVs in 8 genes (STS, CYP2D6, ARSF, GUSB, CLDN3,
CYP2R1, SLC3A2 and SULT2B1) were found exclusively in
ASD subjects, while CNVs in 7 genes (CSH1, MAGEA8,
CYP4X1, CHST5, CSH2, GH2 and ABCC1) were more
frequent in ASD individuals than in controls, after correc-
tion for multiple testing (6.04x10-13<P < 1.37x10-5). All
these genes also carried detrimental loss-of-function or
missense variants. Rare de novo loss-of-function or mis-
sense SNVs were further identified in the AR and ANKRD11
ASD candidate genes, and also in the CBS, CES1, GUSB
and JUP genes. Most of these genes interact with toxicants
implicated in ASD, namely bisphenol A, heavy metals and
benzo(α)pyrene, and are key players in detoxification pro-
cesses or regulation of blood-brain barrier and placenta
permeability, which are crucial in controlling exposure
during development. Notably, the hormonal regulation
functions of molecules encoded by STS, AR, CSH1, CSH2
may link toxicant-related endocrine disruptions with the
high ASD male/female ratio. These findings thus reinforce
the hypothesis that gene-environment interactions con-
tribute to ASD. Therefore, we hypothesized that children with ASD have
distinct facial characteristics that could facilitate diagnosis
of the disorder. Methods: Cases included children with ASD from the
Negev HUB autism database. Frontal facial photos of these
children were compared to those of normally developed
children (matched by age, sex and ethnicity at a 2:1 ratio). A
deep convolutional neural network (DCNN) architecture
with batch normalization was used to evaluate these photos. Classification accuracy between the groups was assessed
using cross-validation approach with 90% of the photos
using for training, and 10% for validation. Permutation
analysis with 1000 replication was used to assess the
statistical significance of group classification. Results: Overall, 82 children with ASD (78% males, and
28% Bedouin) with a mean age of 4.78 ± 2.03 years
participated in the study. The FDNA algorithm could
distinguish between ASD and controls with a 96.6%
accuracy (AUC=0.966; 95%CI=0.964-0.968(. This was
remarkably better than the classification accuracy of gender
or ethnicity (AUC=0.706 and AUC=0.790 respectively). Analysis of upper face achieved better separation between
cases and controls than lower face (AUC=0.892 vs. 1Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisbon,
Portugal, 2Biosystems and Integrative Sciences Institute
(BioISI), Lisbon, Portugal, 3Faculdade de Ciências da
Universidade de Lisboa (FCUL), Lisbon, Portugal,
4Faculdade de Medicina da Universidade de Coimbra,
Coimbra, Portugal, 5Centro de Investigação e Formação
Clinica do HP-CHUC, Coimbra, Portugal, 6Instituto
Gulbenkian de Ciência, Oeiras, Portugal P09.019C AUC=0.728), with eyes and nose being the most distinct
facial
characteristics
(AUC=0.912
and
AUC=0.919
respectively). Conclusions: Our findings suggest that children with
ASD have unique facial characteristics that could be used as
biomarkers for the disorder. I. Menashe: None. M. Ragoler: None. A. Algrabili: A. Employment (full or part-time); Significant; FDNA. O. Bar: A. Employment (full or part-time); Significant;
FDNA. I. Dinstein: None. G. Meiri: None. J.X. Santos: None. A.R. Marques: None. C. Rasga:
None. M. Asif: None. J. Vilela: None. H. Martiniano:
None. G. Oliveira:
None. A. Nunes:
None. A.M. Vicente: None. P09.019C Significance of submicroscopic chromosomal copy-number
variants in etiopathogenesis of autism spectrum disorders 1Ben-Gurion University of the Negev, Beer Sheva, Israel,
2FDNA, Herzelia, Israel, 3Soroka University Medical Center,
Beer Sheva, Israel 1Ben-Gurion University of the Negev, Beer Sheva, Israel,
2FDNA, Herzelia, Israel, 3Soroka University Medical Center,
Beer Sheva, Israel Introduction: Current diagnosis of autism spectrum dis-
order ASD is based on behavioral assessment that compli-
cates
diagnosis. Many
genetic
syndrome
that
share
comorbidities with ASD, have unique facial dysmorphisms. 258 J. del Picchia Autism Spectrum Disorder (ASD) is a complex neurode-
velopmental disorder with multifactorial etiology. Genetic
factors are strongly implicated in ASD, while environmental
toxicant exposure early in development is a documented
risk factor, suggesting a role for gene-environment inter-
actions. We thus examined whether CNVs and SNVs in
genes involved in regulation of toxicant exposure, namely
in detoxification processes and physiological permeability
barriers, occur more frequently in individuals with ASD
than in control subjects. For this purpose, publicly available
genomic datasets (AGP, SSC, ARRA, DGV) and the
Comparative Toxicogenomics Database were analyzed. CNVs in 8 genes (STS, CYP2D6, ARSF, GUSB, CLDN3,
CYP2R1, SLC3A2 and SULT2B1) were found exclusively in
ASD subjects, while CNVs in 7 genes (CSH1, MAGEA8,
CYP4X1, CHST5, CSH2, GH2 and ABCC1) were more
frequent in ASD individuals than in controls, after correc-
tion for multiple testing (6.04x10-13<P < 1.37x10-5). All
these genes also carried detrimental loss-of-function or
missense variants. Rare de novo loss-of-function or mis-
sense SNVs were further identified in the AR and ANKRD11
ASD candidate genes, and also in the CBS, CES1, GUSB
and JUP genes. Most of these genes interact with toxicants
implicated in ASD, namely bisphenol A, heavy metals and
benzo(α)pyrene, and are key players in detoxification pro-
cesses or regulation of blood-brain barrier and placenta
permeability, which are crucial in controlling exposure
during development. Notably, the hormonal regulation
functions of molecules encoded by STS, AR, CSH1, CSH2
may link toxicant-related endocrine disruptions with the
high ASD male/female ratio. These findings thus reinforce
the hypothesis that gene-environment interactions con-
tribute to ASD. Autism Spectrum Disorder (ASD) is a complex neurode-
velopmental disorder with multifactorial etiology. Genetic
factors are strongly implicated in ASD, while environmental
toxicant exposure early in development is a documented
risk factor, suggesting a role for gene-environment inter-
actions. P09.021A A role for gene-environment interactions in Autism
Spectrum Disorder is suggested by an excess of potentially
pathogenic variants in genes regulating exposure to
toxicants P09.022B
miRNA and lncRNA gene variants in Autism Spectrum
Disorder Deletion of 22q13.3 detected in two
patients (6.7%) are common pathogenic anomalies in ASD. The LRCC7, NRXN1 (intronic region) and MACROD2
genes identified in VUS-LP regions have previously been
shown to be related with autism. So, we suppose that VUS-
LPs detected in this study may be a direct cause or low
penetrating risk factor for ASD. Autism Spectrum Disorder (ASD) is a clinically hetero-
geneous neurodevelopmental disorder. Genetic factors are
estimated to account for 50 to 80% of the familial ASD risk,
but most of the genetic determinants are still not known and
a role for epigenetic factors is likely. Results: Pathogenic CNVs (P) were found in 4 (13%)
patients, clinically uncertain CNVs (VUS) in 2 (6,7%) and
VUS/likely pathogenic CNVs (VUS-LP) in 3 (10%; Table). Conclusions: The presence of P/ VUS-LPs in 23% of
patients indicated the importance of CNVs in ASD etiology
and that microarray should be used as the first step in the
diagnostic algorithm. Deletion of 22q13.3 detected in two
patients (6.7%) are common pathogenic anomalies in ASD. The LRCC7, NRXN1 (intronic region) and MACROD2
genes identified in VUS-LP regions have previously been
shown to be related with autism. So, we suppose that VUS-
LPs detected in this study may be a direct cause or low
penetrating risk factor for ASD. In this study we explored the potential role of noncoding
RNAs in ASD by comparing the frequency of Copy
Number Variants (CNVs) targeting microRNA (miRNA) or
long noncoding (lncRNA) genes in ASD patients (n =-
3570) with control subjects (n = 9649), using the Fisher’s
exact test corrected for multiple testing. We found 22 miRNA genes exclusively targeted by
CNVs in ASD subjects and 14 miRNA genes more
frequently disrupted by CNVs in ASD patients than in
controls. Two miRNA were previously associated with
ASD in serum miRNA profiling studies, while 5 novel
miRNAs for ASD have been described in schizophrenia, a
disorder that phenotypically and genetically overlaps with
ASD. Many putative targets of these 36 miRNAs are
reported ASD risk genes. Gene-target enrichment analysis
identified 6 significant pathways, 2 of which, the PI3K-Akt
and MAPK signalling pathways, have been implicated in
ASD. We further identified 102 novel lncRNA genes more
frequently targeted by CNVs in ASD, 3 of which are
antisense to ASD candidate genes. Copy number variation analysis in autism spectrum
disorders G. Kayhan1, E. Guney2, E. Iseri2, M. A. Ergun1, E. F. Percin1 G. Kayhan1, E. Guney2, E. Iseri2, M. A. Ergun1, E. F. Percin1 P09.022B
miRNA and lncRNA gene variants in Autism Spectrum
Disorder J. X. Santos1,2,3, A. R. Marques1,2,3, C. Rasga1,2, M. Asif1,2,3,
J. Vilela1,2,3, H. Martiniano1,2,3, G. Oliveira4,5, A. Nunes2,3, A. M. Vicente1,2,3,6 A. R. Marques1,2, H. Martiniano1,2,3, J. X. Santos1,2, J. Vilela1,2,
M. Asif1,2, G. Oliveira4,5, L. Romão6,2, A. M. Vicente1,2,7 1Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisbon,
Portugal, 2Biosystems and Integrative Sciences Institute
(BioISI), Lisbon, Portugal, 3Faculdade de Ciências da
Universidade de Lisboa (FCUL), Lisbon, Portugal,
4Faculdade de Medicina da Universidade de Coimbra,
Coimbra, Portugal, 5Centro de Investigação e Formação
Clinica do HP-CHUC, Coimbra, Portugal, 6Instituto
Gulbenkian de Ciência, Oeiras, Portugal 1Health Promotion and non Communicable Disease
Prevention, National Institute of Health Doutor Ricardo Jorge,
Lisboa, Portugal, 2BioISI - Biosystems & Integrative Sciences
Institute, Faculdade de Ciências, Universidade de Lisboa,
Lisboa, Portugal, 3Department of Informatics, Faculdade de
Ciências, Universidade de Lisboa, Lisboa, Portugal, 4Unidade
de Neurodesenvolvimento e Autismo (UNDA), Serviço do
Centro de Desenvolvimento da Criança, Centro de Abstracts from the 51st European Society of Human Genetics Conference: Posters 259 Investigação e Formação Clínica, Hospital Pediátrico, Centro
Hospitalar e Universitário de Coimbra, Coimbra, Portugal,
5Institute for Biomedical Imaging and Life Sciences, Faculty of
Medicine, Universidade de Coimbra, Coimbra, Portugal,
6Human Genetics Departament, National Institute of Health
Doutor Ricardo Jorge, Lisboa, Portugal, 7Instituto Gulbenkian
de Ciência, Oeiras, Portugal Introduction: Although the proportion of heredity in aut-
ism spectrum disorders (ASD) is estimated to be as high as
90%, genetic factors can only be detected in 20-25% of
cases because of their heterogeneity and complexity. Among these factors, CNVs come to the forefront with a
high level of 10% and being easily detectable. Introduction: Although the proportion of heredity in aut-
ism spectrum disorders (ASD) is estimated to be as high as
90%, genetic factors can only be detected in 20-25% of
cases because of their heterogeneity and complexity. Among these factors, CNVs come to the forefront with a
high level of 10% and being easily detectable. Methods: Array-CGH analysis (Agilent ISCA 8x60K)
was performed in 30 patients with non-syndromic ASD. p
p
y
Results: Pathogenic CNVs (P) were found in 4 (13%)
patients, clinically uncertain CNVs (VUS) in 2 (6,7%) and
VUS/likely pathogenic CNVs (VUS-LP) in 3 (10%; Table). Conclusions: The presence of P/ VUS-LPs in 23% of
patients indicated the importance of CNVs in ASD etiology
and that microarray should be used as the first step in the
diagnostic algorithm. P09.022B
miRNA and lncRNA gene variants in Autism Spectrum
Disorder Table: CNVs Identified With Array-CGH in Patients with
ASD Patient No Cytoband Start-
Stop (bp)
Size (kb) Genes
Del /
Dup
Inheritance Interpretation
Pt2
22q13.33 50241153-
51178264
930
ALG12, MLC1
SBF1, SCO2,
ARSA,
SHANK3, ACR... Del
De novo
Pathogenic
Pt5
1p31.1
70059561-
70386514
326
LRRC7, PIN1P1
Del
Unknown
VUS/ Likely
pathogenic
Pt10
2q36.3
230479578-
230951771
472
DNER, TRIP12,
FBXO36,
SLC16A14
Dup
Paternal
VUS
Pt13
9p24.3
p24.2
204193-
3221675
3,017
DOCK8, KANK1,
DMRT1, DMRT2,
SMARCA2,
VLDLR…
Del
Maternal
(mildly
affected)
Pathogenic
Pt19
22q13.33 51123491-
51178264
55
SHANK3, ACR
Del
De novo
Pathogenic
Pt22
4q25q26
113762831-
114217645
455
ANK2, MIR1243
Del
Unknown
VUS
Pt23
2p16.3
50937444-
51054432
117
NRXN1 (intronic) Del
Maternal
VUS/ Likely
pathogenic
Pt27
20p12.1
14567155-
14700099
133
MACROD2
Del
Unknown
VUS/ Likely
pathogenic
Pt28
1q21.1
q21.2
146507518-
148545520
2,038
FMO5, CHD1L,
BCL9, GJA5,
GJA8, GPR89B…
Del
De novo
Pathogenic These results support our hypothesis that genetic variants
targeting noncoding regulatory RNAs are involved in ASD
pathophysiology. This systems biology integrative strategy
will provide a better understanding of the biological
processes underlying ASD, and contribute to biomarker
and drug target discovery. A.R. Marques: None. H. Martiniano: None. J.X. Santos: None. J. Vilela: None. M. Asif: None. G. Oliveira: None. L. Romão: None. A.M. Vicente: None. 1Medical Genetics Department, Gazi University Hospital,
Ankara, Turkey, 2Child and Adolescent Psychiatry
Department, Gazi University Hospital, Ankara, Turkey P09.023C P09.023C
Copy number variation analysis in autism spectrum
disorders
G. Kayhan1, E. Guney2, E. Iseri2, M. A. Ergun1, E. F. Percin1
1Medical Genetics Department, Gazi University Hospital,
Ankara, Turkey, 2Child and Adolescent Psychiatry
Department, Gazi University Hospital, Ankara, Turkey G. Kayhan: None. E. Guney: None. E. Iseri: None. M. A. Ergun: None. E.F. Percin: None. P09.024D
Accessing mRNA and miRNA expression in binge eating
disorder
P. N. Moretti1, A. Simabucuro2, V. K. Ota2, N. M. Estella2,
M. Maranhao2, M. G. Cury2, A. Claudino2, S. I. Belangero2 G. Kayhan: None. E. Guney: None. E. Iseri: None. M. A. Ergun: None. E.F. Percin: None. Copy number variation analysis in autism spectrum
disorders P. N. Moretti1, A. Simabucuro2, V. K. Ota2, N. M. Estella2,
M. Maranhao2, M. G. Cury2, A. Claudino2, S. I. Belangero2 P09.024D Accessing mRNA and miRNA expression in binge eating
disorder P. N. Moretti1, A. Simabucuro2, V. K. Ota2, N. M. Estella2,
M. Maranhao2, M. G. Cury2, A. Claudino2, S. I. Belangero2 J. del Picchia 260 1Universidade de Brasilia, Brasilia, Brazil, 2Universidade
Federal de Sao Paulo, Sao Paulo, Brazil 1Universidade de Brasilia, Brasilia, Brazil, 2Universidade
Federal de Sao Paulo, Sao Paulo, Brazil Psychiatry, University of California San Diego, San Diego,
CA, United States, 9Department of Psychiatry, Dalhousie
University, Halifax, NS, Canada, 10National Institute of Mental
Health, Klecany, Czech Republic, 11Intramural Research
Program, National Institute of Mental Health, National
Institutes of Health, US Dept of Health & Human Services,
Bethesda, MD, United States, 12Institute of Psychiatric
Phenomics and Genomics (IPPG), University Hospital,
Munich, Germany, 13Department of Psychiatry and
Psychotherapy, University Medical Center (UMG), Georg-
August University Göttingen, Göttingen, Germany,
14Department of Genetic Epidemiology in Psychiatry, Central
Institute of Mental Health, Medical Faculty Mannheim,
University of Heidelberg, Mannheim, Germany Binge Eating Disorder (BED) is a disorder commonly
associated with obesity, diabetes, cardiovascular diseases
and psychiatric disorders such as depression and anxiety. Although the disorder presents a genetic influence, genes
involved in risk for BED remain unknown. The aim of the
current project was to analyze mRNA expression of neu-
roplasticity and neurotransmission genes (SLC6A4 and
BDNF) and miRNAs (miR16 and miR206) possibly related
to their regulation in BED patients and healthy controls. Fifty five patients with BED and twenty eight control sub-
jects, matched for age and sex, were analyzed. Peripheral
blood of all individuals was collected and total RNA was
extracted from white blood cells using commercial tools. The assessments of mRNA and miRNAs were performed
using qRT-PCR technique with TaqMan detection method. We did not detect miR206 expression in the blood samples
of both groups. In the comparison between BED and control
group, BMI differences were detected, with increased
values of BMI being observed in BED subjects. We
observed differences in BDNF gene expression between
BED and healthy controls, however, these results did not
remain different after correction for BMI values. No dif-
ferences in SLC6A4 and miR16 expression were observed
between groups. Although these target genes were pointed
as candidates involved in BED patophysiology, the absence
of differences in gene expression between groups does not
indicate the use of these genes as BED biomarkers. P09.024D Bipolar disorder (BD) is a common, highly heritable neu-
ropsychiatric disease characterized by recurrent episodes of
mania
and
depression. Lithium
represents
the
best-
established long-term treatment for BD, even though indi-
vidual response is highly variable. The largest GWAS of
lithium response to date conducted by the International
Consortium on Lithium Genetics (ConLiGen) provides
evidence for the genetic basis of this variability. The first
genome-wide analysis of involvement of miRNAs in BD
identified nine BD-associated miRNAs, however it is
unknown whether these miRNAs are also associated with
lithium response in BD. We therefore tested whether com-
mon variants at these nine candidate miRNAs contribute to
the variance in lithium response. Furthermore, we system-
atically analyzed whether any other miRNA is implicated in
the response to lithium. We performed gene-based tests for
all known miRNAs in the ConLiGen GWAS dataset (n =
2,563 patients) using a set-based testing approach adapted
from VEGAS2. In the candidate approach, miR-499a
showed nominally significant association with lithium
response, thus providing evidence for involvement in both
development and treatment of BD. In the genome-wide
miRNA analysis, 71 miRNAs showed nominally significant
associations with the dichotomous and 106 with the con-
tinuous trait for treatment response. 15 miRNAs revealed
nominal significance in both phenotypes with miR-633
showing the strongest association with the continuous (p
= 9.80E-04) and miR-607 with the dichotomous phenotype
(p = 5.79E-04). No association between miRNAs and
treatment response to lithium withstood multiple testing
correction. Given the limited power of our study, the
investigation of miRNAs in larger samples of BD and
lithium response is warranted. P.N. Moretti: None. A. Simabucuro: None. V.K. Ota:
None. N.M. Estella: None. M. Maranhao: None. M.G. Cury: None. A. Claudino: None. S.I. Belangero: None. Analysis of the influence of microRNAs in Lithium
Response in Bipolar Disorder Analysis of the influence of microRNAs in Lithium
Response in Bipolar Disorder C. S. Reinbold1,2, A. J. Forstner3,4,1, J. Hecker5, J. M. Fullerton6,7, The International Consortium on Lithium Genetics,
J. Kelsoe8, M. Alda9,10, F. J. McMahon11, T. G. Schulze11,12,13,
M. Rietschel14, M. M. Nöthen3,4, S. Cichon1,2,3 1Human Genomics Research Group, Department of
Biomedicine, University of Basel, Basel, Switzerland, 2Institute
of Medical Genetics and Pathology, Basel, Switzerland,
3Institute of Human Genetics, University of Bonn, Bonn,
Germany, 4Department of Genomics, Life & Brain Center,
University of Bonn, Bonn, Germany, 5Department of
Biostatistics, Harvard T.H. Chan School of Public Health,
Boston, MA, United States, 6Neuroscience Research Australia,
Sydney, Australia, 7School of Medical Sciences, University of
New South Wales, Sydney, Australia, 8Department of C.S. Reinbold: None. A.J. Forstner: None. J. Hecker:
None. J.M. Fullerton: None. J. Kelsoe: None. M. Alda:
None. F.J. McMahon: None. T.G. Schulze: None. M. Rietschel: None. M.M. Nöthen: None. S. Cichon: None. P09.027C Exome sequencing of multiplex bipolar disorder families
and follow-up resequencing implicate rare variants in
neuronal genes contributing to disease etiology R. Bozhilova1, I. Popov1, M. Penchev2, G. Dzhebir1,
O. Beltcheva1, R. Elliot3, V. Stoyanova2, V. Mitev1, M. Owen3,
M. O’Donovan3, G. Kirov3, V. Milanova2, R. Kaneva1 A. Maaser1,2, J. Strohmaier3, K. U. Ludwig1,2, L. Henschel1,2,
F. Streit3, F. Degenhardt1,2, S. Sivalingam1,2, L. M. Schenk1,2,
A. C. Koller1,2, S. B. Fischer4, H. Thiele5, P. Nürnberg5,
J. Guzman Parra6, G. Orozco Diaz7, G. Auburger8, M. Albus9,
M. Borrmann-Hassenbach9, M. José González6, S. Gil Flores10,
F. J. Cabaleiro Fabeiro11, F. del Río Noriega12, F. Perez Perez13,
J. Haro González14, F. Rivas6, F. Mayoral6, S. Herms1,2,4,
P. Hoffmann1,2,4, S. Cichon1,2,4, M. Rietschel3, M. M. Nöthen1,2,
A. J. Forstner1,2,4 A. Maaser1,2, J. Strohmaier3, K. U. Ludwig1,2, L. Henschel1,2,
F. Streit3, F. Degenhardt1,2, S. Sivalingam1,2, L. M. Schenk1,2,
A. C. Koller1,2, S. B. Fischer4, H. Thiele5, P. Nürnberg5,
J. Guzman Parra6, G. Orozco Diaz7, G. Auburger8, M. Albus9,
M. Borrmann-Hassenbach9, M. José González6, S. Gil Flores10,
F. J. Cabaleiro Fabeiro11, F. del Río Noriega12, F. Perez Perez13,
J. Haro González14, F. Rivas6, F. Mayoral6, S. Herms1,2,4,
P. Hoffmann1,2,4, S. Cichon1,2,4, M. Rietschel3, M. M. Nöthen1,2,
A. J. Forstner1,2,4 1Molecular Medicine Center, Medical University - Sofia, Sofia,
Bulgaria, 2Clinic of Psychiatry, Alexandrovska University
Hospital, Medical University of Sofia, Sofia, Bulgaria, 3MRC
Centre for Neuropsychiatric Genetics and Genomics, Cardiff
University, Cardiff, United Kingdom Background: Genes associated with ion channels were
shown to contribute to schizophrenia (SCZ) and bipolar
affective disorder (BAD). Targeted NGS of a panel of
genes, including ion channel genes, was performed to
investigate the genetic architecture of Bulgarian patients
with BAD and SCZ. P09.026B
Rare genetic variants in ion channels genes identified by
NGS contribute to both schizophrenia and bipolar disorder P09.026B
Rare genetic variants in ion channels genes identified by
NGS contribute to both schizophrenia and bipolar disorder P09.027C P09.027C 1Institute of Human Genetics, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
2Department of Genomics, Life&Brain Center, University of
Bonn, Bonn, Germany, 3Department of Genetic Epidemiology
in Psychiatry, Central Institute of Mental Health, Medical
Faculty Mannheim/University of Heidelberg, Mannheim,
Germany, 4Human Genomics Research Group, Department of
Biomedicine, University of Basel, Basel, Switzerland, 5Cologne
Center for Genomics, University of Cologne, Cologne,
Germany, 6Department of Mental Health, University General
Hospital of Málaga, Biomedical Research Institute of Málaga
IBIMA, Málaga, Spain, 7Unidad de Gestión Clínica del
Dispositivo de Cuidados Críticos y Urgencias del Distrito
Sanitario Málaga, Málaga, Spain, 8Experimental Neurology,
Department of Neurology, Goethe University Hospital,
Frankfurt, Germany, 9kbo Isar Amper Klinikum München Ost,
Haar, Germany, 10Department of Mental Health, University
Hospital Reina Sofía, Córdoba, Spain, 11Department of Mental
Health, Hospital of Jaén, Jaén, Spain, 12Department of Mental
Health, Hospital of Jerez de la Frontera, Jerez de la Frontera,
Spain, 13Department of Mental Health, Hospital of Puerto
Real, Puerto Real, Spain, 14Department of Mental Health,
Hospital Punta de Europa, Algeciras, Spain Methods: A total of 300 individuals with BAD, 151 with
SCZ, 15 SAD, diagnosed with DSMIV and ICD-10, 85
controls and 40 healthy relatives were recruited. Sequencing
was done on Ion PROTON platform. The panel comprised
of 187 candidate genes, 39 of them coding ion channel
proteins. Only samples with coverage >95% of the target
region at 20x were included in the analyses. Case-control
association testing was done using PLINK. Prediction tools
(SIFT&PolyPhen2) were used. Methods: A total of 300 individuals with BAD, 151 with
SCZ, 15 SAD, diagnosed with DSMIV and ICD-10, 85
controls and 40 healthy relatives were recruited. Sequencing
was done on Ion PROTON platform. The panel comprised
of 187 candidate genes, 39 of them coding ion channel
proteins. Only samples with coverage >95% of the target
region at 20x were included in the analyses. Case-control
association testing was done using PLINK. Prediction tools
(SIFT&PolyPhen2) were used. Results: The case-control analysis revealed no significant
association with SCZ, SAD and BAD, that survived the
correction for multiple testing. However, 681 rare variants
present in affected only were found. 260 of them were
recurrent and 421 were singletons. Of these, 4LoF, 218
missense, 63 of which potentially damaging, 30 splice and 7
regulatory variants were detected. The genes with most
detected
variants
were
CACNA1H,CACNA1B
and
CACNA2D4. Abstracts from the 51st European Society of Human Genetics Conference: Posters 261 P09.027C Discussion:
No
significant
association
of
common
variants with the diseases was found, probably due to
limited power. However, recurrent and unique rare variants
with potential functional relevance were detected, that
deserve further investigation. This adds to the accumulating
evidence that ion channelopathies may be involved in the
pathogenesis of SCZ and BAD. The work was supported by
projects DUNK01-2/2009 and B02/6/2014 funded by
NSF, MYES Bipolar disorder (BD) is a highly heritable mood disorder
with a lifetime prevalence of around 1%. Models of illness
are most consistent with a polygenic contribution of com-
mon and rare variants to disease susceptibility. As the
cumulative impact of common alleles may only explain
around 25-38% of the phenotypic variance, rare variants of
high penetrance have been suggested to contribute to BD
susceptibility. R. Bozhilova: None. I. Popov: None. M. Penchev:
None. G. Dzhebir: None. O. Beltcheva: None. R. Elliot:
None. V. Stoyanova: None. V. Mitev: None. M. Owen:
None. M. O’Donovan: None. G. Kirov: None. V. Milanova: None. R. Kaneva: None. In this study, we performed whole-exome sequencing in
226 individuals of 68 large multiplex BD families of
European
origin. We
filtered
for
rare
(minor
allele
frequency < 0.1%), non-synonymous, potentially functional
and segregating variants. 262 J. del Picchia We identified 1214 variants implicating 1122 different
genes. Gene enrichment analysis of 294 genes that were
among
the
20%
most
“intolerant”
genes
showed
a
significant enrichment for 18 pathways (p < 0.001) includ-
ing neuron projection, axon development and cell-adhesion. chemical. The potential cytotoxic and genotoxic effects of
BPA have been thoroughly studied on neuroblastoma cells
(SH-SY5Y). Materials And Methods: SH-SY5Y cells were treated
with 50 μM and 100 μM concentrations of Bisphenol for
48 hours. After RNA isolation and cDNA synthesis, Real-
time PCR reactions were performed for GRP78, XBP1,
BECN1 and ATG5 genes. For follow up analyses, we prioritized genes that were
found in at least two unrelated families in the present study,
previously reported in next generation sequencing or
GWAS studies of BD, or predominantly driving the
significant pathways in our gene enrichment analysis. The
different approaches of prioritization yielded 42 promising
candidate genes including SYNE1 which is a genome-wide
significant BD risk gene. Results: In the present study, we aimed to investigate the
potential effect of Bisphenol on unfolded protein response
induced by Endoplasmic Reticulum Stress and Endoplasmic
Reticulum Stress-Mediated Autophagy in neuron like cell
line (SH-SY5Y). P09.027C In accordance with this purpose we
exposed to SH-SY5Y cell line with both different doses of
bisphenol. After 48h exposure, we measured mRNA levels
of the genes that play a role unfolded protein response and
autophagy. The 42 prioritized candidate genes are currently being
followed up by resequencing in larger cohorts of 2000
independent BD patients and 2000 controls of European
ancestry using the single molecule molecular inversion
probes technology. Conclusion: We observed a significant decrease in
GRP78 (Glucose-Regulated Protein) mRNA level and a
significant increase in XBP1(X-Box Binding Protein 1),
BECN1(Beclin 1), ATG5 (Autophagy Related 5) mRNA
level at low dose (50μM) Bisphenol exposure. According
with this result we can conclude that 50 μM dose of
Bisphenol induced the autophagy via ER stress in SH-
SY5Ycell line. Conclusion: We observed a significant decrease in
GRP78 (Glucose-Regulated Protein) mRNA level and a
significant increase in XBP1(X-Box Binding Protein 1),
BECN1(Beclin 1), ATG5 (Autophagy Related 5) mRNA
level at low dose (50μM) Bisphenol exposure. According
with this result we can conclude that 50 μM dose of
Bisphenol induced the autophagy via ER stress in SH-
SY5Ycell line. Our preliminary results suggest that rare and highly
penetrant variants in neuronal and cell-adhesion genes
contribute to BD etiology. Grant: BMBF IntegraMent. A. Maaser: None. J. Strohmaier: None. K.U. Ludwig:
None. L. Henschel: None. F. Streit: None. F. Degen-
hardt: None. S. Sivalingam: None. L.M. Schenk: None. A.C. Koller: None. S.B. Fischer: None. H. Thiele: None. P. Nürnberg: None. J. Guzman Parra: None. G. Orozco
Diaz: None. G. Auburger: None. M. Albus: None. M. Borrmann-Hassenbach: None. M. José González: None. S. Gil Flores: None. F.J. Cabaleiro Fabeiro: None. F. del
Río Noriega: None. F. Perez Perez: None. J. Haro
González: None. F. Rivas: None. F. Mayoral: None. S. Herms: None. P. Hoffmann: None. S. Cichon: None. M. Rietschel:
None. M.M. Nöthen:
None. A.J. Forstner: None. Ü.H. Lüleyap: None. A. YoldaŞ: None. G. Cömertpay:
None. G. Ay: None. G. Evyapan: None. A. Pazarbaşı:
None. D. Alptekin: None. P09.029A Cerebral MR imaging based genetic assessment of brain
malformations S. Hinreiner1, T. Roedl1, T. Geis2, M. Melter2, G. Schuierer3,
U. Hehr1 S. Hinreiner1, T. Roedl1, T. Geis2, M. Melter2, G. Schuierer3,
U. Hehr1 1Center for Human Genetics, Regensburg, Germany,
2Childrens Hospital University of Regensburg, Regensburg,
Germany, 3Center for Neuroradiology, University Hospital
Regensburg, Regensburg, Germany Ü. H. Lüleyap1, A. YOLDAŞ2, G. Cömertpay1, G. AY1,
G. Evyapan1, A. Pazarbaşı1, D. Alptekin1 Ü. H. Lüleyap1, A. YOLDAŞ2, G. Cömertpay1, G. AY1,
G. Evyapan1, A. Pazarbaşı1, D. Alptekin1 Introduction:
Structural
brain
malformations
are
an
important cause of early psychomotor retardation, intellec-
tual disability and seizures. Identification of the underlying
mutations allows more precise genetic counseling of the
affected families and may also provide additional informa-
tion directly relevant for further diagnostic workup or
therapeutic decisions. 1Cukurova University, Medical Faculty, ADANA, Turkey,
2Kahramanmaraş Sütçü imam University, Medical Faculty,
KAHRAMANMARAŞ, Turkey Introduction: Bisphenol A (BPA) is a chemical produced
in large quantities for use primarily in the production of
polycarbonate plastics and epoxy resins. While BPA is
found in a number of consumer products such as hard
plastic drinking containers and the linings of infant formula
and food cans, human are regularly exposed to this Methods: Initial evaluation of available cMR images (56
patients) or imaging data, clinical data and individual
genetic testing by either Sanger sequencing/MLPA (14
patients) or multi-gene panel sequencing (204 patients) after
Nextera
Enrichment
(Illumina)
and
bioinformatic Abstracts from the 51st European Society of Human Genetics Conference: Posters 263 CNRS UMR 5310 - INSERM U1217, Lyon, France, 11Institute
of Human Genetics, University Medical Center Hamburg-
Eppendorf, Hamburg, Germany assessment of called variants with our in house pipeline
including SeqNext (JSI medical systems) and evaluation for
CNV using an in house JAVA-based skript. Results and Discussion: For 18 patients with typical
cMRI pattern a single gene analysis was employed allowing
to identify the causal L1CAM (6), LIS1/PAFAH1B1 (7),
DCX (2) or TUBA1A (2) mutations. Overall causal
mutations (ACMG class 5 or 4) were identified for 76
patients (35%) including: hydrocephalus 10/37; Walker-
Warburg syndrome 9/12; Periventricular nodular hetero-
topia 2/7; Lissencephaly/Double cortex 20/28; Polymicro-
gyria
6/30;
(Ponto)cerebellar
hypoplasia
10/19;
Holoprosencephaly spectrum 12/51 and Microcephaly 7/
37. NGS panel sequencing substantially increased the
number of identified variants of unknown significance
(ACMG class 3), which were observed in 27% of the
overall patient cohort. Three holoprosencephaly patients
with heterozygous mutation in one of the core genes were
heterozygous for an additional variant, suggestive for
digenic inheritance. Our data emphasize the importance of
the individual clinical and imaging data for the individual
testing strategy and for the final clinical classification of
identified sequence variants. In 2012, Puffenberger et al., reported the first description of
a lethal neonatal rigidity and multifocal seizures syndrome
(OMIM# 614498) in 5 patients from the Amish community. P09.030B Epileptic encephalopathy due to BRAT1 pathogenic
variants: report of eight new patients J. Piard1, D. J. Moris-Rosendahl2, A. Putoux3, G. Delplancq1,
C. Cabrol1, J. Belleville Goffeney4, L. Pasquier5, E. Brischoux-
Boucher1, B. Wollnik6, G. Yigit6, B. Albrecht7, G. Lesca3,
L. Villard8, P. Edery3, D. Sanlaville9, N. Streichenberger10,
C. Altuzarra4, F. L. Harms11, K. Kutsche11, L. Van Maldergem1 J. Piard: None. D.J. Moris-Rosendahl: None. A. Putoux: None. G. Delplancq: None. C. Cabrol: None. J. Belleville Goffeney: None. L. Pasquier: None. E. Brischoux-Boucher: None. B. Wollnik: None. G. Yigit:
None. B. Albrecht: None. G. Lesca: None. L. Villard:
None. P. Edery: None. D. Sanlaville: None. N. Strei-
chenberger: None. C. Altuzarra: None. F.L. Harms:
None. K. Kutsche: None. L. Van Maldergem: None. J. Piard: None. D.J. Moris-Rosendahl: None. A. Putoux: None. G. Delplancq: None. C. Cabrol: None. J. Belleville Goffeney: None. L. Pasquier: None. E. Brischoux-Boucher: None. B. Wollnik: None. G. Yigit:
None. B. Albrecht: None. G. Lesca: None. L. Villard:
None. P. Edery: None. D. Sanlaville: None. N. Strei-
chenberger: None. C. Altuzarra: None. F.L. Harms:
None. K. Kutsche: None. L. Van Maldergem: None. 1Centre de génétique humaine, University Hospital, University
of Franche-Comté, Besançon, France, 2Clinical Genetics and
Genomics, Royal Brompton and Harefield NHS Trust and
Imperial College London, London, United Kingdom,
3Department of Pediatrics, Lyon University Hospitals, Lyon,
France, 4Department of Pediatrics, University Hospital,
University of Franche-Comté, Besançon, France, 5Service de
Génétique Médicale – Centre Référence « Déficiences
Intellectuelles de causes Rares », Hôpital Sud - CHU Rennes,
Rennes, France, 6Institute of Human Genetics, University of
Cologne, Cologne, Germany, 7Institute of Human Genetics,
University of Essen, Essen, Germany, 8Département de
Génétique Médicale, Hôpital d'Enfants de La Timone,
Marseille, France. Inserm, Aix Marseille Université, UMR-S
1251, Marseille, France, 9Department of Genetics, Lyon
University Hospitals, Lyon, France, 10Hospices Civils de Lyon,
University of Claude Bernard Lyon1, Institut NeuroMyogène Ü. H. Lüleyap1, A. YOLDAŞ2, G. Cömertpay1, G. AY1,
G. Evyapan1, A. Pazarbaşı1, D. Alptekin1 This syndrome is caused by bi-allelic mutations in BRAT1
and characterized by early-onset epilepsy, arrest of head
growth, severe muscular hypertonia, frequent apnea and
bradycardia, feeding difficulties and early death due to
cardiopulmonary failure. Microcephaly is inconsistent at
birth but progresses quickly over time accompanied by
progressive cerebral atrophy. EEG may show a suppression-
burst pattern. Beside this severe presentation, a milder form
has been described in 2016 by Srivastava et al., in 5
patients. It is characterized by ataxia and cerebellar atrophy,
prolonged survival, less severe microcephaly and inconstant
epilepsy. Twenty-eight individuals have been reported so
far with bi-allelic mutations in BRAT1. We report here 8
new patients: two Algerian brothers with a homozygous
c.2068G>T (p.Glu690*) variant; three German siblings
compound heterozygous for c.228insA(p.Leu77Thrfs*114)
and c.638insA (p.Val214Glyfs*189); and three unrelated
French female girls, compound heterozygous for the fol-
lowing combination of variants c.458A>C (p.Gln153Pro)
and c.294dupA (p.Leu99fs), c.294dupA(p.Leu99fs) and
c.2125_2128del(p.Phe709fs), and c.359C>A(p.Arg120His)
and c.1313_1314delAG(p.Gln4348Argfs*51). All but one
patient died during the first 14 months of life. The surviving
patient, aged 7 years, has a milder form. Five variants are
novel: 3 nonsense and 2 missense. Although, no clear
genotype-phenotype correlation has emerged, the presence
of two nonsense mutations seems to produce a more severe
phenotype than bi-allelic missense mutations. S. Hinreiner: None. T. Roedl: None. T. Geis: None. M. Melter: None. G. Schuierer: None. U. Hehr: None. G. Lesca1,2, A. Poisson3,4, N. Chatron1,2, A. Labalme1, M. Till1,
E. Broussolle5,6, C. Demily3,4, D. Sanlaville1,2 1Department of Medical Genetics, University Hospital of Lyon,
Lyon, France, 2GENDEV, Centre de Recherche en P09.031C Regressive autism spectrum disorder expands the
phenotype of BSCL2-associated neurodegeneration 1Department of Medical Genetics, University Hospital of Lyon,
Lyon, France, 2GENDEV, Centre de Recherche en J. del Picchia 264 Neurosciences de Lyon, INSERM U1028, CNRS UMR529,
UCBL1, Lyon, France, 3GenoPsy, Reference Center for
Diagnosis and Management of Genetic Psychiatric Disorders,
Centre Hospitalier le Vinatier, Lyon, France, 4EDR-Psy Team
(CNRS and Lyon 1 Claude Bernard University), Lyon, France,
5Service de Neurologie C, Hospices Civils de Lyon, Hôpital
Neurologique Pierre Wertheimer, University Hospital of Lyon,
Lyon, France, 6Institut des Sciences Cognitives Marc
Jeannerod, CNRS, UMR 5229, University of Lyon, Lyon,
France Neurosciences de Lyon, INSERM U1028, CNRS UMR529,
UCBL1, Lyon, France, 3GenoPsy, Reference Center for
Diagnosis and Management of Genetic Psychiatric Disorders,
Centre Hospitalier le Vinatier, Lyon, France, 4EDR-Psy Team
(CNRS and Lyon 1 Claude Bernard University), Lyon, France,
5Service de Neurologie C, Hospices Civils de Lyon, Hôpital
Neurologique Pierre Wertheimer, University Hospital of Lyon,
Lyon, France, 6Institut des Sciences Cognitives Marc
Jeannerod, CNRS, UMR 5229, University of Lyon, Lyon,
France 1Eskisehir Osmangazi University, Faculty of Medicine,
Department of Medical Genetics, Eskisehir, Turkey, 2Acibadem
Hospital Neurology Clinic, Eskisehir, Turkey, 3Istanbul
University, Istanbul Faculty of Medicine, Behavioural
Neurology and Movement Disorders Unit, Department of
Neurology, Istanbul, Turkey, 4Eskisehir Osmangazi University,
Faculty of Medicine, Department of Psychiatry, Eskisehir,
Turkey FTLD describes a group of progressive brain disorders. The
expansion of a noncoding GGGGCC hexanucleotide repeat
in the C9orf72 gene is a major cause of both familial FTLD
and amyotrophic lateral sclerosis (ALS). Loss-of-function variants of BSCL2, encoding seipin, were
reported in congenital generalized lipodystrophy type 2,
whereas two closely localized gain-of-function variants are
linked to two distinct neurological phenotypes: distal her-
editary motor neuropathy type V and hereditary spastic
paraplegia type 17. In 2013, six Spanish patients affected
with progressive encephalopathy death during infancy, and
homozygous or compound heterozygous for a rare BSCL2
exon 7 skipping variant (c.985C>T), were reported. We
report on a female patient with regressive autism spectrum
disorder who developed atypical parkinsonism in adult-
hood. She walked at the age of 11 months and began to
associate words at 18 months. Slight behavioral disorders
appeared by 3 years of age, evolving to invasive rituals, loss
of communication and language skills, and sleep disorders. At the age of 6, a clinical examination disclosed motor
stereotypies, trichotillomania and lower limb hypertonia. P09.031C At
the age of 16, Bichat’s fat pads, strabismus, bilateral wor-
sening of dystonic hypertonia, and extrapyramidal and
pyramidal features were noted. At the age of 23, falls,
dysphagia and a marked frontal lobe syndrome appeared. She died of a pulmonary infection at 28 years of age. Brain
MRI and CIT SPECT showed bilateral dopaminergic
denervation of the caudate nucleus. Trio-based whole-
exome sequencing showed two BSCL2 transitions. In
addition to the c.985C>T transition previously reported she
was compound heterozygous for the c.1004A>C transition
that was also predicted to favor exon 7 skipping. The pre-
sent observation shows that BSCL2 pathogenic variants can
cause severe autistic regression in infancy and lethal aty-
pical parkinsonism in adulthood. The study was aimed to determine the prevalence of
C9orf72
GGGGCC
repeat
expansion
in
the
Turkish
population with FTLD and to determine the effects on the
phenotype. The G4C2 expansion in C9orf72 gene were
analysed in 100 FTLD cases without mutations of MAPT,
PGRN,CHMP2B,VCP,TARDBP,FUS genes and a hundred
age-matched healthy controls by repeat-primed (RP-PCR)
and size-PCR techniques. The pathogenic expansion (>30) was found in one of the
familial cases(1/33) but none of sporadic cases. The allele
length difference between the cases and controls was
statistically significant (p < 0.01). An intermediate (20-30)
repeats was detected in 4% of our cases. The dominancy of
intermediate/pathogenic repeats was seen in the cases with
psychotic symptoms. This is the first study in our knowledge to evaluate the
C9orf72 GGGGCC repeat expansion for the Turkish FTLD
spectrum. As a result of our study, it is thought that C9orf72
repeat expansion is not common in Turkish FTLD cases,
but intermediate repeat may be an increased risk factor for
FTLD or may act as a modifying gene. In addition, we
believe that the correlation of these intermediate/pathogenic
repeats with psychotic symptoms is prognostically impor-
tant. Our data should be supported by further studies in
different ethnic groups of FTLD patients from Turkey. This
study
was
supported
by
The
Scientific
and
Technological
Research
Council
of
Turkey
(TUBI-
TAK1001-114S346) E. Erzurumluoglu: None. O. Cilingir: None. B.D. Ozbabalik Adapinar: None. B. Bilgic: None. S. Kocagil:
None. B. Durak Aras: None. C. Yenilmez: None. S. Artan: None. E. Erzurumluoglu: None. O. Cilingir: None. B.D. Ozbabalik Adapinar: None. B. Bilgic: None. S. Kocagil:
None. B. Durak Aras: None. C. Yenilmez: None. S. Artan: None. G. Lesca: None. A. Poisson: None. N. Chatron: None. A. P09.031C Labalme: None. M. Till: None. E. Broussolle: None. C. Demily: None. D. Sanlaville: None. C. Angelini1, J. Van Gils1, D. Lacombe1, S. Moutton2, F. Riant3,
G. Sole4, E. Tournier-Lasserve5, A. Trimouille1, C. Goizet6 E. Erzurumluoglu1, O. Cilingir1, B. D. Ozbabalik Adapinar2,
B. Bilgic3, S. Kocagil1, B. Durak Aras1, C. Yenilmez4, S. Artan1 P09.033A Major intra-familial phenotypic heterogeneity
encompassing epileptic encephalopathy due to a CACNA1A
missense variant Major intra-familial phenotypic heterogeneity
encompassing epileptic encephalopathy due to a CACNA1A
missense variant Comparison of phenotypic variability with C9orf72 gene
GGGGCC hexanucleotide repeat expansion in
frontotemporal lobar degeneration spectrum C. Angelini1, J. Van Gils1, D. Lacombe1, S. Moutton2, F. Riant3,
G. Sole4, E. Tournier-Lasserve5, A. Trimouille1, C. Goizet6 265 Abstracts from the 51st European Society of Human Genetics Conference: Posters 1Service de Génétique Médicale, CHU Bordeaux and
Laboratoire MRGM, INSERM U1211, Univ. Bordeaux,
Bordeaux, France, 2Service de génétique médicale, Dijon,
France, 3Service de génétique moléculaire, Hopital
Lariboisière, Paris, France, 4Service de neurologie médicale,
Bordeaux, France, 5Service de génétique moléculaire -
Lariboisière, Paris, France, 6Service de génétique médicale,
Centre de référence neurogénétique, Bordeaux, France 1Guangzhou Institute of Pediatrics,Guangzhou Women and
Children's Medical Center, Guangzhou, China, 2Department of
Neuropediatrics,Guangzhou Women and Children's Medical
Center, Guangzhou, China Cerebral palsy (CP) is an umbrella concept spanning a range
of symptoms albeit with the common feature, i.e., an inborn
disturbance of human movement and posture. CP is
observed with consistent prevalence regardless of the
socioeconomic advancement, and familial CP cases were
reported. Seminal studies have confirmed the involvement
of genetic changes, in the forms of copy number variant
(CNV), insertion and deletion (Indel), and single-nucleotide
variant (SNV). As a major children’s medical center in
China, we recruited a CP cohort of >500 individuals with
multiple subtypes and severity. To elucidate the genetic
causality, we initiated a pilot study of 120 idiopathic CP
patients with their parents and siblings, with a comprehen-
sive genomic assessment. We considered possible etiologic
mechanisms from chromosomes to genes via multiple high-
throughput platforms. We found that up to 45% of CP cases
could be due to detrimental mutations. Causative de novo
mutations were more prevalent than inherited ones, and
post-zygotic mutations (PZM) took a nonnegligible portion. Major factions of the causal mutations were SNVs and
indels in nuclear genome, while mitochondrial genome
harbored several putative mutations, and large segmental
alterations accounted for 8% of CP cases. Strikingly,
besides the conventional nonsynonymous and splicing-site
mutations, we observed by whole-genome sequencing a
host of mutations residing in the regulatory regions which
might serve as culprit for disease. Our work is of critical
significance to reveal the underlying reasons of CP, which
is among the major causes for children referred to the
neuropediatric departments and rehabilitation institutions. P09.033A The CACNA1A gene encodes a calcium-dependent voltage
channel, localized in neuronal cells. Pathogenic variants in
this gene are known to lead to a broad clinical spectrum
including episodic ataxia type 2 (EA2), spinocerebellar
ataxia type 6, familial hemiplegic migraine, and more
recently epileptic encephalopathy. We report a large family
revealing wide variability of neurological manifestations
associated with a missense mutation. The index case had
early-onset epileptic encephalopathy with progressive cer-
ebellar
atrophy,
although his
mother
and his
great-
grandmother suffered from paroxystic episodic ataxia. His
grandfather and great grand-aunt reported no symptoms, but
two of her sons displayed progressive late-onset spinocer-
ebellar ataxia. Two of her little daughters also suffered from
epilepsy. All these relatives were carriers of a heterozygous
missense variant in CACNA1A: c.835C>T, p.(Arg279Cys)
(NM_023035.2). This CACNA1A variant has already been
described associated with EA2 phenotype. Prediction soft-
wares suggested a damaging effect on the protein, and this
variant was absent from GnomAD. Mutations in CACNA1A
may lead to several different phenotypes, including severe
epileptic encephalopathy which was only recently descri-
bed. Loss-of-function mutations in CACNA1A associated
with large phenotypic heterogeneity have rarely been
described. We report here the first missense mutation seg-
regating in a large family and leading to major clinical
variability with incomplete penetrance. Environmental fac-
tors and modifier genes may have influences on the phe-
notype. Our
family
highlights
difficulties
to
provide
accurate genetic counselling concerning prenatal diagnosis
in regard to highly variable severity of the clinical spectrum
and incomplete penetrance. N. Li: None. L. He: None. H. Tang: None. K. Xu:
None. H. Hu: None. Replication stress-mediated chromosome instability in the
Alzheimer’s disease brain Replication stress-mediated chromosome instability in the
Alzheimer’s disease brain C. Angelini: None. J. Van Gils: None. D. Lacombe:
None. S. Moutton: None. F. Riant: None. G. Sole: None. E. Tournier-Lasserve: None. A. Trimouille: None. C. Goizet: None. C. Angelini: None. J. Van Gils: None. D. Lacombe:
None. S. Moutton: None. F. Riant: None. G. Sole: None. E. Tournier-Lasserve: None. A. Trimouille: None. C. Goizet: None. Y. B. Yurov1,2, S. G. Vorsanova1,2, T. Liehr3, I. Y. Iourov1,2,4 Y. B. Yurov1,2, S. G. Vorsanova1,2, T. Liehr3, I. Y. Iourov1,2,4 1FSBSI «Mental Health Research Center», Moscow, Russian
Federation, 2Academician Yu.E. Veltishchev Research Clinical
Institute of Pediatrics, N.I. Pirogov Russian National Research
Medical University, Ministry of Health of the Russian
Federation, Moscow, Russian Federation, 3Institute of Human
Genetics, Jena, Germany, 4FSBEI FPE «Russian Medical
Academy of Postgraduate Education» of the Ministry of
Healthcare of the Russian Federation, Moscow, Russian
Federation 1FSBSI «Mental Health Research Center», Moscow, Russian
Federation, 2Academician Yu.E. Veltishchev Research Clinical
Institute of Pediatrics, N.I. Pirogov Russian National Research
Medical University, Ministry of Health of the Russian
Federation, Moscow, Russian Federation, 3Institute of Human
Genetics, Jena, Germany, 4FSBEI FPE «Russian Medical
Academy of Postgraduate Education» of the Ministry of
Healthcare of the Russian Federation, Moscow, Russian
Federation P09.035C Comprehensive genetic analyses implicate highly
heterogeneous etiology of idiopathic cerebral palsy
spectrum disorders N. Li1, L. He2, H. Tang2, K. Xu2, H. Hu1 266 J. del Picchia We retrospectively reviewed the results of diagnostic
array-CGH test on 700 cases with isolated or syndromic
forms of ID and ASDs. We assessed pathogenicity of
identified CNVs by combining mining of literature and
searching in databases reporting deleterious and benign
variants, and ID/ASD-associated genes. VOUS variants
encompassing no genes or genes without any apparent brain
expression/function were further investigated for potential
long
range
position
effects
through
involvement
of
Topologically Associating Domains (TADs), Lamina Asso-
ciated Domains (LADs), and other chromatin activation
signatures by using the web-based 3D Genome Browser and
UCSC Genome Browser. Introduction. Chromosome instability (CIN) was recently
suggested as a mechanism for neurodegeneration. However,
origins and underlying causes of CIN in the brain remain
obscure. To gain further insights into origins of brain-
specific CIN, we have addressed intercellular and inter-
individual genome variations in the Alzheimer’s disease
(AD) brain. Introduction. Chromosome instability (CIN) was recently
suggested as a mechanism for neurodegeneration. However,
origins and underlying causes of CIN in the brain remain
obscure. To gain further insights into origins of brain-
specific CIN, we have addressed intercellular and inter-
individual genome variations in the Alzheimer’s disease
(AD) brain. Materials and Methods. Using FISH-based techniques
(multiprobe/quantitative FISH and interphase chromosome-
specific multicolor banding), we have analyzed 14 AD post-
mortem brain samples. Additionally, we have evaluated
CNV by array CGH and an original bioinformatic technique
(Iourov et al., 2014; Yurov et al., 2017) for identifying
possible CIN origins. We identified non-benign CNVs in 314 patients. Among
the pathogenetic and the probably pathogenetic variants
(19%),
we
found
CNVs
spanning
known
ID/ASDs-
associated genes, including two different de novo deletions
and one duplication affecting genes whose mutations,
recently identified by whole exome sequencing (WES)
studies, were thought to act in ASDs with loss or gain of
function mechanisms, respectively. We further identified 15
cases with de novo VOUS variants involving novel
candidate genes and/or regions of regulatory expression of
flanking ASD-associated genes as one deletion and one
duplication spanning potential TAD boundaries and LADs. Results. CIN affected 5.2-20.3% of brain cells in the
samples. CIN essentially manifested as aneuploidy, struc-
tural chromosome rearrangements affecting mainly chromo-
some 21, chromosomal breaks resulting in the presence of
1-4 acentric chromosomal fragments in a nucleus. P09.035C Further
genomic analyses have shown that a CNV burden enriched
for cell cycle and similar pathways is observed in the AD
brain. Moreover, the distribution of genes affected by CNV
has indicated that there is a significant bias toward genes
involved in the DNA replication pathway. Conclusions. Previously, we proposed the DNA replica-
tion stress hypothesis of AD (Yurov et al., 2011). Here, an
empirical support of this hypothesis seems to be provided. Nonetheless, further studies are required to highlight
replication stress as a mechanism and a therapeutic target
for neurodegeneration treatment. Supported by ERA.Net
RUS Plus Programme. The updating of diagnostic array-CGH CNVs in the light
of new candidate genes and pathogenetic mechanisms
allowed us to unravel complex cases, and revealing
unexpected position effects. S. Bossi: None. M.T. Divizia: None. L. Pisciotta: None. E. Tassano: None. G. Rosti: None. I. Serio: None. M. Lerone: None. E. Veneselli: None. P. Ronchetto: None. A. Puliti: None. S. Bossi: None. M.T. Divizia: None. L. Pisciotta: None. E. Tassano: None. G. Rosti: None. I. Serio: None. M. Lerone: None. E. Veneselli: None. P. Ronchetto: None. A. Puliti: None. Y.B. Yurov: None. S.G. Vorsanova: None. T. Liehr:
None. I.Y. Iourov: None. P09.040D To confirm a position
effect event and assess the pathogenic role of the identified
CNV quantitative COL4A3BP gene expression analysis is
ongoing. Introduction: Hereditary congenital facial palsy (HCFP) is
a rare congenital cranial dysinnervation disorder, recogni-
sable by non-progressive isolated facial nerve palsy (cranial
nerve VII). It is caused by developmental abnormalities of
the facial nerve nucleus and its nerve. So far, 4 homozygous
mutations have been identified in 5 unrelated families (12
patients) with HCFP worldwide. Introduction: Hereditary congenital facial palsy (HCFP) is
a rare congenital cranial dysinnervation disorder, recogni-
sable by non-progressive isolated facial nerve palsy (cranial
nerve VII). It is caused by developmental abnormalities of
the facial nerve nucleus and its nerve. So far, 4 homozygous
mutations have been identified in 5 unrelated families (12
patients) with HCFP worldwide. Materials and Methods: a large Iranian consanguineous
kindred with 5 members affected by HCFP underwent
thorough clinical and genetic evaluation. The candidate
gene HOXB1 was screened and analysed by Sanger
sequencing. As in previous cases, the most remarkable
findings in the affected members of the family were mask-
like faces, bilateral facial palsy with variable sensorineural
hearing loss, and some dysmorphic features. Results: Direct sequencing of the candidate gene HOXB1
identified
a
novel
homozygous
frameshift
mutation
(c.296_302del; p.Y99Wfs * 20) which cosegregated with
the disease phenotype within the extended family. Conclusions: Our findings expand the mutational spec-
trum of HOXB1 involved in HCFP and consolidate the role
of the gene in the development of autosomal recessive
HCFP. Moreover, the truncating mutation identified in this
family leads to a broadly similar resentation and severity
observed in previous patients with nonsense and missense
mutations. This study haracterises and defines the pheno-
typic features of this rare syndrome in a larger family than
has previously been reported. G. Tolva: None. M. Crippa: None. D. Zimbalatti:
None. R. Silipigni: None. I. Bestetti: None. A. Sironi:
None. S. Guerneri:
None. D. Milani:
None. P. Finelli: None. M. Vahidi Mehrjardi: None. R. Maroofian: None. M. Dehghan Tezerjani: None. E. Zare Mehrjardi: None. H. Hozhabri:
None. M. Jaafarinia:
None. M. Dehghani: None. P09.040D Evaluation of CNVs variants in a cohort of isolated and
syndromic intellectual disability/autism spectrum disorders
reveals novel position effects and candidate disease genes Possible position effect on COL4A3BP gene regulation in a
family affected by neurological impairment Possible position effect on COL4A3BP gene regulation in a
family affected by neurological impairment Possible position effect on COL4A3BP gene regulation in a
family affected by neurological impairment p
g G. Tolva1, M. Crippa2, D. Zimbalatti2, R. Silipigni3, I. Bestetti2,4,
A. Sironi2,4, S. Guerneri3, D. Milani1, P. Finelli2,4 G. Tolva1, M. Crippa2, D. Zimbalatti2, R. Silipigni3, I. Bestetti2,4,
A. Sironi2,4, S. Guerneri3, D. Milani1, P. Finelli2,4 S. Bossi1, M. T. Divizia2, L. Pisciotta1, E. Tassano2, G. Rosti1,
I. Serio1,3, M. Lerone2, E. Veneselli1,3, P. Ronchetto2,
A. PULITI2,1 S. Bossi1, M. T. Divizia2, L. Pisciotta1, E. Tassano2, G. Rosti1,
I. Serio1,3, M. Lerone2, E. Veneselli1,3, P. Ronchetto2,
A. PULITI2,1 S. Bossi1, M. T. Divizia2, L. Pisciotta1, E. Tassano2, G. Rosti1,
I. Serio1,3, M. Lerone2, E. Veneselli1,3, P. Ronchetto2,
A. PULITI2,1 1Pediatric Highly Intensive Care Unit, Department of
Pathophysiology and Transplantation,University of Milan,
Fondazione IRCCS Ca' Granda, Milan, Italy, 2Lab. of Medical
Cytogenetics and Molecular Genetics, IRCCS Istituto
Auxologico Italiano, Milan, Italy, 3Laboratory of Medical
Genetics, Fondazione IRCCS Ca´ Granda, Ospedale Maggiore
Policlinico, Milan, Italy, 4Dpt. of Medical Biotechnology and
Translational Medicine, University of Milan, Milan, Italy 1DiNOGMI, University of Genova, Genova, Italy, 2Medical
Genetics Unit, Istituto Giannina Gaslini, Genova, Italy, 3Child
Neuropsychiatry Unit, Istituto Giannina Gaslini, Genova, Italy 1DiNOGMI, University of Genova, Genova, Italy, 2Medical
Genetics Unit, Istituto Giannina Gaslini, Genova, Italy, 3Child
Neuropsychiatry Unit, Istituto Giannina Gaslini, Genova, Italy Array-comparative genomic hybridization (array-CGH) is
widely used to detect copy number variants (CNVs) asso-
ciated with intellectual disability (ID) and autism spectrum
disorders (ASDs). We describe a 46,XX child aged 3 years, who came to our
attention because of slight psychomotor delay (GQ=77),
nystagmus, esotropia, and facial dysmorphisms, namely However, some CNVs have no clear causative effects and
remain of uncertain significance (VOUS). Abstracts from the 51st European Society of Human Genetics Conference: Posters 267 brachycephaly,
round
face,
and
palpebral
upslanting. Family history shows psychomotor retardation, mild school
difficulties (IQ =67), and nystagmus in her older brother,
partial cryptogenic epilepsy and esotropia in the mother,
and Rendu-Osler-Weber Syndrome both in the father and in
the brother. P09.042B A novel loss-of-function mutation in HOXB1 associated
with autosomal recessive hereditary congenital facial palsy
in a large iranian family Congenital Variant of Rett Syndrome: Three siblings with
FOXG1 mutation due to maternal gonodal mosaicism in a
Turkish family O. F. Karacorlu1,2, H. Bagis2, I. Guney3,2, M. Z. Kara4,
S. Ceylaner5 M. Vahidi Mehrjardi1, R. Maroofian2, M. Dehghan Tezerjani3,
E. Zare Mehrjardi4, H. Hozhabri5, M. Jaafarinia6,
M. Dehghani3 P09.040D Array-CGH analysis (60K) identified a rare
microdeletion of 70 kb in the child, in the mother and in the
brother,
mapping
in
5q13.3
region
(chr5:74822073-
74892303). This deletion involves the POLK gene, not
currently known as a disease gene. Interestingly, the dele-
tion proximal breakpoint maps about 20 kb from the 5’
UTR of COL4A3BP gene, which leads to mental retarda-
tion, autosomal dominant 34 (MRD34) [MIM:616351]. There is evidence of few cases with de novo missense
mutations in COL4A3BP gene reported as pathognomonic
for a phenotype characterized by psychomotor and global
development delay, intellectual disability, epilepsy, cranio-
facial dysmorphisms, and skeletal features. The subsequent
high resolution 1M a-CGH analysis finely maps the prox-
imal deletion bkp upstream the COL4A3BP promoter,
located in a region with several predicted regulatory ele-
ments (promoter region, enhancer and transcriptional elon-
gation). In our hypothesis the lack of mentioned regulatory
elements may compromise COL4A3BP gene expression by
altering the gene regulatory domain. To confirm a position
effect event and assess the pathogenic role of the identified
CNV quantitative COL4A3BP gene expression analysis is
ongoing. brachycephaly,
round
face,
and
palpebral
upslanting. Family history shows psychomotor retardation, mild school
difficulties (IQ =67), and nystagmus in her older brother,
partial cryptogenic epilepsy and esotropia in the mother,
and Rendu-Osler-Weber Syndrome both in the father and in
the brother. Array-CGH analysis (60K) identified a rare
microdeletion of 70 kb in the child, in the mother and in the
brother,
mapping
in
5q13.3
region
(chr5:74822073-
74892303). This deletion involves the POLK gene, not
currently known as a disease gene. Interestingly, the dele-
tion proximal breakpoint maps about 20 kb from the 5’
UTR of COL4A3BP gene, which leads to mental retarda-
tion, autosomal dominant 34 (MRD34) [MIM:616351]. There is evidence of few cases with de novo missense
mutations in COL4A3BP gene reported as pathognomonic
for a phenotype characterized by psychomotor and global
development delay, intellectual disability, epilepsy, cranio-
facial dysmorphisms, and skeletal features. The subsequent
high resolution 1M a-CGH analysis finely maps the prox-
imal deletion bkp upstream the COL4A3BP promoter,
located in a region with several predicted regulatory ele-
ments (promoter region, enhancer and transcriptional elon-
gation). In our hypothesis the lack of mentioned regulatory
elements may compromise COL4A3BP gene expression by
altering the gene regulatory domain. O. F. Karacorlu1,2, H. Bagis2, I. Guney3,2, M. Z. Kara4,
S. Ceylaner5 It
is the most severe form of atypical Rett syndrome, is gen-
erally caused by mutations in the FOXG1 gene which is
located on chromosome 14q12. Case Presentation: A 8-year-old girl with had severe
mental and motor retardation referred to our clinic because
of
two
siblings
also
had
similar
findings. Delayed
psychomotor development was noted at 6 months of age
and she sit, crawl or walk all late. She could spaek only a
few words. She also had bruxisim, tongue thrusting and
sialorrhea. Brain MRI was normal except cavum septum
pellucidum vergae. Karyotype was 46,XX and subtelomeric
FISH was normal. Whole exome sequencing of patient
reported with
a heterozygous
c.799G>A(p.Gly267Ser)
variant at FOXG1 gene. Variant classification was class 3
uncertain significance, according to in silico parameters
variant was 4/4 damaging and prediction was disease
causing. Due to clinical manifestations and heterozygous
mutation confirmed by sanger sequence analysis at FOXG1,
congenital variant of Rett syndrome (OMIM#613454)
diagnosed. Parents were not consangious and they had also
a 5-year-old girl and a 3-year-old boy have the same clinic
and mutation. While father had no mutation at FOXG1 gene
mother had 19% mutation at DNA obtained from blood
sample. A. Arlt: None. S. Käseberg: None. M. Linke: None. J. Winter: None. O. Bartsch: None. S. Davydenko: None. S. Schweiger: None. O. Tüscher: None. K. Komlosi: None. Conclusion: Totally three Congenital variant of Rett
syndrome described clinically and genetically at this family. Possible segregation analyses stated that all siblings have
disease due to maternal gonadal mosaicism. Dosage imbalance of the chromatin remodeler CHD1L is
responsible for the neurodevelopmental and under/
overgrowth phenotypes of the 1q21.1 CNVs O.F. Karacorlu: None. H. Bagis: None. I. Guney:
None. M.Z. Kara: None. S. Ceylaner: None. P09.044D Dosage imbalance of the chromatin remodeler CHD1L is
responsible for the neurodevelopmental and under/
overgrowth phenotypes of the 1q21.1 CNVs P09.043C M. Loviglio, C. Weber, C. Golzio M. Loviglio, C. Weber, C. Golzio M. Loviglio, C. Weber, C. Golzio O. F. Karacorlu1,2, H. Bagis2, I. Guney3,2, M. Z. Kara4,
S. Ceylaner5 M. Vahidi Mehrjardi1, R. Maroofian2, M. Dehghan Tezerjani3,
E. Zare Mehrjardi4, H. Hozhabri5, M. Jaafarinia6,
M. Dehghani3 1Ministery of Health, University of Health Sciences, Haseki
Training and Research Hospital, Diagnostic Center of Genetic
Diseases, Istanbul, Turkey, 2Adiyaman University Medical
Faculty Medical Genetics Department, Adiyaman, Turkey,
3Adana Numune Training and Research Hospital Medical
Genetics Department, Adana, Turkey, 4Adiyaman University
Training and Research Hospital Child and Adolescent
Psychiatry Department, Adiyaman, Turkey, 5Intergen Genetic
Diagnosis Center, Ankara, Turkey 1Medical Genetics Research Center, Yazd, Iran, Islamic
Republic of, 2RILD Wellcome Wolfson Centre, Royal Devon
and Exeter NHS Foundation Trust, Exeter, United Kingdom,
3Medical Genetics Research Center, Shahid Sadoughi
University of Medical Sciences, Yazd, Iran, Islamic Republic
of, 4Medical Biotechnology Research Center, Ashkezar
Branch, Islamic Azad University, Ashkezar, Yazd, Iran, Islamic
Republic of, 5Department of Experimental Medicine, Sapienza
University, Rome, Italy, 6Department of Genetic, Marvdasht
branch, Islamic Azad University, Marvdasht, Marvdasht, Iran,
Islamic Republic of Background: Congenital variant of Rett syndrome is a
severe neurodevelopmental disorder with features of classic J. del Picchia 268 history was significant for learning disability but diagnosis
of ADHD was only achieved at age 30. His father is being
treated for depression and anxiety disorder and his younger
sister has had a psychotic episode at age 18 and is currently
on anti-depressant treatment. After NGS analysis for con-
nective tissue diseases was normal in the proband, SNP
array (Affymetrix CytoScan® HD) identified a heterozygous
microduplication of 554 kb at 16p11.2. qPCR analysis
confirmed segregation also for the father and sister. Among
the 28 genes duplicated KIF22 and TBX6 are associated
with joint laxity, for other candidate genes behavioral
aberrations have been reported in mouse models. From
those we selected KIF22, DOC2A and SEZ6 and are per-
forming qPCR studies in fibroblasts and iPSCs of the
affected sister and unaffected mother. Our case demon-
strates the broad intrafamilial spectrum of neuropsychiatric
disturbances and adds data to the expression pattern of
candidate genes contributing to the mental phenotypes of
microduplication carriers. Interestingly, when comparing
the phenotype to another family with a CNV associated
with neuropsychiatric symptoms (16p13.2 deletion) affected
women seem to be more vulnerable to later onset psychotic
manifestations and carrier men rather to early onset neuro-
developmental disturbances. Rett syndrome, but earlier onset in the first months of life. Germany Recurrent
microduplications
on
chromosome
16p11.2
(OMIM #614671) have been implicated in childhood-onset
developmental disorders including language delay, cogni-
tive impairment, ADHD besides neuropsychiatric symp-
toms such as depression, anxiety, schizophrenia and bipolar
disorder. We report a German family whose son had initi-
ally
been
investigated
for
connective
tissue
disease
(ascending aortic aneurysm with 21 and joint laxity). Patient Intrafamilial variability of neuropsychiatric symptoms
associated with the microduplication of chromosome Intrafamilial variability of neuropsychiatric symptoms
associated with the microduplication of chromosome Intrafamilial variability of neuropsychiatric symptoms
associated with the microduplication of chromosome
16p11.2 Institut de Génétique et Biologie Moléculaire et Cellulaire
(IGBMC), Illkirch-Graffenstaden, France A. Arlt1, S. Käseberg1, M. Linke1, J. Winter1, O. Bartsch1,
S. Davydenko2, S. Schweiger1, O. Tüscher2, K. Komlosi1 Recurrent reciprocal 1q21.1 deletions and duplications have
been found in individuals with syndromic autism. Variable
phenotypes have been reported, including congenital heart
defects, autism, schizophrenia, head circumference and
height defects. The deletion is associated with microcephaly
and short stature, whereas the reciprocal duplication is
associated with increased risk of macrocephaly and carriers
tend to be in the upper height percentiles, suggesting a
possible undergrowth/overgrowth phenotype. We modeled
the 1q21.1 duplication by overexpressing each of the eight
1q21.1 genes in zebrafish. Strikingly, we found that the
overexpression of the chromatin remodeler CHD1L induces
an increased number of both brain proliferating cells and
post-mitotic neurons in the anterior forebrain at 2dpf,
resulting in macrocephaly at later stages. Consistently, 1Institute of Human Genetics, University Medical Center,
Mainz, Germany, 2Department of Psychiatry and
Psychotherapy,University Medical Center Mainz, Mainz,
Germany 1Institute of Human Genetics, University Medical Center,
Mainz, Germany, 2Department of Psychiatry and
Psychotherapy,University Medical Center Mainz, Mainz,
Germany CYP2C19 and CYP3A4 gene variants and schizophrenia in
Armenian patients CYP2C19 and CYP3A4 gene variants and schizophrenia in
Armenian patients CYP2C19 and CYP3A4 gene variants and schizophrenia in
Armenian patients E. V. Butenko1, R. F. O. Mamedov1, H. K. Ghazaryan2, R. V. Zakharyan3 M. Loviglio: None. C. Weber: None. C. Golzio: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 269 suppression of the zebrafish ortholog of CHD1L by
CRISPR/Cas9 led to a significantly decreased head size at
5dpf. We further evaluated whether CHD1L could be
implicated in the growth abnormalities observed in 1q21.1
CNV carriers; its overexpression indeed led to a significant
increase of the total body length and inter-somites distance
at 5dpf. We also showed that the combinatorial over-
expression of CHD1L and GJA8, another 1q21.1 gene,
exacerbates the neurodevelopmental alterations induced by
CHD1L overexpression alone, but does not impact further
the body growth, suggesting a genetic interaction between
CHD1L and GJA8 on some, but not all phenotypic com-
ponents. Our results suggest that CHD1L is a major con-
tributor of the 1q21.1 CNV-associated neurodevelopmental
phenotypes and indicate that CHD1L has a potential role in
the control of human growth via an epigenetic regulatory
mechanism. conservation among species they cannot be judged without
additional clinical information and feedback from the
specialist. In conclusion, the WES approach for malformations of
the cerebal cortex is a powerful tool for DNA diagnostics. In order to increase the diagnostic yield of cortical brain
malformations, close collaboration between laboratory and
referring specialist is mandatory. M. Wilke: None. G.M. Bolman:
None. M. van
Tienhoven: None. W.G. de Valk: None. R. Schot: None. R. van Minkelen: None. G.M.S. Mancini: None. M.A. van Slegtenhorst: None. M. Wilke: None. G.M. Bolman:
None. M. van
Tienhoven: None. W.G. de Valk: None. R. Schot: None. R. van Minkelen: None. G.M.S. Mancini: None. M.A. van Slegtenhorst: None. P09.045A 1Southern Federal University, Rostov-on-Don, Russian
Federation, 2Institute of Molecular Biology NAS RA, Yerevan,
Armenia, 3Institute of Molecular Biology NAS RA,Russian-
Armenian University, Yerevan, Armenia Malformations of the cerebal cortex: from targeted next
generation sequencing to whole exome sequencing Malformations of the cerebal cortex: from targeted next
generation sequencing to whole exome sequencing Malformations of the cerebal cortex: from targeted next
generation sequencing to whole exome sequencing M. Wilke, G. M. Bolman, M. van Tienhoven, W. G. de Valk,
R. Schot, R. van Minkelen, G. M. S. Mancini, M. A. van
Slegtenhorst Introduction: Genetic variations play an important role in
antipsychotic drug treatment response in several mental
disorders, including schizophrenia. Clinical studies sug-
gested that antipsychotic drug metabolizing enzymes of
cytochrome P450 family contribute to reduction of disease
symptoms and manifestation of side effects. In this study we
aimed to investigate the potential of schizophrenia with two
single nucleotide polymorphisms (SNPs) of genes, coding
CYP2C19 and CYP3A4 enzymes (CYP2C19 rs4244285
and CYP3A4 rs2740574, respectively). Department of Clinical Genetics, Rotterdam, Netherlands A missense variant in PER2 is associated with delayed
sleep-wake phase disorder 1Humanitas University, Pieve Emanuele (MI), Italy,
2University of Milan, Milan, Italy, 3Humanitas Clinical and
Research Center, Rozzano, Italy T. Miyagawa1,2, M. Shimada1,2, A. Hida3, Y. Honda4,
K. Mishima3, K. Tokunaga2, M. Honda1,4 Introduction:
We
recently
described
biallelic
null-
mutations in the DNAJC12 gene in two probands of unre-
lated families with early-onset, dopa-responsive, and non-
progressive parkinsonism. This gene encodes a member of
DNAJ/Hsp40 family. It was suggested that DNAC12
interacts
with the aromatic amino
acid hydroxylases
involved in the dopamine and serotonin metabolism. Despite this hypothesis, its function is still unclear. Understanding DNAJC12 physiological role could shed
light on new pathways potentially involved in Parkinson
disease pathogenesis and progression. 1Tokyo Metropolitan Institute of Medical Science, Tokyo,
Japan, 2The University of Tokyo, Tokyo, Japan, 3National
Center of Neurology and Psychiatry, Tokyo, Japan,
4Neuropsychiatric Research Institute, Tokyo, Japan Delayed sleep-wake phase disorder (DSWPD) is a circadian
rhythm sleep disorder, and is characterized by an inability to
fall asleep until very late at night and awaken at a socially
acceptable morning time. The pathogenesis of DSWPD is
poorly understood. Recently, several large scale GWASs of
chronotype have reported genetic variants associated with
variation in chronotype. Several associated variants were
located in genes related to circadian rhythms. This study
was performed to identify variants associated with DSWPD
from known circadian genes. We focused on low-frequency
missense variants. We utilized data obtained from databases
of genetic variations (whole exome-/ genome- sequencing). Candidates were extracted by integrating the data and in
silico assessment. DNA samples from 236 patients with
DSWPD and 1,436 controls were genotyped to examine
whether the candidates are associated with DSWPD. A
missense variant (p.Val1205Met) in PER2 showed a sig-
nificant association with DSWPD (minor allele frequency
(MAF) of 2.5% in cases and 1.1% in controls, P=0.026,
odds ratio = 2.32). In addition, MAF of the variant in 222
patients with idiopathic hypersomnia was significantly
higher than that in 3,554 controls (MAF of 2.3% in cases
and 1.1% in controls, P=0.038, odds ratio = 2.07). PER2 is
noted for its major role in circadian rhythms. PER2 forms a
heterodimer with CRY, and the heterodimer plays an
important role in the regulation of the circadian rhythm. The
p.Val1205Met substitution was located in the PER2 CRY-
binding domain. The substitution could be a potential
genetic marker for circadian rhythms and sleep phenotypes. Department of Clinical Genetics, Rotterdam, Netherlands Conclusion:
Despite this
pilot
study
identified
no
association between schizophrenia and genetic variants
within the CYP2C19 and CYP3A4 genes, further studies in
large sample size and independent research centers are
required to clarify these findings.Funding: the basic part of
the Ministry of education and science of the Russian
Federation, state task project No. 6.6762.2017/BT. P09.048D
A novel role for DNAJC12, a gene recently associated with
hyperphenylalaninemia and early-onset dopa-responsive
parkinsonism, in brain development A novel role for DNAJC12, a gene recently associated with
hyperphenylalaninemia and early-onset dopa-responsive
parkinsonism, in brain development E.V. Butenko: None. R.F.O. Mamedov: None. H.K. Ghazaryan: None. R.V. Zakharyan: None. D. Facchi1, A. Ghilardi2, L. Straniero1, V. Rimoldi1, E. Saba1,
G. Soldà1,3, R. Asselta1,3, S. Duga1,3, L. Del Giacco2 Department of Clinical Genetics, Rotterdam, Netherlands Cortical brain malformations (rare disorders of proliferation,
neuronal migration or cortical organization) have been
associated with mutations in a rapidly growing number of
genes. This complicates molecular diagnostic by Sanger
sequencing. Until 2015, we used a targeted Next Generation
Sequencing (NGS) based work flow with a panel of 103
genes for routine diagnostic. Nowadays our flow is based on
whole exome sequencing (WES) using a filter for a panel of
175 relevant genes. This approach has the advantage that
novel genes can be easily included and expansion to full
exome analysis is possible. Materials and Methods: Here patients with paranoid
form of schizophrenia and healthy subjects of Armenian
population were enrolled. DNA was isolated using salting
out method with a simple introduction of chloroform step. Genotyping was performed using PCR-SSP. Distribution of
genotypes corresponded to Hardy-Weinberg equilibrium. Statistical analysis was performed using Pearson’s Chi-
squared test. The WES work-flow involved the DNA enrichment using
the Agilent SureSelect CRE Capture. The detected variants
are filtered and annotated with the Cartagenia software and
classified with Alamut Visual Results: We found that genotypes and allele frequency of
the CYP2C19 gene rs4244285 polymorphism was equally
distributed among the study groups (CYP2C19 681A allele
frequency in cases vs. controls: 0.11 vs. 0.17, p = 0.46). The same applies for the CYP3A4 rs2740574*G allele
frequency (0.017 vs. 0.019, p = 0.96). Interestingly, the
minor allele frequencies obtained significantly differ from
those in 1000 Genomes that might reflect ethnic differences
in the populations enrolled. DNA samples of 108 individuals were tested with the
WES based panel. Eight patients (7,4%) received a direct
diagnosis and 12 (11.1%) patients were solved after
additional investigations. With the previous used targeted
NGS approach in total 192 patients were tested with a
diagnostic yield of 12,5%. However, most of the identified alterations are variants of
unknown clinical relevance. Despite the use of in silico
prediction programs, usage of frequencies, evaluation of the 270 J. del Picchia T. Miyagawa: None. M. Shimada: None. A. Hida:
None. Y. Honda: None. K. Mishima: None. K. Toku-
naga: None. M. Honda: None. Conclusion:
Despite this
pilot
study
identified
no
association between schizophrenia and genetic variants
within the CYP2C19 and CYP3A4 genes, further studies in
large sample size and independent research centers are
required to clarify these findings.Funding: the basic part of
the Ministry of education and science of the Russian
Federation, state task project No. 6.6762.2017/BT. Instituto de Genética Médica y Molecular (INGEMM), Madrid,
Spain Instituto de Genética Médica y Molecular (INGEMM), Madrid,
Spain Materials and Methods: We established a disease model
of SCN1A associated early onset epilepsy, i.e. Dravet
Disease, using induced pluripotent stem cells (iPSC) from
three patients with distinct and heterozygous SCN1A
variants, and from three independent control individuals. The iPSCs were differentiated into cortical GABAergic
interneuron-like cells over 65 days and subsequently
analysed by whole-cell patch clamp recordings and RNA
sequencing. Introduction: Dravet syndrome (DS, MIM 607208) is a
severe early-onset infantile epilepsy that is mainly due to
heterozygous mutations in SCN1A, and other genes. The
application of a comprehensive NGS panel of genes related
to epilepsy may allow identifying other genes and epi-
leptogenic pathways involved in DS. Materials and Methods: 125 patients with a presumptive
diagnosis of DS were studied. The NGS panel Epilep-
sy_v5.0, with 425 genes related to epilepsy, was applied by
Roche
Nimblegen
SeqCap
EZ
capture
and
Illumina
NextSeq. The in-house bioinformatics analysis tool was
applied to the alignment and annotation of variants. Filtering of variants, variant interpretation by ACMG/
AMP and subsequent validation by Sanger sequencing or
MPLA were performed. Materials and Methods: 125 patients with a presumptive
diagnosis of DS were studied. The NGS panel Epilep-
sy_v5.0, with 425 genes related to epilepsy, was applied by
Roche
Nimblegen
SeqCap
EZ
capture
and
Illumina
NextSeq. The in-house bioinformatics analysis tool was
applied to the alignment and annotation of variants. Filtering of variants, variant interpretation by ACMG/
AMP and subsequent validation by Sanger sequencing or
MPLA were performed. q
g
Results: Patch-clamp recordings showed decreased fast
sodium currents in neuron-like cells from the three patients. Transcriptome profiling of neuron-like cells from Dravet
patients revealed persistent upregulation of genes involved
in
chromatin
remodeling. Additionally,
several
genes
belonging to an epilepsy gene network were dysregulated
supporting functional convergence for a group of genetic
epilepsies. Results: Patch-clamp recordings showed decreased fast
sodium currents in neuron-like cells from the three patients. Transcriptome profiling of neuron-like cells from Dravet
patients revealed persistent upregulation of genes involved
in
chromatin
remodeling. Additionally,
several
genes
belonging to an epilepsy gene network were dysregulated
supporting functional convergence for a group of genetic
epilepsies. Results: Epilepsy_v5.0 applied over 125 DS patients
allowed the molecular diagnosis of 47 patients carrying
SCN1A pathogenic (P, 25), likely pathogenic (LP, 15) or
variants of unknown significance (VOUS, 7). P09.051C Expanding the clinical and molecular spectrum of
DYRK1A-related disorder: report on novel mutations in
three patients with syndromic intellectual disability,
microcephaly, febrile seizures, distinctive facial
dysmorphisms and cerebellar vermis hypoplasia Grants: Health Research Institute (FIS) PI14-01753
(2014-2018). E. Barroso: None. A. Rubio: None. A. del Pozo: None. P. Lapunzina: None. S. Barresi1, M. Dentici1, A. Ciolfi1, E. Agolini1, M. Macchiaiolo1,
S. Pizzi1, C. Leoni2, M. Niceta1, F. Pantaleoni1, F. Radio1,
A. Capuano1, R. Onesimo2, M. Digilio1, A. Novelli1,
G. Zampino2, A. Bartuli1, B. Dallapiccola1, M. Tartaglia1 P. Lapunzina: None. Instituto de Genética Médica y Molecular (INGEMM), Madrid,
Spain Moreover, 39
SD patients without mutations in SCN1A carry P variants
(6), LP (2) or VOUS (31) in 35 additional genes that could
explain their molecular defect. Conclusions: Our data suggest chromatin remodeling to
be perturbed in neurons with SCN1A variants from Dravet
disease patients thus providing candidate pathways for the
development of antiepileptic drugs. Grants: This work was supported by grants from the
Swedish Research Council (2015-02424) and AstraZeneca. Conclusions: 68.8% of the tested patients (86/125)
presented variants in SCN1A and additional genes that
could explain DS. The application of Epilepsy_v5.0 has
improved
the
mutational
rate
of
SCN1A
by
Sanger
sequencing and MLPA in DS patients, established in our
laboratory in 44.3%. Genes identified in this study and not
previously related to DS may open a new way for the
molecular diagnosis and treatment of DS patients. S. Jens: None. L. Laan: None. J. Klar: None. Z. Jin:
None. M. Huss: None. S. Korol: None. F.H. Norradin:
None. B. Birnir: None. N. Dahl: None. S. Jens: None. L. Laan: None. J. Klar: None. Z. Jin:
None. M. Huss: None. S. Korol: None. F.H. Norradin:
None. B. Birnir: None. N. Dahl: None. S. Jens1, L. Laan1, J. Klar1, Z. Jin1, M. Huss2, S. Korol1, F. H.
Norradin1, B. Birnir1, N. Dahl1 S. Jens1, L. Laan1, J. Klar1, Z. Jin1, M. Huss2, S. Korol1, F. H. Norradin1, B. Birnir1, N. Dahl1 1Uppsala University, Uppsala, Sweden, 2Stockholm University,
Uppsala, Sweden 1Uppsala University, Uppsala, Sweden, 2Stockholm University,
Uppsala, Sweden A missense variant in PER2 is associated with delayed
sleep-wake phase disorder (Grants: KAKENHI and AMED) Materials and Methods: Immunofluorescence experi-
ments were performed in HepG2 and in differentiated SH-
SY5Y cell lines to assess DNAJC12 cellular localization
and co-localization with potential cellular partners. To
better characterize its function, the DNAJC12-zebrafish
orthologue (dnajc12) has been cloned and the expression
analyzed during embryogenesis. Gene functional ablation
assays were carried out in zebrafish embryos using mRNA-
specific antisense morpholino oligonucleotides. Histological
analyses were performed to evaluate the effects of the loss-
of-function approach. Results: Our immunofluorescence experiments showed
that
DNAJC12
does
not
present
a
specific
cellular
localization but is present both in the cytoplasmic and the
nuclear compartments. Concerning the in-vivo experiments
in zebrafish, the dnajc12-morphants were characterized by a
severe brain developmental phenotype. In particular, the
morpholino-injected embryos displayed a marked expan-
sion of the cerebral ventricles. Conclusion: Our results suggest the existence of other
unknown functions for DNAJC12 beyond dopamine and
serotonin metabolism. Further studies will help shedding
light on the specific role of this gene during early brain
development. Abstracts from the 51st European Society of Human Genetics Conference: Posters 271 D. Facchi: None. A. Ghilardi: None. L. Straniero:
None. V. Rimoldi: None. E. Saba: None. G. Soldà: None.
R. Asselta: None. S. Duga: None. L. Del Giacco: None. D. Facchi: None. A. Ghilardi: None. L. Straniero:
None. V. Rimoldi: None. E. Saba: None. G. Soldà: None. R. Asselta: None. S. Duga: None. L. Del Giacco: None. E. Barroso, A. Rubio, A. del Pozo, P. Lapunzina E. Barroso, A. Rubio, A. del Pozo, P. Lapunzina E. Barroso, A. Rubio, A. del Pozo, P. Lapunzina 1Genetics and Rare Diseases Research Division, Bambino
Gesù Children's Hospital, IRCSS, Rome, Italy, 2Rare Diseases S. Barresi1, M. Dentici1, A. Ciolfi1, E. Agolini1, M. Macchiaiolo1,
S. Pizzi1, C. Leoni2, M. Niceta1, F. Pantaleoni1, F. Radio1,
A. Capuano1, R. Onesimo2, M. Digilio1, A. Novelli1,
G. Zampino2, A. Bartuli1, B. Dallapiccola1, M. Tartaglia1 P09.049A
Identification of the genetic defect in patients with Dravet
syndrome by a NGS gene panel for epilepsy Introduction: Genetic epilepsies are devastating conditions
for patients and their families. Existing anticonvulsant drugs
do not provide satisfactory seizure control in one third of all
patients. One approach to advance the development of
novel antiepileptic drugs is to identify druggable molecular
mechanism underlying the disease. P09.050B Co-dysregulation of epilepsy genes and disrupted
chromatin architecture in an iPSC-derived model of Dravet
syndrome 1Genetics and Rare Diseases Research Division, Bambino
Gesù Children's Hospital, IRCSS, Rome, Italy, 2Rare Diseases 272 J. del Picchia and Genetic Disorders, Fondazione Policlinico Universitario
Agostino Gemelli, Rome, Italy and Genetic Disorders, Fondazione Policlinico Universitario
Agostino Gemelli, Rome, Italy The association between the KIAA0319 gene and dyslexia
was reported almost 15 years ago. While the association has
been consistently replicated, KIAA0319 function remains
poorly understood. Initial characterizations showed a spe-
cific expression in the human developing cortex and in utero
shRNA experiments in rats suggested a role in neuronal
migration. Conversely, recent studies in mice reported
effects in the auditory system but not in neuronal migration. To further elucidate the function of KIAA0319 we used a
range of approaches. Gene expression analysis in zebrafish
revealed a specific pattern restricted to particular developing
structures, confirming a role in the brain and in the auditory
system as well as in the visual system and the notochord. The dyslexia associated genetic variants reside in regulatory
sequences and might therefore affect this tightly regulated
spatial/temporal pattern. To investigate the function of
KIAA0319, we generated and characterised stable cellular
knockouts with different assays. These included the newly
developed Elastic Resonator Interference Stress Microscopy
(ERISM) system which allows the study of mechanical
forces, such as cell-substrate interactions, at single cell
level. This approach shows that KIAA0319 plays a role in
cell motility, migration and attachment, three processes
mediated by the cytoskeleton. Taken together, these data
support a role for KIAA0319 in cytoskeleton dynamics,
consistent with an involvement in neuronal migration. However, such role is likely to extend beyond brain
development and contribute also to the development of
sensory organs and the notochord. Supported by the Royal
Society, Northwood Trust, EPSRC and the ERC. Background: Haploinsufficiency of DYRK1A underlies a
recognizable Intellectual Disability (ID) syndrome (MIM
#614104). To date, about 50 patients have been reported
with clinical features including microcephaly, intrauterine
growth restriction, global developmental delay, ID, feeding
difficulties, and distinctive facial dymorphisms. Other
common finding include febrile seizures, and behavioral
disturbances
encompassing
autism
spectrum
disorder,
attention deficit disorder, and stereotypic movements. Aspecific
brain
malformations
have
been
described,
including enlarged ventricles, cortical atrophy, thin brain-
stem and thin corpus callosum. Results: We report on three Italian patients heterozygous
for previously unreported de novo DYRK1A mutations
identified by exome sequencing. P09.050B The three variants were
predicted to elicit a disruptive effect, being frameshift/
nonsense changes. In one subject, the impact on transcript
processing was confirmed experimentally.The three patients
presented clinical features within the phenotypic spectrum
associated with DYRK1A mutations, including intrauterine
growth restriction, microcephaly, severe speech impair-
ment, ID, feeding difficulties, febrile seizures, behavioral
issues and suggestive facies (prominent metopic appear-
ance, deep set eyes, blepharophimosis, microretrognathia). Of note, all patients presented brain abnormalities, includ-
ing cerebellar vermis hypoplasia, which have not previously
been reported to occur in individuals with DYRK1A
mutations. S. Paracchini: None. R. Diaz: None. M. Gostic: None. A. Martinelli: None. N. Kronenberg: None. K. Sillar:
None. J. Tello: None. M. Gather: None. S. Paracchini: None. R. Diaz: None. M. Gostic: None. A. Martinelli: None. N. Kronenberg: None. K. Sillar:
None. J. Tello: None. M. Gather: None. Conclusion: Our findings provide further information on
the clinical phenotype spectrum associated with truncating
mutations in DYRK1A, adding cerebellar vermis hypoplasia
as
a
previously
unappreciated
feature
of
DYRK1A
haploinsufficiency. S. Paracchini, R. Diaz, M. Gostic, A. Martinelli, N. Kronenberg,
K. Sillar, J. Tello, M. Gather Dopaminergic dysfunction in a rat model for DYT6
dystonia Dopaminergic dysfunction in a rat model for DYT6
dystonia S. Barresi: None. M. Dentici: None. A. Ciolfi: None. E. S. Barresi: None. M. Dentici: None. A. Ciolfi: None. E. Agolini: None. M. Macchiaiolo: None. S. Pizzi: None. C. Leoni: None. M. Niceta: None. F. Pantaleoni: None. F. Radio: None. A. Capuano: None. R. Onesimo: None. M. Digilio: None. A. Novelli: None. G. Zampino: None. A. Bartuli:
None. B. Dallapiccola:
None. M. Tartaglia: None. F. Cheng1, P. Bonsi2, N. Casadei1, L. Yu-Traeger1, T. Ott1,
O. Riess1, A. Pisani3, H. Nguyen1, K. Grundmann-Hauser1 F. Cheng1, P. Bonsi2, N. Casadei1, L. Yu-Traeger1, T. Ott1,
O. Riess1, A. Pisani3, H. Nguyen1, K. Grundmann-Hauser1 1Institute of Medical Genetics and Applied Genomics,
Tübingen, Germany, 2Fondazione Don Gnocchi, Milan, Italy,
3Istituto di Ricovero e Cura a Carattere Scientifico Fondazione
Santa Lucia, Neurophysiology and Plasticity Lab, Rome, Italy 1Institute of Medical Genetics and Applied Genomics,
Tübingen, Germany, 2Fondazione Don Gnocchi, Milan, Italy,
3Istituto di Ricovero e Cura a Carattere Scientifico Fondazione
Santa Lucia, Neurophysiology and Plasticity Lab, Rome, Italy University of St Andrews, St Andrews, United Kingdom P09.052D The KIAA0319 dyslexia susceptibility gene presents a
highly specific expression pattern during the development
of different organs & plays a role in cytoskeleton dynamics The KIAA0319 dyslexia susceptibility gene presents a
highly specific expression pattern during the development
of different organs & plays a role in cytoskeleton dynamics Mutations in THAP1 (thanatos-associated protein domain-
containing apoptosis- associated protein 1) cause autosomal
dominant primary dystonia 6 (DYT6 dystonia). To date,
more than eighty different mutations / variations have been
identified in the THAP1 gene in different ethnic populations
with unknown pathogenic mechanism. Recent studies of
Thap1 mouse models showed a delay in myelination or Abstracts from the 51st European Society of Human Genetics Conference: Posters 273 dysfunction of pathways related to eIF2α Signaling and
mitochondrial dysfunction. However, the neuropathological
and neurophysiological changes in rats due to THAP1
dysfunction are unknown. Using the CRISPR/cas9 techni-
que, we generated a novel model for THAP1 dystonia in a
different species. As observed in THAP1 mouse models
homozygous Thap1 knock-out rats are not viable. RNA-seq
and protein analysis in heterozygous KO rats revealed
expression changes of genes involved in doparminergic
functios. Electrophysiological
studies
in
the
striatum
revealed abnormal firing frequency in Thap1 heterozygous
knock-out rats stimulated with amphetamine. Taken toge-
ther, our novel THAP1 dytonia rat model showed a dystonia
related
phenotype
associated
with
dopaminergic
dysfunction. performed using in-house pipelines. In 2017 molecular
genetic study for trio (proband with parents) performed at
Birmingham Women’s Hospital (England) confirmed de
novo mutation in STXBP1 for proband. performed using in-house pipelines. In 2017 molecular
genetic study for trio (proband with parents) performed at
Birmingham Women’s Hospital (England) confirmed de
novo mutation in STXBP1 for proband. performed using in-house pipelines. In 2017 molecular
genetic study for trio (proband with parents) performed at
Birmingham Women’s Hospital (England) confirmed de
novo mutation in STXBP1 for proband. Conclusions: Presently, the boy 3y/o is under supervision
and
drug
therapy;
he
has
some
temporal
delay
of
psychomotor and speech development. The authors are
grateful to MD Zhilina S. for clinical genetics consulting. The research was supported by the Department of Health of
Moscow (project 2014-2015). M. Belenikin:
None. E. Lukyanova:
None. S. Ayvazyan: None. M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 1Moscow Institute of Physics and Technology (State
University), Dolgoprudny, Russian Federation, 2Research
Center for Children Medical Care, Moscow, Russian
Federation Introduction: Early-onset epilepsy (EOE) is characterized
by seizures of variable intensity, cognitive impairment and
unusual behavior. Approximately twelve forms of epilepsy
have genetic etiology, however, in more than a half of the
EOE patients, the basis remains unknown. Thus, an
unequivocal
molecular
diagnosis in
these patients is
essential to develop targeted-disease therapies. Background: Early Infantile Epileptic Encephalopathy
Type 4 (MIM:612164) is autosomal dominant disease
caused by heterozygous mutation in the STXBP1 gene. Here
we present the clinical case report. The boy (birth year
2013): at the 3rd week of life a single focal seizure attack
was appeared. In 4 months resumed seizures (complex
partial and secondary generalized tonic-clonic). In 5 months
there were seizures on the type of infantile spasms. At 2014
we have performed the molecular-genetic study. Material and Methods: We studied 147 patients with
seizures and/or myoclonus whose started between 2 and 4
years of age using a customized panel (Agilent SureSelect
technologies / MiSeq Illumina, CA). A total of 51 genes
associated to
epilepsy
in
previous publications
were
selected for investigation. The analysis was performed
using
VariantStudio
(Illumina,
CA)
and
SureCall
(Agilent, CA). Material and Methods: We studied 147 patients with
seizures and/or myoclonus whose started between 2 and 4
years of age using a customized panel (Agilent SureSelect
technologies / MiSeq Illumina, CA). A total of 51 genes
associated to
epilepsy
in
previous publications
were
selected for investigation. The analysis was performed
using
VariantStudio
(Illumina,
CA)
and
SureCall
(Agilent, CA). Methods and Results: NGS sequencing (454 GSJunior;
NimbleGen SeqCap target enrichment; testing mutations for
genes,
associated
with
epileptic
encephalopathy):
in
STXBP1 gene we have found heterozygous mutation
c.1235_1236delCC
(NM_003165.3)
or,
using
uniprot
canonical transcript, c.1038_1039delCC (CCDS35146.1/
CCDS6874.1). This two-nucleotide deletion results to
frame-shift
joined
with
nonsense
mutation,
changing
protein sequence: "...THLHL..." => "...TPAPC*". Further
clinical exome analysis (Illumina) did not revealed any
other
candidate-mutations. Clinical
picture
of
disease
allowed to assume de novo mutation. Data analysis was Methods and Results: NGS sequencing (454 GSJunior;
NimbleGen SeqCap target enrichment; testing mutations for
genes,
associated
with
epileptic
encephalopathy):
in
STXBP1 gene we have found heterozygous mutation
c.1235_1236delCC
(NM_003165.3)
or,
using
uniprot
canonical transcript, c.1038_1039delCC (CCDS35146.1/
CCDS6874.1). This two-nucleotide deletion results to
frame-shift
joined
with
nonsense
mutation,
changing
protein sequence: "...THLHL..." => "...TPAPC*". Multi-gene panel testing improves diagnosis in Brazilian
patients with Early-Onset Epilepsy Multi-gene panel testing improves diagnosis in Brazilian
patients with Early-Onset Epilepsy F. Cheng: None. P. Bonsi: None. N. Casadei: None. L. Yu-Traeger: None. T. Ott: None. O. Riess: None. A. Pisani:
None. H. Nguyen:
None. K. Grundmann-
Hauser: None. G. M. Novo Filho1, A. T. Dias1, A. M. Nascimento1, J. G. Damasceno1, É. A. Zanardo1, S. N. Chehimi1, F. A. R. Madia1,
O. S. Akl1, O. S. Akl1, M. M. Montenegro1, Y. G. Oliveira1, L. L. Vieira1, M. L. G. Manreza2, L. D. Kulikowski1 P09.054B A novel de novo nonsense mutation in STXBP1 found in
child diagnosed with early infantile epileptic
encephalopathy type 4 A novel de novo nonsense mutation in STXBP1 found in
child diagnosed with early infantile epileptic
encephalopathy type 4 1Laboratório de Citogenômica LIM 03 – Departamento de
Patologia – Faculdade de Medicina da Universidade de São
Paulo – São Paulo-SP, Brazil, São Paulo, Brazil,
2Departamento de Neurologia - Faculdade de Medicina da
Universidade de São Paulo – São Paulo-SP, Brazil, São Paulo,
Brazil M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 Diagnostic yield of aCGH and NGS gene panels in epilepsy Diagnostic yield of aCGH and NGS gene panels in epilepsy Gain-of-function variants in the CASR gene in genetic
generalized epilepsy L. A. Alcaraz, F. Galán, V. Penacho, I. Manchón, D. Amorós,
S. González-Reig, N. Castejón L. A. Alcaraz, F. Galán, V. Penacho, I. Manchón, D. Amorós,
S. González-Reig, N. Castejón L. A. Alcaraz, F. Galán, V. Penacho, I. Manchón, D. Amorós,
S. González-Reig, N. Castejón M. Kaur1, P. Satishchandra2, S. Sinha2, A. Kapoor1, A. Anand1 1Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore, India, 2National Institute of Mental Health and
Neurosciences, Bangalore, India Bioarray, SL, Elche, Spain Bioarray, SL, Elche, Spain Introduction: genetic testing is a very powerful tool for the
diagnosis of epilepsy. Copy-number variations (CNVs) or
point mutations cause epileptic disorders or predispose to
such heterogeneous pathology. Detection of CNVs require
one method such as microarray CGH (aCGH), while point
mutations within related genes could be detected by next
generation sequencing technologies (NGS). We performed
a retrospective cohort study, comparing the diagnostic yield
of aCGH and NGS gene panels in epilepsy. Introduction: Genetic generalized epilepsies (GGE) are a
common form of human epilepsies with substantial genetic
basis to their etiology. The EIG8 (3q13.3-q21) locus for
GGE was identified in a three-generation family from south
India (Kapoor et al. Ann Neurol 2008). Methods: Sequence analysis of the 64 Mb haplotype at
3p14.2-q21, being shared by all affected members of the
family was conducted by whole-exome sequencing (WES). CASR was sequence analyzed in unrelated 480 GGE/JME
patients
and
504
control
chromosomes. Further,
the
functional implications of rare non-synonymous mutations
identified were evaluated on the CASR-regulated signaling
pathways. Methods: Sequence analysis of the 64 Mb haplotype at
3p14.2-q21, being shared by all affected members of the
family was conducted by whole-exome sequencing (WES). CASR was sequence analyzed in unrelated 480 GGE/JME
patients
and
504
control
chromosomes. Further,
the
functional implications of rare non-synonymous mutations
identified were evaluated on the CASR-regulated signaling
pathways. Material and Methods: we performed a retrospective
cohort study of a series of neuropediatric patients with
seizures as the main symptom of epilepsy. In 158 cases,
aCGH was performed, while 231 cases were studied by
Illumina targeted-exome sequencing and specific NGS
panels. Results: From the 158 cases studied by aCGH we
detected pathogenic or likely pathogenic CNVs in 26
(16.4%) patients. As for NGS panels, we identified
deleterious mutations in 27 (17.7%) cases with 46-gene
panel and 22 cases (27.8%) with 543-gene panel. M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 Further
clinical exome analysis (Illumina) did not revealed any
other
candidate-mutations. Clinical
picture
of
disease
allowed to assume de novo mutation. Data analysis was Results: Our results revealed 72 patients with 113
relevant variants in 42 different genes, highlighting ATM,
CLN6, MSFD8, MECP2 and TPP1 genomic variants. Of
the 113 relevant variants detected by NGS panel, 20 were
classified as pathogenic, 31 likely pathogenic, 41 were a
variant of uncertain significance (VUS) and 21 likely
benign. The most frequent variant was single nucleotide
variants (SNVs) and InDels, being 91 in heterozygosis e 22
in homozygosis. J. del Picchia 274 Competitiveness (MINECO), with specific projects PTQ-
12-05686 for Amoros D, PTQ-12-05687 for Penacho V and
PTQ-13-06028
for
González-Reig
S. The
MINECO
Industrial Ph.D. grant with specific project DI-14-06922
for Castejón N. Conclusions: A total of ~49% of patients obtained a
potential genetic diagnosis evidencing the efficiency of our
customized panel, which allows a high detection rate with a
lower cost than a whole exome. A customized multi-gene
panel allows detect genetic variants and also could further
improve genetic diagnosis in patients with early-onset
Epilepsy. L.A. Alcaraz: A. Employment (full or part-time);
Significant; Bioarray, SL. F. Galán: A. Employment (full
or part-time); Significant; Bioarray, SL. V. Penacho: A. Employment (full or part-time); Significant; Bioarray, SL. I. Manchón: A. Employment (full or part-time); Significant;
Bioarray, SL. D. Amorós: A. Employment (full or part-
time); Significant; Bioarray, SL. S. González-Reig: A. Employment (full or part-time); Significant; Bioarray, SL. N. Castejón: A. Employment (full or part-time); Signifi-
cant; Bioarray, SL. Grant References: FAPESP 2016/09452-0; CAPES; Grant References: FAPESP 2016/09452-0; CAPES;
OMIKA Consultoria em Genética and BioMarin. OMIKA Consultoria em Genética and BioMarin. G.M. Novo Filho: None. A.T. Dias: None. A.M. Nascimento:
None. J.G. Damasceno:
None. É.A. Zanardo: None. S.N. Chehimi: None. F.A.R. Madia:
None. O.S. Akl: None. O.S. Akl: None. M.M. Montene-
gro: None. Y.G. Oliveira: None. L.L. Vieira: None. M.L. G. Manreza: None. L.D. Kulikowski: None. Detection of copy number variations in epilepsy using
exome data N. Matsumoto, N. Tsuchida N. Matsumoto, N. Tsuchida N. Matsumoto, N. Tsuchida Department of Human Genetics, Yokohama City University
Graduate School of Medicine, Yokohama, Japan Epilepsy comprises a wide range of etiologically very het-
erogeneous clinical conditions ranging from benign forms
to treatment-refractory progressive encephalopathies, which
clinical features, seizure type, age of onset, electro-
encephalographic features and response to anti-epileptic
drugs are very diverse and may vary over time. At present,
targeted sequencing of genes associated with genetically
heterogeneous conditions seems to be the elective choice for
early and efficient etiological diagnoses. 155 individuals
have been subjected to NGS investigation, using a custo-
mized panel of 31 genes, on the Ion PGM™sequencing
platform. Sequence variants were interpreted according to
the ACMG guidelines. The average sequencing depth of
coverage was 331.6X, with 97.6% of the reads on-target
and 93.2% of reads uniformity. On average, 114 variants
per patient have been detected. In the overall, we were able
to identify disease-causing variants in 27 individuals
(17.4%) although, since the panel mainly targeted EIEE/
early epilepsy genes, the diagnostic yield stratified accord-
ing to age of seizure onset resulted 27% in cases with onset
within 6 months of life, 22.7% within 12 months of life,
19.2% in cases with seizure onset before 24 months of life. Out of 27 pathogenic/likely pathogenic variants 19 were
found to alter five voltage-gated ion channels: SCN1A (7),
SCN2A (4), SCN8A (4), KCNQ2 (2) and HCN1 (2). Our
work further strengthens the importance of a careful phe-
notype characterization coupled with the power of the NGS
technology and indicates in a subset of few genes the major
players for epilepsy with very early onset. Epilepsies are neurological disorders and genetic factors
contribute to their pathogenesis. Copy number variations
(CNVs) are increasingly recognized as an important etiol-
ogy of many human diseases including epilepsy. Whole
exome sequencing (WES) is becoming a standard tool for
detecting pathogenic mutations and has recently been
applied to detecting CNVs. Here, we analyzed 294 families
with epilepsy using WES, and focused on 168 families with
no causative single nucleotide variants (SNVs) in known
epilepsy-associated genes to further validate CNVs using
two different CNV detection tools using WES data. We
confirmed 18 pathogenic CNVs, and two deletions and two
duplications at chr15q11.2 of clinically unknown sig-
nificance. Gain-of-function variants in the CASR gene in genetic
generalized epilepsy Results: In WES analysis, five disease co-segregating
rare variants in the EPHA6, ABI3BP, KIAA1407, IQCB1
and CASR genes were found. Of these, c.2693G>A (p. Arg898Gln) in CASR fulfilled the criteria of being a
causative mutation. CASR sequence analysis, identified five
additional rare non-synonymous mutations, p.Glu354Ala, p. Asp433His, p.Ser580Asn, p.Ile686Val and p.Ala988Val in
14 unrelated GGE/JME patients. In cell signaling assays,
the mutant CASR receptors exhibited leftward shifts in the
dose-response curves showing an enhanced responsiveness
to extracellular calcium concentrations as compared to the
wild-type receptor, thereby suggesting, activating nature of
these variants. Conclusions: we achieved an effective diagnostic two-
step method for neuropediatric patients with epilepsy. It
consists of aCGH analysis followed by the analysis of
specific NGS panels. Diagnostic rate increased by 35% due
to the application of NGS panels, in an efficient, accurate
and cost-effective strategy. Study
funding/competing
interest(s):
Funding
by
national/international organization(s): The Torres Quevedo
Program from the Spanish Ministry of Economy and 275 Abstracts from the 51st European Society of Human Genetics Conference: Posters Conclusion: Our findings indicate a role for CASR in
predisposition to GGE/JME based on evidence provided at
the gene- and variant-level. Based on observed enhanced
calcium responsiveness of CASR alleles in the cell
signaling assays, we propose that gain-of-function effects
of these mutations may alter CASR-regulated functions in
the brain and contribute to pathophysiology of epilepsy. The use of Next Generation Sequencing for the diagnosis of
early onset epilepsy and epileptic encephalopathies E. Bettella1, R. Polli1, E. Leonardi1, F. Cesca1, M. C. Aspromonte1, M. Vecchi2, I. Toldo3, C. Boniver2, D. Baldo4,
S. Negrin5, S. Sartori3, A. Murgia1 E. Bettella1, R. Polli1, E. Leonardi1, F. Cesca1, M. C. Aspromonte1, M. Vecchi2, I. Toldo3, C. Boniver2, D. Baldo4,
S. Negrin5, S. Sartori3, A. Murgia1 The study was supported by funds from DAE, Mumbai. M. Kaur: None. P. Satishchandra: None. S. Sinha:
None. A. Kapoor: None. A. Anand: None. 1Laboratory of Molecular Genetics of Neurodevelopment,
Department of Women’s and Children’s Health, University of
Padova, Italy, Padua, Italy, 2Pediatric Neurophysiology Unit,
Department of Women’s and Children’s Health, University of
Padua, Italy, Padua, Italy, 3Pediatric Neurology Unit,
Department of Women’s and Children’s Health, University of
Padua, Italy, Padua, Italy, 4Clinical Genetics Unit, Hospital of
Treviso, Treviso, Italy, Treviso, Italy, 5Scientific Institute
IRCCS E. Medea, Conegliano Research Center, 31015
Conegliano, Italy, Padua, Italy The use of Next Generation Sequencing for the diagnosis of
early onset epilepsy and epileptic encephalopathies The use of Next Generation Sequencing for the diagnosis of
early onset epilepsy and epileptic encephalopathies P09.059C Detection of copy number variations in epilepsy using
exome data N. Matsumoto: None. N. Tsuchida: None. P09.061A
De novo variants in neurodevelopmental disorders with
epilepsy D. Kluckova1, B. Tarabova2, M. Kolnikova3, A. Ficek1,
L. Lacinova2, L. Kadasi1,4, A. Soltysova1,4 H. O. Heyne1, T. Singh1, H. Stamberger2, EuroEPINOMICS
RES Consortium & Epilepsy DNV studygroup, A. Poduri3,
Y. Weber4, S. Weckhuysen2, S. M. Sisodiya5, M. J. Daly1,
I. Helbig6, D. Lal1, J. R. Lemke7 1Department of Molecular Biology, Faculty of Natural
Sciences, Comenius University, Bratislava, Slovakia, 2Institute
of Molecular Physiology and Genetics, Slovak Academy of
Sciences, Bratislava, Slovakia, 3Department of Child
Neurology, Children´s Faculty Hospital, Bratislava, Slovakia,
4Institute for Clinical and Translational Research, Biomedical
Research Center, Slovak Academy of Sciences, Bratislava,
Slovakia 1Massachusetts General Hospital, Boston, MA, United States,
2University of Antwerp, Antwerp, Belgium, 3Boston Children's
Hospital, Boston, MA, United States, 4University of Tübingen,
Tübingen, Germany, 5UCL, Institute of Neurology, London,
United Kingdom, 6Children's Hospital of Philadelphia,
Philadelphia, PA, United States, 7University of Leipzig,
Leipzig, Germany 1Massachusetts General Hospital, Boston, MA, United States,
2University of Antwerp, Antwerp, Belgium, 3Boston Children's
Hospital, Boston, MA, United States, 4University of Tübingen,
Tübingen, Germany, 5UCL, Institute of Neurology, London,
United Kingdom, 6Children's Hospital of Philadelphia,
Philadelphia, PA, United States, 7University of Leipzig,
Leipzig, Germany Mutations in SCN1A, the gene encoding voltage-gated
sodium channel NaV1.1, cause a spectrum of epilepsy dis-
orders that range from genetic epilepsy with febrile seizures
plus to severe disorders such as Dravet syndrome. To date,
more than 1,250 mutations in SCN1A have been linked to
epilepsy but only a small number of them has been func-
tionally characterised. We identified several novel muta-
tions (p.E78D, p.D249E, p.W384X, p.E777K, p.T1923I) in
SCN1A gene in Slovak epilepsy patients, which were sub-
sequently subjected to functional characterisation in het-
erologous expression system. EGFP gene was inserted to
pCDM8-hNaV1.1 to visualise the eukaryotic cells expres-
sing protein of interest. All identified mutations were
introduced to pCDM8-hNaV1.1-EGFP construct and were
propagated in TOP10/P3 E.Coli grown at 28°C to minimize
the rearrangements. The entire coding sequence was
sequenced after each propagation. The pathogenic effect of
each mutation on protein function was tested in transiently
transfected HEK293T cells by whole-cell patch clamp
configuration. Cells were also transfected with each of
accessory β subunits to test whether mutant channels can be
rescued by molecular interactions with these modulatory
proteins. Detection of copy number variations in epilepsy using
exome data Of note, we were able to identify small CNVs less
than 10 kb in size, which might be difficult to detect by
conventional microarray. We revealed two cases with
pathogenic CNVs that one of the two CNV detection tools
failed to find, suggesting that different CNV tools are
recommended to increase diagnostic yield. Considering a
relatively high discovery rate of CNVs (18 out of 168
families, 10.7%) and successful detection of CNV with <10
kb in size, CNV detection by WES may be able to surro-
gate, or at least complement, conventional microarray ana-
lysis. Acknowledgements: Nakashima M, Kato M, Heyman
E, Inui T, Haginoya K, Watanabe S, Chiyonobu T, Mor-
imoto M, Ohta M, Kumakura A, Kubota M, Kumagai Y,
Hamano S-I, Lourenco CM, Yahaya NA, Ch'ng G-S, Ngu
L-H, Fattal-Valevski A, Hubshman MW, Orenstein N,
Marom D, Cohen L, Goldberg-Stern H, Nakajima H, Saitsu
H, Miyatake S. E. Bettella: None. R. Polli: None. E. Leonardi: None. F. Cesca: None. M.C. Aspromonte: None. M. Vecchi: None. I. Toldo: None. C. Boniver: None. D. Baldo: None. S. Negrin: None. S. Sartori: None. A. Murgia: None. E. Bettella: None. R. Polli: None. E. Leonardi: None. F. Cesca: None. M.C. Aspromonte: None. M. Vecchi: None. I. Toldo: None. C. Boniver: None. D. Baldo: None. S. Negrin: None. S. Sartori: None. A. Murgia: None. 276 J. del Picchia D. Kluckova: None. B. Tarabova: None. M. Kolni-
kova: None. A. Ficek: None. L. Lacinova: None. L.
Kadasi: None. A. Soltysova: None. Assesment of candidate genes in patients with
frontotemporal lobar degeneration spectrum: preliminary
findings A. Todorova1,2,3, T. Todorov1, S. Sarafov4, T. Chamova4,
M. Gospodinova5, I. Tournev4,6 A. Todorova1,2,3, T. Todorov1, S. Sarafov4, T. Chamova4,
M. Gospodinova5, I. Tournev4,6 A. Todorova1,2,3, T. Todorov1, S. Sarafov4, T. Chamova4,
M. Gospodinova5, I. Tournev4,6 S. Artan1, E. Erzurumluoglu1, O. Cilingir1, B. D. Ozbabalik
Adapinar2, F. Tepgec3, H. Bas1, H. A. Hanagası4, I. H. Gurvit4,
G. Toksoy3, Z. O. Uyguner3, B. Durak Aras1, C. Yenilmez5 1Genetic Medico-Diagnostic Laboratory Genica, Sofia,
Bulgaria, 2IMDL Genome Centre Bulgaria, Sofia, Bulgaria,
3Department of Medical Chemistry and Biochemistry, Medical
University Sofia, Sofia, Bulgaria, 4Clinic of Nervous Diseases,
UMBAL Aleksandrovska, Department of Neurology, Medical
University Sofia, Sofia, Bulgaria, 5Clinic of Cardiology, MVR
hospital, Sofia, Bulgaria, 6Department for cognitive science
and psychology, New Bulgarian University, Sofia, Bulgaria 1Genetic Medico-Diagnostic Laboratory Genica, Sofia,
Bulgaria, 2IMDL Genome Centre Bulgaria, Sofia, Bulgaria,
3Department of Medical Chemistry and Biochemistry, Medical
University Sofia, Sofia, Bulgaria, 4Clinic of Nervous Diseases,
UMBAL Aleksandrovska, Department of Neurology, Medical
University Sofia, Sofia, Bulgaria, 5Clinic of Cardiology, MVR
hospital, Sofia, Bulgaria, 6Department for cognitive science
and psychology, New Bulgarian University, Sofia, Bulgaria 1Eskisehir Osmangazi University, Faculty of Medicine,
Department of Medical Genetics, Eskisehir, Turkey, 2Acibadem
Hospital Neurology Clinic, Eskisehir, Turkey, 3Istanbul
University, Istanbul Faculty of Medicine, Department of
Medical Genetics, Istanbul, Turkey, 4Istanbul University,
Istanbul Faculty of Medicine, Behavioural Neurology and
Movement Disorders Unit, Department of Neurology, Istanbul,
Turkey, 5Eskisehir Osmangazi University, Faculty of Medicine,
Department of Psychiatry, Eskisehir, Turkey Monoallelic gene expression is a phenomenon in which
only one allele from a homologous pair is transcribed. Transthyretin (TTR) amyloidosis is an autosomal dominant
systemic disorder caused by mutations in the TTR gene. Markedly different penetrance according to the gender of
the transmitting parent as well as a variable manifestation
between monozygotic twins was observed in Bulgarian
families. The aim of the present study was to better
understand the difference in the disease penetrance by
evaluating the mutant versus wild type transcripts. The
RNA was extracted from plasma and urine and the TTR RT-
PCR products were sequenced by Sanger. Apart from the
expected traditional biallelic transcription a monoallelic
expression signature of only mutant or only wild type
alleles was observed. For some patients tissue-specific
transcription profile was detected, which corresponds to
multiple tissues and organs involvement in the disease
manifestation. P09.061A
De novo variants in neurodevelopmental disorders with
epilepsy Finally two antiepileptic drugs, phenytoin and
carbamazepine, and an antiarrhythmic drug, mexiletine,
which probably act by stabilizing the correct folding con-
formation were tested, if they can prevent the degradation of
the mutant protein and reduce the loss of function effect. These findings may contribute to the understanding of
mechanisms of the epileptogenesis and to the effective
therapy. Epilepsy is a frequent feature of neurodevelopmental dis-
orders (NDD) but little is known about genetic differences
between NDD with and without epilepsy. We analyzed de
novo variants (DNV) in 6753 parent-offspring trios ascer-
tained for different NDD. In the subset of 1942 individuals
with NDD with epilepsy including 529 individuals with
epileptic encephalopathy, we identified 33 genes with a
significant excess of DNV. Of these, SNAP25 and GABRB2
had previously only limited evidence for disease associa-
tion. Joint analysis of all individuals with NDD also
implicated CACNA1E as a novel disease gene. Comparing
NDD with and without epilepsy, we found missense DNV,
DNV in specific genes, age of recruitment and severity of
intellectual disability to be associated with epilepsy. 24
routinely used diagnostic panels would only have detected
on average 59% of DNV in the 33 genes with exome-wide
DNV burden. We further found low evidence for disease
association for genes frequently used on diagnostic epilepsy
panels. 5% of DNV in our study were in eight genes for
which we could confirm therapeutic consequences with
established evidence-based medicine criteria emphasizing
the benefit of accurate genetic diagnosis in NDD with
epilepsy. This work has been supported by the Eurocores program
EuroEPINOMICS and grants from the German Research
Foundation (DFG), German Federal Ministry of Education
and Research (BMBF) and Institute for Science and
Technology (IWT)-Flanders. H.O. Heyne: None. T. Singh: None. H. Stamberger:
None. A. Poduri: None. Y. Weber: None. S. Weckhuy-
sen: None. S.M. Sisodiya: None. M.J. Daly: None. I. Helbig: None. D. Lal: None. J.R. Lemke: None. D. Kluckova: None. B. Tarabova: None. M. Kolni-
kova: None. A. Ficek: None. L. Lacinova: None. L. Kadasi: None. A. Soltysova: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 277 P09.064D
Assesment of candidate genes in patients with
frontotemporal lobar degeneration spectrum: preliminary
findings A. Todorova: None. T. Todorov: None. S. Sarafov:
None. T. Chamova: None. M. Gospodinova: None. I.
Tournev: None. Assesment of candidate genes in patients with
frontotemporal lobar degeneration spectrum: preliminary
findings Based on our results we propose a model of
natural selection, which includes age-related allele sup-
pression: predominant expression of a wild type allele (at an
early age) and mutant allele (at the process of ageing). Different regulatory mechanisms at molecular level (histone
modifications, chromatin remodelling, transcription factors,
and epigenetic alterations) might be involved and combined
in different manner in different individuals, which explains
interfamilial differences and phenotypic differences in
monozygotic twins with identical genotypes. Further studies
on monoallelic expression of the TTR gene will facilitate a
better understanding of the TTR gene transcriptional reg-
ulation. Acknowledgement: The study was supported by
Pfizer: Grant №WI220557/15.11.2016. Frontotemporal lobar degeneration (FTLD) is a hetero-
geneous disorder group associated with degeneration in
the frontal/temporal lobes of brain. This study is aimed
to determine the frequencies of mutations in MAPT,
PGRN,CHMP2B,VCP,TARDBP and FUS genes, which
are considered as the main genetic causes of FTLD in
Turkish population and to investigate the genotype-
phenotype correlations in cases with pathogenic/ likely-
pathogenic variants. The exon/exon-intron junctions for
related genes in gDNAs of 100 FTLD cases and 100 age-
matched controls were sequenced by using IonTor-
rentS5, then analyzed with bioinformatics pipeline. NGS
results were confirmed by the Sanger Sequencing and are
shown in table without variants considered as benign. It was identified 2 novel variants in MAPT and
CHMP2B genes that are intronic and missense, respec-
tively. Additionally, 2 missense and 2 frameshift muta-
tions were detected in GRN and 1 missense mutation in
TARDBP. Interestingly
c.759_760delTG
GRN
and
c.389A>G CHMP2B variants were identified in the same
patient
who
has
died
a
year
after
the
diagnosis. Segregation studies of family members are in progress. This study indicates that GRN mutations are more
common causative genetic factors for FTLD (%4) and this
ratio is% 12 in cases with positive family history. To our
knowledge, this is first report evaluating the genetic
backround in Turkish FTLD. This study was supported by
The Scientific and Technological Research Council of
Turkey (TUBITAK1001-114S346) A. Todorova: None. T. Todorov: None. S. Sarafov:
None. T. Chamova: None. M. Gospodinova: None. I. Tournev: None. J. del Picchia 278 Table: NGS Results
Genes
cDNA
rs ID
Protein
In siliko
prediction
MAF % Subclass
of FTD
Age
of
Onset
Family
Hist
Classifi-
cation
GRN
c.415T>C
rs763841075
p. Cys139Arg
PD/Del/
DC
0.018
bvFTD
55
+
Pathogenic**
GRN
c.430G>A
rs200591137
p. P09.065A
Role of mitochondrial DNA variants in the development of
FXTAS M. Alvarez-Mora1, C. Santos2, L. Carreño-Gago3, I. Madrigal1,
M. Tejada4, F. Martinez5, S. Izquierdo-Alvarez6, E. Garcia-
Arumi7, M. Mila1, L. Rodriguez-Revenga1 Assesment of candidate genes in patients with
frontotemporal lobar degeneration spectrum: preliminary
findings Gurvit: None. G. Toksoy: None. Z.O. Uyguner: None. B. Durak Aras:
None. C. Yenilmez: None. S. Artan: None. E. Erzurumluoglu: None. O. Cilingir:
None. B.D. Ozbabalik Adapinar: None. F. Tepgec: None. H. Bas: None. H.A. Hanagası: None. I.H. Gurvit: None. G. Toksoy: None. Z.O. Uyguner: None. B. Durak Aras:
None. C. Yenilmez: None. M. Alvarez-Mora: None. C. Santos: None. L. Carreño-
Gago: None. I. Madrigal: None. M. Tejada: None. F. Martinez: None. S. Izquierdo-Alvarez: None. E. Garcia-
Arumi:
None. M. Mila:
None. L. Rodriguez-
Revenga: None. Assesment of candidate genes in patients with
frontotemporal lobar degeneration spectrum: preliminary
findings Asp144Asn
PD/Del/
DC
0.00081 SD
59
+
VUS***
GRN
c.102delC
rs63751073
p. Gly35Glufs
DC
NA
bvFTD
58
+
Pathogenic**
GRN*
c.759_760delTG
rs63751035
p. Cys253Terfs
DC
0.00041 bvFTD
56
+
Likely
Pathogenic***
CHMP2B* c.389A>G
Novel
p. Lys130Arg
PD/Tol/
DC
NA
bvFTD
56
+
VUS***
TARDBP
c.1213A>G
rs762209110
p. Met405Val
PD/Tol/
DC
NA
bvFTD
72
−
VUS***
MAPT
c.1828-3A>C
Novel
p.? −
NA
bvFTD
42
+
VUS***
MAF: Minor allel frequency from gnomAD (The Genome Aggrega-
tion Database),
In silico prediction: Polyphen, SIFT, Mutation Tester respectively
PD: Probably Damaging, Del: Deleterious, DC: Disease Causing, Tol:
Tolareted
*Same patient
**Defined in HGMD (Human Gene Mutation Database)
*** According to ACMG (American College of Medical Genetics and
Genomics) criteria FXTAS. The aim of this study is to elucidate the role of
mtDNA variation in the pathogenesis of FXTAS. FXTAS. The aim of this study is to elucidate the role of
mtDNA variation in the pathogenesis of FXTAS. Table: NGS Results Materials and Methods: Two independent sets of FMR1
premutation carriers were recruited. In the first set (13
FXTAS and 13 no-FXTAS) the entire mitogenome was
sequenced using massively parallel sequencing technolo-
gies. In the second set (39 FXTAS and 67 no-FXTAS),
mitochondrial haplogroups were determined. Materials and Methods: Two independent sets of FMR1
premutation carriers were recruited. In the first set (13
FXTAS and 13 no-FXTAS) the entire mitogenome was
sequenced using massively parallel sequencing technolo-
gies. In the second set (39 FXTAS and 67 no-FXTAS),
mitochondrial haplogroups were determined. Results:
We
identified
haplogroup
T
differentially
enriched in FMR1 premutation carriers and significantly
underrepresented in FXTAS patients. Analysis of mtDNA
sequences revealed an association between disease and the
burden of heteroplasmic variants and their distribution. The
FXTAS group presented 3-fold more low-level heteroplas-
mic variants in compromised regions of the mitochondrial
genome. Conclusions: Our results suggest that haplogroup T
might be a potential protective factor for FXTAS. In
addition, FXTAS individuals accumulate higher rates of
heteroplasmic variants in compromised regions of the
mitochondrial genome. These results may explain, in part,
the role of mtDNA in the development of FXTAS. This
work was supported by the Instituto de Salud Carlos III
(PI12/00879; PI17/01067), co-financed by Fondo Europeo
de Desarrollo Regional (FEDER) “una manera de hacer
Europa”
and
AGAUR
(2014SGR603;
2014SGR1420;
2017SGR1134). S. Artan: None. E. Erzurumluoglu: None. O. Cilingir:
None. B.D. Ozbabalik Adapinar: None. F. Tepgec: None. H. Bas: None. H.A. Hanagası: None. I.H. L. Rodriguez-Revenga1, M. Alvarez-Mora1, P. Podlesniy2,
E. Gelpi3, J. Pagonabarraga4, I. Madrigal1, R. Trullas2, M. Mila1
1Servei de Bioquímica i Genetica Molecular, Barcelona, Spain,
2Neurobiology Unit, Instituto de Investigaciones Biomedicas
de Barcelona, Consejo Superior de Investigaciones Cientificas,
CSIC, Barcelona, Spain, 3Neurological Tissue Bank of the
Biobanc-Hospital Clinic-IDIBAPS,, Barcelona, Spain,
4Neurology Service, Hospital Sant Pau,, Barcelona, Spain Decreased mitochondrial DNA copy number is associated
with clinical manifestations of FXTAS Decreased mitochondrial DNA copy number is associated
with clinical manifestations of FXTAS 1Hospital Clinic/CIBERER/IDIBAPS, Barcelona, Spain,
2Universitat Autònoma de Barcelona, Cerdanyola del Vallès,
Spain, 3Hospital Universitari Vall d'Hebron Institut de Recerca
(VHIR), Barcelona, Spain, 4Cruces University Hospital/
Biocruces Health Research Institute/CIBERER, Barakaldo,
Spain, 5Hospital Universitario y Politecnico La Fe, Valencia,
Spain, 6University Hospital Miguel Servet, Zaragoza, Spain,
7Hospital Universitari Vall d'Hebron/VHIR/CIBERER,
Barcelona, Spain L. Rodriguez-Revenga1, M. Alvarez-Mora1, P. Podlesniy2,
E. Gelpi3, J. Pagonabarraga4, I. Madrigal1, R. Trullas2, M. Mila1
1Servei de Bioquímica i Genetica Molecular, Barcelona, Spain,
2Neurobiology Unit, Instituto de Investigaciones Biomedicas
de Barcelona, Consejo Superior de Investigaciones Cientificas,
CSIC, Barcelona, Spain, 3Neurological Tissue Bank of the
Biobanc-Hospital Clinic-IDIBAPS,, Barcelona, Spain,
4Neurology Service, Hospital Sant Pau,, Barcelona, Spain L. Rodriguez-Revenga1, M. Alvarez-Mora1, P. Podlesniy2,
E. Gelpi3, J. Pagonabarraga4, I. Madrigal1, R. Trullas2, M. Mila1
1Servei de Bioquímica i Genetica Molecular Barcelona Spain 1Servei de Bioquímica i Genetica Molecular, Barcelona, Spain,
2Neurobiology Unit, Instituto de Investigaciones Biomedicas
de Barcelona, Consejo Superior de Investigaciones Cientificas,
CSIC, Barcelona, Spain, 3Neurological Tissue Bank of the
Biobanc-Hospital Clinic-IDIBAPS,, Barcelona, Spain,
4Neurology Service, Hospital Sant Pau,, Barcelona, Spain Introduction: Fragile X-associated tremor/ataxia syndrome
(FXTAS) is a late-onset neurodegenerative disorder that
appears in at least one-third of adult carriers of a premuta-
tion (55-200 CGG repeats) in the FMR1 gene. Although
several studies have described the impairment of mito-
chondrial function in FXTAS patients, to our knowledge
there are no data regarding the involvement of mtDNA in Introduction: Fragile X-associated tremor/ataxia syndrome
(FXTAS) is a late-onset neurodegenerative disorder with
reduced penetrance that appears in adult FMR1 premutation
carriers (55-200 CGGs). There are several studies support-
ing a role for mitochondrial dysfunction in the pathogenesis Abstracts from the 51st European Society of Human Genetics Conference: Posters 279 of FXTAS. However, the mtDNA copy number has been
poorly studied. Stress-Mediated Autophagy in neuron like cell line (SH-
SY5Y). Material and Methods: mtDNA copy number was
studied in multiple tissues from FXTAS patients and
matched control subjects (post-mortem human brains, blood
samples and skin fibroblasts cultures). The results were
compared to age-matched controls. Digital droplet PCR and
Real Time quantitative PCR was performed was used to
determine the mtDNA copy number. Materials And Methods: SH-SY5Y cells were treated
with 100ng/ml dexamethasone at acute stress dose for
4 hours. After RNA isolation and cDNA synthesis, Real-
time PCR reactions were performed for GRP78, ATF4,
XBP1 and ATG5 genes. P09.067C Introduction: GLUT1 deficiency syndrome is defined as a
metabolic encephalopathy that usually caused by patho-
genic variations in the SLC2A1 gene. The aim of the study
is to determine the presence of single nucleotide and copy
number changes in SLC2A1 gene. C. Ornek1, O. Ozdemir1, Y. Kesim1, S. A. Ugur İseri1, B. Kara2 C. Ornek1, O. Ozdemir1, Y. Kesim1, S. A. Ugur İseri1, B. Kara2 L. Rodriguez-Revenga:
None. M. Alvarez-Mora:
None. P. Podlesniy: None. E. Gelpi: None. J. Pagona-
barraga: None. I. Madrigal: None. R. Trullas: None. M. Mila: None. 1Aziz Sancar Institute of Experimental Medicine, Istanbul,
Turkey, 2Kocaeli University Faculty of Medicine, Kocaeli,
Turkey Decreased mitochondrial DNA copy number is associated
with clinical manifestations of FXTAS Results: We found that the expression level of Autop-
hagy
related
5
(ATG5),
which
has
been
previously
characterized as a protein required for autophagy, was
significantly decreased in SH-SY5Y cell lines that were
exposed to Dexmethasone. However, we did not find any
differences between genes (GRP78,ATF4,XBP1s) which
are key players of unfolded protein response. Results: The analysis of mtDNA levels in human tissues
evidenced reduced mtDNA content in cerebellar vermis,
dentate nucleus, parietal and temporal cortex areas. The fact
that no mtDNA copy number alteration was detected in any
other brain regions or in any other tissues analyzed,
suggests that its potential effect is restricted to clinically
relevant regions explaining why FXTAS clinical manifesta-
tions are associated with gait ataxia and tremor. Conclusion: Relationship between decreased autophagy
and neurodegenerative diseases in neuronal cells has been
well studied. For this reason, our result may suggests that
low dose of GCs can contribute to the pathogenesis of the
neurodegenerative diseases through decreasing the expres-
sion of ATG5. Supported by CUBAP/TSA-2017-8116. Conclusions: Our study indicates that reduced mtDNA
copy number is restricted to the affected brain tissue and
provides new insights into the role of mitochondrial
dysfunction in the pathogenesis of FXTAS. Acknowl-
edgements: This work was supported by the Instituto de
Salud Carlos III (PI17/01067), co-financed by Fondo
Europeo de Desarrollo Regional (FEDER) “una manera
de hacer Europa” and AGAUR from the Autonomous
Catalan Government (2017 SGR1134). The CIBER de
Enfermedades Raras is an initiative of the Instituto de Salud
Carlos III. Conclusions: Our study indicates that reduced mtDNA
copy number is restricted to the affected brain tissue and
provides new insights into the role of mitochondrial
dysfunction in the pathogenesis of FXTAS. Acknowl-
edgements: This work was supported by the Instituto de
Salud Carlos III (PI17/01067), co-financed by Fondo
Europeo de Desarrollo Regional (FEDER) “una manera
de hacer Europa” and AGAUR from the Autonomous
Catalan Government (2017 SGR1134). The CIBER de
Enfermedades Raras is an initiative of the Instituto de Salud
Carlos III. D. Alptekin: None. H. Luleyap: None. A. Yoldas:
None. G. Comertpay: None. P. Pazarci: None. G. Ay:
None. G. Evyapan: None. A. Pazarbasi: None. D. Alptekin: None. H. Luleyap: None. A. Yoldas:
None. G. Comertpay: None. P. Pazarci: None. G. Ay:
None. G. Evyapan: None. A. Pazarbasi: None. Genetic analysis in GLUT1 deficiency syndrome Genetic analysis in GLUT1 deficiency syndrome C. LAM1, C. Ko2, W. Cheng2, C. Law1 C. LAM1, C. Ko2, W. Cheng2, C. Law1 1The University of Hong Kong, Hong Kong, China,
2Department of Paediatrics & Adolescent Medicine/
Developmental Disabilities Unit, Caritas Medical Centre,
Hong Kong, China Gene-environment interaction, through abnormal intestinal
adsorption, has been proposed as possible mechanism for
autism pathogenesis in those patients lacking of causative
genetic variants. Haptoglobin (HP) is a haemoglobin bind-
ing and acute-phase plasma protein, encoded by two co-
dominant alleles, HP-1 and HP-2, producing pre-HP-1 and
pre-HP-2 proteins that mature in HP-1 and HP-2, respec-
tively. HP-2 allele contains a 1.7Kb tandem duplication that
includes two extra exons with respect to HP-1. Introduction: There is no effective treatment for GNAO1-
related movement disorder (MD). Here, we describe the
novel use of folinic acid to control MD in a patient who
carried an activating GNAO1 pathogenic variant. Materials and Methods: The patient is a 13-month-old
Chinese girl who presented with global delay and dystonia. She had unremarkable birth history with no family history
of consanguinity or neuromuscular disorders. She had poor
truncal tone and persistent fisting at 5 months. At age one
she could only vocalize. Physical examinations showed
axial hypotonia with extremity hypertonia and brisk jerks. Biochemical and imaginings findings were unremarkable. Subsequent genetic analysis revealed a de novo hetero-
zygous
activating
pathogenic
variant,
NM_020988.2
(GNAO1):c.736G>A; p.Glu246Lys. Endogenous pre-HP-2 protein deregulates intestinal tight-
junctions through EGFR and PAR2 activation, increases
intestinal
permeability
and
has
been
associated
with
autoimmune and inflammatory diseases as well as with
psychiatric conditions. Since the association between HP alleles and autism has
never been investigated, we genotyped, by PCR analysis,
HP in a cohort of Italian patients with autism (n = 406) and
in controls (n = 367). The aim was to evaluate the possible
role of HP-2 in enhancing macromolecular intestinal
trafficking in these patients. Results: Patient’s MD remained intractable. It persisted
throughout the day, and only temporarily ceased during
sleep. She was unresponsive to combined treatment, i.e. risperidone, nitrazepam, tetrabenazine and clonazepam, and
required deep sedation with midazolam infusion. Folinic
acid was administered for suspected secondary cerebral
folate deficiency. Surprisingly, there was a significant
reduction in MD within 2 days with improvement in
awareness and motor functions (video). Pre-treatment CSF
5-methyltetrahydrofolate (MTHF) level was normal, mea-
suring 75 nmol/L (reference interval: 40-128). Her MD
remained well controlled with folinic acid (75 mg daily),
low dose nitrazepam, carbamazepine and risperidone. Association of Haptoglobin-1 allele with Autism A. Mezzelani1, F. A. Cupaioli1, E. Mosca1, C. Magri2,
M. Gennarelli2,3, M. E. Raggi4, M. Landini1, N. Galluccio1,
F. Chiappori1, M. Moscatelli1, M. Gnocchi1, C. Villa4,
M. Molteni4, A. Bonfanti4, F. Ciceri4, A. Marabotti5, L. Milanesi A. Mezzelani1, F. A. Cupaioli1, E. Mosca1, C. Magri2, C. Ornek: None. O. Ozdemir: None. Y. Kesim: None. S.A. Ugur İseri: None. B. Kara: None. A. Mezzelani , F. A. Cupaioli , E. Mosca , C. Magri ,
M. Gennarelli2,3, M. E. Raggi4, M. Landini1, N. Galluccio1, M. Gennarelli2,3, M. E. Raggi4, M. Landini1, N. Galluccio1, F. Chiappori1, M. Moscatelli1, M. Gnocchi1, C. Villa4, Low Dose Dexmethasone Decrease the Level of ATG5 in
SH SY5Y cell line Screening
of this gene is important for understanding genetic basis of
the disease and providing genetic counselling for patients. P09.069A M. Molteni4, A. Bonfanti4, F. Ciceri4, A. Marabotti5, L. Milanesi1 An activating GNAO1 mutation causing refractory chorea
suppressed by folinic acid therapy An activating GNAO1 mutation causing refractory chorea
suppressed by folinic acid therapy 1National Research Council, Segrate, Italy, 2Dept. of
Molecular and Translational Medicine, University of Brescia,
Brescia, Italy, 3Genetic Unit, IRCCS Centro S. Giovanni di Dio
Fatebenefratelli, Brescia, Italy, 4Scientific Institute, IRCCS
Eugenio Medea, Bosisio Parini (LC), Italy, 5Dept. Chemistry
and Biology, “A. Zambelli”, University of Salerno, Fisciano
(SA), Italy Low Dose Dexmethasone Decrease the Level of ATG5 in
SH SY5Y cell line D. Alptekin1, H. Luleyap1, A. Yoldas2, G. Comertpay1,
P. Pazarci1, G. Ay1, G. Evyapan1, A. Pazarbasi1
1Cukurova University, Medical Faculty, Adana, Turkey,
2Kahraman Maras Sutci Imam University, Medical Faculty,
Kahramanmaras, Turkey D. Alptekin1, H. Luleyap1, A. Yoldas2, G. Comertpay1,
P. Pazarci1, G. Ay1, G. Evyapan1, A. Pazarbasi1 Method: In this study, all of the 10 exons in 13 patients
with GLUT1 deficiency syndrome were sequenced by the
Sanger method. Results were analyzed as in-silico with
various bioinformatics tools, phenotype-genotype correla-
tions were revealed. According to the ACMG Standards and
Guidelines published in 2015, necessary to demonstrate that
family members have blood connections with the patient for
the confirmation of de novo variants. For this reason, SNP
Array analysis was performed for 2 patients and their family
members. In 7 patients who do not have any variation in
Sanger analysis, quantitative real-time PCR was applied to
each of the 10 exons in the SLC2A1 gene to determine the
presence of copy number variations. 1Cukurova University, Medical Faculty, Adana, Turkey,
2Kahraman Maras Sutci Imam University, Medical Faculty,
Kahramanmaras, Turkey Introduction: Glucocorticoids (GCs) have a significant role
in the adaptive response of the brain to stress. Increasing
evidence has demonstrated that an increase of GC levels
may induce neuronal cell death via apoptotic pathways. In
the present study, we aimed to investigate whether the
Dexmethasone, a synthetic glucocorticoid, at physiologic
dose plays a role in unfolded protein response induced by
Endoplasmic Reticulum Stress and Endoplasmic Reticulum Result: Sanger sequencing method, 8 variants were
identified. 2 of them are novel and de novo variants. When J. del Picchia 280 an activating GNAO1 mutation. A new inhibitory mechan-
ism between folate and GNAO1 signaling was suggested by
the clinical findings. examined via databases, one of the novel and de novo
variants is a splice-side variant, the other is a frame shift
variant, and the phenotype effects were assessed as
pathogenic by in-silico tools. qRT-PCR calculations for
CNV detection are still ongoing. examined via databases, one of the novel and de novo
variants is a splice-side variant, the other is a frame shift
variant, and the phenotype effects were assessed as
pathogenic by in-silico tools. qRT-PCR calculations for
CNV detection are still ongoing. C. Lam: None. C. Ko: None. W. Cheng: None. C. Law: None. Conclusion:%90 of individuals with Glut1 DS caused by
de novo heterozygous variants in SLC2A1 gene. Leucocyte Telomere Length in Huntington's Disease.
Preliminary data Retrospective study of symptomatic carriers of an
Intermediate Allele (IA) in Huntingtin (HTT) gene A. Ruiz de Sabando1, A. Martínez Descals2, V. Álvarez
Martinez3, I. Legarda Ramírez4, K. Bergazo Corrales5, A. López-
de-Munain6, M. Milà7, M. A. Ramos-Arroyo1 D. Scarabino1, L. Veneziano2, M. Peconi3, M. Frontali2, R. M. Corbo3,1, E. Mantuano2 1Institute of Molecular Biology and Pathology. National
Research Council, Rome, Italy, 2Institute of Translational
Pharmacology. National Research Council, Rome, Italy,
3Department of Biology and Biotechnology, La Sapienza
University, Rome, Italy 1Institute of Molecular Biology and Pathology. National
Research Council, Rome, Italy, 2Institute of Translational
Pharmacology. National Research Council, Rome, Italy,
3Department of Biology and Biotechnology, La Sapienza
University, Rome, Italy 1Complejo Hospitalario de Navarra, Pamplona, Spain,
2Hospital Universitario Fundación Jiménez Díaz, Madrid,
Spain, 3Hospital Universitario Central de Asturias, Oviedo,
Spain, 4Hospital Universitari Son Espases, Palma, Spain,
5Hospital Universitario Cruces, Baracaldo, Spain, 6Hospital
Universitario Donostia, San Sebastián, Spain, 7Hospital Clinic
Barcelona, Barcelona, Spain Introduction: Huntington’s disease (HD) is an autosomal
dominant, fully penetrant, neurodegenerative disease caused
by an expanded CAG repeat in the first exon of the HTT
gene. The onset of symptoms most commonly occurs at
midlife and inversely correlates with the CAG repeat
expansion. However, age of clinical onset, progression rate,
and severity of symptoms can vary between individuals. Leukocyte telomere length (LTL) has been widely investi-
gated in neurodegenerative diseases such as Alzheimer’s
and Parkinson diseases, but very few data on LTL in HD
have been reported. In the present preliminary study, we
investigated the relationship between LTL and HD devel-
opment,
including
premanifest
and
symptomatic
HD
patients. Introduction: Huntington’s Disease (HD) is an autosomal
dominant
neurodegenerative
disorder
characterized
by
uncontrollable movements (chorea) and subtle changes in
cognition and behaviour caused by an expansion of CAG
repeats (n≥36) in Huntingtin (HTT) gene. In this study we
focus on symptomatic patients, carriers of intermediate
alleles (IAs; n = 27-35), in an attempt to describe a possible
phenotypic association. Methodology: We reviewed the available data (pheno-
type and genotype) of symptomatic patients referred for HD
genetic testing. Frequencies of IAs were compared with the
general population. Clinical symptoms of IAs carriers were
classified in three groups: motor, cognitive and behavioural. Methods: LTL (T/S ratio) was measured in HD patients
(n= 46) or pre-HD (n= 31), compared with LTL of age-
matched controls (n= 60). C. LAM1, C. Ko2, W. Cheng2, C. Law1 Contrary to what we expected, HP-1 allele distribution
was different between patients and controls (36.3% and
29.4%, respectively) and significantly associated with
autism (P=0.0041). Since a subgroup of patients and controls have already
been genotyped by Illumina Human Omni-15-8 v.1.0 and
Affy-6.0 chips, respectively, we are trying to impute HP
alleles from flanking SNP haplotypes. HP alleles will
therefore be predicted in publicly available large cohorts of
patients with autism. Conclusions: This is the first reported case of successful
pharmacological control for intractable MD in a patient with Abstracts from the 51st European Society of Human Genetics Conference: Posters 281 in cognition and 32.2% in behaviour (n = 59). A positive
family history could be confirmed in 21% of patients. in cognition and 32.2% in behaviour (n = 59). A positive
family history could be confirmed in 21% of patients. Acknowledgements. Projects: IRCCS Eugenio Medea,
GR-2009-1570296, Interomics-PB05, Acknowledgements. Projects: IRCCS Eugenio Medea,
GR-2009-1570296, Interomics-PB05, Telethon Network of Genetic Biobanks (project No. GTB12001), funded by Telethon-Italy, which provided us
with part of specimens. Telethon Network of Genetic Biobanks (project No. GTB12001), funded by Telethon-Italy, which provided us
with part of specimens. Conclusion: Non-HD symptomatic patients seem to be
more likely to carry an IA. However, except for a late age of
onset
of
symptoms,
they
do
not
show
a
common
recognizable phenotype. Further follow-up studies of IAs
carriers may help understand the possible effect and
penetrance of these alleles. A. Mezzelani: None. F.A. Cupaioli: None. E. Mosca:
None. C. Magri: None. M. Gennarelli: None. M.E. Raggi: None. M. Landini: None. N. Galluccio: None. F. Chiappori: None. M. Moscatelli: None. M. Gnocchi:
None. C. Villa: None. M. Molteni: None. A. Bonfanti:
None. F. Ciceri:
None. A. Marabotti:
None. L. Milanesi: None. A. Ruiz de Sabando: None. A. Martínez Descals: None. Á V. Álvarez Martinez: None. I. Legarda Ramírez: None. K. Bergazo Corrales: None. A. López-de-Munain: None. M. Milà: None. M.A. Ramos-Arroyo: None. Joubert Syndrome:New genes described! A new allelic
phenotypes achieved! U. Abur, G. Ogur, E. Altundag, A. Yilmaz, O. S. Akar, A. Sanri,
H. Mutlu Albayrak 1L'institut du thorax, Inserm, CNRS, Univ Nantes, CHU
Nantes, Nantes, France, 2Department of Neurosurgery, CHU
Nantes, Nantes, France, 3Université de Bretagne Occidentale,
Inserm UMR1078, CHRU Brest, Etablissement Français du
Sang, Brest, France, 4Centre National de Recherche en
Génomique Humaine, CEA, Evry, France P09.073A
ANGPTL6 and Familial Susceptibility to Intracranial
Aneurysm R. Bourcier1,2, S. Le Scouarnec1, S. Bonnaud1, M. Karakachoff1,
E. Bourcereau1, S. Heurtebise Chrétien1, C. Menguy1, C. Dina1,
F. Simonet1, A. Moles2, C. Lenoble2, P. Lindenbaum1,
S. Chatel1, B. Isidor1, E. Génin3, J. F. Deleuze4, J. J. Schott1,
H. Le Marec1, ICAN Study Group, G. Loirand1, H. Desal1,2,
R. Redon1 ONDOKUZ MAYIS UNIVERSITY MEDICAL FACULTY
DEPARTMENT OF GENETICS, Samsun, Turkey Introduction: Joubert syndrome (JS) is characterized by
hypotonia, ataxia, psychomotor delay, ‘molar tooth sign’.JS
is clinically and genetically heterogeneous. Intracranial aneurysms (IA) are acquired cerebrovascular
abnormalities characterized by a localized dilation and wall
thinning in intracranial arteries. The main IA complication
is the rupture, resulting in subarachnoid haemorrhage and
possibly leading to severe outcome. IA pathogenesis is still
largely unknown, with no reliable risk marker available so
far. We have recently identified one rare nonsense variant
(c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-
Like 6) shared by the 4 tested affected members of a large
pedigree with multiple IA cases. Since ANGPTL6 encodes a
circulating pro-angiogenic factor mainly secreted from the
liver, we showed a 50% reduction of ANGPTL6 serum
concentration
in
individuals
heterozygous
for
the
c.1378A>T allele compared to relatives homozygous for the
normal allele, probably due to the non-secretion of the
truncated protein produced by the c.1378A>T transcripts. By sequencing ANGPTL6 in additional index cases with
familial IA, we detected a significant enrichment in rare
coding variants within this gene among 95 affected subjects
compared to a reference population of 404 individuals with
French ancestry. We observed a higher rate of individuals
with a history of high blood pressure among affected versus
healthy individuals carrying ANGPTL6 variants, suggesting
that ANGPTL6 could trigger cerebrovascular lesions when
combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in Methods: NGS and array-CGH were used.An attempt of
genotype-phenotype correlation has been created. All
patients had dysmorphism, developmental delay and ‘molar
tooth sign’ in MRI. Case1:7 year-old patient had hypotonia,
microphtalmia,
pytosis,aganglionic
colon.Homozygous
mutation (p.Arg563His) in INPP5E gene was detected. Case2:16 month-old patient had lobule tongue,bifid uvula,
short thorax,tetramelic postaxial polydactyly.Homozygous
2-bp deletion in IFT80 gene was detected. Case3:8 day-old
baby had cleft palate, coloboma and pytosis.Array-CGH
showed microdeletion of 136 kb at 16q22.1(This region
included a PDPR gene). Case4:12 year-old patient had
sleep disturbance, nephronophthisis and coloboma.Genetic
analysis revealed compound heterozygous mutation c.6012-
2A>G in splice site and c.5668 G>A (p.Gly1890X) in
CEP290 gene. Case5:3 year-old patient had breathing
abnormalities, polysyndactyly.Genetic analysis revealed
homozygote
mutation
(p.Arg154Ter)
in
KIF7
gene. Case6:3
month-old
patient
had
hypotonia,breathing
abnormalities,broad hallux and bilateral syndactyly of toes. Case
7:15
year-old
patient
had
broad
hallux,partial
syndactyly of toes.Genetic analysis of case 6 and 7 revealed
homozygous mutation (p.Arg973Ter) in KIF7 gene. Results: There are more then 20 genes in JS. Leucocyte Telomere Length in Huntington's Disease.
Preliminary data Results: Frequency of IAs was significantly higher
(X2=6.77, p = 0.01) among symptomatic patients (3.69%)
when compared to individuals of the general population
(2.12%). Family and clinical data were available in 73 IA-
symptomatic patients: 64.38% had 29-27 CAG repeats and
35.61% had 30-35 CAG repeats. Mean age at diagnosis was
61.27 (n = 71, SD=20.1), with signs starting 3.27 years
earlier (n = 22, SD=2.46). Symptoms included abnormal
movements, chorea, ataxia, dyskinesia, cognitive impair-
ment and anxiety & depressive disorder. When classified in
groups, 86.44% showed alterations in movement, 23.73% Results: significant LTL differences among controls, pre-
HD and HD subjects were observed (p < 0.0001), with
mean LTL values in the following order: HD patients < pre-
HD < controls. After adjusting LTL for age, the differences
in LTL across the three groups remained highly significant
(p < 0.0001). Conclusion: current data indicate that shortened LTL are
observed in HD patients as found in other neurodegenera-
tive disorders. The analysis of LTL in pre-HD patients
(never examined to date) suggests that a progressive 282 J. del Picchia telomere erosion may occur in the pre-manifest stage. The
possible use of LTL as biomarker of disease progression is
discussed. telomere erosion may occur in the pre-manifest stage. The
possible use of LTL as biomarker of disease progression is
discussed. telomere erosion may occur in the pre-manifest stage. The
possible use of LTL as biomarker of disease progression is
discussed. ANGPTL6 are causally related to familial forms of IA. We
have now generated the knock-in mouse model for the
ANGPTL6 c.1378A>T variant: the pathophysiological
consequences of this truncating substitution are currently
under further investigations. This work was supported by Grant 2016 La Sapienza
University of Rome to RMC D. Scarabino: None. L. Veneziano: None. M. Peconi:
None. M. Frontali:
None. R.M. Corbo: None. E. Mantuano: None. R. Bourcier:
None. S. Le
Scouarnec:
None. S. Bonnaud: None. M. Karakachoff: None. E. Bourcereau:
None. S. Heurtebise Chrétien: None. C. Menguy: None. C. Dina: None. F. Simonet: None. A. Moles: None. C. Lenoble: None. P. Lindenbaum: None. S. Chatel: None. B. Isidor: None. E. Génin: None. J.F. Deleuze: None. J.J. Schott: None. H. Le Marec: None. G. Loirand: None. H. Desal: None. R. Redon: None. P09.076D Expanding the phenotypic spectrum in neurological
disorders associated with mutations in KARS gene (lysyl-
tRNA synthetase) by the identification of a novel mutation F. Consoli1, M. Marano2, S. Migliore2, S. Maffi2, I. Mazzante3,
A. De Luca1, F. Squitieri2 F. Consoli1, M. Marano2, S. Migliore2, S. Maffi2, I. Mazzante3,
A. De Luca1, F. Squitieri2 F. Consoli1, M. Marano2, S. Migliore2, S. Maffi2, I. Mazzante3,
A. De Luca1, F. Squitieri2 S. Scheidecker1, S. Bär2, C. Stoetzel1, V. Geoffroy1, B. Lannes3,
B. Rinaldi2, S. Kremer4, M. Mirande5, C. Tranchant6, J. Muller1,
S. Friant2, H. Dollfus1,7 1Molecular Genetics Unit, Casa Sollievo della Sofferenza
Hospital, IRCCS, San Giovanni Rotondo, Italy, 2Casa Sollievo
della Sofferenza Hospital, IRCCS, San Giovanni Rotondo,
Italy, 3LIRH (Lega Italiana Ricerca Huntington) Foundation,
Rome, Italy 1Medical Genetics Laboratory, INSERM U1112, Institute of
Genetics and medicine of Alsace (IGMA), Université de
Strasbourg, Strasbourg, France, 2Department of Molecular
and Cellular Genetics, UMR7156, Centre National de
Recherche Scientifique (CNRS), Université de Strasbourg,
Strasbourg, France, 3Service d’Anatomo-pathologie, Hôpitaux
Universitaires de Strasbourg, Hôpital de Hautepierre,
Strasbourg, France, 4Service de Neuroradiologie/Imagerie 2,
CHU de Strasbourg, Hôpital de Hautepierre, Strasbourg,
France, 5Institute for Integrative Biology of the Cell (I2BC),
CEA, CNRS, Université Paris-Sud, Université Paris-Saclay,
Paris, France, 6Service de Neurologie Hôpitaux Universitaires
de Strasbourg, Hôpital de Hautepierre, Strasbourg, France,
7Centre de Référence pour les affections rares en génétique
ophtalmologique, CARGO, Filière SENSGENE, Hôpitaux
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Our objective is to characterize the rare occurrence of
clinical manifestations in children carrying mutations in the
low-mild size, generally causing adult Huntington disease
(HD). We are following up a subgroup of young subjects
with HD mutation who manifested with disabling psychia-
tric condition since infancy or adolescence. Among 60
juvenile Huntington disease (JHD) patients we currently
follow-up, four of them with mild mutation size showed
neurological signs or movement disorders suggestive of HD
in adulthood. All patients were genetically (e.g. CAG size
analysis) and clinically (e.g. total motor score within the
Unified HD Rating Scale) characterized. All four subjects
presented a CAG expansion size <45 repeats. Two patients
manifested a schizophrenia-like disturbance during the
adolescence, with the later appearance of motor signs after
age 20. P09.075C Children with CAG expansion in the mild repeat range of
Huntingtin gene showing psychiatric but not neurological
presentation: is it one more shade of the disease? ONDOKUZ MAYIS UNIVERSITY MEDICAL FACULTY
DEPARTMENT OF GENETICS, Samsun, Turkey Here we
suggested two new genes for JS, PDPR, IFT80. PDPR has
not been associated with any disease;It is ought to be Abstracts from the 51st European Society of Human Genetics Conference: Posters 283 responsible for cleft palate and molar tooth sign and
possibly presents a new JS fenotype (Alazami et al.,2017). IFT80 has been previously described in ATD-2 and we
describe its correlation with JS firstly.KIF7 seems to be the
third new gene of JS with a possible founder effect in
Northern Turkey. currently manifesting an autistic disorder in absence of
others neurological signs. The description of JHD is
sometime including children with psychiatric manifestations
associated with adult motor onset. We advise to pay careful
attention to such rare conditions that might represent either
psychiatric conditions erreounously classified as JHD or
prodromic adult HD cases. <!--EndFragment--> U. Abur: None. G. Ogur: None. E. Altundag: None. A. Yilmaz: None. O.S. Akar: None. A. Sanri: None. H. Mutlu Albayrak: None. F. Consoli: None. M. Marano: None. S. Migliore:
None. S. Maffi: None. I. Mazzante: None. A. De Luca:
None. F. Squitieri: None. F. Consoli: None. M. Marano: None. S. Migliore:
None. S. Maffi: None. I. Mazzante: None. A. De Luca:
None. F. Squitieri: None. P09.076D In the other two cases, patients presented symptoms
of autistic spectrum disorder, since infancy. One of them
showed also a schizophrenia-like disturbance and, later, HD
onset with motor signs after 20. One 4-years old patient is Mutations in genes encoding aaRSs (aminoacyl-tRNA
synthetases), an essential enzyme family for protein synth-
esis, were reported in several neurological disorders. aaRSs
can be divided into 3 groups according to the cellular
localization of the aminoacylation: cytoplasmic, mitochon-
drial or both. KARS is one of the 3 aaRSs with a bifunc-
tional role and its cytosolic fraction is a component of a
multiple aminoacyl-tRNA synthetase (MARS) complex. Biallelic mutations in the KARS gene, encoding the lysyl-
tRNA synthetase, were described in peripheral neuropathy,
non syndromic hearing loss and more complex phenotypes
evocating a mitochondrial disorder. We performed whole exome sequencing in a patient
presenting with severe neurological and neurosensory
disorder
and
her
healthy
parents
and
we
identified
compound heterozygous variants in KARS, with one novel J. del Picchia 284 S4 segment which functions as a voltage sensor. KCND3
pathogenic variants have been associated with Spinocer-
ebellar Ataxia (SCA19/22) with reported ataxia onset
ranging from the age of ten to adulthood. Smets et al. 2015 have reported on a patient with a de novo KCND3
variant affecting the S4 segment who showed develop-
mental delay, epilepsy, oral apraxia and attention deficit
hyperactivity and first presented with ataxia symptoms at
the age of 3 years. mutation. To demonstrate the pathogenic effect of the two
mutations, we studied the expression of both KARS
isoforms in patient’s skin fibroblasts and used a double
hybrid interaction study. We showed a different expression of both KARS
isoforms with a decrease of cytoplasmic KARS and an
increase of mitochondrial isoform in the patient’s fibroblasts
compared to a control. With the double hybrid interaction
study we showed a defect of interaction between the two
mutant forms of KARS and the p38 protein, a core protein
responsible for assembly of the MARS complex, which
directly interacts with KARS. Conclusion: This case supports the findings of Smets
et al. that de novo variants should be considered in SCA. Both reported cases suggest that KCND3-associated ataxia
should be taken into consideration even in cases with early
childhood manifestation and that variants in the segment S4
of Kv4.3 might result in earlier clinical manifestation of
ataxia. P09.076D To our knowledge this is the earliest reported ataxia
onset in KCND3-associated ataxia. In conclusion, we report a patient carrying two mutations
in KARS gene, with one novel mutation, and presenting in
addition to neurosensory deafness and neurological features
previously
described,
cerebellar
ataxia
and
optic
neuropathy. S. Scheidecker: None. S. Bär: None. C. Stoetzel: None. S. Scheidecker: None. S. Bär: None. C. Stoetzel: None. V. Geoffroy: None. B. Lannes: None. B. Rinaldi: None. S. Kremer: None. M. Mirande: None. C. Tranchant:
None. J. Muller:
None. S. Friant:
None. H. Dollfus: None. S. Scheidecker: None. S. Bär: None. C. Stoetzel: None. V. Geoffroy: None. B. Lannes: None. B. Rinaldi: None. S. Kremer: None. M. Mirande: None. C. Tranchant:
None. J. Muller:
None. S. Friant:
None. H. Dollfus: None. A. Gazou: None. R. Buchert: None. A. Riess: None. A. Dufke: None. U. Grasshoff: None. D. Gauck: None. J. Magg: None. V. Horber: None. I. Kraegeloh- Mann:
None. O. Riess: None. T. Haack: None. A. Gazou: None. R. Buchert: None. A. Riess: None. A. Dufke: None. U. Grasshoff: None. D. Gauck: None. J. A. Gazou: None. R. Buchert: None. A. Riess: None. A. Dufke: None. U. Grasshoff: None. D. Gauck: None. J. Magg: None. V. Horber: None. I. Kraegeloh- Mann:
None. O. Riess: None. T. Haack: None. S. Scheidecker: None. S. Bär: None. C. Stoetzel: None. V. Geoffroy: None. B. Lannes: None. B. Rinaldi: None. S. Kremer: None. M. Mirande: None. C. Tranchant:
None. J. Muller:
None. S. Friant:
None. H. Dollfus: None. Magg: None. V. Horber: None. I. Kraegeloh- Mann:
None. O. Riess: None. T. Haack: None. S. Kremer: None. M. Mirande: None. C. Tranchant:
None. J. Muller:
None. S. Friant:
None. H. Dollfus: None. An NGS approach to the genetic diagnosis of hereditary
leukodystrophies Case report on a boy with early-onset ataxia and a novel de
novo KCND3 variant affecting the S4 segment of the Kv4.3
channel D. Di Bella1, S. Magri1, E. Sarto1, D. Tonduti2, A. Simonati3,
I. Moroni2, C. Gellera1, E. Salsano4, F. Taroni1 A. Gazou1, R. Buchert1, A. Riess1, A. Dufke1, U. Grasshoff1,
D. Gauck1, J. Magg2, V. Horber2, I. Kraegeloh- Mann2,
O. Riess1, T. Haack1 A. Gazou1, R. Buchert1, A. Riess1, A. Dufke1, U. Grasshoff1,
D. Gauck1, J. Magg2, V. Horber2, I. Kraegeloh- Mann2,
O. Riess1, T. Haack1 1Unit of Genetics of Neurodegenerative and Metabolic
Disease, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milano, Italy, 2Child Neurology Unit, Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milano, Italy, 3Department
of Neuroscience, Biomedicine, Movement Neurology (Child
Neurology and Psychiatry), University of Verona, Verona,
Italy, 4Neurology Unit 10, Fondazione IRCCS Istituto
Neurologico Carlo Besta, Milano, Italy 1Institute of Medical Genetics and Applied Genomics,
University Hospital Tuebingen, Tuebingen, Germany,
2Department of Neuropediatrics, Developmental Neurolgy,
Social Pediatrics, University Hospital Tuebingen, Tuebingen,
Germany 1Institute of Medical Genetics and Applied Genomics,
University Hospital Tuebingen, Tuebingen, Germany,
2Department of Neuropediatrics, Developmental Neurolgy,
Social Pediatrics, University Hospital Tuebingen, Tuebingen,
Germany Introduction: Hereditary white matter disorders (WMDs)
are a heterogeneous group of disorders affecting myelin in
the central nervous system, with abnormalities in myelin
formation (hypomyelinating leukodystrophies, HLD) or
myelin
degeneration
(demyelinating
leukodystrophies,
DLD). Although >100 conditions have been identified,
<50% of patients receive a genetic diagnosis because of the
heterogeneity and complexity of these disorders. Aim of
this study was to employ a comprehensive NGS gene panel
to study both childhood-onset (EO) and adult-onset (AO)
WMD patients negative for the most common genes. Introduction: The boy presented at the age of two years
with delayed motor, cognitive and language development,
dysarthria and postural as well as gait ataxia. Metabolic
workup revealed no abnormalities. Today he is 7 ½ years
old, receives supportive therapy and attends a special
school. Methods:
Whole
exome
sequencing
(SureSelectXT
Human All Exon v6) identified the following novel variant
in the KCND3 gene: c.910T>C: p.Ser304Pro; segregation
analysis showed a de novo status. No other pathogenic
variants were identified. The variant was classified as likely
pathogenic. Materials and Methods: A probe-based customized
panel covering 142 WMD disease-genes was used to screen
81 index cases with HLD (17 AO; 19 EO) or DLD (21 AO;
24 EO). An NGS approach to the genetic diagnosis of hereditary
leukodystrophies Materials and Methods: A probe-based customized
panel covering 142 WMD disease-genes was used to screen
81 index cases with HLD (17 AO; 19 EO) or DLD (21 AO;
24 EO). Discussion: KCND3 encodes Kv4.3, a voltage-gated
potassium channel with six transmembrane segments, S1-
S6, and two intracellular tails. The above variant affects the Abstracts from the 51st European Society of Human Genetics Conference: Posters 285 Results: Pathogenic mutations were identified in 24,7%
of probands (20/81) and likely pathogenic mutations in
other 8 probands (9,9%). Overall, the mutation score was
higher in the hypomyelinating forms: 47% (9/19) in EO and
24% (4/17) in AO subjects. Interestingly, in AO-HLD
forms, we identified mutations in genes usually associated
with the more severe EO-HLD forms or spastic paraplegia. Mutation scores in DLD were 21% (5/24) in EO and only
10% (2/21) in AO. Copy number variation (CNV) analysis
allowed the identification of deletions/duplications in 6%
(5/81) of probands. Methods: Trio/Quad whole-genome sequencing (WGS)
was performed on 27 patients from 22 families with
leukodystrophy and a non-diagnostic MRI pattern or non-
diagnostic WES. Results: A genetic diagnosis was achieved in 11/23
families (47%). Implicated genes included six typically
associated
with
leukodystrophy:
DARS2,
NDUFV1,
BOLA3, COL4A1, TUBB4A, SLC17A5 and five genes not
previously-associated with leukodystrophy: HMBS, FIG4,
STAG2, SCN2A, SCN8A. The latter group includes genes
associated with porphyria, epileptic encephalopathy, per-
ipheral neuropathy and congenital malformation. Trio WGS
was effective in identifying de novo variants as well as
variants that had been missed by exome sequencing due to
poor coverage. Conclusions: Our approach allowed to identify the
genetic cause in ~30% of patients, extending the phenotypic
spectrum associated to different genes and establishing
novel genotype/phenotype correlations. Notably, the high
mutation score in AO-HLD patients reveal a genetic cause
also for these neglected forms. (Italian MoH grant to CG)
D. Di Bella: None. S. Magri: None. E. Sarto: None. D. Tonduti: None. A. Simonati: None. I. Moroni: None. C. Gellera: None. E. Salsano: None. F. Taroni: None. Conclusions: Our approach allowed to identify the
genetic cause in ~30% of patients, extending the phenotypic
spectrum associated to different genes and establishing
novel genotype/phenotype correlations. Notably, the high
mutation score in AO-HLD patients reveal a genetic cause
also for these neglected forms. P09.079C
Trio whole-genome sequencing for patients with
unclassified leukodystrophies Trio whole-genome sequencing for patients with
unclassified leukodystrophies Trio whole-genome sequencing for patients with
unclassified leukodystrophies C. A. Stutterd1,2,3,4, M. Delatycki1,2,3, P. Lockhart1,3, R. Taft5,
A. Vanderver6,7, C. Simons1,8, R. J. Leventer1,3,4 An NGS approach to the genetic diagnosis of hereditary
leukodystrophies (Italian MoH grant to CG) Conclusions: Results from this cohort advocate the use of
trio WGS for the diagnosis of CNS white matter disease as
the genetic aetiologies are diverse and may include de novo
dominant developmental genes as well as recessive house-
keeping, metabolic and mitochondrial genes. NHMRC
project grant:1068278. D. Di Bella: None. S. Magri: None. E. Sarto: None. D. Tonduti: None. A. Simonati: None. I. Moroni: None. C. Gellera: None. E. Salsano: None. F. Taroni: None. D. Di Bella: None. S. Magri: None. E. Sarto: None. D. Tonduti: None. A. Simonati: None. I. Moroni: None. C. Gellera: None. E. Salsano: None. F. Taroni: None. P09.079C P09.079C
Trio whole-genome sequencing for patients with
unclassified leukodystrophies C.A. Stutterd: None. M. Delatycki: None. P. Lockhart:
None. R. Taft: A. Employment (full or part-time); Modest;
lllumina Inc. A. Vanderver: None. C. Simons: None. R.J. Leventer: None. P09.080D We identified three frameshift, one missense,
one extension and one nonsense mutations. Three of the
intragenic mutations were novel. The LIS1 gene was
affected in 4 patients characterized by agyria/pachygyria. A DCX mutation was found in subcortical band heterotopia
and the identified single TUBA1A mutation was associated
with cerebellar hypoplasia and agenesis of the corpus
callosum. FISH analysis detected 17p13.3 microdeletion in
2 patients which confirmed Miller-Dieker syndrome. Conclusion: Previously reported mutations of LAMB1 are
localized to the EGFLAM and Laminin-IV domains. Our
findings for the first time show that a missense mutation in
the ‘Domain alpha’ of LAMB1 can cause a similar
phenotype. Moreover, heterogeneity in the clinical pheno-
type of patients with LAMB1 mutations is an interesting
finding that will require functional studies. Conclusions: These are the first results of genetic testing
for lissencephaly in Hungarian population. We confirmed
the clinical diagnosis in more, than half of the patients. The
precise description of the pattern of gyral malformation and
associated abnormalities may predict the most likely
causative gene in a patient with lissencephaly. E. Yılmaz: None. E. Sönmezler: None. A. Töpf: None. S. Balaraju: None. A. Yaramış: None. S. Güngör: None. E. Yılmaz: None. E. Sönmezler: None. A. Töpf: None. S. Balaraju: None. A. Yaramış: None. S. Güngör: None. B. Bessenyei: None. A. Mokanszki: None. O. Nagy:
None. K. Szakszon: None. A. Zimmermann: None. M. Zombor: None. E. Horvath: None. A. Ujfalusi: None. I. Balogh: None. L. Sztriha: None. S. Laurie: None. R. Horvath: None. H. Lochmüller:
None. Y. Oktay: None. S. Hız: None. P09.081A Genetic investigation of the LIS1, DCX and TUBA1A genes
in patients with lissencephaly Genetic investigation of the LIS1, DCX and TUBA1A genes
in patients with lissencephaly P09.080D A novel mutation in the coiled-coil interaction domain of
LAMB1 extends the molecular basis of laminin-related
cortical malformation phenotypes A novel mutation in the coiled-coil interaction domain of
LAMB1 extends the molecular basis of laminin-related
cortical malformation phenotypes 1Murdoch Children's Research Institute, Parkville, Australia,
2Victorian Clinical Genetics Service, Parkville, Australia,
3University of Melbourne, Parkville, Australia, 4Royal
Children's Hospital, Parkville, Australia, 5Illumina Inc, San
Diego, CA, United States, 6Children's Hospital of
Philadelphia, Philadelphia, PA, United States, 7University of
Pennsylvania, Philadelphia, PA, United States, 8Institute for
Molecular Bioscience, University of Queensland, St. Lucia,
Australia E. Yılmaz1, E. Sönmezler1, A. Töpf2, S. Balaraju2, A. Yaramış3,
S. Güngör4, S. Laurie5, R. Horvath2, H. Lochmüller2,
Y. Oktay1,6, S. Hız7,8 1Izmir Biomedicine and Genome Center (IBG), Izmir, Turkey,
2John Walton Muscular Dystrophy Research Centre, Institute
of Genetic Medicine, Newcastle University, Newcastle upon
Tyne, UK, Newcastle upon Tyne, United Kingdom, 3Diyarbakır
Memorial Hospital, Diyarbakır, Turkey, 4İnönü University,
Malatya, Turkey, 5CNAG-CRG, Centre for Genomic
Regulation, Barcelona Institute of Science and Technology,
Barcelona, Spain, Barcelona, Spain, 6Dokuz Eylul University,
Faculty of Medicine, Dept. of Medical Biology, Izmir, Turkey,
Izmir, Turkey, 7Dokuz Eylul University, Faculty of Medicine,
Dept. of Pediatric Neurology, Izmir, Turkey, Izmir, Turkey,
8Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul
University Health Campus, Izmir, Turkey, Izmir, Turkey Background: Leukodystrophies are a genetically diverse
group of disorders that have in common the selective
involvement of the central nervous system (CNS) white
matter. They most commonly present in childhood and
usually follow a progressive course, with high morbidity
and mortality and limited life span. A genetic diagnosis is
key to providing accurate prognostic information and
reproductive counselling to families, and appropriate clin-
ical management of the patient. Magnetic resonance ima-
ging (MRI) pattern recognition and exome sequencing
currently achieve a diagnosis for 70% of patients. Aim: To
identify the genetic causes underlying a cohort of patients
with unclassified leukodystrophy on MRI or negative whole
exome sequencing (WES). Introduction: Laminins are major components of the basal
laminae. LAMB1 mutations have been reported in only 3
families with lissencephaly and largely overlapping but J. del Picchia 286 distinct phenotypes. Here we present a case with a novel
mutation in LAMB1 gene. Introduction: Lissencephaly is a rare brain malformation
caused by abnormal neuronal migration. The main clinical
symptoms of the condition are developmental delay, intel-
lectual disability and seizures. P09.080D Several genes have been
implicated in lissencephaly, the most frequently affected are
LIS1 (PAFAH1B1), DCX and TUBA1A. The encoded pro-
teins play an essential role in the formation or function of
microtubules. Materials and Methods: The patient, 17 year-old girl
from a consanguineous marriage, never developed the
ability to walk or speak and had drug-resistant seizures. Her
physical examination revealed dysmorphic facial features,
scoliosis, ocular abnormalities, increased deep tendon
reflexes and generalized weakness. The EEG demonstrated
generalized epileptic discharges. Cranial MRI showed
bilateral perisylvian polymicrogyria. Whole-exome sequen-
cing (WES) was performed using Illumina exome capture
(38 Mb) at MIT BROAD Institute. Data analysis was
carried out on the RD-Connect Genome-Phenome Analysis
Platform. Standard filtering criteria with MAF<1% and
high/moderate VEP were used. Materials and Methods: We studied Hungarian patients
with isolated (n = 13) and syndromic (n = 2) lissencephaly. Diagnosis was based on clinical evaluation and magnetic
resonance imaging. Genetic testing involved sequencing of
the LIS1, DCX and TUBA1A genes in the isolated cases and
fluorescence in situ hybridization (FISH) for the detection
of 17p13.3 microdeletion in patients with Miller-Dieker
syndrome. Materials and Methods: We studied Hungarian patients
with isolated (n = 13) and syndromic (n = 2) lissencephaly. Diagnosis was based on clinical evaluation and magnetic
resonance imaging. Genetic testing involved sequencing of
the LIS1, DCX and TUBA1A genes in the isolated cases and
fluorescence in situ hybridization (FISH) for the detection
of 17p13.3 microdeletion in patients with Miller-Dieker
syndrome. Results: We identified a homozygous missense variant
(p.Gly1413Glu) in LAMB1, associated with lissencephaly 5
(OMIM# 615191). This variant is predicted to be highly
pathogenic (CADD=34) and is extremely rare in the control
population (0.000824%). The affected residue is localized
to the ‘Domain alpha’, which is between Domain-I and
Domain-II that mediate the interaction of laminin chains to
form the coiled-coil structure. Results: Genetic analysis revealed pathogenic alterations
in 8 patients. We identified three frameshift, one missense,
one extension and one nonsense mutations. Three of the
intragenic mutations were novel. The LIS1 gene was
affected in 4 patients characterized by agyria/pachygyria. A DCX mutation was found in subcortical band heterotopia
and the identified single TUBA1A mutation was associated
with cerebellar hypoplasia and agenesis of the corpus
callosum. FISH analysis detected 17p13.3 microdeletion in
2 patients which confirmed Miller-Dieker syndrome. Results: Genetic analysis revealed pathogenic alterations
in 8 patients. 1Institute of Humangenetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
Bonn, Bonn, Germany, 3Human Genomics Research Group,
Department of Biomedicine, University of Basel, Basel,
Switzerland, 4Institute of Medical Genetics and Pathology, S. Sivalingam1,2, A. Forstner1,2, S. Herms1,2,3, A. Maaser1,2,
A. Koller1,2, C. Reinbold3,4, S. Fischer3,4, T. Andlauer5, F. Streit6,
J. Frank6, H. Dukal6, S. Witt6, S. Heilmann-Heimbach1,2,
K. Ludwig1,2, F. Degenhardt1,7, A. Krug8, U. Dannlowski9,
T. Kircher8, S. Cichon1,2,3, M. Rietschel6, P. Hoffmann1,2,3,
M. Nöthen1,2 P09.082B Integrated analysis of genetic and epigenetic data of healthy
individuals with different risk factors for affective disorders B. Bessenyei1, A. Mokanszki2, O. Nagy1, K. Szakszon3,
A. Zimmermann4, M. Zombor4, E. Horvath5, A. Ujfalusi1,
I. Balogh1, L. Sztriha4 B. Bessenyei1, A. Mokanszki2, O. Nagy1, K. Szakszon3,
A. Zimmermann4, M. Zombor4, E. Horvath5, A. Ujfalusi1,
I. Balogh1, L. Sztriha4 B. Bessenyei1, A. Mokanszki2, O. Nagy1, K. Szakszon3,
A. Zimmermann4, M. Zombor4, E. Horvath5, A. Ujfalusi1,
I. Balogh1, L. Sztriha4 S. Sivalingam1,2, A. Forstner1,2, S. Herms1,2,3, A. Maaser1,2,
A. Koller1,2, C. Reinbold3,4, S. Fischer3,4, T. Andlauer5, F. Streit6,
J. Frank6, H. Dukal6, S. Witt6, S. Heilmann-Heimbach1,2,
K. Ludwig1,2, F. Degenhardt1,7, A. Krug8, U. Dannlowski9,
T. Kircher8, S. Cichon1,2,3, M. Rietschel6, P. Hoffmann1,2,3,
M. Nöthen1,2 1Division of Clinical Genetics, Department of Laboratory
Medicine, Faculty of Medicine, University of Debrecen,
Debrecen, Hungary, 2Department of Pathology, Faculty of
Medicine, University of Debrecen, Debrecen, Hungary,
3Department of Pediatrics, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary, 4Department of Pediatrics and
Pediatric Health Center, University of Szeged, Szeged,
Hungary, 5Department of Medical Genetics, Faculty of
Medicine, University of Szeged, Szeged, Hungary 1Institute of Humangenetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
Bonn, Bonn, Germany, 3Human Genomics Research Group,
Department of Biomedicine, University of Basel, Basel,
Switzerland, 4Institute of Medical Genetics and Pathology, Two patients with PNKP mutations presenting
microcephaly, seizure, and oculomotor apraxia M. Taniguchi-Ikeda1, N. Morisada2, H. Inagaki3, N. Okamoto4,
T. Toda5, I. Morioka1, H. Kurahashi3, K. Iijima1 1Department of Pediatrics, Kobe University Graduate School
of Medicine, Kobe Hyogo, Japan, 2Clinical Genetics, Hyogo
prefectural Childrens Hospital, Kobe Hyogo, Japan,
3Department of Molecular Genetics, Fujita Health University,
Toyoake,Aichi, Japan, 4Department Medical Genetics, Osaka
Medical Center and Research Institute for Maternal and Child
Health, Izumi, Osaka, Japan, 5Department of Neurology, The
University of Tokyo, Tokyo, Japan Affective disorders (major depressive disorder, bipolar
disorder) are genetically complex and heterogeneous dis-
orders. Both genetic and environmental risk factors con-
tribute to the etiology of the diseases. However, the
neurobiological correlates by which these risk factors
influence disease development are hardly understood. Increasing evidence suggests that epigenetic modifications
such as DNA methylation have important implications on
the development of psychiatric diseases including affective
disorders. Several studies revealed that genetic variants can
alter DNA methylation at specific loci (methylation quan-
titative trait loci, meQTLs). To investigate this, we exam-
ined
the
association
between
genetic
variants
and
methylation levels in whole blood of 44 individuals with
different risk factors for affective disorders (genetic/envir-
onmental risk). Genotyping of the 44 individuals was con-
ducted using the Illumina Infinium PsychArray. Imputation
of the genotypes was performed using IMPUTE2 and 1000
Genomes phase 3 reference haplotypes. DNA methylation
was assessed using the Infinium MethylationEPIC Bead-
Chip spanning 850,000 CpG sites. FastQTL using a cis-
window size of ± 500 MB and a linear regression model
was applied to identify associations between imputed gen-
otypes and methylation levels. In total, we investigated
551,275 SNP-CpG pairs and identified 1,460 significant cis-
meQTLs for an FDR of 5% (p-value < 9.06x10-8). One of
the top meQTLs rs4880352 (p-value < 1.72x10-10) was
associated with the methylation level at cg01458105 located
nearby STK32C. This gene was differentially methylated in
a former study of depression in monozygotic discordant
twins (Dempster et al., 2014). The meQTLs identified in the
present study might improve the interpretation of the
functional relevance of genetic variants associated with
affective disorders. Microcephaly with early-onset, intractable seizures and
developmental delay (MCSZ, OMIM #613402) is an auto-
somal recessive group of heterogeneous disorders, which
can also be associated with other severe neurological
defects. Mutations in polynucleotide kinase 3’-phosphatase
(PNKP) have been suggested to cause MCSZ. Additionally,
PNKP mutations also cause ataxia-oculomotor apraxia type
4 (AOA4) without symptoms of epilepsy or microcephaly. Two patients with PNKP mutations presenting
microcephaly, seizure, and oculomotor apraxia The reported AOA4 cases carried mutations in the kinase
domain of PNKP. Here we identified three new mutations in
two Japanese MCSZ patients who gradually exhibited
AOA4 symptoms. All mutations resided within the kinase
domain and both patients showed severe epilepsy and
microcephaly with congenital anomalies. A 38-year old
MCSZ patient showed apparent AOA symptoms with spi-
nocerebellar degeneration. Another patient with MCSZ
showed lissencephaly and frequent horizontal headshaking
from age 1, suggestive of oculomotor apraxia. Kinase
domain mutations in PNKP may manifest as a wide spec-
trum of overlapping phenotypes of MCSZ and AOA4. Therefore, we suggest that each MCSZ and AOA may be a
sequential phenotypes of PNKP
mutations in kinase
domain. M. Taniguchi-Ikeda: None. N. Morisada: None. H. Inagaki: None. N. Okamoto: None. T. Toda: None. I. Morioka: None. H. Kurahashi: None. K. Iijima: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 287 University Hospital of Basel, Basel, Switzerland, 5Max Planck
Institute of Psychiatry, Munich, Munich, Germany,
6Department of Genetic Epidemiology in Psychiatry, Central
Institute of Mental Health, Medical Faculty Mannheim,
University of Heidelberg, Mannheim, Germany, 7Department
of Genomics, Life & Brain Center, Bonn, Bonn, Switzerland,
8Department of Psychiatry, University of Marburg, Marburg,
Germany, 9Department of Psychiatry, University of Muenster,
Muenster, Germany J. Stephen1, S. Nampoothiri2, A. Banerjee3, N. J. Tolman4,
J. Penninger5, U. Elling5, C. A. Agu5, J. D. Burke1,
K. Devadathan6, R. Kannan7, Y. Huang8, P. J. Steinbach9, W. A.
Gahl1,4,8, M. V. Malicdan1,4,8 Clinical and molecular study of Tunisian families with
autosomal recessive primary microcephaly Conclusions:Bi-allelic mutations in aARSs are well
known for their role in neurodegenerative disorders, yet
human disorders associated with VARS mutations haven’t
yet been clinically well characterized. Our study describes
the phenotype associated with recessive VARS mutations
and further functional delineation of the novel mutations
that widens the clinical and genetic spectra of individuals
with progressive microcephaly. Results: WES identified novel compound heterozygous
mutations in VARS, encoding ValRS, one of the aARSs; a
missense (c.3192G>A;p.Met1064Ile) and splice site muta-
tion (c.1577-2A>G), that segregated with the affected
status. cDNA analysis revealed that the splice site mutation
led to nonsense mediated decay, thus resulting in a null
allele. Three-dimensional modeling of ValRS predicts that
missense mutation lies in the highly-conserved region and
could alter side chain packing, thus affecting tRNA binding
or destabilizing the interface between the catalytic and
tRNA binding domains. Further quantitation of VARS
expression showed remarkably reduced level of mRNA
and protein in patient cells. Aminoacylation experiments
revealed markedly reduced enzyme activity of ValRS
suggesting the mutations to be loss of function. Results: WES identified novel compound heterozygous
mutations in VARS, encoding ValRS, one of the aARSs; a
missense (c.3192G>A;p.Met1064Ile) and splice site muta-
tion (c.1577-2A>G), that segregated with the affected
status. cDNA analysis revealed that the splice site mutation
led to nonsense mediated decay, thus resulting in a null
allele. Three-dimensional modeling of ValRS predicts that
missense mutation lies in the highly-conserved region and
could alter side chain packing, thus affecting tRNA binding
or destabilizing the interface between the catalytic and
tRNA binding domains. Further quantitation of VARS
expression showed remarkably reduced level of mRNA
and protein in patient cells. Aminoacylation experiments
revealed markedly reduced enzyme activity of ValRS
suggesting the mutations to be loss of function. Materials and Methods: We report the clinical and
genetic study of 15 patients belonging to 12 unrelated
families presenting congenital microcephaly (OFC between
-2 and - 6 SD), an intellectual disability of variable severity,
epileptic seizures in 9 patients, and abnormalities on
cerebral MRI in 6 patients. We performed 2 multigene
panel analyses using next generation sequencing of multiple
MCPH-causing genes. Results: Only two different heterozygous mutations were
found in two patients respectively in LIG4 and STIL genes. These variants are not the only causative mutations
explaining the observed clinical features. P09.084D P09.084D
Recessive mutations in VARS encoding cytoplasmic valyl
tRNAsynthetase cause microcephaly, seizures and
progressive cerebral atrophy
J. Stephen1, S. Nampoothiri2, A. Banerjee3, N. J. Tolman4,
J. Penninger5, U. Elling5, C. A. Agu5, J. D. Burke1,
K. Devadathan6, R. Kannan7, Y. Huang8, P. J. Steinbach9, W. A. Gahl1,4,8, M. V. Malicdan1,4,8 S. Sivalingam: None. A. Forstner: None. S. Herms:
None. A. Maaser: None. A. Koller: None. C. Reinbold:
None. S. Fischer: None. T. Andlauer: None. F. Streit:
None. J. Frank: None. H. Dukal: None. S. Witt: None. S. Heilmann-Heimbach:
None. K. Ludwig:
None. F. Degenhardt: None. A. Krug: None. U. Dannlowski:
None. T. Kircher: None. S. Cichon: None. M. Rietschel:
None. P. Hoffmann: None. M. Nöthen: None. S. Sivalingam: None. A. Forstner: None. S. Herms:
None. A. Maaser: None. A. Koller: None. C. Reinbold:
None. S. Fischer: None. T. Andlauer: None. F. Streit:
None. J. Frank: None. H. Dukal: None. S. Witt: None. S. Heilmann-Heimbach:
None. K. Ludwig:
None. F. Degenhardt: None. A. Krug: None. U. Dannlowski:
None. T. Kircher: None. S. Cichon: None. M. Rietschel:
None. P. Hoffmann: None. M. Nöthen: None. Recessive mutations in VARS encoding cytoplasmic valyl
tRNAsynthetase cause microcephaly, seizures and
progressive cerebral atrophy J. del Picchia 288 Funding:This study was supproted by the Intramural
Research Program of NHGRI, NIH, USA Funding:This study was supproted by the Intramural
Research Program of NHGRI, NIH, USA 1Medical Genetics Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD, United
States, 2Department of Pediatric Genetics, Amrita Institute of
Medical Sciences and Research Center, Cochin, India,
3Department of Biochemistry, University of Illinois at Urbana-
Champaign, Urbana, IL, United States, 4Office of the Clinical
Director, NHGRI, and the NIH Undiagnosed Diseases
Program, NHGRI, National Institutes of Health, Bethesda,
MD, United States, 5Institute of Molecular Biotechnology of
the Austrian Academy of Science (IMBA), Vienna Biocenter
(VBC), Dr. Bohr Gasse 3, Vienna, Austria, 6Department of
Pediatric Neurology, KIMS Hospital, Thiruvananthapuram,
India, 7Department of Radiology, Amrita Institute of Medical
Sciences and Research Center, Cochin, India, 8NIH
Undiagnosed Diseases Program, NHGRI, National Institutes
of Health, Bethesda, MD, United States, 9Center for Molecular
Modeling, Center for Information Technology, National
Institutes of Health, Bethesda, MD, United States J. Stephen: None. S. Nampoothiri: None. A. Banerjee:
None. N.J. Tolman: None. J. Penninger: None. U. Elling:
None. C.A. Agu: None. J.D. Burke: None. K. Deva-
dathan: None. R. Kannan: None. Y. Huang: None. P.J. Steinbach:
None. W.A. P09.084D Gahl:
None. M.V. Malicdan: None. Clinical and molecular study of Tunisian families with
autosomal recessive primary microcephaly Clinical and molecular study of Tunisian families with
autosomal recessive primary microcephaly I. Rejeb1, H. Sassi1, H. Jilani1, S. Hizem1, Y. Elaribi1,
S. Bourgou2, A. Belhadj2, A. Meherzi3, I. Selmi3, N. Pouvreau4,
S. Drunat5, A. Verloes4, L. Benjemaa1 1Department of Genetics, Mongi Slim Hospital, Tunis, Tunisia,
2Department of Child Psychiatry, Mongi Slim Hospital, Tunis,
Tunisia, 3Department of Pediatrics, Mongi Slim Hospital,
Tunis, Tunisia, 4Departement of Genetics, Hôpital
Universitaire Robert Debré, APHP, Paris, France, 5Genetic
department, Robert Debre, Paris, France Introduction: Mutations in genes involved in human
transcriptional and translational machinery, including the
amino acyl-tRNA synthetases (aARSs) family of genes
largely account for postnatal neurodegenerative diseases. Herein we investigated the genetic etiology of two siblings
with severe early onset neurological manifestations. Introduction: Mutations in genes involved in human
transcriptional and translational machinery, including the
amino acyl-tRNA synthetases (aARSs) family of genes
largely account for postnatal neurodegenerative diseases. Herein we investigated the genetic etiology of two siblings
with severe early onset neurological manifestations. Introduction:
Autosomal
recessive
microcephaly
or
MicroCephaly Primary Hereditary (MCPH) is a genetic
heterogeneous disorder, characterized by a reduction in
brain volume, with an occipitofrontal circumference (OFC)
at birth equal to or less than -2 SD below the mean for sex,
age, and ethnicity. An MCPH phenotype has been asso-
ciated with mutations in at least 18 loci, MCPH1-18. Among them, ASPM (MCPH5 locus) is the most frequently
mutated gene reported (60%). In Tunisia, there are no data
on genetic variations in this entity. Materials
and
Methods:Whole
exome
sequencing
(WES), homology modeling, RT-PCR, Immunoblotting,
Vars-/- mouse cell generation and valyl tRNA synthetase
(ValRS) enzyme assay were employed during the course of
this study. y
Results: WES identified novel compound heterozygous
mutations in VARS, encoding ValRS, one of the aARSs; a
missense (c.3192G>A;p.Met1064Ile) and splice site muta-
tion (c.1577-2A>G), that segregated with the affected
status. cDNA analysis revealed that the splice site mutation
led to nonsense mediated decay, thus resulting in a null
allele. Three-dimensional modeling of ValRS predicts that
missense mutation lies in the highly-conserved region and
could alter side chain packing, thus affecting tRNA binding
or destabilizing the interface between the catalytic and
tRNA binding domains. Further quantitation of VARS
expression showed remarkably reduced level of mRNA
and protein in patient cells. Aminoacylation experiments
revealed markedly reduced enzyme activity of ValRS
suggesting the mutations to be loss of function. Clinical and molecular genetic analysis of a case of familial
multiple sclerosis in the Republic of Bashkortostan Introduction: Increasing bodies of evidence support a
potential role of iron metabolism in multiple sclerosis (MS). Previous studies examining the association of hemochro-
matosis (HFE) gene polymorphisms and susceptibility to
MS yield inconsistent results. Y. R. Timasheva1, O. V. Zaplakhova2, K. Z. Bakhtiyarova2, I. A. Tuktarova1, O. E. Mustafina1 1Institute of Biochemistry and Genetics Ufa Science Centre
Russian Academy of Sciences, Ufa, Russian Federation,
2Bashkir State Medical University, Ufa, Russian Federation Materials and Methods: We performed a meta-analysis
of six studies conducted in populations of Caucasian origin
(1871 patients and 2030 controls) using the Comprehensive
Meta-analysis 3.0 software. The strength of association
between the HFE C282Y and H63D polymorphisms and
MS risk was estimated by odds ratios with 95% confidence
intervals. Cochran’s Q-statistic and I-squared tests were
applied to quantify heterogeneity among studies. Egger’s
test was used to estimate the publication bias. Multiple sclerosis (MS) is a complex disease, and genetic
predisposition plays an important role in its development. Familial cases comprise 2% to 5% of all MS patients. In the
Republic of Bashkortostan, located in the Volga-Ural region
of Russian Federation (RF), 1145 patients with MS have
been documented in MS Register; familial cases accounted
for 6.1%. Our study focused on a rare case of familial MS -
a family of Russian ethnic origin from the Republic of
Bashkortostan (RF) that included six individuals with MS in
four generations. Pedigree analysis and genotyping of the
MS candidate loci were performed. Clinical features of MS
in the studied family reflected the most common patterns -
matrilineal transmission of the disease, earlier debut and
benign course of MS in the younger generations. We
observed obvious clinical polymorphism of the disease, in
particular, various symptoms of MS debut in affected
family members. The absence of clinical exacerbations and
lack of active MS foci according to neuroimaging in pro-
band was probably due to the timely administration of
immunomodulatory therapy. We found the accumulation in
the family of the alleles that were associated with auto-
immune diseases according to the results of genome-wide
association
studies:
ASAP2
rs1109670*C,
GPC5
rs9523762*G,
IL7R
rs10624573*D
and
rs1494558*I,
STAT3
rs2293152*G,
IL2RA
rs1570538*Т
and
rs12722580*I, IL2 rs2069772*А. Our results corroborate
the hypothesis that several strong-effect genetic variants
may be responsible for familial aggregation of MS cases. The study was supported by the RFBR grant No. 17-44-
020735. Clinical and molecular genetic analysis of a case of familial
multiple sclerosis in the Republic of Bashkortostan Results: The results demonstrated that the HFE C282Y
and H63D polymorphisms had no statistically significant
association with an increased MS risk (all P > 0.05) under
subsequent genetic comparison models: dominant model
(YY+CY vs. CC or DD+HD vs. HH) and allelic contrast
(Y vs. C or D vs. H). No evident publication bias or
significant heterogeneity among studies was detected. Conclusions: The present study indicates that the HFE
C282Y and H63D polymorphisms are not associated with
susceptibility to MS in populations of Caucasian origin. Further studies should be conducted to estimate the
contribution of HFE polymorphisms to the progression of
MS. National research grants: Genetic analysis of Multiple
Sclerosis 13.06.1.1.10 and The role of iron in pathogenesis
of multiple sclerosis 13.06.2.2.61 Conclusions: The present study indicates that the HFE
C282Y and H63D polymorphisms are not associated with
susceptibility to MS in populations of Caucasian origin. Further studies should be conducted to estimate the
contribution of HFE polymorphisms to the progression of
MS. National research grants: Genetic analysis of Multiple
Sclerosis 13.06.1.1.10 and The role of iron in pathogenesis
of multiple sclerosis 13.06.2.2.61 N. Starcevic Cizmarevic: None. B. Ćurko-Cofek:
None. V. Barac-Latas: None. B. Peterlin: None. S. Ristić: None. P09.089A I. Rejeb: None. H. Sassi: None. H. Jilani: None. S. Hizem: None. Y. Elaribi: None. S. Bourgou: None. A. Belhadj: None. A. Meherzi: None. I. Selmi: None. N. Pouvreau: None. S. Drunat: None. A. Verloes: None. L. Benjemaa: None. N. Starcevic Cizmarevic1, B. Ćurko-Cofek1, V. Barac-Latas1,
B. Peterlin2, S. Ristić1 N. Starcevic Cizmarevic1, B. Ćurko-Cofek1, V. Barac-Latas1,
B. Peterlin2, S. Ristić1 1Faculty of Medicine, Rijeka, Croatia, 2Clinical Institute of
Medical Genetics, Ljubljana, Slovenia 1Faculty of Medicine, Rijeka, Croatia, 2Clinical Institute of
Medical Genetics, Ljubljana, Slovenia Aziz Sancar Institute of Experimental Medicine (AS-DETAE),
Istanbul, Turkey S. Sozer, T. Gurbuz, S. B. Tunç, K. Ateş P09.089A
Hemochromatosis gene polymorphisms in multiple
sclerosis: a meta-analysis P09.089A
Hemochromatosis gene polymorphisms in multiple
sclerosis: a meta-analysis Clinical and molecular study of Tunisian families with
autosomal recessive primary microcephaly We suggest that
other mutations would be present in the regulatory regions
of the explored genes, in the non coding regions, or in other
genes that were not present in the used panels. WES is
planned for these families. Conclusions:Bi-allelic mutations in aARSs are well
known for their role in neurodegenerative disorders, yet
human disorders associated with VARS mutations haven’t
yet been clinically well characterized. Our study describes
the phenotype associated with recessive VARS mutations
and further functional delineation of the novel mutations
that widens the clinical and genetic spectra of individuals
with progressive microcephaly. Conclusions: MCPH is a very heterogeneous disorder. Many MCPH families have not yet been ascribed to any of Conclusions: MCPH is a very heterogeneous disorder. Many MCPH families have not yet been ascribed to any of Abstracts from the 51st European Society of Human Genetics Conference: Posters 289 the known genes, suggesting that additional MCPH genes
are still to be discovered. P09.091C P. Sparber1, A. Marakhonov1,2, A. Filatova1, I. Sharkova1,
M. Skoblov1,2 P09.092D Supported by grants from Istanbul University BAP;
Yüksek Lisans 3582 and BAP THZ-2016-21839. In vitro Investigation for the role of IGF-I and MGF in
High Glucose Environment on Neural Stem Cell
Proliferation In vitro Investigation for the role of IGF-I and MGF in
High Glucose Environment on Neural Stem Cell
Proliferation Y.R. Timasheva: None. O.V. Zaplakhova: None. K.Z. Bakhtiyarova:
None. I.A. Tuktarova:
None. O.E. Mustafina: None. 290 J. del Picchia Neural stem cells (NSC) generate neurons, astrocytes, and
oligodendrocytes of the nervous system. Mechano-Growth
Factor (MGF) is a splice variant of Insulin-like Growth
Factor–I (IGF-I), known as a tissue repair factor in different
tissues and expressed in brain and heart during ischemic
conditions. High glucose levels are harmful to cells. This
study aims to determine the intrinsic and extrinsic effects of
IGF-I and MGF on NSCs and to observe their proliferative
and
neuroprotective
ability
with
varying
glucose
concentrations. Pontocerebellar hypoplasia is a group of neurodegenera-
tive disorders. There are 10 known subtyped (PCH1-10)
with common characteristics of pontine and cerebellar
hypoplasia and atrophy, neocortical atrophy, ventriculome-
galy and microcephaly. To date, recessive mutations have
been noted in PCH1 in the EXOSC3 gene, in the tRNA
splicing endonuclease homolog 54 (TSEN54), mitochon-
drial arginyl-transfer RNA synthetase (RARS2), and in the
vaccinia-related kinase 1 (VRK1) gene. We present the cases of 2 siblings from a consanguineous
Moslem
Arabic
family
with
unique
combination
of
progressive cerebellar atrophy and SMA-like anterior horn
cell degeneration due to homozygous mutation in the
PLA2G6 gene in both siblings. Rat NSC cell-line was applied. Cells were subjected to
different glucose levels including 17.5mM: normoglycemia,
27.75mM:diabetes mellitus, 41.75mM:diabetic ketoacidosis
and
83.75mM:hyperglycemia-hyperosmolar-state
with/
without IGF-I ± MGF. NSC proliferation was determined
by flow cytometric analysis of Bromodeoxyuridine, and
expressions were detected by Real-Time-RT-PCR analysis. The PLA2G6 gene encodes phospholipase A2 beta, which
is involved in the remodelling of membrane phospholipids,
signal transduction and calcium signalling, cell proliferation
and apoptosis. High glucose levels were inhibited NSC proliferation
(85,16%, 57,51% and 35,64%, respectively). IGF-I ± MGF
were enhanced cell proliferation and re-acquisition of NSC
proliferation (p≤0.0005). There was a negative correlation
between IGF-I ± MGF expression and glucose levels. The
highest expression with normoglycemia and a dramatic
decrease (100 fold) with hyperglycemia were detected. Even at the level of hyperglycemia, increased expressions
of IGF-I and MGF (0.5 and 3 fold) compared to the controls
were determined. Mutations in the PLA2G6 are known to cause three main
clinical
syndromes:
infantile
neuroaxonal
dystrophy
(INAD), atypical neuroaxonal dystrophy of childhood-
onset (atypical NAD) and adult-onset PLA2G6-related
dystonia- parkinsonism . Our cases have some similarities with INAD. In vitro Investigation for the role of IGF-I and MGF in
High Glucose Environment on Neural Stem Cell
Proliferation Both
syndromes are characterized by rapidly progressive psy-
chomotor regression and cerebellar atrophy. INAD does not
exhibit anterior horn cell degeneration. Though our cases have some similarities with INAD,
SMA-like phenotype has never been described in patients
with PLA2G6 mutations. IGF-I and MGF influence NSC proliferation and increase
even at the high glucose concentrations. This study
enlightens the IGF-I and MGF role in neuroprotective and
neuroproliferative ability of NSC and also for their possible
applications in treatment for the patients having diabetic
complications. M. Michelson Kerman: None. L. Dorit: None. K. Yosovich: None. M. Gurevitch: None. Functional analysis of a splicing region variant in С19orf12
in neurodegeneration with brain iron accumulation 4
(NBIA 4) Functional analysis of a splicing region variant in С19orf12
in neurodegeneration with brain iron accumulation 4
(NBIA 4) Functional analysis of a splicing region variant in С19orf12
in neurodegeneration with brain iron accumulation 4
(NBIA 4) S. Sozer: None. T. Gurbuz: None. S.B. Tunç: None. K. Ateş: None. P09.091C
Novel phenotype associated with mutations in the PLA2G6
gene Novel phenotype associated with mutations in the PLA2G6
gene 1Research center of medical genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology,
Dolgoprudny, Russian Federation M. Michelson Kerman, L. Dorit, K. Yosovich, M. Gurevitch
Wolfson Medical Center, Holon, Israel
Novel
phenotype
associated
with
mutations
in
the
PLA2G6 gene. M. Michelson(1,2),
D. Lev(1,2),
K. Yosovich(1),M. Gurevitch(1). 1) Inst Med Genetics, Wolfson Medical Ctr, Holon,
Israel; 2) Metabolic Neurogenetic clinic, Wolfson Medical
Ctr, Holon, Israel. M. Michelson Kerman, L. Dorit, K. Yosovich, M. Gurevitch M. Michelson Kerman, L. Dorit, K. Yosovich, M. Gurevitch Wolfson Medical Center, Holon, Israel Introduction: NBIA4 is an autosomal recessive disorder,
caused by homozygous or compound heterozygous patho-
genic variants in C19orf12 gene and characterized by
impaired gait, Parkinsonism, behavior and psychiatric
symptoms, optic nerve atrophy. We present a case report of
an 11-y.o. girl with signs of neurodegeneration with brain
MRI typical for iron accumulation. Novel
phenotype
associated
with
mutations
in
the
PLA2G6 gene. M. Michelson(1,2),
D. Lev(1,2),
K. Yosovich(1),M. Gurevitch(1). 1) Inst Med Genetics, Wolfson Medical Ctr, Holon,
Israel; 2) Metabolic Neurogenetic clinic, Wolfson Medical
Ctr, Holon, Israel. 291 Abstracts from the 51st European Society of Human Genetics Conference: Posters pN77K) in CLN6 gene. The amino acid is perfectly con-
served among species. In silico analysis, the mutation is
predicted to be probably damaging. Moreover, gene ana-
lysis for unrelated second Kufs patient was performed, who
had a same homozygous mutation. These data suggest that
the mutation must be pathogenic one. As they lived in the
same district, they seemed to be distant relative or might be
inherited founder effect mutation. Gene analysis of the third
NCL patient (Ueda T et al. Intern Med, 2013) was per-
formed of CLN6 and 13, however failed to detect any gene
mutation. Therefore, we performed whole exome sequen-
cing (WES). We identified a novel heterozygous CLN3
mutation (c313A>G, p. I105V) and autophagy related gene
mutation. Since NCLs are very rare disease in Japan and
awareness of the adult form of NCL is insufficient, WES
will be useful to find causative genes for NCL. Materials and methods: Whole exome sequencing
(WES) was performed in “Genomed” laboratory, Moscow,
Russia. Identified variants were confirmed by Sanger
sequencing. DNA was isolated from whole blood using
the phenol-chloroform extraction. HEK293T cells were
transfected with a minigene plasmid vector containing the
splicing region variant. Splicing change was validated by
RT-PCR with further Sanger sequencing. Results: WES identified two variants in the 19orf12 gene,
a common pathogenic deletion c.204_214del11 and a novel
splicing region variant c.193+5G>A in the intron 2. Functional analysis in HEK293T cells using a minigene
plasmid vector showed that c.193+5G>A variant leads to
skipping of the exon 2. c.193+5G>A variant disrupts the
splicing donor site of the intron 2. This leads to the
C19orf12 exon 2 skipping and results in a frame shift and a
formation of the premature stop codon. Hence, a truncated
protein is formed which length is less than 25% from the
natural. Results: WES identified two variants in the 19orf12 gene,
a common pathogenic deletion c.204_214del11 and a novel
splicing region variant c.193+5G>A in the intron 2. Functional analysis in HEK293T cells using a minigene
plasmid vector showed that c.193+5G>A variant leads to
skipping of the exon 2. c.193+5G>A variant disrupts the
splicing donor site of the intron 2. This leads to the
C19orf12 exon 2 skipping and results in a frame shift and a
formation of the premature stop codon. Hence, a truncated
protein is formed which length is less than 25% from the
natural. T. Inazu: None. M. Onodera: None. S. Tsujimoto:
None. S. Katayama:
None. T. Ueda:
None. T. Makifuchi: None. Conclusion: Therefore, we classify c.193+5G>A variant
in the 19orf12 gene as pathogenic and disease-causing in
our patient. To our knowledge, this is the first reported
pathogenic splicing region variant in NBIA4. P09.096D Identification of a Novel Silent Exonic Point Mutation in
the NF1 Gene Causing Partial Exon 9 Skipping Identification of a Novel Silent Exonic Point Mutation in
the NF1 Gene Causing Partial Exon 9 Skipping P. Sparber: None. A. Marakhonov: None. A. Filatova:
None. I. Sharkova: None. M. Skoblov: None. P. Sparber: None. A. Marakhonov: None. A. Filatova:
None. I. Sharkova: None. M. Skoblov: None. A. T. Hoejland1,2, I. Lolas3, T. Diemer1, H. Okkels3, M. B. Petersen1,2 j
Petersen1,2 Biallelic mutations in the homeodomain of NKX6-2
underlie a severe hypomyelinating leukodystrophy Biallelic mutations in the homeodomain of NKX6-2
underlie a severe hypomyelinating leukodystrophy P09.095C del Picchia phenotype
(HSPB3
mutation
in
Amyotrophic
lateral
sclerosis, and MYH14 mutation in patient mitochondrial
myopathy). phenotype
(HSPB3
mutation
in
Amyotrophic
lateral
sclerosis, and MYH14 mutation in patient mitochondrial
myopathy). spanning exon 7 and 8 of the NF1 gene and a reverse primer
spanning exon 10 and 11. Conclusion: The heterozygous c.987A>G mutation in the
NF1 gene was detected in the index patient and two other
family members with neurofibromatosis 1 and not identified
in a healthy family member. cDNA analysis of the index
patient showed, that the mutation causes a partial skipping
of exon 9, specifically the last 75bp. We speculate that this
silent mutation creates a cryptic donor splice site within
exon 9 leading to the production of truncated dysfunctional
protein. Conclusion: These results signify the importance of CES
in better diagnosis of obscure cases with neurodegenerative
disorders and gave us better insight in complexity of
genetics of these disorders. M. Brankovic: None. V. Dobricic: None. M. Svetel:
None. S. Peric: None. E. Stefanova: None. A. Marja-
novic: None. I. Petrovic: None. I. Novakovic: None. V. Kostic: None. A.T. Hoejland: None. I. Lolas: None. T. Diemer: None. H. Okkels: None. M.B. Petersen: None. Use of clinical exome analysis in rare neurodegenerative
disorders in Serbian population: Firs experience Use of clinical exome analysis in rare neurodegenerative
disorders in Serbian population: Firs experience C. Aiello1, I. Dorboz2, C. Simons3, R. Stone4, M. Niceta1,
M. Elmaleh4, M. Abuawad2, D. Doummar5, A. Bruselles6,
N. Wolf7, L. Travaglini1, O. Boespflug-Tanguy4, M. Tartaglia1,
A. Vanderver8, D. Rodriguez9, E. Bertini1 M. Brankovic1,2, V. Dobricic2, M. Svetel1,2, S. Peric1,2, M. Brankovic1,2, V. Dobricic2, M. Svetel1,2, S. Peric1,2,
E. Stefanova1,2, A. Marjanovic1,2, I. Petrovic1,2, I. Novakovic1,2,
V. Kostic1,2 E. Stefanova1,2, A. Marjanovic1,2, I. Petrovic1,2, I. Novakovic1,2,
V. Kostic1,2 E. Stefanova1,2, A. Marjanovic1,2, I. Petrovic1,2, I. Novakovic1,2,
V. Kostic1,2 1Bambino Gesu' Children's Hospital, Rome, Italy, 2Paris
Diderot University, Paris, France, 3The University of
Queensland, St Lucia, Australia, 4Robert Debré Hospital,
Paris, France, 5Hôpital Armand-Trousseau,, Paris, France,
6Istituto Superiore di Sanità, Rome, Italy, 7VU University
Medical Center,, Amsterdam, Netherlands, 8Children's
Hospital of Philadelphia, Philadelphia, PA, United States,
9Hôpital Armand-Trousseau, Paris, France 1Bambino Gesu' Children's Hospital, Rome, Italy, 2Paris
Diderot University, Paris, France, 3The University of
Queensland, St Lucia, Australia, 4Robert Debré Hospital,
Paris, France, 5Hôpital Armand-Trousseau,, Paris, France,
6Istituto Superiore di Sanità, Rome, Italy, 7VU University
Medical Center,, Amsterdam, Netherlands, 8Children's
Hospital of Philadelphia, Philadelphia, PA, United States,
9Hôpital Armand-Trousseau, Paris, France 1Faculty of Medicine, Belgrade, Serbia, 2Neurology Clinic,
Belgrade, Serbia 1Faculty of Medicine, Belgrade, Serbia, 2Neurology Clinic,
Belgrade, Serbia Introduction: Neurodegenerative diseases encompass a
heterogeneous group of disorders. The clinical diagnosis of
neurodegenerative disorders based on phenotype is difficult
in conditions with overlapping symptoms. Most of these
diseases have a genetic basis and thus are expected to be
amenable to genetic or genomic analysis by next-generation
sequencing (NGS). Introduction:
Hypomyelinating
leukodystrophies
are
genetically heterogeneous disorders with overlapping clin-
ical and neuroimaging features reflecting variable abnorm-
alities in myelin formation. The homeobox protein NKX6-2
is a transcription factor regulating multiple developmental
processes with a main role in oligodendrocyte differentia-
tion and regulation of myelin-specific gene expression. Introduction:
Hypomyelinating
leukodystrophies
are
genetically heterogeneous disorders with overlapping clin-
ical and neuroimaging features reflecting variable abnorm-
alities in myelin formation. The homeobox protein NKX6-2
is a transcription factor regulating multiple developmental
processes with a main role in oligodendrocyte differentia-
tion and regulation of myelin-specific gene expression. Material and Methods: Study included 20 patients with
various neurodegenerative diseases, negative after standard
molecular-genetic testing. Preference was given to family
cases
with
early
presentation
or
complex
phenotype
suggesting genetic heterogeneity. P09.095C 1Department of Clinical Genetics, Aalborg University
Hospital, Aalborg, Denmark, 2Department of Clinical
Medicine, Aalborg University, Aalborg, Denmark, 3Section of
Molecular Diagnostics, Department of Clinical Biochemistry,
Aalborg University Hospital, Aalborg, Denmark Identification of CLN6 and CLN3 genes mutations in three
unrelated Japanese neuronal ceroid lipofuscinosis patients Identification of CLN6 and CLN3 genes mutations in three
unrelated Japanese neuronal ceroid lipofuscinosis patients T. Inazu1, M. Onodera1, S. Tsujimoto1, S. Katayama1, T. Ueda2,
T. Makifuchi3,4 T. Inazu1, M. Onodera1, S. Tsujimoto1, S. Katayama1, T. Ueda2,
T. Makifuchi3,4 1College of Pharmaceutical Sciences, Ritsumeikan university,
Shiga, Japan, 2Department of Emergency Medicine and
General Internal Medicine, Rakuwakai Marutamachi
Hospital,, Kyoto, Japan, 3Department of Clinical Research,
National Hospital Organization Saigata Medical Center,
Niigata, Japan, 4Department of Laboratory Medicine, Joetsu
General Hosipital, Niigata, Japan Introduction: Neurofibromatosis 1 is a genetic disorder
caused by heterozygous mutations in the NF1 gene. The
main features of neurofibromatosis 1 are neurofibromas,
café-au-lait macules, axillary or inguinal freckling, lisch
noduli, and optic gliomas. We report the identification of a
novel silent exonic mutation, which causes partial exon
skipping and co–segregate with the disease. Materials and Methods: We report a family with four
family members that fulfils the diagnostic criteria for
neurofibromatosis 1. All exons and intron-exon boundaries
of the NF1, NF2, and SPRED1 genes were sequenced at
Department of Molecular Medicine, Aarhus University
Hospital by massive parallel sequencing on DNA from the
index patient. Carrier testing was performed on three family
members by direct sequencing analysis of exon 9. Total
RNA extraction was performed from total blood of the
index patient using PAXgene Blood RNA Kit 50 v2, and
reverse transcribed to cDNA using random hexames. Direct
cDNA sequencing was performed using a forward primer The neuronal lipofuscinoses (NCL) are a family of inher-
ited, neurodegenerative disorders that are accumulated with
ceroid lipofuscins in neurons, leading to the progressive loss
of vision and neuronal impairment. The NCLs have been
categorized into four classes based on the age of onset. Kufs
disease is an adult onset NCL, which is autosomal recessive
progressive lysosomal disorders and responsible genes are
CLN6 and CLN13. Here we present the mutational report
for adult NCL patients. We have reported a Kufs patient,
whose parents were consanguineous marriage (Sakajiri K
et al. Intern Med, 1995). We performed gene analysis and
found a known but homozygous mutation (c231C>G, 292 J. Use of clinical exome analysis in rare neurodegenerative
disorders in Serbian population: Firs experience In the third
family, whole exome sequencing established compound
heterozygosity for a non-conservative missense change Abstracts from the 51st European Society of Human Genetics Conference: Posters 293 affecting a key residue participating in DNA binding
(c.599G>A; p.Arg200Gln) and a nonsense substitution
(c.589C>T; p.Gln197*), in both affected siblings. The
clinical presentation was homogeneous, with four subjects
having severe motor delays, nystagmus and absent head
control, and one individual showing gross motor delay at
the age of 6 months. All exhibited neuroimaging that was
consistent with hypomyelination. affecting a key residue participating in DNA binding
(c.599G>A; p.Arg200Gln) and a nonsense substitution
(c.589C>T; p.Gln197*), in both affected siblings. The
clinical presentation was homogeneous, with four subjects
having severe motor delays, nystagmus and absent head
control, and one individual showing gross motor delay at
the age of 6 months. All exhibited neuroimaging that was
consistent with hypomyelination. affecting a key residue participating in DNA binding
(c.599G>A; p.Arg200Gln) and a nonsense substitution
(c.589C>T; p.Gln197*), in both affected siblings. The
clinical presentation was homogeneous, with four subjects
having severe motor delays, nystagmus and absent head
control, and one individual showing gross motor delay at
the age of 6 months. All exhibited neuroimaging that was
consistent with hypomyelination. (Cys67Gly and Gly89Cys) were first described in Serbian
CADASIL patients and characterized as pathogenic accord-
ing to in silico prediction. Positive family history was
reported in 10 of 19 mutation carriers. Interestingly, parents
of one mutation carrier were tested and found negative for
NOTCH3 mutation. Conclusions: Two novel mutations and one confirmed de
novo mutation in our study are implicating that estimation
of NOTCH3 gene mutation frequency and type in different
populations, as well as consideration of possible de novo
mutations, is important for efficient genetic testing strategy
for CADASIL. Conclusion: The finding of individuals with a severe
neurodevelopemental
phenotype
with
hypomyelination
associated with biallelic mutations in NKX6-2 provides
direct evidence of the relevant role of NKX6-2 in CNS
development in humans. M.Z. Jankovic: None. V. Dobricic: None. A. Marja-
novic: None. M. Brankovic: None. A. Pavlovic: None. I. Dujmovic: None. M. Mijajlovic: None. I. Novakovic:
None. V. Kostic: None. C. Aiello: None. I. Dorboz: None. C. Simons: None. R. Stone: None. M. Niceta: None. M. Elmaleh: None. M. Abuawad: None. D. Doummar: None. A. Bruselles:
None. N. Wolf: None. L. Travaglini: None. O. Boespflug-
Tanguy: None. M. Tartaglia: None. A. Vanderver: None. D. Rodriguez: None. E. Bertini: None. A novel case of severe neurodevelopmental delay in a
patient with Pitt-Hopkins like 2 syndrome associated with
compound heterozygous deletion in NRXN1 A novel case of severe neurodevelopmental delay in a
patient with Pitt-Hopkins like 2 syndrome associated with
compound heterozygous deletion in NRXN1 NOTCH3 mutations in Serbian CADASIL patients P. Castronovo1, M. Baccarin1, A. Ricciardello2, C. Picinelli1,
P. Tomaiuolo1, M. Lamberti2, F. Cucinotta2, M. Frittoli1,
C. Lintas3, R. Sacco3, A. Persico2,1 P. Castronovo1, M. Baccarin1, A. Ricciardello2, C. Picinelli1,
P. Tomaiuolo1, M. Lamberti2, F. Cucinotta2, M. Frittoli1,
C. Lintas3, R. Sacco3, A. Persico2,1 M. Z. Jankovic1, V. Dobricic1, A. Marjanovic1, M. Brankovic1,
A. Pavlovic1, I. Dujmovic1, M. Mijajlovic1, I. Novakovic2,
V. Kostic1 1Mafalda Luce Center for Pervasive Developmental Disorders,
Milan, Italy, 2Interdepartmental Program “Autism 0-90”, “G. Martino” University Hospital, University of Messina, Messina,
Italy, 3Unit of Child and Adolescent NeuroPsychiatry &
Laboratory of Molecular Psychiatry and Neurogenetics,
University “Campus Bio-Medico”, Rome, Rome, Italy 1Neurology Clinic, Clinical Center of Serbia, School of
Medicine, University of Belgrade, Belgrade, Serbia, 2Institute
for Human Genetics, School of Medicine, University in
Belgrade, Belgrade, Serbia Introduction: Cerebral autosomal dominant arteriopathy
with
subcortical
infarcts
and
leucoencephalopathy
(CADASIL) is one of the most common inherited small-
vessel disease, presenting with recurrent subcortical stroke
episodes, migraine, mood disorders and dementia. CADA-
SIL is caused by NOTCH3 gene mutations, and majority of
confirmed pathogenic variants are alterating number of
cysteine residues in EGFr domains of the NOTCH3 protein. CADASIL patients usually have positive family history and
de novo mutations are not commonly reported. Introduction: Neurexin 1 is a key organizer of the synapse. Only nine cases with compound heterozygous NRXN1
deletions/mutations have been reported so far, all of which
share a Pitt-Hopkins like 2 syndrome phenotype, char-
acterized by a severe global developmental delay. We
describe a 5 y.o. child with a compound heterozygous
deletion in NRXN1 gene and typical phenotype. Materials and Methods: The patient was evaluated by
GMDS and VABS Scales. CGH-array (180 K, Agilent
Technologies) was performed on the patient and the
NRXN1 deletions and familial segregation were confirmed
by qPCR . Materials and Methods: The patient was evaluated by
GMDS and VABS Scales. CGH-array (180 K, Agilent
Technologies) was performed on the patient and the
NRXN1 deletions and familial segregation were confirmed
by qPCR . Materials and Methods: Total genomic DNA was
extracted from peripheral blood leukocytes of 249 Serbian
patients with clinical diagnosis of CADASIL (with or
without family members with similar symptoms) and family
members of patients with confirmed mutations. PCR
amplification and direct sequencing of exons 2-6 of
NOTCH3 gene were performed. Use of clinical exome analysis in rare neurodegenerative
disorders in Serbian population: Firs experience Clinical exome sequen-
cing (CES) was performed using TruSight One Panel on
Illumina MiSeq NGS platform. Variants in genes related to
neurodegenerative diseases were analysed using Illumina
Variant Studio v3; confirmation by Sanger sequencing
was done. Material and Methods: Study included 20 patients with
various neurodegenerative diseases, negative after standard
molecular-genetic testing. Preference was given to family
cases
with
early
presentation
or
complex
phenotype
suggesting genetic heterogeneity. Clinical exome sequen-
cing (CES) was performed using TruSight One Panel on
Illumina MiSeq NGS platform. Variants in genes related to
neurodegenerative diseases were analysed using Illumina
Variant Studio v3; confirmation by Sanger sequencing
was done. Materials
and
Methods:
Whole-exome
sequencing
(WES) and homozygosity mapping of selected patients
from three unrelated families was undertaken. The variants
identified
were
validated
by
Sanger
sequencing
and
cosegregation analysis. Results: We revealed 10 pathogenic or likely pathogenic
variants in 9 different genes related to rare neurodegenera-
tive disorders in 9 patients. Mutations in PANK2, PDGFB,
DCTN1 and PSEN1 genes causing neurodegeneration with
brain iron accumulation, Fahr's, Perry syndrome and
Alzheimer's disease respectively, are compatible with the
phenotype of patients. In five cases DNA diagnosis remain
unclear. Only one recessive mutation was found in SPG7
and AP5Z1 gene in cases of spastic paraplegia, and in
COL6A3 gene in case of dystonia. In another two cases
detected variants were not directly related to patient’s Results: Five affected subjects (three unrelated families)
were documented to share biallelic inactivating mutations
affecting the NKX6-2 homeobox domain. A trio-based
whole exome sequencing analysis in the first family
detected a homozygous frameshift change [c.606delinsTA;
p.(Lys202Asnfs*?)]. In the second family, homozygosity
mapping coupled to whole exome sequencing identified a
homozygous nucleotide substitution (c.565G>T) introdu-
cing a premature stop codon (p.Glu189*). In the third
family, whole exome sequencing established compound
heterozygosity for a non-conservative missense change Results: Five affected subjects (three unrelated families)
were documented to share biallelic inactivating mutations
affecting the NKX6-2 homeobox domain. A trio-based
whole exome sequencing analysis in the first family
detected a homozygous frameshift change [c.606delinsTA;
p.(Lys202Asnfs*?)]. In the second family, homozygosity
mapping coupled to whole exome sequencing identified a
homozygous nucleotide substitution (c.565G>T) introdu-
cing a premature stop codon (p.Glu189*). V. McNiven1, S. Mamane2, G. Zai3, J. So2,3,4 V. McNiven1, S. Mamane2, G. Zai3, J. So2,3,4 V. McNiven1, S. Mamane2, G. Zai3, J. So2,3,4 Parkinson disease (PD) is a chronic, debilitating and pro-
gressive neurodegenerative disorder characterized by bra-
dykinesia, resting tremor, rigidity and postural instability. 1Division of Clinical and Metabolic Genetics, The Hospital for
Sick Children, Toronto, ON, Canada, 2The Fred A. Litwin
Family Centre in Genetic Medicine, University Health Network
and Mount Sinai Hospital, Toronto, ON, Canada, 3Centre for
Addiction and Mental Health, Department of Psychiatry,
University of Toronto, Toronto, ON, Canada, 4Department of
Medicine, University of Toronto, Toronto, ON, Canada 1Division of Clinical and Metabolic Genetics, The Hospital for
Sick Children, Toronto, ON, Canada, 2The Fred A. Litwin
Family Centre in Genetic Medicine, University Health Network
and Mount Sinai Hospital, Toronto, ON, Canada, 3Centre for
Addiction and Mental Health, Department of Psychiatry,
University of Toronto, Toronto, ON, Canada, 4Department of
Medicine, University of Toronto, Toronto, ON, Canada About 5-10% of patients suffer from a monogenic form of
PD caused by highly penetrant mutations. Currently, sixteen
PARK loci have been identified with autosomal dominant
or autosomal recessive genes. We report here a 48 years old patient with PD. Resting
tremor was initially limited to the left upper limb. As it is
commonly observed in early onset PD patients (Mehanna),
symptoms other than tremor were more evident at the
beginning of the disease with decreased arm swing
movements,
depression,
bradykinesia
and
urinary
incontinence. Olfactory reference syndrome (ORS) is a psychiatric dis-
order in which individuals hold a false belief that they emit
an offensive body odour. ORS leads to significant distress,
and yet remains poorly characterized with limited recogni-
tion and characterization of its management. Patients with
ORS rarely seek psychiatric attention as first-line treatment,
and instead seek an organic diagnosis, such as trimethyla-
minuria (TMAU). TMAU is an inherited disorder in which
there is a failure to break down trimethylamine, creating a
pungent fishy odour. This study characterizes the clinical
and demographic features of a cohort who meet the defi-
nition of ORS, and yet who presented to a Canadian
genetics clinic for query TMAU. Data was obtained via a
retrospective chart review over a 7 year span (N=54). Only
two individuals who presented for query TMAU had this
diagnosis, while 83% had a likely diagnosis of ORS. NOTCH3 mutations in Serbian CADASIL patients Materials and Methods: Total genomic DNA was
extracted from peripheral blood leukocytes of 249 Serbian
patients with clinical diagnosis of CADASIL (with or
without family members with similar symptoms) and family
members of patients with confirmed mutations. PCR
amplification and direct sequencing of exons 2-6 of
NOTCH3 gene were performed. Results: the patient shows a severe global developmental
delay with intellectual disability, poor speech, motor
stereotypies, autistic features and cranio-facial dysmorph-
ism. In addition, the child suffers from celiac disease with
chronic constipation and positive anti-gliadin IgG. Beha-
vior, as assessed by GDMS, is equivalent to an age of
<12 months. Array-CGH revealed a complex alteration of
the region 2p16.3 due to the presence of two inherited, Results: the patient shows a severe global developmental
delay with intellectual disability, poor speech, motor
stereotypies, autistic features and cranio-facial dysmorph-
ism. In addition, the child suffers from celiac disease with
chronic constipation and positive anti-gliadin IgG. Beha-
vior, as assessed by GDMS, is equivalent to an age of
<12 months. Array-CGH revealed a complex alteration of
the region 2p16.3 due to the presence of two inherited, Results: Nineteen heterozygous mutation carriers were
identified in our cohort and mutation frequency was 7,6%
(19/249). Eight previously described, cysteine-alterating
mutations were identified in 13 cases. Two changes J. del Picchia 294 partly overlapping, 467 and 269 kb deletions, both
disrupting the NRXN1 gene. diverse group of patients who fit the definition of ORS. Based on this phenotypic characterization, we suggest
clinical criteria to assist in the recognition of ORS. Improving the recognition and diagnosis of ORS will lead
to more targeted management, reduce psychological distress
and stigma, and save in health care costs. The unique
phenotypic features also suggest a possible as-yet undefined
genetic basis for this syndrome, which future genomic
studies could help to elucidate. Conclusions: We found significant overlap in phenotypic
severity between our case and the nine previously reported
patients with biallelic defects in NRXN1. Our report
confirms the evidence that NRXN1 nullisomy is associated
with the clinical diagnosis of Pitt-Hopkins like 2 syndrome. Carriers of an heterozygous NRXN1 deletion/mutation
showing a particularly severe phenotype should be screened
for possible mutations or microdeletions of the second
allele. V. McNiven: None. S. Mamane: None. G. Zai: None. J. So: None. Funding information: Italian Ministry of Health (NET-
2013-02355263); European Union Innovative Medicines
Initiative Joint Undertaking (EU-AIMS, n. NOTCH3 mutations in Serbian CADASIL patients 115300) Funding information: Italian Ministry of Health (NET-
2013-02355263); European Union Innovative Medicines
Initiative Joint Undertaking (EU-AIMS, n. 115300) P09.104D
Identification of a new candidate gene of monogenic
Parkinkison disease: MTIF3 Identification of a new candidate gene of monogenic
Parkinkison disease: MTIF3 P. Castronovo: None. M. Baccarin: None. A. Ricciar-
dello: None. C. Picinelli: None. P. Tomaiuolo: None. M. Lamberti: None. F. Cucinotta: None. M. Frittoli: None. C. Lintas: None. R. Sacco: None. A. Persico: None. J. J. Telleria1,2,3, E. Bueno-Martínez1, R. González-Sarmiento1,4 J. J. Telleria1,2,3, E. Bueno-Martínez1, R. González-Sarmiento1,4 1Instituto de Investigación Biomedica de Salamanca (IBSAL),
Salamanca, Spain, 2Hospital Clínico Universitario de
Valladolid, Valladolid, Spain, 3IBGM (UVa / CSIC),
Valladolid, Spain, 4Unidad de Medicina Molecular. Universidad de Salamanca, Salamanca, Spain Identification of new genes involved in autosomal dominant
forms of Parkinson’s disease 1Department of Medical Chemistry and Biochemistry,
Molecular Medicine Center, Medical University, Sofia, Sofia,
Bulgaria, 2Neurodegenerative Brain Diseases Group, Center
for Molecular Neurology, VIB, Antwerp, Belgium, 3Laboratory
of Neurogenetics, Institute Born Bunge, University of Antwerp,
Antwerp, Belgium, 4Clinic of Neurology, University Hospital
"Alexandrovska", Department of Neurology, Medical
University-Sofia, Sofia, Bulgaria, 5Molecular Neurogenomics
Group, Center for Molecular Neurology, VIB, University of
Antwerp, Antwerp, Belgium C. Tesson, C. Condroyer, L. Ruaud, V. Drouet, S. Lesage,
A. Brice C. Tesson, C. Condroyer, L. Ruaud, V. Drouet, S. Lesage,
A. Brice - INSERM U1127, CNRS UMR 7225, UPMC Université Paris
06 UMR S1127, Sorbonne Université Institut du C, Paris,
France Parkinson disease (PD) is the second most frequent neu-
rodegenerative disorder, affecting 1% of the population
above 65 years. To date, the identified genes associated
with AD PD only explain 10-15%, so many genes remain
to be discovered. We selected 73 affected relatives from
20 multiplex AD PD families for exome sequencing. We
looked for rare heterozygous variants, predicted to be
pathogenic using CADD and M-CAP scores, shared by all
affected relatives within the family. Replication studies
were done using the available exome data from ~1,500 PD
cases and 560 controls from the IPDGC consortium. Pathway analyses were realized using the R package
Clusterprofiler and the web interface ConsensuspathDB. For each family, we obtain a list of candidate genes (2 to
30). To prioritize them, we applied filtering criteria which
in order were 1) expression in brain; 2) replication of
these possible candidate genes in additional families; 3)
sharing common physiopathological pathways. None of
the identified candidate genes was replicated, using home
and IPDGC consortium exome databases. On the other
hand, using Clusterprofiler we found that some of these
genes can be grouped in the same biological process,
which is actin metabolism. Furthermore, using Con-
sensuspathDB we highlighted that some of these candi-
dates’ genes were able to interact with genes involved in
PD. Conclusion Although we identified a substantial
number of multiplex families with AD PD, none of these
families was explained by possible shared candidate
genes, highlighting the genetic heterogeneity of PD. However, these genes were enriched in a common
regulating. Parkinson disease (PD) is the second most common neu-
rodegenerative disease resulting from the interplay of mul-
tiple genes with environmental risk factors. Inheritance is in
an autosomal dominant, autosomal recessive, or X-linked
manner and monogenic forms are rarely found. P09.106B Targeted genetic analysis of Parkinson disease in Bulgarian
patients K. Mihova1, A. Verstraeten2,3, J. Theuns2,3, R. Pavlova4,
S. Mehrabian4, M. Petrova4, S. Skelina4, R. Kaneva1, V. Mitev1,
A. Jordanova1,5, C. Van Broeckhoven2,3, L. Traykov4 J.J. Telleria: None. E. Bueno-Martínez: None. R. González-Sarmiento: None. C. Tesson: None. C. Condroyer: None. L. Ruaud:
None. V. Drouet: None. S. Lesage: None. A. Brice: None. V. McNiven1, S. Mamane2, G. Zai3, J. So2,3,4 Of this
ORS group, 62% were female, 73% had a psychiatric his-
tory, and 77% were seen by multiple specialties. This study
is the first to systematically examine a large, ethnically Once discarded the presence of pathogenic mutations
within the known genes causing PD. The analysis of whole
exome data applying a prioritization scheme identified two
pathogenic variants which encode premature stop codons in
MTIF3 gene that interacts with PINK1, a known PD
autosomal recessive causing gene. Mitochondrial damage plays a central role in PD
pathogenesis and several PD causing genes are known to
regulate mitochondrial function and homeostasis Moreover,
a known allele of the mitochondrial factor 3 (MTIF3) which
causes a significant reduction of MTIF3 mRNA expression,
has been associated to PD in patient-control series. If paucity of MTIF3 increases the risk of PD, Its complete
absence should be related to significantly higher risk. Abstracts from the 51st European Society of Human Genetics Conference: Posters 295 P09.106B
Targeted genetic analysis of Parkinson disease in Bulgarian
patients Our data suggest that MTIF3 could be a new PD
causing gene. Further functional and population studies
are needed to assess the exact role of MTIF3 in the
pathogenesis of PD. P09.107C
Bridging mitochondrial and lysosomal dysfunction in
Parkinson Disease: clues of oligogenic inheritance C. Pereira1,2, J. Sequeiros1,2,3, R. Gassmann4, I. Alonso1,2,3 C. Pereira1,2, J. Sequeiros1,2,3, R. Gassmann4, I. Alonso1,2,3 1UnIGENe, Instituto de Biologia Molecular e Celular (IBMC),
Instituto de Investigação e Inovação em Saúde (i3S),
Universidade do Porto, Porto, Portugal, 2Instituto de Ciências
biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal, 3Centro de Genética Preditiva e Preventiva
(CGPP), Instituto de Biologia Molecular e Celular (IBMC),
Instituto de Investigação e Inovação em Saúde (i3S),
Universidade do Porto, Porto, Portugal, 4Cell Division
Mechanisms, Instituto de Biologia Molecular e Celular
(IBMC), Instituto de Investigação e Inovação em Saúde (i3S),
Universidade do Porto, Porto, Portugal S. Smolders1,2, D. Crosiers1,2,3, P. Cras2,3, C. Van
Broeckhoven1,2, BELNEU Consortium 1VIB Center for Molecular Neurology, University of Antwerp,
Antwerp, Belgium, 2Institute Born-Bunge, University of
Antwerp, Antwerp, Belgium, 3Department of Neurology,
Antwerp University Hospital, Edegem, Belgium Introduction: Mutations in PARK2, PINK1, DJ-1 and
VPS13C cause autosomal recessive Parkinson Disease (PD)
and impair mitochondrial quality control pathways. More-
over, substantial evidence highlights the importance of
lysosomal mechanisms in PD, including excessive burden
of lysosomal storage disorder (LSD) gene variants in PD
patients. Parkinson disease (PD) results from the loss of dopami-
nergic neurons and represents one of the most common
neurodegenerative diseases. Models for LRRK2-mediated
PD in C. elegans have relied on multi-copy overexpression
of human transgenes from extrachromosomal arrays or
random genomic integrations. Although successful in
reproducing the neurotoxic effects of human LRRK2
mutants, the variability of expression between worm strains
in these models complicates the interpretation of LRRK2
mutant phenotypes. To produce a closer physiological
model of PD in C. elegans we generated new strains in
which transgenes are integrated in a defined genomic locus
in single copy. This ensures that human wild-type LRRK2
and mutant transgenes are expressed at identical levels in
every animal. We also used CRISPR-Cas9-mediated gen-
ome editing to introduce the equivalent of the human
LRRK2 G2019S variant into the worm orthologue lrk-1. We
show that expression of either human mutant LRRK2 (at
similar levels of endogenous lrk-1) or the worm lrk-1
mutant does not lead to a dopaminergic neurodegenerative
phenotype or motility problems, both a major hallmark of
C. elegans PD models, even in severely aged animals. In
fact, mutant strains show a minor increase in the thrashing
rate along with the cat-2 mutant. Other lesser behavior
deficits were also present, such as reduced life span and
hyperactive egg laying. C. Pereira: None. J. Sequeiros: None. R. Gassmann:
None. I. Alonso: None. P09.108D
MosSCI and CRISPR C. elegans models of LRRK2 related
Parkinson’s disease fail to produce the expected phenotype P09.108D Identification of new genes involved in autosomal dominant
forms of Parkinson’s disease The main
goal of the current study is to evaluate the frequency and
type of mutations in genes associated with Parkinson dis-
ease in Bulgarian patients. Altogether, 69 patients with PD
and detailed clinical assessment and 108 healthy controls,
matched by age, gender and ethnicity (NC) were recruited. All individuals were analyzed with a custom panel includ-
ing known and candidate PD genes on an Illumina NGS
platform. All pathogenic variants were confirmed with
Sanger sequencing. 14 patients were analyzed with MLPA
P051-D1 Parkinson kit. In 52% (36/69) of the patients
variants in LRRK2, PARK2, PINK1, PARK7, ATP13A2,
FBXO7, PSEN1, PSEN2, CHMP2B, GRN, MAPT, EIF4G1
were identified. Altogether 14 new variants were found (10
missense, 2 splice site, 2 frameshift) and 14 VUS (13
missense, 1 splice site). Four pathogenic variants, including
two novel ones, were identified in PARK2, PARK7, PSEN2. No mutations were found with MLPA analysis. This is the
first Bulgarian study including a cohort of 69 patients
showing the frequency and distribution of novel and known
variants in genes associated with Parkinson disease. Our
findings contribute to a better understanding of the mole-
cular basis of Parkinson disease and have implications for
diagnostic testing and genetic counseling in Bulgarian
population. Acknowledgements:
D35/27.05.2016,
D77/
02.05.2017 of SF, MU-Sofia, DUNK01-2/2009, NSF. C. Tesson: None. C. Condroyer: None. L. Ruaud:
None. V. Drouet: None. S. Lesage: None. A. Brice: None. K. Mihova: None. A. Verstraeten: None. J. Theuns:
None. R. Pavlova: None. S. Mehrabian: None. M. Petrova: None. S. Skelina: None. R. Kaneva: None. V. 296 J. del Picchia Mitev: None. A. Jordanova: None. C. Van Broeckhoven:
None. L. Traykov: None. P09.107C
Bridging mitochondrial and lysosomal dysfunction in
Parkinson Disease: clues of oligogenic inheritance
S. Smolders1,2, D. Crosiers1,2,3, P. Cras2,3, C. Van
Broeckhoven1,2, BELNEU Consortium P09.108D
MosSCI and CRISPR C. elegans models of LRRK2 related
Parkinson’s disease fail to produce the expected phenotype LUXEMBOURG CENTRE FOR SYSTEMS BIOMEDICINE,
Esch-sur-Alzette, Luxembourg 1Institut de Recerca Sant Joan de Déu, Barcelona, Spain,
2Instituto de Investigación Sanitaria Fundación Jiménez Díaz,
Madrid, Spain, 3Hospital Sant Joan de Déu, Barcelona, Spain,
4Instituto Ramón y Cajal de Investigación Sanitaria, Madrid,
Spain, 5Hospital Universitario Fundación Alcorcón, Madrid,
Spain, 6Hospital Universitario y Politécnico La Fe, Valencia,
Spain, 7Hospital Universitario Infanta Sofía, Madrid, Spain,
8CIBERER, Madrid, Spain, 9CIBERSAM, Madrid, Spain Introduction: Polygenic risk scores (PRS) are widely used
in the field of genetics to associate a group of SNPs to a
disease/trait. However, majority of the studies use only the
common genetic variants to generate the PRS. In our study,
in addition to the common genetic variants, we also used the
number of private loss of function variants per sample
(singleton_score). Aims: Next-generation sequencing (NGS) has proven to be
very useful for the diagnosis of heterogeneous disorders in
both their etiology and clinical expression. The aim of this
study is to evaluate whether genetic diagnosis of patients
with early-onset parkinsonism (EOP) can benefit from a
panel that contains both coding and regulatory regions of
genes related to these disorders. Materials and Methods: The data was provided by
Parkinson’s Progression Markers Initiative consortium. We
used the NeuroX array data to generate the PRS based on
common genetic variants and Whole exome sequencing
data to generate singleton_score. Machine learning models
were built based on state of the art methods and we
performed a comprehensive assessment of the contribution
of singleton_score in the prediction models. Materials and Methods: The data was provided by
Parkinson’s Progression Markers Initiative consortium. We
used the NeuroX array data to generate the PRS based on
common genetic variants and Whole exome sequencing
data to generate singleton_score. Machine learning models
were built based on state of the art methods and we
performed a comprehensive assessment of the contribution
of singleton_score in the prediction models. Materials and methods: we have recruited 96 EOP
patients (<55 years of age) from the Movement Disorders
Units at tertiary centers. The custom genetic panel of
Nextera® Rapid Capture Enrichment technology (Illumina)
contains 63 genes related to the following: i) familiar
Parkinson
disease,
ii)
atypical
Parkinsonisms,
iii)
Parkinsonism-pyramidal syndrome and iv) risk factors for
Parkinson disease. MiniSeq Sequencing System (Illumina)
was used for NGS and bioinformatics analyses and filtering
were performed. Materials and methods: we have recruited 96 EOP
patients (<55 years of age) from the Movement Disorders
Units at tertiary centers. S. Petrucci1,2,3, M. Ginevrino1,4, A. Di Fonzo5,6, M. T.
Pellecchia7, M. C. Altavista8, F. Bove9, A. Cozzolino7,
C. Criscuolo10, A. De Rosa10, G. Fabbrini2, G. Fabbrini2,11,
M. Moccia10, M. Petracca9, M. Squillante12, I. Trezzi5,6,
G. Volpe12, A. R. Bentivoglio9, A. Berardelli2,11, P. Barone7,
F. Morgante13,14, E. M. Valente1,4 LUXEMBOURG CENTRE FOR SYSTEMS BIOMEDICINE,
Esch-sur-Alzette, Luxembourg The custom genetic panel of
Nextera® Rapid Capture Enrichment technology (Illumina)
contains 63 genes related to the following: i) familiar
Parkinson
disease,
ii)
atypical
Parkinsonisms,
iii)
Parkinsonism-pyramidal syndrome and iv) risk factors for
Parkinson disease. MiniSeq Sequencing System (Illumina)
was used for NGS and bioinformatics analyses and filtering
were performed. Results: We confirm that the addition of singleton_score
significantly improved the performance of the model
(AUC=0.72) compared to the model built on common
variants alone (AUC=0.62). Additionally, we show that the
inclusion of singleton_score along with the clinical scores
improved the overall predictive ability of the model. Conclusions: Although PRS generated by using common
variants is a very useful approach in order to understand the
disease mechanism. It is interesting to see that rare/ultra-rare
variants also contributing to the association signal, provid-
ing evidence that rare/ultra-rare might add to missing
heritability of PD. Conclusions: Although PRS generated by using common
variants is a very useful approach in order to understand the
disease mechanism. It is interesting to see that rare/ultra-rare
variants also contributing to the association signal, provid-
ing evidence that rare/ultra-rare might add to missing
heritability of PD. Results: Until date, we have sequenced 48 patients. We
found 91.2% coverage of targeted genomic regions at 50-
fold mean coverage. These results show 71 previously
documented rare variants and 19 new variants in 35 out of
63 genes of the panel. 13 rare variants where reported as
pathogenic and 19 novel variants had in silico prediction of
being pathogenic. We also have detected Copy Number
Variations (CNVs) in 28 samples. Currently, we are
analyzing regulatory regions variants. D.R. Bobbili: None. P. Banda: None. R. Krueger:
None. P. May: None. D.R. Bobbili: None. P. Banda: None. R. Krueger:
None. P. May: None. P09.110B
Generation of prediction models based on rare and
common genetic variants in PD M. Osuna-López1, P. García-Ruiz2, G. Fernandez3, J. Martínez-
Castrillo4, L. Vela5, J. Maynou3, I. Martínez-Torres6, C. Feliz
Feliz2, X. Castro-Martínez1, M. Mata7, F. Palau1,3,8,
J. Hoenicka1,9 P09.109A P09.109A
Genomic analysis of coding and regulatory regions using
NGS in early-onset parkinsonism: a multicentric study Feliz Feliz: None. X. Castro-Martínez: None. M. Mata:
None. F. Palau: None. J. Hoenicka: None. Feliz Feliz: None. X. Castro-Martínez: None. M. Mata:
None. F. Palau: None. J. Hoenicka: None. P09.107C
Bridging mitochondrial and lysosomal dysfunction in
Parkinson Disease: clues of oligogenic inheritance While the newly generated models
do address the issue of variable expression neither mutant
human LRRK2 nor mutant lrk-1 produce a robust easy to
score phenotype, and therefore cannot be used for genetic or
pharma screens. Materials and Methods: We analyzed WES data of 66
PD patients, including 34 patients with a single rare
heterozygous mutation in an autosomal recessive PD gene
and 2 related patients, to investigate a role for different
genetic factors in disease etiology. Variants in autosomal
recessive genes associated with PD, atypical parkinsonian
syndromes and LSD were prioritized based on quality,
frequency in public databases and impact (splice site and
non-synonymous
variants
with
Combined
Annotation
Dependent Depletion score >20). Materials and Methods: We analyzed WES data of 66
PD patients, including 34 patients with a single rare
heterozygous mutation in an autosomal recessive PD gene
and 2 related patients, to investigate a role for different
genetic factors in disease etiology. Variants in autosomal
recessive genes associated with PD, atypical parkinsonian
syndromes and LSD were prioritized based on quality,
frequency in public databases and impact (splice site and
non-synonymous
variants
with
Combined
Annotation
Dependent Depletion score >20). Results: The WES data analysis revealed the presence of
oligogenic inheritance through known pathogenic and rare
novel heterozygous mutations in multiple genes. We
identified 1 patient carrier of compound mutations in
PARK2/DJ-1 and 1 patient with compound mutations in
PARK2/VPS13C. Coexistence of mitochondrial and lyso-
somal pathways is established by the observation in 16
patients of multiple mutations in PD and LSD genes,
including 7 known pathogenic LSD gene mutations. Of
note, the compound mutations PARK2 p.P437L and HEXA
p.R247W are both present in an affected mother and
daughter. Additionally, we found 4 carriers of compound
mutations in different LSD genes. Conclusions: Our results underpin the potential oligo-
genic complexity of Mendelian genes in PD etiology and
highlight the crosstalk between mitochondria and lyso-
somes in the pathophysiology of PD. C. Pereira: None. J. Sequeiros: None. R. Gassmann:
None. I. Alonso: None. C. Pereira: None. J. Sequeiros: None. R. Gassmann:
None. I. Alonso: None. S. Smolders: None. D. Crosiers: None. P. Cras: None. C. Van Broeckhoven: None. S. Smolders: None. D. Crosiers: None. P. Cras: None. C. Van Broeckhoven: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 297 P09.111C GBA-related Parkinson disease: mutational frequency and
clinical- biochemical correlates in the Italian population Conclusions: Our preliminary findings show that NGS
panels can be useful to identify gene variations in EOP
patients
and
it
shows
novel
genotype/phenotype
relationships. Funding: Instituto de Salud Carlos III/FEDER, grant no. PI15/01013. Funding: Instituto de Salud Carlos III/FEDER, grant no. PI15/01013. M. Osuna-López: None. P. García-Ruiz: None. G. Fernandez: None. J. Martínez-Castrillo: None. L. Vela:
None. J. Maynou: None. I. Martínez-Torres: None. C. 298 J. del Picchia 1CERC Neurogenetic Unit, IRCCS Santa Lucia Foundation,
Rome, Italy, 2Department of Human Neurosciences, Sapienza
University, Rome, Italy, 3IRCCS Casa Sollievo della
Sofferenza, Medical Genetics Unit, San Giovanni Rotondo,
Foggia, Italy, 4Department of Molecular Medicine, University
of Pavia, Pavia, Italy, 5IRCCS Foundation Ca' Grande
Ospedale Maggiore Policlinico, Dino Ferrari Center, Milan,
Italy, 6Neuroscience Section, Department of Pathophysiology
and Transplantation, University of Milan, Milan, Italy, 7Center
for Neurodegenerative Diseases (CEMAND), Department of
Medicine and Surgery, Neuroscience Section, University of
Salerno, Salerno, Italy, 8San Filippo Neri Hospital, ASL Roma
1, Rome, Italy, 9Department of Neurology, Catholic University,
Rome, Italy, 10Department of Neurosciences, Reproductive and
Odontostomatological Sciences, Federico II University,
Naples, Italy, 11IRCCS Neuromed, Pozzilli, Italy, 12Department
of Neuroscience, AOU San Giovanni di Dio e Ruggi
d'Aragona, Salerno, Italy, 13Department of Clinical and
Experimental Medicine, University of Messina, Messina, Italy,
14Institute of Molecular and Clinical Sciences, St George's
University of London, London, United Kingdom for counselling, appropriate management of non-motor
complications, and selection of patients amenable to enter
ongoing clinical trials. for counselling, appropriate management of non-motor
complications, and selection of patients amenable to enter
ongoing clinical trials. S. Petrucci: None. M. Ginevrino: None. A. Di Fonzo:
None. M.T. Pellecchia: None. M.C. Altavista: None. F. Bove: None. A. Cozzolino: None. C. Criscuolo: None. A. De Rosa: None. G. Fabbrini: None. G. Fabbrini: None. M. Moccia: None. M. Petracca: None. M. Squillante:
None. I. Trezzi: None. G. Volpe: None. A.R. Bentivoglio:
None. A. Berardelli:
None. P. Barone:
None. F. Morgante: None. E.M. Valente: None. P09.112D
Parkinson’s disease: a disruption of nuclear/mitochondrial
DNA communication H. Lowes1, A. Hudson1, M. Santibanez-Koref2, G. Hudson1 1Wellcome Trust Centre for Mitochondrial Research,
Newcastle upon Tyne, United Kingdom, 2Institute of Genetic
Medicince, Newcastle upon Tyne, United Kingdom Background: The role of mitochondrial function in Par-
kinson’s disease (PD) is established, however the precise
role of mtDNA (mtDNA) variation in PD remained unclear. Work in our laboratory has focused in the role that inher-
ited mtDNA variants play in developing PD, identifying
both low- and high-risk mtDNA alleles associated with PD. Based on this, and supported by our work in other diseases,
we hypothesised that inherited mtDNA variation disturbs
the transcriptomic balance within vulnerable brain regions,
causing either a direct mitochondrial phenotype or a dis-
ruption of the delicate mitochondrial/nuclear synergy con-
tributing to cellular vulnerability and ultimately leading to
PD. Introduction: Heterozygous variants in the GBA gene,
encoding β-glucocerebrosidase (GCase), are major genetic
risk factors for Parkinson Disease (PD). Their frequency
and phenotypic correlates have been assessed in many large
cohorts of different ethnicities, being identified in ~10%
PD. However, in the two published Italian studies, only two
common alleles (N370S and L444P) were investigated, with
an overall frequency of 4,4%. Patients and Methods: The whole GBA coding region
(11 exons) was Sanger-sequenced in 850 Italian PD
probands, who underwent deep phenotyping for motor
and non-motor signs. Clinical features and, in a subset,
GCase activity were compared between GBA-PD and not
mutated-PD (NM-PD), and among carriers of complex,
severe, mild and risk variant alleles. Patients and Methods: The whole GBA coding region
(11 exons) was Sanger-sequenced in 850 Italian PD
probands, who underwent deep phenotyping for motor
and non-motor signs. Clinical features and, in a subset,
GCase activity were compared between GBA-PD and not
mutated-PD (NM-PD), and among carriers of complex,
severe, mild and risk variant alleles. Methodology: To address this hypothesis, we interro-
gated the transcriptome of vulnerable regions of the brain
in post-mortem PD tissue using RNAseq, stratifying our
results using background mtDNA sequence data and
comparing transcript abundances to matched controls. Methodology: To address this hypothesis, we interro-
gated the transcriptome of vulnerable regions of the brain
in post-mortem PD tissue using RNAseq, stratifying our
results using background mtDNA sequence data and
comparing transcript abundances to matched controls. Results: Our results indicate the background mtDNA
sequence modulates the expression of key protein coding
transcripts. Genotype-phenotype correlations in an Italian sample of
patients with Phelan-McDermid syndrome Genotype-phenotype correlations in an Italian sample of
patients with Phelan-McDermid syndrome J. Trinh1, A. Grunewald2, K. Wasner2, A. A. Hicks3, P. Bauer4,
S. Imhoff1, K. K. Kandaswamy4, N. Ouzren2, M. Werber4, M. E. R. Weiss4, A. Rolfs4, P. P. Pramstaller3, P. Seibler1,
K. Lohmann1, C. Klein1 A. Ricciardello1, F. Cucinotta1, L. Turriziani1, M. Lamberti1,
M. Briguglio1, P. Tomaiuolo2, M. Baccarin2, C. Picinelli2,
P. Castronovo2, M. Boncoddo1, F. Bellomo1, G. Turturo1,
M. Canali3, R. Sacco3, C. Lintas3, A. M. Persico1,2
1University of Messina, Messina, Italy, 2Mafalda Luce Center
for Pervasive Developmental Disorders, Milan, Italy,
3University Campus Bio-Medico, Rome, Italy A. Ricciardello1, F. Cucinotta1, L. Turriziani1, M. Lamberti1,
M. Briguglio1, P. Tomaiuolo2, M. Baccarin2, C. Picinelli2,
P. Castronovo2, M. Boncoddo1, F. Bellomo1, G. Turturo1,
M. Canali3, R. Sacco3, C. Lintas3, A. M. Persico1,2 1Institute of Neurogenetics, Luebeck, Germany, 2Luxembourg
Centre for Systems Biomedicine, Luxembourg, Luxembourg,
3Institute for Biomedicine, Eurac Research, Bolzano, Italy,
4Centogene AG, Rostock, Germany 1University of Messina, Messina, Italy, 2Mafalda Luce Center
for Pervasive Developmental Disorders, Milan, Italy,
3University Campus Bio-Medico, Rome, Italy Background: Biallelic mutations in Parkin and PINK1 are
fully penetrant and cause recessively inherited Parkinson’s
disease (PD). On the other hand, heterozygous mutations in
these genes may be considered as a risk factor for PD or
even act in a dominant manner with highly reduced pene-
trance. Since both Parkin and PINK1 function in the
removal of dysfunctional mitochondria, we hypothesize that
mitochondrial DNA (mtDNA) mutations are both a con-
sequence of dysfunction in these genes, and also able to
influence age-dependent penetrance and thus the onset of
disease symptoms if not cleared sufficiently over time (i.e. a
‘second hit’). Introduction: Phelan-McDermid syndrome (PMS), is a
neurodevelopmental disorder characterized by intellectual
disability (ID), hypotonia, delayed or absent speech, and
autism spectrum disorder (ASD). PMS is due to de novo
chr. 22q13 terminal deletions or point mutations involving
the SHANK3 gene, crucial to the formation and plasticity of
excitatory
synapses. Disruption
of
SHANK3
causes
approximately 0.5% of ASD cases, with higher rates
reported in ASD co-morbid with ID. This study aims to
assess genotype-phenotype correlations in 42 Italian PMS
patients. Materials and Methods: For each patient, we collected
medical history and performed neurological examination,
behavioral observation, medical work-up and psychodiag-
nostic testing (Leiter or Raven, ADOS, ADI-R, VABS,
VAS, CGI, CBCL, SCQ, SSP, WHOQOL, QOL-A, ABC
and RBS-R). P09.112D
Parkinson’s disease: a disruption of nuclear/mitochondrial
DNA communication can impact cellular vitality in neuronal tissue; making a
significant contribution to the observed mitochondrial
dysfunction and neuronal cell death seen in PD. J. Trinh: None. A. Grunewald: None. K. Wasner:
None. A.A. Hicks: None. P. Bauer: None. S. Imhoff:
None. K.K. Kandaswamy: None. N. Ouzren: None. M. Werber: None. M.E.R. Weiss: None. A. Rolfs: None. P.P. Pramstaller: None. P. Seibler: None. K. Lohmann: None. C. Klein: None. H. Lowes: None. A. Hudson: None. M. Santibanez-
Koref: None. G. Hudson: None. P09.112D
Parkinson’s disease: a disruption of nuclear/mitochondrial
DNA communication PD cases carrying high-risk alleles showed
differential expression of components of the lipid metabo-
lism or vesicular trafficking pathways; supporting our own
recent metabolomic observations in PD. Conversely, PD
cases carrying low-risk alleles showed differential expres-
sion of components of the calcium homeostasis or apoptosis
pathways, supporting recent expression and proteomic
observations in PD. Results: In this large cohort, the frequency of GBA
heterozygous variants was 14,5%, much higher than
previously reported in Italy. 32 distinct variants were
identified, of whom N370S and L444P accounted only for
48% GBA-PD. Results: Our results indicate the background mtDNA
sequence modulates the expression of key protein coding
transcripts. PD cases carrying high-risk alleles showed
differential expression of components of the lipid metabo-
lism or vesicular trafficking pathways; supporting our own
recent metabolomic observations in PD. Conversely, PD
cases carrying low-risk alleles showed differential expres-
sion of components of the calcium homeostasis or apoptosis
pathways, supporting recent expression and proteomic
observations in PD. GBA-PD significantly differed from NM-PD for earlier,
prevalent bradykinetic onset and higher occurrence of non-
motor features, including cognitive, psychiatric and auto-
nomic dysfunctions. A more aggressive course and a higher
predisposition to dementia correlated with complex/severe
alleles and with risk variants, respectively. GCase activity
was significantly reduced in PD-GBA, without significant
differences among sub-classes. GBA-PD significantly differed from NM-PD for earlier,
prevalent bradykinetic onset and higher occurrence of non-
motor features, including cognitive, psychiatric and auto-
nomic dysfunctions. A more aggressive course and a higher
predisposition to dementia correlated with complex/severe
alleles and with risk variants, respectively. GCase activity
was significantly reduced in PD-GBA, without significant
differences among sub-classes. Comments: GBA heterozygous variants are a common
risk factor for PD in Italy, and a complete gene screening is
warranted to properly identify gene carriers. This is relevant Conclusion: Our experiments, for the first time, reveal a
link between inherited mtDNA variation and the nuclear
transcriptome - suggesting that inherited mtDNA variation Abstracts from the 51st European Society of Human Genetics Conference: Posters 299 health, may partially explain the modified penetrance in
(heterozygous) Parkin/PINK1 mutation carriers. health, may partially explain the modified penetrance in
(heterozygous) Parkin/PINK1 mutation carriers. can impact cellular vitality in neuronal tissue; making a
significant contribution to the observed mitochondrial
dysfunction and neuronal cell death seen in PD. Genotype-phenotype correlations in an Italian sample of
patients with Phelan-McDermid syndrome Array-CGH using the Human Genome CGH
Microarray 4 x 180K or 400K Kit (Agilent) or targeted
Sanger sequencing were performed. Method: We performed deep mtDNA sequencing in 124
individuals with Illumina NextSeq in blood-derived DNA to
assess mitochondria mutational load including somatic
mosaicism and individual mtDNA variants. Patients were
recruited from Germany and Italy and comprised carriers of
PINK1 (n = 29) and Parkin mutations (n = 52), idiopathic
PD patients (n = 23) and controls (n = 20). Materials and Methods: For each patient, we collected
medical history and performed neurological examination,
behavioral observation, medical work-up and psychodiag-
nostic testing (Leiter or Raven, ADOS, ADI-R, VABS,
VAS, CGI, CBCL, SCQ, SSP, WHOQOL, QOL-A, ABC
and RBS-R). Array-CGH using the Human Genome CGH
Microarray 4 x 180K or 400K Kit (Agilent) or targeted
Sanger sequencing were performed. Results: A mean coverage of >10,000X was achieved
with high sensitivity of detecting low level heteroplasmic
(<15%) variants. Parkin mutation carriers have more
variants including both single nucleotide variants (SNV)
and copy number variations (CNV) compared to controls
and idiopathic PD (p = 0.005). Lastly, comparing early and
late onset Parkin mutation carriers, we identified a potential
protective variant in ATP6 (p.A177T). Results: A mean coverage of >10,000X was achieved
with high sensitivity of detecting low level heteroplasmic
(<15%) variants. Parkin mutation carriers have more
variants including both single nucleotide variants (SNV)
and copy number variations (CNV) compared to controls
and idiopathic PD (p = 0.005). Lastly, comparing early and
late onset Parkin mutation carriers, we identified a potential
protective variant in ATP6 (p.A177T). Results: The clinical phenotype of PMS patients displays
great interidividual variability. Larger deletions are asso-
ciated with more severe clinical phenotypes and develop-
mental delays. However, similar deletions result at times in
phenotypes differing significantly in severity. Some pheno-
typic features are highly correlated with a positive family
history. Conclusion: Parkin/ PINK1 mutations may predispose to
an increased mitochondrial mutational load. Replication and
random segregation of heteroplasmic mitochondrial DNA,
alongside cellular mechanisms to correct mitochondrial Conclusions: Interindividual differences in the PMS
phenotype are sizable and may stem from at least three
different sources: (a) deletion size involving other func-
tional genes in addition to SHANK3, (b) additional
mutations, microdeletions, or epigenetic influences in the 300 J. del Picchia she has not achieved any developmental milestones. Brain
imaging showed diffuse brain atrophy. She was born from
healthy parents. Genotype-phenotype correlations in an Italian sample of
patients with Phelan-McDermid syndrome This variant is reported in dbSNP 150 as
rs770671752 and has a minor allele frequency (MAF) of
1.422e-5 (3/211,000) in gnomAD. No homozygotes have
been reported on public databases. PIGC pathogenic
mutations were recently identified in probands of two
unrelated families with epilepsy and intellectual disability. Moreover, our analyses of patient’s leukocytes showed that
the expression of CD16 (GPI-anchored membrane receptor)
and FLAER (marker for all GPI-APs) was significantly
decreased providing further proof that PIGC variants affect
membrane expression of GPI-APs. In conclusion, our
family is the third reported with PIGC mutations, helping to
better delineate the genotype-phenotype correlation and
further
corroborating
the
role
of
this
protein
in
neurodevelopment. non-deleted 22q13 allele, (c) greater penetrance of familial
genetic loading for neuro-behavioral disturbances in the
presence of a SHANK3 synaptopathy. Funding:
Italian
Ministry
of
Health
(NET-2013-
02355263); EU-IMI (EU-AIMS, n. 115300) A. Ricciardello: None. F. Cucinotta: None. L. Turri-
ziani: None. M. Lamberti: None. M. Briguglio: None. P. Tomaiuolo: None. M. Baccarin: None. C. Picinelli: None. P. Castronovo: None. M. Boncoddo: None. F. Bellomo:
None. G. Turturo: None. M. Canali: None. R. Sacco:
None. C. Lintas: None. A.M. Persico: None. A. Ricciardello: None. F. Cucinotta: None. L. Turri-
ziani: None. M. Lamberti: None. M. Briguglio: None. P. Tomaiuolo: None. M. Baccarin: None. C. Picinelli: None. P. Castronovo: None. M. Boncoddo: None. F. Bellomo:
None. G. Turturo: None. M. Canali: None. R. Sacco:
None. C. Lintas: None. A.M. Persico: None. Further delineation of the genotype-phenotype correlation
associated with PIGC mutations Further delineation of the genotype-phenotype correlation
associated with PIGC mutations E. Riberi1, D. Carli1, T. T. M. Nguyen2, A. Bruselles3, F. C. Radio4, E. F. Belligni1, E. Grosso5, L. Tornetta1, E. Biamino1,
E. Giorgio6, E. Di Gregorio5,6, S. Cavalieri6, E. Chierto6,
M. Ferrero6, C. Mancini6, E. Pozzi6, B. Pasini5,6, S. De Rubeis7,8,
M. Tartaglia9, P. M. Campeau2, A. Brusco5,6, G. B. Ferrero1 E. Riberi: None. D. Carli: None. T.T.M. Nguyen:
None. A. Bruselles: None. F.C. Radio: None. E.F. Belligni: None. E. Grosso: None. L. Tornetta: None. E. Biamino: None. E. Giorgio: None. E. Di Gregorio: None. S. Cavalieri: None. E. Chierto: None. M. Ferrero: None. C. Mancini: None. E. Pozzi: None. B. Pasini: None. S. De
Rubeis: None. M. Tartaglia: None. P.M. Campeau:
None. A. Brusco: None. G.B. Ferrero: None. E. Riberi: None. D. Carli: None. T.T.M. Nguyen:
None. A. Bruselles: None. F.C. Radio: None. E.F. Belligni: None. E. Grosso: None. L. Tornetta: None. E. Biamino: None. E. Giorgio: None. E. Di Gregorio: None. S. Cavalieri: None. E. Chierto: None. M. Ferrero: None. C. Mancini: None. E. Pozzi: None. B. Pasini: None. S. De
Rubeis: None. M. Tartaglia: None. P.M. Campeau:
None. A. Brusco: None. G.B. Ferrero: None. Radio4, E. F. Belligni1, E. Grosso5, L. Tornetta1, E. Biamino1,
E. Giorgio6, E. Di Gregorio5,6, S. Cavalieri6, E. Chierto6, M. Ferrero6, C. Mancini6, E. Pozzi6, B. Pasini5,6, S. De Rubeis7,8,
M. Tartaglia9, P. M. Campeau2, A. Brusco5,6, G. B. Ferrero1 1Department of Public Health and Pediatrics, University of
Torino, Torino, Italy, 2Centre Hospitalier Universitaire Sainte
Justine Research Center, University of Montreal, Montreal,
QC, Canada, 3Department of Oncology and Molecular
Medicine, Istituto Superiore di Sanità, Roma, Italy, 4Medical
Genetics, Molecular Medicine Department, Sapienza
University of Rome, San Camillo-Forlanini Hospital, Roma,
Italy, 5Medical Genetics Unit, Città della Salute e della Scienza
University Hospital, Torino, Italy, 6Department of Medical
Sciences, University of Torino, Torino, Italy, 7Seaver Autism
Center for Research and Treatment, Icahn School of Medicine
at Mount Sinai, New York, NY, United States, 8Department of
Psychiatry, Icahn School of Medicine at Mount Sinai, New
York, NY, United States, 9Genetics and Rare Diseases
Research Division, Ospedale Pediatrico Bambino Gesù, Roma,
Italy P09.119C Association between Inflammatory and Metabolic markers
and the Polygenic Risk Score of Schizophrenia in First
Episode Psychosis Mutations of KIF14, encoding a kinesin-3 family member of
microtubule motors, cause primary microcephaly Mutations of KIF14, encoding a kinesin-3 family member of
microtubule motors, cause primary microcephaly M. S. Hussain1,2,3, S. M. Baig4, A. Moawia1,4, R. Shaheen5,
N. Ewida5, M. Al-Owain6, S. Rasool1,7, B. Budde1, A. Hahn8,
A. A. Noegel2,3, F. S. Alkuraya5,9, P. Nürnberg1,3 C. Maj1,2, S. Tosato3,4, M. Ruggeri3,4, M. Gennarelli2,5,
L. Bocchio-Chiavetto2,6 1Cologne center for Genomics, Cologne, Germany, 2Institute of
Biochemistry I, Medical Faculty, University of Cologne,
Cologne, Germany, 3Center for Molecular Medicine Cologne,
University of Cologne, Cologne, Germany, 4Human Molecular
Genetics Laboratory, Health Biotechnology Division, National
Institute for Biotechnology and Genetic Engineering (NIBGE),
PIEAS, Faisalabad, Pakistan, 5Department of Genetics, King
Faisal Specialist Hospital and Research Center, Riyadh, Saudi
Arabia, 6Department of Medical Genetics, King Faisal
Specialist Hospital, Riyadh, Saudi Arabia, 7Institute of
Biochemistry and Biotechnology, Quaid-e-Azam Campus,
University of the Punjab, Lahore, Pakistan, 8Department of
Child Neurology, University of Giessen, Giessen, Germany,
9Saudi Human Genome Program, King Abdulaziz City for
Science and Technology, Riyadh, Saudi Arabia 1Cologne center for Genomics, Cologne, Germany, 2Institute of
Biochemistry I, Medical Faculty, University of Cologne, 1Cologne center for Genomics, Cologne, Germany, 2Institute of
Biochemistry I, Medical Faculty, University of Cologne, 1Institute for Genomic Statistics and Bioinformatics (IGSB),
Bonn, Germany, 2Genetics Unit, IRCCS Centro S. Giovanni di
Dio Fatebenefratelli, Brescia, Italy, 3Unit of Psychiatry,
Azienda Ospedaliera Universitaria Integrata Verona, Verona,
Italy, 4Department of Neurosciences, Biomedicine and
Movement Sciences, Section of Psychiatry, University of
Verona, Verona, Italy, 5Department of Molecular and
Translational Medicine, University of Brescia, Brescia, Italy,
6Faculty of Psychology, eCampus University, Novedrate,
Como, Como, Italy There is an increasing interest in the clarification of the
different components that characterize the genetic vulner-
ability risk for psychotic disorders. In this work, we ana-
lyzed the association between Polygenic Risk Score (PRS)
for schizophrenia and the serum levels of 19 different
inflammatory/metabolic markers in 33 controls and 83 First-
Episode psychosis (FEP) patients. The diagnosis of FEP
was schizophrenia (n = 46), bipolar disorder (n = 35) and
delusional disorder (n = 2). The PRS was calculated
according to the summary association results for schizo-
phrenia available from the Psychiatric Genomic Consortium
(PGC). Genome-wide and pathway specific PRS regarding
186 KEGG pathways were computed. SLP-2 rescues PINK1 deficiency in a cellular model and in
Drosophila A. Zanon1, S. Meschini1, S. Kalvakuri2, A. A. Lavdas1,
R. Bodmer2, P. P. Pramstaller1, A. A. Hicks1, I. Pichler1
1Eurac Research, Bolzano, Italy, 2Development, Aging and
Regeneration Program, Sanford Burnham Prebys Medical
Discovery Institute, La Jolla, CA, United States A. Zanon1, S. Meschini1, S. Kalvakuri2, A. A. Lavdas1,
R. Bodmer2, P. P. Pramstaller1, A. A. Hicks1, I. Pichler1 1Eurac Research, Bolzano, Italy, 2Development, Aging and
Regeneration Program, Sanford Burnham Prebys Medical
Discovery Institute, La Jolla, CA, United States Introduction: Deficiencies of complex I activity have been
observed in the substantia nigra of Parkinson's disease (PD)
patients, and loss of Parkin and PINK1 results in the
reduction of complex I activity shown in cell and animal
models. The two key PD genes related to mitochondrial
function
are
Parkin
(PARK2)
and
PINK1
(PARK6). Recently, we have shown that Parkin interacts with mito-
chondrial Stomatin-like protein 2 (SLP-2), which functions
in the assembly of respiratory chain proteins. Induced
overexpression of SLP-2 was able to correct for mito-
chondrial alterations caused by Parkin deficiency. The aim
of the present study was to extend this work to test whether
SLP-2 is also able to rescue mitochondrial dysfunction
induced by PINK1 knockdown in multiple systems. Phosphatidylinositol glycan anchor biosynthesis class C
(PIGC) encodes for an endoplasmic reticulum protein
essential for the first step of the biosynthesis of the glyco-
sylphosphatidylinositol (GPI) that anchors more than 150
proteins to the cell surface (GPI-anchored proteins, GPI-
APs). These proteins are important for development, neu-
rogenesis, and immunity. Inherited GPI deficiencies (IGDs)
cause intellectual disability, epilepsy, coarse facial features,
and multiple organ anomalies and are inherited as auto-
somal recessive traits. Whole exome sequencing identified a
homozygous variant in PIGC gene (NM_153747.1: c. 859G>T, p. E287*) in a 3-year-old female patient with
macrostomia, tented upper lip, low-set ears, drug-resistant
epilepsy and severe global developmental delay. At present Materials and Methods: PINK1 deficiency was induced
by
siRNA-mediated
knockdown
in
SH-SY5Y 301 Abstracts from the 51st European Society of Human Genetics Conference: Posters neuroblastoma cells and pink1 RNAi in Drosophila. Several
mitochondrial phenotypes were assessed. neuroblastoma cells and pink1 RNAi in Drosophila. Several
mitochondrial phenotypes were assessed. considering p-value and R2 of linear regression analyses. SLP-2 rescues PINK1 deficiency in a cellular model and in
Drosophila We identified a significant positive association of schizo-
phrenia PRS with the inflammatory marker C-C Motif
Chemokine Ligand 4 (CCL4, R2=0.10, adjusted p = 7.86e-
3) and a significant negative association with the hormone
ghrelin (R2=0.11, adjusted p = 6.84e-3). Noteworthy, in
our sample CCL4 levels were increased in patients com-
pared to controls, while ghrelin levels were decreased. Interestingly, pathway specific PRS analysis showed that
genes involved in different pivotal metabolic pathways
(e.g., related to amino acids and nucleotides synthesis/
degradation) are the ones leading the associations for both
CCL4 and ghrelin. These results suggest the presence of a
potential correlation between the genetic component for
schizophrenia and immune/metabolic serum markers levels
at the onset of FEP, indicating an involvement of these
processes in the psychosis vulnerability. Results: The PINK1 siRNA cells exhibited significantly
increased mitochondrial superoxide levels and decreased
mitochondrial membrane potential and complex I activity. Induced overexpression of SLP-2 significantly rescued the
identified mitochondrial dysfunction. In-vivo Drosophila
studies showed a genetic interaction of PINK1 and SLP-2,
and further, overexpression of SLP-2 transgenes rescued
pink1 mutant phenotypes, in particular loss of dopaminergic
neurons. Conclusions: These results highlight a protective effect
of SLP-2 not only in Parkin-deficient cells but also in
PINK1 deficiency, pointing to SLP-2 as a novel molecular
target able to boost mitochondrial function and neuronal
survival in multiple PD genes that disrupt mitochondria. A. Zanon: None. S. Meschini: None. S. Kalvakuri:
None. A.A. Lavdas: None. R. Bodmer: None. P.P. Pramstaller: None. A.A. Hicks: None. I. Pichler: None. C. Maj: None. S. Tosato: None. M. Ruggeri: None. M. Gennarelli: None. L. Bocchio-Chiavetto: None. P09.120D Whole exome sequencing identifies novel etiologies in a
cohort of patients with progressive myoclonus epilepsy Identification of novel mutations in the PRNP gene in
patients with Creutzfeldt Jacob disease C. Courage1,2, M. Muona1, K. Oliver3, J. Cameron3,
S. Berkovic3, A. Lehesjoki1 C. Courage1,2, M. Muona1, K. Oliver3, J. Cameron3,
S. Berkovic3, A. Lehesjoki1 M. G. Tovar-Ayala1, L. M. Gonzalez-Huerta2, R. Vega-Gama2,
N. Xilotl-DeJesus2, V. Martínez-Montoya2, M. R. Rivera-Vega2,
S. A. Cuevas-Covarrubias3 M. G. Tovar-Ayala1, L. M. Gonzalez-Huerta2, R. Vega-Gama2,
N. Xilotl-DeJesus2, V. Martínez-Montoya2, M. R. Rivera-Vega2,
S. A. Cuevas-Covarrubias3 M. G. Tovar-Ayala1, L. M. Gonzalez-Huerta2, R. Vega-Gama2,
N. Xilotl-DeJesus2, V. Martínez-Montoya2, M. R. Rivera-Vega2,
S. A. Cuevas-Covarrubias3 N. Xilotl-DeJesus2, V. Martínez-Montoya2, M. R. Rivera-Vega2,
S. A. Cuevas-Covarrubias3 1Folkhälsan Institute of Genetics, Helsinki, Finland, 2Research
Programs Unit, Molecular Neurology, University of Helsinki,
Helsinki, Finland, 3Epilepsy Research Centre, Department of
Medicine, University of Melbourne, Austin Health, Heidelberg,
Melbourne, Australia 1Folkhälsan Institute of Genetics, Helsinki, Finland, 2Research
Programs Unit, Molecular Neurology, University of Helsinki,
Helsinki, Finland, 3Epilepsy Research Centre, Department of
Medicine, University of Melbourne, Austin Health, Heidelberg,
Melbourne, Australia 1Genetica, 5Hospital General de Mexico, Mexico DF, Mexico,
2Genetica, Hospital General de Mexico, Mexico DF, Mexico,
3Genetica, Hospital General de Mexico, Facultad de Medicina,
Universidad Nacional Autonoma de Mexico, Mexico DF,
Mexico 1Genetica, 5Hospital General de Mexico, Mexico DF, Mexico,
2Genetica, Hospital General de Mexico, Mexico DF, Mexico,
3Genetica, Hospital General de Mexico, Facultad de Medicina,
Universidad Nacional Autonoma de Mexico, Mexico DF,
Mexico Progressive myoclonus epilepsies (PMEs) comprise a group
of clinically and genetically heterogeneous rare disorders
manifesting with action myoclonus, tonic-clonic seizures,
ataxia and progressive decline with typical onset in child-
hood. We carried out WES in a cohort of PME patients
negative for the minisatellite expansion mutation in CSTB
and the recurrent de novo mutation in KCNC1 as well as for
various PME associated genes. Introduction: Transmissible spongiform encephalopathies
(TSEs), also known as prion diseases, are a group of neu-
rodegenerative disorders that affect 1-2 per million habi-
tants. TSEs are classified into three diseases based on their
clinical
and
neuropathological
characteristics:
familial
Creutzfeldt-Jakob disease, Gerstmann-Straßussler-Schein-
ker disease, and familial fatal insomnia. According to their
presentation they are classified into three groups: a) spora-
dic of unknown etiology (85%), b) genetics, caused by
mutations in the PRNP gene (10-15%), and c) acquired
(<1%). P09.119C Conclusions: Through the sequencing study, four muta-
tions in the PRNP gene were identified in the four patients. With the corresponding ethical considerations, the con-
firmation of the mutations is useful to analyze later the
descendants or siblings of the affected patients, in order to
provide an adequate genetic counseling. M.G. Tovar-Ayala: None. L.M. Gonzalez-Huerta:
None. R. Vega-Gama: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. M.R. Rivera-Vega: None. S.A. Cuevas-Covarrubias: None. M.S. Hussain: None. S.M. Baig: None. A. Moawia:
None. R. Shaheen: None. N. Ewida: None. M. Al-Owain: M.G. Tovar-Ayala: None. L.M. Gonzalez-Huerta:
None. R. Vega-Gama: None. N. Xilotl-DeJesus: None. M.S. Hussain: None. S.M. Baig: None. A. Moawia:
None. R. Shaheen: None. N. Ewida: None. M. Al-Owain: None. S. Rasool: None. B. Budde: None. A. Hahn: None. A.A. Noegel:
None. F.S. Alkuraya:
None. P. Nürnberg: None. V. Martínez-Montoya: None. M.R. Rivera-Vega: None. S.A. Cuevas-Covarrubias: None. P09.119C Thus, in keeping with
previous findings on CIT mutations, our data underline the
role of an impaired cytokinesis for the etiology of primary
and syndromic microcephaly. affected individuals each, in which we identified homo-
zygous mutations in KIF14 (NM_014875.2;c.263T>A;
pLeu88* and c.4071G>A;p.Gln1357=, respectively) as the
likely cause. Further, in a German patient presenting with a
severe form of primary microcephaly and short stature, we
identified compound heterozygous missense mutations in
KIF14
(NM_014875.2;c.2545C>G;p.His849Asp
and
c.3662G>T;p.Gly1221Val). Interestingly, all but one (p. His849Asp) of the identified mutations impaired splicing
and resulted in a truncated protein. Intriguingly, Kif14
knockout mice also showed primary microcephaly. Human
KIF14, localizes at the midbody to finalize cytokinesis by
interacting with CRIK (Citron Rho-interacting kinase). We
found impaired localization of both KIF14 and CRIK at the
midbody
in
patient-derived
fibroblasts. Further,
we
observed a large number of binucleated and apoptotic cells
— signs of failed cytokinesis that we also observed in
experimentally KIF14-depleted cells. Thus, in keeping with
previous findings on CIT mutations, our data underline the
role of an impaired cytokinesis for the etiology of primary
and syndromic microcephaly. rapidly progressive dementia with motor characteristics and
a short survival time from the beginning (usually 1 year). In
the present work the genealogical, clinical and molecular
study of four cases of familial Creutzfeldt-Jacob disease is
analyzed. Objective: To molecularly describe for unrelated
familial cases with Creutzfeldt-Jacob disease. rapidly progressive dementia with motor characteristics and
a short survival time from the beginning (usually 1 year). In
the present work the genealogical, clinical and molecular
study of four cases of familial Creutzfeldt-Jacob disease is
analyzed. Objective: To molecularly describe for unrelated
familial cases with Creutzfeldt-Jacob disease. Materials and Methods: From peripheral blood, the
genomic DNA of four patients was obtained by conven-
tional techniques. The coding region of PRNP was
amplified and sequenced through PCR and DNA automated
sequencing. Results were compared with 100 healthy
controls and world databases. Results: Four heterozygous mutations were identified in
the PRNP gene: c.598G>A, c.586G>A, 3 c.586G>A and
c.598G>A. Results: Four heterozygous mutations were identified in
the PRNP gene: c.598G>A, c.586G>A, 3 c.586G>A and
c.598G>A. Conclusions: Through the sequencing study, four muta-
tions in the PRNP gene were identified in the four patients. With the corresponding ethical considerations, the con-
firmation of the mutations is useful to analyze later the
descendants or siblings of the affected patients, in order to
provide an adequate genetic counseling. P09.119C The associations
between markers serum levels and PRS were evaluated Autosomal recessive primary microcephaly (MCPH) is a
rare condition characterized by a reduced cerebral cortex
accompanied with mild to severe intellectual disability. Mutations in 17 different genes have been shown to cause
this phenotype. Recently, mutations in CIT encoding a
component of the central spindle matrix were described in
MCPH families as well as in syndromic cases of micro-
cephaly. Here, we report on two MCPH families, one from
Pakistan and the other from Saudi Arabia, with three J. del Picchia 302 affected individuals each, in which we identified homo-
zygous mutations in KIF14 (NM_014875.2;c.263T>A;
pLeu88* and c.4071G>A;p.Gln1357=, respectively) as the
likely cause. Further, in a German patient presenting with a
severe form of primary microcephaly and short stature, we
identified compound heterozygous missense mutations in
KIF14
(NM_014875.2;c.2545C>G;p.His849Asp
and
c.3662G>T;p.Gly1221Val). Interestingly, all but one (p. His849Asp) of the identified mutations impaired splicing
and resulted in a truncated protein. Intriguingly, Kif14
knockout mice also showed primary microcephaly. Human
KIF14, localizes at the midbody to finalize cytokinesis by
interacting with CRIK (Citron Rho-interacting kinase). We
found impaired localization of both KIF14 and CRIK at the
midbody
in
patient-derived
fibroblasts. Further,
we
observed a large number of binucleated and apoptotic cells
— signs of failed cytokinesis that we also observed in
experimentally KIF14-depleted cells. Thus, in keeping with
previous findings on CIT mutations, our data underline the
role of an impaired cytokinesis for the etiology of primary
and syndromic microcephaly. affected individuals each, in which we identified homo-
zygous mutations in KIF14 (NM_014875.2;c.263T>A;
pLeu88* and c.4071G>A;p.Gln1357=, respectively) as the
likely cause. Further, in a German patient presenting with a
severe form of primary microcephaly and short stature, we
identified compound heterozygous missense mutations in
KIF14
(NM_014875.2;c.2545C>G;p.His849Asp
and
c.3662G>T;p.Gly1221Val). Interestingly, all but one (p. His849Asp) of the identified mutations impaired splicing
and resulted in a truncated protein. Intriguingly, Kif14
knockout mice also showed primary microcephaly. Human
KIF14, localizes at the midbody to finalize cytokinesis by
interacting with CRIK (Citron Rho-interacting kinase). We
found impaired localization of both KIF14 and CRIK at the
midbody
in
patient-derived
fibroblasts. Further,
we
observed a large number of binucleated and apoptotic cells
— signs of failed cytokinesis that we also observed in
experimentally KIF14-depleted cells. P09.120D The abnormal accumulation in the neurons of the
prion protein, leads to apoptosis and cell death, presenting a We studied 41 independent cases including 31 with no
family history. To enhance identification of de novo
mutations a trio approach was used in 24 cases. The overall
bioinformatic analysis included 44 additional cases in
whom an earlier singleton WES study was unrevealing. Four patients had pathogenic variants in known but rare P09.122B
Differential DNA methylation associated with the onset of
psychiatric symptoms L. M. Spindola1, M. Santoro1, P. Pan1, F. Talarico1, G. Xavier1,
C. M. Carvalho1, A. Gadelha1, G. A. Salum2, L. A. Rohde2, E. C. Miguel3, R. Pellegrino4, R. A. Bressan1, V. K. Ota1,
H. Hakonarson4, S. I. Belangero1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 303 PME-associated genes, ASAH1 and CERS1, both involved
in the sphingolipid pathway, as well as in NEU1, coding for
a lysosomal sialidase. Furthermore, identification of novel
pathogenic variants in two neurodevelopmental genes
previously not associated with PME, CHD2 and NAXE,
were confirmed. PME-associated genes, ASAH1 and CERS1, both involved
in the sphingolipid pathway, as well as in NEU1, coding for
a lysosomal sialidase. Furthermore, identification of novel
pathogenic variants in two neurodevelopmental genes
previously not associated with PME, CHD2 and NAXE,
were confirmed. Results: In categorical comparison, we found 619 DMPs/
66 DMRs. Among TOP10 DMRs, we identified a region
mapping to DHX30 associated with neurodevelopmental
disorders. No enrichment was identified. In continuous
comparison, we found 38 DMPs/4 DMRs. We identified a
region mapping CERS3 that was found to be differentially
methylated in brains of schizophrenia patients. We found
the
following
GO
pathways:
glycoprotein
metabolic
process, glycosylation, presynapse and dendrite. We also
identified the neuronal system REACTOME pathway. However, only the first two GO pathways remained
significant after BH correction. In addition, through identification of pathogenic variants
in NUS1 and DHDDS, this study extends the pathomecha-
nistic etiology of PMEs to protein glycosylation, with
NUS1 directly interacting with DHDDS. Functional assays
in fibroblasts of a patient with a de novo frameshift
alteration in NUS1 confirmed the underlying protein
glycosylation defect. Likely pathogenic variants identified
in further genes provide novel insights into the molecular
basis of PMEs and imply that the as yet unsolved cases of
PME are a highly heterogeneous group of ultra-rare
disorders. Conclusions: Our study suggests an association of DNA
methylation and PS using two different comparisons
(categorical and continuous). We found an enrichment of
several pathways related with brain, that supports previous
studies that suggested psychiatric disorders to have an early
neurodevelopmental component. C. Courage: None. M. Muona: None. K. Oliver: None. J. Cameron:
None. S. Berkovic:
None. A. Lehesjoki: None. C. Courage: None. M. Muona: None. K. Oliver: None. J. Cameron:
None. S. Berkovic:
None. A. Lehesjoki: None. L.M. Spindola: None. M. Santoro: None. P. Pan: None. F. Talarico: None. G. Xavier: None. C.M. Carvalho:
None. A. Gadelha: None. G.A. Salum: None. L.A. Rohde: None. E.C. Miguel: None. R. Pellegrino: None. R.A. Bressan: None. V.K. Ota: None. H. Hakonarson:
None. S.I. Belangero: None. A compound heterozygous mutations in ALDH7A1 at
pyridoxine-dependent epilepsy (PDE): a case report A compound heterozygous mutations in ALDH7A1 at
pyridoxine-dependent epilepsy (PDE): a case report M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 Low penetrance in RANBP2-related autosomal dominant
acute necrotizing encephalopathy (ADANE) Low penetrance in RANBP2-related autosomal dominant
acute necrotizing encephalopathy (ADANE) 1Dipartimento di Biologia, Università di Padova, Padova,
Italy, 2Dipartimento di Medicina Molecolare, Università di
Padova, Padova, Italy 1Dipartimento di Biologia, Università di Padova, Padova,
Italy, 2Dipartimento di Medicina Molecolare, Università di
Padova, Padova, Italy C. Engel1, C. Cabrol1, L. Burglen2, A. Munnich3, D. Amsallem4,
L. Van Maldergem1 Background: Hundreds of common alleles have been
implicated in schizophrenia (SCZ) and bipolar disorder
(BPD), but recently a role for rare, high-penetrant variants
has been also suggested in both disorders. 1Centre de Génétique Humaine, Université de Franche-Comté,
Besançon, France, 2Service de Génétique, GHU Armand
Trousseau, Paris, France, 3Service de Génétique, Hôpital
Necker - Enfants Malades, Paris, France, 4Service de
Pédiatrie, CHU, Université de Franche Comté, Besançon,
France Methods: This study investigated a familial cohort of
SCZ and BPD patients from a closed population, where the
high recurrence of the disorders indicated a possible
enrichment in rare risk alleles. A total of 230 subjects were
genetically investigated through a strategy that integrated
identity-by-descent
(IBD)
mapping
and
whole-exome
sequencing (WES). Autosomal Dominant Acute Necrotizing Encephalopathy
(ADANE) is a recently described condition determining
frequent severe sequelae after an acute encephalitis most
commonly occurring after a symptom-free interval of sev-
eral years. Described in 2009 by Neilson, only less than
sixty patients have been described until now, either sporadic
cases or small families with two or three affected patients. Only two large pedigrees are reported in literature, with an
intriguing lack of penetrance in most relatives with large
sibships of unaffected individuals and occasional index
patients. We described another large pedigree with two
severly affected children aged 6 and 9 years at the time of
presentation and important post-critic encephalopathy with
a frontal syndrome in both. Interestingly, some relatives
were heterozygous for the c.1754C>T (p.Thr585Met)
RANBP2 familial mutation without manifesting any clinical
symptom, except seizures in two, developmental delay or
learning disability in one and an acute pseudo-Quincke
facial and laryngeal edema in one. In addition 4 histories of
meningitis in infancy were noted, without any proof that Results:
IBD
analysis
allowed
to
track
high
risk
haplotypes shared exclusively by patients from different
families and possibly carrying the most penetrant alleles. A
total of 444 non-synonymous sequence variants, of which
137 disruptive, were identified in these haplotypes by WES. Interestingly, gene sets previously implicated in SCZ (i.e. M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 1Interdisciplinary Laboratory of Clinical Neurosciences
(LiNC), São Paulo, Brazil, 2Hospital de Clínicas de Porto
Alegre, Porto Alegre, Brazil, 35. Department & Institute of
Psychiatry - Faculdade de Medicina da Universidade de São
Paulo (FMUSP), São Paulo, Brazil, 4Center for Applied
Genomics, Children's Hospital of Philadelphia, Philadelphia,
PA, United States 1Moscow Institute of Physics and Technology (State
University), Dolgoprudny, Russian Federation, 2Research
Center for Children Medical Care, Moscow, Russian
Federation PDE(MIM:266100) is autosomal recessive disease caused
by mutations in the gene ALDH7A1. The main criteria of
PDE is the response to admission of pyridoxine, resistance
to treatment by anti-epileptic drugs. Here we report the
clinical case. Newborn, on first day of life developed tonic-
clonic convulsions. Negative response to initial pyridoxine
admission. Later was polymorphism of convulsive seizures:
focal clonic, epileptic spasms, myoclonic seizures. Correc-
tion of anticonvulsant therapy was no effect. In dynamics,
the frequency and severity of seizures increased to appear-
ance of epileptic status. CMA&Karyotyping did not reveal
abnormalities. Molecular genetic study for proband and his
mother revealed heterozygous variants: (1)NM_199037.4:
c.769G>A(SCN1B)(p.Gly257Arg);
(2)NM_001182.4:
c.1279G>C(ALDH7A1)(p.Glu399Gln);
(3)NM_001182.4:
c.328C>T(ALDH7A1)(p.Arg82Ter). An unaffected mother
has only 1&2 variants. Presently, girl 3y/o delays in Background: The study of psychiatric symptoms (PS)
instead of diagnosis could help to better understand the
basis of psychiatric disorders. We aimed to identify differ-
entially methylated positions (DMPs) and regions (DMRs)
associated with PS. For that, we compared: 1) children and
adolescents with low PS at baseline and with high PS after 3
years of follow-up (categorical comparison); 2) PS as a
score (continuous comparison). Methods: PS were assessed using Child Behavior
Checklist (CBCL). For the present study, we selected from
a large Brazilian study 24 subjects with CBCL score < 30 at
baseline that increased CBCL more than 16 after 3 years of
follow-up. We generated methylation data using EPIC
BeadChip. In continuous comparison, we used Delta CBCL
as independent variable. J. del Picchia 304 psychomotor development: she fixes the sight for a short
time, turns on its side, does not sit, not crawl, not walk, not
babbling. Individual attacks persist despite daily intake of
B6 and P-5-P. We suppose the increased resistance to pri-
mary pyridoxine administration could be partly owing to
variants in both, ALDH7A1 and SCN1B. P09.125A P09.125A
Identity-by-descent mapping and whole-exome sequencing
implicates neuronal development pathways in
schizophrenia and bipolar disorder M. Belenikin:
None. E. Lukyanova:
None. S. Ayvazyan: None. M. Belenikin:
None. E. Lukyanova:
None. S. Ayvazyan: None. M. Belenikin:
None. E. Lukyanova:
None. S. Ayvazyan: None. C. Salvoro1, S. Bortoluzzi2, A. Coppe2, G. Valle1, E. Feltrin1,
M. Mostacciuolo1, G. Vazza1 M. Belenikin1,2, E. Lukyanova2, S. Ayvazyan2 Compound het-
erozygous in ALDH7A1 gene causes PDE, while SCN1B
variant could contribute to severity of the proband condi-
tion, complicating the PDE recognition by routine manner
after birth (in literature the same compound heterozygous
mutations were described for Dutch boy 6y/o only). It
should be noted the clinical picture of proband's seizures
was uncharacteristic for SCN1B mutations. However, the
quantitative assessment of individual gene contribution still
remains unclear. The authors are grateful to MD Zhylina S. for help. these episodes occurred in mutated individuals. In sum-
mary, this condition thought to be determined by an
uncontrolled
inflammatory
process
remains
apparently
triggered by environmental or epigenetic factors at large
making it a singular condition among mendelian disorders. Further studies are required to elucidate the underpinning
mechanisms. Interestingly, autosomal dominant transmis-
sion mode has been demonstrated in three large pedigrees
with a high percentage of non-penetrance. C. Engel: None. C. Cabrol: None. L. Burglen: None. A. Munnich:
None. D. Amsallem:
None. L. Van
Maldergem: None. P09.126B
Allele-specific X chromosome inactivation in Rett syndrome
patients C. Xiol Viñas1,2, S. Vidal Falcó1,2, N. Brandi Tarrau3,
P. Pacheco Fernández4, M. Pineda Marfa1, J. Armstrong
Morón4,2,5 C. Xiol Viñas1,2, S. Vidal Falcó1,2, N. Brandi Tarrau3, 1UNIRIO, Rio de Janeiro, Brazil, 2Fiocruz, Rio de Janeiro,
Brazil P. Pacheco Fernández4, M. Pineda Marfa1, J. Armstrong
Morón4,2,5 1Sant Joan de Déu Research Foundation, Barcelona, Spain,
2Institut de Recerca Pediàtrica Hospital Sant Joan de Déu,
Barcelona, Spain, 3Facultat de Medicina, Universitat de
Barcelona, Barcelona, Spain, 4Molecular and Genetics
Medicine Section, Hospital Sant Joan de Déu, Barcelona,
Spain, 5CIBER-ER (Biomedical Network Research Center for
Rare Diseases), Instituto de Salud Carlos III, Madrid, Spain Introduction: Rett syndrome is a neurodevelopmental
disorder that affects mostly females and is related, in the
majority of cases, to MECP2 mutations. It is reported that
affected patients have a higher probability of sudden death
mostly due to a prolonged corrected QT interval (QTc). Introduction: Rett syndrome is a neurodevelopmental
disorder that affects mostly females and is related, in the
majority of cases, to MECP2 mutations. It is reported that
affected patients have a higher probability of sudden death
mostly due to a prolonged corrected QT interval (QTc). Materials and Methods: We included 21 patients
diagnosed with Rett syndrome, 19 of those tested for
MECP2 mutations. The patients' medical notes were
analysed and used for the classification of their clinical
forms and their clinical findings described. The patients
were also submitted to electrocardiogram (ECG) evaluation
for QTc measurement, which were considered prolonged
when >450ms, and their risk for Long QT Syndrome
(LQTS) was calculated. Introduction: Rett syndrome (RTT; MIM#312750) is a
severe neurological disorder which mainly affects young
females and is the second most common cause of severe
intellectual disability in women worldwide. In most cases, it
is caused by mutations in MECP2 (MIM*300005), the gene
encoding MeCP2 (methyl CpG binding protein 2), which is
located on the X chromosome. Many studies have been
carried out to assess whether X chromosome inactivation
(XCI) plays a role in the wide range of phenotypic variation
of these patients. However, classical methylation-based
protocols to evaluate XCI were only able to determine
whether the XCI pattern was biased or random, but not if
the preferentially inactivated X chromosome was the one
carrying the mutant or the wildtype allele. Results: The classic clinical form was more prevalent in
our cohort (85%). In the clinical findings, the mean age was
18 years old. P09.127C M. Kossmann Ferraz1, A. Salgueiro Nascimento Monteiro1,
W. Norat Siqueira1, C. M. Motta Stofell de Siqueira1, O. Ferreira
de Siqueira1, G. Sousa Domingos1, A. P. Casseta dos Santos
Nucera1, L. Schuindt Monnerat2, F. Regla Vargas1,2 P09.126B
Allele-specific X chromosome inactivation in Rett syndrome
patients Stereotypical hand movements, epilepsy, and
bruxism were the most prevalent symptoms (80%, 76%, and
76% respectively). The average QTc was 401ms and
prolonged QTc was found in 1 patient. However, none
were taking medications known to prolong QTc and none
had a higher risk for LQTS. Materials and Methods: We have developed an allele-
specific methylation-based assay to evaluate methylation on
the loci of several recurrent MECP2 mutations in blood
samples of RTT patients. Conclusions: The classic form of Rett syndrome prevails
in our cohort and in most of the cases, the management of
their seizures required medication. We found no correlation
between prolonged QTc and MECP2 mutations in our
study. None of the patients investigated exhibited a high
risk for LQTS. Nevertheless, a follow-up with ECG is
highly recommended. Materials and Methods: We have developed an allele-
specific methylation-based assay to evaluate methylation on
the loci of several recurrent MECP2 mutations in blood
samples of RTT patients. Results: The aim of this study is to provide data to
effectively
correlate
XCI
patterns
to
the
phenotypic
presentation of RTT. Conclusions: If this correlation is strong enough, it could
be used in the future as a molecular tool to predict the
severity of the clinical presentation of genetically diagnosed
RTT patients. M. Kossmann Ferraz: None. A. Salgueiro Nascimento
Monteiro: None. W. Norat Siqueira: None. C.M. Motta
Stofell de Siqueira: None. O. Ferreira de Siqueira: None. G. Sousa Domingos: None. A.P. Casseta dos Santos
Nucera: None. L. Schuindt Monnerat: None. F. Regla
Vargas: None. Grant: The work was supported by grants from the
Spanish Ministry of Health (Instituto de Salud Carlos III/
FEDER, PI15/01159); Crowdfunding program of Catalan
Association for Rett Syndrome; Fondobiorett and Mi
Princesa Rett. C. Salvoro: None. S. Bortoluzzi: None. A. Coppe:
None. G. Valle: None. E. Feltrin: None. M. Mostac-
ciuolo: None. G. Vazza: None. C. Salvoro: None. S. Bortoluzzi: None. A. Coppe:
None. G. Valle: None. E. Feltrin: None. M. Mostac-
ciuolo: None. G. Vazza: None. P09.127C
Rett syndrome: Correlation between clinical, molecular
and QTc evaluation in 21 patients P09.127C
Rett syndrome: Correlation between clinical, molecular
and QTc evaluation in 21 patients Characterization of large deletions of the MECP2gene in
Rett syndrome patients by gene dosage analysis Low penetrance in RANBP2-related autosomal dominant
acute necrotizing encephalopathy (ADANE) post-synaptic density (PSD) proteins, voltage-gated calcium
channels (VGCCs) and fragile X mental retardation protein
(FMRP) targets) were significantly enriched in genes
carrying these rare variants. Further, IBD variants were
preferentially affecting genes involved in the extracellular
matrix (ECM) biology and axon guidance processes. Conclusions: Results confirmed rare risk variants as key
factors in SCZ and BPD pathogenesis and highlighted an
involvement of ECM biology and development of neuronal
projections in the etiology of both disorders. Abstracts from the 51st European Society of Human Genetics Conference: Posters 305 P09.129A S. Vidal: None. N.M. Brandi: None. P. Pacheco: None. E. Gerotina:
None. A. Garcia-Cazorla:
None. M. O’Callaghan:
None. M. Pineda:
None. J. Armstrong: None. The most recurrent monogenic disorders that overlap with
the phenotype of Rett syndrome The most recurrent monogenic disorders that overlap with
the phenotype of Rett syndrome S. Vidal1, N. M. Brandi2, P. Pacheco3, E. Gerotina1, A. Garcia-
Cazorla4,5,6, M. O’Callaghan4,5,6, M. Pineda1, J. Armstrong3,5,6 P09.128D Introduction: Rett syndrome (RTT) is a neuromaintenance
disease that affects 1:12000 newborn girls becoming the
second cause of mental retardation in women after Down
syndrome. In more than 96% of the cases of classic RTT a
mutation
affecting
the
Methil-CpG-binding
protein
2
(MECP2) has been found and in ~15% of the cases, the
alteration is a big deletion within it. We carried out the
characterization of the break points of the deletions found in
17 classical RTT patients. Introduction: Rett syndrome (RTT) is a neuromaintenance
disease that affects 1:12000 newborn girls becoming the
second cause of mental retardation in women after Down
syndrome. In more than 96% of the cases of classic RTT a
mutation
affecting
the
Methil-CpG-binding
protein
2
(MECP2) has been found and in ~15% of the cases, the
alteration is a big deletion within it. We carried out the
characterization of the break points of the deletions found in
17 classical RTT patients. Material and Methods: It has been studied 396 patients
with Rett-like clinical diagnosis. It has been performed: 242
patients by custom panel with 17 gens related to Rett-like
clinic through HaloPlex Target Enrichment System and 154
patients by commercial panel, TruSightOne Sequencing
Panel. Material and Methods: It has been studied 396 patients
with Rett-like clinical diagnosis. It has been performed: 242
patients by custom panel with 17 gens related to Rett-like
clinic through HaloPlex Target Enrichment System and 154
patients by commercial panel, TruSightOne Sequencing
Panel. Materials and Methods: MLPA was performed in all of
them to detect the alteration. Then, the allele containing the
deletion was narrowed down via DNA-qPCRs and long-
PCRs until Sanger sequencing of it could be done. Materials and Methods: MLPA was performed in all of
them to detect the alteration. Then, the allele containing the
deletion was narrowed down via DNA-qPCRs and long-
PCRs until Sanger sequencing of it could be done. Results: 35 patients had Rett-like clinical features and
pathogenic variants have been found in six different genes:
eleven in STXBP1 (Epileptic encephalopathy, early infan-
tile,4. OMIM#612164), nine in TCF4 (Pitt-Hopkins syn-
drome. OMIM#610954),
six
in
SCN2A
(Epileptic
encephalopathy,early infantile,11. OMIM#613721), four
in MEF2C (Mental retardation. OMIM#613443), three in
SYNGAP1 (Mental retardation. OMIM#612621) and two in
KCNQ2
(Epileptic
encephalopathy,early
infantile,7. OMIM#613720). S. Vidal1, N. M. Brandi2, P. Pacheco3, E. Gerotina1, A. Garcia-
Cazorla4,5,6, M. O’Callaghan4,5,6, M. Pineda1, J. Armstrong3,5,6 P09.128D Results: Following this methodology we could confirm
the presence of the deletion in every case and determine the
area, sometimes even the exact nucleotide, where the large
rearrangement
has
occurred. Further
analysis
of
the
sequences surrounding the break points showed that most
of them happened in regions full of repetitive elements such
as Alus. Conclusions: We therefore provide more evidence to
support this former theory regarding the possible cause of
these rearrangements. Besides, the X chromosome inactiva-
tion pattern was determined and along with clinical data a
possible correlation between genotype and phenotype has
been searched. Conclusions: We therefore provide more evidence to
support this former theory regarding the possible cause of
these rearrangements. Besides, the X chromosome inactiva-
tion pattern was determined and along with clinical data a
possible correlation between genotype and phenotype has
been searched. Conclusions: The genetic study by NGS allows to study
a larger number of genes associated with Rett-like clinic
simultaneously, providing a genetic study to a wider group
of patients. These variants identified by NGS may modify
the initial clinical diagnosis to other neurodevelopmental
syndromes, or determine new candidate genes related to
RTT-like symptoms, providing the clinician with more
information and clues that could help in the prevention of
future symptoms or in the pharmacologic therapy. Grants: The work was supported by grants from the
Spanish Ministry of Health (Instituto de Salud Carlos III/
FEDER, PI15/01159); Crowdfunding program of Catalan
Association for Rett Syndrome; Fondobiorett and Mi
Princesa Rett. Grants: The work was supported by grants from the
Spanish Ministry of Health (Instituto de Salud Carlos III/
FEDER, PI15/01159); Crowdfunding program of Catalan
Association for Rett Syndrome; Fondobiorett and Mi
Princesa Rett. S. Vidal: None. A. Pascual-Alonso: None. M. Rabaza: S. Vidal: None. A. Pascual-Alonso: None. M. Rabaza: Grant: from the Spanish Ministry of Health (Instituto de
Salud
Carlos
III/FEDER,
PI15/01159);
Crowdfunding
program of Catalan Association for RTT; Fondobiorett
and Mi Princesa Rett. None. E. Gerotina: None. N. Brandi: None. P. Pacheco: None. E. Gerotina: None. N. Brandi: None. P. Pacheco:
None. M. Pineda: None. J. Armstrong: None. None. M. Pineda: None. J. Armstrong: None. None. M. Pineda: None. J. Armstrong: None. P09.128D Characterization of large deletions of the MECP2gene in
Rett syndrome patients by gene dosage analysis C. Xiol Viñas: None. S. Vidal Falcó: None. N. Brandi
Tarrau: None. P. Pacheco Fernández: None. M. Pineda Marfa: None. J. Armstrong Morón: None. 306 J. del Picchia S. Vidal1,2, A. Pascual-Alonso1,2, M. Rabaza1,2, E. Gerotina1,2,
N. Brandi3, P. Pacheco4, M. Pineda1, J. Armstrong4,2,5 1Fundació Sant Joan de Déu, Esplugues de Llobregat, Spain,
2Facultat de Medicina, Universitat de Barcelona, Barcelona,
Spain, 3Molecular and Genetics Medicine Section, Hospital
Sant Joan de Déu, Esplugues de Llobregat, Spain, 4Neurology
Service, Hospital Sant Joan de Déu, Barcelona, Esplugues de
Llobregat, Spain, 5Institut de Recerca Pediàtrica, Hospital
Sant Joan de Déu, Barcelona, Spain, 6CIBER-ER (Biomedical
Network Research Center for Rare Diseases), Instituto de
Salud Carlos III, Madrid, Spain 1Fundació Sant Joan de Déu, Esplugues de Llobregat, Spain,
2Facultat de Medicina, Universitat de Barcelona, Barcelona,
Spain, 3Molecular and Genetics Medicine Section, Hospital
Sant Joan de Déu, Esplugues de Llobregat, Spain, 4Neurology
Service, Hospital Sant Joan de Déu, Barcelona, Esplugues de
Llobregat, Spain, 5Institut de Recerca Pediàtrica, Hospital
Sant Joan de Déu, Barcelona, Spain, 6CIBER-ER (Biomedical
Network Research Center for Rare Diseases), Instituto de
Salud Carlos III, Madrid, Spain 1Sant Joan de Déu Research foundation, Barcelona, Spain,
2Institut de Recerca Pediàtrica Hospital Sant Joan de Déu,
Barcelona, Spain, 3Facultat de Medicina, Universitat de
Barcelona, Barcelona, Spain, 4Molecular and Genetics
Medicine Section, Hospital Sant Joan de Déu, Barcelona, 1Sant Joan de Déu Research foundation, Barcelona, Spain,
2Institut de Recerca Pediàtrica Hospital Sant Joan de Déu,
Barcelona, Spain, 3Facultat de Medicina, Universitat de
Barcelona, Barcelona, Spain, 4Molecular and Genetics
Medicine Section, Hospital Sant Joan de Déu, Barcelona,
Spain, 5CIBER-ER (Biomedical Network Research Center for
Rare Diseases) Instituto de Salud Carlos III, Madrid, Spain 1Sant Joan de Déu Research foundation, Barcelona, Spain,
2Institut de Recerca Pediàtrica Hospital Sant Joan de Déu,
Barcelona, Spain, 3Facultat de Medicina, Universitat de
Barcelona, Barcelona, Spain, 4Molecular and Genetics
Medicine Section, Hospital Sant Joan de Déu, Barcelona, Spain, 5CIBER-ER (Biomedical Network Research Center for
Rare Diseases) Instituto de Salud Carlos III, Madrid, Spain Introduction: Rett syndrome (RTT) is an early-onset neu-
rodevelopmental disorder that is caused by mutations in
MECP2, but defects in a handful of other genes (CDKL5
and FOXG1) can lead to presentations that resemble clas-
sical RTT, but do not completely identical. Here, we
attempted to identify other monogenic disorders that share
features with RTT. Mutation screening and global gene expression analyses of P09.131C
Implications of an admixed Brazilian population in
Schizophrenia polygenic risk score F. Talarico1,2, M. L Santoro3,4, V. K Ota1,2, R. Pellegrino5,
A. Gadelha3,2, R. A Bressan3,4, H. Hakonarson5, S. I
Belangero1,3,2 1King Faisal Specialist Hospital and Research Center, Riyadh,
Saudi Arabia, Riyadh, Saudi Arabia, 2King Fahad National
Guard Hospital, Riyadh, Saudi Arabia, Riyadh, Saudi Arabia,
3King Saud University, Riyadh, Saudi Arabia, Riyadh, Saudi
Arabia 1King Faisal Specialist Hospital and Research Center, Riyadh,
Saudi Arabia, Riyadh, Saudi Arabia, 2King Fahad National
Guard Hospital, Riyadh, Saudi Arabia, Riyadh, Saudi Arabia,
3King Saud University, Riyadh, Saudi Arabia, Riyadh, Saudi
Arabia 1Genetics Division of Department of Morphology and Genetics
of Universidade Federal de São Paulo (UNIFESP), São Paulo,
Brazil, 2LiNC - Interdisciplinary Laboratory of Clinical
Neurosciences of UNIFESP, São Paulo, Brazil, 3Department of
Psychiatry of Universidade Federal de São Paulo (UNIFESP),
São Paulo, Brazil, 4 LiNC - Interdisciplinary Laboratory of
Clinical Neurosciences of UNIFESP, São Paulo, Brazil, 5The
Children's Hospital of Philadelphia, Philadelphia, PA, United
States Rett syndrome (RS) is a rare neurodevelopmental disorder
that is found in a variety of racial and ethnic groups with a
notable female predilection. MECP2 mutations are parti-
cularly known to cause RS phenotype, however, involve-
ment of other genes has also been reported. We herein
present a detailed molecular characterization of a cohort of
32 patients with RS Mutation screening revealed that 13
patients had MECP2 mutations, three of which were novel. One patient had a novel FOXG1 mutation and another one
had a novel CDKL5 mutation. All patients were females
except one male who carries FOXG1 mutation. Patients
screened for cytogenetic abnormalities did not carry any
gross chromosomal abnormality except for singleton who
had a gain on chromosome X harboring two genes includ-
ing MECP2. Whole-transcriptome analysis of RS patients
with MECP2 mutation and sex- and age-matching controls
revealed alterations in a number of mitochondria related
pathways, including oxidative phosphorylation and mito-
chondrial dysfunction. We sequenced mtDNA on the
patients who did not have any tested genetic defect which
revealed two novel mtDNA alterations in two patients. Furthermore, we identified common genes and pathways
possibly leading to autistic phenotype by performing net-
work analysis of RS significant genes with the multi-
disorder autism geneset that is associated with autism and at
least one of 13 other autism sibling disorders. P09.130B Mutation screening and global gene expression analyses of Abstracts from the 51st European Society of Human Genetics Conference: Posters 307 Saudi Rett patients implicate mitochondrial dysfunction in
the pathogenesis of Rett syndrome None. O. AlHarazi: None. A. Al-Odaib: None. N. Kaya: None. None. O. AlHarazi: None. A. Al-Odaib: None. N. Kaya: None. D. Colak1, M. Aldosary1, A. AlBakheet1, O. M. Mustafa1,
R. Almass1, M. Alsagob1, L. AlQuait1, H. Al-Dhalaan1,
M. Alfadhel2, Z. Rahbeeni1, A. Chedrawi1, M. Al-Dosari1, Z. Al-
Hassnan1, M. Bulbul1, M. Salih3, M. Al-Owain2, H. Al-Zaidan1,
M. Al-Muheiza1, M. AlSayed1, O. AlHarazi1, A. Al-Odaib1,
N. Kaya1 Genome wide DNA methylation analysis in a longitudinal
cohort of antipsychotic naive first episode of psychosis
patients 1UNIFESP, Sao Paulo, Brazil, 2King’s College London,
London, United Kingdom, 3Irmandade da Santa Casa de
Misericórdia de São Paulo, Sao Paulo, Brazil M. L. Santoro1, V. K. Ota2, F. Talarico2, S. de Jong3, C. Noto1,
L. Spindola2, A. Gadelha1, Q. Cordeiro4, R. Bressan1, G. Breen3,
S. Belangero2 M. L. Santoro1, V. K. Ota2, F. Talarico2, S. de Jong3, C. Noto1,
L. Spindola2, A. Gadelha1, Q. Cordeiro4, R. Bressan1, G. Breen3,
S. Belangero2 In this study, we aimed to test if the schizophrenia (SCZ)
polygenic risk score (PRS) was associated with clinical
symptoms at: a) the first episode of psychosis pre-treatment
(FEP), b) nine weeks after initiation of risperidone treatment
(FEP-9W) and c) with the response to risperidone. We
performed a detailed clinical assessment of 60 antipsychotic
naïve patients in their FEP and, again, after nine weeks of
standardized treatment with Risperidone. Blood derived
DNA was genotyped using the Illumina PsychArrayChip,
along with 59 controls, and then imputed. We used the
latest available GWAS summary statistics from the Psy-
chiatric Genomics Consortium wave-2 SCZ group as a
training set to calculate their PRS for schizophrenia. We
used Poisson Regression to test association between the
PRS and clinical measures adjusting for four ancestry
principal components. We considered as significant a p-
value < 0.001 (Bonferroni correction). First, we verified that
the schizophrenia PRS was also able to distinguish cases
from control in this south-eastern Brazilian sample, with a
similar variance explained (~0.19, observed scale) to that
seen in Northern European populations. In addition, within-
cases, we found that PRS is significantly positively corre-
lated with baseline (pre-treatment) symptoms as measured
by the PANSS-excitement factor. After standardized treat-
ment for 9 weeks, this correlation disappeared and the
depressive symptoms (CDSS) became negatively associated
with PRS. These results highlight the importance of
studying schizophrenia, and other disorders, pre-treatment
to understand the relationship between polygenic risk and
phenotypic features. 1Escola Paulista de Medicina, São Paulo, Brazil, 2UNIFESP,
São Paulo, Brazil, 3King's College London, London, United
Kingdom, 4Santa Casa de Misericordia de São Paulo, São
Paulo, Brazil 1Escola Paulista de Medicina, São Paulo, Brazil, 2UNIFESP,
São Paulo, Brazil, 3King's College London, London, United
Kingdom, 4Santa Casa de Misericordia de São Paulo, São
Paulo, Brazil Identifying the genetic and molecular changes of drug
response is the first step through personalized medicine. P09.133A V. K. Ota1, M. L. Santoro1, S. de Jong2, C. Noto1, L. M. N. Spindola1, F. Talarico1, P. Moretti1, C. M. Carvalho1,
A. Gadelha1, Q. Cordeiro3, R. A. Bressan1, S. I. Belangero1,
G. Breen2 V. K. Ota1, M. L. Santoro1, S. de Jong2, C. Noto1, L. M. N. Spindola1, F. Talarico1, P. Moretti1, C. M. Carvalho1,
A. Gadelha1, Q. Cordeiro3, R. A. Bressan1, S. I. Belangero1,
G. Breen2 P09.131C
Implications of an admixed Brazilian population in
Schizophrenia polygenic risk score The gene
signatures and novel alterations that were found in this
study along with the observed disturbance of the expression
of mitochondrial pathways may indicate the involvement of
mitochondria in the Rett disease pathogenesis. The Polygenic Risk Score (PRS) tool compiles data from
hundreds to millions of common variants into a single
measure, making it a valuable tool to investigate genetic
risk of complex diseases, like Schizophrenia (SCZ). To
calculate the PRS-SCZ, a reference sample must be defined,
but most subjects from such samples are Caucasian and
doubts remain about the reliability of PRS-SCZ in admixed
samples. We verified if PRS-SCZ could differentiate
patients with schizophrenia and healthy controls in a Bra-
zilian sample and if miscegenation could influence the
results. We used the Psychiatric Genomics Consortium-
SCZ summary statistics GWAS as reference. As target
sample, we genotyped 177 patients with schizophrenia and
242 healthy controls. The Sanger Imputation Service plat-
form was used to impute genomic regions. To perform the
quality control and to generate the PRS we used PRSice and
PLINK software. We could explain up to 5.2% of the var-
iance between cases and controls when including all indi-
viduals. A similar result was found when selecting only
individuals with large African component mixed with
Caucasian and Native Amerindian components (named as
Latin
1). Considering
only
Caucasians,
the
variance
explained raised to 11%, with the PRS-SCZ significantly
higher in patients with SCZ than controls. The same was
observed in a sample with large Native Amerindian com-
ponent mixed with Caucasian component (named as Latin
2). We found more robust results after restraining the
sample to Caucasian subjects, but PRS-SCZ was still cap-
able to differentiate cases from controls even in a highly
mixed population. D. Colak: None. M. Aldosary: None. A. AlBakheet:
None. O.M. Mustafa: None. R. Almass: None. M. Alsagob: None. L. AlQuait: None. H. Al-Dhalaan: None. M. Alfadhel: None. Z. Rahbeeni: None. A. Chedrawi:
None. M. Al-Dosari: None. Z. Al-Hassnan: None. M. Bulbul: None. M. Salih: None. M. Al-Owain: None. H. Al-Zaidan: None. M. Al-Muheiza: None. M. AlSayed: Grants: FAPESP, CAPES 308 J. del Picchia P09.133A
Polygenic risk score analyses of symptoms and treatment
response in an antipsychotic-naïve first episode of psychosis
cohort P09.133A
Polygenic risk score analyses of symptoms and treatment
response in an antipsychotic-naïve first episode of psychosis
cohort F. Talarico: None. M. L Santoro: None. V. K Ota:
None. R. Pellegrino: None. A. Gadelha: None. R. A
Bressan:
None. H. Hakonarson:
None. S. I
Belangero: None. P09.134B P09.134B
A molecular analysis of SDCCAG8, a schizophrenia risk
gene that functions in the centrosome Background: Epilepsy is a common clinical and genetic
heterogeneous neurological disorder, with a large number of
cases caused by genetic factors. To understand the mole-
cular basis of epilepsy, 234 epileptic patients were studied
using
a
targeted
sequencing
of
223
epilepsy-
associated genes. M. Flynn, L. Whitton, G. Donohoe, C. Morrison, D. Morris
National University Ireland, Galway, Galway, Ireland M. Flynn, L. Whitton, G. Donohoe, C. Morrison, D. Morris National University Ireland, Galway, Galway, Ireland Schizophrenia affects 1% of adults and is a major global
health problem. The focus of my project is the potential role
of the centrosome in schizophrenia. The centrosome, an
organelle within cells, plays a crucial role in brain devel-
opment where it directs cell shape, polarity and motility. The centrosome also seeds the growth of antenna-like sig-
nalling structures called primary cilia. Rare mutations in
centrosome genes cause disorders that present with severe
cognitive deficits and variable neuropsychiatric phenotypes. Material and Methods: We performed exome sequen-
cing using the Ion AmpliSeqTM Exome RDY, combined
with an AmpliSeq panel design and SureSelectXT technol-
ogy. Sequencing reads were analyzed using Torrent Suite
software and an in-house pipeline, respectively. Annotated
variants using ION Reporter were prioritized with an in-
house analytical pipeline. Results: Among the 234 cases, 152 patients were referred
as Early Infantile Epileptic Encephalopathy (EEIE). On this
group a diagnostic yield of 35% was obtained. SCN1A,
CDKL5 KCNT1, KCNQ2, and SPTAN1 were the most
frequently mutated genes. On the 82 remaining patients,
33% of them with an associated neurodevelopmental
disorder, potential diagnostic variants were detected in
25% of the cases, being IQSEC2, identified in 3 cases, the
only recurrent mutated gene on this group. Out of the 281
identified
variants,
171
(61%)
were
associated
to
autosomal-dominant inheritance pattern diseases. Variants
of uncertain significant category were identified in RYR3,
ARHGEF15, FASN and RELN genes. Recurrent updating of
targeted genes and the familial segregation studies has
shown to be essential to identify causal variants. GWAS data has implicated many genes in schizophrenia. We have shown that seven schizophrenia risk genes encode
proteins with centrosomal functions. Of these, SDCCAG8
is also associated with educational attainment in GWAS and
the genome-wide significant SNPs for the two phenotypes
are in high linkage disequilibrium indicating a pleiotropic
effect. Genome wide DNA methylation analysis in a longitudinal
cohort of antipsychotic naive first episode of psychosis
patients In
this study we aimed to identify DNA methylation markers
in blood of an antipsychotic-naive First Episode of Psy-
chosis (anFEP) cohort before and after two months of ris-
peridone treatment (FEP-2M), furthermore, we investigated
overlaps between these markers and post-mortem schizo-
phrenia brain datasets. Sixty anFEP were recruited for this
study. We used the Human_Illumina-450K microarray. We
covariated the data for sex, age, smoking and cell propor-
tions, considering as statistically significant differentially
methylated positions (DMPs) with a p-value < 0.05 after a
FDR and as differentially methylated regions (DMRs) those
with at least two DMPs. E-GEOD-61107, E-GEOD-61380
and E-GEOD-61431 brain datasets were used to investigate
for DMP overlaps between brain and blood. We searched
for enriched pathways using the WebGestalt software. We
found 14 DMRs between anFEP and FEP-2M, most related
to treatment response (ANKRD33) or side effects such as
male fertility (BRDT, TEX14, ASZ1, STARD6) and meta-
bolism (CPN1, ERLIN1, ASB3). Comparing results with
brain datasets, we found 27 DMPs overlap in RNF39 gene,
which lies within the MHC region. No biological pathway
were enriched for blood or brain studies. To our knowledge,
this is the first study to find DMPs and DMRs in a long-
itudinal cohort of anFEP. Collectively, we identified DMRs
that seem to be related to the adverse effects of risperidone
and a large overlap between blood and brain studies close to
the most associated genomic region in schizophrenia, the
MHC region. Grants: FAPESP, MRC ref NEWTON001 Grants: FAPESP, MRC ref NEWTON001 V.K. Ota: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; FAPESP. M.L. Santoro:
None. S. de Jong: None. C. Noto: None. L.M.N. Spindola: None. F. Talarico: None. P. Moretti: None. C.M. Carvalho: None. A. Gadelha: None. Q. Cordeiro:
None. R.A. Bressan: None. S.I. Belangero: None. G. Breen:
B. Research
Grant
(principal
investigator, M.L. Santoro: None. V.K. Ota: None. F. Talarico:
None. S. de Jong: None. C. Noto: None. L. Spindola:
None. A. Gadelha: None. Q. Cordeiro: None. R. Bressan:
None. G. Breen: None. S. Belangero: None. M.L. Santoro: None. V.K. Ota: None. F. Talarico:
None. S. de Jong: None. C. Noto: None. L. Spindola:
None. A. Gadelha: None. Q. Cordeiro: None. R. Bressan:
None. G. Breen: None. S. Belangero: None. P09.135C P09.135C
Targeted WES study, an effective tool for the genetic
diagnosis of epilepsy P09.134B We have found that a schizophrenia risk SNP in
SDCCAG8 is significantly associated with poorer perfor-
mance in a social cognition task, in a large Irish dataset of
schizophrenia patients and controls (p = 0.001). To analyse the molecular function of SDCCAG8 we have
used genome editing to knock it out in neuronal and retinal
cells. Loss of SDCCAG8 impairs cells’ ability to make
primary cilia and their capacity to repair genome damage. Nuclear lobulation has also been observed. Current work is
addressing whether SDCCAG8 affects cell signalling using
RNA-Seq analysis. This could identify molecular mechan-
isms by which SDCCAG8 mutations contribute to schizo-
phrenia risk and cognition and help uncover the processes
that implicate centrosome genes in neurodevelopmental
phenotypes. Conclusions: Targeted sequencing based on whole
exome sequencing in epilepsy patients provide a cost
effective and comprehensive strategy that accelerates the
identification of a definitive clinical diagnosis on EEIE and
on patients with seizures associated to other neurological
disorders. M. Martinez-Garcia: None. I. Diez: None. C. Rodri-
guez: None. R. Perez-Carro: None. I. Sanchez-Navarro:
None. R. Sanchez-Alcudia: None. E. Mata: None. M. Carcajona: None. E. Fernandez-Tabanera: None. D. Rodriguez: None. G. Benito: None. N. Sánchez-Bolivar:
None. L. De la Vega: None. J. Botet: None. P. Maietta:
None. S. Alvarez: None. M. Martinez-Garcia: None. I. Diez: None. C. Rodri-
guez: None. R. Perez-Carro: None. I. Sanchez-Navarro:
None. R. Sanchez-Alcudia: None. E. Mata: None. M. Carcajona: None. E. Fernandez-Tabanera: None. D. Rodriguez: None. G. Benito: None. N. Sánchez-Bolivar:
None. L. De la Vega: None. J. Botet: None. P. Maietta:
None. S. Alvarez: None. Funded by Irish Research Council. M. Flynn: None. L. Whitton: None. G. Donohoe: None. C. Morrison: None. D. Morris: None. M. Martinez-Garcia, I. Diez, C. Rodriguez, R. Perez-Carro,
I. Sanchez-Navarro, R. Sanchez-Alcudia, E. Mata,
M. Carcajona, E. Fernandez-Tabanera, D. Rodriguez, G. Benito, Genome wide DNA methylation analysis in a longitudinal
cohort of antipsychotic naive first episode of psychosis
patients Abstracts from the 51st European Society of Human Genetics Conference: Posters 309 collaborator or consultant and pending grants as well as
grants already received); Modest; MRC ref NEWTON001. N. Sánchez-Bolivar, L. De la Vega, J. Botet, P. Maietta,
S. Alvarez N. Sánchez-Bolivar, L. De la Vega, J. Botet, P. Maietta,
S. Alvarez Unidad de secuenciación, NIMGenetics S.L., Madrid, Spain,
Madrid, Spain P09.135C
Targeted WES study, an effective tool for the genetic
diagnosis of epilepsy M. Martinez-Garcia, I. Diez, C. Rodriguez, R. Perez-Carro, P09.137A High incidence of SHANK3 loss of function mutations in
individuals with intellectual disability and autistic traits 310 J. del Picchia M. C. Aspromonte1, A. Gasparini2, R. Polli1, E. Bettella1,
F. Cesca1, F. Benedicenti3, S. Boni4, O. Carlet5, F. Rivieri6,
F. Vittorini7, M. Carraro2, S. Sartori8, S. C. E. Tosatto2,9,
A. Murgia1, E. Leonardi1 M.C. Aspromonte: None. A. Gasparini: None. R. Polli:
None. E. Bettella: None. F. Cesca: None. F. Benedicenti:
None. S. Boni: None. O. Carlet: None. F. Rivieri: None. F. Vittorini: None. M. Carraro: None. S. Sartori: None. S.C.E. Tosatto:
None. A. Murgia:
None. E. Leonardi: None. M.C. Aspromonte: None. A. Gasparini: None. R. Polli:
None. E. Bettella: None. F. Cesca: None. F. Benedicenti:
None. S. Boni: None. O. Carlet: None. F. Rivieri: None. F. Vittorini: None. M. Carraro: None. S. Sartori: None. S.C.E. Tosatto:
None. A. Murgia:
None. E. Leonardi: None. 1Molecular Genetics of Neurodevelopment, Dept. of Women's
and Children's Health, University of Padova, Padova, Italy,
2Dept. of Biomedical Sciences and CRIBI Biotechnology
Center, University of Padova, Padova, Italy, 3Genetic
Counseling Service – Dept. of Pediatrics - Regional Hospital of
Bolzano, Bolzano, Italy, 4Medical Genetic Service, S. Martino
Hospital of Belluno, Belluno, Italy, 5Child Psychiatry Unit –
Scientific Institute E. Medea of Conegliano, Treviso, Italy,
6Medical Genetic Service, Department of Laboratory,
S. Chiara Hospital, Trento, Italy, 7S.C. Neuropsichiatria
Infantile Dipartimento di Pediatria e Specialità Pediatriche, A. O.U. Città della Salute e della Scienza Torino, Presidio OIRM,
Torino, Italy, 8Pediatric Neurology Unit, Dept. of Woman's
and Child's Health, University Hospital of Padova, Padova,
Italy, 9CNR Institute of Neuroscience, Padova, Padova, Italy Serotonin transporter polymorphism, cortisol and
hippocampal volume in post-stroke patients E. Ben Assayag1,2, D. Amar1,2, E. Kliper1, S. Usher1,
H. Hallevi1,2, L. Shopin1, J. Molad1, A. Korczyn2, N. M. Bornstein1,2, S. Shenhar-Tsarfaty1,2 E. Ben Assayag1,2, D. Amar1,2, E. Kliper1, S. Usher1,
H. Hallevi1,2, L. Shopin1, J. Molad1, A. Korczyn2, N. M. Bornstein1,2, S. Shenhar-Tsarfaty1,2 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Tel Aviv
University, Tel Aviv, Israel 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Tel Aviv
University, Tel Aviv, Israel 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Tel Aviv
University, Tel Aviv, Israel 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Tel Aviv
University, Tel Aviv, Israel Background: The s allele variant of the serotonin trans-
porter gene (5-HTT) has been related to hypothalamic-
pituitary-adrenal (HPA)-axis reactivity to stress, depressio-
nand negatively impact on memory. Acute strokeis asso-
ciated with elevated cortisol levels as part of the body's
reaction to a stress provoking event. We investigated
whether 5-HTT genotype interacts with physiological stress
to impact on cognitive function and hippocampal structural
measures in stroke patients. SHANK3 deletions or loss of function mutations cause
Phelan-McDermid syndrome and have been found in 2% of
individuals with intellectual disability (ID) and 0.5% of
individuals with autism spectrum disorders (ASD). We
analyzed SHANK3 coding sequence (NM_033517; hg19)
with amplicon-based next-generation sequencing in 163
individuals, negative to aCGH and Fragile X test, with non-
specific ID with or without autistic traits. Due to high GC
content, 7% of the gene was not covered by the analysis. We identified six novel SHANK3 variants, of which four de
novo frameshift or nonsense mutations. Individuals carrying
truncating mutations had global developmental delay with
moderate to severe ID, severely delayed or absent speech,
and abnormal behavior. Although some features were pre-
sent, ASD was specifically referred only for one of them. The other two variants were missense with discordant pre-
diction of pathogenicity; one inherited from an unaffected
parent was found in a girl with Rett-like phenotype. The
other missense, apparently homozygous, was found in a boy
with ID, ASD, epilepsy, speech delay and macrocephaly,
who carried other four apparently homozygous SHANK3
variants. We are currently investigating the possible pre-
sence of a previously missed intragenic deletion. The higher
incidence of SHANK3 mutations (2,5%) we report in indi-
viduals with ID and autistic traits indicates SHANK3 hap-
loinsufficiency as one of the most prevalent monogenic
causes of ID and ASD. We suggest routine screening of
SHANK3 for the diagnosis of non-specific ID in individuals
with or without reported autistic traits. Funding: Italian
Ministry of health Young Investigator Grant GR-2011-
02347754 to E.L. Methods Data from 182 cognitively intact stroke patients
from the TABASCO study were available. Patients under-
went 3T MRI scans, saliva cortisol measure and compre-
hensive cognitive and depression assessments at admission,
6, 12 and 24 months thereafter. Results Carriers of the 5-HTT s allele had significantly
higher admission bedtime salivary cortisol and reduced
hippocampal
volume
than
non-carriers. 1Guy's Hospital, London, United Kingdom, 2Evelina Childrens
Hospital, London, United Kingdom Bi-allelic variants in SPATA5 (spermatogenesis-associated
protein 5, MIM: 613940) are associated with severe global
developmental delay, congenital sensorineural hearing loss,
seizures, cortical visual impairment and microcephaly. SPATA5 is vital for mitochondrial function and morphol-
ogy in the cortical neurons. The absence of functional
protein prevents the normal neuronal development and
interferes with axonal growth. Congenital sensorineural
hearing impairment is often the first presenting symptom,
followed by seizures and motor delay on the back ground of
abnormal neurological phenotype including core hypotonia,
increased peripheral tone. A slowly progressive hyperki-
netic movement disorder evolves from early childhood. Most patients have microcephaly, although brain imaging is
non-specific, demonstrating brain atrophy and/or delayed
myelination. In partnership with the patient support group,
we have had access to an international cohort of patients
with confirmed SPATA5 mutations. We provide a detail
clinical description of the breadth and variability of the
clinical phenotype, alongside the already reported cases in
the literature. SPATA5 should be considered in cases sug-
gestive of mitochondrial disorders especially in young
infants whose clinical picture is often less recognisable. Autism spectrum disorders (ASD) and epilepsies are het-
erogeneous conditions that frequently coexist with other
developmental disabilities. Genetic bases are prominent risk
factors for both disorders. Among others, loss of function
mutations in CHD8 gene represents a recurrent risk factor
for ASD, while CHD2 is more frequently mutated in epi-
lepsy. Thus, the sole reduction in CHD8 or CHD2 expres-
sion is able to cause cellular and molecular phenotypes that
are key hallmarks to follow and rescue in assessing new
therapeutic approaches. Particularly, we aim to test SINEUPs, a novel class of
synthetic antisense long non-coding RNAs - able to increase
the translation of target proteins to physiological level
without affecting transcription - to rescue the phenotypes
caused by CHD8 or CHD2 haploinsufficiency. D.J. Josifova: None. K. Bradbury: None. R.L. Jones:
None. V. Govender: None. Since the activity of SINEUP depends on two domains,
an effector domain required for translation enhancement
and a binding domain conferring target specificity, we
designed SINEUP molecules able to recognize the initial
and internal methionines of CHD8 and CHD2 proteins. We
then proceeded to test the efficacy of different SINEUPs on
neural progenitors. 1Guy's Hospital, London, United Kingdom, 2Evelina Childrens
Hospital, London, United Kingdom From our preliminary observations,
SINEUPs targeting internal methionines are more efficient
in stimulating CHD8 and CHD2 protein production, thus
representing a valid target to be further tested in patients’
derived cell lines and in zebrafish, an in vivo model of the
disorders. Delineation of SPATA5 related epilepsy, hearing loss, and
mental retardation syndrome (EHLMRS) F. Di Leva1, M. Arnoldi1, G. Alvari1, A. Messina2, S. Casarosa2,3,
G. L. Carvill4, S. Zucchelli5,6, S. Gustincich5,7, M. Biagioli1 D. J. Josifova1, K. Bradbury1, R. L. Jones1, V. Govender2 1Neuro Epigenetics laboratory, Centre for Integrative Biology,
Trento, Italy, 2Laboratory of Neural Development and
Regeneration, Centre for Integrative Biology, Trento, Italy,
3CNR Neuroscience Institute, Pisa, Italy, 4Ken and Ruth Davee
Department of Neurology, Feinberg School of Medicine,
Northwestern University, Chicago, IL, United States, 5Area of
Neuroscience, Scuola Internazionale Superiore di Studi
Avanzati (SISSA), Trieste, Italy, 6Department of Health
Sciences, Università del Piemonte Orientale, Novara, Italy,
7Department of Neuroscience and Brain Technologies, Istituto
Italiano di Tecnologia (IIT), Genova, Italy 1Guy's Hospital, London, United Kingdom, 2Evelina Childrens
Hospital, London, United Kingdom 1Guy's Hospital, London, United Kingdom, 2Evelina Childrens
Hospital, London, United Kingdom 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Tel Aviv
University, Tel Aviv, Israel Patients
with
smaller hippocampi had lower cognitive scores at all
timepoints
post-stroke,
and
higher
depression
scores. Carriers of the 5-HTT s allele displayed strong negative
association of admission cortisol with hippocampal volume
and cognitive scores at all timepoints, while non-carriers
displayed no such association. Conclusions Carriers of the 5-HTT s allele had lower
hippocampal volume and higher admission salivary cortisol
. Their cortisol levels negatively correlated with post-stroke
cognitive function. . The interactive effects of the s allele
and cortisol levels on reduced hippocampal volume,lower
cognitive scores and higher depression imply that the
negative effect of 5-HTT-s on cognition involves the HPA
axis. Since genetic factors may influence vulnerability to the
adverse effects of stress, serotonin receptors may provide a
novel target for therapeutics to prevent dementia in stroke
patients. Abstracts from the 51st European Society of Human Genetics Conference: Posters 311 E. Ben Assayag: None. D. Amar: None. E. Kliper:
None. S. Usher: None. H. Hallevi: None. L. Shopin:
None. J. Molad:
None. A. Korczyn:
None. N.M. Bornstein: None. S. Shenhar-Tsarfaty: None. V. MUTO1, E. Flex2, Z. Kupchinsky3, G. Primiano4,
H. Galehdari5, M. Dehghani6, S. Cecchetti2, G. Carpentieri1,
T. Rizza1, N. Mazaheri5, A. Sedaghat7, M. Mehrjardi6,
A. Traversa8, M. Di Nottia1, M. Kousi3, Y. Jamshidi9, A. Ciolfi1,
V. Caputo10, R. Malamiri11, F. Pantaleoni1, S. Martinelli2, E. Ben Assayag: None. D. Amar: None. E. Kliper:
None. S. Usher: None. H. Hallevi: None. L. Shopin:
None.
J.
Molad:
None.
A.
Korczyn:
None.
N.M.
Bornstein: None. S. Shenhar-Tsarfaty: None. In conclusion, our studies represents the first step towards
the development of new types of RNA-based therapy, with
implications for a large repertory of presently incurable
genetic diseases. F. Di Leva: None. M. Arnoldi: None. G. Alvari: None. A. Messina: None. S. Casarosa: None. G.L. Carvill:
None. S. Zucchelli: None. S. Gustincich: None. M. Biagioli: None. Biallelic SQSTM1 mutations in early-onset, variably
progressive neurodegeneration Biallelic SQSTM1 mutations in early-onset, variably
progressive neurodegeneration V. MUTO1, E. Flex2, Z. Kupchinsky3, G. Primiano4,
H. Galehdari5, M. Dehghani6, S. Cecchetti2, G. Carpentieri1,
T. Rizza1, N. Mazaheri5, A. Sedaghat7, M. Mehrjardi6,
A. Traversa8, M. Di Nottia1, M. Kousi3, Y. Jamshidi9, A. Ciolfi1,
V. Caputo10, R. Malamiri11, F. Pantaleoni1, S. Martinelli2, 312 J. del Picchia A. Jeffries12, J. Zeighami7, A. Sherafat13, D. Di Giuda14,
G. Shariati7, R. Carrozzo1, N. Katsanis3, R. Maroofian9,
S. Servidei15, M. Tartaglia1 phenotype characterized by cerebellum anomalies ranging
from depletion of axonal connections to complete atrophy. Italian Ministry of Health (R. C. 2017) V. Muto: None. E. Flex: None. Z. Kupchinsky: None. G. Primiano: None. H. Galehdari: None. M. Dehghani:
None. S. Cecchetti: None. G. Carpentieri: None. T. Rizza: None. N. Mazaheri: None. A. Sedaghat: None. M. Mehrjardi: None. A. Traversa: None. M. Di Nottia:
None. M. Kousi: None. Y. Jamshidi: None. A. Ciolfi:
None. V. Caputo:
None. R. Malamiri:
None. F. Pantaleoni: None. S. Martinelli: None. A. Jeffries: None. J. Zeighami: None. A. Sherafat: None. D. Di Giuda:
None. G. Shariati:
None. R. Carrozzo:
None. N. Katsanis: None. R. Maroofian: None. S. Servidei: None. M. Tartaglia: None. 1Ospedale Pediatrico Bambino Gesù, ROMA, Italy, 2Istituto
Superiore di Sanità, ROMA, Italy, 3Duke University School of
Medicine, Durham, NC, United States, 4Policlinico
Universitario A. Gemelli, ROMA, Italy, 5Shahid Chamran
University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 6Shahid
Sadoughi University of Medical Sciences, Yazd, Iran, Islamic
Republic of, 7Narges Medical Genetics and Prenatal Diagnosis
Laboratory, Ahvaz, Iran, Islamic Republic of, 8Università
“Sapienza", ROMA, Italy, 9St George’s, University of London,
London, United Kingdom, 10Università “Sapienza”, ROMA,
Italy, 11Jundishapur University of Medical Sciences, Ahvaz,
Iran, Islamic Republic of, 12University of Exeter Medical
School, Exeter, United Kingdom, 13Kerman University of
Medical Sciences, Kerman, Iran, Islamic Republic of,
14Fondazione Policlinico A. Gemelli, ROMA, Italy,
15Fondazione Policlinico Universitario A. Gemelli, ROMA,
Italy Compound heterozygosity for a frameshift and a missense
mutation in the SURF1 gene in a patient without Leigh
syndrome Compound heterozygosity for a frameshift and a missense
mutation in the SURF1 gene in a patient without Leigh
syndrome E. Nibbeling1, D. Kamphuis2, j. knijnenburg1, s. bollen1,
M. Laurense-Bik1, I. Fokkema1, M. Kriek1, C. Ruivenkamp1
1LUMC, Leiden, Netherlands, 2Reinier de Graaf Gasthuis,
Delft, Netherlands E. Nibbeling1, D. Kamphuis2, j. knijnenburg1, s. bollen1,
M. Laurense-Bik1, I. Fokkema1, M. Kriek1, C. Ruivenkamp1 Intracellular clearance of damaged cellular constituents,
including protein aggregates and dysfunctional organelles,
is necessary for proper neuronal function and long-term
survival of neuronal cells. Autophagy contributes sig-
nificantly to this process, and its defective function has been
implicated in a number of neurodegenerative disorders. Here, we describe clinically and molecularly a recently
recognized early-onset, variably progressive, neurodegen-
erative disorder caused by loss of function of SQSTM1, a
multidomain protein serving as a selective autophagy
receptor. Eleven affected individuals from three con-
sanguineous families shared a homogeneous phenotype
characterized by ataxia, hypotonia, dysmetria, dysarthria,
ophthalmoplegia, dyskenesia, and cognitive decline as
major features. Whole exome sequencing (WES) in two
families, and a combined approach based on homozygosity
mapping analysis in six affected individuals of the third
family coupled to WES performed in a single affected
member allowed to identify three homozygous inactivating
variants, including a splice site substitution (c.301+2T>A)
causing aberrant transcript processing and accelerated
degradation of a resulting protein lacking exon 2, and two
truncating
changes
(c.875_876insT
and
c.934_936delinsTGA). In vitro studies directed to char-
acterize the consequences of loss of SQSTM1 function on
autophagy provided evidence of a decelerated autophagic
flux and impaired production of ubiquitin-positive protein
aggregates in response to misfolded protein stress. The
impact of sqstm1 down-modulation on the structural
integrity of the cerebellum was analyzed in vivo, using
zebrafish as model, documenting a variable but reproducible Intracellular clearance of damaged cellular constituents,
including protein aggregates and dysfunctional organelles,
is necessary for proper neuronal function and long-term
survival of neuronal cells. Autophagy contributes sig-
nificantly to this process, and its defective function has been
implicated in a number of neurodegenerative disorders. Here, we describe clinically and molecularly a recently
recognized early-onset, variably progressive, neurodegen-
erative disorder caused by loss of function of SQSTM1, a
multidomain protein serving as a selective autophagy
receptor. Eleven affected individuals from three con-
sanguineous families shared a homogeneous phenotype
characterized by ataxia, hypotonia, dysmetria, dysarthria,
ophthalmoplegia, dyskenesia, and cognitive decline as
major features. Compound heterozygosity for a frameshift and a missense
mutation in the SURF1 gene in a patient without Leigh
syndrome Whole exome sequencing (WES) in two
families, and a combined approach based on homozygosity
mapping analysis in six affected individuals of the third
family coupled to WES performed in a single affected
member allowed to identify three homozygous inactivating
variants, including a splice site substitution (c.301+2T>A)
causing aberrant transcript processing and accelerated
degradation of a resulting protein lacking exon 2, and two
truncating
changes
(c.875_876insT
and
c.934_936delinsTGA). In vitro studies directed to char-
acterize the consequences of loss of SQSTM1 function on
autophagy provided evidence of a decelerated autophagic
flux and impaired production of ubiquitin-positive protein
aggregates in response to misfolded protein stress. The
impact of sqstm1 down-modulation on the structural
integrity of the cerebellum was analyzed in vivo, using
zebrafish as model, documenting a variable but reproducible P09.145A L. Corrado1, L. M. Genovese2, E. Mangano3, A. Di Pierro1,
N. Barizzone1, R. Bordoni3, F. Geraci4, R. D'Aurizio2, R. Croce1,
F. De MArchi5, L. Mazzini5, R. Cantello5, G. De Bellis3,
G. Manzini6,7, M. Severgnini3, M. Pellegrini2, S. D'Alfonso1 A. T. Midro1, B. Panasiuk1, L. Cooper2, S. E. Scherer3,
P. Stankiewicz2,4 A. T. Midro1, B. Panasiuk1, L. Cooper2, S. E. Scherer3,
P. Stankiewicz2,4 1Department of Clinical Genetics, Bialystok, Poland, 2Baylor
Genetics, Houston, TX, United States, 3Human Genome
Sequencing Center, Baylor College of Medicine, Houston, TX,
United States, 4Dept of Molecular & Human Genetics, Baylor
College of Medicine, Houston, TX, United States 1Lab. of Human Genetics, Dep. of Healty Sciences, UPO,
NOvara, Italy, 2Institute for Informatics and Telematics (IIT),
National Research Council (CNR), Pisa, Italy, 3Institute for
Biomedical Technologies, National Research Council (CNR-
ITB), Segrate (MI), Italy, 4Institute for Informatics and
Telematics (IIT), National Research Council (CNR),, Pisa (PI),
Italy, 5ALS Center AOU Maggiore della Carità, Novara,
NOvara, Italy, 6University of Eastern Piedmont UPO, Vercelli,
Italy, 7Institute for Informatics and Telematics (IIT), National
Research Council (CNR), Pisa (PI), Pisa, Italy Introduction: We present a 24-year follow-up of a 34-year-
old male with a de novo apparently balanced chromosomal
translocation t(1;17)(q31;q25) reported in 1993 with the
features
of
Russell-Silver
syndrome
(RSS,
PMID:
8403458). The chromosomal region 17q25 was also
involved in another apparently balanced translocation t
(17;20)(q25;q13) associated with RSS (PMID: 1633648). Molecular studies indicated a disruption of KPNA2. How-
ever, subsequent screenings for mutations in KPNA2 in 31
unrelated individuals with RSS revealed no disease-related
variants (PMID: 11735022). Introduction: We present a 24-year follow-up of a 34-year-
old male with a de novo apparently balanced chromosomal
translocation t(1;17)(q31;q25) reported in 1993 with the
features
of
Russell-Silver
syndrome
(RSS,
PMID:
8403458). The chromosomal region 17q25 was also
involved in another apparently balanced translocation t
(17;20)(q25;q13) associated with RSS (PMID: 1633648). Molecular studies indicated a disruption of KPNA2. How-
ever, subsequent screenings for mutations in KPNA2 in 31
unrelated individuals with RSS revealed no disease-related
variants (PMID: 11735022). The C9ORF72 gene repeat expansion is the most frequent
cause of amyotrophic lateral sclerosis (ALS). Long repeats
alleles in ATXN-1, ATXN-2, and NIPA1 genes are asso-
ciated to ALS susceptibility. Thus, Tandem Repeat Poly-
morphisms
(TRPs)
are
good
candidates
for
missing
hereditability in ALS, although they were never system-
atically analyzed because challenging to NGS. 1LUMC, Leiden, Netherlands, 2Reinier de Graaf Gasthuis,
Delft, Netherlands 1LUMC, Leiden, Netherlands, 2Reinier de Graaf Gasthuis,
Delft, Netherlands The SURF1 gene encodes an assembly factor of the mito-
chondrial respiratory complex IV. Recessive mutations in
the SURF1 gene are associated with Leigh syndrome (MIM
256000) and a few patients with Charcot-Marie-Tooth dis-
ease type 4K (MIM 616684). The majority of the reported
mutations lead to premature termination codons. Missense
mutations are reported to cause a milder phenotype and
longer survival. Here we report an adult patient showing
episodes of balance and coordination problems, tremor,
short stature and stuttering who was referred to our clinic
for whole exome sequencing. We identified compound
heterozygosity for a missense and a frameshift mutation in
the SURF1 gene. Both mutations have previously been
reported in patients with Leigh syndrome, however, the
clinical phenotype of the patient did not fit Leigh syndrome. The clinical phenotype of our patient and those reported
with the same mutations is compared and will be presented. Overall, mutations in the SURF1 gene comprise a broad
clinical phenotype and should also be considered in adult
patients with movement disorders. E. Nibbeling: None. D. Kamphuis: None. J. knijnen-
burg: None. S. bollen: None. M. Laurense-Bik: None. I. Fokkema:
None. M. Kriek:
None. C. Ruivenkamp: None. 313 Abstracts from the 51st European Society of Human Genetics Conference: Posters L. Corrado: None. L.M. Genovese: None. E. Man-
gano: None. A. Di Pierro: None. N. Barizzone: None. R.
Bordoni: None. F. Geraci: None. R. D'Aurizio: None. R.
Croce: None. F. De MArchi: None. L. Mazzini: None. R. P09.145A The general
aim of this study is to perform a systematic analysis of
TRPs in ALS by combining NGS and novel bioinformatics
tools. TRPs from whole genome sequencing data (WGS,
Illumina HiSeq X Ten, avg. coverage 30X, 2x 150 bp
length) of a cohort of 70 ALS cases enriched in FALS cases
were evaluated by means of two software developed within
our consortium and by a literature software (lobSTR) to
detect either expansions with a repeat size within the NGS
reads sizes (short TRPs), and repeat expansions larger than
the NGS reads sizes. The analysis of short tandem repeat
expansion for about 600K loci in 70 ALS cases and 300
controls led to the selection of 20 TRPs showing a sig-
nificant distribution among patients and controls. The
validation of these loci by traditional methods revealed a
high technical consistency (70%). However, we failed to
replicate this data in an independent sample (208 Italian
ALS patients and 229 matched controls). For large repeat
expansions detection, the analysis of 700K TRPs identified
16 loci with potential very large repeat expansion observed
in 1 or 2 of the 70 patients, and whose validation and
replication is ongoing. Material and
Methods:
Phenotypic analyses were
performed
according
to
the
Munich
Dysmorphology
Database (MDDB) methodology. Chromosomal microarray
analysis and FISH with BAC and fosmid clones were used
to narrow the 17q breakpoint. Results: We found that the translocation breakpoint maps
to 17q24.2 and disrupts BPTF encoding the largest subunit
of a nucleosome remodeling factor (NURF), a member of
ISWI chromatin remodeling complex. No non-polymorphic
CNVs were identified. Phenotypic analyses showed short
stature, hemihypotrophy, microcephaly, triangular shape of
face, prominent forehead, hypertelorism, protruding eye-
balls, broad palpebral fissures, long eye lashes, long nasal
bridge, short philtrum, thin lips microretrogenia, and
bilateral 5th finger clinodactyly. Muscle hypotonia, intel-
lectual disability, vision problems, speech delay, and the
defect of phonemic audition have improved during 24 years
of treatment. Conclusion: Observed phenotypic changes are similar to
those seen in the recently described patients with Neuro-
developmental disorder with dysmorphic facies and distal
limb
anomalies
(NEDDFL,
OMIM
617755)
due
to
haploinsufficiency of BPTF, further demonstrating its
pathogenicity. Our data expand the clinical spectrum of
human disorders caused by ablation of chromatin remodel-
ing complexes. L. Corrado: None. L.M. Genovese: None. E. Man-
gano: None. A. Di Pierro: None. N. Barizzone: None. R. Bordoni: None. F. Geraci: None. R. D'Aurizio: None. P09.146B A. Kariminejad1, M. Dahl-Halvarsson2, G. Ravenscroft3,
F. Afroozan1, E. Keshavarz4, M. Faraji Zonooz1, H. Najmabadi1,
H. Goullée3, M. Davis5, N. Laing3, H. Tajsharghi6 Generation and in-depth characterization of 20 induced
pluripotent stem cell (iPSC) lines from 10 dystonia patients
and healthy carriers of THAP1 mutations 1Kariminejad Najmabadi Pathology and Genetics Center,
Tehran, Iran, Islamic Republic of, 2Department of Pathology,
University of Gothenburg, Sahlgrenska University Hospital,
Sweden, Gothenburg, Sweden, 3Centre for Medical Research,
The University of Western Australia and the Harry Perkins
Institute for Medical Research, Nedlands, Western Australia,
Australia, Nedlands, Australia, 4Department of Radiology,
Mahdieh Hospital, Shahid Beheshti University of Medical
Science, Tehran, Iran, Tehran, Iran, Islamic Republic of,
5Department of Diagnostic Genomics, Pathwest, QEII Medical
Centre, Nedlands, Western Australia, Australia, Nedlands,
Australia, 6School of Health and Education, Division
Biomedicine and Public Health, University of Skovde, SE-541
28, Skovde, Sweden, Skovde, Sweden H. Baumann1, M. Trilck1, M. Jahn1, A. Muenchau1, V. Kostic2,
C. Klein1, P. Seibler1, K. Lohmann1 1Institute of Neurogenetics, Luebeck, Germany, 2Clinic of
Neurology, Belgrade, Serbia 1Institute of Neurogenetics, Luebeck, Germany, 2Clinic of
Neurology, Belgrade, Serbia Introduction: Mutations in THAP1 have been linked to
dystonia (DYT-THAP1, DYT6) with reduced penetrance. THAP1 encodes a transcription factor that regulates its own
expression and the expression of TOR1A, another dystonia
gene. To date, little data is available on the expression of
THAP1 and TOR1A in mutant THAP1 induced pluripotent
stem cells (iPSCs). Introduction: Mutations in THAP1 have been linked to
dystonia (DYT-THAP1, DYT6) with reduced penetrance. THAP1 encodes a transcription factor that regulates its own
expression and the expression of TOR1A, another dystonia
gene. To date, little data is available on the expression of
THAP1 and TOR1A in mutant THAP1 induced pluripotent
stem cells (iPSCs). Material and Methods: Cultured skin fibroblasts were
reprogrammed into iPSCs using Sendai virus. Two clones
per patient were comprehensively characterized by testing
for the mutation using Sanger sequencing, by expression
analysis of four pluripotency markers using quantitative
PCR and immunocytochemistry, and by their ability to
differentiate into all three germ layers. Genomic rearrange-
ments were excluded by single nucleotide polymorphism
(SNP) array analysis. Expression of THAP1 and TOR1A
was tested by quantitative PCR compared to 10 iPSC
controls while ß-Actin served as a reference gene. P09.147C
TOR1A variants cause a severe arthrogryposis with
developmental delay, strabismus and tremor Cantello: None. G. De Bellis: None. G. Manzini: None. M. Severgnini:
None. M. Pellegrini:
None. S. D'Alfonso: None. P09.145A R. Croce: None. F. De MArchi: None. L. Mazzini: None. R. A.T. Midro: None. B. Panasiuk: None. L. Cooper:
None. S.E. Scherer: None. P. Stankiewicz: None. 314 J. del Picchia P09.147C
TOR1A variants cause a severe arthrogryposis with
developmental delay, strabismus and tremor H. Baumann: None. M. Trilck: None. M. Jahn: None.
A. Muenchau: None. V. Kostic: None. C. Klein: None. P.
Seibler: None. K. Lohmann: None. Abstract Background: Autosomal dominant torsion dystonia-1 is
a disease with incomplete penetrance most often caused by
an in-frame GAG deletion (p.Glu303del) in the endoplas-
mic reticulum luminal protein torsinA encoded by TOR1A. Methods: We report an association of the homozygous
dominant disease-causing TOR1A p.Glu303del mutation,
and a novel homozygous missense variant (p.Gly318Ser)
with a severe arthrogryposis phenotype with developmental
delay, strabismus and tremor in three unrelated families. Results: We generated 20 iPSC lines of 10 affected and
unaffected members of three families carrying pathogenic
THAP1 variants (p.Arg13His [4 lines], p.Ser21Cys [10
lines], p.Leu159fs180X [6 lines]). THAP1 expression was
reduced for Ser21Cys and TOR1A expression in Arg13His
and Ser21Cys mutant cell lines compared to controls. Results: All parents who were carriers of the TOR1A
variant showed no evidence of neurological symptoms or
signs, indicating decreased penetrance similar to families
with autosomal dominant torsion dystonia-1. The results
from cell assays demonstrate that the p.Gly318Ser substitu-
tion causes a redistribution of torsinA from the endoplasmic
reticulum to the nuclear envelope, similar to the hallmark of
the p.Glu303del mutation. Conclusion: We report the generation and characteriza-
tion of 20 lines from 10 human THAP1 iPSC lines as well
as alterations in THAP1 and TOR1A expression in these
cells. These stem cells can further serve as an ideal model to
investigate the mechanism of reduced penetrance by
transcriptomic analysis in affected and unaffected THAP1
mutation carriers on the stem cell and differentiated
neuron level. Conclusion: Our study highlights that TOR1A mutations
should be considered in patients with severe arthrogryposis
and further expands the phenotypic spectrum associated
with TOR1A. Keywords: TOR1A; Endoplasmic reticulum luminal
protein torsinA; DYT1 dystonia; TOR1A p.Glu303del;
Severe arthrogryposis H. Baumann: None. M. Trilck: None. M. Jahn: None. A. Muenchau: None. V. Kostic: None. C. Klein: None. P. Seibler: None. K. Lohmann: None. A. Kariminejad: None. M. Dahl-Halvarsson: None. G. Ravenscroft: None. F. Afroozan: None. E. Keshavarz:
None. M. Faraji Zonooz: None. H. Najmabadi: None. H. Goullée: None. M. Davis: None. N. Laing: None. H. Tajsharghi: None. 315 Abstracts from the 51st European Society of Human Genetics Conference: Posters 1UnIGENe, IBMC - Institute for Molecular and Cell Biology,
i3S - Instituto de Investigação e Inovação em Saúde, Porto,
Portugal, 2CGPP, IBMC - Institute for Molecular and Cell
Biology, i3S - Instituto de Investigação e Inovação em Saúde,
Porto, Portugal, 3ICBAS - Instituto de Ciências Biomédicas
Abel Salazar, Porto, Portugal P09.148D X. Yang1, M. A. Thomas1,2, A. M. Innes1,2 1Department of Medical Genetics, Cumming School of
Medicine, University of Calgary, Calgary, AB, Canada,
2Alberta Children's Hospital Research Institute, University of
Calgary, Calgary, AB, Canada 1Department of Medical Genetics, Cumming School of
Medicine, University of Calgary, Calgary, AB, Canada,
2Alberta Children's Hospital Research Institute, University of
Calgary, Calgary, AB, Canada SCA11 is a rare autosomal dominant form of cerebellar
ataxia, characterized by early-onset cerebellar ataxia and
nystagmus. SCA11 is caused by variants in TTBK2; the
ones reported are heterozygous truncating variants. Never-
theless, the disease mechanism linking TTBK2 and SCA11
remains unclear. TTBK2 encodes tau tubulin kinase 2 pro-
tein, a protein kinase involved in different cellular pro-
cesses, namely, ciliogenesis, microtubule dynamics, and tau
and TDP-43 phosphorylation. Increasingly whole exome sequencing (WES) is used as an
early diagnostic tool in patients with undiagnosed rare
diseases. Particularly in the setting of consanguinity, hun-
dreds of emerging disease genes, usually with private
mutations, have been identified. Typically, these require
replication to validate as bonafide disease genes to facilitate
management of such families. The transport protein particle
(TRAPP) family of protein complexes regulates intracel-
lular trafficking between the endoplasmic reticulum and
Golgi apparatus. Variants in several TRAPP subunits have
been implicated in diverse human diseases. Harripaul
(2017) reported a homozygous nonsense variant in one such
subunit, TRAPPC6B, in two consanguineous individuals
with non-syndromic intellectual disability. A homozygous
founder splice variant in TRAPPC6B was recently described
by Marin-Valencia (2018) in 3 Egyptian sibships with
microcephaly, global developmental delay, autism, and
epilepsy (OMIM 617862). We report two consanguineous
Pakistani sisters with microcephaly (-8 SDs) and severe
global developmental delay. Additionally, the younger sis-
ter had a movement disorder while the older sister had
epilepsy. Trio WES of the parents and younger sister
revealed a homozygous novel variant in TRAPPC6B at a
splice donor site (c.149+2T>A), carried by both parents. This variant is predicted to cause skipping of exon 2, and
has been reported once, in heterozygous form, in gnomAD. Therefore, we report the third “family” with homozygous
TRAPPC6B variants. These findings support the role of
recessive mutations in TRAPPC6B in severe disease. We
will review the phenotype associated with recessive muta-
tions in TRAPPC6B as well as those associated with other
members of the TRAPP family. P09.150B Severe speech delay in Cohen Syndrome: three novel
mutations and the long-term follow-up of nine patients X. Yang:
None. M.A. Thomas:
None. A.M. Innes: None. X. Yang:
None. M.A. Thomas:
None. A.M. Innes: None. P09.148D Our group has previously identified a novel heterozygous
missense variant in TTBK2 in two Portuguese siblings with
a diagnosis of ataxia. Therefore, we aim to characterize the
potential pathogenic effect of this variant in SCA11. For
that, we generated TTBK2 clones (wild-type and different
mutants) in fusion with EGFP-tag that were transfected in
cultured cells. The subcellular localization was accessed by
immunofluorescence and subcellular fractioning; however,
TTBK2 clones did not display significant changes. TTBK2
kinase activity was evaluated by measuring the phosphor-
ylation state of TTBK2 substrates and potential new
interactors
of
TTBK2
were
analyzed
by
co-
immunoprecipitation. Our results showed that the TTBK2
missense variant impairs phosphorylation activity against
TDP-43 and may lead to altered protein-protein interactions,
namely with ataxin-2. In addition, we created a cellular
model expressing the endogenous TTBK2 missense variant,
by CRISPR/Cas9 technology. In conclusion, we showed that the newly identified
TTBK2 missense variant confers different biochemical
properties, which may result in abnormal protein phosphor-
ylation in SCA11. The study of the novel CRISPR/Cas9
cellular model should contribute to a better understanding of
the molecular and cellular mechanisms underlying SCA11. M. Santos: None. J. Sequeiros: None. I. Alonso: None. 1İstanbul University, Cerrahpasa Medical Faculty, İstanbul,
Turkey, 2Yale School of Medicine, New Haven, CT, United
States B. Akdeniz1, N. Gunes1, D. Uludağ1, G. Ercan-Şençiçek2,
O. Çağlayan2, K. Bilguvar2, B. Tüysüz1 M. Santos1, J. Sequeiros1,2,3, I. Alonso1,2 Functional characterization of a new TTBK2 missense
variant: uncovering the molecular basis of SCA11 P09.149A Functional characterization of a new TTBK2 missense
variant: uncovering the molecular basis of SCA11 M. Santos1, J. Sequeiros1,2,3, I. Alonso1,2 316 J. del Picchia cognitive dysfunction and ongoing epileptiform activity. A
genetic aetiology can be identified in a significant propor-
tion of patients. Whole exome sequencing (WES) analysis
by tools like Denovogear and Exomiser has proven to be
very effective. Introduction: Cohen Syndrome (CS) is a rare autosomal
recessive disorder caused by VSP13B mutations. Char-
acteristic features are hypotonia, mild dysmorphic facies at
infantile period, microcephaly, retinitis pigmentosa, devel-
opmental delay with positive social behavior and inter-
mittent neutropenia in childhood. Here, we investigate the
genetic defects and follow-up findings of CS patients. Introduction: Cohen Syndrome (CS) is a rare autosomal
recessive disorder caused by VSP13B mutations. Char-
acteristic features are hypotonia, mild dysmorphic facies at
infantile period, microcephaly, retinitis pigmentosa, devel-
opmental delay with positive social behavior and inter-
mittent neutropenia in childhood. Here, we investigate the
genetic defects and follow-up findings of CS patients. cognitive dysfunction and ongoing epileptiform activity. A
genetic aetiology can be identified in a significant propor-
tion of patients. Whole exome sequencing (WES) analysis
by tools like Denovogear and Exomiser has proven to be
very effective. Materials and methods: We performed WES in a cohort
of 18 unrelated individuals (9 males and 9 females) with
epileptic encephalopathies. All the patients were thoroughly
assessed by medical specialists and counselled by a clinical
geneticist, in order to provide detailed and complete clinical
information. These patients were previously tested by gene
panel and the cause of epilepsy was not identified. Ten trios
were analysed by Denovogear and eight single samples
were analysed with Exomiser also with help of proper HPO
terms, OMIM database and HGMD professional database)
Manual filtering for all samples was also performed. Materials and methods: We performed WES in a cohort
of 18 unrelated individuals (9 males and 9 females) with
epileptic encephalopathies. All the patients were thoroughly
assessed by medical specialists and counselled by a clinical
geneticist, in order to provide detailed and complete clinical
information. These patients were previously tested by gene
panel and the cause of epilepsy was not identified. Ten trios
were analysed by Denovogear and eight single samples
were analysed with Exomiser also with help of proper HPO
terms, OMIM database and HGMD professional database)
Manual filtering for all samples was also performed. P09.149A Materials and methods: Clinical findings of nine
patients from five families were evaluated during the
follow-up period of 1-14 years. Mutations were identified
by using Sanger sequencing of VSP13B gene. Results: While 4 patients were diagnosed at age 3-38
months, 5 patients at 4.5-9.5 years of age. Hypotonia,
microcephaly, joint laxity, almond shaped eyes, and
micrognathia were present in 4 patients admitted during
the infantile period. Patients admitted in childhood had
typical facial appearance and microcephaly (5/5), hypotonia
(5/5) and also pigmentary retinopathy (2/5), neutropenia (2/
5), truncal obesity (2/5) at the first examination. Three novel
mutations (1 splice site, 1 nonsense and 1 frameshift) were
found. During the follow-up, retinopathy and neutropenia in
2, growth hormone deficiency in 1, hypothyroidism in 2,
hyperinsulinemia in 1 patient were detected. Interestingly,
severe speech delay has developed in eight patients over 5
years of age. Results: After running analyses, we were able to identify
pathogenic variants in 28% of patients. Pathogenic variants
were found in: HUWE1, UBTF, NARS2, PPP2R5D,
SETBP1. All these variants were highly prioritised by
algorithms and confirmed by segregation analysis in the
family. The analysis time per sample was approx. 15 min -
instead of hours/days by previous algorithms based on
manual filtering. With manual filtering, no other variants of
interest were found. Conclusions: New approaches in WES analysis helps to
reduce amount of time spent per sample. With this approach
we were able to identify a causal variant in 28% samples. Conclusions: We aim to contribute to the literature three
novel VSP13B mutations without any distinct genotype-
phenotype relationship. In infantile patients with hypotonia,
joint laxity, and typical facial features, CS should be kept in
mind. The observation of severe speech delay in our
patients over the age of 5 suggests that this finding should
be added to diagnostic criteria. Supported by: AZV 15-33041 Supported by: AZV 15-33041 D. Stanek: None. P. Lassuthova: None. L. Sedlackova:
None. J. Neupauerova: None. K. Sterbova: None. M. Vlckova: None. P. Seeman: None. B. Akdeniz: None. N. Gunes: None. D. Uludağ: None. G. Ercan-Şençiçek: None. O. Çağlayan: None. K. Bilguvar: None. B. Tüysüz: None. B. Akdeniz: None. N. Gunes: None. D. Uludağ: None. G. Ercan-Şençiçek: None. O. Çağlayan: None. K. Bilguvar: None. B. Tüysüz: None. Analysis of WES data in 15 minutes - new methods
override manual filtering Around 400 cases have been reported in the
literature, but the disorder is thought to be underdiagnosed
because its features can resemble those of other conditions
such as cerebral palsy or epilepsy. In this study, we inves-
tigated the cause of neuromotor development delay and
dystonia in a 3,5 years old female patient. overlap. Since >100 genes are known for each group,
molecular definition represents a challenging task, with
>50% of patients undiagnosed. NGS technology allows a
comprehensive and systematic approach for genetic testing. Methods: We developed and validated different disease-
specific gene panels covering >98% of target region at
>20X: 1) 205 HSP and ataxia genes; 2) 177 CMT and
related neuropathies genes; 3) 143 WMD genes. We
analyzed 710 probands (142 HSP, 155 ATAXIA, 332
CMT and 81 WMD) negative for the most frequent forms. Results: Pathogenic mutations were identified in 25%
(178/710) of patients (22% HSP, 21% ATAXIA, 28% CMT
and 25% WMD). In particular, we identified mutations in
challenging genes difficult to be studied by conventional
sequencing because of their length (eg, SYNE1, SACS,
SETX, CACNA1A). Mutations in extremely rare genes were
identified in 10% of cases. Panel design allowed CNV
analysis that detected pathogenic CNVs in 13/283 patients
and CNV of unknown significance in 9/283 patients. In 10
patients, we identified mutations in genes unexpected based
on the clinical diagnosis, thus expanding the phenotypic
spectrum. Moreover, pathogenic mutations in more than
one gene were identified in 3 patients, thus challenging
diagnosis and genetic counseling. Materials and methods: We performed whole exome
sequencing in the patient with central hypotonia and facial
myokymia. All biochemical and metabolic screening results
were normal. Chromosome analysis did not show any
abnormalities. Results: Whole exome sequencing analysis identified a
novel heterozygous missense mutation (c.2090G>T; p. Gly697Val) in ADCY5. p.Gly697Val occurs in a conserved
domain whose function is unknown. In silico tools a
deleterious
effect
on
the
protein. The
family
study
confirmed that it is a de novo mutation. Conclusions: The use of high-coverage panels for the
genetic definition of highly heterogeneous neurodegenera-
tive diseases is a reliable (high detection rate, no incidental
findings) and cost-efficient approach (Italian MoH-RF-
2011-02351165 grant to FT). Conclusion: Given its genetic heterogeneity and variable
phenotype, molecular diagnosis of dystonia and dyskinesia
is difficult. Whole exome sequencing is a powerful
diagnostic tool for patients with these phenotypes. P09.154B Whole exome sequencing in a cohort of 48 trios with
neurodevelopmental disorders Analysis of WES data in 15 minutes - new methods
override manual filtering Muta-
tions in ADCY5 should be considered in cases with complex
movement disorders and with or without a family history. S. Magri: None. D. Di Bella: None. E. Sarto: None. F. S. Magri: None. D. Di Bella: None. E. Sarto: None. F. Balistreri: None. S. Baratta: None. D. Tonduti: None. L. Nanetti: None. E. Salsano: None. I. Moroni: None. C. Pisciotta: None. D. Pareyson: None. C. Mariotti: None. C. Gellera: None. F. Taroni: None. B. Cavdarli: None. N. Esen: None. V. Topcu: None. A. Aksoy: None. A. Danis: None. B. Anlar: None. B. Cavdarli: None. N. Esen: None. V. Topcu: None. A. Aksoy: None. A. Danis: None. B. Anlar: None. Balistreri: None. S. Baratta: None. D. Tonduti: None. L. Nanetti: None. E. Salsano: None. I. Moroni: None. C. Pisciotta: None. D. Pareyson: None. C. Mariotti: None. C. Gellera: None. F. Taroni: None. Pisciotta: None. D. Pareyson: None. C. Mariotti: None. C. Gellera: None. F. Taroni: None. Analysis of WES data in 15 minutes - new methods
override manual filtering Seeman1 1Unit of Genetics of Neurodegenerative and Metabolic
Disease, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy, 2Child Neurology Unit, Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milan, Italy, 3Neurology Unit
10, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy 1Unit of Genetics of Neurodegenerative and Metabolic
Disease, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy, 2Child Neurology Unit, Fondazione IRCCS
Istituto Neurologico Carlo Besta, Milan, Italy, 3Neurology Unit
10, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy 1DNA lab, Department of Paediatric Neurology, 2nd Faculty of
Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 2Department of Paediatric Neurology, 2nd Faculty
of Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 3Department of Biology and Medical Genetics, 2nd
Faculty of Medicine, Charles University in Prague a, Prague
5, Czech Republic 1DNA lab, Department of Paediatric Neurology, 2nd Faculty of
Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 2Department of Paediatric Neurology, 2nd Faculty
of Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 3Department of Biology and Medical Genetics, 2nd
Faculty of Medicine, Charles University in Prague a, Prague
5, Czech Republic Introduction:
Hereditary
ataxias,
spastic
paraplegias
(HSP), white matter disorders (WMD) and peripheral neu-
ropathies
(Charcot-Marie-Tooth
(CMT)
disease)
are
genetically highly heterogeneous and exhibit phenotypic Introduction: Epileptic encephalopathies are severe, early
onset disorders associated with global developmental delay, Abstracts from the 51st European Society of Human Genetics Conference: Posters 317 Introduction: Familial dyskinesia with facial myokymia
(FDFM) or ADCY5-related dyskinesia is an autosomal
dominant movement disorder characterized by early-onset
of involuntary choreiform or dystonic movements. ADCY5
belongs to the adenylate cyclase family of enzymes
responsible for the synthesis of cAMP. Heterozygous mis-
sense mutations in this gene are primarily known to cause
the disease. Around 400 cases have been reported in the
literature, but the disorder is thought to be underdiagnosed
because its features can resemble those of other conditions
such as cerebral palsy or epilepsy. In this study, we inves-
tigated the cause of neuromotor development delay and
dystonia in a 3,5 years old female patient. Introduction: Familial dyskinesia with facial myokymia
(FDFM) or ADCY5-related dyskinesia is an autosomal
dominant movement disorder characterized by early-onset
of involuntary choreiform or dystonic movements. ADCY5
belongs to the adenylate cyclase family of enzymes
responsible for the synthesis of cAMP. Heterozygous mis-
sense mutations in this gene are primarily known to cause
the disease. Analysis of WES data in 15 minutes - new methods
override manual filtering Analysis of WES data in 15 minutes - new methods
override manual filtering S. Magri1, D. Di Bella1, E. Sarto1, F. Balistreri1, S. Baratta1,
D. Tonduti2, L. Nanetti1, E. Salsano3, I. Moroni2, C. Pisciotta3,
D. Pareyson3, C. Mariotti1, C. Gellera1, F. Taroni1 override manual filtering
D. Stanek1, P. Lassuthova1, L. Sedlackova1, J. Neupauerova1,
K. Sterbova2, M. Vlckova3, P. Seeman1
1DNA lab, Department of Paediatric Neurology, 2nd Faculty of
Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 2Department of Paediatric Neurology, 2nd Faculty
of Medicine, Charles University in Prague a, Prague 5, Czech
Republic, 3Department of Biology and Medical Genetics, 2nd
Faculty of Medicine, Charles University in Prague a, Prague
5, Czech Republic
Introduction: Epileptic encephalopathies are severe, early
onset disorders associated with global developmental delay, D. Stanek1, P. Lassuthova1, L. Sedlackova1, J. Neupauerova1,
K. Sterbova2, M. Vlckova3, P. P09.153A Salina: None. F. Lombardo: None. G. Salzano: None. F. De Luca: None. G. D'Annunzio: None. C. Bianchini: None. A. Vetro: None. D. Mei: None. E. Cellini: None. D. Pucatti: None. D. Rutigliano: None. S. Virdò: None. D. De Vita: None. V. Cetica: None. C. Mandorlini: None. C. Barba: None. T. Pisano: None. F. Mari: None. S. Chiari: None. M. Montomoli: None. V. Doccini: None. M. Donati: None. C. Marini: None. E. Parrini: None. R. Guerrini: None. P09.153A In
patients with NDDs, analyzed with whole exome sequen-
cing (WES), the molecular yield ranges from 25 to 57%. disability. Next generation sequencing increased to over
450 the number of genes associated with NDDs, underling
their high genetic and phenotypic heterogeneity. Such a
high number of genes as well as genetic and phenotypic
heterogeneity make panel based diagnostics unsuitable. In
patients with NDDs, analyzed with whole exome sequen-
cing (WES), the molecular yield ranges from 25 to 57%. Materials and methods: We performed WES in 48 trios
with NDDs who were mutation-negative to previous genetic
investigations. Results: We identified pathogenic variants in known
disease-genes in 22 patients (22 out of 48: 46%). In three of
these patients, we expanded the phenotypic spectrum
previously associated with the causative gene. In 9 patients
(9 out of 48: 19%), we identified variants in candidate genes
(not yet reported as disease-causing genes), possibly
explaining clinical symptoms. Finally, in 17 patients (17
out of 48: 35%), we detected variants of unknown
significance that were could not be correlated to the clinical
condition. In about one-third of all patients the analysis
revealed a recessive condition. This was an unexpected
result considering the reported excess of de novo variants in
patients with NDDs. Results: DM and OA have been found in all patients
(100%), DI in 26 (57.7%), D in 27 (60%), renal tract
abnormalities in 10 (22.2%), neuro-psychiatric symptoms in
20 (44.4%), and endocrinopathies in 3 (6.6%). Six patients
(13.3%) have died of whom 5 for respiratory failure and one
for chronic renal failure. We have found 34 different
mutations in WFS1 which were all known except for 2
missense substitutions, c.1523 A>G and c.1514 G>A, both
located in exon 8. In 2/45 patients (4.5%), we have detected
a mutation of WFS1 in only one chromosome. We have
found 23 patients (54%) in group 1 mutations, 10 (23%) in
group 2 and 10 (23%) in group 3. Conclusions:
Although
a
clear
genotype-phenotype
correlation is difficult to establish in WS1, we have found
several correlations between severe phenotypes and the type
of WFS1 mutations. Conclusions: Our results, although obtained in a small
series, indicate WES as a first-line diagnostic option to
provide genetic counseling and facilitate personal medical
care in NDDs without a clear syndromic picture and
characterized by high genetic heterogeneity. L. Rigoli: None. P. Bramanti: None. C. Di Bella: None. C. Aloi: None. A. P09.153A Whole exome sequencing identifies a novel mutation in
ADCY5 in a patient with developmental delay and dystonia C. Bianchini1, A. Vetro1, D. Mei1, E. Cellini1, D. Pucatti1, C. Bianchini1, A. Vetro1, D. Mei1, E. Cellini1, D. Pucatti1, D. Rutigliano1, S. Virdò1, D. De Vita1, V. Cetica1, D. Rutigliano1, S. Virdò1, D. De Vita1, V. Cetica1, B. Cavdarli1, N. Esen1, V. Topcu1, A. Aksoy2, A. Danis3,
B. Anlar4 B. Cavdarli1, N. Esen1, V. Topcu1, A. Aksoy2, A. Danis3,
B. Anlar4 C. Mandorlini1, C. Barba1, T. Pisano1, F. Mari1, S. Chiari1, C. Mandorlini1, C. Barba1, T. Pisano1, F. Mari1, S. Chiari1, M. Montomoli1, V. Doccini1, M. Donati2, C. Marini1, E. Parrini1,
R. Guerrini1 1Department of Medical Genetics, Ankara Numune Education
and Research Hospital, Ankara, Turkey, 2Department of
Pediatric Neurology, Dr. Sami Ulus Research and Training
Hospital of Women's and Children's Health and Diseases,
Ankara, Turkey, 3Department of Pediatric Neurology, Dr. Sami
Ulus Research and Training Hospital of Women's and
Children's Health and Diseases,, Ankara, Turkey, 4Department
of Pediatric Neurology, Hacettepe University Faculty of
Medicine, Ankara, Turkey 1Pediatric Neurology Unit, Neurogenetics and Neurobiology
Laboratories, Neuroscience Department, A. Meyer Pediatric
Hospital, University of Florence, Firenze, Italy, 2Pediatric
Neurology Unit, Neuroscience Department, A. Meyer Pediatric
Hospital, University of Florence, Firenze, Italy 1Pediatric Neurology Unit, Neurogenetics and Neurobiology
Laboratories, Neuroscience Department, A. Meyer Pediatric
Hospital, University of Florence, Firenze, Italy, 2Pediatric
Neurology Unit, Neuroscience Department, A. Meyer Pediatric
Hospital, University of Florence, Firenze, Italy Introduction: Neurodevelopmental disorders (NDDs) are a
heterogeneous group of neurological phenotypes diagnosed
during early in life and including epilepsy, brain mal-
formations, autism spectrum disorder, and intellectual J. del Picchia 318 Materials and methods: Genomic DNA has been
extracted from 45 Italian WS1 patients (20 males and 25
females) aged 25-45 years. WFS1 exons have been
amplified by PCR and, then, subjected to automatic
sequencing. The mutations have been subdivided in 3
groups according to their predicted functional conse-
quences: Group 1 (complete depletion of wolframin);
Group 2 (milder degradation of WFS1 protein than group
1); Group 3 (compound heterozygous mutations not found
in 1 and 2 groups). disability. Next generation sequencing increased to over
450 the number of genes associated with NDDs, underling
their high genetic and phenotypic heterogeneity. Such a
high number of genes as well as genetic and phenotypic
heterogeneity make panel based diagnostics unsuitable. Messina, Messina, Italy, 3Pediatric Clinic, IRCCS, Istituto G.
Gaslini, Genova, Italy P09.156D Virdò: None. D. De Vita: None. V. Cetica: None. C. Mandorlini: None. C. Barba: None. T. Pisano: None. F. Is microduplication Xp22.31 a possible synergic factor in
MECP2 defect for Rett Syndrome? Is microduplication Xp22.31 a possible synergic factor in
MECP2 defect for Rett Syndrome? Mandorlini: None. C. Barba: None. T. Pisano: None. F. Mari: None. S. Chiari: None. M. Montomoli: None. V. Doccini: None M. Donati: None C. Marini: None E. Mari: None. S. Chiari: None. M. Montomoli: None. V. Doccini: None. M. Donati: None. C. Marini: None. E. Parrini: None. R. Guerrini: None. E. Candelo, D. Ramirez, H. Pachajoa E. Candelo, D. Ramirez, H. Pachajoa Fenotype genotype correlations in Italian patients with
Wolfram Syndrome 1 Fenotype genotype correlations in Italian patients with
Wolfram Syndrome 1 Introduction: Rett syndrome (RS) is a neurodevelopmental
infant disease characterized by early normal psychomotor
development followed by a regression in the acquisition of
developmental stages. In the majority of cases, it leads to a
sporadic mutation in the MECP2 gene, which is located on
the X chromosome. However, this syndrome has also been
associated with microdeletion, gene translocations, and
other gene mutations. Here we described a classic Rett
syndrome associated with Xp22.31 microduplication. L. Rigoli1, P. Bramanti2, C. Di Bella1, C. Aloi3, A. Salina3,
F. Lombardo1, G. Salzano1, F. De Luca1, G. D'Annunzio3
1Department of Human Pathology, University of Messina,
Messina, Italy, 2IRCCS Centro Neurolesi "Bonino-Pulejo",
Messina, Messina, Italy, 3Pediatric Clinic, IRCCS, Istituto G. Gaslini, Genova, Italy L. Rigoli1, P. Bramanti2, C. Di Bella1, C. Aloi3, A. Salina3,
F. Lombardo1, G. Salzano1, F. De Luca1, G. D'Annunzio3 F. Lombardo1, G. Salzano1, F. De Luca1, G. D'Annunzio3 1Department of Human Pathology, University of Messina,
Messina, Italy, 2IRCCS Centro Neurolesi "Bonino-Pulejo",
Messina, Messina, Italy, 3Pediatric Clinic, IRCCS, Istituto G. Gaslini, Genova, Italy 1Department of Human Pathology, University of Messina,
Messina, Italy, 2IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Messina, Italy, 3Pediatric Clinic, IRCCS, Istituto G. Gaslini, Genova, Italy Materials and Method: A Colombian patient who is 12
years old and is a product of quarter gestation, non-
consanguinity parents. Initially, her neurological develop-
ment was normal until 11 months, when she started a
seizure syndrome with difficult treatment and regression in
the
acquisition
of
developmental
stages
(especially Introduction: Wolfram syndrome 1 (WS1) is a rare, auto-
somal recessive, neurodegenerative, and progressive disease
characterized by diabetes mellitus (DM), bilateral optic
atrophy (OA), diabetes insipidus (DI), deafness (D), renal
tract or neuropsychiatric abnormalities. P10.01A
Whole exome sequencing of patients with amyotrophic
lateral sclerosis C. Eitan1, T. Olender1, E. Barkan1, R. van der Spek2, S. Pulit2,
E. Chapnik1, K. R. van Eijk2, L. Kool2, J. van Vugt2,
W. Sproviero3, D. Rotschild1, O. Weissbrod1, E. Segal1, C. E. Shaw3, L. H. van den Berg2, A. Al-Chalabi3, J. E. Landers4,
R. H. Brown Jr4, J. H. Veldink2, E. Hornstein1 K. Tripolszki1, D. Nagy1, Z. F. Nagy1, J. I. Engelhardt2,
P. Klivényi2, M. Széll1 1The Weizmann Instiute of Science, Rehovot, Israel, 2University
Medical Center Utrecht, Utrecht, Netherlands, 3King's
College, London, United Kingdom, 4University of
Massachusetts Medical School, Worcester, MA, United States 1The Weizmann Instiute of Science, Rehovot, Israel, 2University
Medical Center Utrecht, Utrecht, Netherlands, 3King's
College, London, United Kingdom, 4University of
Massachusetts Medical School, Worcester, MA, United States 1University of Szeged, Department of Medical Genetics,
Szeged, Hungary, 2University of Szeged, Department of
Neurology, Szeged, Hungary 1University of Szeged, Department of Medical Genetics,
Szeged, Hungary, 2University of Szeged, Department of
Neurology, Szeged, Hungary Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal
neurodegenerative disease. Genetic factors play a key role
in ALS and uncovering its genetic background may bring us
closer to fully understand its pathomechanism, therefore the
aim was to identify rare damaging variants in major and
minor genes involved in pathways annotated to ALS. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegen-
erative disease of the human motor neuron system. Although the disease etiology remains unclear, ~25 genes
with ALS underlying protein-coding mutations have been
discovered. The mutations explain mostly familial cases,
and imply genetics as an important driver of sporadic cases
too. Recent studies demonstrated dysregulation of micro-
RNAs (miRNAs) in ALS patients, suggesting a role of
miRNAs in ALS pathogenesis. However, systematic search
for disease-causing variants in miRNA genes was yet not
performed. Here, we screened whole-genome sequencing
data from 4281 ALS sporadic patients and 1838 controls,
and explored genomic regions of 1,872 miRNAs genes and
3′ untranslated regions (3′UTRs) of 295 ALS and miRNA-
related genes. The data was generated by Project MinE
consortium and analyzed under collaboration. First, we
developed a pipeline for functional annotation of 3’UTR
and miRNA variants and called qualified variants. The Patients
and
methods:
The
investigated
sporadic
patients fulfilled the revised El Escorial criteria for ALS. Whole exome sequencing of 21 Hungarian patients affected
by ALS was carried out. The patients were prescreened for
C9ORF72 repeat expansion, SOD1, ANG, FUS, SETX,
TARDBP and UBQLN2 genes. 319 Abstracts from the 51st European Society of Human Genetics Conference: Posters language with motor and verbal stereotypes, hyperactivity,
and autistic spectrum disorder). Comparative genomic
hybridization
array-CGH
(750K)
and
MECP2
genes
sequence were performed. Results: Exome sequencing revealed a novel non-
synonymous variant (T338I) in NEFH gene that encodes
neurofilament heavy polypeptide; a previously described
nonsense mutation (G1177X) in the alsin (ALS2) gene that
leads to premature stop codon and may affect endosomal
and vesicle transport; and finally a recurrent variant
(R261H) in the NEK1 gene, encoding NIMA related
kinase-1, that has recently been associated with ALS in
the Caucasian population. Results:
Array-CGH
detected
Xp22.31
duplication
(6866889-8115153) with a size of 1.248Mb. Due to clinical
criteria of RS a MECP2 gene sequence was performed,
which showed de novo pathogenic variant C.338C>G (p. Pro113Arg) associated with this disease. Conclusion: RS makes a part of the intellectual disability,
developmental delay, and autism. These characteristics are
associated with copy number variations (CNVs) in the X
chromosome such as Xp22. 31 microduplication. This is the
first case report in the literature that shows a CNV and
MECP2 pathogenic mutation simultaneously in the context
of RS. We propose that both DNA alteration might have a
synergy effect and could lead to variable expressivity of
phenotype. Conclusion: Disease causing variants have been detected
in approximately 28% (3/21) of this sporadic cohort. Our
study contributed to the better understanding of the genetic
background of the disorder and indicated that complex
approaches are needed to understand the genetic hetero-
geneity of this disease. Conclusion: Disease causing variants have been detected
in approximately 28% (3/21) of this sporadic cohort. Our
study contributed to the better understanding of the genetic
background of the disorder and indicated that complex
approaches are needed to understand the genetic hetero-
geneity of this disease. Funding: Hungarian Brain Research Program (Grant No. KTIA_13_NAP-A-II/15) K. Tripolszki: None. D. Nagy: None. Z.F. Nagy: None. J.I. Engelhardt: None. P. Klivényi: None. M. Széll: None. K. Tripolszki: None. D. Nagy: None. Z.F. Nagy: None. J.I. Engelhardt: None. P. Klivényi: None. M. Széll: None. E. Candelo:
None. D. Ramirez:
None. H. Pachajoa: None. E. Candelo:
None. D. Ramirez:
None. H. Pachajoa: None. P10.01A
Whole exome sequencing of patients with amyotrophic
lateral sclerosis Exome sequencing was
performed using Illumina NextSeq sequencer and data
analysis was performed according to the best practices to
identify single nucleotide variants and small insertions/
deletions. The detected variants were confirmed by Sanger
sequencing. Patients
and
methods:
The
investigated
sporadic
patients fulfilled the revised El Escorial criteria for ALS. Whole exome sequencing of 21 Hungarian patients affected
by ALS was carried out. The patients were prescreened for
C9ORF72 repeat expansion, SOD1, ANG, FUS, SETX,
TARDBP and UBQLN2 genes. Exome sequencing was
performed using Illumina NextSeq sequencer and data
analysis was performed according to the best practices to
identify single nucleotide variants and small insertions/
deletions. The detected variants were confirmed by Sanger
sequencing. 320 J. del Picchia period. Through the exploration of these two objectives,
necessary steps were taken to further the application of
C9orf72 yeast models in ALS research. S. Kim: None. pipeline predicts loss and gain of 3´UTR miRNA binding
sites and assigns miRNA variants with decreased patho-
genicity score based on location in the seed, mature miRNA
or precursor, respectively. Region-based rare variant asso-
ciation uncovered a significant association with a relevant
inflammatory gene, suggesting that its tight regulation is
critical. The association signal was replicated using >60,000
controls from a different cohort. This is the first report of a
rare protective mutations in ALS and the first association
study that implies noncoding regulation by miRNAs. The
work emphasizes the ability to interrogate whole-genome
sequencing for discovery of new non-coding genetic
mechanisms and suggests neuroprotective immunomodula-
tory targets for therapy development. period. Through the exploration of these two objectives,
necessary steps were taken to further the application of
C9orf72 yeast models in ALS research. S. Kim: None. S. Kim: None. Biallelic mutation in CHP1 causes human autosomal
recessive ataxia by impairing NHE1 membrane targeting Biallelic mutation in CHP1 causes human autosomal
recessive ataxia by impairing NHE1 membrane targeting N. Mendoza Ferreira1, M. Coutelier2,3, E. Janzen1,
S. Hosseinibarkooie1, H. Löhr4, S. Schneider1, J. Milbradt1,
M. Karakaya1, M. Riessland5,1, C. Pichlo6, L. Torres-Benito1,
A. Singleton7, S. Zuchner8, A. Brice2,9, A. Durr2,9,
M. Hammerschmidt4, G. Stevanin2,3,9, B. Wirth1 C. Eitan: None. T. Olender: None. E. Barkan: None. R. van der Spek: None. S. Pulit: None. E. Chapnik:
None. K.R. van Eijk: None. L. Kool: None. J. van Vugt:
None. W. Sproviero: None. D. Rotschild: None. O. Weissbrod: None. E. Segal: None. C.E. Shaw: None. L. H. van den Berg: None. A. Al-Chalabi: None. J.E. Landers: None. R.H. Brown Jr: None. J.H. Veldink:
None. E. Hornstein: None. 1Institute for Human Genetics, Centre for Molecular Medicine,
Center for Rare Diseases. University of Cologne, Cologne,
Germany, 2Institut du Cerveau et de la Moelle épinière
INSERM, Sorbonne Universités, Paris, France, 3Ecole
Pratique des Hautes Etudes, PSL Research University, Paris,
France, 4Institute for Zoology, Developmental Biology,
University of Cologne, Cologne, Germany, 5Laboratory of
Molecular and Cellular Neuroscience, The Rockefeller
University, New York, NY, United States, 6Institute of
Biochemistry, University of Cologne, Cologne, Germany,
7Laboratory of Neurogenetics, National Institute on Aging,
National Institutes of Health, Bethesda, MD, United States,
8John P. Hussman Institute for Human Genomics, University of
Miami, Miami, FL, United States, 9APHP, Hôpital de la Pitié-
Salpêtrière, Centre de réference de neurogénétique, Paris,
France S. Kim S. Kim Not Applicable, Concord, NH, United States France The GGGGCC hexanucleotide repeat expansion located in
the first intron of the C9orf72 gene is the most common
genetic cause for amyotrophic lateral sclerosis (ALS) as
well as frontotemporal dementia (FTD). The repeat expan-
sion is hypothesized to cause repeat-associated translation
(RAN-translation), which results in toxicity by aggregation
of dipeptide repeat proteins (DRPs). Though there have
been many studies focusing on the toxicity of RAN-
translated DPRs, the specific factors that contribute to RAN
translation have not yet been identified. This study employs
yeast models, which share high gene conservation with
humans, to examine the mechanisms of the C9orf72
mutation in ALS. In particular, this study focused on the
cloning of 149 repeat expansions of C9orf72 (C9149R). 149
repeat expansions ligated into yeast-expression vectors,
p416 GAL1 and GPD, were transformed in Stbl3 E.coli and
subsequently tested for stability of the 149R size. Further-
more, this study also observed stability of repeat expansions
in yeast over various time periods. The sizes of 2 repeat
expansions (C92R) and 40 repeat expansions (C940R) of
C9orf72 in yeast were examined to observe how much
shrinkage the expansions had undergone over each time Autosomal recessive cerebellar ataxias (ARCAs) comprise a
heterogeneous group of neurodegenerative disorders that
affect
the
cerebellum,
brain
stem
and
spinal
cord. Approximately 40% of the ARCA-affected patients remain
genetically unresolved. Following a combination of WES and linkage analysis,
we identified a biallelic 3-bp deletion (p.K19del) in CHP1
(Calcineurin-like EF-hand protein-1) that co-segregates
with motor neuropathy, cerebellar atrophy and spastic
paraparesis in two siblings of a consanguineous family. Focused screening for CHP1 variants in two cohorts
(ARCA: N=319 and NeurOmics: N=657) and Gene-
Matcher interrogation did not yield additional variants, thus
revealing the scarcity of CHP1 mutations. CHP1 plays a
crucial role in pH regulation and ion homeostasis, by
controlling
the
function
of
the
Sodium/Hydrogen
Exchanger-1 (NHE1, encoded by SLC19A1). Reduced
CHP1 expression as well as NHE1 depletion cause Purkinje
cell degeneration and ataxia in mouse. Moreover, loss-of-
function mutation in NHE1 causes ataxia in human. Abstracts from the 51st European Society of Human Genetics Conference: Posters 321 Here we demonstrate that mutant CHP1 fails to integrate
into functional protein complexes and is prone to aggrega-
tion, thereby leading to diminished levels of soluble CHP1
and reduced membrane targeting of NHE1. Rome, Italy, 3Child Neurology and Psychiatry Unit, Tor
Vergata University, Rome, Italy Introduction: ATP1A3 gene encodes for the alpha-3 cat-
alytic subunit of the Na+/K+ ATPase transmembrane ion
pump. Gene mutations are associated with alternating
hemiplegia of childhood, rapid-onset dystonia with par-
kinsonism or dystonia 12, cerebellar ataxia, areflexia, pes
cavus, optic atrophy and sensorineural hearing loss syn-
drome and catastrophic early life epilepsy. Methods: medical charts and videos of patients were
retrospectively evaluated. All patients were genetically
proven for ATP1A3 gene mutations. Results: Five patients with “atypical” phenotype were
selected. Three patients were sporadic, while two were
directly related. Patient one had an early-onset epileptic
encephalopathy and later developed paroxysmal episodes of
non-epileptic origin, monocular nystagmus and episodes of
apnoea. He had a multi drug resistance epilepsy associated
to developmental delay. Patients 2 and 3 represent a familial
case. The proband developed at 18 months dysmetria,
hypotonia and upper limbs tremor and lost the standing and
sitting position. Her mother had an episode of hypotonia
and seizures at the same age and after 5 years developed
generalized
dystonia. Patient
four
initially
presented
episodes of intermittent flaccidity and bulbar symptoms
and then developed sudden onset of severe generalized
dystonia, hypothonia, bradykinesia, and ataxic gait. Patient
five exhibited an early onset infantile epilepsy with episodes
of non-epileptic origin of hemiparesis. After several years,
she abruptly developed a RDP phenotype and recently
psychotic symptoms and anorexic behaviour associated. N. Mendoza Ferreira: E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant;
N. Mendoza-Ferreira
co-holds
patent
17172826.4-1401 (European patent office) for Calcineurin
B homologous protein 1 inhibitors and therapeutic and non-
therapeutic uses thereof.. M. Coutelier: None. E. Janzen:
E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; E. Janzen co-holds patent
17172826.4-1401 (European patent office) for Calcineurin
B homologous protein 1 inhibitors and therapeutic and non-
therapeutic uses thereof. S. Hosseinibarkooie: E. Owner-
ship
Interest
(stock,
stock
options,
patent
or
other
intellectual property); Significant; SM. Hosseinibarkooie
co-holds patent 17172826.4-1401 (European patent office)
for Calcineurin B homologous protein 1 inhibitors and
therapeutic and non-therapeutic uses thereof.Seyyedmoh-
sen. H. Löhr: None. S. Schneider: None. J. Milbradt:
None. M. Karakaya: None. M. Riessland: None. C. Pichlo: None. L. Torres-Benito: None. A. Singleton:
None. S. Zuchner: None. A. Brice: None. A. Durr: None. M. Hammerschmidt: None. G. Stevanin: None. B. Wirth: E. Ownership Interest (stock, stock options, patent
or other intellectual property); Significant; B. Rome, Italy, 3Child Neurology and Psychiatry Unit, Tor
Vergata University, Rome, Italy Wirth co-
holds patent 17172826.4-1401 (European patent office) for
Calcineurin
B
homologous
protein
1
inhibitors
and
therapeutic and non-therapeutic uses thereof.. Discussion: here we present five different cases of
ATP1A3 mutations showing intermediate and overlapping
phenotypes compared to the classic ones. This series expand
the continuum phenotype spectrum of ATP1A3. L. Travaglini: None. F. Graziola: None. E. Bertini:
None. M. Valeriani: None. P. Curatolo: None. F. Vigevano: None. A. Capuano: None. L. Travaglini: None. F. Graziola: None. E. Bertini:
None. M. Valeriani: None. P. Curatolo: None. F. Vigevano: None. A. Capuano: None. 1Unit of Neuromuscular and Neurodegnerative Disorders,
Bambino Gesù Children’s Hospital - IRCCS, Rome, Italy,
2Neurology Unit, Bambino Gesù Children’s Hospital - IRCCS, Expanding the histopathological spectrum of CFL2-related
myopathies Expanding the histopathological spectrum of CFL2-related
myopathies 1Neuromuscular Diseases, Genetics and Rare Diseases
Research Division, Bambino Gesù Children’s Hospital, Rome,
Italy, 2Department of Neurosciences, Rehabilitation,
Ophthalmology, Genetics, Maternal and Child Health, L. Travaglini1, F. Graziola2,3, E. Bertini1, M. Valeriani2,
P. Curatolo2, F. Vigevano2, A. Capuano2 France Moreover, we
show
that
morpholino-mediated
chp1
knockdown
in
zebrafish resembles the phenotype of ARCA-affected
humans, leading to spastic movements, cerebellar hypopla-
sia and motor axon abnormalities. These defects were
ameliorated by co-injection with WT, but not mutant,
human CHP1 mRNA. Collectively, our results identified
CHP1 as an ataxia-causative gene in humans, further
expanding the spectrum of ARCA-associated loci, and
corroborate NHE1 mistargeting as a key event underlying
neuronal degeneration in the context of inherited cerebellar
ataxias. Rome, Italy, 3Child Neurology and Psychiatry Unit, Tor
Vergata University, Rome, Italy P10.09A
A report of a family of intermediate Charcot-Marie-Tooth
disease with concomitant mutations in the GNB4 and
DNM2 genes B. Burnyte1,2, A. Morkuniene1,2, L. Ambrozaityte1,2,
V. Regelskyte3, A. Vaitkevicius4,5, V. Kucinskas1, A. Utkus1,2 B. Burnyte1,2, A. Morkuniene1,2, L. Ambrozaityte1,2,
V. Regelskyte3, A. Vaitkevicius4,5, V. Kucinskas1, A. Utkus1,2 1Institute of Biomedical Sciences, Faculty of Medicine, Vilnius
University, Vilnius, Lithuania, 2Centre for Medical Genetics,
Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 3Faculty of Medicine, Vilnius University, Vilnius,
Lithuania, 4Institute of Clinical Medicine, Faculty of Medicine,
Vilnius University, Vilnius, Lithuania, 5Center of Neurology,
Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, Vilnius, Lithuania Introduction: Congenital
myopathy (CM) caused
by
mutation in cofilin-2 gene (CFL2) is a rare neuromuscular
disorder. The few reported cases show phenotypic hetero-
geneity ranging from early onset and rapid progressive
forms to milder myopathy characterized by slow pro-
gressive limb girdle and axial muscles weakness. Muscle
histology shows features of nemaline or myofibrillar myo-
pathy or the coexistence of both histopathological changes. We describe three new cases, from two unrelated families,
of severe CM related to novel loss-of-function mutations in
CFL2. Charcot-Marie-Tooth disease (CMT) is the most common
inherited neuromuscular disorder with an estimated pre-
valence of 1 in 2500 people. Hallmarks include symmetric
foot deformities, slowly progressive weakness and wasting
in the distal parts of upper and lower limbs, and length-
dependent sensory loss. Positive symptoms such as para-
esthesias and pain may occur. Hereditary neuropathies are
ideal candidates for diagnosis by Next-generation sequen-
cing (NGS) approaches because similar phenotype is caused
by variants of different genes. We report the unusual
occurrence of CMT, caused by mutations c.847C>T (p. Arg283Cys) in GNB4 and c.1609G>A (p.Gly537Ser) in
DNM2, in a dominant geneaology. The phenotype of all
affected family members was rather uniform with symp-
toms starting in the lower limbs. Initial signs were foot
deformity and gait abnormalities in early childhood. Nevertheless severity of progressive distal weakness of
upper limbs, postural tremor and hand deformities were
different in some of the affected family members. The
digenic effect of two pathogenic variants in different genes
may modulate the phenotype, since both gene products are
involved in endosomal sorting and cell signalling, possibly
interacting in similar pathways. This study and previously
reported cases of CMT caused by concomitant gene
alterations is a notice to look for several causative mutations
in
families
having
phenotypic
variability
or
unusual
expression. ATP1A3 related disease: a series of new mutations
expanding clinical phenotype F. Fattori1, C. Fiorillo2, C. Rodolico3, G. Tasca4, M. Verardo1,
E. Bellacchio5, S. Pizzi5, A. Ciolfi5, G. Fagiolari6, A. Lupica3,
P. Broda2, M. Pedemonte2, M. Moggio6, C. Bruno2,
M. Tartaglia5, E. Bertini1, A. D'Amico1 L. Travaglini1, F. Graziola2,3, E. Bertini1, M. Valeriani2,
P. Curatolo2, F. Vigevano2, A. Capuano2 J. del Picchia 322 University of Genoa, Genoa, Italy, 3Department of Clinical
and Experimental Medicine, University of Messina, Messina,
Italy, 4Istituto di Neurologia, Università Cattolica del Sacro
Cuore, Fondazione Policlinico Universitario "A. Gemelli",
Rome, Italy, 5Molecular Genetics and Functional Genomics,
Genetics and Rare Diseases Research Division, Bambino Gesù
Children’s Hospital, Rome, Italy, 6Neuromuscular and Rare
Disease Unit, Department of Neuroscience, IRCCS Foundation
Ca' Granda Ospedale Maggiore Policlinico, University of
Milan,, Milan, Italy P10.09A
A report of a family of intermediate Charcot-Marie-Tooth
disease with concomitant mutations in the GNB4 and
DNM2 genes Materials and methods: Whole Exome Sequencing and
targeted resequencing using a custom gene panel for
muscular diseases were performed respectively in Patient-
1 and Patient-2. Studies of muscle biopsies were performed
in all patients using standard technique on quadriceps
muscles. Results: Next Generation Sequencing analysis revealed
novel mutations in CFL2: p.(Asp86His), homozygous in
Pt1, and p.(Asp79Tyr) and p.(Ser94LeufsTer6) in com-
pound heterozygosity in Pt2 and in her older brother (Pt3). All babies presented severe neonatal muscle weakness
needing continuous respiratory and nutritional support since
birth. Muscle biopsies showed features consistent of
nemaline
myopathy
with
thin
filament
accumulations
together to myofibrillar changes. Results: Next Generation Sequencing analysis revealed
novel mutations in CFL2: p.(Asp86His), homozygous in
Pt1, and p.(Asp79Tyr) and p.(Ser94LeufsTer6) in com-
pound heterozygosity in Pt2 and in her older brother (Pt3). All babies presented severe neonatal muscle weakness
needing continuous respiratory and nutritional support since
birth. Muscle biopsies showed features consistent of
nemaline
myopathy
with
thin
filament
accumulations
together to myofibrillar changes. Conclusions: Muscle biopsies in our patients showed
features
evocative
of
the
histopathological
findings
observed in Cfl2-/- knockout mouse model. Structural
modeling analysis supports the pathogenicity of the three
CFL2 mutations and indicates that the mutated residues are
involved in correct folding of cofilin-2 confirming that the
activity of the protein might be important for the postnatal
maintenance of sarcomeric structures. Our report expands
the clinical and histopathological spectrum of CFL2-related
myopathies. B. Burnyte: None. A. Morkuniene: None. L. Ambro-
zaityte: None. V. Regelskyte: None. A. Vaitkevicius:
None. V. Kucinskas: None. A. Utkus: None. B. Burnyte: None. A. Morkuniene: None. L. Ambro-
zaityte: None. V. Regelskyte: None. A. Vaitkevicius:
None. V. Kucinskas: None. A. Utkus: None. F. Fattori: None. C. Fiorillo: None. C. Rodolico: None. G. Tasca: None. M. Verardo: None. E. Bellacchio: None. S. Pizzi: None. A. Ciolfi: None. G. Fagiolari: None. A. Lupica: None. P. Broda: None. M. Pedemonte: None. M. Moggio: None. C. Bruno: None. M. Tartaglia: None. E. Bertini: None. A. D'Amico: None. Comprehensive copy number variant profiling in 234
diagnosis-resistant myopathic patients P10.11C Abstracts from the 51st European Society of Human Genetics Conference: Posters 323 T. Giugliano1, M. Savarese2, A. Garofalo1, A. Torella1,3,
L. Politano4, G. Piluso1, V. Nigro1,3 1Health Sciences Univesity, Kanuni Sultan Suleyman Research
and Training Hospital, Department of Medical Genetics,
Istanbul, Turkey, 2Istanbul University, Istanbul Medical
Faculty, Department of Medical Genetics, Istanbul, Turkey 1Laboratorio di Genetica Medica, Dipartimento di Medicina di
Precisione, Università degli Studi della Campania “Luigi
Vanvitelli”, Napoli, Italy, 2Folkhälsan Institute of Genetics,
Helsinki, Finland, 3TIGEM (Telethon Institute of Genetics and
Medicine), Pozzuoli, Italy, 4Cardiomiologia e Genetica
Medica, Dipartimento di Medicina Sperimentale, Università
degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy 1Laboratorio di Genetica Medica, Dipartimento di Medicina di
Precisione, Università degli Studi della Campania “Luigi
Vanvitelli”, Napoli, Italy, 2Folkhälsan Institute of Genetics,
Helsinki, Finland, 3TIGEM (Telethon Institute of Genetics and
Medicine), Pozzuoli, Italy, 4Cardiomiologia e Genetica
Medica, Dipartimento di Medicina Sperimentale, Università
degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy Introduction: Sohar-Crisponi Syndrome with the other
name cold induced sweating syndrome was first recognized
by Sohar in 1978 and then renamed in 1996 by Crisponi. Today it is defined as an autosomal recessive disorder
characterized by muscular spasms of facial muscles, tetany,
difficulty in swallowing and excess salivation, attacks of
fever during first two years of life. During childhood tetany
and fever attacks ceases but cold induced sweating and
thermodysregulation follows. In most patients typical facial
features, camptodactyly with tapering fingers and kyphos-
coliosis are also present. Most of the patients have muta-
tions in CRLF1 gene, in recent years two additional genes
are defined: CLCF1 and KLHL7. Introduction: Sohar-Crisponi Syndrome with the other
name cold induced sweating syndrome was first recognized
by Sohar in 1978 and then renamed in 1996 by Crisponi. Today it is defined as an autosomal recessive disorder
characterized by muscular spasms of facial muscles, tetany,
difficulty in swallowing and excess salivation, attacks of
fever during first two years of life. During childhood tetany
and fever attacks ceases but cold induced sweating and
thermodysregulation follows. In most patients typical facial
features, camptodactyly with tapering fingers and kyphos-
coliosis are also present. Most of the patients have muta-
tions in CRLF1 gene, in recent years two additional genes
are defined: CLCF1 and KLHL7. Next generation sequencing (NGS) has led to an increase in
the diagnosis of non-specific skeletal muscle disorders with
a detection rate of single nucleotide variants or small ins/
dels of 40%-60%. P10.11C Mutations in unknown genes, multi-
factorial or polygenic conditions, or elusive variants such as
deep intronic mutations, variants in regulatory elements,
trinucleotide repeat expansions or copy number variants
(CNVs) may occur in the patients remaining undiagnosed. An extensive NGS study of 504 genetically undetermined
patients with clinical diagnosis of muscular dystrophies,
congenital myopathies or other conditions affecting muscles
was performed, identifying putative causative mutations in
218/504 cases. We recruited 234/286 unsolved patients to
study the impact of CNVs in skeletal muscle disorders and
potentially to improve the diagnostic rate. All patients were
analyzed by Motor Chip, a custom CGH-array to identify
deletions and duplications in neuromuscular disorders. We
found non-polymorphic CNVs in 22 patients (9.4%). In 12
patients (5.1%), the identified CNVs were considered
responsible for the observed phenotype. Other 10 patients
(4.3%)
had
CNVs
of
uncertain
significance
(VUS). Although these VUS may not act as primary disease drivers,
we cannot exclude that some of them may act as modifiers,
contributing to the observed phenotype. Our study has
allowed the genetic diagnosis in unsolved patients and
identification of previously undescribed rearrangements in
muscle genes. It confirms that deletions and duplications
account for 5-10% of patients affected by a skeletal muscle
disorder without a molecular diagnosis, explaining a not
exiguous number of unsolved cases. Materials and Methods: We collected the datae of 6
families with 7 affected members. All were evaluated for
neurological, metabolic and ophthalmologic pathologies. CRLF1 gene analysis was done in all families. Results: Four of the patients were diagnosed during first
months of life, two were one month old, the oldest one was
10 years old during diagnosis. All have typical face,
camptodactyly, abnormal finger and hand morphology and
difficulty in swallowing. Fever attacks were present in
infants. Thermodysregulation, and hyperhydrosis was pre-
sent in older patients. All the patients had CRLF1
mutations. Two of the mutations were novel. Conclusion: As new cases as our series with novel and
known mutations are described, more information will be
available about this syndrome and this will help clinicians
to diagnose and follow the patients more efficiently. E. Yilmaz Gulec: None. A. Gezdirici: None. A. Ayaz:
None. U. Altunoglu: None. Z.O. Uyguner: None. Deletion & duplication mutations in Duchenne muscular
dystrophy in southern Iranian children Deletion & duplication mutations in Duchenne muscular
dystrophy in southern Iranian children Deletion & duplication mutations in Duchenne muscular
dystrophy in southern Iranian children A. Saberi1,2, G. Shariati1,2, M. Mohammadi anaei2,
N. Abdorasouli2, F. Nanvazed2 T. Giugliano: None. M. Savarese: None. A. Garofalo:
None. A. Torella: None. L. Politano: None. G. Piluso:
None. V. Nigro: None. 1Ahvaz Jundishpour university of medical sciences, Ahvaz,
Iran, Islamic Republic of, 2Narges Genetic & PND Lab, Ahvaz,
Iran, Islamic Republic of E. Yilmaz Gulec1, A. Gezdirici1, A. Ayaz1, U. Altunoglu2, Z. O.
2 A. Saberi1,2, G. Shariati1,2, M. Mohammadi anaei2,
N. Abdorasouli2, F. Nanvazed2 E. Yilmaz Gulec1, A. Gezdirici1, A. Ayaz1, U. Altunoglu2, Z. O.
Uyguner2 P10.12D Crisponi / Cold-induced sweating syndrome: Seven new
cases and two novel mutations Duchenne muscular dystrophy (DMD) and Becker muscular
dystrophy (BMD) are X-linked disorders caused by muta-
tion within the dystrophin gene. Our study has identified 28
Iranian
families collected
from
Narges Genetic Lab,
southwest of Iran, Ahvaz. All cases were subjected to E. Yilmaz Gulec1, A. Gezdirici1, A. Ayaz1, U. Altunoglu2, Z. O. U
2 J. del Picchia 324 complete clinical evaluation pedigree analysis, electro-
myography studies, estimation of serum creatin phospho-
kinase (CPK) level and DNA analysis. Sample's DNA was
analyzed by multiplex ligation-dependent probe amplifica-
tion (MLPA). In this study deletion rate was 75% (21/28)
and was more frequent in the distal end exons whereas
duplication rate was 25% (7/28) and it was more frequent at
proximal exons. Majority of the deletion (11/21, 52%) were
located on the distal hot spot region that encompasses exons
45-55 and 33% of the deletion (7/21) were located at the
proximal hot spot region (exon 1-16). Majority of exon
duplication 71.5% (5/7) located at the proximal hot spot
region (exon 1-16) and 28.5% (2/7) located at the distal hot
spot region (exon 50-62). Single exon deletions were pre-
sent in 8/28 families (28.5%) with the most common in
exon 45 (2/21, 9.5%). One family had a de novo single exon
deletion (exon 51) and whole dystrophin gene was deleted
in only one family. This finding indicate that as with
deletions, duplications occur nonrandomly but with a dra-
matically different distribution. Duplication frequency is
highest near the 5' end of the gene. The breakpoint is in intron2 in case2. The insertion is an
inverted duplication of exon5-7 followed by a duplication
of exon1-2 started further upstream of the DMD gene. Thus,
their DMD reading-frame would be disrupted. Both CGRs
involved microhomology and small insertions at the
breakpoints. In Case1, SNP sequencing results indicated
that the de novo duplication mutation arose in the allele that
originated from the grandfather. Conclusions: This study has report a method to uncover
CGRs in nucleotide level and revealed a novel type of
DMD CGR. Knowing the genetic alteration help to predict
the consequence of the CGRs and provides insight into the
molecular basis of this genomic rearrangement. Y. Xu: None. H. Wang: None. B. Xiao: None. W. Wei:
None. Y. Liu: None. H. Ye: None. X. Ying: None. Y. Chen: None. X. Liu: None. X. Ji: None. P10.12D Y. Sun: None. Duchenne muscular dystrophy: gene therapies in low-
income counties Duchenne muscular dystrophy: gene therapies in low-
income counties K. Hovhannesyan, T. Sargsyan A. Saberi: None. G. Shariati: None. M. Mohammadi
anaei: None. N. Abdorasouli: None. F. Nanvazed: None. Center of medical genetics and primary health care, Yerevan,
Armenia DMD noncontiguous duplications in duplication-normal-
inversed duplication (Dup-Nml-Dup/inv) manner revealed
by targeted sequencing Duchenne muscular dystrophy (DMD; OMIM ref. 310200)
is an X-linked disease that affects 1 in 3600-6000 live male
births. DMD occurs as a result of mutations in the dystro-
phin gene (Xp21.2). Mutations cause a dysfunction in, or
lack of, protein dystrophin that is essential for muscle cell
stability. Milder allelic forms of the disease are intermediate
muscular dystrophy and Becker muscular dystrophy. This is
a descriptive study of 87 patients diagnosed with DMD at
the CMG of Armenia from 2011 to 2017. Patients were
reviewed by neurologist and a clinical geneticist; the clin-
ical diagnosis was established and followed by other sup-
portive investigations. A diagnosis of DMD was confirmed
by molecular testing using multiplex PCR (in Armenia) and
dmd gene sequencing (in France). From 2012 the manage-
ment of DMD patients in Armenia is done according to
DMD Care Considerations Working Group’s recommen-
dations. Here we presented the spectrum of DMD mutations
which were observed among 87 patients with muscular
dystrophies in Armenia. 48% of these cases are with con-
firmed DMD mutations, mainly deletion(s), as well as
duplication(s) and point mutations. Our available data
suggest that up to 40% of DMD patients have genotypes
amenable to exon skipping or Ataluren therapies. Many
promising therapeutic strategies have since been developed,
however availability of those therapies is restricted for low-
income counties such as Armenia. Having no access to
advanced therapies has fatal consequences for the patients, Y. Xu, H. Wang, B. Xiao, W. Wei, Y. Liu, H. Ye, X. Ying,
Y. Chen, X. Liu, X. Ji, Y. Sun Xin Hua Hospital, Shanghai Jiao Tong University School of
Medicine, Shanghai, China Xin Hua Hospital, Shanghai Jiao Tong University School of
Medicine, Shanghai, China A. Ferlini1, E. O'Rourke2, M. Pastore3, A. Martin4 V. Ilinsky1,2, A. Krasnenko2, E. Okuneva2, K. Tsukanov2, P. Shatalov3,2, I. Fedonyuk4, E. Ilina4, S. Artemyeva3 1University of Ferrara, Ferrara, Italy, 2Sarepta Therapeutics,
Inc., Cambridge, MA, United States, 3Nationwide Children's
Hospital, Columbus, OH, United States, 4Parent Project
Muscular Dystrophy, Hackensack, NJ, United States 1Institute of Biomedical Chemistry, Moscow, Russian
Federation, 2Genotek Ltd., Moscow, Russian Federation, 3The
Research and Clinical Institute for Pediatrics named after
Academician Yuri Veltischev of the Pirogov Russian National
Research Medical University of the Russian Ministry of Health,
Moscow, Russian Federation, 4Department of
Psychoneurology No.2, Russian Children's Clinical Hospital,
Ministry of Health Russian Federation, Moscow, Russian
Federation Introduction: Mutation-specific therapies for Duchenne
muscular dystrophy (DMD) are in clinical trials worldwide. More than 60% of DMD patients have DMD gene muta-
tions amenable to two current approaches: stop codon read-
through and single exon skipping. Methods: We identified gaps in DMD genotyping and
examined potential solutions for genetic counselors to help
overcome barriers in determining patient eligibility for
mutation-specific therapies. Mutations in DYNC1H1 gene cause several autosomal
dominant diseases including type 20 Charcot-Marie-Tooth,
type
13
mental
retardation
and
lower
extremity-
predominant spinal muscular atrophy 1. These diseases
have several common clinical symptoms. DYNC1H1 gene
encodes a large (over 530 kD) crucial subunit of cyto-
plasmic dynein complex, a cytoskeleton motor. Results: Access to genetic testing is one barrier. A survey
of 27 physicians who managed >2,000 DMD patients
showed substantial variability in genotyped patients (≈25%-
100%). In Europe, multiplex ligation-dependent probe
amplification is widely adopted, while availability of
sequencing procedures is nonhomogenous, especially in
Eastern countries. Awareness of financially viable genetic
testing
options
is
essential
(eg,
International
DMD,
University of Ferrara [Europe]; Decode Duchenne [USA,
Canada]. Accurate, unequivocal mutation interpretation
presents a second barrier. While nonsense mutations are
amenable to stop codon read-through, other DMD deletion
mutations vary in amenability to exon skipping, depending
on the exons involved. To direct patients to appropriate
therapy, clinicians must discern if a mutation is appropriate
for exon skipping and amenable to a particular exon-
skipping therapeutic. This pinpoints the importance of
correct genotyping and genetic counselor involvement in
mutation interpretation. We did exome sequencing of 4 blood samples using
Agilent FocusedExome enrichment system and Illumina
HiSeq2500. For variant calling and pathogenicity scoring
we followed ACMG guidelines. We also confirmed disease-
causing mutations by Sanger sequencing. Xin Hua Hospital, Shanghai Jiao Tong University School of
Medicine, Shanghai, China Introduction: Complex Genomic Rearrangements(CGRs)
have been demonstrated in DMD. However, few CGRs in
DMD have been described at the nucleotide level thus
difficult to predict the consequence of the CGRs. Materials and methods: Case1, a DMD patient, harbors
duplications of exon53-55 and exon57-79 in DMD gene. Case2 is a female with growth retardation and craniosy-
nostosis. SNP array identified a duplication fragment
included DMD gene. MLPA revealed the patient had
duplications of Dp427c, exon1-2 and exon5-7. They were
sequenced by whole DMD target sequencing. Breakpoints
of the CGRs were detected by split-read method to
reconstruct the structural changes of the gene and to
investigate the mechanism of the CGRs. Results: Both cases shared the same duplication event
(Dup-Nml-Dup/inv). In case1, a fragment comprised of
exon57-79 and two upstream genes(FTHL17, TAB3) in an
inverted orientation and exon53-55 is inserted in intron55. 325 Abstracts from the 51st European Society of Human Genetics Conference: Posters as well as for the society, forcing affected families to seek
help outside of their country. as well as for the society, forcing affected families to seek
help outside of their country. A. Ferlini: None. E. O'Rourke: A. Employment (full or
part-time);
Significant;
Sarepta
Therapeutics,
Inc. M. Pastore: None. A. Martin: None. K. Hovhannesyan: None. T. Sargsyan: None. Identifying and counseling patients amenable to mutation-
specific therapies in Duchenne muscular dystrophy:
knowledge of resources will fuel genetic counselors’ impact V. Ilinsky1,2, A. Krasnenko2, E. Okuneva2, K. Tsukanov2, Whole-genome sequencing detects a large genomic
inversion disrupting the DMD gene in a Becker muscular
dystrophy patient CHP grant reference 336-13(196-DEFI/285-CES). J. Oliveira1,2, A. Gonçalves1,2, Y. Ariyurek3, J. T. den Dunnen4,3,
M. Sousa5,2,6, R. Santos1,2,7 J. Oliveira1,2, A. Gonçalves1,2, Y. Ariyurek3, J. T. den Dunnen4,3,
M. Sousa5,2,6, R. Santos1,2,7 J. Oliveira: None. A. Gonçalves: None. Y. Ariyurek:
None. J.T. den Dunnen: None. M. Sousa: None. R. Santos: None. 1Centro Hospitalar do Porto, Porto, Portugal, 2Unidade
Multidisciplinar de Investigação Biomédica (UMIB), Instituto
de Ciências Biomédicas Abel Salazar (ICBAS), Universidade
do Porto, Porto, Portugal, 3Leiden Genome Technology
Center, Leiden University Medical Center, Leiden,
Netherlands, 4Departments of Human Genetics and Clinical
Genetics, Leiden University Medical Center, Leiden,
Netherlands, 5Departamento de Microscopia, Laboratório de
Biologia Celular, Instituto de Ciências Biomédicas Abel
Salazar (ICBAS), Universidade do Porto, Porto, Portugal,
6Centro de Genética da Reprodução Prof. Alberto Barros,
Porto, Portugal, 7UCIBIO/REQUIMTE, Departamento de
Ciências Biológicas, Laboratório de Bioquímica, Faculdade
de Farmácia, Universidade do Porto, Porto, Portugal P10.19C P10.19C
Whole-genome sequencing detects a large genomic
inversion disrupting the DMD gene in a Becker muscular
dystrophy patient region of dystrophin. Besides expanding the DMD muta-
tional spectrum, this report reinforces the importance of
WGS in clinical genetics, having the potential to detect a
wide variety of mutation types. A. Ferlini1, E. O'Rourke2, M. Pastore3, A. Martin4 Of 4 studied patients one had symptoms of spinal
muscular atrophy and 3 others had mental retardation with
seizures and polymicrogyria. We identified heterozygous mutations in DYNC1H1
gene. Two
variants
(c.9749_9751delAAG
and
c.2029G>A)
were
previously
described
as
uncertain
significance
alleles. Two
others
(c.751C>T
and
c.5882A>T) were not listed in dbNSFP, Clinvar, OMIM,
HGMD, 1000Genomes and ExAC databases. This
work
was
funded
by
Fundamental
Scientific
Research Program of the Russian Academy of Sciences
for 2013-2020. Conclusions: Access to interpretation tools and guidance
is paramount for genetic counselors to advise both patients
and providers. We will review the DMD gene exon map,
Leiden DMD database, and Decode Duchenne mutation-
specific resource as tools to help genetic counselors
determine DMD patients’ eligibility for exon skipping
trials, with the goal of increasing genotyped patients. V. Ilinsky: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Modest; Russian Academy of
Sciences. A. Krasnenko: None. E. Okuneva: None. K. Tsukanov: None. P. Shatalov: None. I. Fedonyuk: None. E. Ilina: None. S. Artemyeva: None. 326 J. del Picchia A non-pathogenic duplication of DMD exon 45-51, inserted
in chromosome 17, in three Danish patients A non-pathogenic duplication of DMD exon 45-51, inserted
in chromosome 17, in three Danish patients M. Faurholdt Lauridsen1, K. Magaard Koldby1, L. Nylansted
Krogh1, J. Graakjaer2, T. Dyrsoe Jensen2, C. Fagerberg1, J. M. Hertz1 1Department of Clinical Genetics, Odense University Hospital,
Odense, Denmark, 2Department of Clinical Genetics, Vejle
Hospital, Vejle, Denmark Introduction: Dystrophinopathies are caused by pathogenic
variants in the DMD-gene. Whole exon deletions or
duplications account for the majority of cases of DMD-
related disease (~65%). When these variants are found it is
important to investigate whether they alter the reading
frame, and special care should be taken when found inci-
dentally or prenatally since not all DMD-duplications or
deletions cause disease. Introduction: Duchenne and Becker muscular dystrophies
(D/BMD) are caused by pathogenic variants in the dystro-
phin (DMD) gene. Being the largest locus of the human
genome (2.3Mb, Xp21.1), DMD is particularly prone to
genomic rearrangements, often intragenic multi-exonic
deletions or duplications (~70% of cases). Single nucleo-
tide variants are identified in most of the remaining D/BMD
cases, whereas complex genomic rearrangements encom-
passing DMD are rarer. Materials and method: Blood and chorionic villus
samples were analyzed using chromosome microarrays
and FISH. Materials and method: Blood and chorionic villus
samples were analyzed using chromosome microarrays
and FISH. Results: We report three cases with incidental finding of
an intragenic duplication encompassing exon 45-51 (out of
79 exons) in the DMD-gene by chromosome microarray
analysis. Case 1 is an asymptomatic 44-year-old male,
investigated as a parental control to an amniotic sample. Case 2 is a male fetus referred because of increased risk of
trisomy 21 at first trimester screen. The duplication was not
inherited from the mother. Unfortunately, the father was
unavailable for study. Case 3 is also a male fetus referred
because of increased risk of trisomy 18 at first trimester
screen. The duplication was inherited from a healthy
mother. The duplicated material was in all cases inserted
in chromosome 17q. Materials and Methods: Routine techniques failed to
detect pathogenic DMD variants in a BMD patient with
progressive muscle weakness, mild intellectual disability
and dystrophic muscle showing irregular dystrophin label-
ling. DMD transcript analysis (RT-PCR and cDNA-MLPA)
was performed, as well as low-coverage whole-genome
sequencing (WGS) using the 10xGenomics Chromium
system. L. N. Luce1,2, M. M. Carcione1,2, C. Mazzanti1,2, I. Szijan1,
F. Giliberto1,2 Materials and methods: We have analyzed 200 boys
with clinical diagnosis of Dystrophinopathy, 12 sympto-
matic women, 240 females at-risk of being carriers and 15
prenatal diagnoses. A diagnostic algorithm was designed for
each case, implementing MLPA, PCR, Whole Exome
Sequencing, Sanger Sequencing, STRs segregation analysis
and HUMARA assay. Materials and methods: We have analyzed 200 boys
with clinical diagnosis of Dystrophinopathy, 12 sympto-
matic women, 240 females at-risk of being carriers and 15
prenatal diagnoses. A diagnostic algorithm was designed for
each case, implementing MLPA, PCR, Whole Exome
Sequencing, Sanger Sequencing, STRs segregation analysis
and HUMARA assay. Results: Sanger sequencing of ECEL1, located in a 24
Mb homozygous region, demonstrated the homozygous
c.2023G>A, p. Ala675Thr variant affecting a highly
conserved amino acid and previously associated with distal
arthrogryposis. Discussion: Our case demonstrates the
classic ECEL1 phenotype. Besides DA, the central atrophy
of the tongue is present in the majority of cases. Congenital
ptosis and limited facial expression are also frequent
findings. Previously unreported, but remarkable findings
in our patient were the vertical grooves on the shins. Long
term complications may include scoliosis and hyperlordo-
sis, as well as restrictive respiratory insufficiency. We
demonstrate that the ECEL1-phenotype is clinically recog-
nizable enabling single gene analysis for the diagnosis of
this specific DA subtype. Results: The selected strategy allowed disease confirma-
tion
in
71.7%
(152/212)
of
the
affected
boys
and
symptomatic females. 12 were candidates for Eteplirsen,
while 22 were suitable for Ataluren. On the other hand, we
were able to establish as carriers 72/255 women/fetuses,
while could exclude from being carriers/affected 143/255. As for gene characterization, we could establish an
association between the most frequent deletion/duplication
intron breakpoints and the abundance of STR loci and, we
have detected 3 haplotypes blocks within the SNPs
identified by the Exome technique. Results: The selected strategy allowed disease confirma-
tion
in
71.7%
(152/212)
of
the
affected
boys
and
symptomatic females. 12 were candidates for Eteplirsen,
while 22 were suitable for Ataluren. On the other hand, we
were able to establish as carriers 72/255 women/fetuses,
while could exclude from being carriers/affected 143/255. As for gene characterization, we could establish an
association between the most frequent deletion/duplication
intron breakpoints and the abundance of STR loci and, we
have detected 3 haplotypes blocks within the SNPs
identified by the Exome technique. L. N. Luce1,2, M. M. Carcione1,2, C. Mazzanti1,2, I. Szijan1,
F. Giliberto1,2 1Dept. of Medical Genetics, Antwerp University Hospital/
Antwerp University, Edegem, Belgium, 2Dept. of Pediatric
Neurology, Antwerp University Hospital, Edegem, Belgium 1Universidad de Buenos Aires, Facultad de Farmacia y
Bioquímica, Cátedra de Genética, Buenos Aires, Argentina,
2CONICET - Universidad de Buenos Aires, Instituto de
Inmunología, Genética y Metabolismo (INIGEM), Buenos
Aires, Argentina 1Universidad de Buenos Aires, Facultad de Farmacia y
Bioquímica, Cátedra de Genética, Buenos Aires, Argentina,
2CONICET - Universidad de Buenos Aires, Instituto de
Inmunología, Genética y Metabolismo (INIGEM), Buenos
Aires, Argentina Introduction: Distal arthrogryposis (DA) a an important
subgroup of arthrogryposis multiplex congenita, character-
ized by congenital joint limitations with involvement of
especially, but not exclusively, the distal extremities. It is a
heterogeneous condition. However, in many DA patients,
the genetic defect remains to be elucidated. Recently,
mutations in ECEL1 were identified as a novel cause of
autosomal recessive distal arthrogryposis. Introduction: Dystrophinopathies are X-linked recessive
diseases caused by mutations in DMD gene. Hitherto there
is no effective treatment for these pathologies, which
enhances the importance of performing genetic assessment
in order to detect mutation carriers and prevent diseased
newborns. However, two mutation-specific gene therapies
were recently approved: Exon 51 Skipping (Eteplirsen) and
Premature Stop Codon Read-through (Ataluren). Therefore,
accurate detection and characterization of the causing
mutation is essential to allow genetic counseling, patient
follow-up and determine the suitable gene therapy. Materials and methods: We report on a girl with
congenital DA with flexed fingers and adducted thumbs,
limited flexion of the knees, abnormal position of the toes
and vertical grooves on both shins. Already at birth, a
striking central atrophy of the tongue was noted. In
addition, she had a right-sided ptosis and limited facial
expression. Radiographs demonstrated dysplastic acetabu-
lae and a right-sided hip luxation. Brain MRI was normal. Family history was unremarkable, apart from parental
consanguinity. Materials and methods: We report on a girl with
congenital DA with flexed fingers and adducted thumbs,
limited flexion of the knees, abnormal position of the toes
and vertical grooves on both shins. Already at birth, a
striking central atrophy of the tongue was noted. In
addition, she had a right-sided ptosis and limited facial
expression. Radiographs demonstrated dysplastic acetabu-
lae and a right-sided hip luxation. Brain MRI was normal. Family history was unremarkable, apart from parental
consanguinity. M. Faurholdt Lauridsen: None. K. Magaard Koldby:
None. L. Nylansted Krogh: None. J. Graakjaer: None. T.
Dyrsoe
Jensen:
None.
C.
Fagerberg:
None.
J.M.
Hertz: None. M. Faurholdt Lauridsen: None. K. Magaard Koldby:
None. L. Nylansted Krogh: None. J. Graakjaer: None. T. Dyrsoe
Jensen:
None. C. Fagerberg:
None. J.M. Hertz: None. L.N. Luce: None. M.M. Carcione: None. C. Mazzanti:
None. I. Szijan: None. F. Giliberto: None. L.N. Luce: None. M.M. Carcione: None. C. Mazzanti:
None. I. Szijan: None. F. Giliberto: None. ECEL1-related distal arthrogryposis: The tale of the tongue M. E. C. Meuwissen1, D. Beysen2, E. Reyniers1, F. Kooy1,
B. Ceulemans2 L. N. Luce1,2, M. M. Carcione1,2, C. Mazzanti1,2, I. Szijan1,
F. Giliberto1,2 A non-pathogenic duplication of DMD exon 45-51, inserted
in chromosome 17, in three Danish patients Materials and Methods: Routine techniques failed to
detect pathogenic DMD variants in a BMD patient with
progressive muscle weakness, mild intellectual disability
and dystrophic muscle showing irregular dystrophin label-
ling. DMD transcript analysis (RT-PCR and cDNA-MLPA)
was performed, as well as low-coverage whole-genome
sequencing (WGS) using the 10xGenomics Chromium
system. Results: RNA analysis showed the absence of exons 75-
79. While automated structural variant calling from WGS
was inconclusive, visual BAM file inspection showed a
possible breakpoint within intron 74 of DMD. Some reads
had homology with a region located upstream of the PRDX4
gene (Xp22.11). Similarly, some reads in that location
showed homology with inverted intron 74 DMD sequences. Breakpoint PCR and Sanger sequencing confirmed the
presence of a ~8Mb inversion. Abnormal DMD transcripts
were subsequently identified, some of which contained
segments from the region upstream of PRDX4. Results: RNA analysis showed the absence of exons 75-
79. While automated structural variant calling from WGS
was inconclusive, visual BAM file inspection showed a
possible breakpoint within intron 74 of DMD. Some reads
had homology with a region located upstream of the PRDX4
gene (Xp22.11). Similarly, some reads in that location
showed homology with inverted intron 74 DMD sequences. Breakpoint PCR and Sanger sequencing confirmed the
presence of a ~8Mb inversion. Abnormal DMD transcripts
were subsequently identified, some of which contained
segments from the region upstream of PRDX4. Conclusion: DMD duplications are a known cause of
DMD-related disease. However, caution should be made
when found incidentally and prenatally, and assumptions
regarding disease prediction should not be made without
further investigations. This is especially important regarding
duplications since they can be inserted in other chromo-
somes and thus do not affect the DMD-reading frame. Conclusions: The patient’s phenotype is explainable by
this DMD inversion with concomitant loss of the C-terminal Abstracts from the 51st European Society of Human Genetics Conference: Posters 327 This study was supported by PTC Therapeutics and
University of Buenos Aires, Argentina. Introduction: Val30Met variant in transthyretin gene (TTR) Introduction: Val30Met variant in transthyretin gene (TTR)
is causative for Familial Amyloid Polyneuropathy (FAP). Substantial phenotypic heterogeneity has been described in
patients with Val30Met, including in age-at-onset. Conse-
quently, other variants in TTR locus, beyond the TTR-FAP
causing variant, could play a regulatory role in its expres-
sion level and modify disease expressivity. We aim to
identify genetic modifiers of disease onset that may con-
tribute to this clinical variability. Materials & Methods: We genotyped the promoter and
coding and flanking regions of TTR gene in Val30Met TTR
carriers. An intensive in silico analysis was performed in
order
to
understand
a
possible
regulation
of
gene
expression. Materials & Methods: We genotyped the promoter and
coding and flanking regions of TTR gene in Val30Met TTR
carriers. An intensive in silico analysis was performed in
order
to
understand
a
possible
regulation
of
gene
expression. Results: We identified 12 known variants in the promoter
region and 2 known intronic (rs36204272, rs1791228), one
3’ UTR (rs62093482) and 2 known exonic variants
(rs28933981 and rs1800458). Importantly, our analysis
revealed variants significantly associated with age-at-onset,
one of which is a CA repeat (rs71383038) in promotor
region. In addition, unreported and very interesting results
in the in silico analysis were found since we observed some
alterations in the mechanism of splicing, transcription
factors binding and miRNAs binding. Results: We identified 12 known variants in the promoter
region and 2 known intronic (rs36204272, rs1791228), one
3’ UTR (rs62093482) and 2 known exonic variants
(rs28933981 and rs1800458). Importantly, our analysis
revealed variants significantly associated with age-at-onset,
one of which is a CA repeat (rs71383038) in promotor
region. In addition, unreported and very interesting results
in the in silico analysis were found since we observed some
alterations in the mechanism of splicing, transcription
factors binding and miRNAs binding. Materials and methods: A detailed clinical description
of a new consanguineous family with two affected children
with fetal akinesia is provided as well as in vitro functional
characterization of the novel CHT1 mutation including a
rescue experiment. Materials and methods: A detailed clinical description
of a new consanguineous family with two affected children
with fetal akinesia is provided as well as in vitro functional
characterization of the novel CHT1 mutation including a
rescue experiment. Results:
While
SLC5A7
cell-surface
biotinylation
showed that the mutant is able to reach the plasma
membrane, no choline transport was detected. L. N. Luce1,2, M. M. Carcione1,2, C. Mazzanti1,2, I. Szijan1,
F. Giliberto1,2 Conclusions: In the present work, we have characterized
a Dystrophinopathy argentine population and contributed to
the understanding of the genetic/molecular basis of these
pathologies M.E.C. Meuwissen:
None. D. Beysen:
None. E. Reyniers: None. F. Kooy: None. B. Ceulemans: None. M.E.C. Meuwissen:
None. D. Beysen:
None. E. Reyniers: None. F. Kooy: None. B. Ceulemans: None. 328 J. del Picchia P10.23C
Phenotypic significance of variants in the TTR promoter:
Familial Amyloid Polyneuropathy (TTR-FAP) onset
M. Alves-Ferreira1,2,3, A. Azevedo1,2,3, T. Coelho4, D. Santos1,2,
J. Abreu-Silva1,2, J. Sequeiros1,2,3, I. Alonso1,2, A. Sousa3,1,2,
C. Lemos3,1,2 Santos: None. J. Abreu-Silva: None. J. Sequeiros: None. I. Alonso: None. A. Sousa: None. C. Lemos: None. University of Alberta, Edmonton, AB, Canada Introduction: Severe fetal akinesia results in a recognizable
deformation sequence with variable pre- and postnatal
phenotype including: polyhydramnios, reduced sponta-
neous movements, arthrogryposis, and pulmonary hypo-
plasia. The primary causes are genetic, heterogeneous, and
due to defects of the motor pathway. A subgroup of these
conditions is characterized by endplate specific mutations of
the neuromuscular junction. Recently, recessive mutations
in the SLC5A7 protein coding for the high affinity choline
transporter CHT1 have been related to a continuum of
phenotypes characterized by congenital myasthenia and
episodic apnea (Bauché et al., 2016, Wang et al., 2017). We
report the independent identification and characterization of
a new family with a lethal form of disease due to a novel
homozygous mutation in SLC5A7 and review the two
previously published families with a similar phenotype
proposing a genotype-phenotype correlation for a new
subclass of lethal fetal akinesia. P10.24D
A new form of fetal akinesia syndrome is due to mutations
in the SLC5A7 gene M. Alves-Ferreira1,2,3, A. Azevedo1,2,3, T. Coelho4, D. Santos1,2,
J. Abreu-Silva1,2, J. Sequeiros1,2,3, I. Alonso1,2, A. Sousa3,1,2,
C. Lemos3,1,2 O. Caluseriu, M. Banerjee, D. Arutyunov, D. Brandwein,
C. Janetzki-Flatt, S. Hume, H. Kolski, N. Leonard, J. Watt,
A. Lacson, M. Baradi, E. M. Leslie, E. Cordat
University of Alberta, Edmonton, AB, Canada 1i3S - Instituto de Investigação e Inovação em Saúde,
Universidade do Porto, Porto, Portugal, 2UnIGENe,IBMC -
Instituto de Biologia Molecular e Celular, Universidade do
Porto, Porto, Portugal, 3ICBAS- Instituto de Ciências
Biomédicas Abel Salazar, Universidade do Porto, Porto,
Portugal, 4Unidade Clínica de Paramiloidose (UCP), Centro
Hospitalar do Porto (CHP), Porto, Portugal Medical Genetics Center MGZ, Munich, Germany Facioscapulohumeral
Muscular
Dystrophy
(FSHD)
is
associated to hypomethylation of the D4Z4 microsatellite,
leading to an increased expression of the DUX4 gene which
is proposed to cause progressive muscle atrophy and ulti-
mately FSHD pathology. Chromatin changes are caused by
contractions to less than 11 units of the D4Z4 repeat
(FSHD1) or by pathogenic variants in chromatin regulatory
proteins (FSHD2). We developed a novel NGS-based
method that allows us to diagnose FSHD and distinguish
FSHD1 from FSHD2 in a single approach. Enrichment of
4qA (A) and 4qAL (AL) associated D4Z4 repeats as well as
all 4q35 D4Z4 repeat units (DUX4) was performed using
nested PCRs after bisulfite conversion of genomic DNA
(EpiTect). DNA libraries were prepared with the NEBNext
Ultra DNA Library Prep Kit and sequenced on an Illumina
MiSeq System. Reads were mapped to A and AL using
bwameth. Mean methylation levels were calculated with an
in-house script. We analyzed 21 patients with a confirmed
FSHD diagnosis and 21 negative controls. We could reli-
ably confirm the hypomethylation status in all patients. Furthermore, the results show a correlation of methylation
levels to the disease status. In addition, we could distinguish
FSHD1 from FSHD2 by DUX4 methylation levels, as due
to a chromatin dysregulation DUX4 is hypomethylated in
FSHD2 as well. In conclusion, sensitive detection of
methylation levels in the D4Z4 array can give insight into
the clinical progression levels more exactly than currently Introduction: The,,floppy child syndrome” is one of the
most unambiguous medical condition presenting at birth or
in early infancy. We aimed to assess the etiology of this
condition with focus on neuromuscular symptoms. Introduction: The,,floppy child syndrome” is one of the
most unambiguous medical condition presenting at birth or
in early infancy. We aimed to assess the etiology of this
condition with focus on neuromuscular symptoms. Material and methods: Till now, 120 probands present-
ing generalized hypotonia at birth or in neonatal period with
excluded major genetic causes (e.g. SMA), were included in
the study. For 75 of them, the exome sequencing (WES)
was performed using QXT Sure Select Human All Exome
v.6. In selected cases, directed analysis using classic Sanger
sequencing / MLPA was performed. Results: Directed analysis revealed the presence of
pathogenic variants in MTM1 in three patients with
myotubular myopathy and in ACTA1 in four patients with
nemaline myopathy. First analysis of WES data included
known genes related to neuromuscular disorders. P10.25A Conclusion: The genetic cause of floppy child syndrome
was found in 27/120 (22.5%) patients. The first-line
analysis for the presence of mutations in known neuromus-
cular genes seems to be a good starting point for further
studies of new genes related to the etiology of “floppy child
syndrome”. Whole exome sequencing in floppy child syndrome patients
with a particular consideration of neuromuscular disorders M. Gos1, E. Dębek1, A. Madej-Pilarczyk2, A. Potulska-Chromik3,
T. Gambin4,1, J. Pilch5, R. Śmigiel6, R. Posmyk7, B. Wojtaś8,
B. Gielniewski8, J. Fijak-Moskal9, A. Kutkowska-Kaźmierczak1,
A. Jakubiuk-Tomaszuk10, A. Kostera-Pruszczyk3, J. Bal1,
M. Jędrzejowska2 The study was supported from National Science Centre
grant no. UMO-2015/17/B/NZ5/01368 M. Gos: None. E. Dębek: None. A. Madej-Pilarczyk:
None. A. Potulska-Chromik: None. T. Gambin: None. J. Pilch: None. R. Śmigiel: None. R. Posmyk: None. B. Wojtaś: None. B. Gielniewski: None. J. Fijak-Moskal:
None. A. Kutkowska-Kaźmierczak: None. A. Jakubiuk-
Tomaszuk: None. A. Kostera-Pruszczyk: None. J. Bal:
None. M. Jędrzejowska: None. 1Department of Medical Genetics, Institute of Mother and
Child, Warsaw, Poland, 2Neuromuscular Department,
Mossakowski Medical Research Centre, PAS, Warsaw, Poland,
3Department of Neurology, Warsaw Medical University,
Warsaw, Poland, 4Institute of Computer Science, Warsaw
University of Technology, Warsaw, Poland, 5Department of
Pediatrics and Neurology for Children and Adolescents,
Medical University of Silesia, Katowice, Poland, 6Department
of Pediatrics and Rare Disorders, Wrocław Medical
University, Wrocław, Poland, 7Department of Clinical
Genetics, Podlaskie Medical Centre, Białystok, Poland,
8Laboratory of Molecular Neurobiology, Neurobiology Center,
Nencki Institute of Experimental Biology, PAS, Warsaw,
Poland, 9Department of Medical Genetics, Jagiellonian
University, Cracow, Poland, 10Department of Pediatric
Neurology and Rehabilitation, Medical University of Białystok,
Białystok, Poland Introduction: Val30Met variant in transthyretin gene (TTR) Functional
rescue of the mutant with different chemical chaperones
failed to compensate the altered function. Conclusions: Our findings raise an interesting possibility
that TTR variants could be genetic modifiers in Val30Met
carriers and might influence disease variability. Further-
more, our findings suggest that variants within promoter
region can modify disease expressivity and have the
potential to be a biomarker to identify disease onset within
asymptomatic carriers at risk of developing TTR-FAP
promoting a better follow-up of Val30Met carriers. Conclusions: Our findings raise an interesting possibility
that TTR variants could be genetic modifiers in Val30Met
carriers and might influence disease variability. Further-
more, our findings suggest that variants within promoter
region can modify disease expressivity and have the
potential to be a biomarker to identify disease onset within
asymptomatic carriers at risk of developing TTR-FAP
promoting a better follow-up of Val30Met carriers. Conclusions: This study brings further clinical and
functional evidence for a novel pathogenic mutation in
CHT1, and proposes that recessive mutations of the
intracytoplasmic protein domains of SLC5A7 are respon-
sible for a lethal form of fetal akinesia. O. Caluseriu: None. M. Banerjee: None. D. Arutyu-
nov: None. D. Brandwein: None. C. Janetzki-Flatt:
None. S. Hume: None. H. Kolski: None. N. Leonard:
None. J. Watt: None. A. Lacson: None. M. Baradi: None. E.M. Leslie: None. E. Cordat: None. MA-F received support from FCT fellowship (SFRH/BD/
101352/2014). MA-F received support from FCT fellowship (SFRH/BD/
101352/2014). M. Alves-Ferreira: None. A. Azevedo: None. T. Coelho: F. Consultant/Advisory Board; Modest; Pfizer. D. M. Alves-Ferreira: None. A. Azevedo: None. T. Coelho: F. Consultant/Advisory Board; Modest; Pfizer. D. Abstracts from the 51st European Society of Human Genetics Conference: Posters 329 Targeted methyl-Seq quantification by NGS technology for
routine diagnostic of FSHD Targeted methyl-Seq quantification by NGS technology for
routine diagnostic of FSHD F. Scharf, S. Bulst, A. Benet-Pagès, J. Romic-Pickl, A. Abicht,
E. Holinski-Feder Diagnostic utility of clinical exome sequencing in a cohort
of patients with hereditary polyneuropathies L. Sedlackova1, P. Lassuthova1, J. Neupauerova1, D. Safka
Brozkova1, D. Stanek1, J. Haberlova2, R. Mazanec3, P. Seeman1 1DNA laboratory, Department of Pediatric Neurology, 2nd
Faculty of Medicine, Charles University in Prague and
University Hospital Motol, Prague, Czech Republic,
2Department of Pediatric Neurology, 2nd Faculty of Medicine,
Charles University in Prague and University Hospital Motol,
Prague, Czech Republic, 3Department of Neurology, 2nd
Faculty of Medicine, Charles University in Prague and
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Introduction:
We
describe clinical characteristics in a cohort of patients with
hereditary polyneuropathies (N=50) and compare them to
the results of clinical exome sequencing (CES). Methods:
A cohort was devided in two clinical groups. First group
with positive CES findings (N=21) and second with
negative CES findings or variants of unknown significance
(N=29). Clinical and genetic characteristics were analyzed. Results: In the first group almost half of the patients
(N=10) had clinical presentation of Charcot-Marie-Tooth
polyneuropathy with positive electromyography findings,
slowly progressive clinical course with moderate severity of
disease, with a beginning as children or as young adults and
positive family history. Four patients with Charcot-Marie-
Tooth polyneuropathy were sporadic cases, four patients
had additional central nervous system findings and three
patients were found to have myopathy based on molecular
results. Negative CES findings were mostly (N=13) corre-
lated to sporadic cases and electromyography findings of
axonal
polyneuropathy. Conclusions:
Clinical
exome
sequencing was found to be conclusive specially in her-
editary polyneuroptahies with Charcot-Marie-Tooth clinical
presentation and positive family history. Yield of CES in
cases of hereditary polyneuropathy was 42%. Sporadic
cases with electromyography findings of axonal poly-
neuropathy were often found negative. <!--EndFragment-->
M Rogac: None A Maver: None K Writzl: None G Introduction: Inherited peripheral neuropathies (IPN) are
the most common monogenic neurological disorder. It is a
clinically and genetically extremely heterogeneous group
with more than 90 genes already involved. Whole-exome
sequencing (WES) was used in patients previously unsolved
using standard diagnostic methods and clinical experience. Patients and Methods: Here we present WES results of
47 undiagnosed patients with IPN from 38 unrelated
families tested previously in our diagnostic lab for better
known IPN types and genes. Medical Genetics Center MGZ, Munich, Germany The most
common mutated genes were: LMNA (4 patients with de
novo
variants)
and
RYR1
(5
patients,
2
compound
heterozygotes, 3 heterozygotes). In single patients, poten-
tially pathogenic variants in LAMA2, PIEZO2, SEPN1,
PMP22, SGCA, PIGA, DNM2, PPP2R1A, COL12A1 or
COL6A1 genes were found, analyzed for the inheritance in
families and confirmed to be related to the patient specific
phenotype. 330 J. del Picchia novel published genes and variants. Supported by: MH CR
AZV 16-30206 available methods. Additionally, a much higher yield and
throughput in FSHD diagnostics can be achieved. F. Scharf: None. S. Bulst: None. A. Benet-Pagès: None. J. Romic-Pickl: None. A. Abicht: None. E. Holinski-
Feder: None. L. Sedlackova: None. P. Lassuthova: None. J. Neu-
pauerova: None. D. Safka Brozkova: None. D. Stanek:
None. J. Haberlova: None. R. Mazanec: None. P. Seeman: None. M. Rogac: None. A. Maver: None. K. Writzl: None. G.
Rudolf: None. B. Peterlin: None. Bambino Gesù Children Hospital, Rome, Italy A. U. Meszarosova1, D. S. Brozkova1, P. Lassuthova1,
D. Stanek1, M. Bittoova2, I. Soldatova2, P. Seeman1,2 A. U. Meszarosova1, D. S. Brozkova1, P. Lassuthova1,
D. Stanek1, M. Bittoova2, I. Soldatova2, P. Seeman1,2 Background: Hereditary spastic paraplegias (HSP) are
clinical and genetic heterogeneous diseases with more than
eighty disease genes identified thus far. Studies on large
cohorts of HSP patients showed that, by means of current
technologies, the percentage of genetically solved cases is
close to 50%. Notably, the percentage of molecularly con-
firmed
diagnoses
decreases
significantly
in
sporadic
patients. 1DNA laboratory, Department of Paediatric Neurology, 2nd
School of Medicine Charles University and Faculty Hospital
Motol, Prague, Czech Republic, 2Centre for Medical Genetics
and Reproductive Medicine GENNET, Prague, Czech Republic 1DNA laboratory, Department of Paediatric Neurology, 2nd
School of Medicine Charles University and Faculty Hospital
Motol, Prague, Czech Republic, 2Centre for Medical Genetics
and Reproductive Medicine GENNET, Prague, Czech Republic Introduction: Hereditary spastic paraplegias (HSP) are
clinically and genetically heterogeneous disorders of the
central motoneuron. Typical clinical feature is progressive
bilateral spasticity and weakness of the lower limbs. All
types of inheritance and causative variants in already more
than 70 genes hase been described in HSP. The most
common cause are pathogenic variants in the SPAST
(SPG4) gene. Objectives: To describe our diagnostic molecular genetic
approach
on
patients
with
pediatric-onset
pure
and
complex HSP. Methods: Forty-seven subjects with HSP from 44
unrelated families underwent molecular screening of 113
known and candidate disease genes by targeted capture and
massively parallel sequencing. Negative cases were succes-
sively analyzed by multiplex ligation-dependent probe
amplification (MLPA) analysis for the SPAST gene, and
high-resolution SNP array analysis for genome-wide CNV
detection. Materials and methods: We aimed to map the genetic
spectrum of HSPs in 72 Czech non-SPG4 uncomplicated
HSP patients. Fourty-three patients were with positive
family history and 29 were sporadic.Targeted Enrichment
of all coding exons of 38 genes associated with uncompli-
cated HSP with our probe design was used for MPS. Results: Diagnosis was molecularly confirmed in 29 out
of 47 (62%) of patients, most of whom had clinical
diagnosis of cHSP. Although SPG11 and SPG4 remain the
most frequent cause of respectively complex and pure HSP,
a large number of pathogenic variants were disclosed in
POLR3A, FA2H, DDHD2, GLUT1, ENTPD1, ERLIN2,
CAPN1, ALS2, ADAR1, RNASEH2B, TUBB4A, ATL1 and
KIF1A. In a subset of these disease genes, phenotypic
expansion and novel genotype-phenotype correlations were
recognized. P10.29A F. Stregapede, L. Travaglini, C. Aiello, V. Alesi, A. D'Amico,
A. Ciofi, A. Bruselles, S. Pizzi, G. Zanni, S. Loddo, S. Barresi,
G. Vasco, M. Tartaglia, E. Bertini, F. Nicita Bambino Gesù Children Hospital, Rome, Italy Notably, SNP array analysis did not provide
any
significant
contribution
in
increasing
the
diagnostic yield. Results: Causal variants were found in 16 patients among
the group of 72 HSP patients (22.2%). Slightly more
patients were detected in the group of familial patients (10/
43; 23.3%) vs. sporadic patients (6/29; 20.1%). Causative
variants were found in SPG11 (4 x), REEP1/SPG31 (3 x),
KIF5A/SPG10 (2 x), SPG7 (2 x), NIPA1/SPG6, CYP7B1/
SPG5, KIAA0196/SPG8, FA2H/SPG35 and SPAST (all 1x). Surprisingly ATL1 variant was found only once and it
finaly turned to not causal because of presence in the
healthy parent. Conclusion: SPG11 seems to be the most common type
of HSP among Czech non-SPG patients. SPG3A and
SPG7 seem not to be so frequent then in other populations. Together nine genetic types of HSP were found among 72
Czech uncomplicated non-SPG4 HSP patients. Project
supported by: Ministry of Health of the Czech Republic
grant nr.15-31899A. Conclusion: Our findings document the high diagnostic
yield of targeted sequencing for patients with pediatric-
onset, complex and pure HSP. MLPA for SPAST and SNP
array should be limited to properly selected cases based on
clinical suspicion. F. Stregapede: None. L. Travaglini: None. C. Aiello:
None. V. Alesi: None. A. D'Amico: None. A. Ciofi: None. A. Bruselles: None. S. Pizzi: None. G. Zanni: None. S. Loddo: None. S. Barresi: None. G. Vasco: None. M. Tartaglia: None. E. Bertini: None. F. Nicita: None. F. Stregapede: None. L. Travaglini: None. C. Aiello:
None. V. Alesi: None. A. D'Amico: None. A. Ciofi: None. A. Bruselles: None. S. Pizzi: None. G. Zanni: None. S. Loddo: None. S. Barresi: None. G. Vasco: None. M. Tartaglia: None. E. Bertini: None. F. Nicita: None. A.U. Meszarosova: None. D.S. Brozkova: None. P. Lassuthova: None. D. Stanek: None. M. Bittoova: None. S
S A.U. Meszarosova: None. D.S. Brozkova: None. P. Lassuthova: None. D. Stanek: None. M. Bittoova: None. I. Soldatova: None. P. Seeman: None. I. Soldatova: None. P. Seeman: None. Diagnostic utility of clinical exome sequencing in a cohort
of patients with hereditary polyneuropathies Patients and Methods: Here we present WES results of
47 undiagnosed patients with IPN from 38 unrelated
families tested previously in our diagnostic lab for better
known IPN types and genes. Results: Causative mutations were found in thirteen
patients (34%) and in twelve genes to date. In six patients
we detected mutations in known IPN genes (SOD1, MFN2,
BSCL2, DNM2, SMN1, AIFM1) and in four of them the
found mutation could confirm previous just novel findings
(MORC2, SLC25A45, DRP2, GNB4). But we also revealed
three novel causative gene-IPN associations in frame of
international collaboration (ATP1A1, HARS). We uncovered
mutations even in well-known genes previously tested in
single gene tests, which highlights that WES may detect
also variants that have been missed by Sanger sequencing
during routine diagnostics. Some of the causative mutations
were identified immediately after publication of their
causality by the WES data reevaluation. Conclusion: WES represents efficient tool for clarifica-
tion of rare, novel or unusual IPN types and it has an
important potential for novel candidate genes in familiar
cases. In unsolved patients WES data should be re-
evaluated every 6 months due to the continual increase of M. Rogac: None. A. Maver: None. K. Writzl: None. G. Rudolf: None. B. Peterlin: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 331 P10.32D
HINT1 gene mutations are frequent cause of CMT in
Russia P10.31C The impact of next generation sequencing on the diagnosis
of pediatric-onset hereditary spastic paraplegias: new
genotype-phenotype correlations for rare HSP-related
genes P10.32D P10.32D
HINT1 gene mutations are frequent cause of CMT in
Russia 332 J. del Picchia Romania, 3Neurology department, Clinical Pediatric Hospital
„Dr.V.Gomoiu”, Bucuresti, Romania Romania, 3Neurology department, Clinical Pediatric Hospital
„Dr.V.Gomoiu”, Bucuresti, Romania Romania, 3Neurology department, Clinical Pediatric Hospital
„Dr.V.Gomoiu”, Bucuresti, Romania T. B. Milovidova1, O. A. Schagina1, E. L. Dadali1, S. A. Kurbatov2, A. V. Polyakov1 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Regional Medical Diagnostic Centre, Voronezh,
Russian Federation Background: Mutations in ATP1A2 gene have been
associated with autosomal dominant familial hemiplegic
migraine type 2 syndrome. We aim to present an atypical
presentation of a published pathogenic variant in the
ATP1A2 gene, in a patient that does not show the classic
episodic attacks of hemiplegia, oculomotor and autonomic
disturbances, nor the progressive cognitive impairment. Background: Mutations in ATP1A2 gene have been
associated with autosomal dominant familial hemiplegic
migraine type 2 syndrome. We aim to present an atypical
presentation of a published pathogenic variant in the
ATP1A2 gene, in a patient that does not show the classic
episodic attacks of hemiplegia, oculomotor and autonomic
disturbances, nor the progressive cognitive impairment. Introduction: Inherited peripheral neuropathies (IPNs) are
neuromuscular and neurodegenerative disorders that affect
the peripheral nervous system (PNS). More than 100 dif-
ferent subtypes have been identified. HINT1 mutations were
revealed as the reason of IPNs and Charcot-Marie-Tooth
neuropathy (CMT). Method: The patient was referred from the neurology
clinic for diagnosis and genetic counselling. Clinical
assessment
and
whole
exome
sequencing(trio)
was
performed. Materials and Methods: Massive parallel sequencing
(MPS), multiplex ligation depended probe amplification
(MLPA) and Sanger’s sequencing have been used. We
investigated 700 patients without mutation in genes of
frequent CMT from 2000 CMT patients. Also 1000
uninspected unrelated persons were tested. Materials and Methods: Massive parallel sequencing
(MPS), multiplex ligation depended probe amplification
(MLPA) and Sanger’s sequencing have been used. We
investigated 700 patients without mutation in genes of
frequent CMT from 2000 CMT patients. Also 1000
uninspected unrelated persons were tested. Results: The 15 years old male patient had a history of
failure to thrive, gastroesophageal reflux since age 2 months,
delay in motor development(sits at age 1 year, walks at 4
years), choreoathetotic movements, difficulty in walking
and dystonia, without seizures. He has normal hearing, but
difficulty in speech. Brain MRI, performed at different
times(age 5,10,13 and 15 years) did not show abnormalities. Brain spectroscopy performed at age 15 years, was
unremarkable. P10.32D Normal nerve speed conduction and EEG
were repeatedly normal. Parents are healthy not consangui-
neous, without significant family history. Whole exome
sequencing revealed a heterozygous variant c.1816G>A p. (Ala606Thr) in the ATP1A2 gene, segregation study
concludes de novo occurrence. No other variant relevant
for the phenotype was detected. The evolution of our patient
was progressive not episodic as described gene associated
diseases. Results: In four of five Russian patients with neuromio-
tonia and axonal neuropathy the homozygous c.110G>C (p. Arg37Pro) HINT1 mutation were identified. Thirty CMT
patients from 700 with c.110G>C homozygous mutation
have
been
revealed. Two
compound-heterozygotes
c.110G>C mutation with earlier not described mutations
(c.112-1delG and c.281A>G (p.Tyr94Cys)) were detected. Three heterozygotes of c.110G>C mutation in HINT1 have
been revealed during the research. We consider that CMT
phenotype of these patients isn't caused by HINT1 mutation
and they are carriers of c.110G>C mutation. The c.110G>C
HINT1 mutation was revealed at 4 of 1000 persons. Therefore the carriage of c.110G>C mutation in the Russia
is 1 of 250 persons. Thus HINT1 mutations are not less than
1,6% of all CMT types in Russia. Three heterozygotes of c.110G>C mutation in HINT1 have
been revealed during the research. We consider that CMT
phenotype of these patients isn't caused by HINT1 mutation
and they are carriers of c.110G>C mutation. The c.110G>C
HINT1 mutation was revealed at 4 of 1000 persons. Therefore the carriage of c.110G>C mutation in the Russia
is 1 of 250 persons. Thus HINT1 mutations are not less than
1,6% of all CMT types in Russia. Conclusions: Although there is a significant candidate
variant in the ATP1A2 gene the phenotype is discordant. Questions that remain: Are they linked? Is this a new
pathogenic implication of the ATP1A2 gene? Conclusions: For the first time in Russia the research of
HINT1 gene mutations at CMT is conducted. The unique
c.110G>C frequency data of heterozygotic carriages in
Russia are obtained. Two new mutations in HINT1 gene
were described. I. Perva: None. A. Chirita Emandi: None. D. Epure:
None. M. Puiu: None. New method for detecting mtDNA deletions from massively
parallel sequencing data T.B. Milovidova: None. O.A. Schagina: None. E.L. Dadali:
None. S.A. Kurbatov:
None. A.V. Polyakov: None. T.B. Milovidova: None. O.A. Schagina: None. E.L. Dadali:
None. S.A. Kurbatov:
None. A.V. Polyakov: None. T. Suominen1, S. Penttilä1, M. Jokela2, J. Palmio2, B. Udd2,3,4 1Victor Babes University of Medicine and Pharmacy, Center of
Genomic Medicine, Timisoara, Timisoara, Romania, 2Louis
Ţurcanu Children’s Clinical Emergency Hospital, Timişoara, An extension to phenotypic spectrum of ATP1A2 gene or
an unsolved case? 1Neuromuscular Research Unit, Tampere, Finland,
2Neuromuscular Research Center, Department of Neurology,
Tampere University Hospital, Tampere, Finland, 3Department
of Neurology, Vaasa Central Hospital, Vaasa, Finland,
4Folkhälsan Institute of Genetics and Department of Medical
Genetics, Medicum, University of Helsinki, Helsinki, Finland An extension to phenotypic spectrum of ATP1A2 gene or
an unsolved case? I. Perva1,2, A. Chirita Emandi1,2, D. Epure3, M. Puiu1,2 I. Perva1,2, A. Chirita Emandi1,2, D. Epure3, M. Puiu1,2 1Victor Babes University of Medicine and Pharmacy, Center of
Genomic Medicine, Timisoara, Timisoara, Romania, 2Louis
Ţurcanu Children’s Clinical Emergency Hospital, Timişoara, Introduction: Human mitochondrial genome (mtDNA) is a
circular DNA of 16 Mb in size. Mutations can cause several Abstracts from the 51st European Society of Human Genetics Conference: Posters 333 Maggiore della Carità, Novara, Italy, 7SC Neurologia, IRCCS
Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy inherited diseases, many of which are neuromuscular. The
mutational rate compared to nuclear DNA is very high. However, because every cell contains several copies of
mtDNA,
mutations
are
heteroplasmic
(0-100%). All
mtDNA mutations have to exceed a threshold on the level
of heteroplasmy to have an effect on the cell. Previously
used methods for detecting mtDNA deletions (real-time
PCRs and Southern blot) do not define the breakpoints
accurately and the heteroplasmic rate is a rough estimation. inherited diseases, many of which are neuromuscular. The
mutational rate compared to nuclear DNA is very high. However, because every cell contains several copies of
mtDNA,
mutations
are
heteroplasmic
(0-100%). All
mtDNA mutations have to exceed a threshold on the level
of heteroplasmy to have an effect on the cell. Previously
used methods for detecting mtDNA deletions (real-time
PCRs and Southern blot) do not define the breakpoints
accurately and the heteroplasmic rate is a rough estimation. Genome Wide Association Studies identified over 100
Multiple Sclerosis (MS) risk loci. However, these studies
are usually focused on common variants, mainly located in
non-coding sequences. We aimed at searching for rare
functional variants in MS associated loci and assessing if
the genes in these regions show an imbalance of rare variant
frequencies (burden) between MS patients and healthy
controls (HC). We sequenced the coding and UTR regions
of 100 MS genes in 588 Italian MS and 408 matched HC,
pooled in groups of 12 individuals using an approach
implemented by our group (Anand 2016). An extension to phenotypic spectrum of ATP1A2 gene or
an unsolved case? Clarelli*: None. E. Man-
gano*: None. C. Basagni: None. M. Zuccalà: None. S. Anand: None. M. Sorosina: None. E. Mascia: None. G. De Bellis: None. F. Esposito: None. D. Vecchio: None. G. Predebon: None. C. Comi: None. R. Cantello: None. V. Martinelli: None. G. Comi: None. M. Leone: None. R. Bordoni*: None. F. Martinelli-Boneschi*: None. S. D'Alfonso*: None. Cumulative effect of rare functional variants in Multiple
Sclerosis associated genes Cumulative effect of rare functional variants in Multiple
Sclerosis associated genes N. Barizzone*1,2, F. Clarelli*3, E. Mangano*4, C. Basagni1,2,
M. Zuccalà1,2, S. Anand4, M. Sorosina3, E. Mascia3, G. De
Bellis4, F. Esposito3,5, PROGEMUS, PROGRESSO, IMSGC,
D. Vecchio6, G. Predebon1,2, C. Comi6, R. Cantello6,
V. Martinelli5, G. Comi5, M. Leone1,2,7, R. Bordoni*4,
F. Martinelli-Boneschi*3,5, S. D'Alfonso*1,2 An extension to phenotypic spectrum of ATP1A2 gene or
an unsolved case? The burden of
rare functional variations in MS susceptibility was assessed
with 3 gene-based statistical tests (Weighted-Sum Statistic,
C-alpha test, Fisher hybrid test). Variants were selected
using various filtering criteria based on allelic frequency,
functional impact according to in-silico prediction and
functional annotation of regulatory regions. Seventeen
genes showed a statistically significant burden and were
thus sequenced in an independent cohort of 504 MS and
504 controls, using the same pipeline developed for the
discovery phase. After meta-analysis of the two cohorts, we
observed a significant burden (p≤0.039) for three genes,
each in a different MS region, with at least one filter and
one burden test. In conclusion, this work suggests that the
association signal in MS loci may be driven by a burden of
rare variants in a single gene and confirms the role of rare
variants in the susceptibility to autoimmune diseases, par-
ticularly to MS. Supporting grant: FISM 2015 Materials and methods: We have analyzed 60 samples
for mtDNA deletions. DNA was isolated from muscle. Samples included controls harboring a single mtDNA
deletion (n = 2) and multiple deletions (n = 4). Amplifica-
tion of the mtDNA was performed using long-range PCR
followed by massively parallel sequencing. Analysis of
mtDNA deletions was done with a new algorithm generated
for detecting both single and multiple mtDNA deletions. Results of the samples were also compared to results from
MLPA analysis. Results: Using this method we were able to detect single
and multiple deletions and also deletions that are within
each other. In addition, the exact breakpoints are disclosed
and the heteroplasmic rate defined. Based on the analysis of
the control samples, the new method seems to be reliable in
detecting all types of mtDNA deletions. Conclusions: Our new detection method sets the mtDNA
analysis to a new level and increases understanding of the
mtDNA deletions’ variety and expectantly their clinical
relevance. T. Suominen: None. S. Penttilä: None. M. Jokela:
None. J. Palmio: None. B. Udd: None. ticularly to MS. Supporting grant: FISM 2015 N. Barizzone*: None. F. Clarelli*: None. E. Man-
gano*: None. C. Basagni: None. M. Zuccalà: None. S. Anand: None. M. Sorosina: None. E. Mascia: None. G. De Bellis: None. F. Esposito: None. D. Vecchio: None. G. Predebon: None. C. Comi: None. R. Cantello: None. V. Martinelli: None. G. Comi: None. M. Leone: None. R. Bordoni*: None. F. Martinelli-Boneschi*: None. S. D'Alfonso*: None. N. Barizzone*: None. F. della Salute e della Scienza di Torino, Torino, Italy, 6The
Neuroscience Institute of Torino, Torino, Italy della Salute e della Scienza di Torino, Torino, Italy, 6The
Neuroscience Institute of Torino, Torino, Italy diseases such as Limb-Girdle muscular dystrophy and
Charcot-Marie-Tooth
are
getting
increasingly
hetero-
geneous as genetic discoveries proliferate and different
genetic testing strategies evolve. Through a 2-year period,
we have offered an expanded gene panel based on Next-
generation Sequencing (NGS) of patients with NMDs. We
performed NGS-analysis with a gene list starting with 256
genes that were expanded to 328 genes during the study on
200 patients where standard testing had yielded no specific
diagnosis. Amyotrophic lateral sclerosis (ALS) is characterized by
motor neuron degeneration in the primary motor cortex,
brainstem and spinal cord. The hexanucleotide repeat
expansion in C9ORF72 gene (C9ORF72-HRE) is the most
frequent genetic cause of ALS. Since many familial pedi-
grees showed incomplete penetrance and heterogeneous
clinical signs, several genetic factors have been analyzed as
possible modifier in ALS. The length of GCG repeat in non-
imprinted in Prader-Willi/Angelman syndrome 1 (NIPA1),
identified as risk factor for ALS susceptibility, has been
investigated as a possible modifier factor for C9ORF72-
HRE ALS patientsNIPA1 long alleles frequency was sig-
nificantly higher in C9ORF72-HRE sporadic ALS carriers
(15.2%) (vs 5.5% in all other sporadic ALS cases and 3.9%
in controls; Dekker 2016) while no difference was observed
in C9ORF72-HRE ALS carriers (3.0%) compared to con-
trols (3.5%) (Van Blitterswijk 2014). Based on this, we
investigate the possible role of NIPA1-GCG repeat length
as modifier of the C9ORF72 phenotype in a large cohort of
558 Italian ALS sporadic cases and 483 Italian controls. To
evaluate the effect of short or long repeat length we
dichotomized NIPA1 alleles as ‘normal’ ((GCG) 7-8), or
‘long’ (>8 GCG repeats). We didn’t observe a higher fre-
quency of NIPA1 long alleles in C9ORF72-HRE carriers
(4%) compared to C9ORF72-HRE negative patients (4.4%)
and healthy controls (5%). This sample size allowed to
replicate the modifier effect observed in the literature (92%
power, p.=0.05). In conclusion, we did not confirm a role of
NIPA1 repeat length as a modifier factor of the C9ORF72
ALS phenotype. Methods and Results: NGS was performed using
Illumina TruSight One Sequencing panel (4813 genes)
and run on an Illumina NextSeq 500 Desktop Sequencer. Analyses were done using Illumina BaseSpace BWA
Enrichment Workflow and annotation, filtration and variant
curation were done using Cartagenia Bench and Alamut
Vision. 1Human Genetics Lab, Department of Health Sciencs,
University of Eastern Piedmont, Novara, Italy, 2"Rita Levi
Montalcini" Department of Neuroscience, Neurology II, ALS
center, University of Torino, Torino, Italy, 3ALS center AOU
Maggiore della Carità, Novara, Italy, 4ALS center, "Rita Levi
Montalcini" Department of Neuroscience, University of Torino,
Torino, Italy, 5The Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Torino, Italy, 6The
Neuroscience Institute of Torino, Torino, Italy Among 200 patients, we received a diagnosis in
approximately 40% of the patients, including some patients
with
variants considered
to
be of uncertain
clinical
significance (VUS). Many of the patients presented with
atypical phenotypes in relation to the genetic diagnosis. Low coverage on exon 1 is a common problem, and
detection of rearrangements is a limitation of the method,
particularly in NMD patients. Methods and Results: NGS was performed using
Illumina TruSight One Sequencing panel (4813 genes)
and run on an Illumina NextSeq 500 Desktop Sequencer. Analyses were done using Illumina BaseSpace BWA
Enrichment Workflow and annotation, filtration and variant
curation were done using Cartagenia Bench and Alamut
Vision. Among 200 patients, we received a diagnosis in
approximately 40% of the patients, including some patients
with
variants considered
to
be of uncertain
clinical
significance (VUS). Many of the patients presented with
atypical phenotypes in relation to the genetic diagnosis. Low coverage on exon 1 is a common problem, and
detection of rearrangements is a limitation of the method,
particularly in NMD patients. Conclusions: An extended gene panel designed for
NMDs resulted in a high diagnostic rate similar to whole
exom sequencing studies (WES) on NMD patients. We
recommend using a broad gene panel for NMDs as
phenotypic overlap is getting more evident from recent
genetic findings. Conclusions: An extended gene panel designed for
NMDs resulted in a high diagnostic rate similar to whole
exom sequencing studies (WES) on NMD patients. We
recommend using a broad gene panel for NMDs as
phenotypic overlap is getting more evident from recent
genetic findings. T. Fagerheim: None. G.Å.M. Hansen: None. B. Nygård: None. K. Arntzen: None. K. Ørstavik: None. M. Rasmussen:
None. Ø. Nilssen:
None. C. Jonsrud: None. L. Corrado *: None. A. Di Pierro: None. M. Brunetti:
None. N. Barizzone *: None. M. Barberis: None. R. Croce: None. E. Bersano: None. F. De Marchi: None. A. Calvo: None. C. Moglia: None. L. Mazzini: None. A. Chiò *: None. S. D' Alfonso *: None. P10.43C P10.43C
SMA-affected individuals with one copy of SMN2 gene P10.41A Evaluation of NIPA1 repeat as potential disease modifier in
Italian amyotrophic lateral sclerosis cases carrying
C9ORF72 expansion L. Corrado *1, A. Di Pierro1, M. Brunetti2, N. Barizzone *1,
M. Barberis2, R. Croce1, E. Bersano3, F. De Marchi3,
A. Calvo4,5,6, C. Moglia4,5, L. Mazzini3, A. Chiò *4,5,6, S. D'
Alfonso *1 P10.40D NGS-testing using an expanded gene panel on
Neuromuscular patients in Norway-results and limitations 1Department of Health Sciences, University of Eastern
Piedmont, Novara, Italy, 2IRCAD (Interdisciplinary Research
Center of Autoimmune Diseases), Novara, Italy, 3Laboratory
of Human Genetics of Complex Neurological Disorders,
Institute of Experimental Neurology (INSPE), Division of
Neuroscience, San Raffaele Scientific Institute, Milan, Italy,
4National Research Council of Italy, Institute for Biomedical
Technologies, Segrate, Milan, Italy, 5Department of
Neurology, Division of Neuroscience, Scientific Institute San
Raffaele, Milan, Italy, 6MS Centre, SCDU Neurology, AOU T. Fagerheim1, G. Å. M. Hansen1, B. Nygård1, K. Arntzen1,
K. Ørstavik2, M. Rasmussen2, Ø. Nilssen1, C. Jonsrud1
1University Hospital of North Norway, Tromsoe, Norway,
2University Hospital of Oslo, Oslo, Norway T. Fagerheim1, G. Å. M. Hansen1, B. Nygård1, K. Arntzen1,
K. Ørstavik2, M. Rasmussen2, Ø. Nilssen1, C. Jonsrud1
1University Hospital of North Norway, Tromsoe, Norway,
2University Hospital of Oslo, Oslo, Norway T. Fagerheim1, G. Å. M. Hansen1, B. Nygård1, K. Arntzen1,
K. Ørstavik2, M. Rasmussen2, Ø. Nilssen1, C. Jonsrud1 T. Fagerheim1, G. Å. M. Hansen1, B. Nygård1, K. Arntzen1,
K. Ørstavik2, M. Rasmussen2, Ø. Nilssen1, C. Jonsrud1 1University Hospital of North Norway, Tromsoe, Norway,
2University Hospital of Oslo, Oslo, Norway Introduction: Neuromuscular disorders (NMDs) contain a
broad group of disorders often with overlapping clinical
signs difficult to differentiate. Some of the more frequent 334 J. del Picchia Carlsbad, CA, United States, 5Center for Rare Diseases,
University Hospital of Cologne, Cologne, Germany Carlsbad, CA, United States, 5Center for Rare Diseases,
University Hospital of Cologne, Cologne, Germany numbers of the centromeric homological copy of SMN gene
(SMN2) producing only 10% of functional protein. On the
contrary in this study an attention has been turned to
patients with one copy of the SMN2 gene. Introduction: Spinal muscular atrophy (SMA) is a first
genetic condition for which ASO therapy has been FDA-
and
EMA-approved. SPINRAZA
are
splice-correcting
ASOs targeting SMN2 RNA which produce increased
SMN levels. Despite impressive clinical improvements in
some patients, there is still a need for additional therapeutic
approaches to treat the full spectrum of SMA patients. Therefore, identifying additional SMN-independent ther-
apeutic approaches are warranted. We identified neurocalcin
delta (NCALD) reduction as a protective genetic modifier in
asymptomatic SMN1-deleted individuals and across species
(Riessland et al., AJHG 2017). Materials and methods: Eight SMA patients with
pathogenic variants in SMN1 gene and one copy of
SMN2 gene have been analyzed. MLPA and sequence
analysis have been performed previously. (Tab. 1) Tab.1
Characteristics of patients in current study Sample
№
Age
SMA Type SMN1
copy number
SMN1,
minor
mutation
SMN2
copy number
690
4 m
1
0
–
1
1485
3 m
1
0
–
1
1510
9 m
1
0
–
1
4291
11 m
1
0
–
1
7600
10 d
0
0
–
1
9664
12 d
0
0
–
1
8589
8 y 8
m
2
1
p.Thr274Ile 1
8826
1 y 7
m
2
1
p.Thr274Ile 1 Materials and methods: In collaboration with IONIS
Pharmaceuticals, Ncald-ASOs were developed to reduce
NCALD level. 30 Ncald-ASOs were generated and tested in
cells and adult mice; the three most efficient were tested for
tolerability and efficiency in the neonatal Taiwanese SMA
mouse model. Ncald3-ASO showed the optimal viability,
with non-toxic effects and the best decrease of protein
expression: 75% in brain and 80% in spinal cord. Results: Upon optimization, we performed a blinded
preclinical study using presymptomatic injection of low-
dose
SMN+Ncald-ASOs
compared
to
SMN+control-
ASOs, where Ncald- or control ASOs were injected ICV
at P2 and SMN-ASOs subcutaneously at P1. Our results
showed a significant increase in compound muscle action
potential, neuromuscular junction size and proprioceptive
input in SMN+Ncald treated SMA mice at P21 and P90
and analyses for P180 are in progress. Carlsbad, CA, United States, 5Center for Rare Diseases,
University Hospital of Cologne, Cologne, Germany Results: The SMA types had varied from Type 0 with
reduced fetal movements, respiratory failure immediately
after birth, lack of the joints mobility and severe muscle
weakness to Type 2 “strong”. Patients with Types 0 and 1
have had homozygous deletion of the SMN1 gene. And
patients with milder SMA type 2 have been compound
heterozygotes (with deletion and pathogenic SMN1 variant
p.Thr274Ile close to a wild type). Conclusion: These findings suggest that a combinatorial
approach using SMN-dependent and SMN-independent
ASO-therapy - resembling a condition found in asympto-
matic individuals - further ameliorates disease symptoms. Conclusion: The variability of the SMA phenotypes is
observed even with a single copy of the SMN2 gene. It can
be important of the new opportunities in the therapy
of SMA. Conclusion: The variability of the SMA phenotypes is
observed even with a single copy of the SMN2 gene. It can
be important of the new opportunities in the therapy
of SMA. Acknowledgments:DFG, CMMC, SMA-Europe and O. Päsel foundation V. Zabnenkova: None. T. Milovidova: None. O. Schagina: None. A. Polyakov: None. V. Zabnenkova: None. T. Milovidova: None. O. Schagina: None. A. Polyakov: None. S. Schneider: None. L. Torres-Benito: None. V. Grysko: None. R. Rombo: None. K.K. Ling: None. F. W. Rigo: None. C.F. Bennett: None. B. Wirth: None. R. Rombo1,2,3, K. K. Ling4, F. W. Rigo4, C. F. Bennett4, S. Schneider1,2,3, L. Torres-Benito1,2,3, V. Grysko1,2,3, 1Institute of Human Genetics, Cologne, Germany, 2Center for
Molecular Medicine (CMMC), Cologne, Germany, 3Institute
for Genetics, Cologne, Germany, 4Ionis Pharmaceuticals, H. Law, G. Tan, C. Yoon, Y. Tan, A. H. M. Lai Combined therapy using low-dose SMN plus Ncald ASOs
further ameliorates disease symptoms in a severe mouse
model for spinal muscular atrophy Combined therapy using low-dose SMN plus Ncald ASOs
further ameliorates disease symptoms in a severe mouse
model for spinal muscular atrophy Prenatal diagnosis of spinal muscular atrophy in 2 couples
with unusual gene arrangement in the SMA region at 5q13 Prenatal diagnosis of spinal muscular atrophy in 2 couples
with unusual gene arrangement in the SMA region at 5q13 H. Law, G. Tan, C. Yoon, Y. Tan, A. H. M. Lai B. Wirth1,2,3,5 S. Schneider1,2,3, L. Torres-Benito1,2,3, V. Grysko1,2,3,
R. Rombo1,2,3, K. K. Ling4, F. W. Rigo4, C. F. Bennett4,
B. Wirth1,2,3,5 P10.43C
SMA-affected individuals with one copy of SMN2 gene L. Corrado *1, A. Di Pierro1, M. Brunetti2, N. Barizzone *1, L. Corrado *1, A. Di Pierro1, M. Brunetti2, N. Barizzone *1,
M. Barberis2, R. Croce1, E. Bersano3, F. De Marchi3,
A. Calvo4,5,6, C. Moglia4,5, L. Mazzini3, A. Chiò *4,5,6, S. D'
Alfonso *1 L. Corrado *1, A. Di Pierro1, M. Brunetti2, N. Barizzone *1,
M. Barberis2, R. Croce1, E. Bersano3, F. De Marchi3,
A. Calvo4,5,6, C. Moglia4,5, L. Mazzini3, A. Chiò *4,5,6, S. D'
Alfonso *1 M. Barberis2, R. Croce1, E. Bersano3, F. De Marchi3,
4 5 6
4 5
3
4 5 6 M. Barberis2, R. Croce1, E. Bersano3, F. De Marchi3, V. Zabnenkova, T. Milovidova, O. Schagina, A. Polyakov
Research Centre for Medical Genetics, Moscow, Russian
Federation V. Zabnenkova, T. Milovidova, O. Schagina, A. Polyakov V. Zabnenkova, T. Milovidova, O. Schagina, A. Polyakov Research Centre for Medical Genetics, Moscow, Russian
Federation Research Centre for Medical Genetics, Moscow, Russian
Federation Introduction: Spinal muscular atrophy (SMA) is a neuro-
degenerative disease characterized by loss of lower motor
neurons of spinal cord. SMA is caused by mutations in the
telomeric copy of the survival motor neuron gene (SMN1). Affected individuals with damaged SMN1 gene seem to
have milder form of the disease with increased copy Abstracts from the 51st European Society of Human Genetics Conference: Posters 335 Carlsbad, CA, United States, 5Center for Rare Diseases,
University Hospital of Cologne, Cologne, Germany .
ombo
,
.
.
ing ,
. W.
igo , C.
.
ennett ,
B. Wirth1,2,3,5 Results: Gene
Father
Mother
SMA child
Fetus
Couple 1
NAIP
1
3
1
2
SMN1
1
1
0
1
SMN2
1
2
2
0
Couple 2
NAIP
1
2
1
2
SMN1
1
1
0
2
SMN2
1
2
3
0 Result: From 150 studied families, a mutation in SMN1
genes identified in 115 families. Homozygosity mapping in
44 families (29.3%) showed linkage in three families to
three different genes. The mutations were in DNAJB2,
SIGMAR1, and PLEKHG5 genes. In 5 families tested by
WES, 3 families had a pathogenic variant in the TNNT1,
TPM3 and TTN genes. ACMG guideline were used to
assess the pathogenicity of the new variants. Result: From 150 studied families, a mutation in SMN1
genes identified in 115 families. Homozygosity mapping in
44 families (29.3%) showed linkage in three families to
three different genes. The mutations were in DNAJB2,
SIGMAR1, and PLEKHG5 genes. In 5 families tested by
WES, 3 families had a pathogenic variant in the TNNT1,
TPM3 and TTN genes. ACMG guideline were used to
assess the pathogenicity of the new variants. Conclusions: Both pregnancies were unaffected. One of
them was a carrier and both carried no SMN2 genes. MLPA
analysis confirmed the phase of gene arrangement for
couple 1, but presented more possibilities for couple 2. This
finding shows the complexity of gene arrangement due to
deletion/gene conversion in the SMA region. Conclusion: The use of MLPA in concomitant with STR
markers in typical SMA cases can increase the rate of
mutation detection with a very low cost. Z. Sharifi: None. H. Noferesti: None. F. Golnabi: None. A. Sarhadi Bandehi: None. F. Forouzesh: None. M. Hashemi: None. M. Abiri: None. S. Zeinali: None. S. Bahramizadegan: None. H. Law: None. G. Tan: None. C. Yoon: None. Y. Tan:
None. A.H.M. Lai: None. Greencross labs, Yongin-si, Korea, Republic of 1Kawsar Human Genetics Research Center (KHGRC), Tehran,
Iran, Islamic Republic of, 2Department of genetics, Islamic
Azad University, Tehran medical sciences branch, Tehran,
Iran, Islamic Republic of, 3Department of Medical Genetics,
School of Medicine, Iran University of Medical Sciences,
Tehran, Iran, Islamic Republic of, 4Molecular Medicine
Department, Biotechnology Research Center, Pasteur Institute
of Iran, Tehran, Iran, Islamic Republic of, 5Kawsar Human
Genetics Research Center (KHGRC), Tehran, Islamic Republic
of Iran, Tehran, Iran, Islamic Republic of Introduction: The Spinocerebellar ataxias (SCA) is auto-
somal dominant diseases characterized by heterogeneous
group of nuerodegerative disorders with variable expres-
sion. CAG repeat expansions in the causative genes have
been used for the diagnoses of patients with ataxia. We
investigated five types of SCAs in 149 unrelated patients
with ataxia to elucidate the distribution of CAG repeats and
the mutation spectrum of SCAs in Korea. Materials and methods: In order to evaluate CAG repeat
size ranges of SCAs by capillary electrophoresis analysis,
Genomic DNA was extracted from peripheral blood using
the Chemagic DNA Blood 200 Kit (Chemagen, Baesweiler, Background: Spinal muscular atrophy (SMA) is highly
heterogeneous disorder and the second most common KK Women's and Children's Hospital, Singapore, Singapore B. Wirth1,2,3,5 Introduction: Spinal muscular atrophy (SMA) is a com-
mon autosomal recessive disease with a carrier rate of about
1/50 worldwide. It is characterized by progressive muscle J. del Picchia 336 diseases after thalassemia in Iran. Autosomal recessive
(AR) forms of diseases are more common in populations
with higher rate of consanguineous marriages. Autozygosity
mapping is a powerful technique to track the defective gene
in consanguineous populations like Iran. weakness and atrophy. There are three major types of SMA
distinguished by the severity and age of onset. More than
94% of SMA patients, regardless of clinical type, have
homozygous absence of SMN1 gene. We report 2 cases of
prenatal diagnosis with previous birth of SMA affected
children due to absence of SMN1 gene. weakness and atrophy. There are three major types of SMA
distinguished by the severity and age of onset. More than
94% of SMA patients, regardless of clinical type, have
homozygous absence of SMN1 gene. We report 2 cases of
prenatal diagnosis with previous birth of SMA affected
children due to absence of SMN1 gene. weakness and atrophy. There are three major types of SMA
distinguished by the severity and age of onset. More than
94% of SMA patients, regardless of clinical type, have
homozygous absence of SMN1 gene. We report 2 cases of
prenatal diagnosis with previous birth of SMA affected
children due to absence of SMN1 gene. Methods: In this study, all suspected patients to SMA
were examined by MLPA to screen for deletion/ duplica-
tions of SMN1,2 genes. Subsequently the families with no
mutation were investigated by homozygosity mapping with
the
help
of
STR
markers
linked
to
the
DNAJB2,
IGHMBP2, SIGMAR1, and PLEHG5 genes. These genes
are the responsible for atypical form of SMA with AR
inheritance. The patients who showed linkage to the
mentioned genes were directly sequence. Finally, Whole
Exome sequencing (WES) was done for the 5 patients who
did not show any linkage with any of genes in this study. Methods: In this study, all suspected patients to SMA
were examined by MLPA to screen for deletion/ duplica-
tions of SMN1,2 genes. Subsequently the families with no
mutation were investigated by homozygosity mapping with
the
help
of
STR
markers
linked
to
the
DNAJB2,
IGHMBP2, SIGMAR1, and PLEHG5 genes. These genes
are the responsible for atypical form of SMA with AR
inheritance. Molecular analysis of CAG repeats in five spinocerebellar
ataxias: The distribution and reference ranges of SCA1, 2,
3, 6 and 7 Molecular analysis of CAG repeats in five spinocerebellar
ataxias: The distribution and reference ranges of SCA1, 2,
3, 6 and 7 Z. Sharifi1,2, H. Noferesti1, F. Golnabi1, A. Sarhadi Bandehi1,
F. Forouzesh2, M. Hashemi2, M. Abiri3, S. Zeinali4,1,
S. Bahramizadegan5 Z. Sharifi1,2, H. Noferesti1, F. Golnabi1, A. Sarhadi Bandehi1,
F. Forouzesh2, M. Hashemi2, M. Abiri3, S. Zeinali4,1,
S. Bahramizadegan5 M. Lee, J. Hyun, E. Kim, E. Lee KK Women's and Children's Hospital, Singapore, Singapore The patients who showed linkage to the
mentioned genes were directly sequence. Finally, Whole
Exome sequencing (WES) was done for the 5 patients who
did not show any linkage with any of genes in this study. Materials and methods: Presence of SMN1 gene exons 7
and 8 in the fetuses was detected by HinfI and DdeI digests. Copy numbers of SMN1, SMN2 and NAIP genes of both
families were further analysed by MLPA (P021 MRC-
Holland) to confirm the results and to determine the phase
of gene arrangement. Materials and methods: Presence of SMN1 gene exons 7
and 8 in the fetuses was detected by HinfI and DdeI digests. Results: Results: 337 Abstracts from the 51st European Society of Human Genetics Conference: Posters Germany). Amplified products were injected into an ABI
3500xL Genetic analyzer (Applied Biosystems, Foster City,
CA, USA). Amplicon length was calculated by comparison
with the GS500-ROX molecular weight standard by using
the GeneMarker v.2.2 software (SoftGenetics, State Col-
lege, PA, USA). was available for this study. Initially, the target region of
MYOcap consisted of exons and UTRs of 180 known or
putative myopathy-causing genes. MYOcap has since been
gradually expanded to target 328 genes. Furthermore,
sensitivity of the assay has been enhanced by optimising
the probes targeting the low covered regions. Results: The normal and pathologic CAG repeat size
ranges were established at five SCA loci. The total
prevalence of the five types of SCAs was 14.8% in the
149 patients with ataxia, regardless of their family history. The most frequent type was SCA 2 (5.6%), followed by
SCA 6 (5.4%). SCA1, SCA7, and SCA 3 were less
frequent, affecting 3.0%, 2.5%, and 0.9% of the cases,
respectively. Results: Irrespective of version, the coverage of MYOcap
reached at least 20X in 94% of the target region. However,
recurrent
low
coverage
(<20X)
areas
were
detected. Optimised probe design improved the coverage for majority
of these regions but some low covered areas remained. These
are
areas
that
are
difficult
to
sequence
or
unambiguously map. Conclusions: There are genomic regions that are resistant
to even targeted MPS. Currently, these areas have to be
studied by complementary methods. Conclusions: We determined the CAG repeat size ranges
of each SCA type on normal and pathologic alleles. This
study will provide the characteristics of the SCA mutations,
and an effective strategy for the molecular diagnosis of
SCAs in Koreans. Conclusions: We determined the CAG repeat size ranges
of each SCA type on normal and pathologic alleles. This
study will provide the characteristics of the SCA mutations,
and an effective strategy for the molecular diagnosis of
SCAs in Koreans. S. Lehtinen: None. S. Penttilä: None. M. Arumilli:
None. P. Hackman:
None. J. Palmio:
None. B. Udd: None. M. Lee: None. J. Hyun: None. E. Kim: None. E. Lee: None. M. Lee: None. J. Hyun: None. E. Kim: None. E. Lee: None. P10.50B A Faroese founder variant in TBCD causes early onset,
progressive encephalopathy with a homogenous clinical
course P10.49A Improving coverage of muscle specific genes in targeted
massively parallel sequencing J. Ek1, S. Grønborg1,2, L. Risom1, K. B. Larsen3,4, D. Scheie3,
Y. Petkov5, V. A. Larsen6, M. Dunø1, F. Joensen7,
E. Østergaard1 S. Lehtinen1, S. Penttilä1, M. Arumilli2, P. Hackman2,
J. Palmio3, B. Udd2,3,4 1Neuromuscular Research Unit, University of Tampere,
Tampere, Finland, 2Folkhälsan Institute of Genetics and
Department of Medical Genetics, Medicum, University of
Helsinki, Helsinki, Finland, 3Neuromuscular Research Center,
Department of Neurology, Tampere University Hospital,
Tampere, Finland, 4Department of Neurology, Vaasa Central
Hospital, Vaasa, Finland 1Department of Clinical Genetics, University Hospital
Copenhagen, Copenhagen, Denmark, 2Center for Rare
Diseases, Department of Pediatrics, University Hospital
Copenhagen, Copenhagen, Denmark, 3Department of
Pathology, University Hospital Copenhagen, Copenhagen,
Denmark, 4Department of Neuropathology and Ocular
Pathology, John Radcliffe Hospital, Oxford University
Hospital, Headington, United Kingdom, 5Department of
Pediatrics, Sydvestjysk Sygehus, Esbjerg, Denmark,
6Department of Radiology, University Hospital Copenhagen,
Copenhagen, Denmark, 7Department of Pediatrics, National
Hospital of the Faroe Islands, Tórshavn, Faroe Islands Introduction: Massively parallel sequencing (MPS) meth-
ods have transformed the genetic analysis of neuromuscular
disorders. These disorders are both genetically and pheno-
typically heterogeneous and therefore laborious to study. However, low coverage regions challenge the comprehen-
siveness of MPS studies. Introduction: Faroe Islands is an archipelago of 18 islands
with around 49,000 inhabitants, which is situated between
Iceland and Norway in the North Atlantic Sea. The Faroese
population originates from a small settlement and several
genetic disorders have an increased incidence in the present
population due to founder effect, e.g. mitochondrial ence-
phalomyopathy due to a SUCLA2 variant and Aicardi
Goutières syndrome. Variants in a host of structural
microtubulin-associated proteins have been identified to
cause progressive neurodegenerative disorders. TBCD is
one of five tubulin-specific chaperones and is required for Our research unit utilises a targeted MPS assay, MYOcap,
in clinical diagnostics of myopathy. The aim of this study
was to systematically survey the target coverage and
especially concentrate on defining the low covered regions
in different versions of MYOcap. Furthermore, we wanted
to examine the effect of probe design optimisation on
boosting the coverage of the target region. Materials and methods: Approximately 1500 samples
had previously been analysed with one of the five versions
of MYOcap. Coverage information from the whole cohort J. del Picchia 338 nystagmus in some. Identical to the Saudi family, the
homozygous
missense
variation
(c.1478A>G
or
p. P10.49A His493Arg) located in the second active site of the TDP1
gene (tyrosyl-DNA phosphodiesterase 1), was found to
segregate in these two families. SNP array analysis in both
probands identified a unique and shared haplotypic back-
ground. The single genetic defect against which SCAN1
occurs would explain the relative phenotypic homogeneity
observed between affected individuals. In line with the
geographic distribution of this rare clinical entity, our
observations suggest a common origin of the TDP1
c.1478A>G molecular alteration. reversible
assembly
of
tubulin-heterodimer. Recently,
mutations in TBCD have been identified in patients with
distinct progressive encephalopathy with a broad clinical
spectrum. reversible
assembly
of
tubulin-heterodimer. Recently,
mutations in TBCD have been identified in patients with
distinct progressive encephalopathy with a broad clinical
spectrum. Materials and methods: Patients with encephalopathy
were collected from families originating from the Faroe
Islands. All were tested negative for the Faroese SUCLA2
founder variant. Whole exome sequencing (WES) was
performed to search for a disease-causing variant. Results: By WES, we found several patients to be
homozygous for a TBCD missense variant, with a high
carrier frequency in the Faroese population. These patients
presented with an early-onset, progressive encephalopathy
with features of primary neurodegeneration and a homo-
genous clinical course. We present a detailed description of
the variant including functional studies, clinical findings,
neuropathology, and MR imaging characteristics of a subset
of these patients, adding insight into the phenotype of
TBCD-related encephalopathy. P. Scott:
None. A. Al-Kindy:
None. R. Nandhagopal: None. P. Scott:
None. A. Al-Kindy:
None. R. Nandhagopal: None. P. Scott:
None. A. Al-Kindy:
None. R. Nandhagopal: None. A new case of rare TRIP4-related neuromuscular disease E. Tsoutsou1, R. Pons2, K. Kekou1, E. A. Makrygianni2,
O. Kenteroglou1, S. Psoni1, S. Amenta3, P. Willems4,
H. Fryssira1 E. Tsoutsou1, R. Pons2, K. Kekou1, E. A. Makrygianni2,
O. Kenteroglou1, S. Psoni1, S. Amenta3, P. Willems4,
H. Fryssira1 Conclusions: The finding of a Faroese founder variant
will allow targeted genetic diagnostics in patients of Faroese
descent as well as improved genetic counseling and testing
of at-risk couples. 1Department of Medical Genetics, Medical School, National
and Kapodistrian University of Athens, "Aghia Sophia"
Children's Hospital, Athens, Greece, 21st Department of
Pediatrics, NKUA, "Aghia Sophia" Children's Hospital,
Athens, Greece, 3"MITERA" Maternity Hospital, Athens,
Greece, 4"GENDIA" Genetic-Diagnostic, Antwerp, Belgium J. Ek: None. S. Grønborg: None. L. Risom: None. K.B. Larsen: None. D. Scheie: None. Y. Petkov: None. V.A. Larsen: None. M. Dunø: None. F. Joensen: None. E. Østergaard: None. J. Ek: None. S. Grønborg: None. L. Risom: None. K.B. Larsen: None. D. Scheie: None. Y. Petkov: None. V.A. Larsen: None. M. Dunø: None. F. Joensen: None. E. Østergaard: None. J. Ek: None. S. Grønborg: None. L. Risom: None. K.B. J. Ek: None. S. Grønborg: None. L. Risom: None. K.B. J. Ek: None. S. Grønborg: None. L. Risom: None. K.B. Larsen: None. D. Scheie: None. Y. Petkov: None. V.A. Larsen: None. M. Dunø: None. F. Joensen: None. E. Østergaard: None. Possible TDP1 founder mutation underlying
spinocerebellar ataxia with axonal neuropathy It was present in heterozygous state in both
parents whose origin is from the same Greek island. software. It was present in heterozygous state in both
parents whose origin is from the same Greek island. We evaluated several tools and propose a unique variant
prioritization score called MPA. MPA is based on curated
interpretation for previously reported variants, biological
assumptions, splice and missense predictors to prioritize all
types of SNV variants. We validated our approach by
comparing
MPA
versus
prediction
tools
in
dbNSFP
including CADD using a dataset composed of DYSF,
DMD, LMNA, NEB and TTN variants extracted from expert-
reviewed (missense n = 246; splice n = 190 pathogenic
variants) and ExAc database (missense n = 603; splice
n = 3441 neutral variants). Conclusion: TRIP4 gene encodes the thyroid receptor-
interacting protein 4, one of the four subunits of the
tetrameric
ASC-1
transcriptional
cointegrator
complex
which is identified to play a significant role in myogenic
differentiation and skeletal myotube growth. The use of
WES
has
been
proven
powerful
in
expanding
the
phenotypical and genetic spectrum of rare congenital
muscle diseases. E. Tsoutsou: None. R. Pons: None. K. Kekou: None. E. A. Makrygianni: None. O. Kenteroglou: None. S. Psoni:
None. S. Amenta:
None. P. Willems:
None. H. Fryssira: None. MPA obtained the best annotation rate for missense and
splice variants. As MPA aggregates results from several
predictors, individual predictors errors are counterweighted,
improving sensitivity and specificity of missense and
splicing variants prédictions, especially in TTN. We propose
a sequential use of MPA, beginning with selection of
variants with higher scores, followed in the absence of
candidate variants, by consideration of variants with lower
scores. Possible TDP1 founder mutation underlying
spinocerebellar ataxia with axonal neuropathy Introduction: Autosomal recessive variants in the TRIP4
gene are known to cause a particular form of congenital
muscular dystrophy, type Davignon-Chauveau (OMIM
#617066) and a congenital spinal muscular atrophy with
bone fractures type 1 (OMIM #616866) as well. Both dis-
orders are very rare. They have been described in only a few
families and they have overlapping phenotypic features. Possible TDP1 founder mutation underlying
spinocerebellar ataxia with axonal neuropathy P. Scott1, A. Al-Kindy2, R. Nandhagopal3 P. Scott1, A. Al-Kindy2, R. Nandhagopal3 1Molecular Genetics and Genomics Laboratory, Sultan
Qaboos University Hospital, Muscat, Oman, 2Genetics and
Developmental Medicine Clinic, Sultan Qaboos University
Hospital, Muscat, Oman, 3Neurology Unit, Sultan Qaboos
University Hospital, Muscat, Oman Material and Methods: We report a new case of TRIP4-
related
neuromuscular
disease. A
14-year-old
female
patient, the first child of two healthy apparently non-
consaquineous parents, presented with extremely low body
weight (BW:23 Κg<<3rd centile), fatigue, muscular weak-
ness and severe scoliosis since the first year of life. Neurological examination revealed muscular weakness,
mainly
involving
truncal
and
proximal
lower
limps. Examination of the respiratory, gastrointestinal and cardi-
ovascular
systems
did
not
reveal
abnormalities. Her
intelligence
was
normal. Whole
Exome
Sequencing
(WES) of the patient and both parents was performed. Spinocerebellar ataxia with axonal neuropathy (SCAN1), an
autosomal recessive form of hereditary ataxia, has so far
been reported in a single extended Saudi Arabian family. We here describe the first independent observation of the
condition in two apparently unrelated Omani families. Clinical presentation consisted of early-adulthood onset of
progressive ataxia with peripheral axonal motor and sensory
neuropathy. Detailed examination revealed significant distal
lower limb weakness, fasciculation, amyotrophy and hypo/
areflexia and variable degrees of such findings in the distal
upper limbs, distal sensory neuropathy in lower limb and
high steppage gait with tandem ataxia, in addition to mild
finger to nose incoordination and ill sustained gaze evoked Results:
A
pathogenic
homozygous
TRIP4:c.1678
+1_1678+2insC novel variant was identified in intron 12
of the TRIP4 gene. The variant is predicted to disrupt the
normal splice site as confirmed by splice site prediction Results:
A
pathogenic
homozygous
TRIP4:c.1678
+1_1678+2insC novel variant was identified in intron 12
of the TRIP4 gene. The variant is predicted to disrupt the
normal splice site as confirmed by splice site prediction Abstracts from the 51st European Society of Human Genetics Conference: Posters 339 software. P10.53A Evaluation of missense and splicing in silico predictions
tools and implementation of an efficient SNV prioritization
NGS pipeline for molecular diagnosis of Myopathies and
Muscular Dystrophies We provide scripts and documentation for an free
academic use validated annotation and prioritization pipe-
line. This pipeline is scaled for panel and exome sequencing
in molecular diagnosis of MMD. K. Yauy1, D. Baux1, H. Pegeot1, C. Van Goethem2, C. Mathieu1,
T. Guignard3, R. Juntas Morales1, D. Lacourt1, M. Krahn4,5,
V. Lektokari6, G. Bonne7, S. Tuffery-Giraud8, M. Koenig1,8,
M. Cossee1,8 K. Yauy1, D. Baux1, H. Pegeot1, C. Van Goethem2, C. Mathieu1,
T. Guignard3, R. Juntas Morales1, D. Lacourt1, M. Krahn4,5,
V. Lektokari6, G. Bonne7, S. Tuffery-Giraud8, M. Koenig1,8,
M. Cossee1,8 K. Yauy: None. D. Baux: None. H. Pegeot: None. C. Van Goethem: None. C. Mathieu: None. T. Guignard:
None. R. Juntas Morales: None. D. Lacourt: None. M. Krahn: None. V. Lektokari: None. G. Bonne: None. S. Tuffery-Giraud:
None. M. Koenig:
None. M. Cossee: None. K. Yauy: None. D. Baux: None. H. Pegeot: None. C. Van Goethem: None. C. Mathieu: None. T. Guignard:
None. R. Juntas Morales: None. D. Lacourt: None. M. Krahn: None. V. Lektokari: None. G. Bonne: None. S. Tuffery-Giraud:
None. M. Koenig:
None. M. Cossee: None. 1Laboratoire de Génétique Moléculaire, CHU Montpellier,
Montpellier, France, 2Laboratoire de Biopathologie Cellulaire
et Tissulaire des Tumeurs, CHU Montpellier, Montpellier,
France, 3Département de Génétique Médicale, Maladies Rares
et Médecine Personnalisée, Centre de Référence Anomalies du
Développement et Syndromes Malformatifs, Plateforme
Recherche de Microremaniements Chromosomiques, Hôpital
Arnaud de Villeneuve, CHU Montpellier, Montpellier, France,
4Université d’Aix Marseille, Inserm, GMGF INSERM-AMU
UMRS910, Marseille, France, 5APHM, Hôpital Timone
Enfants, Département de Génétique Médicale, Marseille,
France, 6The Folkhalsan Institute of Genetics and the
Department of Medical Genetics, Haartman Institute,
University of Helsinki, Helsinki, Finland, 7Sorbonne
Universités, UPMC Univ Paris 06; INSERM U974; Center of
Research in Myology; Institut de Myologie, Paris, France,
8Laboratoire de Génétique des Maladies Rares, EA7402,
Université de Montpellier, Montpellier, France P10.54B Exome trio analysis
revealed
a
UNC13A
variant,
c.1188delC
p. (Asp397Thrfs*107), homozygous and in trans. UNC13A
protein is located in cholinergic neuromuscular synapses
and in the majority of glutamanergic synapses in the brain. Mice with Munc13a homozygous null mutations are
paralysed and die. Two patients with homozygous muta-
tions have been reported in the literature, with features that
may include global delay, microcephaly, seizures, fatal
myasthenia with respiratory failure, hypotonia, myopathy. Our patient had progressive hypotonia, myopathy and sei-
zures, and a fatal course with features strikingly similar to
the two reported patients. Umbilical hernia, Morgagni her-
nia, normocephaly and newly reported pathological findings
add to the clinical phenotype and confirm homozygous null
mutations in UNC13A cause a unique, recognizable, and
severe phenotype in humans. kyphoscoliosis developed in infancy. She had severe con-
stipation and urinary retention. She died at 8 months of
respiratory failure and had not met any significant devel-
opmental milestones. Autopsy revealed an unusual pattern
of myelination of the brain not previously documented. EM
of skin revealed lysosomal inclusions. Exome trio analysis
revealed
a
UNC13A
variant,
c.1188delC
p. (Asp397Thrfs*107), homozygous and in trans. UNC13A
protein is located in cholinergic neuromuscular synapses
and in the majority of glutamanergic synapses in the brain. Mice with Munc13a homozygous null mutations are
paralysed and die. Two patients with homozygous muta-
tions have been reported in the literature, with features that
may include global delay, microcephaly, seizures, fatal
myasthenia with respiratory failure, hypotonia, myopathy. Our patient had progressive hypotonia, myopathy and sei-
zures, and a fatal course with features strikingly similar to
the two reported patients. Umbilical hernia, Morgagni her-
nia, normocephaly and newly reported pathological findings
add to the clinical phenotype and confirm homozygous null
mutations in UNC13A cause a unique, recognizable, and
severe phenotype in humans. V. Martínez-Montoya: None. L.M. Gonzalez-Huerta:
None. M.R. Rivera-Vega: None. J.M. Valdes-Miranda:
None. R. Vega-Gama: None. N. Xilotl-DeJesus: None. M. G. Tovar-Ayala: None. A. Martínez: None. S.A. Cuevas-
Covarrubias: None. S. Dyack: D. Speakers Bureau/Honoraria (speakers
bureau, symposia, and expert witness); Modest; Shire,
Alexion. F. Consultant/Advisory Board; Modest; Biomarin. N. Snow: None. M. Gregoire: None. A. Oveido: None. P10.54B UNC13A causes a severe and recognizable phenotype in
humans S. Dyack1,2, N. Snow1, M. Gregoire1,2, A. Oveido1,3 S. Dyack1,2, N. Snow1, M. Gregoire1,2, A. Oveido1,3 1IWK Health Centre, Halifax, NS, Canada, 2Department of
Pediatrics, Dalhousie University, Halifax, NS, Canada,
3Department of Pathology and Laboratory Medicine,
Dalhousie University, Halifax, NS, Canada We report an infant with encephalopathy, seizures, neuro-
muscular features, and progressive respiratory failure asso-
ciated with a homozygous null mutation in UNC13A,
further refining the phenotype and confirming its role as a
disease causing gene in humans. Our patient was non dys-
morphic with normal growth parameters who presented
with hypertonia and encephalopathy immediately after
birth. EEG revealed burst suppression. MRI of brain non
diagnostic. Seizures were uncontrolled and severe global
hypotonia developed, with limited movement. A Morgagni
diaphragmatic
hernia,
an
umbilical
hernia,
and Interpretation of Next Generation Sequencing huge amount
of data constitutes the main limitation in molecular genetics
diagnosis. In diagnosis of Myopathies and Muscular Dys-
trophies (MMD), another major issue is to efficiently predict
pathogenicity of variants identified in large genes, espe-
cially TTN, since current in silico prediction tools show
limitations to predict and rank the numerous variants of
such genes. 340 J. del Picchia 25% of the patients presented positive findings with the
NGS panel and in 15% the diagnosis of the dystrophy
subtype was confirmed by molecular study. Of the 20
patients, 2 homozygous patients were identified for dys-
functional dysfunction and an atypical family case with a
heterozygous mutation in DYSF. One of the patients was
compound heterozygote for calpain and another presented
variants of uncertain significance in the CALPN3 and DYSF
genes in spite of despite the fact that they fulfilled clinical
criteria for muscular dystrophy. NGS is a useful tool for the
diagnosis of patients with clinical symptoms of LGMD,
even in patients with atypical clinical characteristics. We
found novel mutations not previously reported in the lit-
erature and a rare mutation in DYSF previously described in
Ashkenazi Jewish population in two members of a family. The NGS should be implemented as a first-line diagnostic
tool in these pathologies kyphoscoliosis developed in infancy. She had severe con-
stipation and urinary retention. She died at 8 months of
respiratory failure and had not met any significant devel-
opmental milestones. Autopsy revealed an unusual pattern
of myelination of the brain not previously documented. EM
of skin revealed lysosomal inclusions. Molecular characterization in Mexican patients with limb-
girdle muscular dystrophy M. Zollino1, S. Frangella1, D. Orteschi1, P. Doronzio1,
S. Lattante1, G. Vento2, G. Zampino3, C. Leoni3, I. Contaldo4,
D. Battaglia4, G. Marangi1 M. Zollino1, S. Frangella1, D. Orteschi1, P. Doronzio1,
S. Lattante1, G. Vento2, G. Zampino3, C. Leoni3, I. Contaldo4,
D. Battaglia4, G. Marangi1 V. Martínez-Montoya1, L. M. Gonzalez-Huerta1, M. R. Rivera-
Vega1, J. M. Valdes-Miranda1, R. Vega-Gama1, N. Xilotl-
DeJesus1, M. G. Tovar-Ayala1, A. Martínez2, S. A. Cuevas-
Covarrubias3 1Institute of Genomic Medicine, Università Cattolica del Sacro
Cuore, Roma, Italy, 2Department for the protection of women's
health and the nascent life, child and adolescent, Fondazione
Policlinico Universitario A. Gemelli, Roma, Italy, 3Center for
Rare Disease and Congenital Defects, Fondazione Policlinico
Universitario A. Gemelli, Roma, Italy, 4Child Neuropsichiatry,
Fondazione Policlinico Universitario A. Gemelli, Roma, Italy 1Genetica, Hospital General de Mexico, Mexico DF, Mexico,
2Genetica, Hospital General de Mexico Facultad de Medicina,
Universidad Nacional Autonoma d Mexico, Mexico DF,
Mexico, 3Genetica, Hospital General de Mexico, Facultad de
Medicina, Universidad Nacional Autonoma de Mexico, Mexico
DF, Mexico The recurrent 1.2-1.5 microdeletion of chromosome region
16p13.11 has long been claimed to be a susceptibility factor
for neurocognitive impairment, autism and epilepsy, with
highly variable phenotypic manifestations, in a double hit
model of pathogenesis. In most cases the deletion is
inherited from a healthy parent. Two patients we observed
with 16p13.11 microdeletion inherited from a healthy par-
ent presented with a severe phenotype, including severe
microcephaly associated with a complex brain malforma-
tion, and microcephaly associated with choreoatetosis and Bone ring muscular dystrophies are neuromuscular dis-
orders of genetic origin. Currently, there are more than
30 subtypes of waist dystrophies. Subtype 2 or LGMD2 is a
group of entities with an autosomal recessive inheritance
pattern. In this study, the genetic profile was characterized
through NGS, DNA direct sequencing and MLPA in
Mexican
patients
with
LGMD. Genomic
DNA
was
obtained from peripheral blood of 20 patients. Variants
were analyzed with SIFT and Mutationtaster softwares;
UMD-DYSF was used for dysfelinopathies . In this work, Abstracts from the 51st European Society of Human Genetics Conference: Posters 341 16p13.11. FISH
reanalysis
on
nuclei
confirmed
the
tetrasomy. dystonia, respectively. The complex brain abnormalities
(corpus callosum agenesis, pachygyria, cerebellar hypo-
plasia and colpocephaly) in the first case prompted us to
perform direct sequencing of the NDE1 gene. A nonsense
variant on the remaining allele was identified, leading to the
diagnosis of autosomal recessive type 4 lissencephaly. Our
second patient was analyzed by WES (trio analysis). We
detected a de novo missense variant in GABRB2, which has
recently been reported in a group of patients with an
overlapping phenotype including choreoatetosis, dystonia,
microcephaly and severe ID with white matter abnormal-
ities. Our results confirm that 16p13.11 deletion itself is not
sufficient to cause the disease, with two possible opposite
scenarios: 1) the microdeletion is involved in the etiology of
the observed condition, when it unmasks a recessive variant
on the remaining allele (i.e.: hemizygosity of a pathogenic
NDE1 variant); 2) the microdeletion acts as innocent
bystander of a different gene variant. Conclusions: We report the first case of 19qter tetrasomy
by intrachromosomal triplication, which was not distin-
guishable from duplication by conventional cytogenetics,
but was clearly identified by microarray analysis. This result
allowed us to interpret the signal pattern of FISH on
metaphases
revealing
that
the
middle
repeat
of
the
triplication was inverted. Cytogenetic and cytogenomic
characterization of the rearrangement supports the mole-
cular mechanisms postulated for the formation of intra-
chromosomal triplications. Microarray analysis allowed the
detection of a cryptic microduplication at 16p. It is difficult
to establish a phenotype-genotype correlation due to the
absence of other reported cases and the probable additive
effect of the 16p13.11 microduplication. V.S. Bugatto: None. W. Montes: None. J.F. Martini:
None. S.P. Duarte: None. S. Massara: None. M. Delea:
None. L. Espeche: None. B. Warszatska: None. A. Solari: None. M. Pérez: None. M.E. Mollica: None. L. Furforo: None. S. Rozental: None. M. Zollino: None. S. Frangella: None. D. Orteschi:
None. P. Doronzio: None. S. Lattante: None. G. Vento:
None. G. Zampino: None. C. Leoni: None. I. Contaldo:
None. D. Battaglia: None. G. Marangi: None. M. Zollino: None. S. Frangella: None. D. Orteschi:
None. P. Doronzio: None. S. Lattante: None. G. Vento:
None. G. Zampino: None. C. Leoni: None. I. Contaldo:
None. D. Battaglia: None. G. Marangi: None. P11.004D
Investigation of genetic variants underlying variability in
patients with 22q11.2 Deletion Syndrome Investigation of genetic variants underlying variability in
patients with 22q11.2 Deletion Syndrome P11.003C
First case of 19q triplication: clinical and cytogenomic
characterization K. Ziemkiewicz1, M. Smyk1, T. Gambin2, A. Kutkowska
Kaźmierczak1, D. M. McDonald-McGinn3, T. B. Crowley3,
M. Piotrowicz4, D. Gieruszczak-Białek5, B. A. Nowakowska1 K. Ziemkiewicz1, M. Smyk1, T. Gambin2, A. Kutkowska
Kaźmierczak1, D. M. McDonald-McGinn3, T. B. Crowley3,
M. Piotrowicz4, D. Gieruszczak-Białek5, B. A. Nowakowska1 V. S. Bugatto, W. Montes, J. F. Martini, S. P. Duarte,
S. Massara, M. Delea, L. Espeche, B. Warszatska, A. Solari,
M. Pérez, M. E. Mollica, L. Furforo, S. Rozental
Centro Nacional de Genética Médica, Buenos Aires, Argentina V. S. Bugatto, W. Montes, J. F. Martini, S. P. Duarte,
S. Massara, M. Delea, L. Espeche, B. Warszatska, A. Solari,
M. Pérez, M. E. Mollica, L. Furforo, S. Rozental 1Department of Medical Genetics, Institute of Mother and
Child, Warsaw, Poland, 2Institute of Computer Science,
Warsaw University of Technology, Warsaw, Poland, 3Human
Genetics, The Children's Hospital of Philadelphia,
Philadelphia, PA, United States, 4Department of Genetics,
Polish Mother’s Memorial Hospital Research Institute, Łódź,
Poland, 5Department of Medical Genetics, Children’s
Memorial Health Institute, Warsaw, Poland Centro Nacional de Genética Médica, Buenos Aires, Argentina Centro Nacional de Genética Médica, Buenos Aires, Argentina Introduction: Intrachromosomal triplications leading to
tetrasomy for the corresponding segment is a rare complex
chromosomal rearrangement. It has been reported for a few
chromosomes but, to our knowledge, there is no case
involving chromosome 19 so far. Here we describe a
13 months old girl with 19q terminal triplication, marked
hypotonia,
developmental
delay,
dysmorphic
features,
abnormal
skull
shape
and
premature
thelarche
and
pubarche. Introduction: The 22q11.2 deletion syndrome (DS) is the
most common microdeletion syndrome with an incidence of
1 in 2000 births. Major clinical characteristics are intellec-
tual disability, congenital heart anomalies, immune system
defects, velopharyngeal abnormalities, facial dysmorphism
and psychiatric disorders. Affected individuals have a
hemizygous 1,5-3Mb deletion at chromosome 22q11.2,
which includes about 50 known genes. Highly variable
expressivity of the clinical features can be observed even in
the patients carrying the same deletions. In the project we
focused on two of the potential mechanisms underlying this
variability: allelic variation within the 22q11.2 region of the
non-deleted chromosome and variants in modifier genes
outside of the deletion. Methods and Results: GTW banding technique (550
band level) showed extra material on 19q. As parental
karyotypes were normal, SKY analysis was performed. It
revealed that the derivative chromosome presented a
uniform coloration corresponding to specific sequences of
chromosome
19
suggesting
a
probable
duplication
19q13.33q13.43. The FISH pattern on metaphase chromo-
somes with 19q subtelomeric probes was not conclusive. Microarray analysis identified a 3.7Mb triplication of the
19q13.42q13.43 segment and a 1.7 Mb microduplication at Methods and Results: GTW banding technique (550
band level) showed extra material on 19q. As parental
karyotypes were normal, SKY analysis was performed. It
revealed that the derivative chromosome presented a
uniform coloration corresponding to specific sequences of
chromosome
19
suggesting
a
probable
duplication
19q13.33q13.43. The FISH pattern on metaphase chromo-
somes with 19q subtelomeric probes was not conclusive. Microarray analysis identified a 3.7Mb triplication of the
19q13.42q13.43 segment and a 1.7 Mb microduplication at 342 J. del Picchia Materials and methods: To identify CNVs in genome,
other
than
22q11.2
deletion,
CNV
calling
algorithm
CoNIFER was implemented on the exome sequencing data
set of 40 patients. Materials and methods: To identify rare and likely
phenotype influencing variants Illumina whole exome
sequencing (WES) in 50 patients with DS was applied. Detailed phenotyping of all patients was performed to
estimate correlation between WES results and specific
phenotypes. Centro Nacional de Genética Médica, Buenos Aires, Argentina Materials and methods: To identify rare and likely
phenotype influencing variants Illumina whole exome
sequencing (WES) in 50 patients with DS was applied. Detailed phenotyping of all patients was performed to
estimate correlation between WES results and specific
phenotypes. Results: In three patients, additionally to 22q11.2 loss,
we found one deletion and three duplication calls that were
verified by aCGH. In the first patient showing, unusual for
22q11.2 DS, regression of cognitive functions and demen-
tia, atypical ~3,34 Mb 22q11 deletion encompassing
TUBA8 gene was present. In second, male patient we
found 365 kb potentially pathogenic duplication of Xq22.3
encompassing the PRPS1 gene and exons 1-5 of MID2 gene
that is associated with X-linked mental retardation. In the
third patient two additional CNVs with uncertain clinical
significance were found: 667 kb deletion of 3p26.3
encompassing exons 1-2 of the CNTN4 gene and 618 kb
duplication of 7q21.3q22.1 including NPTX2 gene. Results: In three patients, additionally to 22q11.2 loss,
we found one deletion and three duplication calls that were
verified by aCGH. In the first patient showing, unusual for
22q11.2 DS, regression of cognitive functions and demen-
tia, atypical ~3,34 Mb 22q11 deletion encompassing
TUBA8 gene was present. In second, male patient we
found 365 kb potentially pathogenic duplication of Xq22.3
encompassing the PRPS1 gene and exons 1-5 of MID2 gene
that is associated with X-linked mental retardation. In the
third patient two additional CNVs with uncertain clinical
significance were found: 667 kb deletion of 3p26.3
encompassing exons 1-2 of the CNTN4 gene and 618 kb
duplication of 7q21.3q22.1 including NPTX2 gene. Results: Analysis of the hemizygous region of 22q11.2
revealed rare nonsynonymous SNVs in HIRA and MRPL40
genes. In the region outside of the deletion we found 14
variants that may be considered as a probable phenotype
modifiers; 7 rare nonsynonymous SNVs in genes associated
with congenital heart malformations were found in the
group of 34 patients with heart defects (CITED2, MED13L,
NKX2-5, NODAL, TLL1, CHD7, MYH6); 6 rare non-
synonymous
SNVs
and
1
frameshift
insertion
were
consistent with patients’ clinical features, including one
variant in gene from genetic pathway of 22q11.2 region
(CDH15). Conclusions: In patients with 22q11.2 DS diagnosed by
MLPA
or
FISH
further
investigation
of
CNVs
is
recommended. This work was granted from National Science Centre
(OPUS NCN 2015/17/B/NZ5/01357 to BN). This work was granted from National Science Centre
(OPUS NCN 2015/17/B/NZ5/01357 to BN) K. Ziemkiewicz: None. Centro Nacional de Genética Médica, Buenos Aires, Argentina M. Smyk: None. T. Gambin:
None. A. Kutkowska Kaźmierczak: None. D.M. McDo-
nald-McGinn: None. T.B. Crowley: None. M. Piotro-
wicz:
None. D. Gieruszczak-Białek:
None. B.A. Nowakowska: None. M. Smyk: None. K. Ziemkiewicz: None. T. Gambin:
None. A. Kutkowska-Kaźmierczak: None. M. Piotro-
wicz: None. D. Gieruszczak-Białek: None. A. Pietrzyk:
None. B.A. Nowakowska: None. M. Smyk: None. K. Ziemkiewicz: None. T. Gambin:
None. A. Kutkowska-Kaźmierczak: None. M. Piotro-
wicz: None. D. Gieruszczak-Białek: None. A. Pietrzyk:
None. B.A. Nowakowska: None. P11.006B Different
clinical features of patients, follow-up, management and
mutation type of MASP1 gene will be discussed in detail. M. Basdemirci:
None. A. Sen:
None. S. Ceylaner: None. synonymous SNVs. To predict the pathogenic potential of
the SNVs, we performed in silico analysis by using the tools
Mutation Taster, FATHMM, PolyPhen 2 and SIFT. The
SNVs found in the CRKL, PI4KA, MAPK1, ZFPM2 and
TANGO2 genes led to aminoacid substitution, changes on
splicing sites or RNA's polyA tail alteration. Of the 51
variants analyzed, 11 variants were predicted as deleterious,
one variant was predicted as protective and 19 variants were
predicted to be tolerable to mutations. describe three new patients of 3MC syndrome in a Turkish
family with a novel mutation of MASP1 gene. Different
clinical features of patients, follow-up, management and
mutation type of MASP1 gene will be discussed in detail. synonymous SNVs. To predict the pathogenic potential of
the SNVs, we performed in silico analysis by using the tools
Mutation Taster, FATHMM, PolyPhen 2 and SIFT. The
SNVs found in the CRKL, PI4KA, MAPK1, ZFPM2 and
TANGO2 genes led to aminoacid substitution, changes on
splicing sites or RNA's polyA tail alteration. Of the 51
variants analyzed, 11 variants were predicted as deleterious,
one variant was predicted as protective and 19 variants were
predicted to be tolerable to mutations. describe three new patients of 3MC syndrome in a Turkish
family with a novel mutation of MASP1 gene. Different
clinical features of patients, follow-up, management and
mutation type of MASP1 gene will be discussed in detail. M. Basdemirci:
None. A. Sen:
None. S. Ceylaner: None. P11.007C
Novel mutation in MASP1 gene in three new patient with
3MC syndrome M. Basdemirci1, A. Sen2, S. Ceylaner3 1Şanlıurfa Education and Research Hospital, Medical
Genetics, Şanlıurfa, Turkey, 2Fırat University School of
Medicine, Medical Genetics, Elazığ, Turkey, 3İntergen Genetic
Center, Medical Genetics, Ankara, Turkey 3MC syndrome encompasses the four autosomal recessive
conditions Malpuech syndrome, Michels syndrome, Min-
garelli syndrome, and Carnevale syndrome. The main fea-
tures of these syndromes are facial dysmorphism that
includes hypertelorism, blepharophimosis, blepharoptosis,
and highly arched eyebrows. Cleft lip and palate, postnatal
growth deficiency, cognitive impairment, and hearing loss
are also consistent findings. Craniosynostosis, radioulnar
synostosis, and genital and vesicorenal anomalies occur in
20 to 30% of cases. 3MC syndrome can be caused by
mutations in either COLEC11or MASP1 genes. Recently,
COLEC10 gene was discovered as the cause of the disease. The 7,5 months old infant was referred to our genetic
policlinic because of her dysmorphic appearance. Frontal
bossing, highly arched eyebrows, hypertelorism, blephar-
ophimosis, flattened nasal root, umbilical hernia, sacral
dimple were observed in physical examination. Horseshoe
kidney was detected on ultrasound. Her sister and brother
also had the similar symptoms. Parents were first degree
cousins. Pedigree analysis showed autosomal recessive trait. Both the patient and her siblings were diagnosed with 3MC
syndrome. Sequence analysis of the MASP1 gene identified
a novel not previously described mutation.In this report, we H. Gaspar: None. S. Gallati: None. J. Sanz: None. H. Gaspar: None. S. Gallati: None. J. Sanz: None. P11.008D Novel frameshift-mutation in EOGT gene in a female
individual with a severe autosomal recessive Adams-Oliver
Syndrome with neurological findings and squamous cell
carcinoma Some of our findings were not previously described in the
literature, which represents a great potential for further
studies and contribution to the field, after validation. The
use of gene panels with candidate genes could accelerate the
search for genetic modifiers and improve the treatment of
22q11.2DS patients. Financial support: FAPESP, Brazil. H. Gaspar, S. Gallati, J. Sanz Division of Human Genetics, Department of Pediatrics,
Inselspital, Bern University Hospital, University of Bern, Bern,
Switzerland Division of Human Genetics, Department of Pediatrics,
Inselspital, Bern University Hospital, University of Bern, Bern,
Switzerland A.G. Dantas: None. N. Nunes: None. C.A. Kim: None. D.C.Q. Soares: None. V.A. Meloni: None. S.I. Belangero:
None. G.G. Carvalheira: None. M.I. Melaragno: None. Adams-Oliver Syndrome (AOS) is defined by aplasia cutis
congenita (ACC) of the scalp and terminal transverse limb
defects (TTLD). The clinical findings are variable, there can
be cardiac, neurologic, renal and ophthalmological findings. Several genes have been identified: ARHGAP31, DLL4,
NOTCH1, and RBPJ are associated with autosomal domi-
nant inheritance, DOCK6 and EOGT with autosomal
recessive inheritance. In autosomal dominant families, the
penetrance can be reduced and neurologic findings are very
rare. To our knowledge, only 8 families have been descri-
bed with mutations in EOGT to date. Three different
mutations were detected so far, 2 missense mutations and a
frameshift mutation, which was found in 6 of the 8 families,
suggesting a founder mutation in consanguineous families
of Arabic ancestry. We here report on a 12 year old girl
from Iraq with consanguineous parents. She was born with
a severe AOS with a large scalp defect (ACC) which was
not treated until recently. Molecular genetic analysis (next
generation sequencing) revealed a novel frameshift muta-
tion in the EOGT gene, most probably in homozygous
constellation. Clinically, the girl showed TTLD on both
hands and feet, and she had neurological findings: spastic
paresis, epilepsy, microcephaly, and suspicion of intellec-
tual deficits. In the course of treatment, a squamous cell
carcinoma was detected, she developed a hydrocephalus. An association between squamous cell carcinoma and AOS
has not been described in literature so far. However, this
case report shows another hint, that autosomal recessive
AOS might be associated with a more severe phenotype. P11.007C P11.007C
Novel mutation in MASP1 gene in three new patient with
3MC syndrome P11.006B Next-generation sequencing (NGS) of nine candidate genes
with custom AmpliSeq in 22q11.2 deletion syndrome
patients Next-generation sequencing (NGS) of nine candidate genes
with custom AmpliSeq in 22q11.2 deletion syndrome
patients M. Smyk1, K. Ziemkiewicz1, T. Gambin1,2, A. Kutkowska-
Kaźmierczak1, M. Piotrowicz3, D. Gieruszczak-Białek4,
A. Pietrzyk5, B. A. Nowakowska1 M. Smyk1, K. Ziemkiewicz1, T. Gambin1,2, A. Kutkowska-
Kaźmierczak1, M. Piotrowicz3, D. Gieruszczak-Białek4,
A. Pietrzyk5, B. A. Nowakowska1 A. G. Dantas1, N. Nunes1, C. A. Kim2, D. C. Q. Soares2, V. A. Meloni1, S. I. Belangero1, G. G. Carvalheira1, M. I. Melaragno1
1Universidade Federal de São Paulo, São Paulo, Brazil,
2Universidade de São Paulo, São Paulo, Brazil 1Institute of Mother and Child, Warsaw, Poland, 2Institute of
Computer Science, Warsaw University of Technology,
Warsaw, Poland, 3Department of Genetics, Polish Mother’s
Memorial Hospital Research Institute, Łódź, Poland,
4Department of Medical Genetics, Children’s Memorial Health
Institute, Warsaw, Poland, 5Department of Genetics and
Pathology, Pomeranian Medical University, Szczecin, Poland 1Universidade Federal de São Paulo, São Paulo, Brazil,
2Universidade de São Paulo, São Paulo, Brazil 22q11.2 deletion syndrome (22q11.2DS) results from
hemizygous 3 Mb deletions of chromosome 22 usually
flanked by low copy repeats (LCR) which represents a
substrate for aberrant recombination. Even though most
22q11.2DS patients have the same size deletions, the phe-
notype is highly variable among individuals. Introduction: The most common microdeletion in humans
is 22q11.2 deletion syndrome (DS) caused by deletion of a
1.5–3 Mb region at chromosome 22q11.2 and occurring in 1
in 2,000 births. This disorder is associated with variable
phenotypes, such as congenital cardiac defects, velophar-
yngeal abnormalities, characteristic facial appearance, renal
anomalies, intellectual disability and psychiatric disorders. One of the potential genetics mechanisms underlying this
variability is the presence of additional copy number variant
(CNV) elsewhere in the genome. The presence of single nucleotide variants (SNVs) on the
remaining allele of 22q11.2 or in other genes outside
22q11.2 region is suggested to have a role in the phenotype
variation observed. We performed Ion Ampliseq in peripheral blood from 30
22q11.2DS patients to sequence the coding regions of nine
candidate genes located in and outside the 22q11.2
hemizygous region. We identified 30 SNVs in 3’UTR
regions and 21 SVNs in exons, being 12 missense and nine Abstracts from the 51st European Society of Human Genetics Conference: Posters 343 describe three new patients of 3MC syndrome in a Turkish
family with a novel mutation of MASP1 gene. P11.009A Unravelling the genetic architecture in an extensive cohort
of Adams-Oliver syndrome patients 344 J. del Picchia J. A. N. Meester1, M. Sukalo2, K. C. Schröder2, D. Schanze2,
G. Vandeweyer1, R. Trembath3, L. Van Laer1, B. L. Loeys1,
M. Zenker2, L. Southgate3,4, W. Wuyts1 1CENTRE FOR DNA FINGERPRINTING AND
DIAGNOSTICS, HYDERABAD, India, 2Kasturba Medical
College, Manipal, India, 3All India Institute of Medical
Sciences, New Delhi, India 1Center of Medical Genetics, University of Antwerp, Antwerp
University Hospital, Edegem, Belgium, 2Institute of Human
Genetics, University Hospital Magdeburg, Magdeburg,
Germany, 3Division of Genetics & Molecular Medicine, King’s
College London, Guy’s Hospital, London, United Kingdom,
4Molecular and Clinical Sciences Research Institute, St
George’s University of London, London, United Kingdom Introduction: In human and other mammalian cells, a
unique large tRNA multi-synthetase complex organizes 9
cytoplasmic aminoacyl-tRNA synthetases consisting of
bifunctional, as well as the monospecific tRNA synthetases
and 3 non-enzyme factors, namely p43 (AIMP1), p38
(AIMP2) and p18 (AIMP3). The p38/AIMP2 protein is a
key component of the multi-ARS complex and is crucial for
the assembly of the complex. AIMP1 has been associated
with hypomyelinating leukodystrophy-3 characterized by
progressive neurodegeneration, microcephaly, spasticity,
coarse facies, progressive contractures, generalized brain
atrophy and early death. Introduction: Adams-Oliver syndrome (AOS) is a rare
developmental disorder, characterized by scalp aplasia cutis
congenita (ACC) and transverse terminal limb defects
(TTLD). Several causative genes have been discovered over
recent years. Autosomal dominant forms of AOS are linked
to mutations in ARHGAP31, DLL4, NOTCH1 or RBPJ,
while DOCK6 and EOGT underlie autosomal recessive
inheritance. Despite these advances, data on the frequency
and distribution of mutations in large cohorts is currently
limited. The purpose of this study was to comprehensively
examine the genetic architecture of AOS in an extensive
European cohort. Introduction: Adams-Oliver syndrome (AOS) is a rare
developmental disorder, characterized by scalp aplasia cutis
congenita (ACC) and transverse terminal limb defects
(TTLD). Several causative genes have been discovered over
recent years. Autosomal dominant forms of AOS are linked
to mutations in ARHGAP31, DLL4, NOTCH1 or RBPJ,
while DOCK6 and EOGT underlie autosomal recessive
inheritance. Despite these advances, data on the frequency
and distribution of mutations in large cohorts is currently
limited. The purpose of this study was to comprehensively
examine the genetic architecture of AOS in an extensive
European cohort. Materials and methods: We ascertained two consangui-
neous families with two affected children each with
microcephaly, refractory seizures, intellectual disability
and spastic quadriparesis. A. DALAL1, A. Shukla2, A. Das Bhowmik1, M. Hebbar2, R. KV2,
G. KM2, N. GUPTA3 1INGEMM-CIBERER-IdiPaz-Hospital Universitario La Paz,
Madrid, Spain, 2The National Human Genome Research
Institute, US National Institutes of Health, Bethesda, MD,
United States P11.009A Brain MR imaging showed
atrophy of cerebrum, cerebellum and spinal cord, prominent
cisterna magna, symmetric T2 hypointensities in the
bilateral basal ganglia and thinning of corpus callosum. Whole exome sequencing was carried out on peripheral
leucocyte DNA of the affected individuals from both
families. Material and Methods: Molecular diagnostic screening
of a cohort of 194 AOS/ACC/TTLD probands and their
families was conducted using a combination of custom-
capture, whole-exome and capillary sequencing analyses. Results: Whole exome sequencing of three affected
individuals from the two unrelated families revealed
c.105C>A [p.(Tyr35Ter)] variant in homozygous state in
AIMP2. The variant is present in a shared homozygous
region, likely due to a founder effect. Results: In total, we identified 63 likely pathogenic
mutations, of which 22 were novel, providing a molecular
diagnosis 30% of patients. In familial cases, the diagnostic
yield was 37%. NOTCH1 is the major contributor, under-
lying 10% of AOS/ACC/TTLD cases, with DLL4 (6%),
DOCK6 (6%), ARHGAP31 (3%), EOGT (3%), and RBPJ
(2%) representing additional causality in this cohort. Conclusion: The phenotype of our patients shares
marked similarity with that of mutations in closely related
gene, AIMP1. We hereby report the first human disease
associated with deleterious mutations in AIMP2. Conclusions: We confirm the relevance of genetic
screening across the AOS/ACC/TTLD spectrum, high-
lighting important but limited genotype-phenotype correla-
tions. The presented cohort offers potential for further in-
depth screening and novel gene identification to address
missing heritability. A. Dalal: None. A. Shukla: None. A. Das Bhowmik:
None. M. Hebbar: None. R. Kv: None. G. Km: None. N. Gupta: None. P11.011C Detection of a somatic mosaic variant in the AKT3 gene in a
patient with clinical diagnosis of Macrocephaly-Capillary
Malformation J.A.N. Meester:
None. M. Sukalo:
None. K.C. Schröder: None. D. Schanze: None. G. Vandeweyer:
None. R. Trembath: None. L. Van Laer: None. B.L. Loeys: None. M. Zenker: None. L. Southgate: None. W. Wuyts: None. Detection of a somatic mosaic variant in the AKT3 gene in a
patient with clinical diagnosis of Macrocephaly-Capillary
Malformation G. Gordo1, N. Agra1, L. Rodriguez-Laguna1, P. Lapunzina1,
M. J. Lindhurst2, L. G. Biesecker2, V. Martinez-Glez1 G. Gordo1, N. Agra1, L. Rodriguez-Laguna1, P. Lapunzina1,
M. J. Lindhurst2, L. G. Biesecker2, V. Martinez-Glez1 P11.010B Recently, mutation of FKHL7 gene was
proved to cause defects of the heart valves. Genetic tests are
in progress. The patient was discharged after bacterial
endocarditis prophylaxis was implemented. Materials and methods: We clinically evaluated one
patient with a diagnosis of MCAP, and screened by NGS a
series of genes associated to the PI3K-AKT-mTOR path-
way in blood, buccal swab and affected tissue samples. Candidate variants were validated by pyrosequencing. Results: The AKT3 variant c.863C>T; p.(Thr288Ile) was
detected in 24% (59/246) of the readings by NGS in skin
sample, 21% (49/232) in buccal swab, and 1% (5/775) in
blood. The variant is not described in control population,
cancer, or patients with PROS. Clinically, the patient had
macrocephaly, prominent forehead, widely spaced eyes,
capillary malformation in philtrum and right knee, 2-3
cutaneous syndactyly in the feet, hypotonia, lower limb
asymmetry, and cerebral anomalies: segmental cortical
dysplasia, polymicrogyria, cavum septum pellucidum and
cavum vergae, supratentorial ventriculomegaly, and hydro-
cephalus. The
combination
of
all
these
features
is
compatible with MCAP. cancer, or patients with PROS. Clinically, the patient had
macrocephaly, prominent forehead, widely spaced eyes,
capillary malformation in philtrum and right knee, 2-3
cutaneous syndactyly in the feet, hypotonia, lower limb
asymmetry, and cerebral anomalies: segmental cortical
dysplasia, polymicrogyria, cavum septum pellucidum and
cavum vergae, supratentorial ventriculomegaly, and hydro-
cephalus. The
combination
of
all
these
features
is
compatible with MCAP. Conclusions: Usually, Mitral and Aortic valve insuffi-
ciency is presented in rheumatic fever, bacterial endocardi-
tis or collagen disease. In this case, after we excluded them,
due to the eye involvement with corectopia pupil of the iris
Axenfeld-Rieger syndrome was the correct diagnose. The
lack of cases reported in literature, determined us to present
this patient. Conclusions: Usually, Mitral and Aortic valve insuffi-
ciency is presented in rheumatic fever, bacterial endocardi-
tis or collagen disease. In this case, after we excluded them,
due to the eye involvement with corectopia pupil of the iris
Axenfeld-Rieger syndrome was the correct diagnose. The
lack of cases reported in literature, determined us to present
this patient. G.S. Doros: None. A. Ardelean: None. A.V. Popoiu:
None. C.I. Olariu: None. A. Dumitrescu: None. R. Stroescu: None. R. Isac: None. M. Gafencu: None. R. M. Steflea: None. Conclusions: We describe the first patient with MCAP
and a somatic mosaic variant in AKT3. Functional analysis
is underway. G. Gordo: None. N. Agra: None. L. Rodriguez-
Laguna: None. P. Lapunzina: None. M.J. P11.013A E. Schaefer1, C. Stoetzel1, V. Geoffroy1, L. Mary2, C. Marks3,
M. Holder4, J. Ghoumid4, H. Dollfus1, J. Muller1,2 Identification of a new mutation confirms the implication of
IFT27 in Bardet-Biedl Syndrome (BBS19) Identification of a new mutation confirms the implication of
IFT27 in Bardet-Biedl Syndrome (BBS19) P11.010B Lindhurst:
None. L.G. Biesecker: None. V. Martinez-Glez: None. P11.010B c.105C>A [p.(Tyr35Ter)] in AIMP2 causes microcephaly,
intellectual disability, seizures and spastic quadriparesis A. DALAL1, A. Shukla2, A. Das Bhowmik1, M. Hebbar2, R. KV2,
G. KM2, N. GUPTA3 Abstracts from the 51st European Society of Human Genetics Conference: Posters 345 Introduction: Activating germ-line and somatic variants in
AKT3 have been reported in 24 patients with hemi/mega-
lencephaly and/or segmental cortical dysplasia. There is
also one patient reported with a germ-line variant in AKT3
and
clinically
diagnosed
with
Macrocephaly-Capillary
Malformation (MCAP), a syndrome caused by somatic
activating PIK3CA variants and included in the PIK3CA
Related Overgrowth Spectrum (PROS). Introduction: Activating germ-line and somatic variants in
AKT3 have been reported in 24 patients with hemi/mega-
lencephaly and/or segmental cortical dysplasia. There is
also one patient reported with a germ-line variant in AKT3
and
clinically
diagnosed
with
Macrocephaly-Capillary
Malformation (MCAP), a syndrome caused by somatic
activating PIK3CA variants and included in the PIK3CA
Related Overgrowth Spectrum (PROS). Methods: A 14 year old boy was admitted for cardiac
murmur and fatigue after minor exertion. Results: The patient had a good general state, with a
Grade II Aortic and Mitral heart murmur. The ECG
revealed sinus rhythm, slight tachycardia. Echocardiogra-
phy showed: a Grade II Aortic and Mitral regurgitation. Lab
tests were normal, without any signs of streptococcal
infection: ASLO, throat swab and blood cultures were
normal. Antibodies for collagen disease were negative. Therefore we excluded rheumatic fever, endocarditis and
collagen disease. The particular facial aspect with discrete
exophthalmia of the left eye, coloboma with a particularly
shaped pupil and iris anisocorya made us send the patient
for an ophthalmic exam, where Axenfeld-Rieger syndrome
was suspected. Recently, mutation of FKHL7 gene was
proved to cause defects of the heart valves. Genetic tests are
in progress. The patient was discharged after bacterial
endocarditis prophylaxis was implemented. Results: The patient had a good general state, with a
Grade II Aortic and Mitral heart murmur. The ECG
revealed sinus rhythm, slight tachycardia. Echocardiogra-
phy showed: a Grade II Aortic and Mitral regurgitation. Lab
tests were normal, without any signs of streptococcal
infection: ASLO, throat swab and blood cultures were
normal. Antibodies for collagen disease were negative. Therefore we excluded rheumatic fever, endocarditis and
collagen disease. The particular facial aspect with discrete
exophthalmia of the left eye, coloboma with a particularly
shaped pupil and iris anisocorya made us send the patient
for an ophthalmic exam, where Axenfeld-Rieger syndrome
was suspected. Axenfeld - Rieger syndrome - etiology for Mitral and Aortic
valve insufficiency Axenfeld - Rieger syndrome - etiology for Mitral and Aortic
valve insufficiency Holder4, J. Ghoumid4, H. Dollfus1, J. Muller1,2 G. S. Doros1,2, A. Ardelean1,2, A. V. Popoiu1,2, C. I. Olariu1,2,
A. Dumitrescu2, R. Stroescu1,2, R. Isac1,2, M. Gafencu1,2, R. M. Steflea1,2 1Laboratoire de Génétique Médicale UMRS_1112, IGMA,
Faculté de Médecine - UDS, Strasbourg, France, 2Laboratoire
de Diagnostic Génétique, Hôpitaux Universitaires de
Strasbourg, Strasbourg, France, 3Service des explorations de
la fonction visuelle, CHRU Lille, Lille, France, 4Hôpital
Jeanne de Flandre, Service de Génétique Clinique, CHRU
Lille, Lille, France 1Laboratoire de Génétique Médicale UMRS_1112, IGMA,
Faculté de Médecine - UDS, Strasbourg, France, 2Laboratoire
de Diagnostic Génétique, Hôpitaux Universitaires de
Strasbourg, Strasbourg, France, 3Service des explorations de
la fonction visuelle, CHRU Lille, Lille, France, 4Hôpital
Jeanne de Flandre, Service de Génétique Clinique, CHRU
Lille, Lille, France 1"Victor Babes" University of Medicine and Pharmacy,
Timisoara, Romania, 2"Louis Turcanu" Emergency Hospital
for Children, Timisoara, Romania Objective: To present a child with features of Axenfeld-
Rieger syndrome, initially suspected of rheumatic fever
carditis. Although rare, Axenfeld-Rieger syndrome is an
autsomal dominat disease, known to be associated with
valvular and other cardiac disease. When the PITX gene is
involved, malformations of several different organs is
possible. Background: Bardet-Biedl Syndrome (BBS; MIM 209900)
is a recessive and genetically heterogeneous ciliopathy
characterized by postaxial polydactyly, retinitis pigmentosa,
obesity, hypogonadism, cognitive impairment and kidney
dysfunction. At the time of this study, 21 BBS genes were
identified, with the last reported ones being found in one or
very few families. J. Axenfeld - Rieger syndrome - etiology for Mitral and Aortic
valve insufficiency This report confirmed also the implication of
IFT-pathway in BBS pathogenesis as previously reported
with mutations in IFT27 and secondarily in IFT172
(Bujakowska et al., 2015). E. Schaefer: None. C. Stoetzel: None. V. Geoffroy:
None. L. Mary: None. C. Marks: None. M. Holder:
None. J. Ghoumid:
None. H. Dollfus:
None. J. Muller: None. Beckwith-Wiedemann syndrome (BWS), a human genomic
imprinting disorder, is characterised by phenotypic varia-
bility
that
might
include
overgrowth,
macroglossia,
abdominal wall defects, neonatal hypoglycaemia, lateralized
overgrowth and predisposition to embryonal tumours. Delineation of the molecular defects within the imprinted
11p15.5 region can predict familial recurrence risks and the
risk (and type) of embryonal tumour. Despite recent
advances inknowledge, there is marked heterogeneity in
clinical diagnostic criteria and care. An international con-
sensus group agreed upon 72 recommendations for the
clinical and molecular diagnosis and management of BWS,
including
comprehensive
protocols
for
the
molecular
investigation, care and treatment of patients from the pre-
natal period to adulthood. The consensus recommendations
apply to patients with Beckwith-Wiedemann spectrum
(BWSp), covering classical BWS without a molecular
diagnosis and BWS-relatedphenotypes with an 11p15.5
molecular anomaly. The consensus group recommended a
tumour surveillance programme targeted by molecular
subgroups (though it recognised that surveillance might
differ according to the local health-care system e.g. in
United States) and the results of targeted and universal
surveillance should be evaluated prospectively. Interna-
tional collaboration,including a prospective audit of the P11.015C International consensus group statement on Beckwith-
Wiedemann syndrome E. R. Maher1, F. Brioude2, J. M. Kalish3, A. Mussa4, A. Foster5,
J. Bliek6, G. B. Ferrero4, S. Boonen7, T. Cole8, R. Baker9,
M. Bertoletti10, G. Cocchi11, C. Coze12, M. De Pellegrin13,
K. Hussain14, A. Ibrahim15, M. D. Kilby5, M. Krajewska-
Walasek16, C. P. Kratz17, E. J. Ladusans18, P. Lapunzina19, Y. Le
Bouc2, S. Maas6, F. Macdonald8, K. Õunap20, L. Peruzzi21,
S. Rossignol22, S. Russo23, C. Shipster24, A. Skórka16, K. Tatton-
Brown25, J. Tenorio19, C. Tortora26, K. Grønskov27, I. Netchine2,
R. C. Hennekam6, D. Prawitt28, Z. Tümer27, T. Eggermann29,
D. J. G. Mackay30, A. Riccio31 1University of Cambridge, Cambridge, United Kingdom,
2Sorbonne Université, Pierre and Marie Curie-Paris VI
University (UPMC) Université, Paris, France, 3Children’s
Hospital of Philadelphia, Philadelphia, PA, United States,
4University of Torino, Torino, Italy, 5University of
Birmingham, Birmingham, United Kingdom, 6University of
Amsterdam, Amsterdam, Netherlands, 7Zealand University 1University of Cambridge, Cambridge, United Kingdom,
2Sorbonne Université, Pierre and Marie Curie-Paris VI
University (UPMC) Université, Paris, France, 3Children’s
Hospital of Philadelphia, Philadelphia, PA, United States,
4University of Torino, Torino, Italy, 5University of
Birmingham, Birmingham, United Kingdom, 6University of
Amsterdam, Amsterdam, Netherlands, 7Zealand University Axenfeld - Rieger syndrome - etiology for Mitral and Aortic
valve insufficiency del Picchia 346 Hospital, Roskilde, Denmark, 8West Midlands Regional
Genetics Service, Birmingham, United Kingdom,
9Beckwith–Wiedemann Support Group UK, Dorset, United
Kingdom, 1010Italian Association of Beckwith–Wiedemann
syndrome (AIBWS), Vergiate, Italy, 11Bologna University,
Bologna, United Kingdom, 12Aix-Marseille Univ et Assistance
Publique Hôpitaux de Marseille (APHM),, Marseille, France,
13Pediatric Orthopaedic Unit IRCCS Ospedale San Raffaele,
Milan, Italy, 14Sidra Medical and Research Center, Doha,
Qatar, 15Southmead Hospital, Bristol, United Kingdom, 16The
Children’s Memorial Health Institute, Warsaw, Poland,
17Hannover Medical School, Hannover, Germany, 18Royal
Manchester Children’s Hospital, Manchester, United
Kingdom, 19CIBERER, Centro de Investigación Biomédica en
Red de Enfermedades Raras, Madrid, Spain, 20University of
Tartu, Tartu, Estonia, 21Regina Margherita Children’s
Hospital, Turin, Italy, 22Hôpitaux Universitaires de
Strasbourg, Strasburg, France, 23Istituto Auxologico Italiano,
Milan, Italy, 24Great Ormond Street Hospital for Children,
London, United Kingdom, 25St George’s University of London,
London, United Kingdom, 26San Paolo University Hospital,
Milan, Italy, 27Copenhagen University Hospital, Copenhagen,
Denmark, 28ohannes Gutenberg University Medical Center,
Mainz, Germany, 29Technical University of Aachen, Aachen,
Germany, 30University of Southampton, Southampton, United
Kingdom, 31University of Campania Luigi Vanvitelli, Caserta,
Naples, Italy Methods and Results: Exome sequencing was per-
formed in a child presenting with typical BBS features
(retinitis pigmentosa, postaxial polydactyly of four extre-
mities, brachydactyly, obesity, hypogonadism with micro-
penis, cognitive impairment, neurosensorial deafness and
atrioventricular
septal
defect)
as
no
mutations
were
identified in known BBS genes by different molecular
approaches (Sanger Sequencing of BBS1, BBS10 and
BBS12; Targeted High-Throughput Sequencing including
BBS1 to BBS16 genes). Two mutations were found in IFT27
gene
(NM_
006860:
c.[107A>G];[352+1G>T],
p. [Tyr36Cys];[?]). Familial segregation was consistent with
autosomal recessive inheritance as each parent carried one
mutation. Functional studies on fibroblast cells of the
patient showed that the c.352+1G>T mutation led to the
exon 5 skipping. IFT27 mutations have been already
reported once in a consanguineous BBS family (Aldahmesh
et al., 2014) with a typical presentation (obesity, mild
intellectual disability, polydactyly of all extremities, renal
failure, retinitis pigmentosa and hypogenitalism). Conclusions: This is the second report of IFT27
mutations in BBS patients confirming IFT27 as a BBS
gene (BBS19). This report confirmed also the implication of
IFT-pathway in BBS pathogenesis as previously reported
with mutations in IFT27 and secondarily in IFT172
(Bujakowska et al., 2015). Conclusions: This is the second report of IFT27
mutations in BBS patients confirming IFT27 as a BBS
gene (BBS19). P11.016D 1Department of Genetics, Center for Molecular Medicine,
University Medical Center Utrecht, Utrecht, Netherlands,
2Hubrecht Institute-KNAW and University Medical Centre
Utrecht, Utrecht, Netherlands, 3Department of Clinical
Genetics, Amsterdam Medical Center and Free University
Medical Center, Amsterdam, Netherlands 1Department of Genetics, Center for Molecular Medicine,
University Medical Center Utrecht, Utrecht, Netherlands,
2Hubrecht Institute-KNAW and University Medical Centre
Utrecht, Utrecht, Netherlands, 3Department of Clinical
Genetics, Amsterdam Medical Center and Free University
Medical Center, Amsterdam, Netherlands Efficient CrispR/Cas9-based nucleotide editing to model
cardiovascular anomalies of Cantú syndrome in zebrafish Efficient CrispR/Cas9-based nucleotide editing to model
cardiovascular anomalies of Cantú syndrome in zebrafish H. I. Roessler1, F. Tessadori2, S. M. Savelberg1, J. Bakkers2,
M. M. van Haelst3, G. van Haaften1 Myopia, developmental delay and a new mutation in
ASXL1: a case report of Bohring-Opitz syndrome F. Guidolin1, A. M. Spinelli2, F. Faletra3, M. Faleschini3,
I. Bruno3, P. Magini3, A. Fabretto3, P. Gasparini3 F. Guidolin1, A. M. Spinelli2, F. Faletra3, M. Faleschini3,
I. Bruno3, P. Magini3, A. Fabretto3, P. Gasparini3 Abstracts from the 51st European Society of Human Genetics Conference: Posters 347 nor described in scientific literature, but considering its
position and type it is considered likely pathogenetic. results of implementing the consensus recommendations, is
required to expand the evidence base for the design of
optimum care pathways results of implementing the consensus recommendations, is
required to expand the evidence base for the design of
optimum care pathways Conclusion: We report a further case of BOS with a
novel mutation in ASXL1 gene. Our case enlarges the
knowledge about the clinical and molecular data of BOS
and confirms the previously reported genotype-phenotype
correlation. We are performing functional studies about this
mutation in order to discover if the RNA transcript is
degraded. E.R. Maher: None. F. Brioude: None. J.M. Kalish:
None. A. Mussa: None. A. Foster: None. J. Bliek: None. G.B. Ferrero: None. S. Boonen: None. T. Cole: None. R. E.R. Maher: None. F. Brioude: None. J.M. Kalish:
None. A. Mussa: None. A. Foster: None. J. Bliek: None. G.B. Ferrero: None. S. Boonen: None. T. Cole: None. R. Baker: None. M. Bertoletti: None. G. Cocchi: None. C. Coze: None. M. De Pellegrin: None. K. Hussain: None. A. Ibrahim: None. M.D. Kilby: None. M. Krajewska-
Walasek: None. C.P. Kratz: None. E.J. Ladusans: None. P. Lapunzina: None. Y. Le Bouc: None. S. Maas: None. F. Macdonald: None. K. Õunap: None. L. Peruzzi: None. S. Rossignol: None. S. Russo: None. C. Shipster: None. A. Skórka: None. K. Tatton-Brown: None. J. Tenorio:
None. C. Tortora:
None. K. Grønskov:
None. I. Netchine: None. R.C. Hennekam: None. D. Prawitt:
None. Z. Tümer: None. T. Eggermann: None. D.J.G. Mackay: None. A. Riccio: None. Baker: None. M. Bertoletti: None. G. Cocchi: None. C. Coze: None. M. De Pellegrin: None. K. Hussain: None. A. Ibrahim: None. M.D. Kilby: None. M. Krajewska-
Walasek: None. C.P. Kratz: None. E.J. Ladusans: None. F. Guidolin: None. A.M. Spinelli: None. F. Faletra:
None. M. Faleschini: None. I. Bruno: None. P. Magini:
None. A. Fabretto: None. P. Gasparini: None. Neuroradiologic abnormalities in CHARGE syndrome and
guidelines for cranial imaging Neuroradiologic abnormalities in CHARGE syndrome and
guidelines for cranial imaging C. M. de Geus1,2, R. H. Free3,1, B. M. Verbist4,5, L. C. Meiners6,1,
C. M. A. van Ravenswaaij-Arts2,1 S. Di Lascio1, R. Benfante1,2, E. Di Zanni3, S. Cardani1,
A. Adamo3, D. Fornasari1, I. Ceccherini3, T. Bachetti3 S. Di Lascio1, R. Benfante1,2, E. Di Zanni3, S. Cardani1,
A. Adamo3, D. Fornasari1, I. Ceccherini3, T. Bachetti3 1University of Groningen, University Medical Center
Groningen, Centre of Expertise for CHARGE syndrome,
Groningen, Netherlands, 2University of Groningen, University
Medical Center Groningen, Department of Genetics,
Groningen, Netherlands, 3University of Groningen, University
Medical Center Groningen, Department of
Otorhinolaryngology, Groningen, Netherlands, 4Leiden
University Medical Center, Department of Radiology, Leiden,
Netherlands, 5Radboud University Nijmegen Medical Center,
Department of Radiology, Nijmegen, Netherlands, 6University
of Groningen, University Medical Center Groningen,
department of Radiology, Groningen, Netherlands 1Department of Medical Biotechnology and Translational
Medicine, Università degli Studi di Milano, Milan, Italy,
2CNR- Neuroscience Institute, Milan, Italy, 3UOC Genetica
Medica, Istituto Giannina Gaslini, Genoa, Italy Introduction: heterozygous mutations in the PHOX2B
gene cause congenital central hypoventilation syndrome
(CCHS), characterised by defective autonomic control of
breathing. CCHS can be isolated or syndromic (i.e., asso-
ciated with other autonomic dysfunctions such as Hirsch-
sprung disease (HSCR) and neuroblastoma (NB)). Among
PHOX2B mutations, polyalanine expansions are mostly
associated with isolated CCHS, whereas frameshift muta-
tions (FS) with syndromic CCHS. Our study aimed at
identifying the molecular mechanisms underlying genotype-
phenotype correlations and the predisposition to the severe
associated diseases. Introduction: CHARGE syndrome is a rare congenital
malformation syndrome (6 per 100,000 newborns) with
high and variable comorbidity. As a result, clinicians may
struggle to provide comprehensive care. As patients with
CHARGE are at risk for peri-anesthetic complications, it is
paramount to combine necessary imaging procedures. To
enable efficient and high quality imaging, we used recom-
mendations from literature and data from our CHARGE
cohort to propose a guideline for cranial imaging in
CHARGE syndrome. Methods: We evaluated MRIs of 38
CHARGE patients. Additionally, we performed a structured
literature review to examine all existing advice regarding
cranial imaging. Materials and methods: PHOX2B FS mutations identi-
fied so far were classified in terms of frame change, protein
translation, inheritance and clinical associations. Further-
more, we analysed the functional effects of FS mutations in
terms of promoter transactivation of the genes involved in
autonomic nervous system (ANS) development and the
pathogenesis of HSCR and NB. Structural and functional differences in PHOX2B
frameshift mutations underlie isolated or syndromic
congenital central hypoventilation syndrome Structural and functional differences in PHOX2B
frameshift mutations underlie isolated or syndromic
congenital central hypoventilation syndrome 1University of Padova -Burlo Garofolo, Trieste, Italy,
2University of Padova - Burlo Garofolo, Trieste, Italy, 3I.R.C.
C.S. Brulo Garofolo, Trieste, Italy del Picchia 348 mutations in the terminal part of the protein are more
severe and penetrant. Accordingly, the transcriptional
dysfunction occurs with the mutations associated with
syndromic and severe CCHS. cerebral vasodilation in a structure resembling the human
circle of Willis. We developed a novel technique to establish CS-specific
zebrafish that closely model cardiovascular features and
therefore open the possibility of phenotyping-based drug
screening potentially repurposing sulfonylureas already
clinically applied to inhibit GOF KATP channels involved
in neonatal diabetes. Consequently, future studies in our
model
will
improve
understanding
and
clinical
management of CS. Conclusions: our classification is useful for the genetic
counselling
and
our
data
show
that
the
functional
differences among the mutations belonging to different
frame subgroups are the underlying cause of the ANS
disorders associated with CCHS. Funded by: Telethon Foundation (GGP13055), AIRC
(No. 11501), Associazione Italiana per la Sindrome da
Ipoventilazione Centrale Congenita (A.I.S.I.C.C.). Funded by: Telethon Foundation (GGP13055), AIRC
(No. 11501), Associazione Italiana per la Sindrome da
Ipoventilazione Centrale Congenita (A.I.S.I.C.C.). E-Rare grant I-2101-B26 H.I. Roessler: None. F. Tessadori: None. S.M. Savel-
berg: None. J. Bakkers: None. M.M. van Haelst: None. G. van Haaften: None. S. Di Lascio: None. R. Benfante: None. E. Di Zanni:
None. S. Cardani: None. A. Adamo: None. D. Fornasari:
None. I. Ceccherini: None. T. Bachetti: None. S. Di Lascio: None. R. Benfante: None. E. Di Zanni:
None. S. Cardani: None. A. Adamo: None. D. Fornasari:
None. I. Ceccherini: None. T. Bachetti: None. 1University of Padova -Burlo Garofolo, Trieste, Italy,
2University of Padova - Burlo Garofolo, Trieste, Italy, 3I.R.C.
C.S. Brulo Garofolo, Trieste, Italy 1University of Padova -Burlo Garofolo, Trieste, Italy,
2University of Padova - Burlo Garofolo, Trieste, Italy, 3I.R.C. C.S. Brulo Garofolo, Trieste, Italy Cantú Syndrome (CS) is a rare genetic disorder caused by
gain-of-function (GOF) mutations in genes encoding the
pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2,
ABCC9) subunits of an ATP-sensitive potassium (KATP)
channel. CS is characterized by facial anomalies, hyper-
trichosis, extensive cardiac abnormalities and dilated cere-
bral blood vessels. CS is debilitating with no specific
therapy available. Hence, we applied a novel CrispR/Cas9-
based genome editing approach to create CS zebrafish
models for therapeutic drug screening. Introduction: Bohring-Opitz syndrome (BOS) is a rare
syndrome characterized by intrauterine growth retardation,
poor feeding, profound mental retardation, trigonocephaly,
dysmorphisms and a typical posture with elbow flexion and
wrist deviation. Only 30 cases have been reported and 20
mutations were found. Based on these cases, diagnostic
criteria have been set up for clinical diagnosis. We efficiently introduced three CS-specific point muta-
tions in abbc9 and kcnj8 of zebrafish by combining the
CrispR/Cas9 system with a short template oligonucleotide
harboring the site of mutation. To demonstrate functional
validity, we performed live high-speed video-imaging of
zebrafish heart and cardinal vein to assess cardiovascular
function at 5 days post fertilization. Additionally, cerebral
blood vessels were examined for dilations in live kcnj8
mutants in a Tg(kdrl:GFP) transgenic background. Case report. Our patient is a 2-year-old boy, born at term
to healthy, non-consanguineous Caucasian parents. During
the pregnancy, a vesicoureteral reflux was found. The post-
natal history was charachterized by: profound neurodeve-
lopment delay and failure to thrive (height on the 10th
centile, weight and HC below the 3rd centile); severe
myopia (diagnosed at three months); microcephaly and
trigonocephaly; hypertelorism, upslanting palpebral fis-
sures, exophthalmus, flat nasal bridge, forehead nevus
flammeus and BOS posture. These findings led to a clinical
diagnosis of BOS. In agreement with the clinical data, the
analysis of the ASXL1 gene (NM_015338.5) revealed a de
novo frameshift mutation (c.4127dupG, p.Pro1377Serfs*3). This mutation was not previously reported on the online
databases for healthy (dbSnp, 1000g, ESP6500, Exac,
gnomAD) and affected (HGMD professional) individuals Analogous to CS patients, knock-in fish reveal signifi-
cantly enlarged ventricles with enhanced cardiac output,
contractility and development of pericardial edema. A
significantly reduced vein blood flow velocity can be
associated with diminished vascular tone reported in
patients. Additionally, kcnj8 mutant fish display distinct J. Neuroradiologic abnormalities in CHARGE syndrome and
guidelines for cranial imaging Results: the type and the position of translational frame
affect the protein structure, the predisposition to HSCR and/
or NB, and pattern of inheritance: the majority of inherited
mutations occur upstream the polyalanine region, and Results: P11.021A Medical management of chromosome 18 abnormalities J. D. Cody1, M. Hasi-Zogaj1, P. Heard1, A. Hill1, D. Rupert1,
C. Sebold1, B. Soileau1, D. Hale2 J. D. Cody1, M. Hasi-Zogaj1, P. Heard1, A. Hill1, D. Rupert1,
C. Sebold1, B. Soileau1, D. Hale2 1University of Texas Health Science Center at San Anotnio,
San Antonio, TX, United States, 2Penn State Milton S. Hershey
Medical Center, Hershey, PA, United States J.D. Cody: None. M. Hasi-Zogaj: None. P. Heard:
None. A. Hill: None. D. Rupert: None. C. Sebold: None. B. Soileau: None. D. Hale: None. Management of genetic conditions is complicated by vari-
able expression and penetrance. These factors are com-
pounded in the case of chromosome abnormalities, making
binary designations such as “pathogenic” and “benign”
wholly insufficient to inform management of these condi-
tions. When assessing the potential implications of copy
number variants involving multiple genes, clinicians must
quickly assess which genes are most relevant and what the
likelihood is of each of the associated findings in order to
devise appropriate management and screening recommen-
dations. With this end in mind, we have developed a set of
annotated maps designed as four separate and customized
tracks on the UCSC Genome Browser: Results: Abstracts from the 51st European Society of Human Genetics Conference: Posters Abstracts from the 51st European Society of Human Genetics Conference: Posters 349 Neuroradiologic abnormalities in CHARGE cohort
Vestibular system a/dysplasia
100%
Clivus abnormalities
84%
Olfactory system a/hypoplasia
76%
Cerebellar abnormalities (vermis hypoplasia, foliation
defects, other)
53%
Wide lateral ventricles
30%
Frontal hypoplasia
27%
abnormalities of corpus callosum (n = 2), of
myelinisation (n = 2), of gyration pattern (n = 4),
hippocampal malrotation (n = 3)
incidental
(<15%) Neuroradiologic abnormalities in CHARGE cohort Gene Classification
# Hemizygous
# Suprazygous
No clinical effect
156
147
Risk factor
17
5
Conditional
45
1
Low penetrance
8
1
Causal
15
1
Lethal
0
0
Unknown
23
108
Phenotype Classification
# Hemizygous
# Suprazygous
Non-dosage mechanism
3
3
Low Penetrance
37
3
Causal
12
2
Lethal
2
0
Unknown
25
25 Recommended imaging from literature comprises a
temporal bone CT and MRI of the brain, labyrinth, olfactory
structures, pituitary gland and basiocciput. Conclusions: Cranial abnormalities are very common in
CHARGE syndrome and may aid in diagnosis or require
specialized treatment. We propose an imaging protocol in
which we recommend combining a temporal bone CT and a
comprehensive MRI in 3 directions and describe dedicated
sequences to assess abnormalities seen in CHARGE
syndrome. Grants: CHARGE syndrome foundation pilot grant and
personal UMCG grant C.M. de Geus: None. R.H. Free: None. B.M. Verbist:
None. L.C. Meiners: None. C.M.A. van Ravenswaaij-
Arts: None. These classifications are refined and updated as new data
become available. This tool will help clinicians quickly sift
through the data available on each of the genes on
chromosome 18, determine their relevance, and formulate
an
appropriate
screening
and
management
plan
for
individuals with chromosome 18 conditions. Supported by
the Chromosome 18 Registry & Research Society. P11.022B Maternal uniparental disomy 22 has possible impact on the
phenotype: A case report and literature review Maternal uniparental disomy 22 has possible impact on the
phenotype: A case report and literature review M. Zelinová, J. Drábová, M. Malíková, D. Novotná,
M. Turnovec, M. Macek jr., M. Havlovicová M. Zelinová, J. Drábová, M. Malíková, D. Novotná,
M. Turnovec, M. Macek jr., M. Havlovicová Department of Biology and Medical Genetics, Charles
University and University Hospital Motol, Prague, Czech
Republic Department of Biology and Medical Genetics, Charles
University and University Hospital Motol, Prague, Czech
Republic Introduction: Only several cases of uniparental disomy of
chromosome 22 (UPD 22) were published, thus far. (1) 350 J. del Picchia Majority of reported patients did not have typical clinical
features, with the exception of two cases, who both had a
ventricular septal defect (VSD). establishing the accurate molecular diagnosis. This study
aims to improve the diagnostic yield by employing whole-
exome sequencing (WES) to study the potential genetic
causes in patients that clinically resemble a ciliopathy
phenotype. p
(
)
Material and Methods: We present a newborn girl, who
was born after first pregnancy of a 40 years old healthy
mother. The pregnancy had been monitored due to high
levels of serum free-hCG detected during antenatal screen-
ing and intrauterine growth retardation of the fetus. The
child was born at 34 weeks of gestation with birth weight of
1490 g. Cardiologic examination revealed VSD and the
child exhibits facial dysmorphism comprising broad nasal
bridge, low set dysplastic ears and preauricular pits. Nonetheless, postnatal neurologic-, ophthalmologic- and
otoacoustic emission examinations did not reveal any other
abnormalities. A full trisomy 22 in placental tissue was
detected, while examination of peripheral blood revealed
karyotype 46,XX with no evidence of trisomy 22. Maternal
UPD
22
was
confirmed
in
follow
up
cytogenetic
examination. Material and Methods: We present a newborn girl, who
was born after first pregnancy of a 40 years old healthy
mother. The pregnancy had been monitored due to high
levels of serum free-hCG detected during antenatal screen-
ing and intrauterine growth retardation of the fetus. The
child was born at 34 weeks of gestation with birth weight of
1490 g. Cardiologic examination revealed VSD and the
child exhibits facial dysmorphism comprising broad nasal
bridge, low set dysplastic ears and preauricular pits. Nonetheless, postnatal neurologic-, ophthalmologic- and
otoacoustic emission examinations did not reveal any other
abnormalities. A full trisomy 22 in placental tissue was
detected, while examination of peripheral blood revealed
karyotype 46,XX with no evidence of trisomy 22. Maternal
UPD
22
was
confirmed
in
follow
up
cytogenetic
examination. P11.023C Whole-exome sequencing in patients suspected to have
ciliary disorders has a high diagnostic yield and reveals
novel candidate genes A. T. Vulto-van Silfhout1, E. A. Faqeih2, C. E. M. de Die-
Smulders3, E. K. Vanhoutte3, J. M. Cobben4, E. A. M. Cornelissen5, J. Schoots1, M. M. Oud1, R. Roepman1,
D. Lugtenberg1, E. M. H. F. Bongers1 M. Zelinová, J. Drábová, M. Malíková, D. Novotná,
M. Turnovec, M. Macek jr., M. Havlovicová Materials and methods: WES was performed in 166
unrelated patients with a clinical phenotype suspected to be
a ciliopathy. In a first step, single-nucleotide and copy-
number variants (CNVs) were analyzed within 140 known
ciliopathy-related genes. Subsequently, exome-wide analy-
sis was offered in unsolved cases. Results: Analysis of the ciliopathy-gene panel revealed
likely causative variants in 42 highly suspicious ciliopathy
patients (diagnostic yield 25%), which included CNVs in
BBS1 and EVC. Exome-wide analysis revealed a diagnosis
in 19 additional patients (11%), in whom there was often a
moderately high suspicion for a ciliopathy. This resulted in
a total diagnostic yield of 37%. Furthermore, various
potentially relevant mutations were identified in an addi-
tional 51 patients (potential total yield up to 67%). This
included various interesting candidate genes including
KCTD3, DNHD1, and KIF14. Conclusions: Based on literature review, and considering
the presence of VSD, dysmorphic features in our case, low
frequency (and eventually tissue specific) trisomy 22 is still
possible. Clinical findings of our patient with full placental
trisomy 22 and maternal UPD 22 in lymphocytes will be
discussed and compared to previously published cases. Conclusions:
In
conclusion,
WES
analysis
has
a
diagnostic yield of 37% in patients suspected to have a
ciliopathy. Moreover, various interesting candidate genes
were identified, that could potentially increase the yield to
up to 67%. This indicates that WES starting with ciliopathy
gene panel analysis followed by exome-wide analysis is a
powerful diagnostic tool for identifying the genetic cause in
these patients. Supported by MH CZ-DRO (FNM, 00064203) and
CZ.2.16/3.1.00/24022. M. Zelinová: None. J. Drábová: None. M. Malíková:
None. D. Novotná: None. M. Turnovec: None. M. Macek
jr.: None. M. Havlovicová: None. A.T. Vulto-van Silfhout: None. E.A. Faqeih: None. C. E.M. de Die-Smulders: None. E.K. Vanhoutte: None. J. M. Cobben:
None. E.A.M. Cornelissen:
None. J. Schoots: None. M.M. Oud: None. R. Roepman: None. D. Lugtenberg: None. E.M.H.F. Bongers: None. Analysis ofRUNX2mutations in four Turkish patients with
Cleidocranial Dysplasia E. Mihci1, B. N. Güzel2, A. Toylu3, V. Karaman4, A. R. Aghayev4, Z. O. Uyguner4 1Department of Human Genetics, Radboud university medical
center, Nijmegen, Netherlands, 2Department of Pediatric
Subspecialties, Children’s Hospital, King Fahad Medical City,
Riyadh, Saudi Arabia, 3Department of Clinical Genetics,
Maastricht University Medical Center, Maastricht,
Netherlands, 4Department of Pediatrics, Academic Medical
Center, Amsterdam, Netherlands, 5Department of Pediatrics,
Amalia Children's Hospital, Radboudumc, Nijmegen,
Netherlands 1Akdeniz University School of Medicine Department of
Pediatrics Genetics, Antalya, Turkey, 2Akdeniz University
School of Medicine Department of Pediatrics, Antalya, Turkey,
3Akdeniz University School of Medicine Department of Medical
Genetics, Antalya, Turkey, 4Istanbul University, Istanbul
School of Medicine Department of Medical Genetics, Istanbul,
Turkey Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia
that presents with classic triad of delayed closure of the Introduction: Ciliopathies are clinically and genetically
highly
heterogeneous
conditions,
which
challenges P11.025A
Identification of clinical and functional disorders associated
to VPS13B mutations in Cohen Syndrome 1Universidade Federal de São Paulo, São Paulo, Brazil,
2Faculdade de Medicina do ABC, Santo André, Brazil,
3Children's Hospital of Philadelphia, Philadelphia, PA, United
States, 4University of Lausanne, Lausanne, Switzerland 1Universidade Federal de São Paulo, São Paulo, Brazil,
2Faculdade de Medicina do ABC, Santo André, Brazil,
3Children's Hospital of Philadelphia, Philadelphia, PA, United
States, 4University of Lausanne, Lausanne, Switzerland L. Duplomb1, R. Da Costa1, V. Lhussiez1, S. El Chehadeh-
Djebbar2, C. Thauvin-Robinet1,3, L. Faivre1,3 Correct molecular characterization at nucleotide resolution
in complex rearrangements is an important strategy to find
genetic mechanisms associated with the patients’ pheno-
types. We describe a female patient, with secondary ame-
norrhea, a reciprocal translocation between chromosomes X
and 2, and two ~200 kb gains from material of both chro-
mosomes involved in rearrangement. Breakpoints were
mapped by 10x Genomics Chromium technology, a NGS-
based method using linked reads, combined to library pre-
paration and target enrichment using a premium bait design
(Agilent SureSelect) containing “bridging baits”, enriched
for exonic regions. Translocation breakpoints were vali-
dated at nucleotide level by low-coverage whole genome
sequencing. Our results suggest a complex rearrangement
structure, including a tandem duplication in the transloca-
tion junction at the derivative X-chromosome. The SEPT6
gene was found disrupted at one of the X-chromosome
breakpoints. SEPT6 is coexpressed with genes related to
oocyte
maturation during metaphase
II,
and the
X-
chromosome gain encompasses two genes (RPL39 and
UPF3B) highly expressed in the ovarian tissue, that could
be also related to the phenotype. However, SEPT6 knockout
mice’s phenotype does not include subfertility, so its
pathogenicity could not be confirmed. Nevertheless, other
mechanisms, as position effect and TAD disruptions, could
be related to the premature ovarian failure found in patient. 1INSERM 1231 GAD, Université Bourgogne- Franche Comté,
Dijon, France, 2Service de Génétique Médicale, Hôpital de
Hautepierre, Strasbourg, France, 3Centre de Génétique et
Centre de Référence Anomalies du Développement et
Syndromes Malformatifs, FHU TRANSLAD, Hôpital d'Enfants,
Dijon, France Cohen syndrome (CS) is a rare autosomal recessive disorder
caused by mutations in the VPS13B gene, which encodes a
protein of the Golgi apparatus membrane. Intellectual
deficiency, retinopathy, abnormal fat distribution around the
waist, and neutropenia are CS main clinical features. For the
last 10 years, thanks to clinical and functional studies, we
focused on the improvement of CS diagnosis and patients’
follow-up. Abstracts from the 51st European Society of Human Genetics Conference: Posters 351 cranial sutures, hypoplastic or aplastic clavicles and dental
abnormalities. The frequency of CCD is one in 1.000.000
and is inherited in an autosomal dominant manner with
mutations in RUNX2 gene. It is reported that 65% of the
mutations occur de novo, though gonadal mosaicism in
either parent could not be ruled out. Diagnosis of CDC is
based on the typical clinical and radiographic findings
supported by determined heterozygous pathogenic variant
in RUNX2. In this presentation, we report four Turkish
children presented with classical findings of CCD and their
molecular genetic test results.Two patients had missense
(c.569G>A, p.R190; c.577C>G, p.R193G), one patient had
novel frame shift(c.443_445delTACCAGATGGGAinsG;
p.V148Gfs*9) and one patient had gross deletion of exons
6-9. The aim of the present report is to increase the
awareness of CCD and discuss genotype and phenotype
correlation. matching the clinical studies and the risk of type 2 diabetes. We also showed that VPS13B deficient cells have a dis-
organized Golgi apparatus, which is associated to a strong
defect in protein glycosylation. Furthermore, absence of
early endosome and presence of enlarged lysosomes sug-
gest a crucial role of VPS13B in endosomal-lysosomal
trafficking. All these results allowed us to edit new clinical
recommendations for the CS patients’ follow-up and will
help us to understand other CS symptoms such as retino-
pathy and neutropenia that we are now exploring. L. Duplomb: None. R. Da Costa: None. V. Lhussiez:
None. S. El Chehadeh-Djebbar: None. C. Thauvin-
Robinet: None. L. Faivre: None. P11.026B Complex chromosomal rearrangement associated to
premature ovarian failure Complex chromosomal rearrangement associated to
premature ovarian failure Complex chromosomal rearrangement associated to
premature ovarian failure
A. Di-Battista1, M. Moyses-Oliveira1, V. Cabral1,
D. Christofolini2, C. Kao3, F. Mafra3, M. Gonzalez3,
R. Pellegrino3, A. Reymond4, H. Hakonarson3, M. I. Melaragno1 Complex chromosomal rearrangement associated to
premature ovarian failure
A. Di-Battista1, M. Moyses-Oliveira1, V. Cabral1,
D. Christofolini2, C. Kao3, F. Mafra3, M. Gonzalez3,
R. Pellegrino3, A. Reymond4, H. Hakonarson3, M. I. Melaragno1 E. Mihci: None. B.N. Güzel: None. A. Toylu: None. V. Karaman:
None. A.R. Aghayev:
None. Z.O. Uyguner: None. E. Mihci: None. B.N. Güzel: None. A. Toylu: None. V. Karaman:
None. A.R. Aghayev:
None. Z.O. Uyguner: None. A. Di-Battista1, M. Moyses-Oliveira1, V. Cabral1,
D. Christofolini2, C. Kao3, F. Mafra3, M. Gonzalez3,
R. Pellegrino3, A. Reymond4, H. Hakonarson3, M. I. Melaragno1 A. Di-Battista1, M. Moyses-Oliveira1, V. Cabral1, D. Christofolini2, C. Kao3, F. Mafra3, M. Gonzalez3, D. Christofolini2, C. Kao3, F. Mafra3, M. Gonzalez3, R. Pellegrino3, A. Reymond4, H. Hakonarson3, M. I. Melaragno1 R. Pellegrino3, A. Reymond4, H. Hakonarson3, M. I. Melaragno1 Disruption of WDR26 by a translocation breakpoint
confirms its causal role in Skraban-Deardorff and 1q41q42
microdeletion syndromes Disruption of WDR26 by a translocation breakpoint
confirms its causal role in Skraban-Deardorff and 1q41q42
microdeletion syndromes Disruption of WDR26 by a translocation breakpoint
confirms its causal role in Skraban-Deardorff and 1q41q42
microdeletion syndromes J. Freixo: None. M. Marques: None. J. Fino: None. M. E. Talkowski:
None. C.C. Morton:
None. D. David: None. J. Freixo: None. M. Marques: None. J. Fino: None. M. E. Talkowski:
None. C.C. Morton:
None. D. David: None. J. Freixo1, M. Marques2, J. Fino2, M. E. Talkowski3,4,5, C. C. Morton4,5,6,7, D. David2 P11.025A
Identification of clinical and functional disorders associated
to VPS13B mutations in Cohen Syndrome Clinical studies allowed us to show that between
2 and 6 years old, CS children already present with typical
facial features, which can help to shift the diagnosis towards
CS. We also demonstrated that patients have a high risk of
developing type II diabetes and cardiovascular diseases, as
70% of them have low HDL level. Functional studies
demonstrated that VPS13B invalidation leads to accelerated
adipocyte
differentiation,
consequent
to
an
increased
response of cells to insulin stimulation. Later fat-full
VPS13B deficient cells (CS fibroblasts or VPS13B-invali-
daded by siRNA technology) became resistant to insulin, J. del Picchia 352 Thus, the junction point sequencing at nucleotide level
provided detailed information about pathogenic mechan-
isms possibly related to ovarian development and function. The improvement of linked-reads sequencing technologies
can
facilitate
rearrangements
elucidation,
representing
remarkable advancement for cytogenomics research and
clinical diagnostics. reported as the causative gene of the autosomal dominant
Skraban-Deardorff syndrome (SKDEAS, OMIM #617616),
with clinical features that almost completely overlap the
1q41q42
microdeletion
syndrome. WDR26
is
WDR
domain-containing protein presumably involved in multiple
disease-associated signalling pathways. The 3p25.3 break-
point disrupts IVS 1 of the ATP2B2 (OMIM *108733),
reported as a modifier of the autosomal recessive deafness-
12 (DFNB12, OMIN #601386). The proband’s clinical
features basically confirm the phenotypic overlaps between
SKDEAS and the 1q41q42 microdeletion syndrome. In
conclusion, disruption of WDR26 by the 1q42.11 break-
point most likely leads to its haploinsufficiency due to
nonsense mediated RNA decay, resulting in a complex
clinical phenotype, basically matching both SKDEAS and
the 1q41q42 microdeletion syndrome. Therefore, we con-
firm its major causative role in these phenocopy syndromes. Research grant: FCT HMSP-ICT/0016/2013. Thus, the junction point sequencing at nucleotide level
provided detailed information about pathogenic mechan-
isms possibly related to ovarian development and function. The improvement of linked-reads sequencing technologies
can
facilitate
rearrangements
elucidation,
representing
remarkable advancement for cytogenomics research and
clinical diagnostics. Financial support: FAPESP#2016/22860-0; Children’s
Hospital of Philadelphia/Center Applied Genomics. A. Di-Battista: None. M. Moyses-Oliveira: None. V. Cabral: None. D. Christofolini: None. C. Kao: None. F. Mafra: None. M. Gonzalez: None. R. Pellegrino: None. A. Reymond:
None. H. Hakonarson:
None. M.I. Melaragno: None. Phenotype Assessment of a Brazilian Cornelia de Lange
Syndrome (CDLS) cohort Introduction: Cornelia de Lange syndrome (CdLS) is a rare
genetic
disorder
affecting
neurodevelopment,
gastro-
intestinal and musculoskeletal systems. CdLS is caused by
mutations within NIPBL, SMC1A, SMC3, RAD21, or
HDAC8 genes. These genes codify for the cohesin complex,
a multiprotein structure playing a role in chromatid adhe-
sion, DNA repair and gene expression regulation. It has
been demonstrated that a strong correlation exists between
cohesin complex function and WNT signalling. Recently, it
has been observed that chemical activation of the WNT
pathway in nipblb-loss-of-function zebrafish embryos and
in NIPBL-mutated patient fibroblasts rescued the adverse
phenotype. Both embryos and fibroblasts present similar
patterns of canonical WNT pathway alterations and CCND1
downregulation. V. E. H. Kim, I. Furquim, J. R. M. Ceroni, P. H. S. Castro,
C. Olivati, C. Berlim, D. M. B. Lopes, L. Pimenta, R. S. Honjo,
D. R. Bertola, C. A. Kim P11.028D 1Central Lisbon Hospital Center, Lisbon, Portugal,
2Department of Human Genetics, National Institute of Health
Doutor Ricardo Jorge, Lisbon, Portugal, 3Center for Human
Genetic Research, Departments of Neurology, Pathology and
Psychiatry, Massachusetts General Hospital, Boston, MA,
United States, 4Harvard Medical School, Boston, MA, United
States, 5Broad Institute of MIT and Harvard, Cambridge, MA,
United States, 6Departments of Obstetrics and Gynecology,
and of Pathology, Brigham and Women’s Hospital, Boston,
MA, United States, 7University of Manchester, Manchester
Academic Health Science Center, Manchester, United
Kingdom Genotype-phenotype correlations in six patients carrying
two large CNVs: A two-hit model as one of the possible
explanations for phenotypic variability in genomic
disorders V. Cejnova1, L. Liskova1, L. Vancova1, V. Harmas1, M. Fiser2,
M. Soukupova1, A. Peckova1, J. Lastuvkova1 1Regional Health Corporation, Masaryk Hospital in Usti nad
Labem, Department of Medical Genetics, Usti nad Labem,
Czech Republic, 2GENVIA, Prague, Czech Republic Genomic rearrangements represent an important source of
genetic variability. Rare, recurrent copy-number variants
(CNVs) of pathogenic significance, termed genomic dis-
orders, were identified in persons with a characteristic set of
clinically recognizable features. Microdeletions or contiguous gene syndromes (CGSs) are
characterized by variable complex clinical phenotypes
caused by hemizygosity of contiguous genes, defined
mainly by a common deletion region, or of a major causal
gene locus. Identification of breakpoints at nucleotide
resolution of balanced chromosomal rearrangements loca-
lized within these CGS regions constitutes a key strategy for
definition of the phenotypically important genes. The aim of
this study is the identification of molecular alterations
responsible for an extremely complex clinical phenotype
resembling 1q41q42 microdeletion syndrome (coarse facial
features, severe developmental delay, congenital heart dis-
ease and congenital microcephaly) presented by an indivi-
dual
with
a
t(1;3)(q42.11;p25.3)dn. Translocation
breakpoints were localized and confirmed by large-insert
whole-genome and Sanger sequencing, respectively. The
1q42.11 breakpoint disrupts exon 12 of WDR26, recently However, some CNVs are associated with genomic
disorders with extreme phenotypic heterogeneity. For
example,
16p11.2
deletion
has
been
associated
with
intellectual disability, obesity, schizophrenia, and 1% of
sporadic cases of autism. Girirajan and Eichler (2010)
proposed a ‘two-hit’ model in which a first hit (e.g. 16p12.1
deletions) in concert with a secondary hit (e.g. genetic,
epigenetic or environmental insult) results in a more severe
phenotype. We
present
genotype-phenotype
correlations
in
six
patients carrying two large CNVs known to be associated
with a genomic disorder or potentially associated with
disease. P11.028D Array CGH revealed one large deletion (1,7 Mb 353 Abstracts from the 51st European Society of Human Genetics Conference: Posters 15q13.2q13.3 deletion, 2,9 Mb 13q21.1q21.2 deletion, 7,8
Mb 5q14.3q15 deletion, 4,8 Mb 15q11.2q13.1 deletion, 1,3
Mb 17p12 deletion, 2,6 Mb 22q11.2 deletion) in all six
patients. As additional CNVs we identified: 506 kb
intragenic
duplication
of
CNTN4
gene,
2
Mb
13q21.33q22.1 deletion, 1,6 Mb 16p13.11 deletion, 445
kb deletion of CHRNA7 gene, 452 kb 15q11.2 duplication
and 1,7 Mb 16p13.11 duplication). All findings were
confirmed by FISH and we also investigated the parental
origin of chromosomal rearrangements. short sentences, and 9/110 (8.2%) are capable of reading
and writing. Conclusion: The questionnaire was a pioneer in CDLS
patients and this study provides better understanding of the
clinical aspects of the disease, improving health assistance. Half patients were first-born child, reinforcing the impor-
tance of genetic counselling. Conclusion: The questionnaire was a pioneer in CDLS
patients and this study provides better understanding of the
clinical aspects of the disease, improving health assistance. Half patients were first-born child, reinforcing the impor-
tance of genetic counselling. V.E.H. Kim: None. I. Furquim: None. J.R.M. Ceroni:
None. P.H.S. Castro: None. C. Olivati: None. C. Berlim:
None. D.M.B. Lopes: None. L. Pimenta: None. R.S. Honjo: None. D.R. Bertola: None. C.A. Kim: None. In our six cases, the second hits were inherited maternally
or were de novo, and more often noted in phenotypically
variable diagnoses than syndromic disorders. In summary,
additional CNVs can act as genetic modifiers, which may
contribute to the variable phehotypes of genomic disorders. Drosophila melanogaster as a model to study WNT pathway
alteration in Cornelia de Lange Syndrome Drosophila melanogaster as a model to study WNT pathway
alteration in Cornelia de Lange Syndrome V. Cejnova: None. L. Liskova: None. L. Vancova:
None. V. Harmas: None. M. Fiser: None. M. Soukupova:
None. A. Peckova: None. J. Lastuvkova: None. P. Grazioli1, A. Selicorni2, C. Gervasini1, T. Vaccari1, V. Massa1 1Università degli Studi di Milano, Milano, Italy, 2ASST
Lariana, Como, Italy 1Università degli Studi di Milano, Milano, Italy, 2ASST
Lariana, Como, Italy M. Pollazzon1, I. Ivanovski1, S. G. Caraffi1, T. M. Strom2,3,
I. Parenti4, F. J. Kaiser4, L. Garavelli1 1AUSL-IRCCS of Reggio Emilia, Reggio Emilia, Italy,
2Technische Universität München, Munich, Germany,
3Helmholtz Zentrum München, Munich, Germany, 4Institute of
Human Genetics, Lübeck, Germany 1Clinical Genetics Unit, Fondazione IRCCS Ca' Granda,
Ospedale Maggiore Policlinico, Milano, Italy, 2Department of
Pediatrics, Castelli Hospital, Verbania, Italy, 3Molecular
Medicine Department, General Biology and Medical Genetics
Unit, University of Pavia, Pavia, Italy, 4Medical Genetics
Laboratory, Fondazione IRCCS Ca' Granda Ospedale
Maggiore Policlinico, Milano, Italy, 5Neuroradiology Unit,
Fondazione IRCCS Ca' Granda Ospedale Maggiore
Policlinico, Milano, Italy, 6NICU, Department of Clinical
Sciences and Community Health, Università degli Studi di
Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore
Policlinico, Milano, Italy, 7Pediatric Physical Medicine &
Rehabilitation Unit, Fondazione IRCCS Ca’ Granda Ospedale
Maggiore Policlinico, Milano, Italy, 8Oasi Research Institute -
IRCCS, Troina, Italy We report a 17 years old patient with an in-frame deletion in
CREBBP but without Rubinstein-Taybi phenotype. We report a 17 years old patient with an in-frame deletion in
CREBBP but without Rubinstein-Taybi phenotype. The patient presented with developmental delay, severe
intellectual disability, ventricular septal defect, talipes,
inguinal hernia, peno-scrotal fusion, brain
anomalies,
feeding
difficulties,
self-injurious
behavior. Our
first
diagnostic hypothesis was Cornelia de Lange syndrome,
but extended sequencing panels of known associated genes
(including
NIPBL, SMC1A,
SMC3, HDAC8,
RAD21)
detected no pathogenic variants. By subsequent whole
genome sequencing we identified a small deletion in exon
31 of CREBBP. Variants in CREBBP were originally
described
as
the
genetic
cause
of
Rubinstein-Taybi
syndrome (RSTS). This in-frame variant, NM_004380:
c.5518_5544del (p.Val1840_His1848del), was found in
32% of the reads and confirmed by Sanger sequencing in
both peripheral blood and fibroblasts. Segregation analysis
on the parents revealed that the variant occurred de novo in
the patient and can be classified as pathogenic according to
ACMG standards. Introduction: Individuals with 5p deletions were first
reported in 1963 by Lejeune as having Cri-du-chat syn-
drome. The incidence is estimated to be 1:15,000 to
1:50,000 live births and 1:350 among individuals referred
for Intellectual Disability (ID). 5p deletions can be telo-
meric or interstitial and occur at different breakpoints,
ranging in size from the telomeric region at band 5p15.3 (5
Mb) to nearly the whole short arm of chromosome 5 (40
Mb). Cri-du-chat syndrome is observed in patients with
deletions encompassing a critical region between 5p15.2
and 5p15.3, first defined by Niebuhr in 1978. The classic
phenotype includes a characteristic cry, peculiar facies,
microcephaly, growth retardation, hypotonia, speech and
psychomotor delay and ID. Instituto da Criança, São Paulo, Brazil Introduction: Cornelia de Lange syndrome (CDLS) is a
rare genetic disorder caused by mutations in five different
genes (NIPBL, SMC1A, HDAC8, SMC3, RAD21). The
disorder has a wide clinical variabilty, thus our objective is
to assess the characteristics of 110 Brazilian patients. Materials and methods: A questionnaire was filled by
parents from the Brazilian CDLS association. Results: Maternal age at conception varied from 15 to
42y (mean 27.4 y) and 50% were the first-born child. 42/
110 (38.2%) had clinical intercurrence during pregnancy. 73/110 (66.4%) were born by c-section, and 47/110 (42.8%)
were premature. Birth weight varied from 643 to 3740g
(mean 2126g) and birth lenght from 28 to 51cm (mean
42.6cm). The age of clinical diagnosis varied from birth to
38y (mean 1.98 y, median 0.5 y). The current age varies
from 1 mo to 43 y (mean 11.2y, median 8y). The main
clinical findings are synophris (88.2%), feeding dificulties
(81.1%), hypertrichosis (76.4%), short stature (72.7%),
gastrointestinal malformations (65.5%), heart anomalies
(36.4%) and limb defects (33.6%). All patients presented
neurologic developmental delay, and 73.6% also had motor
developmental
delay. 76/110
(69.1%)
have
listening
comprehension and 44/110 (40%) have speaking ability. 22/110 (20%) are capable of using words, 22/110 (20%) Materials and methods: Drosophila melanogaster is an
inexpensive model to study CdLS and to screen in vivo for
therapeutic compounds. Therefore, we have selected fly
strains mutated in nipped-B and hdac3 genes (respectively
NIPBL and HDAC8 in humans) for assessing the existing
correlation between cohesin complex and WNT pathway
and to screen for chemicals that revert the CdLS associated-
phenotypes efficiently. Results: We have confirmed that mutated flies weight 5%
less than wild type. Moreover, we have tested lithium
chloride (LiCl) as WNT activator, demonstrating that
250mM is the highest concentration tolerated. Conclusions: Hence, we hypothesize that WNT pathway
activation could improve mutant phenotype. We will be
testing different doses of LiCl and other WNT activator to
assess whether some of those chemical compounds could
revert the syndrome-associated phenotype. Conclusions: Hence, we hypothesize that WNT pathway
activation could improve mutant phenotype. We will be
testing different doses of LiCl and other WNT activator to
assess whether some of those chemical compounds could
revert the syndrome-associated phenotype. 354 J. del Picchia Grants: This work has been supported by Fondazione
Cariplo, grant 2015-0783 to Valentina Massa. M. Pollazzon: None. I. Ivanovski: None. S.G. Caraffi:
None. T.M. Strom: None. I. Parenti: None. F.J. Patient with a novel variant in CREBBP exon 31 and
without a typical Rubinstein-Taybi phenotype V. G. C. Fergnani1, A. Guala2, C. Danesino3, R. Silipigni4,
S. Guerneri4, E. Scola5, C. Cinnante5, M. Fumagalli6, S. Gangi6,
O. Picciolini7, D. Greco8, L. Castiglia8, G. A. Cagnoli1, R. Villa1,
F. Lalatta1, C. Romano8, M. F. Bedeschi1 M. Pollazzon1, I. Ivanovski1, S. G. Caraffi1, T. M. Strom2,3,
I. Parenti4, F. J. Kaiser4, L. Garavelli1 Instituto da Criança, São Paulo, Brazil Kaiser:
None. L. Garavelli: None. P. Grazioli: None. A. Selicorni: None. C. Gervasini:
None. T. Vaccari: None. V. Massa: None. Mutations in epigenetic and transcriptional regulation
genes cause majority of human developmental disorders Novel Mutations in CTNND1 Cause Cranial Neural Crest
Associated Anomalies and Neurodevelopment Disorders D. Li1, E. Bhoj1, S. Vergano2, T. Wenger3, M. Harr1, Y. Zarate4,
W. Tan5, P. Mark6, S. White7, M. Falk1, C. Murali1, J. Barea8,
T. Tan7, H. Stalker9, K. Hill-Harfe9, J. Mueller9, C. Bupp6,
A. Hing3, E. Zackai1, H. Hakonarson1 R. Alharatani1, W. Ji2, A. Ververi3, S. Lakhani2, J. Hurst3,
M. Hosey1, R. Newbury-Ecob4, R. Scott3, M. Khokha2, A. Beleza-
Meireles5,1, K. Liu1 1Centre for Craniofacial & Regenerative Biology, King's
College London, London, United Kingdom, 2Pediatric Critical
Care Yale University School of Medicine, New Haven, CT,
United States, 3Clinical Genetics Unit, Great Ormond Street
Hospital, Great Ormond Street, London, United Kingdom,
4Department of Clinical Genetics, University Hospitals Bristol
St Michael's Hospital, Bristol, United Kingdom, 5Guy's
Hospital, London, United Kingdom 1The Children's Hospital of Philadelphia, Philadelphia, PA,
United States, 2Children's Hospital of The King's Daughters,
Norfolk, VA, United States, 3Seattle Children's Hospital,
Seattle, WA, United States, 4University of Arkansas for Medical
Sciences, Little Rock, AR, United States, 5Boston Children's
Hospital, Boston, MA, United States, 6Spectrum Health, Grand
Rapids, MI, United States, 7Murdoch Children's Research
Institute, Melbourne, Australia, 8University of California San
Diego, La Jolla, CA, United States, 9University of Florida,
Gainesville, FL, United States The aim of this project was to identify novel pathogenic
gene variants in children with multiple unexplained phe-
notypes, as part of an on-going Cleft-Tooth Anomalies
Study taking place at the South Thames Cleft Unit at St
Thomas’ Hospital, using whole exome sequencing. We
have identified four unrelated patients with novel de novo
variants in the CTNND1 gene. Multiple congenital anomaly with dysmorphic features,
heart defects and/or neurodevelopmental manifestations is a
group of phenotypically and genetically heterogeneous
developmental disorders (DD) affecting ~2-5% of children. We recruited 228 unrelated patients via broad-reaching
collaboration, systematically phenotyped these individuals,
and undertook a focused study using exome sequencing. We successfully identified causal mutations in known
Mendelian genes for 35.1% of kindreds (n = 80). In addi-
tion, we discovered likely pathogenic variants in 5 genes
not previously implicated in DD in 3.1% of patients (n = 7). The identified mutations were mostly de novo dominant
(n = 56, 76%), consistent with the majority of cases arise
sporadically without familial occurrence, but also X-linked
or autosomal recessive (n = 21, 24%). M. Pollazzon1, I. Ivanovski1, S. G. Caraffi1, T. M. Strom2,3,
I. Parenti4, F. J. Kaiser4, L. Garavelli1 M. Khokha:
None. A. Beleza-Meireles: None. K. Liu: None. R. Alharatani: None. W. Ji: None. A. Ververi: None. S. Lakhani: None. J. Hurst: None. M. Hosey: None. R. Newbury-Ecob: None. R. Scott: None. M. Khokha:
None. A. Beleza-Meireles: None. K. Liu: None. M. Pollazzon1, I. Ivanovski1, S. G. Caraffi1, T. M. Strom2,3,
I. Parenti4, F. J. Kaiser4, L. Garavelli1 There is a wide spectrum of
clinical manifestations that can be attributed to differences
in size and localization of the 5p deletion. Recently, individuals with missense variants in exons 30-
31 of CREBBP without typical RSTS phenotype were
described. All of them show atypical facies and variable
features including short palpebral fissures, telecanthus, short
nose
with
depressed
bridge,
anteverted
nares,
short
columella and long philter. They also show psychomotor
development delay (11/11), behavioral anomalies including
self-injury (8/11), microcephaly (7/11), feeding difficulties
(7/11), transmissible hypoacusia (7/11), short stature (5/11),
recurrent infections (5/11), large 1st toe (3/11). Results: Ten patients with 5p deletions have been
included in the present study (6 males and 4 females, age
ranging from birth to 28 years). Brain and brainstem MRI
was
performed
on
all
patients. The
deletions
were
characterized
by
array-CGH. MRI
findings
included
isolated pontine hypoplasia, vermian hypoplasia, ventricular
anomalies, abnormal basal angle, widening of cavum sellae, In conclusion, we report a novel variant in CREBBP
resulting in an atypical phenotype, different from RSTS and
with only some resemblance to the 11 atypical cases
described with missense mutations in exons 30-31. Abstracts from the 51st European Society of Human Genetics Conference: Posters 355 craniofacial dysmorphism, ventricular septal defect and
neurodevelopmental problems. increased signal from white matter, corpus callosum
anomalies, abnormal cortical development. As a group, these children with CTNND1 variants present
with neurodevelopmental disorders and craniofacial dys-
morphisms, including orofacial clefts, and heart defects;
their phenotype is different from BCD. Moreover, the
results of our functional assays suggest that CTNND1 is a
neurocristopathy gene due to its effect on structures of
neural crest origin, and potentially a key oral clefting gene. Conclusions: Several critical regions related to some of
the main features (such as the cry, the peculiar facies, the
developmental delay) have been identified. The aim of this
study is to further define the genotype-phenotype correla-
tions in 5p deletion syndrome with particular regards to the
specific neuroradiological findings. V.G.C. Fergnani: None. A. Guala: None. C. Danesino:
None. R. Silipigni: None. S. Guerneri: None. E. Scola:
None. C. Cinnante: None. M. Fumagalli: None. S. Gangi:
None. O. Picciolini: None. D. Greco: None. L. Castiglia:
None. G.A. Cagnoli: None. R. Villa: None. F. Lalatta:
None. C. Romano: None. M.F. Bedeschi: None. R. Alharatani: None. W. Ji: None. A. Ververi: None. S. Lakhani: None. J. Hurst: None. M. Hosey: None. R. Newbury-Ecob: None. R. Scott: None. P11.033A Mutations in epigenetic and transcriptional regulation
genes cause majority of human developmental disorders P11.035 V. López González: None. A. Serrano-Antón: None. M. Sánchez-Soler: None. M. Ballesta-Martínez: None. L. Rodríguez-Peña: None. R. Gil-Ferrer: None. S. Ibáñez-
Micó: None. H. Alarcón-Martínez: None. E. Martínez-
Salcedo: None. R. Domingo-Jiménez: None. M. García-
Hoyos: A. Employment (full or part-time); Significant;
Instituto de Medicina Genómica (IMEGEN). Valencia. Spain. E. Guillén-Navarro: None. V. López González: None. A. Serrano-Antón: None. M. Sánchez-Soler: None. M. Ballesta-Martínez: None. L. Rodríguez-Peña: None. R. Gil-Ferrer: None. S. Ibáñez-
Micó: None. H. Alarcón-Martínez: None. E. Martínez-
Salcedo: None. R. Domingo-Jiménez: None. M. García-
Hoyos: A. Employment (full or part-time); Significant;
Instituto de Medicina Genómica (IMEGEN). Valencia. Spain. E. Guillén-Navarro: None. CIntellectual disability caused by de novo heterozygous
mutations in DYRK1A gene: first clinically diagnosed
patients and a new hotspot mutation V. López González1,2, A. Serrano-Antón1, M. Sánchez-Soler1,
M. Ballesta-Martínez1,2, L. Rodríguez-Peña1, R. Gil-Ferrer1,
S. Ibáñez-Micó3, H. Alarcón-Martínez3, E. Martínez-Salcedo3,
R. Domingo-Jiménez3, M. García-Hoyos4, E. Guillén-Navarro1,2 V. López González1,2, A. Serrano-Antón1, M. Sánchez-Soler1,
M. Ballesta-Martínez1,2, L. Rodríguez-Peña1, R. Gil-Ferrer1,
S. Ibáñez-Micó3, H. Alarcón-Martínez3, E. Martínez-Salcedo3,
R. Domingo-Jiménez3, M. García-Hoyos4, E. Guillén-Navarro1,2 Mutations in epigenetic and transcriptional regulation
genes cause majority of human developmental disorders This diagnosis made
us think about the second patient, who had been discharged
without diagnosis. DYRK1A analysis revealed a new variant
c.665-5_665-4delTT. First patient´s mother told us about a
third girl attending early intervention program with physical
resemblance to her daughter. The same variant as in patient
2 was found. All variants occurred de novo. retrognathia, narrow thorax, slender digits and anterior
placement of anus. First patient also has a heart defect and
tibial osteochondrosis. Tentative diagnosis of MRD7 was
put forward and a known heterozygous variant c.613C>T
(p.R205*) was identified in DYRK1A. This diagnosis made
us think about the second patient, who had been discharged
without diagnosis. DYRK1A analysis revealed a new variant
c.665-5_665-4delTT. First patient´s mother told us about a
third girl attending early intervention program with physical
resemblance to her daughter. The same variant as in patient
2 was found. All variants occurred de novo. Discussion: we present the first patients clinically
diagnosed with MRD7, mainly characterized by ID, autism,
epilepsy, microcephaly and recognizable craniofacial fea-
tures. Uncommon features such as anteriorly placed anus
and tibial osteochondrosis could help to better delineate its
phenotype. The identification of the same variant in two
unrelated patients points to a new hostpot mutation. Haploinsufficiency of DYRK1A results in a clinically
distinct and probably underdiagnosed entity, which should
be considered in individuals with Angelman syndrome-like
syndromes. p
D. Li: None. E. Bhoj: None. S. Vergano: None. T. Wenger: None. M. Harr: None. Y. Zarate: None. W. Tan: None. P. Mark: None. S. White: None. M. Falk:
None. C. Murali: None. J. Barea: None. T. Tan: None. H. Stalker: None. K. Hill-Harfe: None. J. Mueller: None. C. Bupp: None. A. Hing: None. E. Zackai: None. H. Hakonarson: None. D. Li: None. E. Bhoj: None. S. Vergano: None. T. Wenger: None. M. Harr: None. Y. Zarate: None. W. Tan: None. P. Mark: None. S. White: None. M. Falk:
None. C. Murali: None. J. Barea: None. T. Tan: None. H. Stalker: None. K. Hill-Harfe: None. J. Mueller: None. C. Bupp: None. A. Hing: None. E. Zackai: None. H. Hakonarson: None. Mutations in epigenetic and transcriptional regulation
genes cause majority of human developmental disorders 58.6% of these 87
resolved kindreds are due to mutations in genes involved in
transcriptional processes and related epigenetic modification
pathway, including TP63 (2), SMARCE1, ACTB, DDX3X,
FOXP1, FOXC1, SMC1A, SF3B4 (2), EFTUD2, EP300, Although CTNND1 is a well-known protein that plays
crucial developmental functions, human variants have not
been discovered until recently, with a report of three
variants in patients with Blepharocheilodontic syndrome
(BCD). Otherwise, little is known about the phenotypes that
are associated with mutations in this gene. We have initially discovered a CTNND1 gene variant in a
patient with unexplained craniofacial anomalies including
cleft lip/palate and oligodontia, cardiac defects, autism
(ASD) and other phenotypes suggestive of a yet unexplored
syndrome. In addition, three more patients with variants in
CTNND1 were found in the Deciphering Developmental
Disorders Study (DDD) UK, one of which shares the same
variant as our patient. He presents with very similar 356 J. del Picchia MED13L (2), KANSL1, SOX2, ASXL3, CTNNB1 (2),
CTNND1, STAG2, TBR1, MAF, CUL4B, SATB2 (2), ADNP
(2), HNRNPK, HIVEP2, TFAP2A (2), MLL, MLL2, DEAF1,
GLI3, KMT5B, KAT6A, KAT6B, H3F3A, MED12 (7),
GTF3C5, NKAP, CHD3, and U2AF2, which highlights the
fundamental roles of global and local regulation of tran-
scription in DD. We first discovered three de novo novel
nonsense and frameshift indels in MED12 in three female
patients with Hardikar syndrome - all having cleft palate,
congenital
heart
disease,
biliary
abnormalities,
renal
abnormalities, and retinal pigmentation. All three indivi-
duals showed extreme skewed X-inactivation (99:1). Our
results reveal insights into genetic control of human
development. MED13L (2), KANSL1, SOX2, ASXL3, CTNNB1 (2),
CTNND1, STAG2, TBR1, MAF, CUL4B, SATB2 (2), ADNP
(2), HNRNPK, HIVEP2, TFAP2A (2), MLL, MLL2, DEAF1,
GLI3, KMT5B, KAT6A, KAT6B, H3F3A, MED12 (7),
GTF3C5, NKAP, CHD3, and U2AF2, which highlights the
fundamental roles of global and local regulation of tran-
scription in DD. We first discovered three de novo novel
nonsense and frameshift indels in MED12 in three female
patients with Hardikar syndrome - all having cleft palate,
congenital
heart
disease,
biliary
abnormalities,
renal
abnormalities, and retinal pigmentation. All three indivi-
duals showed extreme skewed X-inactivation (99:1). Our
results reveal insights into genetic control of human
development. retrognathia, narrow thorax, slender digits and anterior
placement of anus. First patient also has a heart defect and
tibial osteochondrosis. Tentative diagnosis of MRD7 was
put forward and a known heterozygous variant c.613C>T
(p.R205*) was identified in DYRK1A. P11.036D This 3 years old boy
was born with dysmorphic features, hypotonia, bilateral
cloudy cornea, heart defect and portosystemic venous shunt,
diagnosed as Abernethy malformation type 2. The oph-
thalmologic evaluation lead to diagnosis of persistent fetal
vasculature (PFV). Karyotype in the boy showed a de novo
translocation
46,XY,t(5:8)(q22q13). Microarray-based
comparative
genomic
hybridization
showed
normal
results. Shallow genome-wide mate-pair library sequencing
was applied to identify the area where the break points were
present. The PCR product was sequenced with Sanger
sequencing. The region containing the break point on
chromosome 5 was found to disrupt the 3rd intron of
EFNA5 gene, while on chromosome 8 it was located in non
coding DNA. To the extent of our knowledge this is the first
report associating EFNA5 defect with human disease. This
study was supported by the NCN Grant No. UMO-2016/21/
B/NZ5/02541 and the most frequent pathogenic mutation in this gene
(NM_001256442.1:c.649dup, p.Arg217Profs*8) was not
detected because it is included in an homopolymer region of
9 Cytosine. From Oct, 2013 until Dec, 2016 only 3
pathogenic mutations have been identified within PRRT2
gene by NGS. Since the implementation of the new method
in Jan, 2017, 14 additional pathogenic mutations were
identified (100% coverage of PRRT2) increasing our
pathogenic mutations detection rate up to 400%. Then,
PRRT2 mutations represent 1.9% of our cohort. Two types
of CNVs have been found, a complete gene deletion and a
complete gene duplication. The most known single base
duplication (c.649dup, p.Arg217Profs*8) was found 7 times
as well as 1 deletion at the same position (c.649del, p. Arg217Glufs*12). A second duplication within an homo-
polymer of 7 Cytosine was also detected once (c.629dup, p. Ala211Serfs*14). The use of an appropriate technology and
a powerful bioinformatics software allowing the simulta-
neous detection of CNVs, SNVs or InDels in simple region
as well as in homopolymer region is highly required for
diagnostic purpose. S. Mary: None. O. Froment: None. D. Lederer: None. N. Simonis: None. A. Tahon: None. C. Colaux: None. E. Bressy: None. M. D'Amico: None. X. Deghorain: None. M. Tubé: None. V. Benoit: None. P. Hilbert: None. K. Dahan: None. A. Skórka: None. M. Młynek: None. V.M. Pienkowski:
None. P. Gasperowicz: None. M. Sykulski: None. J. Kosińska: None. M. Rydzanicz: None. M. Białecka:
None. D. Gieruszczak-Białek: None. M. Kucharczyk:
None. R. Płoski: None. A novel mutation in MYCN gene causes an unusual
presentation of Feingold syndrome A novel mutation in MYCN gene causes an unusual
presentation of Feingold syndrome A. Peleg1, L. Sagi-Dain1, V. Adir1, A. Kurolap2, C. Gonzaga-
Jauregui3, J. Overton3, A. Mori2, A. Shuldiner3, H. Baris2,
R. Wolstein1 A. Peleg1, L. Sagi-Dain1, V. Adir1, A. Kurolap2, C. Gonzaga-
Jauregui3, J. Overton3, A. Mori2, A. Shuldiner3, H. Baris2,
R. Wolstein1 P11.036D Balanced de novo translocation disrupting EFNA5 in a
patient with dysmorphic features, bilaterally cloudy cornea
and portosystemic venous shunt Balanced de novo translocation disrupting EFNA5 in a
patient with dysmorphic features, bilaterally cloudy cornea
and portosystemic venous shunt 1Sección de Genética Médica. Servicio de Pediatría. Hospital
Clínico Universitario Virgen de la Arrixaca. IMIB-Arrixaca,
Murcia, Spain, 2Grupo Clínico Vinculado al Centro de
Investigación Biomédica en Red de Enfermedades Raras
(CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid,
Spain, 3Sección de Neuropediatría. Servicio de Pediatría. Hospital Clínico Universitario Virgen de la Arrixaca. IMIB
Arrixaca, Murcia, Spain, 4Instituto de Medicina Genómica
(IMEGEN), Valencia, Spain A. Skórka1,2, M. Młynek3, V. M. Pienkowski4,5, P. Gasperowicz4,
M. Sykulski6, J. Kosińska4, M. Rydzanicz4, M. Białecka2,
D. Gieruszczak-Białek1, M. Kucharczyk2, R. Płoski4 1Department of Pediatrics Medical University of Warsaw,
Warsaw, Poland, 2Department of Medical Genetics, Children's
Memorial Health Institute, Warsaw, Poland, 3Department of
Medical Genetics, Children's Memorial Health Institute,
Warsaw, Poland, 4Department of Medical Genetics, Warsaw
Medical University, Warsaw, Poland, 5Postgraduate School of
Molecular Medicine, Warsaw Medical University, Warsaw,
Poland, 6Department of Medical Informatics and
Telemedicine, Medical University of Warsaw, Warsaw, Poland Introduction: Pathogenic variants in DYRK1A resulting in
gene haploinsufficiency are responsible for autosomal
dominant intellectual disability (ID) type 7 (MRD7, MIM
614104). It accounts for 0.5% of individuals with ID and/or
autism. Hitherto 61 molecularly confirmed patients have
been published, all identified by exome sequencing without
a clinical suspicion. Clinical cases: we report on three unrelated female
patients aged 8, 9 and 5. They all present with micro-
cephaly, ID, autism, stereotypies, seizures and hyperopia. Their
clinical
phenotype
includes
deep-set
eyes, Primary vitreous regression is a critical event in mammalian
eye development required for proper ocular maturity and
unhindered vision. Failure of this event results in persistent Abstracts from the 51st European Society of Human Genetics Conference: Posters 357 hyperplastic primary vitreous (PHPV), also identified as
persistent fetal vasculature (PFV). In 2014 Son AI et al. suggested a critical role of ephrin-A5 in regulating proper
cell migration into the primary vitreous during early eye
morphogenesis in mice. So far there were no reports of
studies of EFNA5 gene in humans. Here we present a boy
born with bilateral cloudy cornea and subsequent diagnosis
of persistent fetal vasculature, in whom de novo balanced
translocation disrupted EFNA5 gene. Analysis of PRRT2 gene : improvement of NGS technology
allows us to detect 400% more mutations within
homopolymer region as well as CNVs Analysis of PRRT2 gene : improvement of NGS technology
allows us to detect 400% more mutations within
homopolymer region as well as CNVs 1Carmel Medical Center, Haifa, Israel, 2Rambam Medical
Center, Haifa, Israel, 3Regeneron Genetics Center, Tarrytown,
New York, NY, United States S. Mary, O. Froment, D. Lederer, N. Simonis, A. Tahon,
C. Colaux, E. Bressy, M. D'Amico, X. Deghorain, M. Tubé,
V. Benoit, P. Hilbert, K. Dahan S. Mary, O. Froment, D. Lederer, N. Simonis, A. Tahon,
C. Colaux, E. Bressy, M. D'Amico, X. Deghorain, M. Tubé,
V. Benoit, P. Hilbert, K. Dahan Background: Feingold syndrome 1 (FS1) (OMIM#164280)
is an autosomal dominant malformation syndrome char-
acterized by digital anomalies, microcephaly, facial dys-
morphism, gastrointestinal atresia and mild to moderate
learning
disability. Mutations
in
the
MYCN
gene
(OMIM#164840) are known to cause FS1. Congenital
Absence of the Flexor Pollicis Longus (CAFPL) tendon is a
rare sporadic hand anomaly. We describe a pedigree with
CAFPL, consistent with an autosomal dominant inheri-
tance. Physical and radiographic examinations of family
members revealed variable features of Feingold syndrome. Rouen, France, 20Service de génétique, Rouen, France,
21Service de foetopathologie, Dijon, France Discussion: CAFPL diagnosis should be considered if a
patient is unable to flex the interphalangeal joint of the
thumb. A hypoplastic thumb or an absent interphalangeal
joint crease may be a diagnostic feature. Most cases are
sporadic and, to the best of our knowledge, no familial cases
have been published so far. Variants in MYCN have not
been previously associated with CAFPL. This report widens
the spectrum of MYCN-related disorders suggesting that
MYCN gene mutations should be included in the differential
diagnosis
of
patients
presenting
with
familial
"isolated" CAFPL. A. Peleg: None. L. Sagi-Dain: None. V. Adir: None. A. Kurolap: None. C. Gonzaga-Jauregui: None. J. Overton:
None. A. Mori: None. A. Shuldiner: None. H. Baris:
None. R. Wolstein: None. A. Peleg: None. L. Sagi-Dain: None. V. Adir: None. A. Kurolap: None. C. Gonzaga-Jauregui: None. J. Overton:
None. A. Mori: None. A. Shuldiner: None. H. Baris:
None. R. Wolstein: None. A. Peleg: None. L. Sagi-Dain: None. V. Adir: None. A. Kurolap: None. C. Gonzaga-Jauregui: None. J. Overton: None. A. Mori: None. A. Shuldiner: None. H. Baris:
None. R. Wolstein: None. P11.039C Contribution of whole exome sequencing in the diagnosis of
syndromic developmental abnormalities in fetuses M. Lefebvre1,2, Y. Duffourd2, A. Bruel2, M. Assoum2, P. Kuentz2,
E. Schaefer3, S. El Cheahadeh3, M. Antal4, J. Mandel3,
D. Lehalle5, N. Jean-Marçais5, S. Moutton5, C. Philippe2,
A. Vitobello2, F. Tran Mau Them2, N. Marle6, L. Lambert7,
L. Lambert7, P. Jonveaux8, B. Foliguet9, J. Mazutti9,
E. Ginglinger10, D. Gaillard11, C. Poirisier11, F. Arbez-Gindre12,
S. Odent13, C. Quelin14, M. Fradin13, M. Willems15, N. Bigi16,
P. Loget14, S. Blesson17, C. Francannet18, A. Beaufrere19,
S. Patrier19, A. Guerrot20, A. Goldenberg20, N. Laurent21,
J. Thevenon2, L. Faivre2,5, C. Thauvin-Robinet2,5 M. Lefebvre1,2, Y. Duffourd2, A. Bruel2, M. Assoum2, P. Kuentz2, M. Lefebvre: None. Y. Duffourd: None. A. Bruel:
None. M. Assoum: None. P. Kuentz: None. E. Schaefer:
None. S. El Cheahadeh: None. M. Antal: None. J. Mandel: None. D. Lehalle: None. N. Jean-Marçais:
None. S. Moutton: None. C. Philippe: None. A. Vitobello:
None. F. Tran Mau Them: None. N. Marle: None. L. Lambert: None. L. Lambert: None. P. Jonveaux: None. B. Foliguet: None. J. Mazutti: None. E. Ginglinger:
None. D. Gaillard: None. C. Poirisier: None. F. Arbez-
Gindre: None. S. Odent: None. C. Quelin: None. M. Fradin: None. M. Willems: None. N. Bigi: None. P. Loget: None. S. Blesson: None. C. Francannet: None. A. Beaufrere: None. S. Patrier: None. A. Guerrot: None. A. Goldenberg: None. N. Laurent: None. J. Thevenon:
None. L. Faivre: None. C. Thauvin-Robinet: None. 1Génétique et embryologie Médicale, Paris, France, 2GAD
EA4271 « Génétique des Anomalies du Développement »
(GAD), FHU-TRANSLAD, Université de Bourgogne, Dijon,
France, 3Service de génétique, Strasbourg, France, 4Service d
efoetopathologie, Strasbourg, France, 5Service de génétique,
Dijon, France, 6Service de cytogénétique, Dijon, France,
7Service de génétique, Nancy, France, 8Service de
cytogénétique, nancy, France, 9Service de foetopathologie,
Nancy, France, 10Service de génétique, Mulhouse, France,
11Service de génétique, Reims, France, 12Service de
foetopathologie, Besançon, France, 13Service de génétique,
Rennes, France, 14Service de foetopathologie, Rennes, France,
15Service de génétique, Montpellier, France, 16Service de
foetopathologie, Montpellier, France, 17Service de
foetopathologie, Tours, France, 18Service de génétique,
Clermont-Ferrand, France, 19Service de foetopathologie, Rouen, France, 20Service de génétique, Rouen, France,
21Service de foetopathologie, Dijon, France Rouen, France, 20Service de génétique, Rouen, France,
21Service de foetopathologie, Dijon, France analysis
focused
inheritance model. analysis
focused
on
an
autosomal
dominant
inheritance model. autosomal an on Results: Whole exome analysis revealed a novel hetero-
zygous mutation (c.1171C>T; p.Arg391Cys) in the MYCN
gene. Full segregation confirmed the association between
the phenotype and the mutation in the family. Multiple Congenital Anomalies (MCA) are defined by the
association of at least 2 congenital malformations. The
etiological diagnosis of these conditions is needed for
genetic counseling and prenatal or preimplantation diag-
nosis. The rate of diagnosis for MCA fetuses is about 30%
with current diagnostic tests. Most parents are not provided
with accurate genetic counseling. We aimed to assess the
contribution of whole exome sequencing (WES) to fetal
MCA diagnosis after conventional genetic testing. We
performed solo WES in 105 fetuses (58 male and 47
female) with MCA and normal array-CGH. Fetal exam-
ination did not suggest any clinical diagnosis. The 105
fetuses presented with facial dysmorphism (50%), and
intrauterine growth retardation (39%), as well as brain
(46%), heart (38%), skeletal (33%), urogenital (27%),
digestive (23%), respiratory (21%) or distal limb (15%)
anomalies. WES identified a pathogenic variant in 17
fetuses (21%), a variant of unknown significance in 4
fetuses (5%) and a candidate gene in 4 fetuses (5%). This
diagnostic yield is lower than in other postnatal MCA
cohorts. This result is probably due to extreme, atypical or
unspecific phenotypes identified in fetuses, the lack of
neurodevelopmental data in this population and the small
number of studies performed in fetuses, which limits data
sharing. To conclude, the efficacy of solo WES in fetuses is
lower than for the postnatal period for MCA. Sporadic
variants could be easily identified through trio analysis of
the negative cases by second-step parental WES, increasing
the rate of candidate variants and the identification of
new genes. Discussion: CAFPL diagnosis should be considered if a
patient is unable to flex the interphalangeal joint of the
thumb. A hypoplastic thumb or an absent interphalangeal
joint crease may be a diagnostic feature. Most cases are
sporadic and, to the best of our knowledge, no familial cases
have been published so far. Variants in MYCN have not
been previously associated with CAFPL. This report widens
the spectrum of MYCN-related disorders suggesting that
MYCN gene mutations should be included in the differential
diagnosis
of
patients
presenting
with
familial
"isolated" CAFPL. P11.040D Another case of Galloway-Mowat Syndrome associated
with a homozygous mutation of the OSGEP gene Another case of Galloway-Mowat Syndrome associated
with a homozygous mutation of the OSGEP gene IPG, Gosselies, Belgium The use of multi-gene panels in routine diagnostic of
complex pathologies like epileptic encephalopathies is more
and more essential. However, using a reliable technology
associated with powerful bioinformatics software is highly
required to improve diagnostic rate. Here, we compare the
use a 150 genes panel (pyrosequencing technology),
involved in epileptic encephalopathies and intellectual
deficiency (since Oct, 2013) and a second capture based 70
genes panel (SophiaGenetics and Illumina sequencing). With the first panel, PRRT2 gene had a coverage of 98%, Methods: We performed whole exome sequencing of 5
related patients from the same family and one unaffected
married-into the family relative (n = 6). Bioinformatic 358 J. del Picchia Abstracts from the 51st European Society of Human Genetics Conference: Posters 359 S. Bulk1, S. d’Otreppe1, M. Tebache2, J. Lombet2, J. Caberg1 S. Bulk1, S. d’Otreppe1, M. Tebache2, J. Lombet2, J. Caberg1 Introduction: The generalized lymphatic anomaly (GLA)
is characterized by lymphatic malformations (LM) and
osteolysis. Its genetic cause is unknown, although its pattern
of distribution is suspected to be a mechanism of genetic
mosaicism, probably limited to lymphatic endothelial cells
(LEC).The isolation of LECs from affected tissue of
patients affected by GLA has not been described so far, and
will allow the detection of possible mutations in mosaic that
cause the disease. Introduction: The generalized lymphatic anomaly (GLA)
is characterized by lymphatic malformations (LM) and
osteolysis. Its genetic cause is unknown, although its pattern
of distribution is suspected to be a mechanism of genetic
mosaicism, probably limited to lymphatic endothelial cells
(LEC).The isolation of LECs from affected tissue of
patients affected by GLA has not been described so far, and
will allow the detection of possible mutations in mosaic that
cause the disease. 1CHU de Liège, Liège, Belgium, 2CHR de la Citadelle, Liège,
Belgium 1CHU de Liège, Liège, Belgium, 2CHR de la Citadelle, Liège,
Belgium Introduction: Galloway-Mowat syndrome (GWS) is a rare
disease entity associating microcephaly and developmental
delay with glomerular proteinuria progressing to cortico-
resistant nephrotic syndrome and end-stage renal disease. Six genes are now known to be implicated in GWS
including the OSGEP gene. Materials and methods: We present a protocol for
isolation of LECs derived from LM both sporadically and
from a patient with GLA. The fresh tissue is enzymatically
digested and the cells are subjected to a Percoll density
gradient. The isolated cells are expanded in culture and
selected by immunomagnetic methods (MACS), using
specific markers of LECs: CD31 and Podoplanin (PDPN). The purified LM-LECs are verified by immunofluorescence
with other markers of LECs (VEGFR3, PROX1 and LYVE-
1). Materials and methods: We present a protocol for
isolation of LECs derived from LM both sporadically and
from a patient with GLA. The fresh tissue is enzymatically
digested and the cells are subjected to a Percoll density
gradient. The isolated cells are expanded in culture and
selected by immunomagnetic methods (MACS), using
specific markers of LECs: CD31 and Podoplanin (PDPN). The purified LM-LECs are verified by immunofluorescence
with other markers of LECs (VEGFR3, PROX1 and LYVE-
1). Here, we describe another case of GWS associated with a
homozygous missense mutation of the OSGEP gene. Results: At 30 weeks gestation, fetal MRI confirmed the
presence of microcephaly associated with bilateral frontal
pachygyria in our patient. Proteinuria was detected soon
after birth (3g/l). At 6 months, the child presented severe
developmental delay, died at the age of 8 months. The index
case
also
presented
several
dysmorphic
features,
as
previously described
associated
with KEOPS-complex
mutations. Results: The enzymatic digestion of the tissue with
collagenase is critical to obtain a high yield and viability of
the starting cell population. The Percoll gradient reduces the
contamination by non-endothelial cells, which facilitates the
subsequent selection by MACs and allows obtaining a
population of pure LECs. These results require further
phenotypic and functional studies: immunohistochemistry,
proliferation and apoptosis assays,, etc. Our index case was the fifth child of consanguineous
parents; the parents had had two children born with ‘a very
small head’ who had died in infancy of terminal renal
failure. SNP-array showed multiple regions of homozygosity
including the 14q11.2 region. Sequencing of the OSGEP
gene revealed the presence of a homozygous c.953C>T
missense mutation. Conclusions: We have established an isolation protocol
and obtained LM-LECs from a patient with GLA. This will
allow us to delve into the genetic causes of the disease, as
well as perform functional studies that reveal possible
targets for new treatments Conclusions: Pathogenic mutations of OSGEP have been
shown to induce defects in the cytoskeleton and to decrease
the migration rate of podocytes, thus linking the major
manifestations of GWS, structural brain anomalies and
nephrotic syndrome. N. Agra: None. L. Rodriguez-Laguna: None. G. Oliva-
Molina: None. G. Herranz: None. G. Gordo: None. J. López-Gutierrez: None. P. Lapunzina: None. V. Martí-
nez-Glez: None. Hereby, we present another case of nephrotic syndrome
with primary microcephaly and brain anomalies, also
known as GWS, associated with a homozygous mutatin
of the OSGEP gene. 1INGEMM-CIBERER-IdiPAZ, Hospital Universitario La Paz,
Madrid, Spain, 2Vascular Anomalies Center, Plastic Surgery,
Hospital Universitario La Paz, Madrid, Spain N. Agra1, L. Rodriguez-Laguna1, G. Oliva-Molina1, G. Herranz1,
G. Gordo1, J. López-Gutierrez2, P. Lapunzina1, V. Martínez-
Glez1 Whole Exome Sequencing identifies WDR47 as a new
candidate gene for heterotaxy syndrome Whole Exome Sequencing identifies WDR47 as a new
candidate gene for heterotaxy syndrome S. Bulk: None. S. d’Otreppe: None. M. Tebache: None. J. Lombet: None. J. Caberg: None. S. Bulk: None. S. d’Otreppe: None. M. Tebache: None. J. Lombet: None. J. Caberg: None. K. Breuer1,2,3, A. C. Hilger1,2, N. Müller3, B. Schaidinger3,
R. Zhang1,2, J. Breuer3, H. Reutter1,2,4, K. M. Riedhammer5,6,
J. Höfele5 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
University of Bonn, Bonn, Germany, 3Department of Pediatric
Cardiology, University of Bonn, Bonn, Germany, 4Department
of Neonatology and Pediatric Intensive Care, University of
Bonn, Bonn, Germany, 5Institute of Human Genetics, Klinikum
rechts der Isar, Technical University of Munich, Munich, Isolation of endothelial lymphatic cells from patients with
generalized lymphatic anomaly 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Department of Genomics, Life & Brain Center,
University of Bonn, Bonn, Germany, 3Department of Pediatric
Cardiology, University of Bonn, Bonn, Germany, 4Department
of Neonatology and Pediatric Intensive Care, University of
Bonn, Bonn, Germany, 5Institute of Human Genetics, Klinikum
rechts der Isar, Technical University of Munich, Munich, 360 J. del Picchia upon Tyne, United Kingdom, Newcastle upon Tyne, United
Kingdom, 3Newcastle upon Tyne NHS Foundation Trust,
Plastic Surgery Department, Newcastle upon Tyne, United
Kingdom, Newcastle upon Tyne, United Kingdom Germany, 6Department of Nephrology, Klinikum rechts der
Isar, Technical University of Munich, Munich, Germany Germany, 6Department of Nephrology, Klinikum rechts der
Isar, Technical University of Munich, Munich, Germany Introduction: Heterotaxy syndromes represent lateraliza-
tion defects. The determination of left-right body axis dur-
ing early embryogenesis depends on a leftward flow
generated by rotating primary cilia of the primitive node. Heterotaxy syndromes comprise complex heart defects
(CHD), abdominal situs abnormalities including intestinal
malrotations, biliary atresia, asplenia, or polysplenia. The
birth prevalence is 1/15.000. The combination of iris heterochromia and patchy hair
depigmentation is strongly suggestive of Waardenburg
syndrome, but should not be considered pathognomonic. We report a 2 years old girl who presented at birth with iris
heterochromia and patchy depigmentation of hair. Oph-
thalmological examination also showed unilateral hypo-
pigmentation of the fundus. Her mother reported early
greying of hair. On physical examination, the patient also
had hypertelorism, epicanthus and left gum and palate
hypertrophy. Length was on the 75th centile and head cir-
cumference on the 98th centile; she had no other body
asymmetries, normal skin and normal development. Genetic
testing for the known genes associated with Waardenburg
syndrome was negative, and kidney ultrasound, performed
because of the gum hemihypertrophy, was normal. A brain
MRI showed multiple bilateral subcortical hyperintensities
suggestive of Tuberous Sclerosis (TSC) and a bony lesion
of the left maxilla, likely due to fibrous dysplasia. Genetic
testing for TSC showed a heterozygous de novo TSC1
pathogenic variant affecting a splice donor site, confirming
the diagnosis of TS. White tufts of the hair and, more rarely,
patchy area of iris depigmentation, have already been
described in patients with TS, but the association of both is
not a typical presentation (McWilliam and Stephenson,
1978; Rowley et al., 2001). An early diagnosis of TSC is
desirable to allow an appropriate management. Isolation of endothelial lymphatic cells from patients with
generalized lymphatic anomaly This case
confirms that the finding of patchy depigmented hair and
iris depigmentation should prompt careful skin examination
and that TSC should considered as a possible differential
diagnosis in these cases. Methods:
We performed whole
exome sequencing
(WES) in five parent-child-trios to identify potentially de
novo or autosomal recessive disease-causing variants. All
patients had CHD and a variable heterotaxy phenotype. All
parents and siblings underwent thorough ultrasound studies
to exclude mild phenotypes. WES filter criteria included:
MAF≤0.1%, protein altering, high conservation and dele-
terious in silico prediction (Sift, PolyPhen-2, CADD). Identified variants were validated using Sanger Sequencing. Results: Filtering of WES data revealed WDR47 as a
novel candidate gene. Here, a de novo variant (c.2077G>A,
p.Val693Ile; NM_001142550) was identified in the affected
child. According to gnomAD browser beta, the variant was
found once in 30978 alleles. WDR47 encodes a protein
known to regulate autophagy and microtubule dynamic
instability. Moreover, it is involved in developmental
disorders of the brain, notably corpus callosum defects. Discussion: WDR47 is a promising candidate gene for
heterotaxy syndromes due to its central role in microtubule
dynamics and function. Screening of the identified candi-
date gene in larger patient cohorts is warranted. Additional
15 families with heterotaxy syndromes will soon be
analyzed using WES and the data will be presented at the
conference. Funded by SciMED Graduate Program, Faculty of
Medicine, University of Bonn Funded by SciMED Graduate Program, Faculty of
Medicine, University of Bonn M. Bertoli: None. M. Clarke: None. A. Henderson:
None. P. Hodgkinson: None. M. Splitt: None. K. Breuer: None. A.C. Hilger: None. N. Müller: None. B. Schaidinger: None. R. Zhang: None. J. Breuer: None. P11.044D A rare case of chromosomal mosaicism with seven cellular
lines that contains a jumping translocation that imply the
chromosome 14 in a newborn with craniofacial dysmorphia
and multiple congenital anomalies H. Reutter:
None. K.M. Riedhammer:
None. J. Höfele: None. H. Reutter:
None. K.M. Riedhammer:
None. J. Höfele: None. 1"Grigore T. Popa" University of Medicine and Pharmacy Iaşi,
6600 IASI, Romania, 2"Cuza Vodă" Obstetrics and Gynecology
Hospital, 6600 IASI, Romania P11.043C Unexpected diagnosis in a patient presenting with iris
heterochromia and white forelock Unexpected diagnosis in a patient presenting with iris
heterochromia and white forelock M. Grămescu1, L. Caba1, R. Popescu1, S. Popa1, V. Martiniuc2,
L. Păduraru1, M. Guzganu2, I. Pădureţ2, E. GORDUZA1 M. Grămescu1, L. Caba1, R. Popescu1, S. Popa1, V. Martiniuc2,
L. Păduraru1, M. Guzganu2, I. Pădureţ2, E. GORDUZA1 M. Bertoli1, M. Clarke2, A. Henderson1, P. Hodgkinson3,
M. Splitt1 M. Bertoli1, M. Clarke2, A. Henderson1, P. Hodgkinson3,
M. Splitt1 1Newcastle upon Tyne NHS Foundation Trust, Northern
Genetics Service, Newcastle upon Tyne, United Kingdom,
Newcastle upon Tyne, United Kingdom, 2Newcastle upon Tyne
NHS Foundation Trust, Ophthalmology department, Newcastle We present a premature boy born at 35 WA that has a
plurimalformative
syndrome
characterised
by: M. Bertoli1, M. Clarke2, A. Henderson1, P. Hodgkinson3,
M. Splitt1 1"Grigore T. Popa" University of Medicine and Pharmacy Iaşi,
6600 IASI, Romania, 2"Cuza Vodă" Obstetrics and Gynecology
Hospital, 6600 IASI, Romania 1"Grigore T. Popa" University of Medicine and Pharmacy Iaşi,
6600 IASI, Romania, 2"Cuza Vodă" Obstetrics and Gynecology
Hospital, 6600 IASI, Romania 1Newcastle upon Tyne NHS Foundation Trust, Northern
Genetics Service, Newcastle upon Tyne, United Kingdom,
Newcastle upon Tyne, United Kingdom, 2Newcastle upon Tyne
NHS Foundation Trust, Ophthalmology department, Newcastle We present a premature boy born at 35 WA that has a
plurimalformative
syndrome
characterised
by: Abstracts from the 51st European Society of Human Genetics Conference: Posters 361 dolicocephaly, sloping forehead, small nose, micrognathia,
abnormal ears (low set and posterior rotated), bilateral
criptorhidy, cardiomegaly with persistent ductus arteriosus,
cerebral ventriculomegaly and agenesis of corpum calosum. We made GTG banding chromosomal analyse and we
discovered a complex mosaicism characterised by the pre-
sence of a jumping translocation that imply the long arm of
chromosome 14. The chromosomal formula was: 45,X,-14,
der(Y)t(14;Y)(q11.2;q12)[76]/45,XY,-14,der(1)t(1;14)(q44;
q11.2)[8]/45,XY,-14,der(5)t(5;14)(q15.3;q11.2)[7]/45,XY,-
14,der(6)t(6;14)(q27;q11.2)[4]/45,XY,-14,der(21)t(21;14)
(q10;q11.2)[3]/45,XY,-14,der(20)t(20;14)(q13.3;q11.2)[1]/
45,XY,-14,der(22)t(22;14)(q10;q11.2)[1]. We applied also
the MLPA with telomere probes (P-036 kit and P-070 kit,
MrcHolland®) and we discovered a deletion of approximate
2,21Mb located on chromosome 14. Using the P358 kit
(MrcHolland®) we confirmed the absence of segment
between the position 19,863,569 and 22,127,740 on chro-
mosome 14. Jumping translocations are extremely rare and
represents the translocation of the same chromosomal
fragment to different other chromosomes in different cell
lines. The majority of cases were described in different type
of cancers, but also jumping translocations were found in
constitutional cytogenetic associated with an abnormal
pattern of development. The breakpoints implied in jumping
translocations are located in chromosomal regions that
contain repetitive DNA. This feature is present also in our
case, when the breakpoint on chromosome 14 is on the long
arm in proximity of centromere while the rest of breakpoints
imply the telomeric region. In conclusion, we presume that
in our case the congenital anomalies were generated by the
absence of a small segment of chromosome 14 produced
during the complex mechanism of jumping translocation. M. Bertoli1, M. Clarke2, A. Henderson1, P. Hodgkinson3,
M. Splitt1 Caba: None. R. Popescu:
None. S. Popa: None. V. Martiniuc: None. L. Păduraru:
None. M. Guzganu:
None. I. Pădureţ:
None. E. Gorduza: None. M. Grămescu: None. L. Caba: None. R. Popescu:
None. S. Popa: None. V. Martiniuc: None. L. Păduraru:
None. M. Guzganu:
None. I. Pădureţ:
None. E. Gorduza: None. M. Bertoli1, M. Clarke2, A. Henderson1, P. Hodgkinson3,
M. Splitt1 Montpellier, France, 4Service de génétique médicale, CHU
d'Angers, Angers, France, 5Service de génétique médicale,
CHU de Besançon, Besançon, France, 6Service de génétique
médicale, CHU de Brest, Brest, France, 7Service de génétique
médicale, CHU de Caen, Caen, France, 8Service de génétique
médicale, CHU de Clemont-Ferrand, Clermont-Ferrand,
France, 9Service de génétique médicale, CHU de Grenoble,
Grenoble, France, 10Service de génétique médicale, CHU de
Marseille, Marseille, France, 11Service de génétique médicale,
CHU de Nancy, Nancy, France, 12Service de génétique
médicale, CHU de Strasbourg, Strasbourg, France, 13Service
de génétique médicale, CHU de Rouen, Rouen, France,
14Service de génétique médicale, CHU de Nantes, Nantes,
France, 15Service de génétique médicale, CHU de Rennes,
Rennes, France, 16Service de génétique médicale, CHU de
Nice, Nice, France, 17Service de génétique médicale, CHU de
Martinique, Fort de France, France, 18Service de génétique
médicale, CHU de la Reunion, Saint-Denis, France, 19Service
de génétique médicale, CHU de Toulouse, Toulouse, France,
20Service de génétique médicale, CHU de Lyon, Lyon, France,
21Service de génétique médicale, CHU de Tours, Tours,
France, 22Service de génétique médicale, CHU de Dijon,
Dijon, France, 23Service de génétique médicale, AP-HP Necker
enfants Malades, Bordeaux, France, 24Service d'onco
hématologie pédiatrique, CHU de Bordeaux, Bordeaux,
France, 25Centre de référence des cytopénies auto-immunes de
l'enfant, CHU de Bordeaux, Bordeaux, France, 26INSERM
U1211, Université de Bordeaux, Bordeaux, France Introduction: The kabuki syndrome (KS) (MIM 3147920
and 300867) is a rare malformative syndrome, including
specific facial features, moderate to severe intellectual dis-
ability and various malformations. A high prevalence of
immunological
manifestations
is
observed
in
Kabuki
patients. Immune function impairment reduces the prog-
nosis whereas this aspect is one of the less studied in the
literature. To prove the importance of the management
those manifestations, we measured the prevalence of
immune manifestations. We analyzed data to detect parti-
cularities of presentation, phenotypic associations and
therapeutic effectiveness on a large cohort. Introduction: The kabuki syndrome (KS) (MIM 3147920
and 300867) is a rare malformative syndrome, including
specific facial features, moderate to severe intellectual dis-
ability and various malformations. A high prevalence of
immunological
manifestations
is
observed
in
Kabuki
patients. Immune function impairment reduces the prog-
nosis whereas this aspect is one of the less studied in the
literature. To prove the importance of the management
those manifestations, we measured the prevalence of
immune manifestations. We analyzed data to detect parti-
cularities of presentation, phenotypic associations and
therapeutic effectiveness on a large cohort. M. Grămescu: None. L. 1Service de génétique médicale, CHU de Bordeaux, Bordeaux,
France, 2Département de génétique médicale, Maladies rares
et médecine personnalisée, CHU de Montpellier, Montpellier,
France, 3INSERM U1183, Université de Montpellier, H. Margot1, G. Boursier2,3, A. Guichet4, M. Serey5, P. Parent6,
S. Weber7, V. Magry8, K. Dieterich9, J. Robbe10, B. Leheup11,
S. Baer12, A. Goldenberg13, S. Conrad14, M. Fradin15,
G. Morel16, E. Sarrazin17, M. Jacquemont18, S. Julia19,
T. Armand20, A. Toutain21, M. Lefevbre22, S. Lyonnet23,
Y. Perel24,25, D. Lacombe1,26, N. Aladjidi24,25, D. Genevieve2,3 P11.045A Kabuki syndrome and immune manifestations: a cohort of
176 patients Methods: The 176 Kabuki patients included were
followed by 30 French centers and molecularly confirmed. (KDM6A or KMT2D). Questionnaires assess the presence of
immune deficit and autoimmune diseases and on clinic and
biological basis. H. Margot1, G. Boursier2,3, A. Guichet4, M. Serey5, P. Parent6,
S. Weber7, V. Magry8, K. Dieterich9, J. Robbe10, B. Leheup11,
S. Baer12, A. Goldenberg13, S. Conrad14, M. Fradin15,
G. Morel16, E. Sarrazin17, M. Jacquemont18, S. Julia19,
T. Armand20, A. Toutain21, M. Lefevbre22, S. Lyonnet23,
Y. Perel24,25, D. Lacombe1,26, N. Aladjidi24,25, D. Genevieve2,3 Results: 44,6% of our patients had repeated infections
(mainly ENT) and 61,6% had hypogammaglobinemia. 13.2% patients had an autoimmune disease and 5% had
two or more. Prevalence in adult patients raised to 25,8%. The most frequent autoimmune manifestation is immune
thrombocytopenic purpura (8,6%, RR: 215). Autoimmune
hemolytic anemia is observed in 4,0% (RR: 358). Among 1Service de génétique médicale, CHU de Bordeaux, Bordeaux,
France, 2Département de génétique médicale, Maladies rares
et médecine personnalisée, CHU de Montpellier, Montpellier,
France, 3INSERM U1183, Université de Montpellier, J. del Picchia 362 but the mutational analysis using customized TruSight One
panel did not confirm it. Nevertheless, NGS revealed a
novel frameshift deletion, c.4160_4161del/p.Tyr1387*, in
approximately 65% of X-linked KDM6A alleles derived
from the patient’s leukocytes. This result might indicate a
diagnosis of KS, that has been finally concluded after
analysis of genotype-phenotype correlation. Characteristic
facial cue, abnormalities of growth and development, as
well skeletal anomalies seen in the presented patient were
consistent with clinical expression of KS. but the mutational analysis using customized TruSight One
panel did not confirm it. Nevertheless, NGS revealed a
novel frameshift deletion, c.4160_4161del/p.Tyr1387*, in
approximately 65% of X-linked KDM6A alleles derived
from the patient’s leukocytes. This result might indicate a
diagnosis of KS, that has been finally concluded after
analysis of genotype-phenotype correlation. Characteristic
facial cue, abnormalities of growth and development, as
well skeletal anomalies seen in the presented patient were
consistent with clinical expression of KS. non-hematological
manifestations,
vitiligo
and
auto-
immune thyroiditis were frequents. Immune deficiency
during
childhood
is
correlated
with
susceptibility
to
autoimmunity in adults Conclusion: We measure a high prevalence of immune
manifestations, demonstrating the importance of an efficient
management of this frequent, treatable and sometimes
severe aspect of KS whereas during the time of our study,
data
where
incomplete. P11.047C Improve Kagami-Ogata syndrome understanding through
induced pluripotent stem cells from two patients with a
deletion not including the imprinting centers 1Department of Medical Genetics, The Children's Memorial
Health Institute, Warsaw, Poland, 2Department of Pediatrics,
Nutrition and Metabolic Diseases, The Children's Memorial
Health Institute, Warsaw, Poland, 3Department of Medical
Genetics, Warsaw Medical University, Warsaw, Poland,
4Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland 1Department of Medical Genetics, The Children's Memorial
Health Institute, Warsaw, Poland, 2Department of Pediatrics,
Nutrition and Metabolic Diseases, The Children's Memorial
Health Institute, Warsaw, Poland, 3Department of Medical
Genetics, Warsaw Medical University, Warsaw, Poland,
4Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland C. Barilla'1,2, S. Suzuki2, K. Chosa2, D. C. Gruenert2, R. G. Sargent2, A. Provenzano3, S. Giglio3,4, O. Zuffardi1 C. Barilla'1,2, S. Suzuki2, K. Chosa2, D. C. Gruenert2, R. G. Sargent2, A. Provenzano3, S. Giglio3,4, O. Zuffardi1 C. Barilla'1,2, S. Suzuki2, K. Chosa2, D. C. Gruenert2, R. G. Sargent2, A. Provenzano3, S. Giglio3,4, O. Zuffardi1 1Department of Molecular Medicine, University of Pavia,
Pavia, Italy, 2Department of Otolaryngology - Head and Neck
Surgery, University of California-San Francisco, San
Francisco, CA, United States, 3Department of Biomedical
Experimental and Clinical Sciences "Mario Serio", University
of Florence, Firenze, Italy, 4Medical Genetics Unit. Meyer
Children's University Hospital, Firenze, Italy 1Department of Molecular Medicine, University of Pavia,
Pavia, Italy, 2Department of Otolaryngology - Head and Neck
Surgery, University of California-San Francisco, San
Francisco, CA, United States, 3Department of Biomedical
Experimental and Clinical Sciences "Mario Serio", University
of Florence, Firenze, Italy, 4Medical Genetics Unit. Meyer
Children's University Hospital, Firenze, Italy Introduction: Kabuki syndrome (KS) is a rare disorder
characterized by distinctive face, congenital anomalies and
intellectual disability caused by mutations in KMT2D and
KDM6A, two interacting chromatin modifiers responsible
for 56-75% and 5-8% of KS, respectively. To date, only six
KS patients with mosaic KMT2D mutations were described. Any mosaicism in KDM6A have been reported so far. Imprinting disorders are associated with the alteration of
genes
differentially
expressed
between
maternal-
and
paternal-inherited chromosomes, as a consequence of
abnormal methylation pattern in regulatory elements called
imprinting centers (ICs). Kagami-Ogata syndrome (KOS) is
a rare and sometime lethal congenital disorder caused by
alteration at the 14q32 maternal region, between DLK1-
DIO3. The region hosts two long-RNAs, MEG3 and MEG8, Methods and Results: A 2.5-years-old male is the first
child of healthy non-consanguineous parents with negative
family history. P11.046B Mosaic mutation in KDM6B gene in patient with Kabuki
syndrome: analysis of genotype-phenotype correlation E. Ciara1, D. Wesół-Kucharska2, D. Wicher1, D. Piekutowska-
Abramczuk1, J. Kosińska3, P. Stawiński3,4, M. Pelc1,
M. Rydzanicz3, J. Gajewska-Jahołowska2, D. Rokicki2,
B. Chałupczyńska1, D. Siestrzykowska1, P. Halat-Wolska1,
P. Kowalski1, A. Madej-Pilarczyk1, D. Jurkiewicz1,
K. Chrzanowska1, R. Płoski3, M. Krajewska-Walasek1 E. Ciara1, D. Wesół-Kucharska2, D. Wicher1, D. Piekutowska-
Abramczuk1, J. Kosińska3, P. Stawiński3,4, M. Pelc1,
M. Rydzanicz3, J. Gajewska-Jahołowska2, D. Rokicki2,
B. Chałupczyńska1, D. Siestrzykowska1, P. Halat-Wolska1,
P. Kowalski1, A. Madej-Pilarczyk1, D. Jurkiewicz1,
K. Chrzanowska1, R. Płoski3, M. Krajewska-Walasek1 P11.045A This
phenomenon
could
be
explained because Kabuki genes are indirectly involved in
B and Treg lymphocytes differentiation. Conclusions: This is the first report of a patient with
mosaic mutation in KDM6A and clinical features of KS. His
KS phenotype is not consistent with previously reported in
KMT2D-positive patients with mosaicism, who had only
mild facial dysmorphism. Genotype-first approach com-
bined with reverse phenotyping has shown to be a powerful
tool in human genetics, especially in the era of next-
generation sequencing. Study was supported by CMHI
project S149/16. Conclusions: This is the first report of a patient with
mosaic mutation in KDM6A and clinical features of KS. His
KS phenotype is not consistent with previously reported in
KMT2D-positive patients with mosaicism, who had only
mild facial dysmorphism. Genotype-first approach com-
bined with reverse phenotyping has shown to be a powerful
tool in human genetics, especially in the era of next-
generation sequencing. Study was supported by CMHI
project S149/16. H. Margot: None. G. Boursier: None. A. Guichet:
None. M. Serey: None. P. Parent: None. S. Weber: None. V. Magry: None. K. Dieterich: None. J. Robbe: None. B. Leheup: None. S. Baer: None. A. Goldenberg: None. S. Conrad: None. M. Fradin: None. G. Morel: None. E. Sarrazin: None. M. Jacquemont: None. S. Julia: None. T. Armand: None. A. Toutain: None. M. Lefevbre:
None. S. Lyonnet: None. Y. Perel: None. D. Lacombe:
None. N. Aladjidi: None. D. Genevieve: None. H. Margot: None. G. Boursier: None. A. Guichet:
None. M. Serey: None. P. Parent: None. S. Weber: None. V. Magry: None. K. Dieterich: None. J. Robbe: None. B. Conrad: None. M. Fradin: None. G. Morel: None. E. Sarrazin: None. M. Jacquemont: None. S. Julia: None. T. Armand: None. A. Toutain: None. M. Lefevbre:
None. S. Lyonnet: None. Y. Perel: None. D. Lacombe:
None. N. Aladjidi: None. D. Genevieve: None. E. Ciara: None. D. Wesół-Kucharska: None. D. Wicher: None. D. Piekutowska-Abramczuk: None. J. Kosińska: None. P. Stawiński: None. M. Pelc: None. M. Rydzanicz: None. J. Gajewska-Jahołowska: None. D. Rokicki: None. B. Chałupczyńska: None. D. Siestrzy-
kowska: None. P. Halat-Wolska: None. P. Kowalski:
None. A. Madej-Pilarczyk: None. D. Jurkiewicz: None. K. Chrzanowska: None. R. Płoski: None. M. Krajewska-
Walasek: None. P11.047C At the age of 1 year he had his first medical
examination
because
of
hypoglycemia
and
muscular
hypotonia. A diagnosis of glycogen storage disease type 0
or fructose-1,6-bisphosphatase deficiency were suggested, Abstracts from the 51st European Society of Human Genetics Conference: Posters 363 and clusters of small-RNAs. Although a few cases of KOS
have been reported with partial deletion of the region, the
specific role of its non-coding genes within the pathogenesis
of KOS has not been yet clarified. KBG syndrome (KBGS) is a disorder characterized by short
stature, distinctive facial features and developmental/cog-
nitive delay, caused by ANKRD11 gene mutations/deletions
at 16q24. Here we describe the utility of ANKRD11 RT-qPCR gene
expression analysis to investigate the effect, at transcript
level, of ANKRD11 sub-microscopic rearrangements in four
patients with KBGS/KBGS-like clinical diagnosis. To improve the understanding of KOS mechanisms, we
generated iPSCs from KOS sibship having a 130Kb
deletion at 14q32.2, which interrupted MEG3 and elimi-
nated several non-coding RNAs, inherited by the healthy
mother. We used a non-integrative system, to not alter
patients’ genotype. RT-qPCR in the first patient, with a de novo deletion
involving the last two ANKRD11 exons, confirmed the
molecular defect and identified not only a halved amount of
wt transcript, which is indicative for KBG diagnosis, but
also an aberrant mRNA in the expected size, likely
truncated and dysfunctional. Mature iPSCs were characterized by Sanger sequencing,
qPCR and karyotyping to confirm rearrangement stability
and
immune-fluorescence
to
test
iPSCs
quality. We
succeeded to obtain patient-derived iPSCs showing stem
cells features such as expression of specific markers and
pluripotency. Further analysis revealed that the methylation
status was not altered. RT-qPCR proved of great clinical utility in evaluating the
pathogenic effect of a partial ANKRD11 duplication in a
patient with a less convincing KBGS phenotype, as it
revealed mRNA levels comparable to controls. We created the first human model of KOS maintaining
patient’s genotype and methylation status. Furthermore, our
model allows to reveal a correlation between phenotype and
individual genes in 14q32 domain because our patients’
rearrangement does not alter ICs functionality. Also, iPSCs
we generated allow to study the rearrangement in adult
tissues by direct differentiation in culture and rescuing the
deleted genes through gene editing. P11.047C ANKRD11 haploinsufficiency was again confirmed by
RT-qPCR in two further patients: the first with a known de
novo molecular defect in IVS1 encompassing part of the
ANKRD11 promoter region and concordant clinical fea-
tures; and the second with a clear KBG clinical diagnosis
but negative molecular investigations. Consistent with the
RT-qPCR data further molecular characterizations were
performed in the second patient and a 1.8 kb deletion
involving the gene promoter was identified. C. Barilla': None. S. Suzuki: None. K. Chosa: None. D. C. Gruenert: None. R.G. Sargent: None. A. Provenzano:
None. S. Giglio: None. O. Zuffardi: None. This preliminary data proved RT-qPCR is a helpful tool
for molecular and clinical diagnosis both in patients with
ANKRD11
sub-microscopic
rearrangements,
in
which
molecular effect is uncertain, and in molecularly unsolved
KBGS patients. P11.048D Molecular deepening by ANKRD11 gene expression
analyses of KBG patients harboring submicroscopic
rearrangements M. Crippa: None. I. Bestetti: None. M. Falkenberg
Smeland: None. S. Naik: None. O. Murch: None. A. Sironi: None. E. Adamo: None. H. Gåmh: None. S. Davies: None. R. Evans: None. D. McMullan: None. D. Milani: None. L. Larizza: None. K. Low: None. P. Finelli: None. M. Crippa1, I. Bestetti1,2, M. Falkenberg Smeland3, S. Naik4,
O. Murch5, A. Sironi1,2, E. Adamo1, H. GÅMH3, S. Davies5,
R. Evans6, D. McMullan6, D. Milani7, L. Larizza1, K. Low8,
P. Finelli1,2 1IRCCS Istituto Auxologico Italiano, Cusano Milanino, Italy,
2Dpt. of Medical Biotechnology and Translational Medicine,
University of Milan, Milan, Italy, 3Dpt. of Medical Genetics,
University Hospital of North Norway, Tromsø, Norway,
4Clinical Genetics Unit, Birmingham Women's Hospital,
Birmingham, United Kingdom, 5Institute of Medical Genetics,
University Hospital of Wales, Cardiff, United Kingdom, 6West
Midlands Regional Genetics Laboratories, Birmingham
Women’s and Children’s NHS Foundation Trust, Birmingham,
United Kingdom, 7Medical Genetic Unit, Pediatric Highly
Intensive Care, Fondazione IRCCS Ca' Granda Ospedale
Maggiore Policlinico, Milan, Italy, 8Hospitals Bristol NHS
Trust, University of Bristol, Bristol, United Kingdom P11.049A P11.049A
Macrophthalmia and large vessels aneurysms: a
coincidence? P11.049A
Macrophthalmia and large vessels aneurysms: a
coincidence? * Both authors contributed equally to this work. Introduction: We describe four individuals with truncat-
ing variants in the paternally expressed allele of the,
maternally
imprinted
MAGEL2
gene,
responsible
for
Schaaf-Yang syndrome (SYS). Patients suffering from
SYS present with hypotonia, global developmental delay
(DD)/intellectual disability (ID) and feeding difficulties. Additional features include higher prevalence of autism
spectrum disorder (ASD), joint contractures, sleep apnea
and lowered bone density. Materials and methods: - Variants were identified by
whole exome sequencing or using a NGS custom panel
containing a set of genes involved in ID, ASD and other
common genetic conditions. - Sanger sequencing was
performed to determine whether the variant was de novo
or inherited. - Methylation-sensitive digestion followed by
PCR amplification was used to ascertain the parental origin
of the variants. Results: MAGEL2 variants were found on the paternal
allele in all four subjects. Two of them were paternally
inherited while the other two were de novo. A detailed
clinical description of these individuals and a review of
previously reported cases will be provided. Conclusions: MAGEL2 truncating variants are a hitherto
unrecognized likely “common” cause of syndromic distal
arthrogryposis but the exact proportion of cases explained
by this gene still needs to be ascertained. We suggest that a
specific analysis pipeline that does not include inheritance
filtering should be used for the analysis of imprinted genes
such as MAGEL2.This work was supported by a grant of the
Spanish Institute of Health Carlos III, ISCIII (PI13/02010). M.G. Serey-Gaut: None. E. Faudi: None. D. Schor-
deret: None. E. Gillis: None. M. Saleh: None. C. Schwartz: None. C. Cabrol: None. A. Verstraeten: None. B. Loeys: None. L. Van Maldergem: None. M.G. Serey-Gaut: None. E. Faudi: None. D. Schor-
deret: None. E. Gillis: None. M. Saleh: None. C. Schwartz: None. C. Cabrol: None. A. Verstraeten: None. B. Loeys: None. L. Van Maldergem: None. M. Pacio Míguez*: None. F. Santos-Simarro*: None. S. García-Miñaúr: None. P. Tirado Requero: None. E. Vallespín: None. Á. del Pozo: None. M. Solís: None. D. Rodríguez Galiano: None. R. Martín Arenas: None. H. González Pecellín: None. V. Rufo Rabadán: None. E. Galán: None. A. Martínez Bermejo: None. L. Salamanca
Fresno: None. P. Lapunzina Badía: None. M. Palo-
mares-Bralo: None. P11.051C P11.051C
Melorheostosis and vascular anomalies associated with
KRAS mosaicism
V. Seidel1, E. Guillén2, V. M. Martínez-Glez3, Á. M. Lancharro
Zapata4, F. Ballesteros Tejerizo5, V. A. Parra Blanco6, A. García
Martín7, A. Salcedo Posadas8, A. Cervantes Pardo9, M. Campos
Domínguez10 Melorheostosis and vascular anomalies associated with
KRAS mosaicism Truncating variants in the paternally expressed allele of
MAGEL2 as a common cause of syndromic distal
arthrogryposis M. Pacio Míguez*1, F. Santos-Simarro*1,2, S. García-Miñaúr1,2,
P. Tirado Requero3, E. Vallespín1,2, Á. del Pozo1,2, M. Solís1,2,
D. Rodríguez Galiano1,2, R. Martín Arenas1,2, H. González
Pecellín1,2, V. Rufo Rabadán1, E. Galán4, A. Martínez Bermejo3,
L. Salamanca Fresno5, P. Lapunzina Badía1,2, M. Palomares-
Bralo1,2 P11.049A
Macrophthalmia and large vessels aneurysms: a
coincidence? Exophthalmia was observed in the acute
phase and progressed regularly thereafter, leading to retinal
detachments and painful disproportionate enlargement of
eyeballs (transverse diameter > 32mm, NR mean 24,5mm). At 12 years, a routine heart ultrasound detected an aortic
dilatation (aortic root +3 SD, ascending aorta +7 SD), also
progressive,
prompting
funnel
replacement
of
aortic
ascending aorta at 18 years. Dilatation of the brachioce-
phalic arterial trunk and bilateral carotid dysplasia are
observed, raising the hypothesis of a connective tissue
disorder. Blindness in the left eye with phtisis bulbum
occurred at 27 years. Transient ischemic attacks occurred at
29 years manifesting by focal neurologic deficits. A 31
genes TAAD panel did not identify any mutation. A trio
WES yielded two relevant Results: a de novo CCDC51
c.634C>T(p.R212X) variant and a BCORL1 compound
heterozygosity c.3158A>G(p.K1530R) and c.185T>C(p. L62P). No disease-causing mutation in BCORL1 has been
identified so far. We suggest that this singular association of
progressive megalophthalmia and dilatation of the great
vessels is not coincidental and represents a new entity
awaiting description of additional cases to be confirmed. Functional studies are underway to confirm this hypothesis. Infantil., Badajoz, Spain, 5Servicio de Endocrinología
pediátrica. Hospital Universitario La Paz., Madrid, Spain P11.050B Truncating variants in the paternally expressed allele of
MAGEL2 as a common cause of syndromic distal
arthrogryposis Truncating variants in the paternally expressed allele of
MAGEL2 as a common cause of syndromic distal
arthrogryposis P11.049A
Macrophthalmia and large vessels aneurysms: a
coincidence? M. G. Serey-Gaut1, E. Faudi1,2, D. Schorderet3, E. Gillis4,
M. Saleh2, C. Schwartz2, C. Cabrol1, A. Verstraeten4, B. Loeys4,
L. Van Maldergem1 M. G. Serey-Gaut1, E. Faudi1,2, D. Schorderet3, E. Gillis4,
M. Saleh2, C. Schwartz2, C. Cabrol1, A. Verstraeten4, B. Loeys4,
L. Van Maldergem1 1Centre de Génétique Humaine, Université de Franche-Comté,
Besançon, France, 2Department of Ophthalmology, University
Hospital, Université de Franche-Comté, Besançon, France,
3Institute for Research in Ophthalmology, Sion, Switzerland,
4Centrum Medische Genetica, University of Antwerp, Antwerp,
Belgium J. del Picchia 364 Dilatation of large thoracic vessels, either in its isolated or
syndromic form, is highly heterogeneous, with disease-
causing mutations in over 30 genes being identified so far. However, none of the associated syndromes presents with
macrophthalmia. We report on a 31-year-old female with a
history of HLAB27-related bilateral anterior uveitis in the
context of severe and rapidly progressive myopia occurring
at 6 years of age. Exophthalmia was observed in the acute
phase and progressed regularly thereafter, leading to retinal
detachments and painful disproportionate enlargement of
eyeballs (transverse diameter > 32mm, NR mean 24,5mm). At 12 years, a routine heart ultrasound detected an aortic
dilatation (aortic root +3 SD, ascending aorta +7 SD), also
progressive,
prompting
funnel
replacement
of
aortic
ascending aorta at 18 years. Dilatation of the brachioce-
phalic arterial trunk and bilateral carotid dysplasia are
observed, raising the hypothesis of a connective tissue
disorder. Blindness in the left eye with phtisis bulbum
occurred at 27 years. Transient ischemic attacks occurred at
29 years manifesting by focal neurologic deficits. A 31
genes TAAD panel did not identify any mutation. A trio
WES yielded two relevant Results: a de novo CCDC51
c.634C>T(p.R212X) variant and a BCORL1 compound
heterozygosity c.3158A>G(p.K1530R) and c.185T>C(p. L62P). No disease-causing mutation in BCORL1 has been
identified so far. We suggest that this singular association of
progressive megalophthalmia and dilatation of the great
vessels is not coincidental and represents a new entity
awaiting description of additional cases to be confirmed. Functional studies are underway to confirm this hypothesis. Dilatation of large thoracic vessels, either in its isolated or
syndromic form, is highly heterogeneous, with disease-
causing mutations in over 30 genes being identified so far. However, none of the associated syndromes presents with
macrophthalmia. We report on a 31-year-old female with a
history of HLAB27-related bilateral anterior uveitis in the
context of severe and rapidly progressive myopia occurring
at 6 years of age. V. Seidel1, E. Guillén2, V. M. Martínez-Glez3, Á. M. Lancharro
Zapata4, F. Ballesteros Tejerizo5, V. A. Parra Blanco6, A. García
Martín7, A. Salcedo Posadas8, A. Cervantes Pardo9, M. Campos
Domínguez10 Melorheostosis and vascular anomalies associated with
KRAS mosaicism 1Instituto de Genética Médica y Molecular (INGEMM)
Hospital Universitario La Paz, IdiPaz., Madrid, Spain,
2CIBERER, Centro de Investigación Biomédica en Red de
Enfermedades Raras, ISCIII., Madrid, Spain, 3Servicio de
Neurología Infantil. Hospital Universitario La Paz., Madrid,
Spain, 4Departamento de Pediatria, Hospital Materno V. Seidel1, E. Guillén2, V. M. Martínez-Glez3, Á. M. Lancharro
Zapata4, F. Ballesteros Tejerizo5, V. A. Parra Blanco6, A. García
Martín7, A. Salcedo Posadas8, A. Cervantes Pardo9, M. Campos
Domínguez10 1Adnan Menderes University, Aydın, Turkey, 2Institute of
Genetics and Molecular Medicine, Edinburgh, United
Kingdom Introduction: Microcephalic Osteodysplastic Primordial
Dwarfism Type 2 (MOPD2) is a rare autosomal recessive
syndrome with extreme prenatal and postnatal growth
retardation, microcephaly, characteristic facial appearance
and skeletal findings, caused by biallelic mutations in the
Pericentrin gene (PCNT). We report a case of MOPD2 with
a novel mutation in PCNT. Introduction: Melorheostosis is a rare sclerosing bone
dysplasia resembling dripping candle-wax along bones on
radiographs and usually follows a sclerotomal distribution. Introduction: Melorheostosis is a rare sclerosing bone
dysplasia resembling dripping candle-wax along bones on
radiographs and usually follows a sclerotomal distribution. Often neighbouring extraosseous anomalies are associated,
i.e. scleroderma-like skin changes. We present a case of
polyostotic melorheostosis with multiple vascular (arterial,
lymphatic) anomalies and a hyperpigmented patch. Often neighbouring extraosseous anomalies are associated,
i.e. scleroderma-like skin changes. We present a case of
polyostotic melorheostosis with multiple vascular (arterial,
lymphatic) anomalies and a hyperpigmented patch. Materials
and
methods:
Targeted
next-generation
sequencing (NGS) including PCNT gene was performed
in the case with the clinical diagnosis of MOPD2 at the
Institute of Genetics and Molecular Medicine, in Edinburgh
University. Materials
and
methods:
Targeted
next-generation
sequencing (NGS) including PCNT gene was performed
in the case with the clinical diagnosis of MOPD2 at the
Institute of Genetics and Molecular Medicine, in Edinburgh
University. Case description: A 12 year old girl was referred for
assessment. Family and prenatal history were unremarkable. She was born preterm with normal birthweight. She had
congenital chylothorax and aortic coarctation. Diffuse
pulmonary lymphangiomatosis caused a moderate restric-
tive lung disease. Stenosis of other arteries (superior
mesenteric artery, celiac trunk and right renal artery,
causing hypertension) were detected. Sclerosing bone
changes became apparent and affected only the upper left
side of her body (from metacarpal bones to scapula,
extending to ribs and vertebral bodies, causing scoliosis),
characteristic of melorheostosis. She had normal growth
parameters and no learning difficulties. On examination,
upper limb and rib cage asymmetry were evident.Also a
large hyperpigmented patch with geographic borders on her
trunk, and a reddened soft plaque at the lower neck
(lymphatic malformation on biopsy). Results: A 17-year-old male who had a history of
repetitive afebrile seizures, was consulted to the genetics
department while being interned at the emergency ward due
to respiratory distress, confusion, and hypertension. The
case had severe growth retardation and microcephaly, with
a height of 98 cm [-11,35 SD], and OFC of 38 cm [-11,64
SD]. 1Adnan Menderes University, Aydın, Turkey, 2Institute of
Genetics and Molecular Medicine, Edinburgh, United
Kingdom Cervantes Pardo: None. M. Campos Domínguez: None. Z. Manav Kabayegit1, E. Can1, B. Kipcak Yuzbasi1, M. Altan1,
A. Jackson2, G. Bozkurt1, A. Tosun1 Z. Manav Kabayegit1, E. Can1, B. Kipcak Yuzbasi1, M. Altan1,
A. Jackson2, G. Bozkurt1, A. Tosun1 1Adnan Menderes University, Aydın, Turkey, 2Institute of
Genetics and Molecular Medicine, Edinburgh, United
Kingdom 1Adnan Menderes University, Aydın, Turkey, 2Institute of
Genetics and Molecular Medicine, Edinburgh, United
Kingdom Dysmorphic features included a prominent nose,
micrognathia,
microdontia,
sparse
hair,
hyper-
hypopigmented skin lesions and clinodactyly. MRI angio-
graphy showed a “cigarette smoke” view that is suggestive
of Moyamoya disease. X-rays showed small iliac wings and
coxa vara. Targeted NGS analysis of PCNT revealed a
novel homozygous variant, c.3465-1G>A, in the patient. Parents were heterozygous for the same variation. It is
predicted to be disease causing by disrupting the splice
region of the exon 18. (lymphatic malformation on biopsy). Results: NGS of the cervical lymphatic malformation and
the
hyperpigmented
patch
revealed
mosaicism
for
a
heterozygous KRAS mutation (Q61H): 40% and 4%,
respectively. The mutation was absent in blood leukocytes. Conclusions: The same KRAS mutation was detected in
another case of melorheostosis with no vascular anomalies. Our case contributes to the hypothesis of a postzygotic
mosaicism as the disease causing mechanism of melorheos-
tosis
and
widens
the
clinical
spectrum
of
mosaic
RASopathies. Results: NGS of the cervical lymphatic malformation and
the
hyperpigmented
patch
revealed
mosaicism
for
a
heterozygous KRAS mutation (Q61H): 40% and 4%,
respectively. The mutation was absent in blood leukocytes. Conclusion: MOPD2 significantly overlaps with the
Primary autosomal recessive microcephaly/Seckel syn-
drome spectrum, but definitive diagnosis is important
regarding follow-up of vascular central neural system
complications, such as Moyamoya disease. Identification
of the disease-causing mutation is important for early
prenatal diagnosis. Conclusions: The same KRAS mutation was detected in
another case of melorheostosis with no vascular anomalies. Our case contributes to the hypothesis of a postzygotic
mosaicism as the disease causing mechanism of melorheos-
tosis
and
widens
the
clinical
spectrum
of
mosaic
RASopathies. Conclusions: The same KRAS mutation was detected in
another case of melorheostosis with no vascular anomalies. Our case contributes to the hypothesis of a postzygotic
mosaicism as the disease causing mechanism of melorheos-
tosis
and
widens
the
clinical
spectrum
of
mosaic
RASopathies. Z. Manav Kabayegit: None. E. Can: None. B. Kipcak
Yuzbasi: None. M. Altan: None. A. Jackson: None. G. Bozkurt: None. A. Tosun: None. V. Seidel: None. E. Guillén: None. V.M. Martínez-
Glez: None. Á.M. Lancharro Zapata: None. F. Balles-
teros Tejerizo: None. V.A. Parra Blanco: None. A. García Martín: None. A. Salcedo Posadas: None. A. Cervantes Pardo: None. M. Campos Domínguez: None. V. Seidel: None. E. Guillén: None. V.M. Martínez-
Glez: None. Á.M. Lancharro Zapata: None. F. Balles-
teros Tejerizo: None. V.A. Parra Blanco: None. A. García Martín: None. A. Salcedo Posadas: None. A. Abstracts from the 51st European Society of Human Genetics Conference: Posters 365 1Clinical Genetics, Hospital MI Gregorio Marañón, Madrid,
Spain, 2Clinical Genetics, Hospital Clínico Universitario
Virgen de la Arrixaca, Murcia, Spain, 3INGEMM, Hospital
Universitario La Paz, Madrid, Spain, 4Radiology Department,
Hospital MI Gregorio Marañón, Madrid, Spain, 5Pediatric
Cardiology, Hospital MI Gregorio Marañón, Madrid, Spain,
6Pathology Department, Hospital GU Gregorio Marañón,
Madrid, Spain, 7Orthopedics, Hospital MI Gregorio Marañón,
Madrid, Spain, 8Pediatric Pneumology, Hospital MI Gregorio
Marañón, Madrid, Spain, 9Pediatrics, Hospital Clínico
Universitario Virgen de la Arrixaca, Murcia, Spain,
10Dermatology, Hospital MI Gregorio Marañón, Madrid,
Spain P11.052D
A microcephalic osteodysplastic primordial dwarfism type
2 case with a homozygous novel mutation in PCNT Growth pattern and morphologicalcharacteristics of the
fingers in Mowat-Wilson syndrome S. MIZUNO1, M. Inaba1, Y. Muramatsu1, H. Taniai1,
K. Yamada2, N. Wakamatsu3 Material & methods: Whole Exome Sequencing were
used to enrich all exons of protein-coding genes as well as
some important other genomic regions. Next generation
sequencing was performed to sequence close to 100 million
reads on Illumina Sequencer. Bioinformatics analysis of the
sequencing
results
was
performed
using
international
databases
and
standard
bioinformatics
software. This
mutation was confirmed in proband and parents by sanger
sequencing. 1Central Hospital Aichi Human Service Center, Kasugai,
Aichi, Japan, 2Institute of Developmental Research, Aichi
Human Service Center, Kasugai, Aichi, Japan, 3Takamatsu
Municipal Hospital, Takamatsu, Kagawa, Japan Mowat-Wilson syndrome (MOWS) is caused by de novo
heterozygous loss of function mutations or deletions of the
ZEB2 gene. Patients present with mental retardation, epi-
lepsy, and characteristic facies. Health care for genetic
syndromes requires data on standard growth patterns;
however, data on patients with MOWS have not been
documented in detail. We report growth patterns and other
physical characteristics of patients with MOWS. This study
collected physical measurements of patients with MOWS
and examined the finger morphology. Our results showed
that the physical findings characteristic of this syndrome
included measurements at birth showing values within the
normal range, with subsequent postnatal growth impair-
ment, microcephaly and thin habitus. Fingers in patients
with MOWS are bamboo-like, long and thin, with promi-
nent joints. The skin is thin, mildly redundant, and hyper-
extensible. Combined with the typical facial characteristics
of MOWS, these findings could be a clue to phenotypic
diagnosis in this syndrome. Result: Whole Exome sequencing identified a novel
stopgain homozygous variant c.C958T p.R320X in exon 9
of the EXOC6B gene. The variant was assessed by
analytical software and multiple databases. Genotype-
Phenotype correlation and co-segregation analysis was
confirmed among family members to confirm the patho-
genicity of the alteration. Discussion: Here we report for the second time a novel
mutation in the EXOC6B gene known to cause this different
type of skeletal dysplasia in an Iranian patient. Till date only
one mutation was reported in this gene (Girish et al.,2016). Discussion: Here we report for the second time a novel
mutation in the EXOC6B gene known to cause this different
type of skeletal dysplasia in an Iranian patient. Till date only
one mutation was reported in this gene (Girish et al.,2016). S. Seyedhassani: None. Z. Ravesh: None. M. Ebra-
himi: None. L. Najafi: None. S. Seyedhassani: None. Z. Ravesh: None. M. Ebra-
himi: None. L. Najafi: None. P11.055C Analysis of mutational load in Joubert syndrome genes in
affected individuals compared to controls S. Mizuno: None. M. Inaba: None. Y. Muramatsu:
None. H. Taniai:
None. K. Yamada:
None. N. Wakamatsu: None. S. Mizuno: None. M. Inaba: None. Y. Muramatsu:
None. H. Taniai:
None. K. Yamada:
None. N. Wakamatsu: None. I. G. Phelps1, J. Dempsey1, M. Grout1, D. Doherty1,
R. Bachmann-Gagescu2 I. G. Phelps1, J. Dempsey1, M. Grout1, D. Doherty1,
R. Bachmann-Gagescu2 R. Bachmann-Gagescu2 P11.053A Growth pattern and morphologicalcharacteristics of the
fingers in Mowat-Wilson syndrome 366 J. del Picchia sever laxity of wrist joints, delayed bone age, flat feet and
cryptorchidism. 1University of Washington, Seattle, WA, United States,
2University of Zurich- Medical Genetics, Zürich, Switzerland Report of a case with multiple joint dislocation syndrome
associated with a homozygous pathogenic mutation in the
EXOC6B gene 1University of Washington, Seattle, WA, United States,
2University of Zurich- Medical Genetics, Zürich, Switzerland Next-generation sequencing frequently uncovers multiple
rare, predicted-deleterious variants (RDVs) in different
genes associated with the same recessive disorder in any
individual, described as “mutational load”. While such
RDVs could contribute to “oligogenic inheritance” or act as
“genetic modifiers”, their clinical significance remains
unclear. Focusing on the genetically highly heterogeneous
recessive ciliopathy Joubert syndrome (JBTS), we under-
took a systematic analysis of RDVs in 25 JBTS genes,
comparing ~400 affected individuals with JBTS to in-house
controls and to UK1958 birth-cohort per-sample exome
data. Using criteria for deleteriousness established in the
JBTS cohort, we identified a surprisingly large number of
controls harboring RDVs in JBTS genes (~30%), whereby
the type/distribution of variants in controls differed sub-
stantially from the causal alleles in affected individuals,
suggesting that despite the predictions, the majority of
variants in controls are not disease-causing. RDVs in ≥2 S. Seyedhassani1,2, Z. Ravesh2, M. Ebrahimi1, L. Najafi3 S. Seyedhassani1,2, Z. Ravesh2, M. Ebrahimi1, L. Najafi3 1Dr. Seyedhassani medical genetic center, Yazd, Iran, Islamic
Republic of, 2Genomic research center, Shahid Beheshti
medical science university, Tehran, Iran, Islamic Republic of,
3Kharazmi university, Tehran, Iran, Islamic Republic of 1Dr. Seyedhassani medical genetic center, Yazd, Iran, Islamic
Republic of, 2Genomic research center, Shahid Beheshti
medical science university, Tehran, Iran, Islamic Republic of,
3Kharazmi university, Tehran, Iran, Islamic Republic of Introduction: Spondyloepimetaphyseal dysplasia (SEMD)
is an inherited disorder characterised by joint dislocation at
birth, thin limbs, joint laxity, poor bone classification and
delayed bone age. Skeletal dysplasia with multiple joint
dislocation are various group of disorders comprising dif-
ferential diagnosis. Case: A 21 month old boy with multiple joint disloca-
tions joint laxity, born to consanguineous parents was
referred to our clinic of genetic. Patient suffered from hip,
knee and elbow dislocation, joint slackness, kyphoscoliosis, Abstracts from the 51st European Society of Human Genetics Conference: Posters 367 genes were common in affected individuals and in controls,
as predicted by allele frequencies in ExAC (probability of
heterozygous RDVs in any 2 of 25 genes =~9%). 38% of
affected individuals carried bi-allelic causal variants in one
gene plus additional RDVs in other gene(s). Phenotypic
discordance was observed between 60% of affected indi-
viduals sharing identical causal alleles, supporting existence
of genetic modifiers. NBAS associated disease: further defining the phenotype of
a recognizable syndrome NBAS associated disease: further defining the phenotype of
a recognizable syndrome I.G. Phelps: None. J. Dempsey: None. M. Grout: None. D. Doherty: None. R. Bachmann-Gagescu: None. I.G. Phelps: None. J. Dempsey: None. M. Grout: None. D. Doherty: None. R. Bachmann-Gagescu: None. D. Carli1, E. Giorgio2,3, F. Pantaleoni4, S. Barresi4,
G. Baldassarre1, E. Riberi1, F. Licciardi1, S. Pizzi4, A. Ciolfi4,
A. Gazzin1, D. Montin1, C. Molinatto1, A. Brusco2,3,
M. Tartaglia4, G. B. Ferrero1 D. Carli1, E. Giorgio2,3, F. Pantaleoni4, S. Barresi4,
G. Baldassarre1, E. Riberi1, F. Licciardi1, S. Pizzi4, A. Ciolfi4, P11.056D A. Gazzin1, D. Montin1, C. Molinatto1, A. Brusco2,3, M. Tartaglia4, G. B. Ferrero1 The co-existence of Nablus Mask-Like Facial Syndrome
and Klinefelter Syndrome 1University of Torino, Department of Public Health and
Pediatrics, Torino, Italy, 2University of Torino, Department of
Medical Sciences, Torino, Italy, 3Città della Salute e della
Scienza University Hospital, Medical Genetics Unit, Torino,
Italy, 4Genetics and Rare Diseases Research Division,
Ospedale Pediatrico Bambino Gesù, Roma, Italy Ö. Anlaş1, B. Sarıkepe2, S. Zeybek1, M. Öztürk1, G. Bağcı1, G. O. Çetin1 Ö. Anlaş1, B. Sarıkepe2, S. Zeybek1, M. Öztürk1, G. Bağcı1, G. O. Çetin1 1Medical School Of Pamukkale University Department Of
Medical Genetics, Denizli, Turkey, 2State Hospital, Adana,
Turkey Introduction: Biallelic pathogenic variants in the NBAS
gene are associated with two different phenotypes: infantile
liver failure syndrome 2 (OMIM 616483) and short stature,
optic nerve atrophy, and Pelger-Huet anomaly (SOPH)
syndrome (OMIM 614800). Introduction: Nablus mask-like facial syndrome (NMLFS)
is a rare microdeletion syndrome. A mask-like facial
appearance is the characterized symptom of disease. Here
we report the co-existence of NMLFS together with Kli-
nefelter Syndrome in a patient, for the first time in the lit-
erature, to the best of our knowledge. Patients and Methods: two unrelated patients, a 4-year-
old female and a 7-year-old male, were referred to our
Genetics Unit. They presented with a highly overlapping
phenotype,
characterized
by
prenatal
hyposomatism
evolved into harmonic short stature, persistent hypertransa-
minasemia, hypogammaglobulinemia and hypovision. The
second patient also presented congenital glaucoma and
primary
hypothyroidism. They
both
presented
slight
psychomotor delay and mild dysmorphic facial features
with hypotelorism, smooth philtrum and narrow mouth. Both patients were enrolled in the Undiagnosed Patients
Program of the Ospedale Pediatrico Bambino Gesù. Whole
exome sequencing and cDNA analysis were performed on
probands’ genomic DNA and RNA extracted from PBMC,
respectively. Patients and Methods: two unrelated patients, a 4-year-
old female and a 7-year-old male, were referred to our
Genetics Unit. They presented with a highly overlapping
phenotype,
characterized
by
prenatal
hyposomatism
evolved into harmonic short stature, persistent hypertransa-
minasemia, hypogammaglobulinemia and hypovision. The
second patient also presented congenital glaucoma and
primary
hypothyroidism. They
both
presented
slight
psychomotor delay and mild dysmorphic facial features
with hypotelorism, smooth philtrum and narrow mouth. Both patients were enrolled in the Undiagnosed Patients
Program of the Ospedale Pediatrico Bambino Gesù. Whole
exome sequencing and cDNA analysis were performed on
probands’ genomic DNA and RNA extracted from PBMC,
respectively. 1University of Washington, Seattle, WA, United States,
2University of Zurich- Medical Genetics, Zürich, Switzerland However, the presence of RDVs in
addition to causal variants did not correlate with phenotypic
severity, indicating that simple addition of RDV numbers
has no predictive value. While interpretation of the pheno-
typic effect of RDVs remains challenging, comparison of
variant types/distribution between causal alleles and con-
trol/additional alleles can provide valuable insights. Syndrome and explains our patient’s clinical findings. Parental karyotypes were normal. Conclusions: Nablus mask-like facial syndrome is a rare
microdeletion syndrome. According to the literature, this is
the first time that NMLFS and Klinefelter’s Syndrome are
together in a patient. Reference: Raas-Rothschild A., Dijkhuizen T. et al.,
European Journal of Medical Genetics 52(2009) 140-144 Ö. Anlaş: None. B. Sarıkepe: None. S. Zeybek: None. M. Öztürk: None. G. Bağcı: None. G.O. Çetin: None. P11.056D pending on variants validation by Sanger; and phenotypic
and genotypic (WES) characterization is finished in 18
cases, of which 13 (72.2%) have been diagnosed. In 77% of
diagnosed cases the causal variant corresponded to a ‘de
novo’ mutation (frameshift and stopgain variants). Conclusion:
The
presence
of
biallelic
inactivating
variants in NBAS in the presently reported cases prompts
the unification of dependent infantile liver failure syndrome
2 and SOPH syndrome in a single autosomal recessive
condition characterized by variable association of hyper-
transaminasemia, recurrent acute liver failure, short stature,
bone fragility, ocular defects and immune system disorders. Conclusions: For the still undiagnosed cases, functional
studies are expected. Moreover, SpainUDP participates in
international initiatives such as the European projects RD-
Connect and Solve RD, the Undiagnosed Diseases Network
International (UDNI), and the MatchMaker Exchange
platform, sharing phenotypic and genotypic data to find
cases with similar profiles to get a diagnosis. Conclusions: For the still undiagnosed cases, functional
studies are expected. Moreover, SpainUDP participates in
international initiatives such as the European projects RD-
Connect and Solve RD, the Undiagnosed Diseases Network
International (UDNI), and the MatchMaker Exchange
platform, sharing phenotypic and genotypic data to find
cases with similar profiles to get a diagnosis. D. Carli: None. E. Giorgio: None. F. Pantaleoni: None. S. Barresi: None. G. Baldassarre: None. E. Riberi: None. F. Licciardi: None. S. Pizzi: None. A. Ciolfi: None. A. Gazzin: None. D. Montin: None. C. Molinatto: None. A. Brusco: None. M. Tartaglia: None. G.B. Ferrero: None. B. Martínez-Delgado: None. E. López: None. S. Monzón: None. I. Cuesta: None. V. Aquino: None. A. Damián: None. I. Gonzalo: None. C. Rodríguez-Martín:
None. G. Gómez-Mariano: None. A. Navarro: None. S. Ramos: None. J. Lara: None. E. Román: None. M.R. Cazorla: None. G. Iglesias: None. P. Ros Pérez: None. P. Tutor: None. S.T. Mellor: None. M.J. Cabrejas: None. C. Jiménez: None. F.J. Alonso: None. E. Bermejo: None. M. Posada: None. B. Martínez-Delgado: None. E. López: None. S. Monzón: None. I. Cuesta: None. V. Aquino: None. A. Damián: None. I. Gonzalo: None. C. Rodríguez-Martín:
None. G. Gómez-Mariano: None. A. Navarro: None. S. Ramos: None. J. Lara: None. E. Román: None. M.R. Cazorla: None. G. Iglesias: None. P. Ros Pérez: None. P. Tutor: None. S.T. Mellor: None. M.J. Cabrejas: None. C. Jiménez: None. F.J. Alonso: None. E. Bermejo: None. M. Posada: None. Trio based exome analysis results at the Spanish
Undiagnosed Rare Diseases Program, SpainUDP Trio based exome analysis results at the Spanish
Undiagnosed Rare Diseases Program, SpainUDP B. Martínez-Delgado1, E. López1, S. Monzón2, I. Cuesta2,
V. Aquino1, A. Damián1, I. Gonzalo1, C. Rodríguez-Martín1,
G. Gómez-Mariano1, A. Navarro1, S. Ramos1, J. Lara3,
E. Román3, M. R. Cazorla3, G. Iglesias3, P. Ros Pérez3,
P. Tutor3, S. T. Mellor3, M. J. Cabrejas3, C. Jiménez3, F. J. Alonso1, E. Bermejo1, M. Posada1 P11.059C Whole exome sequencing analysis candidates MRVI1 as a
potential susceptibility gene for Moyamoya syndrome and
cerebral arteriopathies in Neurofibromatosis type 1 1Instituto de Investigación de Enfermedades Raras. Instituto de
Salud Carlos III (IIER / ISCIII), Majadahonda, Madrid, Spain,
2Bioinformatics Unit. Instituto de Salud Carlos III (ISCIII),
Majadahonda, Madrid, Spain, 3Hospital Puerta de Hierro,
Majadahonda, Madrid, Spain 1Instituto de Investigación de Enfermedades Raras. Instituto de
Salud Carlos III (IIER / ISCIII), Majadahonda, Madrid, Spain,
2Bioinformatics Unit. Instituto de Salud Carlos III (ISCIII),
Majadahonda, Madrid, Spain, 3Hospital Puerta de Hierro,
Majadahonda, Madrid, Spain C. Santoro1, T. Giugliano2, M. Kraemer3, A. Torella2,
J. Schwitalla4, M. Cirillo5, P. Berlit6, V. Nigro6, S. Perrotta6,
G. Piluso6 C. Santoro1, T. Giugliano2, M. Kraemer3, A. Torella2, J. Schwitalla4, M. Cirillo5, P. Berlit6, V. Nigro6, S. Perrotta6,
G. Piluso6 1Department of Woman, Child and General and Specialistic
Surgery,Università degli Studi della Campania “Luigi
Vanvitelli”, Naples, Italy, 2Dipartimento di Medicina di
Precisione, Università degli Studi della Campania “Luigi
Vanvitelli”, Naples, Italy, 3Department of Neurology,Alfried
Krupp Hospital,, Essen, Germany, 4Department of Neurology,
Alfried Krupp Hospital, Essen, Italy, 5eDipartimento di Scienze
Mediche, Chirurgiche, Neurologiche, Metaboliche e
dell'Invecchiamento,Università degli Studi della Campania
“Luigi Vanvitelli”, Naples, Italy, 6Università degli Studi della
Campania “Luigi Vanvitelli”, Naples, Italy Introduction: SpainUDP is the Spanish Undiagnosed Rare
Diseases Program, implemented by the Institute of Rare
Diseases Research (IIER) of the Institute of Health Carlos
III (ISCIII). Since 2015 works in collaboration with Hos-
pital Puerta de Hierro (Madrid) which supports detailed
clinical examination and complementary studies of patients. SpainUDP aims to find a definitive diagnosis to patients
with undiagnosed rare diseases, through a multidisciplinary
approach (by clinicians, geneticists, bioinformaticians and
researchers). Introduction: SpainUDP is the Spanish Undiagnosed Rare
Diseases Program, implemented by the Institute of Rare
Diseases Research (IIER) of the Institute of Health Carlos
III (ISCIII). Since 2015 works in collaboration with Hos-
pital Puerta de Hierro (Madrid) which supports detailed
clinical examination and complementary studies of patients. SpainUDP aims to find a definitive diagnosis to patients
with undiagnosed rare diseases, through a multidisciplinary
approach (by clinicians, geneticists, bioinformaticians and
researchers). Materials and methods: Taking advantage of next
generation sequencing techniques, mainly whole exome
analysis (WES) is performed by trio analysis. In addition,
Phenotips software is used for an accurate and standardized
description of phenotypes (through HPO, Human Pheno-
type Ontology). P11.056D Clinical Report: A five years old male patient was
referred to our clinic because of speech delay, growth
retardation, mental retardation and dysmorphic features. He
was
a
born
at
38
weeks
gestational
age
to
non-
consanguineous parents. His weight, height and head
circumference percentiles were <3p at the time of physical
examination. Microcephaly, hyperthelorism, upper epi-
canthus, wide nasal base, high arched plate, micrognatia,
protruding ears and hypomimic, mask-like face were the
other physical examination findings. His electrocardiogram,
hearing test, abdominal USG and cranial MR were normal
and
Denver
II
developmental
screening
test
showed
development delay. The patient’s karyotype was 47,XXY,
compatible with Klinefelter syndrome. Since this karyotype
was not enough to explain the patient’s dysmorphic features
and motor and mental retardation we performed microarray
analysis. The microarray analyses revealed a 5,024 Kb
deletion on 8q21.3q22.1 which contains 17 OMIM genes. This deleted region is the region associated with NMLF Clinical Report: A five years old male patient was
referred to our clinic because of speech delay, growth
retardation, mental retardation and dysmorphic features. He
was
a
born
at
38
weeks
gestational
age
to
non-
consanguineous parents. His weight, height and head
circumference percentiles were <3p at the time of physical
examination. Microcephaly, hyperthelorism, upper epi-
canthus, wide nasal base, high arched plate, micrognatia,
protruding ears and hypomimic, mask-like face were the
other physical examination findings. His electrocardiogram,
hearing test, abdominal USG and cranial MR were normal
and
Denver
II
developmental
screening
test
showed
development delay. The patient’s karyotype was 47,XXY,
compatible with Klinefelter syndrome. Since this karyotype
was not enough to explain the patient’s dysmorphic features
and motor and mental retardation we performed microarray
analysis. The microarray analyses revealed a 5,024 Kb
deletion on 8q21.3q22.1 which contains 17 OMIM genes. This deleted region is the region associated with NMLF Results: Both patients carried a nonsense mutation in
NBAS (NM_015909.3; p.Arg501* and p.Ser230fs*4) in
compound heterozygosity with the synonymous variant
c.6840G>A (p.Thr2280Thr). This variant (MAF< 0.00001,
ExAC) affects the last nucleotide of exon 51, and cDNA J. del Picchia 368 analysis demonstrated its pathogenicity, causing skipping of
the exon. analysis demonstrated its pathogenicity, causing skipping of
the exon. pending on variants validation by Sanger; and phenotypic
and genotypic (WES) characterization is finished in 18
cases, of which 13 (72.2%) have been diagnosed. In 77% of
diagnosed cases the causal variant corresponded to a ‘de
novo’ mutation (frameshift and stopgain variants). P11.059C Materials and methods: Taking advantage of next
generation sequencing techniques, mainly whole exome
analysis (WES) is performed by trio analysis. In addition,
Phenotips software is used for an accurate and standardized
description of phenotypes (through HPO, Human Pheno-
type Ontology). Introduction: Moyamoya disease (MMD) is a progressive
cerebral vasculopathy. The p.(Arg4810Lys) substitution in
RNF213 is linked to MMD in Asians. Recently several rare
variants in RNF213 have been associated to MMD in eur-
opeans. People with neurofibromatosis type 1 (NF1) are
particularly prone to develope this angiopathy thus called
syndrome (MMS). Intriguingly, most cases of NF1-related
MMS have been described in Caucasians, inverting the
population ratio for MMD observed in Asians. Additive Results: In 2015-2017, 135 cases were accepted in
SpainUDP. During this time, 37 cases (27.4%) dropped out
the program due to diverse reasons. The remaining 98 cases
are distributed as follows: 40 cases are in a deep phenotypic
characterization; WES is ongoing for 33 cases; 7 cases are Abstracts from the 51st European Society of Human Genetics Conference: Posters 369 genetic factors, independent of the NF1 locus, may con-
tribute to the pathogenesis of MMS. Patients and Results: We present the molecular profile
of 45 unrelated Polish NF1 and neurofibromatosis-Noonan
patients (and 26 affected family members), expanded by
analysis of potential modifiers within other nuclear genes
and mtDNA. Using MLPA and NGS sequencing we
identified
40
different
pathogenic/probably
pathogenic
NF1 variants (including whole-gene deletions/duplications),
nearly half of them novel. Mutation detection rate in the
entire study cohort was 92%, familial cases constituted
56%. To evaluate the potential influence of molecular
modifiers, clinical exomes of 37 NF1-positive probands
were analysed, revealing variants in other clinically relevant
genes
(oncologic,
cardiac,
neurologic,
cutaneous
or
immune),
including
DNA
repair-related
genes. Co-
occurrence
of
another
Ras/MAPK
gene
variant
and
concomitance of Silver-Russell syndrome were noted in 6
and 1 patients, respectively. Mitochondrial DNA analysis in
59 NF1 cases excluded known mutations, but did show
several secondary/synergistic/modifier alterations or risk
factors for neurological, cardiovascular, metabolic disorders
or cancerogenesis. Methods: We carried out a whole exome study in a large
Italian family with MMS-NF1 co-occurrence in two first
cousins and minor cerebral vasculopathies in NF1 relatives. Results: None of the RNF213 variants already reported or
other rare variants were found yet the p.(Pro186Ser)
substitution
(rs35857561)
in
MRVI1
segregated
with
MMS and other minor cerebral vasculopathies in our Italian
family. P11.060D Supported by CMHI projects: S140/2014, 234/15. The molecular profile of Polish patients with
neurofibromatosis type 1 and neurofibromatosis-Noonan
syndrome: do additional genetic factors affect the
phenotypic variability among NF1-positive patients? M. Pelc: None. P. Halat-Wolska: None. D. Wicher:
None. E. Ciara: None. J. Kosińska: None. P. Stawiński:
None. A. Cieślikowska: None. D. Piekutowska-Abramc-
zuk: None. D. Jurkiewicz: None. D. Siestrzykowska:
None. B. Chałupczyńska: None. M. Rydzanicz: None. P. Kowalski: None. M. Jędrzejowska: None. R. Płoski:
None. M. Krajewska-Walasek:
None. K. Chrzanowska: None. M. Pelc1, P. Halat-Wolska1, D. Wicher1, E. Ciara1, J. Kosińska2,
P. Stawiński2,3, A. Cieślikowska1, D. Piekutowska-Abramczuk1,
D. Jurkiewicz1, D. Siestrzykowska1, B. Chałupczyńska1,
M. Rydzanicz2, P. Kowalski1, M. Jędrzejowska1, R. Płoski2,
M. Krajewska-Walasek1, K. Chrzanowska1 M. Pelc1, P. Halat-Wolska1, D. Wicher1, E. Ciara1, J. Kosińska2,
P. Stawiński2,3, A. Cieślikowska1, D. Piekutowska-Abramczuk1,
D. Jurkiewicz1, D. Siestrzykowska1, B. Chałupczyńska1,
M. Rydzanicz2, P. Kowalski1, M. Jędrzejowska1, R. Płoski2,
M. Krajewska-Walasek1, K. Chrzanowska1 P11.059C Conclusions: MRVI1 is a functional partner of ITPR1,
PRKG1 and GUCY1A3, differently related to MMS and
other vasculopathies, all involved in response to NO. The
rs35857561
substitution
has
got
a
higher
MAF
in
Europeans than in Asians. The variant segregated with
two more patients with MMS form a further NF1 German
family. The 11p15.4 cytoband, where MRVI1 is located, has
been linked to retinal vessel diameter, with the D11S1999
marker closely located to the 5'UTR of MRVI1. These
reasons support the hypothesis that MRVI, and the p. (Pro186Ser) substitution, might really represent a suscept-
ibility factor for MMS in Caucasians with NF1. Conclusions: Our study contributes to further delineation
of the NF1 molecular profile and presents new data
concerning potential factors influencing clinical variability
of the disease. More detailed correlation/differentiation
studies for NF1-related phenotypes may lead to an update of
the diagnostic criteria and future therapeutics. C. Santoro: None. T. Giugliano: None. M. Kraemer:
None. A. Torella: None. J. Schwitalla: None. M. Cirillo:
None. P. Berlit: None. V. Nigro: None. S. Perrotta: None. G. Piluso: None. C. Santoro: None. T. Giugliano: None. M. Kraemer:
None. A. Torella: None. J. Schwitalla: None. M. Cirillo:
None. P. Berlit: None. V. Nigro: None. S. Perrotta: None. G. Piluso: None. Neurofibromatosis-Noonan syndrome diagnosed in an
infant of a three-generation NF1 family Neurofibromatosis-Noonan syndrome diagnosed in an
infant of a three-generation NF1 family 1Department of Medical Genetics, The Children’s Memorial
Health Institute, Warsaw, Poland, 2Department of Medical
Genetics, Warsaw Medical University, Warsaw, Poland,
3Department of Genetics, Institute of Physiology and
Pathology of Hearing, Warsaw, Poland J. Kárteszi1, B. Elmont2, J. Bene3, M. Buzogány2, M. Tihanyi1
1Genetic Laboratory, Zalaegerszeg, Hungary, 2Department of
Paediatrics, Zalaegerszeg, Hungary, 3Department of Medical
Genetics, Pécs, Hungary J. Kárteszi1, B. Elmont2, J. Bene3, M. Buzogány2, M. Tihanyi1 1Genetic Laboratory, Zalaegerszeg, Hungary, 2Department of
Paediatrics, Zalaegerszeg, Hungary, 3Department of Medical
Genetics, Pécs, Hungary Introduction: Neurofibromatosis type 1 (NF1) is a RASo-
pathy characterized by neuro-cutaneous abnormalities and
predisposition to tumorigenesis. Despite molecular homo-
geneity (NF1 gene mutations), significant phenotypic
variability is observed, not only among unrelated NF1
patients, but also within affected families. Consequently, a
possible correlation of NF1 clinical spectrum with addi-
tional genetic factors (“modifiers”) has been inferred. Neurofibromatosis-Noonan
syndrome
(NFNS,
MIM:
601321) is a peculiar entity characterized by the clinical
signs of both Neurofibromatosis type 1 (NF1) and Noonan
syndrome (NS). The features of Noonan syndrome mainly
facial dysmorphism and short stature accompany the char-
acteristics of NF1 as café-au-lait spots and skeletal changes. 370 J. del Picchia also been identified in 17 patients referred to as Sotos 2 or
Malan syndrome (MS, MIM614753). Neurofibromas and Lisch nodules are less frequently
observed in this NF1 variant. In most of the cases published
until now a heterozygous mutation in NF1 gene was
determined. It was also demonstrated that RAS-MAPK
pathway is affected and neurofibromin is a negative reg-
ulator of this signal transduction pathway. Therefore, NFNS
belongs to the so called RASopathies with Noonan and
Noonan-like syndromes. Classical mutations causing NF1
phenotype can also cause NFNS. Neurofibromas and Lisch nodules are less frequently
observed in this NF1 variant. In most of the cases published
until now a heterozygous mutation in NF1 gene was
determined. It was also demonstrated that RAS-MAPK
pathway is affected and neurofibromin is a negative reg-
ulator of this signal transduction pathway. Therefore, NFNS
belongs to the so called RASopathies with Noonan and
Noonan-like syndromes. Classical mutations causing NF1
phenotype can also cause NFNS. We present two brothers aged 23 and 7 years with ID,
craniofacial dysmorphism (macrodolichocephaly, high fore-
head
and
anterior
hairline,
long
face,
downslanting
palpebral fissures, low-set dysplastic ears, prominent chin)
and musculoskeletal, ocular and CNS abnormalities. Neurofibromatosis-Noonan syndrome diagnosed in an
infant of a three-generation NF1 family Exome
sequencing identified an identical de novo NFIX exon 2
variant NM_001271043.2:c.370C>T p.(Arg124Trp) in both
brothers. The variant was also in 1/95 reads in the father. This could be an error or contamination, but it may indicate
mosaicism (including germline mosaicism). Paternal origin
of the variant was supported also by haplotype analysis: the
brothers carried different copies of maternal 19p13 but
shared a paternal haplotype. The variant was predicted to be
deleterious and was absent from all databases (ESP, ExAC,
gnomAD, GEEVS). A substitution affecting the neighbour-
ing residue, p.(Arg123Trp), has been reported in one MS
patient. We present a three-generation family with the clinical and
genetic diagnosis of NF1 following autosomal dominant
inheritance (all affected family members meet the NIH-
consensus criteria for NF1). The proband is a 6-months-old
boy with multiple café-au-lait spots, failure to thrive,
relative macrocephaly and facial minor anomalies resem-
bling Noonan syndrome. Mutation was determined in the
family to be c.499_502del4bp in heterozygous form. As far
as we know this mutation was not published previously in
connection with NFNS. The origin of this interesting phenotype is still debated,
the most accepted concept keeps it an NF1 variant and a
recent publication suggests that fetal environmental factors
may also play a role in the evolvement. We discuss in our
poster the current knowledge about the pathogenesis of
RASopathies and focus on the unanswered questions
regarding NFNS. These two new cases of MS help to define the clinical
picture of MS and underscore the recent notion that a
significant fraction (4-10%) of de novo mutations can
originate from germline mosaics, with consequences for
recurrence risks. Supported by 17-29423A and 00064203. M. Havlovicová: None. M. Hančárová: None. J. Vetička: None. D. Prchalová: None. V. Stránecký: None. Z. Sedláček: None. J. Kárteszi: None. B. Elmont: None. J. Bene: None. M. Buzogány: None. M. Tihanyi: None. P11.064D P11.064D Application of panel next generation sequencing in the
diagnosis and clinical differentiation of patients with
Noonan syndrome clinical suspicion Aim: Determine the pathogenicity of novel variants in
order to give the correct genetic counseling to families. Aim: Determine the pathogenicity of novel variants in
order to give the correct genetic counseling to families. Materials and Methods: 80 non related cases were
analyzed using NGS panels. Detailed clinical data and
genealogies were noted. Written informed consent was
obtained from all families before testing. The NGS capture
panels were designed and validated by Sistemas Genómicos
with CE marking certificate for diagnosis, and performed
with Illumina technology. The variants were analyzed using
GeneSystems software. Families were assessed and advised
by clinical geneticists. N. Bezniakow1, M. Gos1, A. Landowska1, A. Abramowicz1,
O. Kordowska1, S. Rzońca1, J. Sawicka1, T. Gambin2,
J. Klapecki1, A. Kutkowska-Kaźmierczak1, J. Wierzba3,4,
R. Śmigiel5, A. Jakubiuk-Tomaszuk6, M. Karpiński7,
A. Doraczyńska-Kowalik8, M. Piotrowicz9, T. Chilarska9,
R. Ślęzak8, E. Kaczorowska10, M. Krygier10, K. Wojciechowska11,
J. Pilch12, R. Posmyk13, E. Obersztyn1, J. Bal1 N. Bezniakow1, M. Gos1, A. Landowska1, A. Abramowicz1,
O. Kordowska1, S. Rzońca1, J. Sawicka1, T. Gambin2,
J. Klapecki1, A. Kutkowska-Kaźmierczak1, J. Wierzba3,4,
R. Śmigiel5, A. Jakubiuk-Tomaszuk6, M. Karpiński7,
A. Doraczyńska-Kowalik8, M. Piotrowicz9, T. Chilarska9,
R. Ślęzak8, E. Kaczorowska10, M. Krygier10, K. Wojciechowska11,
J. Pilch12, R. Posmyk13, E. Obersztyn1, J. Bal1 N. Bezniakow1, M. Gos1, A. Landowska1, A. Abramowicz1,
O. Kordowska1, S. Rzońca1, J. Sawicka1, T. Gambin2,
J. Klapecki1, A. Kutkowska-Kaźmierczak1, J. Wierzba3,4,
R. Śmigiel5, A. Jakubiuk-Tomaszuk6, M. Karpiński7,
A. Doraczyńska-Kowalik8, M. Piotrowicz9, T. Chilarska9,
R. Ślęzak8, E. Kaczorowska10, M. Krygier10, K. Wojciechowska11,
J. Pilch12, R. Posmyk13, E. Obersztyn1, J. P11.064D Bal1 1Department of Medical Genetics, Institute of Mother and
Child, Warsaw, Poland, 2Institute of Computer Science,
Warsaw University of Technology, Warsaw, Poland,
3Department and Clinic of Pediatrics, Hematooncology,
Oncology and Endocrinology, Medical University of Gdańsk,
Gdańsk, Poland, 4Department of General Nursing, Medical
University of Gdańsk, Gdańsk, Poland, 5Department of Social
Pediatrics, Wroclaw Medical University, Wrocław, Poland,
6Department of Pediatric Neurology and Rehabilitation,
Medical University of Białystok, Białystok, Poland, 7Institute of
Cardiology, Jagiellonian University School of Medicine,
Cracow, Poland, 8Department of Genetics, Wroclaw Medical
University, Wrocław, Poland, 9Department of Genetics, Polish
Mother’s Memorial Hospital - Research Institute, Łódź,
Poland, 10Department of Biology and Genetics, Medical
University of Gdańsk, Gdańsk, Poland, 11Department of
Children Hematology, Oncology and Transplantology,
Children’s University Hospital, Lublin, Poland, 12Department
of Pediatric Neurology, Medical University of Silesia,
Katowice, Poland, 13NZOZ Genetics, Center for Clinical
Genetics, Białystok, Poland Results: Novel likely pathogenic variants were found in 5
families, and confirmed by Sanger sequencing. The table describes the variants found in patients, and
their clinical diagnosis. Case Panel
Novel Variation
Gene
Clinical
diagnosis
zigocity
1
Intellectual
disability
NM_004456.4:
c.2030A>G
p.Asp677Gly
EZH2
Weaver
Syndrome,
(autosomal
dominant)
heterozygous
2
Neurology
NM_014946.3:
c.165C>A
p.Tyr55*
SPAST
Spastic
Paraplegia 4,
(autosomal
dominant)
heterozygous
3
Cardiology
NM_000116.4:
c.202A>T
p.Asn68Tyr
TAZ
Left ventricular
noncompaction,
(X linked
recessive)
hemizygous
4
Epilepsy
NM_001165963.1:
c.2665G>A
p.Ala889Thr
NM_000068.3:
c.6739C>T
p.Arg2247Trp
SCN1A
CACNA1A
Epilepsy with
Ataxia,
(autosomal
dominant)
heterozygous
5
Skeletal
dysplasia
NM_001287.5:
c.2229dupC
p. Ser744LeufsTer183
CLCN7
Osteopetrosis,
(autosomal
dominant/
recessive)
homozygous
(Allele
deletion was
ruled out
by aCGH) Introduction: The implementation of the next generation
sequencing (NGS) technique allowed not only to identify
novel genes related to disease etiology, but also sig-
nificantly improved molecular diagnosis of Noonan syn-
drome (NS) and related disorders. The aim of the study was
the identification of pathogenic variants in novel and can-
didate genes related to NS syndrome pathogenesis. After deep molecular, clinical and genealogy analysis,
further complementary tests in relatives were performed. After deep molecular, clinical and genealogy analysis,
further complementary tests in relatives were performed. These results allowed the assessment of pathogenicity of all
novel variants. Genetic counseling of families will be
discussed. These results allowed the assessment of pathogenicity of all
novel variants. Genetic counseling of families will be
discussed. Conclusion: A detailed clinical history in addition to the
panel-based NGS technology, might allow the determina-
tion of the pathogenicity of novel variants, improving the
genetic counseling of the families involved. P11.063C Novel
variants` pathogenicity assignment is one of the biggest, but 371 Abstracts from the 51st European Society of Human Genetics Conference: Posters in some cases, it might be clarify by a detailed clinical
history and a multidisciplinary approach. P11.063C Panel based next generation sequencing: pathogenicity
assessment of novel variants and their impact in genetic
counseling Panel based next generation sequencing: pathogenicity
assessment of novel variants and their impact in genetic
counseling Two brothers with Malan syndrome and identical
seemingly de novo missense NFIX variant due to probable
paternal germline mosaicism identified using exome
sequencing F. Cantarella1, G. Moya1, N. Loreti1, M. Capelli1, L. Espeche1,
M. Samara1, S. López1, M. Obregón2, M. Villanueva3, A. Solari4,
P. Vega5, S. Santillán Garzón6, C. Moya6, M. Gil6, V. Ferreiro1 M. Havlovicová1, M. Hančárová1, J. Vetička2, D. Prchalová1,
V. Stránecký3, Z. Sedláček1 M. Havlovicová1, M. Hančárová1, J. Vetička2, D. Prchalová1,
V. Stránecký3, Z. Sedláček1 1Department of Biology and Medical Genetics, 2nd Medical
Faculty, Charles University and University Hospital Motol,
Prague, Czech Republic, 2Genetika Ostrava s. r. o., Ostrava,
Czech Republic, 3Department of Pediatrics and Adolescent
Medicine, Diagnostic and Research Unit for Rare Diseases,
Charles University 1st Faculty of Medicine and General
University Hospital, Prague, Czech Republic 1Genos S.A., Buenos Aires, Argentina, 2Sección Genética. Departamento de Pediatría. Hospital Italiano de Buenos Aires,
Buenos Aires, Argentina, 3FLENI. Fundación de Lucha para
las Enfermedades Neurológicas de la Infancia., Buenos Aires,
Argentina, 4Clínica Zabala., Buenos Aires, Argentina,
5Consultorio y laboratorio de neurogenética. Centro
universitario de neurología. Hospital J.M. Ramos Mejía.,
Buenos Aires, Argentina, 6Sistemas Genómicos S. L., Valencia,
Spain 1Genos S.A., Buenos Aires, Argentina, 2Sección Genética. Departamento de Pediatría. Hospital Italiano de Buenos Aires,
Buenos Aires, Argentina, 3FLENI. Fundación de Lucha para
las Enfermedades Neurológicas de la Infancia., Buenos Aires,
Argentina, 4Clínica Zabala., Buenos Aires, Argentina,
5Consultorio y laboratorio de neurogenética. Centro
universitario de neurología. Hospital J.M. Ramos Mejía.,
Buenos Aires, Argentina, 6Sistemas Genómicos S. L., Valencia,
Spain Overgrowth syndromes combine height greater than two
SDs above the mean and other features. The best recognised
is Sotos syndrome (MIM117550), an autosomal dominant
disorder with distinctive facial features, intellectual dis-
ability (ID), body overgrowth in early life and macro-
cephaly. Mutations in NSD1 are found in 90% of cases. De
novo mutations in the initial exons of NFIX in 19p13 have Introduction: Identification of disease-causing mutations
has been tremendously accelerated by Next Generation
Sequencing (NGS) implementation. However, the benefits
offered by NGS come with a number of challenges. P11.065A 1Centre for Medical Genetics, Vilnius University Hospital
Santaros Klinikos, Vilnius, Lithuania, 2Department of Human
and Medical Genetics, Institute of Biomedical Sciences,
Faculty of Medicine, Vilnius University, Vilnius, Lithuania,
3Faculty of Medicine, Vilnius University, Vilnius, Lithuania,
4Lithuanian Academy of Sciences, Vilnius, Lithuania P11.064D The NGS panel also included genes
related to disorders clinically similar to NS and pathogenic/
potentially pathogenic variants in these genes (particularly
KMT2D, ARDI1A, ARID1B, CREBBP, KAT6B) were found
in 24 patients. Also, variants in RASA2, MAP3K8, LZTR1 or
A2ML1 genes were found in single patients. No variants in
SOS2 or PPP1CB were found. Conclusions: The implementation of panel testing is
beneficial in the diagnosis of Noonan syndrome and other
RASopathies, although it seems that mutations in “novel”
genes are incidental. Supported from NCN research projects no. 2013/09/B/
NZ2/03164. M. Zamariolli: None. L.C. Santos: None. M.E.S. Colovati: None. A.B. Perez: None. S. Bragagnolo: None. M.I. Melaragno: None. Supported from NCN research projects no. 2013/09/B/
NZ2/03164. M. Zamariolli: None. L.C. Santos: None. M.E.S. Colovati: None. A.B. Perez: None. S. Bragagnolo: None. M.I. Melaragno: None. N. Bezniakow: None. M. Gos: None. A. Landowska: N. Bezniakow: None. M. Gos: None. A. Landowska:
None. A. Abramowicz: None. O. Kordowska: None. S. Rzońca: None. J. Sawicka: None. T. Gambin: None. J. Klapecki: None. A. Kutkowska-Kaźmierczak: None. J. Wierzba: None. R. Śmigiel: None. A. Jakubiuk-Tomas- N. Bezniakow: None. M. Gos: None. A. Landowska:
None. A. Abramowicz: None. O. Kordowska: None. S. Rzońca: None J Sawicka: None T Gambin: None J None. A. Abramowicz: None. O. Kordowska: None. S. R
ń
N
J S
i k
N
T G
bi
N
J Three unrelated Lithuanian cases of oculodentodigital
dysplasia: phenotypic analysis and comparison to the
literature Three unrelated Lithuanian cases of oculodentodigital
dysplasia: phenotypic analysis and comparison to the
literature A. Matulevičienė1,2, K. iaurytė3, L. Cimbalistienė1,2,
B. Burnytė1,2, L. Ambrozaitytė1,2, R. Mekienė1,2, V. Kučinskas2,4,
A. Utkus2 Ten candidate genes sequenced in patients with oculo-
auriculo-vertebral spectrum M. Zamariolli, L. C. Santos, M. E. S. Colovati, A. B. Perez,
S. Bragagnolo, M. I. Melaragno Introduction: Oculodentodigital dysplasia (ODDD) is a
rare autosomal dominant syndrome, caused by a hetero-
zygous mutation in the GJA1 gene on chromosome 6q22. Around 300 cases have been described in the scientific lit-
erature. Features of ODDD include facial, skeletal, neuro-
logical, cardiac, and ocular anomalies with high penetrance,
intra- and interfamilial phenotypic variability, and advanced
paternal age in sporadic cases. P11.064D Materials and Methods: One hundred twenty-eight
patients with Noonan syndrome clinical diagnosis and
excluded mutations in PTPN11, RAF1, SOS1 and KRAS
genes were tested with custom designed NGS panel
(SeqCap EZ Choice Library, Roche Diagnostics). Func-
tional in silico and in vitro (when possible) studies of new
mutations were performed. F. Cantarella: None. G. Moya: None. N. Loreti: None. M. Capelli: None. L. Espeche: None. M. Samara: None. S. López: None. M. Obregón: None. M. Villanueva:
None. A. Solari: None. P. Vega: None. S. Santillán
Garzón: None. C. Moya: None. M. Gil: None. V. Ferreiro: None. Results: Panel testing allowed to identify pathogenic /
potentially pathogenic variants in 67 (52.3%) probands. Thirty of them had mutation in known RASopathies-related
genes including: NF1 (10pts, all patients besides facial 372 J. del Picchia MAPK1, NKX3-2, HMX1, MYT1, OTX2, GSC, PUF60, and
HOXA2,
by
Ion
PGM
System
for
Next-Generation
Sequencing (Thermo Fisher Scientific). In 89 individuals
studied (77 patients and 12 relatives), we identified a total of
194 variants in DNA extracted from peripheral blood. In
order to infer the potential pathogenicity of these variants,
in silico analysis was performed using the prediction tools
Mutation Taster, FATHMM, PolyPhen 2, and SIFT, as well
as clinical databases such as ClinVar. Using these approa-
ches, seven SNVs, found in genes YPEL1, MAPK1, CRKL,
OTX2, and MYT1, were considered potentially pathogenic. Also, 44 SNVs with unknown significance were found. Our
data confirms the possible genetic heterogeneity in OAVS. Financial Support: FAPESP 2016/18781-7. MAPK1, NKX3-2, HMX1, MYT1, OTX2, GSC, PUF60, and
HOXA2,
by
Ion
PGM
System
for
Next-Generation
Sequencing (Thermo Fisher Scientific). In 89 individuals
studied (77 patients and 12 relatives), we identified a total of
194 variants in DNA extracted from peripheral blood. In
order to infer the potential pathogenicity of these variants,
in silico analysis was performed using the prediction tools
Mutation Taster, FATHMM, PolyPhen 2, and SIFT, as well
as clinical databases such as ClinVar. Using these approa-
ches, seven SNVs, found in genes YPEL1, MAPK1, CRKL,
OTX2, and MYT1, were considered potentially pathogenic. Also, 44 SNVs with unknown significance were found. Our
data confirms the possible genetic heterogeneity in OAVS. Financial Support: FAPESP 2016/18781-7. dysmorphy and other NS related symptoms presented CAL
spots), BRAF (6pts), RIT1 (4pts, including twin sisters
suspected for NF1/NFNS), CBL (4pts) and SHOC2 (2pts,
c.4A>G variant). P11.067C Rzońca: None. J. Sawicka: None. T. Gambin: None. J. Klapecki: None. A. Kutkowska-Kaźmierczak: None. J. Wierzba: None. R. Śmigiel: None. A. Jakubiuk-Tomas-
zuk: None. M. Karpiński: None. A. Doraczyńska-
Kowalik: None. M. Piotrowicz: None. T. Chilarska:
None. R. Ślęzak: None. E. Kaczorowska: None. M. Krygier: None. K. Wojciechowska: None. J. Pilch: None. R. Posmyk: None. E. Obersztyn: None. J. Bal: None. Universidade Federal de São Paulo, São Paulo, Brazil Introduction: Oculodentodigital dysplasia (ODDD) is a
rare autosomal dominant syndrome, caused by a hetero-
zygous mutation in the GJA1 gene on chromosome 6q22. Around 300 cases have been described in the scientific lit-
erature. Features of ODDD include facial, skeletal, neuro-
logical, cardiac, and ocular anomalies with high penetrance,
intra- and interfamilial phenotypic variability, and advanced
paternal age in sporadic cases. Oculo-auriculo-vertebral spectrum (OAVS) is a craniofacial
developmental disorder that mainly affects the structures
derived from the first and second pharyngeal arches. The
phenotype is heterogeneous and typically characterized by
abnormal mandibular, oral, and ear development. The
spectrum’s etiology is complex and heterogeneous since
genetic, epigenetic and environmental factors seem to be
involved; however, the mechanisms are still not clear. Structure variations have been described as potentially
pathogenic for the disorder, but evidences of single
nucleotide variants (SNVs) on specific genes are still scarce. So far, MYT1 gene has been the only gene implicated in
some patients with OAVS. Therefore, the investigation of
single nucleotide variants (SNVs) on more candidate genes
is crucial to understanding this complex disorder. We
investigated the coding and UTR regions of ten candidate
genes that may be relevant to OAVS: CRKL, YPEL1, Materials and Methods: Three unrelated Lithuanian
patients with genetically confirmed ODDD are reported. A
girl, now aged 10, a boy, now aged 9, and a girl, now aged
2, with typical features of ODDD presented bilateral
epicanthus, prominent columella, hypoplastic alae nasi,
and enamel hypoplasia. Sanger sequencing of coding
regions of the GJA1 gene was performed for all cases. All
three identified variants were analyzed with previously
reported ODDD phenotypes. Materials and Methods: Three unrelated Lithuanian
patients with genetically confirmed ODDD are reported. A
girl, now aged 10, a boy, now aged 9, and a girl, now aged
2, with typical features of ODDD presented bilateral
epicanthus, prominent columella, hypoplastic alae nasi,
and enamel hypoplasia. Sanger sequencing of coding
regions of the GJA1 gene was performed for all cases. All
three identified variants were analyzed with previously
reported ODDD phenotypes. Results:
Molecular
genetic
analysis of the coding
sequences
of
GJA1
(NM_000165.4,
NP_000156.1) Abstracts from the 51st European Society of Human Genetics Conference: Posters 373 identified such pathogenic variants c.412G>A (p.G138S) in
the first (CM086823), c.75G>T (p.W25C) in the second
(CM120084), and c.338T>C (p.L113P) in the third case
(CM040074). Facial phenotype was consistent throughout
the cases. Universidade Federal de São Paulo, São Paulo, Brazil In the literature these variants were associated
with neurological anomalies, whereas in our cases such
phenotype was not observed. Ocular findings as described
for p.L113P, and p.W25C were only present in our case 2. Cardiac phenotype for p.W25C has not been previously
reported in association with ODDD, but was presented in
our case. hypotrophy, growth retardation, GH deficiency, and osteo-
tendinous areflexia. The 2 brothers developed spinocer-
ebellar ataxia, with cerebellar atrophy on MRI. Two
heterozygous mutations of PNPLA6 (NM_006702) were
identified [c.3241G>A / p.(Gly1081Arg) and c.3088C>T /
p.(Gln1030*)]. Recent identification of biallelic mutations
of PNPLA6 gene in OMFS, confirmed the autosomal
recessive inheritance suspected from many years (Hufnagel
et al., 2015). Biallelic PNPLA6 mutations also induce 3
other overlapping conditions: Boucher-Neuhauser syn-
drome (OMIM 215470), Laurence-Moon syndrome (OMIM
245800), and Gordon-Holmes syndrome (OMIM 212840). Mutations of PNPLA6 also caused an autosomal recessive
form of spastic paraplegia (OMIM 612020). Now, the term
of « PNPLA6-related disorders » is used to designate this
group of diseases. Conclusions: Thorough examination, analysis of litera-
ture and molecular diagnosis are crucial for care improve-
ment and further disease management, especially when
patients with rare syndromes are concerned. Conclusions: Thorough examination, analysis of litera-
ture and molecular diagnosis are crucial for care improve-
ment and further disease management, especially when
patients with rare syndromes are concerned. A. Matulevičienė: None. K. iaurytė: None. L. Cimba-
listienė: None. B. Burnytė: None. L. Ambrozaitytė:
None. R. Mekienė: None. V. Kučinskas: None. A. Utkus: None. A. Matulevičienė: None. K. iaurytė: None. L. Cimba-
listienė: None. B. Burnytė: None. L. Ambrozaitytė:
None. R. Mekienė: None. V. Kučinskas: None. A. Utkus: None. G. Jedraszak: None. N. de Roux: None. F. Jobic: None. R. Desailloud: None. H. Bony: None. S. Milazzo: None. M. Mathieu-Dramard: None. G. Morin: None. G. Jedraszak: None. N. de Roux: None. F. Jobic: None. R. Desailloud: None. H. Bony: None. S. Milazzo: None. M. Mathieu-Dramard: None. G. Morin: None. G. Jedraszak: None. N. de Roux: None. F. Jobic: None. R. Desailloud: None. H. Bony: None. S. Milazzo: None. M. Mathieu-Dramard: None. G. Morin: None. Oliver-McFarlane syndrome: mutations of PNPLA6 and
follow-up of 30 years in two brothers 14 years of experience of de Spanish Overgrowth Registry
Consortium in the diagnostic of overgrowth disorders G. Jedraszak1, N. de Roux2, F. Jobic3, R. Desailloud4, H. Bony5,
S. Milazzo6, M. Mathieu-Dramard3, G. Morin3 J. Tenorio, P. Arias, V. Romanelli, G. Gordo, S. García-Miñaur,
F. Santos, I. Dapía, L. Rodríguez-Laguna, E. Vallespín,
M. Palomares, A. Del Pozo, F. García-Santiago, E. Mansilla,
M. Solís, K. Heath, Á. Campos, V. Martínez-Glez,
T. Consortium, J. Nevado, P. Lapunzina 1Laboratory of Human Genetics, Amiens, France, 2Laboratoire
de Biochimie-Hormonologie - Hôpital Robert Debré, Paris,
France, 3Clinical Genetics, Amiens, France, 4Service of
Endocrinology, Amiens, France, 5Pediatric Endocrinology,
Amiens, France, 6Service of Ophthalmology, Amiens, France P11.069A Oliver-McFarlane syndrome: mutations of PNPLA6 and
follow-up of 30 years in two brothers Oliver-McFarlane syndrome: mutations of PNPLA6 and
follow-up of 30 years in two brothers Medical and Molecular Genetics Institute (INGEMM), Madrid,
Spain Conclusions: Overall, the molecular confirmation of an
initial clinical suspicious was about 50% in the SOGRI
cohort. Three new clinical entities and their underlying
molecular defects were described, highlighting the impor-
tance of the application of new technology to study the
negatives cases. There are a relatively high number of
patients without molecular confirmation, suggesting the
existence
of
new
molecular
mechanism
that
remain
unknown and must be explore in the future. M.G. Serey-Gaut: None. C. Cabrol: None. J. Vallat:
None. L. Tatu:
None. B. Loeys:
None. L. Van
Maldergem: None. M.G. Serey-Gaut: None. C. Cabrol: None. J. Vallat:
None. L. Tatu:
None. B. Loeys:
None. L. Van
Maldergem: None. A new recognizable syndrome caused by mutations in the
PITX1 gene J. Tenorio: None. P. Arias: None. V. Romanelli: None. G. Gordo: None. S. García-Miñaur: None. F. Santos:
None. I. Dapía: None. L. Rodríguez-Laguna: None. E. Vallespín: None. M. Palomares: None. A. Del Pozo:
None. F. García-Santiago: None. E. Mansilla: None. M. Solís: None. K. Heath: None. Á. Campos: None. V. Martínez-Glez: None. T. Consortium: None. J. Nevado:
None. P. Lapunzina: None. M. Rossi1,2, G. Lesca1,2, N. Chatron1,2, A. Labalme1,
D. Sanlaville1,2, A. Fassier3, F. Escande4, S. Manouvrier5,
L. Faivre6, P. Edery1,2, D. Geneviève7 1Service de génétique, Centre de Référence Anomalies du
Développement et Centre de Compétence Maladies Osseuses
Constitutionnelles, Hospices Civils de Lyon, Bron, France,
2INSERM U1028, CNRS UMR5292, Centre de Recherche en
Neurosciences de Lyon, GENDEV Team, Bron, France,
3Service d’orthopédie pédiatrique, Centre de Compétence
Maladies Osseuses Constitutionnelles, Hospices Civils de
Lyon, Bron, France, 4Institut de Biochimie et Génétique
moléculaire, CBP, CHRU de Lille et EA7364 Université de
Lille, Lille, France, 5Clinique de Génétique médicale, Hôpital
Jeanne de Flandre, CHRU de Lille et EA7364 Université de
Lille, Lille, France, 6Service de Génétique et Centre de
Référence Anomalies du Développement, Hôpital d'Enfants,
CHU de Dijon, Dijon, France, 7Département de génétique,
maladies rares et médecine personnalisée, Centre de Référence
Maladies Rares SORO, Inserm U1183, Université Montpellier,
CHU Montpellier, Montpellier, France Medical and Molecular Genetics Institute (INGEMM), Madrid,
Spain Oliver-McFarlane syndrome (OMIM 275400) (OMFS), first
described in 1965, is a rare genetic condition associating
multiple and congenital pituitary hormone deficiencies (GH,
TSH, gonadotropins), trichomegaly, precocious and sever-
echorioretinal atrophy chorioretinal atrophy chorioretinal
atrophy chorioretinal atrophy. If untreated, thyroid and GH
abnormalities result in short stature and intellectual defi-
ciency. Most of patients have hypogonadism, congenital or
revealed during adolescence, impacting their reproduction
capacity. Half of cases have progressive neurological trou-
bles (spinocerebellar ataxia, peripheral neuropathy, spastic
paraplegia). We report on 2 brothers, suspect of OMFS
from childhood (Mathieu et al., 1991). The eldest had
severe temporal and occipital alopecia, long eyelashes and
eyebrows, hypolasia of tooth enamel, early chorioretinal
atrophy with low vision, peripheral neuropathy, hypogo-
nadotropic hypogonadism, GH deficiency, and short stature. The youngest had the same physical appearance, tricho-
megaly, less severe alopecia, enamel tooth anomalies, ret-
inal degeneration with low vision, micropenis, testicular Introduction: Overgrowth syndromes (OGS) comprise a
heterogeneous group in which the main feature is the gen-
eralized or partial increase of growth above 2SD. There is a
high overlap of the clinical features among the OGS,
making the clinical diagnosis a challenge. Since the estab-
lishment of the Spanish Overgrowth Registry (SOGRI) in
2004, more than 2,000 patients have been studied. Thus, the
aim of this project is to reflect our experience in of this
group of patients. Material and Methods: This project was approved by
the ethical committee of the hospital. To study the initial
clinical suspicious, a battery of different methodologies
were applied. For those patients who were negative for the
targeted analysis, screening for other genomic alterations
was performed through SNP-arrays, cGH-arrays, NGS and
functional validation if necessary. Results: The average diagnostic yield in the total
syndromic and non-syndromic overgrowth was 50% and
25%, respectively. Up to date, three new entities were
described:
CLAPO
and
Tenorio
syndromes
and
a J. del Picchia 374 careful search in medical literature identified seven similar
additional families, all of them being compatible with an
AD mode of inheritance. Unfortunately, the corresponding
papers were too old to allow a reevaluation. Based on WES
and segregation analysis performed in the current two
pedigrees, a variant in an interesting candidate gene was
identified. Additional families will be necessary to confirm
this preliminary result. ch19p13.3 microdeletion/microduplication syndrome. Also,
our group was involved in the development of several
clinical guidelines for overgrowth syndromes. P11.071C Grants: FIS-PI15/01481 Grants: FIS-PI15/01481 A new recognizable syndrome caused by mutations in the
PITX1 gene A new recognizable syndrome caused by mutations in the
PITX1 gene P11.072D
Poland Sequence - long time follow-up of 21 cases Poland Sequence - long time follow-up of 21 cases G. E. Girnet1, R. Popescu2, M. Gramescu2, I. Resmerita2,
M. Panzaru2,3, L. Butnariu2,3, E. Gorduza2, C. Rusu2,3 G. E. Girnet1, R. Popescu2, M. Gramescu2, I. Resmerita2,
M. Panzaru2,3, L. Butnariu2,3, E. Gorduza2, C. Rusu2,3 1”St.Mary” Children's Hospital Iasi, Iasi, Romania, 2”Gr. T. Popa” University of Medicine and Pharmacy Iasi, Iasi,
Romania, 3”St.Mary” Children's Hospital, Iasi, Romania 1”St.Mary” Children's Hospital Iasi, Iasi, Romania, 2”Gr. T. Popa” University of Medicine and Pharmacy Iasi, Iasi,
Romania, 3”St.Mary” Children's Hospital, Iasi, Romania Poland sequence is a rare disorder that associates unilateral
defect of pectoralis muscle and syndactyly of hand on the
same side. The disorder is considered “a non-specific
developmental field defect” occurring at 6 weeks of fetal
development, for the moment the cause being unknown. It
is suggested that diminished blood flow through the sub-
clavian artery that goes to the arm may be the precipitating
cause. Rare cases are thought to be caused by a genetic
change that can be passed down in families, but no related
genes have been identified. We have performed a clinical
study on 21 cases of Poland sequence diagnosed in Iasi
Medical Genetics Center, aiming to identify defects asso-
ciated to the main features, as well as the clinical evolution. Our group included 13 males and 8 females. Thirteen
patients had right-sided Poland's syndrome, eight left-sided. Two patients were related (brothers). Major clinical findings
include: hypoplasia/aplasia of pectoralis muscle 21/21
cases, symbrachydactyly 12/21, hypoplastic/absent nipple
5/21, hand hypoplasia 4/21, scoliosis 3/21 and forearm Introduction: Pontocerebellar hypoplasias (PCH) comprise
a heterogeneous group of inherited autosomal recessive or
X-linked disorders characterized by concurrent hypoplasia
of the pons and the cerebellum and variable clinical and
imaging features. The current classification includes 11
PCH subtypes, and 20 associated genes are known to date. Materials and methods: We performed deep clinical and
imaging phenotyping in 62 Italian probands (39 females)
with neuroradiological diagnosis of PCH, who underwent
NGS-based panel sequencing of all known PCH-associated
genes (TruSeq Custom Amplicon technology on a MiSeq
platform) and MLPA for CASK exon rearrangements. Materials and methods: We performed deep clinical and
imaging phenotyping in 62 Italian probands (39 females)
with neuroradiological diagnosis of PCH, who underwent
NGS-based panel sequencing of all known PCH-associated
genes (TruSeq Custom Amplicon technology on a MiSeq
platform) and MLPA for CASK exon rearrangements. Autosomal dominant recurrent parotitis: an under-
reported entity In con-
clusion, Poland anomaly is more common in boys than
girls, and the right side is affected approximately twice as
often as the left. Most cases arise sporadically. However,
because
we
have
identified
an
affected
sibship,
we
appreciate
that
in-depth
genetic
testing
should
be
performed. Whole exome sequencing detected heterozygous mis-
sense mutations in the homeobox transcription factor PITX1
gene (c.793G>T in patients 1 and 2; c.412A>C in patient 3
and, in a mosaic status, in his asymptomatic father). PITX1
anomalies have been reported to cause different syndromes:
i) Liebenberg syndrome, characterized by malformations of
upper limbs that acquire radiological features of the legs,
caused by 5q31.1 rearrangements involving putative PITX1
regulatory elements by disruption of a TAD; ii) Mirror-
image polydactyly/tibial agenesis, caused by intragenic
deletion; iii) Congenital clubfoot with or without preaxial
polydactyly and right tibial hemimelia, caused by dominant
negative mutation. The patients here described showed a
distinct recognizable allelic picture, whose hallmarks are
mandibular hypoplasia, narrow iliac wings, patellar hypo-
aplasia leading to knee flexion deformity, and possible short
stature. G.E. Girnet: None. R. Popescu: None. M. Gramescu:
None. I. Resmerita: None. M. Panzaru: None. L. Butnariu: None. E. Gorduza: None. C. Rusu: None. G.E. Girnet: None. R. Popescu: None. M. Gramescu:
None. I. Resmerita: None. M. Panzaru: None. L. Butnariu: None. E. Gorduza: None. C. Rusu: None. P11.073A Redefining the mutational spectrum and gene-phenotype
correlates in pontocerebellar hypoplasia: results of a
multicentric Italian study A. Micalizzi1, R. Romaniello2, S. Nuovo1,3, F. Arrigoni4,
M. Ginevrino1,5, A. Casella1, T. Mazza6, G. Zanni7, E. Bertini7,
R. Borgatti2, E. Valente1,5 M. Rossi: None. G. Lesca: None. N. Chatron: None. A. Labalme: None. D. Sanlaville: None. A. Fassier: None. F. Escande: None. S. Manouvrier: None. L. Faivre: None. P. Edery: None. D. Geneviève: None. 1Neurogenetics Unit, IRCCS Santa Lucia Foundation, Rome,
Italy, 2Neuropsychiatry and Neurorehabilitation Unit,
Scientific Institute IRCCS Eugenio Medea, Bosisio Parini,
Italy, 3Department of Medicine and Surgery, University of
Salerno, Salerno, Italy, 4Neuroimaging Unit, Scientific Institute
IRCCS Eugenio Medea, Bosisio Parini, Italy, 5Department of
Molecular Medicine, University of Pavia, Pavia, Italy,
6Laboratory of Bioinformatics, IRCCS Casa Sollievo della
Sofferenza, San Giovanni Rotondo, Italy, 7Laboratory of
Molecular Medicine, Unit of Neuromuscular and
Neurodegenerative Disorders, Department of Neurosciences,
IRCCS Bambino Gesù Children’s Hospital, Rome, Italy Autosomal dominant recurrent parotitis: an under-
reported entity M. G. Serey-Gaut1, C. Cabrol1, J. Vallat2, L. Tatu3, B. Loeys4,
L. Van Maldergem1 M. G. Serey-Gaut1, C. Cabrol1, J. Vallat2, L. Tatu3, B. Loeys4,
L. Van Maldergem1 1Centre de Génétique Humaine, CHU Besançon, Université de
Franche-Comté, Besançon, France, 2Service de neurologie,
Centre de Référence Neuropathies Périphérique Rares, CHU
Limoges, Limoges, France, 3Service de Neurologie, CHU
Besançon, Université de Franche-Comté, Besançon, France,
4Centrum Medische Genetica, University of Antwerp, Antwerp,
Belgium Familial salivary glands inflammation is a rare condition of
unknown aetiology, affecting mostly parotid glands. Only a
single British pedigree is referenced in OMIM. By evalu-
ating a 34 y-old French female patient suffering chronic
recurrent parotitis since the age of 6 years, we uncovered a
similar involvement in her father and her son, making
autosomal dominant (AD) inheritance likely. In addition the
patient had a progressive distal four limbs muscle wasting,
paresis and camptodactyly corresponding on nerve biopsy
to focal alterations of the myelin sheet suggestive of a
demyelinating
peripheral
neuropathy,
contrasting
with
apparently normal NCV. We are unable to conclude if the
motor impairment is coincidental or belonging to the clin-
ical spectrum of AD chronic parotitis since her affected
relatives had no motor impairment. Another family eval-
uated in Belgium was also suggestive of AD inheritance. A We report the clinical and molecular characterization of a
previously undescribed syndrome observed in a family
(mother and son) and an unrelated child. Patient 1, aged 3.5 years, presented with mandibular
hypoplasia, knee flexion deformity, hyperlordosis, and
prenatal-onset short stature (-2,5SD). Psychomotor devel-
opment was normal as were cardiac and renal ultrasounds
scans, and chromosomal microarray. His mother (patient 2)
showed a similar phenotype: mandibular hypoplasia, knee
instability requiring surgery, hyperlordosis, short stature
treated by growth hormone (adult height: 160 cm), recurrent
otitis during childhood, and myopia. Patient 3 was an
unrelated child presenting with Pierre-Robin sequence and
patellar
agenesis
leading
to
knee
flexion
deformity. Psychomotor development and cardiac/renal ultrasounds 375 Abstracts from the 51st European Society of Human Genetics Conference: Posters scans were normal. Radiological features included a striking
mandibular obtuse angle and narrow iliac wings. hypoplasia and rib defect 1/21. Occasional findings include:
congenital heart defect found in 7/21 cases, spasmophylia 2/
21 and cleft lip/palate in 1/21 cases. Genetic tests have been
normal. The evolution in time has been constant. P11.072D
Poland Sequence - long time follow-up of 21 cases Results: The responsible genetic defect was identified in
44 probands (71%). Interestingly, the commonest causative
gene was CASK (40%), which harbored both SNVs and
CNVs and was mutated in females and males, with striking
genotype-phenotype
correlates. The
European
founder 376 J. del Picchia mutation TSEN54 p.A53T and pathogenic variants in
EXOSC3 only accounted for 18% and 8% cases, respec-
tively. Single patients with peculiar phenotypes were
mutated in RARS2, VLDLR and TOE1. We confirmed some
previously reported
associations, e.g. retinopathy and
microcephaly with CASK, lower motor-neuron signs with
EXOSC3 and cerebellar cysts with TSEN54. However, we
could not replicate other gene-phenotype correlates: for
instance, we failed to observe univocal correlations of
TSEN54 p.A53T with hyperkinetic movement disorders,
nor with the typical “dragonfly appearance” of cerebellar
hemispheres. Copy Number Variants (CNVs) has been poorly investi-
gated so far, making it difficult to counsel families as regard
this genetic test. mutation TSEN54 p.A53T and pathogenic variants in
EXOSC3 only accounted for 18% and 8% cases, respec-
tively. Single patients with peculiar phenotypes were
mutated in RARS2, VLDLR and TOE1. We confirmed some
previously reported
associations, e.g. retinopathy and
microcephaly with CASK, lower motor-neuron signs with
EXOSC3 and cerebellar cysts with TSEN54. However, we
could not replicate other gene-phenotype correlates: for
instance, we failed to observe univocal correlations of
TSEN54 p.A53T with hyperkinetic movement disorders,
nor with the typical “dragonfly appearance” of cerebellar
hemispheres. Materials and methods: CNVs were assessed either by
CGH- or SNP-array in a cohort of 111 probands with
various PFMs. Detailed neuroimaging assessment led to
classify the patients in the following groups: Dandy-Walker
malformation (DMW, n = 10), isolated vermis hypoplasia
(IVH, n = 12), whole cerebellar hypoplasia (WCH, n = 12),
cerebellar dysplasia (CD, n = 18), ponto-cerebellar hypo-
plasia (PCH, n = 18), non-progressive cerebellar atrophy
(NPCA, n = 13) and other PMFs (n = 28). A logistic
regression model was used to assess the proportion of
CNVs in PFM classes. Conclusions: CASK represents the major gene causative
of PCH in Italy. Phenotypic variability of PCH subtypes is
wider than previously thought, with significant clinical and
neuroimaging overlap among distinct conditions. Results: Pathogenic heterozygous CNVs (all deletions)
were identified in 9 (8%) probands, including 5 (50%)
DWM, 1 (8%) IVH, 1 (8%) WCH and 2 (11%) CD. No
CNVs were detected in the PCH and NPCA groups. Assessment of genome-wide burden of rare genic CNVs in
posterior fossa malformations Conclusions: Screening is being extended to a larger
cohort of PFMs. CNVs analysis is advised in patients with
DWM, and in other PFMs when associated to a syndromic
phenotype. S. Nuovo1,2, C. Cesario3, L. Bernardini3, R. Battini4,5, Funding: ERC Starting Grant 260888; Ricerca Finaliz-
zata NET-2013-02356160. F. Arrigoni6, R. Romaniello7, G. Zanni8, E. Del Giudice9,
9 10
7
8
1 11 F. Arrigoni6, R. Romaniello7, G. Zanni8, E. Del Giudice9,
G. Vitiello9,10, R. Borgatti7, E. Bertini8, E. Valente1,11 G. Vitiello9,10, R. Borgatti7, E. Bertini8, E. Valente1,11 S. Nuovo: None. C. Cesario: None. L. Bernardini:
None. R. Battini: None. F. Arrigoni: None. R. Roma-
niello: None. G. Zanni: None. E. Del Giudice: None. G. Vitiello: None. R. Borgatti: None. E. Bertini: None. E. Valente: None. 1Neurogenetics Unit, IRCCS Santa Lucia Foundation, Rome,
Italy, 2Department of Medicine and Surgery, University of
Salerno, Salerno, Italy, 3Cytogenetics Unit, Casa Sollievo della
Sofferenza Hospital, San Giovanni Rotondo FG, Italy,
4Department of Developmental Neuroscience, IRCCS Stella
Maris Foundation, Pisa, Italy, 5Department of Clinical and
Experimental Medicine, University of Pisa, Pisa, Italy,
6Neuroimaging Laboratory, Scientific Institute, IRCCS
Eugenio Medea, Bosisio Parini, Lecco, Italy, 7Neuropsychiatry
and Neurorehabilitation Unit, Scientific Institute IRCCS
Eugenio Medea, Bosisio Parini, Lecco, Italy, 8Department of
Neurosciences, Unit of Neuromuscular and Neurodegenerative
Disorders, Bambino Gesù Children's Hospital, IRCCS, Rome,
Italy, 9Department of Translational Medicine-Section of
Pediatrics, Federico II University, Naples, Italy, 10CEINGE-
Advanced Biotechnologies, Naples, Italy, 11Department of
Molecular Medicine, University of Pavia, Pavia, Italy 1Neurogenetics Unit, IRCCS Santa Lucia Foundation, Rome,
Italy, 2Department of Medicine and Surgery, University of
Salerno, Salerno, Italy, 3Cytogenetics Unit, Casa Sollievo della
Sofferenza Hospital, San Giovanni Rotondo FG, Italy,
4Department of Developmental Neuroscience, IRCCS Stella
Maris Foundation, Pisa, Italy, 5Department of Clinical and
Experimental Medicine, University of Pisa, Pisa, Italy,
6Neuroimaging Laboratory, Scientific Institute, IRCCS
Eugenio Medea, Bosisio Parini, Lecco, Italy, 7Neuropsychiatry
and Neurorehabilitation Unit, Scientific Institute IRCCS
Eugenio Medea, Bosisio Parini, Lecco, Italy, 8Department of
Neurosciences, Unit of Neuromuscular and Neurodegenerative
Disorders, Bambino Gesù Children's Hospital, IRCCS, Rome,
Italy, 9Department of Translational Medicine-Section of
Pediatrics, Federico II University, Naples, Italy, 10CEINGE-
Advanced Biotechnologies, Naples, Italy, 11Department of
Molecular Medicine, University of Pavia, Pavia, Italy P11.072D
Poland Sequence - long time follow-up of 21 cases CNVs
were significantly commoner in DWM than in non-DWM
(log-odds = 3.078, p < 0.001), while their frequency was
not significantly enriched in the other PFM categories. The
identified genomic deletions were in most cases associated
to a syndromic phenotype. Deletion of 3q24 region was
confirmed as a DWM-associated CNV. Funding: ERC Starting Grant 260888; Ricerca Finaliz-
zata NET-2013-02356160. A. Micalizzi: None. R. Romaniello: None. S. Nuovo:
None. F. Arrigoni: None. M. Ginevrino: None. A. Casella: None. T. Mazza: None. G. Zanni: None. E. Bertini: None. R. Borgatti: None. E. Valente: None. Co-occurrence of two recurrent copy number alterations in
a child with complex phenotype Á. Till1,2, K. Hadzsiev1,2, A. Szabó1,2, Z. Bánfai1,2, B. Melegh1,2,
J. Berente Bene1,2, M. Czakó1,2 1University of Pécs, Medical School, Pécs, Hungary,
2Szentagothai Research Center, Pécs, Hungary 1University of Pécs, Medical School, Pécs, Hungary, Introduction: two copy number variations observed on the
same chromosome in a patient is a very rare condition. In
our patient beside of the 17p11.2 microduplication an
additional deletion of 17q12 region has been detected. Introduction:
Posterior
fossa
malformations
(PFMs)
include a wide spectrum of congenital abnormalities, het-
erogeneous with respect to neuroimaging findings, genetic
cause and recurrence risk. The contribution of genomic Methods: routine cytogenetic investigation was indicated
in a 8 months old baby because of neonatal hypotonia,
developmental delay, renal cysts, hearing loss and minor Abstracts from the 51st European Society of Human Genetics Conference: Posters 377 anomalies which resulted in normal karyotype. Because of
the complex clinical symptoms array CGH analysis using
Agilent Sureprint 8x60K oligo-array (ISCA v.2) was
performed. anomalies which resulted in normal karyotype. Because of
the complex clinical symptoms array CGH analysis using
Agilent Sureprint 8x60K oligo-array (ISCA v.2) was
performed. médicale, Centre Hospitalier Universitaire, Clermont Ferrand,
France, 12Service de Génétique - Hôpital Bretonneau, Tours,
France, 13Service de génétique médicale, Centre Hospitalier
Universitaire de Bordeaux-GH Pellegrin, Bordeaux, France,
14Unité de génétique chromosomique ou cytogénétique, Centre
Hospitalier Universitaire, Nantes, France, 15Service de
génétique médicale, Nantes, France, 16Service de Génétique
Médicale, Centre Hospitalier Bretagne Atlantique, Vannes,
France, 17Centre de Génétique et Centre de référence
Anomalies du Développement et Syndromes Malformatifs,
CHU de Dijon, Dijon, France, 18UMR-Inserm 1231 GAD
team, Génétique des Anomalies du développement, Université
de Bourgogne Franche-Comté, Dijon, France, 19Fédération
Hospitalo-Universitaire Médecine Translationnelle et
Anomalies du Développement (FHU TRANSLAD), Centre
Hospitalier Universitaire de Dijon et Université de Bourgogne-
Franche Comté, Dijon, France Results: array CGH detected a 3,573 Mb recurrent
microduplication of 17p11.2 and additionally a 1,638 Mb
deletion affecting 17q12. The unexpected results provided
explanation of the complex clinical features allowing for a
more detailed genotype-phenotype analysis. Our patient has
been diagnosed as having Potocki-Lupski syndrome and
Renal cysts and diabetes syndrome at the same time. Conclusions: the high-resolution array CGH analysis in
patients with a complex phenotype is of great importance
and it is recommended to carry-out this test first. The
application
of
array
CGH
in
patients
with
unusual
compound phenotype may enable more accurate estimates
of the incidence of similar cases and can lead to the
exploration of development of multiple copy number
alterations in the same patient. Primrose syndrome is characterized by variable intellectual
deficiency, behavior disorders and facial dysmorphism with
macrocephaly. The phenotype is progressive with distal
muscle wasting, contractures, hearing loss and ectopic cal-
cifications of the ears and brain. In 2014, ZBTB20 variants
were identified as responsible of Primrose syndrome. Indeed, ZBTB20 plays an important role in cognition,
memory, and learning processes, and has a transcription
repressive effect on numerous genes. A more severe, pro-
gressive phenotype was found in a small number of patients
with single nucleotide variants (SNVs) than in those with
large deletions. Here, we report on the clinical and mole-
cular results of 14 patients, seven carrying ZBTB20 SNV,
and seven carrying 3q13.31 deletions, recruited through the
French AnDDI-Rares network. We compared their pheno-
types and reviewed data in the literature, in order to
establish more powerful phenotype-genotype correlations
between the two groups of patients. All patients presented
mild-to-severe ID and/or a psychomotor delay. Facial fea-
tures were similar with a prominent forehead, down-slanting
palpebral fissures, ptosis and large ears but macrocephaly
was more frequent in patient with SNVs (p = 0.026). Hearing loss (p = 0.004), pinna calcification and pro-
gressive muscular wasting and contractures were observed
only in patients with SNVs. Corpus callosum dysgenesis
(p = 0.003), diabetes and hypothyroidism were more fre-
quent in this group. However, the median age was 9.7 years
in patients with deletions compared with 17.5 years in those
with SNVs. Longer follow-up will be necessary to deter-
mine whether the phenotype of patients with deletions is
also progressive, and to adapt information given to families. A Juven: None S Nambot: None A Piton: None P Á. Till: None. K. Hadzsiev: None. A. Szabó: None. Z. Bánfai: None. B. Melegh: None. J. Berente Bene: None. M. Czakó: None. P11.077A
PTK7-a candidate gene for a novel human malformation
phenotype P11.078B
Pura syndrome: an emerging neurodevelopmental disorder P11.078B
Pura syndrome: an emerging neurodevelopmental disorder S. Bigoni1, G. Garani2, G. Santen3, M. Della Monica4,
C. Graziano5, C. Ruivenkamp6, E. Ballardini2, R. Guerrini7,
P. Magini8, E. Procopio9, E. Parrini10, A. Suppiej11,
D. Colavito12, V. Maritan13, M. Hoffer6, D. Ognibene14,
A. Ferlini14 N. Meier1,2,3, E. Bruder4,3, O. Lapaire5,3, S. Tercanli6,3,
I. Filges1,2,3 N. Meier1,2,3, E. Bruder4,3, O. Lapaire5,3, S. Tercanli6,3,
I. Filges1,2,3 C. Graziano5, C. Ruivenkamp6, E. Ballardini2, R. Guerrini7, P. Magini8, E. Procopio9, E. Parrini10, A. Suppiej11, D. Colavito12, V. Maritan13, M. Hoffer6, D. Ognibene14,
A. Ferlini14 1Medical Genetics, Institute of Medical Genetics and
Pathology, University Hospital Basel, Basel, Switzerland,
2Department of Clinical Research, University Hospital Basel,
Basel, Switzerland, 3University of Basel, Basel, Switzerland,
4Pathology, Institute of Medical Genetics and Pathology,
University Hospital Basel, Basel, Switzerland, 5Department of
Obstetrics and Gynecology, Basel University Hospital, Basel,
Switzerland, 6Centre for Prenatal Ultrasound, Basel, Basel,
Switzerland 1UOL of Medical Genetics, University-Hospital S.Anna,
Ferrara, ferrara, Italy, 2NICU, University-Hospital S.Anna,
Ferrara, ferrara, Italy, 3LDGA,, leiden, Netherlands, 4Medical
Genetics SOD, Meyer University Chldren Hospital, florence,
Italy, 5Medical Genetics Unit, Sant'Orsola Malpighi Polyclinic,
Bologna, Italy, 6LDGA, Leiden, Netherlands, 7Pediatric
Neurology Unit and Laboratories, Meyer Children University
Hospital, florence, Italy, 8Medical Genetic Unit, S.Orsola-
Malpighi Polyclinic, Bologna, Italy, 9Unit of Metabolic and
Muscular Disease, Meyer Children University Hospital,
Florence, Italy, 10Pediatric Neurology Unit and Laboratories,
Meyer Children University Hospital, Florence, Italy, 11Child
Neurology Unit, Paediatric University Hospital, Padua, Italy,
12Research & Innovation SRL Genetic Laboratory, Padua,
Italy, 13Regional Centre for Low Vision, Children Pediatric
University Hospital, Padua, Italy, 14Section of Microbiology
and Medical Genetics, Ferrara University, ferrara, Italy Introduction: Prenatal ultrasound identifies an increasing
number of fetal malformations. We report on a fetus with
brain malformations, Müllerian duct aplasia, bile duct
atresia, unilateral anophthalmia and bilateral cleft palate. Our aim was to identify the genetic cause for this novel
multiple congenital anomaly (MCA) phenotype. Methods: We perform trio whole exome sequencing
(WES) and prioritize variants according to their frequency,
disruptive potential and their presence in genes involved in
early developmental processes. We correlate the malforma-
tion pattern, confirmed by autopsy, to the candidate
genotypes. The additional comparison of human and animal
morphology validates potential candidate genes. Using RT-
PCR and qPCR we study expression on fetal FFPE-RNA. PURA Syndrome is a neurodevelopmental disorder char-
acterized by moderate/severe cognitive impairment, delayed
or absent speech and difficulties in acquiring independent
ambulation. P11.076D Fédération Hospitalo-Universitaire Médecine Transla-
tionnelle et Anomalies du Développement (FHU TRANS-
LAD),
Centre
Hospitalier
Universitaire
de
Dijon
et
Université
de
Bourgogne-Franche
Comté,
Dijon,,
4. UMR-Inserm 1231 GAD team, Génétique des Anomalies
du développement, Université de Bourgogne Franche-
Comté, F-21000 Dijon, France.. We are further investigating the specific mutations in a cell
culture expression model. Results: We identified candidate missense mutations in
the PTK7 gene which encodes a membrane receptor
tyrosine kinase acting in the canonical and non-canonical
Wnt-signaling as well as the planar cell polarity pathways. So far, the gene is known to play a role in Müllerian duct
and neural tube development. Knockout mice embryos
present with a similar severe malformation pattern. qPCR of
liver
tissue
of
the
fetus
confirmed
a
decrease
of
PTK7 mRNA. Conclusions: PTK7 is a candidate gene for a novel
human MCA syndrome. WES can be used in individual
families with undiagnosed lethal MCA syndromes to
discover novel disease genes, provided that the fetal
phenotype can be correlated to a particular developmental
pathway in embryogenesis. Proof of pathogenicity of the
mutations in such ultrarare disorders can be challenging. N. Meier: None. E. Bruder: None. O. Lapaire: None. S. Tercanli: None. I. Filges: None. P11.076D Primrose syndrome: a phenotypic comparison of patients
with a ZBTB20 single-nucleotide variant versus a 3q13.31
microdeletion including ZBTB20 Primrose syndrome: a phenotypic comparison of patients
with a ZBTB20 single-nucleotide variant versus a 3q13.31
microdeletion including ZBTB20 A. Juven1, S. Nambot1, A. Piton2, P. Callier3, C. Philippe4,
N. Jean-Marçais1, A. Munnich5, M. Rio6, L. Colleaux7,
S. Rondeau6, J. Steffann7, T. Attie-Bitach5, S. Thomas5, S. El
Chehadeh8, C. Vincent Delorme9, S. Bouquillon9,
C. Francannet10, F. Laffargue10, L. Gouas11, S. Blesson12,
D. Lacombe13, M. Vincent14, B. Isidor15, H. Journel16,
C. Thauvin17,18, L. Faivre1,19,18 1Centre de génétique du C.H.U. de Dijon, Dijon, France,
2Unité de Biologie et de Génétique Moléculaire, Centre
Hospitalier Universitaire de Strasbourg, Strasbourg, France,
3Laboratoire de Génétique Moléculaire, C.H.U de Dijon,
Dijon, France, 4Laboratoire de Génétique Moléculaire, UF
Innovation en diagnostic génomique des maladies rares,
Plateau Technique de Biologie, Centre Hospitalier
Universitaire de Dijon, France., Dijon, France, 5Institut
Imagine, Paris, France, 6Département de Génétique, Hôpital
Necker-Enfants Malades,, Paris, France, 7Département de
Génétique, Hôpital Necker-Enfants Malades, Paris, France,
8Service de Génétique Médicale, Centre Hospitalier
Universitaire de Strasbourg, Strasbourg, France, 9Service de
Génétique Médicale, Hôpital Jeanne de Flandre, Centre
Hospitalier Régional Universitaire, Lille, France, 10Service de
Génétique Médicale, Centre Hospitalier Universitaire Estaing,
Clermont Ferrand, France, 11Service de cytogénétique A. Juven: None. S. Nambot: None. A. Piton: None. P. Callier: None. C. Philippe: None. N. Jean-Marçais:
None. A. Munnich: None. M. Rio: A. Employment (full or
part-time); Modest; 5. Département de Génétique, Hôpital 378 J. del Picchia We are further investigating the specific mutations in a cell
culture expression model. We are further investigating the specific mutations in a cell
culture expression model. We are further investigating the specific mutations in a cell
culture expression model. Necker-Enfants Malades, Paris, France, Institut Imagine
Paris. L. Colleaux: A. Employment (full or part-time);
Modest;
Département
de
Génétique,
Hôpital
Necker-
Enfants Malades, Paris, France, Inserm U781, Hôpital
Necker-Enfants Malades, Paris, France. S. Rondeau: None. J. Steffann: None. T. Attie-Bitach: None. S. Thomas:
None. S. El Chehadeh: None. C. Vincent Delorme: None. S. Bouquillon: None. C. Francannet: None. F. Laffar-
gue: None. L. Gouas: None. S. Blesson: None. D. Lacombe: None. M. Vincent: None. B. Isidor: None. H. Journel:
None. C. Thauvin:
None. L. Faivre:
A. Employment
(full
or
part-time);
Modest;
Centre
de
génétique,
Centre
hospitalier
universitaire
de
Dijon,
3. P11.077A
PTK7-a candidate gene for a novel human malformation
phenotype At birth, newborns present central hypotonia,
hypothermia,
lethargy,
swelling-feeding
difficulties, Abstracts from the 51st European Society of Human Genetics Conference: Posters 379 frequent hiccups and respiratory abnormalities that may
need intensive care for life-threatening risk. In the 90% of
the patients a heterozygous mutation in PURA gene is
present, revealed mostly through WES analysis; in the
remaining 10% a deletion of 5p31.3 encompassing the
PURA gene is detected using Array-CGH. Until last year,
71 patients had been described, but the number is rapidly
growing. We report 4 Italian patients (age range 6 month -
11 years). At birth, all of them exhibited axial hypotonia,
swallowing difficulties and hypoventilation. Two had neo-
natal respiratory failure requiring intensive care and recur-
rent
hiccups. Recurrent
dysmorphic
features
were
anteverted nostrils, thin and sparse eyebrows. Neurodeve-
lopment was delayed in all patients, with absent speech and
limited walking autonomy in older patients. De novo het-
erozygous mutations in PURA gene were found in 2/4
patients by WES, using an NGS panel in 1 patient and a de
novo 2,6 Mb deletion in 5q31.2q31.3 was detected using
array-CGH in the remaining patient. PURA Syndrome, is
now a frequently recognized neurodevelopmental disorder
which should be considered in the differential diagnosis of
syndromes manifesting with the neonatal hypotonia and
early respiratory abnormalities. on PSIQUIQ library, using molecular interaction databases
(Biogrid, IntAct, MINT, Reactome, UniProt, etc.) regarding
to previous 13 genes. Results: We detected pathogenic/probably pathogenic
variants (P/PP) in the 35%(23/66) of the patients, in the
41% (27/66) were detected one or several variant of
unknown significance (VUS), and in the 24% (16/66) no
variants of interest (VI) were identified. 88 VI (P/PP or
VUS) were detected in 26 different genes, PTPN11 was the
most mutated gene (15% of variants, 12 P/P and 1 VSI)
followed by ANKRD11 (10%, 9 VSI), SRCAP (9%, 8 VSI)
and SOS1 (8%, 3 P/PP and 4 VSI). The 65% of VI (all
classified as VUS) was detected in candidate genes. Conclusions: We show the results of applying an
extended NGS panel in a cohort of 66 patients. According
to the literature, PTPN11 and SOS1 were the most mutated
RASopathies related genes. Remarkably, we detected a high
percentage of VUS in candidate genes that could have
clinical implications. However, further familiar and func-
tional analysis must be done to confirm this. J. López Montiel: None. J. Lezana Rosales: None. C. Torres Fernández: None. P11.077A
PTK7-a candidate gene for a novel human malformation
phenotype S. Franco Freire: None. C. Sánchez Linares: None. C. Benito López: None. J. López
Siles: None. S. Bigoni: None. G. Garani: None. G. Santen: None. M. Della
Monica:
None. C. Graziano:
None. C. Ruivenkamp: None. E. Ballardini: None. R. Guerrini:
None. P. Magini: None. E. Procopio: None. E. Parrini:
None. A. Suppiej: None. D. Colavito: None. V. Maritan:
None. M. Hoffer:
None. D. Ognibene:
None. A. Ferlini: None. The smallest SATB2 exon-deletion detected by arrayCGH.
Further delineation of a new emerging syndrome The smallest SATB2 exon-deletion detected by arrayCGH. Further delineation of a new emerging syndrome E. Lloveras, L. Barranco, A. Canellas, M. Costa, B. Mendez,
N. Palau, M. Piqué, D. Yeste, S. Martin, C. Pérez R. Urreizti, L. Castilla-Vallmanya, H. Franco-Valls, G. Cunill,
D. Grinberg, S. Balcells R. Urreizti, L. Castilla-Vallmanya, H. Franco-Valls, G. Cunill,
D. Grinberg, S. Balcells Department of Genetics, Microbiology and Statistics, Faculty
of Biology, University of Barcelona, IBUB, IRSJD, CIBERER,
Barcelona, Spain Department of Genetics, Microbiology and Statistics, Faculty
of Biology, University of Barcelona, IBUB, IRSJD, CIBERER,
Barcelona, Spain Schaaf-Yang syndrome (SHFYNG) is due to truncating
mutations in MAGEL2, a gene included in the Prader-Willi
region (15q11-q13). This syndrome is characterized mainly
by neurodevelopmental delay, contractures and craniofacial
dysmorphology and around 100 patients have been identi-
fied worldwide so far. MAGEL2 is an essential component
of the retromer, involved in the retrograde transport (back to
the trans-Golgi network). Previous studies have shown that
the depletion of MAGEL2 leads to a change in the sub-
cellular localization of integrin alpha-5 and that the altera-
tion of VPS35, a MAGEL2 partner, leads to alterations in
the APP transport in the endosome. We have analysed
different biomarkers in fibroblasts of 3 SHFYNG patients in
comparison with 6 healthy controls. Intracellular localiza-
tion of Integrin alpha-5 was assessed by immunocy-
tochemistry while the levels of the amyloid-beta (1-40)
were measured by ELISA. Cell viability was measured by
the MTT assay. We observed no difference in the viability
of the fibroblasts of the patients when compared with con-
trols and no alteration in the integrin alpha-5 levels in the
cellular membrane while we notice a slight increase in
integrin alpha-5 co-localization with early endosomes y
p
g
Case Report: Female patient with polymalformative
syndrome, fetal hypokinesia, arthrogryposis, small feet,
hypotonia,
and
psychomotor
retardation. At[AZC1]
22 months of age, she was not able to keep standing
without support, had not speech development, clamp nor
steps. She developed trunk obesity, not associated with
increased intake. She did not show hyperphagia and
presented feeding problems. No alterations in brain, spinal,
abdominal, renal or cardiac MR imaging studies. Normal
array-CGH. Metabolic
study
including
HBA1c,
lipid
metabolism, liver function and cortisol normal. Genetic
study was performed by PCR amplification of exonic and
intronic regions of MAGEL2 gen, and analyzed by Sanger
sequencing method. An heterozygous variant was detected,
c.1850G>A (p.Thr617Ter; NM_01966) and it was de novo
since
parents
were
not
carriers. Reported
cases
of
SCHYNGS inherited usually a paternal mutated allele. Identification of new biomarkers for MAGEL2 truncating
mutations in Schaaf-Yang syndrome Identification of new biomarkers for MAGEL2 truncating
mutations in Schaaf-Yang syndrome Introduction: Shaaf-Yang syndrome (SHFYNGS; #MIM
615547) is an autosomal dominant multisystem disorder
characterized by delayed psychomotor development, intel-
lectual disability, hypotonia, and behavioral abnormalities. Additional features include contractures, feeding difficul-
ties, and variable dysmorphic facial features. MAGEL2 is
located in the Prader-Willi critical region 15q11-13, and
have been reported to cause SHFYNGS. Individuals are
affected only if the mutation occurs on the paternal allele,
since MAGEL2 is a maternally imprinted gene. Departament de Genètica. SYNLAB International Group,
Esplugues de Llobregat, Spain Analyzing 66 cases of RASopathies with an extended NGS
panel J. López Montiel1, J. Lezana Rosales1, C. Torres Fernández1,
S. Franco Freire2, C. Sánchez Linares1, C. Benito López2,
J. López Siles1 Introduction: Few cases of recurrent 2q33.1 deletion with
size variability have been reported in the literature. Hap-
loinsufficiency of one gene, SATB2, within the deleted
region has been suggested to be responsible for most of the
features of the affected patients and a new syndrome named
SATB2-associated syndrome (SAS) has been proposed. Herein, we present a case with the smallest SABT2 deletion
detected by arrayCGH, to our knowledge. 1M.G.C. Genetaq, Málaga, Spain, 2UGC Laboratorio H.R.U. Málaga, Málaga, Spain 1M.G.C. Genetaq, Málaga, Spain, 2UGC Laboratorio H.R.U. Málaga, Málaga, Spain Introduction: RASopathies are a group of genetic diseases
caused by mutations in genes encoding proteins in the Ras/
MAPK pathway. RASopathies include several syndromes:
neurofibromatosis-1, Noonan/LEOPARD, Costello, Legius
and cardio-facio-cutaneous syndromes, etc. The phenotypic
spectrum is wide and overlapping. Patient and Results: The present case is a boy evaluated
in the Pediatric Neurology consultation at the age of 3 years
with
developmental
growth
retardation. Conventional
chromosome analysis of the patient revealed a normal
karyotype. X-fragile was normal and an array CGH analysis
was performed. The CytoSure™Constitutional v3 4x180k
(Oxford Gene Technology, UK) was used, scanned using
the Agilent Microarray Scanner according to the manufac-
turer’s protocol. High-resolution microarray analysis of the
patient detected a 5,24 Kb deletion in the region 2q33.1, arr Material and Methods: We present a cohort of 66
patients from different populations with suspicion of
RASopathies. We used an extended NGS panel (TruSight-
One, Illumina) including 13 RASopathies associated genes. Other 32 candidate genes were selected by R custom script J. del Picchia 380 [GRCh37]2q33.1(200212030_200217267x1)
including
exon 8 of SATB2 gene. The main features of the patient
were concordant with those related to SAS: intellectual
deficit, autistic traits, hypotonia, elongated face, atypical
teeth, micrognathia, local hypertonia, atypical thumbs and
nails, sparse hair. (marked with EEA1). We have observed a significant
decrease of the AB1-40 in the patient’s fibroblasts when
compared with controls. In conclusion, we have identified a
promising biomarker (AB1-40) that could help to better
understand the pathophysiology of the SHFYNG syndrome
and to monitor the effect of therapeutic drugs. Grants:
Spanish
Ministerio
de
Economía
y
Competitividad
(SAF2014-56562-R; FECYT, crowdfunding PRECIPITA). G. Pi1, A. Caro2, A. Zúñiga2, F. Martínez2, J. Cervera2 1Serv. Pediatría. Hospital de la Ribera., Alzira, Spain, 2Unidad
de Genética. HUP La Fe., Valencia, Spain Departament de Genètica. SYNLAB International Group,
Esplugues de Llobregat, Spain Catalan Government (2014SGR932) Comments: - Intragenic SATB2 deletions are very rare and only four
cases have been described. Review of clinical records
showed similar clinical features among these patients,
including severe developmental delay and tooth abnormal-
ities as the present one. R. Urreizti: None. L. Castilla-Vallmanya: None. H. Franco-Valls: None. G. Cunill: None. D. Grinberg:
None. S. Balcells: None. R. Urreizti: None. L. Castilla-Vallmanya: None. H. Franco-Valls: None. G. Cunill: None. D. Grinberg:
None. S. Balcells: None. - CytoSure™Constitutional v3 array offers an enhanced
exon-level coverage and has allowed the detection of the
smallest SATB2 deletion described until now. P11.082B
CASE REPORT OF SHAAF-YANG SYNDROME WITH
A DE NOVO MUTATION IN MAGEL2 GEN E. Lloveras: None. L. Barranco: None. A. Canellas:
None. M. Costa: None. B. Mendez: None. N. Palau:
None. M. Piqué: None. D. Yeste: None. S. Martin: None. C. Pérez: None. Carmel medical center, Haifa, Israel For the two individuals with variants
outside of the ATPase/helicase domains, SIHIWES was the
second and fifth top syndrome suggested by the software
supporting these variants result in a similar phenotype. G. Larom-Khan: None. A. Peleg: None. L. Sagi-Dain:
None. A. Harari-Shaham: None. Carmel medical center, Haifa, Israel K. Weiss1, T. Paperna1, H. Baris1, S. Whalen2, S. Whalen2,
S. Heidi2, B. Keren2, K. Lachlan3 Introduction: STAR syndrome (Syndactyly, Telecanthus,
Anogenital malformations, Renal malformations) is a very
rare x-linked dominant disorder. Loss-of-function mutations
of the FAM58A gene were previously reported to be
associated with STAR syndrome. Less than 20 patients
have been described thus far, two of them displaying mild
developmental delay. 1Genetics Institute, Rambam Health Care Campus, Haifa,
Israel, 2AP-HP, Département de Génétique, Hôpital de la Pitié
Salpêtrière, Paris, France, 3Wessex Clinical Genetics Service,
Southampton University Hospitals NHS Foundation Trust,
Princess Anne Hospital, Southhampton, United Kingdom Case presentation: The parents were referred to genetic
counseling at 27 weeks of gestation during their first
pregnancy due to a sonographic demonstration of absent
gallbladder. No consanguinity or abnormal medical condi-
tions were reported, and the pregnancy was uneventful. As
part of genetic evaluation, the parents were tested for Cystic
Fibrosis, and fetal Chromosomal Microarray Analysis
(CMA) was recommended. Case presentation: The parents were referred to genetic
counseling at 27 weeks of gestation during their first
pregnancy due to a sonographic demonstration of absent
gallbladder. No consanguinity or abnormal medical condi-
tions were reported, and the pregnancy was uneventful. As
part of genetic evaluation, the parents were tested for Cystic
Fibrosis, and fetal Chromosomal Microarray Analysis
(CMA) was recommended. Introduction: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a
recently described form of syndromic intellectual disability
associated with congenital heart defects, hypogonadism,
macrocephaly and additional features. The condition is
caused by de novo missense variants in CHD4, which
encodes an ATP-dependent chromatin remodeler. Most
reported variants are missense and lie within the ATPase/
helicase domains. A few variants were identified outside of
this hotspot region and therefore their effect is unclear. The
aim of this study was to investigate whether facial recog-
nition software can be utilized for facial gestalt recognition
and aid in variant interpretation in SIHIWES. Results: CMA testing yielded a female karyotype with
Xq28 deletion sized 117kb, which included 3 clinically
significant genes - FAM58A gene (raising a suspicion of
STAR syndrome) and two genes related to recessive
disorders. Parental CMA testing was normal. Magnetic
Resonance Imaging was performed, yielding no abnormal
findings. Following a throughout discussion, the parents
decided to continue the pregnancy. The newborn girl was
delivered at 38 weeks of gestation. She was diagnosed with
unilateral
2-4
toe
syndactyly,
with
no
evidence
of
dysmorphism or renal/anogenital malformations. Carmel medical center, Haifa, Israel At the
age of 2 months the parents report normal congenital and
motor development. Results: CMA testing yielded a female karyotype with
Xq28 deletion sized 117kb, which included 3 clinically
significant genes - FAM58A gene (raising a suspicion of
STAR syndrome) and two genes related to recessive
disorders. Parental CMA testing was normal. Magnetic
Resonance Imaging was performed, yielding no abnormal
findings. Following a throughout discussion, the parents
decided to continue the pregnancy. The newborn girl was
delivered at 38 weeks of gestation. She was diagnosed with
unilateral
2-4
toe
syndactyly,
with
no
evidence
of
dysmorphism or renal/anogenital malformations. At the
age of 2 months the parents report normal congenital and
motor development. Methods:
We used
the
deep
convolutional
neural
network architecture provided by Face2Gene (FDNA Inc,
USA) on 16 photographs of individuals with variants in the
ATPase/helicase domain of CHD4. In addition, a compar-
ison of SHINWES individuals to control groups was
conducted using across-validation approach. Then, we
evaluated the ability of the software to recognize the
SIHIWES gestalt in two individuals with de novo variants
outside of the hotspot region. Methods:
We used
the
deep
convolutional
neural
network architecture provided by Face2Gene (FDNA Inc,
USA) on 16 photographs of individuals with variants in the
ATPase/helicase domain of CHD4. In addition, a compar-
ison of SHINWES individuals to control groups was
conducted using across-validation approach. Then, we
evaluated the ability of the software to recognize the
SIHIWES gestalt in two individuals with de novo variants
outside of the hotspot region. Conclusions: To the best of our knowledge, we describe
the first prenatal diagnosis of STAR syndrome, with mild
abnormal features. This case is an interesting example of a
challenging genetic counseling in the face of very limited
clinical information. Results: The cohort of individuals with SIHIWES and
variants in the ATPase/helicase domain differed signifi-
cantly from a control group of 32 healthy individuals (AUC
0.917, p-value 0.009). For the two individuals with variants
outside of the ATPase/helicase domains, SIHIWES was the
second and fifth top syndrome suggested by the software
supporting these variants result in a similar phenotype. Results: The cohort of individuals with SIHIWES and
variants in the ATPase/helicase domain differed signifi-
cantly from a control group of 32 healthy individuals (AUC
0.917, p-value 0.009). K. Weiss: None. T. Paperna: None. H. Baris: None. S.
Whalen: None. S. Whalen: None. S. Heidi: None. B.
Keren: None. K. Lachlan: None. G. Pi: None. A. Caro: None. A. Zúñiga: None. F. Martínez: None. J. Cervera: None. distal arthrogryposis, newborns with severe undiagnosed
central hypotonia, or children for whom PWS is clinically
suspected. [AZC1] distal arthrogryposis, newborns with severe undiagnosed
central hypotonia, or children for whom PWS is clinically
suspected. [AZC1] distal arthrogryposis, newborns with severe undiagnosed
central hypotonia, or children for whom PWS is clinically
suspected. [AZC1] K. Weiss: None. T. Paperna: None. H. Baris: None. S. Whalen: None. S. Whalen: None. S. Heidi: None. B. Keren: None. K. Lachlan: None. R. Urreizti, L. Castilla-Vallmanya, H. Franco-Valls, G. Cunill,
D. Grinberg, S. Balcells In
the absence of paternal deletion of 15q11-15q13 or maternal
uniparental disomy 15, a search for intragenic mutations on
the paternal allele of MAGEL2 should be proposed for
fetuses with reduced movements, polyhydramnios, and Abstracts from the 51st European Society of Human Genetics Conference: Posters 381 D. Villela, S. Da Costa, A. Vianna-Morgante, A. Krepischi,
C. Rosenberg P11.087C Sweeney-Cox syndrome: report of the third patient affected
by this new delineated syndrome harboring a novel variant
in TWIST1 D. Bertola1,2, C. M. Musso2, K. Rocha2, G. L. Yamamoto1,2,
M. Passos-Bueno2 D. Bertola1,2, C. M. Musso2, K. Rocha2, G. L. Yamamoto1,2,
M. Passos-Bueno2 G. Morin: None. V. Li Thiao Te: None. G. Jedraszak:
None. F. Jobic: None. M. Mathieu-Dramard: None. B. Roussel: None. J. Bordet: None. J. Rochette: None. 1Instituto da Criança - University of São Paulo, São Paulo,
Brazil, 2Instituto de Biociências da Universidade de São
Paulo, São Paulo, Brazil 1Instituto da Criança - University of São Paulo, São Paulo,
Brazil, 2Instituto de Biociências da Universidade de São
Paulo, São Paulo, Brazil P11.085A Roussel4, J. Bordet5, J. Rochette3 1Clinical Genetics, Amiens, France, 2Pediatric Hematology,
Amiens, France, 3Laboratory of Human Genetics, Amiens,
France, 4Laboratory of Hematology, Amiens, France,
5Laboratory of haemostasis, Lyon, France In 1985, Stormorken et al. reported a unique family with a
new phenotype associating thrombocytopathia, thrombo-
cytopenia, muscle fatigue, asplenia, miosis, migraine, dys-
lexia and ichthyosis. In 2000, Mizobuchi et al. found
tubular aggregates on the muscle biopsy in another family
with this phenotype. In 2014, three groups including ours,
found that « Stormorken syndrome » (OMIM 185070) was
related to a specific gain-of-function mutation in STIM1
gene [c.910C>T/p.(Arg304Trp)]. STIM1 encodes a calcium
sensor involved in Store-operated calcium entry. With the
mutation, this system seems permanently activated, even
when intracellular stocks of calcium are full. Independently,
White et al. reported from 2003 a new platelet disease,
characterized in electronic microscopy by the presence of 2
types of platelet organelles (opaque and target organelles). In 2015, they found that “York platelet syndrome” was also
caused
by
mutations
of
STIM1
gene,
including
the
c.910C>T [p.(Arg304Trp)] (Markello et al., 2015). The
patients shared several symptoms with patients affected by
Stormorken syndrome: thrombocytopathia, thrombocyto-
penia, muscle weakness, rimmed vacuoles on muscle
biopsy, and sometimes miosis or splenic hypoplasia. As the
2 conditions were secondary to the same mutation of STIM1
gene, and patients had a rather similar phenotype, we
assumed that they constituted one unique disease, only
investigated by different approaches. In this work, we
showed for the first time that ultrastructural platelet
anomalies described in York platelet syndrome, were also
found in platelets of patients with Stormorken syndrome. This result suggests that York platelet syndrome and Stor-
morken syndrome are one and the same disease. D. Villela: None. S. Da Costa: None. A. Vianna-
Morgante:
None. A. Krepischi:
None. C. Rosenberg: None. University of São Paulo, São Paulo, Brazil P11.085A Conclusions: Facial gestalt recognition is sometimes
used by clinicians in the interpretation of novel variants in
disease causing genes. Here we show this can also be
applied by facial recognition software. Conclusions: Facial gestalt recognition is sometimes
used by clinicians in the interpretation of novel variants in
disease causing genes. Here we show this can also be
applied by facial recognition software. Stormorken syndrome and York platelet syndrome share
the same mutation of STIM1 and the same ultrastructural
platelet anomalies 382 J. del Picchia G. Morin1, V. Li Thiao Te2, G. Jedraszak3, F. Jobic1,
M. Mathieu-Dramard1, B. Roussel4, J. Bordet5, J. Rochette3
1Clinical Genetics, Amiens, France, 2Pediatric Hematology,
Amiens, France, 3Laboratory of Human Genetics, Amiens,
France, 4Laboratory of Hematology, Amiens, France,
5Laboratory of haemostasis, Lyon, France Mosaicism is defined as the presence of two or more
genetically distinct cell lines. With the introduction of
increasingly sensitive technologies for DNA mutation
detection such as microarrays and next‐generation sequen-
cing, the importance of mosaicism for human diseases is
now being more fully appreciated. However, extracting
information on mosaicism from targeted sequencing, espe-
cially exome data, can be challenging because read cover-
age varies between regions and the target is limited to ~ 3%
of the genome. In the present study, we aimed to evaluate
the efficiency of a new targeted approach to detect mosai-
cism involving structural/numerical chromosomal altera-
tions of DNA samples previously characterized by SNP-
array. We used a focused exome panel supplemented by
backbone and SNPs probes that express a larger repre-
sentation of the human genome. A total of 9 DNA muta-
tions ranging from 400 Kb to 35 Mb and with different level
of mosaicism (10-50%) were used as positive control for
targeted sequencing analysis. Our approach correctly iden-
tified 7 alterations (7/9), pointing to a sensitivity of 81% of
our test. The two cases that were not detected are below
30% of mosaicism. Although in both cases it was possible
to observe a greater dispersion of probes that deviated from
the 50% heterozygosis line, the algorithms did not correctly
integrate the information of copy number and B allele fre-
quency to make a call. Our results point out that it is pos-
sible to improve the methodology for the identification of
structural mosaicism from sequencing data based on the
analysis of allelic frequency. G. Morin1, V. Li Thiao Te2, G. Jedraszak3, F. Jobic1,
M. Mathieu-Dramard1, B. P11.086B Detection of mosaicism involving structural and numerical
chromosome aberrations from targeted sequencing data Introduction: Sweeney-Cox syndrome (SCoS) is a new
syndrome reported in two individuals presenting hetero-
zygous missense variants in residue 117 in TWSIT1,
showing a dominant-negative effect. Previously, hetero-
zygous mutations in this gene, leading to happloinsuffi-
ciency, were known to cause Saethre-Chotzen syndrome
(SCS). In
SCoS,
the
craniofacial
involvement
is University of São Paulo, São Paulo, Brazil Abstracts from the 51st European Society of Human Genetics Conference: Posters 383 fundamentally different from the one seen in SCS and
premature closure of cranial sutures has not been a
hallmark. fundamentally different from the one seen in SCS and
premature closure of cranial sutures has not been a
hallmark. cause aberrant processing of the transcript with complete
loss of the RBM10 mRNA. Conclusions: To our knowledge, this is the second
individual with TARP syndrome who survived at the first
decade of life, allowing a first depiction of the natural
history of this disorder. These data indicate that an adequate
intensive neonatal care can overcome the supposed lethality
of TARP. This should to be taken into consideration in
counselling. Casuistic and Methods: the proband is a 4 year-old male
patient showing typical facial features of SCoS and coronal
craniosynostosis. A targeted gene panel for craniosynostosis
was performed in the proband, followed by Sanger
sequencing in the proband and his parents. Results:
a
novel,
de
novo,
missense
variant
(p. Asp141Glu) in TWIST1 was found in the proband. Results:
a
novel,
de
novo,
missense
variant
(p. Asp141Glu) in TWIST1 was found in the proband. Funding information: Fondazione Bambino Gesù (Vite
Coraggiose), Italian Ministry of Health (RC 2017). Conclusions: This is the third patient reported with
SCoS. Although the facial features are typical for this new
recognized syndrome, the patient presented with coronal
craniosynostosis, similar to what is seen in SCS, suggesting
that this specific variant could cause a phenotype compris-
ing clinical characteristics of both SCoS and SCS. The
variant found in TWIST1 changes the aminoacid aspartate
for a glutamin in position 141. Different variants in the
same residue have been reported associated with SCS. It is
possible to speculate that a substitution for a more similar
aminoacid, as it is the case here, could lead to a dominant-
negative effect, similar to what was proposed for the
variants in residue 117. Further functional studies are
required to prove this preliminary impression. P11.086B FAPESP
2015/21783-9; 13/08028-1; CNPq 304130/2016-8 M. Niceta: None. S. Barresi: None. F. Pantaleoni:
None. M.L. Dentici: None. R. Capolino: None. S. Pizzi:
None. A. Ciolfi: None. B. Dallapiccola: None. M. Tartaglia: None. M.C. Digilio: None. Whole Exome Sequencing reveals novel biallelic UBE3B
mutations in two unrelated patients with undiagnosed
condition Whole Exome Sequencing reveals novel biallelic UBE3B
mutations in two unrelated patients with undiagnosed
condition A. Pagliazzi1, A. Provenzano1, F. Peluso1, S. Bargiacchi2,
M. Della Monica2, G. Carignani1, G. Forzano1, S. Ciabattoni3,
E. Agostini4, P. Fiorini4, A. La Barbera1, S. Guarducci2,
M. Pantaleo2, B. Lucherini2, S. Giglio5 D. Bertola: None. C.M. Musso: None. K. Rocha: None. G.L. Yamamoto: None. M. Passos-Bueno: None. 1Medical Genetics Unit, University of Florence, Florence,
Italy, 2Medical Genetics Unit, Meyer Children's University
Hospital, Florence, Italy, 3Medical Genetics Unit, Santa Maria
Nuova Hospital, Florence, Italy, 4Neonatal Intensive Care
Unit, Meyer Children's University Hospital, Florence, Italy,
5Medical Genetics Unit, University of Florence, Meyer
Children's University Hospital, Florence, Italy P11.090B
Overlapping phenotypes in patients harboring
heterozygous mutations in KAT6A and KAT6B Overlapping phenotypes in patients harboring
heterozygous mutations in KAT6A and KAT6B E. F. Percin1, G. Kayhan1, A. Sezer1, A. Koc1,2, M. A. Ergun1 1Medical Genetics Department, Gazi University Hospital,
Ankara, Turkey, 2Genetic Diagnostic Center, Tepecik Training
and Research Hospital, Izmir, Turkey S. Estrella, D. Bertola, G. L. Yamamoto, R. Honjo, J. R. Ceroni,
C. A. Kim S. Estrella, D. Bertola, G. L. Yamamoto, R. Honjo, J. R. Ceroni,
C. A. Kim C. A. Kim TARP syndrome: clinical characteristics of a 10 year-
surviving patient and review of the literature Kim: None. P11.091C Dual overlapping phenotype recessively inherited due to
paternal unipaternal disomy of chromosome 2 (pUPD2) in
a patient TARP syndrome: clinical characteristics of a 10 year-
surviving patient and review of the literature KOS represents a rare disease that,
despite showing a characteristic phenotype, represents a
challenging clinical diagnosis based only on clinical
aspects. WES allowed us to make a diagnosis of KOS in our
cases reducing cost and time of the diagnostic pathway,
demonstrating, once again, its capability in establishing the
clinical diagnostic rate, and its impact on medical man-
agement in a large paediatric centre. WES, moreover, pro-
vides a unique glimpse into the complexity of genetic
disorders. features, like blepharophimosis, hypertelorism, depressed
nasal bridge, low-set and posteriorly rotated ears and
micrognathia. To date only 19 patients from 16 unrelated
families were reported and in all cases an initial different
diagnosis were made. KOS represents a rare disease that,
despite showing a characteristic phenotype, represents a
challenging clinical diagnosis based only on clinical
aspects. WES allowed us to make a diagnosis of KOS in our
cases reducing cost and time of the diagnostic pathway,
demonstrating, once again, its capability in establishing the
clinical diagnostic rate, and its impact on medical man-
agement in a large paediatric centre. WES, moreover, pro-
vides a unique glimpse into the complexity of genetic
disorders. Results: frameshift variants in KAT6B, (p.S1303Vfs*31)
and (p.K1199Rfs*26), were found in the female patients. A
nonsense variant in KAT6A (p.R1129*) was found in the
male patient. Sanger sequencing confirmed a de novo event
in the latter. Conclusions: this is the first patient with KAT6A
mutation showing patellar hypoplasia. Due to the similarity
of function between KAT6A and KAT6B, this result could
expand the phenotype of this autosomal dominant mental
retardation syndrome, suggesting the disorder has over-
lapping features with GPS. Moreover, one of the proband
with GPS presented with preaxial polydactyly, which has
not been previously reported. FAPESP 2015/21783-9;
CNPq 304130/2016-8 A. Pagliazzi: None. A. Provenzano: None. F. Peluso:
None. S. Bargiacchi: None. M. Della Monica: None. G. Carignani: None. G. Forzano: None. S. Ciabattoni:
None. E. Agostini: None. P. Fiorini: None. A. La
Barbera: None. S. Guarducci: None. M. Pantaleo: None. B. Lucherini: None. S. Giglio: None. A. Pagliazzi: None. A. Provenzano: None. F. Peluso:
None. S. Bargiacchi: None. M. Della Monica: None. G. Carignani: None. G. Forzano: None. S. Ciabattoni:
None. E. Agostini: None. P. Fiorini: None. A. La
Barbera: None. S. Guarducci: None. M. Pantaleo: None. B. Lucherini: None. S. Giglio: None. S. Estrella: None. D. Bertola: None. G.L. Yamamoto:
None. R. Honjo:
None. J.R. Ceroni:
None. C.A. TARP syndrome: clinical characteristics of a 10 year-
surviving patient and review of the literature TARP syndrome: clinical characteristics of a 10 year-
surviving patient and review of the literature TARP syndrome: clinical characteristics of a 10 year-
surviving patient and review of the literature M. NICETA, S. Barresi, F. Pantaleoni, M. L. Dentici,
R. Capolino, S. Pizzi, A. Ciolfi, B. Dallapiccola, M. Tartaglia,
M. C. Digilio We report two unrelated patients with severe congenital
malformations and distinctive facial appearance, for whom
we have primarily hypothesized Marden-Walker syndrome
in the first case and CHARGE syndrome in the second one. In both cases, exome sequencing (WES) was performed in
order to validate our clinical suspicion. WES revealed novel
biallelic variants in the UBE3B gene: in the first case we
found homozygosity for a novel truncating mutation
(c.2169_2170insG p.Ile725Aspfs) within the HECT domain
of UBE3B protein, while in the second case compound
heterozygosity
for
two
novel
missense
mutations
(c.214A>G p.Ser72Gly; c.838T>C p.Ser280Pro). Biallelic
mutations of UBE3B have recently been associated with
Kaufman oculocerebrofacial syndrome (KOS), a rare entity
within the blepharophimosis-metal retardation syndromes,
characterized by typical clinical features but often mis-
diagnosed. Affected subjects present microcephaly, poor
growth, respiratory difficulties, gastrointestinal and geni-
tourinary anomalies and a peculiar constellation of facial Genetics and Rare Diseases Research Division, Ospedale
Pediatrico Bambino Gesù, Rome, Italy, Roma, Italy Introduction: TARP syndrome, comprising Talipes equi-
novarus, atrial septal defect, Robin sequence, and persis-
tence of the left superior vena cava, is a severe X-linked
condition that has been considered lethal in affected males. The disease is caused by inactivating mutations in RBM10,
which encodes for a RNA binding motif protein involved in
regulation
of
alternative
splicing
of
diverse
mRNA
precursors. The disease is caused by inactivating mutations in RBM10,
which encodes for a RNA binding motif protein involved in
regulation
of
alternative
splicing
of
diverse
mRNA
precursors. Materials, Methods and Results: By using a trio-based
WES approach, coupled with an in-house implemented
pipeline, we identified a maternally inherited RBM10
frameshift variant in a survived 10-year-old male with
clinical features fitting TARP syndrome. The RBM10
variant was confirmed by Sanger and was documented to 384 J. del Picchia features, like blepharophimosis, hypertelorism, depressed
nasal bridge, low-set and posteriorly rotated ears and
micrognathia. To date only 19 patients from 16 unrelated
families were reported and in all cases an initial different
diagnosis were made. S. Estrella, D. Bertola, G. L. Yamamoto, R. Honjo, J. R. Ceroni,
C. A. Kim P11.092D Towards a novel diagnostic strategy using patient-derived
URECs to diagnose ciliopathies 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Anatomical Institute, University of Bonn, Bonn,
Germany, 3Department of Pediatrics, Children's Hospital,
University of Bonn, Bonn, Germany, 4Department of
Neonatology, University of Bonn, Bonn, Germany M. M. Oud, D. Lugtenberg, E. M. H. F. Bongers, R. Roepman,
L. E. L. M. Vissers M. M. Oud, D. Lugtenberg, E. M. H. F. Bongers, R. Roepman,
L. E. L. M. Vissers Radboudumc, Nijmegen, Netherlands Radboudumc, Nijmegen, Netherlands The acronym VATER/VACTERL association refers to the
rare, non-random co-occurrence of the following compo-
nent features (CFs): vertebral defects (V), anorectal mal-
formations
(ARM)
(A),
cardiac
defects
(C),
tracheoesophageal fistula with or without esophageal atresia
(TE), renal malformations (R), and limb defects (L). Patients may present with additional congenital anomalies
however, the clinical diagnosis requires the presence of at
least three CFs. The pathogenesis of VATER/VACTERL
association is heterogeneous and remains to further be
elucidated. Introduction: Diagnostic exome analysis frequently leads
to an inconclusive molecular diagnosis due to the detection
of variants of unknown significance. Functional tests eval-
uating the effect of genetic variants on protein level could
elucidate their pathogenicity. Urine-derived renal epithelial
cells (URECs) provide a non-invasive source of patient
material. We used patient-derived URECs to functionally
evaluate the pathogenicity of genetic variants potentially
associated with ciliopathies (disorders caused by cilium
dysfunction). In a multiplex family with VATER/VACTERL associa-
tion, we performed whole exome sequencing (WES). Pedigree
information
suggested
an
underlying
X-
chromosomal recessive mode of inheritance which appeared
likely since the index female patient showed skewed X-
inactivation. We detected her maternally-derived X chro-
mosome being activated in 84% of lymphocytes, while
array-based analyses were normal. Consecutive WES and
filtering for rare X-chromosomal variants prioritized one
novel variant in SHROOM4 (p.E314K). Materials and methods: URECs were obtained from
patient’s urine and used for functional tests: (1) the ciliary
phenotype was analyzed using immunofluorescence cyto-
chemistry and (2) URECs and blood cells were used to
study mRNA splicing. Results: We tested the pathogenicity of two heterozygous
variants of unknown significance in IFT140, identified by
WES in a Mainzer-Saldino syndrome patient. IFT140
encodes a protein involved in ciliary intraflagellar transport
(IFT). Patient-derived URECs showed abnormal IFT in
comparison to healthy controls, which confirmed the
pathogenicity of both variants and validated the patient’s
diagnosis. Instituto da Criança, São Paulo, Brazil Introduction: Blending of two different disease phenotypes
in a single patient may apparently suggest a new clinical
phenotype or prevent the diagnosis of the other. Whole-
exome sequencing (WES) can provide insight into the
relationship between observed clinical phenotypes and
underlying genotypes. Here we present, a patient with both
Warburg Micro syndrome-1 (WARBM1) and Hypotonia,
infantile, with psychomotor retardation and characteristic
facies 2 syndrome (IHPFR2) caused by pUPD2 detected by
WES analysis. Introduction: KAT6A and KAT6B encode a lysine acetyl-
transferase, playing a role in chromatin regulation. Hetero-
zygous variants in KATB have been associated with Say-
Barber-Biesecker-Young-Simpson and genitopatellar syn-
dromes (GPS), both showing developmental delay/mental
retardation and dysmorphic features. Although these two
disorders were well characterized syndromes, some patients
present overlapping clinical features, hampering a straight-
forward
genotype-phenotype
correlation. Heterozygous
variants in KAT6A have been recently associated with
mental retardation, microcephaly and variable dysmorphic
features. Patellar anomaly is considered a cardinal feature of
GPS within this group. Patient and Results: WES analysis of a 14-year-old male
patient whose parents are unrelated, with characteristic
WARMB findings revealed two novel homozygous muta-
tions in both of the RAB3GAP1 (c.664delC) and UNC80
genes (c.C1459A). Sanger sequencing confirmed that the
same mutations was present in the patient’s heterozygous
father but not in the patient’s mother. In the homozygosity
analysis, it was found that pUPD2. In the patient, because of
overlapping findings of the both syndrome, the presence of
IHPFR2 syndrome in the patient was undiagnosed until the
end of the analysis. Casuistic and Methods: we report on three non-related
individuals, two female patients with typical findings of
GPS, one of them with preaxial polydactyly, and no family
history for polydactyly, and a male patient with severe
developmental delay, absent speech, microcephaly, facial
dysmorphisms and patellar hypoplasia. A targeted gene
panel and sanger sequencing was performed in the two
female probands and whole-exome sequencing in the male
proband. Conclusion: To date, the UPD2, especially of paternal
origin, has been very rarely described in the literature. When considering literature data there are no paternally Abstracts from the 51st European Society of Human Genetics Conference: Posters 385 procedure to facilitate accurate diagnosis of ciliopathy
patients. procedure to facilitate accurate diagnosis of ciliopathy
patients. imprinted genes on chromosome 2 that have a major effect
on growth or development. With the exception of various
autosomal recessive disorders their phenotype is normal. P11.093A
SHROOM4 as a candidate gene for VATER/VACTERL
association and characterization in a zebrafish model E.F. Percin: None. G. Kayhan: None. A. Sezer: None. A. Koc: None. M.A. Ergun: None. G. C. Dworschak1,2,3, F. Thieme1, K. U. Ludwig1, B. Odermatt2,
H. Reutter1,4 G. C. Dworschak1,2,3, F. Thieme1, K. U. Ludwig1, B. Odermatt2,
H. Reutter1,4 P11.092D
Towards a novel diagnostic strategy using patient-derived
URECs to diagnose ciliopathies Instituto da Criança, São Paulo, Brazil In
patient with multiple molecular diagnoses, the phenotypic
complexity of disorder can make it difficult for doctors to
diagnose it. Genomewide analyses may reveal more than
one Mendelian disease that is relevant for a patient and the
patient’s family. M.M. Oud: None. D. Lugtenberg: None. E.M.H.F. Bongers:
None. R. Roepman:
None. L.E.L.M. Vissers: None. Novel phenotypic and molecular insights in families with
Waardenburg syndrome Novel phenotypic and molecular insights in families with
Waardenburg syndrome A. Shukla1, P. Somashekar1, S. Nampoothiri2,
K. Gowrishankar3, R. Rama Devi4, N. Gupta5, D. Lakshmi N6,
A. Kaur7, S. Bajaj8, S. Jagadeesh9, G. KM1 1Kasturba Medical College and Hospital, Manipal, India,
2Amrita Institute of Medical Sciences & Research Centre,
Cochin, India, 3Apollo Children’s Hospitals, Chennai, India,
4Sandor Proteomics Pvt Ltd, Hyderabad, India, 5All India
Institute of Medical Sciences, New Delhi, India, 6Nizam's
Institute of Medical Sciences, Hyderabad, India, 7PGIMER,
Chandigarh, India, 8Seth G.S. Medical College and KEM
Hospital, Mumbai, India, 9Mediscan Systems, Chennai, India 1Kasturba Medical College and Hospital, Manipal, India,
2Amrita Institute of Medical Sciences & Research Centre,
Cochin, India, 3Apollo Children’s Hospitals, Chennai, India,
4Sandor Proteomics Pvt Ltd, Hyderabad, India, 5All India
Institute of Medical Sciences, New Delhi, India, 6Nizam's
Institute of Medical Sciences, Hyderabad, India, 7PGIMER,
Chandigarh, India, 8Seth G.S. Medical College and KEM
Hospital, Mumbai, India, 9Mediscan Systems, Chennai, India We report the case of a male newborn come to our obser-
vation for the evidence, at birth, of a white forelock of hair. We report the case of a male newborn come to our obser-
vation for the evidence, at birth, of a white forelock of hair. ,
,
Family history reported several cases of unspecific
premature graying of hair (onset in childhood-adolescence). The parents were first degree cousins. During pregnancy,
bilateral hydroureteronephrosis, megacystis and ureteral
stenosis were observed at the XXth ww prenatal ultrasounds
examination. Karyotype and Array CGH on amniotic fluid
were normal. In the first days of life, abdominal distension
became evident and failure of meconium passage was
observed. He underwent several multiple surgery interven-
tions due to subsequent intestinal blocks. Intestinal biopsy
was suggestive for Hirschprung disease. Ophtalmologic
evaluation was normal. Otoemissions revealed bilateral
profound sensorineural hearing loss. Renal and urinary tract
instrumental investigations confirmed the prenatal findings. He underwent a surgical intervention of right ureteral
reimplantation. At 50 days he developed epileptic seizures. Central hypotonia and psychomotor delay were observed. Introduction: Waardenburg syndrome (WS) is a type of
neurocristopathy. It is clinically and genetically hetero-
geneous disorder characterized by auditory and pigmentary
abnormalities. Materials and methods: We investigated a cohort of 15
patients from 13 Indian families clinically diagnosed with
WS. Candidate gene sequencing for PAX3 in four families
and whole exome sequencing for twelve families was
carried out. Urinary tract abnormalities associated with Waardenburg
Syndrome due to mutations in EDN3 gene: a new
phenotype D. Ognibene: None. G. Garani: None. L. Raimondi:
None. S. Melchionda: None. E. Di Muro: None. P. Gamba: None. P. Greco: None. D. Morano: None. A. Franchella: None. E. Cacciatore: None. A. Ferlini: None. S. Bigoni: None. D. Ognibene1, G. Garani2, L. Raimondi2, S. Melchionda3, E. Di
Muro3, P. Gamba4, P. Greco5, D. Morano5, A. Franchella6,
E. Cacciatore2, A. Ferlini1,7, S. Bigoni7 1Section of Microbiology and Medical Genetics, Department of
Medical Science, Ferrara University, Ferrara, Italy, 2Neonatal
Intensive Care Unit, Department of Reproduction and Growth,
University-Hospital St Anna, Ferrara, Italy, 3Division of
Medical Genetics, IRCCS Casa Sollievo della Sofferenza, San
Giovanni Rotondo, Italy, 4Pediatric Surgery Unit, Woman and
Children's Health Department, University of Padua, Padova,
Italy, 5Obstetric and Gynecology Unit, Department of
Reproduction and Growth, University-Hospital St Anna,
Ferrara, Italy, 6Pediatric Surgery Unit, Department of
Reproduction and Growth, University-Hospital St Anna,
Ferrara, Italy, 7UOL of Medical Genetics, Department of
Reproduction and Growth and Department of Medical Science,
University-Hospital St Anna, Ferrara, Italy G.C. Dworschak: None. F. Thieme: None. K.U.
Ludwig: None. B. Odermatt: None. H. Reutter: None. G.C. Dworschak: None. F. Thieme: None. K.U. Ludwig: None. B. Odermatt: None. H. Reutter: None. This report aims to show new features in WS caused by
EDN3 mutations, that may lead to a deeper understanding
of genotype-phenotype correlations and better clinical
management at early stage. P11.092D We also studied mRNA splicing of DYNC2H1 in
another ciliopathy patient with a heterozygous synonymous
change. DYNC2H1 encodes a subunit of the IFT dynein
motor. mRNA
splicing
revealed
exon
skipping
and
premature transcription termination. Together with the
pathogenic variant on the second allele, this functional
assay helped to diagnose this patient. We are currently re-sequencing SHROOM4 in 310 male
patients with VATER/VACTERL, VATER/VACTERL-
like, A only, or TE only phenotype utilizing a targeted re-
sequencing approach with molecular inversion probes. Expression studies of shroom4 in zebrafish larvae (zfl)
showed expression from 48 until 72 hours post fertilisation
in the region of the terminal gut. For functional analysis, we
have initiated Morpholino-knock-down (MO) experiments
in zflto describe morphological changes in Shroom4 MO-
morphants. Preliminary data suggests increased death rates
and a clear morphologic alteration of the cloaca, as well as
differences in fluorescent in vivo dye uptake and excretion Conclusion: The increasing number of variants with
uncertain
pathogenicity
requires
functional
testing
to
improve diagnostics. Functional tests using urine-derived
patient cells have proven to be an attractive non-invasive 386 J. del Picchia EDN3 gene, likely pathogenic but never described in
literature, inherited from both his parents. assay. However, the findings are not yet fully conclusive
and further analysis is warranted. Grant: Bonfor-O-120.0001.1 This is the first case of WS due to EDN3 mutation with
urinary tract involvement, psychomotor delay and epileptic
seizures. Grant: Bonfor-O-120.0001.1 Novel phenotypic and molecular insights in families with
Waardenburg syndrome Extended exome analysis and Real-time PCR
were carried out for copy number variant analysis. Results: Nine novel variants in PAX3, MITF, SOX10,
EDNRB, EDN3 and a reported pathogenic variant in MIFT
were identified. Intra-familial phenotypic variability was
observed in one a family and non-penetrance was observed
in two families. We observed low level germline mosaicism
in an asymptomatic father of two affected siblings with
pathogenic variant, c.256A>T in PAX3. Biallelic novel
missense variant, c.1021C>G in MITF was identified in a
patient with
WS
2
for
the
first
time
in
literature. Homozygous c.673G>A in EDNRB was identified in a
patient without any signs of Hirschsprung disease. Extended In the strong suspicious of a Waardenburg Syndrome
(WS) type 4, molecular analysis of EDN3, EDNRB and
SOX10 genes was performed. This analysis revealed an
homozygous nonsense variant c.364G>T (p.Glu122*) in Abstracts from the 51st European Society of Human Genetics Conference: Posters 387 exome analysis for copy number variations revealed 0.17
Mb heterozygous deletion encompassing SOX10 in a patient
with WS 4. Additionally, in two patients fulfilling the
diagnostic criteria for WS, we identified a homozygous
known stop-gain variant, c.71G>A in GJB2, which causes
Deafness, autosomal recessive 1A and a novel biallelic
stop-gain variant, c.1608C>G in ADGRV1, known to cause
Usher syndrome 2C. exome analysis for copy number variations revealed 0.17
Mb heterozygous deletion encompassing SOX10 in a patient
with WS 4. Additionally, in two patients fulfilling the
diagnostic criteria for WS, we identified a homozygous
known stop-gain variant, c.71G>A in GJB2, which causes
Deafness, autosomal recessive 1A and a novel biallelic
stop-gain variant, c.1608C>G in ADGRV1, known to cause
Usher syndrome 2C. Based on clinical signs, elevated CK and radiological
findings with brain and eye malformations, the tentative
diagnosis WWS was established. Molecular panel analysis
revealed a homozygous nonsense mutation in POMK
(OMIM*615247) in both twins. These molecular findings
confirmed the diagnosis of a POMK associated WWS
(OMIM#615247). Based on clinical signs, elevated CK and radiological
findings with brain and eye malformations, the tentative
diagnosis WWS was established. Molecular panel analysis
revealed a homozygous nonsense mutation in POMK
(OMIM*615247) in both twins. These molecular findings
confirmed the diagnosis of a POMK associated WWS
(OMIM#615247). So far, with only five different POMK mutations
published in three families (Jae et al., 2013, von Rennesse
et al. 2014, Di Constanzo et al., 2014), POMK mutations
represent a very rare cause of alpha-dystroglycanopathies. Novel phenotypic and molecular insights in families with
Waardenburg syndrome Meningo/encephaloceles have been reported so far as a rare
finding in WWS including one patient with POMK-related
WWS (Jae et al., 2013). The observation of occipital
meningoceles at identical positions in both twins appears
interesting and might point to a more important role of
POMK in the pathogenesis of neural tube defects. Conclusion: We herein present novel phenotypic and
molecular insights into Waardenburg Syndrome. Funding: Science and Engineering Research Board,
Government of India, India (YSS/2015/002009) A. Shukla: None. P. Somashekar: None. S. Nam-
poothiri: None. K. Gowrishankar: None. R. Rama Devi:
None. N. Gupta: None. D. Lakshmi N: None. A. Kaur:
None. S. Bajaj: None. S. Jagadeesh: None. G. Km: None. POMK-associated Walker-Warburg Syndrome (WWS) in
monozygotic twins with occipital meningocele POMK-associated Walker-Warburg Syndrome (WWS) in
monozygotic twins with occipital meningocele A. Kuechler1, M. Elgizouli1, K. Rupprich2, A. Stein2,
M. Dzietko2, A. Iannaccone3, A. Köninger3, B. Schweiger4,
H. Kölbel2, U. Schara2, U. Hehr5 A. Kuechler1, M. Elgizouli1, K. Rupprich2, A. Stein2,
M. Dzietko2, A. Iannaccone3, A. Köninger3, B. Schweiger4,
H. Kölbel2, U. Schara2, U. Hehr5 A. Kuechler1, M. Elgizouli1, K. Rupprich2, A. Stein2, P11.097A M. Dzietko2, A. Iannaccone3, A. Köninger3, B. Schweiger4,
H. Kölbel2, U. Schara2, U. Hehr5 Whole exome sequencing in Polish patients as an
experience of one Genetic Out-patients Clinic 1Institut für Humangenetik, Universitätsklinikum Essen, Essen,
Germany, 2Klinik für Kinderheilkunde I, Universitätsklinikum
Essen, Essen, Germany, 3Klinik für Frauenheilkunde und
Geburtshilfe, Universitätsklinikum Essen, Essen, Germany,
4Institut für Diagnostische und Interventionelle Radiologie und
Neuroradiologie, Universitätsklinikum Essen, Essen, Germany,
5Praxis für Humangenetik und Zentrum für Humangenetik,
Universitätsklinikum Regensburg, Regensburg, Germany R. Smigiel1, A. Doraczynska-Kowalik1, M. Biela1, M. Bloch1,
E. Szmida1, A. Walczak2, G. Kostrzewa2, J. Kosinska1,
M. Rydzanicz2, P. Stawinski2, A. Biernacka2, M. Sasiadek1,
M. Gos3, R. Ploski2 R. Smigiel1, A. Doraczynska-Kowalik1, M. Biela1, M. Bloch1, R. Smigiel1, A. Doraczynska-Kowalik1, M. Biela1, M. Bloch1, E. Szmida1, A. Walczak2, G. Kostrzewa2, J. Kosinska1, M. Rydzanicz2, P. Stawinski2, A. Biernacka2, M. Sasiadek1,
M. Gos3, R. Ploski2 M. Rydzanicz2, P. Stawinski2, A. Biernacka2, M. Sasiadek1,
M. Gos3, R. Ploski2 1Medical University, Wroclaw, Poland, 2Medical University,
Warsaw, Poland, 3Institute of Mother i Child, Warsaw, Poland P11.096D A. Kuechler: None. M. Elgizouli: None. K. Rupprich:
None. A. Stein: None. M. Dzietko: None. A. Iannaccone:
None. A. Köninger: None. B. Schweiger: None. H. Kölbel: None. U. Schara: None. U. Hehr: None. A. Kuechler: None. M. Elgizouli: None. K. Rupprich:
None. A. Stein: None. M. Dzietko: None. A. Iannaccone:
None. A. Köninger: None. B. Schweiger: None. H. Kölbel: None. U. Schara: None. U. Hehr: None. POMK-associated Walker-Warburg Syndrome (WWS) in
monozygotic twins with occipital meningocele Clinical whole exome sequencing and its potential as a
diagnostic and disclosing tool S. Moutton, F. Tran-Mau-Them, C. Philippe, A. Vitobello,
A. Bruel, J. Thevenon, D. Lehalle, N. Jean, V. Carmignac,
C. Poe, T. Jouan, M. Chevarin, N. Houcinat, P. Kuentz, J. Saint-
Onge, A. Masurel, S. El Chehadeh, A. Mosca, N. Marle,
J. Rivière, P. Vabres, P. Callier, Y. Duffourd, L. Faivre,
C. Thauvin-Robinet S. Moutton, F. Tran-Mau-Them, C. Philippe, A. Vitobello,
A. Bruel, J. Thevenon, D. Lehalle, N. Jean, V. Carmignac,
C. Poe, T. Jouan, M. Chevarin, N. Houcinat, P. Kuentz, J. Saint-
Onge, A. Masurel, S. El Chehadeh, A. Mosca, N. Marle,
J. Rivière, P. Vabres, P. Callier, Y. Duffourd, L. Faivre,
C. Thauvin-Robinet S. M. Al Tala1, N. Nahavandi2, C. Betz2, O. Birkholz2,
S. Neuber2, C. Neuhaus2, E. Decker2, S. Lenzner2, S. Brunck2,
M. Hartig2, K. Haug2, M. Drasdo2, O. Brandau2, N. Bachmann2,
M. H. Elsunni1, C. Bergmann2 1Medical University, Wroclaw, Poland, 2Medical University,
Warsaw, Poland, 3Institute of Mother i Child, Warsaw, Poland The
most common diagnoses were: 13 cases (12%) - early
infantile epileptic encephalopathy, 5 - metabolic defects
including mitochondrial disorders, 3 - Schaaf-Yang syn-
drome, 2 - Joubert syndrome, 2 - GPI, 2- NBIA5, 2 -
MICPCH and 2 - spastic paraplegia 47. diagnosis was established in 58 of 78 (74%) patients with
clinical suspicion of a multigenic syndromes or spectrum of
syndromes and in 16 of 29 (55%) patients with completely
unspecific manifestations. Final diagnoses were in accor-
dance with clinical suspicions in 31 of 58 (53%) cases. The
most common diagnoses were: 13 cases (12%) - early
infantile epileptic encephalopathy, 5 - metabolic defects
including mitochondrial disorders, 3 - Schaaf-Yang syn-
drome, 2 - Joubert syndrome, 2 - GPI, 2- NBIA5, 2 -
MICPCH and 2 - spastic paraplegia 47. Conclusion: Trio-based WES analysis is a powerful
second step strategy for patients affected by DD/ID. It
reduces analysis time, limits Sanger sequencing validations,
and promises to be an invaluable approach for translational
research and identifying new genes. Conclusions: In our experience WES proved to be
especially useful in patients with infantile onset of non-
specific and severely debilitating neurological syndromes. R. Smigiel: None. A. Doraczynska-Kowalik: None. M. Biela: None. M. Bloch: None. E. Szmida: None. A. Walczak: None. G. Kostrzewa: None. J. Kosinska: None. M. Rydzanicz: None. P. Stawinski: None. A. Biernacka:
None. M. Sasiadek:
None. M. Gos:
None. R. Ploski: None. R. Smigiel: None. A. Doraczynska-Kowalik: None. M. Biela: None. M. Bloch: None. E. Szmida: None. A. Walczak: None. G. Kostrzewa: None. J. Kosinska: None. M. Rydzanicz: None. P. Stawinski: None. A. Biernacka:
None. M. Sasiadek:
None. M. Gos:
None. R. Ploski: None. S. Moutton: None. F. Tran-Mau-Them: None. C. Philippe: None. A. Vitobello: None. A. Bruel: None. J. Thevenon: None. D. Lehalle: None. N. Jean: None. V. Carmignac: None. C. Poe: None. T. Jouan: None. M. Chevarin: None. N. Houcinat: None. P. Kuentz: None. J. Saint-Onge: None. A. Masurel: None. S. El Chehadeh:
None. A. Mosca: None. N. Marle: None. J. Rivière: None. P. Vabres: None. P. Callier: None. Y. Duffourd: None. L. Faivre: None. C. Thauvin-Robinet: None. P11.098B Trio whole exome sequencing as an efficient second step
strategy to decipher molecular basis of developmental
disorders after negative first-tier solo clinical WES 1Medical University, Wroclaw, Poland, 2Medical University,
Warsaw, Poland, 3Institute of Mother i Child, Warsaw, Poland Whole exome sequencing (WES) is an universal diagnostic
test that can be carried out in relatively short time. WES is
commonly used in clinical practice, especially in patients
with ambiguous manifestations. Walker-Warburg syndrome (WWS) represents the most
severe end of a phenotypic spectrum of disorders caused by
defective
glycosylation
of
alpha-dystroglycan. Alpha-
dystroglycanopathies are genetically heterogeneous auto-
somal recessive disorders; mutations in 16 underlying genes
have been identified so far. Materials and methods: WES was performed using
SureSelect V5 kit and HiSeq1500 sequencing in 107 Polish
patients from single Genetic Out-Patients Clinic: 1 foetus, 6
newborns, 10 infants, 84 children and 6 adults. The most
common findings were psychomotor development delay,
intellectual disability, epilepsy or epileptic encephalopathy,
dysmorphic syndromes, athrogryposis, ataxia syndromes,
manifestations
of
neuromuscular
disorders,
connective
tissue diseases as well as central nervous, cardiovascular,
urinary,
skeletal
and
eye
congenital
anomalies. The
suspicion of a multigenic syndromes or spectrum of
syndromes was established in 78 persons. 29 patients
showed highly unspecific clinical symptoms. We report on monozygotic male twins with strikingly
similar manifestations of WWS in prenatal ultrasonography
(hydrocephalus, occipital meningocele, hypoplastic cere-
bellum). Postnatal examination confirmed the meningocele
and revealed eye anomalies in both. CK was increased
>1000U/l. cMRI scans showed an occipital meningocele
with dorsally enlarged fourth ventricle, hypo-/aplasia of
cerebellar vermis, cortical malformation with generalized
polymicrogyria-like cobblestone malformation, temporo-
occipital subcortical band heterotopia, and eye malforma-
tions (microphthalmia with coloboma and caudal cyst /
persistent hyperplastic primary vitreous body and posterior
staphyloma). Results: Pathogenic mutations consistent with patients’
phenotypes were detected in 64 of 107 (60%) cases. The J. del Picchia 388 (38%) in 12 genes affected by de novo variants and 3 genes
affected by biallelic variants (11/15 new genes). Interna-
tional datasharing allowed to identify additional patients
carrying variants in the same gene with a consistent
genotype-phenotype correlation, confirming implication in
human phenotypes. Moreover, 5 patients were found to
carry a VUS (9%), but datasharing and literature review
were insufficient to better classify these variants. diagnosis was established in 58 of 78 (74%) patients with
clinical suspicion of a multigenic syndromes or spectrum of
syndromes and in 16 of 29 (55%) patients with completely
unspecific manifestations. Final diagnoses were in accor-
dance with clinical suspicions in 31 of 58 (53%) cases. Service de génétique médicale - CHU de Dijon, Dijon, France 1Armed Forces Hospital, Khamis Mushayt, Saudi Arabia,
2Bioscientia, Center for Human Genetics, Ingelheim, Germany 1Armed Forces Hospital, Khamis Mushayt, Saudi Arabia,
2Bioscientia, Center for Human Genetics, Ingelheim, Germany 1Armed Forces Hospital, Khamis Mushayt, Saudi Arabia,
2Bioscientia, Center for Human Genetics, Ingelheim, Germany 1Armed Forces Hospital, Khamis Mushayt, Saudi Arabia,
2Bioscientia, Center for Human Genetics, Ingelheim, Germany Purpose: Developmental disorders (DD) and intellectual
disability (ID) are frequent disorders affecting around 1-3%
of the worldwide population. Genetic defects are estimated
to account for approximately 80% of DD/ID etiology, but
the diagnosis yield is currently 30-40% when clinical
assessment, array-CGH, sequencing of single genes or gene
panels and clinical whole exome sequencing (cWES)
focusing on known disease-causing genes are combined. In
laboratories using solo cWES strategy as a first-tier analysis,
a subsequent trio-based strategy is expected to facilitate the
selection of candidate variants thanks to rapid identification
of de novo and compound heterozygous variants, and
therefore increase diagnostic yield. Whole-exome sequencing (WES) is increasingly used as an
effective diagnostic tool for patients with complex pheno-
types. Here we report our experience with WES in 282
index patients and assess the result in respect to the rate of
molecular diagnosis among phenotypic groups and the
ability to introduce novel disease variants and genes. The
patients were primarily pediatric (259 [91%] younger than
10 years old; 113 females [40%], 169 males [60%])
demonstrating diverse clinical manifestations, most often
including neurological dysfunctions. We could genetically
diagnose 122 patients (43%) based on a pathogenic or likely
pathogenic variant from which 58 have not been previously
reported. These variants are detected in disease genes (e. g. GLRB,
HPS1,
ASNS,
PLA2G6,
SERAC1,
MYO18B,
PLOD1, SUOX, KMT2D, CASK and TBX1) with significant
phenotypic overlap with probands’ clinical picture. Given
the parental consanguinity of the majority of the examined
individuals, recessively inherited phenotypes were most Methods: We selected patients affected by DD/ID who
did not get a molecular diagnosis after multiple investiga-
tions including solo cWES, and the data were re-analyzed in
a trio-based strategy. Results: Second step trio WES analysis of 53 DD/ID
patients selected likely pathogenic variants in 15 individuals Results: Second step trio WES analysis of 53 DD/ID
patients selected likely pathogenic variants in 15 individuals Abstracts from the 51st European Society of Human Genetics Conference: Posters 389 frequent (73%). However several variants (24%) causative
for dominantly inherited diseases such as Kabuki, Cowden,
DiGeorge and Coffin-Siris syndrome have been also
observed. In further 63 patients a variant of unknown sig-
nificance was identified. Several new disease candidate
genes have been also observed, namely PTK7 for holo-
prosencephaly. Our results once again highlight the feasi-
bility and usefulness of the whole-exome sequencing in the
clinical setting for timely medical interventions. 1Armed Forces Hospital, Khamis Mushayt, Saudi Arabia,
2Bioscientia, Center for Human Genetics, Ingelheim, Germany The high
yield of variants from dominant diseases in a largely con-
sanguineous cohort underlines the need for unbiased eva-
luation
and
highlights
subsequent
TRIO
analyses as
reasonable next step in negative cases. efficiently and effectively establish genetic diagnoses for
children with medical complexity, and that cohorts of these
children are enriched for novel genetic disorders. Screening 542 children with medical complexity from a
single Complex Care Program yielded 126 children (23%)
suspected of having an undiagnosed genetic condition
despite previous genetic testing. Eligible participants were
evaluated through a clinical genetic assessment; WGS was
performed in parallel with any outstanding conventional
genetic testing. In the first 21 probands with WGS data (including 13
trios, 1 dyad, and 7 singletons), 11 have reportable primary
diagnostic variants. We identified seven pathogenic/likely
pathogenic variants in known disease genes, including one
small structural variant, one intronic variant predicted to
affect splicing, and one mosaic variant; as well as a
promising variant of uncertain significance. De novo
missense variants were also identified in three genes
without published disease associations: FBXW7, H3F3B,
and RAC3. Importantly, by using public databases (DECI-
PHER, Matchmaker Exchange, and ClinVar), similarly
affected individuals were rapidly identified around the globe
and collaborations have been established to publish our
joint findings. S.M. Al Tala: None. N. Nahavandi: None. C. Betz:
None. O. Birkholz: None. S. Neuber: None. C. Neuhaus:
None. E. Decker: None. S. Lenzner: None. S. Brunck:
None. M. Hartig: None. K. Haug: None. M. Drasdo:
None. O. Brandau: None. N. Bachmann: None. M.H. Elsunni: None. C. Bergmann: None. S.M. Al Tala: None. N. Nahavandi: None. C. Betz:
None. O. Birkholz: None. S. Neuber: None. C. Neuhaus:
None. E. Decker: None. S. Lenzner: None. S. Brunck:
None. M. Hartig: None. K. Haug: None. M. Drasdo:
None. O. Brandau: None. N. Bachmann: None. M.H. Elsunni: None. C. Bergmann: None. P11.100D Whole genome sequencing as a tool for genetic diagnosis
and gene discovery in children with medical complexity G. Costain1,2, R. Z. Hayeems3,4, M. Snell5, M. Marano6,
M. Reuter7, D. Veenma7, S. Walker7, R. Basran8, E. Cohen6,1,
R. D. Cohn6,1, C. R. Marshall5,8,7, S. W. Scherer7,9,10,
C. Shuman2,9, D. J. Stavropoiulos8,11, J. Orkin6,3, M. Meyn12,2,9 G. Costain1,2, R. Z. Hayeems3,4, M. Snell5, M. Marano6,
M. Reuter7, D. Veenma7, S. Walker7, R. Basran8, E. Cohen6,1,
R. D. Cohn6,1, C. R. Marshall5,8,7, S. W. Scherer7,9,10,
C. Shuman2,9, D. J. Stavropoiulos8,11, J. Orkin6,3, M. Meyn12,2,9 G. Costain1,2, R. Z. Hayeems3,4, M. Snell5, M. Marano6,
M. Reuter7, D. Veenma7, S. Walker7, R. Basran8, E. Cohen6,1,
R. D. Cohn6,1, C. R. Marshall5,8,7, S. W. Scherer7,9,10,
C. Shuman2,9, D. J. Stavropoiulos8,11, J. Orkin6,3, M. Meyn12,2,9 Our initial experience applying WGS to children with
medical complexity suggests that trio-based genome-wide
sequencing is a high yield testing strategy for this patient
population, which appears to be enriched for de novo
mutations
and
novel
genetic
disorders. (Funded
by
University of Toronto and Hospital for Sick Children
grants). C. Shuman2,9, D. J. Stavropoiulos8,11, J. Orkin6,3, M. Meyn12,2,9 1Department of Paediatrics, University of Toronto, Toronto,
ON, Canada, 2Division of Clinical and Metabolic Genetics,
Hospital for SIck Children, Toronto, ON, Canada, 3The
Institute of Health Policy Management and Evaluation,
University of Toronto, Toronto, ON, Canada, 4Program in
Child Health Evaluative Sciences, Hospital for SIck Children,
Toronto, ON, Canada, 5Centre for Genetic Medicine, Hospital
for SIck Children, Toronto, ON, Canada, 6Department of
Paediatrics, Hospital for SIck Children, Toronto, ON, Canada,
7The Centre for Applied Genomics, Hospital for SIck Children,
Toronto, ON, Canada, 8Department of Paediatric Laboratory
Medicine, Hospital for SIck Children, Toronto, ON, Canada,
9Department of Molecular Genetics, University of Toronto,
Toronto, ON, Canada, 10Program in Genetics and Genome
Biology, Hospital for Sick Children, Toronto, ON, Canada,
11Department of Laboratory Medicine and Pathology,
University of Toronto, Toronto, ON, Canada, 12UW Center for
Human Genomics and Precision Medicine, University of
Wisconsin, Madison, WI, United States G. Costain: None. R.Z. Hayeems: None. M. Snell:
None. M. Marano: None. M. Reuter: None. D. Veenma:
None. S. Walker: None. R. Basran: None. E. Cohen:
None. R.D. Cohn: None. C.R. Marshall: None. S.W. Scherer: None. C. Shuman: None. D.J. Stavropoiulos:
None. J. Orkin: None. M. Meyn: None. 1Federal State Budgetary Institution “Research Centre for
Medical Genetics” of the Russian Academy of, Moscow,
Russian Federation, 2Moscow State University of Medicine and
Dentistry, Moscow, Russian Federation, 3Genomed Ltd.,
Moscow, Russian Federation Singapore, 13Reproductive Biology Laboratory, Academic
Medical Center, Amsterdam, Netherlands Kozlova: None. E. Dadali: None. Z. Markova:
None. N. Shilova: None. D. Khmelkova: None. F. Konovalov: None. I. Kanivets: None. R-SPONDIN2 inhibition of RNF43/ZNRF3 Dictates Limb
Numbers Independentlyof LGR4/5/6 R-SPONDIN2 inhibition of RNF43/ZNRF3 Dictates Limb
Numbers Independentlyof LGR4/5/6 T. Naert1,2, E. Szenker-Ravi2, U. Altunoglu3, M. Leushacke2,
C. Bosso-Lefèvre2,4, M. Khatoo2, T. Hong1, R. Noelanders1,
A. Hajamohideen2, C. Beneteau5, S. Bernardo de Sousa6,7,
B. Karaman3, X. Latypova5, S. Başaran3, E. Börklü Yücel8,
T. Thong2, L. Vlaeminck1, S. S. Nayak9, G. Katta Mohan9, C. Le
Caignec5,10, N. Soshnikova11, Z. O. Uyguner3, N. Barker2,
H. Kayserili3,7, K. Vleminckx1, B. Reversade2,4,8,12,13 T. Naert1,2, E. Szenker-Ravi2, U. Altunoglu3, M. Leushacke2,
C. Bosso-Lefèvre2,4, M. Khatoo2, T. Hong1, R. Noelanders1,
A. Hajamohideen2, C. Beneteau5, S. Bernardo de Sousa6,7,
B. Karaman3, X. Latypova5, S. Başaran3, E. Börklü Yücel8,
T. Thong2, L. Vlaeminck1, S. S. Nayak9, G. Katta Mohan9, C. Le
Caignec5,10, N. Soshnikova11, Z. O. Uyguner3, N. Barker2,
H. Kayserili3,7, K. Vleminckx1, B. Reversade2,4,8,12,13 1Department of Biomedical Molecular Biology, Ghent,
Belgium, 2Institute of Medical Biology, A* STAR, Singapore,
Singapore, 3Medical Genetics Department, Istanbul Medical
Faculty, Istanbul University, Istanbul, Turkey, 4Department of
Paediatrics, National University of Singapore, Singapore,
Singapore, 5CHU Nantes, Service de Génétique Médicale,
Nantes, France, 6Medical Genetics Unit, Hospital Pediátrico,
Centro Hospitalar e Universitário de Coimbra, Coimbra,
Portugal, 7Faculdade de Ciências da Saúde, Universidade da
Beira Interior, Covilhã, Portugal, 8Medical Genetics
Department, Koç University School of Medicine (KUSOM),
Istanbul, Turkey, 9Department of Medical Genetics, Kasturba
Medical College, Manipal University, Manipal, India,
10INSERM, UMR 957, Laboratoire de Physiopathologie de la
Résorption Osseuse et Thérapie des Tumeurs Osseuses
Primitives, Université de Nantes, Natues, France, 11Institute of
Molecular Biology (IMB) GmbH, Mainz, Germany, 12Institute
of Molecular and Cellular Biology, A* STAR, Singapore, T. Naert: None. E. Szenker-Ravi: None. U. Altunoglu:
None. M. Leushacke: None. C. Bosso-Lefèvre: None. M. Khatoo: None. T. Hong: None. R. Noelanders: None. A. Hajamohideen: None. C. Beneteau: None. S. Bernardo
de Sousa: None. B. Karaman: None. X. Latypova: None. S. Başaran: None. E. Börklü Yücel: None. T. Thong:
None. L. Vlaeminck: None. S.S. Nayak: None. G. Katta
Mohan: None. C. Le Caignec: None. N. Soshnikova:
None. Z.O. Uyguner: None. N. Barker: None. H. Kayserili:
None. K. Vleminckx:
None. B. Reversade: None. Singapore, 13Reproductive Biology Laboratory, Academic
Medical Center, Amsterdam, Netherlands Singapore, 13Reproductive Biology Laboratory, Academic
Medical Center, Amsterdam, Netherlands describe 4 y.o. boy with mild dysmorphic features, devel-
opmental and speech delay and seizures. Materials and metods, Results: The first performed test
with 561-gene customized NGS-platform for the most
frequent genes associated with congenital epilepsy using
Illumina NextSeq 500 amended to suggest 4p deletion on a
coverage basis. Subsequently Microarray analysis with
Affymerix Cytoscan Optima array confirmed 1,5 Mb
deletion in region 4p without critical WHS region (NSD2
gene) involvement. The same deletion was also confirmed
in proband’s phenotypically normal mother. Although her
hybridization profile was suggestive for mosaicism, a 4p-
subtelomeric FISH in blood samples was performed, but did
not confirm this version. The amphibian Xenopus tropicalis is extremely well posi-
tioned for modeling human disease. It shares with zebrafish
the
easy
manipulations
associated
with
its
external
embryonic development, but it manifests unique features
making it a more favorable organism. (1) Unlike zebrafish,
Xenopus tropicalis has a true diploid genome. Hence, gene
disruption studies are not suffering from redundancy and (2)
its genome shows high synteny with humans, greatly
facilitating identification of human disease gene orthologs. We employed a CRISPR/Cas9 based pipe-line for rapid
identification of genes influencing limb development and
collaborated on a human clinical study on WNT signaling
associated amelia. The four R-SPONDIN secreted ligands
are considered to act via their cognate LGR4/5/6 and
RNF43/ZNRF3 co-receptors to amplify WNT signaling. Here we document an allelic series of recessive RSPO2
mutations in humans causing Tetra-Amelia Syndrome. CRISPR/Cas9 mediated knockout of rspo2 in Xenopus
tropicalis also caused tetra-amelia, confirming earlier find-
ings in mouse knockouts. Unexpectedly, the triple and
ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not
recapitulate the known Rspo2 LOF phenotypes. Instead, we
found that TALEN mediated concurrent deletion of rnf43
and znrf3 in Xenopus embryos was sufficient to induce
super
numerous
limbs. Our
results
establish
that
RSPO2 serves as a direct ligand of RNF43/ZNRF3 but,
surprisingly, acts independently of LGR4/5/6. This signal
constitutes a master switch that governs limb numbers in the
embryo. In conclusion, the combined data clearly demon-
strates that the currently accepted paradigm for the LGR-
dependent R-SPONDIN signaling pathway is incorrect in
the context of limb formation. Conclusion: Thus, discrepancy in phenotypes of mother
and proband could be explained by possible existence of
undescribed imprinted genes in this region or different
expression profiles which are to be determined. Y. Familial case of Wolf-Hirschhorn syndrome with atypical
deletion and asymptomatic carrier Familial case of Wolf-Hirschhorn syndrome with atypical
deletion and asymptomatic carrier Y. Kozlova1,2, E. Dadali1, Z. Markova1, N. Shilova1,
D. Khmelkova3, F. Konovalov3, I. Kanivets3 Y. Kozlova1,2, E. Dadali1, Z. Markova1, N. Shilova1,
D. Khmelkova3, F. Konovalov3, I. Kanivets3 1Federal State Budgetary Institution “Research Centre for
Medical Genetics” of the Russian Academy of, Moscow,
Russian Federation, 2Moscow State University of Medicine and
Dentistry, Moscow, Russian Federation, 3Genomed Ltd.,
Moscow, Russian Federation Children with medical complexity have ≥1 chronic condi-
tion(s), functional limitations, multiple subspecialist invol-
vement, and high healthcare utilization. We hypothesized
that whole-genome sequencing (WGS) has the potential to Introduction: Inheritance of Wolf-Hirschhorn syndrome
(WHS) due to familial deletions is reported quite rare. We 390 J. del Picchia P11.103C Broader spectrum of OBSL1 mutations Broader spectrum of OBSL1 mutations Erasmus Medical Centre, Rotterdam, Netherlands Abstracts from the 51st European Society of Human Genetics Conference: Posters 391 Case: A 2.5-year-old boy presented with cleft palate,
developmental delay and dysmorphic features. He was born
term with birth weight of 4242 gram. The cleft palate was
seen shortly after birth. Over the years he developed growth
retardation, a unilateral conductive hearing loss and a global
developmental delay. His facial features showed long eye
lashes, broad nasal bridge, vertical line in lower lip, dental
problems, prominent ears with broad and bifid earlobes,
micrognathia. He also had fleshy hands and feet and flat
feet. He is the second child of a Polish father and Thai
mother. Family history is non-contributory. Case: A 2.5-year-old boy presented with cleft palate,
developmental delay and dysmorphic features. He was born
term with birth weight of 4242 gram. The cleft palate was
seen shortly after birth. Over the years he developed growth
retardation, a unilateral conductive hearing loss and a global
developmental delay. His facial features showed long eye
lashes, broad nasal bridge, vertical line in lower lip, dental
problems, prominent ears with broad and bifid earlobes,
micrognathia. He also had fleshy hands and feet and flat
feet. He is the second child of a Polish father and Thai
mother. Family history is non-contributory. Case: A 2.5-year-old boy presented with cleft palate,
developmental delay and dysmorphic features. He was born
term with birth weight of 4242 gram. The cleft palate was
seen shortly after birth. Over the years he developed growth
retardation, a unilateral conductive hearing loss and a global
developmental delay. His facial features showed long eye
lashes, broad nasal bridge, vertical line in lower lip, dental
problems, prominent ears with broad and bifid earlobes,
micrognathia. He also had fleshy hands and feet and flat
feet. He is the second child of a Polish father and Thai
mother. Family history is non-contributory. mortality. Still, a significant number of patients die within
few years from the disease onset, mainly due to the resis-
tance to post-operative radioiodine treatment. There is a
need for markers allowing for stratification of low and high-
risk PTC patients. In this study we analyzed impact of the germline
rs2910164 polymorphism in miR-146a-3p on the overall
survival of PTC patients and on the expression of the
sodium-iodide transporter NIS. rs2910164 in miR-146a-3p is associated with increased
mortality in thyroid cancer patients M. Kotlarek1,2, A. Kubiak1,2, M. Czetwertyńska3, M. Świerniak1,2,
W. Gierlikowski2, M. Kolanowska1,2, E. Bakuła Zalewska4, S. M. Jhiang5, K. Jażdżewski1,2, A. Wójcicka1,2 P11.103C The study included 2441 patients (2163 women, 278
men), including 359 cases with follicular variant of
papillary thyroid carcinoma (fvPTC). Tumor/blood DNA
was used for rs2910164 genotyping. Overall survival was
assessed retrospectively (median follow–up 10.25 years). The miR:NIS interactions were analyzed using in vivo and
radioactive iodine uptake assays. DNA testing: A ‘congenital anomaly’ gene panel showed
compound heterozygosity for variants of unknown sig-
nificance (missense) in the OBSL1 gene. Both parents are
carriers. The variants have not been described before. At an
earlier stage DNA testing for Kabuki syndrome did not
show any abnormalities. Rs2910164 functional variant within miR-146a-3p is
associated with increased overall mortality among fvPTC
female patients. The deaths per 1000-person-years were
29.7 in CC vs. 5.08 in GG/GC-carriers (HR=6.21,
P=0.006). Higher mortality of CC vs. GG/GC carriers
was also observed in patients with lower clinical stage
(HR=22.72,P < 0.001),
smaller
tumor
(HR=25.05,
P<0.001), lack of extrathyroidal (HR=9.03,P=0.02) and
nodular (HR=7.84,P=0.002) invasion, lack of metastases
(HR=6.5,P=0.005) and older (HR=7.8,P=0.002). We also
showed that miR-146a-3p directly regulates NIS. Inhibition
of mir-146a-3p restores the expression and function of NIS,
increasing radioactive iodine uptake. Discussion: OBSL1 gene mutations are involved in 3M
syndrome type 2. 3M syndrome is characterized with
extremely short stature, skeletal abnormalities and in
general normal intelligence. Our patient, however, does
not have extremely short stature and does not show
radiological findings seen in 3M syndrome. Additionally,
our patient has a developmental delay. The question rose if
these variants are causing this boy’s symptoms. Are OBSL1
gene mutations causing a broader spectrum than skeletal
problems? Are
there
other
patients
with
a
similar
phenotype? K. Stuurman: None. M. van Slegtenhorst: None. K. Stuurman: None. M. van Slegtenhorst: None. We propose a novel molecular marker of the clinical
outcome of PTCfv patients. Rs2910164 increases the
overall mortality with inhibition of NIS and disruption of
radioiodine uptake as a possible mechanism. P12 Cancer genetics P12 Cancer genetics P12.001A
rs2910164 in miR-146a-3p is associated with increased
mortality in thyroid cancer patients P12.001A
rs2910164 in miR-146a-3p is associated with increased
mortality in thyroid cancer patients M. Kotlarek: None. A. Kubiak: None. M. Czetwer-
tyńska: None. M. Świerniak: None. W. Gierlikowski:
None. M. Kolanowska: None. E. Bakuła Zalewska:
None. S.M. Jhiang: None. K. Jażdżewski: None. A. Wójcicka: None. P12.002B
A novel translocation of t(2;14)(q31;q32) in B-cell
precursor acute lymphoblastic leukemia A novel translocation of t(2;14)(q31;q32) in B-cell
precursor acute lymphoblastic leukemia 1Centre of New Technologies, Warsaw, Poland, 2Genomic
Medicine, Medical University of Warsaw, Warsaw, Poland,
3Department of Nuclear Medicine & Endocrine Oncology,
Maria Sklodowska-Curie Memorial Cancer Center and
Institute of Oncology, Warsaw, Poland, 4Department of
Pathology, Maria Sklodowska-Curie Memorial Cancer Center
and Institute of Oncology, Warsaw, Poland, 5Department of
Physiology and Cell Biology, The Ohio State University,
Columbus, OH, United States M. S. Yildirim1, O. Ceneli2, L. Simsek1, A. G. Zamani1 M. S. Yildirim1, O. Ceneli2, L. Simsek1, A. G. Zamani1 M. S. Yildirim1, O. Ceneli2, L. Simsek1, A. G. Zamani1 1Department of Medical Genetics, Meram Medical Faculty,
Necmettin Erbakan University, Konya, Turkey, 2Department of
Hematology, Meram Medical Faculty, Necmettin Erbakan
University, Konya, Turkey Introduction: Cytogenetics is among most useful tools of
predicting prognosis and choosing appropriate therapy for
haematological diseases. Translocations of 14q32 where Papillary thyroid carcinoma (PTC), the most common
thyroid cancer subtype, is considered a disease of low 392 J. del Picchia Results: Mutations were found in 343/620 (55.3%)
patients and were more often detected in patients with
normal karyotype (p = 0.001). The presence of FLT3-ITD
mutation was associated with adverse overall survival (OS)
and relapse-free survival (RFS) (p = 0.005 and p = 0.009,
respectively). Increasing FLT3-ITD allelic ratio (≥0.5)
correlated with low OS (p = 0.028). Patients with single
NPM1 mutation reached significantly better OS and RFS
compared to other patients (p = 0.040, p = 0.049, respec-
tively). The negative influence (tendency) of DNMT3A
mutations and IDH1 polymorphism rs11554137 was found
(p = 0.112, p = 0.186, respectively), whereas the presence
of IDH1 mutations correlated with better outcome com-
pared to group without mutations (p = 0.092). In 144
patients various combinations of mutations (from 2 to 5)
were detected. The presence of 2 mutations in one patient
significantly reduced OS compared to patients with one
mutation (p = 0.003). The worst prognosis was for patients
with combinations of NPM1+/FLT3-ITD+, NPM1+/FLT3-
ITD+/DNMT3A+, DNMT3A+/FLT3-ITD+ mutations. Results: Mutations were found in 343/620 (55.3%)
patients and were more often detected in patients with
normal karyotype (p = 0.001). The presence of FLT3-ITD
mutation was associated with adverse overall survival (OS)
and relapse-free survival (RFS) (p = 0.005 and p = 0.009,
respectively). Increasing FLT3-ITD allelic ratio (≥0.5)
correlated with low OS (p = 0.028). P12.002B
A novel translocation of t(2;14)(q31;q32) in B-cell
precursor acute lymphoblastic leukemia Patients with single
NPM1 mutation reached significantly better OS and RFS
compared to other patients (p = 0.040, p = 0.049, respec-
tively). The negative influence (tendency) of DNMT3A
mutations and IDH1 polymorphism rs11554137 was found
(p = 0.112, p = 0.186, respectively), whereas the presence
of IDH1 mutations correlated with better outcome com-
pared to group without mutations (p = 0.092). In 144
patients various combinations of mutations (from 2 to 5)
were detected. The presence of 2 mutations in one patient
significantly reduced OS compared to patients with one
mutation (p = 0.003). The worst prognosis was for patients
with combinations of NPM1+/FLT3-ITD+, NPM1+/FLT3-
ITD+/DNMT3A+, DNMT3A+/FLT3-ITD+ mutations. IGH gene resides, are found approximately 5% of acute
lymphoblastic leukemias. Among this group of transloca-
tions, t(2;14)(q31;q32) with B-cell precursor acute lym-
phoblastic leukemia has not been reported in literature
so far. Materials and methods: Here we present a 27 years old
female patient who referred to our clinic with diagnosis of
B-cell precursor acute lymphoblastic leukemia. GRAALL-
2003 protocol initiated after evaluation. During chemother-
apy, she developed mucormycosis in nasal region. Four-
nier's gangrene developed at 36th day of the patient’s
admission and debridement surgery with colostomy per-
formed immediately. Results:Karyotype from bone marrow resulted as:46,XX
[6]/46,XX,t(2;14)(q31;q32)[9], FISH analysis from same
sample revealed signal patterns consistent with IGH
rearrangement in 50% of interphases. Probes targeted to
cMYC, p16 del, MLL and BCR/ABL showed no abnormal
signal. RT-PCR of BCR/ABL was negative. Histopatholo-
gical and flow cytometry from bone marrow were consistent
with B-cell precursor acute lymphoblastic leukemia. Conclusions: Mutations in analyzed genes are frequent in
intermediate risk group of AML patients. They significantly
affect the prognosis, wherein it is important to consider the
type of mutation, its allele ratio and the presence of
additional mutations. Complex analysis of genetic aberra-
tions in AML patients provides the most accurate prognosis
prediction and planning of targeted therapy. Conclusion: The patient died a day after debridement
surgery due to respiratory failure. Considering detrimental clinical course, short survival
and age of this patient, it seems reasonable that this
translocation is concordant with the putative knowledge of
14q32 translocations occuring at an early age and leading
poor prognosis. P12.002B
A novel translocation of t(2;14)(q31;q32) in B-cell
precursor acute lymphoblastic leukemia In view of being first case reported, we
hope this case would be a contribution to literature and a
further step for comprehension of mechanisms lying behind
this disease and ultimately, to discovery of an effective
therapy for patients affected. E. Motyko: None. I. Martynkevich: None. O. Blau:
None. L. Polushkina: None. L. Martynenko: None. M. Bakaj: None. N. Cybakova: None. Y. Ruzhenkova:
None. E. Kleina:
None. N. Pavlenko:
None. A. Chechetkin: None. E. Motyko: None. I. Martynkevich: None. O. Blau:
None. L. Polushkina: None. L. Martynenko: None. M. Bakaj: None. N. Cybakova: None. Y. Ruzhenkova:
None. E. Kleina:
None. N. Pavlenko:
None. A. Chechetkin: None. E. Motyko: None. I. Martynkevich: None. O. Blau:
None. L. Polushkina: None. L. Martynenko: None. M. Bakaj: None. N. Cybakova: None. Y. Ruzhenkova:
None. E. Kleina:
None. N. Pavlenko:
None. A. Chechetkin: None. M.S. Yildirim: None. O. Ceneli: None. L. Simsek:
None. A.G. Zamani: None. NIPBL a new player with NPMc+ in the onset of acute
myeloid leukemia NIPBL a new player with NPMc+ in the onset of acute
myeloid leukemia P12.005A Molecular mutations, their cooccurrences & prognostic
value in acute myeloid leukemia Molecular mutations, their cooccurrences & prognostic
value in acute myeloid leukemia M. Mazzola1, G. Fazio2, G. Deflorian3, L. Ferrari3,
M. Spreafico1, C. Saitta2, L. Ferrari1, E. Bresciani4, A. Biondi2,
F. Cotelli5, M. Fumagalli6, M. Parma6, P. Riva1, A. Marozzi1,
G. Cazzaniga2, A. Pistocchi1 M. Mazzola1, G. Fazio2, G. Deflorian3, L. Ferrari3,
M. Spreafico1, C. Saitta2, L. Ferrari1, E. Bresciani4, A. Biondi2,
F. Cotelli5, M. Fumagalli6, M. Parma6, P. Riva1, A. Marozzi1,
G. Cazzaniga2, A. Pistocchi1 M. Mazzola1, G. Fazio2, G. Deflorian3, L. Ferrari3,
M. Spreafico1, C. Saitta2, L. Ferrari1, E. Bresciani4, A. Biondi2,
F. Cotelli5, M. Fumagalli6, M. Parma6, P. Riva1, A. Marozzi1,
G. Cazzaniga2, A. Pistocchi1 E. Motyko1, I. Martynkevich1, O. Blau2, L. Polushkina1,
L. Martynenko1, M. Bakaj1, N. Cybakova1, Y. Ruzhenkova1,
E. Kleina1, N. Pavlenko1, A. Chechetkin1
1Russian research institute of hematology and transfusiology,
St.-Petersburg, Russian Federation, 2Charite clinic, Berlin,
Germany
The aim of the research was to analyze the prognostic effect
of typical for AML patients mutations. Materials and methods: The study included 620 patients
from Germany and Russia. Screening of mutations in genes
FLT3, NPM1, DNMT3A, IDH1/2 was performed by PCR
and sequencing. E. Motyko1, I. Martynkevich1, O. Blau2, L. Polushkina1,
L. Martynenko1, M. Bakaj1, N. Cybakova1, Y. Ruzhenkova1,
E. Kleina1, N. Pavlenko1, A. Chechetkin1 1Dip. Biotecnologie Mediche e Medicina Traslazionale,
Università degli Studi di Milano, Milan, Italy, 2Centro Ricerca
Tettamanti, Clinica Pediatrica Università di Milano-Bicocca,
Centro Maria Letizia Verga, Monza, Italy, 3Istituto Fondazione
FIRC di Oncologia Molecolare IFOM, Milan, Italy,
4Oncogenesis and Development Section, National Human
Genome Research Institute, National Institutes of Health,
Bethesda, MD, United States, 5Dip. Bioscienze, Università
degli Studi di Milano, Milan, Italy, 6Haematology Division and
BMT Unit, Ospedale San Gerardo, Monza, Italy 1Russian research institute of hematology and transfusiology,
St.-Petersburg, Russian Federation, 2Charite clinic, Berlin,
Germany The aim of the research was to analyze the prognostic effect
of typical for AML patients mutations. The aim of the research was to analyze the prognostic effect
of typical for AML patients mutations. Materials and methods: The study included 620 patients
from Germany and Russia. Screening of mutations in genes
FLT3, NPM1, DNMT3A, IDH1/2 was performed by PCR
and sequencing. 393 Abstracts from the 51st European Society of Human Genetics Conference: Posters Cohesins form a multimeric protein complex (SMC1A,
SMC3, RAD21, STAG and additional proteins NIPBL,
MAU2, ESCO1, HDAC8) involved in the cohesion of sister
chromatids, post-replicative DNA repair and transcriptional
regulation. Recently, recurrent somatic mutations and
deletions of cohesins have been reported in the 10% of the
patients with Acute Myeloid Leukemia (AML) or other
myeloid neoplasms. Frequently, mutations in cohesin genes
co-occurred with the known AML-associated gene nucleo-
phosmin (NPM1) that, when mutated, aberrantly relocates to
the cytoplasm (NPMc+). Forced NPMc+ expression in
zebrafish embryos causes an expansion of hematopoietic
stem cells (HSCs) according with AML patient features. Introduction: allogeneic hematopoietic stem-cell trans-
plantation represents the most effective treatment for
patients with high-risk Acute Myeloid Leukemia, but post-
transplant
relapses
remain
frequent. Next
generation
sequencing allows the investigation of relapse mechanisms,
but tumor heterogeneity, clonality and genomic complexity
demands for ad-hoc bioinformatics solutions. Introduction: allogeneic hematopoietic stem-cell trans-
plantation represents the most effective treatment for
patients with high-risk Acute Myeloid Leukemia, but post-
transplant
relapses
remain
frequent. Next
generation
sequencing allows the investigation of relapse mechanisms,
but tumor heterogeneity, clonality and genomic complexity
demands for ad-hoc bioinformatics solutions. Methods: we combined whole exome sequencing and
RNA-Seq to characterize AML samples collected and
purified from 14 patients before (preTx) and after allo-
HSCT (postTx). We used alt-aware aligners to efficiently
map reads to hg38DH reference, and alignment-free
quantification of RNA-Seq data to detect differentially
regulated transcripts. Somatic variant-call was performed
both on DNA/RNA sequencing data. In our cohort of adult AML patients, we observed a
specific and significative reduction of the NIPBL expression
in NPMc+ patients. We generated a zebrafish model of
nipblb haploinsufficiency to investigate the hematopoietic
phenotype and the interactions between NPMc+ and
nipblb. In nipblb-loss-of-function zebrafish embryos, we
observed an increase in myeloid progenitors, a phenotype
resembling the NPMc+ zebrafish model. Therefore, we
characterize the functional interaction between NPMc+ and
NIPBL in the onset of the aberrant hematopoietic phenotype
in zebrafish and showed the involvement of the canonical
Wnt pathway in this process. Results: we detected an average of 15 damaging somatic
mutations per leukemic sample, a total of 54 exclusive to
relapses. The mutational burden increased significantly
from pre-to-postTx (p = 0.02, Wilcoxon) and consistently,
clonal-analysis evidenced the emergence of new clones at
relapse in 8 patients. Relapse-specific mutations encom-
passed known AML driver-genes including WT1 and
KRAS but no gene evidently related to immune function. By linear-model analysis of RNA-Seq data we found ~500
genes significantly deregulated in blasts at postTx-relapse. In contrast to the genomic data, most of these genes belong
to immune-related categories, including antigen-processing
and presentation via HLA Class II and T cell costimulation
pathways. We demonstrate for the first time a role for NIPBL during
zebrafish hematopoiesis and that its decreased expression,
due
to
NPM1
mutations,
might
play
a
role
in
leukemia onset. Funding grant: My First AIRC grant (MFAG) 18714 Funding grant: My First AIRC grant (MFAG) 18714 Conclusions: postTx-relapses originate upon a complex
process, in which leukemia clonal evolution intertwines
with immune-driven changes in patient-specific combina-
tions, on which precision treatments should be tailored. Conclusions: postTx-relapses originate upon a complex
process, in which leukemia clonal evolution intertwines
with immune-driven changes in patient-specific combina-
tions, on which precision treatments should be tailored. Funding grant: My First AIRC grant (MFAG) 18714
M. Mazzola: None. G. Fazio: None. G. Deflorian:
None. L. Ferrari: None. M. Spreafico: None. C. Saitta:
None. L. Ferrari: None. E. Bresciani: None. A. Biondi:
None. F. Cotelli: None. M. Fumagalli: None. M. Parma:
None. P. Riva: None. A. Marozzi: None. G. Cazzaniga:
None. A. Pistocchi: None. M. Mazzola: None. G. Fazio: None. G. Deflorian:
None. L. Ferrari: None. M. Spreafico: None. C. Saitta: None. L. Ferrari: None. E. Bresciani: None. A. Biondi:
N
F C t lli N
M F
lli N
M P G. Bucci: None. L. Zanotti: None. F. Santaniello:
None. D. Biancolini:
None. D. Cittaro:
None. D. Lazarevic: None. G. Tonon: None. F. Ciceri: None. L. Vago: None. G. Bucci: None. L. Zanotti: None. F. Santaniello:
None. D. Biancolini:
None. D. Cittaro:
None. D. Lazarevic: None. G. Tonon: None. F. Ciceri: None. L. Vago: None. None. P. Riva: None. A. Marozzi: None. G. Cazzaniga:
None. A. Pistocchi: None. 1Center for Translational Genomics and Bioinformatics,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Unit of
Immunogenetics, Leukemia Genomics and Immunobiology,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 3Unit of
Hematology and Bone Marrow Transplantation, IRCCS San
Raffaele Scientific Institute, Milan, Italy 1University of Medicine and Pharmacy Tirgu Mures, Tirgu
Mures, Romania, 2University of Medicine and Pharmacy "Iuliu
Hatieganu", Cluj-Napoca, Romania, 3Center for Advanced
Medical and Pharmaceutical Research, University of Medicine
and Pharmacy Tirgu Mures, Tirgu Mures, Romania P12.008D Acknowledgement:This work was supported by a grant of
the Romanian National Authority for Scientific Research
and Innovation,CNCS/CCCDI-UEFISCDI,project number
PN-III-P2-2.1- PED-2016-1076 within PNCDI III, contract
no.147PED/2017. C. Banescu: None. A. Crauciuc: None. V. Moldovan:
None. A. Boglis: None. E. Lazar: None. F. Tripon: None. P12.010B Integrating multiple genetic analysis into the clinical
diagnosis of Acute Myeloid Leukemia: a case report A. Pansa1, S. Salmoiraghi2, R. Cavagna2, K. Buklijas2,
A. Michelato2, L. Zannino2, G. Cassina1, A. Rambaldi2,
P. Fruscella1, B. Facchinetti1, O. Spinelli2, U. Giussani1 A. Pansa1, S. Salmoiraghi2, R. Cavagna2, K. Buklijas2, A. Pansa1, S. Salmoiraghi2, R. Cavagna2, K. Buklijas2, F. Tripon: None. A. Crauciuc: None. E. Lazar: None. A. Trifa: None. C. Banescu: None. A. Michelato2, L. Zannino2, G. Cassina1, A. Rambaldi2, A. Michelato2, L. Zannino2, G. Cassina1, A. Rambaldi2, A. Michelato2, L. Zannino2, G. Cassina1, A. Rambaldi2, P. Fruscella1, B. Facchinetti1, O. Spinelli2, U. Giussani1 P. Fruscella1, B. Facchinetti1, O. Spinelli2, U. Giussani1 1Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo,
Italy, 2Hematology and Bone Marrow Transplantation Unit,
Papa Giovanni XXIII Hospital, Bergamo, Italy P12.008D The aim of our study was to evaluate if
there are any correlation between the mentioned poly-
morphism, the risk of developing acute myeloid leukemia
(AML),FLT3 ITD mutation status, overall survival and
cytogenetic risk. Materials and methods: One hundred and forty-three
AML patients, and one hundred seventy-three healthy
subjects with no history of any malignancy were included in
the present case-control study. Materials and methods: One hundred and forty-three
AML patients, and one hundred seventy-three healthy
subjects with no history of any malignancy were included in
the present case-control study. Material and Methods: A number of 143 patients and
375 healthy people were enrolled in the study. The FLT3
ITD status was determined by RFLP-PCR and HRM
techniques. ABI 7500 fast real-time PCR system and
TaqMan assay were used for rs1534309 genotyping. Results: No association between variant allele of the
TERT rs 2853669 and AML risk was observed (OR = 1.55,
p = 0.71). We noticed a slightly shorter overall survival in
the cases with homozygous variant genotypes compared to
those with wild type homozygous genotype (p = 0.048). In
addition, we investigated the relation of variant genotype of
mentioned SNP and NPM1 and DNMT3A mutations in
AML cases but no association was observed. In conclusion,
we consider that TERT rs2853669 SNP is not a risk factor
for the development of AML in our cohort. Results: For rs1534309 we found the following geno-
types: 86 wild type in patients group and 252 in control
group,52 heterozygous in patients group and 114 in control
group, 5 homozygous with the variant allele in patients
group and 9 in control group. In patients group a number of
23 patients were positive for FLT3 ITD mutation (14 were
wild type for rs1534309, 8 heterozygous and 1 homozygous
with the variant allele). No associations were found between
this polymorphism, AML risk and FLT3 ITD status. No
significant differences were detected between sex,ages,
overall survival, cytogenetic risk or demographic character-
istics and genotypes. Acknowledgement: This work was supported by a grant
of the Romanian National Authority for Scientific Research
and Innovation, CNCS/CCCDI-UEFISCDI, project number
PN-III-P2-2.1- PED-2016-1076 within PNCDI III, contract
no.147 PED/2017 Conclusion: For our population rs1534309 polymorph-
ism is not a risk factor for AML, is not associated with
FLT3 ITD mutation and overall survival in AML patients. P12.008D Genomic and transcriptional profiling of acute myeloid
leukemia by next-generation sequencing unravels patient-
specific patterns of post-transplantation relapse No association between rs1534309 gene polymorphism and
FLT3 ITD mutation in patients with acute myeloid
leukemia No association between rs1534309 gene polymorphism and
FLT3 ITD mutation in patients with acute myeloid
leukemia G. Bucci1,2, L. Zanotti2, F. Santaniello1,2, D. Biancolini1,
D. Cittaro1, D. Lazarevic1, G. Tonon1, F. Ciceri3, L. Vago2,3 G. Bucci1,2, L. Zanotti2, F. Santaniello1,2, D. Biancolini1, F. Tripon1, A. Crauciuc1, E. Lazar1, A. Trifa2, C. Banescu1,3
1University of Medicine and Pharmacy Tirgu Mures, Tirgu
Mures, Romania, 2University of Medicine and Pharmacy "Iuliu
Hatieganu", Cluj-Napoca, Romania, 3Center for Advanced
Medical and Pharmaceutical Research, University of Medicine
and Pharmacy Tirgu Mures, Tirgu Mures, Romania
Introduction: Rs1534309, one of the polymorphism of
minichromosome
maintenance
complex
component
7 F. Tripon1, A. Crauciuc1, E. Lazar1, A. Trifa2, C. Banescu1,3 D. Cittaro1, D. Lazarevic1, G. Tonon1, F. Ciceri3, L. Vago2,3 D. Cittaro1, D. Lazarevic1, G. Tonon1, F. Ciceri3, L. Vago2,3 1University of Medicine and Pharmacy Tirgu Mures, Tirgu
Mures, Romania, 2University of Medicine and Pharmacy "Iuliu
Hatieganu", Cluj-Napoca, Romania, 3Center for Advanced
Medical and Pharmaceutical Research, University of Medicine
and Pharmacy Tirgu Mures, Tirgu Mures, Romania 1University of Medicine and Pharmacy Tirgu Mures, Tirgu
Mures, Romania, 2University of Medicine and Pharmacy "Iuliu
Hatieganu", Cluj-Napoca, Romania, 3Center for Advanced
Medical and Pharmaceutical Research, University of Medicine
and Pharmacy Tirgu Mures, Tirgu Mures, Romania 1Center for Translational Genomics and Bioinformatics,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Unit of
Immunogenetics, Leukemia Genomics and Immunobiology,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 3Unit of
Hematology and Bone Marrow Transplantation, IRCCS San
Raffaele Scientific Institute, Milan, Italy Introduction: Rs1534309, one of the polymorphism of
minichromosome
maintenance
complex
component
7 394 J. del Picchia functional single nucleotide polymorphism (SNP), namely
rs2853669, in the gene that encodes telomerase reverse
transcriptase (TERT) with the risk of acute myeloid leuke-
mia in a Romanian population. (MCM7) gene, was reported to be associated with several
types of cancer. The aim of our study was to evaluate if
there are any correlation between the mentioned poly-
morphism, the risk of developing acute myeloid leukemia
(AML),FLT3 ITD mutation status, overall survival and
cytogenetic risk. (MCM7) gene, was reported to be associated with several
types of cancer. P12.009A No association between TERT rs2853669 polymorphism
and NPM1, DNMT3A gene mutations and acute myeloid
leukemia risk No association between TERT rs2853669 polymorphism
and NPM1, DNMT3A gene mutations and acute myeloid
leukemia risk Introduction: Testing for genetic markers is strongly
recommended by all international guidelines as an essential
step for proper diagnosis, prognosis and subsequent mon-
itoring of acute leukemias. In the absence of genetic lesions
the decision-making process might become difficult. We
report a case of Acute Myeloid Leukemia (AML) in a
young adult in which genetic analysis, performed at diag-
nosis according to European LeukemiaNet, showed no
alterations. C. Banescu1, A. Crauciuc1, V. Moldovan1, A. Boglis1, E. Lazar2,
F. Tripon1 1Genetics Laboratory, Center for Advanced Medical and
Pharmaceutical Research,University of Medicine and
Pharmacy Tg. Mures, Romania, Tg. Mures, Romania,
2University of Medicine and Pharmacy Tg. Mures, Romania,
Tg. Mures, Romania 1Genetics Laboratory, Center for Advanced Medical and
Pharmaceutical Research,University of Medicine and
Pharmacy Tg. Mures, Romania, Tg. Mures, Romania,
2 2University of Medicine and Pharmacy Tg. Mures, Romania,
Tg. Mures, Romania Methods: Conventional cytogenetic analysis, fluores-
cence in situ hybridization (FISH), molecular assays as
polymerase chain reaction (PCR) amplification and frag-
ments analysis, Next Generation Sequencing (NGS) and
array comparative genomic hybridization (CGH) were Introduction:
Telomerase
reverse
transcriptase
gene
(TERT) which is important for the maintenance of chro-
mosome stability and it was reported to be significantly
associated with different cancer types, including malignant
hemopathies. We
investigated
the
association
of
a A. Pansa: None. S. Salmoiraghi: None. R. Cavagna:
None. K. Buklijas: None. A. Michelato: None. L. Zannino: None. G. Cassina: None. A. Rambaldi: None. P. Fruscella: None. B. Facchinetti: None. O. Spinelli:
None. U. Giussani: None. Z. Nozhat: None. M. Hedayati: None. M. Zarkesh:
None. S. Mohammadi-Yeganeh: None. K. Tabaei: None. F. Azizi: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 395 applied
for
the
identification
of
genetic
lesions
in
leukemia cells. and migration were investigated by MTT assay, microscopy
observation, AnexinV-PI and wound healing assay, respec-
tively. The mRNA levels of PI3K and AKT were evaluated
using qRTPCR. The phosphorylated levels of PI3K and
AKT were determined by ELISA. Results: Cytogenetic analysis was unsuccessful and FISH
assay of TP53 and KMT2A provided normal results. Molecular
evaluation
of
RUNX1-RUNX1T1,
CBFB-
MYH11, BCR-ABL1 fusion genes and NPM1, CEBPA,
FLT3 mutations, proved negative. NGS analysis suggested
a duplication of TET2 and KIT genes and a partial deletion
of RUNX1. Array CGH results confirmed a trisomy of
chromosome 4 and a partial deletion of chromosome 21
where the above genes are mapped. In addition, a partial
tetrasomy of chromosome 13 and a partial deletion of
chromosome 17 were identified. The treatment of the
patient was modified accordingly. Results: Metformin inhibited the cells proliferation and
migration in a significant time- and dose-dependent manner. It induced apoptosis and caused morphological changes in
all examined cells. qRTPCR results showed that the PI3K
and AKT mRNA levels were inhibited by metformin
(P<0.05). There was no change in the mRNA level of AKT
following metformin treatment in C643 cell line (P>0.05). The ELISA results showed that metformin treatment had no
effects on the phosphorylated levels of PI3K and AKT
(P>0.05). Conclusions: The laboratory evaluation of leukemias is
complex and has evolved significantly with the incorpora-
tion of advanced techniques. The combination of multiple
genetic approaches helped identifying prognostic markers
leading to proper risk category stratification and a better
patient management. Conclusions: The laboratory evaluation of leukemias is
complex and has evolved significantly with the incorpora-
tion of advanced techniques. The combination of multiple
genetic approaches helped identifying prognostic markers
leading to proper risk category stratification and a better
patient management. Conclusuions: The inhibition of proliferation by metfor-
min strongly was associated with the downregulation of the
molecules involved in the PI3K/AKT pathway. The exact
molecular mechanism of the metformin on the inhibition of
PI3K/AKT pathway and subsequent suppression of cell
proliferation has remained unclear and further studies are
required to its clarification. A. Pansa: None. S. Salmoiraghi: None. R. Cavagna:
None. K. Buklijas: None. A. Michelato: None. L. Zannino: None. G. Cassina: None. A. Rambaldi: None. P. Fruscella: None. B. Facchinetti: None. O. Spinelli:
None. U. Giussani: None. The Effects of Metformin on the PI3K/AKT Pathway in
Anaplastic Thyroid Cancer Cell Lines Transcriptional profiling reveals a potent activity of
anticancer imidazoacridinone C-1311 against prostate
cancer cells expressing androgen receptor Z. Nozhat1,2, M. Hedayati1, M. Zarkesh1, S. Mohammadi-
Yeganeh2, K. Tabaei1, F. Azizi3 Z. Nozhat1,2, M. Hedayati1, M. Zarkesh1, S. Mohammadi-
Yeganeh2, K. Tabaei1, F. Azizi3 Z. Nozhat1,2, M. Hedayati1, M. Zarkesh1, S. Mohammadi-
Yeganeh2, K. Tabaei1, F. Azizi3 M. Niemira1, A. Bielska1, Z. Mazerska2, M. Kwasniewski3,
A. Kretowski1, A. Skwarska2 M. Niemira1, A. Bielska1, Z. Mazerska2, M. Kwasniewski3,
A. Kretowski1, A. Skwarska2 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Tehran, Iran, Islamic
Republic of, 2Biotechnology Department, School of Advanced
Technologies in Medicine, Shahid Beheshti University of
Medical Sciences, Tehran, Iran, Islamic Republic of,
3Endocrine Research Center, Research Institute for Endocrine
Sciences, Shahid Beheshti University of Medical Science,
Tehran, Iran, Islamic Republic of 1Centre Clinical Research, Medical University of Bialystok,
Bialystok, Poland, 2Department of Pharmaceutical Technology
and Biochemistry, Chemical Faculty, Gdansk University of
Technology, Gdansk, Poland, 3Centre for Bioinformatics and
Data Analysis, Medical University of Bialystok, Bialystok,
Poland Introduction: The androgen receptor (AR) plays critical
role in the poor response of androgen-dependent and
castrate-resistant prostate cancers to chemotherapy. Antic-
ancer imidazoarcidinone C-1311 (Symadex™) is a new
inhibitor of topisomerase II and receptor tyrosine kinase
FLT3 that has been tested in phase II studies against
metastatic breast cancer. Here, we assessed the effect of C-
1311 on the transcriptional profiles in prostate cancer cell
lines with different AR status. Introduction: Association between T2D with the thyroid
cancers was shown and it has been attributed to the insulin
resistance. Insulin has relevance to tumor growth and
metastasis and it can exert its cell growth-stimulating effects
via PI3K/AKT signaling pathway. The aim of the present
study was to indicate whether metformin could affect
insulin-promoting cell growth by regulation of the PI3K/
AKT pathway. Materials and Methods: Prostate cancer cells were
exposed to C-1311 for 24 h. Global transcriptome profiling
for LNCaP (AR+) and DU-145 (AR-) cells was performed Material
and
Methods:
Anaplastic
thyroid
cancer
(ATC)-derived cells were treated with 0-60 mM metformin
for 24, 48 and 72h. Cell viability, morphology, apoptosis, J. del Picchia 396 6633), Bacillus cereus (ATCC 11778), and Staphylococcus
aureus ATCC 29213, and the Gram-negative Escherichia
coli (XL1), Xanthomonas campestris (ATCC 33013). using
Illumina
Hiseq
4000
platform. The
biological
relevance of dysregulated transcripts was assessed by
Ingenuity Pathway Analysis (IPA) software. Results: IPA analysis of transcripts uniquely expressed in
LNCaP and DU-145 cells exposed to C-1311 revealed
activation of diverse signalling pathways depending on AR
status. The main enriched top canonical pathways identified
in LNCaP (AR+) cells were associated with multiple cell
cycle control and survival processing genes like ATM and
breast cancer signalling whereas glycolysis, gluconeogen-
esis and lipid metabolism pathways were predominantly
dysregulated in DU-145 (AR-) cells. P12.013A O1 (CuFeO2) and O2 (Cu2O) Nanoparticles (NPs) induced
cytogenetic and genotoxic effects on human cultured
lymphocytes, antimicrobial activity on three bacterial
strains and damaging effects on pDNA S. del Carmen1, J. Sayagués1, O. Bengoechea1, J. Alcazar1,
R. Gervas1, J. Garcia1, A. Orfao2, M. Sarasquete1, M. Abad1 M. Niemira1, A. Bielska1, Z. Mazerska2, M. Kwasniewski3,
A. Kretowski1, A. Skwarska2 Furthermore, the main
upstream regulators and their target genes in both cell lines
were involved in the different biological processes. Results: These NPs at various concentrations demon-
strated high and significant genotoxic, cytostatic and
cytotoxic effects while the frequency of SCEs/cell was
increased
4-6
times
over
the
control
level. Strong
antibacterial activity of O2 was also observed against all
the bacterial strains and furthermore the effect of increasing
concentrations of the newly synthesized NPs on the
integrity and electrophoretic mobility of pDNA showed
that both NPs mimic topoisomerase I enzymatic nicking
activity. Conclusion: These results suggest that the O1 and O2
NPs exert strong genotoxic, cytogenetic, antimicrobial and
pDNA damaging activity, providing a significant property
that might support its possible involvement in DNA
damaging phenomena and their possible clinical use as a
new potent anticancer agent. Conclusion: Taken together, our finding suggest that AR
status may significantly affect the efficacy of C-1311
against prostate
cancer
cells. Association
of specific
canonical pathways indicates the opposite mechanism of
action of imidazoacridinone C-1311 in both androgen-
dependent and independent prostate cancer. Founded by the
National Science Centre, Grant No 2013/09/D/NZ7/04185. Conclusion: Taken together, our finding suggest that AR
status may significantly affect the efficacy of C-1311
against prostate
cancer
cells. Association
of specific
canonical pathways indicates the opposite mechanism of
action of imidazoacridinone C-1311 in both androgen-
dependent and independent prostate cancer. Founded by the
National Science Centre, Grant No 2013/09/D/NZ7/04185. M. Niemira: None. A. Bielska: None. Z. Mazerska:
None. M. Kwasniewski: None. A. Kretowski: None. A. Skwarska: None. K. Lafazanis: None. O. Antonoglou: None. A. Den-
drinou-Samara:
None. T.S. Lialiaris:
None. A. Pantazaki: None. M. Niemira: None. A. Bielska: None. Z. Mazerska:
None. M. Kwasniewski: None. A. Kretowski: None. A. Skwarska: None. P12.014B Heterogeneity of KRAS, NRAS, PIK3CA and BRAF
mutational status in primary tumor, lymph nodes and liver
metastases obtained from patients with metastatic
colorectal cancer Heterogeneity of KRAS, NRAS, PIK3CA and BRAF
mutational status in primary tumor, lymph nodes and liver
metastases obtained from patients with metastatic
colorectal cancer BAP1 germline variants in Australia N. K. Hayward1, S. Walpole1,2, J. Palmer1, A. Pritchard1,3,
M. Howlie1, J. Symmons1, H. Hamilton1, O. Rolfe4, A. Stark4,
M. D'Mellow4, S. Warrier4, W. Glasson4 K. Nebral1, S. Haslinger1, A. Inthal1, M. König1, D. Schinnerl1,
A. Attarbaschi2, G. Mann2, S. Strehl1, O. A. Haas1,2 1Children's Cancer Research Institute, St. Anna
Kinderkrebsforschung, Vienna, Austria, 2St. Anna Children’s
Hospital, Medical University of Vienna, Vienna, Austria 1QIMR Berghofer Medical Research Institute, Brisbane,
Australia, 2University of Queensland, Brisbane, Australia,
3The University of the Highlands and Islands, Inverness,
United Kingdom, 4Queensland Ocular Oncology Service,
Brisbane, Australia 1Children's Cancer Research Institute, St. Anna
Kinderkrebsforschung, Vienna, Austria, 2St. Anna Children’s
Hospital, Medical University of Vienna, Vienna, Austria 1Children's Cancer Research Institute, St. Anna
Kinderkrebsforschung, Vienna, Austria, 2St. Anna Children’s
Hospital, Medical University of Vienna, Vienna, Austria The subdivision of childhood B-cell precursor acute lym-
phoblastic leukemia (BCP-ALL), based on specific genetic
features, such as gene fusions or ploidy and copy number
aberrations, provides the basis for treatment stratification
and decisions. The so-called "B-other" group embraces all
cases with rare recurrent abnormalities that are hitherto less
well-defined. Because they include potential candidates for
targeted and personalized therapies, they are currently the
main focus of interest. One of these recently identified
subgroups that accounts for approximately 4% of BCP-ALL
and up to 10% of B-other cases, involves the ZNF384 gene,
which is fused to at least ten different partners. These cases
have commonly a CD10 negative (pro-B/BI) or CD10low
immunophenotype with myeloid markers and a distinct
gene expression pattern. To search for ZNF384 positive
cases we screened all B-other cases that were enrolled in the
ALL-BFM 2009 study with SNP/CGH arrays as well as an
additional selected cohort with a ZNF384-specific dual
color break apart FISH probe set. We found nine patients
with a ZNF384 fusion, which make-up approximately 5%
of all B-other cases. Five of them had a EP300-ZNF384 and
two a TCF3-ZNF384 fusion. The remaining two had novel
fusion partners, one of which was ascertained as CCAR1. The other one will be hopefully identified with whole
transcriptome RNA-sequencing, which is currently per-
formed in all cases. Three of them had IKZF1 deletions and Introduction: Germline mutations in BAP1 are linked to a
spectrum of cancers, defined as the BAP1 tumour predis-
position syndrome (BAP1-TPDS). K. Lafazanis1,2, O. Antonoglou3, A. Dendrinou-Samara3, T. S.
Lialiaris1, A. Pantazaki2 1Hospital Universitario de Salamanca, Salamanca, Spain,
2Universidad de Salamanca, Salamanca, Spain 1Demokrition University of Thrace, Medical School, Dept of
Genetics, Alexandroupolis, Greece, 2Aristotle University of
Thessaloniki, Dept of Chemistry, Lab of Biochemistry,
Thessaloniki, Greece, 3Aristotle University of Thessaloniki,
Dept of Chemistry, Lab of Inorganic Chemistry, Thessaloniki,
Greece 1Demokrition University of Thrace, Medical School, Dept of
Genetics, Alexandroupolis, Greece, 2Aristotle University of
Thessaloniki, Dept of Chemistry, Lab of Biochemistry,
Thessaloniki, Greece, 3Aristotle University of Thessaloniki,
Dept of Chemistry, Lab of Inorganic Chemistry, Thessaloniki,
Greece It is well known that activating mutations in the KRAS and
NRAS genes are associated with poor response to anti-
EGFR therapies in patients with metastatic colorectal cancer
(mCRC). Approximately half of the patients with wild-type
KRAS colorectal carcinoma do not respond to these thera-
pies. This could be because the treatment decision is
determined by the mutational profile of the primary tumor,
regardless of the presence of small tumor subclones har-
boring RAS mutations in lymph nodes or liver metastases. We analyzed the mutational profile of the KRAS, NRAS,
BRAF and PI3KCA genes in samples of 26 paired primary
tumors, 16 lymph nodes and 34 liver metastases from 26
untreated mCRC patients. The most frequent mutations
found in primary tumors were KRAS and PI3KCA, followed
by NRAS and BRAF. The distribution of the mutations in the
16 lymph node metastases analyzed was as follows: 4 in
KRAS gene, 3 in NRAS gene and 1 mutation each in
PI3KCA and BRAF. The most prevalent mutation in liver <META
NAME="author"
CONTENT="Χρήστης
των
Windows"> Introduction: O1 (CuFeO2) and O2 (Cu2O) nanoparticles
(NPs) have been tested at various concentrations in human
peripheral blood cells in vitro in order to investigate their
genotoxic, cytotoxic and cytostatic effects. DNA damaging
action was estimated by the Sister Chromatid Exchanges
(SCEs) methodology, a method for controlling genotoxicity
of human exposure to different mutagenic agents and by
DNA electrophoretic mobility experiments on pDNA
(pUC18). Their antimicrobial activity was evaluated against
the Gram-positive bacterial strains Bacillus subtilis (ATCC Abstracts from the 51st European Society of Human Genetics Conference: Posters 397 metastasis was in the KRAS gene, followed by PI3KCA and
BRAF. Of the 26 cases studied, 15 displayed an overall
concordance in the mutation status detected in the lymph
node metastases and liver metastases compared with pri-
mary tumor, suggesting no clonal evolution. K. Lafazanis1,2, O. Antonoglou3, A. Dendrinou-Samara3, T. S.
Lialiaris1, A. Pantazaki2 The mutation
profiles differed in the primary tumor and lymph node/
metastases samples of the remaining 11 patients, suggesting
intertumoral clonal evolution. Our results suggest the need
to perform mutational analysis in all available tumor sam-
ples of patients before deciding to commence anti-EGFR
treatment. family with BAP1-TPDS was observed, however, in the
Australian
family,
incomplete
family
history
for
the
proband did not allow for assessment of cancer history. Conclusions: Detection of four definite and two likely
deleterious variants in this focused assessment of BAP1 in
Australia suggests a need for a comprehensive worldwide
database of germline BAP1 variants and cancers present in
carriers and development of refined guidelines for clinical
testing. N.K. Hayward: None. S. Walpole: None. J. Palmer:
None. A. Pritchard:
None. M. Howlie:
None. J. Symmons: None. H. Hamilton: None. O. Rolfe: None. A. Stark: None. M. D'Mellow: None. S. Warrier: None. W. Glasson: None. S. del Carmen: None. J. Sayagués: None. O. Bengoe-
chea: None. J. Alcazar: None. R. Gervas: None. J. Garcia: None. A. Orfao: None. M. Sarasquete: None. M. Abad: None. P12.017A 1The Gurdon Institute, University of Cambridge, Cambridge,
United Kingdom, 2Department of Genetics, University of
Cambridge, Cambridge, United Kingdom, 3Cancer Research
Centre, Oncology Department, Sheba Medical Centre
Hospital, Ramat Gan, Israel, 4Oncology Department, Sackler
Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel,
5School of Clinical Medicine, University of Cambridge,
Cambridge, United Kingdom Hallym University Kangnam Sacred Heart Hospital, Seoul,
Korea, Republic of Hallym University Kangnam Sacred Heart Hospital, Seoul,
Korea, Republic of Cancer immunotherapy is becoming increasingly popular in
both research and clinical medicine. The potential to har-
ness the patient’s immune system to selectively destroy
cancer cells is promising in theory, and indeed already
yielded some impressive results in the clinic. However, the
individual success rate of such therapies is still very low. Many patients do not respond to treatment, while a small
percentage of others suffer from catastrophic side effects. The immune response is mediated by immunological
synapse: an interface between lymphocytes and antigen
presenting cells (APCs), which consists of co-inhibitory and
co-stimulatory checkpoint proteins. Therefore, a better
understanding of immunological synapse will enable us to
better design clinical trials and will provide us with better
diagnostics and therapeutics. Introduction: The BCR-ABL1 oncogenic fusion gene is a
hallmark of chronic myeloid leukemia (CML). Poly-
cythemia vera (PV) belong to a BCR-ABL1 negative mye-
loproliferative neoplasm. Transformation of PV to CML has
been rarely reported. Here, we present a secondary CML
case
progressed
from
long-standing
JAK2
V617F
positive PV. Materials and Methods: A 68-year-old woman pre-
sented with easy bruising. Complete blood count (CBC)
showed elevated hemoglobin level (hemoglobin concentra-
tion, 17.9 g/dL), leukocytosis (white blood cell count, 28.48
x 109/L), and marked thrombocytosis (platelet count, 1779
x 109/L). Bone marrow was hypercellular with a marked
myeloid and megakaryocytic hyperplasia. Materials and Methods: A 68-year-old woman pre-
sented with easy bruising. Complete blood count (CBC)
showed elevated hemoglobin level (hemoglobin concentra-
tion, 17.9 g/dL), leukocytosis (white blood cell count, 28.48
x 109/L), and marked thrombocytosis (platelet count, 1779
x 109/L). Bone marrow was hypercellular with a marked
myeloid and megakaryocytic hyperplasia. Results: Allele-specific polymerase chain reaction (PCR)
revealed heterozygous JAK2 V617F mutation and reverse-
transcriptase
(RT)-PCR
was
negative
for
BCR-ABL1
rearrangement. G-banding showed normal karyotype. The
patient was diagnosed with PV and treated with hydro-
xyurea. Seven years following diagnosis, CBC revealed re-
occurrence of neutrophilia and thrombocytosis, and newly
developed basophilia (16% of white blood cells; basophil
count, 5.45 x 109/L). G-banding revealed Philadelphia
chromosome in all analyzed metaphases. RT-PCR showed
positivity for BCR-ABL1 transcript and the patient was
diagnosed with CML. The patient has been followed with
imatinib and hydroxyurea. P12.019C all but one are in remission, supporting the notion that
ZNF384 positive cases seem to respond well to current
therapies. K. Nebral: None. S. Haslinger: None. A. Inthal: None. M. König: None. D. Schinnerl: None. A. Attarbaschi:
None. G. Mann: None. S. Strehl: None. O.A. Haas: None. K. Nebral: None. S. Haslinger: None. A. Inthal: None. M. König: None. D. Schinnerl: None. A. Attarbaschi:
None. G. Mann: None. S. Strehl: None. O.A. Haas: None. P. Stempor1,2, P. Dobosz3,4,5, R. Leibowitz-Amit3,4 S. Shin, J. Kim, M. Park, W. Song Hallym University Kangnam Sacred Heart Hospital, Seoul,
Korea, Republic of BAP1 germline variants in Australia The most commonly
associated cancers are uveal melanoma (UM), mesothe-
lioma, cutaneous melanoma, renal cell carcinoma, cho-
langiocarcinoma and meningioma, however, there is likely a
‘long tail’ of rare cancers also associated with the
syndrome. Materials and Methods: To assess the prevalence of
BAP1-TPDS in Australia, we screened Queensland UM
probands for BAP1 mutations (n = 64), identified variants
from published studies from Australia, and liaised with
national clinical genetics services to ascertain families with
BAP1 germline variants. Variants with a population
frequency of <0.0005 were considered for a potential role
in BAP1-TPDS. Materials and Methods: To assess the prevalence of
BAP1-TPDS in Australia, we screened Queensland UM
probands for BAP1 mutations (n = 64), identified variants
from published studies from Australia, and liaised with
national clinical genetics services to ascertain families with
BAP1 germline variants. Variants with a population
frequency of <0.0005 were considered for a potential role
in BAP1-TPDS. Results: We identified 4 truncating variants, all in
kindreds with classical features of BAP1-TPDS. Of the 11
missense variants, only 1 (p.T173C), is from a family with
typical BAP1-TPDS features, and is predicted to impair
ubiquitin hydrolase activity. In silico prediction suggests 8/
9 unique missense variants either alter splicing or damage
protein structure, though all require functional confirmation. A promoter variant identical to that reported in an Italian 398 J. del Picchia P12.019C
Expression patterns analyses of TCGA datasets reveal new
interactions and regulatory factors of immune response in
bladder cancer P12.019C
Expression patterns analyses of TCGA datasets reveal new
interactions and regulatory factors of immune response in
bladder cancer P. Stempor: None. P. Dobosz: None. R. Leibowitz-
Amit: None. S. Shin: None. J. Kim: None. M. Park: None. W.
Song: None. Hallym University Kangnam Sacred Heart Hospital, Seoul,
Korea, Republic of In this study we focus on The Cancer Genome Atlas
expression datasets and computational analyses to reveal the
regulatory patterns linking checkpoint genes to miRNA or
other regulatory factors that might enhance or switch off
their activity. The method can be generally applied to all types of cancer
with sufficient expression datasets. To showcase this
method, we have applied it to a bladder cancer (BLCA)
cohort. The graphical Gaussian model shows co-expression
of several checkpoint genes and anti-correlated expression
profile of many miRNAs, most prominently mir-15a and
miR-15b. This model predicts miRNA as negative regula-
tors of immunological response, potentially serving as a
post-transcriptional regulator of checkpoint genes expres-
sion. We have validated these interactions using the
calculation of free-energy of miRNA-mRNA binding, and
independently in cell lines using qPCR expression profiling. Conclusions:
In
conclusion,
we
report
a
case
of
secondary BCR-ABL1 positive CML progressed from
JAK2 V617F positive PV. Although the development of
CML in PV is a rare event, careful examination including
molecular studies should be considered in patient with PV
who showed severe leukocytosis and/or basophilia. GrantRefs: https://orcid.org/0000-0002-9464-7475 P. Stempor: None. P. Dobosz: None. R. Leibowitz-
Amit: None. S. Shin: None. J. Kim: None. M. Park: None. W. Song: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 399 P12.020D 4University of Manchester, Manchester, United Kingdom,
5Nanyang Technological University, Singapore, Singapore Background: Results from BRCA1/2 germline mutation
testing are important in guiding clinicians on cancer man-
agement and surveillance strategies. The Manchester scor-
ing system (MSS) helps identify patients indicated for
germline BRCA1/2 testing. The recent third iteration of
MSS showed improvements after including further adjust-
ments for triple negative breast cancer, high grade serous
ovarian cancer and HER2 receptor status. This study eval-
uates the relative effectiveness of MSS1-3 in a Southeast
Asian population. O. Cilingir1, M. Dincer2, S. Arslan1, B. Durak Aras1,
E. Erzurumluoglu1, O. Kutlay1, S. Aynacı1, S. Artan1 O. Cilingir1, M. Dincer2, S. Arslan1, B. Durak Aras1,
E. Erzurumluoglu1, O. Kutlay1, S. Aynacı1, S. Artan1 O. Cilingir1, M. Dincer2, S. Arslan1, B. Durak Aras1,
E. Erzurumluoglu1, O. Kutlay1, S. Aynacı1, S. Artan1 1Eskisehir Osmangazi University, Faculty of Medicine,
Department of Medical Genetics, Eskisehir, Turkey, 2Eskisehir
Osmangazi University, Faculty of Medicine, Department of
Medical Oncology, Eskisehir, Turkey The BRAF gene encodes a protein with serine/threonine
kinase activity involved in the mitogen activated protein
kinase signaling pathway (MAPs). It causes cell prolifera-
tion through the ras pathway. Somatic BRAF gene muta-
tions were detected in 80% melanomas, 15% colorectal
cancers(CRC), 45% papillary thyroid cancers(PLT) and
15% nonsmall cell lung cancers(NSCLC). Mutations are
frequently observed in the codon 600 (V600A, V600D,
V600E and V600K,R,M), exons 15 and 11. The advent of
therapeutics targeting the MAPK signal pathway has led to
great advances in the treatment of metastatic melanoma. Methods. We carried out a retrospective study of 330
index patients that were genetically tested using next
generation sequencing (NGS) panels which included full
gene sequencing and coverage for large deletions/duplica-
tions
in
BRCA1/2. Clinicopathological
features
(e.g.,
tumour histology, grade and age of diagnosis) and family
cancer history were collected to calculate MSS1-3 scores
and evaluated against genetic results with ROC analysis. Calculations were performed using Medcalc17. Results In total, 47 (14.2%) patients were tested positive
for BRCA1 or BRCA2 germline mutation. Based on a 10%
likelihood of BRCA1/2 germline mutation as a threshold for
genetic testing, 43.0% (142/330) of patients were indicated
for testing under MSS3, compared to 35.8% (118/330) for
MSS1 and 36.4% (120/330) for MSS2. In terms of model
effectiveness, MSS3 had a statistically significant improve-
ment over MSS1 (p = 0.037) and MSS2 (p = 0.032), with
91.5% sensitivity and 65.0% specificity at the 10%
threshold. P12.022B Assessing the effectiveness of the Manchester Scoring
System in predicting Southeast Asian patients with BRCA1/
2germline mutations W. Chew1, R. B. Moorakonda2,3, J. C. Allen2, E. Courtney1,
H. Soh1, L. Shao Tzu1, Y. Chen1, T. Shaw1, G. Evans4,
J. Ngeow1,5,2 P12.020D This study was designed to investigate the mutation status
of 11th and 15th exons of BRAF gene in 83 melanoma
patients. Sequence analysis from paraffin embedded tissue
samples was performed by pyrosequencing method The
overall incidence of somatic mutations within the BRAF
gene was 37,35%. The clinical subtypes of the melanomas
were compared with the mutation types and ratio. The most
prevalent
type
of
BRAF
mutation
was
c.1799T>A
(27,71%). According to the literature, about 40-60% of
melanoma patients have mutations. The findings of our
study were supported the literature. The detected mutations
considered as potential drug targets in advanced melanoma
therapy. Conclusion. Compared to previous iterations, the latest
MSS3 is a better performing model and relative to the
United
Kingdom
population,
is
equally
effective
in
distinguishing patients with BRCA1/2 germline mutations
in a Southeast Asian population. Conclusion. Compared to previous iterations, the latest
MSS3 is a better performing model and relative to the
United
Kingdom
population,
is
equally
effective
in
distinguishing patients with BRCA1/2 germline mutations
in a Southeast Asian population. O. Cilingir: None. M. Dincer: None. S. Arslan: None. B. Durak Aras: None. E. Erzurumluoglu: None. O. Kutlay: None. S. Aynacı: None. S. Artan: None. W. Chew: None. R.B. Moorakonda: None. J.C. Allen:
None. E. Courtney: None. H. Soh: None. L. Shao Tzu: W. Chew: None. R.B. Moorakonda: None. J.C. Allen:
None. E. Courtney: None. H. Soh: None. L. Shao Tzu:
None. Y. Chen: None. T. Shaw: None. G. Evans: None. J. Ngeow: None. None. Y. Chen: None. T. Shaw: None. G. Evans: None. J. Ngeow: None. None. Y. Chen: None. T. Shaw: None. G. Evans: None. J. Ngeow: None. 1Medical Genetics, Department of Experimental and Clinical
Biomedical Sciences “Mario Serio”, University of Florence,
Firenze, Italy, 2Medical Genetics Unit, Careggi University
Hospital, Firenze, Italy 1National Cancer Centre Singapore, Singapore, Singapore,
2Duke-NUS Medical School, Singapore, Singapore,
3Singapore Clinical Research Institute, Singapore, Singapore, P12.023C Detection of BRCA1 and BRCA2 variants in circulating free
DNA by using a commercial kit G. L. Capone1, A. L. Putignano1, A. Patuelli1, I. Paganini1,
R. Sestini1, F. Gensini1, A. Marozza2, B. Porfirio1, L. Papi1 J. del Picchia 400 Introduction: Patients with high-grade serous ovarian
cancer with germline or somatic mutations in BRCA1/2
genes can benefit from PARP inhibitors treatment. While
germline mutational screening has become a routine prac-
tice by Next Generation Sequencing, detection of somatic
mutation is challenging. The major advantage of using
liquid biopsy, rather than tissue biopsy as tool for mutation
screening in cancer, is to scan allele status and frequencies
of a patient over time. In this preliminary study, we
demonstrated that detection of both germline and somatic
BRCA1/2 mutations in circulating free DNA (cfDNA) with
targeted amplicon sequencing is feasible and might be
helpful in the standard diagnostic procedures. Introduction: Patients with high-grade serous ovarian
cancer with germline or somatic mutations in BRCA1/2
genes can benefit from PARP inhibitors treatment. While
germline mutational screening has become a routine prac-
tice by Next Generation Sequencing, detection of somatic
mutation is challenging. The major advantage of using
liquid biopsy, rather than tissue biopsy as tool for mutation
screening in cancer, is to scan allele status and frequencies
of a patient over time. In this preliminary study, we
demonstrated that detection of both germline and somatic
BRCA1/2 mutations in circulating free DNA (cfDNA) with
targeted amplicon sequencing is feasible and might be
helpful in the standard diagnostic procedures. Reducing-Bilateral-Salpingo-Oophorectomy
(RRBSO)
is
widely recommended for mutation carriers by professional
organizations (NCCN, NCI etc.). RR-BSO reduces ovarian
cancer by 95%, and decreases all-cause mortality. Various
aspects influence decision-making-process regarding sur-
gery. By understanding these considerations, we could
increase RRBSO-rate. Objective:
to
estimate
RRBSO-rate
among
BRCA
mutation carriers, and examine effect of demographic
factors and cancer history on RRBSO-rate Methods: Data on 543 female carriers of BRCA1/2
mutations from age 35 were collected from medical records
and attendance to our specialized-high-risk-clinic . Chi-
squared, Fisher-exact tests and student-t-test were used for
categorical, continuous variables, respectively. Materials and Methods: cfDNA of patients, previously
screened for BRCA1/2 mutations, was extracted using
QIAamp Circulating Nucleic Acid Kit, while BRCA1/2
mutation screening was performed using the Devyser
BRCA kit. P12.023C In addition, two known reference standards
(BRCA Somatic Multiplex I FFPE and Structural Multiplex
cfDNA Reference Standard) carrying different pathogenic
and not pathogenic variants with known percentage of
mutated allele were analyzed. Results: Overall RRBSO-rate was 83.5% . There was
positive correlation between mean age and RRBSO-rate:
53.27( ± 9.66)yrs. in those with RR-BSO vs. 44.17( ±
8.56)yrs. in those without, p < 0.001. Parous carriers were
more inclined to have RR-BSO than nulliparas: 84.5% vs. 61.5%, respectively, p = 0.045. These results are consistent
with common medical recommendations based on age,
birth-planning. No association was found between marrital
status and RRBSO. There was positive correlation between
breast cancer and RRBSO: 92.2% with positive history, vs. 81% without, p = 0.033. Familial cancer history had no
significant impact. Results: We confirmed the presence of all germline
mutations in cfDNA and the entire set of variants with
known percentage of mutated alleles in the two reference
standards achieving a sensitivity as low as 5% for somatic
variants. Conclusions: the vast majority of BRCA mutation
carriers that are attend our specialized-high-risk-clinic,
choose to undergo RRBSO, in line with acceptable medical
recommendations. Age, parity, and personal history of
breast cancer are positive predictors of RRBSO. Conclusions:
Our
results
demonstrate
that
cfDNA
mutation screening with a commercial kit allows the
detection of both germline and somatic BRCA1/2 mutations
at the same time, permitting to monitor the presence of
possible reverse mutations, which could affect the efficacy
of PARP inhibitors. GRANT: RICATEN2017 R. Michaelson-Cohen: None. O. Reichman: None. T. Goldstein: None. P. Mor: None. E. Levi: None. S. Lieberman:
None. U. Beller:
None. E. Levy-
Lahad: None. G.L. Capone:
None. A.L. Putignano:
None. A. Patuelli: None. I. Paganini: None. R. Sestini: None. F. Gensini: None. A. Marozza: None. B. Porfirio: None. L. Papi: None. Factors involved in decision to undergo risk reduction
bilateral salpingo-oophorectomy in carriers of BRCA
mutations Factors involved in decision to undergo risk reduction
bilateral salpingo-oophorectomy in carriers of BRCA
mutations R. Michaelson-Cohen, O. Reichman, T. Goldstein, P. Mor,
E. Levi, S. Lieberman, U. Beller, E. Levy-Lahad Swift Biosciences, Ann Arbor, MI, United States Shaare Zedek Medical Center, Hebrew University, Jerusalem
Israel, Jerusalem, Israel The use of next-generation sequencing (NGS) to assess
genetic variation in genes involved in both inherited dis-
eases, as well as cancer, is critical. However, obstacles
including
homology
to
pseudogenes
and
difficult
to
sequence motifs arise, making genes like CFTR, BRCA1,
and BRCA2 challenging to sequence with short-read (SR)
chemistry. Long-read (LR) assays overcome these issues, The lifetime risk of ovarian cancer among BRCA1,2
mutation carriers is 36 times higher than general population
(50% vs. 1.4%, respectively), and risk of breast cancer is
increased by 5.8 (70% vs 12%, respectively). Risk- C. A. Schumacher, A. M. Wood, S. Sandhu, J. Lenhart,
L. Kurihara, V. Makarov Abstracts from the 51st European Society of Human Genetics Conference: Posters 401 but are restricted by limited available technologies. To
enhance the benefits of each platform, we developed tar-
geted, single-tube, multiplexed amplicon assays that allow
for overlapping, contiguous coverage. mediated genetic testing, PM implemented changes to
pathology reporting, appointment scheduling, and in-service
education for referring physicians. Methods: Following the implementation of new referral
and scheduling processes, a list of women diagnosed with
SOC at PM between 2010-2016 was obtained from the PM
Cancer Registry and cross-referenced against the genetics
database. DNA from MUTCF-2 (Coriell Institute) was used in both
the SR and LR assays. In the SR assay, an 87-amplicon
panel comprehensively covered all exons (~10Kb) in
CFTR. The LR assay covered all the same exons, and 6
complete intronic regions, using 21 amplicons (~54Kb). DNA from BC01 (Coriell Institute) was used in the 246-
amplicon (~23Kb) SR assay and the 35-amplicon LR assay
(~86kB). While both comprehensively cover all exons, the
LR assay additionally covers 20 complete introns. For all
genes, SR libraries were sequenced on an Illumina MiniSeq
and LR libraries were sequenced on a Pacific Biosciences
RSII. Greater than 95% on-target and coverage uniformity
was seen in libraries from both SR and LR assays, and the
same number of variants were detected. SR assays allow for screening of a wide range of targets
for immediate and broad use. LR assays enable compre-
hensive sequencing of entire gene coding regions and
neighboring introns, gaining additional insight into variants
in difficult-to-sequence regions, large insertion/deletions,
and structural variations. DNA from MUTCF-2 (Coriell Institute) was used in both
the SR and LR assays. In the SR assay, an 87-amplicon
panel comprehensively covered all exons (~10Kb) in
CFTR. The LR assay covered all the same exons, and 6
complete intronic regions, using 21 amplicons (~54Kb). DNA from BC01 (Coriell Institute) was used in the 246-
amplicon (~23Kb) SR assay and the 35-amplicon LR assay
(~86kB). While both comprehensively cover all exons, the
LR assay additionally covers 20 complete introns. For all
genes, SR libraries were sequenced on an Illumina MiniSeq
and LR libraries were sequenced on a Pacific Biosciences
RSII. Greater than 95% on-target and coverage uniformity
was seen in libraries from both SR and LR assays, and the
same number of variants were detected. Results: Of 724 women with SOC, 68% were referred for
GC and 93% attended their scheduled appointment. Of
those who received GC, 96% proceeded with genetic
testing, 22% of whom were found to have a pathogenic
variant in BRCA1/2. Notably, 25% of women with a
pathogenic variant reported no family history of breast or
ovarian cancer. Conclusion: Despite substantial improvements in referral
rates,
many
eligible
women
are
not
receiving
GC. Considering the high rate of pathogenic variants, therapeu-
tic implications, and the value of identifying high-risk
families, it is imperative that all women with SOC are
offered genetic testing, irrespective of family history or age
at diagnosis. Our study demonstrates that even simple
interventions can address barriers to GC. SR assays allow for screening of a wide range of targets
for immediate and broad use. LR assays enable compre-
hensive sequencing of entire gene coding regions and
neighboring introns, gaining additional insight into variants
in difficult-to-sequence regions, large insertion/deletions,
and structural variations. R. Demsky: None. S. Randall
Armel:
None. J. McCuaig: None. N. Maani: None. R.H. Kim: None. A. Volenik: None. R. Demsky: None. S. Randall
Armel:
None. J. McCuaig: None. N. Maani: None. R.H. Kim: None. A. Volenik: None. C.A. Schumacher: A. Employment (full or part-time);
Significant; Swift Biosciences. A.M. Wood: A. Employ-
ment (full or part-time); Significant; Swift Biosciences. S. Sandhu: A. Employment (full or part-time); Significant;
Swift Biosciences. J. Lenhart: A. Employment (full or
part-time); Significant; Swift Biosciences. L. Kurihara: A. Employment (full or part-time); Significant; Swift Bios-
ciences. V. Makarov: A. Employment (full or part-time);
Significant; Swift Biosciences. C.A. Schumacher: A. Employment (full or part-time);
Significant; Swift Biosciences. A.M. Wood: A. Employ-
ment (full or part-time); Significant; Swift Biosciences. S. Sandhu: A. Employment (full or part-time); Significant;
Swift Biosciences. J. Lenhart: A. Employment (full or
part-time); Significant; Swift Biosciences. L. Kurihara: A. Employment (full or part-time); Significant; Swift Bios-
ciences. V. Makarov: A. Employment (full or part-time);
Significant; Swift Biosciences. Still not 100%: A 15 year evaluation of genetic counselling
rates for serous ovarian cancer at a single Canadian cancer
centre Still not 100%: A 15 year evaluation of genetic counselling
rates for serous ovarian cancer at a single Canadian cancer
centre Introduction: Male breast cancer (MBC) accounts for <1%
of breast cancers cases, with an incidence of ~230/year in
Canada. The limited data available on MBC suggests that
~18% are hereditary, that most are diagnosed with advanced
(stage 3-4) disease, and are typically ER/PR+, Her2-. No
studies have examined Canadian MBC characteristics. A
better understanding of the genetic and pathologic profiles
of MBC would improve risk assessment and genetic
counselling. Introduction: Male breast cancer (MBC) accounts for <1%
of breast cancers cases, with an incidence of ~230/year in
Canada. The limited data available on MBC suggests that
~18% are hereditary, that most are diagnosed with advanced
(stage 3-4) disease, and are typically ER/PR+, Her2-. No
studies have examined Canadian MBC characteristics. A
better understanding of the genetic and pathologic profiles
of MBC would improve risk assessment and genetic
counselling. R. Demsky, S. Randall Armel, J. McCuaig, N. Maani, R. H. Kim,
A. Volenik Features of male breast cancer: Review from a single
Canadian hereditary cancer clinic Features of male breast cancer: Review from a single
Canadian hereditary cancer clinic S. Randall Armel1, J. McCuaig1, R. Demsky1, S. Neil2, R. H. Kim1, A. Volenik1 S. Randall Armel1, J. McCuaig1, R. Demsky1, S. Neil2, R. H. Kim1, A. Volenik1 1Princess Margaret Cancer Centre, Toronto, ON, Canada,
2University of Toronto, Toronto, ON, Canada Princess Margaret Cancer Centre, Toronto, ON, Canada Introduction: An estimated 15-20% of invasive serous
ovarian cancers (SOC) are related to pathogenic variants in
BRCA1/2. Guidelines recommend that all women with SOC
are offered genetic counselling (GC) irrespective of family
history or age at diagnosis. Despite this, most centers report
suboptimal referral rates. Prior to 2009, only 23% of women
with SOC were seen for GC at Princess Margaret Cancer
Center (PM) in Toronto, Canada. While some centres have
approached this issue with automatic referrals or oncologist- Methods: A retrospective chart review examined the
characteristics of males diagnosed with MBC between 2000
and 2017, and who received genetic testing at Princess
Margaret Cancer Centre. Methods: A retrospective chart review examined the
characteristics of males diagnosed with MBC between 2000
and 2017, and who received genetic testing at Princess
Margaret Cancer Centre. Results: A total of 29 men were identified; 20 received
BRCA1/2 genetic testing and 9 received multi-gene panel
testing (MGPT). No pathogenic variants were identified in J. del Picchia 402 BRCA1/2; however, 1 CHEK2 pathogenic variant was
detected. The average age of diagnosis was 64.1 years. The
majority of men were diagnosed with invasive ductal
carcinoma (86%), of which 55% were stage 1. 100% of
cancers were ER+, 83% PR+, and 96% Her2-. 62% of men
reported a family history of breast and/or ovarian cancer,
21% a second non-breast primary, and 7% a second breast
primary. mutation sites which were proximal to apoptotic domains of
ankyrin and BRCT repeats respectively. For BRCA2
variants, homologous recombination was impaired for
BRCA2 c.9154C>T but not BRCA2 c.440A>G. To facilitate in reclassification of variants, RAD51
nuclear localization assay can be performed on homologous
recombination VUS. For BARD1, the right assay should be
chosen based on the affected functional domain - DNA
repair or apoptosis. Inappropriate use of the assays may
misinform the variant’s pathogenic status. Conclusions: Our data supports the current literature that
most MBC are ER/PR+ and HER2-. In contrast, our study
found an earlier stage of cancer and a relatively low
mutation rate. The absence of BRCA1/2 mutations in this
unselected Canadian cohort of MBC demonstrates the role
of MGPT to increase the diagnostic yield of genetic testing. S. Randall Armel: None. J. McCuaig: None. R. Demsky: None. S. Neil: None. R.H. Kim: None. A. Volenik: None. M. Toh: None. S. Chan: None. N. Ishak: None. S. Chong: None. J. Ngeow: None. Germline loss-of-function variants in the BARD1 gene are
associated with familial breast cancer Germline loss-of-function variants in the BARD1 gene are
associated with familial breast cancer N. Weber-Lassalle1, K. Weber-Lassalle1, J. Borde1,
G. Neidhardt1, C. Ernst1, B. Blümcke1, K. Klonowska2, A. E. Volk3, C. Kubisch3, R. Baber4, C. Engel4, P. Kozlowski2,
E. Hahnen1, R. K. Schmutzler1, J. Hauke1 N. Weber-Lassalle1, K. Weber-Lassalle1, J. Borde1, M. Toh1, S. Chan2, N. Ishak2, S. Chong2, J. Ngeow2,1 M. Toh1, S. Chan2, N. Ishak2, S. Chong2, J. Ngeow2,1 M. Toh1, S. Chan2, N. Ishak2, S. Chong2, J. Ngeow2,1 1Duke-NUS Medical School, Singapore, Singapore, 2National
Cancer Centre, Singapore, Singapore With the advent of personalized medicine, genetic testing
has become increasingly prevalent. Detection of pathogenic
variants facilitates enhanced screening measures, early
intervention and modified treatments. Unfortunately, a sig-
nificant portion of the genetic testing results are the non-
actionable variants of unknown significance (VUS). Func-
tional studies specific to the altered functional domains are
needed to ascertain their pathogenicity. Here, we function-
ally assessed the pathogenicity of BARD1 and BRCA2
clinical VUS identified among young colorectal cancer
patients. Introduction: Recent studies revealed a weak association
of BARD1 germline loss-of-function (LoF) variants with
breast cancer (BC). We determined the BARD1 mutation
prevalence
in
n = 3,348
well-characterized
BC
index
patients of the German descent and geographically-matched
female control individuals (GMCs; n = 2,196). Introduction: Recent studies revealed a weak association
of BARD1 germline loss-of-function (LoF) variants with
breast cancer (BC). We determined the BARD1 mutation
prevalence
in
n = 3,348
well-characterized
BC
index
patients of the German descent and geographically-matched
female control individuals (GMCs; n = 2,196). Introduction: Recent studies revealed a weak association
of BARD1 germline loss-of-function (LoF) variants with
breast cancer (BC). We determined the BARD1 mutation
prevalence
in
n = 3,348
well-characterized
BC
index
patients of the German descent and geographically-matched
female control individuals (GMCs; n = 2,196). Methods: Female BC index patients and GMCs were
screened for LoF variants in the BARD1 gene by next
generation sequencing. All patients met the inclusion
criteria of the German Consortium for Hereditary Breast
and Ovarian Cancer for germline testing. The SOPHiA
DDM® platform (SOPHiA GENETICS®) was applied for
the detection of copy number variations (CNVs) in a subset
of 2,810 BC patients. BARD1 and BRCA2 harbor domains for homologous
recombination
via
RAD51
nuclear
localization
while
BARD1 has additional apoptotic domains. RAD51 nuclear
localization was assessed using nuclear-cytoplasmic frac-
tions of patient-derived lymphoblastoid cells upon induc-
tion of DNA breaks. Apoptotic function was determined by
immunoblotting of activated p53 and downstream marker,
activated caspase-3. BARD1 and BRCA2 harbor domains for homologous
recombination
via
RAD51
nuclear
localization
while
BARD1 has additional apoptotic domains. RAD51 nuclear
localization was assessed using nuclear-cytoplasmic frac-
tions of patient-derived lymphoblastoid cells upon induc-
tion of DNA breaks. Functional evaluation of variants of unknown significance
in the homologous recombination genes BARD1 and
BRCA2 Functional evaluation of variants of unknown significance
in the homologous recombination genes BARD1 and
BRCA2 1Center for Familial Breast and Ovarian Cancer, University
Hospital Cologne, Cologne, Germany, 2Department of
Molecular Genetics, Institute of Bioorganic Chemistry, Polish
Academy of Sciences, Poznan, Poland, 3Institute of Human
Genetics, Medical Center Hamburg-Eppendorf, Hamburg,
Germany, 4LIFE-Leipzig Research Center for Civilization
Diseases, University of Leipzig, Leipzig, Germany M. Toh1, S. Chan2, N. Ishak2, S. Chong2, J. Ngeow2,1
1Duke-NUS Medical School, Singapore, Singapore, 2National
Cancer Centre, Singapore, Singapore M. Toh1, S. Chan2, N. Ishak2, S. Chong2, J. Ngeow2,1 Apoptotic function was determined by
immunoblotting of activated p53 and downstream marker,
activated caspase-3. Results: We identified 14 LoF variants (excluding CNVs)
in 3,348 female BC index patients (carrier frequency
[CF]:0.42%) compared with 36 in a total of 36,694 controls
(CF:0.10%, OR=4.276, 95%CI=2.30-7.94, p = 0.00004). The CF in each control dataset was 0.07% (GMCs;
n = 2,196), 0.11% (FLOSSIES; n = 7,325) and 0.10%
(ExAC; n = 27,173), respectively. The BARD1 p.Gln564*
variant was found in 6/14 patients. CNVs affecting the
BARD1 gene were identified in 3/2,810 BC index patients Two BARD1 and one BRCA2 VUS were tested. DNA
repair was unaffected in BARD1 variants, evident from the
normal
RAD51
nuclear
localization
and
previous
homology-directed repair studies. However, their apoptotic
function was impaired with failure of caspase-3 activation
upon induction of apoptosis. This correlated well with their Abstracts from the 51st European Society of Human Genetics Conference: Posters 403 (CNV CF=0.11%), which is higher than that observed in
the FLOSSIES database (2/7,325; CF=0.03%). For the 17
BARD1 mutation carriers, the mean age of first BC
diagnosis was 46 years (range 24-60) compared with 47
years (range 17-92) in the overall sample (n = 3,348). Karamzade: None. M. Saberi: None. N. Nourpour:
None. R. Behdad: None. (CNV CF=0.11%), which is higher than that observed in
the FLOSSIES database (2/7,325; CF=0.03%). For the 17
BARD1 mutation carriers, the mean age of first BC
diagnosis was 46 years (range 24-60) compared with 47
years (range 17-92) in the overall sample (n = 3,348). 1Center for Hereditary Breast and Ovarian Cancer, Cologne,
Germany, 2Genomic Vision, Paris, France 1Center for Hereditary Breast and Ovarian Cancer, Cologne,
Germany, 2Genomic Vision, Paris, France Approximately 24% of breast (BC) and/or ovarian cancer
(OC) cases who met the inclusion criteria of the German
Consortium for Hereditary Breast and Ovarian Cancer (GC-
HBOC) for germline testing are caused by pathogenic
mutations in the BRCA1 and BRCA2 gene. About 1% of
patients carry deleterious CNVs (copy number variations) in
BRCA1 or BRCA2. According to the guidelines of the GC-
HBOC, confirmed CNVs of every size are estimated as
pathogenic, if they result in a frameshift and thereby disrupt
functional protein domains. While the pathogenicity of
deletions is less complicated to determine, the situation for
duplications is much more complex, because duplications
can appear either intragenic or extragenic. Here, we present
a case (BC at age 26) carrying a complex duplication in
BRCA1. Initial MLPA analysis showed a duplication of
exon 1-8 and 11-12 of BRCA1. RNA and molecular
combing experiments could not confirm an intragenic
duplication but gave evidence for a translocation of the
duplicated sequence. The patient is carrying a pathogenic
mutation in PALB2 as well (c.654del, p.(Asp219Thrfs*4)),
which is most probably the cause of the BC disease. This is
also supported by the fact that her healthy mother (57)
carries only the BRCA1 variant and not the PALB2 muta-
tion. The majority of duplications appear in tandem, but this
case shows the great importance of verifying this assump-
tion. When the duplicated fragment is translocated else-
where in the genome, the mutation is most likely not
pathogenic, which has a great impact on clinical con-
sequences and predictive testing of relatives. P12.032D Experience learned from BRCA1 and BRCA2 screening in
Iranian women Experience learned from BRCA1 and BRCA2 screening in
Iranian women M. Keramatipour, Z. Golchehre, A. Nasrollahzadeh,
M. Arabpour, A. Karamzade, M. Saberi, N. Nourpour,
R. Behdad M. Keramatipour, Z. Golchehre, A. Nasrollahzadeh,
M. Arabpour, A. Karamzade, M. Saberi, N. Nourpour,
R. Behdad Tehran University of Medical Sciences, Department of Medical
Genetics, Tehran, Iran, Islamic Republic of Tehran University of Medical Sciences, Department of Medical
Genetics, Tehran, Iran, Islamic Republic of Breast cancer is the most common malignancy and second
leading cause of cancer-related death among women. 5-10%
of breast cancer cases are hereditary and several associated
loci have been identified. BRCA1 and BRCA2 genes are the
most important genes involved in breast cancer. Here we
investigated BRCA1 and BRCA2 genes in 240 women with
clinical presentations of breast cancer (n = 141) or asymp-
tomatic females with positive family history of this cancer
(n = 99). All exons plus flanking regions were enriched and
sequencing was performed by Illumina platform. Classifi-
cation of detected variants was done based on ACMG
guideline for variant interpretation 2015. 52 samples out of
240 samples (21.6%) have at least one VUS (Variant of
Uncertain Significance), likely pathogenic, or pathogenic
variant. Among these 52 variants, 13 were pathogenic, 9
were likely pathogenic and 30 variants were VUS. 23 var-
iants were found in BRCA1 and 29 in BRCA2 gene. 36 of 52
variants were detected in symptomatic and 16 variants in
asymptomatic women. Among 52 detected variants, 29
were missense, 16 frameshift, 4 nonsense, and 3 splicing
variants. Full data and analysis will be presented in the
meeting. M. Larsen: None. E. Pohl: None. C. Ernst: None. K. Keupp: None. S. Reichstein-Gnielinski: None. Y. Delpu:
None. A. Petit: None. J. Bertho: None. B. Wappensch-
midt: None. E. Hahnen: None. R. Schmutzler: None. P12.033A
Importance of verifying the location of duplicated
sequences - example of a benign complex duplication in the
BRCA1 gene in a breast cancer patient Conclusion: We observed a significant association of
deleterious BARD1 variants with the BC phenotype and
confirm BARD1 as a moderately-penetrant risk gene. The
inclusion of CNVs is crucial for a comprehensive genetic
screening of BC patients. M. Larsen1, E. Pohl1, C. Ernst1, K. Keupp1, S. Reichstein-
Gnielinski1, Y. Delpu2, A. Petit2, J. Bertho2, B. Wappenschmidt1,
E. Hahnen1, R. Schmutzler1 N. Weber-Lassalle: None. K. Weber-Lassalle: None. J. Borde: None. G. Neidhardt: None. C. Ernst: None. B. Blümcke: None. K. Klonowska: None. A.E. Volk: None. C. Kubisch: None. R. Baber: None. C. Engel: None. P. Kozlowski: None. E. Hahnen: None. R.K. Schmutzler:
None. J. Hauke: None. 1Center for Hereditary Breast and Ovarian Cancer, Cologne,
Germany, 2Genomic Vision, Paris, France BRCA1 and BRCA2 mutational spectrum in breast cancer
patients from the Republic of Macedonia P12.034B BRCA1 and BRCA2 mutational spectrum in breast cancer
patients from the Republic of Macedonia M. Keramatipour: None. Z. Golchehre: None. A. Nasrollahzadeh:
None. M. Arabpour:
None. A. BRCA1 and BRCA2 mutational spectrum in breast cancer
patients from the Republic of Macedonia M. Keramatipour: None. Z. Golchehre: None. A. Nasrollahzadeh:
None. M. Arabpour:
None. A. 404 J. del Picchia L. Lepkes, B. Blümcke, M. Larsen, A. Baasner, B. Versmold,
J. Driesen, K. Keupp, C. Ernst, G. Neidhardt, J. Hauke,
B. Wappenschmidt, E. Hahnen, R. Schmutzler 1Research Centre for Genetic Engineering and Biotechnology
“Georgi D. Efremov”, Skopje, Macedonia, The Former
Yugoslav Republic of, 2Clinical Hospital Acibadem Sistina,
Skopje, Macedonia, The Former Yugoslav Republic of, 3Re-
Medika General Hospital, Skopje, Macedonia, The Former
Yugoslav Republic of, 4University Clinic of Radiotherapy and
Oncology, Medical Faculty, University “Ss Cyril and
Methodius”, Skopje, Macedonia, The Former Yugoslav
Republic of, 5University Clinic of Radiology, Medical Faculty,
University “Ss Cyril and Methodius”, Skopje, Macedonia, The
Former Yugoslav Republic of L. Lepkes, B. Blümcke, M. Larsen, A. Baasner, B. Versmold,
J. Driesen, K. Keupp, C. Ernst, G. Neidhardt, J. Hauke,
B. Wappenschmidt, E. Hahnen, R. Schmutzler Center for Familial Breast and Ovarian Cancer, Cologne,
Germany Blümcke: None. M. Larsen: None. A. Baasner: None. B. Versmold: None. J. Driesen: None. K. Keupp: None. C. Ernst: None. G. Neidhardt: None. J. Hauke: None. B. Wappenschmidt: None. E. Hahnen:
None. R. Schmutzler: None. R. M. Rodrigues Peres1, M. Anurag2, J. T. Lei2, M. J. Ellis2,
L. O. Z. Sarian1 Center for Familial Breast and Ovarian Cancer, Cologne,
Germany Introduction: Next Generation Sequencing (NGS)-based
detection of copy number variations (CNVs) is widely used
in a routine diagnostic setting. The prevalence of CNVs in
BRCA1/2 and other risk genes in patients with familial
breast cancer (BC) or ovarian cancer (OC) remains elusive. Introduction: Next Generation Sequencing (NGS)-based
detection of copy number variations (CNVs) is widely used
in a routine diagnostic setting. The prevalence of CNVs in
BRCA1/2 and other risk genes in patients with familial
breast cancer (BC) or ovarian cancer (OC) remains elusive. Identification of the spectrum of mutations in BRCA1 and
BRCA2 genes in specific populations allows for imple-
mentation of a cost-effective genetic screening. Thus far,
the BRCA1/2 mutations present among the BC patients from
the Republic of Macedonia have been largely unknown. Therefore, we used targeted next-generation sequencing,
Sanger DNA sequencing and multiplex ligation probe
amplification analysis to search for point mutations and
deletions/duplications involving BRCA1 and BRCA2-cod-
ing regions. We have analyzed a total of 337 BC patients,
enriched for family history of cancer, early age of onset and
bilateral and/or triple negative BC. A total of 28 pathogenic
mutations were observed in 55 unrelated BC patients (55/
337, 16.3%) with the predominance of BRCA2 mutations
(29/55, 52.7%). Nine novel mutations were identified; four
in BRCA1 and five in BRCA2. The most prevalent mutations
were c.181T>G, c.1102G>T, c.3700_3704del5, c.5212G>A
and
c.5266dupC
in
BRCA1
and
c.5722_5723delCT,
c.5851_5854delAGTT, c.7879A>T and c.8317_8330del14
in
BRCA2
gene. Thus
far,
BRCA2
c.7879A>T
and
c.8317_8330del14 mutations have been described in several
isolated cases however, our study is the first one showing
that they have a founder effect among Macedonian popu-
lation. Nine recurrent mutations account for 63.6% of all of
the detected mutations allowing for implementation of a fast
first-step BRCA1/2 mutational screening strategy. In con-
clusion, this study provides a comprehensive view of
known and novel BRCA1/2 mutations in BC patients from
the Republic of Macedonia and contributes to the global
spectrum of BRCA1/2 mutations in BC. Identification of the spectrum of mutations in BRCA1 and
BRCA2 genes in specific populations allows for imple-
mentation of a cost-effective genetic screening. Thus far,
the BRCA1/2 mutations present among the BC patients from
the Republic of Macedonia have been largely unknown. Therefore, we used targeted next-generation sequencing,
Sanger DNA sequencing and multiplex ligation probe
amplification analysis to search for point mutations and
deletions/duplications involving BRCA1 and BRCA2-cod-
ing regions. P12.035C M. Jakimovska1, I. Maleva Kostovska1, K. Popovska-Jankovic1,
K. Kubelka-Sabit2, M. Karadjozov2, L. Stojanovska3,
A. Arsovski3, S. Smichkoska4, E. Lazarova4, M. Jakimovska
Dimitrovska5, D. Plaseska-Karanfilska1 Center for Familial Breast and Ovarian Cancer, Cologne,
Germany We have analyzed a total of 337 BC patients,
enriched for family history of cancer, early age of onset and
bilateral and/or triple negative BC. A total of 28 pathogenic
mutations were observed in 55 unrelated BC patients (55/
337, 16.3%) with the predominance of BRCA2 mutations
(29/55, 52.7%). Nine novel mutations were identified; four
in BRCA1 and five in BRCA2. The most prevalent mutations
were c.181T>G, c.1102G>T, c.3700_3704del5, c.5212G>A
and
c.5266dupC
in
BRCA1
and
c.5722_5723delCT,
c.5851_5854delAGTT, c.7879A>T and c.8317_8330del14
in
BRCA2
gene. Thus
far,
BRCA2
c.7879A>T
and
c.8317_8330del14 mutations have been described in several
isolated cases however, our study is the first one showing
that they have a founder effect among Macedonian popu-
lation. Nine recurrent mutations account for 63.6% of all of
the detected mutations allowing for implementation of a fast
first-step BRCA1/2 mutational screening strategy. In con-
clusion, this study provides a comprehensive view of
known and novel BRCA1/2 mutations in BC patients from
the Republic of Macedonia and contributes to the global
spectrum of BRCA1/2 mutations in BC. Methods: We used the TruRisk® gene panel (Agilent
SureSelect) which covers 34 cancer risk genes/candidate
risk genes for BC/OC. The Sophia Genetics DDM platform
was employed to predict CNVs. conspicuous findings were
verified by MLPA. Results: A cohort of 4,418 index patients (3,846 BC, 445
OC, and 127 BC/OC patients) was included. All cases met
the inclusion criteria of the German Consortium Hereditary
Breast- and Ovarian cancer for germline testing. Most
CNVs predicted by DDM platform were detected in the
BRCA1, BRCA2 and CHEK2 genes. CNVs were also
observed in further (candidate) cancer predisposition genes
ATM, BARD1, FANCA, MLH1, MSH2, PALB2, PMS2,
RAD50, RAD51C, RAD51D, TP53, but not in MSH6,
CDH1, NBN, FANCM, PTEN, STK11. For BRCA1, 43
CNVs were predicted in 4,418 patients, of which 40 (93%)
could be confirmed by MLPA. For BRCA2, 9 CNVs were
predicted, of which 5 could be confirmed (56%). In total,
about 1% of all index patients carried CNVs affecting the
BRCA1/2 genes. Conclusions: In summary, bioinformatic analysis of NGS
gene panel data detects CNVs. However, it remains to be
determined whether sensitivity/specificity of in silico CNV
detection meets diagnostic criteria. L. Lepkes: None. B. Blümcke: None. M. Larsen: None. A. Baasner: None. B. Versmold: None. J. Driesen: None. L. Lepkes: None. B. Blümcke: None. M. Larsen: None. A. Baasner: None. B. Versmold: None. J. Driesen: None. L. Lepkes: None. B. P12.036D M. Jakimovska: None. I. Maleva Kostovska: None. K. Popovska-Jankovic: None. K. Kubelka-Sabit: None. M. Karadjozov: None. L. Stojanovska: None. A. Arsovski:
None. S. Smichkoska: None. E. Lazarova: None. M. Jakimovska
Dimitrovska:
None. D. Plaseska-
Karanfilska: None. M. Jakimovska: None. I. Maleva Kostovska: None. K. Popovska-Jankovic: None. K. Kubelka-Sabit: None. M. Karadjozov: None. L. Stojanovska: None. A. Arsovski:
None. S. Smichkoska: None. E. Lazarova: None. M. Jakimovska
Dimitrovska:
None. D. Plaseska-
Karanfilska: None. Identification of differential genes in luminal breast tumors
by comparison of Brazilian and American population data Identification of differential genes in luminal breast tumors
by comparison of Brazilian and American population data R. M. Rodrigues Peres1, M. Anurag2, J. T. Lei2, M. J. Ellis2,
L. O. Z. Sarian1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 405 1UNICAMP, Campinas, Brazil, 2Baylor College of Medicine,
Houston, TX, United States Maggiore Policlinico, Milano, Italy, 3Unit of Bioinformatics
and Biostatistics, Department of Applied Research and
Technological Development, Fondazione IRCCS Istituto
Nazionale dei Tumori, Milano, Italy, 4Immunohematology &
Transfusion Medicine Service, Fondazione IRCCS Istituto
Nazionale Tumori, Milano, Italy, 5Medical Genetics,
Department of Health Sciences, Università degli Studi di
Milano, Milano, Italy, 6Department of Pathology and
Laboratory Medicine, Fondazione IRCCS Istituto Nazionale
dei Tumori, Milano, Italy Introduction: Luminal breast tumors are the most common
type of diagnosed breast cancers, having worse prognosis in
the long-term. Chemotherapy is less effective in this group,
which makes research in this field necessary. The objective
of this study was to compare data from Brazilian and
American populations of luminal breast carcinomas to
evaluate the differences in the amplified or deleted genes
between these sets of samples. Introduction: Early age at onset of breast cancer (eoBC) is
considered to be suggestive of an increased genetic risk. Although genetic testing of known high penetrance genes is
offered to all eoBC-affected women, in the absence of a
positive family history the detection rate of pathogenic
variants is <10%. This study aimed at assessing the role of
constitutive promoter methylation at BC-associated loci as
an underlying predisposing event in women with eoBC and
negative family history. Subjects and Methods: 49 samples of luminal breast
tumors from Brazilian women were selected by histopatho-
logical characterization of biopsy specimens. We performed
the chromosome microarray (CMA) technique to assess
Copy Number Variations (CNVs) and indels from these
tumors. Raw data was segmented by PSCBS followed by
normalization. P12.036D Conclusions: In isolated eoBC patients, BRCA1 consti-
tutive promoter methylation appears to be an underlying
predisposing event. Further studies are required to define
the impact of slight methylation changes involving other
BC-predisposing genes. Results: Only one patient, with mean methylation value of
26%, exceeds the threshold by 0.15 at the BRCA1 promoter. Interestingly, analyses on FFPE BC from the patient reveal a
mean BRCA1 methylation of 60-70% and the loss of the
unmethylated allele. Another patient, with mean methylation
level of 21%, exceeds the threshold by 0.05 at the RAD51C
promoter, though its biological significance is yet to be defined. Financial Support: Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Brazil. R.M. Rodrigues Peres: None. M. Anurag: None. J.T. Lei: None. M.J. Ellis: None. L.O.Z. Sarian: None. Conclusions: In isolated eoBC patients, BRCA1 consti-
tutive promoter methylation appears to be an underlying
predisposing event. Further studies are required to define
the impact of slight methylation changes involving other
BC-predisposing genes. P12.038B P12.038B
Constitutive promoter methylation analysis of breast
cancer associated genes in women with isolated early onset
breast cancer J. Azzollini1, C. Pesenti2, S. Pizzamiglio3, L. Fontana2,
C. Guarino4, B. Peissel1, M. Plebani3, S. Tabano2, S. Sirchia5,
B. Paolini6, P. Verderio3, M. Miozzo2, S. Manoukian1 J. Azzollini1, C. Pesenti2, S. Pizzamiglio3, L. Fontana2,
C. Guarino4, B. Peissel1, M. Plebani3, S. Tabano2, S. Sirchia5,
B. Paolini6, P. Verderio3, M. Miozzo2, S. Manoukian1 J. Azzollini1, C. Pesenti2, S. Pizzamiglio3, L. Fontana2, None. M. Plebani: None. S. Tabano: None. S. Sirchia: C. Guarino4, B. Peissel1, M. Plebani3, S. Tabano2, S. Sirchia5,
6
3
2
1 None. B. Paolini: None. P. Verderio: None. M. Miozzo: None. B. Paolini: None. P. Verderio: None. M. Miozzo:
None. S. Manoukian: None. C. Guarino4, B. Peissel1, M. Plebani3, S. Tabano2, S. Sirchia5,
B. Paolini6, P. Verderio3, M. Miozzo2, S. Manoukian1 1Unit of Medical Genetics, Department of Medical Oncology
and Hematology, Fondazione IRCCS Istituto Nazionale dei
Tumori, Milano, Italy, 2Department of Pathophysiology &
Transplantation, Università degli Studi di Milano; Division of
Pathology, Fondazione IRCCS Ca' Granda Ospedale 1Unit of Medical Genetics, Department of Medical Oncology
and Hematology, Fondazione IRCCS Istituto Nazionale dei
Tumori, Milano, Italy, 2Department of Pathophysiology &
Transplantation, Università degli Studi di Milano; Division of
Pathology, Fondazione IRCCS Ca' Granda Ospedale P12.036D Copy number aberration (CNA) analysis was
performed using GISTIC2. The Brazilian genes data found
to be significantly amplified or deleted were compared to
TCGA data obtained for American samples. Materials and methods: Promoter methylation at 12 loci
(BRCA1, BRCA2, ATM, CDH1, FANCM, STK11, NBN,
PALB2, PTEN, RAD51C, RECQL and TP53) was assessed
by the EpiTYPER-MassARRAY assay in blood from 110
BRCA1/2 negative patients with eoBC and negative family
history, and 60 healthy donors (controls). Hypermethylation
was determined, within each promoter, by comparing the
patient’s mean methylation value with thresholds based on
the upper limit of the 95% bootstrap confidence interval of
the controls’ mean. Materials and methods: Promoter methylation at 12 loci
(BRCA1, BRCA2, ATM, CDH1, FANCM, STK11, NBN,
PALB2, PTEN, RAD51C, RECQL and TP53) was assessed
by the EpiTYPER-MassARRAY assay in blood from 110
BRCA1/2 negative patients with eoBC and negative family
history, and 60 healthy donors (controls). Hypermethylation
was determined, within each promoter, by comparing the
patient’s mean methylation value with thresholds based on
the upper limit of the 95% bootstrap confidence interval of
the controls’ mean. Results: We found significantly amplified or deleted
genes that belong mainly to the FGF and Wnt signaling
pathways exclusively in the cohort of Brazilian breast
tumors. Conclusions: These data suggest that there are differ-
ences between Brazilian and American luminal breast
tumors at the genomic level, potentially affecting tumor
biology, influencing their prognosis and response to
treatment. This comparison should bring light to the
question of population differences in terms of genomics,
personal habits and the environment, having an effect on
tumor genetics leading to resistance to the available
treatments. Conclusions: These data suggest that there are differ-
ences between Brazilian and American luminal breast
tumors at the genomic level, potentially affecting tumor
biology, influencing their prognosis and response to
treatment. This comparison should bring light to the
question of population differences in terms of genomics,
personal habits and the environment, having an effect on
tumor genetics leading to resistance to the available
treatments. Results: Only one patient, with mean methylation value of
26%, exceeds the threshold by 0.15 at the BRCA1 promoter. Interestingly, analyses on FFPE BC from the patient reveal a
mean BRCA1 methylation of 60-70% and the loss of the
unmethylated allele. Another patient, with mean methylation
level of 21%, exceeds the threshold by 0.05 at the RAD51C
promoter, though its biological significance is yet to be defined. P12.039C Large case-control study and functional analyses show that
FANCM truncating mutations are associated with a breast
cancer risk magnitude that varies depending on their
location J. del Picchia 406 S. SENECA1,2, J. De Greve3,4, M. Bonduelle1, S. Joris3,
K. Keymolen1, M. De Rademaeker1, K. Stouffs1, A. Gheldof1,2
1UZ Brussel, Center for Medical Genetics, Brussels, Belgium,
2Neurogenetics Research Group, Reproduction Genetics and
Regenerative Medicine Research Group, Vrije Universiteit
Brussel, 1050, Belgium, 3UZ Brussel, Department of Oncology,
Brussels, Belgium, 4Vrije Universiteit Brussel, 1050, Belgium G. Figlioli1, M. Bogliolo2, L. Caleca3, BCAC collaborators,
P. Radice3, J. Surralles2, P. Peterlongo1 Logistic regression analyses indicated that the p.Arg658* is
a moderate risk factor for ER-negative and triple negative
BC (TNBC): OR=2.44 (1.12-5.34) and OR=3.79 (1.56-
9.18), respectively. Similar analyses showed that the p. Gln1701* and p.Arg1931* may confer a lower risk in
TNBC
[OR=2.15
(1.05-4.38)]
and
ER-negative
BC
[OR=1.98 (1.26-3.13)], respectively. Results: A retrospective data analysis of the fastq files for
the gene pool proposal was performed for 700 patient
samples previously analyzed. An additional 13 families
were identified, with the following diversification: 6 ATM, 3
BRIP1, 1 RAD51C, 1 RAD51D, 1 MLH1 and 1 MSH6 gene
families. These patients were all negative for class 4 and 5
core gene panel variants. Conclusion: Broadening of the present core gene panel,
as proposed, will result in the identification of only a limited
extra number (1.85%) of patients with germ line mutations
as the possible molecular cause of their cancer. However,
this molecular diagnosis can have major influences on the
treatment and follow-up of the index cases, while it also
offers
possibilities
for
predictive
testing
and
family
planning in at risk family members. We are now testing these and other two patient-derived
truncating
mutations
subjecting
transfected
human
FANCM-/- cells to DNA-ICL inducing agents to measure
survival rates and chromosome fragility. Consistently with
our genetic and clinical data, our initial functional results
support the hypothesis that upstream FANCM truncation
could be associated with a higher BC risk and more severe
clinical phenotypes. These results will allow a better BC
risk estimate in FANCM-mutation carriers from families and
general population. S. Seneca: None. J. De Greve: None. M. Bonduelle:
None. S. Joris: None. K. Keymolen: None. M. De
Rademaeker: None. K. Stouffs: None. A. Gheldof: None. G. Figlioli1, M. Bogliolo2, L. Caleca3, BCAC collaborators,
P. Radice3, J. Surralles2, P. Peterlongo1 S. SENECA1,2, J. De Greve3,4, M. Bonduelle1, S. Joris3,
K. Keymolen1, M. De Rademaeker1, K. Stouffs1, A. Gheldof1,2 1UZ Brussel, Center for Medical Genetics, Brussels, Belgium,
2Neurogenetics Research Group, Reproduction Genetics and
Regenerative Medicine Research Group, Vrije Universiteit
Brussel, 1050, Belgium, 3UZ Brussel, Department of Oncology,
Brussels, Belgium, 4Vrije Universiteit Brussel, 1050, Belgium 1Genome Diagnostics Program, IFOM, The FIRC Institute of
Molecular Oncology, Milan, Italy, 2Department of Genetics
and Microbiology, Genetics Department of Hospital de les
Santes Creus i Sant Pau, Universitat Autònoma de Barcelona,
and Centro de Investigación Biomédica en Red de
Enfermedades Raras (CIBERER), Barcelona, Spain, 3Unit of
Molecular Bases of Genetic Risk and Genetic Testing,
Department of Preventive and Predictive Medicine,
Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Introduction: The Belgium core gene panel for analysis of
Hereditary breast and ovarian cancer (HBOC) patients
consists of 4 genes BRCA1, BRCA2, PALB2, TP53, along
with the c.1100delC mutation in CHECK2 and inspection
for copy number variations in BRCA1/2. Patients are
selected according to national guidelines (www.BeSHG. be). Broadening the analysis with an identification of
mutations in the Lynch genes MLH1, MSH2 and MSH6, and
screening for truncating mutations in the ATM, BRIP1,
RAD51C/D genes has been proposed recently. Breast cancer (BC) is the most common female oncological
disease. About 50% of the familial cases are explained by
rare mutations in BRCA1 and BRCA2 and other high-risk
genes, by mutations in moderate-risk genes including
PALB2, ATM and CHEK2, and by common low-risk alleles. Previously, other and we showed that truncating mutations
within the FANCM gene are associated with BC risk, par-
ticularly with ER-negative subtype. Also, we recently
observed that upstream mutations cause more severe clin-
ical phenotypes than those located in the C-terminus. Methods: Mutation analysis was performed with the
BRCA Hereditary Cancer MASTR Plus kit (Multiplicom). Deletion/duplication analysis was performed with the
MLPA kits P002-D1 and P045-C1 (MRC Holland). The
presence of reported variants is confirmed with Sanger
sequencing. Methods: Mutation analysis was performed with the
BRCA Hereditary Cancer MASTR Plus kit (Multiplicom). Deletion/duplication analysis was performed with the
MLPA kits P002-D1 and P045-C1 (MRC Holland). The
presence of reported variants is confirmed with Sanger
sequencing. In this study, the three most common FANCM truncating
mutations p.Arg658*, p.Gln1701* and p.Arg1931* were
genotyped in 67,112 BC cases and 53,776 controls
collected within the Breast Cancer Association Consortium. P12.041A Funded by AIRC (IG-16732 to P.P.) and by a FUV
fellowship (to G.F.). Identification of ultra-rare loss of function variants in a
west of Ireland breast cancer population G. Figlioli: None. M. Bogliolo: None. L. Caleca: None. P. Radice: None. J. Surralles: None. P. Peterlongo: None. U. M. McVeigh1, T. P. McVeigh2, N. Miller1, D. W. Morris3,
M. J. Kerin1 U. M. McVeigh1, T. P. McVeigh2, N. Miller1, D. W. Morris3,
M. J. Kerin1 Germ line gene panel analysis in a HBOC population 1Discipline of Surgery, Lambe Institute for Translational
Research, NUIG, Galway, Ireland, 2Department of Clinical P12.040D Germ line gene panel analysis in a HBOC population 1Discipline of Surgery, Lambe Institute for Translational
Research, NUIG, Galway, Ireland, 2Department of Clinical Abstracts from the 51st European Society of Human Genetics Conference: Posters 407 Genetics, Our Lady’s Children’s Hospital, Crumlin, Dublin,
Ireland, 3Discipline of Biochemistry, NUIG, Galway, Ireland team with 128 breast cancer patients having the same 17-
gene panel. Our philosophy thusfar has been to initiate most
testing with BRCA 1-2, followed by reflex testing to a
panel; this approach will underestimate the number of
patients harboring more than one mutation. The finding of
multiple germline mutations in a few patients has led us to
reflect on the relative contribution of the mutations to their
clinical phenotypes and to risk assessment strategies for
their families. team with 128 breast cancer patients having the same 17-
gene panel. Our philosophy thusfar has been to initiate most
testing with BRCA 1-2, followed by reflex testing to a
panel; this approach will underestimate the number of
patients harboring more than one mutation. The finding of
multiple germline mutations in a few patients has led us to
reflect on the relative contribution of the mutations to their
clinical phenotypes and to risk assessment strategies for
their families. Breast cancer is the most common female cancer globally. Approximately 5-10% of breast cancers are due to inherited
variants in numerous breast cancer susceptibility genes, of
which 20-30% are in BRCA1/BRCA2. Variants in other
genes demonstrate reduced penetrance. The genetic het-
erogeneity of breast cancer means that next-generation
sequencing(NGS) using multi-gene panels is a cost- and
time-efficient way to identify causative germline variants. We aimed to use NGS to identify pathogenic variants
contributing to breast cancer susceptibility in an Irish
population. A multi-gene panel was designed on a Roche-
Nimblegen platform. Sequencing was performed on an
Illumina NextSeq. GATK best practices (2016) were fol-
lowed for bioinformatics analysis. Samples were confirmed
to be unrelated using Plink1.9; population stratification
using 1000 Genomes data confirmed ethnicity. Variants in
90 patients with breast cancer and 68 unaffected controls
were annotated using Snpeff, VEP, and Annovar. Con-
flicting annotation was clarified by manual interpretation via
UCSC genome browser. One loss-of-function(LOF) variant
was identified in BRCA1. P12.040D Ultra-rare variants (MAF ≤1.5x10-
5) were identified in genes frequently appearing on breast
cancer risk panels(n = 3); genes implicated in breast cancer
susceptibility in GWAS (n = 2); genes reported to be
somatically mutated/hypermethylated in breast cancers (n
= 2). Pathogenic LOF variants were identified in nine other
genes including RECQL4, OBSCN, TTN, NOTCH3. Our
results show that NGS increases diagnostic yield, but also
increases the yield of variants in genes with weak/uncon-
firmed association with breast cancer. Further analysis is
required to determine if these variants are incidental find-
ings, or causal variants. Results: Two patients had BRCA mutations only (panel
testing done because of family history). Six patients had
mutations in other panel genes: ATM, BRIP1, PALB2,
CHEK2. 18% of patients had a VUS in one or two genes. Two
young
patients
with
metastatic
invasive
ductal
carcinoma at diagnosis (grade 3, ER/PR-positive, Her-2
negative), neither of whom had a close family history of
cancer, had multiple mutated genes. Patient #1, age 37, had
a synchronous renal cell carcinoma. She had germline
mutations in BARD-1, CHEK-2, and PALB2. Patient #2,
age 23, has Neurofibromatosis type I, without prior
significant medical complications. She was found to have
mutations in both the NF-1 and MUTYH genes. As
literature on such cases is scant, multicenter reporting is
needed to develop management strategies for such patients. Results: Two patients had BRCA mutations only (panel
testing done because of family history). Six patients had
mutations in other panel genes: ATM, BRIP1, PALB2,
CHEK2. 18% of patients had a VUS in one or two genes. Two
young
patients
with
metastatic
invasive
ductal
carcinoma at diagnosis (grade 3, ER/PR-positive, Her-2
negative), neither of whom had a close family history of
cancer, had multiple mutated genes. Patient #1, age 37, had
a synchronous renal cell carcinoma. She had germline
mutations in BARD-1, CHEK-2, and PALB2. Patient #2,
age 23, has Neurofibromatosis type I, without prior
significant medical complications. She was found to have
mutations in both the NF-1 and MUTYH genes. As
literature on such cases is scant, multicenter reporting is
needed to develop management strategies for such patients. C.D. DeLozier: None. C. Stoehr: None. R. Kliewer:
None. I. Adelaja: None. P12.043C Hereditary breast cancer gene variants- multigene panel
testing outcome from Sri Lanka P. M. Padeniya1,2, M. Abayasekara2, C. Thanaseelan2,
V. Gnanam2 P. M. Padeniya1,2, M. Abayasekara2, C. Thanaseelan2,
V. Gnanam2 1Faculty of Medicine, Ragama, Sri Lanka, 2Credence
Genomics Private Limited, Colombo, Sri Lanka 1Faculty of Medicine, Ragama, Sri Lanka, 2Credence
Genomics Private Limited, Colombo, Sri Lanka U.M. McVeigh: None. T.P. McVeigh: None. N. Miller:
None. D.W. Morris: None. M.J. Kerin: None. Introduction: Globally one in eight women develops a
breast cancer (BC) in her lifetime. As per the national sta-
tistics, 38.7% Sri Lankan women aged 39-46 years account
for BC which is ranked number one among all cancers. About 5–10% of BCs are clustered in families owing to
germline
mutations. In
the
era
of
Next
Generation
Sequencing (NGS), a panel based genetic testing for her-
editary BC is feasible and cost effective. Multiple germline mutations in breast cancer patients-
useful? Multiple germline mutations in breast cancer patients-
useful? C. D. DeLozier1, C. Stoehr2, R. Kliewer1, I. Adelaja2 Digenic inheritance of RASSF1A and KLK3 mutations in an
Iranian multiple-case breast cancer family H. Radmanesh1,2, A. Sadr-Nabavi2, F. Homaei Shandiz2,
S. Ardalan Khales2, T. Park-Simon1, P. Hillemanns1, D. Liu1,
A. Riahi1,3, R. Geffers4, T. Dörk1 1Hannover Medical School, Hannover, Germany, 2Mashhad
University of Medical Sciences, Mashhad, Iran, Islamic
Republic of, 3Tunis Medical School, Tunis, Tunisia, 4Helmholtz
Center for Infection Research, Braunschweig, Germany Conclusions: Of the pathogenic variants detected, p. Leu28Argfs mutation has been previously reported in Asian
patients. There is no reliable data for the incidence of the
other mutations in the Asian population. P.M. Padeniya: None. M. Abayasekara: None. C. Thanaseelan: None. V. Gnanam: None. P.M. Padeniya: None. M. Abayasekara: None. C. Thanaseelan: None. V. Gnanam: None. Introduction: Much of the hereditary breast cancer risk in
families is still unexplained. Additional breast cancer sus-
ceptibility genes might be detectable via exome sequencing
in multiple-case breast cancer families. C. D. DeLozier1, C. Stoehr2, R. Kliewer1, I. Adelaja2 1Community Regional Medical Center, Fresno, CA, United
States, 2Central California Faculty Medical Group, Fresno,
CA, United States 1Community Regional Medical Center, Fresno, CA, United
States, 2Central California Faculty Medical Group, Fresno,
CA, United States Materials and Methodology: Credence Genomics pro-
vides NGS based screening for inherited predisposition to
BC. A multi gene panel consists of 18 genes, including
BRCA1, BRCA2, TP53, ATM, BARD1, BRIP1, CDH1,
CHEK2, MRE11A, MUTYH, NBN, NF1, PAL B2, PTEN,
RAD50, RAD51C, RAD51D and STK11 are incorporated in
the panel. A descriptive, retrospective study was carried out
from July 2014–December 2017 of all the patients referred The use of multigene “panels” has become common in
hereditary cancer testing, the result being more fortuitous
findings, variants of unknown significance (VUS) and “low
risk” mutations, which may not be clinically relevant. We
report here the experience of our multidisciplinary breast 408 J. del Picchia E.P. Silveira-lacerda: None. R.M. Goveia: None. B.S. Gamba: None. T.B. Texeira: None. C.E. Anunciação:
None. R. Freitas-junior: None. for
hereditary
BC
screening
at
Credence
Genomics
laboratory. Results: A total of 53 patients either who have been
affected or with a family history of BC were included to the
study. Pathogenic mutations were detected in 6(11%)
patients of which two were affected with BC and three
had a strong family history of BC. Four patients had BRCA1
gene variants(p.Arg1203Ter, p.Glu907Ter, p.Leu28Argfs
and
p.Glu23Valfs),
one
had
BRCA2
gene
variant(p. Phe12Leufs), one had TP53 gene variant(p.Arg248Gln). One patient had a benign deletion in BARD1 gene(p. Leu359_Pro365del). P12.044D Analysis of gene rearrangements in the BRCA1 and
BRCA2genes in patients with suspected hereditary breast
and ovarian cancer syndrome in the Brazil-central region Methods: To elucidate the molecular basis of breast
cancer in the Iranian population, we carried out exome
sequencing of genomic DNA from an Iranian patient with a
strong family history of breast cancer (4 first-degree family
members affected) but negative for BRCA1 and BRCA2
mutations. We then investigated specific mutations in
twelve other members of this family. Furthermore, we
directly genotyped specific mutations in a breast cancer
case-control series from Iran. Methods: To elucidate the molecular basis of breast
cancer in the Iranian population, we carried out exome
sequencing of genomic DNA from an Iranian patient with a
strong family history of breast cancer (4 first-degree family
members affected) but negative for BRCA1 and BRCA2
mutations. We then investigated specific mutations in
twelve other members of this family. Furthermore, we
directly genotyped specific mutations in a breast cancer
case-control series from Iran. E. P. SILVEIRA-LACERDA, R. M. GOVEIA, B. S. GAMBA,
T. B. TEXEIRA, C. E. ANUNCIAÇÃO, R. FREITAS-JUNIOR
FEDERAL UNIVERSITY OF GOIAS, GOIAS, Brazil E. P. SILVEIRA-LACERDA, R. M. GOVEIA, B. S. GAMBA, T. B. TEXEIRA, C. E. ANUNCIAÇÃO, R. FREITAS-JUNIOR P12.046B
Exome sequencing and case-control analyses identify RCC1
as a candidate breast cancer susceptibility gene 1UNIVERSITY OF BOLOGNA, BOLOGNA, Italy, 2Unit of
Medical Genetics, Policlinico S. Orsola-Malpighi, BOLOGNA,
Italy, 3Unit of Oncology, Policlinico S. Orsola-Malpighi,
BOLOGNA, Italy, 4Unit of Medical Genetics, Policlinico S. Orsola-Malpighi, Bologna, Italy A. Riahi1,2, H. Radmanesh1,3, P. Schürmann1, N. Bogdanova1,
R. Geffers4, R. Meddeb2, M. Kharrat2, T. Dörk1 1Hannover Medical School, Hannover, Germany, 2Tunis
Medical University, Tunis, Tunisia, 3Mashhad University of
Medical Sciences, Mashhad, Iran, Islamic Republic of,
4Helmholtz Center for Infection Research, Braunschweig,
Germany Introduction: The two major genes BRCA1 and BRCA2,
together with rare high-penetrance genes (p53, PTEN,
CDH1, STK11) and moderate-penetrance genes such as
PALB2 collectively explain ~30% of familial Breast Cancer
(BC) risk. Expanding our knowledge to additional genes is
crucial to extend the benefits of targeted surveillance/pre-
vention to a larger population of high-risk women. Introduction: Breast cancer is a genetic disease but the
known genes explain a minority of cases. RCC1, the Reg-
ulator of Chromosome Condensation 1, is important for
replication control and proper mitotic spindle formation but
has not previously been associated with hereditary cancer. Materials and methods: Whole Exome Sequencing
(WES) was performed on constitutional DNA with Nextera
coding exome library enrichment and annotated using an in-
house pipeline. Mutation screening was performed via
TruSeq
Custom
Amplicon
panel
and
VariantStudio. Expression studies were performed on MCF10A and
MCF7 breast cell lines. Methods: To elucidate the molecular basis of breast
cancer in the Tunisian population, we performed exome
sequencing
on
six
BRCA1/BRCA2
mutation-negative
patients with familial breast cancer. We then directly
genotyped a Tunisian breast cancer case-control series for
the identified RCC1 mutation, and further analysed tumors
from six mutation carriers for loss of heterozygosity. Methods: To elucidate the molecular basis of breast
cancer in the Tunisian population, we performed exome
sequencing
on
six
BRCA1/BRCA2
mutation-negative
patients with familial breast cancer. We then directly
genotyped a Tunisian breast cancer case-control series for
the identified RCC1 mutation, and further analysed tumors
from six mutation carriers for loss of heterozygosity. Results:
WES
on
first-degree
affected
cousin-pairs
identified detrimental variants in ROS1, RASAL1, POLN,
and NPL genes. Analysis with a target custom-made kit in
131 unrelated patients with familial and/or early onset BC
who had tested negative for BRCA1 and 2 revealed rare/
novel variants with a significant allele frequency difference
between cases and controls (p < 10-3), in particular in ROS1. P12.047C Identification of variants predisposing to breast cancer
through a WES approach Identification of variants predisposing to breast cancer
through a WES approach P12.046B
Exome sequencing and case-control analyses identify RCC1
as a candidate breast cancer susceptibility gene MCF10A cells expressed ROS1 mRNA, whereas MCF7
cells did not, suggesting that ROS1 absence may be
correlated to tumor development. Moreover, a ROS1 splice-
site variant, detected in two affected cousins and in one
unrelated patient, resulted in altered splicing, demonstrated
via minigene approach, and inserted a premature stop-codon
in the protein. yg
y
Results: Exome sequencing identified a novel frameshift
mutation RCC1*c.1067_1086del19. Subsequent genotyp-
ing detected the 19-bp deletion in additional 5 out of 153
(3%) breast cancer patients but in none of 400 female
controls (p = 0.0015). The deletion was enriched in patients
with a positive family history (5%, p = 0.0009) and co-
segregated with breast cancer in the initial pedigree. The
mutant allele was lost in 4/6 breast tumors from mutation
carriers which may be consistent with the hypothesis that
RCC1 dysfunction provides a selective disadvantage at the
stage of tumor progression. Conclusions: The results suggest RCC1 as a novel breast
cancer susceptibility gene and encourage further search for
germline RCC1 mutations in cancer patients from other
populations. Grant reference: This work was funded by the
German Ministry of Education and Research and the
Tunisian Ministry for Higher Education and Scientific
Research (TUNGER-70). Conclusions: The results suggest RCC1 as a novel breast
cancer susceptibility gene and encourage further search for
germline RCC1 mutations in cancer patients from other
populations. Grant reference: This work was funded by the
German Ministry of Education and Research and the
Tunisian Ministry for Higher Education and Scientific
Research (TUNGER-70). Conclusion: We identified novel/rare damaging variants
in genes not previously associated to BC risk. Replication in
independent samples and further functional analysis are
ongoing to elucidate their role in BC development. Supported
by
Italian
Ministry
of
Health-
grant
DIANE to EB. A. Riahi: None. H. Radmanesh: None. P. Schürmann:
None. N. Bogdanova: None. R. Geffers: None. R. Meddeb: None. M. Kharrat: None. T. Dörk: None. I. Bozzarelli: None. F. Isidori: None. C. Diquigiovanni:
None. F. Buscherini: None. R. Zuntini: None. L. Godino:
None. S. Miccoli:
None. D. Turchetti:
None. E. Bonora: None. FEDERAL UNIVERSITY OF GOIAS, GOIAS, Brazil FEDERAL UNIVERSITY OF GOIAS, GOIAS, Brazil The present study aimed to identify the prevalence of gene
rearrangements in BRCA1/2 in patients diagnosed with
breast cancer in central Brazil. We evaluated 47 patients
with breast cancer who met the criteria of the National
Health Agency published in the document "Annex II:
guidelines for use to cover supplementary health proce-
dures” for research on HBOC syndrome. A 4mL blood
sample was collected for DNA extraction using a com-
mercial kit and the MLPA (Multiplex Ligation dependent
Probe Amplification) technique was performed using the
SALSA MLPA P002 BRCA1 and SALSA MLPA P045
BRCA2 / CHECK2 kits. The majority of the patients were
female (95.75%) and the mean age of the patients was 39
years. The most common molecular subtype was luminal A
(50%) followed by triple negative tumors (27.27%). No
patients were found with BRCA1 gene rearrangements. In
BRCA2, one patient (2.12%) presented deletion in hetero-
zygosity
of
the
exon
27,
being
this
female,
with
HER2 superexpressor tumor diagnosed at 24 years old and
with a family history of prostate and stomach cancer. We
can conclude that the frequency of gene rearrangements in
the Central-Brazilian population is low. Results: Exome sequencing identified novel mutations
with predicted pathogenicity in the three candidate genes
KLK3, RASSF1A, and FAM81B. The KLK3 truncating
mutation segregated with breast cancer in the family. The
KLK3 gene product, PSA, has been implicated in both
breast and prostate cancer. However, a supportive segrega-
tion pattern was also observed for a novel missense
mutation in RASSF1A (p.S135F). This mutation eliminates
the ATM phosphorylation site on the RASSF1A protein, a
known
tumor
suppressor
in
breast
carcinomas. The
truncating mutation in FAM81B did not segregate with
breast cancer in this family. The KLK3 and RASSF1A
mutations were not observed in further 235 Iranian breast
cancer patients and 260 controls. Conclusions: Our findings illustrate the difficulties to
identify the causal gene if candidate mutations are restricted
to the single family and show a similarly plausible
segregation pattern. Digenic or oligogenic inheritance may
contribute to breast cancer risk in such cases. H. Radmanesh: None. A. Sadr-Nabavi: None. F. Homaei Shandiz: None. S. Ardalan Khales: None. T. Park-Simon: None. P. Hillemanns: None. D. Liu: None. A Riahi: None R Geffers: None T Dörk: None A. Riahi: None. R. Geffers: None. T. Dörk: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 409 Important Effect of BIBR1532 and Rapamycin
Combination on Breast Cancer Stem Cells Identification of variants predisposing to breast cancer
through a WES approach P12.046B I. Bozzarelli1, F. Isidori1, C. Diquigiovanni1, F. Buscherini2,
R. Zuntini1, L. Godino3, S. Miccoli2, D. Turchetti1,4,
E. BONORA1 I. Tournier1 1Inserm U1245, UNIROUEN, Normandy Univ, and
Department of Genetics, Rouen University Hospital, Normandy
Center for Genomics and Personalized Medicine, Rouen,
France, ROUEN, France, 2Inserm U1245, UNIROUEN,
Normandy Univ, and Department of Metabolic Biochemistry,
Rouen University Hospital, Normandy Center for Genomics
and Personalized Medicine, Rouen, France, ROUEN, France,
3UMR 6014 Laboratory of Organic and Analytical Chemistry
COBRA, IRCOF, UNIROUEN, Normandy Univ, Mont-Saint-
Aignan, France, ROUEN, France 1Inserm U1245, UNIROUEN, Normandy Univ, and
Department of Genetics, Rouen University Hospital, Normandy
Center for Genomics and Personalized Medicine, Rouen,
France, ROUEN, France, 2Inserm U1245, UNIROUEN,
Normandy Univ, and Department of Metabolic Biochemistry,
Rouen University Hospital, Normandy Center for Genomics
and Personalized Medicine, Rouen, France, ROUEN, France,
3UMR 6014 Laboratory of Organic and Analytical Chemistry
COBRA, IRCOF, UNIROUEN, Normandy Univ, Mont-Saint-
Aignan, France, ROUEN, France Conclusion: Consequently we show that treatment of
BIBR1532 and rapamycin is effective on mTOR pathway,
moreover, it is important to target to BCSCs. Ç. Biray Avcı: None. F. DoĞan: None. N. Özateş:
None. B. Göker Bağca: None. C. Gündüz: None. Ç. Biray Avcı: None. F. DoĞan: None. N. Özateş:
None. B. Göker Bağca: None. C. Gündüz: None. P12.048D Important Effect of BIBR1532 and Rapamycin
Combination on Breast Cancer Stem Cells J. del Picchia 410 (TSGs). Computational variant impact prediction (CVIP)
tools such as SIFT, PolyPhen, LINSIGHT, CADD and
FATHMM can classify SNVs as benign or deleterious,
where deleteriousness suggests an alteration to protein
structure or function. We sought to determine if CVIP can
distinguish between GoF and LoF SNVs, including both
smSNVs and germline mSNVs (gmSNVs). In order to
answer this question, a database of n = 28,744 GoF and
LoF smSNVs in n = 530 cancer causing genes (CCGs) from
COSMIC were scored using FATHMM and CADD. A
linear relationship was observed between scores (R2=0.05,
p < 1x10-10), and CADD scores were lower (less deleter-
ious) for variants predicted “CANCER” by FATHMM (p
< 1x10-10). A similar trend was observed in n = 7,664,768
gmSNVs, where CADD scores were lower in CCGs
(17.0 ± 0.05) versus all genes
(18.1 ± 0.07; p < 10-10). Scores were higher for gmSNVs in TSGs (19.7 ± 0.2) ver-
sus OGs (18.7 ± 0.2; p < 10-10). These results suggest that
CVIP produces higher “deleteriousness” scores for LoF
SNVs than for GoF SNVs, which can lead to inaccurate
classification of causative GoF SNVs in cancer. (TSGs). Computational variant impact prediction (CVIP)
tools such as SIFT, PolyPhen, LINSIGHT, CADD and
FATHMM can classify SNVs as benign or deleterious,
where deleteriousness suggests an alteration to protein
structure or function. We sought to determine if CVIP can
distinguish between GoF and LoF SNVs, including both
smSNVs and germline mSNVs (gmSNVs). In order to
answer this question, a database of n = 28,744 GoF and
LoF smSNVs in n = 530 cancer causing genes (CCGs) from
COSMIC were scored using FATHMM and CADD. A
linear relationship was observed between scores (R2=0.05,
p < 1x10-10), and CADD scores were lower (less deleter-
ious) for variants predicted “CANCER” by FATHMM (p
< 1x10-10). A similar trend was observed in n = 7,664,768
gmSNVs, where CADD scores were lower in CCGs
(17.0 ± 0.05) versus all genes
(18.1 ± 0.07; p < 10-10). Scores were higher for gmSNVs in TSGs (19.7 ± 0.2) ver-
sus OGs (18.7 ± 0.2; p < 10-10). These results suggest that
CVIP produces higher “deleteriousness” scores for LoF
SNVs than for GoF SNVs, which can lead to inaccurate
classification of causative GoF SNVs in cancer. J. L. Rodriguez-Flores, R. G. Crystal Weill Cornell Medical College, New York, NY, United States Weill Cornell Medical College, New York, NY, United States EGE UNİVERSİTY MEDİCAL BİOLOGY, TURKEY, Turkey Introduction: Cancer stem cells (CSCs) have features of
self-renewal, proliferation, differentiation similar to normal
stem cells. Targeting CSCs has been considered as a new
approach in cancer therapy. The stemness and replicative
properties of CSCs are related to telomerase activity. BIBR1532 has been used as a quite effective inhibitor of
hTERT. It is known that mTOR regulates telomerase
activity at the translational and post-translational level. PI3K/Akt/mTOR pathway is common in breast cancer and
the interaction between the mTOR pathway and hTERT is
important for the survival of cancer cells. Introduction: Cancer stem cells (CSCs) have features of
self-renewal, proliferation, differentiation similar to normal
stem cells. Targeting CSCs has been considered as a new
approach in cancer therapy. The stemness and replicative
properties of CSCs are related to telomerase activity. BIBR1532 has been used as a quite effective inhibitor of
hTERT. It is known that mTOR regulates telomerase
activity at the translational and post-translational level. PI3K/Akt/mTOR pathway is common in breast cancer and
the interaction between the mTOR pathway and hTERT is
important for the survival of cancer cells. Materials and Methods: We performed cell culture in
this study. The WST-1 solution was used to determine the
cytotoxicity of BIBR1532. IC50 doses of Rapamycin and
BIBR1532 on BCSCs were calculated via CalcuSyn
Version 2.0 software Annexin-FITC Detection Kit was
used for apoptosis, Cycletest Plus DNA Reagent Kit was
used for cell cycle. RNA Isolation was performed and Real-
time PCR was used for gene expression analysis (Qiagen). Furthermore, hTERT gene expression was investigated. Materials and Methods: We performed cell culture in
this study. The WST-1 solution was used to determine the
cytotoxicity of BIBR1532. IC50 doses of Rapamycin and
BIBR1532 on BCSCs were calculated via CalcuSyn
Version 2.0 software Annexin-FITC Detection Kit was
used for apoptosis, Cycletest Plus DNA Reagent Kit was
used for cell cycle. RNA Isolation was performed and Real-
time PCR was used for gene expression analysis (Qiagen). Furthermore, hTERT gene expression was investigated. J.L. Rodriguez-Flores: None. R.G. Crystal: None. Computational Variant Impact Prediction for Gain-of-
Function Somatic Missense SNVs Computational Variant Impact Prediction for Gain-of-
Function Somatic Missense SNVs Computational Variant Impact Prediction for Gain-of-
Function Somatic Missense SNVs The identification of the constitutional mutation responsible
for a genetic predisposition to cancer is essential to the
clinical management of the patient and its relatives. With
the implementation of high-throughput sequencing to the
diagnostic routine of these pathologies, the challenge no
longer lies in the detection of alterations but in their bio-
logical and clinical interpretation. While specific treatments
are emerging, simple functional assays to help with the
interpretation of the detected variants are needed. In this
context, we are currently evaluating the relevance of a J. L. Rodriguez-Flores, R. G. Crystal P12.050B Identification by a multi-omic approach of new biomarkers
indicative of a constitutional defect in the TP53 and BRCA
tumor suppressor genes Results: The IC50 doses of Rapamycin and BIBR1532
were detected as 7.87 nM and 23 μM, respectively for in
48th hour on BCSCs. The combination was increased
apoptosis 4.79 fold compared to control. We observed
significant changes in expression levels of mTOR related
genes. BIBR1532 reduced hTERT activity compared to
control. In addition, Rapamycin and BIBR1532 further
reduced hTERT activity compared to only BIBR1532
treatment. S. Raad1, A. Tebani2, R. Lanos1, E. Kasper1, C. Derambure1,
S. Coutant1, C. Afonso3, G. Bougeard1, T. Frebourg1, S. Bekri2,
I. Tournier1 Ç. Biray Avcı, F. DOĞAN, N. Özateş, B. Göker Bağca,
C. Gündüz Weill Cornell Medical College, New York, NY, United States The majority of human mutations that cause cancer are
somatic missense single nucleotide variants (smSNVs), and
tumors evolve through positive selection on somatic gain-
of-function (GoF) smSNVs in oncogenes (OGs) and loss-
of-function (LoF) smSNVs in tumor suppressor genes Abstracts from the 51st European Society of Human Genetics Conference: Posters 411 of Turin, Turin, Italy, 12Interdepartmental Center for Studies
on Asbestos and other Toxic Particulates “G. Scansetti”,
University of Turin, Turin, Italy, 13Medical Genetics Unit,
AOU Città della Salute e della Scienza, Turin, Italy multi-omic approach combining high-throughput analysis
of the transcriptome and the metabolome to identify new
biomarkers able to discriminate cells with a deleterious
heterozygous mutation from wild-type cells. Using geno-
toxic drugs, we exacerbated the differences between the
lymphocytes of 4 control subjects, 4 Li-Fraumeni patients
carrying a deleterious mutation in the TP53 gene and 4
patients carrying mutations in the BRCA genes involved in
hereditary breast and ovarian cancers. We performed a total
RNA-Seq experiment to analyze both coding and non-
coding RNA transcripts. This analysis revealed a list of
genes
differentially
expressed
between
wild-type
and
mutant conditions. In parallel, we will perform non-targeted
analysis of the metabolome (UHPLC-IM-MS) of these cells. The data will then be integrated to target the key pathways
and biological actors of these cellular responses. The
identified biomarkers will be integrated to simple functional
tests fitted to the diagnostic routine. This work is supported
by the Ligue contre le Cancer, the Canceropôle Nord-Ouest
(Emerging
Project),
the
Normandy
Region
and
Europe (ERDF) Introduction: A direct correlation between the amount of
asbestos exposure and the risk of malignant pleural meso-
thelioma (MPM) is apparent, but not all individuals exposed
to high level of asbestos develop MPM. This observation
and the reports of families with multiple MPM cases sug-
gest a role for inherited predisposition. BAP1 tumor pre-
disposition syndrome (TPDS) includes mesothelioma in its
tumor constellation, but we found BAP1 mutations in only
3/28 MPM probands with familial MPM. We hypothesized
that genes involved in other TPDS could also predispose
to MPM. Materials and Methods: We investigated the prevalence
of germline variants in 94 cancer-predisposing genes in 93
MPM
patients
with
a
quantified
asbestos
exposure
using NGS. Results: Ten pathogenic truncating variants (PTVs) were
identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2,
FANCC, FANCF, PMS1 and XPC, all genes involved in
DNA repair. Germline mutations in DNA repair genes predispose
asbestos-exposed patients to malignant pleural
mesothelioma Germline mutations in DNA repair genes predispose
asbestos-exposed patients to malignant pleural
mesothelioma Conclusions: These data suggest that patients with
germline mutations in DNA repair genes show increased
susceptibility to asbestos-induced MPM. Our preliminary
findings (Betti 2017) preceeded a paper (Robinson 2017),
that reported PTVs in 12.2% of 500 patients with metastatic
tumors (75% in DNA repair genes). Therefore, our
observation
likely
reflects
a
general
phenomenon
of
carcinogenesis. According to the concept of BRCAness,
patients with germline mutations in homologous recombi-
nation repair genes may respond to drugs that induce
synthetic lethality. Grants: AIRC 2015-IG17464(GM),
IIGM(GM), ISS2013-14(CM). M. Betti1, E. Casalone2,3, D. Ferrante4, A. Aspesi1, G. Morleo1,
A. Biasi1, M. Sculco1, G. Mancuso5, S. Guarrera3,2, L. Righi6,
F. Grosso7, R. Libener8, M. Pavesi9, N. Mariani8, C. Casadio10,
D. Mirabelli11,12, B. Pasini3,13, C. Magnani4,12, G. Matullo3,2,12,13,
I. Dianzani1,12 1Department of Health Sciences, Novara, Italy, 2Italian
Institute for Genomic medicine (IIGM), Turin, Italy,
3Department of Medical Sciences, University of Turin, Turin,
Italy, 4CPO-Piemonte and Unit of Medical Statistics and
Epidemiology, Department of Translational Medicine,
University of Piemonte Orientale, Novara, Italy, 5Department
of Health Sciences, Section of Pathological Anatomy,
University of Piemonte Orientale, Novara, Italy, 6Department
of Oncology, University of Turin at San Luigi Hospital,
Orbassano, Turin, Italy, 7Division of Medical Oncology, SS. Antonio e Biagio General Hospital, Alessandria, Italy,
8Pathology Unit, SS. Antonio e Biagio General Hospital,
Alessandria, Italy, 9Pathological Anatomy Unit, Santo Spirito
Hospital, Casale Monferrato, Alessandria, Italy, 10Thoracic
Surgery Unit, AOU Maggiore della Carità, Novara, Italy,
11Unit of Cancer Epidemiology, CPO-Piemonte and University M. Betti: None. E. Casalone: None. D. Ferrante: None. A. Aspesi: None. G. Morleo: None. A. Biasi: None. M. Sculco: None. G. Mancuso: None. S. Guarrera: None. L. Righi: None. F. Grosso: None. R. Libener: None. M. Pavesi: None. N. Mariani: None. C. Casadio: None. D. Mirabelli: None. B. Pasini: None. C. Magnani: None. G. Matullo: None. I. Dianzani: None. M. Betti: None. E. Casalone: None. D. Ferrante: None. A. Aspesi: None. G. Morleo: None. A. Biasi: None. M. Sculco: None. G. Mancuso: None. S. Guarrera: None. L. Righi: None. F. Grosso: None. R. Libener: None. M. Pavesi: None. N. Mariani: None. C. Casadio: None. D. Mirabelli: None. B. Pasini: None. C. Magnani: None. G. Matullo: None. I. Dianzani: None. Weill Cornell Medical College, New York, NY, United States Mutated patients (9.7%) had a significantly
lower asbestos exposure than non-mutated patients (p =-
0.0015). This result remains significant (p < 0.0001), when
four
patients
carrying
BAP1
germline
mutations
are
included in the analysis. S. Raad: None. A. Tebani: None. R. Lanos: None. E. Kasper: None. C. Derambure: None. S. Coutant: None. C. Afonso: None. G. Bougeard: None. T. Frebourg:
None. S. Bekri: None. I. Tournier: None. S. Raad: None. A. Tebani: None. R. Lanos: None. E. Kasper: None. C. Derambure: None. S. Coutant: None. C. Afonso: None. G. Bougeard: None. T. Frebourg:
None. S. Bekri: None. I. Tournier: None. I. Drejeriene1,2, A. Krasauskas1,3, J. Kasnauskiene1,2,
D. Stanciute3, A. Laurinavicius1,4, V. Sapoka1,5, S. Cicenas1,3 Hotspot mutations in 22
oncogenes (KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2,
PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1,
MET, TP53, SMAD4, FBX7, FGFR3, NOTCH1, ERBB4,
EGFR1, FGFR2) were detected using NGS (Ion Torrent™
PGM) Ion AmpliSeq colon and lung cancer research panel
(ThermoFisher). NGS was performed from FFPE DNA and
plasma cfDNA for every patient. Samples were taken before
treatment. Conclusions: CHEK2 mutations seem to be associated
with ER- and PR-positive disease, family history of cancer
and early age of disease onset in Slovene BC patients. Although our analysis is severely limited by small sample
size and ascertainment bias, our findings are compatible
with what is currently known about CHEK2-positive BC
patients. Results: Mutations in EGFR, KRAS, TP53, ALK and
MET genes were identified most frequently. Mutations were
observed in 5 (11%) females tumor and plasma samples. For 2 females mutations were detected only in plasma
sample and for 27 (59%) females only in tumor samples. The remaining 12 (26%) females had no mutations detected
in both types of samples. Deletion, insertion and duplication
in EGFR and ERBB2 genes were detected in 11 tumor
samples, however, only one deletion in EGFR gene was
detected in plasma sample. A. Blatnik: None. K. Strojnik: None. V. Stegel: None. V. etrajčič Drago: None. G. Klančar: None. S. Nova-
ković: None. M. Krajc: None. A. Blatnik, K. Strojnik, V. Stegel, V. etrajčič Drago, G. Klančar,
S. Novaković, M. Krajc I. Drejeriene1,2, A. Krasauskas1,3, J. Kasnauskiene1,2,
D. Stanciute3, A. Laurinavicius1,4, V. Sapoka1,5, S. Cicenas1,3 Introduction:
Pathogenic
variants
in
CHEK2
which
encodes a cell-cycle-checkpoint kinase are associated with
moderate risk of developing breast cancer (BC). We col-
lected and analysed data on clinical characteristics, family
history and mutation spectrum in Slovene CHEK2 mutation
carriers with BC. 1Vilnius University, Vilnius, Lithuania, 2Klaipeda University
Hospital, Klaipeda, Lithuania, 3National Cancer Institute,
Vilnius, Lithuania, 4National Center of Pathology, Affiliate of
Vilnius University Hospital Santaros Klinikos, Vilnius,
Lithuania, 5Vilnius University Life Science Center, Vilnius,
Lithuania Vilnius University Hospital Santaros Klinikos, Vilnius, Materials and Methods: 25 BC patients from 21
different families who developed 29 tumours and were
verified or obligatory CHEK2 mutation carriers were
included in our analysis. Clinical information was obtained
from their medical records and family pedigrees. Lithuania, 5Vilnius University Life Science Center, Vilnius,
Lithuania Objectives: The aim of this study was to assess non-small
cell lung cancer (NSCLC) female patients’ hotspot muta-
tions in oncogenes and compare it between formalin-fixed,
paraffin-embedded (FFPE) tumor DNA and plasma cfDNA
samples. Results: We detected four recurrent CHEK2 pathogenic
variants in our families: c.444+1G>A (23.8%), c.349A>G
(23.8%), c.1100delC (18.2%) and deletion of exons 9-10
(13.6%). 62.5% of our patients had a first or second degree
relative with BC and 87.5% had a first or second degree
relative with any cancer. A male patient developed
oestrogen receptor (ER)-positive, progesterone receptor
(PR)-positive and human epidermoid growth factor receptor
(HER2)-positive invasive ductal carcinoma (IDC) at the age
of 49. Of the 24 female patients, four developed bilateral
BC. Median age of onset for female BC patients was 42.5
(range 21-64). 22.7% of their cancers had lobular histology,
the rest being IDCs. 91.1% were ER-positive, 81.8% were
PR-positive, 22.7% were HER2-positive and 9% were triple
negative. Materials
and
Methods:
46
female
patients
with
NSCLC were included in the study. 15 (33%) patients
were heavy smokers. The dominant morphology was
adenocarcinoma - 33 (72%). Hotspot mutations in 22
oncogenes (KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2,
PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1,
MET, TP53, SMAD4, FBX7, FGFR3, NOTCH1, ERBB4,
EGFR1, FGFR2) were detected using NGS (Ion Torrent™
PGM) Ion AmpliSeq colon and lung cancer research panel
(ThermoFisher). NGS was performed from FFPE DNA and
plasma cfDNA for every patient. Samples were taken before
treatment. Materials
and
Methods:
46
female
patients
with
NSCLC were included in the study. 15 (33%) patients
were heavy smokers. The dominant morphology was
adenocarcinoma - 33 (72%). P12.056D Conclusion: The mutations were detected in 32 (70%)
tumor samples and in 7 (15%) plasma samples. Detection of
mutations using plasma cfDNA samples could be only an
additional assay in a routine clinical work. Conclusion: The mutations were detected in 32 (70%)
tumor samples and in 7 (15%) plasma samples. Detection of
mutations using plasma cfDNA samples could be only an
additional assay in a routine clinical work. Co-existence of multiple subclones in ETV6/RUNX1 at
diagnosis of B-cell lymphoblastic leukemia Co-existence of multiple subclones in ETV6/RUNX1 at
diagnosis of B-cell lymphoblastic leukemia V. Caner1, N. Sen Turk2, G. Çetin1, B. Albuz1, Y. Ay3 V. Caner1, N. Sen Turk2, G. Çetin1, B. Albuz1, Y. Ay3 I. Drejeriene: None. A. Krasauskas: None. J. Kas-
nauskiene: None. D. Stanciute: None. A. Laurinavicius:
None. V. Sapoka: None. S. Cicenas: None. I. Drejeriene: None. A. Krasauskas: None. J. Kas-
nauskiene: None. D. Stanciute: None. A. Laurinavicius:
None. V. Sapoka: None. S. Cicenas: None. 1Pamukkale University School of Medicine Department of
Medical Genetics, Denizli, Turkey, 2Pamukkale University
School of Medicine Department of Medical Pathology, Denizli,
Turkey, 3Pamukkale University School of Medicine
Department of Pediatric Hematology, Denizli, Turkey Detection of oncogenes hotspot mutations in female NSCLC
tumor DNA and cell-free DNA Detection of oncogenes hotspot mutations in female NSCLC
tumor DNA and cell-free DNA 412 J. del Picchia Institute of Oncology, Ljubljana, Slovenia P12.055C Cytogenetic analysis was performed at the time
of diagnosis on bone marrow culture by trypsin-G-banding. FISH panel testing for ALL which includes eight different
FISH
probes
(MYC
rearrangement,
CDKN2A,
E2A
rearrangement, MLL rearrangement, ETV6/RUNX1 trans-
location, BCR/ABL1 translocation, IGH rearrangement,
and hyperdiploidy probes consisting of CHIC2, D10Z1, and
D17Z1) (Cytocell, Cambridge, UK) was performed on the
bone marrow cytogenetic pellet. The analysis was done in
200 interphase nuclei. The patient was diagnosed with B-
ALL. There was no bone marrow metaphase spreads
available for conventional cytogenetic analysis, however
FISH examination revealed 3-6 copies of ETV6/RUNX1
fusion signals together with ETV6 deletion in 54% of cells. The classical ETV6/RUNX1 translocation was found in
15% of cells. Clinical
Report:
The
patient,
9-year-old-boy,
was
admitted to the Department of Pediatric Haematology at
Pamukkale University Hospital in November 2017 with
inappetence, progressive fatigue and neutropenia. The bone
marrow biopsy was performed for evaluation of blood
cytopenias. Cytogenetic analysis was performed at the time
of diagnosis on bone marrow culture by trypsin-G-banding. FISH panel testing for ALL which includes eight different
FISH
probes
(MYC
rearrangement,
CDKN2A,
E2A
rearrangement, MLL rearrangement, ETV6/RUNX1 trans-
location, BCR/ABL1 translocation, IGH rearrangement,
and hyperdiploidy probes consisting of CHIC2, D10Z1, and
D17Z1) (Cytocell, Cambridge, UK) was performed on the
bone marrow cytogenetic pellet. The analysis was done in
200 interphase nuclei. The patient was diagnosed with B-
ALL. There was no bone marrow metaphase spreads
available for conventional cytogenetic analysis, however
FISH examination revealed 3-6 copies of ETV6/RUNX1
fusion signals together with ETV6 deletion in 54% of cells. The classical ETV6/RUNX1 translocation was found in
15% of cells. Conclusion: Report of rare numerical and structural
genetic aberrations contributes to our understanding of the
disease classification, prognosis and clinical management. However, a long-term follow-up is required to verify the
possible prognostic effect of the ETV6/RUNX1 fusions
amplification. F.T. Papa: None. A.M. Pinto: None. F.C. Lorenzetti:
None. E. Frullanti: None. I. Meloni: None. R. Tita: None. R. Caselli: None. C. Fallerini: None. D. Lopergolo: None. M.A. Mencarelli: None. M. Bocchia: None. A. Gozzetti:
None. A. Renieri: None. F. Mari: None. V. Caner: None. N. Sen Turk: None. G. Çetin: None. B. Albuz: None. Y. Ay: None. The effects of the therapeutic agents Ponatinib and VS-5584
on Chronic Myeloid Leukemia leukemogenesis The effects of the therapeutic agents Ponatinib and VS-5584
on Chronic Myeloid Leukemia leukemogenesis P12.055C Breast cancer in Slovene CHEK2 mutation carriers Introduction: The chromosomal translocation t(12;21)
(p13;q22) which results in the formation of the ETV6/
RUNX1 fusion gene is the most common structural genetic
aberration in childhood B-cell lymphoblastic leukemia (B- Institute of Oncology, Ljubljana, Slovenia 413 Abstracts from the 51st European Society of Human Genetics Conference: Posters ALL). Co-existence of multiple subclones in ETV6/
RUNX1 is rare with limited clinical information available. Herein is described a case of B-ALL with multiple sub-
clones in ETV6/RUNX1. refractory clones. In order to assess the timing of somatic
acquisition of clonal origin of TP53 mutation, we performed
a longitudinal deep next generation sequencing (NGS) in a
patient with familial CLL. A clone and the subclone bearing
deletion 11q and 13q switched from 80% and 45% at
diagnosis to
undetectable after
chemotherapy (started
31 months after a therapy-free period), and TP53-mutated
subclone with deletion on the 13q shifted from undetectable
at the diagnosis to 80% after chemotherapy. A TP53
pathogenic mutation (c.548C>G;p.Ser183*) was detected
with a load of 93.8% in post-chemotherapy sample. Inter-
estingly, the same mutation was present in a blood sample
collected 16 months before clinical manifestation of disease
with a mutational load of 15%. Risk stratification based on
genetic prognostic markers is highly recommended at
diagnosis to determine the most appropriate strategy for
clinical management. Analysis of copy number variations,
IGHV and TP53 mutations can predict the aggressive
clinical course of disease. Given the positive family history
for CLL, pre-clinical stage NGS TP53 analysis would have
ranked the patient as a high-risk, low-responder individual,
leading to opt for the last generation targeted therapies
available. Given the low TP53 mutational load, NGS test
should be included in current clinical practice to ensure the
best clinical management being the optimal technique to
detect low mosaic mutations. Clinical
Report:
The
patient,
9-year-old-boy,
was
admitted to the Department of Pediatric Haematology at
Pamukkale University Hospital in November 2017 with
inappetence, progressive fatigue and neutropenia. The bone
marrow biopsy was performed for evaluation of blood
cytopenias. Cytogenetic analysis was performed at the time
of diagnosis on bone marrow culture by trypsin-G-banding. FISH panel testing for ALL which includes eight different Clinical
Report:
The
patient,
9-year-old-boy,
was
admitted to the Department of Pediatric Haematology at
Pamukkale University Hospital in November 2017 with
inappetence, progressive fatigue and neutropenia. The bone
marrow biopsy was performed for evaluation of blood
cytopenias. Low level of TP53 mutation can be detected by NGS years
before CLL clinical/laboratory diagnosis Low level of TP53 mutation can be detected by NGS years
before CLL clinical/laboratory diagnosis C. Kayabasi1, B. Ozmen Yelken1, A. Asik1, T. Balci Okcanoglu2,
F. Sogutlu1, R. Gasimli1, S. Yilmaz Susluer1, C. Biray Avci1,
C. Gunduz1 C. Kayabasi1, B. Ozmen Yelken1, A. Asik1, T. Balci Okcanoglu2,
F. Sogutlu1, R. Gasimli1, S. Yilmaz Susluer1, C. Biray Avci1,
C. Gunduz1 F. T. Papa1, A. M. Pinto1,2, F. C. Lorenzetti1, E. Frullanti1,
I. Meloni1, R. Tita2, R. Caselli2, C. Fallerini1, D. Lopergolo1,2,
M. A. Mencarelli2, M. Bocchia3, A. Gozzetti3, A. Renieri1,2,
F. Mari1,2 F. T. Papa1, A. M. Pinto1,2, F. C. Lorenzetti1, E. Frullanti1,
I. Meloni1, R. Tita2, R. Caselli2, C. Fallerini1, D. Lopergolo1,2,
M. A. Mencarelli2, M. Bocchia3, A. Gozzetti3, A. Renieri1,2,
F. Mari1,2 1Ege University, Department of Medical Biology, IZMIR,
Turkey, 2Near East University, Vocational School of Health
Sciences, Nicosia, Cyprus 1Medical Genetics, Siena, Italy, 2Genetica Medica, Azienda
Ospedaliera Universitaria Senese, Siena, Italy, 3Department of
Medicine and Immunological Sciences, Hematology Unit,
Azienda Ospedaliera Universitaria Senese and University of
Siena, Siena, Italy Chronic myeloid leukemia (CML) is characterized by cells
with BCR-ABL. Leukemia stem cells (LSCs) are malignant
derivatives of hematopoietic stem cells (HSCs) and cause
resistance to chemotherapy. Ponatinib is ATP-competitive
tyrosine kinase inhibitor (TKI) capable of inhibiting all
BCR-ABL autophosphorylation, including T315I-mutant. VS-5584 is selective PI3K-mTOR dual-inhibitor, preferably
killing cancer stem cells. We aim to investigate changes in
apoptosis,
cell
cycle,
oncogenic
Akt
pathway TP53 mutations are present in 10% of Chronic Lymphocytic
Leukemia (CLL) patients at the time of diagnosis, and are
associated with impaired survival and poor therapy response
due
to
a
selection
of
TP53-mutated
chemotherapy- J. del Picchia 414 phosphorylation, and transcription factor (TF) activity by
targeting K562 and LSC with VS-5584 and/or ponatinib. Cytotoxic effects of VS-5584 and/or ponatinib on cell-lines
were measured with WST-8. Combination indices were
evaluated by isobologram analysis. Apoptotic effects were
evaluated with AnnexinV and Caspase3 assays, effects on
cell cycle were assessed using PI assay with flow-cytometry
and by cyclinD1, p27 antibodies with western blot. Changes
in TF activities and phosphorylated proteins were measured
with dual-luciferase quantitation after transfection of con-
structs and PathScan Antibody-Array, respectively. It was
determined that combinations were synergistic. In addition
to ponatinib-induced apoptosis, VS-5584 was shown to
cause marked G0/G1 arrest. Ponatinib in combination with
VS-5584 was able to suppress Akt pathway through inhi-
biting the phosphorylation of several proteins including S6,
S6K, BAD. Low level of TP53 mutation can be detected by NGS years
before CLL clinical/laboratory diagnosis In leukemic cells, suppression of SRE/Elk-1,
AP-1, NFkB, CREB, myc/max, E2F/DP-1 and activation of
C/EBP, FOXO, p53 TFs were more intense with VS-5584
treatment, compared to ponatinib treatment. HSCs were
least affected. VS-5584 mediated suppression of BCR-ABL
independent oncogenic pathways known to be active in
CML, promises hope for the elimination of LSCs that can’t
be targeted with traditional TKI therapy. the highest level of their expression in the initial stage of
ccRCC was revealed. Significant reducing in expression
levels of the genes at low degree of differentiation in rela-
tion to high degree was found for all three genes by U-test. The association study was carrying out using ROC analysis
and the exact Fisher test. An association of the studied gene
expression levels with the degree of tumor cell differentia-
tion was significant by both tests: in the ROC analysis (p =
0.0015 - 0.035) and in Fisher test (p = 0.008 - 0.015). The
application of the FDR correction to the multiplicity of
comparisons retains the significance of association for all
three genes. Thereby, a decrease of the expression level of
the genes ANGPTL4, BHLHE41, IGFBP3 in tumors of
ccRCC is associated with a decrease in the degree of tumor
cell differentiation, which becomes more malignant. N.V. Apanovich: None. M.V. Peters: None. P.V. Apanovich: None. B.S. Kamolov: None. V.B. Matveev:
None. A.V. Karpukhin: None. G. Asgari, M. Sadeghizadeh C. Kayabasi: None. B. Ozmen Yelken: None. A. Asik:
None. T. Balci Okcanoglu: None. F. Sogutlu: None. R. Gasimli: None. S. Yilmaz Susluer: None. C. Biray Avci:
None. C. Gunduz: None. TMU, Tehran, Iran, Islamic Republic of Introduction: Chronic myeloid leukemia (CML) is a
hematological disorder originated from a single oncoprotein
encoded by BCR-ABL fused gene. Recently, the leukemia
incidence has been grown meanwhile drug resistance and
cancer recurrence have been occurred. Aberrant expression
of some lncRNAs and consequently their association with
tumorigenesis has been demonstrated in numerous cancers
containing leukemia. Furthermore, recent studies introduced
them as a noticeable target for therapy. The large intergenic
non-coding RNA, H19, is abundantly expressed in CML
cells and plays a meaningful role in leukemogenesis. Mentioned oncogenic lincRNA is tightly regulated by BCR-
ABL transcript. Solanine is a natural glycoalkaloid with
anti-proliferative effects has reported in diverse cancers. To
investigate if solanine and DNS (dendrosomal nano sola-
nine) have any considerable effect on H19 expression level,
we designed further study. P12.059C The expression of some HIF-1a regulated genes is
connected with the differentiation of clear-cell renal cell
carcinoma cells N. V. Apanovich1, M. V. Peters2, P. V. Apanovich1, B. S. Kamolov2, V. B. Matveev2, A. V. Karpukhin1 N. V. Apanovich1, M. V. Peters2, P. V. Apanovich1, B. S.
Kamolov2, V. B. Matveev2, A. V. Karpukhin1 N. V. Apanovich1, M. V. Peters2, P. V. Apanovich1, B. S. Kamolov2, V. B. Matveev2, A. V. Karpukhin1 1Research Centre For Medical Genetics, Moscow, Russian
Federation, 2NN Blokhin Russian Cancer Research Centre,
Moscow, Russian Federation Clear-cell renal cell carcinoma (ccRCC) is the most com-
mon (70-80%) and aggressive among malignant neoplasms
of the kidney. The mechanisms of its development are not
fully known. It is important to obtain additional information
on the molecular genetics mechanisms of ccRCC progres-
sion. In this study, the expression levels of 21 genes in the
sample of 65 paired tumor/ normal renal tissue from patients
with ccRCC were investigated by RT-qPCR. When ana-
lyzing the functional processes associated with the genes on
Gene Ontology, three genes involved in the cell differ-
entiation process (ANGPTL4, BHLHE41, IGFBP3) has
been identified. These genes are activated by HIF-1α and Materials and Methods: BCR-ABL expressing cell line,
K562,
was
cultured
and
treated
with
semi-cytotoxic
concentration of solanine and DNS and incubated for 48
h. RNA extraction and cDNA synthesis was performed. The
expression level of H19 was evaluated by real-time RT-
PCR. Results: ANOVA analysis revealed the down-expression
of H19 in K562 cell line in only DNS treated group (P< Abstracts from the 51st European Society of Human Genetics Conference: Posters 415 0.028). Down-expression was 1.8 times less in treated
group in comparison to control group. somatic mutation affecting the active site of the exonuclease
domain of POLD1. In addition, three cases presented with
potentially pathogenic mutations in genes involved in the
extracellular matrix. Conclusion: Suspending solanine in dendrosomal parti-
cles makes it more impressive in order to down-regulate
H19 expression in k562 CML cells. Likewise DNS can be
proposed as an effective alternative for targeted therapy if it
brings about the same result in in vivo as well as
in vitro model. Sequencing of tumour and paired germline samples and
selective screening for de novo mutations provides a
powerful strategy to investigate known and novel genes
that predispose to early onset CRC. Our findings indicate
that a genetic predisposition is frequent and heterogeneous
in these patients. G. Asgari: None. M. Sadeghizadeh: None. G. Asgari: None. M. Sadeghizadeh: None. P12.063C M.C.J. Jongmans:
None. J. Zhang:
None. A.R. Mensenkamp: None. R.D.A. Weren: None. L. Spruijt:
None. C.M. Kets: None. W.A. van Zelst-Stam: None. M. I. Schouten: None. M.J.W. Olderode-Berends: None. J. C. Oosterwijk: None. M.M. Hitzert: None. M.G.E. Ausems: None. E.J. Kamping: None. F.N. van Leeuwen:
None. L. Yuniati: None. H.K. Schackert: None. R.S. van
der Post: None. M.R. Teixeira: None. H. Liu: None. J. Wang: None. R.P. Kuiper: None. A. Geurts van Kessel:
None. N. Hoogerbrugge: None. M.J.L. Ligtenberg:
None. R.M. de Voer: None. M.C.J. Jongmans:
None. J. Zhang:
None. A.R. Mensenkamp: None. R.D.A. Weren: None. L. Spruijt:
None. C.M. Kets: None. W.A. van Zelst-Stam: None. M. I. Schouten: None. M.J.W. Olderode-Berends: None. J. C. Oosterwijk: None. M.M. Hitzert: None. M.G.E. Ausems: None. E.J. Kamping: None. F.N. van Leeuwen:
None. L. Yuniati: None. H.K. Schackert: None. R.S. van
der Post: None. M.R. Teixeira: None. H. Liu: None. J. Wang: None. R.P. Kuiper: None. A. Geurts van Kessel:
None. N. Hoogerbrugge: None. M.J.L. Ligtenberg:
None. R.M. de Voer: None. Germline cancer susceptibility in adolescents and young
adults with colorectal cancer M. C. J. Jongmans1,2,3, J. Zhang4, A. R. Mensenkamp1, R. D. A. Weren1, L. Spruijt1, C. M. Kets1, W. A. van Zelst-Stam1, M. I. Schouten1, M. J. W. Olderode-Berends5, J. C. Oosterwijk5, M. M. Hitzert5, M. G. E. Ausems3, E. J. Kamping1, F. N. van Leeuwen1,
L. Yuniati1, H. K. Schackert6, R. S. van der Post1, M. R. Teixeira7, H. Liu4, J. Wang4, R. P. Kuiper1,2, A. Geurts van
Kessel1, N. Hoogerbrugge1, M. J. L. Ligtenberg1, R. M. de Voer1 M. C. J. Jongmans1,2,3, J. Zhang4, A. R. Mensenkamp1, R. D. A. Weren1, L. Spruijt1, C. M. Kets1, W. A. van Zelst-Stam1, M. I. Schouten1, M. J. W. Olderode-Berends5, J. C. Oosterwijk5, M. M. Hitzert5, M. G. E. Ausems3, E. J. Kamping1, F. N. van Leeuwen1,
L. Yuniati1, H. K. Schackert6, R. S. van der Post1, M. R. Teixeira7, H. Liu4, J. Wang4, R. P. Kuiper1,2, A. Geurts van
Kessel1, N. Hoogerbrugge1, M. J. L. Ligtenberg1, R. M. de Voer1 Evidence for GALNT12 as a moderate penetrance gene for
colorectal cancer Evidence for GALNT12 as a moderate penetrance gene for
colorectal cancer P12.064D 1Radboudumc, Nijmegen, Netherlands, 2Princess Maxima
Center for Pediatric Oncology, Utrecht, Netherlands,
3University Medical Center Utrecht, Utrecht, Netherlands,
4Sun Yat-Sen University, Guangzhou, China, 5University
Medical Center Groningen, Groningen, Netherlands,
6Technische Universität Dresden, Dresden, Germany,
7Portuguese Oncology Institute of Porto (IPO-Porto), Porto,
Portugal D. R. Evans Memorial University of Newfoundland, St. Johns, NL, Canada Memorial University of Newfoundland, St. Johns, NL, Canada P12.066B Identification of mismatch repair-deficient colorectal
cancers using a molecular inversion probe based
sequencing assay of short mononucleotide repeats 1Microbiology Department, Hospital General Universitario de
Elche, Elche, Spain, 2Sequencing and Bioinformatics Service,
Foundation for the Promotion of Health and Biomedical
Research of Valencia Region (FISABIO), Valencia, Spain,
3Centro de Investigaciones Biológicas (CIB) - CSIC, Madrid,
Spain, 4Molecular Genetics Laboratory, Hospital General
Universitario de Elche, Alicante Institute for Health and
Biomedical Research (ISABIAL - FISABIO Foundation),
Alicante, Spain, 5Molecular Genetics Laboratory, Hospital
General Universitario de Elche, Elche, Spain, 6Department of
Public Health, History of Science and Gynecology, Miguel
Hernández University, Alicante, Spain H. Sheth1, R. Gallon1, C. Hayes1, L. Redford1, G. Alhilal1,
A. Miguel Alonso2, S. Moreno Laguna3, M. Arends4, A. Oniscu4,
O. O'Brien2, S. Needham5, M. S. Jackson1, M. Santibanez-
Koref1, J. Burn1 H. Sheth1, R. Gallon1, C. Hayes1, L. Redford1, G. Alhilal1,
A. Miguel Alonso2, S. Moreno Laguna3, M. Arends4, A. Oniscu4,
O. O'Brien2, S. Needham5, M. S. Jackson1, M. Santibanez-
Koref1, J. Burn1 1Newcastle University, Newcastle upon Tyne, United Kingdom,
2Northern Genetics Service, Newcastle upon Tyne, United
Kingdom, 3Servicio de Genetica Medica, Pamplona, Spain,
4Western General Hospital, Edinburgh, United Kingdom,
5Royal Victoria Infirmary, Newcastle upon Tyne, United
Kingdom Introduction: Cancer is a heterogeneous and complex set
of multifactorial diseases that have in common an (epi)
genetic origin. Colorectal cancers (CRC) with microsatellite
instability (MSI) are a subset of hypermutated tumors
generated by DNA mismatch repair deficiency. MSI tumors
have specific pathological and clinical characteristics. There
is a growing interest in knowing the putative role of the
microbiota in the colorectal oncogenesis. We aimed to
explore potential differences in microbiota in CRC patients
with and without MSI. Introduction: UK’s NICE guidelines recommend mis-
match repair (MMR) deficiency testing of colorectal cancers
(CRCs) to identify Lynch syndrome, a hereditary predis-
position for CRC. Uptake of MMR deficiency testing has
been poor due to the unsuitability of current assays -
immunohistochemistry and fragment analysis - to high
throughput testing, including manual workflows and results
interpretation. We aimed to develop a next generation
sequencing-based assay of short mononucleotide repeats to
assess microsatellite instability (MSI), a biomarker of MMR
deficiency, using single molecule-molecular inversion probe
(smMIP) technology, with a view to improving the clinical
uptake of MMR deficiency testing. Methods: 24 short
mononucleotide repeats, together with clinically-actionable
markers in BRAF and KRAS, were amplified in multiplex
using smMIPs. Colorectal cancer bacteria microenvironment in different
tumor genetic contexts M. Parra-Grande: None. V. Sánchez-Hellín: None. L. Martínez: None. E. Larriba: None. V. Barbera: None. G. D'Auria: None. M. Castillejo: None. B. Lumbreras:
None. J. Soto: None. M. Parra-Grande1, V. Sánchez-Hellín1, L. Martínez2,
E. Larriba3, V. Barbera4, G. D'Auria2, M. Castillejo5,
B. Lumbreras6, J. Soto4 D.R. Evans: None. D.R. Evans: None. Funding: Supported by Conselleria d’Educació General-
itat Valenciana, Spain (GV/2016/175). Abstract Characterizing moderate penetrance susceptibility genes
is an emerging frontier in colorectal cancer (CRC) research. GALNT12 is a strong candidate CRC-susceptibility gene
given
previous
linkage
and
association
studies,
and
inactivating somatic and germline alleles in CRC patients. We previously found rare segregating germline GALNT12
variants in a clinic-based cohort (N=118) with predisposi-
tion for CRC. Here, we screened a new population-based
cohort of incident CRC cases (N=479) for rare (MAF <1%)
deleterious germline GALNT12 variants. GALNT12 screen-
ing revealed 8 rare variants. Two variants were previously
described (p.D303N, p.R297W), and additionally, we found
6 other rare variants: five missense (p.H101Q, p.I142T, p. E239Q, p.T286M, p.V290F) and one putative splice-
altering variant (c.732-8 G>T). Sequencing of population-
matched controls (N=400) revealed an over-representation
of these variants in CRC cases compared to healthy controls
(P=0.04). We then functionally characterized the impact of
these substitutions on GALNT12 enzyme activity using
in vitro-derived peptide substrates. Three of the newly
identified GALNT12 missense variants (p.H101Q, p.I142T,
p.V290F) demonstrated a marked loss (>2-fold reduction) Colorectal cancer (CRC) at young age (≤25 years) is a rare
condition. Two CRC syndromes causing such an early onset
are familial adenomatous polyposis and constitutional
mismatch-repair
deficiency
(CMMRD). However,
the
majority of CRCs ≤25 years are microsatellite-stable, are
not associated with polyposis and show an increased
mucinous histology compared to adult-onset CRC. These
differences suggest that CRC ≤25 years represents a distinct
clinical CRC entity. We performed whole-exome sequencing (WES) on
germline DNA from patients with CRC ≤25 years (n=40;
for 15 cases trio-based sequencing was performed) and
paired tumour samples (n=19) to unravel the underlying
genetics of CRC at young age. Six cases carried a pathogenic mutation in a known
cancer predisposing gene: BRCA2, NF1, POLD1, PALB2
(n=1 each), and TP53 (n=2). One case carried a de novo
mutation in the RAS-MAPK gene SOS2. In vitro expression
of this mutant revealed an increase in ERK phosphorylation,
suggesting a gain-of-function effect. The mutation in
POLD1 resulted in a frameshift and this patient presented
with a hypermutated cancer resulting from an in trans J. del Picchia 416 family and genus levels. Taxa differences among MSI vs
MSS tumors were in families Veilloneacea (p = 0.028) and
Verrucomicrobiae and genus Akkermansia (p = 0.04). family and genus levels. Taxa differences among MSI vs
MSS tumors were in families Veilloneacea (p = 0.028) and
Verrucomicrobiae and genus Akkermansia (p = 0.04). Abstract of enzymatic activity compared to wild-type (P≤0.05), while
p.E239Q
exhibited
a
~2-fold
reduction
in
activity
(P=0.077). These findings provide strong, independent
evidence for the association of GALNT12 defects with
CRC-susceptibility; underscoring implications for glycosy-
lation pathway defects in CRC. Conclusions:
Differences
on
the
colon
microbiota
composition associated to MSI/MSS tumor molecular
phenotype were observed. Further studies are needed for a
better understanding of these findings. T. Shapochka, D. Shapochka, O. Selezniov, A. Turkina,
O. Sulaieva T. Shapochka, D. Shapochka, O. Selezniov, A. Turkina,
O. Sulaieva P12.066B Amplicons were sequenced using the Illu-
mina MiSeq platform, aligned to reference genome hg19
using BWA, and analysed using custom R scripts. An MSI
classifier was trained on short mononucleotide repeat
sequences from 98 CRCs collected in pathology labora-
tories in Edinburgh and Spain, and validated in 98 CRCs
collected at Newcastle. Materials and Methods: Ninety-six DNAs from frozen
tissues (tumor and normal mucosa) of 48 CRC patients (24
MSI and 24 microsatellite-stable-tumors (MSS)) were used
to performed metagenomic 16S analysis (V3 and V4
region). Sequencing was conducted using a paired-end
2×300 bp cycle run on Illumina MiSeq. RDP Classifier was
used for taxonomical assignment. Groups of data were
compared using a paired and unpaired t test for normal vs
tumor and MSI vs MSS, respectively. Results: Globally, lower bacteria diversity (Shannon and
Simpson indexes) was observed in tumor vs normal tissues. Diversity of MSI tumors vs their normal counterparts
showed significant differences at genus level. Similarly,
diversity of MSS tumors vs their normals were also lower at Results: Globally, lower bacteria diversity (Shannon and
Simpson indexes) was observed in tumor vs normal tissues. Diversity of MSI tumors vs their normal counterparts
showed significant differences at genus level. Similarly,
diversity of MSS tumors vs their normals were also lower at 417 Abstracts from the 51st European Society of Human Genetics Conference: Posters Results: The MSI classifier showed 100% sensitivity and
specificity, relative to fragment analysis, using either a 6 or
a 24 marker panel. Frequencies of BRAF and KRAS
mutations concurred with previous observations. Cost
estimate for sample analysis using the proposed assay is
£4.28/sample in contrast to £11.65/sample with fragment
analysis. patients with MSI demonstrated significantly lower associa-
tion with distant metastasis (P<0.0001). Results: The MSI classifier showed 100% sensitivity and
specificity, relative to fragment analysis, using either a 6 or
a 24 marker panel. Frequencies of BRAF and KRAS
mutations concurred with previous observations. Cost
estimate for sample analysis using the proposed assay is
£4.28/sample in contrast to £11.65/sample with fragment
analysis. patients with MSI demonstrated significantly lower associa-
tion with distant metastasis (P<0.0001). Conclusions: Thus, in Ukrainian population the fre-
quency of MSI CRC is 14.7% and, as in other studies MSI
was associated with male sex, younger age, right-sided
location of tumor and high grade. T. Shapochka:
None. D. Shapochka:
None. O. Selezniov: None. A. Turkina: None. O. Sulaieva: None. P12.066B Conclusions: Our novel MSI assay could streamline
CRC molecular diagnostics by providing cheap and high
throughput detection of MMR deficiency, acting as a
companion diagnostic for immunotherapy and improving
the identification of Lynch syndrome patients. Conclusions: Our novel MSI assay could streamline
CRC molecular diagnostics by providing cheap and high
throughput detection of MMR deficiency, acting as a
companion diagnostic for immunotherapy and improving
the identification of Lynch syndrome patients. Cytogenetic and molecular characterization of 33 complex
variant Ph translocations diagnosed in patients with
chronic myeloid and acute lymphoblastic leukemia Cytogenetic and molecular characterization of 33 complex
variant Ph translocations diagnosed in patients with
chronic myeloid and acute lymphoblastic leukemia H. Sheth: None. R. Gallon: None. C. Hayes: None. L. Redford: None. G. Alhilal: None. A. Miguel Alonso:
None. S. Moreno Laguna: None. M. Arends: None. A. Oniscu: None. O. O'Brien: None. S. Needham: None. M. S. Jackson:
None. M. Santibanez-Koref:
None. J. Burn: None. H. Sheth: None. R. Gallon: None. C. Hayes: None. L. Redford: None. G. Alhilal: None. A. Miguel Alonso:
None. S. Moreno Laguna: None. M. Arends: None. A. Oniscu: None. O. O'Brien: None. S. Needham: None. M. S. Jackson:
None. M. Santibanez-Koref:
None. J. Burn: None. D. Costa1, M. López1, A. Arias1, C. Gómez1, B. Espinet2,
J. Grau3, M. Nomdedeu1, F. Cervantes1, F. Cobo4, D. Colomer1 Costa1, M. López1, A. Arias1, C. Gómez1, B. Espinet2, D. Costa1, M. López1, A. Arias1, C. Gómez1, B. Espinet2,
J. Grau3, M. Nomdedeu1, F. Cervantes1, F. Cobo4, D. Colomer1
1Hospital Clinic, Barcelona, Spain, 2Hospital Mar, Barcelona,
Spain, 3Hospital Germans Trias i Pujol, Badalona, Spain,
4Hospital Nostra Senyora de Meritxell, Escaldes Engordany,
Andorra D. Costa , M. López , A. Arias , C. Gómez , B. Espinet ,
J. Grau3, M. Nomdedeu1, F. Cervantes1, F. Cobo4, D. Colomer1 1Hospital Clinic, Barcelona, Spain, 2Hospital Mar, Barcelona,
Spain, 3Hospital Germans Trias i Pujol, Badalona, Spain,
4Hospital Nostra Senyora de Meritxell, Escaldes Engordany,
Andorra P12.067C Clinicopathological characteristics of patients with
colorectal cancer in regards with microsatellite instability
status in Ukrainian population: the pilot study Chronic myeloid leukemia (CML) is a myeloproliferative
disease. The hallmark of the disease is the presence of a
Philadelphia (Ph1) chromosome produced by a reciprocal
translocation t(9;22)(q34;q11). In 5-10% of the cases, the
Ph1 chromosome is generated by variant rearrangements,
involving 9q34, 22q11, and one or more genomic regions. We report 33 cases of complex variant Ph1 translocations
diagnosed in 32 patients affected with CML and one patient
affected with acute lymphoblastic leukemia (ALL). Cyto-
genetic and FISH studies were carried out in bone marrow
samples. The third chromosome involved in the 33 complex
variant translocations was the chromosome 1 (n = 7), 5 (n-
= 5), 12 (n = 4), 3, 11 (n = 3), 2,6,15 (n = 2), and 7,13, 17,
19, 20, 21 (n = 1). The total number of breakpoints were 34
(one of the translocations involved 4 chromosomes), and 26
were different. The q arm chromosome was the most fre-
quently involved in the translocations (62%). The break-
points were located in 81% of the cases in the G-light
bands. Seven out of the 22 karyotypes showed one or 2
additional chromosomal abnormalities. FISH studies using
the LSI BCR/ABL (VYSIS) probe were carried out in 24
out of the 33 cases allowing the detection of the fusion
genes BCR/ABL on chromosome Ph1 in all the cases. After
FISH studies using LSI, CEP and WCP probes the kar-
yotype was modified in 3 cases. Most of the breakpoints in
our series are in the G-light bands. The combination of
conventional cytogenetics and FISH studies allow us to
identify these complex variant translocations. Z. Bo1, S. Ho1, X. Zhu1, X. Zhang1, N. Spies1, S. Byeon2, J. G.
Arthur1, R. Pattni1, N. Ben-Efraim1, M. S. Haney1, R. R.
Haraksingh1, G. Song2, D. Perrin3, W. H. Wong1, A. Abyzov4,
A. E. Urban1 1Simon Fraser University, Burnaby, BC, Canada, 2Princess
Margaret Cancer Centre, Toronto, ON, Canada, 3Queens
University, Kingston, ON, Canada, 4BC Cancer Agency,
Vancouver, BC, Canada 1Stanford University, Palo Alto, CA, United States, 2Pusan
National University, Busan, Korea, Democratic People's
Republic of, 3Queensland University of Technology, Brisbane,
Australia, 4Mayo Clinic, Rochester, MN, United States Introduction: Non-Hodgkin’s lymphoma (NHL) is the 9th
most common cancer in Europe, with 93,000 new cases and
38,000 deaths in 2012 alone. Diffuse Large B-Cell Lym-
phoma (DLBCL) is the most common subtype of NHL, and
for DLBCL patients who fail primary treatment (rrDLBCL),
prognosis is extremely poor, with a 5-year survival rate of
around 10%. Thus, the genetic mechanism which lead to
treatment failure must be identified to aid in the develop-
ment of new molecular-based treatments. The chronic myelogenous leukemia cell line K562 is one of
the most widely used in biomedical research. It is one of
three tier-one cell lines of ENCODE, and one of the cell
lines most commonly used for large-scale CRISPR/Cas9
gene-editing screens. Although the functional genomic and
epigenomic characteristics of K562 are extensively studied,
its genome sequence has never been comprehensively
analyzed and higher-order structural features of its genome
beyond its karyotype were only cursorily known. The high
degree of aneuploidy in K562 renders traditional genome
variant analysis methods challenging and partially ineffec-
tive. Correct and complete interpretation of the extensive
functional genomics data from K562 requires an under-
standing of the cell line's genome sequence and genome
structure. We performed deep whole-genome sequencing,
mate-pair sequencing and linked-read sequencing to iden-
tify a wide spectrum of genome characteristics in K562:
copy numbers of chromosomal segments, SNVs and Indels
(both corrected for copy-number), phased haplotype blocks,
structural variants (SVs) including complex genomic rear-
rangements, and novel mobile element insertions. A large
number of SVs were phased, sequence assembled and
experimentally
validated. Several
chromosomes
show
striking loss of heterozygosity. We re-analyzed K562 RNA-
Seq and whole-genome bisulfite sequencing data for allele-
specific expression and phased DNA methylation. We show
examples where deeper insights into genomic regulatory
complexity could be gained by taking knowledge of geno-
mic structural contexts into account. Furthermore, we used
the haplotype information to produce an allele-specific
CRISPR targeting map. This comprehensive whole-genome
analysis serves as a resource for future studies that
utilize K562. Laboratory of Pathology CSD Health Care, Kiev, Ukraine Cervantes: None. F. Cobo: None. D. Colomer: None. J. Kuruvilla: None. M. Crump: None. M. Jain: None. L. Shepherd: None. D. Scott: None. R. Morin: None. J. Kuruvilla: None. M. Crump: None. M. Jain: None. L. Shepherd: None. D. Scott: None. R. Morin: None. Laboratory of Pathology CSD Health Care, Kiev, Ukraine Introduction: Identification of colorectal cancer (CRC)
subtypes, especially microsatellite instability (MSI), is of
great clinical importance because of its role in prognosis
and predictioin of sensitivity to therapy. The lack of data on
the prevalence of MSI in Ukrainian population, as well as
its relationship with the clinical and morphological char-
acteristics of patients makes actual to perform this study. Materials and Methods: 177 patients with CRC who
underwent MSI (26), or MMR (151 patients) testing were
enrolled in the study. Demographic and clinical data were
assessed in patients with MSI and MSS. Materials and Methods: 177 patients with CRC who
underwent MSI (26), or MMR (151 patients) testing were
enrolled in the study. Demographic and clinical data were
assessed in patients with MSI and MSS. p
Results: The overall incidence of MSI among observed
patients was 14.7%, while the incidence among men
(25.35%) was significantly higher (p = 0.0369) than in
women (10%). There were significant differences in age of
patients with MSI (46.9 years) and MSS (57.1 years)
(P=0,0020). Strong relation was found between MSI and
tumor location in the proximal part of large intestine
(p < 0.0001), regardless of sex. MSI was more often
associated with medullary (100%) and mucinous (15.4%)
histological types (P=0.0008), and these subtypes in 88.9%
were detected in men. The strong relation of MSI with high
grade was found - about 50% of patients with MSI had
Grade 3 carcinomas independently of sex. However, Results: The overall incidence of MSI among observed
patients was 14.7%, while the incidence among men
(25.35%) was significantly higher (p = 0.0369) than in
women (10%). There were significant differences in age of
patients with MSI (46.9 years) and MSS (57.1 years)
(P=0,0020). Strong relation was found between MSI and
tumor location in the proximal part of large intestine
(p < 0.0001), regardless of sex. MSI was more often
associated with medullary (100%) and mucinous (15.4%)
histological types (P=0.0008), and these subtypes in 88.9%
were detected in men. The strong relation of MSI with high
grade was found - about 50% of patients with MSI had
Grade 3 carcinomas independently of sex. However, D. Costa: None. M. López: None. A. Arias: None. C. Gómez: None. B. Espinet: None. J. Grau: None. M. D. Costa: None. M. López: None. A. Arias: None. C. Gómez: None. B. Espinet: None. J. Grau: None. M. 418 J. del Picchia Nomdedeu: None. F. Z. Bo: None. S. Ho: None. X. Zhu: None. X. Zhang:
None. N. Spies: None. S. Byeon: None. J.G. Arthur:
None. R. Pattni: None. N. Ben-Efraim: None. M.S. C. Rushton1, M. Alcaide1, J. Davidson1, M. Cheung1,
K. Bushell1, S. Yu1, J. Kuruvilla2, M. Crump2, M. Jain2,
L. Shepherd3, D. Scott4, R. Morin1,4 Z. Bo1, S. Ho1, X. Zhu1, X. Zhang1, N. Spies1, S. Byeon2, J. G. Arthur1, R. Pattni1, N. Ben-Efraim1, M. S. Haney1, R. R. Haraksingh1, G. Song2, D. Perrin3, W. H. Wong1, A. Abyzov4,
A. E. Urban1 Z. Bo1, S. Ho1, X. Zhu1, X. Zhang1, N. Spies1, S. Byeon2, J. G. Arthur1, R. Pattni1, N. Ben-Efraim1, M. S. Haney1, R. R. Haraksingh1, G. Song2, D. Perrin3, W. H. Wong1, A. Abyzov4,
A. E. Urban1 The use of circulating tumor DNA to study the genetic basis
of treatment failure in rrDLBCL Comprehensive, integrated, and phased whole-genome
analysis of the primary ENCODE cell line K562 C. Rushton1, M. Alcaide1, J. Davidson1, M. Cheung1,
K. Bushell1, S. Yu1, J. Kuruvilla2, M. Crump2, M. Jain2,
L. Shepherd3, D. Scott4, R. Morin1,4 Haney: None. R.R. Haraksingh: None. G. Song: None.
D. Perrin: None. W.H. Wong: None. A. Abyzov: None.
A.E. Urban: None. Haney: None. R.R. Haraksingh: None. G. Song: None. D. Perrin: None. W.H. Wong: None. A. Abyzov: None. A.E. Urban: None. Haney: None. R.R. Haraksingh: None. G. Song: None. D. Perrin: None. W.H. Wong: None. A. Abyzov: None. A.E. Urban: None. P12.073A
Whole transcriptome sequencing > Kazakhstani patients
with esophageal squamous cell carcinoma Results: According to the histological type of ESCC was
prevalent moderately differentiated squamous cell carci-
noma with infiltrative growth and without keratinization. In
our patients the third stage of the disease (51.8%) was more
often detected. Paired analysis of cancer and normal tissues
identified 287 down-regulated and 192 up-regulated genes. Among up-regulated genes PPAR signaling pathway (p-
value = 0.01), cytokine-cytokine receptor interaction (p-
value = 0.05) and metabolism of lipids and lipoproteins (p-
value = 0.03) have been identified. Whereas the most
significant pathways among down-regulated genes are
metabolism of xenobiotics by cytochrome P450 (p-value-
= 1.31E-4), retinol metabolism P450 (p-value = 0.01), O-
Glycan biosynthesis (p-value = 0.02). Results: According to the histological type of ESCC was
prevalent moderately differentiated squamous cell carci-
noma with infiltrative growth and without keratinization. In
our patients the third stage of the disease (51.8%) was more
often detected. Paired analysis of cancer and normal tissues
identified 287 down-regulated and 192 up-regulated genes. Among up-regulated genes PPAR signaling pathway (p-
value = 0.01), cytokine-cytokine receptor interaction (p-
value = 0.05) and metabolism of lipids and lipoproteins (p-
value = 0.03) have been identified. Whereas the most
significant pathways among down-regulated genes are
metabolism of xenobiotics by cytochrome P450 (p-value-
= 1.31E-4), retinol metabolism P450 (p-value = 0.01), O-
Glycan biosynthesis (p-value = 0.02). Conclusions: Our results suggest that serum possesses
extra information of exosomal miRNAs from platelets, and
therefore
may
be
a
better
resource
while
applying
repositories
for
studying
diseases
related
to
platelet
functions.This study is supported by Research University
Network on precision medicine in Thailand (MURA2017/
747) Conclusion: Using whole transcriptome sequencing we
could identify molecular pathways involved in esophageal
tumorigenesis to understanding pathogenesis of ESCC and
develop new diagnostic markers. This work was supported
by grants of the Ministry of education and science
#AP05134722 and # AP05135430 M. Shiao: None. T. Thammasorn: None. T. Pisitkun:
None. N. Jinawath: None. D. G. R. Evans1,2,3,4, E. M. van Veen1,5, H. J. Byers1,5, A. J.
Wallace5, J. M. Ellingford1,5, G. Beaman1,5, J. Santoyo-Lopez6, Inherited BRCA1 epimutation as a novel cause of breast
and ovarian cancer Z. Bo1, S. Ho1, X. Zhu1, X. Zhang1, N. Spies1, S. Byeon2, J. G.
Arthur1, R. Pattni1, N. Ben-Efraim1, M. S. Haney1, R. R.
Haraksingh1, G. Song2, D. Perrin3, W. H. Wong1, A. Abyzov4,
A. E. Urban1 Materials and methods: We have analyzed temporal
samples obtained from 35 patients enrolled in two ongoing
clinical trials. For each patient enrolled, a tumour biopsy
sample is obtained prior to investigational treatment, as well
as several blood samples as treatment progresses. Through a
combination of whole exome and targeted sequencing of
biopsy samples and circulating tumor DNA, the genetic
landscape of rrDLBCL, and how it evolves in response to
treatment, can be characterized. Results: Prior to investigational treatment, we observed
recurrent mutations in well-described lymphoma-associated
genes (KMT2D, EP300, EZH2, EP300) as well as several
genes associated with treatment failure (CYP2A6, ABCA12,
AHNAK2) and metastasis (NFBP1) in other cancers. Following investigational therapy, mutations in several
genes (S1PR2 and FOXO1) were enriched overall, and sub-
clonal populations containing these mutations expanded in
several patients who failed treatment. Conclusions: Mutations in lymphoma-associated genes
may either directly contribute to salvage treatment failure,
or act as a possible biomarker. Analysis of additional
samples as they become available may identify other
mutations. Grants: Terry Fox New Frontiers Program Project
Grant #1061 Z. Bo: None. S. Ho: None. X. Zhu: None. X. Zhang:
None. N. Spies: None. S. Byeon: None. J.G. Arthur:
None. R. Pattni: None. N. Ben-Efraim: None. M.S. Z. Bo: None. S. Ho: None. X. Zhu: None. X. Zhang:
None. N. Spies: None. S. Byeon: None. J.G. Arthur:
None. R. Pattni: None. N. Ben-Efraim: None. M.S. C. Rushton: None. M. Alcaide: None. J. Davidson:
None. M. Cheung: None. K. Bushell: None. S. Yu: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 419 P12.075C
Exosomal miRNA profiles in serum, plasma and platelets in
healthy donors M. Shiao1, T. Thammasorn1, T. Pisitkun2, N. Jinawath1 M. Shiao1, T. Thammasorn1, T. Pisitkun2, N. Jinawath1 P12.073A
Whole transcriptome sequencing > Kazakhstani patients
with esophageal squamous cell carcinoma 1Faculty of Medicine Ramathibodi Hospital, Bangkok,
Thailand, 2Chulalongkorn University Systems Biology Center,
Bangkok, Thailand S. Rakhimova1, A. Molkenov1, U. Kairov1, Y. Zhukov2,
M. Omarov2, A. Akilzhanova1 Introduction: Repository of human clinical specimen
(biobanking) is the key to study genetic diseases. Among
those, blood samples are the best resource of discovering
serum/plasma biomarkers for liquid biopsies. Increasing
evidence shows that exosomes, which can be found in all
biofluids, play important roles in various kinds of diseases
and are therefore good biomarkers. Besides, recent studies
showed that platelets are able to intake and transport exo-
somes associated with several diseases, such as cancer. Hence, we aim to identify large-scale miRNA profiles of
exosomes in serum, plasma, as well as those released by
activated platelets. 1National Laboratory Astana, Nazarbayev University, Astana,
Kazakhstan, 2Oncology center, Astana, Kazakhstan Introduction: Esophageal cancer is the eighth most com-
mon cancer in the world. The incidence rate in Kazakhstan
is 10.1: 100 000. The aim of the project is to identify
genetic basis of ESCC by performing whole transcriptome
sequencing in Kazakhstani patients. Introduction: Esophageal cancer is the eighth most com-
mon cancer in the world. The incidence rate in Kazakhstan
is 10.1: 100 000. The aim of the project is to identify
genetic basis of ESCC by performing whole transcriptome
sequencing in Kazakhstani patients. Materials and Methods: 54 patients with ESCC under-
went surgery at Oncology center (Astana) between 2013
and 2016. Fresh frozen cancer tissue and its adjacent normal
tissue specimen were obtained from each patient. Whole
transcriptome sequencing was performed on Illumina
platform using TruSeq RNA protocol. STAR software and
DESeq package have been used for mapping and defining
differentially expressed genes. MSigDB and KEGG data-
bases were processed for analysis of signaling networks. Material and Methods: Serum, plasma, and activated
platelets from 12 healthy donors are collected and used in
this study. Exosomal miRNAs were isolated with ExoR-
Neasy kit (Qiagen) and quantified using Human miRNA
panel v3 on Nanostring nCounter system. Results: A total of ~250 exosomal miRNAs are detected
from all three resources: serum, plasma and activated
platelets. Three groups of miRNAs are identified: higher
expression in serum and plasma, higher expression in
platelet, higher expression in serum and platelets. Overall,
platelets have a distinct expression pattern comparing to
serum and plasma. Interestingly, platelets show more
overlapped genes with serum comparing to plasma, likely
due to coagulation process in serum collection. Reduced familial fertility in carriers of mutations in the
BRCA1 and BRCA2 genes H. Akopyan1,2, N. Kitsera2, A. Siekierzynska3, T. Nguyen-
Dumont4, F. Hammet4, H. Tsimiklis4, D. J. Park4,5, B. J. Pope5,6,
M. C. Southey4, D. Blonioarz1, A. Myszka1 1Institute of Experimental and Clinical Medicine, University of
Rzeszow, Rzeszow, Poland, 2Institute of Hereditary Pathology
of National Academy of Medical Sciences, Lviv, Ukraine,
3Department of Biotechnology and Plant Physiology,
University of Rzeszow, Rzeszow, Poland, 4Genetic
Epidemiology Laboratory, Department of Pathology, The
University of Melbourne, Melbourne, Australia, 5Victorian Life
Sciences Computation Initiative, Melbourne, Victoria,
Australia, 6Department of Computing and Information
Systems, The University of Melbourne, Victoria, Australia Introduction: Pathogenic variants in BRCA1 or BRCA2 are
identified in ~20% of families with multiple individuals
with early-onset breast/ovarian cancer. Extensive searches
for additional highly penetrant genes or alternative muta-
tional mechanisms altering BRCA1/2 have not explained the
missing heritability. For the first time, we report transge-
nerational epigenetic silencing of BRCA1 due to promoter
hypermethylation
in
two
families
with
breast/ovarian
cancer. Methods: BRCA1 promoter methylation of ten CpG
dinucleotides in breast/ovarian cancer families without
germline BRCA1/2 pathogenic variants was assessed by
pyrosequencing and clonal bisulfite sequencing. RNA and
DNA sequencing of BRCA1 from lymphocytes was under-
taken to establish allelic expression and the presence of
germline variants. The frequency of founder mutations in BRCA1 and BRCA2
genes influences the genetic testing strategy for breast and/
or ovarian cancer susceptibility in many populations. The
aim of this study was assess clinical data associated with
BRCA1 and BRCA2 mutation-associated familial breast and/
or ovarian cancer in the West Ukraine population. Previous
genetic testing in a group of 125 women with familial breast
and/or ovarian cancer identified 25 and 9 pathogenic
mutations in BRCA1 and BRCA2 genes respectively. Clin-
ical parameters were compared between groups of carriers
and non-carriers of pathogenic mutations (34 and 91,
respectively).Thirty three (97%) of the mutation carriers had
at least one family members with the same gynecological
cancer. In contrast, 24 (26%) of the non-carrier cases were
the first breast and or ovarian cancer case in a family
(χ2=7,09, p < 0,01; OR=11.82 [95% CI: 1.53-91,22]). In
families
with
BRCA1/2
mutation
carriers
there
were
numerous incidences of familial infertility and infertility of
mutation carrier’s children (p < 0,05). This study also Results: BRCA1 promoter hypermethylation was identi-
fied in two of 49 families with multiple women affected
with grade 3 breast/high grade serous ovarian cancer. P12.076D Inherited BRCA1 epimutation as a novel cause of breast
and ovarian cancer S. Rakhimova: None. A. Molkenov: None. U. Kairov:
None. Y. Zhukov:
None. M. Omarov:
None. A. Akilzhanova: None. D. G. R. Evans1,2,3,4, E. M. van Veen1,5, H. J. Byers1,5, A. J. Wallace5, J. M. Ellingford1,5, G. Beaman1,5, J. Santoyo-Lopez6, 420 J. del Picchia T. J. Aitman6, D. M. Eccles7, F. I. Lalloo5, M. J. Smith1,5, W. G. Newman1,5 5’UTR variant in two independent families. We propose
that methylation analyses are indicated in all families
affected by early onset breast/ovarian cancer without a
BRCA1/2 pathogenic variant. 1Division of Evolution and Genomic Sciences, School of
Biological Sciences, Faculty of Biology, Medicine and Health,
University of Manchester, Manchester Academic Health
Science Centre, Manchester, United Kingdom, 2Prevention
Breast Cancer Centre and Nightingale Breast Screening
Centre, University Hospital of South Manchester, Manchester,
United Kingdom, 3The Christie NHS Foundation Trust,
Manchester, United Kingdom, 4Manchester Breast Centre,
Manchester Cancer Research Centre, University of
Manchester, Manchester, United Kingdom, 5Manchester
Centre for Genomic Medicine, St. Mary’s Hospital,
Manchester University NHS Foundation Trust, Manchester
Academic Health Science Centre, Manchester, United
Kingdom, 6Centre for Genomic and Experimental Medicine,
and Edinburgh Genomics, University of Edinburgh,
Edinburgh, United Kingdom, 7Cancer Sciences Academic Unit
and Southampton Clinical Trials Unit, Faculty of Medicine,
University of Southampton and University Hospital
Southampton Foundation Trust, Southampton, United
Kingdom Funded by Prevent Breast Cancer (GA 12-006, GA 15-
002) and the Manchester NIHR Biomedical Research
Centre (IS-BRC-1215-20007). Funded by Prevent Breast Cancer (GA 12-006, GA 15-
002) and the Manchester NIHR Biomedical Research
Centre (IS-BRC-1215-20007). D.G.R. Evans: None. E.M. van Veen: None. H.J. Byers: None. A.J. Wallace: None. J.M. Ellingford: None. G. Beaman: None. J. Santoyo-Lopez: None. T.J. Ait-
man: D. Speakers Bureau/Honoraria (speakers bureau,
symposia, and expert witness); Modest; Illumina. D.M. Eccles: None. F.I. Lalloo: None. M.J. Smith: None. W.G. Newman: None. FFPE testing in deceased family members: implications for
clinical management of patients seen in the genetics clinic S. Bennett, E. Alexander, H. Fraser, A. J. Wallace, S. Huang,
H. Schlecht, A. Quinn, G. Evans, F. Lalloo, E. Woodward,
N. Bowers S. Bennett, E. Alexander, H. Fraser, A. J. Wallace, S. Huang,
H. Schlecht, A. Quinn, G. Evans, F. Lalloo, E. Woodward,
N. Bowers 1i3S - Instituto de Investigação e Inovação em Saúde, Porto,
Portugal, 2IPATIMUP, Porto, Portugal, 3Department of
Pathology and Laboratory Medicine, University of British
Columbia, Vancouver, BC, Canada, 4Department of General
Surgery and Surgical Oncology, University of Siena, Siena,
Italy, 5Genetic Pathology Evaluation Centre, University of
British Columbia and Vancouver General, Vancouver, BC,
Canada, 6Centre for Translational and Applied Genomics
(CTAG), BC Cancer Agency, Vancouver, BC, Canada,
7Faculty of Medicine of the University of Porto, Porto,
Portugal Manchester Centre for Genomic Medicine, St Mary's Hospital,
Manchester University NHS Foundation Trust, Manchester,
United Kingdom Introduction: Archived pathology tissue can be a source of
germline DNA for genomic analysis in families with no
living affected individuals where the determination of
mutation status can assist in the clinical management of
living relatives. Introduction: Familial Intestinal Gastric Cancer (FIGC)
remains genetically unexplained; despite has its autosomal
dominant inheritance pattern. However, FIGC pedigrees
often display generations without affected individuals, and
late onset intestinal gastric cancer, indicating the presence
of low or moderate risk alleles that may increase cancer
susceptibility in FIGC. Introduction: Familial Intestinal Gastric Cancer (FIGC)
remains genetically unexplained; despite has its autosomal
dominant inheritance pattern. However, FIGC pedigrees
often display generations without affected individuals, and
late onset intestinal gastric cancer, indicating the presence
of low or moderate risk alleles that may increase cancer
susceptibility in FIGC. Method: The Manchester Genomic Diagnostics Labora-
tory has developed and validated Next Generation Sequen-
cing (NGS) based gene panels optimised for the detection of
point and small insertion/deletion mutations in formalin
fixed paraffin embedded (FFPE) tissue DNA. At present,
the laboratory has analysed 80 FFPE samples from
deceased breast and/or ovarian cancer index cases for
variants in the BRCA1 and BRCA2 genes and 23 FFPE
samples from deceased colorectal cancer index patients for
variants in a panel of 13 genes implicated in inherited
colorectal cancer. We aimed at identifying the germline cause of FIGC and
at characterizing the 2nd hits in potentially causative genes
and other somatic events in FIGC tumours. P12.079C
Familial Intestinal Gastric Cancer: a polygenic or a
monogenic disease? Familial Intestinal Gastric Cancer: a polygenic or a
monogenic disease? Reduced familial fertility in carriers of mutations in the
BRCA1 and BRCA2 genes Soma-
wide BRCA1 promoter hypermethylation was confirmed in
blood, buccal mucosa and hair follicles. Methylation levels
were ~50%, consistent with the silencing of one allele and
confirmed by clonal bisulfite sequencing. RNA sequencing
revealed allelic loss of BRCA1 expression in both families
and this segregated with a novel heterozygous variant c.-
107A>T in the BRCA1 5’UTR. Conclusion: Our results indicate a novel mechanism for
familial breast/ovarian cancer, caused by epigenetic silen-
cing of the BRCA1 promoter, segregating with an in cis 421 Abstracts from the 51st European Society of Human Genetics Conference: Posters revealed a significantly higher incidence of medical abor-
tion and spontaneous miscarriage (OR=7.84 [95% CI: 1.71-
35.91]) that may be an independent risk factor for gyne-
cological cancer in the group of non-carriers. In conclusion,
the occurrence of the same gynecological cancer in relatives
and reduced familial fertility may significantly increase the
probability of carrying mutations in BRCA1/2 genes in
patients from West Ukraine. screening recommendations and the availability of pre-
dictive genetic testing. screening recommendations and the availability of pre-
dictive genetic testing. Conclusion: Our data demonstrates how FFPE testing in
deceased relatives is an accurate, informative and valuable
tool in the clinical management of patients seen in the
genetics clinic. Conclusion: Our data demonstrates how FFPE testing in
deceased relatives is an accurate, informative and valuable
tool in the clinical management of patients seen in the
genetics clinic. S. Bennett: None. E. Alexander: None. H. Fraser:
None. A.J. Wallace: None. S. Huang: None. H. Schlecht:
None. A. Quinn: None. G. Evans: None. F. Lalloo: None. E. Woodward: None. N. Bowers: None. S. Bennett: None. E. Alexander: None. H. Fraser:
None. A.J. Wallace: None. S. Huang: None. H. Schlecht: H. Akopyan: None. N. Kitsera: None. A. Siekier-
zynska: None. T. Nguyen-Dumont: None. F. Hammet:
None. H. Tsimiklis: None. D.J. Park: None. B.J. Pope:
None. M.C. Southey: None. D. Blonioarz: None. A. Myszka: None. None. A. Quinn: None. G. Evans: None. F. Lalloo: None. E. Woodward: None. N. Bowers: None. P12.078B C. São José1,2, J. Carvalho1,2, H. Pinheiro1,2, P. Oliveira1,2,
J. Senz3, S. Hansford3, A. S. Valente1,2, D. Lemos1,2,
F. Roviello4, D. Huntsman3,5,6, C. Oliveira1,2,7 FFPE testing in deceased family members: implications for
clinical management of patients seen in the genetics clinic FFPE testing in deceased family members: implications for
clinical management of patients seen in the genetics clinic Increased telomere length was observed in
gallstone (inflamed) tissues in comparison to both adjacent
non-tumor and tumor tissues. Significant down-regulation
of TERF1, POT1 and TINF2 genes was observed in
inflamed tissues as compared to non-tumor and tumor
tissues. Further, POT1 was found significantly up-regulated
in tumor tissues and specifically in tumor tissues with gall
stones, compared to inflamed tissues. Conclusion: In conclusion, this work pinpointed FIGC as
a likely polygenic rather than a monogenic disease in 42%
of FIGC families. Co-occurrence of low or moderate risk
alleles may interact with family history and other non-
genetic factors, increasing the risk of cancer. Conclusion: Thus, our study suggests that telomere
length remains unaffected in inflamed tissues, however,
decreased expression of some shelterin genes may lead to
improper telomere capping, paving the way for tumorigen-
esis. Also, POT1 expression in gallstones patients could act
as a diagnostic marker for GBC after further validations in
future. Funding:
1)FEDER/COMPETE,FCT/MEC/FEDER/
PT2020,"PEst-C/SAU/LA0003/2013”;2) ON.2–ONovoNorte/FEDER/QREN:NORTE-07-0162-
FEDER-000118,NORTE-07-0162-FEDER-000067);3)No
Stomach for Cancer Foundation S.S. Poojary: None. G. Mishra: None. S. Gupta: None. B.R. Shrivastav: None. P.K. Tiwari: None. S.S. Poojary: None. G. Mishra: None. S. Gupta: None. B.R. Shrivastav: None. P.K. Tiwari: None. C. São José: None. J. Carvalho: None. H. Pinheiro:
None. P. Oliveira: None. J. Senz: None. S. Hansford: C. São José: None. J. Carvalho: None. H. Pinheiro:
None. P. Oliveira: None. J. Senz: None. S. Hansford:
None. A.S. Valente: None. D. Lemos: None. F. Roviello:
None. D. Huntsman: None. C. Oliveira: None. None. A.S. Valente: None. D. Lemos: None. F. Roviello:
None. D. Huntsman: None. C. Oliveira: None. FFPE testing in deceased family members: implications for
clinical management of patients seen in the genetics clinic Materials and methods: Normal and tumour DNA from
52 FIGC probands were screened using a multiplex custom-
panel of 67 cancer-associated genes. Somatic 2nd mutation
and promoter methylation were searched by PCR-Sanger-
sequencing. Results: Successful analysis was achieved in 80% of
cases and both pathogenic variants and variants of unknown
clinical significance in the BRCA1 and BRCA2 genes,
mismatch repair genes and the APC gene were identified. FFPE samples from as far back as 1988 have been
successfully analysed. In total, of the 71 successful analyses
with clinical data available, 55% of the results (17
pathogenic variants and 22 negative results) directly
impacted the clinical management of relatives seen in the
genetics clinic. This included changes to risk assessments, Results: Successful analysis was achieved in 80% of
cases and both pathogenic variants and variants of unknown
clinical significance in the BRCA1 and BRCA2 genes,
mismatch repair genes and the APC gene were identified. FFPE samples from as far back as 1988 have been
successfully analysed. In total, of the 71 successful analyses
with clinical data available, 55% of the results (17
pathogenic variants and 22 negative results) directly
impacted the clinical management of relatives seen in the
genetics clinic. This included changes to risk assessments, Results: Twenty-four out of 52 FIGC families harboured
germline variants. From these, 36 germline variants were
found, affecting 17 genes, being MSH6, SDHD, MAP3K6
and ATM the most frequently altered. Of notice, 42% of the
families carried co-occurrence of germline variants. In fact,
four families display germline and somatic variants with
common features between different signaling pathways. 422 J. del Picchia Further,
two
families
displayed
a 2nd
mutation
that
potentially inactivates MSH6 and FAT4 genes at the somatic
level. The
somatic
landscape
revealed
115
variants,
affecting 23 genes, found in 36 families. TP53, MSH3,
ARID1A and FAT4 were mutated more frequently. Conclusion: In conclusion, this work pinpointed FIGC as
a likely polygenic rather than a monogenic disease in 42%
of FIGC families. Co-occurrence of low or moderate risk
alleles may interact with family history and other non-
genetic factors, increasing the risk of cancer. Further,
two
families
displayed
a 2nd
mutation
that
potentially inactivates MSH6 and FAT4 genes at the somatic
level. The
somatic
landscape
revealed
115
variants,
affecting 23 genes, found in 36 families. TP53, MSH3,
ARID1A and FAT4 were mutated more frequently. gallstones. Differential expression of shelterin genes and telomere
length variation in gallstone and gallbladder cancer
patients of North Central India E. Machackova, M. Navratilova, E. Stahlova Hrabincova,
P. Vasickova, J. Hazova, M. Svoboda, L. Foretova
Masaryk Memorial Cancer Institute, Brno, Czech Republic E. Machackova, M. Navratilova, E. Stahlova Hrabincova,
P. Vasickova, J. Hazova, M. Svoboda, L. Foretova S. S. Poojary1,2, G. Mishra1,3, S. Gupta4, B. R. Shrivastav5, P. K. Tiwari1 P12.081A
Point mutation in exon 1B of APC in Czech families with
GAPPS Point mutation in exon 1B of APC in Czech families with
GAPPS Masaryk Memorial Cancer Institute, Brno, Czech Republic Masaryk Memorial Cancer Institute, Brno, Czech Republic Gastric adenocarcinoma and proximal polyposis of the
stomach (GAPPS) is an autosomal-dominant cancer-pre-
disposition syndrome with a significant risk of gastric
adenocarcinoma. Li et al., 2016 described a few point
mutations in the YY1 binding site of the APC gene 1B
promoter associated with GAPPS syndrome. We performed
Sanger Sequencing of APC promoter 1B (by Li et al., 2016)
in 35 Czech families with previously unsolved cases of
hereditary predisposition to familiar polyposis, gastric
polyposis or gastric adenocarcinoma. The only pathogenic
variant
we
detected
in
1B
promoter
was
APC
NM_001127511:
c.-191T>C
mutation
associated
with
GAPPS. Nine severely affected individuals from 7 unre-
lated families all of them carpeting of more than 100 fundic
gland polyps and 4 of them already died of adenocarci-
noma. We have performed predictive testing on asympto-
matic family members and detected other 5 carriers of APC
c.-191T>C mutation. Three of these carriers were diagnosed
with more than 100 asymptomatic fundic gland polyps
(born at 1988, 1983, 1968) and were recommended for
prophylactic gastrectomy. Prophylactic gastrectomy was
done in a carrier born 1988 and gastric adenocarcinoma was
already present. No polyps were currently detected in one
carrier woman born at 1987. Carrier mother (born at 1967)
of the patient who died of generalized tubular adenocarci-
noma at 28 years of age was ordered to gastroscopy. We 1Centre for Genomics, Jiwaji University, Gwalior, India,
2Amity Institute of Molecular Medicine and Stem Cell
Research, Amity University, Noida, India, 3ICMR-National
Institute of Pathology, New Delhi, India, 4Department of
Pathology, Cancer Hospital and Research Institute, Gwalior,
India, 5Department of Surgical Oncology, Cancer Hospital
and Research Institute, Gwalior, India Introduction: Gallbladder cancer (GBC) has a very high
prevalence in North Central India with an equally high
mortality rate due to poor prognosis and lack of screening
markers. Presence of gallstone/s is viewed as an important
risk factor for GBC. A functional telomere, bound with
shelterin protein complex, is central to maintaining genomic
stability, failing which, may result in carcinogenesis. In this
study we analyzed the role of telomere length variation and
shelterin complex gene expression in GBC pathogenesis. Materials and methods: Monochrome multiplex qPCR
was performed to analyze telomere length and RT-qPCR
was performed for expression analysis of shelterin genes
(TERF1, TERF2, POT1, TERF2IP and TINF2) in 15 GBC
and 10 gallstone patients. Statistical analysis was done
using SigmaPlot v.11 software. O. Calvete1, H. Valdés-Socin2, J. Reyes3, J. Benitez1 A. F. T. Rossi1, J. C. Contiero1, G. C. Moraes1, F. E. Severino2,
A. E. Silva1 1Centro Nacional de Investigaciones Oncologicas (CNIO),
Madrid, Spain, 2Service d' Endocrinologie. CHU de Liège.,
Liège, Belgium, 3Department of Gastroenterology, Hospital
INCA., Majorca, Spain A. F. T. Rossi1, J. C. Contiero1, G. C. Moraes1, F. E. Severino2,
A. E. Silva1 1São Paulo State University - UNESP, São José do Rio Preto,
SP, Brazil, 2São Paulo State University - UNESP, Botucatu,
SP, Brazil Gastric neuroendocrine tumors (gNET) arise from enter-
ochromaffin cell hyperplasia, which classically occurs in
patients with autoimmune atrophic gastritis. Tumor pro-
gression entails gastric achlorhydria due to the destruction
of parietal cells, which acidifies the stomach through the
ATP4A proton pump. However, we have recently identified
a mutation in the ATP4A gene that explained achlorhydria
in a studied familial gNET. The knockin mouse model
confirmed achlorhydria as the main effector of the tumor
development. A second studied familial gNET case that
segregated with two autoimmune diseases (Hashimoto
hypothyroidism and rheumatoid arthritis) allowed us to
describe a second mutated gene. The three individuals
affected with gNET had two heterozygous mutations in the
ATP4A and PTH1R genes (digenic model). The PTH1R
gene, which directly regulates the gastric cells homeostasis
and gastric chlorhydria, was also found involved in the
regulation of Ca2+ by thyrotrophin (TSH) in the thyroid
gland regulation pathway. We found that the cumulative
effect of both mutations explained the gNET but also
allowed us to link the gastric disease with both autoimmune
pathologies (hypothyroidism and arthritis) with a common
genetic origin. In order to deeper investigate this relation-
ship, we have studied by WES five families with thyr-
ogastric
disease
(chronic
atrophic
gastritis
and
hypothyroidism). In all families, we have found germinal
deleterious mutations in new genes expressed in parietal
cells that are involved in the regulation of gastric chlorhy-
dria and Ca2+ homeostasis. This data has allowed us to
compose a comprehensive genetic landscape of the biology
of the gastric achlorhydria in thyrogastric disease. Introduction: Tumor necrosis factor (TNF)-α, a proin-
flammatory cytokine, can induce cell survival or apoptosis
due to binding to TNFR1 or TNFR2 receptors. Both
receptors may lead to cell survival, but only TNFR1 results
in apoptosis, so deregulation in this pathway can promotes
malignant progression. Thus, we investigated the mRNA
and miRNAs expression involved in the TNF-α signaling
pathway in gastric carcinogenesis and mRNA:miRNA
interaction networks. Deregulation of TNF-a signaling pathway in gastric
carcinogenesis mediated by miRNAs Deregulation of TNF-a signaling pathway in gastric
carcinogenesis mediated by miRNAs Masaryk Memorial Cancer Institute, Brno, Czech Republic Results: Telomere length was found to be significantly
reduced in tumor tissues, irrespective of the presence of Results: Telomere length was found to be significantly
reduced in tumor tissues, irrespective of the presence of Abstracts from the 51st European Society of Human Genetics Conference: Posters 423 confirmed the presence of APC c.-191T>C mutation in
several Czech high risk patients with gastric adenocarci-
noma and/or proximal polyposis of the stomach. Supported
by Czech Ministry of Health: MH CZ - DRO (MMCI,
00209805) confirmed the presence of APC c.-191T>C mutation in
several Czech high risk patients with gastric adenocarci-
noma and/or proximal polyposis of the stomach. Supported
by Czech Ministry of Health: MH CZ - DRO (MMCI,
00209805) miRNAs are deregulated and may influence the early stages
of gastric carcinogenesis, suggesting a new altered pathway
in gastric cancer. Financing: FAPESP, CNPq A.F.T. Rossi:
None. J.C. Contiero:
None. G.C. Moraes: None. F.E. Severino: None. A.E. Silva: None. E. Machackova: None. M. Navratilova: None. E. Stahlova Hrabincova: None. P. Vasickova: None. J. Hazova: None. M. Svoboda: None. L. Foretova: None. O. Calvete: None. H. Valdés-Socin: None. J. Reyes:
None. J. Benitez: None. O. Calvete1, H. Valdés-Socin2, J. Reyes3, J. Benitez1 To understand one of these
molecular mechanisms, we forced to overexpress CD24 in
U87 GBM like cells in which CD133 and CD24 expression
are low and then we performed CSC array by quantitative
real-time PCR in CD24+ and CD24- U87 cells. We found
that mRNAs of epidermal growth factor (EGF), integrin
alpha-2 (ITGA2) or CD49b and mucin 1 (MUC1) were
increased 2 folds, while DNA-binding protein inhibitor ID-
1, jagged 1 (JAG1) and snail 1(SNAIL1) were increased 4
folds compared to those expressed in CD24- cells. These
results suggest that CD24 induces activation of the EGFR,
NOTCH signaling pathways and Epithelial Mesenchymal
Transition. This research has been supported by The Sci-
entific and Technological Research Council of Turkey
(No:114S189). Gliomas are the most prevalent intracranial tumors and
glioblastoma (GBM) is the most malign type among these
tumors due to average short patient survival (12-15 months)
and patients’ limited response to the treatments. Therefore,
efforts to better understand the biology of GBM require the
development of effective therapies and prognostic markers
that provide patient survival. One of these efforts is the
discovery of the presence of cancer stem cells (CSC) which
is correlated with the malignity of GBM. CD24, one of the
most studied GBM stem cell markers, has been found to be
prominently elevated in GBM tumors, indicating its invol-
vement in tumor propogation. However, the molecular
mechanisms underlying the tumor promotion in CD24+
GBM cells is still unclear. To understand one of these
molecular mechanisms, we forced to overexpress CD24 in
U87 GBM like cells in which CD133 and CD24 expression
are low and then we performed CSC array by quantitative
real-time PCR in CD24+ and CD24- U87 cells. We found
that mRNAs of epidermal growth factor (EGF), integrin
alpha-2 (ITGA2) or CD49b and mucin 1 (MUC1) were
increased 2 folds, while DNA-binding protein inhibitor ID-
1, jagged 1 (JAG1) and snail 1(SNAIL1) were increased 4
folds compared to those expressed in CD24- cells. These
results suggest that CD24 induces activation of the EGFR,
NOTCH signaling pathways and Epithelial Mesenchymal
Transition. This research has been supported by The Sci-
entific and Technological Research Council of Turkey
(No:114S189). A. Sunguroglu1, D. A. Yildiz2, Y. Hekmatshoar1, T. Ozkan1,
Y. Dilek1, H. C. Ugur3 1Seegene medical foundation, Seoul, Korea, Republic of, 2Kangwon National University, Chuncheon, Korea, Republic Background: The aim of this study is to dertermine the
frequency and type of KIT gene mutation in Korean patients
with gastrointestinal stromal tumors (GISTs). Methods: Fourty four cases of GISTs were examined. DNA samples were extracted from hematoxylin/eosin-
stained sections of representative paraffin-embedded blocks
using a QIAamp DNA Mini Kit (QUIAGEN, Hilden,
Germany). Bidirectional sequencing was performed using
the BigDye Terminator v1.1 kit (Applied Biosystems,
Foster city, CA, USA). Exons 9, 11, 13 and 17 of the KIT
gene were amplified by PCR and sequenced. Results: We detected mutations in 38 (86.4%) of the 44
patients with GISTs by direct sequencing. KIT exon 11
mutations were detected in 31 (70.5%) of 44 GISTs (19
deletions, 10 missense mutations, and 2 duplications);
followed by KIT exon 9 mutations (5 duplications); and
KIT exon 13 and exon 17 mutations in 1 each (2 missense
mutations). Most of mutations consisted of the in-frame
deletion from 3 to 51 bp in heterozygous fashion. Frame
deletions
and
point
mutations
were
most
frequently
observed at codons 550-580, but duplications were most
concentrated at codons 502-503. Double mutation (exons 9
and 17) in metastatic GIST patient was detected in 1 (2.3%)
of 44. KIT mutations were slightly more frequent in
metastatic than in nonmetastatic GISTs (100% vs. 83.8%). Results: We detected mutations in 38 (86.4%) of the 44
patients with GISTs by direct sequencing. KIT exon 11
mutations were detected in 31 (70.5%) of 44 GISTs (19
deletions, 10 missense mutations, and 2 duplications);
followed by KIT exon 9 mutations (5 duplications); and
KIT exon 13 and exon 17 mutations in 1 each (2 missense
mutations). Most of mutations consisted of the in-frame
deletion from 3 to 51 bp in heterozygous fashion. Frame
deletions
and
point
mutations
were
most
frequently
observed at codons 550-580, but duplications were most
concentrated at codons 502-503. Double mutation (exons 9
and 17) in metastatic GIST patient was detected in 1 (2.3%)
of 44. KIT mutations were slightly more frequent in
metastatic than in nonmetastatic GISTs (100% vs. 83.8%). A. Sunguroglu: None. D.A. Yildiz: None. Y. Hekmat-
shoar: None. T. Ozkan: None. Y. Dilek: None. H.C. Ugur: None. 1Ankara University, School of Medicine, Department of
Medical Biology, Ankara, Turkey, 2Department of Biology,
Faculty of Arts and Sciences, Mehmet Akif Ersoy University,,
Burdur, Turkey, 3Department of Neurosurgery, Faculty of
Medicine, Ankara University, Ankara, Turkey O. Calvete1, H. Valdés-Socin2, J. Reyes3, J. Benitez1 Introduction: Tumor necrosis factor (TNF)-α, a proin-
flammatory cytokine, can induce cell survival or apoptosis
due to binding to TNFR1 or TNFR2 receptors. Both
receptors may lead to cell survival, but only TNFR1 results
in apoptosis, so deregulation in this pathway can promotes
malignant progression. Thus, we investigated the mRNA
and miRNAs expression involved in the TNF-α signaling
pathway in gastric carcinogenesis and mRNA:miRNA
interaction networks. Materials and Methods: Relative quantification (RQ) of
gene expression (TNFA, TNFR1, TNFR2, TRADD, TRAF2,
CFLIP, CASP8, CASP3, NFKB1, NFKB2) and miRNAs
(miR-19a, miR-34a, miR-103a, miR-130a, miR-181c) was
assessed by the 2(-ΔΔCt) method after RT-qPCR (TaqMan®
assays) in 31 gastric cancer (GC) and 30 intestinal
metaplasia (IM) tissue samples using normal mucosa pool
as calibrator. ACTB/GAPDH and RNU6B/RNU48 normal-
ized the mRNA and miRNA expression, respectively. Interation network were obtained by Cytoscape v3.1.1. Results: IM showed downregulation of various mRNA as
TNFR1, TNFR2, NFKB1, NFKB2 and miR-19a, miR-103a
and miR-130a. Contrary, GC showed upregulation most of
the TNFA signaling pathway genes involved in cell survival
as TNFA, TNFR2, TRAF2, TRADD, CFLIP, NFKB2 and
miR-34a, except TNFR1 and CASP3 that were down-
regulated. Correlation analyzes and interaction network
showed
various
relationship
between
miRNAs:mRNA
highlighting the miR-103a that target various TNF-α
pathway genes. O. Calvete: None. H. Valdés-Socin: None. J. Reyes:
None. J. Benitez: None. O. Calvete: None. H. Valdés-Socin: None. J. Reyes:
None. J. Benitez: None. Conclusion: TNF-α participates in the gastric carcino-
genesis, favoring cell survival through the TNF-α/TNFR2/
NF-κB pathway and blocking of TNFR1-mediated apopto-
sis,
with
predominance
of
anti-apoptotic
mediators. 424 J. del Picchia Gliomas are the most prevalent intracranial tumors and
glioblastoma (GBM) is the most malign type among these
tumors due to average short patient survival (12-15 months)
and patients’ limited response to the treatments. Therefore,
efforts to better understand the biology of GBM require the
development of effective therapies and prognostic markers
that provide patient survival. One of these efforts is the
discovery of the presence of cancer stem cells (CSC) which
is correlated with the malignity of GBM. CD24, one of the
most studied GBM stem cell markers, has been found to be
prominently elevated in GBM tumors, indicating its invol-
vement in tumor propogation. However, the molecular
mechanisms underlying the tumor promotion in CD24+
GBM cells is still unclear. Gene expression with association of estrogen receptor
status and tumor stage in breast cancer Gene expression with association of estrogen receptor
status and tumor stage in breast cancer Conclusions: In most cases, the mutations were found at
KIT exon 11, and in-frame deletions were the most
common type. K. Grishina1, T. Muzaffarova1, N. Pospekhova1, U. Krumin2,
V. Khaylenko2, A. Karpukhin1 K. Grishina1, T. Muzaffarova1, N. Pospekhova1, U. Krumin2,
V. Khaylenko2, A. Karpukhin1 S. Jung: None. K. Lee: None. 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Blokhin Russian Cancer Research Centre,
Moscow, Russian Federation P12.086B S. Jung1, K. Lee2 1Seegene medical foundation, Seoul, Korea, Republic of,
2Kangwon National University, Chuncheon, Korea, Republic
of 1Seegene medical foundation, Seoul, Korea, Republic of,
2Kangwon National University, Chuncheon, Korea, Republic
of 1Seegene medical foundation, Seoul, Korea, Republic of, P12.087C P12.087C
CD24 gene overexpression induces EGFR and NOTCH
signaling pathways and EMT in Glioblastoma cells P12.087C
CD24 gene overexpression induces EGFR and NOTCH
signaling pathways and EMT in Glioblastoma cells The choice of optimal therapy for breast cancer (BC) can
depend on tumor features. In this connection, the level of 74
genes quantitative expression, selected bioinformationally
as the most significant for the BC development, was per-
formed in tumor in relation to the normal breast tissue. The
sample of BC consists of infiltrative ductal BC on I-II
stages. All patients of the sample were not subject to
radiation, neither to chemotherapy. The gene differential
expression on II stages in relation to stage I was revealed for
eight genes: BIRC5, CD151, mTOR, uPAR, ZEB1, FOXO1, Abstracts from the 51st European Society of Human Genetics Conference: Posters 425 KLF8, PDGFR (p = 0.008-0.04). The gene expression was
studied in groups with estrogen-positive and estrogen-
negative tumors. An increased expression of the CSF1R
gene in the group with estrogen-positive BC (OR = 17.3, p
=
0.01)
was
revealed. The
CSF1R
is
a
key
pro-
inflammatory cytokine that is involved in the recruitment
and activation of tissue macrophages. Tumor-associated
macrophages have been identified as regulators that enhance
angiogenic, invasive and metastatic programming of neo-
plastic tissue. Our data suggest the increased recruitment of
macrophages in estrogen-positive in comparison with
estrogen-negative BC tumors. Methods: The 3248 individuals analysed for TA insertion
in UGT1A1 promotor were divided into three groups: 1)
group of patients with different types of carcinomas
classified in subgroups, 2) control group and 3) group of
individuals older than 60 years without personal and family
history of cancer. Genotypic differences in these groups
were statistically evaluated by chi square test. Methods: The 3248 individuals analysed for TA insertion
in UGT1A1 promotor were divided into three groups: 1)
group of patients with different types of carcinomas
classified in subgroups, 2) control group and 3) group of
individuals older than 60 years without personal and family
history of cancer. Genotypic differences in these groups
were statistically evaluated by chi square test. Results: In all cancer subgroups and in group of
individuals older than 60 years without carcinomas were
found different frequencies of TA genotypes compared to
the control group. Conclusion: Herein we discuss the protective effect of
bilirubin and UGT1A1 activity against the development of
different types of cancers. P12.089A
Gilbert syndrome and cancers Gilbert syndrome and cancers A. Boday: None. S. Tavandzis: None. M. Skrovina: A. Boday: None. S. Tavandzis: None. M. Skrovina:
None. J. Syrovatka: None. J. Kollarova: None. E. Pal:
None. J. Roubec: None. G. Ondrejka: None. R. Vrtel:
None. O. Urban: None. A. Boday1, S. Tavandzis1, M. Skrovina2, J. Syrovatka3,
J. Kollarova4, E. Pal5, J. Roubec6, G. Ondrejka7, R. Vrtel8,
O. Urban9 None. J. Syrovatka: None. J. Kollarova: None. E. Pal: None. J. Roubec: None. G. Ondrejka: None. R. Vrtel: None. J. Roubec: None. G. Ondrejka: None. R. Vrtel:
None. O. Urban: None. 1AGEL Laboratories, Laboratory of Molecular Biology, Novy
Jicin, Czech Republic, 2Comprehensive Cancer Center, AGEL,
Department of Surgery, Novy Jicin, Czech Republic,
3Comprehensive Cancer Center, AGEL, Department of ORL,
Novy Jicin, Czech Republic, 4Komarno Hospital, Department
of Internal Medicine, Komarno, Slovakia, 5University Hospital
L. Pasteur, Department of Laboratory Medicine, Kosice,
Slovakia, 6Vitkovice Hospital, AGEL, Department of
Pneumology, Ostrava, Czech Republic, 7Comprehensive
Cancer Center, AGEL, Department of Pneumology, Novy
Jicin, Czech Republic, 8Institute of Medical Genetics, Faculty
Hospital, Olomouc, Czech Republic, 9Vitkovice Hospital,
AGEL, Department of Gastroenterology, Ostrava, Czech
Republic P12.087C The variances found in the
genotypes of cancer subgroups are explained by their
different etiology, as well as by genetic background of the
organism, the influence of environment and lifestyle of the
individuals. K. Grishina: None. T. Muzaffarova: None. N. Pospe-
khova: None. U. Krumin: None. V. Khaylenko: None. A. Karpukhin: None. K. Grishina: None. T. Muzaffarova: None. N. Pospe-
khova: None. U. Krumin: None. V. Khaylenko: None. A. Karpukhin: None. Impact of miRNA-138 in infiltrative and migratory ability
and MMMI profiles of primary glioblastoma Impact of miRNA-138 in infiltrative and migratory ability
and MMMI profiles of primary glioblastoma L. Muñoz Hidalgo1, C. Lòpez Ginés2, A. Carratalá1, R. Gil
Benso2, J. Megías2, T. San Miguel2, M. Cerdá Nicolás2 L. Muñoz Hidalgo1, C. Lòpez Ginés2, A. Carratalá1, R. Gil
Benso2, J. Megías2, T. San Miguel2, M. Cerdá Nicolás2 Background: Understanding the genetic alterations that
drive glioma formation and progression may help improve
patient’s prognosis and treatment. Here, we investigated
gliomas samples from Northern Brazil to identify recurrent
oncogenic copy number alterations (CNAs) and gene
mutations. 1Foundation for research HCU-INCLIVA, Valencia, Spain,
2Department of Pathology, Medical School, University of
Valencia, Valencia, Spain Methods: In the present study, thirty-seven samples of
gliomas were retrospectively analyzed in detail by mole-
cular approaches, i.e. array-comparative genomic hybridi-
zation (aCGH), polymerase chain reaction-single-strand
conformation polymorphism (PCR-SSCP) followed by
DNA sequencing and fluorescence in situ hybridization
(FISH). Introduction: Glioblastoma (GB) is the most common and
aggressive entity from the primary tumors of the central
nervous system shows a high infiltrative capacity. In pre-
vious studies, we have identified in primary GB, significant
alterations in the expression of several miRNAs related to
the different models of EGFR amplification and with
MMMI changes and angiogenesis processes. In the present
work we show the downregulation of miRNA-138 expres-
sion which, through its HIF-1 modulating action, acts as a
transcription factor activated in hypoxia situations, with the
ability of modulating behavior of neoplastic cells playing a
role in tumorgenesis. Results: CNAs were detected in 28/37 gliomas. The most
frequent CNAs were loss of #9, #10, 13q, 22q and gain of
#4, #7, #19 and #20. Of special interest among the detected
CNAs are the following findings: gain of BRAF and
amplification of EGFR genes (8.1% and 16.2%, respec-
tively) and loss of TP53, PTEN and deletion of CDKN2A
genes (8.1%, 16.2% and 24.3%, respectively). In addition,
we found occurrence of mutations in hotspot regions of
TP53 in 12.2% of the gliomas, while no mutation was
identified in PTEN. Material and Methods: We propose the development an
in vitro model of study based on GB cell cultures, Allowing
comparative analyses in cell lines and primary cultures of
primary GB, with different levels of EGFR amplification: (i)
in situations of miRNA-138 overexpression and silencing,
(ii) in situations of EG FR and HIF mRNA inhibition (iii) in
different situations of hypoxia. Genetic alterations of gliomas I. A. Pessôa1,2, M. A. K. Othman1, C. K. N. Amorim2, W. A. S. Ferreira2, R. J. R. N. Brito3, T. Liehr1, E. H. C. de Oliveira2 M. Rinelli: None. G. Milano: None. R. De Vito: None. I. Russo: None. F. Locatelli: None. A. Novelli: None. E. Agolini: None. Ferreira2, R. J. R. N. Brito3, T. Liehr1, E. H. C. de Oliveira2
1Jena University Hospital, Jena, Germany, 2Tissue Culture and
Cytogenetics Laboratory, Evandro Chagas Institute, Belém,
Pará, Brazil., Brazil, 3Ophir Loyola Hospital, Belém, Pará,
Brazil., Brazil 1Jena University Hospital, Jena, Germany, 2Tissue Culture and
Cytogenetics Laboratory, Evandro Chagas Institute, Belém,
Pará, Brazil., Brazil, 3Ophir Loyola Hospital, Belém, Pará,
Brazil., Brazil Carney-Stratakis syndrome in two patients with SDHB/
SDHC genes mutations and Gastrointestinal Stromal
Tumor (GIST) M. Rinelli1, G. Milano2, R. De Vito2, I. Russo2, F. Locatelli2,
A. Novelli1, E. Agolini1 1UOC Laboratory of Medical Genetics, Children's Hospital
Bambino Gesù, Rome, Italy, 2Department of Pediatric
Hematology and Oncology, Children's Hospital Bambino
Gesù, Rome, Italy 1UOC Laboratory of Medical Genetics, Children's Hospital
Bambino Gesù, Rome, Italy, 2Department of Pediatric
Hematology and Oncology, Children's Hospital Bambino
Gesù, Rome, Italy Gastrointestinal Stromal Tumors (GISTs) are the most
common mesenchymal tumors of the gastrointestinal tract. KIT and PDGFRA mutations account for 85-90% of GIST
carcinogenesis. However, the remaining 10-15% of GISTs
present loss of function mutations in genes encoding for the
subunits of the succinate dehydrogenase (SDH) complex:
SDHA, SDHB, SDHC and SDHD. The occurrence of SDH-
deficient GISTs is restricted to stomach, and they typically
occur in children and young adults representing a spectrum
of clinical behavior from indolent to progressive. SDH-
deficient GISTs can be due to either genetic or epigenetic
changes. We report on two patients who were diagnosed
with a gastric GIST at 14 years of age. Both tumors were c-
kit-negative based on immunohistochemical staining. Next
generation sequencing analysis on the tumor DNA samples
revealed two homozygous mutations, a maternally inherited
splicing variant and a de novo 1-bp frameshift deletion, Introduction: UDP-glucuronosyltransferase 1A isoform 1
is phase II xenobiotic biotransformation enzyme. Mutations
in UGT1A1 gene can cause insufficient glucuronidation of
various endogenous (e.g. bilirubin) and exogenous sub-
stances (carcinogens and xenobiotics). Bilirubin is one of
the strongest antioxidant in the body. Gilbert syndrome
(GS) is one of the genetic disorders of bilirubin catabolism
which is characterized by a moderate increase of total
bilirubin. GS frequency is high (8-15%) in Caucasian,
African and Afro-American populations due to the occur-
rence of TA dinucleotide insertion in UGT1A1 gene pro-
motor. The aim of this study was to determine whether
people with GS with higher levels of total bilirubin are
better protected against carcinomas than others. J. del Picchia 426 the changes in the MMMI, suggesting a potential role for
these molecules in the pathogenesis of GB.FIS P114/01669
and PROMETEO II/2015/009 the changes in the MMMI, suggesting a potential role for
these molecules in the pathogenesis of GB.FIS P114/01669
and PROMETEO II/2015/009 respectively in the SDHB and SDHC genes. The same
mutations were confirmed in heterozygous state in the
constitutional DNA from peripheral blood leukocytes. Carney-Stratakis syndrome in two patients with SDHB/
SDHC genes mutations and Gastrointestinal Stromal
Tumor (GIST) Germline mutations in SDHB, SDHC, and SDHD were seen
in patients with the Carney-Stratakis syndrome (CSS), a
very rare condition inherited in an autosomal dominant
manner with incomplete penetrance, who are predisposed to
developing paraganglioma and GIST. Here we report on
two new cases of the CSS, with a gastric GIST, expanding
the clinical and molecular spectrum of the disease. the changes in the MMMI, suggesting a potential role for
these molecules in the pathogenesis of GB.FIS P114/01669
and PROMETEO II/2015/009 respectively in the SDHB and SDHC genes. The same
mutations were confirmed in heterozygous state in the
constitutional DNA from peripheral blood leukocytes. Germline mutations in SDHB, SDHC, and SDHD were seen
in patients with the Carney-Stratakis syndrome (CSS), a
very rare condition inherited in an autosomal dominant
manner with incomplete penetrance, who are predisposed to
developing paraganglioma and GIST. Here we report on
two new cases of the CSS, with a gastric GIST, expanding
the clinical and molecular spectrum of the disease. L. Muñoz Hidalgo: None. C. Lòpez Ginés: None. A. Carratalá: None. R. Gil Benso: None. J. Megías: None. T. San Miguel: None. M. Cerdá Nicolás: None. F. Ganci1, F. Spinella2, G. Cottone2, E. Cotroneo2, A. Nuccitelli2,
A. Biroccio1, A. Sacconi1, V. Manciocco3, M. Pallocca3,
F. Rollo4, R. Covello4, M. Benevolo4, G. Spriano3,
F. Fiorentino2, G. Blandino1 HSEIT/SEEBMO, Angra do Heroismo, Portugal Germline
mutations
in
CDH1,
encoding
E-cadherin,
account for around 30% of cases of Hereditary Diffuse
Gastric Cancer (HDGC). Individuals carrying a pathogenic
mutation in CDH1 have a high lifetime risk of developing
diffuse gastric cancer, estimated to be 70% in men and 56%
in woman. Female carriers also present an elevated risk to
develop lobular breast cancer. Introduction: Breast cancer is the most prevalent cancer
among women. Pathogenic variants in BRCA genes are the
main cause of hereditary breast and ovarian cancer syn-
drome (HBOC); 7% of all breast cancers (BC) and 11-15%
of ovarian cancers (OC) are associated with inherited pre-
disposition, with a 40-80% lifetime risk of developing BC,
and 11-50% of developing OC. The identification of carriers
can significantly impact their and at-risk family members
medical management. Identification of causal mutation allows predictive testing
of family members and preventive care of carriers. Here we
describe the identification of a large unpublished genomic
rearrangement in CDH1 gene. Materials and Methods: The mutational profile and
prevalence of BRCA variants were evaluated among 139
Azorean probands (cases) fulfilling the NCCN HBOC
testing criteria. Variants were first scanned using HRM and
the polymorphic fragments were Sanger sequenced. The
clinical significance of the detected variants was assessed
using ClinVar database. Variants not found in ClinVar were
assessed in Ensembl, dbSNP, LOVD and BRCA Share
databases. Molecular testing of the index patient by sequencing and
MLPA methods revealed a deletion encompassing exons 10
and 11, leading to an expected frameshift and premature
stop codon. RNA study was performed to exclude false
positive result of the MLPA method and to confirm the
pathogenicity of the mutation. The presence of a premature
stop codon was confirmed and the mutation was considered
as likely pathogenic. Familial screening was therefore
proposed in this large family. Results: Seventy-four variants were detected in the
samples analyzed: 33 in BRCA1 gene and 41 in BRCA2
gene. Three were pathogenic (BRCA1 c.2717delA and
BRCA2 c.1138delA, c.2808_2811delAAAC), six were of
uncertain significance, two had conflicting interpretations of
pathogenicity and one, intronic with no significant splicing
motif alteration, was not found in consulted databases
(c.441+51T>C). The remaining 61 were classified as
benign or likely benign. During this familial screening, the mutation was identified
in several “old” healthy patients. I.A. Pessôa: None. M.A.K. Othman: None. C.K.N.
Amorim: None. W.A.S. Ferreira: None. R.J.R.N. Brito:
None. T. Liehr: None. E.H.C. de Oliveira: None. SEEBMO. J. Bruges-Armas: A. Employment (full or
part-time); Significant; HSEIT / SEEBMO. HSEIT/SEEBMO, Angra do Heroismo, Portugal This unexpected result
leads us to the hypothesis that the mutation may show
variable penetrance which complicate the follow-up and
prophylactic recommendations to give to the family. After multiple discussions with geneticists, CDH1 experts
and gastro-enterologists, prophylactic gastrectomy was
proposed to mutation carriers of the family. At this time,
several carriers undergo surgery and pre-tumoral cells were
identified in at least two of the carriers. Conclusion: The low percentage (2,2%) of pathogenic
variants detected, in a group fulfilling NCCN HBOC testing
criteria, suggests a strong contribution of genes other than
BRCAs. To achieve a more efficient detection a broad panel
of genes implicated should be applied. K.A. segers: None. V. Bours: None. A. Bynens: None. P. Leclercq: None. M. R. Santos, A. R. Couto, F. Rocha, B. Parreira, P. Sousa,
J. Bruges-Armas chu de liège, liège, Belgium HSEIT/SEEBMO, Angra do Heroismo, Portugal Impact of miRNA-138 in infiltrative and migratory ability
and MMMI profiles of primary glioblastoma Conclusion:
High
resolution
molecular
approaches
combined in a cost-efficient way are an important tool to
help in grading and understanding in future associations
between genetic alteration in gliomas and prognosis. Higher
rates of CNAs in highly malignant compared to low grade
gliomas were identified. In addition, the TP53 pathway is
impaired, either by mutation or chromosomal loss, while
allelic
loss
of
PTEN
could
represent
an
alternative
mechanism
for
its
protein
inactivation
in
gliomas
development. Results: The miR-138 expression levels were stable
under
hypoxia
conditions
but
decresed
in
normoxia
conditions. miR-138 overexpression, an increase in the
miR-138 values and a decrease of VEGF and HIF1a mRNA
in both normal and hypoxic conditions were observed. Results: The miR-138 expression levels were stable
under
hypoxia
conditions
but
decresed
in
normoxia
conditions. miR-138 overexpression, an increase in the
miR-138 values and a decrease of VEGF and HIF1a mRNA
in both normal and hypoxic conditions were observed. Conclusion: Our findings indicate that miR-138 is
deregulated in GB and changed their expression levels
within the different grades of GB. We reaffirm the
possibility of miR-138 being able to act by modulating p
This work partial supported from DAAD and CNPq This work partial supported from DAAD and CNPq Abstracts from the 51st European Society of Human Genetics Conference: Posters 427 I.A. Pessôa: None. M.A.K. Othman: None. C.K.N. Amorim: None. W.A.S. Ferreira: None. R.J.R.N. Brito:
None. T. Liehr: None. E.H.C. de Oliveira: None. k. A. segers, V. Bours, A. Bynens, P. Leclercq M. R. Santos, A. R. Couto, F. Rocha, B. Parreira, P. Sousa,
J. Bruges-Armas P12.095C
Genetics of breast cancer in the Azores islands - BRCA
genes k. A. segers, V. Bours, A. Bynens, P. Leclercq P12.097A A mutational chip to detect TP53, CDKN2A and FAT1
mutations in circulating cell free-DNA of head and neck
squamous cell carcinoma patients:a pilot study M.R. Santos:
A. Employment
(full
or
part-time);
Significant; HSEIT / SEEBMO. A.R. Couto: A. Employ-
ment (full or part-time); Significant; HSEIT / SEEBMO. F. Rocha: A. Employment (full or part-time); Significant;
HSEIT / SEEBMO. B. Parreira: A. Employment (full or
part-time); Significant; HSEIT / SEEBMO. P. Sousa: A. Employment (full or part-time); Significant; HSEIT / A mutational chip to detect TP53, CDKN2A and FAT1
mutations in circulating cell free-DNA of head and neck
squamous cell carcinoma patients:a pilot study 428 J. del Picchia Technology Research and Application Center, Selcuklu,
Konya, Turkey 1Oncogenomic and Epigenetic Unit, Italian National Cancer
Institute “Regina Elena”, Rome, Italy, 2Genoma Group, Rome,
Italy, 3Otolaryngology Department, Italian National Cancer
Institute “Regina Elena”, Rome, Italy, 4Pathology Department,
Italian National Cancer Institute “Regina Elena”, Rome, Italy 1Oncogenomic and Epigenetic Unit, Italian National Cancer
Institute “Regina Elena”, Rome, Italy, 2Genoma Group, Rome,
Italy, 3Otolaryngology Department, Italian National Cancer Introduction: Endothelial cells constitute an important part
of the tumor microenvironment (TME). However, the
underlying mechanisms of their functions within the
microenvironment of head and neck squamous cell carci-
noma (HNSCC) remain poorly understood. Here we per-
formed an in vitro non-contact co-cultivation system to
analyze the influence of microenvironment in both tumor
(laryngeal cancer cell line HEp-2) and healthy cells (endo-
thelial cell line HUVEC cells). Introduction: Head and neck squamous cell carcinoma
(HNSCC) is characterized by a high incidence of relapse,
which is the common cause of death in HNSCC patients. The identification of biomarkers supporting the manage-
ment of HNSCC disease is still an unmet need in clinical
oncology. Materials and Methods: We prepared conditioned
medium (CM) from cell cultures for migration assay. CM
has components secreted by cells such as specific cytokines,
chemokines, growth factors. CM provides a co-culture
microenvironments for the cells. We have used in vitro
strach assay to examine cell migration and cell interactions. Photos were taken at intervals of 0, 3, 6, 9, 12 and 24 h. To
assess the exact speed of cell migration, ImageJ software
was used to measure the change in the cell-covered area
over time, which is the characteristic parameter in migration
assays. P12.097A Meterials and Methods: To this end, we designed a
mutational chip for next generation sequencing (NGS)
analysis to detect mutations in tumor tissues and matched
plasma of HNSCC patients. The analysis of the HNSCC
TCGA cohort revealed that TP53 (72%), CDKN2A (22%)
and FAT1 (24%) are the most frequently mutated genes in
HNSCCs. Notably TP53 mutations associate with poor
outcome. A cohort of 250 fresh frozen tissues from HNSCC
patients has been collected. Specifically, for each patient
three biopsies representing non-tumoral (resection margin),
peritumor (histologically tumor-free tissue at ≥1cm from the
tumor) and tumor tissues were collected. Materials and Methods: We prepared conditioned
medium (CM) from cell cultures for migration assay. CM
has components secreted by cells such as specific cytokines,
chemokines, growth factors. CM provides a co-culture
microenvironments for the cells. We have used in vitro
strach assay to examine cell migration and cell interactions. Photos were taken at intervals of 0, 3, 6, 9, 12 and 24 h. To
assess the exact speed of cell migration, ImageJ software
was used to measure the change in the cell-covered area
over time, which is the characteristic parameter in migration
assays. Results: The wound healing assay showed that HEp-2
cells grown in CM from HUVEC have less capacity for
migration and mobility compared with HEp-2 cells grown
in the culture medium alone. HUVEC cells grown in CM
from HEp-2 have less capacity for migration and mobility
compared
with
HUVEC
cells
grown
in
the
culture
medium alone. Results:
This
cohort
has
been
challenged
with
a
customized mutational chip for NGS analysis of mutations
that includes the entire CDS of the TP53, CDKN2A and
FAT1 genes. We found that the TP53 (743%) and
CDKN2A (243%) mutation frequency in tumors of our
cohort was similar to that of TGCA. While the frequency of
FAT1 (38,6%) mutations was higher in our dataset. Many
of the identified FAT1 mutations have not been annotated
yet. Since we have also collected matched plasma at the
surgery and during follow-up, our analysis is progressing
toward the identification of mutations in ccf-DNA from
patients. Conclusions: In vitro co-culture cell system for investi-
gation of interaction between tumor cells and the tumor
microenvironment was used. We believe that this study will
contribute to our understanding of tumor microenvironment
effect in head and neck carcinoma. Z.B. Sari: None. E. Yavuz: None. T. Cora: None. P12.097A Conclusions: This analysis is ongoing and the related
data will be presented. Pathologies of helicases and premature ageing: study by
derivation of induced pluripotent stem cells F. Ganci: None. F. Spinella: None. G. Cottone: None. E. Cotroneo: None. A. Nuccitelli: None. A. Biroccio:
None. A. Sacconi: None. V. Manciocco: None. M. Pallocca: None. F. Rollo: None. R. Covello: None. M. Benevolo: None. G. Spriano: None. F. Fiorentino: None. G. Blandino: None. Pathologies of helicases and premature ageing: study by
derivation of induced pluripotent stem cells V. Gatinois1, R. Desprat2, C. Corsini3, J. Puechberty4, J. B. Gaillard1, A. Schneider1, C. Lemattre1, T. Guignard1, J. M. Lemaitre2, F. Pellestor1 Z. B. Sari1, E. Yavuz2, T. Cora1 P12.098B 1Unité de génétique chromosomique - Hôpital A. de Villeneuve,
Montpellier, France, 2Plateforme SAFE-iPS - Hôpital St Eloi,
Montpellier, France, 3Service d'Oncogénétique - Hôpital A. de
Villeneuve, Montpellier, France, 4Service de génétique
médicale - Hôpital A. de Villeneuve, Montpellier, France Impact of cross-talk between laryngeal cancer cells and
endothelial cells on cell migration and interactions Helicases process the double-stranded DNA dissociation. They
are
involved
in
replication,
DNA
repair
and 1Department of Medical Genetics, Selcuk University, Medical
Faculty, Selcuklu, konya, Turkey, 2Selcuk University Advanced Abstracts from the 51st European Society of Human Genetics Conference: Posters 429 maintenance of telomeres. In human, 3 helicases display
mutations responsible for clinical syndromes: WRN for the
Werner syndrome, BLM for the Bloom syndrome and
RECQL4 for the Rothmund-Thomson syndrome. All these
diseases cause premature ageing and high risk of cancer. Molecular and cellular mechanisms involved in these dis-
eases are not well defined. Particularly, little is known
concerning the link between genomic instability and ageing. During this project, we used blood samples and skin
biopsies of affected
patients to
generate models
by
reprogramming cells to induced pluripotent stem cells
(iPSCs). These cells have the advantage of self-renewing
and theoretically could be differentiated in all cell types. At
the same time, an iPSC senescence control was performed
from cells of a Hutchinson-Gilford Progeria syndrome
patient. therapy. Next generation sequencing (NGS) of the tran-
scriptome facilitates a more comprehensive expression
profile of both host and viral genes including viral-host
chimeric transcripts. In this study, deep sequencing of the
transcriptome of HCC patients revealed that differentially
expressed host transcripts are enriched in the cell-cycle,
DNA damage response, cell-survival and apoptosis signal-
ling and several of these were significantly associated with
various clinical characteristics. An average of ~230 tumor-
specific mutations were identified for each patients with
most of these mutations being missense mutations within
coding regions. Majority of the tumor-specific mutations
were unique with only ~5% being found in more than a one
patient. Genes harbouring somatic mutatio ns are primarily
in cancer pathways affecting phosphatidyl inositol signal-
ling and ubiquitin-mediated proteolysis. HBV-human chi-
meric transcripts were also observed in these HCC patients
with majority of the HBV transcripts mapping to either the
Pre-S or X gene. Interestingly fewer chimeric transcripts
were observed in the tumor compared to the non-tumorous
tissues. Comprehensive molecular characterization of the
transcriptome of HCC patients Comprehensive molecular characterization of the
transcriptome of HCC patients F. FOSTIRA, I. Konstantopoulou, M. S. Papamentzelopoulou,
P. Apostolou, D. Kalfakakou, A. Delimitsou, A. Vagena, I. S. Vlachos, D. Yannoukakos F. FOSTIRA, I. Konstantopoulou, M. S. Papamentzelopoulou, P. Apostolou, D. Kalfakakou, A. Delimitsou, A. Vagena, I. S. Vlachos, D. Yannoukakos C. G. Lee1, W. Lee2, J. Yu3, S. Toh2, C. Tennakoon4, H. Toh3,
P. Chow3, A. Chung5, L. L. P. J. Ooi5, S. S. Chong2, K. Sung2 C. G. Lee1, W. Lee2, J. Yu3, S. Toh2, C. Tennakoon4, H. Toh3,
P. Chow3, A. Chung5, L. L. P. J. Ooi5, S. S. Chong2, K. Sung2 Multi-gene testing of a high-risk group of Greek breast
cancer patients reveals known and novel gene associations Multi-gene testing of a high-risk group of Greek breast
cancer patients reveals known and novel gene associations P12.098B Gaillard: None. A. Schneider: None. C. Lemattre: None. T. Guignard:
None. J.M. Lemaitre: None. F. Pellestor: None. V. Gatinois: None. R. Desprat: None. C. Corsini:
None. J. Puechberty: None. J.B. Gaillard: None. A. Schneider: None. C. Lemattre: None. T. Guignard:
None. J.M. Lemaitre: None. F. Pellestor: None. P12.098B Majority of the HBV-human chimeric transcripts
were primarily fusion of the HBx gene and introns or
intergenic regions of human genes suggesting that favoured
sites of integration within HBV remains at the C-terminal of
the
HBx
gene. Acknowledgement:
National
Medical
Research Council (NMRC) (CBRG14nov034 and NMRC/
CBRG/0095/2015). maintenance of telomeres. In human, 3 helicases display
mutations responsible for clinical syndromes: WRN for the
Werner syndrome, BLM for the Bloom syndrome and
RECQL4 for the Rothmund-Thomson syndrome. All these
diseases cause premature ageing and high risk of cancer. Molecular and cellular mechanisms involved in these dis-
eases are not well defined. Particularly, little is known
concerning the link between genomic instability and ageing. During this project, we used blood samples and skin
biopsies of affected
patients to
generate models
by
reprogramming cells to induced pluripotent stem cells
(iPSCs). These cells have the advantage of self-renewing
and theoretically could be differentiated in all cell types. At
the same time, an iPSC senescence control was performed
from cells of a Hutchinson-Gilford Progeria syndrome
patient. iPSCs were characterized for pluripotency. In the aim of
recapitulate these pathologies in vitro, we identified sets of
cellular and molecular phenotypes. We also engaged
differentiation of iPSCs in cell pathways closed to the
affected tissues in vivo. Finally, we studied the genomic
stability of iPSCs and derived cells. We observed that
Bloom cells are susceptible to frequent recombinations and
are characterized by a genome instability through all studied
cell types. Werner cells showed an instability of telomeres
length. Finally, all premature ageing diseases displayed
mitochondrial defects. This work was supported by an internal public hospital
fellowship “AOI Jeunes chercheurs, CHU de Montpellier”. V. Gatinois: None. R. Desprat: None. C. Corsini:
None. J. Puechberty: None. J.B. Gaillard: None. A. Schneider: None. C. Lemattre: None. T. Guignard:
None. J.M. Lemaitre: None. F. Pellestor: None. This work was supported by an internal public hospital
fellowship “AOI Jeunes chercheurs, CHU de Montpellier”. C.G. Lee: None. W. Lee: None. J. Yu: None. S. Toh:
None. C. Tennakoon: None. H. Toh: None. P. Chow:
None. A. Chung: None. L.L.P.J. Ooi: None. S.S. Chong:
None. K. Sung: None. C.G. Lee: None. W. Lee: None. J. Yu: None. S. Toh:
None. C. Tennakoon: None. H. Toh: None. P. Chow:
None. A. Chung: None. L.L.P.J. Ooi: None. S.S. Chong:
None. K. Sung: None. V. Gatinois: None. R. Desprat: None. C. Corsini:
None. J. Puechberty: None. J.B. NSCR DEMOKRITOS, ATHENS, Greece 1National Cancer Centre and National University of
Singapore, Singapore, Singapore, 2National University of
Singapore, Singapore, Singapore, 3National Cancer Centre,
Singapore, Singapore, 4Genome Institute of Singapore,
Singapore, Singapore, 5Singapore General Hospital,
Singapore, Singapore Introduction: Although BRCA1 and BRCA2 mutations
dominate among breast cancer (BrCa) patients, additional
genes are associated with BrCa susceptibility, which are
nowadays analyzed simultaneously in multigene panels. The limitation in the implementation of such panels in the
clinical setting is the uncertain clinical implications required
in the case of mutations detected in genes other than
BRCA1/2. Therefore, we sought to investigate the con-
tribution and association of predisposing alleles in highly
selected cohort of Greek BrCa patients. Hepatocellular carcinoma (HCC) is one of the deadliest
cancer of the liver mainly due to late detection and limited
interventional options. There is thus a need to better
understand HCC to facilitate the development of better
biomarkers and identification of potential targets for J. del Picchia 430 San Carlos, Madrid, Spain, 9Laboratorio de Oncología
Molecular, Instituto Universitario de Oncología del
Principado de Asturias, Hospital Universitario Central de
Asturias, Oviedo, Spain, 10Familial Cancer Unit, Department
of Medical Oncology, Instituto Universitario de Oncología del
Principado de Asturias, Hospital Universitario Central de
Asturias, Oviedo, Spain, 11Molecular Biology Laboratory, 12
de Octubre University Hospital, Madrid, Spain, 12Instituto de
Biología y Genética Molecular, IBGM-UVA-CSIC, Valladolid,
Spain, 13Unit of Biomarkers and Susceptibility, Catalan
Institute of Oncology, IDIBELL and CIBERESP, Hospitalet de
Llobregat, Spain, 14Department of Experimental Therapeutics,
The University of Texas MD Anderson Cancer Center,
Houston, TX, United States Materials and methods: The sequences of 94 cancer
genes were targeted by applying Trusight cancer panel in a
high-risk group of 1382 BrCa Greek patients, diagnosed at
very young age (<35years) and/or having strong family
history (at least three breast, ovarian or pancreatic cancer
cases). Case-control analysis was performed comparing the
frequency of loss-of-function (LoF) mutations to Exome
Aggregation Consortium controls. Materials and methods: The sequences of 94 cancer
genes were targeted by applying Trusight cancer panel in a
high-risk group of 1382 BrCa Greek patients, diagnosed at
very young age (<35years) and/or having strong family
history (at least three breast, ovarian or pancreatic cancer
cases). Case-control analysis was performed comparing the
frequency of loss-of-function (LoF) mutations to Exome
Aggregation Consortium controls. P12.103C P12.103C
BRF1, a new gene associated with hereditary colorectal
cancer P12.103C
BRF1, a new gene associated with hereditary colorectal
cancer P. Mur1, N. Sowada2, F. Bellido3, C. Lázaro1, T. Pons4,
R. Valdés-Mas5, M. Pineda1, G. Aiza1, S. Iglesias1, J. Soto6,
M. Urioste7, T. Caldés8, M. Balbín9, P. Blay10, D. Rueda11,
M. Durán12, A. Valencia4, V. Moreno13, J. Brunet1, I. Blanco3,
M. Navarro1, G. A. Calin14, G. Borck2, X. S. Puente5,
G. Capellá1, L. Valle1 P. Mur1, N. Sowada2, F. Bellido3, C. Lázaro1, T. Pons4,
R. Valdés-Mas5, M. Pineda1, G. Aiza1, S. Iglesias1, J. Soto6,
M. Urioste7, T. Caldés8, M. Balbín9, P. Blay10, D. Rueda11,
M. Durán12, A. Valencia4, V. Moreno13, J. Brunet1, I. Blanco3,
M. Navarro1, G. A. Calin14, G. Borck2, X. S. Puente5,
G. Capellá1, L. Valle1 1Catalan Institute of Oncology, IDIBELL and CIBERONC,
Barcelona, Spain, 2Institute of Human Genetics, University of
Ulm, Ulm, Germany, 3Catalan Institute of Oncology, IDIBELL,
Barcelona, Spain, 4Structural Biology and Biocomputing
Program, Spanish National Cancer Research Center (CNIO),
Madrid, Spain, 5Departamento de Bioquímica y Biología
Molecular, Instituto Universitario de Oncología del
Principado de Asturias, Universidad de Oviedo, Oviedo,
Spain, 6Molecular Genetics Laboratory, Elche University
Hospital, Elche, Spain, 7Familial Cancer Clinical Unit, Human
Cancer Genetics Program, Spanish National Cancer Research
Centre (CNIO) and Center for Biomedical Network Research
on Rare Diseases, Madrid, Spain, 8Laboratorio de Oncología
Molecular, Servicio de Oncología Médica, Hospital Clínico P. Mur: None. N. Sowada: None. F. Bellido: None. C. Lázaro: None. T. Pons: None. R. Valdés-Mas: None. M. Pineda: None. G. Aiza: None. S. Iglesias: None. J. Soto:
None. M. Urioste: None. T. Caldés: None. M. Balbín:
None. P. Blay: None. D. Rueda: None. M. Durán: None. A. Valencia: None. V. Moreno: None. J. Brunet: None. I. Blanco: None. M. Navarro: None. G.A. Calin: None. G. Borck: None. X.S. Puente: None. G. Capellá: None. L. Valle: None. NSCR DEMOKRITOS, ATHENS, Greece Results: In total, 31.6% of patients tested carried LoF
mutations in 26 genes; 6.7% carried LoF alleles in the post-
BRCA genes, of which 4.5% involved in CHEK2, ATM and
PALB2 mutations. Case-control analysis showed estab-
lished associations of TP53, PALB2, ATM and CHEK2
truncating mutations (odds ratios 3.4-8.0), as well as novel
associations of RAD51C and missense CHEK2 mutations
(odds
ratios
6.19
&
3.79,
respectively)
to
BrCa
predisposition. The identification of genes associated with hereditary col-
orectal cancer (CRC) facilitates the management of families
and individuals carrying pathogenic mutations, having a
direct impact in the processes of genetic testing and coun-
seling. However, much of the genetic predisposition to CRC
remains
unexplained. We
performed
whole-exome
sequencing in 3 CRC-affected relatives of an Amsterdam
I CRC family. Variants located in 38 genes were shared by
all affected relatives. A splice-site mutation in BRF1 (sub-
unit of RNA polymerase III transcription initiation factor),
stood up as potential causal mutation. BRF1 mutational
screening was performed in 547 additional familial CRC
cases using pooled DNA and targeted next generation
sequencing. Ten novel or rare (population MAF<1%) BRF1
variants were identified in 11 independent CRC families. The deleterious nature of the identified BRF1 mutations
were demonstrated for seven of them (1 detected in two
families): BRF1 c.1459+2T>C and 6 missense variants, p. T12M, p.V75M, p.S81T, p.C140S, p.P405R and p.R572G,
which led to the alteration of protein function and/or protein
expression in functional studies carried out in yeast and/or
human CRC cell lines. The frequency of mutations in
familial CRC cases was significantly higher to the fre-
quency observed in control population. Germline hetero-
zygous mutations in BRF1 may contribute for at least 1.4%
of unexplained familial colorectal cancer cases. If validated
in independent series, BRF1 mutation carrier families could
benefit in the future from a clinical management based on
carrier status and personalized risk assessment. Conclusions: Among a high-risk group of Greek BrCa
patients, where there are strong founder effects, the
mutational spectrum is heterogeneous, giving rise to useful
associations on known and candidate genes. Through such
approaches, clinical actionability of genes, frequently
included in panels, will be determined. <!--EndFragment-->
F. Fostira: None. I. Konstantopoulou: None. M.S. Papamentzelopoulou: None. P. Apostolou: None. D. Kalfakakou: None. A. Delimitsou: None. A. Vagena:
None. I.S. Vlachos: None. D. Yannoukakos: None. NSCR DEMOKRITOS, ATHENS, Greece Conclusions: Among a high-risk group of Greek BrCa
patients, where there are strong founder effects, the
mutational spectrum is heterogeneous, giving rise to useful
associations on known and candidate genes. Through such
approaches, clinical actionability of genes, frequently
included in panels, will be determined. <!--EndFragment--> F. Fostira: None. I. Konstantopoulou: None. M.S. Papamentzelopoulou: None. P. Apostolou: None. D. Kalfakakou: None. A. Delimitsou: None. A. Vagena:
None. I.S. Vlachos: None. D. Yannoukakos: None. F. Fostira: None. I. Konstantopoulou: None. M.S. Papamentzelopoulou: None. P. Apostolou: None. D. Kalfakakou: None. A. Delimitsou: None. A. Vagena:
None. I.S. Vlachos: None. D. Yannoukakos: None. Abstract Introduction: Early onset and family history of cancer
are the strongest predictors of hereditary colorectal cancer
(CRC) risk. Mendelian CRC predisposition syndromes
underlie about 5% of all cases. Despite this knowledge, the
remaining CRC heritability is still unexplained and may be
caused by rare or population-specific variants in known
candidate genes or other cancer-related genes. Here we aim
to identify known or novel colorectal cancer predisposing
gene variants in early-onset and familial CRC cases. Materials and Methods: After genomic DNA isolation
from
peripheral
blood,
Trusight
Rapid
Capture
Kit
(Illumina Inc., San Diego, CA) has been used for the
Sequencing reactions according to manufacturer’s instruc-
tions. MiSeq Reporter (v2.5.1; Illumina Inc.) was used for
fastq generation and Genomize Seq Platform was used for
variant calling and filtering. ClinVar and HGMD databases
were considered for known variant interpretation, Varsome
was used for in silico analysis and ACMG 2015 guidelines
were followed for the interpretation of novel variants. Materials and Methods: After genomic DNA isolation
from
peripheral
blood,
Trusight
Rapid
Capture
Kit
(Illumina Inc., San Diego, CA) has been used for the
Sequencing reactions according to manufacturer’s instruc-
tions. MiSeq Reporter (v2.5.1; Illumina Inc.) was used for
fastq generation and Genomize Seq Platform was used for
variant calling and filtering. ClinVar and HGMD databases
were considered for known variant interpretation, Varsome
was used for in silico analysis and ACMG 2015 guidelines
were followed for the interpretation of novel variants. Materials and Methods: Forty-eight CRC patients with
family history or early age of onset were enrolled in this
study. Genomic DNA was extracted from blood samples,
library construction and targeted capture of 94 cancer genes
were performed using the Illumina Trusight cancer panel
followed by sequencing at approximately 80x coverage on
an Illumina MiSeq instrument. Results: In total, 16 (9 novel) pathogenic and 3 likely
pathogenic variations have been determined in 18 out of 28
patients (64.2%). Segregation analysis of the the variants
classified as unknown significance is ongoing in the patients
who have available family members. Results: In total, 65 variants in 30 genes were identified. Seventeen patients (35,4%) had a pathogenic mutation in
one of the known CRC predisposing genes including:
MLH1 (3 cases), MSH2 (4 cases, with two novel variants),
MSH6 (2 cases), MUTYH (3 cases, biallelic) and APC (4
cases, with one novel variant). P12.104D None. &. Çiçin: None. H.C. Karlıkaya: None. Y.A. Karamustafaoğlu: None. Detection of a variety of mutations in cancer predisposing
genes in familial & early-onset colorectal cancer patients
using targeted multigene panels S. Demir1, H. Tozkır1, H. Gürkan1, E. Atlı1, D. Eker1, H. A. Tezel2, Y. A. Sezer3, &. Çiçin4, H. C. Karlıkaya5, Y. A. Karamustafaoğlu1 F. MOUFID1,2, M. Dieng3, I. EL bouchikhi1,2, M. Iraqui
Houssaini4, L. Bouguenouch1, E. Benjelloun5, K. Ouldim1,
Y. Idaghdour3 F. MOUFID1,2, M. Dieng3, I. EL bouchikhi1,2, M. Iraqui
Houssaini4, L. Bouguenouch1, E. Benjelloun5, K. Ouldim1,
Y. Idaghdour3 1Trakya University Faculty of Medicine Department of Medical
Genetics, Edirne, Turkey, 2Trakya University Faculty of
Medicine Department of Gastroenterology, Edirne, Turkey,
3Trakya University Faculty of Medicine Department of General
Surgery, Edirne, Turkey, 4Trakya University Faculty of
Medicine Department of Medical Oncology, Edirne, Turkey,
5Trakya University Faculty of Medicine Department of Chest
Diseases, Edirne, Turkey 1Medical genetics and Oncogenetics Department, Hassan II
University Hospital of Fez, Morocco, Fez, Morocco,
2Microbial Biotechnology Laboratory, Faculty of Sciences and
Technologies, Sidi Mohamed Benabdellah University, Fez,
Morocco, 3Biology Program, Division of Science and
Mathematics, New York University Abu Dhabi, Abu Dhabi,
United Arab Emirates, 4Microbial Biotechnology Laboratory,
Faculty of Sciences and Technologies, Sidi Mohamed
Benabdellah University,., Fez, Morocco, 5Visceral Suregery
Department, Hassan II University Hospital, Fez, Morocco Introduction: Cancer genetics is a developing area of
cancer researches. Next Generation Sequencing (NGS) is a
powerfull technology, becoming an important tool offering
opportunities to solve the complexity and multigenic nature
of cancers and hereditary cancer predisposition syndromes
(HCPS). In this study we aimed to summarise the results of
genetic analysis performed in a population of patients with
hereditary
colorectal
(n = 6),
hereditary
breast/ovarian
cancers (n = 7), neurofibromatosis type I/II (n = 9), para-
ganglioma (n = 2), pheochromocytoma (n = 1), tubero-
sclerosis (n = 1) and malign mesothelioma (n = 1). Abstracts from the 51st European Society of Human Genetics Conference: Posters 431 Institute of Medical Genetics, Medical University of Vienna,
Vienna, Austria Conclusions: HLRCC-associated renal cell carcinoma
can occur at early age and uniform recommendations are
warranted for genetic testing and surveillance of young FH
mutation carriers. The presence of complex renal cysts
seems to require careful monitoring, and resection when
solid components appear. Based on the examples of our families we discuss various
counseling issues and more general aspects of this rare
tumor syndrome. These include clinical criteria, predictive
genetic testing of children, renal cancer surveillance,
possible underdiagnosis of HLRCC in the general popula-
tion and prospects of immunohistochemical screening of
HLRCC-associated tumors. J.A. Hol: None. M.C.J. Jongmans: None. M.E.M. Van
der Perk: None. A.S. Littooij: None. R.R. De Krijger:
None. J.J.T. Van Harssel: None. A.R. Mensenkamp:
None. M.M. Van den Heuvel-Eibrink: None. M. Van
Grotel: None. J.A. Hol: None. M.C.J. Jongmans: None. M.E.M. Van
der Perk: None. A.S. Littooij: None. R.R. De Krijger:
None. J.J.T. Van Harssel: None. A.R. Mensenkamp:
None. M.M. Van den Heuvel-Eibrink: None. M. Van
Grotel: None. M. Gerykova Bujalkova: None. F. Laccone: None. M. Gerykova Bujalkova: None. F. Laccone: None. Abstract In addition, rare and novel
pathogenic variants were detected in 25 genes known to
play a role in different cancer pathways. Conclusion: We suggest that multi-gene panels with
NGS is a powerfull tool for determining genetic background
of cancers and cancer-related syndromes. Besides some
disadvantages like the probability of false positivity, NGS is
holding a great advantage in terms of time and cost
effectiveness according to conventional methods. Conclusions: Our findings demonstrate the power of
using targeted sequencing of a broad panel of genes in
clinical diagnosis of hereditary CRC and highlight the
potential role of other cancer-related genes in the disease as S. Demir: None. H. Tozkır: None. H. Gürkan: None. E. Atlı: None. D. Eker: None. H.A. Tezel: None. Y.A. Sezer: 432 J. del Picchia well as the potential pleiotropic effects in promoting
germline predisposition to CRC. 1Princess Máxima Center for Paediatric Oncology, Utrecht,
Netherlands, 2University Medical Center Utrecht / Wilhelmina
Children's Hospital, Utrecht, Netherlands, 3Radboud
University Medical Center Nijmegen, Nijmegen, Netherlands,
4University Medical Center Utrecht, Utrecht, Netherlands well as the potential pleiotropic effects in promoting
germline predisposition to CRC. F. Moufid: None. M. Dieng: None. I. EL bouchikhi:
None. M. Iraqui Houssaini: None. L. Bouguenouch:
None. E. Benjelloun: None. K. Ouldim: None. Y. Idaghdour: None. Introduction: Hereditary leiomyomatosis and renal cell
cancer (HLRCC) is a rare, autosomal dominant syndrome
caused by pathogenic germline mutations in the fumarate
hydratase (FH) gene. It is characterized by cutaneous and
uterine leiomyomas and an increased risk of developing
renal cell carcinoma. Currently, there is no consensus on
what age to start surveillance for renal tumours in FH
mutation carriers, and whether the presence of renal cysts is
an indication for more intensive surveillance or resection. J. A. Hol1, M. C. J. Jongmans2,3, M. E. M. Van der Perk1, A. S.
Littooij2, R. R. De Krijger1, J. J. T. Van Harssel4, A. R.
Mensenkamp3, M. M. Van den Heuvel-Eibrink1, M. Van Grotel1 Institute of Medical Genetics, Medical University of Vienna,
Vienna, Austria Institute of Medical Genetics, Medical University of Vienna,
Vienna, Austria Institute of Medical Genetics, Medical University of Vienna,
Vienna, Austria Materials & Methods: We report two paediatric patients
with HLRCC-associated renal cell carcinoma and/or renal
cysts and reviewed the available literature for HLRCC-
associated renal tumours occurring in patients <20 years. Materials & Methods: We report two paediatric patients
with HLRCC-associated renal cell carcinoma and/or renal
cysts and reviewed the available literature for HLRCC-
associated renal tumours occurring in patients <20 years. Hereditary leiomyomatosis and renal cell cancer (HLRCC)
is an autosomal dominant inherited tumor syndrome pre-
disposing to the development of multiple cutaneous pilo-
leiomyomas, early onset uterine leiomyomas (myomatous
uterus) and also to a clinically aggressive form of type 2
papillary renal cell cancer in approximately 15% of affected
individuals. HLRCC is caused by heterozygous germline
mutations in the fumarate hydratase (FH) gene. At present
more than 300 families with HLRCC have been described. Results: Localized HLRCC-associated papillary type II
renal cell carcinoma was successfully resected in a 15-year-
old female patient with a family history of leiomyomas. In
an unrelated 14-year-old female with a confirmed FH
mutation (c.1210G>T(p.(Glu404*)), complex renal cysts
were detected upon first screening and carefully monitored
by regular MRI's. Nine previously described cases of
HLRCC-associated renal cell carcinoma were identified in
patients aged 10-18 years: four papillary type II, one
collecting
duct
tumour,
four
unspecified. Five
were
metastatic at diagnosis. Atypical cells have been reported
in the lining of resected renal cysts in adult FH mutation
carriers, suggesting a potential preneoplastic lesion. We report three independent families that underwent
genetic testing and counseling for HLRCC at our institute. The renal cell cancer occurred in two families, one family
presented with cutaneous and uterine leiomyomas only. Each family harbored a different mutation in the FH gene. Two
mutations
(out
of
frame
deletion
c.912_918delTTTTGTC and splice site mutation c.905-
1G>A) were definitely pathogenic. One missense mutation
(c.977G>A; p.Gly326Glu) was novel, very likely patho-
genic
and
a
further
segregation
analysis
has
been
recommended. Conclusions: HLRCC-associated renal cell carcinoma
can occur at early age and uniform recommendations are
warranted for genetic testing and surveillance of young FH
mutation carriers. The presence of complex renal cysts
seems to require careful monitoring, and resection when
solid components appear. 1Department of Human Genetics, Faculty of Medicine,
University of Debrecen, Debrecen, Hungary, 2Institute of miR-15a, miR-15b and checkpoint gene expression among
bladder cancer cell lines as a model of tumour
heterogeneity miR-15a, miR-15b and checkpoint gene expression among
bladder cancer cell lines as a model of tumour
heterogeneity B. Soltesz: None. J. Lukács: None. E. Szilágyi: None. R. Póka: None. B. Nagy: None. B. Soltesz: None. J. Lukács: None. E. Szilágyi: None. R. Póka: None. B. Nagy: None. P. Dobosz1,2,3, P. A. Stempor4,5, A. Layani1, T. Meninghier1,
D. Avni1, Y. Sidi1,2, R. Leibowitz-Amit1,2 P12.107C ACTB was used as reference gene. Student t-test was
applied for the statistical calculations. Materials and Methods: Twenty previously untreated
ovarian cancer patients (60.60 ± 10.84y; FIGO stages III or
IV) and 20 healthy controls (56.70 ± 14.51y) were involved
in the study. EDTA blood was drawn; RNA was isolated;
cDNA was synthesised and the HOTAIR lncRNA expres-
sion were determined by using ExiLERATE LNA™qPCR,
for mRNA and long non-coding RNA (Exiqon, USA). ACTB was used as reference gene. Student t-test was
applied for the statistical calculations. Results: Higher expression of HOTAIR was detected in
the samples of patients with ovarian cancer (1.89 ± 2.39)
than in healthy controls (1.39 ± 1.48) but the difference was
no significant (p = 0.432). Conclusions: These findings together with the two
previously identified cases define immunoglobulin defi-
ciency, cellular radiosensitivity and increased AFP levels as
the hallmarks of RNF168 deficiency. The failure of 53BP1
recruitment after irradiation may serve as a useful marker to
screen for further patients. Conclusion: We determined the concentration of the cell
free HOTAIR lncRNA from the plasma of ovarian cancer
patients and healthy controls. There was no significant
difference in the expression of the analysed lncRNA; but
this is the first study to analyse the cell-free HOTAIR
expression among Hungarian ovarian cancer patients. We
would like to extend our study on higher number of cases as
this lncRNA seem to be novel biomarker for the diagnosis
of ovarian cancer. B. Pietrucha: None. E. Heropolitańska-Pliszka: None. R. Geffers: None. J. Enßen: None. B. Wieland: None. N. Bogdanova: None. T. Dörk: None. B. Pietrucha: None. E. Heropolitańska-Pliszka: None. R. Geffers: None. J. Enßen: None. B. Wieland: None. N. Bogdanova: None. T. Dörk: None. P12.107C Cell-free, long non-coding RNA as novel biomarker in the
diagnosis of ovarian cancer HLRCC-associated renal cell carcinoma and renal cysts in
paediatric patients: two case reports and review of the
literature B. Soltesz1, J. Lukács2, E. Szilágyi1, R. Póka2, B. Nagy1 J. A. Hol1, M. C. J. Jongmans2,3, M. E. M. Van der Perk1, A. S. Littooij2, R. R. De Krijger1, J. J. T. Van Harssel4, A. R. Mensenkamp3, M. M. Van den Heuvel-Eibrink1, M. Van Grotel1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 433 Obstetrics and Gynecology, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary Only two patients have hitherto been described with
somewhat different phenotypes. Methods: We performed exome sequencing on two
Polish
siblings
who
presented
with
immunoglobulin
deficiency,
telangiectasia,
cellular
radiosensitivity
and
increased alpha-fetoprotein levels. We investigated the
levels of RNF168, ATM and ATM target phosphorylation
using Western blotting, and we monitored the DNA damage
response after 6 Gy irradiation using immunocytochemical
analyses of 53BP1 and pSer139-H2AX. Background: Ovarian cancer is the fifth leading cause of
cancer-associated mortality among women. A reliable, non-
invasive diagnostic method may contribute to the early
detection of this malignancy. The long non-coding RNAs
(lncRNAs) have a significant role in the oncogenesis,
metastasis and chemoresistance. The upregulation of Hox
transcript antisense intergenic RNA (HOTAIR) was deter-
mined in the development of ovarian cancer cells, however,
the cell-free HOTAIR expression was not detected from the
plasma until now. Results: Exome sequencing identified homozygosity for
a novel frameshift mutation, c.295delG, in the RNF168
gene for both siblings. The younger sibling with a more
pronounced neurological and morphological phenotype also
carried an ATM gene mutation in the heterozygous state. Immunoblot analyses showed absence of RNF168 protein,
whereas ATM levels and function were proficient in LCLs
from both patients. 53BP1 recruitment to DNA double-
strand breaks after irradiation was undetectable in cell lines
from either of the two patients. pSer139-H2AX foci
accumulated normally but they disappeared with significant
delay, indicating a severe defect in DNA double-strand
break repair. Materials and Methods: Twenty previously untreated
ovarian cancer patients (60.60 ± 10.84y; FIGO stages III or
IV) and 20 healthy controls (56.70 ± 14.51y) were involved
in the study. EDTA blood was drawn; RNA was isolated;
cDNA was synthesised and the HOTAIR lncRNA expres-
sion were determined by using ExiLERATE LNA™qPCR,
for mRNA and long non-coding RNA (Exiqon, USA). P12.109A
Clinical and biological manifestation of RNF168 deficiency
in two Polish siblings Clinical and biological manifestation of RNF168 deficiency
in two Polish siblings 1Cancer Research Centre, Oncology Department, Sheba
Medical Centre Hospital, Tel Hashomer, 52621 Ramat Gan,
Israel, Tel Aviv, Israel, 2Oncology Department, Sackler
Faculty of Medicine, Tel Aviv University, 6997801 Tel Aviv,
Israel, Tel Aviv, Israel, 3School of Clinical Medicine,
University of Cambridge, The Old Schools, Trinity Ln,
Cambridge CB2 1TN, UK, Cambridge, United Kingdom, 4The
Wellcome Trust/CRUK Gurdon Institute, University of
Cambridge, Tennis Court Rd, Cambridge, CB2 1QN, UK,
Cambridge, United Kingdom, 5Department of Genetics,
University of Cambridge, Downing Street, Cambridge, CB2
3EH, UK, Cambridge, United Kingdom B. Pietrucha1, E. Heropolitańska-Pliszka1, R. Geffers2,
J. Enßen3, B. Wieland3, N. Bogdanova3, T. Dörk3 B. Pietrucha1, E. Heropolitańska-Pliszka1, R. Geffers2,
J. Enßen3, B. Wieland3, N. Bogdanova3, T. Dörk3 1The Children’s Memorial Health Institute, Warsaw, Poland,
2Helmholtz Institute for Infection Research, Braunschweig,
Germany, 3Hannover Medical School, Hannover, Germany 1The Children’s Memorial Health Institute, Warsaw, Poland,
2Helmholtz Institute for Infection Research, Braunschweig,
Germany, 3Hannover Medical School, Hannover, Germany Introduction: Germline mutations in the RING finger
protein gene RNF168 have been identified in a combined
immunodeficiency disorder called RIDDLE syndrome. 434 J. del Picchia Introduction:
The
complex
immunological
synapse
between T lymphocytes and cancer cells contains check-
point proteins, which modulate the signal transmitted to T
lymphocytes. The role of miRNAs from the miR-15/16
family in bladder cancer remains unknown, although some
research suggests its downregulation. Only a small fraction
of bladder cancer patients respond to immunotherapy. The
present study is aimed at the evaluation of the correlation
between the expression of these miRNAs and the expres-
sion of checkpoint mRNAs in bladder cancer. Introduction:
The
complex
immunological
synapse
between T lymphocytes and cancer cells contains check-
point proteins, which modulate the signal transmitted to T
lymphocytes. The role of miRNAs from the miR-15/16
family in bladder cancer remains unknown, although some
research suggests its downregulation. Only a small fraction
of bladder cancer patients respond to immunotherapy. The
present study is aimed at the evaluation of the correlation
between the expression of these miRNAs and the expres-
sion of checkpoint mRNAs in bladder cancer. same gene. Such data are not avalaible for Romanian
patients. P12.109A
Clinical and biological manifestation of RNF168 deficiency
in two Polish siblings The
expression level of checkpoint genes together with their
interconnections might be crucial for successful immu-
notherapy. Revealing such novel interconnections may aid
in the development of new diagnostic tools, outcome
predictors or immunotherapeutic drugs or combinations
thereof. Conclusions: Many checkpoint genes are co-expressed
together and the expression of mir-15a and miR-15b is
negatively correlated with them. This suggests that these
miRNA negatively regulate the expression of checkpoint
genes at the synapse, at the post-transcriptional level. The
expression level of checkpoint genes together with their
interconnections might be crucial for successful immu-
notherapy. Revealing such novel interconnections may aid
in the development of new diagnostic tools, outcome
predictors or immunotherapeutic drugs or combinations
thereof. P. Apostol: None. S. Dinu: None. P. Gurban: None. F. Iordache: None. D. Blendea: None. G. Cardos: None. Pathogenic alterations in advanced HPV-negative cell
squamous laryngeal carcinoma revealed by Next
Generation Sequencing S. Giragosyan1, T. Popov2, V. Petkova1, G. Stancheva1,
D. Kachakova1, K. Mihova1, J. Rangachev2, V. Mitev1,
D. Popova2, R. Kaneva1 D. Kachakova1, K. Mihova1, J. Rangachev2, V. Mitev1,
D. Popova2, R. Kaneva1 P. Dobosz: None. P.A. Stempor: None. A. Layani:
None. T. Meninghier: None. D. Avni: None. Y. Sidi:
None. R. Leibowitz-Amit: None. 1Molecular Medicine Center, Department of Medical
Chemistry and Biochemistry, Medical University – Sofia, Sofia,
Bulgaria, 2Clinic of ENT, Department of ENT, UMHAT
“Tsaritsa Yoanna-ISUL”, Medical University – Sofia, Sofia,
Bulgaria P12.109A
Clinical and biological manifestation of RNF168 deficiency
in two Polish siblings The present study reports a case of coexistence of
two different somatic mutations in exon 2 (codons 12 and
13) of KRAS gene, in a 68 years old Caucasian woman
suffering from mixt adenoma (tubulo-papillary).Genomic
DNA was isolated from FFPE primary tumor, from the
same selected area, with 58% tumor cells content, and tested
for KRAS exon 2 mutations by PCR-RFLP method fol-
lowed by capillary sequencing. From 2013 we have performed the screening of KRAS
exon 2 for 550 patients suffering from advanced CRC. To
our knowledge, this is the first Romanian case reported with
coexistence of two different somatic mutations, in an early
stage of colorectal carcinoma. One mutation was detected
and confirmed in codon 12 as c.34G>T (p.Gly12Cys). The
other mutation was detected in codon 13 but could not be
confirmed by capillary sequencing, being probably under
LOD,
which
is
20%
in
our
laboratory. Based
on
epidemiological data, KRAS mutations in adenoma stage
represents a very rare event compared with adenocarcinoma
stage. Our results suggest the need for KRAS exon
2 screening even in early stages of colorectal cancer,
contributing in this way to effective selection of CRC
patients prior to personalising treatment decision. Materials & Methods: Three cell lines: MGHU4, 5637
and SW1710 (low to high level of genome instability), were
used as a model of bladder cancer. The expression was
analysed using RT-PCR, statistical and graphical analyses
(Spearman Rho correlation, Benjamini-Hochberg FDR)
were performed using R tools and XLstat. Materials & Methods: Three cell lines: MGHU4, 5637
and SW1710 (low to high level of genome instability), were
used as a model of bladder cancer. The expression was
analysed using RT-PCR, statistical and graphical analyses
(Spearman Rho correlation, Benjamini-Hochberg FDR)
were performed using R tools and XLstat. Results: The expression of most checkpoint genes is
positively correlated to the expression of any other analysed
checkpoint gene. The expression level of miRNAs is
negatively correlated with several checkpoint genes, show-
ing high level of heterogeneity between cell lines. There is a
negative correlation between genome instability within the
cell line and expression of mir-15b. Conclusions: Many checkpoint genes are co-expressed
together and the expression of mir-15a and miR-15b is
negatively correlated with them. This suggests that these
miRNA negatively regulate the expression of checkpoint
genes at the synapse, at the post-transcriptional level. A rare case of colorectal cancer with coexistance of two
different KRAS mutations A rare case of colorectal cancer with coexistance of two
different KRAS mutations P. Apostol1, S. Dinu1, P. Gurban1, F. Iordache1, D. Blendea2,
G. Cardos1 P. Apostol1, S. Dinu1, P. Gurban1, F. Iordache1, D. Blendea2,
G. Cardos1 Introduction: There is still no enough data on the genetic
alterations in laryngeal squamous cell cancer (LSCC),
detected by NGS. The aim of our study was to explore the
somatic status of patients, diagnosed with advanced lar-
yngeal carcinoma by NGS techniques and its clinical
impact. 1Personal Genetics, Bucharest, Romania, 2Targu-Jiu County
Hospital, Targu-Jiu, Romania 1Personal Genetics, Bucharest, Romania, 2Targu-Jiu County
Hospital, Targu-Jiu, Romania Driving mutations in the RAS genes represent a negative
predictor for personalized treatment decision in patients
with colorectal cancer (CRC). Although mutations in these
genes are mutually exclusive, few cases (2%) have been
described with coexistence of two different mutations in the Methods: In the current study were included 38 HPV-
negative patients with advanced LSCC. The isolated DNA
was used for Next Generation Sequencing of 48 tumour-
associated genes (TSACP) by MiSeq (Illumina). The data
was analyzed by VarSeq Software (v.1.4.6). Abstracts from the 51st European Society of Human Genetics Conference: Posters 435 Results: The results showed 60 pathogenic mutations in
23 tumour associated genes. The most commonly mutated
gene was TP53, with 28 (46.67%) variants with pathogenic
prediction. We found 7 new pathogenic variants in TP53
gene in 7 patients. The second most mutated genes were
PIK3CA and FBWX7 with 4 pathogenic variants each. We
found all together 27 (45%) non published variants with
pathogenic predictions in CDH1, CDKN2A, ERBB4,
FBXW7, NOTCH1, PIK3CA, SMARCB1, TP53, FGFR2,
FLT3, GNAQ, GNAS, KRD, NRAS, SMAD4, STK11,
VHL genes. One benign and known pathogenic variants,
relevant to cancer treatment, were found in KRAS, PIK3CA
and TP53. Results: The results showed 60 pathogenic mutations in
23 tumour associated genes. The most commonly mutated
gene was TP53, with 28 (46.67%) variants with pathogenic
prediction. We found 7 new pathogenic variants in TP53
gene in 7 patients. The second most mutated genes were
PIK3CA and FBWX7 with 4 pathogenic variants each. We
found all together 27 (45%) non published variants with
pathogenic predictions in CDH1, CDKN2A, ERBB4,
FBXW7, NOTCH1, PIK3CA, SMARCB1, TP53, FGFR2,
FLT3, GNAQ, GNAS, KRD, NRAS, SMAD4, STK11,
VHL genes. One benign and known pathogenic variants,
relevant to cancer treatment, were found in KRAS, PIK3CA
and TP53. A rare case of colorectal cancer with coexistance of two
different KRAS mutations been shown that they are through the effect of thymoqui-
none. Introducing the apoptotic activity of thymoquinone
on laryngeal cancer provides basis to investigate the ther-
apeutic efficacy of this ingredient. Materials
and
Methods:
The
apoptotic
effect
of
thymoquinone on the laryngeal cancer cell line was
determined by MTT and Sulphurodamine B cytotoxicity
tests. Its effect on the mutant KRAS gene transfected cells
was observed through MTT and intracellular ATP analysis. FBXW7, NOTCH1, PIK3CA, SMARCB1, TP53, FGFR2,
FLT3, GNAQ, GNAS, KRD, NRAS, SMAD4, STK11,
VHL genes. One benign and known pathogenic variants,
relevant to cancer treatment, were found in KRAS, PIK3CA
and TP53. Results: Thymoquinone has cytotoxic effect on the
laryngeal cancer cells and cell death was decreased in the
cells carrying mutation in the coding region of KRAS gene. However, cell death increase was observed in cells having
mutation
in
the
coding
and
non-coding
region
of
KRAS gene. Conclusion:
In
conclusion,
molecular
profiling
of
laryngeal cancer with NGS could reveal the genetic
architecture of LSCC and offer opportunities for prediction
of response to existing target therapies and finding new
therapeutic targets, which is a step forward in individualized
medicine. Conclusions: It was observed that thymoquinone inter-
acts with KRAS oncogene. Conclusions: It was observed that thymoquinone inter-
acts with KRAS oncogene. V.B. Yenigun: None. H. Acar: None. T. Cora: None. A. Yenigun: None. A. Kocyigit: None. V.B. Yenigun: None. H. Acar: None. T. Cora: None. A. Yenigun: None. A. Kocyigit: None. Acknowledgements: This work was supported by Grants
550/21.01.2016/Contract №30/2016/MU-Sofia; DUNK01/
2/2009/NSF. Acknowledgements: This work was supported by Grants
550/21.01.2016/Contract №30/2016/MU-Sofia; DUNK01/
2/2009/NSF. P12.116D Expression of LINC01330,long intergenic non-protein
coding RNA 1330, in serous ovarian carcinoma Expression of LINC01330,long intergenic non-protein
coding RNA 1330, in serous ovarian carcinoma S. Giragosyan: None. T. Popov: None. V. Petkova:
None. G. Stancheva: None. D. Kachakova: None. K. Mihova: None. J. Rangachev: None. V. Mitev: None. D. Popova: None. R. Kaneva: None. S. Valeh Sheida1, S. Mowla1, S. Azizmohamadi2 1TMU, Tehran, Iran, Islamic Republic of, 2Hajar Hospital,
Tehran, Iran, Islamic Republic of 1TMU, Tehran, Iran, Islamic Republic of, 2Hajar Hospital,
Tehran, Iran, Islamic Republic of Investigation of thymoquinone effect on laryngeal cancer
cells with KRAS mutant Introduction: Among common cancers in women, ovarian
cancer has the lowest incidence; however, mortality rates
are more than other gynecological cancers due to the lack of
specific symptoms during early stages and absence of
diagnostic markers. Comparative genomic hybridization
(CGH) demonstrated high frequency of 3q25-26 copy
number gains (CNGs) in ovarian carcinomas. Serous
papillary carcinoma as a common type of epithelial ovarian
cancer shows singular copy number variations (CNV) of
this region. This chromosomal region contains many protein
coding genes and some of them have an oncogenic role,
including
PIK3CA,
Sox2,
and
HTERC. LINC01330
(LRRC77P) which is a Pseudogene, located in this con-
troversial region and led us to make more extensive studies
of its expression alteration in serous ovarian cancer. According to following study, LINC01330 has shown a
remarkable change. V. B. Yenigun1,2, H. Acar3, T. Cora2, A. Yenigun4, A. Kocyigit1 1Bezmialem Vakif University, Faculty of Medicine, Department
of Medical Biochemistry, Istanbul, Turkey, 2Selcuk University,
Faculty of Medicine, Department of Medical Genetics, Konya,
Turkey, 3Diagen Biotechnology, Ankara, Turkey, 4Bezmialem
Vakif University, Faculty of Medicine, Department of
Otorhinolaryngology, Istanbul, Turkey Introduction: Larynx cancer constitutes about 2% of all
cancers and 25% of head and neck cancers. KRAS oncogene
is a member of the RAS gene family. When KRAS is
mutated, the gene functions as "open" as a result. KRAS
mutation has been shown to be effective in the development
of head and neck cancer. Phyto-therapy practices are subject
to rapid cancer research in the world. Since active ingre-
dients of many drugs are plant original, the interest to plants
increases. Many plants used in the early conventional
treatments have become the subject of numerous important
molecular research studies. Thymoquinone is one of the
active ingredients of Nigella sativa known also as the black
cumin. Many of the biological effects of these seeds have Materials and Methods: In this study, expression of
LINC01330 was evaluated in fresh frozen tissues. Total
RNA was isolated from the tissues and cDNA synthesis was
performed. Semi-quantitative RT-PCR technique was used
and the validity of PCR products was determined by Sanger
sequencing. The expression level of LINC01330 was
evaluated by real-time PCR. Result: Our finding revealed Materials and Methods: In this study, expression of
LINC01330 was evaluated in fresh frozen tissues. Total
RNA was isolated from the tissues and cDNA synthesis was
performed. Monitoring the EGFR T790M mutation in liquid biopsy in
patients with lung cancer Monitoring the EGFR T790M mutation in liquid biopsy in
patients with lung cancer Development of a noninvasive method for assessing
circulating tumour DNA in patients with solid tumours G. Cardos1, R. Motoc1, D. G. N. Leonte1,2, G. Simion1,3,
P. Gurban1, F. Iordache1,4, P. Iorga3, P. P. Apostol1 G. Cardos1, R. Motoc1, D. G. N. Leonte1,2, G. Simion1,3,
P. Gurban1, F. Iordache1,4, P. Iorga3, P. P. Apostol1 A. Eliades, C. Loizides, M. Ioannides, A. Achilleos, P. Mina,
K. Tsangaras, E. Kypri, G. Koumbaris, P. C. Patsalis A. Eliades, C. Loizides, M. Ioannides, A. Achilleos, P. Mina,
K. Tsangaras, E. Kypri, G. Koumbaris, P. C. Patsalis 1Personal Genetics, Bucharest, Romania, 2National
Pneumology Institute "M.Nasta", Bucharest, Romania,
3Emergency University Hospital, Bucharest, Romania,
4Institute of Cellular Biology and Pathology "Nicolae
SImionescu", Bucharest, Romania NIPD Genetics, Nicosia, Cyprus NIPD Genetics, Nicosia, Cyprus Introduction: It is estimated that more than 14 million
people are diagnosed with cancer each year. In 2012, over
583,000 deaths were caused by lung, colorectal, breast,
prostate, and ovarian cancer accounting for over 45% of all
cancer-related deaths in Europe. Current diagnostic methods
rely on molecular and histopathology results derived from
invasive tissue biopsy, which has several inherent draw-
backs; it is time-intensive, costly and not always accessible. Furthermore, it is associated with risks to the patient while
localised sampling misses cancer heterogeneity. However,
circulating tumour DNA (ctDNA) in plasma may provide a
novel non-invasive means for early cancer detection, diag-
nosis, and monitoring not otherwise possible with conven-
tional testing. Molecular EGFR analysis from liquid biopsy is increasingly
being used to monitor response to tyrosine kinase inhibitor
(TKIs) therapy in lung cancer patients. Our ongoing study aimed to monitor the EGFR T790M
mutation status in patients with non-small cell lung cancer
(NSCLC), which is the most common mechanism of
resistance to TKIs. The molecular EGFR analysis was performed by real-
time PCR on plasma DNA collected from 34 patients (aged
33-81 years) with advanced NSCLC, who received EGFR-
TKIs therapy based on EGFR activating mutation detected
at diagnosis (by the same method, applied on DNA
extracted from FFPE tissues between 2012-2017). Blood
samples were collected from patients at the time of disease
progression (3-58 months, median of 13.5 months) after the
initial EGFR analysis (exon 19 deletion (19del) in 27
patients, L858R mutation in 7 patients and S768I mutation
in 1 patient). Investigation of thymoquinone effect on laryngeal cancer
cells with KRAS mutant Semi-quantitative RT-PCR technique was used
and the validity of PCR products was determined by Sanger
sequencing. The expression level of LINC01330 was
evaluated by real-time PCR. Result: Our finding revealed J. del Picchia 436 or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Signifi-
cant; NIPD Genetics. P. Mina: A. Employment (full or
part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant;
NIPD Genetics. G. Koumbaris: A. Employment (full or
part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Significant; NIPD Genetics. the down-expression of LINC01330 in tumor tissues
(P<0.01). Down-expression was 2.5 times less in tumor
group in comparison to control group. the down-expression of LINC01330 in tumor tissues
(P<0.01). Down-expression was 2.5 times less in tumor
group in comparison to control group. Conclusion: Lack of specific markers in ovarian cancer is
the
vital
barrier
against
early
detection
and
finding
molecular markers with specific expression alteration will
help us for better diagnosis. Therefore LINC01330 can be
proposed as an effective and sensitive biomarker. S. Valeh
Sheida:
None. S. Mowla:
None. S. Azizmohamadi: None. S. Valeh
Sheida:
None. S. Mowla:
None. S. Azizmohamadi: None. P12.121A T. Burjanivova1, B. Malicherova1, M. Grendar2, E. Minarikova3,
M. Bobrovska4, B. Vanova1, I. Kasubova1, E. Jezkova5,
I. Homola6, T. Pecova3, Z. Lasabova1, L. Plank7 Identification of tumor suppressor microRNAs by
integrative miRNA and mRNA sequencing of matched
tumor normal pairs in lung adenocarcinoma n. yu1, s. yong1, y. jung1, y. lee1, j. seo1, j. kim1, j. kim2, s. lee1 n. yu1, s. yong1, y. jung1, y. lee1, j. seo1, j. kim1, j. kim2, s. lee1 1Biomedical Center Martin JFM CU, Division of Oncology
JFM CU, Martin, Slovakia, 2Biomedical Center Martin JFM
CU,Bioinformatic unit, Martin, Slovakia, 3Clinic of
Dermatovenerology, Jessenius Faculty of Medicine and
University Hospital in Martin, Martin, Slovakia, 4Department
of Pathological Anatomy, Jessenius Faculty of Medicine and
University Hospital in Martin,, Martin, Slovakia, 5Department
of Histology and Embryology, Jessenius Faculty of Medicine,
Comenius University in Bratislava, Martin, Slovakia,
6Department of Plastic Surgery, Jessenius Faculty of Medicine
and University Hospital in Martin, Martin, Slovakia,
7Department of Pathological Anatomy, Jessenius Faculty of
Medicine and University Hospital in Martin,Division of
Oncology JFM CU, Martin, Slovakia 1Biomedical Center Martin JFM CU, Division of Oncology
JFM CU, Martin, Slovakia, 2Biomedical Center Martin JFM
CU,Bioinformatic unit, Martin, Slovakia, 3Clinic of
Dermatovenerology, Jessenius Faculty of Medicine and
University Hospital in Martin, Martin, Slovakia, 4Department
of Pathological Anatomy, Jessenius Faculty of Medicine and
University Hospital in Martin,, Martin, Slovakia, 5Department
of Histology and Embryology, Jessenius Faculty of Medicine,
Comenius University in Bratislava, Martin, Slovakia,
6Department of Plastic Surgery, Jessenius Faculty of Medicine
and University Hospital in Martin, Martin, Slovakia,
7Department of Pathological Anatomy, Jessenius Faculty of
Medicine and University Hospital in Martin,Division of
Oncology JFM CU, Martin, Slovakia 1Ewha Woman's University, Seoul, Korea, Republic of,
2Sungkyunkwan University School of Medicine, Seoul, Korea,
Republic of Roles of microRNAs (miRNAs) have not yet been explored
systematically at the genome scale in lung cancer biology
despite their important regulatory functions. Here, we report
an integrative analysis of microRNA and mRNA sequen-
cing data for the matched tumor and normal samples from
110 Korean female patients with non-small-cell lung ade-
nocarcinoma. We produced miRNA-Seq and RNA-Seq data
for 49 patients and RNA-Seq data only for additional 61
patients. Differential expression analysis with stringent
criteria yielded 44 miRNAs and 2,322 genes. Integrative
gene set analysis of differentially expressed miRNAs and
genes using miRNA-target information yielded several
processes related to cell cycle regulation that were targeted
by tumor suppressor miRNAs. Monitoring the EGFR T790M mutation in liquid biopsy in
patients with lung cancer Additionally, the initial EGFR activating
mutation was also detected in liquid biopsy in 28 (82.35%)
patients (21 with 19del, 6 with L858R mutation and 1 with
S768I mutation). Materials and Methods: We developed an assay based
on a proprietary form of targeted in-solution hybridisation
for the detection of somatic mutations in the patient’s
plasma. A total of 523 driver and clinically actionable
mutations across 49 genes are targeted. Reference material
was used for the proof of concept study and plasma and
matched-tissue samples from non-metastatic breast cancer
patients were used for pre-clinical validation. Results: Our proof of concept study exhibited LOD
between
0.1-1%
MAF. Preliminary
experiments
with
clinical samples from patients with breast cancer showed
concordance of the mutational profile of the tissue and
matched plasma. Results: Our proof of concept study exhibited LOD
between
0.1-1%
MAF. Preliminary
experiments
with
clinical samples from patients with breast cancer showed
concordance of the mutational profile of the tissue and
matched plasma. Our results suggested that the EGFR T790M mutation
occurs more frequently in NSCLC patients with EGFR
19del than in patients with L858R, possibly due to the fact
that EGFR 19del mutants of NSCLC have a distinct
biological phenotype that would favor the acquisition of
T790M
mutation,
as
other
literature
studies
have
hypothesized. Conclusions: We aim to use circulating cell free DNA as
a biomarker coupled with our targeted NGS-based approach
to develop a liquid biopsy assay that provides safe and
highly accurate non-invasive testing for solid cancers. A. Eliades: A. Employment (full or part-time); Sig-
nificant; NIPD Genetics. C. Loizides: A. Employment (full A. Eliades: A. Employment (full or part-time); Sig-
nificant; NIPD Genetics. C. Loizides: A. Employment (full 437 Abstracts from the 51st European Society of Human Genetics Conference: Posters Our results are potentially important for clinical decision-
making in NSCLC patients with EGFR mutation. Study on
larger group of NSCLC patients is ongoing to establish a
certain statistical significance. supported by the "Biomedical Center Martin (BioMed
Martin)" ITMS code 26220220187 project which is co-
financed from EU sources and by the Slovak Research and
Development Agency under the contracts no.APVV-16-
0066 and no. APVV-14-0273. G. Cardos: None. R. Motoc: None. D.G.N. Leonte:
None. G. Simion: None. P. Gurban: None. F. Iordache:
None. P. Iorga: None. P.P. Apostol: None. T. Burjanivova: None. B. Malicherova: None. M. Grendar: None. E. Minarikova: None. M. Bobrovska:
None. B. Vanova: None. I. Kasubova: None. Detection of BRAF V600E mutation by droplet digital PCR
in plasma of patients with malignant melanoma Detection of BRAF V600E mutation by droplet digital PCR
in plasma of patients with malignant melanoma Monitoring the EGFR T790M mutation in liquid biopsy in
patients with lung cancer E. Jezkova:
None. I. Homola: None. T. Pecova: None. Z. Lasabova:
None. L. Plank: None. P12.121A We performed the colony
formation assay in A549 and NCI-H460 cell lines to test the
tumor suppressive activity of down-regulated miRNAs in
cancer and identified 7 novel tumor suppressor miRNAs
(miR-144-5p, miR-218-1-3p, miR-223-3p, miR-27a-5p,
miR-30a-3p, miR-30c-2-3p, miR-338-5p). Two miRNAs of
miR-30a-3p and miR-30c-2-3p showed differential survival
characteristics in the TCGA LUAD patient cohort, sug-
gesting their prognostic value. Our study not only provides
a massive dataset of miRNA and mRNA sequencing from
the matched tumor-normal samples but also reports several
novel tumor suppressor miRNAs that could be further
developed into prognostic biomarkers or RNA therapeutic
targets. Introduction: Melanoma is the most aggresive form of skin
cancer. About 50% of melanomas have BRAF V600Emu-
tation. This mutation is an attractive therapeutic target. Detection of BRAF V600E mutation is a potential prog-
nostic factor in malignant melanoma. Here, we studied
BRAF V600E mutations in circulating cell-free DNA in
plasma (,,liquid biopsy“) by droplet digital PCR (ddPCR). Materials and Methods: We analyzed 100 patients with
malignant melanoma. Circulating DNA from plasma was
isolated by using QIAamp DSP Virus Kit (Qiagen, Hilden,
Germany). DNA quantification was performed in a Qubit
2.0 fluorometer. ddPCR was performed with QX200 system
(BIO-RAD®, Hercules, USA). All samples were tested in
duplicate. Materials and Methods: We analyzed 100 patients with
malignant melanoma. Circulating DNA from plasma was
isolated by using QIAamp DSP Virus Kit (Qiagen, Hilden,
Germany). DNA quantification was performed in a Qubit
2.0 fluorometer. ddPCR was performed with QX200 system
(BIO-RAD®, Hercules, USA). All samples were tested in
duplicate. Results: The Limit of Detection was determined by the
method of Tzonev. The LOD was found to be 5 events per
well. The BRAF V600E mutation was detected in 37/
100 samples. Results: The Limit of Detection was determined by the
method of Tzonev. The LOD was found to be 5 events per
well. The BRAF V600E mutation was detected in 37/
100 samples. N. yu: None. S. yong: None. Y. jung: None. Y. lee:
None. J. seo: None. J. kim: None. J. kim: None. S. lee: None. Conclusion: ddPCR is a highly sensitive method and
could be use for routine laboratory detection of BRAF
V600E mutation as well as follow-up to treatment response
in patients with malignant melanomas. N. yu: None. S. yong: None. Y. jung: None. Y. lee:
None. J. seo: None. J. kim: None. J. kim: None. S.
lee: None. 1Chulabhorn Graduate Institute, Bangkok, Thailand,
2Chulabhorn Research Institute, Bangkok, Thailand,
3Chulabhon Hospital, Bangkok, Thailand The structure of LD block will
be shown in the poster. Results
and
Conclusions:
CHRNA5
polymorphism
showed association with both nicotinic consumption and
risk for lung cancer development as well in Thai population. Heavy metal levels was different between groups of
patients, however the association between nicotinic receptor
polymorphisms and heavy metal metabolism should be
explored with larger studies. The structure of LD block will
be shown in the poster. S. Boonvisut: None. T. Suriyo: None. S. Chotirat:
None. N. Triphutidet: None. CHRNA3, CHRNA5 and CHRNB4 Polymorphisms
Mapping and Correlation with Lung Cancer Susceptibility
in Thai Population 1Chulabhorn Graduate Institute, Bangkok, Thailand,
2Chulabhorn Research Institute, Bangkok, Thailand,
3Chulabhon Hospital, Bangkok, Thailand 1Chulabhorn Graduate Institute, Bangkok, Thailand,
2Chulabhorn Research Institute, Bangkok, Thailand,
3Chulabhon Hospital, Bangkok, Thailand I. Donner1, R. Katainen1, L. J. Sipilä1, M. Aavikko1,
E. Pukkala2,3, L. A. Aaltonen1 I. Donner1, R. Katainen1, L. J. Sipilä1, M. Aavikko1,
E. Pukkala2,3, L. A. Aaltonen1 Introduction: Several risk factors of lung cancer were
known, for example; air pollutions, smoking and genetic
variations. However, the information about environmental-
gene interaction associated with lung cancer in Thai popu-
lation is limited. Recent genome wide association studies in
Europeans identified a few regions related to lung cancer
susceptibility including 15q25.1 region, containing nico-
tinic acetylcholine receptor genes. This study aimed to
investigate the association between genetic variations,
exposure to environmental risk factor and lung cancer
occurred in Thailand, and for further study of some clinical
significance, the onset of disease evaluation in heavy
smokers, and the prognosis in lung cancer. 1Department of medical and clinical genetics, Medicum, and
Genome-scale biology research program, Research programs
unit, Faculty of medicine, University of Helsinki, Helsinki,
Finland, 2Finnish cancer registry, Institute for statistical and
epidemiological cancer research, Helsinki, Finland, 3Faculty
of social sciences, University of Tampere, Tampere, Finland 1Department of medical and clinical genetics, Medicum, and
Genome-scale biology research program, Research programs
unit, Faculty of medicine, University of Helsinki, Helsinki,
Finland, 2Finnish cancer registry, Institute for statistical and
epidemiological cancer research, Helsinki, Finland, 3Faculty
of social sciences, University of Tampere, Tampere, Finland Objectives: Although the primary cause of lung cancer is
smoking, a considerable proportion of all lung cancers
occur in never smokers. Gender influences the risk and
characteristics of lung cancer and women are over-
represented among never smokers with the disease. Young
age at onset and lack of established environmental risk
factors suggest genetic predisposition. In this study, we
used whole population-based sampling of the youngest
patients in Finland to discover candidate predisposition
variants for lung adenocarcinoma in never-smoking women. Objectives: Although the primary cause of lung cancer is
smoking, a considerable proportion of all lung cancers
occur in never smokers. Gender influences the risk and
characteristics of lung cancer and women are over-
represented among never smokers with the disease. Young
age at onset and lack of established environmental risk
factors suggest genetic predisposition. In this study, we
used whole population-based sampling of the youngest
patients in Finland to discover candidate predisposition
variants for lung adenocarcinoma in never-smoking women. Analysis of somatic mutations in lung cancer with targeted
next generation sequencing I. Donner: None. R. Katainen: None. L.J. Sipilä: None. M. Aavikko:
None. E. Pukkala:
None. L.A. Aaltonen: None. I. Donner: None. R. Katainen: None. L.J. Sipilä: None. M. Aavikko:
None. E. Pukkala:
None. L.A. Aaltonen: None. V. Y. Petkova1, A. Mitkova1, D. Kachakova1, G. Stancheva1,
K. Mihova1, D. Marinova2,3, Y. Slavova-Marinova2,4, V. Mitev1,
R. Kaneva1 1Chulabhorn Graduate Institute, Bangkok, Thailand,
2Chulabhorn Research Institute, Bangkok, Thailand,
3Chulabhon Hospital, Bangkok, Thailand Materials and Methods: Participants were recruited
from healthy volunteers, heavy smokers and lung cancer
patients who attended hospitals in Bangkok from 2012-
2016. All samples were examined serum heavy metal levels
and genotyped for CHRNA3, CHRNA5, and CHRNB4
polymorphisms
such
as
rs1051730,
rs6495309,
rs16969968, and rs17486278 using TaqMan Genotyping
Assay Systems. The association between each minor allele
frequencies and lung cancer and heavy metal levels were
assessed by logistic regression analysis. Age, sex, and
smoking consumption were included as covariates. Haplo-
type frequencies were calculated and LD blocks were drawn
by Haploview software. Materials and Methods: Participants were recruited
from healthy volunteers, heavy smokers and lung cancer
patients who attended hospitals in Bangkok from 2012-
2016. All samples were examined serum heavy metal levels
and genotyped for CHRNA3, CHRNA5, and CHRNB4
polymorphisms
such
as
rs1051730,
rs6495309,
rs16969968, and rs17486278 using TaqMan Genotyping
Assay Systems. The association between each minor allele
frequencies and lung cancer and heavy metal levels were
assessed by logistic regression analysis. Age, sex, and
smoking consumption were included as covariates. Haplo-
type frequencies were calculated and LD blocks were drawn
by Haploview software. Materials and Methods: We employed archival tissue
material from 21 never-smoker women who had been
diagnosed with lung adenocarcinoma before the age of 45,
and exome sequenced their germline DNA. Results and Conclusion: Potentially pathogenic variants
were found in eight Cancer Gene Census germline genes:
BRCA1,
BRCA2,
ERCC4,
EXT1,
HNF1A,
PTCH1,
SMARCB1 and TP53. A nonsense variant in TP53, and
missense variants in BRCA1 and BRCA2, are likely to have
contributed to the early onset lung cancer in the respective
patients. This supports the notion that lung adenocarcinoma
can be a component of certain cancer predisposition
syndromes. Fifteen genes displayed potentially pathogenic
mutations in at least two patients: ABCC10, ATP7B,
CACNA1S, CFTR, CLIP4, COL6A1, COL6A6, GCN1,
GJB6, RYR1, SCN7A, SEC24A, SP100, TTN and USH2A. Four patients showed a mutation in COL6A1, three in
CLIP4 and two in the rest of the genes. Some of these
candidate genes may explain a subset of female lung
adenocarcinoma predisposition. Results
and
Conclusions:
CHRNA5
polymorphism
showed association with both nicotinic consumption and
risk for lung cancer development as well in Thai population. Heavy metal levels was different between groups of
patients, however the association between nicotinic receptor
polymorphisms and heavy metal metabolism should be
explored with larger studies. P12.124D Analysis of somatic mutations in lung cancer with targeted
next generation sequencing P12.121A This work was Conclusion: ddPCR is a highly sensitive method and
could be use for routine laboratory detection of BRAF
V600E mutation as well as follow-up to treatment response
in patients with malignant melanomas. This work was J. del Picchia 438 P12.122B 1Chulabhorn Graduate Institute, Bangkok, Thailand,
2Chulabhorn Research Institute, Bangkok, Thailand,
3Chulabhon Hospital, Bangkok, Thailand P12.123C 1Molecular Medicine Center,Department of Medical Chemistry
and Biochemistry, Medical Faculty, Sofia, Bulgaria, 2Clinical Abstracts from the 51st European Society of Human Genetics Conference: Posters 439 Center for Lung Diseases, Medical Faculty, Medical
University of Sofia, Sofia, Bulgaria, 3Department of
Bronchology, University Hospital for Pulmonary Diseases “St. Sofia", Sofia, Bulgaria, 4Department of Pathomorphology,
University Hospital for Pulmonary Diseases “St. Sofia", Sofia,
Bulgaria Center for Lung Diseases, Medical Faculty, Medical
University of Sofia, Sofia, Bulgaria, 3Department of
Bronchology, University Hospital for Pulmonary Diseases “St. Sofia", Sofia, Bulgaria, 4Department of Pathomorphology,
University Hospital for Pulmonary Diseases “St. Sofia", Sofia,
Bulgaria S. Bonfiglio1, D. Cittaro1, I. Vanni2, D. Lazarevic1, M. Mora3,
V. Rossella4, C. Genova2, A. Truini2, M. G. Dal Bello2,
E. Rijavec2, F. Grossi2, S. Coco2 1Centre for Translational Genomics and Bioinformatics,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Lung
Cancer Unit, IRCCS Ospedale Policlinico San Martino,
Genoa, Italy, 3Department of Pathology, IRCCS Ospedale
Policlinico San Martino, Genoa, Italy, 4Swiss Stem Cell
Biotech, Vacallo, Switzerland Introduction: Small cell lung cancer(SCLC), adenocarci-
noma(ADC) and squamous cell lung carcinoma(SCC) could
be discriminated at molecular level by specific “driver
mutations” in many tumour-associated genes. Targeted next
generation sequencing(NGS) allows simultaneous analysis
of many genes and more complete characterization of the
somatic mutations in tumours. Introduction: About 10% of women with breast cancer
(BC) develop a second unrelated tumor including lung
cancer (LC). The risk increases in radiotherapy-treated
patients and in smokers. Since the current radiotherapy does
not fully justify this risk, we hypothesize that genetic pre-
disposition could enhance LC development and performed
whole exome sequencing to unravel it. Materials and Methods: In the current study DNA
isolated from fresh-frozen tumour tissues of 12 patients with
ADC, 12 with SCC and 13 SCLC were included. Hotspot
somatic mutations in a panel of 48 tumour-associated genes
(TSACP) were analysed by NGS platform MiSeq(Illumina). Materials and Methods: At Ospedale Policlinico San
Martino, Genova, 28 women with LC after BC (Study
Population, SP) and 32 women with BC only (Control
Population,
CP)
were
enrolled. Genomic
DNA
was
extracted from FFPE tumor and normal samples. Libraries
were prepared with Agilent SureSelect All Exon and
sequenced on Illumina HiSeq 2500. Variant calling was
performed
with
FreeBayes. Somatic
signatures
were
calculated on all nucleotidic substitutions and burden tests
were computed using WSS and C-α statistics. Materials and Methods: At Ospedale Policlinico San
Martino, Genova, 28 women with LC after BC (Study
Population, SP) and 32 women with BC only (Control
Population,
CP)
were
enrolled. Genomic
DNA
was
extracted from FFPE tumor and normal samples. Libraries
were prepared with Agilent SureSelect All Exon and
sequenced on Illumina HiSeq 2500. Variant calling was
performed
with
FreeBayes. Somatic
signatures
were
calculated on all nucleotidic substitutions and burden tests
were computed using WSS and C-α statistics. Results: The performed analysis showed 23 pathogenic
variants in ADC samples.The most frequently mutated gene
was TP53 with 12 pathogenic mutations(52,17%).Eight
activating mutations(34,78%) were found in KRAS. P12.125A Whole exome sequencing to discover lung tumor
predisposition in women with previous breast cancer S. Bonfiglio1, D. Cittaro1, I. Vanni2, D. Lazarevic1, M. Mora3,
V. Rossella4, C. Genova2, A. Truini2, M. G. Dal Bello2,
E. Rijavec2, F. Grossi2, S. Coco2 Other
pathogenic variants were observed in APC,RB1,BRAF with
frequency of 4,35% each.In SCC samples 21 pathogenic
variants were detected:12 variants in TP53(57,14%),5
activating mutations in KRAS(23.82%) and mutations in
the genes GNAS,PIK3CA,SMO,STK11 with frequency of
4,76% each.In SCLC 23 pathogenic variants were observed. The most frequently mutated gene was TP53 with 12
pathogenic variants(52,17%),followed by Rb1(30,76%) and
BRAF,
EGFR,FGFR2,NOTCH1,PIC3CA,PTPN1,
SMARCB1 with one mutation each-(4,35%). Around 50%
of all analysed samples harboured more than 1 pathogenic
mutation. Results: Two mutational signatures were extracted: S1,
similar to COSMIC-S30 (unknown etiology), included all
SP-BC and 16/28 LC; S2, enriched in 12/28 LC, positively
correlated with smoking and matched with COSMIC-S4,
linked to tobacco use. These signatures may reflect two
distinct mutagenic processes underlying LC development:
smoking could have played a major role in S2-LC subgroup
while genetic predisposition could enhance LC develop-
ment in S1-LC patients. Therefore, we performed a gene-
based burden test over rare germline variants in S1-LC
versus
CP
and
we
identified
249
candidate
genes
(FDR<0.05). Conclusion: Our results suggest that the NGS analysis of
lung cancer using a panel of tumour-associated genes is able
to detect somatic mutations that are currently used as
predictive biomarkers for targeted therapies.In addition it
reveals the spectra of specific “driver” mutations and
provides opportunities for the discovery of new therapeutic
targets and personalized treatment. Acknowledgements:
This work was supported by Grants 508/21.01.2016/
Contract №24/2016/MU-Sofia;DUNK01/2/2009/NSF. Conclusions: Our results show two mutational signatures
underlying LC development. Germline data validation step
is ongoing to confirm them in an independent cohort. Italian Ministry of Health supported this work (GR-2011-
02350922). Italian Ministry of Health supported this work (GR-2011-
02350922). V.Y. Petkova: None. A. Mitkova: None. D. Kacha-
kova: None. G. Stancheva: None. K. Mihova: None. D. Marinova: None. Y. Slavova-Marinova: None. V. Mitev:
None. R. Kaneva: None. S. Bonfiglio: None. D. Cittaro: None. I. Vanni: None. D. Lazarevic: None. M. Mora: None. V. Rossella: None. C. Genova: None. A. Truini: None. M.G. Dal Bello:
None. E. Rijavec: None. F. Grossi: None. S. Coco: None. S. Bonfiglio: None. D. Cittaro: None. I. Vanni: None. D. Lazarevic: None. M. Mora: None. V. Rossella: None. C. Genova: None. A. Truini: None. M.G. Dal Bello:
None. E. Rijavec: None. F. Grossi: None. S. Coco: None. P12.126B Birth order affects risk of multiple lymphoid cancers and
allergies in lymphoid cancer families 440 J. del Picchia 1Our Lady's Children's Hospital Crumlin, Dublin, Ireland,
2National University of Ireland Galway, Galway, Ireland,
3Cork University Hospital, Cork, Ireland S. J. Jones1,2, O. S. Uvarov3, D. Salema1, J. M. Connors4,
A. Brooks-Wilson1,3 1BC Cancer Research Centre, Vancouver, BC, Canada,
2University of British Columbia, Vancouver, BC, Canada,
3Simon Fraser University, Burnaby, BC, Canada, 4British
Columbia Cancer Agency, Vancouver, BC, Canada Pathogenic variants in mismatch repair genes MLH1,
MSH2, MSH6, PMS2 and more rarely EPCAM cause Lynch
syndrome (LS), conferring variable risks of endometrial,
colorectal, upper gastro-intestinal, urinary and biliary tract
cancers, depending on gene and type of variant. Evidence
for associations between LS and Breast Cancer is conflict-
ing1, with a recent report suggesting the risk may be limited
to pathogenic variants in MSH6 or PMS22. We report a
female patient with multi-focal breast cancer associated
with a germline variant in MSH2. A 37-year old female,
presented with multifocal, ER-positive, invasive ductal
breast cancer. Her father (deceased) developed colon cancer
at 37 years of age. Analysis of his tumour identified
microsatellite instability, and immunohistochemical (IHC)
analysis identified absent staining for MSH2 and MSH6
proteins. IHC of our patient’s breast tumour confirmed
mismatch repair deficiency, and subsequent germline test-
ing identified a likely pathogenic splice-site variant (c.2210
+1G>C) in MSH2. We have objectively demonstrated a
clear causal relationship between an MSH2 variant and
breast cancer in one individual. Considering the frequency
of breast cancer, it is unsurprising that a proportion of
families with LS include members affected by the condi-
tion. We plan to analyse pedigrees of other Irish families
with LS to further elucidate the frequency of breast cancer
in this cohort. The penetrance of such variants for breast
cancer is unclear, and additional surveillance for this cancer
in this population may not be cost-effective. 1. Win et al.,
Breast Cancer Research, 2013, 2. Roberts et al., Genetics in
Medicine, 2018 Introduction: Lymphoid cancers are a heterogeneous group
of neoplasms that arise from immune cells. Familial clus-
tering of lymphomas support a genetic contribution to
cancer predisposition; however, infectious diseases and the
immune system have also been implicated. The hygiene
hypothesis proposes that a lower infectious burden during
early life inhibits the immune system from maturing opti-
mally. M. Duff1, N. Cody1, T. P. McVeigh2, C. Clabby1, S. O'Reilly3,
A. J. Green1 P12.126B Consequently, such individuals are more susceptible
to atopic disorders, including allergies and autoimmune
disease, and some lymphoid cancers. Methods: We characterized atopic conditions in lym-
phoid affected sibships of 182 families with a history of
lymphoid cancers. Early life data was collected from
telephone interviews and questionnaires from multiple
family members. Results: 314 sibships had 405 lymphoid affected and 927
unaffected siblings. An inverse relationship between birth
order and risk of cancer was observed for all lymphoid
cancers collectively (p<0.0001), and separately for multiple
myeloma
(p=0.0015),
non-Hodgkin
lymphoma
(p <-
0.0001) and individual B-cell subtypes, including chronic
lymphocytic leukemia (p=0.0124), follicular (p=0.0217)
and
marginal
zone
lymphoma
(p=0.0169). We
also
observed an inverse relationship between birth order and
risk of allergies (p=0.0284), for both environmental
allergies (p=0.0465) and multiple allergies (p=0.0114) in
lymphoid affected individuals. Results: 314 sibships had 405 lymphoid affected and 927
unaffected siblings. An inverse relationship between birth
order and risk of cancer was observed for all lymphoid
cancers collectively (p<0.0001), and separately for multiple
myeloma
(p=0.0015),
non-Hodgkin
lymphoma
(p <-
0.0001) and individual B-cell subtypes, including chronic
lymphocytic leukemia (p=0.0124), follicular (p=0.0217)
and
marginal
zone
lymphoma
(p=0.0169). We
also
observed an inverse relationship between birth order and
risk of allergies (p=0.0284), for both environmental
allergies (p=0.0465) and multiple allergies (p=0.0114) in
lymphoid affected individuals. M. Duff: None. N. Cody: None. T.P. McVeigh: None. C. Clabby: None. S. O'Reilly: None. A.J. Green: None. M. Duff: None. N. Cody: None. T.P. McVeigh: None. C. Clabby: None. S. O'Reilly: None. A.J. Green: None. Conclusions: Childhood exposures to infectious diseases
may play a role in immune dysregulation and subsequent
risk of multiple types of lymphoid cancers, and allergies. The familial nature of the cancers implies shared genetic
and/or environmental factors. There is a need for further
evaluation of lifestyle factors that may protect against
lymphoid cancers even in the familial context. Comprehensive genotypic and phenotypic characterization
of heterozygous versus homozygous MMR gene mutation
carriers in an Indian cohort Comprehensive genotypic and phenotypic characterization
of heterozygous versus homozygous MMR gene mutation
carriers in an Indian cohort Comprehensive genotypic and phenotypic characterization
of heterozygous versus homozygous MMR gene mutation
carriers in an Indian cohort A. Lipsa1,2, R. R. Kunduru3, R. Sarin1,2 S.J. Jones: None. O.S. Uvarov: None. D. Salema:
None. J.M. Connors: None. A. Brooks-Wilson: None. 1Sarin Lab, Advanced Centre for Treatment Research and
Education in Cancer (ACTREC), Tata Memorial Centre
(TMC), Kharghar, Navi Mumbai, India, 2Homi Bhabha
National Intitute (HBNI), Mumbai, India, 3Cancer Genetics
Clinic, Tata Memorial Hospital, TMC, Parel, India P12.127C Breast cancer in Irish patients with Lynch syndrome Introduction: MMR gene mutations when heterozygous
cause adult onset autosomal dominant Lynch Syndrome
(LS), in homozygous state they cause autosomal recessive Abstracts from the 51st European Society of Human Genetics Conference: Posters 441 Constitutional MisMatch Repair Deficiency (CMMRD)
syndrome characterized by a variety of childhood cancers
and café-au-lait spots. The MMR gene mutation phenotype,
which is strikingly different depending on its zygosity, has
not been described in any Asian population. somatic changes remain obscure. This study defines DNA
methylation changes at different stages of LS-associated
CRC, and somatic mutations in adenomas. Colorectal biopsies including normal mucosae, adenomas
and carcinomas were prospectively collected from 104 LS
patients, supplemented with retrospective tumor specimens. DNA methylation was analysed using methylation-specific
multiplex ligation-dependent probe amplification test (MS-
MLPA) for selected tumor suppressor genes (TSGs), for
CpG island methylator phenotype (CIMP)-associated mar-
kers, and LINE-1. Immunohistochemistry was used to
detect MMR protein expression in tumors. As an ongoing
study, mutation statuses of adenomas are investigated using
the Ion AmpliSeqTM Colon and Lung Cancer Panel. Method: Based on personal and family history of cancer,
phenotyping and tumour IHC, 50 LS and 10 CMMRD
families were identified. One or more MMR genes (MLH1,
MSH2, MSH6 and PMS2) were sequenced followed by
large genomic rearrangements (LGR) analysis. To avoid
pseudogene amplification, long range PCR was done for
PMS2. If no mutation / LGR was identified in these 4 genes,
extended gene panel test was done which included MLH3. Result: In the 50 families with suspected LS, hetero-
zygous pathogenic mutations (including 6 LGRs) were
detected in 46 (92%) families (31-MLH1, 14-MSH2 and 1-
MSH6) and a homozygous MLH3 mutation in a 30 yr old
female with endometrial cancer and family history of colon
cancer. In the 10 CMMRD suspected families, biallelic
germline PMS2 mutations were identified in 4 families
(novel homozygous frameshift mutations in 3, compound
heterozygous mutation in 1). Result: In the 50 families with suspected LS, hetero-
zygous pathogenic mutations (including 6 LGRs) were
detected in 46 (92%) families (31-MLH1, 14-MSH2 and 1-
MSH6) and a homozygous MLH3 mutation in a 30 yr old
female with endometrial cancer and family history of colon
cancer. In the 10 CMMRD suspected families, biallelic
germline PMS2 mutations were identified in 4 families
(novel homozygous frameshift mutations in 3, compound
heterozygous mutation in 1). P12.127C The MMR protein expression decreases along with
dysplasia but occurs relatively late in tumor progression,
suggesting other somatic events to drive tumorigenesis. Methylation of TSGs and frequency of CIMP increases and
LINE-1
methylation
decreases in
tumors
along
with
dysplasia. In
MMR-proficient
(MMR-P)
and
MMR-
deficient
(MMR-D)
adenomas,
LINE-1
methylation
decreases along the loss of MMR protein expression, and
interestingly, higher methylation of SFRP1 is observed in
MMR-P adenomas. Furthermore, certain CRC-associated
somatic mutations (e.g. KRAS) appear prevalent in MMR-P
adenomas. Conclusion: While phenotypic manifestations of hetero-
zygous MMR mutations are in concordance with previous
reports, we expand the CMMRD spectrum with the first
report of an adult onset endometrial caner in a biallelic
MLH3 carrier. This largest single centre study of CMMRD
suspected families identifies a high prevalence of biallelic
PMS2 mutations. Concluding, similarly to sporadic CRC, early appearance
of epigenetic changes and somatic mutations are important
in LS-associated tumorigenesis. A. Lipsa: None. R.R. Kunduru: None. R. Sarin: None. Grant
support:
Jane
and Aatos
Erkko Foundation,
Academy of Finland, the Finnish Cancer Organizations,
the Sigrid Jusélius Foundation, and HiLIFE. P12.129A DNA methylation changes and somatic mutations as early
events in Lynch syndrome-associated colorectal cancer S. Mäki-Nevala: None. S. Valo: None. A. Ristimäki:
None. V.K. Sarhadi: None. S. Knuutila: None. M. Nyström: None. L. Renkonen-Sinisalo: None. A. Lepistö:
None. J. Mecklin: None. P. Peltomäki: None. S. Mäki-Nevala1, S. Valo1, A. Ristimäki2,3, V. K. Sarhadi2,
S. Knuutila2, M. Nyström4, L. Renkonen-Sinisalo5, A. Lepistö5,
J. Mecklin6, P. Peltomäki1 P12.130B
High cancer risk in two cases of constitutional MLH1
epimutation Introduction: Male breast cancer (MBC) is a rare disease,
whose etiology appears to be associated with genetic fac-
tors. Inherited mutations in BRCA1/2 genes account for
about 10-15% of all cases. Biallelic germ-line mutations in
the DNA repair gene MUTYH cause MUTYH-associated
polyposis (MAP) syndrome, whereas monoallelic mutations
are reported in families with both colorectal and breast
cancer. We aimed to test if MUTYH germ-line mutations
may contribute to MBC susceptibility. All cancers but urothelial one of both patients showed
high microsatellite instability (MSI-H) and loss of immu-
nohistochemical MLH1/PMS2 protein expressions. No
germline MLH1 and PMS2 pathogenic variants were
diagnosed
using
both
NGS
and
MLPA
technical
approaches. All cancers but urothelial one of both patients showed
high microsatellite instability (MSI-H) and loss of immu-
nohistochemical MLH1/PMS2 protein expressions. No
germline MLH1 and PMS2 pathogenic variants were
diagnosed
using
both
NGS
and
MLPA
technical
approaches. Materials and Methods: We screened the entire coding
region of MUTYH in 560 MBC cases by a multi-gene panel
analysis. The presence of all variants detected was also
analyzed in 1540 male controls using a TaqMan approach. Results:
Biallelic
MUTYH
pathogenic
variants
(c.536A>G and c.721C>T) were identified in one case
with phenotypic manifestation of adenomatous polyposis. Monoallelic pathogenic variants were identified in 14
(2.5%) MBC cases, in particular: c.536A>G in 7 cases,
c.1187G>A in 5 cases, c.734G>A and c.933+3A>C in 1
case, respectively. Increased MBC risk in association with
c.536A>G emerged (OR = 4.60; 95% CI: 1.19–17.81; p =
0.027). Conclusions:
The LS
flow-chart
including
somatic
genetic and epigenetic analyses is crucial for diagnosis of
this subset of Lynch-Like patients. The identification of
constitutional primary epimutation has an important clinical
impact for carriers showing a high risk of developing LS-
related cancers. On the contrary, their relatives, as it is not
an
inherited
condition,
have
a
general
population’s
cancer risk. Conclusions:
The LS
flow-chart
including
somatic
genetic and epigenetic analyses is crucial for diagnosis of
this subset of Lynch-Like patients. The identification of
constitutional primary epimutation has an important clinical
impact for carriers showing a high risk of developing LS-
related cancers. On the contrary, their relatives, as it is not
an
inherited
condition,
have
a
general
population’s
cancer risk. Conclusion: Our results suggest that MUTYH pathogenic
variants may have a role in MBC, in particular MUTYH
c.536A>G variant may be a low/moderate penetrance risk
allele for MBC. P12.130B
High cancer risk in two cases of constitutional MLH1
epimutation 1Department of Medical and Clinical Genetics, University of
Helsinki, Helsinki, Finland, 2Department of Pathology, 1Department of Medical and Clinical Genetics, University of
Helsinki, Helsinki, Finland, 2Department of Pathology,
University of Helsinki, Helsinki, Finland, 3HUSLAB, Helsinki
University Hospital, Helsinki, Finland, 4Department of
Biosciences, University of Helsinki, Helsinki, Finland,
5Department of Surgery, Helsinki University Hospital,
Helsinki, Finland, 6Department of Surgery, Central Finland
Central Hospital and University of Eastern Finland, Jyväskylä,
Finland 1Department of Medical and Clinical Genetics, University of
Helsinki, Helsinki, Finland, 2Department of Pathology,
University of Helsinki, Helsinki, Finland, 3HUSLAB, Helsinki
University Hospital, Helsinki, Finland, 4Department of
Biosciences, University of Helsinki, Helsinki, Finland,
5Department of Surgery, Helsinki University Hospital,
Helsinki, Finland, 6Department of Surgery, Central Finland
Central Hospital and University of Eastern Finland, Jyväskylä,
Finland M. T. Ricci1, D. Furlan2, A. M. Chiaravalli2, V. Pensotti3,4,
S. Volorio3,4, S. Signoroni1, M. Vitellaro1, M. G. Tibiletti2 1Hereditary Digestive Tract Tumours Unit, Fondazione IRCCS
Istituto Nazionale dei Tumori, Milano, Italy, 2Unit of
Pathology, Ospedale di Circolo ASST Settelaghi, Varese, Italy,
3Cogentech Cancer Genetics Test Laboratory, Milano, Italy,
4IFOM, Fondazione Istituto FIRC di Oncologia Molecolare,
Milano, Italy 1Hereditary Digestive Tract Tumours Unit, Fondazione IRCCS
Istituto Nazionale dei Tumori, Milano, Italy, 2Unit of
Pathology, Ospedale di Circolo ASST Settelaghi, Varese, Italy,
3Cogentech Cancer Genetics Test Laboratory, Milano, Italy,
4IFOM, Fondazione Istituto FIRC di Oncologia Molecolare,
Milano, Italy Lynch syndrome (LS) is caused by germline mutations in
DNA mismatch repair (MMR) genes resulting in increased
cancer risk, including colorectal cancer (CRC). Early events
of multistep tumorigenesis accelerated by germline and Introduction: Constitutional epigenetic inactivation of the
MLH1 gene represents a minor cause of Lynch syndrome 442 J. del Picchia IRCCS, Padua, Italy, 11Fondazione IRCCS Istituto Nazionale
Tumori (INT), Milan, Italy, 12Cancer Research and Prevention
Institute (ISPO), Florence, Italy, 13The FIRC (Italian
Foundation for Cancer Research) Institute of Molecular
Oncology, Milan, Italy (LS), but the identification of this condition is relevant for
cancer risk assessment and clinical management of the
patients. We describe two cases of constitutional MLH1
hypermethylation without any apparent linked variants in
the MLH1 promoter. Case descriptions: A 30-year-old woman with colorectal
cancers (CRC) and a man with multiple cancers (urothelial
cancer at age 48, two CRCs at ages 59 and 69, sebaceous
adenomas at age 61) referred to genetic counselling as
suspected for LS. The family history in both cases was
negative for Amsterdam criteria. P12.130B
High cancer risk in two cases of constitutional MLH1
epimutation Moreover, our results suggest that MBC
may be part of the tumor spectrum associated with MAP
syndrome, with implications in the clinical management of
the patients and their relatives. M.T. Ricci: None. D. Furlan: None. A.M. Chiaravalli:
None. V. Pensotti: None. S. Volorio: None. S. Signoroni:
None. M. Vitellaro: None. M.G. Tibiletti: None. Study supported by AIRC (IG 16933) to L.O. Study supported by AIRC (IG 16933) to L.O. Contribution of MUTYH variants to male breast cancer
risk: results from a multicenter study in Italy Contribution of MUTYH variants to male breast cancer
risk: results from a multicenter study in Italy Contribution of MUTYH variants to male breast cancer
risk: results from a multicenter study in Italy P. Rizzolo1, V. Silvestri1, A. Bucalo1, V. Zelli1, V. Valentini1,
A. Spinelli2, S. Tommasi3, M. Tibiletti4, A. Russo5, L. Varesco6,
G. Giannini1, D. Calistri7, L. Cortesi8, A. Viel9, M. Montagna10,
P. Radice11, D. Palli12, P. Peterlongo13, L. Ottini1 P. Rizzolo1, V. Silvestri1, A. Bucalo1, V. Zelli1, V. Valentini1,
A. Spinelli2, S. Tommasi3, M. Tibiletti4, A. Russo5, L. Varesco6,
G. Giannini1, D. Calistri7, L. Cortesi8, A. Viel9, M. Montagna10,
P. Radice11, D. Palli12, P. Peterlongo13, L. Ottini1 Radice: None. D. Palli: None. P. Peterlongo: None. L. Ottini: None. GERMLINE INVESTIGATION IN DNA REPAIR 1Sapienza University, Rome, Italy, 2IRCCS, Burlo Garofolo,
Trieste, Italy, 3Istituto Tumori, Bari, Italy, 4Ospedale di
Circolo, Varese, Italy, 5University, Palermo, Italy, 6IRCCS
AOU San Martino, Genoa, Italy, 7Istituto Scientifico
Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola
(FC), Italy, 8University of Modena and Reggio Emilia,
Modena, Italy, 9CRO Aviano, National Cancer Institute,,
Aviano (PN), Italy, 10Veneto Institute of Oncology IOV - 1Sapienza University, Rome, Italy, 2IRCCS, Burlo Garofolo,
Trieste, Italy, 3Istituto Tumori, Bari, Italy, 4Ospedale di
Circolo, Varese, Italy, 5University, Palermo, Italy, 6IRCCS
AOU San Martino, Genoa, Italy, 7Istituto Scientifico
Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola
(FC), Italy, 8University of Modena and Reggio Emilia,
Modena, Italy, 9CRO Aviano, National Cancer Institute,,
Aviano (PN), Italy, 10Veneto Institute of Oncology IOV - GENES OF MALE BREAST CANCER BY NEXT-
GENERATION SEQUENCING M. A. Caligo1, R. Scarpitta1, G. Gambino1, P. Aretini2, B. Mei1,
K. Zavaglia1, E. Falaschi1, F. Bonci3, F. Bonci3, E. Landucci3,
S. Gana4, C. Congregati4, M. Ghilli5, E. Rossetti5, G. Allegrini6,
G. Arrighi6, I. Zanna7, M. Roncella5, A. Naccato8, D. Palli7 P12.131C P. Rizzolo: None. V. Silvestri: None. A. Bucalo: None. P. Rizzolo: None. V. Silvestri: None. A. Bucalo: None. V. Zelli: None. V. Valentini: None. A. Spinelli: None. S. Tommasi: None. M. Tibiletti: None. A. Russo: None. L. Varesco: None. G. Giannini: None. D. Calistri: None. L. Cortesi: None. A. Viel: None. M. Montagna: None. P. Radice: None. D. Palli: None. P. Peterlongo: None. L. Ottini: None. 1Sapienza University, Rome, Italy, 2IRCCS, Burlo Garofolo,
Trieste, Italy, 3Istituto Tumori, Bari, Italy, 4Ospedale di
Circolo, Varese, Italy, 5University, Palermo, Italy, 6IRCCS
AOU San Martino, Genoa, Italy, 7Istituto Scientifico
Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola
(FC), Italy, 8University of Modena and Reggio Emilia,
Modena, Italy, 9CRO Aviano, National Cancer Institute,,
Aviano (PN), Italy, 10Veneto Institute of Oncology IOV - P. Rizzolo1, V. Silvestri1, A. Bucalo1, V. Zelli1, V. Valentini1,
A. Spinelli2, S. Tommasi3, M. Tibiletti4, A. Russo5, L. Varesco6,
G. Giannini1, D. Calistri7, L. Cortesi8, A. Viel9, M. Montagna10,
P. Radice11, D. Palli12, P. Peterlongo13, L. Ottini1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 443 1Section of Molecular Genetics, Pisa, Italy, 2Lab. of Genomics,
Pisa Science Foundation, Pisa, Italy, 3Medical Oncology, Dep. of Oncology, Pisa, Italy, 4Lab. of Medical Genetics, Pisa, Italy,
5Dep. of Senology, Pisa, Italy, 6Dep. Of Oncology, ATNO,
Pontedera, Italy, 7Molecular and Nutritional Epidemiology
Unit, Cancer Research and Prevention Institute (ISPO),
Firenze, Italy, 8Dep. of Pathology, Pisa, Italy 1Engelhardt Institute of Molecular Biology, Russian Academy
of Sciences, Moscow, Russian Federation, 2N.N. Blokhin
Medical Research Center of Oncology, Moscow, Russian
Federation 1Section of Molecular Genetics, Pisa, Italy, 2Lab. of Genomics,
Pisa Science Foundation, Pisa, Italy, 3Medical Oncology, Dep. of Oncology, Pisa, Italy, 4Lab. of Medical Genetics, Pisa, Italy,
5Dep. of Senology, Pisa, Italy, 6Dep. Of Oncology, ATNO,
Pontedera, Italy, 7Molecular and Nutritional Epidemiology
Unit, Cancer Research and Prevention Institute (ISPO),
Firenze, Italy, 8Dep. of Pathology, Pisa, Italy Tumors continuously evolve to maintain their growth; sec-
ondary mutations increase the proliferation, resulting in
high heterogeneity of tumor cells. To study the clonal
architecture
of
melanoma
samples
the
whole-exome
sequencing of three patient’s primary tumors and meta-
chronous
metastases
was
performed
using
NextSeq
500 system. Somatic mutations were annotated and clus-
tered based on their frequency. The clusters were ordered
regarding to their cellular prevalence to reconstruct the
tumor clonal evolution. One patient carried BRAF V600E
mutation in primary tumor and in a metastasis after targeted
therapy with vemurafenib. Additional mutated genes in
metastatic cells were PMS2 (16%) and CYP21A2 (19%). The second patient had BRAF V600E (27%), PMS2 c.706-
4delT (23%), CTNNB1 c.95A>G (15%) both in tumor and
metastasis, but TERT c.835G>A was presented in 64% of
primary tumor cells and only in 15% of metastasis. The
third case was presented by tumor (M0) and three metas-
tases differed by localization and time from initial diagnosis
(M1, M2, M3). BRAF V600E was identified in all samples
except М1. However, in M1 the mutated MICAL1 gene
was presented almost in all cells comparing with 4% in M0. The MICAL1 gene is known to control cell growth and
survival of V600E melanoma cells. The last in time M3
carried additional somatic mutations in ANK3, DCDC1,
STAB2, FLT1, ZNF638, ACVR1C, SNAP91 genes. Thus,
reconstruction of tumor clonal evolution is important for
understanding further tumor progression and mechanisms of
resistance to anticancer therapy.The work was supported by
the Russian Science Foundation (grant # 14−35-00107). Male breast cancer (MBC) is a rare disease, its incidence is
1/105 year and it represents less than 1% of all breast can-
cers. MBC tends to occur between 60-70 years old and to
express oestrogen and progesterone receptors frequently. Subsequently, luminal subtype is the most common phe-
notype with occasionally epidermal growth factor receptor 2
amplification. The rarity of MBC has precluded the large
clinical trials, thus genetic predisposition remains not well
understood. In order to better define genetic risk factors in
men, a germline investigation in MBC cases was performed
through the screening of 24 genes involved in breast cancer
predisposition, genome stability maintenance and DNA
repair mechanisms by Next Generation Sequencing. Clin-
ical pathological data and family history of 81 MBC cases
were collected. The average age of onset was 61.3 years and
35 men showed breast cancer family history. Our results let
us to attribute a genetic cause to breast cancer in 23% of
cases. In total, 19 patients carried a pathogenic mutation in
4 genes: BRCA2, BRIP1, MUTYH and PMS2. As expected,
a positive family history is a strong predictor of germline
BRCA2 mutations. Moreover, 14 variants of unknown
clinical significance (VUS) in 9 genes (BARD1, BRCA1,
BRCA2, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1)
were predicted as potentially pathogenic by in-silico ana-
lysis leading to 40% the mutation detection rate. Under-
standing the potential pathogenicity of VUS represents an
extremely urgent question for the implementation of breast
cancer risk management in MBC cases and their own
families. T. Nasedkina: None. I. Abramov: None. G. Krasnov:
None. M. Emelyanova: None. O. Ryabaya: None. K. Orlova: None. L. Demidov: None. T. Nasedkina: None. I. Abramov: None. G. Krasnov:
None. M. Emelyanova: None. O. Ryabaya: None. K. Orlova: None. L. Demidov: None. T. Nasedkina: None. I. Abramov: None. G. Krasnov:
None. M. Emelyanova: None. O. Ryabaya: None. K. Orlova: None. L. Demidov: None. M.A. Caligo: None. R. Scarpitta: None. G. Gambino:
None. P. Aretini: None. B. Mei: None. K. Zavaglia: None. E. Falaschi: None. F. Bonci: None. F. Bonci: None. E. Landucci: None. S. Gana: None. C. Congregati: None. M. Ghilli: None. E. Rossetti: None. G. Allegrini: None. G. Arrighi: None. I. Zanna: None. M. Roncella: None. A. Naccato: None. D. Palli: None. T. Nasedkina1, I. Abramov1, G. Krasnov1, M. Emelyanova1,
O. Ryabaya2, K. Orlova2, L. Demidov2 C. Badenas1,2, M. Potrony2,3, J. Puig-Butille1,2, J. M. Farnham4,
P. Gimenez-Xavier3,2, G. Tell-Marti2,3, P. Aguilera3,2,
C. Carrera3,2, J. Malvehy3,2, C. C. Teerlink4, S. Puig3,2
1Servei de Bioquimica i Genetica Molecular. Hospital Clinic
de Barcelona, Barcelona, Spain, 2Centro de Investigacion
Biomedica en Red en Enfermedades Raras (CIBERER),
Barcelona, Spain, 3Dermatology Department. Melanoma Unit.
Hospital Clinic de Barcelona, IDIBAPS, Universitat de 1Servei de Bioquimica i Genetica Molecular. Hospital Clinic
de Barcelona, Barcelona, Spain, 2Centro de Investigacion
Biomedica en Red en Enfermedades Raras (CIBERER),
Barcelona, Spain, 3Dermatology Department. Melanoma Unit.
Hospital Clinic de Barcelona, IDIBAPS, Universitat de P12.133A
Intratumoral heterogeneity of melanoma as revealed by
whole-exome sequencing P12.133A
Intratumoral heterogeneity of melanoma as revealed by
whole-exome sequencing P12.134B New familial melanoma susceptibility locus at 11q identified
by genome-wide linkage analysis in Spanish melanoma-
prone families C. Badenas1,2, M. Potrony2,3, J. Puig-Butille1,2, J. M. Farnham4,
P. Gimenez-Xavier3,2, G. Tell-Marti2,3, P. Aguilera3,2,
C. Carrera3,2, J. Malvehy3,2, C. C. Teerlink4, S. Puig3,2 P12.133A P12.133A
Intratumoral heterogeneity of melanoma as revealed by
whole-exome sequencing J. del Picchia 444 MicroRNAs
are
short,
non
coding
RNAs. Aberrant
expression of miRs is observed in numerous cancers,
leading to tumor development, growth and progression. With use of the next-generation sequencing we discovered,
a putative non-coding RNA (miR-TG) encoded within the
sequence of thyroglobulin (TG). In silico and in vitro ana-
lysis confirmed that putative miR-TG is microRNA. Next,
the downregulation of miR-TG and TG in papillary thyroid
carcinoma (PTC) was confirmed. The expression of the
novel miR-TG and TG was decreased to 44% (p = 0.04)
and to 48% (p = 0.001) in PTC compared with unaffected
tissue. Using in silico tools we identified MAP4K4 as target
gene for miR-TG. To confirm direct interaction between the
miR-TG and 3’UTR MAP4K4 the Dual Luciferase System
was used. Analysis of transcriptome by RNA-seq proved
that overexpression of miR-TG in PTC-derived cell line
downregulates several genes, including MAP4K4 (fold
change 0.82; p = 0.036). We confirmed this result by SQ-
PCR. The level of MAP4K4 was lowered to 0.71 (p =
0.004). PTC-derived cell line transfected with miR-TG
reveals increased proliferation.We propose that miR-TG
plays a fine-tunning role in proper function of thyroid gland
and its downregulation potentially can lead to activation of
MAPK kinases, underlying initiation and progression of
thyroid carcinogenesis. This work was supported: Pre-
ludium DEC-2012/07/N/NZ3/02033 (to M.K.); Opus DEC-
2013/11/B/NZ3/00193 (to K.J.); LIDER/017/299/L-5/13/
NCBR/2014) (to A.W.) Barcelona, Barcelona, Spain, 4Division of Genetic
Epidemiology, Department of Medicine, University of Utah
School of Medicine, Salt Lake City, UT, United States Melanoma etiology is complex and involves environmental,
phenotypic and genetic factors. Approximately 10% of
melanoma cases occur in a familial context, but the main
genetic factors for familial melanoma remain unknown in
more than 75% of families. CDKN2A is mutated in around
20% of melanoma-prone families, while other high-risk
melanoma susceptibility genes explain less than 3% of
families studied to date.We performed the first genome-
wide
linkage
analysis
in
CDKN2A-negative
Spanish
melanoma-prone families to identify novel melanoma sus-
ceptibility loci. We included 68 individuals from 2, 3 and 6
families with 2, 3 and at least 4 melanoma cases. P12.133A Subjects
were genotyped on either the HumanOmni2.5 (Illumina)
array versions v1.0 (81% of subjects) or v1.1 (19% of
subjects).We detected a locus with significant linkage evi-
dence at 11q14.1-q14.3, with a maximum het-TLOD of
3.449 (rs12285365:A>G), using evidence from multiple
pedigrees. The genes contained by the subregion with the
strongest linkage evidence were: DLG2, PRSS23, FZD4
and TMEM135. We also detected several regions with
suggestive linkage evidence (TLOD>1.9) (1q, 6p, 7p, 11q,
12p, 13q) including the region previously detected in
melanoma-prone families from Sweden at 3q29. The family
specific analysis revealed three loci with suggestive linkage
evidence for family #1: 1q31.1-q32.1 (max. TLOD 2.447),
6p24.3-p22.3 (max. TLOD 2.409) and 11q13.3-q21 (max. TLOD 2.654). Future next generation sequencing studies of
these regions may allow the identification of new melanoma
susceptibility genetic factors.Acknowledgments: Instituto
de Salud Carlos III (15/00716,15/00483), AGAUR 2017
SGR1134, “CERCA Programme / Generalitat de Catalu-
nya”, NCI of the US NIH (CA83115). M. Kolanowska:
None. A. Wójcicka:
None. A. Kubiak: None. M. Świerniak: None. M. Kotlarek: None. M. Maciąg: None. P. Gaj: None. &. Koperski: None. B. Górnicka: None. K. Jażdżewski: None. Association of microRNA expression with metastases in
gastric cancer C. Badenas: None. M. Potrony: None. J. Puig-Butille:
None. J.M. Farnham: None. P. Gimenez-Xavier: None. G. Tell-Marti: None. P. Aguilera: None. C. Carrera:
None. J. Malvehy: None. C.C. Teerlink: None. S. Puig: None. C. Badenas: None. M. Potrony: None. J. Puig-Butille:
None. J.M. Farnham: None. P. Gimenez-Xavier: None. G. Tell-Marti: None. P. Aguilera: None. C. Carrera:
None. J. Malvehy: None. C.C. Teerlink: None. S. Puig: None. F. Kipkeeva1, T. Muzaffarova1, P. Apanovich1, M. Nikulin2,
S. Nered2, M. Narimanov2, O. Malekhova2, I. Stilidi2,
A. Karpukhin1 F. Kipkeeva1, T. Muzaffarova1, P. Apanovich1, M. Nikulin2,
S. Nered2, M. Narimanov2, O. Malekhova2, I. Stilidi2,
A. Karpukhin1 1Research Centre for Medical Genetics, Moskow, Russian
Federation, 2NN Blokhin Russian Cancer Research Centre,
Moskow, Russian Federation 1Medical University of Warsaw, Warsaw, Poland, 2Centre of
New Technologies, University of Warsaw, Warsaw, Poland M. Kolanowska1, A. Wójcicka1, A. Kubiak1, M. Świerniak1,
M. Kotlarek2, M. Maciąg1, P. Gaj2, &. Koperski1, B. Górnicka1,
K. Jażdżewski1 P12.136D Novel, thyroglobulin-embedded microRNA gene
deregulated in papillary thyroid carcinoma Novel, thyroglobulin-embedded microRNA gene
deregulated in papillary thyroid carcinoma The association of microRNA expression with metastasis in
gastric cancer (GC) was studied. Two approaches were
used: a hybridization of separate from tissue microRNA on
NanoString chip containing more than 800 microRNAs, and
expression investigation by quantitative real time PCR of
bioinformationally selected a set of microRNAs important
for tumor development. The sixty paired tumor / normal
samples from patients with early-stage GC and metastatic M. Kolanowska1, A. Wójcicka1, A. Kubiak1, M. Świerniak1,
M. Kotlarek2, M. Maciąg1, P. Gaj2, &. Koperski1, B. Górnicka1,
K. Jażdżewski1 1Medical University of Warsaw, Warsaw, Poland, 2Centre of
New Technologies, University of Warsaw, Warsaw, Poland 445 Abstracts from the 51st European Society of Human Genetics Conference: Posters GC were analyzed. The decreased expression of mir34a,
mir146a and mir335 was associated with distant metastases
among gastric cancer patients. These microRNAs act as
tumor suppressors and a decline of their expression level
can affect the tumor progression. Expression of miRNA
among patients with the same tumor size (T4) with varying
degrees of regional lymph node involvement in metastasis
(from 0 to 15) was investigated using the NanoString CSO /
Human v3 miRNA chip, which allows selection without
preliminary amplification of the studied microRNAs. It was
found that mir146a expression was significantly reduced
(by more than 10 times) in tumors of patients with a high
degree of regional lymph node involvement (10-15) in
comparison with tumors without metastases. In summary,
reduced expression level of mir34a, mir146a and mir335 is
associated with distant metastases. The decreased expres-
sion level of mir146a is also associated with early metas-
tasis of the GC tumor that can suggest its possible
involvement in starting of metastasis process. A participa-
tion of microRNA in distant metastasis may not be obli-
gatory connected with early-stage metastasis. Results: We identified MSI in 7% of all examined
malignancies (present in 7 tumour types; table 1). MSI
phenotype was frequently observed in EC (20.2%) and
STAD (16.7%). Most notably, MSI was detected in ALL
(5%), LIHC (4.9%), KIRC (3.5%) and MESO (2.6%) in
which MSI has not yet been well described. MSI-high GBM
had the highest mutational load among all MSI-positive
samples (mean, 16,432
±
standard deviation, 16,301;
p < 0.001) suggesting that such tumours will better respond
to checkpoint blockade immunotherapy. P12.138B Frequency and molecular fingerprints of microsatellite
instability across multiple cancer types V. Khammad1, A. Arkhipov1, I. Nikitin1, I. Abramov2,
E. Khammad1, M. Filimonov3, N. Zhuchenko3 V. Khammad1, A. Arkhipov1, I. Nikitin1, I. Abramov2,
E. Khammad1, M. Filimonov3, N. Zhuchenko3 1Medical Rehabilitation Center under the Ministry of Health of
Russian Federation, Moscow, Russian Federation,
2Engelhardt Institute of Molecular Biology, Moscow, Russian
Federation, 3I.M. Sechenov First State Moscow Medical
University, Moscow, Russian Federation Introduction: Microsatellite instability (MSI) is a hyper-
mutable phenotype caused by defective DNA mismatch
repair system. Although MSI has been well described in
colorectal adenocarcinoma, less is known about the pre-
valence and distinct molecular features of MSI among other
types of cancer. V. Khammad: None. A. Arkhipov: None. I. Nikitin:
None. I. Abramov: None. E. Khammad: None. M. Filimonov: None. N. Zhuchenko: None. P12.136D We also discov-
ered that MSI-H ALL and EC harbour significant number of
somatic fremeshift mutations in DNA repair genes. Conclusion: Our findings reveal the landscape of MSI
across different malignancies and underscore the need for
further studies of this tumour phenotype. Prevalence of MSI across 14 cancer types
Cancer Type
№of
samples
MSI-
H
MSI-
L
% MSI
1)Endometrial carcinoma (EC)
89
14
4
20.22
2)Stomach adenocarcinoma (STAD)
78
9
4
16.67
3)Glioblastoma multiforme (GBM)
57
4
1
8.77
4)Pediatric acute lymphoblastic
leukaemia (ALL)
39
2
0
5.13
5)Liver hepatocellular
carcinoma (LIHC)
41
1
1
4.88
6)Kidney renal clear cell
carcinoma (KIRC)
29
1
0
3.45
7)Mesothelioma (MESO)
38
1
0
2.63
8)Breast invasive carcinoma (BRCA) 54
0
0
0.00
9)Ovarian serous
cystadenocarcinoma (OV)
45
0
0
0.00
10)Prostate adenocarcinoma (PRAD) 32
0
0
0.00
11)Head and neck squamous cell
carcinoma (HNSC)
29
0
0
0.00
12)Lung adenocarcinoma (LUAD)
28
0
0
0.00
13)Skin cutaneous
melanoma (SKCM)
20
0
0
0.00
14)Thyroid carcinoma (THCA)
17
0
0
0.00 F. Kipkeeva: None. T. Muzaffarova: None. P. Apano-
vich: None. M. Nikulin: None. S. Nered: None. M. Narimanov: None. O. Malekhova: None. I. Stilidi: None. A. Karpukhin: None. M. Florczuk1, A. Szpechcinski1, K. Duk1, M. Komorowski1,
W. Kupis1, P. Rudzinski1, M. Bryl2, T. Orlowski1,
J. Chorostowska-Wynimko1 P12.139C Materials and Methods: We examined MSI across 14
cancer types (n= 596) by employing the Promega MSI
Analysis System which used 5 mononucleotide markers
(BAT-25, BAT-26, NR-21, NR-24 and MONO-27) to
identify MSI in a tumour and normal tissue DNA. Within a
subset of samples with detected MSI we assessed mutation
burden and distinct molecular signatures associated with
this tumour phenotype. Circulating plasma miRNAs expression alterations as
promising marker in discrimination of EGFR mutation
status in NSCLC patients J. Chorostowska-Wynimko1 J. del Picchia 446 1National Institute of Tuberculosis and Lung Diseases,
Warsaw, Poland, 2E.J. Zeyland Wielkopolska Center of
Pulmonology and Thoracic Surgery, Poznan, Poland Urology, Istanbul, Turkey, 3Istanbul Medeniyet University,
Faculty of Medicine,Phase IV, Istanbul, Turkey, 4Istanbul
Medeniyet University Faculty of Medicine Department of
Pathology, Istanbul, Turkey, 5Istanbul Medeniyet University,
Faculty of Medicine,Phase III, Istanbul, Turkey, 6Istanbul
Medeniyet University, Faculty of Medicine, Department of
Biostatistics, Istanbul, Turkey Presently, the only predictive biomarker of response to
EGFR-TKI therapy in non-small cell lung cancer (NSCLC)
patients is the EGFR mutation status. The aberrant expres-
sion of miRNAs play a key role in lung carcinogenesis. Herein, we evaluated the potential diagnostic usefulness of
several circulating plasma miRNAs expression under dif-
ferent normalization approaches that might have dis-
criminative value for EGFRm+ and EGFRm- NSCLC
patients. Total RNA was extracted from 100μL of plasma;
material with hemolysis was excluded. Expression of miR-
504, miR-122, miR-195, miR-10b, miR-21, and UniSP6
(extraction efficacy control) in plasma of 60 non-squamous
NSCLC patients (31 patients EGFR+ in paired tumor tissue
and plasma specimen) was investigated using RT-qPCR. Similar procedure was applied for in vitro NSCLC cell lines
(HCC4006, PC-9, H1975, and H2347). Next, association
between circulating miRNA expression and EGFR mutation
status was analyzed according to different data normal-
ization approaches (miR-16 and miR-191 as normalizers). Only plasma miR-504 expression was significantly asso-
ciated with EGFR mutation status in NSCLC patients
regardless the normalizer used (p = 0.0158 and p = 0.002;
for adenocarcinoma patients p = 0.0004 and p = 0.001; for
miR-16 and miR-191 normalization, respectively). The
highest discriminatory power of circulating miR-504 was
shown for patients with exon 19 deletions versus wild-type
EGFR normalized to miR-191 (AUC=0.807 p < 0.0001). Aforementioned relations were not observed for in vitro cell
lines. Our study demonstrated the feasibility and potential
diagnostic value of miR-504 expression analysis in plasma
for
discrimination
between
EGFRm+
and
EGFRm-
NSCLC patients. P12.140D I. Akalin: None. B. Erol: None. E. Aslan: None. S.S. Ozkanli: None. O. Efiloglu: None. S. Yildirim: None. H. Güclü: None. T. Caskurlu: None. M.I. Karaman: None. Aggressive prostate cancer development might be under the
regulation of both Tregs and miRNAs Aggressive prostate cancer development might be under the
regulation of both Tregs and miRNAs I. Akalin1, B. Erol2, E. Aslan3, S. S. Ozkanli4, O. Efiloglu2,
S. Yildirim5, H. Güclü6, T. Caskurlu2, M. I. Karaman2
1Istanbul Medeniyet University Faculty of Medicine
Department of Medical Genetics, Istanbul, Turkey, 2Istanbul
Medeniyet University Faculty of Medicine Department of I. Akalin1, B. Erol2, E. Aslan3, S. S. Ozkanli4, O. Efiloglu2,
S. Yildirim5, H. Güclü6, T. Caskurlu2, M. I. Karaman2 I. Akalin1, B. Erol2, E. Aslan3, S. S. Ozkanli4, O. Efiloglu2,
S. Yildirim5, H. Güclü6, T. Caskurlu2, M. I. Karaman2 P12.139C Szpechcinski: None. K. Duk:
None. M. Komorowski: None. W. Kupis: None. P. Rudzinski: None. M. Bryl: None. T. Orlowski: None. J. Chorostowska-Wynimko: None. References:Song, C.J et al. J Cell Biochem. 2018. 119
(3):2763-2786 P12.139C However, the normalization strategy is of
key importance, strongly impacting circulating miRNA
analysis outcome. Introduction: The progress of prostate cancer comprises
complex contemporaneous tumor developmental events in
diverse stages are still yet to be clarified. miRNAs might
accompany to balance between regulatory (FOXP3+) and
cytotoxic T cells in tumors. Here, we investigated miRNAs
and FOXP3 expressions in patients with prostate cancer
spectrum. Material and Method:38 prostate cancer patients enrolled
within two groups as having Gleason Score up to 7; 8 or
more and 19 benign prostate hyperplasia (BPH) controls. 12
miRNAs expressions were analyzed by real time PCR from
paraffin embedded prostate tissue samples. Correlation
analyses were made between serum PSA levels, immuno-
histochemical staining of CD3, CD4, FOXP3 and miRNA
expressions. Results: We found, hsa-let7c-3p significantly 1,52 (p =-
0,018) and 1,84 (p = 0,0095) fold down-regulated whereas,
miR-141-3p was significantly 2,36 (p = 0,0006) and 2,24
(p = 0,001) fold upregulated in the prostate cancer patients
compared to BPH in group 1 and group 2, respectively. Only CD4 (p = 0,004) and PSA (p < 0,001) have statisti-
cally significant differences among groups when compared
to BPH. The Treg marker FOXP3 expressions were
significantly correlated with miR-143-p, miR-221-3p, hsa-
let7c-3p and miR-17-3p expressions. No significant correla-
tions
were
found
between
CD3,
CD4
and
miRNA
expressions or between PSA and FOXP3 expressions. Results: We found, hsa-let7c-3p significantly 1,52 (p =-
0,018) and 1,84 (p = 0,0095) fold down-regulated whereas,
miR-141-3p was significantly 2,36 (p = 0,0006) and 2,24
(p = 0,001) fold upregulated in the prostate cancer patients
compared to BPH in group 1 and group 2, respectively. Only CD4 (p = 0,004) and PSA (p < 0,001) have statisti-
cally significant differences among groups when compared
to BPH. The Treg marker FOXP3 expressions were
significantly correlated with miR-143-p, miR-221-3p, hsa-
let7c-3p and miR-17-3p expressions. No significant correla-
tions
were
found
between
CD3,
CD4
and
miRNA
expressions or between PSA and FOXP3 expressions. Conclusions: We for the first time reported significantly
altered expressions of miRNAs (miR-let7c, miR221, miR-
146a, miR-141, miR-143, miR17) and correlations between
Treg marker FOXP3 in the prostate cancer patients
suggesting that prostate cancer progression might be under
the regulation of both Tregs and miRNAs. M. Florczuk: None. A. Szpechcinski: None. K. Duk:
None. M. Komorowski: None. W. Kupis: None. P. Rudzinski: None. M. Bryl: None. T. Orlowski: None. J. Chorostowska-Wynimko: None. M. Florczuk: None. A. C. J. Pérez-Amado1, H. Tovar2, V. Bautista-Piña3, L. A. Alfáro-
Rauíz2, A. Hidalgo-Miranda2, S. Jiménez-Morales2 I. Akalin1, B. Erol2, E. Aslan3, S. S. Ozkanli4, O. Efiloglu2,
S. Yildirim5, H. Güclü6, T. Caskurlu2, M. I. Karaman2 1Istanbul Medeniyet University Faculty of Medicine
Department of Medical Genetics, Istanbul, Turkey, 2Istanbul
Medeniyet University Faculty of Medicine Department of P12.141A
Mitochondrial DNA mutation analysis in molecular
subtypes of breast cancer Mitochondrial DNA mutation analysis in molecular
subtypes of breast cancer 1Istanbul Medeniyet University Faculty of Medicine
Department of Medical Genetics, Istanbul, Turkey, 2Istanbul
Medeniyet University Faculty of Medicine Department of C. J. Pérez-Amado1, H. Tovar2, V. Bautista-Piña3, L. A. Alfáro-
Rauíz2, A. Hidalgo-Miranda2, S. Jiménez-Morales2 447 Abstracts from the 51st European Society of Human Genetics Conference: Posters 1Biochemistry Sciences Program, Universidad Nacional
Autónoma de México, Mexico City, Mexico, 2Instituto Nacional
de Medicina Genómica, Mexico City, Mexico, 3Instituto de
Enfermedades de la Mama y Fundación del Cáncer de Mama,
Mexico City, Mexico Introduction: Breast cancer is the commonest female
cancer, globally, as well as in Sri Lanka. Several mtDNA
mutations are reported to be associated with breast cancer,
but there are no data for Sri Lanka. Methods: Thirty patients with sporadic primary breast
cancer and their age, BMI and menopausal status matched
controls were studied. Macro-haplogroups M and N were
determined using coding region SNP based PCR-RFLP. A
900 bp region covering the hypervariable region I of
mtDNA was PCR-amplified, sequenced and analysed. Introduction. Breast cancer (BC) is the is the commonest
cancer type in women worldwide. Many studies have sug-
gested that variants in mitochondrial genome (mtDNA) are
risk factors to develop BC, are associated with severity,
relapse or treatment response. To know the landscape of the
mtDNA mutations in BC tumors from Mexican women we
analyzed mtDNA from paired peripheral blood (PB)-tumor. Materials and methods. We included 39 tumor matched in
PB samples from Mexican women with BC. Tumors were
classified
immunohistochemically
and
molecularly
(PAM50). The entire DNAmt was sequenced through the
MiSeq platform (Illumina) and using two overlapping pri-
mer sets. Results. 38.5% of the tumors were luminal A
(LA), 25.6% luminal B (LB), 10.3% HER2, 10.5% basal
(B) and 15.4% normal-like (NL). A total of 349 and 364
variants were identified in SP and tumor, respectively, of
which, 225 variants were shared among both tissue. 139 somatic mutaciones were identified in tumors, being the
single base substitutions the most common (94%) and the
remainder variants were insertions (3%) and deletions (3%). The CYB gene showed the highest number of somatic
mutations (18.7%) in comparison with other mitochondrial
genes. Stratification analysis by molecular subtype showed
that HER subtype displays the highest number of mutations
(average: 11 ± 8.2) than the other tumor subtypes. Con-
clusions. P12.141A
Mitochondrial DNA mutation analysis in molecular
subtypes of breast cancer mtDNA from HER subtype showed the highest
mutation number suggesting that there is an association
between the rate of DNAmt mutations with the molecular
subtype of BC and with the prognosis of the disease. Acknowledgments: CONACyT FOSISS 2016-272618 Introduction. Breast cancer (BC) is the is the commonest
cancer type in women worldwide. Many studies have sug-
gested that variants in mitochondrial genome (mtDNA) are
risk factors to develop BC, are associated with severity,
relapse or treatment response. To know the landscape of the
mtDNA mutations in BC tumors from Mexican women we
analyzed mtDNA from paired peripheral blood (PB)-tumor. Materials and methods. We included 39 tumor matched in
PB samples from Mexican women with BC. Tumors were
classified
immunohistochemically
and
molecularly
(PAM50). The entire DNAmt was sequenced through the
MiSeq platform (Illumina) and using two overlapping pri-
mer sets. Results. 38.5% of the tumors were luminal A
(LA), 25.6% luminal B (LB), 10.3% HER2, 10.5% basal
(B) and 15.4% normal-like (NL). A total of 349 and 364
variants were identified in SP and tumor, respectively, of
which, 225 variants were shared among both tissue. 139 somatic mutaciones were identified in tumors, being the
single base substitutions the most common (94%) and the
remainder variants were insertions (3%) and deletions (3%). The CYB gene showed the highest number of somatic
mutations (18.7%) in comparison with other mitochondrial
genes. Stratification analysis by molecular subtype showed
that HER subtype displays the highest number of mutations
(average: 11 ± 8.2) than the other tumor subtypes. Con-
clusions. mtDNA from HER subtype showed the highest
mutation number suggesting that there is an association
between the rate of DNAmt mutations with the molecular
subtype of BC and with the prognosis of the disease. Acknowledgments: CONACyT FOSISS 2016-272618 Results: 18 patients and 17 controls belonged to the M
macro-haplogroup and the remainder to the N macro-
haplogroup. In total 72 and 45 mutations were identified in
the patients and controls respectively, with 38 and 15 of
them occurring exclusively in each group. Prevalence of
mutations previously reported to be associated with breast
cancer,
namely
T16189C,
T16519C,
T16311C
and
C16207T were not significantly different between patients
and controls. Exclusive mutations seen in 23 patients were
distributed as follows: 05 (N=3), 04 (N=2), 03 (N=3), 02
(N=7) and 01mutation (N=8). Some patients had more than
one exclusive mutation with the highest number co-existing
being five. P12.141A
Mitochondrial DNA mutation analysis in molecular
subtypes of breast cancer Conclusion:
Mitochondrial
DNA
D-loop
mutations
previously unreported to be associated with breast cancer
observed in the present study require further analysis
especially since several such mutations co-exist in some
patients. Prevalence of HVI mutations reported to be
associated
with
breast
cancer
or
M
and
N
macro-
haplogroup status did not significantly differ between
patients and controls in present study. Supported by National Science Foundation, Sri-Lanka
(NSF/SCH/2016/04) J.T. Kotelawala: None. K.H. Tennekoon: None. R. Ranasinghe: None. H.A.C.I. K. Rodrigo: None. G.K.S. De Silva: None. C.J. Pérez-Amado: None. H. Tovar: None. V. Bau-
tista-Piña: None. L.A. Alfáro-Rauíz: None. A. Hidalgo-
Miranda: None. S. Jiménez-Morales: None. C.J. Pérez-Amado: None. H. Tovar: None. V. Bau-
tista-Piña: None. L.A. Alfáro-Rauíz: None. A. Hidalgo-
Miranda: None. S. Jiménez-Morales: None. P12.144D RET Genotype & MEN2 Phenotype correlation in a large
Indian Medullary Thyroid Carcinoma (MTC) cohort &
influence of SNPs in 3 genetic pathways on MTC behavior RET Genotype & MEN2 Phenotype correlation in a large
Indian Medullary Thyroid Carcinoma (MTC) cohort &
influence of SNPs in 3 genetic pathways on MTC behavior 1Institute of Biochemistry, Molecular Biology and
Biotechnology, University of Colombo, Colombo, Sri Lanka,
2National Cancer Institute, Maharagama, Sri Lanka P12.142B Mitochondrial DNA (mtDNA) haplogroups and
hypervariable region I (HVI) mutations in a cohort of Sri
Lankan sporadic breast cancer patients - A preliminary
analysis V. Mishra1,2, P. Kowtal1,2, R. Reddy1,2, N. Mahida1,2, R. Sarin1,2 1Advanced Centre for Treatment Research & Education in
Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai,
India, 2Homi Bhabha National Intitute (HBNI), Mumbai, India J. T. Kotelawala1, K. H. Tennekoon1, R. Ranasinghe1, H. A. C. I. K. Rodrigo1, G. K. S. De Silva2 J. T. Kotelawala1, K. H. Tennekoon1, R. Ranasinghe1, H. A. C. I. K. Rodrigo1, G. K. S. De Silva2 Introduction: MTC is an aggressive cancer of Thyroid
parafollicular cells. Around 25% MTC cases occur as
Multiple Endocrine Neoplasia Type2 (MEN2) syndrome
due to germline mutation in RET protooncogene. However
phenotypic heterogeneity in individuals harboring the same 448 J. del Picchia (MM) into extramedullary disease and short overall survival
(OS=23 months). I-FISH investigation revealed presence of
gain 1q21 and hyperdiploidy (+5,+9,+15) in 82% and
86%, respectively, while IgH rearrangements, del(17)(p13)
and del(13)(q14) were evaluated as negative. Whole-
genome
profiling
using
array-CGH
showed
complex
genomic changes including hyperdiploidy (+3,+5,+9,
+11, +15,+19), monosomy X, structural gains (1q21-
1q23.1, 1q32-1q44, 16p13.13-16p11.2) and losses (1q23.1-
1q32.1; 8p23.3-8p11.21) of genetic material and chromo-
thripsis in chromosome 18 with 6 breakpoint areas. Next-
generation sequencing showed a total of 338 variants with
1.8%
(6/338)
of
pathological
mutations
in
NRAS
(c.181C>A; p.Gln61Lys) or variants of unknown signifi-
cance in TP53, CUX1 and POU4F1. (MM) into extramedullary disease and short overall survival
(OS=23 months). I-FISH investigation revealed presence of
gain 1q21 and hyperdiploidy (+5,+9,+15) in 82% and
86%, respectively, while IgH rearrangements, del(17)(p13)
and del(13)(q14) were evaluated as negative. Whole-
genome
profiling
using
array-CGH
showed
complex
genomic changes including hyperdiploidy (+3,+5,+9,
+11, +15,+19), monosomy X, structural gains (1q21-
1q23.1, 1q32-1q44, 16p13.13-16p11.2) and losses (1q23.1-
1q32.1; 8p23.3-8p11.21) of genetic material and chromo-
thripsis in chromosome 18 with 6 breakpoint areas. Next-
generation sequencing showed a total of 338 variants with
1.8%
(6/338)
of
pathological
mutations
in
NRAS
(c.181C>A; p.Gln61Lys) or variants of unknown signifi-
cance in TP53, CUX1 and POU4F1. RET mutation suggests the role of additional genetic events
in MTC. The aim is to identify novel and recurrent germline
RET mutations in a cohort of 400 Indian MTC cases and to
study the role of 13 different SNPs in genes of distinct
pathways on MTC behavior. P12.142B Materials
and
Methods:
Germline
RET
mutation
analysis was performed on genomic DNA by Sanger/Next
Generation
Sequencing. For
SNP
genotyping
RFLP
approach was used followed by correlation of SNP
genotype with different clinico pathological parameters of
patients. Results:
Pathogenic
germline
RET
mutations
were
identified in 62 families (120 mutation carriers). Double
mutation in RET was seen in 3 families & novel mutation in
1 family. In 3 cases with classical MEN2B phenotype
Sanger Sequencing could not identify RET mutation. Whole Exome NGS analysis identified M918T mutation
in RET indicating allele dropout on Sanger Sequencing. Three of the 13 SNPs studied (Cyp1A1m1, CDKN2A,
NAT2) showed significant protective association with lesser
metastatic spread and calcitonin levels. Conclusions: Our findings suggest that presence of
chromothripsis should be considered as another important
genetic hallmark of poor prognosis in MM patients and
utilization of genome-wide screening techniques such as
array-CGH and NGS improves the clinical diagnostics of
the disease. This project was supported by MUNI/A/0824/
2017. Conclusions: Our findings suggest that presence of
chromothripsis should be considered as another important
genetic hallmark of poor prognosis in MM patients and
utilization of genome-wide screening techniques such as
array-CGH and NGS improves the clinical diagnostics of
the disease. This project was supported by MUNI/A/0824/
2017. in RET indicating allele dropout on Sanger Sequencing. Three of the 13 SNPs studied (Cyp1A1m1, CDKN2A,
NAT2) showed significant protective association with lesser
metastatic spread and calcitonin levels. Discussion: This is the first large report on genotype-
phenotype association in Indian MTC cohort revealing
several distinct associations with double or novel RET
mutation and modifier effect of few SNPs on MTC clinical
outcome. The not so infrequent occurrence of allele dropout
is highlighted in this study. J. Smetana: None. J. Oppelt: None. M. Štork: None. L. Pour: None. P. Kuglik: None. J. Smetana: None. J. Oppelt: None. M. Štork: None. L. Pour: None. P. Kuglik: None. High prevalence of pathogenic PTEN germline mutations in
population data Grant & Fellowship: ICMR for funding, DST-Inspire
for Fellowship J. R. Vos1, R. M. de Voer1, C. M. Kets1, A. R. Mensenkamp1,
M. J. L. Ligtenberg1,2, N. Hoogerbrugge1
1Department of Human Genetics, Radboud university medical
center, Nijmegen, Netherlands, 2Department of Pathology,
Radboud university medical center, Nijmegen, Netherlands J. R. Vos1, R. M. de Voer1, C. M. Kets1, A. R. Mensenkamp1,
M. J. L. Ligtenberg1,2, N. Hoogerbrugge1 V. Mishra: None. P. Kowtal: None. R. Reddy: None. N. Mahida: None. R. Sarin: None. V. Mishra: None. P. Kowtal: None. R. Reddy: None. N. Mahida: None. R. Sarin: None. 1Department of Human Genetics, Radboud university medical
center, Nijmegen, Netherlands, 2Department of Pathology,
Radboud university medical center, Nijmegen, Netherlands P12.148D High prevalence of pathogenic PTEN germline mutations in
population data Coexistance of APC and NF1 pathogenic mutations in the
same patient NAT2 polymorphisms are risk factor to acute
lymphoblastic leukemia in children from Mexico P. Vázquez1,2,3, E. Bueno Martínez1,2, N. Gestoso-Uzal1,2,3,
J. Perez-Garcia1,2,3, M. Zafra Cobo4, R. González-Sarmiento1,2,5 E. Hurtado-Córdova1, D. A. Bárcenas-López1,2, J. C. Núñez-
Enríquez3, A. Medina-Sanzón4, J. M. Mejía-Aranguré3,
E. Jiménez-Hernández3, J. Flores-Lujano3, J. G. Peñaloza-
González5, L. E. Merino-Pasaye6, K. A. Solis-Labastida3, J. R. Torres-Nava7, M. L. Pérez-Saldívar3, A. Hidalgo-Miranda2,
S. Jiménez-Morales2 E. Hurtado-Córdova1, D. A. Bárcenas-López1,2, J. C. Núñez-
Enríquez3, A. Medina-Sanzón4, J. M. Mejía-Aranguré3,
E. Jiménez-Hernández3, J. Flores-Lujano3, J. G. Peñaloza-
González5, L. E. Merino-Pasaye6, K. A. Solis-Labastida3, J. R. Torres-Nava7, M. L. Pérez-Saldívar3, A. Hidalgo-Miranda2,
S. Jiménez-Morales2 1Molecular Medicine Unit, Salamanca, Spain, 2Institute of
Biomedical Research of Salamanca (IBSAL), Salamanca,
Spain, 3Institute of Molecular and Cellular Biology of Cancer
(IBMCC). University of Salamanca-CSIC, Salamanca, Spain,
4Dermatology Unit, Hospital Virgen de la Concha, Zamora,
Spain, 5Institute of Molecular and Cellular Biology of Cancer. Univeristy of Salamanca-CSIC, Salamanca, Spain 1Programa de Maestría en Ciencias Biológicas UNAM,
Mexico, Mexico, 2Instituto Nacional de Medicina Genómica,
Mexico, Mexico, 3Centro Médico Nacional Siglo XXI, IMSS,
Mexico, Mexico, 4Hospital Infantil de México, Mexico, Mexico,
5Hospital Juárez de México, Mexico, Mexico, 6Centro Médico
Nacional 20 de Noviembre, ISSSTE, Mexico, Mexico, 7Hospital
Pediátrico de Moctezuma, Mexico, Mexico Introduction: Type I Neurofibromatosis is a rare genetic
disease that causes benign nerve tumors, and cutaneous and
skeletal manifestations. Mutations in the NF1 gene have
been found to cause the disease. Familial Adenomatous
Polyposis (FAP) is an uncommon hereditary disease char-
acterized by the appearance of more than a hundred ade-
nomatous polyps. Mutations in the gene APC greatly
increase the risk of suffering FAP. Mutations in either of
these genes is uncommon, much more so the simultaneous
mutation of both genes in the same patient Acute lymphoblastic leukemia (ALL) is the leading cause of
pediatric cancer worldwide that display a disparity inci-
dence among ethnic groups. Mexican is one of the most
affected population, which also has a high childhood mor-
tality rate due to ALL. Evidences suggest that single
nucleotide polymorphisms (SNPs) in genes involved in the
metabolism of xenobiotics, such as N-acetyltransferase 2
(NAT2) gene, could contribute to the ALL risk. To know
wether SNPs in NAT2 gene are associated with ALL in The case of a 20-year-old patient with rose-colored
polypoid lesions in the dorso-lumbar region associated to a
possible type I neurofibromatosis is presented. Chromothripsis 18 in multiple myeloma patient with rapid
extramedullary relapse Results:
In gnomAD, 13 pathogenic
(10
variants,
AF=2.62e-5) and 14 predicted pathogenic (12 variants,
AF=2.69e-5) PTEN mutations were detected. In ExAC, 5
pathogenic (5 variants, AF=4.12e-5) and 6 predicted
pathogenic
(5
variants,
AF=4.94e-5)
mutations
were
identified. When in ExAC cancer patients (i.e. TCGA data)
were excluded to minimize selection bias, 2 pathogenic (2
variants, AF=1.88e-5) and 5 predicted pathogenic (5
variants, AF=4.72e-5) mutations were identified. This
suggests a mutation prevalence of 8-36/200,000. Results:
In gnomAD, 13 pathogenic
(10
variants,
AF=2.62e-5) and 14 predicted pathogenic (12 variants,
AF=2.69e-5) PTEN mutations were detected. In ExAC, 5
pathogenic (5 variants, AF=4.12e-5) and 6 predicted
pathogenic
(5
variants,
AF=4.94e-5)
mutations
were
identified. When in ExAC cancer patients (i.e. TCGA data)
were excluded to minimize selection bias, 2 pathogenic (2
variants, AF=1.88e-5) and 5 predicted pathogenic (5
variants, AF=4.72e-5) mutations were identified. This
suggests a mutation prevalence of 8-36/200,000. Conclusion: The prevalence of pathogenic germline
PTEN mutations is 8-16 times higher than expected based
on current estimates. When including predicted pathogenic
mutations, this was 21-36 times higher. E. Hurtado-Córdova: None. D.A. Bárcenas-López:
None. J.C. Núñez-Enríquez: None. A. Medina-Sanzón:
None. J.M. Mejía-Aranguré: None. E. Jiménez-Hernán-
dez: None. J. Flores-Lujano: None. J.G. Peñaloza-
González:
None. L.E. Merino-Pasaye:
None. K.A. Solis-Labastida: None. J.R. Torres-Nava: None. M.L. Pérez-Saldívar: None. A. Hidalgo-Miranda: None. S. Jiménez-Morales: None. This data substantiates that many PHTS patients are still
unrecognized, which might be related to varying disease
penetrance. With the increasing use of gene panels, this data
provides relevant expected detection rates in unselected
patients. J.R. Vos: None. R.M. de Voer: None. C.M. Kets: None. A.R. Mensenkamp: None. M.J.L. Ligtenberg: None. N. Hoogerbrugge: None. Chromothripsis 18 in multiple myeloma patient with rapid
extramedullary relapse Chromothripsis 18 in multiple myeloma patient with rapid
extramedullary relapse J. Smetana1, J. Oppelt2, M. Štork3, L. Pour3, P. Kuglik1 J. Smetana1, J. Oppelt2, M. Štork3, L. Pour3, P. Kuglik1 Introduction:
PTEN
Hamartoma
Tumour
Syndrome
(PHTS)
is
a
severe
inherited
cancer
risk
syndrome
–including malignant and benign neoplasms, overgrowth
and autism– caused by germline mutations in PTEN. The
prevalence of pathogenic germline PTEN mutations is
unclear, and estimated at ~1/200,000. The aim of this study
was to assess the population prevalence of pathogenic
PTEN mutations. 1Laboratory of Molecular Cytogenetics, Institute of
Experimental Biology, Faculty of Science, Masaryk University,
Brno, Czech Republic, 2CEITEC-Central European Institute of
Technology, Masaryk University, Brno, Czech Republic,
3Department of Internal Medicine-Hematooncology, University
Hospital Brno, Brno, Czech Republic Methods: The prevalence of pathogenic PTEN mutations
was assessed using the genome Aggregation Database
(gnomAD) and Exome Aggregation Collaboration (ExAC)
database,
including
138,632
and
60,706
individuals,
respectively. Background: Catastrophic chromosomal event known as
chromothripsis was proven to be a significant hallmark of
poor prognosis in several cancer diseases. While this phe-
nomenon is very rare in among multiple myeloma (MM)
patients, its presence in karyotype is associated with very
poor prognosis. Mutations were classified as pathogenic (i.e. multiple
times reported as pathogenic in ClinVar and/or truncating
variants) or predicted pathogenic (i.e. CADD score >25). Case presentation: In our case, we report a 62 year
female patient with rapid progression of multiple myeloma Abstracts from the 51st European Society of Human Genetics Conference: Posters 449 For each group, the combined allele frequency (AF) was
determined. For each group, the combined allele frequency (AF) was
determined. Mexican children we performed a case control-study. We
included 311 (152 cases and 159 controls) individuals aged
<17 years old. Cases were newly diagnosed and risk clas-
sified base on National Cancer Institute (NCI) criteria. DNA
was extracted from peripheral blood and saliva and geno-
typing was performed using Taqman probes. No statistical
differences were detected to rs1799930 SNP between cases
and controls, but rs1801280 (OR 2.2, CI 1.6-4.8, p = 4.4-
07), rs1789929 (OR 3.53, CI 2.58-4.84, P= 9.9-16), rs1208
(OR0 5.18, CI 3.77-7.10, P= 8.23-26) and rs1799931G
(OR=2.48, CI 1.72-3.57, P= 6.08-7) were associated with
ALL. In addition the rs1041983 showed the TT genotype
(OR 2.048, CI 1.047-4.0, P= 0.03) increased the risk to this
malignancy. Our results suggest that SNPs in NAT2 confer
susceptibility to develop ALL in children from Mexico. Coexistance of APC and NF1 pathogenic mutations in the
same patient Her clinical
history stood out because of a total colectomy to prevent 450 J. del Picchia Conclusions: We found a different spectrum of NF2
somatic
mutations
in
the
schwannomatosis-associated
schwannomas compared to the germinal mutations found
in NF2 patients, as already demonstrated in sporadic
schwannomas. The preponderance of indels as somatic
mutations and of single mucleotide substitutions as germ-
line ones, suggest the existence of distinct mechanisms of
mutagenesis during mitosis and meiosis. This work was
funded by grants from Ministero della Salute and Istituto
Toscano Tumori. Conclusions: We found a different spectrum of NF2
somatic
mutations
in
the
schwannomatosis-associated
schwannomas compared to the germinal mutations found
in NF2 patients, as already demonstrated in sporadic
schwannomas. The preponderance of indels as somatic
mutations and of single mucleotide substitutions as germ-
line ones, suggest the existence of distinct mechanisms of
mutagenesis during mitosis and meiosis. This work was
funded by grants from Ministero della Salute and Istituto
Toscano Tumori. FAP (several cases of polyposis colorectal cancer were
described in her maternal line), several surgeries to remove
trichilemmal and epidermic cysts in different locations and
an abdominal laparotomy to remove a desmoid tumor. FAP (several cases of polyposis colorectal cancer were
described in her maternal line), several surgeries to remove
trichilemmal and epidermic cysts in different locations and
an abdominal laparotomy to remove a desmoid tumor. Material and Methods: DNA extracted from peripheral
blood and from each type of tumor underwent heteroduplex
analysis Material and Methods: DNA extracted from peripheral
blood and from each type of tumor underwent heteroduplex
analysis Results:
In
NF1,
a
pathogenic
alteration
(c.5418_5422delGGGC/ p.Q1806QfsX), which generates
a truncated protein and causes neurofibromatosis, was found
in DNA extracted from the neurofibroma but not from
peripheral
blood. In
APC,
a
pathogenic
mutation
(c.3783_3785delTT/p.T1261TfsX), which generates a trun-
cated protein that increases the risk of FAP, was found. Neither of these mutations had been previously described I. Paganini: None. G.L. Capone: None. A. Putignano:
None. R. Sestini: None. F. Gensini: None. B. Porfirio:
None. A. Marozza: None. L. Papi: None. P12.153A Conclusion: The patient presented two rare pathogenic
mutations in genes responsible for diseases with unrelated
phenotypes Conclusion: The patient presented two rare pathogenic
mutations in genes responsible for diseases with unrelated
phenotypes Fifty first tests and beyond: a real world experience of
cancer genetic testing in a high risk cancer genetic clinic of
a university based urban hospital in Thailand, an upper
middle income country This project was funded by FIS PI 10/00219 This project was funded by FIS PI 10/00219 P. Vázquez: None. E. Bueno Martínez: None. N. Gestoso-Uzal: None. J. Perez-Garcia: None. M. Zafra
Cobo: None. R. González-Sarmiento: None. Schwannomatosis associated schwannomas show a
different NF2 mutational spectrum compared to
Neurofibromatosis type 2 patients I. Paganini1, G. L. Capone1, A. Putignano1, R. Sestini1,
F. Gensini1, B. Porfirio1, A. Marozza2, L. Papi1 Methods: We reviewed cases at the high-risk CGC at the
Chulalongkorn Hospital in 2017. 1University of Florence, Firenze, Italy, 2A.O.U.Careggi,
Firenze, Italy P12.151C Introduction: Cancer genetic testing (CGT) in low- to-
middle-income countries is less accessible and has mostly
been done in the research. We reported here the real-world
situation in a high risk cancer genetics clinic (CGC) at a
tertiary-center in Thailand. 1University of Florence, Firenze, Italy, 2A.O.U.Careggi,
Firenze, Italy Results: Of 69 cases, fifty cases (50/69=72.5%) under-
went CGC. The indications for patients tested were personal
history of cancer (47/50=94%). Only three unaffected
probands with family history of cancers (3/50=6%) were
tested. Breast (20/50=40%) and ovarian (6/50=12%)
cancers were the indication for the majority of tested
patients. We identified fifteen pathogenic variants (PV) or
likely
PV
in
BRCA1(5),BRCA2(3),APC(2),MLH1(1),
FANCA(1),PTEN(1),NF1(1),
and
monoallelic
PV
in
MUTYH(1)
in
14
patients
(14/50=28%:
one
patient
harbored 2 PV in BRCA1 and NF1). We identified eleven
variants of unknown significance in eleven patients (11/
50=22%).4 patients underwent prophylactic surgery after
testing. Nineteen cases refused(8) or were not offered
receiving (clinical diagnoses (2), or in the low risk group
(9)) the CGT. The reasons of refusal were financial
concerns and the perception of no benefit to themselves. The median age was 40 years old compared to 47 years old
in the tested patients. 36.8% (7/19) of patients were
unaffected. Results: Of 69 cases, fifty cases (50/69=72.5%) under-
went CGC. The indications for patients tested were personal
history of cancer (47/50=94%). Only three unaffected
probands with family history of cancers (3/50=6%) were
tested. Breast (20/50=40%) and ovarian (6/50=12%)
cancers were the indication for the majority of tested
patients. We identified fifteen pathogenic variants (PV) or
likely
PV
in
BRCA1(5),BRCA2(3),APC(2),MLH1(1),
FANCA(1),PTEN(1),NF1(1),
and
monoallelic
PV
in
MUTYH(1)
in
14
patients
(14/50=28%:
one
patient
harbored 2 PV in BRCA1 and NF1). We identified eleven
variants of unknown significance in eleven patients (11/
50=22%).4 patients underwent prophylactic surgery after
testing. Nineteen cases refused(8) or were not offered
receiving (clinical diagnoses (2), or in the low risk group
(9)) the CGT. The reasons of refusal were financial
concerns and the perception of no benefit to themselves. The median age was 40 years old compared to 47 years old
in the tested patients. 36.8% (7/19) of patients were
unaffected. Introduction: Two genetically distinct entities, Neurofi-
bromatosis type 2 (NF2) and schwannomatosis, predispose
to development of multiple schwannomas. Thus far, two
genes are involved in schwannomatosis predisposition:
SMARCB1 and LZTR1, both located on the long arm of
chromosome 22. NF2 gene, responsible for NF2, is soma-
tically involved in the four hits/three steps mechanism
described in schwannomatosis-associated schwannomas. However,
the
mutational
spectrum
of
NF2
gene
in
schwannomatosis tumors is not well defined. Pathogenic variants (PVs) detection in a 19-gene core panel
and yield of opportunistic screening in BRCA1/2 and MMR
genes in a cohort of 1121 hereditary cancer patients Through NGS Trusight Pan-Cancer analysis, we
identified variants in cohesin genes, in particular in exon 46
of NIPBL, in heterozygosity. This variant is a new mutation
that causes frameshift and a premature stop codon. This
anomaly was confirmed on the patient's bone marrow DNA
both at diagnosis and in remission of leukemia and in a
buccal smear sample. Both parents and brother are negative,
phenotypically normal and not affected by hematological
diseases. We further analyzed 86 pediatric BCP-ALL cases
considering NIPBL, RAD21, SMC1A, SMC3, STAG2. We
detected 36 variants, identifying recurrent known variants in
addition to 10 novel variants in cohesin genes, mainly
affecting NIPBL, which seems peculiar of ALL, since it has
never been reported as altered in AML. Results and Conclusions: At disease onset, patient did
not present any prognostically relevant cytogenetic abnorm-
ality and was enrolled to high risk treatment group for MRD
analysis. Through NGS Trusight Pan-Cancer analysis, we
identified variants in cohesin genes, in particular in exon 46
of NIPBL, in heterozygosity. This variant is a new mutation
that causes frameshift and a premature stop codon. This
anomaly was confirmed on the patient's bone marrow DNA
both at diagnosis and in remission of leukemia and in a
buccal smear sample. Both parents and brother are negative,
phenotypically normal and not affected by hematological
diseases. We further analyzed 86 pediatric BCP-ALL cases
considering NIPBL, RAD21, SMC1A, SMC3, STAG2. We
detected 36 variants, identifying recurrent known variants in
addition to 10 novel variants in cohesin genes, mainly
affecting NIPBL, which seems peculiar of ALL, since it has
never been reported as altered in AML. Results: Overall, 1015 female and 106 male were
studied, mean age at cancer diagnosis was 47 years old,
579 had breast cancer (BC), 258 ovarian cancer (OC), 124
colorectal cancer, and 27 were unaffected. A BC, OC, or
BC/OC panel was requested in 66% and a HNPCC panel in
12%. One hundred and fifty-one (13%) probands carried at
least one PV with the customized diagnostic panel. All
BRCA1/2 carriers fulfilled BC, OC or BC/OC criteria, while
among the MMR carriers, 50 (89%) PV were identified in
the HNPCC panel and 6 (11%) were opportunistic, all 6 in
MSH6. The 19-gene research panel provided 22 (2%)
additional PV beyond the customized panel according to the
clinical phenotype: 5 BARD1, 4 NBN, 3 BRIP1, 3 ATM, 2
CHEK2, 2 RAD51C, 2 RAD51D, 1 CDH1. Pathogenic variants (PVs) detection in a 19-gene core panel
and yield of opportunistic screening in BRCA1/2 and MMR
genes in a cohort of 1121 hereditary cancer patients Conclusions: The yield of PV detection in different
actionable genes identified by multiplex testing is clinically
relevant. Eleven percent of MMR mutation carriers (all
carrying MSH6 PVs) were identified through opportunistic
screening. V. Massa: None. G. Fazio: None. A. Grioni: None. V. Bystry: None. S. Rigamonti: None. C. Saitta: None. C. Rizzari: None. C. Consarino: None. A. Biondi: None. A. Selicorni: None. G. Cazzaniga: None. P12.155C
NIPBL pathogenic variant in a Cornelia de Lange
Syndrome patient with Acute Lymphoblastic Leukemia P. Phowthongkum: None. W. Kamolvisit: None. V. Massa1, G. Fazio2, A. Grioni2,3, V. Bystry3, S. Rigamonti1,2,
C. Saitta2, C. Rizzari2, C. Consarino4, A. Biondi2, A. Selicorni5,
G. Cazzaniga2 Pathogenic variants (PVs) detection in a 19-gene core panel
and yield of opportunistic screening in BRCA1/2 and MMR
genes in a cohort of 1121 hereditary cancer patients Pathogenic variants (PVs) detection in a 19-gene core panel
and yield of opportunistic screening in BRCA1/2 and MMR
genes in a cohort of 1121 hereditary cancer patients 1Università degli Studi di Milano, Milano, Italy, 2Università
degli Studi di Milano-Bicocca, Monza, Italy, 3Masaryk
University, Brno, Czech Republic, 4Presidio Ospedaliero
Ciaccio-De Lellis, Catanzaro, Italy, 5ASST Lariana, Como,
Italy C. Lazaro1, L. Feliubadaló1, A. Stradella1, O. Díez2, G. Capellá1,
S. Gutiérrez2, J. del Valle1, J. Brunet1, J. Balmaña2, Hereditary
Cancer Program Catalan Cancer Network University, Brno, Czech Republic, 4Presidio Ospedaliero
Ciaccio-De Lellis, Catanzaro, Italy, 5ASST Lariana, Como,
Italy 1Hereditary Cancer Program, ICO-IDIBELL-CIBERONC,
Hospitalet de Llobregat, Spain, 2Hospital Vall d'Hebron-
VHIO, Barcelona, Spain Background: Cornelia de Lange syndrome (CdLS) is a rare
genetic disorder characterized by pre- and post-natal growth
retardation, mental retardation, facial dysmorphism and
upper limb abnormalities. The main causes of the disease
are mutations in the NIPBL, SMC1A, SMC3, HDAC8 and
RAD21 genes, which encode proteins of cohesin complex. Mutations in the cohesin genes have recently been identified
in AML, CML and myelodysplastic syndromes. Background: Multigene panels provide a powerful tool for
analyzing several genes simultaneously and identifying
cancer susceptibility beyond the suspected clinical pheno-
type. We evaluated the PVs frequency in customized pre-
defined panels according to phenotype and extended the
analysis to our 19-gene research panel. We also investigated
the yield of opportunistic screening in the BRCA1/2 and
MMR genes in all patients. Background: Multigene panels provide a powerful tool for
analyzing several genes simultaneously and identifying
cancer susceptibility beyond the suspected clinical pheno-
type. We evaluated the PVs frequency in customized pre-
defined panels according to phenotype and extended the
analysis to our 19-gene research panel. We also investigated
the yield of opportunistic screening in the BRCA1/2 and
MMR genes in all patients. Objectives: Here, we report the description of the first
case of a CdLS pediatric patient who developed precursors
B Acute Lymphoblastic Leukemia (BCP-ALL). Further-
more, we investigated the presence of cohesin genes
variants in pediatric ALL patients not affected by CdLS. Patients: Overall, 1121 unrelated probands underwent
multigene testing with customized pre-defined panels
according to their phenotype in addition to BRCA1/2 and
MMR genes, and a 19-gene research. Results and Conclusions: At disease onset, patient did
not present any prognostically relevant cytogenetic abnorm-
ality and was enrolled to high risk treatment group for MRD
analysis. 1University of Florence, Firenze, Italy, 2A.O.U.Careggi,
Firenze, Italy Materials and Methods: We analysed NF2 gene in
116 schwannomas from 46 schwannomatosis patients with
or
without
LZTR1
or
SMARCB1
germline
mutation,
comparing this mutational spectrum with the one of 117
NF2 patients and 25 mosaic NF2. In schwannomas we also
assessed LOH. Materials and Methods: We analysed NF2 gene in
116 schwannomas from 46 schwannomatosis patients with
or
without
LZTR1
or
SMARCB1
germline
mutation,
comparing this mutational spectrum with the one of 117
NF2 patients and 25 mosaic NF2. In schwannomas we also
assessed LOH. Results: We demonstrated a statistically significant
increase of indel mutations in schwannomatosis-associated
tumors versus the germinal ones. Discussion: We presented the CGT in the high risk CGC
in the real world setting since the more affordable CGT has Abstracts from the 51st European Society of Human Genetics Conference: Posters 451 P12.155C
NIPBL pathogenic variant in a Cornelia de Lange
Syndrome patient with Acute Lymphoblastic Leukemia
V. Massa1, G. Fazio2, A. Grioni2,3, V. Bystry3, S. Rigamonti1,2,
C. Saitta2, C. Rizzari2, C. Consarino4, A. Biondi2, A. Selicorni5,
G. Cazzaniga2 been offered in the last few years. Patients refused or not-
offered to be tested were younger and were more unaffected
cases than the tested group. National Institute of Tuberculosis and Lung Diseases, Warsaw,
Poland National Institute of Tuberculosis and Lung Diseases, Warsaw,
Poland Background: Alpha-1 antitrypsin (AAT) is encoded by the
highly polymorphic SERPINA1 gene. Mutations in this
gene can lead to AAT deficiency (AATD) which is a risk
factor for number of chronic lung disorders. The causative
link between AATD and non-small cell lung cancer
(NSCLC) is controversial due to insufficient research data. We evaluated the frequency of major pathogenic SERPINA1
alleles among Polish NSCLC patients. Methods: blood samples were collected from 468
NSCLC patients. The serum AAT concentration was
measured by nephelometry and the phenotype was analyzed
by isoelectric focusing. The SERPINA1 variants were
identified by DNA sequencing. The frequency of SER-
PINA1 alleles was estimated by means of Hardy-Weinberg
equilibrium. Results: 446 out of 468 (95.2%) NSCLC patients
presented normal Pi*MM phenotype. AATD alleles were
found in 22 patients. 10 (2.1%) carried Pi*S allele, 10
(2.1%)
-
Pi*Z
allele,
and
3
(0.6%)
-
Pi*F
allele. Accordingly, the frequency of major AATD alleles was:
Pi*S 10.7/1000 (95%CI:4.1-17.3) and Pi*Z 10.7/1000
(95%CI:4.1-17.3). The mean serum AAT concentration in
NSCLC patients was 180 mg/dL in Pi*MM individuals and
148 mg/dL in AATD allele carriers. Conclusions: The frequency of Pi*S and Pi*Z alleles in
NSCLC patients differ noticeably from the actual data of
general Polish population study (Pi*Z 13.7/1000 and Pi*S
7.6/1000): the frequency of Pi*Z allele is lower whereas the
frequency of Pi*S is higher than normal reference values. The NSCLC patients who carry AATD allele do not present
reduced AAT concentration in blood as compared to the
reference values from population studies. Conclusions: The frequency of Pi*S and Pi*Z alleles in
NSCLC patients differ noticeably from the actual data of
general Polish population study (Pi*Z 13.7/1000 and Pi*S
7.6/1000): the frequency of Pi*Z allele is lower whereas the
frequency of Pi*S is higher than normal reference values. The NSCLC patients who carry AATD allele do not present
reduced AAT concentration in blood as compared to the
reference values from population studies. A. Wood: A. Employment (full or part-time); Significant;
Swift Biosciences. S. Sandhu: A. Employment (full or part-
time); Significant; Swift Biosciences. M. Pezeshkian: A. Employment (full or part-time); Significant; Swift Bios-
ciences. V. Kelchner: A. Employment (full or part-time);
Significant; Swift Biosciences. J. RoseFigura: A. Employ-
ment (full or part-time); Significant; Swift Biosciences. J. Lenhart: A. Employment (full or part-time); Significant;
Swift Biosciences. L. Kurihara: A. P12.156D C. Lazaro: None. L. Feliubadaló: None. A. Stradella:
None. O. Díez: None. G. Capellá: None. S. Gutiérrez:
None. J. del
Valle:
None. J. Brunet:
None. J. Balmaña: None. The allelic frequency for S and Z mutations in the
SERPINA1 gene in NSCLC patients from Poland The allelic frequency for S and Z mutations in the
SERPINA1 gene in NSCLC patients from Poland 452 J. del Picchia A. Szpechcinski, K. Duk, A. Zdral, M. Florczuk, P. Rudzinski,
W. Kupis, J. Zaleska, R. Langfort, T. Orlowski, K. Roszkowski-
Sliz, J. Chorostowska-Wynimko Swift Biosciences, Ann Arbor, MI, United States The growing use of liquid biopsy for early detection and
monitoring of disease necessitates accurate variant detection
at <1% allele frequencies due to a low population of disease
DNA within circulating, cell-free DNA (cfDNA). Reliable,
low-frequency
variant
detection
by
next-generation
sequencing (NGS) is challenging due to background noise
from PCR and sequencing errors. We employed molecular
identifiers (MIDs) to uniquely label individual DNA
molecules prior to amplification, facilitating the distinction
of true variants from PCR and sequencing errors. We
incorporated MIDs in both our Accel-Amplicon library prep
that uses multiplex PCR for targeted NGS and our Accel-
NGS 2S whole genome library prep followed by targeting
with hybridization capture using an 800kb pan-cancer
panel. We performed low frequency spike-in experiments at
<1% allele frequencies. We prepared MID libraries from
amplicon panels including a 17 amplicon EGFR pathway
panel and a 104 amplicon SNP panel. Deep sequencing to
>30,000x was done to maximize MID family size (number
of PCR duplicates) and optimize generation of a consensus
sequence. This analysis identified all known variants pre-
sent at 1%, 0.5%, and 0.25% allele frequencies. Next, the
targeted 2S libraries were prepared with low-frequency
spike-in samples, sequenced to >8000x, and all known
variants at 1% and 0.5% allele frequencies were maintained
in the consensus data. In both cases, the number of false
positives was reduced, resulting in improved specificity. This study highlights the ability of MID technology to
enable low frequency variant detection, critical to track
known variants and identify novel pathogenic mutations in
cfDNA samples. P12.157A Use of molecular identifiers in targeted NGS to detect
mutations below one percent allele frequencies in
circulating cell-free DNA Use of molecular identifiers in targeted NGS to detect
mutations below one percent allele frequencies in
circulating cell-free DNA National Institute of Tuberculosis and Lung Diseases, Warsaw,
Poland Employment (full or
part-time); Significant; Swift Biosciences. V. Makarov: A. Employment
(full
or
part-time);
Significant;
Swift
Biosciences. A. Szpechcinski: None. K. Duk: None. A. Zdral: None. M. Florczuk: None. P. Rudzinski: None. W. Kupis:
None. J. Zaleska: None. R. Langfort: None. T. Orlowski:
None. K. Roszkowski-Sliz: None. J. Chorostowska-
Wynimko: None. N. P. Özateş Ay, K. Öztürk, &. Kaya, B. Sezgin, N. Akyıldız, Ü.
Uluöz, A. Veral, C. Çalışkan Kurt, B. Göker Bağca, Ç. Biray
Avcı A. Wood, S. Sandhu, M. Pezeshkian, V. Kelchner,
J. RoseFigura, J. Lenhart, L. Kurihara, V. Makarov J. RoseFigura, J. Lenhart, L. Kurihara, V. Makarov A. Wood, S. Sandhu, M. Pezeshkian, V. Kelchner, EGE ÜNİVERSİTESİ, İZMİR, Turkey might effectively identify patients eligible for PARP inhi-
bitor therapy and serve as a pre-selection for germline
BRCA1/2 testing. might effectively identify patients eligible for PARP inhi-
bitor therapy and serve as a pre-selection for germline
BRCA1/2 testing. Head and neck cancers, which constitute 2-5% of all
malignancies worldwide and in our society, can be diag-
nosed early by increasing the use of endoscopy and have a
high survival rate with appropriate treatment. There is no
definitive tumor marker used in the diagnosis and follow-up
of head and neck cancer. Genetic factors involved in the
formation of head and neck cancers have not been identi-
fied. The MET gene encodes the proto-oncogen MET, a
member of the protein receptor tyrosine kinase family. Mutations in these gene have been associated with papillary
renal cell carcinoma, hepatocellular carcinoma, and various
head and neck cancers. In this study, we aimed to investi-
gate the expression changes of MET gene in oral cavity
cancers which are thought to be effective in many cancers. RNA isolation was performed from tumoral and normal
tissue specimens stored in paraffin blocks of 30 patients
under the age of 40 years, with a mean age of 32-41 and
who did not smoke and whose oral cavity tumors were
present between January 2016 and August 2016. MET gene
expression levels were analyzed by real-time quantitative
reverse transcription in comparison with normal tissues. Postoperative pathology of all patients was squamous cell
carcinoma. According to the results, when compared to
normal tissues, the expression levels of MET genes were
increased by 8.47 fold in tumor tissue. The obtained data
will guide the molecular recognition and mechanism of the
disease and provide prognostically valuable information by
associating it with recurrence / survival. Materials and Methods: Newly diagnosed OCs patients
form 8 hospitals were included by pathologists. Tumour
DNA BRCA1/2 testing was performed in formalin fixed,
paraffin embedded (FFPE) samples using single molecule
Molecular Inversion Probe-based targeted next generation
sequencing
and
BRCA1
Multiplex
Ligation-dependent
Probe Amplification. Gynaecologists advised patients with
a tumour BRCA1/2 mutation to have genetic counselling
and germline BRCA1/2 testing. Results: Of 301 consecutive OC patients, 53 (18%) had a
tumour DNA BRCA mutation of which 56% was diagnosed
with a germline BRCA1/2 mutation. EGE ÜNİVERSİTESİ, İZMİR, Turkey More than half (58%)
of the families with a germline BRCA1/2 mutation did not
comply with criteria for germline BRCA1/2 testing based on
family history prior to the OC diagnosis. 81% of all newly
diagnosed OC patients were included by the pathologists for
tumour DNA BRCA testing. Median turnaround time of
tumour DNA BRCA testing was 14 days. The workflow was
positively reviewed by all patients and 83% of the
gynaecologists. Discussion: Tumour DNA BRCA1/2 testing in all newly
diagnosed OC patients as pre-selection for PARP inhibitor
therapy and germline testing is feasible and effective. More
than half of the patients with a germline BRCA1/2 mutation
would not have been identified by family history. N. Hoogerbrugge: B. Research Grant (principal inves-
tigator, collaborator or consultant and pending grants as
well as grants already received); Significant; AstraZeneca. I. E. Fakkert: None. I.D. Nagtegaal: None. E.M. Leter:
None. A.R. Mensenkamp: None. G. Woldringh: None. M. Simons: None. C.M. Kets: None. H. Bulten: None. J. A. de Hullu: None. M.J. Ligtenberg: B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; AstraZeneca. N. Hoogerbrugge: B. Research Grant (principal inves-
tigator, collaborator or consultant and pending grants as
well as grants already received); Significant; AstraZeneca. I. E. Fakkert: None. I.D. Nagtegaal: None. E.M. Leter:
None. A.R. Mensenkamp: None. G. Woldringh: None. M. Simons: None. C.M. Kets: None. H. Bulten: None. J. A. de Hullu: None. M.J. Ligtenberg: B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; AstraZeneca. N.P. Özateş Ay: None. K. Öztürk: None. &. Kaya:
None. B. Sezgin: None. N. Akyıldız: None. Ü. Uluöz:
None. A. Veral: None. C. Çalışkan Kurt: None. B. Göker
Bağca: None. Ç. Biray Avcı: None. N.P. Özateş Ay: None. K. Öztürk: None. &. Kaya:
None. B. Sezgin: None. N. Akyıldız: None. Ü. Uluöz:
None. A. Veral: None. C. Çalışkan Kurt: None. B. Göker
Bağca: None. Ç. Biray Avcı: None. P12.158B Investigation of the effect of MET gene expression
alteration on oral cavity tumors Avcı Abstracts from the 51st European Society of Human Genetics Conference: Posters 453 1Section of Molecular Genetics, Department of Laboratory
Medicine, University Hospital of Pisa, Pisa, Italy, 2Department
of Clinical and Experimental Medicine, Division of
Gynecology and Obstetrics, University of Pisa, Pisa, Italy, Tumour DNA BRCA1/2 testing in newly diagnosed ovarian
cancer as pre-selection for PARP inhibitor therapy and
germline testing is feasible and effective Tumour DNA BRCA1/2 testing in newly diagnosed ovarian
cancer as pre-selection for PARP inhibitor therapy and
germline testing is feasible and effective Tumour DNA BRCA1/2 testing in newly diagnosed ovarian
cancer as pre-selection for PARP inhibitor therapy and
germline testing is feasible and effective P12.160D P12.160D
BRCA1 AND BRCA2 GENES MUTATIONAL
SCREENING IN FFPE OVARIAN CANCER TISSUES N. Hoogerbrugge1, I. E. Fakkert1, I. D. Nagtegaal1, E. M. Leter2,
A. R. Mensenkamp1, G. Woldringh1, M. Simons1, C. M. Kets1,
H. Bulten1, J. A. de Hullu1, M. J. Ligtenberg1 G. Gambino1, R. Scarpitta1, M. Farnocchia1, K. Zavaglia1,
M. Guerrieri2, E. Falaschi1, M. Tancredi1, F. Bonci3,
C. Congregati4, S. Gana4, S. Pistolesi5, F. Carbone5,
G. Naccarato5, A. Gadducci2, M. Caligo1 1Radboud university Medical Center, Nijmegen, Netherlands,
2Maastricht university Medical Center, Maastricht,
Netherlands 1Radboud university Medical Center, Nijmegen, Netherlands,
2Maastricht university Medical Center, Maastricht,
Netherlands Introduction: Ovarian cancer (OC) patients with either
somatic or germline BRCA1/2 mutations may benefit from
PARP inhibitor therapy. Tumour DNA BRCA1/2 testing
initiated by the pathologist in newly diagnosed OC patients J. del Picchia 454 3UO Medical Oncology, Department of Oncology, University
Hospital of Pisa, Pisa, Italy, 4Laboratory of Medical Genetics,
A.O.U. Pisana, Ospedale S. Chiara, Pisa, Italy, 5Dep. of
Pathology, University Hosp. of Pisa, Pisa, Italy Introduction: Ovarian cancer is the fifth most common
form of cancer death among women. Developing a fast,
reliable blood test would facilitate the early detection of
ovarian cancer,
which
would
also
contribute to the
improvement of survival chances. Screening circulating
miRNAs proved to be reliable biomarkers in various can-
cers. The dysregulation of miRNAs is also known in
ovarian cancer, however, only a few publications focus on
their diagnostic role. Introduction: Ovarian cancer is the fifth most common
form of cancer death among women. Developing a fast,
reliable blood test would facilitate the early detection of
ovarian cancer,
which
would
also
contribute to the
improvement of survival chances. Screening circulating
miRNAs proved to be reliable biomarkers in various can-
cers. The dysregulation of miRNAs is also known in
ovarian cancer, however, only a few publications focus on
their diagnostic role. Ovarian cancer patients with germline or somatic patho-
genic variants in BRCA genes benefit from treatment with
poly ADP ribose polymerase inhibitors (iPARP1). To select
patients who may benefit from these treatments, assessment
of the mutation status of BRCA1 and BRCA2 in the tumor is
required. Tumor BRCA1/2 testing is more challenging than
germline testing as the majority of samples are formalin-
fixed paraffin embedded (FFPE), the tumor genome is
complex, and the allelic fraction of somatic variants can be
low. P12.162B Mir200a, miR200b and miR200c as candidate biomarkers
in the diagnosis of ovarian cancer Mir200a, miR200b and miR200c as candidate biomarkers
in the diagnosis of ovarian cancer A. Vagena, M. Papamentzelopoulou, D. Kalfakakou,
D. Yannoukakos, I. Konstantopoulou, F. Fostira M. Szilágyi-Bónizs1, É. Márton1, J. Lukács2, B. Soltész1,
E. Janka3, R. Póka2, B. Nagy1 M. Szilágyi-Bónizs1, É. Márton1, J. Lukács2, B. Soltész1,
E. Janka3, R. Póka2, B. Nagy1 C
PALB2 c.2257 C>T mutation is a Greek founder and is
associated with early breast cancer diagnosis PALB2 c.2257 C>T mutation is a Greek founder and is
associated with early breast cancer diagnosis P12.160D Nagy: None. 1University of Debrecen, Faculty of Medicine, Department of
Human Genetics, Debrecen, Hungary, 2University of
Debrecen, Faculty of Medicine, Department of Obstetrics and
Gynecology, Debrecen, Hungary, 3University of Debrecen,
Faculty of Medicine, Department of Dermatology, Debrecen,
Hungary P12.160D There is the need for efficient and timely methods to
detect both somatic and germline mutations using FFPE
tissues and commercially available technology. We used
commercial kits to explore all exons and 50bp exon-intron
junctions
of
BRCA1
and
BRCA2
in
next-generation
sequencing (NGS) on DNA from 40 FFPE ovarian serous
carcinoma tissues. In total, 10/40 patients (25%) carried 11
mutations (9 in BRCA1 and 2 in BRCA2). Pathogenic var-
iants were confirmed by Sanger sequencing in tumor DNA. One novel frameshift BRCA1 mutations was found. The
germline or somatic status of the mutations will be assessed
in DNA from blood sample or from FFPE non neoplastic
tissues. This study evaluates the relevance of standardiza-
tion in tumor BRCA testing particularly when the test results
dictate clinical decisions regarding life extending therapies
of patients. Materials and Methods: We screened several miRNAs
(miR200a, miR200b, miR200c, miR205, miR483, let7f) in
the
plasma
samples
of
healthy
(n = 30),
previously
untreated serous epithelial ovarian carcinoma patients
(n = 17, FIGO stages III or IV) and patients with benign
masses (n = 14). The relative amount of miRNAs was
detected by qRT-PCR. Results: The expression levels of miR200a, miR200b and
miR200c were elevated in the carcinoma samples compared
to the healthy donors (p < 0.00001). The level of miR200a
was also higher in cancerous than in benign samples
(p < 0.01). However, no significant difference was detected
in the case of miR205, miR483 and let7f. Diagnostic
accuracy was the highest in the case of miR200a: 87.23%
with the power area under the curve (AUC) of 0.91 (95%
CI=0.79-1). The diagnostic accuracy was also promising in
the case of miR200b and miR200c: 80.85% and 74.47%
with AUC 0.82 (95% CI=0.67-0.97) and 0.8 (95%
CI=0.64-0.96) respectively. The correlation was relatively
high between the test results of miR200b and miR200c. Conclusions: MiR200a, miR200b and miR200c are
applicable biomarkers in ovarian cancer. G. Gambino: None. R. Scarpitta: None. M. Farnoc-
chia: None. K. Zavaglia: None. M. Guerrieri: None. E. Falaschi: None. M. Tancredi: None. F. Bonci: None. C. Congregati: None. S. Gana: None. S. Pistolesi: None. F. Carbone: None. G. Naccarato: None. A. Gadducci:
None. M. Caligo: None. M. Szilágyi-Bónizs:
None. É. Márton:
None. J. Lukács: None. B. Soltész: None. E. Janka: None. R. Póka: None. B. Nagy: None. M. Szilágyi-Bónizs:
None. É. Márton:
None. J. Lukács: None. B. Soltész: None. E. Janka: None. R. Póka: None. B. A. Vagena, M. Papamentzelopoulou, D. Kalfakakou,
D. Yannoukakos, I. Konstantopoulou, F. Fostira P12.165A
Polymorphic pre-miR-146a and synergistic action of all of
its products on NTRK2 gene in Papillary Thyroid
Carcinoma Polymorphic pre-miR-146a and synergistic action of all of
its products on NTRK2 gene in Papillary Thyroid
Carcinoma Polymorphic pre-miR-146a and synergistic action of all of
its products on NTRK2 gene in Papillary Thyroid
Carcinoma A. Vagena: None. M. Papamentzelopoulou: None. D. Kalfakakou: None. D. Yannoukakos: None. I. Konstan-
topoulou: None. F. Fostira: None. A. Kubiak1,2, A. Wójcicka1,2, W. Wiechno3, G. Niewiński4,
K. Jażdżewski1,2 A. Kubiak1,2, A. Wójcicka1,2, W. Wiechno3, G. Niewiński4,
K. Jażdżewski1,2 NCSR Demokritos, Athens, Greece The discordance of
genetic and clinical data in some patients suggests that a
deeper biological understanding of this tumor entity is
required. Conclusions: This study demonstrates that ctDNA testing
is technically feasible for high throughput sequencing and
therefore has great potential as a noninvasive monitoring
tool for PDAC patients. Nevertheless, refinement of the
cancer gene list is necessary since several cases revealed no
mutation in the 51 analyzed genes. The discordance of
genetic and clinical data in some patients suggests that a
deeper biological understanding of this tumor entity is
required. S. Balendran-Braun: None. M. Kieler: None. S. Liebmann-Reindl:
None. G.W. Prager:
None. B. Streubel: None. S. Balendran-Braun: None. M. Kieler: None. S. Liebmann-Reindl:
None. G.W. Prager:
None. B. Streubel: None. Discussion: Herein, the PALB2 c.2257 C>T mutation is
shown to be a Greek founder mutation, statistically
significantly associated with earlier breast cancer diagnosis,
while
families had
strong
family
history
of
cancer,
specifically enriched for pancreatic cancer incidence. NCSR Demokritos, Athens, Greece Introduction: Germline mutations in PALB2 (Partner And
Localizer of BRCA2) are rare, attributing for approximately
1–2% of familial breast cancer (BrCa) cases; their frequency
can be possibly influenced by founder effects. Deleterious
PALB2 variants are clinically important, predisposing for
breast, pancreatic and possibly, ovarian cancer. Ten,
apparently unrelated, Greek families carried the PALB2
c.2257C>T (p.Arg753*) mutation; we therefore sought to 1University of Debrecen, Faculty of Medicine, Department of
Human Genetics, Debrecen, Hungary, 2University of
Debrecen, Faculty of Medicine, Department of Obstetrics and
Gynecology, Debrecen, Hungary, 3University of Debrecen,
Faculty of Medicine, Department of Dermatology, Debrecen,
Hungary Abstracts from the 51st European Society of Human Genetics Conference: Posters 455 clarify its possible founder effect and to investigate asso-
ciations with cancer diagnoses. Treatment response was evaluated via computer tomogra-
phy before and 3 months after therapy. Results: Technically, all 63 samples were sequenced
successfully with both library kits. NGS resulted in average
3.65 Mio passed filter reads for TST15 and 1.07 Mio for
Ampliseq with mean amplicon coverages of 21718 and
4368, respectively. The ctDNA mutation frequencies were
between 1.2% and 21.7%. 8/21 patients showed KRAS and/
or TP53 driver mutations. Concerning correlation with
therapy responsiveness, computer tomography and cfDNA
analysis revealed concordant results in 17/21 patients. Methods: Genotyping for the mutation was pursued
either by multi-gene panel testing, Sanger sequencing or
Real-Time
PCR,
followed
by
haplotype
analysis
of
mutation carriers. Genotyping was performed on 2791
breast (median age 47.8 years ± 11.23), and 436 ovarian
cancer (median age 52 years, ± 13) patients. Approximately
one third of patients tested had family history (at least one
relative diagnosed with breast, ovarian or pancreatic
cancer). Results: The mutation prevalence was 0.36% (10/2791)
and 0.23% (1/436) in breast and ovarian cases, respectively. Targeted testing on family members revealed five more
carriers. All eleven families had strong family history, while
pancreatic cancer diagnosis among close relatives was
reported in two families. Median age at breast cancer
diagnosis among carriers was 41.8 years (range 33 – 58),
suggesting association with earlier breast cancer diagnosis
(p =
0.02). Haplotype
analysis
revealed
a
common
haplotype spanning 0.7Mb. Conclusions: This study demonstrates that ctDNA testing
is technically feasible for high throughput sequencing and
therefore has great potential as a noninvasive monitoring
tool for PDAC patients. Nevertheless, refinement of the
cancer gene list is necessary since several cases revealed no
mutation in the 51 analyzed genes. S. Balendran-Braun, M. Kieler, S. Liebmann-Reindl, G. W.
Prager, B. Streubel Medical University of Vienna, Vienna, Austria Cell-free circulating tumor DNA monitoring in pancreatic
cancer patients Cell-free circulating tumor DNA monitoring in pancreatic
cancer patients 1Genomic Medicine, Medical University of Warsaw, Warsaw,
Poland, 2Centre of New Technologies, University of Warsaw,
Warsaw, Poland, 3Department of General and Thoracic
Surgery, Medical University of Warsaw, Warsaw, Poland,
42nd Department of Anaesthesiology and Intensive Therapy,
Medical University of Warsaw, Warsaw, Poland 1Genomic Medicine, Medical University of Warsaw, Warsaw,
Poland, 2Centre of New Technologies, University of Warsaw,
Warsaw, Poland, 3Department of General and Thoracic
Surgery, Medical University of Warsaw, Warsaw, Poland,
42nd Department of Anaesthesiology and Intensive Therapy,
Medical University of Warsaw, Warsaw, Poland S. Balendran-Braun, M. Kieler, S. Liebmann-Reindl, G. W. Prager, B. Streubel S. Balendran-Braun, M. Kieler, S. Liebmann-Reindl, G. W. Prager, B. Streubel V. Rudaitis1, G. Januka1, R. Janavicius2,3 Introduction: Despite the remarkable improvement in
survival, cancer remains the leading cause of mortality in
childhood. In this work, we introduce the development and
clinical validation of a pediatric solid tumour gene panel,
based on NGS. This gene panel allows the characterisation
of somatic and germline mutations in solid tumours in
primary or relapsing malignancies. The aim of the study is
to provide clinically relevant information in diagnostics,
prognostics and personalised therapy. Introduction: Despite the remarkable improvement in
survival, cancer remains the leading cause of mortality in
childhood. In this work, we introduce the development and
clinical validation of a pediatric solid tumour gene panel,
based on NGS. This gene panel allows the characterisation
of somatic and germline mutations in solid tumours in
primary or relapsing malignancies. The aim of the study is
to provide clinically relevant information in diagnostics,
prognostics and personalised therapy. 1Vilnius University Hospital Santara Klinikos, Gynecology
department, Vilnius, Lithuania, 2Vilnius University Hospital
Santara Klinikos, Hematology, oncology and transfusion
medicine center, Vilnius, Lithuania, 3State Reasearch Institute
Innovative Medicine Center, Vilnius, Lithuania Objective: to evaluate the incidence of occult neoplasia in
specimen collected during PBSO and to determine the
significance of this operation in BRCA1/2 mutation carriers. Objective: to evaluate the incidence of occult neoplasia in
specimen collected during PBSO and to determine the
significance of this operation in BRCA1/2 mutation carriers. Methods: An NGS multigene panel of 254 full-sequence
genes is presented in this work. Tumour and blood paired
samples were sequenced on a NextSeq 500 System
(Illumina) according to the manufacturer's instructions
(Agilent, SureSelect QXT protocol). FASTQ files were
analysed by means of a self-developed pipeline for the
detection of somatic variants, indels and CNVs. Methods: Between January 2010 and October 2016 a
total of 564 new germline BRCA1/2 mutation positive
women were identified in VULSK. Laparoscopic PBSO
was performed for 71 eligible women, who opted for this
procedure and were included in this perspective study. Fifty
nine women (83,1%) were BRCA1 and 12 (16,9%) were
BRCA2 mutation carriers; study patients harboured 22
different BRCA1/2 germline mutations. Results and Conclusions: A set of 40 samples were
analysed in this study. First, we worked with fresh-frozen
samples, belonging to a retrospective cohort of 16 primary
tumours including the ten most-frequent pediatric tumour
subtypes. Medical University of Vienna, Vienna, Austria and NTRK2. The analysis conducted in 60 pairs of PTC
tumor and adjacent control tissue showed that the expres-
sion of NTRK2 is significantly decreased in PTC, with even
higher reduction in patients heterozygous for rs2910164
(61% decrease in 84% of tumors, p = 6.25-4). The analysis
of miR:NTRK2 interaction in luciferase assay confirmed
that all the polymorphic miR-146a products interact with
target gene and this effect is additive, giving a 26%(p =
3.49-11) reduction of luciferase activity by the synergistic
binding of all isomiRs compared with 21%(p = 1.96-6) for
the main miR-146a-5p isoform.This is the first functional
study of the synergistic effect exerted by polymorphic
miRNAs produced from a single precursor. The results may
give a strong basis for better understanding of the role of
miR-146a and NTRK2 gene in development of PTC. This
work was supported by the Polish National Science Centre
Grant DEC-2012/07/N/NZ2/01333 carriers (out of 27) of exclusively BRCA1 c.4035delA
mutation (22.2%; 95% CI 10.26 - 41.10), a common Baltic
founder mutation. The difference between STIC detected in
c.4035delA (6/27) and other mutations carriers (1/44) was
statistically significant (P=0.0105). Conclusion: Detection rate of precursor-only lesions
(STIC) in our study (9.85%) was slightly higher than
previously reported (5-8%). We found statistically signifi-
cant enrichment of BRCA1 c.4035delA mutation carriers in
STIC group, which may indicate that c.4035delA mutation
carriers may have an increased tumorigenesis rate in
fallopian tubes. V. Rudaitis:
None. G. Januška:
None. R. Janavicius: None. V. Rudaitis:
None. G. Januška:
None. R. Janavicius: None. P12.167C Clinical validation of a gene panel refines diagnostics and
tailors personalised treatment in pediatric cancer patients A. Kubiak: None. A. Wójcicka: None. W. Wiechno:
None. G. Niewiński: None. K. Jażdżewski: None. D. A. Garcia-Dios, C. Perez-Garcia, M. Gonzalez-Acera,
M. Garcia-Ruiz, P. Marin-Garcia, C. Ruiz, C. de Torres,
J. Garcia-Planells Medical University of Vienna, Vienna, Austria Medical University of Vienna, Vienna, Austria Introduction: Pancreatic ductal adenocarcinoma (PDAC) is
a leading cause of cancer death worldwide. 80-85% of
patients are diagnosed at an advanced stage since clinical
presentation often occurs very late in the disease process. In
this study, we evaluated cell-free circulating tumor DNA
(ctDNA) of 21 PDAC patients before treatment and under
chemotherapy in serial plasma samples. Numerous studies confirm the deregulation of microRNAs
in various human cancers, including Papillary Thyroid
Carcinoma (PTC). The G/C heterozygosity in rs2910164 in
miR-146a-3p predisposes to PTC and occurs as a somatic
mutation
in
thyroid
tumors. Deleterious
function
of
rs2910164 results from the fact that the SNP is located in
the seed region of miR-146a-3p, thus heterozygous carriers
of the SNP produce 3 mature miRs: miR-146a-5p, miR-
146a-3p(G) and 146a-3p(C). Interestingly, there is only one
gene (NTRK2 - involved in control of differentiation and
programmed cell death), identified in silico as concertedly
targeted by the three isoforms. The aim of this study was to
analyze the interaction between all the miR-146a isoforms Material and Methods: Three serial plasma samples per
patient were obtained. Library preparation of 63 liquid
biopsies was conducted using Illumina TruSight Tumor 15
(TST15) and the brand-new AmpliSeq for Illumina Cancer
HotSpot Panel, which covers 50 cancer-related genes. J. del Picchia 456 and NTRK2. The analysis conducted in 60 pairs of PTC
tumor and adjacent control tissue showed that the expres-
sion of NTRK2 is significantly decreased in PTC, with even
higher reduction in patients heterozygous for rs2910164
(61% decrease in 84% of tumors, p = 6.25-4). The analysis
of miR:NTRK2 interaction in luciferase assay confirmed
that all the polymorphic miR-146a products interact with
target gene and this effect is additive, giving a 26%(p =
3.49-11) reduction of luciferase activity by the synergistic
binding of all isomiRs compared with 21%(p = 1.96-6) for
the main miR-146a-5p isoform.This is the first functional
study of the synergistic effect exerted by polymorphic
miRNAs produced from a single precursor. The results may
give a strong basis for better understanding of the role of
miR-146a and NTRK2 gene in development of PTC. This
work was supported by the Polish National Science Centre
Grant DEC-2012/07/N/NZ2/01333 carriers (out of 27) of exclusively BRCA1 c.4035delA
mutation (22.2%; 95% CI 10.26 - 41.10), a common Baltic
founder mutation. The difference between STIC detected in
c.4035delA (6/27) and other mutations carriers (1/44) was
statistically significant (P=0.0105). J. Garcia-Planells Instituto de Medicina Genomica (IMEGEN), Paterna
(Valencia), Spain Instituto de Medicina Genomica (IMEGEN), Paterna
(Valencia), Spain V. Rudaitis1, G. Januka1, R. Janavicius2,3 P12.166B The incidence of occult ovarian neoplasia and cancer in
BRCA1/2 mutation carriers after the bilateral prophylactic
salpingo-oophorectomy (PBSO): a single center prospective
study J. Garcia-Planells D. A. Garcia-Dios, C. Perez-Garcia, M. Gonzalez-Acera,
M. Garcia-Ruiz, P. Marin-Garcia, C. Ruiz, C. de Torres,
J. Garcia-Planells V. Rudaitis1, G. Januka1, R. Janavicius2,3 In a second stage, we included 24 patient cases
which directly benefited from an accurate diagnostic and
personalised
treatment
based
on
the
present
genetic
findings. Finally, we screened matching formalin-fixed
paraffin-embedded (FFPE) tissue when this was available. Here,
we
were
able
to
validate
and
fine-tune
the
performance of our gene panel in low-quality fragmented Results: STIC was diagnosed in seven (9.85%; 95% CI
4.58 - 19.26) women and occult ovarian cancer (OC) was
found in four (5.6%; 95% CI 1.80 - 14.03) women. The
median age of women that were diagnosed with STIC or
OC at the time PBSO was 45,9 years ( ± 6,68; P=0.7314),
the youngest patient being 42 and the oldest 64 years. All
11 women who were diagnosed with STIC or OC had
BRCA1 mutations. Interestingly, STIC was detected in 6 Results: STIC was diagnosed in seven (9.85%; 95% CI
4.58 - 19.26) women and occult ovarian cancer (OC) was
found in four (5.6%; 95% CI 1.80 - 14.03) women. The
median age of women that were diagnosed with STIC or
OC at the time PBSO was 45,9 years ( ± 6,68; P=0.7314),
the youngest patient being 42 and the oldest 64 years. All
11 women who were diagnosed with STIC or OC had
BRCA1 mutations. Interestingly, STIC was detected in 6 457 Abstracts from the 51st European Society of Human Genetics Conference: Posters DNA. Future studies include the testing in tissue obtained
from minimally invasive tests such as liquid biopsy. This is
of special interest in those pediatric patients with unresect-
able cancers or otherwise challenging to biopsy. DNA. Future studies include the testing in tissue obtained
from minimally invasive tests such as liquid biopsy. This is
of special interest in those pediatric patients with unresect-
able cancers or otherwise challenging to biopsy. including STK11. Neither of the parents carried this
alteration. Conclusions: We present an additional case of 19p13.3
deletion encompassing STK11 gene and forming a dis-
tinctive phenotype. Muscular symptoms, beside hypotonia,
are not described before in 19p13.3 deletion patients. Therefore, it is not precisely known which gene in 19p13.3
region is responsible for muscular phenotype. Finding the
most efficient diagnostic algorithm for each patient is
challenged by large clinical and molecular variability of
Peutz-Jeghers syndrome. D.A. Garcia-Dios: A. Employment (full or part-time);
Significant; Imegen. C. Perez-Garcia: A. Employment (full
or part-time); Significant; Imegen. M. Gonzalez-Acera: A. Employment (full or part-time); Significant; Imegen. V. Rudaitis1, G. Januka1, R. Janavicius2,3 M. Garcia-Ruiz: A. Employment (full or part-time); Signifi-
cant; Imegen. P. Marin-Garcia: None. C. Ruiz: A. Employment (full or part-time); Significant; Imegen. C. de Torres: None. J. Garcia-Planells: A. Employment (full
or part-time); Significant; imegen. D.A. Garcia-Dios: A. Employment (full or part-time);
Significant; Imegen. C. Perez-Garcia: A. Employment (full
or part-time); Significant; Imegen. M. Gonzalez-Acera: A. Employment (full or part-time); Significant; Imegen. M. Garcia-Ruiz: A. Employment (full or part-time); Signifi-
cant; Imegen. P. Marin-Garcia: None. C. Ruiz: A. Employment (full or part-time); Significant; Imegen. C. de Torres: None. J. Garcia-Planells: A. Employment (full
or part-time); Significant; imegen. L. Roht: None. S. Pajusalu: None. T. Kahre: None. O. ilina: None. K. Õunap: None. L. Roht: None. S. Pajusalu: None. T. Kahre: None. O. ilina: None. K. Õunap: None. L. Roht1, S. Pajusalu1,2, T. Kahre1,2, O. ilina1,3, K. Õunap1,2 L. Roht1, S. Pajusalu1,2, T. Kahre1,2, O. ilina1,3, K. Õunap1,2 L. Roht1, S. Pajusalu1,2, T. Kahre1,2, O. ilina1,3, K. Õunap1,2 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia, 3Department of Biotechnology, Institute of
Molecular and Cell Biology, University of Tartu, Tartu,
Estonia 1Medical Genetics Unit, S. Orsola-Malpighi Hospital,
University of Bologna, Bologna, Italy, 2General Surgery Unit,
S. Orsola-Malpighi Hospital, University of Bologna, Bologna,
Italy, 3Pathology Unit, S.Orsola-Malpighi Hospital, University
of Bologna, Bologna, Italy, 4Endocrinology Unit, S. Orsola-
Malpighi Hospital, University of Bologna, Bologna, Italy,
5Internal Medicine Unit, University of Bologna, Bologna, Italy Introduction: Peutz-Jeghers syndrome is an autosomal
dominant disease associated with gastrointestinal polyposis
and mucocutaneous pigmentation. It usually results from
germline point mutations in STK11 gene, large deletions are
rare. Patients with 19p13.3 deletions encompassing STK11
gene have been reported to constitute a distinctive pheno-
type of intellectual disability, hypotonia and dysmorphic
features. Introduction: Germline mutations in PHD1, PHD2 genes
and either germline and somatic mutations in EPAS1 have
been detected in some patients diagnosed with the rare
pheochromocytoma/paraganglioma
with
polycythemia
syndrome. Materials and Methods: A man developed symptomatic
arterial hypertension at age 29 and was subsequently
diagnosed with unilateral pheochromocytoma and multi-
focal paragangliomas. Polycythemia had been reported as
an incidental finding at age 13 and confirmed afterwards. JAK2 gene sequencing found no mutations. Family history
was irrelevant; polycythemia was excluded in the patient’s
parents and younger brother. Whole Exome Sequencing
(WES) was performed on constitutional DNA of the patient
and his parents and on DNA from fresh-frozen paragan-
glioma tissue collected at surgery, using the MedExome
library enrichment method (Roche) and the Illumina
Next500 platform. Data were aligned and filtered using
our
internal
pipeline. Variants
were
annotated
using
ANNOVAR and validated by Sanger sequencing. Materials and Methods: We are presenting a 12-year old
girl with mild intellectual disability, tremor in hands,
clumsy gait with muscle weakness and hypotonia, positive
Gowers’ sign, dysmorphic facial features and mucocuta-
neous pigmentation. At first NGS gene panel (~4800 genes)
was sequenced, which showed no alterations associated
with her phenotype. Due to muscular phenotype muscle
biopsy was done, which showed lipid accumulation. In
suspicion of inherited myopathy trio exome analysis was
performed
and
unexpectedly
copy
number
algorithm
CoNIFER identified a deletion of chromosomal region
19p13.3, which was confirmed by chromosomal microarray
analysis (CMA). Exome sequencing in a case of pheochromocytoma/
paraganglioma-polycythemia syndrome Exome sequencing in a case of pheochromocytoma/
paraganglioma-polycythemia syndrome A. Tranchina1, E. Bonora1, F. Isidori1, C. Gurioli2, C. Ceccarelli3, D. Santini3, V. Vicennati4, E. Cosentino5, F. Minni2,
D. Turchetti1 A. Tranchina1, E. Bonora1, F. Isidori1, C. Gurioli2, C. Ceccarelli3, D. Santini3, V. Vicennati4, E. Cosentino5, F. Minni2,
D. Turchetti1 L. Roht1, S. Pajusalu1,2, T. Kahre1,2, O. ilina1,3, K. Õunap1,2 We suppose, that
these genomic alterations could play a role in tumor
dissemination and might lead to identification of new
therapeutic targets. P12.170B Complex analysis of genomic and transcriptomic
alterations in tumor tissue associated with presence of
various subpopulations of circulating tumor cells in
primary breast cancer Acknowledgements: This work was supported by Slovak
Research and Development Agency by grants APVV-14-
0327 and APVV-16-0010. G. Repiska: None. G. Minarik: None. T. Tokar: None. M. Hajduk: None. M. Karaba: None. J. Benca: None. L. Krasnicanova: None. J. Macuch: None. G. Sieberova:
None. J. Pindak: None. M. Cristofanilli: None. J. Reuben: None. I. Jurisica: None. J. Mardiak: None. M. Mego: None. G. Repiska: None. G. Minarik: None. T. Tokar: None. M. Hajduk: None. M. Karaba: None. J. Benca: None. L. Krasnicanova: None. J. Macuch: None. G. Sieberova:
None. J. Pindak: None. M. Cristofanilli: None. J. Reuben: None. I. Jurisica: None. J. Mardiak: None. M. Mego: None. G. Repiska1, G. Minarik2, T. Tokar3, M. Hajduk4, M. Karaba5, J. Benca5,6, L. Krasnicanova2, J. Macuch5, G. Sieberova5, J. Pindak5,7, M. Cristofanilli8, J. Reuben9, I. Jurisica7, J. Mardiak1,5, M. Mego1,5 G. Repiska1, G. Minarik2, T. Tokar3, M. Hajduk4, M. Karaba5, J. Benca5,6, L. Krasnicanova2, J. Macuch5, G. Sieberova5, J. Pindak5,7, M. Cristofanilli8, J. Reuben9, I. Jurisica7, J. Mardiak1,5, M. Mego1,5 1Comenius University in Bratislava Faculty of Medicine,
Bratislava, Slovakia, 2Comenius University in Bratislava
Faculty of Natural Sciences, Bratislava, Slovakia, 3University
Health Network and University of Toronto, Toronto, ON,
Canada, 4Institute of Molecular Biology of Slovak Academy of
Sciences, Bratislava, Slovakia, 5National Cancer Institute,
Bratislava, Slovakia, 6St. Elizabeth University Department of
Medicine, Bratislava, Slovakia, 7Slovak Medical University,
Bratislava, Slovakia, 8Division of Hematology-Oncology at
Northwestern University Feinberg School of Medicine,
Chicago, IL, United States, 9University of Texas MD Anderson
Cancer Center, Houston, TX, United States L. Roht1, S. Pajusalu1,2, T. Kahre1,2, O. ilina1,3, K. Õunap1,2 Results: The deletion of 19p13.3 (1,152,656-2,120,154)
seen on exome analysis and later confirmed by CMA was
~1 Mb long and comprised eight disease associated genes Results: The deletion of 19p13.3 (1,152,656-2,120,154)
seen on exome analysis and later confirmed by CMA was
~1 Mb long and comprised eight disease associated genes Results: WES failed to detect mutations in EPAS1, PHD1
and
PHD2,
or
in
genes
associated
to
hereditary
pheochromocytoma-paraganglioma (VHL, SDHA, SDHB, Results: WES failed to detect mutations in EPAS1, PHD1
and
PHD2,
or
in
genes
associated
to
hereditary
pheochromocytoma-paraganglioma (VHL, SDHA, SDHB, Results: WES failed to detect mutations in EPAS1, PHD1
and
PHD2,
or
in
genes
associated
to
hereditary
pheochromocytoma-paraganglioma (VHL, SDHA, SDHB, 458 J. del Picchia Results: Mutations in BRCA1/2 genes in tumors were
more common in patients with EP_CTC compared to
patients without EP-CTC in peripheral blood (23.5% vs. 0%, p = 0.02), while there were no mutation in specific
gene associated with CTC_EMT. Further, 90 genes and 7
miRs were expressed at significantly different levels in
EP_CTCs
tumors
and
199
genes
and
13
miRs
in
CTC_EMT tumors when compared to tumors without
detectable CTCs. Moreover 39 overlapping genes and 7
miRs were found to be expressed at significantly different
levels
in
tumors
with
EP_CTCs
and/or
CTC_EMT
compared to tumors without detectable CTCs. SDHC,
SDHD,
SDHAF2,
TMEM127,
MAX,
KIF1B,
TCEB1). Compound heterozygosity for two very rare
variants (p.W342X and p.R571H) in OVGP1 (encoding
for Oviductal Glycoprotein 1, expressed in ovary but also in
male gonads and in some cancers) were found in patient's
constitutional DNA, inherited from the parents, while in the
tumor a somatic frameshift variant in CHST15 (encoding for
carbohydrate sulfotransferase 15) was detected. Conclusions: Clinical manifestations in this patient seem
unrelated to known genes; studies are ongoing to assess the
potential role of the variants detected. A. Tranchina: None. E. Bonora: None. F. Isidori:
None. C. Gurioli: None. C. Ceccarelli: None. D. Santini:
None. V. Vicennati: None. E. Cosentino: None. F. Minni:
None. D. Turchetti: None. Conclusions: We identified for the first time various
genomic alterations in tumor tissue associated with different
CTCs subpopulation in PBC patients. We suppose, that
these genomic alterations could play a role in tumor
dissemination and might lead to identification of new
therapeutic targets. Conclusions: We identified for the first time various
genomic alterations in tumor tissue associated with different
CTCs subpopulation in PBC patients. The in-vitro evaluation of anticancer effect of DMU-212, a
novel resveratrol analog, on prostate cancer cells The in-vitro evaluation of anticancer effect of DMU-212, a
novel resveratrol analog, on prostate cancer cells B. Ozmen Yelken1, C. Kayabaşi1, A. Aşik1, T. Balci Okcanoğlu2,
F. Soğutlu1, R. Gasimli1, S. Yilmaz Susluer1, C. Biray Avci1, C. Gunduz1 P12.173A Genome editing to map the role of 7p14.3 locus in prostate
cancer Genome editing to map the role of 7p14.3 locus in prostate
cancer B. Ozmen Yelken: None. C. Kayabaşi: None. A. Aşik:
None. T. Balci Okcanoğlu: None. F. Soğutlu: None. R. Gasimli: None. S. Yilmaz Susluer: None. C. Biray Avci:
None. C. Gunduz: None. B. Stringa, S. Garritano, A. Romanel, P. Gasperini, G. Petris, A. Cereseto, F. Demichelis Gunduz1 1Ege University Medical School, Department of Medical
Biology, İzmir, Turkey, 2Near East University, Vocational
School of Health Sciences, Nicosia, Cyprus Prostate cancer (PCa) is the second leading cause of cancer
among men in developed countries. It is known that Epi-
thelial Mesenchymal Transition (EMT) plays a crucial role
in the progression and metastasis of PCa. It has been
reported that DMU-212, which is one of the resveratrol
analogs, induces G2/M arrest. In our study, we focused on
the effect of DMU-212 on the cell cycle, apoptosis,
migration, and invasion of prostate cancer cells. The cyto-
toxicity effect of DMU-212 on LNCaP and PC-3 cells was
determined by WST-8 test. While the apoptotic effect of
determined IC50 dosages of DMU-212 was tested by
Annexin V, PI analysis and flow cytometry were used to
evaluate its effect on cell cycle. The invasion and migration Background: CTCs play major role in tumor dissemination
and progression and are one of the key components of
metastatic cascade. The aim of this study was to identify
signaling pathways associated with presence of CTCs in
primary breast cancer (PBC) patients using comprehensive
genomics approach. Methods: This study included 78 patients with PBC. CTCs were detected before surgery by quantitative RT-PCR
assays for expression of epithelial (EP) or epithelial-
mesenchymal transition (EMT) genes. Total RNA was
used for expression profiling by microarray approach and
total
DNA
for
breast
cancer
related
gene
panel
resequencing. Abstracts from the 51st European Society of Human Genetics Conference: Posters 459 of PCa cells were analyzed by “Cell Biolabs CytoSelect 96
well Cell Invasion Assay Kit” and “Wound Healing Assay”,
respectively. PCR Arrays were assessed 84 EMT-related
gene. E-cadherin and vimentin level changes were assessed
by immunofluorescence. Additionally, the protein expres-
sions of cyclin B1, E-cadherin, and β-catenin were tested by
western blot. It was observed that the IC50 dosage of DMU-
212 induced apoptosis, reduced the invasion and migration,
and induced predominantly G2/M arrest in LNCaP and PC-
3 cells. It was shown that DMU-212 inhibited the EMT by
suppressing the WNT signaling pathway, especially, on PC-
3 cells. In conclusion, compatible with the hypothesis of
that the inhibition of EMT may prevent metastasis in PCa; it
is believed that the DMU-212 has a potential role in the
treatment of cancer. to have pathogenic mutations: BRCA2(6), CHEK2(2),
PMS2(1), BRCA1(1), and TP53(1). Gunduz1 Conclusions: Genetic predisposition to prostate cancer
risk was confirmed in 23.2% (16/69) of patients seen as part
of the PCGC service. This provides an opportunity to
advocate for the clinical importance of germline genetic
testing in men with metastatic prostate cancer, not only for
their
personal
oncologic
treatment,
but
also
for
the
identification of at-risk family members. L.V. Naylor: None. M.Y. Laurino: None. B.A. Sjoding:
None. H.H. Cheng: None. P12.172D Prostate cancer (PCa), the second most common cancer
among men, is a highly heritable molecularly and clinically
heterogeneous disease. In this work we focused on the study
of germline variants as putative responsible for early
somatic events in PCa. In silico analysis by our group
identified a non-coding polymorphic regulatory element at
the 7p14.3 locus that is associated with DNA repair and
hormone regulated transcript levels and with a prostate
cancer specific subclass with high genomic instability. In
vitro studies confirmed the enhancer activity of the locus
and the binding affinity for two transcription factors (TFs);
androgen receptor (AR) and CCAAT/Enhancer Binding
Protein (C/EBP) beta (CEBPB). To first proof a functional
role of the 7p14.3 locus I deleted 731bp of the genomic area
of interest in PC-3 cells by using CRISPR-Cas9. RNA-seq
analyses
performed
upon
AR
overexpression
and/or
CEBPB silencing revealed significant deregulation of the
transcriptome in edited versus non-edited cells (control
cells). To well-characterize TFs binding motifs flaking the
locus, I am performing the disruption of AR and CEBPB
motifs via CRISPR-Cas9 in prostate cell lines. Moreover, to
better study the role of minor allele of the polymorphism on
genomic instability, I am currently working on the estab-
lishment of isogenic PC-3 cells via CRISPR-Cas9. Once
clones with the three possible genotypes are selected, I plan
on testing DNA damage response under relevant conditions. This work is a proof of concept of germline predisposition
to molecularly distinct cancer subclasses and has the
potential
to
nominate
new
mechanisms
of
cancer
development. Prostate cancer genetics clinic: First 16-month report of
establishing a multidisciplinary service Prostate cancer genetics clinic: First 16-month report of
establishing a multidisciplinary service L. V. Naylor1, M. Y. Laurino1, B. A. Sjoding1, H. H. Cheng1,2,3 1Seattle Cancer Care Alliance, Seattle, WA, United States,
2University of Washington, Seattle, WA, United States, 3Fred
Hutchinson Cancer Research Center, Seattle, WA, United
States 1Seattle Cancer Care Alliance, Seattle, WA, United States,
2University of Washington, Seattle, WA, United States, 3Fred
Hutchinson Cancer Research Center, Seattle, WA, United
States Introduction: Recent studies show that over 10% of men
with metastatic prostate cancer harbor a pathogenic germ-
line mutation. To address this patient population, the Seattle
Cancer Care Alliance established the prostate cancer
genetics clinic (PCGC) in order to (1) help identify patients
who meet criteria for clinical genetic testing, (2) ensure
appropriate oncologic follow up for patients who carry a
pathogenic mutation, (3) support cascade testing of at-risk
family members and (4) inform and connect patients with
research and clinical trial opportunities. Materials and Methods: From October 2016 to January
2018, 60.9% (42/69) of patients seen in PCGC had germline
genetic testing ordered at the time of their clinic visit. Another 23.2% (16/69) of patients presented to the clinic
with germline genetic testing already complete. Materials and Methods: From October 2016 to January
2018, 60.9% (42/69) of patients seen in PCGC had germline
genetic testing ordered at the time of their clinic visit. Another 23.2% (16/69) of patients presented to the clinic
with germline genetic testing already complete. Results: A total of 5 (11.9%; 5/42) germline mutations
that were presumed to be pathogenic were detected in
BRCA2(2), HOXB13(1), MSH6(1), and ATM(1). Analysis
also showed 9 variants of uncertain significance, and two
patients were found to have an incidental finding (i.e. heterozygous for a pathogenic MUTYH mutation). For
those previously tested, 11/16 (68.8%) patients were found 460 J. del Picchia Comenius University, Bratislava, Slovakia, 3Geneton s.r.o.,
Bratislava, Slovakia, 4Slovak Center of Scientific and
Technical Information, Bratislava, Slovakia, 5Comenius
University Science Park, Bratislava, Slovakia B. Stringa: None. S. Garritano: None. A. Romanel:
None. P. Gasperini: None. G. Petris: None. A. Cereseto:
None. F. Demichelis: None. B. Stringa: None. S. Garritano: None. A. Romanel:
None. P. Gasperini: None. G. Petris: None. A. Cereseto:
None. F. Demichelis: None. Liquid biopsy for detection of genomic instability in
prostate cancer O. Pös1, J. Budiš2,3, J. Radvansky1,3, M. Haršaniová1,3, L. Striešková1,3, F. Ďuri4,3, M. Böhmer1, J. Gazdarica1,3, I. Gazdaricová1, J. Turňa4,1, T. Szemes1,5,3 P12.174B
HER2 gene amplification in patients with prostate cancer:
Evaluating a CISH-based method Introduction: According to statistical data, cancer is among
the main causes of morbidity and mortality in the Slovak
Republic. Since genomic instability plays a crucial role in
the malignant progression of prostate cancer, it may be used
as a potential biomarker. In current clinical practice in
Slovakia, histological examination of tumor tissue is used
for diagnosis and staging of cancer. This approach is far
from a perfect solution because it requires invasive proce-
dure. In addition, the obtained information is not always
sufficiently representative. On the other hand, genomic
instability plays a crucial role in the malignant progression
of prostate cancer, so it may be used as a potential bio-
marker. A non-invasive method based on liquid biopsy for
detection of genomic instability represented by copy num-
ber variations, may lead to more precise diagnosis and may
replace traditional, painful procedures on patients. N. Sharifi Employment (full or part-time); Significant;
Geneton s.r.o., Bratislava, Slovakia. M. Haraniová: A. Employment (full or part-time); Significant; Geneton s.r.o.,
Bratislava, Slovakia. L. Strieková: A. Employment (full or
part-time); Modest; Geneton s.r.o., Bratislava, Slovakia. F. Ďuri: A. Employment (full or part-time); Modest; Geneton
s.r.o., Bratislava, Slovakia. M. Böhmer: None. J. Gazdar-
ica:
A. Employment
(full
or
part-time);
Significant;
Geneton s.r.o., Bratislava, Slovakia. I. Gazdaricová: None. J. Turňa: None. T. Szemes: A. Employment (full or part-
time); Significant; Geneton s.r.o., Bratislava, Slovakia. O. Pös: None. J. Budiš: A. Employment (full or part-
time); Significant; Geneton s.r.o., Bratislava, Slovakia. J. Radvansky: A. Employment (full or part-time); Significant;
Geneton s.r.o., Bratislava, Slovakia. M. Haraniová: A. Employment (full or part-time); Significant; Geneton s.r.o.,
Bratislava, Slovakia. L. Strieková: A. Employment (full or
part-time); Modest; Geneton s.r.o., Bratislava, Slovakia. F. Ďuri: A. Employment (full or part-time); Modest; Geneton
s.r.o., Bratislava, Slovakia. M. Böhmer: None. J. Gazdar-
ica:
A. Employment
(full
or
part-time);
Significant;
Geneton s.r.o., Bratislava, Slovakia. I. Gazdaricová: None. J. Turňa: None. T. Szemes: A. Employment (full or part-
time); Significant; Geneton s.r.o., Bratislava, Slovakia. N. Sharifi: None. N. Sharifi Department of Medical Genetics, Cancer Institute of Iran,
Tehran University of Medical Sciences, Teh, Tehran, Iran,
Islamic Republic of Department of Medical Genetics, Cancer Institute of Iran,
Tehran University of Medical Sciences, Teh, Tehran, Iran,
Islamic Republic of Prostate cancer (PCa) is one of the most widespread
malignancies in the world. The role of the human epidermal
growth factor receptor 2 (HER2) in the pathogenesis and
progression of human PCa remains poorly understood. In
contradiction with breast cancer, studies on HER2 over-
expression and gene amplification in PCa have produced
varying results, although the HER2 oncogene has been
implicated in the biology of numerous tumor types, and
serves as a prognostic marker and therapeutic target in
breast cancer. Technical challenges are considered the main
reasons for data discrepancies. Amplification of the HER2
gene has previously been reported in PCa, in which it was
associated with tumor progression. The present study aimed
to evaluate the prevalence and clinical significance of HER2
amplification in PCa. A total of 32 biopsy samples obtained
from human prostate adenocarcinomas were evaluated by
chromogenic in situ hybridization (CISH) to determine the
frequency of patients with HER2 gene amplifications. High
copy numbers of HER2 were detected in 19 of the prostate
tumors analyzed. The results of the present study suggested
that, in patients without amplification of HER2, high levels
of prostate-specific antigen or a high Gleason score were
not significantly correlated with a high pathologic stage. Furthermore, amplification levels of the HER2 gene were
directly associated with pathologic stage in patients with
PCa. Therefore, the potential use of HER2 as a prognostic
factor or therapeutic target for PCa warrants further study. Materials and Methods: We performed whole genome
sequencing analysis of cfDNA from set of patients with
positive PSA screening result (10) and healthy individuals
(10). We compared sequencing profiles of these groups to
identify cancer specific copy number aberrations. Results: Identified differences between prostate cancer
patients and healthy individuals were summarized and
further analyzed to trace recurrent patterns. Conclusions: Our results suggest that prostate cancer
specific copy number aberrations can be used as potential
non-invasive biomarker. Introduction of this approach to
routine clinical practice would be a tremendous contribution
to preventing or monitoring of cancer and personalizing
anticancer therapy. O. Pös: None. J. Budiš: A. Employment (full or part-
time); Significant; Geneton s.r.o., Bratislava, Slovakia. J. Radvansky: A. 1Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 2Faculty of Mathematics, Physics and Informatics, P12.177A
Genetic, pre and post-test findings in a survey of PTEN
mutation carriers 1Kulu State Hospital, Medical Biochemistry Laboratory,
Konya, Turkey, 2Bilecik Şeyh Edebali University, Faculty of
Science and Letters, Department of Molecular Biology and
Genetics, Bilecik, Turkey, 3Bilecik Sehy Edebali University,
Biotechnology Research and Application Center, Bilecik,
Turkey 1Kulu State Hospital, Medical Biochemistry Laboratory,
Konya, Turkey, 2Bilecik Şeyh Edebali University, Faculty of
Science and Letters, Department of Molecular Biology and
Genetics, Bilecik, Turkey, 3Bilecik Sehy Edebali University,
Biotechnology Research and Application Center, Bilecik,
Turkey S. Ekram1,2, V. Cusin1,3,4, C. Colas1,5, S. Tezenas du Montcel6, F. Caux7, M. Warcoin1, E. Guillerm1, M. Eyries1, F. Coulet1, F. Soubrier1 1Département de Génétique, GH Pitié-Salpêtrière, Paris,
France, 2Department of Medical Genetics, Umm Al-Qura
University faculty of medicine, Makkah, SA, Saudi Arabia,
3Hôpital Privé d'Antony, Antony, France, 4Hôpital Privé des
Peupliers, Paris, France, 5Institut Curie, Service de Génétique,
Paris, France, 6Département de Biostatistique, Santé Publique
et Information Médicale (BIOSPIM) GH Pitié-Salpêtrière,
Paris, France, 7Service de Dermatologie, CHU Paris Seine-
Saint-Denis - Hôpital Avicenne, Bobigny, France 1Département de Génétique, GH Pitié-Salpêtrière, Paris,
France, 2Department of Medical Genetics, Umm Al-Qura
University faculty of medicine, Makkah, SA, Saudi Arabia,
3Hôpital Privé d'Antony, Antony, France, 4Hôpital Privé des
Peupliers, Paris, France, 5Institut Curie, Service de Génétique,
Paris, France, 6Département de Biostatistique, Santé Publique
et Information Médicale (BIOSPIM) GH Pitié-Salpêtrière,
Paris, France, 7Service de Dermatologie, CHU Paris Seine-
Saint-Denis - Hôpital Avicenne, Bobigny, France Introduction: The study purpose is to examine the diag-
nostic competence of miR-375 and miR-93-5p serum levels
in prostate cancer differential diagnosis. It is also aimed to
examine whether there is an interfering situation in the
differentiation
of
chronic
prostatitis,
benign
prostatic
hyperplasia (BPH) and prostate cancer, considering the
oncogenic and tumor suppressor properties of miRNAs
together with the proinflammatory characteristics. Materials and Methods: 25 Patients with BPH, 10
patients with chronic prostatitis and 33 patients with
prostate cancer were included in the study. RNA isolation,
cDNA synthesis and qRT-PCR steps were performed with
Qiagen branded kits based on SYBR Green method, using
the protocol of the commercial company. For statistical
analysis, -ΔCt values, obtained from the use of ce-miR-39
Ct values in normalization, were used. Differences between
groups were tested by independent sample t test and
variance analysis. ROC analysis was performed to calculate
the diagnostic competence. "Fold change" calculations were
performed online on Qiagen webpage. In all analyzes, alpha
error level was accepted as 0.05. P12.177A
Genetic, pre and post-test findings in a survey of PTEN
mutation carriers Introduction: Germline heterozygous mutations of PTEN
are responsible for hamartoma tumour syndrome (PHTS), a
wide spectrum of phenotypes characterised by high tumour
risk in diverse tissues. PTEN loss of function also leads to
macrocephaly, risk of autism, psychomotor delay (PD) and
vascular anomalies. Methods, Patients and Results: We analysed a cohort of
index cases referred to our laboratory for PTEN gene
mutation testing (SNV, indel and CNV analysis) : among
151 children referred, 30 were found to have a PTEN
mutation, and 47 % were estimated de novo. All positive
cases presented with macrocephaly and 91% had a PD. The
positive predictive value (PPV) for macrocephaly is 24%
(CI28-48) and 23% (CI16-31) for PD/autism. Methods, Patients and Results: We analysed a cohort of
index cases referred to our laboratory for PTEN gene
mutation testing (SNV, indel and CNV analysis) : among
151 children referred, 30 were found to have a PTEN
mutation, and 47 % were estimated de novo. All positive
cases presented with macrocephaly and 91% had a PD. The
positive predictive value (PPV) for macrocephaly is 24%
(CI28-48) and 23% (CI16-31) for PD/autism. Results: The results are summarized in Table-1. Conclusions: It was observed that miR-375 and miR-93-
5p differ in significantly between malignant and non-
malignant disease groups. It was assessed that the sensitivity
of miR-375 to differentiate non-malignant diseases from
malignant diseases was higher than prostate specific antigen
(PSA) and chronic prostatitis may be an interfering condition
in BPH and cancer differential diagnosis. Adults were referred for Cowden syndrome suspicion
(n=122) and presented with diverse clinical findings
considered as criteria for the syndrome. The PPV were
important for Lhermitte-Duclos syndrome (89%, CI 52-
100%), cutaneous manifestations (55%, CI: 36-74), macro-
cephaly (38%:CI28-48), and intestinal polyps (42%, CI:23-
61). Although poorly specific, benign breast lesions (58%,
CI: 32-86) and benign thyroid lesions (46%, CI:31-69)
show high PPV. in BPH and cancer differential diagnosis. Results
Compared Groups
miRNA
P value Fold Change AUC Specificity Sensitivity
Malignant-Non-
malignant
miR-375
<0.001
-4.48
0.781 91%
46%
Malignant-Non-
malignant
miR-93-
5p
0.045
-1.79
0.662 91%
23%
BPH-Chronic prostatitis
miR-375
0.324
–
–
–
–
BPH-Chronic prostatitis
miR-93-
5p
0.338
–
–
–
–
BPH-Cancer
miR-375
<0.001
-5.60
0.829 91%
56%
BPH-Cancer
miR-93-
5p
0.392
–
–
–
–
Chronic prostatitis-
cancer
miR-375
0.169
–
–
–
–
Chronic prostatitis-
cancer
miR-93-
5p
0.046
-2.78
0.744 91%
50%
Y. Dulgeroglu: None. O. Abstracts from the 51st European Society of Human Genetics Conference: Posters 461 Y. Dulgeroglu1, O. Eroglu2,3 P12.176D Diagnostic competence of mir-375 and mir-93-5p serum
levels in malignant and non-malignant prostate diseases 1Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 2Faculty of Mathematics, Physics and Informatics, Abstracts from the 51st European Society of Human Genetics Conference: Posters Abstracts from the 51st European Society of Human Genetics Conference: Posters P12.178B
Bannayan-Riley-Ruvalcaba syndrome due to a new PTEN
gene mutation Meantime she undervent a subtotal thyroidectomy for
unilateral thyroid adenoma, she had a right sided ovarial
cyst resection, and an endovascular correction of a right
sided arterio-venous fistula at the transition of sinus
transversus and sigmoideus. Kariotyping, Sotos syndrome
FISH testing and PTEN gene Sanger sequencing were
performed. Results: We identified four pathogenic mutations in four
unrelated individuals with family history of BC and/or OC. Results: We identified four pathogenic mutations in four
unrelated individuals with family history of BC and/or OC. Three of the carriers had developed BC while the other one
was an OC patient, thus accounting for a mutation
frequency of 0.16% in the OC cohort, and 0.53% in the
BC cohort. One of the detected mutations is novel (c.738
+1G>A), while the rest had been detected previously (p. Gln151Ter, p.Arg186Ter, p.Arg300Ter). It is noteworthy
that among family members of the four carriers, thirteen BC
cases were reported and only four OC cases, suggesting a
BC association to RAD51D mutations. Results: She had normal kariotype, Sotos syndrome
FISH testing turned out negative. A new heterozygous
missense mutation c.2104G>T (p.Val249Gly) was detected
in the PTEN gene. In silico analyses predicted it to be
deleterious. Conclusions: RAD51D should be implemented into the
multigene panel testing offered for genetic screening of BC
or OC families, since RAD51D carriers may benefit from
the administration of PARP inhibitors. Conclusions: PTEN mutations disrupt the mTOR cell
signalling pathway, thus leading to a longer cell survival
and proliferation. Treatment with mTOR inhibitors should
be tested for this cathegory of patients. Conclusions: PTEN mutations disrupt the mTOR cell
signalling pathway, thus leading to a longer cell survival
and proliferation. Treatment with mTOR inhibitors should
be tested for this cathegory of patients. I. Konstanta: None. F. Fostira: None. P. Apostolou:
None. E. Stratikos: None. D. Kalfakakou: None. P. Kollia:
None. I. Konstantopoulou:
None. D. Yannoukakos: None. I. Konstanta: None. F. Fostira: None. P. Apostolou:
None. E. Stratikos: None. D. Kalfakakou: None. P. Kollia:
None. I. Konstantopoulou:
None. D. Yannoukakos: None. J. Zima: None. Á. Till: None. R. Ripszám: None. A. Szabó: None. Z. Bánfai: None. Z. Laufer: None. K. Hadzsiev: None. J. Zima: None. Á. Till: None. R. Ripszám: None. A. Szabó: None. Z. Bánfai: None. Z. Laufer: None. K. Hadzsiev: None. P12.177A
Genetic, pre and post-test findings in a survey of PTEN
mutation carriers Eroglu: None. Among 46 adult mutation carriers, post-test examination
identified a Lhermitte-Duclos in 3 cases and vascular
anomalies were identified in 6 more patients. Two breast
and 2 thyroid cancers were also detected within a two-year
follow-up. Vascular anomalies were also found in children. Conclusions: These results confirm the frequency of de
novo mutations found in children and emphasize the
importance of a systematic search for all features of PTEN
syndrome in mutation carriers enabling an appropriate
management. S. Ekram: None. V. Cusin: None. C. Colas: None. S. Tezenas du Montcel: None. F. Caux: None. M. Warcoin: S. Ekram: None. V. Cusin: None. C. Colas: None. S. Tezenas du Montcel: None. F. Caux: None. M. Warcoin: Y. Dulgeroglu: None. O. Eroglu: None. 462 J. del Picchia None. E. Guillerm: None. M. Eyries: None. F. Coulet:
None. F. Soubrier: None. P12.178B
Bannayan-Riley-Ruvalcaba syndrome due to a new PTEN
gene mutation 1Molecular Diagnostics Laboratory, INRaSTES, National
Center for Scientific Research "Demokritos", Athens, Greece,
2Protein Chemistry Laboratory, INRaSTES, National Center
for Scientific Research "Demokritos", Athens, Greece,
3Department of Genetics & Biotechnology, Faculty of Biology,
National and Kapodistrian University of Athens, Athens,
Greece, 4Molecular Diagnostics Laboratory, INRaSTES,
National Center for Scientific Research, Athens, Greece None. E. Guillerm: None. M. Eyries: None. F. Coulet:
None. F. Soubrier: None. I. Konstanta1, F. Fostira1, P. Apostolou1, E. Stratikos2, D.
Kalfakakou1, P. Kollia3, I. Konstantopoulou1, D. Yannoukakos4 P12.178B
Bannayan-Riley-Ruvalcaba syndrome due to a new PTEN
gene mutation J. Zima1, Á. Till1, R. Ripszám1, A. Szabó1, Z. Bánfai1, Z. Laufer2,
K. Hadzsiev1 J. Zima1, Á. Till1, R. Ripszám1, A. Szabó1, Z. Bánfai1, Z. Laufer2,
K. Hadzsiev1 Introduction: RAD51D is a member of the RAD51 protein
family and its protein product is known to be involved in
the DNA repair mechanism by homologous recombination. RAD51D germline mutations have been associated with
ovarian (OC) and breast cancer (BC) predisposition. Although the association to OC is established, BC risk
conferred by RAD51D mutations is still questionable. Our
aim was to assess the frequency of RAD51D mutations in a
large cohort of Greek patients. 1Department of Medical Genetics & Szentagothai Research
Center, University of Pécs, Clinical Center, Medical School,
Pécs, Hungary, 2Department of Child Neurology, University of
Pécs, Clinical Center, Medical School, Pécs, Hungary Introduction: Bannayan-Riley-Ruvalcaba syndrome is part
of PTEN Hamartoma Tumor Syndrome along with Cowden
syndrome, PTEN-related Proteus syndrome and Proteus-
like syndrome. It is characterized by macrocephaly, multi-
ple noncancerous tumors, and tumor-like growths. 60% of
cases are associated with PTEN gene mutations. Introduction: Bannayan-Riley-Ruvalcaba syndrome is part
of PTEN Hamartoma Tumor Syndrome along with Cowden
syndrome, PTEN-related Proteus syndrome and Proteus-
like syndrome. It is characterized by macrocephaly, multi-
ple noncancerous tumors, and tumor-like growths. 60% of
cases are associated with PTEN gene mutations. Materials and Methods: We screened 609 patients
diagnosed with OC, unselected for age or family history and
569 BC patients diagnosed under age 55 with a relative with
BC or OC, for RAD51D germline mutations. All patients
screened by NGS, were previously tested negative for
BRCA1 and BRCA2 mutations. Materials and Methods: We present the case of a 15
year old girl we have been investigated since she was 2
years old for macrosomia. She had a spina bifida, right sided
facial nerve palsy, and developed deep vein thrombosis. Meantime she undervent a subtotal thyroidectomy for
unilateral thyroid adenoma, she had a right sided ovarial
cyst resection, and an endovascular correction of a right
sided arterio-venous fistula at the transition of sinus
transversus and sigmoideus. Kariotyping, Sotos syndrome
FISH testing and PTEN gene Sanger sequencing were
performed. Materials and Methods: We present the case of a 15
year old girl we have been investigated since she was 2
years old for macrosomia. She had a spina bifida, right sided
facial nerve palsy, and developed deep vein thrombosis. A. Castillejo1,2, M. I. Castillejo1,2, A. B. Sánchez-Heras3, N.
Garrigós4, G. Capellá5, C. Lázaro5, E. Serra6, J. L. Soto1,7 E. Prosperi1, C. Gervasini2, C. Scalera1, I. Dutto1, M. Thillon1,
G. Ticli1, O. Cazzalini3, M. Savio1, L. Larizza4 An alternative possibility is that the patient carries two
mosaic alleles in genes responsible for Familial Melanoma
and Li-Fraumeni syndromes, respectively. Although this
scenario is very unlikely, it would explain the previous
neoplasias as caused by genetic predisposition Results: RSTS cells were more sensitive than normal
LCLs to oxidative DNA damage, but also to agents
inducing replication stress such as the Topoisomerase I
inhibitor camptothecin. In addition, we have analyzed the
acetylation levels of factors participating in DNA base
excision repair (BER), such as OGG1, and found that these
levels were decreased in RSTS cells. Conclusions: We found BER defects in RSTS cells using
the Comet and DNA incision assays. Finally, EP300
mutated cells were transfected with p300-plasmid to
complement the diminished protein levels. In the comple-
mented cells the sensitivity to potassium bromate was
restored to levels approaching those observed in normal
LCLs, indicating that DNA repair defects in RSTS cells are
associated to p300 or CBP protein haploinsufficiency. In
some
special
circumstances,
differential
genetic
diagnosis might be highly difficult. Further studies are
needed to clarify this dilema. E. Prosperi: None. C. Gervasini: None. C. Scalera:
None. I. Dutto: None. M. Thillon: None. G. Ticli: None. O. Cazzalini: None. M. Savio: None. L. Larizza: None. A. Castillejo:
None. M.I. Castillejo:
None. A.B. Sánchez-Heras: None. N. Garrigós: None. G. Capellá:
None. C. Lázaro: None. E. Serra: None. J.L. Soto: None. A. Castillejo:
None. M.I. Castillejo:
None. A.B. Sánchez-Heras: None. N. Garrigós: None. G. Capellá:
None. C. Lázaro: None. E. Serra: None. J.L. Soto: None. Fluorescence in situ hybridization as a tool in the diagnosis
of soft tissue sarcomas P12.179C A challenging clinical case. Patient with multiple primary
neoplasias and no family history of cancer: Richter
syndrome versus Familial Melanoma plus Li-Fraumeni
syndrome Contribution of RAD51D germline mutations in breast and
ovarian cancer in Greece Contribution of RAD51D germline mutations in breast and
ovarian cancer in Greece I. Konstanta1, F. Fostira1, P. Apostolou1, E. Stratikos2, D. Kalfakakou1, P. Kollia3, I. Konstantopoulou1, D. Yannoukakos4 A. Castillejo1,2, M. I. Castillejo1,2, A. B. Sánchez-Heras3, N. Garrigós4, G. Capellá5, C. Lázaro5, E. Serra6, J. L. Soto1,7 Abstracts from the 51st European Society of Human Genetics Conference: Posters 463 1Molecular Genetics Unit. Elche University Hospital, Elche,
Spain, 2Foundation for the Promotion of Biomedical and
Sanitary Research (FISABIO), Valencia, Spain, 3Genetic
Counseling in Cancer Unit. Elche University Hospital, Elche,
Spain, 4Alicante Immunology Center, San Juan, Spain,
5Hereditary Cancer Program, Joint Program on Hereditary
Cancer, Catalan Institute of Oncology, IDIBELL campus,
Hospitalet de Llobregat, Spain, 6Hereditary Cancer Group,
Program on Predictive and Personalized Medicine of Cancer
(PMPPC), Germans Trias i Pujol Research Institute (IGTP),
Can Ruti Campus, Badalona, Spain, 7Alicante Institute for
Health and Biomedical Research (ISABIAL - FISABIO
Foundation), Alicante, Spain E. Prosperi1, C. Gervasini2, C. Scalera1, I. Dutto1, M. Thillon1,
G. Ticli1, O. Cazzalini3, M. Savio1, L. Larizza4 E. Prosperi1, C. Gervasini2, C. Scalera1, I. Dutto1, M. Thillon1,
G. Ticli1, O. Cazzalini3, M. Savio1, L. Larizza4 1Istituto di Genetica Molecolare del CNR, Pavia, Italy,
2Medical Genetics, Dept. Health sciences, Università degli
Studi di Milano, Milano, Italy, 3Dipartimento di Medicina
Molecolare ; Unità di Immunologia e Patologia generale;
Università di Pavia, Pavia, Italy, 4Laboratory of Medical
Cytogenetics and Molecular Genetics, IRCCS Istituto
Auxologico Italiano, Milano, Italy Common risk factors for multiple primary neoplasias are
inherited predisposition to cancer; cancer-promoting aspects
of lifestyle; hormonal and environmental factors; treatment
of the previous primary cancer and increased surveillance of
cancer survivors. Introduction: The Rubinstein–Taybi syndrome (RSTS) is a
rare genetic disease characterized by growth defects, intel-
lectual disability, and an increased tumor formation. RSTS
patients
carry
heterozygous
mutation/deletion
of
the
CREBBP or EP300 gene encoding CBP or p300 lysine
acetyltransferases, essential proteins playing a key role in
transcription and in epigenetic regulation through histone
acetylation. In addition, both proteins acetylate p53 and
DNA repair factors involved in nucleotide (NER) and base
excision repair (BER). A case with multiple primary neoplasias and no relevant
family history of cancer was tested to search for a putative
genetic etiology of her neoplasias. The patient under study was diagnosed of endometrial
cancer, melanoma, breast cancer and chronic lymphatic
leukemia (CLL) at age 58, 60, 76 and 79 years, respectively. Blood DNA was analyzed with a NGS custom panel of 135
hereditary cancer genes (I2HCPv2.2). SureCall-Cartagenia-
Agilent tools were used for the bioinformatic analysis. AMG criteria were used for variant classification. Patho-
genic variants were confirmed by Sanger sequencing. Methods: In order to investigate whether a possible
altered DNA damage response (DDR) may concur to RSTS
pathogenesis we have analyzed DNA repair efficiency in
RSTS lymphoblastoid cells (LCLs). To this end, RSTS cells
were treated with potassium bromate, a chemical inducing
oxidative DNA damage, and their viability assessed. We
found
two
pathogenic
mutations:
CDKN2A:
c.152dupT;p.V51fs (not previously reported) and TP53:
c.637C>T;p.R213* (recurrently reported as somatic muta-
tion). The fact that the mutant allele frequency of these two
pathogenic variants was around 0.20, suggests the possibi-
lity of being somatic mutations as a consequence of an early
Richter syndrome not clinically evidenced. This is a CLL
form with a highly aggressive phenotype. In this situation,
no genetic cause could justify the personal history of cancer. P12.184D 464 J. del Picchia 1Department of Medical Genetics, University Hospital of North
Norway, Tromsø, Norway, 2Department of Medical Genetics,
Oslo University Hospital, Oslo, Norway, 3Department of
Medical Genetics, Telemark Hospital Trust, Skien, Norway,
4Centre for Genomic Medicine, St Mary’s hospital,
Manchester, United Kingdom, 5Department of Medical
Genetics, St. Olavs Hospital, Trondheim, Norway J. molíkova, I. Urbanovska, M. Uvírová, S. Pitronová, R. Skalikova, J. imová, P. Heranova, R. Mech, D. iak, J. Dvořáčková, D. Konvalinka, M. Smolikova 1Department of Medical Genetics, University Hospital of North
Norway, Tromsø, Norway, 2Department of Medical Genetics,
Oslo University Hospital, Oslo, Norway, 3Department of
Medical Genetics, Telemark Hospital Trust, Skien, Norway,
4Centre for Genomic Medicine, St Mary’s hospital,
Manchester, United Kingdom, 5Department of Medical
Genetics, St. Olavs Hospital, Trondheim, Norway CGB,a.s., ostrava, Czech Republic CGB,a.s., ostrava, Czech Republic Soft tissue sarcomas (STS) are rare solid cancers of
mesenchymal cell origin accounting for <1 % of adult
cancers and they represent histologically and molecularly
heterogeneous group of tumors.The diagnostics of STS is
difficult because of heterogeneity and low incidence, but it
is necessary to diagnose the highly malignant sarcomas
early and accurately. Specific genetic findings as translo-
cations of SS18, COL1A/PDGFB, EWSR1, DDIT3 genes
have been found in 30 % STS, and they can be detected by
fluorescence in situ hybridization (FISH).Between January
2014 and January 2017, in our laboratory, 17 formalin-
fixed, paraffin-embedded (FFPE) tissues were examined by
FISH using probes:Kreatech ON SYT (18q11) Break, ON
EWSR1 (22q12) Break, ON CHOP (12q13) Break, Zyto-
Vysion SPEC COL1A1/PDGFB Dual color.A total of 17
cases were evaluated, namely a Ewing sarcoma (7 cases),
Synovial sarcoma (8 cases), Liposarcoma (1) and Derma-
tofibrosarcoma (1).Rearrangements of investigated genes
were identified in 11 samples (64,7 %): rearrangement of
SS18 in 3 cases of synovial sarcoma, rearrangement of
EWSR1 in 4 cases of Ewing sarcoma, rearrangement of
genes
COL1A1/PDGFB
in
one
case
of
dermatofi-
brosarcoma and rearrangement of gene DDIT3 in one case
of liposarcoma.In 2 cases of suspect Ewing sarcoma with
rearrangement of EWSR1 the final histopathological diag-
nosis
were
olfactory
neuroblastoma
and
extraskeletal
myxoid chondrosarcoma.6 FISH negative cases (35,3 %)
were reported as stromal endometrial carcinoma, malignant
peripheral nerve sheath tumor, leiomyosarcoma, adeno-
carcinoma,
sarcomatoid
carcinoma.Rearrangements
of
investigated genes have been identified by FISH in 64,7 %
cases, the findings were corresponding with the final diag-
nosis and with literature. S. Briskemyr1, C. Jonsrud1, C. F. Rustad2, T. E. Prescott3, G.
Evans4, M. J. Smith4, M. Nystad1, H. Hjellnes1, A. S. Bø3, Ø. L.
Holla3, C. Schmidt5, R. Østern5, G. Å. M. Hansen1 P12.185A None. Ø.L. Holla: None. C. Schmidt: None. R. Østern:
None. G.Å.M. Hansen: None. None. Ø.L. Holla: None. C. Schmidt: None. R. Østern:
Å None. Ø.L. Holla: None. C. Schmidt: None. R. Østern:
None. G.Å.M. Hansen: None. Further support for the 4-hit/3-step model of tumorigenesis
in LZTR1- associated schwannomatosis None. G.Å.M. Hansen: None. P12.184D Introduction: Schwannomatosis (OMIM# 615670) is a
tumor predisposition syndrome characterized by benign
intracranial and spinal nerve root tumors as well as per-
ipheral nerve sheath tumors that rarely undergo malignant
transformation. Mosaic neurofibromatosis type 2 (NF2 -
OMIM# 101000) and schwannomatosis may have similar
clinical manifestations. Treatment, follow-up and genetic
counseling are dependent upon correct diagnosis. Case
report: This 61-year-old man presented with a spinal
schwannoma at age 51 years and was diagnosed with a
unilateral vestibular schwannoma at age 60. Additional
findings include four café-au-lait spots and multiple non-
tender subcutaneous tumors, one verified as a lipoma. His
father has non-tender subcutaneous tumors on both fore-
arms that have not been biopsied, family history is other-
wise non-contributory. Methods/Results: Genetic analyses were performed on
DNA extracted from blood and tumor tissue. In blood,
MLPA and Sanger sequencing of DNA and cDNA for NF1
and NF2 was normal and an exome-based panel for
schwannomatosis revealed a novel heterozygous variant in
LZTR1 (NM_006767.3) c.2463dup p.(Asp822Argfs*29). In
DNA extracted from a single tumor, MLPA identified a
heterozygous deletion of chromosome 22q11q12 which
included LZTR1, SMARCB1 and NF2, and Sanger sequen-
cing detected a hemizygous variant in NF2 (NM_000268.3)
c.222del p.(Trp74Cysfs*49) as well as the constitutional
LZTR1 variant in the hemizygous state. Parental analyses
are pending. Conclusions: Results of tumor and germline analyses in
this individual support the 4-hit/3-step model of tumorigen-
esis in LZTR1-associated schwannomatosis. Comprehensive
tumor and germline testing may facilitate differentiation of
mosaic NF2 and schwannomatosis. J. molíkova: None. I. Urbanovska: None. M. Uvírová:
None. S. Pitronová: None. R. Skalikova: None. J. Šimová: None. P. Heranova: None. R. Mech: None. D. iak: None. J. Dvořáčková: None. D. Konvalinka: None. M. Smolikova: None. S. Briskemyr: None. C. Jonsrud: None. C.F. Rustad: None. T.E. Prescott: None. G. Evans: None. M.J. Smith: None. T.E. Prescott: None. G. Evans: None. M.J. Smith: None. M. Nystad: None. H. Hjellnes: None. A.S. Bø: P12.186B Expanded sequencing capacity for increased depth and
plexy using the Ion Torrent S5 platform to analyze
oncogenic markers Abstracts from the 51st European Society of Human Genetics Conference: Posters 465 J. Gioia, J. Veitch, E. Lagier, C. Parikh, K. Atehortua-Khalsa
Thermo Fisher Scientific, South San Francisco, CA, United
States J. Gioia, J. Veitch, E. Lagier, C. Parikh, K. Atehortua-Khalsa Experimental and Molecular Medicine, Academic Medical
Center Amsterdam, Amsterdam, Netherlands, 5Center for
Hereditary Tumor Syndromes, University of Bonn, Bonn,
Germany Thermo Fisher Scientific, South San Francisco, CA, United
States Thermo Fisher Scientific, South San Francisco, CA, United
States Background: Serrated polyposis syndrome (SPS) is a
poorly defined colorectal cancer predisposition syndrome
characterized by the occurrence of multiple and/or large
serrated lesions throughout the colon. To date, only few
molecular signatures have been described and the etiology
of the syndrome has not been identified in the vast majority
of patients. Introduction: Use of Next Generation Sequencing tech-
nology for analysis of genetic markers related to specific
cancer types is now a common laboratory technique. The
sequencing capacity per instrument use is a limiting factor
in the application of sequencing for oncogenic analysis. Here we report system improvements to the Ion S5 Gen-
eStudio platform that double the capacity of the number of
samples analyzed per sequencing run. Methods: To uncover causative germline mutations,
exomes of 31 SPS patients have been sequenced (Illumina
HiSeq) using leukocyte DNA. The variants were filtered for
rare (MAF: biallelic ≤1%, heterozygous ≤0.1% according
to dbSNP, EVS, and ExAC), truncating, and missense
variants (pathogenic in ≥2/3 prediction tools). For data
analysis
and
filtering,
the
GATK
software
and
the
Cartagenia
Bench
Lab
NGS
Software
were
applied;
functional scores were used to prioritize the variants. Methods: To uncover causative germline mutations,
exomes of 31 SPS patients have been sequenced (Illumina
HiSeq) using leukocyte DNA. The variants were filtered for
rare (MAF: biallelic ≤1%, heterozygous ≤0.1% according
to dbSNP, EVS, and ExAC), truncating, and missense
variants (pathogenic in ≥2/3 prediction tools). For data
analysis
and
filtering,
the
GATK
software
and
the
Cartagenia
Bench
Lab
NGS
Software
were
applied;
functional scores were used to prioritize the variants. Materials and Methods: The Oncomine Comprehensive
Assay v3 (OCAv3) is an assay that targets 161 genes
relevant to solid tumors and is compatible with FFPE DNA
and RNA. P12.186B Spier: None. S. Aretz: None. S. Peters: None. C. Trueck: None. J. Altmueller: None. K. Kayser: None. E. Mangold: None. R. Adam: None. H. Thiele: None. I. Spier: None. S. Aretz: None. M. Schmidt1, E. Svobodova2, V. Semanekova2, M. Klabanova3, J.
Mares2 1Urology Clinic, 2nd Medical Faculty, Prague, Czech
Republic, 2Institute of Biology and Medical Genetics, 2nd
Medical Faculty, Prague, Czech Republic, 3Diana Lucina,
Prague, Czech Republic 1Institute of Human Genetics, University of Bonn, Bonn,
Germany, 2Cologne Center for Genomics, University of
Cologne, Cologne, Germany, 3Institute of Human Genetics,
University of Cologne, Cologne, Germany, 4Center for P12.186B We used this panel as a test case for the newly
released Ion 550 chip, which has double the capacity of the
540 chip. Results: The 550 chip allowed for simultaneous analysis
of 32 samples (16 DNA + 16 RNA) in one sequencing run. Greater than 99% concordance was observed between the
550 chip and the 540 chip with respect to recognition of
variants in commercially available controls and tissue
samples of known truth. We also used a set of control
samples to optimize variant calling parameters for this
panel, reducing the frequency of false positive variant calls. Results: After stringent filtering steps, potentially bialle-
lic variants were found in 60 genes, with seven genes
functioning in known cancer-associated pathways such as
DNA-repair and AKT1-signaling. Heterozygous variants in
at least two patients were found in 334 genes. These
encompass
four
regulators
of
the
oncogene
induced
senescence pathway, three genes involved in DNA repair
and two genes known to be causal for other polyposis
syndromes. The most interesting finding was a hetero-
zygous RNF43 splice-site mutation identified in an index
patient and his affected daughter. Conclusions: Use of the Ion 550 chip for oncogenic
analysis by DNA sequencing supports double the capacity
of the 540 chip with no decrease in accuracy or quality. Furthermore, the greater sequencing capacity achieved can
also be applied to increasing the throughput or detection
limit of other assays, such as liquid biopsies. Conclusions: The data indicate that exome sequencing
might identify causative variants for SPS. The current work-
up includes testing of relatives to determine the zygosity of
assumed biallelic variants, analyzing the segregation with
the phenotype, and functional analyses of the most
interesting variants. Conclusions: The data indicate that exome sequencing
might identify causative variants for SPS. The current work-
up includes testing of relatives to determine the zygosity of
assumed biallelic variants, analyzing the segregation with
the phenotype, and functional analyses of the most
interesting variants. J. Gioia: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. J. Veitch: A. Employment (full
or part-time); Significant; Thermo Fisher Scientific. E. Lagier: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. C. Parikh: A. Employment (full
or part-time); Significant; Thermo Fisher Scientific. K. Atehortua-Khalsa: A. Employment (full or part-time);
Significant; Thermo Fisher Scientific. S. Peters: None. C. Trueck: None. J. Altmueller: None. K. Kayser: None. E. Mangold: None. R. Adam: None. H. Thiele: None. I. Exome sequencing identified potential candidate genes for
serrated polyposis syndrome Diagnostic value of SHB expression in human prostate
cancer tissue and in benign prostatic hyperplasia Diagnostic value of SHB expression in human prostate
cancer tissue and in benign prostatic hyperplasia S. Peters1, C. Trueck1, J. Altmueller2,3, K. Kayser1, E. Mangold1,
R. Adam4, H. Thiele2, I. Spier1,5, S. Aretz1,5 M. Schmidt1, E. Svobodova2, V. Semanekova2, M. Klabanova3, J. Mares2 J. del Picchia 466 The general diagnosis for metastatic CaP remains poor and
locally advanced disease is difficult to treat successfully. Except the PSA, no specific diagnostic test for CaP is
currently available. SHB protein takes part in regulation
system of apoptosis, angiogenesis and cell cycle. Reduced
tumour growth in vivo and increased c-Abl activity in PC3
prostate cancer cells overexpressing SHB was described in
mice. In this first-ever study evaluating SHB expression in
human prostate, transcription levels of SHB in prostate
cancer and benign prostate hyperplasia was compared. Isolation of total RNA from prostate tissue in 127 patients
with histologically proved prostate cancer and 55 patients
with benign prostate hyperplasia was performed. After
qRT-PCR, comparisons of transcriptional levels in prostate
cancer and BPH and in prostate cancer groups classified by
staging, Gleason score, age and PSA were evaluated. Underexpression of SHB in prostate cancer tissue compar-
ing to benign prostate hyperplasia (p< 0.001) and decreased
expression in locally advanced disease (T2 v.s. T3-4) The general diagnosis for metastatic CaP remains poor and
locally advanced disease is difficult to treat successfully. Except the PSA, no specific diagnostic test for CaP is
currently available. SHB protein takes part in regulation
system of apoptosis, angiogenesis and cell cycle. Reduced
tumour growth in vivo and increased c-Abl activity in PC3
prostate cancer cells overexpressing SHB was described in
mice. In this first-ever study evaluating SHB expression in
human prostate, transcription levels of SHB in prostate
cancer and benign prostate hyperplasia was compared. Isolation of total RNA from prostate tissue in 127 patients
with histologically proved prostate cancer and 55 patients
with benign prostate hyperplasia was performed. After
qRT-PCR, comparisons of transcriptional levels in prostate
cancer and BPH and in prostate cancer groups classified by
staging, Gleason score, age and PSA were evaluated. Underexpression of SHB in prostate cancer tissue compar-
ing to benign prostate hyperplasia (p< 0.001) and decreased
expression in locally advanced disease (T2 v.s. T3-4) assay. Exome sequencing identified potential candidate genes for
serrated polyposis syndrome Gerstner: None. E. Schreiber: None. S.`. Higdon:
None. S. Jackson: None. H. Leong: None. H. Sangha:
None. S. Schneider: None. K. Varma: None. Exome sequencing identified potential candidate genes for
serrated polyposis syndrome The ready-to-use plates are preloaded with primers
for all the eight frequently studied hotspot regions of KRAS
and NRAS genes thus only the PCR mix and templates need
to be added. Minimized hands-on time, extra convenience
and flexibility gained by using the SeqStudio Genetic
Analyzer which utilizes an all-in-one cartridge and provides
simple plate setup with easy-to-use touch-screen interface
and real-time/remote data monitoring. The general diagnosis for metastatic CaP remains poor and
locally advanced disease is difficult to treat successfully. Except the PSA, no specific diagnostic test for CaP is
currently available. SHB protein takes part in regulation
system of apoptosis, angiogenesis and cell cycle. Reduced
tumour growth in vivo and increased c-Abl activity in PC3
prostate cancer cells overexpressing SHB was described in
mice. In this first-ever study evaluating SHB expression in
human prostate, transcription levels of SHB in prostate
cancer and benign prostate hyperplasia was compared. Isolation of total RNA from prostate tissue in 127 patients
with histologically proved prostate cancer and 55 patients
with benign prostate hyperplasia was performed. After
qRT-PCR, comparisons of transcriptional levels in prostate
cancer and BPH and in prostate cancer groups classified by
staging, Gleason score, age and PSA were evaluated. Underexpression of SHB in prostate cancer tissue compar-
ing to benign prostate hyperplasia (p< 0.001) and decreased
expression in locally advanced disease (T2 v.s. T3-4) Results: The performance of the assay on the SeqStudio
instrument was tested in the laboratory of Dr. Quagliata
(University of Basel, Switzerland) on twenty-two FFPE
DNA samples derived from colon cancer biopsies. Variants
(ranging from 5.25% to 91.1%) were successfully detected
even from research samples where less than 1 ng of input
DNA per reaction was used. Only limited amount of
microdissected FFPE sections were available for 6 research
samples where DNA concentrations were below 1 ng (down
to 0.094 ng). Conclusions: RAS mutations down to 5% level can be
detected even from less than 1 ng FFPE DNA with
simplified workflow, fast turnaround time and low cost per
sample. (p = 0.0066) were detected. There were no differences in
comparison of the groups distributed by Gleason score, age
and PSA. SHB was underexpressed in prostate cancer tissue
compared to BPH and its expression was lower in locally
advanced tumours. This data may be used to make the
diagnosis of prostate cancer more precise. Research was
supported by Diana Lucina, GAUK 200 090 and IG
00064203. A. A novel method for detection of somatic L1HS
retropositional events in tumor cells A novel method for detection of somatic L1HS
retropositional events in tumor cells A novel method for detection of somatic L1HS
retropositional events in tumor cells M. Schmidt: None. E. Svobodova: None. V. Semane-
kova: None. M. Klabanova: None. J. Mares: None. M. V. Saliutina1,2, A. Y. Komkov1, G. A. Nugmanov1, Y. B. Lebedev1, I. Z. Mamedov1 P12.189A Improved performance and workflow using Sanger
sequencing for low-level somatic mutation detection from
low amount of formalin-fixed paraffin-embedded (FFPE)
DNA 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of
Russian Academy of Science, Moscow, Russian Federation,
2Lomonosov Moscow State University, Moscow, Russian
Federation A. Gerstner, E. Schreiber, S. `. Higdon, S. Jackson, H. Leong, H. Sangha, S. Schneider, K. Varma A. Gerstner, E. Schreiber, S. `. Higdon, S. Jackson, H. Leong, H. Sangha, S. Schneider, K. Varma Introduction: Retroelement activity is one of the important
causes of the human genome instability. In previous studies
the somatic insertions of L1HS retroelements were revealed
in neurons and in different types of tumors. Modern
approaches for somatic L1HS detection are based on ana-
lysis of whole genome sequences or target massive
sequencing of fragments adjusted to retroelement’s inser-
tions. However, it is still hard to distinguish true somatic
retroelement’s insertions and various artifacts. Here we
describe an advanced artifact-resistant method for the
detection of somatic L1HS insertions. Thermo Fisher Scientific, South San Francisco, CA, United
States Introduction: Sanger sequencing is often used in oncology
research applications for molecular profiling of cancers. Applied Biosystems™Minor Variant Finder (MVF) Soft-
ware now enables low-level variant detection in Sanger
sequencing traces. RAS mutational testing is frequently
performed by clinical researchers due to the strong corre-
lation between RAS mutational profiles of colorectal can-
cers and their anti-epidermal growth factor receptor (EGFR)
response. Materials and Methods: UMI-containing adapters were
ligated to restriction digested DNA from 5000 seminoma
cells and 5000 normal cells of testicle tissues. Tumor and
control libraries were prepared by multiplex PCR with Materials
and
Methods:
We
have
improved
and
simplified the workflow for the extended RAS research Abstracts from the 51st European Society of Human Genetics Conference: Posters 467 biotinylated L1HS-specific primers covering 3'- and 5'-
flanks of full-sized and truncated L1HS insertions. Obtained
amplicons were extracted by streptavidin magnetic beads
capture. The filtration of possible artifacts was based on
statistical models built on known fixed L1HS insertions. ACMG and ENIGMA criteria, our results support the re-
classification of 12 variants from VUS to pathogenic (c.67
+1G>T,
c.67+3A>G,
c.426-12del5,
c.441A>G,
c.451G>A, c.475+3A>T, c.516+4delAA, c.517-1G>A,
c.517G>T, c.631+1G>A, c.632+3A>G and c.632-3G>C)
and 3 from VUS to likely pathogenic (c.97G>A, c.100G>A,
c.476-3C>A). Results: The use of restriction endonuclease instead of
random fragmentation enables to distinguish true flanks
from chimeras. P12.191C K. Anoshkin1,2, K. Mosyakova3, Y. Shpot3, D. Zaletaev3, A. Vinarov3, K. Karandasheva1, V. Strelnikov1,2 Classification of 15 new BRCA2 exons 2-9 splicing variants
by hybrid minigenes Classification of 15 new BRCA2 exons 2-9 splicing variants
by hybrid minigenes 1Reasearch Centre for Medical Genetics, Moscow, Russian
Federation, 2Pirogov Russian National Research Medical
University, Moscow, Russian Federation, 3I.M. Sechenov First
Moscow State Medical University, Moscow, Russian
Federation E. Fraile-Bethencourt1, A. Valenzuela-Palomo1, B. Díez-
Gómez1, A. Acedo2, E. Velasco1 1Reasearch Centre for Medical Genetics, Moscow, Russian
Federation, 2Pirogov Russian National Research Medical University, Moscow, Russian Federation, 3I.M. Sechenov First
Moscow State Medical University, Moscow, Russian
Federation 1Instituto de Biología y Genética Molecular (CSIC/UVa),
Valladolid, Spain, 2AC-GEN Reading Life SL, Valladolid,
Spain 1Instituto de Biología y Genética Molecular (CSIC/UVa),
Valladolid, Spain, 2AC-GEN Reading Life SL, Valladolid,
Spain Novel regions with LOH in sporadic renal angiomyolipoma The clinical classification of BRCA2 variants of uncertain
significance (VUS) poses a challenge in human genetics. Historically, variants have been catalogued from the protein
outlook, however, upstream gene-expression mechanisms,
such as splicing, could be impaired by changes in the DNA
sequence. Disrupted splicing variants could alter the ORF
provoking the BRCA2 truncation and be involved in cancer
development. Here, we have evaluated the splicing effect of
83 BRCA2 exons 2-9 variants by functional assays using
the minigene MGBR2_2-9. The MGBR2_2-9 was built based on the splicing vector
pSAD
(Patent-P201231427,
CSIC). In
silico
selected
BRCA2 variants, from BIC and UMD databases, were
introduced into MGBR2_2-9 and assayed in MCF-7 cells. Transcripts were analyzed by capillary electrophoresis and
sequenced. After the bioinformatic analysis of 302 BRCA2 variants,
83 were functionally tested. Results showed that 53 variants
impaired splicing (26 intronic and 27 exonic). Among them,
36 provoked ≥2/3 of aberrant transcripts. According to the g
p
g
y
p
Introduction: Sporadic renal angiomyolipoma (RA) is a
common benign neoplasm, which occurs in 13 from 10000
people, predominantly in women. Recent researches show
contradictory results of mutational profiling of sporadic
RAs. According to COSMIC database, 45% RAs have
mutations in TSC2, which is involved in PI3k/akt/mTOR
pathway. Here we demonstrate two novel regions with LOH
in 15q14-q15.1 and 16q21-q22.1, apart from six cases with
LOH in 16p13.3 in sporadic RAs (table). The clinical classification of BRCA2 variants of uncertain
significance (VUS) poses a challenge in human genetics. P12.189A Sequencing of captured amplified copies of
L1HS insertions allows to use template DNA for further
insertion validation. UMI provide accurate count of cells
bearing particular somatic insertions. Sequencing of both
flanks
enables
to
identify
target
site
duplications
-
characteristic features of transpositional events. The reproducibility of this technique was supported by
previous studies based on minigenes or/and patient RNA. MGBR2_2-9 is a robust tool which provides RNA data for
the clinical interpretation of splicing variants. Acknowledgements: Projects FIS PI13/01749 and PI17/
00227 (ISCIII, Spanish Ministry of Economy and Compe-
titivity), BIO/VA34/15 (Junta Castilla-León); EF-B is
supported by a predoctoral fellowship (University of
Valladolid/Banco Santander). Conclusions: The developed method is highly sensitive,
resistant to various artifacts and is able to detect somatic
retroelement insertions in bulk DNA as well as in single
cells. Funds: RSF grant #18-14-00244, RFBR #17-04-
01280, #16-04-00779. E. Fraile-Bethencourt: None. A. Valenzuela-Palomo:
None. B. Díez-Gómez: None. A. Acedo: None. E. Velasco: None. E. Fraile-Bethencourt: None. A. Valenzuela-Palomo:
None. B. Díez-Gómez: None. A. Acedo: None. E. Velasco: None. M.V. Saliutina: None. A.Y. Komkov: None. G.A. Nugmanov:
None. Y.B. Lebedev:
None. I.Z. Mamedov: None. P12.191C Historically, variants have been catalogued from the protein
outlook, however, upstream gene-expression mechanisms,
such as splicing, could be impaired by changes in the DNA
sequence. Disrupted splicing variants could alter the ORF
provoking the BRCA2 truncation and be involved in cancer
development. Here, we have evaluated the splicing effect of
83 BRCA2 exons 2-9 variants by functional assays using
the minigene MGBR2_2-9. Introduction: Sporadic renal angiomyolipoma (RA) is a
common benign neoplasm, which occurs in 13 from 10000
people, predominantly in women. Recent researches show
contradictory results of mutational profiling of sporadic
RAs. According to COSMIC database, 45% RAs have
mutations in TSC2, which is involved in PI3k/akt/mTOR
pathway. Here we demonstrate two novel regions with LOH
in 15q14-q15.1 and 16q21-q22.1, apart from six cases with
LOH in 16p13.3 in sporadic RAs (table). Materials and Methods: 409 tumor related genes were
sequenced by NGS in 20 samples of sporadic angiomyo-
lipoma tissues from 19 women and one man. For two
samples (#6 and #7) micro-array analysis was performed to
validate NGS findings. Materials and Methods: 409 tumor related genes were
sequenced by NGS in 20 samples of sporadic angiomyo-
lipoma tissues from 19 women and one man. For two
samples (#6 and #7) micro-array analysis was performed to
validate NGS findings. The MGBR2_2-9 was built based on the splicing vector
pSAD
(Patent-P201231427,
CSIC). In
silico
selected
BRCA2 variants, from BIC and UMD databases, were
introduced into MGBR2_2-9 and assayed in MCF-7 cells. Transcripts were analyzed by capillary electrophoresis and
sequenced. Results: We have identified in total eight LOH regions,
were six were localized in 16p13.3 encompassing TSC2
gene, and two were novel, one in 15q14-q15.1 and one in
16q21-q22.1. In samples #6 and #7 micro-array analysis
revealed
uniparental
disomy
(UPD)
and
deletion
respectively. After the bioinformatic analysis of 302 BRCA2 variants,
83 were functionally tested. Results showed that 53 variants
impaired splicing (26 intronic and 27 exonic). Among them,
36 provoked ≥2/3 of aberrant transcripts. According to the After the bioinformatic analysis of 302 BRCA2 variants,
83 were functionally tested. Results showed that 53 variants
impaired splicing (26 intronic and 27 exonic). Among them,
36 provoked ≥2/3 of aberrant transcripts. According to the 468 J. del Picchia control’s molecules. Using this pipeline, whose runtime is
only a few hours, we can efficiently focus on several dozen
significant SV candidates. P12.195C
Variants in genes related to oncogenesis in
supercentenarians D. Serbezov1, L. Balabanski1, D. Nikolova1, M. Mihaylova1, V. Damyanova1, Z. Hammoudeh1, D. Nesheva1, S. Karachanak-
Yankova1, R. Staneva1, O. Antonova1, R. Vazharova2, S. Hadjidekova1, D. Toncheva1 K. Anoshkin: None. K. Mosyakova: None. Y. Shpot:
None. D. Zaletaev:
None. A. Vinarov:
None. K. Karandasheva: None. V. Strelnikov: None. Simple, automated somatic structural variation detection
and annotation using Bionano Genome Mapping Simple, automated somatic structural variation detection
and annotation using Bionano Genome Mapping A. Pang, J. Lee, A. Hastie, E. Lam, G. Papoutsoglou, H. Cao, M. Borodkin, S. Bocklandt P12.191C Conclusions: Apart from six LOH regions in 16p13.3,
for the first time we demonstrate two novel regions with
LOH in sporadic cases of renal angiomyolipoma (15q14-
q15.1; 16q21-q21.1). In these two cases, no mutations in
TSC2 or TSC1 were found, as well as in other 407 tumor
related genes that were analyzed. We have applied Bionano’s DLS labeling chemistry to
multiple samples originating from solid and hematologic
tumors. Bionano mapping is able to assemble genomes with
unprecedented contiguity and accuracy for simple under-
standing of somatic structural variation. We compared
variants from a human breast mammary duct cell line
against a matched lymphoblastoid cell line. We identified
45 translocations events, impacting genes such as RUNX1. We discovered known rearrangements such as the t(9;22) in
chronic myeloid leukemia (CML). Moreover, we uncovered
novel mutations ranging in size from a small 4.2 kbp
insertion disrupting an acute lymphoblastic leukemia (ALL)
gene CNOT3 to a large 9.6 Mbp deletion at 17q25. In
conclusion, Bionano mapping is a cost effective method to
detect a broad range of structural variations in leukemias
and solid tumors. Table. Regions with LOH in sporadic renal angiomyoli-
poma identified in this study. Sample #
Coordinates of LOH regions (ISCN 2016) Sample #
Coordinates of LOH regions (ISCN 2016) 4
seq[CRch37/hg19]hmz(15)(q14-q15.1)
chr15:g.39884882_40494960hmz
6
arr[CRch37/hg19]hmz(16)(p13.3)
chr16:g.(1800000_2700000)x2hmz
7
arr[CRch37/hg19] hmz(16)(p13.3)
chr16:g.1925000_2120000del
9
seq[CRch37/hg19]hmz(16)(p13.3-p13.11)
chr16:g.2129637_15820863hmz
11
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2110608_23646191hmz
12
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2105335_2138422hmz
19
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2136360_3779115hmz
20
seq[CRch37/hg19]hmz(16)(q21-q22.1)
chr16:g.65025718_68857441hmz 4
seq[CRch37/hg19]hmz(15)(q14-q15.1)
chr15:g.39884882_40494960hmz
6
arr[CRch37/hg19]hmz(16)(p13.3)
chr16:g.(1800000_2700000)x2hmz
7
arr[CRch37/hg19] hmz(16)(p13.3)
chr16:g.1925000_2120000del
9
seq[CRch37/hg19]hmz(16)(p13.3-p13.11)
chr16:g.2129637_15820863hmz
11
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2110608_23646191hmz
12
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2105335_2138422hmz
19
seq[CRch37/hg19] hmz(16)(p13.3)
chr16:g.2136360_3779115hmz
20
seq[CRch37/hg19]hmz(16)(q21-q22.1)
chr16:g.65025718_68857441hmz A. Pang: None. J. Lee: None. A. Hastie: None. E. Lam:
None. G. Papoutsoglou: None. H. Cao: None. M. Borodkin: None. S. Bocklandt: None. 20 seq[CRch37/hg19]hmz(16)(q21-q22.1)
chr16:g.65025718_68857441hmz seq[CRch37/hg19]hmz(16)(q21-q22.1)
chr16:g.65025718_68857441hmz P12.194B 1Medical University of Sofia, Sofia, Bulgaria, 2Sofia University,
Sofia, Bulgaria 1Medical University of Sofia, Sofia, Bulgaria, 2Sofia University,
Sofia, Bulgaria A. Pang, J. Lee, A. Hastie, E. Lam, G. Papoutsoglou, H. Cao, M.
Borodkin, S. Bocklandt Introduction: Supercentenarians are individuals aged 110
or more, and their worldwide number is estimated at no
more than a few hundred individuals. It can be assumed that
their genome is free of pathogenic variants related to
oncogenesis and/or contains protective variants. Discovery
of such variants will be of great significance to elucidating
the pathohenetic mechnisms of the diseases of the elderly. We tested this hypothesis by analyzing the genomic data of
supercentenarians and a control group. Bionano Genomics, San Diego, CA, United States Bionano Genomics, San Diego, CA, United States Structural variation (SV) detection is fundamental to
understanding cancer. While karyotyping and conventional
approaches are robust, they can be manually intensive, and
biased towards targeted loci. Bionano Genomics’ Saphyr System generates genome
maps and detects large SVs. Bionano’s variant annotation
workflow can uncover rare and sample-specific mutations. To determine variant frequency in a tumor-normal experi-
mental design, it compares the cancer sample’s calls against
>600,000 SVs from >150 humans with no reported
diseases. To identify somatic mutations, the workflow
compares against a control sample, and examines whether
the cancer mutations are present in low fraction among the Materials and Methods: We analyze the publicly
available whole-genome sequence database from 17 super-
centenarians and 34 controls. We filtered the dataset for
nonsynonymous variants in genes associated with oncogen-
esis. We then compared the frequency in variants defined as
pathogenic, protective and variants of unclear clinical
significance in a freely accessible, public archive of reports
of the relationships among human variations and pheno-
types ClinVar. Investigation of telomerase activity in ulcerative colitis and
colorectal cancer patients G. C. COZARU, M. Aschie, A. F. Mitroi, C. Brinzan, G. I. Baltatescu, A. Nicolau, M. Enciu T. Akin Duman1, M. S. Yildirim2, M. Cakir3, H. Ataseven4, A. G. Zamani2 T. Akin Duman1, M. S. Yildirim2, M. Cakir3, H. Ataseven4, A. G. Zamani2 CEDMOG - “Ovidius” University of Constanta, “Sf Apostol
Andrei” Emergency Clinical County Hospital of Constanta,
CONSTANTA, Romania 1Department of Medical Genetics, Haseki Research and
Training Hospital, Health Sciences University, İstanbul,
Turkey, 2Department of Medical Genetics, Meram Medical
Faculty,Necmettin Erbakan University, Konya, Turkey,
3Department of General Surgery, Meram Medical Faculty,
Necmettin Erbakan University, Konya, Turkey, 4Department of
Gastroenterology, Meram Medical Faculty,Necmettin Erbakan
University, Konya, Turkey 1Department of Medical Genetics, Haseki Research and
Training Hospital, Health Sciences University, İstanbul,
Turkey, 2Department of Medical Genetics, Meram Medical
Faculty,Necmettin Erbakan University, Konya, Turkey,
3Department of General Surgery, Meram Medical Faculty,
Necmettin Erbakan University, Konya, Turkey, 4Department of
Gastroenterology, Meram Medical Faculty,Necmettin Erbakan
University, Konya, Turkey Introduction: Determination of the specific type of thyroid
cancer is crucial for the prognosis and selection of treatment
of this malignancy. Medullary thyroid carcinoma (MTC) is
a histopathological type derived from parafollicular "C"
cells of the thyroid, which synthesizes calcitonin. MTC is
more aggressive than papillary or folliculary type, com-
prises 3-5% of thyroid cancers. Introduction: Telomerase, a RNA-dependent DNA poly-
merase which adds telomeric DNA at the 3' ends of
eukaryotic chromosomes.Its activity appear elevated in
>80% of human cancers. Colorectal cancer is one of the
leading cause of cancer-related mortality in most of the
world. In the present study, we evaluated telomerase
activity in colorectal cancer, ulserative colitis patients and
aimed to investigate whether telomerase activity can be
used as a malignant marker in premalign lesions such as
ulcerative colitis. Normal tissues adjacent to the lesions of
the same patients were used as the control group. Materials and Methods: Tissue samples were obtained
from 28 colorectal cancer and 27 ulserative colitis patients
during colonoscopy and surgery.Telomerase activity was
measured by quantitative telomeric repeat amplification
protocol(TRAP)-eze assay. Introduction: Telomerase, a RNA-dependent DNA poly-
merase which adds telomeric DNA at the 3' ends of
eukaryotic chromosomes.Its activity appear elevated in
>80% of human cancers. Colorectal cancer is one of the
leading cause of cancer-related mortality in most of the
world. Molecular profiles in medullary thyroid carcinomas Molecular profiles in medullary thyroid carcinomas H. Ataseven: None. A.G. Zamani: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 469 Results: Surprisingly, we found pathological variants in
the genomes of supercentenarian as we did in the control
group. Generally, there was no statistically significant
difference in the amount of surveyed variants between the
two groups. The genotype frequencies of the variants
examined are in Hardy-Weinberg equilibrium, indicating
absence of natural selection aginst these genetic variants. Results: When we compared the three groups, difference
in telomerase activity was detected between colorectal
cancer, ulcerative colitis and normal tissue groups. In binary
comparisons of ulcerative colitis patients, there was no
significant difference in telomerase activity between the
diseased and normal tissues(p = 0.113). However, there was
a statistically significant difference between normal and
diseased tissues of colorectal cancer patients(p = 0.044). Discussion: The results of our analysis is in concordance
with other recent studies that find similar composition of
pathologic variants between the genomes of long-lived
individuals and controls. We discuss possible explanations
of the observed variant frequencies, and we emphasize the
need for further studies to clarify the role of variants of
unknown clinical significance. Discussion: The results of our analysis is in concordance
with other recent studies that find similar composition of
pathologic variants between the genomes of long-lived
individuals and controls. We discuss possible explanations
of the observed variant frequencies, and we emphasize the
need for further studies to clarify the role of variants of
unknown clinical significance. Conclusions: There was no association between ulcera-
tive colitis and telomerase activity and we showed that
telomerase activity could not be a malignancy marker for
precancerous lesions of colon. Our data suggest that
telomerase activity detection in colorectal cancer patients
may serve as a diagnostic or prognostic marker for
malignancy. Further studies are warranted to confirm the
diagnostic significance of telomerase activity by quantita-
tive TRAP-eze assay in colorectal cancer. D. Serbezov: None. L. Balabanski: None. D. Nikolova:
None. M. Mihaylova: None. V. Damyanova: None. Z. Hammoudeh: None. D. Nesheva: None. S. Karachanak-
Yankova: None. R. Staneva: None. O. Antonova: None. R. Vazharova:
None. S. Hadjidekova:
None. D. Toncheva: None. D. Serbezov: None. L. Balabanski: None. D. Nikolova:
None. M. Mihaylova: None. V. Damyanova: None. Z. Hammoudeh: None. D. Nesheva: None. S. Karachanak-
Yankova: None. R. Staneva: None. O. Antonova: None. R. Vazharova:
None. S. Hadjidekova:
None. D. Toncheva: None. T. Akin Duman: None. M.S. Yildirim: None. M. Cakir: None. Diagnosis of Li-Fraumeni Syndrome: Differentiating TP53
germline mutations from clonal hematopoiesis - Results of
the observational AGO-TR1 trial Diagnosis of Li-Fraumeni Syndrome: Differentiating TP53
germline mutations from clonal hematopoiesis - Results of
the observational AGO-TR1 trial Investigation of telomerase activity in ulcerative colitis and
colorectal cancer patients The research was made possible following completion of
the project POS CCE 2.21., ID 1844, SMIS 48750,
CEDMOG Conclusions: In conclusion, our results, together with
previous genetic studies on thyroid neoplasms, are con-
sistent with the concept that histopathological patterns could
be supported by molecular biomarkers to confirm the
prognostic
and
to
improve
understanding
of
thyroid
tumorigenesis. Conclusions: In conclusion, our results, together with
previous genetic studies on thyroid neoplasms, are con-
sistent with the concept that histopathological patterns could
be supported by molecular biomarkers to confirm the
prognostic
and
to
improve
understanding
of
thyroid
tumorigenesis. Funding: Astra Zeneca Germany; Federal Institute of
Drugs and Medical Devices (V-16698/68502/2016-2020) K. Weber-Lassalle:
B. Research
Grant
(principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Significant; Federal
Institute for Drugs and Medical Devices. P. Harter: F. Consultant/Advisory Board; Modest; Astra Zeneca, Ger-
many. J. Hauke: None. B. Ataseven: None. J.C. Stingl:
None. R.K. Schmutzler: F. Consultant/Advisory Board;
Modest; Astra Zeneca, Germany. E. Hahnen: F. Con-
sultant/Advisory Board; Modest; Astra Zeneca, Germany. The research was made possible following completion of
the project POS CCE 2.21., ID 1844, SMIS 48750,
CEDMOG. The research was made possible following completion of
the project POS CCE 2.21., ID 1844, SMIS 48750,
CEDMOG. G.C. Cozaru: None. M. Aschie: None. A.F. Mitroi:
None. C. Brinzan: None. G.I. Baltatescu: None. A. Nicolau: None. M. Enciu: None. Investigation of telomerase activity in ulcerative colitis and
colorectal cancer patients In the present study, we evaluated telomerase
activity in colorectal cancer, ulserative colitis patients and
aimed to investigate whether telomerase activity can be
used as a malignant marker in premalign lesions such as
ulcerative colitis. Normal tissues adjacent to the lesions of
the same patients were used as the control group. Methods:
12
postoperative
tissue
collected
during
thyroidectomy
and
stored
as
formalin-fixed
paraffin-
embedded material was used in the study. Immunohisto-
chemical examination it was also essential. RAS and BRAF
mutations were characterized by an enhanced polymerase
chain reaction followed by restriction fragment length
polymorphism analysis (PCR-RFLP). Methods:
12
postoperative
tissue
collected
during
thyroidectomy
and
stored
as
formalin-fixed
paraffin-
embedded material was used in the study. Immunohisto-
chemical examination it was also essential. RAS and BRAF
mutations were characterized by an enhanced polymerase
chain reaction followed by restriction fragment length
polymorphism analysis (PCR-RFLP). Results: In all cases, by immunohistochemical diagnostic
methods were positive reactions to calcitonin, CEA antigen
and thyroglobulin. Chromogranin A and B were moderately
positive, neuronal specific enolase and synaptophisine were
positive in 15-20% of the tumor cells. Note that both p53
and
Ki67,
immunohistochemical
markers
of
nuclear
proliferation were weak positive. The KRAS mutation
was more frequently found than the HRAS mutation,
comprising 19.7 % of the RAS mutations, and the V600E Materials and Methods: Tissue samples were obtained
from 28 colorectal cancer and 27 ulserative colitis patients
during colonoscopy and surgery.Telomerase activity was
measured by quantitative telomeric repeat amplification
protocol(TRAP)-eze assay. Materials and Methods: Tissue samples were obtained
from 28 colorectal cancer and 27 ulserative colitis patients
during colonoscopy and surgery.Telomerase activity was
measured by quantitative telomeric repeat amplification
protocol(TRAP)-eze assay. 470 J. del Picchia mutation of BRAF was found in 65.2% MTC samples
tested. purely in blood-derived DNA but not in the tumor of the
patient seeking advice may have severe implications for
genetic counselling. Our findings may help to avoid false-
positive genetic diagnoses of LFS1. mutation of BRAF was found in 65.2% MTC samples
tested. Conclusions: In conclusion, our results, together with
previous genetic studies on thyroid neoplasms, are con-
sistent with the concept that histopathological patterns could
be supported by molecular biomarkers to confirm the
prognostic
and
to
improve
understanding
of
thyroid
tumorigenesis. The role of Trp53 in chemical-induced lung
adenocarcinoma K. Weber-Lassalle1, P. Harter2, J. Hauke1, B. Ataseven2, J. C. Stingl3, R. K. Schmutzler1, E. Hahnen1 M. Oplopoiou1, D. Kati1, G. Ntaliarda1, V. Papaleonidopoulos1,
I. Giopanou1, I. Lilis1, G. T. Stathopoulos1,2 1Center for Familial Breast and Ovarian Cancer, Cologne,
Germany, 2Department of Gynecology & Gynecologic
Oncology, Kliniken Essen-Mitte, Essen, Germany, 3Federal
Institute for Drugs & Medical Devices, Bonn, Germany 1Laboratory for Molecular Respiratory Carcinogenesis,
Department of Physiology, Faculty of Medicine, University of
Patras, Rion, Greece, 2Comprehensive Pneumology Center,
Helmholtz Zentrum München, Member of the German Center
for Lung Research (DZL), Munich, Germany Introduction: The Li-Fraumeni cancer predisposition syn-
drome (LFS1) presents with a variety of tumor types and the
TP53 gene is therefore covered by most diagnostic cancer
gene panels. While inherited, heterozygous germline var-
iants usually show a variant fraction (VF) of approximately
50%, TP53 variants with a VF below 50% were described,
suggesting de novo somatic mosaic variants. Introduction: Lung adenocarcinoma (LADC), the prime
cancer killer worldwide, features frequent loss-of-function
of tumor protein 53 (TP53) that portends disease dis-
semination and poor survival. However, the exact timing
and functional role of TP53 loss in LADC progression is
unknown. Objectives: To time and functionally characterize Trp53
mutations in murine LADC induced by chemical constitu-
ents of tobacco smoke. Methods: Using blood-derived DNA, we screened 523
unselected patients with ovarian cancer (OC) for deleterious
variants in cancer predisposition genes, including TP53, by
hybridization capture-based next-generation sequencing. Materials and Methods: Trp53 was deleted in the lungs
of Trp53-conditional mice (Trp53f/f) via intratracheal
adenovirus (Ad)-CRE or intercrosses with lung lineage-
restricted CRE-drivers. Autochthonous LADC were trig-
gered by intraperitoneal urethane and were examined after
six months. LADC cell lines were derived from LADC of
Wt and Trp53-conditional mice and were recombined by
Ad CRE in vitro Results: Potentially deleterious TP53 missense variants
were identified in blood-derived DNA of 6 patients with
OC. The VFs of 3 TP53 mutations detected in 3 patients
were 50%, 49% and 55%, respectively, compatible with
VFs usually observed for germline variants. These TP53
mutations were also present in the corresponding tumor
samples. In the remaining 3 patients, 4 TP53 variants with
lower VFs of 34%, 26%, 17% and 7%, respectively, were
observed. None of these mutations were detected in the
corresponding tumor samples. TRIM8 participates to the mitotic spindle organization:
implication in human diseases and cancer S. Venuto1,2, L. Monteonofrio3, F. Cozzolino4, M. Monti4, I. Appolloni5, T. Mazza6, D. Canetti4, C. Fusco1, G. M. Squeo1, A. I. Croce1, V. Giambra7, P. Panelli7, P. Pucci4, P. Malatesta5,8, S. Soddu3, G. Merla1, L. Micale1 S. Venuto: None. L. Monteonofrio: None. F. Cozzo-
lino: None. M. Monti: None. I. Appolloni: None. T. Mazza: None. D. Canetti: None. C. Fusco: None. G.M. Squeo: None. A.I. Croce: None. V. Giambra: None. P. Panelli: None. P. Pucci: None. P. Malatesta: None. S. Soddu: None. G. Merla: None. L. Micale: None. S. Venuto: None. L. Monteonofrio: None. F. Cozzo-
lino: None. M. Monti: None. I. Appolloni: None. T. Mazza: None. D. Canetti: None. C. Fusco: None. G.M. Squeo: None. A.I. Croce: None. V. Giambra: None. P. Panelli: None. P. Pucci: None. P. Malatesta: None. S. Soddu: None. G. Merla: None. L. Micale: None. 1IRCCS Casa Sollievo della Sofferenza, San Giovanni
Rotondo, Foggia, Italy, 2PhD Program, Experimental and
Regenerative Medicine, University of Foggia, Foggia, Italy,
3Unit of Cellular Networks and Molecular Therapeutic
Targets, Regina Elena National Cancer Institute - IRCCS,
Rome, Italy, 4CEINGE Advanced Biotechnology and
Department of Chemical Sciences Federico II University,
Naples, Italy, 5U.O. Medicina Rigenerativa, Ospedale
Policlinico San Martino, Genoa, Italy, 6Bioinformatics Unit,
IRCCS Casa Sollievo della Sofferenza, Mendel Institute, Rome,
Italy, 7Institute of Stem Cells Biology, Regenerative Medicine
and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo
della Sofferenza Hospital, San Giovanni Rotondo, Foggia,
Italy, 8Department of Experimental Medicine (DiMES),
University of Genova, Genoa, Italy Ad-CRE in vitro. Results: 5/62 LADC (8%) and 6/9 LADC cell lines
(67%) displayed Trp53 loss. Trp53 deletion in airway cells
had no effect on primary tumor development and growth,
but selectively functioned as a tumor promoter in LYZ2+
alveolar type II cells. Trp53 deletion in LADC cells in vitro
induced marked changes in gene expression, abnormalities
in cell cycling, and, astonishingly, marked epithelial-to- Conclusions: Deleterious TP53 variants identified in
blood-derived DNA of patients with OC were not causally
related to the patients´ cancer in 3/6 TP53-positive cases. The evidence for deleterious TP53 mutations identified Abstracts from the 51st European Society of Human Genetics Conference: Posters 471 mesenchymal transition (EMT) increasing the metastatic
potential of LADC cells. silencing induced a significant accumulation of monopolar
spindles in HeLa, human Fibroblasts and in glioma U87MG
cells. Time-lapse live-cell imaging, western blot, and
cytofluorimetric analysis confirmed that TRIM8-silencing
caused a slow-down of the mitosis progression showing
cells that stall in G2/M phases taking up to 113 minutes to
enter telophase, compared to 38 minutes taken by control
cells. Additional studies revealed that TRIM8 affects
chromosomal stability, with a significant increase of
micronuclei and aneuploidy (near- haploid cells) in TRIM8-
silencend cells. Conclusions: Trp53 loss in LADC functions in a lineage-
restricted fashion to perturb cellular proliferation, but also to
promote EMT and metastasis. Our murine data explain the
dismal role of TP53 loss in human LADC. Acknowledgements: This work was supported by the
European Research Council. M. Oplopoiou: None. D. Kati: None. G. Ntaliarda:
None. V. Papaleonidopoulos: None. I. Giopanou: None. I. Lilis: None. G.T. Stathopoulos: None. Our data confirmed that TRIM8 plays a crucial role in the
mitotic process and may contribute to the glioma develop-
ment and neurological disease onset, through a not yet
identified mechanism that can control KIF11 and KIFC1
activity and/or protein level. P12.203C TRIM8 participates to the mitotic spindle organization:
implication in human diseases and cancer Genetic Susceptibility to Triple-Negative Breast Cancer in
Cyprus; an NGS panel-testing approach M. Zanti1,2,3, M. A. Loizidou2, K. Michailidou2,1, P. Pirpa2, C. Machattou2, Y. Marcou4, F. Kyriakou4, E. Kakouri4, I. Zouvani5,
K. Kyriacou2,1, A. Hadjisavvas2,1 1The Cyprus School of Molecular Medicine, The Cyprus
Institute of Neurology and Genetics, Nicosia, Cyprus,
2Department of Electron Microscopy/Molecular Pathology,
The Cyprus Institute of Neurology and Genetics, Nicosia,
Cyprus, 3Bioinformatics Group, The Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus, 4Departments of
Medical and Radiation Oncology, Bank of Cyprus Oncology
Center, Nicosia, Cyprus, 5Department of Histopathology,
Nicosia General Hospital, Nicosia, Cyprus TRIM8 is a Ring-E3 ubiquitin ligase with a role in cancer
pathogenesis and central nervous system-human diseases. TRIM8 is a Ring-E3 ubiquitin ligase with a role in cancer
pathogenesis and central nervous system-human diseases. TRIM8 is a Ring-E3 ubiquitin ligase with a role in cancer
pathogenesis and central nervous system-human diseases. We recently demonstrated the role of TRIM8 in controlling
cell growth in glioma. Perturbing TRIM8 expression in
Neural Stem Cells (NSC) we explored TRIM8-interactome
by immunoprecipitation and LC-MS/MS. A number of
identified TRIM8 potential interactors, 11/50 (22%), are
related to mitotic spindle formation and cytoskeleton
organization. We validated four members of the kinesin-like
protein family by co-immunoprecipitation assays, including
KIF11 and KIFC1, both involved in mitosis and, if aber-
rantly
expressed,
in
monopolar
spindle
organization. Immunofluorescence assays in HeLa cells showed that
TRIM8 co-localizes with centrosomes and midbodies,
confirming a possible role of TRIM8 in the mitotic spindle
machinery functions. We further demonstrated that TRIM8- Introduction: Triple Negative breast cancer (TNBC) is a
very aggressive form of breast cancer (BC), characterized
by lack of expression of the estrogen and progesterone
receptors (ER/PR), as well as the human epidermal growth
factor receptor 2 (HER2). The aim of this study was to
assess the distribution of germline mutations in cancer
susceptibility genes in Cypriot TNBC patients that tested
negative for the BRCA1/2 genes. Materials and Methods: Genomic DNA from 84 TNBC
patients was sequenced using the TruSight Cancer panel. We followed GATK guidelines and all variants were
verified by Sanger Sequencing. Rare variants of uncertain 472 J. del Picchia also located after the core promoter and upstream of the
EGFP gene. significance (VUS) were evaluated by seven pathogenicity
prediction algorithms, and those predicted as pathogenic
from
at
least
five
tools,
were
selected
for
further
investigation. B. Nami, Z. Wang B. Nami, Z. Wang P. Chanani1,2, N. Sanei Ata-abadi3,4, K. Dormiani3, N. Rezaei2,
M. H. Nasr-Esfahani2 P. Chanani1,2, N. Sanei Ata-abadi3,4, K. Dormiani3, N. Rezaei2,
M. H. Nasr-Esfahani2 Department of Medical Genetics, Faculty of Medicine and
Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Genetics, Faculty of Medicine and
Dentistry, University of Alberta, Edmonton, AB, Canada 1ACECR Institute of Higher Education (Isfahan Branch),
Isfahan, Iran, Islamic Republic of, 2Cell Science Research
Center, Royan Institute for Biotechnology, ACECR, Isfahan,
Iran, Islamic Republic of, 3Cell Science Research Center,
Royan Institute for Biotechnology, Isfahan province, Iran,
Islamic Republic of, 4Department of Genetics, Faculty of Basic
Sciences, Tarbiat Modarres University, Tehran, Iran, Islamic
Republic of Introduction: Taxanes are a class of chemotherapeutic
agents that inhibit cell division by disrupting mitotic spindle
through the stabilization of microtubule. Most of breast
cancers (BC) tumors show resistance against taxanes par-
tially due to alterations in tubulin genes. In this study, using
genomics databases, we analyze genetic alteration, mRNA
expression and the activity of the cis-regulatory elements of
28 tubulin genes in BC subtypes and taxane-resistant BCs. Introduction: Preparation of an ideal vector for efficient,
safe and targeted expression of transgene is a perfect goal in
cancer gene therapy. One of the best approaches for targeted
expression in tumor cells is using tumor specific promoters,
although the weakness of these promoters limits their suc-
cess. Two-step transcriptional activation system (TSTA)
system can augment the transgene expression using a strong
trans-activation factor such as VP16. In this study a tumor
specific minicircle, named TSTA-minicircle was prepared
that contained hTERT as a tumor specific promoter. VP16
coding sequence and GAL4 DNA binding domain were Materials and Methods: Whole exome sequencing, and
expression array profiles were obtained from cBioPortal and
Gene Expression Omnibus (GEO) databases. Cistrome
Dataset Browser was used to analyze H3K4me1 and
H3K4me3 mark enrichment. Data from 529 breast tumors,
8 normal breast biopsies and 5 BC cell lines were analyzed. Materials and Methods: Whole exome sequencing, and
expression array profiles were obtained from cBioPortal and
Gene Expression Omnibus (GEO) databases. Cistrome
Dataset Browser was used to analyze H3K4me1 and
H3K4me3 mark enrichment. Data from 529 breast tumors,
8 normal breast biopsies and 5 BC cell lines were analyzed. Genetic Susceptibility to Triple-Negative Breast Cancer in
Cyprus; an NGS panel-testing approach Materials and Methods: For construction of the TSTA-
minicircle, two expression cassettes including hTERT/
GAL4/VP16 and G5/EGFP were prepared using PCR and
restriction cloning. They were cloned into a proper parental
plasmid, which was transformed into ZYCY10P3S2T E. coli strain to generate TSTA-minicircle. Resulted minicircle
was transfected to MDA-MB-231 and SKBR31 as human
breast cancer cell lines, as well as MCF10A1 as a normal
mammary cell line. At last, the expression of EGFP was
assessed using qPCR and flow cytometry. Results: Six pathogenic mutations were identified in
three BC susceptibility genes; PALB2 (4), TP53 (1) and
FANCL (1). Presumed pathogenic germline variants were
identified in PRF1 and ERCC2 genes; 2 truncating PRF1
mutations and 1 missense ERCC2 mutation. Additionally, a
novel frameshift mutation was found in ERCC5 gene, a
promising BC candidate. Predicted damaging VUS were
identified in several genes including ATM (2), BRIP1 (2),
PALB2 (2), CDH1 (1), STK11 (1) and TP53 (1) BC
susceptibility genes. Results: The TSTA-minicircle with the length of 2934 bp
was successfully constructed. The vector showed a robust
and persistent EGFP expression in MDA-MB-231 and
SKBR3
cells
compared
to
normal
mammary
MCF10A cells. Conclusions: These results suggest that pathogenic and
presumed pathogenic variants in cancer predisposition
genes can be detected by large scale panel testing. A larger
sample size and functional studies, and/or co-segregation
analysis are needed for further conclusions. Conclusions: Our results showed that combination of the
minicircle as a safe vector with TSTA element results in an
ideal vector for more safe and robust expression of the
transgene specifically in cancer cells. Conclusions: Our results showed that combination of the
minicircle as a safe vector with TSTA element results in an
ideal vector for more safe and robust expression of the
transgene specifically in cancer cells. Grant:
EU
H2020;
669026;
Establishment
of
the
Bioinformatics ERA Chair at the CING (BIORISE) P. Chanani: None. N. Sanei Ata-abadi: None. K. Dormiani:
None. N. Rezaei:
None. M.H. Nasr-
Esfahani: None. P. Chanani: None. N. Sanei Ata-abadi: None. K. Dormiani:
None. N. Rezaei:
None. M.H. Nasr-
Esfahani: None. M. Zanti: None. M.A. Loizidou: None. K. Michailidou:
None. P. Pirpa: None. C. Machattou: None. Y. Marcou: None. F. Kyriakou: None. E. Kakouri: None. I. Zouvani:
None. K. Kyriacou: None. A. Hadjisavvas: None. None. F. Kyriakou: None. E. Kakouri: None. I. Zouvani: None. K. Kyriacou: None. A. Hadjisavvas: None. Preparation of TSTA.tumor specific minicircle DNA vector
for targeted expression in cancer cells Preparation of TSTA.tumor specific minicircle DNA vector
for targeted expression in cancer cells VISIon: Von Hippel-Lindau Information Technology-
Sharing International Consortium Conclusions: Different genetic and expression profile of
tubulin genes were found in the four subtypes of BCs and in
the taxane-sensitive and the -resistant BC. This study was
supported by Women and Children’s Health Research
Institute (WCHRI). S. Albanyan1, G. Bullivant1, L. Krimus2, K. Gomez-Hernandez3,
M. Sukhai3, Z. Lau3, H. Druker1, J. Wasserman1, K. Clark4, D. Azzariti5, M. Griffith4, H. Rehm5, A. Finelli2, T. Stockley2, M. Jewett2, R. Giles6, R. Kim1, M. Szybowska7 B. Nami: None. Z. Wang: None. B. Nami: None. Z. Wang: None. 1The Hospital for Sick Children, Toronto, ON, Canada,
2University Health Network, Toronto, ON, Canada,
3University of Toronto, Toronto, ON, Canada, 4Washington
University, St. Louis, MO, United States, 5ClinGen, Bethesda,
MD, United States, 6University Medical Centre, Utrecht,
Netherlands, 7University Health network, Toronto, ON,
Canada B. Nami, Z. Wang Results: Comparative expression levels of tubulin genes
were determined in: BC versus normal breast biopsies,
PAM50 luminal A, B, HER2-enriched and basal-like BC
tumors and cell lines, taxane-resistant versus taxane-
sensitive BC tumors and cell line, and the tumors with Abstracts from the 51st European Society of Human Genetics Conference: Posters 473 was adjusted to the elevated risk for other primary
malignancies both for the patient and her siblings. was adjusted to the elevated risk for other primary
malignancies both for the patient and her siblings. pathologic complete response (pCR) to taxane treatment
compared to non-pCR. We found that mRNA down-
regulation and gene amplification were the most frequent
alteration in the tubulin genes. Frequency of mutations in
each tubulin gene was analyzed in the four BC subtypes. Enrichment of H3K4me1 and H3K4me3 marks were
studied for the all tubulin genes in different BC cell lines. A. Semyanikhina: None. M. Fillipova: None. O. Arkhipova: None. L. Lyubchenko: None. A rare case of hereditary uveal melanoma as part of Li-
Fraumeni-like syndrome A rare case of hereditary uveal melanoma as part of Li-
Fraumeni-like syndrome A. Semyanikhina, M. Fillipova, O. Arkhipova, L. Lyubchenko A. Semyanikhina, M. Fillipova, O. Arkhipova, L. Lyubchenko A. Semyanikhina, M. Fillipova, O. Arkhipova, L. Lyubchenko
FSBI "NN Blokhin National Medical Research Centre of
oncology", Moscow, Russian Federation FSBI "NN Blokhin National Medical Research Centre of
oncology", Moscow, Russian Federation FSBI "NN Blokhin National Medical Research Centre of
oncology", Moscow, Russian Federation Introduction: Von Hippel-Lindau (VHL) disease is an
inherited cancer predisposition condition with diverse
clinical manifestations spanning multiple organ systems. Germline variants in the VHL gene identify most VHL
families. Certain VHL variants have been shown to pre-
dispose individuals to distinct manifestations. To collate the
genetic impact of variants in VHL, we initiated an inter-
national data-sharing consortium to develop a standardized
genomic and phenotypic data capturing protocol. Introduction: Uveal melanoma (UM) is the most common
primary malignant tumour of the uveal tract with distinct
molecular-genetic features that allow to distinguish UM
from other subtypes of melanoma. Somatic mutations in
UM affect some genes - BAP1, GNA11, GNAQ et al., that
determine the biology and behaviour of a tumour and
appear to be the predictors of disease. In about 2-5% of
cases the manifestation of UM is associated with a heredi-
tary condition and could be a result of germline mutations in
the genes responsible for some syndromes. Currently sev-
eral syndromes have been described such as BAP1-asso-
ciated, FAMMM, Li-Fraumeni and others. Methods: Our initial data set comprises 2251 cases from
existing cohorts in the Netherlands and Canada, as well as
published literature and publicly-accessible databases. We
have developed a standardized data collection protocol that
captures all aspects of the VHL phenotype to collect
genotypic and phenotypic data from the collaborating sites. We have worked closely with the National Institutes of
Health Clinical Genome Resource to define a platform to
share our data. We will store the data in CIVic (Clinical
Interpretation of Variants in Cancer), an open source
database for genotype-phenotype correlations, and then
contribute this data to the NIH’s ClinVar, a freely-available
public datasharing resource that facilitates collaboration
with scientists in VHL. Methods: Our initial data set comprises 2251 cases from
existing cohorts in the Netherlands and Canada, as well as
published literature and publicly-accessible databases. A rare case of hereditary uveal melanoma as part of Li-
Fraumeni-like syndrome We
have developed a standardized data collection protocol that
captures all aspects of the VHL phenotype to collect
genotypic and phenotypic data from the collaborating sites. We have worked closely with the National Institutes of
Health Clinical Genome Resource to define a platform to
share our data. We will store the data in CIVic (Clinical
Interpretation of Variants in Cancer), an open source
database for genotype-phenotype correlations, and then
contribute this data to the NIH’s ClinVar, a freely-available
public datasharing resource that facilitates collaboration
with scientists in VHL. Materials and Methods: We present here the case of a
62-year-old woman with Li-Fraumeni-like syndrome (LFL)
who had late UM of the left eye. Pedigree analysis identified
5 first-degree and second-degree relatives with different
tumours including a 73-year-old sister who had been
previously treated from melanoma of horioidea and a 52-
year-old brother suffered from renal cancer. Given the
extensive family history of late-onset malignancies, the
sample of peripheral blood lymphocytes of the patient was
assessed for BRCA2 and CHEK2 mutation using Real-time
PCR followed by Sanger sequencing. We have worked closely with the National Institutes of
Health Clinical Genome Resource to define a platform to
share our data. We will store the data in CIVic (Clinical
Interpretation of Variants in Cancer), an open source
database for genotype-phenotype correlations, and then
contribute this data to the NIH’s ClinVar, a freely-available
public datasharing resource that facilitates collaboration
with scientists in VHL. Conclusions: By collating VHL patients and mutations,
we plan to create the most comprehensive VHL data-
sharing mechanism to best understand VHL. We hope to
define new patterns of phenotypic expressions of known
mutations. We invite collaborators with VHL databases or
cohorts to contribute cases to improve the characterization
of
genotype-phenotype
relationships
in
this
complex
disorder, and will make the data collection tools freely Conclusions: By collating VHL patients and mutations,
we plan to create the most comprehensive VHL data-
sharing mechanism to best understand VHL. We hope to
define new patterns of phenotypic expressions of known
mutations. We invite collaborators with VHL databases or
cohorts to contribute cases to improve the characterization
of
genotype-phenotype
relationships
in
this
complex
disorder, and will make the data collection tools freely Results: The analysis revealed a germline heterozygotic
CHEK2 mutation (с.470Т/С, р.Ile157Thr, rs17879961). The patient's sister underwent genetic screening as well and
was found to carry the same mutation. P12.212D Using archived FFPE samples for retrospective miRNA
expression profiling of blastemal Wilms’ tumors by qRT-
PCR A rare case of hereditary uveal melanoma as part of Li-
Fraumeni-like syndrome Conclusions: We suggest LFL for the family with late-
onset cancer and low penetrance. The surveillance program Conclusions: We suggest LFL for the family with late-
onset cancer and low penetrance. The surveillance program J. del Picchia 474 available to interested investigators. Source of Funding:
VHL Alliance. available to interested investigators. Source of Funding:
VHL Alliance. demonstrate the potential of MIP-based assay towards the
identification and mapping of important CNAs and LOH
involved in WT for its prognostication. demonstrate the potential of MIP-based assay towards the
identification and mapping of important CNAs and LOH
involved in WT for its prognostication. S. Albanyan: None. G. Bullivant: None. L. Krimus:
None. K. Gomez-Hernandez: None. M. Sukhai: None. Z. Lau: None. H. Druker: None. J. Wasserman: None. K. Clark: None. D. Azzariti: None. M. Griffith: None. H. Rehm: None. A. Finelli: None. T. Stockley: None. M. Jewett: None. R. Giles: None. R. Kim: None. M. Szybowska: None. L. Toh: None. M. Tan: None. H. Yon: None. W. Seow:
None. C. Kuick: None. H. Chen: None. K. Chang: None. M. Yong: None. KK Women's and Children's Hospital, Singapore, Singapore Introduction: Wilms tumour (WT) is the most common
renal tumour in children. Loss of heterozygosity (LOH) for
chromosomes 1p and 16q is an adverse prognostic factor in
WT. The aim of this study is to characterize WTs using
Molecular Inversion Probe-based (MIP) Array to identify
copy number aberrations (CNAs) and LOH. Introduction: Wilms tumour (WT) is the most common
renal tumour in children. Loss of heterozygosity (LOH) for
chromosomes 1p and 16q is an adverse prognostic factor in
WT. The aim of this study is to characterize WTs using
Molecular Inversion Probe-based (MIP) Array to identify
copy number aberrations (CNAs) and LOH. Materials and Methods: We extracted miRNA from two
FFPE samples per patient: a tumor sample and a tumor-free
region of the same kidney to be used as control. A PCR
array was used to reveal miRNAs of interest in the first
patient and the expression of selected miRNAs was studied
in seven other patients by individual qRT-PCR primers. Materials and Methods: Genomic DNA were extracted
from 8 formalin-fixed paraffin-embedded (FFPE) sections
of WTs using Promega Maxwell RSC Instrument and
purified as input into Affymetrix OncoScan FFPE Assay
Kit. Data was analysed with Chromosome Analysis Suite
software version 3.2.0.1252. Results: MiRNA expression patterns obtained from
FFPE samples showed a close resemblance to the results
of previous profiling reports using fresh-frozen tissue. Relevant differences include miR-184 found to be more
underexpressed than expected, and miR-203a, which we
report to be downregulated in Wilms’ tumor for the first
time to our knowledge. Results: MiRNA expression patterns obtained from
FFPE samples showed a close resemblance to the results
of previous profiling reports using fresh-frozen tissue. Relevant differences include miR-184 found to be more
underexpressed than expected, and miR-203a, which we
report to be downregulated in Wilms’ tumor for the first
time to our knowledge. Results: We saw gains of chromosomes 1q and 7q;
segmental gains of 1q21.1-q32.1, 2p24.3, 4q31.3, 11p14.3,
14q11.2,
15q21.3,
16q22.2,
17q24.1,
17p11.2-q25.3,
19p13.11, 22q13.33 and Xp22.33; losses of 1p, 7p, 10p,
11 and 14; and segmental losses of 3p25.2, 4q12-q13.1,
4q13.3-q35.2,
8q24.3,
10p11.23,
11p15.4,
16p13.3,
16q22.2,
19q13.2,
19p13.2,
19p13.3,
22q12.3-13.33,
22q11.23-q12.1 and Xq11.1-q11.2. The most common
aberrations were gains of chromosomes 12(5/8), 13(4/8),
7(3/8), 7q(2/8) and 8(3/8); and loss of 7p(2/8). Frequent
LOH seen were 1p36.11-p35.3(2/8), 1p31.1(2/8), 1p33-
p32.3(3/8),
1q21.2-q21.3(3/8),
3p21.31-p21.31(6/8),
3p12.2p12.1(3/8),
6p22.1(2/8),
7q11.1-q11.21(3/8),
10q11.21-q11.23(2/8),
11p(3/8),
12q21.3-q21.33(3/8),
17p11.2-p11.1(5/8) and 17q23.1-q24.1(2/8). P12.211C Detection of copy number aberrations and loss of
heterozygosity in wilms tumour DNA samples from
formalin-fixed paraffin-embedded tissue using molecular
inversion probe-based array G. Buglyó1, Z. Magyar2, É. Görbe2, M. Csóka2, P. Varga2, R. Bánusz2, T. Micsik2, Z. Berki1, B. Nagy1 1University of Debrecen, Debrecen, Hungary, 2Semmelweis
University, Budapest, Hungary 1University of Debrecen, Debrecen, Hungary, 2Semmelweis
University, Budapest, Hungary L. Toh, M. Tan, H. Yon, W. Seow, C. Kuick, H. Chen, K. Chang,
M. Yong Introduction: Wilms’ tumor is the most frequent geni-
tourinary malignancy in young children. A high percentage
of blastema after pre-operative chemotherapy (according to
the SIOP protocol) is a sign of poor prognosis. Differences
between miRNA signatures of blastemal and regressive
tumor subtypes may hold relevant information on genetic
factors underlying chemo-responsiveness. KK Women's and Children's Hospital, Singapore, Singapore G. Buglyó: None. Z. Magyar: None. É. Görbe: None.
M. Csóka: None. P. Varga: None. R. Bánusz: None. T.
Micsik: None. Z. Berki: None. B. Nagy: None. KK Women's and Children's Hospital, Singapore, Singapore Conclusions: Despite favorable reviews in the literature
with respect to various diseases, miRNA expression
analyses from FFPE samples are still rarely reported. We
hereby demonstrate the usefulness of pathological archives
in Wilms’ tumor profiling. Putting our results in perspective
with literature data, miR-184 seems to be underexpressed in
a subset of blastemal and possibly other Wilms’ tumors. Downregulation of miR-203a may or may not be specific to
blastemal tissue, but it could explain the overexpression of
E2F3 thought to be important in the pathogenesis of the
disease. Conclusions: Of 8 WTs, we identified LOH at 11p in 3
cases, a frequency consistent with reported literature, and
found LOH in 1p and 16q(1/8). Our data also supports
published reports that trisomies 6, 7, 8, 12, 13 and 18 are the
most
common
aneuploidies
in
WT. Our
findings G. Buglyó: None. Z. Magyar: None. É. Görbe: None. M. Csóka: None. P. Varga: None. R. Bánusz: None. T. Micsik: None. Z. Berki: None. B. Nagy: None. G. Buglyó: None. Z. Magyar: None. É. Görbe: None. M. Csóka: None. P. Varga: None. R. Bánusz: None. T. Micsik: None. Z. Berki: None. B. Nagy: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 475 1Department of Biomedical Molecular Biology, Ghent,
Belgium, 2Cancer Research Institute Ghent, Ghent, Belgium,
3Center for Medical Genetics Ghent, Ghent, Belgium D. Dimitrakopoulou1,2, T. Naert1,2, D. Tulkens1,2, R.
Noelanders1, T. Van Nieuwenhuyse1, P. Van Vlierberghe2,3, K.
Vleminckx1,2,3 Laboratory Medicine, Lund, Sweden Introduction: Acute myeloid leukemia (AML) is the most
common form of acute leukemia in adults. Mutations in
Wilms' tumor gene 1 (WT1) are present in 10-15% of
cytogenetically normal AML and are reported as indepen-
dent negative prognostic factors. The recurrent WT1
(R394W) mutation changes the basic arginine to an
uncharged tryptophane. Since Arg394 contacts DNA, the
binding of WT1(R394W) may be adversely affected. The
aim of this project was to characterize the binding pattern of
WT1(R394W) as compared to the binding of the wild type
WT1-KTS and WT1+KTS isoforms. Material and Methods: From leukemic K562 cells
expressing
BIO-tagged
WT1(R394W),
we
performed
chromatin precipitation by streptavidin capture, followed
by deep sequencing. Results: Three independent experiments yielded 470
overlapping peaks. We found that the R394W mutation in a
WT1-KTS background deprived WT1-KTS of its binding
close to transcription start sites of target genes. WT1
(R394W) shows a reduced binding affinity and, similar to
WT1+KTS, binds mainly within the gene bodies of target
genes. Upon transient overexpression in HEK293 cells,
only WT1-KTS showed evidence of transcriptional activa-
tion of target genes common to WT1(R394W) and the wild
type -KTS and +KTS isoforms. While overexpression of
WT1-KTS in K562 cells conferred resistance to the tyrosine
kinase inhibitor Imatinib, overexpression of WT1R394W
conferred only partial resistance. D. Dimitrakopoulou:
None. T. Naert:
None. D. Tulkens: None. R. Noelanders: None. T. Van Nieuwen-
huyse:
None. P. Van
Vlierberghe:
None. K. Vleminckx: None. D. Dimitrakopoulou:
None. T. Naert:
None. D. Tulkens: None. R. Noelanders: None. T. Van Nieuwen-
huyse:
None. P. Van
Vlierberghe:
None. K. Vleminckx: None. Conclusions: Taken together, our results support the
notion of WT1(R394W) as a loss of function mutation. The
functional consequences of the acquisition of binding to the
gene body within target genes, in a manner similar to WT1
+KTS, remain to be clarified. P12.215C
Young breast cancer: Predisposed to aggression? T. Ullmark:
None. G. Montano:
None. U. Gullberg: None. T. Ullmark:
None. G. Montano:
None. U. Gullberg: None. T. Ullmark:
None. G. Montano:
None. U. Gullberg: None. Young breast cancer: Predisposed to aggression? S. Randall Armel1, A. Volenik1, R. Demsky1, S. Neil2, R. H. Kim1, J. McCuaig1 P12.213A
Global binding pattern of mutant WT1(R394W) to the
genome in leukemic cells T. Ullmark, G. Montano, U. Gullberg CRISPR/Cas9 and TALEN mediated genome editing cre-
ates unique and unmatched opportunities for modeling
human disease in non-mammalian model organisms. The
amphibian Xenopus tropicalis is extremely well positioned
for this approach. It shares with zebrafish the aquatic habitat
and easy manipulations associated with its external devel-
opment. However, it manifests unique features making it a
powerful organism for modeling human genetic disease. (1)
unlike zebrafish, Xenopus tropicalis has a true diploid
genome, precluding redundancy. (2) Its genome shows high
synteny with humans, greatly facilitating identification of
disease orthologs. (3) Gene manipulations can be restricted
to specific tissues and organs via targeted blastomere
injection. We have recently generated the first genetic
cancer models in Xenopus tropicalis. Via mosaic targeting
of the tumor suppressor gene apc we generated tadpoles that
rapidly (< 1.5 months) and efficiently (>90%) developed a
range of neoplasia characteristic for Familial Adenomatous
Polyposis (Van Nieuwenhuysen et al., Oncoscience 2015). Similarly, we found that rb1/rbl1 double mosaic mutant
tadpoles rapidly develop retinoblastoma (Naert et al. Sci. Rep. 2016). More recent work also indicates the possibility
of modeling Li-Fraumeni, T-cell acute lymphoblastic leu-
kemia and pancreatic neuroendocrine cancer. The rapid
kinetics of tumor development in Xenopus pave the way for
their use as pre-clinical model, providing unique possibi-
lities for fast identification of modifier genes and novel drug
targets. We present our first promising results with multi-
plexed gene targeting in desmoid tumors. We believe that
these models offer a unique experimental platform to con-
tribute to the field of human cancer and medical genetics. S. Randall Armel1, A. Volenik1, R. Demsky1, S. Neil2, R. H.
Kim1, J. McCuaig1 P12.214B Modeling human hereditary cancer syndromes using
CRISPR/Cas9 mediated genome editing in Xenopus
tropicalis 1Princess Margaret Cancer Centre, Toronto, ON, Canada,
2University of Toronto, Toronto, ON, Canada 1Princess Margaret Cancer Centre, Toronto, ON, Canada,
2University of Toronto, Toronto, ON, Canada Introduction: 2% of breast cancers occur in women ≤35
years of age. Limited data suggests that up to 15% may be
hereditary, a high proportion are aggressive (triple-negative,
TN or HER2+), and frequently diagnosed at an advanced 476 J. del Picchia stage. Canadian-specific data of this population is lacking
and would serve to improve risk assessment and genetic
counselling. been reported. Several studies showed that alterations in the
type I versus type II mRNA ratio of nf1 gene can be
associated with the presence of malignancies including
colon cancer, neuroectodermal tumors and ovarian cancer in
patients not affected by NF1. Methods: A retrospective chart review examined the
characteristics of women diagnosed with breast cancer ≤35
years and who received treatment/genetic counselling at
Princess Margaret Cancer Centre (PM) in Toronto, Canada
after 2000. Materials and Methods: The study was conducted on a
cohort of 144 NF1 patients, age 1-57 years, and 144 age and
sex matched controls. The patients were divided into three
groups (mild, moderate and severe phenotype). The mRNA
levels of isoform 1 and 2 of the nf1 gene in peripheral blood
leukocytes of NF1 patients and of controls was assessed by
real-time PCR. Statistical analysis was performed using t-
test. Results: Of 226 women identified, 207 received testing at
PM. Overall rate of pathogenic variants was 19% for
BRCA1/2 and 7% for other genes. Most women had stage 2
disease (42% overall); however, 33% of carriers compared
to 24% of non-carriers were ≥stage 3. Most tumors were
ER+/PR+/HER2- (45% overall); however, 61% of carriers
presented with aggressive disease (45% TN, 16% HER2+)
as compared to only 46% of non-carriers (21% TN, 25%
HER2+). Of note, 86% of BRCA1 were TN, 75% of
BRCA2 were ER+/PR+, and 60% of TP53 were HER2+. Additionally, 22% of carriers reported no family history of
breast/ovarian cancer. Results: Molecular analysis showed a wide distribution
of each mutation along the NF1 gene. Expression levels of
isoform 1 were lower in patients than in controls (0,00066
vs 0,0012, p = 0,0000055), and the patients with severe
phenotype showed levels lower than mild patients (0,0005
vs 0,00074, p = 0,02). P12.216D Neurofibromatosis and Related Tumors: molecular
markers associated to high risk for tumor-development P12.214B Expression levels of isoform 2 were
lower in patients than in controls (0,0024 vs 0,01, p =
0,0004). Conclusions: This study demonstrates that early-onset
breast cancer has distinct features that vary based on genetic
status. Overall, carriers tend to present with later stage and
more aggressive disease as compared to non-carriers. Given
the high mutation rate, and that many carriers report no
family history, it is important that all women with this
diagnosis be offered genetic testing. Conclusions:
The
identification
of
an
association
between specific NF1 expression pattern and severity of
phenotype might help to establish a specific prognosis and
consequently to start a specific follow-up program and
eventually a specific therapeutic approach in NF1 patients. A. Assunto: None. U.P. Ferrara: None. C. Pivonello:
None. L. Lombardo: None. A.P. Piscitelli: None. C. Tortora: None. M. Chicone: None. V. Pinna: None. A. De Luca: None. R. Pivonello: None. A. Colao: None. V. Donofrio: None. G. Cinalli: None. F. Schonauer: None. F.D. Andrea:
None. P. Strisciuglio:
None. D. Melis: None. A. Assunto: None. U.P. Ferrara: None. C. Pivonello:
None. L. Lombardo: None. A.P. Piscitelli: None. C. Tortora: None. M. Chicone: None. V. Pinna: None. A. De Luca: None. R. Pivonello: None. A. Colao: None. V. Donofrio: None. G. Cinalli: None. F. Schonauer: None. F.D. Andrea:
None. P. Strisciuglio:
None. D. Melis: None. S. Randall Armel: None. A. Volenik: None. R. Demsky: None. S. Neil: None. R.H. Kim: None. J. McCuaig: None. P13 Basic mechanisms in molecular and cytogenetics A. Assunto1, U. P. Ferrara1, C. Pivonello2, L. Lombardo1, A. P. Piscitelli1, C. Tortora1, M. Chicone3, V. Pinna4, A. De Luca4, R. Pivonello2, A. Colao2, V. Donofrio3, G. Cinalli5, F. Schonauer6,
F. D. Andrea6, P. Strisciuglio1, D. Melis1 P13.01A
Comparison between functional predictors and in vitro
effect of alpha-1 antitrypsin missense variants P13.01A
Comparison between functional predictors and in vitro
effect of alpha-1 antitrypsin missense variants 1Department of Traslational Medical Sciences, Naples, Italy,
2Department of Clinical Medicine and Surgery, Naples, Italy,
3Hospital Company of National Importance Santobono,
Naples, Italy, 4IRCCS Casa Sollievo della Sofferenza Hospital,
Naples, Italy, 5S.C. Pediatric Neurosurgery, Hospital
Company of National Importance Santobono, Naples, Italy,
6Department of Public Healthy, Section of Aesthetic Plastic
Surgery and Reconstructive, University of Naples, Naples, Italy N. Matamala, G. Gomez-Mariano, S. Martínez, A. Damián, S. Ramos, V. Aquino, I. Gonzalo, A. Navarro, B. Martínez-Delgado
Institute of Health Carlos III (ISCIII), Madrid, Spain N. Matamala, G. Gomez-Mariano, S. Martínez, A. Damián, S. Ramos, V. Aquino, I. Gonzalo, A. Navarro, B. Martínez-Delgado N. Matamala, G. Gomez-Mariano, S. Martínez, A. Damián, S. Ramos, V. Aquino, I. Gonzalo, A. Navarro, B. Martínez-Delgado Institute of Health Carlos III (ISCIII), Madrid, Spain Institute of Health Carlos III (ISCIII), Madrid, Spain Introduction: The alpha-1 antitrypsin (AAT) coding gene
SERPINA1 is highly polymorphic with more than one
hundred variants described in databases. Although the
functional implications of the most common mutations S
and Z have been well characterized, the effect of many
newly
identified
variants
has
not
been
empirically Introduction: Neurofibromatosis type 1 (NF1) is an auto-
somal dominant disorder, caused by inactivating mutations
of neurofibromin gene (17q11.2). Nf1 gene encodes for five
different isoform. Few cases of genotype-phenotype have 477 Abstracts from the 51st European Society of Human Genetics Conference: Posters demonstrated, and it is unclear whether bionformatic soft-
wares are able to correctly predict their pathogenicity. demonstrated, and it is unclear whether bionformatic soft-
wares are able to correctly predict their pathogenicity. Familial apparently balanced translocations (ABTs) segre-
gating with discordant phenotypes are extremely challen-
ging for interpretation and counseling. We report four
families, each including individuals with identical ABTs
and discordant phenotypes. All mechanisms underlying
differential phenotypes were thoroughly investigated using
FISH, array-CGH, and whole-genome mate-pair sequen-
cing; however, no associations were determined, therefore
whole-exome sequencing (WES) was performed. Materials and Methods: We have analyzed the coding
sequence of SERPINA1 gene in patients with AAT
deficiency (AATD) and have identified 12 previously
undescribed missense variants. Different algorithms were
used to predict the effects of these substitutions. In addition,
mutant proteins were expressed in vitro and functional
assays
were
performed
to
empirically
determine
the
pathogenicity of the variants (western blot analysis, PAS
staining,
elastase
inhibitory
assay,
pulse-chase
experiments). In this study, the same families were revisited using WES. Disease-candidate variants were validated with Sanger
sequencing and their pathogenic impact was assessed with
in silico tools. Results: Most of the variants had a functional impact
when overexpressed in a cellular model: intracellular
polymerization, impaired secretion, intracellular stabiliza-
tion and/or reduced anti-elastase activity. Only in one case
no pathogenic effect was identified. For most of the variants
the functional assay was consistent with the bioinformatic
prediction, although in some cases discrepancies between
algorithms were observed and for one variant the predictors
failed to identify the pathogenic effect of the amino acid
substitution. Results: Most of the variants had a functional impact
when overexpressed in a cellular model: intracellular
polymerization, impaired secretion, intracellular stabiliza-
tion and/or reduced anti-elastase activity. Only in one case
no pathogenic effect was identified. For most of the variants
the functional assay was consistent with the bioinformatic
prediction, although in some cases discrepancies between
algorithms were observed and for one variant the predictors
failed to identify the pathogenic effect of the amino acid
substitution. In family 1, WES revealed a novel, patient-specific
heterozygous
splice
donor
variant
in
STXBP1
(NM_003165.3: c.1110+2T>G), which is essential for
neurotransmitter release through syntaxin regulation. Simi-
lar patients with epilepsy and intellectual disability have
been reported carrying heterozygous STXBP1 disruptions. The variant identified is predicted to disrupt normal
STXBP1 splicing. In family 2, WES identified a novel,
patient-specific heterozygous missense variant in TUBA1A
(NM_006009.3: c.875C>T), one of the main microtubule
components with important roles in neuronal migration. Similar patients with intellectual disability, microcephaly,
and lissencephaly have been reported carrying heterozygous
TUBA1A variants. In families 3 and 4, a novel, patient-
specific heterozygous missense variant in SCN1A, and a
compound heterozygous variant in C5orf42 were identified,
respectively; however, further investigation is required to
determine pathogenicity. Conclusions: Functional studies are essential to unravel
the molecular mechanisms affected by newly identified
genetic variants. Although helpful, functional prediction
algorithms are not always correct in their predictions. N. Matamala: None. G. Gomez-Mariano: None. S. Martínez: None. A. Damián: None. S. Ramos: None. V. Aquino: None. I. Gonzalo: None. A. Navarro: None. B. Martínez-Delgado: None. J. Bokajeva1, M. Männistu1, M. Nõukas1, O. Tšuiko1,2, R. Mägi3,
A. Salumets2, A. Kurg1 1Institute of Molecular and Cell Biology, Tartu, Estonia,
2Institute of Biomedicine and Translational Medicine, Tartu,
Estonia, 3Estonian Genome Center, Tartu, Estonia Patient-specific variants identified by whole-exome
sequencing underlie discordant phenotypes in familial
apparently balanced translocations Patient-specific variants identified by whole-exome
sequencing underlie discordant phenotypes in familial
apparently balanced translocations C. Aristidou1,2, A. Theodosiou1, A. Alexandrou1, I. Papaevripidou1, P. Evangelidou1, Z. Kosmaidou-Aravidou3, F. Behjati4, G. A. Tanteles5, V. Christophidou-Anastasiadou6, C. Sismani1,2 C. Aristidou: None. A. Theodosiou: None. A. Alexan-
drou: None. I. Papaevripidou: None. P. Evangelidou:
None. Z. Kosmaidou-Aravidou: None. F. Behjati: None. G.A. Tanteles: None. V. Christophidou-Anastasiadou:
None. C. Sismani: None. 1Cytogenetics and Genomics Department, The Cyprus Institute
of Neurology and Genetics, Nicosia, Cyprus, 2The Cyprus
School of Molecular Medicine, The Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus, 3Department of
Genetics, Alexandra Hospital, Athens, Greece, 4Cytogenetics
Unit, University of Social Welfare and Rehabilitation Sciences,
Genetics Research Center, Tehran, Iran, Islamic Republic of,
5Clinical Genetics Clinic, The Cyprus Institute of Neurology
and Genetics, Nicosia, Cyprus, 6Clinical Genetics Clinic, The
Cyprus Institute of Neurology and Genetics and Archbishop
Makarios III Medical Centre, Nicosia, Cyprus P13.02B In conclusion this study supports our previous publication
demonstrating that in the majority of familial ABTs with
discordant phenotypes, translocations are coincidental to
phenotype, unlike de novo ABTs. Instead, patient-specific
variants identified by WES seem to underlie phenotype
associations in these families. Influence of human X chromosome structural variations
and aberrations on X chromosome inactivation Two groups of women
were selected from the Estonian Genome Center at the
University of Tartu population-based biobank, genotyped
with
high-density
whole-genome
SNP
BeadArrays. PennCNV programme was used for CNV calling according
to the manufacturer's protocol. Subjects group was com-
prised of 436 women with X chromosome aberrations over
50 kb in size spanning the whole X chromosome, while the
controls group was comprised of 436 women without X
chromosome defects of the mentioned size. Both sample
sets were further divided into age groups, taking into con-
sideration the increased rate of skewing in older women. XCI ratio was assessed using HUMARA method. No sig-
nificant difference between XCI ratios of women with and
without X chromosome aberrations has been found so far. However, as expected, an increase in XCI skewing is seen
in older women. Turin, Italy, 7Department of Public Health and Pediatrics,
turin, Italy Turin, Italy, 7Department of Public Health and Pediatrics,
turin, Italy Background: Ataxia-Telangiectasia (A-T) is a rare auto-
somal recessive disease affecting cerebellum, immune sys-
tem, lungs, liver and characterised by an enhanced tumor
risk. Despite the cerebellar degeneration, the major cause of
mortality in A-T is due to respiratory failure caused by
recurrent bacterial infections of the upper-respiratory tract
and oral tissues .Since inflammation is emerging as an
important hallmark in A-T, we hypothesized that A-T
patients could exhibit impaired innate immune response due
to defective pattern-recognition receptors (PRRs) such as
Toll-like receptors (TLRs) by microbes. Methods: Primary skin fibroblasts from A-T and healthy
controls and Hela cell lines ATM -/- were assessed for genes encoding inflammatory
response
factors
using
RT-qPCR
and
citofluorimetric
analysis, before and after stimulation with E.Coli lipopoly-
saccharide (LPS). LPS and TNF-α mediated Nf-kβ activa-
tion was measured by western blot analysis. Results: Cells lacking ATM protein were less responsive
than controls as shown by the defective gene expression of
TLR-4 and IL-6 and a significant reduction of IL-6 secreted
protein both at basal level and after LPS or TNF-α stimuli. In the same conditions, the Results: Cells lacking ATM protein were less responsive
than controls as shown by the defective gene expression of
TLR-4 and IL-6 and a significant reduction of IL-6 secreted
protein both at basal level and after LPS or TNF-α stimuli. In the same conditions, the Nf-kβ activation pathway was less activated in A-T cells
respect to healthy LCLs. Influence of human X chromosome structural variations
and aberrations on X chromosome inactivation Conclusions: Our studies indicate for the firt time a
defective trafficking of TLR-4 in response to LPS stimuli. This defect could contribute to hyposensitive response of A-
T patients to immunogenic challenge. Further investigations
in this pathways coul provide a potential target for
therapeutic clinical intervention in A-T. J. Bokajeva: None. M. Männistu: None. M. Nõukas:
None. O. Tšuiko: None. R. Mägi: None. A. Salumets:
None. A. Kurg: None. Defective trafficking of inflammatory response factors
exhibits hyposensitive immunogenic response in skin
fibroblasts from Ataxia Telangiectasia patients Defective trafficking of inflammatory response factors
exhibits hyposensitive immunogenic response in skin
fibroblasts from Ataxia Telangiectasia patients E. Pozzi1, S. Cannito2, M. Parola2, M. Vinciguerra3, S. Augeri4,
C. Mariotti5, E. Giorgio1, C. Mancini1, E. Di Gregorio6, M. Ferrero1, E. Riberi7, A. Brusco1,6, S. Cavalieri1 1Department of Medical Sciences, Genetic Unit, Turin, Italy,
2Department of Clinical and Biological sciences, Unit of
Experimental Medicine and Clinical pathology, Turin, Italy,
3DNA Metabolism Laboratory,the FIRC Institute of Molecular
Onclogy, IFOM, Milan, Italy, 4Department of Medical
Sciences, Immunogenetic Unit, turin, Italy, 5Unit of Genetics of
Neurodegenerative and Metabolic Diseases, IRCCS
Neurologic Institute “Carlo Besta”, Milan, Italy, 6S.C.D.U.
Medical Genetics, A.O.U. "Citta della Salute e della Scienza", Influence of human X chromosome structural variations
and aberrations on X chromosome inactivation Influence of human X chromosome structural variations
and aberrations on X chromosome inactivation 478 J. del Picchia X chromosome inactivation (XCI) balances the expression
of X-linked genes between females and males. Early in
female development, cells transcriptionally silence one
randomly chosen X chromosome. This leads to a general
ratio of 50:50 cells, where half the cells inactivate the
maternal and half the paternal X chromosome. The ratio
varies among women, although significant deviations from
it are relatively rare among phenotypically normal women
and are known as skewed XCI. Skewing is more common
among older women or women with certain X-linked dis-
eases or X chromosome aberrations. The latter is often
marked by inactivation of the defective X chromosome. Our
study focuses on detecting the influence of X chromosome
deletions and duplications on XCI. Two groups of women
were selected from the Estonian Genome Center at the
University of Tartu population-based biobank, genotyped
with
high-density
whole-genome
SNP
BeadArrays. PennCNV programme was used for CNV calling according
to the manufacturer's protocol. Subjects group was com-
prised of 436 women with X chromosome aberrations over
50 kb in size spanning the whole X chromosome, while the
controls group was comprised of 436 women without X
chromosome defects of the mentioned size. Both sample
sets were further divided into age groups, taking into con-
sideration the increased rate of skewing in older women. XCI ratio was assessed using HUMARA method. No sig-
nificant difference between XCI ratios of women with and
without X chromosome aberrations has been found so far. However, as expected, an increase in XCI skewing is seen
in older women. X chromosome inactivation (XCI) balances the expression
of X-linked genes between females and males. Early in
female development, cells transcriptionally silence one
randomly chosen X chromosome. This leads to a general
ratio of 50:50 cells, where half the cells inactivate the
maternal and half the paternal X chromosome. The ratio
varies among women, although significant deviations from
it are relatively rare among phenotypically normal women
and are known as skewed XCI. Skewing is more common
among older women or women with certain X-linked dis-
eases or X chromosome aberrations. The latter is often
marked by inactivation of the defective X chromosome. Our
study focuses on detecting the influence of X chromosome
deletions and duplications on XCI. M. Moyses-Oliveira1, A. Di-Battista1, M. Zamariolli1, V. Meloni1,
S. Bragagnolo1, D. Christofolini2, C. Steiner3, L. Sisdelli1, N.
Kosyakova4, T. Liehr4, A. Reymond5, M. Melaragno1
1Universidade Federal de São Paulo, São Paulo, Brazil,
2Faculdade de Medicina do ABC, São Paulo, Brazil,
3Universidade Estadual de Campinas, Campinas, Brazil,
4Friedrich Schiller University, Jena, Germany, 5University of
Lausanne, Lausanne, Switzerland P13.04D E. Pozzi: None. S. Cannito: None. M. Parola: None. M. Vinciguerra: None. S. Augeri: None. C. Mariotti: None. E. Pozzi: None. S. Cannito: None. M. Parola: None. M. Vinciguerra: None. S. Augeri: None. C. Mariotti: None. E. Giorgio: None. C. Mancini: None. E. Di Gregorio:
None. M. Ferrero: None. E. Riberi: None. A. Brusco:
None. S. Cavalieri: None. 1Universidade Federal de São Paulo, São Paulo, Brazil,
2Faculdade de Medicina do ABC, São Paulo, Brazil,
3Universidade Estadual de Campinas, Campinas, Brazil,
4Friedrich Schiller University, Jena, Germany, 5University of
Lausanne, Lausanne, Switzerland M. Moyses-Oliveira1, A. Di-Battista1, M. Zamariolli1, V. Meloni1,
S. Bragagnolo1, D. Christofolini2, C. Steiner3, L. Sisdelli1, N.
Kosyakova4, T. Liehr4, A. Reymond5, M. Melaragno1 P13.05A Breakpoint mapping at nucleotide resolution in balanced
translocations associated with clinical phenotypes Abstracts from the 51st European Society of Human Genetics Conference: Posters 479 Precise breakpoint mapping of balanced chromosomal
rearrangements is crucial to identify disease genes. We
evaluated 11 female patients with balanced reciprocal
translocations associated with phenotypic alterations. We
mapped and sequenced their breakpoints, assessed the
rearrangements’ impacts on expression of disrupted genes,
addressed candidate genes to position effect, and inferred
mechanisms of formation. Four out of 11 patients presented
one of the chromosomal breaks in heterochromatic and
highly repetitive DNA segments, such as centromeres or
short arm of acrocentric chromosomes. We demonstrated
that nucleotide resolution characterization of breakpoints at
gaps in the reference genome is feasible when cytogenomic
methods and short-read sequencing are associated. Most of
the
rearrangements
were
possibly
formed
by
non-
homologous end joining with breakpoints within repeat
elements. Seven of the 11 patients presented with break-
points within a total of nine genes. Seven of which showed
altered expression levels and the functional impairment of
two of them, e.g. KIAA2022, IL1RAPL1, could be con-
sidered causative of the patients’ phenotypes. The disrup-
tion of a promoter region triggered the description of a
novel X-linked syndrome caused by AMMECR1 loss of
function. In four patients, there was no gene disruption at
the breakpoints, suggesting other pathogenic mechanisms. Four candidate genes were considered potentially affected
by position effect and expression abrogation of one of them,
e.g. TSPAN7, was confirmed. We emphasize the importance
of breakpoint-junction characterization at nucleotide reso-
lution in balanced chromosomal rearrangements to reveal
the genetic mechanisms associated to the patients’ pheno-
types, mechanisms of formation, and genomic nature of the
disrupted DNA sequences. with a function of oxidative stress resistance by scavenging
reactive oxygen species (ROS), but the gene function
remains to be explored. The aim of this study was to elu-
cidate X gene function, mechanism and its role in eye
development using Drosophila and mice as a model
organism. Materials and Methods: The GAL4-UAS system were
used to specifically knockdown homologue of X in the eye
of Drosophila and resulted in the small eye phenotype. Various genetic crosses were performed to analyze the
phenotype were mediated through apoptotic or autophagy
pathway. The mice homologous gene knockout was
generated using CRISPR/Cas9 technology. Results: Knockdown of X in Drosophila resulted in
severe small eye phenotype, and the knockout mice were
blind. P13.05A In Drosophila, P35 baculovirus overexpression
(active caspase inhibitor) and knockdown of pro-apoptotic
genes showed complete rescue of small eye phenotype. To
further identify the exact apoptotic pathway, various players
involved in JUN N-terminal kinase (JNK) apoptosis
pathway rescue the small eye phenotype. Conclusions: The small eye phenotype of X could be
involved in the JNK-mediated apoptosis pathway. Our
future studies would be to prove JNK dependent apoptosis,
the role of ROS, mutagenesis study to explore more about
the function of X. D. Suresh: None. Y. Ching: None. M. Lin: None. B. Liao: None. P13.07C Whole Genome Sequencing of 9 patients allowed a better
understanding of complex chromosomal rearrangements N. Chatron1,2, F. Diguet1, P. Rollat-Farnier1, K. Uguen1,3, J. Lauer Zillhardt4, A. Sorlin5,6, J. Andrieux7, S. Chantot-
Bastaraud8, P. Callier9, M. Cordier1, C. Dubourg10,11, F. Girard12, S. Jaillard13, B. Keren14, J. Lespinasse15, N. Marle5, A. Masurel5, M. Mathieu16, C. Metay17, M. Portnoï8, F. Prieur18, M. Rio19, J. Siffroi8, C. Schluth-Bolard1,2, D. Sanlaville1,2 M. Moyses-Oliveira: None. A. Di-Battista: None. M. Zamariolli: None. V. Meloni: None. S. Bragagnolo:
None. D. Christofolini: None. C. Steiner: None. L. Sisdelli: None. N. Kosyakova: None. T. Liehr: None. A. Reymond: None. M. Melaragno: None. hatron1,2, F. Diguet1, P. Rollat-Farnier1, K. Uguen1,3, 1Service de Génétique, Hospices Civils de Lyon, Lyon, France,
2Equipe GENDEV, CRNL, INSERM U1028, CNRS UMR 5292
UCBL1, Lyon, France, 3Service de Génétique, CHU Brest,
Brest, France, 4Unité de Diagnostic Préimplantatoire,
Laboratoires de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France, 5Service de
génétique, CHU Dijon, Dijon, France, 6Equipe GAD, INSERM
U1231, Dijon, France, 7Service de génétique, Hôpital Jeanne
de Flandre, CHRU de Lille, Lille, France, 8AP-HP,
Département de Génétique médicale, UF de Génétique
chromosomique, hôpital d’Enfants Armand Trousseau, Paris,
France, 9Service de génétique, CHU Dijon, Dijon, Dijon,
France, 10Service de Génétique Moléculaire et Génomique, P13.06B 1Service de Génétique, Hospices Civils de Lyon, Lyon, France,
2Equipe GENDEV, CRNL, INSERM U1028, CNRS UMR 5292
UCBL1, Lyon, France, 3Service de Génétique, CHU Brest,
Brest, France, 4Unité de Diagnostic Préimplantatoire,
Laboratoires de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France, 5Service de
génétique, CHU Dijon, Dijon, France, 6Equipe GAD, INSERM
U1231, Dijon, France, 7Service de génétique, Hôpital Jeanne
de Flandre, CHRU de Lille, Lille, France, 8AP-HP,
Département de Génétique médicale, UF de Génétique
chromosomique, hôpital d’Enfants Armand Trousseau, Paris,
France, 9Service de génétique, CHU Dijon, Dijon, Dijon,
France, 10Service de Génétique Moléculaire et Génomique, 1Service de Génétique, Hospices Civils de Lyon, Lyon, France,
2Equipe GENDEV, CRNL, INSERM U1028, CNRS UMR 5292
UCBL1, Lyon, France, 3Service de Génétique, CHU Brest,
Brest, France, 4Unité de Diagnostic Préimplantatoire,
Laboratoires de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France, 5Service de
génétique, CHU Dijon, Dijon, France, 6Equipe GAD, INSERM
U1231, Dijon, France, 7Service de génétique, Hôpital Jeanne
de Flandre, CHRU de Lille, Lille, France, 8AP-HP,
Département de Génétique médicale, UF de Génétique
chromosomique, hôpital d’Enfants Armand Trousseau, Paris,
France, 9Service de génétique, CHU Dijon, Dijon, Dijon,
France, 10Service de Génétique Moléculaire et Génomique, 1Service de Génétique, Hospices Civils de Lyon, Lyon, France,
2Equipe GENDEV, CRNL, INSERM U1028, CNRS UMR 5292
UCBL1, Lyon, France, 3Service de Génétique, CHU Brest,
Brest, France, 4Unité de Diagnostic Préimplantatoire,
Laboratoires de Diagnostic Génétique, Hôpitaux
Universitaires de Strasbourg, Strasbourg, France, 5Service de
génétique, CHU Dijon, Dijon, France, 6Equipe GAD, INSERM
U1231, Dijon, France, 7Service de génétique, Hôpital Jeanne
de Flandre, CHRU de Lille, Lille, France, 8AP-HP,
Département de Génétique médicale, UF de Génétique
chromosomique, hôpital d’Enfants Armand Trousseau, Paris,
France, 9Service de génétique, CHU Dijon, Dijon, Dijon,
France, 10Service de Génétique Moléculaire et Génomique, Functional analysis of a novel gene X causing small eye
phenotype through JNK-dependent apoptosis pathway D. Suresh1, Y. Ching1, M. Lin1, B. Liao2 1TzuChi University, Hualien city, Taiwan, 2National Health
Research institute, Miaoli, Taiwan 1TzuChi University, Hualien city, Taiwan, 2National Health
Research institute, Miaoli, Taiwan Introduction: A bioinformatics work comprising co-
expression gene network in the mouse was built based on
microarray and RNA-Seq platforms, revealed an unex-
plored gene X which might play a role in retinal develop-
ment. X contains an evolutionary conserved protein domain 480 J. P13.08D Contribution of CMA to genetic diagnosis of individuals
with dysmorphisms: a collaborative study of the SIGU
(Italian Society of Human Genetics) Cytogenetic and
Cytogenomic working group Chromosomal rearrangements are used to be considered as
complex when involving at least 3 breakpoints on two
different chromosomes. Cytogenetic microarrays and whole
genome sequencing (WGS) revealed rare much more
complex situations with numerous breakpoints on a single
chromosome overshooting the first definition. Henceforth
grouped under the chromoanagenesis term mechanisms
generating such rearrangements remain misunderstood and
their definition elusive especially since such constitutional
complex chromosome rearrangement (CCR) are rare. M. Garzo1, I. Catusi1, M. Recalcati1, M. Alfonsi2, A. Alghisi3, S. Cappellani4, R. Casalone5, R. Caselli6, C. Ceccarini7, C. Ceglia8,
A. Ciaschini9, D. Coviello10, F. Crosti11, A. D'Aprile7, A. Fabretto4, R. Genesio12, M. Giagnacovo13, P. Granata5, I. Longo6, M. Malacarne10, G. Marseglia14, A. Montaldi3, A. Nardone15, C. Palka16, V. Pecile4, C. Pessina5, D. Postorivo15, S. Redaelli17, A. Renieri6, C. Rigon18, F. Tiberi9, M. Tonelli19, C. Valtorta1, N. Villa11, A. Zilio3, D. Zuccarello18, A. Novelli20, L. Larizza1, D. Giardino1 M. Garzo1, I. Catusi1, M. Recalcati1, M. Alfonsi2, A. Alghisi3, S. Cappellani4, R. Casalone5, R. Caselli6, C. Ceccarini7, C. Ceglia8,
A. Ciaschini9, D. Coviello10, F. Crosti11, A. D'Aprile7, A. Fabretto4, R. Genesio12, M. Giagnacovo13, P. Granata5, I. Longo6, M. Malacarne10, G. Marseglia14, A. Montaldi3, A. Nardone15, C. Palka16, V. Pecile4, C. Pessina5, D. Postorivo15, S. Redaelli17, A. Renieri6, C. Rigon18, F. Tiberi9, M. Tonelli19, C. Valtorta1, N. Villa11, A. Zilio3, D. Zuccarello18, A. Novelli20, L. Larizza1, D. Giardino1 1IRCCS Istituto Auxologico Italiano, Milano, Italy, 2Ospedale
SS Annunziata, U.O.C. di Genetica medica, Chieti, Italy,
3Azienda ULSS 6, U.O.S. Genetica e Biologia Molecolare,
Vicenza, Italy, 4IRCCS Burlo Garofolo, S.C. Genetica Medica,
Trieste, Italy, 5ASST Sette Laghi, Osp. di Circolo e Fond. Macchi,SMeL specializzato Citogenetica e Genetica Medica,
Varese, Italy, 6Azienda Ospedaliera Universitaria Senese, U.O. C. Genetica Medica, Siena, Italy, 7A.O.U. Ospedali Riuniti,
Lab. di Citogenetica, Foggia, Italy, 8AORN SG Moscati,
UOSD Genetica Medica, Avellino, Italy, 9A.O.U. Ospedali
Riuniti Umberto I-G.M.Lancisi-G.Salesi, Lab. Genetica
Medica SOS Malattie Rare, Ancona, Italy, 10E.O. Ospedali
Galliera, Lab.di Genetica Umana, Genova, Italy, 11Ospedale
San Gerardo- ASST Monza, U.S. Genetica Medica, Monza,
Italy, 12A.O.U. Federico II, U.O.C. di Citogenetica, Napoli,
Italy, 13ASST Lariana - Ospedale Sant' Anna, Lab. di Genetica,
Como, Italy, 14A.O.U. Careggi, S.O.D. Diagnostica Genetica,
Firenze, Italy, 15Policlinico Tor Vergata, U.O.C. Laboratorio
di Genetica Medica, Roma, Italy, 16Università G. P13.06B del Picchia CHU Rennes, Rennes, France, 11IGDR, CNRS UMR 6290,
Université de Rennes 1, Rennes, France, 12Laboratoire de
cytogénétique constitutionnelle et prénatale, Strasbourg,
France, 13Service de cytogénétique et biologie cellulaire, CHU
Rennes, Rennes, France, 14Département de Génétique et
Centre de Référence Déficiences Intellectuelles de Causes
Rares, Hôpital de la Pitié-Salpêtrière, Assistance Publique -
Hôpitaux de Paris, Paris, France, 15Service de génétique, CH
Métropole Savoie, Chambéry, France, 16Service de génétique
clinique et oncogénétique, CHU Amiens-Picardie, Amiens,
France, 17AP-HP, Hôpital Henri Mondor, Creteil, France,
18Service de Génétique Clinique, Chromosomique et
Moléculaire, CHU Hôpital Nord, Saint-Etienne, France, 19AP-
HP, Service de Génétique Médicale, Hôpital Necker-Enfants
Malades, Paris, France N. Chatron: None. F. Diguet: None. P. Rollat-Farnier:
None. K. Uguen: None. J. Lauer Zillhardt: None. A. Sorlin: None. J. Andrieux: None. S. Chantot-Bastaraud:
None. P. Callier: None. M. Cordier: None. C. Dubourg:
None. F. Girard: None. S. Jaillard: None. B. Keren:
None. J. Lespinasse: None. N. Marle: None. A. Masurel:
None. M. Mathieu: None. C. Metay: None. M. Portnoï:
None. F. Prieur: None. M. Rio: None. J. Siffroi: None. C. Schluth-Bolard: None. D. Sanlaville: None. P13.08D D’Annunzio,
Dipartimento di Pediatria, Chieti-Pescara, Italy, 17Università
di Milano-Bicocca, Dipartimento di Medicina e Chirurgia,
Monza, Italy, 18A.O.U. di Padova, U.O.C. Genetica e
Epidemiologia Clinica, Padova, Italy, 19Università di Brescia,
LCGM Dipartimento di Medicina Molecolare e Traslazionale,
Brescia, Italy, 20Ospedale Pediatrico del Bambino Gesù, U.O. C. Laboratorio di Genetica Medica, Roma, Italy We
performed
WGS
for
9
probable
constitutional
chromoanagenesis: 4 cases with a minimum of 4 Copy
Number Variations on a single chromosome and 5 cases
with at least 10 chromosome breakpoints identified in
patients with balanced chromosomal rearrangements char-
acterized with WGS (ANI project). We analyzed paired-end
WGS data using BreakDancer and ERDS respectively for
breakpoint and CNV calling. Our Svagga pipeline was used
for filtering and annotation. All rearrangements appeared to be more complex than
initially thought with a total of 232 breakpoints and a
maximum of 74 breakpoints clustered in 4 hotspots for a
single patient. No statistical significant imbalance was
observed compared to random distribution regarding TAD
disruption or purine/pyrimidine nucleotide at breakpoint. A
statistical depletion of gene-disrupting breakpoints was
observed compared to theoretical distribution (p = 0,0016). Nucleotide resolution showed the combination of several
repairing
mechanisms
within
a
rearrangement
adding
complexity to complexity. Gathering several exceptional observations we help to
delineate the chromoanagenesis phenomenon. Breakpoint
distribution compared to simpler rearrangements will help
understand
its
origins
and
provide
new
insights
in
cytogenomics such as guidelines for structural variant
pathogenicity classification. 481 Abstracts from the 51st European Society of Human Genetics Conference: Posters Chromosomal microarray analysis (CMA) significantly
increased the possibility to identify copy number variations
(CNVs) associated with a wide range of genomic disorders. 1National Institutes of Biomedical Innovation, Ibaraki, Japan,
2University of Cambridge, Cambridge, United Kingdom Chromosome translocations can be detected by cytogenetic
analysis, but this is independent of expression data which
do not identify the origin of alleles. It is known that the
number of abnormal chromosomes in tumor cells is gen-
erally increased during progression in vivo or serial passage
in vitro due to chromosome instability. This raises a fun-
damental question about whether chromosome rearrange-
ments can cause effects on gene expression in rearranged
chromosomes in addition to fusion genes. Chromosome
sorting by flow cytometry produces flow karyotypes that
enable the distinction between normal and abnormal chro-
mosomes. P13.08D Garzo: None. I. Catusi: None. M. Recalcati: None. M. Alfonsi: None. A. Alghisi: None. S. Cappellani: None. R. Casalone: None. R. Caselli: None. C. Ceccarini: None. C. Ceglia: None. A. Ciaschini: None. D. Coviello: None. F. Crosti: None. A. D'Aprile: None. A. Fabretto: None. R. Genesio: None. M. Giagnacovo: None. P. Granata:
None. I. Longo:
None. M. Malacarne:
None. G. Marseglia: None. A. Montaldi: None. A. Nardone: None. C. Palka: None. V. Pecile: None. C. Pessina: None. D. Postorivo: None. S. Redaelli: None. A. Renieri: None. C. Rigon: None. F. Tiberi: None. M. Tonelli: None. C. Valtorta: None. N. Villa: None. A. Zilio: None. D. Zuccarello: None. A. Novelli: None. L. Larizza: None. D. Giardino: None. F. Kasai: None. J.C. Pereira: None. A. Kohara: None. M.A. Ferguson-Smith: None. F. Kasai: None. J.C. Pereira: None. A. Kohara: None. M.A. Ferguson-Smith: None. P13.10B P13.10B
Very short DNA segments can be detected and handled by
the repair machinery during germline chromothriptic
chromosome reassembly Very short DNA segments can be detected and handled by
the repair machinery during germline chromothriptic
chromosome reassembly M. Hančárová1, L. Nazaryan-Petersen2, M. M. Mehrjouy2, Z. Slamova1, J. Drabova1, T. Marikova1, D. Novotna1, M. Vlckova1,
Z. Vlckova3, M. Bak2, Z. Zemanova4, N. Tommerup2, Z. Sedlacek1 M. Hančárová1, L. Nazaryan-Petersen2, M. M. Mehrjouy2, Z. Slamova1, J. Drabova1, T. Marikova1, D. Novotna1, M. Vlckova1,
Z. Vlckova3, M. Bak2, Z. Zemanova4, N. Tommerup2, Z. Sedlacek1 M. Hančárová1, L. Nazaryan-Petersen2, M. M. Mehrjouy2, Z. Slamova1, J. Drabova1, T. Marikova1, D. Novotna1, M. Vlckova1,
Z. Vlckova3, M. Bak2, Z. Zemanova4, N. Tommerup2, Z. Sedlacek1 P13.08D In this study, a derivative chromosome t(9;14)
and its homologous normal chromosomes 9 from the Ishi-
kawa 3-H-12 cell line were sorted to collect homologue-
specific samples. Chromosome sequencing of the der(9)
identified the breakpoint junction at 9p24.3 and 14q13.1
and uncovered the formation of a fusion gene, WASH1-
NPAS3. Of 293,903 amplicons in the Ion Ampliseq exome
panel, 11,809 amplicons are localized in chromosome 9,
corresponding to 749 genes. Chromosome-specific exome
sequencing of sorted chromosomes demonstrated that 87%
of the chromosome 9 exome was amplified in der(9), which
include 982 SNVs. This permits the assignment of allelic
variants and can lead to comparisons between normal and
abnormal chromosomes. Compared to the RNA sequencing
data with the allele-specific variant profiles, each gene
expression along chromosome 9 is assigned to both or one
of alleles. We show that allele-specific chromosome
sequencing of homologues is a robust technique for dis-
tinguishing alleles and this provides a valuable approach for
the investigation of chromosome instability. (
)
g
g
Here we report on a collaborative study of the SIGU(Italian
Society of Human Genetics) Cytogenetic and Cytogenomic
working group, on 780 patients referred to CMA for dys-
morphisms as the only clinical manifestation(21%) or
associated with intellectual disability/developmental delay
(57%), congenital malformation(s)(10%), autism spectrum
disorders(6%), epilepsy(3%) or growth anomalies(3%). Overall 329 non-polymorphic CNVs have been identified in
266 patients(34%) of which 78 CNVs have been detected in
56 patients(21%) with dysmorphisms as the only clinical
manifestation. In 36% of these probands the CNVs have
been classified as pathogenic(pCNVs), with 15% associated
to known syndromes, and in 64% as variants of uncertain
clinical significance(VOUS). The remaining 251 CNVs
have been detected in 210 patients referred for dysmorph-
isms combined to another clinical manifestation and clas-
sified as pCNVs in 38% of cases, with 21% associated to
known syndromes, and 62% as VOUS. The average size
was 2,9 Mb for all CNVs, >6 Mb for pCNVs and <1 Mb for
VOUS. This study allowed us to assess the detection rate of
CMA for patients referred for dysmorphisms as the only
clinical manifestation, hence informing about the consistent
or possible underlying genetic causes to validate/explore in
the distinct patients groups by 3d-facial-analysis. Dys-
morphisms combined to other clinical findings could be
evaluated in the same study and carriers of pCNVs received
their diagnosis, while those with VOUS remain amenable to
be solved by novel literature insights. M. F. Kasai1, J. C. Pereira2, A. Kohara1, M. A. Ferguson-Smith2 P13.09A Allelic gene expression profiles revealed by homologue
specific exome sequencing Allelic gene expression profiles revealed by homologue
specific exome sequencing 1Department of Biology and Medical Genetics, Charles
University 2nd Faculty of Medicine and University Hospital
Motol, Prague, Czech Republic, 2Wilhelm Johannsen Centre
for Functional Genome Research, Department of Cellular and
Molecular Medicine, University of Copenhagen, Copenhagen,
Denmark, 3GHC Genetics, Prague, Czech Republic, 4Center of F. Kasai1, J. C. Pereira2, A. Kohara1, M. A. Ferguson-Smith2 482 J. del Picchia Pavia, Italy, 3Pediatrics Department, Papa Giovanni XXIII
Hospital, Bergamo, Italy Oncocytogenetics, Institute of Medical Biochemistry and
Laboratory Diagnostics, General University Hospital and
Charles University 1st Faculty of Medicine, Prague, Czech
Republic Introduction: Chromothripsis is a one-step genome-shat-
tering catastrophe resulting from disruption of one or few
chromosomes in multiple fragments and consequent ran-
dom rejoining and repair leading to complex chromosomal
rearrangements. While chromotripsis has been extensively
observed in cancers, few investigations documented a
similar phenomenon in congenital disorders. We report a
case of a newborn, conceived using a donor egg, with a
complex karyotype and phenotypic abnormalities including
congenital cardiomyopathy, genital ambiguity, agenesis of
corpus callosum, bilateral ocular abnormalities and absence
of left 12th rib. Detailed analyses down to the nucleotide resolution reveal
unexpected complexity of seemingly simpler and balanced
chromosomal rearrangements. This concerns also chromo-
thripsis, a rare type of complex rearrangement involving
local shattering of one or more chromosomes and random
reassembly of the resulting segments. Chromothripsis can
influence expression of many genes and cause abnormal
phenotypes. We studied the structure and mechanism of a
seemingly balanced de novo chromosome rearrangement in
a boy with developmental and growth delay. Karyotyping
and mFISH identified 11 segments from four chromosomes
to participate in the rearrangement. Microarray analysis
revealed two de novo deletions of 0.7 and 2.5 Mb at two of
the breakpoints in 1q24.3 and 6q24.1-q24.2, respectively. They
affected
paternal
chromosomes
and
possibly
explained most symptoms of the patient. Subsequent whole-
genome mate-pair sequencing revealed that the four chro-
mosomes were in fact broken into 29 segments longer than
one kb. Sanger sequencing of all junctions showed addi-
tional complexity compatible with the involvement of dif-
ferent repair pathways. A translocation of a 33 bp long
fragment to one of the junctions was observed which may
have implications for the definition of the lower size limit of
structural variants. P13.12D Medical consequences of pathogenic CNVs in adults P13.09A Our observations and review of pub-
lished chromothripsis events indicate that even very small
fragments from the shattered chromosomes can be detected
and handled by the repair machinery during germline
chromothriptic chromosome reassembly. Supported by 17-
29423A, 00064203, 00064165, LM2015091 (Czech Min-
istries of Health and Education), 2013-14290 (Lundbeck
Foundation), Global Genes, Local Concerns (University of
Copenhagen) and 4183-00482B (Danish Council for Inde-
pendent Research). Methods:
Conventional
cytogenetic
analysis,
array
comparitive genomic hybridization, whole genome sequen-
cing (WGS), and polymerase chain reaction were used to
identify the chromosome rearrangement and characterize
the breakpoints in our patient and his father. Results: Conventional and molecular cytogenetic analy-
sis showed a complex karyotype with structural variations
including deletions of three regions on chromosome 2,
deletions of two regions on chromosome 4 with inversion of
the fragment between the two breakpoints, and a pericentric
inversion of chromosome 11. WGS analysis detected
further
multiple
complex
balanced
intra-chromosomal
rearrangements of chromosomes 2 and 4, with 17 break-
points resulting in the breakage of 11 genes. Paternal origin
of the abnormal chromosomes has been confirmed. Conclusions: The pattern of random joining of chromo-
somal fragments observed in our case suggests that a
chromothripsis event might have driven the formation of
these complex rearrangements. The rearranged chromo-
somes were demonstrated to be of paternal origin suggest-
ing that a chromothripsis occurred during spermatagenesis
although the onset in the preimplantation embryo cannot be
excluded. M. Hančárová: None. L. Nazaryan-Petersen: None. M. M. Mehrjouy: None. Z. Slamova: None. J. Drabova:
None. T. Marikova: None. D. Novotna: None. M. Vlckova: None. Z. Vlckova: None. M. Bak: None. Z. Zemanova:
None. N. Tommerup:
None. Z. Sedlacek: None. A. Pansa: None. N. Kurtas: None. A. Cereda: None. B. Facchinetti: None. G. Cassina: None. F. Comi: None. C. Perico: None. D. Nicoli: None. O. Zuffardi: None. U. Giussani: None. 1Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo,
Italy, 2Department of Molecular Medicine, University of Pavia, New evidence of chromothripsis in congenital disorder G. Kirov, K. Crawford, M. Smith, K. Kendall, E. Rees, V. Escott-
Price, J. Walters, M. J. Owen, M. C. O'Donovan New evidence of chromothripsis in congenital disorder G. Kirov, K. Crawford, M. Smith, K. Kendall, E. Rees, V. Escott-
Price, J. Walters, M. J. Owen, M. C. O'Donovan A. Pansa1, N. Kurtas2, A. Cereda3, B. Facchinetti1, G. Cassina1,
F. Comi1, C. Perico1, D. Nicoli1, O. Zuffardi2, U. Giussani1
1Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo,
Italy, 2Department of Molecular Medicine, University of Pavia, A. Pansa1, N. Kurtas2, A. Cereda3, B. Facchinetti1, G. Cassina1,
F. Comi1, C. Perico1, D. Nicoli1, O. Zuffardi2, U. Giussani1 A. Pansa1, N. Kurtas2, A. Cereda3, B. Facchinetti1, G. Cassina1,
F. Comi1, C. Perico1, D. Nicoli1, O. Zuffardi2, U. Giussani1 Cardiff University, Cardiff, United Kingdom Cardiff University, Cardiff, United Kingdom Background: Copy number variants (CNVs) increase risk
for learning difficulties and early-onset neurodevelopmental 1Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo,
Italy, 2Department of Molecular Medicine, University of Pavia, Abstracts from the 51st European Society of Human Genetics Conference: Posters 483 disorders but their role in medical outcomes in middle- and
old age is limited. The UK Biobank, with half a million well
phenotyped adults, presents an opportunity to study the
medical consequences of CNV in the general population. 7Serviço de Genética Médica, Hospital Egas Moniz, Lisbon,
Portugal, 8Serviço de Cirurgia Pediátrica, Hospital de Santo
António, Porto, Portugal, 9Serviço de Genética Médica, Centro
Hospitalar e Universitário de Coimbra, Coimbra, Portugal,
10Serviço de Endocrinologia Pediátrica, Centro Hospitalar e
Universitário de Coimbra, Coimbra, Portugal, 11ToxOmics -
Centro de Toxicogenómica e Saúde Humana, Nova Medical
School, Lisbon, Portugal, 12This work was partially funded by
Project: UID/BIM/0009/2016 from Fundação para a Ciência e
a Tecnologia, Lisbon, Portugal Methods: We analysed 54 pathogenic CNVs in all
Biobank participants. We used logistic regression analysis
to test CNVs for associations with 58 broad medical
phenotypes, present in at least 2000 participants. Results: CNV carriers had an increased risk to develop
37 of the 58 phenotypes at nominal levels of statistical
significance, with 19 of these associations surviving
Bonferroni correction for 58 tests. Individual comparisons
of each of the 54 CNVs against the 58 phenotypes produced
18 associations that survived Bonferroni correction for 3132
tests and a further 57 that were significant at a false
discovery rate of 0.1. New evidence of chromothripsis in congenital disorder Thirteen CNV loci had three or more
significant associations at FDR=0.1, with 16p11.2 deletions
leading the list with 15 significant results. The most
common CNVs (at 0.5-0.7% frequency) have none or
minimal impact on medical outcomes in adults. Introduction: Congenital adrenal hyperplasia(CAH) is due
to 21-hidroxilase deficiency(21-OHD) in about 95% of the
cases. 21-OH is encoded by CYP21A2 gene, and most
frequent mutations occurring in CYP21A2 are due to gene
conversions originated from its pseudogene(CYP21A1P). The clinical severity of CAH is associated with the
impairment of 21-OH activity, which is directly related with
the molecular defect. CAH is classified as classic salt-
wasting(SW) and simple virilising(SV) forms, and non-
classic(NC) form of the disease. SW and SV are usually
diagnosed after birth or during the first years of life,
respectively, while most cases of NC-CAH are diagnosed
during infancy, puberty or until adult age. Here we present
the molecular results performed in paediatric patients
with CAH. Conclusions: Some of the 54 CNVs proposed to be
pathogenic have profound effects on physical health, even
in people who have largely escaped early neurodevelop-
mental outcomes. Our work provides clinicians with a
morbidity map of potential outcomes among carriers of
these CNVs, which will be made available on a dedicated
web resource, updated as new data is released by the
Biobank. Methodology: molecular analysis (using genomic DNA)
included mini-sequencing, restriction enzyme digestion,
Sanger sequencing, Southern-blotting and/or multiplex
ligation-dependent probe amplification(MLPA). G. Kirov: None. K. Crawford: None. M. Smith: None. K. Kendall: None. E. Rees: None. V. Escott-Price: None. J. Walters:
None. M.J. Owen:
None. M.C. O'Donovan: None. G. Kirov: None. K. Crawford: None. M. Smith: None. K. Kendall: None. E. Rees: None. V. Escott-Price: None. J. Walters:
None. M.J. Owen:
None. M.C. O'Donovan: None. Results: We analysed 265 patients with CAH (65 with
SW, 51 with SV and 149 with NC). In 211 patients (80%)
the genotypes were in agreement with their phenotypes,
while in the remaining 20%, only one pathogenic allele was
identified or their genotype was normal. In the SW group
the most frequent variant was the splicing mutation
g.655A>G(28.5%), in the SV was g.999T>A(25.5%), and
in NC was g.1683G>T(61%). P13.15C
Partial trisomy 21 map: ten cases further supporting the
highly restricted Down syndrome critical region (HR-
DSCR) on human chromosome 21 1Service de Génétique, CHU-Reims, Reims, France, 2Service
de Génétique, CHU-Dijon, Dijon, France, 3Service de
Génétique, CHU-Lyon, Lyon, France, 4Service de Génétique,
CHU-Marseilles, Marseilles, France, 5Service de Génétique,
CHU-Besançon, Besançon, France, 6Service de Génétique,
CHU-Cochin, Paris, France, 7Service de Génétique, CHU-
Nantes, Nantes, France, 8Service de Génétique, CHU-le Mans,
le Mans, France, 9Service de Génétique, CHU-Clermont-
Ferrand, Clermont-Ferrand, France, 10Service de Génétique,
CHU-Robert Debré, Paris, France, 11Service de Génétique,
CHU-Brest, Brest, France, 12Service de Génétique, CHU-
Caen, Caen, France, 13Service de Génétique, CHU-Versailles,
Versailles, France M. C. Pelleri1, E. Cicchini1, M. B. Petersen2,3, L. Tranebjærg4,5,
T. Mattina6, P. Magini7, F. Antonaros1, M. Caracausi1, L. Vitale1, C. Locatelli8, M. Seri9, P. Strippoli1, A. Piovesan1, G. Cocchi10 1Department of Experimental, Diagnostic and Specialty
Medicine (DIMES), Bologna, Italy, 2Department of Genetics,
Aalborg University Hospital, Aalborg, Denmark, 3Department
of Clinical Genetics, Aalborg University, Aalborg, Denmark,
4Department of Clinical Genetics/Rigshospitalet, The Kennedy
Centre, Glostrup, Denmark, 5University of Copenhagen,
Institute of Clinical Medicine, The Panum Institute,
Copenhagen, Denmark, 6Department of Pediatrics, Medical
Genetics University of Catania, Catania, Italy, 7Medical
Genetics Unit, St. Orsola-Malpighi Polyclinic, Bologna, Italy,
8Neonatology Unit, St. Orsola-Malpighi Polyclinic, Bologna,
Italy, 9Medical Genetics Unit, St. Orsola-Malpighi Polyclinic,
Department of Medical and Surgical Sciences (DIMEC),
University of Bologna, Bologna, Italy, 10Neonatology Unit, St. Orsola-Malpighi Polyclinic, Department of Medical and
Surgical Sciences (DIMEC), University of Bologna, Bologna,
Italy The purpose of our study was to review the prevalence of
1p36 deletion diagnosed in France in comparison with the
same work made for 22q11 deletion (700 patients). The
1p36 deletion has a described incidence of 1/5000 to 1/
10,000 births living. It is also a common condition within
the microdeletions and potentially the most frequent after
22q11 deletion any age. Following a national ACLF survey
with 15 centers, we report a cohort of 70 patients born
living,at least 30 girls and 20 boys diagnosed between 2004
and 2017. The age of patients is 2 days to 31 years. The
diagnosis was possible in the first years by the FISH tech-
nique, MLPA and finally thanks array-CGH in priority. This
is a deletion interstitial in the majority of cases. For relatives
tested more than 28 cases occurred de novo. Patients had
clinical signs consistent with those already described in
literature. They had growth retardation, hypotonia and/or a
delay in acquisitions, or Intellectual disability (51/66
responses), IUGR and/or stunting (32/32 responses), a facial
dysmorphism (53/56). Microdeletion 1p36 diagnostic follow-up of a cohort of 70
patients diagnosed in France Microdeletion 1p36 diagnostic follow-up of a cohort of 70
patients diagnosed in France M. Doco-Fenzy1, C. Jacquin1, P. Callier2, D. Sanlaville3, C. Missirian4, P. Kuentz5, P. Jaeger3, A. Heddar6, C. Lecaignec7, C. Poirsier1, E. Landais1, D. Martin8, C. Richard9, a. Tabet10, S. Redon11, N. Gruchy12, F. Vialard13 P13.13A Congenital adrenal hyperplasia in paediatric age<:>
molecular analysis of the CYP21A2 gene and implications
for genetic counselling S. Gomes1, J. Silva1, I. Pereira-Caetano1, L. Lopes2, C. Limbert2,
D. Amaral2, R. Pina2, T. Kay3, L. Sampaio4, C. Pereira4, O. Moldovan5, A. B. Sousa5, I. Rebelo6, I. Gaspar7, J. C. Rodrigues8, F. Ramos9, L. Ramos9, I. Dinis10, R. C. Cardoso10,
A. Mirante10, J. Goncalves1,11,12 S. Gomes1, J. Silva1, I. Pereira-Caetano1, L. Lopes2, C. Limbert2,
D. Amaral2, R. Pina2, T. Kay3, L. Sampaio4, C. Pereira4, O. Moldovan5, A. B. Sousa5, I. Rebelo6, I. Gaspar7, J. C. Rodrigues8, F. Ramos9, L. Ramos9, I. Dinis10, R. C. Cardoso10,
A. Mirante10, J. Goncalves1,11,12 S. Gomes1, J. Silva1, I. Pereira-Caetano1, L. Lopes2, C. Limbert2, Conclusions: Knowing the molecular bases of CAH is
essential
for
a
correct
genetic
counselling;
prenatal
diagnosis and treatment during pregnancy can be offered
to couples at risk of having a female child with SW or SV in
order to avoid sexual ambiguity of the newborns. D. Amaral2, R. Pina2, T. Kay3, L. Sampaio4, C. Pereira4, O. Moldovan5, A. B. Sousa5, I. Rebelo6, I. Gaspar7, J. C. Rodrigues8, F. Ramos9, L. Ramos9, I. Dinis10, R. C. Cardoso10,
A. Mirante10, J. Goncalves1,11,12 S. Gomes: None. J. Silva: None. I. Pereira-Caetano:
None. L. Lopes: None. C. Limbert: None. D. Amaral:
None. R. Pina: None. T. Kay: None. L. Sampaio: None. C. Pereira: None. O. Moldovan: None. A.B. Sousa: None. I. Rebelo: None. I. Gaspar: None. J.C. Rodrigues: None. F. Ramos: None. L. Ramos: None. I. Dinis: None. R.C. Cardoso: None. A. Mirante: None. J. Goncalves: None. S. Gomes: None. J. Silva: None. I. Pereira-Caetano:
None. L. Lopes: None. C. Limbert: None. D. Amaral: 1Departamento Genetica Humana, Instituto Nacional de Saude
DR Ricardo Jorge, Lisbon, Portugal, 2Serviço de
Endocrinologia Pediátrica, Hosp. D. Estefânia, Lisbon,
Portugal, 3Serviço de Genética Médica, Hosp. D. Estefânia,
Lisbon, Portugal, 4Serviço de Endocrinologia Pediátrica,
Departamento de Pediatria, Hospital de Santa Maria, Lisbon,
Portugal, 5Serviço de Genética Médica, Departamento de
Pediatria, Hospital de Santa Maria, Lisbon, Portugal, 6Serviço
de Pediatria, Hospital S. Francisco Xavier, Lisbon, Portugal, F. Ramos: None. L. Ramos: None. I. Dinis: None. R.C. Cardoso: None. A. Mirante: None. J. Goncalves: None. 484 J. del Picchia Lecaignec: None. C. Poirsier: None. E. Landais: None. D. Martin: None. C. Richard: None. A. Tabet: None. S. Redon: None. N. Gruchy: None. F. Vialard: None. The activation of FGFR3 signalling has different
consequences in the transmission of mutations the male
germline 1Research Center for Medical Genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology
(State University), Dolgoprudny, Russian Federation B. Arbeithuber1,2, E. Palzenberger1, R. Reinhard1, T. Ebner1, P. Calabrese3, L. Muresan4, I. Tiemann-Boege1 Introduction:
The
development
of
Next-Generation
Sequencing technology revealed that significant part of
Mendelian disease-associated mutations is located in non-
coding regions and may affect splicing. Because of the
complexity of splicing regulation, it is not possible to pre-
dict accurately the effect of genomic variants on splicing
events and RNA structure. In this work, we focused on
functional analysis of genomic variants affecting splicing in
a variety of Mendelian disorders. 1Johannes Kepler University Linz, Linz, Austria, 2Penn State
University, State College, PA, United States, 3University of
Southern California, Los Angeles, CA, United States,
4University of Cambridge, Cambridge, United Kingdom The majority of new mutations originate in the male
germline. However, to date we lack information on a unique
type of mutagenesis—expansion of driver mutations in the
male germline. These are associated with congenital dis-
orders, occur at thousand-fold higher frequencies than other
mutations, and drastically increase with paternal-age. To
date, the mechanisms propagating these germline driver
mutations are not completely understood. Here we exam-
ined the origin and expansion of four driver mutations in
sperm and a dissected testis differing in mutation rates and
the strength of dysregulation of the mutant FGFR3 receptor:
c.1138G>A,
c.1138G>C
both
causing
achondroplasia
(ACH), c.1948A>G associated with thanatophoric displasia
II (TDII), and c.1948A>C causing hypochondroplasia
(HCH). Only two out of four mutations (c.1138G>A and
c.1948A>G) showed that mutant DNA concentrated within
different clusters of the old donor’s testis, resulting from the
growth advantage of mutant stem cells caused by the acti-
vation of FGFR3. Interestingly, no measurable clustering
was observed for the ACH mutation with the lower muta-
tion frequency (c.1138G>C). We also observed that muta-
tions with a stronger effect on FGFR3 signaling (TDII)
showed a reduced transmission into sperm. In contrast,
mildly activating mutations (HCH) were measured at a very Materials and Methods: To determine the effect of
mutations we used two approaches: (1) RT-PCR from
available patient’s samples and (2) in-vitro minigene assay. For different cases, we performed one or both methods. Results: We analyzed >25 previously uncharacterized
genetic variants in >12 genes, associated with different
Mendelian disorders. These variants are located in both
exons and introns and mostly were classified as variant of
unknown significance (VUS). Functional analysis of sequence variants affecting splicing
in Mendelian disorders Functional analysis of sequence variants affecting splicing
in Mendelian disorders M. Y. Skoblov1,2, A. Marakhonov1,2, Y. Vyakhireva1, P. Sparber1,
M. Freire1, A. Filatova1 P13.15C
Partial trisomy 21 map: ten cases further supporting the
highly restricted Down syndrome critical region (HR-
DSCR) on human chromosome 21 Our analysis forms a basis for understanding this type of
mutagenesis and the associated risks of delayed parenthood
in
our
society. Funded
by:
LIT213201001
and
FWFP25525000 high frequency in sperm of old donors by ultra-sensitive
sequencing. Thus, there are important regulatory mechan-
isms at different developmental stages of spermatogenesis
that affect the downstream transmission of driver mutations. Our analysis forms a basis for understanding this type of
mutagenesis and the associated risks of delayed parenthood
in
our
society. Funded
by:
LIT213201001
and
FWFP25525000 Conclusions: This prospective work further support the
association of the HR-DSCR with the diagnosis of DS,
representing an unbiased validation of the original model. Further studies are needed to identify genetic determinants
presumably located in the HR-DSCR and functionally
associated to the critical manifestations of DS. B. Arbeithuber: None. E. Palzenberger: None. R. Reinhard: None. T. Ebner: None. P. Calabrese: None. L. Muresan: None. I. Tiemann-Boege: None. M.C. Pelleri: None. E. Cicchini: None. M.B. Petersen:
None. L. Tranebjærg: None. T. Mattina: None. P. Magini: None. F. Antonaros: None. M. Caracausi: None. L. Vitale: None. C. Locatelli: None. M. Seri: None. P. Strippoli: None. A. Piovesan: None. G. Cocchi: None. P13.15C
Partial trisomy 21 map: ten cases further supporting the
highly restricted Down syndrome critical region (HR-
DSCR) on human chromosome 21 Cardiac malformations or large ves-
sels (> 33/53 responses), seizures (29/36 responses), brain
abnormalities (30/43 responses), behavioral disorders (15/
16 responses). Children were too young to have all signs. The size of the deletion evaluated in most cases and ranged
from 4100bp to 47Mb. Two groups with a distal or prox-
imal deletion were detected. This study is not exhaustive but
raises the need to create registers for a better evaluation of
the prevalence of microdeletions for better management. Background: Down syndrome (DS) is characterized by the
presence of an extra full or partial human chromosome 21
(Hsa21). An
invaluable
model
to
define
genotype-
phenotype correlations in DS is the study of the extremely
rare cases of partial (segmental) trisomy 21 (PT21). A
systematic retrospective reanalysis of 125 PT21 cases
allowed the identification of a 34-kb highly restricted DS
critical region (HR-DSCR) as the minimal region whose
duplication is shared by all PT21 DS subjects. Material
and
Methods:
We
reanalyzed
at
higher
resolution three cases previously published and we searched
for any new PT21 report in order to verify whether HR-
DSCR limits could prospectively be confirmed and possibly
refined. Material
and
Methods:
We
reanalyzed
at
higher
resolution three cases previously published and we searched
for any new PT21 report in order to verify whether HR-
DSCR limits could prospectively be confirmed and possibly
refined. Results:
Hsa21
partial
duplications
of
three
PT21 subjects were refined through array-CGH and resulted
fully consistent with the previous reports and with the
presence of a duplicated HR-DSCR only in DS subjects and
not in non-DS individuals. Seven additional PT21 cases
have been incorporated into the PT21 map. The PT21 map
now integrates 132 subjects onto a common framework M. Doco-Fenzy: None. C. Jacquin: None. P. Callier:
None. D. Sanlaville: None. C. Missirian: None. P. Kuentz: None. P. Jaeger: None. A. Heddar: None. C. Abstracts from the 51st European Society of Human Genetics Conference: Posters 485 fully consistent with the presence of a duplicated HR-
DSCR, on distal 21q22.13 sub-band, only in DS subjects
and not in non-DS individuals. No documented exception to
the HR-DSCR model was found. high frequency in sperm of old donors by ultra-sensitive
sequencing. Thus, there are important regulatory mechan-
isms at different developmental stages of spermatogenesis
that affect the downstream transmission of driver mutations. P13.16D The activation of FGFR3 signalling has different
consequences in the transmission of mutations the male
germline Interstitial microduplication Xp22.2 in two brothers with
developmental delay and mild facial dysmorphism 1University of Nicosia, Nicosia, Cyprus, 2Aristotle University
of Thessaloniki, Thessaloniki, Greece B. Krabichler1, S. Scholl-Bürgi2, U. Albrecht2, J. Zschocke1, C. Fauth1 Colorectal cancer (CRC) is the third leading cause of death
in the world. Due to its slow development from pre-
malignant lesions, perspectives to reduce the burden of
disease by early detection and treatments are particularly
promising for this disease. Although, a list with the cancer-
related miRNAs has been created, their role in CRC remains
to be elucidated. There appears to be a clear link between
Ribosomal Proteins (RPs) and cancer as deregulation of
RPs was shown to interfere with basic biological processes
such as cell cycle regulation, apoptosis, genome integrity
and tumorogenesis. This attribute requires a tight and
careful regulation of RPs expression which might tend to
cycle through carcinogenesis and cancer progression. We
hypothesize that this cycling is at least partially controlled
by miRNAs. Preliminary in silico analysis demonstrated
that five miRNAs (miR-129-5p, miR-19b-1-5p, miR-193b-
5p, miR-1207-5p, miR-663a) regulate more than 85% of the
human RPs turning those miRNAs into potential crucial
players in RP expression regulation. Interestingly, those
miRNAs appear to also regulate genes that are involved in
proliferation and cancer related pathways. Expression ana-
lysis in a panel of CRC cell lines with increasing aggres-
siveness, demonstrated differential expression of the above
miRNAs. The aim of this project is to explore the con-
nection between RPs and miRNAs in relevance to CRC,
with the goal to expose promising miRNA therapeutic tar-
gets at different stages of carcinogenesis. Furthermore, it is
expected that alternation of miRNA expression in colorectal
tissue through an exosomal-based drug delivery system will
improve disease outcome. 1Division of Human Genetics, Medical University of Innsbruck,
Innsbruck, Austria, 2Clinical Department of Pediatrics I,
Medical University of Innsbruck, Innsbruck, Austria 1Division of Human Genetics, Medical University of Innsbruck,
Innsbruck, Austria, 2Clinical Department of Pediatrics I,
Medical University of Innsbruck, Innsbruck, Austria Interstitial microduplications affecting chromosome band
Xp22.2 are very rare with only a few cases described so far. Reported symptoms include developmental delay, intellec-
tual disability, and minor dysmorphism. While all affected
individuals are males, carrier females usually have a normal
phenotype, which suggests that intellectual disability is
probably due to a dosage effect of one or more duplicated
genes. The activation of FGFR3 signalling has different
consequences in the transmission of mutations the male
germline We determined the effect of
these variants on mRNA structure; it allowed us to classify
most of them as pathogenic and to make assumption of the
mechanisms involved in the molecular pathogenesis of
diseases (e.g. RNA degradation by NMD, disruption of
functional domain of protein). Additionally, we compared
our experimental data with prediction tools for splicing
events and revealed that it is not always possible to predict
accurately the effect of mutation on splicing. Conclusions: Although it is now known that mutations
affecting splicing can cause the Mendelian diseases,
however their contribution may be underrepresented due
to limitation of diagnostic procedures. To prove the 486 J. del Picchia pathogenicity of these mutations, additional functional
analysis is often required. pathogenicity of these mutations, additional functional
analysis is often required. Interstitial microduplication Xp22.2 in two brothers with
developmental delay and mild facial dysmorphism However, due to the rarity of Xp22.2 micro-
duplications, their heterogeneous size and variable locali-
zation the contribution of single genes to the clinical
phenotype is difficult to evaluate. Here we report on male siblings with mild to moderate
global developmental delay, minor dysmorphism and
measurements within normal ranges. Family history is
unremarkable, both parents and the older sister are healthy. Molecular karyotyping revealed an interstitial microdupli-
cation Xp22.2-Xp22.31 of 5 megabases in both affected
brothers (arr[hg18] Xp22.31p22.2(9301848_14415165)x2). The duplication, which was probably inherited from the
healthy
mother
contains
36
OMIM-annotated
genes,
including CLCN4, MID1, HCCS, ARGHAP6, FRMPD4,
and OFD1. Hemizygous loss of function mutations of
CLCN4 (encoding the chloride/hydrogen ion exchanger
ClC-4) and FRMPD4 (encoding a neural scaffolding
protein)
cause
X-linked
intellectual
disability
type
MRX15 and MRX104, respectively. Both genes are highly
expressed in brain and undergo X inactivation in females. It
is tempting to speculate that increased expression of genes
like CLCN4 and FRMPD4, which are subject to dosage
compensation, may contribute to intellectual disability in
males with X chromosomal duplications. Analysis of
further patients with overlapping Xp microduplications will
be required to elucidate the contribution of these genes to
the clinical phenotype. C. Savva: None. D. Avramopoulou: None. Z. Ellinas:
None. D. Fatouros: None. I. Vizirianakis: None. K.N. Felekkis: None. P13.20D M.Y. Skoblov: None. A. Marakhonov: None. Y. Vyakhireva: None. P. Sparber: None. M. Freire: None. A. Filatova: None. L. Ferrari1, E. Mangano2, M. Bonati3, I. Monterosso1, I.
Brambilla4, F. Chiapparoni2, C. Battaglia1, R. Bordoni2, P. Riva1 P13.21A Genetic heterogeneity & di/oligogenic inheritance
involvement in variable expressivity of Noonan syndrome L. Ferrari1, E. Mangano2, M. Bonati3, I. Monterosso1, I. Brambilla4, F. Chiapparoni2, C. Battaglia1, R. Bordoni2, P. Riva1 B. Krabichler:
None. S. Scholl-Bürgi:
None. U. Albrecht: None. J. Zschocke: None. C. Fauth: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 487 1Department of Medical Biotechnology and Translational
Medicine University of Milan, Milan, Italy, 2Institute of
Biomedical Technologies, National Research Council, Milan,
Italy, Milan, Italy, 3Istituto Auxologico Italiano, Milan, Italy,
4St. Matteo Hospital, Pavia, Italy Clinical and Experimental Biomedical Sciences 'Mario Serio',
University of Florence, Florence, Italy, 3Department of
Science’s Health, Anna Meyer Children’s University Hospital,
Florence, Italy Obesity, with its complications, emerges as a major con-
tributor to the global health burden assuming the status of a
pandemic. It’s extremely complex disorder resulting of
interaction of biological, social and behavioural factors that
cause increase in food intake and reduction in energy
expenditure. Although rare monogenic forms, several genes
and regions of susceptibility have been described, the
genetic causes underlying remain largely unknown, despite
the role of genetic background is indisputable. GWAS
revealed consistent association between SNPs with BMI
and fat-mass, but cannot demonstrate the undoubted caus-
ality, and elucidating the culprit events continues to be
challenging, especially when it’s not known the way in
which variants primarily act. To expand our knowledge, we
performed WES in 30 strictly clinical classified Caucasian
probands, with severe early-onset obesity. We screened a
set of 80 genes responsible/susceptibility for syndromic/
monogenic forms, including pathways of obesity develop-
ment. We identified potentially pathogenic variants in 75%. 5 cases presented a single variant in genes related to
increase of BMI/WHR, 3 patients had variants in hypo-
thalamic leptin-melacortin pathway, whereas remaining
cases showing complex genetic background with substitu-
tions in 2/more genes. Interesting, in light of the genetic
background we planned personalized treatment in patient
with family history of diabetes, hepatic steatosis and severe
obesity who presented pathogenic variant in SH2B1, leaded
significant weight loss. P. Kuentz1,2,3, B. Keren4, D. Sanlaville5,6, A. Masurel7, A.
Mosca8,2,3, N. Marle8,2,3, M. Payet8, C. Ragon8, M. Pouleau8, C.
Thauvin-Robinet7,2,3, L. Faivre7,2,3, C. Schluth-Bolard5,6, P.
Callier8,2,3 P13.22B
Genomic knowledge as the powerful tool to understand the
obesity R. Artuso1, V. Palazzo1, L. Giunti1, S. Landini2, A. Provenzano2,
A. La Barbera2, S. Guarducci1, M. Pantaleo1, B. Lucherini1, I. Sani1, S. Bargiacchi1, P. Reho2, E. Bosi2, F. Peluso2, A. Pagliazzi2, L. Dosa1, G. Traficante1, M. Della Monica1, S. Stagi3,
S. Giglio1,2 1Medical Genetic Unit, Meyer's Children University Hospital,
Florence, Italy, 2Medical Genetics Unit, Department of P13.21A The systematic discovery of rare
variants in complex diseases suggests that the reverse-
strategy is fruitful for assigning pathogenic effects of sev-
eral genes simultaneously: the genotype-first approach will
be able to identify clinically recognizable phenotypes Noonan syndrome (NS) is characterized by an autosomal
dominant inheritance, variable expressivity and genetic
heterogeneity, given the high number of variants in
PTPN11, SOS1 RAF1, NRAS, KRAS, BRAF, MEK1 and
SHOC2 genes. Despite the increasing application of NGS in
30% of patients pathogenetic mutations remain unknown. Further
NS
genes
and
pathogenetic
mechanisms
are
expected. We search for new NS genes by a targeted NGS
analysis using a panel of 26 RAS pathway genes, in nine
NS patients negative after genetic screenings. New poten-
tially pathogenetic variants were detected in NS genes: c. T355C (p.Y119H) in LZTR1 and c.A2882G (p.D961G) in
A2ML1 genes. We also observed in the same patient
mutations in A2ML1 (p.K110T) and SOS2 (p.Q742X) and
identified two new NS candidate genes in three patients. All
of them have healthy parents and inherited a missense
mutation in a new NS gene from one parent and the second
one in a known NS gene from the other parent. The co-
presence of both mutated genes probably contributes to the
NS phenotype. Functional studies on lymphoblastoid cells
from the three trios are ongoing. We propose an additive
effect of subclinical mutations leading to the RAS pathway
activation, causing NS. According to this hypothesis NS
could be classified as mono/di/oligenic disease, a model that
could explain, with genetic heterogeneity, the variable
expressivity. Subclinical expression of specific variants,
escaping unfavorable selective pressure, show high allelic
frequency and therefore could be co-inherited. This model
could also explain the missing of pathogenetic mutation in
30% of NS patients. L. Ferrari: None. E. Mangano: None. M. Bonati:
None. I. Monterosso: None. I. Brambilla: None. F. Chiapparoni: None. C. Battaglia: None. R. Bordoni:
None. P. Riva: None. R. Artuso: None. V. Palazzo: None. L. Giunti: None. S. Landini: None. A. Provenzano: None. A. La Barbera:
None. S. Guarducci: None. M. Pantaleo: None. B. Lucherini: None. I. Sani: None. S. Bargiacchi: None. P. Reho: None. E. Bosi: None. F. Peluso: None. A. Pagliazzi: None. L. Dosa: None. G. Traficante: None. M. Della Monica: None. S. Stagi: None. S. Giglio: None. P13.23C Atypical recombinant chromosomes arising from parental
paracentric inversions: report of three patients Atypical recombinant chromosomes arising from parental
paracentric inversions: report of three patients Callier8,2,3 488 J. del Picchia question and may guide future recommendations for genetic
counseling. 1Génétique Biologique, PCBio, Centre Hospitalier
Universitaire de Besançon, Besançon, France, 2Fédération
Hospitalo-Universitaire Médecine Translationnelle et
Anomalies du Développement (FHU TRANSLAD), Centre
Hospitalier Universitaire de Dijon et Université de Bourgogne
Franche-Comté, Dijon, France, 3UMR-Inserm 1231 GAD,
Génétique des Anomalies du développement, Université de
Bourgogne Franche-Comté, Dijon, France, 4Département de
génétique, APHP, UPMC Inserm, UMR7225, ICM, GH Pitié-
Salpêtrière, Paris, France, 5Service de Génétique, Laboratoire
de Cytogénétique Constitutionnelle, Groupement Hospitalier
Est, Hospices Civils de Lyon, Bron, France, 6Lyon
Neuroscience Research Center, GENDEV Team, INSERM
U1028; CNRS UMR5292; UCBL1, Bron, France, 7Centre de
Génétique et Centre de reference "Anomalies du
Développement et Syndromes Malformatifs", Hôpital
d’Enfants, Centre Hospitalier Universitaire de Dijon, Dijon,
France, 8Laboratoire de Génétique chromosomique et
moléculaire, Plateau Technique de Biologie, Centre
Hospitalier Universitaire de Dijon, Dijon, France question and may guide future recommendations for genetic
counseling. P. Kuentz: None. B. Keren: None. D. Sanlaville: None. A. Masurel: None. A. Mosca: None. N. Marle: None. M. Payet: None. C. Ragon: None. M. Pouleau: None. C. Thauvin-Robinet: None. L. Faivre: None. C. Schluth-
Bolard: None. P. Callier: None. P13.24D A complicated WES diagnosis: hereditary spherocytosis
due to autosomal recessively inherited mutation in EPB42
associated with mosaic genome-wide paternal uniparental
isodisomy A. Walczak1, A. Biernacka1,2, P. Gasperowicz1, A. Koppolu1,2, J. Kosińska1, G. Kostrzewa3, V. Murcia-Pieńkowski1,2, M. Rydzanicz1, M. Biela4, J. Kozłowska5, M. Sąsiadek5, R. Śmigiel4,
R. Płoski1 1Department of Medical Genetics, Warsaw Medical University,
Warsaw, Poland, 2Postgraduate School of Molecular
Medicine, Warsaw Medical University, Warsaw, Poland,
3Department of Forensic Medicine, Medical University of
Warsaw, Warsaw, Poland, 4Department of Paediatrics and
Rare Disorders, Wroclaw Medical University, Wroclaw,
Poland, 5Department of Genetics, Wroclaw Medical
University, Wroclaw, Poland Introduction: Paracentric inversions can generate aneu-
somic gametes with duplication-deficiency distal to the
inversion breakpoints and the formation of unstable dicen-
tric and acentric derivatives leading to low embryonic via-
bility. We
report
atypical
recombinant
monocentric
chromosomes arising from parental paracentric inversions
in three patients with syndromic intellectual disability. Introduction: Genome-wide uniparental disomy is very
rare phenomenon. In over 10 patients described so far the
manifestations were mostly due to known imprinting dis-
orders. We report a case of a child without imprinting
defects features with mosaic paternal genome-wide uni-
parental isodisomy (GWUPiD) and symptomatic autosomal
recessively inherited mutation in EPB42. Materials
and
Methods
-
Results:
Chromosomal
microarray showed similar rearrangements characterized
by interstitial duplications-deficiencies proximal to the
breakpoints of a parental paracentric inversion. The study
of genotypes in the three patients using SNP microarray
showed that: (i) the parental origins of the duplications and
the deletions were from the parents with the paracentric
inversions; (ii) there were three haplotypes in the duplica-
tions showing that the gain of copy came from both
homologous chromosomes of the parent with the inversion;
(iii) for each patient, there was a copy neutral region
between the deletion and the duplication, flanked by low
copy repeats causing reported cryptic inversions. We thus
hypothesized the presence of an additional cryptic para-
centric inversion in the same parent, either on the
chromosome bearing the initial paracentric inversion, or
on the homologous chromosome. In both cases, the
speculated mechanism may involve a crossingover within
the small loop of a double inversion loop. Materials and Methods: 3y old girl was referred for
whole exome sequencing (WES) due to chronic anemia,
chronic liver failure, cysts of the bile ducts treated by Kasai
procedure. After delivery, she developed jaundice, anemia
and hepatopathy. Many metabolic disorders were excluded. P13.24D Grants: FINEP-CT INFRA
0160/12 SP8, FAPESP 2016/09452-0 Conclusions: To the extent of our knowledge this is the
first case of a recessive disease occurring in the mechanism
of GWUPiD diagnosed by WES. Conclusions: To the extent of our knowledge this is the
first case of a recessive disease occurring in the mechanism
of GWUPiD diagnosed by WES. Support: National Science Centre, Poland 2017/25/N/
NZ4/00250, Wroclaw Medical University ST- E160.17.055 A. Walczak: None. A. Biernacka: None. P. Gasper-
owicz: None. A. Koppolu: None. J. Kosińska: None. G. Kostrzewa: None. V. Murcia-Pieńkowski: None. M. Rydzanicz: None. M. Biela: None. J. Kozłowska: None. M. Sąsiadek: None. R. Śmigiel: None. R. Płoski: None. Repetitive elements associated with breakpoints of distal 5p
deletions suggest mechanisms mediating these
rearrangements Repetitive elements associated with breakpoints of distal 5p
deletions suggest mechanisms mediating these
rearrangements S.N. Chehimi: None. E.A. Zanardo: None. F.A.R. Madia: None. A.T. Dias: None. G.M. Novo-Filho: None. M.M. Montenegro: None. A.M. Nascimento: None. J.G. Damasceno: None. Y.G. Oliveira: None. L.L. Vieira:
None. C.A. Kim: None. L.D. Kulikowski: None. S. N. Chehimi1, E. A. Zanardo2, F. A. R. Madia1, A. T. Dias2, G. M. Novo-Filho2, M. M. Montenegro2, A. M. Nascimento1, J. G. Damasceno2, Y. G. Oliveira2, L. L. Vieira2, C. A. Kim3, L. D. Kulikowski2 P13.24D WES (DNA from blood, repeated 3 times, also from
independently collected blood sample) was performed on
HiSeq1500. To identify GWUPiD a panel of forensic STRs
(short tandem repeats) was analyzed. Materials and Methods: 3y old girl was referred for
whole exome sequencing (WES) due to chronic anemia,
chronic liver failure, cysts of the bile ducts treated by Kasai
procedure. After delivery, she developed jaundice, anemia
and hepatopathy. Many metabolic disorders were excluded. WES (DNA from blood, repeated 3 times, also from
independently collected blood sample) was performed on
HiSeq1500. To identify GWUPiD a panel of forensic STRs
(short tandem repeats) was analyzed. Results: Bioinformatics analysis of WES revealed lower
than average number of variants. We also observed
“pseudo” homozygous variants with low percentage of
reference
allele
(~15%),
in
particular
we
found
p. (Arg310Gln)/c.929G>A in EPB42 (associated with spher-
ocytosis) at 85% level mosaic (confirmed by amplicon deep
sequencing). The variant was observed in father’s sample in
heterozygous state. STR analysis confirmed GWUPiD in
patient’s blood with biparental inheritance (BPI) presented Conclusions: It is currently thought that heterozygotes
for paracentric inversions have a risk of abnormal offspring
comparable to that of the general population, and that
prenatal diagnosis should not be offered systematically. The
observations of this study have called this dogma into Abstracts from the 51st European Society of Human Genetics Conference: Posters 489 at low level. DNA extracted from buccal cells, nails, hair
follicles and urine sediment showed normal BPI. at low level. DNA extracted from buccal cells, nails, hair
follicles and urine sediment showed normal BPI. Conclusions: The most common type of SINEs are Alu
elements that are associated with NAHR and breakpoints
with large LCRs (>10 kb) with high sequence homology
also promote NAHR. Furthermore, we can assume that
other mechanisms may be involved in the stabilization of
DNA double-strand breaks in these cases, as NHEJ, that
result in non-recurrent rearrangements leading to variation
in deletions sizes and breakpoints location. The breakpoints
investigated were in regions with repetitive elements, with
the exception of two samples, suggesting an important role
in mediating terminal rearrangements, previously seen in
other terminal deletions but not reported in 5p distal
deletions. The elucidation of the breakpoints can suggest
mechanisms underlying structural variants involved in
terminal deletions syndromes. Functional retrogene at the RB1 locus C. Dehainault1, M. Boutte1, L. Golmard1, A. Fievet1, H. C. Dehainault1, M. Boutte1, L. Golmard1, A. Fievet1, H. Pacquement1, L. Lumbroso Le Rouic1, M. Gauthier Villars1, C. Houdayer1,2 C. Dehainault1, M. Boutte1, L. Golmard1, A. Fievet1, H. Pacquement1, L. Lumbroso Le Rouic1, M. Gauthier Villars1, C. Houdayer1,2 Pacquement1, L. Lumbroso Le Rouic1, M. Gauthier Villars1, C. Houdayer1,2 P13.26B
Functional retrogene at the RB1 locus 1Laboratorio de Citogenomica, Departamento de Pediatria,
Faculdade de Medicina FMUSP, Universidade de Sao Paulo,
Sao Paulo, Brazil, 2Laboratorio de Citogenomica,
Departamento de Patologia, Faculdade de Medicina FMUSP,
Universidade de Sao Paulo, Sao Paulo, Brazil, 3Unidade de
Genetica, Departamento de Pediatria, Instituto da Crianca,
Hospital das Clinicas HCFMUSP, Faculdade de Medicina,
Universidade de Sao Paulo, Sao Paulo, Brazil 1Laboratorio de Citogenomica, Departamento de Pediatria,
Faculdade de Medicina FMUSP, Universidade de Sao Paulo,
Sao Paulo, Brazil, 2Laboratorio de Citogenomica,
Departamento de Patologia, Faculdade de Medicina FMUSP,
Universidade de Sao Paulo, Sao Paulo, Brazil, 3Unidade de
Genetica, Departamento de Pediatria, Instituto da Crianca,
Hospital das Clinicas HCFMUSP, Faculdade de Medicina,
Universidade de Sao Paulo, Sao Paulo, Brazil P13.27C C. Gervasini1, G. Negri1, P. Magini2, D. Milani3, M. C. Gandini1,
E. Di Fede1, M. Crippa4, E. Biamino5, G. B. Ferrero5, M. Piccione6, S. Sotgiu7, C. Perria7, G. Vitiello8, M. Frontali9, E. A. Colombo1, A. Boni10, M. L. Cavaliere11, M. A. Pisanti11, L. Giordano12, M. J. Bamshad13, D. A. Nickerson13, J. D. Smith13,
I. Loddo14, P. Finelli4,15, L. Larizza4, T. Pippucci2 Clinical and molecular characterization of an almost
complete ring chromosome 4 in two sisters, with recurrence
due to gonadal mosaicism E. Phillips1, O. Caluseriu2, K. Schlade-Bartusiak3, J. Chernos1,
R. McLeod1, M. Thomas1 1Genetica Medica, Dipartimento di Scienze della Salute,
Università degli Studi di Milano, Milano, Italy, Milano, Italy,
2U.O. Genetica Medica, Policlinico S. Orsola-Malpighi,
Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy,
Bologna, Italy, 3Unità di Pediatria ad alta Intensità di Cura,
Fondazione IRCCS Ca' Granda, Milano, Italy, Milano, Italy,
4Medical Cytogenetics and Molecular Genetics Lab, IRCCS
Istituto Auxologico Italiano, Milan, Italy, Milano, Italy,
5Dipartimento di Pediatria, Università di Torino, Torino, Italy,
Torino, Italy, 6Dipartimento Materno Infantile, Azienda
Ospedali Riuniti Villa Sofia Cervello, Università di Palermo,
Italy, Palermo, Italy, 7Dipartimento di Medicina Clinica e
Sperimentale. U.O.C. Neuropsichiatria Infantile, A.O.U. di
Sassari, Sassari, Italy, Sassari, Italy, 8Dipartimento di
Medicina Traslazionale, Sezione di Pediatria, Università
Federico II, Napoli, Italy, Napoli, Italy, 9Istituto di
Farmacologia Traslazionale CNR, Roma, Italy, Roma, Italy,
10Unità di Neurologia Infantile, IRCCS Istituto delle Scienze
Neurologiche, Bologna, Italy, Bologna, Italy, 11Service of
Medical Genetics, Ospedale Cardarelli, Napoli, Italy, Napoli,
Italy, 12Neuropsychiatric Department, Spedali Civili Brescia,
Brescia, Italy, Brescia, Italy, 13Department of Genome
Sciences, University of Washington, Seattle, Washington, USA,
Seattle, WA, United States, 14Dipartimento di Medicina di
Laboratorio e Biotecnologie Avanzate, IRCCS ISMETT,
Palermo, Italy, Palermo, Italy, 15Department of Medical
Biotechnology and Translational Medicine, Università degli
Studi di Milano, Milan, Italy, Milano, Italy 1Cumming School of Medicine, Calgary, AB, Canada,
2University of Alberta, Edmonton, AB, Canada, 3University of
British Columbia, Vancouver, BC, Canada Introduction:
Autosomal
ring
chromosomes
are
rare
cytogenetic findings that arise from breakage and fusion of
the chromosome ends. Rings are mitotically unstable,
usually sporadic, and associated with a “ring chromosome
syndrome”, characterized by a variable phenotype of
growth retardation, few or no minor anomalies, and intel-
ligence ranging from normal to moderate intellectual dis-
ability. We describe the clinical features and molecular
characterization of two sisters with ring chromosome 4. Materials
and
Methods:
Karyotype
analysis
was
performed on both sisters and parents. 1Institut Curie, Paris, France, 2Université Paris Descartes,
Sorbonne Paris Cité, Paris, France Beyond the identification of the hidden causal mutation, we
describe here a very rare phenomenon as functional retro-
genes have an estimated frequency of one retrogene per
million year [Marques et al. PLoS Biology 2005]. E. Phillips: None. O. Caluseriu: None. K. Schlade-
Bartusiak: None. J. Chernos: None. R. McLeod: None. M. Thomas: None. 1Institut Curie, Paris, France, 2Université Paris Descartes,
Sorbonne Paris Cité, Paris, France 1Institut Curie, Paris, France, 2Université Paris Descartes,
Sorbonne Paris Cité, Paris, France Retrotransposons are a major class of mobile elements
accounting for 45% of our genome. The DNA of a retro-
transposon is transcribed into RNA then reverse-transcribed
into a cDNA copy which is reinserted into the genome at a
new location. Retrotranspositions are frequent and usually
lead to inactive elements due to the lack of promoter. However,
we
report
a
rare
phenomenon
of
retro-
transposition leading to a functional retrogene, segregating
in a retinoblastoma family. A father and his son were
affected by retinoblastoma, a malignant tumor of the eye
due to RB1 mutation. Given the absence of RB1 mutation by
DNA sequencing, we embarked upon RNA analyses
demonstrating an abnormal RB1 transcript including 2
exons of the HPF1 gene. Oddly enough, HPF1 is located on
another chromosome. Following genomic analyses showed
the insertion of HPF1 full cDNA into the large intron 17 of
RB1. This insertion was present in the two affected patients
and not in unaffected individuals from the family. Hence the
retrotransposition of the HPF1 gene into RB1 leads to a
chimeric fusion transcript RB1-HPF1-RB1 by using new
splice sites. The phenomenon keeps the frame but disrupts a Introduction: Cytogenomic techniques, such as single
nucleotide polymorphism (SNP) microarrays, allow the
detection of copy number variants and structural char-
acterization of breakpoint sites in several genomic diseases. Mechanisms have been proposed to explain genomic rear-
rangements, including nonallelic homologous recombina-
tion
(NAHR),
nonhomologous
end
joining
(NHEJ),
replicative mechanisms and long interspersed element
(LINE)-mediated retrotransposition. Material and Methods: We used Illumina Infinium
CytoSNP-850K and UCSC Genome Browser (GRCh37/
hg19) in order to map the breakpoints in 14 patients with 5p
distal deletion. Results: The results revealed breakpoint patterns on
chromosome
5
ranging
position
chr5:
17,235,998-
34,402,152. The structural characterization of breakpoints
reveals that 10 of the 14 cases presented predominantly
LINEs than SINEs. We only detected one patient with large
low copy repeats (LCRs >10 kb), having 98 - 99%
similarity. 490 J. del Picchia instance of a ring 4 chromosome recurring in siblings after
extensive testing on parents, which suggests this was due to
maternal gonadal mosaicism. major functional domain of pRb and is likely to cause
retinoblastoma. As the grand parents are unaffected, it
suggests a recent retransposition e.g. in their gametes. P13.28D C. Dehainault: None. M. Boutte: None. L. Golmard:
None. A. Fievet: None. H. Pacquement: None. L. Lumbroso Le Rouic: None. M. Gauthier Villars: None. C. Houdayer: None. Exploring by whole exome sequencing patients with initial
diagnosis of Rubinstein-Taybi syndrome: the
interconnections of epigenetic machinery disorders P13.27C Chromosome
microarray was performed on both sisters to delineate the
imbalance at the breakpoints. Clinical correlation including
physical
examination
and
formal
neurodevelopmental
assessments are compared for both sisters, as one sister
received growth hormone therapy. Results: Both sisters had a large ring 4 chromosome in
the majority of cells analyzed on karyotype (97% and 83%
respectively). Microarray results were identical in the
sisters, showing a 55.8 kb duplication on the terminal 4p
arm and a 1.5 Mb deletion on the terminal 4q arm. No genes
of interest were identified in these regions. Parental
karyotypes on lymphocytes and fibroblasts were normal,
with no finding of mosaicism for the ring 4 chromosome. Polymorphic marker analysis revealed maternal origin of
the ring. Background: Rubinstein-Taybi syndrome (RSTS) is an
autosomal dominant neurodevelopmental disease affecting
1:125,000 newborns characterized by intellectual disability,
growth
retardation,
facial
dysmorphisms
and
skeletal
abnormalities. RSTS is caused by mutations in genes Conclusions: We describe the clinical features and
molecular imbalances of two teenage girls with almost
complete ring 4. To our knowledge, this is the first reported Abstracts from the 51st European Society of Human Genetics Conference: Posters 491 Italy, 4Neuroscience Institute Cavalieri Ottolenghi, Orbassano,
Italy, 5Institute of Protein Biochemistry, National Research
Council, Napoli, Italy, 6Centre for Integrative Biology,
University of Trento, Trento, Italy, 7University of Turin,
Department of Public Health and Pediatrics, Turin, Italy,
8Department of Pharmacological and Biomolecular Sciences,
University of Milan, Milano, Italy, 9Department of Medical,
Oral and Biotechnological Sciences, University G. D'Annunzio, Chieti, Italy encoding for writers of the epigenetic machinery: CREBBP
(~60%) or its homologous EP300 (~10%). However, no
causative mutation is identified in up to 30% of patients. Methods: To identify novel candidate genes for RSTS,
we performed whole-exome sequencing (WES) on eight
individuals with a diagnosis of RSTS who had normal high-
resolution array CGH testing and were CREBBP- and
EP300- mutation-negative. Methods: To identify novel candidate genes for RSTS,
we performed whole-exome sequencing (WES) on eight
individuals with a diagnosis of RSTS who had normal high-
resolution array CGH testing and were CREBBP- and
EP300- mutation-negative. Results: In four families, we identified putatively causal
variants in three genes (ASXL1, KMT2D and KMT2A)
encoding members of the epigenetic machinery known to be
associated
with
the
Bohring-Opitz,
Kabuki
and
Wiedemann-Steiner syndromes. Each variant is novel, arose
de novo and was predicted to result in loss-of-function. P13.27C Grants: This work has been supported by Dotazione
d’Ateneo-Linea 2 to CG Grants: This work has been supported by Dotazione
d’Ateneo-Linea 2 to CG C. Gervasini: None. G. Negri: None. P. Magini: None. D. Milani: None. M.C. Gandini: None. E. Di Fede: None. M. Crippa: None. E. Biamino: None. G.B. Ferrero:
None. M. Piccione: None. S. Sotgiu: None. C. Perria:
None. G. Vitiello: None. M. Frontali: None. E.A. Colombo: None. A. Boni: None. M.L. Cavaliere: None. M.A. Pisanti: None. L. Giordano: None. M.J. Bamshad:
None. D.A. Nickerson: None. J.D. Smith: None. I. Loddo: None. P. Finelli: None. L. Larizza: None. T. Pippucci: None. P13.29A
ER stress as pathogenic mechanism in Spinocerebellar
Ataxia 38 (SCA38) P13.29A
ER stress as pathogenic mechanism in Spinocerebellar
Ataxia 38 (SCA38) E. Di Gregorio: None. M. Ferrero: None. M. Manes:
None. E. Hoxha: None. C. Costanzi: None. A. Di Campli:
None. D. Tripathy: None. S. Cavalieri: None. E. Giorgio:
None. C. Mancini: None. E. Pozzi: None. E. Riberi:
None. E. Chierto: None. N. Mitro: None. D. Caruso:
None. M. Basso: None. M. Sallese: None. F. Tempia:
None. B. Borroni: None. A. Brusco: None. E. Di Gregorio: None. M. Ferrero: None. M. Manes:
None. E. Hoxha: None. C. Costanzi: None. A. Di Campli:
None. D. Tripathy: None. S. Cavalieri: None. E. Giorgio:
None. C. Mancini: None. E. Pozzi: None. E. Riberi:
None. E. Chierto: None. N. Mitro: None. D. Caruso:
None. M. Basso: None. M. Sallese: None. F. Tempia:
None. B. Borroni: None. A. Brusco: None. E. Di Gregorio1, M. Ferrero2, M. Manes3, E. Hoxha4, C. Costanzi3, A. Di Campli5, D. Tripathy6, S. Cavalieri2, E. Giorgio2, C. Mancini2, E. Pozzi2, E. Riberi7, E. Chierto2, N. Mitro8, D. Caruso8, M. Basso6, M. Sallese9, F. Tempia4, B. Borroni3, A. Brusco1 E. Di Gregorio1, M. Ferrero2, M. Manes3, E. Hoxha4, C. Costanzi3, A. Di Campli5, D. Tripathy6, S. Cavalieri2, E. Giorgio2, C. Mancini2, E. Pozzi2, E. Riberi7, E. Chierto2, N. Mitro8, D. Caruso8, M. Basso6, M. Sallese9, F. Tempia4, B. Borroni3, A. Brusco1 1S.C.D.U. Medical Genetics, Città della Salute e della Scienza,
Turin, Italy, 2Department of Medical Sciences, University of
Turin, Turin, Italy, 3Neurology Unit, Department of Clinical
and Experimental Sciences, University of Brescia, Brescia, P13.27C In
the remaining patients additional candidate variants in
XRN2 or in PLXNB2, not yet related to any human disease,
and in XYLT2 or PLCB4
associated
respectively to
spondyloocular and auriculocondylar type 2 syndromes
are identified. ELOVL5 gene is associated with autosomal dominant Spi-
nocerebellar Ataxia 38 (SCA38, MIM#611805), a rare
adult-onset
cerebellar
neurodegeneration. This
gene
encodes for an elongase, an enzyme localized in the endo-
plasmic reticulum (ER) where it is involved in the synthesis
of a subset of polyunsaturated fatty acids. We explored
pathogenic
mechanism
of
SCA38,
studying
aberrant
ELOVL5-p.Gly230Val protein. We demonstrated a subcellular mislocalization in peri-
nuclear area of aberrant protein in different cellular models. We demonstrated a subcellular mislocalization in peri-
nuclear area of aberrant protein in different cellular models. Based on these, we hypothesized p.Gly230Val-ELOVL5 is
a misfolded protein able to activate the cellular unfolded
protein response (UPR). Supporting that idea, we showed a
significant increase of ELOVL5 protein in SCA38 fibroblasts
after a treatment with the proteasome inhibitor MG-132. In
COS7 cells stably expressing p.Gly230Val ELOVL5 we
demonstrated the activation of ER-stress response by a
significant increase of UPR markers CHOP, ATF-4 and
XBP1 and an alteration of ER homeostasis by a slow-down
protein transport from ER to the Golgi. Moreover, the use of
the
chemical
chaperone PBA,
acting on unfolded p. Gly230Val-ELOVL5 in COS7 cells, led to a physiological
ER relocalization of the protein. To determine whether the
activation of UPR was associated with neuronal degeneration,
we measured cell viability of primary cortical neurons
overexpressing wild type or aberrant ELOVL5. Preliminary
data suggested a slightly increased of cells death in p. Gly230Val ELOVL5 neurons. In conclusion, our results
support a role for altered ER-stress response in SCA38
pathogenesis, suggesting chemical chaperones might be
useful in the treatment. Telethon Grant: GGP14225 Conclusions: These results underscore the broad clinical
spectrum of RSTS and other Mendelian disorders of the
epigenetic apparatus. The overlapping features of distinct
intellectual disability syndromes herein underlined reflect
common pathogenic molecular mechanisms affecting the
complex regulation of balance between open and closed
chromatin. Conclusions: These results underscore the broad clinical
spectrum of RSTS and other Mendelian disorders of the
epigenetic apparatus. The overlapping features of distinct
intellectual disability syndromes herein underlined reflect
common pathogenic molecular mechanisms affecting the
complex regulation of balance between open and closed
chromatin. Bionano Genomics, San Diego, CA, United States Current methods for detection of balanced structural varia-
tion can be broken down into two categories: traditional
cytogenetics and molecular methods. Cytogenetics may
include chromosomal karyotyping, fluorescence in situ
hybridization (FISH), chromosomal microarray and adap-
tions of them. Molecular methods primarily include NGS
sequencing based methods. Bionano genome mapping, an
optical mapping approach, is a method that combines the
advantages of different categories while solving many of the
limitations. Compared to cytogenetics, Bionano mapping is
high throughput and removes manual interpretation, it also
has much higher resolution, detecting balanced events as
small as about 30 kbp compared to multi-megabases needed
for cytogenetic approaches, and unbalanced events starting
at 500 bp. NGS based methods often are limited by read
lengths that cannot provide unambiguous information
across repeat elements longer than individual reads. This
limitation results in significantly reduced sensitivity for
balanced variation and abnormalities as well as for inser-
tions and even some categories of larger deletions. This is
particularly true in highly medically relevant locations
where
segmental
duplications
mediate
chromosomal
abnormalities. Heterozygous beta-thalassemia individuals, inheriting a
single defective allele, are usually asymptomatic, but in
extremely
rare
cases
a
transfusion
dependent
beta-
thalassemia intermedia develops later in life. We report a
rare case of late-onset thalassemia intermedia caused by
inheritance of 1 thalassemic HBB variant along with an
acquired somatic deletion of the normal trans HBB locus in
peripheral blood cells, resulting in hemizygosity for the
beta-thalassemia mutation in erythrocytes. Direct Sanger
sequencing characterized the beta-thalassemia mutation in
HBB (HBB:c.315+1G>A) in DNA isolated from leuco-
cytes, buccal cells and hair. Leucocyte DNA was analysed
for genomic copy number variations (CNVs) using the
Affymetrix CytoScan HD Array with Chromosome Ana-
lysis Suite (ChAS, version 3.0) and software, according to
the manufacturer’s instructions (Thermo Fisher Scientific,
Santa Clara, CA, USA). Sanger sequencing of leucocyte
DNA gave a skewed ratio of normal (G) versus variant (A),
although DNA from buccal cells and hair showed classic
heterozygosity. Array analysis showed a 4.97 Mb deletion
(hg19/GRCh37: 1,313,791-6,287,277) on the short arm of
chromosome 11 of maternal origin, which included the
patient’s normal beta-globin gene cluster, causing hemi-
zygosity of the paternally inherited beta-thalassemia muta-
tion, explaining the predominant hemizygosity for the beta-
thalassemia mutation and her evolving clinical phenotype. P13.30B Adult-onset beta-thalassemia intermedia caused by a 5 Mb
somatic clonal segmental deletion in hemopoietic stem cells
involving the HBB locus Adult-onset beta-thalassemia intermedia caused by a 5 Mb
somatic clonal segmental deletion in hemopoietic stem cells
involving the HBB locus 492 J. del Picchia A.L. Ruivenkamp: None. E. Kanavakis: None. C. Kattamis: None. J. Traeger-Synodinos: None. C. L. Harteveld1, C. A. J. Bosch2, C. Vrettou3, L. Maragoudaki3,
J. Apostilidis4, S. G. J. Arkesteijn1, M. J. V. Hoffer2, C. A. L. Ruivenkamp5, E. Kanavakis6, C. Kattamis7, J. Traeger-
Synodinos3 A. Hastie: None. A. Pang: None. J. Lee: None. E.T.
Lam: None. X. Zhang: None. T. Anantharaman: None.
S. Marin: None. H. Sadowski: None. M. Borodkin: None.
H. Cao: None. Chromosome arm scale de novo genome assemblies better
detect and resolve structural variation and chromosomal
abnormalities related to and causing genetic disease Chromosome arm scale de novo genome assemblies better
detect and resolve structural variation and chromosomal
abnormalities related to and causing genetic disease 1Hemoglobinopathy Expert Centre, Leiden University Medical
Centre, Leiden, Netherlands, 2Dpt of Clinical Genetics, Leiden
University Medical Centre, Leiden, Netherlands, 3Department
of Medical Genetics, National & Kapodistrian University of
Athens, St. Sophia’s Children’s Hospital, Athens, Greece,
4Department of Hematology and Bone Marrow
Transplantation, Evangelismos Hospital, Athens, Greece, 5Dpt
of Clinical genetics, Leiden University Medical Centre, Leiden,
Netherlands, 6Genesis Genoma Lab, Athens, Greece,
7Emeritus Professor, National & Kapodistrian University of
Athens, St. Sophia’s Children’s Hospital, Greece, Greece A. Hastie, A. Pang, J. Lee, E. T. Lam, X. Zhang, T. Anantharaman, S. Marin, H. Sadowski, M. Borodkin, H. Cao
Bionano Genomics, San Diego, CA, United States A. Hastie, A. Pang, J. Lee, E. T. Lam, X. Zhang, T. Anantharaman, S. Marin, H. Sadowski, M. Borodkin, H. Cao N. V. Shilova, M. E. Minzhenkova, Z. G. Markova Federal State Budgetary Institution "Research Centre for
Medical Genetics", Moscow, Russian Federation Federal State Budgetary Institution "Research Centre for
Medical Genetics", Moscow, Russian Federation Results: Eleven out of 13 KS patients selected for this
work presented karyotype 47,XXY, one 49,XXXXY and
another 48,XXYY. Among the genes analyzed, XIST
(Xq13.2) and GTPBP6 (Xp22.33/Yp11.32) showed dif-
ferential expression in peripheral blood by RT-qPCR. As
expected, XIST differed between all patients with KS and
male controls (P=0.0046). Differently, GTPBP6 had a
higher expression in
all KS patients than controls
(P=0.0005, females; P=0.0167, males). Among these,
46.1% (n=6) present ID, in which the GTPBP6 expres-
sion is also high (P=0.0012, females; P=0.0152, males). In addition, seven out of 13 KS patients revealed
informative HUMARA tests. All four KS patients, with
IQ value above 79 (IQ above the borderline), showed
skewed X inactivation (SXI) but GTPBP6 expression did
not differ between controls. In contrast, in three KS
patients, whose IQ values were below 75 (low IQ), was
observed random X inactivation (RXI) and the GTPBP6
expression
was
increased
(P=0.00167,
females;
P=0.00476, males). Introduction: It has now been estimated that in 0,23% of
patients with developmental delay and intellectual disability
unbalanced de novo translocations are detected. The
mechanisms underlying unbalanced de novo translocations
have been intensively studied, but still enigmatic. Materials and Methods: We have analyzed 5 de novo
translocations, ascertained either by conventional GTG-
banded chromosome analysis or by array analysis, con-
firmed with targeted FISH. All samples were analyzed by
multicolor banding (MCB) FISH technique with probe sets
XCyte for chromosomes 4, 8, 9, 10, 12, 13, 18, 21 and X. Results: The translocations were present in all cells. FISH with corresponding subtelomeric DNA-probes con-
firmed the deleted and duplicated segments previously
found on arrays. MCB pattern indicated inverted duplica-
tion of the terminal region of the centric segment of
derivative chromosome in all cases. Inverted duplications
were not detected on translocated segments in any of
the cases. Conclusions: These data suggest the trend that SXI can
negatively regulate the GTPBP6 expression and, therefore,
likely improves the neuropsychological performance of KS
patients. Conclusions: Our findings suggest that unbalanced de
novo translocations are formed by inverted duplications on
derivative chromosomes. A ”fold-back” mechanism of large
inverted duplications formation may be the first step of de
novo translocation origin. N. V. Shilova, M. E. Minzhenkova, Z. G. Markova Following end-joining between of
free end of the inverted duplication and another chromo-
some would give rise to an inverted duplication transloca-
tion chromosome. A.C. Vidi: None. J.S. Souza: None. L. Simonetti: None. M.I. Melaragno: None. I.S. Kunii: None. C.B. Mello:
None. G.M.G. Carvalheira:
None. M.R. Dias
da
Silva: None. N.V. Shilova: None. M.E. Minzhenkova: None. Z.G. Markova: None. Bionano Genomics, San Diego, CA, United States Typical heterozygosity for the beta-thalassemia mutation in
DNA in other tissues (buccal cells, hair) suggests a somatic
origin of the segmental deletion affecting the hemopoietic Genome mapping using Bionano Genomics’ Saphyr
System and new direct labeling chemistry (DLS) facil-
itates economical whole genome and highly sensitive
structural variation detection. We present genome assem-
blies of individuals with genetic disease caused by
extremely
long
tandem
repeat
array
expansion
and
collapse,
and
by
rearrangements
mediated
by
large segmental duplications that can be completely
resolved by Bionano mapping. Bionano mapping is a fast
and cost effective method for the detection of a broad
range of traditionally refractory SVs across the human
genome. Typical heterozygosity for the beta-thalassemia mutation in
DNA in other tissues (buccal cells, hair) suggests a somatic
origin of the segmental deletion affecting the hemopoietic
stem cells. The late onset of expression of beta-thalassemia
intermedia indicates a preferential survival of the hemo-
poietic cells containing the deleted region, which con-
tributes to the majority of erythropoiesis. A. Hastie: None. A. Pang: None. J. Lee: None. E.T. Lam: None. X. Zhang: None. T. Anantharaman: None. S. Marin: None. H. Sadowski: None. M. Borodkin: None. H. Cao: None. C.L. Harteveld:
None. C.A.J. Bosch:
None. C. Vrettou: None. L. Maragoudaki: None. J. Apostilidis:
None. S.G.J. Arkesteijn: None. M.J.V. Hoffer: None. C. C.L. Harteveld:
None. C.A.J. Bosch:
None. C. Vrettou: None. L. Maragoudaki: None. J. Apostilidis:
None. S.G.J. Arkesteijn: None. M.J.V. Hoffer: None. C. Abstracts from the 51st European Society of Human Genetics Conference: Posters 493 1Medical Genetics, Department of Precision Medicine,
University of Campania ‘Luigi Vanvitelli’, Napoli, Italy,
2Telethon Institute of Genetics and Medicine (TIGEM),
Pozzuoli, Italy, 3Department of Translational Medicine,
Section of Pediatrics, Federico II University, Napoli, Italy A. Torella1,2, F. Musacchia2, F. Del Vecchio Blanco1, G.
Esposito1, G. Casari2, R. Castello2, T. Giugliano1, M. Pinelli2,3,
M. Mutarelli2, V. Nigro1,2 P13.33A
Skewed X inactivation seems to regulate the GTPBP6
expression and be associated with neuropsychological
performance in Klinefelter patients Skewed X inactivation seems to regulate the GTPBP6
expression and be associated with neuropsychological
performance in Klinefelter patients P13.32D neuropsychological
deficits,
including
intellectual
disability (ID). Materials and Methods: In this study, we analyzed: (1)
expression of eight X-chromosome genes; (2) X inactiva-
tion pattern by the HUMARA technique, and (3) the
neuropsychological IQ using Wechsler Adult Intelligence
and Raven General Matrix scales. P14.002B 1AI duPont Hospital, Wilmington, DE, United States,
2Children’s Mercy Hospital, Kansas City, MO, United States,
3Advocate Children’s Hospital, Park Ridge, IL, United States,
4FDNA, Boston, MA, United States P14.001A
Ultra-exome:a new approach to solve the unsolved The recently
developed
linked-read
sequencing
technology
from
10XGenomics combines a novel barcoding strategy with
Illumina sequencing. Genomic DNA fragments are parti-
tioned into separate micro-reactions, where the same index
sequence is incorporated into each of the sequencing frag-
ment inserts derived from a given long fragment. However,
WES provides little information for genes with long introns. To overcome this limitation, we redesigned our WES cap-
ture target including spaced polymorphic regions located in
deep introns (>30kb). These WES barcoded reads contain
linked exons with long-range sequence information that is
advantageous for identification intragenic structural variants
and phasing. In addition, we are able to distinguish very
similar genes, such as SMN1 and SMN2. We are applying
this technology to solve the WES-negative patients affected
by a number of different genetic disorders We show that the use of an exon-focused array design
results in an increased diagnostic yield, which will even
further increase with the implementation of whole genome
sequencing, as this enables the simultaneous detection of
CNVs and single nucleotide variants (SNVs) for this group
of patients. We show that the use of an exon-focused array design
results in an increased diagnostic yield, which will even
further increase with the implementation of whole genome
sequencing, as this enables the simultaneous detection of
CNVs and single nucleotide variants (SNVs) for this group
of patients. Miller DT, Adam MP, Aradhya S, Biesecker LG, et al.,
Consensus statement: chromosomal microarray is a first-tier
clinical diagnostic test for individuals with developmental
disabilities or congenital anomalies. Am J Hum Genet. 2010
May 14;86(5):749-64. To overcome this limitation, we redesigned our WES cap-
ture target including spaced polymorphic regions located in
deep introns (>30kb). These WES barcoded reads contain
linked exons with long-range sequence information that is
advantageous for identification intragenic structural variants
and phasing. In addition, we are able to distinguish very
similar genes, such as SMN1 and SMN2. We are applying
this technology to solve the WES-negative patients affected
by a number of different genetic disorders S. Altıner: None. A. Boogaerts: None. J. Vermeesch:
None. K. Van Den Bogaert: None. Training a Facial Analysis Software to Recognize a Very
Rare Condition: Aymé-Gripp Syndrome Training a Facial Analysis Software to Recognize a Very
Rare Condition: Aymé-Gripp Syndrome Training a Facial Analysis Software to Recognize a Very
Rare Condition: Aymé-Gripp Syndrome A. Torella: None. F. Musacchia: None. F. Del Vecchio
Blanco: None. G. Esposito: None. G. Casari: None. R. Castello: None. T. Giugliano: None. M. Pinelli: None. M. Mutarelli: None. V. Nigro: None. A. Torella: None. F. Musacchia: None. F. Del Vecchio
Blanco: None. G. Esposito: None. G. Casari: None. R. Castello: None. T. Giugliano: None. M. Pinelli: None. M. Mutarelli: None. V. Nigro: None. K. W. Gripp1, S. M. Amudhavalli2, R. Hanson2, B. Angle3, K. Bontempo3, N. Fleischer4 P14.001A
Ultra-exome:a new approach to solve the unsolved A. Torella1,2, F. Musacchia2, F. Del Vecchio Blanco1, G. Esposito1, G. Casari2, R. Castello2, T. Giugliano1, M. Pinelli2,3,
M. Mutarelli2, V. Nigro1,2 A. C. Vidi, J. S. Souza, L. Simonetti, M. I. Melaragno, I. S. Kunii, C. B. Mello, G. M. G. Carvalheira, M. R. Dias da Silva A. C. Vidi, J. S. Souza, L. Simonetti, M. I. Melaragno, I. S. Kunii, C. B. Mello, G. M. G. Carvalheira, M. R. Dias da Silva Unifesp, São Paulo, Brazil Unifesp, São Paulo, Brazil Introduction: Klinefelter syndrome (KS) displays a broad
reproductive and endocrinological spectrum, in which the
most common karyotype is 47,XXY. KS patients may
present
dysmorphological
features
and
develop 494 J. del Picchia Trio WES-negative patients may carry very elusive variants
that require additional testing. Trio WGS, high resolution
array CGH, as well as transcriptomic studies can detect a
part of missing variants. However, some structural infor-
mation is lost anyway and the cost/workload of these
additional approaches may be limiting factors. The recently
developed
linked-read
sequencing
technology
from
10XGenomics combines a novel barcoding strategy with
Illumina sequencing. Genomic DNA fragments are parti-
tioned into separate micro-reactions, where the same index
sequence is incorporated into each of the sequencing frag-
ment inserts derived from a given long fragment. However,
WES provides little information for genes with long introns. To overcome this limitation, we redesigned our WES cap-
ture target including spaced polymorphic regions located in
deep introns (>30kb). These WES barcoded reads contain
linked exons with long-range sequence information that is
advantageous for identification intragenic structural variants
and phasing. In addition, we are able to distinguish very
similar genes, such as SMN1 and SMN2. We are applying
this technology to solve the WES-negative patients affected
by a number of different genetic disorders microarray platform (Oxford Gene Technology), while this
CNV was under the detection limit of the previously used
v2 platform. The v3 platform combines the most up-to-date
content information from ClinGen and the DDD project
with exon-focused probe design for single-exon CNV
resolution in disease-relevant genes. Trio WES-negative patients may carry very elusive variants
that require additional testing. Trio WGS, high resolution
array CGH, as well as transcriptomic studies can detect a
part of missing variants. However, some structural infor-
mation is lost anyway and the cost/workload of these
additional approaches may be limiting factors. NUCLEIC-CARDTM as innovative blood collection device
for NGS genetic analysis 1Molecular Biology Laboratory, Fundació Puigvert, Instituto
de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau),
Universitat Autònoma de Barcelona, REDinREN, Instituto de
Investigación Carlos III, Barcelona, Spain, 2Nephrology
Department, Fundació Puigvert, Instituto de Investigaciones
Biomédicas Sant Pau (IIB-Sant Pau), Universitat Autònoma de
Barcelona, REDinREN, Instituto de Investigación Carlos III,
Barcelona, Spain, 3Pediatric Nephrology Department,
Hospital de la Santa Creu i Sant Pau, Barcelona, Spain,
4Pediatric Nephrology Department, Hospital Vall d’Hebron,
Universitat Autònoma de Barcelona, Barcelona, Spain,
5Genetics Department, Hospital Vall d’Hebron, Universitat
Autònoma de Barcelona, Barcelona, Spain, 6Nephrology
Department, Pediatrics Service, Hospital Universitario Virgen
de la Arrixaca, Murcia, Spain, 7Genetics Department, Hospital
Universitario Cruces, Biocruces Health Research Institute,
Centre for Biomedical Research on Rare Diseases (CIBERER),
Barakaldo-Bizkaia, Spain, 8Department of Pediatrics,
Nephrology Unit, Hospital Universitario Cruces, Barakaldo-
Bizkaia, Spain, 9Barcelona Supercomputing Center (BSC),
Joint BSC-CRG-IRB Research Program in Computational
Biology, Barcelona, Spain NUCLEIC-CARDTM as innovative blood collection device
for NGS genetic analysis M. Rosso1, A. Gervasoni1, A. Squassina1, A. Breda Klobus2, S. Castriciano1 1Copan Italia Spa, Brescia, Italy, 2Breda Genetics Srl, Brescia,
Italy 1Copan Italia Spa, Brescia, Italy, 2Breda Genetics Srl, Brescia,
Italy Background:
Clinical
genetic
analysis
and
research,
including
Next-Generation
Sequencing
(NGS),
have
increased in recent years. Fresh blood has been always
considered the gold standard, but it is prone to handling and
biological risks during shipping. Copan developed the
NUCLEIC-CARDTM (NC) for collection, preservation,
stabilization, transport and long-term storage at room tem-
perature of blood samples. The objective of this study was
to report the performance of the NC, as analytical accuracy
of clinical (~6,300 clinically relevant genes) and whole
(WES) exome sequencing of DNA extracted from blood
spots on NC. Background:
Clinical
genetic
analysis
and
research,
including
Next-Generation
Sequencing
(NGS),
have
increased in recent years. Fresh blood has been always
considered the gold standard, but it is prone to handling and
biological risks during shipping. Copan developed the
NUCLEIC-CARDTM (NC) for collection, preservation,
stabilization, transport and long-term storage at room tem-
perature of blood samples. The objective of this study was
to report the performance of the NC, as analytical accuracy
of clinical (~6,300 clinically relevant genes) and whole
(WES) exome sequencing of DNA extracted from blood
spots on NC. Methods: DNA, extracted from the entire NC blood spot
of 16 donors using a modified QIAamp® DNA mini kit with
the Copan NAO® (nucleic acid optimizer) baskets protocol,
were used for this study. Increased diagnostic yield with exon-focused array
platforms required by the tool and AGS is now included as a
recognized condition. Comparison between the 3 cohorts
yielded an accuracy of 89.1%, double the random accuracy
of 37.7%. In binary comparisons, the analysis correctly
differentiated between AGS and controls with an AUC of
0.99 (STD 0.01). M. Rosso: None. A. Gervasoni: None. A. Squassina:
None. A. Breda Klobus: None. S. Castriciano: None. M. Rosso: None. A. Gervasoni: None. A. Squassina:
None. A. Breda Klobus: None. S. Castriciano: None. Conclusions: Even few images can be sufficient for an
automated facial analysis tool to identify characteristic
facial features of rare syndromes such as AGS, and thus
allow for targeted molecular testing if clinically indicated. Genetic testing approach for patients with congenital
anomalies of the kidney and urinary tract (CAKUT) Genetic testing approach for patients with congenital
anomalies of the kidney and urinary tract (CAKUT) Genetic testing approach for patients with congenital
anomalies of the kidney and urinary tract (CAKUT) K.W. Gripp: A. Employment (full or part-time); Modest;
FDNA. S.M. Amudhavalli: None. R. Hanson: None. B. Angle: None. K. Bontempo: None. N. Fleischer: A. Employment (full or part-time); Significant; FDNA. A. Domingo Gallego1, G. Bullich1, P. Ruiz1, L. Lorente
Grandoso1, M. Furlano2, G. Fraga3, Á. Madrid4, G. Ariceta4, L. Martínez Ribot5, J. Piñero Fernández6, I. Llano Rivas7, M. Aguirre Meñica8, L. Guirado2, D. Torrents9, R. Torra2, E. Ars1 Increased diagnostic yield with exon-focused array
platforms S. Altıner1,2, A. Boogaerts2, J. Vermeesch2, K. Van Den Bogaert2 S. Altıner1,2, A. Boogaerts2, J. Vermeesch2, K. Van Den Bogaert2 Introduction: Subjective impressions of an identifiable
facial phenotype arise during clinical practice. It is chal-
lenging to assess this impression objectively, particularly
for very rare syndromes. Aymé-Gripp syndrome (AGS) is
such a rare condition, resulting from specific MAF muta-
tions and characterized by developmental delay and ID,
cataracts, hearing loss, short stature, pericardial effusion and
skeletal anomalies. We evaluated an automated facial ana-
lysis tool for its ability to distinguish between patients with
AGS and hundreds other rare genetic syndromes. 1Trabzon Kanuni Training and Research Hospital, Department
of Medical Genetics, Trabzon, Turkey, 2Department of Human
Genetics, KU Leuven, Leuven, Belgium Array-Comparative Genomic Hybridisation (Array-CGH) is
the first-tier genetic test for patients with unexplained
developmental delay, intellectual disability, autism spec-
trum disorders or multiple congenital anomalies. Although
pathogenic genomic imbalances are detected in a substantial
subset of this group (15%-20%), the underlying cause of the
phenotype cannot be determined in almost 50% of patients
(Miller et al., 2010). Methods: Frontal facial photographs of 13 individuals
with molecularly confirmed AGS were used to train the
Face2Gene (FDNA Inc, USA) tool against 216 other
syndromes. In addition, we compared to images from 20
unaffected individuals and 20 individuals with Down
syndrome (DS), chosen for its similarities to the facial
features
of
AGS. Classification
statistics
were
used,
Receiver Operator Characteristic (ROC) curves and their
Area Under the Curve (AUC) calculated. In order to increase the diagnostic yield of array-CGH,
platforms are being adjusted in accordance with the
discovery of novel disease-causing genes. Moreover, an
increased probe density across exons enables the detection
of exonic copy number variants (CNVs) in addition to
whole gene or multiple exon variation. Results: The training of the technology yielded and AUC
of 0.97 which is above the significance level threshold We present six cases for which a pathogenic variant could
be detected by means of the CytoSure™Constitutional v3 Abstracts from the 51st European Society of Human Genetics Conference: Posters 495 clinical and whole exome sequencing. NUCLEIC-CARDTM
allows long-term storage and stability of dry blood samples
for easy samples shipment at room temperature to testing
laboratories. clinical and whole exome sequencing. NUCLEIC-CARDTM
allows long-term storage and stability of dry blood samples
for easy samples shipment at room temperature to testing
laboratories. NUCLEIC-CARDTM as innovative blood collection device
for NGS genetic analysis Besides clinical exome analyses,
two of the samples were also used for WES. Introduction: Congenital anomalies of the kidney and
urinary tract (CAKUT) are a heterogeneous group of
embryonic malformations representing the most common
cause of chronic kidney disease in the first 3 decades of life. About 40 monogenic causes of CAKUT are known
explaining <20% of CAKUT cases. We aim to improve the
genetic diagnosis of this nephropathy. Results: High-throughput sequencing, performed on
DNA extracted from dried blood spots onto NC gave
excellent yield, reaching high quality levels, comparable
with the ones obtainable from EDTA-blood samples. Also,
it has been possible to achieve overlapping values in terms
of final average coverage on target, target percentages at
10x, 20x and 30x, and base call quality. Methods: Ninety unrelated patients with CAKUT (21%
with perinatal death) mostly bilateral (86%), with a
suspicion of monogenic cause due to extrarenal defects
(29%) and/or positive familial history of CAKUT (46%)
underwent a two-step genetic analysis. 1) HNF1B gene
mutation screening, for CNV and SNV; and if negative; 2)
targeted next generation sequencing (NGS) of a >200 Methods: Ninety unrelated patients with CAKUT (21%
with perinatal death) mostly bilateral (86%), with a
suspicion of monogenic cause due to extrarenal defects
(29%) and/or positive familial history of CAKUT (46%)
underwent a two-step genetic analysis. 1) HNF1B gene
mutation screening, for CNV and SNV; and if negative; 2)
targeted next generation sequencing (NGS) of a >200 Conclusions: Data obtained in this study demonstrated
that good quality DNA can be recovered from blood spotted
on Copan NUCLEIC-CARDTM, and that such DNA is
perfectly suitable for high-throughput analyses such as 496 J. del Picchia Results: HaplotypeCaller’s weighted QUAL field pro-
vides the most powerful metric for filtering. Sequencing
depth (DP), while widely used, has relatively little to do
with call accuracy. For Illumina HiSeq platform and GATK
Best Practices-like processing pipeline, we report the
optimal filtering parameters to be: DP≥6 & QUAL≥75. This set of cutoffs achieves a tradeoff by calling reasonably
many variants (45 361 using targeted sequencing kit) while
minimizing false positive rate (0.34%). In comparison,
naive depth-only filtering (DP>12) called only 36 171
variants with a FPR of 0.71%. kidney-disease gene panel including 62 genes associated
with CAKUT. Results: Causative mutations were identified in 38% (34/
90) of patients [9% (3/34) with perinatal death] involving
the following genes: HNF1B (27), PAX2 (5) and ACE (3). NUCLEIC-CARDTM as innovative blood collection device
for NGS genetic analysis Probably pathogenic variants were detected in 3% (3/90) of
patients in JAG1, NEK8 and DYNC2H1 genes. Variants of
uncertain significance were detected in 8% (7/90) of
patients in the: SIX2, RET, PAX2, GLI3, NOTCH2 and
PAX8 genes. Conclusions: HNF1B screening allowed the etiologic
diagnosis in one-quarter (27/90) of our CAKUT patients, of
whom only one presented with perinatal death. NGS of our
kidney-disease gene panel identified a likely pathogenic
variant in an additional 12% (11/90) of patients. Our
approach achieved a 42% diagnostic yield (63% with
positive familial history/extrarenal defects). Whole exome
sequencing is undergoing in selected cases. Conclusions: HNF1B screening allowed the etiologic
diagnosis in one-quarter (27/90) of our CAKUT patients, of
whom only one presented with perinatal death. NGS of our
kidney-disease gene panel identified a likely pathogenic
variant in an additional 12% (11/90) of patients. Our
approach achieved a 42% diagnostic yield (63% with
positive familial history/extrarenal defects). Whole exome
sequencing is undergoing in selected cases. Conclusions: We present a formal yet simple approach to
determine the most optimal quality filtering cutoffs for NGS
calls. This allows to get the most out of a NGS sample
without compromising accuracy of the results. K. Tsukanov: None. O. Klimchuk: None. D. Plakhina:
None. A. Krasnenko: None. V. Ilinsky: None. Germline analysis using FFPE-tissue in cancer families
with high mortality rates Germline analysis using FFPE-tissue in cancer families
with high mortality rates A. Domingo Gallego: None. G. Bullich: None. P. Ruiz:
None. L. Lorente Grandoso: None. M. Furlano: None. G. Fraga: None. Á. Madrid: None. G. Ariceta: None. L. Martínez Ribot: None. J. Piñero Fernández: None. I. Llano Rivas: None. M. Aguirre Meñica: None. L. Guirado: None. D. Torrents: None. R. Torra: None. E. Ars: None. K. Keupp1, M. Larsen1, B. Bluemcke1, B. Versmold1, A. Waha1,
J. Driesen1, A. Baasner1, L. Buelow1, C. Eßer1, B. Markiefka2,
B. Wappenschmidt1, E. Hahnen1, R. K. Schmutzler1 1Center for Hereditary Breast and Ovarian Cancer, Cologne,
Germany, 2Institute for Pathology, Cologne, Germany Determination of optimal call quality cutoffs for clinical
applications of NGS The identification of disease-causing germline mutations in
families with hereditary cancer is essential for predictive
testing of unaffected family members and estimation of
individual lifetime risks. Germline testing usually requires
blood- or saliva-derived DNA of the index patient. How-
ever, due to high rate of mortality living affected family
members may not be available in cancer families. The
establishment of Next Generation Sequencing (NGS) in
genetic diagnostics enables comprehensive risk gene ana-
lyses even in DNA isolated from formalin-fixed-paraffin-
embedded (FFPE) tissue. Here, we show successful post
mortem germline analysis in high risk breast (BC) and
ovarian cancer (OC) families by using DNA from non-
cancerous FFPE-tissue derived from tumor surgery. Of
particular importance is the exact discrimination between
tumor and surrounding healthy tissue because only analysis
in non-cancerous tissue assures the identification of germ-
line mutations. So far, in 111 families without a living index
patient, we performed NGS using FFPE-derived DNA
samples by employing the TruRisk® gene panel and iden-
tified a mutation prevalence of about 29 % within 15 ana-
lyzed cancer predisposition genes. This result is comparable
to the mutation prevalence identified using blood-derived
DNA in familial BC and OC cases. Based on our K. Tsukanov1,2, O. Klimchuk1,2, D. Plakhina1,2, A. Krasnenko1,2,
V. Ilinsky1,2 K. Tsukanov1,2, O. Klimchuk1,2, D. Plakhina1,2, A. Krasnenko1,2,
V. Ilinsky1,2 1Genotek Ltd., Moscow, Russian Federation, 2Genotek IT,
Moscow, Russian Federation P14.007C Funding: Instituto Carlos III/FEDER:(PI15/01824-PI16/
01998). P14.008D
Cell-free DNA analysis with the 2100 Bioanalyzer system H. Mundi1, E. Graf2, R. Nitsche2 H. Mundi1, E. Graf2, R. Nitsche2 1Agilent Technologies LDA, Cheadle, United Kingdom,
2Agilent Technologies, Waldbronn, Germany 1Agilent Technologies LDA, Cheadle, United Kingdom,
2Agilent Technologies, Waldbronn, Germany Methods: Female genomic DNA from a patient with
Jacobsen syndrome and from a single normal source were
used in this study. Sensitivity was assessed by preparing
different percentages of artificial mosaicism (5%, 8%, 10%,
and 15%), labeling 50 ng of DNA with CYTAG®
SuperCGH labeling kit (Enzo, ENZ-GEN120), and hybri-
dizing the labeled DNA onto SurePrint® G3 human CGH
4x180k microarrays. Sequencing of cell-free DNA (cfDNA) extracted from blood
specimens or other body fluids is possible due to the
establishment of low input library protocols for next-
generation sequencing workflows. Accurate quantification
of cfDNA samples is essential to determine suitable input
amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mono-
nucleosome with a size around 170 bp, sometimes with
additional
species representing
nucleosome multimers. Further, cfDNA samples may contain larger DNA frag-
ments dependent on the donor’s health status, preanalytical
sample treatment, or extraction method. High molecular
weight material can negatively influence library preparation
and subsequently result in lower sequencing depth. There-
fore, reliable quantification of cfDNA requires a method
that separates DNA fragments by size, such as electro-
phoresis. This poster shows the use of a new cell-free DNA
assay for the 2100 Bioanalyzer system as an add-on for the
High Sensitivity DNA assay. The cell-free DNA assay
enables automated cfDNA quantification with pre-defined
regions. Moreover, the results include sample purity as a
score to qualify cfDNA samples according to their con-
tamination level with high molecular weight material. The
features of the cfDNA assay are described with examples of
typical sample patterns. Results: DLRS were 0.115, 0.104, 0.102, and 0.105 for
samples with 5%, 8%, 10%, and 15% artificial mosaicism,
respectively. Female genomic DNA with Jacobsen syn-
drome has a 12.5Mb terminal deletion on the q arm of
chromosome 11. With a setting for the Average Log Ratio
in the CytoGenomics software of 0.0 or 0.05, the 12.5Mb
deletion was visualized and detected in samples with as low
as 8% of artificial mosaicism. Conclusions: Upon labeling of 50 ng of DNA with
CYTAG® SuperCGH labeling kit, the lowest limit of
detection was deemed to be 8%. P14.008D
Cell-free DNA analysis with the 2100 Bioanalyzer system The results demonstrated
that aCGH is an invaluable tool to detect segmental
aneuploidy with length around 10Mb in mosaic samples
with low DNA input thereby offering new possibilities for
aCGH analysis in prenatal or oncology. P. Pande: None. J. Coleman: None. N. Rana: None. M. Mathieu: None. 1Genotek Ltd., Moscow, Russian Federation, 2Genotek IT,
Moscow, Russian Federation Introduction: NGS generates large number of variants of
varying quality. It is important to decide which calls to
retain for downstream, but few formalized, practical
recommendations exist on how to do it. Materials and Methods: We sequenced two platinum
genome samples, NA12877 and NA12878, using Agilent
FocusedExome enrichment kit on Illumina HiSeq 2500
platform. The data was called using in-house pipeline based
on GATK Best Practices. We tested multiple combinations
of quality metrics (depth of coverage, B allele count, B
allele fraction, and HaplotypeCaller’s QUAL score) to filter
the resulting calls. By comparing the remaining calls with
platinum genome reference callsets, sensitivity and false
positive rate were calculated for each combination of cutoff Abstracts from the 51st European Society of Human Genetics Conference: Posters 497 1Enzo Life Sciences, Farmingdale, NY, United States, 2Enzo
Life Sciences, Lausen, Switzerland K. Keupp: None. M. Larsen: None. B. Bluemcke:
None. B. Versmold: None. A. Waha: None. J. Driesen:
None. A. Baasner: None. L. Buelow: None. C. Eßer:
None. B. Markiefka: None. B. Wappenschmidt: None. E. Hahnen: None. R.K. Schmutzler: None. Background: aCGH is a powerful clinical diagnostic tool
allowing the genome-wide assessment of CNVs with high
resolution. Despite several studies validating aCGH for the
detection of cytogenetic abnormalities, only a few have
reported on array sensitivity. Here, we determined the
sensitivity of whole-genome aCGH by labeling low DNA
inputs with CYTAG® SuperCGH labeling kit and detecting
low levels of artificial mosaicism. P. Pande1, J. Coleman1, N. Rana1, M. Mathieu2 P. Pande1, J. Coleman1, N. Rana1, M. Mathieu2 1Enzo Life Sciences, Farmingdale, NY, United States, 2Enzo
Life Sciences, Lausen, Switzerland P14.009A
Detection of low-level mosaicism by array CGH using low
amounts of DNA labeled with CYTAG SuperCGH labeling
kit P14.009A
Detection of low-level mosaicism by array CGH using low
amounts of DNA labeled with CYTAG SuperCGH labeling
kit P14.009A experience, we highly recommend using non-cancerous
FFPE material for germline analysis in families without a
living index in a routine diagnostic setting. Particularly, in
hereditary cancer families this procedure is of substantial
relevance for subsequent predictive gene analysis, risk
calculation
and
cancer
prevention
in
further
family
members. P. Pande1, J. Coleman1, N. Rana1, M. Mathieu2 P14.010B Integrated genetic analysis: Harnessing the power of NGS H. Mundi: A. Employment (full or part-time); Signifi-
cant; Agilent Technologies LDA. E. Graf: A. Employment
(full or part-time); Significant; Agilent Technologies. R. Nitsche: A. Employment (full or part-time); Significant;
Agilent Technologies. H. Mundi: A. Employment (full or part-time); Signifi-
cant; Agilent Technologies LDA. E. Graf: A. Employment
(full or part-time); Significant; Agilent Technologies. R. Nitsche: A. Employment (full or part-time); Significant;
Agilent Technologies. A. J. Obregon-Tito, C. Lee, J. Li, M. Teguh, Y. Shen
Fulgent Genetics, Temple City, CA, United States A. J. Obregon-Tito, C. Lee, J. Li, M. Teguh, Y. Shen
Fulgent Genetics, Temple City, CA, United States A. J. Obregon-Tito, C. Lee, J. Li, M. Teguh, Y. Shen
Fulgent Genetics, Temple City, CA, United States Fulgent Genetics, Temple City, CA, United States Fulgent Genetics, Temple City, CA, United States Introduction: The study of copy number variants is often
equated to microarray platforms. Microarray platforms are 498 J. del Picchia Methods: We analyzed adult patients consulted to the
adult genetic service in 2017, who underwent CES. Methods: We analyzed adult patients consulted to the
adult genetic service in 2017, who underwent CES. appropriate to detect genetic imbalances but present chal-
lenges that might be overcome by NGS technologies. We
propose an NGS based approach to chromosomal analysis
with an increased sensitivity and an unbiased representation
of the whole genome. appropriate to detect genetic imbalances but present chal-
lenges that might be overcome by NGS technologies. We
propose an NGS based approach to chromosomal analysis
with an increased sensitivity and an unbiased representation
of the whole genome. Results: CES revealed a diagnosis in 10 of the 28 patients
(35.7%) (age range: 20 to 104 years old) (Table 1). 14
pathogenic variants (PVs) or likely PVs were identified in
12
genes:
CPT1A,CPT2,MMAHC,CXCR4,ACVRL1,
FANCA,PTEN,NF1,BRCA1,SLC26A2,HBB,TGFBR1. Two
patients carried two diagnoses (Patient 8 and 9 in table 1). Two patients (2/28 = 7.1%) with autosomal recessive
disorders (Citrin Deficiency, Autosomal recessive Poly-
cystic Kidney Disease with Hepatic Fibrosis) were found to
harbor only one mutant allele in the causative genes
(SLC25A3 and PKHD1 respectively). We also identified
secondary findings in two cases (2/28 = 7.1%) without
primary molecular diagnosis (Hb Constant Spring carrier
and G-6-PD deficiency carrier). P14.010B NGS-based
CNV analysis can be part of a step-wise testing algorithm
that includes multigene panels and exomes, resulting in a
cost and time effective alternative to current strategies. Conclusions: Compared to microarray platforms, NGS-
based chromosomal analysis exhibits an increased sensitiv-
ity and an option to streamline genetic testing. NGS-based
CNV analysis can be part of a step-wise testing algorithm
that includes multigene panels and exomes, resulting in a
cost and time effective alternative to current strategies. A.J. Obregon-Tito: A. Employment (full or part-time);
Significant; Fulgent Genetics. C. Lee: A. Employment (full
or part-time); Significant; Fulgent Genetics. J. Li: A. Employment
(full
or
part-time);
Significant;
Fulgent
Genetics. M. Teguh: A. Employment (full or part-time);
Significant; Fulgent Genetics. Y. Shen: A. Employment
(full or part-time); Significant; Fulgent Genetics. P. Phowthongkum: None. V. Shotelersuk: None. K. Suphapeetiporn: None. C. Srijomtong: None. C. Ittiwut:
None. W. Kamolvisit: None. P. Damrongphol: None. P14.012D
High Yield of Clinical Exome Sequencing as a primary
molecular diagnostic tool in Adults High Yield of Clinical Exome Sequencing as a primary
molecular diagnostic tool in Adults A. Narravula1, N. Kienle1, I. Hövel1, A. Romito1, A. Bertoli-
Avella1, Z. Yüksel1, O. Paknia1, S. Nampoothiri2, Z. Hadipour3,
F. Hadipour3, G. Oprea1, S. Kishore1, P. Bauer1,4, A. Rolfs1,5 P. Phowthongkum, V. Shotelersuk, K. Suphapeetiporn, C. Srijomtong, C. Ittiwut, W. Kamolvisit, P. Damrongphol P. Phowthongkum, V. Shotelersuk, K. Suphapeetiporn, C. Srijomtong, C. Ittiwut, W. Kamolvisit, P. Damrongphol
Faculty of Medicine, Bangkok, Thailand 1CENTOGENE AG, Rostock, Germany, 2Department of
Pediatric Genetics, Amrita Institute of Medical Sciences &
Research Centre, Cochin, India, 3Medical Genetics
Department, Sarem Cell Research Center & Hospital, Tehran,
Iran, Islamic Republic of, 4Institute of Medical Genetics and
Applied Genomics, University of Tübingen, Tübingen,
Germany, 5Albrecht-Kossel-Institute, Rostock, Germany P14.010B Furthermore, we identified
the variants in the genes that strongly support the clinical
diagnosis, but the pathogenicity cannot be confirmed solely
by CES in 21.4% (6/28) of the patients. Results: CES revealed a diagnosis in 10 of the 28 patients
(35.7%) (age range: 20 to 104 years old) (Table 1). 14
pathogenic variants (PVs) or likely PVs were identified in
12
genes:
CPT1A,CPT2,MMAHC,CXCR4,ACVRL1,
FANCA,PTEN,NF1,BRCA1,SLC26A2,HBB,TGFBR1. Two
patients carried two diagnoses (Patient 8 and 9 in table 1). Two patients (2/28 = 7.1%) with autosomal recessive
disorders (Citrin Deficiency, Autosomal recessive Poly-
cystic Kidney Disease with Hepatic Fibrosis) were found to
harbor only one mutant allele in the causative genes
(SLC25A3 and PKHD1 respectively). We also identified
secondary findings in two cases (2/28 = 7.1%) without
primary molecular diagnosis (Hb Constant Spring carrier
and G-6-PD deficiency carrier). Furthermore, we identified
the variants in the genes that strongly support the clinical
diagnosis, but the pathogenicity cannot be confirmed solely
by CES in 21.4% (6/28) of the patients. Materials and Methods: We compare the results of
microarray analysis and pair-ended, low-coverage whole
genome sequencing to test 31 samples from cell lines and
19 deidentified individuals. All variants were verified by
orthologous methods. Results: The NGS-based approach identified all variants
identified by microarray plus 52 additional copy number
variants (CNVs). The NGS-based test exhibits increased
sensitivity with a detection limit of up to 200bp when
average sequencing coverage is 1X. Identification of exact
breakpoints requires higher coverage. Furthermore, the
NGS-based chromosomal analysis allows the identification
of structural abnormalities that microarray is unable to
detect. Primer design for segmental duplication is extremely
difficult for areas of high homology, a limitation shared by
microarray, MLPA or qPCR. In contrast, NGS-based
chromosomal analysis and stringent bioinformatic protocols
allow precise mapping of reads with up to 1 base difference
in 300 bp DNA block (~99.7% identity) correctly comput-
ing CNVs in areas of segmental duplication. Discussion: We report the high yield of diagnosis and
secondary findings (14/28 = 50%) from CES in adult
patients. The yield was increased to 71.4% (20/28) if all
possible/actionable
results
were
included. Our
results
demonstrated the efficacy of CES as a primary diagnostic
tool in adults. Conclusions: Compared to microarray platforms, NGS-
based chromosomal analysis exhibits an increased sensitiv-
ity and an option to streamline genetic testing. 1CENTOGENE AG, Rostock, Germany, 2Department of
Pediatric Genetics, Amrita Institute of Medical Sciences &
Research Centre, Cochin, India, 3Medical Genetics
Department, Sarem Cell Research Center & Hospital, Tehran,
Iran, Islamic Republic of, 4Institute of Medical Genetics and
Applied Genomics, University of Tübingen, Tübingen,
Germany, 5Albrecht-Kossel-Institute, Rostock, Germany Faculty of Medicine, Bangkok, Thailand Faculty of Medicine, Bangkok, Thailand Introduction:
Clinical
exome
sequencing
(CES)
is
increasingly used as a molecular diagnostic tool for genetic
disorders. We presented here the use of CES as a primary
diagnostic tool in adults. Abstracts from the 51st European Society of Human Genetics Conference: Posters 499 Introduction: Clinical exome sequencing (CES) involves
the sequencing and analysis of the coding regions of only
the clinically-relevant genes in the genome compared to
sequencing all 20,000 genes as in a whole exome
sequencing (WES). other intellectual property); Significant; CENTOGENE AG. A. Rolfs: A. Employment (full or part-time); Significant;
CENTOGENE AG. B. Research Grant (principal investi-
gator, collaborator or consultant and pending grants as well
as grants already received); Significant; Albrecht-Kossel-
Institute. E. Ownership Interest (stock, stock options, patent
or
other
intellectual
property);
Significant;
CENTOGENE AG. Aim: To assess the clinical utility of CES (CentoDx
Plus™) in patients tested to date using diagnostic yield as a
measure. Methods: Previously reported 91 CES cases were
analyzed to identify cases with diagnostic variants (patho-
genic/ likely pathogenic) and those with variants of
unknown significance (VUS). Sidra Medicine, Doha, Qatar Background: Clinical Exome Sequencing (CES) is rapidly
becoming a standard molecular diagnostic tool in rare and
diverse genetic disorders such as neurocognitive/neurode-
velopmental, neuromuscular disorders, and multiple con-
genital anomalies. The clinical indications recommended
for CES, the overall molecular diagnostic yield for diverse
phenotypes, along with differences in molecular diagnostic
yields between CES as first-tier test or reflex test, will be
determined in a cohort of 508 patients from a highly con-
sanguineous population. Conclusions: The high diagnostic yield of CES (49.4%)
when compared to WES can likely be attributed to CES
being used to diagnose cases with known, suspected
diagnoses as well as undiagnosed cases with unknown,
complex phenotypes. CES is a versatile test with high
clinical utility for a variety of diagnostic indications. Due to
its cost- and time-efficient nature and high diagnostic rate,
CES is a good alternative to sequential testing, NGS panels
or WES. Especially in regions with economic barriers to
genetic testing, CES should be considered a prime test of
choice. Methods: A clinical cohort of 508 probands from Qatar
underwent singleton or trio CES from April 2014 to
December 2016. Cases were considered ‘solved’ when
mono- or bi-allelic pathogenic or likely pathogenic variants
(collectively “PV”), including pathogenic CNVs, were
present in a disease gene, consistent with the known mode
of inheritance of a disorder. A. Narravula: A. Employment (full or part-time);
Significant; CENTOGENE AG. N. Kienle: A. Employment
(full or part-time); Significant; CENTOGENE AG. I. Hövel: A. Employment (full or part-time); Significant;
CENTOGENE AG. A. Romito: A. Employment (full or
part-time); Significant; CENTOGENE AG. A. Bertoli-
Avella: A. Employment (full or part-time); Significant;
CENTOGENE AG. Z. Yüksel: A. Employment (full or
part-time); Significant; CENTOGENE AG. O. Paknia: A. Employment (full or part-time); Significant; CENTOGENE
AG. S. Nampoothiri: A. Employment (full or part-time);
Significant;
Amrita
Institute
of
Medical
Sciences
&
Research Centre. Z. Hadipour: A. Employment (full or
part-time); Significant; Sarem Cell Research Center &
Hospital. F. Hadipour: A. Employment (full or part-time);
Significant; Sarem Cell Research Center & Hospital. G. Oprea: A. Employment (full or part-time); Significant;
CENTOGENE AG. S. Kishore: A. Employment (full or
part-time); Significant; CENTOGENE AG. P. Bauer: A. Employment (full or part-time); Significant; CENTOGENE
AG. E. Ownership Interest (stock, stock options, patent or A. Narravula: A. Employment (full or part-time);
Significant; CENTOGENE AG. N. Kienle: A. Employment
(full or part-time); Significant; CENTOGENE AG. I. Hövel: A. Clinical exome sequencing in 508 Middle Eastern families
with Mendelian diseases provides a high diagnostic yield
and discovery of novel genes Results: In 45 (49.4%) of 91 cases tested, CES resulted in
a (likely) confirmed diagnosis. Additionally, in 25 cases
(27.5%), one or more VUS were detected. Out of these,
parental testing was recommended for 13 cases to help
reclassify the VUS as it matched the phenotype of the
patient. Seven (7.7%) patients were (likely) carriers of one
diagnostic variant or VUS. Fourteen (15.4%) cases were
negative and no phenotype-related variant was detected. P14.014B Clinical exome sequencing in 508 Middle Eastern families
with Mendelian diseases provides a high diagnostic yield
and discovery of novel genes Copy Number Variations calling from Whole Genome
Sequencing data as a substitute to array CGH 1Laboratório de Citogenômica, Departamento de Patologia,
Faculdade de Medicina da Universidade de Sao Paulo, Sao
Paulo, Brazil, 2Laboratório de Citogenômica, Departamento
de Pediatria, Faculdade de Medicina da Universidade de Sao
Paulo, Sao Paulo, Brazil, 3Mendelics Análise Genômica, Sao
Paulo, Brazil, 4Unidade de Genética, Departamento de
Pediatria, Instituto da Criança do HCFMUSP, Sao Paulo,
Brazil M. Coutelier1, M. Holtgrewe2, M. Jäger3, R. Flöttman1, M. Mensah1, M. Spielmann1, P. Krawitz4, D. Horn1, D. Beule2, S. Mundlos1 M. Coutelier1, M. Holtgrewe2, M. Jäger3, R. Flöttman1, M. Mensah1, M. Spielmann1, P. Krawitz4, D. Horn1, D. Beule2, S. Mundlos1 M. Coutelier1, M. Holtgrewe2, M. Jäger3, R. Flöttman1, M. Mensah1, M. Spielmann1, P. Krawitz4, D. Horn1, D. Beule2, S. Mundlos1 1Institute of Medical and Human Genetics, Berlin, Germany,
2Core Unit Bioinformatics, Berlin Institute of Health, Berlin,
Germany, 3Core Unit Genomis, Berlin Institute of Health,
Berlin, Germany, 4Institut für Genomische Statistik und
Bioinformatik, Bonn, Germany 1Institute of Medical and Human Genetics, Berlin, Germany,
2Core Unit Bioinformatics, Berlin Institute of Health, Berlin,
Germany, 3Core Unit Genomis, Berlin Institute of Health,
Berlin, Germany, 4Institut für Genomische Statistik und
Bioinformatik, Bonn, Germany Over the last years the identification of CNVs from whole
exome sequencing (WES) data has become a common
practice for research. In this study, we evaluated 32 patients
with multiple congenital malformations and developmental
delay with WES (Illumina) technique using the Exome-
Depth software and with array (Illumina) using the Blue-
Fuse
software
for
CNVs
evaluation. According
to
information from Illumina, the average of the minimum
resolution for CNVs detection by array is about 18 kb, thus
we selected only CNVs larger than this value to measure the
consistency between the both methodologies. Array analy-
sis for all patients showed 44 CNVs, among then 29
alterations were deletions and 15 were duplications. Exome
analysis, of the same samples, showed 60 CNVs wherein 39
were deletions and 21 duplications. So, the overlapping rate
of genomic changes was 13.8% between the techniques. The extraction of CNVs information obtained from the
WES is an advantageous approach since it can improve the
cost-effectiveness and reduce the number of genomic tests
required to diagnosis. However, WES is not developed for
the evaluation of CNVs, and shows a high rate of false-
positive and false-negative results. On the other hand, the
array is a gold standard technique with high accuracy for
detection of deletions and duplications. Copy Number Variations calling from Whole Genome
Sequencing data as a substitute to array CGH Thus, to date, the
screening by arrays allows yet a better detection of the
CNVs making possible the conclusion of diagnosis for most
patients, suggesting array performance present an out-
standing level when compared to exome in routine diag-
nostics. Grants: FINEP-CT INFRA 0160/12 SP8, CAPES. É Along with Single Nucleotide Variants (SNV), Copy
Number Variations (CNV) are a major player in genetic
diseases, and can be detected in up to 15% of patients with
heterogeneous diseases. They can affect the coding part of
the genome and exert a gene dosage effect, or be located in
non-coding regions, where they can alter the DNA tri-
dimensional organization and the enhancer-promoter con-
tacts. If Whole Exome and Genome Sequencing are now
classical high-throughput approaches for SNP detection,
array-CGH remains the gold-standard for genome-wide
study of CNV. Efficiently combining the detection of both
types of variants with a single technique would prove to be
a speed- and cost-effective approach towards the determi-
nation of the causative variant. In 27 index cases with limb
malformations, we compare array-CGH results with calls
from WGS with different algorithms, either based on split-
reads and abnormal paired-end insert size (Delly), or on
coverage variations (CNVnator, cnvkit, FREEC). With
default parameters, 13 to 89% (on average 59%) of array-
CGH deletions can be detected by at least one algorithm
with 50% reciprocal identity. This is only the case for 0 to
45% of amplifications (on average 31%). Many false
positive variants are also called, and filters, including cohort
genotyping, need to be adapted to improve detection effi-
ciency. Further tuning is hence required to reach an efficient
pipeline in terms of both sensitivity and specificity, but
CNV detection from WGS data is a promising alternative to
array-CGH. É.A. Zanardo:
None. S.N. Chehimi:
None. F.P. Monteiro: None. F.A.R. Madia: None. G.M. Novo-Filho:
None. A.T. Dias: None. M.M. Montenegro: None. Y.G. Oliveira: None. L.L. Vieira: None. M. Rocha: None. A.S. Brasil: None. A.M. Nascimento: None. T.V.M.M. Costa:
None. J.G. Damasceno: None. F. Kok: None. C.A. Kim:
None. L.D. Kulikowski: None. É.A. Zanardo:
None. S.N. Chehimi:
None. F.P. Monteiro: None. F.A.R. Madia: None. G.M. Novo-Filho:
None. A.T. Dias: None. M.M. Montenegro: None. Y.G. Oliveira: None. L.L. Vieira: None. M. Rocha: None. A.S. Brasil: None. A.M. Nascimento: None. T.V.M.M. Costa:
None. J.G. Damasceno: None. F. Kok: None. C.A. Kim:
None. L.D. Kulikowski: None. M. Coutelier: None. M. Holtgrewe: None. M. Copy Number Variations calling from Whole Genome
Sequencing data as a substitute to array CGH Jäger:
None. R. Flöttman: None. M. Mensah: None. M. Spielmann: None. P. Krawitz: None. D. Horn: None. D. Beule: None. S. Mundlos: None. P14.017A Investigating the CNVs in routine diagnostics using WES
and array in Brazilian patients É. A. Zanardo1, S. N. Chehimi2, F. P. Monteiro3, F. A. R.
Madia2, G. M. Novo-Filho1, A. T. Dias1, M. M. Montenegro1, Y. Sidra Medicine, Doha, Qatar Employment (full or part-time); Significant;
CENTOGENE AG. A. Romito: A. Employment (full or
part-time); Significant; CENTOGENE AG. A. Bertoli-
Avella: A. Employment (full or part-time); Significant;
CENTOGENE AG. Z. Yüksel: A. Employment (full or
part-time); Significant; CENTOGENE AG. O. Paknia: A. Employment (full or part-time); Significant; CENTOGENE
AG. S. Nampoothiri: A. Employment (full or part-time);
Significant;
Amrita
Institute
of
Medical
Sciences
&
Research Centre. Z. Hadipour: A. Employment (full or
part-time); Significant; Sarem Cell Research Center &
Hospital. F. Hadipour: A. Employment (full or part-time);
Significant; Sarem Cell Research Center & Hospital. G. Oprea: A. Employment (full or part-time); Significant;
CENTOGENE AG. S. Kishore: A. Employment (full or
part-time); Significant; CENTOGENE AG. P. Bauer: A. Employment (full or part-time); Significant; CENTOGENE
AG. E. Ownership Interest (stock, stock options, patent or Results: The CES diagnostic yield for our cohort of
patients was 48.0% (n=245). Consanguinity and a positive
family history predicted a higher diagnostic yield up to
56%. Eleven patients (2.1%) had two distinct genetic
disorders. 18 novel candidate genes were identified. As 65%
of families were consanguineous, a considerable number of
probands (56%) were homozygous for a PV in a gene
associated with an autosomal recessive trait, and another
9% had PV in genes with AR or autosomal dominant
inheritance. Nevertheless, a considerable number of patients
had PVs in AD (29%) or X-linked genes (5%), or de novo
copy number variants (1.8%). Conclusions:Due to its high diagnostic yield in this
population group, CES is recommended as a first-line
diagnostic approach in the genetics clinic. T.I.M.M. Ben-Omran: None. T.I.M.M. Ben-Omran: None. 500 J. del Picchia P14.016D G. Oliveira1, L. L. Vieira1, M. Rocha1, A. S. Brasil1, A. M. Nascimento2, T. V. M. M. Costa1, J. G. Damasceno1, F. Kok3, C. A. Kim4, L. D. Kulikowski1,2 1MGC Genetaq, Malaga, Spain, 2Hospital Universitario
Virgen del Rocío, Sevilla, Spain 1MGC Genetaq, Malaga, Spain, 2Hospital Universitario
Virgen del Rocío, Sevilla, Spain Introduction: Congenital Adrenal Hyperplasia (CAH)
related to CYP21A2 gene is an autosomal recessive dis-
orders affecting steroidogenesis. Material and Methods: We present a cohort of 33
patients: asymptomatic (AP):9, clinical/biochemical pheno-
type
(CBP):20
(15
with
clear
diagnosis),
unknown
phenotype (UPP):4. All DNA samples were sequenced using NEXTflex®
Congenital Adrenal Hyperplasia Amplicon Panel. In some
samples: SALSA-MLPA P050-C1-CAH probemix and
Sanger sequencing were performed. Introduction: The clinical noninvasive prenatal testing
(NIPT) is mainly focused on detecting whole-chromosome
aneuploidies using low-coverage whole genome sequen-
cing. Nowadays, advanced versions of NIPT are able to
identify even various subchromosomal copy number var-
iants (CNV) such as microdeletions and microduplications. However, these CNVs are being detected only on autosomal
chromosomes, since it is quite challenging to normalize and
train parameters on gonosomal chromosomes due to vari-
able amount of reads mapped to gonosomal chromosomes
according to fetal fraction and sex of the fetus. Results: 11 samples showed a profile compatible with
presence of multiple copies and chimeric alleles. AP had a concordant genotype in 77,78% (7/9). NEXTflex and Sanger sequencing had concordant results
in 81,82% (9/11). NEXTflex and MLPA data were concordant for IVS2-13
status in 68,18% (15/22) and for copy number status in 50%
(11/22). Materials and Methods: We extended our in-house
CNV detector with gonosomal CNV feature. The normal-
ization on autosomal chromosomes is done per chromo-
some, while autosomes are normalized relative to all
autosomes. This allows us to train and normalize the
algorithm parameters and predict local CNVs on gonosomal
chromosomes. Training was performed on 790 samples
with more than 7M reads. Genotype-phenotype concordance in CBP was obtained
in 80% (12/15). Genotype-phenotype concordance in CBP was obtained
in 80% (12/15). Conclusions: NEXTflex is a great method to detect
chimaeras and multimodular RCCX state, whose detection
was not possible by Sanger or MLPA. NEXTflex also
potentially allows to distinguish multiple copies. Results: In addition to artificial testing samples with
introduced sex chromosomal microaberration, we were able
to identify three subchromosomal aberrations on chromo-
some X in clinical samples. The lower limit of detection
was 1.66Mb. The discrepancies in AP and CBP could be due to allele
dropouts, caused by the presence of a copy of CYP21A2 on
3' end followed by TNXA, or by formation of super-
secondary
structures
close
to
I2-13. These
potential
limitations will be checked by no LR-PCR. P14.018B Preliminary evaluation of a CYP21A2 genotyping by NGS Preliminary evaluation of a CYP21A2 genotyping by NGS Abstracts from the 51st European Society of Human Genetics Conference: Posters 501 1MGC Genetaq, Malaga, Spain, 2Hospital Universitario
Virgen del Rocío, Sevilla, Spain Conclusions: The CNV detection software currently used
in NIPT can be extended to detect local aberrations on
gonosomal chromosomes. However, due to the normal-
ization only for single chromosome, it is unable to detect
whole chromosome aneuploidies (monosomies, trisomies,
⋯) - a different method is employed to detect these. Moreover, large scale aberrations are indistinguishable from
their counterparts - for example deletion of the whole short
arm (p) is indistinguishable from a duplication of whole
longer arm (q). It is necessary to perform further analyses (trio analysis,
check with no LR-PCR, test other genes related to CAH) in
complex or not explained cases. This study points out the importance of a multiapproach
analysis based on complementary genetic tests, clinical and
biochemical data to give an accurate diagnosis of CAH. J. Lezana Rosales: None. J. Lopez Montiel: None. C. Sanchez Linares: None. P. Remon Ruiz: None. M. Dovis
Costarelli: None. J. Lopez Siles: None. J. Lezana Rosales: None. J. Lopez Montiel: None. C. Sanchez Linares: None. P. Remon Ruiz: None. M. Dovis
Costarelli: None. J. Lopez Siles: None. J. Lezana Rosales: None. J. Lopez Montiel: None. C. Sanchez Linares: None. P. Remon Ruiz: None. M. Dovis
Costarelli: None. J. Lopez Siles: None. M. Böhmer: None. M. Kucharík: A. Employment (full
or part-time); Significant; Medirex a.s. J. Budiš: A. Employment (full or part-time); Significant; Geneton Ltd. G. Minárik:
A. Employment
(full
or
part-time); J. Lezana Rosales1, J. Lopez Montiel1, C. Sanchez Linares1, P.
Remon Ruiz2, M. Dovis Costarelli1, J. Lopez Siles1 1Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 2Medirex a.s., Bratislava, Slovakia, 3Geneton Ltd.,
Bratislava, Slovakia, 4Department of Computer Science,
Faculty of Mathematics, Physics and Informatics, Comenius
University, Bratislava, Slovakia, 5Slovak Centre of Scientific
and Technical Information, Bratislava, Slovakia, 6Trisomytest
s.r.o., Bratislava, Slovakia, 7Department of Molecular Biology,
Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 8Comenius University Science Park, Bratislava,
Slovakia amplicon method based on LR-PCR enrichment for
CYP21A2 and chimaeras M. Böhmer1, M. Kucharík2, J. Budiš3,4,5, G. Minárik2,6, T. Szemes3,7,8 J. Lezana Rosales1, J. Lopez Montiel1, C. Sanchez Linares1, P. Remon Ruiz2, M. Dovis Costarelli1, J. Lopez Siles1 Copy number variant caller for gonozomal chromosomes S. Nikolaev1, L. Lemmens1, T. Koessler2, J. Blouin1, T.
Nouspikel1 Aim:
Increasing
CNV
detection
resolution
should
increase diagnostic yield, but to our knowledge there are
no published studies on the clinical utility of genome wide
analysis of CNV below the resolution of microarray. To
identify variants previously undetectable by other methods,
and determine the clinical utility of high resolution genome-
wide CNV analysis, we have begun a retrospective review
of previously reported uninformative cases (n=161) using
ClinSV. 1Molecular and Genomic Diagnostics Laboratory, Geneva
University Hospitals, Geneva, Switzerland, 2Department of
Oncology, Geneva University Hospitals, Geneva, Switzerland 1Molecular and Genomic Diagnostics Laboratory, Geneva
University Hospitals, Geneva, Switzerland, 2Department of
Oncology, Geneva University Hospitals, Geneva, Switzerland We present the results of our technical validation process in
establishing analysis of circulating tumor DNA (ctDNA) as
a diagnostic tool in our laboratory. Results: Initial assessment of a small number of previous
negative WGS cases has yielded diagnostic CNVs includ-
ing: 1kb homozygous deletion of SPG7 exon 6 in a patient
with Spastic Paraplegia and a compound heterozygous stop-
gain plus exon 1 deletion of DST in two deceased neonates. Like most cells in our body, tumor cells shed DNA in the
blood flow. Detection of ctDNA and analysis of its
mutational content can provide invaluable information on
the genetic makeup of a tumor, and assist oncologists in
deciding on therapy or in following the evolution of residual
disease. However, the low absolute amounts of circulating
DNA, and the minor fraction of ctDNA within it, constitute
formidable analytical challenges. A key step into ctDNA
detection is to avoid any contamination with genomic DNA
resulting from cell lysis. Several brands of specialized blood
collection tubes are available to prevent leukocyte lysis, but
we found that they are not equally efficient, depending on
storage temperature and on time before plasma preparation. Conclusions: Genome-wide CNV analysis has wider
breadth and higher resolution than any other test available
and increases the diagnostic yield of WGS. It is particularly
relevant to those patients with previous uninformative
genome and exome testing. B. Lundie: A. Employment (full or part-time); Signifi-
cant; Genome.One. A.E. Minoche: A. Employment (full or
part-time);
Significant;
Garvan
Institute
of
Medical
Research. V. Gayevskiy: A. Employment (full or part-
time); Significant; Garvan Institute of Medical Research. E. Lee: A. Employment (full or part-time); Significant;
Genome.One. L. Ewans: A. Employment (full or part-
time); Significant; Genome.One, NSW Health. G. P14.021A P14.021A
Validation of circulating tumor DNA analysis in a
diagnostic laboratory: pre-analytical factors and quality
control S. Nikolaev1, L. Lemmens1, T. Koessler2, J. Blouin1, T. Nouspikel1 P14.019C Copy number variant caller for gonozomal chromosomes Copy number variant caller for gonozomal chromosomes Copy number variant caller for gonozomal chromosomes 502 J. del Picchia A. Employment (full or part-time); Significant; Genone. One. T. Ohnesorg: A. Employment (full or part-time);
Significant; Genome.One. A. Sherstyuk: A. Employment
(full or part-time); Significant; Genome.One. M. Dinger: A. Employment (full or part-time); Significant; Genome.One,
Garvan Institute of Medical Research. B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Garvan Institute of Medical Research. M.J. Cowley: A. Employment (full or part-time); Significant;
Garvan Institute of Medical Research. B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Garvan Institute of Medical Research. L. Burnett: A. Employment (full or part-time); Significant;
Genome.One. B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Shire International. F. Consultant/Advisory Board; Modest; Royal College of
Pathologists Australiasia. A. Employment (full or part-time); Significant; Genone. One. T. Ohnesorg: A. Employment (full or part-time);
Significant; Genome.One. A. Sherstyuk: A. Employment
(full or part-time); Significant; Genome.One. M. Dinger: A. Employment (full or part-time); Significant; Genome.One,
Garvan Institute of Medical Research. B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Garvan Institute of Medical Research. M.J. Cowley: A. Employment (full or part-time); Significant;
Garvan Institute of Medical Research. B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Garvan Institute of Medical Research. L. Burnett: A. Employment (full or part-time); Significant;
Genome.One. B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Shire International. F. Consultant/Advisory Board; Modest; Royal College of
Pathologists Australiasia. Significant; Medirex a.s., Trisomytest s.r.o. T. Szemes: A. Employment (full or part-time); Significant; Geneton Ltd.. P14.020D
Clinical utility of copy number variant (CNV) detection by
whole genome sequencing (WGS) B. Lundie1, A. E. Minoche2, V. Gayevskiy2, E. Lee1, L. Ewans1,
G. Hollway1, T. Ohnesorg1, A. Sherstyuk1, M. Dinger1,2, M. J. Cowley2, L. Burnett1 1Genome.One, Darlinghurst, Australia, 2Garvan Institute of
Medical Research, Darlinghurst, Australia Background: We have recently completed clinical valida-
tion of ClinSV, an analysis pipeline that identifies regions of
CNV utilising evidence from split-reads, discordant pairs
and depth-of-coverage to obtain a comprehensive high
confidence CNV call-set. Clinical validation was performed
against clinical microarray, MLPA and NA12878 gold
standard. No lower limit for size was applied, allowing for
unprecedented detection of CNV. Analytical sensitivity as
assessed against NA12878 was 86.6% and 99.1% for
deletions <500bp and >500bp respectively. Results were
100% concordant for reportable variants previously detec-
ted by microarray and MLPA. Gene testing in CYLD cutaneous syndrome: 5-year data
from the first European service Gene testing in CYLD cutaneous syndrome: 5-year data
from the first European service Pathogenic variants in the Cystic fibrosis (CF) transmem-
brane conductance regulator (CFTR) gene have been linked
to CF, one of the most common life-threatening genetic
diseases. We provide CFTR genotyping by “cascade”
strategy. Initially, we test the most common CF-causing
variants by in-house methods. In next step we use com-
mercial kit (Elucigene® CF-EU2v1). These routine methods
are followed by sequencing to detect rare and unknown
CFTR variants. V. Wilson1, A. Dubois2, M. Arefi3, J. Burn3, D. Bourn1, N. Rajan3 V. Wilson1, A. Dubois2, M. Arefi3, J. Burn3, D. Bourn1, N. Rajan3 1Northern Genetics Service, Newcastle upon Tyne, United
Kingdom, 2Department of Dermatology, Newcastle upon Tyne,
United Kingdom, 3Institute of Genetic Medicine, Newcastle
upon Tyne, United Kingdom Aim: To develop and validate a fast, accurate, cost-
effective method for detecting prevalent CFTR pathogenic
variants based on LightCyclerTechnology (Roche®) with
SimpleProbe® probes (LightSNiP assays, TIB Molbiol). Aim: To develop and validate a fast, accurate, cost-
effective method for detecting prevalent CFTR pathogenic
variants based on LightCyclerTechnology (Roche®) with
SimpleProbe® probes (LightSNiP assays, TIB Molbiol). Patients with CYLD cutaneous syndrome develop multiple
cylindromas,
spiradenomas
or
trichoepitheliomas. The
occurrence of these rare skin tumours that are related to the
hair follicle has been associated with germline mutations in
CYLD. We established a UK wide gene testing service to
detect CYLD mutations in patients fulfilling testing criteria. Here we report outcomes of CYLD gene testing using
Sanger sequencing of coding exons. We received a total of
56 referrals of novel probands for CYLD gene testing over 5
years. We detected pathogenic CYLD mutations in 39 (69%)
cases. Of these, 17 were novel mutations. Predictive cas-
cade testing was done in 13 further cases, of which 6
confirmed the familial mutation. Previous estimates of
mutation positivity ranged from 44-85% depending on the
phenotype of CYLD cutaneous syndrome. Here, using
conservative inclusive criteria, we report a rate of germline
exonic and splice site mutation of 69%. Gene testing served
to confirm the clinical diagnosis, inform genetic testing of
unaffected relatives and in one case to facilitate pre-
implantation genetic diagnosis. At risk patients who were
confirmed to be mutation negative were able to plan their
families without the concern of disease transmission. In
summary, we highlight the experience of a single national
centre performing CYLD testing. S. Nikolaev1, L. Lemmens1, T. Koessler2, J. Blouin1, T.
Nouspikel1 Hollway: We report our analysis of preanalytical factors pertaining
to ctDNA analysis (collection tubes, transportation time and
temperature) and our conclusions in terms of instructions
being provided to prescribing physicians and medical Abstracts from the 51st European Society of Human Genetics Conference: Posters 503 professionals. We also stress the importance of proper DNA
quality control and compare several methods to this aim,
including a differential amplicon length PCR technique
which allows us to determine multiple QC parameters from
minimal amounts of circulating DNA. professionals. We also stress the importance of proper DNA
quality control and compare several methods to this aim,
including a differential amplicon length PCR technique
which allows us to determine multiple QC parameters from
minimal amounts of circulating DNA. Gene testing in CYLD cutaneous syndrome: 5-year data
from the first European service The ability to solve the
majority of cases by sequencing coding exons suggest that
additional assays, required to detect copy number changes
and rearrangements, can be reserved for the investigation of
mutation-negative cases, thus allowing a more judicious use
of resources. Methods: We developed the CFTR RealTime Panel for
use with LightSNiP assays enabling scoring of 11 prevalent
CFTR
pathogenic
variants
(newborn
screening
data). LightSNiP assays were designed by TIB Molbiol (Berlin,
Germany). Panel contains LightSNiP assays pre-plated and
dried-down for convenient storage and stability in Light-
Cycler® 480 Multiwell Plates, 96. LightSNiP assays carried
out RealTime PCR in the LightCycler®480 Instrument. Results: From amplification and detection with specific
probes by melting curve analysis, it is possible to obtain a
visual discrimination of normal and pathogenic alleles in the
homozygous and heterozygous status. The cost of the CFTR
RealTime Panel and Elucigene® is 19€ and 46€ per patient,
respectively. The detection rate of CF-causing variants is
86% and 90%, respectively. Conclusions: Based on the features and the ease of use of
SimpleProbe® technology, we suggest that the LightSNiP
assay is a good strategy for detecting prevalent CFTR
pathogenic variants to provide the clinician correct results in
a short time. I. Valaskova: None. J. Vinohradska: None. R.M. Selingerova: None. M. Ricankova: None. R. Spesna:
None. R. Gaillyova: None. I. Valaskova: None. J. Vinohradska: None. R.M. Selingerova: None. M. Ricankova: None. R. Spesna:
None. R. Gaillyova: None. P14.023C
CFTR RealTime Panel using pre-plated LightSNiP assays,
a good strategy for CFTR genotyping Altogether, these data should provide a useful practical
guide to other diagnostic laboratories wishing to implement
the assay of ct DNA in clinical practice. I. Valaskova1,2, J. Vinohradska1, R. M. Selingerova1, M.
Ricankova1, R. Spesna1, R. Gaillyova1 S. Nikolaev: None. L. Lemmens: None. T. Koessler:
None. J. Blouin: None. T. Nouspikel: None. A. Bruel1,2, A. Vittobello1,2, F. Tran Mau Them1,2, S. Nambot1,2,3,
V. Quéré1,2, P. Kuentz1, J. Thevenon1, M. Assoum1, S.
Moutton1,3, N. Houcinat1,3, N. Jean-Marçais1,3, M. Lefebvre1,2,
A. Mosca-Boidron1,2, P. Callier1,2, C. Philippe1,2, L. Faivre1,3, C.
Thauvin-Robinet1,2,3 Sapporo medical university, Sapporo, Japan Whole-exome sequencing (WES) has proven to be a pow-
erful tool to identify the molecular bases of heterogeneous
conditions such as intellectual disability (ID) and/or multi-
ple congenital abnormalities (MCA). A large number of
results remain non-conclusive, especially for ultra-rare
conditions
that
limit
genotype-phenotype
correlations. International data-sharing was used to identify additional
patients carrying variants in the same gene, in order to draw
definitive conclusions on their implication in the disease. Here, we report our experience using the GeneMatcher
initiative, a data-sharing platform designed to enable con-
nections between clinicians and researchers to help solve
'unsolved' exomes and to identify new genes. Over the last
two years, we have shared 70 candidate genes identified by
WES performed in individuals affected by ID/MCA. We
evaluated the ability to determine the involvement of these
genes and the necessary timeframe: 59/70 genes (84%) were
matched to at least one other mutated individual, and 23
genes recurring in additional affected individuals were
identified as the probable cause of a developmental disease
(39%). The waiting period between submission and the first
match varied, with an overall median of 4 hours. When a
match occurred, the median response time between the first
email to contact a submitter and the response was estimated
at 31 hours. The rapid identification of these new genes
remains essential for clinical characterization, genetic
counselling and for translation to the diagnostic field. GeneMatcher appears to be a very efficient tool to identify
new genes in highly heterogeneous conditions. Introduction: Approximately 25% of congenital disorders
are caused by chromosome abnormalities. The conventional
karyotyping methods such as G-banding and fluorescence
in situ hybridization (FISH) have been used for the diag-
nosis of chromosomal abnormalities. However, they have
several limitations including possible false-negative results,
time-consuming, and requiring an expert’s reading. We
evaluated the low-coverage whole-genome sequencing
(WGS) using a semi-conductor next generation sequencing
technique for genome-wide detection of aneuploidies. Materials and Methods: Genomic DNA was extracted
from peripheral blood or buccal cells from 9 patients
diagnosed with congenital chromosomal abnormalities by
conventional cytogenetic method. WGS library preparation
and low-coverage genome sequencing (0.01-0.04X) were
performed using Ion Torrent platform. Aneuploidies were
detected using Ion Reporter Low-pass whole-genome
aneuploidy analysis pipelines. Results: We detected whole-chromosomal and sub-
chromosomal duplications/deletions from genomic DNA
by low-pass sequencing. We also detected the deletion in
the end of chromosome and duplication at the adjacent
region in one patient. P14.025A Our Genematcher data sharing experience: 10 days on
average to confirm the pathogenicity of a candidate gene H. Fukushima, Y. Sasaki, M. Tamura, A. Ishikawa, M.
Mizukami, T. Tokino, A. Sakurai H. Fukushima, Y. Sasaki, M. Tamura, A. Ishikawa, M. Mizukami, T. Tokino, A. Sakurai H. Fukushima, Y. Sasaki, M. Tamura, A. Ishikawa, M. Mizukami, T. Tokino, A. Sakurai Sapporo medical university, Sapporo, Japan By comparison of the results from
conventional (G-banding) and novel (low-pass WGA)
method, the origin of unknown fragment attached to the
end of the chromosome has been suggested. Conclusions: This study demonstrates that low-coverage
WGS provides highly accurate, low-cost diagnosis of
aneuploidy in cells from patients with congenital chromo-
somal abnormalities. A. Bruel: None. A. Vittobello: None. F. Tran Mau
Them: None. S. Nambot: None. V. Quéré: None. P. Kuentz: None. J. Thevenon: None. M. Assoum: None. S. Moutton: None. N. Houcinat: None. N. Jean-Marçais:
None. M. Lefebvre: None. A. Mosca-Boidron: None. P. Callier: None. C. Philippe: None. L. Faivre: None. C. Thauvin-Robinet: None. H. Fukushima: None. Y. Sasaki: None. M. Tamura:
None. A. Ishikawa: None. M. Mizukami: None. T. Tokino: None. A. Sakurai: None. P14.024D NGS-based approach to detect aneuploidies and large 504 J. del Picchia 1UMR1231 GAD, Inserm, University of Burgundy-Franche
Comté, Dijon, France, 2FHU-TRANSLAD, Université de
Bourgogne/CHU Dijon, Dijon, France, 3Centre of Reference
for Rare Diseases: Development disorders and malformation
syndromes, Genetics Department, FHU-TRANSLAD, Dijon
University Hospital, Dijon, France chromosome aberrations in patients with congenital
cytogenetic abnormalities E. Maurer1, J. Dumfarth2, S. Rudnik1, J. Zschocke1, M. Witsch-
Baumgartner1 Challenging detection of a novel deletion at the exon-intron
junction in MYH11 Challenging detection of a novel deletion at the exon-intron
junction in MYH11 E. Maurer1, J. Dumfarth2, S. Rudnik1, J. Zschocke1, M. Witsch-
Baumgartner1 Thauvin-Robinet1,2,3 Abstracts from the 51st European Society of Human Genetics Conference: Posters 505 1Division of Human Genetics, Medical University Innsbruck,
Innsbruck, Austria, 2University Hospital for Cardiac Surgery,
Medical University Innsbruck, Innsbruck, Austria Introduction: Although complicated by the fragmented
nature of the next-generation targeted resequencing data,
copy number variants (CNVs) may be detected by read-
depth analysis. Along with whole exome sequencing,
sequencing of large panels of disease-associated genes
(Mendeliomes) are widely used in clinical practice. The
diagnostic utility of CNV detection from Mendeliome
sequencing, however, remains underreported. Introduction: Although complicated by the fragmented
nature of the next-generation targeted resequencing data,
copy number variants (CNVs) may be detected by read-
depth analysis. Along with whole exome sequencing,
sequencing of large panels of disease-associated genes
(Mendeliomes) are widely used in clinical practice. The
diagnostic utility of CNV detection from Mendeliome
sequencing, however, remains underreported. Introduction: The detection of deletions comprising mainly
intronic and only small exonic regions are challenging as
they may be missed in exome and gene panel analysis with
standard software (including copy number variation tools)
and analysis settings. Here we report an analysis approach
to overcome this limitation. Materials and Methods: TruSight One panels (Illumina
Inc., San Diego, CA) targeting 4813 genes were sequenced
as a diagnostic test in 1407 consecutive patients at our
department. The indications for testing were not restricted to
any disease group; many patients had undergone previous
molecular testing including chromosomal microarrays. Raw
sequencing reads were mapped to hg19 reference genome. Materials and Methods: TruSight One panels (Illumina
Inc., San Diego, CA) targeting 4813 genes were sequenced
as a diagnostic test in 1407 consecutive patients at our
department. The indications for testing were not restricted to
any disease group; many patients had undergone previous
molecular testing including chromosomal microarrays. Raw
sequencing reads were mapped to hg19 reference genome. In addition to small variants, CNVs were called using
CoNIFER software. All reported CNVs were validated as
appropriate. Patient: We investigated a 37 y old man who was
currently hospitalized due to an iliac artery aneurysm. At
the age of 19 y the patient had undergone surgical
correction of an acute type A aortic dissection. Family
history was negative. Challenging detection of a novel deletion at the exon-intron
junction in MYH11 Method: Massive parallel sequencing of 10 genes:
ACTA2, FBN1, TGFBR1, TGFBR2, TGFB2, SMAD3,
COL3A1,
MYH11,
MYLK,
TGFB3
(GRCh37,
hg19);
library: TruSightTMOne, Illumina; sequencer: NextSeq,
Illumina; data analysis incl. CNV: SeqNext [JSI] and
cnMOPS. Breakpoint detection by long range PCR and
sequencing; RNA analysis after cell culture. Results:
Among
1407
patients
conclusive
genetic
diagnosis was made after Mendeliome sequencing in 327
(23.2%), accompanied by additional 10.8% patients in
whom
variants
of
unclear
clinical
significance
were
reported. Rare CNVs were reported for 30 patients. The
detected CNVs ranged from single exon to contiguous gene
deletions. In additional two cases, X-chromosome aneu-
ploidies were suspected after noticing variant read ratio
discrepancies. In 18 (1.3%) patients, the CNVs were
classified as disease causing, while others remained of
unclear significance. In 4/18 cases, the CNV was identified
in trans with pathogenic small variant. Result: At the exon-intron 29 border of MYH11,
SeqNext called a delins variant followed by unaligned
sequence in a small fraction of reads (26%). The aberrant
sequence was manually aligned to a 3´ intronic sequence,
representing a heterozygous deletion c.3878_3880-85del. This deletion comprises 1773 bp and include the last two
bases of exon 29 and a large part of intron 29. RNA
analysis is ongoing to determine the exact effect on
transcript level. Conclusions: CNV detection improved diagnostic yield
of Mendeliome sequencing by over 1% without increasing
costs significantly, and thus should be encouraged in all
clinical laboratories. Conclusions: The detection of large “mainly” intronic
deletions by panel/exom sequencing is challenging as CNV-
tools are not easily able to characterize them. Heightened
awareness and additional methods such as RNA analysis are
required
for
identification
and
confirmation
of
such
deletions. Conclusions: The detection of large “mainly” intronic
deletions by panel/exom sequencing is challenging as CNV-
tools are not easily able to characterize them. Heightened
awareness and additional methods such as RNA analysis are
required
for
identification
and
confirmation
of
such
deletions. Funding: Estonian Research Council PUT355 Funding: Estonian Research Council PUT355 S. Pajusalu: None. H. Roomere: None. T. Kahre:
None. Ü. Murumets:
None. V. Pata:
None. K. Õunap: None. E. Maurer: None. J. Dumfarth: None. S. Rudnik:
None. J. Zschocke:
None. M. Witsch-
Baumgartner: None. E. Maurer: None. J. Dumfarth: None. S. Rudnik:
None. J. Zschocke:
None. M. Witsch-
Baumgartner: None. S. Pajusalu1,2, H. Roomere1, T. Kahre1,2, Ü. Murumets1, V.
Pata1, K. Õunap1,2 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia P14.028D Developing a Test Directory for Rare and Inherited Disease
Genomic Testing in the National Health Service in England P14.030B R.H. Scott: None. E.R.A. Thomas: None. L. Modiano:
None. C. Turnbull:
None. Z.C. Deans:
None. A. Prudhoe: None. T. Fowler: None. E. Graham: None. M.J. Caulfield: None. S.L. Hill: None. A new powerful PCR-based assay for the molecular
diagnosis of myotonic dystrophy type I A new powerful PCR-based assay for the molecular
diagnosis of myotonic dystrophy type I 1Univ. of Iceland, Reykjavik, Iceland, 2Landspitali - National
University Hospital, Reykjavik, Iceland, 3Lifeind ehf.,
Reykjavik, Iceland, 4Lifeind ehf, Reykjavik, Iceland S. Rondeau, S. Paisley, M. Simon, A. Munnich, J. Steffann, J. Bonnefont S. Rondeau, S. Paisley, M. Simon, A. Munnich, J. Steffann, J. Bonnefont Introduction: Northern Lights Assay (NLA) is a versatile
technique for detecting various types of DNA damage in
body
fluids
including
single-stranded
breaks, double-
stranded breaks, intrastrand and interstrand DNA cross-
links (ICL), single-stranded DNA and bulky lesions. With
NLA we tested whether specific DNA lesions in body fluids
are seen in colon cancer patients treated with a combination
therapy of a platinum crosslinking agent and a thymidylate
synthase inhibitor. Interstrand crosslinked DNA in body fluids of cancer
patients treated with a platinum agent Interstrand crosslinked DNA in body fluids of cancer
patients treated with a platinum agent J. J. Jonsson1,2, H. G. Thormar1,3, B. Gudmundsson1,2,4, H. I. Ragnarsson1, D. Gudmundsson1, H. Sigurdsson1,2 P14.027C
Copy number variant detection increases diagnostic yield of
Mendeliome sequencing The directory contains 350 clinical indications with 452
test method components including 24 indications for whole
genome sequencing. It includes 61 ‘core’ indications,
covering the bulk of testing by volume, to be performed
at each of the new laboratory hubs and 289 ‘specialised’
indications to be performed by a subset. The directory will be a component of a National
Genomics Informatics System that captures all test requests
and results to facilitate monitoring of activity and equity of
access to testing and provide data crucial to ongoing
assessment of optimal testing approaches and other quality
improvement
activities
and,
for
consented
patients,
research. S. Rondeau: C. Other Research Support (supplies,
equipment, receipt of drugs or other in-kind support);
Modest; Asuragen. S. Paisley: None. M. Simon: None. A. Munnich: None. J. Steffann: None. J. Bonnefont: None. P14.027C
Copy number variant detection increases diagnostic yield of
Mendeliome sequencing R. H. Scott1, E. R. A. Thomas1, L. Modiano2, C. Turnbull1, Z. C. Deans2, A. Prudhoe2, T. Fowler1, E. Graham2, M. J. Caulfield1,
S. L. Hill2 1Genomics England, London, United Kingdom, 2NHS England,
London, United Kingdom 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia To enable the National Health Service (NHS) to fully
benefit from the advances in genomics, building on its long
history of laboratory and clinical genetics, NHS England is 506 J. del Picchia DNA
were
isolated
from
different
tissues
(blood,
chorionic villi and amniotic fluid) and amplified by PCR
(DMPK gene specific PCR (GS-PCR) and TP-PCR). FAM-
labeled amplicons were resolved by capillary electrophor-
esis (CE). The AmplideX® PCR/CE assay gave expected
results for the 49 samples (100% sensitivity and specificity)
including resolution of homozygous and heterozygous
samples, and those with large CTG expansions (57-1800
repeats). Furthermore, previously unidentified mosaicism
was revealed in 6 patients. establishing a new national Genomic Medicine Service. The
service will deliver a nationally agreed repertoire of tests set
out in a single, annually refreshed, ‘Test Directory’, cov-
ering the range of genomic testing for Rare and inherited
disease and Cancer from highly targeted testing to whole
genome sequencing. We describe the structured approach used to create the
Rare and inherited disease directory and the contents of the
resulting directory. With the support of experts in each area, ‘Clinical
indications’ for testing were linked to ‘Test scope’ (relevant
variant types and loci) and then ‘Test method(s)’ capable of
testing across the defined scope. A previously presented
evaluative framework was used to inform the optimal
approach in each setting (Scott et al ASHG 2017). Ten samples from DM1-patients (85-1500 repeats) were
further sized by GS-PCR and agarose gel electrophoresis. In
one sample, the expanded allele (1300 CTG) revealed by
Southern blot analysis, was not detected. The AmplideX® PCR/CE DMPK technology is therefore
able to efficiently detect expanded alleles and differentiate
homozygous genotypes from others. It is able to size alleles
with a high resolution from 5 to 200 repeats, detect
expanded alleles up to at least 1,800 repeats, and size
expanded alleles up to at least 1,500 repeats. AmplideX®
PCR/CE DMPK technology appears to be a simpler and
faster approach than conventional techniques for the
molecular DM1 diagnosis. Medical Genetic Unit, Necker-Enfants Malades Hospital,
Paris, France Myotonic dystrophy type 1 (DM1) is caused by expansion
of the CTG repeats in the 3’ UTR of DMPK gene. The
conventional diagnostic strategy is based on a fluorescent
PCR (amplification up to 110 CTG), followed by Southern
Blot for apparently homozygous patients. It is therefore
expensive and time-consuming. We have assessed the
sensitivity and specificity of a new prototype diagnostic
procedure (Asuragen’s AmplideX® PCR/CE DMPK tech-
nology) on 49 samples (7 controls and 42 DM1-patients). Materials and Methods: Randomly selected 27 colon
cancer patients treated with standard CapOx infusion
including capecitabine and oxaliplatin (125 mg/m2). We
collected a complete set of plasma, saliva and urine from
each patient immediately after the infusion, isolated DNA Abstracts from the 51st European Society of Human Genetics Conference: Posters 507 and analyzed with NLA. NLA based on Two-Dimensional
Strandness-Dependent
Electrophoresis
(2D-SDE)
in
microgels. malignancies. Here we describe the comparison of two B-
cell mitogens, lipopolysaccharide (LPS) and DSP30 com-
bined with IL2 as mitogens in a range of common B-cell
disorders excluding CLL. The results showed that DSP30/
IL2 was an effective mitogen in mature B-cell disorders,
revealing abnormal cytogenetic results in a range of B-cell
malignancies. The abnormality rate increased when com-
pared to the use of LPS to 64% (DSP30/IL2) from 14%
(LPS). In a number of cases the disease burden was pro-
portionally very low, less than 10% of white cells. In 37%
of these cases, the DSP30 culture revealed abnormal results. Importantly, we also obtained abnormal conventional
cytogenetics results in 3 bone marrow cases in which
immunophenotyping showed an absence of an abnormal B-
cell clone. In these cases the cytogenetics results correlated
with the provisional diagnosis and altered their staging
level. The use of DSP30 and IL2 is recommended for use in
many B-cell malignancies as an effective mitogen and their
use has been shown to enable successful culture of the
malignant clone, even at very low levels of disease Results: We detected ICL in urinary sediments in all
patients analyzed. ICL were detected in cfDNA in plasma in
7
patients
and
nicked,
double-stranded,
nucleosomal
cfDNA was detected in 9 patients. Of those patients 4 had
both types of DNA lesions. Conclusions: Treatment with the platinum agent oxali-
platin commonly results in interstrand crosslink DNA in
urinary sediment cells and sometimes in plasma cfDNA
right after infusion. Medical Genetic Unit, Necker-Enfants Malades Hospital,
Paris, France The finding of nicked, double- stranded,
nucleosomal cfDNA could result from treatment with the
thymidylate inhibitor capecitabine comprising de novo
DNA synthesis, oxaliplatin and/or from cellular stress. These DNA lesions at different time points after therapy
could be potential biomarkers in cancer theragnostics to
evaluate response to treatment or risk of side-effects. The Icelandic Centre for Research Technology Develop-
ment Fund 142709-0612 J.J. Jonsson: C. Other Research Support (supplies,
equipment, receipt of drugs or other in-kind support);
Significant; Lifeind ehf.. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Significant;
Lifeind ehf. H.G. Thormar: A. Employment (full or part-
time); Significant; Lifeind ehf.. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Lifeind ehf. B. Gudmundsson: A. Employ-
ment (full or part-time); Significant; Lifeind ehf. B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Lifeind ehf. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Lifeind ehf. H.I. Ragnarsson: None. D. Gudmundsson: None. H. Sigurdsson: None. J.J. Jonsson: C. Other Research Support (supplies,
equipment, receipt of drugs or other in-kind support);
Significant; Lifeind ehf.. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Significant;
Lifeind ehf. H.G. Thormar: A. Employment (full or part-
time); Significant; Lifeind ehf.. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Lifeind ehf. B. Gudmundsson: A. Employ-
ment (full or part-time); Significant; Lifeind ehf. B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Lifeind ehf. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Lifeind ehf. H.I. Ragnarsson: None. D. Gudmundsson: None. H. Sigurdsson: None. K. Dun: None. K. Dun: None. P14.031C DSP30 as a mitogen in B-cell malignancies including those
of a low disease burden A gene-agnostic trio exome sequencing strategy
outperforms gene panel analysis A gene-agnostic trio exome sequencing strategy
outperforms gene panel analysis J. Baptista1,2, K. Stals1, E. De Franco1,2, V. Fryer1, M. Wakeling2, A. Parrish1, L. Mallin1, A. Bussell1, J. Settle1, A. C. Gunning1, R. Caswell2, C. Tysoe1, E. Baple1,2, S. Ellard1,2 1Royal Devon and Exeter NHS Foundation Trust, Exeter,
United Kingdom, 2University of Exeter Medical School, Exeter,
United Kingdom The identification of the genetic aetiology in children with
non-specific/heterogeneous phenotypes, or without the full
spectrum of syndromic features, poses a challenge. For
these cases, selection of a gene panel for next generation
sequencing (NGS) can be restrictive whereas a gene-
agnostic inheritance-based whole exome trio offers a more
comprehensive analysis. We analysed 266 consecutive trios
referred for diagnostic testing. Whole exome sequencing
was performed using Agilent (v5/6) capture and Illumina
sequencing (HiSeq2500/ NextSeq500). Rare potentially
deleterious variants were filtered by mode of inheritance
and classified using ACMG-AMP guidelines. We ascer-
tained whether the disease gene was included in at least one
Genomics England PanelApp gene panel. Likely patho-
genic/pathogenic variant(s) were identified in 43% (113/
266) of cases. For 9 patients the disease gene was not
included within a PanelApp gene panel and the maximal
diagnostic yield for a phenotype-driven panel analysis
would have been 39%. In 6 cases the genes were very recent Twist Bioscience, San Francisco, CA, United States Twist Bioscience, San Francisco, CA, United States Introduction: We recently successfully explored rapid
diagnostic whole-genome sequencing in critically ill new-
borns, aiming to improve their clinical care and replace
time-consuming and/or invasive diagnostic testing. Now,
we implemented rapid sequencing in standard diagnostics,
with adapted procedures and an expanded age-range to
include all critically ill children. Whole Exome Sequencing and Target Enrichment approa-
ches provide a powerful solution for effectively focusing
sequencing costs on genomic regions of interest. However,
hybridization-based enrichment can pose various challenges
leading to context-specific biases and lack of uniformity
affecting quality and efficiency. Here we present data
obtained
using
Twist
Bioscience's
new
NGS
Target
Enrichment System, which shows substantial improvements
in multiplexing capacity with single-plex performance, high
uniformity with fold 80 base penalties as low as 1.3, and
near optimal capture across low to high GC content regions. Altogether, these improvements lead to a target coverage of
up to >98% and >95% of bases at 20x and 30x respectively
with only 4.5GB sequencing, a much wider linearly scalable
cost to coverage relation for high-coverage applications,
and >99% precision and sensitivity in variant calling on
both hg38 and the hg19 human assemblies which are
natively supported. These results are possible due to a
carefully designed system including double-stranded bioti-
nylated probes, an optimized stringency workflow, and
efforts focusing on optimizing bait design and target pro-
files. This allows capturing 99% of potentially pathogenic
clinVar variants in just 33MB of coding sequence targets
based on a CCDS exome, or can be incorporated in custom
user designs taking advantage of Twist's unique ability, Materials and Methods: Twenty-five critically ill or
deceased patients under the age of 16 years were included
between April 2017 and February 2018.Trio exome
sequencing was performed using Agilent SureSelect XT
exome v6 capturing . After filtering using a virtual gene-
panel of 3850 monogenic disease genes and inheritance-
mode, selected variants were discussed in a multi-
disciplinary
team. Pathogenic
or
likely
pathogenic
variants matching the patients’ phenotype were reported. Variants
in
genes
not
matching
the
patient’s
phenotype but considered as actionable, were reported
as incidental findings. Materials and Methods: Twenty-five critically ill or
deceased patients under the age of 16 years were included
between April 2017 and February 2018.Trio exome
sequencing was performed using Agilent SureSelect XT
exome v6 capturing . P14.034B
Rapid whole exome sequencing in critically ill children W. S. Kerstjens-Frederikse, A. H. van der Hout, Y. J. Vos, T. Dijkhuizen, J. B. G. M. Verheij, I. M. van Langen, B. Sikkema-
Raddatz, M. J. de Groot, H. H. J. Drok, R. Kinds, G. Beunders,
E. Zonneveld-Huijssoon, J. C. Herkert, F. Vansenne, J. S. Klein
Wassink-Ruiter, E. Gerkes, K. J. van der Velde, M. A. Swertz, R. J. Sinke, C. C. van Diemen K. Dun Cytogenetics, Hobart, Australia Chromosome abnormalities detected during cytogenetic
investigations
for
B-cell
malignancy
offer
prognostic
information that can have wide ranging clinical impacts on
patients. These impacts may include monitoring frequency,
treatment type and disease staging level. The use of the
synthetic oligonucleotide DSP30 combined with interleukin
2 (IL2) has been described as an effective mitotic stimulant
in B-cell disorders, predominantly in chronic lymphocytic
leukaemia
(CLL)
but
also
a
range
of
other
B-cell 508 J. del Picchia flexibility, and fast turnaround time in high quality DNA
synthesis. flexibility, and fast turnaround time in high quality DNA
synthesis. discoveries where our findings have provided further evi-
dence to support the disease gene association (e.g. KLH7,
RHOBTB2 and AIMP2). We identified three strong biolo-
gical candidate genes (NXN, NAXD and PIGB) and via
GeneMatcher found additional cases/collaborations that
subsequently confirmed these as novel disease genes. The
gene-agnostic trio approach using inheritance filtering
enabled a diagnosis for an additional 9 patients (8% of
diagnoses). We will discuss how the fast pace of gene
discovery in the NGS era coupled with the clinical/genetic
heterogeneity provides a strong case for using this strategy
combined with GeneMatcher and future re-analysis to
maximise diagnostic yield. L. Arbiza: A. Employment (full or part-time); Signifi-
cant; Twist Bioscience. S. Chen: A. Employment (full or
part-time); Significant; Twist Bioscience. C. Thompson: A. Employment
(full
or
part-time);
Significant;
Twist
Bioscience. K. Butcher: A. Employment (full or part-
time); Significant; Twist Bioscience. R. Zeitoun: A. Employment
(full
or
part-time);
Significant;
Twist
Bioscience. R. Gantt: A. Employment (full or part-time);
Significant; Twist Bioscience. H. Chilton: A. Employment
(full or part-time); Significant; Twist Bioscience. L. Arbiza: A. Employment (full or part-time); Signifi-
cant; Twist Bioscience. S. Chen: A. Employment (full or
part-time); Significant; Twist Bioscience. C. Thompson: A. Employment
(full
or
part-time);
Significant;
Twist
Bioscience. K. Butcher: A. Employment (full or part-
time); Significant; Twist Bioscience. R. Zeitoun: A. Employment
(full
or
part-time);
Significant;
Twist
Bioscience. R. Gantt: A. Employment (full or part-time);
Significant; Twist Bioscience. H. Chilton: A. Employment
(full or part-time); Significant; Twist Bioscience. P14.034B J. Baptista: None. K. Stals: None. E. De Franco: None. V. Fryer: None. M. Wakeling: None. A. Parrish: None. L. Mallin: None. A. Bussell: None. J. Settle: None. A.C. Gunning: None. R. Caswell: None. C. Tysoe: None. E. Baple: None. S. Ellard: None. J. Baptista: None. K. Stals: None. E. De Franco: None. V. Fryer: None. M. Wakeling: None. A. Parrish: None. L. Mallin: None. A. Bussell: None. J. Settle: None. A.C. Gunning: None. R. Caswell: None. C. Tysoe: None. E. Baple: None. S. Ellard: None. J. Baptista: None. K. Stals: None. E. De Franco: None. V. Fryer: None. M. Wakeling: None. A. Parrish: None. L. Mallin: None. A. Bussell: None. J. Settle: None. A.C. Gunning: None. R. Caswell: None. C. Tysoe: None. E. Baple: None. S. Ellard: None. L. Arbiza, S. Chen, C. Thompson, K. Butcher, R. Zeitoun, R.
Gantt, H. Chilton University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands L. Arbiza, S. Chen, C. Thompson, K. Butcher, R. Zeitoun, R. Gantt, H. Chilton .033
Improving the performance, cost, and flexibility of custom
target enrichment & whole exome sequencing Improving the performance, cost, and flexibility of custom
target enrichment & whole exome sequencing University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands Twist Bioscience, San Francisco, CA, United States After filtering using a virtual gene-
panel of 3850 monogenic disease genes and inheritance-
mode, selected variants were discussed in a multi-
disciplinary
team. Pathogenic
or
likely
pathogenic
variants matching the patients’ phenotype were reported. Variants
in
genes
not
matching
the
patient’s
phenotype but considered as actionable, were reported
as incidental findings. Results: Turnaround time was less than 14 days in all
samples. Pathogenic or likely pathogenic mutations explain-
ing the phenotypes were detected in 9 of the 25 children
(36%)
and
involved
the
MTM1,
PDHA1,
MAP3K7,
KCNQ2, PTPN11, KCNT1, TBCK and CRLF1 and CHD7
genes. Incidental findings with clinical consequences were
found and communicated in two families. 509 Abstracts from the 51st European Society of Human Genetics Conference: Posters Conclusions: Rapid trio-exome sequencing is feasible
and has a high yield (36%) and a speed comparable with
whole genome sequencing with targeted panel analysis. Conclusions: Our results show that deep WES (100x)
with least biased technologies can provide similar coverage
(97% of 10x bases) and CDS variant discovery to the
standard 30x WGS. Both WES and WGS are effectively
incapable of variant discovery in low mappability regions of
the exome due to limitations of short read technology. Our
study may help to select the resequencing approach for
human genetics studies. W.S. Kerstjens-Frederikse: None. A.H. van der Hout:
None. Y.J. Vos: None. T. Dijkhuizen: None. J.B.G.M. Verheij: None. I.M. van Langen: None. B. Sikkema-
Raddatz: None. M.J. de Groot: None. H.H.J. Drok:
None. R. Kinds: None. G. Beunders: None. E. Zonne-
veld-Huijssoon: None. J.C. Herkert: None. F. Vansenne:
None. J.S. Klein Wassink-Ruiter: None. E. Gerkes:
None. K.J. van der Velde: None. M.A. Swertz: None. R. J. Sinke: None. C.C. van Diemen: None. This study was supported by RSF grant no. 14 50 00069. Y. Barbitoff: None. D. Polev: None. I. Shcherbakova:
None. A. Kiselev: None. A. Glotov: None. E. Serebrya-
kova: None. A. Kostareva: None. O. Glotov: None. A. Predeus: None. A. Maver, B. Peterlin A. Maver, B. Peterlin P14.036D Systematic dissection of biases in whole exome and whole
genome strategies for human genome resequencing reveals
major determinants of human coding sequence coverage Increased burden of possibly pathogenic variants in
disease-associated genes in patients with negative exome
sequencing result Increased burden of possibly pathogenic variants in
disease-associated genes in patients with negative exome
sequencing result Y. Barbitoff1,2,3, D. Polev1, I. Shcherbakova1, A. Kiselev4, A. Glotov1, E. Serebryakova1, A. Kostareva4, O. Glotov5, A. Predeus2 Y. Barbitoff1,2,3, D. Polev1, I. Shcherbakova1, A. Kiselev4, A. Glotov1, E. Serebryakova1, A. Kostareva4, O. Glotov5, A. Predeus2 Clinical institute of Medical Genetics, Ljubljana, Slovenia Clinical institute of Medical Genetics, Ljubljana, Slovenia 1Biobank of the Research Park, Saint-Petersburg State
University, Saint Petersburg, Russian Federation,
2Bioinformatics Institute, Saint Petersburg, Russian
Federation, 3Department of Genetics and Biotechnology,
Saint-Petersburg State University, Saint Petersburg, Russian
Federation, 4Almazov National Medical Research Centre,
Saint Petersburg, Russian Federation, 5City Hospital #40,
Saint Petersburg, Russian Federation 1Biobank of the Research Park, Saint-Petersburg State
University, Saint Petersburg, Russian Federation,
2Bioinformatics Institute, Saint Petersburg, Russian
Federation, 3Department of Genetics and Biotechnology,
Saint-Petersburg State University, Saint Petersburg, Russian
Federation, 4Almazov National Medical Research Centre,
Saint Petersburg, Russian Federation, 5City Hospital #40,
Saint Petersburg, Russian Federation Although exome sequencing has considerably improved
diagnostic yield in patients with suspected genetic diseases,
it is still not possible to identify a conclusively causative
genetic variant in a notable proportion of patients. In these
cases, we nevertheless observe multiple residual findings in
genes associated with referral phenotype, including variants
of unknown significance and carriership of known patho-
genic and known risk factor variants. To assess the rele-
vance of these findings, we studied if patients with negative
or inconclusive diagnostic exome result carry an excess of
variants in genes associated with referral diagnosis. We
selected 196 patients with negative diagnostic exome result
from five broad disease groups, including hypertrophic
cardiomyopathy, skeletal myopathies, hearing loss, epilepsy
and Parkinson disease (PD). For each group, we system-
atically assessed the burden of variants in disease-associated
genes and compared this rate in patients versus controls. We
observed a consistent excess of known pathogenic and loss-
of-function variants in disease-associated gene panel for all
five surveyed disease categories. The difference was most
notable in case of PD, where patients had on average 5.2
times more variants in PD-associated gene panel versus
controls, followed by hypertrophic cardiomyopathy (1.7x),
myopathies (1.4x), hearing loss (1.4x) and epilepsy (1.1x). In conclusion, we observed an overall excess of possibly
pathogenic variants in disease-associated genes in patients
with negative or inconclusive exome results. This suggests
that residual findings in exome data might be relevant in
these patients and further stresses the need for improvement Introduction: Advantages and diagnostic effectiveness of
whole exome (WES) vs. whole genome (WGS) sequencing
in research and clinical practice are still heavily debated. In
our study, we took an effort to systematically assess cov-
erage of CDS regions provided by several modern WES
platforms and a PCR-free WGS kit. P14.037A Y. Gurovich:
A. Employment
(full
or
part-time);
Significant; FDNA Inc. Y. Hanani: A. Employment (full
or part-time); Significant; FDNA Inc. O. Bar: A. Employ-
ment (full or part-time); Significant; FDNA Inc. L. Basel-
Salmon: F. Consultant/Advisory Board; Significant; FDNA
Inc. P.M. Krawitz: A. Employment (full or part-time);
Modest; FDNA inc. S.B. Kamphausen: None. M. Zenker:
None. N. Fleischer: A. Employment (full or part-time);
Significant; FDNA Inc. D. Gelbman: A. Employment (full
or part-time); Significant; FDNA Inc. L.M. Bird: F. Consultant/Advisory Board; Modest; FDNA Inc. K.W. Gripp:
A. Employment
(full
or
part-time);
Modest;
FDNA inc. Y. Gurovich:
A. Employment
(full
or
part-time);
Significant; FDNA Inc. Y. Hanani: A. Employment (full
or part-time); Significant; FDNA Inc. O. Bar: A. Employ-
ment (full or part-time); Significant; FDNA Inc. L. Basel-
Salmon: F. Consultant/Advisory Board; Significant; FDNA
Inc. P.M. Krawitz: A. Employment (full or part-time);
Modest; FDNA inc. S.B. Kamphausen: None. M. Zenker:
None. N. Fleischer: A. Employment (full or part-time);
Significant; FDNA Inc. D. Gelbman: A. Employment (full
or part-time); Significant; FDNA Inc. L.M. Bird: F. Consultant/Advisory Board; Modest; FDNA Inc. K.W. Gripp:
A. Employment
(full
or
part-time);
Modest;
FDNA inc. Y. Gurovich1, Y. Hanani1, O. Bar1, L. Basel-Salmon2,3, P. M.
Krawitz4, S. B. Kamphausen5, M. Zenker5, N. Fleischer1, D.
Gelbman1, L. M. Bird6, K. W. Gripp7 Y. Gurovich1, Y. Hanani1, O. Bar1, L. Basel-Salmon2,3, P. M. Krawitz4, S. B. Kamphausen5, M. Zenker5, N. Fleischer1, D. Gelbman1, L. M. Bird6, K. W. Gripp7 1FDNA Inc, Boston, MA, United States, 2Recanati Genetic
Institute, Rabin Medical Center & Schneider Children’s
Medical Center, Petach Tikva, Israel, 3Sackler Faculty of
Medicine, Tel Aviv, Israel, 4Institute for Genomic Statistic and
Bioinformatics, University Hospital Bonn,Rheinische-
Friedrich-Wilhelms University, Bonn, Germany, 5Institute of
Human Genetics, University Hospital Magdeburg, Magdeburg,
Germany, 6Department of Pediatrics, University of California,
San Diego, CA, United States, 7Division of Medical Genetics,
A. I. du Pont Hospital for Children/Nemours, Wilmington, DE,
United States Clinical institute of Medical Genetics, Ljubljana, Slovenia Materials and Methods: We performed bioinformatic
analysis of 312 WES samples sequenced with four
technologies
(Agilent
SureSelect,
Roche
SeqCap
EZ
MedExome, Illumina Nextera Rapid Capture, and Illumina
TruSeq Exome), as well as 17 PCR-free WGS samples. Materials and Methods: We performed bioinformatic
analysis of 312 WES samples sequenced with four
technologies
(Agilent
SureSelect,
Roche
SeqCap
EZ
MedExome, Illumina Nextera Rapid Capture, and Illumina
TruSeq Exome), as well as 17 PCR-free WGS samples. Results: We identified the ~400 kb region of human
exome that could not be effectively characterized using
short (2x150) read technology and b37 human genome
reference. Using several novel metrics to characterize exon
coverage in WES and WGS, we showed that some of the
WES
platforms
(SureSelect
and
MedExome)
achieve
substantially less biased CDS coverage than others, with
lower within- and between-interval variation and GC-
content bias. We also showed that the overall power for
SNP and indel discovery in CDS region is indistinguishable
for WGS and best WES platforms. 510 J. del Picchia of variant interpretation process, especially by sharing data
across multiple institutions. of variant interpretation process, especially by sharing data
across multiple institutions. Conclusions: We suggest that this form of artificial
intelligence is ready to support medical genetics in clinical
and laboratory practices and will play a key role in the
future of precision medicine. A. Maver: None. B. Peterlin: None. A. Maver: None. B. Peterlin: None. A. Maver: None. B. Peterlin: None. Extraction and sequencing of DNA from human tissue fixed
and stored in formalin Extraction and sequencing of DNA from human tissue fixed
and stored in formalin A. Alqahtani1, A. Skelton2, L. Eley1, S. Annavarapu1,3, D. Henderson1, B. Chaudhry1 Introduction: Advances in machine-learning, computer
vision and deep-learning enable computerized systems to
prioritize variants through recognition of syndromic phe-
notypes. One of the main challenges for such a system is
being able to generalize well for hundreds of (individually
rare) genetic syndromes from small amounts of data. 1Cardiovascular Research Centre, Newcastle upon Tyne,
United Kingdom, 2Bioinformatics Support Unit, Newcastle
upon Tyne, United Kingdom, 3Department of Cellular
Pathology, Newcastle upon Tyne, United Kingdom Introduction: Formalin is widely used to preserve human
tissues for pathological investigation. However, the DNA
isolated is usually fragmented and corrupted, limiting its use
for diagnosis or research. We wanted to extract high-quality
DNA from formalin-fixed tissue suitable for use in Sanger
and next-generation exome sequencing. Methods: DeepGestalt is a machine-learning framework
that uses deep convolutional neural networks (DCNNs) to
score genetic syndromes by analyzing frontal facial 2D-
photos. The framework was trained stepwise: First, face
location and facial landmarks were automatically detected,
followed by face alignment and cropping into multiple
regions. Then a general face representation was learned
using DCNNs and used to train a specialized multiple
DCNN. Results are aggregated and sorted to obtain the final
ranked list of syndromes. DeepGestalt was trained on a
phenotype-genotype database curated by clinical geneticists
using a community-driven tool called Face2Gene (FDNA
Inc, USA) and is the framework powering this tool. Methods: Using human tissue stored in formalin for
12 months, we created a novel protocol to extract high-
quality DNA, combining proteinase-K and heating with
Chelex resin. We also evaluated the effect of uracil-DNA-
glycosylase (UDG), an enzyme suggested to remove
formalin artefacts. The yield, purity, fragment size distribu-
tion, efficacy as a template for PCR and sequences of PCR
products
were
compared. Using
this
protocol,
next-
generation whole exome sequencing was performed on
DNA extracted from an explanted heart that had been stored
in formalin for over two years. The results were compared
with those from freshly obtained DNA (blood) and the
utility of Sanger sequencing as a confirmatory strategy was
also evaluated. Results: 1. Multiclass: 502 images of randomly sampled
diagnosed cases, yielded 91% top-10-accuracy for 216
different syndromes. 2. Extraction and sequencing of DNA from human tissue fixed
and stored in formalin Noonan genes: Clinicians and
DeepGestalt were asked to classify photos of patients
diagnosed with any of 5 gene-mutations in Noonan
Syndrome, yielding no recognition by clinicians while
DeepGestalt had 64%-top-1-accuracy, compared to a 20%-
random chance. 3. Yes/No type testing recognizing Angel-
man Syndrome, yielding 71%-accuracy by clinicians while
DeepGestalt identified 92%. Results: 1. Multiclass: 502 images of randomly sampled
diagnosed cases, yielded 91% top-10-accuracy for 216
different syndromes. 2. Noonan genes: Clinicians and
DeepGestalt were asked to classify photos of patients
diagnosed with any of 5 gene-mutations in Noonan
Syndrome, yielding no recognition by clinicians while
DeepGestalt had 64%-top-1-accuracy, compared to a 20%-
random chance. 3. Yes/No type testing recognizing Angel-
man Syndrome, yielding 71%-accuracy by clinicians while
DeepGestalt identified 92%. Results
and
Conclusions:
High-quality
DNA
was
obtained from formalin-fixed tissue using our protocol. This increased the PCR product length obtained from 200 to Abstracts from the 51st European Society of Human Genetics Conference: Posters 511 400bp. However, whilst UDG removed some artefacts it
introduced others. Results of NGS exome sequencing on
formalin-fixed DNA compared favourably to fresh DNA
with 94% specificity, but 73% sensitivity. Thus, it is
possible
to
obtain
high-quality
DNA
template
from
formalin-fixed samples, but limited sensitivity to detect all
variants limits its use to confirmation of known, rather than
discovery of novel, variants.Supported by Saudi Arabia
Cultural Bureau and Ministry of Education Conclusions: We show that single-tube, long-range RP-
PCR can be adapted to different repeat sequences using
high-resolution, easy-to-use fragment sizing platforms. This
flexibility may accelerate the development and adoption of
STR-based assays for clinical research and molecular
diagnostics. *For Research Use Only, Not for Use in Diagnostic
Procedures *For Research Use Only, Not for Use in Diagnostic
Procedures G.J. Latham:
A. Employment
(full or part-time);
Significant; Asuragen, Inc. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Asuragen, Inc. C. Redmond: A. Employment
(full or part-time); Significant; Asuragen, Inc. K. Nichol-
son: A. Employment (full or part-time);
Significant;
Asuragen, Inc. E. Lagow: A. Employment (full or part-
time); Significant; Asuragen, Inc. J. Wallace: A. Employ-
ment (full or part-time); Significant; Thermo Fisher, Inc. E. Schreiber: A. Employment (full or part-time); Significant;
Thermo Fisher, Inc. S. Higdon: A. Employment (full or
part-time); Significant; Thermo Fisher, Inc. E. Bram: A. Employment (full or part-time); Significant; Asuragen, Inc. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Asuragen, Inc. G.J. Extraction and sequencing of DNA from human tissue fixed
and stored in formalin Latham:
A. Employment
(full or part-time);
Significant; Asuragen, Inc. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Asuragen, Inc. C. Redmond: A. Employment
(full or part-time); Significant; Asuragen, Inc. K. Nichol-
son: A. Employment (full or part-time);
Significant;
Asuragen, Inc. E. Lagow: A. Employment (full or part-
time); Significant; Asuragen, Inc. J. Wallace: A. Employ-
ment (full or part-time); Significant; Thermo Fisher, Inc. E. Schreiber: A. Employment (full or part-time); Significant;
Thermo Fisher, Inc. S. Higdon: A. Employment (full or
part-time); Significant; Thermo Fisher, Inc. E. Bram: A. Employment (full or part-time); Significant; Asuragen, Inc. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Asuragen, Inc. A. Alqahtani: None. A. Skelton: None. L. Eley: None. S. Annavarapu:
None. D. Henderson:
None. B. Chaudhry: None. P14.039C Generalization of an Assay System using Repeat-primed
PCR and Capillary Electrophoresis to Resolve Multiple
AT- and GC-rich Short Tandem Repeats Associated with
Neurological Diseases G. J. Latham1, C. Redmond1, K. Nicholson1, E. Lagow1, J. Wallace2, E. Schreiber2, S. Higdon2, E. Bram1 1Asuragen, Inc., Austin, TX, United States, 2Thermo Fisher,
Inc., South San Francisco, CA, United States 1Asuragen, Inc., Austin, TX, United States, 2Thermo Fisher,
Inc., South San Francisco, CA, United States Impact of improving gene annotation on diagnostic yield of
Mendelian disorders Impact of improving gene annotation on diagnostic yield of
Mendelian disorders Introduction: DNA repeat sequences constitute roughly
50% of the human genome. Short tandem repeats (STRs)
impact human disease through variable length effects on
gene expression, splicing, and translation. Over 30 Men-
delian disorders are associated with STR expansions, yet
diagnostic applications have often been thwarted by a lack
of rapid, simple, and accurate assay systems. Here we
describe the analytical performance of multiple repeat-
primed (RP) PCR assays* resolved on the SeqStudio
Genetic Analyzer. D. Zhang1, S. Guelfi1, A. E. Jaffe2, L. Collado-Torres2, M. Ryten1 D. Zhang1, S. Guelfi1, A. E. Jaffe2, L. Collado-Torres2, M. Ryten1 1Institute of Neurology (UCL), London, United Kingdom,
2Lieber Institute for Brain Development, Baltimore, MD,
United States 1Institute of Neurology (UCL), London, United Kingdom,
2Lieber Institute for Brain Development, Baltimore, MD,
United States Genetic diagnosis of Mendelian disorders requires clinical
scientists to distinguish pathogenic variants from many
potentially functional variants present in any human gen-
ome. Variant interpretation is reliant on gene annotation;
however, as our understanding of transcriptomic complexity
improves it is apparent that existing annotation is incom-
plete. Comparison of Ensembl and AceView annotations
reveals over 15,800 genes that lie in intronic or intergenic
regions according to the other database. Consequently,
incorrect or incomplete annotation may cause pathogenic
variants to be misassigned, hidden amongst false positives
or overlooked within mis-annotated non-coding regions. To
address this problem, we used publicly available tran-
scriptomic data to improve the annotation of 2806 OMIM-
morbid genes. We obtained annotation-agnostic quantifi-
cation of transcription from 54 GTEx tissues using recount2
and combined this with RNA-seq reads spanning exon-exon
junctions to link putatively transcribed regions to known Materials and Methods: Mono-, tri-, and hexa- nucleo-
tide repeat expansions from AT- and GC-rich STR disease
markers (TOMM40, FMR1 and C9orf72) were amplified
from clinically-derived samples (n=49) using AmplideX®
PCR/CE kits* (Asuragen). FAM-labeled amplicons were
resolved
on
SeqStudio
and
3500
Genetic
Analyzers
(Thermo Fisher). Results: All genotype results were concordant between
instruments and deviations were at most one repeat within
the sizeable range. Tunable run conditions extended repeat
quantification by 25% (to 180 C9orf72 G4C2 repeats) from
baseline conditions, enhanced expanded peak detection
sensitivity by up to 100-fold, and enabled rapid run times as
short as 20 min. Further, different assays could be analyzed
on the same plate to improve batch run efficiency. A. Berndt, C. Oberkanins, H. Puehringer, S. Németh ViennaLab Diagnostics GmbH, Vienna, Austria D. Zhang: None. S. Guelfi: None. A.E. Jaffe: None. L. Collado-Torres: None. M. Ryten: None. Introduction: PCR is known to work well on crude and
complex templates unless interfering substances are present. In case of blood, it is essential to remove heme, which is a
potent inhibitor of most DNA polymerases. Impact of improving gene annotation on diagnostic yield of
Mendelian disorders Studies
are ongoing to assess STRs in myotonic dystrophy type 1
and Huntington’s disease. J. del Picchia 512 OMIM genes. We characterised these transcribed regions
using sequence properties (leveraging information from
ENCODE and using ORFfinder) and their expression in
relation to the reference gene. Despite OMIM genes having
been extensively studied, we discovered over 5Mb of
additional unannotated transcription within 50Kb of an
OMIM gene, which predominantly fell within intronic
regions (72%). Restricting our analysis to non-overlapping
genes, we find that 0.35Mb of transcribed regions connect
to known OMIM isoforms, many of which are expressed
within disease relevant tissues. Overall, we improve the
annotation of OMIM genes, a vital step for the accurate
assignment of variant pathogenicity, and anticipate that this
will lead to improvements in diagnostic yield from whole
genome sequencing. myopathy
(n=1,
ISPD),
Myelination
disorder
(n=1,
EIF2B5), congenital disorder of deglycosylation (n=1,
NGLY1), and Axenfeld-Rieger syndrome (n=1, PITX2). Therefore, in this study, we demonstrated the possibility of
shortening the diagnosis duration of WES for ICU patients
with potentially genetic disease. This project is funded by
the Mistry of Health and Welfare. W. Hwu: None. Y. Chien: None. N. Lee: None. T. Chen: None. J. Hsu: None. F. Lai: None. P14.041A
Rapid genetic diagnosis employing whole exome sequencing
for critical illness in infants and children Rapid genetic diagnosis employing whole exome sequencing
for critical illness in infants and children Material and Methods: We have developed a simple,
inexpensive and time-saving method to skip DNA extrac-
tion when using Taqman-based ViennaLab RealFast assays
for genotyping and variant detection on fresh and frozen
blood. After a 10 min heat denaturation and quick
centrifugation step, the resulting supernatant is diluted into
direct-to-PCR (D2PCR) buffer and directly used as template
in PCR. W. Hwu1, Y. Chien1, N. Lee1, T. Chen2, J. Hsu2, F. Lai3 W. Hwu1, Y. Chien1, N. Lee1, T. Chen2, J. Hsu2, F. Lai3 1Department of Pediatrics, National Taiwan University
Hospital, Taipei, Taiwan, 2Department of Computer Science &
Information Engineering, National Taiwan University, Taipei,
Taiwan, 3Department of Pediatrics, Taipei, Taiwan Critical illness in infants and children is one of the most
challenging fields in maternal and children health. Because
precise managements in respective to the diseases are often
not possible in patients with genetic diseases, the prognosis
can be poor even though a large amount of resources are
consumed. This project employs rapid whole exome
sequencing (WES) and artificial intelligence-assisted data
processing to fit the needs of acute disease managements. The inclusion criteria include patients admitted to intensive
care unit and suspecting to have genetic etiologies, and/or
inborn error of metabolism. A familial trio were sampled for
WES analysis. From May 2017 to Dec 2017, 23 patients
were enrolled for WES trio analysis. The clinical diagnosis
of
enrollment
includes
shock
(n=4),
seizure
(n=7),
Respiratory failure (n=5), Sudden infant death syndrome
(n=1), Abnormal newborn screening (n=2), and Undiag-
nosed disease with multiple systemic involvement (n=4). Of them, 13 (56.5%) had molecular diagnosis. The mean
duration of turnaround time to have tentative diagnosis is
was 6.3±1.4 days (range 4.3-9.9 days). Disease causing
genes identified included Immunodeficiency (n=2; CD3E
and C3), Cardiomyopathy (n=2; 2 COQ4 cases), Channo-
pathy (n=3; KCNQ5, RYR2, and SCN8A), Peroxisomal
biogenesis disorder (n=2, PEX1 and PEX7), congenital Critical illness in infants and children is one of the most
challenging fields in maternal and children health. Because
precise managements in respective to the diseases are often
not possible in patients with genetic diseases, the prognosis
can be poor even though a large amount of resources are
consumed. This project employs rapid whole exome
sequencing (WES) and artificial intelligence-assisted data
processing to fit the needs of acute disease managements. A simple and fast method for direct-to-PCR genotyping A simple and fast method for direct-to-PCR genotyping A. Berndt, C. Oberkanins, H. Puehringer, S. Németh Widespread uptake of the Human Phenotype Ontology
(HPO) in the National Health Service (NHS) in England as
part of the 100,000 Genomes Project Widespread uptake of the Human Phenotype Ontology
(HPO) in the National Health Service (NHS) in England as
part of the 100,000 Genomes Project 1Inserm UMR 1231 GAD team, Genetics of Developmental
disorders, Université de Bourgogne-Franche Comté, Dijon,
France, 2. Service de Génétique, CHU de Grenoble, Grenoble,
France, 3CHU Dijon, FHU TRANSLAD, Dijon, France,
4Centre National de Recherche en Génomique Humaine
(CNRGH), CEA, Evry, France, 5Service de Génétique,
Laboratoire de Cytogénétique, CHU de Lyon, HCL, Lyon,
France, 6CHU Dijon, Centre de référence Anomalies du
Développement et Syndromes Malformatifs, Centre de
Génétique, Dijon, France 1Inserm UMR 1231 GAD team, Genetics of Developmental
disorders, Université de Bourgogne-Franche Comté, Dijon,
France, 2. Service de Génétique, CHU de Grenoble, Grenoble,
France, 3CHU Dijon, FHU TRANSLAD, Dijon, France,
4Centre National de Recherche en Génomique Humaine
(CNRGH), CEA, Evry, France, 5Service de Génétique,
Laboratoire de Cytogénétique, CHU de Lyon, HCL, Lyon,
France, 6CHU Dijon, Centre de référence Anomalies du
Développement et Syndromes Malformatifs, Centre de
Génétique, Dijon, France E. R. A. Thomas1, A. Devereau1, H. Brittain1, A. Tucci1, M. Ryten1,2, D. Smedley1, A. Rendon1, M. J. Caulfield1, R. H. Scott1 1Genomics England, Queen Mary University of London,
London, United Kingdom, 2Institute of Neurology, University
College London, London, United Kingdom The 100,000 Genomes Project is an NHS transformation
project which is founding a national research database
linking genomic and clinical data from thousands of
patients with rare disease or cancer. The rare disease pro-
gramme recruits individuals across >200 recruitment cate-
gories, spanning the majority of monogenic diseases. Phenotype data have been collected primarily as HPO
terms. For each recruitment category, a ‘questionnaire’ of
relevant HPO terms (1 to 82 per category, median 26.5) is
presented to clinicians in an electronic data capture tool,
with the facility to add extra terms where relevant. Pheno-
typic data have been collected on >20,000 participants with
a mean of 6 positive and 20 negative terms from around
1,000 contributing clinicians. HPO terms have directed
application of gene panels beyond the recruited disorder,
with 24% of diagnostic variants lying within these addi-
tional panels. On review of the data, 2% of families needed
further clarification or enrichment of the terms initially
entered to inform the analysis. HPO functions better in
some clinical contexts (e.g. paediatric developmental dis-
orders) than in others (e.g. inherited cancer, due to limited
capture of histological subtypes and precise age of onset). P14.041A
Rapid genetic diagnosis employing whole exome sequencing
for critical illness in infants and children Results: The D2PCR approach for RealFast assays can be
performed on any common qPCR instrument capable of
detecting FAM/HEX (single-plex assays) or FAM/HEX/
ROX/Cy5 (duplex assays) fluorescence. Optimal time
saving is achieved only when combining D2PCR testing
with fast mode thermocycling on the Magnetic Induction
Cycler (MIC; Bio Molecular System). Using magnetic
induction technology for heating and fan forced air for
cooling, 40 cycle PCR runs can be shortened to 40 min, and
the analysis of samples from drawing blood to final result
can be completed in less than one hour. Results: The D2PCR approach for RealFast assays can be
performed on any common qPCR instrument capable of
detecting FAM/HEX (single-plex assays) or FAM/HEX/
ROX/Cy5 (duplex assays) fluorescence. Optimal time
saving is achieved only when combining D2PCR testing
with fast mode thermocycling on the Magnetic Induction
Cycler (MIC; Bio Molecular System). Using magnetic
induction technology for heating and fan forced air for
cooling, 40 cycle PCR runs can be shortened to 40 min, and
the analysis of samples from drawing blood to final result
can be completed in less than one hour. Conclusions: Reduction of the turnaround, and in
particular the hands-on time for molecular genetic assays
is key to increasing the capacities and daily throughput of a
diagnostic laboratory. As a consequence, costs can be
reduced
and
delivering
reports
to
patients
can
be
accelerated. A. Berndt: A. Employment (full or part-time); Signifi-
cant; ViennaLab Diagnostics. C. Oberkanins: A. Employ-
ment
(full
or
part-time);
Significant;
ViennaLab
Diagnostics. H. Puehringer: A. Employment (full or part-
time);
Significant;
ViennaLab
Diagnostics. S. Németh: None. 513 Abstracts from the 51st European Society of Human Genetics Conference: Posters P14.043C
Integrated functional characterization of a non-coding
GPC3 allele in a family with recurrent Simpson-Golabi-
Behmel syndrome condition with clear clinical delineation and negative or
inconclusive routine laboratory results. condition with clear clinical delineation and negative or
inconclusive routine laboratory results. A. Vitobello: None. J. Thevenon: None. P. Callier:
None. T. Jouan: None. L. Duplomb: None. M. Bordes-
soules: None. Y. Duffourd: None. E. Tisserand: None. A. Boland: None. R. Olaso: None. J. Deleuze: None. D. Sanlaville: None. C. Thauvin: None. L. Faivre: None. A. Vitobello1, J. Thevenon2, P. Callier1,3, T. Jouan1, L. Duplomb1, M. Bordessoules1, Y. Duffourd1, E. Tisserand1, A. Boland4, R. Olaso4, J. Deleuze4, D. Sanlaville5, C. Thauvin1,3,6,
L. Faivre1,3,6 Widespread uptake of the Human Phenotype Ontology
(HPO) in the National Health Service (NHS) in England as
part of the 100,000 Genomes Project While the validity of genetic imputa-
tion algorithms has been determined, the limitations to
imputation reference panels from short-read sequencing
inaccessibility has not been systematically assessed. Here,
we investigated these limitations and their impact on
GWAS in the Cooperative Health Research In South Tyrol
(CHRIS) study, a population based study with 4,661 gen-
otyped and phenotyped individuals. By comparing imputed
genotypes to microarray genotypes in the CHRIS study, we
demonstrated a low quality of imputation for variants pre-
sent in regions defined by the 1000 Genomes Project Phase
3 as inaccessible to short-read sequencing. Screening the
summary statistics of 58 biochemical trait GWASs per-
formed in the CHRIS study, we detected an aspartate ami-
notransferase associated locus in an inaccessible region that
is driven by microarray genotyped variants, and cannot be
reproduced
through
1000
Genomes
imputation. Con-
sistently, in the publicly available NHGRI-EBI GWAS
catalog and in UK Biobank GWASs, we observed a lower
density of trait-associated variants in inaccessible regions
when compared to accessible regions. In summary, we
show that the nature of short-read sequencing affects the
ability of GWASs to discover trait associated loci in inac-
cessible regions. Widespread uptake of the Human Phenotype Ontology
(HPO) in the National Health Service (NHS) in England as
part of the 100,000 Genomes Project Using set HPO questionnaires standardises the pattern of
data capture between users and encourages depth of capture
of positive and negative features; however, this approach
may limit the capture of additional relevant phenotypes and
introduce bias into the dataset. Our experience indicates that
HPO is an intuitive and rich ontology which can be used at
scale in frontline healthcare. Sipson-Golabi-Behmel syndrome type 1 (SGBS1 -MIM
312870) is an X-linked condition occurring primarily in
males, characterized by pre- and postnatal overgrowth,
coarse facial features, variable intellectual disability, con-
genital heart defects, supernumerary nipples, hepatomegaly
and increased risk to develop neoplasia such as Wilms
tumor. SGBS1 is usually caused by deletion or mutation in
the gene encoding glypican-3 (GPC3), an outer membrane
glycoprotein implicated in WNTs, Hedgehogs (Hh), fibro-
blast growth factors (FGF), and bone morphogenetic pro-
teins (BMP) signaling modulation. To identify the genetic
variant responsible for recurrent cases of Simpson-Golabi-
Behmel syndrome type 1 within a family with negative
whole exome sequencing (WES) results, we performed an
integrated genomic and transcriptional analysis. Whole
genome sequencing (WGS) coupled with patient-derived
fibroblasts RNA expression analysis was performed in order
to interrogate non-coding regulatory mechanisms, poten-
tially accounting for dysregulation of GPC3, GPC4,
CXORF5 or other candidate genes implicated in overgrowth
syndromes for a differential analysis. WGS allowed us to
identify the presence of a complex rearrangement resulting
in a duplication of Xq24 with concomitant deletion in
Xq26.2 corresponding to a portion of the second intron of
the gene GPC3. RNA expression not only revealed GPC3
loss of expression, but also allowed the identification of
SGBS-specific transcriptional pathways associated with
glypican-3 knockout. We provide evidence of the utility of
genome-wide genetic and expression analyses as com-
plementary approaches to interrogate the transcriptional
readout of non-coding variants in an ultra-rare genetic E.R.A. Thomas:
None. A. Devereau:
None. H. Brittain: None. A. Tucci: None. M. Ryten: None. D. Smedley: None. A. Rendon: None. M.J. Caulfield: None. R.H. Scott: None. E.R.A. Thomas:
None. A. Devereau:
None. H. Brittain: None. A. Tucci: None. M. Ryten: None. D. Smedley: None. A. Rendon: None. M.J. Caulfield: None. R.H. Scott: None. 514 J. del Picchia Imputation is now a routine step in genome-wide associa-
tion studies (GWASs) and has facilitated the discovery of
many novel signals. J. S. Mitchell, E. König, M. Gögele, C. Pattaro, P. P.
Pramstaller, C. Fuchsberger P14.047C X-chromosomal INDELs examined by ddPCR for female
sex determination in NIPD and in forensic science X-chromosomal INDELs examined by ddPCR for female
sex determination in NIPD and in forensic science M. Korabecna1,2, B. Veselá1, I. Svobodova1, E. Pazourkova1 M. Korabecna1,2, B. Veselá1, I. Svobodova1, E. Pazourkova1 1First Faculty of Medicine, Charles University, Prague, Czech
Republic, 2General Faculty Hospital in Prague, Prague, Czech
Republic 1First Faculty of Medicine, Charles University, Prague, Czech
Republic, 2General Faculty Hospital in Prague, Prague, Czech
Republic Introduction: In families with gonosomal recessive dis-
eases, fetal sex is determined prenatally by detection of Y-
chromosomal sequences in cell-free fetal DNA in maternal
plasma. When these sequences are not found, female sex of
the fetus is reported. Here, we present a methodology
allowing the detection of paternal X-chromosomal alleles
on maternal background and the confirmation of female sex
by positive amplification signals. S.C. Yau: None. K. Brown: None. A. Bond: None. G.R. Taylor: None. Viapath Analytics, London, United Kingdom Detection of repeat expansions is challenging for short
read next generation sequencing and fluorescent PCR
assays are limited by their inability to amplify large
expansion alleles. Long read sequencing technology such
as Oxford Nanopore Technology (ONT) has the potential
to overcome these limitations. We investigated the per-
formance of ONT sequencing in Huntington Disease
using a 2.1kb amplicon that includes the unstable patho-
genic CAG and polymorphic CCG repeats. 48 samples
tested by fluorescent PCR spanning 15 to 101 CAG
repeats (including all alleles in the intermediate and
reduced penetrance ranges) were analysed. Amplicons
were prepared using the Nanopore 1D barcoding protocol
and analysed on MinION Flow Cell (R9.4). FASTQ files
were generated using Albacore (ONT) to perform both
basecalling and demultiplexing. BAM files were gener-
ated using minimap2. Allele calls were performed by
RepeatHMM* (with the predefined HTT model) using
FASTQ and BAM inputs. Repeat length estimates by both
Nanopore sequencing and fluorescent PCR showed a
concordance (R2) >0.9957. RepeatHMM analysis with
BAM inputs produced the best results with a specificity of
0-1 repeat for alleles with 20-39 repeats. However, alleles
with <20 repeats were overestimated by 1-2 repeats. The
2.1kb amplicon reduced the preferential amplification of
the smaller alleles in relation to large expansion alleles,
thereby increasing the specificity of the assay. Provided
sequencing depth was 100-fold or greater, we achieved
high accuracy of repeat calling. This study shows that
Nanopore long-read sequencing has the potential to pro-
vide a diagnostic assay for repeat expansions. *Lui et al. Genome Medicine (2017) 9:65 J.S. Mitchell: None. E. König: None. M. Gögele: None. C. Pattaro:
None. P.P. Pramstaller:
None. C. Fuchsberger: None. J.S. Mitchell: None. E. König: None. M. Gögele: None. C. Pattaro:
None. P.P. Pramstaller:
None. C. Fuchsberger: None. P14.045A
Molecular Diagnosis of Huntington Disease using Nanopore
Sequencing S. C. Yau, K. Brown, A. Bond, G. R. Taylor S. C. Yau, K. Brown, A. Bond, G. R. Taylor Viapath Analytics, London, United Kingdom Viapath Analytics, London, United Kingdom Institute for Biomedicine, Eurac Research, Affiliated Institute
of the University of Lübeck, Bolzan, Bolzano, Italy The impact of reference panel short-read sequencing
inaccessibility on genotype imputation We confirmed the presence of
paternal X-chromosome in 12 out of 13 female bearing
pregnancies (92.31% sensitivity). Conclusions: We developed the ddPCR approach allow-
ing the determination of paternal X-chromosomal alleles on
maternal background for non-invasive prenatal diagnostics. This approach may be used for prenatal paternity exclusion
and for determination of female sex in forensic samples. Supported by grants no. VI20172020102 by Ministry of
Interior of the Czech Republic, no. Progres Q25/LF1 and
no. SVV 260 263 of the Ministry of Education, Youth and
Sport of the Czech Republic, and by the grant RVO-VFN
64165 of the Ministry of Health of the Czech Republic. Y. Zheng: A. Employment (full or part-time); Modest;
Integrated DNA Technologies. Z. Zwirko: A. Employment
(full or part-time); Modest; Integrated DNA Technologies. M. Light: A. Employment (full or part-time); Modest;
Integrated DNA Technologies. K. Lai: A. Employment
(full or part-time); Modest; Integrated DNA Technologies. U. Chakravarty: A. Employment (full or part-time);
Modest; Integrated DNA Technologies. K. Bryan: A. Employment (full or part-time); Modest; Integrated DNA
Technologies. S. Rose: A. Employment (full or part-time);
Modest;
Integrated
DNA
Technologies. Y. Bao:
A. Employment (full or part-time); Modest; Integrated DNA
Technologies. M. Jarosz: A. Employment (full or part-
time); Modest; Integrated DNA Technologies. D. Kupec:
A. Employment (full or part-time); Modest; Integrated DNA
Technologies. Y. Wang: A. Employment (full or part-
time); Modest; Integrated DNA Technologies. C. Chen: A. Employment (full or part-time); Modest; Integrated DNA
Technologies. Y. Zheng: A. Employment (full or part-time); Modest;
Integrated DNA Technologies. Z. Zwirko: A. Employment
(full or part-time); Modest; Integrated DNA Technologies. M. Light: A. Employment (full or part-time); Modest;
Integrated DNA Technologies. K. Lai: A. Employment
(full or part-time); Modest; Integrated DNA Technologies. U. Chakravarty: A. Employment (full or part-time);
Modest; Integrated DNA Technologies. K. Bryan: A. Employment (full or part-time); Modest; Integrated DNA
Technologies. S. Rose: A. Employment (full or part-time);
Modest;
Integrated
DNA
Technologies. Y. Bao:
A. Employment (full or part-time); Modest; Integrated DNA
Technologies. M. Jarosz: A. Employment (full or part-
time); Modest; Integrated DNA Technologies. D. Kupec:
A. Employment (full or part-time); Modest; Integrated DNA
Technologies. Y. Wang: A. Employment (full or part-
time); Modest; Integrated DNA Technologies. C. Chen: A. Employment (full or part-time); Modest; Integrated DNA
Technologies. M. Korabecna: None. B. Veselá: None. I. Svobodova:
None. E. Pazourkova: None. A novel duplex-ready library construction method for
improved conversion of low-input and highly degraded
DNA A novel duplex-ready library construction method for
improved conversion of low-input and highly degraded
DNA Y. Zheng, Z. Zwirko, M. Light, K. Lai, U. Chakravarty, K. Bryan,
S. Rose, Y. Bao, M. Jarosz, D. Kupec, Y. Wang, C. Chen
Integrated DNA Technologies, Redwood City, CA, United
States Y. Zheng, Z. Zwirko, M. Light, K. Lai, U. Chakravarty, K. Bryan,
S. Rose, Y. Bao, M. Jarosz, D. Kupec, Y. Wang, C. Chen Y. Zheng, Z. Zwirko, M. Light, K. Lai, U. Chakravarty, K. Bryan,
S. Rose, Y. Bao, M. Jarosz, D. Kupec, Y. Wang, C. Chen Integrated DNA Technologies, Redwood City, CA, United
States The impact of reference panel short-read sequencing
inaccessibility on genotype imputation The impact of reference panel short-read sequencing
inaccessibility on genotype imputation Material and Methods: Using ddPCR we examined X-
chromosomal
INDELs:
rs2307932,
rs16397,
rs16637,
rs3048996, rs16680 in buccal swabs of 50 females to
receive the population data. For all examined INDELs, we
tested the performance of ddPCR for mixtures 20%, 10%, Institute for Biomedicine, Eurac Research, Affiliated Institute
of the University of Lübeck, Bolzan, Bolzano, Italy 515 Abstracts from the 51st European Society of Human Genetics Conference: Posters 5% and 2.5% of one homozygote on the background of the
opposite one. We examined the cell-free plasma DNA from
13 pregnant women bearing Y-chromosome negative
fetuses. 5% and 2.5% of one homozygote on the background of the
opposite one. We examined the cell-free plasma DNA from
13 pregnant women bearing Y-chromosome negative
fetuses. increase ligation efficiency and suppress chimera formation
as well as adaptor dimers. We tested the performance of our
method using three sample types: sheared genomic DNA,
cfDNA, and FFPE DNA. We also tested its performance in
liquid biopsy applications using libraries from mixtures of
NA12878 and NA24385 cell line DNA with mutant allele
fractions (MAFs) down to 0.2%. When compared to
commercially available methods, our approach yielded
significant increase in sensitivity and PPV, especially in
low input range (1-25ng). Our results demonstrate that our
methodology provides a useful tool for applications where
high conversion of input DNA samples is needed. increase ligation efficiency and suppress chimera formation
as well as adaptor dimers. We tested the performance of our
method using three sample types: sheared genomic DNA,
cfDNA, and FFPE DNA. We also tested its performance in
liquid biopsy applications using libraries from mixtures of
NA12878 and NA24385 cell line DNA with mutant allele
fractions (MAFs) down to 0.2%. When compared to
commercially available methods, our approach yielded
significant increase in sensitivity and PPV, especially in
low input range (1-25ng). Our results demonstrate that our
methodology provides a useful tool for applications where
high conversion of input DNA samples is needed. Results: We detected the minor fraction (representing the
paternal X-chromosomal allele on the maternal background)
in all artificial mixtures. We confirmed the presence of
paternal X-chromosome in 12 out of 13 female bearing
pregnancies (92.31% sensitivity). Results: We detected the minor fraction (representing the
paternal X-chromosomal allele on the maternal background)
in all artificial mixtures. High-throughput SMRTSequencing of clinically relevant
targets Diagnostic tools based on next generation sequencing
(NGS) are fundamentally transforming clinical oncology. Current library preparation strategies only allow relatively
low library conversion, especially when the input is low,
making the detection of low-frequency mutations by NGS
particularly challenging. In addition, mutation artifacts arise
from various sample storage and preparation processes,
which can significantly lower the positive predictive value
(PPV) of a clinical NGS diagnostic assay. These artifacts
can be identified by “duplex sequencing”, where strand-
specific unique molecular identifiers (UMIs) are used to
filter out artefactual DNA damages. The duplex sequencing
strategy requires high conversion in library preparation to
enable sequencing and pairing both strands of the same
DNA molecule. S. Ranade, L. A. Aro, I. McLaughlin, C. Heiner, P. Baybayan, A. Toepfer, B. Bowman, R. Hall Pacific Biosciences, Menlo Park, CA, United States P14.051C C
Standard Sanger re-sequencing protocols achieve 5% limit
of detection for single nucleotide polymorphisms and
insertion and deletion variants using a novel signal
processing algorithm H. M. F. Leong1, E. Schreiber1, W. George1, S. Berosik1, J. Marks2, S. Schneider1 H. M. F. Leong1, E. Schreiber1, W. George1, S. Berosik1, J. Marks2, S. Schneider1 1thermo fisher scientific, south san francisco, CA, United
States, 2thermo fisher scientific, South san francisco, CA,
United States Pacific Biosciences, Menlo Park, CA, United States (CCS) or Long Amplicon Analysis (LAA) tools to support
the targeted SMRT Sequencing applications. CCS generates
a highly accurate (QV30) consensus read from each single
intra-molecular multi-pass polymerase read. The CCS
approach is highly reliable for identification of minor
variants with allelic frequencies as low as 1 %. On the other
hand, Long Amplicon Analysis (LAA) derives highly
accurate consensus reads (>QV50) by de novo clustering
subreads originating from multiple copies of the targets. LAA generates phased, full-length consensus sequences for
various genes in a single sequencing run and is most useful
for imputation free allele segregation. In this work, we demonstrate SMRT Sequencing work-
flows for efficient and cost-effective sequencing of a broad
range of clinically relevant targets from 250 bp to >10 kb. Specifically, we illustrate the advantage of SMRT Sequen-
cing to overcome the challenges of traditional methods
when genotyping CYP2D6 and CYP2D7 as well as high
resolution four field HLA typing. In this work, we demonstrate SMRT Sequencing work-
flows for efficient and cost-effective sequencing of a broad
range of clinically relevant targets from 250 bp to >10 kb. Specifically, we illustrate the advantage of SMRT Sequen-
cing to overcome the challenges of traditional methods
when genotyping CYP2D6 and CYP2D7 as well as high
resolution four field HLA typing. S. Ranade: A. Employment (full or part-time); Sig-
nificant; Pacific Biosciences. L.A. Aro: A. Employment
(full or part-time); Significant; Pacific Biosciences. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Pacific Biosciences. I. McLaughlin: A. Employment (full or part-time); Signifi-
cant; Pacific Biosciences. C. Heiner: A. Employment (full
or part-time); Significant; Pacific Biosciences. P. Bayba-
yan: A. Employment (full or part-time); Significant; Pacific
Biosciences. A. Toepfer: A. Employment (full or part-
time); Significant; Pacific Biosciences. B. Bowman: A. Employment (full or part-time); Significant; Pacific Bios-
ciences. R. Hall: A. Employment (full or part-time);
Significant; Pacific Biosciences. K. Neveling: None. R. Derks: None. M. Kwint: None. M. van de Vorst: None. T. Gardeitchik: None. M. Nelen: None. Long-read sequencing goes clinical Introduction: Detecting minor genetic variants is essential
to cancer and infectious disease management. Many have
turned to next generation sequencing for this based on an
assumption that the limit of detection for Sanger sequencing
is a variant allele frequency (VAF) of 20%. We have
recently developed algorithmic methods to reduce this to
5% for single nucleotide polymorphisms (SNPs) and
insertion/deletion variants (indels). K. Neveling, R. Derks, M. Kwint, M. van de Vorst, T. Gardeitchik, M. Nelen Pacific Biosciences, Menlo Park, CA, United States High throughput sequencing of targeted genomic DNA or
cDNA to access disease associated causal variants using
NGS approaches is a standard study practice in translation
research. However, short-read data is prone to mis-mapping
and coverage bias posing many challenges during variant
analysis beyond simple SNPs. We explore the use of long-
reads delivered by SMRT Sequencing for complete char-
acterization of a range of genomic variants including
structural variations, haplotype phasing, low-frequency
SNPs, repeat expansions and GC-rich promoter regions. To achieve high library conversion for low input DNA
samples and to increase sensitivity and PPV, we developed
a
novel
library
construction
chemistry. The
method
provides superior library conversion efficiency using a
unique, mutant DNA ligase and sequencing adapters that We have developed flexible multiplexing options as well
as analysis pipelines using Circular Consensus Sequencing 516 J. del Picchia started with amplicon-based sequencing on the Sequel
(PacBio). We sequenced amplicons up to 16kb, and learned
that long-read sequencing works well for HLA typing,
mtDNA, or long range amplicons designed to avoid pseu-
dogene regions. We also reasoned that small amplicons
(<1kb) can be sequenced accurately on a long-read
sequencer and started to transfer our amplicon-based
workflows from the IonTorrent towards the Sequel. Ulti-
mately, we aim to combine all different workflows into one
amplicon-based sequencing run. A LIMS-based automated
workflow and an automated bioinformatic pipeline thereby
facilitate streamlined sample processing and data analysis,
and assure highest flexibility. We believe that long-read
amplicon sequencing is a first step to make use of the
advantages of long reads in NGS-based diagnostics, thereby
allowing to combine different sequencing approaches in one
test. Once the price for (long-read) genomes is at a range
acceptable for routine diagnostics, targeted sequencing
approaches
will
ultimately
be
replaced
by
genome
sequencing. (CCS) or Long Amplicon Analysis (LAA) tools to support
the targeted SMRT Sequencing applications. CCS generates
a highly accurate (QV30) consensus read from each single
intra-molecular multi-pass polymerase read. The CCS
approach is highly reliable for identification of minor
variants with allelic frequencies as low as 1 %. On the other
hand, Long Amplicon Analysis (LAA) derives highly
accurate consensus reads (>QV50) by de novo clustering
subreads originating from multiple copies of the targets. LAA generates phased, full-length consensus sequences for
various genes in a single sequencing run and is most useful
for imputation free allele segregation. Targeted sequencing analysis of commonly mutated genes
in myelodysplastic syndromes using NGS: Impact and
clinical implications in a single center Thermo Fisher Scientific, South San Francisco, CA, United
States Thermo Fisher Scientific, South San Francisco, CA, United
States MicroRNAs (miRNAs) are a class of small non-coding
RNAs of approximately 22 nucleotides transcribed from
genomes of plants, animals, and viruses. They are highly
conserved and are thought to be micro-managers of gene
regulation, controlling at least one-third of human genes,
therefore having a profound impact on almost every cellular
pathway. Publications showing the diverse function of
miRNAs have continued to increase significantly: miRNAs
are involved in the developmental stages of cells, natural
cell death, and major diseases such as cancer, diabetes, and
cholesterol biosynthesis. MiRNAs have also been shown to
be important biomarkers. Expression of miRNAs is the key
step to understanding their diverse functions. We have
developed a method for the detection and quantification of
miRNAs that is highly specific and sensitive. By extending
the miRNAs on both the 5’ and 3’ ends via single strand
ligation and poly A tailing with universal reverse tran-
scription, respectively, we were able to incorporate 1)
unbiased pre-amplification utilizing the universal adaptor
sequences and 2) flexible assay design to provide the most
optimal and versatile miRNA detection. The universality of
the template library from the upstream chemistry allows
downstream
real-time
TaqMan
qPCR
miRNA-specific
detection requiring only 1 to 10 ng of input sample. This S. Palchetti1, A. Valencia2, S. Bonifacio3, L. Falai3, D. Parrini3,
M. Betti3, B. Boschi3, B. Minuti3, E. Contini3, E. Masala2, V. Santini2, E. Pelo3 S. Palchetti1, A. Valencia2, S. Bonifacio3, L. Falai3, D. Parrini3,
M. Betti3, B. Boschi3, B. Minuti3, E. Contini3, E. Masala2, V. Santini2, E. Pelo3 1AOU Careggi, Florence, Italy, 2Dipartimento di Medicina
Sperimentale e Clinica, Università degli studi di Firenze, AOU
Careggi, Florence, Italy, 3SOD Diagnostica Genetica, AOU
Careggi, Florence, Italy The pathogenetic role of mutations in myelodysplastic
syndromes (MDS) is presently investigated, and important
clinical implications of these mutations are apparent. Clin-
ical guidelines suggest including mutation evaluation in
current practice. We sequenced a panel of 22 genes using
Haloplex system and the Miseq platform to assess the
mutational landscape of MDS patients. We evaluated
98 sequential MDS patients diagnosed in our center. At
least one genomic alteration was observed in 89% of
patients. The most frequently mutated genes were TET2
(26%), SF3B1 (24%), DNMT3A (22%), SRSF2 (21%),
ASXL1 (21%), RUNX1 (9%), and TP53 (7%). Department of Human Genetics, Nijmegen, Netherlands Next generation sequencing has revolutionized the field of
human genetics by offering new possibilities to unravel
human diseases. Due to limitations of short reads however,
various traditional tests including single gene sequencing,
MLPA or genescan are still performed, leading to a com-
plex landscape of different available genetic tests running
on a variety of platforms. A generic ‘one test fits all’
strategy would be a more efficient and cost-effective way to
perform genetic testing. Long-read sequencing does provide
an opportunity to move a step closer towards this objective. To get a feel for both platform and underlying chemistry we Materials and Methods: Samples with indels spanned
33 different amplicons from 21 different genes; SNPs
spanned 22 different amplicons from eight different genes. Samples were from DNA reference standards, genomic
DNA, and DNA extracted from formalin-fixed, paraffin-
embedded tissues. DNA was quantified using the RNase-P
quantitative polymerase chain reaction assay, and serially
diluted. Allelic proportions spanned 0.6125% to 50% for 517 Abstracts from the 51st European Society of Human Genetics Conference: Posters in patients with normal karyotype (n = 73) where DNMT3A
mutations conferred shorter OS (49 months vs 23 months;
P= 0.02). A significant difference in OS was observed
when two groups: OS of pts with ≥2 mutations was
25 months vs 49 months; P= 0.003), consistent with what
shown previously. Our experience confirms that the pre-
sence of even isolated RUNX1 mutations, although rare,
identify patients with shorter survival. Patients with normal
karyotype when carrying DNMT3A mutations or >2 somatic
mutations have a significantly shorter survival than pre-
dicted by IPSS-R. Our observations, although carried out in
a limited group of cases, strongly confirm the prognostic
importance of mutations, and support the implementation of
NGS analysis of somatic mutation, especially for MDS
patients without cytogenetic abnormalities. SNPs and 2.5% to 50% for indels. Samples were amplified,
sequenced, and pre-processed using standard protocols and
tools for Sanger sequencing from Applied BiosystemsTM. Results: 121 samples had deletions from 1 to 48 base
pairs (bp) and 82 samples had insertions from 1 to 6-bp. 1130 samples contained 0 to 16 SNP variants. For SNPs, a
detection sensitivity of 95.9% and specificity of 99.8% was
observed at 5% VAF. For indel detection, there were zero
false positives and zero false negatives down to 2.5% VAF. P14.052D L. Wong, C. Liu, F. Hu, S. Dong L. Wong, C. Liu, F. Hu, S. Dong Department of Human Genetics, Nijmegen, Netherlands For indel characterization accuracy, type was 100%, length
100%, location 93%, sequence 93%, and VAF 92% within
3% of the expected value. Conclusions: With new algorithms to process data from
Sanger sequencing, it is possible to reliably and auto-
matically detect 5% minor variants. S. Palchetti: None. A. Valencia: None. S. Bonifacio:
None. L. Falai: None. D. Parrini: None. M. Betti: None. B. Boschi: None. B. Minuti: None. E. Contini: None. E. Masala: None. V. Santini: None. E. Pelo: None. H.M.F. Leong: A. Employment (full or part-time);
Significant; thermo fisher scientific. E. Schreiber: A. Employment (full or part-time); Significant; thermo fisher
scientific. W. George: A. Employment (full or part-time);
Significant; thermo fisher scientific. S. Berosik: A. Employ-
ment (full or part-time); Significant; thermo fisher scientific. J. Marks: None. S. Schneider: A. Employment (full or
part-time); Significant; thermo fisher scientific. A versatile and highly sensitive method for microRNA
detection with real-time TaqMan® assays A versatile and highly sensitive method for microRNA
detection with real-time TaqMan® assays Parental origin of deletions and duplications; about the
necessity to check for cryptic inversions Parental origin of deletions and duplications; about the
necessity to check for cryptic inversions T. Liehr1, I. Schreyer1,2, A. Kuechler3, E. Manolakos4, S. Singer5, A. Dufke5 T. Liehr1, I. Schreyer1,2, A. Kuechler3, E. Manolakos4, S. Singer5, A. Dufke5 1Jena University Hospital, Friedrich Schiller University,
Institute of Human Genetics, Jena, Germany, 2Jena University
Hospital, Center for Ambulant Medicine, Jena, Germany,
3Universitätsklinikum Essen, Institut für Humangenetik, Jena,
Germany, 4Access to Genome, ATG Labs, Athens, Greece,
5Institut für Medizinische Genetik und angewandte Genomik,
Tübingen, Germany 1Jena University Hospital, Friedrich Schiller University,
Institute of Human Genetics, Jena, Germany, 2Jena University
Hospital, Center for Ambulant Medicine, Jena, Germany,
3Universitätsklinikum Essen, Institut für Humangenetik, Jena,
Germany, 4Access to Genome, ATG Labs, Athens, Greece,
5Institut für Medizinische Genetik und angewandte Genomik,
Tübingen, Germany Methods: The method utilizes a system of target-specific
labelled probes allowing each to be read out by subsequent
melting curve analysis, allowing more than 5 probes per
fluorophore channel, thereby greatly increasing the level of
multiplexing achievable. By utilizing meltcurve readout of
modified probes - one for each target - rather than only of
the PCR amplicons, the system also adds an extra level of
specificity to meltcurve analysis. Copy number variants (CNVs) are the genetic bases for
microdeletion/microduplication syndromes (MMSs). Cou-
ples with an affected child and desire to have further chil-
dren are tested for a potential parental origin of the CNV
routinely either by molecular karyotyping or two color
fluorescence in situ hybridization (FISH), yet. In the latter
case a critical region probe (CRP) is combined with a
control probe for identification of the involved chromo-
some. However, CNVs can also appear due to other rea-
sons, like a recombination of a submicroscopic, cryptic
inversion in one of the parents. 74 patients with different
MMSs and overall 81 CNVs were studied here by a novel
three color FISH. The way how three locus-specific probes
are selected (one in the CRP and two flanking it in a dis-
tance of 5-10 Mb) enables to detect or exclude two possible
parental conditions as origins of the CNV in the index: (i)
direct parental origin of the CNV (deletion or duplication)
or (ii) a parental cryptic inversion. Thus, for overall 51/81
CNVs (63%) a parental origin could be determined. P14.054B In many clinical settings, achieving multiplex answers from
the same sample provide benefits both in terms of cost,
speed, added clinical value as well as preservation of lim-
ited samples. In cases such as cancer susceptibility testing,
sepsis, RSV-testing, gastrointestinal testing and many oth-
ers, a broad spectrum of targets are relevant for testing. However, PCR readout is commonly limited to the current
maximum of 4-5 fluorophores on most instruments. We
have developed a homogenous assay method to allow read-
out of more than 20 answers from a single PCR reaction. Parental origin of deletions and duplications; about the
necessity to check for cryptic inversions 36/51
(70.5%) inherited the CNV directly from one of the parents,
but 15/51 (29.5%) were due to a detectable parental inver-
sion by three color FISH. A 2:1 ratio of maternal versus
paternal inheritance was found. The new, here suggested
three color FISH approach increased the detection rate for
parental origin in this study by 140%. Still, for 30/81 cases Result: To test the system, we designed probes based on
previously designed TaqMan probes targeting 17 different
hemorrhagic fever viruses and tested them against artificial
DNA targets. Testing was perfomed in single wells
containing all probes labeled with different flourophors
and was able to separately detect all 17 targets. PCR was
performed on a Bio-Rad CFX instrument, a MIC PCR
cycler as well as the Agilent AriaMX. Conclusions: The method comprise a robust, high-
multiplex, homogeneous system to provide 20+ readouts
per PCR reaction on most PCR platforms in use in current
clinical diagnostics Conclusions: The method comprise a robust, high-
multiplex, homogeneous system to provide 20+ readouts
per PCR reaction on most PCR platforms in use in current
clinical diagnostics S.M. Echwald: A. Employment (full or part-time);
Significant; Anapa Biotech A/S. Targeted sequencing analysis of commonly mutated genes
in myelodysplastic syndromes using NGS: Impact and
clinical implications in a single center The uni-
variate analysis showed that only RUNX1 mutations were
associated with a shorter OS (49 months (WT) vs 12 months
(mut); P= 0.001). We also analysed the impact of mutations 518 J. del Picchia (37%) no reason for the ‘de novo’ MMS in the affected
index patient could be found. (37%) no reason for the ‘de novo’ MMS in the affected
index patient could be found. versatile approach allows detection of a large number of
miRNAs for profiling or detection of a smaller set of
miRNAs for screening and validation using the same uni-
versal cDNA template. Data for differential expression
between normal brain tissue and the various cancer cell
lines will be presented. versatile approach allows detection of a large number of
miRNAs for profiling or detection of a smaller set of
miRNAs for screening and validation using the same uni-
versal cDNA template. Data for differential expression
between normal brain tissue and the various cancer cell
lines will be presented. T. Liehr: None. I. Schreyer: None. A. Kuechler: None. E. Manolakos: None. S. Singer: None. A. Dufke: None. P14.055C L. Wong: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. C. Liu: A. Employment (full or
part-time); Significant; Thermo Fisher Scientific. F. Hu: A. Employment (full or part-time); Significant; Thermo Fisher
Scientific. S. Dong: A. Employment (full or part-time);
Significant; Thermo Fisher Scientific. T. Singer, P. Capek, E. S. Allen, T. Ghosh, P. Taylor, S. B.
Greene, K. Bojanovic Machado, M. Chu, Y. Sun, S. Lee, G. J. Detection of 17 targets in a single PCR tube by a novel
probe system combining melting curves and Taqman
probes Detection of 17 targets in a single PCR tube by a novel
probe system combining melting curves and Taqman
probes S. M. Echwald Anapa Biotech A/S, Hoersholm, Denmark Swift Biosciences, Ann Arbor, MI, United States Demand for improved indexing capabilities has increased
due to higher sequencing output, increased multiplexing to
lower cost, and to avoid misassignment of reads from
indexing errors. Sequencing reads may be misassigned due
to “index hopping” on Illumina patterned flow cells. This
misassignment can lead to false positives in ultra-sensitive
variant detection, which is detrimental to analysis of liquid
biopsy results and detection of low frequency (<1%) alleles. It is also necessary to eliminate errors associated with
insufficient edit distances between index sequences. We
have observed misassignment due to insufficient edit dis-
tance among Illumina TruSeq HT indices at a frequency up
to 1.5%. Therefore, we developed 96 single (i7) 8 base
indices, which can be paired with the TruSeq HT i5 indices
to achieve 768-plex high throughput dual combinations. These indices were validated using a novel method on both
Illumina’s 2 and 4-channel technologies. This method
involved the use of 96 libraries with unique, non-
overlapping inserts to facilitate tracking of index mis-
assignment. This allowed us to assess not only which index
has misassigned library molecules, but to also pinpoint the
origin and rate of misassignment. With our 96 i7 indices,
misassignment was observed at rates <0.1%. For even
higher fidelity de-multiplexing, we have paired our 96
indices as single use in both the i5 and i7 positions, known
as Unique Dual Indices (UDIs). We are validating the 96
new indices as UDIs for avoidance of index hopping, and
for eliminating PCR-induced chimerism during multiplexed
library amplification performed during hybridization cap-
ture workflows. T. Singer: A. Employment (full or part-time); Modest;
Illumina is employer. P. Capek: A. Employment (full or
part-time); Modest; Illumina is employer. E.S. Allen: A. Employment
(full or part-time);
Modest;
Illumina
is
employer. T. Ghosh: A. Employment (full or part-time);
Modest; Illumina is employer. P. Taylor: None. S.B. Greene: A. Employment (full or part-time); Modest;
Illumina is employer. K. Bojanovic Machado: A. Employ-
ment (full or part-time); Modest; Illumina is employer. M. Chu: A. Employment (full or part-time); Modest; Illumina
is employer. Y. Sun: A. Employment (full or part-time);
Modest; Illumina is employer. S. Lee: A. Employment (full
or part-time); Modest; Illumina is employer. G.J. Bean: A. Employment
(full or part-time);
Modest;
Illumina
is
employer. I. Khrebtukova: A. Employment (full or part-
time); Modest; Illumina is employer. J. Bruand: A. T. Singer: A. Employment (full or part-time); Modest;
Illumina is employer. P. Capek: A. Bean, I. Khrebtukova, J. Bruand, D. M. Agius, R. M. Kelley, G.
P. Schroth Illumina Inc., San Diego, CA, United States Illumina Inc., San Diego, CA, United States A challenge for any multiplex PCR assay is combining
many amplicons in one reaction and achieving high target
specificity. AmpliSeqTM biochemistry is the industry
leading assay that allows for efficient amplification of a
high number of amplicons, enabling high specificity and
uniformity of coverage across a wide range of targets. We
modified the AmpliSeqTM library preparation workflow so
that the resulting amplicon libraries are amenable to
sequencing on Illumina platforms. We designed specific
dual index adapter sequences to ensure optimal perfor-
mance and demultiplexing on Illumina® sequencing sys-
tems
(MiSeq™,
MiniSeq™,
NextSeq™,
iSeq™,
HiSeq™) and to allow pooling of up to 96 samples into
one sequencing run. We tested multiple cancer specific
DNA panels as well as the Exome panel with this new
workflow and investigated sequencing performance, as
well as somatic and germline variant detection sensitivity
and specificity on a wide variety of samples, including
formalin-treated and degraded FFPE tissue samples. Fur-
ther, we assessed detection of known gene fusions and
evaluated gene expression measurements with RNA-
specific panels. Custom panels were designed and tested
over a wide range of amplicons. We observed high uni-
formity of coverage for all panels tested with high cor-
relation between Illumina’s sequencing platforms. Single
nucleotide variants (SNVs) were reliably detected at 5%
and down to 1%, even in very degraded FFPE samples
with only 10 ng DNA input. Detection of CNVs was
demonstrated with the Comprehensive v3 and Focus
panels. We assessed variant call sensitivity and false
positive rate in Platinum genome NA12878 and results
will be discussed. Improved indices for high fidelity de-multiplexing on
Illumina instruments L. Kurihara, J. RoseFigura, S. Sandhu, B. Lahann, J. Irish, V. Makarov L. Kurihara, J. RoseFigura, S. Sandhu, B. Lahann, J. Irish, V. Makarov Swift Biosciences, Ann Arbor, MI, United States P14.056D P14.056D
Illuminizing the AmpliSeqTM Assay Illuminizing the AmpliSeqTM Assay Abstracts from the 51st European Society of Human Genetics Conference: Posters 519 Bean, I. Khrebtukova, J. Bruand, D. M. Agius, R. M. Kelley, G. P. Schroth Employment
(full or part-time);
Modest;
Illumina
is
employer. D.M. Agius: A. Employment (full or part-time);
Modest; Illumina is employer. R.M. Kelley: A. Employ-
ment (full or part-time); Modest; Illumina is employer. G.P. Schroth: A. Employment (full or part-time); Modest;
Illumina is employer. 1SOD Diagnostica Genetica, AOU Careggi, Florence, Italy,
2Centro Regionale di Riferimento Fibrosi Cistica, AOU Meyer,
Florence, Italy Materials and Methods: Results from 100 exome-based
analyses using a panel of 34 HCTD genes were compared to
information in medical records and requisition forms. Materials and Methods: Results from 100 exome-based
analyses using a panel of 34 HCTD genes were compared to
information in medical records and requisition forms. Cystic Fibrosis (CF) is the most common life threatening
autosomal recessive disease in Caucasian population and
it is caused by mutations in the Cystic Fibrosis Trans-
membrane Regulator (CFTR) gene. Since an early diag-
nosis is known to improve the outcome of CF patients, a
newborn screening (NBS) has been implemented in many
countries with different strategies. In Tuscany, molecular
analysis of selected CFTR variants has been introduced
into NBS since 2011 for those newborns with an elevate
immunoreactive trypsinogen (IRT) concentration on dried
blood spots (DBSs). To improve the screening procedure,
a Next Generation Sequencing (NGS) assay able to detect
a panel of 276 CF-causing variants, according to CFTR2
project, has been introduced. The aforementioned NGS
approach has proven to be a reliable and fast method for
the identification of the CFTR variants on DNA from
DBS. So far, 180 samples have been analyzed: 155 cases
were negative, one variant has been detected in 22 cases
whereas two variants have been identified in 3 cases. Among the identified variants, one would not have been
detected by the previously used panel. The opportunity to
test a larger number of variants could increase the chance
to identify two CF-causing variants improving the clinical
management of newborns. Furthermore, according to
clinical indications, the re-analysis of NGS data con-
cerning the whole gene sequence could be performed
improving turnaround time and reducing time-consuming
procedures. Results: In 51 % of patients, Sanger sequencing of
selected genes had previously been performed. Gene panel
analysis identified a clinically relevant and likely patho-
genic sequence variant in 18 % and a relevant, unclassified
variant (VUS) in 10 % of samples. A likely pathogenic variant or VUS was identified in 28
(82.4%) of genes in the panel. Likely pathogenic variants
were identified in nine genes (26.5%), especially genes
associated with Loeys-Dietz syndrome, all were requested
from a university hospital. Requisitions indicating cardio-
vascular symptoms were associated with higher rate of
pathogenic findings than other requisitions (19.6 % vs 6.8
%). Certain clinical symptoms were frequently noted in
medical records but infrequently in requisitions. Swift Biosciences, Ann Arbor, MI, United States Employment (full or
part-time); Modest; Illumina is employer. E.S. Allen: A. Employment
(full or part-time);
Modest;
Illumina
is
employer. T. Ghosh: A. Employment (full or part-time);
Modest; Illumina is employer. P. Taylor: None. S.B. Greene: A. Employment (full or part-time); Modest;
Illumina is employer. K. Bojanovic Machado: A. Employ-
ment (full or part-time); Modest; Illumina is employer. M. Chu: A. Employment (full or part-time); Modest; Illumina
is employer. Y. Sun: A. Employment (full or part-time);
Modest; Illumina is employer. S. Lee: A. Employment (full
or part-time); Modest; Illumina is employer. G.J. Bean: A. Employment
(full or part-time);
Modest;
Illumina
is
employer. I. Khrebtukova: A. Employment (full or part-
time); Modest; Illumina is employer. J. Bruand: A. L. Kurihara:
A. Employment
(full
or
part-time);
Significant; Swift Biosciences. J. RoseFigura: A. Employ-
ment (full or part-time); Significant; Swift Biosciences. S. Sandhu: A. Employment (full or part-time); Significant;
Swift Biosciences. B. Lahann: A. Employment (full or
part-time); Significant; Swift Biosciences. J. Irish: A. Employment
(full
or
part-time);
Significant;
Swift L. Kurihara:
A. Employment
(full
or
part-time);
Significant; Swift Biosciences. J. RoseFigura: A. Employ-
ment (full or part-time); Significant; Swift Biosciences. S. Sandhu: A. Employment (full or part-time); Significant;
Swift Biosciences. B. Lahann: A. Employment (full or
part-time); Significant; Swift Biosciences. J. Irish: A. Employment
(full
or
part-time);
Significant;
Swift 520 J. del Picchia Biosciences. V. Makarov: A. Employment (full or part-
time); Significant; Swift Biosciences. Biosciences. V. Makarov: A. Employment (full or part-
time); Significant; Swift Biosciences. 1University of Oslo, Oslo, Norway, 2Oslo University Hospital,
Oslo, Norway 1University of Oslo, Oslo, Norway, 2Oslo University Hospital,
Oslo, Norway 1University of Oslo, Oslo, Norway, 2Oslo University Hospital,
Oslo, Norway M. K. Pope1, H. M. Johnsen2, K. B. Rypdal1, Y. Sejersted2, B.
Paus2,1 1SOD Diagnostica Genetica, AOU Careggi, Florence, Italy,
2Centro Regionale di Riferimento Fibrosi Cistica, AOU Meyer,
Florence, Italy Interpretation: Many relevant and likely pathogenic
sequence variants were identified, however in HCTD the
findings of VUS remains a significant challenge. A multi-
gene panel as first tier test would reduce costs compared to
Sanger sequencing preceding analysis. The likelihood of
identifying a pathogenic sequence variant depended on the
requisitioning instance and indication for analysis. M.K. Pope: None. H.M. Johnsen: None. K.B. Rypdal:
None. Y. Sejersted: None. B. Paus: None. Implementation of a new automated sample quality control
tool in a whole exome sequencing workflow Implementation of a new automated sample quality control
tool in a whole exome sequencing workflow R. Nitsche1, E. Viering1, J. Petersen2, M. Beckhaus2, S. Wolf2 R. Nitsche1, E. Viering1, J. Petersen2, M. Beckhaus2, S. Wolf2 F. Gerundino: None. B. Minuti: None. C. Centrone:
None. C. Pescucci: None. M. Trafeli: None. F. Buchi:
None. P. Santelli: None. M. Cavicchi: None. 1Agilent Technologies, Waldbronn, Germany, 2DKFZ
Genomics and Proteomics Core Facility, High Throughput
Sequencing Unit, Heidelberg, Germany P14.058B
CFTR Mutation detection from newborn dry blood spots
by using NGS technology Introduction: The identification of a causative mutation in
hereditary connective tissue disorders (HCTD) enables
correct follow-up and treatment of patients and relatives. Compared
to
conventional
methods,
next
generation
sequencing (NGS) is expected to increase the rate of
molecular findings. The pretest likelihood of findings may
depend on the clinical indication as well as on the requisi-
tioning physician. Introduction: The identification of a causative mutation in
hereditary connective tissue disorders (HCTD) enables
correct follow-up and treatment of patients and relatives. Compared
to
conventional
methods,
next
generation
sequencing (NGS) is expected to increase the rate of
molecular findings. The pretest likelihood of findings may
depend on the clinical indication as well as on the requisi-
tioning physician. F. Gerundino1, B. Minuti1, C. Centrone1, C. Pescucci1, M. Trafeli1, F. Buchi1, P. Santelli1, M. Cavicchi2 1SOD Diagnostica Genetica, AOU Careggi, Florence, Italy,
2Centro Regionale di Riferimento Fibrosi Cistica, AOU Meyer,
Florence, Italy 1SOD Diagnostica Genetica, AOU Careggi, Florence, Italy,
2Centro Regionale di Riferimento Fibrosi Cistica, AOU Meyer,
Florence, Italy P14.059C Application of next generation sequencing in the diagnosis
of hereditary connective tissue disorders Objectives: The High Throughput Sequencing Unit of the
DKFZ Genomics and Proteomics Core Facility provides
general sequencing services. This project demonstrates the
use of an automated electrophoresis system as a quality
control (QC) tool in a whole exome sequencing workflow. M. K. Pope1, H. M. Johnsen2, K. B. Rypdal1, Y. Sejersted2, B. Paus2,1 521 Abstracts from the 51st European Society of Human Genetics Conference: Posters Mandatory for the experimental success of whole exome
sequencing is the quality of the incoming genomic DNA
(gDNA) material and the DNA samples at various stages of
the library preparation workflow. Mandatory for the experimental success of whole exome
sequencing is the quality of the incoming genomic DNA
(gDNA) material and the DNA samples at various stages of
the library preparation workflow. Mandatory for the experimental success of whole exome
sequencing is the quality of the incoming genomic DNA
(gDNA) material and the DNA samples at various stages of
the library preparation workflow. is use of hybridisation-based approaches such as SureSelect
enrichment protocol (Agilent). Materials and Methods: Two separate panels were
adopted, optimised and tested as a proof-of-concept:
ClearSeq Comprehensive Cancer (4 samples) and a custom
SureSelect panel designed for diagnostics of syndromic and
non-syndromic forms of craniosynostoses (14 samples). The Next Generation Sequencing was performed using
IonTorrent S5 platform. Methodology: Exome libraries were prepared according
to the Agilent Low Input Sure-SelectXT Human All Exon v5
protocol from FFPE tumor tissue samples. To ensure
success quality control was verified with an Agilent 4200
TapeStation system of the received gDNA samples and
during the library preparation. Results: The following quality control parameters were
achieved for both panels: coverage - 97%, on target – 74%,
mean depth – 274, ISP loading – 80%, 97% aligned
specifically. Bioinformatic analysis was performed on the
Ion Reporter™Software and variants were classified based
on the results obtained from several predictors (e.g. PolyPhen-2, SIFT, Mutation Taster, CADD). Previously
known
mutations
were
detected
across
both
panels,
confirming their effective performance. Results: Intermediate QC steps were taken throughout
the protocol to monitor library preparation for sequencing,
such as evaluation of DNA after fragmentation, analysis of
adapter-ligated and amplified DNA, and lastly, qualification
of the final library. The initial QC of incoming gDNA was
determined based on the DNA integrity number (DIN). P14.059C All
samples had a low DNA integrity, what is usual for DNA
extracted from FPPE material. Conclusions: Quality control is an important part of NGS
workflows, library preparation protocols recommend quan-
tification and qualification of the DNA samples at various
stages. The increasing sample throughput creates a need for
automation especially in a core facility where many
precious samples are proceeded with time pressure. The
implementation of the automated electrophoresis system
enabled to increase the efficiency of the workflow and
ensure good sequencing results. Conclusions: Quality control is an important part of NGS
workflows, library preparation protocols recommend quan-
tification and qualification of the DNA samples at various
stages. The increasing sample throughput creates a need for
automation especially in a core facility where many
precious samples are proceeded with time pressure. The
implementation of the automated electrophoresis system
enabled to increase the efficiency of the workflow and
ensure good sequencing results. Conclusions: Hybridisation-based enrichment is recog-
nized as a valid alternative for amplification-based panels in
diagnosis of complex disorders. Further optimisation of
cost-effectiveness ratio should increase the viability of the
approach and allow for better parallelisation of sequencing
multiple samples. Presented results clearly indicate that it is
possible to adopt hybridisation-based approaches such as
SureSelect enrichment protocol (Agilent) on the IonTorrent
S5 platform, what significantly widens the options for
semiconductor based sequencing of clinical samples. R. Nitsche: A. Employment (full or part-time); Sig-
nificant; Agilent Technologies. E. Viering: A. Employment
(full or part-time); Significant; Agilent Technologies. J. Petersen: None. M. Beckhaus: None. S. Wolf: None. D. Popiel: None. A. Dawidziuk: None. G. Koczyk:
None. B. Wojciechowicz: A. Employment (full or part-
time); Modest; Perlan Technologies. M. Socha: None. E. Olech:
None. A. Sowińska-Seidler:
None. A. Jamsheer: None. P14.062B Optimization of a next generation sequencing innovative
protocol for the accurate detection of punctual mutations
and copy number variants in children with intellectual
disability and obesity D. Popiel1, A. Dawidziuk1, G. Koczyk1,2, B. Wojciechowicz3, M. Socha4, E. Olech4,1, A. Sowińska-Seidler4, A. Jamsheer4,1 1Centers for Medical Genetics GENESIS, Poznań, Poland,
2Institute of Plant Genetics of the Polish Academy of Sciences,
Poznań, Poland, 3Perlan Technologies, Poznań, Poland,
4Department of Medical Genetics, Poznan University of
Medical Sciences, Poznań, Poland P14.061A Adopting hybridisation-based enrichment in molecular
diagnostics of complex disorders via next-generation ion
semiconductor sequencing M. Derhourhi UMR8199 - EGID, Lille, France UMR8199 - EGID, Lille, France Introduction: Whole-exome sequencing has considerably
benefited the molecular diagnosis of patients with suspected
Mendelian disorders, but its poor ability to accurately detect
copy number variations (CNVs) remains a major limitation
for sensitive genetic screening. We developed a next-
generation sequencing strategy (CoDE-seq) enabling the
accurate detection of both CNVs and punctual mutations in
one step, and assessed CoDE-seq for the molecular diag-
nosis of intellectual disability and obesity. Introduction: In current paradigm of next-generation
sequencing, the ease of use and hands-on time in labora-
tory settings are the main factors in adopting IonTorrent
semiconductor-based sequencing platform. The enrichment
strategies for diagnostic panels are amplification-based
(AmpliSeq), which creates a potential for pseudogene
interference or compositional biases. An alternative choice 522 J. del Picchia Methods: CoDE-seq is based on an augmented whole-
exome sequencing protocol, with probes distributed uni-
formly throughout the genome in addition to exome. This
new method was validated in 40 patients for whom
chromosomal DNA microarray was available. Subse-
quently, CNVs and punctual mutations were assessed in
82 children or young adults with suspected monogenic
obesity and/or intellectual disability, and their available
parents. 1 195 387 for MCF7/M and 1 376 487 for MCF7/T. The
software automatically performed a filtering of the frag-
ments obtained on quality, adapter trimming and mapping
of the fragments obtained according to reference genome
hg19. Gene variants with statistical significance (p = 1.0 E-
4) were selected by manual software. The NGS sequencing of PIK3CA, ALK, EGFR, EGFR-
AS1, ERBB2, ESR1 genes has revealed identical genetic
aberrations - mutations in the cell lines (Table 1). The
presence of PIK3CA mutation (c.1633G>A) may be
associated to tamoxifen and metformin resistance in both
sublines and parental line. Results: CoDE-seq not only detected all CNVs (n=97)
identified by chromosomal DNA microarrays but also found
84 additional CNVs, due to a better resolution. When
compared to CoDE-seq and chromosomal DNA micro-
arrays, whole-exome sequencing failed to detect 37% and
14% of CNVs, respectively. In the 82 patients with
suspected Mendelian obesity and/or intellectual disability,
a likely molecular diagnosis was achieved in more than
30% of the patients. Half of the genetic diagnoses were
explained by pathogenic CNVs while the other half by
pathogenic punctual mutations. Results: CoDE-seq not only detected all CNVs (n=97)
identified by chromosomal DNA microarrays but also found
84 additional CNVs, due to a better resolution. UMR8199 - EGID, Lille, France When
compared to CoDE-seq and chromosomal DNA micro-
arrays, whole-exome sequencing failed to detect 37% and
14% of CNVs, respectively. In the 82 patients with
suspected Mendelian obesity and/or intellectual disability,
a likely molecular diagnosis was achieved in more than
30% of the patients. Half of the genetic diagnoses were
explained by pathogenic CNVs while the other half by
pathogenic punctual mutations. Table 1. Concordance of mutational status in cell lines
MCF7, MCF7/M, MCF7/T. Gene
MCF7
MCF7/М
MCF7/Т
PIK3CA
c.1633G>A
Allele fraction:
66% (of 17311
reads)
c.1633G>A
Allele fraction:
67% (of 12976
reads)
c.1633G>A
Allele fraction:
65% (of 11224
reads)
ALK
c.2535T>C
Allele fraction:
100% (of 8773
reads)
c.2535T>C
Allele fraction:
100% (of 2210
reads)
c.2535T>C
Allele fraction:
100% (of 3463
reads)
EGFR
c.474C>T
Allele fraction:
99% (of 1121
reads)
c.474C>T
Allele fraction:
99% (of 2520
reads)
c.474C>T
Allele fraction:
99% (of 2606
reads)
EGFR,
EGFR-
AS1
c.2361G>A
Allele fraction:
99% (of 1365
reads)
c.2361G>A
Allele fraction:
100% (of 1489
reads)
c.2361G>A
Allele fraction:
99% (of 2174
reads)
EGFR
c.2982C>T
Allele fraction:
51% (of 4994
reads)
c.2982C>T
Allele fraction:
41% (of 2128
reads)
c.2982C>T
Allele fraction:
47% (of 1581
reads)
ERBB2
c.1963A>G
Allele fraction:
100% (of 3402
reads)
c.1963A>G
Allele fraction:
100% (of 2800
reads)
c.1963A>G
Allele fraction:
100% (of 2231
reads)
ERBB2
c.3508C>G
Allele fraction:
99% (of 1188
reads)
c.3508C>G
Allele fraction:
99% (of 1076
reads)
c.3508C>G
Allele fraction:
99% (of 2903
reads)
ESR1
c.30T>C
Allele fraction:
50% (of 941
reads)
c.30T>C
Allele fraction:
53% (of 735
reads)
c.30T>C
Allele fraction:
46% (of 625
reads)
ESR1
c.1782G>A
Allele fraction:
53% (of 23574
reads)
c.1782G>A
Allele fraction:
51% (of 8793
reads)
c.1782G>A
Allele fraction:
53% (of 6958
reads) Conclusions: CoDE-seq has proven cost-efficient for the
accurate detection of CNVs and punctual mutations. It
avoids the sequential genetic screening approaches cur-
rently used in clinical practice, and is highly effective for
the molecular diagnosis of intellectual disability and
obesity. M. Derhourhi: None. The study of mutational status of estrogen-dependent
MCF-7 breast cancer cells V. Safronova, D. Golovina, S. Semina, A. Scherbakov, M. Gudkova, M. Krasil’nikov, L. Lyubchenko FSBI «N.N. Blokhin national medical research centre of
oncology», Moscow, Russian Federation, Moscow, Russian
Federation P14.064D NGS in a clinical diagnostic setting evolved routine testing
for many genetic diseases. The identification of single
nucleotide polymorphisms (SNVs) and small insertions/
deletions (InDels) in protein coding regions is straight
ahead. However, reliable detection of larger genomic dele-
tions and insertions from NGS enrichment data remains
sophisticated, especially for those presenting as mosaicism
and/or are located in regulatory regions. As structural var-
iations (SVs) may vary significantly in size, their detection
from short reads and distinction from NGS errors is chal-
lenging. Somatic mosaicism also present in leukocyte DNA
is underestimated. NGS methods have the capacity to
identify pathogenic mosaic variants, providing insights into
their spectrum and underlining their importance for clinical
molecular diagnosis. For a cohort of 400 patients with
connective tissue diseases (290 cases, 34 genes) and neu-
rocutaneous syndromes (50 cases, NF1/SPRED1 gene; 60
cases, TSC1/2 gene) the coding and to some extend the
promotor regions were enriched in a custom design and
analyzed by NGS (Agilent Sure Select QXT, Illumina
NextSeq500). For the detection of SVs emerging as copy
number variations (CNVs) affecting at least one target the
analysis combined the application of ExomeDepth, CoN-
VaDING and XHMM. The intersection of at least two tools
is highly sensitive for heterozygous and homozygous CNVs
whereas pooled results of all tools indicate SVs present as
mosaics. Preliminary results identified SVs representing one
heterozygous COL3A1 deletion and one heterozygous
TGFB3 duplication. Moreover, three SVs suspected to
imply mosaics comprise one COL5A1 two exon duplica-
tion, one complete FLNA gene deletion and one TSC2
promoter deletion. FSBI «N.N. Blokhin national medical research centre of
oncology», Moscow, Russian Federation, Moscow, Russian
Federation The experiments were performed on in vitro cultured
estrogen-dependent MCF-7 breast cancer cells and MCF-7
sublines resistant to antiestrogen tamoxifen and/or metfor-
min - MCF-7/T and MCF-7/M. Next generation sequencing
(NGS) using commerсial panel for gene targeted enrich-
ment - Gene Reader Actionable Insights Tumor Panel
(GRTP - 101X)- was performed on the platform QCI
Analyser version 1.1. For reliable evaluation of gene
mutations 1.5% of tumor cells in the sample are enough. DNA libraries under study were normalized by concentra-
tion followed by pooling. The quality of the reads obtained
after sequencing was higher than Q25 for more than 70% of
studied DNA samples. The numbers of the reads for DNA
samples analyzed after filtering were 1 490 465 for MCF7, V. Safronova: None. D. Golovina: None. S. Semina:
B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Russian Science Foundation, grant 14- Abstracts from the 51st European Society of Human Genetics Conference: Posters 523 15-00362, and Russian Foundation for Basic Research,
grant 16-04-00347. A. Scherbakov: B. Research Grant
(principal investigator, collaborator or consultant and
pending grants as well as grants already received);
Modest;
Russian
Science
Foundation,
grant
14-15-
00362, and Russian Foundation for Basic Research, grant
16-04-00347. M. Gudkova: B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Modest; Russian
Science Foundation, grant 14-15-00362, and Russian
Foundation for Basic Research, grant 16-04-00347. M. Krasil’nikov: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants
already
received);
Modest;
Russian
Science
Foundation, grant 14-15-00362, and Russian Foundation
for
Basic
Research,
grant
16-04-00347. L. Lyubchenko: None. genetic variants detected due to misleading reads and to
detect new ones that were not previously detected due to a
seemingly low allele frequency. Thus we can increase the
quality of interpretation of the NGS data, which is
especially important for DNA diagnostics. K. Karandasheva:
None. A. Tanas:
None. V. Strelnikov: None. K. Karandasheva:
None. A. Tanas:
None. V. Strelnikov: None. Exclusion of unreliably mapped reads from the results of
Ion AmpliSeq targeted NGS K. Karandasheva1, A. Tanas1,2, V. Strelnikov1,2 P14.065A Improvement of the molecular diagnostics in disease genes
by detection of genomic deletions/insertions including
pathogenic mosaic and promoter variants from NGS data K. Mayer, M. Ziegler, D. Becker, J. Schiller, H. Klein Center for Human Genetics and Laboratory Diagnostics
(AHC), Martinsried, Germany K. Mayer: None. M. Ziegler: None. D. Becker: None.
J. Schiller: None. H. Klein: None. Clinical Genetics, Erasmus Medical Center, Rotterdam,
Netherlands Whole exome sequencing (WES) has proven to be a suc-
cessful approach for solving a wide range of genetic dis-
orders. We have offered this test since 2014 on a large scale
in a diagnostic setting for various indications. Some of the
limiting steps in the diagnostic setting are the high costs and
a turnaround time of approximately 4 months, resulting in
non-optimal clinical management, especially for children in
the neonatal intensive care unit (NICU). In the past year we
have set up a workflow for rapid WES testing in children
from the NICU in Sophia Children’s hospital. Initially, we
included 9 children with different indications to optimize
the workflow. The samples consisted of 3 trios, 1 duo and
5 single patients. Although different analysis pipelines had
to be used, in 4 out of 9 cases we found the explanatory
pathogenic mutation. The turnaround time was relatively
high
(average
8
weeks),
but
workflow
adjustments
decreased this to 4 weeks maximum. In the past 3 months
we performed trio WES on 7 children from the NICU, using
one uniform workflow with a multiple congenital anomaly
panel of 2764 genes. When requested, we expanded the
analysis to full exome. One certain diagnosis was made and
at least 3 full exome analyses provided promising candidate
genes. All reports were completed within 3 weeks. In
conclusion, fast WES analysis for NICU children helps to
establish an early definitive diagnosis. This experience will
help us to implement a comparable workflow for prenatal
testing later this year. Methods: We present a cohort of four patients with
euploid twin pregnancies in which fetal reduction was
performed. Maternal blood was obtained prior to the
procedure and at sequential time points. cfDNA was
isolated from maternal plasma and polymorphic DANSR
assays were used to determine the fetal fraction in each
sample. Results: Fetal fraction was observed to decrease between
time points in all cases but no consistent pattern was
observed. In contrast to the average increase in fetal fraction
over time previously reported in singleton pregnancies, no
patient had a higher fetal fraction at the end of the series
than the start, likely reflecting the loss of contributory fetal
fraction from the co-twin. Conclusions: In this small cohort, the fetal fraction of
cfDNA dropped in the weeks following fetal reduction. P14.067C P14.067C
Fetal fraction following selective reduction in twin
pregnancies K. Karandasheva1, A. Tanas1,2, V. Strelnikov1,2 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Pirogov Russian National Research Medical
University, Moscow, Russian Federation Introduction: The use of the NGS is fraught with errors:
not all of the identified genetic variants are true and can be
confirmed by alternative methods. Incorrectly mapped reads
are among the causes of NGS analysis errors. We believe
that using Ion AmpliSeq Targeted Sequencing Technology
enables
to
eliminate
unreliably
mapped
reads
algor-
ithmically utilizing additional information about the geno-
mic coordinates of targeted regions and primers used for
amplification. Thus alignments that do not correspond to the
experiment design can be excluded from the downstream
analysis. Materials and Methods: We have analyzed NGS data of
30 patients (lymphocytes DNA has been used to prepare Ion
AmpliSeq Comprehensive Cancer Panel libraries) and
compared variant calling results based on the initial set of
reads and on the revised set obtained by excluding
unreliable reads. Results: We identified several groups of genetic variants:
(1) observed in both sets, 6072 variants; (2) detected
exclusively in the original data, 127 (systematic, false
positive); detected in the revised data only, 63 (false
negative, previously undetectable). Results: We identified several groups of genetic variants:
(1) observed in both sets, 6072 variants; (2) detected
exclusively in the original data, 127 (systematic, false
positive); detected in the revised data only, 63 (false
negative, previously undetectable). Conclusions: We conclude, that the use of additional
information about the designed start and end of the targeted
regions allows to reduce the number of false-positive K. Mayer: None. M. Ziegler: None. D. Becker: None. J. Schiller: None. H. Klein: None. 524 J. del Picchia 1Department of Obstetrics and Gynecology, University
Hospital Brugmann, Université Libre de Bruxelles, Brussels,
Belgium, 2Ariosa Diagnostics Inc., Roche Sequencing
Solutions Inc., San Jose, CA, United States E. Bevilacqua1, J. C. Jani1, L. H. Kunz2, V. Valmeekam2, M.
Holstrom2, E. Wang2, M. Schmid2 Clinical Genetics, Erasmus Medical Center, Rotterdam,
Netherlands Because a similar pattern may occur in pregnancies
complicated by a spontaneous fetal reduction, a larger
study, in terms of number of pregnancies followed and
measurements per pregnancy, may provide additional data
to more comprehensively describe these patterns. E. Bevilacqua: None. J.C. Jani: B. Research Grant
(principal
investigator,
collaborator
or
consultant
and
pending
grants
as
well
as
grants
already
received);
Significant; Ariosa Diagnostics Inc., Roche Sequencing
Solutions Inc. L.H. Kunz: A. Employment (full or part-
time); Significant; Ariosa Diagnostics Inc., Roche Sequen-
cing Solutions Inc. V. Valmeekam: A. Employment (full or
part-time); Significant; Ariosa Diagnostics Inc., Roche
Sequencing Solutions Inc. M. Holstrom: A. Employment
(full or part-time); Significant; Ariosa Diagnostics Inc.,
Roche Sequencing Solutions Inc. E. Wang: A. Employ-
ment (full or part-time); Significant; Ariosa Diagnostics
Inc., Roche Sequencing Solutions Inc. M. Schmid: None. M. van Slegtenhorst: None. M. Wilke: None. H.T. Brüggenwirth: None. J.N.R. Kromosoeto: None. F. Sleutels: None. W.G. de Valk: None. Y. van Bever:
None. A.S. Brooks: None. G.M.S. Mancini: None. M.W. Wessels: None. M. Joosten: None. R.J.H. Galjaard:
None. L.H. Hoefsloot: None. P14.067C
Fetal fraction following selective reduction in twin
pregnancies M. van Slegtenhorst, M. Wilke, H. T. Brüggenwirth, J. N. R.
Kromosoeto, F. Sleutels, W. G. de Valk, Y. van Bever, A. S.
Brooks, G. M. S. Mancini, M. W. Wessels, M. Joosten, R. J. H.
Galjaard, L. H. Hoefsloot Objectives: Loss of a fetus in a multiple pregnancy is
recognized to be a complicating factor when using maternal
plasma for aneuploidy screening. Cell-free DNA (cfDNA)
from a non-viable embryo or fetus is released into the
maternal bloodstream and may lead to an increased chance
of a discordant NIPT result; however, the nature of this
biological process is not well understood. The objective of
this study is to observe changes in fetal cfDNA over time in
pregnancies with one non-viable twin. Clinical Genetics, Erasmus Medical Center, Rotterdam,
Netherlands Clinical Genetics, Erasmus Medical Center, Rotterdam,
Netherlands P14.068D 1CENTOGENE AG, Rostock, Germany, 2Molecular Medicine
Section, King Abdulaziz Medical City, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 3King Saud Bin
Abdulaziz University For Health Sciences, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 4Pediatric
Neurology Section, King Abdulaziz Medical City, Ministry of
National Guard Health Affairs, Jeddah, Saudi Arabia,
5Department of Pediatrics, King Abdulaziz Medical City,
Ministry of National Guard Health Affairs, Jeddah, Saudi
Arabia, 6Kuwait Medical Genetics Centre, Kuwait, Kuwait,
7Genatak Center for Genomic Medicine, Kuwait, Kuwait,
8King Abdullah International Medical Research Centre, King
Saud bin Abdulaziz Uiversity for Health Sciences, Neurology
Division, Department of Pediatrics, King Abdulaziz Medical
City, Ministry of National Guard Health Affairs (NGHA),
Riyadh, Saudi Arabia, 9Department of Genetics, Sultan
Qaboos University Hospital, Muscat, Oman, 10Department of
Paediatrics, Tawam Hospital, Al-Ain, United Arab Emirates,
11Department of Pathology, College of Medicine and Health
Sciences, United Arab Emirates University, Al-Ain, United
Arab Emirates, 12Department of Pediatrics, College of
Medicine and Health Sciences, United Arab Emirates
University, Al-Ain, United Arab Emirates, 13Department of
Medical Genetics, Kasturba Medical College, Manipal
University, Manipal, India, 14Department of Medical Genetics,
Faculty of Medical Sciences, Tarbiat Modares University,
DeNA Laboratory, Tehran, Iran, Islamic Republic of,
15Genetic and Metabolic Department, King Fahad Specialist
Hospital, Dammam, Saudi Arabia, 16John Hopkins Aramco
Health Care, Pediatric Services, Dhahran, Saudi Arabia,
17Albrecht-Kossel-Institute for Neuroregeneration, Medical
University Rostock, Rostock, Germany 1CENTOGENE AG, Rostock, Germany, 2Molecular Medicine
Section, King Abdulaziz Medical City, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 3King Saud Bin
Abdulaziz University For Health Sciences, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 4Pediatric
Neurology Section, King Abdulaziz Medical City, Ministry of
National Guard Health Affairs, Jeddah, Saudi Arabia,
5Department of Pediatrics, King Abdulaziz Medical City,
Ministry of National Guard Health Affairs, Jeddah, Saudi
Arabia, 6Kuwait Medical Genetics Centre, Kuwait, Kuwait,
7Genatak Center for Genomic Medicine, Kuwait, Kuwait,
8King Abdullah International Medical Research Centre, King
Saud bin Abdulaziz Uiversity for Health Sciences, Neurology
Division, Department of Pediatrics, King Abdulaziz Medical
City, Ministry of National Guard Health Affairs (NGHA),
Riyadh, Saudi Arabia, 9Department of Genetics, Sultan
Qaboos University Hospital, Muscat, Oman, 10Department of
Paediatrics, Tawam Hospital, Al-Ain, United Arab Emirates,
11Department of Pathology, College of Medicine and Health
Sciences, United Arab Emirates University, Al-Ain, United
Arab Emirates, 12Department of Pediatrics, College of
Medicine and Health Sciences, United Arab Emirates
University, Al-Ain, United Arab Emirates, 13Department of
Medical Genetics, Kasturba Medical College, Manipal
University, Manipal, India, 14Department of Medical Genetics,
Faculty of Medical Sciences, Tarbiat Modares University,
DeNA Laboratory, Tehran, Iran, Islamic Republic of,
15Genetic and Metabolic Department, King Fahad Specialist
Hospital, Dammam, Saudi Arabia, 16John Hopkins Aramco
Health Care, Pediatric Services, Dhahran, Saudi Arabia,
17Albrecht-Kossel-Institute for Neuroregeneration, Medical
University Rostock, Rostock, Germany 1CENTOGENE AG, Rostock, Germany, 2Molecular Medicine
Section, King Abdulaziz Medical City, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 3King Saud Bin
Abdulaziz University For Health Sciences, Ministry of National
Guard Health Affairs, Jeddah, Saudi Arabia, 4Pediatric
Neurology Section, King Abdulaziz Medical City, Ministry of
National Guard Health Affairs, Jeddah, Saudi Arabia,
5Department of Pediatrics, King Abdulaziz Medical City,
Ministry of National Guard Health Affairs, Jeddah, Saudi
Arabia, 6Kuwait Medical Genetics Centre, Kuwait, Kuwait,
7Genatak Center for Genomic Medicine, Kuwait, Kuwait,
8King Abdullah International Medical Research Centre, King
Saud bin Abdulaziz Uiversity for Health Sciences, Neurology
Division, Department of Pediatrics, King Abdulaziz Medical
City, Ministry of National Guard Health Affairs (NGHA),
Riyadh, Saudi Arabia, 9Department of Genetics, Sultan
Qaboos University Hospital, Muscat, Oman, 10Department of
Paediatrics, Tawam Hospital, Al-Ain, United Arab Emirates,
11Department of Pathology, College of Medicine and Health
Sciences, United Arab Emirates University, Al-Ain, United
Arab Emirates, 12Department of Pediatrics, College of
Medicine and Health Sciences, United Arab Emirates
University, Al-Ain, United Arab Emirates, 13Department of
Medical Genetics, Kasturba Medical College, Manipal
University, Manipal, India, 14Department of Medical Genetics,
Faculty of Medical Sciences, Tarbiat Modares University,
DeNA Laboratory, Tehran, Iran, Islamic Republic of,
15Genetic and Metabolic Department, King Fahad Specialist
Hospital, Dammam, Saudi Arabia, 16John Hopkins Aramco
Health Care, Pediatric Services, Dhahran, Saudi Arabia,
17Albrecht-Kossel-Institute for Neuroregeneration, Medical
University Rostock, Rostock, Germany Conclusions: We confirmed the pathogenicity of bi-
allelic GTPBP2, NUDT2, NKX6-2 and ACER3 variants and
broaden the related phenotypic spectra. P14.068D Therefore, these
genes should be added to the growing list of causative genes
for hereditary neurodevelopmental disorders to be consid-
ered during molecular diagnosis. Neurology Section, King Abdulaziz Medical City, Ministry of
National Guard Health Affairs, Jeddah, Saudi Arabia, # families
# patients
# disease
causing
variants
gene
first
association
with
monogenic
disease
Previously
published
Centogene
data
Previously
published
Centogene
data
Previously
published
Centogene
data
Extension
of
phenotype
GTPBP2
2016
1
5
3
5
1
5
yes
NUDT2
2017
1
3
2
5
1
1
yes
NKX6-2
2016
7
8
15
12
7
4
yes
ACER3
2016
1
2
2
2
1
2
yes
Total
2016-2017
10
18
22
24
10
12
for every
gene A.M. Bertoli Avella: A. Employment (full or part-time);
Modest; Centogene. Z. Yüksel: A. Employment (full or
part-time); Modest; Centogene AG. H. Yavuz: A. Employ-
ment (full or part-time); Modest; Centogene AG. C. Baldi:
A. Employment (full or part-time); Modest; Centogene AG. A. Marais: A. Employment (full or part-time); Modest;
Centogene AG. J.M. Garcia-Aznar: A. Employment (full
or part-time); Modest; Centogene AG. N.M. Grüning: A. Employment (full or part-time); Modest; Centogene AG. L. Abbasi Moheb:
A. Employment
(full
or part-time);
Modest; Centogene AG. O. Paknia: A. Employment (full
or part-time); Modest; Centogene AG. K.K. Kandaswamy:
A. Employment (full or part-time); Modest; Centogene AG. M. Werber: A. Employment (full or part-time); Modest;
Centogene AG. M. Weiss: A. Employment (full or part-
time); Modest; Centogene AG. G.E. Oprea: A. Employ-
ment (full or part-time); Modest; Centogene AG. F. Al-
Hakami: None. N. Alshaikh: None. S. Alameer: None. A. Shawli: None. M.J. Marafi: None. A.A. Elmonairy:
None. F. Al-Mulla: None. W. Al-Twaijri: None. K. Al-
Thihli: None. A.M. Al Shamsi: None. H.A. Abdelrah-
man: None. L. Al-Gazali: None. A. Shukla: None. K.M. Girisha: None. M. Garshasbi: None. Y. Housawi: None. Introduction: Many newly suggested disease-gene asso-
ciations result from findings in single patients or families. Independent confirmation/validation as well as definition of
the corresponding clinical and mutational spectra is neces-
sary before these variants/genes can be considered as
causative. Introduction: Many newly suggested disease-gene asso-
ciations result from findings in single patients or families. Independent confirmation/validation as well as definition of
the corresponding clinical and mutational spectra is neces-
sary before these variants/genes can be considered as
causative. P14.068D E. Bevilacqua1, J. C. Jani1, L. H. Kunz2, V. Valmeekam2, M. Holstrom2, E. Wang2, M. Schmid2 Validation of gene causality for neurological disorders by
WES/WGS analyses in a diagnostic setting Abstracts from the 51st European Society of Human Genetics Conference: Posters 525 A. M. Bertoli Avella1, Z. Yüksel1, H. Yavuz1, C. Baldi1, A. Marais1, J. M. Garcia-Aznar1, N. M. Grüning1, L. Abbasi
Moheb1, O. Paknia1, K. K. Kandaswamy1, M. Werber1, M. Weiss1, G. E. Oprea1, F. Al-Hakami2,3, N. Alshaikh3,4, S. Alameer3,5, A. Shawli3, M. J. Marafi6, A. A. Elmonairy6, F. Al-
Mulla7, W. Al-Twaijri8, K. Al-Thihli9, A. M. Al Shamsi10, H. A. Abdelrahman11, L. Al-Gazali12, A. Shukla13, K. M. Girisha13, M. Garshasbi14, Y. Housawi15, F. Al Mutairi8, N. Al-Sannaa16, M. Alfadhel8, O. Brandau1, A. Rolfs1,17, P. Bauer1 stored in our database (CentoMD®) we aimed to validate the
role of newly identified genes as causative for a specific
disorder. Results: Based on application of an ad-hoc bioinfor-
matics pipeline, Sanger confirmation and evaluation of
clinical information, we have identified bi-allelic causative
variants for: 1) GTPBP2 related to a neurodevelopmental
phenotype with severe, early onset motor and cognitive
impairment,
2)
NUDT2
causing
a
hypotonia-
neurodevelopmental disorder, 3) NKX6-2 related to spastic
ataxia with hypomyelinating leukodystrophy and 4) ACER3
related to early onset, progressive leukodystrophy. Results: Based on application of an ad-hoc bioinfor-
matics pipeline, Sanger confirmation and evaluation of
clinical information, we have identified bi-allelic causative
variants for: 1) GTPBP2 related to a neurodevelopmental
phenotype with severe, early onset motor and cognitive
impairment,
2)
NUDT2
causing
a
hypotonia-
neurodevelopmental disorder, 3) NKX6-2 related to spastic
ataxia with hypomyelinating leukodystrophy and 4) ACER3
related to early onset, progressive leukodystrophy. P14.068D Patients and Methods: We have performed whole
exome sequencing (WES) and whole genome sequencing
(WGS) as diagnostic tool for 10,859 and 1,118, respec-
tively, index cases. With a systematic analysis of the data as 526 J. del Picchia possibility
of
in-the-field
sequencing
experiments
for
population genetics studies. A. Trimouille: None. C. Mouden: None. C. Boury:
None. A. Courrèges: None. P. Fergelot: None. M. Martin-Négrier: None. possibility
of
in-the-field
sequencing
experiments
for
population genetics studies. A. Trimouille: None. C. Mouden: None. C. Boury:
None. A. Courrèges: None. P. Fergelot: None. M. Martin-Négrier: None. possibility
of
in-the-field
sequencing
experiments
for
population genetics studies. possibility
of
in-the-field
sequencing
experiments
for
population genetics studies. F. Al Mutairi: None. N. Al-Sannaa: None. M. Alfadhel:
None. O. Brandau: A. Employment (full or part-time);
Modest; Centogene AG. A. Rolfs: A. Employment (full or
part-time); Modest; Centogene AG. P. Bauer: A. Employ-
ment (full or part-time); Modest; Centogene AG. A. Trimouille: None. C. Mouden: None. C. Boury:
None. A. Courrèges: None. P. Fergelot: None. M. Martin-Négrier: None. A. Trimouille: None. C. Mouden: None. C. Boury:
None. A. Courrèges: None. P. Fergelot: None. M. Martin-Négrier: None. Detection of phage nucleic acid in human blood plasma in
data from NIPT protocol Nanopore MinION A. Trimouille1,2, C. Mouden3, C. Boury3, A. Courrèges4, P. Fergelot1,2,5, M. Martin-Négrier4,6 I. Gazdaricová1, D. Smoľak1,2, A. Balá3,2, J. Budi3,2, J. Gazdarica1,2, M. Kajsik1,4, J. Turňa1,4,5, T. Szemes1,2,4 1CHU Bordeaux - Service de Génétique Médicale, Bordeaux,
France, 2Univ. Bordeaux - INSERM 1211, Bordeaux, France,
3INRA, UMR Biodiversity of Genes and Ecosystems,
Transcriptomics and Genomics Platform, Cestas, France,
4CHU Bordeaux - Service de Pathologie, Bordeaux, France,
5Plateforme Génome Transcriptome, Centre de Génomique
Fonctionnelle de Bordeaux, Université de Bordeaux,
Bordeaux, France, 6Univ. Bordeaux, CNRS, Institut des
Maladies Neurodégénératives, UMR 5293, Bordeaux, France 1Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 2Geneton Ltd., Bratislava, Slovakia, 3Faculty of
Mathematics, Physics, and Informatics, Comenius University,
Bratislava, Slovakia, 4Science Park, Bratislava, Slovakia,
5Slovak Center of Scientific and Technical Information,
Bratislava, Slovakia Introduction: Whole genome NIPT designed to detect
chromosomal or subchromosomal aberrations in the fetus. NIPT assays based on whole genome sequencing are
actually analyzing total present cell free DNA isolated from
human plasma. Obtained sequencing data contains reads
mapped to reference human genome, but also a significant
number of unmapped reads, some of them belonging to
viruses. The main objective of this study was to identify
nucleic acids of phages, the most abundant organisms in
biosphere, in NIPT data and to try to build viral contigs
from the unmapped reads. Mitochondrial cytopathies can be caused by mitochondrial
DNA (mtDNA) anomalies such as point variants or large-
scale single deletions. In addition to these causative
anomalies, multiple deletions of mtDNA can arise due to
pathogenic variants in nuclear genes involved in the main-
tenance of mtDNA. These 3 types of anomalies (point
variants, single and multiple deletions) are investigated for
diagnostic purpose thanks to amplification of mtDNA by 2
long-range PCR, and sequencing by NGS short reads
technology. However, the use of short reads does not allow
to easily and accurately identify the deletion breakpoints at
the nucleotide level. Materials and Methods: We sequenced total DNA and
transcribed RNA (cDNA) from plasma of both healthy
individuals and individuals with bacterial infection. In
addition, we analyzed samples from the surface of mucous
membrane for both groups. In analysis, human reads were
removed and all remaining fragments were taxonomically
labeled using metagenomic classifiers and phage mappers. Presence and composition of phages in samples were
visualized using Krona graphs. Development and clinical utility ofINGEMM custom panels 1Children's Hospital Westmead, Westmead, Australia, 2NSW
Health Pathology, Randwick, Australia, 3Royal Brisbane
Hospital, Brisbane, Australia, 4NSW Health Pathology,
Newcastle, Australia, 5SA Pathology, Adelaide, Australia,
6Wellington Hospital, Wellington, New Zealand, 7Western
Genome Diagnostics, Perth, Australia, 8ASDG Quality
Assessment Program, Alexandria, Australia, 9Royal Hobart
Hospital, Hobart, Australia, 10Mater Health Services,
Brisbane, Australia, 11Queensland Fertility Group, Brisbane,
Australia, 12Retired, formely at Pathwest Laboratory Medicine,
Nedlands, Australia, 13St Vincent's Hospital, Melbourne,
Australia, 14Canterbury Health Laboratories, Christchurch,
Australia, 15Monash Medical Centre, Melbourne, Australia,
16Retired, formely at NSW Health Pathology, Newcastle,
Australia, 17Murdoch Children's Research Institute,
Melbourne, Australia, 18Retired, formely at Children's Hospital
Westmead, Westmead, Australia R. Martin Arenas1, A. Escudero1, A. Campos1, A. del Pozo
Mate2, C. Prior1, C. Peña1, C. Gomez-Gonzalez1, E. Barroso1, E. Galvez1, F. Santos-Simarro1, F. Nieto1, G. Gordo1, H. Gonzalez-
Pecellin1, M. Solis2, I. Dapia1, J. Tenorio1, J. Nevado1, J. Moreno1, J. Solera1, K. Heath1, K. Ibañez2, L. Rodriguez-
Laguna1, L. Fernadez Garcia-Moya1, M. Bravo1, M. Palomares-
Bralo1, R. Mena1, M. Azca1, P. Lapunzina1, P. Arias1, P. Martinez1, R. Lleuger-Pujol1, S. Benito1, S. Garcia-Miñaur1, S. Rodriguez-Novoa1, V. F. Montaño1, L. Stark1, V. Ruiz1, V. Martinez-Glez1, I. Ibañez1, E. Vallespin1 R. Martin Arenas1, A. Escudero1, A. Campos1, A. del Pozo
Mate2, C. Prior1, C. Peña1, C. Gomez-Gonzalez1, E. Barroso1, E. Galvez1, F. Santos-Simarro1, F. Nieto1, G. Gordo1, H. Gonzalez-
Pecellin1, M. Solis2, I. Dapia1, J. Tenorio1, J. Nevado1, J. Moreno1, J. Solera1, K. Heath1, K. Ibañez2, L. Rodriguez-
Laguna1, L. Fernadez Garcia-Moya1, M. Bravo1, M. Palomares-
Bralo1, R. Mena1, M. Azca1, P. Lapunzina1, P. Arias1, P. Martinez1, R. Lleuger-Pujol1, S. Benito1, S. Garcia-Miñaur1, S. Rodriguez-Novoa1, V. F. Montaño1, L. Stark1, V. Ruiz1, V. Martinez-Glez1, I. Ibañez1, E. Vallespin1 1INGEMM NGS Diagnostic Group - Instituto de Genética
Médica y Molecular (INGEMM) - Hospital La Paz - IdiPaz –
CIBERER, Madrid, Spain, 2Bionformatics Unit - Instituto de
Genética Médica y Molecular (INGEMM) - Hospital La Paz -
IdiPaz - CIBERER, Madrid, Spain Chromosome microarrays are an essential tool for investi-
gation of copy number changes in children with congenital
anomalies and intellectual deficit. Attempts to standardise
microarray testing have focused on establishing technical
and clinical quality criteria, however external quality
assessment programs are still needed. We report on a
microarray proficiency testing program for Australasian
laboratories. Quality metrics evaluated included analytical
accuracy, result interpretation, report completeness, and
laboratory performance data: sample numbers, success and
abnormality rate and reporting times. Chromosome microarray proficiency testing and analysis
of quality metric data trends through an external quality
assessment program for Australasian laboratories Chromosome microarray proficiency testing and analysis
of quality metric data trends through an external quality
assessment program for Australasian laboratories D.C. Wright: None. L. Carey: None. N. Adayapalam:
None. N. Bain: None. S.M. Bain: None. A. Brown: None. N. Buzzacott: None. J. Cross: None. K. Dun: None. C. Joy: None. C. McCarthy: None. S. Moore: None. A.R. Murch: None. F. O'Malley: None. E. Parker: None. J. Watt: None. H. Wilkin: None. K. Fagan: None. M.D. Pertile: None. G.B. Peters: None. D. C. Wright1, L. Carey2, N. Adayapalam3, N. Bain4, S. M. Bain5, A. Brown6, N. Buzzacott7, J. Cross8, K. Dun9, C. Joy10, C. McCarthy11, S. Moore5, A. R. Murch12, F. O'Malley13, E. Parker14, J. Watt14, H. Wilkin15, K. Fagan16, M. D. Pertile17, G. B. Peters18 Detection of phage nucleic acid in human blood plasma in
data from NIPT protocol Kajsik:
None. J. Turňa: None. T. Szemes: A. Employment (full or
part-time); Significant; Geneton Ltd., Comenius University
Science Park, Bratislava, Slovakia. (full or part-time); Significant; Geneton Ltd., Comenius
University Science Park, Bratislava, Slovakia. M. Kajsik:
None. J. Turňa: None. T. Szemes: A. Employment (full or
part-time); Significant; Geneton Ltd., Comenius University
Science Park, Bratislava, Slovakia. results. Data trends showed a positive correlation with
improvement for all these quality metrics, however only
‘report completeness’ and reporting times reached statistical
significance. Whether the overall improvement in laboratory
performance was due to participation in this program, or
from accumulated laboratory experience over time, is not
clear. Either way, the outcome is likely to assist referring
clinicians and improve patient care. Detection of phage nucleic acid in human blood plasma in
data from NIPT protocol Materials and Methods: We sequenced total DNA and
transcribed RNA (cDNA) from plasma of both healthy
individuals and individuals with bacterial infection. In
addition, we analyzed samples from the surface of mucous
membrane for both groups. In analysis, human reads were
removed and all remaining fragments were taxonomically
labeled using metagenomic classifiers and phage mappers. Presence and composition of phages in samples were
visualized using Krona graphs. In order to determine if long read sequencing technolo-
gies overcome this limitation, we perform mtDNA sequen-
cing of several controls and patients with single or multiple
deletions on Oxford Nanopore MinION. This technology allowed an accurate identification at the
nucleotide level of the breakpoints of the deletions, even in
the case of complex multiple deletions. This breakpoint
determination is useful for family testing in the case of
single deletion. Furthermore, studies could also take
advantage of this method to decipher the mechanisms of
occurrence and to track the accumulation of mtDNA
multiple deletions in mitochondrial or degenerative dis-
eases, but also during normal aging. Results: We detected the presence of phage sequences in
a small number of samples in both groups. We hypothesize
that phages can be used as indicators of certain pathophy-
siological conditions. Results: We detected the presence of phage sequences in
a small number of samples in both groups. We hypothesize
that phages can be used as indicators of certain pathophy-
siological conditions. Results: We detected the presence of phage sequences in
a small number of samples in both groups. We hypothesize
that phages can be used as indicators of certain pathophy-
siological conditions. Conclusions: NIPT data offers a lot of additional
information and various bioinformatic approaches can be
used to obtain additional results. Our results demonstrate
the detection of phage sequences in the data which are
usually discarded in common NIPT analyses. In addition, despite a higher per base error rate in
comparison with other NGS technologies, the accuracy of
Oxford Nanopore MinION sequencing is sufficient to
correctly determine the mitochondrial haplogroup of each
patient. Due to the portability of MinION, it raises the I. Gazdaricová: None. D. Smoľak: None. A. Balá:
None. J. Budi: A. Employment
(full or part-time);
Significant; Geneton Ltd., Comenius University Science
Park, Bratislava, Slovakia. J. Gazdarica: A. Employment Abstracts from the 51st European Society of Human Genetics Conference: Posters 527 (full or part-time); Significant; Geneton Ltd., Comenius
University Science Park, Bratislava, Slovakia. M. Development and clinical utility ofINGEMM custom panels Between 2009 and
2014 nine samples were dispatched with variable results for
analytical accuracy (30-100%), correct interpretation (32-
96%), and report completeness (30-92%). Laboratory per-
formance data (2007-2014) showed an overall mean success
rate of 99.2% and abnormality rate of 23.6%. Reporting
times decreased from >90 days to <30 days for normal
results and from >102 days to <35 days for abnormal Introduction: There are many ways to tackle the genetic
study in patients in NGS. Custom panels offer better base-
pair coverage, running times, costs and dataset handling
than other NGS applications such as whole genome
sequencing and whole exome sequencing. We present our
workflow and results in more than 10,000 patients studied
with 25 different INGEMM NGS custom panels. Patients and Methods: Sequencing libraries are prepared
with SeqCap EZ system (RocheNimblegen) using NEXT-
flex- DNA-barcodes (Bioo Scientific) and sequenced on a
NextSeq500
platform
(Illumina). The
researcher
has
selected a group of genes involved in their pathologies. Each panel has a particular size, and in our workflow we
combine different panels using NEXTflex-DNA-barcodes
in the same run. Bioinformatic analysis was carried out by
the Clinical Bioinformatics Unit of INGEMM center. J. del Picchia 528 covered samples, or increases per sample data requirements
to eliminate variations. UV absorption, intercalating dyes,
capillary electrophoresis and quantitative PCR are widely
used for library quantification. In this study we compared
accuracy of these popular methods with ultra-low coverage
whole-genome sequencing (ULC-WGS). covered samples, or increases per sample data requirements
to eliminate variations. UV absorption, intercalating dyes,
capillary electrophoresis and quantitative PCR are widely
used for library quantification. In this study we compared
accuracy of these popular methods with ultra-low coverage
whole-genome sequencing (ULC-WGS). Results and Discussion: The diagnostic yield can change
between the different custom panels and pathologies, but in
general we observe a high level of diagnostic yield. Our
biggest custom panel is the Clinical one, it contains 1,588
genes involved in intellectual disability, autism spectrum
disorders and other common genetic disorders, the diag-
nostic yield is 50%. “BAF-complex panel” includes 96
genes involved in BAF-complex (Coffin-Siris syndrome,
Nicolaides-Baraitser syndrome) and other genetic disorders
with overlapping features: 45%. “Cardio-panel”, 325 genes
related with cardiovascular diseases, 29%. “Hepato panel”
with 125 genes involved in genetic liver diseases: 28% and
“Oft-panel”, genetic ophthalmological diseases and 298
genes, 60%. Development and clinical utility ofINGEMM custom panels To optimize the analyses, panel testing should
be performed by bioinformaticians, clinicians and labora-
tory staff in close collaboration. Materials and Methods: DNA was extracted using
QIAmp DNA mini kit (Qiagen). Libraries were prepared
using NEBNext Ultra DNA Kit (NEB). We quantified
libraries by Bioanalyzer 2100 (Agilent Technologies),
different qPCR approaches, Qubit 3.0 (Life Technologies),
300PE sequencing at nano flowcell on Miseq (Illumina)
with followed bioinformatic quantification. Libraries were
pooled together and enriched using Focused Exome kit
(Agilent
Technologies)
and
sequenced
on
Illumina
HiSeq 2500. tory staff in close collaboration. R. Martin Arenas: None. A. Escudero: None. A. Campos: None. A. del Pozo Mate: None. C. Prior: None. C. Peña: None. C. Gomez-Gonzalez: None. E. Barroso:
None. E. Galvez: None. F. Santos-Simarro: None. F. Nieto: None. G. Gordo: None. H. Gonzalez-Pecellin:
None. M. Solis: None. I. Dapia: None. J. Tenorio: None. J. Nevado: None. J. Moreno: None. J. Solera: None. K. Heath: None. K. Ibañez: None. L. Rodriguez-Laguna:
None. L. Fernadez Garcia-Moya: None. M. Bravo: None. M. Palomares-Bralo: None. R. Mena: None. M. Azca:
None. P. Lapunzina: None. P. Arias: None. P. Martinez:
None. R. Lleuger-Pujol: None. S. Benito: None. S. Garcia-Miñaur: None. S. Rodriguez-Novoa: None. V. F. Montaño: None. L. Stark: None. V. Ruiz: None. V. Martinez-Glez:
None. I. Ibañez:
None. E. Vallespin: None. Results: We found that ULC-WGS is the most accurate
library quantification method. This method significantly
decrease sample to sample variations thus being the most
cost-effective. We also found that insert size significantly
influences capture efficiency. Results: We found that ULC-WGS is the most accurate
library quantification method. This method significantly
decrease sample to sample variations thus being the most
cost-effective. We also found that insert size significantly
influences capture efficiency. Conclusions: Our results show that ultra-low coverage
sequencing overcomes qPCR and other popular library
quantification methods in accuracy and cost-effectiveness. Conclusions: Our results show that ultra-low coverage
sequencing overcomes qPCR and other popular library
quantification methods in accuracy and cost-effectiveness. A. Krasnenko: None. I. Stetsenko: None. N. Plotnikov:
None. K. Tsukanov: None. O. Klimchuk: None. V. Ilinsky: None. P14.075C Reinterpretation and reclassification in diagnostic Next-
Generation Sequencing: current daily practices and
dilemmas of Dutch clinical genomics laboratory
professionals P14.074B J. el Mecky1, L. Johansson1, T. Dijkhuizen1, A. van der Hout1,
M. Plantinga1, A. Fenwick2, A. Lucassen2, I. van Langen1
1University Medical Centre Groningen, Groningen,
Netherlands, 2University of Southampton, Southampton,
United Kingdom J. el Mecky1, L. Johansson1, T. Dijkhuizen1, A. van der Hout1,
M. Plantinga1, A. Fenwick2, A. Lucassen2, I. van Langen1 Ultra-low coverage sequencing is the most accurate method
for library quantification prior to post-pooling exome
capture Ultra-low coverage sequencing is the most accurate method
for library quantification prior to post-pooling exome
capture J. el Mecky1, L. Johansson1, T. Dijkhuizen1, A. van der Hout1,
M. Plantinga1, A. Fenwick2, A. Lucassen2, I. van Langen1 1University Medical Centre Groningen, Groningen,
Netherlands, 2University of Southampton, Southampton,
United Kingdom 1University Medical Centre Groningen, Groningen,
Netherlands, 2University of Southampton, Southampton,
United Kingdom A. Krasnenko, I. Stetsenko, N. Plotnikov, K. Tsukanov, O. Klimchuk, V. Ilinsky Introduction: Information on genetic findings is in rapid
flux: as new evidence becomes available, genetic results
may turn out more/less pathogenic than initially commu-
nicated by the laboratory, which can have important con-
sequences for patients. How laboratory professionals deal
with this constantly changing information remains unclear;
therefore, this topic merits attention. Genotek Ltd., Moscow, Russian Federation Genotek Ltd., Moscow, Russian Federation Introduction: Exome sequencing is a powerful tool for
inherited disease diagnostics. The price of exome sequen-
cing has reduced dramatically, but library preparation and
target enrichment are still expensive. Post-pooling target
capture decreases enrichment price per sample, but in this
case pre-pooling quantification becomes crucial for accurate
estimation of required sequencing data. Equal distribution
of reads per sample gives a possibility to sequence more
samples in a single run. On the other hand, sample to
sample variation can result in either dropout of under This is the first qualitative research into how reinterpreta-
tion (reviewing new evidence on variants) and reclassifica-
tion (changing variants’ initial classifications, e.g. VUS to
pathogenic) functions in everyday genomics laboratory Abstracts from the 51st European Society of Human Genetics Conference: Posters 529 triplex PCR reaction. The IntelliQube utilizes the Array
Tape® consumable and combines liquid handling with real-
time qPCR analysis in 1.6 μL reaction volumes. practice, and into laboratory professionals’ perspectives on
ideal (future) practice. Methods: Fifteen laboratory specialists from all (nine)
Dutch genomics laboratories participated in a 7-day online
focus group. Brief follow-up telephone interviews were
conducted with participants. Using TBP and GAPDH as reference genes, it was
determined that Muc1 was upregulated in kidney 143-fold
and lung 297-fold in comparison to liver, which correlated
with previously published data. Using a three way ANOVA,
it was shown that there was no significant difference in
results between singleplex and triplex reaction formats, or
between multiple runs on the IntelliQube. Results: • No formal local/national reinterpretation policies or
guidelines exist, despite participants’ expressed. • Participants identified a lack of phenotypic information
updates from clinic to laboratory This study demonstrates the ability of this instrument and
the associated Array Tape technology to successfully
multiplex BHQ qPCR assays in a one-step RT-PCR format
for gene expression studies, without compromising data
quality. Combined with the automated inline process and
economic benefits of Array Tape, the IntelliQube and
associated BHQ probe chemistry may prove to be a very
useful platform for multiplex qPCR applications in Human
Genetics. • Practices among different laboratories appear similar: 1. Variants are reinterpreted ad hoc (commonly at request
of clinicians or following identification of a previously
detected VUS in a new proband) and never periodically. 2. Variants are reclassified depending on new evidence. 3.Clinicians are recontacted by laboratories when reclas-
sifications have a potentially clinically relevant impact on
patients’ treatment/monitoring. M. Freeman: None. L. P14.076D Multiplex, low-volume one-step RT-qPCR for gene
expression analysis using the IntelliQube® and BHQ®
probes Background: Since Next-Generation Sequencing (NGS)
and buccal samples are more and more used in Genetic
field, Copan developed buccal collection and preservation
devices for Genetic applications: human DNA (hDNA) free
FLOQSwabs® (FS) available W/ or W/O Active Drying
System; eNAT®, a molecular medium preserving nucleic
acids at room temperature, and NAO® (nucleic acid opti-
mizer) basket for complete sample recovery from the swab. The objective of this study was to demonstrate the perfor-
mance of Copan collection and preservation devices for
Genetic diagnosis and NGS analysis, including clinical
exome (~6,300 genes). M. Freeman1, L. Linz1, K. Lind2, V. Novosadova2, S. H. Bauer3
1LGC Douglas Scientific, Alexandria, MN, United States,
2TATAA Biocenter, Goteborg, Sweden, 3LGC Genomics,
Hoddesdon, United Kingdom M. Freeman1, L. Linz1, K. Lind2, V. Novosadova2, S. H. Bauer3 M. Freeman1, L. Linz1, K. Lind2, V. Novosadova2, S. H. Bauer3 1LGC Douglas Scientific, Alexandria, MN, United States,
2TATAA Biocenter, Goteborg, Sweden, 3LGC Genomics,
Hoddesdon, United Kingdom 1LGC Douglas Scientific, Alexandria, MN, United States,
2TATAA Biocenter, Goteborg, Sweden, 3LGC Genomics,
Hoddesdon, United Kingdom One-step reverse transcription-PCR (RT-PCR) is widely
used for gene expression analysis, RNA virus detection, and
routine RNA quantification experiments. In this study, we evaluated the IntelliQube® real-time PCR
instrument for multiplex gene expression analysis using
commercially available RNA from human liver, lung, and
kidney tissue, a one-step RT-PCR master mix, and BHQ®
probe-based assays from LGC Biosearch Technologies. BHQ probes and primers were designed to target the mucin
1
(MUC1),
glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH), and the TATA-box binding protein (TBP) in a Methods: Buccal swabs (BS), collected with dry FS and
with FS stored in eNAT®, were extracted using QIAamp®
DNA Mini Kit (Qiagen) and processed using the NAO®
basket. Extracted hDNA was analyzed by Real-Time PCR,
by electrophoresis on agarose gel and by NanoDrop
Spectrophotometer (Thermo Fisher). DNA extracted from
3 dry FS and 3 in eNAT® BS, tested 4 weeks post collection,
were used to analyze clinical exome using NGS technique. Methods: Buccal swabs (BS), collected with dry FS and
with FS stored in eNAT®, were extracted using QIAamp®
DNA Mini Kit (Qiagen) and processed using the NAO®
basket. Extracted hDNA was analyzed by Real-Time PCR,
by electrophoresis on agarose gel and by NanoDrop
Spectrophotometer (Thermo Fisher). Copan buccal collection devices for Next Generation
Sequencing analysis J. el Mecky: None. L. Johansson: None. T. Dijkhuizen:
None. A. van der Hout: None. M. Plantinga: None. A. Fenwick:
None. A. Lucassen:
None. I. van
Langen: None. J. el Mecky: None. L. Johansson: None. T. Dijkhuizen:
None. A. van der Hout: None. M. Plantinga: None. A. Fenwick:
None. A. Lucassen:
None. I. van
Langen: None. A. Squassina1, A. Gervasoni1, M. Rosso1, S. Castriciano1, A. Breda Klobus2 A. Squassina1, A. Gervasoni1, M. Rosso1, S. Castriciano1, A. Breda Klobus2 1COPAN ITALIA, BRESCIA, Italy, 2Breda Genetics srl,
BRESCIA, Italy Genotek Ltd., Moscow, Russian Federation Linz: None. K. Lind: None. V. Novosadova: None. S.H. Bauer: Other; Significant; LGC
Genomics. • However, important nuances are discernible: e.g. laboratory specialists differ in what is considered sufficient
evidence for a reclassification, and what are deemed
clinically relevant reclassifications that should be commu-
nicated to clinicians. This has important implications for
guideline development, as it demonstrates that policy in this
context may differ widely in its implementation. P14.076D DNA extracted from
3 dry FS and 3 in eNAT® BS, tested 4 weeks post collection,
were used to analyze clinical exome using NGS technique. 530 J. del Picchia Results: The median error rate ranged from 0.25-0.48%
in Q20-trimmed de-novo sequencing, depending on sequen-
cing chemistry and short(350bp)-, medium(500bp)-, and
long(800bp)-read
sequencing
options. The
percent
of
correctly called mixed bases in resequencing data was
greater than 99%; the false negative rate was 0.6%. Homopolymer stretches of length 31 were sequenced
without error. The 5’ end of amplicons sequenced with
BigDye Direct chemistry were sequenced flawlessly starting
at the first base. Results: All the samples gave appropriate quantity and
purity of hDNA (low 16, medium 40 and high 77 μg of
hDNA/swab) and no DNA degradation on gel. High-
throughput NGS performed on the extracted DNA gener-
ated results at quality levels as NGS results performed using
EDTA-blood. Results: All the samples gave appropriate quantity and
purity of hDNA (low 16, medium 40 and high 77 μg of
hDNA/swab) and no DNA degradation on gel. High-
throughput NGS performed on the extracted DNA gener-
ated results at quality levels as NGS results performed using
EDTA-blood. Conclusions: Data obtained in this study demonstrated
that good quantity and quality of DNA can be recovered
from buccal swabs collected with the Copan hDNA free
FLOQSwabs® stored dry or in eNAT®medium. DNA
recovered is also optimal for NGS analyses such, as clinical
exome sequencing. Conclusions: Testing demonstrated robust performance
for basecalling and QV accuracy on a wide variety of
sequences
generated
on
the
new
SeqStudio
Genetic
Analyzer. A. Squassina: A. Employment (full or part-time);
Significant; COPAN ITALIA. A. Gervasoni: A. Employ-
ment (full or part-time); Significant; COPAN ITALIA. M. Rosso: A. Employment (full or part-time); Significant;
COPAN ITALIA. S. Castriciano: A. Employment (full or
part-time); Significant; COPAN ITALIA. A. Breda Klo-
bus: A. Employment (full or part-time); Significant; Breda
Genetics srl. A. Squassina: A. Employment (full or part-time);
Significant; COPAN ITALIA. A. Gervasoni: A. Employ-
ment (full or part-time); Significant; COPAN ITALIA. M. Rosso: A. Employment (full or part-time); Significant;
COPAN ITALIA. S. Castriciano: A. Employment (full or
part-time); Significant; COPAN ITALIA. A. Breda Klo-
bus: A. Employment (full or part-time); Significant; Breda
Genetics srl. Y. Chu: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. S. Schneider: A. Employment
(full or part-time); Significant; Thermo Fisher Scientific. S. Berosik: A. Accuracy of DNA Sequence and Quality Values for a new
Sanger Sequencing Instrument Y. Chu, S. Schneider, S. Berosik, J. Bodeau, S. Chang, D. Rodriguez, D. Woo, D. A. Hom P14.076D Employment (full or part-time); Significant;
Thermo Fisher Scientific. J. Bodeau: A. Employment (full
or part-time); Significant; Thermo Fisher Scientific. S. Chang: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. D. Rodriguez: A. Employment
(full or part-time); Significant; Thermo Fisher Scientific. D. Woo: A. Employment (full or part-time); Significant;
Thermo Fisher Scientific. D.A. Hom: None. Expanding the spectrum of SHOX mutations in idiopathic
short stature patients through a custom array-CGH Expanding the spectrum of SHOX mutations in idiopathic
short stature patients through a custom array-CGH Thermo Fisher Scientific, South San Francisco, CA, United
States Thermo Fisher Scientific, South San Francisco, CA, United
States A. Fanelli1, D. Babu1, S. Mellone1, G. Stress1, S. Bellone2, F. Prodam2, G. Bona2, S. Vannelli3, M. Giordano1 A. Fanelli1, D. Babu1, S. Mellone1, G. Stress1, S. Bellone2, F. Prodam2, G. Bona2, S. Vannelli3, M. Giordano1 Introduction: Sanger Sequencing with capillary electro-
phoresis is a cost-effective, time-efficient, and highly
accurate DNA sequencing method to interrogate DNA for
Mendelian variants and minor variants present at 5% or
greater, using Minor Variant Finder software. With the
introduction of the easy-to-use Applied Biosystems Seq-
Studio Genetic Analyzer, featuring updated KB Basecaller,
new spectral auto-calibration algorithm, and integrated
cartridge containing a new separation polymer, testing the
accuracy of basecalls and Quality Values of SeqStudio
sequence data is important. 1Laboratory of Human Genetics, Department of Health
Sciences, University of Eastern Piedmont, Novara, Italy,
2Pediatric Unit, University of Eastern Piedmont, Novara, Italy,
3Ospedale Regina Margherita, Turin, Italy The short
stature
homeobox-containing
gene
(SHOX)
resides within the pseudoautosomal region 1 (PAR1) on the
sex chromosomes, and SHOX defects are responsible for 2-
15% of idiopathic short stature (ISS) cases. Molecular
diagnosis for SHOX deficiency is carried out by sequencing
the coding exons and by performing MLPA for deletions/
duplications. We screened 700 ISS patients for SHOX
defects for diagnostic purposes; fifty-four patients (7.7%)
carried mutations. Among these, one patient showed a
single probe deletion at homozygous state that did not
involve any of the known enhancers. Since MLPA probes
are enriched in the exons and in the known enhancers, we
hypothesised that they might be other non-coding elements
with potential regulatory activity not yet described. To test
our hypothesis, we developed a high-resolution custom Materials and Methods: Statistically sound validation of
basecalling and QV accuracy requires generation of a
significant number of diverse sequences. We used purified
plasmids derived from shotgun sequencing of NA12878
NIST genome reference standard for de-novo sequencing as
well as PCR resequencing of 38 amplicon targets, ranging
in length from 143-873 bases, in up to 50 human specimens
for mixed-base calling. 10,000 samples were generated,
totaling 6,400,000 basecalls. Sequencing reads were tested
for basecalling and quality value accuracy. P14.081A
Accurate calls for copy number variation using the
Novallele™HRM Analyzer Spontaneous miscarriages occur in 10-40% of pregnancies. The high level of miscarriage can be related to difficulties in
embryonic development caused by anomalies in the embryo
itself. It is well known that a large proportion of first-
trimester spontaneous abortions is caused by chromosomal
disorders (40-64%): autosomal trisomies are most fre-
quently detected, followed by monosomy X and poly-
ploidies. The results of conventional cytogenetic studies of
spontaneous abortions depend on tissue culturing and are
associated with a significant cell culture failure rate related
to necrotic tissues, low mitotic rate and maternal cell con-
tamination. Cytogenetic studies show high rates of failure
and chromosomal analysis is possible only in a low number
of samples (20 - 30%). Extended QF-PCR analysis of
chromosomes 13,15,16,18,21,22, X and Y was used for the
rapid analysis of the most frequent chromosomal disorders
in spontaneous miscarriage in order to overcome the pro-
blems connected with karyotyping such as culture failure,
external contamination and selective growth of maternal
cells. The results of QF-PCR have been compared with
those of classical karyotyping. Since 2009 karyotyping in
first-trimester miscarriage has been replaced by DNA ana-
lysis by QF-PCR. In about 98% of cases a report has been
produced; in almost 50% of them a genetic aneuploidy has
been identified as the genetic cause of miscarriage. In order
to estimate the recurrence risk of those women in which the
genetic cause of the miscarriage has not been detected by
QF-PCR, we are taking into consideration a CGH Array
analysis. Y. Machida, Z. Kabir, L. Jiang, S. Paquerault, S. Kanderian, J. Huuskonen Y. Machida, Z. Kabir, L. Jiang, S. Paquerault, S. Kanderian, J. Huuskonen Canon U.S. Life Sciences, Inc., Rockville, MD, United States Canon U.S. Life Sciences, Inc., Rockville, MD, United States Introduction: The Novallele HRM Analyzer is web-based
software with comprehensive algorithms capable of ana-
lyzing and displaying high-resolution melting (HRM) data
for copy number variation (CNV) assessment. We investi-
gated two analysis methods using the software. Methods: The first method requires multiple, character-
ized DNA controls from cell lines. Review of the difference
plot using the software with the unknown samples overlaid
on the DNA controls suffices to assign a copy number. The
second method requires only one characterized 2-copy
control prepared uniformly with the samples. Expanding the spectrum of SHOX mutations in idiopathic
short stature patients through a custom array-CGH Abstracts from the 51st European Society of Human Genetics Conference: Posters 531 array-CGH platform with 8000 probes within the PAR1 and
we screened 50 individuals that tested negative with the
standard methods. Two deletions of 12.3 kb (1 patient) and
7 kb (2 patients) downstream SHOX not present in 300
controls were identified. These rearrangements shared a 1.8
kb region, while the 12.3 kb deletion included a 1 kb region
conserved among different species. A functional assay to
test if these sequences affect the gene expression was per-
formed in NIH3T3 cells. We observed an increase of luci-
ferase activity (45%) for the construct harbouring the 1.8 kb
region compared to the promoter vector (p = 0,0003). In
conclusion, we identified a novel region downstream of
SHOX with potential regulatory activity that might be
responsible for ISS cases undiagnosed through standard
procedures. a single, characterized control prepared with the samples,
which avoids requiring multiple control wells. Both CNV
analysis methods using the Novallele HRM Analyzer
software generate accurate CNV calls. This product is only
available in the U.S.A. The products mentioned are for
Research Use Only. Not for use in diagnostic procedures. a single, characterized control prepared with the samples,
which avoids requiring multiple control wells. Both CNV
analysis methods using the Novallele HRM Analyzer
software generate accurate CNV calls. This product is only
available in the U.S.A. The products mentioned are for
Research Use Only. Not for use in diagnostic procedures. Y. Machida: None. Z. Kabir: None. L. Jiang: None. S. Paquerault:
None. S. Kanderian:
None. J. Huuskonen: None. SOD Diagnostica genetica, AOU Careggi, Florence, Italy SOD Diagnostica genetica, AOU Careggi, Florence, Italy SOD Diagnostica genetica, AOU Careggi, Florence, Italy Quantitative fluorescent polymerase chain reaction (QF-
PCR) for rapid and culture independent aneuploidy
analysis in spontaneous miscarriages Quantitative fluorescent polymerase chain reaction (QF-
PCR) for rapid and culture independent aneuploidy
analysis in spontaneous miscarriages L. Falai, S. Bernabini, M. Betti, F. Sbernini, R. Ciampi, S. Fibbi,
C. Romolini, E. Pelo, C. Pescucci L. Falai, S. Bernabini, M. Betti, F. Sbernini, R. Ciampi, S. Fibbi,
C. Romolini, E. Pelo, C. Pescucci L. Falai, S. Bernabini, M. Betti, F. Sbernini, R. Ciampi, S. Fibbi,
C. Romolini, E. Pelo, C. Pescucci A. Fanelli: None. D. Babu: None. S. Mellone: None. G. Stress: None. S. Bellone: None. F. Prodam: None. G. Bona: None. S. Vannelli: None. M. Giordano: None. P14.082B Quantitative fluorescent polymerase chain reaction (QF-
PCR) for rapid and culture independent aneuploidy
analysis in spontaneous miscarriages L. Falai: None. S. Bernabini: None. M. Betti: None. F.
Sbernini: None. R. Ciampi: None. S. Fibbi: None. C.
Romolini: None. E. Pelo: None. C. Pescucci: None. Bionano Genomics, San Diego, CA, United States Diverse genetic variations exits among individuals and are
crucial to the understanding of human biology and diseases. Numerous efforts are underway to generate high-quality
reference genomes for different ethnic groups. However,
human genomes are complex and contain large fractions of
repetitive sequences that make generating high-quality
assemblies difficult. Bionano genome mapping provides a
solution to visualize genome structure. The synergistic
approach of combining next-generation sequencing (NGS)
data with Bionano maps produces affordable, contiguous,
and accurate de novo genome assemblies. Repetitive DNA sequence represented a big technical
challenge for NGS technologies. Their analysis istradition-
ally based on PCR-amplification followed by fluorescence
capillary electrophoresis to identifyfragment length differ-
ences. Theoretically, NGS technology could offer several
potential advantages forTandem Repeat Polymorphisms
(TRPs) genotyping.Our aim was to test the feasibility of the
analysis of TRPs in NGS data. To do this we compared 16
differentdisease-associated TRPs genotypes from short-read
NGS data to those derived through fluorescencefragment
analysis approach.We sequenced and analyzed 14 DNA
samples affected by various neurodegenerative diseases
with repeatexpansions of different lengths for 16 disease
associated TRPs by Illumina MiSeq sequencing platform
(300+300 bp reads). We performed a repeat specific cap-
ture probes design (Duitama 2014) which uses theflanking
regions of the repeats to design enrichment probes in unique
regions. For the target regionsenrichment, we used Sur-
eSelect XT Kit, (Agilent technologies). Besides a known
bioinformatics tool(lobSTR), a new software procedure was
successfully implemented to precisely genotype TRPs from
NGSdata to achieve genotype accuracy and efficiency. Overall, we were able to compare 224 genotypes. Locus-
specific TRP typing demonstrated a very good correlation
(up to 80%) between genotypes derived byfluorescence
fragment analysis and those measured by our NGS
approach. Even for very large expansionswe have been able
to identify the samples carrying a pathological expansion. This pilot experimentdemonstrated the feasibility of the
analysis of TRPs in NGS data for a large number of loci. Here, we present an improvement to Bionano’s traditional
nickase-based approach by employing an enzymatic direct
labeling approach. This new method doesn't introduce any
undesirable breaks in the DNA and allows us to create very
contiguous Bionano maps which can then be used to
scaffold NGS sequence assemblies to produce highly
contiguous and structurally accurate hybrid assemblies that
can span most repeat regions. This direct labeling is
compatible with a vast array of various organisms. We validated our approach with the well-studied human
NA12878 genome. P14.081A
Accurate calls for copy number variation using the
Novallele™HRM Analyzer A copy
number is assigned to each sample using both the number of
melt peaks on the derivative plot and sample fluorescence
relative to the control on the difference plot. Samples were
extracted
using
four
different
commercial
kits. Both
methods were investigated using SMN1 (n=1462) and
SMN2 (n=1557) melt curves generated on five different
types of thermocyclers. Results: Both methods were 100% accurate. CNV calls
were accurately assigned using all five thermocyclers and
all four sample extraction kits. y
L. Falai: None. S. Bernabini: None. M. Betti: None. F. Sbernini: None. R. Ciampi: None. S. Fibbi: None. C. Romolini: None. E. Pelo: None. C. Pescucci: None. L. Falai: None. S. Bernabini: None. M. Betti: None. F. Sbernini: None. R. Ciampi: None. S. Fibbi: None. C. Romolini: None. E. Pelo: None. C. Pescucci: None. Conclusions: The first method is simpler as compared to
the second method and allows a user to immediately
perform their experiments using commercially available
controls. The advantage of the second method is that it uses 532 J. del Picchia Disease associated tandem repeat genotyping from NGS
target sequencing data Bionano Genomics, San Diego, CA, United States Starting with NGS assemblies with N50
ranging from 0.18 - 14.5 Mbp, we produced hybrid
assemblies with N50 from 70 to 80 Mbp. Chromosome-
arm length scaffolds were assembled in 20 out of 23
chromosomes,
and
alignments
show
that
they
were
consistent with the hg19 reference. The hybrid assemblies
incorporated 80-97% of total NGS sequences with over
99% scaffolding accuracy. The scaffolds generated with this
data have set a new standard for genome assembly that can
be accomplished in less than one week and starting at 500
dollars. J. Wang: None. A. Pang: None. E. Lam: None. T. Anantharaman: None. A. Hastie: None. H. Sadowski:
None. Z. Zhu: None. R. Prins: None. M. Austin: None. H. Cao: None. M. Borodkin: None. G. Papoutsoglou: None. L. Corrado *: None. F. Geraci *: None. M. Severgnini
*: None. A. Di Pierro: None. V. Frontini: None. E. Mangano: None. L.M. Genovese: None. N. Barizzone:
None. R. D'Aurizio: None. R. Croce: None. F. De
Marchi: None. L. Mazzini: None. A. Brusco: None. G. De Bellis: None. G. Manzini: None. R. Bordoni *: None. M. Pellegrini *: None. S. D' Alfonso *: None. P14.083C
Building high quality, chromosome-scale, de novo genome
assemblies by Bionano genome mapping and NGS
sequencing L. Corrado *1, F. Geraci *2, M. Severgnini *3, A. Di Pierro1, V. Frontini1, E. Mangano3, L. M. Genovese2, N. Barizzone1, R. D'Aurizio2, R. Croce1, F. De Marchi4, L. Mazzini4, A. Brusco5,
G. De Bellis3, G. Manzini2,6, R. Bordoni *3, M. Pellegrini *2, S. D' Alfonso *1 J. Wang, A. Pang, E. Lam, T. Anantharaman, A. Hastie, H.
Sadowski, Z. Zhu, R. Prins, M. Austin, H. Cao, M. Borodkin, G.
Papoutsoglou J. Wang, A. Pang, E. Lam, T. Anantharaman, A. Hastie, H. Sadowski, Z. Zhu, R. Prins, M. Austin, H. Cao, M. Borodkin, G. Papoutsoglou 1Human Genetics Lab, Department of Health Sciencs,
University of Eastern Piedmont, Novara, Italy, 2Institute of
informatics and telematics of CNR, Pisa, Italy, 3instute for
biomedical technologies, CNR-ITB, Segrate, Italy, 4ALS center
AOU Maggiore della Carità, Novara, Italy, 5Medical sciences,
University of Torino, Torino, Italy, 6Univeristy of Eastern
Piedmont (UPO), Vercelli, Italy Bionano Genomics, San Diego, CA, United States Bionano Genomics, San Diego, CA, United States CMGG Ghent University Hospital, Ghent, Belgium Specific point mutations in HFE or MTHFR give rise to
Haemochromatosis type 1 or Homocystinuria respectively,
for both genes it is not feasible nor cost efficient to routinely
screen the complete coding region. Although the CFTR
mutation spectrum is more variable, a population bias is
observed and targeted mutation analysis of preselected
mutations is frequently offered as a first line test. Numerous
commercially available kits were launched to analyse pre-
defined mutations in these genes. In an era where the
majority of routine diagnostic analyses moved to Next
generation sequencing, we developed a (multiplex) NGS
approach to include CFTR, HFE and MTHFR targeted
mutation analysis in our general workflow. In this workflow
we specifically target two HFE polymorphisms, MTHFR
c.677 C>T and the 50 most frequent CFTR mutations
detected in the Belgian population. Library preparation is
done with a modified Nextera XT protocol and sequencing
on a Miseq. An intensive pooling strategy decreased the
sequencing cost, down to the limit were single nucleotide
sequencing with NGS becomes cost effective. With in
house developed scripts analyzing only the nucleotides of
interest, we generate patient specific reports that can easily
be interpreted. A thorough validation of this workflow was
performed with hundreds of samples resulting in an overall
sensitivity (>99%) similar to Sanger sequencing and a
positive predictive value of >99%. Furthermore, the inde-
pendency of commercially available kits allows us to easily
change or add targets to our panels. The
Italian
Telethon
Undiagnosed
Diseases
Program
(TUDP) mission is bestowing a molecular diagnosis to rare
undiagnosed patients collected through a countrywide net-
work of pediatric hubs. Patients are prioritized according to
well defined criteria, described using standardized and
shared tools (i. e. Human Phenotype Ontology, HPO) and
made “matchable” with other cases stemming from other
Undiagnosed Diseases Programs all over the world, in order
to favor the finding of “second cases” and to confirm ultra
rare causal variants. We focus on pediatric patients with
complex or syndromic diseases, who are subjected to in-
depth exome sequencing. A tabular output provides anno-
tations of genes and variants with multiple predictions of
their potential impact and observed frequency in external
and internal control datasets. Selected variants of uncertain
pathogenic significance are tested through functional studies
to elucidate the disease causality and the pathogenetic
mechanism. P14.084D Disease associated tandem repeat genotyping from NGS
target sequencing data Abstracts from the 51st European Society of Human Genetics Conference: Posters 533 Y. Matsubara1, K. Hata1, T. Kaname1, K. Kosaki2, the IRUD-P
Consortium 1Telethon Institute of Genetics and Medicine - TIGEM,
Pozzuoli (NA), Italy, 2Telethon Institute of Genetics and
Medicine - TIGEM and ICAR-CNR, Naples, Italy, Pozzuoli CMGG Ghent University Hospital, Ghent, Belgium For instance, depending on the supposed phe-
notypic effect, cellular model created by gene-editing or
non-mammalian vertebrate models such as medaka fish are
developed and characterized. Among the 200 patients we
have gathered discussed, approximately two thirds have
been prioritized and subjected to whole exome sequencing
as “trios” (case(s) and parents). Overall, the success rate of
diagnosing of TUDP is about 50%. Thanks to a new phased
exome sequencing technology, we will now be also able to
determine separately the maternal and paternal haplotypes
in order to detect elusive mutations such as single exon
deletions, polymorphic repeats and others. T. Rosseel: None. K. Claes: None. E. Imant: None. E. d'Haese: None. E. Debals: None. P. Coucke: None. E. De
Baere: None. A. De Jaegher: None. K. De Leeneer: None. R. Castello: None. A. Torella: None. F. Musacchia:
None. M. Mutarelli: None. M. Dionisi: None. G. Oliva:
None. M. Pizzo: None. G. Mancano: None. S. Maitz:
None. F. Grilli: None. G. Sgroi: None. G. Cappuccio:
None. M. Pinelli: None. G. Esposito: None. G. Parenti:
None. S. Banfi: None. A. Selicorni: None. N. Brunetti-
Pierri: None. V. Nigro: None. G. Casari: None. R. Castello: None. A. Torella: None. F. Musacchia:
None. M. Mutarelli: None. M. Dionisi: None. G. Oliva:
None. M. Pizzo: None. G. Mancano: None. S. Maitz:
None. F. Grilli: None. G. Sgroi: None. G. Cappuccio:
None. M. Pinelli: None. G. Esposito: None. G. Parenti:
None. S. Banfi: None. A. Selicorni: None. N. Brunetti-
Pierri: None. V. Nigro: None. G. Casari: None. P14.087C P14.087C
The Italian Telethon Undiagnosed diseases Program .08 C
The Italian Telethon Undiagnosed diseases Program R. Castello1, A. Torella1, F. Musacchia1, M. Mutarelli1, M. Dionisi1, G. Oliva2, M. Pizzo1, G. Mancano3, S. Maitz3, F. Grilli4,
G. Sgroi3, G. Cappuccio5, M. Pinelli5, G. Esposito6, G. Parenti5,
S. Banfi6, A. Selicorni7, N. Brunetti-Pierri5, V. Nigro6, G. Casari1,8, Telethon Undiagnosed Diseases Program P14.085A
A Next generation analysis of mutation hot spots in CFTR,
MTHFR and HFE (NA), Italy, 3MBBM Foundation, Monza, Italy, 4Sant’Anna
Hospital, Como, Italy, 5Telethon Institute of Genetics and
Medicine - TIGEM and DISMET, Federico II University,
Naples, Italy, Pozzuoli (NA), Italy, 6Telethon Institute of
Genetics and Medicine - TIGEM and University of Campania
Luigi Vanvitelli, Naples, Italy, Pozzuoli (NA), Italy, 7MBBM
Foundation, Monza and Sant’Anna Hospital, Como, Italy, 8San
Raffaele University, Milan, Italy T. Rosseel, K. Claes, E. Imant, E. d'Haese, E. Debals, P. Coucke,
E. De Baere, A. De Jaegher, K. De Leeneer T. Rosseel, K. Claes, E. Imant, E. d'Haese, E. Debals, P. Coucke,
E. De Baere, A. De Jaegher, K. De Leeneer T. Rosseel, K. Claes, E. Imant, E. d'Haese, E. Debals, P. Coucke,
E. De Baere, A. De Jaegher, K. De Leeneer CMGG Ghent University Hospital, Ghent, Belgium P14.088D Initiative on Rare and Undiagnosed Diseases in Pediatrics
(IRUD-P) in Japan: recent achievement and statistics 534 J. del Picchia put together by a core group of four European Reference
Networks. Solve-RD will deliver 7 main implementation
steps: (i) Collect Phenotypes, (ii) New phenotype patterns,
(iii) Re-analyse exomes / genomes, (iv) Novel molecular
strategies, (v) Functional analysis, (iv) Clinical utility and
(vii) Towards therapy. For analysis Solve-RD will apply
data driven and expert driven approaches. We anticipate to
increase diagnostic yield from 19.000 unsolved exomes/
genomes by about 3-5%. Cohort specific innovative -omis
strategies will be pursued, also addressing cost-effective
issues. Analysis of more than 800 patients with highly
peculiar phenotypes will highly increase the chance to find
novel disease genes and novel disease mechanisms. We
anticipate to solve more than 2.000 cases. Finding further
matching patients will be secured by newly developed
matchmaking approaches and by screening using MIPs
technology in the more than 20.000 unclassified patients. For the first time in Europe we will also implement a novel
brokerage structure connecting clinicians, gene discoverer
and basic researcher to quickly verify novel genes and
disease mechanisms. 1NCCHD, Tokyo, Japan, 2Keio University, Tokyo, Japan The Initiative on Rare and Undiagnosed Diseases (IRUD) is
a national consortium designed to help patients and their
families suffering from rare and undiagnosed disease con-
ditions in Japan. The project started in July 2015. The aims
of the project are to make diagnosis on patients with rare
and undiagnosed diseases, to construct their genome data-
base with clinical information, and to make banking system
of precious specimens. The pediatric version of IRUD
(IRUD-P) is coordinated by dual centers, the National
Center for Child Health and Development (NCCHD) and
Keio University. The IRUD-P developed a nation-wide
network for patient recruitment involving 17 regional core
clinical centers, and mainly performed whole exome
sequencing on children with undiagnosed diseases and their
parents. Till now, more than 2,000 patients, who passed the
first screening in the core clinical centers, were consulted to
the IRUD-P centers. Specimens accompanied with medical
information (n=6,600) were collected from patients and
their families, mainly in trios, and sent to the centers. Of
882 patients analyzed, genetically confirmed diagnosis in
295 patients (diagnostic yield was 33.4%). Nine patients
were found to have pathogenic variants in novel genes. 1Parseq Lab, St-Petersburg, Russian Federation, 2Genomed
Ltd, London, United Kingdom Introduction: Most MPS-based assays yield false-positive
and false-negative results. According to the CLIA and CAP
recommendations laboratories have to validate the perfor-
mance of tests before reporting patient results. This analytic
validation includes wet-lab procedures as well as data
analysis. Moreover, laboratories should perform revalida-
tion after any assay alteration. This complex process of
proving that an assay works as expected and consistently
achieves the expected result has not yet been thoroughly
described. Y. Matsubara: None. K. Hata: None. T. Kaname:
None. K. Kosaki: None. Solve-RD. Solving the unsolved Rare Diseases Solve-RD. Solving the unsolved Rare Diseases O. Riess1, O. Solve-RD Consortium2 O. Riess1, O. Solve-RD Consortium2 P14.088D Patient cohort represents children with a wide range of
undiagnosed disorders (malformation syndromes 49.4%,
neuromuscular disorders 29.9%, skeletal and connective
tissue diseases 6.6%, renal diseases 1.6%, liver diseases
1.3%, others 11.1%). For the remaining undiagnosed
patients, the IRUD-P consortium has started data-sharing
network system (IRUD-Exchange) and disease model ana-
lysis. The IRUD-P also built international collaboration and
data sharing on rare and undiagnosed diseases. O. Riess: None. O. Solve-RD Consortium: None. O. Riess: None. O. Solve-RD Consortium: None. P14.090BAnalytical validation of MPS diabetes assay with
NIST and 1000G project reference materials T. Simakova1, M. Glushkova1, N. Pilshchikova1, A. Korosteleva1,
A. Slepchenkov1, C. Gülsev2, A. Pavlov1 1Parseq Lab, St-Petersburg, Russian Federation, 2Genomed
Ltd, London, United Kingdom 1Parseq Lab, St-Petersburg, Russian Federation, 2Genomed
Ltd, London, United Kingdom 1Universität Tübingen, Tuebingen, Germany, 2Solve-RD
partners, Europe, Germany 1Universität Tübingen, Tuebingen, Germany, 2Solve-RD
partners, Europe, Germany Material and Methods: We have sequenced NIST RM-
8398 and 17 GBR (1000G project) reference materials with
24-genes diabetes panel (104,3kbp). Alignment and variant
calling have been performed using VariFind™Software. For the reference and experimental data, we have applied
developed algorithm that selects regions with sequencing
quality above the given threshold. The algorithm calculates
common, reference and experimental data-specific variants
in these regions. All calculations have been made for the
diploid genome, i.e. homozygous variants have been
counted as two independent variants. Solve-RD is a H2020 funded flagship EU project that brings
together 21 partners from 10 countries and which will be
running from 2018 to 2022. The main ambitions are (i) to
solve large numbers of RD, for which a molecular cause is
not know yet, by sophisticated combined Omics approa-
ches, and (ii) to improve diagnostics of RD patients through
a “genetic knowledge web”. Solve-RD will pursue a clear
visionary and integrated “beyond the exome” approach. The
entire Solve-RD project has been motivated, designed and 535 Abstracts from the 51st European Society of Human Genetics Conference: Posters Results: For the diabetes panel we have identified 629
common variants, 9 false-negative and 4 false-positive
variants. Sensitivity and specificity are 98.1% and 98.9%
respectively (95% CI). We have found 15 discordant bases
among 1 174 744 total analyzed bases in 18 reference
samples. General accuracy of the assay is above 99.9%. Results: For the diabetes panel we have identified 629
common variants, 9 false-negative and 4 false-positive
variants. Sensitivity and specificity are 98.1% and 98.9%
respectively (95% CI). We have found 15 discordant bases
among 1 174 744 total analyzed bases in 18 reference
samples. General accuracy of the assay is above 99.9%. builds up a collective, high-quality knowledge base with high
transparency and traceability, with the ability to share further
to international databases. Discordances in classification are
flagged and resolution facilitated through communication
with the relevant partners. builds up a collective, high-quality knowledge base with high
transparency and traceability, with the ability to share further
to international databases. Discordances in classification are
flagged and resolution facilitated through communication
with the relevant partners. Conclusions: Harmonization of variant classification is a
priority for multi-site healthcare systems where equal access
to
quality
healthcare
is
a
goal. TVX
enables
such
harmonization while continuously curating and accumulat-
ing expertise. Trusted Variant eXchange: a database for secure sharing
of variant classifications between trusted partners Materials and Methods: Requirements for TVX were
collected and collated through a series of workshops and
feedback cycles with our clinical partners through BigMed, a
research project funded by the Norwegian Research Council. The database was designed with the following in mind:
scalability, for varying data volumes; and adaptability, for
evolving technological and regulatory needs. The database
was built using components and microservices, based around
blob and table storage, queues and functions. Materials and Methods: Requirements for TVX were
collected and collated through a series of workshops and
feedback cycles with our clinical partners through BigMed, a
research project funded by the Norwegian Research Council. The database was designed with the following in mind:
scalability, for varying data volumes; and adaptability, for
evolving technological and regulatory needs. The database
was built using components and microservices, based around
blob and table storage, queues and functions. Conclusions: Comprehensive phenotyping of individuals
is crucial step in identifying candidate phenotype-related
variants. We outline the benefit obtained from access to the
patient’s medical records and communication with referring
physicians. Keywords: report, variants, classification, vcf, reassess-
ment, genomic, genetic, counseling, NGS. Keywords: report, variants, classification, vcf, reassess-
ment, genomic, genetic, counseling, NGS. L. Alsubaie: None. M. Alfadhel: None. S. Alturki:
None. A. Alothaim: None. A. Alfares: None. L. Alsubaie: None. M. Alfadhel: None. S. Alturki:
None. A. Alothaim: None. A. Alfares: None. Results: TVX database facilitates sharing of evidence-
based classification of interpreted variants between trusted
clinical diagnostic partners, focusing on data quality and
conflict reporting. After authentication, partners can submit
and search for variants with their associated classifications
through a secure API. Entering all new variant classifications Trusted Variant eXchange: a database for secure sharing
of variant classifications between trusted partners Trusted Variant eXchange: a database for secure sharing
of variant classifications between trusted partners 1King Abdulaziz Medical City, Riyadh, Saudi Arabia, 2Qassim
University, Qassim, Saudi Arabia 1King Abdulaziz Medical City, Riyadh, Saudi Arabia, 2Qassim
University, Qassim, Saudi Arabia S. Alagaratnam1, T. Søreng1, T. Håndstad2, V. Wirta3, B. Ray-
Sannerud1 S. Alagaratnam1, T. Søreng1, T. Håndstad2, V. Wirta3, B. Ray-
Sannerud1 Background: Next generation sequencing has been leading
the genetic study of human diseases for the past ten years,
generating a huge amount of sequence variant data, which
are stored in variant call format files. To our knowledge,
there has been little discussion about the utility of VCF files
for reanalysis. 1Life Sciences programme, Group Technology and Research,
DNV GL, Oslo, Norway, 2Department of Medical Genetics,
Oslo University Hospital, Oslo, Norway, 3Clinical Genomics
facility, Science for Life Laboratory, Karolinska Institutet,
Stockholm, Sweden Results: In this study, twenty samples were clinically
reassessed using variant interpretation software. We reported
seven cases (n = 20) differently from the outside laboratory. This accounts for almost 35% of all cases and is mainly due
to the ability to gather more information about the patient’s
phenotype. One whole genome sequence case changed from
inconclusive to negative. In addition, we identified variants
related to the patient’s phenotype in six cases; two of them
were whole genome sequence and four were whole exome
sequence, all reported as negative before the reanalysis. Results: In this study, twenty samples were clinically
reassessed using variant interpretation software. We reported
seven cases (n = 20) differently from the outside laboratory. This accounts for almost 35% of all cases and is mainly due
to the ability to gather more information about the patient’s
phenotype. One whole genome sequence case changed from
inconclusive to negative. In addition, we identified variants
related to the patient’s phenotype in six cases; two of them
were whole genome sequence and four were whole exome
sequence, all reported as negative before the reanalysis. Introduction: Guidelines aim to standardize the classifica-
tion of genetic variants for rare diseases and cancer. How-
ever, adherence to guidelines can vary greatly within a
single healthcare system or country, leading to discordance
in variant classification. International classification data-
bases such as ClinVar can support the variant interpretation
process, but issues of data incompleteness and incon-
sistency remain. To address this, we have developed the
Trusted Variant eXchange, or TVX. Clinical reassessment of post-laboratory variant call
format<VCF>files Clinical reassessment of post-laboratory variant call
format<VCF>files 1Universität Tübingen, Tuebingen, Germany, 2Solve-RD
partners, Europe, Germany Conclusions: We have developed streamlined automated
validation approach for targeted MPS-based assays that is
suitable to perform fast assay revalidation on any alteration
in
the
bioinformatic
pipeline
or
laboratory
protocol. Developed validation algorithm has been tested on different
sequencing platforms and multiple targeted panels. S. Alagaratnam: None. T. Søreng: None. T. Håndstad:
None. V. Wirta: None. B. Ray-Sannerud: None. S. Alagaratnam: None. T. Søreng: None. T. Håndstad:
None. V. Wirta: None. B. Ray-Sannerud: None. T. Simakova: None. M. Glushkova: None. N. Pilshchi-
kova: None. A. Korosteleva: None. A. Slepchenkov:
None. C. Gülsev: None. A. Pavlov: None. T. Simakova: None. M. Glushkova: None. N. Pilshchi-
kova: None. A. Korosteleva: None. A. Slepchenkov:
None. C. Gülsev: None. A. Pavlov: None. P14.091C L. Alsubaie1, M. Alfadhel1, S. Alturki1, A. Alothaim1, A. Alfares2 Trusted Variant eXchange: a database for secure sharing
of variant classifications between trusted partners P14.093A To figure out the percentage of heterozygous SNVs, the
ratio number of heterozygous SNVs to total number of
SNVs is calculated for chromosome X. In order to identify
heterozygosity rates were calculated for 101 female and 89
male individuals. WES heterozygosity rates of sex chromo-
somes can be used for quality control or identify anomalies
of sex chromosomes. L. Pezzoli: None. A. Pansa: None. D. Marchetti: None. B. Facchinetti: None. A.R. Lincesso: None. L. Perego:
None. L. Pezzani: None. A. Cereda: None. U. Giussani:
None. M. Iascone: None. L. Pezzoli: None. A. Pansa: None. D. Marchetti: None. B. Facchinetti: None. A.R. Lincesso: None. L. Perego:
None. L. Pezzani: None. A. Cereda: None. U. Giussani:
None. M. Iascone: None. This work was supported by grants from the Scientific
Research Projects Coordination Unit of Istanbul University,
Project Number 26646. Measuring coverage and accuracy of whole exome
sequencing in clinical context Measuring coverage and accuracy of whole exome
sequencing in clinical context N.H. Akcakaya: None. B. Salman: None. Y. Tarkan
Argüden: None. Z. Görmez: None. R. Dönmez: None. B. Colakoglu: None. Z. Yapici: None. S. Hacıhanefioğlu:
None. U. Ozbek: None. S.A. Ugur Iseri: None. How many homozygous mutations in exome sequencing
data are true homozygous? P14.093A .093
Identification of the post-zygotic mosaic nonsense mutation
in WDR45gene leading to beta-propeller protein-associated
neurodegeneration and defining sex chromosomal
mosaicism at whole exome sequencing 536 J. del Picchia L. Pezzoli1, A. Pansa1, D. Marchetti1, B. Facchinetti1, A. R. Lincesso1, L. Perego1, L. Pezzani1, A. Cereda2, U. Giussani1, M. Iascone1 N. H. Akcakaya1, B. Salman1, Y. Tarkan Argüden2, Z. Görmez3,
R. Dönmez4, B. Colakoglu4, Z. Yapici5, S. Hacıhanefioğlu2, U. Ozbek6, S. A. Ugur Iseri1 1Laboratory of Medical Genetics, ASST Papa Giovanni XXIII,
Bergamo, Italy, 2Department of Pediatrics, ASST Papa
Giovanni XXIII, Bergamo, Italy 1Department of Genetics, Aziz Sancar Institute of Experimental
Medicine, Istanbul University, Istanbul, Turkey, 2Department
of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul
University, Istanbul, Turkey, 3Istinye University, Istanbul,
Turkey, 4Department of Neurology, Dokuz Eylul Faculty of
Medicine, Izmir, Turkey, 5Department of Neurology, Istanbul
Faculty of Medicine, Istanbul University, Istanbul, Turkey,
6Department of Medical Genetics, Acıbadem Faculty of
Medicine, Acıbadem University, Istanbul, Turkey Introduction: Compound heterozigosity for a point
mutation and a deletion is a well-known pathogenic
mechanism for recessive conditions, but its overall inci-
dence has never been investigated. The aim of this
study was to assess retrospectively the frequency of this
genetic
alteration
in
a
large
exome
dataset
from
unselected cases. Introduction: Beta-propeller protein-associated neurode-
generation (BPAN) is a very rare X-linked dominant dis-
ease. BPAN has been associated with WDR45 gene and
identified almost exclusively in females. The clinical pre-
sentation of BPAN is marked with a biphasic disease
course. Herein, we set out to characterize the genetic defect
underlying the complex pheonotype in describe a novel,
WDR45 mutation by using WES and also aim to define the
mosaic/heterozygous ratios of WES on sex chromosomes. Introduction: Beta-propeller protein-associated neurode-
generation (BPAN) is a very rare X-linked dominant dis-
ease. BPAN has been associated with WDR45 gene and
identified almost exclusively in females. The clinical pre-
sentation of BPAN is marked with a biphasic disease
course. Herein, we set out to characterize the genetic defect
underlying the complex pheonotype in describe a novel,
WDR45 mutation by using WES and also aim to define the
mosaic/heterozygous ratios of WES on sex chromosomes. Materials and Methods: We reviewed the exome data of
642 patient-parents trios analysed for diagnostic purpose
during the last three years. The findings on the techniques
employed to confirm the genetic alterations are discussed. S. Kong1,2, I. Lee1, X. Liu1, J. N. Hirschhorn1,3,2, K. D. Mandl1,2
1Boston Children's Hospital, Boston, MA, United States,
2Harvard Medical School, Boston, MA, United States, 3Broad
Institute, Cambridge, MA, United States P14.093A Results: Among the 642 patient-parents trios exome
analyses, 9 probands (1,4%) showed a pathogenic homo-
zygous point mutation although only one of the two parents
in each trio proved to be a carrier. This findings suggested
there must be another genetic alteration underpinning the
homozygous status of the probands. Indeed, 6 patients
showed deletions spanning one exon up to a whole gene
that were confirmed by MLPA, 2 patients carried large
deletions
and
one
showed
uniparental
isodisomy
of
chromosome
2,
detected
by
CGH-
and
SNP-array
respectively. Results: Among the 642 patient-parents trios exome
analyses, 9 probands (1,4%) showed a pathogenic homo-
zygous point mutation although only one of the two parents
in each trio proved to be a carrier. This findings suggested
there must be another genetic alteration underpinning the
homozygous status of the probands. Indeed, 6 patients
showed deletions spanning one exon up to a whole gene
that were confirmed by MLPA, 2 patients carried large
deletions
and
one
showed
uniparental
isodisomy
of
chromosome
2,
detected
by
CGH-
and
SNP-array
respectively. Case: 34 years old male patient with a medical history of
delayed psychomotor development and intellectual disabil-
ity. After age of 24 years he developed moving difficulty,
slowness and contraction in the legs. The neurological
examination revealed dystonia-parkinsonism. Results and Discussion: WES was performed to patient
and mosaic c.873C>G; p.Y291*(NM_007075) mutation
was found. The variant was confirmed in mosaic state only
in the patients’ blood but other family members did not
carry the mutation. However, it has been observed that there
are other mosaic/heterozygous variants on X chromosome
in exome data. Results and Discussion: WES was performed to patient
and mosaic c.873C>G; p.Y291*(NM_007075) mutation
was found. The variant was confirmed in mosaic state only
in the patients’ blood but other family members did not
carry the mutation. However, it has been observed that there
are other mosaic/heterozygous variants on X chromosome
in exome data. Conclusions: Our data show that a small but significant
percentage of recessive conditions can be caused by
compound heterozigosity for point mutations and genomic
rearrangements. This pathogenic mechanism can be sus-
pected in exome data but need confirmation by different
techniques, that should be tailored on a case by case basis. A unifying wide method to detect all possible genomic
alterations is currently not available. P14.094B How many homozygous mutations in exome sequencing
data are true homozygous? 537 Abstracts from the 51st European Society of Human Genetics Conference: Posters Purpose: To evaluate the coverage and accuracy of whole
exome sequencing (WES) across vendors. Purpose: To evaluate the coverage and accuracy of whole
exome sequencing (WES) across vendors. We tested our method using a cohort of 20 solved cases,
who on average harbor 312 variants with a high CADD
score and a low minor allele frequency (of which many are
classified as variants of unknown significance). We show
that our method successfully captures the true causal variant
in 85% of the cases within the top 10 of prioritized variants. This indicates that our ranking algorithm helps to reduces
the number of variants that need manual interpretation. We
expect our algorithm to be particularly helpful for unsolved
cases, where the causal mutation might not be in known
disease-causing genes, but rather in other genes that are co-
regulated with the known disease-causing genes and so-far
remain ignored in current analyses. Methods: Blood samples from three trios underwent
WES at three vendors. Relative performance of the three
WES services was measured for breadth and depth of
coverage in clinically implicated genes. The false negative
rates were estimated using the segregation pattern within
each trio. Results: Mean depth of coverage for all genes was 189.0,
124.9 and 38.3 for the three vendor services. Fifty-five of
the ACMG 56 genes, but only 56 of 63 pharmacogenes
were 100% covered at 10x in at least one of the nine
individuals for all three vendors; however, there was
substantial inter-individual variability. For the two vendors
with mean depth of coverage >120x, analytic positive
predictive values (aPPV) exceeded 99.1% for SNVs and
homozygous indels, and sensitivities were 98.9 - 99.9%;
however, heterozygous indels showed lower accuracy and
sensitivity. Among the trios, false-negative rates in the
offspring were 0.07 - 0.62% at well-covered variants
concordantly called in both parents. Our web-based gene function predictions and HPO based
gene prioritization method is freely available at http://www. genenetwork.nl/. GeneNetwork.nl also provides lookup
functionality for predicted GO-terms, Reactome pathways
and KEGG pathways, based on the co-regulation network
of 31,995 samples. P. Deelen: None. S. van Dam: None. J.M. Karjalainen:
None. H. Brugge: None. P. Folkertsma: None. C.C. van
Diemen: None. W.S. Kerstjens-Frederikse: None. J.C. Herkert: None. T. Gillett: None. M.A. Swertz: None. L. Franke: None. P14.097A Power efficiency of research reanalyze of negative clinical
WES data to identify new genes in Intellectual disability or
Congenital anomalies S. Kong: None. I. Lee: None. X. Liu: None. J.N. Hirschhorn: None. K.D. Mandl: None. A. Bruel1,2, S. Nambot1,3, V. Quéré1,2, M. Assoum1,2, A. Vittobello1,2, S. Moutton1,2, N. Houcinat1,2, D. Lehalle1,2, N. Jean-Marçais1,2, J. Thevenon1,2, Orphanomix Group, M. Chevarin1,2, T. Jouan1,2, C. Poë1,2, P. Callier1,2,4, E. Tisserand1,2,
C. Philippe1,2, F. Tran Mau Them1,2, Y. Duffourd1,2, L. Faivre1,2,
C. Thauvin-Robinet1,2 P14.094B Conclusions: The current standard of 120x coverage for
clinical WES may be insufficient for consistent breadth of
coverage across the exome, and additional measures of
sensitivity may be useful. Ordering clinicians and research-
ers would benefit from vendors' reports that estimate
sensitivity and aPPV, including depth of coverage across
the exome and across pre-specified genes of interest. P14.096D Improving diagnostic yield of exome-sequencing through
prioritization of genes with predicted HPO assignments P. Deelen, S. van Dam, J. M. Karjalainen, H. Brugge, P. Folkertsma, C. C. van Diemen, W. S. Kerstjens-Frederikse, J. C. Herkert, T. Gillett, M. A. Swertz, L. Franke 1Inserm UMR 1231 GAD team, Genetics of Developmental
disorders, Université de Bourgogne-Franche Comté, Dijon,
France, 2FHU-TRANSLAD, Université de Bourgogne/CHU
Dijon, Dijon, France, 3FHU-TRANSLAD, Université de
Bourgogne/CHU Dijon, France, Dijon, France, 4Laboratoire
de génétique moléculaire et de Cytogénétique, Plateau
Technique de Biologie, CHU Dijon, Dijon, France 1Inserm UMR 1231 GAD team, Genetics of Developmental
disorders, Université de Bourgogne-Franche Comté, Dijon,
France, 2FHU-TRANSLAD, Université de Bourgogne/CHU
Dijon, Dijon, France, 3FHU-TRANSLAD, Université de
Bourgogne/CHU Dijon, France, Dijon, France, 4Laboratoire
de génétique moléculaire et de Cytogénétique, Plateau
Technique de Biologie, CHU Dijon, Dijon, France P14.099C Precise breakpoint detection of balanced and unbalanced
structural variation in whole genome sequencing data using
haplotype blocks created by linked-reads University Medical Center Groningen, Groningen,
Netherlands In addition,
we elucidate limitations of short-read HTS on exemplary
cases including the influence of homologous/repetitive
regions (mappability <1) on variant calling and the impact
of sequence composition on read depth, as well as show
differences in the performance of WGS analysis pipelines. With this strategy, we identified or contributed for the
identification of 45 disease-causing genes or candidates. The genes were classified in five categories: 1) new genes
(17 genes); 2) new genotype-phenotype correlations for
known genes supported by data-sharing (5 genes); 3) ultra-
rare disorders with low recurrence (8 genes); 4) non-OMIM
genes recently published (5 genes); 5) candidate genes (10
genes). This study demonstrates the power of research
reanalysis after negative clinical WES and shows that
screening results can rapidly lead to diagnosis. Despite
using omics technologies, such as whole-genome sequen-
cing, many new genes implicated in rare human diseases
remain to be identified. The performance of WES will
certainly improve in the future. Conclusions: We recommend to select the HTS method
with care and to combine more than one independent
bioinformatics pipeline for the most comprehensive data
analysis. The use of PCR-free WGS (>60×) instead of WES
or panels and the inclusion of CNV analysis can contribute
to increased diagnostic yield in molecular diagnosis with
lifetime value. As long-read HTS may overcome limitations
of short-read HTS, it is envisioned as the future of (clinical)
sequencing. A. Bruel: None. S. Nambot: None. V. Quéré: None. M. Assoum: None. A. Vittobello: None. S. Moutton: None. N. Houcinat: None. D. Lehalle: None. N. Jean-Marçais:
None. J. Thevenon: None. M. Chevarin: None. T. Jouan:
None. C. Poë: None. P. Callier: None. E. Tisserand:
None. C. Philippe: None. F. Tran Mau Them: None. Y. Duffourd:
None. L. Faivre:
None. C. Thauvin-
Robinet: None. J. Meienberg: None. A.M. Kopps: None. M. Plüss:
None. S.M. Caspar: None. N. Dubacher: None. G. Matyas: None. J. Meienberg: None. A.M. Kopps: None. M. Plüss:
None. S.M. Caspar: None. N. Dubacher: None. G. Matyas: None. J. Meienberg: None. A.M. Kopps: None. M. Plüss:
None. S.M. Caspar: None. N. Dubacher: None. G. Matyas: None. High-throughput sequencing of Mendelian disorders: From
raw data to diagnosis with lifetime value J. Knijnenburg, M. Kriek, H. A. van Duyvenvoorde, N. Pappas,
Y. Ariyurek, H. P. J. Buermans, M. E. Y. Laurense-Bik, K. Kuipers-Heijboer, F. Baas High-throughput sequencing of Mendelian disorders: From
raw data to diagnosis with lifetime value J. Knijnenburg, M. Kriek, H. A. van Duyvenvoorde, N. Pappas,
Y. Ariyurek, H. P. J. Buermans, M. E. Y. Laurense-Bik, K. Kuipers-Heijboer, F. Baas J. Meienberg1, A. M. Kopps1, M. Plüss1,2, S. M. Caspar1, N. Dubacher1, G. Matyas1 J. Meienberg1, A. M. Kopps1, M. Plüss1,2, S. M. Caspar1, N. Dubacher1, G. Matyas1 University Medical Center Groningen, Groningen,
Netherlands Interpretation of genetic variants identified through whole
exome sequencing remains challenging, due to many var-
iants with unknown clinical significance. In order to
improve the diagnostic yield, we first developed a method
that can predict per gene what the likely phenotypic con-
sequences are when mutated, by integrating gene expression
data on 31,995 public RNA-seq samples. We subsequently
used this information, along with phenotype information per
patient, to prioritize genes that, when mutated, likely cause
each of these phenotypes. The diagnostic yield of clinical whole-exome sequencing
(WES) in intellectual disability (ID) and/or congenital
anomalies (CA) is now about 30%, which means that 70%
of patients remain without a molecular etiology. To go
beyond the stringent criteria of the ACMG recommenda-
tions in variant interpretation, which are limited to estab-
lished human disease genes, a further analysis in a research
environment appears essential to identify novel disease 538 J. del Picchia genes. Thus, we performed a systematic research analysis of
negative WES of 500 patients. The solo WES interpretation
was extended through a translational research approach to
all variants in order to identify candidate variants, by
studying protein functions, model organisms, and a litera-
ture review. International data-sharing was used to identify
additional patients carrying variants in the same gene, in
order to draw definitive conclusions on their implication in
the disease. Materials and Methods: We address the chances and
challenges of HTS in the molecular diagnosis of Mendelian
disorders as well as assess the sensitivity/recall, precision,
computation time, and disk footprint of four corresponding
HTS analysis pipelines. Results: We exemplify the limitations of targeted (gene
panel) and whole-exome sequencing (WES) as well as
emphasize the potential of whole-genome sequencing
(WGS) in the detection of single nucleotide variants
(SNVs) and copy number variations (CNVs). In addition,
we elucidate limitations of short-read HTS on exemplary
cases including the influence of homologous/repetitive
regions (mappability <1) on variant calling and the impact
of sequence composition on read depth, as well as show
differences in the performance of WGS analysis pipelines. Results: We exemplify the limitations of targeted (gene
panel) and whole-exome sequencing (WES) as well as
emphasize the potential of whole-genome sequencing
(WGS) in the detection of single nucleotide variants
(SNVs) and copy number variations (CNVs). Blueprint Genetics, Helsinki, Finland Blueprint Genetics, Helsinki, Finland Utility of Whole Exome Sequencing (WES) in clinical
diagnostics has been limited by the non-uniform sequencing
coverage across exons, leaving a substantial proportion of
the regions with shallow coverage that prevents accurate
variant detection. We evaluated a WES assay that is spe-
cifically designed for clinical use, enables wide breadth of
coverage
resembling
high-coverage
gene-panel
based
assays, and provides high sensitivity in variant detection. We performed WES capture experiments using an assay
with
boosted
clinical
content,
namely
xGen
Exome
Research Panel (IDT) assay that was spiked-in with custom
designed clinical content. Sequencing was performed using
an Illumina NovaSeq sequencing system and data was
down-sampled to 100M reads. Performance of the WES
assay was demonstrated by using reference samples with
high-quality variant calls (The Genome In a Bottle Con-
sortium and Platinum Genome samples for SNVs and
INDELs and Coriell samples for assessing Del/Dups and
complex genetic variants). In clinically associated CCDS
genes, the assay achieved high average sequencing depth
(183x) and coverage (99.7% of regions covered >20x). Sensitivity to detect SNVs was 0.998, and for INDELs 0.97. Utility of Whole Exome Sequencing (WES) in clinical
diagnostics has been limited by the non-uniform sequencing
coverage across exons, leaving a substantial proportion of
the regions with shallow coverage that prevents accurate
variant detection. We evaluated a WES assay that is spe-
cifically designed for clinical use, enables wide breadth of
coverage
resembling
high-coverage
gene-panel
based
assays, and provides high sensitivity in variant detection. Materials and Methods: Peripheral blood purified DNA
samples were enriched with a reference kit (SureSelect-
QXT, Agilent) and with various Illumina solutions: TruSeq-
Nano, Nextera-DNA Exome with standard protocol (Nx-
Std) and with various modifications (Nx-Mod), including
insert sizes, input DNA and post-enrichment PCR cycles. Pools of 5-7 samples at 2 nM library concentrations were
sequenced with 75 bp paired-end reads. Bcl2fastq (v2.18),
BWA
(v0.7.15),
Samtools
(v1.3),
Picard
(v2.10.10),
QualiMap (v2.2.1), and GATK (v3.8) software were used
for analysis. Materials and Methods: Peripheral blood purified DNA
samples were enriched with a reference kit (SureSelect-
QXT, Agilent) and with various Illumina solutions: TruSeq-
Nano, Nextera-DNA Exome with standard protocol (Nx-
Std) and with various modifications (Nx-Mod), including
insert sizes, input DNA and post-enrichment PCR cycles. Pools of 5-7 samples at 2 nM library concentrations were
sequenced with 75 bp paired-end reads. LUMC, Leiden, Netherlands Muona: None. M. Valori: None. V. Kytölä: None. P. Salmenperä: None. M. Rantanen:
None. M. Gentile: None. S. Myllykangas: None. Conclusions: Structural variant analysis using WGS with
linked-reads promises to be a feasible technique for
detection of balanced and unbalanced structural variants
in the genome and may be a next step in replacing classical
cytogenetic techniques for genome wide screening. Assessment of in-solution enrichment capture protocols for
whole-exome sequencing in HiSeq 4000 System Assessment of in-solution enrichment capture protocols for
whole-exome sequencing in HiSeq 4000 System A. Díaz-de Usera1,2, J. Lorenzo-Salazar1, A. Mendoza Álvarez1,2,
R. González-Montelongo1, C. Flores1,2,3 A. Díaz-de Usera1,2, J. Lorenzo-Salazar1, A. Mendoza Álvarez1,2,
R. González-Montelongo1, C. Flores1,2,3 J. Knijnenburg: None. M. Kriek: None. H.A. van
Duyvenvoorde: None. N. Pappas: None. Y. Ariyurek:
None. H.P.J. Buermans: None. M.E.Y. Laurense-Bik:
None. K. Kuipers-Heijboer: None. F. Baas: None. J. Knijnenburg: None. M. Kriek: None. H.A. van
Duyvenvoorde: None. N. Pappas: None. Y. Ariyurek:
None. H.P.J. Buermans: None. M.E.Y. Laurense-Bik:
None. K. Kuipers-Heijboer: None. F. Baas: None. 1Genomics Division, Instituto Tecnológico y de Energías
Renovables (ITER) S.A., Granadilla de Abona, S/C de Tenerife,
Spain, 2Research Unit, Hospital Universitario N.S. de
Candelaria, Universidad de La Laguna, Santa Cruz de
Tenerife, Spain, 3CIBER de Enfermedades Respiratorias,
Instituto de Salud Carlos III, Madrid, Spain LUMC, Leiden, Netherlands 1Center for Cardiovascular Genetics and Gene Diagnostics,
Foundation for People with Rare Diseases, Schlieren-Zurich,
Switzerland, 2Institute of 4D Technologies, University of
Applied Sciences and Arts Northwestern Switzerland,
Windisch, Switzerland Introduction:
Routine
diagnostics
of
genome
wide
balanced
and
unbalanced
structural
variation
is
still
dependent on classical techniques such as karyotyping and
(SNP-)array. Both techniques result in a rough breakpoint
detection where especially karyotyping can result in sig-
nificant misinterpretation of chromosomal breakpoints. New
sequencing technologies such as long range haplotyping by
mapping linked-reads to generate haplotype blocks (Chro-
mium Genome Solution, 10x Genomics, San Francisco
USA) are expected to overcome this problem, although
repeat sequence elements at breakpoints in the genome may
occasionally interfere with a precise detection. Introduction: High-throughput sequencing (HTS) is widely
used for clinical applications such as the molecular diag-
nosis of Mendelian disorders. As the applied technology/
workflow substantially affects the diagnostic yield, knowl-
edge about the pitfalls and advantages of HTS technologies
and analysis pipelines is crucial for the successful applica-
tion of hitherto unprecedented large-scale genetic testing. Materials and Methods: To test whether this NGS based
approach
with
linked-reads
is
suitable
for
routine Abstracts from the 51st European Society of Human Genetics Conference: Posters 539 Sensitivity to detect 1 exon deletions and duplications was
0.93 and 0.99 for 5 exon deletions and duplications. The
assay was observed to provide a uniform coverage over
difficult-to-sequence regions (e.g. the RPGR gene) and GC-
rich 1st exons. Our results demonstrate that WES assay with
boosted clinical content provide high sequencing coverage
and allows high variant calling sensitivity for different
genetic variations, which makes it well-suited for clinical
diagnostics of inherited disorders. diagnostics, two patients, one with a de novo balanced
translocation (8;17) and one with a duplication detected
with SNP-array, were analysed. Results: The expected structural variants based on
previous knowledge were identified and in both patients
precise breakpoints of these variants could be detected. In
the translocation patient, the breakpoint differed at least 15
Mb from the estimated breakpoint and proved SOX9 to be
causative for the phenotype of the patient. In the second
patient the orientation of the duplication fragment could be
defined to be in tandem, making a gene disruption to be
causal for the phenotype unlikely. M. Mehine: None. M. Muona: None. M. Valori: None. V. Kytölä: None. P. Salmenperä: None. M. Rantanen:
None. M. Gentile: None. S. Myllykangas: None. M. Mehine: None. M. Comparison of whole exome sequencing assays with
boosted clinical content Comparison of whole exome sequencing assays with
boosted clinical content M. Mehine, M. Muona, M. Valori, V. Kytölä, P. Salmenperä, M. Rantanen, M. Gentile, S. Myllykangas Introduction:
Whole-exome
sequencing
(WES)
has
become a central application in research and disease diag-
nosis. A performance comparison of the most common in
solution-capture enrichment alternatives was needed in
order to develop adapted cost-effective workflows for
HiSeq 4000 System (Illumina). Outcome of Whole Exome Sequencing for diagnostic cases
of a clinic pediatric center: the Medical Genetic Unit of
MeyerChildren’s hospital experience Outcome of Whole Exome Sequencing for diagnostic cases
of a clinic pediatric center: the Medical Genetic Unit of
MeyerChildren’s hospital experience N. Corsten-Janssen, J. el Mecky, K. Bouman, Y. J. Vos, J. B. G. M. Verheij, H. Westers, R. Kinds, A. J. Scheper, B. Sikkema-
Raddatz, R. J. Sinke, I. M. van Langen, R. H. Sijmons, C. C. van
Diemen A. Provenzano1, A. La Barbera1, V. Palazzo2, R. Artuso2, S. Landini1, L. Giunti2, P. Reho1, E. Bosi1, A. Pagliazzi1, F. Peluso1, S. Bargiacchi2, G. Traficante2, L. Dosa2, E. Andreucci2,
B. Rinaldi1, S. Guarducci2, M. Pantaleo2, B. Lucherini2, I. Sani2,
D. Formicola1, P. Romagnani3, S. Stagi4, B. Giusti5, M. Della
Monica2, P. Fiorini6, E. Agostini6, R. Biagiotti7, C. De Filippi8,
S. Toni9, L. Genitori10, A. Iolascon11, O. Zuffardi12, S. Giglio1,2 A. Provenzano1, A. La Barbera1, V. Palazzo2, R. Artuso2, S. Landini1, L. Giunti2, P. Reho1, E. Bosi1, A. Pagliazzi1, F. Peluso1, S. Bargiacchi2, G. Traficante2, L. Dosa2, E. Andreucci2,
B. Rinaldi1, S. Guarducci2, M. Pantaleo2, B. Lucherini2, I. Sani2,
D. Formicola1, P. Romagnani3, S. Stagi4, B. Giusti5, M. Della
Monica2, P. Fiorini6, E. Agostini6, R. Biagiotti7, C. De Filippi8,
S. Toni9, L. Genitori10, A. Iolascon11, O. Zuffardi12, S. Giglio1,2 University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands Introduction: Identifying the cause of fetal anomalies seen
on ultrasound provides important information to improve
perinatal management. The conventional test (chromosomal
microarray) leads to a diagnosis in approximately 25% of
the fetuses with multiple congenital anomalies (MCA). Whole Exome Sequencing (WES) is a promising technique
to improve diagnostic yield. Implementing WES in prenatal
setting is challenging due to uncertainties around fetal
phenotyping, variant interpretation, ethical issues of inci-
dental findings and variants of unknown clinical sig-
nificance and the requirement of short turnaround times. 1Medical Genetics Unit, Dpt. Experimental and Clinical
Biomedical Sciences, Firenze, Italy, 2Medical Genetics Unit,
Meyer Children's University Hospital, Firenze, Italy,
3Nephology Unit, Meyer Children's University Hospital,
Firenze, Italy, 4Auxoendocrinology Unit, Dpt. Healt’s
Medicine, Meyer Children's University Hospital,, Firenze,
Italy, 5Atherothrombotic Disease, Dpt. Blueprint Genetics, Helsinki, Finland Bcl2fastq (v2.18),
BWA
(v0.7.15),
Samtools
(v1.3),
Picard
(v2.10.10),
QualiMap (v2.2.1), and GATK (v3.8) software were used
for analysis. Results and Conclusions: The best average enrichment
(reads on target) was obtained for the Nx-Std solution
(69%), although at distance from that provided by the
reference kit (89%). However, Nx-Mod with 350 bp insert
size had lower mean proportion of duplicate reads than the
reference (23% vs. 25%) while returning a comparable
proportion of target bases at >10X depth of coverage (84%
vs. 91%). TruSeq-Nano
performed
the
worst
in
all
comparisons, except in the proportion of duplicate reads 540 J. del Picchia a theoretical turn-around time of 10 days after the invasive
procedure. (17%). At least for the proportion of duplicate reads, these
results support that performance of HiSeq 4000 is not
comparable to that of previous HiSeq systems, providing
guidance for the design of WES projects. Phase 2: The prospective study has just started. Conclusions: Retrospective analysis and modeling of our
pipeline show that implementing WES as a routine test in
the prenatal setting is technically feasible. Conclusions: Retrospective analysis and modeling of our
pipeline show that implementing WES as a routine test in
the prenatal setting is technically feasible. Funding: Fellowship by Spanish Ministry of Education,
Culture, and Sports to ADU (FPU16/01435). CEDeI
fellowship (Cabildo de Tenerife) to AMA. N. Corsten-Janssen: None. J. el Mecky: None. K. Bouman: None. Y.J. Vos: None. J.B.G.M. Verheij: None. H. Westers: None. R. Kinds: None. A.J. Scheper: None. B. Sikkema-Raddatz: None. R.J. Sinke: None. I.M. van
Langen:
None. R.H. Sijmons:
None. C.C. van
Diemen: None. A. Díaz-de Usera: None. J. Lorenzo-Salazar: None. A. Mendoza
Álvarez:
None. R. González-Montelongo:
None. C. Flores: None. Outcome of Whole Exome Sequencing for diagnostic cases
of a clinic pediatric center: the Medical Genetic Unit of
MeyerChildren’s hospital experience Experimental Medicine,
University of Florence, Firenze, Italy, 6Neonatal Intensive
Care Unit, Medical Surgical Feto-Neonatal Department,
Meyer Children's University Hospital, Firenze, Italy, 7Prenatal
Diagnosis Unit, Meyer Children's University Hospital, Firenze,
Italy, 8Department of Diagnostic Imaging, Meyer Children's
University Hospital, Firenze, Italy, 9Tuscany Regional Centre
of Pediatric Diabetes, Meyer University Children's Hospital,
Firenze, Italy, 10Neurosurgery Unit, Neuroscience Department,
Meyer Children's University Hospital, Firenze, Italy, 11Medical
Genetics Unit, Dpt. Molecular Medicine and Medical
Biotechnology, Univeristy Federico II, Napoli, Italy, 12Medical
Genetics Unit, Dpt Melecular Medicine, University of Pavia,
Pavia, Italy P14.102B Implementing fast whole exome sequencing sequencing as
diagnostic test for fetal multiple congenital anomalies on
ultrasound Method: Phase 1: Retrospective WES analysis (blindly) of six
fetuses (including parents) with known postnatal genetic
diagnosis to test if this diagnosis could be made on the fetal
phenotype only. Variants were filtered
using
human
phenotype ontology (HPO) or using our custom virtual
gene panel (about 3850 disease genes, excluding late-onset
diseases genes). Phase 1: Retrospective WES analysis (blindly) of six
fetuses (including parents) with known postnatal genetic
diagnosis to test if this diagnosis could be made on the fetal
phenotype only. Variants were filtered
using
human
phenotype ontology (HPO) or using our custom virtual
gene panel (about 3850 disease genes, excluding late-onset
diseases genes). Phase 2: Prospective study of fast trio WES analysis in
addition to conventional genetic tests for twenty-five fetuses
with two or more congenital malformations. Phase 2: Prospective study of fast trio WES analysis in
addition to conventional genetic tests for twenty-five fetuses
with two or more congenital malformations. Result: Result: WES has shown an unprecedented success rate in the
identification of disease causing genes in projects ranging
from tailored sequencing, used to discover the molecular
bases of a recognizable syndrome in a homogeneous group
of patients, to the systematic application of pan-genomic
sequencing in large heterogeneous cohorts. The usefulness WES has shown an unprecedented success rate in the
identification of disease causing genes in projects ranging
from tailored sequencing, used to discover the molecular
bases of a recognizable syndrome in a homogeneous group
of patients, to the systematic application of pan-genomic
sequencing in large heterogeneous cohorts. The usefulness Phase 1: WES analysis on fetal DNA resulted in a
coverage > 95%. Five of six known diagnoses could be
confirmed using our gene panel. HPO was not helpful. One
causal pathogenic PTPN11 mutation was missed due to low
coverage of the variant. Modeling the WES pipeline shows 541 Abstracts from the 51st European Society of Human Genetics Conference: Posters 4UMR1231 GAD, Inserm - Université Bourgogne-Franche
Comté, Dijon, France, 5Service de Cytogénétique, Hôpital
Necker-Enfants Malades, APHP,, Paris, France, 6Institut
Cochin, INSERM U1016 ; Université Paris Descartes - Faculté
de Médecine ; APHP, HUPC, site Cochin, Laboratoire de
Cytogénétique, Paris, France, 7HCL, Service de Génétique,
BRON Cedex, France, 8Unité Fonctionnelle d'Innovation en
Diagnostic génomique des maladies rares, FHU-TRANSLAD,
CHU Dijon Bourgogne, Dijon, France, 9HCL, Cellule
bioinformatique de la plateforme NGS du CHU Lyon, BRON
Cedex, France, 10Université Lyon 1, CNRS, Laboratoire de
Biométrie et Biologie Evolutive UMR5558,, Villeurbanne,
France, 11Service de Génétique, Hôpital Necker Enfants
Malades, APHP et Institut Imagine,, Paris, France,
12Laboratoire de génétique chromosomique et moléculaire,
FHU-TRANSLAD, CHU Dijon Bourgogne, Dijon, France,
13Centre de génétique, Hôpital Couple-Enfant, CHU Grenoble-
Alpes, F-38700 La Tronche, Grenoble, France, 14Centre de
génétique, FHU-TRANSLAD, CHU Dijon Bourgogne, Dijon,
France of an unbiased sequencing approach has been highlighted in
various heterogeneous disorders, including intellectual dis-
ability, developmental delay, kidney diseases and con-
genital heart defects. We analyzed 400 WES and 20 WGS
on pediatric patients affected by different rare diseases. Result: In
particular, we studied patients with renal diseases, including
pediatric kidney tumors, MODY, pediatric glioblastoma,
Chiari Malformation-type 1, cardiomyopathies and patients
with isolated/syndromic intellectual disability.In 54 trios
with different clinical conditions (excluding renal diseases,
MODY and Chiari malformation that showed greater
diagnostic success) we obtained a diagnosis in more than a
half cases, with a detection rate of 54%, while in a singleton
analysis WES approach helped us to identify variants in
causative genes on 15/21 cases. We showed that WES
identified significantly more conclusive diagnoses than the
standard care pathway without incurring higher costs helped
also by the use of genotype-driven approach, as a comple-
ment to the traditional phenotype-driven one.Deep pheno-
typing and WES end a diagnostic odyssey, allows for
precise genetic counselling and has the potential to change
clinical management. It is also the start point for the
development of targeted pharmacologic therapies, which
can translate these discoveries into efficacious novel treat-
ments to achieve a personalized genomic medicine. Structural variants (SV) include copy number variants
(CNV) and balanced chromosomal abnormalities (BCA). Whole-genome sequencing (WGS) enables to detect SV at
base-pair resolution. However, CNV and BCA are difficult
to detect using a short-read sequencing, and long-read
approaches are not yet available for diagnosis. Recently,
10XGenomics proposed a pseudo long-read technology,
using linked-reads to reconstruct long DNA fragments. Since long read methods remain very expensive, 10X-
Genomics could be a good alternative. The main aim of this
work is to determine if 10X-Genomics solution enables a
more sensitive detection and better comprehension of SV
than a short read WGS. We included 13 patients with
known or suspected SV, with signed informed consent: 5
possible chromoanagenesis, 7 reciprocal translocations and
1 roberstonian translocation. Whole-genome analyses were
performed using Chromium (10X-Genomics) library pre-
paration before HiSeq X (Illumina) sequencing, and com-
pared to a classical HiSeq X sequencing. Two different
pipelines were used, using BreakDancer for classical WGS
and LongRanger for 10X-Genomics WGS. For Break-
Dancer, we used a local database filter out recurrent SV. The variant interpretations were blinded for bothtechnolo-
gies. The classical WGS pipeline allowed the diagnosis of
known SV in 10/13 patients. The 10X-Genomics pipeline
found 1/13 SV tagged as “high confident”, and 9/13 as “low
confident”. None of them detected the roberstonian trans-
location. In conclusion, 10X-Genomics solution didn’t
improve the SV detection in this small cohort. Result: To increase
the SV detection we point out the importance of local
databases to filter recurrent SVs and the improvement of
informatics pipelines using 10X-Genomics data. A. Provenzano: None. A. La Barbera: None. V. Palazzo: None. R. Artuso: None. S. Landini: None. L. Giunti: None. P. Reho: None. E. Bosi: None. A. Pagliazzi: None. F. Peluso: None. S. Bargiacchi: None. G. Traficante: None. L. Dosa: None. E. Andreucci: None. B. Rinaldi: None. S. Guarducci: None. M. Pantaleo:
None. B. Lucherini: None. I. Sani: None. D. Formicola:
None. P. Romagnani: None. S. Stagi: None. B. Giusti:
None. M. Della Monica: None. P. Fiorini: None. E. Agostini: None. R. Biagiotti: None. C. De Filippi: None. S. Toni: None. L. Genitori: None. A. Iolascon: None. O. Zuffardi: None. S. Giglio: None. 1Centre National de Recherche en Génomique Humaine
(CNRGH), CEA, Evry, France, 2Labex GenMed, Evry, France,
3Service de génétique, CHRU de Brest, Brest, France, 1Medical Genetics Unit, Dpt. Experimental and Clinical
Biomedical Sciences "Mario Serio", University of Florence,
Florence, Italy, 2Pediatric Endocrinology Unit, IRCCS
Policlinico “San Matteo” Foundation, Pavia, Italy, 3Medical
Genetics Unit, Dpt. Molecular Medicine, University of Pavia,
Pavia, Italy, 4Medical Genetics Unit, Meyer Children's
University Hospital, Florence, Italy P14.105A Does 10X Genomics technology improve the identification
andcharacterization of structural variants? C. Jubin1,2, K. Uguen3, Y. Duffourd4, V. Malan5, J. Dupont6, N. Chatron7, A. Vitobello4,8, P. Rollat-Farnier9, C. Baulard1,2, M. Lelorch5, A. Leduc1,2, E. Tisserand4, F. Tran Mau-Them4,8, M. Delepine1,2, M. Till7, V. Meyer1,2, C. Bardel9,10, S. Lyonnet11, A. Mosca-Boidron4,12, J. Thevenon13, L. Faivre4,14, C. Thauvin-
Robinet4,14, C. Schluth-Bolard7, A. Boland1,2, R. Olaso1,2, P. Callier4,12, S. Romana5, J. Deleuze1,2, D. Sanlaville7 542 J. del Picchia C. Jubin: None. K. Uguen: None. Y. Duffourd: None. V. Malan: None. J. Dupont: None. N. Chatron: None. A. Vitobello: None. P. Rollat-Farnier: None. C. Baulard:
None. M. Lelorch: None. A. Leduc: None. E. Tisserand:
None. F. Tran Mau-Them: None. M. Delepine: None. M. Till: None. V. Meyer: None. C. Bardel: None. S. Lyonnet: None. A. Mosca-Boidron: None. J. Thevenon:
None. L. Faivre: None. C. Thauvin-Robinet: None. C. Schluth-Bolard: None. A. Boland: None. R. Olaso: None. P. Callier: None. S. Romana: None. J. Deleuze: None. D. Sanlaville: None. incremental inclusion of mitochondrial DNA analysis,
automated
data
reinterpretation,
and
availability
of
secondary analysis (such as pharmacogenomics and
Mendelian predisposition panels) to enhance the utility
of genomic analysis. B. Lundie: A. Employment (full or part-time); Signifi-
cant; Genome.One. L. Ewans: A. Employment (full or part-
time); Significant; NSW Health, Genome.One. E. Lee: A. Employment (full or part-time); Significant; Genome.One. G. Hollway: A. Employment (full or part-time); Signifi-
cant; Genome.One. T. Ohnesorg: A. Employment (full or
part-time); Significant; Genome.One. A. Statham: A. Employment (full or part-time); Significant; Genome.One. L. Burnett: A. Employment (full or part-time); Significant;
Genome.One. B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Shire International. F. Consultant/Advisory
Board;
Modest;
(Honorary
-
no
financial compensation) Royal College of Pathologists of
Australasia, Genetics Advisory Committee, Honorary - no
financial
compensation)
NSW
Government.-
Genetics
Services Network Executive Committee. M. Young: A. Employment (full or part-time); Significant; Genome.One. H. Taouk: A. Employment (full or part-time); Significant;
Genome.One. E. Richardson: A. Employment (full or part-
time); Significant; Genome.One. M. Dinger: A. Employ-
ment (full or part-time); Significant; Genome.One, Garvan
Institute of Medical Research. B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Significant; National
Health and Medical Research Council of Australia. F. Consultant/Advisory Board; Modest; Australian Genomic
Health Alliance. B. Lundie: A. Employment (full or part-time); Signifi-
cant; Genome.One. L. Ewans: A. P14.105A Employment (full or part-
time); Significant; NSW Health, Genome.One. E. Lee: A. Employment (full or part-time); Significant; Genome.One. G. Hollway: A. Employment (full or part-time); Signifi-
cant; Genome.One. T. Ohnesorg: A. Employment (full or
part-time); Significant; Genome.One. A. Statham: A. Employment (full or part-time); Significant; Genome.One. L. Burnett: A. Employment (full or part-time); Significant;
Genome.One. B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Shire International. F. Consultant/Advisory
Board;
Modest;
(Honorary
-
no
financial compensation) Royal College of Pathologists of
Australasia, Genetics Advisory Committee, Honorary - no
financial
compensation)
NSW
Government.-
Genetics
Services Network Executive Committee. M. Young: A. Employment (full or part-time); Significant; Genome.One. H. Taouk: A. Employment (full or part-time); Significant;
Genome.One. E. Richardson: A. Employment (full or part-
time); Significant; Genome.One. M. Dinger: A. Employ-
ment (full or part-time); Significant; Genome.One, Garvan
Institute of Medical Research. B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Significant; National
Health and Medical Research Council of Australia. F. Consultant/Advisory Board; Modest; Australian Genomic
Health Alliance. Utility of clinical Whole Genome Sequencing (WGS):
diagnostic success factors now and into the future Utility of clinical Whole Genome Sequencing (WGS):
diagnostic success factors now and into the future B. Lundie1, L. Ewans1,2, E. Lee1, G. Hollway1, T. Ohnesorg1, A. Statham1, L. Burnett1,3,4,5, M. Young1, H. Taouk1, E. Richardson1, M. Dinger1,3 B. Lundie1, L. Ewans1,2, E. Lee1, G. Hollway1, T. Ohnesorg1, A. Statham1, L. Burnett1,3,4,5, M. Young1, H. Taouk1, E. Richardson1, M. Dinger1,3 1Genome.One, Darlinghurst, Australia, 2NSW Health,
Camperdown, Australia, 3Garvan Institute of Medical
Research, Darlinghurst, Australia, 4Sydney Medical School, St
Leonards, Australia, 5St Vincent's Clinical School, UNSW,
Darlinghurst, Australia Introduction: Application of genomic technologies to
genetically heterogeneous complex disorders significantly
increases diagnostic yields. WGS further increases diag-
nostic success over other genomic technologies. Methods: Genome.One is the world's first ISO15189
clinically-accredited WGS laboratory. In establishing this
clinical laboratory we solved various challenges and
identified potential directions to further leverage WGS for
increasing clinical diagnoses. Association of ADORA3 gene polymorphisms with efficacy
and toxicity of methotrexate Association of ADORA3 gene polymorphisms with efficacy
and toxicity of methotrexate M. B. Grk1, B. Jekić1, V. Milić2, V. Dolzan3, N. Maksimović1, T. Damnjanović1, M. Duanović Pjević1, M. Peić1 P14.107C Our results show that, in clinical suspicion of TS, lc-WGS
in cf-DNA is a valuable screening test for the detection of
low-level mosaicism and complex structural chromosome
abnormalities, in the face of extremely content cost. T. Skaric-Juric: None. N. Smolej Narancic: None. B. Janicijevic: None. M. Zajc Petranovic: None. Z. Tomas:
None. M. Pericic Salihovic: None. y
P. Reho: None. D. Larizza: None. C. Montalbano:
None. D. Vergani: None. M. Carella: None. A. Proven-
zano: None. A. La Barbera: None. V. Palazzo: None. S. Landini: None. E. Bosi: None. R. Artuso: None. A. Pagliazzi: None. L. Giunti: None. D. Formicola: None. B. Lucherini: None. M. Pantaleo: None. I. Sani: None. S. Guarducci: None. S. Giglio: None. O. Zuffardi: None. P. Reho: None. D. Larizza: None. C. Montalbano:
None. D. Vergani: None. M. Carella: None. A. Proven-
zano: None. A. La Barbera: None. V. Palazzo: None. S. Landini: None. E. Bosi: None. R. Artuso: None. A. Pagliazzi: None. L. Giunti: None. D. Formicola: None. B. Lucherini: None. M. Pantaleo: None. I. Sani: None. S. Guarducci: None. S. Giglio: None. O. Zuffardi: None. P14.107C We detected low-level mosaicism for XX or XY cell
lines, partial deletions/duplications of sex chromosomes
indicating an iso-chromosome, X chromosome partial
deletions, some of them suggesting a ring/marker chromo-
some. WGS confirmed SK results in 50/64 cases; we
detected nine X chromosome rearrangements at low-level
mosaic that, although hidden at SK, were confirmed by a-
CGH in all but 2 cases, and previously unreported Y
chromosome material was found in 5 patients. Our results show that, in clinical suspicion of TS, lc-WGS
in cf-DNA is a valuable screening test for the detection of
low-level mosaicism and complex structural chromosome
abnormalities, in the face of extremely content cost. Turner syndrome (TS) is characterized by complete or
partial absence of the second sex chromosome, either in
mosaic or in all cells, in phenotypic female patients. Introduction: The ADME (absorption, distribution, meta-
bolism and excretion) genes' variation is markedly related to
ethnicity and shows distinct geographic patterns. Generally,
the knowledge on distribution of ADME genes in isolated
populations is limited, particularly in the Roma, transna-
tional minority population of Indian origin. The aim of this
study is to determine the allele frequencies ADME “core
list” markers and to compare them with world-wide data in
order to elucidate the position of Roma in the global ADME
genetic landscape. Gonadoblastoma risk is increased in TS with Y chromo-
some, thus gonadectomy is strongly recommended before
starting growth-hormone treatment. The detection of Y
chromosome, at low-level mosaic or as marker chromo-
some, may be tricky by standard karyotype (SK) and array-
CGH (a-CGH). Thus, further molecular screening to detect
Y-chromosomal sequences is mandatory in TS individuals
who are negative by these approaches. Methods: The 95 loci from 32 ADME genes were
genotyped using KASP method in 440 Croatian Roma
DNA samples. Data were analyzed using standard statistical
population-genetics methods. We
performed
low-coverage
(0.2x)
whole
genome
sequencing (lc-WGS) of plasma cell-free DNA (cf-DNA)
to determine its potential role in TS diagnosis. Results: The analysis of genetic vs. geographic distances
placed Croatian Roma among European populations but
their proximity to South-Asian populations is also evident. Results: The analysis of genetic vs. geographic distances
placed Croatian Roma among European populations but
their proximity to South-Asian populations is also evident. P15 Personalized/Predictive Medicine and
Pharmacogenomics 1Institute of Human Genetics, Belgrade, Serbia, 2Institute of
Rheumatology, Belgrade, Serbia, 3Institute of Biochemistry,
Phaemacogenetics Laboratory, Ljubljana, Slovenia P15.01A P15.01A
Position of Croatian Roma in the global ADME core
markers’ variation landscape
T. Skaric-Juric, N. Smolej Narancic, B. Janicijevic, M. Zajc
Petranovic, Z. Tomas, M. Pericic Salihovic
Institute for Anthropological Research, Zagreb, Croatia P14.107C Next, Roma show the outlying position on the global scale
in minor allele frequencies of 12 loci: for 9 loci within 8
genes
(rs1128503,
rs1138272,
rs1799853,
rs1902023,
rs3758581,
rs8192709,
rs10509681,
rs12248560,
rs34059508) they have the highest frequency while for
three
loci
in
three
genes
(rs28399433,
rs28371725,
rs4149117) have the lowest frequencies of the minor allele. Our study was performed on 64 TS patients, previously
characterized by SK and a-CGH analysis on genomic DNA,
and 42 healthy controls (21 females, 21 males). Genome
coverage information from controls was used as reference to
identify structural variants. Our study was performed on 64 TS patients, previously
characterized by SK and a-CGH analysis on genomic DNA,
and 42 healthy controls (21 females, 21 males). Genome
coverage information from controls was used as reference to
identify structural variants. Next, Roma show the outlying position on the global scale
in minor allele frequencies of 12 loci: for 9 loci within 8
genes
(rs1128503,
rs1138272,
rs1799853,
rs1902023,
rs3758581,
rs8192709,
rs10509681,
rs12248560,
rs34059508) they have the highest frequency while for
three
loci
in
three
genes
(rs28399433,
rs28371725,
rs4149117) have the lowest frequencies of the minor allele. We detected low-level mosaicism for XX or XY cell
lines, partial deletions/duplications of sex chromosomes
indicating an iso-chromosome, X chromosome partial
deletions, some of them suggesting a ring/marker chromo-
some. WGS confirmed SK results in 50/64 cases; we
detected nine X chromosome rearrangements at low-level
mosaic that, although hidden at SK, were confirmed by a-
CGH in all but 2 cases, and previously unreported Y
chromosome material was found in 5 patients. We detected low-level mosaicism for XX or XY cell
lines, partial deletions/duplications of sex chromosomes
indicating an iso-chromosome, X chromosome partial
deletions, some of them suggesting a ring/marker chromo-
some. WGS confirmed SK results in 50/64 cases; we
detected nine X chromosome rearrangements at low-level
mosaic that, although hidden at SK, were confirmed by a-
CGH in all but 2 cases, and previously unreported Y
chromosome material was found in 5 patients. Conclusions: The outlying positon in almost 13% of
ADME core markers results from the specific genetic
history of the Roma population. This finding may be helpful
in developing personalized medicines’ protocols in drug
therapies for this specific population. The research was
funded by Croatian Science Foundation grant (HRZZ-IP-
2014-09-4454) to MPS. P14.107C Results: We reviewed clinical referrals from 400+
pedigrees (600+ individuals, 2/3 singleton referrals), with
phenotypes ranging from intellectual disability to single
organ disease. Many cases had extensive investigation
(including
microarray
and
exome
sequencing)
before
referral. 52% had reportable findings and 32% met ACMG
class 4 or 5. Incidental findings were reported in 3% of
cases. Multidisciplinary review meetings enabled collabora-
tion between geographically distant groups, enhancing
variant curation and patient care. Low-coverage whole genome sequencing in plasma
circulating cell-free DNA analysis: the Turner syndrome
experience P. Reho1, D. Larizza2, C. Montalbano2, D. Vergani3, M. Carella3,
A. Provenzano1, A. La Barbera1, V. Palazzo4, S. Landini1, E. Bosi1, R. Artuso4, A. Pagliazzi4, L. Giunti4, D. Formicola1, B. Lucherini4, M. Pantaleo4, I. Sani4, S. Guarducci4, S. Giglio1,4, O. Zuffardi3 P. Reho1, D. Larizza2, C. Montalbano2, D. Vergani3, M. Carella3,
A. Provenzano1, A. La Barbera1, V. Palazzo4, S. Landini1, E. Bosi1, R. Artuso4, A. Pagliazzi4, L. Giunti4, D. Formicola1, B. Lucherini4, M. Pantaleo4, I. Sani4, S. Guarducci4, S. Giglio1,4, O. Zuffardi3 Conclusions: We have largely solved issues relating to
library preparation, sequencing and primary bioinfor-
matics. Variant prioritisation and interpretation remain
challenges we are addressing with filter automation and
custom software solutions. The most promising diagnostic
improvement (currently being implemented) is inclusion
of structural/copy number variant detection with no lower
size
limits. Further
improvements
are
likely
with 543 Abstracts from the 51st European Society of Human Genetics Conference: Posters Turner syndrome (TS) is characterized by complete or
partial absence of the second sex chromosome, either in
mosaic or in all cells, in phenotypic female patients. Gonadoblastoma risk is increased in TS with Y chromo-
some, thus gonadectomy is strongly recommended before
starting growth-hormone treatment. The detection of Y
chromosome, at low-level mosaic or as marker chromo-
some, may be tricky by standard karyotype (SK) and array-
CGH (a-CGH). Thus, further molecular screening to detect
Y-chromosomal sequences is mandatory in TS individuals
who are negative by these approaches. We
performed
low-coverage
(0.2x)
whole
genome
sequencing (lc-WGS) of plasma cell-free DNA (cf-DNA)
to determine its potential role in TS diagnosis. Our study was performed on 64 TS patients, previously
characterized by SK and a-CGH analysis on genomic DNA,
and 42 healthy controls (21 females, 21 males). Genome
coverage information from controls was used as reference to
identify structural variants. T. Skaric-Juric, N. Smolej Narancic, B. Janicijevic, M. Zajc
Petranovic, Z. Tomas, M. Pericic Salihovic Position of Croatian Roma in the global ADME core
markers’ variation landscape Position of Croatian Roma in the global ADME core
markers’ variation landscape Introduction: Adenosine A3 receptor (ADORA3) is part of
the adenosine-mediated antiinflammatory pathway. These
receptors are over-expressed in peripheral blood leukocytes
and synovia of patients with rheumatoid arthritis (RA). Methotrexate (MTX), the anchor drug and part of the first
treatment strategy in RA patients, exerts its antiinflamatory
effects
via
increased
release
of
adenosine
into
the T. Skaric-Juric, N. Smolej Narancic, B. Janicijevic, M. Zajc
Petranovic, Z. Tomas, M. Pericic Salihovic
Institute for Anthropological Research, Zagreb, Croatia T. Skaric-Juric, N. Smolej Narancic, B. Janicijevic, M. Zajc
Petranovic, Z. Tomas, M. Pericic Salihovic 544 J. del Picchia COL6A1 as a genetic cause of Collagen VI-related dystro-
phies (COL6-RD). The variant, located in intron 11, leads
to the insertion of a dominantly-acting pseudoexon that
disrupts the gene’s critical motif. They estimate that ~25%
of all cases with COL6-RD, but negative by exon sequen-
cing are due to this mutation. This variant was found in a de
novo pattern in over 30 patients, and we recently identified
it in our lab in a 4 year old girl with COL6-RD. As part of in
silico machine learning tools we are developing in our lab,
we designed 16 AONs with 2’-OMe modifications and a
phosphorothioate backbone to correct the c.904+189C>T
variant. The most effective AONs were able to reduce the
mutant allele by 95% in a dose dependent manner. The
AONs designed in this work, as well as those designed in
the independent work by Bolduc et al., may offer a treat-
ment for children suffering from the collagen VI-like dys-
trophy. We expect the knowledge gained in this project to
be applicable to a wide range of splice mutations. Grant
support: Thrasher Research Fund extracellular space. Adenosine binds to ADORA2a and A3
and initiates an antiinflammatory response. Therefore
ADORA3 gene polymorphisms could have an impact on
MTX therapy outcome. Materials and Methods: We have analyzed 118 RA
patients on MTX monotherapy whose diagnoses were based
on ACR/EULAR criteria. Genotypisation within ADORA3
gene (rs3393, rs1544223, rs2298191) was performed using
the KASP genotyping system. MTX efficacy was assessed
based on the changes in the Disease activity score (DAS28)
after 6 months of treatment according to the EULAR
response criteria. Patients with good and moderate response
were classified as ‘’responders’’, whereas patients with poor
response
were
considered
‘’nonresponders’’. Data
of
adverse effects were collected during this period. Position of Croatian Roma in the global ADME core
markers’ variation landscape MTX
efficacy and toxicity were compared among patients with
different genotypes. Results: Among patients 106 (89,8%) were responders. Adverse effects were reported in 24 (20,3%) patients. There
was no significant difference in therapy response between
patients with different genotypes. Two patients had bone
marrow complications, both were carriers of rare alleles of
all three polymorphisms (p = 0,02). Results: Among patients 106 (89,8%) were responders. Adverse effects were reported in 24 (20,3%) patients. There
was no significant difference in therapy response between
patients with different genotypes. Two patients had bone
marrow complications, both were carriers of rare alleles of
all three polymorphisms (p = 0,02). D. Yagel: None. Y. Anikster: None. A. Veber: None. M. Shohat: None. Reporting genetic risks to participants of the Estonian
Biobank, Genome Center Reporting genetic risks to participants of the Estonian
Biobank, Genome Center Conclusions: According to our results, ADORA3 gene
polymorphisms may influence bone marrow toxicity in RA
patients treated with MTX. M. Palover1,2, L. Leitsalu1, K. Krebs1,2, K. Läll1,3, M. Kals1,3, T. Nikopensius1, A. Reigo1, L. Milani1, K. Fischer1, R. Mägi1, A. Allik1, B. Kolk1, K. Metsalu1, M. Puusepp1, K. Mikkel1, M. Tammesoo1, E. Pintsaar1, T. Temberg1, S. Reisberg4,5,6, J. Vilo4,5,6, T. Esko1,7, N. Tõnisson1,8, A. Metspalu1 M.B. Grk: None. B. Jekić: None. V. Milić: None. V. Dolzan: None. N. Maksimović: None. T. Damnjanović:
None. M. Duanović Pjević: None. M. Peić: None. M.B. Grk: None. B. Jekić: None. V. Milić: None. V. Dolzan: None. N. Maksimović: None. T. Damnjanović:
None. M. Duanović Pjević: None. M. Peić: None. P15.03C Correction of splice mutation in COL6A1 gene with novel
antisense oligonucleotides as prototype for other orphan
geneticdiseases 1Estonian Genome Center, Institute of Genomics, Tartu,
Estonia, 2Institute of Molecular and Cell Biology, University of
Tartu, Tartu, Estonia, 3Institute of Mathematics and Statistics,
University of Tartu, Tartu, Estonia, 4Institute of Computer
Science, University of Tartu, Tartu, Estonia, 5Software
Technology and Applications Competence Center, Tartu,
Estonia, 6Quretec Ltd, Tartu, Estonia, 7Broad Institute,
Cambridge, MA, United States, 8Department of Genetics, Tartu
University Hospital, Tartu, Estonia D. Yagel1, Y. Anikster1,2, A. Veber1, M. Shohat3 D. Yagel1, Y. Anikster1,2, A. Veber1, M. Shohat3 1Metabolic Disease Unit, Edmond and Lily Safra Children's
Hospital, Sheba Medical Center, Tel-Hashomer, Ramat Gan,
Israel, 2The Wohl Institute for Translational Medicine, Sheba
Medical Center, Tel-Hashomer, Ramat Gan, Israel, 3Sheba
Cancer Research Center, Sheba Medical Center,Tel-
Hashomer, Ramat Gan, Israel Introduction: The Estonian Genome Center at the Institute
of Genomics, University of Tartu, Estonia (EGCUT) holds
genotype and health data of nearly ~52,000 individuals. At
the end of 2017, EGCUT has started offering personalised
genetic risk predictions to its participants. The reports
include genetic risk scores for common diseases, carrier
status, other hereditary risk factors, and pharmacogenetic
recommendations. Premessenger RNA splicing is a necessary step in the pro-
duction of a functional protein product. Many genetic var-
iants lead to aberrant splicing and cause genetic diseases. One prominent technique for correcting splicing-related
mutations is through the use of splice-switching antisense
oligonucleotides (AONs). The antisense drug Nusinersen
for SMA, recently approved by the FDA, is one notable
example. Cummings et al. recently identified a highly
recurrent pathogenic splice variant (c.904+189C>T) in Methods: To date, 47,000 biobank participants have been
genotyped with the Illumina Global Screening Array and
nearly 5,000 individuals have whole genome or exome Abstracts from the 51st European Society of Human Genetics Conference: Posters 545 topotecan, vinblastine, and vinorelbine. These are com-
prised of 3 - 17 biochemically-relevant genes, and have
misclassification rates from 0% to 26.1%. Validation is
performed in patients with breast, bladder, ovarian, and
colon cancers. Signatures derived from tumour cell lines
predicted complete remission from paclitaxel treatment in
84% of breast cancer patients (10% more accurate than
differential expression analysis). Expression of MAPT
correlated with survival in paclitaxel-treated breast cancer
patients. Cisplatin
and
hydroxycyclophosphamide-
resistance were respectively predicted with 71% and 66%
accuracy in bladder and breast cancer patients. Analysis of a
comprehensive set ML-based gene signatures for a panel of
drugs in primary tumours would be feasible to carry out
prior to treatment, and could influence selection of therapy. If current treatment plans are not adequate, ML-based
genomic profiling may also offer alternative tailored stra-
tegies for adjuvant chemotherapies. 1Mol.Oncol. 10:85-100,
2016; 2F1000Res. 5:2124, 2017; 3bioRxiv. https://doi.org/
10.1101/231712. Funding:NSERCDiscovery RGPIN-2015-
06290. sequencing data available. Digital semi-automated report
assembly and participant portal have been set up for signing
an informed consent for return of results, scheduling the
counselling appointment and updating one’s records. For
common complex diseases genetic risk scores (GRS) are
calculated. For type 2 diabetes, 10-year risk estimates
incorporating both GRS and classical risk factors are
provided. Clinically actionable genetic variants are reported
based on ACMG guidelines. The immediate and long-term
feedback of the participants is surveyed to understand the
impact of the return of results. sequencing data available. Digital semi-automated report
assembly and participant portal have been set up for signing
an informed consent for return of results, scheduling the
counselling appointment and updating one’s records. For
common complex diseases genetic risk scores (GRS) are
calculated. For type 2 diabetes, 10-year risk estimates
incorporating both GRS and classical risk factors are
provided. Clinically actionable genetic variants are reported
based on ACMG guidelines. The immediate and long-term
feedback of the participants is surveyed to understand the
impact of the return of results. Results:
1283
participants
have
expressed
interest
through logging into the patient portal, 499 have registered
for feedback visit and 209 have attended to the genetic
counselling session from November 2017 to mid-February
2018, with an average of 24 participants in per week. Conclusions: The EGCUT initiative of return of results
to population biobank participants provides insight on how
healthy individuals respond to personalised genetic risk
information. The long-term aim of the project is to promote
the introduction of personalised medicine in Estonia. M. Palover: None. L. Leitsalu: None. K. Krebs: None. K. Läll: None. M. Kals: None. T. Nikopensius: None. A. Reigo: None. L. Milani: None. K. Fischer: None. R. Mägi: None. A. Allik: None. B. Kolk: None. K. Metsalu:
None. M. Puusepp:
None. K. Mikkel:
None. M. Tammesoo: None. E. Pintsaar: None. T. Temberg: None. S. Reisberg: None. J. Vilo: None. T. Esko: None. N. Tõnisson: None. A. Metspalu: None. M. Palover: None. L. Leitsalu: None. K. Krebs: None. K. Läll: None. M. Kals: None. T. Nikopensius: None. A. M. Palover: None. L. Leitsalu: None. K. Krebs: None. K. Läll: None. M. Kals: None. T. Nikopensius: None. A. P.K. Rogan: Other; Significant; CytogGnomix. E.J. Mucaki: None. J.Z.L. Zhao: None. J.H.M. Knoll: Other;
Significant; CytoGnomix. Novel copy-number variations in pharmacogenes
contribute to interindividual differences in drug
pharmacokinetics Novel copy-number variations in pharmacogenes
contribute to interindividual differences in drug
pharmacokinetics M. Santos1, M. Niemi2, M. Hiratsuka3, M. Kumondai3, M. Ingelman-Sundberg4, V. M. Lauschke4, C. Rodríguez-Antona1,5
1Spanish National Cancer Research Centre (CNIO), Madrid,
Spain, 2University of Helsinki, Helsinki, Finland, 3Tohoku
University, Sendai, Japan, 4Karolinska Institutet, Stockholm,
Sweden, 5ISCIII Center for Biomedical Research on Rare
Diseases, Madrid, Spain M. Santos1, M. Niemi2, M. Hiratsuka3, M. Kumondai3, M. Ingelman-Sundberg4, V. M. Lauschke4, C. Rodríguez-Antona1,5 Comprehensive prediction of responses to chemotherapies
by biochemically-inspired machine learning Comprehensive prediction of responses to chemotherapies
by biochemically-inspired machine learning 1Spanish National Cancer Research Centre (CNIO), Madrid,
Spain, 2University of Helsinki, Helsinki, Finland, 3Tohoku
University, Sendai, Japan, 4Karolinska Institutet, Stockholm,
Sweden, 5ISCIII Center for Biomedical Research on Rare
Diseases, Madrid, Spain P. K. Rogan, E. J. Mucaki, J. Z. L. Zhao, J. H. M. Knoll P. K. Rogan, E. J. Mucaki, J. Z. L. Zhao, J. H. M. Knoll
University of Western Ontario, London, ON, Canada University of Western Ontario, London, ON, Canada Chemotherapy response varies significantly among cancer
patients, and drug resistance is responsible for significant
amount of this mortality. The patterns of gene expression
and copy number changes in the tumour can predict treat-
ment outcomes after chemotherapy by supervised machine
learning (ML)1-3. Computational models based on tran-
scriptome gene signatures of tumor cell lines were used
predict chemosensitivity and resistance for 25 cancer drugs. This learned set of genes was used to predict clinical out-
comes from tumor transcriptomes. Gene signatures have
been derived for 5-fluorouracil, bortezomib, carboplatin,
cisplatin, docetaxel, doxorubicin, erlotinib, epirubicin, eto-
poside, gefitinib, gemcitabine, hydroxycyclophosphamide,
imatinib, irinotecan, Ixabepilone, methotrexate, oxaliplatin,
paclitaxel, pemetrexed, rapamycin, sorafenib, tamoxifen, Introduction: Variability in pharmacokinetics and drug
response is shaped by single-nucleotide variants (SNVs) as
well as copy-number variants (CNVs) in genes with
importance for drug absorption, distribution, metabolism,
and excretion (ADME). While SNVs have been extensively
studied, a systematic assessment of the CNV landscape in
ADME genes is lacking. Methods: We integrated data from 2,504 whole genomes
from the 1000 Genomes Project and 59,898 exomes from
the Exome Aggregation Consortium to identify CNVs in
208 relevant pharmacogenes. Results:
We
describe
novel
exonic
deletions
and
duplications in 201 (97%) of the pharmacogenes analyzed. The deletions are population-specific and frequencies range J. del Picchia 546 (BioRad) and QuantStudio™3D (Applied Biosystems)
platforms. Quantification and detection limits and false
positive
rates
were
defined. To
establish
genotypic
concordance designs were tested in Horizon lines and/or
FFPE samples previously quantified. from singletons up to 1%, accounting for >5% of all loss-of-
function alleles in up to 42% of the genes studied. We
experimentally confirmed novel deletions in CYP2C19,
CYP4F2,
and
SLCO1B3
by
Sanger
sequencing
and
validated their allelic frequencies in selected populations. Conclusions: CNVs are an additional source of pharma-
cogenetic variability with important implications for drug
response and personalized therapy. P15.07C Innovative, high-sensitive and short-term Digital Droplet
PCR (ddPCR) methodology development, for oncologic
therapy response associated biomarkers quantification A. Andújar1, Y. Moreno1, S. Valverde1, L. Arocas1, R. Martínez1,
L. Pérez-Cabornero1, C. Buades1, S. Santillán1, V. Calabuig2, R. Miñambres2, C. M. Moya1, D. M. Valero-Hervás1 A. Andújar: None. Y. Moreno: None. S. Valverde:
None. L. Arocas: None. R. Martínez: None. L. Pérez-
Cabornero: None. C. Buades: None. S. Santillán: None. V. Calabuig: None. R. Miñambres: None. C.M. Moya:
None. D.M. Valero-Hervás: None. 1Medical Genetics Unit, Sistemas Genómicos, Paterna,
Valencia, Spain, 2I+D+I Department, Sistemas Genómicos,
Paterna, Valencia, Spain Introduction: Outstanding advances in pharmacogenetics
knowledge and its influence in oncological therapy success
has impelled the need of a high-sensitive and short
turnaround-time methodologies for quantification of opti-
mal drug treatment biomarkers. ddPCR technique has
demonstrated the best sensitivity and turnaround-time/cost
ratio. The aim of this work was to develop a ddPCR assay
for biomarker quantification in NRAS, KRAS, EGFR and
BRAF genes. Comprehensive prediction of responses to chemotherapies
by biochemically-inspired machine learning This, together with the
important contribution of rare alleles to the variability of
pharmacogenes, emphasizes the necessity of comprehensive
next-generation sequencing–based genotype identification
for an accurate prediction of the genetic variability of drug
pharmacokinetics. Results: The design detected the complete biomarker-set. False positive rate, and detection and quantification means
are described in the table. Selected variants were identified
in both Horizon lines and clinical samples within a 0.4-
1.6% quantification concordance. Description of the obtained technical parameters
ddPCR
Sensitivity Mean False
positive Rate
Mean
Limit of
Detection
Mean Limit
of
Quantification
Turnaround Time
KRAS
0.001%
0.045%
0.657%
0.813%
1 day
NRAS
0.810%
1.190%
1.190%
EGFR indels
0.084%
0.359%
0.459%
EGFR SNVs
0.063%
0.187%
0.285%
BRAF
0.279%
0.513%
0.513%
Pyrosequencing (data
described in published
literature)
5%
1-5%
5-10%
5%
2 days
Sanger Sequencing
(data described in
published literature)
15%
5%
5%
5-10%
2-3 days Description of the obtained technical parameters This work was supported by the Spanish Ministry of
Economy and Competiveness (grant SAF2015-64850-R),
by the Spanish Ministry of Education, Culture and Sport
(grant FPU16/05527), by the European Union’s Horizon
2020 research and innovation program U-PGx under grant
agreement 668353, and by the Swedish Research Council
(grant agreements 2015-02760, 2016-01153, and 2016-
01154). M. Santos: None. M. Niemi: None. M. Hiratsuka:
None. M. Kumondai: None. M. Ingelman-Sundberg:
None. V.M. Lauschke:
None. C. Rodríguez-
Antona: None. Conclusions: Our results show ddPCR as an ultrasensi-
tive
low
time-processing
technology
with
affordable
requirements
(simple designs, manageable
procedures,
suitability for complex samples). These features make
ddPCR the optimal technique for biomarker quantification
in oncological therapy, providing useful genetic information
for drug selection. This testing translation to clinical routine
would increase therapy effectiveness, shorten elapsed-time
until treatment administering, and prevent adverse effects
development. P15.08D Pipeline for knowledge curation and decision support in
pharmacogenomics S. U. Gjerald1, N. Vethe1, S. Skogstad Tuv1, K. Nordal1, S. Alagaratnam2, T. Håndstad1, S. Bergan1,3 1Oslo University Hospital, Oslo, Norway, 2DNV GL, Høvik,
Norway, 3University of Oslo, Oslo, Norway Materials and Methods: Mutant control fragments for
twenty-nine
selected
high
frequency-biomarkers
were
generated through directed mutagenesis and confirmed by
Sanger Sequencing. The methodology was developed using
allele
frequency
positive
controls
mixes
for
QX200 The introduction of whole-genome sequencing in clinical
practice has opened a path for preemptive, opportunistic
pharmacogenomics (PGx) testing. The Pharmacogenomics 547 Abstracts from the 51st European Society of Human Genetics Conference: Posters Knowledgebase (PharmGKB) distributes clinical dosing
guidelines, and provides a link between these guidelines
and genomic variants. Before applying this knowledge in
healthcare, it is necessary to verify and adapt the guidelines
to local practices. We describe a pipeline for translating
knowledge from knowledge databases such as PharmGKB
into a system that is valid for PGx decision support in our
clinic. Germany, 8Department of Neurology, St. Elisabeth Hospital,
Willemstad, Curaçao, Netherlands Antilles, 9Department of
Biomedical and Clinical Sciences "Luigi Sacco", University of
Milan, Milano, Italy Diabetic patients can develop painful (PDN) or painless
(PLDN) neuropathy irrespective of the severity or dura-
tion of diabetes. Understanding the underlying genetic
background would allow developing personalized thera-
pies. We examined the incidence of variants in ten
Voltage-Gated Sodium Channels (VGSCs) genes in PDN/
PLDN patients and analyzed if differences in frequency,
protein localization or pathogenicity correlated with the
phenotype stratification. VGSCs genes were sequenced by
Molecular Inversion Probe-NGS and run on NextSeq500
Illumina©. The variant selection and the pathogenicity
classification were performed according to the ACGS
guidelines. We analyzed 547 patients: PDN (41.5%) and
PLDN (58.5%). We identified 33 rare variants exclusively
carried by PDN (N=38) and 42 exclusively carried by
PLDN (N=47). The distribution of the mutations among
the VGSCs genes is reported in the table. 6 PDN patients
with more than 1 mutation were prevalently carrying
SCN9A mutations whereas the 8 plurimutated PLDN
patients were prevalently carrying SCN10A mutations. According to pathogenicity classification, 84.9% was
represented by Class 3 mutations and 12.1% by Class 4 in
the PDN cohort, whereas in the PLDN were 83.3% and
9.5% respectively. Considering the protein localization,
PDN mostly carried variants located in the linker DII-DIII
(83.3%), PLDN in the C-terminal (87.5%). P15.08D This study
describes the genetic profiling in PDN/PLDN neuropathy
suggesting a preliminary approach for the phenotype
stratification based on VGSCs genetic characterization. Grant: PROPANE Health-602273. We implement the pipeline using the Web Ontology
Language (OWL) in order to keep the semantics of the
decision
support
system compatible
with
PharmGKB
knowledge. OWL ontologies allow for automatic testing
of ontology coherence, and the inference of dosing
recommendations based on the genomic variants in each
patient. We demonstrate how we have translated the semantics of
the PharmGKB linked data model into an OWL ontology. We emphasize the strategy for aligning the PharmGKB
definitions of indel and structural variants with patient
variants. Then we outline how we use a web-based tool for
collaborative curation of PGx knowledge. Finally, we show
how we can provide curated dosing recommendations to
whole-genome sequenced patients, exemplified by the drugs
azathioprine and clopidogrel. The work received funding from project number 247965
of the IKTPLUSS-programme of the Research Council of
Norway. S.U. Gjerald: None. N. Vethe: None. S. Skogstad Tuv:
None. K. Nordal: None. S. Alagaratnam: None. T. Håndstad: None. S. Bergan: None. Detection of Familial Chylomicronemia Syndrome in a
cohort of patients with severe hypertriglyceridemia
through a Next Generation Sequencing approach 1Institut de Pharmacologie Moléculaire et Cellulaire,
Valbonne, France, 2University Roma 3, Roma, Italy, 3Institut
de Biologie Paris Seine, Paris, France, 4Università di Catania,
Catania, Italy 1Institut de Pharmacologie Moléculaire et Cellulaire,
Valbonne, France, 2University Roma 3, Roma, Italy, 3Institut
de Biologie Paris Seine, Paris, France, 4Università di Catania,
Catania, Italy A. Di Costanzo, L. D'Erasmo, F. Cassandra, I. Minicocci, L. Polito, M. Arca A. Di Costanzo, L. D'Erasmo, F. Cassandra, I. Minicocci, L. Polito, M. Arca Polito, M. Arca Fragile X syndrome (FXS), the most common form of
inherited intellectual disability and a leading cause of aut-
ism spectrum disorder (ASD). It is due to the functional
deficiency of the Fragile X Mental Retardation Protein
(FMRP), an RNA-binding protein involved in translational
regulation of many proteins having key roles in synaptic
morphology and plasticity. No specific and effective treat-
ment for FXS is available. We searched for FMRP targets
by HITS-CLIP during early development of multiple mouse
brain regions (hippocampus, cortex and cerebellum) at a
time when FMRP is most highly expressed and synapto-
genesis peaks. Our data point out one specific phospho-
diesterase mRNA as a prominent target of FMRP which
negatively modulates its translation and intracellular trans-
port. Since the abundance of this protein and activity are
increased in Fmr1-KO cortex and hippocampus impacting
the homeostasis of cAMP/cGMP, we propose here a new
therapeutic approach for FXS, based on the specific phar-
macological inhibition of this protein. We will present data
showing pharmacological inhibition of this enzyme rescues
some behavioral deficits in newborn and adolescent Fmr1-
null mice such as social communication, discrimination and
interaction. Importantly, chronic blockade in newborn
Fmr1-KO mice, followed by a wash-out interval, results in
the rescue of the altered social behavior in adolescent mice,
showing that the beneficial effects of early pharmacological
blockade of our target are long-lasting. La Sapienza, Roma, Italy La Sapienza, Roma, Italy Aims: Familial Chylomicronemia syndrome (FCS) is a rare
recessive disease caused by mutations in LPL, APOC2,
APOA5, LMF1 and GPIHBP1 genes. It is characterized by
very severe hypertriglyceridemia (HTG) with or without
episodes of abdominal pain and recurrent acute pancreatitis. FCS diagnosis is often difficult due to its phenotypic
similarity with other forms of severe hypertriglyceridemia. The aim of our study was to detect pathogenic mutations in
candidate genes in patients with suspected FCS based on
specific clinical criteria and evaluate clinical differences
between genotypes. Detection of Familial Chylomicronemia Syndrome in a
cohort of patients with severe hypertriglyceridemia
through a Next Generation Sequencing approach Methods: By examining 3000 clinical records, 31
patients were classified as suspected FCS on the following
criteria: a) plasma trigliceridemia (TG) levels >1000 mg/dl
in multiple determinations; b) resistance to pharmacological
therapy; c) history of acute pancreatitis. All patients
underwent fully clinical examination and their information
were
collected
retrospectively. Candidate
genes
were
sequenced
using
Next
generation
sequencing
(NGS)
technique. Results: 51.6% subjects were carriers of FCS causing
mutations, the majority in LPL gene (56.2%). Compared to
non-carriers, FCS patients showed higher prevalence of
history of acute pancreatitis (P=0.04) and early onset of
HTG (P=0.0008). Comparing homozygous carriers of
mutations in other genes with LPL homozygotes, the latter
group showed higher TG levels (P < 0.001) and lower TG
reduction during treatment (Padj=0.03). B. Bardoni: None. V. Trezza: None. S. Martin: None. P. Vincent: None. L. Ciranna: None. M. Jarjat: None. S. Delhaye: None. S. Castagnola: None. F. Melancia: None. T. Maurin: None. Conclusions: Our data suggests that the proposed
diagnostic criteria are highly predictive of FCS diagnosis
in severe HTG patients. Mutations in LPL gene are the most
common cause of FCS and homozygous carriers of
mutations in LPL gene have the more severe clinical
phenotype. P15.11C None. R. Malik: None. D. Ziegler: None. G. Bönhof:
None. I. Merkies:
None. C. Faber:
None. G. Lauria: None. B. Bardoni1, V. Trezza2, S. Martin1, P. Vincent3, L. Ciranna4, M. Jarjat1, S. Delhaye1, S. Castagnola1, F. Melancia2, T. Maurin1 E. M. Quinn1, D. Crognale2, A. W. Ryan1, A. Butler2, J. Dolan1,
J. Pratt2, T. Magalhaes1, R. Carr2, R. Moran1, K. O’Connell2, J.
Conroy1, G. DeVito3, C. Boreham2,3, S. Ennis1,4 P15.09A Genetic profiling of voltage-gated sodium cannels in painful
and painless diabetic neuropathy M. Marchi1, M. Molasy2, R. Almomani2, M. Gerrits2, E. Salvi1,3,
I. D'Amato1, R. Malik4,5, D. Ziegler6,7, G. Bönhof6, I. Merkies2,8,
C. Faber2, G. Lauria1,9 SYMBOL
PDN
PLDN
Total
SCN3A
2 (50 %)
2 (50 %)
4
SCN7A
7 (50 %)
7 (50 %)
14
SCN8A
1 (33.3 %)
2 (66.7 %)
3
SCN9A
4 (50 %)
4 (50 %)
8
SCN10A
7 (33.3%)
14 (66.7%)
21
SCN11A
5 (41.7 %)
7 (58.3 %)
12
SCN1B
2 (50 %)
2 (50 %)
4
SCN2B
2 (40 %)
3 (60 %)
5
SCN3B
2 (66.7 %)
1 (33.3 %)
3
SCN4B
1 (100 %)
0 (0 %)
1
Total
33 (44 %)
42 (56 %)
75
M. Marchi: None. M. Molasy: None. R. Almomani:
None. M. Gerrits: None. E. Salvi: None. I. D'Amato: SYMBOL
PDN
PLDN
Total
SCN3A
2 (50 %)
2 (50 %)
4
SCN7A
7 (50 %)
7 (50 %)
14
SCN8A
1 (33.3 %)
2 (66.7 %)
3
SCN9A
4 (50 %)
4 (50 %)
8
SCN10A
7 (33.3%)
14 (66.7%)
21
SCN11A
5 (41.7 %)
7 (58.3 %)
12
SCN1B
2 (50 %)
2 (50 %)
4
SCN2B
2 (40 %)
3 (60 %)
5
SCN3B
2 (66.7 %)
1 (33.3 %)
3
SCN4B
1 (100 %)
0 (0 %)
1
Total
33 (44 %)
42 (56 %)
75 1Neuroalgology Unit, IRCCS Foundation “Carlo Besta”
Neurological Institute, Milano, Italy, 2Clinical Genetics and
Department of Neurology, Maastricht University Medical
Center, Maastricht, Netherlands, 3Department of Health
Sciences, University of Milan, Milano, Italy, 4Institute of
Human Development, Centre for Endocrinology and Diabetes,
University of Manchester and Central Manchester NHS
Foundation Trust, Manchester Academic Health Science
Center, Manchester, United Kingdom, 5Department of
Medicine, Weill Cornell Medicine, Doha, Qatar, 6Institute for
Clinical Diabetology, German Diabetes Center, Leibniz Center
for Diabetes Research, Heinrich Heine University, Düsseldorf,
Germany, 7Department of Endocrinology and Diabetology,
Medical Faculty, Heinrich Heine University, Düsseldorf, M. Marchi: None. M. Molasy: None. R. Almomani:
None. M. Gerrits: None. E. Salvi: None. I. D'Amato: 548 J. del Picchia P15.11C
Modulation of cGMP and cAMP as a new therapeutic
target for Fragile X Syndrome
B. Bardoni1, V. Trezza2, S. Martin1, P. Vincent3, L. Ciranna4, M. Jarjat1, S. Delhaye1, S. Castagnola1, F. Melancia2, T. Maurin1 Preliminary genomic analysis of the GenoFit cohort: a
health and fitness study P15.12D In addition to the elucidation of complex disease risk,
genome wide association studies (GWAS) have potential to
uncover the genetic components of health, fitness and
wellness. Genetic variants associated with quantitative traits
such as height, adiposity (BMI/waist hip ratio/visceral fat),
blood pressure and hand grip strength have been uncovered
in multiple studies. GenoFit is a recently initiated study of
the genetic basis of health and fitness associated variation,
collected among attendees of a university fitness centre. GenoFit participants are physically assessed for general
health and fitness measures including bone and muscle
health. Participants also complete a detailed lifestyle ques-
tionnaire. The age profile of the participants varies from the
student population to locally resident individuals in their
70s and 80s. Fitness and adiposity metrics are approxi-
mately representative of the general population. The final
target population is 5,000 individuals. The genomes of all
enrolled individuals will be whole genome sequenced to a
depth of 30x and genotyped using an Affymetrix precision
medicine research array containing over 800,000 markers. To date, 277 individuals have been genotyped, and analysed
using linear regression for quantitative trait association
using PLINK v1.9. Analyses have yielded nominally sig-
nificant (uncorrected P < 0.05) association signals for
established genetic loci for adiposity (BMI/waist hip ratio:
FTO, MC4R, GNPDAP2, GRB14, CPEB4, FAM13A). With
the current sample size, no loci reached genome-wide sig-
nificance. This non-obese Irish population sample is cur-
rently under active ongoing sample collection, phenotypic
characterisation and molecular analysis with the aim of
developing a valuable resource for future genomic studies. Background & Aims: Hepatotoxicity is one of the most
common drug-related toxicity during the treatment of
childhood acute lymphoblastic leukemia (ALL). During
induction, this toxicity may be due to asparaginase while in
consolidation is typically associated with methotrexate
(MTX). Pharmacogenetic studies have identified SNPs
strongly associated with this toxicity in children with ALL
during the induction while during consolidation, most of the
studies have analyzed few variants in MTX pathway genes,
with no clear markers. Recently, we found association
between another MTX-related toxicity, mucositis, and a
SNP in miR-1206. Many genes involved in liver-specific
signaling pathways are tightly controlled by miRNAs. In
consequence, we hypothesized that variants in miRNAs
targeting ASP-related genes in induction and MTX-related
genes in consolidation could be also associated with drug-
induced hepatotoxicity. P15.12D Preliminary genomic analysis of the GenoFit cohort: a
health and fitness study A. Di
Costanzo:
None. L. D'Erasmo:
None. F. Cassandra: None. I. Minicocci: None. L. Polito: None. M. Arca: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 549 1Genomics Medicine Ireland, Dublin, Ireland, 2UCD Institute
for Sport and Health, University College Dublin, Dublin,
Ireland, 3UCD School of Public Health, Physiotherapy and
Sports Science, University College Dublin, Dublin, Ireland,
4University College Dublin, Dublin, Ireland 1Department of Genetics, Physical Anthropology and Animal
Physiology, University of the Basque Country, UPV/EHU,
Leioa, Spain, 2Department of Pediatrics, University Hospital
Donostia, San Sebastian, Spain, 3Department of Pediatrics,
University of the Basque Country, UPV/EHU, Leioa, Spain,
4Department of Oncohematology, University Hospital La Paz,
Madrid, Spain, 5Department of Pediatrics, University Hospital
Cruces, Barakaldo, Spain, 6BioCruces Health Research
Institute, Barakaldo, Spain In addition to the elucidation of complex disease risk,
genome wide association studies (GWAS) have potential to
uncover the genetic components of health, fitness and
wellness. Genetic variants associated with quantitative traits
such as height, adiposity (BMI/waist hip ratio/visceral fat),
blood pressure and hand grip strength have been uncovered
in multiple studies. GenoFit is a recently initiated study of
the genetic basis of health and fitness associated variation,
collected among attendees of a university fitness centre. GenoFit participants are physically assessed for general
health and fitness measures including bone and muscle
health. Participants also complete a detailed lifestyle ques-
tionnaire. The age profile of the participants varies from the
student population to locally resident individuals in their
70s and 80s. Fitness and adiposity metrics are approxi-
mately representative of the general population. The final
target population is 5,000 individuals. The genomes of all
enrolled individuals will be whole genome sequenced to a
depth of 30x and genotyped using an Affymetrix precision
medicine research array containing over 800,000 markers. To date, 277 individuals have been genotyped, and analysed
using linear regression for quantitative trait association
using PLINK v1.9. Analyses have yielded nominally sig-
nificant (uncorrected P < 0.05) association signals for
established genetic loci for adiposity (BMI/waist hip ratio:
FTO, MC4R, GNPDAP2, GRB14, CPEB4, FAM13A). With
the current sample size, no loci reached genome-wide sig-
nificance. This non-obese Irish population sample is cur-
rently under active ongoing sample collection, phenotypic
characterisation and molecular analysis with the aim of
developing a valuable resource for future genomic studies. A. Gutierrez-Camino1, M. Umerez1, B. Santos1, I. Martín-
Guerrero1, E. Lopez-Lopez1, N. García de Andoin2,3, A. Sastre4,
A. Navajas5,6, I. Astigarraga3,5,6, A. García-Orad1,6 P15.13A P15.13A
rs2648841 in miR-1208 is involved in hepatotoxicity during
consolidation in childhood acute lymphoblastic leukemia
treatment A. Gutierrez-Camino: None. M. Umerez: None. B. Santos: None. I. Martín-Guerrero: None. E. Lopez-
Lopez: None. N. García de Andoin: None. A. Sastre:
None. A. Navajas: None. I. Astigarraga: None. A. García-Orad: None. Lopez: None. N. García de Andoin: None. A. Sastre:
None. A. Navajas: None. I. Astigarraga: None. A. García-Orad: None. Generation of patient-specific hiPSCs model to study
POLD1-related MDPL syndrome P15.12D Methods: Therefore, we analyzed all the SNPs in
miRNAs that could target these drug-related genes in a
large cohort of Spanish children with ALL homogeneously
treated. Results: A total of 5 SNPs in 5 miRNAs during induction
were associated with hepatotoxicity and 11 different SNPs
in 10 miRNAs during consolidation, which suggests
different mechanisms. Among them, we pointed out
rs2648841 in miR-1208 which was the most significant
SNP associated with hepatotoxicity during consolidation
phase after Bonferroni correction, probably through a
higher
expression
of
its
target
genes
in
the
MTX
pharmacodynamic pathway, DHFR, MTR and MTHFR. Conclusions: These results support the importance of
miRNAs studies to understand the differences in toxicity to
chemotherapy in children diagnosed with ALL. A. Gutierrez-Camino: None. M. Umerez: None. B. Santos: None. I. Martín-Guerrero: None. E. Lopez- Results: A total of 5 SNPs in 5 miRNAs during induction
were associated with hepatotoxicity and 11 different SNPs
in 10 miRNAs during consolidation, which suggests
different mechanisms. Among them, we pointed out
rs2648841 in miR-1208 which was the most significant
SNP associated with hepatotoxicity during consolidation
phase after Bonferroni correction, probably through a
higher
expression
of
its
target
genes
in
the
MTX
pharmacodynamic pathway, DHFR, MTR and MTHFR. E.M. Quinn: None. D. Crognale: None. A.W. Ryan:
None. A. Butler: None. J. Dolan: None. J. Pratt: None. T. Magalhaes: None. R. Carr: None. R. Moran: None. K. O’Connell: None. J. Conroy: None. G. DeVito: None. C. Boreham: None. S. Ennis: None. E.M. Quinn: None. D. Crognale: None. A.W. Ryan:
None. A. Butler: None. J. Dolan: None. J. Pratt: None. T. Magalhaes: None. R. Carr: None. R. Moran: None. K. O’Connell: None. J. Conroy: None. G. DeVito: None. C. Boreham: None. S. Ennis: None. Conclusions: These results support the importance of
miRNAs studies to understand the differences in toxicity to
chemotherapy in children diagnosed with ALL. Conclusions: These results support the importance of
miRNAs studies to understand the differences in toxicity to
chemotherapy in children diagnosed with ALL. P15.13A
rs2648841 in miR-1208 is involved in hepatotoxicity during
consolidation in childhood acute lymphoblastic leukemia
treatment P15.15C Generation of patient-specific hiPSCs model to study
POLD1-related MDPL syndrome Generation of patient-specific hiPSCs model to study
POLD1-related MDPL syndrome 550 J. del Picchia The study of human development and diseases relies on
analysis of samples obtained and manipulated ex vivo. It is
critical in such studies to know the provenance and confirm
that the identity of these materials is correct. Analysis of
highly variable short tandem repeats (STRs), provides a
simple, inexpensive and highly specific genetic “finger-
print” of a human sample. In this study, we describe a
complete workflow for cell line authentication and chimeric
receptor T-cell (CAR T) and induced pluripotent stem cell
(iPSC) sample matching. Analysis of serial dilutions of
purified genomic DNA from isolated human cell lines
identified the correct alleles from as little as 0.1ng of pur-
ified DNA. Samples immobilized onto Copan NUCLEIC
Cards were correctly identified when a suspension of 5-10 x
105 cells were dried onto the cards. Contaminating HeLa
alleles could be detected in a mixture when they were
present in as little as 1% of genomic DNA from this
population. Blinded samples of donor and iPSCs, as well as
manipulated CAR T cells, were correctly matched in every
case tested. Together, these results describe a facile and
consolidated workflow for establishing the provenance,
authenticity
and
identity
of
ex
vivo-obtained
human
samples. P. Spitalieri1, R. V. Talarico1, M. Murdocca1, C. De Masi1, E. Campione2, A. Serafino3, M. D'Adamo2,4, P. Sbraccia2,4, M. R. D'Apice4, G. Novelli1, F. Sangiuolo1 1Dept. of Biomedicine and Prevention,Tor Vergata University,
Rome, Italy, 2Dept. of Systems Medicine,Tor Vergata
University, Rome, Italy, 3IFT, Italian National Research
Council, Rome, Italy, 4University Hospital Policlinico Tor
Vergata, Rome, Italy Mandibular hypoplasia, Deafness and Progeroid features
with concomitant Lipodystrophy and insulin resistance,
define a multisystem disorder named MDPL syndrome. MDPL has been associated to de novo heterozygous
mutations in POLD1 gene which encodes the active site of
DNA polymerase δ, involved in DNA replication and
repair
mechanisms. Recent
cellular
studies
revealed
complex aetiology of this disorder, with an important role
for POLD1 function in adipose homeostasis. To date, 22
cases with POLD1-related MDPL syndrome have been
reported worldwide. In order to improve our under-
standing on the molecular mechanisms of the MDPL
disease, we generated human induced pluripotent stem
cells (hiPSCs) from dermal fibroblasts obtained from a
patient harbouring the recurrent heterozygous in-frame
deletion p.Ser605del within polymerase domain. K. Goričar1, V. Kovač2, V. Dolan1 K. Goričar1, V. Kovač2, V. Dolan1 1Pharmacogenetics Laboratory, Institute of Biochemistry,
Faculty of Medicine, University of Ljubljana, Ljubljana,
Slovenia, 2Institute of Oncology Ljubljana, Ljubljana, Slovenia 1Pharmacogenetics Laboratory, Institute of Biochemistry,
Faculty of Medicine, University of Ljubljana, Ljubljana,
Slovenia, 2Institute of Oncology Ljubljana, Ljubljana, Slovenia P. Spitalieri: None. R.V. Talarico: None. M. Mur-
docca: None. C. De Masi: None. E. Campione: None. A. Serafino: None. M. D'Adamo: None. P. Sbraccia: None. M.R. D'Apice:
None. G. Novelli:
None. F. Sangiuolo: None. Introduction: Immune checkpoints such as programmed
cell death 1 (PD-1) or programmed cell death 1 ligand 1
(PD-L1) are key modulators of a balanced physiological
immune response. Cancer cells can exploit these mechan-
isms to evade immune response and PD-1 or PD-L1 inhi-
bitors have recently proved successful in several tumors. However, PD-1 and PD-L1 expression may also confer
resistance to cisplatin-based chemotherapy, commonly used
in treatment of malignant mesothelioma (MM). Our aim
was therefore to determine whether polymorphisms in
immune checkpoint genes influence treatment outcome in
MM patients. Confirming the identity of human cell lines and other ex
vivo-manipulated human samples Confirming the identity of human cell lines and other ex
vivo-manipulated human samples P15.15C The cells
displayed expression of stemness markers by immuno-
fluorescence studies and normal pluripotent stem cell
morphology. In addition, despite MDPL- hiPSCs showed
normal phenotypes without significant nuclear abnorm-
alities, we highlighted the presence of micronuclei, that
correlates with DNA damage repair defects and genome
instability. Their subsequent differentiation into adipose
tissue, that is mainly involved in the MDPL disease, could
represent an outstanding opportunity to yield additional
insights into the pathophysiology of fat loss. Finally, this
new tool might be used for drug screening and further
development of targeted therapeutic approaches. S. Jackson: A. Employment (full or part-time); Sig-
nificant; Thermo Fisher Scientific. D. Yoder: A. Employ-
ment
(full
or
part-time);
Significant;
Thermo
Fisher
Scientific. K. Varma: A. Employment (full or part-time);
Significant; Thermo Fisher Scientific. Thermo Fisher Scientific, South San Francisco, CA, United
States P15.18B Immune checkpoints PD-1 and PD-L1 polymorphisms and
outcome of cisplatin-based chemotherapy in malignant
mesothelioma S. Jackson, D. Yoder, K. Varma Goričar: None. V. Kovač: None. V. Dolan: None. K. Goričar: None. V. Kovač: None. V. Dolan: None. P15.19C Detection of cfBRAFV600E in plasma from melanoma
patients and non-melanoma individuals Detection of cfBRAFV600E in plasma from melanoma
patients and non-melanoma individuals S. Jackson, D. Yoder, K. Varma Thermo Fisher Scientific, South San Francisco, CA, United
States 551 Abstracts from the 51st European Society of Human Genetics Conference: Posters melanoma individuals and 65 melanoma patients (25 stage
III melanoma patients, 6 IV melanoma patients and 32
disease-free melanoma patients). Nevus count information
and presence of clinically atypical nevi was available in
97.2% and 91.5% individuals, respectively. We detect
cfBRAFV600E in plasma of 83.3% of stage IV melanoma
patient, 14.8% of stage III melanoma patients, 3.1% disease
free
melanoma
patients
and
4.1%
of
non-melanoma
patients. The amount of cfBRAFV600E plasma was higher
in stage IV melanoma patients (377.8±5224 copies/mL) and
stage III melanoma patients (16.7±214.8 copies/mL) com-
pare to non-melanoma individuals (5.28±3.64 copies/mL). In addition, percentage of mutation differ between mela-
noma patients (>0.65% cfBRAFV600E) and disease free
melanoma patients and non-melanoma patients (<0.65%
cfBRAFV600). Association between nevus count or clinically
atypical nevi and cfBRAFV600E was not statistically assessed
due to the limited number of cfBRAFV600E subjects. How-
ever, individuals with high number of nevi showed higher
concentration of cfBRAFV600E. In conclusion, cfBRAFV600E
can be detected in individuals without melanoma. Thus, is
necessary to stablish normality threshold of cfBRAFV600E
for the implementation of the analysis into clinical practice. This study was funded by Grant PI15/00956 from Fondo
Investigaciones Sanitarias, Spain. Materials and Methods: MM patients treated with
gemcitabine/cisplatin or pemetrexed/cisplatin doublet che-
motherapy were genotyped for six polymorphisms in genes
coding for PD-1 (PDCD1) and PD-L1 (CD274). Cox and
logistic regression were used to assess their influence on
treatment outcome. Results: Among 171 MM patients, carriers of at least one
polymorphic CD274 rs4742098 (c.*2635A>G) allele were
more likely to achieve partial or complete response after
gemcitabine/cisplatin treatment compared to carriers of two
wild-type alleles (OR=2.19, 95% CI=1.02-4.71, P=0.045). They also had significantly longer progression-free (9.1 vs
7.1 months, HR=0.64, 95% CI=0.43-0.94, P=0.025) and
overall survival (21.5 vs 14.0 months, HR=0.59, 95%
CI=0.39-0.91,
P=0.016). Regarding
adverse
events,
CD274 rs4742098 was associated with increased risk of
nausea/vomiting
(P=0.024)
and
alopecia
(P=0.036). CD274 rs4143815 and PDCD1 rs10204525 were also
associated with increased risk of nausea/vomiting (P=0.023
and P=0.025, respectively). Conclusions: Genetic variability of PD-1 and PD-L1
immune checkpoints may influence response to gemcita-
bine/cisplatin chemotherapy and could support personalized
treatment in MM. Acknowledgements: ARRS-L3-8203 project grant. Acknowledgements: ARRS-L3-8203 project grant. J.A. Puig-Butillé: None. N. Calbet: None. M. Potrony:
None. P. Aguilera: None. C. Carrera: None. S. Puig:
None. J. Malvehy: None. K. P15.22B Colin8, D. Haye4, A. Verloes9, S. Marlin3, S. Manouvrier10, P. Edery2, N. Philip11, D. Geneviève12, D. Lacombe13, S. Odent8, J. Clayton-Smith14, S. Douzgou14, M. Smith14, B. Arveiller15, A. Piton16, P. Saugier-
Veber17, B. Gérard16, AnDDI-Rares, BRAIN-TEAM, Cardiogen,
DéfiScience, FAI²R, FAVA-Multi, Filfoie, Filnemus, Filslan,
Fimarad, Fimatho, Firendo, G2M, MaRiH, MCGRE, Mhémo,
Muco CFT, NeuroSphinx, Orkid, Oscar, RespiFIL, Sensgene,
Tetecou, C. Thauvin-Robinet1, L. Faivre1 Twenty-four RRMS patients were sampled at baseline
and after 6 months of FTY treatment. CD3+ T cells and
CD20+ B cells were sorted with MACS MicroBeads
system and RNA sequencing performed using Illumina
NextSeq500
platform. Differentially
expressed
genes
(DEGs) were identified for each cell type using DESeq2
R package. Genes modulated by FTY (fold change [FC]>2
or FC<0.5 and false discovery rate [FDR]<5%) were
considered for pathway analysis based on KEGG database. We observed a marked up-regulation in both T and B
lymphocytes (313 up- and 240 down-regulated genes in
T cells; 400 up- and 104 down-regulated genes in B cells),
with evidence of significant overlap between the two cell
types. Most of the DEGs had immune-related functions:
among them, CX3CR1 was strongly up-regulated (padjus-
ted=6.4x10-26, FC=5.96) and CCR7 was down-regulated in
both cell types (padjusted=5.4x10-31, FC=0.18). DEGs were
enriched of genes involved in immune-related pathways
(e.g. Cytokine-cytokine Receptor Interaction, Chemokine
Signaling Pathway). Network analysis elicited CD44 as a
hub gene involved in cell migration. 1CLAD-Est, Dijon, France, 2CLAD-Sud-Est, Lyon, France,
3Service de génétique, Necker, Paris, France, 4CLAD-Ile de
France, Paris, France, 5CLAD-Est, Strasbourg, France,
6CLAD-Nord-Ouest, Rouen, France, 7CLAD-Ouest, Nantes,
France, 8CLAD-Ouest, Rennes, France, 9CLAD-Il de France,
Paris, France, 10CLAD-Nord-Ouest, Lille, France, 11CLAD-
Sud-Est, Marseille, France, 12CLAD-Sud-Ouest-Occitanie-
Réunion, Montpellier, France, 13CLAD-Sud-Ouest-Occitanie-
Réunion, Bordeaux, France, 14Manchester Centre for Genomic
Medicine, Manchester, United Kingdom, 15ANPGM, Bordeaux,
France, 16ANPGM, Strasbourg, France, 17ANPGM, Paris,
France Our data suggest that fingolimod induces major transcrip-
tional changes in genes with immune and cell migration
functions; this modulation is shared between T and B
lymphocytes. Over the previous decades, geneticists have had to adapt to
the various technological and medical advances in their
field. France is preparing for the arrival of next generation
sequencing (NGS) in the care context, particularly for rare
diseases and cancer. It was therefore mandatory for clinical
geneticists (CG) to think about how they see their future
role. A survey was distributed via Google Forms to 4 dis-
tinct groups of professionals. P15.22B Consultant/Advisory
Board; Modest; Teva, Sanofi-Genzyme, Novartis, Merck,
Biogen-Idec, Excemed, Roche, Almirall, Chugai, Receptos,
Forward Pharma. F. Martinelli Boneschi: D. Speakers
Bureau/Honoraria (speakers bureau, symposia, and expert
witness); Modest; Teva, Biogen-Idec, Merck. F. Esposito:
D. Speakers Bureau/Honoraria (speakers bureau, symposia,
and expert witness); Modest; Teva, Merck. P15.22B Overall, we received 126
questionnaires from CGs, 130 from molecular biologists,
172 from medical specialists, and 20 from ERN ITHACA
coordinators. 60% of respondents think that only CGs
should prescribe NGS tests, and even 100% for the pre-
scription of exomes or genomes. Nevertheless, 80% think
that a specialist who has acquired skills in genetics could
prescribe gene panel analysis. The commonly raised issues
were the limited time dedicated to the implications of test
results, secondary data, management of VUS, and genetic
counselling. It should be noted that CGs see this primary
role in particular for polymalformative syndromes (75%),
but less systematically for nonsyndromic intellectual dis-
ability (44%), single organ diseases in adults (retinitis pig-
mentosa 24%, MODY diabetes 6%). CGs are worried about
the increase in the number of consultations, and the possible
reduced quality of patient information if growing numbers
of tests are prescribed by non-geneticists. These findings are
compared with the results of the 3 other surveys, including
the ERN, surveyed in order to learn how the transition had
been managed in other European countries. Supported by the “Fondazione Italiana Sclerosi Multipla”,
project 2013/R/13. Supported by the “Fondazione Italiana Sclerosi Multipla”,
project 2013/R/13. p
j
F. Clarelli: None. P. Provero: None. L. Ferrè: None. G. Sferruzza: None. E. Mascia: None. M. Sorosina: None. L. Moiola:
D. Speakers
Bureau/Honoraria
(speakers
bureau, symposia, and expert witness); Modest; Sanofi-
Genzyme, Biogen-Idec, Novartis, Teva. V. Martinelli: D. Speakers Bureau/Honoraria (speakers bureau, symposia,
and expert witness); Modest; Biogen-Idec, Sanofi-Gen-
zyme, Novartis, Teva, Merck, Bayer. G. Comi: D. Speakers
Bureau/Honoraria (speakers bureau, symposia, and expert
witness); Modest; Teva, Sanofi-Genzyme, Novartis, Merck,
Biogen-Idec, Excemed, Roche. F. Consultant/Advisory
Board; Modest; Teva, Sanofi-Genzyme, Novartis, Merck,
Biogen-Idec, Excemed, Roche, Almirall, Chugai, Receptos,
Forward Pharma. F. Martinelli Boneschi: D. Speakers
Bureau/Honoraria (speakers bureau, symposia, and expert
witness); Modest; Teva, Biogen-Idec, Merck. F. Esposito:
D. Speakers Bureau/Honoraria (speakers bureau, symposia,
and expert witness); Modest; Teva, Merck. F. Clarelli: None. P. Provero: None. L. Ferrè: None. G. Sferruzza: None. E. Mascia: None. M. Sorosina: None. L. Moiola:
D. Speakers
Bureau/Honoraria
(speakers
bureau, symposia, and expert witness); Modest; Sanofi-
Genzyme, Biogen-Idec, Novartis, Teva. V. Martinelli: D. Speakers Bureau/Honoraria (speakers bureau, symposia,
and expert witness); Modest; Biogen-Idec, Sanofi-Gen-
zyme, Novartis, Teva, Merck, Bayer. G. Comi: D. Speakers
Bureau/Honoraria (speakers bureau, symposia, and expert
witness); Modest; Teva, Sanofi-Genzyme, Novartis, Merck,
Biogen-Idec, Excemed, Roche. F. P15.22B Transcriptional modulation induced by fingolimod
treatment in Relapsing Remitting Multiple Sclerosis
patients J. A. Puig-Butillé1, N. Calbet2, M. Potrony3, P. Aguilera3, C. Carrera3, S. Puig3, J. Malvehy3 F. Clarelli1, P. Provero2,3, L. Ferrè1,4, G. Sferruzza1,4, E. Mascia1, M. Sorosina1, L. Moiola1, V. Martinelli4, G. Comi4, F. Martinelli Boneschi5, F. Esposito1,4 1Molecular Biology CORE, Biochemistry and Molecular
Genetics Department, Hospital Clínic, IDIBAPS, University of
Barcelona, Centro de Investigación Biomédica en Red en
Enfermedades Raras, Instituto de Salud Carlos III, Barcelona,
Spain, 2Melanoma Unit, Department of Dermatology, Hospital
Clínic de Barcelona, University of Barcelona,, Barcelona,
Spain, 3Melanoma Unit, Department of Dermatology, Hospital
Clínic de Barcelona, University of Barcelona, Centro de
Investigación Biomédica en Red en Enfermedades Raras,
Instituto de Salud Carlos III, Barcelona, Spain 1Laboratory of Human Genetics of Neurological Disorders,
CNS Inflammatory Unit & INSPE, San Raffaele Scientific
Institute, Milan, Italy, 2Department of Molecular
Biotechnology and Health Sciences, University of Turin, Turin,
Italy, 3Center for Translational Genomics and BioInformatics,
San Raffaele Scientific Institute, Milan, Italy, 4Department of
Neurology, San Raffaele Scientific Institute, Milan, Italy,
5Department of Neurology, IRCCS Policlinico San Donato,
San Donato Milanese, Milan, Italy Approximately, 50% melanomas and 65% of benign nevi
harbor the somatic alteration p.V600E in BRAF gene. Detection of this mutation in plasma (cfBRAFV600E) is
currently used for diagnosis and disease monitoring in
melanoma. However, there are no information whether host
factors may impact into cfBRAFV600E detection. In this
study, we quantified the cfBRAFV600E by ddPCR in 211
individuals
including
146
clinically
confirmed
non- Fingolimod (FTY) is a second-line drug approved for
Relapsing Remitting Multiple Sclerosis (RRMS), known to
prevent lymphocyte egress outside lymph nodes, thus
reducing peripheral lymphocytes counts. We investigated
transcriptional changes induced by the drug in immune cell J. del Picchia 552 subtypes, to better elucidate its mechanism of action at the
molecular and pathway levels. L. Demougeot1, E. Gautier1, M. Rossi2, G. Baujat3, A. Lapointe4,
E. Schaefer5, A. Goldenberg6, B. Isidor7, E. Colin8, D. Haye4, A. Verloes9, S. Marlin3, S. Manouvrier10, P. Edery2, N. Philip11, D. Geneviève12, D. Lacombe13, S. Odent8, J. Clayton-Smith14, S. Douzgou14, M. Smith14, B. Arveiller15, A. Piton16, P. Saugier-
Veber17, B. Gérard16, AnDDI-Rares, BRAIN-TEAM, Cardiogen,
DéfiScience, FAI²R, FAVA-Multi, Filfoie, Filnemus, Filslan,
Fimarad, Fimatho, Firendo, G2M, MaRiH, MCGRE, Mhémo,
Muco CFT, NeuroSphinx, Orkid, Oscar, RespiFIL, Sensgene,
Tetecou, C. Thauvin-Robinet1, L. Faivre1 L. Demougeot1, E. Gautier1, M. Rossi2, G. Baujat3, A. Lapointe4,
E. Schaefer5, A. Goldenberg6, B. Isidor7, E. Danube Hospital, Vienna, Wien, Austria p { margin-bottom: 0.1in; line-height: 120%; } Morbid
obesity is a serious epidemic that is on the rise in all parts of
the world. Roux-en-Y gastric bypass and modifications
thereof enable short-term weight reductions and improve
metabolic conditions in morbidly obese patients, but the
response varies widely between individuals. We propose
that these differences may in part be due to genetic differ-
ences. We studied a Central European population of mor-
bidly obese patients who underwent gastric bypass (n=99). Our genetic study was performed using next-generation-
sequencing (NGS) with a panel that included several genes
and polymorphisms associated with obesity and other
obesity-related phenotypes. One genetic polymorphism that
stood out in particular was a single-nucleotide polymorph-
ism (SNP, rs738409) located in the gene coding for the
palatin-like phospholipase domain-containing protein 3
(PNPLA3, adiponutrin): one in three of the obese collective
was a homozygous carrier of PNPLA3-148Met allele
compared to one in thirteen in the general population. Interestingly, the number of heterozygous carriers was
markedly lower (one in six vs. one in three) in the obese
collective. Further, heterozygous carriers had difficulties
sustaining their weight, one year post surgery. Our results
demonstrate how a genetic variant can predict the long term
benefit of a surgical procedure. Introduction: Tobacco smoking is one of the major risk
factors for many chronic diseases and is the leading cause of
preventable deaths. Varenicline was approved by the FDA
in 2006 as an aid to smoking cessation. However, not all the
smokers are benefit from the efficacy of Varenicline. Only a
few of pharmacogenetic studies were available and were
primarily European Americans or of European origin. We
aimed to assess cessation effect of nicotinic acetylcholine
receptor subunit genes on smokers receiving treatment of
Varenicline. Methods: A total of 303 current smokers receiving
Varenicline treatment were recruited from the smoking
cessation clinics and were followed up for abstinence at 3,
6, 9, 12 months. Sixty-five single nucleotide polymorph-
isms on ten nicotinic acetylcholine receptor subunit genes
were genotyped. Methods: A total of 303 current smokers receiving
Varenicline treatment were recruited from the smoking
cessation clinics and were followed up for abstinence at 3,
6, 9, 12 months. Sixty-five single nucleotide polymorph-
isms on ten nicotinic acetylcholine receptor subunit genes
were genotyped. P15.25A M. Lin: None. C. Wu: None. T. Wu: None. Y.W. Wu:
None. Y. Chang: None. C. Lai: None. K. Hsuch: None. H. Leu: None. L. Chen: None. Y.I. Tsai: None. K. Liang: None. M. Lin: None. C. Wu: None. T. Wu: None. Y.W. Wu:
None. Y. Chang: None. C. Lai: None. K. Hsuch: None. H. Leu: None. L. Chen: None. Y.I. Tsai: None. K. Liang: None. Pharmacogenetic study of the nicotinic acetylcholine
receptor subunit genes and smoking cessation among
smokers receiving Varenicline treatment M. Lin1, C. Wu1, T. Wu2, Y. W. Wu1, Y. Chang1, C. Lai3, K. Hsuch4, H. Leu5, L. Chen6, Y. I. Tsai7, K. Liang1 M. Lin1, C. Wu1, T. Wu2, Y. W. Wu1, Y. Chang1, C. Lai3, K. Hsuch4, H. Leu5, L. Chen6, Y. I. Tsai7, K. Liang1 P15.24D The evolving role of the clinical geneticist facing new
technologies: the opinion of French clinical geneticists,
molecular biologists and specialists, and the views of the
European Reference Network ITHACA Abstracts from the 51st European Society of Human Genetics Conference: Posters 553 L. Demougeot: None. E. Gautier: None. M. Rossi:
None. G. Baujat: None. A. Lapointe: None. E. Schaefer:
None. A. Goldenberg: None. B. Isidor: None. E. Colin:
None. D. Haye: None. A. Verloes: None. S. Marlin: None. S. Manouvrier: None. P. Edery: None. N. Philip: None. D. Geneviève: None. D. Lacombe: None. S. Odent: None. J. Clayton-Smith: None. S. Douzgou: None. M. Smith:
None. B. Arveiller: None. A. Piton: None. P. Saugier-
Veber: None. B. Gérard: None. C. Thauvin-Robinet:
None. L. Faivre: None. CHRNA3 rs472054 (HR=1.93, 95% C.I.: 1.06-3.51) and
rs578776 (HR=1.96, 95% C.I.: 1.09-3.51) were signifi-
cantly associated with time to smoking relapse after quitting
smoking. Conclusions: Our study demonstrated that smokers
carrying G allele of CHRNB4 rs1316971 would quit
smoking easily with Varenicline. This study was the first
report of the association between nicotinic acetylcholine
receptor
subunit
genes
and
smoking
cessation
with
Varenicline treatment in Chinese population. Grant No.:
NHRI-106-10304PI & NHRI-107-10304PI The homozygous Ile148Met Variant of PNPLA3 confers
high risk for morbid obesity in a central european
population 1Institute of Public Health, National Yang-Ming University,
Taipei, Taiwan, 2Department of Public Health, Chung-Shan
Medical University, Taichung, Taiwan, 3Department of Family
Medicine, Taipei Veterans General Hospital, Taipei, Taiwan,
4Department of Family Medicine, Kaohsiung Veterans General
Hospital, Kaohsiung, Taiwan, 5Healthcare Center, Taipei
Veterans General Hospital, Taipei, Taiwan, 6Department of
Psychiatry, Washington University, St. Louis, MO, United
States, 7Institute of Health Policy and Welfare, National Yang-
Ming University, Taipei, Taiwan A. Robubi, N. Loibner-Ott, P. Patri, A. Schmalzbauer, M.
Mansfeld, N. Vock, U. Knaack, I. Klinghofer, K. R. Huber, S.
Kriwanek, W. Krugluger A. Robubi, N. Loibner-Ott, P. Patri, A. Schmalzbauer, M. Mansfeld, N. Vock, U. Knaack, I. Klinghofer, K. R. Huber, S. Kriwanek, W. Krugluger P15.26B The homozygous Ile148Met Variant of PNPLA3 confers
high risk for morbid obesity in a central european
population The homozygous Ile148Met Variant of PNPLA3 confers
high risk for morbid obesity in a central european
population Danube Hospital, Vienna, Wien, Austria Results: We found that smokers carrying one additional
G allele of CHRNB4 rs1316971 had significantly higher
probability of abstinence (OR=1.62, 95% C.I.: 1.08-2.42;
OR=1.59, 95% C.I.: 1.06-2.39 at 6 or 9 month follow-up,
respectively)
when
receiving
Varenicline. Moreover,
rs1316971 was also found to be associated with time to
quit smoking (HR=1.33, 95% C.I.: 1.03-1.73). Besides, 554 J. del Picchia Establishment of tumor-derived organoids: an approach to
personalized medicine These cultures
have shown to mimic three-dimensional structure of the
origin tissue and could be a perfect source to determine
genetic alterations in these patients. Conclusions: Organoids can be obtained from different
cancer tissues. For their development, it is essential to
choose the right cytokines to grow them in. These cultures
have shown to mimic three-dimensional structure of the
origin tissue and could be a perfect source to determine
genetic alterations in these patients. T. Brunet: None. S. Konrad: None. M. Kiechle: None. A. Meindl: None. J. Ramser: None. E. Gross: None. T. Brunet: None. S. Konrad: None. M. Kiechle: None. A. Meindl: None. J. Ramser: None. E. Gross: None. Establishment of tumor-derived organoids: an approach to
personalized medicine 1Department of Gynecology and Obstetrics, Technische
Universität München, Munich, Germany, 2Department of
Gynecology and Obstetrics, University of Munich, Munich,
Germany 1Department of Gynecology and Obstetrics, Technische
Universität München, Munich, Germany, 2Department of
Gynecology and Obstetrics, University of Munich, Munich,
Germany M. Ovejero-Sánchez1,2,3, J. Fernández-Mateos1,2, P. Vázquez-
Cárdenas1,3, R. González-Sarmiento1,2,3 1Molecular Medicine Unit. Department of Medicine. University
of Salamanca., Salamanca, Spain, 2Institute of Biomedical
Research of Salamanca (IBSAL), Salamanca, Spain, 3Institute
of Molecular and Cellular Biology of Cancer (IBMCC). University of Salamanca-CSIC, Salamanca, Spain Introduction: The majority of patients with ovarian cancer
is still diagnosed at an advanced stage of the disease gen-
erally leading to poor outcome. With PARP inhibitors being
approved as a targeted therapy option for BRCA-mutated
advanced ovarian cancer, the concept of “BRCAness“ has
become increasingly important. “BRCAness“ describes the
phenotypic characteristics that BRCA1/2-mutated tumors
share with sporadic, non BRCA1/2 mutated tumors, such as
sensitivity to DNA double-strand break inducing agents. Our purpose was to search for other genes involved in
homologous recombination repair that might be associated
with “BRCAness“ and/or better survival. Introduction: Organoids are three-dimensional in vitro
grown structures derived from induced pluripotent stem
cells, adult stem cells or embryonic stem cells capable of
self-renewal and self-organization, that exhibit the same
organ functionality as the original tissue. They present
organ-specific differential cells types and tissue compart-
mentalization. Additionally, they can be used to detect
genetic alterations in patients. In this work we studied the
potential of this type of culture for modeling cancer. Material and Methods: We performed a qPCR-based
mRNA expression analysis of six candidate genes in 48
ovarian cancer tissues. Optimal cut-off points for gene
expression values were calculated with the Maximally
Selected Rank Statistics in R. Overall survival (OS) was
illustrated by Kaplan-Meier curves. Univariate and multi-
variate Cox regression analyses were performed with R. Material and Methods: We performed a qPCR-based
mRNA expression analysis of six candidate genes in 48
ovarian cancer tissues. Optimal cut-off points for gene
expression values were calculated with the Maximally
Selected Rank Statistics in R. Overall survival (OS) was
illustrated by Kaplan-Meier curves. Univariate and multi-
variate Cox regression analyses were performed with R. Material and Methods: Organoids from fresh tumor
tissues of 40 patients with several types of cancer (colon
(CRC), endometrial, ovary, kidney, lung, head and neck
squamous cell carcinoma) were established. Establishment of tumor-derived organoids: an approach to
personalized medicine Tumor tissues
were dissociated into functional units, seeded in Matrigel
and cultured with the appropriate medium. Material and Methods: Organoids from fresh tumor
tissues of 40 patients with several types of cancer (colon
(CRC), endometrial, ovary, kidney, lung, head and neck
squamous cell carcinoma) were established. Tumor tissues
were dissociated into functional units, seeded in Matrigel
and cultured with the appropriate medium. Results: Patients with low mRNA expression of BRCA1,
BRIP1 and RAD51C showed significantly improved OS. Moreover, BRIP1 gene expression persisted as an indepen-
dent predictive factor for OS (HR: 4.38 [CI:1.56; 12.32], p
=0.005) in multivariate Cox regression analyses. Results: We established organoids from six types of
cancer
and
determined
their
culture
conditions. The
cytokines needed for each cell type were different but all
of them include EGF, Noggin, Y-27632 and Wnt-3A. For
organoids derived from CRC, R-spondin, FGF10, FGF2,
prostaglandin E2, nicotinamide, A83-01 and gastrin are
needed. Organoids derived from kidney cancer, also need
VEGF. Those derived from lung cancer require VEGF and
progesterone and β-estradiol are necessary for organoids
derived from gynecological cancers. Discussion: Our data suggest a predictive role of BRCA1,
BRIP1 and RAD51C for the survival of ovarian cancer
patients. Results may indicate that, besides BRCA1/2,
patients with aberrant expression of BRIP1 and RAD51C
might also benefit from PARP inhibitors. Furthermore, they
contribute to the understanding of the complex concept of
“BRCAness”. To confirm the results, candidate protein
expression in tumor tissues needs to be explored. Discussion: Our data suggest a predictive role of BRCA1,
BRIP1 and RAD51C for the survival of ovarian cancer
patients. Results may indicate that, besides BRCA1/2,
patients with aberrant expression of BRIP1 and RAD51C
might also benefit from PARP inhibitors. Furthermore, they
contribute to the understanding of the complex concept of
“BRCAness”. To confirm the results, candidate protein
expression in tumor tissues needs to be explored. Conclusions: Organoids can be obtained from different
cancer tissues. For their development, it is essential to
choose the right cytokines to grow them in. These cultures
have shown to mimic three-dimensional structure of the
origin tissue and could be a perfect source to determine
genetic alterations in these patients. Conclusions: Organoids can be obtained from different
cancer tissues. For their development, it is essential to
choose the right cytokines to grow them in. P15.28D A. Robubi: None. N. Loibner-Ott: None. P. Patri:
None. A. Schmalzbauer: None. M. Mansfeld: None. N. Vock: None. U. Knaack: None. I. Klinghofer: None. K.R. Huber: None. S. Kriwanek: None. W. Krugluger: None. T. Brunet1, S. Konrad1, M. Kiechle1, A. Meindl2, J. Ramser1, E. Gross2 T. Brunet1, S. Konrad1, M. Kiechle1, A. Meindl2, J. Ramser1, E. Gross2 P15.29A The effect of inflammation-related genetic polymorphisms
on the occurrence of non-motor adverse events of
dopaminergic treatment in Parkinson's disease This project was funded by PI16/01920 M. Ovejero-Sánchez: None. J. Fernández-Mateos:
None. P. Vázquez-Cárdenas:
None. R. González-
Sarmiento: None. S. Redenšek1, M. Trošt2, V. Dolan1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 555 1Pharmacogenetics Laboratory, Institute of Biochemistry,
Faculty of Medicine, University of Ljubljana, Ljubljana,
Slovenia, 2Department of Neurology, University Medical
Centre Ljubljana, Ljubljana, Slovenia Universitat Pompeu Fabra (UPF), Barcelona, Spain, 2Institut
Hospital del Mar d'Investigacions Mèdiques (IMIM),
Barcelona, Spain, 3Pediatric Neurology Research Group, Vall
d'Hebron Research Institute, Universitat Autònoma de
Barcelona, Barcelona, Spain, 4Department of Genetic and
Molecular Medicine, Hospital Sant Joan de Déu and Institut de
Recerca Sant Joan de Déu Universitat de Barcelona,
Barcelona, Spain, 5Centro de Investigación Biomédica en Red
de Enfermedades Raras, CIBERER, Madrid, Spain, 6Secció
d’Errors Congènits del Metabolisme-IBC, Servei de
Bioquimica i Genètica Molecular, Hospital Clínic de
Barcelona, IDIBAPS, Barcelona, Spain, 7Departament de
Ciències Experimentals i de la Salut, Universitat Pompeu
Fabra, Barcelona, Spain, 8Women’s and Children’s Health
Network & University of Adelaide, Adelaide, Australia Introduction: The role of inflammation in pathogenesis of
Parkinson's disease (PD) is supported by candidate-gene
and GWAS studies. It is widely accepted that pathways
involved in disease pathogenesis may also influence treat-
ment outcome. However, the influence of genetic variability
in inflammatory pathways on treatment outcome has not
been studied in PD, yet. Our aim was to investigate the
association of selected single nucleotide polymorphisms
(SNPs) in inflammatory pathways with non-motor adverse
events (AEs) of dopaminergic treatment in PD. Materials and Methods: We enrolled 223 PD patients
and collected their demographic and clinical data including
non-motor AEs (nausea/vomiting, orthostatic hypotension,
peripheral oedema, sleep attacks, visual hallucinations, and
impulse
control
disorders). DNA
was
isolated
from
peripheral blood samples and genotyped for NLRP3
rs35829419,
CARD8
rs2043211,
IL1β
rs16944,
IL1β
rs1143623, IL6 rs1800795, and TNFα rs1800629. Statistical
analysis using logistic regression was performed. Almost 40 million people in Europe are affected by one of
the ~7000 rare diseases (RDs). Unfortunately, a significant
percentage of patients remain undiagnosed after years of
investigation, representing a real burden to the health care
system. Almost 40 million people in Europe are affected by one of
the ~7000 rare diseases (RDs). Unfortunately, a significant
percentage of patients remain undiagnosed after years of
investigation, representing a real burden to the health care
system. P15.29A The URDCat (Undiagnosed Rare Disease Program of
Catalonia) project was launched in 2017 and aims to
enable the Catalan Health System to provide personalised
genomic medicine as a fully integrated service for patients
with RDs, initially as a pilot project for RDs with
neurologic involvement. URDCat comprises a collabora-
tion between 16 groups, associated with 7 hospitals across
Catalonia. Results: The study included 58% males and 42%
females, with median age at PD onset 62 (55-71) years
and median disease duration 7.5 (4-14) years. IL1β
rs1143623 (c.-1591G>C) was associated with occurrence
of orthostatic hypotension (OH). Heterozygotes had a lower
chance of developing OH (p = 0.028; OR=0.510; 95%
CI=0.279-0.931) compared to homozygotes for wild-type
allele. This association remained significant after adjusting
for gender and disease duration (p = 0.026; OR=0.500;
95% CI=0.272-0.920). No other SNP showed any associa-
tion with other studied AEs. The project has several overarching objectives: (i)
standardise the process of analysis and integration of
clinical and genomic data; (ii) implement a platform for
analysis of genomics data that is suitable for clinical
practice; (iii) identify causative genetic variants of undiag-
nosed RD patients; (iv) highlight the utility of genomics,
and advance its usage as a tool of personalised medicine for
RD diagnostics and (v) educate stakeholders in the value of
genomic data. Conclusions: Our pilot study indicates a link between
inflammatory pathways and OH as AE of dopaminergic
treatment in PD. Further studies are needed to explain the
detected association. Deep phenotyping data has been collated from more than
750 families with a customised PhenoTips form using HPO,
ORDO and OMIM. Over 220 associated genomic datasets
have been re-analysed in detail using the URDCat platform,
based on RD-Connect. Pathogenic or candidate causative
variants have been identified for almost 20% of the re-
analysed cases, which are currently being followed up in the
laboratory. In order to solve remaining cases, whole exome
or genome sequencing has been undertaken for around 500
prioritised index cases. Grant: ARRS, grant P1-0170 Grant: ARRS, grant P1-0170 S. Redenšek: None. M. Trošt: None. V. Dolan: None. S. Redenšek: None. M. Trošt: None. V. Dolan: None. 1CNAG-CRG, Centre for Genomic Regulation (CRG),
Barcelona Institute of Science and Technology (BIST), A. Macaya3, F. Palau4,5, A. Ribes6,5, L. Pérez-Jurado7,8,5, S.
Beltran1, URDCat Consortium T. Blagus1, S. Soldat1, M. Goričar1, K. Goričar1, S. Redenek1, V.
Dolan1, L. Konta2, Ubiquitos Pharmacogenomics Consortium T. Blagus1, S. Soldat1, M. Goričar1, K. Goričar1, S. Redenek1, V. Dolan1, L. Konta2, Ubiquitos Pharmacogenomics Consortium 1Institute of Biochemistry, Ljubljana, Slovenia, 2bio.logis
Zentrum fur Humangenetik, Frankufurt am Main, Germany 1Institute of Biochemistry, Ljubljana, Slovenia, 2bio.logis
Zentrum fur Humangenetik, Frankufurt am Main, Germany Introduction: In 2015 European Union approved Eliglustat
as first-line oral treatment for adult patient with type 1
Gaucher disease. It is important for these patients to geno-
type the gene that encode the main metabolizer enzyme of
this drug (CYP2D6). The clinical trials of Eliglustat shows
that the concomitant use of this drug with CYP3A inducers
is not recommended in this patients. For this reason, it is
also important to know the genotype of the other genes that
are involved in metabolism of this drug such as CYP3A4
and ABCB1 genes. Introduction: The Horizon 2020 UPGx (Ubiquitous Phar-
macogenomics:
www.upgx.eu)
project
aims
to
make
actionable pharmacogenomics data and effective treatment
optimization accessible to every European citizen in order
to improve patient treatment outcome and consequently
lower the expenses of the treatment. According to the
existing
data
published
and
Dutch
Pharmacogenetics
Working Group (https://www.pharmgkb.org/page/dpwg),
the guidelines made by the UPGx Consortium revealed that
actionable drug prescribing for 55 drugs is most strongly
linked to 13 pharmacogenes (www.pharmgkb.org/cpic/pa
irs). One of the aims of the project was to implement genetic
testing for a panel of pharmacogenes to support a clinical
study on pre-emptive pharmacogenomic testing in seven
European countries, including Slovenia. Material and Methods: DNA samples of 61 Spanish
patients with type 1 Gaucher disease were genotyped using
the xTAG® CYP2D6 kit v3 protocol for CYP2D6 gene,
PCR- RFLP for CYP3A4*1B and ABCB1 haplotype, and
RT-PCR for CYP3A4*22. Material and Methods: DNA samples of 61 Spanish
patients with type 1 Gaucher disease were genotyped using
the xTAG® CYP2D6 kit v3 protocol for CYP2D6 gene,
PCR- RFLP for CYP3A4*1B and ABCB1 haplotype, and
RT-PCR for CYP3A4*22. Results: The metabolizer status of CYP2D6 was 4.9%,
13.1% and 82% for poor, intermediate and extensive,
respectively. The genotype of CYP3A4*22 showed that
94.8% of patients were wild-type for this SNP and 5.2%
heterozygous. The SNPs of ABCB1 gene (rs1045642,
rs2032582, rs1128503) related with low expression of P-
glyprotein are infrequent in our cohort. This is important
because
it
prevents
the
eliglustat
accumulation
in
the brain. T. Blagus1, S. Soldat1, M. Goričar1, K. Goričar1, S. Redenek1, V.
Dolan1, L. Konta2, Ubiquitos Pharmacogenomics Consortium Methods: 1.Setting up a panel of common functional
variants within pharmacogenes with major influence on
interpatient variability in drug response and existing
evidence based treatment guidelines. 2.Setting up the
infrastructure and validation of methodology for prospec-
tive genotyping for selected variants. Results: In total 48 common functional SNPs along with
the CYP2D6 deletion and duplication were identified as
targets
for
pharmacogenetics
testing. Real-time
PCR
fluorescence-based endpoint genotyping (KASPar) was
chosen as the genotyping method and SNPline (LCG
Group) genotyping system was set up at the Pharmacoge-
netics Laboratory. The respective KASPar assays were
designed by LCG Group and Biologis and introduced,
validated and tested at the Pharmacogenetics Laboratory. LR-PCR amplification followed by SNP genotyping was
used to detect CYP2D6 polymorphisms. Conclusions: In the patients who have ultra-rapid
genotype for CYP2D6, the use of Eliglustat is not approved. For the rest of patients it is important to have genotyping
information about CYP3A4 and ABCB1 and to assess
concomitant use of medications and herbal supplements to
ensure a precision personalized medicine. A. Almeida: None. J. Concha Mayayo: None. E. Sangüesa Sangüesa: None. L. López de Frutos: None. B. Medrano Engay: None. M. Andrade Campos: None. M. P. Giraldo Castellano: None. M.P. Ribate Molina: None. C.B. García García: None. Conclusions: We have successfully implemented the
genotyping methodology and infrastructure for pre-emptive
pharmacogenomics testing within the UPGx Project. P15.30B The Undiagnosed Rare Disease Program of Catalonia
(URDCat) - illustrating the value of NGS for personalised
medicine The Undiagnosed Rare Disease Program of Catalonia
(URDCat) - illustrating the value of NGS for personalised
medicine L. Matalonga1, D. Ovelleiro1, S. Laurie1, M. Gut1, M. Pujadas2,
A. Macaya3, F. Palau4,5, A. Ribes6,5, L. Pérez-Jurado7,8,5, S. Beltran1, URDCat Consortium L. Matalonga1, D. Ovelleiro1, S. Laurie1, M. Gut1, M. Pujadas2,
A. Macaya3, F. Palau4,5, A. Ribes6,5, L. Pérez-Jurado7,8,5, S. Beltran1, URDCat Consortium L. Matalonga1, D. Ovelleiro1, S. Laurie1, M. Gut1, M. Pujadas2,
A. Macaya3, F. Palau4,5, A. Ribes6,5, L. Pérez-Jurado7,8,5, S. Beltran1, URDCat Consortium L. Matalonga: None. D. Ovelleiro: None. S. Laurie:
None. M. Gut: None. M. Pujadas: None. A. Macaya:
None. F. Palau: None. A. Ribes: None. L. Pérez-Jurado:
None. S. Beltran: None. 1CNAG-CRG, Centre for Genomic Regulation (CRG),
Barcelona Institute of Science and Technology (BIST), 556 J. del Picchia P15.34B Acknowledgements: Horizon 2020 UPGx Project: Grant
No 668353. Pharmacogenetics of everolimus in the treatment of the
rare disease tuberous sclerosis Pharmacogenetics of everolimus in the treatment of the
rare disease tuberous sclerosis T. Blagus: None. S. Soldat: None. M. Goričar: None. K. Goričar: None. S. Redenek: None. V. Dolan: None. L. Konta: None. J. Concha Mayayo1, E. Sangüesa Sangüesa1, J. L. Peña
Segura2, M. P. Ribate Molina1, C. B. García García1 P15.33APharmacogenetics of Eliglustat in Gaucher disease J. Concha Mayayo1, E. Sangüesa Sangüesa1, J. L. Peña
Segura2, M. P. Ribate Molina1, C. B. García García1 P15.31C A. Almeida1,2, J. Concha Mayayo1, E. Sangüesa Sangüesa1, L. López de Frutos2, B. Medrano Engay2, M. Andrade Campos2, M. P. Giraldo Castellano2, M. P. Ribate Molina1, C. B. García
García1 1Faculty of health sciences, San Jorge University, Villanueva
de Gallego, Spain, 2Grupo de Enfermedades Hematológicas y
Metabólicas, Instituto Investigación Sanitaria Aragón (IIS
Aragón). CIBERER: Instituto Carlos III, Zaragoza, Spain P15.33APharmacogenetics of Eliglustat in Gaucher disease Introduction:
Everolimus
is
an
immunosuppressant
recently accepted to treat adjacent symptoms to the rare
disease tuberous sclerosis (or Bourneville syndrome), as
renal angiomyolipoma and giant cell astrocytoma. The
prevalence of this pathology is between 1:6000 and
1:10000. Most genes have shown to be close relationated
with pharmacokinetics and pharmacodynamics of ever-
olimus, so their polymorphisms can cause a great inter-
individual variability in its plasmatic levels. The narrow
therapeutic window of this drug generates the risk of
occurrence adverse drug effects or lack of effectiveness. The Single Nucleotide Polymorphisms (SNP) analyzed to
study its genotype-phenotype relation are CYP3A5*3
(6986 A>G), CYP3A4*1B (g-290 A>G), CYP3A4*22
(15389
C>T),
ABCB1
(c.1236C>T),
ABCB1
(c.2677G>T), ABCB1 (c.3435C>T), PXR (c.44477T>C),
PXR (c.63396C>T), PXR (c.69789A>G), CYP2C8*3 (416
G>A), CYP2C8*3’ (1196 A>G) and CYP2C8*4 (792
C>G). Materials and Methods: Pharmacy students of the
subject “Pharmacogenetics and Pharmacogenomics” have
selected the most important information about the different
sections of the guideline: glossary of terms, basic concepts
about
pharmacokinetics
and
pharmacodynamics,
main
genes involved in metabolism, transport and receptors of
drugs with pharmacogenetic relevance and frequently asked
questions. The
content
is
revised
and
corrected
by
Pharmacy students of “Pharmacology” and “Pharmacoki-
netics” subjects. In order to get an attractive aspect of the
guideline, Architecture students of “Analysis of Architec-
tonic forms” develop its graphic design. Materials and Methods: Pharmacy students of the
subject “Pharmacogenetics and Pharmacogenomics” have
selected the most important information about the different
sections of the guideline: glossary of terms, basic concepts
about
pharmacokinetics
and
pharmacodynamics,
main
genes involved in metabolism, transport and receptors of
drugs with pharmacogenetic relevance and frequently asked
questions. The
content
is
revised
and
corrected
by
Pharmacy students of “Pharmacology” and “Pharmacoki-
netics” subjects. In order to get an attractive aspect of the
guideline, Architecture students of “Analysis of Architec-
tonic forms” develop its graphic design. Material and Methods: A total of 8 voluntary patients
who suffer tuberous sclerosis in treatment with everolimus
participate in this study. Blood samples were collected in
FTATMcards. RFLP-PCR has been performed to genotype
the selected SNPs, being CYP3A4*22 genotyped by RT-
PCR. Material and Methods: A total of 8 voluntary patients
who suffer tuberous sclerosis in treatment with everolimus
participate in this study. Blood samples were collected in
FTATMcards. RFLP-PCR has been performed to genotype
the selected SNPs, being CYP3A4*22 genotyped by RT-
PCR. P15.33APharmacogenetics of Eliglustat in Gaucher disease Results: After the edition period, the guidelines will be
distributed to the different pharmacies of the area close to
San
Jorge
University. Its
usefulness
will
be
tested
among them. Results: The TTT haplotype of ABCB1, CYP3A5*3,
CYP2C8*3, CYP2C8*4 and CYP3A4*22 would increase
the drug levels and thus produce a higher risk of adverse
effects. On the other hand, the CYP3A4*1B and PXR SNPs
would decrease drug levels and thus produce lack of
therapeutic effectiveness. Conclusions: It is hoped that through this activity,
knowledge about pharmacogenetics will be accessible for
pharmacists so that this science could be more understood. Conclusions: It is hoped that through this activity,
knowledge about pharmacogenetics will be accessible for
pharmacists so that this science could be more understood. C.B. García: None. E. Sanguesa: None. J. Concha:
None. L. Lomba: None. V. López: None. J.N. Gutiérrez:
None. A. Estepa: None. M.P. Ribate: None. C.B. García: None. E. Sanguesa: None. J. Concha:
None. L. Lomba: None. V. López: None. J.N. Gutiérrez:
None. A. Estepa: None. M.P. Ribate: None. Conclusions: The genotyping of patients treated with
everolimus
is
a
useful
tool
to
predict
and
explain
interindividual dose variations and thus offer relevant
information to improve the treatment of patients. Conclusions: The genotyping of patients treated with
everolimus
is
a
useful
tool
to
predict
and
explain
interindividual dose variations and thus offer relevant
information to improve the treatment of patients. Pharmacogenomic recommendations based on different
sequencing and genotyping platforms: challenges and
solutions for using existing guidelines Pharmacogenomic recommendations based on different
sequencing and genotyping platforms: challenges and
solutions for using existing guidelines J. Concha Mayayo: None. E. Sangüesa Sangüesa:
None. J.L. Peña Segura: None. M.P. Ribate Molina:
None. C.B. García García: None. K. Krebs1,2, S. Reisberg3,4,5, M. Kals1, R. Mägi1, J. Vilo3,4,5, L. Milani1,6 K. Krebs1,2, S. Reisberg3,4,5, M. Kals1, R. Mägi1, J. Vilo3,4,5, L.
Milani1,6 P15.33APharmacogenetics of Eliglustat in Gaucher disease Abstracts from the 51st European Society of Human Genetics Conference: Posters 557 1Faculty of health sciences, San Jorge University, Villanueva
de Gallego, Spain, 2Hospital Universitario Miguel Servet,
Zaragoza, Spain Introduction: Pharmacogenetics is a relatively new science
that has not been studied widely in health sciences degree
programmes. Nevertheless, it is considered as a tool that
contributes to precision medicine since it provides relevant
information about the patients and could predict the effec-
tiveness of the drugs or the occurrence of adverse drug
reactions. Health Sciences professionals, mainly the phar-
macists, should know the clinical applications of this area of
knowledge or at least they should understand its main
concepts and basic vocabulary. For this reason, the first aim
of this project was to develop a guideline about Pharma-
cogenetics for pharmacists who have not received any for-
mation about it. Introduction: Pharmacogenetics is a relatively new science
that has not been studied widely in health sciences degree
programmes. Nevertheless, it is considered as a tool that
contributes to precision medicine since it provides relevant
information about the patients and could predict the effec-
tiveness of the drugs or the occurrence of adverse drug
reactions. Health Sciences professionals, mainly the phar-
macists, should know the clinical applications of this area of
knowledge or at least they should understand its main
concepts and basic vocabulary. For this reason, the first aim
of this project was to develop a guideline about Pharma-
cogenetics for pharmacists who have not received any for-
mation about it. Introduction:
Everolimus
is
an
immunosuppressant
recently accepted to treat adjacent symptoms to the rare
disease tuberous sclerosis (or Bourneville syndrome), as
renal angiomyolipoma and giant cell astrocytoma. The
prevalence of this pathology is between 1:6000 and
1:10000. Most genes have shown to be close relationated
with pharmacokinetics and pharmacodynamics of ever-
olimus, so their polymorphisms can cause a great inter-
individual variability in its plasmatic levels. The narrow
therapeutic window of this drug generates the risk of
occurrence adverse drug effects or lack of effectiveness. The Single Nucleotide Polymorphisms (SNP) analyzed to
study its genotype-phenotype relation are CYP3A5*3
(6986 A>G), CYP3A4*1B (g-290 A>G), CYP3A4*22
(15389
C>T),
ABCB1
(c.1236C>T),
ABCB1
(c.2677G>T), ABCB1 (c.3435C>T), PXR (c.44477T>C),
PXR (c.63396C>T), PXR (c.69789A>G), CYP2C8*3 (416
G>A), CYP2C8*3’ (1196 A>G) and CYP2C8*4 (792
C>G). Strengthening pharmacogenetics 1Estonian Genome Center, Institute of Genomics,University of
Tartu, Tartu, Estonia, 2Institute of Molecular and Cell Biology,
University of Tartu, Tartu, Estonia, 3Institute of Computer
Science, University of Tartu, Tartu, Estonia, 4Software
Technology and Applications Competence Centre, Tartu,
Estonia, 5Quretec Ltd, Tartu, Estonia, 6Science for Life
Laboratory, Department of Medical Sciences, Uppsala,
Sweden Strengthening pharmacogenetics C. B. García1, E. Sanguesa1, J. Concha1, L. Lomba1, V. López1,
J. N. Gutiérrez2, A. Estepa2, M. P. Ribate1 C. B. García1, E. Sanguesa1, J. Concha1, L. Lomba1, V. López1,
J. N. Gutiérrez2, A. Estepa2, M. P. Ribate1 1Faculty of Health Science, San Jorge University, Villanueva
de Gállego-Zaragoza, Spain, 2School of Architecture, San
Jorge University, Villanueva de Gállego-Zaragoza, Spain 558 J. del Picchia differences. Despite the evidence for pharmacogenomic
(PGx) tests to provide precision medicine, clinical use is
still limited. We conducted a study on the prevalence of
drug-gene-interactions (DGIs) in the Dutch Lifelines Cohort
(www.lifelines.nl) and are exploring the practical barriers
and success factors of pre-emptive PGx-testing in a
clinical pilot. Introduction: The past decade has shown an increase in the
amount of genetic information that could be translated into
clinically actionable decisions tailoring medical therapy. The Clinical Pharmacogenetics Implementation Consortium
has prepared curated genotype-based guidelines for trans-
lating genetic information into drug prescription recom-
mendations for 14 genes associated with 36 different drugs. Here, we investigated how successfully we could translate
existing genotype data into informative recommendations. Introduction: The past decade has shown an increase in the
amount of genetic information that could be translated into
clinically actionable decisions tailoring medical therapy. The Clinical Pharmacogenetics Implementation Consortium
has prepared curated genotype-based guidelines for trans-
lating genetic information into drug prescription recom-
mendations for 14 genes associated with 36 different drugs. Here, we investigated how successfully we could translate
existing genotype data into informative recommendations. Methods: Genotypes in Lifelines were identified using
Illumina
CytoSNP12
array
data
and
GoNL
(www. nlgenome.nl) imputed, translated to PGx star-alleles and
predicted phenotypes following national PGx guidelines. Due to the absence of copy number variance (CNV),
genotypes for CYP2D6 were not available. DGIs were
estimated using Lifelines questionnaire’s medication his-
tory. In addition to the Lifelines study, a clinical pilot
(n=300) is being conducted in our hospital’s outpatient
clinics. The prevalence of DGIs will also be assessed for
this clinical cohort. Strengthening pharmacogenetics Materials and Methods: We surveyed the genotype data
of 44 448 Estonian Biobank participants: 2420 with whole
genome sequence (WGS), 2445 with whole exome sequen-
cing (WES), 8132 genotyped on the HumanOmniExpress
beadchip (OMNI), and 33157 on the Global Screening
Array (GSA) from Illumina. The genotype data obtained on
both arrays were phased and imputed. For detecting star
alleles, we used gene-specific star allele definition tables
from the PharmGKB database. Results: 12.792 individuals, 95,5% of the genotyped
Lifelines Cohort, have at least one clinically relevant PGx
variant. Prescription drug use was linked to the genetic data
in 6.415 (47,9%) individuals where 435 DGIs were
identified amongst 420 individuals (6,55%). We observed
similar prevalence in the preliminary data of the clinical
cohort. Results: The greatest challenge that we faced was large
allele definition tables that do not appear to cover all
possible allelic combinations, and limited guidance on how
to prioritize the variants in cases of multiple or no matching
star alleles. When comparing the proportion of multiple
matching star alleles, WGS clearly stands out with 2.8%
ambiguous calls, followed by 32% for GSA, 35% for
OMNI and 61% for WES as several important variants fall
outside the coding region of the genome. Conclusions: PGx variants and estimated DGIs are
abundantly present in the Lifelines Cohort. Prevalence is
expected to become even higher when CYP2D6 genotypes
are taken into account and when DGIs are identified based
on drug prescription data which will become available in
the near future. Conclusions: Translating existing genotype and WGS
data into pharmacogenomic recommendations for biobank
participants
illustrates
the
importance
and
innovative
potential of curated guidelines: a crucial step in advancing
the whole process of personalised medicine in general. P. Lanting: None. B.R. Comello: None. F. van Dijk:
None. B. Wilffert: None. R.H. Sijmons: None. P. Lanting: None. B.R. Comello: None. F. van Dijk:
None. B. Wilffert: None. R.H. Sijmons: None. K. Krebs: None. S. Reisberg: None. M. Kals: None. R. Mägi: None. J. Vilo: None. L. Milani: None. K. Krebs: None. S. Reisberg: None. M. Kals: None. R. Mägi: None. J. Vilo: None. L. Milani: None. P15.38B
SLCO1B1 c.521T>C genotype, sex, and initial statin
treatment as contributing factors to continuous simvastatin
or atorvastatin treatment in a Dutch cohort M. E. Jansen1,2, T. Rigter1,2, T. M. C. Fleur1,3, P. Souverein3, S.
J. H. Vijverberg3,4, W. Rodenburg2, M. C. Cornel1 1VU University Medical Center, Section Community Genetics,
Department of Clinical Genetics and Amsterdam Public Health
research institute, Amsterdam, Netherlands, 2National Institute
for Public Health and the Environment, Centre for Health
Protection, Bilthoven, Netherlands, 3Utrecht University,
Division of Pharmacoepidemiology & Clinical Pharmacology,
Utrecht Institute of Pharmaceutical Sciences, Utrecht,
Netherlands, 4Amsterdam Medical Center and Amsterdam
Public Health research institute, Department of Pulmonology,
Amsterdam, Netherlands P15.37A P15.37A
The prevalence of predicted drug-gene-interactions in
12.792 individuals from the Dutch Lifelines Cohort The prevalence of predicted drug-gene-interactions in
12.792 individuals from the Dutch Lifelines Cohort M. E. Jansen1,2, T. Rigter1,2, T. M. C. Fleur1,3, P. Souverein3, S. J. H. Vijverberg3,4, W. Rodenburg2, M. C. Cornel1 P. Lanting1, B. R. Comello2, F. van Dijk1, B. Wilffert2,3, R. H. Sijmons1 P. Lanting1, B. R. Comello2, F. van Dijk1, B. Wilffert2,3, R. H. Sijmons1 1University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands,
2University of Groningen, Groningen Research Institute of
Pharmacy, Department of PharmacoTherapy, -Epidemiology
& -Economics, Groningen, Netherlands, 3University of
Groningen, University Medical Center Groningen, Department
of Clinical Pharmacy and Pharmacology, Groningen,
Netherlands 1VU University Medical Center, Section Community Genetics,
Department of Clinical Genetics and Amsterdam Public Health
research institute, Amsterdam, Netherlands, 2National Institute
for Public Health and the Environment, Centre for Health
Protection, Bilthoven, Netherlands, 3Utrecht University,
Division of Pharmacoepidemiology & Clinical Pharmacology,
Utrecht Institute of Pharmaceutical Sciences, Utrecht,
Netherlands, 4Amsterdam Medical Center and Amsterdam
Public Health research institute, Department of Pulmonology,
Amsterdam, Netherlands Introduction: (Adverse) effects of drug treatment for
similar
indications
can
show
striking
interindividual 559 Abstracts from the 51st European Society of Human Genetics Conference: Posters Introduction: Statins reduce low-density-lipoprotein cho-
lesterol levels and lower the risk for cardiovascular events. Although statins are generally well-tolerated, patients can
experience adverse drug events (ADE), and discontinue
their treatment. In this study we aim to investigate if and
how pre-emptive pharmacogenomic SLCO1B1 c.521T>C
screening would have influenced the statin treatment out-
comes for patients in primary care. Introduction: Statins reduce low-density-lipoprotein cho-
lesterol levels and lower the risk for cardiovascular events. Although statins are generally well-tolerated, patients can
experience adverse drug events (ADE), and discontinue
their treatment. In this study we aim to investigate if and
how pre-emptive pharmacogenomic SLCO1B1 c.521T>C
screening would have influenced the statin treatment out-
comes for patients in primary care. chromosome 15q11-q13 region. We aim to improve the
quality of life and healthcare of patients and families
with PWS. Method: PWS patients under 18 had been coordinated in
SJD-Hospital between March 2016 and December 2017. Multidisciplinary medical following is needed due to the
multisystemic involvement. American Academy of Pedia-
tric recommendations were followed and coordinated by a
Case Manager (CM). We assess patients’ experience,
consultation adherence and extraprotocolized needs. Methods: The amount of ADE experienced by 1159
patients treated with statins in a Dutch cohort were
measured
through three
proxies: mean
dose
change,
discontinuing treatment or switching statins and, time to
establish stable dosing. Information on their statin use was
evaluated. For every outcome, patients were compared on
their SLCO1B1 genotype, sex, and type of statin. Analyses
were corrected for relevant confounders. Results: 37 patients were attended coordinately 2-3 times
per year at the hospital, thanks to the consecutive-
concentrated visits and scheduled coordination of the CM. Lost days of work and school absences were reduced. Meetings with all the involved physicians to discuss the best
personalized therapeutic attitudes were scheduled at the end
of the day. CM and the Social Worker were also included. The degree of satisfaction of the family increased in
all cases. Results: At baseline, female simvastatin users received a
lower dose compared to male simvastatin users. Regardless
of determinants, approximately half of patients discontinued
their statin treatment within three years follow-up of starting
their treatment. The proxies for ADE showed no statistically
significant differences between SLCO1B1 genotypes TT
versus TC and CC, men versus women or simvastatin
versus atorvastatin. P15.40D Experience within the UPGx project M.E. Jansen: None. T. Rigter: None. T.M.C. Fleur:
None. P. Souverein: None. S.J.H. Vijverberg: None. W. Rodenburg: None. M.C. Cornel: None. V. Dolzan1, T. Blagus1, S. Redenšek1, K. Goricar1, G. Mlinek2, . Vivod Pečnik3, B. Mazej Poredo3, A. krinjar3, A. Zidanek3, B. Rajter3, A. Čau3, K. Jeras4, M. Saje4, J. Klen5, M. Juterek6, M. Kolek uterič3, A. Poplas Susič3, M. Prica3, T. Stegne Ignjatovič3,
R. Jakopič lahtič3, T. Terzič4, M. Vrbnjak4, M. Bokalič4, L. Bukovec4, M. Rus Makovec4, S. Kokot6, S. Doblekar7, R. Verboten Kopriva8, B. Ejupi6, B. Kores Plesničar4, Ubiquitous
Pharmacogenomics Consortium V. Dolzan1, T. Blagus1, S. Redenšek1, K. Goricar1, G. Mlinek2, . Vivod Pečnik3, B. Mazej Poredo3, A. krinjar3, A. Zidanek3, B. Rajter3, A. Čau3, K. Jeras4, M. Saje4, J. Klen5, M. Juterek6, M. Kolek uterič3, A. Poplas Susič3, M. Prica3, T. Stegne Ignjatovič3,
R. Jakopič lahtič3, T. Terzič4, M. Vrbnjak4, M. Bokalič4, L. Bukovec4, M. Rus Makovec4, S. Kokot6, S. Doblekar7, R. Verboten Kopriva8, B. Ejupi6, B. Kores Plesničar4, Ubiquitous
Pharmacogenomics Consortium V. Dolzan1, T. Blagus1, S. Redenšek1, K. Goricar1, G. Mlinek2, . Vivod Pečnik3, B. Mazej Poredo3, A. krinjar3, A. Zidanek3, B. Rajter3, A. Čau3, K. Jeras4, M. Saje4, J. Klen5, M. Juterek6, M. Kolek uterič3, A. Poplas Susič3, M. Prica3, T. Stegne Ignjatovič3,
R. Jakopič lahtič3, T. Terzič4, M. Vrbnjak4, M. Bokalič4, L. Bukovec4, M. Rus Makovec4, S. Kokot6, S. Doblekar7, R. Verboten Kopriva8, B. Ejupi6, B. Kores Plesničar4, Ubiquitous
Pharmacogenomics Consortium Conclusions: The PWS functional plan improves the
patient’s care. CM plays an essential role to coordinate the
assistance of each patient to interfere as less as possible in
their lives: 1) grouping consultations to the hospital
avoiding unnecessary journeys, 2) coordinating with other
areas such as school or primary care to ensure continuity of
care and 3) collaborating with other specialties for a global
management of the patient and their families. Conclusions:
The
results
indicate
that
SLCO1B1
c.521T>C screening would not have contributed to less
discontinuation and switches in simvastatin and atorvastatin
prescription for patients in our Dutch regional cohort. Factors that do contribute to approximately half of patients
discontinuing their statin treatment should be studied further
to gain insight in determinants of unsuccessful implementa-
tion of statin treatment.Grant: RIVM (S/132001/01/PD),
Project: “Personalised medicine: eligible or not?” about
eligiblility of pharmacogenomics in primary care M. Bolasell Girgas: None. A. Martinez-Monseny:
None. M. Serrano: None. C. Arjona: None. M. Jabalera:
None. A. Bosque: None. F. Palau: None. New horizons for Prader-Willi patient’s assistance:
families’ centered health care New horizons for Prader-Willi patient’s assistance:
families’ centered health care M. Bolasell Girgas, A. Martinez-Monseny, M. Serrano, C. Arjona, M. Jabalera, A. Bosque, F. Palau Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain DNA
samples from patients were genotyped for a panel of 50
genetic variants in 13 pharmacogenes. Within 3-5 working
days genotype data and DPWG guidelines were available to
physicians to modify the treatment with the index drug if
recommended. Treatment outcomes were followed for
12 weeks. The most commonly prescribed index drugs
were statins (40%), tacrolimus (20%) and antidepressants
(23%). The number of polymorphic pharmacogenes was
from 1 to 8 per patient, with the average of 4.2. In total 74
patients (50%) carried polymorphic CYP2D6, which is the
gene with the highest number of therapeutic recommenda-
tions. In 30.5% of patients the therapeutic recommendations
included the index drug. The 2016 BBMRI-LPC WES Call offered a unique free-of-
charge opportunity to genetically diagnose rare disease
patients with biological samples deposited within the
EuroBioBank network. 800 whole exomes were sequenced
from 17 distinct projects, each having 2-3 principal inves-
tigators from different countries. The projects spanned a
wide range of rare disease phenotypes, including neuro-
muscular disorders, inborn errors of metabolism, albinism,
and sudden cardiac depth, amongst others, and informed
consent had to permit data sharing for research purposes
through controlled access repositories such as the EGA
(https://ega.crg.eu/) and RD-Connect (http://rd-connect.eu/). Clinical and phenotypic information for every case was
collected in RD-Connect’s customised PhenoTips instance,
using standards such as the Human Phenotype Ontology
(HPO), OMIM and Orpha codes, and sequencing under-
taken at the CNAG in Spain, and the WTSI in the UK. Sequencing data was processed using the RD-Connect
standard analysis pipeline and results made available
through the RD-Connect Genome-Phenome Analysis Plat-
form (https://platform.rd-connect.eu/) once all call require-
ments had been met. The Platform allows researchers to
analyse and interpret their genotype:phenotype data pri-
vately for up to 6 months before it is shared with other
authorised users. It also facilitates anonymised data sharing
through APIs from initiatives such as the GA4GH/IRDiRC
MatchMaker Exchange (http://www.matchmakerexchange. org/) and Beacon Network (https://beacon-network.org). We report on the challenges and lessons learned from
conducting such a complex transnational collaborative
initiative and present an up-to-date diagnostic yield of the
project as a whole, with some illustrative success stories. Conclusions: Within the U-PGx project and the PRE-
PARE study panel-based pre-emptive pharmacogenomic
was successfully introduced in Slovenia. Acknowledgements: Horizon 2020 UPGx Project – grant
No 668353. V. Dolzan: None. T. Blagus: None. S. Redenšek: None. K. Goricar: None. G. Mlinšek: None. . Vivod Pečnik:
None. B. Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain Mazej Poredo: None. A. krinjar: None. A. Zidanek: None. B. Rajter: None. A. Čau: None. K. Jeras:
None. M. Saje: None. J. Klen: None. M. Juterek: None. M. Kolek uterič: None. A. Poplas Susič: None. M. Prica:
None. T. Stegne Ignjatovič: None. R. Jakopič lahtič:
None. T. Terzič: None. M. Vrbnjak: None. M. Bokalič:
None. L. Bukovec: None. M. Rus Makovec: None. S. Kokot: None. S. Doblekar: None. R. Verboten Kopriva:
None. B. Ejupi: None. B. Kores Plesničar: None. V. Dolzan: None. T. Blagus: None. S. Redenšek: None. K. Goricar: None. G. Mlinšek: None. . Vivod Pečnik:
None. B. Mazej Poredo: None. A. krinjar: None. A. Zidanek: None. B. Rajter: None. A. Čau: None. K. Jeras:
None. M. Saje: None. J. Klen: None. M. Juterek: None. M. Kolek uterič: None. A. Poplas Susič: None. M. Prica:
None. T. Stegne Ignjatovič: None. R. Jakopič lahtič:
None. T. Terzič: None. M. Vrbnjak: None. M. Bokalič:
None. L. Bukovec: None. M. Rus Makovec: None. S. Kokot: None. S. Doblekar: None. R. Verboten Kopriva:
None. B. Ejupi: None. B. Kores Plesničar: None. Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain 1University of Ljubljana, Faculty of Medicine, Institute of
Biochemistry, Pharmacogenetics Laboratory, Ljubljana,
Slovenia, 2University Medical Center, Ljubljana, Slovenia,
3Health Care Center Ljubljana, Ljubljana, Slovenia,
4University Psychiatric Clinic Ljubljana, Ljubljana, Slovenia,
5Health Care Center Kočevje, Kočevje, Slovenia, 6Health Care
Center Litija, Litija, Slovenia, 7Health Care Center Litija,, Background: Prader-Willi Syndrome (PWS) is a genetic
condition with recognizable pattern of physical findings
with significant neurocognitive, endocrine and behavioral
abnormalities. It is caused by lack of expression of genes
from an imprinted region of the paternally inherited 560 J. del Picchia S. Laurie1, S. Beltran1, M. Bayes1, B. Fuste1, M. Gut1, L. Matalonga1, D. Piscia1, J. Dawson2, R. Thompson2, E. López-
Martín3, M. Posada3, L. Monaco4, C. Wang4, G. B. van Ommen5,
S. Sims6, E. Zeggini6, H. Löchmuller2, I. Gut1, BBMRI-LPC
consortium Litja, Slovenia, 8Health Care Center Litija, Slovenia;, Litija,
Slovenia Introduction: The clinical study PREPARE within the
Horizon2020
Ubiquitous
Pharmacogenomics
(U-PGx)
project (www.upgx.eu) aims to establish if implementation
of PGx-guided drug prescribing for a panel of drug-
pharmacogene
pairs
reduces
drug-genotype
associated
adverse drug reactions (ADRs) in comparison to patients
receiving standard of care treatment in seven European
countries, including Slovenia. 1Centro Nacional de Análisis Genómico (CNAG-CRG), Center
for Genomic Regulation; Barcelona Institute of Science and
Technology (BIST); Universitat Pompeu Fabra (UPF),
Barcelona, Spain, 2Institute of Genetic Medicine, MRC Centre
for Neuromuscular Diseases, Newcastle, United Kingdom,
3Institute of Rare Diseases Research, IIER-ISCIII; Centre for
Biomedical Network Research on Rare Diseases, CIBERER,
Madrid, Spain, 4Fondazione Telethon, Milano, Italy,
5Department of Human Genetics, Leiden University Medical
Center, Leiden, Netherlands, 6Wellcome Trust Sanger Institute,
Hinxton, United Kingdom Methods and Results: Within a prospective, block-
randomized, controlled clinical study, Slovenia was rando-
mized to start with PGx-guided prescribing (study arm) and
will switch to standard of care (control arm) after 18 months. Methods and Results: Within a prospective, block-
randomized, controlled clinical study, Slovenia was rando-
mized to start with PGx-guided prescribing (study arm) and
will switch to standard of care (control arm) after 18 months. Methods and Results: Within a prospective, block-
randomized, controlled clinical study, Slovenia was rando-
mized to start with PGx-guided prescribing (study arm) and
will switch to standard of care (control arm) after 18 months. Up to now, 150 patients participated in the study. They
were invited when having a drug with DPWG guideline
(index drug) first prescribed in the routine care. Novel candidate gene associated with mTOR inhibitor
response in Renal Cell Carcinoma Conclusions: We propose a novel candidate gene
associated with mTORi response, which may help to select
patients sensitive to mTORi therapy. J. Roldán-Romero1, M. Santos-Romero1, J. Maroto2, G. Anguera2, F. Algaba3, S. Ruiz-Llorente4, C. Montero-Conde1, A. Cascon1,5, G. Roncador6, N. Djouder7, M. Robledo1,5, C. Rodriguez-Antona1,5 J. Roldán-Romero: None. M. Santos-Romero: None. J. Maroto: None. G. Anguera: None. F. Algaba: None. S. Ruiz-Llorente: None. C. Montero-Conde: None. A. Cascon: None. G. Roncador: None. N. Djouder: None. M. Robledo: None. C. Rodriguez-Antona: None. 1Hereditary Endocrine Cancer Group, Human Cancer
Genetics Programme, Spanish National Cancer Research
Centre (CNIO), Madrid, Spain, 2Department of Medical
Oncology, Hospital de la Santa Creu I Sant Pau, Barcelona,
Spain, 3Section of Pathology, Fundació Puigvert-Universitat
Autonoma de Barcelona, Barcelona, Spain, 4CNIO-Lilly
Epigenetics Laboratory, Spanish National Cancer Research
Center (CNIO), Madrid, Spain, 5Centro de Investigación
Biomédica en Red de Enfermedades Raras (CIBERER),
Madrid, Spain, 6Monoclonal Antibodies Core Unit,
Biotechnology Programme, Spanish National Cancer Research
Centre (CNIO), Madrid, Spain, 7Growth Factors, Nutrients
and Cancer Group, Cancer Cell Biology Programme, Spanish
National Cancer Research Centre (CNIO), Madrid, Spain P15.41A P15.41A
800 exomes for rare disease research: outcomes of the
transnational BBMRI-LPC WES call in collaboration with
EuroBioBank and RD-Connect P15.41A
800 exomes for rare disease research: outcomes of the
transnational BBMRI-LPC WES call in collaboration with
EuroBioBank and RD-Connect S. Laurie: None. S. Beltran: None. M. Bayes: None. B. Fuste: None. M. Gut: None. L. Matalonga: None. D. Piscia: None. J. Dawson: None. R. Thompson: None. E. Abstracts from the 51st European Society of Human Genetics Conference: Posters 561 López-Martín: None. M. Posada: None. L. Monaco:
None. C. Wang: None. G. B. van Ommen: None. S. Sims:
None. E. Zeggini: None. H. Löchmuller: None. I.
Gut: None. TCGA (2 out of 66). MTT assays revealed that depletion of
the protein through shRNAs, or treatment with an specific
small-molecule inhibitor, sensitized HeLa cells to rapamy-
cin. mTOR pathway activation, measured by p-S6/p-S6K1/
p-AKT, was not altered in shRNA-treated cells, suggesting
mTORi sensitizing effects occurred through alternative
effectors. TCGA (2 out of 66). MTT assays revealed that depletion of
the protein through shRNAs, or treatment with an specific
small-molecule inhibitor, sensitized HeLa cells to rapamy-
cin. mTOR pathway activation, measured by p-S6/p-S6K1/
p-AKT, was not altered in shRNA-treated cells, suggesting
mTORi sensitizing effects occurred through alternative
effectors. Framework for clinical assessment of Mendelian inherited
risk alleles O. Senol-Cosar1,2, D. C. Hoskinson1,3, M. Lebo1,2, H. Mason-
Suares1,2 1Laboratory for Molecular Medicine/Partners Personalized
Medicine, Cambridge, MA, United States, 2Harvard Medical
School/Brigham and Women Hospital, Boston, MA, United
States, 3Tempus Labs, Chicago, IL, United States There is an increased interest and demand in clinical
genomics to interpret and return Mendelian-inherited risk
alleles to the electronic medical records. Although this is
emerging as part of routine clinical practice, there is no
formal terminology nor a framework for classification of
these low-penetrant variants. Therefore, we established an
evidence-based framework to clinically classify risk alleles
into three categories recommended by ACMG: established
risk allele, likely risk allele, and uncertain risk allele. Evi-
dence used for risk allele classification was collected by a
thorough literature search for case/control studies, meta-
analyses, functional data, and included cohort size, pheno-
typing, statistical significance, and replication of results
over time. For example, to be classified as an established
risk allele, a variant must meet the following criteria: 1)
significant results after Bonferroni, false discovery rate or
permutation correction; 2) odds ratio > 2; 3) results repli-
cated in at least 5 studies by different laboratories using
underlying cohorts, including through meta-analyses. To
test this method, we classified seven well-established risk
alleles all of which met our criteria for established risk
variant. In addition, we classified five previously reported
risk alleles. Of these, two were established risk alleles, one
was likely risk allele, and two were uncertain risk alleles. This framework establishes a standardized approach to Introduction: Inhibitors of the mammalian target of rapa-
mycin (mTORi) are used to treat several cancers, including
renal cell carcinoma (RCC). Although response to these
drugs can be partially explained by mutations activating
mTOR pathway, the majority of responders have no
mutations identified. This suggests that additional genes
may contribute to mTORi response. In this study, genomic
screening of chromophobe RCC (chRCC) patients sensitive
to mTORi, together with in vitro functional studies, iden-
tified a novel candidate predictive gene. Materials and Methods: Whole exome sequencing
(WES) was applied to three chRCC patients sensitive to
mTORi. An independent chRCC series was collected for
targeted sequencing and immunohistochemistry (IHC) of
candidate genes. Rapamycin sensitivity and mTOR pathway
activation was assessed in cells with shRNA-mediated gene
silencing. Materials and Methods: Whole exome sequencing
(WES) was applied to three chRCC patients sensitive to
mTORi. Framework for clinical assessment of Mendelian inherited
risk alleles del Picchia Center, American Hospital of Paris, Neuilly, France, 21Service
de Génétique, CHU Angers, Angers, France, 22Centre
PREDIR, Hôpital de Bicêtre, AP-HP, Paris-Sud University, Le
Kremlin-Bicêtre, France, 23UMR 7268-ADÉS, Faculté de
Médecine de Marseille, Aix-Marseille Université-EFS-CNRS,
Marseille, France, 24Oncology, Hopital Pitie-Salpetriere,
Paris, France, 25Département de Génétique Assistance
Publique-Hôpitaux de Paris, Hôpital Européen Georges
Pompidou, Paris, France, 26Department of Digestive
Oncology, Beaujon University Hospital, AP-HP and University
Paris 7 – Denis Diderot, Clichy, France, 27Institut de
Cancérologie de Lorraine Alexis Vautrin, Nancy, France,
28Clinique Beau Soleil, EA2415, Association française
d’urologie, Montpellier, France, 29ICFuro, intergroupe
coopérateur francophone de recherche en onco-urologie,
Paris, France, 30UMR 1027, Inserm, Université Toulouse III-
Paul Sabatier, Toulouse, France, 31Plateforme Sociétale
Genotoul, 37 allées Jules Guesde, Toulouse, France,
32Psychoanalytic Psychopathology, Department of Psychology,
Strasbourg University, Strasbourg, France, 33Laboratoire de
Biopathologie Cellulaire et Tissulaire des Tumeurs, CHU
Montpellier, Montpellier, France, 34Réseau TenGen, Paris,
France, 35Laboratoire d’Ethique Médicale et Médecine Légale
EA4569, Faculté de Médecine, Université Paris Descartes,
Paris, France, 36Zorn & Associés law firm, Strasbourg,
France, 37APHP, INSERM U938, IUC-UPMC, Sorbonne
Université, Paris, France, 38Department of Radiation
Oncology, Radiobiology Unit, Biometric and Bio-informatic
Divisions, Montpellier Cancer Institute (ICM), IRCM, INSERM
U1194, Montpellier, France, 39Département de Biologie et
Pathologie Médicales, Gustave Roussy, Université Paris-
Saclay, Villejuif, France, 40Université Paris Descartes, PRES
Sorbonne Paris Cité, Faculté de Médecine, Paris, France,
41INSERM, UMR970, Paris-Cardiovascular Research Center,
Paris, France, 42Institut Gustave-Roussy, department of
medical oncology, Villejuif cedex, France, 43Institut Français
du Sein, 15 rue Jean Nicot, Paris, France, 44Department of
Urology, Tenon Academic Hospital, Assistance Publique-
Hôpitaux de Paris, Pierre et Marie Curie Medical School,
Sorbonne Universités, Paris, France, 45Groupe de recherche
clinique-UPMC n°5, Oncotype-Uro, Institut Universitaire de
Cancérologie de l’UPMC, Pierre and Marie Curie Medical
School, Sorbonne Universités, Paris, France, 46Réseau
TenGen, France, Paris, France, 47Laboratoire de Génétique
Moléculaire, CHU Lyon, Lyon, France, 48Ethique médicale –
EA 4569 – Université Paris Descartes, Sorbonne Paris Cité,
Faculté de Médecine, Coordination Associations Filières de
Santé AnDDI-Rares, VML (Vaincre les Maladies Lysosomales)
association, Paris, France, 49Assistance Publique-Hôpitaux de
Marseille, Centre Hospitalier Universitaire la Timone, Service
d’Oncologie Medicale (P N ) Marseille France
50CHU classifying risk variants with high individual odds ratios and
provides clinicians and patients with important, con-
textualizable information. This process will be incorporated
into our laboratory for additional risk allele interpretations
and will be extended to a similar framework for protective
alleles. O. Senol-Cosar: None. D.C. Hoskinson: None. M. Lebo: None. H. Framework for clinical assessment of Mendelian inherited
risk alleles Mason-Suares: None. Framework for clinical assessment of Mendelian inherited
risk alleles An independent chRCC series was collected for
targeted sequencing and immunohistochemistry (IHC) of
candidate genes. Rapamycin sensitivity and mTOR pathway
activation was assessed in cells with shRNA-mediated gene
silencing. Results: WES revealed one single mutated gene, not
related to mTOR pathway, shared by the three chRCC
tumors. Two of the somatic mutations were loss-of-
function, one was a misssense variant. IHC staining and
Sanger sequencing validated the results. Inactivation of the
gene was detected at low frequency in an independent
chRCC cohort (3 out of 96 tumors), in agreement with 562 J. P15.44D Guidelines for reporting secondary findings of genome
sequencing in cancer genes: the SFMPP recommendations P. Pujol1,2, P. Van de Perre1,3, L. Faivre4, D. Sanlaville5,6,7, C. Corsini1, B. Baertschi8,9, M. Anahory10, D. Vaur11,12, S. Olschwang13,14,15, N. Soufir16,17, N. Bastide18, S. Amar10,19, M. Vintraud20, O. Ingster21, S. Richard22, P. Le Coz23, J. Spano24, O. Caron25, P. Hammel26, E. Luporsi27, A. Toledano20, X. Rebillard28,29, A. Cambon-Thomsen30,31, O. Putois32, J. Rey33,34,
C. Hervé35, C. Zorn36, K. Baudry1, V. Galibert1, J. Gligorov37, D. Azria38, B. Bressac-De Paillerets39, N. Burnichon40,25,41, M. Spielmann42, D. Zarca43, I. Coupier1, O. Cussenot44,45, A. Gimenez-Roqueplo46,25, S. Giraud46,47, A. Lapointe48, P. Niccoli49, I. Raingeard50, M. Le Bidan51, T. Frebourg52, A. Rafii53, D. Geneviève54 1Department of Cancer Genetics, University of Montpellier
and University Hospital (CHU), Montpellier, France,
2Université Montpellier 1, Montpellier, France, 3Université
Toulouse III Paul Sabatier, Toulouse, France, 4Fédération
Hospitalo-Universitaire Médecine Translationnelle et
Anomalies Du Développement (TRANSLAD), Centre
Hospitalier Universitaire Dijon, Dijon, France, 5Department
of Genetics, Lyon University Hospitals, Lyon, France, 6Lyon
Neuroscience Research Centre, CNRS UMR5292, Inserm
U1028, Lyon, France, 7Claude Bernard Lyon I University,
Lyon, France, 8INSERM Ethics Committee, Paris, France,
9University of Geneva, Geneva, Switzerland, 10Pech de
Laclause, Bathmanabane & Associés law firm, Paris, France,
11Department of Cancer Biology and Genetics, CLCC
François Baclesse, Normandy Centre for Genomic and
Personalized Medicine, Caen, France, 12INSERM U1079-IRIB,
Normandy Centre for Genomic and Personalized Medicine,
University of Rouen, Rouen, France, 13Aix Marseille
Université, INSERM GMGF UMR S_910, Marseille, France,
14Département de Génétique Médicale, Hôpital d’enfants de la
Timone, Marseille, France, 15Groupe Ramsay Générale de
Santé, Hôpital Clairval, Marseille, France, 16Department of
Genetics, Bichat Hospital, Paris, France, 17INSERM U976
Saint-Louis Hospital, Paris, France, 18BRCA France
association, Paris, France, 19Cancer Immunotherapy Lab,
Neurosurgery Department, Tel-Aviv Medical Center, Tel Aviv,
Israel, 20Department of radiotherapy, Hartmann Radiotherapy P15.45A
Secondary findings on multigene panels: A new frontier for
clinical utility in hereditary cancer genes E. D. Esplin1, S. Yang1, E. Haverfield1, S. T. Michalski1, J. Sotelo2, L. Kipnis2, A. Chittenden2, J. Stopfer2, K. Schneider2, R. Sacca2, S. Stickevers2, D. Koeller2, S. Gaonkar2, E. Vaccari2, S. Cochrane2, W. Espinel3, M. Champine3, J. Jeter4, K. Sweet4, R. Pilarski4, R. Pearlman4, K. Shane4, P. Brock4, J. Westman4, H. Hampel4, S. E. Lincoln1, S. Aradhya1, R. L. Nussbaum1 Large-scale genomic sequencing for clinical purposes has
led to an increase discovery of secondary findings (SF). The
increasing use of multi-gene panel analysis to explore
familial cancer predisposition and of tumor genome analysis
rise important questions regarding SF in cancer suscept-
ibility genes. The American College of Medical Genetics
and Genomics (ACMG) published a policy statement for SF
management for a list of genes including 29 cancer genes. There are currently no equivalent recommendations in
Europe. From June 2016 to May 2017, the French Society
of Predictive and Personalized Medicine (SFMPP) establish
a working group included 47 experts to elaborate guidelines
about management of SF in cancer genes presented here. A
first workgroup including ethicists, lawyers, representatives
of patients’ associations, and psychologists, provides ethical
reflection, information guidelines and information materials
(written consent form and video). A second group dedicated
to medical expertise including oncologists, clinical and
molecular geneticists provides independent evaluation and
classification of 60 genes. The main criterions were the
“actionability” (access to validate screening or prevention
strategies), the risk evaluation (severity, penetrance, and
disease’s age of onset), and the level of evidence from
published data. Genes were divided into 3 subgroups: class
1 for which it is recommended to return the results (n=28),
class 2 for which the restitution remains questionable
(n=18) and class 3 for which it is not recommended to
return the result (n=14). These first European guidelines on
SF in cancer predisposing genes may help clinicians and
laboratories to standardize and guide practices. 1Invitae, San Francisco, CA, United States, 2Dana Farber
Cancer Institute, Boston, MA, United States, 3Huntsman
Cancer Institute, Salt Lake City, UT, United States, 4The Ohio
State University, Columbus, OH, United States Medically actionable secondary findings in whole exome/
genome sequencing (WES/WGS) are reported independent
of WES/WGS indication. Multigene panels can cover 100s
of genes and reveal hereditary cancer gene secondary
findings. P15.45A
Secondary findings on multigene panels: A new frontier for
clinical utility in hereditary cancer genes We report the prevalence of secondary findings
identified via multigene panel in 3000 individuals of mul-
tiple ethnicities undergoing cardiovascular genetic testing
and the clinical utility of these cancer-risk gene findings.We
analyzed de-identified data for 47 cancer-risk genes in 3679
patients referred for hereditary cardiovascular genetic test-
ing. Frameshift, nonsense, and splice-site disruptions were
classified as pathogenic variants (PV).We observed 141
PVs in cancer-risk genes, for a prevalence of 6.03%,
including PVs with established management guidelines in
ATM, BRCA1, BRCA2, CHEK2, MUTYH, PALB2, PMS2. The prevalence of secondary findings by ethnicity in our
cohort was: African American (1.73%), Asian (8%), His-
panic (4.98%), Caucasian (6.43%).Multigene panel cancer-
risk secondary findings prevalence is higher than studies
using WES/WGS. The distribution of findings by ethnicity
suggests pathogenic missense variants from lower pre-
valence populations are underrepresented in current data-
bases, and the true prevalence of secondary findings is
underreported. Follow-on analysis assessed the clinical
utility of cancer-risk gene panels in breast cancer patients. In 80% of breast cancer cases clinical management for
patients and/or their family members was changed based on
PVs in ATM, CHEK2, MUTYH, PALB2, PMS2 and others,
impacting the outcome of almost half of patients. These data
support the clinical utility of PVs in cancer-risk genes and
suggest a role for multigene panels in the detection of
clinically actionable secondary findings. g
p
P. Van de Perre: None. L. Faivre: None. D. Sanlaville:
None. C. Corsini:
None. B. Baertschi:
None. M. Anahory: None. D. Vaur: None. S. Olschwang: None. N. Soufir: None. N. Bastide: None. S. Amar: None. M. Vintraud: None. O. Ingster: None. S. Richard: None. P. Le Coz: None. J. Spano: None. O. Caron: None. P. Hammel: None. E. Luporsi: None. A. Toledano: None. X. Rebillard: None. A. Cambon-Thomsen: None. O. Putois: None. J. Rey: None. C. Hervé: None. C. Zorn:
None. K. Baudry: None. V. Galibert: None. J. Gligorov:
None. D. Azria: None. B. Bressac-De Paillerets: None. N. Burnichon: None. M. Spielmann: None. D. Zarca: None. I. Coupier: None. O. Cussenot: None. A. Gimenez-
Roqueplo: None. S. Giraud: None. A. Lapointe: None. P. P. Van de Perre: None. L. Faivre: None. D. Sanlaville:
None. C. Corsini:
None. B. Baertschi:
None. M. Anahory: None. D. Vaur: None. S. Olschwang: None. N. Soufir: None. N. Bastide: None. S. Amar: None. M. Vintraud: None. O. Ingster: None. S. Richard: None. P. Le Coz: None. J. Spano: None. O. 563 Abstracts from the 51st European Society of Human Genetics Conference: Posters APTEPF association, Paris, France, 52Department of
Genetics, Rouen University Hospital, Normandy Centre for
Genomic and Personalized Medicine, Rouen, France,
53Department of Genetic Medicine, Weill-Cornell Medical
College, New York, NY, United States, 54Department of
Genetics, University of Montpellier and University Hospital
(CHU), Montpellier, France APTEPF association, Paris, France, 52Department of
Genetics, Rouen University Hospital, Normandy Centre for
Genomic and Personalized Medicine, Rouen, France,
53Department of Genetic Medicine, Weill-Cornell Medical
College, New York, NY, United States, 54Department of
Genetics, University of Montpellier and University Hospital
(CHU), Montpellier, France Niccoli: None. I. Raingeard: None. M. Le Bidan: None. T. Frebourg: None. A. Rafii: None. D. Geneviève: None. P15.45A
Secondary findings on multigene panels: A new frontier for
clinical utility in hereditary cancer genes Caron: None. P. Hammel: None. E. Luporsi: None. A. Toledano: None. X. Rebillard: None. A. Cambon-Thomsen: None. O. Putois: None. J. Rey: None. C. Hervé: None. C. Zorn:
None. K. Baudry: None. V. Galibert: None. J. Gligorov:
None. D. Azria: None. B. Bressac-De Paillerets: None. N. Burnichon: None. M. Spielmann: None. D. Zarca: None. I. Coupier: None. O. Cussenot: None. A. Gimenez-
Roqueplo: None. S. Giraud: None. A. Lapointe: None. P. E.D. Esplin:
A. Employment
(full
or
part-time);
Significant; Invitae. E. Ownership Interest (stock, stock E.D. Esplin:
A. Employment
(full
or
part-time);
Significant; Invitae. E. Ownership Interest (stock, stock E.D. Esplin:
A. Employment
(full
or
part-time);
Significant; Invitae. E. Ownership Interest (stock, stock 564 J. del Picchia options, patent or other intellectual property); Modest;
Invitae. S. Yang: A. Employment (full or part-time);
Significant; Invitae. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest;
Invitae. E. Haverfield: A. Employment (full or part-time);
Significant; Invitae. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest;
Invitae. S.T. Michalski: A. Employment (full or part-time);
Significant; Invitae. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest;
Invitae. J. Sotelo: None. L. Kipnis: None. A. Chittenden:
None. J. Stopfer: None. K. Schneider: None. R. Sacca:
None. S. Stickevers:
None. D. Koeller:
None. S. Gaonkar: None. E. Vaccari: None. S. Cochrane: None. W. Espinel: None. M. Champine: None. J. Jeter: None. K. Sweet: None. R. Pilarski: None. R. Pearlman: None. K. Shane: None. P. Brock: None. J. Westman: None. H. Hampel: None. S.E. Lincoln: None. S. Aradhya: A. Employment (full or part-time); Significant; Invitae. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; Invitae. R.L. Nussbaum: A. Employment (full or part-time); Significant; Invitae. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; Invitae. workflow does not necessitate upfront library preparation,
and instead converts the captured molecules into libraries
compatible with Illumina sequencing. We have designed
and optimized baits specific to the full exonic content of a
growing library genes associated with cancer, neurological
disorders, autism, cardiac disease, and other conditions. These are designed, balanced, and pooled on a per gene
basis, and can be combined into customized panels,
allowing rapid turnaround of specific custom gene subsets. Novel approach enables rapid deployment of high quality,
flexible content target enrichment panels J. Barry1, K. M. Patel2, A. B. Emerman2, S. Adams2, S. Bowman2, E. Mauceli2, B. Textor3, C. Elfe2, F. Stewart1, E. Dimalanta1, S. Russello1, T. B. Davis1, C. L. Hendrickson2
1New England Biolabs, Ipswich, MA, United States, 2Directed
Genomics, Ipswich, MA, United States, 3New England Biolabs,
GmbH, Ipswich, MA, United States J. Barry1, K. M. Patel2, A. B. Emerman2, S. Adams2, S. Bowman2, E. Mauceli2, B. Textor3, C. Elfe2, F. Stewart1, E. Dimalanta1, S. Russello1, T. B. Davis1, C. L. Hendrickson2 1New England Biolabs, Ipswich, MA, United States, 2Directed
Genomics, Ipswich, MA, United States, 3New England Biolabs,
GmbH, Ipswich, MA, United States 1New England Biolabs, Ipswich, MA, United States, 2Directed
Genomics, Ipswich, MA, United States, 3New England Biolabs,
GmbH, Ipswich, MA, United States P15.45A
Secondary findings on multigene panels: A new frontier for
clinical utility in hereditary cancer genes Here, we present the ability to rapidly deploy custom gene
panels across a variety of panel sizes and content, while
maintaining high specificity, uniformity of coverage across
target content, and sensitivity to detect nucleic acid variants
from clinically relevant samples. y
p
A.J. Barry: A. Employment (full or part-time); Sig-
nificant; New England Biolabs. K.M. Patel: A. Employ-
ment (full or part-time); Significant; Directed Genomics. A. B. Emerman: A. Employment (full or part-time); Sig-
nificant; New England Biolabs. S. Adams: A. Employment
(full or part-time); Significant; Directed Genomics. S. Bowman: A. Employment (full or part-time); Significant;
Directed Genomics. E. Mauceli: A. Employment (full or
part-time); Significant; Directed Genomics. B. Textor: A. Employment (full or part-time); Significant; New England
Biolabs. C. Elfe: A. Employment (full or part-time);
Significant; Directed Genomics. F. Stewart: A. Employ-
ment (full or part-time); Significant; New England Biolabs. E. Dimalanta: A. Employment (full or part-time); Sig-
nificant; New England Biolabs. S. Russello: A. Employ-
ment (full or part-time); Significant; New England Biolabs. T.B. Davis: A. Employment (full or part-time); Significant;
New England Biolabs. C.L. Hendrickson: A. Employment
(full or part-time); Significant; Directed Genomics. Targeted high-throughput sequencing in thyroid cancer
diagnostics Efficient utilization of targeted gene panels for clinical
research is challenged by the wide variation in gene con-
stituents specific to a given study. While focused gene
panels efficiently provide the necessary depth of coverage
for low frequency variant detection, the high costs and
technical requirements associated with panel design present
challenges. The NEBNext Direct technology utilizes a
novel approach to selectively enrich nucleic acid targets
ranging from a single gene to several hundred genes,
without sacrificing specificity. The approach rapidly hybri-
dizes both strands of genomic DNA with biotinylated
probes
prior
to streptavidin bead capture, enzymatic
removal of off-target sequences, and conversion of captured
molecules into sequence-ready libraries. This results in an
exceptionally uniform coverage profile for a given target. Unlike
alternative
hybridization
methods,
this
1-day V. Yakushina1,2, L. Lerner3, T. Kazubskaya4, T. Kondrat’ieva4,
A. Lavrov1,5 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology,
Moscow, Russian Federation, 3PreMed-European
Technologies, Moscow, Russian Federation, 4N.N. Blokhin
Russian Cancer Research Center, Moscow, Russian
Federation, 5Russian National Research Medical University,
Moscow, Russian Federation 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology,
Moscow, Russian Federation, 3PreMed-European
Technologies, Moscow, Russian Federation, 4N.N. Blokhin
Russian Cancer Research Center, Moscow, Russian
Federation, 5Russian National Research Medical University,
Moscow, Russian Federation Introduction:
Identification
of
driver
and
secondary
mutations with targeted high-throughput sequencing is a
promising approach in diagnosis and management of thyr-
oid cancer. 565 Abstracts from the 51st European Society of Human Genetics Conference: Posters Materials and Methods: Point mutations, fusions and
CNV known to be associated with sporadic thyroid cancer
were selected from COSMIC and TCGA project data. Additionally, exons of RET gene harboring germline
mutations were included. Design of primers for targeted
amplification of regions with pathogenic point mutations
was done with AmpliSeq Designer v. 5.4.1, the length of
amplicons was set in range of 125-375 b.p. The technical
validation was performed on cell lines SW620, MCF7. FNA
samples of papillary and follicular carcinomas (15 samples)
and surgical samples of normal thyroid tissue (11 samples)
were analyzed. Analytical sensitivity was assessed via
introduction of DNA with known mutations into DNA of
normal tissue, which did not harbor pathogenic mutations. Materials and Methods: Point mutations, fusions and
CNV known to be associated with sporadic thyroid cancer
were selected from COSMIC and TCGA project data. Additionally, exons of RET gene harboring germline
mutations were included. Targeted high-throughput sequencing in thyroid cancer
diagnostics Design of primers for targeted
amplification of regions with pathogenic point mutations
was done with AmpliSeq Designer v. 5.4.1, the length of
amplicons was set in range of 125-375 b.p. The technical
validation was performed on cell lines SW620, MCF7. FNA
samples of papillary and follicular carcinomas (15 samples)
and surgical samples of normal thyroid tissue (11 samples)
were analyzed. Analytical sensitivity was assessed via
introduction of DNA with known mutations into DNA of
normal tissue, which did not harbor pathogenic mutations. intelligence, and a 5-year-old girl with moderate mental
retardation of unknown cause, speech delay and light skin,
hair and eyes were tested with the TruSight One gene panel
on a MiSeq platform. Consequently, the younger brother of
the WS patient, who had bilateral deafness, pigmentation
changes, CNS malformations and borderline IQ, was tested
by targeted amplicon NGS sequencing for the mutation
detected in his relative. The family of the older brother
subsequently decided to undergo preimplantation genetic
diagnosis. Four day-3 embryos were biopsied and tested for
carrier status of the mutation through targeted NGS. intelligence, and a 5-year-old girl with moderate mental
retardation of unknown cause, speech delay and light skin,
hair and eyes were tested with the TruSight One gene panel
on a MiSeq platform. Consequently, the younger brother of
the WS patient, who had bilateral deafness, pigmentation
changes, CNS malformations and borderline IQ, was tested
by targeted amplicon NGS sequencing for the mutation
detected in his relative. The family of the older brother
subsequently decided to undergo preimplantation genetic
diagnosis. Four day-3 embryos were biopsied and tested for
carrier status of the mutation through targeted NGS. Results: Despite their wide phenotypic variability all
three patients were found to be heterozygous carriers of the
same non-sense mutation in PAX3: Chr2:g.223160283T>A,
NM_181459.3:c.415A>T, NP_852124.1:p.Lys139Ter. Per-
forming PGD we established that two of the four embryos
tested were not carriers and were suitable for transfer. Results: The design included over 450 point mutations in
25 genes, 3 regions of CNVs and 25 types of fusions. Mutations expected in cell lines (according COSMIC CLP
and Cancer Cell Line Encyclopedia) were detected: APC p. Q1338* and KRAS p.G12V – in SW620; PIK3CA p.E545K
– in MCF7. The panel is highly sensitive and successfully
identifies as low as 3% of alleles. In PTC samples following
mutations were detected: BRAF p.V600E (5 samples);
KRAS
p.G12V;
fusions
ETV6-NTRK3,
PAX8-PPARG,
NCOA4-RET. Observation of wide phenotypic variability in 3 individuals
with the same pathogenic mutation in PAX3 Observation of wide phenotypic variability in 3 individuals
with the same pathogenic mutation in PAX3 Targeted high-throughput sequencing in thyroid cancer
diagnostics In the sample with ETV6-NTRK3, secondary
mutation IDH1 p.V178I was found. Supported by The
Foundation for Assistance to Small Innovative Enterprises
in Science and Technology (№442ГС2/9119 - С3-40846). V Y k
hi
N
L L
N
T K
b Conclusions: Variable expressivity complicates not only
the clinical identification of a particular syndrome and
subtype, but also makes genetic counselling in regard to
prenatal diagnosis challenging and risky. Performing PGD
gives the opportunity to avoid the moral dilemma whether
to keep the embryo in conditions where the phenotype
could vary from debilitating to less severe. L.T. Balabanski: None. R.V. Vazharova: None. S. Ivanov: None. M. Atanasoska: None. A. Kalchev: None. G. Nenkova: None. S. Dzhuneva: None. M. Malinov:
None. D. Toncheva: None. V. Yakushina: None. L. Lerner: None. T. Kazubs-
kaya: None. T. Kondratieva: None. A. Lavrov: None. V. Yakushina: None. L. Lerner: None. T. Kazubs-
kaya: None. T. Kondratieva: None. A. Lavrov: None. Whole Genome Sequencing in healthy people Whole Genome Sequencing in healthy people SAMPLE ID GENE/ MUTATION Results: At baseline, both CPD and eCO were associated
with polymorphisms in the CHRNA5 locus (rs503464,
rs55853698, rs55781567 and rs16969968; P < 0.01). rs503464, a variant in the 5′-UTR of CHRNA5, was also
associated with 1-, 3- and 12-month responses to therapy (P
= 0.011, P = 0.0043, P = 0.020, respectively), although
after correction for multiple testing only the association at
the 3-month assessment remained significant. Conclusions: These data support the role of individual
genetic makeup in the ability to quit smoking. Conclusions: These data support the role of individual
genetic makeup in the ability to quit smoking. In
part
supported
by
the
5x1000
contribution
of
Fondazione IRCCS Istituto Nazionale dei Tumori, Milan. F. Colombo is recipient of a Fondazione Umberto Veronesi
fellowship. In
part
supported
by
the
5x1000
contribution
of
Fondazione IRCCS Istituto Nazionale dei Tumori, Milan. F. Colombo is recipient of a Fondazione Umberto Veronesi
fellowship. Conclusions: WGS should be the method of choice not
only in cases with designated phenotype but also in the
context of carrier screening. G. Pintarelli: None. A. Galvan: None. P. Pozzi: None. S. Noci: None. G. Pasetti: None. F. Sala: None. U. Pastorino: None. R. Boffi: None. F. Colombo: None. G. Pintarelli: None. A. Galvan: None. P. Pozzi: None. S. Noci: None. G. Pasetti: None. F. Sala: None. U. Pastorino: None. R. Boffi: None. F. Colombo: None. M. Chatzidaki: None. G. Thodi: None. Y. L. loukas:
None. E. Molou:
None. O. Triantafilli:
None. Y. Dotsikas: None. K. Schoulpis: None. M. Chatzidaki: None. G. Thodi: None. Y. L. loukas:
None. E. Molou:
None. O. Triantafilli:
None. Y. Dotsikas: None. K. Schoulpis: None. M. CHATZIDAKI1,2, G. THODI3, Y. L. LOUKAS1,2, E.
MOLOU3, O. TRIANTAFILLI3, Y. DOTSIKAS1, K.
SCHOULPIS4 L. T. Balabanski1,2, R. V. Vazharova1,3, S. Ivanov1, M. Atanasoska1, A. Kalchev1, G. Nenkova1, S. Dzhuneva1, M. Malinov1, D. Toncheva1,2 1Department of Pharmacy, National and Kapodistrian
University of Athens, ATHENS, Greece, 2NEOSCREEN LTD,
Athens, Greece, 3NEOSCREEN LTD, ATHENS, Greece,
4Department of Mental Health and Social Welfare, Hospital
Paidon “Agia Sofia”, ATHENS, Greece 1Department of Pharmacy, National and Kapodistrian
University of Athens, ATHENS, Greece, 2NEOSCREEN LTD,
Athens, Greece, 3NEOSCREEN LTD, ATHENS, Greece,
4Department of Mental Health and Social Welfare, Hospital
Paidon “Agia Sofia”, ATHENS, Greece 1Gynecology and Assisted Reproduction Hospital “Malinov
MD”, Sofia, Bulgaria, 2Department of Medical Genetics,
Medical University of Sofia, Sofia, Bulgaria, 3Sofia University
St Kliment Ohridski, Faculty of Medicine, Department of
Biology, Medical genetics and Microbiology, Sofia, Bulgaria Introduction: Whole genome sequencing (WGS) provides
the most comprehensive collection of an individual’s
genetic variations compared to the human reference gen-
ome. WGS can be a useful diagnostic and research tool, as it
allows the detection of pathogenic and susceptibility var-
iants enabling the diagnosis of an associated syndrome as
well as the identification of variants explaining the observed
genetic diversity. Introduction: The PAX3 gene encodes an important tran-
scription factor regulating the expression of many genes and
playing a crucial role during foetal development. Patho-
genic mutations in this gene lead to autosomal dominant
forms
of
Waardenburg
syndrome
and
Craniofacial-
deafness-hand syndrome. Methods and Materials: A 29-year-old male diagnosed
with Waardenburg syndrome, who exhibited unilateral
deafness,
pigmentation
changes
and
above-average Purpose: The purpose of this study was to evaluate the
clinical significance of WGS in healthy people regarding the
detection of mutations associated with clinical disorders, 566 J. del Picchia predisposition to various clinical conditions and response
to drugs. fraction of smokers. There is growing evidence that genetic
variations in nicotinic acetylcholine receptor (nAChR)
subunits influence the risk of nicotine dependence and the
ability to quit smoking. Materials/Methods: 50 healthy people were subjected to
WGS conducted on BGISEQ-500 sequencing platform
which offers high quality data with average coverage 30x. The vcf files were filtered based on the following exclusive
criteria MAF≥1% and assignment by ClinVar as benign/
likely benign. Materials and Methods: 337 smokers who underwent
pharmacotherapy with varenicline, bupropion, nicotine
replacement therapy (NRT) alone, or NRT plus bupropion
were genotyped for 7 germline polymorphisms in genes
encoding the nAChR subunits CHRNA4, CHRNA5, and
CHRNB2 by pyrosequencing. Pharmacogenetic study of seven polymorphisms in three
nicotinic acetylcholine receptor subunits in smoking-
cessation therapies I. Jang1, D. Yang2, I. Jung2, B. Lee1 I. Jang1, D. Yang2, I. Jung2, B. Lee1 G. Pintarelli1, A. Galvan2, P. Pozzi1,3, S. Noci1, G. Pasetti1, F. Sala1, U. Pastorino1, R. Boffi1, F. Colombo1
1Fondazione IRCCS Istituto Nazionale dei Tumori, Milan,
Italy, 2Formerly Fondazione IRCCS Istituto Nazionale dei
Tumori, Milan, Italy, 3Azienda Sociosanitaria Territoriale
Lariana Sant’Antonio Abate Hospital, Cantù, Italy
Introduction: Smoking-cessation therapy reduces the risk
of smoking-related diseases, but is successful only in a 1Korea Research Institute of Bioscience and Biotechnology,
Daejeon, Korea, Republic of, 2Korea Advanced Institute of
Science and Technology, Daejeon, Korea, Republic of G. Pintarelli1, A. Galvan2, P. Pozzi1,3, S. Noci1, G. Pasetti1, F. Sala1, U. Pastorino1, R. Boffi1, F. Colombo1 1Fondazione IRCCS Istituto Nazionale dei Tumori, Milan,
Italy, 2Formerly Fondazione IRCCS Istituto Nazionale dei
Tumori, Milan, Italy, 3Azienda Sociosanitaria Territoriale
Lariana Sant’Antonio Abate Hospital, Cantù, Italy M. CHATZIDAKI1,2, G. THODI3, Y. L. LOUKAS1,2, E.
MOLOU3, O. TRIANTAFILLI3, Y. DOTSIKAS1, K.
SCHOULPIS4 Smoking habit and abstention
were assessed from the number of cigarettes smoked
per day (CPD) and the exhaled CO (eCO), at four time
points (baseline, 1, 3 and 12 months). Statistical tests were
carried out with PLINK software using sex, therapy, and
eCO as covariates. Significance was set at P < 0.05. Multiple testing was done using the Benjamini-Hochberg
procedure. Results: Several mutations with clinical implications
were identified in all healthy participants. Table 1 depicts
some of our findings. TABLE 1:
SAMPLE ID GENE/ MUTATION
25008
CFTR:c.1521_1523delCTT
Cystic fibrosis (INHERITED)
25972
CYP4F2:c.1297G>A
DRUG RELATED
25987
MEFV:c.2082G>A
Mediterranian syndrome
(INHERITED)
30007
BCHE:c.1256G>T
DRUG RELATED
30125
BRCA1:c.181T>G
PREDISPOSITION to cancer
30982
ΜΥΗ7:c.3645G>C
PREDISPOSITION to familial
hypertrophic cardiomyopathy
30999
DYSF:c.386G>A
“Miyoshi myopathy”
(INHERITED)
32039
CFTR:c.489+1G>T
Cystic fibrosis (INHERITED)
31033
GJB2: c.35delG
Nonsyndromic hearing loss
(INHERITED)
35000
HBB:c.93-21G>A
Beta thalassemia (INHERITED)
35022
BRCA2:
c.3554_3563delCAGTTGAAAT
PREDISPOSITION to cancer
31025
ABCA4:c.2690C>T
Stargart macular degeneration
(INHERITED)
35064
BRCA2:c.5722_5723delCT
PREDISPOSITION to cancer Improved accuracy of ultra-low frequency mutation
detection by novel duplex sequencing adapters and
consensus read reconstruction J. Wang, K. Lai, M. Light, W. Lee, K. Giorda, M. Jarosz, Y. Bao,
D. Kupec, C. Walworth, C. Chen J. Wang, K. Lai, M. Light, W. Lee, K. Giorda, M. Jarosz, Y. Bao,
D. Kupec, C. Walworth, C. Chen J. Wang, K. Lai, M. Light, W. Lee, K. Giorda, M. Jarosz, Y. Bao,
D. Kupec, C. Walworth, C. Chen 3DIV: A 3D-genome Interaction Viewer and database Abstract: Three-dimensional (3D) chromatin structure is
an emerging paradigm for understanding gene regulation
mechanisms. Hi-C (high-throughput chromatin conforma-
tion capture), a method to detect long-range chromatin
interactions, allows extensive genome-wide investigation of Introduction: Smoking-cessation therapy reduces the risk
of smoking-related diseases, but is successful only in a 567 Abstracts from the 51st European Society of Human Genetics Conference: Posters 3D chromatin structure. However, broad application of Hi-
C data have been hindered by the level of complexity in
processing Hi-C data and the large size of raw sequencing
data. In order to overcome these limitations, we constructed
a database named 3DIV (a 3D-genome Interaction Viewer
and database) that provides a list of long-range chromatin
interaction partners for the queried locus with genomic and
epigenomic annotations. 3DIV is the first of its kind to
collect all publicly available human Hi-C data to provide 66
billion uniformly processed raw Hi-C read pairs obtained
from 80 different human cell/tissue types. In contrast to
other
databases,
3DIV
uniquely
provides
normalized
chromatin interaction frequencies against genomic distance
dependent background signals and a dynamic browsing
visualization tool for the listed interactions, which could
greatly advance the interpretation of chromatin interactions. ‘3DIV’ is available at http://kobic.kr/3div. ground truths, Duplex Seq Adapters offered >30-50 fold
better specificity than standard adapters at the same level of
detection sensitivity, regardless of sample type. For exam-
ple, using Duplex Seq Adapters paired with consensus read
construction, >90% detection sensitivity was achieved for
genomic and cfDNA samples at 0.5% MAF with 0 false
positives, resulting in 100% specificity and >40-fold error
suppression compared to standard adapters. Such advances
in detection accuracy are key to refining diagnostics and
improving precision cancer care. J. Wang: A. Employment (full or part-time); Significant;
Integrated DNA Technologies. K. Lai: A. Employment
(full or part-time); Significant; Integrated DNA Technolo-
gies. M. Light: A. Employment (full or part-time);
Significant; Integrated DNA Technologies. W. Lee: A. Employment (full or part-time); Significant; Integrated
DNA Technologies. K. Giorda: A. Employment (full or
part-time); Significant; Integrated DNA Technologies. M. Jarosz: A. Employment (full or part-time); Significant;
Integrated DNA Technologies. Y. Bao: A. Employment
(full or part-time); Significant; Integrated DNA Technolo-
gies. D. Kupec: A. Employment (full or part-time);
Significant; Integrated DNA Technologies. C. Walworth:
A. Employment (full or part-time); Significant; Integrated
DNA Technologies. C. Chen: A. Employment (full or part-
time); Significant; Integrated DNA Technologies. I. Jang: None. D. Yang: None. I. 3DIV: A 3D-genome Interaction Viewer and database Jung: None. B. Lee: None. J. Ravel1,2, L. Monraz Gomez3,4,5, E. Barillot3,4,5, A. Zinovyev3,4,5,
I. Kuperstein3,4,5 P16.02B Improved accuracy of ultra-low frequency mutation
detection by novel duplex sequencing adapters and
consensus read reconstruction P16.03C Integrated DNA Technologies, Redwood City, CA, United
States Comprehensive map of regulated cell death signalling
network: a powerful analytical tool for studying diseases Col-
lectively, our single-cell data indicates that clonal popula-
tions inferred from VAFs obtained from bulk sequencing
data may not fully resolve the heterogeneity within tumors;
moreover, the single-cell nature of our approach enabled the
unambiguous identification of multiple co-occurring muta-
tions within subclones that is not possible with bulk mea-
surements. Collectively, our results show a greater degree of
heterogeneity in AML tumor samples than is commonly
appreciated with traditional sequencing paradigms and
demonstrate the value of single-cell analysis for AML. contextually in the whole map, or as individual diagrams. The map contains more than 1200 proteins and genes, 2020
biochemical reactions and is based on 600 scientific papers. It is an open source platform facilitated by the NaviCell web
tool for map navigation (https://navicell.curie.fr/pages/ma
ps_rcd.html). The RCD map was used for functional
interpretation of the differences in cell death regulation
between Alzheimer’s disease and lung cancer. These
diseases were demonstrated to have a reverse comorbidity. Interestingly, we observed that metabolism-related modules
are deregulated in Alzheimer’s disease. Moreover, in lung
cancer, these modules are disrupted in an opposite way. Conclusions: This approach will help study dynamic
molecular mechanisms of diseases, especially cancers. A
comprehensive
reconstruction
of
regulated
cell
death
signaling network will bring a better understanding of
regulatory circuits and, in a near future, can be applied for
therapeutic
target
identification
and
for
treatment
approaches optimization. This will also be useful to medical
geneticists,
both
researchers
and
clinicians,
who
are
constantly dealing with a great variety of new genes. g
y
D.J. Eastburn: A. Employment (full or part-time);
Significant; Mission Bio. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Mission Bio. M. Pellegrino: E. Ownership
Interest (stock, stock options, patent or other intellectual
property);
Significant;
Mission
Bio. A. Sciambi:
A. Employment (full or part-time); Significant; Mission Bio. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Mission Bio. S. Treusch:
A. Employment (full or part-time); Significant; Mission
Bio. E. Ownership Interest (stock, stock options, patent or
other intellectual property); Significant; Mission Bio. L. Xu: None. R. Durruthy-Durruthy: A. Employment (full
or part-time); Significant; Mission Bio. E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Significant; Mission Bio. K. Gokhale: A. Employment (full or part-time); Significant; Mission Bio. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Mission Bio. J. Jacob:
A. Comprehensive map of regulated cell death signalling
network: a powerful analytical tool for studying diseases Comprehensive map of regulated cell death signalling
network: a powerful analytical tool for studying diseases Detection of low to ultra-low frequency mutations, enabled
by advances in next generation sequencing (NGS), is often
confounded by errors introduced during standard NGS
workflows, even those using unique-molecular-identifiers
(UMIs). We have developed xGen® Duplex Seq Adapters,
which permit double-stranded DNA tagging and consensus
read calling among duplicated sequences. These novel
adapters are compatible with standard library preparation
and enrichment methods, yet enable duplex sequencing and
enhanced error correction. Well-established genomes, or
commercially acquired and individually genotyped FFPE
and cfDNA samples, were mixed to mimic low-frequency
variant samples with MAF down to 0.1%. Libraries from
these sample mixtures were prepared for deep sequencing
using Duplex Seq Adapters and a 75 kb custom, target-
capture panel. A bioinformatic tool suite was developed to
create UMI-aware consensus reads. Compared to standard
libraries, libraries made using the Duplex Seq Adapters,
with their unique tagging structure, had comparable or
better yields and mean deduplicated sequencing depth for
all sample types and input masses tested. For variant calling
accuracy, evaluated against published cell-line or genotyped J. Ravel1,2, L. Monraz Gomez3,4,5, E. Barillot3,4,5, A. Zinovyev3,4,5,
I. Kuperstein3,4,5 1Laboratoire de génétique, Centre Régional Hospitalier
Universitaire de Nancy, Nancy, France, 2INSERM UMR 954,
Université de Lorraine, Vandœuvre-lès-Nancy, France,
3Institut Curie, 26 rue d'Ulm, Paris, France, 4PSL Research
University, Paris, France, 5Mines Paris Tech, Fontainebleau,
France Introduction: The Cell death process draws special atten-
tion, due to frequent perturbations of its machinery in var-
ious diseases. It can be described into three highly
interconnected steps: initiation, signaling and execution of
death signals. Materials and Methods: We used a systems biology
approach to graphically represent biological processes in a
seamless comprehensive map of signaling network that is
accessible for visualization, computational analysis and
applications. Results: The Regulated Cell Death (RCD) map is divided
into
27
functional
modules
that
can
be
visualized J. del Picchia 568 sensitively identify SNV and indel-defined clones within
AML samples and assess their distribution at the time of
diagnosis, remission and relapse. We also used single cell
SNVs to monitor host and donor cell populations during
bone marrow transplantation (BMT), which allowed us to
accurately evaluate engraftment and disease relapse. P16.04D Single-cell analysis of mutational heterogeneity in acute
myeloid leukemia tumors with high-throughput droplet
microfluidics D. J. Eastburn1, M. Pellegrino1, A. Sciambi1, S. Treusch1, L. Xu2, R. Durruthy-Durruthy1, K. Gokhale1, J. Jacob1, T. X. Chen1, W. Oldham1, J. Matthews3, H. Kantarjian3, A. Futreal3,
K. Patel3, J. Zehnder2, K. Jones1, K. Takahashi3 1Mission Bio, Inc., South San Francisco, CA, United States,
2Stanford School of Medicine, Stanford, CA, United States,
3MD Anderson Cancer Center, University of Texas, Houston,
TX, United States To make possible the routine characterization of genetic
diversity within cancer cell populations, we developed a
novel two-step microfluidic droplet workflow that enables
efficient
and
massively-parallel
single-cell
PCR-based
genomic barcoding for single-cell DNA sequencing appli-
cations. We demonstrate that the two-step microfluidic
approach is required for robust DNA amplification on
thousands of individual cells per run with high coverage
uniformity and low allelic dropout of targeted genomic loci. To apply our single-cell sequencing technology to human
tumor samples, we developed a targeted panel to partially
sequence 26 genes frequently mutated in acute myeloid
leukemia (AML) including TP53, DNMT3A, FLT3, NPM1,
NRAS, IDH1 and IDH2. Using this panel, we were able to Comprehensive map of regulated cell death signalling
network: a powerful analytical tool for studying diseases Employment (full or part-time); Significant; Mission
Bio. E. Ownership Interest (stock, stock options, patent or
other intellectual property); Significant; Mission Bio. T.X. Chen: A. Employment (full or part-time); Significant;
Mission Bio. E. Ownership Interest (stock, stock options,
patent or other intellectual property); Significant; Mission
Bio. W. Oldham: A. Employment (full or part-time);
Significant; Mission Bio. J. Matthews: None. H. Kantar-
jian: None. A. Futreal: None. K. Patel: None. J. Zehnder: None. K. Jones: A. Employment (full or part-
time); Significant; Mission Bio. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Mission Bio. K. Takahashi: None. J. Ravel: None. L. Monraz Gomez: None. E. Barillot:
None. A. Zinovyev: None. I. Kuperstein: None. J. Ravel: None. L. Monraz Gomez: None. E. Barillot:
None. A. Zinovyev: None. I. Kuperstein: None. Phenopolis: an open platform for harmonisation of genetic
and phenotypic data M. Asif1,2,3, H. Martiniano1,3, C. Rasga2,3, A. R. Marques2,3, J. X.
Santos2,3, G. Oliveira4,5, F. M. Couto1, A. M. Vicente2,3,6 M. Asif1,2,3, H. Martiniano1,3, C. Rasga2,3, A. R. Marques2,3, J. X. Santos2,3, G. Oliveira4,5, F. M. Couto1, A. M. Vicente2,3,6 1UCL Institute of Ophthalmology, London, United Kingdom,
2Moorfields Eye Hospital, London, United Kingdom, 3UCL
Cancer Institute, London, United Kingdom, 4Nuffield
Department of Clinical Neurosciences, University of Oxford,
Oxford, United Kingdom, 5UCL Genetics Institute, London,
United Kingdom 1Departamento de Informática, Faculdade de Ciências da
Universidade de Lisboa, Lisbon, Portugal, 2Instituto Nacional
de Saúde Doutor Ricardo Jorge, Lisbon, Portugal, 3BioISI -
Biosystems & Integrative Sciences Institute, Faculdade de
Ciências, Universidade de Lisboa, Lisbon, Portugal,
4Faculdade de Medicina da Universidade de Coimbra,
Coimbra, Portugal, 5Centro de Investigação e Formação
Clinica do HP-CHUC, Coimbra, Portugal, 6Instituto
Gulbenkian de Ciência, Oeiras, Lisbon, Portugal 1UCL Institute of Ophthalmology, London, United Kingdom,
2Moorfields Eye Hospital, London, United Kingdom, 3UCL
Cancer Institute, London, United Kingdom, 4Nuffield
Department of Clinical Neurosciences, University of Oxford,
Oxford, United Kingdom, 5UCL Genetics Institute, London,
United Kingdom 1Departamento de Informática, Faculdade de Ciências da
Universidade de Lisboa, Lisbon, Portugal, 2Instituto Nacional
de Saúde Doutor Ricardo Jorge, Lisbon, Portugal, 3BioISI -
Biosystems & Integrative Sciences Institute, Faculdade de
Ciências, Universidade de Lisboa, Lisbon, Portugal,
4Faculdade de Medicina da Universidade de Coimbra,
Coimbra, Portugal, 5Centro de Investigação e Formação
Clinica do HP-CHUC, Coimbra, Portugal, 6Instituto
Gulbenkian de Ciência, Oeiras, Lisbon, Portugal Introduction: The molecular diagnosis of rare genetic
diseases requires detailed clinical phenotypes and proces-
sing of large amounts of genetic data. This motivates large-
scale collaborations between clinicians, geneticists and
bioinformaticians across multiple sites where patient data
are pooled together to boost the chances of solving rare
cases, and validating novel genes. This motivated the
development of Phenopolis, a platform for harmonisation of
genetic and phenotypic data. Autism Spectrum Disorder (ASD) is characterized by highly
heterogeneous clinical phenotypes and complex genetic
architecture, rendering early diagnosis and prognosis diffi-
cult. CNVs in many genes have been implicated in ASD,
disrupting specific Biological Processes (BP) eventually
associated with distinct clinical phenotypes. Here we sought
to identify and predict clinical patterns associated with BP
disrupted by specific brain gene CNVs, using a machine
learning-based
integrative
approach. Firstly,
clustering
analysis of clinical records from 2446 ASD patients from
the Autism Genome Project identified two consistent and
highly stable clusters, differing in ASD severity, adaptive
behaviour
and
cognitive
ability. M. Asif1,2,3, H. Martiniano1,3, C. Rasga2,3, A. R. Marques2,3, J. X.
Santos2,3, G. Oliveira4,5, F. M. Couto1, A. M. Vicente2,3,6 Secondly,
functional
enrichment
analysis
of rare
CNVs,
disrupting brain-
expressed genes in these ASD subjects, identified 15 sig-
nificant BPs, including nervous system development, cog-
nition, and protein polyubiquitination. Random Forest
feature importance analysis showed that all these BP con-
tributed positively to the classification of ASD severity, as
defined by the identified clusters. Finally, a Naive Bayes
classifier was trained using cluster and BP information from
a subset of data, comprising the 325 individuals with
highest BP information content scores. A stratified five-fold
cross validated model predicted the severity of ASD phe-
notype from BPs, with precision of 0.82 and recall of 0.39. This study thus shows that severity predictions can be
attained from BP putatively disrupted by brain-gene CNVs. However, precise predictions are only achieved in sub-
groups with high BP information content, and specificity is
generally low. A clinical application will thus require fur-
ther analysis, in much larger datasets that include detailed
phenotypic information. FCT-Portugal (SFRH/BD/52485/
2014) Methods: Using Human Phenotype Ontology (HPO)
encoded phenotypes, Phenopolis is able to prioritise
causative genes using different sources of evidence, such
as literature search and model organism phenotype ontology
analysis. Additionally,
Phenopolis
uncovers
gene-
phenotype relationships within the stored patient data
through variant filtering and statistical enrichment of HPO
terms. The database is implemented using a graph database
for scalability which allows efficient linking of HPO, genes
and variants. Methods: Using Human Phenotype Ontology (HPO)
encoded phenotypes, Phenopolis is able to prioritise
causative genes using different sources of evidence, such
as literature search and model organism phenotype ontology
analysis. Additionally,
Phenopolis
uncovers
gene-
phenotype relationships within the stored patient data
through variant filtering and statistical enrichment of HPO
terms. The database is implemented using a graph database
for scalability which allows efficient linking of HPO, genes
and variants. Results: Phenopolis is an open-source web server
providing an intuitive interface to genetic and phenotypic
databases. Phenopolis is also an ideal platform for studying
the pleiotropy of genes. The online version available at
www.phenopolis.org, includes four example patients with
inherited retinal dystrophies, to illustrate our methods. Discussion: The Phenopolis platform accelerates clinical
diagnosis, gene discovery and encourages wider adoption of
the HPO in the study of rare genetic diseases. We plan on
extending Phenopolis to interface with the Genomics
England GenePanel app to retrieve relevant genes and
contribute novel disease genes. M. Asif1,2,3, H. Martiniano1,3, C. Rasga2,3, A. R. Marques2,3, J. X.
Santos2,3, G. Oliveira4,5, F. M. Couto1, A. M. Vicente2,3,6 Discussion: The Phenopolis platform accelerates clinical
diagnosis, gene discovery and encourages wider adoption of
the HPO in the study of rare genetic diseases. We plan on
extending Phenopolis to interface with the Genomics
England GenePanel app to retrieve relevant genes and
contribute novel disease genes. N. Pontikos: None. I. Moghul: None. J. Yu: None. A. Fiorentino: None. G. Arno: None. A.J. Hardcastle: None. A.R. Webster: None. V. Plagnol: A. Employment (full or
part-time); Significant; Inivata. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Inivata. M. Asif: None. H. Martiniano: None. C. Rasga: None. A.R. Marques: None. J.X. Santos: None. G. Oliveira:
None. F.M. Couto: None. A.M. Vicente: None. P16.06B An integrative approach to correlate clinical presentation
patterns in Autism Spectrum Disorder with biological
processes disrupted by Copy Number Variants (CNV) in
brain genes P16.07C
BEA: a web tool for BioMark gene expression analysis P16.05A Abstracts from the 51st European Society of Human Genetics Conference: Posters 569 N. Pontikos1,2, I. Moghul3, J. Yu4, A. Fiorentino1, G. Arno1,2, A.
J. Hardcastle1, A. R. Webster1,2, V. Plagnol5 N. Pontikos1,2, I. Moghul3, J. Yu4, A. Fiorentino1, G. Arno1,2, A. J. Hardcastle1, A. R. Webster1,2, V. Plagnol5 B. Cadenas1,2,3, I. Cebrián2,4, E. Sánchez1, M. Calle3, C.
Tornador2,5,4, M. Sánchez1,6,5 Fresh-frozen PTs and non-
tumoral specimens from blood were collected from 8
patients at IRCCS-INT; BCICs were isolated at early
in vitro passages; the resulting samples (PTs, BCICs and
blood) were whole-exome sequenced; mutations detected
by at least two variant callers were considered. Functional
interactions were collected from STRING. The network
proximity of altered genes was calculated by network
diffusion. BRCA genes were collected from COSMIC,
OMIM, INTOGEN and DOCM. Materials and Methods: TCGA data (49 subjects) were
collected using TCGAbiolinks. Fresh-frozen PTs and non-
tumoral specimens from blood were collected from 8
patients at IRCCS-INT; BCICs were isolated at early
in vitro passages; the resulting samples (PTs, BCICs and
blood) were whole-exome sequenced; mutations detected
by at least two variant callers were considered. Functional
interactions were collected from STRING. The network
proximity of altered genes was calculated by network
diffusion. BRCA genes were collected from COSMIC,
OMIM, INTOGEN and DOCM. To meet this need, we are developing BEA, Biomark
Expression Analysis, a web tool designed to analyse Real
Time qPCR data from Biomark HD System across multiple
conditions and replicates. Analysis includes relative quanti-
fication using normalization against a reference gene (ΔΔCT
method), and appropriate statistical testing to identify
differential expressed genes between tested groups, taking
into account test of normality and number of groups. The
web application is written in R using the Shiny platform. BEA has been tested using Bimark 96.96 dynamic array
data from myelodisplastic syndrome patients. Results: The network-based analysis increased the over-
lap between the significant genes associated with each
omics. The proposed multi-omics gene score prioritized a
higher number of BRCA genes than the analysis of each
omics on its own. The score underlined potential members
of pathways that suggest functional links between mutations
in BCICs and PTs. Results: The network-based analysis increased the over-
lap between the significant genes associated with each
omics. The proposed multi-omics gene score prioritized a
higher number of BRCA genes than the analysis of each
omics on its own. The score underlined potential members
of pathways that suggest functional links between mutations
in BCICs and PTs. BEA
will
help
scientists
perform
gene
expression
analyses
by
interactively
visualizing
each
step
with
customizable options and intuitive graphical user interface. The program provide comprehensive results visualization,
downloadable plots and a summarizing report, and it would
not require programming knowledge. BEA will be an open
source software freely available online. B. Cadenas1,2,3, I. Cebrián2,4, E. Sánchez1, M. Calle3, C.
Tornador2,5,4, M. Sánchez1,6,5 Conclusions: Network diffusion-based integrative analy-
sis underlines links among different types of evidences,
prioritizing omics-specific and common players within the
human interactome. This research was supported by grant SAF2015-70412-R
(MINECO), DJCLS R14/04 from Deutsche José Carreras
leukämie Stiftung, 2017 SGR 288 GRC (GRE) Generalitat
de Catalunya and from Fundació Internacional Josep
Carreras and Obra Social “la Caixa” Spain to M.S. Funding: MIUR (INTEROMICS PB05, PRIN 2015
20157ATSLF); FRRB (LYRA 2015-0010). N. Di Nanni: None. V. Appierto: None. C. De Marco:
None. E. Ortolan: None. L. Milanesi: None. M. Daidone:
None. E. Mosca: None. B. Cadenas: None. I. Cebrián: None. E. Sánchez:
None. M. Calle:
None. C. Tornador:
None. M. Sánchez: None. B. Cadenas: None. I. Cebrián: None. E. Sánchez:
None. M. Calle:
None. C. Tornador:
None. M. Sánchez: None. P16.09A Increased BRCA1/2 classification confidence: comparison
of ACMG to public database classification - call for
systematic molecular profiling B. Cadenas1,2,3, I. Cebrián2,4, E. Sánchez1, M. Calle3, C.
Tornador2,5,4, M. Sánchez1,6,5 N. Di Nanni1,2, V. Appierto3, C. De Marco3, E. Ortolan3, L. Milanesi1, M. Daidone3, E. Mosca1 B. Cadenas1,2,3, I. Cebrián2,4, E. Sánchez1, M. Calle3, C. Tornador2,5,4, M. Sánchez1,6,5 1Institute of Biomedical Technologies, CNR, Milano, Italy,
2Dipartimento di Informatica e Sistemistica, University of
Pavia, Pavia, Italy, 3IRCSS Istituto Nazionale dei Tumori,
Milano, Italy 1Iron Metabolism: Regulation and Diseases Group, Josep
Carreras Leukaemia Research Institute (IJC), Badalona,
Spain, 2Whole Genix S.L, Barcelona, Spain, 3Universitat de
Vic-Universitat Central de Catalunya, Vic, Spain, 4Fundación
Teresa Moretó, Esplugues de Llobregat, Spain,
5BloodGenetics S.L., Esplugues de Llobregat, Spain, 6Program
of Program of Predictive and Personalized Medicine of Cancer
(PMPPC), Institutd'Investigació Germans TriasiPujol (IGTP),
Badalona, Spain 1Iron Metabolism: Regulation and Diseases Group, Josep
Carreras Leukaemia Research Institute (IJC), Badalona,
Spain, 2Whole Genix S.L, Barcelona, Spain, 3Universitat de
Vic-Universitat Central de Catalunya, Vic, Spain, 4Fundación
Teresa Moretó, Esplugues de Llobregat, Spain,
5BloodGenetics S.L., Esplugues de Llobregat, Spain, 6Program
of Program of Predictive and Personalized Medicine of Cancer
(PMPPC), Institutd'Investigació Germans TriasiPujol (IGTP),
Badalona, Spain Introduction: The generation of multiple omics from the
same biological system poses challenges in data analysis
and interpretation. We propose a network-based approach
that prioritizes genes considering their network (functional)
proximity to altered genes, according to different types of
experimental evidence. We compare our multi-omics gene
score with those of single omics analyses, using breast
cancer (BRCA) somatic mutation and gene expression from
TCGA. We also identify functionally related genes ana-
lysing somatic mutations in primary tumors (PT) and PT-
derived BRCA initiating cells (BCICs). Biomark HD system is a high-throughput technology that
performs more than nine thousands of real-time quantitative
PCR (qPCR) reactions simultaneously. Although numerous
statistical tools are available in the public domain for the
analysis of conventional qPCR experiments, this is not the
case when many qPCR dataset from high-throughput
experiments have to be analysed. Therefore, there is a
need to provide a user-friendly software able to analyze
Biomark output data. Biomark HD system is a high-throughput technology that
performs more than nine thousands of real-time quantitative
PCR (qPCR) reactions simultaneously. Although numerous
statistical tools are available in the public domain for the
analysis of conventional qPCR experiments, this is not the
case when many qPCR dataset from high-throughput
experiments have to be analysed. Therefore, there is a
need to provide a user-friendly software able to analyze
Biomark output data. Materials and Methods: TCGA data (49 subjects) were
collected using TCGAbiolinks. Network diffusion for the integrative analysis of multiple
“-omics”: case studies on breast cancer Increased BRCA1/2 classification confidence: comparison
of ACMG to public database classification - call for
systematic molecular profiling P16.07C 570 J. del Picchia Molecular Health, Heidelberg, Germany Introduction: Detection of certain BRCA1/2 mutations
may be indicative of risk for breast or ovarian cancer. Confident determination of the clinical significance of
BRCA1/2 variants is therefore critical to geneticists,
patients, and medical socioeconomics. Lack of molecular
characterization for many variants and unknown association
to disease complicate genetic test reporting. One approach
to interpret germline variants is provided by ACMG
guidelines based on five-class clinical categorization. Introduction: Detection of certain BRCA1/2 mutations
may be indicative of risk for breast or ovarian cancer. Confident determination of the clinical significance of
BRCA1/2 variants is therefore critical to geneticists,
patients, and medical socioeconomics. Lack of molecular
characterization for many variants and unknown association
to disease complicate genetic test reporting. One approach
to interpret germline variants is provided by ACMG
guidelines based on five-class clinical categorization. Generation of the Cancer Pathway Prototype - a platform
for predictive cancer pathway modeling Generation of the Cancer Pathway Prototype - a platform
for predictive cancer pathway modeling T. Sorg1, Y. Herault1, J. Jonkers2, A. Ploubidou3, L. Frappart3,
J. Hasenauer4, J. Banga5, O. Rinner6, V. Naumova7, D. Koubi8,
B. Lange9 Methods: We investigated the ACMG classification of a
breast and ovarian cancer study with respect to criteria that
affect ACMG-interpretation. We (a) compared variant-
annotations
from
public
databases,
(b)
assessed
the
contribution
of
expert-validated
publications,
and
(c)
calculated an independent ACMG-classification. Methods: We investigated the ACMG classification of a
breast and ovarian cancer study with respect to criteria that
affect ACMG-interpretation. We (a) compared variant-
annotations
from
public
databases,
(b)
assessed
the
contribution
of
expert-validated
publications,
and
(c)
calculated an independent ACMG-classification. 1Phenomin-ICS, Illkirch, France, 2Netherlands Cancer
Institute, Amsterdam, Netherlands, 3Leibniz Institute on Aging
- Fritz-Lipmann Institute, Jena, Germany, 4Helmholtz Zentrum
München, Munchen, Germany, 5Spanish National Research
Council, CSIC, Vigo, Spain, 6Biognosys, Schlieren,
Switzerland, 7Simula Research Laboratory, Fornebu, Norway,
8Finovatis, Lyon, France, 9Alacris Theranostics, Berlin,
Germany 1Phenomin-ICS, Illkirch, France, 2Netherlands Cancer
Institute, Amsterdam, Netherlands, 3Leibniz Institute on Aging
- Fritz-Lipmann Institute, Jena, Germany, 4Helmholtz Zentrum
München, Munchen, Germany, 5Spanish National Research
Council, CSIC, Vigo, Spain, 6Biognosys, Schlieren,
Switzerland, 7Simula Research Laboratory, Fornebu, Norway,
8Finovatis, Lyon, France, 9Alacris Theranostics, Berlin,
Germany Results: Combined assessment of variant pathogenicity
derived from public database annotations requires caution. Pairwise comparison of public database content showed up
to 15 and 22% discrepancy among BRCA1/2 variant-
annotations, respectively. While public database consensus
was
comparable
to
the
ACMG-classification
of
the
investigated study, incorporation of additional information
from
published
functional
studies
further
ascertained
confidence. Incorporation of our curated content to ACMG
guidelines revealed up to 84 % and 78 % less ‘likely-
benign’ or ‘likely-pathogenic’ BRCA1/2 classifications,
respectively. CanPathPro is developing a bioinformatics concept and the
associated computational tools required for the translation
of highly complex and heterogeneous omics data into pre-
dictive modelling of cancer signaling. The approach is uti-
lizing existing data and databases relevant to cancer
signaling as well as CanPathPro-generated mouse omics
data obtained from mouse cancer models, for practical
testing and validation of the system. These data are inte-
grated by an existing modelling computational platform, to
identify signaling networks critical for breast and lung
cancer development. The in silico-generated predictions are
validated using (i) organotypic systems recapitulating breast
and
lung
lesions,
(ii)
well-phenotyped,
existing
and
CanPathPro-generated mouse models. S. Kaduthanam, U. Schmidt-Edelkraut, S. Santiago-Mozos, M.
Hartenfeller, R. Narang, J. Hermanns, M. Stein, D. Jackson, M.
Weber, S. Hettich, S. Brock, T. Soldatos part-time); Significant; Molecular Health. M. Weber: F. Consultant/Advisory Board; Significant; BRIDGES Con-
sulting Group. S. Hettich: A. Employment (full or part-
time); Significant; Molecular Health. S. Brock: A. Employ-
ment (full or part-time); Significant; Molecular Health. T. Soldatos: A. Employment (full or part-time); Significant;
Molecular Health. part-time); Significant; Molecular Health. M. Weber: F. Consultant/Advisory Board; Significant; BRIDGES Con-
sulting Group. S. Hettich: A. Employment (full or part-
time); Significant; Molecular Health. S. Brock: A. Employ-
ment (full or part-time); Significant; Molecular Health. T. Soldatos: A. Employment (full or part-time); Significant;
Molecular Health. P16.08D Network diffusion for the integrative analysis of multiple
“-omics”: case studies on breast cancer Abstracts from the 51st European Society of Human Genetics Conference: Posters 571 S. Kaduthanam, U. Schmidt-Edelkraut, S. Santiago-Mozos, M. Hartenfeller, R. Narang, J. Hermanns, M. Stein, D. Jackson, M. Weber, S. Hettich, S. Brock, T. Soldatos Institute of Genetic Medicine, Newcastle, United Kingdom Institute of Genetic Medicine, Newcastle, United Kingdom 1MSMSU, Moscow, Russian Federation, 2xGen Cybernetics,
Moscow, Russian Federation, 3Federal state scientific
budgetary Institution «Research Centre for Medical
Genetics»,, Moscow, Russian Federation, 4Filatov Children's
clinical hospital, Moscow, Russian Federation, 5Ministry of
Health of Russian Federation, Separated Structural Unit
“Clinical Research Institute of Pediatrics” at Pirogov Russian
National Research Medical University, Moscow, Russian
Federation Introduction: Previous research has indicated that cell-free
mitochondrial DNA (ccf-mtDNA) copy number (CN) is
significantly depleted in PD and AD CSF versus controls,
suggesting this may serve as a viable biomarker of broad
neurodegeneration. Therefore further study, broadening the
clinical spectrum are warranted. To address this we inves-
tigated the role and integrity of ccf-mtDNA in the devel-
opment and progression of several neurodegenerative
diseases (NDD) including; PD, DLB, AD and MS. Introduction: The existence of many forms of rare mono-
genic syndromes and unbalanced chromosome aberrations
with similar phenotypes create difficulties in diagnosis and
require the development of tools for differential diagnosis
before the appointment of genetic testing to confirm the
final diagnosis. Methods: We have used quantitative PCR to measure
ccf-mtDNA CN in ventricular and lumbar CSF samples
from various NDD, to assess the viability of ccf-mtDNA as
a disease biomarker. Secondly, we have investigated both
mitochondrial and cell-death marker protein levels of these
samples using western blotting and ELISA, correlating
these to ccf-mtDNA, to determine the origin and effect of
ccf-mtDNA. Finally we have assessed the integrity of ccf-
mtDNA using NGS, comparing ccf-mtDNA to reference
tissue samples. Materials and Methods: Available public INTERNET
sources were used to fill the knowledge base of the decision
support system. For the formation of the catalogs of
monogenic forms the information from OMIM, ORPHA-
NET was analyzed. Description of the clinical picture
according to the literature data was carried out on the basis
of modified and extended ontology HPO. The catalog of
chromosome aberrations was formed on the basis of dbVar,
OMIM and ISCA. The program was implemented on the
basis of the platform xGEN IDS and xGenCloud service. Results: Our results support our hypothesis that low ccf-
mtDNA is a biomarker for neurodegeneration, particularly
in PD. However, we found no correlation to ccf-mtDNA
and mitochondrial or neuronal protein levels, despite
significantly reduced levels in PD versus controls. Further-
more, Braak stage and dementia severity did not correlate to
ccf-mtDNA. Clinical decision support system for differential diagnosis
of monogenic syndromes and microdeletion and
microduplication syndromes Clinical decision support system for differential diagnosis
of monogenic syndromes and microdeletion and
microduplication syndromes Generation of the Cancer Pathway Prototype - a platform
for predictive cancer pathway modeling A. Sharkov: None. N. Ivanov: None. I. Ugarov: None. V. Chernykh: None. I. Sharkova:
None. O. Novoselova: None. A. Sharkov: None. N. Ivanov: None. I. Ugarov: None. V. Chernykh: None. I. Sharkova:
None. O. Novoselova: None. A. Sharkov: None. N. Ivanov: None. The role of cell-free mitochondrial DNA in
neurodegenerative disease The role of cell-free mitochondrial DNA in
neurodegenerative disease The role of cell-free mitochondrial DNA in
neurodegenerative disease I. Ugarov1,2, V. Chernykh3,2, I. Sharkova3,2, O. Novoselova4,2, A. Sharkov5,2, N. Ivanov2 I. Ugarov1,2, V. Chernykh3,2, I. Sharkova3,2, O. Novoselova4,2, A. Sharkov5,2, N. Ivanov2 H. Lowes, M. Kurzawa-Akanbi, A. Pyle, G. Hudson Generation of the Cancer Pathway Prototype - a platform
for predictive cancer pathway modeling Conclusions: Our results invite initiating prospective
studies that characterize the observed confidence improve-
ment. Overall, consolidated standards with harmonized
BRCA1/2 variant-annotations are essential for adept clinical
classification. While patient information is a key- parameter,
one other challenge is supporting the geneticist’s expertise
with curated evidence. We anticipate that tools reliably
combining
these
factors
will
systematically
augment
rational management of associated medical treatment- and
cost-options by further ascertaining decision-making. The validated bioinformatics concept will be built into the
CanPathPro prototype: A tripartite tool for the (i) generation
and (ii) integration of quantitative mouse omics data,
followed by (iii) predictive in silico modelling of cancer
signaling pathways and networks in mouse models. S. Kaduthanam: A. Employment (full or part-time);
Significant; Molecular Health. U. Schmidt-Edelkraut:
None. S. Santiago-Mozos: A. Employment (full or part-
time); Significant; Molecular Health. M. Hartenfeller: A. Employment (full or part-time); Significant; Molecular
Health. R. Narang: A. Employment (full or part-time);
Significant; Molecular Health. J. Hermanns: A. Employ-
ment (full or part-time); Significant; Molecular Health. M. Stein: A. Employment (full or part-time); Significant;
Molecular Health. D. Jackson: A. Employment (full or CanPathPro has made good progress towards expanding
and building a bioinformatics modelling platform. Well
defined mouse models were amplified and omics analysis
was carried out of mouse models, mouse model derived
cells and organoid models for validation of the predictive
modelling
platform. The
combined
experimental-
&
systems biology validation platform, will be utilized in
generating and testing cancer signaling hypotheses in
biomedical research. 572 J. del Picchia algorithm in the dialog mode ensures the elimination of
syndromes and brings the user to the final diagnosis. algorithm in the dialog mode ensures the elimination of
syndromes and brings the user to the final diagnosis. The innovative approach taken by CanPathPro is set to
have broad and significant impact on diverse areas, from
cancer research and personalized medicine to drug dis-
covery and development, and ultimately improving out-
comes for cancer patients. Conclusion: The using of CDSS can significantly
optimize the work of the doctor, simplifying differential
diagnosis and, therefore, improve the effectiveness of
medical and genetic counseling of patients with monogenic
syndromes and chromosome aberrations. T. Sorg: None. Y. Herault: None. J. Jonkers: None. A. Ploubidou: None. L. Frappart: None. J. Hasenauer:
None. J. Banga: None. O. Rinner: None. V. Naumova:
None. D. Koubi: None. B. Lange: None. I. Ugarov: None. V. Chernykh: None. I. Sharkova:
None. O. Novoselova: None. Comparison of clinical yield between a single NGS assay vs.
combined array and NGS approach for clinical testing Comparison of clinical yield between a single NGS assay vs. combined array and NGS approach for clinical testing 1Department of Experimental Medicine, Sapienza University of
Rome, Rome, Italy, 2Clinical Genomics Unit, IRCCS Casa
Sollievo della Sofferenza, Rome, Italy, 3Bioinformatics Unit,
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo,
Italy, 4Cytogenetics Unit, IRCCS Casa Sollievo della
Sofferenza, San Giovanni Rotondo, Italy, 5Medical Genetics,
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo,
Italy I. Simonic1, S. Verma2, S. Shams2 1Addenbrooke’s Hospital, Cambridge, United Kingdom,
2BioDiscovery, Inc., El Segundo, CA, United States We present an approach that utilizes NGS technology to
effectively and simultaneously extract CNV, AOH and
SNV data using a single assay and integrate this data into a
single analysis pipeline. This approach is contrasted with
the more traditional way of using Chromosomal Micro-
Arrays (CMAs) for CNV and AOH detection and the use of
NGS technology for detection of Sequence Variants. We
have demonstrated the utility of such an integrated filtering
and visualization framework on a set of anonymized clinical
samples referred for various constitutional conditions. These samples have been processed for molecular testing
using the Illumina TruSight One panel with targeted cov-
erage across ~ 4800 genes. The established laboratory
informatics pipeline is used for sequence alignment and
variant calling followed by identification of CNV and AOH
regions via the BAM MultiScale Reference (MSR) algo-
rithm. Results of CNV analysis when compared with
Affymetrix 750k array data for the same samples has gen-
erated over 80% concordance with the remaining 20%
being attributed to the differences in probe coverage and/or
targeted sequencing read-depth. The whole genome CNV
and AOH analysis has been combined with HPO directed
SNV analysis and compared with the lab’s existing gene-
panel testing pipeline. The results of this analysis show an
overall increase in clinical yield of over 10% over the tra-
ditional approach. A detailed comparison of the two
approaches will be presented. Introduction: Copy Number Variants (CNVs) are defined
as deletions or duplications of stretches of contiguous
nucleotides typically larger than 1,000 base pairs. Human
CNVs are known to be involved in several diseases such as
intellectual disability, autism, schizophrenia and congenital
anomalies, generally due to the presence of dosage-sensitive
genes. However, CNVs frequently do not target protein-
coding genes, thereby hitting interspersed functional DNA
regions, with clinical significance that need to be largely
explored. P16.13A
InCAS: an integrative annotator of human copy number
variants using functional genomic features A. Giovannetti1, A. Traversa2, S. Castellana3, M. Truglio3, C. Fusilli3, M. Goldoni4, M. L. Genovesi1, N. Panzironi1, G. Napoli1, P. Palumbo5, O. Palumbo5, L. Bernardini4, A. Pizzuti1,
M. Carella5, V. Caputo1, T. Mazza3 Comparison of clinical yield between a single NGS assay vs.
combined array and NGS approach for clinical testing Materials and Methods: InCAS is an annotation tool
that draws information on gene and functional DNA
elements as well as of known and clinically significant
CNVs from several third-party databases. Gene annotations
are obtained from RefSeq and GENCODE databases. Non-
coding genes (microRNAs and lnRNAs) annotations are
retrieved from miRBase and lncRNAdb. Functional ele-
ments (e.g. conserved transcription factor-binding sites,
microRNA regulatory sites, ultraconserved region, promo-
ter
and
enhancer
predictions)
are
annotated
through
different other sources of annotations. Polymorphic CNVs
are retrieved from the Database of Genomic Variants
(DGV). CNV-related clinically significant data are obtained
from
OMIM,
Deciphering
Developmental
Disorders
(DECIPHER) and ClinVar databases. The whole frame-
work is implemented in Python and Flask and deployed to a
running instance of Nginx. Data are stored in MongoDB
and accessible through both a mobile-first web interface and
a RESTful interface for programmatic access. I. Simonic: None. S. Verma: A. Employment (full or
part-time); Significant; BioDiscovery, Inc. S. Shams: None. Institute of Genetic Medicine, Newcastle, United Kingdom Results: We developed a clinical decision support system
(CDSS), including 10934 monogenic syndromes and 17713
aberrations
with
clinical
picture. For
each
symptom
described the clinical picture of the syndrome, the presence
information indicates the frequency of occurrence among
the patients in five categories (obligatory, very frequent,
frequent,
rare,
very
rare),
type
of
symptom
in
the
manifestation of symptoms (congenital, acquired), the time
of occurrence of the symptom. The differential diagnostic Conclusions: Low CSF ccf-mtDNA is a viable biomarker
for neurodegeneration, raising the potential that this could
be used as a diagnostic predictor of disease onset and
progression, however more work is needed to validate this. In addition, our work sheds light on potential pathomechan-
isms of disease development of age-related neurodegenera-
tive disorders. Abstracts from the 51st European Society of Human Genetics Conference: Posters 573 H. Lowes: None. M. Kurzawa-Akanbi: None. A. Pyle:
None. G. Hudson: None. H. Lowes: None. M. Kurzawa-Akanbi: None. A. Pyle:
None. G. Hudson: None. significant CNVs. It can be advantageous for the assessment
of the functional effect and clinical significance of CNVs. significant CNVs. It can be advantageous for the assessment
of the functional effect and clinical significance of CNVs. A. Giovannetti: None. A. Traversa: None. S. Castel-
lana: None. M. Truglio: None. C. Fusilli: None. M. Goldoni: None. M.L. Genovesi: None. N. Panzironi:
None. G. Napoli: None. P. Palumbo: None. O. Palumbo:
None. L. Bernardini: None. A. Pizzuti: None. M. Carella:
None. V. Caputo: None. T. Mazza: None. Validation of an ultra-fast CNV calling tool for Next
Generation Sequencing data using MLPA-verified copy
number alterations Advances in computer-assisted syndrome recognition by
the example of inborn errors of metabolism Advances in computer-assisted syndrome recognition by
the example of inborn errors of metabolism B. Tolhuis: A. Employment (full or part-time); Sig-
nificant; Genalice Core BV. H. Karten: A. Employment
(full or part-time); Significant; Genalice Core BV. J. T. Pantel1,2, M. Zhao1, M. A. Mensah1,2, N. Hajjir1, T. Hsieh3,
Y. Hanani4, N. Fleischer4, T. Kamphans5, S. Mundlos1, Y. Gurovich4, P. M. Krawitz3,5 GENALICE BV, Nijkerk, Netherlands GENALICE BV, Nijkerk, Netherlands GENALICE BV, Nijkerk, Netherlands DNA Copy Number Variations (CNVs) are often associated
with human disease and, as such, important for clinical
diagnostics
using
genetic
testing. Next
Generation
Sequencing (NGS) has caused a massive increase in genetic
testing, but accurate calling of CNVs has remained chal-
lenging. Mahamdallie et al (2017) has published high
quality sequencing data from a targeted NGS assay (Tru-
Sight Cancer Panel) together with Multiplex Ligation-
dependent Probe Amplification (MLPA) validated positive
and negative CNV results for 96 independent samples
(ICR96). This is a highly reliable resource to evaluate the
accuracy of NGS CNV calling tools. We validated the
GENALICE MAP CNV calling tool with this ICR96
benchmark dataset. Our results show that GENALICE MAP
has high sensitivity and detected 65 out of 68 MLPA-
verified CNVs. The verified CNVs occur in 66 samples and
include 20 cancer predisposition genes, such as BRCA1,
BRCA2, TP53, PTEN and others. We also assessed its
specificity by examining 26 genes with normal copy num-
bers across 69 samples. Compared to other CNV calling
tools, GENALICE MAP CNV calling is exceptionally fast. We observed turnaround times of less than 10 seconds per
sample. Processing exome and full genome samples takes
less than 10 seconds and 2 minutes respectively. In con-
clusion, GENALICE MAP CNV calling provides highly
accurate results in almost real time. J.A. Sánchez: None. R.H. Reynolds: None. J. Hardy:
None. M. Ryten: None. J.A. Botía: None. Mahamdallie et al. Wellcome Open Research 2017, 2:35
(https://doi.org/10.12688/wellcomeopenres.11689.1) P16.15C Validation of an ultra-fast CNV calling tool for Next
Generation Sequencing data using MLPA-verified copy
number alterations Validation of an ultra-fast CNV calling tool for Next
Generation Sequencing data using MLPA-verified copy
number alterations Results: InCAS allows to comprehensively annotate
CNVs using continuously updated information of protein-
coding and non-coding elements as well as of clinically J. del Picchia 574 not necessarily the most important. With this in mind, we
pursue the identification of secondary networks. We focus
on
secondary
cell
type-specific
networks
since
CE
(WGCNA-based) networks are highly driven by cell type
and we know that genes vary considerably in terms of their
cellular specificity. We understand gene expression as an
additive model of eigengenes that we use for regression-
based cell type-specific signal correction. We create a co-
expression network from GTExV6 frontal cortex samples
and use its eigengenes to “clean” gene expression and create
a new network for each cell type. Using this approach
secondary cell type associations were identified for neurons
(403 genes), astrocytes (205 genes), oligodendrocytes (190
genes) and microglia (115 genes) to generate a 2.8 - 11.0%
increase in the predicted cell type-specific gene sets. We
assess whether these secondary gene assignments were
significantly enriched for true cell type-specific associations
using public transcriptomic data generated by immuno-
panning of human frontal cortex (http://web.stanford.edu/
group/barres_lab/). We detect significant enrichments in cell
type-specific gene expression for genes newly assigned to
astrocyte, neuron, microglia and oligodendrocyte-specific
modules, with amongst the most significant enrichments
detected for the reassignment of astrocytic genes to the
neuronal set (empirical p-value <9.9 x 10-5). Thus, we
demonstrate the utility of secondary co-expression networks
for identifying additional and potentially important novel
gene clusters. B. Tolhuis, H. Karten B. Tolhuis, H. Karten Secondary co-expression networks for the study of gene
roles in multiple cell types 1Institute of Medical Genetics and Human Genetics, Berlin,
Germany, 2Berlin Institute of Health, Berlin, Germany,
3Institute for Genomic Statistics and Bioinformatics, Bonn,
Germany, 4FDNA, Boston, MA, United States, 5GeneTalk,
Bonn, Germany J. A. Sánchez1, R. H. Reynolds2, J. Hardy2, M. Ryten2, J. A. Botía1,2 1Departamento de Ingeniería de la Información y las
Comunicaciones. Universidad de Murcia, Murcia, Spain,
2Department of Molecular Neuroscience, Institute of
Neurology, University College London, London, United
Kingdom 1Departamento de Ingeniería de la Información y las
Comunicaciones. Universidad de Murcia, Murcia, Spain,
2Department of Molecular Neuroscience, Institute of
Neurology, University College London, London, United
Kingdom Significant improvements in automated image analysis have
been achieved over the recent years and tools are now
increasingly being used in computer-assisted syndromol-
ogy. However, the ability to recognize a syndromic facial
gestalt might depend on the syndrome and may also be
confounded by severity of phenotype, size of available
training
sets,
ethnicity,
age,
and
sex. Therefore, Gene co-expression (CE) network analysis is an effective
approach for discovering gene clusters of functional sig-
nificance. Normally, CE assumes a gene has a single set of
“co-relationships”, and captures the most evident clustering, 575 Abstracts from the 51st European Society of Human Genetics Conference: Posters benchmarking and comparing the performance of deep-
learned classification processes is inherently difficult. enriching the data through mapping it to relevant biome-
dical ontologies, such as SNOMED CT and Human
Phenotype Ontology. Efficient ontology encoding of data,
in turn, is key to e.g. recently developed methods for using
hierarchical
structure
between
phenotypes
in
genetic
association analyses. For a systematic analysis of these influencing factors we
chose the lysosomal storage diseases Mucolipidosis as well
as Mucopolysaccharidosis type I and II, that are known for
their wide and overlapping phenotypic spectra. For a
dysmorphic comparison we used Smith-Lemli-Opitz syn-
drome
as
another
inborn
error
of
metabolism
and
Nicolaides-Baraitser syndrome. A classifier that was trained
on these five cohorts, comprising 288 patients in total,
achieved a mean accuracy of 62%. To solve the most common issues in clinical and
phenotypic data curation, we have developed the Accurate
(TM) data curation and ontology mapping solution. We
present
results
on
preliminary
benchmarking
of
the
narrative text analytics on the MIMIC III data set to
suggest good specificity and sensitivity for identification of
biomedically relevant concepts. Secondary co-expression networks for the study of gene
roles in multiple cell types Additionally, we present
results for benchmarking our ontology mapping approach
for structured data, illustrating the large gains achieved in
ontology mapping efficiency. We also developed a simulation framework to analyze the
effect of potential confounders, such as cohort size, age,
sex, or ethnic background on the distinguishability of
phenotypes. We found that the true positive rate increases
for all analyzed disorders for growing cohorts (n= [10...40])
while ethnicity and sex have no significant influence. In summary, we here present an intuitive and highly
efficient solution for curating clinical and phenotypic data,
emphasizing enriching it through ontology mapping of both
structured data and narrative text. The dynamics of the accuracies strongly suggest that the
maximum distinguishability is a phenotype-specific value,
that hasn’t been reached yet for any of the studied disorders. This should also be a motivation to further intensify data
sharing efforts, as computer-assisted syndrome classifica-
tion can still be improved by enlarging the available
training sets. H. Edgren: A. Employment (full or part-time); Sig-
nificant; MediSapiens Ltd. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; MediSapiens Ltd. B. Mano: A. Employment
(full or part-time); Significant; MediSapiens Ltd. M. Laaksonen: A. Employment (full or part-time); Significant;
MediSapiens Ltd. M. Roccato: A. Employment (full or
part-time); Significant; MediSapiens Ltd. H. Lodenius: A. Employment
(full
or
part-time);
Significant;
MediSapiens Ltd. J.T. Pantel: None. M. Zhao: None. M.A. Mensah:
None. N. Hajjir: None. T. Hsieh: None. Y. Hanani: A. Employment (full or part-time); Significant; FDNA. N. Fleischer: A. Employment (full or part-time); Significant;
FDNA. T. Kamphans: None. S. Mundlos: None. Y. Gurovich: A. Employment (full or part-time); Significant;
FDNA. P.M. Krawitz: None. Zenome as a new blockchain solution for storing,
processing and exchanging of genetic data Zenome as a new blockchain solution for storing,
processing and exchanging of genetic data Efficient curation and ontology mapping of clinical and
phenotypic data N. Kulemin1,2, A. Gorbachev2, V. Naumov2, A. Gubina2, S. Popov2 H. Edgren, B. Mano, M. Laaksonen, M. Roccato, H. Lodenius
Medisapiens Ltd, Helsinki, Finland H. Edgren, B. Mano, M. Laaksonen, M. Roccato, H. Lodenius
Medisapiens Ltd, Helsinki, Finland H. Edgren, B. Mano, M. Laaksonen, M. Roccato, H. Lodenius H. Edgren, B. Mano, M. Laaksonen, M. Roccato, H. Lodenius
Medisapiens Ltd, Helsinki, Finland 1Federal Research Clinical Center for Physical-Chemical
Medicine FMBA, Moscow, Russian Federation, 2Zenome.io ltd,
Moscow, Russian Federation Medisapiens Ltd, Helsinki, Finland G.G. Varenikov: None. M.Y. Skoblov: None. A bioinformatics framework to identify cell subpopulations
from bulk gene expression data of cancer samples A bioinformatics framework to identify cell subpopulations
from bulk gene expression data of cancer samples A. Grilli1,2, C. Battaglia3, S. Bicciato1 A. Grilli1,2, C. Battaglia3, S. Bicciato1 N. Kulemin: None. A. Gorbachev: None. V. Naumov:
None. A. Gubina: None. S. Popov: None. 1Department of Life Sciences, Center for Genome Research,
University of Modena and Reggio Emilia, Modena, Italy, 2PhD
Program of Molecular and Translational Medicine,
Department of medical Biotechnology and Traslational
Medicine, University of Milan, Segrate (MI), Italy,
3Department of Medical Biotechnology and Translational
Medicine (BIOMETRA), University of Milan, Milano, Italy Medisapiens Ltd, Helsinki, Finland Medisapiens Ltd, Helsinki, Finland Heterogeneity within and between data sources is one of the
primary impediments to assembling rich sets of phenotypic
and clinical data. Such heterogeneity can arise from multi-
ple sources: data collection practices evolve over time,
standards for data encoding may be missing, or the data
(e.g. EHR data) may simply originally have been collected
for another purpose. Additionally, narrative text, such as
medical statements, are inherently unstructured and require
curation before analysis. The main problem faced by researchers in the field of
human genetics is the accession to the structured genomic
and related data. In addition, most of the analyzed para-
meters are medical, and access to them is regulated by local
rules. But, although the banks of genomic data are already
actively developing, until there is no network with the real
time feedback operation.In order to solve the tasks and
significantly simplify the work with the data, a Zenome
project was created. Irrespective of where heterogeneity derives from, curation
of the data
is necessary
for efficient,
accurate
and
reproducible data analysis. In addition to correcting errors
and inconsistencies in the data, curation extends into It is a decentralized p2p network in which everyone can
place their data and update information about themselves, J. del Picchia 576 and those who wish to use this data can request permission. The technical basis of the network is the DHT Kademlia
decentralized data storage protocol, and the protocol of
blocking is used to fix the interaction. Also, the network
will use a token-based incentive system in which research-
ers will pay for access to the data, while the user who
provided the data will receive funds for their provision. To
defeat user's privacy, a self-developed algorithm for storing
open statements signed with identifiers is used. In such a
network, everyone can get information about the presence
of participants with a particular mutation or with one or
another response, but only the owner knows the connection
between the statements. Our service will make the costs for
GWAS and family genetic tests significantly lower. those mutations which can be edited without conversion of
nearby nucleotides. In total 220 T(A)>C(G) and 2002 C(G)
>T(A) mutations can be targeted by dCas9-deaminase for
single nucleotide editing. Conclusion: The list of 2222 ClinVar mutations provides
a valuable tool for the search of perspective targets for
targeted direct DNA editing with dCas9-deaminase tools. A.V. Lavrov: None. P16.20D Targeted single nucleotide editing allows correction of
hundreds of pathogenic variants in hereditary diseases A. V. Lavrov1,2, G. G. Varenikov3, M. Y. Skoblov1,3 A. V. Lavrov1,2, G. G. Varenikov3, M. Y. Skoblov1,3 A. V. Lavrov1,2, G. G. Varenikov3, M. Y. Skoblov1,3 1Research Center of Medical Genetics, Moscow, Russian
Federation, 2The Russian National Research Medical
University Named after N.I. Pirogov, Moscow, Russian
Federation, 3Moscow Institute of Physics and Technology
(State University), Moscow, Russian Federation The expression levels of biological samples are affected by
the intrinsically heterogeneous cell and tissue composition. Nevertheless, when analyzing transcriptional profiles, each
sample is generally evaluated in bulk without considering
the presence of multiple subpopulations. This limitation
might be extremely critical when analyzing gene expression
profiles from cancer samples, where dissecting the mixed
cell population could shed light on the intratumoral het-
erogeneity and on the molecular mechanisms shaping dif-
ferent cancer behaviors. Introduction: Recently new CRISPR/Cas9 tools have been
developed. Briefly nucleotide deaminases (ND) fused to
dCas9 allow targeted direct conversion from C(G) to T(A)
and from T(A) to C(G) without double-stranded brakes. It
means that in total four types of mutations in hereditary
diseases can be corrected: T>C, A>G, C>T, G>A. However
ND are active in a window of several nucleotides which
limits their potential usage. We built on Cibersort, a recently published deconvolution
algorithm (Newman et al., 2015), to design a framework for
the identification of cellular subpopulations from bulk gene
expression of cancer samples. After testing the efficacy of
the approach on mouse gene expression data, we applied the
framework to a set of clinically-defined Triple Negative
Breast Cancer (TNBC). Specifically, we defined a novel
gene signature starting from 55 samples characterized as
Luminal A, Luminal B, Her2, and TNBC subtypes by IHC. Afterward, we applied the gene signature to quantify the
fractions of each subtype in a set of 357 TNBC samples. Results confirmed that 71.4% of samples was indeed
enriched in the TNBC-like cell subpopulation, but also
evidenced that the remaining 28.6% of samples were not
TNBC-like. We are currently evaluating the correlation
between the detected subpopulation heterogeneity and the
clinical outcome reported for this cohort of TNBCs. Methods: ClinVar database was used to extract all known
pathogenic mutations. Nucleotide surrounding of each
mutation was analyzed for the available PAM sequence
and for the absence of other nucleotides which can be
nonspecifically changed by ND. P16.22B
Identification of Disease Causal Variant using Artificial
Intelligence same consortium. In an effort to solve this problem, we
developed TFC (Trusted Friend Computing), an extension
to the POP-Java programming language, enabling devel-
opers to create applications that can share data and com-
puting
power
in
a
secure
way. We
integrated
this
technology into GensearchNGS, an existing NGS data
analysis software developed by Phenosystems SA, to enable
users to share variant data in an easy and secure way. W. Li Breakthrough Genomics, Arcadia, CA, United States Breakthrough Genomics, Arcadia, CA, United States Breakthrough Genomics, Arcadia, CA, United States Genomic sequencing is important for diagnosing rare
genetic diseases or cancer. Typically, a patient carries
hundreds of thousands of genetic variants in the protein
coding regions in the genome. Since variant classification is
the basis for clinical diagnosis, it is often the bottle neck for
patients’ genomic data analysis, and for clinical reporting. In order to speed up the variant classification process, we set
out to see if it is possible to use artificial intelligence/
machine learning method to find disease causal variants
with satisfactory precision. Three sets of variants are iden-
tified with known labels, namely pathogenic, unknown
significance and benign, respectively. Feature engineering
techniques are used to figure out the best feature set to feed
into the model. Total eleven selected type of variant feature
families including 200 features are fed into the model for
training. Proprietary machine learning algorithm based on
random forest is used to learn a multi-label classification
model. The learned model achieves 72.0% accuray on the
held-out 20% dataset (48,211 variants) across all three
labels. Particularly for pathogenic variants, we achieve
74.9% precision and 64.3% recall (F-measure 69.2%). The
features that can dramatically increase F-measure for
pathogenic variant classification will be presented. In con-
clusion, machine learning method can take into account of
tens or hundreds of variant features for the variant classi-
fication process, which is labor intensive if it is done
manually. A well-trained model will speed up the causal
variant identification and clinical diagnosis. The lessons
learned and the future direction will be discussed. Methods: We extended the open source POP-Java
language to include the features needed to implement
TFC. Those include the ability to create a friend network (in
which computing resources can be shared) as well as the
integration of encrypted communications. P16.20D p
y
g
y
Results: We performed analysis of all variants registered
in ClinVar and found that 54957 T(A)>C(G) substitutions
constitute 18.2% of all records and 112740 C(G)>T(A)
other 37.4%. 7084 (12.9%) and 18702 (16.6%) of them
respectively were classified as pathogenic and likely
pathogenic variants. For 3744 T(A)>C(G) and 5550 C(G)
>T(A) mutations PAM sequence for Cas9 (NGG) was
found in 11-23 nucleotides up or downstream which enables
their conversion by ND fused to dCas9. Since deaminase is
active in the window of 12 nucleotides for T>C and 6
nucleotides for C>T mutations it’s important to select only Results: We performed analysis of all variants registered
in ClinVar and found that 54957 T(A)>C(G) substitutions
constitute 18.2% of all records and 112740 C(G)>T(A)
other 37.4%. 7084 (12.9%) and 18702 (16.6%) of them
respectively were classified as pathogenic and likely
pathogenic variants. For 3744 T(A)>C(G) and 5550 C(G)
>T(A) mutations PAM sequence for Cas9 (NGG) was
found in 11-23 nucleotides up or downstream which enables
their conversion by ND fused to dCas9. Since deaminase is
active in the window of 12 nucleotides for T>C and 6
nucleotides for C>T mutations it’s important to select only A. Grilli: None. C. Battaglia: None. S. Bicciato: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 577 P16.22B
Identification of Disease Causal Variant using Artificial
Intelligence We also modified
GensearchNGS to make use of those features to let
laboratories share both summary variant statistics (e.g is a
variant known by a certain laboratory) as well share
computing power through distributed sequence alignment. Results: We produced a version of GensearchNGS which
lets users share their data and computing power with a
selected list of friends. The underlying technology, POP-
Java, has been released as open-source software. Grants:
CTI no. 18781.1 PFES-ES B. Wolf: None. P. Kuonen: F. Consultant/Advisory
Board; Modest; Phenosystems SA. D. Mazzoleni: None. J. Stoppani: None. D. Atlan: A. Employment (full or part-
time); Significant; Phenosystems SA. Identification and characterization of 3ʹUTR mutations
affecting NFKBIZ in non-Hodgkin lymphoma S. Arthur1, B. Grande1, A. Jiang1, R. Cojocaru1, M. Alcaide1, C. Rushton1, P. K. Lat1, D. Ennishi2, S. Jessa1, P. Pararajalingam1,
L. Chong2, M. Boyle2, B. Meissner2, A. Mottok2, L. Sehn2, P. Farinha2, A. Mungall1, R. Gascoyne2, M. Marra2, D. Sen1, J. Connors2, T. Audas1, P. Unrau1, D. W. Scott2, R. D. Morin1
1Simon Fraser University, Burnaby, BC, Canada, 2BC Cancer,
Vancouver, BC, Canada S. Arthur1, B. Grande1, A. Jiang1, R. Cojocaru1, M. Alcaide1, C. Rushton1, P. K. Lat1, D. Ennishi2, S. Jessa1, P. Pararajalingam1,
L. Chong2, M. Boyle2, B. Meissner2, A. Mottok2, L. Sehn2, P. Farinha2, A. Mungall1, R. Gascoyne2, M. Marra2, D. Sen1, J. Connors2, T. Audas1, P. Unrau1, D. W. Scott2, R. D. Morin1 1Simon Fraser University, Burnaby, BC, Canada, 2BC Cancer,
Vancouver, BC, Canada 1Simon Fraser University, Burnaby, BC, Canada, 2BC Cancer,
Vancouver, BC, Canada W. Li: None. W. Li: None. P16.23C
Trusted Friend Computing: Securely share OMICs data P16.23C
Trusted Friend Computing: Securely share OMICs data Introduction: Diffuse large B-cell lymphoma (DLBCL) is
an aggressive cancer originating from mature B-cells. Most
somatic driver mutations characterized to date affect the
coding sequence of proteins or induce loss of function or
deregulated expression through deletion, translocation or
amplification. DLBCL has two molecular subgroups with
distinct genetic features and prognosis. A relative lack of
drivers unique to the activated B-cell (ABC) subgroup,
which suggests incomplete understanding of its molecular
aetiology. B. Wolf1, P. Kuonen1, D. Mazzoleni1, J. Stoppani1, D. Atlan2
1University of applied sciences and arts western switzerland,
Fribourg, Switzerland, 2Phenosystems SA, Wallonia, Belgium B. Wolf1, P. Kuonen1, D. Mazzoleni1, J. Stoppani1, D. Atlan2 B. Wolf1, P. Kuonen1, D. Mazzoleni1, J. Stoppani1, D. Atlan2 1University of applied sciences and arts western switzerland,
Fribourg, Switzerland, 2Phenosystems SA, Wallonia, Belgium Introduction: Sharing OMICs data is becoming increas-
ingly essential when analysing patient data. Yet many legal
and ethical constraints slow down or make it impossible to
share the data. This leads to a situation where many
laboratories store their valuable patient data only in internal
databases, unable to share them even with even others in the Materials and Methods: Using a combination of data
modalities including RNA-seq, copy number arrays, whole
genome sequencing and targeted sequencing of over 700 J. del Picchia 578 individual feature is a combined interaction evidence score
ranging from 0 for lowest interaction evidence to 0.999 for
highest. Different types of support vector machine algo-
rithms (SVMs) built with Python 3.5 and scikit-learn library
were used to classify proteins based on interaction data as
either epilepsy related or not. 1665 randomly selected
protein interaction data was used for training and 331 for
testing. individual feature is a combined interaction evidence score
ranging from 0 for lowest interaction evidence to 0.999 for
highest. Different types of support vector machine algo-
rithms (SVMs) built with Python 3.5 and scikit-learn library
were used to classify proteins based on interaction data as
either epilepsy related or not. 1665 randomly selected
protein interaction data was used for training and 331 for
testing. cases, we searched for patterns of noncoding mutations that
may affect gene expression or mRNA structure in DLBCL. We implemented a new algorithm to identify discrete loci
enriched for somatic noncoding single nucleotide variants
(SNVs) genome-wide. Reference-free eQTL analysis reveals the importance of
unannotated transcribed regions in neurological and
neuropsychiatric disorders Reference-free eQTL analysis reveals the importance of
unannotated transcribed regions in neurological and
neuropsychiatric disorders S. Guelfi1, D. Zhang1, K. D'Sa1, R. H. Reynolds1, J. A. Botia1,2,
UKBEC consortium, J. Hardy1, M. E. Weale3, M. Ryten1 P16.23C
Trusted Friend Computing: Securely share OMICs data Results: This analysis identified numerous genes and
non-coding loci that are commonly mutated in DLBCL,
with many of these attributable to aberrant somatic
hypermutation. Of particular interest were recurrent muta-
tions in the 3′UTR of NFKBIZ. We found these to cause
elevated mRNA and protein levels in lymphoma samples. Using a combination of techniques, we demonstrate that the
most commonly observed variants affect a conserved
structure in the mRNA. Moreover, we show that these
changes
increase
mRNA
abundance
by
reducing
its
cleavage by the RNAse MCPIP1. Results:
C-Support
Vector
Classification
algorithm
showed best performance on the testing set with an area
under curve (AUC) of 0.88, specificity of 0.84 and
sensitivity of 0.93. Similar performance was observed with
Nu-Support Vector Classification (AUC 0.86, specificity
0.79, sensitivity 0.92) and Linear Support Vector Classifi-
cation
(AUC
0.84,
specificity
0.87,
sensitivity
0.79)
algorithms. Results:
C-Support
Vector
Classification
algorithm
showed best performance on the testing set with an area
under curve (AUC) of 0.88, specificity of 0.84 and
sensitivity of 0.93. Similar performance was observed with
Nu-Support Vector Classification (AUC 0.86, specificity
0.79, sensitivity 0.92) and Linear Support Vector Classifi-
cation
(AUC
0.84,
specificity
0.87,
sensitivity
0.79)
algorithms. Conclusions: SVMs show a good performance on
epilepsy associated protein detection. Machine learning
approaches can be useful in novel epilepsy causing protein
discovery. Conclusions: Our results inform on mechanisms of NF-
κB pathway activation in ABC DLBCL and demonstrate the
importance of noncoding mutations in contributing to
oncogene over-expression in this cancer. K. ablauskas:
None. B. Tumienė:
None. A. Utkus: None. K. ablauskas:
None. B. Tumienė:
None. A. Utkus: None. S. Arthur: None. B. Grande: None. A. Jiang: None. R. Cojocaru: None. M. Alcaide: None. C. Rushton: None. P. K. Lat: None. D. Ennishi: None. S. Jessa: None. P. Pararajalingam: None. L. Chong: None. M. Boyle: None. B. Meissner: None. A. Mottok: None. L. Sehn: None. P. Farinha: None. A. Mungall: None. R. Gascoyne: None. M. Marra: None. D. Sen: None. J. Connors: None. T. Audas: None. P. Unrau: None. D.W. Scott: None. R.D. Morin: None. P16.25A
Machine learning approach for detecting epilepsy causing
proteins using protein interaction data Machine learning approach for detecting epilepsy causing
proteins using protein interaction data 1Institute of Neurology (UCL), London, United Kingdom,
2Departamento de Ingeniería de la Información y las
Comunicaciones, Universidad de Murcia, Murcia, Spain,
Murcia, Spain, 3Department of Medical & Molecular Genetics,
King's College London, London, United Kingdom K. Šablauskas1, B. Tumienė2, A. Utkus2 1Faculty of Medicine, Vilnius University, Vilnius, Lithuania,
2Institute of Biomedical Sciences, Faculty of Medicine, Vilnius
University, Vilnius, Lithuania 1Faculty of Medicine, Vilnius University, Vilnius, Lithuania,
2Institute of Biomedical Sciences, Faculty of Medicine, Vilnius
University, Vilnius, Lithuania Accurate gene annotation is required for the interpretation
of risk loci identified by genome-wide association studies
(GWAS). However, the complexity of splicing in the
human brain has made annotation of brain-expressed genes
a non-trivial task, and this may contribute to why the
majority of risk loci for neurological disorders remain
poorly characterised, even when relevant eQTL data exist. To address this issue, we used eQTL analysis in a reference-
free manner to improve GWAS interpretation. We used the
R package derfinder to identify and quantify transcribed
regions from RNA-sequencing data generated from human
non-diseased
post-mortem
brain
tissue
(hippocampus,
putamen and substantia nigra). After stringent quality con-
trol, cis-eQTL analysis was conducted on ~460,000 tran-
scribed regions. Strikingly, we identified a 2.1-fold increase
in eQTL detection as compared to an equivalent analysis Introduction: Genetic factors play a role in more than half
of all epilepsies. Proteins expressed by epilepsy associated
genes are known to be clustered in specific biochemical
pathways. This knowledge can be applied to find similar
proteins and genes that are part of these pathways. Materials and Methods: 998 expert reviewed epilepsy
genes and 998 random non-epilepsy genes expressed in
fetal
temporal
tissue
(ENCODE
accession
number:
ENCDO064AAA) were selected. Protein interaction data
expressed by the selected genes was accessed from
STRING database with a minimal interaction score of
0.151. Each protein was assigned 998 features based on
interaction data with 998 epilepsy related proteins. Each P16.27C P16.27C
Frailomic Project: Genetics in frailty Founded by: FP7-HEALTH-2012-INNOVATION-1 Founded by: FP7-HEALTH-2012-INNOVATION-1 L. Bernad Palomares: None. B. Cortina Gil: None. D. Valero Hervas: None. C. Moya: None. F. Garcia Garcia:
None. K. Peres-Bouchet: None. L. Rodriguez Mañas:
None. B. Herraez: None. G. Pita: None. G. Olaso
González: None. J. Gambini Buchón: None. D. Gomez
Cabrero: None. F. Rodriguez Artalejo: None. M. Cesari:
None. I. Ara Royo: None. R. Miñambres Herraiz: None. L. Bernad Palomares1, B. Cortina Gil1, D. Valero Hervas1, C. Moya2, F. Garcia Garcia3, K. Peres-Bouchet4, L. Rodriguez
Mañas5, B. Herraez6, G. Pita6, G. Olaso González7, J. Gambini
Buchón7, D. Gomez Cabrero8, F. Rodriguez Artalejo9, M. Cesari10, I. Ara Royo11, R. Miñambres Herraiz1 1Sistemas Genomicos, Paterna, Spain, 2Sistemas Genomicos,
Valencia, Spain, 3Geriatrics department. Toledo Hospital
Complex, Toledo, Spain, 4Bordeaux Health Research Center. Bordeaux University, Bordeaux, France, 5Geriatric
department. Getafe University Hospital, Getafe, Spain,
6CEGEN, Madrid, Spain, 7Physiology department. Valencia
University, Valencia, Spain, 8YouHealth, Stockholm, Sweden,
9Universidad Automoma de Madrid, Madrid, Spain,
10University of Toulouse, Toulouse, France, 11Castilla La
Mancha University, Toledo, Spain Abstracts from the 51st European Society of Human Genetics Conference: Posters 579 performed on annotated regions of the genome alone. Using
our own R package annotatER, which leverages reads
spanning exon-exon junctions, we found that a significant
proportion of unannotated transcribed regions were spliced
to known genes. Combining eQTLs with GWAS hits from
the NHGRI-EBI catalogue demonstrated a significant
enrichment of risk SNPs for neurological disorders amongst
eQTLs tagging unannotated transcribed regions. We further
characterised these findings with eQTL-GWAS co-locali-
sation analyses, and identified several eQTLs tagging
unannotated regions and co-localising with GWAS hits for
neurological and neuropsychiatric disorders. Our findings
confirm the utility of performing eQTL analyses in a
reference-free manner and demonstrate that unexplored
regions of the genome can be crucial for the understanding
of complex diseases. performed on annotated regions of the genome alone. Using
our own R package annotatER, which leverages reads
spanning exon-exon junctions, we found that a significant
proportion of unannotated transcribed regions were spliced
to known genes. Combining eQTLs with GWAS hits from
the NHGRI-EBI catalogue demonstrated a significant
enrichment of risk SNPs for neurological disorders amongst
eQTLs tagging unannotated transcribed regions. We further
characterised these findings with eQTL-GWAS co-locali-
sation analyses, and identified several eQTLs tagging
unannotated regions and co-localising with GWAS hits for
neurological and neuropsychiatric disorders. Our findings
confirm the utility of performing eQTL analyses in a
reference-free manner and demonstrate that unexplored
regions of the genome can be crucial for the understanding
of complex diseases. genotyped in a total of 1515 samples (TOLEDO and AMI
cohorts) and expression analysis of candidate genes was
performed in 819 samples (TOLEDO cohort). Results were
harmonized using a minimal model to identify and select
significant biomarkers. Results:
From
the
exploratory
phase,
4
SNPs
(rs17286758, rs2067051, rs6657616, rs2568958) and 5
candidate genes (NFE2L2, TP53, TXNRD1, HMOX2,
HIF1A) statistically significant associated to frailty were
selected to be tested in the validation phase in a second
independent series formed by a total of 1000 samples from
ENRICA, EXERNET, TOLEDO cohorts for genotyping
and 700 samples from EXERNET and MAPT Cohorts from
expression
analysis. The
results
are
currently
being
analysed. Conclusion: The results obtained in this set of genetic
testing would enable the performance of a close following-
up of these patients, or even prevent possible health-related
problems conditions, thus increasing its quality of life,
while reducing direct and indirect derived cost of the
national health care system. Conclusion: The results obtained in this set of genetic
testing would enable the performance of a close following-
up of these patients, or even prevent possible health-related
problems conditions, thus increasing its quality of life,
while reducing direct and indirect derived cost of the
national health care system. S. Guelfi: None. D. Zhang: None. K. D'Sa: None. R.H. S. Guelfi: None. D. Zhang: None. K. D'Sa: None. R.H. S. Guelfi: None. D. Zhang: None. K. D'Sa: None. R.H. Reynolds: None. J.A. Botia: None. J. Hardy: None. M.E. Weale: A. Employment (full or part-time); Significant;
Genomics plc. M. Ryten: None. S. Guelfi: None. D. Zhang: None. K. D'Sa: None. R.H. Reynolds: None. J.A. Botia: None. J. Hardy: None. M.E. Weale: A. Employment (full or part-time); Significant;
Genomics plc. M. Ryten: None. P16.29A
CYP21A2 mutation update: Comprehensive analysis of
databases and published genetic variants We have developed predictive models that use prior biolo-
gical information on genomic features that more accurately
predict genetic predisposition. Genomic features, consisting
of a set of genetic markers, are regions of the genome that
are linked to some external information (e.g. prior QTL
information, genes, biological pathways, gene ontologies,
sequence annotation). The main idea underlying this mod-
eling approach is that these regions are enriched for causal
variants affecting a specific trait. This is accounted for in
our model by using prior information on the relative con-
tribution of different genome regions to the overall trait
heritability. This
information
is
used
for
differential
shrinkage of single marker effects allowing markers to
contribute differently to the overall genetic predisposition
which in turn leads to more accurate predictions. The pre-
dictions are based on a genomic feature best linear unbiased
prediction (GFBLUP) model implemented using a Gauss
Seidel (GS) method. In order to handle very large genotype-
phenotype data sets (e.g. UK Biobank) a matrix-free GS
method based on adjustment of residuals was used for
solving the linear equation systems. Our simulation studies
show that it is possible to further increase the accuracy of
genomic prediction for complex traits using this model,
provided the genomic features are enriched for causal var-
iants. Our GFBLUP model using prior information on
genomic features enriched for causal variants can increase
the accuracy of genomic predictions in populations of
unrelated individuals and provides a formal statistical fra-
mework for leveraging and evaluating information across C. D. Bruque1,2, L. Simonetti3, C. S. Fernandez1, B. Belen1, D. Marisol1, J. E. Kolomenski2, L. D. Espeche1, N. D. Buzzalino1,
A. D. Nadra4,5, L. Dain1,2,5 1Centro Nacional de Genética Médica - ANLIS-Malbrán,
CABA, Argentina, 2Instituto de Biología y Medicina
Experimental, CONICET., Caba, Argentina, 3Uppsala
Universit - Department of Chemistry - BMC, Biochemistry,
Uppsala, Sweden, 4Departamento de Química Biológica,
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, IQUIBICEN-CONICET.., CABA, Argentina,
5Departamento de Fisiología, Biología Molecular y Celular,
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires., Caba, Argentina Congenital adrenal hyperplasia (CAH) is a group of auto-
somal recessive disorders of adrenal steroidogenesis. Dis-
orders in steroid 21-hydroxylation account for over 95% of
patients with CAH. Clinically, the 21-hydroxylase defi-
ciency has been classified in a broad spectrum of clinical
forms, ranging from severe or classical, to mild late onset or
non-classical. Aarhus University, Tjele, Denmark Aarhus University, Tjele, Denmark Bioinformatic pipeline validation for the application of next
generation sequencing in genetic testing Bioinformatic pipeline validation for the application of next
generation sequencing in genetic testing Bioinformatic pipeline validation for the application of next
generation sequencing in genetic testing S. Merella1, S. Benedetti1, C. Di Resta2,3, G. Pipitone1, M. Ferrari1,2,3, P. Carrera1,2 S. Merella1, S. Benedetti1, C. Di Resta2,3, G. Pipitone1, M. Ferrari1,2,3, P. Carrera1,2 Ferrari1,2,3, P. Carrera1,2 1IRCCS San Raffaele Scientific Institute, Clinical Molecular
Biology Laboratory, Milan, Italy, 2IRCCS San Raffaele
Scientific Institute, Unit of Genomics for human disease
diagnosis, Milan, Italy, 3University of Vita San Salute San
Raffaele, Milan, Italy 1IRCCS San Raffaele Scientific Institute, Clinical Molecular
Biology Laboratory, Milan, Italy, 2IRCCS San Raffaele
Scientific Institute, Unit of Genomics for human disease
diagnosis, Milan, Italy, 3University of Vita San Salute San
Raffaele, Milan, Italy Introduction: The forecasted increase in the number of
older people will be accompanied by an increase of those
with disabilities. Disability is preceded by frailty, a disorder
that encompasses changes associated with ageing, life style. OSR Hospital introduced NGS for genetic testing but
accurate identification of genomic variants is a critical factor
which may affect sensitivity and specificity.We present the
validation of a bioinformatic pipeline to detect germline
variants in inherited diseases. The pipeline was first applied
to 66 patients with known genotype sequenced with 4 dif-
ferent Illumina panels using Illumina platforms. We focused
our attention to genes specific for the patient disease con-
sidering a total of 9 different disorders. The pipeline per-
forms all the steps necessary to generate a diagnostic report: OSR Hospital introduced NGS for genetic testing but
accurate identification of genomic variants is a critical factor
which may affect sensitivity and specificity.We present the
validation of a bioinformatic pipeline to detect germline
variants in inherited diseases. The pipeline was first applied
to 66 patients with known genotype sequenced with 4 dif-
ferent Illumina panels using Illumina platforms. We focused
our attention to genes specific for the patient disease con-
sidering a total of 9 different disorders. The pipeline per-
forms all the steps necessary to generate a diagnostic report: Objective: Characterize clinical and omics-based labora-
tory biomarkers related with frailty to diagnosis, prognosis
and monitoring of frailty. Material
and
Methods:
A
two-phase
study
was
designed: Exploratory and Validation phase in a well-
established prospective cohort of elderly people. 256 SNPs
and 20 candidates genes were selected taking into account
published literature related with frailty. Selected SNPs were J. Bioinformatic pipeline validation for the application of next
generation sequencing in genetic testing del Picchia 580 alignment, variant calling, filtering for high quality variants
and high resolution evaluation of coverage statistics. The
pipeline is able to detect very small regions (<20 bp) with
mean coverage <20X. We tested different variant callers
and we highlighted differences in called variants. In the end
we decided to include 3 different callers in our pipeline:
Freebayes, GATK HaplotypeCaller and Illumina commer-
cial software. We obtained a specificity ≥94% and a sen-
sitivity ≥99% with differences between variant caller and
sequencing panels. In particular Illumina pipeline performs
better in SNV detection while HaplotypeCaller and Free-
bayes in INDELs (up to 40 bp). Combining the results of
these three softwares we were able to detect all the 427
known variants in our dataset. Moreover, 4 samples were
previously sequenced with a different NGS method and we
had a high confidence SNV dataset with 100% concordance
with previous results.Overall our pipeline allows high
confidence in variant detection with a low probability of
mutations loss. database. Nevertheless, a large number of variants are being
described in massive genome projects, many of which are
found in dbSNP, but lack functional implications and/or
their phenotypic effect. In this work, we gathered a total of
1,340 GVs in the CYP21A2 gene, from which 899 variants
were unique and 230 have an effect on human health, and
compiled all this information in an integrated database. We
also connected CYP21A2 sequence information to pheno-
typic effects for all available mutations, including double
mutants in cis. Data compiled in the present work could
help physicians in the genetic counseling of families
affected with 21-hydroxylase deficiency. C.D. Bruque: None. L. Simonetti: None. C.S. Fernan-
dez: None. B. Belen: None. D. Marisol: None. J.E. Kolomenski: None. L.D. Espeche: None. N.D. Buzzalino:
None. A.D. Nadra: None. L. Dain: None. P. Duun Rohde, I. Sørensen, P. Sørensen P. Duun Rohde, I. Sørensen, P. Sørensen P. Duun Rohde, I. Sørensen, P. Sørensen Genomic Feature Prediction Models Genomic Feature Prediction Models S. Merella: None. S. Benedetti: None. C. Di Resta:
None. G. Pipitone:
None. M. Ferrari:
None. P. Carrera: None. P16.29A
CYP21A2 mutation update: Comprehensive analysis of
databases and published genetic variants Known allelic variants in the disease causing
CYP21A2 gene are spread among different sources. Until
recently, most variants reported have been identified in the
clinical setting, which presumably bias described variants to
pathogenic ones, as those found in the CYPAlleles Abstracts from the 51st European Society of Human Genetics Conference: Posters 581 multiple experimental studies to provide more accurate
predictions. members of signaling pathways related to carcinogenesis
process. P. Duun
Rohde:
None. I. Sørensen:
None. P. Sørensen: None. Conclusion: This genomic predictive model for recur-
rence and metastasis development may help in a more
practical and individualized patient management and,
eventually contribute to a more targeted drug design. Predictive model for metastasis and recurrence in head and
neck cancer using amplified genes of tumor samples I.P. Ribeiro: None. L. Esteves: None. F. Marques:
None. L. Barroso:
None. I.P. Baptista:
None. F. Caramelo: None. J.B. Melo: None. I.M. Carreira: None. I. P. Ribeiro1,2, L. Esteves1, F. Marques3,2, L. Barroso4,2, I. P. Baptista3,2, F. Caramelo5, J. B. Melo1,2, I. M. Carreira1,2 Dante - genotyping of complex and expanded short tandem
repetitions 1Cytogenetics and Genomics Laboratory, Faculty of Medicine,
University of Coimbra, Coimbra, Portugal, 2iCBR-CIMAGO -
Center of Investigation on Environment Genetics and
Oncobiology - Faculty of Medicine, University of Coimbra,
Coimbra, Portugal, 3Department of Dentistry, Faculty of
Medicine, University of Coimbra, Coimbra, Portugal,
4Maxillofacial Surgery Department, Coimbra Hospital and
University Centre, CHUC, EPE, Coimbra, Portugal,
5Laboratory of Biostatistics and Medical Informatics, iCBR -
Faculty of Medicine, University of Coimbra, Coimbra,
Portugal J. Budiš1,2,3, M. Kucharík4, F. Ďuri2,3, J. Gazdarica2,5, M. Zrubcová5, A. Ficek5, T. Szemes2,5,6, B. Brejová1, M. Lichvár2, R. Hekel2,3, J. Radvanszky2,5,7 1Department of Computer Science, Faculty of Mathematics,
Physics and Informatics, Comenius University, Bratislava,
Slovakia, 2Geneton Ltd., Bratislava, Slovakia, 3Slovak Centre
of Scientific and Technical Information, Bratislava, Slovakia,
4Medirex a.s., Bratislava, Slovakia, 5Department of Molecular
Biology, Faculty of Natural Sciences, Comenius University,
Bratislava, Slovakia, 6Comenius University Science Park,
Bratislava, Slovakia, 7Institute for Clinical and Translational
Research, Biomedical Research Centre, Slovak Academy of
Sciences, Bratislava, Slovakia Introduction: There is an urgent need to develop effective
diagnostic and prognostic approaches for head and neck
squamous cell carcinoma (HNSCC). This study aimed to
implement a model of prediction of recurrence and metas-
tasis using a genome-wide characterization of HNSCC
samples. Introduction: Short tandem repeats (STRs) are DNA
stretches in which short sequences (2-6 nucleotides) are
repeated several times. Since STRs have many important
biological roles and belong to the most polymorphic parts of
the human genome, they became utilized in several
molecular-genetic applications. Genotyping of STR alleles,
therefore, was of high relevance during the last decades. Despite this, massively parallel sequencing (MPS) still lacks
the analysis methods to fully utilize the information value of
STRs in genome scale assays. Material and Methods: The genomic characterization of
tumor samples in a 102 HNSCC patients’ cohort was
performed using array comparative genomic hybridization
technique,
Agilent
4x180K. We
identified
the
most
statistically significant signalling pathways for amplification
and deletion and the respective altered genes, using the
Homo.sapiens package in R and the overrepresented
pathways were determined, using the limma package. A
genomic signature that distinguishes between patients with
metastasis/recurrence from those without, was determined
using the LASSO regression analysis. The selected genes
were then used to calculate the model’s performance and
prediction accuracy in a logistic regression classifier with
balanced training and test sets. This genomic signature was
validated in an independent cohort of 176 patients from the
TCGA database. Dante - genotyping of complex and expanded short tandem
repetitions Material and Methods: The genomic characterization of
tumor samples in a 102 HNSCC patients’ cohort was
performed using array comparative genomic hybridization
technique,
Agilent
4x180K. We
identified
the
most
statistically significant signalling pathways for amplification
and deletion and the respective altered genes, using the
Homo.sapiens package in R and the overrepresented
pathways were determined, using the limma package. A
genomic signature that distinguishes between patients with
metastasis/recurrence from those without, was determined
using the LASSO regression analysis. The selected genes
were then used to calculate the model’s performance and
prediction accuracy in a logistic regression classifier with
balanced training and test sets. This genomic signature was
validated in an independent cohort of 176 patients from the
TCGA database. Materials and Methods: We propose an alignment-free
algorithm for genotyping and characterizing STR alleles
based on sequence reads originating from STR loci of
interest. The method accounts for natural deviations from
the expected sequence, such as variation in the repeat count,
sequencing errors, ambiguous bases, and complex loci
containing several different motifs. We implemented a
correction
for
copy
number
defects
caused
by
the
polymerase induced stutter effect and prediction of STR
expansions that, according to the conventional view, cannot
be fully captured by inherently short MPS reads. Results: The developed predictive model using 17
amplified genes showed a good accuracy (78%, CI95%
[54; 88] %) and was validated in an independent population
(TCGA) (66%, CI95% [50; 80.3] %). This genomic
predictive model comprises regions from chromosomes 4,
5, 7, 8, 10, 11, 16, 17, 18 and 22, where are mapped several Results: We tested Dante on simulated data sets and on
data sets obtained by targeted sequencing of protein coding
parts of thousands of selected clinically relevant genes. In 582 J. del Picchia both these data sets, Dante outperformed HipSTR and
GATK genotyping tools. Furthermore, Dante was able to
predict allele expansions in all tested clinical cases. both these data sets, Dante outperformed HipSTR and
GATK genotyping tools. Furthermore, Dante was able to
predict allele expansions in all tested clinical cases. data, such as the method of faecal DNA purification and the
selected 16S gene variable region, and confirmed that this
pipeline successfully reduced the technical biases between
the cohorts. A preliminary analysis on ten most abundant
bacterial genera successfully replicated well-established
microbe-SNP associations, such as the functional LCT-
Bifidobacterium linkage, and identified five novel genome-
wide associations. Dante - genotyping of complex and expanded short tandem
repetitions To our knowledge, this is the largest
consortium devoted to microbiome GWAS, and we aim to
bring new knowledge to the rapidly expanding field of
microbiome research. The presented work was supported by the “REVOGENE
- Research centre for molecular genetics” project (ITMS
26240220067) supported by the Operational Programme
Research and Development funded by the ERDF. J. Budiš: A. Employment (full or part-time); Significant;
Geneton Ltd., Bratislava, Slovakia. M. Kucharík: A. Employment (full or part-time); Significant; Medirex a.s.,
Bratislava, Slovakia. F. Ďuri: A. Employment (full or part-
time); Significant; Geneton Ltd., Bratislava, Slovakia. J. Gazdarica: A. Employment (full or part-time); Significant;
Geneton Ltd., Bratislava, Slovakia. M. Zrubcová: None. A. Ficek: None. T. Szemes: A. Employment (full or part-
time); Significant; Geneton Ltd., Bratislava, Slovakia. B. Brejová: None. M. Lichvár: A. Employment (full or part-
time); Significant; Geneton Ltd., Bratislava, Slovakia. R. Hekel: A. Employment (full or part-time); Significant;
Geneton Ltd., Bratislava, Slovakia. J. Radvanszky: A. Employment (full or part-time); Modest; Geneton Ltd.,
Bratislava, Slovakia. A. Kurilshikov: None. J. Wang: None. D. Radjabza-
deh: None. L. Franke: None. J. Raes: None. R. Kraaij:
None. A. Zhernakova: None. A. Buniello1,2, O. Vrousgou1,2, M. Ghoussaini2, L. J. W. Harris1,
J. A. L. MacArthur1, T. Burdett1, H. Parkinson1, F.
Cunningham1, I. Dunham2, J. Barrett2 1European Molecular Biology Laboratory, European
Bioinformatics Institute, Hinxton, Cambridge, United
Kingdom, 2Open Targets, an academic-industrial
collaboration between the Wellcome Sanger Institute,
European Bioinformatics Institute (EMBL-EBI),
GlaxoSmithKline plc., Biogen Inc. and Takeda
Pharmaceuticals, Wellcome Genome Campus, Hinxton,
Cambridge, United Kingdom Expanding the scope of the GWAS Catalog to improve
drug target identification and prioritisation Expanding the scope of the GWAS Catalog to improve
drug target identification and prioritisation A. Buniello1,2, O. Vrousgou1,2, M. Ghoussaini2, L. J. W. Harris1,
J. A. L. MacArthur1, T. Burdett1, H. Parkinson1, F. Cunningham1, I. Dunham2, J. Barrett2 MiBioGen: a new consortium for meta-analysis of human
genome-microbiome association A. Kurilshikov1, J. Wang2, D. Radjabzadeh3, MiBioGen
Consortium Initiative, L. Franke1, J. Raes2, R. Kraaij3, A. Zhernakova1 A. Kurilshikov1, J. Wang2, D. Radjabzadeh3, MiBioGen
Consortium Initiative, L. Franke1, J. Raes2, R. Kraaij3, A. Zhernakova1 A. Kurilshikov1, J. Wang2, D. Radjabzadeh3, MiBioGen
Consortium Initiative, L. Franke1, J. Raes2, R. Kraaij3, A. Zhernakova1 1University Medical Center Groningen, Groningen,
Netherlands, 2KU Leuven–University of Leuven, Leuven,
Belgium, 3Erasmus Medical Center, Rotterdam, Netherlands 1University Medical Center Groningen, Groningen,
Netherlands, 2KU Leuven–University of Leuven, Leuven,
Belgium, 3Erasmus Medical Center, Rotterdam, Netherlands The identification of disease-associated genetic loci and
validation of drug targets are overarching drivers of scien-
tific research. Open Targets provides robust integration of
evidence from different public data sources to associate
genes, proteins and pathways with diseases, with the aim of
prioritising targets for drug discovery. A major source of
genetic evidence is the NHGRI-EBI GWAS Catalog, a
manually curated open resource repository of all published
GWAS results. As of January 2018, the GWAS Catalog
contains 4,847 studies and 56,935 unique SNP-trait asso-
ciations from 3,280 publications. Increasing evidence that the gut microbiome plays a role in
human health calls for further investigations into the factors
that determine the structure of the symbiotic relation
between host and microbes. As demonstrated by the initial
genome-wide association studies on microbiome composi-
tion published in recent years, the host genome is clearly
one of these factors. Even so, high inter-individual varia-
bility in the gut microbiome and its capacity to respond to
environmental
exposures
requires
large,
multi-ethnic,
population-based study designs to ensure sufficient statis-
tical power for detecting genotype-microbe associations. To
this end, we have established a collaborative initiative,
MiBioGen, that joins 18 population-based cohorts com-
prising more than 20,000 samples for which both genetic
(genome-wide genotyping platforms) and microbiome (16S
rRNA gene taxonomic profiling) are available. We devel-
oped a standardized pipeline to harmonize the different
platforms and methods used to generate the microbiome To date, curation has been limited to array-based GWAS
including association analysis of >100,000 SNPs with
genome-wide coverage and SNP-trait associations with a p-
value <1x10-5. Together with Open Targets, we are working
on expanding the scope of the Catalog to: 1) include association studies carried out using targeted
arrays (such as MetaboChip, ImmunoChip and exome
arrays) to increase the number of causal variants that focus
on immunologic, metabolic and oncologic phenotypes (key
therapeutic areas for Open Targets). Institute of Clinical Molecular Biology, Kiel, Germany Results: The male nuclear diploid genome extends for
6.27 Gigabase pairs (Gbp), is 205.00 centimeters (cm) long
and weighs 6.41 picograms (pg). Female values are 6.37
Gbp, 208.23 cm, 6.51 pg. The individual variability and the
implication for the DNA informational density in terms of
bits/volume were discussed. The genomic GC content is
40.9%. Following analysis in different transcriptomes and
species, we showed that the greatest deviation was observed
in the pathological condition analysed (trisomy 21 leukemic
cells) and in Caenorhabditis elegans. Conclusions: Our results may represent a solid basis for
further investigations on human structural and functional
genomics while also providing a framework for other
genome comparative analysis. A. Piovesan: None. M.C. Pelleri: None. F. Antonaros:
None. P. Strippoli: None. M. Caracausi: None. L. Vitale: None. MiBioGen: a new consortium for meta-analysis of human
genome-microbiome association Curation of fifty-five 583 Abstracts from the 51st European Society of Human Genetics Conference: Posters high accuracies, making the use of this dataset for future
research promising. high accuracies, making the use of this dataset for future
research promising. high accuracies, making the use of this dataset for future
research promising. targeted array publications for inclusion in the GWAS
Catalog is ongoing. targeted array publications for inclusion in the GWAS
Catalog is ongoing. 2) develop a comprehensive database of all available
GWAS
summary
statistics
(with
no
p-value
cut-off)
harmonised across studies to enable easy comparison and
downstream analysis. These data will be made publicly
available via the GWAS Catalog website and, in the future,
through an application programming interface (API). 2) develop a comprehensive database of all available
GWAS
summary
statistics
(with
no
p-value
cut-off)
harmonised across studies to enable easy comparison and
downstream analysis. These data will be made publicly
available via the GWAS Catalog website and, in the future,
through an application programming interface (API). F. Degenhardt: None. M. Wendorff: None. M. Wittig:
None. A. Franke: None. 2) develop a comprehensive database of all available
GWAS
summary
statistics
(with
no
p-value
cut-off)
harmonised across studies to enable easy comparison and
downstream analysis. These data will be made publicly
available via the GWAS Catalog website and, in the future,
through an application programming interface (API). F. Degenhardt: None. M. Wendorff: None. M. Wittig:
None. A. Franke: None. Institute of Clinical Molecular Biology, Kiel, Germany Genotype imputation of the Human Leukocyte Antigen
region (HLA), has become increasingly popular in the last
years. In the research setting, imputation helps avoid costs
for wetlab-based HLA typing and thus renders large-cohort
analyses of the HLA feasible. Here, we present a high-
quality multiethnic reference dataset based on genotypes
measured with the Illumina Immunochip genotyping array
including samples from Europe, China, India, Iran, Japan,
Korea and Malta and an admixed African American (AA)
dataset. We typed more than 1,300 samples from the afore-
mentioned eight different ethnic backgrounds using an in-
house high-resolution NGS-based approach and our allele-
calling tool HLAssign (Wittig et al., 2015). Manually
curated 4 digit-level alleles for the classical class I and II
loci were included in the reference panel. Using extensive
cross validation and different combinations of reference
datasets, we analysed imputation outputs of HIBAG (Zheng
et al. 2014) and Beagle (Browning et al., 2016). While
HIBAG had a mean imputation accuracy across all loci of
0.96 ±0.03 for the AA, 0.99 ±0.02 for Malta and Europe
and 0.98 ±0.2 to 0.4 for the remaining cohorts, Beagle had
an accuracy 0.97 ±0.02 for the AA, 0.99 ± 0.02 and
0.98 ±0.02 to 0.03 for the other cohorts. While median
accuracies across the loci are higher for HIBAG than for
Beagle, mean accuracies are slightly higher for Beagle
using our in-house dataset only. In summary, both Beagle
and HIBAG are able to impute our multiethnic dataset with Genotype imputation of the Human Leukocyte Antigen
region (HLA), has become increasingly popular in the last
years. In the research setting, imputation helps avoid costs
for wetlab-based HLA typing and thus renders large-cohort
analyses of the HLA feasible. Here, we present a high-
quality multiethnic reference dataset based on genotypes
measured with the Illumina Immunochip genotyping array
including samples from Europe, China, India, Iran, Japan, Results: The male nuclear diploid genome extends for
6.27 Gigabase pairs (Gbp), is 205.00 centimeters (cm) long
and weighs 6.41 picograms (pg). Female values are 6.37
Gbp, 208.23 cm, 6.51 pg. The individual variability and the
implication for the DNA informational density in terms of
bits/volume were discussed. The genomic GC content is
40.9%. Following analysis in different transcriptomes and
species, we showed that the greatest deviation was observed
in the pathological condition analysed (trisomy 21 leukemic
cells) and in Caenorhabditis elegans. Construction of a multiethnic HLA-imputation reference
panel Construction of a multiethnic HLA-imputation reference
panel Materials and Methods: By using updated data and
original software we determined these values to the best of
our knowledge as standard reference for the whole human
nuclear genome, for each chromosome and for mitochon-
drial DNA. We also devised a method to calculate the
relative GC content in the whole messenger RNA sequence
set and in transcriptomes by multiplying the GC content of
each gene by its mean expression level. F. Degenhardt, M. Wendorff, M. Wittig, TE HLA Working
Group of the IBDGC, A. Franke Institute of Clinical Molecular Biology, Kiel, Germany Department of Experimental, Diagnostic and Specialty
Medicine (DIMES), Bologna, Italy Department of Experimental, Diagnostic and Specialty
Medicine (DIMES), Bologna, Italy A. Buniello: None. O. Vrousgou: None. M. Ghoussaini:
None. L.J.W. Harris: None. J.A.L. MacArthur: None. T. Burdett: None. H. Parkinson: None. F. Cunningham:
None. I. Dunham: None. J. Barrett: None. Introduction: Basic parameters commonly used to describe
genomes including length, weight and relative guanine-
cytosine (GC) content are widely cited in absence of a
primary source. On the length, weight and GC content of the human
genome This collaboration aims to vastly increase the number of
available
disease
associations,
ultimately
leading
to
improved target identification and disease prognosis. This collaboration aims to vastly increase the number of
available
disease
associations,
ultimately
leading
to
improved target identification and disease prognosis. A. Piovesan, M. C. Pelleri, F. Antonaros, P. Strippoli, M. Caracausi, L. Vitale E. Tsare1,2, A. Gioutlakis1,2, M. I. Klapa2, N. K. Moschonas1,2 A. Piovesan, M. C. Pelleri, F. Antonaros, P. Strippoli, M.
Caracausi, L. Vitale Grant reference: 40237 OTAR034-02 Grant reference: 40237 OTAR034-02 Department of Experimental, Diagnostic and Specialty
Medicine (DIMES), Bologna, Italy E. Tsare1,2, A. Gioutlakis1,2, M. I. Klapa2, N. K. Moschonas1,2 Fleischer5, H. David-Eden4, Y. Hanani4, T. Kamphans6, D.
Horn2, S. Mundlos2, P. Krawitz1 These pathways contribute to the: (i)
regulation of myocardial contraction, (ii) blood circulation,
(iii)
cardiac
tissue
morphogenesis
and
(iv)
pressure-
natriuresis mechanism. Results: A prominent cluster was formed by BRAF,
PTPN11, NRAS and other genes of the MAPKinase
pathway that result in phenotypes such as Noonan,
LEOPARD or cardiofaciocutaneous syndrome and which
are considered similar. Likewise, many genes linked to
inborn errors of metabolism also clustered, mirroring a
higher-level feature that is referred to by clinicians as
‘coarse facies’. Furthermore, we also found genes involved
in chromatin remodeling to be near neighbors, even if no
superordinate joint feature exists in medical terminology to
describe the associated diseases. Conclusions: This study furthers our understanding on
the molecular architecture of hypertension, elucidates
disease modules with a central role in abnormal blood
pressure regulation and prioritizes hypertension predispos-
ing genes. Supported by: Hellenic Foundation for Research
and Innovation (HFRI) PhD scholarship grant; NSRF 2014-
2020 ELIXIR-GR (MIS 5002780) and BITAD (MIS
5002469) projects. Conclusion: We were able to reproduce molecular
interactions by information encoded in the facial gestalt
by using NGP tools. Thus, the phenotype space exploration
is a promising subject in the characterization of gene
function. E. Tsare: None. A. Gioutlakis: None. M.I. Klapa:
None. N.K. Moschonas: None. T. Hsieh: None. N. Hajjir: None. J.T. Pantel: None. M. Mensah: None. M. Zhao: None. J. Hertzberg: None. M. Schubach: None. S. Köhler: None. Y. Gurovich: A. Employment (full or part-time); Significant; FDNA. N. Fleischer: A. Employment (full or part-time); Significant;
FDNA. H. David-Eden: A. Employment (full or part-time);
Significant; FDNA. Y. Hanani: A. Employment (full or
part-time); Significant; FDNA. T. Kamphans: None. D. Horn: None. S. Mundlos: None. P. Krawitz: None. Fleischer5, H. David-Eden4, Y. Hanani4, T. Kamphans6, D.
Horn2, S. Mundlos2, P. Krawitz1 Fleischer5, H. David-Eden4, Y. Hanani4, T. Kamphans6, D. Horn2, S. Mundlos2, P. Krawitz1 Fleischer5, H. David-Eden4, Y. Hanani4, T. Kamphans6, D. Horn2, S. Mundlos2, P. Krawitz1 1Medical School, University of Patras, Rio, Greece, 2Institute
of Chemical Engineering Sciences, Foundation for Research
and Technology – Hellas (FORTH/ICE-HT), Patras, Greece 1Institute for Genomic Statistics and Bioinformatics, Bonn,
Germany, 2Charité Universitätsmedizin Berlin, Berlin,
Germany, 3Berlin Institute of Health, Berlin, Germany,
4FDNA, Boston, MA, United States, 5FDNA, Bonn, MA, United
States, 6GeneTalk, Bonn, Germany Introduction: Hypertension is a major cardiovascular dis-
ease and stroke risk factor. To-date, many genes have been
suggested as associated to blood pressure regulation
through polycentric GWAS. These findings can be vali-
dated and extended, if analyzed in the context of the protein
interaction network where proteins encoded by disease-
associated genes may form functional subnetworks. Our
objective is to combine hypertension GWAS with relevant
protein interactοme data for the elucidation of blood pres-
sure regulation mechanisms and prioritization of hyperten-
sion predisposing genes. Introduction: Recent advances in computer vision and
machine learning resulted in the next-generation pheno-
typing (NGP) tools for syndromology. Deep convolutional
neural networks such as DeepGestalt in Face2Gene have
been trained on thousands of patient photos with confirmed
molecular diagnoses and learned phenotype representations
of multiple disorders. These systems of artificial intelligence
support clinicians in the diagnostic workup of patients and
even excel human experts in certain classification tasks. We
therefore wondered whether this unprecedented sensitivity
to detect mutation-specific patterns in the facial gestalt
could also be used to reveal information about gene
function. Materials and Methods: Primary hypertension GWAS
data were retrieved from recent studies referring collectively
to approximately 1,200,000 samples. The hypertension-
related protein interactome was reconstructed using the
PICKLE
meta-database
(www.pickle.gr)
and
analyzed
using the Cytoscape software. Gene clustering and func-
tional
annotation
was
performed
using
the
DAVID
platform. Materials and Methods: We designed the knowledge
base Deep Phenotyping for Deep Learning (DPDL) as a
public resource to compile similarity scores from NGP tools
and performed a cluster analysis on currently 1216 cases
with monogenic disorders. Results: About 350 hypertension-associated genes were
retrieved from GWAS. The hypertension-associated protein
interactome analysis revealed a subnetwork of 81 inter-
connected proteins; some, e.g. NPR1, GJA1, MME, are
known anti-hypertension drug targets. Topological &
functional analysis of this subnetwork revealed four path-
ways connected through a core of 10 proteins including
NOS3 and CRK. T. Hsieh1, N. Hajjir2, J. T. Pantel2, M. Mensah2, M. Zhao2, J.
Hertzberg2, M. Schubach3, S. Köhler2, Y. Gurovich4, N. P16.39C P16.39C
Investigating the genetic architecture of hypertension
through combined analysis of genome-wide association
studies (GWAS) data and the human protein interaction
network E. Tsare1,2, A. Gioutlakis1,2, M. I. Klapa2, N. K. Moschonas1,2 J. del Picchia 584 J. Halvardson, M. Danielsson, H. Davies, B. Torabi Moghadam,
J. Dumanski, L. A. Forsberg J. Halvardson, M. Danielsson, H. Davies, B. Torabi Moghadam,
J. Dumanski, L. A. Forsberg Department of Immunology, Genetics and Pathology, Uppsala
University, Uppsala, Sweden 1Institute for Cardiogenetics, Lübeck, Germany, 2DZHK
(German Research Center for Cardiovascular Research),
partner site Hamburg-Lübeck-Kiel, Lübeck, Germany,
3University Heart Center Lübeck, Lübeck, Germany, 4Charité-
University Medicine Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität zu Berlin, and Berlin
Institute of Health, Institute for Dental and Craniofacial
Sciences, Dept. Periodontology and Synoptic Dentistry, Berlin,
Germany Men live shorter lives than women however the factors
contributing to this difference remains elusive. Recent
research indicates that the loss of the Y chromosome (LOY)
in blood can explain parts of this difference, as it have been
associated both to cancer mortality and Alzheimer's disease. In order to investigate the effect of losing the Y
chromosome we have performed single cell sequencing of
12 elderly males using the 10X genomics chromium
system. Using the transcriptomes of each sequenced single
cell we are able to use clustering techniques together with t-
Distributed Stochastic Neighbor Embedding (tSNE) to
determine the fraction of LOY cells in each major PBMC
type. We can show that the results from this analysis
compares well with the results from the Illumina Infinium
QC Array-24 kit on FACS sorted cell fractions from the
same individuals. As cohort sizes keep increasing (>500,000), it has become
vital to avoid data redundancy, data duplication, reduce
the influence chromosomal artifacts, and facilitate repli-
cation. Linkage disequilibrium (LD) pruning is used to
decrease an initial variant list to a few representatives and
can also be leveraged to find variants that were not gen-
otyped.A limited number of software tools perform LD-
pruning, however, of those available we were unable to
find a tool that did not require one or more of the fol-
lowing steps: [i] prior ranking of SNPs according to most
significant p-value; [ii] need to provide the actual geno-
type dataset or reference panel; [iii] perform multiple LD-
pruning analysis with different correlation coefficient
values; [iv] require either chromosome number and/or
position and if absent, then have to create a dummy
variable; and [v] must require variants to be on the same
chromosome. We have so far sequenced approximately 37 000 cells and
have assigned cell-type and LOY status to each of these. Gene interaction discovery in myelodysplastic syndromes Gene interaction discovery in myelodysplastic syndromes M. Munoz Venegas: None. M. Munz: None. J. Erdmann: None. A. Demartini1, F. Vitali2, E. Sauta3, M. G. Della Porta4, R. Bellazzi1, S. Marini5 J. Halvardson, M. Danielsson, H. Davies, B. Torabi Moghadam,
J. Dumanski, L. A. Forsberg This allows us to use the information from a large amount
of sequenced single cells to find genes which have altered
gene
expression
in
LOY
cells
in
specific
PBMC
populations. The work presented here shows that single cell RNA
sequencing can be used as a viable technique to identify the
fraction of LOY in the PBMC populations of an individual. Furthermore it gives insight into the genes and pathways
affected when losing the Y chromosome in blood. We introduce reduSNP a lightweight, stand-alone, freely
available command line tool that responds to each of the
aforementioned “shortcomings” with the highlight on
minimum to no pre-processing of input information. The project is funded by an ERC-StG as well as other
sources J. Halvardson: None. M. Danielsson: None. H. Davies:
None. B. Torabi Moghadam: None. J. Dumanski: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; CRAY Innovation. L.A. Forsberg: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; CRAY
Innovation. J. Halvardson: None. M. Danielsson: None. H. Davies:
None. B. Torabi Moghadam: None. J. Dumanski: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; CRAY Innovation. L.A. Forsberg: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; CRAY
Innovation. It prunes SNPs and short (insertions/deletions) InDels
based on the European ancestry 1000Genomes Project
Phase3 LD values. To execute reduSNP through a series of
automated steps a minimum of two parameters are required
namely a list of non-ordered variants; and (multiple)
normalized coefficient of LD (D’) or correlation coefficients
(r2). All output results are then comprehensively logged
featuring suggested reasons to why variants could not be
mapped. A. Demartini1, F. Vitali2, E. Sauta3, M. G. Della Porta4, R.
Bellazzi1, S. Marini5 1University of Pavia, Pavia, Italy, 2University of Arizona,
Tucson, AZ, United States, 3University of Michigan, Ann
Arbor, MI, United States, 4Humanitas University, Milano,
Italy, 5Department of Computational Medicine and P16.40D Exploring molecular interactions by clustering analysis of
similarity scores from next-generation phenotyping
approaches Exploring molecular interactions by clustering analysis of
similarity scores from next-generation phenotyping
approaches Abstracts from the 51st European Society of Human Genetics Conference: Posters 585 P16.41A
reduSNP: used for linkage disequilibrium-pruning
M. Munoz Venegas1,2,3, M. Munz1,2,3,4, J. Erdmann1,2,3
1Institute for Cardiogenetics, Lübeck, Germany, 2DZHK
(German Research Center for Cardiovascular Research),
partner site Hamburg-Lübeck-Kiel, Lübeck, Germany,
3University Heart Center Lübeck, Lübeck, Germany, 4Charité-
University Medicine Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität zu Berlin, and Berlin
Institute of Health, Institute for Dental and Craniofacial
Sciences, Dept. Periodontology and Synoptic Dentistry, Berlin,
Germany P16.41A
reduSNP: used for linkage disequilibrium-pruning
M. Munoz Venegas1,2,3, M. Munz1,2,3,4, J. Erdmann1,2,3
1Institute for Cardiogenetics, Lübeck, Germany, 2DZHK
(German Research Center for Cardiovascular Research),
partner site Hamburg-Lübeck-Kiel, Lübeck, Germany,
3University Heart Center Lübeck, Lübeck, Germany, 4Charité-
University Medicine Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität zu Berlin, and Berlin
Institute of Health, Institute for Dental and Craniofacial
Sciences, Dept. Periodontology and Synoptic Dentistry, Berlin,
Germany P16.42B 1University of Pavia, Pavia, Italy, 2University of Arizona,
Tucson, AZ, United States, 3University of Michigan, Ann
Arbor, MI, United States, 4Humanitas University, Milano,
Italy, 5Department of Computational Medicine and Functional effects from loss of chromosome Y (LOY) in
single cells estimated by genome-wide transcriptional
analyses using the 10X Chromium platform J. del Picchia 586 standard method for germline variant interpretation has
been proposed by the American College of Medical
Genetics and Genomics (ACMG) with the Association for
Molecular Pathology (AMP) (PMID 25741868). Actual
implementation of such guidelines in clinical routine
requires the development of automated systems solving
both complexity and reproducibility issues. standard method for germline variant interpretation has
been proposed by the American College of Medical
Genetics and Genomics (ACMG) with the Association for
Molecular Pathology (AMP) (PMID 25741868). Actual
implementation of such guidelines in clinical routine
requires the development of automated systems solving
both complexity and reproducibility issues. Bioinformatics, University of Michigan, Ann Arbor, MI, United
States The characterization of genetic interactions underlying
myelodysplastic syndrome (MDS) pathology is the key to
understand its molecular machinery and, as consequence, to
design viable treatments. Many studies have been carried on
in this direction, including MDS expression profiling and
analysisof somatic mutations affecting MDS stems cells. In
this study we underpin MDS-specific gene-gene interac-
tions, i.e., gene interactions characterizing MDS cells, either
because they are absent is healthy cells, or because the gene
coexpression is significantly altered in MDS. We applied a
matrix
factorization-based,
multi-omic,
data-agnostic
approach to predict MDS-related gene-gene interactions,
integrating MDS somatic mutation data from TCGA,
KEGG pathways, BioGRID gene-gene interactions, Dis-
GeNET gene-disease relations, and Disease Ontology
disease-disease similarity. Our method unveiled 320 MDS-
specific gene-gene interactions as output. To validate these
results, we (a) assessed if our predicted gene-gene interac-
tions were confirmed at the protein level by using the
STRING repository, and (b) verified if the predicted inter-
actions were reflected in gene co-expression on external
MDS cohorts. We found 173 (54%) of our predictions
confirmed as protein-protein interactions in STRING. Even
more interestingly, more than half of them (98/173) have a
combined score greater than 0.7, which is the level indi-
cated by the STRING curators as high confidence. Next, by
combining four MDS cohorts from Gene Expression
Omnibus data, we calculated the paired co-expression dis-
tribution of the predicted interactions in cases and controls. An automatic implementation of ACMG/AMP variant
interpretation guidelines G. Nicora1, I. Limongelli2, P. Gambelli3, M. Memmi3, C. Napolitano3, A. Malovini4, A. Mazzanti3, S. Priori3, R. Bellazzi1
1Dept. of Electrical, Computer and Biomedical Engineering,
University of Pavia, Pavia, Italy, 2enGenome S.r.l, Pavia, Italy,
3Molecular Cardiology, IRCCS Istituti Clinici Maugeri, Pavia,
Italy, 4Laboratory of Informatics and Systems Engineering,
University of Pavia, Pavia, Italy
Introduction: Variant pathogenicity assessment in the
diagnosis of genetic diseases is a complex process. A G. Nicora1, I. Limongelli2, P. Gambelli3, M. Memmi3, C. Napolitano3, A. Malovini4, A. Mazzanti3, S. Priori3, R. Bellazzi1 P16.42B A Wilcoxon signed rank test found a significant difference
(p-value 4.1*10-18). Materials and Methods: We have developed eVAI
(enGenome VAriant Interpreter), an automatic variant
interpretation system based on ACMG/AMP guidelines. Seventeen out of twenty-eight ACMG/AMP criteria are
implemented integrating data from different omic-resources. We
tested
eVAI
performance
on
CLINVITAE,
a
collection of clinically-observed benign and pathogenic
variants related to a broad set of disorders. We also
validated our system on 60 MYH7-related variants from
ClinGen’s
Inherited
Cardiomyopathy
Panel
(PMID
29300372). We
tested
eVAI
performance
on
CLINVITAE,
a
collection of clinically-observed benign and pathogenic
variants related to a broad set of disorders. We also
validated our system on 60 MYH7-related variants from
ClinGen’s
Inherited
Cardiomyopathy
Panel
(PMID
29300372). Results: In CLINVITAE, eVAI achieved a concordance
of 76.2% (4310/5651) on pathogenic variants and of 88.5%
(7499/8462) on benign variants. On the same data we
compared eVAI to InterVar, another similar tool, showing a
reduction of VUS of about 45.3%. eVAI concordance was
about 85.38% (289/299) and of 96.66% (168/200) on
pathogenic and benign cardiovascular-related variants in
CLINVITAE. Compared
to
CardioClassifier,
a
tool
designed specifically for variants interpretation in cardio-
vascular diseases, eVAI showed a VUS reduction of 64%. Finally, 44/60 MYH7 validated variants were correctly
interpreted. Conclusions: Complexity of standard guidelines and the
ever-growing number of variants detected from sequencing-
based analysis hampers manual pathogenicity assessment. eVAI represents an automated support in guidelines-based
variant interpretation in clinical practice. Conclusions: Complexity of standard guidelines and the
ever-growing number of variants detected from sequencing-
based analysis hampers manual pathogenicity assessment. eVAI represents an automated support in guidelines-based
variant interpretation in clinical practice. A. Demartini: None. F. Vitali: None. E. Sauta: None. M.G. Della
Porta:
None. R. Bellazzi:
None. S. Marini: None. G. Nicora: None. I. Limongelli: A. Employment (full or
part-time); Significant; enGenome S.r.l. P. Gambelli: None. M. Memmi: None. C. Napolitano: None. A. Malovini:
None. A. Mazzanti: None. S. Priori: None. R. Bellazzi: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; enGenome S.r.l, Biomeris S. r.l. 1RCU Genomics, Hannover, Germany, 2Pediatric
Pneumology, Medical School Hannover, Hannover, Germany P16.45A Airways microbial metagenomes of individuals with
immune deficiency, asthma, cystic fibrosis or COPD 1Dept. of Electrical, Computer and Biomedical Engineering,
University of Pavia, Pavia, Italy, 2enGenome S.r.l, Pavia, Italy,
3Molecular Cardiology, IRCCS Istituti Clinici Maugeri, Pavia,
Italy, 4Laboratory of Informatics and Systems Engineering,
University of Pavia, Pavia, Italy 1Dept. of Electrical, Computer and Biomedical Engineering,
University of Pavia, Pavia, Italy, 2enGenome S.r.l, Pavia, Italy,
3Molecular Cardiology, IRCCS Istituti Clinici Maugeri, Pavia,
Italy, 4Laboratory of Informatics and Systems Engineering,
University of Pavia, Pavia, Italy L. Wiehlmann1, K. Pienkowska2, M. Gessner2, M. Dorda1, S. Hedtfeld2, T. Scheithauer1, C. Davenport1, B. Tümmler2
1RCU Genomics, Hannover, Germany, 2Pediatric
Pneumology, Medical School Hannover, Hannover, Germany L. Wiehlmann1, K. Pienkowska2, M. Gessner2, M. Dorda1, S. Hedtfeld2, T. Scheithauer1, C. Davenport1, B. Tümmler2 1RCU Genomics, Hannover, Germany, 2Pediatric
Pneumology, Medical School Hannover, Hannover, Germany Introduction: Variant pathogenicity assessment in the
diagnosis of genetic diseases is a complex process. A 587 Abstracts from the 51st European Society of Human Genetics Conference: Posters Airway infections cause exacerbations of the underlying
disease in patients with immune deficiency (ID), asthma or
COPD and determine course and prognosis in most indi-
viduals with cystic fibrosis. To identify the composition of
the upper and lower airway metagenomes in these patient
groups during clinically stable condition, nasal lavage,
throat swabs and sputa were collected from 142 CF, 35
asthma, 11 COPD and 10 ID patients. Genomic DNA was
extracted and sequenced on SOLiD and Illumina platforms. In an automated pipeline, raw data were filtered, mapped
onto a microbial pangenome (1,892 bacteria, 4,193 fungi/
moulds and 1,153 DNA viruses) and normalized (GC-bias,
genome size, human DNA). The data were analyzed for
phylogeny, taxonomic classification, diversity and correla-
tions. Disease-associated bacterial metagenomes were seen
in patients with CF or ID, but not in patients with asthma or
COPD. The prevalent phyla in human airways are Actino-
bacteria,
Bacteroidetes,
Firmicutes
and
Proteobacteria
which make up more than 90% of the metagenome. COPD
patients exhibited an airway metagenome dominated by
Streptococci, Rothia and Prevotella similar to healthy
smokers and non-smokers. Pre-school wheezers were
mainly carrying a normal, but low-diversity microbial flora
with Rothia mucilaginosa as prime and Actinomyces as
diagnostic
organisms. Besides
numerous
aerobes
and
anaerobes, patients with ID were harbouring Moraxella and
Haemophilus as major pathogens CF patients exhibited a
disease-specific flora, primarily Firmicutes (Staphylococcus
spp.), alpha- and gamma- Proteobacteria (P. aeruginosa, H. influenzae). P16.46B 1European Molecular Biology Laboratory, European
Bioinformatics Institute (EMBL-EBI), Cambridge, United
Kingdom, 2Medical Research Council Harwell (Mammalian
Genetics Unit and Mary Lyon Centre), Oxford, United
Kingdom, 3Queen Mary, University of London, London, United
Kingdom The Mouse Genome Informatics (MGI) resource: new
features facilitating accessibility and presentation of mouse
models of human disease S. M. Bello, A. V. Anagnostopoulos, C. L. Smith, The MGI Staff
and Software Team S. M. Bello, A. V. Anagnostopoulos, C. L. Smith, The MGI Staff
and Software Team P16.45A PCA uncovered clusters of species associated
with severity and chronicity of CF lung disease. reinforces the semantic acquisition, annotation, integration
and display of phenotype and disease model information,
and facilitates comparison of disease-related data across all
model organism databases of the Alliance of Genome
Resources (AGR). Recent enhancements include a new
MGI Human Phenotype Ontology browser, incorporation of
human disease-phenotype relationships from Orphanet, and
new mouse embryonic lethal phenotype data from the
DMDD consortium. Importantly, MGI has adopted the
Disease Ontology (DO), a hierarchical classification of
standardized disease concepts with cross-references to
OMIM, MeSH, SNOMED and other clinical terminologies. MGI disease model annotations have been translated from
OMIM terms to DO terms to optimize searches, and
grouping and display of disease classes in relevant detail
pages. The MGI Quick Search tool, Human-Mouse Disease
Connection (HMDC), and advanced query forms now
support searches by DO terms or IDs. Furthermore, the
HMDC grid groups phenotype and disease data by top-level
terms to enhance visualization of these data. Finally, an
improved DO Browser allows users to traverse the ontology
tree or graphical view, examine a selected term’s definition,
IDs and hierarchical relationships, identify all genes and
mouse models annotated to that disease or any of its sub-
types, and access the unified AGR disease report. Supported
by NIH grant HG000330. S.M. Bello: None. A.V. Anagnostopoulos: None. C.L. Smith: None. P16.47C The IMPC: mouse high-throughput phenotyping
contributes to understanding the role of genes in human
disease L. Wiehlmann: None. K. Pienkowska: None. M. Gessner: None. M. Dorda: None. S. Hedtfeld: None. T. Scheithauer:
None. C. Davenport:
None. B. Tümmler: None. V. Munoz Fuentes1, T. Meehan1, A. Mallon2, H. Parkinson1, D. Smedley3, on behalf of the IMPC consortium MIDAS GPMS- A flexible gene panel management system MIDAS GPMS- A flexible gene panel management system P16.48D Targeted analysis of whole genome sequence data to
identify the link between sensory-immune system and
Neuroticism E. Bounda Ndinga, R. Brumm, M. Schmuck, D. Schmitz, H. Klein, S. H. Eck The Jackson Laboratory, Bar Harbor, ME, United States In allelic model and trend model, we
found the associated variants in 13 olfactory receptor genes
and 5 genes related with DAMP which have high and
moderate impact in low and high Neuroticism groups. Conclusions:
This
study
provide
insights
for
Neuroticism-linking genetic variations of sensory and
immune system. This research was supported by the National Research
Foundation of Korea, funded by the Ministry of Education
(NRF-2016R1A6A3A11932719
and
NRF-
2017R1D1A1B03035501). Results: Following GATK best practices and VQSR
filtering, 17,843,140 SNVs, 2,246,819 Ins and 2,662,425
Dels were retained. In allelic model and trend model, we
found the associated variants in 13 olfactory receptor genes
and 5 genes related with DAMP which have high and
moderate impact in low and high Neuroticism groups. Conclusions:
This
study
provide
insights
for
Neuroticism-linking genetic variations of sensory and
immune system. This research was supported by the National Research
Foundation of Korea, funded by the Ministry of Education
(NRF-2016R1A6A3A11932719
and
NRF-
2017R1D1A1B03035501). This research was supported by the National Research
Foundation of Korea, funded by the Ministry of Education
(NRF-2016R1A6A3A11932719
and
NRF-
2017R1D1A1B03035501). H. Kim: None. Y. Yun: None. E. Lee: None. E. Park:
None. Y. Chang: None. S. Ryu: None. H. Kim: None. V. Munoz Fuentes: None. T. Meehan: None. A. Mallon: None. H. Parkinson: None. D. Smedley: None. The Jackson Laboratory, Bar Harbor, ME, United States Introduction: The International Mouse Phenotyping Con-
sortium (IMPC) is a world-wide initiative that has so far
characterized over 4,000 knockout mouse lines, many for
poorly understood genes (the ignorome), to better under-
stand mammalian gene function and human disease. Mouse Genome Informatics (MGI) is the premier commu-
nity knowledgebase for the laboratory mouse. MGI inte-
grates mouse genotype-phenotype datasets from biomedical
publications and large-scale projects with genomic, muta-
tion, expression, functional and human disease model data
to accelerate correlative discoveries and inform genetic
disease etiology and therapeutics. New MGI functionality Materials and Methods: Dedicated phenotyping, statis-
tical and bioinformatic pipelines are collecting, analyzing
and displaying data from more than 250 phenotypic
parameters of embryos, young adult and aged mice. J. del Picchia 588 sequencing data (30X coverage) in 185 women, who have
the extreme scores neuroticism (≤10th percentile and ≥90th
percentile). All sequenced DNA reads were mapped to the
human genome reference (hg19) and SNV and indel VCFs
were create by multi-sample joint aggregation using the
gVCFs. Targeted analysis was focused on the olfactory
receptor
genes,
neuroinflammation,
inflammasome-,
PAMP-, and DAMP-related genes. Results and Conclusions: We will present how the latest
data are contributing to previously observed biological
insights and discovering new ones. First, we continue to
observe a wide-ranging prevalence of sexual dimorphism,
highlighting the importance of including both males and
females in biomedical research. An embryonic phenotyping
pipeline continues finding that approximately one-third of
mammalian genes are essential for life, and these are highly
correlated with human disease genes. Further, the overlap of
adult mouse phenotypes with human clinical features has
identified over 400 new disease models. Specialized studies
have led to the identification of 52 genes associated to
deafness for the first time, and a separate study found 974
genes associated to the glucose and lipid metabolism,
including 23 genes associated with human diabetes via
GWAS. These models fill the gap between GWAS and
functional validation of complex traits, and shared regula-
tory features may potentially allow functional predictions
for other metabolic genes via co-regulation networks. In
addition, over 1500 publications indicate that IMPC mouse
models or data are being widely used by the research
community, for instance in cholesterol, bone formation, and
halitosis research. Results: Following GATK best practices and VQSR
filtering, 17,843,140 SNVs, 2,246,819 Ins and 2,662,425
Dels were retained. P16.51C 1European Bioinformatics Institute (EMBL-EBI), Hinxton,
United Kingdom, 2The Jackson Laboratory Mouse Genome
Informatics, Bar Harbor, ME, United States Integration of large-scale genetic and functional datasets
enable mechanistic insights into craniofacial development
and disease J. Welzenbach1,2, E. Mangold1, M. Knapp3, K. Ludwig1,2 J. Welzenbach1,2, E. Mangold1, M. Knapp3, K. Ludwig1,2 Introduction:
Patient-derived
tumor
xenograft
(PDX)
mouse models are an important research platform to study
tumor evolution, drug response and for tailoring che-
motherapeutic approaches to individual patients. PDX
models are produced and made available in repositories
managed by academic labs, large research consortia and
contract research organizations. The heterogeneous nature
of PDX repositories makes finding relevant models of
interest challenging. 1Institute of Human Genetics, Bonn, Germany, 2Department of
Genomics, Life & Brain Center, University of Bonn, Bonn,
Germany, 3Institute of Medical Biometry, Informatics and
Epidemiology, University of Bonn, Bonn, Germany GWAS for nonsyndromic cleft lip with or without cleft
palate (nsCL/P) have identified 40 risk loci, mostly in non-
coding regions. The functional effects of these associations
remain largely unclear. Here, we applied comprehensive
data integration approaches of genetic and epigenetic data
sets to 1) identify potential regulatory effects of risk var-
iants, 2) characterize potential novel variants located in
functional candidate regions, and 3) to specify the manner
and timing of regulatory processes affected by known
variants. Methods: To address this issue, The Jackson Laboratory
and EMBL-EBI have co-developed the PDX Finder, a
comprehensive open global catalogue of PDX models and
their
associated
data. In
support
this
initiative,
we
coordinated the community initiative to develop the PDX
models Minimal Information standard (PDX-MI) that
defines the information necessary for describing key
elements of a PDX model including the clinical attributes
of a patient’s tumor, host strain, implantation and model
validation methods. PDX-MI is the basis for PDX Finder’s
search and filtering options (e.g., tumor histology, mole-
cular variant, drug response). Within PDX Finder, model
attributes are integrated into a cohesive ontological data
model that supports consistent searching across various
resources. From PDX Finder, direct links to the relevant
institution are provided for further collaboration. PDX
Finder is collaborating with several worldwide consortia
including PDXnet and EurOPDX to increase “findability”
of PDX models and to advance cancer research and drug
discovery. We used relevant published ChIP-seq datasets from
different stages of craniofacial development, i.e., human
embryonic craniofacial tissue (Carnegie stages (CS) 13-17)
/ neural crest cells, and in-house nsCL/P GWAS summary
statistics. P16.54B PDX Finder: An Open and Global Catalogue of Patient
Tumor Derived Xenograft Models E. Bounda Ndinga: None. R. Brumm: None. M. Schmuck: None. D. Schmitz: None. H. Klein: None. S. H. Eck: None. Tumor Derived Xenograft Models
N. Conte1, T. Meehan1, J. Mason1, C. Halmagyi1, A. Mosaku1, S. Neuhauser2, D. Krupke2, D. Begley2, H. Parkinson1, C. Bult2
1European Bioinformatics Institute (EMBL-EBI), Hinxton,
United Kingdom, 2The Jackson Laboratory Mouse Genome
Informatics, Bar Harbor, ME, United States N. Conte1, T. Meehan1, J. Mason1, C. Halmagyi1, A. Mosaku1, S. Neuhauser2, D. Krupke2, D. Begley2, H. Parkinson1, C. Bult2 Center for human genetics, Munich, Germany H. Kim1, Y. Yun1, E. Lee1, E. Park1, Y. Chang2, S. Ryu2, H. Kim1 H. Kim1, Y. Yun1, E. Lee1, E. Park1, Y. Chang2, S. Ryu2, H. Kim1 The implementation of Next-Generation Sequencing in a
clinical diagnostic setting opens vast opportunities through
the ability to simultaneously sequence all genes contributing
to a certain indication. However, this approach remains
challenging and requires complex algorithms and significant
computational resources to annotate, classify and store data. Furthermore, the need of a comprehensive system managing
patient information, gene panels, samples, sequencing data
and diagnostic reports becomes crucial. 1Department of Biochemistry, School of Medicine, Ewha
Womans University, Seoul, Korea, Republic of, 2Center for
Cohort Studies, Total Healthcare Center, Kangbuk Samsung
Hospital, School of Medicine, Sungkyunkwan University,
Seoul, Korea, Republic of Introduction: Neuroticism is a personality trait character-
ized by a tendency toward various negative emotions
including anxiety, depression, and anger. Accordingly, this
trait has been associated with several psychiatric disorders,
and considered one of the risk factors for developing major
depression and anxiety disorders. According to the theory,
neurotic tendencies derive from an innate predisposition
toward
low-threshold,
high-intensity,
and
long-lasting
autonomic activation in response to sensory stimuli. Recently, we found that Neuroticism was associated with
the olfactory receptor genes and gut microbiota in our
previous study. To solve these issues we develop MIDAS (Multiple
Integration of Data Annotation Software) a desktop based
and
modular
constructed
javafx
application
for
data
integration. MIDAS consists of many independent modules, here we
introduce
the
gene-panel-management-system
(GPMS)
module. The GPMS allows the user through a graphical
user
interface
(GUI)
to
perform
“CRUD-operations”
(Create, Read, Update and Delete) on gene panels and their
content by respecting all database constraints. Considering a
gene panel may consist of many subpanels whose content Materials: This research was performed using the
Revised NEO Personality Inventory and the whole genome Abstracts from the 51st European Society of Human Genetics Conference: Posters 589 can overlap and a gene can belong to different panels,
performing such operation become a non-trivial task. (rs7590268) is located within an enhancer that is active in
CS15/17, while being non-active in the earlier CS13/14. This correlates with a specifically strong expression of the
adjacent THADA gene in CS15, potentially reflecting a
contribution of the apoptosis-related THADA gene in fusion
processes in facial development. (rs7590268) is located within an enhancer that is active in
CS15/17, while being non-active in the earlier CS13/14. Center for human genetics, Munich, Germany This correlates with a specifically strong expression of the
adjacent THADA gene in CS15, potentially reflecting a
contribution of the apoptosis-related THADA gene in fusion
processes in facial development. Furthermore, the GPMS provides meta-information about
all panels such as gene-, exon-, CDS- and base-count,
department and the panel status. Since Panel diagnostics rely on target enrichment, GPMS
can also provide information about all enriched and not
enriched genes of the selected panel version. Our “functional genomics” approach will help to close the
gap in the functional understanding of genetic risk loci for
nsCL/P and other complex traits. MIDAS aims to aid molecular diagnostics by simplifying
and accelerating data analysis and interpretation, improving
patient care. MIDAS is a part of a prospective multicentric
study including clinical, diagnostic and software develop-
ment partners and is funded by a grant by the Bavarian
Ministry of Economy. J. Welzenbach: None. E. Mangold: None. M. Knapp:
None. K. Ludwig: None. J. Welzenbach: None. E. Mangold: None. M. Knapp:
None. K. Ludwig: None. Who am I? The Personal Genome Project UK Who am I? The Personal Genome Project UK L. Marks1, N. Pontikos2, A. Webster3, the PGP-UK Consortium L. Marks1, N. Pontikos2, A. Webster3, the PGP-UK Consortium 1University College London, London, United Kingdom, 2UCL
Institute of Ophthalmology, University College London, London, United Kingdom, 3UCL Cancer Institute, University
College London, London, United Kingdom Background: The Personal Genome Project UK (PGP-
UK), part of the global PGP network, creates freely avail-
able genetic, epigenetic, phenotypic, health and associated
trait data from volunteer participants. Methods: By using a multi-omics approach combined
with a robust recruitment process based on open consent,
we provide whole genome, methylome and transcriptome
data as an open-access resource. Alongside raw data, PGP-
UK provides individual variant reports for each donated
genome based on multi-omic analyses. The genomic variant
reports
include information
about
ancestry,
alongside
genetic risk for various diseases and traits. For the first
time, we have reported epigenetic variants to participants in
reports including prediction of epigenetic age and smoking
status based on DNA methylation markers. Results: The PGP-UK pilot study focused on ten
volunteer participants as the first collaborative citizen
scientists. Alongside these, the unique genome donation
framework was developed and trialed on three participants. Results: The PGP-UK pilot study focused on ten
volunteer participants as the first collaborative citizen
scientists. Alongside these, the unique genome donation
framework was developed and trialed on three participants. In an additional demonstration of citizen science, a
biomedical postgraduate student has become the first to
analyse their own genome and epigenome as part of PGP-
UK. Results from all three project areas will be presented. M. Kaakinen: None. C.J. Rhodes: None. M. Humbert:
None. I. Prokopenko: None. M. Wilkins: None. Discussion: PGP-UK demonstrates that citizen science is
possible, and is a key to advancing public understanding of
personal genomics and health. The project has demonstrated
the importance of data sharing and public collaboration in
the scientific process. We conclude that the PGP-UK
process and citizen science collaborative approach to
genomic data creation is critical in the advancement of
genomics as a tool in personalised medicine, and in public
perception of personal genomics. P16.51C After epigenetic imputation of histone marks, a
joint bioinformatics pipeline (QC, peak calling) was
applied, and chromatin segmentation procedures were
performed. This provided insights into the particular
involvement of different activity states at different stages
and could help to differentiate which molecular processes of
facial development (e.g, cell migration/fusion, or tissue
formation) might be influenced by which genetic risk loci. As one preliminary example, the lead SNP at 2p21 590 J. del Picchia P16.57A
Genome-wide association study of 1,124 protein levels in
pulmonary arterial hypertension patients identifies a novel
trans-pQTL at ELK2AP for Death Receptor 3
M. Kaakinen1, C. J. Rhodes1, NIHR Bioresource for Rare
Diseases, M. Humbert2, I. Prokopenko1, M. Wilkins1
1Imperial College London, London, United Kingdom,
2Université Paris-Sud, Paris, France Genome-wide association study of 1,124 protein levels in
pulmonary arterial hypertension patients identifies a novel
trans-pQTL at ELK2AP for Death Receptor 3 M. Kaakinen1, C. J. Rhodes1, NIHR Bioresource for Rare
Diseases, M. Humbert2, I. Prokopenko1, M. Wilkins1 M. Kaakinen1, C. J. Rhodes1, NIHR Bioresource for Rare
Diseases, M. Humbert2, I. Prokopenko1, M. Wilkins1 N. Conte: None. T. Meehan: None. J. Mason: None. C. Halmagyi: None. A. Mosaku: None. S. Neuhauser: None. D. Krupke: None. D. Begley: None. H. Parkinson: None. C. Bult: None. 1Imperial College London, London, United Kingdom,
2Université Paris-Sud, Paris, France 1University of Lübeck, Lübeck, Germany, 2Charité - University
Medicine Berlin, Berlin, Germany P16.55C
Who am I? The Personal Genome Project UK Recent genome-wide association studies (GWAS) on blood
plasma proteome in healthy individuals have identified
hundreds of protein quantitative trait loci, pQTLs, both in
cis and in trans. Dissecting the mechanisms of protein level
variability in unhealthy individuals may help to reveal novel
disease-specific pathways. Pulmonary arterial hypertension,
PAH, is a rare disease leading to premature death. We
conducted a GWAS using whole-genome sequencing data
and 1,124 blood plasma protein levels measured with the
SOMAscan platform in 128 British (discovery) and 79
French (replication) PAH patients. Each protein level was
natural logarithm transformed, adjusted for age, sex and
four principal components, and the resulting residuals were
further inverse-normal transformed to assure normality. We
discovered 21 cis and 6 trans-pQTLs at genome-wide sig-
nificance corrected for multiple testing (P < 4.45×10-11) that
replicated with nominal significance and directional con-
sistency. These contain four novel trans-pQTLs at/near
ELK2AP for death receptor 3 (DR3); MIR4435−1 for
Complement component 1 subcomponent r (C1r); RETNLB
for Hepatocyte growth factor activator (HGFA); and
TMEM215 for Properdin. Animal studies have shown DR3,
a cell surface receptor of the tumor necrosis factor receptor
superfamily (TNFRSF), to emerge as a major regulator of
inflammatory and autoimmune disease, and therapeutic
agonists of TNFRSF25 can be used to simulate Treg
expansion. ELK2AP is on the border of immunoglobulin
heavy locus, further suggesting the role of this novel pQTL
to be inflammation and immunity related. Our results pro-
vide support for previous research suggesting that inflam-
mation
and
altered
immune
processes
underlie
the
development of PAH. P16.57A Results/conclusion: PDX Finder is currently displaying
over 1800 PDX models for a wide variety of cancers and is
actively recruiting more models.The community is invited
to explore and provide feedback on our portal at: www. pdxfinder.org. Genome-wide association study of 1,124 protein levels in
pulmonary arterial hypertension patients identifies a novel
trans-pQTL at ELK2AP for Death Receptor 3 M. Munz1,2, A. Schäfer2, J. Erdmann1 P16.58B Qtlizer: Comprehensive QTL annotation of GWAS results M. Munz1,2, A. Schäfer2, J. Erdmann1 L. Marks: None. N. Pontikos: None. A. Webster: None. 591 Abstracts from the 51st European Society of Human Genetics Conference: Posters Exploration of genetic variant to gene relationships can help
to identify candidate causal variants and genes in Post-
GWAS analyses. In this work, we introduce Qtlizer, a novel
web-based tool to annotate common variants in human with
associated changes in gene expression using the, to-date,
most comprehensive database of published quantitative trait
loci (QTL). sequencing of messenger RNA (RNA-seq) extracted from
available patients’ tissues, blood and fibroblasts, to search
for pathogenic variants. Eight patients with known muta-
tions, affecting splicing or leading to the loss of expression
of one allele (heterozygote deletion, nonsense mRNA
decay, etc) were included, as well as ten patients with ID or
RP without diagnosis after TS or WES. We performed a
combined analysis of RNAseq data including 1) a variant
detection comparison between RNASeq and DNAseq data
using VaRank 2) the detection of splicing alterations using
three different programs (rMATS, JunctionSeq and Leaf-
Cutter) 3) a differential expression study using DEseq. This
3-way analysis allowed us to retrieve the known mutations
in 8/10 cases and identified interesting candidate variations
in some of the other patients. A significant number of genes
involved in ID and RP are expressed either in fibroblasts or
blood. We therefore confirm that RNAseq from patient’s
skin or blood cells can be a useful approach to identify
pathogenic variants in coding and noncoding regions. sup-
ported by Fondation Maladies Rares, Fondation Jérome
Lejeune More precisely, we integrated 169 tissue-specific QTL
datasets (one response expression QTLs, one protein
abundance QTLs, all other expression QTLs) from the
public domain and further included two inhouse datasets on
monocytes
and
macrophages
from
the
Cardiogenics
Consortium. The QTL table resulting from up to 100 inputted rsIDs
(optionally, LD variants can be included) consists of
columns for variant, gene, type of QTL, distance in base
pairs, tissue, effect size, significance information, and
origin. Additionally, we added three more attributes: (1)
Co-localization: instead of defining fixed size of e.g. one
mega base pair, we utilized the TAD boundaries to
categorize QTLs into cis (i.e. University of Michigan, Ann Arbor, MI, United States University of Michigan, Ann Arbor, MI, United States M. Munz: None. A. Schäfer: None. J. Erdmann: None. P16.58B variant and gene remain in
the same TAD) and trans (2) Relevance flags and counts:
these were added to each QTL to draw conclusions about its
reproducibility across tissues and studies as well as its
causality (3) GWAS Catalog: variants and genes are marked
if listed in the catalog. F. MAttioli: None. C. Stoezel: None. D. Plassard:
None. H. Dollfus: None. J. Mandel: None. C. Keime:
None. J. Muller: None. A. Piton: None. Seekmer: fast and accurate transcript level quantification
at low sequencing depth Seekmer: fast and accurate transcript level quantification
at low sequencing depth In summary, Qtlizer provides a web-based solution for
annotating lists of genetic variants with QTLs from a large
number of published datasets as well as from two recently
released eQTL datasets. Our tool is freely available at
http://www.genehopper.de/qtlizer. Use of RNA sequencing for the diagnosis of heterogenous
genetic diseases Use of RNA sequencing for the diagnosis of heterogenous
genetic diseases Hongjiu Zhang1†, Yifan Wang
1,2, †, Ebrahim Azizi,
Gilbert S. Omenn
1,2,3, Ryan E. Mills1,2,*, Yuanfang
Guan1,3,4,* Hongjiu Zhang1†, Yifan Wang
1,2, †, Ebrahim Azizi,
Gilbert S. Omenn
1,2,3, Ryan E. Mills1,2,*, Yuanfang
Guan1,3,4,* F. MAttioli1, C. Stoezel2, D. Plassard1, H. Dollfus2, J. Mandel1,
C. Keime1, J. Muller2, A. Piton1 1 Department of Computational Medicine and Bioinfor-
matics,
University
of
Michigan,
Ann
Arbor,
MI,
USA 48109 1IGBMC, Strasbourg, France, 2INSERM U1112, Strasbourg,
France 1IGBMC, Strasbourg, France, 2INSERM U1112, Strasbourg,
France 2 Department of Human Genetics, University of Michi-
gan, Ann Arbor, MI, USA 48109 Use of DNA high throughput sequencing (Whole‐exome
sequencing, WES or targeted sequencing, TS) in clinical
practices have greatly improved molecular diagnosis of
genetically heterogenous diseases, such as Intellectual
Disability (ID) or Retinitis pigmentosa (RP). However, an
important portion of the patients remains undiagnosed after
the exploration of genomic coding regions. To identify
additional variants not seen by WES, whole genome
sequencing (WGS) is a straightforward strategy but still
remains heavy and expensive. Thus, we used paired-end 3
Department
of
Internal
Medicine,
University
of
Michigan, Ann Arbor, MI, USA 48109 3
Department
of
Internal
Medicine,
University
of
Michigan, Ann Arbor, MI, USA 48109 4 Department of Electronic Engineering and Computer
Science,
University
of
Michigan,
Ann
Arbor,
MI,
USA 48109 † Zhang and Wang equally contributed to the work. † Zhang and Wang equally contributed to the work. *
To
whom
correspondence
should
be
addressed:
Yuanfang Guan <gyuanfan@umich.edu> and Ryan E. Mills <remills@umich.edu> Abstract J. del Picchia 592 Achieving both fast and accurate quantification of
transcript abundance in RNA-Seq experiments remains an
elusive goal, with trade-offs between the high accuracy of
alignment-based
methods
and
the
short
runtime
of
alignment-free approaches for estimating the abundance of
individual transcripts. We present Seekmer, a highly
efficient quantification tool that leverages the relative
advantages of each approach without their corresponding
shortcomings. Seekmer incorporates a modified de Bruijn
approach
for
quick
sequence
assignments
into
an
alignment-free mapping, resulting in a 95% increase in
speed over alignment-based tools while retaining a high
accuracy as compared to both molecular and simulated data
sets. We further show increased transcript discrimination at
lower expression levels, thus enabling the analysis of subtle
changes between different cellular systems as well as
individual cells. Seekmer thus provides scalable and robust
transcript quantification for current full-length bulk and
single-cell RNA sequencing methods. which reflect the levels in isolated T Cells (R2=0.62). Furthermore, LRRN3 LS-TA measured in Whole Blood
samples
differentiated
patients
on
immunosuppressant
therapy from healthy controls (sensitivity >85%). There-
fore, it is feasible to translate this direct LS-TA assay on
Whole Blood Samples in routine hospital applications. N.L. Tang: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; patent
prosecution. C. Szeto: None. N.L. Tang: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; patent
prosecution. C. Szeto: None. University of Michigan, Ann Arbor, MI, United States Y. Wang: None. H. Zhang: None. R.E. Mills: None. Y. Guan: None. Our study tackles three challenges facing high-dimensional
sparse
counts
data:
mathematical
theory,
algorithmic
development, and applications. With single-cell RNA
sequencing, the transcript counts contain low integers and
very high rates of 0's due to shallow sampling and “drop-
outs”. Since cell number (100's-1000's) is usually smaller
than gene number (20,000-40,000), statistical inference
tends to be under-determined; yet much information is
conveyed by subsets of genes. Most current theories on
statistical learning assume real-valued measurements in
well-behaved distributions. We developed new theoretical
support for the unique distribution properties of “double-
sparse” data, with (1) low-rank structure and (2) low inte-
gers with zero-inflation. One recent effort is to adopt the
Gini Index to assess if a cell devotes most of its transcripts
on a small spectrum of genes, or spread them over many
regulatory programs. Yet the Gini varies with the cell's
sequencing depth; in fact, both the alpha and beta diversity
measures exhibit dependencies with abundance and dis-
persion properties of the counts data. We modify classic
distance measures and critically reassess existing algorithms
for clustering, imputation, marker selection, and benchmark
them in reusable, extendable, truth-known simulations. We
apply our workflow to analyze >36K single cells from the
mouse testis, finding that the transition from spermatocytes
to mature sperms follow a smooth transition; four identified
spermatogonia subtypes map to known differentiation pro-
cesses; and nine subtypes of Sertoli cells are interspersed
with spatially defined histological stages. Our experience
highlights the value of iterative discovery cycles through
theory-methods-applications. (Funding: 1R21HD090371-
01A1, 1DP2HD091949-01, Michigan Institute for Data
Science.) P16.62B Sparse discrete data analytics for single-cell genomics and
applications to spermatogenesis A. C. Gilbert, C. Green, Q. Ma, X. Zheng, S. S. Hammoud,
Michigan Center for Single-Cell Genomic Data Analytics, J. Z. Li Erasmus Medical Centre, Rotterdam, Netherlands Introduction: Research into the contribution of somatic
mutations to human diseases has been mostly confined to
cancer genetics. Recently, studies have emerged regarding
somatic mutations in neurological diseases. Most use can-
didate gene panel sequencing or sequencing of single cells. These methods are either limited to a subset of genes or a
subset of cells. We measured all coding somatic mutations
in two specific brain regions of six Semantic Dementia (SD)
patients and report affected genes. Introduction: Autoinflammatory diseases represent a pri-
vileged scenario to the study of the somatic variation. First,
somatic mutations are suspected to cause autoinflammatory
diseases in a considerable fraction of patients. Second, DNA
from blood is easily obtained. And third, separating dif-
ferent cell populations is also possible with standard flow
cytometry procedures. However, the detection of somatic
mutations from NGS presents some difficulties. Low fre-
quency somatic mutations are at risk of being undetected
with standard procedures. Also, most of the algorithms for
somatic mutation analyses are optimized for cancer studies,
where a tumour sample is compared with the healthy tissue. Thus, higher coverages and the modification of experi-
mental designs and pipelines are needed to detect these
variants. Methods: We performed whole exome sequencing on
dentate gyrus (mean coverage 250x), middle temporal gyrus
(500x) and blood (120x) of six SD patients. Using Mutect
and GATK’s Unified Genotyper, we extracted a total of
193.250 candidate mutation sites. Through a custom
filtering pipeline, we retained 2.312 somatic mutations. Each mutation has a CADD score of at least 10 and is
completely absent from the ExAC database. Results: Of 2.312 somatic mutations, 736 are detected in
dentate gyrus, 1.571 in middle temporal gyrus and 5 in
both. Between 24 and 479 somatic mutations were found
per tissue per patient. In total 2.033 genes were affected by
somatic mutations. 254 genes were affected in multiple
tissues or samples. The most affected gene was TTN,
carrying seven unique somatic mutations in six tissues of
five patients. Materials and Methods: We sequenced the whole
exomes from blood samples of five patients with known
somatic variants with a mean coverage 260X. We used
somatic variant callers, VarScar2, MuTect2 and VarDict, to
generate a list of possible somatic mutations. For two
patients, we also separated blood cell populations in order to
establish their frequencies. CNV analysis was performed
using CoNIFER and XHMM software. Erasmus Medical Centre, Rotterdam, Netherlands Materials and Methods: We sequenced the whole
exomes from blood samples of five patients with known
somatic variants with a mean coverage 260X. We used
somatic variant callers, VarScar2, MuTect2 and VarDict, to
generate a list of possible somatic mutations. For two
patients, we also separated blood cell populations in order to
establish their frequencies. CNV analysis was performed
using CoNIFER and XHMM software. Conclusions: Deep WES allows measuring a wide range
of coding somatic mutations within a selected tissue. Large
variation exist in the number of potentially damaging
mutations per patient and tissue. We identified 254 genes
with mutations in multiple patients or tissues. Results: We found 4 out of 5 variants, all but the one at
lower frequency (2.8%). All others (ranging from 7.7%-
31.3%) were detected by, at least, VarScan2. Estimated
frequencies by experimental methods and by the NGS
approach were similar in general. Results: We found 4 out of 5 variants, all but the one at
lower frequency (2.8%). All others (ranging from 7.7%-
31.3%) were detected by, at least, VarScan2. Estimated
frequencies by experimental methods and by the NGS
approach were similar in general. J. van Rooij: None. S. Melhem: None. D. Salkovic:
None. P. Arp: None. A. Uitterlinden: None. H. Seelaar:
None. J. van Meurs: None. J. van Swieten: None. A.C. Gilbert: None. C. Green: None. Q. Ma: None. X.
Zheng: None. S.S. Hammoud: None. J.Z. Li: None. A.C. Gilbert: None. C. Green: None. Q. Ma: None. X. Zheng: None. S.S. Hammoud: None. J.Z. Li: None. A.C. Gilbert: None. C. Green: None. Q. Ma: None. X. Zheng: None. S.S. Hammoud: None. J.Z. Li: None. M. Solis-Moruno: None. A. Mensa-Vilaro: None. L. Batlle-Maso: None. T. Marques-Bonet: None. J. Aroste-
gui: None. F. Casals: None. Somatic Mutation detection using deep Whole Exome
Sequencing on post-mortem brain tissue M. Solis-Moruno1, A. Mensa-Vilaro2, L. Batlle-Maso1, T. Marques-Bonet1, J. Arostegui2, F. Casals3 J. van Rooij, S. Melhem, D. Salkovic, P. Arp, A. Uitterlinden, H. Seelaar, J. van Meurs, J. van Swieten 1Institut de Biologia Evolutiva (CSIC-UPF), Departament de
Ciències Experimentals i de la Salut, Univ, Barcelona, Spain,
2Department of Immunology, Hospital Clínic-IDIBAPS,
Barcelona, Spain / Functional Unit of Clinical Immunology
Hospital Sant Joan de Déu-Hospital Clínic, Barcelona, Spain.,
Barcelona, Spain, 3Genomics Core Facility, Departament de
Ciències Experimentals i de la Salut, Universitat Pompeu
Fabra, Parc de Recerca Biomèdica de Barcelona, 08003
Barcelona, Spain., Barcelona, Spain P16.61A Determination of CD3 subpopulation specific gene
expression from whole blood samples and its applications N. L. Tang, C. Szeto N. L. Tang, C. Szeto The Chinese University of Hong Kong, Shatin, Hong Kong The Chinese University of Hong Kong, Shatin, Hong Kong Traditional approach to determine transcript abundance(TA)
of one cell type in cell-mixture samples (e.g. Whole blood)
require cell-isolation, like magnetic cell-sorting to single out
target cell-types for quantification of TA. The cell-sorting
procedure is labour-intense and/or expensive (e.g. single
cell RNA-seq). An in-vitro diagnostics that can report TA of
single cell-type without cell-sorting is needed for routine
hospital laboratory operation. In this example, we used CD3
T-cell as the target leukocyte subpopulation in which tran-
script abundance was determined in Whole Blood samples. A list of T-cells cell-type specific genes were defined as
genes
whose
transcripts
in
the
Whole
Blood
were
predominantly contributed from T-cells (over 50%). From
such T cells cell-type specific gene list, a functional reporter
gene of interest was chosen as a target gene. Another cell-
type specific gene with low biological variation and not
affected by the disease condition was chosen as reference
gene. We used real time PCR to quantify the 2 genes in
Whole Blood Samples and ddCT ratio of them provided an
index of the target gene expression level in T Cells. LRRN3 was T cells cell-type specific gene. Using
PRKCQ as the T cells cell-type specific reference gene,
LRRN3 LS-TA was determined in Whole Blood samples Abstracts from the 51st European Society of Human Genetics Conference: Posters 593 M. Grant SAF2015-68472-C2-2-R from Mineco (Spain)
and FEDER to FC. Pacific Biosciences, Menlo Park, CA, United States Most of the base pairs that differ between human genomes
are in structural variants (differences ≥50 bp), which are
broadly missing from variant databases built with short-read
DNA sequencing. To support use of structural variants in
rare disease and common trait association studies, it is
necessary to perform population-scale surveys with a
technology effective at detecting structural variants, such as
PacBio SMRT sequencing. After sample collection, the
challenges to constructing population-scale structural var-
iant databases are: 1. library preparation; 2. data collection;
and 3. data processing. For library preparation, we devel-
oped SMRTbell Express Template Preparation, a 3-hour
single-tube protocol. For data collection, prior studies have
demonstrated that low coverage per sample (~5-fold) is
sufficient for surveys of variation. The PacBio Sequel
System generates low-coverage for a single sample in one
day. For data processing, we developed pbsv, the first joint
structural variant caller which combines reads from multiple
individuals to identify variants. We first applied these tools
to evaluate a human trio. At 5-fold coverage per sample, we
discover most of the variants found by high coverage
sequencing, with a 25% increase in sensitivity from joint
variant calling. We further applied joint variant calling to a
Mendelian disease case and identified a pathogenic, de novo
structural variant. Finally, we developed a mathematical
model to suggest how to power studies that apply 5-fold
coverage to discover structural variants at a desired popu-
lation frequency.Together, the tools and model support the
creation of the first population-scale database of human
structural variation. Our methods and visualisation tools will be made
available as a part of our graph-based toolset for structural
variant calling (https://github.com/illumina/paragraph). The
validated calls and raw sequence data will be made
available publicly for over 210 samples that have been
sequenced to at least 30x depth as part of the Platinum
Genomes
(https://github.com/illumina/platinumgenomes)
and Polaris (https://github.com/illumina/polaris) resources. P. Krusche: A. Employment (full or part-time); Sig-
nificant; Illumina Cambridge Ltd.. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Illumina Inc. S. Chen: A. Employment (full or
part-time); Significant; Illumina Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Illumina Inc. E. Dolzhenko: A. Employment
(full or part-time); Significant; Illumina Inc. E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Significant; Illumina Inc. B.L. Moore: A. Employment (full or part-time); Significant; Illumina Cam-
bridge Ltd.. E. P. Krusche1, S. Chen2, E. Dolzhenko2, B. L. Moore1, M. A.
Bekritsky1, A. Gross2, D. R. Bentley1, M. A. Eberle2 P. Krusche1, S. Chen2, E. Dolzhenko2, B. L. Moore1, M. A. Bekritsky1, A. Gross2, D. R. Bentley1, M. A. Eberle2 (stock, stock options, patent or other intellectual property);
Significant; Illumina Inc. D.R. Bentley: A. Employment
(full or part-time); Significant; Illumina Cambridge Ltd.. E. Ownership Interest (stock, stock options, patent or other
intellectual property);
Significant;
Illumina
Inc. M.A. Eberle: A. Employment (full or part-time); Significant;
Illumina Inc. E. Ownership Interest (stock, stock options,
patent
or
other
intellectual
property);
Significant;
Illumina Inc. 1Illumina Cambridge Ltd., Saffron Walden, United Kingdom,
2Illumina Inc., San Diego, CA, United States Accurate detection and annotation of complex variants (e.g. paralogs and SVs) is a critical part of the clinical variant
calling process. A challenge with SV calling is that the same
event is often represented differently between samples. Nearby
variants
and
sequence
instability
around
the
breakpoints can further complicate these issues. Thus,
complex variants can be difficult to aggregate across sam-
ples leading to Mendelian conflicts, or underestimation of
variant frequencies. P16.66B
Population-scale discovery of structural variants with
PacBio SMRT sequencing
A. Wenger, R. Vogelsang, B. Galvin, L. Hickey, Y. Li, P. Peluso,
J. Wilson, M. Sonnested
Pacific Biosciences Menlo Park CA United States P16.65A Conclusion: Next generation sequencing is a tool with
potential application to detect somatic mutations in autoin-
flammatory diseases. MDM-2014-0370-16-3 grant to M.S.- Graph methods for validating complex variants in
pedigrees and large sample cohorts J. del Picchia 594 P. Krusche1, S. Chen2, E. Dolzhenko2, B. L. Moore1, M. A. Bekritsky1, A. Gross2, D. R. Bentley1, M. A. Eberle2
1Illumina Cambridge Ltd., Saffron Walden, United Kingdom,
2Illumina Inc., San Diego, CA, United States P16.66B
Population-scale discovery of structural variants with
PacBio SMRT sequencing A. Wenger, R. Vogelsang, B. Galvin, L. Hickey, Y. Li, P. Peluso,
J. Wilson, M. Sonnested A. Wenger, R. Vogelsang, B. Galvin, L. Hickey, Y. Li, P. Peluso,
J. Wilson, M. Sonnested We have developed a joint genotyping method for
complex variants based on realigning sequence reads
overlapping variant locations or breakpoints to a variant
graph. This method enables us to find a canonical variant
representation and use it to consistently genotype a variant
across many samples. Once a set of variants is consistently
represented and genotyped across a large cohort of samples,
we validate it using Mendelian inheritance, Hardy Wein-
berg statistics and other approaches. The resulting sets of
high-quality variant annotations are essential for clinical
applications where the variant calling is performed on one
or few samples. Pacific Biosciences, Menlo Park, CA, United States Pacific Biosciences, Menlo Park, CA, United States Ownership Interest (stock, stock options,
patent or other intellectual property); Significant; Illumina
Inc. M.A. Bekritsky: A. Employment (full or part-time);
Significant; Illumina Cambridge Ltd.. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Illumina Inc. A. Gross: A. Employment (full or
part-time); Significant; Illumina Inc. E. Ownership Interest A. Wenger: A. Employment (full or part-time); Sig-
nificant; Pacific Biosciences. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant;
Pacific
Biosciences. R. Vogelsang:
A. Abstracts from the 51st European Society of Human Genetics Conference: Posters 595 Employment (full or part-time); Significant; Pacific Bios-
ciences. E. Ownership Interest (stock, stock options, patent
or other intellectual property); Significant; Pacific Bios-
ciences. B. Galvin: A. Employment (full or part-time);
Significant; Pacific Biosciences. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Pacific Biosciences. L. Hickey: A. Employ-
ment (full or part-time); Significant; Pacific Biosciences. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Pacific Biosciences. Y. Li: A. Employment (full or part-time); Significant; Pacific
Biosciences. E. Ownership Interest (stock, stock options,
patent or other intellectual property); Significant; Pacific
Biosciences. P. Peluso: A. Employment (full or part-time);
Significant; Pacific Biosciences. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Pacific Biosciences. J. Wilson: A. Employment
(full or part-time); Significant; Pacific Biosciences. E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; Pacific Biosciences. M. Sonnested: None. pregnancy, a difficult issue of prognosis due to the risk of
intellectual disability. The advent of new technologies such
as Whole Genome Sequencing (WGS) makes it possible to
consider the detection and characterization of SVs. The goal
is to use the WGS to identify and characterize SVs, and to
secondarily improve our pipeline. A short-reads WGS was
performed in 19 patients with de novo SVs (8 transloca-
tions, 7 complex chromosomal rearrangements (CCRs) and
4 sSMCs) that had been found in postnatal (11/19 patients)
or prenatal (8/19 patients) periods. Bioinformatic analysis of
the raw data was performed with Lumpy algorithms for
translocations and inversions, and ControlFreec for CNVs. The analyses identified 7/8 translocations, one of which
interrupts the GRIN2B gene at a breakpoint compatible with
the patient’s phenotype. The undetected translocation
involves pericentromeric breakpoints. The 7 CCRs were
also identified with, sometimes, the discovery of more than
10
breakpoints,
in
favor
of
a
chromothrypsis
type
mechanism. P16.67C Prenatal and postnatal molecular characterization by
Whole Genome Sequencing (WGS) of de novo structural
variations (SVs) N. Marle: None. A. Vitobello: None. J. Puechberty:
None. J. Thevenon: None. E. Tisserant: None. A. Guerrot: None. P. Chambon: None. G. Joly-Hélas:
None. A. Guichet: None. B. Keren: None. S. Brisset:
None. L. Van Maldergem: None. P. Khau Van Kien:
None. M. Doco: None. E. Pipiras: None. C. Beneteau:
None. J. Deleuze: None. R. Olaso: None. A. Boland:
None. M. Poulleau: None. T. Jouan: None. C. Poe: None. C. Thauvin-Robinet: None. L. Faivre: None. Y. Duf-
fourd: None. P. Callier: None. N. Marle1, A. Vitobello1, J. Puechberty2, J. Thevenon1, E. Tisserant1, A. Guerrot3, P. Chambon4, G. Joly-Hélas4, A. Guichet5, B. Keren6, S. Brisset7, L. Van Maldergem8, P. Khau
Van Kien9, M. Doco10, E. Pipiras11, C. Beneteau12, J. Deleuze13,
R. Olaso13, A. Boland13, M. Poulleau1, T. Jouan1, C. Poe1, C. Thauvin-Robinet1, l. Faivre1, Y. Duffourd1, P. Callier1 N. Marle1, A. Vitobello1, J. Puechberty2, J. Thevenon1, E. Tisserant1, A. Guerrot3, P. Chambon4, G. Joly-Hélas4, A. Guichet5, B. Keren6, S. Brisset7, L. Van Maldergem8, P. Khau
Van Kien9, M. Doco10, E. Pipiras11, C. Beneteau12, J. Deleuze13,
R. Olaso13, A. Boland13, M. Poulleau1, T. Jouan1, C. Poe1, C. Thauvin-Robinet1, l. Faivre1, Y. Duffourd1, P. Callier1 1CHU le Bocage, DIJON, France, 2Service de Génétique
Médicale, Montpellier, France, 3Service de Génétique, Rouen,
France, 4Laboratoire de Cytogénétique, Rouen, France,
5Service de Génétique, Angers, France, 6Département de
Génétique, Hôpital Pitié-Salpêtrière, Paris, France, 7Service
d’Histologie Embryologie Cytogénétique, Clamart, France,
8Centre de Génétique Humaine, Besançon, France, 9Centre de
Génétique Médicale et Cytogénétique, Nimes, France,
10Service de Génétique Médicale, Reims, France,
11Laboratoire de Cytogénétique, AP-HP, Hôpital Robert
Debré, Paris, France, 12Service de Génétique Médicale,
Nantes, France, 13Centre National de Recherche en
Génomique Humaine (CNRGH), Evry, France Pacific Biosciences, Menlo Park, CA, United States Of the 4 sSMCs, the 2 with euchromatin were
detected since the 2 with heterochromatin were not. This
project allowed us to improve our WGS pipeline and
identify SVs. It so demonstrates that short-reads WGS is
particularly effective in characterizing the breakpoints of
SVs (16/19) in both prenatal and postnatal periods. Open Targets: Integrating genetics and functional biology
to identify and prioritise gene targets for disease Open Targets: Integrating genetics and functional biology
to identify and prioritise gene targets for disease E. M. Schmidt1,2 E. M. Schmidt1,2 1Wellcome Sanger Institute, Hinxton, Cambridgeshire, United
Kingdom, 2Open Targets, an academic-industrial
collaboration between the Wellcome Sanger Institute,
European Bioinformatics Institute (EMBL-EBI),
GlaxoSmithKline plc., Biogen Inc. and Takeda
Pharmaceuticals, Hinxton, Cambridgeshire, United Kingdom In the pre- and postnatal periods, de novo structural variants
(SVs),
such
as
supernumerary
chromosomal
markers
(sSMCs)
and
apparently
balanced
translocations
and
inversions, are usually evidenced by standard karyotype. The presence of these SVs raises, especially during Over the past decade, the genetic aetiology of many human
diseases and complex phenotypes has been deeply char-
acterised. However, attributing causal genes to genetic
associations and developing effective therapeutics remains
challenging. 596 J. del Picchia are still unclear, making it difficult to link a non-coding
mutation with the patient's phenotype. In Open Targets, we integrate large-scale genetics and
genomics with drug information to influence the way drug
targets are identified and prioritised. We also generate new
data using human cell models (e.g. organoids, iPSCs) and
genome editing (CRISPR/Cas9) to identify drug targets for
three main therapeutic areas: oncology, immunology, and
neurodegeneration. The Open Targets Platform enables
users to identify and investigate links between genes,
pathways, and diseases, and can be accessed via a web
interface or REST-API. We compute, score and rank target-
to-disease associations using biological evidence integrated
from the NHGRI-EBI GWAS Catalog, Genomics England,
PheWAS, ClinVar, expression and eQTL resources, Uni-
Prot, ChEMBL, and many others. We have recently
developed POSTGAP, a pipeline which merges common
genetic associations curated from literature with functional
genomics, epigenetic and expression data to resolve the
association signals at each trait-associated locus and link
each variant to its target gene(s). Functional evidence from
multiple sources including GTEx data, promoter capture Hi-
C data, DNase hypersensitivity sites, and FANTOM5 is
incorporated into an evidence score, providing a single
point of reference to explore the underlying biology of
conditions and prioritise potential targets. We propose here a supervised machine learning strategy
using random forests, adapted to complex and heteroge-
neous datasets, to classify and select non-coding mutations
potentially involved in the deregulation of disease genes. A
notable innovation of our approach is to take into account
association
data
between
non-coding
regions
and
target genes. We apply 3 classifiers, trained on different sets of
experimentally predicted regulatory regions, on more than
40,000 non-coding mutations in 48 patients affected with
X-linked intellectual disabilities from the FP7-funded
project "NeuroXsys". P16.70B ExpansionHunter: A software tool to detect long repeat
expansions from PCR-free whole-genome sequence data Aggregation and integration of functional data and
annotation from multiple heterogeneous sources in Open
Targets offers a robust solution to prioritising genes,
quantifying their biological significance, and assessing their
potential as pharmaceutical targets. M. A. Eberle1, E. Dolzhenko1, J. J. F. A. van Vugt2, G. Narzisi3,
J. H. Veldink2, D. A. Bentley4 1Illumina, Inc, San Diego, CA, United States, 2University
Medical Center Utrecht, Utrecht, Netherlands, 3New York
Genome Center, New York, NY, United States, 4Illumina
Cambridge Ltd, Little Chesterford, United Kingdom E.M. Schmidt: None. E. M. Schmidt1,2 Selected mutations were shown to
segregate with the disease in affected families, and to
deregulate the predicted target gene in animal models. We
discuss the results in light of their genome-wide application
to larger cohorts of patients. L. Moyon: None. C. Berthelot: None. H. Roest
Crollius: None. P16.69A Context-specific prioritization of non-coding variants
implicated in human diseases Introduction: Identifying large repeat expansions such as
those that cause amyotrophic lateral sclerosis (ALS) and
fragile X syndrome (FXS) is challenging for short-read
(100-150 bp) whole genome sequencing (WGS) data. A
solution to this problem is an important step towards inte-
grating WGS into precision medicine. To this end, we
developed a software tool called ExpansionHunter that,
using PCR-free WGS short-read data, can genotype repeats
at a locus of interest even if the expanded repeat is larger
than the read length. L. Moyon, C. Berthelot, H. Roest Crollius L. Moyon, C. Berthelot, H. Roest Crollius Institut de Biologie de l'École Normale Supérieure, CNRS,
INSERM, PSL Research University, Paris, France Whole genome sequencing is increasingly being used for
patients with rare genetic diseases as a diagnostic tool. However, for a large proportion of sequenced patients, no
coding mutation is found in a gene associated with the
phenotype. In these cases, a non-coding mutation, located in
a cis-regulatory region, may affect the expression of a gene
involved in the disease. Despite the existence of methods
for annotating and predicting regulatory sequences on the
basis of biochemical and epigenetic properties, defining
objective criteria remains difficult to effectively select
candidates among the millions of non-coding mutations
present in each patient. Moreover, the mechanisms of action
and interaction between regulatory regions and target genes Materials and Methods: We applied ExpansionHunter
to a set of 144 samples harboring repeat expansions
associated with Huntington’s disease, fragile X syndrome,
Friedreich’s ataxia and five other genetic disorders. In
addition, we tested the C9orf72 repeat in a cohort of 3,001
ALS samples and compared our calls with RP-PCR results. Results: All but one of the repeat expansions in the
Coriell samples were detected by our method even though
many of the repeats were significantly longer than the read
lengths. In the ALS cohort, ExpansionHunter correctly Materials and Methods: We applied ExpansionHunter
to a set of 144 samples harboring repeat expansions
associated with Huntington’s disease, fragile X syndrome,
Friedreich’s ataxia and five other genetic disorders. In
addition, we tested the C9orf72 repeat in a cohort of 3,001
ALS samples and compared our calls with RP-PCR results. Results: All but one of the repeat expansions in the
Coriell samples were detected by our method even though
many of the repeats were significantly longer than the read
lengths. In the ALS cohort, ExpansionHunter correctly Abstracts from the 51st European Society of Human Genetics Conference: Posters 597 classified all (212/212) of the expanded samples as either
expansions (208) or potential expansions (4). Additionally,
99.9% (2,786/2,789) of the wild type samples were
correctly classified as wild type by our method with the
remaining three identified as possible expansions. Conclusions: ExpansionHunter now enables researchers
to identify many known pathogenic repeat expansions from
whole genome sequencing data. classified all (212/212) of the expanded samples as either
expansions (208) or potential expansions (4). L. Moyon, C. Berthelot, H. Roest Crollius Additionally,
99.9% (2,786/2,789) of the wild type samples were
correctly classified as wild type by our method with the
remaining three identified as possible expansions. to complexity/severity of the disease to the patient’s age. The estimated cost of each year of diagnostic delay was
2146 € (range 48 – 18,320 €). Conclusions: While a subset of individuals affected by
rare genetic diseases receives a diagnosis at the first clinical
evaluation, a significant number remains undiagnosed. This
study substantiates the cost-effectiveness of WES as a first
line diagnostic tool in these patients. Conclusions: ExpansionHunter now enables researchers
to identify many known pathogenic repeat expansions from
whole genome sequencing data. F. Radio: None. A. Bartuli: None. A. Novelli: None. M. Tartaglia: None. B. Dallapiccola: None. M.A. Eberle:
A. Employment
(full
or
part-time);
Significant; Illumina, Inc. E. Ownership Interest (stock,
stock
options,
patent
or
other
intellectual
property);
Significant; Illumina, Inc. E. Dolzhenko: A. Employment
(full or part-time); Significant; Illumina, Inc. E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Significant; Illumina, Inc. J.J.F.A. van Vugt:
None. G. Narzisi: None. J.H. Veldink: None. D.A. Bentley: A. Employment (full or part-time); Significant;
Illumina, Inc. E. Ownership Interest (stock, stock options,
patent
or
other
intellectual
property);
Significant;
Illumina, Inc. GENALICE Core BV, Nijkerk, Netherlands With a growing use of Next Generation Sequencing (NGS)
in human genetics it is evermore important to benchmark
data analysis pipelines. Benchmark datasets exist, such as
NIST-GIAB and Illumina’s Platinum Genomes, holding
“truth” variants to determine sensitivity and specificity of
NGS analysis pipelines. Although highly valuable, those
benchmarks have drawbacks as NGS data analysis pipelines
determined their truth calls. In addition, they include only a
few individual samples that could lead to overfitting
through training sets. Such drawbacks introduce biases that
should be avoided in benchmark studies. Ruark et al (2016)
have developed high quality whole exome sequencing data
from 142 samples together with Sanger sequencing data at
730 loci (ICR142). This dataset allows NGS pipeline eva-
luation with orthogonal verified truth variants across a
cohort of unrelated samples and avoids biases associated
with other benchmark sets. We used the ICR142 dataset to
validate the GENALICE MAP population calling tool. We
observed correct calling of all 123 true SNPs and 260 out of
268 true INDELs. In addition, it called 6 SNPs and 18
INDELs at the remaining Sanger negative loci. We com-
pared the results to those obtained with the bcbio platform
(https://github.com/bcbio/icr142-validation) and observed
that GENALICE MAP produces the lowest numbers of
false negative and false positive calls. This makes GENA-
LICE MAP best in class next to being extraordinarily fast: it
took less than 3 minutes per sample to go from FASTQ
to VCF. Cost-effectiveness of whole exome sequencing to solve the
unsolved: an Italian pilot study F. Radio, A. Bartuli, A. Novelli, M. Tartaglia, B. Dallapiccola F. Radio, A. Bartuli, A. Novelli, M. Tartaglia, B. Dallapiccola QIAGEN, Redwood City, CA, United States Introduction: Gathering the most current and accurate
information is critical to variant interpretation and classifi-
cation. The QIAGEN knowledgebase includes the most
comprehensive database of variant specific publications,
and data from public and proprietary databases. This
resource is the cornerstone of QIAGEN Clinical Insight –
Interpret (QCI-I), which facilitates rapid variant filtering,
interpretation, and reporting. Here, QCI-I quickly identifies
candidate causal variants in two cases from the Harvard
Personal Genome Project (PGP). Results: GARFIELD-NGS consists of 4 distinct models
tested on NA12878 gold-standard exome variants dataset
(NIST v.3.3.2): Illumina INS/DELs, Illumina SNVs, ION
INS/DELs, and ION SNVs. AUROC values for each
variants category are 0.9269, 0.7998, 0.9464, and 0.9757,
respectively. GARFIELD-NGS is robust on low coverage
data down to 30X and on Illumina two-colour data, as well. Our tool outperforms previous hard-filters, and calculates
for each variant a score from 0.0 to 1.0, allowing application
of different thresholds based on desired level of sensitivity
and specificity. GARFIELD-NGS processes standard VCF
file input using Perl and Java scripts and produces a regular
VCF output. Thus, it can be easily integrated in existing
analysis pipeline. Materials and Methods: Health records and VCFs were
obtained from PGP (https://my.pgp-hms.org/users). Case 1
presented with dilated cardiomyopathy and case 2 exhibited
autosomal dominant non-syndromic hearing loss. Filtering
was performed in QCI-I to exclude poor quality bases,
common variants, and those classified as benign or likely
benign. VCFs were uploaded to QCI-I and respective
phenotypes were entered. Materials and Methods: Health records and VCFs were
obtained from PGP (https://my.pgp-hms.org/users). Case 1
presented with dilated cardiomyopathy and case 2 exhibited
autosomal dominant non-syndromic hearing loss. Filtering
was performed in QCI-I to exclude poor quality bases,
common variants, and those classified as benign or likely
benign. VCFs were uploaded to QCI-I and respective
phenotypes were entered. Results: Case 1 harbored a single candidate causal
variant in LMNA (c.176T>G), which was classified as likely
pathogenic based on several lines of evidence in the
QIAGEN knowledgebase. Review of curated publications
revealed that this LMNA variant is novel in the context of
cardiomyopathy. Upon analysis of case 2, QCI-I identified a
compelling
candidate
causal
variant
in
HOMER2
(c.587G>C). While this variant is classified as a variant of
uncertain significance, HOMER2 has been mutated in
autosomal
dominant
non-syndromic
hearing
loss
previously. Availability:
GARFIELD-NGS
available
at
https://
github.com/gedoardo83/GARFIELD-NGS EG has been supported by “Fondazione Cariplo” and
“Regione Lombardia”, Grant Emblematici Maggiori 2015-
1080. V. Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy,
Rome, Italy Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy,
Rome, Italy Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy,
Rome, Italy Introduction: Rare, ultrarare and orphan diseases are het-
erogeneous disorders with a prevalence of less than 1/2000
persons, affecting some hundred millions of people world-
wide. The needs of these patients and their families repre-
sent a medical, economical and societal challenge. The wide
application of next generation sequencing has allowed a
dramatic increase in the speed of genome sequencing with a
direct impact in terms of diagnostic rate and a decrease
in cost. Materials & Methods: 300 patients were enrolled
between 2014 and 2017 in the Undiagnosed Patients
Program at Bambino Gesù Children Hospital (OPBG) in
Rome. Each subject was suspected to have a mendelian
disorder and remained undiagnosed despite extensive
multidisciplinary clinical and instrumental evaluation, and
targeted genetic testing. The cost-effectiveness analysis was
performed on 211 patients, aged between 1 months and 43
years, w/wo a conclusive diagnosis following mendeliome/
WES analysis. The total costs and costs for each year of
diagnostic delay for the National Health System were
compared with the cost of mendeliome/WES analysis. Ruark et al. F1000Research 2016, 5:386 (https://doi.org/
10.12688/f1000research.8219.1) L. Baarspul:
A. Employment
(full
or
part-time);
Significant; Genalice Core BV. B. Tolhuis: A. Employment
(full or part-time); Significant; Genalice Core BV. H. Results: The average total cost for each undiagnosed
patient was 11,572 € (range 160 – 75,840 €) and was related Results: The average total cost for each undiagnosed
patient was 11,572 € (range 160 – 75,840 €) and was related 598 J. del Picchia 1Dep. of Molecular and Translaional Medicine, University of
Brescia, Brescia, Italy, 2Unit of Molecular Neurogenetics,
Fondazione IRCCS Istituto Neurologico 'Carlo Besta', Milan,
Italy Karten: A. Employment (full or part-time); Significant;
Genalice Core BV. QIAGEN, Redwood City, CA, United States Ravasio: None. M. Ritelli: None. A. Legati: None. E. Giacopuzzi: None. Karolinska Institutet, Solna, Sweden J.L. Poitras: None. Background: We have implemented a healthcare region-
wide strategy for diagnosing patients with rare genetic
disorders using WG (>3500 samples). This is result of
collaboration between healthcare (Karolinska University
Hospital) and academic settings, represented by Science for
Life Laboratory. P16.75C
WGS based workflow for identification of disease causing
variants in rare diseases Conclusions: Driven by content, QCI-I rapidly filtered
VCFs and identified candidate causal variants in two PGP
cases in under an hour. This work also highlights the
importance of collaborative research efforts like PGP in
furthering our understanding of genetics and disease. V. Ravasio1, M. Ritelli1, A. Legati2, E. Giacopuzzi1 GARFIELD-NGS: Genomic vARiants FIltering by dEep
Learning moDels in NGS P16.73A
QIAGEN Clinical Insight - Interpret (QCI-I) drives rapid
identification and interpretation of candidate causal
variants in two Personal Genome Project cases Motivation: Exome sequencing approach is extensively
used in research and diagnostic laboratories to discover
pathological variants and study genetic architecture of
human diseases. Even if present platforms produce high
quality sequencing data, false positives variants remain an
issue and can confound subsequent analysis and results
interpretation. Here,
we propose a new tool named
GARFIELD-NGS (Genomic vARiants FIltering by dEep
Learning moDels in NGS), which is based on deep learning
models to dissect false and true variants in single sample
exome sequencing experiments performed with Illumina or
ION platforms. J. L. Poitras V. Wirta Karolinska Institutet, Solna, Sweden J. L. Poitras QIAGEN, Redwood City, CA, United States QIAGEN, Redwood City, CA, United States P16.74B GARFIELD-NGS: Genomic vARiants FIltering by dEep
Learning moDels in NGS V. Ravasio1, M. Ritelli1, A. Legati2, E. Giacopuzzi1 599 Abstracts from the 51st European Society of Human Genetics Conference: Posters Results: Approximately 100 samples are processed
monthly. Turnaround time from DNA to results ready for
clinical interpretation is 11 days (range 4 to 16). Using
NIST samples, we estimate that SNV in known disease-
causing genes are detected at >99.7% sensitivity and with
>99.6% PPV. Indel detection is at 94% sensitivity. Detection of CNV and other SV is achieved using multiple
variant callers. An in-house established frequency database
is used to filter out technical artifacts. Variants are ranked according to their disease causing
potential using a rank sum model considering various
sources of annotation, including observed inheritance
pattern vs. expected pattern, CADD score, frequency,
quality,
consequence,
clinical
significance
and
other
sources. A suite of informatics solutions consisting of Chanjo (QC
tool), Genmod (variant prioritization), and Scout (GUI),
enable clinical experts without in-depth bioinformatic
knowhow to evaluate the results. The workflow is quality assured using ISO17025. Data
sharing is being established through ClinVar and Beacon. Integration with multi-omic data is being evaluated for
assisting in functional interpretation of clinical significance
of variants outside coding regions and linked canonical
splice sites. Results: Approximately 100 samples are processed
monthly. Turnaround time from DNA to results ready for
clinical interpretation is 11 days (range 4 to 16). Using
NIST samples, we estimate that SNV in known disease-
causing genes are detected at >99.7% sensitivity and with
>99.6% PPV. Indel detection is at 94% sensitivity. Detection of CNV and other SV is achieved using multiple
variant callers. An in-house established frequency database
is used to filter out technical artifacts. Materials
and
Methods:
We
created
GeneHancer
(PMID:28605766), a novel regulatory element database
with >250,000 enhancers and promoters. Information is
amalgamated
from
six
sources:
ENCODE,
Ensembl,
FANTOM5, VISTA, dbSUPER and EPDnew. Target genes
are gleaned via GTEx expression QTLs, Capture Hi-C,
expression
correlation
between
genes
and
FANTOM
enhancer-transcribed ncRNAs, expression correlation of
genes to enhancer-contained transcription factors, and
genomic distance. In parallel, >100,000 unified ncRNAs
are consolidated from 21 general and specialized databases
(PMID:23172862). Results: The widely-used GeneCards Suite has now been
remodeled, creating an indispensable WGS disease inter-
pretation platform. This is based on the abovementioned
data and on >20,000 deeply annotated disease entries in
MalaCards (PMID:27899610). P16.74B This knowledgebase feeds
the Suite’s NGS tools: VarElect, the phenotype interpreter,
and TGex, the VCF-to-report analyzer (PMID:27357693),
augmented to prioritize WGS variants with respect to
diseases and phenotypes. g
g
g
Integration with multi-omic data is being evaluated for
assisting in functional interpretation of clinical significance
of variants outside coding regions and linked canonical
splice sites. Conclusions: The GeneCards Suite provides a compre-
hensive route to clinical significance of variants, coding and
non-coding
single
nucleotide
and
structural
genomic
variations, often elucidating unsolved cases. In summary, we have introduced at large scale a
healthcare region wide implementation of WGS for patients
with rare genetic diseases. A key aspect has been the
collaboration between academia and healthcare. Support: LifeMap Sciences grant S. Fishilevich: None. R. Barshir: None. M. Twik:
None. N. Rosen: None. T. Iny Stein: None. M. Safran:
None. D. Lancet: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; LifeMap Sciences
Inc., Marshfield, MA 02050, USA. V. Wirta: None. Yield of Clinically Relevant Candidates in Family Genomes
in the UK 100,000 Genomes Project Using the Fabric
Genomics Platform S. Fishilevich, R. Barshir, M. Twik, N. Rosen, T. Iny Stein, M. Safran, D. Lancet S. Fishilevich, R. Barshir, M. Twik, N. Rosen, T. Iny Stein, M. Safran, D. Lancet M. Babcock1, C. Son Rigby1, M. Falcioni1, A. Guo1, J. Grigonis1,
K. Hart1, M. Yandell2, M. Reese1 Weizmann Institute of Science, Rehovot, Israel Weizmann Institute of Science, Rehovot, Israel Weizmann Institute of Science, Rehovot, Israel Introduction: Whole genome sequencing (WGS) identifies
50 times more variants than exome sequencing, most
residing in the genomic non-coding “dark matter”. Three
classes of functional genomic elements that thus become
amenable to variant analyses are promoters, enhancers and
ncRNAs, all central to tissue-related gene expression. Together they amount to >20% of the new DNA territories. Enhancers and some ncRNAs moderate spatiotemporal
orchestration of embryonic development and cell differ-
entiation, with many underlying diseases. The WGS chal-
lenge is disease interpretation of this new avalanche of dark
matter variants. 1Fabric Genomics, Oakland, CA, United States, 2University of
Utah, Salt Lake City, UT, United States The 100,000 Genomes Project, spearheaded by Genomics
England (GeL), is a UK National Health Service sponsored
study aimed at identifying disease-causing genetic variants
in patients and families with rare genetic diseases and
cancer using a WGS approach. For this study, clinical his-
tory was used to recruit patients into specific disease cate-
gories, each of which were associated with gene panels
curated in the GeL PanelApp tool. 600 J. del Picchia performance of this approach. Probands where individuals
with rare genetic diseases that failed single-gene based
diagnosis. We identify the VAAST and Phevor score rank
for 644 candidate causative variants ultimately selected by
clinical reporting. Reports were generated with Opal Clin-
ical software, which provides a rich set of annotations and
both filtering widgets and VAAST and Phevor scores. We
show that candidate causative variants reported reside in the
top 10 VAAST rank in 35% of cases, whereas they are
found in 70% of the cases when ranked by Phevor. When
the candidate causative variant fell out of the top 20 rank,
fewer and more ambiguous provided phenotypes are
observed. In summary, the use of phenotype-driven variant
prioritization scores can reduce significantly the interpreta-
tion
turnaround
time,
ultimately
reducing
costs
and
increasing diagnostic rates. Fabric Genomics, a clinical interpretation partner for the
100,000 Genomes Project, has analyzed over 1220 clinical
cases using Opal Clinical. The variant filtering and
prioritization protocols utilized for case analysis include
GeL’s variant tiering methodology, ClinVar, and Fabric
Genomics’ proprietary variant and gene ranking algorithms
VAAST (Variant Annotation, Analysis and Selection Tool)
and Phevor (Phenotype Driven Variant Ontological Re-
ranking Tool). We report results showing that by applying
VAAST and Phevor we increase the clinical candidate yield
compared to using the GeL tiering system alone. P16.79C 1Fabric Genomics, Oakland, CA, United States, 2Stanford
University School of Medicine, Stanford, CA, United States,
3University of Utah, Salt Lake City, UT, United States Weizmann Institute of Science, Rehovot, Israel We
identified candidate causal genes/variants in 42.3% of the
cases. In 23.3% of these cases (9.8% overall) candidates
were only obtained by using the VAAST/Phevor top 20
ranked genes/variants. In a small subset of ~300 cases, we reviewed the effects
of providing parental genomes in the analysis and return
rate of results. Interestingly, there was a small difference in
clinical candidate yield between solo cases and trios. Further analysis will be carried out to see if this difference
is observed in a larger cohort. F.M. De La Vega: A. Employment (full or part-time);
Significant; Fabric Genomics Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant;
Fabric
Genomics,
Inc. M. Babcock:
A. Employment (full or part-time); Significant; Fabric Geno-
mics, Inc. E. Kiruluta: A. Employment (full or part-time);
Significant; Fabric Genomics, Inc. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; Fabric Genomics, Inc. M. Yandell: E. Owner-
ship
Interest
(stock,
stock
options,
patent
or
other
intellectual property); Modest; Fabric Genomics, Inc. F. Consultant/Advisory Board; Modest; Fabric Genomics, Inc. M. Reese: A. Employment (full or part-time); Significant;
Fabric Genomics, Inc. E. Ownership Interest (stock, stock
options, patent or other intellectual property); Significant;
Fabric Genomics, Inc. M. Babcock: None. C. Son Rigby: None. M. Falcioni:
None. A. Guo: None. J. Grigonis: None. K. Hart: None. M. Yandell: None. M. Reese: None. P16.78B Phenotype-driven variant prioritization significantly
improves over impact and prevalence scores in a large-scale
analysis of 1,963 cases of Mendelian disease diagnostics by
whole-genome sequencing F. M. De La Vega1,2, M. Babcock1, E. Kiruluta1, M. Yandell3, M. Reese1 P17.02B
Allele specific expression identifies rare variants as cause
for extreme allelic imbalance A. Stoccoro1, R. Gallo1, L. Mosca2, C. Tarlarini2, C. Lunetta2, A. Marocchi2, V. Carnicelli1, L. Migliore1, F. Coppedè1
1University of Pisa, Pisa, Italy, 2ASST Grande Ospedale
Metropolitano Niguarda, Milan, Italy A. Stoccoro1, R. Gallo1, L. Mosca2, C. Tarlarini2, C. Lunetta2, A. Marocchi2, V. Carnicelli1, L. Migliore1, F. Coppedè1 A. Stoccoro1, R. Gallo1, L. Mosca2, C. Tarlarini2, C. Lunetta2, A. Marocchi2, V. Carnicelli1, L. Migliore1, F. Coppedè1 N. de Klein1, F. van Dijk1,2, A. Claringbould1, P. Deelen1,2, U. Võsa1, J. Verlouw3, R. Monajemi4, R. Sinke1, M. Swertz1,2, P. A. C. 't Hoen5, L. Franke1, BIOS Consortium N. de Klein1, F. van Dijk1,2, A. Claringbould1, P. Deelen1,2, U. Võsa1, J. Verlouw3, R. Monajemi4, R. Sinke1, M. Swertz1,2, P. A. C. 't Hoen5, L. Franke1, BIOS Consortium 1University of Pisa, Pisa, Italy, 2ASST Grande Ospedale
Metropolitano Niguarda, Milan, Italy Several
evidences
suggest
that
aberrant
epigenetic
mechanisms could be involved the etiology of amyo-
throphic lateral sclerosis (ALS). Particularly, methylation
alterations in several nuclear genes have been detected in
whole blood and spinal cord DNA of ALS subjects. Recently it has been reported also an involvement of epi-
genetic deregulation in mitochondrial DNA (mtDNA) in
patients affected by neurodegenerative diseases, but evi-
dences in ALS is limited.In the current study we investi-
gated
DNA
methylation
levels
of
the
mitochondrial
displacement loop (D-loop) region, which regulates mito-
chondrial DNA replication and transcription, in peripheral
blood DNA samples collected from 40 ALS patients with
mutations in one of the ALS associated genes, including
SOD1, FUS, TARDBP and C9ORF72, and from 60 healthy
individuals, some of which carriers of the same mutation. D-loop methylation levels were significantly lower in blood
DNA of ALS patients than in healthy individuals (P < 0.05). Moreover a significant inverse correlation between D-loop
methylation levels and mtDNA copy number (r=-0.33; P <
0.001), as well as a higher amount of mtDNA copy number
in ALS patients compared to healthy individuals (P < 0.001) 1University of Groningen, University Medical Center
Groningen, Department of Genetics, Groningen, Netherlands,
2Genomics Coordination Center, University of Groningen,
University Medical Center Groningen, Groningen,
Netherlands, 3Dept. of Internal Medicine, Erasmus Medical
Center Rotterdam, Rotterdam, Netherlands, 4Dept. Z. Gormez1,2, I. Erserim1, D. Erdogan1 Z. Gormez:
None. I. Erserim:
None. D. Erdogan: None. Z. Gormez1,2, I. Erserim1, D. Erdogan1 We have used 11 automatable
evidences (PVS1, PM2, PP2, PP3, PP5, BA1, BS1, BS2,
BP1, BP4, BP6) and an inhouse variant database for
Turkish population to provide a genetic analysis summary
report for MEFV, BRCA1, BRCA2 and CFTR genes. into 5 groups as likely benign, benign, VUS, likely
pathogenic and pathogenic. We have used 11 automatable
evidences (PVS1, PM2, PP2, PP3, PP5, BA1, BS1, BS2,
BP1, BP4, BP6) and an inhouse variant database for
Turkish population to provide a genetic analysis summary
report for MEFV, BRCA1, BRCA2 and CFTR genes. median. For the genes that have at least one outlier we find
that there is an enrichment of rare variants within the outlier
genes (p = 2e-31) and known pathogenic variants (p = 4e-
72). When looking at high impact variants we observe that
mendelian disease genes show stronger ASE effects in the
case of nonsense mutations (p = 1.66e-74). We found that
when a sample has a nonsense mutation in a mendelian
disease gene this gene shows outlier ASE expression in
11% of the cases. Thus unbiased and reliable results can be generated by
reducing the laborious time of variant curation to a
minimum. Genetic analysis summary reports of 80 patients
can be generated and be ready for review in a few minutes. The results of this study show that rare variants are more
likely to have a high impact and cause extreme allelic
imbalance, underlining the importance of investigating
allelic imbalance in the classification of variants of
unknown clinical significance. GenerAVI can use the manually curated variant database. Resources and data developed by Maxwell et al and by
expert panels mentioned in ClinVar were used as a
reference for variant classification. Also, users can interpret
and store their variants. In addition, they can import variants
interpretation of an expert panel into their database. GeneraAVI uses this variant database to generate genetic
analysis summary report automatically. N. de Klein: None. F. van Dijk: None. A. Claring-
bould: None. P. Deelen: None. U. Võsa: None. J. Verlouw: None. R. Monajemi: None. R. Sinke: None. M. Swertz:
None. P.A.C. 't
Hoen:
None. L. Franke: None. N. de Klein: None. F. van Dijk: None. A. Claring-
bould: None. P. Deelen: None. U. Võsa: None. J. Verlouw: None. R. Monajemi: None. R. Sinke: None. M. Swertz:
None. P.A.C. 't
Hoen:
None. L. Franke: None. Z. Gormez:
None. I. Erserim:
None. D. Erdogan: None. P17.03C Decreased methylation of the mitochondrial D-Loop region
in peripheral blood DNA of amyotrophic lateral sclerosis
patients Z. Gormez1,2, I. Erserim1, D. Erdogan1 Z. Gormez1,2, I. Erserim1, D. Erdogan1 Next-generation sequencing of genomes or exomes is
becoming pervasive in the clinical diagnostics of Mendelian
syndromes, idiopathic disease and fast diagnostics for
newborns. Pressures remain to demonstrate the clinical
utility of WGS and to reduce the cost of the diagnostic
process. Traditionally, analysis consists in iteratively per-
forming filtering steps using predicted impact and variant
population prevalence and reviewing literature for candidate
variants. We
have
previously
developed
probabilistic
approaches to integrate variant impact and population pre-
valence (VAAST), and in combination with patient phe-
notype, leveraging the HuPO hierarchy (Phevor), to provide
scores to prioritize variants for review clinicians. Here we
present a large-scale analysis of 1,963 reviewed clinical
WGS
diagnostics
cases
allowing
to
quantify
the 1Gen Era Diagnostic, Istanbul, Turkey, 2Faculty of
Engineering, Istinye University, Istanbul, Turkey 1Gen Era Diagnostic, Istanbul, Turkey, 2Faculty of
Engineering, Istinye University, Istanbul, Turkey We have developed a software that generates genetic ana-
lysis summary report by using VCF files are standard output
files of variant identification programs. GenerAVI pro-
grammed by using java. It can be run on three must com-
mon operating systems: Windows, Linux, Mac-OSX. There
is no installation requirement. The software generates a genetic analysis summary report
based on the evidence classification of the article of
American College of Genetics and Genomics (ACMG)
published for sequenced variants of clinical interpretation in
2015. Using 28 of evidence of ACMG, it classifies variants Abstracts from the 51st European Society of Human Genetics Conference: Posters 601 into 5 groups as likely benign, benign, VUS, likely
pathogenic and pathogenic. We have used 11 automatable
evidences (PVS1, PM2, PP2, PP3, PP5, BA1, BS1, BS2,
BP1, BP4, BP6) and an inhouse variant database for
Turkish population to provide a genetic analysis summary
report for MEFV, BRCA1, BRCA2 and CFTR genes. into 5 groups as likely benign, benign, VUS, likely
pathogenic and pathogenic. We have used 11 automatable
evidences (PVS1, PM2, PP2, PP3, PP5, BA1, BS1, BS2,
BP1, BP4, BP6) and an inhouse variant database for
Turkish population to provide a genetic analysis summary
report for MEFV, BRCA1, BRCA2 and CFTR genes. into 5 groups as likely benign, benign, VUS, likely
pathogenic and pathogenic. P17.02B
Allele specific expression identifies rare variants as cause
for extreme allelic imbalance of Human
Genetics, Leiden University Medical Center, Leiden,
Netherlands, 5Centre for Molecular and Biomolecular
Informatics, Radboud Institute for Molecular Life Sciences,
Radboud University Medical Center, Nijmegen, Netherlands Large eQTL studies have mostly been used to map the
regulatory effects of common SNPs on gene expression. However, with current sample sizes it is difficult to identify
eQTLs for rare variants. To address this we have used
RNA-sequencing to genotype 4,001 blood samples from
BIOS, a large Dutch biobank consortium, and used this to
measure Allele Specific Expression (ASE) to assess the
effects of rare variants on allelic imbalance. Per gene we defined outlier samples as those that show an
allelic imbalance that is 3 absolute deviations from the J. del Picchia 602 chromatin landscape leading to a generalized reduction in
the number of peaks in actively transcribed (H3K36me3)
and enhancer regions (H3K4me1 and H3K27ac). Particu-
larly, transcriptional initiation and elongation chromatin
states result the most significantly affected, highlighting
genes implicated in “mitotic nuclear division”, “cell cycle”
and “mRNA processing” as the major dysregulated targets
following CHD8 suppression. Moreover, by overlaying
histone marks data with CHD8 binding sites, we observe
that most chromatin changes seem to intervene in regions
not-bound by CHD8, thus suggesting an ‘indirect mode’ of
chromatin remodeling. In summary, our results point toward
broad regulatory consequence of CHD8 suppression, pos-
sibly implicating altered RNA processing, as well as asso-
ciation with specific biological pathways of relevance to
ASD pathogenesis. were observed. Interestingly, when we considered methy-
lation levels in relation to the mutations of ALS associated
genes, lower D-loop methylation levels were detected in
carriers of SOD1 mutations, respect to carriers of mutations
in the three other genes considered and non-carriers of
mutations (P < 0.05).Present results indicate that mito-
chondrial D-loop region is hypomethylated in peripheral
blood DNA of ALS patients and in carriers of SOD1mu-
tations, and that this epigenetic signature is related to
mitochondrial deregulation. A. Stoccoro: None. R. Gallo: None. L. Mosca: None. C. Tarlarini: None. C. Lunetta: None. A. Marocchi: None. V. Carnicelli:
None. L. Migliore:
None. F. Coppedè: None. A. Stoccoro: None. R. Gallo: None. L. Mosca: None. C. Tarlarini: None. C. Lunetta: None. A. Marocchi: None. V. Carnicelli:
None. L. Migliore:
None. F. Coppedè: None. CHD8 suppression in neuronal progenitors correlates with
alterations in chromatin landscape especially affecting
transcriptional initiation and elongation CHD8 suppression in neuronal progenitors correlates with
alterations in chromatin landscape especially affecting
transcriptional initiation and elongation E. Kerschbamer: None. T. Tripathi: None. S. Erdin:
None. F. Di Leva: None. M. Benelli: None. S. Piazza:
None. J.F. Gusella: None. F. Ferrari: None. F. Demiche-
lis: None. M.E. Talkowski: None. M. Biagioli: None. E. Kerschbamer: None. T. Tripathi: None. S. Erdin:
None. F. Di Leva: None. M. Benelli: None. S. Piazza:
None. J.F. Gusella: None. F. Ferrari: None. F. Demiche-
lis: None. M.E. Talkowski: None. M. Biagioli: None. E. Kerschbamer1, T. Tripathi1, S. Erdin2,3, F. Di Leva1, M. Benelli4, S. Piazza5, J. F. Gusella2,3, F. Ferrari6, F. Demichelis7,
M. E. Talkowski2,3, M. Biagioli1 P17.05A BWS in caused by defects at Abstracts from the 51st European Society of Human Genetics Conference: Posters 603 chromosome 11p15.5 including Loss of Methylation (LoM)
at the Imprintig Center 2 (IC2), Gain of Methylation at the
Imprinting Center 1 (IC1), paternal uniparental disomy of
11p15.5 region and maternally inherited pathogenic variant
of CDKN1C. In about 30% of BWS, MLID (Multilocus
Methylation Imprinting Disturbance) is also reported. We
present a case of monozygotic, monochorionic and dia-
mniotic female twins discordant for BWS clinical mani-
festations (only one twin presented with macrosomia,
macroglossia and nevus flammeus). At birth, the phenoty-
pically affected twin had IC2 LoM and MLID on both
blood and buccal smear samples, whereas the unaffected
one showed IC2 LoM, without MLID, only on blood
sample. Shared placental circulation and twin-to-twin
transfusion have been hypotesized to explain the presence
of epimutations in blood of the phenotypically normal twin. A two years clinical follow-up confirmed the complete
absence of BWS signs in the unaffected twin, despite the
maintenance of IC2 LoM in blood. We are currently
investigating the methylation profile of placenta (collected
at delivery) in order to deepen the insight into the epigenetic
constitution of the fetal/placental compartment. shows that the absence of Kcnk9 leads to impaired working
memory, reduced acoustic startle response and abnormal
sensorimotor gating. Investigations of circadian rhythms
revealed selectively increased locomotor activity during the
dark phase in Kcnk9KOhom and, to a significantly smaller
extent, in Kcnk9KOmat mice compared to controls. Using
Quantification of Allele-Specific Expression by Pyrose-
quencing (QUASEP) and Allele-Specific RT-qPCR in
wildtype
(C57BL/6xCast/Ei)F1
hybrid
mice,
biallelic
Kcnk9 expression from the repressed paternal allele (1-
17% of transcripts) was observed in all analyzed brain
regions and was particularly strong in the locus coeruleus
(LC). Slice patch-clamp recordings revealed wildtype-like
pacemaker activity during the dark phase in LC neurons
from Kcnk9KOmat but not from Kcnk9KOhom mice, which
discharged at significantly higher frequencies. The neuronal
data are in line with the locomotor phenotype and
demonstrate the functional relevance of paternal Kcnk9
expression. Through epigenetic manipulation with CI994, a specific
histone deacetylase inhibitor, we could induce a significant
up-regulation of the paternal Kcnk9 allele in several
analyzed brain regions after injections in Kcnk9KOmat mice. Together with this we observed a significant behavioral
improvement of Kcnk9KOmat mice after CI994 treatment. This novel approach shall open new avenues for treatment
of cognitive dysfunctions in Birk-Barel syndrome and other
imprinting disorders. P17.05A P17.05A
Female monozygotic twins phenotypically discordant for
Beckwith-Wiedemann Syndrome sharing hypomethylation
of IC2 1Neuro Epigenetics laboratory, Centre for Integrative Biology,
University of Trento, Trento, Italy, 2Center for Genomic
Medicine, Massachusetts General Hospital, Boston, MA,
United States, 3Department of Neurology, Harvard Medical
School, Boston, MA, United States, 4Bioinformatics Unit,
Hospital of Prato, Istituto Toscano Tumori, Prato, Italy,
5Bioinformatic facility, Centre for Intergrative Biology,
University of Trento, Trento, Italy, 6Computational genomics
laboratory, IFOM, Milan, Italy, 7Laboratory of Computational
and Functional Oncology, Centre for Integrative Biology,
University of Trento, Trento, Italy M. F. Bedeschi1, G. A. Cagnoli1, R. Villa1, V. G. C. Fergnani1,
F. Lalatta1, S. Gangi2, F. Mosca2, N. Persico3, M. Porro4, L. Fontana5,6, S. Tabano5,6, M. Miozzo5,6 1Clinical Genetics Unit, Fondazione IRCCS Ca’ Granda,
Ospedale Maggiore Policlinico, Milan, Italy, 2NICU,
Department of Clinical Sciences and Community Health,
Università degli Studi di Milano, Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico, Milan, Italy,
3Department of Obstetrics and Gynecology, Fondazione
IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan,
Italy, 4Pediatric Physical Medicine & Rehabilitation Unit,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, Milan, Italy, 5Department of Pathophysiology &
Transplantation, Università degli Studi di Milano, Milan, Italy,
6Division of Pathology, Fondazione IRCCS Ca’ Granda
Ospedale Maggiore Policlinico, Milan, Italy Autism Spectrum Disorders (ASD) is a collection of het-
erogeneous neurodevelopmental disorders defined by social
impairment and repetitive behaviors with significant geno-
typic and phenotypic complexity. Mutations in several
hundred loci have been associated with the disease, but the
chromodomain helicase DNA binding protein 8 (CHD8)
represents a recurrent and independently validated ASD-
risk gene. All ASD mutations in CHD8 gene have been
reported to be disruptive, leading to haploinsufficiency. Here we investigate how chromatin landscape reacts to
CHD8 suppression by analyzing different histone mod-
ifications through Chromatin Immunoprecipitation and
Sequencing (ChIP-seq) in iPS-derived neural progenitors
after CHD8 knock-down. We interrogated transcriptionally
active and repressed regions as well as active and poised
enhancers to explore the potential impact of loss-of-function
mutations. CHD8 suppression mildly affects the overall Beckwith-Wiedemann
Syndrome
is
an
overgrowth
imprinting syndrome characterized by pre- and post-natal
macrosomia, congenital abnormalities (such as macro-
glossia, omphalocele or umbilical hernia, visceromegaly,
nephrourologic malformatios, ear anomalies) and tumor
predisposition. The prevalence is about 1:10,500 live births,
with an equal incidence in males and females, except for
monozygotic twins, for which a significant female pre-
ponderance is reported. P17.06B A. Cooper: None. S. Jagannath: None. T. Butto: None. M. Linke: None. F. Lesage: None. K. Radyushkin: None. J. Roeper: None. S. Schweiger: None. U. Zechner: None. Inhibition of histone deacetylation up-regulates the
repressed paternal allele of the imprinted Kcnk9 gene and
improves the behavioral phenotype of a mouse model of
Birk-Barel syndrome P17.05A M.F. Bedeschi: None. G.A. Cagnoli: None. R. Villa:
None. V.G.C. Fergnani: None. F. Lalatta: None. S. Gangi: None. F. Mosca: None. N. Persico: None. M. Porro: None. L. Fontana: None. S. Tabano: None. M. Miozzo: None. P17.07C Micro-
RNAs (miRNAs) are aberrantly expressed in BC, and may
be isolated from various biological specimens, including
urine. We performed a miRNA profiling in urine samples
from 66 BC male patients (10 muscle invasive BC (MIBC)
and 56 non-muscle invasive BC (NMIBC)) and 48 healthy
controls using a Next Generation Sequencing approach that
could accurately distinguish BC patients and predict disease
outcome. Specific urinary miRNA signatures could distin-
guish the different types of BC patients from healthy con-
trols in one of the best and closest surrogate tissue for BC. Twenty-three miRNAs (21 target and 2 reference miRNAs)
were validated by qPCR on 177 urine samples from 113 BC
case and 64 controls . MiRNA signatures were also eval-
uated in concomitance with the presence of a panel of
mutations in DNA from exfoliated urinary cells from the
same samples. Forty-one mutations in TERT, FGFR3,
PIK3CA, and RAS were analyzed in relation to clinical
outcome. We will present the results on the possibility to
use urinary miRNAs and exfoliated urinary cell mutational
status as diagnostic, prognostic and predictive biomarkers
for BC. identification of new biomarkers for early BC detection,
recurrence/progression is urgently needed to both improve
patient outcomes and decrease health care costs. Micro-
RNAs (miRNAs) are aberrantly expressed in BC, and may
be isolated from various biological specimens, including
urine. We performed a miRNA profiling in urine samples
from 66 BC male patients (10 muscle invasive BC (MIBC)
and 56 non-muscle invasive BC (NMIBC)) and 48 healthy
controls using a Next Generation Sequencing approach that
could accurately distinguish BC patients and predict disease
outcome. Specific urinary miRNA signatures could distin-
guish the different types of BC patients from healthy con-
trols in one of the best and closest surrogate tissue for BC. Twenty-three miRNAs (21 target and 2 reference miRNAs)
were validated by qPCR on 177 urine samples from 113 BC
case and 64 controls . MiRNA signatures were also eval-
uated in concomitance with the presence of a panel of
mutations in DNA from exfoliated urinary cells from the
same samples. Forty-one mutations in TERT, FGFR3,
PIK3CA, and RAS were analyzed in relation to clinical
outcome. We will present the results on the possibility to
use urinary miRNAs and exfoliated urinary cell mutational
status as diagnostic, prognostic and predictive biomarkers
for BC. P17.07C Methylation in
breast cancer
and/or adjacent
nonmalignant
samples (%)
Methylation
in normal
mammary
gland from
autopsy (%)
Presence(+) / absence(-) of
methylation in breast cancer
cell lines
Association with
clinicopathological
features
ZR MCF7 T47D BT HBL HS
LAMA1
29,4
0
+
+
+
+
+
-
-
LAMA2
27
0
+
+
+
+
+
-
HER2=3+
Luminal B type
LAMB1
26
0
+
+
+
+
-
-
HER2=3+
Luminal B type
ITGA1
15,2
0
-
-
-
-
-
-
-
ITGA4
30
0
+
+
+
+
-
-
HER2=3+
ITGA7
3,4
0
-
-
-
+
-
-
-
ITGA9
41,3
0
+
+
+
+
+
-
Unmethylated
status associated
with triple-negative
type, lack of ER
NID1
38,7
0
+
+
+
-
-
+
Unmethylated
status associated
with triple-
negative type
NID2
41,2
0
-
+
+
+
-
-
-
CDH2
8,8
0
-
-
-
-
-
-
-
CDH3
41,5
0
+
+
+
+
+
-
Lack of ER
MMP2
7,7
0
+
+
+
-
-
-
-
MMP23B 17
0
+
+
+
-
+
+
Lack of ER, PR
HER2=3+
HER2-positive type
MMP24
12
0
-
-
-
+
+
-
-
MMP25
15,4
0
-
+
+
-
+
-
-
MMP28
4,9
0
+
+
+
+
+
-
- Methylation in
breast cancer
and/or adjacent
nonmalignant
samples (%)
Methylation
in normal
mammary
gland from
autopsy (%)
Presence(+) / absence(-) of
methylation in breast cancer
cell lines
Association with
clinicopathological
features
ZR MCF7 T47D BT HBL HS
LAMA1
29,4
0
+
+
+
+
+
-
-
LAMA2
27
0
+
+
+
+
+
-
HER2=3+
Luminal B type
LAMB1
26
0
+
+
+
+
-
-
HER2=3+
Luminal B type
ITGA1
15,2
0
-
-
-
-
-
-
-
ITGA4
30
0
+
+
+
+
-
-
HER2=3+
ITGA7
3,4
0
-
-
-
+
-
-
-
ITGA9
41,3
0
+
+
+
+
+
-
Unmethylated
status associated
with triple-negative
type, lack of ER
NID1
38,7
0
+
+
+
-
-
+
Unmethylated
status associated
with triple-
negative type
NID2
41,2
0
-
+
+
+
-
-
-
CDH2
8,8
0
-
-
-
-
-
-
-
CDH3
41,5
0
+
+
+
+
+
-
Lack of ER
MMP2
7,7
0
+
+
+
-
-
-
-
MMP23B 17
0
+
+
+
-
+
+
Lack of ER, PR
HER2=3+
HER2-positive type
MMP24
12
0
-
-
-
+
+
-
-
MMP25
15,4
0
-
+
+
-
+
-
-
MMP28
4,9
0
+
+
+
+
+
-
- P17.07C Urine microRNA profiling by next generation sequencing
and multiple mutations in urinary exfoliated cells in
bladder cancer A. Cooper1, S. Jagannath2, T. Butto1, M. Linke1, F. Lesage3, K. Radyushkin4, J. Roeper2, S. Schweiger1, U. Zechner1 A. Cooper1, S. Jagannath2, T. Butto1, M. Linke1, F. Lesage3, K. Radyushkin4, J. Roeper2, S. Schweiger1, U. Zechner1 B. Pardini1,2, F. Cordero3, A. Naccarati1, R. Critelli1, C. Viberti1,2, M. Oderda4, M. Allasia4, A. Allione1,2, C. Sacerdote5,
P. Gontero4, P. Vineis6, G. Matullo1,2 1Institute of Human Genetics, University Medical Center,
Johannes Gutenberg University, Mainz, Germany, 2Institute of
Neurophysiology, Goethe University, Frankfurt, Germany,
32LabEx ICST, Institut de Pharmacologie Moléculaire et
Cellulaire, CNRS and Université de Nice-Sophia Antipolis,
Valbonne, France, 4Focus Program Translational
Neuroscience, University Medical Center of the Johannes
Gutenberg-University, Mainz, Germany 1Institute of Human Genetics, University Medical Center,
Johannes Gutenberg University, Mainz, Germany, 2Institute of
Neurophysiology, Goethe University, Frankfurt, Germany,
32LabEx ICST, Institut de Pharmacologie Moléculaire et
Cellulaire, CNRS and Université de Nice-Sophia Antipolis,
Valbonne, France, 4Focus Program Translational
Neuroscience, University Medical Center of the Johannes
Gutenberg-University, Mainz, Germany 1Italian Institute for Genomic Medicine (IIGM), Turin, Italy,
2Department of Medical Sciences, University of Turin, Turin,
Italy, 3Department of Computer Science, University of Turin,
Turin, Italy, 4Department of Surgical Sciences, University of
Turin, Turin, Italy, 5Center for Cancer Prevention (CPO-
Piemonte), Turin, Italy, 6School of Public Health, Imperial
College London, London, United Kingdom 1Italian Institute for Genomic Medicine (IIGM), Turin, Italy,
2Department of Medical Sciences, University of Turin, Turin,
Italy, 3Department of Computer Science, University of Turin,
Turin, Italy, 4Department of Surgical Sciences, University of
Turin, Turin, Italy, 5Center for Cancer Prevention (CPO-
Piemonte), Turin, Italy, 6School of Public Health, Imperial
College London, London, United Kingdom Kcnk9/KCNK9 is a maternally expressed imprinted gene,
whose mutations are causative for the maternally inherited
Birk-Barel mental retardation syndrome. It encodes a K2P-
channel that controls resting membrane potential and
excitability of neurons. Bladder cancer (BC) is one of the most aggressive malig-
nancies of the urinary tract. Because of the high rate of
recurrences requiring continuous surveillance, BC is the
most
expensive
cancer
for
the
health
system.The By analyzing WT, Kcnk9KOmat and Kcnk9KOhom mice in
a behavioral test battery during the light phase, our data 604 J. del Picchia identification of new biomarkers for early BC detection,
recurrence/progression is urgently needed to both improve
patient outcomes and decrease health care costs. P17.07C Materials and Methods: We analyzed methylation status
of 12 laminin-encoding genes (LAMA1, LAMA2, LAMA3A,
LAMA3B, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3,
LAMC1, LAMC2, LAMC3), 8 integrins (ITGA1, ITGA2,
ITGA3, ITGA4, ITGA6, ITGA7, ITGA9, ITGB1), 2 nidogens
(NID1,NID2), 2 cadherins (CDH2,CDH3), the dystroglycan
gene DAG1 and 11 matrix metalloproteinases-encoding genes
(MMP2, MMP11, MMP14, MMP15, MMP16, MMP17,
MMP21, MMP23B, MMP24, MMP25, MMP28) and 4 tissue
inhibitors
of
matrix
metalloproteinases
genes
(TIMP1,
TIMP2, TIMP3, TIMP4) in 206 breast cancer samples, 206
paired adjacent nonmalignant samples, 6 samples of normal
mammary gland from autopsy and 6 samples of breast cancer
cell lines by MSRE PCR and bisulfite sequencing. (
)
(
)
y
g y
gene DAG1 and 11 matrix metalloproteinases-encoding genes
(MMP2, MMP11, MMP14, MMP15, MMP16, MMP17,
MMP21, MMP23B, MMP24, MMP25, MMP28) and 4 tissue
inhibitors
of
matrix
metalloproteinases
genes
(TIMP1,
TIMP2, TIMP3, TIMP4) in 206 breast cancer samples, 206
paired adjacent nonmalignant samples, 6 samples of normal
mammary gland from autopsy and 6 samples of breast cancer
cell lines by MSRE PCR and bisulfite sequencing. Results: Promoters of 17 genes (LAMA1, LAMA2,
LAMB1, LAMC1 NID1, NID2, ITGA1, ITGA4, ITGA7,
ITGA9, CDH2,CDH3, MMP2, MMP23B, MMP24,MMP25,
MMP28) have shown abnormal methylation in 3,4% to
41,5% samples of breast cancer and adjacent tissues. Further statistical analysis with additional data have
demonstrated, that methylation of 15 gens LAMA1, LAMA2,
LAMB1, NID1, NID2, ITGA1, ITGA4, ITGA7, ITGA9,
CDH2,CDH3, MMP2, MMP24, MMP25, MMP28 was
strongly associated with highly methylated breast cancer
type. Based on this information we designed a system of
markers which discriminate hyper- and hypomethylated
subtypes of breast cancer with sensitivity and specificity of
0,79 and 0,78, respectively; AUC=0,83. Acknowledgements:
Fondazione
Umberto
Veronesi
(FUV) “Post-doctoral fellowship Years 2014-2017”, FUV
Grant 2013, AIRC-IG 17464. B. Pardini: None. F. Cordero: None. A. Naccarati:
None. R. Critelli: None. C. Viberti: None. M. Oderda:
None. M. Allasia: None. A. Allione: None. C. Sacerdote:
None. P. Gontero:
None. P. Vineis:
None. G. Matullo: None. B. Pardini: None. F. Cordero: None. A. Naccarati:
None. R. Critelli: None. C. Viberti: None. M. Oderda:
None. M. Allasia: None. A. Allione: None. C. Sacerdote:
None. P. Gontero:
None. P. Vineis:
None. G. Matullo: None. B. Pardini: None. F. Cordero: None. A. Naccarati:
None. R. Critelli: None. C. Viberti: None. M. Oderda:
None. M. Allasia: None. A. Allione: None. C. Sacerdote:
None. P. Gontero:
None. P. Vineis:
None. G. Matullo: None. . C te
:
C. P17.07C V be t :
. Ode da:
None. M. Allasia: None. A. Allione: None. C. Sacerdote:
None. P. Gontero:
None. P. Vineis:
None. G. Matullo: None. P17.08D
Breast cancer epigenetics: abnormal promoter methylation
of matrix and transmembrane proteins encoding genes
O. A. Simonova1, E. B. Kuznetsova1,2, V. V. Rudenco1, E. V. Poddubskaya3, R. A. Kerimov3, A. S. Tanas1,4, V. V. Strelnikov1,4, D. V. Zaletaev1,2
1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Sechenov First Moscow State Medical University,
Moscow, Russian Federation, 3Blokhin Russian Research
Center for Oncology (FSBI N.N.Blokhin), Moscow, Russian
Federation, 4Pirogov Russian National Research Medical
University, Moscow, Russian Federation
Extracellular matrix molecules and transmembrane recep-
tors organize tissue structure and provide its adequate
function. During cancer progression both these features
suffer disruption. 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan 1Department of Medical Sciences. University of Girona,
Girona, Spain, 2Biomedical Research Institute of Girona
(IDIBGI), Salt, Spain, 3Department of Medicine, University of
California, San Diego (UCSD), La Jolla, CA, United States Introduction: CAD is a tri-functional enzyme comprise the
first three steps of total six steps of de novo pyrimidine
biosynthesis with unknown role during mammalian devel-
opment. Zebrafish Cad mutants exhibited reduced eye size,
growth, tectum, jaw, and pectoral fins abnormalities and the
gene is highly expressed the in the retina. Through a three-
generation ENU program, two mouse Cad mutant families
were generated: the L5Jcs24 (missense mutation) and the
L5Jcs27 (nonsense mutation). Homozygotes were embryo-
nic lethal, and heterozygotes exhibiting small eyes with
degenerated retina. Due to the phenotypic similarities
between well-studied eye-specific transcription factors,
Pax6 and Crx, we speculated that these genes might func-
tion in the same pathway in controlling ocular development. In silico transcription factor binding site analysis predicted
multiple potential binding sites for both PAX6 and CRX. Introduction: CAD is a tri-functional enzyme comprise the
first three steps of total six steps of de novo pyrimidine
biosynthesis with unknown role during mammalian devel-
opment. Zebrafish Cad mutants exhibited reduced eye size,
growth, tectum, jaw, and pectoral fins abnormalities and the
gene is highly expressed the in the retina. Through a three-
generation ENU program, two mouse Cad mutant families
were generated: the L5Jcs24 (missense mutation) and the
L5Jcs27 (nonsense mutation). Homozygotes were embryo-
nic lethal, and heterozygotes exhibiting small eyes with
degenerated retina. Due to the phenotypic similarities
between well-studied eye-specific transcription factors,
Pax6 and Crx, we speculated that these genes might func-
tion in the same pathway in controlling ocular development. In silico transcription factor binding site analysis predicted
multiple potential binding sites for both PAX6 and CRX. Genetic variation within transcription factor binding sites
(TFBS) generates diversity in gene expression regulation,
and may lead to increased or decreased risk for human
disease. Here, we aim to characterize genetic variation at
genomic locations candidate to host TFBS nearby genes
associated with Brugada Syndrome (BrS). BrS is an
arrhythmogenic disease that has been extensively associated
with genetic variations in genes encoding cardiac ion
channels (25-30%), but the contribution of genetic variation
at
nearby
TFBS
remains
unknown. H. Liang-shuan1, L. Cheng-der1, C. Jia-ying2, C. Chi-shuan1, H.
Shun-ping1, C. Yung-hao1 M. Pinsach-Abuin1,2,3, B. del Olmo1,2, J. Mates1,2, A. Pérez1,2, D. Merkurjev3, C. Allegue1,2, S. Konovalov3, R. Brugada1,2, I. Garcia-Bassets3, S. Pagans1,2 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan 1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua
University, Hsinchu, Taiwan We
integrated
ENCODE data of topological organization, chromatin
accessibility, histone marks, and transcription factor binding
in human cardiac cells and annotated 1,300 genomic loci
that potentially host TFBS nearby BrS-associated genes. To
characterize genetic variation in these regions, we selec-
tively captured and sequenced in depth (x100) this set of
1,300 loci in a cohort of 89 BrS cases (BrS cohort), and
compared them with genetic variations in the Wellderly
cohort (healthy-aging individuals). In the BrS cohort, we
identified 5,382 single-nucleotide variants (SNVs) and 737
insertions and deletions (Indels). We observe 4,231 SNVs
in common between both cohorts, while the remaining
1,888 SNVs are BrS patient-specific. Our current analyses
are focused on the integration of these data to determine the
potential simultaneous contribution of multiple TFBS to the
disease. To investigate the potential functional relevance of
our findings, we also integrate data of experimentally vali-
dated CTCF-TFBS, and machine learning-based predictions
of TFBS for cardiac transcription factors. Collectively, our
study represents the most exhaustive analysis up-to-date of
genetic variation associated with BrS. Materials and Methods: Luciferase assay were used to
evaluate whether Cad promoter is under transcriptional
control of PAX6 or CRX. We also use shRNA knockdown
combined with Western blotting and qRT-PCR, to further
evaluate our hypothesized that PAX6 and CRX might be
involved in the regulation of Cad gene expression. Results: The luciferase activities of Cad promoter were
up-regulated by PAX6 and CRX for six-fold when
transfected alone, and the promoter activities were up-
regulated for 14 times when PAX6 and CRX were co-
transfected to the Cad promoter. Results: The luciferase activities of Cad promoter were
up-regulated by PAX6 and CRX for six-fold when
transfected alone, and the promoter activities were up-
regulated for 14 times when PAX6 and CRX were co-
transfected to the Cad promoter. Conclusions: We have confirmed that PAX6 and CRX
bind to the promoter of Cad region and up-regulated gene
expression in additive fashion. H. Liang-shuan: None. L. Cheng-der: None. C. Jia-
ying: None. C. Chi-shuan: None. H. Shun-ping: None. C. Yung-hao: None. Developing CAR-T cells therapies : Is the European law
adequate ? Genetic variation on transcription factor binding sites
nearby human genes associated with Brugada syndrome H. Liang-shuan1, L. Cheng-der1, C. Jia-ying2, C. Chi-shuan1, H. Shun-ping1, C. Yung-hao1 P17.08D Breast cancer epigenetics: abnormal promoter methylation
of matrix and transmembrane proteins encoding genes Abstracts from the 51st European Society of Human Genetics Conference: Posters 605 O.A. Simonova: None. E.B. Kuznetsova: None. V.V. Rudenco: None. E.V. Poddubskaya: None. R.A. Keri-
mov: None. A.S. Tanas: None. V.V. Strelnikov: None. D. V. Zaletaev: None. P17.11C Funding: SAF2015-70823-R (MINECO/FEDER-UE) Funding: SAF2015-70823-R (MINECO/FEDER-UE) Funding: SAF2015-70823-R (MINECO/FEDER-UE) Developing CAR-T cells therapies : Is the European law
adequate ? M. Pinsach-Abuin: None. B. del Olmo: None. J. Mates:
None. A. Pérez: None. D. Merkurjev: None. C. Allegue: M. Pinsach-Abuin: None. B. del Olmo: None. J. Mates:
None. A. Pérez: None. D. Merkurjev: None. C. Allegue: D. Pichereau1, C. Chabannon2, E. Rial-Sebbag1 J. del Picchia 606 1INSERM UMR 1027, Paul Sabatier University, Toulouse,
France, 2Centre d'Investigations Cliniques en Biothérapies,
Aix-Marseille University, Institut Paoli-Calmettes, AP-HM,
INSERM CBT-1409, Marseille, France pathogenesis
of
neuroblastomas,
and
heterozygous
PHOX2B mutations cause Congenital Central Hypoventi-
lation Syndrome (CCHS), a life-threatening neurocristo-
pathy characterised by failure of autonomic respiratory
control. A partial recovery of ventilation has been observed
in some CCHS patients using the potent contraceptive
progestin desogestrel. Moreover, previous findings have
shown that progesterone suppressed the growth of neuro-
blastoma in vitro and in vivo. In December 2017, at the American Society of Hematology,
the successful use of CAR-T cells in several specialized
worldwide centres was presented. This therapy consists in
the development of T cells (type of immune cells) in
laboratory, inserting into them, the gene of a special
receptor called chimeric antigen receptor (CAR). This
manipulation will modify the immune cells, so that they
carry the same target of cancer cells, that will be attacked. This method requires rigorous organisation since it should
imply serious side effects. Thus, it is relevant to analyse the
framework of this revolutionary method of treatment first
concerning their legal qualification which defines the reg-
ulatory pathway to seek for marketing authorization and
second the bioethical principles this innovation could
challenge. According to the innovative characteristics of
CAR-T cells, EU strictly regulates their use by considering
them like gene therapy and the Committee for Advanced
Therapies has provided specific guidance for their devel-
opment. So professionals, using CAR-T cells should
implement specific safety rules for the use of cells and for
patients from sampling to the infusion of the modified cells. Moreover, professionals and medical establishments will
have to adapt their facilities and to develop specific medical
expertise to implement the technique in practice. P17.11C Finally,
beyond these technical aspects, the transformation of cells
into a drug questions the bioethical principles beyond the
use of the elements of the human body such as property,
autonomy or justice The latter is of major importance due to
the high cost of the treatments. Materials and Methods: To investigate the effects of
desogestrel on the expression of PHOX2B and its target
genes, we generated a progesterone-responsive neuroblas-
toma cell line. Results: Our findings demonstrate that, through proges-
terone nuclear receptor PR-B, desogestrel down-regulates
PHOX2B
gene
expression,
by
a
post-transcriptional
mechanism, and its target genes, thus reinforcing the role
that PHOX2B has in the pathogenesis of CCHS and in
therapy response.They also further support the view that the
drugs and molecules that are effective in counteracting
neuroblastoma cell growth act through the down-regulation
of PHOX2B gene expression, and open up the possibility
that this mechanism may contribute to the positive effects
observed in some CCHS patients. Conclusions: These findings have a strong proof of
concept value, in the perspective of a pharmacological
intervention in CCHS, at least for ameliorating respiratory
symptoms. Fundings: Telethon Foundation [Grant No. GGP13055];
Associazione Italiana per la Sindrome da Ipoventilazione
Centrale Congenita (A.I.S.I.C.C.). S. Cardani: None. S. Di Lascio: None. D. Belperio:
None. E. Di Biase: None. I. Ceccherini: None. R. Benfante: None. D. Fornasari: None. D. Pichereau: None. C. Chabannon: None. E. Rial-
Sebbag: None. P17.13A Where SNPs, coexpression and TADs intersect: new
insights in the regulation of coexpression in celiac disease P17.12D
Molecular bases of desogestrel effects in congenital central
hypoventilation syndrome (CCHS) I. Romero-Garmendia1, N. Fernandez-Jimenez1, K. Garcia-
Etxebarria2, A. Jauregi-Miguel1, A. Olazagoitia-Garmendia1, I. Santin1, A. Castellanos-Rubio1, J. Bilbao1 S. Cardani1, S. Di Lascio1, D. Belperio1, E. Di Biase1, I. Ceccherini2, R. Benfante1,3, D. Fornasari1,3 1UPV/EHU-Biocruces-CIBERDEM, Leioa, Spain,
2Biodonostia, Donostia-San Sebastian, Spain 1Department of Medical Biotechnology and Translational
Medicine, Università degli Studi di Milano, Milan, Italy, 2UOC
Genetica Medica, Istituto Giannina Gaslini, Genoa, Italy,
3CNR- Neuroscience Institute, Milan, Italy Introduction: Alterations in expression and coexpression
patterns have been shown in celiac disease (CD), but the
mechanisms behind those changes remain unclear. Topo-
logically Associating Domains (TADs) are functional
domains of gene expression coordination and could explain
those changes. Thus, the aim of this study is to identify
SNPs that could be altering TAD structures and thus Introduction:
The
paired-like
homeobox
2B
gene
(PHOX2B) encodes a key transcription factor that plays a
role in the development of the autonomic nervous system. In humans, its over-expression is associated with the Abstracts from the 51st European Society of Human Genetics Conference: Posters 607 of Cagliari, Cagliari, Italy, 8Independent Researcher, Machine
Learning, Lucca, Italy, 9Unit of Hereditary Cancer, IRCCS
AOU San Martino-IST, Genova, Italy, 10Department of
Clinical and Experimental Medicine, University of Sassari,
Sassari, Italy deregulate (co)expression in CD, using data from coex-
pression
experiments,
location
of
TADs
and
CD-
associated SNPs. Materials and Methods: We used RNAseq data from the
epithelial fractions of intestinal biopsies from 10 CD
patients and 12 control individuals to build coexpression
matrixes; next, we checked whether coexpressed genes
overlapped with conserved TADs, and search for associated
SNPs that could alter those TADs. A candidate region was
deleted from HCT15 and 293FT cell lines using CRISPR-
Cas9 technology, and the expression of neighbouring genes
was assesed. Introduction: Colorectal cancer (CRC) develops through
the accumulation of both genetic and epigenetic alterations. While the former are used as prognostic and predictive
biomarkers, the latter are less characterized. The aims of the
study were to identify signature alterations in the CRC
methylome, to test whether they represent early events in
CRC development and to explore the use of non-invasive
techniques to reveal altered methylation. Results: Thirty-eight putative TAD fusions and disrup-
tions were identified when we compared celiac and control
groups and considered TAD and CD-associated SNP
coordinates. Deletion of an inter-TAD region spanning
chr20: 34026832 - 34027214 (Hg19), including a CTCF
binding site and a DNase hypersensitivity site, located near
the associated risk SNP rs224436 resulted in the coexpres-
sion of adjacent genes PROCR and ROMO1 resembling
active CD. Materials
and
Methods:
methylome
analysis
was
conducted by HumanMethylation450 BeadChips and raw
data were analysed using RnBeads. Pathways enrichment
was evaluated using ToppGene. In silico validation was
performed in publicly available datasets. Methylation of
three selected and validated markers was analysed in
24 stool DNAs and 45 plasma DNAs by digital PCR. Materials
and
Methods:
methylome
analysis
was
conducted by HumanMethylation450 BeadChips and raw
data were analysed using RnBeads. Pathways enrichment
was evaluated using ToppGene. In silico validation was
performed in publicly available datasets. Methylation of
three selected and validated markers was analysed in
24 stool DNAs and 45 plasma DNAs by digital PCR. Conclusions: This study shows that changes in TAD
structure could functionally explain part of the genetic
associations in complex diseases through gene (co)expres-
sion regulation. Results: we identified in 39 samples and validated in over
500 samples a panel of 74 altered CpG islands. The panel
discriminates CRCs and adenomas from peritumoral and
normal mucosa, with very high specificity (100%) and
sensitivity (99.9%). To establish the usefulness of these
findings as non-invasive markers for detection of CRC, we
selected and tested three biomarkers in stool DNA and
plasma cell-free circulating DNA, confirming the presence
of altered methylation in affected patients. Funding: ISCIII PI13/01201 and PI16/0258, co-funded
by ERDF/ESF, a way to make Europe. Funding: ISCIII PI13/01201 and PI16/0258, co-funded
by ERDF/ESF, a way to make Europe. I. Romero-Garmendia: None. N. Fernandez-Jimenez: I. Romero-Garmendia: None. N. Fernandez-Jimenez:
None. K. Garcia-Etxebarria: None. A. Jauregi-Miguel:
None. A. Olazagoitia-Garmendia: None. I. Santin: None. A. Castellanos-Rubio: None. J. Bilbao: None. A. Castellanos-Rubio: None. J. Bilbao: None. Conclusions: our study identifies a panel of genes with
strongly altered methylation in both adenomas and CRCs,
candidating its use as biomarker for adenomas and early
CRC detection through non-invasive techniques. F. Coppedè1, A. Stoccoro1, A. Lazzarotti1, R. Spisni2, L.
Migliore1 Discovery and validation of altered methylation loci as
early biomarkers of colorectal cancer Discovery and validation of altered methylation loci as
early biomarkers of colorectal cancer Grants: Fondazione Banco di Sardegna (2012), Regione
Autonoma Sardegna (CRP-79303), AIRC IG n. 17707,
AIRC 2010 Special Program Molecular Clinical Oncology
5 per mille, project no. 9970. Grants: Fondazione Banco di Sardegna (2012), Regione
Autonoma Sardegna (CRP-79303), AIRC IG n. 17707,
AIRC 2010 Special Program Molecular Clinical Oncology
5 per mille, project no. 9970. A. Fadda1, D. Gentilini2,3, L. Moi1, L. Barault4,5, V. P. Leoni6, P. Sulas6, L. Zorcolo7, A. Restivo7, F. Cabras7, F. Fortunato7, C. Zavattari8, L. Varesco9, V. Gismondi9, M. De Miglio10, A. M. Scanu10, F. Colombi4, P. Lombardi4, I. Sarotto5, E. Loi1, F. Leone4,5, S. Giordano4,5, F. Di Nicolantonio4,5, A. Columbano6,
P. Zavattari1 A. Fadda: None. D. Gentilini: None. L. Moi: None. L. Barault: None. V.P. Leoni: None. P. Sulas: None. L. Zorcolo: None. A. Restivo: None. F. Cabras: None. F. Fortunato: None. C. Zavattari: None. L. Varesco: None. V. Gismondi: None. M. De Miglio: None. A.M. Scanu:
None. F. Colombi: None. P. Lombardi: None. I. Sarotto:
None. E. Loi: None. F. Leone: None. S. Giordano: None. F. Di Nicolantonio: None. A. Columbano: None. P. Zavattari: None. 1Unit of Biology and Genetics, Department of Biomedical
Sciences, University of Cagliari, Cagliari, Italy, 2Department
of Brain and Behavioral Sciences, University of Pavia, Pavia,
Italy, 3Bioinformatics and Statistical Genomics Unit, Istituto
Auxologico Italiano IRCCS, Cusano Milanino, Milano, Italy,
4Department of Oncology, University of Torino, Torino, Italy,
5Candiolo Cancer Institute-FPO, IRCCS, Torino, Italy, 6Unit
of Oncology and Molecular Pathology, Department of
Biomedical Sciences, University of Cagliari, Cagliari, Italy,
7Department of Surgery, Colorectal Surgery Center, University P17.15C
GHSR methylation as a biomarker of colorectal cancer
F. Coppedè1, A. Stoccoro1, A. Lazzarotti1, R. Spisni2, L. Migliore1 P17.15C P17.15C
GHSR methylation as a biomarker of colorectal cancer J. del Picchia 608 1Dept of Translational Research and New Technologies in
Medicine and Surgery, University of Pisa, Pisa, Italy,
2Department of Surgery, Medical, Molecular & Critical Area
Pathology, University of Pisa, Pisa, Italy Introduction: The Human Leucocyte Antigen (HLA)
explains around 40% of the heritability of celiac disease
(CD). However, the pathogenesis of CD could be driven by
other layers of genomic information independent from
inherited sequence variation, such as DNA methylation,
also in this region. Introduction: The Human Leucocyte Antigen (HLA)
explains around 40% of the heritability of celiac disease
(CD). However, the pathogenesis of CD could be driven by
other layers of genomic information independent from
inherited sequence variation, such as DNA methylation,
also in this region. Introduction: Epigenetic events contribute significantly to
colorectal cancer (CRC) pathogenesis. DNA methylation
signatures have been proposed as potential biomarkers in
CRC diagnosis and in measuring response to therapy. Recently, hypermethylation of the growth hormone secre-
tagogue receptor (GHSR) gene has been proposed as a
common epigenetic alteration of high diagnostic value in a
broad spectrum of cancers. Regarding CRC, only a single
study addressed this issue in a small sample of CRC
specimens. Methods: DNA methylation landscape is expected to be
different among cell types. Therefore, we analyzed the
methylome and the transcriptome of sorted epithelial and
immune cells of duodenal biopsies in 10 CD-patients and
10 controls. Methylation differences were confirmed by
next generation sequencing (NGS) in bisulfite-treated, PCR-
amplified fragments spanning TAP1 and HLA-B promoters
in an independent cohort of 14 inactive-CD and 14 control
individuals. Results: TAP1 was hypomethylated in the CpG island
shores surrounding its promoter in both epithelial and
immune cells, and so was HLA-B in the epithelia. This was
confirmed in complete biopsies from inactive-CD patients. Additionally, TAP1 and its overlapping gene PSMB9 were
overexpressed in the epithelial and immune fractions, while
the upregulation of HLA-B and its overlapping pseudogene
was observed only in epithelia. Both TAP1-PSMB9 and
HLA-B-pseudogene pairs were coexpressed. Materials and Methods: In order to validate GHSR
hypermethylation as a CRC biomarker we compared 73
DNA samples extracted from CRC tissues and 73 DNA
samples obtained from the healthy colonic mucosa of the
same patients. Results: Very interestingly we observed a statistically
significant hypermethylation of GHSR in tumor tissues than
in healthy mucosa (51.3% vs. 20.5%, P < 5 x 10-7). P17.15C ROC
analysis revealed a high degree of both sensitivity and
specificity for discriminating tumor tissue and correspond-
ing healthy mucosa (AUC value of 0.819). No correlation
between GHSR hypermethylation in tumor DNA samples
and tumor stage, size, location, or patient’s age and gender
was observed, indicating that this is an early epigenetic
event already observable at the adenoma stage. Moreover,
no correlation between GHSR methylation and polymorphic
variants of genes involved in DNA methylation reactions,
including DNA methyltransferases and folate-related genes,
was observed. In the normal colonic mucosa tissue a
significant positive correlation between GHSR methylation
and age (r=0.34; P=0.003) was observed. Conclusions: The confirmation of the differential methy-
lation in inactive-CD suggests that alterations were acquired
early in life. Expression results are coherent with the cell
type-specificity of the methylation alterations.We propose
the identified genes as novel CD candidates and particularly
TAP1, an important HLA class-I peptide whose exacervated
expression suggests an anomalous boost of adaptive
immunity. Funding:
Basque
Government-POSDOC
Fellowship
(POS_2016_2_0026); ISCIII PI13/01201 and PI16/0258,
co-funded by ERDF/ESF, a way to make Europe. N. Fernandez-Jimenez: None. I. Romero-Garmendia:
None. K. Garcia-Etxebarria: None. A. Jauregi-Miguel:
None. I. Irastorza: None. J.R. Bilbao: None. Conclusions: Present results confirm GHSR methylation
as a strong and highly accurate epigenetic biomarker
of CRC. P17.18B
Analysis of endothelin-1 (EDN-1) promoter region P17.18B
Analysis of endothelin-1 (EDN-1) promoter region Analysis of endothelin-1 (EDN-1) promoter region C. López Solarat1, M. Lago-Docampo1, A. Baloira2, D. Valverde1
1University of Vigo, Vigo, Spain, 2Servicio de Neumología,
Complejo Hospitalario de Pontevedra, Vigo, Spain P17.17A
TAP1 and HLA-B genes in the HLA region are
hypomethylated and overexpressed in celiac duodenal cell
populations C. López Solarat1, M. Lago-Docampo1, A. Baloira2, D. Valverde1 1UPV/EHU, BioCruces, CIBERDEM, Leioa, Spain, 2Cruces
University Hospital, Leioa, Spain P17.18B F. Coppedè: None. A. Stoccoro: None. A. Lazzarotti:
None. R. Spisni: None. L. Migliore: None. Epigenetic role of DNA transposable elements (TEs) in
shaping CD4+ T cell identity and plasticity in health and
disease Epigenetic role of DNA transposable elements (TEs) in
shaping CD4+ T cell identity and plasticity in health and
disease FFPE preserved tumors are a valuable resource for retro-
spective research on clinical samples. Clinical information,
treatments and outcomes are often available for these
samples providing an opportunity to link molecular biology
research data to disease, diagnosis and biomarker discovery. Unlike fresh tumor samples, which have been used in ChIP-
Seq-based epigenomic profiling experiments to discovery
novel cancer subtypes, FFPE samples are more challenging
due to extensive crosslinking which hampers chromatin
extraction. However, recent advances at Active Motif have
enabled the routine generation of high quality ChIP-Seq
data sets from limited amounts of FFPE preserved tumors. FFPE ChIP-Seq data shows 1) extracted chromatin can be
used with multiple different histone modification specific
antibodies resulting in the expected genome-wide pattern
for each histone mark 2) FFPE ChIP-Seq epigenetic profiles
are highly concordant with profiles generated from matched
freshly frozen tumors 3) FFPE ChIP-Seq profiles reveal
tumor specific, differential histone occupancy patterns 4)
More challenging transcription factor targets, such as
Estrogen Receptor, can be profiled efficiently using FFPE
ChIP-Seq. It is anticipated that the availability of a reliable
ChIP-Seq method for profiling FFPE preserved patient
samples will result in advancements in our understanding of FFPE preserved tumors are a valuable resource for retro-
spective research on clinical samples. Clinical information,
treatments and outcomes are often available for these
samples providing an opportunity to link molecular biology
research data to disease, diagnosis and biomarker discovery. F. Marasca1, S. Sinha1, R. Bonnal1, C. Cordiglieri1, P. Nastaly2,
E. Provasi1, P. Maiuri2, M. Pagani1, S. Abrignani1, B. Bodega1
1INGM, Milan, Italy, 2IFOM, Milan, Italy F. Marasca1, S. Sinha1, R. Bonnal1, C. Cordiglieri1, P. Nastaly2,
E. Provasi1, P. Maiuri2, M. Pagani1, S. Abrignani1, B. Bodega1 1INGM, Milan, Italy, 2IFOM, Milan, Italy CD4+
T
lymphocytes
coordinate
adaptive
immune
responses differentiating into functionally different subsets
that exert an extraordinary degree of functional plasticity,
novel epigenetic players are still being searched to explain
immune related phenotypes. Notably, among non coding
genome, Transposable Elements account roughly for the
45% of the genome and although ignored for decades, are
nowadays robustly emerging as novel key molecules acting
at different level of cell type specific genome regulation.We
are dissecting TEs function in the epigenetic regulation of
human primary CD4+ lymphocytes. P17.21A Using ChIP-Seq to Profile FFPE Preserved Tumors for
Epigenetic Alterations Using ChIP-Seq to Profile FFPE Preserved Tumors for
Epigenetic Alterations S. CHLAMYDAS1, A. Blattler2, T. Yen2, P. Lebhart2, B. Egan2
1ACTIVE MOTIF, LA HULPE, Belgium, 2ACTIVE MOTIF,
Carlsbad, CA, United States S. CHLAMYDAS1, A. Blattler2, T. Yen2, P. Lebhart2, B. Egan2 C. López Solarat: None. M. Lago-Docampo: None. A. Baloira: None. D. Valverde: None. 1ACTIVE MOTIF, LA HULPE, Belgium, 2ACTIVE MOTIF,
Carlsbad, CA, United States P17.17A B. Bodega: None. F. Marasca: None. S. Sinha: None. R. Bonnal: None. C. Cordiglieri: None. P. Nastaly: None. E. Provasi: None. P. Maiuri: None. M. Pagani: None. S. Abrignani: None. B. Bodega: None. F. Marasca: None. S. Sinha: None. R. Bonnal: None. C. Cordiglieri: None. P. Nastaly: None. E. Provasi: None. P. Maiuri: None. M. Pagani: None. S. Abrignani: None. B. Bodega: None. In conclusion, this SNP in the promoter region of END1
could be related with gene expression levels. Even more,
the epigenetic regulation could be also related to the
methylation state of this region. All these data have to be
taken carefully as these are preliminary data from 13
patients. Deciphering the regulation of EDN1 expression
could shed light in the molecular basis of this disease. P17.17A P17.17A
TAP1 and HLA-B genes in the HLA region are
hypomethylated and overexpressed in celiac duodenal cell
populations 1University of Vigo, Vigo, Spain, 2Servicio de Neumología,
Complejo Hospitalario de Pontevedra, Vigo, Spain N. Fernandez-Jimenez1, I. Romero-Garmendia1, K. Garcia-
Etxebarria1, A. Jauregi-Miguel1, I. Irastorza2, J. R. Bilbao1 N. Fernandez-Jimenez1, I. Romero-Garmendia1, K. Garcia-
Etxebarria1, A. Jauregi-Miguel1, I. Irastorza2, J. R. Bilbao1 Endothelin-1 (ET-1) is a peptide secreted by the endothe-
lium of blood vessels that promotes vasoconstriction. A
deregulation in the synthesis of endothelin-1, increasing its
secretion, is a triggering factor for Pulmonary Arterial
Hypertension (PAH). We carried out the characterization of
the promoter region of endothelin-1gene (EDN-1), in order 609 Abstracts from the 51st European Society of Human Genetics Conference: Posters to determine possible variations that may be associated with
this disease and therefore target possible treatments. rapidly depleted from the nuclei in a TCR activation
dependent manner. Furthermore, chromatin associated L1
RNAs localize on genomic regions different from L1 DNA,
enriched
in
euchromatic
marker
(e.g. H3K4me3,
H3K36me3) avoiding heterochromatin (e.g. H3K9me3),
foreseeing a possible ncRNAs regulatory function. We have
also collected preliminary observations on CD4+ T naïve
cells derived from new born and old individuals, indicating
that L1 transcriptional deregulation is a hallmark of immune
system aging. We are now dissecting with novel, custom
Next Generation Sequencing technologies L1 relevance at
transcriptional and genomics level in order to dissect the
mechanisms by which L1 elements might contribute to
human CD4+ T lymphocytes cell identity, plasticity and
specialization in healthy and disease conditions. The genetic analysis was carried out in 13 patients with
Idiopathic PAH (IPAH), analysing a fragment of 2 kb
promoter region. First, an in silico analysis was performed
to evaluate binding transcription factors and CpG islands. Sequencing data was aligned to the reference Ensembl
EDN-1 sequence. Luciferase assay was done to evaluate
in vitro the SNP influence in gene expression. A
deletion
in
the
promoter
region
was
found
(rs397751713). The distribution of the genotype frequencies
in our IPAH patients were: A/A: 0.15; A/-: 0.31; -/-: 0.54. This variation is located in a KLF4 binding sequence, a
transcription factor related to PAH development. A CpG
island was also detected that comprise the aforementioned
variation. Future
methylation
pattern
studies
will
be
performed. F. Marasca: None. S. Sinha: None. R. Bonnal: None. C. Cordiglieri: None. P. Nastaly: None. E. Provasi: None. P. Maiuri: None. M. Pagani: None. S. Abrignani: None. P17.23C S. Chlamydas: None. A. Blattler: None. T. Yen: None. P. Lebhart: None. B. Egan: None. Epigenetic role of DNA transposable elements (TEs) in
shaping CD4+ T cell identity and plasticity in health and
disease We found that CD4+
T cells subsets show a specific and dynamic expression of
LINE1 (L1) elements, being Naïve and Treg enriched in
chromatin associated L1 transcripts in respect to T effectors,
that are less enriched (i.e. Th1, Th2). Interestingly, L1
RNAs show a peculiar and timely specific dynamics being 610 J. del Picchia disease, lead to the discovery of new biomarkers and
eventually impact therapeutic decisions. disease, lead to the discovery of new biomarkers and
eventually impact therapeutic decisions. A. Cortesi1, M. Pesant1, S. Sinha1, F. Gregoretti2, L. Antonelli2,
G. Oliva2, G. Soldà3,4, B. Bodega1 Our study provides insight into the epigenetic role of
DNA repeats in fine-tuning gene transcription by orches-
trating
the
crosstalk
between
chromatin
folding
and
structure, which deregulation maybe central in human
genetic diseases pathophysiology. This work has been supported by the following grants to
B.B.: EPIGEN Italian flagship program, Italian Ministry
of
Health,
Association
Française
contre
les
Myopathies (AFM). A. Cortesi: None. M. Pesant: None. S. Sinha: None. F. Gregoretti: None L. Antonelli: None G. Oliva: None G. Despite increasing insights in genome structure organiza-
tion, the role of DNA repetitive elements, accounting for
more than two thirds of the human genome, remains elu-
sive.Facioscapulohumeral Dystrophy (FSHD) is associated
with deletion of D4Z4 repeat array below 11 units at
4q35.2. It is known that the deletion alters chromatin
structure in cis, leading to gene upregulation. Here we show
a genome-wide role of 4q-D4Z4 array in modulating gene
expression via 3D nuclear contacts. We have developed an
integrated strategy of 4q-D4Z4 specific 4C-seq and chro-
matin segmentation analysis, showing that D4Z4 3D inter-
actome and chromatin states of interacting genes are
impaired in FSHD; in particular, genes which have lost the
D4Z4 interaction and with a more active chromatin state are
enriched for muscle atrophy signature. Among these, we
further characterized the muscle atrophy marker Atrogin 1
(8q24), showing by 4C-seq and 3C strengthened enhancer-
promoter chromatin loops at the locus and transcriptional
upregulation
during
FSHD
myogenic
differentiation. Expression level of these genes is restored by an ectopic
wild type 4q-D4Z4 array, suggesting that the repeat directly
modulates the transcription of contacted targets. Results: YMM was used to train the methylation-based
fetal fraction estimation model. Statistical analysis resulted
in the final optimal methylation assay which employs 4
DMRs (FFMM). High correlation between FFMM and
YMM fetal fraction measurements was observed in 85 male
pregnancies (r=0.86 CI:0.80-0.91) and confirmed using
additional 53 male pregnancies. A final validation on 234
pregnancies using FFMM and fetal fraction measurements
obtained from the VERACITY NIPT test showed strong
correlation. Results: YMM was used to train the methylation-based
fetal fraction estimation model. Statistical analysis resulted
in the final optimal methylation assay which employs 4
DMRs (FFMM). High correlation between FFMM and
YMM fetal fraction measurements was observed in 85 male
pregnancies (r=0.86 CI:0.80-0.91) and confirmed using
additional 53 male pregnancies. A. Cortesi1, M. Pesant1, S. Sinha1, F. Gregoretti2, L. Antonelli2,
G. Oliva2, G. Soldà3,4, B. Bodega1 A. Cortesi1, M. Pesant1, S. Sinha1, F. Gregoretti2, L. Antonelli2,
G. Oliva2, G. Soldà3,4, B. Bodega1 Introduction: Accurate fetal fraction assessment is very
important in non-invasive prenatal testing (NIPT). Affected
samples with low fetal fraction have an increased risk for
misdiagnosis. We present a multiplex droplet digital PCR
(ddPCR) assay for fetal fraction estimation using methyla-
tion sensitive restriction enzymes (MSREs) and a robust set
of novel fetal-specific differentially methylated regions
(DMRs). 1Istituto Nazionale di Genetica Molecolare, Milan, Italy, 2CNR
Institute for High Performance Computing and Networking,
Naples, Italy, 3Department of Biomedical Sciences, Humanitas
University, Milan, Italy, 4Humanitas Clinical and Research
Center, Milan, Italy Methods: We discovered 38 fetal-specific DMRs which
can potentially be used for fetal fraction estimation in NIPT. Eight biomarkers (7 DMRs and 1 reference control) were
selected for further analysis. An assay comprising MSRE
digestion followed by multiplexed (octaplex) ddPCR was
developed for fetal fraction estimation. A chromosome Y-
multiplex ddPCR assay (YMM) was also developed for
fetal fraction estimation in male fetuses. YMM was used to
test the robustness of the methylation-based fetal fraction
estimation assay in 138 male pregnancy samples. A final
validation was performed on 234 pregnancy samples. Despite increasing insights in genome structure organiza-
tion, the role of DNA repetitive elements, accounting for
more than two thirds of the human genome, remains elu-
sive.Facioscapulohumeral Dystrophy (FSHD) is associated
with deletion of D4Z4 repeat array below 11 units at
4q35.2. It is known that the deletion alters chromatin
structure in cis, leading to gene upregulation. Here we show
a genome-wide role of 4q-D4Z4 array in modulating gene
expression via 3D nuclear contacts. We have developed an
integrated strategy of 4q-D4Z4 specific 4C-seq and chro-
matin segmentation analysis, showing that D4Z4 3D inter-
actome and chromatin states of interacting genes are
impaired in FSHD; in particular, genes which have lost the
D4Z4 interaction and with a more active chromatin state are
enriched for muscle atrophy signature. Among these, we
further characterized the muscle atrophy marker Atrogin 1
(8q24), showing by 4C-seq and 3C strengthened enhancer-
promoter chromatin loops at the locus and transcriptional
upregulation
during
FSHD
myogenic
differentiation. Expression level of these genes is restored by an ectopic
wild type 4q-D4Z4 array, suggesting that the repeat directly
modulates the transcription of contacted targets. K. Kankava, T. Kvaratskhelia, E. Kvaratskhelia, M. Zarandia, G.
Burkadze, E. Abzianidze Allele specific chromatin signals, 3D interactions, and
refined motif predictions for immune and B cell related
diseases To elucidate possible regulatory mechanisms we integrated
long-range 3D interactions data (HiC and HiCap) to identify
putative target genes and motif predictions based on
Parsimonious Markov Models (PMMs) to identify TFs
whose binding may be affected by AS-SNPs yielding a
collection of 173 AS-SNPs associated to gene expression
and 60 to B cell related traits. Results: We identified 17,293 unique AS-SNPs across 7
lymphoblastoid cell lines of which 237 were associated to
immune GWAS traits and 714 to gene expression in B cells. To elucidate possible regulatory mechanisms we integrated
long-range 3D interactions data (HiC and HiCap) to identify
putative target genes and motif predictions based on
Parsimonious Markov Models (PMMs) to identify TFs
whose binding may be affected by AS-SNPs yielding a
collection of 173 AS-SNPs associated to gene expression
and 60 to B cell related traits. Funding: Japan Society for the Promotion of Science
(JSPS); Japan Agency for Medical Research and Develop-
ment (AMED); National Center for Child Health and
Development (NCCHD); Ministry of Health, Labour and
Welfare (MHLW); Gunma University. H. Soejima: None. F. Matsuhisa: None. S. Kitajima:
None. K. Nishioka: None. K. Higashimoto: None. H. Yatsuki: None. T. Kono: None. H. Koseki: None. K. Joh: None. Conclusions: We present a systems strategy to find
functional gene regulatory variants, the TFs that bind
differentially between alleles and novel strategies to detect
the regulated genes. A. Cortesi1, M. Pesant1, S. Sinha1, F. Gregoretti2, L. Antonelli2,
G. Oliva2, G. Soldà3,4, B. Bodega1 A final validation on 234
pregnancies using FFMM and fetal fraction measurements
obtained from the VERACITY NIPT test showed strong
correlation. Conclusions: We developed a robust methylation-based
assay for accurate fetal fraction estimation using a novel set
of fetal-specific DMRs. This simple method can be used as
an accurate fetal fraction estimation tool in NIPT. Our study provides insight into the epigenetic role of
DNA repeats in fine-tuning gene transcription by orches-
trating
the
crosstalk
between
chromatin
folding
and
structure, which deregulation maybe central in human
genetic diseases pathophysiology. G. Koumbaris: A. Employment (full or part-time);
Significant; NIPD Genetics. S. Kyriakou: A. Employment
(full or part-time); Significant; NIPD Genetics. A. Achil-
leos: A. Employment (full or part-time); Significant; NIPD
Genetics. C. Loizides: A. Employment (full or part-time);
Significant; NIPD Genetics. L. Constantinou: A. Employ-
ment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant;
NIPD Genetics. K. Tsangaras: A. Employment (full or
part-time); Significant; NIPD Genetics. P. MIna: A. This work has been supported by the following grants to
B.B.: EPIGEN Italian flagship program, Italian Ministry
of
Health,
Association
Française
contre
les
Myopathies (AFM). A. Cortesi: None. M. Pesant: None. S. Sinha: None. F. Gregoretti: None. L. Antonelli: None. G. Oliva: None. G. Soldà: None. B. Bodega: None. A. Cortesi: None. M. Pesant: None. S. Sinha: None. F. Gregoretti: None. L. Antonelli: None. G. Oliva: None. G. Soldà: None. B. Bodega: None. 611 Abstracts from the 51st European Society of Human Genetics Conference: Posters Employment (full or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Sig-
nificant; NIPD Genetics. P.C. Patsalis: A. Employment
(full or part-time); Significant; NIPD Genetics. Allele specific chromatin signals, 3D interactions, and
refined motif predictions for immune and B cell related
diseases 1Saga University, Saga, Japan, 2Tokyo University of Agriculture,
Tokyo, Japan, 3RIKEN Center for Integrative Medical Science,
Yokohama, Japan M. Cavalli1, N. Baltzer1, H. M. Umer1, J. Grau2, I. Lemnian2, G. Pan1, O. Wallerman1, R. Spalinskas3, P. Sahlen3, I. Grosse4, J. Komorowski1, C. Wadelius1 Zrsr1 is a paternally expressed imprinted gene located in the
first intron of Commd1, and the Zrsr1 promoter resides in a
differentially methylated region (DMR) that is maternally
methylated in the oocyte. Commd1 is transcribed in the
opposite
direction
to
Zrsr1
and
shows
predominant
expression of the maternal allele, especially in the adult
brain. A mechanism for the establishment of methylation at
Zrsr1-DMR in the oocyte has not been well established. Commd1 is expressed in the growing oocyte; therefore,
Zrsr1-DMR transcription occurs when methylation is
established. Zrsr1-DMR methylation was abolished by
inserting a poly(A) signal cassette into Commd1, which
prevents transcription through the DMR. Methylation did
not occur at the artificially unmethylated maternal Zrsr1-
DMR during embryonic development when transcription at
the DMR was restored by deleting the truncation cassette in
the zygote. Loss of methylation at the maternal Zrsr1-DMR
resulted in biallelic Zrsr1 expression and reduced the extent
of the predominant maternal expression of Commd1. These
results indicate that the establishment of methylation at
Zrsr1-DMR occurs in a transcription-dependent and oocyte-
specific manner, and caused Zrsr1 imprinting by repressing
maternal expression. The predominant maternal expression
of Commd1 is likely caused by transcriptional interference
by paternal Zrsr1 expression. 1Uppsala University, Uppsala, Sweden, 2Martin Luther
University HalleWittenberg, Halle, Germany, 3KTH Royal
Institute of Technology, Stockholm, Sweden, 4Martin Luther
University HalleWittenberg, Halle, Sweden Introduction: Several Genome Wide Association Studies
(GWAS) have reported variants associated to immune
system diseases. However the identified variants are rarely
the real drivers of the associations due to the heterogeneity
in and between the study groups and most of the molecular
mechanisms behind the genetic contributions to immune
diseases remain poorly understood. ChIP-seq data for TFs
and histone modifications provide snapshots of protein-
DNA interactions allowing the identification of hetero-
zygous SNPs with significant allele specific binding (AS-
SNPs). These variants, which can affect a TF binding site
resulting in altered gene regulation, are primary candidates
to explain associations observed in GWAS and expression
studies. Results: We identified 17,293 unique AS-SNPs across 7
lymphoblastoid cell lines of which 237 were associated to
immune GWAS traits and 714 to gene expression in B cells. P17.26B Transcription-dependent de novo DNA methylation at the
imprinted Zrsr1-DMR occurs in the growing oocyte, but not
in early embryonic cellsH. Soejima1, F. Matsuhisa1, S. Kitajima1, K. Nishioka1, K. Higashimoto1, H. Yatsuki1, T. Kono2, H. Koseki3, K. Joh1; A study on global DNA methylation in white blood cells of
patients with breast cancer The higher IL6 and lower IL-1Ra production persisted for
up to 3 days after treatment. Genome wide assessment of
two main candidate histone posttranslational modifications
(histone 3 lysine 4 trimethylation, H3K4me3, and histone 3
lysine 27 acetylation, H3K27ac) did not show significant
differences in the epigenetic landscape for these marks after
in vitro urate treatment in human monocytes. The results showed that methylation level in genomic
DNA from white blood cells correlated neither with the
grade nor the stage of tumor. There was no relationship of
global DNA methylation level with the size or histological
parameters (duct formation, nuclear pleomorphism, mitotic
activity) of the tumor, nor with lymph node status. The only
variable found do be related with methylation level was the
age - patients older than 75 showed to have significantly
higher global methylation level compared to patients
under 50. The higher IL6 and lower IL-1Ra production persisted for
up to 3 days after treatment. Genome wide assessment of
two main candidate histone posttranslational modifications
(histone 3 lysine 4 trimethylation, H3K4me3, and histone 3
lysine 27 acetylation, H3K27ac) did not show significant
differences in the epigenetic landscape for these marks after
in vitro urate treatment in human monocytes. Conclusion: Our data suggests the involvement of
methylation regulatory pathways in response to uric acid
and excludes H3K4me3 and H3K27ac as regulatory marks. Future perspectives for genetic studies using hyperuricemic
controls
are
warranted
for
gout
research,
while
the
exploration of urate induced epigenetic regulation is likely
to help understand the variability in the gout phenotype. The results support our previous investigation data
showing tumor-specific methylation changes (like LINE-1
methylation) to be much better pronounced in tumor tissue
and normal ductal epithelial cells from adjacent area rather
than in blood. Epigenetic changes in cancer tissue and
normal breast tissues will further be studied in this group to
get a better understanding of whether or not global or site-
specific methylation plays a role in determining histopatho-
logical characteristics of cancer. T. Crisan: None. M. Cleophas: None. V. Kluck: None. R. Davar: None. E. Habibi: None. H. Stunnenberg:
None. M. Netea: None. L.A.B. Joosten: None. T. Crisan: None. M. Cleophas: None. V. Kluck: None. R. Davar: None. E. Habibi: None. H. Stunnenberg:
None. M. Netea: None. L.A.B. Joosten: None. The research was funded by Shota Rustaveli National
Science Foundation. A study on global DNA methylation in white blood cells of
patients with breast cancer M. Cavalli: None. N. Baltzer: None. H.M. Umer: None. A study on global DNA methylation in white blood cells of
patients with breast cancer M. Cavalli: None. N. Baltzer: None. H.M. Umer: None. J. Grau: None. I. Lemnian: None. G. Pan: None. O. Wallerman: None. R. Spalinskas: None. P. Sahlen: None. J. Grau: None. I. Lemnian: None. G. Pan: None. O. Wallerman: None. R. Spalinskas: None. P. Sahlen: None. I. Grosse:
None. J. Komorowski:
None. C. Wadelius: None. 612 J. del Picchia Tbilisi State Medical University, Tbilisi, Georgia Introduction: Metabolic triggers are important modulators
of inflammation in gout. Hyperuricemia predisposes to
monosodium urate (MSU) crystallization and also primes
inflammatory responses in relation to TLR stimulation. Genetic studies have identified 28 loci associated to gout
mainly based on urate outcomes, whereas inflammatory and
epigenetic factors are potential additional factors in the gout
phenotype. Introduction: Metabolic triggers are important modulators
of inflammation in gout. Hyperuricemia predisposes to
monosodium urate (MSU) crystallization and also primes
inflammatory responses in relation to TLR stimulation. Genetic studies have identified 28 loci associated to gout
mainly based on urate outcomes, whereas inflammatory and
epigenetic factors are potential additional factors in the gout
phenotype. Methylation changes have long been studied to trigger
initiation and progression of tumors. The aim of our
research was to compare changes in global methylation
levels in blood cells from patients having invasive ductal
carcinoma of breast with different histological parameters
and stage. 20 patients with biopsy-proven invasive ductal carcinoma
of breast and no pre-operative chemotherapy were randomly
selected for the study. Blood was collected preoperatively. Global DNA methylation was quantitatively measured
using ELISA-based assay. Post-operative specimen was
processed
routinely
for
investigation
of
histological
characteristics. Materials
and
Methods:
Monocytes
from
healthy
volunteers were pre-treated with urate for 24h and then
subjected to increasing resting periods, followed by 24h
stimulation with TLR2 or TLR4 ligands in the presence or
absence of MSU. Cytokine production was assessed by
ELISA. ChIP sequencing was performed in treated cells. Results: We show in in vitro and in vivo studies that high
levels of uric acid enhance inflammation and that broad
spectrum methylation inhibitors reverse uric acid effects. Results: We show in in vitro and in vivo studies that high
levels of uric acid enhance inflammation and that broad
spectrum methylation inhibitors reverse uric acid effects. P17.29A Variation in microbiome composition impacts human gene
expression by changing chromatin accessibility K. Kankava:
None. T. Kvaratskhelia:
None. E. Kvaratskhelia: None. M. Zarandia: None. G. Burkadze:
None. E. Abzianidze: None. A. L. Richards1, A. L. Muehlbauer2, A. Alazizi1, M. B. Burns2, T. J. Gould2, C. Cascardo1, R. Pique-Regi1, R. Blekhman2, F. Luca1, F. Messina1 A. L. Richards1, A. L. Muehlbauer2, A. Alazizi1, M. B. Burns2, T. J. Gould2, C. Cascardo1, R. Pique-Regi1, R. Blekhman2, F. Luca1, F. Messina1 1Department of Medical Genetics, University of Medicine and
Pharmacy, Cluj-Napoca, Romania, 2Department of Internal
Medicine and Radboud Institute for Molecular Life Sciences
(RIMLS), Nijmegen, Netherlands, 3Department of Molecular
Biology, Faculty of Science, Radboud University,, Nijmegen,
Netherlands A. L. Richards1, A. L. Muehlbauer2, A. Alazizi1, M. B. Burns2, T.
J. Gould2, C. Cascardo1, R. Pique-Regi1, R. Blekhman2, F.
Luca1, F. Messina1 P17.30B L. Ferrari1, C. Bragato2,3, L. Brioschi1, S. Esposito1, M. Mazzola1, A. Pezzotta1, F. Pizzetti4, A. Moreno-Fortuny5,6, P. Riva1, F. Frabetti4, P. Viani1, G. Cossu5, M. Mora2, A. Marozzi1,
A. Pistocchi1 P17.28D Persistent effects of urate on cytokine production with
implications for epigenetic regulation in gout 1Wayne State University, Detroit, MI, United States,
2University of Minnesota, Minneapolis, MN, United States 1Wayne State University, Detroit, MI, United States,
2University of Minnesota Minneapolis MN United States T. Crisan1,2, M. Cleophas2, V. Kluck2, R. Davar3, E. Habibi3, H. Stunnenberg3, M. Netea2, L. A. B. Joosten1,2 Variation in gut microbiome is associated with human dis-
ease, yet the underlying molecular mechanisms are not well
understood. A likely mechanism is through changes in gene
regulation in interfacing host epithelial cells. Here, we
treated colonic epithelial cells with live microbiota from five
healthy individuals and quantified changes in transcriptional
regulation and chromatin accessibility in host cells. We 1Department of Medical Genetics, University of Medicine and
Pharmacy, Cluj-Napoca, Romania, 2Department of Internal
Medicine and Radboud Institute for Molecular Life Sciences
(RIMLS), Nijmegen, Netherlands, 3Department of Molecular
Biology, Faculty of Science, Radboud University,, Nijmegen,
Netherlands Abstracts from the 51st European Society of Human Genetics Conference: Posters 613 identified over 5,000 host genes that change expression,
including 588 distinct associations between specific taxa
and host genes. The taxa with the strongest influence on
gene expression alter the response of genes associated with
complex traits. Further, using ATAC-seq, we show that
these changes in gene expression are likely the result of
changes in host chromatin accessibility induced by expo-
sure to gut microbiota. We then created a manipulated
microbial community with titrated doses of Collinsella,
demonstrating that both natural and controlled microbiome
composition
leads
to
distinct,
and
predictable,
gene
expression profiles in the host.Together, our results suggest
that specific microbes play an important role in regulating
expression of individual host genes involved in human
complex traits. Our work also supports the hypothesis that
one of the mechanisms by which the microbiome regulates
host gene expression is through changes in chromatin
accessibility in host cells. Finally, the ability to fine tune the
expression of host genes by manipulating the microbiome
suggests future therapeutic routes for human wellness. using ELISA kit(Abcam) and global DNA methylation was
measured using the Methylated DNA Quantification Kit
(Abcam). Results: Animals exhibited hyperalgesia during formalin
test and had high HDAC levels. Treatment with either
NSAIDs or 3-day pretreatment with HDAC inhibitors
reduce nociceptive mechanical and thermal responses of
the formalin test independently. P17.28D NSAIDs used in combina-
tion with SAHA delay the establishment of hyperalgesia
and significantly reduce its intensity, also decrease HDAC
amount. HDAC and global DNA methylation levels were
observed to be different in RVM, CeA and ACC under
given conditions. Conclusions: Our data provide evidence that HDAC
inhibitors cause analgesia and are potential targets for
epigenetic treatment of pain. Study supported by Tbilisi State Medical University. Study supported by Tbilisi State Medical University. E. Abzianidze: None. E. Kvaratskhelia: None. T. Tkemaladze: None. M. Zarandia: None. N. Tsiklauri:
None. G. Gurtskaia: None. M. Nebieridze: None. M.G. Tsagareli: None. A.L. Richards: None. A.L. Muehlbauer: None. A. Alazizi: None. M.B. Burns: None. T.J. Gould: None. C. Cascardo: None. R. Pique-Regi: None. R. Blekhman:
None. F. Luca: None. F. Messina: None. A.L. Richards: None. A.L. Muehlbauer: None. A. Alazizi: None. M.B. Burns: None. T.J. Gould: None. C. Cascardo: None. R. Pique-Regi: None. R. Blekhman:
None. F. Luca: None. F. Messina: None. P17.31C The discovery of a new role of HDAC8 in skeletal muscle
differentiation and in centronuclear myopathy insurgence Are HDAC potential targets for the epigenetic treatment of
pain L. Ferrari1, C. Bragato2,3, L. Brioschi1, S. Esposito1, M. Mazzola1, A. Pezzotta1, F. Pizzetti4, A. Moreno-Fortuny5,6, P. Mazzola1, A. Pezzotta1, F. Pizzetti4, A. Moreno-Fortuny5,6, P. Riva1, F. Frabetti4, P. Viani1, G. Cossu5, M. Mora2, A. Marozzi1,
A. Pistocchi1 E. Abzianidze1,2, E. Kvaratskhelia1, T. Tkemaladze1, M. Zarandia1, N. Tsiklauri2, G. Gurtskaia2, M. Nebieridze2, M. G. Tsagareli2 E. Abzianidze1,2, E. Kvaratskhelia1, T. Tkemaladze1, M. Zarandia1, N. Tsiklauri2, G. Gurtskaia2, M. Nebieridze2, M. G. Tsagareli2 1Dip. Biotecnologie Mediche e Medicina Traslazionale,
Università degli Studi di Milano, Milan, Italy, 2Fondazione
IRCCS Istituto Neurologico C. Besta, Milan, Italy, 3PhD
program in Neuroscience, University of Milano-Bicocca,
Milan, Italy, 4Department of Experimental, Diagnostic and
Specialty Medicine, University of Bologna, Bologna, Italy,
5Division of Cell Matrix Biology & Regenerative Medicine,
FBMH, University of Manchester, Manchester, United
Kingdom, 6Developmental Genetics, Department of
Biomedicine, University of Basel, Basel, Switzerland 1Tbilisi State Medical University, Dept. of Molecular and
Medical Genetics, Tbilisi, Georgia, 2Ivane Beritashvili Centre
of Experimental Biomedicine, Dept of Pain and Analgesia,
Tbilisi, Georgia Introduction: Objective of the present study was to eval-
uate the changes of a subset of histone deacetylases
(HDAC) and global DNA methylation level in pain-matrix
structures of the brain as the rostroventromedial medulla
(RVM), central nucleus of amygdale (CeA) and anterior
cingulate cortex (ACC) of formalin test. It involves study of
epigenetic agents as potentially therapeutic drugs for man-
agement of inflammatory pain. Histone Deacetylase 8 (HDAC8) is a member of class I
HDACs with peculiar characteristics: it is subjected to
posttranslational phosphorylation and presents differences
in substrate specificity, suggesting a unique role for HDAC8
across the HDACs family. For example, HDAC8 activity is
specifically inhibited by molecules such as PCI-34051, with
a 200-fold higher selectivity than other HDACs. Here, we
describe for the first time a specific expression for HDAC8
during skeletal muscle differentiation. In C2C12 myoblasts
the expression of HDAC8 increases during differentiation
and
in
RD/18
and
RD/12
rhabdomyosarcoma
cells, Methods: All animal studies conformed to the guidelines
of IASP regarding investigations. Adult rats receiving
intraplantar
formalin
were
tested
for
antinociception
following injection of NSAIDs or/and SAHA in thermal
and mechanical tests. The levels of global DNA methylation
and HDAC were measured in nuclear extracts of RVM,
CeA and ACC neurons. HDAC amount was measured J. del Picchia 614 repeats. Alterations in linear and back splicing as new players in
Huntington’s Disease pathogenesis Alterations in linear and back splicing as new players in
Huntington’s Disease pathogenesis Are HDAC potential targets for the epigenetic treatment of
pain F. Pizzetti: None. A. Moreno-Fortuny: None. P. Riva:
None. F. Frabetti: None. P. Viani: None. G. Cossu: None. M. Mora: None. A. Marozzi: None. A. Pistocchi: None. F. Pizzetti: None. A. Moreno-Fortuny: None. P. Riva:
None. F. Frabetti: None. P. Viani: None. G. Cossu: None. M. Mora: None. A. Marozzi: None. A. Pistocchi: None. M. Biagioli: None. D. Ayyildiz: None. E. Dassi: None. T. Tripathi: None. A. Monziani: None. E. Kerschbamer:
None. F. Di Leva: None. J. Zasso: None. L. Conti: None. V. Mattis: None. C. Dieterich: None. S. Piazza: None. Are HDAC potential targets for the epigenetic treatment of
pain To discover alterations in linear splicing, we
analyzed genome-wide transcriptomic profiles for stria-
tum, cortex and liver at different developmental time
points, quantifying the total number of differential AS
events. Our analysis demonstrated that, specifically for the
striatum, the total number of differential AS events
increased significantly with increasing CAG expansion. Interestingly, only EXON SKIPPING, a specific AS
subtype emerged to be strongly and specifically affected. Additionally, we identified the first circRNA originating
from the HTT locus, expressed in the brain and presenting
augmented expression levels with increasing HTT CAG
size. At genome-wide level, a decreased circRNAs pro-
duction correlated with mutant HTT expression, revealing
14 circRNAs in neuronal progenitor cells with CAG-
length-dependent decreased expression. In conclusion,
our results support the idea that AS machinery is
responding to HD mutational process altering both linear
and back-splicing events locally at the HTT locus, but also
at the genome-wide level. This knowledge will pave the
way to new trials of therapeutic intervention aimed to
possibly target spliceosomal-circRNAs alterations. respectively with low and high tumorigenic potential con-
sistent with their differentiating capability, its expression
correlates with the differentiation degree. Furthermore, we
demonstrate that PCI-34051-mediated-HDAC8-inhibition
impairs myogenesis in C2C12 myoblasts and in zebrafish
embryos. In zebrafish, when HDAC8 activity is inhibited,
only type I fibres are reduced. Moreover, preliminary data
show altered expression of HDAC8 in muscle derived from
centronuclear myopathy (CNM) affected patients in com-
parison with healthy donors. CNM is a condition char-
acterized by skeletal muscles weakness, hypotonia and
atrophy mainly in type 1 fibres. Mutations in MTM1,
DNM2, BIN1, RYR1 or TTN genes have been associated
with CNM, although some patients do not carry mutations
in these genes. Hence the need to identify additional genes
associated with CNM. In this work, we demonstrate a new
role for HDAC8 activity in normal skeletal muscle differ-
entiation, in rhabdomyosarcoma and we suggest a potential
involvement of HDAC8 in CNM insurgence. L. Ferrari: None. C. Bragato: None. L. Brioschi: None. S. Esposito: None. M. Mazzola: None. A. Pezzotta: None. L. Ferrari: None. C. Bragato: None. L. Brioschi: None. S. Esposito: None. M. Mazzola: None. A. Pezzotta: None. F. Pizzetti: None. A. Moreno-Fortuny: None. P. Riva:
None. F. Frabetti: None. P. Viani: None. G. Cossu: None. M. Mora: None. A. Marozzi: None. A. Pistocchi: None. L. Ferrari: None. C. Bragato: None. L. Brioschi: None. S. Esposito: None. M. Mazzola: None. A. Pezzotta: None. Promoter methylation status of interleukin-8 in cystic
fibrosis M. Biagioli1, D. Ayyildiz2, E. Dassi3, T. Tripathi1, A. Monziani1,
E. Kerschbamer1, F. Di Leva1, J. Zasso4, L. Conti4, V. Mattis5, C. Dieterich6, S. Piazza2 E. Kvaratskhelia1, M. Gagua1, E. Maisuradze1, M. Ghughunishvili2, E. Abzianidze3 E. Kvaratskhelia1, M. Gagua1, E. Maisuradze1, M. Ghughunishvili2, E. Abzianidze3 E. Kvaratskhelia1, M. Gagua1, E. Maisuradze1, M.
Ghughunishvili2, E. Abzianidze3 Methylation status of the IL-8
gene was determined by methylation-specific polymerase
chain reaction (MSP) analysis. PCR products were electro-
phoresed on 3% agarose gel and visualized. Level of IL-8 in
supernatants of activated CD4+ T-cells was measured using
ELISA method. 7q31.1 microdeletion of maternal origin were detected in a
patient. The 7q31.1 microdeletion affected included only
exons 1-2 of the IMMP2L gene. IMMP2L gene is located in
the critical region for the autistic disorder locus on
chromosome 7q (AUTS1). The bisulfite sequencing of 87
intragenic CpG-sites revealed the comparable level of
methylation
in
the
proband,
healthy
sibling
without
microdeletion and father. Whereas a reduced methylation
level and increased IMMP2L expression were observed in
healthy mother’s peripheral blood lymphocytes in compar-
ison with a proband. Results: Our results indicate that individuals with CF
have a significantly higher percentage of hypomethylation
of IL-8 gene promoter regions in CD4+ T-cells compared
to healthy controls and expression level of IL-8 was higher
than in individuals without CF. Conclusion: Obtained results provide evidence for a
possible compensation of IMMP2L haploinsuffiency in a
healthy mother with 7q31.1 microdeletion due to epigenetic
mechanisms. This study was supported by Russian Science
Foundation, grant 16-15-10229. Conclusions: Our results suggest that methylation status
of the IL-8 promoter region might be a useful predictor of
the complication of Cystic Fibrosis. The study was
supported by a grant from the Ministry of Science and
Education of Georgia (N59; 2015-30-01). N.A. Skryabin: None. S.A. Vasilyev: None. E.N. Tolmacheva: None. R.R. Savchenko: None. D.I. Zhiga-
lina: None. A.A. Kashevarova: None. A.A. Zarubin:
None. E.O. Belyaeva: None. L.P. Nazarenko: None. I.N. Lebedev: None. E. Kvaratskhelia: None. M. Gagua: None. E. Maisur-
adze:
None. M. Ghughunishvili:
None. E. Abzianidze: None. E. P17.35C Epigenetic modifications associated with intrauterine
growth restriction Role of intragenic DNA methylation in incomplete
penetrance of IMMP2Lgene microdeletion C. Caniçais1, J. Marques1,2, C. Ramalho3,4, S. Dória1,5 C. Caniçais1, J. Marques1,2, C. Ramalho3,4, S. Dória1,5 N. A. Skryabin, S. A. Vasilyev, E. N. Tolmacheva, R. R. Savchenko, D. I. Zhigalina, A. A. Kashevarova, A. A. Zarubin,
E. O. Belyaeva, L. P. Nazarenko, I. N. Lebedev
Research Institute of Medical Genetics, Tomsk National
Research Medical Center, Russian Academy of Sciences,
Tomsk, Russian Federation N. A. Skryabin, S. A. Vasilyev, E. N. Tolmacheva, R. R. Savchenko, D. I. Zhigalina, A. A. Kashevarova, A. A. Zarubin,
E. O. Belyaeva, L. P. Nazarenko, I. N. Lebedev 1Genetics Unit, Department of Pathology, Faculty of Medicine,
University of Porto, Porto, Portugal, 2i3S – Instituto de
Investigação e Inovação em Saúde, University of Porto, Porto,
Portugal, Porto, Portugal, 3Department of Obstetrics and
Gynecology, Centro Hospitalar São João and Faculty of
Medicine, Porto, Portugal., Porto, Portugal, 4i3S – Instituto de
Investigação e Inovação em Saúde, University of Porto, Porto,
Portugal, Porto, Poland, 5i3S – Instituto de Investigação e
Inovação em Saúde, University of Porto, Porto, Portugal E. Kvaratskhelia1, M. Gagua1, E. Maisuradze1, M.
Ghughunishvili2, E. Abzianidze3 1NeuroEpigenetics laboratory, Centre for Integrative Biology,
Povo, Italy, 2Bioinformatic facility, Centre for Integrative
Biology, Povo, Italy, 3Computational biology laboratory,
Centre for Integrative Biology, Povo, Italy, 4Laboratory of
Stem Cell Biology, Centre for Integrative Biology, Povo, Italy,
5The Board of Governors Regenerative Medicine Institute,
Cedars-Sinai Medical Center, Los Angeles, CA, United States,
6Section of Bioinformatics and Systems Cardiology, University
Hospital Heidelberg, Heidelberg, Germany 1Tbilisi State Medical University, Institute of Medical
Biotechnology, Tbilisi, Georgia, 2Tbilisi State Medical
University, Zhvania Clinic of Pediary, Tbilisi, Georgia, 3Tbilisi
State Medical University, Dept. of Molecular and Medical
Genetics, Tbilisi, Georgia Introduction: Cystic Fibrosis (CF) is an inherited disorder
characterised
by
a
chronic
infection
and
neutrophil-
dominated airway inflammation. This inflammation is
characterised
by
an
increased
production
of
pro-
inflammatory cytokines in the lung. Interleukin-8 (IL-8), a
member of the CXC chemokine family that attracts neu-
trophils and other leukocytes, has been elevated in Cystic
Fibrosis (CF) disease. Epigenomic disregulation has been
reported to play a role for the development of chronic
inflammation in CF. In the present work we analyzed
expression of IL-8 by the CD4+ T-cells from CF patients
and DNA methylation status in the promoter region of the
IL-8 gene. Recent findings revealed that alternative splicing (AS)
might be compromised in Huntington’s Disease (HD), a
fatal neurodegenerative disorder, caused by ‘CAG’ tri-
nucleotide repeat expansion in the Huntingtin (HTT) gene. AS regulation is crucial to the establishment of protein
coding isoforms, but also to the biogenesis of circular
RNAs (circRNA), stable non-coding RNAs produced by
the circularization of exons. The goal of our research was
to discover alterations in linear and back-splicing events
in in vivo and in vitro models bearing normal or expanded 615 Abstracts from the 51st European Society of Human Genetics Conference: Posters Materials and Methods: Genomic DNA from activated
CD4+ T-cells of 7 CF patients and 7 controls were purified
and subjected to bisulfite modification using EpiTect Fast
Bisulfite Kit (Qiagen, USA). Methylation status of the IL-8
gene was determined by methylation-specific polymerase
chain reaction (MSP) analysis. PCR products were electro-
phoresed on 3% agarose gel and visualized. Level of IL-8 in
supernatants of activated CD4+ T-cells was measured using
ELISA method. Materials and Methods: Genomic DNA from activated
CD4+ T-cells of 7 CF patients and 7 controls were purified
and subjected to bisulfite modification using EpiTect Fast
Bisulfite Kit (Qiagen, USA). Research Institute of Medical Genetics, Tomsk National
Research Medical Center, Russian Academy of Sciences,
Tomsk, Russian Federation Introduction: Most of CNVs in the human genome exhibit
incomplete penetrance with unknown underlined mechan-
isms. One of such mechanisms may be epigenetic mod-
ifications of DNA, in particular, DNA methylation. Here,
we report about differential methylation of intragenic CpGs
of IMMP2L gene in a proband with maternal 7q31.1
microdeletion and de novo 15q11-q13 microdeletion. Intrauterine growth restriction is a condition in which the
fetus is not able to reach its normal growth potential. The
abnormal imprinted gene expression is associated with fetal
growth restriction and may be controlled by DNA methy-
lation. DNA hydroxymethylation was recently described
and associated with fetal size at birth. The aim of this study
was to find biomarkers that allow the prediction of IUGR,
studying
the
effects
of
imprinted
genes
(PHLDA2,
CDKN1C, KCNQ1, H19, IGF2, PEG10 and MEST), the
effects
of
KvDMR1
methylation
and
the
global
hydroxymethylation. Materials and Methods: The proband with Prader-Willi
phenotype with signs of autism spectrum disorders was
analyzed using aCGH. The presence of 7q31.1 and 15q11-
q13 microdeletions in his parents and healthy sibling was
investigated by real-time PCR. DNA methylation level in
intragenic CpGs of IMMP2L gene was measured by
bisulfite amplicon next generation sequencing. Genome-
wide expression profile was assessed by microarray analysis
(Agilent Technologies). Quantitative Real-Time PCR, Combined Bisulfite and
Restriction
Analysis
and
5-hmC
DNA
ELISA
were
performed in twenty-one term placental samples with IUGR
and in nine normal placental samples. Bisulfite sequencing Results: De novo 15q11-q13 microdeletion consistent
with Prader-Willi phenotype (OMIM: 176270) as well as 616 J. del Picchia was performed to confirm KvDMR1 methylation level in
five samples. Results: In SALS patients, a total of 775 deregulated
mRNAs (45% down and 55% up) and 235 lncRNAs (76 %
down and 24% up) were found. 84 of lncRNAs are reported
as antisense, 91 as lincRNAs, while remaining 60 RNAs
classified as processed transcripts or intronic sense RNAs. Interesting, the analysis of SALS patients classified by fat
and slow progression (Gomeni et al., 2013) showed a most
important deregulation and the involvement of different
pathways in faster-progressing patients. About mutated
patients, the most interesting data are about FUS patients
that showed the major number of deregulated genes. Gene expression analysis demonstrated a significantly
increased expression of CDKN1C, PHLDA2 and PEG10
genes in IUGR. The results for CDKN1C and PHLDA2
genes are consistent with literature and with parental
conflict theory. Peripheral blood DNA methylation as potential biomarker
of malignant pleural mesothelioma in asbestos-exposed
subjects Peripheral blood DNA methylation as potential biomarker
of malignant pleural mesothelioma in asbestos-exposed
subjects Research Institute of Medical Genetics, Tomsk National
Research Medical Center, Russian Academy of Sciences,
Tomsk, Russian Federation However, the result for PEG10 gene
suggests a compensatory response to IUGR. The COBRA
and BS-sequencing results demonstrated the KvDMR1
hypermethylation in IUGR. The global hydroxymethylation
analysis exhibited the presence of 5-hydroxymethylcytosine
in the placenta. However, the results showed non-significant
changes between the two groups samples. Conclusions:
This
investigation
has
confirmed
the
importance of extending our knowledge on molecular
alterations
of
transcriptome
in
ALS
disease
offering
numerous starting points for new investigations about
pathogenic mechanism involved in ALS disease.Grants:
ALS-ARiSLA, FRRB 2015-0023 In this study, CDKN1C and PHLDA2 genes were
identified as potential IUGR biomarkers. In addition, the
study confirms the presence of 5-hydroxymethylcytosine in
placenta and show the KvDMR1 hypermethylation in IUGR
samples. The imprinted genes expression increase may
result
from
hypermethylation
of
KvDMR1,
leading
to IUGR. S. Gagliardi: None. S. Zucca: None. C. Pandini: None. D. Sproviero: None. O. Pansarasa: None. R.A. Calogero:
None. C. Cereda: None. C. Caniçais: None. J. Marques: None. C. Ramalho:
None. S. Dória: None. P17.37A P17.37A
Analysis of coding and long non-coding RNA expression
profiles in Peripheral Blood Mononuclear Cells from
Amyotrophic Lateral Sclerosis patients
S. Gagliardi1, S. Zucca1, C. Pandini1,2, D. Sproviero1, O. Pansarasa1, R. A. Calogero3, C. Cereda1 M. Ustinova1, I. Silamiķelis1, I. Elbere1, I. Kalniņa1, L.
Zaharenko1, R. Pečulis1, I. Konrāde2, V. Pīrāgs3, J. Kloviņ1 M. Ustinova1, I. Silamiķelis1, I. Elbere1, I. Kalniņa1, L. Zaharenko1, R. Pečulis1, I. Konrāde2, V. Pīrāgs3, J. Kloviņ1 Introduction: Malignant pleural mesothelioma (MPM) is
an aggressive tumour strongly associated with asbestos
exposure. MPM prognosis is poor, highlighting the need for
non-invasive tests to monitor asbestos-exposed people
aiming at an early MPM diagnosis. Introduction: Malignant pleural mesothelioma (MPM) is
an aggressive tumour strongly associated with asbestos
exposure. MPM prognosis is poor, highlighting the need for
non-invasive tests to monitor asbestos-exposed people
aiming at an early MPM diagnosis. 1Latvian Biomedical Research and Study Centre, Riga, Latvia,
2Riga East University Hospital, Riga, Latvia, 3Pauls Stradiņ
Clinical University Hospital, Riga, Latvia Methods: We investigated genome-wide DNA methyla-
tion
(HumanMethylation450-BeadChip)
in
163
MPM
cases/137 cancer-free controls (82/68 Training-Set; replica-
tion 81/69 Test-Set), all of them with asbestos exposure
assessment. Introduction: Metformin is the first-line antidiabetic agent
used in pharmacotherapy of type 2 diabetes to improve
glucose homeostasis. Nevertheless, additional therapeutic
directions such as cancer prevention, treatment of polycystic
ovary syndrome and neurodegenerative diseases have been
highlighted lately justifying the pleiotropic effect of the
drug. Despite the identification of AMPK as the major
mediator of its effects, exact mechanisms of metformin
action remain obscure. Results: We found case/controls differential methylation
(>800 CpG sites, Pfdr<0.05) mainly in immune system related
genes, and accelerated methylation age of white blood cells in
MPM cases (extrinsic epigenetic age acceleration, p = 0.009). Considering the “top” differentially methylated signals, 7
single-CpGs and 5 genomic regions replicated with similar
effect size in the Test Set (pfdr<0.05). The top hypomethylated
single-CpG (cases vs controls effect size<-0.15, pfdr <0.05 in
both Training and Test sets) was detected in FOXK1
(Forkhead-box K1) gene, an interactor of BAP1 which was
found mutated in MPM tissue and as germline mutation in
familial MPM. In the Test set, comparison of receiver
operating characteristic (ROC) curves analysis of two models,
including/excluding
methylation,
showed
a
significant
increase in case/control discrimination when considering
DNA
methylation
together
with
asbestos
exposure
(AUC=0.81 vs AUC=0.89, DeLong’s test p = 0.0013). Additionally, preliminary results from mediation analysis
suggest asbestos-related differential methylation. Materials and Methods: The longitudinal study enrolled
25 healthy volunteers of European descent, receiving oral
850mg dose of metformin twice-daily for seven days. RNA
was isolated from whole blood samples, collected at three
consecutive time points: before metformin administration,
10 hours after the first dose and at the end of metformin
treatment. M. Ustinova1, I. Silamiķelis1, I. Elbere1, I. Kalniņa1, L.
Zaharenko1, R. Pečulis1, I. Konrāde2, V. Pīrāgs3, J. Kloviņ1 RNA-seq was performed on Ion Proton™
System and Ion PI™Chip. For bioinformatic analysis
Trimmomatic 0.36, STAR 2.5.3a, edgeR and DAVID 6.8. tools were applied. Results: In total 681 differentially expressed genes were
identified (FDR<0.05), among them genes related to
inflammatory responses, promoting enrichment of intestinal
immune network for IgA production and cytokine-cytokine
receptor interaction pathways. Four additional functional
gene clusters were revealed after consideration of subject-
specific effects including ribosomal genes, snoRNAs and
genes relevant to insulin production (HNF1B, HNF1A,
HNF4A, GCK, INS, NEUROD1, PAX4, PDX1, ABCC8,
KCNJ11) and cholesterol homeostasis (APOB, LDLR,
PCSK9). Conclusions: We identified signatures of differential
methylation in DNA from whole blood between asbestos
exposed MPM cases and controls. Our results suggest the
potential use of DNA methylation profiles in blood to
develop non-invasive tests for MPM detection in asbestos-
exposed subjects. Grants:
AIRC
2015-IG17464(GM),
IIGM(GM),
Ricerca-Scientifica-Applicata(DM), ISS2013-14(CM). Conclusion: In healthy individuals universal metformin-
induced alterations of global gene expression profiles in
white blood cells are associated with immune responses,
while subject-specific effects, which tendency to be more
permanent are related to energy metabolism. Funded by
ERDF Project Nr.1.1.1.1/16/A/091. S. Guarrera: None. C. Viberti: None. G. Cugliari:
None. A. Allione: None. E. Casalone: None. M. Betti:
None. D. Ferrante: None. A. Aspesi: None. C. Casadio:
None. F. Grosso: None. R. Libener: None. E. Piccolini:
None. D. Mirabelli: D. Speakers Bureau/Honoraria (speak-
ers bureau, symposia, and expert witness); Modest; DM
served as expert witness for the public prosecution office in
asbestos–related deaths court trials. I. Dianzani: None. C. Magnani: D. Speakers Bureau/Honoraria (speakers bureau,
symposia, and expert witness); Modest; CM served as
expert
witness
for
the
public
prosecution
office
in
asbestos–related deaths court trials. G. Matullo: None. S. Guarrera: None. C. Viberti: None. G. Cugliari:
None. A. Allione: None. E. Casalone: None. M. Betti: M. Ustinova: None. I. Silamiķelis: None. I. Elbere:
None. I. Kalniņa: None. L. Zaharenko: None. R. Pečulis:
None. I. Konrāde:
None. V. Pīrāgs:
None. J. Kloviņ: None. P17.40D Correlation status of BRAF V600E mutation and miR-137
and miR-181b expression in Iranian PTC patients Correlation status of BRAF V600E mutation and miR-137
and miR-181b expression in Iranian PTC patients Analysis of coding and long non-coding RNA expression
profiles in Peripheral Blood Mononuclear Cells from
Amyotrophic Lateral Sclerosis patients Kloviņ1
1Latvian Biomedical Research and Study Centre, Riga, Latvia,
2Riga East University Hospital, Riga, Latvia, 3Pauls Stradiņ
Clinical University Hospital, Riga, Latvia Correlation status of BRAF V600E mutation and miR-137
and miR-181b expression in Iranian PTC patients Analysis of coding and long non-coding RNA expression
profiles in Peripheral Blood Mononuclear Cells from
Amyotrophic Lateral Sclerosis patients S. Guarrera1,2, C. Viberti1,2, G. Cugliari1,2, A. Allione1,2, E. Casalone1,2, M. Betti3, D. Ferrante4,5, A. Aspesi3, C. Casadio6, F. Grosso7, R. Libener8, E. Piccolini9, D. Mirabelli10,11,12, I. Dianzani3,13, C. Magnani4,5,13, G. Matullo1,2,14,13 S. Gagliardi1, S. Zucca1, C. Pandini1,2, D. Sproviero1, O. Pansarasa1, R. A. Calogero3, C. Cereda1 1Italian Institute for Genomic Medicine (IIGM), Turin, Italy,
2Department of Medical Sciences, University of Turin, Turin,
Italy, 3Department of Health Sciences, University of Piemonte
Orientale, Novara, Italy, 4Medical Statistics and Cancer
Epidemiology Unit, Department of Translational Medicine,
University of Piemonte Orientale, Novara, Italy, 5Cancer
Epidemiology Unit, CPO-Piemonte, Novara, Italy, 6Thoracic
Surgery Unit, AOU Maggiore Della Carità, Novara, Italy,
7Division of Medical Oncology, SS. Antonio e Biagio General
Hospital, Alessandria, Italy, 8Pathology Unit, SS. Antonio e
Biagio General Hospital, Alessandria, Italy, 9Pneumology
Unit, Santo Spirito Hospital, Casale Monferrato (AL), Italy,
10Cancer Epidemiology Unit, Department of Medical Sciences,
University of Turin, Turin, Italy, 11Cancer Epidemiology Unit,
CPO Piemonte, Turin, Italy, 12nterdepartmental Center for
Studies on Asbestos and Other Toxic Particulates "G. Scansetti", University of Turin, Turin, Italy,
13Interdepartmental Center for Studies on Asbestos and Other
Toxic Particulates "G. Scansetti", University of Turin, Turin,
Italy, 14Medical Genetics Unit, AOU Città della Salute e della
Scienza, Turin, Italy 1IRCCS Mondino Foundation, Genomic and Post Genomic
Center, Pavia, Italy, 2University of Pavia, Department of
Biology and Biotechnology “L. Spallanzani”, Pavia, Italy,
3Department of Molecular Biotechnology and Health Sciences,
Bioinformatic and Genomic Unit, University of Turin, Torino,
Italy Background: Alteration in RNA metabolism, concerning
both coding and long non coding RNAs (lncRNAs), may
play an important role in Amyotrophic Lateral Sclerosis
(ALS) pathogenesis (Gagliardi et al., 2018). We have ana-
lyzed the regulation of mRNAs and lncRNAs in Sporadic
(SALS) and mutated ALS patients and in matched controls
in peripheral blood mononuclear cells (PBMCs) and we
have correlated the founded RNA regulation with ALS
onset and disease progression. Materials and Methods: RNAs from PBMCs of SALS,
SOD1, FUS, TARDBP mutated patients and matched
controls have been used for RNA-seq experiments (TruSeq
Stranded RNA Library Prep, Illumina) that have been
analyzed by R package EBSeq. RNA-seq data has been
validated by Real time PCR. 617 Abstracts from the 51st European Society of Human Genetics Conference: Posters M. Ustinova1, I. Silamiķelis1, I. Elbere1, I. Kalniņa1, L. Zaharenko1, R. Pečulis1, I. Konrāde2, V. Pīrāgs3, J. Metformin-induced alterations of transcriptome profile in
healthy individuals Institue of Biomedicine and Translational Meidicine, Tartu,
Estonia Introduction: microRNAs drive coordinated expressional
changes of their target genes and trigger functional shift in
cells. Placental microRNAs are specifically involved in
trophoblast
differentiation
and
function. Additionally,
miRNAs transferred by extracellular vesicles released from
trophoblastic cells exhibit critical immunomodulatory role
at the maternal-fetal interphase. Single nucleotide variants
(SNVs) associated with the expression level of genes are
defined as expression quantitative trait loci (eQTLs). The
aim of my PhD project is to identify placental eQTLs
modulating the expression of microRNAs and to understand
their downstream effect on the placental transcriptome. Introduction: Papillary thyroid carcinoma (PTC) as well-
differentiated thyroid carcinoma is the most frequent
endocrine malignancy. Abnormal expression of micro-
RNAs (miRs) is related to numerous carcinomas and
malignancy. We planned to assess the miR-137 and -181b
expressions and their association with BRAF V600E
mutation
and
clinicopathological
features
in
Iranian
PTC cases. Materials and Methods: In total, 90 primary thyroid
samples (60 PTC and 30 benign with multinodular goiter
(MNG)) and two cell lines including human PTC (B-CPAP)
and human embryonic kidney (HEK293) were surveyed. All clinicopathological information of patients was obtained
and a pathologist confirmed specimens. T1799A mutation
of exon 15 in the BRAF gene was identified. MiRs
expression was assessed by quantitative reverse transcrip-
tase real-time PCR. Materials and Methods: Placental miRSeq (unpubl. data) and genotyping (Kasak et al 2015) datasets were
subjected to genetic association testing for eQTL discovery,
implicated in PLINK v1.07 (Purcell et al 2007). microRNA
target genes were predicted using the BCmicrO database
(Yue et al 2012). Association testing between identified
miRNA eQTLs and placental expression levels of predicted
target genes were tested in PLINK. Correlations between
the expression profile of placental miRNAs (miRSeq
dataset) and transcripts (RNA-Seq dataset; Sõber et al
2015)
were
analyzed
using
DESeq2
platform
(Love
et al 2014). Materials and Methods: Placental miRSeq (unpubl. data) and genotyping (Kasak et al 2015) datasets were
subjected to genetic association testing for eQTL discovery,
implicated in PLINK v1.07 (Purcell et al 2007). microRNA
target genes were predicted using the BCmicrO database
(Yue et al 2012). Association testing between identified
miRNA eQTLs and placental expression levels of predicted
target genes were tested in PLINK. Correlations between
the expression profile of placental miRNAs (miRSeq
dataset) and transcripts (RNA-Seq dataset; Sõber et al
2015)
were
analyzed
using
DESeq2
platform
(Love
et al 2014). P17.39C Metformin-induced alterations of transcriptome profile in
healthy individuals 618 J. del Picchia Institue of Biomedicine and Translational Meidicine, Tartu,
Estonia Results: Of 60 PTC cases and 30 MNG subjects, 75 and
83.3 % were female, respectively. MiR-137 and -181b were
upregulated in PTC compared to MNG (P=0.010 and
0.007, respectively). Their levels were upregulated in B-
CPAP compared to HEK293 cell line (P<0.001). Moreover,
these two miRs were upregulated in PTC patients with
BRAF V600E mutation (P=0.036 and 0.002, respectively),
tumor size ≥2cm (P=0.002 and 0.010, respectively),
extracapsular invasion (P=0.034 and 0.027, respectively),
lymphovascular invasion (P=0.038 and 0.032, respec-
tively). The miR-181b expression was elevated in patients
with multifocal tumors (P=0.045) and the miR-137 level
was increased in PTC cases with lymph node metastasis
(P=0.011). Results: In total, 11 placental microRNAs were detected
that were expressionally modulated by SNVs. Research was
focused to six miRNAs (hsa-miR-130b, hsa-miR-490-3p,
hsa-miR-3927, hsa-miR-941, hsa-miR-301b, hsa-miR-152)
that have been implicated in the placental function and/or
pregnancy complications. For each prioritized miRNA
several novel target genes and involved biological pathways
were identified. Results: In total, 11 placental microRNAs were detected
that were expressionally modulated by SNVs. Research was
focused to six miRNAs (hsa-miR-130b, hsa-miR-490-3p,
hsa-miR-3927, hsa-miR-941, hsa-miR-301b, hsa-miR-152)
that have been implicated in the placental function and/or
pregnancy complications. For each prioritized miRNA
several novel target genes and involved biological pathways
were identified. Conclusions: miRNA eQTLs may represent additional
modulators of the placental transcriptome, placental func-
tion and pregnancy course. Conclusions: Upregulation of miR-137 and -181b have
been confirmed in PTC tumors and cell line. These two
miRs associated with BRAF V600E mutation and malig-
nancy of PTC. Conclusions: Upregulation of miR-137 and -181b have
been confirmed in PTC tumors and cell line. These two
miRs associated with BRAF V600E mutation and malig-
nancy of PTC. Grant: IUT34-12 (Estonian Research Council) R. Inno: None. S. Sõber: None. M. Laan: None. R. Inno: None. S. Sõber: None. M. Laan: None. M. Zarkesh1, Z. Nozhat1, A. Zadeh-Vakili1, A. Fanaei2, M.
Akbarzadeh1, A. Daneshafrooz1, M. Hedayati1, F. Azizi3 M. Zarkesh1, Z. Nozhat1, A. Zadeh-Vakili1, A. Fanaei2, M. Akbarzadeh1, A. Daneshafrooz1, M. Hedayati1, F. Azizi3 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Iran, Islamic Republic of,
2Association Professor of General Surgery, Erfan Hospital,
Tehran, Iran, Islamic Republic of, 3Endocrine Research
Center, Research Institute for Endocrine Sciences, Shahid
Beheshti University of Medical Science, Tehran, Iran, Islamic
Republic of 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Iran, Islamic Republic of,
2Association Professor of General Surgery, Erfan Hospital,
Tehran, Iran, Islamic Republic of, 3Endocrine Research
Center, Research Institute for Endocrine Sciences, Shahid
Beheshti University of Medical Science, Tehran, Iran, Islamic
Republic of R. Inno, S. Sõber, M. Laan R. Inno, S. Sõber, M. Laan Institue of Biomedicine and Translational Meidicine, Tartu,
Estonia P17.44D O.M. Plotnikova: None. M.Y. Skoblov: None. P17.42B Totally, we identified 156 thousand miRNA binding
regions and formed a subset of 45 thousand high-confidence
regions that were identified in at least two different
experiments. The analysis of high-confidence miRNA
binding sites revealed tissue-specific interactions for two
predominate cells: HEK293 and Huh7.5. On the other hand,
we obtained a group of 13,6 thousand “house-keeping”
miRNA
binding
regions
that
were
identified
in
predominant cells. Totally, we identified 156 thousand miRNA binding
regions and formed a subset of 45 thousand high-confidence
regions that were identified in at least two different
experiments. The analysis of high-confidence miRNA
binding sites revealed tissue-specific interactions for two
predominate cells: HEK293 and Huh7.5. On the other hand,
we obtained a group of 13,6 thousand “house-keeping”
miRNA
binding
regions
that
were
identified
in
predominant cells. Results: RNA-seq analysis showed different profiles
between ALS, AD and CTR. In EXOs, we have detected 8
deregulated genes (DE) in ALS patients and 72 in AD
group. In MVs, we found 17 DE in ALS, 173 DE genes in
AD. In AD patients, 3 DE miRNA were detected in EXOs,
while 65 miRNA DE in MVs. In ALS group 5 miRNA were
DE in exosomes while 9 were altered in MVs. Conclusions: About ALS, the most interesting data
concern the exosome coding transcriptome where we
identified 8 coding RNAs. Regarding AD, a distinct
miRNA signature (65 miRNA) was identified in micro-
vesicles. Our
results
hypothesized
a
mRNA/miRNA
signature in plasma derived EVs that may be an specific
biomarker for neurodegenerative diseases. For analyzing nucleotide variants from patients with an
inherited disease which could be caused by breaking in the
miRNA-mRNA interactions, we developed a tool based on
the high-confidence regions. We identified the subset of 45 thousand high-confident
human miRNA binding regions that were arranged in the
tool. Hence, it will be a valuable resource that should
provide additional insights into the identification new
molecular mechanisms of hereditary diseases caused by
breaking in the miRNA-mRNA interactions. C. Cereda: None. D. Sproviero: None. S. Zucca: None. M. Giannini: None. S. Gagliardi: None. M. Arigoni:
None. O. Pansarasa: None. R.A. Calogero: None. P17.42B Tool for pathogenesis analysis of nucleotide variants based
on high-confidence human miRNA-mRNA interactions M. Zarkesh: None. Z. Nozhat: None. A. Zadeh-Vakili:
None. A. Fanaei: None. M. Akbarzadeh: None. A. Daneshafrooz: None. M. Hedayati: None. F. Azizi: None. M. Zarkesh: None. Z. Nozhat: None. A. Zadeh-Vakili:
None. A. Fanaei: None. M. Akbarzadeh: None. A. Daneshafrooz: None. M. Hedayati: None. F. Azizi: None. O. M. Plotnikova1, M. Y. Skoblov2,1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 619 1Moscow Institute of Physics and Technology, Moscow,
Russian Federation, 2Research Centre for Medical Genetics,
Moscow, Russian Federation 1Moscow Institute of Physics and Technology, Moscow,
Russian Federation, 2Research Centre for Medical Genetics,
Moscow, Russian Federation Bioinformatic and Genomic Unit, University of Turin, Torino,
Italy Bioinformatic and Genomic Unit, University of Turin, Torino,
Italy Bioinformatic and Genomic Unit, University of Turin, Torino,
Italy Introduction: Exploring robust biomarkers is essential for
early diagnosis of neurodegenerative diseases. Blood con-
tains microvesicles (MVs) and exosomes (EXOs), extra-
cellular vesicles of different sizes and biological functions,
which transfer mRNA, miRNAs, or proteins among dif-
ferent cell types. Aim of our study was to investigate
mRNA/miRNA signature in plasma derived MVs and
EXOs of Amyotrophic Lateral Sclerosis (ALS) and Alz-
heimer’s Disease (AD) patients. miRNAs play a key role in the regulation of gene expres-
sion, while a majority of miRNA-mRNA interactions
remain unidentified. Different miRNA prediction pro-
grammes have a lack of consensus about predicted miRNA
binding sites. Crosslinking immunoprecipitation (CLIP)-seq
experimental techniques allow revealing transcriptome-
wide binding sites of RBPs. Today there are many data of
CLIP-seq experiments with AGO2 protein which allow to
use it for high-throughput characteristics of miRNA-mRNA
interactome of human. Materials and Methods: MVs and EXOs were isolated
from plasma of 5 sALS, 5 AD patients and 5 healthy
volunteers (CTR) by ultracentrifugation and whole RNA
was extracted. mRNA libraries were prepared by TruSeq
Stranded Total RNA kit (Illumina) (60 million reads). miRNA libraries were prepared by TruSeq Small RNA
Library
kit
(Illumina)
(5-8
millionreads). Data
were
analyzed with ad hoc Bioconductor packages. We collected results of 79 AGO2-CLIP-seq data (18
PAR-CLIP and 61 HITS-CLIP) from 11 studies in 9 cell
types. We also took data from two modified CLIP-seq
studies (CLASH, CLEAR-CLIP) that straightforwardly
detect miRNA–mRNA pairs as chimeric reads. 1Genomic and post-Genomic Center, IRCCS Mondino
Foundation, Pavia, Italy, 2Department of Brain and
Behavioral Science, University of Pavia, Pavia, Italy,
3Department of Molecular Biotechnology and Health Sciences, P17.43C mRNA/microRNAin extracellular vesicles from ALS and
AD patients: a specific signature for Neurodegenerative
Diseases M. V. Golubenko1,2, A. V. Markov1, R. R. Salakhov1,2, A. A. Zarubin1,3, A. V. Frolov2, A. A. Sleptcov1, M. S. Nazarenko1,2,3,
O. L. Barbarash2, V. P. Puzyrev1,3 C. Cereda1, D. Sproviero1, S. Zucca1, M. Giannini1,2, S. Gagliardi1, M. Arigoni3, O. Pansarasa1, R. A. Calogero3 1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk,
Russian Federation, 2Research Institute for Complex Issues of
Cardiovascular Diseases, Kemerovo, Russian Federation,
3Siberian State Medical University, Tomsk, Russian Federation 1Genomic and post-Genomic Center, IRCCS Mondino
Foundation, Pavia, Italy, 2Department of Brain and
Behavioral Science, University of Pavia, Pavia, Italy,
3Department of Molecular Biotechnology and Health Sciences, Introduction: Mitochondrial DNA is controlled by nuclear
genes, and there is increasing evidence that epigenetic 620 J. del Picchia Garofolo, Trieste, Italy, 6UOC Pediatria ASST Lariana, Como,
Italy, 7Clinical Pedriatric Genetics Unit, Pediatric Clinics,
MBBM Foundation, San Gerardo Hospital, Monza, Italy,
8Medical Genetics Unit, Papa Giovanni XXIII Hospital,
Bergamo, Italy, 9Pediatric Highly Intensive Care Unit,
Fondazione IRCCS Ca' Granda Ospedale Maggiore
Policlinico, Milan, Italy, 10Medical Genetics Unit, Fondazione
IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan,
Italy, 11Department of Pathophysiology and Transplantation,
Università degli Studi di Milano; Division of Pathology,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, Milan, Italy mechanisms play significant role in mtDNA function and
maintenance. The aim of the study was to explore epige-
netic
links
between
nucleus
and
mitochondrion
in
atherosclerosis. Materials and Methods: We studied mtDNA copy
number (by real time PCR), mtDNA D-loop methylation
(by bisulfite treatment, PCR and Illumina sequencing) and
LINE-1 methylation (by pyrosequencing) in blood and
carotid atherosclerotic plaques of patients subjected to
endarterectomy (N=50). Results: We registered very low level of mtDNA
methylation
in
the
D-loop
(0.5%-2%,
depending
on
position), both in CpG sites and for non-CpG cytosines. However, rate of A and G calls in the reference C positions,
and C calls in the reference T positions (indicating PCR/
sequencing error rate) was even less (about 0.3%). Blood
samples and plaques did not differ by mtDNA methylation
level. Median value of mtDNA copy number per cell was
higher in plaques than in blood cells. There was negative
correlation of mtDNA copy number with LINE-1 methyla-
tion level in plaques (Spearman r=-0.54), but not in blood. P17.43C In our cohort, MLID appears higher
frequent in females, with a male-to-female ratio of 1:4. No significant differences in clinical features between
single- and multilocus patients were observed. However, a
number of clinical features appeared more frequent in BWS
MLID patients, i.e. macroglossia, outer ears anomalies and
facial nevus flammeus. The study was supported by RFBR (No.16-04-01481-A). y
pp
y
M.V. Golubenko: None. A.V. Markov: None. R.R. Salakhov: None. A.A. Zarubin: None. A.V. Frolov:
None. A.A. Sleptcov: None. M.S. Nazarenko: None. O.L. Barbarash: None. V.P. Puzyrev: None. M.V. Golubenko: None. A.V. Markov: None. R.R. Salakhov: None. A.A. Zarubin: None. A.V. Frolov:
None. A.A. Sleptcov: None. M.S. Nazarenko: None. O.L. Barbarash: None. V.P. Puzyrev: None. P17.43C Introduction: Several patients with imprinting disorders
(IDs) exhibit epigenetic alterations not only at the disease-
specific loci but extended to other imprinted genes, the so
called
multilocus
methylation
imprinting
disturbance
(MLID). Causative mutations in genes involved in methy-
lation establishment have been identified only in few cases
and in the majority of MLID cases the causative defect
remains unknown. Conclusions: The results indicate that there might be
some epigenetic control of mtDNA replication in the
atherosclerotic lesions. Taking into account that decreased
overall genomic methylation is considered as negative
factor, the increased mtDNA copy number in atherosclerotic
plaques and its negative correlation with LINE-1 methyla-
tion can be explained by compensatory amplification in
response to mitochondrial dysfunction and oxidative stress
in the affected vessels. Materials and Methods: We developed a quantitative
methylation test by MassARRAY to detect alterations at
12 imprinted regions in 21 pre-and post-natal Beckwith-
Wiedemann (BWS) and 7 post-natal Silver-Russell (SRS)
patients. Targeted
NGS
analysis
was
performed
to
identify possible causative mutations in a panel of 25
genes
involved
in
methylation
establishment
and
maintenance. Conclusions: The results indicate that there might be
some epigenetic control of mtDNA replication in the
atherosclerotic lesions. Taking into account that decreased
overall genomic methylation is considered as negative
factor, the increased mtDNA copy number in atherosclerotic
plaques and its negative correlation with LINE-1 methyla-
tion can be explained by compensatory amplification in
response to mitochondrial dysfunction and oxidative stress
in the affected vessels. Materials and Methods: We developed a quantitative
methylation test by MassARRAY to detect alterations at
12 imprinted regions in 21 pre-and post-natal Beckwith-
Wiedemann (BWS) and 7 post-natal Silver-Russell (SRS)
patients. Targeted
NGS
analysis
was
performed
to
identify possible causative mutations in a panel of 25
genes
involved
in
methylation
establishment
and
maintenance. Results: About 50% of BWS and 29% of SRS patients
showed MLID, with loss of methylation confined to
maternally imprinted loci. NGS allowed the detection of
two novel mutations in NLRP2 and ZFP42 genes in two
MLID patients. In our cohort, MLID appears higher
frequent in females, with a male-to-female ratio of 1:4. Results: About 50% of BWS and 29% of SRS patients
showed MLID, with loss of methylation confined to
maternally imprinted loci. NGS allowed the detection of
two novel mutations in NLRP2 and ZFP42 genes in two
MLID patients. Molecular diagnosis and genetic bases of multilocus
methylation defects in Beckwith-Wiedemann and Silver-
Russell syndromes Molecular diagnosis and genetic bases of multilocus
methylation defects in Beckwith-Wiedemann and Silver-
Russell syndromes Conclusions: MLID appeared a frequent finding in BWS/
SRS patients. The hypomethylation confined to maternally
imprinted loci, suggests alterations in trans-acting factors
involved in methylation establishment/maintenance in the
oocyte, as confirmed by mutations found in MLID patients. In silico analysis and bioinformatics modelling confirm the
pathogenic effects of these mutations. L. Fontana1, C. Faré2, A. Seresini3, F. Cortini3, S. M. Sirchia4,
V. Pecile5, A. Selicorni6, S. Maitz7, A. Cereda8, D. Milani9, F. Lalatta10, M. F. Bedeschi10, M. Miozzo11, S. Tabano1 1Department of Pathophysiology and Transplantation,
Università degli Studi di Milano, Milan, Italy, 2Division of
Pathology, Fondazione IRCCS Ca'Granda Ospedale Maggiore
Policlinico, Milan, Italy, 3Molecular Genetics Laboratory,
Fondazione IRCCS Ca' Granda, Ospedale Maggiore
Policlinico, Milan, Italy, 4Department of Health Sciences,
Università degli Studi di Milano, Milan, Italy, 5Department of
Genetics, Institute for Maternal and Child Health IRCCS Burlo L. Fontana: None. C. Faré: None. A. Seresini: None. F. Cortini: None. S.M. Sirchia: None. V. Pecile: None. A. Selicorni: None. S. Maitz: None. A. Cereda: None. D. Milani: None. F. Lalatta: None. M.F. Bedeschi: None. M. Miozzo: None. S. Tabano: None. L. Fontana: None. C. Faré: None. A. Seresini: None. F. Cortini: None. S.M. Sirchia: None. V. Pecile: None. A. Selicorni: None. S. Maitz: None. A. Cereda: None. D. Milani: None. F. Lalatta: None. M.F. Bedeschi: None. M. Miozzo: None. S. Tabano: None. Milani: None. F. Lalatta: None. M.F. Bedeschi: None. M. Miozzo: None. S. Tabano: None. Milani: None. F. Lalatta: None. M.F. Bedeschi: None. M. Miozzo: None. S. Tabano: None. 1Department of Pathophysiology and Transplantation,
Università degli Studi di Milano, Milan, Italy, 2Division of
Pathology, Fondazione IRCCS Ca'Granda Ospedale Maggiore
Policlinico, Milan, Italy, 3Molecular Genetics Laboratory,
Fondazione IRCCS Ca' Granda, Ospedale Maggiore
Policlinico, Milan, Italy, 4Department of Health Sciences,
Università degli Studi di Milano, Milan, Italy, 5Department of
Genetics, Institute for Maternal and Child Health IRCCS Burlo P17.46B M. Elbracht, I. Kurth, M. Begemann, T. Eggermann Institute for Human Genetics, Aachen, Germany Institute for Human Genetics, Aachen, Germany Imprinting disorders comprise a group of clinical entities
sharing parent-specific changes in the epigenetic signature
of specific genomic loci. The imprinting disorder Silver-
Russell syndrome (SRS) is caused by loss of methylation
(LOM) of the imprinting center region 1 (ICR1) on
chromosome 11p15.5 in 30-60% of patients. In nearly
10% of these patients, aberrant methylation on additional
chromosomes have been detected, establishing a spectrum
of multilocus imprinting disorders (MLIDs). By investi-
gating different tissues, MLID cases in the ICR1 LOM
cohort increases up to 38%. Maternally-provided factors
in the ooplasm are postulated for the establishment and
maintenance of imprinting marks in the embryo, leading
to MLIDs by so-called maternal effect mutations. Several
of these maternal effect genes are members of the NLRP
gene family1. Objective: Osteogenesis Imperfecta(OI) is associated with
long bone deformities and fractures, accompanied by a
genetic connective tissue disorder. According to their clin-
ical, inherited, and radiological characteristics 17different
types of OI has been reported. There are autosomal domi-
nant(OD)
and
autosomal
recessive(OR)
transmission,
although inheritance patterns differ according to types. MicroRNAs(miRNAs) play important roles in processes
such as ossification, osteoporosis, osteoblastic proliferation,
differentiation. In this study we aimed to identify the role of
microRNAs in the clinical heterogenity of OI, contribute to
the understanding of their utility as biomarker,and also to
determine the expression differences of bone-associated
miRNAs(miR-26a,miR29a,miR-133a) between the patients
and healthy controls in order to search for their possible use
as a new therapeutic target. This study questions whether maternal effect mutations
are also cause of apparently isolated ICR1 LOM. We
analysed peripheral blood lymphocytes from 21 mothers of
ICR1 LOM patients by a targeted next-generation sequen-
cing approach (NLRP2-14, TRIM28, KHDC3L). Hetero-
zygosity for variants in NLRP5 were identified in two
mothers:
NM_153447.4:c.68T>A
(p.(Val23Asp);
rs753824534)
and
NM_153447.4:c.3259G>A
(p. (Glu1087Lys), both listed as rare/pathogenic, compatible
with maternal-effect mutations. It has to be discussed
whether our findings are in line with an association of
isolated ICR1 LOM and maternal effect mutations in
NLRP5 or whether MLID remained undetected in the
children of these two mothers. Thus, identification of further
factors causing ICR1 LOM and studies of additional tissues
with more sensitive approaches should help distinguishing
between both possibilities. Abstracts from the 51st European Society of Human Genetics Conference: Posters 621 1Akdeniz University Medical School, Department of Pediatrics,
Antalya, Turkey, 2Akdeniz University Medical School,
Department of Pediatric Genetics, Antalya, Turkey, 3Akdeniz
University Medical School, Department of Medical Biology,
Antalya, Turkey, 4Akdeniz University Medical School,
Department of Pediatric Endocrinology, Antalya, Turkey,
5Akdeniz University Medical School, Department of Physical
Medicine and Rehabilitation, Antalya, Turkey E. Loi1, M. Antonelli2, A. Fadda1, L. Moi1,3, C. Zavattari4, P.
Sulas5, D. Gentilini6,7, C. Cameli8, E. Bacchelli8, M. Badiali3, A.
Arcella9, I. Morra10, F. Giangaspero2,9, P. Zavattari1 P17.46B Method: 0-18 years age matched 26 OI patients that
followed by pediatric genetics, endocrinology and physical
rehabilitation clinic and 16 healty control group were
involved. MiRNeasy Serum / Plasma Kit (Qiagen, 217184)
was used for the isolation of miRNA(mir-26a,mir-29a,mir-
133a)
from
the
plasma. The
values
determined
by
quantitativePCR were normalized using REST programme. Results: There was a significant difference between the
miRNA levels ofthe patient and control groups and the
miRNA levels of the patient group werehigher. Serum
calcium and vitamin D levels of patients with higher
mir133alevels were lower than those of the other groups. Conclusion: OI has no definitive treatment. Identification
of some biomarkers may be important in the detection of the
disease at an early stage, monitoring the response to
treatment, and determining miRNAs that are likely to be a
new therapeutic targets. Monk, D. et al. Reproduction 154, R161-R170 (2017). L. Oz: None. B. Nur: None. A. Toylu: None. G. Celmeli: None. H. Nur: None. E. Mihci: None. Monk, D. et al. Reproduction 154, R161-R170 (2017). M. Elbracht: None. I. Kurth: None. M. Begemann:
None. T. Eggermann: None. M. Elbracht: None. I. Kurth: None. M. Begemann:
None. T. Eggermann: None. P17.46B
Do maternal effect mutations in NLRP5 cause ICR1
hypomethylation in Silver-Russell syndrome patients? 1Akdeniz University Medical School, Department of Pediatrics,
Antalya, Turkey, 2Akdeniz University Medical School,
Department of Pediatric Genetics, Antalya, Turkey, 3Akdeniz
University Medical School, Department of Medical Biology,
Antalya, Turkey, 4Akdeniz University Medical School,
Department of Pediatric Endocrinology, Antalya, Turkey,
5Akdeniz University Medical School, Department of Physical
Medicine and Rehabilitation, Antalya, Turkey L. Oz1, B. Nur2, A. Toylu3, G. Celmeli4, H. Nur5, E. Mihci2 Badiali: None. A. Arcella: None. I. Morra: None. F.
Giangaspero: None. P. Zavattari: None. Badiali: None. A. Arcella: None. I. Morra: None. F. Giangaspero: None. P. Zavattari: None. Badiali: None. A. Arcella: None. I. Morra: None. F. Giangaspero: None. P. Zavattari: None. 1Unit of Biology and Genetics, Department of Biomedical
Sciences, University of Cagliari, Cagliari, Italy, 2Department
of Radiological, Oncological and Anatomo-Pathological
Sciences, University Sapienza of Rome, Rome, Italy, 3Bone
Marrow Transplantation Unit, Microcitemico Children's
Hospital, Cagliari, Italy, 4Independent Researcher, Machine
Learning, Lucca, Italy, 5Unit of Oncology and Molecular
Pathology, Department of Biomedical Sciences, University of
Cagliari, Cagliari, Italy, 6Department of Brain and Behavioral
Sciences, University of Pavia, Pavia, Italy, 7Bioinformatics and
Statistical Genomics Unit, Istituto Auxologico Italiano IRCCS,
Cusano Milanino, Milan, Italy, 8Department of Pharmacy and
Biotechnology, University of Bologna, Bologna, Italy, 9IRCCS
Neuromed, Pozzilli, Italy, 10Department of Pathology OIRM-S. Anna Hospital, A.O.U. City of Health and Science, Turin, Italy P17.50B
Epigenetic signature of preterm birth in adult twins Gene expression levels
of selected differentially methylated genes, evaluated by
qRT-PCR,
were
compared
to
commercially
available
human normal brain samples and to GTEx expression data
observed in different normal brain sites. Results:
we
identified
distinct
methylation
profiles
characterizing PA from different locations (supratentorial
vs infratentorial) and tumors with onset before and after 3
years of age. Gene expression analysis of TOX2 and IRX2,
hypermethylated in supratentorial PA, revealed a decreased
expression in supratentorial compared to infratentorial PAs. The expression level of IRX2 in the tumor samples resulted
in line with that observed in the normal brain sites. In
contrast, TOX2 showed an opposite expression pattern in
PA compared to that in normal brain sites. Q. Tan: None. S. Li: None. P17.50B
Epigenetic signature of preterm birth in adult twins Q. Tan1, S. Li2
1Epidemiology and Biostatistics, Department of Public Health,
Odense, Denmark, 2Unit of Human Genetics, Department of
Clinical Research, University of Southern Denmark, Odense,
Denmark Q. Tan1, S. Li2 1Epidemiology and Biostatistics, Department of Public Health,
Odense, Denmark, 2Unit of Human Genetics, Department of
Clinical Research, University of Southern Denmark, Odense,
Denmark 1Epidemiology and Biostatistics, Department of Public Health,
Odense, Denmark, 2Unit of Human Genetics, Department of
Clinical Research, University of Southern Denmark, Odense,
Denmark Preterm birth is a leading cause of perinatal mortality and
long-term health consequences. Epigenetic mechanisms
may have been at play in preterm birth survivors and these
could be persistent and detrimental to health later in life. We
performed a genome-wide DNA methylation profiling adult
twins of premature birth to identify genomic regions under
differential epigenetic regulation in 144 twins with a median
age of 33 years (age range: 30-36). Association analysis
detected three genomic regions annotated to the SDHAP3,
TAGLN3, and GSTT1 genes on chromosomes 5, 3, and 22
(FWER: 0.01, 0.02 and 0.04) respectively. These genes
display strong involvement in neurodevelopmental dis-
orders,cancer susceptibility and premature delivery. The
three identified significant regions were successfully repli-
cated in an independent sample of twins of even older age
(median age 66, range: 56-80) with similar regulatory pat-
terns and nominal p values <5.05e-04. Biological pathway
analysis detected 5 significantly enriched pathways all
explicitly involved in immune responses. The study pro-
vides novel epigenetic evidence for the association between
important genes/pathways and premature delivery and
meanwhile reveals that preterm birth, as an early life event,
could be related to differential methylation regulation pat-
terns observable in adults and even at high ages which
could potentially mediate susceptibility to age-related dis-
eases and adult health. Introduction: Pilocytic astrocytoma (PA) is the most
common pediatric brain tumor. Although genetic and epi-
genetic alterations characterizing PA from different locali-
zations
have
been
reported,
the
role
of
methylome
alterations in PA development is still not clear. In order to
investigate whether distinctive methylation patterns may
define biological relevant subgroups of PAs, we character-
ized the DNA methylation profiles of 20 tumors and 4 non-
tumoral brain samples. Materials and Methods: genome-wide DNA methyla-
tion analysis was performed using Illumina Infinium
HumanMethylation27K BeadChips. 1Moscow Institute of Physics and Technology (State
University), Dolgoprudny, Russian Federation, 2Federal
Research and Clinical Center of Physical Chemical Medicine
of the Federal Medical and Biological Agency of the Russian P17.47C Integrated DNA methylation analysis identifies
topographical and tumoral biomarkers in pilocytic
astrocytomas Evaluation of Serum miRNA (mir-26a, mir-29a, mir-133a)
Expression Levels in Patients with Osteogenesis Imperfecta L. Oz1, B. Nur2, A. Toylu3, G. Celmeli4, H. Nur5, E. Mihci2 622 J. del Picchia R. I. Sultanov1,2,3, G. P. Arapidi3,1, K. Babalyan1,2, V.
Gordeeva1,2, E. V. Generozov2, V. M. Govorun2,1 Lecumberri22, M. Levine23, O. Mäkitie24, R. Martin25, G. Martos-
Moreno26, M. Minagawa27, P. Murray28, A. Pereda29, R.
Pignolo30, L. Rejnmar31, R. Rodado13, A. Rothenbuhler6, V.
Saraff32, A. Shoemaker33, E. Shore34, C. Silve35, S. Turan36, P.
Woods19, M. Zillikens37, G. Perez de Nanclares29, A. Linglart6,17 Lecumberri22, M. Levine23, O. Mäkitie24, R. Martin25, G. Martos-
Moreno26, M. Minagawa27, P. Murray28, A. Pereda29, R. Pignolo30, L. Rejnmar31, R. Rodado13, A. Rothenbuhler6, V. Saraff32, A. Shoemaker33, E. Shore34, C. Silve35, S. Turan36, P. Woods19, M. Zillikens37, G. Perez de Nanclares29, A. Linglart6,17 Introduction: Aggressive forms of prostate cancer (PCa)
are often associated with epithelial-mesenchymal transition
(EMT). Thus, the study of the master regulators of epithelial
fate (TP63) can help to understand the potential mechan-
isms of formation non-indolent forms of PCa. Lecumberri22, M. Levine23, O. Mäkitie24, R. Martin25, G. Martos-
Moreno26, M. Minagawa27, P. Murray28, A. Pereda29, R.
Pignolo30, L. Rejnmar31, R. Rodado13, A. Rothenbuhler6, V.
Saraff32, A. Shoemaker33, E. Shore34, C. Silve35, S. Turan36, P.
Woods19, M. Zillikens37, G. Perez de Nanclares29, A. Linglart6,17 Further, we found the
association between the cluster and the EMT in prostate cell
lines (data from Olsen et al. 2013). The master regulator of
this cluster was TP63 because promoters were significantly
enriched with TP63 binding sites (p-value=5e-26), and
TP63-knockdown altered expression of cluster genes. The
expression of cluster genes was highly correlated (Spear-
man < -0.8) with methylation level of 556 CpG-sites located
in the enhancers and super-enhancers (eCpG-sites, signifi-
cant enrichment with H3K27ac and H3K4me peaks). This
eCpG-sites interacted with promoters of the genes asso-
ciated with EMT. Results: We obtained a cluster of genes and CpG-sites
downregulated in PCa samples. Further, we found the
association between the cluster and the EMT in prostate cell
lines (data from Olsen et al. 2013). The master regulator of
this cluster was TP63 because promoters were significantly
enriched with TP63 binding sites (p-value=5e-26), and
TP63-knockdown altered expression of cluster genes. The
expression of cluster genes was highly correlated (Spear-
man < -0.8) with methylation level of 556 CpG-sites located
in the enhancers and super-enhancers (eCpG-sites, signifi-
cant enrichment with H3K27ac and H3K4me peaks). This
eCpG-sites interacted with promoters of the genes asso-
ciated with EMT. Conclusions: We found the cluster of genes and CpG-
sites that associated with EMT and were regulated by
expression of TP63. Expression of cluster genes highly
correlated with eCpG-sites. We speculate that expression of
TP63 effects methylation level of eCpG-sites because TP63
is a pioneer transcription factor. This work was supported
by the Russian Foundation for Basic Research (project No. 17-29-06063). R.I. Sultanov: None. G.P. Arapidi: None. K. Babal-
yan: None. V. Gordeeva: None. E.V. Generozov: None. V.M. Govorun: None. Lecumberri22, M. Levine23, O. Mäkitie24, R. Martin25, G. Martos-
Moreno26, M. Minagawa27, P. Murray28, A. Pereda29, R.
Pignolo30, L. Rejnmar31, R. Rodado13, A. Rothenbuhler6, V.
Saraff32, A. Shoemaker33, E. Shore34, C. Silve35, S. Turan36, P.
Woods19, M. Zillikens37, G. Perez de Nanclares29, A. Linglart6,17 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, Endocrinology Unit, Department of Clinical
Sciences and Community Health, University of Milan, Milan,
Italy, 2Endocrine Unit, Department of Medicine,
Massachusetts General Hospital and Harvard Medical School,
Boston, MA, United States, 3Imprinting and Cancer group,
Cancer Epigenetic and Biology Program (PEBC), Institut
d'Investigació Biomedica de Bellvitge (IDIBELL), Barcelona,
Spain, 4Pediatric Endocrinology Unit, Department of Public
Health and Pediatric Sciences, University of Torino, Torino,
Italy, 5Division of Pediatric Endocrinology and Diabetes,
Department of Pediatrics, University of Lübeck, Lübeck,
Germany, 6APHP, Reference Center for Rare Disorders of
Calcium and Phosphate Metabolism, Platform of Expertise
Paris-Sud for Rare Diseases and Filière OSCAR; APHP,
endocrinology and diabetes for children, Bicêtre Paris-Sud
Hospital, Le Kremlin-Bicêtre, France, 7Developmental
Endocrinology Research Group, School of Medicine, Dentistry
and Nursing Studies, University of Glasgow, Glasgow, United
Kingdom, 8IPOHA, Italian Progressive Osseous Heteroplasia
Association, Cerignola, Foggia, Italy, 9K20, French PHP and
related disorders patient association, Jouars Pontchartrain,
France, 10APHP, Department of medicine for adolescents,
Bicêtre Paris Sud Hospital, Le Kremlin-Bicêtre, France,
11Insitute of Human Genetics, Technical University of Aachen,
Aachen, Germany, 12Department of Cardiovascular Sciences,
Center for Molecular and Vascular Biology, Gasthuisberg,
University of Leuven, Leuven, Belgium, 13AEPHP, Spanish
PHP and related disorders patient association, Huércal-
Overa, Almeria, Spain, 14Albright Center & Center for Rare
Bone Disorders, Division of Pediatric Endocrinology &
Diabetes, Connecticut Children's Medical Center and
Department of Pediatrics, University of Connecticut School of
Medicine, Farmington, CT, United States, 15APHP,
Department of Endocrinology, Cochin Hospital; Université
Paris Descartes, Sorbonne Paris Cité, Paris, France,
16Department of Medicine, Division of Endocrinology &
Centre for Bone Quality, Leiden University Medical Center,
Leiden, Netherlands, 17INSERM U1169, Bicêtre Paris Sud,
Université Paris Sud Paris Saclay, Le Kremlin-Bicêtre,
France, 18APHP, Reference Center for Rare Disorders of
Calcium and Phosphate Metabolism, Platform of Expertise
Paris-Sud for Rare Diseases and Filière OSCAR; APHP,
Department of Endocrinology and Reproductive Diseases,
Bicêtre Paris Sud Hospital; INSERM U1185, Le Kremlin-
Bicêtre, France, 19UK acrodysostosis patients’ group, London,
United Kingdom, 20Department of Genetics, Caen University Materials and Methods: Whole-genome methylation,
RNA-sequencing, and copy number variation data of
patients with PCa from The Cancer Genome Atlas (TCGA)
portal were used for this research. The regulatory networks
were reconstructed from EWAS (epigenome-wide associa-
tion study) based on mixed linear models. ChIP-seq data
were downloaded from GEO. Results: We obtained a cluster of genes and CpG-sites
downregulated in PCa samples. P17.51C Conclusions: our study revealed brain-region and age-
related specific methylation patterns in PA and identified
IRX2 as a topographic biomarker and TOX2 as a promising
tumoral biomarker. The effect of TP63 expression on the methylation level of
CpG-sites located in enhancers of the genes associated with
epithelial-mesenchymal transition in prostate cancer Grants: Fondazione Neuroblastoma, Fondazione Banco
di Sardegna, Regione Autonoma della Sardegna (CRP-
79303) and Fondo per la Ricerca Locale (ex 60%),
Università di Cagliari. E. Loi: None. M. Antonelli: None. A. Fadda: None. L. Moi: None. C. Zavattari: None. P. Sulas: None. D. Gentilini: None. C. Cameli: None. E. Bacchelli: None. M. Abstracts from the 51st European Society of Human Genetics Conference: Posters 623 Federation, Moscow, Russian Federation, 3Shemyakin-
Ovchinnikov Institute of Bioorganic Chemistry of the Russian
Academy of Sciences, Moscow, Russia, Moscow, Russian
Federation P17.52D First international consensus statement on diagnosis and
management of pseudohypoparathyroidism and related
disorders G. Mantovani1, M. Bastepe2, D. Monk3, L. de Sanctis4, S. Thiele5, A. Usardi6, F. Ahmed7, R. Bufo8, T. Choplin9, G. DeFilippo10, G. Devernois9, T. Eggermann11, F. Elli1, K. Freson12, A. Garcia-Ramirez13, E. Germain-Lee14, L. Groussin15,
N. Hamdy16, P. Hanna17, O. Hiort5, H. Jüppner2, P. Kamenický18, N. Knigh19, M. Kottle20, E. Le Norcy21, B. 624 J. del Picchia Hospital, Caen, France, 21APHP, Department of Odontology,
Bretonneau Hospital PNVS, Paris; Faculty of Dentistry, Paris
Descartes Universit, Montrougeu Hospital PNVS, Paris;
Faculty of Dentistry, Paris Descartes University, Montrouge,
France, 22Department of Endocrinology and Nutrition, La Paz
University Hospital, Madrid, Spain, 23Department of
Pediatrics, Division of Endocrinology and Diabetes and
Center for Bone Health, Children's Hospital of Philadelphia,
University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA, United States, 24University of Helsinki and
Helsinki University Hospital, Children's Hospital., Helsinki,
Finland, 25Laboratório de Metabolismo e Endocrinologia do
Departamento de Fisiologia e Biofísica do Instituto de
Ciências Biomédicas da Universidade de São Paulo (ICB-
USP), São Paulo, Brazil, 26Department of Endocrinology, Hospital, Caen, France, 21APHP, Department of Odontology,
Bretonneau Hospital PNVS, Paris; Faculty of Dentistry, Paris
Descartes Universit, Montrougeu Hospital PNVS, Paris;
Faculty of Dentistry, Paris Descartes University, Montrouge,
France, 22Department of Endocrinology and Nutrition, La Paz
University Hospital, Madrid, Spain, 23Department of
Pediatrics, Division of Endocrinology and Diabetes and
Center for Bone Health, Children's Hospital of Philadelphia,
University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA, United States, 24University of Helsinki and
Helsinki University Hospital, Children's Hospital., Helsinki,
Finland, 25Laboratório de Metabolismo e Endocrinologia do
Departamento de Fisiologia e Biofísica do Instituto de
Ciências Biomédicas da Universidade de São Paulo (ICB-
USP), São Paulo, Brazil, 26Department of Endocrinology, diagnosis, to the molecular confirmation up to the man-
agement of the most frequent manifestations of these rare
diseases. Methods: A consensus statement was prepared for 2
years
to
produce
recommendations
for
clinical
and
molecular diagnosis and management of patients with
PHP and related disorders. The approach comprised 2
pre-consensus meetings, an expert consensus meeting, and a
Delphi-like methodology, adjusted to rare diseases. Methods: A consensus statement was prepared for 2
years
to
produce
recommendations
for
clinical
and
molecular diagnosis and management of patients with
PHP and related disorders. The approach comprised 2
pre-consensus meetings, an expert consensus meeting, and a
Delphi-like methodology, adjusted to rare diseases. P17.52D CIBERobn, ISCIII, Madrid, Spain, 27Division of
Endocrinology, Chiba Children's Hospital, Chiba, Japan,
28Department of Paediatric Endocrinology, Royal Manchester
Children's Hospital, Manchester University NHS Foundation
Trust, Manchester, United Kingdom, 29Molecular (Epi)
Genetics Laboratory, BioAraba National Health Institute,
Hospital Universitario Araba-Txagorritxu, Vitoria-Gasteiz,
Araba, Spain, 30Department of Medicine, Mayo Clinic,
Rochester, MN, United States, 31Department of Endocrinology
and Internal Medicine, Aarhus University Hospital, Aarhus,
Denmark, 32Department of Endocrinology and Diabetes,
Birmingham Children’s Hospital, Birmingham, United
Kingdom, 33Pediatric Endocrinology and Diabetes, Vanderbilt
University Medical Center, Nashville, TN, United States,
34Departments of Orthopaedic Surgery and Genetics, Center
for Research in FOP and Related Disorders, Perelman School
of Medicine, University of Pennsylvania, Philadelphia, PA,
United States, 35APHP, service de biochimie et génétique
moléculaires, Hôpital Cochin, Paris, France, 36Department of
Pediatrics, Division of Endocrinology and Diabetes, Marmara
University, Istanbul, Turkey, 37Department of Internal
Medicine, Bone Center Erasmus MC - University Medical
Center Rotterdam, Rotterdam, Netherlands Conclusion:
Overall,
a
coordinated
approach
from
infancy through adulthood should help us to improve the
care of patients affected by these disorders. Funding: This consensus meeting was supported by
several
patients’
associations
and
scientific
societies,
including ESHG. G. Mantovani: None. M. Bastepe: None. D. Monk:
None. L. de Sanctis: None. S. Thiele: None. A. Usardi:
None. F. Ahmed: None. R. Bufo: None. T. Choplin:
None. G. DeFilippo: None. G. Devernois: None. T. Eggermann: None. F. Elli: None. K. Freson: None. A. Garcia-Ramirez:
None. E. Germain-Lee:
None. L. Groussin: None. N. Hamdy: None. P. Hanna: None. O. Hiort: None. H. Jüppner: None. P. Kamenický: None. N. Knigh: None. M. Kottle: None. E. Le Norcy: None. B. Lecumberri: None. M. Levine: None. O. Mäkitie: None. R. Martin: None. G. Martos-Moreno: None. M. Mina-
gawa: None. P. Murray: None. A. Pereda: None. R. Pignolo: None. L. Rejnmar: None. R. Rodado: None. A. Rothenbuhler: None. V. Saraff: None. A. Shoemaker:
None. E. Shore: None. C. Silve: None. S. Turan: None. P. Woods: None. M. Zillikens: None. G. Perez de Nan-
clares: None. A. Linglart: None. Knigh: None. M. Kottle: None. E. Le Norcy: None. B. Lecumberri: None. M. Levine: None. O. Mäkitie: None. R. Martin: None. G. Martos-Moreno: None. M. Mina-
gawa: None. P. Murray: None. A. Pereda: None. R. P17.52D Results: After literature search using PubMed, >800
papers published since 1990 to 2016 have been reviewed
and
recommendations
on
clinical
and
diagnosis
and
management on PHP and related disorders have been voted
and
approved
with
different
levels
of
evidence:
14
recommendations on clinical diagnosis, 11 on molecular
diagnosis and 39 on management and treatment. Helsinki University Hospital, Children's Hospital., Helsinki,
Finland, 25Laboratório de Metabolismo e Endocrinologia do
Departamento de Fisiologia e Biofísica do Instituto de
Ciências Biomédicas da Universidade de São Paulo (ICB-
USP), São Paulo, Brazil, 26Department of Endocrinology, Hospital Infantil Universitario Niño Jesús. IIS La Princesa. Department of Pediatrics, Universidad Autónoma de Madrid. CIBERobn, ISCIII, Madrid, Spain, 27Division of
Endocrinology, Chiba Children's Hospital, Chiba, Japan,
28Department of Paediatric Endocrinology, Royal Manchester
Children's Hospital, Manchester University NHS Foundation
Trust, Manchester, United Kingdom, 29Molecular (Epi)
Genetics Laboratory, BioAraba National Health Institute,
Hospital Universitario Araba-Txagorritxu, Vitoria-Gasteiz,
Araba, Spain, 30Department of Medicine, Mayo Clinic,
Rochester, MN, United States, 31Department of Endocrinology
and Internal Medicine, Aarhus University Hospital, Aarhus,
Denmark, 32Department of Endocrinology and Diabetes,
Birmingham Children’s Hospital, Birmingham, United
Kingdom, 33Pediatric Endocrinology and Diabetes, Vanderbilt
University Medical Center, Nashville, TN, United States,
34Departments of Orthopaedic Surgery and Genetics, Center
for Research in FOP and Related Disorders, Perelman School
of Medicine, University of Pennsylvania, Philadelphia, PA,
United States, 35APHP, service de biochimie et génétique
moléculaires, Hôpital Cochin, Paris, France, 36Department of
Pediatrics, Division of Endocrinology and Diabetes, Marmara
University, Istanbul, Turkey, 37Department of Internal
Medicine, Bone Center Erasmus MC - University Medical
Center Rotterdam, Rotterdam, Netherlands Hospital Infantil Universitario Niño Jesús. IIS La Princesa. Department of Pediatrics, Universidad Autónoma de Madrid. P17.53A
Functional validation of an epigenomic variant associated
to retinitis pigmentosa Functional validation of an epigenomic variant associated
to retinitis pigmentosa Introduction: Pseudohypoparathyroidism (PHP) and rela-
ted disorders lead to a wide spectrum of abnormal physical
characteristics, neurocognitive and endocrine abnormalities
that share a common PTH/PTHrP signaling pathway. The
clinical and molecular overlap of these disorders leads to
difficulties in clinical and molecular diagnosis which
prompt to the possibility of incorrect management of these
patients. Over the past 30 years, incredible progress has
been made on the pathophysiology of these disorders by
physicians and research networks. However, caregivers and
patients are lacking guidelines for the daily life management
of patients. Our aim was to help them from the clinical A. V. Vig1,2, M. Liang1,2, A. Mollica1,2, E. Tavares2, M. Wilson1,2,
V. Mennella1,2, A. Vincent2,1, E. Heon1,2 1University of Toronto, Toronto, ON, Canada, 2The Hospital
for Sick Children, Toronto, ON, Canada 1University of Toronto, Toronto, ON, Canada, 2The Hospital
for Sick Children, Toronto, ON, Canada Introduction: Recently, an intronic variant in OFD1
(chrX:13768358 A>G), which decreases the amount of
properly spliced OFD1 transcript, was reported in an Abstracts from the 51st European Society of Human Genetics Conference: Posters 625 individual with a subtype of non-syndromic retinal degen-
eration (RD) called retinitis pigmentosa 23 (RP23). Our lab
has identified an individual with a similar X-linked phe-
notype who has a novel intergenic variant within a DNase I
hypersensitive site (DHS) upstream of OFD1. We hypo-
thesize that this variant is affecting expression levels of
OFD1. Materials and Methods: The CD4+ T cells from 82
Korean patients with RA and 40 healthy controls were
examined by using the Illumina HT-12 expression and
Infinium methylation 450K beadchips, respectively. After a
series
of
standard
quality
control
and
normalization
procedures, differentially expressed genes (DEGs) and
differentially methylated positions (DMPs) were assessed
using a linear model adjusting for the slide batch effect, age,
sex and/or relative cell composition. Materials and Methods: OFD1 mRNA and protein
expression levels of the proband relative to male controls
were measured in primary fibroblast cell lines using droplet
digital PCR (ddPCR) and immunofluorescence. 4C-seq was
also used to capture physical interactions between the DHS
and OFD1 in an adult retinal pigment epithelial cell line
(RPE1). Results: We identified 55 DEGs and 91,067 DMPs at the
5% FDR threshold (e.g., P=1.61x10-5 for ASCL2 in the
DEG analysis; P=9.69x10-4 for cg11373604 in the DMP
analysis). P17.55C A.V. Vig: None. M. Liang: None. A. Mollica: None. E. Tavares: None. M. Wilson: None. V. Mennella: None. A. Vincent: None. E. Heon: None. A.V. Vig: None. M. Liang: None. A. Mollica: None. E. Tavares: None. M. Wilson: None. V. Mennella: None. A. Vincent: None. E. Heon: None. Genome-wide analysis of RNA editing levels in human
blood identified interactions with mRNA processing genes
and suggested correlations with biological and drug-related
variables P17.53A
Functional validation of an epigenomic variant associated
to retinitis pigmentosa In a joint analysis for DEGs and DMPs, higher
expression levels in 15 DEGs were explained by lowered
DNA methylation at neighboring DMPs (P < 0.05 for a
DEG-DMP correlation). For example, the expression level
of ASCL2 and the methylation level at cg11373604
represented a strong negative correlation (P=8.27x10-6;
r=-0.424). Results: Preliminary ddPCR results suggest that the
proband expresses lower levels of OFD1 than controls. However, 4C-seq results suggest that the DHS does not
interact with the OFD1 promoter in adult RPE. Therefore,
further functional validation is required to determine the
specificity of our variant to OFD1 expression and to
determine how and at which developmental stage our
variant alters the activity of the DHS. Results: Preliminary ddPCR results suggest that the
proband expresses lower levels of OFD1 than controls. However, 4C-seq results suggest that the DHS does not
interact with the OFD1 promoter in adult RPE. Therefore,
further functional validation is required to determine the
specificity of our variant to OFD1 expression and to
determine how and at which developmental stage our
variant alters the activity of the DHS. Conclusions: This study revealed a number of RA-
specific expression and DNA methylation in an RA-relevant
immune cell type, CD4+ T cells, and identified plausible
RA-specific
DMPs
that
lead
expressional
variances
in DEGs. Conclusions: This study revealed a number of RA-
specific expression and DNA methylation in an RA-relevant
immune cell type, CD4+ T cells, and identified plausible
RA-specific
DMPs
that
lead
expressional
variances
in DEGs. Conclusions: Although preliminary results suggest that
the proband expresses lower levels of OFD1, the mechan-
ism by which disruption of the DHS causes disease is
inconclusive. Additional functional assays will elucidate the
regulatory and functional consequences of our variant, and
will provide insight about the ways epigenetics and
regulatory genetic mechanisms can influence RD. *This study was supported from the National Research
Foundation of Korea (2017R1E1A1A01076388) and Korea
National Institute of Health. J. Lim: None. S. Bang: None. H. Lee: None. B. Kim:
None. S. Bae: None. K. Kim: None. Funding: Vision Science Research Program, Restracomp DNA methylation and gene expression profiling of CD4+
T cells in rheumatoid arthritis DNA methylation and gene expression profiling of CD4+
T cells in rheumatoid arthritis E. Giacopuzzi, M. Gennarelli, C. Sacco, C. Magri, A. Barbon E. Giacopuzzi, M. Gennarelli, C. Sacco, C. Magri, A. Barbon E. Giacopuzzi, M. Gennarelli, C. Sacco, C. Magri, A. Barbon
Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy J. Lim1, S. Bang2, H. Lee2, B. Kim3, S. Bae2, K. Kim1 J. Lim1, S. Bang2, H. Lee2, B. Kim3, S. Bae2, K. Kim1 Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy 1Department of Biology, Kyung Hee University, Seoul, Korea,
Republic of, 2Department of Rheumatology, Hanyang
University Hospital for Rheumatic Diseases, Seoul, Korea,
Republic of, 3Center for Genome Science, Korea National
Institute of Health, Chungcheongbuk-do, Korea, Republic of A-to-I RNA editing is a post-transcriptional modification
catalyzed by ADAR enzymes, that deaminate Adenosines
(A) into Inosines (I). Most of known editing events are
located within inverted ALU repeats, but they also occur in
coding sequences and may alter the function of encoded
proteins. RNA
editing
contributes
to
generate
tran-
scriptomic diversity and it is found altered in cancer,
autoimmune and neurological disorders. However, little is
known about how editing process could be influenced by
genetic variations, biological and environmental variables. We analyzed the dynamic of RNA editing in human blood
using RNA-seq data from 459 healthy individuals and Republic of, 3Center for Genome Science, Korea National Institute of Health, Chungcheongbuk-do, Korea, Republic of Introduction: Rheumatoid arthritis (RA) is an autoimmune
disease causing chronic inflammation preferentially in
joints. Altered function in CD4+ T cells has been well
documented for its association with the development of RA. In this study, we investigated RA-specific gene expression
and DNA methylation in CD4+ T cells. 626 J. del Picchia half of the control level. Moreover, levels of H2B15K were
found to be significantly lower in RTS patients than in
controls. obtained detectable editing levels for 2,079 sites. Analysis
of gene expression showed that in blood ADAR expression
accounts for ~13% of observed variability in overall editing
level. After removing ADAR effect, we found significant
associations for 1,122 genes, mainly involved in RNA
processing and identified 276 potential ADARs interactors,
including 9 ADARs direct partners. In addition, association
analysis between editing levels principal components and
35 biological and drugs intake variables revealed 24 factors
potentially influencing RNA editing in blood, including sex,
age, BMI, drugs and medications. Finally, we identified
genetic loci associated to global editing levels, including
known ADAR eQTLs and a small region on chromosome 7,
containing LOC730338 lincRNA gene. Our data provides a
detailed picture of RNA editing and its variability in human
blood, giving interesting insights on the mechanisms behind
this
post-transcriptional modification
and
genetic
and
environmental factors involved in its regulation. Conclusions: A wide spectrum of CREBBP mutations in
RTS patients was associated with a general deficit in
specific histone acetylation by CBP, providing additional
understanding of the molecular etiology of the syndrome. P17.56D
Histone acetylation is reduced in Rubinstein-Taybi
patients: epigenetics role in the syndrome Histone acetylation is reduced in Rubinstein-Taybi
patients: epigenetics role in the syndrome Runt related transcription factor 2 (RUNX2) encodes the
master transcription factor in skeletal development. RUNX2
affects skull ossification and is thought to be involved in the
progressive skull “globularization” of anatomically-modern
humans (AMHs) compared with extinct species. RUNX2
mutations cause disorders of skull development, including
craniosynostosis. The aim of our study was to compara-
tively characterize the genomic structure of RUNX2 in
AMH and extinct hominins, and to infer putative functional
correlations for human skull morphogenesis, using non-
syndromic craniosynostosis (NCS) as a disease model. RUNX2 counts 11 splice variants, alternatively transcribed
from two promoters, and featuring two alternative 3’UTRs. In silico analysis allowed detecting all nt changes between
AMH and Neanderthal/Denisova, within noncoding reg-
ulatory sequences and predicting the effect of changes in the
3’UTRs on miRNA binding. miRNA and RUNX2 splice
isoforms were analyzed by qPCR, during the in vitro
osteogenic differentiation of suture-derived cells. We found
that miRNA expression was modulated during the ossifi-
cation process and correlated with RUNX2 expression. Particularly the isoform containing the second 3'UTR was
apparently stabilized by miRNA binding. In silico protein
modeling showed the presence of an additional DNA-
binding leucine-zipper domain which is lacking in the iso-
form with the first 3’UTR. Our data suggest that RUNX2
genomic evolution may have affected miRNA binding
leading to changes in epigenetic regulation of the gene,
mostly affecting a specific splice isoform. This isoform is
apparently able to bind the target DNA with increased V. Pérez-Grijalba1, M. López1, L. M. Valor2, E. Domínguez-
Garrido1 1Fundación Rioja Salud, Logroño, Spain, 2Hospital
Universitario Puerta del Mar, Cádiz, Spain 1Fundación Rioja Salud, Logroño, Spain, 2Hospital
Universitario Puerta del Mar, Cádiz, Spain Introduction: Rubinstein-Taybi (RTS) is a rare autosomal-
dominant disorder characterized by growth retardation,
cognitive impairment, facial abnormalities and high inci-
dence of neoplasia. CREBBP mutations account for 50-
60% of RTS cases. CBP, encoded by CREBBP, is a tran-
scriptional co-activator with lysine acetyltransferase (KAT)
activity, targeting specific positions on histones H2B
(H2BK15) and H3 (H3K18 and H3K25). The aim of this
study was to assess the relationship of CREBBP mutations
and impaired KAT activity of CBP in RTS patients. Materials and Methods: Histones were purified from
nuclear blood cells of 18 RTS patients with a representative
variety of CREBBP mutations, and 6 age-matched controls. Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy These preliminary findings may also extend epigenetics as a
key factor and potential therapeutic target in RTS. V. Pérez-Grijalba: None. M. López: None. L.M. Valor:
None. E. Domínguez-Garrido: None. P17.57A Post-transcriptional regulation of RUNX2 gene during
evolution: implications in the ossification process in
nonsyndromic craniosynostosis evolution: implications in the ossification process in
nonsyndromic craniosynostosis
L. Di Pietro1, M. Barba1, P. Frassanito1, M. Baranzini1, V. Orticelli1, C. Prampolini1, A. Benitez-Burraco2, W. Lattanzi1
1Università Cattolica del Sacro Cuore, Rome, Italy,
2Universidad de Sevilla, Sevilla, Spain L. Di Pietro1, M. Barba1, P. Frassanito1, M. Baranzini1, V. Orticelli1, C. Prampolini1, A. Benitez-Burraco2, W. Lattanzi1 L. Di Pietro1, M. Barba1, P. Frassanito1, M. Baranzini1, V. Orticelli1, C. Prampolini1, A. Benitez-Burraco2, W. Lattanzi1
1Università Cattolica del Sacro Cuore, Rome, Italy,
2Universidad de Sevilla, Sevilla, Spain E. Giacopuzzi: None. M. Gennarelli: None. C. Sacco:
None. C. Magri: None. A. Barbon: None. 1Università Cattolica del Sacro Cuore, Rome, Italy,
2Universidad de Sevilla, Sevilla, Spain 1Università Cattolica del Sacro Cuore, Rome, Italy,
2Universidad de Sevilla, Sevilla, Spain V. Pérez-Grijalba1, M. López1, L. M. Valor2, E. Domínguez-
Garrido1 A CTCF- dependent chromatin interaction ensures robust
enhancer - promoter communication at the Shh locus XPO1, located in the Immunochip region 2p16.1, mediates
the nuclear export of other proteins and some RNAs. The
SNP rs3087898, in the 5’UTR of the XPO1 transcript, has
been associated to celiac disease (CeD), an immune dis-
order of the small intestine. N’6 methyladenosine (m6A) is
the most frequent methylation mark in mRNAs and
lncRNAs, affecting several aspects of RNA metabolism. Interestingly, XPO1 shows m6A methylation signals in the
5’UTR, very near the associated SNP, which could influ-
ence the function of the protein. Thus, we wanted to
investigate whether the CeD-associated SNP could affect
the methylation levels of XPO1 mRNA and subsequently,
its protein levels. m6A immunoprecipitation in intestinal
cells confirmed the presence of mRNA methylation on the
5’ UTR of XPO1. Moreover, cells heterozygous for the
rs3087898 SNP showed preferential methylation in the
CeD-associated allele. After treatment with cycloleucine, an
m6A inhibitor, both XPO1 mRNA and protein amounts
tend to decrease, suggesting that there will be lower levels
of XPO1 in the individuals with the protection genotype. Concordantly, when we quantified the amounts of XPO1 in
intestinal biopsies from individuals with different geno-
types, we observed that XPO1 levels were higher in the
presence of the risk allele. These data suggest that the SNP
rs3087898, associated with CeD, influences the methylation
levels of XPO1 mRNA and regulates XPO1 protein trans-
lation, which could in turn affect the nuclear export of
certain proteins that have been related with disease devel-
opment as STAT3 or IKBa. Funding: ACM; PI2018007
Basque
Government;
ISCIII
PI13/0120-PI16/0258;
CIBERDEM C. Paliou1, P. Guckelberger1, I. Jerković2, V. Heinrich1, S. Haas1, S. Mundlos1,3,4, G. Andrey1 1Max Planck for Molecular Genetics, Berlin, Germany,
2Institute of Human Genetics, Montpellier, France, 3Institute
for Medical and Human Genetics, Charité Universitätsmedizin,
Berlin, Germany, 4Berlin-Brandenburg Center for
Regenerative Therapies (BCRT), Charité Universitätsmedizin,
Berlin, Germany Sonic Hedgehog (Shh) is expressed in the distal-posterior
part of developing limb buds and controls digit growth,
number and identity. The ZRS enhancer, which regulates
Shh transcription in developing limbs, is located in the
intron 5 of the constitutively transcribed gene Lmbr1 and
has been involved in numerous patient cases with limb
malformations. Shh and the ZRS communicate through a
1Mb-large
stable
chromatin
interaction. However
the
mechanism facilitating this long-range contact is not known
yet. P17.56D
Histone acetylation is reduced in Rubinstein-Taybi
patients: epigenetics role in the syndrome Percentage of acetylation (Ac%) of specific positions
H3K18 and H3K23 was determined by ELISA. Addition-
ally, quantification of H2B15KAc relative to total H2B was
carried out by Western Blot in 6 RTS and 4 controls. Results: RTS patients showed a 35- 40% of the control
acetylation level on H3K18 and H3K25, which implied a
statistically significant reduction compared to controls. More than 70% of the RTS patients showed a consistent
Ac% reduction in the two specific H3 acetylation sites to Abstracts from the 51st European Society of Human Genetics Conference: Posters 627 affinity, thus influencing the expression of bone-specific
genes plausibly involved in "globularization". L. Di Pietro: None. M. Barba: None. P. Frassanito:
None. M. Baranzini: None. V. Orticelli: None. C. Prampolini:
None. A. Benitez-Burraco:
None. W. Lattanzi: None. affinity, thus influencing the expression of bone-specific
genes plausibly involved in "globularization". L. Di Pietro: None. M. Barba: None. P. Frassanito:
None. M. Baranzini: None. V. Orticelli: None. C. Prampolini:
None. A. Benitez-Burraco:
None. W. Lattanzi: None. affinity, thus influencing the expression of bone-specific
genes plausibly involved in "globularization". A CTCF- dependent chromatin interaction ensures robust
enhancer - promoter communication at the Shh locus Using a series of CRISPR/Cas9 engineered alleles and
4C-seq experiments in mouse embryos, we aim to elucidate
the role of the Lmbr1 constitutive transcription as well as
CTCF in the establishment of the Shh-ZRS chromatin
interaction. Deletion of the Lmbr1 promoter abolishes the
transcription over the ZRS, yet 4C-seq experiments showed
that the contact frequency between Shh and its enhancer
remains unchanged in these embryos allowing for normal
Shh expression. In contrast, the removal of CTCF binding
sites around and within the ZRS results in the emergence of
compensatory CTCF binding sites as well as in altered 3D
chromatin architecture, diminished Shh transcription and
skeletal abnormalities. Our results suggest that CTCF acts
to support a robust and permissive enhancer-promoter
interaction that ensures normal Shh expression. This CTCF-
dependent regulatory mechanism provides a framework to
understand the pathomechanism of unsolved structural
variants described at the Shh locus in patients with skeletal
abnormalities. A. Olazagoitia-Garmendia: None. J. Rodriguez: None. J. Bilbao: None. A. Castellanos-Rubio: None. C. Paliou: None. P. Guckelberger: None. I. Jerković:
None. V. Heinrich: None. S. Haas: None. S. Mundlos:
None. G. Andrey: None. An inflammation-related SNP in XPO1 5’UTR regulates
protein amount by altering m6A mRNA methylation L. Di Pietro: None. M. Barba: None. P. Frassanito:
None. M. Baranzini: None. V. Orticelli: None. C. Prampolini:
None. A. Benitez-Burraco:
None. W. Lattanzi: None. A. Olazagoitia-Garmendia1, J. Rodriguez2, J. Bilbao1, A. Castellanos-Rubio1 A. Olazagoitia-Garmendia1, J. Rodriguez2, J. Bilbao1, A.
Castellanos-Rubio1 1UPV-EHU, Biocruces, Ciberdem, Leioa, Spain, 2UPV-EHU,
Leioa, Spain An inflammation-related SNP in XPO1 5’UTR regulates
protein amount by altering m6A mRNA methylation An inflammation-related SNP in XPO1 5’UTR regulates
protein amount by altering m6A mRNA methylation P17.61A miR-146a single nucleotide polymorphism rs2910164
(G>C) regulates miR-146a expression in osteoarthritic
chondrocytes I. Papathanasiou1, E. Mourmoura1, K. Malizos2, A. Tsezou1 C. Paliou: None. P. Guckelberger: None. I. Jerković:
None. V. Heinrich: None. S. Haas: None. S. Mundlos:
None. G. Andrey: None. 1Laboratory of cytogenetics and Medical Genetics, Larissa,
Greece, 2Department of Orthopaedics, Larissa, Greece 1Laboratory of cytogenetics and Medical Genetics, Larissa,
Greece, 2Department of Orthopaedics, Larissa, Greece 628 J. del Picchia Materials and Methods: 9 datasets assessed with
Affymetrix Human Genome U133 Plus 2.0 Array and
published in GEO were selected. Samples with known
radiation
exposure
were
excluded. The
analyzed
set
included normal thyroid tissue (106 samples); tumor tissue
of classic papillary carcinoma (67 samples); follicular
variant
of
papillary
carcinoma
(35
samples);
poorly
differentiated carcinoma (18 samples); anaplastic carcinoma
(45 samples). Datasets of RNAseq from SRA and TCGA
repositories
were
selected
for
validation. Differential
expression of 4398 non-coding genes (including 1982
lincRNA and 1518 antisense RNA) was assessed. Genes
with FDR adjusted p-value ≤0.01 and Fold Change > 2
were considered to be differentially expressed. Introduction: Deregulation of microRNAs is involved in
osteoarthritis (OA) pathogenesis. MiR-146a contributes to
inflammation and cartilage degradation observed in OA
joints. Single polynucleotide polymorphisms in miRNAs
genes may influence miRNAs expression and have often
been associated with diseases. In our study, we investigated
whether miR-146a rs2910164 (G>C) predisposes to OA
susceptibility and its possible functional role to miR-146a
expression in osteoarthritic chondrocytes. Introduction: Deregulation of microRNAs is involved in
osteoarthritis (OA) pathogenesis. MiR-146a contributes to
inflammation and cartilage degradation observed in OA
joints. Single polynucleotide polymorphisms in miRNAs
genes may influence miRNAs expression and have often
been associated with diseases. In our study, we investigated
whether miR-146a rs2910164 (G>C) predisposes to OA
susceptibility and its possible functional role to miR-146a
expression in osteoarthritic chondrocytes. Material and Methods: Genetic association analysis was
performed using a cohort of 900 Greek OA patients and 750
healthy controls. Genomic DNA was extracted from blood
and genotyped using PCR-RFLP. Articular cartilage was
obtained from 25 patients with primary osteoarthritis
undergoing total knee replacement surgery and 15 healthy
individuals with no history of joint disease. Total RNA was
extracted from cultured chondrocytes and expression levels
of miR-146a were evaluated using real-time PCR. Results: In classic PTC 140 lncRNAs were found to be
differentially expressed (including 54 lincRNAs and 44
antisense RNAs). P17.61A In follicular PTC differential expression
was revealed for 88 lncRNAs (including 36 lincRNAs and
29 antisense RNAs). In poorly differentiated thyroid
carcinomas 146 lncRNAs were differentially expressed
(including 62 lincRNAs and 42 antisense RNAs). The
highest amount of differently expressed genes with out-
standing statistical significance was observed in anaplastic
carcinomas: 354 lncRNAs, including 140 lincRNAs and
109 antisense RNAs. In total, 26 lncRNAs were differently
expressed in all histological subtypes. Results: miR-146a rs2910164-GC and CC genotypes
were not associated with an elevated risk of OA compared
to GG genotype. Moreover, we observed that miR-146a
expression levels were significantly decreased in osteoar-
thritic chondrocytes compared to normal and that the
relative expression levels of miR-146a in chondrocytes of
OA patients carrying the rs2910164-GC genotype were
significantly lower than that ones with GG genotype. Conclusion: Common and specific patterns of lncRNA
expression are found in distinct subtypes of thyroid cancer. Conclusion: Our study demonstrates, for the first time,
that
although
miR-146a
rs2910164
(G>C)
is
not
a
susceptibility factor for OA, miR-146a rs2910164-GC
genotype is associated with reduced miR-146a expression
levels in osteoarthritic chondrocytes. These data provide
strong evidence that genetic variations could regulate the
expression levels of microRNAs that are linked to OA
pathogenesis. V. Yakushina: None. A. Lavrov: None. V. Yakushina: None. A. Lavrov: None. Genome-wide microRNA analysis of transient focal
ischemia in rat brain Genome-wide microRNA analysis of transient focal
ischemia in rat brain I. B. Filippenkov1, V. V. Stavchansky1, A. E. Denisova2, L. V. Dergunova1,2, S. A. Limborska1,2 I. Papathanasiou: None. E. Mourmoura: None. K. Malizos: None. A. Tsezou: None. 1Institute of Molecular Genetics, Russian Academy of Sciences,
Moscow, Russian Federation, 2Pirogov Russian National
Research Medical University, Moscow, Russian Federation P17.62B
Differential expression of long non-coding RNAs in distinct
histological type of thyroid cancer V. Yakushina1,2, A. Lavrov1,3 Introduction: Ischemic stroke is one of the most serious
diseases leading to death or disability of the population. The
study of non-coding RNAs in ischemia has exceptional
importance for the development of new strategies for neu-
roprotection and reconstruction of nerve tissues. It is known
that microRNAs can play a role both as neuroprotective
agents and also contribute to pathological processes in
ischemia. The work is devoted to the study of the expres-
sion of miRNAs and their possible mRNA targets in rat
model of cerebral ischemia with reperfusion (rMCAO),
which resembles events in human ischemic stroke. 1Research Centre for Medical Genetics, Moscow, Russian
Federation, 2Moscow Institute of Physics and Technology,
Moscow, Russian Federation, 3Russian National Research
Medical University, Moscow, Russian Federation Introduction: Long non-coding RNAs (lncRNAs) are
assumed to participate in cancer pathogenesis and can be
considered as potential markers or therapeutic targets. Investigations of lncRNA in thyroid cancer are not suffi-
cient and limited mostly to papillary thyroid cancer. 629 Abstracts from the 51st European Society of Human Genetics Conference: Posters Materials
and
Methods:
rMCAO
in
rats,
high-
throughput RNA sequencing (RNA-Seq), real-time RT-
PCR, bioinformatics. Introduction: WNT signaling is fundamental to bone
health and its aberrant activation leads to skeletal patholo-
gies. A heterozygous missense mutation p.C218G in WNT1,
a key WNT pathway ligand, leads to severe early-onset and
progressive osteoporosis with multiple peripheral and spinal
fractures. Results: A comparative analysis of the expression of
microRNAs and mRNAs was performed in subcortical
structures of the brain containing a damage focus in
rMCAO. 24 h after occlusion, the expression level of 407
microRNAs was changed (Fold change>2, padj<0.05). Also, the expression level of mRNAs of 108 genes as
potential targets for those microRNAs was changed. They
included collagen (Col3a1, Col4a1), integrin (Itgb2, Itga5),
dopamine receptor (Drd1, Drd2) mRNAs, and other genes
associated mainly with signaling pathways Focal adhesion
(p = 1.4E-3), ECM-receptor interaction (p = 1.8E-3), Gap
junction (p = 0.015), and others. Subjects and Methods: A cross-sectional cohort study
on 12 mutation-positive (35 years, range 9-59 years) and 12
mutation-negative (35 years, range 9-59 years) subjects
from two Finnish families with a heterozygous p.C218G
WNT1 mutation. Serum samples were screened with a
custom-designed panel of 192 miRNAs using qPCR. Findings were compared between the two groups. Altered microRNA profile in osteoporosis caused by
impaired WNT signaling Altered microRNA profile in osteoporosis caused by
impaired WNT signaling R. E. Mäkitie1, M. Hackl2, R. Niinimäki3, S. Kakko4, J. Grillari5,
O. Mäkitie6 R. E. Mäkitie1, M. Hackl2, R. Niinimäki3, S. Kakko4, J. Grillari5,
O. Mäkitie6 1Folkhälsan Institute of Genetics and University of Helsinki,
Helsinki, Finland, 2TAmiRNA GmbH, Vienna, Austria,
3Department of Children and Adolescents, Oulu University
Hospital, and PEDEGO Research Unit, University of Oulu,
Oulu, Finland, 4Internal Medicine and Clinical Research
Center, University of Oulu, Oulu, Finland, 5Christian Doppler
Laboratory on Biotechnology of Skin Aging, Department of
Biotechnology, BOKU - University of Natural Resources and
Life Sciences Vienna, Vienna, Austria, 6Folkhälsan Institute of
Genetics and University of Helsinki; Children's Hospital,
University of Helsinki and Helsinki University Hospital; and
Center for Molecular Medicine, Karolinska Institutet, and
Clinical Genetics, Karolinska University Hospital, Helsinki,
Finland R.E. Mäkitie: None. M. Hackl: A. Employment (full or
part-time); Significant; TAmiRNA. R. Niinimäki: None. S. Kakko: None. J. Grillari: F. Consultant/Advisory Board;
Modest; TAmiRNA. O. Mäkitie: None. P17.62B
Differential expression of long non-coding RNAs in distinct
histological type of thyroid cancer Results: The pattern of 192 circulating miRNAs is
significantly different in the mutation-positive subjects: two
were
upregulated
(miR-18a-3p,
miR-223-3p)
and
six
downregulated (miR-22-3p, miR-31-5p, miR-34a-5p, miR-
143-5p miR-423-5p, miR-423-3p) in the WNT1 mutation-
positive subjects (p-values=0.001-0.053). Three of these
(miR-22-3p, miR-34a-5p, and miR-31-5p) are known
inhibitors of WNT signaling: miR-22-3p and miR-34a-5p
target WNT1 mRNA and miR-31-5p is predicted to bind to
WNT1 3’UTR. Conclusions: RNA-Seq analysis revealed the active
involvement of microRNAs in the regulation of inflamma-
tion, stress and neurotransmission in rMCAO. Further
analysis of non-coding RNAs will facilitate the under-
standing of the mechanisms of post-transcriptional regula-
tion of gene expression in brain ischemia. Conclusions: RNA-Seq analysis revealed the active
involvement of microRNAs in the regulation of inflamma-
tion, stress and neurotransmission in rMCAO. Further
analysis of non-coding RNAs will facilitate the under-
standing of the mechanisms of post-transcriptional regula-
tion of gene expression in brain ischemia. This work was supported by grant from the Russian
Science Foundation 17-74-10189. I.B. Filippenkov: B. Research Grant (principal investi-
gator, collaborator or consultant and pending grants as well
as grants already received); Modest; Grant from the Russian
Science
Foundation
17-74-10189. V.V. Stavchansky:
None. A.E. Denisova: None. L.V. Dergunova: None. S. A. Limborska: None. I.B. Filippenkov: B. Research Grant (principal investi-
gator, collaborator or consultant and pending grants as well
as grants already received); Modest; Grant from the Russian
Science
Foundation
17-74-10189. V.V. Stavchansky:
None. A.E. Denisova: None. L.V. Dergunova: None. S. A. Limborska: None. Conclusions: The miRNA profile reflects WNT1 muta-
tion status. The results suggest that WNT1 mutation disrupts
a feed-back regulation between these miRNAs and WNT1,
providing new insights into the pathogenesis of WNT-
related bone disorders. These miRNAs could offer future
potential in diagnosis and treatment of osteoporosis. Grant references: Sigrid Jusélius Foundation, Folkhäl-
san Research Foundation, Academy of Finland, Foundation
for Pediatric Research, Helsinki University Research Funds,
Swedish Research Council, Novo Nordisk Foundation,
Helsinki University Hospital (KLTO), Finnish Medical
Foundation,
Jalmari
and
Rauha
Ahokas
Foundation,
Swedish Childhood Cancer Foundation, Stockholm County
Council (ALF project). Age-dependent patterns of X chromosome DNA
methylation in the elderly Age-dependent patterns of X chromosome DNA
methylation in the elderly 1Unit of Human Genetics, Department of Clinical Research,
University of Southern Demark, Odense M, Denmark,
2Epidemiology and Biostatistics, Department of Public Health,
University of Southern Demark, Odense M, Denmark Analysis of bones ancient mtDNA from the medieval
archeological site of Amiternum (L’Aquila), Italy Analysis of bones ancient mtDNA from the medieval
archeological site of Amiternum (L’Aquila), Italy P18 Genetic epidemiology/Population genetics/Statistical
methodology and evolutionary genetics S. Li1, Q. Tan2 Fernandez: None. Z. De Barbieri: None. L. Carvajal-Carmona:
None. J. Cazier:
None. D.F. Newbury: None. S. Li: None. Q. Tan: None. S. Li: None. Q. Tan: None. S. Li1, Q. Tan2 Taking advantage of large sample
sizes of DNA methylation data available on the elderly, we
performed X-chromosome-wide association study on X-
linked DNA methylation changes during aging by analyzing
male and female samples separately and comparing the rate
of change in DNA methylation between sexes to identify
significant CpGs sites differentially or consistently methy-
lated in males and females. We detected 2300 significant
CpGs
(False
Discovery
Rate<0.05)
displaying
sex-
independent methylation patterns and 286 CpGs showing
sex-dependent methylation changes in the Lothian birth
cohort born in 1936 in Scotland (732 males with mean age
71.27 and 732 females with mean age 71.23). As a repli-
cation approach, a similar analysis was performed in an
independent sample of middle aged Danish twins (226
males with mean age 67.26 and 226 females with mean age
66.04) reporting 136 CpGs consistently and 59 CpGs dif-
ferentially methylated in males and females with over-
lapping rates of 38.08% and 5.1% with the discovery
results. In conclusion, we have shown significant DNA
methylation patterns of aging on the X-chromosome
dominated by sex-independent regulation highly replicable
in independent samples. descended from the original founder families. The geo-
graphical isolation of RCI means few outsiders have
migrated to the island. As a result, islanders share a high
degree of consanguinity (14.9%), and most islander unions
are at least second cousins (Villanueva et al 2014). RCI islanders show an exceptionally high occurrence rate
of developmental language disorder, 10-fold the rate seen in
mainland
Chile. Near
complete
islander
genealogical
records have shown that 90% of affected children are direct
descendants of a pair of original founder brothers, who
likely carried a susceptibility variant for language disorders
(Villanueva et al 2014). To understand the genetic contribution to developmental
language disorder on RCI, we have investigated the
underlying population structure of the islanders. Extensive
pedigree data suggest the current islander population are of
recently admixed European and Chilean backgrounds. Recent
studies
have
shown
a
higher
proportion
of
indigenous South American ancestry in the mainland
Chilean population than previously thought (Lorenzo-
Bermejo et al 2017), and this may therefore be directly
relevant to the RCI population. Using high density genome-wide SNP genotyping data
from 154 islanders, and whole genome sequencing from 24
of the most distantly related islanders, we have performed
the first in-depth genetic analysis of the population structure
of RCI. H.S. Mountford: None. P. Villanueva: None. L. Jara:
None. M.A. S. Li1, Q. Tan2 1Unit of Human Genetics, Department of Clinical Research,
University of Southern Demark, Odense M, Denmark,
2Epidemiology and Biostatistics, Department of Public Health,
University of Southern Demark, Odense M, Denmark 630 J. del Picchia The age-dependent patterns of DNA methylation in the
elderly population have been intensively studied on the
autosomal
chromosomes,
revealing
large
numbers
of
genomic sites under significant epigenetic remodeling dur-
ing the aging process. However, DNA methylation changes
on the X-chromosomes have been routinely ignored due to
analytical difficulties in dealing with difference in X chro-
mosome content of females and males and X chromosome
inactivation in females. Taking advantage of large sample
sizes of DNA methylation data available on the elderly, we
performed X-chromosome-wide association study on X-
linked DNA methylation changes during aging by analyzing
male and female samples separately and comparing the rate
of change in DNA methylation between sexes to identify
significant CpGs sites differentially or consistently methy-
lated in males and females. We detected 2300 significant
CpGs
(False
Discovery
Rate<0.05)
displaying
sex-
independent methylation patterns and 286 CpGs showing
sex-dependent methylation changes in the Lothian birth
cohort born in 1936 in Scotland (732 males with mean age
71.27 and 732 females with mean age 71.23). As a repli-
cation approach, a similar analysis was performed in an
independent sample of middle aged Danish twins (226
males with mean age 67.26 and 226 females with mean age
66.04) reporting 136 CpGs consistently and 59 CpGs dif-
ferentially methylated in males and females with over-
lapping rates of 38.08% and 5.1% with the discovery
results. In conclusion, we have shown significant DNA
methylation patterns of aging on the X-chromosome
dominated by sex-independent regulation highly replicable
in independent samples. The age-dependent patterns of DNA methylation in the
elderly population have been intensively studied on the
autosomal
chromosomes,
revealing
large
numbers
of
genomic sites under significant epigenetic remodeling dur-
ing the aging process. However, DNA methylation changes
on the X-chromosomes have been routinely ignored due to
analytical difficulties in dealing with difference in X chro-
mosome content of females and males and X chromosome
inactivation in females. P18 Genetic epidemiology/Population genetics/Statistical
methodology and evolutionary genetics A. M. G. Poma1,2, O. Zarivi1,2, S. Colafarina1,2, G. Vecchiotti1,2,
P. Piccone3, A. Iannarelli3, F. Savini1,4, F. Redi1,4 Prenatal diagnostics of anencephaly in the Czech Republic:
20 years of population wide surveillance Prenatal diagnostics of anencephaly in the Czech Republic:
20 years of population wide surveillance A. Sipek1,2,3, V. Gregor1,2, J. Jirova4, A. Sipek Jr1,5, M. Maly6,7,
J. Klaschka6,8 1Department of Medical Genetics, Thomayer Hospital, Prague,
Czech Republic, 2Department of Medical Genetics, Pronatal
Sanatorium, Prague, Czech Republic, 3Institute of Medical
Genetics, Third Faculty of Medicine, Charles University,
Prague, Czech Republic, 4Institute of Health Information and
Statistics of the Czech Republic, Prague, Czech Republic,
5Institute of Biology and Medical Genetics, First Faculty of
Medicine, Charles University, Prague, Czech Republic,
6Institute of Computer Science of the Czech Academy of
Sciences, Prague, Czech Republic, 7National Institute of Public
Health, Prague, Czech Republic, 8Institute of Biophysics and
Informatics, First Faculty of Medicine, Charles University,
Prague, Czech Republic P18.02B
Investigating the population structure of Robinson Crusoe
Island, Chile Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic, grant AZV 17-29622A. V. Gregor: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. J. Jirova: None. A. Sipek Jr: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic Grant nr. AZV 17-29622A. M. Maly:
B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. J. Klaschka: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. A. Sipek: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic, grant AZV 17-29622A. V. Gregor: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Significant; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. J. Jirova: None. A. Sipek Jr: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic Grant nr. AZV 17-29622A. M. Maly:
B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. J. Klaschka: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. P18.02B
Investigating the population structure of Robinson Crusoe
Island, Chile The study of the
haplogroups, the analysis of genetic variability and the
studies of phylogeny on the obtained sequences show a
genetic proximity between individuals of Amiternum, the
current population of north/central Italy and the Germanic
tribe of Longobards, which dominated the Italian peninsula
between 568 and 774 A. D. The match of ancient
Byzantines sequences with one of the Amiternum samples
highlights also a Byzantine genetic trait in the populations
of Amiternum and L’Aquila. Grants RIA Univaq 2017 to
Poma A and Redi F Results: During the selected 20 years a total of 592 cases
of anencephaly were diagnosed (2.89 cases per 10 000 live
births). Only 27 of those cases were diagnosed in births
(0.13 per 10 000), the rest of cases were diagnosed
prenatally (565 cases, 2.76 per 10 000). The average
gestation week at the time of diagnosis was 17.21 in 1996
and 13.00 in 2015. The average maternal age changed from
23.68 years in 1996 to 28.64 in 2015. Results and Conclusions: This work provides prelimin-
ary information on the correlation between the inhabitants
of Amiternum and the Longobards populations of northern
Italy and the Byzantines, migrant peoples transited and/or
allocated in the territory of Amiternum. The study of the
haplogroups, the analysis of genetic variability and the
studies of phylogeny on the obtained sequences show a
genetic proximity between individuals of Amiternum, the
current population of north/central Italy and the Germanic
tribe of Longobards, which dominated the Italian peninsula
between 568 and 774 A. D. The match of ancient
Byzantines sequences with one of the Amiternum samples
highlights also a Byzantine genetic trait in the populations
of Amiternum and L’Aquila. Grants RIA Univaq 2017 to
Poma A and Redi F Discussion: The majority of anencephaly cases are
diagnosed prenatally in the Czech Republic. The overall
percentage of terminations of pregnancy is high (95.44%). We also observed an improvement in ultrasound based
prenatal diagnosis along the study period, leading to a
significant decrease in the average gestation week at the
time of diagnosis. A.M.G. Poma: None. O. Zarivi: None. S. Colafarina:
None. G. Vecchiotti:
None. P. Piccone:
None. A. Iannarelli: None. F. Savini: None. F. Redi: None. Acknowledgements: Supported by Ministry of Health of
the Czech Republic, grant AZV 17-29622A A. Sipek: B. R. van de Putte1, C. H. W. Wijers1, H. Reutter2, C. L. M.
Marcelis1, E. Brosens3, P. M. A. Broens4, E. Jenetzky5,6, C. E. J. P18.02B
Investigating the population structure of Robinson Crusoe
Island, Chile 1University of L'Aquila, L'Aquila, Italy, 2Dept of Life Health
and Environmental Sciences, L'Aquila, Italy, 3ARTA Abruzzo,
Regional Agency for Environmental Protection, L’Aquila,
Italy, L'Aquila, Italy, 4Department of Human Sciences,
L'Aquila, Italy H. S. Mountford1, P. Villanueva2, L. Jara2, M. A. Fernandez2, Z. De Barbieri2, L. Carvajal-Carmona3, J. Cazier4, D. F. Newbury1 H. S. Mountford1, P. Villanueva2, L. Jara2, M. A. Fernandez2, Z. De Barbieri2, L. Carvajal-Carmona3, J. Cazier4, D. F. Newbury1 1Oxford Brookes University, Oxford, United Kingdom,
2University of Chile, Santiago, Chile, 3UC Davis, Davis, CA,
United States, 4University of Birmingham, Birmingham, United
Kingdom 1Oxford Brookes University, Oxford, United Kingdom,
2University of Chile, Santiago, Chile, 3UC Davis, Davis, CA,
United States, 4University of Birmingham, Birmingham, United
Kingdom Introduction: The study of ancient DNA allows to analyze
genetic relationships between individuals and populations
of the past and the present. In this work we analyzed human
bones remains datable between the 6th-9th century D.C. from burials of the archaeological site of Amiternum,
L’Aquila. Robinson Crusoe (RCI) is a geographically and socially
isolated island located 670km west of San Antonio, Chile. It
was founded in 1876 by a group of eight founder families. It
is now home to over 800 inhabitants, most of whom are Materials and Methods: As a genetic marker, the
hypervariable 1 region of mitochondrial DNA (HVR1) Abstracts from the 51st European Society of Human Genetics Conference: Posters 631 has been chosen. The HVR1 marker has been amplified by
PCR, the amplicon have been cloned and sequenced. Sequences of the HVR1 region were compared with
Anderson's sequence for the identification of polymorph-
isms. The data obtained were analyzed with different
software and phylogenetic methods. For inter-populations
comparisons, the known sequences in literature and found
in ancient and modern databases have been used. delivery. Our goal was to analyse the efficiency of prenatal
diagnosis of this congenital anomaly in the Czech Republic. Methods:
We
present
retrospective
epidemiological
analysis of the prevalence of anencephaly in the Czech
Republic. We used population based data from the National
Registry of Congenital Anomalies, stored in the Institute of
Health Information and Statistics of the Czech Republic
(1996 - 2015 time period). Results and Conclusions: This work provides prelimin-
ary information on the correlation between the inhabitants
of Amiternum and the Longobards populations of northern
Italy and the Byzantines, migrant peoples transited and/or
allocated in the territory of Amiternum. A. Sipek1,2,3, V. Gregor1,2, J. Jirova4, A. Sipek Jr1,5, M. Maly6,7,
J. Klaschka6,8 Genetic and molecular analysis of urinary magnesium
concentration in Scottish and Croatian populations 1Radboudumc, Nijmegen, Netherlands, 2University of Bonn,
Bonn, Germany, 3Erasmus Medical Centre, Rotterdam,
Netherlands, 4University Medical Center Groningen,
Groningen, Netherlands, 5German Cancer Research Center,
Heidelberg, Germany, 6Johannes-Gutenberg University,
Mainz, Germany, 7Maastricht University Medical Centre,
Maastricht, Netherlands 1Radboudumc, Nijmegen, Netherlands, 2University of Bonn,
Bonn, Germany, 3Erasmus Medical Centre, Rotterdam,
Netherlands, 4University Medical Center Groningen, C. B. Joseph1, C. Drake1, C. M. Stanton1, T. S. Boutin1, O. Devuyst2, T. Hurd1, C. Hayward1 1MRC Human Genetics Unit, Institute of Genetics and
Molecular Medicine, Edinburgh, United Kingdom, 2Institute of
Physiology, Zurich Center for Integrative Human Physiology,
Zurich, Switzerland 1MRC Human Genetics Unit, Institute of Genetics and
Molecular Medicine, Edinburgh, United Kingdom, 2Institute of
Physiology, Zurich Center for Integrative Human Physiology,
Zurich, Switzerland Mainz, Germany, 7Maastricht University Medical Centre,
Maastricht, Netherlands Introduction: Anorectal malformation (ARM) is a rare
birth defect, resulting from disturbed development of the
hindgut. Although suspected, evidence of a genetic etiology
is still scarce. The aim of this study is to identify rare
variants in ARM etiology. Introduction: Electrolyte imbalance, including changes in
magnesium concentration and other electrolyte ratios in the
body can result in dizziness, arrhythmia and if left untreated
can result in serious illness or even death. Magnesium is the
second most abundant bivalent cation in the body and is
essential for many cellular processes. Renal magnesium
handling plays an important role in maintaining magnesium
homeostasis, however the exact biological mechanisms
remain unclear. A recent genome-wide association study
(GWAS) identified an association between urinary magne-
sium concentration (uMg) and variants in the ARL15 gene
on chromosome 5. ARL15 encodes a GTP-binding protein
that interacts with the magnesium transporter channel
TRPM6, and other proteins involved in magnesium home-
ostasis in physiologically relevant cell lines. Materials and Methods: We genotyped 568 Caucasian
ARM patients and 1,860 population-based controls using
the Illumina ExomeChip, which contains >240,000 rare
coding variants. GenomeStudio clustering and calling was
followed by re-calling of ‘no-calls’ using Zcall, for patients
and controls simultaneously. Single variant analyses were
performed
to
identify
statistically
significant
variants
(Bonferroni corrected threshold p<1.15*10-6). Based on
the calling quality in the clusterplots and distribution of
minor alleles for the variants among patients and controls,
variants
were
identified
for
validation
using
Sanger
Sequencing. Materials and Methods: We genotyped 568 Caucasian
ARM patients and 1,860 population-based controls using
the Illumina ExomeChip, which contains >240,000 rare
coding variants. Genetic and molecular analysis of urinary magnesium
concentration in Scottish and Croatian populations GenomeStudio clustering and calling was
followed by re-calling of ‘no-calls’ using Zcall, for patients
and controls simultaneously. Single variant analyses were
performed
to
identify
statistically
significant
variants
(Bonferroni corrected threshold p<1.15*10-6). Based on
the calling quality in the clusterplots and distribution of
minor alleles for the variants among patients and controls,
variants
were
identified
for
validation
using
Sanger
Sequencing. Materials and Methods: We conducted meta-analyses
for urinary magnesium, magnesium to creatinine (uMg/cre)
ratio, as well as magnesium concentration to other clinically
relevant electrolyte ratios in 11, 617 individuals from
Scottish and Croatian populations to determine possible
genes involved in these traits. Results: In total, 13 single variants reached statistical
significance. However, the majority of minor alleles for
these variants were absent in controls, and some patients
showed 3 or more minor alleles for these 13 variants,
which is highly unlikely. We identified the 3 most
promising candidate variants with acceptable calling
quality that remained statistically significant in the single
variant analysis after exclusion of patients with 3 or more
minor alleles. However, Sanger sequencing did not
confirm presence of the minor alleles for the variants in
patients as indicated based on ExomeChip data. Results: We observed genome-wide significant peaks in
the ARL15 locus in our meta-analyses conducted for uMg,
magnesium to creatinine (uMg/cre), magnesium to phos-
phate (uMg/uPh) and magnesium to potassium (uMg/uK)
ratios. The top SNP associated with uMg and uMg/cre in
the meta-analyses lies within a transcription factor binding
site in an enhancer region of ARL15. Conclusions: We did not find evidence for associations
between ARM and rare coding variants present on the
ExomeChip. Furthermore, we would like to emphasize the
importance of validation of ExomeChip data before large
replication studies are initiated. Conclusions: We hypothesise that the variant found
affects the expression of ARL15, which, in turn modulates
magnesium transport. We are performing functional studies
to elucidate the role ARL15 plays in magnesium home-
ostasis both in vitro and in vivo. R. van de Putte: None. C.H.W. Wijers: None. H. Reutter: None. C.L.M. Marcelis: None. E. Brosens:
None. P.M.A. Broens: None. E. Jenetzky: None. C.E.J. Sloots: None. T.E. Galesloot: None. I. de Blaauw: None. H.G. Brunner: None. N. Roeleveld: None. I.A.L.M. van
Rooij: None. C.B. Joseph: None. C. Drake: None. C.M. Stanton:
None. T.S. Boutin: None. O. Devuyst: None. T. Hurd:
None. C. Hayward: None. P18.06B Rare coding variants and the risk of congenital anorectal
malformations: an exome chip association study Rare coding variants and the risk of congenital anorectal
malformations: an exome chip association study Introduction: Anencephaly is a lethal congenital anomaly
from the group of neural tube defects. Because of its dis-
tinctive phenotype, it is always diagnosed, either during
pregnancy by ultrasonography, or clinically after the 632 J. del Picchia Sloots3, T. E. Galesloot1, I. de Blaauw1, H. G. Brunner1,7, N. Roeleveld1, I. A. L. M. van Rooij1 Identification of incompletely penetrant variants by a
population genetic approach Genetic and molecular analysis of urinary magnesium
concentration in Scottish and Croatian populations Genetic and molecular analysis of urinary magnesium
concentration in Scottish and Croatian populations P18.10B Identification of incompletely penetrant variants by a
population genetic approach Abstracts from the 51st European Society of Human Genetics Conference: Posters 633 Á. Mikó1,2, A. Kaposi1, K. Schnabel1, D. Seidl1, K. Tory1,2
1MTA-SE Lendület Nephrogenetic Laboratory, Hungarian
Academy of Sciences, Budapest, Hungary, 2First Department
of Pediatrics, Semmelweis University, 1083, Budapest,
Hungary Á. Mikó1,2, A. Kaposi1, K. Schnabel1, D. Seidl1, K. Tory1,2 1Institute of Human Genetics, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
2Department of Genomics, Life&Brain Center, University of
Bonn, Bonn, Germany, 3Institute for Medical Biometry,
Informatics, and Epidemiology, University of Bonn, School of
Medicine & University Hospital Bonn, Bonn, Germany,
4Department of Medicine II, Sana Klinikum, Offenbach,
Germany, 5Department of Internal Medicine II, Evangelisches
Krankenhaus, Düsseldorf, Germany, 6Department of Visceral,
Transplant, Thoracic and Vascular Surgery, University
Hospital of Leipzig, Leipzig, Germany, 7Department of
General, Visceral and Transplantation Surgery, University of
Heidelberg, Heidelberg, Germany, 8Department of General,
Visceral and Thoracic Surgery, University Medical Center
Hamburg-Eppendorf, University of Hamburg, Hamburg,
Germany, 9Department of Gastroenterology, Radboud
University Nijmegen Medical Center, Nijmegen, Netherlands,
10Department of Interdisciplinary Endoscopy, University
Hospital Hamburg-Eppendorf, Hamburg, Germany 1MTA-SE Lendület Nephrogenetic Laboratory, Hungarian
Academy of Sciences, Budapest, Hungary, 2First Department
of Pediatrics, Semmelweis University, 1083, Budapest,
Hungary Autosomal recessive (AR) disorders are typically com-
pletely penetrant. We recently identified the first variant
(NPHS2, p.R229Q), that is pathogenic only with specific
trans-associated mutations. We aimed to identify other
incompletely penetrant variants (IPV) in AR disorders. We
collected clinical and genotype data from 9.283 Caucasian
patients with biallelic HGMD mutations in 19 genes
responsible for frequent AR disorders (ASL, ATP7B,
CAPN3, CFTR, CTNS, DHCR7, GAA, GALNS, GALT,
IDUA, MUT, NPHS1, NPHS2, PAH, PCCB, PKHD1,
PMM2, SLC26A4, TYR) in the medical literature. Variants
that were less frequent in the patient population than in the
general European non-Finnish population of gnomAD were
considered non-pathogenic and were excluded (n=28). The
penetrance of each pathogenic variant (n=2141) was cal-
culated as its enrichment in the patient population as com-
pared to that of the loss-of-function (LOF) mutations. DHCR7 and PMM2 LOF mutations were underrepresented
in the patient population suggesting in utero lethality. Out
of the 82 variants that were frequent enough to assess their
penetrance, we found 23 (28%) ASL, CAPN3, CFTR, GAA,
NPHS2, PAH and PKHD1 variants to be incompletely
penetrant, including the known IPVs (NPHS2 R229Q,
CFTR R117H and L997F). P18.10B No other but the R229Q variant
was subject of interallelic interactions. Based on the asso-
ciated phenotype, most of the IPVs are hypomorphic (16/
23, 70%) vs. 19/59, 32% of completely penetrant variants,
p = 0.0002). Frequent IPVs can be identified by this com-
puterized population-genetic approach and are more com-
mon than expected. Proper genetic counseling requires their
knowledge. This work was supported by MTA-SE Lendulet
Research Grant (LP2015-11/2015). Barrett’s esophagus (BE) is a metaplasia at the lower eso-
phagus and a precancerous condition for the development of
esophageal adenocarcinoma (EA). The etiology of both BE
and EA is multifactorial. A recent GWAS meta-analysis has
led to the identification of 14 susceptibility loci for BE/EA. The aim of the present study was (i) to link the genome-
wide genetic data of the meta-analysis with gene expression
levels through expression quantitative trait loci (eQTLs) and
(ii) to analyse identified eQTL-SNPs in an independent
cohort. For this, the meta-analysis data were systematically
integrated with the eQTL data from the GTEx tissues from
gastroesophageal junction and esophageal mucosa. We
selected SNPs that (i) revealed a p-value of 5×10-5>p>5×10-
8 in the BE/EA meta-analysis and (ii) were an eQTL in at
least one of the esophageal GTEx tissues. Here, we
identified 35 eQTLs. Of these, 32 were integrated into an
independent genotyping study, consisting of 1,406 BE/EA
cases and 992 controls. Following quality control, in which
9 SNPs were excluded, four SNPs showed a nominal
significant association. In a next step, we performed a fixed-
effect meta-analysis. The best association P-value was
observed for the SNP rs37050130 (p = 9.22×10-8), an
eQTL for AF131215.9, which is an uncharacterized gene. The second best associated eQTL mapped to SLC22A3,
encoding an organic cation transporter that improves
chemotherapy drug absorption. Á. Mikó: None. A. Kaposi: None. K. Schnabel: None. D. Seidl: None. K. Tory: None. P18.11C Barrett’s esophagus and esophageal adenocarcinoma:
systematic integration of eQTL data and candidate loci
association study J. Schröder1,2, V. Schüller3, N. Ishorst1,2, N. Fricker1,2, M.
Knapp3, A. May4, C. Gerges5, N. Kreuser6, T. Schmidt7, Y.
Vashist8, W. H. M. Peters9, H. Neuhaus5, T. Rösch10, C. Ell4, M.
M. Nöthen1,2, I. Gockel6, J. Schumacher1, A. C. Böhmer1,2 Potential adaptive role of the human-specific duplication at
16p11.2 in iron homeostasis and immune response M. Kaakinen1, R. Mägi2, V. Lagou3,4, A. Claringbould5, K. Gaulton6, BIOS consortium, K. Fischer2, A. P. Morris7, I. Prokopenko1 M. Kaakinen1, R. Mägi2, V. Lagou3,4, A. Claringbould5, K. Gaulton6, BIOS consortium, K. Fischer2, A. P. Morris7, I. Prokopenko1 G. Giannuzzi1,2, D. Risso3, X. Nuttle3, E. Porcu1,2, 16p11.2
Consortium, G. Willemin1,4, K. Hoekzema3, J. Chrast1, Y. Herault5, Z. Kutalik6,2, R. Bernier7, E. Eichler3,8, A. Reymond1 G. Giannuzzi1,2, D. Risso3, X. Nuttle3, E. Porcu1,2, 16p11.2
Consortium, G. Willemin1,4, K. Hoekzema3, J. Chrast1, Y. 1Imperial College London, London, United Kingdom,
2University of Tartu, Tartu, Estonia, 3VIB Center for Brain &
Disease Research, Leuven, Belgium, 4KU Leuven, Leuven,
Belgium, 5University Medical Centre Groningen, Groningen,
Netherlands, 6Stanford University, Stanford, CA, United
States, 7University of Liverpool, Liverpool, United Kingdom 1Imperial College London, London, United Kingdom,
2University of Tartu, Tartu, Estonia, 3VIB Center for Brain &
Disease Research, Leuven, Belgium, 4KU Leuven, Leuven,
Belgium, 5University Medical Centre Groningen, Groningen,
Netherlands, 6Stanford University, Stanford, CA, United
States, 7University of Liverpool, Liverpool, United Kingdom Herault5, Z. Kutalik6,2, R. Bernier7, E. Eichler3,8, A. Reymond1 1Center for Integrative Genomics, University of Lausanne,
Lausanne, Switzerland, 2Swiss Institute of Bioinformatics,
Lausanne, Switzerland, 3Department of Genome Sciences,
University of Washington School of Medicine, Seattle, WA,
United States, 4Mouse Metabolic Evaluation Facility, Center
for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland, 5Institut de Génétique et de Biologie Moléculaire
et Cellulaire, Illkirch, France, 6Institute of Social and
Preventive Medicine, Lausanne University Hospital (CHUV),
Lausanne, Switzerland, 7Department of Psychiatry and
Behavioral Sciences, University of Washington, Seattle, WA,
United States, 8Howard Hughes Medical Institute, University
of Washington, Seattle, WA, United States 1Center for Integrative Genomics, University of Lausanne,
Lausanne, Switzerland, 2Swiss Institute of Bioinformatics,
Lausanne, Switzerland, 3Department of Genome Sciences,
University of Washington School of Medicine, Seattle, WA,
United States, 4Mouse Metabolic Evaluation Facility, Center
for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland, 5Institut de Génétique et de Biologie Moléculaire
et Cellulaire, Illkirch, France, 6Institute of Social and
Preventive Medicine, Lausanne University Hospital (CHUV),
Lausanne, Switzerland, 7Department of Psychiatry and
Behavioral Sciences, University of Washington, Seattle, WA,
United States, 8Howard Hughes Medical Institute, University
of Washington, Seattle, WA, United States Serum lipid levels and obesity share biochemical pathways,
suggesting overlapping causal genetic factors. Genome-
wide association studies (GWAS) of correlated phenotypes
have been developed to identify such shared genetic effects
with increased power. Barrett’s esophagus and esophageal adenocarcinoma:
systematic integration of eQTL data and candidate loci
association study J. Schröder1,2, V. Schüller3, N. Ishorst1,2, N. Fricker1,2, M. Knapp3, A. May4, C. Gerges5, N. Kreuser6, T. Schmidt7, Y. Vashist8, W. H. M. Peters9, H. Neuhaus5, T. Rösch10, C. Ell4, M. M. Nöthen1,2, I. Gockel6, J. Schumacher1, A. C. Böhmer1,2 Our study is the first systematic approach that identifies
specific eQTLs at BE/EA candidate loci and it provides first
insights into the biological mechanism contributing to the
development of BE/EA. J. Schröder: None. V. Schüller: None. N. Ishorst:
None. N. Fricker: None. M. Knapp: None. A. May: J. Schröder: None. V. Schüller: None. N. Ishorst:
None. N. Fricker: None. M. Knapp: None. A. May: 634 J. del Picchia None. C. Gerges: None. N. Kreuser: None. T. Schmidt:
None. Y. Vashist: None. W.H.M. Peters: None. H. Neuhaus: None. T. Rösch: None. C. Ell: None. M.M. Nöthen: None. I. Gockel: None. J. Schumacher: None. A. C. Böhmer: None. The MP-GWAS is a powerful approach to detect shared
genetic effects on correlated phenotypes, as demonstrated
by our analysis of lipids and BMI. Funding: EU-FP7-MARVEL (PIEF-GA-2013-626461),
WT205915. C. Böhmer: None. P18.12D
Multi-phenotype genome-wide meta-analysis of lipid levels
and BMI in 64,736 Europeans suggests shared genetic
architecture
M. Kaakinen1, R. Mägi2, V. Lagou3,4, A. Claringbould5, K. Gaulton6, BIOS consortium, K. Fischer2, A. P. Morris7, I. Prokopenko1 M. Kaakinen: None. R. Mägi: None. V. Lagou: None. M. Kaakinen: None. R. Mägi: None. V. Lagou: None. A Claringbould: None K Gaulton: None K Fischer: M. Kaakinen: None. R. Mägi: None. V. Lagou: None. A. Claringbould: None. K. Gaulton: None. K. Fischer:
None. A.P. Morris: None. I. Prokopenko: None. E. Grechanina, Y. Grechanina, L. Molodan, D. Oleinyk, A.
Zabelina E. Grechanina, Y. Grechanina, L. Molodan, D. Oleinyk, A. Zabelina Potential adaptive role of the human-specific duplication at
16p11.2 in iron homeostasis and immune response Similarly,
mouse models carrying the 16p11.2 orthologous deletion Abstracts from the 51st European Society of Human Genetics Conference: Posters 635 Conclusions: 15q11.2 (BP1-BP2) deletions and duplica-
tions are common findings among affected and unaffected
populations, suggesting a low pathogenicity of these CNVs. and,
more
specifically,
Bola2
knockout
mice
show
significantly lower blood iron and hemoglobin levels than
their wild-type littermates. Corroboratingly, gene dosage of
the 16p11.2 BP4-BP5 CNV is positively associated with
blood lymphocyte count in the UK biobank cohort
(P=0.007). and,
more
specifically,
Bola2
knockout
mice
show
significantly lower blood iron and hemoglobin levels than
their wild-type littermates. Corroboratingly, gene dosage of
the 16p11.2 BP4-BP5 CNV is positively associated with
blood lymphocyte count in the UK biobank cohort
(P=0.007). I. Maya: None. M. Shohat: None. S. Kahana: None. S. Yacobson: None. T. Tenne: None. I. Agmon-Fishman:
None. L. Basel-Salmon: None. R. Sukenik-Halevy: None. Taken together, our results suggest a role of the genes
mapping to this human-specific duplication in iron home-
ostasis and lymphocyte level and a potential adaptation in
improving iron metabolism and/or immune response. Potential adaptive role of the human-specific duplication at
16p11.2 in iron homeostasis and immune response p
We performed a multi-phenotype GWAS (MP-GWAS)
on three blood lipids (high-/low-density lipoprotein choles-
terol and triglycerides, HDL-C/LDL-C/TG) and body-mass
index (BMI) in 22 European-ancestry studies (N=64,736)
imputed to the 1000 Genomes reference panel (Phase 1). We fitted a “reverse regression” model between each single-
nucleotide variant (SNV) and the linear combination of
HDL-C/LDL-C/TG/BMI using the SCOPA software, i.e. for the ith variant SNVi=β1i×HDL-C+β2i×LDL-C+β3i×TG
+β4i×BMI+εi, where εi~N(0,ơ2). Study-specific effect sizes
and variance-covariance matrices for each variant were
combined in a meta-analysis using the META-SCOPA
software. Recurrent pathogenic copy-number variation (CNV) at
human chromosome 16p11.2 is mediated by Homo sapiens-
specific duplications of a segment including the BOLA2/2B
and SLX1A/1B genes. This segment is copy-number variant
in humans (3-8 copies per genome) and its duplication is
almost fixed most likely due to positive selection. BOLA2-
GLRX3 heterotrimers participate in iron homeostasis. SLX1, together with SLX4, MUS81 and EME1, forms a
complex involved in the control of genome stability. SLX4
mutations cause Fanconi anemia, characterized by the
inability to produce blood cells. We identified 14 novel and 41/9 established lipid/BMI
loci, respectively, at genome-wide significance (P < 5x10-8). Nine novel (SDC1, SLC8A1, EPHA6, SPATA4, MAGI2,
CTSB, BC014119, SMCO4 and CNTN5) and 32 established
loci showed effects on both BMI and lipids in the joint
model, suggesting shared genetic architecture. This obser-
vation was supported through hierarchical cluster analysis,
which resulted in six clades representing a mixture of lipid-
and BMI-associated variants. We detected significant eQTL
effects in whole blood, subcutaneous/visceral fat and liver
at 16 of the identified loci, and enrichment of association
signals at HDAC6 binding sites, indicating a critical role of
associated loci in various cellular events. To gain insights into the potential adaptive role of this
human-specific duplication, we analyzed hematological and
blood iron data in 16p11.2 deletion carriers with varying
copies of this segment. We found that 50% of individuals
with the lowest number of copies, i.e. three, need iron
supplementation and/or are anemic, and 4 out of 6 have low
lymphocyte counts. When compared to individuals with
more copies, i.e. four and five (n=14), the frequency of the
iron phenotype was not significantly different (P=0.13)
while the low lymphocyte count was (P=0.004). Study of the character of psychiatric symptoms with
hereditary metabolic diseases. Symptom complex and
comorbidity G. Giannuzzi: None. D. Risso: None. X. Nuttle: None. E. Porcu: None. G. Willemin: None. K. Hoekzema:
None. J. Chrast: None. Y. Herault: None. Z. Kutalik:
None. R. Bernier:
None. E. Eichler:
None. A. Reymond: None. E. Grechanina, Y. Grechanina, L. Molodan, D. Oleinyk, A. Zabelina What is the significance of 15q11.2 BP1-BP2 deletions and
duplications? Introduction: F.Sedel's classification of CMD according to
the type of psychiatric symptoms in disease onset allows to
adequately estimate the nature of polyorganous affections,
clarify the diagnosis and, normalizing metabolism, relieve
the patient from psychiatric disorders. I. Maya1, M. Shohat2, S. Kahana3, S. Yacobson1, T. Tenne4, I. Agmon-Fishman5, L. Basel-Salmon1, R. Sukenik-Halevy4 I. Maya1, M. Shohat2, S. Kahana3, S. Yacobson1, T. Tenne4, I. Agmon-Fishman5, L. Basel-Salmon1, R. Sukenik-Halevy4 The Objective: to study CMD spectrum, accompanied by
psychiatric disorders. 11Recanati Genetic Institute, Rabin Medical Center, Petah
Tikva, Israel, 2Asuta Genetic Institute,,Ashdod, Israel, Results: among first applied 132,309 patients, 1056
nosological forms were revealed. Children with mental
retardation - 863 cases - 0.65% among the first-applied, and
81% among the nosological forms. Psychiatric diseases - in
the case of urea formation cycle defects, methionine
remineralization defects, porphyria. Chronic psychiatric
symptoms - in patients with homocystinuria, Wilson's
disease, adrenal leukodystrophy and lysosomal accumula-
tion diseases. Homocystinuria, spinal-tendinous xanthoma-
tosis, non-ketone hyperglycinemia, monoamine oxidase
insufficiency, insufficient dehydrogenase of half-aldehyde
of succinic acid and creatine transporter deficiency - in
patients with mild mental retardation and behavioral
changes. The greatest number of patients with these changes
- in porphyria and methionine remethylation disorder. In
severe cases of tryptophan, methionine, heme metabolism,
comprehensive
treatment
has
allowed
to
get
mental
recovery. Tikva, Israel, 2Asuta Genetic Institute,,Ashdod, Israel,
3Recanati Genetic Institute, Rabin Medical Center, Petah
Tikva, Israel, Israel, 4Meir Medical Center, Kfar Saba, Israel,
5M1Recanati Genetic Institute, Rabin Medical Centerl, Petah
Tikva, Israel 3Recanati Genetic Institute, Rabin Medical Center, Petah Tikva, Israel, Israel, 4Meir Medical Center, Kfar Saba, Israel, Tikva, Israel, Israel, 4Meir Medical Center, Kfar Saba, Israel,
5M1Recanati Genetic Institute, Rabin Medical Centerl, Petah
Tikva, Israel 5M1Recanati Genetic Institute, Rabin Medical Centerl, Petah
Tikva, Israel Purpose: To assess the detection rate of copy number
variations (CNVs) of 15q11.2 (BP1-BP2) in relation to
indications for testing, and other clinical characteristics. Purpose: To assess the detection rate of copy number
variations (CNVs) of 15q11.2 (BP1-BP2) in relation to
indications for testing, and other clinical characteristics. Methods: We analyzed 11,004 chromosomal microarray
tests performed in a single referral lab during a four-year
period (7,596 prenatal, 3,408 postnatal). Methods: We analyzed 11,004 chromosomal microarray
tests performed in a single referral lab during a four-year
period (7,596 prenatal, 3,408 postnatal). Results: Deletions were detected in 66 cases (0.6%); 39
in prenatal tests (0.51%) and 27 in postnatal tests (0.79%). P18.15C Study of the character of psychiatric symptoms with
hereditary metabolic diseases. Symptom complex and
comorbidity F. Thieme1,2, L. Henschel1,2, N. Ishorst1,2, E. Mangold1, A.
Hoischen3,4,5, K. U. Ludwig1,2 F. Thieme1,2, L. Henschel1,2, N. Ishorst1,2, E. Mangold1, A. Hoischen3,4,5, K. U. Ludwig1,2 1Institute of Human Genetics, University Hospital Bonn, Bonn,
Germany, 2Department of Genomics, Life&Brain Center,
Bonn, Germany, 3Department of Human Genetics, Radboud
University Medical Center, Nijmegen, Netherlands,
4Department of Internal Medicine, Radboud University
Medical Center, Nijmegen, Netherlands, 5Radboud Institute for
Molecular Life Sciences, Radboud University Medical Center,
Nijmegen, Netherlands 1Institute for Molecular Medicine Finland FIMM, University of
Helsinki, Helsinki, Finland, 2Stanley Center for Psychiatric
Research, The Broad Institute of Harvard and MIT,
Cambridge, MA, United States, 3Psychiatric and
Neurodevelopmental Genetics Unit, Department of Psychiatry,
Massachusetts General Hospital, Boston, MA, United States,
4National Institute for Health and Welfare, Helsinki, Finland Copy Number Variations (CNVs) are associated with a
myriad of syndromic and severe developmental disorders,
often
overlapping
developmentally
important
genes. Usually considered high-impact variants, CNVs are never-
theless also observed in the general population, where their
impact is poorly characterised and knowledge of their
impact to health and well-being is limited. To investigate
the effect of CNVs on the health, risk for neurodevelop-
mental phenotypes and socioeconomic and educational
attainment in the population in general, we employ long-
itudinal health record data together with genotypic infor-
mation from 20 098 individuals in the FINRISK population
cohort. Phenotype information includes: neurodevelop-
mental diagnoses from health record data spanning over 35
years; use of select classes of prescription drugs (data
spanning 20 years); and socioeconomic factors reported on
participation forms. Genotypic information entails intensity
and allelic frequency data retrieved using 542 585 probes in
the Illumina HumanCoreExome DNA MicroArray. CNVs
are identified using both PennCNV and iPsychCNV,
focusing initially on CNVs identified by both algorithms. All calls will be manually curated for validity. We have
analysed the dataset with PennCNV, the iPsychCNV ana-
lysis and phenotype association analyses are ongoing. In
our preliminary analysis, carrying a large deletion (>1Mb)
increased the risk for a neurodevelopmental or neu-
ropsychiatric phenotype almost 2-fold (18.6% vs 9.8%). For
intellectual disability, the risk ratio was 9.6 (2.3% vs
0.24%). We will also perform enrichment analyses for
neurodevelopmental phenotypes, use of prescription drugs,
socioeconomic status, educational attainment and overall
health status. The genetic etiology of complex traits is largely character-
ized by associations of common risk variants located in
non-coding regions. These regions might also harbor puta-
tive causative, rare variants in individual families, which
can be identified by targeted resequencing using smMIPs. E. Grechanina: None. Y. Grechanina: None. L.
Molodan: None. D. Oleinyk: None. A. Zabelina: None. None. O. Pietiläinen:
None. M. Daly:
None. A. Palotie: None. None. O. Pietiläinen:
None. M. Daly:
None. A. Palotie: None. What is the significance of 15q11.2 BP1-BP2 deletions and
duplications? Duplications were detected in 94 cases (0.85%); 62 in
prenatal tests (0.82%), 32 in postnatal tests (0.94%). The
prevalence of deletions and duplications among clinically-
indicated prenatal tests was 0.57% and 0.9% respectively,
which was not statistically different from tests performed
without an indication; 0.49% and 0.78% (p = 0.6, 0.59,
respectively). The prevalence of deletions and duplications
among postnatal tests performed due to a clinical indication
was 0.73% and 1%, respectively, which was similar to the
prevalence in healthy individuals; 0.98% and 0.74%,
respectively; (p = 0.497, 0.67). There was no difference
between fetal sex, the prevalence of additional CNVs and
the size of the deletion/duplication involving 15q11.2
between indicated and unindicated tests. Results: Deletions were detected in 66 cases (0.6%); 39
in prenatal tests (0.51%) and 27 in postnatal tests (0.79%). Duplications were detected in 94 cases (0.85%); 62 in
prenatal tests (0.82%), 32 in postnatal tests (0.94%). The
prevalence of deletions and duplications among clinically-
indicated prenatal tests was 0.57% and 0.9% respectively,
which was not statistically different from tests performed
without an indication; 0.49% and 0.78% (p = 0.6, 0.59,
respectively). The prevalence of deletions and duplications
among postnatal tests performed due to a clinical indication
was 0.73% and 1%, respectively, which was similar to the
prevalence in healthy individuals; 0.98% and 0.74%,
respectively; (p = 0.497, 0.67). There was no difference
between fetal sex, the prevalence of additional CNVs and
the size of the deletion/duplication involving 15q11.2
between indicated and unindicated tests. Conclusions: since 2007 - the conduction of selective
screening for methionine metabolism, investigating the
polymorphisms of MTHFR, MTRR, MTR and RFC1 folate
carrier. 11 579 patients were examined in whom heredity is
burdened by cardiovascular, psychiatric, oncological dis-
orders. Population
frequency of homozygotes
C677T
MTHFR - 7.04%, among patients - 10.1%; the allele
frequency of MTRR A66G - 57%. These features increase
the chances of combining any form of CMD with folate-
methionine cycle disorder. Currently we determine comor-
bidity frequency. 636 J. del Picchia E. Grechanina: None. Y. Grechanina: None. L. Molodan: None. D. Oleinyk: None. A. Zabelina: None. F. Thieme: None. L. Henschel: None. N. Ishorst: None.
E.
Mangold:
None.
A.
Hoischen:
None.
K.U.
Ludwig: None. Targeted resequencing of non-coding risk loci using single-
molecule molecular inversion probes (smMIPs) E. Saarentaus1, M. Kurki1,2,3, L. Urpa1, A. S. Havulinna1,4, M. Perola4, V. Salomaa4, M. Peltonen4, O. Pietiläinen2, M. Daly2,3,
A. Palotie1,2,3 P18.20D Significant; A European DNA bank for deciphering the
missing heritability of Alzheimer's disease (EADB). I.B. Mathijssen: A. Employment (full or part-time); Significant;
Clinical Genetics, Academic Medical Center, Amsterdam. D. Posthuma: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; NWO Vidi, NWO
Aspasia grant, Scott Fuller Memorial Award from the
International Behavior Genetics Association, NWO VICI. H.J. Meijers-Heijboer: B. Research Grant (principal
investigator, collaborator or consultant and pending grants
as well as grants already received); Significant; NWO Vidi,
NutsOhra, CCA/V-ICI, Dutch Cancer Society, Erasmus MC
Revolving Fund “Top down". P18.20D
Genetic homogeneity in DARWIN, an isolated population
in the Netherlands E. S. van Walree1,2, I. E. Jansen2,3, I. B. Mathijssen1, D. Posthuma2, H. J. Meijers-Heijboer1,4 1Department of Clinical Genetics, Academic Medical Center,
Amsterdam, Netherlands, 2Department of Complex Trait
Genetics, Center for Neurogenomics and Cognitive Research,
VU University, Amsterdam, Netherlands, 3Department of
Neurology, Alzheimer Center, Amsterdam Neuroscience, VU
University Medical Center, Amsterdam, Netherlands,
4Department of Clinical Genetics, VU University Medical
Center, Amsterdam, Netherlands Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe Introduction: Isolated populations have the potential to
facilitate gene mapping of complex traits. The number of
haplotypes shared among inhabitants is smaller, while the
length of the shared haplotypes is longer, as they have been
derived from a handful of relatively recent founders. Together with genetic drift and restricted migration this
leads to the enrichment of rare alleles and genetic homo-
geneity. Combined
with
environmental
homogeneity
resulting from a comparable lifestyle, population isolation
increases power to detect genetic risk loci. Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe M. Brkušanin1, J. Peović1, S. Perić2, J. Radvanszky3, S. Mairević1, V. Kovčić1, Z. Musova4, K. Stehlíková5, L. Leonardis6,
K. Kekou7, V. Jovanović8, G. Brajuković1, L. P. Ranum9,10,11, V. Rakočević Stojanović2, D. Savić-Pavićević1 1University of Belgrade-Faculty of Biology, Centre for Human
Molecular Genetics, Belgrade, Serbia, 2University of
Belgrade-School of Medicine, Neurological Clinic, Clinical
Centre of Serbia, Belgrade, Serbia, 3Institute for Clinical and
Translational Research, Biomedical Research Centre, Slovak
Academy of Sciences, Bratislava, Slovakia, 4Department of
Biology and Medical Genetics, Charles University, 2nd
Faculty of Medicine and University Hospital Motol, Prague,
Czech Republic, 5Centre of Molecular Biology and Gene
Therapy, University Hospital Brno, Brno, Czech Republic,
6Institute of Clinical Neurophysiology, University Clinical
Center, Ljubljana, Slovenia, 7Department of Medical Genetics,
University of Athens, "Aghia Sophia Children's Hospital",
Athens, Greece, 8University of Belgrade-Institute for
Biological Research “Sinia Stanković, Department of Genetic
Research, Belgrade, Serbia, 9Center for NeuroGenetics,
University of Florida, Gainesville, FL, United States,
10Department of Molecular Genetics & Microbiology, College
of Medicine, University of Florida, Gainesville, FL, United
States, 11Department of Neurology, University of Florida,
Gainesville, FL, United States Methods and Results: The DARWIN (DNA Analysis in
Residents Within an Isolate in the Netherlands) village is an
isolated population (~22.000 inhabitants) that was founded
in the 14th century by 7-20 families, and remained in
religious and cultural isolation ever since. By using whole
genome sequencing data of 20 DARWIN trio’s, we show
that the DARWIN population has a high inbreeding
coefficient, expressed as FROH: the percentage of the
autosomal genome that is
in runs
of homozygosity
(ROH). DARWIN shows features of ancient inbreeding
(FROH 3.2% with an ROH threshold of 0.5 Mb) and recent
inbreeding (FROH 1.3% with a threshold of 5 Mb). These
numbers are ~6-14 times greater than the FROH of the
overall Dutch population, represented by 748 trio’s from the
Genome of the Netherlands study. F. Thieme1,2, L. Henschel1,2, N. Ishorst1,2, E. Mangold1, A.
Hoischen3,4,5, K. U. Ludwig1,2 These are 75 bp-long molecules composed of two region-
specific primers that are linked by a common backbone
sequence. Upon contact with genomic DNA, smMIPs form
a circular DNA fragment around the target region. Barcoded
primers enable multiplexing, and unique molecular tags in
the smMIPs backbone reduce the number of PCR artifacts
in downstream analyses. In a first assay, 19 non-coding
candidate regions at four risk loci for non-syndromic cleft
lip with/without cleft palate were selected for resequencing
in 1,061 cases and 1,591 controls. Data analysis included
smMIP-design by MIPGEN, read alignment with BWA and
variant calling with UnifiedGenotyper. Downstream anno-
tation was based on rarity of variants, annotation scores
(e.g. CADD, FATHMM-MKL, LINSIGHT, DANN, and
Eigen), and co-segregation analyses in family members. We
also
assessed
concordance
rates
between
genotypes
obtained by array genotyping and smMIPs, and identified a
concordance of 100% for each of three analyzed common
SNPs in 1,400 samples, after filtering out low-quality
smMIPs variant calls. First annotation results show varying
degrees of correlation between the different scores, sug-
gesting that no single score is sufficient for assessing the
potential pathogenicity of variants. We suggest that smMIPs
resequencing is a powerful tool for targeted sequence ana-
lysis beyond the protein-coding regions, in large cohorts, in
a cost-efficient way. F. Thieme: None. L. Henschel: None. N. Ishorst: None. E. Mangold:
None. A. Hoischen:
None. K.U. Ludwig: None. M. Kurki: None. L. Urpa: None. A.S. Havulinna:
None. M. Perola: None. V. Salomaa: None. M. Peltonen: M. Kurki: None. L. Urpa: None. A.S. Havulinna:
None. M. Perola: None. V. Salomaa: None. M. Peltonen: Abstracts from the 51st European Society of Human Genetics Conference: Posters 637 Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe Introduction: Down, Edwards and Patau syndrome are the
most common chromosomal abnormalities diagnosed dur-
ing the prenatal diagnostics. The major goal of this study
was to identify any possible trends that can be associated
with the gradual implementation of non-invasive prenatal
genetic testing in the Czech Republic. Introduction: Down, Edwards and Patau syndrome are the
most common chromosomal abnormalities diagnosed dur-
ing the prenatal diagnostics. The major goal of this study
was to identify any possible trends that can be associated
with the gradual implementation of non-invasive prenatal
genetic testing in the Czech Republic. Materials and Methods: CL3N122, CL3N99, CL3N59,
CL3N119, CL3N19 and CL3N23 loci were genotyped in
401 individuals from Serbian, Greek, Slovenian, Slovakian
and Czech DM2 families. 320 healthy and 79 DM2
haplotypes phased by family segregation analysis, and 53
DM2 published German haplotypes were used for the
coalescent modeling of intra-allelic variability in DMLE+. The maximum likelihood estimation (MLE) of the mutation
age was determined for the population growth rate set at
0.025-0.05, assuming DM2 mutation frequency in Finland
(1/1830). Methods:
The
population-based
data
on
prenatally
diagnosed cases were obtained from the National Registry
of Congenital Anomalies of the Czech Republic (2012-2016
time period). The proportion of the number of the three
major autosomal trisomies was compared to the proportion
of all other chromosomal aberrations that were identified
during prenatal diagnostics. The annual number of live
births in the Czech Republic was used as a denominator. The ratios were subjected to the logistic regression. Statistical evaluation was carried out by the statistical
software R. Results: The estimated DM2 mutation age is 170-280
generations (~3400-5600 years assuming 20 years/genera-
tion). It dates back to the Late Neolith and the early Bronze
Age when massive migrations of Yamnaya individuals from
the Pontic-Caspian steppe to Europe occurred (3500-2200
BCE), accompanied by an expansion of the Corded Ware
individuals (2800-2200 BCE). Results: Down, Edwards and Patau syndromes together
presented 62.6% of all prenatally diagnosed chromosomal
aberrations. The overall frequency (per 10 000 live births)
of major autosomal trisomies have increased from 30.86 in
2012 to 37.41 in 2016 (Poisson regression: p = 0.014). Also
the proportion of major autosomal trisomies increased from
57.22 % (of all prenatally diagnosed aberrations) in 2012 to
66.09 % in 2016 (logistic regression: p < 0.001). Conclusion: Presented results bring a novel insight into
the origin and spreading of DM2 mutation throughout
Europe. Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe According to epidemiological data, distribution of
the DM2 mutation seems to reflect a decreasing Yamnaya
ancestry from the north to the south in the present-day
Europeans. Discussion: We confirmed that the overall frequency of
major
autosomal
trisomies
(and
their
proportion)
is
increasing. The possible explanations are the improving
methods of prenatal diagnostics and also the increasing
maternal age in the Czech Republic. Acknowledgements:
Grant
No. 173016,
MESTD,
Republic of Serbia. M. Brkušanin: None. J. Peović: None. S. Perić: None. J. Radvanszky: None. S. Mairević: None. V. Kovčić:
None. Z. Musova: None. K. Stehlíková: None. L. Leonardis: None. K. Kekou: None. V. Jovanović: None. G. Brajuković: None. L.P. Ranum: None. V. Rakočević
Stojanović: None. D. Savić-Pavićević: None. Acknowledgements: Supported by Ministry of Health of
the Czech Republic, grant AZV 17-29622A A. Sipek Jr: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic Grant nr. AZV 17-29622A . V. Gregor: B. Research Grant (principal investigator, colla-
borator or consultant and pending grants as well as grants
already received); Significant; Ministry of Health of the
Czech Republic Grant nr. AZV 17-29622A. J. Klaschka:
B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. M. Maly: B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. A. Sipek: B. Research
Grant (principal investigator, collaborator or consultant and
pending
grants
as
well
as
grants
already
received);
Significant; Ministry of Health of the Czech Republic
Grant nr. AZV 17-29622A. A. Sipek Jr: B. Research Grant (principal investigator,
collaborator or consultant and pending grants as well as
grants already received); Significant; Ministry of Health of
the Czech Republic Grant nr. AZV 17-29622A . V. Gregor: B. Research Grant (principal investigator, colla-
borator or consultant and pending grants as well as grants
already received); Significant; Ministry of Health of the
Czech Republic Grant nr. AZV 17-29622A. J. Klaschka:
B. Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. M. Maly: B. P18.22B
Changing frequencies of main autosomal trisomies in the
Czech Republic: population-based data A. Sipek Jr1,2, V. Gregor2,3, J. Klaschka4,5, M. Maly4,6, A. Sipek2,3,7 1Institute of Biology and Medical Genetics, First Faculty of
Medicine, Charles University, Prague, Czech Republic,
2Department of Medical Genetics, Thomayer Hospital, Prague,
Czech Republic, 3Department of Medical Genetics, Pronatal
Sanatorium, Prague, Czech Republic, 4Institute of Computer
Science of the Czech Academy of Sciences, Prague, Czech
Republic, 5Institute of Biophysics and Informatics, First
Faculty of Medicine, Charles University, Prague, Czech
Republic, 6National Institute of Public Health, Prague, Czech
Republic, 7Institute of Medical Genetics, Third Faculty of
Medicine, Charles University, Prague, Czech Republic Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe Research Grant (principal investigator, collaborator or
consultant and pending grants as well as grants already
received); Modest; Ministry of Health of the Czech
Republic Grant nr. AZV 17-29622A. A. Sipek: B. Research
Grant (principal investigator, collaborator or consultant and
pending
grants
as
well
as
grants
already
received);
Significant; Ministry of Health of the Czech Republic
Grant nr. AZV 17-29622A. Age, origin and spreading of the myotonic dystrophy type 2
mutation throughout Europe Furthermore, the FROH is
equal to or even higher than that of previously described
population isolates, such as Sardinia. Methods and Results: The DARWIN (DNA Analysis in
Residents Within an Isolate in the Netherlands) village is an
isolated population (~22.000 inhabitants) that was founded
in the 14th century by 7-20 families, and remained in
religious and cultural isolation ever since. By using whole
genome sequencing data of 20 DARWIN trio’s, we show
that the DARWIN population has a high inbreeding
coefficient, expressed as FROH: the percentage of the
autosomal genome that is
in runs
of homozygosity
(ROH). DARWIN shows features of ancient inbreeding
(FROH 3.2% with an ROH threshold of 0.5 Mb) and recent
inbreeding (FROH 1.3% with a threshold of 5 Mb). These
numbers are ~6-14 times greater than the FROH of the
overall Dutch population, represented by 748 trio’s from the
Genome of the Netherlands study. Furthermore, the FROH is
equal to or even higher than that of previously described
population isolates, such as Sardinia. Conclusion: The DARWIN village is a population isolate
whose genetic homogeneity offers great potential for future
genetic studies. Conclusion: The DARWIN village is a population isolate
whose genetic homogeneity offers great potential for future
genetic studies. Introduction: Myotonic dystrophy type 2 (DM2) is mainly
a disease of the European Caucasians. Haplotype analyses
suggested a founder mutation ~4000-11000 years old,
which coincides with the Neolithic Age, when the Near
Eastern farmers, and afterwards herders from the Pontic-
Caspian steppe, had settled in Europe, shaping the genomic
architecture of the Europeans. We aimed to estimate the age Funding: Amsterdam Neuroscience, Alliance projects Funding: Amsterdam Neuroscience, Alliance projects Funding: Amsterdam Neuroscience, Alliance projects E.S. van Walree: B. Research Grant (principal investi-
gator, collaborator or consultant and pending grants as well
as grants already received); Significant; Amsterdam Neu-
roscience, Alliance projects. I.E. Jansen: B. Research
Grant (principal investigator, collaborator or consultant and
pending
grants
as
well
as
grants
already
received); E.S. van Walree: B. Research Grant (principal investi-
gator, collaborator or consultant and pending grants as well
as grants already received); Significant; Amsterdam Neu-
roscience, Alliance projects. I.E. Jansen: B. Research
Grant (principal investigator, collaborator or consultant and
pending
grants
as
well
as
grants
already
received); 638 J. del Picchia and
origin
of
DM2
mutation
and
reconstruct
its
dispersal route. J. F. Martini, M. P. Bidondo, S. P. Duarte, R. Liascovich, P.
Barbero, B. Groisman Conclusion: The results don’t confirm the effect of
elevated inbreeding as couse of small Ne/N in the
Lithuanian population. However, more detailed analysis is
needed. This work supported by the LITGEN project (VP1-3.1-
ŠMM-07-K-01-013), funded by the European Social Fund
under the Global Grant Measure. A. Urnikyte:
None. A. Molytė:
None. V. Kučinskas: None. A. Urnikyte:
None. A. Molytė:
None. V. Kučinskas: None. Conclusions: DS prevalence in Argentina is similar to
that reported previously in the region. A higher prevalence
and percentage of prenatal diagnosis was observed in
private hospitals. The association between DS and lower
birth weight is consistent with previous findings. Knowl-
edge of the epidemiological characteristics will allow the
implementation of health policies for this pathology. J. F. Martini, M. P. Bidondo, S. P. Duarte, R. Liascovich, P.
Barbero, B. Groisman J. F. Martini, M. P. Bidondo, S. P. Duarte, R. Liascovich, P. Barbero, B. Groisman Red Nacional de Anomalías Congénitas (RENAC), Centro
Nacional de Genética Médica, ANLIS, Ministerio de Salud de
la Nación, Buenos Aires, Argentina Introduction: Down syndrome (DS) is the most common
chromosomal disorder in children. Purpose: To describe the
epidemiologic characteristics of DS in Argentina based on
data from the National Network of Congenital Anomalies
(RENAC). Methods: 295 randomly selected DNA samples were
genotyped using the Illumina 770K HumanOmniExpress-
12v1.0 array. The Ne values were obtained for 50
generations assuming a generation time of 25 years by
using inferred long segments of identity by descent (IBD)
from high-density genotyping data. The inbreeding coeffi-
cient in generation t was estimated as Materials and Methods: The prevalence of DS by
province and by maternal age for the period 2009-2015 was
calculated, and compared between healthcare system sectors
(private vs. public) and complexity levels of public
hospitals. The proportion of cases with prenatal diagnosis
was established. The association between DS and lower
birth weight and gestational age was analyzed. Materials and Methods: The prevalence of DS by
province and by maternal age for the period 2009-2015 was
calculated, and compared between healthcare system sectors
(private vs. public) and complexity levels of public
hospitals. The proportion of cases with prenatal diagnosis
was established. The association between DS and lower
birth weight and gestational age was analyzed. Results: The estimated Inbreeding coefficient range from
0.00209 in generation 50 to 1.305 ·10-6 in generation one,
with average value of 0.000645. As was expected we
observed Ft decrease as Ne increased. Statistically sig-
nificant difference between Ft values of generations and
average value was not found (p = 0.5964, Single sample
Wilcoxon Test). Results: From 1,358,158 births examined, 2,344 cases of
DS were registered. The prevalence -expressed per 10,000
births- was 17.26; among provinces it varied between 10.99
and 23.71. By maternal age, the prevalence ranged from
10.32 in <20 years, to 158.06 in those ≥45. In public
hospitals the prevalence was 16.25 and 21.55 in private
hospitals,
prenatal
detection
was
14.1%
and
30.5%
respectively. In high complexity public hospitals the
prevalence was 17.45 and 14.62 in hospitals with lower
complexity. There was a negative correlation between birth
weight and DS (B:-362.22, p<0.01). The mean gestational
age was 0.28 weeks lower in DS newborns than in the
general population. Abstracts from the 51st European Society of Human Genetics Conference: Posters 639 The eQTLs Catalog and LinDA browser: a platform for
prioritising target genes of GWAS variants The eQTLs Catalog and LinDA browser: a platform for
prioritising target genes of GWAS variants The eQTLs Catalog and LinDA browser: a platform for
prioritising target genes of GWAS variants 1Dipartimento di Scienze Biomediche - Università degli Studi
di Sassari, Sassari, Italy, 2Istituto di Ricerca Genetica e
Biomedica - Consiglio Nazionale delle Ricerche, Cagliari,
Italy J.F. Martini: None. M.P. Bidondo: None. S.P. Duarte:
None. R. Liascovich: None. P. Barbero: None. B. Groisman: None. A. Urnikyte, A. Molytė, V. Kučinskas Vilnius University, Vilnius, Lithuania P18.23C
Epidemiology of Down syndrome in Argentina Introduction: In previous studies, we have reported low
effective population size (Ne) and census population size
(N) ratio in the Lithuanian population. The estimates of Ne
were approximately one-tenth of the Lithuanian census size
compared with other genetics-based estimates of between
0.21–0.65. Natural levels of fluctuations such as variance in
size, reproduction, sex ratio, the degree to which genera-
tions overlap, and possibly inbreeding caused the small
values of Ne/N. The main interest of this study was to
evaluate the potential impact of small Ne/N on inbreeding,
estimating the inbreeding coefficient (Ft) for 50 generations
from effective population size data of Lithuania. S. Onano1,2, F. Cucca1,2, M. Pala2 Relationship between effective population size and
inbreeding in the Lithuanian population The expression Quantitative Traits Loci (eQTLs) are genetic
polymorphisms associated with changes in gene expression
levels. They have been successfully used to prioritize the
target genes of the variants associated with complex traits
and diseases (GWAS variants). Up to date a few eQTLs Relationship between effective population size and
inbreeding in the Lithuanian population Vilnius University, Vilnius, Lithuania Vilnius University, Vilnius, Lithuania 640 J. del Picchia databases exist and they collect only a small portion of the
available datasets. physiological cardiac conduction traits. From an analysis of
4342 subjects from the Cooperative Health Research In
South Tyrol study, we reported association of two Junction
Plakoglobin (JUP) SNPs with P wave length and one
Desmoplakin (DSP) SNP with QRS interval. We thus planned to build the largest publically available
catalog of eQTLs, coupled with a browser, to optimize and
simplify their interrogation. We collected and manually curated 51 eQTL public
studies ranging from 2007 to date, corresponding to more
than 100 sample types and 25 human populations for a total
of 259176 cis-eQTLs and 32929 genes with at least one cis-
eQTL (cis-eGenes). Most of the eQTLs studies were
conducted in blood samples from healthy individuals of
European ancestry. We found that for 93% of the known
protein-coding genes were eGenes, 20% of them intersect-
ing (r2≥0.8) with the NHGRI-EBI GWAS Catalog and 26%
of whom considered as druggable. Furthermore, for those
GWAS variants for which an eGene was known, we found
that the NHGRI-EBI GWAS Catalog proposed the same
gene as candidate target only for the 60% of the times. Material and Methods: In this subsequent study, we
assessed replication in the MICROS (N=636), an indepen-
dent study from the same geographical region, and in the
Northern Germany SHIP/SHIP Trend (N=3797) studies. We characterized the variants and their genomic context
using the SCREEN/ENCODE tool, the GTeX and methyla-
tion QTL (meQTL) databases, and Framingham Heart
Study meQTL data (N=4170). We tested few biological
hypotheses using two-sample Mendelian randomization
(MR) based on Wald estimator, with standard errors
computed via delta method. Results: The DSP rs2744389-QRS association was
replicated in MICROS (p = 0.010) but not in SHIP/SHIP
Trend (p = 0.505), suggesting genetic heterogeneity. We
did not replicate the P wave associations. Falling in the DSP
promoter, the rs2744389 is an eQTL of the antisense RNA
RP3-512B11.3, and a meQTL of cg02643433. Bioinformatic and mendelian randomization analyses
characterize variants in desmosomal genes associated with
ECG traits in the general population Bioinformatic and mendelian randomization analyses
characterize variants in desmosomal genes associated with
ECG traits in the general population Conclusions: While needing to understand the reasons
for the partial lack of replication, MR results suggest an
antisense RNA-mediated mechanism of DSP expression
control to be validated with laboratory experiments. Conclusions: While needing to understand the reasons
for the partial lack of replication, MR results suggest an
antisense RNA-mediated mechanism of DSP expression
control to be validated with laboratory experiments. L. Foco1, F. Del Greco M1, C. Fuchsberger1, M. Gögele1, F. Murgia1, T. Huan2,3, D. B. Levy2,3, A. Teumer4,5, M. Dörr5,6, G. Schmidt7, A. Rossini1, P. P. Pramstaller1,8,9, C. Pattaro1 L. Foco: None. F. Del Greco M: None. C. Fuchsberger:
None. M. Gögele: None. F. Murgia: None. T. Huan:
None. D.B. Levy: None. A. Teumer: None. M. Dörr:
None. G. Schmidt:
None. A. Rossini:
None. P.P. Pramstaller: None. C. Pattaro: None. L. Foco: None. F. Del Greco M: None. C. Fuchsberger:
None. M. Gögele: None. F. Murgia: None. T. Huan:
None. D.B. Levy: None. A. Teumer: None. M. Dörr:
None. G. Schmidt:
None. A. Rossini:
None. P.P. Pramstaller: None. C. Pattaro: None. Murgia1, T. Huan2,3, D. B. Levy2,3, A. Teumer4,5, M. Dörr5,6, G. Schmidt7, A. Rossini1, P. P. Pramstaller1,8,9, C. Pattaro1 1Institute for Biomedicine, Bolzano, Italy, 2Framingham Heart
Study, Framingham, MA, United States, 3The Population
Studies Branch, National Heart, Lung, and Blood Institute of
the National Institutes of Health, Bethesda, MD, United States,
4Institute for Community Medicine, University Medicine
Greifswald, Greifswald, Germany, 5DZHK (German Center for
Cardiovascular Research), partner site Greifswald,
Greifswald, Germany, 6Department of Internal Medicine B,
University Medicine Greifswald, Greifswald, Germany,
7Medizinische Klinik und Poliklinik, Klinikum rechts der Isar,
Technische Universität München, Munich, Germany,
8Department of Neurology, General Central Hospital, Bolzano,
Italy, 9Department of Neurology, University of Lübeck, Lübeck,
Germany Relationship between effective population size and
inbreeding in the Lithuanian population MR analysis
showed evidence of a causal
effect
of cg02643433
methylation on QRS (p = 0.001), of RP3-512B11.3 expres-
sion on QRS (p = 0.030), but lacking evidence of a causal
effect of RP3-512B11.3 expression on cg02643433 methy-
lation (p = 0.090). Our eQTL-Catalog can be used as a reference to measure
the degree of novelty for future eQTLs studies; it is
provided within a platform with a web interface (LinDA)
that we plan to implement with other types of quantitative
traits (i.e. epigenetic, proteomic, metabolomics and micro-
biota) to better dissect the pleiotropy of the GWAS variants. S Onano: None F Cucca: None M Pala: None S. Onano: None. F. Cucca: None. M. Pala: None. 1Department of Molecular Biotechnology and Health Sciences,
University of Turin, Turin, Italy, 2Genome Biology Unit,
European Molecular Biology Laboratory, Heidelberg,
Germany, 3Department of Medical and Molecular Genetics,
King’s College, London, United Kingdom, 4Center for
Translational Genomics and Bioinformatics, San Raffaele
Scientific Institute, Milan, Italy P18.27C
A functional strategy to characterize expression
Quantitative Trait Loci E. Grassi1, E. Mariella1, M. Forneris2, F. Marotta1, M. Catapano3, I. Molineris4, P. Provero1,4 1Department of Molecular Biotechnology and Health Sciences,
University of Turin, Turin, Italy, 2Genome Biology Unit,
European Molecular Biology Laboratory, Heidelberg,
Germany, 3Department of Medical and Molecular Genetics,
King’s College, London, United Kingdom, 4Center for
Translational Genomics and Bioinformatics, San Raffaele
Scientific Institute, Milan, Italy Introduction: Arrhythmogenic cardiomyopathy is caused
by mutations in desmosomal genes. We previously tested
association of common variants in such genes with Abstracts from the 51st European Society of Human Genetics Conference: Posters 641 Introduction: Expression Quantitative Trait Loci (eQTL)
studies represent an important step in the functional char-
acterization of genetic variants that have been associated
with diseases by Genome Wide
Association Studies
(GWAS). We propose a new strategy to detect eQTLs
leveraging the idea that changes in gene expression often
pass through the alteration of transcription factors (TFs)
binding on regulatory regions. at-onset (AO), including within some families. We hypo-
thesized that candidate genes associated with TTR path-
ways, such as C1QA and C1QC, might act as genetic
modifiers of AO in TTR-FAP Val30Met Portuguese
patients. Subjects & Methods: We analysed DNA samples of 267
patients (117 families). To search for variants, all exons and
flanking regions were genotyped by automated bidirectional
sequencing. We used generalized estimating equations
(GEEs) to take into account the non-independency of AO
among relatives. Intensive in silico analyses were per-
formed, using various software to assess miRNAs target
sites, splicing sites, transcription factor binding sites
alterations and gene-gene interactions. Material and Methods: The Total Binding Affinity
(TBA) measures the propensity of a TF to bind a sequence. In each model, we compute TBA values for 640 human TFs
on a regulatory region after the reconstruction of its
sequence in several individuals and then correlate this
TBA profile with the expression of a target gene through
principal component regression. This approach was applied
to the analysis of a dataset including genome and coupled
gene expression data for 344 European individuals. Results: Two variants for C1QA gene: GA genotype of
rs201693493 (P<0.001) and CT genotype of rs149050968
(P<0.001) were significantly associated with later AO (≥50
years). In silico analysis demonstrated, that rs201693493
may alter splicing activity. Regarding C1QC, we found
three statistically significant variants: GA genotype of
rs2935537
(P=0.003),
GA
genotype
of
rs201241346
(P<0.001) and GA genotype (P<0.001) of rs200952686
(P<0.001). The first two were associated with earlier AO (≤
40 years), while the third was associated with late-onset. Results:
Our
TBA-based
inference
detected
3,781
eQTLs, of which 1,543 weren’t revealed by the traditional
single-SNP method. In addition it permitted the formulation
of mechanistic hypotheses pinpointing for each gene the
most relevant TFs in each associated regulatory region and
we showed that these outcomes are fairly consistent with
the presence of binding QTLs (bQTLs). Future perspectives: We want to integrate the TBA
model with new approaches, collectively named Transcrip-
tome Wide Association Studies (TWAS), that combine
GWAS and eQTL data to identify susceptibility genes. At
least in principle, TBA-based TWAS have the advantage of
considering also the effect of rare variants that cannot be
measured in standard eQTL studies. Conclusions: C1QA was associated with later. C1QC
may have a double role: variants may confer earlier or later
AO,
depending
on
the
associated
pathophysiological
mechanisms. We thus confirmed the role of C1Q comple-
ment genes as modulator of AO in TTR-FAP Val30Met
Portuguese patients. M. Alves-Ferreira received a FCT fellowship grant
[SFRH/BD/101352/2014] E. Grassi: None. E. Mariella: None. M. Forneris:
None. F. Marotta:
None. M. Catapano:
None. I. Molineris: None. P. Provero: None. A. Dias: None. D. Santos: None. T. Coelho: F. Consultant/Advisory Board; Modest; Pfizer Inc. M. Alves-
Ferreira: None. J. Sequeiros: None. I. Alonso: None. C. Lemos: None. A. Sousa: None. A. Dias: None. D. Santos: None. T. Coelho: F. Consultant/Advisory Board; Modest; Pfizer Inc. M. Alves- Ferreira: None. J. Sequeiros: None. I. Alonso: None. C. Lemos: None. A. Sousa: None. Ferreira: None. J. Sequeiros: None. I. Alonso: None. C. Lemos: None. A. Sousa: None. 1UnIGENe, IBMC - Instituto de Biologia Molecular e Celular,
Universidade do Porto, Porto, Portugal, 2i3S - Instituto de
Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal, 3FCUP, Faculdade de Ciências da
Universidade do Porto, Porto, Portugal, 4ICBAS - Instituto
Ciências Biomédicas Abel Salazar, Universidade do Porto,
Porto, Portugal, 5UCA, Unidade Corino de Andrade, Centro
Hospitalar do Porto (CHP), Porto, Portugal Association of genetic markers of coagulation and
fibrinolysis with prematurity complication Recent studies
showed that FV Leiden and PAI-1 4G/5G gene poly-
morphisms are associated with spontaneous abortion and
prematurity. Our objective was to analyse the FV Leiden
and PAI-1 4G/5G variants in order to determine their
association with complications in premature babies. retinopathy of prematurity (ROP) and sepsis. Recent studies
showed that FV Leiden and PAI-1 4G/5G gene poly-
morphisms are associated with spontaneous abortion and
prematurity. Our objective was to analyse the FV Leiden
and PAI-1 4G/5G variants in order to determine their
association with complications in premature babies. Material
and
Methods:
The
study
included
136
premature babies, mean GA= 30,46±1,98 and BW=1294
±278,3. Genotyping of FV Leiden variant was performed
by Real-time PCR analysis with specific TaqMan assay. PAI-1 4G/5G variants genotyping was performed by PCR-
RFLPs method. Results: We present three large pedigrees spanning seven
generations, with data from group pedigrees with good and
bad self-reported health status we show whether consan-
guinity reflects the outcome, and trace its origins to the
different islands on the Faroe Islands. We present 15 family
pedigrees with specific disease status, which may contribute
to our understanding of the pathogenesis of these diseases. Results: The frequency of complications in premature
babies was: 73,7% RDS, 19,1% BDP, 14,6% IVH, 46,7%
ROP and 35,8% sepsis. The frequency of risk allele A for
FV Leiden variant is 6.7%. FV Leiden variant is associated
with development of BPD (X2=5,245, p = 0,022; OR
0,302, 95% CI 0,104-0,877). The frequency of risk allele
4G for PAI-1 4G/5G variant is 60,6%. PAI-1 variant is a
associated with IVH expression (X2=6,700, p = 0,035;
0,257, 95% CI 0,088-0,775). There was no significant
association of analyzed variants with RDS, ROP or sepsis. Perspectives: The pedigrees will be used in combination
with genotype data to gain information on population
stratification and the demographical history of the Faroe
Islands. Funding:
The
project
is
funded
by
the
Faroese
Government. Funding:
The
project
is
funded
by
the
Faroese
Government. Conclusion: Our findings suggest that FV Leiden
variantis a risk factor for development of BPD while the
presence of 4G variant is associated with occurrence
of IVH. Ó. Mortensen: None. L.N. Lydersen: None. B. á Steig:
None. G. Andorsdóttir: None. N.O. Gregersen: None. Ó. Mortensen: None. L.N. Lydersen: None. B. á Steig:
None. G. Andorsdóttir: None. N.O. Gregersen: None. T.M. Damnjanovic: None. M. Grk: None. T. Varljen: Households and Family Projections: Tehran Lipid and
Glucose Study: 1999 to 2016 Households and Family Projections: Tehran Lipid and
Glucose Study: 1999 to 2016 None. J. Pantelic: None. N. Maksimovic: None. B. Jekic: None. I. Novakovic: None. K. Guity1, B. SedaghatiKhayat1, A. Momenan2, A. Seyedhamzehzadeh1, N. Sarbazi1, F. Azizi3, M. Daneshpour1 Association of genetic markers of coagulation and
fibrinolysis with prematurity complication A. Dias1,2,3, D. Santos1,2,4, T. Coelho5, M. Alves-Ferreira1,2,4, J. Sequeiros1,2,4, I. Alonso1,2, C. Lemos4,1,2, A. Sousa4,1,2 T. M. Damnjanovic1, M. Grk1, T. Varljen2, J. Pantelic3, N. Maksimovic1, B. Jekic1, I. Novakovic1 1UnIGENe, IBMC - Instituto de Biologia Molecular e Celular,
Universidade do Porto, Porto, Portugal, 2i3S - Instituto de
Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal, 3FCUP, Faculdade de Ciências da
Universidade do Porto, Porto, Portugal, 4ICBAS - Instituto
Ciências Biomédicas Abel Salazar, Universidade do Porto,
Porto, Portugal, 5UCA, Unidade Corino de Andrade, Centro
Hospitalar do Porto (CHP), Porto, Portugal 1UnIGENe, IBMC - Instituto de Biologia Molecular e Celular,
Universidade do Porto, Porto, Portugal, 2i3S - Instituto de
Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal, 3FCUP, Faculdade de Ciências da
Universidade do Porto, Porto, Portugal, 4ICBAS - Instituto
Ciências Biomédicas Abel Salazar, Universidade do Porto,
Porto, Portugal, 5UCA, Unidade Corino de Andrade, Centro
Hospitalar do Porto (CHP), Porto, Portugal 1Institute of Human Genetics, Belgrade, Serbia, 2Institute of
Forensic Medicine "Milovan Milovanovic", Belgrade, Serbia,
3Clinic for eye disease, Belgrade, Serbia Introduction: The risk of complications in premature
babies increases with lower gestational age (GA) and lower
birth weight (BW). Most frequent complications are
respiratory distress syndrome (RDS), bronchopulmonary
dysplasia
(BPD),
intraventricular
hemorrhage
(IVH), Introduction: Transthyretin (TTR) familial amyloid poly-
neuropathy (FAP) (OMIM 176300) shows a variable age- 642 J. del Picchia Materials and Methods: In accordance with the FarGen
project pedigrees were made from all 1530 individuals who
had registered as FarGen participants. All the participants
are in the Multi-Generation register and have answered a
questionnaire about health status. Three different pedigrees
were constructed: 1) all 1530 participants, 2) participants
with “good” health status, and 3) participants with “bad”
health status. From the 21 different ICD-10 groups of
diseases reported in the FarGen project, pedigrees were
made using 15 specific diseases. Materials and Methods: In accordance with the FarGen
project pedigrees were made from all 1530 individuals who
had registered as FarGen participants. All the participants
are in the Multi-Generation register and have answered a
questionnaire about health status. Three different pedigrees
were constructed: 1) all 1530 participants, 2) participants
with “good” health status, and 3) participants with “bad”
health status. From the 21 different ICD-10 groups of
diseases reported in the FarGen project, pedigrees were
made using 15 specific diseases. retinopathy of prematurity (ROP) and sepsis. P18.32D T.M. Damnjanovic: None. M. Grk: None. T. Varljen:
None. J. Pantelic: None. N. Maksimovic: None. B. Jekic:
None. I. Novakovic: None. P18.31C Use of large pedigrees from the FarGen project to gain
information on consanguinity and inbreeding within the
Faroese population 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid, Tehran, Iran, Islamic
Republic of, 2Prevention of Metabolic Disorders Research
Center, Research Institute for Endocrine Sciences, Shahid,
Tehran, Iran, Islamic Republic of, 3Endocrine Research
Center, Research Institute for Endocrine Sciences, Shahid,
Tehran, Iran, Islamic Republic of Ó. Mortensen1, L. N. Lydersen1, B. á Steig2, G. Andorsdóttir1, N. O. Gregersen1 1FarGen, The Genetic Biobank of the Faroe Islands, Tórshavn,
Faroe Islands, 2General Medical Department, National
Hospital of the Faroe Islands, Tórshavn, Faroe Islands 1FarGen, The Genetic Biobank of the Faroe Islands, Tórshavn,
Faroe Islands, 2General Medical Department, National
Hospital of the Faroe Islands, Tórshavn, Faroe Islands Introduction: During last decade, along with developing
high throughput sequencing technology and mapping the
human genome, social and biological relationships are
considered as pivotal tools in medical genetics. Family-
based studies are suitable for population stratification con-
trol, investigations of parent-of-origin; X-linked transmitted
inheritance and identification of disease risk factors. The
aim of the current study was to describe the family struc-
tures of Tehran Lipid and Glucose Study (TLGS) popula-
tion, and rates of its consanguineous marriage. Introduction: The Faroe Islands is a north Atlantic isolate
with a current population size of 50.000 individuals. The
Multi-Generation register of the Genetic Biobank (www. biobank.gov.fo) comprises hereditary records of all indivi-
duals registered in the Faroe Islands since 1800, moreover
the majority of the individuals (85%) have records dating
back to 1650. Together with the FarGen project (www.fa
rgen.fo) we are using this valuable resource of genealogy to
show consanguinity and inbreeding within the Faroese
population. Materials and methods: The TLGS is one of the oldest
longitudinal cohort study, which has been performed on a Abstracts from the 51st European Society of Human Genetics Conference: Posters 643 population residents of the region district 13 of Tehran
municipality, in two main and four follow up phases. Between 1999-2016, the complete genealogy data of 20,077
participants were documented and collected. Pedigree of
each participant was drawn on paper sheet based on
standardized human pedigree nomenclature and checked by
ChIP-Typed data from the Tehran Cardiometabolic Genetic
Study (TCGS). wide penalties according to marginal p-value were applied
for two-step methods. Results: We identified five loci near ABCG8, ATP2B1,
CWC27 including the genetic interaction with smoking. P18.31C The
heritability explained by ABCG8 was 0.9% by a GWAS but
was increased to 5.7% when we considered GxSmoking
interactions. We also identified 11 significant loci near
CCDC63, SH2B3, CUX2 for GxAlcohol interactions. About
11.6% of the heritability has been elucidated further due to
the interplay between identified loci and alcohol. We
identified
several
suggestive
loci
that
synergistically
increase the risk of HBP with obesity. Results: Genealogy data of participants and their relatives
overall for 23259 individuals were checked through 5434
households. Before pedigree drawing and splicing unrelated
individuals, the mean of cluster was 4.2 ± 1.9 (Min 1, Max
18). After taking into account ChIP genotyping data, 1741
clusters which their members had a common ancestor,
joined together to make extended families by the appro-
priated methods. With splicing and adding disjointed,
dummy individuals, 25642 remained in 3724 ± 6.9 (Min
3, Max 52) pedigrees. Family structures of the TLGS along
with demographic, anthropometrics, biochemical, genetics,
and well-phenotyped data can explore more genetics
different. Conclusion: The identified loci might be used as genetic
markers to give a personalized guideline for those who are
particularly susceptible to smoking, alcohol, and obesity. Some amount of missing heritability would be explained by
discovering more GxE interactive loci. M. Kang: None. J. Sung: None. M. Kang: None. J. Sung: None. A genome-wide gene by lifestyle interaction study on high
blood pressure Eurac Research, Institute for Biomedicine, Bolzano, Italy P18.34B
Genetic landscape of kidney function: results from a trans-
ethnic genome-wide association meta-analysis of >750,000
individuals Genetic landscape of kidney function: results from a trans-
ethnic genome-wide association meta-analysis of >750,000
individuals K. Guity:
None. B. SedaghatiKhayat:
None. A. Momenan: None. A. Seyedhamzehzadeh: None. N. Sarbazi: None. F. Azizi: None. M. Daneshpour: None. K. Guity:
None. B. SedaghatiKhayat:
None. A. Momenan: None. A. Seyedhamzehzadeh: None. N. Sarbazi: None. F. Azizi: None. M. Daneshpour: None. Eurac Research, Institute for Biomedicine, Bolzano, Italy Introduction: Chronic kidney disease (CKD) is a public
health threat, which affects >10% adult population in
Western countries and is associated with increased risk of
end-stage renal disease, cardiovascular disease, and all-
cause mortality. In the absence of therapies for CKD pre-
vention, understanding the biological regulators of glo-
merular filtration rate (GFR), the key element that defines
CKD, is of the highest relevance. M. Kang1, J. Sung2 Multi-phenotype epigenome-wide association analysis of
fasting glucose and insulin in 981 Finnish individuals Multi-phenotype epigenome-wide association analysis of
fasting glucose and insulin in 981 Finnish individuals M. Kang1, J. Sung2 1Department of Epidemiology, School of Public Health, Seoul
National University, Seoul, Korea, Republic of, 2Department of
Epidemiology, School of Public Health and Institute of Health
and Environment, Seoul National University, Seoul, Korea,
Republic of 1Department of Epidemiology, School of Public Health, Seoul
National University, Seoul, Korea, Republic of, 2Department of
Epidemiology, School of Public Health and Institute of Health
and Environment, Seoul National University, Seoul, Korea,
Republic of Materials and Methods: Within the CKDGen Consor-
tium, 121 studies applied standardized protocols and scripts
to run genome-wide association scans (GWAS) of estimated
GFR (eGFR) on ~9 million high-quality SNPs, imputed on
the 1000 Genomes ph3v5 or Haplotype Reference Con-
sortium datasets. We performed fixed-effect meta-analysis
of GWAS data on >750,000 participants of European, East-
Asian, South-Asian, Hispanic, and African ancestries. Effect heterogeneity due to ancestry was investigated using
trans-ethnic genome-wide meta-regression as implemented
in MR-MEGA. Materials and Methods: Within the CKDGen Consor-
tium, 121 studies applied standardized protocols and scripts
to run genome-wide association scans (GWAS) of estimated
GFR (eGFR) on ~9 million high-quality SNPs, imputed on
the 1000 Genomes ph3v5 or Haplotype Reference Con-
sortium datasets. We performed fixed-effect meta-analysis
of GWAS data on >750,000 participants of European, East-
Asian, South-Asian, Hispanic, and African ancestries. Effect heterogeneity due to ancestry was investigated using
trans-ethnic genome-wide meta-regression as implemented
in MR-MEGA. Introduction: Epidemiologic studies showed synergistic
effects on the risk of atherosclerotic cardiovascular disease
due to the interplay between high blood pressure (HBP) and
lifestyle including smoking, alcohol, and obesity. Although
several GWASs and candidate-gene studies have found out
GxE interactions on hypertension, relatively fewer studies
have been performed to scan the entire genome to identify
variants that modify the effects of lifestyle on the risk
of HBP. Methods: 21,504 individuals from three Korean cohorts
were involved. Blood pressure was measured following the
American Heart Association (AHA) guideline. We con-
ducted exhaustive GxE interaction analyses for 4.1 million
SNPs imputed using the 1000 genomes GRCh37 reference
panel. We applied various exhaustive scans and two-step
methods for detecting GxE interactions in parallel: step- Results: Genomic inflation was negligible: lambda =
1.05 and LD score regression intercept = 1.04. Across
ethnicities, we identified 308 1-Mb segments containing ≥1
SNP associated with eGFR (p value ≤5E-08): 228 of these
loci were novel and 80 validated previously identified
signals. Trans-ethnic
meta-regression
revealed
limited 644 J. del Picchia involving this marker attained p=0.13 and p=4.0×10-3,
respectively. M. Kang1, J. Sung2 involving this marker attained p=0.13 and p=4.0×10-3,
respectively. heterogeneity correlated with ancestry for most genomic
regions. Approximate conditional analysis of the European-
ancestry dataset identified 269 independent variants, which
together doubled the eGFR variance explained compared to
previous estimates. Conclusion: We extended our MP approach to MP-
EWAS, and demonstrated its enhanced power over single-
trait analysis for detection of MP epigenetic effects in large-
scale data. Conclusions: This study represents the largest screen of
kidney function genetic loci to date. The large number of
identified loci will contribute to increase our understanding
of kidney function’s biology. Funding:
DYNAhealth-H2020-PHC-2014-633595,
WT205915, Academy of Finland, EGEA, 285547. H. Draisma: None. M. Wielscher: None. S. Hassan:
None. Z. Balkhiyarova: None. M. Jarvelin: None. M. Kaakinen: None. I. Prokopenko: None. C. Pattaro: None. Imperial College London, London, United Kingdom Imperial College London, London, United Kingdom Background: Multi-phenotype genome-wide association
studies (MP-GWAS) of correlated traits have greater power
to detect genotype–phenotype associations than single-trait
GWAS. In our previously-developed MP-GWAS method,
implemented in the SCOPA software, single-nucleotide
polymorphism
(SNP)
genotype
dosage
is
‘reversely’
regressed on a linear combination of phenotypes. Con-
sidering the epidemiologic correlations between fasting
plasma glucose (FG) and fasting insulin (FI) levels, we
aimed to use a SCOPA adaptation for multi-phenotype
epigenome-wide association analysis (MP-EWAS) for these
two traits in non-diabetic individuals. 1University of Leicester, Leicester, United Kingdom,
2Wellcome Sanger Institute, Cambridge, United Kingdom,
3Infectious Disease Unit, Department of Medicine Solna,
Karolinska Institutet, Stockholm, Sweden, 4Nyamisati Malaria
Research, Rufiji, National Institute for Medical Research, Dar-
es-Salaam, Tanzania, United Republic of, 5School of Medicine,
University of Leeds, Leeds, United Kingdom 1University of Leicester, Leicester, United Kingdom,
2Wellcome Sanger Institute, Cambridge, United Kingdom,
3Infectious Disease Unit, Department of Medicine Solna,
Karolinska Institutet, Stockholm, Sweden, 4Nyamisati Malaria
Research, Rufiji, National Institute for Medical Research, Dar-
es-Salaam, Tanzania, United Republic of, 5School of Medicine,
University of Leeds, Leeds, United Kingdom Human glycophorins A and B are proteins expressed on the
surface of erythrocytes, and are receptors for invasion of the
Plasmodium falciparum parasite, which causes malaria in
sub-Saharan Africa. The proteins are encoded by the genes
GYPA and GYPB which, together with GYPE, reside on a
tandemly-duplicated
repeat
region
on
chromosome
4q31.21. Previous genome-wide analysis has shown that a
structural variant within this region (DUP4), is identical to
the blood group antigen Dantu NE+, and confers a
clinically-important protective effect, is common in East
Africans and is strongly protective against severe malaria,
reducing the risk of severe malaria by up to 40%. DUP4 is a
complex structural genomic variant that carries hybrid
(GYPA/GYPB) fusion genes. Using fibre-FISH, we validate
the structural arrangement of the glycophorin locus in the
DUP4 variant, and provide evidence of somatic variation in
the number of GYPA/GYPB fusion genes. Subsequently, we
have developed a paralogue-specific junction fragment PCR
to genotype DUP4. We demonstrate association of DUP4
variant with hemoglobin levels - a phenotype related to
malaria - in 348 unrelated DNA samples from a Tanzanian
village holoendemic for malaria using a family-based
association
test. H. Draisma, M. Wielscher, S. Hassan, Z. Balkhiyarova, M.
Jarvelin, M. Kaakinen, I. Prokopenko W. Algady1, S. Gomes2, D. Carpenter1, P. Brajer1, A. Färnert3, I. Rooth4, M. Shaw5, F. Yang2, E. J. Hollox1 P18.36D Characterisation and somatic mosaicism of the human
glycophorin DUP4 structural variant, and association with
hemoglobin levels in a malaria-endemic village H. Draisma, M. Wielscher, S. Hassan, Z. Balkhiyarova, M. Jarvelin, M. Kaakinen, I. Prokopenko H. Draisma, M. Wielscher, S. Hassan, Z. Balkhiyarova, M. Jarvelin, M. Kaakinen, I. Prokopenko Imperial College London, London, United Kingdom Using
the
family-based
association
approach implemented in (QTDT), we have found a sta-
tistically significant association of the DUP4 variant with Methods: We developed the “methylSCOPA” software,
extending the SCOPA approach to MP-EWAS using DNA
[hyper/hypo]methylation data, and applied it to FG, FI and
Illumina Infinium HumanMethylation450K BeadChip array
data from the Northern Finland Birth Cohorts (NFBC)
1966/1986. We subjected all data to stringent quality
control
and
corrected
them
for
measured
(potential)
confounders by linear regression analysis, and normalized
the methylation signal intensity and FI data. The MP-
EWAS included data for 635/346 individuals and 459,378/
466,290
methylation
probes
from
NFBC1966
and
NFBC1986, respectively. We meta-analyzed the study-
specific MP-EWAS results, mapped genomic locations to
CGCh37/hg19, and adopted p<1×10-7 to denote epigenome-
wide significance. Methods: We developed the “methylSCOPA” software,
extending the SCOPA approach to MP-EWAS using DNA
[hyper/hypo]methylation data, and applied it to FG, FI and
Illumina Infinium HumanMethylation450K BeadChip array
data from the Northern Finland Birth Cohorts (NFBC)
1966/1986. We subjected all data to stringent quality
control
and
corrected
them
for
measured
(potential)
confounders by linear regression analysis, and normalized
the methylation signal intensity and FI data. The MP-
EWAS included data for 635/346 individuals and 459,378/
466,290
methylation
probes
from
NFBC1966
and
NFBC1986, respectively. We meta-analyzed the study-
specific MP-EWAS results, mapped genomic locations to
CGCh37/hg19, and adopted p<1×10-7 to denote epigenome-
wide significance. Results: In meta-analysis, the MP-EWAS association
involving a methylation marker located at chromosome
22:49,088,813 and annotated to FAM19A5 attained the
lowest p-value (p=1.4×10-7) epigenome-wide. In single-
trait FG and FI EWAS meta-analyses, the associations Results: In meta-analysis, the MP-EWAS association
involving a methylation marker located at chromosome
22:49,088,813 and annotated to FAM19A5 attained the
lowest p-value (p=1.4×10-7) epigenome-wide. In single-
trait FG and FI EWAS meta-analyses, the associations Abstracts from the 51st European Society of Human Genetics Conference: Posters 645 hemoglobin levels (p = 0.0054). Our study confirms the
importance of the DUP4 variant in malaria protection, and
raises the intriguing possibility of heightened somatic
instability and somatic mosaicism at this locus in DUP4
carriers, which might confer added protection against
malaria. variants in ABCG2/BCRP, including rs72552713 (Q126X)
and rs2231142 (Q141K). However, the effects of rare
ABCG2 variants on gout is unknown. Therefore, we
investigated their effects on gout susceptibility. Materials and Methods: We sequenced all exons of
ABCG2 in 480 gout patients and 480 healthy controls
(Japanese males). P18.37A
Both common and rare genetic variants of ABCG2 are risks
for gout Matsuo1 1Department of Integrative Physiology and Bio-Nano
Medicine, National Defense Medical College, Tokorozawa,
Japan, 2Department of Pharmacy, The University of Tokyo
Hospital, Faculty of Medicine, The University of Tokyo, Tokyo,
Japan, 3Division of Human Genetics, Department of Integrated
Genetics, National Institute of Genetics, Mishima, Japan,
4First Faculty of Medicine, Charles University and General
University Hospital in Prague, Institute of Inherited Metabolic
Disorders, Prague, Czech Republic, 5Institute of
Rheumatology, Prague, Czech Republic, 6Department of
Preventive Medicine and Public Health, National Defense
Medical College, Tokorozawa, Japan, 7Laboratory for
Mathematics, National Defense Medical College, National
Defense Medical College, Tokorozawa, Japan, 8Department of
Preventive Medicine, Nagoya University Graduate School of
Medicine, Nagoya, Aichi, Japan, 9Department of Medical
Chemistry, Kurume University School of Medicine, Kurume,
Japan, 10Department of Bioinformatics and Genomics,
Graduate School of Medical Sciences, Kanazawa University,
Ishikawa, Japan, 11Division of Kidney and Hypertension,
Department of Internal Medicine, Jikei University School of
Medicine, Tokyo, Japan, 12Department of Pathophysiology and
Therapy in Chronic Kidney Disease, Jikei University School of
Medicine, Tokyo, Japan, 13Department of Pathophysiology,
Tokyo University of Pharmacy and Life Sciences, Hachioji,
Tokyo, Japan, 14Ryougoku East Gate Clinic, Tokyo, Japan,
15Department of Biochemisty, University of Otago, Dunedin,
New Zealand 1Department of Integrative Physiology and Bio-Nano
Medicine, National Defense Medical College, Tokorozawa,
Japan, 2Department of Pharmacy, The University of Tokyo
Hospital, Faculty of Medicine, The University of Tokyo, Tokyo,
Japan, 3Division of Human Genetics, Department of Integrated
Genetics, National Institute of Genetics, Mishima, Japan,
4First Faculty of Medicine, Charles University and General
University Hospital in Prague, Institute of Inherited Metabolic
Disorders, Prague, Czech Republic, 5Institute of
Rheumatology, Prague, Czech Republic, 6Department of
Preventive Medicine and Public Health, National Defense
Medical College, Tokorozawa, Japan, 7Laboratory for
Mathematics, National Defense Medical College, National
Defense Medical College, Tokorozawa, Japan, 8Department of
Preventive Medicine, Nagoya University Graduate School of
Medicine, Nagoya, Aichi, Japan, 9Department of Medical
Chemistry, Kurume University School of Medicine, Kurume,
Japan, 10Department of Bioinformatics and Genomics,
Graduate School of Medical Sciences, Kanazawa University,
Ishikawa, Japan, 11Division of Kidney and Hypertension,
Department of Internal Medicine, Jikei University School of
Medicine, Tokyo, Japan, 12Department of Pathophysiology and
Therapy in Chronic Kidney Disease, Jikei University School of
Medicine, Tokyo, Japan, 13Department of Pathophysiology,
Tokyo University of Pharmacy and Life Sciences, Hachioji,
Tokyo, Japan, 14Ryougoku East Gate Clinic, Tokyo, Japan,
15Department of Biochemisty, University of Otago, Dunedin,
New Zealand Conclusions: We showed that both common and rare
variants of ABCG2 are independent risks for gout. P18.37A
Both common and rare genetic variants of ABCG2 are risks
for gout Results: We genotyped 3 common and 19 rare non-
synonymous
variants
of
ABCG2. SIFT
scores
were
significantly correlated with the urate transport function. After the effects of common variants were removed by
stratification, the rare ABCG2 variants were significantly
associated with gout susceptibility (odds ratio [OR]=3.2, p
= 6.4×10-3). Additionally, multivariate logistic regression
analysis clarified that these rare ABCG2 variants (OR=2.7,
p = 3.0×10-3) were
similar
in
effect
size
to
Q126X
(OR=3.4,
p = 3.2×10-6)
and
Q141K
(OR=2.3,
p =
2.7×10-16). T. Higashino1, T. Takada2, H. Nakaoka3, Y. Toyoda2, B. Stiburkova4,5, H. Miyata2, Y. Ikebuchi2, H. Nakashima6, S. Shimizu1, M. Kawaguchi1, M. Sakiyama1, A. Nakayama1, A. Akashi1, Y. Tanahashi1, Y. Kawamura1, T. Nakamura7, K. Wakai8, R. Okada8, K. Yamamoto9, K. Hosomichi3,10, T. Hosoya11,12, K. Ichida12,13, H. Ooyama14, H. Suzuki2, I. Inoue3,
T. R. Merriman15, N. Shinomiya1, H. Matsuo1 Hosoya11,12, K. Ichida12,13, H. Ooyama14, H. Suzuki2, I. Inoue3,
T. R. Merriman15, N. Shinomiya1, H. Imperial College London, London, United Kingdom We also performed functional assay of
non-synonymous
ABCG2
variants
and
analyzed
the
correlation between urate transport function and scores
from the protein prediction algorithms (SIFT and PolyPhen-
2). Stratified association analyses and multivariate logistic
regression analysis were used to evaluate the effects of
ABCG2 variants on gout susceptibility. W. Algady: None. S. Gomes: None. D. Carpenter:
None. P. Brajer: None. A. Färnert: None. I. Rooth: None. M. Shaw: None. F. Yang: None. E.J. Hollox: None. S. Im Department of Biochemistry and Molecular Biology, Seoul
National University College of Medicine, Seoul, Korea,
Republic of 1Division of Hygiene and Medical Microbiology, Medical
University of Innsbruck, Innsbruck, Austria, 2Istitute for
Biomedicine, Eurac Research, Affiliated Institute of the
University of Lübeck, Bolzano, Italy, 3Division of Genetic
Epidemiology, Medical University of Innsbruck, Innsbruck,
Austria, 4Department of Neurology, General Central Hospital,
Bolzano, Italy, 5Department of Neurology, University of
Lübeck, Lübeck, Germany Elevated serum uric acid (SUA) contributes to diverse
health outcomes including gout, hypertension, diabetes
mellitus and cardiovascular disease. The incidence and
prevalence of hyperuricemia have been increasing. The
proportion of heritability contributing to SUA levels is
estimated to be about 40~70%. We aimed to identify
genetic variants involved in the regulation of SUA using
genome-wide
association
study
(GWAS)
in
Korean. Genetic association was assessed with 4,414,664 single
nucleotide polymorphisms (SNPs) after genomic imputation
in 1905 unrelated men. We identified four loci (FRMD8,
OVOL1, ABCG2 and NRXN2) that reached genome-wide
significance (<p<5×10-8). The strongest association was
found with a SNP in FRMD8 (rs184521656, p = 3.02×10-
18), which has not been reported to be associated with SUA
in the other studies. The other two loci (ABCG2 and
NRXN2) were frequently reported in previous studies of
several ethnic groups. In addition, we conducted replication
study of the SNP, rs184521656 in 2912 Koreans including
both men and women. Rs184521656 (p = 1.50×10-10) was
successfully validated. Rs184521656 is found only in East
Asian with a minor allele frequency (MAF) of 0.01 and
functions as a protective allele in the direction of lowering
the SUA in our study. This study validated previously
published results and suggested new possibilities. The complement system (CS), a fundamental component of
the innate immune response, can be activated via the clas-
sical (CP), lectin (LP), and alternative (AP) pathways. We
conducted a genome-wide association study (GWAS) of the
three pathways in a general population framework. CS
activation was assessed in 4990 participants of the CHRIS
study, using the WIESLAB commercial assay. For each
pathway, GWAS were performed on >7 million dosage
levels, imputed on the Haplotype Reference Consortium
dataset, and selected for minor allele frequency of ≥0.01 and
imputation quality of ≥0.3. Association models accounted
for sex, age, plate and date of experiment, and relatedness. For significant SNPs, SNP-by-sex interaction was tested via
linear mixed models. SNP-based heritability was estimated
using GCTA-GREML. P18.37A
Both common and rare genetic variants of ABCG2 are risks
for gout These
results could support not only “Common Disease, Common
Variant”
but
also
“Common
Disease,
Multiple
Rare
Variant” hypotheses for the association between ABCG2
and gout. Fundings: Japan (MEXT [Nos. 25293145, 15K15227,
17015018,
221S0001,
221S0002,
16H06277
and
16H06279], MHLW, MOD, the Kawano Masanori Memor-
ial Foundation for Promotion of Pediatrics, and the Gout
Research Foundation) T. Higashino: None. T. Takada: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; a patent pending based on the work. H. Nakaoka:
None. Y. Toyoda: None. B. Stiburkova: None. H. Miyata: None. Y. Ikebuchi: None. H. Nakashima: None. S. Shimizu: None. M. Kawaguchi: None. M. Sakiyama:
None. A. Nakayama: None. A. Akashi: None. Y. Tanahashi: None. Y. Kawamura: None. T. Nakamura:
E. Ownership Interest (stock, stock options, patent or other
intellectual property); Modest; a patent pending based on
the work. K. Wakai: None. R. Okada: None. K. Yamamoto: None. K. Hosomichi: None. T. Hosoya:
None. K. Ichida: E. Ownership Interest (stock, stock
options, patent or other intellectual property); Modest; a
patent pending based on the work. H. Ooyama: None. H. Suzuki: E. Ownership Interest (stock, stock options, patent
or other intellectual property); Modest; a patent pending
based on the work. I. Inoue: None. T.R. Merriman: None. T. Higashino: None. T. Takada: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; a patent pending based on the work. H. Nakaoka: 15Department of Biochemisty, University of Otago, Dunedin,
New Zealand Introduction: Previous studies unveiled the association
between gout susceptibility and common dysfunctional J. del Picchia 646 to be involved in CS activation. Further characterization is
warranted to define their pleiotropic landscape and involve
CS activation in complex disease etiology. to be involved in CS activation. Further characterization is
warranted to define their pleiotropic landscape and involve
CS activation in complex disease etiology. N. Shinomiya: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; a patent
pending based on the work. H. Matsuo: E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Modest; a patent pending based on the work. N. Shinomiya: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; a patent
pending based on the work. H. Matsuo: E. Ownership
Interest (stock, stock options, patent or other intellectual
property); Modest; a patent pending based on the work. D. Noce: None. L. Foco: None. C. Fuchsberger: None. M. Gögele: None. F. Kronenberg: None. M. Yakoreva1,2, T. Kahre1,2, E. Õiglane-Shlik3,4, M. Vals1,3,4, P.
Mee5, K. Õunap1,2 S. Im All SNPs explained 14%, 13%, and
45%, of the CP, AP, and LP variance, respectively. We
identified associations at 8 loci carrying 12 independent
variants with large effects: for AP, at chromosomes 1q31 (p
= 8.8e-10), 5p13.1 (p = 1.8e-17), 6p22.1 (p = 4.7e-14), and
12p12.2 (p = 1.8e-9); for CP, at chromosome 6p22.1 (p =
1.4e-35); and for LP, at chromosomes 1p36.22 (p = 7.3e-
11), 9q34.2 (p = 7.6e-45), and 10q21.1 (p = 1.4e-260). Six
of these loci carry genes known for their relation with CS
activation, as partially confirmed by summary-data-based
Mendelian randomization analysis. SNP-by-sex interaction
analysis identified a possible interaction between the
1p36.22 SNP and sex in association with LP (p = 0.023). We identified eight loci, most of which carry genes known The complement system (CS), a fundamental component of
the innate immune response, can be activated via the clas-
sical (CP), lectin (LP), and alternative (AP) pathways. We
conducted a genome-wide association study (GWAS) of the
three pathways in a general population framework. CS
activation was assessed in 4990 participants of the CHRIS
study, using the WIESLAB commercial assay. For each
pathway, GWAS were performed on >7 million dosage
levels, imputed on the Haplotype Reference Consortium
dataset, and selected for minor allele frequency of ≥0.01 and
imputation quality of ≥0.3. Association models accounted
for sex, age, plate and date of experiment, and relatedness. = 8.8e-10), 5p13.1 (p = 1.8e-17), 6p22.1 (p = 4.7e-14), and
12p12.2 (p = 1.8e-9); for CP, at chromosome 6p22.1 (p =
1.4e-35); and for LP, at chromosomes 1p36.22 (p = 7.3e-
11), 9q34.2 (p = 7.6e-45), and 10q21.1 (p = 1.4e-260). Six
of these loci carry genes known for their relation with CS
activation, as partially confirmed by summary-data-based
Mendelian randomization analysis. SNP-by-sex interaction
analysis identified a possible interaction between the
1p36.22 SNP and sex in association with LP (p = 0.023). We identified eight loci, most of which carry genes known S. Im: None. S. Im: None. Genome-Wide Association Study of Serum Uric Acid in
Korean D. Noce1,2, L. Foco2, C. Fuchsberger2, M. Gögele2, F. Kronenberg3, R. Würzner1, C. Lass-Flörl1, P. P. Pramstaller2,4,5,
D. Orth-Höller1, C. Pattaro2 D. Noce1,2, L. Foco2, C. Fuchsberger2, M. Gögele2, F. Kronenberg3, R. Würzner1, C. Lass-Flörl1, P. P. Pramstaller2,4,5,
D. Orth-Höller1, C. Pattaro2 P18.37A
Both common and rare genetic variants of ABCG2 are risks
for gout R. Würzner:
None. C. Lass-Flörl: None. P.P. Pramstaller: None. D. Orth-Höller: None. C. Pattaro: None. A retrospective analysis of the prevalence of imprinting
disorders in Estonia: updated results A retrospective analysis of the prevalence of imprinting
disorders in Estonia: updated results Abstracts from the 51st European Society of Human Genetics Conference: Posters 647 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia, 3Department of Pediatrics, Faculty of
Medicine, University of Tartu, Tartu, Estonia, 4Children’s
Clinic, Tartu University Hospital, Tartu, Estonia, 5United
Laboratories, Tartu University Hospital, Tartu, Estonia 1Department of Clinical Genetics, United Laboratories, Tartu
University Hospital, Tartu, Estonia, 2Department of Clinical
Genetics, Institute of Clinical Medicine, University of Tartu,
Tartu, Estonia, 3Department of Pediatrics, Faculty of
Medicine, University of Tartu, Tartu, Estonia, 4Children’s
Clinic, Tartu University Hospital, Tartu, Estonia, 5United
Laboratories, Tartu University Hospital, Tartu, Estonia 1V. N. Karazin Kharkiv National University, Kharkiv, Ukraine,
2Regional Clinical Dermatovenerologic Dispensary No. 1,
Kharkiv, Ukraine, 3Medical Center IGR, Kyiv, Ukraine,
4Kharkiv National Medical University, Kharkiv, Ukraine,
5Kharkiv Medical Academy of Postgraduate Education,
Kharkiv, Ukraine, 6National Scientific Center “Institute of
Experimental and Clinical Veterinary Medicine”, Kharkiv,
Ukraine Introduction: Imprinting disorders (IDs) are a group of rare
congenital diseases affecting growth, development, meta-
bolism and behaviour. Because of high clinical and genetic
heterogeneity, the exact prevalence of IDs is not known. Introduction: The trend to assortative marriages in Eur-
opean
countries
leads
to
population
subdivision and
increased inbreeding level. It results in decreased population
diversity, loci similarity, intensified recombination pro-
cesses associated with chromosome rearrangements. The
study was aimed to assess the gene linkage extent at dif-
ferent exogamy degrees of subjects. Methods: In this study we retrospectively reviewed
records of all Estonian patients with both molecularly and
clinically diagnosed IDs found during the period 1998-
2017. Results: A total of 83 individuals with IDs were
identified. 31% (26) of them had Prader-Willi syndrome
(PWS), 18% (15) Silver-Russell syndrome (SRS), 17% (14)
Angelman
syndrome
(AS),
14,5%
(12)
Beckwith-
Wiedemann
syndrome
(BWS),
11%
(9)
pseudo-
or
pseudopseudohypoparathyroidism (PHP/PPHP), 5%
(4)
central
precocious
puberty
(CPP),
2,5%
(2)
Temple
syndrome (TS14) and 1% (1) transient neonatal diabetes
mellitus (TNDM). 1/3 of all SRS and BWS cases fulfilled
diagnostic criteria for these disorders, but were negative for
genetic abnormalities. Age at diagnosis varied from prenatal
to 83 years. During the period 2004-2017 birth prevalence
of PWS in Estonia was 1/14,565 live births (1/10,000-1/
25,000
worldwide),
AS
1/29,130
(1/12,000-1/20,000),
BWS 1/22,656 (~1/15,000), SRS 1/16,992 (1/75,000-1/
100,000) and PHP/PPHP 1/33,985 (prevalence unknown). The total prevalence of IDs in Estonia is 6.3/100,000. P18.43C P18.43C
Effects of inbreeding on linkage disequilibrium for SNPs of
MTHFR, MTR, F5, LCT and VDR3 genes in Ukrainian
population P18.43C
Effects of inbreeding on linkage disequilibrium for SNPs of
MTHFR, MTR, F5, LCT and VDR3 genes in Ukrainian
population Materials and Methods: The genotyping was carried for
MTHFR 677C-T, 1298A-C, 1p36.3 (n=105), MTR 2756A-
G, 1q43, F5 1691G-A, 1q24.2 (n=50), LCT C/T(-13910),
G/A(-22018),
2q21.3
(n=34),
VDR3
61888G>T,
61968T>C, 12q13.11 (n=100). The chromosome structure
was analyzed using classical cytogenetics methods, GTG,
FISH (n=6156). Exogamy degree scale: from 1 - parentage
originated from one settlement to 4 - interethnic marriage. Inbreeding level was measured with FST. The linkage
disequilibrium (LD) was estimated by D’, r2. Materials and Methods: The genotyping was carried for
MTHFR 677C-T, 1298A-C, 1p36.3 (n=105), MTR 2756A-
G, 1q43, F5 1691G-A, 1q24.2 (n=50), LCT C/T(-13910),
G/A(-22018),
2q21.3
(n=34),
VDR3
61888G>T,
61968T>C, 12q13.11 (n=100). The chromosome structure
was analyzed using classical cytogenetics methods, GTG,
FISH (n=6156). Exogamy degree scale: from 1 - parentage
originated from one settlement to 4 - interethnic marriage. Inbreeding level was measured with FST. The linkage
disequilibrium (LD) was estimated by D’, r2. Results: FST in urban and rural Ukrainian populations in
2016 were 81.6×10-6 and 124.1×10-6, increased over past 30
years up to 4 times. D’ (r2) for SNPs analyzed were: 1p36.3
- 0.045 (0.010), 1q43/1q24.2 - 0.025 (0.010), 2q21.3 -
0.209
(0.554),
12q13.11
-
0.039
(0.006). Increased
exogamy degree was associated with increased D’ (r2) in
loci 1p36.3: 0.027 - 0.081 (0.003 - 0.025), 1q43/1q24.2:
0.015 - 0.027 (0.002 - 0.004) and 12q13.11: 0.043 - 0.073
(0.008 - 0.023). Among all detected balanced translocations
(n=69, 1.12%), 24.6% were in: 1p36.3 (2.9%), 1q43
(5.8%), 1q24.2 (4.4%), 2q21.3 (10.1%), 12q13.11 (1.4%). There were no translocations in other loci of chromosomes
analyzed. Conclusions: The birth prevalence of PWS has been
found to be as expected, but the prevalence of AS and BWS
was ~1.5 times lower. Probably the real worldwide
prevalence of SRS is underestimated and may be at least
4 times higher than expected. The GNAS-gene-related IDs
(PHP/PPHP) may also be relatively frequent disorders. This
work was supported by the Estonian Research Council grant
PUT355. Conclusions: The birth prevalence of PWS has been
found to be as expected, but the prevalence of AS and BWS
was ~1.5 times lower. Probably the real worldwide
prevalence of SRS is underestimated and may be at least
4 times higher than expected. The GNAS-gene-related IDs
(PHP/PPHP) may also be relatively frequent disorders. This
work was supported by the Estonian Research Council grant
PUT355. Conclusions: Considering pleiotropic effects of genes
studied, increased FST leads to multifactorial diseases. O. M. Fedota1, L. V. Roshenyuk2, Y. V. Gontar3, N. G. Lysenko4,
V. O. Babalian5, T. V. Tyzhnenko1, Y. A. Sadovnychenko4, V. M.
Vorontsov2, P. P. Ryzhko2, A. P. Gerilovych6 LD
could be used as indirect inbreeding evaluation. M. Yakoreva: None. T. Kahre: None. E. Õiglane-
Shlik:
None. M. Vals:
None. P. Mee:
None. K. Õunap: None. M. Yakoreva: None. T. Kahre: None. E. Õiglane-
Shlik:
None. M. Vals:
None. P. Mee:
None. K. Õunap: None. O.M. Fedota: None. L.V. Roshenyuk: None. Y.V. Gontar: None. N.G. Lysenko: None. V.O. Babalian:
None. T.V. Tyzhnenko: None. Y.A. Sadovnychenko:
None. V.M. Vorontsov: None. P.P. Ryzhko: None. A.P. Gerilovych: None. P18.43C
Effects of inbreeding on linkage disequilibrium for SNPs of
MTHFR, MTR, F5, LCT and VDR3 genes in Ukrainian
population Linkage disequilibrium maps in sub-Saharan African
populations Vorontsov2, P. P. Ryzhko2, A. P. Gerilovych6 R. Jabalameli, R. J. Pengelly, W. J. Tapper, S. Ennis, A. Collins R. Jabalameli, R. J. Pengelly, W. J. Tapper, S. Ennis, A. Collins Linkage disequilibrium maps in sub-Saharan African
populations The weaker LD in
African populations achieves unprecedented resolution for
functional analysis of LD maps and provides a basis for
developing integrated models of gene essentiality and
pathogenicity in the context of human disorders. Identifying patterns of linkage disequilibrium (LD) is of
paramount importance for effective application of GWAS in
mapping human complex traits. The LD structure in each
population reflects the historical impact of recombination
(along with other evolutionary forces) that shapes the cur-
rent human population. Using the Malécot-Morton model
we constructed LD maps across six sub-Saharan African
(SSA) populations using the LDMAP program. The LD
maps are constructed on the scale of linkage disequilibrium
units (LDU) in which the decline of LD to ‘background’
levels across a variable physical distance corresponds to one
LDU. Our results demonstrate that although population-
specific LDU-maps present highly concordant contours, the
magnitude of LDU steps differs across populations and is
reflective of the duration since the last effective bottleneck. Furthermore, regions of the genome that have been influ-
enced by various effective evolutionary forces such as
mutation, selection or drift may define the remaining dif-
ferences between population-specific LDU maps. Cross-
population comparison of LD maps suggests that recombi-
nation hotspots are localised in all populations and can be
leveraged in models of gene essentiality for disease gene
prediction. We posited here that recombination rate and
haplotype diversity can be considered as a proxy for gene
essentiality
and
establish
the
interesting
relationship
between gene function and LD strength. The weaker LD in
African populations achieves unprecedented resolution for
functional analysis of LD maps and provides a basis for
developing integrated models of gene essentiality and
pathogenicity in the context of human disorders. LDSR in the sex-combined analysis revealed a genetic
correlation (rg) of 0.13 (standard error, se=0.04; p = 2×10-
4) between BE/EA and BMI and 0.12 (se=0.05, p = 0.01)
between BE/EA and WHR. The sex-specific analysis
revealed a stronger correlation with BMI in women,
whereas the correlation with WHR was stronger in men. Analysis on single risk marker level revealed a statistical
significant enrichment of BMI/WHR associated variants in
BE/EA GWAS data (P=0.0086). Our results provide evidence that the WHR in men and
the BMI in women are genetically correlated with BE/EA. Our data point towards sex-specific mechanisms by which
obesity mediates the risk for developing BE/EA. Further
research into elucidation of these mechanisms is required. A.C. Böhmer: None. Genetic footprints and functional analysis of
polymorphisms in the PKLR gene Genetic footprints and functional analysis of
polymorphisms in the PKLR gene R. Jabalameli: None. R.J. Pengelly: None. W.J. Tapper: None. S. Ennis: None. A. Collins: None. O. C. L. Bezerra1, N. Mabunda2, G. Salomé2, L. A. Arnez1, F. S. G. Kehdy1, F. S. N. Manta1, R. M. Silva1, L. P. Ferreira1, R. O. Pinheiro1, A. P. Latini3, E. M. T. Santos4, E. F. Carvalho5, A. G. Pacheco1, I. V. Jani2, M. O. Moraes1 Linkage disequilibrium maps in sub-Saharan African
populations J. Hecker: None. H. Fier: None. J. Schumacher: None. Linkage disequilibrium maps in sub-Saharan African
populations Linkage disequilibrium maps in sub-Saharan African
populations O. M. Fedota1, L. V. Roshenyuk2, Y. V. Gontar3, N. G. Lysenko4,
V. O. Babalian5, T. V. Tyzhnenko1, Y. A. Sadovnychenko4, V. M. Vorontsov2, P. P. Ryzhko2, A. P. Gerilovych6 R. Jabalameli, R. J. Pengelly, W. J. Tapper, S. Ennis, A. Collins R. Jabalameli, R. J. Pengelly, W. J. Tapper, S. Ennis, A. Collins J. del Picchia 648 elucidate the overall shared genetic etiology between BE/
EA and obesity, one of the major risk factors. We applied
cross-trait LD score regression (LDSC) to summary level
results of our recently published BE/EA GWAS meta-
analysis and the GIANT consortium. For GIANT data, we
extracted genetic data for BMI, as a proxy for general
obesity, and waist-hip-ratio
(WHR), as a proxy for
abdominal obesity. LDSRs were performed both sex-
specific and sex-combined. We also performed analyses
on single marker level and compared risk alleles for all
genome-wide significant SNPs in the BMI and WHR stu-
dies with those reported in BE/EA meta-analysis. University of Southampton, Southampton, United Kingdom Identifying patterns of linkage disequilibrium (LD) is of
paramount importance for effective application of GWAS in
mapping human complex traits. The LD structure in each
population reflects the historical impact of recombination
(along with other evolutionary forces) that shapes the cur-
rent human population. Using the Malécot-Morton model
we constructed LD maps across six sub-Saharan African
(SSA) populations using the LDMAP program. The LD
maps are constructed on the scale of linkage disequilibrium
units (LDU) in which the decline of LD to ‘background’
levels across a variable physical distance corresponds to one
LDU. Our results demonstrate that although population-
specific LDU-maps present highly concordant contours, the
magnitude of LDU steps differs across populations and is
reflective of the duration since the last effective bottleneck. Furthermore, regions of the genome that have been influ-
enced by various effective evolutionary forces such as
mutation, selection or drift may define the remaining dif-
ferences between population-specific LDU maps. Cross-
population comparison of LD maps suggests that recombi-
nation hotspots are localised in all populations and can be
leveraged in models of gene essentiality for disease gene
prediction. We posited here that recombination rate and
haplotype diversity can be considered as a proxy for gene
essentiality
and
establish
the
interesting
relationship
between gene function and LD strength. P18.45A Shared genetic etiology obesity with Barrett esophagus and
esophageal adenocarcinoma: Insights from large genome-
wide association studies 1FIOCRUZ, Rio de Janeiro, Brazil, 2National Institute of
Health, Maputo, Mozambique, 3Lauro de Souza Lima Institut
(ILSL), Bauru, Brazil, 4Federal University of Minas Gerais
(UFMG), Belo Horizonte, Brazil, 5State University of Rio de
Janeiro (UERJ), Rio de Janeiro, Brazil A. C. Böhmer1,2, J. Hecker3, H. Fier3, J. Schumacher1 A. C. Böhmer1,2, J. Hecker3, H. Fier3, J. Schumacher1 A. C. Böhmer1,2, J. Hecker3, H. Fier3, J. Schumacher1 1Institute of Human Genetics, University Bonn, Bonn,
Germany, 2Life&Brain Research Center, Department of
Genomics, University Bonn, Bonn, Germany, 3Department of
Biostatistics, Harvard TH Chan School of Public Health,
Boston, MA, United States PKLR encodes for pyruvate kinase, a red blood cell enzyme
that catalyzes ATP in glycolysis. This gene is under selec-
tive pressure by Plasmodium in Africa. Mutations for PK
deficiency have a dual role: association with resistance to
malaria and susceptibility to Salmonella typhimurium. We
hypothesized that malaria selective pressure in Africa
shaped PKLR resistant genotypes in humans and those
could be an evolutionary trade off for intracellular patho-
gens. Thus, aiming to investigate the PKLR polymorphisms Barrett esophagus (BE) is a precancerous condition of
esophageal adenocarcinoma (EA), characterized by the
replacement of normal squamous epithelium by metaplastic
columnar epithelium in the distal esophagus. BE and EA are
multifactorial disorders. The aim of the present study was to Abstracts from the 51st European Society of Human Genetics Conference: Posters 649 and its association to mycobacterial pathogens, we per-
formed two independent case-control studies with leprosy
and tuberculosis in Rio de Janeiro and Mozambique. Our
results highlighted G allele of the four tested SNPs and the
haplotype G/G/G/G as associated with leprosy susceptibility
in individuals from Rio de Janeiro (haplotype frequency in
cases – 0.39 – and controls – 0.27). Subsequently, the
association was confirmed in a Mozambican TB case-
control study. The frequency of the haplotype G/G/G/G was
higher in cases (0.48) than in controls (0.38), suggesting the
same direction of association in Mozambique. Interestingly,
the frequency of the susceptibility haplotype was aug-
mented in African-ancestry cohorts from EPIGEN, Salvador
(0.56), and 1000Genomes, YRI (0.87), compared to
European-ancestry cohorts, Pelotas (0.32) and CEU (0.27). In addition, GG-genotype was correlated to higher ferritin
and haptoglobin loads in healthy subjects and leprosy
patients, which supports a relevance role of the PKLR gene
with mycobacterial infections. Finally, we evidenced a
locus of evolutionary trade off in which individuals carrying
resistant selected genotypes to malaria infection were more
susceptible to intracellular pathogens. 597 individuals from the Wellderly study. LD distances
were computed according to the Malecot-Morton model
which defines LD maps in LD units (LDUs). Results: The results suggest that LDU/cM ratios of
chromosome lengths are nearly constant in human popula-
tions, where the recombination is the primary determinant
of LD structure. A. C. Böhmer1,2, J. Hecker3, H. Fier3, J. Schumacher1 LD is more extensive in genic than
intergenic regions demonstrating raised natural selection
and
decreased
recombination
in
genes. The
overall
difference in the extent of LD between exons and introns
is small but LD is more extensive in exons. Conclusions: LD structure contains important insights
into genome function to improve understanding of disease-
causing variation. Therefore, gene-specific LD structure
may be useful to improve filtering of NGS variant lists of
disease candidates. Conclusions: LD structure contains important insights
into genome function to improve understanding of disease-
causing variation. Therefore, gene-specific LD structure
may be useful to improve filtering of NGS variant lists of
disease candidates. A. Vergara-Lope: None. R.J. Pengelly: None. A. Collins: None. P18.47C Introduction: Genetic imputation works well with frequent
alleles (minor allele frequency>1%), however, its predictive
accuracy drops for rare genetic variants which can also play
a role for eventual development of diseases. Exceptions to
such limitations are endogamous populations like Estonia. Currently, 50,000 participants of Estonian biobank have
been genotyped, with additional 100,000 being collected
within next year, and their genetic profiles will be added to
electronic health records. We performed a study to predict
and verify rare mutation carriers for severe disease predis-
positions such as familial breast cancer (in genes BRCA1
and BRCA2) and familial hypercholesterolemia (APOB)
using long range haplotyping (LRH) and “surrogate parent”
theory. Fine-scale linkage disequilibrium structure of functional
elements within genic and sub-genic sequences Fine-scale linkage disequilibrium structure of functional
elements within genic and sub-genic sequences A. Vergara-Lope, R. J. Pengelly, A. Collins A. Vergara-Lope, R. J. Pengelly, A. Collins University of Southampton, Southampton, United Kingdom University of Southampton, Southampton, United Kingdom Predicting rare allele carriers from genotyping-array data
using whole genome sequencing data in the Estonian
population O.C.L. Bezerra: None. N. Mabunda: None. G. Salomé:
None. L.A. Arnez: None. F.S.G. Kehdy: None. F.S.N. Manta: None. R.M. Silva: None. L.P. Ferreira: None. R. O. Pinheiro: None. A.P. Latini: None. E.M.T. Santos:
None. E.F. Carvalho: None. A.G. Pacheco: None. I.V. Jani: None. M.O. Moraes: None. T. Sikka1, M. Palover1, T. Nikopensius2, M. Alver1,2, M. Nelis2,
A. Metspalu1,2, N. Tõnisson2, T. Esko1,2 1University of Tartu, Tartu, Estonia, 2Estonian Genome
Center, Tartu, Estonia 1University of Tartu, Tartu, Estonia, 2Estonian Genome
Center, Tartu, Estonia Mosaic loss of chromosome Y (LOY) in blood helps explain
why men live shorter lives Mosaic loss of chromosome Y (LOY) in blood helps explain
why men live shorter lives University of Southampton, Southampton, United Kingdom Introduction: Next-generation sequencing (NGS) technol-
ogies have become high-throughput methods to identify
disease-causing
variations. However,
NGS
involves
sequencing of millions of DNA pieces simultaneously
leading to big data analyses, which may detect hundreds or
thousands of apparently deleterious, but false positive,
variants per sample. The integration of gene-specific prop-
erties, including patterns of linkage disequilibrium (LD),
may help prioritise genes most likely to be involved in
disease and reduce the number of the false positives. The
aim of this study is to analyse LD maps of the autosomal
genome at a very fine scale, down to the exonic and intronic
level, providing novel insights into the impact of recombi-
nation and selection on genome structure and function. Materials and Methods: We used whole genome
sequence data of 2,244 and genotyped data of 15,416
participants of the Estonian biobank. Whole genome
sequencing (WGS) with 30x coverage was carried out at
Broad Institute using Illumina HiSeq xTen platform. Results: WGS identified 14, 4 and 6 mutations carriers
for BRCA1, BRCA2 and APOB, respectively. We identified
16 mutations carriers for BRCA1 and 3 for BRCA2, and 5
carriers for APOB using LRH among genotyped samples. Materials and Methods: We analysed single nucleotide
polymorphism data from whole genome sequence data for 650 J. del Picchia could become a predictive biomarker for increased risk of
common disease in middle-aged and aging men. We failed to find carriers for additional 6 out of 9 mutations,
highlighting these as recent mutations and only present in
limited historical lineages. The project is funded by an ERC-StG as well as other
sources. Conclusion: LRH is a cost-effective approach to predict
additional rare mutation carriers for different disease predis-
positions from genotyped data in endogamous populations and
will be important in the process of reporting clinically relevant
mutations to Estonian biobank participants. L.A. Forsberg: A. Employment (full or part-time);
Modest; CRAY Innovation AB. E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; CRAY Innovation AB. F. Consultant/Advisory
Board; Modest; CRAY Innovation AB. J. Halvardson:
None. M. Danielsson: None. E. Ryshlicka: None. B. Torhabi
Moghadam:
None. H. Davies:
None. J. Dumanski: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; CRAY
Innovation AB. F. Consultant/Advisory Board; Modest;
CRAY Innovation AB. L.A. Forsberg: A. Employment (full or part-time);
Modest; CRAY Innovation AB. E. University of Southampton, Southampton, United Kingdom Ownership Interest
(stock, stock options, patent or other intellectual property);
Modest; CRAY Innovation AB. F. Consultant/Advisory
Board; Modest; CRAY Innovation AB. J. Halvardson:
None. M. Danielsson: None. E. Ryshlicka: None. B. Torhabi
Moghadam:
None. H. Davies:
None. J. Dumanski: E. Ownership Interest (stock, stock options,
patent or other intellectual property); Modest; CRAY
Innovation AB. F. Consultant/Advisory Board; Modest;
CRAY Innovation AB. Grants: PUT1660 & IUT20-60 (Estonian Research
Council) T. Sikka: None. M. Palover: None. T. Nikopensius:
None. M. Alver: None. M. Nelis: None. A. Metspalu:
None. N. Tõnisson: None. T. Esko: None. Genotype-phenotype correlation inMarfan spectrum:
application of NGS to study the role of rare and common
genetic variants L. A. Forsberg1, J. Halvardson2, M. Danielsson2, E. Ryshlicka2,
B. Torhabi Moghadam2, H. Davies2, J. Dumanski2 1Department of Immunology, Genetics and Pathology, Science
for Life Laboratory, Beijer Laboratory of Genome Research,
Uppsala University, Uppsala, Sweden, 2Department of
Immunology, Genetics and Pathology, Uppsala, Sweden D. Gentilini1, A. Oliveri2, T. Fazia2, A. Pini3, S. Marelli3, R. Gaetano3, L. Bernardinelli2, A. M. Di Blasio4 1Bioinformatics and Statistical Genomics Unit, Istituto
Auxologico Italiano, Cusano Milanino, Italy, 2Department of
Brain and Behavioural Sciences. University of Pavia, Pavia,
Italy, 3Centro Malattie Rare, Cardiologia, Fatebenefratelli-
Sacco, Milano, Italy, 4Medical genetics Laboratory, Istituto
Auxologico Italiano, Cusano Milanino, Italy Introduction: A growing number of papers shows that
LOY in blood cells is associated with increased risk for all-
cause mortality and disorders such as cancer, Alzheimer’s
disease, and cardiovascular disease. Known risk factors to
be affected with LOY include age, smoking and inherited
genetic variants. How much of the increased mortality of
men is associated with LOY? How can losing chromosome
Y in blood cells increase risk for disease in other organs? Introduction: Diagnosis of Marfan Spectrum is based
several clinical criteria. Although identification of patho-
genic variants contributes to the diagnostic process, its
value for the prediction of clinical outcomes is still limited. The aim of the study was to extend the genotype-phenotype
correlation starting from genetic data obtained by NGS
analysis. Materials and Methods: To answer these questions we
are studying functional consequences of LOY at DNA-,
RNA- and protein-levels. LOY-induced transcriptional
dysregulation are estimated in single cells using 10X
Chromium system and changes in plasma proteins by
Olinks proximity extension assays (PEA). FACS sorting are
used to compare levels of LOY in different immune cells in
men with cancer, Alzheimer’s and controls. Materials and Methods: A cohort of 181 patients were
analyzed. NGS panel included 10 known causative genes for
Marfan spectrum (FBN1, TGFBR1-2, COL1A1-A2, COL3A1,
COL5A1-A2, MYH11, ACTA2). An algorithm was developed
to obtain a quality control checked pedigree file containing
genotypes. Rare and common variants association analysis was
performed using the appropriate statistical method and results
were adjusted for multiple testing. Results: Our data suggests that the mechanism(s) behind
associations between LOY and a various diseases could be
related to disrupted immune cell functions in cells with
LOY. Institute of Biomedical Sciences, Taipei, Taiwan The relationships between triglyceride (TG) and elevated
glucose /HbA1c/type 2 diabetes (T2D) have been reported
in many studies. The causal relationship is ambiguous. We
used Mendelian randomization analysis to explore a causal
relation between elevated TG leading to elevated HbA1c in
16,000 participants of Taiwan Biobank Cohort. TG showed
a high correlation in T2D and hyperlipidemia, it was gen-
erate a hypothesis that TG may play an important role in
T2D and HbA1c. We selected 7 SNPs and a genetic risk
score (GRS) composed of these 7 SNPs, which were
associated with TG specifically in East Asian populations. The intercept of MR-egger regression was with a confidence
interval including the null (intercept, 0.143; 95% CI -0.868-
1.154; P=0.78), suggesting that pleiotropic effect did not
influence the result. Furthermore, the GRS was not sig-
nificantly associated with confounding factors. Based on
GRS, the association between TG and HbA1c shows that
one standard-deviation increase in TG-associated GRS was
significantly associated with a 9% increase in odds of being
high HbA1c (>6.8%) (OR: 1.09, 95% CI: 1.001-1.196,
P=0.048). The finding suggests a causal role of high TG
level leading to HbA1c. To confirm our results, we per-
formed bidirectional MR analysis. It show that the genetic
determinants of HbA1c don’t contribute to TG, suggested
by a non-significant beta value. (beta:1.748, 95% CI:-
11.497-14.9), and indicating no causal role of HbA1c level
to affect TG level. The present study supports a causal
relationship of high elevated TG resulting in high HbA1c,
and, by inference, the development of T2D. Material and Methods: All participants (10,635 unre-
lated) were selected from the TCGS (male 46.2%) with the
average age of 41.29±19.1 years which classified by JIS
definition. DNA
samples
were
genotyped
with
HumanOmniExpress-24-v1-0 bead chips. SNPs were set
according to their LD, categorized thru Haploview software
and their association was tested via kernel association test
(SKAT). For
the
purpose
of
single
analysis,
linear
regression analysis was used under recessive inheritance
model after confounders adjustment. Material and Methods: All participants (10,635 unre-
lated) were selected from the TCGS (male 46.2%) with the
average age of 41.29±19.1 years which classified by JIS
definition. DNA
samples
were
genotyped
with
HumanOmniExpress-24-v1-0 bead chips. SNPs were set
according to their LD, categorized thru Haploview software
and their association was tested via kernel association test
(SKAT). Genotype-phenotype correlation inMarfan spectrum:
application of NGS to study the role of rare and common
genetic variants Furthermore, men live on average about 6 years
shorter compared to women and new analyses of epide-
miological data shows that LOY explains up to 70% of the
increased mortality. Results: Our data suggests that the mechanism(s) behind
associations between LOY and a various diseases could be
related to disrupted immune cell functions in cells with
LOY. Furthermore, men live on average about 6 years
shorter compared to women and new analyses of epide-
miological data shows that LOY explains up to 70% of the
increased mortality. Results: More than 62% genetic variants were rare
(MAF<0.01). The overall number of rare variants was
significantly
associated
with
the
number
of
clinical
manifestations (p < 0.008). Four common variants in the
COL1A1 were significantly associated with skeletal out-
comes (p< 0.05). Both rare and common variants were
found to significantly contribute to clinical spectrum. Conclusions: The causality still remain an open question
but new data suggest that disrupted immune system
function(s) in cells with LOY could mediate reduced
protection from different disease processes. LOY in blood 651 Abstracts from the 51st European Society of Human Genetics Conference: Posters Conclusions: The genotype-phenotype correlation was
extended to the overall common and rare genetic variability
observed in NGS data. This approach increased the
percentage of phenotypic variability explained by genetic
factors. B. Sedaghati-khayat1, N. Javanrouh1, M. Hedayati1, K. Guity1,
F. Azizi2, M. S Daneshpour1 B. Sedaghati-khayat1, N. Javanrouh1, M. Hedayati1, K. Guity1,
F. Azizi2, M. S Daneshpour1 B. Sedaghati-khayat1, N. Javanrouh1, M. Hedayati1, K. Guity1,
F. Azizi2, M. S Daneshpour1 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Iran, Islamic Republic of,
2Endocrine Research Center, Research Institute for Endocrine
Sciences, Shahid Beheshti University of Medical Sciences,
Tehran, Iran, Islamic Republic of 1Cellular and Molecular Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Iran, Islamic Republic of,
2Endocrine Research Center, Research Institute for Endocrine
Sciences, Shahid Beheshti University of Medical Sciences,
Tehran, Iran, Islamic Republic of D. Gentilini: None. A. Oliveri: None. T. Fazia: None. A. Pini: None. S. Marelli: None. R. Gaetano: None. L. Bernardinelli: None. A.M. Di Blasio: None. D. Gentilini: None. A. Oliveri: None. T. Fazia: None. A. Pini: None. S. Marelli: None. R. Gaetano: None. L. Bernardinelli: None. A.M. Di Blasio: None. Institute of Biomedical Sciences, Taipei, Taiwan For
the
purpose
of
single
analysis,
linear
regression analysis was used under recessive inheritance
model after confounders adjustment. Results: The result of SKAT model among 3 SNPs sets
with the size of 5, 1 and 12 shown the last set in 3.3 kb has
significant association after multiple testing corrections
(FDR=0.0002). The result of single SNP association
analysis has shown that TG has the most significant
associated signals in this region and the remarkable lowest
p-values belonged to two variants of ZPR1 (S.E±ß;p-value,
rs2266788
“5.5±57.2;8.84E-25”
and
rs651821
“6.3
±55.6;1.91E-18”). Conclusions: This study indicates the notable association
of ZPR1 SNPs with TG blood concentration among the
Iranian population. Although most investigations of lipid
profile associated with chromosome 11 belongs to Apo A
cluster, this study suggests that neighborhood regions are
very likely to have significant effects on TG metabolism. B. Sedaghati-khayat: None. N. Javanrouh: None. M. Hedayati: None. K. Guity: None. F. Azizi: None. M. S
Daneshpour: None. B. Sedaghati-khayat: None. N. Javanrouh: None. M. Hedayati: None. K. Guity: None. F. Azizi: None. M. S
Daneshpour: None. P18.53A P.E. Wu: None. C.Y. Shen: None. P.E. Wu: None. C.Y. Shen: None. A metabolomic comparison between the ischaemic strokes
and coronary artery diseases: A Mendelian Randomization
approach A metabolomic comparison between the ischaemic strokes
and coronary artery diseases: A Mendelian Randomization
approach Mendelian Randomization Analysis to Explore a Causal
Relation between Triglyceride Levels and HbA1c Levels Introduction: Metabolic syndrome (MetS) plays the main
role as one of the risk factors of cardiovascular disease. Some studies demonstrated that 11q23-25 region played a
potential role in the pathogenesis of MetS. This study was
performed to estimate the association of 20 tagged SNPs in
BUD13/ZPR1 cluster with MetS components on Tehran
Cardio-metabolic Genetics Study (TCGS). P. E. Wu, C. Y. Shen P18.52D Conclusions: For the first time a genetic interaction was
found among NRXN2, one of GABAA-receptors and CASK
genes showing a synergetic effect of interaction between
these genes in migraine susceptibility. These genes interactions may be a small part of a higher
network of genes, allowing us to better understand migraine
etiology and leading to the development of new therapeutic
approaches. Conclusions: For the first time a genetic interaction was
found among NRXN2, one of GABAA-receptors and CASK
genes showing a synergetic effect of interaction between
these genes in migraine susceptibility. These genes interactions may be a small part of a higher
network of genes, allowing us to better understand migraine
etiology and leading to the development of new therapeutic
approaches. M. Alves-Ferreira received FCT fellowship grant [SFRH/
BD/101352/2014] M. Alves-Ferreira received FCT fellowship grant [SFRH/
BD/101352/2014] M. Alves-Ferreira: None. J.L. Neto: None. J. Pereira-
Monteiro: None. J. Sequeiros: None. I. Alonso: None. A. Sousa: None. C. Lemos: None. P18.54B A study of Kibbutzim in Israel reveals risk factors for
cardiometabolic traits and subtle population structure 1IBMC and ICBAS, Porto, Portugal, 2IBMC, Porto, Portugal Introduction: Migraine is a common neurological disorder
affecting about 15% of the general population. In the last
years, we have centered our attention to the synaptic vesi-
cles’ molecular machinery and life cycle, with a central role
in neurotransmitter release and its regulation.Neurexin
(NRXN2), a component of the synaptic vesicle machinery, P18.52D Some of the
metabolites showed associations specifically with CAD
(ApoA1/ Esterified.Cholesterol/ Fatty Acid Len/ Fatty Acid
(FA) Omega-3, Medium-LDL Phospholipd (PL) / Mono
Unsaturated FA/ Sphingomyelin /Extrasmall VLDL Parti-
cles/ Extrasmall Extrasmall VLDL triglyceride, extremely
large VLDL Lipids, extremely large VLDL PL, extremely
large VLDL particles, extremely large VLDL Triglyceride)
S. Lee: None. J. Ahn: None. J. Sung: None. Methods: Four tagging single nucleotide polymorphisms
(SNPs) of NRXN2 were analyzed in 183 cases and 265
controls. To evaluate association between NRXN2 SNPs and
migraine, a multivariable-logistic regression was performed. Allelic and haplotypic frequencies were estimated. Interac-
tion between NRXN2-SYT, NRXN2-GABRE and NRXN2-
CASK was assessed by a multivariable-logistic regression
and confirmed by a multifactor dimensionality reduction
analysis. An NMR-based metabolomics analysis was performed
for 1,000 Korean adult individuals (with ~ 230 metabolites)
with full genetic, epidemiologic and clinical information. We performed a genome-wide scan for all metabolomics
traits, and compared the results with existing compatible
metabolomics-genomics analysis. We systematically identi-
fied IVs of each metabolite using following standards: 1)
SNPs which was previous reported to have genome-wide
significance with metabolite 2) markers among 1), which
were replicated (p<0.01) in the Korean studies. We
examined metabolites’ genetic IVs for their roles in the IS
(Metastroke) with CCS subtypes, and also in CAD (using
GWAS results (CardiOgramPlusC4D). The associations
were analyzed using different threshold p-values. Results: We found two strong and significant synergistic
interactions between migraine liability and the following
gene
pairs:
NRXN2-GABRE
and
NRXN2-CASK
that
remained significant after 1000-fold permutation-based
correction. Results: We found two strong and significant synergistic
interactions between migraine liability and the following
gene
pairs:
NRXN2-GABRE
and
NRXN2-CASK
that
remained significant after 1000-fold permutation-based
correction. Among 230 metabolites, 140 overlapped with previous
metabolomics-genomics studies and were included. A total
of 114 metabolites, mainly those related to lipid metabo-
lisms were associated either or IS and CAD at p = 0.01
level. Notably, all the metabolites causally associated with
IS showed associations with CAD too. Some of the
metabolites showed associations specifically with CAD
(ApoA1/ Esterified.Cholesterol/ Fatty Acid Len/ Fatty Acid
(FA) Omega-3, Medium-LDL Phospholipd (PL) / Mono
Unsaturated FA/ Sphingomyelin /Extrasmall VLDL Parti-
cles/ Extrasmall Extrasmall VLDL triglyceride, extremely
large VLDL Lipids, extremely large VLDL PL, extremely
large VLDL particles, extremely large VLDL Triglyceride)
S. Lee: None. J. Ahn: None. J. Sung: None. Neurexin (NRXN2) and other components of the synaptic
vesicle machinery: the importance of gene-gene interaction
in migraine susceptibility E. Granot-Hershkovitz1, D. Karasik2, Y. Friedlander1, L. Rodriguez-Murillo3, R. Dorajoo4, J. Liu4, A. Sewda5, I. Peter5, S. Carmi1, H. Hochner1 Rodriguez-Murillo3, R. Dorajoo4, J. Liu4, A. Sewda5, I. Peter5, S. Carmi1, H. Hochner1 M. Alves-Ferreira1, J. L. Neto2, J. Pereira-Monteiro2, J. Sequeiros1, I. Alonso2, A. Sousa1, C. Lemos1
1IBMC and ICBAS, Porto, Portugal, 2IBMC, Porto, Portugal M. Alves-Ferreira1, J. L. Neto2, J. Pereira-Monteiro2, J. Sequeiros1, I. Alonso2, A. Sousa1, C. Lemos1 1Hebrew University-Hadassah Medical Center, Jerusalem,
Israel, 2Faculty of Medicine in the Galilee, Bar-Ilan
University, Zefat, Israel, 33Department of Genetics and
Genomic Sciences, Icahn School of Medicine at Mount Sinai,
New York, NY, United States, 4Genome Institute of Singapore,
Agency for Science, Technology and Research, Singapore,
Singapore, Singapore, 5Department of Genetics and Genomic
Sciences, Icahn School of Medicine at Mount Sinai, New York,
NY, United States 1IBMC and ICBAS, Porto, Portugal, 2IBMC, Porto, Portugal E. Granot-Hershkovitz1, D. Karasik2, Y. Friedlander1, L.
Rodriguez-Murillo3, R. Dorajoo4, J. Liu4, A. Sewda5, I. Peter5, S.
Carmi1, H. Hochner1 1Hebrew University-Hadassah Medical Center, Jerusalem,
Israel, 2Faculty of Medicine in the Galilee, Bar-Ilan
University, Zefat, Israel, 33Department of Genetics and
Genomic Sciences, Icahn School of Medicine at Mount Sinai,
New York, NY, United States, 4Genome Institute of Singapore,
Agency for Science, Technology and Research, Singapore,
Singapore, Singapore, 5Department of Genetics and Genomic
Sciences, Icahn School of Medicine at Mount Sinai, New York,
NY, United States P18.52D Genetic risk variants of ZPR1/BuD13 for metabolic
syndrome and metabolic components identified in Tehran
Cardio-metabolic Genetics Study (TCGS) S. Lee, J. Ahn, J. Sung 652 J. del Picchia forms connections between the fusion proteins of intracel-
lular and synaptic vesicles, interacting with other important
components of this mechanism as synaptotagmin, GABAA-R
or CASK. Objective: Our aim is to further explore the role and
interaction of these proteins involved in the regulatory
mechanisms
of
neurotransmitter
release,
in
migraine
susceptibility. Seoul National University, Seoul, Korea, Republic of forms connections between the fusion proteins of intracel-
lular and synaptic vesicles, interacting with other important
components of this mechanism as synaptotagmin, GABAA-R
or CASK. forms connections between the fusion proteins of intracel-
lular and synaptic vesicles, interacting with other important
components of this mechanism as synaptotagmin, GABAA-R
or CASK. Dyslipidemia is well-established, and one of the strongest
risk factors of two major cardiovascular diseases - ischae-
mic strokes (IS) and coronary artery diseases (CAD). Although IS and CAD share many risk factors, differences
are also evident; A direct comparisons between IS and CAD
has been difficult despite its potential value. Objective: Our aim is to further explore the role and
interaction of these proteins involved in the regulatory
mechanisms
of
neurotransmitter
release,
in
migraine
susceptibility. Objective: Our aim is to further explore the role and
interaction of these proteins involved in the regulatory
mechanisms
of
neurotransmitter
release,
in
migraine
susceptibility. ;
p
has been difficult despite its potential value. An NMR-based metabolomics analysis was performed
for 1,000 Korean adult individuals (with ~ 230 metabolites)
with full genetic, epidemiologic and clinical information. We performed a genome-wide scan for all metabolomics
traits, and compared the results with existing compatible
metabolomics-genomics analysis. We systematically identi-
fied IVs of each metabolite using following standards: 1)
SNPs which was previous reported to have genome-wide
significance with metabolite 2) markers among 1), which
were replicated (p<0.01) in the Korean studies. We
examined metabolites’ genetic IVs for their roles in the IS
(Metastroke) with CCS subtypes, and also in CAD (using
GWAS results (CardiOgramPlusC4D). The associations
were analyzed using different threshold p-values. Among 230 metabolites, 140 overlapped with previous
metabolomics-genomics studies and were included. A total
of 114 metabolites, mainly those related to lipid metabo-
lisms were associated either or IS and CAD at p = 0.01
level. Notably, all the metabolites causally associated with
IS showed associations with CAD too. 653 Abstracts from the 51st European Society of Human Genetics Conference: Posters Genetic studies in isolated populations have provided
increased power for identifying loci associated with com-
plex diseases and traits. We present here the Kibbutzim
Family Study (KFS), initiated for investigating environ-
mental and genetic determinants of cardiometabolic traits in
extended Israeli families living in communes characterized
by long-term social stability and homogeneous environ-
ment. Extensive information on cardiometabolic traits, as
well as genome-wide genetic data, was collected on 901
individuals, making this study, to the best of our knowl-
edge, the largest of its kind in Israel. We have thoroughly
characterized the KFS genetic structure, observing that most
participants were of Ashkenazi Jewish (AJ) origin, and
confirming a recent severe bottleneck in their recent history
(point estimates: effective size ≈450 individuals, 23 gen-
erations ago). Focusing on genetic variants enriched in KFS
compared with non-Finnish Europeans, we demonstrated
that AJ-specific variants are largely involved in cancer-
related pathways. Using linear mixed models, we conducted
an association study of these enriched variants with 16
cardiometabolic traits. We found 24 variants to be sig-
nificantly
associated
with
cardiometabolic
traits. The
strongest association,
which
we
also
replicated,
was
between a variant upstream of the MSRA gene, ≈200-fold
enriched in KFS, and weight (P=3.6∙10-8). In summary, the
KFS is a valuable resource for the study of the population
genetics of Israel as well as the genetics of cardiometabolic
traits in a homogeneous environment. MP-GWAS framework, and compared them with the CC
analysis using simulation studies. We simulated genetic data for 5,000/50,000/500,000
individuals using Hapgen2, and highly (r=0.64) and
moderately correlated (r=0.33) phenotypes (3/30/120) for
these individuals in the statistical package R. We randomly
chose common (minor allele frequency (MAF)> 5%), low-
frequency (1%<MAF<5%) and rare (MAF<1%) variants to
be associated with the simulated phenotypes. We consid-
ered 1/10/20/50% missingness under the three mechanisms:
missing completely at random (MCAR), missing at random
(MAR), and missing not at random (MNAR). The resulting betas and standard errors (SEs) from the
MP-GWAS after applying the selected methods were
compared to the true values from full data analysis as well
as to those from the CC analysis. These analyses showed
that MI/KNN/RF perform the best, followed by SI, even
under the scenario of MNAR, although SI/MI assume at
least MAR. A whole-genome sequencing study associates GRAMD1B
with Multiple Sclerosis risk and disease activity A whole-genome sequencing study associates GRAMD1B
with Multiple Sclerosis risk and disease activity E. Granot-Hershkovitz: None. D. Karasik: None. Y. Friedlander: None. L. Rodriguez-Murillo: None. R. Dorajoo: None. J. Liu: None. A. Sewda: None. I. Peter:
None. S. Carmi: None. H. Hochner: None. A. Osiceanu1, F. Esposito1,2, L. Ottoboni3, B. Bollman4, M. Sorosina1, S. Santoro1, A. Zauli1, F. Clarelli1, E. Mascia1, A. Calabria5, D. Zacchetti6, D. Lazarevic7, D. Cittaro7, P. Carrera8,
D. Toniolo9, A. Sadovnik10, G. Comi2,1, E. Stupka7, C. Vilariño-
Güell10, L. Piccio4, F. Martinelli Boneschi11,1,12 For the CC analysis, the performance worsened
when the number of phenotypes was increased, while the
other approaches were not influenced by the number of
phenotypes
nor
differences
in
phenotype
correlations
or MAF. In conclusion, we recommend MI/KNN/RF imputation
approaches over the commonly applied CC analysis,
especially KNN/RF under MNAR. Funding: DYNAhealth-H2020-PHC-2014-633595. Funding: DYNAhealth-H2020-PHC-2014-633595. M.D. Anasanti:
None. M. Kaakinen:
None. I. Prokopenko: None. This study was supported by Israeli Science Foundation
grant 201/98-1 and partially by National Institutes of Health
research grant R01HL088884. M. D. Anasanti, M. Kaakinen, I. Prokopenko M. D. Anasanti, M. Kaakinen, I. Prokopenko 1Laboratory of Human Genetics of Neurological Disorders,
Institute of Experimental Neurology (INSPE), Division of
Neuroscience, San Raffaele Scientific Institute, Milan, Italy,
2Department of Neurology, Institute of Experimental
Neurology (INSPE), Division of Neuroscience, San Raffaele
Scientific Institute, Milan, Italy, 3Unit of Neuroimmunology,
Institute of Experimental Neurology (INSpe), Division of
Neuroscience, San Raffaele Scientific Institute,, Milan, Italy,
4Department of Neurology, Washington University School of
Medicine,, St Louis, MO, United States, 5San Raffaele Telethon
Institute for Gene Therapy (SR-Tiget), Milan, Italy, 6Unit of
Cellular Neurophysiology, Division of Neuroscience, San
Raffaele Scientific Institute,, Milan, Italy, 7Center for Modern approaches to address missing data in multi-
phenotype genome-wide association studies Modern approaches to address missing data in multi-
phenotype genome-wide association studies Imperial College London, London, United Kingdom Multi-phenotype genome-wide association studies (MP-
GWAS) play an important role in improving the power for
locus discovery. However, joint analysis of multiple phe-
notypes increases the proportion of missingness, leading the
standardly applied complete case (CC) analysis to become
ineffiecient. We investigated the properties of single
imputation
(SI),
multiple
imputation
(MI),
k-Nearest
Neighbour (k-NN), and Random Forest (RF) within the J. del Picchia 654 M. Vikkula1, N. Revencu2, A. Dompmartin3, E. Khoury4, A.
Mendola4, N. Limaye4, R. Helaers4, E. Fastré4, M. Schlögel4, N.
Sepehr4, P. Brouillard4, L. Boon5 Methods: Data was collected from genetic analyses
performed on >1800 vascular anomaly tissues obtained
during surgical treatments or as biopsies. This represented
about 1000 individuals. Mutations were screened for using
targeted NGS for high coverage to identify low frequency
somatic mutations in heterogeneous samples. Clinical
diagnosis was compared to the genetic results. Results:
We
identified
a
missense
c.1801T>C
(p. Ser601Pro) variant in GRAMD1B gene located at the
linkage peak (LOD: 2.194). Target sequencing of the gene
revealed additional rare missense and splice-site variants, 2
of which (rs755488531 and rs769527838) absent in 1000
Italian healthy controls, and an additional missense variant
(rs199604534) in 4 MS Canadian families. Functional
studies demonstrated that GRAMD1B, a gene with unknown
function, is expressed by different CNS and peripheral
immune cells. Notably, GRAMD1B was downregulated in
vessel-associated astrocytes of active multiple sclerosis
lesions in autopsied brains (p<0.05) and by inflammatory
stimuli in primary astrocytes (p = 0.007) and peripheral
monocytes (p = 0.0002). Results: A causative mutation was identified in > 45% of
patients; sometimes it was a germline mutation. PIK3CA
was the most frequently mutated: >80% of isolated and
syndromic LMs, and 20% of VMs. Somatic TIE2 mutation
was identified in 60% of VMs, and GNAQ or GNA11
mutation in 70% of SWS and CMs. The genetic result fit
with the clinico-radiologic diagnosis in most cases (+/- 920/
1000 = 92%), but resulted in revised diagnosis in others. Importantly, it was helpful for differential diagnosis of
lesions of small size, deeper localization and unusual
presentation. Series of lesions without any mutation were
also identified. They often clinically differed from those
with a mutation. Conclusions:
These
findings
suggest
a
role
of
GRAMD1B in inflammation and disease pathophysiology
and open new avenues of investigation. Conclusions: Genetic testing has become a valuable tool
for management of vascular anomalies. It is useful for
confirming and guiding towards correct diagnosis, for
recognising rare entities, and for identifying persons with
a familial risk. It also allows to identify unclassified entities,
which need further clinico-genetic characterisation. A. Osiceanu: None. F. Esposito: None. L. Ottoboni:
None. B. Bollman: None. M. Sorosina: None. S. Santoro: A. Osiceanu: None. F. Esposito: None. L. Ottoboni:
None. B. Bollman: None. M. Sorosina: None. S. Santoro:
None. A. Zauli: None. F. Clarelli: None. E. Mascia:
None. A. Calabria: None. D. Zacchetti: None. D. Lazarevic: None. D. Cittaro: None. P. Carrera: None. D. Toniolo: None. A. Sadovnik: None. G. M. Vikkula1, N. Revencu2, A. Dompmartin3, E. Khoury4, A.
Mendola4, N. Limaye4, R. Helaers4, E. Fastré4, M. Schlögel4, N.
Sepehr4, P. Brouillard4, L. Boon5 1de Duve Institute, Université catholique de Louvain, Brussels,
Belgium, 2Center for Human Genetics, Cliniques universitaires
Saint-Luc, Université catholique de Louvain, Brussels,
Belgium, 3Department of Dermatology, Université de Caen
Basse Normandie, CHU Caen, Caen, France, 4Human
Molecular Genetics, de Duve Institute, Université catholique
de Louvain, Brussels, Belgium, 5Center for Vascular
Anomalies, Division of Plastic Surgery, Cliniques
universitaires Saint-Luc, Université catholique de Louvain,
Brussels, Belgium Milan, Italy Introduction: While the role of common genetic variants in
multiple sclerosis (MS) has been elucidated, the contribu-
tion of rare variants remains unclear. We performed a
whole-genome sequencing (WGS) study in an Italian family
with four relatives with MS. Materials: A 40x-10x coverage WGS was performed in 4
affected and 4 unaffected relatives on an Illumina® HiSeq
2000 instrument, and a bioinformatic pipeline used to filter
and prioritize rare functional variants. We used HumanOm-
niexpress array and the Merlin software for linkage
analyses. A target sequencing of GRAMD1B was applied
in 91 probands of familial MS cases. GRAMD1B gene
expression in rat cells and tissues and in human peripheral
blood immune cells was evaluated by qRT-PCR, while the
protein expression was evaluated in immune and brain cells
and brain tissues by immunofluorescence. Materials: A 40x-10x coverage WGS was performed in 4
affected and 4 unaffected relatives on an Illumina® HiSeq
2000 instrument, and a bioinformatic pipeline used to filter
and prioritize rare functional variants. We used HumanOm-
niexpress array and the Merlin software for linkage
analyses. A target sequencing of GRAMD1B was applied
in 91 probands of familial MS cases. GRAMD1B gene
expression in rat cells and tissues and in human peripheral
blood immune cells was evaluated by qRT-PCR, while the
protein expression was evaluated in immune and brain cells
and brain tissues by immunofluorescence. Introduction: Differential diagnosis of vascular mal-
formations can be difficult. The identification of somatic
genetic causes for many has opened the door for systematic
genetic testing as an aide in clinical assessment. We eval-
uated the usefulness of such testing. Methods: Data was collected from genetic analyses
performed on >1800 vascular anomaly tissues obtained
during surgical treatments or as biopsies. This represented
about 1000 individuals. Mutations were screened for using
targeted NGS for high coverage to identify low frequency
somatic mutations in heterogeneous samples. Clinical
diagnosis was compared to the genetic results. P18.59C
Genetic testing in the diagnostic workup of vascular
anomalies Translational Genomics and Bionformatics, IRCCS San
Raffaele Scientific Institute, Milan, Italy, 8Unit of Genomics for
human disease diagnosis, Department of Genetics and Cell
Biology, San Raffaele Scientific Institute, Milan, Italy,
9Department of Genetics and Cell Biology, San Raffaele
Scientific Institute,, Milan, Italy, 10Department of Medical
Genetics, University of British Columbia, Vancouver, BC,
Canada, vancouver, BC, Canada, 11Department of Biomedical
Sciences for Health, University of Milan, Milan, Italy,
12Department of Neurology, IRCCS Policlinico San Donato,
Milan, Italy P18.59C M. Vikkula1, N. Revencu2, A. Dompmartin3, E. Khoury4, A. Mendola4, N. Limaye4, R. Helaers4, E. Fastré4, M. Schlögel4, N. Sepehr4, P. Brouillard4, L. Boon5 S. Karachanak-Yankova1, Z. Hammoudeh1, D. Nesheva1, A. S.
Galabov2, D. Toncheva1 1Federal State Budgetary Institution «Research Centre for
Medical Genetics», Moscow, Russian Federation, 2Research
Institute of Clinical Medicine, Tbilisi, Georgia, 3Childrens New
Hospital, Tbilisi, Georgia, 4New Vision University, Tbilisi,
Georgia, 5Georgian Foundation for Genetic and Rare
Diseases, Tbilisi, Georgia 1Department of Medical Genetics, Medical Faculty, Medical
University-Sofia, Sofia, Bulgaria, 2The Stephan Angeloff
Institute of Microbiology, Bulgarian Academy of Sciences,
Sofia, Bulgaria 1Department of Medical Genetics, Medical Faculty, Medical
University-Sofia, Sofia, Bulgaria, 2The Stephan Angeloff
Institute of Microbiology, Bulgarian Academy of Sciences,
Sofia, Bulgaria Introduction: Georgia is located in the Caucasus region of
Eurasia and populated mostly by Georgians. Due to the
remoteness of Georgians from the main routes of incursions
and migrations, the territory of Georgia was the object of
great demographic homogeneity. This allows us to assume
the existence of a unique mutation spectrum in monogenic
disease genes in this country. Introduction: Although mitochondrial DNA (mtDNA)
variants that define haplogroups are generally considered as
neutral, they may modify the impact of drugs, toxic envir-
onmental exposures or other factors. The possible linkage of
mtDNA haplogroups with sensitivity to certain drugs and
adverse drug reactions was established in several studies. The aim of our research was to determine the mtDNA
haplogroups with pharmacogenetic effect found in the
Bulgarian population. Introduction: Although mitochondrial DNA (mtDNA)
variants that define haplogroups are generally considered as
neutral, they may modify the impact of drugs, toxic envir-
onmental exposures or other factors. The possible linkage of
mtDNA haplogroups with sensitivity to certain drugs and
adverse drug reactions was established in several studies. The aim of our research was to determine the mtDNA
haplogroups with pharmacogenetic effect found in the
Bulgarian population. Materials and Methods: DNA samples of 135 PKU
patients from Georgia were analyzed for the presence of 25
common PAH gene mutations using allele-specific MLPA
method. Сalculations were made for 124 unrelated probands
(248 chromosomes). Materials and Methods: DNA samples of 135 PKU
patients from Georgia were analyzed for the presence of 25
common PAH gene mutations using allele-specific MLPA
method. Сalculations were made for 124 unrelated probands
(248 chromosomes). Materials and Methods: The analyzed sample com-
prised 855 healthy unrelated Bulgarian subjects. Their
mtDNA
haplogroup
classification
was
performed
by
sequencing of the control region of mtDNA in blood
DNA samples, followed by genotyping of coding region
markers for confirmation of haplogroup assignment. Results: PAH gene mutations were detected on 85.1% of
chromosomes. P18.61A
Mitochondrial DNA haplogroups in Bulgarians with
potential pharmacogenetic effect P. Gundorova1, I. A. Kuznetsova1, D. Agladze2, L. Margvelashvili3, E. Kldiashvili4, O. Kvlividze5, A. V. Polyakov1 P. Gundorova1, I. A. Kuznetsova1, D. Agladze2, L. Margvelashvili3, E. Kldiashvili4, O. Kvlividze5, A. V. Polyakov1 S. Karachanak-Yankova1, Z. Hammoudeh1, D. Nesheva1, A. S. Galabov2, D. Toncheva1 S. Karachanak-Yankova1, Z. Hammoudeh1, D. Nesheva1, A. S.
Galabov2, D. Toncheva1 Agladze: None. L. Margvelashvili: None. E. Kldiashvili:
None. O. Kvlividze: None. A.V. Polyakov: None. P. Gundorova: None. I.A. Kuznetsova: None. D. Agladze: None. L. Margvelashvili: None. E. Kldiashvili:
None. O. Kvlividze: None. A.V. Polyakov: None. S. Karachanak-Yankova1, Z. Hammoudeh1, D. Nesheva1, A. S.
Galabov2, D. Toncheva1 At least one pathogenic allele was detected
in 94.4% cases. The most common PAH gene mutation
among patients from Georgia is P281L (33.5% of alleles). The following frequent variants are IVS10-11G>A (20.2%),
R261* (7.7%), L48S (4.8%), R261Q (3.6%), R408W
(3.2%), E390G
(2.8%),
IVS2+5G>C
(2.4%),
E280K
(1.6%), R252W (1.2%), A300S (1.2%). Also 7 different
pathogenic variants were found in single cases. Results: The Bulgarian mtDNA gene pool is over-
whelmingly represented by Western Eurasian haplogroups
(H, U, JT, W, X, V, I). The most common haplogroup H
(41.9%) can lead to toxicity of highly active anti-retroviral
therapy (HAART) with increased lipoatrophy. Haplogroup
T, found in 10.6% of modern Bulgarians, may be protective
against lipoatrophy in HAART, but it is associated with
peripheral neuropathy in treatment with nucleoside reverse
transcriptase inhibitors. The carriers of non-haplogroup H
mtDNA lineages have better respond to riboflavin in
migraine treatment. MtDNA haplogroup J (7.9%) shows
linkage with cisplatin-induced hearing impairment. therapy (HAART) with increased lipoatrophy. Haplogroup
T, found in 10.6% of modern Bulgarians, may be protective
against lipoatrophy in HAART, but it is associated with
peripheral neuropathy in treatment with nucleoside reverse
transcriptase inhibitors. The carriers of non-haplogroup H
mtDNA lineages have better respond to riboflavin in
migraine treatment. MtDNA haplogroup J (7.9%) shows
linkage with cisplatin-induced hearing impairment. Conclusions: Due to the PAH gene mutation spectrum,
we can conclude, that Georgians are genetically far from
nations living in contiguous territories. This feature is
characteristic of most of the ethnic groups living in the
Caucasus - they are quite original and preserved their
national
identity. Nevertheless,
the
frequent
mutation
method that was originally created for patients from Russia
is suitable for diagnosing patients with PKU from Georgia. Conclusions: Due to the PAH gene mutation spectrum,
we can conclude, that Georgians are genetically far from
nations living in contiguous territories. This feature is
characteristic of most of the ethnic groups living in the
Caucasus - they are quite original and preserved their
national
identity. Nevertheless,
the
frequent
mutation
method that was originally created for patients from Russia
is suitable for diagnosing patients with PKU from Georgia. Conclusion: The accumulation of knowledge about the
prevalence of mtDNA pharmacogenetic variants including
those that are haplogroup defining in healthy and in subjects
with adverse drug reactions in different populations will
improve mitochondrial pharmacogenomics - a lagging area
in the improvement of drug therapy. P. Gundorova: None. I.A. Kuznetsova: None. D. M. Vikkula1, N. Revencu2, A. Dompmartin3, E. Khoury4, A.
Mendola4, N. Limaye4, R. Helaers4, E. Fastré4, M. Schlögel4, N.
Sepehr4, P. Brouillard4, L. Boon5 Comi: None. E. Stupka: None. C. Vilariño-Güell: None. L. Piccio: None. F. Martinelli Boneschi: None. M. Vikkula: None. N. Revencu: None. A. Dompmar-
tin: None. E. Khoury: None. A. Mendola: None. N. Limaye: None. R. Helaers: None. E. Fastré: None. M. Abstracts from the 51st European Society of Human Genetics Conference: Posters 655 P18.62B
Mutation spectrum of PAH gene in phenylketonuria
patients from Georgia Schlögel: None. N. Sepehr: None. P. Brouillard: None. L. Boon: None. 1IRCCS-Foundation Neurological Institute "C.Besta", Milan,
Italy, 2University "Federico II", Naples, Italy 1IRCCS-Foundation Neurological Institute "C.Besta", Milan,
Italy, 2University "Federico II", Naples, Italy 1IRCCS-Foundation Neurological Institute "C.Besta", Milan,
Italy, 2University "Federico II", Naples, Italy Introduction: NIPT for common aneuploidies is one of
the most rapidly adopted and relatively low-cost DNA
tests. However, the information from the resulting data are
not used up to their full potential. High throughput
sequencing on pooled samples, each sample from differ-
ent individual, is a strategy to identify genetic variability
at a small fraction of the cost required to do population
scale studies. Here we describe the possible re-use of the
data generated during NIPT for genome scale population
specific frequency determination of single nucleotide
variants (SNV). Introduction: Polyglutamine (PolyQ) diseases are inherited
neurodegenerative disorders, commonly manifesting in
adulthood, and caused by CAG repeat expansions in the
coding regions of the respective disease-genes. Alleles with
CAG
repeat
number
slightly
below
the
pathological
threshold are defined as intermediate alleles (IAs). IAs are
associated with reduced penetrance and meiotic instability,
and may be responsible for new diagnosis in subjects
without family history. In Italy the most common polyQ
diseases are Spinocerebellar Ataxia type 1-2-17 (SCA1-2-
17) and Huntington disease (HD). We aimed to investigate
the frequencies of IAs in HD, SCA 1-2-17 in healthy Italian
subjects. Introduction: Polyglutamine (PolyQ) diseases are inherited
neurodegenerative disorders, commonly manifesting in
adulthood, and caused by CAG repeat expansions in the
coding regions of the respective disease-genes. Alleles with
CAG
repeat
number
slightly
below
the
pathological
threshold are defined as intermediate alleles (IAs). IAs are
associated with reduced penetrance and meiotic instability,
and may be responsible for new diagnosis in subjects
without family history. In Italy the most common polyQ
diseases are Spinocerebellar Ataxia type 1-2-17 (SCA1-2-
17) and Huntington disease (HD). We aimed to investigate
the frequencies of IAs in HD, SCA 1-2-17 in healthy Italian
subjects. Materials and Methods: To evaluate the limitations of
this approach, data generated by massively parallel low
coverage whole-genome sequencing of plasma DNA of
1,548 pregnant women
undergoing NIPT
procedure
was analysed. We used statistical methods, such as
PCA, for visualization and comparison our data to other
studies. Materials and Methods: To evaluate the limitations of
this approach, data generated by massively parallel low
coverage whole-genome sequencing of plasma DNA of
1,548 pregnant women
undergoing NIPT
procedure
was analysed. We used statistical methods, such as
PCA, for visualization and comparison our data to other
studies. 1IRCCS-Foundation Neurological Institute "C.Besta", Milan,
Italy, 2University "Federico II", Naples, Italy A. Castaldo: None. A. Mongelli: None. L. Sarro: None. A. Castaldo: None. E. Rizzo: None. C. Gellera: None. E. Salvatore: None. F. E. Rizzo: None. C. Gellera: None. E. Salvatore: None. F. Taroni: None. C. Mariotti: None. L. Nanetti: None. 1Faculty of Natural Sciences, Comenius University, Bratislava,
Slovakia, 2Geneton Ltd., Bratislava, Slovakia, 3Faculty of
mathematics, physics and informatics, Comenius University, 1IRCCS-Foundation Neurological Institute "C.Besta", Milan,
Italy, 2University "Federico II", Naples, Italy Materials and Methods: We enrolled 366 healthy Italian
subjects (177M/189F, aged 19-84) without familiar history. All subjects were informed of the purpose of the study and
gave their written consent for CAG repeat screening in
SCA1-2-17 and HD genes, according to European guide-
lines EMQN. Genetic counseling was offered to the
individuals with IAs. Results: We detected 6,622,893 (0.21% of the genome)
distinct potential SNVs. Nearly 98% of the variants were
found to be already known in dbSNP allowing verification
of our results on several levels. Allelic frequency distribu-
tions of our population (Central Europe) were compared to
the six ExAC populations placing our sample set most
closely to the two European ExAC populations that is
consistent with the geographical and genetic relationships
between the compared populations. Results: One SCA1-IA (35 CAG; 0.1%) and three
SCA17-IAs (42-44 CAG; 0.4%) were found in 4 unrelated
individuals. Two subjects carried a SCA2-IA with 31 CAG
repeats (0.3%), and 16 subjects had HD-IAs associated with
meiotic instability (27-35 CAG; 2.2%). Conclusions: The allelic frequencies of IAs in SCA1-2-
17 and HD diseases in Italian population were similar to
those previously reported. SCA1-2-17 IAs may be asso-
ciated with extremely late-onset symptomatology. More-
over, SCA2-IAs are recognized as risk factors for ALS
susceptibility. The high frequency of HD-IAs may represent
a reservoir for fully pathological expanded alleles. Conclusion: Re-using NIPT data to determine population
frequencies of small DNA variants can be a real low-cost
alternative to costly population scale studies. J. Gazdarica: A. Employment (full or part-time);
Significant; Geneton Ltd., Bratislava, Slovakia. J. Budiš:
A. Employment (full or part-time); Significant; Geneton
Ltd., Bratislava, Slovakia. J. Radvanszky: A. Employment
(full or part-time); Modest; Geneton Ltd., Bratislava,
Slovakia. M. Haršanyová: A. Employment (full or part-
time); Modest; Geneton Ltd., Bratislava, Slovakia. I. Gazdaricová: None. L. Striešková: A. Employment (full
or part-time); Modest; Geneton Ltd., Bratislava, Slovakia. F. Duriš: A. Employment (full or part-time); Modest;
Geneton Ltd., Bratislava, Slovakia. O. Pös: None. M. Böhmer: None. G. Minárik: A. Employment (full or part-
time); Significant; Medirex a.s., Bratislava, Slovakia. J. Turňa: None. T. Szemes: A. Employment (full or part-
time); Significant; Geneton Ltd., Bratislava, Slovakia. M. Lichvár: A. Employment (full or part-time); Significant;
Geneton Ltd.. A. Mongelli: None. L. Sarro: None. A. Castaldo: None. E. Rizzo: None. C. Gellera: None. E. Salvatore: None. F. Taroni: None. C. Mariotti: None. L. Nanetti: None. A. Mongelli: None. L. Sarro: None. J. Gazdarica1,2, J. Budiš3,2,4, J. Radvanszky1,2, M. Haršanyová1,2,
I. Gazdaricová1, L. Striešková1,2, F. Duriš4,2, O. Pös1, M.
Böhmer1, G. Minárik5,6, J. Turňa1,4,7, T. Szemes1,2,7, M. Lichvár2 P18.63C Neurodegenerative disorders associated with polyglutamine
expansions: an Italian study to investigate the prevalence of
Intermediate polyQ alleles in healthy subjects Neurodegenerative disorders associated with polyglutamine
expansions: an Italian study to investigate the prevalence of
Intermediate polyQ alleles in healthy subjects S. Karachanak-Yankova:
None. Z. Hammoudeh:
None. D. Nesheva: None. A.S. Galabov: None. D. Toncheva: None. S. Karachanak-Yankova:
None. Z. Hammoudeh:
None. D. Nesheva: None. A.S. Galabov: None. D. Toncheva: None. 656 J. del Picchia A. Mongelli1, L. Sarro1, A. Castaldo1, E. Rizzo1, C. Gellera1, E. Salvatore2, F. Taroni1, C. Mariotti1, L. Nanetti1 Bratislava, Slovakia, 4Slovak centre of scientific and technical
information, Bratislava, Slovakia, 5Medirex a.s., Bratislava,
Slovakia, 6Trisomy Test Ltd., Bratislava, Slovakia, 7Comenius
University Science Park, Bratislava, Slovakia P18.66B J. Giemza1, M. Karakachoff1,2, E. Charpentier1,2, K. Rouault3,4,5,
A. Saint-Pierre3,4,5, F. Simonet1,2, S. Lecointe1,2, P. Lindenbaum1, C. Férec3,4,5, H. Le Marec1,2, S. Chatel1,2, J. Schott1,2, E. Génin3,4,5, R. Redon1,2, C. Dina1,2 C. L. Hernández1, G. Pita2, B. Cavadas3, J. Dugoujon4, A. Novelletto5, L. Pereira3, R. Calderón1 1Departamento de Biodiversidad, Ecología y Evolución. Facultad de Biología. Universidad Complutense de Madrid,
Madrid, Spain, 2Centro Nacional de Investigaciones
Oncológicas (CNIO), Madrid, Spain, 3Instituto de Patologia e
Imunologia Molecular da Universidade do Porto (IPATIMUP),
Porto, Portugal, 4Laboratoire d’Anthropologie, FRE 2960,
Centre National de la Recherche Scientifique (CNRS),
Université Paul Sabatier, Toulouse, France, 5Dipartimento di
Biologia, Università Tor Vergata, Rome, Italy 1l'institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes,
France, 2l'institut du thorax, CHU Nantes, Nantes, France,
3Université de Bretagne Occidentale, Brest, France, 4Inserm
UMR1078, Brest, France, 5Centre Hospitalier Régional
Universitaire de Brest, Brest, France Characterising the genetic structure of human populations
provides insight into demographical history and informs
research on disease association studies, especially on rare
recent variants which tend to be geographically clustered. In
this study, we examine the fine-scale genetic structure of
Brittany, Anjou, Poitou and Maine in western France. Introduction: In the last few years, genome-wide (GW)
studies have become essentials in the fields of human
genetics and genomic anthropology. Here, we performed a
GW screening of a key geographic area: the western
Mediterranean. Its
metapopulation,
at
the
crossroads
between Europe and Africa, is a target for the study of
human evolutionary history across continents. y
j
We genotyped 2,500 individuals whose 4 grandparents
were born within a small distance in western France on
Affymettrix Axiom PMRA array plates. Principal Compo-
nents are highly correlated with geographical coordinates of
grandparents' birthplaces (p-value < 2e-16). Visualisation of
single principal components' values (from PC1 to PC5) on
the map reveals patterns of local genetic structure. Those
patterns are confirmed when samples are assigned their
most probable source populations from ADMIXTURE. Loire River and its tributaries, Erdre and Sèvre Nantaise,
seem to be at the limits of observed genetic subpopulations. Using Identity By Descent sharing (IBDNe), we show that
identified subgroups may have followed different trajec-
tories of effective population size evolution in the last 25
generations. Materials and Methods: We have analyzed six popula-
tions from southern Iberia and Morocco. Altogether,
142 samples were genotyped on Illumina’s Omni2.5 array. A systematic pipeline was performed to merge our dataset
with previously reported GW information. Non-invasive prenatal testing as a valuable source of
population specific allelic frequencies Non-invasive prenatal testing as a valuable source of
population specific allelic frequencies J. Gazdarica1,2, J. Budiš3,2,4, J. Radvanszky1,2, M. Haršanyová1,2,
I. Gazdaricová1, L. Striešková1,2, F. Duriš4,2, O. Pös1, M. Böhmer1, G. Minárik5,6, J. Turňa1,4,7, T. Szemes1,2,7, M. Lichvár2 657 Abstracts from the 51st European Society of Human Genetics Conference: Posters Department of Paediatrics, The University of Hong Kong,
Hong Kong, Hong Kong N. Maksimovic1, V. Vidovic2, T. Damnjanovic1, B. Jekic1, D. Perovic1, S. Vidovic2, I. Milovac2, I. Novakovic1 The healthcare burden of rare diseases (RD) is important but
difficult to estimate. Walker et al. (2017) made use of cross-
referencing between ORPHA codes and ICD-10 in health
administrative datasets to identify RD-related admissions in
Western Australia. Such methodology was adopted in Hong
Kong which has recently awakened to the needs of RD
patients. 1Institute of Human Genetics, Faculty of Medicine, University
of Belgrade, Belgrade, Serbia, 2Faculty of Medicine,
University of Banja Luka, Banja Luka, Bosnia and
Herzegovina Introduction: Brown adipose tissue (BAT) presence in
adolescents and adults has raised many questions about its
physiology and possible influence on human health. In
healthy adults, presence of BAT was associated with lower
glucose levels, total cholesterol and LDL and levels. The
key
regulator
of
BAT
development
is
PR
domain-
containing protein 16 (PRDM16). It induces brown fat
phenotype by interaction with PGC1α, PPARα, PPARγ, C/
EBP and represses white adipose tissue specific genes
through
the
association
with
C-terminal
binding
co-
repressor proteins (CtBP1 and CtBP2). Our aim was to
analyze the association of PRDM16 gene (rs12409277) and
CtBP2 gene (rs1561589) polymorphisms with BMI, fasting
glucose level and lipid profile of adolescents. Methods: We extracted from the local public healthcare
database admission records of all patients coded with one or
more of the 1084 ICD-10 codes cross referenced with 467
ORPHA codes from 2005 to 2016. We further analyzed
RD-related inpatient healthcare cost using a subset of
patients admitted during 2015 -2016. Results: A total of 546,673 admissions were identified,
representing 3.2% of total admission in 2005-2016. About
109,000 patients were alive at the end of the study,
representing 1.5% of the Hong Kong population. The most
common RD category in the pediatric age group was ‘rare
developmental defects during embryogenesis’; whereas that
amongst adults was ‘rare haematological disease’. The
aforementioned
subset
for
2015-2016
accounted
for
330,091 patient-days, placing the estimated total inpatient
cost for RD at HKD$1,594,339,530 (~EUR 163,612,000)
i.e. 4.3% of total inpatient cost. Material and Methods: Our study included 300 healthy
school children, 146 boys (48.7%) and 154 girls (51.3%),
15 years of age. Genotypes for the selected polymorphisms
were detected by the Real-time PCR method. Age, gender,
height,
weight,
lipid
profile
(total
cholesterol,
HDL
cholesterol, LDL cholesterol, triglycerides) and fasting
glucose were recorded. Healthcare burden of rare diseases in Hong Kong - the use
of ORPHA codes in an ICD-10 based healthcare
administrative dataset Association of PRDM16 and CtBP2 genes polymorphisms
with lipid profile of adolescents Department of Paediatrics, The University of Hong Kong,
Hong Kong, Hong Kong P18.69A
Explaining RLS families using risk SNPs from GWAS Conclusion: Our study suggests that rs12409277 and
rs1561589 polymorphism might have influence on total and
LDL cholesterol levels in adolescents. E. Tilch1,2, C. Zhao1, A. Salminen1, A. Antic1, B. Schormair1, K. Oexle1, EU-RLS-Gene consortium, J. B. Sampson3, B. Müller-
Myhsok4,5,6, J. Winkelmann1,2,5 N. Maksimovic: None. V. Vidovic: None. T. Damnja-
novic: None. B. Jekic: None. D. Perovic: None. S. Vidovic: None. I. Milovac: None. I. Novakovic: None. 1Institute of Neurogenomics, Helmholtz Zentrum München,
Neuherberg, Germany, 2Neurologische Klinik und Poliklinik,
Klinikum rechts der Isar der Technischen Universität
München, Munich, Germany, 3Neurology, Stanford University,
Stanford, CA, United States, 4Statistical Genetics, Max Planck P18.69A P18.69A
Explaining RLS families using risk SNPs from GWAS Department of Paediatrics, The University of Hong Kong,
Hong Kong, Hong Kong Conclusion: Cross referencing between ICD-10 and
ORPHA codes may be adopted in different healthcare
datasets for international comparison. Despite differences in
the prevalence of individual diseases, the disparity between
RD prevalence and associated inpatient cost is consistent
across settings reflects the importance of RD in healthcare
policies. Results: We did not find statistically significant associa-
tion between rs12409277 and rs1561589 polymorphisms
with BMI, fasting glucose and lipid profile of adolescents. We further analysed combine effect of the two SNPs. Statistical analysis has shown that carriers of CT genotype
of
rs12409277
polymorphism
and
GG
genotype
of
rs1561589 polymorphism had significantly lower total
cholesterol (p = 0.001) and LDL cholesterol (p = 0.008)
level when compared to all other groups of genotypes. Acknowledgement: Thanks the Society for the Relief of
Disabled Children (Hong Kong) for funding support B. Chung: None. A. Chiu: None. C. Chung: None. W. Wong: None. P18.67C Association of PRDM16 and CtBP2 genes polymorphisms
with lipid profile of adolescents P18.66B Depending on
the SNP density (1.8M and 84k markers), two resolution
levels were stablished. By using bioinformatic tools,
demographic histories, admixture dynamics and genetic
structure of the Mediterranean populations was assessed,
along with global/local ancestries, admixture time model-
ling and the magnitude of population relationships. Results: Our data showed a latitudinal gradient from
northern to southern Europe with respect to the African
genomic contribution. African ancestral proportion in the
southwestern end of Iberia differs twofold than that found in
most Iberian populations. The analysis of the tract-length
distribution showed the presence of very short African
ancestry tracts in southern Iberia. This scenario would be
reflecting ancient African gene flow to Europe through the
Iberian Peninsula. Our findings are compatible with two
temporally spaced migratory events. We here report existence of a fine-scale structure across
western France, with evidence of distinct demographic
histories between subpopulations. These results support the
need for a genetically matched panel of controls from
France, to avoid confounding effects of fine-scale popula-
tion structure. We will subsequently verify and extend our findings
methods based on haplotype structure and IBD, on an
extended population of 5,700 individuals. Further study of
demographic
models will yield not only
insight
on
population history, but also provide a null model for tests
of selection. Conclusions: Genomic analysis provide clues to under-
stand the complex role of southern Iberia and northwestern
Africa as transition areas of old, recurrent human migrations
across
their
respective
continents. Financial
support:
Spanish Ministry MINECO (CGL2014-53985-R). J. Giemza: None. M. Karakachoff: None. E. Charpen-
tier: None. K. Rouault: None. A. Saint-Pierre: None. F. Simonet: None. S. Lecointe: None. P. Lindenbaum:
None. C. Férec: None. H. Le Marec: None. S. Chatel:
None. J. Schott: None. E. Génin: None. R. Redon: None. C. Dina: None. C.L. Hernández: None. G. Pita: None. B. Cavadas:
None. J. Dugoujon: None. A. Novelletto: None. L. Pereira: None. R. Calderón: None. 658 J. del Picchia B. Chung, A. Chiu, C. Chung, W. Wong P18.68D Healthcare burden of rare diseases in Hong Kong - the use
of ORPHA codes in an ICD-10 based healthcare
administrative dataset Abstracts from the 51st European Society of Human Genetics Conference: Posters 659 Institute of Psychiatry, Munich, Germany, 5Munich Cluster for
Systems Neurology (SyNergy), Munich, Germany, 6Institute of
Translational Medicine, University of Liverpool, Liverpool,
United Kingdom myopia becomes high with the beginning of the school
remains unanswered. On the one hand, we must search for
an environmental risk factor of myopia changed by spend-
ing time at school. On the other hand, while all children
visit the school, not all of them develop myopia, therefore
some children are more genetically sensitive to the envir-
onmental factor we are looking for. So, if we find genetic
factor of the myopia, and this factor is regulated by some
environmental factor, then we can confirm this environ-
mental factor to be a risk factor of myopia. In our work, we
found out that the vitamin D receptor gene polymorphism
А-3731G was associated with myopia in schoolchildren,
and the carriers of (-3731A)-allele were more sensitive to
the myopia. As the VDR gene signaling pathway is regu-
lated by UV light we concluded that UV, or rather it’s lack
during the stay in the school building during the daytime,
can cause the school myopia. This means that it is necessary
to change hygienic norms of staying at school to prevent the
development of myopia in children in the period of eye
growth and vision formation. Because despite of the fact
that correction of myopia is quite simple, myopia still
worsens the quality of life and finally can lead to blindness. Introduction: Restless legs syndrome (RLS) is as disease
of the nervous system. At rest in the evening RLS patients
experience an urge to move the legs which impacts sleep
and quality of life. RLS has a strong family history. Here,
we investigated whether RLS GWAS risk SNPs might
explain RLS inheritance patterns in families. Materials and Methods: Published RLS risk loci from
GWAS were genotyped/imputed in 79 families of European
descent (843 individuals) using the Affymetrix Axiom
genotyping array. Logistic regression was applied to each
single family for individual SNPs and polygenic risk scores
(PRS), including the pedigree structure as a random effect
to correct for relatedness in the association test. A. Voitovich, R. Bannour, V. Larionova A. Voitovich, R. Bannour, V. Larionova We selected functional variants both benign and patho-
genic from the analyzed samples which yielded nearly 94
exonic non-synonymous variants per sample, of which over
43 variants per sample were considered rare, present only in
a single individual in our database. Furthermore, aggregat-
ing detected CNVs, nonconsensus splice site variants,
mitochondrial variants and genomic breakpoints provided
additional
information
on distinct Slovenian genomic
variability. In total, we discovered over 28 thousand Academy of Molecular Medicine, Saint-Petersburg, Russian
Federation Clinical Institute of Medical Genetics, UMC Ljubljana,
Ljubljana, Slovenia Clinical Institute of Medical Genetics, UMC Ljubljana,
Ljubljana, Slovenia E. Tilch: None. C. Zhao: None. A. Salminen: None. A. Antic: None. B. Schormair: None. K. Oexle: None. J.B. Sampson: None. B. Müller-Myhsok: None. J. Winkel-
mann: D. Speakers Bureau/Honoraria (speakers bureau,
symposia, and expert witness); Modest; UCB. E. Owner-
ship
Interest
(stock,
stock
options,
patent
or
other
intellectual property); Modest; patent pending (17 15
6438.8). F. Consultant/Advisory Board; Modest; UCB. Population specific genetic variability represents a corner-
stone of interpretation of massive amounts of data generated
by next-generation sequencing approaches. Large interna-
tional sequencing initiatives have generated good coverage
of genetic variation in major databases, but the utility of
those is limited in distinct world populations. To tackle this
issue we report our findings from clinical exome and whole
exome sequencing of 1626 patients submitted to our insti-
tution and the implementation of locally gathered data for
variant filtration in diagnostic NGS interpretation. P18.68D The model
fit
was
quantified
using
Nagelkerke’s
R²
confidence
intervals and significances were assessed empirically. Materials and Methods: Published RLS risk loci from
GWAS were genotyped/imputed in 79 families of European
descent (843 individuals) using the Affymetrix Axiom
genotyping array. Logistic regression was applied to each
single family for individual SNPs and polygenic risk scores
(PRS), including the pedigree structure as a random effect
to correct for relatedness in the association test. The model
fit
was
quantified
using
Nagelkerke’s
R²
confidence
intervals and significances were assessed empirically. Results: In family-wise meta-analyses across SNPs
(Fisher’s method), three families showed a significant
association after correcting for multiple testing of families. In one family, two SNPs alone almost reached significance
at the MEIS and BTBD9 locus after correcting for multiple
testing of SNPs and families. No family reached significant
association using a PRS. Results: In family-wise meta-analyses across SNPs
(Fisher’s method), three families showed a significant
association after correcting for multiple testing of families. A. Voitovich:
None. R. Bannour:
None. V. Larionova: None. A. Voitovich:
None. R. Bannour:
None. V. Larionova: None. In one family, two SNPs alone almost reached significance
at the MEIS and BTBD9 locus after correcting for multiple
testing of SNPs and families. No family reached significant
association using a PRS. Utility of Slovene genomic variation database in diagnostic
NGS Utility of Slovene genomic variation database in diagnostic
NGS Utility of Slovene genomic variation database in diagnostic
NGS Conclusion: We hypothesize that risk SNPs might
explain only some RLS inheritance patterns, either through
tagging of a causal variant or interaction with other family-
specific RLS triggering genetic/environmental factors. A. Voitovich, R. Bannour, V. Larionova Howard University, Washington, DC, United States Violence exposure has long-lasting social and biological
impacts on African American (AA) health and can lead to
physiological changes, including shorter telomere length
(TL). While studies have investigated the relationship
between TL and life stress, few focus on African American
young adults and their direct nexus to violence. This study
examines the effect of violence and gender on both stress
biomarkers and TL in AA young adults. We examine the
relationship between violence exposure, seven stress bio-
markers (IgA/G/E/M, C Reactive Protein, Cortisol, Epstein
Barr Virus Antigen) and TL in a cross sectional analysis of
50 buccal samples (N=25 males & 25 females) of AA 18-
25 years old in Washington DC who experience differential
violence exposure (physical, threat, witnessed, and sexual). Average TL was measured by qPCR. Mann-Whitney tests
identified differences between males and females in expo-
sure to violence, stress biomarkers, and the measures of TL. Correlations were calculated between TLs, biomarker
levels, and violence measures. Elevated sexual violence
exposure was positively correlated (RRANGE= 0.22 to 0.55)
with all elevated stress biomarkers except IgE. TL in the
high violence exposure group was negatively correlated to
all stress biomarker levels (RRANGE= -0.09 to -0.31) except
for IgG. There was no significant difference in TL among
females exposed and not exposed to violence. Violence
exposed females had a longer TL than men (MeanF:M=1.87
v 1.62, p≤.054). Male sexual violence exposure was cor-
related to TL (R=0.575, p≤0.03). High violence levels
correlate to shorter TLs and higher stress biomarker levels
in AA young adults. Methods: First, we estimated the evolutionary age of
each region of the human genome by applying maximum
parsimony to genome-wide alignments with 100 verte-
brates; we then studied the age distribution of several types
of functional regions, focusing on regulatory elements. Results: The age distribution of regulatory elements
revealed the extensive use of newly formed genomic
sequence in the evolution of regulatory interactions: many
transcription factors have expanded their repertoire of
targets through waves of genomic expansion that can be
traced to specific evolutionary events. Repetitive elements
greatly contributed to such expansion: several classes of
these elements are enriched in binding sites of one or a few
specific transcription factors, with such binding sites being
localized
in
particular
portions
of
the
element
and
characterized by distinctive motif words. Academy of Molecular Medicine, Saint-Petersburg, Russian
Federation Since the formation of the compulsory education system for
children worldwide many researchers observe an increase of
myopia incidence in children attending schools, so called
school myopia. Nowadays, the question of why the risk of J. del Picchia 660 D. Marnetto1, F. Mantica2, I. Molineris3, E. Grassi1, I. Pesando4,5, P. Provero1,3 variants previously unreported in population variation
databases, which also proved beneficial in the diagnostic
process by having a positive effect on the overall workload
of variant interpretation. Overall, our data provided us with
18,4%
increased
variant
filtering
power,
significantly
supplementing the sequencing data of Genome Aggregation
Database (gnomAD). 1Department of Molecular Biotechnology and Health Sciences,
University of Turin, Turin, Italy, 2European Molecular Biology
Laboratory (EMBL), Genome Biology Unit, Heidelberg,
Germany, 3Center for Translational Genomics and
Bioinformatics, San Raffaele Scientific Institute IRCCS, Milan,
Italy, 4Department of Physics, University of Turin, Turin, Italy,
5I.N.F.N., Section of Turin, Turin, Italy G. Bergant: None. A. Maver: None. B. Peterlin: None. G. Bergant: None. A. Maver: None. B. Peterlin: None. P18.75C L.F. Jackson: None. F. Saadatmand: None. L.F. Jackson: None. F. Saadatmand: None. Identification of founder effect and haplotype
reconstruction in transthyretin amyloidosis patients with
Glu89Gln mutation in the TTR gene from endemic region
in Bulgaria Howard University, Washington, DC, United States Conclusions: Taken together, these features suggest that
the binding sites were available as soon as the new sequence
entered the genome, rather than being created later by
accumulation of point mutations. By comparing the age of
regulatory regions to the evolutionary shift in expression of
nearby genes, we show that rewiring through genome
expansion played an important role in shaping current
human regulatory networks. D. Marnetto: None. F. Mantica: None. I. Molineris:
None. E. Grassi:
None. I. Pesando:
None. P. Provero: None. D. Marnetto: None. F. Mantica: None. I. Molineris:
None. E. Grassi:
None. I. Pesando:
None. P. Provero: None. Violence exposure, stress biomarkers and gender
differences on buccal telomere length in African American
young adults Introduction: Divergence at the regulatory level underlies a
significant fraction of the phenotypic differences between
extant species, with genome expansion potentially playing a
key role in the evolution of gene regulation. Thus, we
investigated how newly arising sequences contributed to the
rewiring of regulatory networks in the human genome
throughout its evolution from the common ancestor of all
Vertebrates. L. F. Jackson, F. Saadatmand Howard University, Washington, DC, United States Evolutionary rewiring of human regulatory networks by
waves of genome expansion Evolutionary rewiring of human regulatory networks by
waves of genome expansion Abstracts from the 51st European Society of Human Genetics Conference: Posters 661 Z. Pavlova1,2, A. Kirov2, T. Todorov1,3,2, S. Sarafov4, T. Chamova4, M. Gospodinova5, I. Tournev4,6, A. Todorova1,2,3 G. Mishra1,2,3, N. Kumar2,3, A. Saxena2, G. Kaur2, S. Jain4, N. K. Mehra5, P. K. Tiwari1 G. Mishra1,2,3, N. Kumar2,3, A. Saxena2, G. Kaur2, S. Jain4, N. K. Mehra5, P. K. Tiwari1 1Centre for Genomics, Jiwaji University, Gwalior, India,
2Department of Transplant Immunology and Immunogenetics,
All India Institute of Medical Sciences, New Delhi, India,
3ICMR-National Institute of Pathology, New Delhi, India,
4District Hospital, Sheopur, India, 5Dr. C G Pandit National
Chair, All India Institute of Medical Sciences, New Delhi, India 1Genetic Medico-Diagnostic Laboratory Genica, Sofia,
Bulgaria, 2IMDL Genome Center “Bulgaria”, Sofia, Bulgaria,
3Department of Medical Chemistry and Biochemistry, Medical
University Sofia, Sofia, Bulgaria, 4Clinic of Nervous Diseases,
UMBAL Aleksandrovska, Department of Neurology, Medical
University Sofia, Sofia, Bulgaria, 5Clinic of Cardiology, MVR
hospital, Sofia, Bulgaria, 6Department for cognitive science
and psychology, New Bulgarian University, Sofia, Sofia,
Bulgaria 1Genetic Medico-Diagnostic Laboratory Genica, Sofia,
Bulgaria, 2IMDL Genome Center “Bulgaria”, Sofia, Bulgaria,
3Department of Medical Chemistry and Biochemistry, Medical
University Sofia, Sofia, Bulgaria, 4Clinic of Nervous Diseases,
UMBAL Aleksandrovska, Department of Neurology, Medical
University Sofia, Sofia, Bulgaria, 5Clinic of Cardiology, MVR
hospital, Sofia, Bulgaria, 6Department for cognitive science
and psychology, New Bulgarian University, Sofia, Sofia,
Bulgaria Introduction: Sahariya, a primitive tribe of Central India,
has shown an increased incidence of TB as compared to
other tribes. It suggests the role of host immunogenetic
factors for such a high incidence of PTB. Thus, in view of
the unique ethnicity of Sahariya, the association of immu-
nogenetic factors like Chemokines and HLA with PTB was
determined. Introduction: Sahariya, a primitive tribe of Central India,
has shown an increased incidence of TB as compared to
other tribes. It suggests the role of host immunogenetic
factors for such a high incidence of PTB. Thus, in view of
the unique ethnicity of Sahariya, the association of immu-
nogenetic factors like Chemokines and HLA with PTB was
determined. Familial transthyretin amyloidosis is an autosomal domi-
nant genetic disorder caused by missense mutations in the
TTR gene resulting in amyloid plaques formation of the
transthyretin protein. Depending on the system affection
the manifestations may be different and high hetero-
geneity in the penetrance is observed. An endemic region
in Bulgaria exists where the TTR mutation Glu89Gln is
found with high frequency. Evolutionary rewiring of human regulatory networks by
waves of genome expansion Additionally, we found that
the CCL5 level may not only be influenced with the
genotypes of -403G>A SNP and bacillary load but also with
the treatment. Thus, these genetic factors could be used as a
diagnostic marker and could be useful in designing drug to
target tuberculosis. G. Mishra: None. N. Kumar: None. A. Saxena: None. G. Kaur: None. S. Jain: None. N.K. Mehra: None. P.K. Tiwari: None. Z. Pavlova: None. A. Kirov: None. T. Todorov: None. S. Sarafov: None. T. Chamova: None. M. Gospodinova:
None. I. Tournev: None. A. Todorova: None. Z. Pavlova: None. A. Kirov: None. T. Todorov: None. S. Sarafov: None. T. Chamova: None. M. Gospodinova:
None. I. Tournev: None. A. Todorova: None. P18.77A Deletions at 63 GWAS catalog loci based on genome-wide
1000 Genomes project CNV-tagging SNPs Evolutionary rewiring of human regulatory networks by
waves of genome expansion This is a rare mutation and
was probably introduced in the population by a common
ancestor. This phenomenon, called “founder effect” was
proved in carrier families with haplotype analysis of
microsatellite markers showing linkage disequilibrium. Allele frequencies were analyzed and haplotype recon-
struction was done with Arlequin v.3.01 software. The
common ancestry of the carriers was demonstrated using
additional data for their genealogies and microsatellite
data from a control group of non-affected individuals. The
results show that the mutation Glu89Gln is linked to one
haplotype, called “hypothetical founder haplotype” which
was compared to haplotipic data from other European
patients and no similarity was found. The fact that the
founder haplotype has been subjected to decay was used
to determine the mutation age using DMLE+ v.2.0 soft-
ware. The results demonstrated that the mutation arouse
850 generations ago or 15 300 years, assuming average
reproductive age of 18 years. The haplotype analysis of
patients with transthyretin amyloidosis could help with
differentiation of subgroups showing the same phenotype
in order to investigate the heterogenic manifestation of the
disorder. Acknowledgement: The study was supported by
Pfizer: Grant №WI220557/15.11.2016. Methods: About 400 individuals from Sahariya tribe
were genotyped for HLA genes and SNPs in Chemokine
using luminex based methods and ARMS-PCR respec-
tively. Plasma level of positively associated chemokine was
also determined using commercial ELISA kits. The data
was statistically analyzed by using SPSSv16.0. Results: The allelic frequencies of HLA-DRB1*15:01
and 15:02 were significantly increased in the PTB patients
than in healthy controls (p=0.04). However, DRB1*16:02
was found to be significantly reduced in PTB patients
(p=0.003). Further, the frequencies of ‘AA’ genotype and
‘A’ allele of -403G/A SNP of CCL5 gene were found
significantly higher in cases than in controls which resulted
in increased plasma CCL5 level. However, the level was
decreased significantly in patients who were on therapy or
have completed their therapy (KWp = 0.04). Conclusions: Our study for the first time, showed a
unique association of HLA-DRB1*16:02 with protection
against pulmonary tuberculosis. Additionally, we found that
the CCL5 level may not only be influenced with the
genotypes of -403G>A SNP and bacillary load but also with
the treatment. Thus, these genetic factors could be used as a
diagnostic marker and could be useful in designing drug to
target tuberculosis. Conclusions: Our study for the first time, showed a
unique association of HLA-DRB1*16:02 with protection
against pulmonary tuberculosis. E. Loizidou1, E. Bellos1, L. Coin2, M. Johnson1, I. Prokopenko1 1Imperial College London, London, United Kingdom,
2University of Queensland, Brisbane, Australia Association of immunogenetic factors with susceptibility to
pulmonary tuberculosis in Sahariya tribe of North Madhya
Pradesh P18.76D Association of immunogenetic factors with susceptibility to
pulmonary tuberculosis in Sahariya tribe of North Madhya
Pradesh 662 J. del Picchia Médecine Vasculaire, CHU Montpellier, Montpellier, France,
6AP-HP, Hôpital européen Georges Pompidou, Service de
Radiologie Cardiovasculaire, Paris, France, 7AP-HP, Hôpital
européen Georges Pompidou, Département de Pharmacologie,
Paris, France Background: Genome-wide association studies (GWAS)
successfully exploit the variability of most abundant DNA
variants, namely single nucleotide polymorphisms (SNPs),
but frequently fail to provide information about causal
mutations. Copy number variation (CNV) impacts pheno-
type variability and disease susceptibility and is one of the
sources for the so-called “missing heritability”. Despite
notable genomic effects of both CNVs and SNPs, the cor-
relation between them is understudied, and the role of
CNVs in SNP-based phenotypic effects is not established. Vascular Ehlers-Danlos syndrome (vEDS) is a rare genetic
connective tissue disorder secondary to pathogenic variants
within the COL3A1 gene, resulting in exceptional arterial
and organ fragility and premature death. No specific med-
ical treatment has been proven as efficient in reducing the
spontaneous arterial dissections/ruptures, but celiprolol, a
specific beta-blocker that was shown beneficial in a limited
open clinical trial. The main objective of this retrospective
study was to analyze the outcomes of a large cohort of
molecularly confirmed vEDS patients followed up to 17
years in a single referral centre, most of them being medi-
cally treated with celiprolol and scheduled for yearly
follow-up. Between 2000 and 2017, 144 patients, (median
age at diagnosis 34.5 years, 87 females, 91 probands) were
eligible for this study. After a median follow-up of 5.3
years, overall patient survival was high (71.6%; 95% CI
0.5-0.9) and dependent on the type of COL3A1 variant, age
at diagnosis and treatment. More than two-thirds of patients
remained asymptomatic, despite a large number of arterial
lesions at the initial follow-up. Patients treated with celi-
prolol had improved survival (p = 0.0004) and the greatest
protection was observed with 400mg/day compared to
<400mg/day (p = 0.003). Hospitalization rates for sympto-
matic arterial events also decreased with the systematic use
of celiprolol. In this long-term survey, vEDS patients
exhibited a high survival rate compared to what was
expected as well as a low annual occurrence of arterial
complications. Despite the absence of a control group,
celiprolol seems to have a significant positive influence on
the rate of arterial complications and survival. P18.76D Methods: We estimated linkage disequilibrium (LD)
between CNVs and SNPs in protein-coding genes using the
1000 Genomes project sequencing data (1000G) from phase
3. We defined CNV-tagging SNPs for variants reported in
the GWAS catalog for disease/phenotype associations (July,
2017) and for recently published DIAGRAM consortium
type 2 diabetes (T2D) 1000G reference panel-imputed
meta-analysis in Europeans (PMID:28566273). Results: We replicated established CNV-tagging SNP
effects at ten loci, including NEGR1, LCE3A/B, CFHR1-3
for obesity, psoriasis and nephropathy, respectively. We
revealed 31 novel CNVs (length 275bp to ~ 6kb), all but
one being deletions. Among novel CNVs fifteen are <1kb,
tagging lead breast cancer and T2D/lupus erythematosus
SNPs at CHST9 and JAZF1 loci among others. Novel
CNVs covered drug-target genes, such as HTR3D/C/E,
PLEKHA1, and MGST1 and tagged SNPs associated with
major depressive disorder, age related macular degenera-
tion, and visceral fat, respectively. Conclusion: This is the most detailed CNV-tagging SNPs
catalog to date, which will help in dissecting the functional
impact of SNP-trait associations and could drive the
development of new drugs. Conclusion: This is the most detailed CNV-tagging SNPs
catalog to date, which will help in dissecting the functional
impact of SNP-trait associations and could drive the
development of new drugs. Funding: WT205915 E. Loizidou: None. E. Bellos: None. L. Coin: None. M. Johnson: None. I. Prokopenko: None. E. Loizidou: None. E. Bellos: None. L. Coin: None. M. Johnson: None. I. Prokopenko: None. S. Adham: None. S. Seigle: None. T. Mirault: None. P. Henneton: None. A. Legrand: None. J. Albuisson: None. N. Denarié: None. J. Mazzella: None. E. Mousseaux:
None. E. Messas: None. P. Boutouyrie: None. X. Jeunemaitre: None. C. Fuchsberger, J. S. Mitchell, E. König, M. Gögele, C. Pattaro,
P. P. Pramstaller P18.78B Improvementof survival in vascular Ehlers-Danlos
syndrome: effect of celiprolol in along-term cohort study M. Frank1,2, S. Adham1,3, S. Seigle1, T. Mirault1,4, P. Henneton1,5, A. Legrand1,2,3, J. Albuisson1,2,3, N. Denarié1, J. Mazzella1, E. Mousseaux2,3,6, E. Messas1,4, P. Boutouyrie2,3,7, X. Jeunemaitre1,2,3 M. Frank1,2, S. Adham1,3, S. Seigle1, T. Mirault1,4, P. M. Frank1,2, S. Adham1,3, S. Seigle1, T. Mirault1,4, P. P18.79C Henneton1,5, A. Legrand1,2,3, J. Albuisson1,2,3, N. Denarié1, J. Henneton1,5, A. Legrand1,2,3, J. Albuisson1,2,3, N. Denarié1, J. 1
2 3 6
1 4
2 3 7 Insights to the genetic architecture of quantitative traits in
the Cooperative Health Research in South Tyrol (CHRIS)
study Insights to the genetic architecture of quantitative traits in
the Cooperative Health Research in South Tyrol (CHRIS)
study Mazzella1, E. Mousseaux2,3,6, E. Messas1,4, P. Boutouyrie2,3,7, X. Jeunemaitre1,2,3 Mazzella1, E. Mousseaux2,3,6, E. Messas1,4, P. Boutouyrie2,3,7, X. Jeunemaitre1,2,3 1AP-HP, Hôpital européen Georges Pompidou, Département
de Génétique, Centre de Référence des Maladies, Paris,
France, 2INSERM, U 970, Paris Centre de Recherche
Cardiovasculaire – PARCC, Paris, France, 3Université Paris
Sorbonne Cité, Faculté de Médecine Paris Descartes, Paris,
France, 4AP-HP, Hôpital européen Georges Pompidou,
Service de Médecine Vasculaire, Paris, France, 5Service de Institute for Biomedicine, Eurac Research, Affiliated Institute
of the University of Lübeck, Bolzano, Italy Institute for Biomedicine, Eurac Research, Affiliated Institute
of the University of Lübeck, Bolzano, Italy Institute for Biomedicine, Eurac Research, Affiliated Institute
of the University of Lübeck, Bolzano, Italy 663 Abstracts from the 51st European Society of Human Genetics Conference: Posters When undertaking an association study of low-frequency
and rare variants, there are compelling reasons to focus on
variation in protein-coding sequence. First, coding variants
are enriched for impact on molecular function and support
more direct biological interpretation than associations
within non-coding sequence. Second, the functional anno-
tation of coding variants allows discovery efforts to benefit
from the improved power offered by the aggregation of rare
alleles presumed to exert broadly similar molecular effects. We sequenced the exomes of 3,549 individuals from the
population-based CHRIS study and identified >389k var-
iants. We then tested 58 quantitative cardiovascular and
metabolic biochemical parameters for genetic associations. We performed single variant analyses using mixed-model
approaches that account for relatedness (EMMAX). To
increase power to detect rare variant effects, we performed
gene-level
tests
using
SKAT-O
focusing
on
non-
synonymous variants with minor allele frequency (MAF)
< 0.05. We identified at genome-wide significance level 31
loci associated with at least one trait, of which 9 were not
reported in the EBI GWAS catalog. The gene-level analysis
revealed at gene-wide significance level 17 association, of
which 10 were novel. These include novel association for
kidney function, anticoagulation, and iron metabolism. Together,
these
results
suggest
the
value
of
exome
sequencing in well-characterized cohorts for gene discovery
experiments. 5Department of Dermatology, Hôpital d’Instruction des
Armées Legouest, Metz, France, 6Pediatric Neurology and
National Reference Center for Neurogenetic Disorders, AP-
HP, Trousseau Hospital, Paris, France, 7Neuropediatrics, Gui
de Chaulliac Hospital, Montpellier, France, 8department of
genetics, Montpellier, France, 9Department of Medical
Genetics, Oslo University Hospital, Oslo, France, 10Division of
Rheumatology, IRCCS Pediatrico Bambino Gesù’ Children’s
Hospital, Rome, Italy, 11Department of Dermatology, Teaching
Hospital Cochin, AP-HP, Paris, France, 12Internal Medicine,
Archet-1 Hospital, Nice, France, 13Internal Medecine, Cochin
Hospital, AP-HP, Paris Descartes University, Paris, France,
14Pediatric Immunology-Hematology and Rheumatology Unit,
Institut Imagine, Necker Enfants Malades Hospital, AP-HP,
Paris, France, 15Internal medicine, CEREMAIA, Tenon
Hospital, AP-HP, University of Pierre et Marie Curie, Paris,
France, 16Department of Paediatric Nephrology,
Rheumatology and Dermatology, RAISE, Lyon University
Hospital, Lyon, France, 17Department of Paediatric
Rheumatology, CEREMAIA, Bicêtre Hospital, AP-HP,
University of Paris Sud, AP-HP, Kremlin-Bicetre, France,
18Department of Paediatrics, CEREMAIA, Versailles Hospital,
Versailles, France Deficiency of adenosine deaminase 2 (DADA2) is a
recently
described
autoinflammatory
disorder. Genetic
analysis is required to confirm the diagnosis. We aimed to
describe the identifying symptoms and genotypes of
patients referred to our reference centres and to improve the
indications for genetic testing. DNA from 66 patients with
clinically suspected DADA2 were sequenced by Sanger or
next-generation
sequencing. Detailed
epidemiological,
clinical and biological features were collected by use of a
questionnaire and were compared between patients with and
without genetic confirmation of DADA2. We identified 13
patients (19.6%) carrying recessively inherited mutations in
ADA2 that were predicted to be deleterious. Eight patients
were compound heterozygous for mutations. Seven muta-
tions were novel (4 missense variants, 2 predicted to affect
mRNA splicing and 1 frameshift). The mean age of the 13
patients with genetic confirmation was 12.7 years at disease
onset and 20.8 years at diagnosis. Phenotypic manifesta-
tions included fever (85%), vasculitis (85%) and neurolo-
gical disorders (54%). Features best associated with a
confirmatory genotype included fever with neurologic or
cutaneous attacks (odds ratio [OR] 10.71, p = 0.003 and OR
10.9, p< 0.001), fever alone (OR 8.1, p = 0.01), and ele-
vated C-reactive protein (CRP) level with neurologic
involvement (OR 6.63, p = 0.017). Our proposed decision
tree may help improve obtaining genetic confirmation of
DADA2 in the context of autoinflammatory symptoms. Prerequisites for quick and low-cost Sanger analysis include
one typical cutaneous or neurological sign, one marker of C. Fuchsberger: None. J.S. Mitchell: None. E. König:
None. M. Gögele:
None. C. Pattaro:
None. P.P. Pramstaller: None. A decision tree for the genetic diagnosis of deficiency of
adenosine deaminase 2 (DADA2) A decision tree for the genetic diagnosis of deficiency of
adenosine deaminase 2 (DADA2) I. Touitou1, M. Rama1, C. Duflos2, I. Melki3, D. Bessis4, A. Bonhomme4, H. Martin5, D. Doummar6, S. Valence5, D. Rodriguez6, E. Carme7, D. Genevieve8, K. Heimdal9, A. Insalaco10, N. Franck11, V. Queyrel12, N. Tieule12, J. London13,
F. Uettwiller14, S. Georgin-Lavialle15, A. Belot16, I. Kone-Paut17,
V. Hentgen18, G. Sarrabay1 I. Touitou1, M. Rama1, C. Duflos2, I. Melki3, D. Bessis4, A. Bonhomme4, H. Martin5, D. Doummar6, S. Valence5, D. Rodriguez6, E. Carme7, D. Genevieve8, K. Heimdal9, A. Insalaco10, N. Franck11, V. Queyrel12, N. Tieule12, J. London13,
F. Uettwiller14, S. Georgin-Lavialle15, A. Belot16, I. Kone-Paut17,
V. Hentgen18, G. Sarrabay1 I. Touitou1, M. Rama1, C. Duflos2, I. Melki3, D. Bessis4, A. Bonhomme4, H. Martin5, D. Doummar6, S. Valence5, D. Rodriguez6, E. Carme7, D. Genevieve8, K. Heimdal9, A. Insalaco10, N. Franck11, V. Queyrel12, N. Tieule12, J. London13,
F. Uettwiller14, S. Georgin-Lavialle15, A. Belot16, I. Kone-Paut17,
V. Hentgen18, G. Sarrabay1 1UMAI laboratoire de génétique, Montpellier, France,
2Medical Information Department, Montpellier, France,
3General Pediatrics, Infectious Disease and Internal Medicine
Department, Robert Debré Hospital, AP-HP, Paris, France;
Pediatric Hematology-Immunology and Rheumatology
Department, Necker-Enfants Malades Hospital, AP-HP,, Paris,
France, 4Department of Dermatology, Saint-Eloi Hospital and
Montpellier University Hospital, Montpellier, France, 664 J. del Picchia inflammation (fever or elevated CRP level), and recurrent or
chronic attacks in adults. University of Manchester, Manchester, United Kingdom Development of next generation sequencing (NGS) tech-
nology has enabled panel, exome and whole genome
sequencing
for
clinical
diagnostics. Clinical
NGS
is
becoming increasing commonplace. Large scale sequencing
projects, such as the 100,000 Genomes project, are building
capacity within the NHS, paving the way for whole genome
sequencing of patients, and a more personalised approach to
the delivery of patient care. The reduced cost of NGS
enables more widespread testing. A recent UK study sug-
gested all women over thirty should be offered sequencing
of genes associated with breast cancer. Widespread inte-
gration of genomics into healthcare requires greater num-
bers of healthcare professionals with specialised data
management and analysis skills, at a time when there is a
bioinformatics skills shortage. Since 2013, the University of
Manchester (UoM) has been delivering Masters pro-
grammes to educate bioinformaticians, clinical scientists
and genetic counsellors working in the NHS. Close links
between UoM and the Centre for Genomic Medicine, St
Mary’s Hospital Manchester enables us to ensure that
training aligns with clinical practice and professional body
standards. We recently trained 55 students in the use of the
ACMG guidelines for variant classification and report here
on how students utilised the guidelines to classify clinical
variants. The skills shortage is a problem worldwide and we
receive increasing interest from overseas students. In
response, we developed a clinical bioinformatics MOOC
(https://www.futurelearn.com/courses/bioinformatics), with
over 11,000 participants to date. We are now developing a
Distance -Learning Post-Graduate Certificate in Clinical
Bioinformatics, with individual modules also available for
CPD, launching in September 2018. S. Whyte: None. N. Ellery: None. C. Berlin: None. A.F. Brady: None. North West Thames Regional Genetics Service, London, United
Kingdom The identification of targeted therapies for cancer has led to
an increase in the demand of rapid gene test results within
cancer MDT’s. The Royal Marsden Hospital piloted
Mainstreaming Cancer Genetics testing and this has now
been rolled out by several Regional Genetics Services. The
advantages of BRCA mainstreaming include fast, accurate
testing within the cancer care pathway, enabling decisions
about best treatments. In addition, it helps identify at risk
relatives giving them the opportunity to decrease their
cancer risk. We were keen to roll out this service and in
2016 following training to Gynaecological Oncologists in
the North West Thames region, mainstream BRCA testing
was offered for patients with non-mucinous ovarian cancer. The BRCA detection rate was 11.1% (7/63) and in addition
two variants of uncertain significance (VUS) were detected. The overall experience has been positive. However the
Gynaecological oncologists recognised an issue around
informed consent as patients who had previously declined
testing through Clinical Genetics then went ahead with
testing. In addition patients given a normal BRCA result
were disappointed when they were not eligible for certain
treatments. Issues also arose around informing next of kin
of results as some patients were terminally ill. The expla-
nation of results such as lifetime risks of cancer and VUSs
has been variable and a more streamlined pathway has been
put in place. The benefits of this service have outweighed
the drawbacks however interesting lessons have been
learned which we felt were important to share. 1University of Edinburgh, Edinburgh, United Kingdom,
2Western General Hospital, Edinburgh, United Kingdom,
3University of Oxford, Oxford, United Kingdom 9.0
Delivering clinical bioinformatics education in the NGS era M. Cornell, A. Brass, A. Davies M. Cornell, A. Brass, A. Davies P19.03C I. Touitou: None. M. Rama: None. C. Duflos: None. I. Melki: None. D. Bessis: None. A. Bonhomme: None. H. Martin: None. D. Doummar: None. S. Valence: None. D. Rodriguez: None. E. Carme: None. D. Genevieve: None. K. Heimdal: None. A. Insalaco: None. N. Franck: None. V. Queyrel: None. N. Tieule: None. J. London: None. F. Uettwiller: None. S. Georgin-Lavialle: None. A. Belot:
None. I. Kone-Paut: None. V. Hentgen: None. G. Sarrabay: None. S. Whyte, N. Ellery, C. Berlin, A. F. Brady S. Whyte, N. Ellery, C. Berlin, A. F. Brady North West Thames Regional Genetics Service, London, United
Kingdom S. Wright1, D. Stirling1, C. Gourlay1, J. Lawton1, O. Young2, M.
Porteous1, N. Hallowell3 P19.03C
Important lessons learned in the first year of
mainstreaming BRCA testing at the north west thames
regional genetics service inflammation (fever or elevated CRP level), and recurrent or
chronic attacks in adults. P19.05A E. Whitmore: None. T.P. McVeigh: None. L. Kelly:
None. T. Clark: None. B. Mullaney: None. D.E. Barton:
None. A. Ward: None. S.A. Lynch: None. E. Whitmore: None. T.P. McVeigh: None. L. Kelly:
None. T. Clark: None. B. Mullaney: None. D.E. Barton:
None. A. Ward: None. S.A. Lynch: None. Mainstreaming BRCA1 and BRCA2 testing: an interview
study of healthcare professionals’ views 33(26%) would recommend discontinuing cardiology
follow-up, while for 44 (35%) management decisions would
depend on phenotype and family history. Most (67, 53%)
respondents thought variant reclassification should be
performed at least annually. Results: 126 respondents (21 countries) completed the
survey, the majority from UK, US or Australia. 91(72%)
were clinical genetics professionals, 12(10%) cardiologists
and 15(12%) clinical scientists. Table 1 outlines circum-
stances where predictive testing would be offered to
relatives of the proband. Considering class 4 variants, 101
(80%) counselled about possible future variant reclassifica-
tion. With negative predictive tests, 88(70%) counselled
about outstanding risks of developing the familial pheno-
type. 33(26%) would recommend discontinuing cardiology
follow-up, while for 44 (35%) management decisions would
depend on phenotype and family history. Most (67, 53%)
respondents thought variant reclassification should be
performed at least annually. Conclusions: Considerable variability in management of
families with class 3 and 4 variants exists. Decision-making
relies on the phenotype, family history and genotype. Close
multi-disciplinary working between cardiology and genetics
is critical to accurately define phenotype and genotype. 1. Richards et al, Genetics in medicine. 2015;17(5):405-
424. Predictive testing
Predictive Testing
Yes
No
Affected relatives only
Class 5
122(97%)
2(2%)
1(1%)
Class 4
99(79%)
2(2%)
23(18%)
Class 3
12(10%)
30(24%)
82(65%) S. Wright: None. D. Stirling: None. C. Gourlay: None. J. Lawton: None. O. Young: None. M. Porteous: None. N. Hallowell: None. Mainstreaming BRCA1 and BRCA2 testing: an interview
study of healthcare professionals’ views Mainstreaming BRCA1 and BRCA2 testing: an interview
study of healthcare professionals’ views 665 Abstracts from the 51st European Society of Human Genetics Conference: Posters The mainstreaming of genetic services in cancer care offers
the promise of streamlined, tailored treatment for indivi-
duals, provided through simplified care pathways. We know
little about how clinicians working outside clinical genetics
view mainstreaming, particularly, how this impacts on their
professional role and responsibilities. We conducted a
qualitative study of healthcare providers at a UK teaching
hospital, focusing on their views and experiences of pro-
viding treatment-focused BRCA 1 and BRCA 2 genetic
testing (TFGT) for breast and ovarian cancer patients. Semi-
structured interviews were undertaken with: two medical
oncologists who offer TFGT to their ovarian cancer
patients, six breast surgeons (and a breast care nurse spe-
cialist) who were responsible for triaging patients for
onward referral to clinical genetics, six medical oncologists
who were about to undergo training and start offering TFGT
to breast cancer patients, and six members of the clinical
genetics team who counsel patients from the ovarian and
breast pathways. Interviewees expressed a range of opi-
nions. Some, primarily oncologists, championed the intro-
duction of TFGT in their clinic, while others, primarily
breast surgeons, saw the introduction of TFGT as a threat to
their professional identity and as negatively impacting their
workload. Support for mainstreaming was related to the
extent to which interviewees regarded this genetic infor-
mation as informing personal treatment plans. We highlight
some of the difficulties that may be encountered when
introducing
genetics
into
mainstream
specialities
and
emphasise the need for further education of non-genetics
specialists. Breast Cancer Now Grant no: [2016MayPR700]
S. Wright: None. D. Stirling: None. C. Gourlay: None. J L
t
N
O Y
N
M P
t
N Methods: We developed an electronic questionnaire
(www.surveymonkey.com/r/cardiacvariants) based on inter-
disciplinary concerns to gauge international practice. This
link
was distributed
to
colleagues
by email
or via
professional bodies. Results: 126 respondents (21 countries) completed the
survey, the majority from UK, US or Australia. 91(72%)
were clinical genetics professionals, 12(10%) cardiologists
and 15(12%) clinical scientists. Table 1 outlines circum-
stances where predictive testing would be offered to
relatives of the proband. Considering class 4 variants, 101
(80%) counselled about possible future variant reclassifica-
tion. With negative predictive tests, 88(70%) counselled
about outstanding risks of developing the familial pheno-
type. Managing uncertainty in cardiac genetics - An
international survey Managing uncertainty in cardiac genetics - An
international survey E. Whitmore1, T. P. McVeigh2, L. Kelly3, T. Clark2, B. Mullaney2, D. E. Barton2, A. Ward2, S. A. Lynch1 P19.06B Patients at high risk of bowel cancer like the opportunity to
receive, store and share confidential documents via a secure
website - www.familyweb.org.uk 1Temple St. Children's University Hospital, Dublin, Ireland,
2Our Lady's Children's Hospital Crumlin, Dublin, Ireland,
3Royal College of Surgeons in Ireland, Dublin, Ireland 1Temple St. Children's University Hospital, Dublin, Ireland,
2Our Lady's Children's Hospital Crumlin, Dublin, Ireland,
3Royal College of Surgeons in Ireland, Dublin, Ireland S. M. A. Goodman1, R. Jones1, L. Jackson2, H. Skirton1
1Plymouth University, Plymouth, United Kingdom, 2Exeter
University, Exeter, United Kingdom S. M. A. Goodman1, R. Jones1, L. Jackson2, H. Skirton1 Introduction: Multi-gene panel testing is useful in geneti-
cally heterogeneous conditions, including inherited cardiac
pathologies. Extended panels increase diagnostic yield,
identifying variants where pathogenicity is certain (ACMG
(1) class 5), probable (class 4) and uncertain (class 3). Concerns exist regarding management of class 3 and 4
variants, particularly for conditions of oligo-genic inheri-
tance or variable expressivity. 1Plymouth University, Plymouth, United Kingdom, 2Exeter
University, Exeter, United Kingdom Many relatives who share a high lifetime risk of cancer
remain unaware of their opportunities for genetic testing or
screening. Providing letters and verbal recommendations to
patients are not necessarily sufficient to support effective 666 J. del Picchia communication in families and methods for disseminating
information to relatives are still under debate. The Family
Web study investigated sharing digital documents securely
online. Data from a cross-sectional survey (n=286) and
telephone interviews (n=14) informed website content. Video recorded Think-Aloud interviews (n=12) elicited
users' reactions to a website through three iterative phases. Patients at high risk of bowel cancer who had been advised
to have regular colonoscopy and were aware that their
diagnosis had implications for their relatives were inter-
viewed while testing the website, commenting on its appeal
and function. Recruitment was via NHS clinics or charity
websites in the UK. Participants welcomed the opportunity
to store and share personal information on the website but
desired more support from health professionals, reporting
the profound effect of the diagnosis on them and their
family relationships. They wanted more information on a
variety of topics to support themselves and inform their
relatives. Key issues were: advice on a healthy lifestyle,
talking to children, gene specific risks and accessing ade-
quate cancer surveillance. The results indicate that use of a
personalised web-based resource is acceptable to patients
and meets a range of their information needs. P19.06B This is
important in the context of growing numbers of patients
diagnosed with Lynch syndrome through tumour screening. Website funded by charity: Bowel Cancer West. training session focussing on considerations for consent in
the 100,000 Genomes Project, followed by the opportunity
to observe and be observed in consent appointments as well
as ongoing mentorship. training session focussing on considerations for consent in
the 100,000 Genomes Project, followed by the opportunity
to observe and be observed in consent appointments as well
as ongoing mentorship. Results: 45 NHS staff members across the West of
England region were trained, including genetic counsellors,
nurses and specialist registrars. Overall, staff members felt
that the framework provided appropriate information and
guidance; however, some expressed concerns regarding the
extent of their scientific knowledge and ability to address
the wider familial implications of genomic testing. Conclusions: Our experience delivering training across a
region for a specific project highlights the skill gaps and
knowledge required for healthcare professionals to address
issues specific to informed consent for genomic testing. This emphasizes the need to embed a competency frame-
work in order to enhance the provision of genomic medicine
for effective patient and family-centred care. A. Pichini: None. C. Carpenter-Clawson: None. A. Watford: None. M. Watson: None. Creation of Ukrainian guideline of cystic fibrosis neonatal
screening N. G. Gorovenko1, Z. I. Rossokha2, N. V. Olkhovych3, S. P. Kyriachenko2 S.M.A. Goodman: None. R. Jones: None. L. Jackson:
None. H. Skirton: None. 1Shupyk National Medical Academy of Postgraduate
Education, Kyiv, Ukraine, 2State Institution Reference-centre
for molecular diagnostic of Public Health Ministry of Ukraine,
Kyiv, Ukraine, 3National Children Hospital «OHMATDIT»,
Kyiv, Ukraine Developing a competency framework to consent patients
for genomic testing Developing a competency framework to consent patients
for genomic testing A. Pichini1, C. Carpenter-Clawson1, A. Watford1, M. Watson2 Awareness of screening and diagnostic tests for Down
syndrome in Bulgarian women at reproductive age Awareness of screening and diagnostic tests for Down
syndrome in Bulgarian women at reproductive age M. J. Nabais Sá1, S. Jansen1, A. van Remortele1, J. Ewals1, M. Verbruggen1, D. A. Koolen1, H. G. Brunner1, E. Eichler2, J. Gecz3, B. B. A. de Vries1 M. Levkova1,2, M. Hachmeriyan1, V. Miteva1, M. Stoyanova1, M. Tsvetkova1, L. Angelova1 M. Levkova1,2, M. Hachmeriyan1, V. Miteva1, M. Stoyanova1, M. Tsvetkova1, L. Angelova1 1Department of Medical Genetics, Medical University Varna,
Varna, Bulgaria, 2University Hospital St Marina, Varna,
Bulgaria 1Department of Human Genetics, Donders Institute for Brain,
Cognition and Behaviour, Radboud University Medical
Center, Nijmegen, Netherlands, 2Department of Genome
Sciences, University of Washington School of Medicine,
Seattle, WA, United States, 3Adelaide Medical School and the
Robinson Research Institute, University of Adelaide, Adelaide,
SA, Australia 1Department of Human Genetics, Donders Institute for Brain,
Cognition and Behaviour, Radboud University Medical
Center, Nijmegen, Netherlands, 2Department of Genome
Sciences, University of Washington School of Medicine,
Seattle, WA, United States, 3Adelaide Medical School and the
Robinson Research Institute, University of Adelaide, Adelaide,
SA, Australia Introduction: NIPT (non-invasive prenatal tests) are fast
coming to the practice and aggressively offered by private
companies. Nevertheless, the question remains - do women
at reproductive age understand the possibilities and limita-
tions of the different tests for Down syndrome and which
are the key factors for preferring a certain test. Human Disease Genes website series (HDG) is an interna-
tional library of websites for professional information on the
clinical consequences of novel variants in the human gen-
ome (http://humandiseasegenes.nl/). HDG is an initiative of
the Human Genetics Department, Nijmegen, in collabora-
tion with the University of Washington and the University
of Adelaide. Each website is moderated by a dedicated team
of clinicians and molecular biologists, and collects and
provides up-to-date mostly unpublished clinical information
about one specific gene or copy number variant. HDG aims
to fill the gap between first publication of several cases and
consecutive publication of a large review paper. Profes-
sionals can use the information of the new and rare disorder
for counseling and will have the opportunity to share clin-
ical data. Patients, parents and care-givers will find useful
information on the disease and have the opportunity to share
detailed clinical information through the website of our
partner GenIDA. HDG is also a platform where researchers
can collect and share (functional)data. Awareness of screening and diagnostic tests for Down
syndrome in Bulgarian women at reproductive age Recently a new
version of the websites has been launched which not only
allows collection of phenotype data using HPO (http://huma
ndiseasegenes.nl/wac/professionals/upload-clinical-informa
tion/) but also provides an up-to-date overview of the
clinical data that have been collected (http://humandisea
segenes.nl/wac/graph-and-chart/). These
tools
will
be Materials and Methods: We present a survey among
300 randomly selected females from 18 to 47 years old,
carried out both online and at the office for genetic
counseling in the Laboratory of Medical genetics, Varna. Of all 59.3% are currently pregnant, 35.8% have been
pregnant before, 4.9% not been pregnant. Materials and Methods: We present a survey among
300 randomly selected females from 18 to 47 years old,
carried out both online and at the office for genetic
counseling in the Laboratory of Medical genetics, Varna. Of all 59.3% are currently pregnant, 35.8% have been
pregnant before, 4.9% not been pregnant. Results: 73.2% of the women receive information from
the obstetrician although high percentage are self-educated -
26.8%. In total 65.7% of the females think that this
information is well or very well delivered. 66.1 % describe
their understanding of biochemical screening for Down
syndrome as good or very good compared to 41.6% who do
not know what NIPT is. They are better informed about the
amniocentesis - 56.2%, though only 13.7% would definitely
undergo the procedure. The main argument when choosing
the method for diagnosing Down syndrome is the accuracy
of the test - 65.1% and only 1.2% point the price. However,
the majority (77.3%) are willing to pay no more than
150 euro. Results: 73.2% of the women receive information from
the obstetrician although high percentage are self-educated -
26.8%. In total 65.7% of the females think that this
information is well or very well delivered. 66.1 % describe
their understanding of biochemical screening for Down
syndrome as good or very good compared to 41.6% who do
not know what NIPT is. They are better informed about the
amniocentesis - 56.2%, though only 13.7% would definitely
undergo the procedure. The main argument when choosing
the method for diagnosing Down syndrome is the accuracy
of the test - 65.1% and only 1.2% point the price. However,
the majority (77.3%) are willing to pay no more than
150 euro. Conclusions: The results from the survey highlight the
lack of knowledge about the offered screening and
diagnostic tests for Down syndrome, especially NIPT. P19.10B Adopting a gene through Human Disease Genes website
series facilitates a clinical diagnosis for rare genetic
disorders Adopting a gene through Human Disease Genes website
series facilitates a clinical diagnosis for rare genetic
disorders A. Pichini1, C. Carpenter-Clawson1, A. Watford1, M. Watson2 DNA test will
be held in two state accredited centers. The development of
a national guideline and the workshop is an important link
in the education of specialists and improvement of their
work quality. highly valuable for the clinical practice. So far, over 60
genes have been adopted (http://humandiseasegenes.nl/
gene-websites/) by more than 60 moderators world-wide
(http://humandiseasegenes.nl/moderators/). If you wish to
adopt a gene and join the team of moderators please, contact
us at the poster or send us a mail to info@humandisease-
genes.com. M.J. Nabais Sá: None. S. Jansen: None. A. van
Remortele: None. J. Ewals: None. M. Verbruggen:
None. D.A. Koolen: None. H.G. Brunner: None. E. Eichler: None. J. Gecz: None. B.B.A. de Vries: None. N.G. Gorovenko: None. Z.I. Rossokha: None. N.V. Olkhovych: None. S.P. Kyriachenko: None. N.G. Gorovenko: None. Z.I. Rossokha: None. N.V. Olkhovych: None. S.P. Kyriachenko: None. A. Pichini1, C. Carpenter-Clawson1, A. Watford1, M. Watson2 Introduction: The first national pilot project of cystic
fibrosis newborn screening (CF NBS) was performed in
2012-2014 years. National program CF NBS is going re-
start in 2018. The aim of the study was to analyze pre-
liminary results and approved strategy of next program for
all specialists from 12 regional genetic centers. Introduction: The first national pilot project of cystic
fibrosis newborn screening (CF NBS) was performed in
2012-2014 years. National program CF NBS is going re-
start in 2018. The aim of the study was to analyze pre-
liminary results and approved strategy of next program for
all specialists from 12 regional genetic centers. 1University Hospitals Bristol NHS Foundation Trust, Bristol,
United Kingdom, 2North Bristol NHS Trust, Bristol, United
Kingdom 1University Hospitals Bristol NHS Foundation Trust, Bristol,
United Kingdom, 2North Bristol NHS Trust, Bristol, United
Kingdom Introduction: Genomic medicine involves unique con-
siderations with regards to informed consent, which have
been highlighted in the 100,000 Genomes Project. Specific
challenges include addressing the needs of different family
members, data sharing protocols, and returning results
including additional findings. It is important to promptly
address the training needs of healthcare professionals that
may be involved in consenting patients for genomic testing
as it becomes increasingly part of standard clinical care. Materials and Methods: IRT level results of 894152
newborns tested in pilot program was analyzed together
with all diagnostic procedure of CF NBS. Results: IRT retest investigated for 6943 newborns in
pilot program. 95 patients with positive second IRT retest
had confirmed CF diagnosis. DNA test was done for 75
patients. CF mutations were found in 92.59% of diagnosed
patients. These results were discussed at national workshop
with specialists from all 25 Ukrainian regions. There were
approved basic principles of CF NBS within this framework
for Ukraine: IRT/IRT/DNA. Materials and Methods: Standardized consent training
materials were developed by the authors and included Good
Clinical Practice training as well as courses developed by
Health Education England’s Genomics Education Pro-
gramme. The competency framework involved a three hour Conclusions: Results analysis showed the advisability
IRT/IRT/DNA scheme to screen CF in newborns in 667 Abstracts from the 51st European Society of Human Genetics Conference: Posters Ukraine. Workshop results will be included in the national
guideline with procedure of internal and external quality
control systems. The increased CFTR mutations spectrum
will be tested at the next CF NBS program. P19.12D
Clinical implication of FMR1 intermediate alleles in a
Spanish population M. Smith1, J. Clayton-Smith1,2, S. Douzgou1,2, B. Kerr1,2, S. Banka1,2, A. Renieri3,4, L. Faivre5, M. Rawson1 1Manchester Centre for Genomic Medicine, Manchester
University NHS Foundation Trust, Health Innovation
Manchester, Manchester, United Kingdom, 2Division of
Evolution and Genomic Sciences, Faculty of Biology, Medicine
and Health, School of Biological Sciences, University of
Manchester, Manchester, United Kingdom, 3Medical Genetics,
University of Siena, Siena, Italy, 4Genetica Medica, Azienda
Ospedaliera Universitaria Senese, Siena, Italy, 5Centre de
Génétique, Centre de Référence Maladies Rares "Anomalies du
Développement et Syndromes Malformatifs" Hôpital d'Enfants,
Dijon, France M. Milà1,2, M. Alvarez-Mora1,3, I. Madrigal1,3, F. Martinez4, M. Tejada5,6, S. Izquierdo7, L. Perez-Jurado8,3, L. Rodriguez-
Revenga1,3 1Servei de Bioquimica i Genética Molecular, Barcelona, Spain,
2Hospital Clinic. CIBERER Centre for Biomedical Research on
Rare Diseases (CIBERER), ISCIII,, Barcelona, Spain, 3Centre
for Biomedical Research on Rare Diseases (CIBERER),
ISCIII,, Barcelona, Spain, 4Hospital Universitario y
Politecnico La Fe,, Valencia, Spain, 5Biocruces Health
Research Institute, Barakaldo-Bizkaia, Spain, 6Centre for
Biomedical Research on Rare Diseases (CIBERER), ISCIII,,
Madrid, Spain, 7Genetics department Clinical Biochemistry
Service. University Hospital Miguel Servet, Zaragoza, Spain,
8Genetics Unit, Universitat Pompeu Fabra, and Hospital del
Mar Research Institute (IMIM),, Barcelona, Spain 1Servei de Bioquimica i Genética Molecular, Barcelona, Spain,
2Hospital Clinic. CIBERER Centre for Biomedical Research on
Rare Diseases (CIBERER), ISCIII,, Barcelona, Spain, 3Centre
for Biomedical Research on Rare Diseases (CIBERER),
ISCIII,, Barcelona, Spain, 4Hospital Universitario y
Politecnico La Fe,, Valencia, Spain, 5Biocruces Health
Research Institute, Barakaldo-Bizkaia, Spain, 6Centre for
Biomedical Research on Rare Diseases (CIBERER), ISCIII,,
Madrid, Spain, 7Genetics department Clinical Biochemistry
Service. University Hospital Miguel Servet, Zaragoza, Spain,
8Genetics Unit, Universitat Pompeu Fabra, and Hospital del
Mar Research Institute (IMIM),, Barcelona, Spain The 2011 EU Directive on Patients’ Rights in Cross-border
Healthcare aims to facilitate access to healthcare resources
from across the EU and thus increase patients’ management
and treatment options. In response to this, 24 thematic
European Reference Networks (ERNs) for rare disorders
were formed in March 2017. FMR1 premutation carriers (55-200 CGGs) are at risk of
developing Fragile X-associated primary ovarian insuffi-
ciency (FXPOI) as well as a progressive neurodegenerative
disorder called Fragile X-associated tremor/ataxia syndrome
(FXTAS). FMR1 premutation alleles are also associated
with a variety of disorders, including psychiatric, develop-
mental, and neurological problems. While the clinical
implications of the FMR1 premutation alleles are well
established, there is major concern regarding smaller CGG
expansions known as intermediate alleles (IA) or gray zone
alleles (45-54 CGG). Awareness of screening and diagnostic tests for Down
syndrome in Bulgarian women at reproductive age Better education and counseling of women during their
pregnancy consultations are recommended. 668 J. del Picchia first step in implementing changes which will have a
positive impact on the care of patients across Europe. M. Smith: None. J. Clayton-Smith: None. S. Douzgou:
None. B. Kerr: None. S. Banka: None. A. Renieri: None. L. Faivre: None. M. Rawson: None. first step in implementing changes which will have a
positive impact on the care of patients across Europe. M. Smith: None. J. Clayton-Smith: None. S. Douzgou:
None. B. Kerr: None. S. Banka: None. A. Renieri: None. L. Faivre: None. M. Rawson: None. M. Levkova: None. M. Hachmeriyan:
None. V. Miteva: None. M. Stoyanova: None. M. Tsvetkova:
None. L. Angelova: None. first step in implementing changes which will have a
positive impact on the care of patients across Europe. M. Smith: None. J. Clayton-Smith: None. S. Douzgou:
None. B. Kerr: None. S. Banka: None. A. Renieri: None. L. Faivre: None. M. Rawson: None. Skills and competencies in clinical genetics for the
European general practitioner Skills and competencies in clinical genetics for the
European general practitioner I. Ejarque-Doménech1, C. Cuenca-Valero2, M. Calviño-
Naveira3, A. Souvirón-Rodríguez4, V. Martín-Gutiérrez5 1Agència Valenciana de Salut, Valencia, Spain, 2Conselleria
de Sanitat. Hospital de Requena., Requena, Spain, 3Servizo
Galego de Saúde (SERGAS)., Santiago de Compostela., Spain,
4Centro de Salud de El Palo. Servicio Andaluz de Salud.,
Málaga., Spain, 5Centro de Salud. Servicio Andaluz de Salud.,
Sevilla., Spain Background: The German Act on Genetic Testing stipu-
lates the requirements of good practice with regard to
safety, informed consent and free decision-making for
people undergoing genetic testing. The act required the
GEKO to issue a guideline on requirements for competence
in genetic counseling within the scope of each medical
subspecialty (2011). No empirical data are currently avail-
able on how the GEKO guideline is applied at point of care. Introduction: The training programs of the specialty of
General Practitioner (GP) remain heterogeneous at a EU
level. Henceforth, in some European countries lack the
skills and competencies in clinical genetics for the GP. This
poster is a proposal of these skills and competencies. Objectives: To provide preliminary data for assessing the
translation of the guideline. Objectives: To provide preliminary data for assessing the
translation of the guideline. Material and Methods: This study has been carried out
through a qualitative group of twelve experts selected by
these three criteria: GP specialists, interested in clinical
genetics and from different Spanish regions. During a four-
hour session was discussed what kind of skills and
competencies should be included in the curriculum of the
trainee for GP. Methods: (i) key-areas for an assessment were identified
by a multidisciplinary expert group; (ii) development of a
questionnaire based upon key-areas (iii) a pre-study/inquiry
guided by the questionnaire. Results: 150 OB/GYN representing different types of
practice participated in the inquiry. 80% had obtained the
qualification for genetic counseling within their specialty. 48% reported changes in the management of patients, 61%
reported more referrals to genetic specialists for genetic
counseling. Genetic counseling provided by OB/GYN
mostly include: counseling before First Trimester Screening
(ETS) (76%) and after ETS with normal results (77%),
before prenatal ultrasound for fetal anomalies (64%), before
NIPT for Trisomy 21 and after NIPT with normal results
(57%). Additional work load, time constraints in busy
practice settings and the guideline conflicting with estab-
lished approaches to delivering care were reported. P19.12D
Clinical implication of FMR1 intermediate alleles in a
Spanish population Although several studies have hypo-
thesized that IA may be involved in the etiology of FMR1
premutation associated phenotypes, this association still
remains unclear. The aim of this study was to provide new
data on the clinical implications of IA. We reviewed a total
of 17,011 individuals: 1,142 with premature ovarian
insufficiency, 478 with movement disorders, 14,006 with
neurodevelopmental disorders and 1,385 controls. Similar
IA frequencies were detected in all the cases and controls
(cases 1.20% vs. controls 1.39%, p = 0.427). When com-
paring the allelic frequencies of IA ≥50CGGs, a greater,
albeit not statistically significant, number of alleles were
detected in all the cohorts of patients. However, statistical
analysis did not detect significant differences between any
group of patients and controls suggesting that IA do not
confer increased risk for primary ovarian insufficiency, The ERN for Congenital Malformations and Intellectual
Disability (ERN-ITHACA) seeks to improve access to
diagnosis, consistent high-quality care, educational and
research resources by facilitating expert exchange, bringing
TeleHealth into clinical practice where required, establish-
ing rare patient registries, encouraging engagement in pan-
European research activities and creating new educational
resources for professionals and patient groups. Here, we
demonstrate some of our key initiatives in delivering patient
benefit as part of ERN-ITHACA. In year 1 there have been tangible benefits: interaction
with
patient
groups
helped
us
formulate
an
all-
encompassing strategy; the web-based Clinical Patient
Management System (CPMS) has been piloted successfully;
expertise on care pathways and training initiatives has been
consolidated and ITHACA has participated in large-scale
EU projects, such as SOLVE-RD and the European Joint
Programme. However, there have also been challenges: the networks
are conservatively financed by the EU and can therefore be
accommodated by Health Care Providers (HCPs) from
countries with existing management frameworks; the CPMS
still presents many technical challenges; inclusion of new
HCPs will strain the ERNs. We have developed strategies to
overcome these challenges and the first year represents the Abstracts from the 51st European Society of Human Genetics Conference: Posters 669 movement disorders, or neurodevelopmental disorder phe-
notypes. This study was supported by ISCIII [PI17/01067],
co-financed by FEDER "una manera de hacer Europa" and
AGAUR 2017 SGR1134 from the Generalitat de Catalunya
and by grants from MINECO [BIO2011-27069] [SAF2013-
49108-R]. I. Nippert1, J. T. Epplen2, A. Gasiorek-Wiens3, R. Glaubitz4, T.
Grimm5, K. Kagan6, R. P. Nippert7, J. Schmidtke8, R.
Schwerdtfeger9, K. Zerres10, H. Toennies11 I. Nippert1, J. T. Epplen2, A. Gasiorek-Wiens3, R. Glaubitz4, T. Grimm5, K. Kagan6, R. P. Nippert7, J. Schmidtke8, R. Schwerdtfeger9, K. Zerres10, H. Toennies11 M. Milà: None. M. Alvarez-Mora: None. I. Madrigal:
None. F. Martinez:
None. M. Tejada:
None. S. Izquierdo: None. L. Perez-Jurado: None. L. Rodri-
guez-Revenga: None. 1Institut für Humangenetik,Universitätsklinikum Münster,
Münster, Germany, 2Institut für Humangenetik, RUB, Bochum,
Germany, 3Campus Charité Mitte, Berlin, Germany, 4amedes
genetics, Hannover, Germany, 5Institut für Humangenetik,
Biozentrum Universität Würzburg, Würzburg, Germany,
6Universitäts-Frauenklinik Tübingen, Tübingen, Germany,
7Universitätsklinikum Münster, Münster, Germany, 8MHH,
Hannover, Germany, 9Zentrum für Pränatalmedizin und
Humangenetik, Hannover, Germany, 10Institut für
Humangenetik, RWTH Aachen, Aachen, Germany, 11Robert
Koch-Institut, Berlin, Germany P19.14B Translating the German Commission on Genetic Testing
(GEKO) guideline for the requirements of the qualifications
and the contents of genetic counseling into OB/GYN
specialist practice - Results from the GenBIn2 project P19.15C Factors affecting compliance to genetic counseling in the
Israeli Bedouin population P19.16D
An alternative pathway for countries without a training
program for genetic counsellors: the Swedish experience An alternative pathway for countries without a training
program for genetic counsellors: the Swedish experience I. Nippert: None. J.T. Epplen: None. A. Gasiorek-
Wiens: None. R. Glaubitz: None. T. Grimm: None. K. Kagan: None. R.P. Nippert: None. J. Schmidtke: None. R. Schwerdtfeger:
None. K. Zerres:
None. H. Toennies: None. K. Svensson1, R. Pestoff2, M. Paneque3, C. Malmgren
Ingvoldstad4 1Office for Medical Services, Division of Laboratory Medicine
Department of Clinical Genetics, Lund, Sweden, 2Department
of Clinical Genetics, Linköping University Hospital, Linköping
Sweden, Linköpping, Sweden, 31i3S – Instituto de Investigação
e Inovação em Saúde, Universidade do Porto, Porto, Portugal,
Porto, Portugal, 4Department of Clinical Science, Intervention
and Technology, Karolinska Institute, Stockholm, Sweden,
Stockholm, Sweden R.O.I. abo rabia: None. Conclusions: By identifying numerous challenges the
inquiry helps to better understand factors influencing the
translation process and provides groundwork for future
assessment studies. r. O. I. abo rabia Clalit Health Services and the Genetics Institute,, beer sheva,
Israel In Sweden there is currently no academic education at
master level in genetic counselling, which is recommended
by the European organizations promoting professional
regulation. As a result, professionals with different back-
grounds and experiences are employed as genetic counsel-
lors in Sweden. There is a need to harmonize the quality and
standards for practice. Background: Genetic counseling is mostly carried out in
the framework of hospitals. With recent massive progress in
genetic technologies and propensity in genetic testing, there
is growing need to make the genetic services more acces-
sible in community medicine in order to increase respon-
siveness. This is of specific interest in inbred rural
communities, where consanguinity leads to high incidence
of hereditary diseases, yet mobility to central medical cen-
ters is cumbersome. The Swedish Society for Genetic Counsellors, SFGV
together
with
the
Swedish
Association
for
Medical
Genetics, SFMG have determined standards, and created a
career development pathway for genetic counsellors. Objective: To assess in the rural Bedouin Israeli
community the compliance of arrival to genetic counseling
in a central medical center following nurse-mediated early
interviews and referral. Objective: To assess in the rural Bedouin Israeli
community the compliance of arrival to genetic counseling
in a central medical center following nurse-mediated early
interviews and referral. The result is an individual training program leading up to
certification for genetic counsellors in clinical practice in
Sweden. Each professional has an educational plan, and
there are three different levels of employment within the
genetic counseling profession. The first level is an employ-
ment according to the individual´s basic profession, the
second level is an assistant genetic counsellor and the third
level, as a genetic counsellor, requires an academic
education equivalent to one year at an advanced level in
the nursing or biomedical field, and approval as a certified
genetic counsellor according to the SFMG-standards. Methods: Prospective study. Data collected from records
of couples visiting genetics-specialized nurses at public
community clinics, including personal interviews, clinical,
demographic and obstetric data, and family history of
genetic disease variables. Methods: Prospective study. Data collected from records
of couples visiting genetics-specialized nurses at public
community clinics, including personal interviews, clinical,
demographic and obstetric data, and family history of
genetic disease variables. Results: Of the 1754 women reaching initial interview by
the genetics-trained nurse, 684 (40%) needed further
professional genetic counseling at Soroka medical center. Skills and competencies in clinical genetics for the
European general practitioner Positive
responses include: increased sensitivity for skills required
for genetic counseling and increased referrals to genetic
specialists. Results: 150 OB/GYN representing different types of
practice participated in the inquiry. 80% had obtained the
qualification for genetic counseling within their specialty. 48% reported changes in the management of patients, 61%
reported more referrals to genetic specialists for genetic
counseling. Genetic counseling provided by OB/GYN
mostly include: counseling before First Trimester Screening
(ETS) (76%) and after ETS with normal results (77%),
before prenatal ultrasound for fetal anomalies (64%), before
NIPT for Trisomy 21 and after NIPT with normal results
(57%). Additional work load, time constraints in busy
practice settings and the guideline conflicting with estab-
lished approaches to delivering care were reported. Positive
responses include: increased sensitivity for skills required
for genetic counseling and increased referrals to genetic
specialists. Results: As a result of the work carried out by a group of
experts,
certain
skills
and
competencies
have
being
identified for the curriculum of the European GP. Results: As a result of the work carried out by a group of
experts,
certain
skills
and
competencies
have
being
identified for the curriculum of the European GP. Conclusions: It is very important to adapt the current
regulation across Europe in order to include specific skills
and competencies in clinical genetic for the curriculum of
the trainee of GP. A rotation of this professional during a
month in a service of clinical genetics complementing the
current competence-knowledge matrix would grant better
tools to improve the quality of the service provided to
patients. Conclusions: It is very important to adapt the current
regulation across Europe in order to include specific skills
and competencies in clinical genetic for the curriculum of
the trainee of GP. A rotation of this professional during a
month in a service of clinical genetics complementing the
current competence-knowledge matrix would grant better
tools to improve the quality of the service provided to
patients. I. Ejarque-Doménech: None. C. Cuenca-Valero: None. M. Calviño-Naveira: None. A. Souvirón-Rodríguez:
None. V. Martín-Gutiérrez: None. 670 J. del Picchia R.O.I. abo rabia: None. r. O. I. abo rabia Reasons
for further formal genetic counseling were:
Medical problems detected during current pregnancy;
Consanguinity; Family history of genetic diseases; pre-
viously known carrier status of couple. Of the 684 women
referred to formal genetic counseling, only 360(52.6%)
arrived. Reasons for failure to arrive were assessed. Results: Of the 1754 women reaching initial interview by
the genetics-trained nurse, 684 (40%) needed further
professional genetic counseling at Soroka medical center. Reasons
for further formal genetic counseling were:
Medical problems detected during current pregnancy;
Consanguinity; Family history of genetic diseases; pre-
viously known carrier status of couple. Of the 684 women
referred to formal genetic counseling, only 360(52.6%)
arrived. Reasons for failure to arrive were assessed. Conclusion: Professional
certification is a way
of
assuring that an individual has the mandatory knowledge,
skills and abilities to provide quality of patient care and safe
practice. We describe an alternative pathway for national
regulation of genetic counsellor career development and
certification process that may support other countries in
providing national regulation of genetic counsellors where
the
profession
is
still
emerging,
and
where
no
educational route. Conclusion: In a population with high incidence of
intermarriage and genetic diseases, it is recommended to
integrate a genetic counseling services in the community
health
system
to
increase
responsiveness
to
genetic
counseling and testing in a timely manner, adapted to
religion and culture. Dedicated teams should be trained and
continuous activity promoting public awareness is essential. K. Svensson: None. R. Pestoff: None. M. Paneque:
None. C. Malmgren Ingvoldstad: None. K. Svensson: None. R. Pestoff: None. M. Paneque:
None. C. Malmgren Ingvoldstad: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 671 E. Dagan1, S. Barnoy2 E. Dagan1, S. Barnoy2 P19.17A
Complementariness between medical geneticists and
genetic counsellors: its added value in genetics services in
Europe 1University of Haifa, Haifa, Israel, 2Tel Aviv University, Tel
Aviv, Israel M. Paneque Herrera1, C. Serra-Juhé2, R. Pestoff3, C. Cordier4,
J. Silva1, R. Moldovan5, C. Ingvoldstad6 M. Paneque Herrera1, C. Serra-Juhé2, R. Pestoff3, C. Cordier4,
J. Silva1, R. Moldovan5, C. Ingvoldstad6 Aim: To study factors associated with genetic skills per-
formance in nursing in highly educated nurses in Israel. Methods: Data were collected during a conference for
professional associations in nursing in Israel. The partici-
pants were asked to complete a set of anonymous
questionnaires which included, genetic knowledge, mathe-
matics literacy, sense of genetic epistemology, openness to
technological innovations, attitude towards the importance
of assimilating genetics skills in nursing, and the extent they
perform these skills in the routine nursing work. 1IBMC, Porto, Portugal, 2Genetics Unit, Universitat Pompeu
Fabra - Hospital del Mar Research Institute (IMIM),,
Barcelona, Spain, 3Department of Clinical Genetics, Linköping
University Hospital, Linköping, Sweden, Linköping,, Sweden,
4Synlab Genetics, Department of Genetics,, Lausanne,
Switzerland, 5Department of Psychology, Babe-Bolyai
University, Cluj-Napoca, Romania, 6Department of Public
Health and Caring Science, Uppsala University, Uppsala,
Sweden 1IBMC, Porto, Portugal, 2Genetics Unit, Universitat Pompeu
Fabra - Hospital del Mar Research Institute (IMIM),,
Barcelona, Spain, 3Department of Clinical Genetics, Linköping
University Hospital, Linköping, Sweden, Linköping,, Sweden,
4Synlab Genetics, Department of Genetics,, Lausanne,
Switzerland, 5Department of Psychology, Babe-Bolyai
University, Cluj-Napoca, Romania, 6Department of Public
Health and Caring Science, Uppsala University, Uppsala,
Sweden p
g
Results: Participants included 81 nurses aged 46.5±10.6
years on average, most of them were Israeli born, Jewish,
secular, working in hospitals, and ~70% holding a Master’s
degree. The mean genetic knowledge was low (53.4%
±13.43), as was the mathematics literacy (61.3%±25.3). Nurses reported of low assimilation of genetic skills in their
routine work (1.9±0.78; range, 1-4), although their overall
attitudes towards the importance of genetics in nursing were
positive. Positive correlations were found between a sense
of epistemology about genetics, and attitudes towards the
importance of genetics in nursing, with assimilation of
genetic skills in routine nursing work. These variables
added 33.4% to the explained variance in the hierarchical
regression model for variables predicting genetic skills
performance in nursing. Additionally, mathematical literacy
correlated with genetic knowledge, and older nurses held
more positive attitudes towards the importance of assimilat-
ing genetics skills in nursing. Results: Participants included 81 nurses aged 46.5±10.6
years on average, most of them were Israeli born, Jewish,
secular, working in hospitals, and ~70% holding a Master’s
degree. P19.17A
Complementariness between medical geneticists and
genetic counsellors: its added value in genetics services in
Europe The mean genetic knowledge was low (53.4%
±13.43), as was the mathematics literacy (61.3%±25.3). Nurses reported of low assimilation of genetic skills in their
routine work (1.9±0.78; range, 1-4), although their overall
attitudes towards the importance of genetics in nursing were
positive. Positive correlations were found between a sense
of epistemology about genetics, and attitudes towards the
importance of genetics in nursing, with assimilation of
genetic skills in routine nursing work. These variables
added 33.4% to the explained variance in the hierarchical
regression model for variables predicting genetic skills
performance in nursing. Additionally, mathematical literacy
correlated with genetic knowledge, and older nurses held
more positive attitudes towards the importance of assimilat-
ing genetics skills in nursing. Clinical genetic services have progressed significantly the
last few decades. This has led to the need for non-medical
healthcare professionals working as genetic counsellors in
Europe and worldwide. However, there is no unified
approach to genetic counsellors’ role in healthcare services
in Europe, as in most countries the profession is still
emerging and the educational backgrounds diverge notice-
ably, within and between countries. This qualitative study
aims to describe the potential added value of genetic
counsellors in clinical genetics teams and to explore their
tasks and responsibilities in different European countries. A
total of 143 participants providing genetic counselling in
Europe at the time of the survey responded. The results
show differences in activities of genetic counsellors,
although there is a wide range of roles which are similar. The ability to establish a quality relationship with con-
sultands was frequently mentioned as one of the strengths of
genetic counsellors, as well as a patient centred approach. It
is believed that genetic counsellors add a more holistic
approach of psychosocial and familial dimensions of
genetic concerns to the multidisciplinary teams. This study
provides examples of successful integration of genetic
counsellors in teams, as complementariness with medical
geneticist became clear in several cases. Although the added
value of genetic counsellors was manifested, professional
recognition of genetic counsellors across Europe is still
needed in order to support the quality of patients care and
safety of practice. Conclusion: Although the nurses in this study were
highly educated, we found disappointingly low scores in
genetic knowledge and in performance of genetic skills in
the nursing routine work. E. Dagan: None. S. Barnoy: None. E. Dagan: None. S. Barnoy: None. P19.20D The evolving role of the clinical geneticist facing new
technologies: the opinion of patients from the AnDDI-Rares
network A. Lapointe1, F. Neuhaus2, C. Biotteau2, C. Dervieux2, I. Marchetti-Waternaux2, S. Marlin3, I. Ben Aissa4, M. Rossi5, A. Goldenberg6, G. Baujat7, C. Thauvin8, E. Gautier8, L. Demougeot9, L. Faivre9 A. Lapointe1, F. Neuhaus2, C. Biotteau2, C. Dervieux2, I. Marchetti-Waternaux2, S. Marlin3, I. Ben Aissa4, M. Rossi5, A. Goldenberg6, G. Baujat7, C. Thauvin8, E. Gautier8, L. Demougeot9, L. Faivre9 M. Paneque Herrera: None. C. Serra-Juhé: None. R. Pestoff: None. C. Cordier: None. J. Silva: None. R. Moldovan: None. C. Ingvoldstad: None. 1Filière AnDDI-Rares, Paris, France, 2CoPIL Filière AnDDI-
Rares, Paris, France, 3Centre de référence des surdités
génétiques, Genetic Department, Hôpital Necker-enfants
malades, Paris, France, 4Filière Sensgene, Strasbourg, 1Filière AnDDI-Rares, Paris, France, 2CoPIL Filière AnDDI-
Rares, Paris, France, 3Centre de référence des surdités
génétiques, Genetic Department, Hôpital Necker-enfants
malades, Paris, France, 4Filière Sensgene, Strasbourg, A. Lapointe1, F. Neuhaus2, C. Biotteau2, C. Dervieux2, I.
Marchetti-Waternaux2, S. Marlin3, I. Ben Aissa4, M. Rossi5, A.
Goldenberg6, G. Baujat7, C. Thauvin8, E. Gautier8, L.
Demougeot9, L. Faivre9 P19.19C In these cases, 70% of the respondents would
like a consultation with a geneticist for transmission of the
results. With NGS development, the whole patient care
pathway needs to be redesigned and professional training
reconsidered to face this the arrival of genomic medicine. In this context, a survey was done to study the preferences
of patients for the circuit of prescription and transmission of
results of NGS tests in a clinical diagnostic setting. The
survey was sent to a group of 50 patients organizations of
the AnDDI-Rares network (developmental disorders and
intellectual disability). 404 questionnaires were collected. More than 80 % of the respondents had met a clinical
geneticist in their diagnostic odyssey. 93% of them agreed
that
geneticists
should
have
a
primary
role
in
the
prescription of pangenomic tests, in particular because
genetic teams offer multidisciplinary cares (genetic coun-
sellors, psychologists, social worker). However, 72%, were
also in favour of NGS tests being prescribed by a non-
geneticist medical specialist, in particular because he knows
better his patient, and have shorter delay for accessing to
consultations. In these cases, 70% of the respondents would
like a consultation with a geneticist for transmission of the
results. With NGS development, the whole patient care
pathway needs to be redesigned and professional training
reconsidered to face this the arrival of genomic medicine. These include: These include: ●
Provision and evaluation of genomic sequencing to
patients through defined flagships providing a multi-
disciplinary environment for experiential learning; ●
A 12 week cross-training program for medical scientists
in variant curation and reporting; ●
Research placements in genomics for advanced trainee
clinical geneticists; ●
Research, clinical and laboratory placements for non-
genetic medical specialists that address an authentic
problem drawn from their practice. These experiential placements are being augmented with These experiential placements are being augmented with ●
Interactive workshops in germline and somatic variant
curation A. Lapointe: None. F. Neuhaus: None. C. Biotteau:
None. C. Dervieux: None. I. Marchetti-Waternaux:
None. S. Marlin: None. I. Ben Aissa: None. M. Rossi:
None. A. Goldenberg: None. G. Baujat: None. C. Thauvin: None. E. Gautier: None. L. Demougeot: None. L. Faivre: None. ●
Development of on-line modules in variant curation and
interpretation Results and Conclusions: Ongoing qualitative evalua-
tion of the program reveals consistently high levels of
engagement
and
learning. P19.19C Genetic professional skills performance in nursing: A
preliminary study in highly educated nurses 672 J. del Picchia Research Institute, Parkville, Australia, 4Victorian Clinical
Genetics Service, Parkville, Australia, 5Peter MacCallum
Cancer Centre, Parkville, Australia, 6Walter and Eliza Hall
Institute, Parkville, Australia France, 5CLAD Sud Est - CHU HCL, Lyon, France, 6CLAD
Nord de France, Rouen, France, 7Centre de référence des
Maladies Osseuses Constitutionnelles, Hôpital Necker-enfants
malades, Paris, France, 8CLAD Est, Dijon, France, 9Filière
AnDDI-Rares, Dijon, France Introduction: Rapid advances in genomics are bringing
benefits to healthcare but not without challenge. Key among
these is the need for a workforce that can deliver the ben-
efits of genomic medicine to patients. The arrival of new diagnostic technologies and genomic
medicine lead to important changes in genetic clinics. An
expected growing number of indications of tests raise the
question of the prescription of pangenomic tests by non-
geneticists. In that case, clinical geneticists are worried of
the possible reduced quality of patient information, in par-
ticular the limited time dedicated to the implications of test
results, genetic counselling, but also new issues including
secondary data and management of VUS. Methods: In the demonstration phase of Melbourne
Genomics, an alliance of 10 leading healthcare and research
organisations, an immediate need, and opportunity, was
identified to develop a workforce to guide genomic
sequencing into healthcare across Victoria. To address this
we have developed a multi-faceted workforce development
program providing opportunities for learning experiences
that enhance and augment clinical and laboratory practice,
focussing on engagement with real-life problems relevant to
practice or skills-based training. In this context, a survey was done to study the preferences
of patients for the circuit of prescription and transmission of
results of NGS tests in a clinical diagnostic setting. The
survey was sent to a group of 50 patients organizations of
the AnDDI-Rares network (developmental disorders and
intellectual disability). 404 questionnaires were collected. More than 80 % of the respondents had met a clinical
geneticist in their diagnostic odyssey. 93% of them agreed
that
geneticists
should
have
a
primary
role
in
the
prescription of pangenomic tests, in particular because
genetic teams offer multidisciplinary cares (genetic coun-
sellors, psychologists, social worker). However, 72%, were
also in favour of NGS tests being prescribed by a non-
geneticist medical specialist, in particular because he knows
better his patient, and have shorter delay for accessing to
consultations. P19.19C Quantitative
evaluation
of
structured workshops measured improved knowledge and
skills for all participants with over 90% reporting high
levels
of
satisfaction. This
success
demonstrates
the
effectiveness of interactive, participant-centred approaches
in developing a sustainable genomics workforce develop-
ment strategy. 1Melbourne Genomics Health Alliance, Parkville, Australia,
2Monash Health, Clayton, Australia, 3Murdoch Childrens F. Cunningham: None. S. Lunke: None. T.Y. Tan:
None. M. Martyn: None. E. Lynch: None. S.N.A.
Roesley: None. N. Thorne: None. C. Gaff: None. F. Cunningham1,2,3, S. Lunke4,3, T. Y. Tan4,3, M. Martyn1,3, E.
Lynch1, S. N. A. Roesley1,5, N. Thorne1,6, C. Gaff1 P19.21A “Experience is the teacher of all things”- upskilling the
genomics workforce in variant interpretation “Experience is the teacher of all things”- upskilling the
genomics workforce in variant interpretation F. Cunningham1,2,3, S. Lunke4,3, T. Y. Tan4,3, M. Martyn1,3, E. Lynch1, S. N. A. Roesley1,5, N. Thorne1,6, C. Gaff1 F. Cunningham: None. S. Lunke: None. T.Y. Tan:
None. M. Martyn: None. E. Lynch: None. S.N.A. Roesley: None. N. Thorne: None. C. Gaff: None. F. Cunningham: None. S. Lunke: None. T.Y. Tan:
None. M. Martyn: None. E. Lynch: None. S.N.A. Roesley: None. N. Thorne: None. C. Gaff: None. 1Melbourne Genomics Health Alliance, Parkville, Australia,
2Monash Health, Clayton, Australia, 3Murdoch Childrens Abstracts from the 51st European Society of Human Genetics Conference: Posters 673 The 100,000 Genomes Project in the UK has established
and refined many of the processes around the delivery of
whole genome sequencing at scale. The next objective is the
implementation of these technologies as an integral part of
routine healthcare in specialities outside genetics thus pro-
moting equitable access to these technologies. This requires
‘mainstream’ clinicians to adopt some tasks which are
currently the province of clinical geneticists. In March
2017, we held a workshop bringing together clinical and
genetics specialists and specialists from other clinical areas
including cardiology, ophthalmology, paediatrics, respira-
tory and renal medicine, to evaluate the particular issues
that arise at two interfaces: patient identification and referral
for testing; and interpretation of results and clinical decision
making. We found that most clinicians will need a lot of
support to identify patients who may benefit from testing
and decide which test to request. The capture of necessary
clinical and phenotypic information at the point of referral is
problematic. Examples of supporting methods were shared. Similarly, barriers to involving mainstream clinicians in
interpretation include practicalities (such as geography,
timing and length of consultation) and lack of required
expertise, which may be outside their clinical area. Support
for clinical decision making based on genomic test results
would be vital for effective implementation. For equitable
implementation of genomic testing across the health system
detailed attention must be paid to developing new clinical
pathways in each speciality and the necessary support sys-
tems. Our report can be downloaded from http://www. phgfoundation.org/report/genomics-mainstream-clinical-pa
thways. Queen Elizabeth University Hospital, Glasgow, United
Kingdom Educational and self-assessment smartphone apps that
explain (and test knowledge of) commonly used genomics
and bioinformatics terms and acronyms, were created by the
authors. These non-profit Clinical Genomics guide and quiz
apps have been made available, completely free of charge,
via the official Apple and Android app stores (search for:
“Clinical
Genomics”). They
provide
immediate
clear
explanations of genomics-related terminology (such as
“FASTQ”, “BAM”, “GVCF”, “BED files”, “GATK”
and “BWA”) that are otherwise not easily accessible. Creating these multi-platform apps was time-consuming. Highly positive feedback has, however, been received from
physicians, genetic counsellors, laboratory scientists and
students, internationally. The apps are used by postgrad-
uate students to assist their understanding eg during
lectures or when reading scientific papers. For instance,
they are used to assist understanding of key Clinical
Genomics concepts and terminology by students on the
internationally popular Medical Genetics & Genomics
Masters programmes at Glasgow University. The
apps
are
also
used
by
professionals,
who
increasingly receive complex genetic laboratory reports
that must be interpreted for patient benefit. H. Burton: None. A. Hall: None. S. Raza: None. M. Kroese: None. The apps have been approved by the ESHG, the
Scottish Genetics Education Network (ScotGEN) and by
DSDnet. They have been downloaded thousands of times,
across approximately 50 countries and have recently been
shortlisted for a national higher education "Innovation
Technology Excellence" award. Further programming and
development of the apps is currently underway, to include
explanations of additional terms and concepts. The authors
would be grateful for further feedback and for suggestions
regarding any more terms that could be incorporated. Please
email: edward.tobias@glasgow.ac.uk. Genetic counselling in hereditary diffuse gastric cancer:
economical and psycho-social impact Genetic counselling in hereditary diffuse gastric cancer:
economical and psycho-social impact L. Garrido1, T. Nércio1, R. Leal2, R. Guimarães1, L. Ferro1, L. Vilarinho1, S. Costa1, A. Magalhães1, A. S. Mesquita1, A. F. Pereira1, I. Gullo1,3,2, H. Pinheiro2, S. Sousa2, B. Carvalho3,2, A. P. Neto3,2, L. Capela3,2, C. Teixeira1, A. Fareleira1, V. Devezas1,
G. Macedo1,3, J. Preto1,3, J. Barbosa1,3, M. Baptista1,3, G. Pinto1,
M. Damasceno1, J. L. Fougo1,3, J. Costa-Maia1, S. Fernandes3,2,
F. Carneiro1,3,2, S. Castedo1,3,2, C. Oliveira2,3 E.S. Tobias: None. A.P. Tobias: None. E.S. Tobias: None. A.P. Tobias: None. P19.22B E. S. Tobias, A. P. Tobias E. S. Tobias, A. P. Tobias Queen Elizabeth University Hospital, Glasgow, United
Kingdom H. Burton, A. Hall, S. Raza, M. Kroese T. M. Aloraini1, A. Gari2, G. AlKarbani2 Introduction: Hereditary Diffuse Gastric Cancer (HDGC)
syndrome is caused by CDH1-germline mutations and
carriers have high-risk to develop early-onset diffuse gastric
cancer (DGC) in both genders and lobular breast cancer
(LBC) in females. HDGC is deadly for those expressing
clinical phenotype, but presymptomatic testing allows dis-
ease prevention or early-diagnosis in mutation-carriers
through risk-reduction gastrectomy and yearly breast MRI,
discharging ~50% of non-carriers. As Health-Care-Provider
of ERN-GENTURIS, we aim to evaluate the economic
impact of genetic counselling, presymptomatic diagnosis
and multidisciplinary care for the National Health Service
(NHS), and the clinical and psycho-social implications for
HDGC families. 1NGHA, Riyadh, Saudi Arabia, 2Alfaisal University; King
Faisal Specialist Hospital And Research Center., Riyadh,
Saudi Arabia There is a consensus that whole-genome sequencing (WGS)
and whole-exome sequencing (WES) will improve in
accuracy and decline in price, eventually becoming an
integral part of clinical medicine. In clinical genome
sequencing, there is a potential for the recognition of inci-
dental findings that are unrelated to the indication for
ordering the sequencing but may nonetheless be of medical
value. Here, we aimed to compare the attitudes of genetics
professionals and patients towards incidental findings from
WGS/WES, including a hypothetical situation in which a
patient/family declines to receive all the incidental findings. A web-based survey was conducted with 20 genetics pro-
fessionals (medical geneticists and genetic counselors) and
30 patients from King Faisal Specialist Hospital and
Research Center in Riyadh, Saudi Arabia. Data collection
took place between the 2nd of April and the 4th of May 2017. The results demonstrated that 55% of genetics professionals
would not share all WES/WGS results with patients and
they preferred to focus on actionable results that yield
benefits such as medical treatment and disease prevention. However, 73.3% of patients were interested in receiving all
the raw genomic data for themselves and their children. References 1- Alkuraya, F. S. (2014). Genetics and genomic
medicine in Saudi Arabia. Molecular Genetics and Genomic
Medicine. 2- Kalia et al., 2017. Recommendations for
reporting of secondary findings in clinical exome and gen-
ome sequencing, 2016 update a policy statement of the
American College of Medical Genetics and Genomics. Material
and
Methods:
We
evaluated
outputs
of
structured oncogenetics and high-risk consultations in 111
individuals from 7 HDGC families by consulting clinical/
financial records, and assessed its psycho-social impact by
applying an emotional-distress-scale (HADS) to 70/111
individuals. P19.26B L. Garrido: None. T. Nércio: None. R. Leal: None. R. Guimarães: None. L. Ferro: None. L. Vilarinho: None. S. L. Garrido: None. T. Nércio: None. R. Leal: None. R. Guimarães: None. L. Ferro: None. L. Vilarinho: None. S. L. Garrido: None. T. Nércio: None. R. Leal: None. R. Guimarães: None. L. Ferro: None. L. Vilarinho: None. S. Costa: None. A. Magalhães: None. A.S. Mesquita: None. A.F. Pereira: None. I. Gullo: None. H. Pinheiro: None. S. Sousa: None. B. Carvalho: None. A.P. Neto: None. L. Capela: None. C. Teixeira: None. A. Fareleira: None. V. Devezas: None. G. Macedo: None. J. Preto: None. J. Barbosa: None. M. Baptista: None. G. Pinto: None. M. Damasceno: None. J.L. Fougo: None. J. Costa-Maia:
None. S. Fernandes: None. F. Carneiro: None. S. Castedo: None. C. Oliveira: None. Informing relatives at risk of inherited cardiac conditions:
attitudes of healthcare professionals, probands and
relatives Costa: None. A. Magalhães: None. A.S. Mesquita: None. A.F. Pereira: None. I. Gullo: None. H. Pinheiro: None. S. Costa: None. A. Magalhães: None. A.S. Mesquita: None. A.F. Pereira: None. I. Gullo: None. H. Pinheiro: None. S. L. M. Van den Heuvel1, M. J. Huisinga1, Y. M. Hoedemaekers2,
A. B. Baas3, M. Plantinga2, L. Henneman4, J. P. van Tintelen1,
E. M. A. Smets1, I. Christiaans1 T. M. Aloraini1, A. Gari2, G. AlKarbani2 Results: From 2011-2016, we screened 111 individuals,
from which 53% tested negative (n=58;30M:28F) and were
discharged from follow-up each costing 200€ to the
Hospital. 47% (n=53;26M:27F) tested positive. From 7
probands, 2 were diagnosed with LBC and remain alive
after curative surgery, while 5 diagnosed with DGC are
deceased. The hospital expenses with probands range from
30-50K€ independently of cancer type. Costs with carriers
opting for prophylactic approaches range from 4-8K€
(higher cost/females), except if disease is identified after
the first high-risk consultation, raising the cost to 27K€ but
life-saving. Those
opting
for
surveillance,
cost
~1K
€/patient. Concerning psychologic impact, carriers demon-
strate higher levels of distress than non-carriers. T.M. Aloraini:
None. A. Gari:
None. G. AlKarbani: None. T.M. Aloraini:
None. A. Gari:
None. G. AlKarbani: None. T.M. Aloraini:
None. A. Gari:
None. G. AlKarbani: None. Conclusion: Structured healthcare in HDGC economic-
ally benefits NHS, is life-saving for carriers, reassuring non-
carriers. Conclusion: Structured healthcare in HDGC economic-
ally benefits NHS, is life-saving for carriers, reassuring non-
carriers. 1Academic Medical Center, Amsterdam, Netherlands,
2University Medical Center Groningen, Groningen,
Netherlands, 3University Medical Center Utrecht, Utrecht,
Netherlands, 4VU University Medical Center, Amsterdam,
Netherlands P19.23C 1Centro Hospitalar São João, Porto, Portugal, 2Ipatimup/i3S,
Institute of Molecular Pathology and Immunology at the
University of Porto (Ipatimup), Porto, Portugal & Instituto de
Investigação e Inovação em Saúde (i3S), University of Porto,
Porto, Portugal, Porto, Portugal, 3FMUP, Faculty of Medicine
of the University of Porto, Porto, Portugal, Porto, Portugal P19.23C
Genomics in mainstream clinical pathways P19.23C
Genomics in mainstream clinical pathways P19.23C
Genomics in mainstream clinical pathways PHG Foundation, Cambridge, United Kingdom J. del Picchia 674 T. M. Aloraini1, A. Gari2, G. AlKarbani2 P19.25A Based on these findings, we are conducting a
randomized clinical trial to evaluate the uptake and accep-
tance of a tailored approach, compared to the family-
mediated approach. In this tailored approach probands can
choose which relatives they will inform themselves and
which relatives they prefer to be informed by the counsellor. K.L. Barr:
None. M. McAllister:
None. M. Votruba: None. P19.29A An interactive application to support practical teaching in
medical genomics and bioinformatics K. Prochazkova1, P. Novotny2, M. Hancarova1, D. Prchalova1, Z. Sedlacek1 L.M. Van den Heuvel: None. M.J. Huisinga: None. Y. M. Hoedemaekers: None. A.B. Baas: None. M. Plan-
tinga: None. L. Henneman: None. J.P. van Tintelen:
None. E.M.A. Smets: None. I. Christiaans: None. 1Department of Biology and Medical Genetics, Charles
University 2nd Faculty of Medicine and UH Motol, Prague,
Czech Republic, 2Department of Biology and Environmental
Studies, Charles University Faculty of Education, Prague,
Czech Republic P19.25A Comparison between the attitudes of genetics professionals
and patients towards incidental findings from whole-
genome or whole-exome sequencing Comparison between the attitudes of genetics professionals
and patients towards incidental findings from whole-
genome or whole-exome sequencing 675 Abstracts from the 51st European Society of Human Genetics Conference: Posters Inherited cardiac conditions (ICCs) can lead to sudden
cardiac death (SCD) at young age. Cardiac monitoring and/
or predictive genetic testing is advised to at-risk relatives to
prevent SCD. Usually, probands are asked to inform their
relatives with a family letter. This study investigated atti-
tudes towards this family-mediated approach in ICCs to use
for potential improvements. A qualitative study design was
used. Two online focus groups with 27 healthcare profes-
sionals (HCPs), including cardiologists, clinical geneticists,
genetic counsellors and psychosocial workers, and 25 face-
to-face semi-structured interviews with probands and rela-
tives were independently analysed by two researchers using
a thematic approach. Main findings show that it is generally
preferred that probands inform their relatives. However,
both HCPs and probands struggle with the dependency and
psychological and practical burden on probands to inform
their relatives. HCPs think a more active role of HCPs in
informing at-risk relatives is needed to overcome these
barriers. Probands and relatives ideally prefer a tailored
information provision strategy to inform at-risk relatives,
adjusted to family dynamics and personality characteristics. In contrast, HCPs prefer uniformity in procedures to restrict
the workload. To summarize, although in most cases it is
preferred that probands inform relatives, several barriers are
perceived. Based on these findings, we are conducting a
randomized clinical trial to evaluate the uptake and accep-
tance of a tailored approach, compared to the family-
mediated approach. In this tailored approach probands can
choose which relatives they will inform themselves and
which relatives they prefer to be informed by the counsellor. Inherited cardiac conditions (ICCs) can lead to sudden
cardiac death (SCD) at young age. Cardiac monitoring and/
or predictive genetic testing is advised to at-risk relatives to
prevent SCD. Usually, probands are asked to inform their
relatives with a family letter. This study investigated atti-
tudes towards this family-mediated approach in ICCs to use
for potential improvements. A qualitative study design was
used. P19.25A Two online focus groups with 27 healthcare profes-
sionals (HCPs), including cardiologists, clinical geneticists,
genetic counsellors and psychosocial workers, and 25 face-
to-face semi-structured interviews with probands and rela-
tives were independently analysed by two researchers using
a thematic approach. Main findings show that it is generally
preferred that probands inform their relatives. However,
both HCPs and probands struggle with the dependency and
psychological and practical burden on probands to inform
their relatives. HCPs think a more active role of HCPs in
informing at-risk relatives is needed to overcome these
barriers. Probands and relatives ideally prefer a tailored
information provision strategy to inform at-risk relatives,
adjusted to family dynamics and personality characteristics. In contrast, HCPs prefer uniformity in procedures to restrict
the workload. To summarize, although in most cases it is
preferred that probands inform relatives, several barriers are
perceived. Based on these findings, we are conducting a
randomized clinical trial to evaluate the uptake and accep-
tance of a tailored approach, compared to the family-
mediated approach. In this tailored approach probands can
choose which relatives they will inform themselves and
which relatives they prefer to be informed by the counsellor. attitudes towards novel therapies for IRD. Interviews were
transcribed verbatim and analysed using thematic analysis. Results: Interviewees’ attitudes were classified into three
broad themes: hope, despondency, and ‘knowledge is
power’. Whilst hope was expressed universally among
participants, this was also accompanied by concerns
regarding the risk involved in therapy uptake and the
lengthy timescale of development. There was also an
appetite identified for access to more information regarding
therapy development from trustworthy, accessible sources. Although participants varied in the extent to which they felt
anticipation versus resignation it was universally acknowl-
edged that all would draw empowerment from being
provided with up-to-date, accurate information. Conclusions: This study suggests there is a need for
improved dissemination of accessible information on novel
IRD therapies through face-to-face as well as online
platforms. The development and validation of an ophthal-
mic genetic-specific PROM may help to ensure the broad
and complex needs of IRD patients are met. A better-
informed IRD patient population could improve supply to
clinical trials and ultimately, aid in the successful develop-
ment and clinical implementation of novel IRD therapies. In contrast, HCPs prefer uniformity in procedures to restrict
the workload. To summarize, although in most cases it is
preferred that probands inform relatives, several barriers are
perceived. P19.27C A qualitative study exploring patient attitudes towards
novel therapies for inherited retinal dystrophies K. L. Barr1, M. McAllister1, M. Votruba2 There was a significant improvement after
the practical (on average by one grade) of knowledge and
skills of the students in medical molecular genetics,
genomics and bioinformatics. The feedback is used to improve potentially problematic
sections of the practical. A pilot version of another teaching
application, illustrating the workflow of whole-genome
analyses, is under development. Funding: Agreement OA17/008 with ITER to strengthen
scientific and technological education, training, research,
development and innovation in Genomics, Personalized
Medicine and Biotechnology; CEDeI fellowship (Cabildo
de Tenerife) to CGN. Supported by 17-29423A. K. Prochazkova: None. P. Novotny: None. M. Hancar-
ova: None. D. Prchalova: None. Z. Sedlacek: None. C. Gonzalez Navasa: None. T. Hernández-Beeftink:
None. H. Rodríguez-Pérez: None. J. Lorenzo-Salazar:
None. R. González-Montelongo: None. C. Flores: None. C. Gonzalez Navasa: None. T. Hernández-Beeftink:
None. H. Rodríguez-Pérez: None. J. Lorenzo-Salazar: K. L. Barr1, M. McAllister1, M. Votruba2 K. L. Barr1, M. McAllister1, M. Votruba2 K. L. Barr1, M. McAllister1, M. Votruba2 Medical students usually find molecular genetics, genomics
and bioinformatics difficult and remote from their future
clinical plans. However, their orientation in these topics is
necessary as genetic testing is rapidly penetrating into all
medical specialties. Patient involvement and practical
laboratory work can enhance teaching, but obstacles limit
their broader implementation. Computer simulations can
instead be used to ameliorate the teaching process. 1Cardiff University, Cardiff, United Kingdom, 2Cardiff and
Vale University Health Board, Cardiff, United Kingdom Introduction: There is currently no treatment or cure for
inherited retinal dystrophies, yet numerous clinical trials are
underway investigating novel therapeutic strategies. This
study aimed to explore the awareness and opinions of new
therapies among IRD patients in South Wales. We created an electronic teaching application simulating
the process of molecular genetic diagnosis of a monogenic
disorder in a family. Thirteen tasks presented on a sequence
of web pages deal with the clinical assessment of pedigrees
and phenotypes, clinical diagnosis, design of a laboratory
test, identification of a mutation using capillary sequencing, Materials and Methods: Ten participants with diagnoses
of IRD were recruited through the Genetic Eye Clinic at
Cardiff and Vale University Health Board (C&V UHB). Semi-structured qualitative interviews explored patients’ J. del Picchia 676 Jupyter notebook-based pipeline with plain contextual
explanations was designed to seamlessly progress from
event-level data and obtain species abundance. assessment of pathogenicity and clinical relevance of the
mutation, and final confirmation of the diagnosis. The tasks
employ publicly available bioinformatic resources used in
the
daily
routine
of
medical
genetics
departments
worldwide. Results and Conclusions: Overall, protocols performed
similarly enriching mtDNA directly from DNA extractions,
both from animal and plant species. We anticipate that the
choice of enrichment will depend on the attendees’
educational level and the available laboratory equipment. Sequencing of a mock specimen (artificial mixture of foods)
after a simple 10-minute library preparation allowed
identifying all species with minimal deviations from the
expected. We envisage this hand-on experience as a
learning-by-doing instrument for transdisciplinary educa-
tion adapted to basic laboratory resource settings. Feedback from students was obtained using a test
administered before and after the practical. Most comments
on the practical were very positive. The answers to specific
test questions were classified as very good, satisfactory, or
unsatisfactory. P19.30B None. R. González-Montelongo: None. C. Flores: None. Tricky situation and diagnostic odyssey solved by
combination of “make a point” genetic counseling and NGS On the basis of these observation it
was fairly difficult to interpret this situation as de novo
autosomal dominant variant. The further analysis by NGS
has been performed, heterozygosity of c.1085G>A (p. Gly362Asp) variant was revealed in 5% of mother’s DNA. All variants have been confirmed by Sanger sequencing. Herein and in n Post-test Genetic Counseling the results and
in depth explanation of germinal mosaicism have been
discussed. information regarding the presence or absence of children. f-tree can assemble a pedigree that comprises data for up to
five generations, and places a couple with and without
children within the third and fourth generation of a given
pedigree, respectively. 2) Next, an interviewer may assist
the user to complete a multiple-choice questionnaire to
collect genealogically pertinent information for the user and
their
relatives
over
five
generations. 3)
f-tree
then
automatically creates a pedigree chart, which can be
reviewed on the screen in real time. Conclusions: The f-tree software program has been made
available for free on the Iwate Tohoku Medical Megabank
Organization
website
(http://iwate-megabank.org/en/
genetic/). Until date, this program has been downloaded
more than 3,000 times since its release on March 19, 2015. T. Tokutomi: None. A. Fukushima: None. K. Yama-
moto: None. F. Nakayama: None. Y. Katsube: None. A. Shimizu: None. M. Sasaki: None. N. Bukvic: None. A. D’Aprile: None. O. Palumbo:
None. C. Cesarano: None. C. Ceccarini: None. P. Palumbo: None. F.C. Susca: None. A. Stella: None. C. Piccoli: None. N. Resta: None. N. Bukvic: None. A. D’Aprile: None. O. Palumbo:
None. C. Cesarano: None. C. Ceccarini: None. P. Palumbo: None. F.C. Susca: None. A. Stella: None. C. Piccoli: None. N. Resta: None. Tricky situation and diagnostic odyssey solved by
combination of “make a point” genetic counseling and NGS Tricky situation and diagnostic odyssey solved by
combination of “make a point” genetic counseling and NGS C. Gonzalez Navasa1, T. Hernández-Beeftink2, H. Rodríguez-
Pérez2, J. Lorenzo-Salazar1, R. González-Montelongo1, C. Flores1,2,3 C. Gonzalez Navasa1, T. Hernández-Beeftink2, H. Rodríguez-
Pérez2, J. Lorenzo-Salazar1, R. González-Montelongo1, C. Flores1,2,3 N. Bukvic1, A. D’Aprile2, O. Palumbo3, C. Cesarano2, C. Ceccarini2, P. Palumbo3, F. C. Susca4, A. Stella4, C. Piccoli5, N. Resta4 1Genomics Division, Instituto Tecnológico y de Energías
Renovables, Granadilla de Abona, Spain, 2Research Unit,
Hospital Universitario N.S. de Candelaria, Universidad de La
Laguna, Santa Cruz de Tenerife, Spain, 3CIBER de
Enfermedades Respiratorias, Instituto de Salud Carlos III,
Madrid, Spain 1U.O.C Genetica Medica Azienda Ospedaliero – Universitaria
Consorziale, Bari, Italy, 2Sez di Citogenetica- Laboratorio
Centrale– Dipartimento di Patologia Clinica, Azienda
Ospedaliero-Universitaria, Foggia, Italy, 3Genetica Medica,
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo
(FG), Italy, 4U.O.C Genetica Medica, D.A.I. Patologia
Diagnostica, Bioimmagini e Sanità Pubblica, Università degli
Studi “Aldo Moro”, Bari, Italy, 5Dipartimento di Medicina
Clinica e Sperimentale - Universita di FG, Foggia, Italy Introduction: Because of the low cost, laptop operability,
and the USB-powered compact design of MinION (Oxford
Nanopore Technologies), using cutting-edge Next-Genera-
tion Sequencing (NGS) technology in field experiments and
the classroom is a reality. By leveraging the benefits of
using mitochondrial DNA (mtDNA), here we devised the
protocols and materials for a NGS hands-on training with
educational purposes. Encephalopathy due to defective mitochondrial and perox-
isomal fission-1 (EMPF1) is characterized by delayed psy-
chomotor development and hypotonia that may lead to
death in childhood. Many patients develop refractory sei-
zures, consistent with an epileptic encephalopathy, and
thereafter show neurologic decline. The age at onset, fea-
tures, and severity are variable. We report on, 40 years old
woman pass through our Genetic Counseling, due to posi-
tive anamnestic data regarding presence of intellectual dis-
ability, transient ischemical episodes and myoclonical
seisures in sibs. These signs and simptoms were present in
first son (exitus at 3 years), as also in second son (exitus at Materials and Methods: We assessed two procedures
for facilitated mtDNA enrichment from foods: QIAprep
Spin Miniprep Kit (QIAGEN) and the classic alkaline lysis
protocol for plasmid isolation. Enrichment of mtDNA vs. nuclear DNA was evaluated by quantitative Real-Time PCR
(CFX96 Touch, BioRad) using primers from eukaryotic
conserved regions. PCR-free libraries were constructed with
Rapid Sequencing Kit and sequenced with MinION. Comparison between different screening strategies for the
detection of all fetal karyotype abnormalities F. R. Grati1, B. Grimi1, K. Bajaj2, L. Marcato1, B. Malvestiti1, F. Maggi1, G. Simoni1, S. J. Gross3, J. C. P. B. Ferreira4,5 Tricky situation and diagnostic odyssey solved by
combination of “make a point” genetic counseling and NGS A Abstracts from the 51st European Society of Human Genetics Conference: Posters 677 11 years; the DNA samples available). The children were
generated by different fathers. A hypothesis of EMPF1 was
advanced. The sequencing of DNM1L gene has been per-
formed on the proband’s, mother’s and DNA of 2nd father. The
known
patogenetic
mutation
c.1085G>A
(p. Gly362Asp) and VUS c.1535T>C (p.Ile512Thr) in the
DNM1L gene were revailed in proband’s DNA. The
sequencing of mother’s DNA resulted in presence of only
VUS c.1535T>C (p.Ile512Thr), while in father’s DNA both
variants were absent. On the basis of these observation it
was fairly difficult to interpret this situation as de novo
autosomal dominant variant. The further analysis by NGS
has been performed, heterozygosity of c.1085G>A (p. Gly362Asp) variant was revealed in 5% of mother’s DNA. All variants have been confirmed by Sanger sequencing. Herein and in n Post-test Genetic Counseling the results and
in depth explanation of germinal mosaicism have been
discussed. 11 years; the DNA samples available). The children were
generated by different fathers. A hypothesis of EMPF1 was
advanced. The sequencing of DNM1L gene has been per-
formed on the proband’s, mother’s and DNA of 2nd father. The
known
patogenetic
mutation
c.1085G>A
(p. Gly362Asp) and VUS c.1535T>C (p.Ile512Thr) in the
DNM1L gene were revailed in proband’s DNA. The
sequencing of mother’s DNA resulted in presence of only
VUS c.1535T>C (p.Ile512Thr), while in father’s DNA both
variants were absent. On the basis of these observation it
was fairly difficult to interpret this situation as de novo
autosomal dominant variant. The further analysis by NGS
has been performed, heterozygosity of c.1085G>A (p. Gly362Asp) variant was revealed in 5% of mother’s DNA. All variants have been confirmed by Sanger sequencing. Herein and in n Post-test Genetic Counseling the results and
in depth explanation of germinal mosaicism have been
discussed. 11 years; the DNA samples available). The children were
generated by different fathers. A hypothesis of EMPF1 was
advanced. The sequencing of DNM1L gene has been per-
formed on the proband’s, mother’s and DNA of 2nd father. The
known
patogenetic
mutation
c.1085G>A
(p. Gly362Asp) and VUS c.1535T>C (p.Ile512Thr) in the
DNM1L gene were revailed in proband’s DNA. The
sequencing of mother’s DNA resulted in presence of only
VUS c.1535T>C (p.Ile512Thr), while in father’s DNA both
variants were absent. f-tree: A family health history tool available from the Iwate
Tohoku Medical Megabank Organization 1TOMA, Advanced Biomedical Assays S.p.A, Busto Arsizio,
Italy, 2Department of Obstetrics & Gynecology, NYC Health +
Hospitals/Jacobi, Bronx, NY, United States, 3Icahn School of
Medicine at Mount Sinai, New York, NY, United States,
4Hospital Central de Maputo, Maputo, Mozambique, 5and
Faculdade de Medicina da Universidade Eduardo Mondlane,
Maputo, Mozambique T. Tokutomi1,2, A. Fukushima1,2, K. Yamamoto1,2, F. Nakayama2, Y. Katsube2,3, A. Shimizu2, M. Sasaki2 T. Tokutomi1,2, A. Fukushima1,2, K. Yamamoto1,2, F. Nakayama2, Y. Katsube2,3, A. Shimizu2, M. Sasaki2 1Department of Clinical Genetics, School of Medicine, Iwate
Medical University, Morioka, Japan, 2Iwate Tohoku Medical
Megabank Organization, Iwate Medical University, Yahaba,
Japan, 3Genetic Counseling Program, Applied Medical
Science, Graduate School of Medical Science, Iwate Medical
University, Morioka, Japan Introduction: For T21,T18,T13 Cell-free DNA (cfDNA)
tests have a higher detection rate (DR) and a much lower
false positive rate (FPR) than traditional serum±ultrasound
screening (TSS). However, TSS can pick up unexpected
additional
‘off-target’
chromosome
abnormalities
con-
sequent to the higher reflex invasive testing rate. The aim of
this study is to compare the detection rates of all (target and
off-target) fetal karyotype abnormalities at birth by cfDNA
and TSS. Science, Graduate School of Medical Science, Iwate Medical
University, Morioka, Japan Introduction: The Tohoku Medical Megabank project aims
to create a next-generation personalized healthcare system. We designed a questionnaire-based pedigree-drawing soft-
ware program named “f-tree” to enable users to accurately
and rapidly collect genealogical information and assemble
pedigrees. Factors influencing the decision whether or not to have
prenatal genetic testing in pregnant women having children
with Down syndrome Introduction: ‘Project Machine’ is a targeted project of our
Medical Genetics department. It’s established from under-
graduate medical students in our university. The aim was to
provide them a systematic way of conducting pilot projects
to
intend
innovation
and
patent
targeting
academic
improvement by teaching them on basic and advanced
laboratory methods in Molecular Genetics. Introduction: ‘Project Machine’ is a targeted project of our
Medical Genetics department. It’s established from under-
graduate medical students in our university. The aim was to
provide them a systematic way of conducting pilot projects
to
intend
innovation
and
patent
targeting
academic
improvement by teaching them on basic and advanced
laboratory methods in Molecular Genetics. E. Kise1, M. Ishikawa1, T. Kojima2, K. Takano1,2, S. Ohira3,1, N. Kikuchi3,1, Y. Fukushima2, T. Kosho1,2 1Center for Medical Genetics, Shinshu University Hospital,
Matsumoto, Nagano, Japan, 2Department of Medical Genetics,
Shinshu University School of Medicine, Matsumoto, Nagano,
Japan, 3Department of Obstetrics and Gynecology, Shinshu
University Hospital, Matsumoto, Nagano, Japan Materials and Methods: Project Machine consists of
8 students from 3 different medical classes (Phase 2,3,4). The Project Machine group received hands-on training in
eleven different subject projects in two years. Trainings
were given by Medical Geneticist, the first Author. All of
these activities were done as a team work particularly
during their free studying times besides active medical
education. Students were evaluated by Medical Scores
(before and after participating machine), Laboratory works
(n=30) and academic skills (n=17) completed. The t test
and Chi square tests were used for statistics. Materials and Methods: Project Machine consists of
8 students from 3 different medical classes (Phase 2,3,4). The Project Machine group received hands-on training in
eleven different subject projects in two years. Trainings
were given by Medical Geneticist, the first Author. All of
these activities were done as a team work particularly
during their free studying times besides active medical
education. Students were evaluated by Medical Scores
(before and after participating machine), Laboratory works
(n=30) and academic skills (n=17) completed. The t test
and Chi square tests were used for statistics. In Japan, non-invasive prenatal testing (NIPT) has been
available as a clinical research since the introduction in
2013. Shinshu University Hospital does not participate in
this clinical research, and offers careful prenatal genetic
counseling (GC) for the decision whether or not to have
amniocentesis-based fetal karyotyping by certified genetic
counselors (CGC) and clinical geneticists specializing in
chromosomal or genetic disorders. P19.37A F.R. Grati: None. B. Grimi: None. K. Bajaj: None. L. Marcato: None. B. Malvestiti: None. F. Maggi: E. Ownership Interest (stock, stock options, patent or other
intellectual property); Significant; TOMA Advanced Bio-
medical Assays S.p.A. G. Simoni: E. Ownership Interest
(stock, stock options, patent or other intellectual property);
Significant; TOMA Advanced Biomedical Assays S.p.A.. S.J. Gross: None. J.C.P.B. Ferreira: None. P19.37A
Project machine: a unique approach for industrial medical
education Project machine: a unique approach for industrial medical
education I. Akalin1, E. Aslan2, E. Akbas2, S. Yilmaz2, U. Telli2, S. Yildirim2, N. Durcanoglu2, G. Aktemur2, A. Karaoglu2, R. Yilmaz2 1Istanbul Medeniyet University, Faculty of Medicine,
Department of Medical Genetics, Istanbul, Turkey, 2Istanbul
Medeniyet University, Faculty of Medicine, Istanbul, Turkey Introduction: The Tohoku Medical Megabank project aims
to create a next-generation personalized healthcare system. resulting in the identification of a greater proportion of off-
target abnormalities; cfDNA-T equals TSSs at a MA of 38y,
when trisomies dominate the risk. Conclusions: Distribution of the chromosome abnormal-
ities is different at different MA. Therefore, although two
strategies may show approximately the same overall DR,
TSSs favour the detection of a broad array of ‘off-target’
abnormalities, at the cost of a consistent individual residual
risk, while universal cfDNA tests efficiently detect common
aneuploidies with a limited view of ‘off-target’ abnormal-
ities. These results are useful for the development of
screening implementation economical models for public
health policy decision-makers. E. Kise: None. M. Ishikawa: None. T. Kojima: None. K. Takano: None. S. Ohira: None. N. Kikuchi: None. Y. Fukushima: None. T. Kosho: None. Introduction: The Tohoku Medical Megabank project aims
to create a next-generation personalized healthcare system. Introduction: The Tohoku Medical Megabank project aims
to create a next-generation personalized healthcare system. Introduction: The Tohoku Medical Megabank project aims
to create a next-generation personalized healthcare system. We designed a questionnaire-based pedigree-drawing soft-
ware program named “f-tree” to enable users to accurately
and rapidly collect genealogical information and assemble
pedigrees. We designed a questionnaire-based pedigree-drawing soft-
ware program named “f-tree” to enable users to accurately
and rapidly collect genealogical information and assemble
pedigrees. Materials and Methods: f-tree is a stand-alone applica-
tion written in ActionScript 3.0. Because it was built as a
cross-platform runtime system using Adobe AIR, it is
important that Adobe AIR be installed prior to the
installation and use of f-tree. Materials and Methods: Using a model derived from
our laboratory data, we predicted the DR of any karyotype
anomalies at birth in pregnancies with no risk factors other
than maternal age (MA) for seven screening strategies,
including different TSSs, cfDNA tests (±SCAs) and
contingent/sequential approaches. Results: f-tree is a bilingual software designed in
Japanese and English, and complies with international
nomenclature guidelines for human pedigrees. It can be
used to assemble pedigrees in the following manner: 1)
Upon system startup, the user is prompted to provide Results: f-tree is a bilingual software designed in
Japanese and English, and complies with international
nomenclature guidelines for human pedigrees. It can be
used to assemble pedigrees in the following manner: 1)
Upon system startup, the user is prompted to provide Results: CfDNA testing for T21,18,13+SCA (cfDNA-
TXY) has the highest DR for all karyotype abnormalities at
all MAs. In young women, cfDNA for T21,18,13only
(cfDNA-T) has a lower DR than any TSS due to the 5%FPR J. del Picchia 678 from their narratives. Reasons for choosing the testing were
described as “cannot raise one more child with disabilities”,
and “request for the testing by the child’s grandparents”. Reasons for not choosing testing were described as “don’t
like to terminate the fetus”, and ”can make a happy family
regardless of disabilities of the child”. In the setting of
prenatal GC for the clients who had children with DS, we
have to aware that they could have ambivalent feelings
about the burden and the value of raising children with DS. resulting in the identification of a greater proportion of off-
target abnormalities; cfDNA-T equals TSSs at a MA of 38y,
when trisomies dominate the risk. Psychiatric Genetic Testing - An emerging,
interdisciplinary field F. Degenhardt1, O. Andreassen2, I. Borg3,4, A. Børglum5,6,7, S. Cichon8,9,10, D. Coviello11, P. M. Czerski12, D. Demontis5,6, C. Foley13, W. Hennah14,15,16, K. Koido17, L. M. Lopez13, M. Mattheisen18,19,20, K. A. McGhee21, A. McQuillin22, A. Miu23, O. Mors24,25, L. Pojskic26, K. Popovska-Jankovic27, K. Tammimies28 Psychiatric disorders such as schizophrenia and bipolar
disorder are common and highly heritable. Despite this high
heritability it has taken many years to begin to identify
underlying genetic risk factors. However, in the last decade
major advances in psychiatric genetics have been driven by
both technological advances (e.g. cost effective array-based
genome-wide genotyping and next-generation sequencing
technologies) and the establishment of large-scale, interna-
tional consortia. Of particular relevance has been the iden-
tification of copy number variants (CNVs), such as 22q11.2
deletions, that confer substantial risk for psychiatric dis-
orders. Findings such as these have sparked an interest in
implementing genetic testing for patients with these dis-
orders in routine clinical care. Psychiatric Genetic Testing
(PsyGT) is a newly emerging and interdisciplinary field. Across Europe experts with varying backgrounds (e.g. Genetics, Epidemiology, Psychiatry) have assembled to
facilitate pan-European research relevant to PsyGT and to
help pave the way for its clinical implementation. Our work
will address: (i) establishing a list of genetic factors for
which it is scientifically/clinically appropriate to offer
genetic testing; (ii) defining patient inclusion criteria for e.g. CNV testing; (iii) discussing the potential challenges of
PsyGT (e.g. informed consent; reporting back incidental
findings to a patient with a psychiatric disorder); (iv) pro-
moting research into the potential positive and negative
effects of PsyGT for patients and their family members; (v)
increasing the “psychiatric genomic literacy” among all
relevant stakeholders as genetic counsellors and psychia-
trists report that they feel ill-equipped to educate patients
and their family members about the genetics of psychiatric
disorders. Factors influencing the decision whether or not to have
prenatal genetic testing in pregnant women having children
with Down syndrome In this study, we report
our experiences about prenatal GC to the clients having a
child with Down syndrome (DS). From medical records of
Center for Medical Genetics, Shinshu University Hospital
from April 2013 to December 2017, we identified 8 preg-
nant women who had a child with DS and received prenatal
GC. The average age of the cohort was 36.3 years (range,
32−41). Four of them (50%) had prenatal genetic testing
and the others declined the testing. We reviewed factors
influencing the decision whether or not to have the testing Results:
The
average
score
of
the
students
who
participated in the project machine increased significantly
(79,24±8,961;min-max:56-98)(p = 0,021). The
average
molecular techniques learned were 17,75±8,598 (min-
max:6-29). The average academic skills they succeed were
8,25±5,523 (2-15) including 3 domestic and 1 international
awards won and 11 projects has finished during project
(min-max:9-31 months) with an accepted publication. Discussion: Participation of medical students in scientific
studies by mutual interaction with academicians during their Discussion: Participation of medical students in scientific
studies by mutual interaction with academicians during their Abstracts from the 51st European Society of Human Genetics Conference: Posters 679 23Cognitive Neuroscience Laboratory, Department of
Psychology, Babes-Bolyai University, Cluj-Napoca, Romania,
24Psychosis Research Unit, Aarhus University Hospital,
Risskov, Denmark, 25The Lundbeck Foundation Initiative for
Integrative Psychiatric Research, iPSYCH Aarhus University,
Aarhus, Denmark, 26University of Sarajevo - Institute for
Genetic Engineering and Biotechnology, Sarajevo, Bosnia and
Herzegovina, 27Research Centre for Genetic Engineering and
Biotechnology “Georgi D. Efremov”, Macedonian Academy of
Sciences and Arts, Skopje, Macedonia, The Former Yugoslav
Republic of, 28Center of Neurodevelopmental Disorders
(KIND), Department of Women's and Children's Health,
Karolinska Institutet, Solna, Sweden medical education might lead more successful, prompt, and
enhanced skills to overcome hard working and time
consuming jobs in an active team work system. Hence,
our study might be pilot model for industrial medical and
genetic education. I. Akalin: None. E. Aslan: None. E. Akbas: None. S. Yilmaz: None. U. Telli: None. S. Yildirim: None. N. Durcanoglu: None. G. Aktemur: None. A. Karaoglu:
None. R. Yilmaz: None. Queens University of Belfast, Belfast, United Kingdom Introduction: Rare diseases, present in <1/2,000 persons with
a genetic cause, affect ~5% of the population in Northern
Ireland (NI). Following public and stakeholder consulta-
tions, a NI Rare Disease Implementation Plan was pub-
lished stressing 51 key commitments to improve medical
and societal challenges specifically associated with rare
diseases. Since 2016, 5 Belgian genetic centres started to provide data
to the CRRD for all patients with a genetic diagnosis
(confirmed or provisional). ORPHA-codes (preferably) or
codes cross-referenced to ORPHA-codes are used. One
objective of the CRRD is data sharing at EU level for
epidemiological and other purposes. Materials and Methods: Mixed methods were employed
to seek views from a range of stakeholders to recognise
good practice, identify where urgent change is required, and
prioritise short/medium/longer term goals for rare diseases
in NI. The establishment of our NI Genomic Medicine
Centre (2015) and applying multi-omics approaches to
improve the speed and accuracy of diagnosis for individuals
with rare diseases has revealed important issues. However, lack of standardised use of appropriate rare
disease (RD) coding across the EU is hampering data
sharing. To allow data sharing, RD-ACTION WP5*
members propose the use of a table, retaining only
ORPHA-codes at disorder level in the Orphanet classifica-
tion of RD. The table is called “Beta-Masterfile”, a tool
serving RD-coding and exploitation purposes prepared by
the above-mentioned group. Results: Differences were noted depending on the
method of data collection. Common themes consistently
included a frequently tortuous path to diagnosis, difficulty
finding details for health and social care contacts, and
challenges
accessing
relevant
information
in
suitable
formats. We analysed the diagnosis-related information from the
current CRRD dataset and evaluated the future exploit-
ability at EU-level using the above-mentioned table. Non-rare
cases
(~4%) were excluded from
further
analysis. For only 11 of 1633 cases analysed, a conversion
into ORPHA-codes was necessary. ~65% of RD diagnoses
had a confirmed status. Conclusions: Current health and social care systems in
NI are not designed to optimise care for rare diseases. The
development
of
our
digital
integrated
care
record
(http://www.hscboard.hscni.net/encompass/)
is
offering
unprecedented opportunities to integrate effective and
sustainable strategies to improve rare disease identification,
optimise care and service provision, provide relevant
information to individuals living and working with rare
diseases, and establish effective multi-disciplinary commu-
nication networks. P19.39C
Improving rare disease identification and coordinating
health and social care priorities P. M. Urbina1, A. Rath2, S. Weber3, V. Van Casteren1, A. Olry2,
M. Marx3, E. Swinnen1 H. McAneney, A. Crowe, J. Miller, K. Kerr, J. C. McMullan, A. J. McKnight 1WIV-ISP, Scientific Institute of Public Health, Brussels,
Belgium, 2Orphanet, INSERM, Paris, France, 33. DIMDI,
German Institute of Medical Documentation and Information,
Cologne, Germany Psychiatric Genetic Testing - An emerging,
interdisciplinary field 1Institute of Human Genetics, Bonn, Germany, 2Institute of
Clinical Medicine; Psychosis Research Centre (TOP), Oslo,
Norway, 3Department of Pathology, Faculty of Medicine and
Surgery, University of Malta, Msida, Malta, 4Department of
Pathology, Medical Genetics Unit, Mater Dei Hospital, Msida,
Malta, 5Department of Biomedicine – Human Genetics, Aarhus
University, Aarhus, Denmark, 6The Lundbeck Foundation
Initiative for Integrative Psychiatric Research, iPSYCH,
Aarhus, Denmark, 7Center for Integrative Sequencing and
Aarhus Genome Center, Aarhus, Denmark, 8Institute of
Medical Genetics and Pathology, University Hospital Basel,
Basel, Switzerland, 9Department of Biomedicine, University of
Basel, Basel, Switzerland, 10Institute of Neuroscience and
Medicine (INM-1), Research Center Juelich, Juelich, Germany,
11Department of Genetic Sciences and I.B.M.D.R.; Galliera
Hospital, Genova, Italy, 12Laboratory of Psychiatric Genetics,
Poznan University of Medical Sciences, Poznan, Poland,
13Department of Psychiatry, Trinity College Dublin, Dublin,
Ireland, 14Institute for Molecular Medicine FIMM, University
of Helsinki, Helsinki, Finland, 15Medicum, University of
Helsinki, Helsinki, Finland, 16Department of Health, Mental
Health Unit, National Institute for Health and Welfare,
Helsinki, Finland, 17Department of Physiology, Institute of
Biomedicine and Translational Medicine, University of Tartu,
Tartu, Estonia, 18Department of Psychiatry, Psychosomatics
and Psychotherapy, Center of Mental Health, University
Hospital Würzburg, Würzburg, Germany, 19Department of
Biomedicine, Aarhus University, Aarhus, Denmark,
20Department of Clinical Neuroscience, Centre for Psychiatry
Research, Karolinska Institutet, Stockholm, Sweden, 21Faculty
of Science & Technology, Bournemouth University,
Bournemouth, United Kingdom, 22Division of Psychiatry,
University College London, London, United Kingdom, F. Degenhardt: None. O. Andreassen: None. I. Borg:
None. A. Børglum: None. S. Cichon: None. D. Coviello:
None. P.M. Czerski: None. D. Demontis: None. C. Foley:
None. W. Hennah: None. K. Koido: None. L.M. Lopez:
None. M. Mattheisen: None. K.A. McGhee: None. A. McQuillin: None. A. Miu: None. O. Mors: None. L. 680 J. del Picchia P19.40D
Testing the ORPHA-code based Beta-Masterfile using data
from the Belgian Central Registry of Rare Diseases
(CRRD) for the use case of rare diseases patient data
sharing at EU level P19.42B
Triage in a Clinical Genetics setting - investigating
consistency within and between units Proportion of Clinicians offering Face-to-Face appointments
Referral
Centre1 Centre2
Centre3
Centre4
Centre5
1
Copy Number Variant
6(67%) 9(100%) 5(100%) 10(100%) 6(100%)
2
Hypermobility
0(0%)
8(89%)
3(60%)
4(40%)
2(33%)
3
Intermediate
FMR1 allele
7(78%) 7(78%)
5(100%) 10(100%) 6(100%)
4
Trisomy 21
0(0%)
9(100%) 6(100%)
9(90%)
5(83%)
5
Adult with Intellectual
Disability
5(56%) 6(67%)
3(50%)
9(90%)
6(100%)
6
Predictive BRCA
(familial variant
information unavailable)
4(44%) 9(100%) 8(89%)
7(70%)
3(50%)
7
Cervical cancer
0(0%)
2(22%)
0(0%)
1(10%)
0(0%)
8
Isolated Cleft lip
0(0%)
2(29%)
1(14%)
7(70%)
3(50%)
9
Congenital Heart Disease
(unspecified)
0(0%)
3(33%)
2(40%)
7(70%)
6(100%)
10 Pregnant woman, Family
history of Duchenne MD
8(89%) 9(100%) 6(100%) 10(100%) 6(100%)
11 Hereditary
Haemochromatosis
3(33%) 3(43%)
0(0%)
6(60%)
4(67%)
12 Mitochondrial disease
(pre-symptomatic)
4(44%) 3(43%)
2(40%)
9(90%)
6(100%)
13 Neural Tube Defect
3(33%) 7(88%)
4(57%)
4(40%)
4(67%) T. P. McVeigh1, M. Porteous2, A. Murray3, E. Jones4, S. Wedderburn5, E. Kinning6, S. Lynch7 1National University of Ireland Galway, Galway, Ireland,
2University of Edinburgh, Edinburgh, United Kingdom,
3University Hospital of Wales, Cardiff, United Kingdom,
4Manchester Cente for Genomic Medicine, Manchester, United
Kingdom, 5NHS GREATER GLASGOW & CLYDE, Glasgow,
United Kingdom, 6NHS Greater Glasgow and Clyde, Glasgow,
United Kingdom, 7Temple Street Children's University
Hospital, Dublin, Ireland Background: The triage process aims to ensure provision
of appropriate care to patients, within an acceptable time-
period, depending on urgency of referral. Clinical Genetics
is unique regarding breadth of types of referrals, and age
range of patients (pre-natal, paediatric or adults). Referrals
are complex, and ensuring consistency of triage can be
difficult. T.P. McVeigh: None. M. Porteous: None. A. Murray:
None. E. Jones: None. S. Wedderburn: None. E. Kinning: None. S. Lynch: None. Aim: To compare triage practices within and between
clinical genetic centres in UK (n=4) and Republic of
Ireland (n=1). Queens University of Belfast, Belfast, United Kingdom Considering that subtype ORPHA-codes are convertible
into their parenting ORPHA-code, data from ~80% of
CRRD patients could be shared at EU level, albeit losing
more detailed coding information for ~6% of them. For both
provisional and confirmed diagnoses, the use of ORPHA-
codes at group-of-disease level is about 20%. The relatively
high use of codes at group-of-disease level for confirmed
diagnoses should be further investigated in order to obtain
better quality of coding. ALC & KK are supported by a PhD studentship from the
Department for the Economy. This work was conducted in
collaboration with the NI Department of Health, Public
Health Agency, and NI Rare Disease Partnership. *WP5: “Steering, maintaining and promoting the adop-
tion of ORPHA-codes for Rare Diseases across member
states”; EU’s joint action ‘677024/RD-ACTION’ P.M. Urbina: None. A. Rath: None. S. Weber: None. V. Van Casteren: None. A. Olry: None. M. Marx: None. E. Swinnen: None. H. McAneney: None. A. Crowe: None. J. Miller: None. K. Kerr:
None. J.C. McMullan:
None. A.J. McKnight: None. H. McAneney: None. A. Crowe: None. J. Miller: None. K. Kerr:
None. J.C. McMullan:
None. A.J. McKnight: None. Abstracts from the 51st European Society of Human Genetics Conference: Posters 681 Young adults' perceptions of the implications of their
hereditary visual impairment: A Cape Town based study Young adults' perceptions of the implications of their
hereditary visual impairment: A Cape Town based study Methods: Thirteen simulated referrals were created
(TMcV, SAL) based on common real-life referrals to
Clinical Genetics services, and distributed to each centre for
"triaging". Individuals were asked to pick from the
following options: offer appointment (face-to-face/tele-
phone); hold referral pending further information; manage
referral by letter/telephone call; or reject referral. For
patients offered appointments, participants were asked to
specify urgency (priority/routine) and clinician (consultant/
counsellor). Data regarding staffing levels and waiting times
for each centre was noted. Methods: Thirteen simulated referrals were created
(TMcV, SAL) based on common real-life referrals to
Clinical Genetics services, and distributed to each centre for
"triaging". Individuals were asked to pick from the
following options: offer appointment (face-to-face/tele-
phone); hold referral pending further information; manage
referral by letter/telephone call; or reject referral. For
patients offered appointments, participants were asked to
specify urgency (priority/routine) and clinician (consultant/
counsellor). Data regarding staffing levels and waiting times
for each centre was noted. K. Popel1, G. Dusterwald2, C. Leisagang3, J. Greenberg1 1Division of Human Genetics, Department of Pathology,
University of Cape Town, Cape Town, South Africa, 2Private
Practice, Cape Town, South Africa, 3Clinical Pharmocology,
Division of Medicine, University of Cape Town, Cape Town,
South Africa vision of Human Genetics, Department of Pathology Introduction: Globally there are 39 million blind indivi-
duals and 246 million with low vision. A third of genetic
conditions involve the eye. Knowing that one’s condition is
genetic presents challenges for individuals and leads to a
sense of obligation regarding life plans. As young adults
with hereditary visual impairment must make choices
regarding relationships, having children, further education
or occupation and independence, which sets the foundation
for adulthood, it is important to focus on them. The study
explored the understanding, perceptions and lived experi-
ences of young adults with genetic based visual impairment. Results: Inconsistencies were noted within and between
units (table). In 9/13(69%) referrals, clinicians in Centre1
were the least likely to offer face-to-face appointments. Centre1 had the lowest number of consultants/head of
population. Conclusions: Clinical triage is a somewhat subjective
process. Institutional factors, including staffing levels,
local practices; and patient factors, including age of
patient/parent (family planning considerations), indication
for, and type of, referral (predictive/diagnostic), can
modify triage decisions. Standardised guidelines for triage
are required to ensure equitable and appropriate service
provision. P20.02B A tremendous distress that goes unnoticed - the anxiety
caused by abnormal results of Down syndrome screening Introduction: Fabry disease is a rare X-linked disorder
caused by a mutation in the GLA gene. Women hetero-
zygous for a mutation may have symptoms. Little is known
about the psychosocial experiences of women heterozygous
for Fabry disease. The aim of the study was to explore
women’s psychosocial experiences of being heterozygous
for Fabry disease. L. Sagi-Dain1, A. Peleg1, S. Sagi2, A. Singer3 1Carmel Medical Center, Haifa, Israel, 2Bnai Zion Medical
Center, Haifa, Israel, 3Community Genetics, Public Health
Services, Ministry of Health, Jerusalem, Israel Materials and Methods:
We used an explorative
qualitative study design and conducted in-depth semi-
structured interviews with ten Norwegian women who were
known heterozygous for Fabry disease. We analyzed the
interviews using inductive thematic analysis. Materials and Methods:
We used an explorative
qualitative study design and conducted in-depth semi-
structured interviews with ten Norwegian women who were
known heterozygous for Fabry disease. We analyzed the
interviews using inductive thematic analysis. Background: Screening tests for Down syndrome are
routinely advised during pregnancy, with recommendation
of invasive chromosomal testing to high-risk women (about
5% of pregnant population). The objective of our survey
was to evaluate the anxiety experienced by women receiv-
ing abnormal screening results, and to explore potential
ways to relieve this distress. Results: Most women reported a relief of getting the
Fabry diagnosis, as an explanation to present end previously
unexplainable symptoms. It was however important for the
women to make a distinction between Fabry disease and
their daily life. The women on enzyme replacement
treatment were grateful for available treatment; however,
the treatment was not without personal cost. A frustration
regarding the lack of health care providers’ knowledge of
Fabry disease was common. Methods: Women who had experienced an abnormal
screening result for Down syndrome were invited to
participate in an electronic anonymous cross-sectional
survey. Anxiety level was evaluated by a six-item short-
form of the Spielberger State-Trait Anxiety Inventory. Methods: Women who had experienced an abnormal
screening result for Down syndrome were invited to
participate in an electronic anonymous cross-sectional
survey. Anxiety level was evaluated by a six-item short-
form of the Spielberger State-Trait Anxiety Inventory. Results: Overall, 559 women have answered the ques-
tionnaire. In 86.04% high anxiety scores were reported
following
abnormal
screening
results. Fabry disease: Female perspectives Fabry disease: Female perspectives K. Popel: None. G. Dusterwald: None. C. Leisagang:
None. J. Greenberg: None. C. von der Lippe1, J. Frich2, A. Harris3, K. N. Solbrække2 1Centre for Rare Disorders, Oslo University Hospital, Oslo,
Norway, 2Institute of Health and Society, University of Oslo,
Oslo, Norway, 3Department of Technology and Society Studies,
Maastricht University, Maastricht, Netherlands Young adults' perceptions of the implications of their
hereditary visual impairment: A Cape Town based study Conclusions: Clinical triage is a somewhat subjective
process. Institutional factors, including staffing levels,
local practices; and patient factors, including age of
patient/parent (family planning considerations), indication
for, and type of, referral (predictive/diagnostic), can
modify triage decisions. Standardised guidelines for triage
are required to ensure equitable and appropriate service
provision. Method: Using qualitative research, fifteen participants
were recruited, through purposive sampling, from various
organisations for visually impaired individuals. In-depth
interviews were conducted and analysed using thematic
analysis. J. del Picchia 682 Results: Many young adults identified the dilemma with
regards to bearing children as the main implication of
having a genetic based visual impairment. Almost all
participants had minimal knowledge of their condition and
felt that they did not understand it well. Many participants
felt misunderstood by society. Social interactions were
greatly impacted on by means of social alienation, support
structures, and treatment by others. Having a disability
created the sense of need and desire to succeed and prove
themselves. An aspect that significantly impacted their lives
was the challenges with mobility, which included the
incapacity of driving and public transportation. Results: Many young adults identified the dilemma with
regards to bearing children as the main implication of
having a genetic based visual impairment. Almost all
participants had minimal knowledge of their condition and
felt that they did not understand it well. Many participants
felt misunderstood by society. Social interactions were
greatly impacted on by means of social alienation, support
structures, and treatment by others. Having a disability
created the sense of need and desire to succeed and prove
themselves. An aspect that significantly impacted their lives
was the challenges with mobility, which included the
incapacity of driving and public transportation. syndrome), and only 4.4% stated they prefer the current
form (e.g. 1 in 100). The participants noted several factors
which could relieve their anxiety, including explanatory
booklet or site (marked as "helpful" by 72.4% and 77.9% of
the women, respectively). Discussion: Women receiving abnormal results of Down
syndrome screening experience significant anxiety, which
seems to be underappreciated by the treating physicians. Efforts must be made to relieve this distress, including
consideration of changing the historical report of the risk as
a ratio to percentage or a combination of options. L. Sagi-Dain: None. A. Peleg: None. S. Sagi: None. A. Singer: None. Young adults' perceptions of the implications of their
hereditary visual impairment: A Cape Town based study Conclusion: This research could assist in improving
genetic counselling services, as well as provide organisa-
tions with information to strengthen support and guidance
for these individuals.The study was funded by the NRF. Ethical decision-making competence regarding the
possibilities of genome editing by CRISPR/Cas Whole Genome Sequencing (WGS) and Whole Exome
sequencing (WES) for diagnostic and research purposes
generate a large volume of raw data. Previous ethical and
legal discussions concerning genomic data management
have mainly focused on concerns related to interpretation of
the results and the return of both primary and secondary
findings from these tests. Yet to date, issues related to the
storage and return of primary sequencing data (such as bam
(Binary Alignment) or fastq files (unaligned reads)) that
allow both the regeneration of primary results and the rea-
nalysis of data have received far less attention, particularly
in the clinical setting. This is despite the huge potential for
long-term data storage to lead to future data re-analysis and
reinterpretation in light of new evidence. In this paper, we
critically appraise three main issues, namely, data storage
policies and practices of clinical laboratories, patients’
access to raw data, and sharing of raw data by individuals. We argue that there is a need for well-established and
transparent raw genomic data retention and returning poli-
cies in order to enable patients to practice their rights to
access raw genomic data. In addition, professional com-
munities could guide laboratories in adopting best practices
for the storage and return of raw data, and introduce uni-
formity to these practices. As WES and WGS become more
embedded in diagnostics, it is timely to consider how cur-
rent data storage policies align with patients’ rights and
interests to access raw data, and how these rights can be
managed in the clinical setting. B. Vajen1, L. Heinisch2, C. Hößle2, U. Krause2, W. Rathje2, B. Schlegelberger1 B. Vajen1, L. Heinisch2, C. Hößle2, U. Krause2, W. Rathje2, B. Schlegelberger1 1Institute of Human Genetics, Hannover, Germany, 2Institute
of Biology and Environmental Science, Oldenburg, Germany The CRISPR/Cas9 technology is proclaimed as one of the
greatest innovations of the last decade and has sparked a
public debate about its ethical issues. To facilitate partici-
pation of adolescents in public debates, the decision-making
competence is fixed as a part of educational standards for
the natural science curriculum in Germany. In the present
study, we will analyze ethical decision-making compe-
tences of medical students and adolescents in the context of
genome editing. The
Oldenburg
model
of
ethical
decision-making
competences includes perception of moral relevance,
assessment, judgement, argumentation, consequence reflec-
tion, change of perspective and basic ethical knowledge. P20.02B Using
logistic
regression analysis, higher anxiety scores were noted in
younger women and in those informed of the abnormal
result by the caregiver vs. written answer. 41.7% of the
respondents preferred the risk reported as percentage (e.g. 1% risk for Down syndrome), 16.6% - as positive
percentage (99% chance for fetus negative for Down Conclusions: Women who are heterozygous for Fabry
disease react to and reflect on their diagnosis differently,
and they should receive personalized information, support,
and management. Health care providers should be aware
and acknowledge feelings of guilt and difficulties in
communicating about Fabry disease in the family. Health
care providers should also recognize the personal cost of
receiving treatment, and support the patients on this. Conclusions: Women who are heterozygous for Fabry
disease react to and reflect on their diagnosis differently,
and they should receive personalized information, support,
and management. Health care providers should be aware
and acknowledge feelings of guilt and difficulties in
communicating about Fabry disease in the family. Health
care providers should also recognize the personal cost of
receiving treatment, and support the patients on this. Abstracts from the 51st European Society of Human Genetics Conference: Posters 683 C. von der Lippe: D. Speakers Bureau/Honoraria
(speakers bureau, symposia, and expert witness); Modest;
SHIRE, SANOFI. J. Frich: None. A. Harris: None. K.N. Solbrække: None. Ethical decision-making competence regarding the
possibilities of genome editing by CRISPR/Cas On a paper-pencil test with open-ended questions on
authentic ethical dilemmata, regarding genome editing for
somatic or germline gene therapy for RUNX1-associated
familial leukemia, the above-mentioned competences are
recorded in a sample of 50 medical students and 50
adolescents of a secondary school. The responses of
participants are analyzed using a structured qualitative
content analysis. Points are awarded for the dimensions of
ethical decision-making competence. M. Shabani: None. D. Vears: None. P. Borry: None. M. Shabani: None. D. Vears: None. P. Borry: None. M. Shabani, D. Vears, P. Borry M. Shabani, D. Vears, P. Borry 1PhD school of medical genetics, Sapienza University, Roma,
Italy, 2Liceo Scientifico Statale Giovanni Keplero, Roma, Italy,
3PhD school of Cognitive Science, University of Messina,
Messina, Italy, 4PhD school of Biomarkers in chronic and
complex diseases, Magna Graecia University, Catanzaro,
Italy, 5Indipendent researcher, Roma, Italy, 6Imago child and
adolescence neurology and psychiatrics clinic, Roma, Italy,
7Orientale University, Naples, Italy, 8Caravaggio laboratories, B. Vajen: None. L. Heinisch: None. C. Hößle: None. U.
Krause:
None.
W.
Rathje:
None.
B.
Schlegelberger: None. P20.06B First results of the study show, that medical students and
adolescents are open-minded towards genome editing in
case of healing heavy diseases. Nevertheless, they have
great fears about a misuse of this technology creating
“designer babies” or “enhanced humans” and that human
dignity might be hurt while tinkering in the DNA of
embryos. None of them agree with the usage in general. The
results of this study can be untilized to improve education
on ethical decision-making competence and/or on basics
about the CRISPR/Cas-based genome editing in school and
university. Incidental findings in genetic testing: a comparison between
US and european guidelines and a bioethical reflection M. Vismara1, V. Meschesi2, G. Pulvirenti3, A. Donato4, S. Vismara5, B. Zepponi6, C. Settanni5, F. Donato7, L. Coltrinari5,
V. Moroni5, A. Valentini8, J. Toaff9, G. Donato10 1PhD school of medical genetics, Sapienza University, Roma,
Italy, 2Liceo Scientifico Statale Giovanni Keplero, Roma, Italy,
3PhD school of Cognitive Science, University of Messina,
Messina, Italy, 4PhD school of Biomarkers in chronic and
complex diseases, Magna Graecia University, Catanzaro,
Italy, 5Indipendent researcher, Roma, Italy, 6Imago child and
adolescence neurology and psychiatrics clinic, Roma, Italy,
7Orientale University, Naples, Italy, 8Caravaggio laboratories, B. Vajen: None. L. Heinisch: None. C. Hößle: None. U. Krause:
None. W. Rathje:
None. B. Schlegelberger: None. 684 J. del Picchia 1Bank of Biological Material, First Faculty of Medicine of
Charles University, Prague, Czech Republic, 2General
University Hospital, Prague, Czech Republic, 3Masaryk
Memorial Cancer Institute, Brno, Czech Republic Roma, Italy, 9Indipendent researcher, Versailles, France,
10Chair of Pathology, Magna Graecia University, Catanzaro,
Italy Next-Generation Sequencing (NGS) use in biomedical
diagnosis and research makes now possible a deeper genetic
and genomic analysis at a reduced cost. However, the
analysis of evidence that is based on such big data can result
in the generation of knowledge that is not relevant to the
clinical question posed because some genes are associated
with multiple medical conditions. These kind of information
are commonly referred to as "incidental findings" and their
management pose new ethical issues and dilemmas, espe-
cially regarding their disclosure to patients as well as the
role of the different clinical figures involved. In this work
we aim to analyze different recommendations and guide-
lines, referring in particular to USA and Europe. P20.06B Numerous
discussions have been conducted in order to reach a con-
sensus on how to handle such findings in line with the legal
and cultural particularities of individual states and general
bio-ethical principles and different guidelines have been
published. These reports mainly focus on the pros and cons
of NGS technology and potential benefits and risks for
reporting of incidental findings.We want to compare these
various statements and try to figure out how patient’s will
could be better included in the decision making and the
disclosure process, in order to respect its autonomy. We
also point out the need for continued debate, research, and
discussion among all stakeholders to improve our under-
standing of the effect that different policies have on
patients, providers, laboratories, and societies. Background: Biobanks provide biomedical research with
high quality samples together with associated data including
genetic data. Since the donors of biological material consent
at the same time to storage and use of samples, to proces-
sing of personal data and to future research whose methods
and aims cannot be clearly identified at moment of consent,
there is currently no consensus on the best consent model. To benefit the donors one of the proposed ways is return of
research results mainly from genetic research, which can
reveal some of the future health risks. Current opinions
differ in this regard taking into account practical but also
ethical consideration like the “right not to know”. The aim
of this study is to explore the experience with and opinions
on consent models and return of research results in practice
of oncological biobanks. Methods: Semi-structured interviews with the staff of 6
recently established oncological research biobanks in Czech
Republic. Results: In Czech oncological research biobanks the
preferred model of consent is broad consent; none of the
biobanks
employs
any
interactive
consent
model
(,,
unethical to remind someone about cancer“). Also return
of
research
results
was
considered
inappropriate
for
oncological patients (,,To whom the information should
be communicated when the donor is dead?“). Results: In Czech oncological research biobanks the
preferred model of consent is broad consent; none of the
biobanks
employs
any
interactive
consent
model
(,,
unethical to remind someone about cancer“). Also return
of
research
results
was
considered
inappropriate
for
oncological patients (,,To whom the information should
be communicated when the donor is dead?“). P20.06B Conclusions: The recently established Czech research
biobanks implemented in their practise broad consent and
no return of research results mainly with regard to specific
issues connected to oncological patients. This study was
supported
by
project
MŠMT-LM2015089
and
RVO
VFN 64165 M. Vismara: None. V. Meschesi: A. Employment (full
or part-time); Significant; LSS "Keplero". F. Consultant/
Advisory Board; Modest; Fondazione Craxi. G. Pulvirenti:
A. Employment (full or part-time); Significant; University
of Messina. A. Donato: A. Employment (full or part-time);
Significant; Magna Graecia University. S. Vismara: None. B. Zepponi: Other; Significant; Owner of Imago clinic. C. Settanni: F. Consultant/Advisory Board; Modest; società
cooperativa Zurigo. F. Donato: None. L. Coltrinari:
Other; Significant; owner of Coltrinari Law firm. V. Moroni: None. A. Valentini: A. Employment (full or
part-time); Significant; Caravaggio laboratories. J. Toaff:
A. Employment (full or part-time); Significant; Blizzard
corporation. G. Donato: A. Employment (full or part-time);
Significant; Magna Graecia University. V. Frankova: None. R. Hermankova: None. D. Valik:
None. K. Jirsova: None. T. Zima: None. P20.08D Moving towards clinical use of New Genome Sequencing
technologies: Understanding the Ethical, Legal and
psychological challenge S. JULIA1,2, M. Rosier3, A. Cambon-Thomsen2,4, M. Guedj3, M. Muñoz Sastre3, E. Rial Sebbag2,5 1Medical genetics department, Toulouse, France, 2U1027
Inserm and University of Toulouse 3, Toulouse, France,
3Centre d’Etudes et de Recherches en Psychopathologie et
Psychologie de la Santé UT2J, Toulouse, France, 4Plateforme
"Génétique et société" Genotoul, Toulouse, France, P20.07C P20.07C
Consent model and return of results in oncological
biobanks: a qualitative study
V. Frankova1,2, R. Hermankova2, D. Valik3, K. Jirsova1, T. Zima1 V. Frankova1,2, R. Hermankova2, D. Valik3, K. Jirsova1, T.
Zima1 S. JULIA1,2, M. Rosier3, A. Cambon-Thomsen2,4, M. Guedj3, M.
Muñoz Sastre3, E. Rial Sebbag2,5 Consent model and return of results in oncological
biobanks: a qualitative study Consent model and return of results in oncological
biobanks: a qualitative study V. Frankova1,2, R. Hermankova2, D. Valik3, K. Jirsova1, T. Zima1 Abstracts from the 51st European Society of Human Genetics Conference: Posters 685 5Plateforme "Génétique et société" Genotoul, Toulouse,
Austria present in this paper the major changes induced by the
adoption of several Regulations for the translation of PGx
tests into clinics. Medical genetics illustrate the link between technological
developments, an evolving scientific knowledge, and prac-
tical decisions which impact patients’ lives. The rapid
development of sequencing technologies marks a historical
turning point in human genetics. Important questions need
to be asked about how to use these new technologies in
order to maximize the translational relevance of genetic
research for patients and for society: How to give adequate
information for patients in a context of uncertainty? How to
deal with incidental/secondary findings? On the blurring
between care and research? The introduction of emerging
technologies in clinic requires technical/clinical validation,
harmonisation of practice, and evolution of the legal and
ethical framework. Drawing on our experience with the
development of genomic technologies and their application
in clinical settings, we analyze the ethical, legal and psy-
chological issues inherent in the sharing, recording and
storing of the medical information provided by these tech-
nologies. This approach takes into account the current
context of genetic tests and provides concrete answers to the
concerns of patients and professionals. Several issues to
consider are identified: the status of large scale genetic
information regarding privacy and confidentiality, clinically
useful information, health related information where no
immediate clinical measure exists. This work highlight the
importance of a combined approaches of research in law,
psychology and empirical ethic in order to describe the
benefits and the limits of the use of new sequencing in
clinics which will determine their ethical and social
acceptability Since the adoption of the IVDR PGx tests and companion
diagnostics (CDx) are now clearly falling under the scope of
the Regulation (Class C according to the IVD’s classifica-
tion) with their respective definitions. Based on the
evaluation of risks (from Class A, less, to D, high), this
classification introduces some new rules regarding proce-
dures. A notified body involvement is now required for the
conformity assessment procedure, with specific provisions
for CDx. Precision medicine and its infrastructures of solidarity.
Probing the social contract in US and EU precision
medicine initiatives S. Julia:
None. M. Rosier:
None. A. Cambon-
Thomsen: None. M. Guedj: None. M. Muñoz Sastre:
None. E. Rial Sebbag: None. I. Van Hoyweghen1, E. Aarden2 1Leuven Institute of human Genomics and Society (LIGAS),
Leuven, Belgium, 2University of Vienna, Vienna, Austria P20.11C Precision medicine and its infrastructures of solidarity. Probing the social contract in US and EU precision
medicine initiatives Consent model and return of results in oncological
biobanks: a qualitative study The performance study for marketing new IVDs
should be based on the clinical benefit of the device
(scientific validity, analytical performance and clinical
performance). New traceability and transparency rules are
also provided, such as the Unique Device Identification
system and a summary of safety and performance. More-
over, manufacturers have to put in place surveillance and
vigilance systems with the obligation to produce several
reports. Additionally, as for the General Data Protection Regula-
tion, a person responsible for regulatory compliance must
be present. And finally, as for clinical trial data, a new database is
introduced:
Eudamed,
including
several
databases
on
medical devices and IVDs. A. Constantin:
None. E. Rial-Sebbag:
None. L. Serres: None. A. Constantin:
None. E. Rial-Sebbag:
None. L. Serres: None. P20.10B
Impact of the new EU in vitrodiagnostics regulation on
pharmacogemonics: the U-PGx project P20.10B
Impact of the new EU in vitrodiagnostics regulation on
pharmacogemonics: the U-PGx project On a global scale, a ‘race for innovation’ between nations
has begun to set off for the building of Precision Medicine
(PM) initiatives. To establish PM, various initiatives call on
‘a new social contract’ between citizens and the health care
system. This contract implies that citizens recognize that
they and everybody else will benefit from medical science if
they allow data about their own genome to be collected and
shared. Policy makers thereby mark PM not just with grand
claims on scientific and economic benefits, but also with the
formation of a new genomic citizenship. In this paper, we
investigate the mobilization of the social contract in the
economic competition between ‘PM nations’. Drawing on a
comparative analysis of the United States and Europe, we
show how these new social contracts for PM are not only A. Constantin, E. Rial-Sebbag, L. Serres A. Constantin, E. Rial-Sebbag, L. Serres INSERM - UMR 1027, Université de Toulouse, Toulouse,
France INSERM - UMR 1027, Université de Toulouse, Toulouse,
France The Ubiquitous Pharmacogenomics (U-PGx) project aims at
addressing the major challenges and obstacles to implement
pharmacogenomics (PGx) testing in European patient rou-
tine medical care. In this context, U-PGx has to comply
with the current European frameworks for performing
genetic testing in practice notably the new in vitro diag-
nostic (IVD) medical devices regulation (IVDR). We will J. del Picchia 686 imagined as a critical part of the successful development of
PM, but are materialized in infrastructures of health care
delivery. We demonstrate how the successfulness of PM
depends on infrastructures of health care delivery that per-
form as trust-generating techniques for the mutualization of
benefits and rights. We consider the role of infrastructures
of health care delivery, which we coin as “Infrastructures of
Solidarity”, as key markers for the implications of efforts to
incorporate Precision Medicine (PM) in contemporary
health care systems. We conclude by a reflection on what
the new social contracts for PM entail for conditions of
citizenship, access to health care, and the future of the
welfare state. remain long-term. According to 6-month follow-up all 12
responders cope with the finding, none regret the decision
made, and all communicated the finding to their physician
and family. Nine of 12 have seen a specialist and five
pursued risk-reducing surgery. remain long-term. According to 6-month follow-up all 12
responders cope with the finding, none regret the decision
made, and all communicated the finding to their physician
and family. Nine of 12 have seen a specialist and five
pursued risk-reducing surgery. Conclusions: The genotype-first approach demonstrated
that currently only a minority of individuals carrying
pathogenic BRCA mutations are under appropriate medical
surveillance. Although the 45 percent response-rate sup-
ports choice regarding disclosure, all participants perceived
results received valuable. Long-term follow-up showed that
information is shared generously and demonstrated increase
in screening and prevention behavior. Conclusions: The genotype-first approach demonstrated
that currently only a minority of individuals carrying
pathogenic BRCA mutations are under appropriate medical
surveillance. Although the 45 percent response-rate sup-
ports choice regarding disclosure, all participants perceived
results received valuable. Long-term follow-up showed that
information is shared generously and demonstrated increase
in screening and prevention behavior. I. Van Hoyweghen: None. E. Aarden: None. L. Leitsalu: None. M. Palover: None. A. Reigo: None. T. Nikopensius: None. H. Alavere: None. P. Padrik:
None. T. Esko:
None. A. Metspalu:
None. N. Population biobank participants’ preferences and response
to disclosure of unexpected genetic findings Population biobank participants’ preferences and response
to disclosure of unexpected genetic findings INSERM - UMR 1027, Université de Toulouse, Toulouse,
France Tõnisson: None. P20.14B L. Leitsalu1, M. Palover1,2, A. Reigo1, T. Nikopensius1, H. Alavere1, P. Padrik3, T. Esko1,4, A. Metspalu1,2, N. Tõnisson1,5 Secondary findings from whole exome or genome Secondary findings from whole exome or genome sequencing: psychological and ethical Issues. Patient point
of view 1Estonian Genome Center, Institute of Genomics, University of
Tartu, Tartu, Estonia, 2Institute of Molecular and Cell Biology,
University of Tartu, Tartu, Estonia, 33Haematology and
Oncology Clinic, Tartu University Hospital, Tartu, Estonia,
4Broad Institute of MIT and Harvard, Cambridge, MA, United
States, 5Department of Genetics, Tartu University Hospital,
Tartu, Estonia 1Estonian Genome Center, Institute of Genomics, University of
Tartu, Tartu, Estonia, 2Institute of Molecular and Cell Biology,
University of Tartu, Tartu, Estonia, 33Haematology and
Oncology Clinic, Tartu University Hospital, Tartu, Estonia, F. Houdayer1, O. Putois2,3, S. Staraci4,5, M. L. Babonneau6, C. C. Michon6, H. Chaumet7, L. Joly8, C. Juif8, A. Chassagne3,9, E. Gautier3,8, C. Thauvin-Robinet3,8, D. Sanlaville1,10, P. Edery1,10,
A. S. Lapointe11, M. Garguilo5,12, L. Faivre3,8 4Broad Institute of MIT and Harvard, Cambridge, MA, United
States, 5Department of Genetics, Tartu University Hospital,
Tartu, Estonia 1Service de génétique, Centre de Référence Maladies du
développement, Bron, France, 2SuLiSoM EA 3071, Strasbourg,
France, 3FHU TRANSLAD, CHU Dijon, Dijon, France,
4Service de génétique, GH Pitié-Salpêtrière, Assistance
Publique et Hôpitaux de Paris, Paris, France, 5Laboratoire de
Psychologie Clinique, Psychopathologie, Psychanalyse
(EA4056), Université Paris Descartes, Sorbonne Paris Cité,
Paris, France, 6Filière Nationale de Santé Cardiogen, GH
Pitié-Salpêtrière, Assistance Publique et Hôpitaux de Paris,
Paris, France, 7Service de génétique, Oncogénétique, Hospices
Civils de Lyon, Lyon, France, 8Service de génétique, Centre de
Référence Maladies du développement, CHU Dijon, Dijon,
France, 9CIC, Inserm 1431, CHRU Besançon, Besançon,
France, 10INSERM U1028, CNRS UMR5292, Centre de
Recherche en Neurosciences de Lyon, GENDEV Team,
Université Claude Bernard Lyon 1, Bron, France, 11Alliance
Maladies Rares, Paris, France, 1212 Institut de Myologie, GH
Pitié-Salpêtrière, Assistance Publique et Hôpitaux de Paris,
Paris, France Introduction: The Estonian biobank legislation gives par-
ticipants the right to receive results. Disclosure of unex-
pected genetic findings to population biobank participants
through genotype-first approach provides valuable infor-
mation on how individuals from the general population
would respond. Methods: A project involving participants carrying
pathogenic variants in BRCA1 and 2 genes associated with
familial breast and ovarian cancer (HBOC) was initiated. The procedural framework includes contacting without
disclosing genetic status, project specific consent, indepen-
dent validation, disclosure with genetic counseling, colla-
boration
with
oncologists,
and
cascade
screening. P20.14B Five ques-
tions were asked regarding access to SF: 1. Favorable or
not. 2. Opinion concerning minors. 3. Concerning equity of
access. 4. Concerning reporting procedures. 5. To FG5
only: opinion on the search for heterozygosity for frequent
recessive diseases. The discussions were recorded and then
analyzed with qualitative research methods. Introduction: Silver-Russell syndrome (SRS) is a rare
disorder characterized by severe intrauterine and postnatal
growth retardation with relative macrocephaly at birth,
typical dysmorphic features, and feeding difficulties. In the
majority of cases, SRS is caused by a methylation defect of
11p15 imprinting center region 1. To our knowledge, only
three studies have documented the cognitive development
of children with SRS, but these are old studies in which
patients were diagnosed only by non-standardized clinical
criteria. Currently, no studies have been published on the
assessment of the intellectual functioning of adults. Results: Overall, 10/25 were favorable to the action-
ability of SF, but 13/25 unfavorable because of the
psychological consequences. 9/25 modified their views
over the course of the discussions. 60% were favorable and
30% ambivalent to the access of SF by minors. Equity of
access to SF led to philosophical discussions on quality of
life. The 4 key information-based issues for patients were:
explanation of the issues of SF, the importance of a
reflection period, autonomy of choice, and quality of
information. Materials and Methods: An evaluation of intellectual
efficiency was performed in 8 patients with SRS (4 males, 4
females), aged 20 to 39 years (mean age 23.75 years)
followed at France from 2016 to 2018. All patients had an
epimutation of the region 11p15. Conclusions: The weight of individual experience was
decisive in patient expectations and fears regarding access
to SF. Longitudinal studies based on real announcements
are necessary to establish guidelines. Results: Mean overall IQ score was 89.13 (14.33 SD)
with a range between 71-107. Verbal comprehension index
was on average higher than other indexes (mean 104.50,
18.55 SD). 50% were treated with growth hormone. Speech
delay in childhood and learning difficulties have been
reported in some patients. 75% have benefited from speech
therapy and/or psychological care. Seven patients had a
bachelor’s degree and five patients completed higher
education. F. Houdayer: None. O. Putois: None. S. Staraci: None. M.L. Babonneau:
None. C.C. Michon:
None. H. Chaumet: None. L. Joly: None. C. Juif: None. A. Chassagne:
None. E. Gautier:
None. C. Thauvin-
Robinet: None. D. Sanlaville: None. Cognitive profile in Silver-Russell syndrome: a first French
study in adults Cognitive profile in Silver-Russell syndrome: a first French
study in adults M. Burgevin1, A. Lacroix1, A. Toutain2,3, D. Martin-Coignard4,
M. Vincent5, C. Crosnier-Schoedel6,7, V. Coutinho8,9, I. Netchine6,7, S. Odent10,11 M. Burgevin: None. A. Lacroix: None. A. Toutain:
None. D. Martin-Coignard: None. M. Vincent: None. C. Crosnier-Schoedel: None. V. Coutinho: None. I. Netch-
ine: None. S. Odent: None. 1Univ Rennes, LP3C (Laboratoire de Psychologie : Cognition,
Comportement, Communication) - EA 1285, Rennes, France,
2CHRU de Tours, Hôpital Bretonneau, Service de Génétique,
Tours, France, 3Université François Rabelais, Faculté de
Médecine, INSERM UMR U930, Tours, France, 4CH du Mans,
Service de Génétique, Le Mans, France, 5CHU de Nantes,
Service de Génétique Médicale, Nantes, France, 6Hôpitaux
Universitaires Paris Est (AP-HP), Hôpital des Enfants Armand
Trousseau, Service d’explorations Fonctionnelles
Endocriniennes, Paris, France, 7Centre de Recherche Saint
Antoine, INSERM UMR S938, Paris, France, 8Hôpital des P20.14B P. Edery: None. A. S. Lapointe: None. M. Garguilo: None. L. Faivre: None. Conclusions: Overall adults with SRS had normal
intellectual efficiency and a fairly positive life course. If
more patients to study are needed to objectify these results,
they can improve knowledge about SRS and point to the
importance of early intervention and multidisciplinary care
from childhood to adulthood in SRS. P20.14B Immediate and long-term follow-up were surveyed to
understand the psychological and behavioral impact of
results disclosed. Methods: A project involving participants carrying
pathogenic variants in BRCA1 and 2 genes associated with
familial breast and ovarian cancer (HBOC) was initiated. The procedural framework includes contacting without
disclosing genetic status, project specific consent, indepen-
dent validation, disclosure with genetic counseling, colla-
boration
with
oncologists,
and
cascade
screening. Immediate and long-term follow-up were surveyed to
understand the psychological and behavioral impact of
results disclosed. Results: Of 49 carriers identified 22 have visited the
biobank and expressed interest towards return of results. Of
these participants, only three had no known family history
of HBOC, and one individual had previously received
genetics evaluation. All 19 participants who received results
perceived this information valuable. Although three parti-
cipants reported feeling worry at first, these feelings did not Introduction: Discovery of secondary findings (SF) in
diagnostic practice is a major psychological and ethical
issue for the introduction of genomic medicine. Materials and Methods: The purpose of this qualitative
study is to analyze patient expectations for SF and to Abstracts from the 51st European Society of Human Genetics Conference: Posters 687 Enfants Armand Trousseau, AP-HP, GUEP, Service de
Neuropédiatrie, Paris, France, 9Centre de Recherche en
Épidémiologie et Santé des Populations, INSERM U1018,
Villejuif, France, 10Hôpital Sud, CHU de Rennes, Service de
génétique clinique, Centre de référence Maladies Rares CLAD-
Ouest, Rennes, France, 11Univ Rennes, CNRS UMR 6290,
Rennes, France identify the reporting procedures necessary for informed
consent, by interrogating patients with no diagnosis, but
also patients facing diseases concerned by SF. Five focus
groups (FG) were led by psychologists: FG1 (n=5) without
diagnosis; FG2 (n=10) oncogenetics; FG3 (n=3) cardio-
genetics; FG4 (n=3) metabolic diseases; FG5 (n=4)
heterozygotes for frequent recessive diseases. Five ques-
tions were asked regarding access to SF: 1. Favorable or
not. 2. Opinion concerning minors. 3. Concerning equity of
access. 4. Concerning reporting procedures. 5. To FG5
only: opinion on the search for heterozygosity for frequent
recessive diseases. The discussions were recorded and then
analyzed with qualitative research methods. identify the reporting procedures necessary for informed
consent, by interrogating patients with no diagnosis, but
also patients facing diseases concerned by SF. Five focus
groups (FG) were led by psychologists: FG1 (n=5) without
diagnosis; FG2 (n=10) oncogenetics; FG3 (n=3) cardio-
genetics; FG4 (n=3) metabolic diseases; FG5 (n=4)
heterozygotes for frequent recessive diseases. 1Auckland District Health Board, Auckland, New Zealand,
2University of Auckland, Auckland, New Zealand, 3Muscular M. J. Rodrigues1,2,3, R. H. Roxburgh1,2, A. Theadom4 M.J. Rodrigues: None. R.H. Roxburgh: None. A.
Theadom: None. P20.16D Attitudes towards reproductive carrier screening among
the genetic muscle disorder community M. J. Rodrigues1,2,3, R. H. Roxburgh1,2, A. Theadom4 1Auckland District Health Board, Auckland, New Zealand,
2University of Auckland, Auckland, New Zealand, 3Muscular 688 J. del Picchia Dystrophy New Zealand, Auckland, New Zealand, 4Auckland
University of Technology, Auckland, New Zealand (74% vs 92%). People diagnosed with a less severe
disorder, such as myotonia congenita, were more likely to
not favour preconception screening compared to those
diagnosed with a more severe condition. All diagnosed with
Duchenne's favoured screening. Methods: We conducted a population-based study of the
prevalence and outcomes of genetic muscle disorders in
New Zealand. All those diagnosed on or before the point
prevalence date of 1st April 2015 were included. All eli-
gible cases were invited to participate in an assessment to
determine the impact of the disorders on people’s lives,
including their attitude towards preconception screening for
genetic muscle disorders. Conclusion: The majority of people living with genetic
muscle disorders are in favour of reproductive carrier
screening for that disorder. By examining sub groups within
the overall cohort (age, severity, inheritance pattern), we are
able to get a more detailed and nuanced view of attitudes
towards reproductive carrier screening. An understanding of
what the community want with respect to the accessibility
and acceptability of screening is important when consider-
ing utilising new genetic technologies to offer the option of
screening individuals to identify carrier status for genetic
disorders. Results: 596 participated in the impact assessment, 83 of
these were a parent of an affected child(ren). The majority
(87%) of those with a disorder were in favour of
reproductive carrier screening for their disorder as were
parents of an affected child (84%). Parents of older children
(11 - 15 years) were less likely to be in favour of
preconception screening than parents of younger children M.J. Rodrigues: None. R.H. Roxburgh: None. A. Theadom: None.
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https://openalex.org/W2027962422
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https://hal.archives-ouvertes.fr/hal-01189739/document
|
English
| null |
Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources
|
BMC genomics
| 2,010
|
cc-by
| 11,299
|
To cite this version: Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet, Catherine Feuillet, et al.. Specific
patterns of gene space organisation revealed in wheat by using the combination of barley and wheat
genomic resources. BMC Genomics, 2010, 11, 10.1186/1471-2164-11-714. hal-01189739 Specific patterns of gene space organisation revealed in
wheat by using the combination of barley and wheat
genomic resources
Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet,
Catherine Feuillet, Robbie Waugh, Etienne Paux Specific patterns of gene space organisation revealed in
wheat by using the combination of barley and wheat
genomic resources
Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet,
Catherine Feuillet, Robbie Waugh, Etienne Paux RESEARCH ARTICLE Open Access © 2010 Rustenholz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Specific patterns of gene space organisation
revealed in wheat by using the combination of
barley and wheat genomic resources Camille Rustenholz1†, Pete E Hedley2†, Jenny Morris2, Frédéric Choulet1, Catherine Feuillet1, Robbie Waugh2,
Etienne Paux1* * Correspondence: etienne.paux@clermont.inra.fr
† Contributed equally
1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100
Clermont-Ferrand, France
Full list of author information is available at the end of the article Abstract Background: Because of its size, allohexaploid nature and high repeat content, the wheat genome has always
been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a
wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between
the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based
markers have been developed. This study aims at combining the unique genomic resources developed in wheat
and barley to decipher the organisation of gene space on wheat chromosome 3B. Results: Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were
hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous
genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were
syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome
3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing
gradient of gene density from the centromere to the telomeres positively correlated with the number of genes
clustered in islands on wheat chromosome 3B. Conclusion: Our study shows that novel structural genomics resources now available in wheat and barley can be
combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to
perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space. HAL Id: hal-01189739 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 A high throughput anchoring method g
t
oug put a c o
g
et od
To assess the efficiency of wheat-barley cross-species
hybridisation for gene-based physical map anchoring, a
barley Agilent 15K unigene microarray was hybridised
with 60 three-dimensional (plate, row, column) BAC
pools from the MTP of the wheat chromosome 3B [17]. After signal quantification and normalisation, hybridisa-
tion data were evaluated with four complementary scor-
ing methods to reliably locate as many barley gene
homologs as possible on the wheat BACs (see Methods). Using the most stringent “automated scoring” method,
3355, 3401 and 3286 probes were identified as positive
with the plate, row and column pools, respectively. Deconvolution of the pool data led to the identification
of 571 unambiguous BAC addresses for 566 unigenes,
defining 561 unique genomic loci and 5 duplicated loci. The less stringent “boxplot scoring” method led to the
identification of 6205, 5103 and 6761 positive probes for
the plate, row and column pools, respectively. With this
method, 770 probes having unambiguous BAC addresses
were identified, including 481 that were already identi-
fied with the “automated” method. Out of the 289 newly
identified probes, we selected 86 probes (100 loci) that
correspond to the most robust data (i.e. located on two
to
three
overlapping
BACs). Finally
the
“semi-
automated” and the “manual scoring” methods added
additional BAC addresses for 13 and 78 probes respec-
tively that showed missing coordinates with the two other
methods due to technical limitations (detailed below). We recently constructed a physical map of chromo-
some 3B, the largest wheat chromosome (1 Gb, 2.5 times
the whole rice genome) [17]. The map consists of 1036
contigs spanning 811 Mb, of which 611 Mb are anchored
with 1443 molecular markers. However, very few contigs
are anchored by gene-derived markers. Indeed despite
the development of genomic resources, such as extensive
marker collections and saturated genetic maps [18-20],
genetic mapping of genes in wheat is still hampered by
the lack of polymorphism and the presence of the three
homoeologous copies of each gene. As a result, no high
density transcript genetic map is available. In contrast,
several gene maps have been constructed for barley
[21-25] (Hordeum vulgare L.) that diverged from wheat
~10-12 MYA [26,27] and belongs to the same tribe (Tri-
ticeae). Background Arabidopsis thaliana (125 Mb), Brachypodium distach-
yon (272 Mb) and Oryza sativa (389 Mb) exhibit fairly
homogenous gene distribution along their chromosomes
[3-5]. The transition from a homogenous to a non-
homogenous gene distribution seems correlated to the
genome size. Indeed, in intermediate size genome, such
as Populus trichocarpa (485 Mb) and Vitis vinifera
(487 Mb), large regions alternating between high and
low gene density were observed [6,7], whereas larger
genomes, such as Glycine max (1115 Mb) and Zea mays
(2300 Mb), display an increasing gradient of gene den-
sity from the centromere to the telomeres [8,9]. The term “gene space” refers to the fraction of the gen-
ome corresponding to protein coding genes and, by
extension, to the distribution of these genes [1]. In large
genomes that contain abundant repetitive DNA, it
encompasses also the notion of regions containing
genes, the so-called gene-rich regions, surrounded by
gene-poor regions composed of repeats [2]. With the growing number of sequenced plant gen-
omes, it becomes obvious that the distribution pattern
of genes is far from random and not universal across
the plant kingdom. Small plant genomes, such as Because of its size (17000 Mb), allohexaploid nature
(A, B and D-genomes) and high repeat content (>80%)
[10], the bread wheat (Triticum aestivum L.) genome is
among the largest and most complex plant genomes
and has always been considered too complex for * Correspondence: etienne.paux@clermont.inra.fr
† Contributed equally
1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100
Clermont-Ferrand, France
Full list of author information is available at the end of the article Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 2 of 12 molecular analyses. As a result, no genome sequence is
available yet and very little is known about the organisa-
tion of the wheat gene space. The first insights were
obtained from the mapping of wheat gene-based mar-
kers in wheat aneuploid genotypes called deletion lines
where fragments of chromosomes or deletion bins are
missing [11]. Based on EST and Pst1 genomic clone
mapping, Erayman et al. [12] suggested a very heteroge-
neous distribution of the genes along the wheat chro-
mosomes, with 94% of the genes being located in only
29% of the entire wheat chromosomes and mostly at
their telomeric parts. In contrast, by EST mapping on
chromosome group 3 deletion bins, Munkvold et al. Background [13] observed a slight gradient of the gene density along
the chromosomes as well as a significant number of
genes in the most proximal bins thereby suggesting a
more homogeneous distribution. More recently, indivi-
dual BAC sequencing [14,15] confirmed a rather homo-
geneous gene distribution in wheat with an average of
one gene per BAC. Finally, Choulet et al. [16] investi-
gated megabase-sized regions from various parts of
chromosome 3B and indicated that the gene-free regions
are much smaller than expected by Erayman et al. [12],
i.e. not larger than 1 Mb. Moreover, they found evidence
for a slight gradient (twofold) of the gene density distri-
bution from the centromere to the telomeres. Thus,
additional whole genome or whole chromosome ana-
lyses are needed to better characterize the gene space
organisation in wheat. microarrays to identify the location of genes along
the wheat 3B physical map. The results show that such
barley-wheat cross-hybridisations represent high-
throughput cost-efficient approaches for anchoring
genes on wheat physical maps and for performing com-
parative genomics studies between wheat and other
grass genomes. In addition, the possibility to locate
genes precisely within BAC contigs that were anchored
by other markers onto the chromosome 3B enabled us
to gain new insights into the distribution of genes along
a wheat chromosome. A high throughput anchoring method With a size of 4.9 Gb [10] and a repeat content of
over 80% [28], the diploid barley genome (2n = 14) is
very similar to the wheat subgenomes and several map-
ping studies have demonstrated a high collinearity
between barley and wheat [26,27,29-32]. In total, the combination of four methods enabled us
to identify 762 unambiguous wheat BAC addresses for
743 barley probes. A BLASTN search [33] against the
Triticeae repeat database TREP [34], indicated that five
probes had high sequence identity (>86%) with TEs and
were removed from further analysis. Each of the remain-
ing 738 non TE-related genes was assigned to one to
three wheat BACs resulting in 757 gene loci identified
on the wheat chromosome 3B physical map [17]. These
barley unigenes were located on 624 wheat BACs
that corresponded to 388 individual contigs of 187 kb to
3.8 Mb and 86 singletons. Here, we wanted to explore the possibility of using
barley transcript genetic maps as a surrogate to anchor
and order the wheat physical contigs. BAC pools repre-
senting the minimal tiling path (MTP) of wheat chro-
mosome 3B were hybridised onto barley expression Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 3 of 12 Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 We tested the reliability of the 757 genes using the
sequenced contigs available on chromosome 3B [16]. We found that 74% (23/31) of the genes located on the
sequenced contigs through hybridisation gave a hit on
the sequenced contigs after a BLASTN analysis. Out of
these genes, 91% (21/23) matched a gene on the
sequenced contigs at their expected location. Eight uni-
genes (26%) were assigned to these contigs but their
position was not supported by sequence information. Several hypotheses can be proposed to explain such dis-
crepancy between hybridization and sequencing data,
including false positives, misassembled MTP BACs or
gaps in the sequence. In addition, 30 out of the 15208
barley Agilent microarray unigenes matched a gene after
a BLASTN analysis against the sequenced contigs but
were not located on a BAC through hybridisation. How-
ever the sequence identity of these 30 unigenes (84%)
was lower than the sequence identity of the 21 unigenes
located on the sequenced contigs through hybridisation
(90%). These unigenes would therefore hardly be located
on a BAC through hybridisation. A high throughput anchoring method These data validated
this cross-species hybridisation approach as a powerful
and reliable method to map genes to BAC contigs. sequence conservation played a key role in the detection
of hybridisation signals and that more than half of the
potentially positive barley probes generated a near unde-
tectable hybridisation signal with the wheat BACs. A second origin of the discrepancy likely originates from
the presence of gene families located at multiple loci. The wheat genome is allohexaploid (three subgenomes:
A, B and D) and at least one copy of each wheat gene is
expected to be present on the three homoeologous chro-
mosomes. In addition, there is increasing evidence for
high level of tandem and interchromosomal duplication
events in wheat and perhaps barley genomes since their
divergence from the other grasses [16,39]. Thus there is a
good probability that some genes are found in multiple
copies on chromosome 3B. Such genes can result in mul-
tiple non-overlapping BAC addresses that cannot be
resolved without ambiguity and are therefore excluded
from our analysis. Another critical point affecting the
efficiency of the approach lies in the putative heterogene-
ity of the BAC pools. Indeed, each three-dimensional
MTP pool contains more than 300 BACs (see Methods),
making it difficult to guarantee equimolarity for all
BACs. In some extreme cases, this heterogeneity in indi-
vidual BAC quantity may lead to weak signal intensity for
positive probes resulting in missing coordinates. These
two limitations could be circumvented by the use of six-
dimension pools of the complete chromosome 3B BAC
library [40]. However, such pools would have required
almost 3 times more hybridisations than the three-
dimensional MTP pools (177 vs. 60) thereby reducing the
cost-efficiency of the approach. g
g
The 738 probes correspond to roughly 40% of the bar-
ley unigenes that were expected to be present on the
wheat chromosome 3B physical map. Indeed, chromo-
some 3 H accounts for approximately 14.8% of the bar-
ley genome [35,36]. Assuming a comparable gene
density for all barley chromosomes, 2250 probes out of
the 15208 unigenes are expected to be located on chro-
mosome 3 H. As the MTP covers 82% of the whole
wheat chromosome 3B, about 1845 probes should in
theory be present on the wheat chromosome 3B physi-
cal map assuming that all barley genes are conserved in
wheat. A high throughput anchoring method The difference of 60% between expected and
observed results could be explained by both biological
and technical limitations of our experiment. First,
sequence divergence between wheat and barley genes
may have significantly impacted the efficiency of this
approach. Letowski et al. [37] estimated that hybridising
a probe and a DNA target sharing 90% of sequence
identity results in 73% to 99% decrease in hybridisation
signal intensity compared to a probe and a DNA sharing
100% of sequence identity. Blasting the barley 60-mer
probes against the 6162 wheat cv. Chinese Spring full-
length cDNA dataset [38] revealed that 56% of the hits
show more than 10% nucleotide divergence (86% iden-
tity on average). Moreover we found that the unigenes
located on the sequenced contigs of the wheat chromo-
some 3B [16] through BLASTN and hybridisation
showed a significantly higher sequence identity (90%)
compared to the ones that were located on the
sequenced contigs through BLASTN only (83%) (T-test,
P-value = 5E-6). Therefore, one can estimate that Despite these limitations, this single experiment per-
mitted the localisation of 738 genes on the wheat chro-
mosome 3B contigs and allowed us to get novel
information for the order of the BAC contigs along the
chromosome based on the barley EST genetic maps. So
far, genetic mapping of genes in wheat has been ham-
pered by the lack of polymorphism in the genic
sequences and the presence of several homoeologous
copies. As a consequence, only a third of the 680 mar-
kers located on the chromosome 3B genetic map con-
structed using the ‘neighbours’ approach correspond to
ESTs [17]. Here, we established a barley whole genome
neighbour map using the same criteria as the IBM
neighbour map of maize [41] and used it to assess the
order of wheat contigs based on the EST order found
on the genetic map. Out of the 738 probes assigned to
BAC contigs of wheat chromosome 3B, 308 (42%) were
mapped to the barley neighbour map including 209 on
chromosome 3 H and 99 on other chromosomes. Using
the barley 3 H mapping data, 151 BAC contigs and
20 singletons from the wheat chromosome 3B physical
map were genetically ordered. Only 30% of these contigs
were previously ordered genetically using the wheat Rustenholz et al. A high throughput anchoring method BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 4 of 12 probes located on the contigs of wheat chromosome 3B,
209 were mapped on barley chromosome 3 H and 99
on other barley chromosomes. Rice orthologous genes
were identified unambiguously for 659 of the 738 probes
located on wheat chromosome 3B, of which 389 are
located on rice chromosome 1and 270 on other rice
chromosomes (Table 1). These results suggest that, at
the whole chromosome scale, 68% and 59% of wheat
chromosome 3B genes are syntenic with genes located
on the orthologous barley chromosome 3 H and rice
chromosome 1, respectively. These results are consistent
with previous studies that estimated between 59% and
74% of the genes were in conserved positions between
wheat chromosome group 3 and rice chromosome 1
and 75% between wheat chromosome group 3 and bar-
ley chromosome 3 H [13,25,43-50]. The genes that are
not syntenic between wheat chromosome 3B and barley
chromosome 3 H mapped on the other barley chromo-
somes with no significant bias towards any other single
chromosome (Chi2 test, P-value = 0.16). In contrast, the
non-syntenic genes between wheat chromosome 3B and
rice chromosome 1 are biased in favour of genes
mapped on the rice chromosomes carrying the highest
number of genes (chromosomes 3 and 5) and against
the rice chromosome carrying the lowest number of
genes (chromosome 9) (Chi2 test, P-value < 10-5). Further analyses confirmed that the distribution of the
non-syntenic genes between wheat chromosome 3B and
rice chromosome 1 on the other rice chromosomes is
correlated with the number of genes per rice chromo-
some (Pearson’s correlation coefficient r = 0.735;
P-value = 0.01) [3]. Thus, no real mapping bias was
identified towards any of the non-syntenic barley or rice
chromosomes. chromosome 3B neighbour map, whereas 44% were
mapped to the wheat chromosome 3B deletion bin map,
but not ordered in bins, and 26% were not anchored at
all [17]. In addition, it is worth noting that 36% of the
151 contigs are only anchored by gene-based markers. This is consistent with the results of Paux et al. [17]
who showed that some regions of the genome can only
be anchored by specific types of markers (ESTs, SSRs,
ISBPs) and that 35% of the contigs were anchored by
ESTs only. A high throughput anchoring method Therefore, we conclude that the cross-hybri-
disations of wheat BAC pools with barley expression
microarrays is a straight forward approach to order
wheat contigs with gene-based markers without the dif-
ficulty of EST genetic mapping in wheat. Moreover, the total cost for these 60 pool hybridisa-
tions on 15 microarrays was approximately 8800 USD. For the same price, PCR screening of individual EST
markers on the same BAC pools (including primers and
amplification) would only have allowed testing of 500
markers. Thus, the method is a cost-efficient alternative
to PCR-based physical map anchoring. However, despite
its convenience and its cost-efficiency, this technique is
still limited in the number of contigs anchored and
ordered but it would be greatly improved by technologi-
cal developments in the near future. First, use of the
barley 44K Agilent expression microarray will signifi-
cantly increase the number of positive probes, regardless
of the experiment efficiency. Second, as large amounts
of barley SNPs are becoming available [22], the number
of genetically mapped genes will increase in the coming
years, therefore improving the efficiency of the anchor-
ing strategy. Finally, wheat-barley cross-species hybridisation is a
convenient, cost-efficient and relatively high-throughput
approach for gene-based physical map anchoring and
ordering of wheat BAC contigs. However, even if the
use of barley genomic resources circumvents the limita-
tions caused by the complexity of the wheat genome,
the divergence between the two species is large enough
to observe synteny breaks. Thus, we performed a com-
parative study between wheat, barley and rice to assess
the extent to which the barley gene order is transferable
to wheat. Interestingly, a number of genes located on wheat
chromosome 3B were not syntenic with barley chromo-
some 3 H but their homologs were syntenic between
barley and rice. For example, 11 wheat chromosome 3B
genes mapped on barley chromosome 2 H and on its
ortholog in rice (chromosome 4). We found another
example with 9 wheat chromosome 3B genes mapping
on barley chromosome 6 H and on the orthologous rice
chromosome 2 [44]. This result indicates that these
genes have undergone rearrangements specifically in
wheat and supports the recent finding of Choulet et al. [16] for extensive interchromosomal duplications in
wheat. Comparative genomics between wheat, barley and rice We calculated the per-
centage of probes that are syntenic to barley chromo-
some 3 H genes for each deletion bin of chromosome
3B and found that the conservation of genes is signifi-
cantly uniform along chromosome 3B (Chi2 test, P-value
= 0.84) with 73% of syntenic genes per bin on average
(Table 1). We performed the same calculation with the
285 genes assigned to wheat chromosome 3B deletion
bins and syntenic to genes on rice chromosome 1. In this case, the distribution of syntenic genes was nega-
tively correlated with the distance to the centromere
(Pearson’s correlation coefficient r = -0.742; P-value =
0.04). In other words, the level of synteny between
wheat chromosome 3B and rice chromosome 1
decreases from the centromere to the telomeres. This is
in complete agreement with the results of Akhunov
et al. [39] who correlated this with the recombination
rate along wheat chromosomes. However, using the data
from Saintenac et al. [51] who performed an analysis of
the distribution of the recombination rate among chro-
mosome 3B, we did not find any correlation between
the synteny level and crossing-over frequency (Pearson’s
correlation coefficient r = -0.378; P-value = 0.36). Com-
parisons between the sequences of 18 Mb sized contigs
of chromosome 3B with the rice and Brachypodium
genomes led to the same conclusions [16]. Moreover,
the authors found a positive correlation between trans-
posable element activity and the number of non syntenic genes. Thus, it is likely that the synteny level between
wheat chromosome 3B and rice chromosome 1 that
decreases from the centromere to the telomeres results
from a combination of factors that have still to be
identified. The links between the barley chromosome 3 H genetic
map, the rice chromosome 1 sequence and the wheat
chromosome 3B deletion bin map were used to analyse
the collinearity, i.e. the order of the genes [42], between
the genes on wheat chromosome 3B and on barley chro-
mosome 3 H and between the genes on wheat chromo-
some 3B and on rice chromosome 1. As the relative
order of wheat genes in a given deletion bin is not
known, we inferred this order from the barley chromo-
some 3 H and rice chromosome 1 data. Comparative genomics between wheat, barley and rice Comparative genomics between wheat, barley and rice
In addition to its interest for anchoring physical maps,
cross-species hybridisation also provides valuable data
for comparative genomics as it allows the mapping of
barley (and to some extent rice) orthologous genes on
wheat chromosomes. We studied synteny, i.e. the con-
servation of the genes on the orthologous chromosomes
of wheat chromosome 3B in barley (chromosome 3H)
and rice (chromosome 1) without the assumption of the
conservation of the gene order [42]. Out of the 738 Out of the 219 gene loci orthologous to barley chro-
mosome 3 H genes, 153 have been located on wheat
BACs assigned to one of the eight deletion bins of
wheat chromosome 3B using the physical map data [17]. Their approximate location on the chromosome arms
was thus inferred from the mapping data of the BAC. This enabled us to study the synteny between wheat, Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 5 of 12 Page 5 of 12 Table 1 Mapping data in wheat chromosome 3B deletion bins
Wheat
Barley
Rice
3B
Deletion
Bin
Bin
size
(Mb)
Number
of loci
Density
(locus/
Mb)
Gene loci
mapped
on 3H
Gene loci mapped
on the other
chromosomes
Collinear gene
loci between
3B and 3H
Gene loci
mapped
on Os01
Gene loci mapped
on the other
chromosomes
Collinear gene
loci between 3B
and Os01
3BS8-
0.78-1.00
44.2
37
0.84
10
9
7
15
14
10
3BS9-
0.57-0.78
43.2
43
1.00
12
4
9
22
16
16
3BS1-
0.33-0.57
94.3
86
0.91
14
11
8
40
33
20
C-3BS1-
0.33
58.3
34
0.58
9
1
4
23
7
16
C-3BL2-
0.22
45.7
36
0.79
15
3
8
27
6
18
3BL2-
0.22-0.50
74.9
78
1.04
25
10
21
47
26
38
3BL10-
0.50-0.63
40.1
40
1.00
13
5
8
18
17
8
3BL7-
0.63-1.00
155.5
165
1.06
55
14
37
93
57
59
Total
assigned
556.2
519
0.93
153
57
102
285
176
185
Not
assigned
438.8
238
/
66
42
/
111
104
/
Total
995
757
/
219
99
/
396
280
/
318
676 Table 1 Mapping data in wheat chromosome 3B deletion bins
Wheat
Barley Table 1 Mapping data in wheat chromosome 3B deletion bins barley and rice at a finer scale. Comparative genomics between wheat, barley and rice This virtual
order was used to define eight synteny blocks on barley
chromosome 3 H and rice chromosome 1 corresponding
to the eight deletion bins of wheat chromosome 3B
(Figure 1). Genes being assigned to a given deletion bin
of wheat and to the corresponding synteny block of bar-
ley or rice were considered as collinear. Those not being
conserved at syntenic positions were considered as non
collinear. In total we found that 102 (67%) genes were
collinear between wheat 3B and barley 3 H and 185
(65%) between wheat 3B and rice 1 (Table 1 & Figure 1). We calculated the percentage of collinear genes between
wheat chromosome 3B and barley chromosome 3 H and
between wheat chromosome 3B and rice chromosome 1 Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 6 of 12 Figure 1 Collinearity between wheat chromosome 3B, barley
chromosome 3 H and rice chromosome 1. Each colour on wheat
chromosome 3B corresponds to a deletion bin. Yellow: 3BS8-0.78-
1.00; light green: 3BS9-0.57-0.78; pink: 3BS1-0.33-0.57; red: C-3BS1-
0.33; orange: C-3BL2-0.22; purple: 3BL2-0.22-0.50; dark green: 3BL10-
0.50-0.63 and brown: 3BL7-0.63-1.00. The black segments
correspond to the heterochromatic regions identified by C-banding,
the coloured segments to the euchromatic regions and the circle to
the centromere. The coloured blocks represent regions where the
genes are collinear between two chromosomes. The lines represent
the genes that are not collinear between two chromosomes. As the
relative order of wheat genes in a given deletion bin is not known,
we inferred this order from the barley chromosome 3 H and from
rice chromosome 1 data. we had to use a barley neighbour map combining these
maps to optimize the anchoring experiment. However,
the gene order is not fully reliable in such maps especially
in the pericentromeric and centromeric parts of the chro-
mosomes where recombination is reduced or totally sup-
pressed [25]. Finally, the use of wheat neighbour genetic
mapping data suggests additional rearrangements in bins
(data not shown) as suggested by Liu et al. [53] and
Chantret et al. [54] and this may also lead to some
discrepancies. Altogether, our results regarding conservation between
wheat chromosome 3B, barley chromosome 3 H and
rice chromosome 1 at the whole chromosome and at
the deletion bins scales are in agreement with previous
studies. Wheat gene space organisation For the first time, we were able to assign precisely a
large number of genes to individual BACs and BAC
contigs whose order is known on wheat chromosome
3B. This led us to analyse the pattern of gene distribu-
tion along the chromosome. Out of the 757 loci mapped
on the wheat chromosome 3B physical map, 519 loci
were assigned to the eight deletion bins (Table 1). The
density of loci per deletion bin was calculated by divid-
ing the number of loci assigned to each deletion bin by
the cumulative length of contigs in the bin [17]. The
density of loci showed a slight increasing gradient from
the centromere to the telomeres (Figure 2) with a posi-
tive correlation with the distance to the centromere
(Pearson’s correlation coefficient r = 0.664), even though
this correlation was not statistically significant using a
5% threshold (P-value = 0.07). The highest density was
found on the most distal 3BL7-0.63-1.00 deletion bin of
the long arm (1.06 probes per megabase), whereas the
lowest density was observed on the most proximal
C-3BS1-0.33 bin of the short arm (0.58 probes per
megabase). Such a gradient could be artificially created
by a difference in gene sequence conservation from the
centromere to the telomeres between wheat and barley. To test this hypothesis, we calculated the coefficient of
correlation between the gene density and the percentage
of sequence identity per bin. No significant correlation
was found (Pearson’s correlation coefficient r = -0.478;
P-value = 0.23), demonstrating that the gene density
gradient is not biased by the differences in the similarity Figure 1 Collinearity between wheat chromosome 3B, barley
chromosome 3 H and rice chromosome 1. Each colour on wheat Figure 1 Collinearity between wheat chromosome 3B, barley
chromosome 3 H and rice chromosome 1. Each colour on wheat for each bin. We found that the distribution of collinear
genes is significantly uniform along chromosome 3B
(Chi2 test, P-value = 0.89 and 0.69 for barley and rice
respectively). Some translocations of genes can be
observed between wheat chromosome 3B and barley
chromosome 3 H and between wheat chromosome 3B
and rice chromosome 1 (Figure 1) that are similar to pre-
vious studies [13,52]. However, we expected a higher col-
linearity between the wheat and barley group 3
chromosomes with regard to previous results [29-32]. Comparative genomics between wheat, barley and rice However, we also noticed some wheat-specific
rearrangements of the genes that disrupt the collinearity
between wheat and barley and between wheat and rice. Thus, globally we expected the genes to be in the same
order between wheat and barley but rearrangements are
likely to be observed locally. So the results of anchoring
and ordering of the wheat BAC contigs along chromo-
some 3B using the barley mapping data should be con-
sidered with caution as they may not be perfectly exact. Wheat gene space organisation We think that this apparent discrepancy may originate
from the construction of the barley neighbour map. None of the five individual barley genetic maps available
to date holds a sufficient number of genes, and therefore Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 7 of 12 Figure 2 Gene density in the eight deletion bins of wheat chromosome 3B. The density of isolated genes is represented by blue bars. The
density of the genes organized in island is represented by red bars. The proportions of genes organized in island per deletion bins are shown
as percentages within the red bars. Figure 2 Gene density in the eight deletion bins of wheat chromosome 3B. The density of isolated genes is represented by blue bars. The
density of the genes organized in island is represented by red bars. The proportions of genes organized in island per deletion bins are shown
as percentages within the red bars. 3B. This resulted in an estimate of 904 loci assigned to
the eight deletion bins compared to the 519 loci identi-
fied by hybridisation in this study. Recently Choulet
et al. [16] estimated that chromosome 3B carries 8400
genes. We then extrapolated the gene density by consid-
ering 8400 loci assigned to the eight deletion bins. We
found that the distal bin 3BL7-0.63-1.00 and the proxi-
mal bin C-3BS1-0.33 would have a gene density of
1 gene per 101 kb and 1 per 185 kb, respectively. There-
fore, even in the least gene-dense regions of the chro-
mosome, our results indicate that there may be one
gene on average every 185 kb and therefore no mega-
base-sized regions devoid of genes. This is consistent
with RNA hybridisations on chromosome 3B MTP
arrays that showed that the largest region without genes
is about 800 kb long and that genes are distributed
across the entire chromosome 3B [16]. between wheat and barley gene sequences. Thus, the
gene density distribution observed here provides a reli-
able snapshot of the gene space organisation along
wheat chromosome 3B and led to a hypothesis where
the gene density is higher in the distal parts than in the
proximal parts of the chromosome (Figure 2). Conclusions Our study demonstrates that hybridisations of the barley
Agilent 15K expression microarray with wheat chromo-
some 3B MTP pools is a convenient and cost-efficient
technique to perform physical map anchoring with
gene-based markers. Our comparative genomics analysis
between wheat, barley and rice confirms good global
collinearity between these species, with a few wheat-spe-
cific rearrangements that could lead to local mis-order-
ing of wheat contigs using the barley gene order. Using
this technique, we also confirmed previous studies that
the gene space organisation follows a gradient of gene
density along chromosome 3B from centromere to telo-
meres without large “gene-free” regions. We also
demonstrated that this gradient was generated by a dif-
ferential accumulation of gene islands between the cen-
tromere and the telomeres with more genes in islands
in the distal parts of the chromosome. Such results have
far-reaching implications in terms of strategies to
sequence the wheat genome. Indeed, our results confirm
that to access the whole wheat gene set, the entire
wheat genome needs to be sequenced. A wheat expres-
sion microarray is currently being utilised to increase
the density of genes at the BAC scale located along
wheat chromosome 3B and to improve our understand-
ing of the wheat gene space organisation. To further study the gene space and especially the
genes clustered in islands in more detail, we considered
gene islands as multiple genes located on the same BAC
or overlapping BACs, i.e. separated by less than 150 kb. Out of the 757 loci mapped on wheat chromosome 3B
physical map, 303 loci, i.e. 40%, were considered part of
gene islands, whereas the 454 remaining genes (60%)
were considered as isolated genes. In contrast to the dis-
tribution of isolated genes that we found significantly
uniform along chromosome 3B (Chi2 test, P-value =
0.97), the distribution of genes organised in islands was
significantly non-uniform along chromosome 3B (Chi2
test, P-value < 10-5) with a positive correlation between
the density of genes in islands and the distance to the
centromere (Pearson’s correlation coefficient r = 0.762;
P-value = 0.03) (Figure 2). We also found a correlation
between the density of genes in islands and the overall
gene density (Pearson’s correlation coefficient r = 0.956,
P-value < 10-3). Wheat gene space organisation As we
found that 87% of the genes were mapped on the 81%
most distal parts of chromosome 3B, our result is in
agreement with the moderate gradient of gene density
along wheat chromosomes suggested by Munkvold et al. [13], Devos et al. [15], Charles et al. [14] and more
recently, Choulet et al. [16]. The discrepancy between
these results and the first suggestions of Erayman et al. [12] that most of the genes are located in the distal
parts of the wheat chromosomes may originate from
their use of a consensus deletion bin map from the A, B
and D chromosomes, where markers were non-systema-
tically assigned to the deletion bins [51]. This likely led
to approximations of the relative positions of the mar-
kers and the postulation that genes are mostly found in
gene-rich regions in wheat. Here, the possibility to
assign genes on physical contigs that cover 82% of a
specific chromosome 3B [17] enabled us to derive more
precise information about the gene distribution. However, our approach suffers from a major limitation
to estimate the gene density precisely. Here, the gene
densities estimated for the 3BS8-0.78-1.00 and 3BL7-
0.63-1.00 telomeric deletion bins were lower than the
ones found through RFLP hybridisation with ESTs by
Munkvold et al. [13] (normalized gene densities: 0.928
versus 1.190 and 1.176 versus 1.421, respectively). One
of the characteristics of telomeric parts of wheat chro-
mosomes is that they accumulate tandemly duplicated
genes at a high rate [16,39]. Thus, it is likely that the
differences in gene density observed between the two
experiments reflect the inability of gene mapping based We then extrapolated the expected gene density per
deletion bin to the whole set of chromosome 3B genes. We first estimated the number of genes per bin by con-
sidering the bins fully covered by contigs and by keeping
the same gene density distribution along chromosome Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 8 of 12 on BAC hybridisation to detect tandemly duplicated
genes. This method is only qualitative and detects the
presence or absence of a gene on a BAC but it does not
indicate whether a gene located on a BAC is present in
single or multiple copies. Wheat gene space organisation Thus, the gene density estab-
lished through gene mapping based on BAC hybridisa-
tion is likely underestimated in the distal regions and
therefore one can expect an even higher gene density gra-
dient. If we consider that the difference in gene density
between the two studies is only due to tandemly dupli-
cated genes, we could estimate that we missed 28% and
21% of genes for 3BS8-0.78-1.00 and 3BL7-0.63-1.00
deletion bins, respectively. This also explains why our
estimation of gene density at telomeres was lower com-
pared to the gene density of sequenced contigs located in
distal regions of chromosome 3B (1 gene per 101 kb ver-
sus 1 gene per 86 kb) whereas our estimation at the cen-
tromere precisely fits the gene density of sequenced
contigs located in proximal regions (1 gene per 185 kb
versus 1 gene per 184 kb). Assuming that we missed 21%
of genes due to tandem duplications in 3BL7-0.63-1.00
deletion bin, the gene density would be 1 gene per 90 kb. This demonstrates that all these studies can give an indi-
cation of the general gene space organisation along
wheat chromosomes but are unable to precisely estimate
the local gene density of specific regions. organisation. We confirmed that the gene density distri-
bution along the chromosome 3B follows a slight gradi-
ent from the centromere to the telomeres and we suggest
that the presence of more gene islands in the distal part
of the chromosome explains this gradient. However, the
ultimate experiment to access the whole set of genes and
confirm the gene density distribution at a high resolution
along a wheat chromosome will be high-quality sequen-
cing and annotation. This is currently underway for chro-
mosome 3B (C. Feuillet, personal communication). Conclusions This strongly suggests that the gradient
of gene density between centromeric and telomeric
regions is due to the differential distribution of genes
organised in islands across the chromosome with pro-
portionately more genes in islands in the distal parts
compared to the proximal parts. Methods
l Golden Promise RNAs
(equal amounts of leaf, root and inflorescence) labelled
with Cy5. RNA (5 μg) was labelled as described by
Ducreux et al. [55]. Amplified BAC pool DNA (200 ng)
was labelled using a modified BioPrime Genomic DNA
Labelling System (Invitrogen, Carlsbad, California USA):
BAC pool DNA in 11 μl was added to 10 μl Random
Primer Reaction Buffer mix and denatured at 95°C for
5 min prior to cooling on ice and to this was added
2.5 μl modified 10× dNTPs buffer (1.2 mM each of
dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris pH8.0;
1 mM EDTA), Cy3 dCTP (1 μl of 1 nM) and 0.5 μl Kle-
now enzyme (20U) followed by incubation for 16 h at
37°C. Labelled samples (BAC DNA & reference RNA)
for each array were combined and unincorporated dyes
removed using the Qiaquick PCR Purification Kit (Qia-
gen, Hilden, Germany) as recommended, eluting with
20 μl EB buffer (Qiagen, Hilden, Germany). Hybridisa-
tions and washing were carried out as recommended
(Agilent Protocol v5.5). Scanning was performed with an
Agilent G2505B scanner using default settings and data
extracted using Agilent FE software (v 9.5.3). All data
has been submitted to ArrayExpress [56] (accession #
E-TABM-1011) under MIAME guidelines [57]. a barley probe. For each barley probe, we identified the
positive pools to determine the original MTP BAC
address on which it is located. Each type of pool does
not contain the same number of BACs (plate: 384
BACs/pool; row: 480 BACs/pool; column: 320 BACs/
pool). The first normalisation step undertaken addressed the
fact that the 60 samples had different hybridisation sig-
nal averages. Medians were calculated for each pool
independently and each value was divided by the median
corresponding to the pool type. This led to comparable
hybridisation values for each pool. A second normalisa-
tion step was undertaken for each probe, based on the
same method. After this second normalisation step,
probe hybridisation values were all comparable, while
pool hybridisation values were not significantly changed. p
y
g
y
g
To identify pools with positive signal, we first used an
automated classical outlier detection method, that we
called the “automated scoring” method. The mean and
the standard deviation were calculated for each probe
and used to define a different threshold for each probe. Blast analyses y
A BLASTN analysis [33] was performed with the
60-mer barley probes against the TREP database [34] to
identify probes that could hybridise with TEs of the
wheat BACs. We considered that a probe could generate
a false positive due to TEs if we found 80% identity on a
minimum 45 nucleotides. Then a BLASTN analysis [33]
was performed with the 15208 60-mer barley probes
against the sequenced contigs of wheat chromosome 3B
[16]. The annotation of the best hit on the sequenced
contigs was viewed using Artemis [58]. The best hit and
the query barley probe were then aligned using Clus-
talW2 [59] and the sequence identity was calculated
using the entire barley probe length. In addition, a
BLASTN analysis [33] was performed with the 60-mer
barley probes against 6162 wheat cv Chinese Spring full-
length cDNAs developed by Kawaura et al. [38]. The
sequence identity between the best hit and the query
barley probe was calculated on the entire barley probe
length as previously described. The most significant rice
homologues to the unigenes used to design the barley
microarray probes were identified by BLASTN searches
of the gene models from the Rice Genome Annotation
Project from Michigan State University (Rice Pseudomo-
lecules v5 database [60]). Following this “automated scoring” method, a “semi-
automated” one was performed to identify missing coor-
dinates from probes having five positive pools (e.g. two
plate coordinates, two row coordinates and one column
coordinate). Here, a combination of all possible coordi-
nates was used to try to identify two overlapping BACs. A “manual scoring” analysis was also performed to iden-
tify the missing coordinate from probes having two posi-
tive pools. The final analysis that we called “boxplot scoring”
method was performed whereby a boxplot was drawn
using R software [61] for each probe for each of the
three pool types and the upper outlier values were con-
sidered as positive pools. This analysis was less stringent
than the “automated scoring” so we kept only the
probes that were located on two overlapping BACs. To rebuild the BAC addresses, we collated the positive
pools for each probe. Some probes gave one positive
pool per pool type which enables us to identify an
unambiguous BAC address. However, some probes gave
more positive pools per pool type. We therefore looked
at every combination and used the chromosome 3B Methods
l Calculation of the thresholds was different for each pool
type (plate: Mean + 2.8 × Standard Deviation; row:
Mean + 2.5 × Standard Deviation; column: Mean + 3 ×
Standard Deviation). All the pools with probe signal
above this threshold were considered positive. We
repeated this step twice by deleting positive signals pre-
viously detected, calculating the mean, standard devia-
tion and the threshold again for each probe and
selecting the new positive signals above the new thresh-
olds. The calculation of the thresholds for the three
pool types remained the same. Methods
l Barley expression microarray and hybridisations
The barley Agilent 15K expression microarray contains
15208 barley 60-mer probes derived from unigenes of
HarvEST assembly #25 used to originally design probe
sets for the 22K Barley1 Affymetrix GeneChip [21]. BACs (7440 in total) arranged in twenty 384-well plates
were selected to build a wheat chromosome 3B Minimal
Tiling Path covering 82% of the whole chromosome
with ~30% overlap as described by Paux et al. [17]. These twenty plates were pooled in three dimensions
(20 plate pools, 16 row pools and 24 column pools) to
generate 60 samples by CNRGV (Toulouse, France) and
the BAC pools were amplified as described by Paux
et al. [17]. Two channels processing of the microarrays
was used, with BAC pool DNA labelled with Cy3 and a In conclusion, our cross-species hybridisation techni-
que allowed us to assign a large number of genes onto
wheat chromosome 3B at the BAC resolution and to
obtain original results on the wheat gene space Rustenholz et al. BMC Genomics 2010, 11:714
http://www.biomedcentral.com/1471-2164/11/714 Page 9 of 12 mixed reference set of barley cv. Golden Promise RNAs
(equal amounts of leaf, root and inflorescence) labelled
with Cy5. RNA (5 μg) was labelled as described by
Ducreux et al. [55]. Amplified BAC pool DNA (200 ng)
was labelled using a modified BioPrime Genomic DNA
Labelling System (Invitrogen, Carlsbad, California USA):
BAC pool DNA in 11 μl was added to 10 μl Random
Primer Reaction Buffer mix and denatured at 95°C for
5 min prior to cooling on ice and to this was added
2.5 μl modified 10× dNTPs buffer (1.2 mM each of
dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris pH8.0;
1 mM EDTA), Cy3 dCTP (1 μl of 1 nM) and 0.5 μl Kle-
now enzyme (20U) followed by incubation for 16 h at
37°C. Labelled samples (BAC DNA & reference RNA)
for each array were combined and unincorporated dyes
removed using the Qiaquick PCR Purification Kit (Qia-
gen, Hilden, Germany) as recommended, eluting with
20 μl EB buffer (Qiagen, Hilden, Germany). Hybridisa-
tions and washing were carried out as recommended
(Agilent Protocol v5.5). Scanning was performed with an
Agilent G2505B scanner using default settings and data
extracted using Agilent FE software (v 9.5.3). All data
has been submitted to ArrayExpress [56] (accession #
E-TABM-1011) under MIAME guidelines [57]. mixed reference set of barley cv. Synteny and collinearity analyses In total, five barley genetic maps [21-25] were used to
establish a barley neighbour map using the same criteria
as the IBM neighbour map of maize [41]. For two maps
[24,25], we had to perform a BLASTN analysis to link the
markers to the barley unigenes mapped on wheat chro-
mosome 3B. The best hits with at least 85% identity over
100 nucleotides were selected for each unigene. The
order of the rice genes along the chromosomes was
established using the rice gene numbering annotation. As
the relative order of wheat genes in a given deletion bin
is not known, we inferred this order from the barley
chromosome 3 H and rice chromosome 1 data. The soft-
ware GenomePixelizer [62] was used for the graphical
display of the collinearity between wheat chromosome
3B, barley chromosome 3 H and rice chromosome 1. manuscript. The final version of the manuscript was approved by all the
authors. Authors’ contributions PH and JM carried out the hybridisations; CR, PH and FC performed data
analysis; RW, CF and EP helped with the interpretation of the results; CR
drafted the manuscript; FC, RW, CF and EP were involved in improving the Author details
1 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100
Clermont-Ferrand, France. 2Scottish Crop Research Institute, Invergowrie,
Dundee, DD2 5DA, UK. After identification of BACs carrying barley orthologs,
we used the physical map [17] to locate them in their
respective contig and possibly in one of the eight chro-
mosome 3B deletion bins used for this study. 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100
Clermont-Ferrand, France. 2Scottish Crop Research Institute, Invergowrie,
Dundee, DD2 5DA, UK. References
1
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Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B,
Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C,
Alaux M, Di Gaspero G, Dumas V, et al: The grapevine genome sequence
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2007, 449:463-467. Data deconvolution Following hybridisation, signals were analysed to rebuild
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http://www.biomedcentral.com/1471-2164/11/714 Page 10 of 12 (FP7/2007-2013) under the grant agreement n°FP7-212019. CR was
financially supported by Région Auvergne. (FP7/2007-2013) under the grant agreement n°FP7-212019. CR was
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The Edinburgh Type 2 Diabetes Study: study protocol
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Edinburgh Research Explorer Digital Object Identifier (DOI):
10.1186/1472-6823-8-18 Document Version:
Publisher's PDF, also known as Version of record Published In:
BMC Endocrine Disorders Published In:
BMC Endocrine Disorders The Edinburgh Type 2 Diabetes Study
study protocol Citation for published version:
Price, JF, Reynolds, RM, Mitchell, R, Williamson, RM, Fowkes, FGR, Deary, IJ, Lee, AJ, Frier, BM, Hayes,
PC & Strachan, MWJ 2008, 'The Edinburgh Type 2 Diabetes Study: study protocol', BMC Endocrine
Disorders, vol. 8, pp. 18. https://doi.org/10.1186/1472-6823-8-18 Citation for published version:
Price, JF, Reynolds, RM, Mitchell, R, Williamson, RM, Fowkes, FGR, Deary, IJ, Lee, AJ, Frier, BM, Hayes,
PC & Strachan, MWJ 2008, 'The Edinburgh Type 2 Diabetes Study: study protocol', BMC Endocrine
Disorders, vol. 8, pp. 18. https://doi.org/10.1186/1472-6823-8-18 Publisher Rights Statement: © 2008 Price et al; licensee BioMed Central Ltd. © 2008 Price et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
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doi:10.1186/1472-6823-8-18 This article is available from: http://www.biomedcentral.com/1472-6823/8/18 © 2008 Price et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
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(page number not for citation purposes) Study protocol Open
Study protocol
The Edinburgh Type 2 Diabetes Study: study protocol
Jackie F Price*1, Rebecca M Reynolds2, Rory J Mitchell1,
Rachel M Williamson3, F Gerald R Fowkes1, Ian J Deary4, Amanda J Lee5,
Brian M Frier6, Peter C Hayes7 and Mark WJ Strachan3 Address: 1Division of Community Health Sciences and Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK, 2Centre
for Cardiovascular Sciences, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK, 3Metabolic Unit, Western General
Hospital, Edinburgh, UK, 4Psychology in the School of Philosophy, Psychology and Language, University of Edinburgh, Edinburgh, UK,
5Department of General Practice and Primary Care, University of Aberdeen, Aberdeen, UK, 6Department of Diabetes, Royal Infirmary of
Edinburgh, Edinburgh, UK and 7Centre for Liver and Digestive Disorders, University of Edinburgh, Edinburgh UK Email: Jackie F Price* - Jackie.Price@ed.ac.uk; Rebecca M Reynolds - rreynold@staffmail.ed.ac.uk; Rory J Mitchell - R.J.Mitchell@ed.ac.uk;
Rachel M Williamson - Rachel.Williamson@luht.scot.nhs.uk; F Gerald R Fowkes - Gerry.Fowkes@ed.ac.uk; Ian J Deary - I.Deary@ed.ac.uk;
Amanda J Lee - A.J.Lee@abdn.ac.uk; Brian M Frier - Brian.Frier@ed.ac.uk; Peter C Hayes - P.Hayes@ed.ac.uk;
Mark WJ Strachan - Mark.Strachan@luht.scot.nhs.uk * Corresponding author Received: 18 November 2008
Accepted: 11 December 2008 Received: 18 November 2008
Accepted: 11 December 2008 Study Population
Sampling frame With the permission of the Lothian Diabetes Services
Advisory Group and the Caldicott Guardian for NHS
Lothian, patients recorded as having type 2 diabetes were
selected from the Lothian Diabetes Register (LDR). The
LDR is a computerised database, which was established in
2001, and contains clinical details on over 20,000
patients with known type 2 diabetes living in Lothian,
Scotland. Comparison of age-sex specific prevalences of
diabetes recorded on the LDR with those from other data
sources in Scotland suggests that the LDR captures almost
everyone with diagnosed diabetes in Lothian (Dr Sarah
Wild, personal communication). Confirmation of diagnosis of type 2 diabetes
Individual patients are recorded on the LDR once the
diagnosis of diabetes has been confirmed according to
WHO criteria. Further classification of the patient as suf-
fering from type 2 diabetes is made by medical staff in
individuals who are not insulin deficient (as indicated by
the presence of ketonuria or ketonaemia) and who have
no evidence of secondary diabetes (endocrinopathies or
exocrine pancreatic insufficiency) or a genetic aetiology
(e.g. Maturity Onset Diabetes of the Young). In order to
confirm the diagnosis of type 2 diabetes and exclude indi-
viduals erroneously recorded as such on the LDR, the
study team reviewed data on potential participants. For
the purposes of the present study, the diagnosis of diabe-
tes was accepted in any individual treated with oral anti-
diabetic agents and/or insulin, and in any subject treated
with dietary modification alone whose HbA1c was > 6.5%
at the research clinic. The clinical records of all subjects
treated with dietary modification alone and with an
HbA1c ≤ 6.5% at the research clinic were reviewed by a
consultant diabetologist (MS) to ensure that the diagnosis
of diabetes was robust. The clinical records of individuals
who either:(i) started on insulin within one year of diag-
nosis of diabetes, (ii) reported evidence of pancreatic sur-
gery/disease at the research clinic or (iii) were treated with The aims of this project were: 1. To determine the association between potentially mod-
ifiable risk factors (including microvascular disease,
inflammatory mediators and hormones of the hypotha-
lamic-pituitary-adrenal (HPA) axis) and cognitive decre-
ments in people with type 2 diabetes. 2. Methods and design The study was designed as a population-based, prospec-
tive cohort study. Ethical permission was obtained from
the Lothian Medical Research Ethics Committee. Background Type 2 diabetes currently affects around two million peo-
ple in the UK and approximately 10% of people aged over
65 years. The prevalence of the condition is predicted to
double over the next 20 years, with a particular increase in
elderly people [1]. Much has been done over the last dec-
ade to try to prevent and treat the well-recognised micro-
and macrovascular complications of diabetes, including
atherosclerotic cardiovascular disease, diabetic retinopa-
thy, nephropathy and peripheral neuropathy. However,
morbidity and mortality from vascular disease remains
high in older people with type 2 diabetes. In addition,
other complications of diabetes are becoming increas-
ingly evident in the ageing diabetic population. These
include the deleterious effects of type 2 diabetes on the
brain, resulting in a greater prevalence of age-related cog-
nitive impairment and an enhanced risk of age-related
cognitive decline, in addition to higher incidences of
stroke and dementia [2-4]. Non-alcoholic fatty liver dis-
ease, a disorder that encompasses a wide spectrum of
hepatic abnormality from fatty infiltration and steatosis to
steatohepatitis, fibrosis and ultimately cirrhosis, is
increasingly recognised to be associated with the diabetic
condition, potentially affecting up to 90% of people with
established type 2 diabetes [5-7]. Relatively little is known
about the risk factors associated with the development
and progression of these less prominent diabetes-related
complications, although factors such as poor glycaemic
control, increased inflammation, abnormal glucocorti-
coid metabolism and microangiopathy may be important
[8-12]. Detailed information on potential risk factors is
crucial to identify causal and modifiable risk factors that
can be targeted for the development of appropriate pre-
ventive and therapeutic interventions, in addition to help-
ing to identify patients who are at increased risk of
developing complications. 4. To establish a well-characterised and compliant popu-
lation sample with extensive phenotyping and the poten-
tial for genotyping, which can be used as the sampling
frame for subsequent nested case control studies (includ-
ing neuroimaging), and as a replication population for
findings arising from genome-wide association studies. Abstract Background: Risk factors underlying the development and progression of some of the less well-
recognised complications of type 2 diabetes, including cognitive impairment and non-alcoholic fatty
liver disease, are poorly understood. The Edinburgh Type 2 Diabetes Study was established in 2006
in order to investigate the role of potential risk factors in these complications, as well as to further
investigate mechanisms underlying the development and progression of micro and macrovascular
disease in type 2 diabetes. Methods and design: The study is designed as a prospective cohort study. Participants recruited
at baseline (2006–2007) constitute 1066 men and women aged 60 to 75 years with established type
2 diabetes, living in the Lothian region of central Scotland. Subjects underwent detailed cognitive
and physical examination, the latter including measures of micro- and macro-vascular disease,
glycaemic control, body fat composition and plasma inflammatory markers, cortisol, lipids and liver
function tests. Participants were re-examined after one year with hepatic ultrasonography and
additional measures of vascular disease. This paper reports the methods of recruitment to the
study and examinations performed at baseline and one year. Follow-up cognitive, vascular and liver
assessments are scheduled for 2010–2011 when subjects will have been in the study for 4 years. Discussion: This study will provide a wealth of epidemiological and biomarker data that should be
invaluable in the identification of potentially modifiable, causal risk factors for diabetes-related
cognitive impairment, liver dysfunction and vascular disease, which can be targeted for the
development of preventive and therapeutic interventions. Page 1 of 10
(page number not for citation purposes) Page 1 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 associated with progression of these complications and/or
(iii) have a potentially causal role in their development. Page 2 of 10
(page number not for citation purposes) Power and sample size The aim was to recruit 1000 subjects in order to provide
around 90% power at the two-sided 5% level of signifi-
cance to detect a Pearson correlation coefficient of ≥ 0.10,
between continuous outcome measures (e.g. cognitive
test scores) and predictor variables. Allowing for deaths
and drop-outs during follow-up, it was estimated that a
sample size of 800 would retain 90% power to detect a
correlation coefficient of ≥ 0.12 between risk factors and
outcome measures. It was also estimated that with this
sample size and with the same levels of power and signif-
icance, it would be possible to detect any risk factor that
contributed 1% or more to the variance in outcome, both
at baseline and at follow-up. http://www.biomedcentral.com/1472-6823/8/18 http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 actually assessed, including payment of travel expenses,
provision of transport to and from the clinic if required, a
choice of days to attend the clinic, multiple contact
attempts from the ET2DS team by both telephone and
mail, and reminder telephone calls on the day preceding
clinic appointments. Of the 1252 subjects who expressed
interest, 1077 attended the baseline research clinic: the
ET2DS team were unable to re-contact 56 individuals, 111
were unable or unwilling to attend for clinical examina-
tion when invited, five repeatedly failed to attend their
research clinic appointment and three had died. Of the
1077 subjects attending the baseline clinic, four were sub-
sequently excluded from the study because they were una-
ble for physical or emotional reasons to complete the
cognitive or physical examinations. In addition, seven
subjects were excluded as they did not meet the criteria for
type 2 diabetes after detailed review of hospital and pri-
mary care notes, searching of electronic databases and,
where necessary, discussion with the general practitioner. Of these seven subjects, two diet-treated patients with
HbA1c < 6.5% at the research clinic could not be con-
firmed as having diabetes (from a total of 70 subjects who
were reviewed for this reason), four subjects were felt to
have type 1 diabetes (from a total of 25 subjects reviewed
due to starting on insulin within 1 year of diagnosis or
being treated with insulin and diagnosed under the age of
35 years), and one subject had a previous pancreatic neu-
roendocrine tumour (from a total of 11 subjects reviewed
due to reporting prior pancreatic disease). This left 1066
subjects who were both willing and eligible to take part in
the ET2DS (Figure 1). insulin and were aged < 35 years at diagnosis were also
reviewed. Such individuals were considered to be at the
greatest risk of mis-classification. Any subject in whom it
was not possible to confirm a clinical diagnosis of type 2
diabetes by review of hospital and/or GP records was
excluded. Exclusion criteria Other exclusion criteria were, (i) non-English speakers
(since fluent English is required for some of the cognitive
tasks), (ii) corrected visual acuity worse than 6/36 for dis-
tance vision or unable to read large print text (as at least
moderate visual function is required to complete some of
the cognitive tasks), (iii) unwilling to give consent (or
judged by clinical research staff to be unable to give fully-
informed consent) (iv) physically unable to complete the
clinical and cognitive examination. Page 3 of 10
(page number not for citation purposes) Study Population
Sampling frame To determine, in older people with type 2 diabetes, (i)
the prevalence of Non-Alcoholic Fatty Liver Disease
(NAFLD), (ii) clinical factors that might permit early
detection of people at increased risk of developing NAFLD
and (iii) potentially causal risk factors for the develop-
ment and progression of NAFLD. 3. To identify circulating biomarkers and other risk factors
which (i) predict the development of symptomatic and
asymptomatic micro- and macrovascular disease, (ii) are Page 2 of 10
(page number not for citation purposes) Page 2 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 Subject Recruitment People aged between 60 and 74 years on 1st August 2006
were selected by sex and 5-year age bands from computer-
randomised lists of eligible subjects extracted from the
LDR. Between 20th June 2006 and 1st June 2007, 5454
invitations to participate in the study were mailed by the
custodians of the Lothian Diabetes Register. Of these, no
response was received from 2104, and a further 64 were
returned as being unknown at the address recorded on the
LDR. Of the 3286 individuals who replied, 1252
expressed interest in participating in the study (Figure 1). Another randomly selected subject from the same sex and
5-year age band replaced subjects not replying to the invi-
tation letter or refusing to participate. To preserve patient
confidentiality, only the names and addresses of subjects
who expressed interest in participating were forwarded to
the ET2DS team, precluding follow-up of individual non-
responders. Each person expressing an interest in the
study was sent an appointment for the research clinic,
together with a questionnaire to be completed and
returned to the research team. Strenuous efforts were
made to ensure that anyone agreeing to participate was At baseline and following an overnight fast, all subjects
attended a dedicated research clinic where they gave writ-
ten informed consent for participation in the study. Each
subject provided a urine specimen, underwent venepunc-
ture, and, following breakfast, completed a cognitive and
physical examination (predominantly for cardiovascular
risk factors and vascular disease). The physical examina-
tion was undertaken by one of six specially trained
research nurses and measurement technicians, using pre-
specified standard operating procedures and a specially
designed form for data collection. Subjects also returned
their completed questionnaires, including questions on
demographic characteristics, educational attainment, dia-
betes history and treatment, cardiovascular disease and
other co-morbidities, medications, alcohol intake, smok-
ing habits, employment/occupation, stress, satisfaction
with life, subjective social status and personality. Approx-
imately two to three weeks after their initial visit, subjects
returned to a separate research clinic for digital retinal
photography. Subject Recruitment Page 3 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 Invited
n = 5454
Replied
n = 3286
No response (n=2104)
Moved address (n=64)
Not interested in participation
(n = 2034)
Interested in
participation
n = 1252
Attended baseline clinic
n = 1077
ET2DS participants
n = 1066
Failed to meet inclusion criteria
(i) unable to complete cognitive/physical
examination (n= 4)
(ii) not type 2 diabetes (n=7)
Did not attend baseline clinic
(i) unable/unwilling to attend (n=111)
(ii) could not be contacted (n=56)
(iii) repeatedly failed to keep appointment (n=5)
(iv) deceased (n=3)
Invited to attend one
year clinic
n = 1054
Not invited to one year clinic
(i) known to have died (n=2)
(ii) refused consent to be contacted for
examinations other than baseline and 4 year
follow-up (n=5)
(iii) deemed unsuitable for contact by study
team (n=3)
(iv) withdrew from any further contact after
baseline examination (n=2)
Attended one year clinic
n = 940
Did not attend one year clinic
(i) unable to contact subject (n=19)
(ii) unable/unwilling to attend on health grounds
(n=23)
(iii) unable/unwilling to attend for non health
related reason (n=38)
(iv) cancelled/did not attend appointment (n=21)
(v) dead (n=13)
Failed inclusion criteria
(i) unable to complete examination (n=1)
One year clinic
participants Invited
n = 5454 No response (n=2104)
Moved address (n=64) Not interested in participation
(n = 2034) Invited to attend one
year clinic
n = 1054 Did not attend one year clinic
(i) unable to contact subject (n=19)
(ii) unable/unwilling to attend on health grounds
(n=23)
(iii) unable/unwilling to attend for non health
related reason (n=38)
(iv) cancelled/did not attend appointment (n=21)
(v) dead (n=13)
Failed inclusion criteria
(i) unable to complete examination (n=1) Attended one year clinic
n = 940 Failed inclusion criteria
(i) unable to complete examination (n=1) One year clinic
participants
n = 939 Recruitment and participation in the Edinburgh Type 2 Diabetes Study (baseline and year 1 examinations)
Figure 1
Recruitment and participation in the Edinburgh Type 2 Diabetes Study (baseline and year 1 examinations). Recruitment and participation in the Edinburgh Type 2 Diabetes Study (baseline and year 1 examinations)
Figure 1
Recruitment and participation in the Edinburgh Type 2 Diabetes Study (baseline and year 1 examinations). Subject Recruitment Page 4 of 10
(page number not for citation purposes) BMC Endocrine Disorders 2008, 8:18 http://www.biomedcentral.com/1472-6823/8/18 ata collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study
gure 2
ata collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study. Baseline clinic
(cognitive and physiological testing)
Cognitive and mood testing
Battery of neuropsychological tests
Mill Hill Vocabulary Scale
Hospital Anxiety and Depression Scale
Physical examination
Blood pressure
BMI, WHR, % body fat
ECG
Ankle brachial index
Neurothesiometry
Questionnaire (including WHO Chest Pain
and Edinburgh Claudication Questionnaires
Blood sample
Inflammatory markers
Cortisol
Lipids
Blood glucose, HbA1c
Liver function tests
Hyaluronic acid
Urinary albumin/creatinine ratio
Baseline Clinic
(digital retinal photography)
Diabetic retinopathy
One year clinic
Questionnaire including sleep apnoea
questionnaires
Abdominal USS
Carotid IMT and plaque measurement
Pulse wave analysis and velocity
Hypoglycaemia questionnaire
‘Routine’ data collection
ISD SMR01 data
Medical and surgical discharges
from Scottish hospitals
LDR data
HbA1c, blood pressure,
cholesterol
2-3 weeks
1 year ‘Routine’ data collection
ISD SMR01 data
Medical and surgical discharges
from Scottish hospitals
LDR data
HbA1c, blood pressure,
cholesterol Data collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study
Figure 2
Data collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study. Data collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study
Figure 2
Data collection at baseline and one year research clinics in the Edinburgh Type 2 Diabetes Study. Page 5 of 10
(page number not for citation purposes) Page 5 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 BMC Endocrine Disorders 2008, 8:18 'Routine' data collection which requires participants to generate as many words as
possible with a specified initial letter (C, F and L), within
a one minute period for each letter (proper nouns disal-
lowed and repeated words scored only once) [16]. Imme-
diate and delayed verbal declarative memory was assessed
using a participant's score on the Logical Memory subtest
of the Wechsler Memory Scale 3rd Edition (WMS-IIIUK)
[17]. This involves the immediate recall of a story with 25
elements ("story units"), which is read aloud. Participants
are informed that they will be questioned about the story
again later, and delayed recall is recorded in the same way
after approximately 40 minutes. One year visit All participants who were alive and who had consented
(at baseline) to be contacted about additional studies,
were invited to return for a further examination, one year
after their recruitment into the study. This visit was prima-
rily for hepatic assessment, including abdominal ultra-
sound examination, but also enabled further vascular
phenotyping (carotid intima media thickness (IMT),
carotid plaque assessment, pulse wave velocity and analy-
sis). A total of 940 subjects attended for this examination. Of the remaining 126 original ET2DS participants, 17 had
died or had withdrawn from further contact since base-
line, 8 had either indicated that they did not wish to be re-
contacted for this examination when seen at baseline or
were deemed unsuitable for contact by the study team, 61
were unable or unwilling to attend when contacted by let-
ter and/or telephone and 40 either could not be contacted
or did not attend their clinic appointment (Figure 1). In
addition to the abdominal ultrasound, vascular examina-
tions and venous blood sampling, all of which were per-
formed after a fast of at least four hours, a second
questionnaire was completed, including updated ques-
tions on alcohol intake, medications and history of liver
disorder, Berlin and Epworth sleep apnoea questionnaires
[14,15], and questions relating to diabetes healthcare pro-
vision in Lothian. Subjects were also provided with short
self-assessment questionnaires on which to record the
occurrence of severe hypoglycaemic episodes over the
subsequent six months. All subjects were asked to complete a combined version of
the Junior and Senior Form A synonyms of the Mill Hill
vocabulary scale [20], an indicator of prior ('best ever' or
pre-morbid) cognitive ability which changes very little
with age [21,22]. Subjects were presented with a word and
asked to identify the closest synonym from six given alter-
natives. The Mini Mental State Examination [23], often
used as a 'screen' for dementia, was included as a general
mental assessment. Subject Recruitment The score is based on the
number of story units recalled correctly. Non-verbal mem-
ory was assessed using the Faces subtest of the WMS-IIIUK. Subjects are shown a series of pictures of faces and then,
in subsequent immediate and delayed tests they are asked
to select these faces from a larger series of pictures. As a
measure of mental flexibility, the Trail Making Test [18]
was administered and the time taken to complete part B
was used in the subsequent analysis. Subjects completed
several subtests of the Wechsler Adult Intelligence Scale
3rd Edition (WAIS-IIIUK) [19]. The Digit Symbol Coding
subtest was used as a measure of speed of information
processing – the number of symbols matched correctly to
their corresponding numbers in 120 seconds was
recorded. The Letter-Number Sequencing subtest was
used to assess working memory – participants listened
while the tester read mixed, and increasingly longer,
strings of numbers and letters. They repeated them to the
tester, with the numbers first, in numerical order, and
then the letters in alphabetical order. In each item of the
Matrix Reasoning subtest (non-verbal reasoning), partici-
pants examined a pattern arrayed in matrix with a piece
missing. The elements of the matrix are arrayed according
to rules. The task is to work out the rules, apply them to
find out what the missing piece should look like, and
choose the correct piece from the answer options. At baseline, data were collected for all participants on dis-
charges from non-psychiatric, non-obstetric wards in
Scottish hospitals between 1981 and September 2007
using record linkage to the SMR01 scheme (acute hospital
admissions) at the Information and Services Division of
NHS Scotland [13]. These data were used to supplement
self-reported history of cardiovascular disease, liver condi-
tions and diabetic complications. Selected primary and
secondary care data held on the Lothian Diabetes Register
(LDR) were also retrieved for participants to provide serial
historical data for key biochemical and clinical variables,
such as HbA1c, blood pressure and serum cholesterol lev-
els. Non-identifiable data on non-participants were also
collected from the LDR, to enable comparison of charac-
teristics with study participants and therefore assess the
representativeness of the study population. Cognitive testing The Hospital Anxiety and Depression Scale (HAD A and
HAD D respectively) [24] was also used for the assessment
of mood states, which can affect performance on the cog-
nitive tests. Corrected visual acuity, near vision and capil-
lary blood glucose were also measured before proceeding
to cognitive testing. A blood glucose > 4 mmol/l was At baseline, a battery of psychometric tests, which pro-
vides a comprehensive and validated assessment of cogni-
tive functions and mood states, was administered. Individual testers were trained and then observed for val-
idation by one investigator (ID). Executive function was
assessed with the Borkowski Verbal Fluency Test (VFT), Page 6 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 required, or subjects were asked to return on another day
for cognitive testing. by clinical members of the research team http://
www.epi.umn.edu/ecg/ (MS, RR, RW). Right and left bra-
chial, posterior tibial and dorsalis pedis systolic pressures
were recorded with the subject in the supine position and
after at least 5 minutes rest, using the aneroid sphyg-
momanometer and a doppler probe (Dopplex® advanced
pocket Doppler, Huntleigh Healthcare Ltd., Cardiff, UK). The ankle brachial pressure index (ABI) was calculated for
each subject by dividing the lowest of the ankle pressures
by the higher of the two arm pressures. To assess inter-
observer variation in ABI (and WHR), a total of 20 sub-
jects had repeat measurements of waist and hip circumfer-
ences and ankle and brachial pressures, done on a single
day by all six of the clinic staff, independently. Questionnaire In the questionnaire applied at baseline, participants
answered questions on medical diagnoses and/or treat-
ment (medical or surgical) for angina, coronary heart dis-
ease/myocardial infarction, stroke, peripheral arterial
disease, carotid stenosis, hypertension and hypercholes-
terolaemia. Details on the year of diagnosis or event, and
hospital or general practice attended, were collected to
enable further validation of diagnoses after comparison
with ISD and LDR data. Subjects also completed a stand-
ard smoking history questionnaire and the WHO Chest
Pain [25] and Edinburgh Claudication Questionnaires
[26]. Bilateral carotid IMT was measured in the supine position
with the neck extended and the chin turned contralateral
to the side being examined. A Sonoline Elegra Ultrasound
Imaging System (Sieman's Medical Systems Inc, Washing-
ton, USA), software version 6, was used. A high frequency
linear transducer (8–12 MHz), set up for maximum reso-
lution, was orientated so that the back wall of the com-
mon carotid artery was parallel to the transducer face and
a double line was clearly seen. IMT was measured on the
far wall of the artery, 1–2 cm below the carotid bifurcation
and in an area free of plaque. The mean of three measure-
ments (to the nearest 0.1 mm) from three separate images
was taken. A longitudinal image was also stored for subse-
quent off-line serial IMT measurements. The common,
internal and external carotid arteries were examined for
the presence or absence of plaque. Plaque was defined as
a focal structure encroaching into the arterial lumen of at
least 0.5 mm or 50% of the surrounding IMT value, or
with a thickness > 1.5 mm as measured from the media-
adventitia interface to the intima-lumen interface. Plaque
at, or within 2 cm, of the bifurcation was recorded bilater-
ally, together with the maximum thickness of the plaque
and the presence or absence of echoluscent plaques. Echo-
luscent plaque was recorded where one or more plaques
appeared as black or almost as black as flowing blood
(compared with echogenic plaque which appeared white
or almost white, similar to the far wall media-adventitia
interface). The presence of heterogeneous plaque was
recorded if there were one or more plaques in which echo-
genicity of more than 20% of the plaque area differed sub-
stantially from the echogenicity of the rest of the plaque. Video images were stored for further assessment, includ-
ing grading of echogenicity. http://www.biomedcentral.com/1472-6823/8/18 http://www.biomedcentral.com/1472-6823/8/18 BMC Endocrine Disorders 2008, 8:18 sphygmomanometer or a mercurial sphygmomanometer
(W.B.I.C, Wenzhou, China) and the mean of each taken. All measurements were taken from the left arm unless this
was undesirable or impossible (e.g. previous stroke or
amputation affecting left arm) in which case the right side
was used. Pulse wave analysis was determined by applana-
tion tonometry of the radial, carotid and femoral arteries
using a high-fidelity micromanometer (SPT-301B; Millar
Inc, Texas, USA) and the SphygmoCor™ system 8.0 ver-
sion (AtCor Medical Pty Ltd, New South Wales, Australia). Data from the radial artery pulse wave were collected
directly into a computer and, after 10 seconds of sequen-
tial waveforms had been acquired, an averaged peripheral
waveform was generated. A corresponding averaged cen-
tral pressure waveform was then generated, using a vali-
dated transfer function. From this, central augmentation
index (both unadjusted and adjusted to a standard heart
rate of 75 bpm) and central aortic blood pressure were
determined using the integral software. For measurement
of pulse wave velocity the arterial pulse waveform was
recorded as above at the common carotid and femoral
arteries. The time delay between the arrival of the foot of
the pulse wave at the two points was obtained by gating to
the peak of the R-wave on electrocardiogram. Separation
of the pulse waveforms was defined as the difference
between the distances from the sternal notch to the
carotid measurement site and from sternal notch to the
femoral measurement site. Pulse wave velocity was calcu-
lated as distance/time (m/s). All measurements were
made in duplicate and the mean values calculated. If there
was variation in the augmentation index of greater than
5% or in pulse wave velocity of greater than 0.5 m/s, a
third reading was obtained if possible. If the latter was
within 5% or 1.0 m/s of one of the first readings then the
mean of these was used; otherwise the mean of all three
readings was used. If only one reading was obtained then
this alone was used. images independently and discrepancies in coding
between the graders were resolved in the first instance by
discussion between the graders. Unresolved discrepancies
at this point were reviewed and arbitrated upon by a con-
sultant ophthalmologist. Assessments of peripheral neuropathy and nephropathy
were undertaken as secondary measures of microvascular
disease. Liver assessment At baseline, liver function was assessed by measurement
of plasma liver function tests (LFTs) (details below) and
hyaluronic acid. In addition a full history of previous liver
disease, alcohol intake and use of prescription medica-
tions was obtained by questionnaire. At one year, LFTs
were repeated, history was updated by questionnaire and
a more detailed examination was undertaken using ultra-
sonography. All ultrasound examinations were performed
by a single ultrasonographer who was unaware of the clin-
ical and laboratory results of the participants, on a Sono-
line Elegra Ultrasound Imaging System (Sieman's Medical
Systems Inc, Washington, USA), software version 6, using
a 3.5 MHz transducer. A phantom (411 LE 0.5, GAMMEX
rmi Ltd, Nottingham, UK) was scanned multiple times
using the different pre-sets prior to the study to ensure
continuity of image contrast at follow-up and the appro-
priate pre-set was chosen for each subject and noted on
the ultrasound data collection sheet. Three graders (one
sonographer, one specialist registrar in radiology and one
consultant radiologist) independently graded the liver for
evidence of fatty infiltration using accepted criteria includ-
ing a bright hepatic echo pattern (compared with the right
kidney), increased attenuation of the echo beam and loss
of intrahepatic architectural details. Participants received
an overall grading of "normal", "indeterminate", "mildly
fatty" or "severely fatty". Evidence of more advanced
forms of liver disease, including cirrhosis and features of
portal hypertension was also sought systematically. In
addition the gallbladder, common bile duct, spleen, pan-
creas, kidneys and aorta were examined. A subgroup of 60
subjects with the full range of liver ultrasound findings
underwent detailed magnetic resonance imaging and
spectroscopy to quantify liver fat and help validate the
ultrasound findings. http://www.biomedcentral.com/1472-6823/8/18 Hand-held neurothesiometer readings were taken
from the apex of the big toe (Horwell Neurothesiometer,
Scientific Laboratory Supplies Ltd, Nottingham, UK), with
subjects sitting or lying, feet elevated and eyes closed. Fol-
lowing a test procedure on the subject's hand, the mini-
mum vibration threshold at which the subject was aware
of vibration sensation was recorded to the nearest 0.5
volts. The means of three recordings from each foot were
taken as the final measurements. Albuminuria was meas-
ured using the urinary albumin/creatinine ratio in an
early-morning specimen of urine. Page 8 of 10
(page number not for citation purposes) Physical examination Systolic and diastolic brachial blood pressures were meas-
ured in the right arm to the nearest 2 mmHg with subjects
in the supine position and with the arm resting at the level
of the mid-sternum, using a standard stethoscope and an
aneroid, 6 inch dial, desk standing sphygmomanometer
(Acceson™, AC Cossor & Son (Surgical) Ltd, Harlow, UK). To assess body mass index, standing height was measured
to the nearest mm, without shoes, using a wall-mounted
vertical rule. Weight was assessed to the nearest 0.1 kg
without outdoor clothing or shoes using SECA 761 elec-
tronic weighing scales. For waist:hip ratio (WHR), waist
circumference was measured during exhalation at the
level midway between the lower rib margin and the iliac
crest (pre-marked at the mid-axillary line on each side of
the subject), with the subject standing with their feet 30
cm apart and with their hands by their sides. Hip circum-
ference was taken with the subject in the same standing
position, by wrapping the tape measure around the but-
tocks and lowering or raising the tape until the maximum
circumference was located and the tape fitted comforta-
bly. Both measurements were made using a non-expand-
able tape measure, and the average of two readings taken
to the nearest 0.5 cm was taken as the final measurement. Body fat percentage was taken as the average of three con-
secutive readings (to the nearest 0.1%), using an OMRON
BF306 Body Fat Monitor (OMRON Healthcare (UK) Ltd.,
Henfield, UK). The monitor was pre-set to the correct
weight, age and gender for each subject. Subjects stood
with their feet 30 cm apart, with their arms at 90° to their
trunk and with the palms of each hand placed firmly on
the top and bottom electrodes. Pulse wave analysis and velocity are established measures
of arterial stiffness whose measurement is increasingly
standardised [27]. Measurements were performed after
subjects had been supine for at least 25 minutes. Systolic
and diastolic brachial blood pressures were measured
twice using either the aneroid, 6 inch dial, desk standing A resting 12-lead electrocardiogram was recorded accord-
ing to standard procedures (Marquette MAC 1200
machine) and coded using the Minnesota coding system Page 7 of 10
(page number not for citation purposes) Page 7 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1472-6823/8/18 Competing interests
h
h
d
l
h
h p
g
The authors declare that they have no competing interests. Data entry, checking and storage Data from the baseline questionnaire and data collection
forms were coded and entered onto a master Microsoft
Access database. The results of plasma assays were entered
onto the same master database, either from paper records
(biochemistry and haematology) or from electronic files
provided by the participating laboratories. For retinopa-
thy grading, data on individual retinal characteristics for
each relevant photographic field were entered directly
onto a specially designed spreadsheet (Microsoft Excel). The overall retinopathy grade for the right and left eyes for
each subject was calculated automatically and these
results were transferred to the master database. The major-
ity of data from paper records were double entered and
discrepancies resolved by reference to the original paper
documentation. For the remaining data a random sample
of records was double-checked. A small amount of base-
line data that was found to be missing was collected at the
one year clinic (primarily clarification of incomplete or
inconsistent personal and clinical details) and used to
update the master database. The principal investigator on the ET2DS grant was JP, and
MS, RR, GF, ID, AL and BF were co-investigators. The prin-
cipal investigator on the liver sub-study was MS, and JP,
RR, BF and PH were co-investigators. The investigators
designed the study, with AL providing statistical input. RM and RW collected, entered and checked the data. JP
drafted the article, with sections contributed by RW, MS
and RM. All authors contributed to revising the article. All
authors read and approved the final article. Assessment of microvascular disease Assessment of diabetic retinopathy by digital retinal pho-
tography was used as the most sensitive and specific meas-
ure of generalised microvascular disease. Standard 7-field
digital retinal colour photographs of both eyes were taken
at 45% by a single specially trained medical photogra-
pher, following pupillary dilatation and using a high res-
olution digital retinal camera (TOPCON TRC-50FX). Retinopathy was graded using the Early Treatment of Dia-
betic Retinopathy Scale modification of the Airlie House
Classification scheme, which assesses the level of retinop-
athy for each eye [28,29]. This grading system has been
used extensively in clinical and epidemiological studies of
diabetic retinopathy, including the Diabetes Control and
Complications Trial and the UK Prospective Diabetes
Study, to assess baseline status of retinopathy and progres-
sion of disease. Two dedicated optometrists graded Page 8 of 10
(page number not for citation purposes) Page 8 of 10
(page number not for citation purposes) BMC Endocrine Disorders 2008, 8:18 http://www.biomedcentral.com/1472-6823/8/18 http://www.biomedcentral.com/1472-6823/8/18 base at the time of grading and these were transferred
directly onto the master database. Pulse wave measure-
ments were exported to a Microsoft Excel file and from
there entered onto the master database. Individuals with an abnormal liver ultrasound scan and/
or abnormal liver function tests underwent further sero-
logical assessment to investigate causes of abnormal liver
function, including hepatitis B and C serology, ferritin,
anti-smooth muscle and anti-mitochondrial antibodies,
anti-nuclear factor and alpha fetoprotein. Individuals
with abnormal tests and/or significant derangement of
liver function were referred to a specialist hepatology
clinic for further investigation and management. All data, including the master database, the retinal photo-
graphs and carotid and abdominal ultrasound scans were
stored securely on dedicated university computers and
backed up on a dedicated university server. Discussion At baseline, venous blood sample were taken after an
overnight fast for DNA extraction, white blood cell
immortalisation, measurement of plasma c-reactive pro-
tein, fibrinogen, interleukin-6, tissue necrosis factor-α,
cortisol and cortisol binding globulin, creatinine, eGFR,
total cholesterol, high density lipoprotein cholesterol,
blood glucose, HbA1c, total protein, aspartate amino
transferase, alanine amino transferase, alkaline phos-
phatase, gamma glutamyl transferase, bilirubin, albumin,
full blood count and hyaluronic acid. At one year, venous
blood samples were taken (after a four-hour fast) for uric
acid, total cholesterol, low and high density lipoprotein
cholesterol, triglycerides, plasma glucose and repeat LFTs
and HbA1c. At both baseline and 1 year, plasma was
stored for future biomarker measurements. The Edinburgh Type 2 Diabetes Study is a large prospec-
tive cohort study that has the potential to identify factors
that are important for the development and progression
of less well-characterised complications of type 2 diabetes. The study protocol is being reported at the point at which
baseline and one year data have been collected and
checked. Analyses are currently underway on this rich
dataset to address the objectives described in this report. Further analyses to fully meet these objectives will be con-
ducted after the follow-up assessments of cognitive func-
tion, vascular parameters and liver structure and function
have been completed in 2010–11. References 1. Wild S, Roglic G, Green A, Sicree R, King H: Global prevalence of
diabetes: estimates for the year 2000 and projections for
2030. Diabetes Care 2004, 27:1047-1053. 27. Laurent S, Cockcroft J, Bortel LV, Boutouyrie P, Giannattasio C,
Hayoz D, Pannier B, Vlachopoulos C, Wilkinson I, Struijker-Boudier
H, on behalf of the European Network for Non-invasive Investigation
of Large Arteries: Expert consensus document on arterial stiff-
ness: methodological issues and clinical applications. Eur
Heart J 2006, 27:2588-2605. 2. Biessels GJ, Staekenborg S, Brunner E, Brayne C, Scheltens P: Risk of
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28. Early Treatment Diabetes Retinopathy Study Group: Grading Dia-
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graphs – An Extension of the Modified Airlie House
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4. Strachan MWJ, Deary IJ, Ewing FME, Frier BM: Is type 2 (non-insu-
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6. Silverman JF, Pories WJ, Caro JF: Liver pathology in diabetes
mellitus and morbid obesity. Clinical, pathological and bio-
chemical considerations. Pathology Ann 1989, 24:275-302. Acknowledgements We thank the participants and staff of the Edinburgh Type 2 Diabetes Study. Also staff at the Wellcome Trust Clinical Research Facility and Princess
Alexandra Eye Pavilion in Edinburgh where research clinics were held. We
also thank collaborators and members of the steering committee who have
contributed to the design and/or management of the study: B. Walker, J.R. Seckl, K. Swa, D.J. Webb, M.C. Whiteman, A. Rumley, G.D.O Lowe, J. McK-
night, P. Halpin, S. Glancy and P.L. Allan. The study is funded by the Medical Research Council, the Chief Scientist
Office of the Scottish Executive and Pfizer plc. JP, ID and BF are members of the University of Edinburgh Centre for Cog-
nitive Aging and Cognitive Epidemiology. The Centre is part of the cross
council Lifelong Health and Wellbeing Initiative. Funding from the BBSRC,
EPSRC, ESRC and MRC is gratefully acknowledged. Questionnaire data and plasma assays collected at the one
year clinic visit were entered initially onto a separate
Microsoft Access database and checked for inconsistencies
prior to transfer onto the master database. Data from the
abdominal ultrasound scan, carotid IMT and assessment
of plaque were entered onto a third Microsoft Access data- Page 9 of 10
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85:1349-1355. The pre-publication history for this paper can be accessed
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here: 8. Price JF, Reynolds RM, Frier BM, Strachan MWJ: Type 2 diabetes
and cognitive impairment: the Edinburgh Type 2 Diabetes
Study. Pract Diab Int 2008, 25:132-133. http://www.biomedcentral.com/1472-6823/8/18/prepub http://www.biomedcentral.com/1472-6823/8/18/prepub y
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13. Scottish public Health observatory. Overview of key data
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UK: The Psychological Corporation; 1998. Pre-publication history Publish with BioMed Central and every
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20. Raven J, Raven JC, Court JH: Manual for Raven's Progressive Matrices
and Vocabulary Scales Oxford: Oxford Psychologists Press Ltd; 1998. y
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21. Schaie KW: Developmental influences on adult intelligence Oxford:
Oxford University Press; 2005. y
22. Salthouse TA: Localizing age-related individual differences in a
hierarchical structure. Intelligence 2004, 32:541-561. g
23. Folstein MF, Folstein SE: Mini Mental State. A practical method
for grading the cognitive state of patients for the clinician. J
Psychiat Res 1975, 12:189-198. y
24. Zigmond AS, Snaith RP: The hospital anxiety and depression
scale. Acta Psychiatr Scand 1983, 67:361-370. y
25. Rose G, McCartney P, Reid DD: Self-administration of a ques-
tionnaire on chest pain and intermittent claudication. Br J
Prev Soc Med 1977, 31:42-48. Page 10 of 10
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English
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Catalytic oxidation of carbon monoxide
|
Applied catalysis
| 1,989
|
public-domain
| 6,027
|
Introduction. This problem was a phase of the War Gas Investigations and was under-
taken at this laboratory2 early in May 1917, under the auspices of the
U. S. Bureau of Mines, with the object of providing a mask ingredient
which would afford protection against carbon monoxide when present in
air. The. desired application and the necessary specifications of such a
material having been described3 in detail, the present article presents, from
the chemical standpoint, the most important features of the development
of a suitable oxidizing catalyst. [CONTRIBUTION
FROM THE DEPARTMENT
OF CHEMISTRY, JOHNS HOPKINS UNIVERSITY
1
THE CATALYTIC OXIDATION OF CARBON MONOXIDE.‘ [CONTRIBUTION
FROM THE DEPARTMENT
OF CHEMISTRY, JOHNS HOPKINS UNIVERSITY
1
THE CATALYTIC OXIDATION OF CARBON MONOXIDE.‘ BY T. H. ROGERS,
C. S. PICGOT,
W. H. BAHLKE
AND J. M. JENNINGS. Received July 12, 1920. BY T. H. ROGERS,
C. S. PICGOT,
W. H. BAHLKE
AND J. M. JENNINGS. Received July 12, 1920. No. 9. No. 9. SEPTEMBER,
1921. No. 9. VOL. 43. American Chemical Journal
(Founded by Ira R e m n ) American Chemical Journal
(Founded by Ira R e m n ) 4 Cf. Gmelin-Kraut, “Handbuch d. anorganischen Chemie,” on carbon monoxide p
3 Lamb, Bray and Frazer, Ref. 2. 2 This investigation was directed by Professor J. C. W. Frazer, who is one of the
authors of a recent general article, Lamb, Bray and Frazer, J. Ind. and Eng. Chem., 12,
213 (1920), concerning the treatment of this problem as a whole by various laboratories
in cooperation with the Central Laboratory at Washington. General Statement. The first work was done in making comparative tests of the materiaIs
described in the literature as capable of oxidizing carbon monoxide at
room temperat~res,~
always with the object in view of ultimately making 1 Published by permission of the Director of the Chemical Warfare Service. 2 This investigation was directed by Professor J. C. W. Frazer, who is one of the
authors of a recent general article, Lamb, Bray and Frazer, J. Ind. and Eng. Chem., 12,
213 (1920), concerning the treatment of this problem as a whole by various laboratories
in cooperation with the Central Laboratory at Washington. 2 This investigation was directed by Professor J. C. W. Frazer, who is one of the
authors of a recent general article, Lamb, Bray and Frazer, J. Ind. and Eng. Chem., 12,
213 (1920), concerning the treatment of this problem as a whole by various laboratories
in cooperation with the Central Laboratory at Washington. T. H. ROGERS, c. s. PIGGOT, w. H. BAHLKE AND J. M. JENNINGS. 1974 the reaction catalytic. It was observed while doing this preliminary
work that many of these substances when intimately mixed gave a material
much more active than any one of its constituents. For example, while
neither chromium trioxide, CrOa nor yellow mercuric oxide oxidized carbon
monoxide appreciably when a low concentration of this gas in air was passed
through a tube containing these materials, a mixture of the oxides con-
taining chromium trioxide, in excess of equivalent proportions was found
to possess considerable oxidizing power and about 50% of the carbon mon-
oxide was oxidized for a s5ort period. Again, silver oxide which oxidizes carbon monoxide only slowly at room
temperatures, when mixed in various proportions with calcium hydroxide
showed markedly greater activity, evidencing the increased activity of
mixtures over that of single constituents. Other important factors,
more or less self-evident, in the use of mixtures of two or more substances
are the fineness of subdivision and intimate admixture of these substances,
the resulting product being essentially a solid solution in certain cases. When a mixture of silver and calcium hydroxides was prepared by the
simultaneous precipitation of the hydroxides from a water solution of the
nitrates of these metals, and the precipitate washed and dried at 120-130"
in a current of oxygen a materi.1 w?.s obtained having marked activity. General Statement. When dried in a current of air or oxygen the color of this mixture at times
changed during the process of drying from a light brown to a slate color,
a color change which indicates the decomposition of the silver oxide. On
examination it was found that globules of metallic silver could be seen
in the material. This decomposition was observed at 100" in the air and
at 130" in oxygen, whereas the decomposition pressure of silver oxide is
but 0 . 2 atmospheres at 130'. This notable increase in the decomposition
pressure of silver oxide is being investigated further to determine whether
the phenomenon is due to fineness of subdivision or to some other cause
connected with the presence of calcium hydroxide, one effect of which may
be to prevent the increase in size of the silver oxide particles. p
p
The activity of these mixtures of silver oxide and calcium hydroxide
was slightly increased by the addition of a small quzntity of sodium hydrox-
ide. The oxidation of the resulting material (about 807, for 20 minutes)
was entirely due to the combined oxygen, as the amount of oxidation
was never more than could be accounted for by the silver oxide present. Manganese dioxide was ultimately found to be an essential constituent
of the best catalysts, and here also the same generel statements made above
apply. Mixtures of manganese dioxide and silver or copper oxide were
found to be much more active towards carbon monoxide than either of the
oxides used separately. The addition of a third component, sodium
hydroxide, was advantageous in the preliminary experiments on manganese
dioxide. The action of these mixtures containing sodium hydroxide was The activity of these mixtures of silver oxide and calcium hydroxide
was slightly increased by the addition of a small quzntity of sodium hydrox-
ide. The oxidation of the resulting material (about 807, for 20 minutes)
was entirely due to the combined oxygen, as the amount of oxidation
was never more than could be accounted for by the silver oxide present. Manganese dioxide was ultimately found to be an essential constituent
of the best catalysts, and here also the same generel statements made above
apply. Mixtures of manganese dioxide and silver or copper oxide were
found to be much more active towards carbon monoxide than either of the
oxides used separately. General Statement. The addition of a third component, sodium
hydroxide, was advantageous in the preliminary experiments on manganese
dioxide. The action of these mixtures containing sodium hydroxide was CATALYTIC OXIDATION OF CARBON MONOXIDE. 19T5 not catalytic. They functioned for only about 40 minutes under the
testing conditions given below and the amount of oxidation was not greater
than could be accounted for by the active oxygen contained in the material. The behavior of these mixtures is shown in Table I. It was not until finely divided manganese dioxide was prepared, and
special precautions, listed later, were used to secure the admixture therewith
of other oxides, that the Catalytic oxidation of carbon monoxide at room
temperature was sztisfactorily solved. The most satisfactory catalysts
were obteined by using a mixture containing either silver oxide or copper
oxide end mangenese dioxide. Of these mixtures those containing silver
oxide seemed to be more active than those containing copper oxide. Other
oxides such as iron oxide may be tolerated in cert:in amounts in the final
products, but such oxides seem to act more as inert diluents and decrease
rather than increase the activity of the material. However, when catalysts
are prepared by properly mixing manganese dioxide with both silver
and copper oxides such catalysts may be quite as active as those containing
either copper or silver oxide alone. The conclusions reached from the preliminary work on manganese
dioxide were, first, that the degree of subdivision was an extremely im-
portant factor in the activity of this material; and, second, that the activity
is also greatly increased by properly incorporating with finely divided
manganese dioxide, other metallic oxides such as copper and silver oxides. g
pp
A comparative study was then made of samples of manganese dioxide
prepared by the following methods. 1. Reduction of Potassium Permanganate by Methyl Alcohol. (a)-When
a
warm solution was reduced, the manganese dioxide particles were comparatively coarse,
settling rapidly, and the mixtures prepared from such materials were not very active
(Table I (a)). When the reduction was brought about in a cold solution a more flocculent
precipitate was obtained which settled more slowly than (a), and it will be seen by re-
ferring to Table I that the silver oxide mixtures were more active than similar mixtures
made from samples (a). We are indebted to W. A. Patrick far
uggesting this method of preparation.
Frkmy, Compt rend., 82, 1213 (1876). General Statement. The mixtures of silver oxide and manganese dioxide made from both (a) and (b)
above were quite efficient in bringing about the oxidation of carbon monoxide for an
hour or more and the oxygen consumed was greater than the available oxygen of the
material, showing these samples to be partially catalytic. The active zone of the
material a t any time during the test could be immediately ascertained by the location
of the warm portion of the tube. This zone moved along gradually as the efficiency of
that portion dropped and finally passed to the bottom of the tube, when carbon mon-
oxide could be detected in the effluent gas. Prior to this, however, the action was
100 yo efficient. The Frdmy Method.6-One hundred g. of finely ground potassium permanganate
is added to 500 g. of conc. sulfuric acid in 150 cc. of water, cooled, carefully stirred, and
allowed to stand, when the permanganic acid formed decomposes, giving off oxygen. Permanganic anhydride, MnzOr, will sometimes separate as an oil. which is extremely
2. The Frdmy Method.6-One hundred g. of finely ground potassium permanganate
is added to 500 g. of conc. sulfuric acid in 150 cc. of water, cooled, carefully stirred, and
allowed to stand, when the permanganic acid formed decomposes, giving off oxygen. Permanganic anhydride, MnzOr, will sometimes separate as an oil. which is extremely
2. We are indebted to W. A. Patrick far
suggesting this method of preparation. Frkmy, Compt rend., 82, 1213 (1876). We are indebted to W. A. Patrick far
suggesting this method of preparation. Frkmy, Compt rend., 82, 1213 (1876). 1976 T. H. ROGERS, C. S. PIGGOT, W. H. BAHLKE AND J. M. JENNINGS. explosive. When all the permanganic acid is decomposed the manganese dioxide
is washed in a large quantity of water by decantation until free from sulfates. At the
final washing the precipitate approaches the colloidal condition and settles very slowly. The manganese dioxide still contains a small quantity of a potassium salt, which is
evidently very strongly adsorbed. The oxide mixtures prepared from this grade of
manganese dioxide were truly catalytic, and, when tested on dry gas, showed that a
layer only about 1 cm. deep was entirely efficient in oxidizing carbon monoxide. This
method of preparation of manganese dioxide was generally used in the work of develop-
ment of the optimum proportions of the constituents. 3. General Statement. Electrolytic Method.6-This
consists in the electrolytic oxidation of manganese
in an ammonium carbonate solution using a ferromanganese alloy as the anode. The
ammonium permanganate formed decomposes with the precipitation of finely divided
manganese dioxide. When a 2-component cell is used this decomposition takes place best
at temperatures slightly above 40'. When a 1-compartment cell is used oxidation and
reduction take place simultaneously, thus permitting lower temperatures and conse-
quently finer manganese dioxide. The material so produced was not uniform but the
best preparations were very active. 4. Reduction of Permanganic Acid by Oxalic Acid.-Permanganic
acid, prepared
from the calcium salt is diluted with a considerable quantity of water, well cooled and a
cold solution of oxalic acid is added in slight excess. As carbon dioxide is the by-product,
no washing is necessary. This manganese dioxide with silver oxide produced a catalytic
material of unlimited life. 4. Reduction of Permanganic Acid by Oxalic Acid.-Permanganic
acid, prepared
from the calcium salt is diluted with a considerable quantity of water, well cooled and a
cold solution of oxalic acid is added in slight excess. As carbon dioxide is the by-product,
no washing is necessary. This manganese dioxide with silver oxide produced a catalytic
material of unlimited life. 5. Decomposition of Ammonium Permanganate.-One
hundred g. of calcium
permanganate is dissolved in 500 cc. of water, and ammonium carbonate solution
containing a little ammonia is added. Calcium carbonate is filtered off, and the am-
monium permanganate, diluted to 4 liters, is reduced slowly in the cold with methyl
alcohol. This method gives very finely divided manganese dioxide and yields uniformly
active catalysts of unlimited life. It is probably superior to that prepared by Method
1, because ammonium salts are less strongly adsorbed than are potassium salts. 5. Decomposition of Ammonium Permanganate.-One
hundred g. of calcium
permanganate is dissolved in 500 cc. of water, and ammonium carbonate solution
containing a little ammonia is added. Calcium carbonate is filtered off, and the am-
monium permanganate, diluted to 4 liters, is reduced slowly in the cold with methyl
alcohol. This method gives very finely divided manganese dioxide and yields uniformly
active catalysts of unlimited life. It is probably superior to that prepared by Method
1, because ammonium salts are less strongly adsorbed than are potassium salts. 6. * This method was developed by B. F. Lovelace. General Statement. Air Oxidation of Manganese Oxide.-The
probable mechanism of catalytic
oxidation by manganese dioxide, niz., oxidation and reduction of the oxides of manganese,
indicated that a catalyst might be prepared from manganous hydroxide and its oxidation
effected before use, by drying in atmospheric oxygen. Manganous and silver oxides,
precipitated simultaneously from a solution of the nitrates, were dried in oxygen at
130'; a number of mixtures of moderate life were thus produced, but the product was
not of uniform activity. Results using copper oxide were less encouraging. Reparation of Catalytic Material.-The
oxide catalysts were prepared
by the following method, which was used quite generally throughout the
investigation whenever the solubilities of the salts used permitted. Man-
ganese dioxide paste of known water content (60-65%) was weighed and
suspended in a large volume of cold water, care being taken to secure
a uniform suspension; to this was added a standard silver nitrate solution
sufficient to give the desired amount of silver oxide in the final mixture. After thorough mixing silver hydroxide was precipitated by a slight excess
of sodium hydroxide solution, with constant stirring. After filtering and
washing free from sodium nitrate, the mixture was dried on a water-bath
and then at about 125" in air or, preferably, in dry oxygen. The drying
* This method was developed by B F Lovelace Reparation of Catalytic Material.-The
oxide catalysts were prepared
by the following method, which was used quite generally throughout the
investigation whenever the solubilities of the salts used permitted. Man-
ganese dioxide paste of known water content (60-65%) was weighed and
suspended in a large volume of cold water, care being taken to secure
a uniform suspension; to this was added a standard silver nitrate solution
sufficient to give the desired amount of silver oxide in the final mixture. After thorough mixing silver hydroxide was precipitated by a slight excess
of sodium hydroxide solution, with constant stirring. After filtering and
washing free from sodium nitrate, the mixture was dried on a water-bath
and then at about 125" in air or, preferably, in dry oxygen. The drying
* Thi
th d
d
l
d b
B F L
l CATALYTIC OXIDATION OF CARBON MONOXIDE. 1977 must be quite thorough, otherwise the mixture will not function catalyti-
cally. must be quite thorough, otherwise the mixture will not function catalyti-
cally. General Statement. The 3-component mixtures (silver oxide,
cupric oxide and manganese dioxide), described above, were developed with
a practical end in view, i. e., to reduce the cost of the catalyst. In this
connection the precipitation as carbonates was especially valuable and,
as stated above, the silver oxide could then be completely replaced by
cupric oxide when using manganese dioxide prepared by the Frdmy method. Special tests on the most active 2-component catalyst (62.5% of man-
ganese dioxide, 37.5% of silver oxide) are of interest. After a 10-hour
test on 1% carbon monoxide with 100% efficiency, the material was allowed
t o cool and a 0.3y0 carbon monoxide mixture run in at the same rate
(500 cc./min.). The warm area (about 0.5 cm. in length) immediately
reappeared at the same place and remained stationary for 10 hours longer,
no carbon monoxide at all being detected in the effluent gases at the end
of this time. The “pick up” of the catalyst was excellent; as the material
was found to function at even lower than room temperature, there was no
initial period of low efficiency while the catalyst was warming up. p
g
g
p
p
y
y
Special tests on the most active 2-component catalyst (62.5% of man-
ganese dioxide, 37.5% of silver oxide) are of interest. After a 10-hour
test on 1% carbon monoxide with 100% efficiency, the material was allowed
t o cool and a 0.3y0 carbon monoxide mixture run in at the same rate
(500 cc./min.). The warm area (about 0.5 cm. in length) immediately
reappeared at the same place and remained stationary for 10 hours longer,
no carbon monoxide at all being detected in the effluent gases at the end
of this time. The “pick up” of the catalyst was excellent; as the material
was found to function at even lower than room temperature, there was no
initial period of low efficiency while the catalyst was warming up. General Statement. Copper oxide cannot be incorporated with manganese dioxide by the
above method and a good catalyst obtained, but by using the method of
precipitating with a sodium carbonate solution as described below an
excellent catalyst may be made containing only manganese dioxide and
copper oxide. An important variation of the above method consisted in the precipita-
tion of silver (or copper) as carbonate, rather than the oxide. During
the subsequent drying the carbonate hydrolyzed almost completely, so
that what was essentially the oxide mixture was finally produced, similar to
the above but more active, especially in the case of mixtures containing
only copper oxide and manganese dioxide. When 3 component mixtures
were prepared (silver oxide, manganese dioxide and cupric oxide) the
nitrates of copper and silver were added to the suspension of manganese
dioxide. It has been stated that Methods 2, 4 and 5 produce the most suitable
manganese dioxide for a catalytic material when mixed with other oxides. The relative activities of the different samples were better shown when the
amount of silver oxide in the material was reduced (Table 111). The
most effective mixture prepared according to Method b contained 62.5%
of silver oxide, while with that made by Methods 2 , 4 and 5 the silver oxide
content was successfully reduced to 37.570, and to 28% when the silver
was precipitated as carbonate. The 3-component mixtures (silver oxide,
cupric oxide and manganese dioxide), described above, were developed with
a practical end in view, i. e., to reduce the cost of the catalyst. In this
connection the precipitation as carbonates was especially valuable and,
as stated above, the silver oxide could then be completely replaced by
cupric oxide when using manganese dioxide prepared by the Frdmy method. It has been stated that Methods 2, 4 and 5 produce the most suitable
manganese dioxide for a catalytic material when mixed with other oxides. The relative activities of the different samples were better shown when the
amount of silver oxide in the material was reduced (Table 111). The
most effective mixture prepared according to Method b contained 62.5%
of silver oxide, while with that made by Methods 2 , 4 and 5 the silver oxide
content was successfully reduced to 37.570, and to 28% when the silver
was precipitated as carbonate. Effect of Humidity. Inasmuch as these catalysts were to be used for military and naval
purposes under all weather conditions with varying temperatures 1978 T. H. ROGERS, C. S. PIGGOT, W. H. BAHLKE AND J. M. JENNINGS. and humidities, the effect of these factors was of considerable im-
portance. p
At low temperatures a poor catalyst was less active, but a good catalyst
would give 100% efficiency immediately, under varying concentrations
of carbon monoxide at all temperatures covered in any tests, -5.0"
being the lowest temperature used. The greater the concentration of
carbon monoxide (the rate of flow being the same) the more the catalyst
would automatically heat up. The most active catalysts were exposed
to both high (2% to 5%) and low (0.08% to 0.1%) concentrations of
carbon monoxide at temperatures ranging from +loo" to -5" without
any detectable loss of efficiency. y
y
As has been indicated
above, the deleterious effect of moisture was recognized early in the in-
vestigations while working out the best conditions of drying the first
catalysts produced. Samples which were not entirely dry possessed little
or no activity, while a good dry sample broke down when exposed to any
appreciable quantity of water vapor. However, a catalyst which had been
rendered inactive in this way could be "regenerated" by drying at 130'
in a stream of dry air or oxygen. Prom the military point of view this
effect of moisture was of great importance and efforts were made to combat
it. This effect of water vapor is a necessary result of the fine structure of
the catalyst, and is obviously due to condensation in the pores of the
material. With water vapor the case was quite different. The ordinary mixture is rather soft and very porous and has a low
apparent density. However, if this material be put in a press while still
moist and subjected to a high pressure (2819 kilograms per sq. cm.)
the apparent density is greatly increased and the resultant cake when
dried, is relatively hard and quite resistant to crumbling, which in
itself is of great importance in the military canister. A material so treated
is more resistant to moisture and will last longer than the same volume of
the unpressed material against a gas mixture of a given humidity. Effect of Humidity. Tests
were made on both pressed and unpressed catalysts at relative humidities
from 25y0 to lOOy& the pressed material in each case having a somewhat
longer life. CA'fALYTIC OXIDATION OE' CARBON MONOXID3& The catalyst before being tested was broken up and screened and that portion which
passed through the 10-mesh sieve and was retained by the 20-mesh was used. Con-
ditions in a canister are more favorable than those of the tube test, since in a layer of
broad cross sectional area there is less channeling of the gases and the loss of heat by
radiation is less. Samples, 500 cc. in volume, of the gases issuing from the test-tubes, were taken in
glass bottles by displacement of water. A sample was taken immediately after the
run was started and at regular intervals (usually 10 minutes) thereafter. These samples
were analyzed by the iodine pentoxide method, consisting of passing the gases over a
considerable quantity of pure iodine pentoxide at 160'. The gases from the sample
bottle were passed successively through standard barium hydroxide, over calcium chlo-
ride, iodine pentoxide, and finally through potassium iodide solution and then through
barium hydroxide. The barium hydroxide was titrated with standard oxalic acid,
and in this way the quantity of carbon monoxide oxidized by the catalyst and that oxi-
dized by the iodine pentoxide was determined, thus furnishing a check on the analysis. This method of analysis is quite accurate and, by using a large quantity of iodine pent-
oxide can be made with reasonable rapidity. Frequent analyses of the gases passing
effluent to the flow meters were made as a check on the flow meters. TABLE I. EFFECT
OF ADDITION
OF SODIUM
HYDROXIDE
T o MnOn-AgzO MIXTURES. . Rate: 500 cc./min. 1.0% CO. MnOz made by Method I a. TABLE I. EFFECT
SO TABLE I. EFFECT
OF ADDITION
OF SODIUM
HYDROXIDE
T o MnOn-AgzO MIXTURES. . Rate: 500 cc./min. 1.0% CO. MnOz made by Method I a. Percentage efficiency,
MnOl,
AgzO,
NaOH,
Time in minutes. %. %. %. %. %. %. %.. %. %. 27.8
69.4
2 . 8
I00
98
88
40
10
.. 27.0
67.6
5 . 4
100
100
92
38
22
.. 26.3
65.6
7.9
100
100
99
98
63
.. 25.6
64.1
10.3
100
100
100
100
88
.. 25.0
62.5
12.5
100
100
100
100
100
98
25.0
62.5
12.5
100
100
100
99.5
95.6
. . 25.0
62.5
12.5
100
100
98
90.1
88.5
. . TABLE 11. Rate:500 cc./cm.*/min. 1.0% CO. RELATION
OF DEGREE OF SUBDIVISION TO CATALYTIC LIFE. Methods of Testing and Analysis. The standard test prescribed by the Bureau of Mines was used throughout, except
in special cases. This consisted in passing the air-gas mixture at the rate of 500 cc. per
sq. cm. of cross section per minute through a layer of the catalyst 10 cm. deep. This
corresponds to a rate of 32 liters per minute for a canister of regulation size, and is about
twice the average rate of breathing. The standard concentration was 1.0%, regulated by mixing pure carbon monoxide
and air in the proper proportion through flow meters. For the catalyst, glass tubes
were used having 1 sq. cm. cross sectional area, the granular material to be tested being
poured in and the tube gently tapped to cause moderate settling. CA'fALYTIC OXIDATION OE' CARBON MONOXID3& 1979 CA'fALYTIC OXIDATION OE' CARBON MONOXID3& MnOz,
AgzO,
Type of
Catalytic life, yo efficiency. %. %. MnOz. 50
50
1 a
98%, end of one hour; loyo, end 2 hrs. 50
50
1 b
97%, end of one hour; SO%, end 2 hrs. 50
50
6
Limited life
55
45
6
loo%, end of 5 hrs; limited life
50
50
2
Catalytic; unlimited life
50
50
5
Catalytic; unlimited life
50
50
4
Catalytic; unlimited life
TABLE 111. MANGANESE
DIOXIDE
AND OTHER OXIDES. Type of
MnOz,
CUO,
Coloi,
MnOz. %. %. %. %. Remarks. 2
62.5
37.5
10.0
.. Unlimited; 7 hours
4
72.0
18.0
.. .. No activity
2
50.0
40.0
10.0
.. Unlimited life
2
50.. 0
30.0
20.0
.. Unlimited life
2
50.0
20.0
30.0
.. Unlimited life Rate:500 cc./cm.*/min. 1.0% CO. RELATION
OF DEGREE OF SUBDIVISION TO CATALYTIC LIFE. MnOz,
AgzO,
Type of
Catalytic life, yo efficiency. %. %. MnOz. 50
50
1 a
98%, end of one hour; loyo, end 2 hrs. 50
50
1 b
97%, end of one hour; SO%, end 2 hrs. 50
50
6
Limited life
55
45
6
loo%, end of 5 hrs; limited life
50
50
2
Catalytic; unlimited life
50
50
5
Catalytic; unlimited life
50
50
4
Catalytic; unlimited life
TABLE 111. MANGANESE
DIOXIDE
AND OTHER OXIDES. Type of
MnOz,
CUO,
Coloi,
MnOz. %. %. %. %. Remarks. 2
62.5
37.5
10.0
.. Unlimited; 7 hours
4
72.0
18.0
.. .. No activity
2
50.0
40.0
10.0
.. Unlimited life
2
50.. 0
30.0
20.0
.. Unlimited life
2
50.0
20.0
30.0
.. Unlimited life 1980 T. H. ROGERS, C. S. PIGGOT, W. H. BAHLKE AND J. M. JENNINGS. 1980 TABLE
I11 (continued). CUO,
COZOJ,
%. %. Remarks. Tgpeof
MnCh,
MnOn. %. 2
50.0
10.0
40.0
. . 1 a
20.0
40.0
. . 40.0
1 b
20.0
40.0
. . 40.0
2
50.0
.. .. 50.0
2
50.0
25.0
.. 25.0
6
72.0
18.0
.. . . 6
62.5
.. 37.5
. . 6
66.6
13.4
20.0
. . loo%, 6 hours
94%, 80 min. loo%, 80 min. loo%, 20 min. Unlimited life
Slight activity
No activity
loo%, 5 hours
TABLE IV. MANGANESE
DIOXIDE AND OXIDES PRECIPITATED
AS CARBONATES. CUO,
Remarks. Typeof
MnOn,
Agio,
MnOz. %. %. %. 2
83.2
5
77.3
5
70.1
2
70.0
2
60.8
2
80.4
2
71.4
2
54.8
2
68.4
16.8
* . 22.7
. . CA'fALYTIC OXIDATION OE' CARBON MONOXID3& 29.9
.. 30.0
.. .. 39.2
.. 19.6
.. 28.6
5.4
39.1
6 . 8
24.8
No activity
loo%, 4 hours; limited
Catalytic; unlimited
Catalytic; unlimited
loo%, 5 hours
95%, 8 hours
Catalytic
Catalytic
lOOo&, 8 hours; limited life
Discussion. The present investigation relates entirely to experiments which were
made for the purpose of affording protection against the poisonous action
of carbon monoxide. It was done under the stress of war conditions when
no opportunity was afforded to investigate the mechanism of the reactions
involved. The physical structure of the oxides in the catalysts described
is an essential factor in their activity; this matter is now being investigated
by a careful study of their adsorption isotherms. However, certain facts
were noted in the course of this work which appear to have relation to a
possible chemical explanation of the reaction. The fundamental assump-
tion of such an explanation is that the oxygen used for the oxidation of the
carbon monoxide comes immediately from the oxide catalyst, and that
in turn the catalyst is reoxidized by the oxygen of the air sufficiently
rapidly to maintain its oxygen content and high activity. As the catalysts
are mixtures of more than one oxide it is a question which of these plays the
principal r61e. Since both silver oxide and manganese dioxide as prepared
above will oxidize carbon monoxide at room temperature, either may be
considered as the initial cause of the reaction. But both of these oxides
when used alone suffer loss of oxygen, soon lose their activity and are not
catalytic in their action. Copper oxide does not oxidize carbon monoxide
at an appreciable rate at room temperatures. As the catalysts described
above all contain manganese dioxide as an essential constituent, and as the
physical condition of the manganese dioxide is of paramount importance it CATALYTIC OXIDATION OF CARBON MONOXIDE. 1981 is assumed on the basis of the above facts that the manganese dioxide is
the initial cause of the oxidation, probably functioning by reason of the
variable valence of the manganese. Manganese dioxide prepared by any
rnethod yielding relatively coarse particles has very slight activity, whereas
iE its particles are practically of colloidal dimensions it uniformly produces
a. satisfactory catalyst. Fine subdivision of the particles of the other
constituents and their intimate contact with the manganese dioxide
particles tend of course toward the production of the most reactive mate-
rial. CA'fALYTIC OXIDATION OE' CARBON MONOXID3& On the basis of this explanation of the reaction, the single oxides
sire not reoxidized by oxygen fast enough to maintain catalytic action ;
the function of the other oxide or oxides is to increase the velocity of re-
oxidation of the manganese dioxide, and for this purpose silver oxide
appears to be more efficient than copper oxide. In support of this view
is the fact that a material prepared by simultaneous precipitation of
manganous and silver hydroxides can be made very reactive by air oxida-
tion at 125”. Of course the alternative is to assume that silver or copper
oxide is the immediate cause of the action and that manganese dioxide
catalyses the reoxidation of the reduced oxides. In favor of this assump-
t ion is the fact that manganese dioxide is a good catalyst for the decompo-
sition of such metallic oxides as those of silver and mercury. The present
work furnishes no proof of either view, and until further observations on
the behavior of these oxide mixtures are available both views are tentative. Another fact related to the intermediate oxide theory is that a mixture
which is partially catalytic, as is the case when the catalyst is not suffi-
ciently active or when the catalyst is poisoned by water vapor, always
loses “available” oxygen. The first explanation of the poisonous effect
of water vapor would be that the loss of activity results from a diminution
of the large internal surface, so essential in these catalysts, which is in
part covered by liquid water. This, however, seems hardly to account for
all the facts, since under these conditions the catalyst suffers reduction,
therefore the carbon monoxide in the mixture has access to the catalyst. ’The loss of activity seems to result, therefore, primarily from the inability
of the catalyst surface to recombine with oxygen. It is possible that this
is due to a condensation of water within the pores of the catalyst which
thereby separates the oxide particles and thus prevents the intimate
contact of the oxides which is essential for the catalytic activity of the
material. Some of these points are under investigation, and it is realized
that a further discussion is premature until these results, especially those
bearing on the adsorption of these catalysts, are available. *An historical review of the investigations connected with the problem of the
carbon monoxide mask giving due credit to the various persons concerned has been
published by A. B. Lamb, W. C. Bray and J. C. W. Frazer in the J . Ind. Eng. Chem.,
12, 213 (1920). CA'fALYTIC OXIDATION OE' CARBON MONOXID3& The use of these oxides as catalysts is naturally not limited to carbon
monoxide, but may be applied to a number of other substances; further
work along these lines is in progress, including the oxidation of ammonia. The range of applicability is quite wide, especially in the field of organic
oxidations, depending of course, on the possibility of carrying out the DAVID R. MERRILL AND CHARLES C. SCALIONG. 1982 reaction in the vapor phase. It has been found that the action is too
energetic in some cases, but the possible variations in physical properties
and in constituents present many possibilities, as is illustrated by the
varying degrees of activity toward carbon monoxide observed in the various
mixtures during the present investigation. Thus the degree of catalytic
activity may be adapted and controlled to suit the conditions of each
oxidation reaction. 1 Published with the permission of General Amos A. Fries, Chief of the Chemical
Warfare Service. Summary. 1. The decomposition temperature of silver oxide when simulteneously
precipitated with calcium hydroxide is considerably lower than that of
silver oxide alone. 2. A number of catalysts have been prepared which cause rapid and
complete oxidation] at ordinary temperatures, of carbon monoxide in
any concentration at which sufficient oxygen is present. 2. A number of catalysts have been prepared which cause rapid and
complete oxidation] at ordinary temperatures, of carbon monoxide in
any concentration at which sufficient oxygen is present. 3. The essential constituent or this class of catalysts is specially prepared
manganese dioxide, upon which is precipitated the oxide of silver or copper
or both. 4. The silver or copper is best precipitated as the carbonate, and sub-
sequently hydrolyzed to the oxide. 4. The silver or copper is best precipitated as the carbonate, and sub-
sequently hydrolyzed to the oxide. q
y y
y
5. The presence of water vapor limits the life of these catalysts. 6. The application of this class of catalysts to a number of oxidation
reactions is suggested. 6. The application of this class of catalysts to a number of oxidation
reactions is suggested. In conclusion the authors desire to pay full tribute to Professor Frazer,
to whom principally the success of this investigation is due, whose resource
and pertinacity were a source of inspiration to those associated with him. BALTIMORE,
MARYLAND [CONTRIBUTION
FROM THE CHEMICAL
WARFARE SERVICE.']
THE CATALYTIC OXIDATION OF CARBON MONOXIDE AT
BY DAVID R. MERRILL
AND CHARLES
C. SCALIONE.
ORDINARY TEMPERATURES.
Received March 2, 1921. The experimental work upon which this paper is based was done by the
authors and their essociates largely for the purpose of developing a suitable
absorbent for carbon monoxide for use in gas masks,2 and, because of the
importance of obtaining production on a large scale without delay, it
was not possible to investigate the scientific aspects of the problem as
thoroughly as could be desired. An endeavor has been made in the prepa-
ration of the present paper to choose, from the large mass of data obtained] 1 Published with the permission of General Amos A. Fries, Chief of the Chemical
Warfare Service.
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https://openalex.org/W1968952516
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https://europepmc.org/articles/pmc3753320?pdf=render
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English
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Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently
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PloS one
| 2,013
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cc-by
| 7,879
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Abstract Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary
functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and
affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore,
regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been
demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically
processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we
report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal
truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2;
that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the
truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that
co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of
cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-
dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration,
and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or
availability during bone growth and development. The similarities and differences of the interactions between Spp24
proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with
respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal
disorders. i C-S, Zhao K-W, Brochmann EJ, et al. (2013) Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone
LoS ONE 8(8): e72645. doi:10.1371/journal.pone.0072645 Editor: Arthur Veis, Northwestern University, United States of America Editor: Arthur Veis, Northwestern University, United States of America Copyright: © 2013 Tian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by the Department of Veterans Affairs Biomedical Laboratory Research (http://www.research.va.gov/services/blrd/
#.UW8XKqV9FFg) and Development and Rehabilitation Research and Development Services (http://www.research.va.gov/services/
rrd.cfm#.UW8XV6V9FFg) (1I01BX000511 and 1I0RX000383). The funders had no role in study design, data collection and analysis, decision to publish, o
preparation of the manuscript. Competing interests: Dr. Jeffrey C. Wang or one of his immediate family own stock or stock options in the following pharmaceutical, biomaterial or
orthopaedic device or equipment company, or suppliers: Fziomed, Promethean spine, Paradigm spine, Benevenue, NexGen, Pioneer, Amedica, Vertiflex,
Electrocore, Surgitech, Axiomed, VG Innovations, Corespine, Expanding orthopaedics, Syndicom, Osprey, Bone biologics, Curative biosciences,
Pearldiver. Dr. Jeffrey C. Wang or a member of his immediate family receive royalties for the following pharmaceutical, biomaterialor orthopaedic product
or devices: Biomet, Stryker, Medtronics, Depuy-Synthes, Amedica, Osprey, Aesculap, Seaspine. Dr. Michael D. Daubs receive royalties and is a paid
consultant for the orthopaedic product or device: Dupuy-Synthes. Dr. Michael D. Daubs receives research or institutional support as a principal investigator
from the orthopaedic device company Stryker. The disclosed competing interests do not alter the authors' adherence to all the PLOS ONE policies on
sharing data and materials. * E-mail: haijuntianmd@gmail.com Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect
TGF-β Induced Bone Formation Differently 1 Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, P. R. China, 2 Department of Ophthalmology, the
309th Hospital of PLA, Beijing, P. R. China, 3 Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, P. R. China,
4 Research Service, Education and Clinical Center, VA Greater Los Angeles Healthcare System, North Hills, California, United States of America, 5 Geriatric
Research, Education and Clinical Center, VA Greater Los Angeles Healthcare System, North Hills, California, United States of America, 6 Department of
Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 7 Department of Physiology, University of California Los
Angeles, Los Angeles, California, United States of America, 8 Department of Orthopaedic Surgery, University of California Los Angeles, Los Angeles, California,
United States of America, 9 Department of Orthopaedic Surgery, the Methodist Hospital System, Houston, Texas, United States of America In vivo bioassay All research involving animals was reviewed and approved
by the VA Greater Los Angeles Healthcare System Institution
Animal Care and Use Committee (IACUC) prior to the initiation
of any experiments. Research on living, non-human subjects at
this institution is in compliance with the guiding principles in the
Guide for the Care and Use of Laboratory Animals (Eighth
Edition). Four-day-old Long Evans rats were divided into four
groups and received 14 daily injections of test materials. Group
I received 10 μl injections of TGF-β2 50 ng; Group II received
10 μl injections of TGF-β2 50 ng and Spp24; Group III received
10 μl injections of TGF-β2 50 ng and Spp14.5; Group IV
received 10 μl PBS only. Proteins were dissolved in PBS, pH
7.4. The quantities of Spp24 and Spp14.5 were devised to be
equimolar at 11.5 μM (the highest common solubility that could
be achieved for the two proteins) and to provide a molar ratio of
about 29 fold molar excess with respect to TGF-β2. We hypothesized that TGF-β would bind to Spp24 and its
derivatives in a manner similar to that in which Spp24 binds to
BMP-2 and that Spp24 and its proteolytic derivatives would
inhibit TGF-β activity to different degrees. In the present study,
we have tested that hypothesis and confirmed that Spp24 and
its C-terminal truncation products bind TGF-βs and modulate
their bioactivity. Therefore, it is likely that the bone matrix
protein Spp24, which influences the activity of both BMPs and
TGF-β, plays a significant role in the overall control of the BMP/
TGF-β economy of the bone environment and that proteolysis
of Spp24 is one mechanism through which more refined levels
of control are imparted to this mechanism. Injections were performed following a modification of the
protocol of Joyce, et al. [17]. A dull 27-gauge needle was used
for the subperiosteal injection technique. Microinjections were
directed into the subperiosteal region of the anterior-superior
surface of the rat femur. This technique was perfected by
injecting dye until injections could be consistently reproduced. All the rats were injected on the left femurs only for 14
consecutive days, and right femurs were left intact to serve as
their own control to quantify new bone formation. Femurs were
harvested on the 15th day after receiving 14 injections. An
incision was made on the superior skin along the femur and the
hip joint, and the knee joint were exposed. In vivo bioassay All ligaments were
dissected away so that the femoral head could be taken out of
the acetabulum and then the femur was also separated from
the tibia and fibula. Great caution was taken when removing
the femur to preserve the intact bone. Some muscle tissue
attached to the femur was left in place because of the
possibility of bone formation extending into the muscle. Surface Plasmon Resonance Surface plasmon resonance analyses of protein interactions
were performed on a Biacore T-100 instrument (G.E. Healthcare, Piscataway, NJ). CM5 chips, HBS-EP running
buffer and amine coupling reagents were obtained from the
manufacturer. BMP and TGF-β were immobilized as the
ligands whereas the four Spp24 proteins were employed as the
analytes. In order to obtain precise measurements of the
concentration of the analytes each Spp24 protein was
dissolved in water at a concentration of 1 mg/ml, mixed
thoroughly, centrifuged at 12,000 x g for one minute, and then
decanted. Concentrations were determined using custom
coefficients obtained from the ProtParam tool of ExPASy
(web.expasy.org; Swiss Institute of Bioinformatics, Lausanne,
CH). In calculating the coefficients, it was assumed that one
half of the cysteine residues were oxidized. The coefficients (in
the form of X units of absorption at 280 nmeter = 1g/L) where:
X= 1.267 for Spp24, X= 1.127 for Spp18.1, X= 1.161 for
Spp16.0, and X= 1.189 for Spp14.5. Five concentration of each
analyte were tested with each ligand. Kinetic constants were
calculated using software supplied by the manufacturer. One matrix protein that has binding affinity for several
members of the TGF-β family of proteins is secreted
phosphoprotein 24 kD (Spp24) [10]. This liver-derived bone
matrix protein is exquisitely labile to proteolysis [12] and
circulates in a protective complex with α-macroglobulins and
anti-thrombin III (Serpin C1) [13]. The protein exists in the bone
environment in several forms ranging in size from 14 kD to 24
kD [14] with a dominant isoform of about 18.5 kD [15]. Degradation of recombinant Spp24 gives rise to well defined
proteins of 18.1 kD, 16.0 kD, and 14.5 kD [12]. These
degradation products retain the N-terminus of the mature
parental protein and have a truncated C-terminus of various
lengths. When co-implanted with BMP-2 in different models of
BMP-induced bone formation, Spp24 and its derivatives inhibit
bone formation. Significantly, the full-length molecule is more
inhibitory than the truncated forms [16]. Introduction [1]. Among these growth factors, bone morphogenetic proteins
(BMPs), especially BMP-2 and -7, and transforming growth
factor-β (TGF-β), are the most significant. These regulatory
molecules have complementary but also opposing activities. In Mature, mineralized bone contains a number of growth
factors that are essential for proper bone remodeling and repair 1 PLOS ONE | www.plosone.org August 2013 | Volume 8 | Issue 8 | e72645 1 Spp24 and Spp14 Affect TGF-β Differently general, TGF-β enhances preosteoblast proliferation and
extracellular matrix synthesis but also opposes the BMP effect
on osteoblast differentiation [2,3]. On the other hand,
recombinant BMPs such as BMP-2 and -7 enhance osteoblast
differentiation [4] but are also capable of inducing the entire
recapitulation of endochondral bone formation as originally
described by Urist [5]. This process involves stem cell
proliferation, chondrogenic differentiation, and replacement of
cartilage by bone. TGF-β and BMPs (BMP-2 and -7) are
present in bone matrix in similar concentrations [6,7]. Bone
also contains a number of extracellular proteins that bind to
members of the TGF-β superfamily of cytokines and regulate
their activity [8–10]. TGF-β has a family of associated proteins
that maintain the active molecule in the matrix in a latent form
[11]. No such group of proteins has yet been described in
relation to the BMPs. Micro-computerized tomography (μCT) Rat femurs were analyzed by high resolution micro-
computed tomography (µCT), using a µCT imaging system
(µCT40, Scanco Medical AG, Brüttisellen, Switzerland) with a
resolution of 16µm and an X-ray energy of 55kVp and 160mA,
calibrated against a hydroxyapatite (HA) phantom. Five
hundred projections were acquired per 180-degree rotation
with an integration time of 300 ms. A threshold of 122 was
used to discriminate bone and soft tissue. The entire femur was
included for every scan. Left femurs were analyzed for bone
formation and the right femur from the same animal subject
was scanned and used as a control. New bone formation
volume (BV) was calculated by subtracting the volume of the
right femur from the volume of the left femur. A 2D contouring
algorithm was used to identify the new bone formation area in
the rat femurs in TGF-β group and TGF-β + Spp14 group, and
relative bone volume (BV/TV) was calculated using the same
software. The BV/TV value was not calculated in the PBS and
TGF-β + Spp24 group because there were not enough new
bone and it was difficult to identify the new bone formation
area. q
p
β
Having established that Spp24 and its derivatives bind TGF-
β1 and -β2 with high affinity, we then assessed the biological
effects of Spp24 and Spp14.5 on TGF-β2-induced bone
formation in newborn rats. All of the rats grew well after the
injections and no nerve injury, vascular injury, or other
complications were observed. Radiographs of representative
subjects taken after completion of the injections are shown in
Figure 2. Visual inspection of X-rays confirmed that all of the
limbs that received an injection of TGF-β2 alone demonstrated
obvious new bone formation whereas none of the limbs in the
PBS only control group showed any new bone formation
(Figure 2, Panel A). These results are consistent with those
reported by Joyce, et al. [17]. The area of the mass of new
bone formation apparent on the radiographs was measured
(Figure 2, Panel B). The new bone formation in the PBS alone
group was very low, while significant bone formation as seen in
the TGF-β2 group. The bone forming activity of TGF-β2 was
inhibited by Spp24 as manifest by a mass area about half of
that of TGF-β2 alone. Materials Recombinant human BMP-2, human TGF-β1, and human
TGF-β2 were purchased from R & D Systems (Minneapolis,
MN). Recombinant Spp24 proteins were produced in a
bacterial expression system and purified by IMAC (immobilized
metal affinity chromatography) chromatography using a
BioLogic chromatography workstation (BioRad, Hercules, CA)
as described in detail previously [12]. August 2013 | Volume 8 | Issue 8 | e72645 PLOS ONE | www.plosone.org 2 Spp24 and Spp14 Affect TGF-β Differently Micro-computerized tomography (μCT) Interestingly, Spp14.5 did not show the
same inhibitory effect as did Spp24, but rather was associated
with a modest increase in bone formation above TGF-β2-
treated levels that was not statistically significant. Analyses of
the images from μCT confirmed the results obtained from the
X-rays (Figure 2, Panels C and D). Average new bone volume
and BV/TV for each of the groups was shown in Table 2. The
relative bone volumes of the TGF-β2 group and TGF-β2 +
Spp14.5 groups were also measured (Figure 2, Panels E and
F), showing that TGF-β2 + Spp14.5 group had a higher BV/TV
value. Figure 3 shows histological sections from specimens
representing each group. This analysis confirmed that the
masses on the femurs from animal subjects from the treatment
groups were composed of bone and cartilage. Only minimal
trauma was visible in the PBS treatment group. A paucity of
cartilage was observed in specimens from the TGF-β2 plus
Spp14.5 group, which was confirmed in histologic sections
stained for cartilage with Safarin oil red O and for sulfated
proteoglycans synthesized by functional chondrocytes with
Alcian blue (Figure 4, Panels A and B). This explains why the Numerical analysis The area of the mass of new bone formation apparent on the
radiographs was measured for each subject. Comparisons of
means of groups by either ANOVA or Student’s t-test were
conducted using IBM SPSS Statistics (version 20, IBM,
Armonk, NY). Histology After imaging was completed, the specimens were fixed in
10% formalin, decalcified, washed with tap water, and then
transferred to 70% ethanol. Serial longitudinal sections were
carefully cut at the site of new bone formation. Specimens were
embedded in paraffin, sectioned, deparaffinized in xylene,
hydrated in graduated ethanols, and stained with hematoxylin
and eosin (H&E), Safranin O and Alcian Blue. Safranin O
sections were stained in 0.2% Fast Green (Sigma, St Louis,
MO, USA), for 5 min, pretreated with 1% acetic acid and then
stained in 0.1% Safranin O stain for 1 hour and then
counterstained with hematoxylin. Alcian Blue sections were
stained with 1% Alcian Blue at pH 2.5 for 30 minutes,
thoroughly rinsed with tap water, and then counterstained with
nuclear fast red for 5 min. Radiographic analyses plasmon resonance (Figure 1, Table 1), and the results are
compared to those obtained with recombinant human BMP-2,
which were previously published [15,18]. Both TGF-β1 and -β2
bound each of the four Spp24 proteins (FL-Spp24, Spp18.1,
Spp16, and Spp14.5). Kinetic analysis demonstrated that FL-
Spp24 had the greatest affinity in both cases, and the affinity of
binding by C-terminally truncated Spp24 derivatives was one to
two orders of magnitude lower than that of FL-Spp24. Qualitatively similar results were obtained for the binding to
BMP-2, although the affinity for BMP was about an order of
magnitude higher than the affinity for TGF-βs [18]. Thus, Spp24
and its C-terminal truncation products bind human TGF-β1 and
–β2 with lower affinity than they bind recombinant human
BMP-2, and C-terminal truncation of Spp24 reduces its binding
affinity. Given the relatively equivalent concentrations of TGF-
βs and BMPs in bone [6,7], the kinetics of binding suggest that
Spp24 and its degradation products would bind BMPs at lower
concentrations than TGF-βs, thus achieving a relative
sequestration of BMPs when compared to free TGF-βs. X-rays were taken using a Faxitron small specimen X-ray
cabinet (Faxitron; Tucson, AZ) with an operating voltage of 35
KV and an exposure time 12.5 s. The area of the mass of new
bone formation apparent on the radiographs was measured for
each subject using ImageJ software (National Institutes of
Health; http://rsb.info.nih.gov/ij/). Results The kinetics of the binding of FL-Spp24 and its degradation
products to TGF-β1 and -β2 were determined by surface PLOS ONE | www.plosone.org August 2013 | Volume 8 | Issue 8 | e72645 3 Spp24 and Spp14 Affect TGF-β Differently Figure 1. Sensogram from the surface plasmon resonance analysis of the interaction between TGF-β1, β2 and different
Spps. The response (RU or response units) is plotted on the Y-axis, while time (seconds) is plotted on the X-axis. doi: 10.1371/journal.pone.0072645.g001 Figure 1. Sensogram from the surface plasmon resonance analysis of the interaction between TGF-β1, β2 and different
Spps. The response (RU or response units) is plotted on the Y-axis, while time (seconds) is plotted on the X-axis. doi: 10.1371/journal.pone.0072645.g001 terminal truncation of Spp24 reduces its binding affinity. TGF-β
induced bone formation was inhibited by FL-Spp24, but
enhanced by Spp14.5. We also previously reported that FL-
Spp24 inhibits BMP-2 and -7-stimulated bone formation, and
C-terminal truncation reduces its inhibitory effects [19,20]. Table 1. Kinetic parameters as determined by surface
plasmon resonance for the interactions of TGF-β1, TGF-β2,
and BMP-2 versus each of the four Spp24 isoforms. spp14.5
spp16.0
spp18.1
spp24
TGF-β1
ka (M-1 s-1×103)
4.18
5.55
10.00
71.9
kd (s-1×10-3)
5.89
12.2
10.6
3.93
KD (kd//ka) (μM)
1.41
3.25
1.39
0.065
TGF-β2
ka (M-1 s-1×103)
2.39
1.34
3.64
32.4
kd (s-1×10-3)
5.39
5.66
10.9
5.86
KD (kd//ka) (μM)
3.22
4.24
3.18
0.194
BMP-2
ka (M-1 s-1×103)
15.3
63.6
263
311
kd (s-1×10-3)
3.60
2.61
1.26
5.50
KD (kd//ka) (μM)
0.236
0.041
0.005
0.018
ka represents “recognition”, kd represent “stability”, and KD stand for the actual
“affinity” of the proteins. The values pertaining to BMP-2 are from reference 16 and
are shown for purposes of comparison. Given that Spp24 and its C-terminal truncation products bind
BMP/TGF-β cytokines with different affinities, does this account
for the differences in their biological effects? The proposed mechanism for the interaction between BMP-2
and Spp24 is the presence of a domain in the middle of the
Spp24 molecule (specifically amino acids 110 through 128 in
the 203 amino acid bovine protein) [10] that shares some
sequence similarity with the TGF-β type II receptor which
would, presumably, bind to the same aspect of the BMP-2
molecule that is involved in physiological receptor binding. This, however, is far from certain. Demetriou, et al. Results [21] first
pointed out the similarity of a region (the TRH1 or TGF-β
receptor II homology-1 domain) in fetuin, which, like Spp24,
contains a cystatin domain within which the areas with
similarity to the TGF-β type II receptor are found. Cystatin
domains are named for their similarity to the cysteine protease
inhibitor, cystatin, but the domains in fetuin and Spp24 do not
have any inhibition of proteolytic activity. Behnam, et al. [15]
located in bovine Spp24 a region that is somewhat similar to
the TRH1 domain in fetuin (Figure 5). The domains in both
fetuin and Spp24 are 19 amino acids long and are bounded by
a loop-forming disulfide bond between cysteine residues. The
degree of similarity between the TRH1 domains in fetuin or
Spp24 and the human TGF-β receptor type II (and related
receptors) is, in actuality, quite small (Figure 5). The hypothesis
of Demetriou, et al. [21] that the TRH1 domain was the binding a
p
g
d
p
y
D
“affinity” of the proteins. The values pertaining to BMP-2 are from reference 16 and
are shown for purposes of comparison. TGF-β2 plus Spp14.5 group has a higher BV/TV volume than
TGF-β2 group. TGF-β2 plus Spp14.5 group has a higher BV/TV volume than
TGF-β2 group. Discussion We hypothesized that Spp24 is an important regulator of the
net balance of available free forms of the BMPs and TGF-β in
skeletal tissue throughout the lifespan and initiated studies to
define the physical interactions of Spp24 with BMP/TGF-β
cytokines. We observed that Spp24 and its C-terminal
truncation products bind human TGF-β1 and –β2 with lower
affinity than they bind recombinant human BMP-2, and C- August 2013 | Volume 8 | Issue 8 | e72645 PLOS ONE | www.plosone.org 4 Spp24 and Spp14 Affect TGF-β Differently Figure 2. Radiographs of femurs of newborn rats treated for 14 consecutive days with TGF-β2 alone or with Spp24 or with
Spp14.5. Representatives of treatment groups as follows: A.X-ray images, with both various treated left femurs and untreated right
femurs; B. Bone area derived from radiographic analysis of femurs of newborn rats with different treatment (**: P<0.01); C. CT
images of treated femurs and untreated control; D. Bone volume of newly formed bone derived from CT images (**: P<0.01); E. 2D
CT image comparing new bone quality of TGF-β2 group and TGF-β2 + Spp14.5 group; F. BV/TV value from the new bone formation
area of TGF-β2 group and TGF-β2 + Spp14.5 group. doi: 10.1371/journal.pone.0072645.g002 Figure 2. Radiographs of femurs of newborn rats treated for 14 consecutive days with TGF-β2 alone or with Spp24 or with
Spp14.5. Representatives of treatment groups as follows: A.X-ray images, with both various treated left femurs and untreated right
femurs; B. Bone area derived from radiographic analysis of femurs of newborn rats with different treatment (**: P<0.01); C. CT
images of treated femurs and untreated control; D. Bone volume of newly formed bone derived from CT images (**: P<0.01); E. 2D
CT image comparing new bone quality of TGF-β2 group and TGF-β2 + Spp14.5 group; F. BV/TV value from the new bone formation
area of TGF-β2 group and TGF-β2 + Spp14.5 group. site of TGF-β and BMP-2 to fetuin was based on sequence
similarities to the TGF-β receptor type II and made no
reference to functional studies such as epitope mapping which
were, in fact, not available at the time. Subsequently, Guimond,
et al. [22,23] produced an extensive series of point and deletion
mutations in the corresponding region of the TGF-β type II
receptor and reported that they had no effect on binding of
TGF-β to the receptor. Similarly, Sun, et al. Discussion [24] reported that a
number of the amino acids of the TRH1 domain of Spp24
(including the two terminal cysteines one at a time) could be
substituted with alanine with minimal impact on the binding of
the protein to BMP-2. Demetriou, et al. [21] concluded that
domains similar to the TRH1 domain existed in both the TGF-β
type II receptor and the BMP type II receptor but not in any of
the type I receptors. They did not explicitly delineate, however,
the sequence in the BMP type II receptor that they felt was
similar to the TRH1 domain. Nevertheless, the likelihood that
these regions of fetuin and Spp24 participate in growth factor
binding is strongly suggested by surface plasmon resonance
binding studies with synthetic peptides the sequence of which
were based on the TRH1 domains of fetuin and Spp24. Demetriou, et al. [21] tested the interaction of a peptide based
on the TRH1 domain of fetuin (CDIHVLKQDGQFSVLFTKCD)
and reported that it bound to rhBMP-2 with a modest KD of 2.4 Table 2. New bone volume (BV) and relative bone volume
(BV/TV) of different groups. x 10-6 M but did not bind to TGF-β1 (“no binding” defined as KD
>1 x 10-5 M). On the other hand, a peptide based on the
corresponding region within the hTGF-β receptor type II
(CVAVWRKNDENITLETVCHD) bound TGF-β1 with a modest
KD of 1 x 10-6 M but did not bind rhBMP-2 (“no binding” defined
as KD > 1 x 10-5 M) [21]. Similarly, Behnam, et al. [15] tested
the interaction between a peptide based on the TRH1 domain
of bovine Spp24 (CRSTVRMSAEQVQNVWVRC) and rhBMP-2
and reported a modest affinity as reflected in a KD of 3 x 10-5 M. Note that this value for the KD is higher, which is to say, reflects
a lower affinity, than the limit for “no binding” employed by
Demetriou, et al. [21]. However, the same group subsequently
reported substantially higher affinities for this peptide and a
Table 2. New bone volume (BV) and relative bone volume
(BV/TV) of different groups. BV (mm3)
BV/TV
PBS
-0.263 ± 1.801
/
TGF-β2
9.370 ± 0.679
0.293 ± 0.015
TGF-β2+Spp24
2.784 ± 1.584
/
TGF-β2+Spp14
9.944 ± 0.376
0.355 ± 0.017 x 10-6 M but did not bind to TGF-β1 (“no binding” defined as KD
>1 x 10-5 M). Discussion On the other hand, a peptide based on the
corresponding region within the hTGF-β receptor type II
(CVAVWRKNDENITLETVCHD) bound TGF-β1 with a modest
KD of 1 x 10-6 M but did not bind rhBMP-2 (“no binding” defined
as KD > 1 x 10-5 M) [21]. Similarly, Behnam, et al. [15] tested
the interaction between a peptide based on the TRH1 domain
of bovine Spp24 (CRSTVRMSAEQVQNVWVRC) and rhBMP-2
and reported a modest affinity as reflected in a KD of 3 x 10-5 M. Note that this value for the KD is higher, which is to say, reflects
a lower affinity, than the limit for “no binding” employed by
Demetriou, et al. [21]. However, the same group subsequently
reported substantially higher affinities for this peptide and a x 10-6 M but did not bind to TGF-β1 (“no binding” defined as KD
>1 x 10-5 M). On the other hand, a peptide based on the
corresponding region within the hTGF-β receptor type II
(CVAVWRKNDENITLETVCHD) bound TGF-β1 with a modest
KD of 1 x 10-6 M but did not bind rhBMP-2 (“no binding” defined
as KD > 1 x 10-5 M) [21]. Similarly, Behnam, et al. [15] tested
the interaction between a peptide based on the TRH1 domain
of bovine Spp24 (CRSTVRMSAEQVQNVWVRC) and rhBMP-2
and reported a modest affinity as reflected in a KD of 3 x 10-5 M. Note that this value for the KD is higher, which is to say, reflects
a lower affinity, than the limit for “no binding” employed by
Demetriou, et al. [21]. However, the same group subsequently
reported substantially higher affinities for this peptide and a PLOS ONE | www.plosone.org 5 August 2013 | Volume 8 | Issue 8 | e72645 Spp24 and Spp14 Affect TGF-β Differently Figure 3. Histologic examination of representative samples from each experimental groups. Stained with H&E. doi: 10.1371/journal.pone.0072645.g003 Figure 3. Histologic examination of representative samples from each experimental groups. Stained with H&E. doi: 10 1371/journal pone 0072645 g003 Figure 3. Histologic examination of representative samples from each experimental groups. Stained with H&E. doi: 10.1371/journal.pone.0072645.g003 comparisons with the “blast” tool (www.NCBI.nlm.nih.gov), the
following pair-wise sequence comparisons (Figure 5) gave
results of “no significant similarity”: bovine fetuin TRH1 vs. hTGF-β R type II; bovine Spp24 TRH1 vs. hTGF-β R type II;
human Spp24 TRH1 vs. hTGF-β R type II; human Spp24 TRH1
vs. human BMP R type II.) The negative conclusion of
Demetriou, et al. Discussion Histological examination of specimens from each treatment groups stained with Alcian Blue or Safranin O. Note
that Spp24 and Spp14.5 decrease the amount of cartilage-specific staining in TGF-β2-treated bone. doi: 10.1371/journal.pone.0072645.g004 were co-implanted with 5 μg of rhBMP-2 in the assay, the
different forms imparted different degrees of inhibition [16]. At
the highest dose of Spp (2.5 mg), Spp24 completely inhibited
BMP-2 induced bone formation, whereas Spp18.1 and Spp16.0
inhibited bone formation by about 30% and Spp14.5 inhibited
bone formation by about 70% [16]. In a rat model of BMP-2
enhanced spine fusion, Spp24 reduced “bone area” by a much
greater degree than did “spp18.5” (Spp18.1) [20]. In that study
the “manual palpation score” (0 to 7) was 7.0 for 10 μg of
BMP-2 alone, 6.0 for BMP-2 plus spp18.5, and 1.5 for BMP-2
plus full-length Spp24 [20]. Therefore, it appeared that Spp24
is a more potent inhibitor of BMP-2 activity than is Spp18.1 optimal binding either through allosteric changes of the binding
site or through provision of a second binding site. As discussed
in detail above, it should be emphasized that the exact nature
of the TGF/BMP binding site of Spp24 has not been defined by
physico-chemical studies, and the binding of growth factors to
aspects of the Spp24 molecule other than the TRH1 domain,
most notably the C-terminus, has not been tested. The affinity
of TGF-β for full-length Spp24 is less than the affinity of BMP-2
for full-length Spp24 but, the values are not greatly different. On the other hand, the affinity of TGF-β for each of the three
smaller size forms of Spp24 is significantly less than that of
BMP-2 (Table 1). is a more potent inhibitor of BMP-2 activity than is Spp18.1
In vivo, TGF-β2 induced bone formation when it was injected
into the periosteum of newborn rats as reported previously by
other investigators [17]. At the concentrations that we
employed, roughly a 30-fold molar excess, Spp24 inhibited the
TGF-β2 induced bone formation but Spp14.5 did not. These
results are similar to our previously reported findings with
respect to BMP-2 in which we demonstrated that full-length
Spp24 more strenuously inhibited the activity of BMP than did
the smaller forms of Spp24 [16,20]. In general, we propose that
Spp24 binds to TGF-β2 and inhibits its activity through a
mechanism of extracellular sequestration. Discussion [21] notwithstanding, it is not unreasonable,
therefore, to examine the BMP receptor type I for a region
similar to the TRH1 domain of Spp24. And, in fact, a domain
does exit in the putative binding region of the BMP receptor
type Ia and type Ib molecules [27,28] that does resemble,
somewhat, the TRH1 domain of Spp24. At this time it can only
be concluded that the exact structural nature of the interaction
between Spp24 and members of the TGF-β family has not
been precisely defined but that the TRH1 domain participates
in some manner. number of growth factors from the TGF-β family [18]. The
differences in reported values were attributed to greater
accuracy in determining the concentration of the peptides,
which are very poorly soluble in aqueous solution. Precise
measurements of concentration are critical for the kinetic
calculations associated with SPR analyses. Specifically, the KD
values for the interaction between the peptide based on the
TRH1 domain of Spp24 (called BMP Binding Peptide or BBP)
and the growth factors were: rhBMP-2(KD = 53.3 nM); rhOP-1
(also called BMP-7) (KD = 11.6 nM); rhTGF-β (KD = 68 nM); and
rmGDF-5 (recombinant murine growth and differentiation
factor-5) (KD = 777 nM) [18]. The situation is made more complex by differences between
the TGF-β receptors and the BMP receptors. While TGF-β
binds with high affinity to the type II receptor and not at all to
the isolated type I receptor, BMP binds with high affinity to the
type I receptor [25] though this high affinity binding may require
the simultaneous presence of the type II receptor [26]. The
degrees of similarity between the TRH1 domains of fetuin or
Spp24 and TGF-β or BMP-2 are relatively small. (In fact, in Our studies have shown that TGF-β binds to all four of the
Spp24 forms. The pattern is, as in the case of BMP-2, that the
full-length form is bound with significantly greater affinity. This
suggests that the C-terminus of the molecule is required for PLOS ONE | www.plosone.org 6 August 2013 | Volume 8 | Issue 8 | e72645 Spp24 and Spp14 Affect TGF-β Differently Figure 4. Histological examination of specimens from each treatment groups stained with Alcian Blue or Safranin O. Note
that Spp24 and Spp14.5 decrease the amount of cartilage-specific staining in TGF-β2-treated bone. doi: 10.1371/journal.pone.0072645.g004 Figure 4. Discussion Spaces between residues were inserted by the authors (or from
references) to achieve alignments are indicated with an “*”. a Human TGF-β receptor type II P37173 Sequence selection spacing alignment and structure assignment from Demetriou et al Figure 5. Sequence and secondary structure comparisons of various receptor and pseudoreceptor molecules proposed to
give information pertaining to the site of interaction of Spp24 with BMP-2 and related cytokines. All sequences are from
Unipro KB. The first residue number in the sequence of each protein is indicated in parentheses. Proposed secondary structural
elements are indicated by underlining, with β-sheet domains underlined once, turn of α-helical domains underlined twice, and
regions of undefined structure (“the rest”) receiving no underlining. Spaces between residues were inserted by the authors (or from
references) to achieve alignments are indicated with an “*”. a. Human TGF-β receptor type II. P37173. Sequence selection, spacing, alignment, and structure assignment from Demetriou, et al. (21). Spacing modified by the authors. b. Bovine fetuin A. P12763. Sequence selection, spacing, alignment and structure assignment from Demetriou, et al. (21).. c. Bovine fetuin A “inactive” loop 2. Sequence selection, alignment and spacing by the authors. d. Bovine Spp24. Q27967. Sequence selection, alignment, spacing, and structure assignment by the authors. e. Human Spp24. Q13103. Sequence selection, alignment, spacing, and structure assignment by the authors. f. Human BMP receptor type II. Q13873. Sequence stated by Demetriou, et al. (21) but not specifically identified. Sequence
selection, spacing and alignment by the authors. g. Human BMP receptor type IA. P36894. Sequence selection by the authors. Structure assignment by Kotzsch, et al. (27). h. Human BMP receptor type IB. O00238. Sequence selection by the authors. Structure assignment by Kotzsch, et al. (27). doi: 10.1371/journal.pone.0072645.g005 tuin A “inactive” loop 2. Sequence selection, alignment and spacing by the authors. pp24. Q27967. Sequence selection, alignment, spacing, and structure assignment by the authors. equence selection, alignment, spacing, and structure assignment by the authors. Spp24. Q13103. Sequence selection, alignment, spacing, and structure assignment by the authors. f. Human BMP receptor type II. Q13873. Sequence stated by Demetriou, et al. (21) but not specifically identified. Sequence
selection, spacing and alignment by the authors. Of interest is the observation that Spp24 and Spp14.5 may
drive osteogenic differentiation in preference to chondrogenic
differentiation (Figures 3-4). Discussion Specifically, we
would hypothesize that the disparity between the effects of
Spp24 and those of Spp14.5 relate to the greater affinity of
Spp24 for TGF-β2 compared with that of Spp14.5 (
)
The inhibition of the osteogenic activity of BMP by Spp24
has been demonstrated in a number of different model
systems. Parenthetically, it is interesting that Spp24 is
inhibitory of BMP-2 mediated bone formation because the 18.1
kD form was isolated during efforts to characterize the active
component of Urist’s “BMP/NCP” [15] and, furthermore,
another early investigator published the sequence of another
allegedly osteogenic protein the sequence of which was nearly
identical to that of Spp24 (that had not been described at the
time) [29]. In female transgenic mice in which full length Spp24
was expressed under the control of the osteocalcin (OC)
promoter, femoral bone mineral density (BMD) was reduced by
about 10% at three months and about 6% at 8 months [19]. It
was not unexpected that the inhibition was limited to female
transgenic mice since OC is expressed at higher levels in
female mice. In the mouse hindquarter ectopic bone formation
model, ectopic bone formation by 5 μg of rhBMP-2 was
completely inhibited by co-implantation with 2.5 mg of full-
length Spp24 but not affected by 0.05 mg of Spp24 [19]. On the
other hand, when various doses of the different forms of Spp24 As outlined above, it appears that the Spp24 molecules and
members of the TGF-β family of cytokines interact through
binding of the growth factors to the receptor-like TRH1 domain. There is, however, a significant amount of information, as
discussed above, that suggest that binding of growth factors to
the TRH1 domain is a less than complete explanation for this 7 PLOS ONE | www.plosone.org August 2013 | Volume 8 | Issue 8 | e72645 Spp24 and Spp14 Affect TGF-β Differently Figure 5. Sequence and secondary structure comparisons of various receptor and pseudoreceptor molecules proposed to
give information pertaining to the site of interaction of Spp24 with BMP-2 and related cytokines. All sequences are from
Unipro KB. The first residue number in the sequence of each protein is indicated in parentheses. Proposed secondary structural
elements are indicated by underlining, with β-sheet domains underlined once, turn of α-helical domains underlined twice, and
regions of undefined structure (“the rest”) receiving no underlining. References Blum B, Moseley J, Miller L, Richelsoph K, Haggard W (2004)
Measurement of bone morphogenetic proteins and other growth factors
in demineralized bone matrix. Orthopedics 27: s161-s165. PubMed:
14763551. 20. Sintuu C, Simon RJ, Miyazaki M, Morishita Y, Hymanson HJ et al. (2011) Full-length spp24, but not its 18.5-kDa proteolytic fragment,
inhibits bone-healing in a rodent model of spine fusion. J Bone Joint
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93:
1022-1032. doi:10.2106/JBJS.J.00081. PubMed:
21655895. 8. Canalis E, Economides AN, Gazzerro E (2003) Bone morphogenetic
proteins, their antagonists, and the skeleton. Endocr Rev 24: 218-235. 8. Canalis E, Economides AN, Gazzerro E (2003) Bone morphogenetic
proteins, their antagonists, and the skeleton. Endocr Rev 24: 218-235. 9. Gazzerro E, Canalis E (2006) Bone morphogenetic proteins and their 21. Demetriou M, Binkert C, Sukhu B, Tenenbaum HC, Dennis JW (1996)
Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type
II receptor mimic and cytokine antagonist. J Biol Chem 271:
12755-12761. doi:10.1074/jbc.271.22.12755. PubMed: 8662721. 9. Gazzerro E, Canalis E (2006) Bone morphogenetic proteins and
antagonists. Rev Endocr Metab Disord 7: 51-65 10. Brochmann EJ, Behnam K, Murray SS (2009) Bone morphogenetic
protein-2 activity is regulated by secreted phosphoprotein-24 kd, an
extracellular pseudoreceptor, the gene for which maps to a region of
the human genome important for bone quality. Metabolism 58:
644-650. doi:10.1016/j.metabol.2009.01.001. PubMed: 19375587. 22. Guimond A, Sulea T, Pepin MC, O’Connor-McCourt MD (1999)
Mapping of putative binding sites on the ectodomain of the type II TGF-
beta receptor by scanning-deletion mutagenesis and knowledge-based
modeling. FEBS Lett 456: 79-84. doi:10.1016/S0014-5793(99)00869-8. PubMed: 10452534. 11. Hyytiäinen M, Penttinen C, Keski-Oja J (2004) Latent TGF-beta binding
proteins: extracellular matrix association and roles in TGF-beta
activation. Crit
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233-264. doi:
10.1080/10408360490460933. PubMed: 15307633. 23. Guimond A, Sulea T, Pen A, Ear P, O’Connor-McCourt MD (2002) Site-
directed mutagenesis of the type II TGF-beta receptor indicates a
ligand-binding site distinct from that of the type II activin receptor. FEBS
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515:
13-19. doi:10.1016/S0014-5793(02)02378-5. PubMed:
11943186. 12. Murray EJ, Murray SS, Simon R, Behnam K (2007) Recombinant
expression, isolation, and proteolysis of extracellular matrix-secreted
phosphoprotein-24 kDa. Connect Tissue Res 48: 292-299. doi:
10.1080/03008200701692404. PubMed: 18075815. 24. Sun A, Murray SS, Simon RJ, Jawien J, Behnam K et al. (2010)
Alanine-scanning mutations of the BMP-binding domain of recombinant
secretory bovine spp24 affect cytokine binding. Connect Tissue Res
51: 445-451. doi:10.3109/03008201003615734. PubMed: 20615094. 13. Discussion The histological examination of the
ectopic bone masses formed in response to TGF-β2, alone or
in combination with Spp24 or Spp14.5, consisted mostly of
bone and cartilage. However, there was a paucity of cartilage
in the specimens from the TGF-β2 plus Spp24 or Spp14.5
treatment groups. Joyce, et al. [17] reported that injections of
“low dose” (20 ng/day) induced the formation of predominantly
(about 78%) intramembranous bone whereas injections with
"high dose" (200 ng/day) TGF-β2 induced the formation of
mostly (80%) cartilage. This is a significant finding because
previous studies have also demonstrated that the kinetics of
the exposure of mesenchymal progenitors to BMPs can
influence whether intramembranous or endochondral bone
formation predominates [30,31]. Therefore, it is possible that
Spp24 and Spp14.5 affects the exposure of mesenchymal interaction. Certainly, the data presented here and our
previously reported data pertaining to the interactions of Spp24
proteins and BMP-2 strongly suggest an important role for a
segment of the Spp24 molecule located near the C-terminus in
the binding of growth factors to Spp24. The review of the
sequence similarities presented above suggests that a β-
pleated sheet/turn/β-pleated sheet motif (formally, a hairpin β
motif) is involved in part of the binding domain. However, while
an oxidized cyseine-cysteine bond is absolutely required for
binding of short peptides with TRH1-like sequences to the
pertinent growth factors, this is not the case for the full length
protein. Furthermore, there appear to be very non-stringent
requirements for specific amino acids in the binding domain. In
summary, a thorough definition of the nature of the interaction
between Spp24 and TGF-β or BMP will require comprehensive
physico-chemical studies. PLOS ONE | www.plosone.org August 2013 | Volume 8 | Issue 8 | e72645 8 Spp24 and Spp14 Affect TGF-β Differently progenitors to TGF-β2 in such a way as to favor osteogenic
differentiation over chrondrogenic differentiation. appear to be one of the forms of Spp24 remains an enigma. Secondly, the concentrations and relative proportions of the
various forms of Spp24 in the active compartment of the bone
environment must impose controls on the absolute and relative
amounts of biologically active (not “latent”) TGF-β and BMPs. These absolute and relative amounts, in turn, govern the
balance of proliferation and differentiation in bone undergoing
growth, repair, or turnover. References 1. Canalis E (2003) Osteogenic growth factors. In: MJ Favus.Primer on
the metabolic bone diseases and disorders of mineral metabolism. 5th
ed. Washington, DC: American Society for Bone and Mineral Research. pp. 28-31. cystatin family of thiol protease inhibitors. J Biol Chem 270: 431-436. doi:10.1074/jbc.270.1.431. PubMed: 7814406. cystatin family of thiol protease inhibitors. J Biol Chem 270: 431-436. doi:10.1074/jbc.270.1.431. PubMed: 7814406. 15. Behnam K, Phillips ML, Silva JD, Brochmann EJ, Duarte ME et al. (2005) BMP binding peptide: a BMP-2 enhancing factor deduced from
the sequence of native bovine bone morphogenetic protein/non-
collagenous protein. J Orthop Res 23: 175-180. doi:10.1016/j.orthres. 2004.05.001. PubMed: 15607890. 2. Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai S et
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Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is
essential for full inhibition of BMP-2 activity. J Orthop Res 28:
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17. Joyce ME, Roberts AB, Sporn MB, Bolander ME (1990) Transforming
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system for bone morphogenetic protein. Clin Orthop Relat Res:
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Bone morphogenetic protein binding peptide mechanism and
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(Phila Pa 1976) 35: 2049-2056. PubMed: 20581758. 6. Murray SS, Brochmann EJ, Harker JO, King E, Lollis RJ et al. (2007) A
statistical model to allow the phasing out of the animal testing of
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length bovine spp24 [spp24 (24-203)] inhibits BMP-2 induced bone
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18253966. 7. Discussion Thirdly, these differences can be
exploited to engineer therapeutic molecules that affect growth
factor activity on a spectrum from inhibition (sequestration) to
enhancement (slow release). In summary, we have demonstrated that Spp24 and the
three natural proteolytic products of Spp24 (Spp18.1, Spp16.0,
and Spp14.5) bind TGF-β. While the affinity of this interaction is
about an order of magnitude less than that of the interactions
between the Spp24 proteins and BMP-2, the affinity is still
sufficient for the form with the greatest affinity, full-length
Spp24, to inhibit the bone inducing activity of TGF-β. The most
truncated of the common forms, Spp14.5, did not inhibit the
bone inducing activity of TGF-β. This disparity in the difference
of inhibiting potential is distinctly different than the case of the
Spp24 proteins and BMP-2 though the bioassays are obviously
quite different. The observed differences in the binding of
different forms of Spp24 to TGF-β and to BMP and the
associated differences in biological effects do have several
important ramifications. First, the fact that two independent
investigators decades ago reported “osteogenic proteins” that Author Contributions Conceived and designed the experiments: HT EJB MDD JCW
SSM. Performed the experiments: HT XB CL KZ EJB SRM BA
DC SSM. Analyzed the data: HT KZ EJB SRM MDD JCW
SSM. Wrote the manuscript: HT KZ EJB SSM. 1. Canalis E (2003) Osteogenic growth factors. In: MJ Favus.Primer on
the metabolic bone diseases and disorders of mineral metabolism. 5th
ed. Washington, DC: American Society for Bone and Mineral Research.
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receptor complexes reveals a mechanism of receptor inactivation in
juvenile polyposis syndrome. J Biol Chem 283: 5876-5887. PubMed:
18160401. 30. Sykaras N, Opperman LA (2003) Bone morphogenetic proteins
(BMPs): how do they function and what can they offer the clinician? J
Oral Sci 45: 57-73. doi:10.2334/josnusd.45.57. PubMed: 12930129. j
31. Janssens K, ten Dijke P, Janssens S, Van Hul W (2005) Transforming
growth factor-beta1 to the bone. Endocr Rev 26: 743-774. doi:
10.1210/er.2004-0001. PubMed: 15901668. 28. Nickel J, Dreyer MK, Kirsch T, Sebald W (2001) The crystal structure of
the BMP-2:BMPR-IA complex and the generation of BMP-2 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 August 2013 | Volume 8 | Issue 8 | e72645
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Inpatient GHB withdrawal management in an inner-city hospital in Sydney, Australia: a retrospective medical record review
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Psychopharmacology/Psychopharmacologia
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cc-by
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* Krista J. Siefried
Krista.Siefried@svha.org.au Abstract Rationale Regular consumption of gamma-hydroxybutyrate (GHB) may result in a dependence syndrome that can lead to
withdrawal symptoms. There are limited data on medications to manage GHB withdrawal. withdrawal symptoms. There are limited data on medications to manage GHB withdrawal. Objectives To examine characteristics associated with delirium and discharge against medical advice (DAMA), in the context
of implementing a GHB withdrawal management protocol at an inner-city hospital in 2020. Methods We retrospectively reviewed records (01 January 2017–31 March 2021), and included admissions that
were ≥ 18 years of age, admitted for GHB withdrawal, and with documented recent GHB use. Admissions were assessed
for demographics, medications administered, features of delirium, ICU admission, and DAMA. Exploratory analyses were
conducted to examine factors associated (p < 0.2) with features of delirium and DAMA. Results We identified 135 admissions amongst 91 patients. Medications administered included diazepam (133 admissions,
98.5%), antipsychotics (olanzapine [70 admissions, 51.9%]), baclofen (114 admissions, 84%), and phenobarbital (8 admis-
sions, 5.9%). Features of delirium were diagnosed in 21 (16%) admissions. Delirium was associated with higher daily GHB
consumption prior to admission, while duration of GHB use, time from presentation to first dose of diazepam, and concomi-
t
t
th
h t
i
i
l
i t d
ith d li i
DAMA
d
t 41 (30%) d i i
d Objectives To examine characteristics associated with delirium and discharge against medical advice (DAMA), in the context
of implementing a GHB withdrawal management protocol at an inner-city hospital in 2020. Methods We retrospectively reviewed records (01 January 2017–31 March 2021), and included admissions that
were ≥ 18 years of age, admitted for GHB withdrawal, and with documented recent GHB use. Admissions were assessed
for demographics, medications administered, features of delirium, ICU admission, and DAMA. Exploratory analyses were
conducted to examine factors associated (p < 0.2) with features of delirium and DAMA.i Results We identified 135 admissions amongst 91 patients. Medications administered included diazepam (133 admissions,
98.5%), antipsychotics (olanzapine [70 admissions, 51.9%]), baclofen (114 admissions, 84%), and phenobarbital (8 admis-
sions, 5.9%). Features of delirium were diagnosed in 21 (16%) admissions. Delirium was associated with higher daily GHB
consumption prior to admission, while duration of GHB use, time from presentation to first dose of diazepam, and concomi-
tant methamphetamine use were inversely associated with delirium. Inpatient GHB withdrawal management in an inner‑city hospital
in Sydney, Australia: a retrospective medical record review Krista J. Siefried1,2,3 · Georgia Freeman3 · Darren M. Roberts4,5,6 · Rhiannon Lindsey7 · C
Nadine Ezard1,2,3,8 · Jonathan Brett4,5,8 Krista J. Siefried1,2,3 · Georgia Freeman3 · Darren M. Roberts4,5,6 · Rhiannon Lindsey7 · Craig
Nadine Ezard1,2,3,8
Jonathan Brett4,5,8 Received: 17 May 2022 / Accepted: 19 November 2022
© The Author(s) 2022
/ Published online: 12 December 2022 1
The National Centre for Clinical Research On Emerging
Drugs (NCCRED), The University of New South Wales,
Randwick Campus 22/32 King St Randwick, Sydney,
NSW 2031, Australia https://doi.org/10.1007/s00213-022-06283-6
Psychopharmacology (2023) 240:127–135 https://doi.org/10.1007/s00213-022-06283-6
Psychopharmacology (2023) 240:127–135 ORIGINAL INVESTIGATION Keywords Gamma-hydroxybutyrate · GHB · Gamma-butyrolactone · GBL · 1,4-butanediol · 1,4BD · Withdrawal ·
Pharmacotherapy · Oxybate Introduction However, most studies report infrequent rates of delirium and
are underpowered to examine for associated factors (Dijkstra
et al. 2017; Kamal et al. 2016a), particularly studies examin-
ing tapering GHB pharmacotherapy, which likely ameliorates
symptoms of withdrawal such as delirium (Wolf et al. 2021). In the absence of randomised clinical trials, the existing
literature reports on case studies and open-label treatment
protocols. Pharmacological interventions for the manage-
ment of GHB withdrawal are guided by the mechanism of
action of GHB on the GABA system, including high-dose
benzodiazepines (see narrative reviews by Busardò and
Jones (2015); Kamal et al. (2016b); Kamal et al. (2017);
Schep et al. (2012); Wood et al. (2011)); titration and taper-
ing with pharmaceutical GHB (sodium oxybate) (not avail-
able for use in Australia) (Dijkstra et al. 2017; Kamal et al. 2017); barbiturates such as phenobarbital (particularly if
symptoms are resistant to benzodiazepines; reported in lim-
ited case reports) (Ghio et al. 2014); or the GABA-B recep-
tor agonist baclofen (reported in case series) (LeTourneau
et al. 2008). Less commonly discussed are gabapentin and
antipsychotics (Kamal et al. 2016b). People who consume GHB regularly may experience a
dependence syndrome that can result in typical withdrawal
symptoms (Kamal et al. 2017). The onset of withdrawal can
be rapid—commencing within an hour of discontinuation and
peaking at 24 h—and can last up to 3 weeks (Schep et al. 2012). While the clinical characteristics of a withdrawal syndrome
have not been well defined in the literature, case studies and
reviews describe similarities between withdrawal symptoms
of GHB and other CNS depressants such as alcohol or ben-
zodiazepines (Wojtowicz et al. 2008; Wolf et al. 2021). The
Diagnostic and Statistical Manual of Mental Disorders, Fifth
Edition (DSM-5), characterises a withdrawal syndrome associ-
ated with sedative, hypnotic, or anxiolytic drugs by two or more
symptoms including autonomic hyperactivity (e.g. increased
temperature, heart rate, respiratory rate, blood pressure), Given the lack of large-scale randomised trials, with-
drawal scales, or treatment protocols, the management of
GHB withdrawal can vary, although there is a pharmacologi-
cal rationale for the aforementioned regimens. We sought
to review the clinical experience at an inner-urban hospital,
where in 2020 a procedure for the management of GHB
withdrawal was introduced in an attempt to standardise prac-
tice. The procedure includes benzodiazepines, baclofen, and
phenobarbital, as outlined below. Introduction A naturally occurring neurotransmitter, gamma-hydroxybu-
tyrate (GHB), is both a precursor and metabolite of GABA
(Schep et al. 2012). It can cause desired effects such as eupho-
ria and disinhibition but, at higher doses, GHB becomes a
potent central nervous system (CNS) depressant (Schep et al. 2012). GHB (and its precursors gamma-butyrolactone [GBL]
and 1,4-butanediol [1,4-BD]) is an illicit substance used for
many reasons, and often in combination with other drugs,
including as a “party drug”, to facilitate sex-based social-
ity (known as “chemsex” or “party and play”) (Bourne et al. 2014), and for self-management of sleep and anxiety. Medi-
cally, the sodium salt of GHB, sodium oxybate, is used in
the USA and some European countries for the treatment of
narcolepsy, and in some countries for the treatment of alco-
hol use disorder and withdrawal (Fuller and Hornfeldt 2003;
Wedin et al. 2006). In the most recent Australian population
household survey (2019), approximately 1% of respondents
14 years and older reported any lifetime use of GHB (Austral-
ian Institute of Health and Welfare 2020). However, amongst
convenience samples of some demographic groups, such as
lesbian, gay, and bisexual people, rates 20–54 times greater
have been recorded (Hammoud et al. 2018; Mooney-Somers
et al. 2020). A global systematic review identified growing
evidence for GHB-related adverse effects amongst people
who use GHB regularly (Dijkstra et al. 2021). Harms associ-
ated with the use of GHB include overdose and use disorder
(Schep et al. 2012). Harms are increasing worldwide (United
Nations Office on Drugs and Crime 2018). This includes in
Australia, where ambulance call-outs (Arunogiri et al. 2020)
and emergency department presentations for GHB overdose
(Harris et al. 2021) have risen over the past decade, and a
national priority setting study identified GHB overdose and
withdrawal as a key clinical research priority in the alcohol
and other drug sectors (Siefried et al. 2021). Due to the potential for severe physical or psychiatric
complications, inpatient medical management of GHB with-
drawal may be warranted for people who regularly consume
high amounts (van Noorden et al. 2015), although outpatient
withdrawal management has been documented. A number of
possible risk factors for GHB withdrawal complications have
been identified, such as frequent intervals of GHB dosing prior
to cessation (Kamal et al. 2017); concomitant psychostimulant
use (Kamal et al. 2016a); or female gender (Wolf et al. 2021). Abstract DAMA occurred amongst 41 (30%) admissions, and
was associated with a longer time from presentation to first dose of baclofen, while being female and receiving a loading
dose of diazepam were inversely associated. p
y
Conclusions This study adds to the literature in support of the safety and feasibility of diazepam and baclofen for the man-
agement of GHB withdrawal. Prospective, randomised trials are required. Keywords Gamma-hydroxybutyrate · GHB · Gamma-butyrolactone · GBL · 1,4-butanediol · 1,4BD · Withdrawal ·
Pharmacotherapy · Oxybate 5
St Vincent’s Clinical School, The University of New South
Wales, Sydney, Australia
6
Drug Health, Royal Prince Alfred Hospital, Sydney,
Australia
7
Faculty of Medicine, Notre Dame University, Sydney,
Australia
8
New South Wales Drug and Alcohol Clinical Research
and Improvement Network (DACRIN), Sydney, NSW,
Australia 5
St Vincent’s Clinical School, The University of New South
Wales, Sydney, Australia 5
St Vincent’s Clinical School, The University of New South
Wales, Sydney, Australia * Krista J. Siefried
Krista.Siefried@svha.org.au Krista.Siefried@svha.org.au
1
The National Centre for Clinical Research On Emerging
Drugs (NCCRED), The University of New South Wales,
Randwick Campus 22/32 King St Randwick, Sydney,
NSW 2031, Australia
2
The National Drug and Alcohol Research Centre (NDARC),
The University of New South Wales, Sydney, Australia
3
Alcohol and Drug Service, St Vincent’s Hospital Sydney,
Darlinghurst, Australia
4
Department of Clinical Pharmacology and Toxicology, St
Vincent’s Hospital Sydney, Darlinghurst, Australia 6
Drug Health, Royal Prince Alfred Hospital, Sydney,
Australia 1
The National Centre for Clinical Research On Emerging
Drugs (NCCRED), The University of New South Wales,
Randwick Campus 22/32 King St Randwick, Sydney,
NSW 2031, Australia 7
Faculty of Medicine, Notre Dame University, Sydney,
Australia 7
Faculty of Medicine, Notre Dame University, Sydney,
Australia 2
The National Drug and Alcohol Research Centre (NDARC),
The University of New South Wales, Sydney, Australia 8
New South Wales Drug and Alcohol Clinical Research
and Improvement Network (DACRIN), Sydney, NSW,
Australia 8
New South Wales Drug and Alcohol Clinical Research
and Improvement Network (DACRIN), Sydney, NSW,
Australia 3
Alcohol and Drug Service, St Vincent’s Hospital Sydney,
Darlinghurst, Australia 4
Department of Clinical Pharmacology and Toxicology, St
Vincent’s Hospital Sydney, Darlinghurst, Australia :(0123
1 23456789)
3 Psychopharmacology (2023) 240:127–135 128 tremor, insomnia, nausea, anxiety, and psychomotor agitation
(American Psychiatric Association 2013). Other symptoms
include audio/visual/tactile hallucinations, paranoid delusions,
and seizures (American Psychiatric Association 2013; Kamal
et al. 2017). Renal failure (Bhattacharya et al. Abstract 2011), cardiac
arrest, and death (Dyer et al. 2001) have all been reported in
GHB withdrawal (Wojtowicz et al. 2008). GHB withdrawal procedure Records were assessed for medications administered, fea-
tures of delirium, ICU admission, discharge against medical
advice, and re-presentation to a hospital (limited only to data
for the admitting hospital) within the study period. Explora-
tory analyses were conducted to examine factors associated
with features of delirium and DAMA. The inpatient GHB withdrawal procedure commences with
a clinical review by a Registered Nurse and Addiction Medi-
cine Specialist. This includes assessment of medical history
and comorbidities, and current drug and alcohol use including
other CNS depressants (e.g. alcohol, benzodiazepines). Where
indicated based on a prediction of risk of withdrawal severity,
planned admission to the inpatient withdrawal unit is facili-
tated. This is based on dosing pattern, duration of continuous
use and volume of GHB consumption, history of complicated
GHB withdrawal, or other medical or psychiatric comorbidities. Patients can remain as inpatients in the specialist withdrawal
treatment unit for 7 days, or longer if medically indicated. The
procedure includes a loading dose of oral diazepam (20 mg
every 1–2 h to a recommended 60 mg or until lightly sedated),
commenced as early as possible relative to the last GHB dose
and ideally prior to the onset of withdrawal symptoms. This is
followed by a maintenance dose of diazepam 10–20 mg hourly
to a maximum of 120 mg in a 24-h period (tapering over the
admission). Baclofen (10–25 mg three times daily) can be com-
menced concurrently with benzodiazepines when a moderate
or severe withdrawal is expected. If a patient requires more
than 200 mg of diazepam in a 24-h period, this prompts an
intensive care review and consideration of barbiturate therapy. An oral loading dose of 30 mg phenobarbital hourly (maxi-
mum 120 mg) until lightly sedated then 30 mg two hourly (max
240 mg) is given to patients in the withdrawal unit or general
medical ward. Intravenous phenobarbital is recommended for
patients admitted to intensive care with boluses of 5-10 mg/kg
two hourly until sedated. Once stabilised on baclofen, patients
are weaned from benzodiazepines by tapering the dose over
7 days. Patients are not discharged on benzodiazepines; these
are ceased upon discharge. If continued on discharge, baclofen
is weaned by tapering the dose over several weeks at the treat-
ing specialist’s discretion. Statistics Included records were manually reviewed and data entered
into a RedCap database (Harris et al. 2009). Descriptive
statistics were used to describe the sample. Logistic regres-
sion analysis was conducted to determine factors associated
with the outcomes of delirium, and discharge against medi-
cal advice. Given patients could re-present, generalised lin-
ear models with generalised estimating equations and logit
link functions were used with an exchangeable correlation
structure to account for within-patient correlations. Bivariate
regression was conducted on all variables and those with a
p-value ≤ 0.2 were included in a multivariate regression analy-
sis. Variables were removed by backward elimination based
on their significance (two-sided alpha p < 0.2) (Heinze et al. 2018) and model fit based on the Akaike information criterion
(AIC). Given that this is an exploratory analysis, all variables
with a p-value < 0.2 were retained in the multivariate analysis
and are reported, irrespective of 95% confidence intervals. Data were analysed in R version 3.6.2 (2019–12-12). GHB withdrawal procedure Demographic and other data collected from the included
medical records were age, sex, gender, sexuality, relation-
ship status, known legal issues, GHB use and history (prior
history of GHB use, prior history of GHB overdose, prior
history of GHB withdrawal, age of first GHB use, date and
time of last GHB use [estimated or actual], GHB use pat-
tern [frequency, duration, dose, nocturnal use]), other sub-
stance use (frequency, dose, age first use, date last use, prior
withdrawal), intravenous drug use, comorbidities, concom-
itant medications, date and time of admission, reason for
admission, number of admissions (e.g. if prior admissions
recorded), pharmacological management of GHB with-
drawal (medication given, first [loading] dose amount, main-
tenance dose amount, time of first dose, total daily dose,
discharge prescription, free text for notations), intensive care
assessment/admission (binary yes/no—if yes: date and time,
discharge date and time, sedation, intubation), delirium diag-
nosis (binary yes/no—if yes: features, resolved, free text for
notations), adverse events (binary yes/no—if yes: details,
resolved), code black called for aggressive/self-harming
behaviour, discharge status (with a plan, or against medical
advice), relapse prevention plan, follow-up post-discharge,
last follow-up date, and known GHB use post-discharge. Introduction The present study aims to
determine the outcomes of patients presenting with GHB
withdrawal including features of delirium and discharge
against medical advice (DAMA) prior to and following
implementation of the procedure, and the characteristics
associated with delirium and DAMA. 1 3 Psychopharmacology (2023) 240:127–135 129 Included patient admissions One-hundred and thirty-five admissions relating to admis-
sions of 91 patients met our eligibility criteria and were
included in our review. Forty-two patients (46.2%) were
male; the median age at first admission was 31 years
(range: 19–53 years). The median age of first GHB use
was 27 years (range: 15–46). The median amount of GHB
consumed daily in the days prior to admission was 30 mL
(range: 9–120 mL). Over half of the admissions involved
nocturnal dosing of GHB (n = 70 [52%]), and 83 patients
(91%) consumed GHB daily. Eighty-seven patients (96%)
consumed other substances, most commonly methampheta-
mine (n = 76, 84%) and benzodiazepines (n = 36, 40%). Ninety-four (70%) admissions were an elective admission
for withdrawal, 41 (30%) were unplanned (i.e. via the emer-
gency department), and seven (5%) of these were related to
a GHB overdose. Demographics are detailed in Table 1, and
presentations over time are presented in Fig. 1. Ethics a Recorded at first presentation
bCalled
hen staff determined personal threat
Sample (n = 91) a
Demographics
Age (years, median [IQR])
31 (19–53)
Sex
Male
42 (46.2)
Female
49 (53.8)
Other
0 (0)
Sexuality
Heterosexual
12 (13.2)
Homosexual
4 (4.4)
Not recorded
75 (82.4)
Relationship status
Single
35 (38.5)
Partnered
23 (25.3)
Not recorded
33 (36.3)
GHB use and history
Age of first GHB use (years, median [IQR])
27 (15–46)
Prior history GHB overdose
No
1 (1.1)
Yes
39 (42.9)
Not documented
51 (56)
Prior GHB withdrawal at home
Yes
23 (25.3)
No
11 (12.1)
Not documented
51 (56)
GHB use frequency
2–4 times per week
3 (3.3)
5–6 times per week
5 (5.5)
Daily
83 (91.2)
GHB use dose (mL, median [IQR])
30 (9–120)
Other substance use
Methamphetamine
76 (83.5)
MDMA
2 (2.2)
Cocaine
10 (11)
Opioids/opiates
9 (9.9)
Cannabis
15 (16.5)
Benzodiazepines
36 (39.6)
Alcohol
25 (27.5)
Nicotine
80 (87.9)
Intravenous drug use
Current
34 (37.4)
Previous
15 (16.5)
Never
42 (46.2)
Hospital admission and outcomes
Delirium
21 (15.6)
Intensive Care Unit admission
3 (2.2)
Code black b
7 (5.2)
Discharge against medical advice
41 (31)
Legal issues
Presentations (n = 135) c
Current
45 (33.3)
Previous
22 (16.3)
Never
20 (14.8)
Not documented
48 (35.6) The St. Vincent’s Hospital Human Research Ethics Commit-
tee approved the study (ETH: 02,221) and given the meth-
odology (retrospective record review) a waiver of consent
was approved. This report follows the STROBE statement
for reporting observational studies (von Elm et al. 2014). Included records We conducted a retrospective medical record review of
patients admitted to an inner-urban tertiary hospital in
Sydney, Australia, with GHB withdrawal for the period of
01 January 2017 to 31 March 2021. Patient records were
identified by a medical record search for admissions that
were coded for withdrawal, which coded GHB as the pri-
mary drug of concern. Patient records were included in
the study if they met the eligibility criteria: admitted as an
inpatient, diagnosed with GHB withdrawal, documented
recent GHB use at least two times per week, and at least
18 years of age. 1 3 Psychopharmacology (2023) 240:127–135 130 Discussion Features of delirium were diagnosed in 21 (16%) admis-
sions. Delirium was present in five admissions (11%)
prior to the procedure implementation and 16 admissions
(18%) following. The median time from admission to the
onset of delirium was 10.1 h (0.6 to 43.7 h). Episodes
of delirium lasted for a median of 40.5 h (range 13.0
to 398.2 h); 44.8 h pre- versus 35.3 h post-procedure
implementation. We analysed 135 admissions for GHB withdrawal, assess-
ing treatments provided and outcomes including features of
delirium and discharge against medical advice. Our sample
was nearly universally administered diazepam, and follow-
ing implementation of a procedure for withdrawal manage-
ment, nearly all received baclofen and almost half were dis-
charged home on ongoing baclofen treatment. The procedure
for dosing diazepam and baclofen was safe and feasible in
this sample; intensive care admission was rare, and delirium
uncommon. In an exploratory analysis, delirium was asso-
ciated proportionally with the amount of GHB consumed,
while the duration of GHB use, concomitant metham-
phetamine use, and earlier diazepam dosing were inversely
related. Discharge against medical advice was less likely
amongst females and those who received a loading dose of
baclofen, while participants who had a longer time from the
presentation to the first dose of baclofen were more likely to
discharge against medical advice. Delirium was associated with higher daily GHB con-
sumption prior to admission (odds ratio [OR] 1.02, 95%
confidence interval [95%CI] 1.00–1.04, p = 0.097), while
the duration of GHB use (OR 0.92, 95%CI 0.83–1.01,
p = 0.085), concomitant methamphetamine use (OR 0.24,
95%CI 0.08–0.73, p = 0.013), and time from presentation
to the first dose of diazepam (OR 0.80, 95%CI 0.66–0.98,
p = 0.032) were inversely associated with delirium. GHB withdrawal management Medications administered were benzodiazepines (diazepam
[133 admissions, 98.5%]), antipsychotics (olanzapine [70 admis-
sions, 51.9%]), and baclofen [114 admissions, 84%]. Load-
ing doses of diazepam were given in 88 (65.2%) admissions. This was more consistent following the implementation of the
procedure (43.5% of admissions prior, and 76.4% following). The median time from presentation to the first diazepam dose
was 1.8 h (range 0 to 80.5 h). Baclofen was more commonly
administered following the implementation of the procedure
(71.7% of admissions prior, and 91.0% of admissions follow-
ing the procedure). The median time from presentation to the
first baclofen dose was 3.2 h (range 0 to 56.5 h). Forty-eight
percent of admissions were discharged on baclofen (21.7% of
admissions prior, and 61.8% following). Twenty-five admissions
(18.5%) indicated that other medications were used, including
phenobarbital (8 admissions, 5.9%), clonidine (8 admissions,
5.9%), and other medications (17 admissions, 12.6%). Twelve admissions (9%) received assessment by an inten-
sive care doctor, three admissions (2.2%) were admitted to
the intensive care unit, and two (1.5%) were intubated. The 1 3 3 131 Psychopharmacology (2023) 240:127–135 Fig. 1 GHB withdrawal presen-
tations over time
Qtr, quarter (i.e. 3 months) Qtr, quarter (i.e. 3 months) Qtr, quarter (i.e. 3 months) Qtr, quarter (i.e. 3 months) median length of stay in the hospital was 3.5 days (range 0.4
to 32.6 days), and there was little change prior to (3.4 days)
and following (3.6 days) the procedure’s implementation. Seventy-four (55%) had a documented relapse to GHB use
following discharge, 21 (16%) within 7 days of discharge;
however, for 41 admissions (30%), these data were unavail-
able, as these patients did not have any further documented
interaction with our hospital. but with a treatment plan. In the 41 admissions who were
documented as discharged against medical advice, this
was associated with time from presentation to the first
dose of baclofen (OR 1.04, 95% CI 0.99–1.08, p = 0.099),
while being female (OR 0.32, 95%CI 0.12–0.82,
p = 0.018) and receiving a loading dose of diazepam (OR
0.45, 95%CI 0.17–1.16, p = 0.097) were inversely associ-
ated with discharge against medical advice. Covariates predictive of discharge against medical
advice However, little to no population-level
data are available on the prevalence and demographics
of those with GHB use disorder. Given ongoing surveil-
lance projects amongst LGBTQ people, particularly gay
or bisexual men (Hammoud et al. 2017), further monitor-
ing of other populations and people who are experiencing
harms associated with GHB (e.g. withdrawal) is required.ii ( g
)
q
While delirium was identified in less than one in five
admissions, other studies have demonstrated similar or
lower rates of delirium, reporting delirium in 2–21% of par-
ticipants (Bell and Collins 2011; Beurmanjer et al. 2020;
Wojtowicz et al. 2008). Episodes of delirium increased over
our study period, and may have reflected that the protocol
encouraged clinicians to examine and document features
of delirium, thereby reflecting an increase in milder forms
of delirium following the implementation of the protocol. While prior studies have identified risks of complicated
withdrawal being concomitant methamphetamine use or
female gender, we did not find this in our sample, while we
did find earlier provision of diazepam to be inversely associ-
ated with delirium. However, given the exploratory analysis
methods and small sample size, this should be interpreted
cautiously. The GHB dose consumed by our sample was
relatively low (30 mL median) and may have contributed to
the lower prevalence of delirium in this study. Larger stud-
ies, enrolling participants with diverse GHB use patterns,
or analysis of larger samples that would allow for disag-
gregation of participants who consumed higher doses, are
warranted. Furthermore, unmeasured factors could result in
more severe withdrawal presentations.fi In Australia, GHB consumption is low in household
surveys (1%) (Australian Institute of Health and Welfare
2020) but up to 50 times greater in people who identify
as LGBTQ (Hammoud et al. 2018; Mooney-Somers et al. 2020). Our study did not collect data on sexuality, as this
is not a minimum data standard for collection by public
services (Freestone et al. 2022). Thus, we are unable to
compare our sample with the underlying population of
LGBTQ people who consume GHB, or identify any emerg-
ing trends of increased use amongst heterosexual people. More broadly, it makes it difficult to ascertain if the cur-
rent public health campaigns, predominantly targeted to
LGBTQ Australians, require expansion to other popula-
tions. Covariates predictive of discharge against medical
advice Our sample enrolled cases that were often com-
plex in terms of complications and polysubstance use,
with high rates of discharge against medical advice and Discharge against medical advice occurred amongst 41
(30%) admissions, while 14 (10%) discharged prematurely 1 3 Psychopharmacology (2023) 240:127–135 132 re-presentation to the hospital for relapse to GHB use, or
subsequent GHB withdrawal support. This may be due to
the inner-urban location of the hospital, where specialist
stimulant treatment services are available and may refer
for services related to GHB, combined with the availabil-
ity of a drug and alcohol withdrawal unit led by addic-
tion medicine specialists. The median length of stay in the
hospital was 3.5 days; this and the proportion of discharge
against medical advice may be related to the inpatient
unit being limited in some respects that may influence a
patient’s choice to remain admitted. Contextual factors
such as tobacco smoking and the use of vaporised nicotine
products not being permitted (although another nicotine
replacement therapy is offered to all inpatients) or the inpa-
tient unit being a closed unit (no outside visitors permitted)
may be factors associated with early discharge, although
this study did not collect the qualitative data required to
assess this. Studies in other settings, multi-centre studies,
and qualitative studies are required to elucidate reasons for
short durations of stay in the inpatient withdrawal unit. We
also note that discharge against medical advice is being re-
interpreted as premature discharge (Ambasta et al. 2020)
and greater exploration is required to understand how
patients’ needs can be met, and the relationship between
premature discharge and treatment outcomes. Cravings for
GHB, urge and the desire to consume GHB could all be
theorised to contribute to premature discharge, but in the
absence of a validated withdrawal scale or further qualita-
tive work with patients, it is unknown the extent to which
these contribute to premature discharge. Furthermore,
given the long half-life of some of the withdrawal medi-
cations provided (e.g. phenobarbital) early discharge and
risk of GHB consumption post-discharge warrant further
follow-up. Dunn 2008) and recent analysis of 74 deaths attributed to
GHB identified 70% as being amongst males (Darke et al. 2020). Our sample, being on average female, older, and
consuming a median of 30 ml GHB daily, does not neces-
sarily reflect the known population of people who consume
GHB in Australia. Covariates predictive of discharge against medical
advice Our sample included a slightly higher proportion
of females to males, consistent with a case series of 12
inpatient GHB withdrawals in Australia (Cappetta and
Murnion 2019), while a 2007 study of people who use
GHB (n = 115) reported 60% were male (Degenhardt and While a retrospective study cannot evaluate the efficacy
of a procedure for GHB withdrawal, the data presented here
demonstrate its feasibility and safety in an inpatient setting
with intensive care availability. This report aims to improve
treatment by transparent reporting to share learning and
improve the evidence base for GHB withdrawal treatment
given the paucity of evidence to date. We also aimed to
inform the future development of GHB withdrawal proce-
dures. This is particularly important given that anecdotally,
the severity of dependence increased over the study period,
although this was not possible to measure retrospectively. While sodium oxybate has been more extensively studied
than other pharmacotherapies for GHB withdrawal (Tay
et al. 2022), and is now commonly used in the Netherlands
(Beurmanjer et al. 2020), it is not currently registered in
Australia, so we have pragmatically evaluated a procedure
implementing pharmacotherapies available in our setting. 1 3 133 Psychopharmacology (2023) 240:127–135 A tool for quantifying the severity of GHB withdrawal which
can guide the dosing of pharmacotherapies is also of particu-
lar interest. Furthermore, a resource to predict withdrawal
severity could streamline management by determining
who is at a higher risk of complications (such as delirium),
thereby ideally managed in a hospital setting, as opposed
to in an outpatient setting. A prospective study to examine
outcomes associated with treatment (for example different
rates of dose escalation of pharmacotherapies) would pro-
vide a valuable evidence base for optimising the treatment
of GHB withdrawal. We present the largest review of baclofen in the context
of GHB withdrawal. Baclofen has a longer half-life than
GHB and is an agonist at GABA-B receptors, and due to
its similarity to GHB has demonstrated effectiveness in
open-label studies of GHB relapse prevention (Beurmanjer
et al. 2018). Establishing the evidence base for administra-
tion concomitantly with high-dose benzodiazepines and in
the context of anticipated complicated or uncomplicated
GHB withdrawal is an important addition to the literature. Declarations Conflict of interest RL is a medical student at Notre Dame University,
Sydney, and contributed to the data collection as part of her independ-
ent research project. JB is funded by an NHMRC Investigator Grant
(1196560) and Medicines Intelligence CRE Fellowship (1196900). All
other authors have no relevant conflicts of interest to declare. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Our study has limitations. The design is a retrospective
data analysis, and the preference would have been to test
the procedure and its components prospectively; however,
the lower incidence of delirium would require a larger
and resource-intensive multi-centre trial of the procedure. Furthermore, the lack of a validated withdrawal measure,
standardised approach, or evidence base would make the
design for a randomised trial challenging, due to the lack of
a “treatment as usual” comparator group. Given the nature
of retrospective data collection, data is sometimes missing
or incomplete—for example, ideally, the last GHB dose
time to the first benzodiazepine time would be recorded but
these data were often not documented, as described. Data
regarding outcomes post-discharge are not always reliably
captured, for example re-presentation to other hospitals is
unknown and abstinence from GHB is unknown. Our study
also has strengths, while it was relatively small and under-
powered, it is the largest case series of GHB withdrawal,
reporting on a large number of GHB withdrawal records
and analysing treatment with a protocolised regimen, and
medical complications, which significantly adds to the
literature that mainly comprises case reports and series. Declarations However, these results should be interpreted with caution,
as there is likely to be unmeasured confounding, and given
the low event rates, regression analyses should be treated as
hypothesis-generating. Covariates predictive of discharge against medical
advice However, due to the retrospective nature, our study was
unable to actively follow up on admitted cases to assess
longer-term outcomes associated with baclofen use, includ-
ing ongoing engagement in care and at services outside
of our institution or psychosocial therapy. Furthermore,
our procedure did not include a protocol for the tapering
of baclofen for cases discharged on baclofen. This study
explored the treatment of withdrawal and the prevalence
of delirium as a complication. Further prospective stud-
ies are required to develop a protocolised management
approach—ideally with a scale to more objectively meas-
ure withdrawal signs and symptoms and the associations
of the treatment of withdrawal and longer-term treatment
outcomes. A tool for quantifying the severity of GHB withdrawal which
can guide the dosing of pharmacotherapies is also of particu-
lar interest. Furthermore, a resource to predict withdrawal
severity could streamline management by determining
who is at a higher risk of complications (such as delirium),
thereby ideally managed in a hospital setting, as opposed
to in an outpatient setting. A prospective study to examine
outcomes associated with treatment (for example different
rates of dose escalation of pharmacotherapies) would pro-
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Homberg JR, Schellekens AFA (2021) Characterization of the Publisher's note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 3
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Impact of visual callosal pathway is dependent upon ipsilateral thalamus
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ARTICLE Results Locating the visual callosal pathway and measuring its role in
visual cortex activity. To locate the visual callosal pathway (vc
pathway), we injected retrograde tracers (alexa-conjugated cho-
lera toxin beta subunit, CTB) into multiple sites in the primary
visual area (Fig. 1a). Injections into the binocular area (V1b)
resulted in retrograde labeling of contralateral hemisphere neu-
ronal somata along a narrow band at the border of primary (V1)
and secondary (V2) visual areas (FWHM in medial–lateral
dimension: 0.72 ± 0.17 mm, mean ± SD, N = 3, Fig. 1a, b). Sepa-
rate injections targeted to surrounding monocular regions did not
result in labeling of neurons in contralateral cortex (Fig. 1a),
supporting previous studies showing callosal projections are
limited to lateral V1 (refs. 6,12). In this labeled region we used
multiphoton imaging of Ca2+ transients to quantify L2/3 neu-
rons’ suprathreshold responses to monocular and binocular visual
stimulations (Fig. 1c, d for experimental setup and terminology,
Fig. 1e, f). Labeled neurons could be statistically classified as ipsi-
laterally or contralaterally responsive only or as binocularly
responsive (Fig. 1g) by first converting the recorded Ca2+-responses
to spiking activity26 (Fig. 1f) and comparing stimulus-locked
responses to baseline spontaneous activity (see “Methods” section
for statistical criteria). All recorded fields of view contained both
visually
responsive
and
non-responsive
neurons
(147/279
responsive, 132/279 non-responsive neurons, FOV = 22, N = 17
animals), with responsive neurons comprising monocular ipsi-
laterally and contralaterally responsive neurons (Fig. 1h, i) and
binocularly responsive neurons (19% ipsilateral, 42.9% con-
tralateral, and 38.1% binocular). Based on intrinsic optical signal
imaging response (see “Methods” section and Supplementary
Fig. 1), all fields of view were overlaid showing that both
monocular and binocular neurons were interspersed and showed
no obvious spatial clustering (two-tailed Fisher’s exact test P =
0.5647, Supplementary Fig. 2). We next quantified functional
properties of projecting and non-projecting neurons. VC pathway mirrors stimulus-evoked activity contralaterally. Responses were recorded from neuronal populations after retro-
gradely labeling VCPNs (Fig. 2a, N = 3 animals, N = 5 FOV, N =
100 neurons). The first co-labeled neurons were located ~140 μm
below the pia (Fig. 2b, Supplementary Fig. 3). Within layer 2/3,
the VCPNs were the minority of the neuronal population (34.76
± 5.67%, mean ± SD, N = 362 neurons, Fig. 2b), and often VCPNs
and non-VCPNs were direct neighbors (Fig. 2a) without showing
specific spatial clustering (pairwise distance distribution: VCPNs
vs. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 A A
t the earliest stages of cortical visual processing visually
responsive neurons can be divided into those only
responsive to one eye (monocular neurons) and those
responsive to both eyes (binocular neurons). For binocular neu-
rons, the most direct anatomical pathway giving rise to responses
is through the ipsilateral lateral geniculate nucleus (LGN)1,2. Cortical responses to contralateral eye stimulation arise primarily
from its crossed projection to the ipsilateral LGN and the sub-
sequent projection from the LGN to visual cortex, while cortical
responses to the ipsilateral eye mainly arise from its uncrossed
projection to the ipsilateral LGN (see Fig. 1 for schematic). An
alternative anatomical pathway giving rise to responses from the
ipsilateral eye results from its tri-synaptic projection involving the
contralateral visual cortex and projection through the corpus
callosum (ipsilateral eye–contralateral LGN–contralateral visual
cortex–ipsilateral visual cortex via the callosal projection)3–5. Such interhemispherically projecting neurons can be found in
many mammalian species4,6–10 including rodents6,11,12, where a
subpopulation of neurons project their axons to the contralateral
visual cortex. These callosal pathway projecting neurons originate
from, and in turn target, the visual cortex regions where receptive
fields are activated by stimuli presented along the vertical
meridian3,4,8,13, in front of the animal. In cats and primates,
where
the
visual
callosal
pathway
has
been
extensively
investigated3,4,10, the callosal pathway has been proposed to
facilitate stereovision3,4 through the binocular pathway. However,
differences in rodent visual system, compared to cat and primate,
at the level of the projections from the retinae14,15 and the central
visual centers16,17, hinder clear comparisons of callosal function. One suggestion for the function of the callosal projection is that it
facilitates lateral interactions between neurons representing
adjacent regions in the visual field across the location where the
visual field is divided between the hemispheres8,18,19. This has
been proposed for primates and cats where the visual field is
divided along the vertical meridian with the left and right halves
of the visual field (hemifields) being represented in the right and
left hemispheres, respectively. However, in many other animals
including rodents8,16,20,21, there is considerably more overlap in
the representation of the visual field around the vertical meridian
in the two cortical hemispheres, and lateral connectivity can in
principle be achieved without the callosal projection. ARTICLE Single-cell
recordings in rodents have suggested that the callosal pathway
contributes ipsilateral eye-derived visual inputs13,22,23 for bino-
cular processing, while other studies have suggested that the
callosal pathway has little if any influence on ipsilateral eye
activation of visual cortex neurons11,24. While numerous studies
have investigated the contribution of the callosal pathway to
stimulus activation of binocular neurons11,13,22,23,25, it is not
known what the relative contribution of the isolated visual
pathway is in generating responses across neuronal populations,
including the activation of monocular neurons. show that while blocking this pathway significantly reduced
spiking across neuronal populations, the callosal projection alone
was not capable of driving suprathreshold activity in V1 neurons,
but required simultaneous input from the ipsilateral LGN. Together this shows that the callosal pathway plays an essential
role in shaping cortical spiking responses to visual stimuli pre-
sented to either eye, but only in conjunction with ipsilateral LGN
activation. Impact of visual callosal pathway is dependent
upon ipsilateral thalamus
https://doi.org/10.1038/s41467-020-15672-4
OPEN shnudev Ramachandra1, Verena Pawlak1, Damian J. Wallace1 & Jason N. D. Kerr1✉ The visual callosal pathway, which reciprocally connects the primary visual cortices, is
thought to play a pivotal role in cortical binocular processing. In rodents, the functional role of
this pathway is largely unknown. Here, we measure visual cortex spiking responses to visual
stimulation using population calcium imaging and functionally isolate visual pathways origi-
nating from either eye. We show that callosal pathway inhibition significantly reduced spiking
responses in binocular and monocular neurons and abolished spiking in many cases. How-
ever, once isolated by blocking ipsilateral visual thalamus, callosal pathway activation alone is
not sufficient to drive evoked cortical responses. We show that the visual callosal pathway
relays activity from both eyes via both ipsilateral and contralateral visual pathways to
monocular and binocular neurons and works in concert with ipsilateral thalamus in generating
stimulus evoked activity. This shows a much greater role of the rodent callosal pathway in
cortical processing than previously thought. 1 NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications Results Note that
injections resulted in retrograde labeling of contralateral neurons (red, white single arrow), and retrograde labeling of neurons in V2 (red, green, blue, white
double arrow). b Reconstruction of left and right occipital poles showing average regions of retrograde labeling of CTB-Alexa 594 showing coronal and
sagittal pixel intensity distributions (N = 3 animals). c Schematic of experimental design. d Visual stimulation convention showing major visual pathways
from eyes (circles) to dorsal LGN (small squares) to cortical V1 (large squares) and visual callosal pathway (vc pathway). Red arrows indicate the two
potential anatomical pathways to ipsilateral V1 from the ipsilateral eye, direct via ipsi-LGN (solid) and via the callosal pathway (dashed). e Overview
example 2-photon image showing functionally labeled neurons (green) and astrocytes (red). f Both spontaneous (between gray boxes) and evoked (gray
boxes) Ca2+-transients from five neurons in response to moving gratings at different angles (angles not shown). Inferred spiking activity within an imaging
frame (red marks for single APs) and the underlying Ca2+-transients shown. g Activity traces from three representative neurons during binocular
stimulation (left), contralateral stimulation (middle) and ipsilateral stimulation (right). Calcium responses to single stimuli are overlaid (gray) with average
(black) and inferred-spike PSTH (lower to each trace). Binocular neuron (cell 1), contralateral stimuli responsive monocular neuron (cell 2) and ipsilateral
stimuli responsive monocular neuron (cell 3). h Overview example 2-photon image (same as e) labeled with response types within a field of view, non-
responders (white), contralateral stimuli responsive monocular (turquoise), ipsilateral stimuli responsive monocular (red) and binocular (black). i Neuronal
response type maps overlaid using intrinsic image responses (N = 17 animals, 22 non-overlapping imaging areas, contains example shown in h). Each
intrinsic imaging area (gray) was centered and associated imaged population overlaid, average intrinsic border (black). Results randomly chosen neurons of the same number as that of
VCPNs, Kolmogorov–Smirnov test P = 0.176; non-VCPNs vs. randomly chosen neurons of the same number as that of non-
VCPNs, Kolmogorov–Smirnov test P = 0.996). The mean spiking
rate of the VCPN subpopulation evoked by drifting grating sti-
muli was not significantly different to that of non-VCPN sub-
population (VCPN vs. non-VCPN, 0.32 ± 0.22 vs. 0.4 ± 0.29 Hz,
mean ± SD, Mann–Whitney U test P = 0.437, N = 28 (VCPN)
and N = 33 (non-VCPN), gratings presented in the visual
space
in
front
of
the
nose,
see
“Methods”
section
for
details). Mean spontaneous firing rates were also not different Here, we used optogenetic and pharmacological manipulation
combined with multiphoton population imaging and retrograde
anatomical tracing, to quantify the role of the visual callosal
pathway on spiking responses to visual stimulation of neurons
located in the binocular cortex. We show that visual cortex
neurons projecting their axons through the callosal pathway can
modulate spiking responses in subpopulations of both monocular
and binocular neurons in the opposite cortex. At the individual
cell level, there were a wide range of possible outcomes of
blocking this pathway during visual stimulation. At one extreme
subpopulations of monocular and binocular neurons completely
lost their ability to generate spiking responses to visual stimuli
upon callosal pathway inactivation, at the other extreme callosal
pathway inactivation had no effect on a subpopulation of
monocular and binocular neurons spiking response. We also NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 *
*
40%
ΔF/F
4 s
100 μm
Anterior
Medial
5 Spikes
20%
ΔF/F
2 s
Binocular
stimulation
Monocular
stimulation
(contra)
Monocular
stimulation
(ipsi)
20 μm
c
e
f
g
20 μm
Cell 1
Cell 2
Cell 3
20X
V1
V1
Contralateral eye
= LGN
Vc-pathway
d
h
i
Time from stim (s)
1 mm
a
0
Max
L
R
1 mm
0.1 P
0.1 P
0.1 P
b
V1
V1
LGN
LGN
Vc-pathway
Ipsilateral eye
Fig. 1 Localizing the visual callosal pathway and experimental setup. a Coronal section of rat brain showing fluorescence from CTB-Alexa 594 (red),
CTB-Alexa 488 (green), and CTB-Alexa 647 (blue) dyes injected into rat visual cortex (asterisks) at 4.1, 3.0, and 2.0 mm lateral to midline. Results Inferred spiking activity within an imaging
frame (red marks for single APs) and the underlying Ca2+-transients shown. g Activity traces from three representative neurons during binocular
stimulation (left), contralateral stimulation (middle) and ipsilateral stimulation (right). Calcium responses to single stimuli are overlaid (gray) with average
(black) and inferred-spike PSTH (lower to each trace). Binocular neuron (cell 1), contralateral stimuli responsive monocular neuron (cell 2) and ipsilateral
stimuli responsive monocular neuron (cell 3). h Overview example 2-photon image (same as e) labeled with response types within a field of view, non-
responders (white), contralateral stimuli responsive monocular (turquoise), ipsilateral stimuli responsive monocular (red) and binocular (black). i Neuronal
response type maps overlaid using intrinsic image responses (N = 17 animals, 22 non-overlapping imaging areas, contains example shown in h). Each
intrinsic imaging area (gray) was centered and associated imaged population overlaid, average intrinsic border (black). Fig. 1 Localizing the visual callosal pathway and experimental setup. a Coronal section of rat brain showing fluorescence from CTB-Alexa 594 (red),
CTB-Alexa 488 (green), and CTB-Alexa 647 (blue) dyes injected into rat visual cortex (asterisks) at 4.1, 3.0, and 2.0 mm lateral to midline. Note that
injections resulted in retrograde labeling of contralateral neurons (red, white single arrow), and retrograde labeling of neurons in V2 (red, green, blue, white
double arrow). b Reconstruction of left and right occipital poles showing average regions of retrograde labeling of CTB-Alexa 594 showing coronal and
sagittal pixel intensity distributions (N = 3 animals). c Schematic of experimental design. d Visual stimulation convention showing major visual pathways
from eyes (circles) to dorsal LGN (small squares) to cortical V1 (large squares) and visual callosal pathway (vc pathway). Red arrows indicate the two
potential anatomical pathways to ipsilateral V1 from the ipsilateral eye, direct via ipsi-LGN (solid) and via the callosal pathway (dashed). e Overview
example 2-photon image showing functionally labeled neurons (green) and astrocytes (red). f Both spontaneous (between gray boxes) and evoked (gray
boxes) Ca2+-transients from five neurons in response to moving gratings at different angles (angles not shown). Inferred spiking activity within an imaging
frame (red marks for single APs) and the underlying Ca2+-transients shown. g Activity traces from three representative neurons during binocular
stimulation (left), contralateral stimulation (middle) and ipsilateral stimulation (right). Calcium responses to single stimuli are overlaid (gray) with average
(black) and inferred-spike PSTH (lower to each trace). Results Binocular neuron (cell 1), contralateral stimuli responsive monocular neuron (cell 2) and ipsilateral
stimuli responsive monocular neuron (cell 3). h Overview example 2-photon image (same as e) labeled with response types within a field of view, non-
responders (white), contralateral stimuli responsive monocular (turquoise), ipsilateral stimuli responsive monocular (red) and binocular (black). i Neuronal
response type maps overlaid using intrinsic image responses (N = 17 animals, 22 non-overlapping imaging areas, contains example shown in h). Each
intrinsic imaging area (gray) was centered and associated imaged population overlaid, average intrinsic border (black). transmits an unbiased representation of the orientation-relevant
stimulus responses (contralateral eye response: VCPN vs. non-
VCPN, 112.0° ± 34.19° vs. 137.98° ± 45.23°, circular mean ± SD,
Kuiper test P > 0.1, N = 14 VCPN and 18 non-VCPN; ipsilateral
eye response: VCPN vs. non-VCPN, 113.22° ± 43.65° vs. 115.81°
± 45.78°, circular mean ± SD, Kuiper test P > 0.1, N = 14 VCPN
and 20 non-VCPN, Fig. 2d). Ocular dominance distributions
were also not significantly different (contralateral bias index;
VCPN vs. non-VCPN, 0.582 ± 0.186 vs. 0.577 ± 0.177, median ±
SD, Kolmogorov–Smirnov test P = 0.734, Fig. 2e). The VCPNs
and non-VCPNs identified by this retrograde tracing method
were functionally indistinguishable from each other, suggesting (VCPN vs. non-VCPN, 0.17 ± 0.14 vs. 0.16 ± 0.16 Hz, mean ± SD,
Mann–Whitney U test P = 0.599, Fig. 2c). While the maximum
response to the preferred orientation was not significantly dif-
ferent (mean of contralateral and ipsilateral eye preferred orien-
tation response for binocular neurons, preferred orientation
response for monocular neurons, VCPN vs. non-VCPN, 1.0 ±
0.46 Hz vs. 1.41 ± 1.08 Hz, mean ± SD, Mann–Whitney U test
P = 0.342, Fig. 2c), a significantly higher fraction of VCPNs were
responsive to drifting grating stimuli (VCPN vs. non-VCPN,
77.46 ± 3.37% vs. 55.91 ± 7.56%, mean ± SEM, two-tailed Fisher’s
exact test P = 0.0329). The distributions of preferred orientations
between the two groups were the same, indicating that this area (VCPN vs. non-VCPN, 0.17 ± 0.14 vs. 0.16 ± 0.16 Hz, mean ± SD,
Mann–Whitney U test P = 0.599, Fig. 2c). While the maximum
response to the preferred orientation was not significantly dif-
ferent (mean of contralateral and ipsilateral eye preferred orien-
tation response for binocular neurons, preferred orientation
response for monocular neurons, VCPN vs. non-VCPN, 1.0 ±
0.46 Hz vs. Results *
*
40%
ΔF/F
4 s
100 μm
Anterior
Medial
5 Spikes
20%
ΔF/F
2 s
Binocular
stimulation
Monocular
stimulation
(contra)
Monocular
stimulation
(ipsi)
20 μm
c
e
f
g
20 μm
Cell 1
Cell 2
Cell 3
20X
V1
V1
Contralateral eye
= LGN
Vc-pathway
d
h
i
Time from stim (s)
1 mm
a
0
Max
L
R
1 mm
0.1 P
0.1 P
0.1 P
b
V1
V1
LGN
LGN
Vc-pathway
Ipsilateral eye 1 mm
a 0
Max
L
R
1 mm
0.1 P
0.1 P
0.1 P
b b 100 μm
Anterior
Medial
20 μm
h
i *
*
c
e
f
20X
V1
V1
Contralateral eye
= LGN
Vc-pathway
Ipsilateral eye h *
40%
ΔF/F
4 s
100 μm
Anterior
Medial
5 Spikes
20%
ΔF/F
2 s
Binocular
stimulation
Monocular
stimulation
(contra)
Monocular
stimulation
(ipsi)
20 μm
e
f
g
20 μm
Cell 1
Cell 2
Cell 3
20X
V1
h
i
Time from stim (s)
V1
LGN
ay
ateral eye 40%
ΔF/F
4 s
20 μm
e
f
0X c g e i i f d
V1
V1
LGN
LGN
Vc-pathway d Time from stim (s) Fig. 1 Localizing the visual callosal pathway and experimental setup. a Coronal section of rat brain showing fluorescence from CTB-Alexa 594 (red),
CTB-Alexa 488 (green), and CTB-Alexa 647 (blue) dyes injected into rat visual cortex (asterisks) at 4.1, 3.0, and 2.0 mm lateral to midline. Note that
injections resulted in retrograde labeling of contralateral neurons (red, white single arrow), and retrograde labeling of neurons in V2 (red, green, blue, white
double arrow). b Reconstruction of left and right occipital poles showing average regions of retrograde labeling of CTB-Alexa 594 showing coronal and
sagittal pixel intensity distributions (N = 3 animals). c Schematic of experimental design. d Visual stimulation convention showing major visual pathways
from eyes (circles) to dorsal LGN (small squares) to cortical V1 (large squares) and visual callosal pathway (vc pathway). Red arrows indicate the two
potential anatomical pathways to ipsilateral V1 from the ipsilateral eye, direct via ipsi-LGN (solid) and via the callosal pathway (dashed). e Overview
example 2-photon image showing functionally labeled neurons (green) and astrocytes (red). f Both spontaneous (between gray boxes) and evoked (gray
boxes) Ca2+-transients from five neurons in response to moving gratings at different angles (angles not shown). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 neuron 1). b Fraction of VCPNs as a function of depth from the pia (N = 3 animals). c Pooled data of spontaneous (gray) and
maximum stimulus response (black, average max. response to preferred orientation for VCPNs (N = 28 neurons) and non-VCPNs (N = 33 neurons)) and
mean ± SD (red) for each group. d Distribution of preferred orientations for orientation selective neurons from both VCPN and non-VCPN groups (ipsi-eye
stimulation, red, contra, turquoise). e Scatter plot comparing mean stimulus response of ipsi- to the mean response from the contralateral eye stimulation
(lower) for VCPNs (orange markers) and non-VCPNs (blue markers) with the distribution of the contralateral bias index shown in upper panels (see
“Methods” section for details). that, at least for the stimulus types tested here, the vc pathway
mirrors stimulus-evoked information to the opposite hemisphere. To test the impact of vc pathway, we next optogenetically
modulated this pathway during visual stimulation. significantly
reduced
both
the
neurons’
average
response
probabilities when taking the response to all presented angles
(0.277 ± 0.166, mean ± SD, paired Wilcoxon signed-rank test P <
0.0001; binocular stimulation 0.34 ± 0.191, ipsilateral stimulation
0.184 ± 0.147, contralateral stimulation 0.308 ± 0.209), as well as
the average response probabilities for the neurons’ maximally
tuned response angle (0.506 ± 0.21, mean ± SD, paired Wilcoxon
signed-rank test P < 0.0001; binocular stimulation 0.688 ± 0.25,
ipsilateral stimulation 0.466 ± 0.269, contralateral stimulation
0.614 ± 0.286, Supplementary Fig. 5a). The vc pathway has been
implicated in the generation of correlated firing responses
between the cortices28. We therefore next investigated the
possibility that the vc pathway is involved in generating network
synchrony by influencing correlated activity. Probability distribu-
tions of pairwise correlations were not significantly changed by vc
pathway inactivation (Supplementary Fig. 5b, P = 0.221), but as
pairwise correlation does not capture a complete view of
correlated population-wide spiking events26, we next quantified
how the visually active neurons represented binocular and
monocular stimulations as a population. Vc pathway modulates monocular and binocular spiking
responses. We used an adeno-associated virus (AAV) to express
the optogenetic activity inhibitor eArchT 3.0 (ref. 27) and a
yellow-fluorescent protein (YFP) tag in neurons in the region
containing the VCPNs (Fig. 3a, Supplementary Fig. 4, N = 29
animals). This simultaneously established the location of the
termination of callosal projections to contralateral hemisphere
(Fig. 3a). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Labeled axonal arbors formed dense arborizations
localized around the V1–V2 border region spanning all cortical
layers (Fig. 3a). We next identified cortical representation of the
binocular visual space directly in front of the animal using
intrinsic optical signal imaging, and made a small injection of
Alexa-conjugated CTB into layer 2/3 in the center of the identi-
fied region (small red spot in Fig. 3a, see “Methods” for full
details). This showed that the V1–V2 border region receiving the
afferent callosal projection overlaps with the representation of the
frontal binocular visual field. Neurons expressing eArchT 3.0
were located across all cortical layers, and juxtasomal electrical
recordings confirmed that activation of eArchT rapidly and
reversibly silenced both spontaneous and stimulus-evoked firing
in neurons in all cortical layers (Fig. 3b, c, Supplementary Fig. 4,
22 recordings, N = 10 animals). We next quantified the extent to
which inhibition of the vc pathway modulated suprathreshold
activity to visual stimuli across neuronal populations (Fig. 3d). d h
b b l
f
ll p p
We quantified, for visually responsive neurons, how many
spikes were elicited from each single stimulus presentation for
both monocular and binocular stimuli as a measure of the
strength of each pathway in generating population-wide corre-
lated spiking events26 (Fig. 3f). Monocular contralateral and
binocular stimuli elicited similar probability distributions, while
ipsilateral stimulation generated significantly less spiking and
more trials with no spikes, which was also significantly more than
observed during spontaneous activity (binocular vs. contralateral,
Kolmogorov–Smirnov test P = 0.299; binocular vs. ipsilateral,
Kolmogorov–Smirnov test P < 0.0001; ipsilateral vs. spontaneous;
Kolmogorov–Smirnov test P < 0.0001, Fig. 3f, N = 30 neurons,
N = 5 populations with six neurons each). Upon inactivation of
the vc pathway, population spiking significantly reduced for all
stimuli indicating that for small populations of visually responsive
neurons,
the
vc
pathway
contributes
significantly
to
the
generation of large population-wide spiking events for both the
ipsilateral
and
contralateral
visual
pathways
(Fig. 3f,
Kolmogorov–Smirnov test: binocular P < 0.0001, contralateral
P < 0.0001, ipsilateral P < 0.0001). As bursting activity from a few
neurons could strongly influence these statistics, we next We compared the response probabilities for visually responsive
neurons to monocular and binocular visual stimulations, with
and without vc pathway inactivation (Fig. 3e, total of 41 neurons,
14 binocular, 9 ipsilateral, 18 contralateral responsive neurons,
N = 6 populations, N = 5 animals). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 ARTICLE Frac. Depth (μm)
0
0.5
0
140
280
Rat 1
Rat 2
Rat 3
2
1
500 μm
L1
L2/3
L4
L5
L6
500 μm
0
2
4
6
VCPN
Non-VCPN
Spikes (Hz)
0
0.2
0.4
0
0.2
0.4
0
0.2
0.4
0
0.2
0.4
VCPN
Non-VCPN
P
P
Pref. ori. Mean rate (Hz)
contra-eye
Mean rate (Hz)
ipsi-eye
0
1
0
1
0
0.5
1
0
0.4
0
0.5
1
0
0.4
P
(CBI)
c
a
b
d
e
VCPN
Non-VCPN
Fig. 2 Functional responses of projecting and non-projecting neurons are indistinguishable. a Coronal rat brain section showing the CTB-Alexa 594
injection site and retrogradely labeled neurons in contralateral V1 (upper) expanded view of retrogradely labeled projection neurons location across cortical
layers (lower left, from upper image). Lower right: Overview of neurons labeled with OGB-1 (green, e.g. neuron 2) with callosal projecting neurons (VCPNs)
co-labeled with CTB (red, e.g. neuron 1). b Fraction of VCPNs as a function of depth from the pia (N = 3 animals). c Pooled data of spontaneous (gray) and
maximum stimulus response (black, average max. response to preferred orientation for VCPNs (N = 28 neurons) and non-VCPNs (N = 33 neurons)) and
mean ± SD (red) for each group. d Distribution of preferred orientations for orientation selective neurons from both VCPN and non-VCPN groups (ipsi-eye
stimulation, red, contra, turquoise). e Scatter plot comparing mean stimulus response of ipsi- to the mean response from the contralateral eye stimulation
(lower) for VCPNs (orange markers) and non-VCPNs (blue markers) with the distribution of the contralateral bias index shown in upper panels (see
“Methods” section for details). 0
0.2
0.4
0
0.2
0.4
0
0.2
0.4
0
0.2
0.4
VCPN
Non-VCPN
P
P
Pref. ori. d Mean rate (Hz)
contra-eye
Mean rate (Hz)
ipsi-eye
0
1
0
1
0
0.5
1
0
0.4
0
0.5
1
0
0.4
P
(CBI)
e
VCPN
Non-VCPN 2
1
500 μm
L1
L2/3
L4
L5
L6
500 μm
a d Mean rate (Hz) Fig. 2 Functional responses of projecting and non-projecting neurons are indistinguishable. a Coronal rat brain section showing the CTB-Alexa 594
injection site and retrogradely labeled neurons in contralateral V1 (upper) expanded view of retrogradely labeled projection neurons location across cortical
layers (lower left, from upper image). Lower right: Overview of neurons labeled with OGB-1 (green, e.g. neuron 2) with callosal projecting neurons (VCPNs)
co-labeled with CTB (red, e.g. Results 1.41 ± 1.08 Hz, mean ± SD, Mann–Whitney U test
P = 0.342, Fig. 2c), a significantly higher fraction of VCPNs were
responsive to drifting grating stimuli (VCPN vs. non-VCPN,
77.46 ± 3.37% vs. 55.91 ± 7.56%, mean ± SEM, two-tailed Fisher’s
exact test P = 0.0329). The distributions of preferred orientations
between the two groups were the same, indicating that this area NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 10−3
10−2
10−1
100
P
5
10
0
Spikes (n)
2
4
6
10−3
10−2
10−1
100
Active neurons (n)
0
P
f
g
e
0.5 mm
1 mm
*
a
d
LED
20X
V1
Contralateral
eye
Ipsilateral
eye
= LGN
*
*
*
−2
4
8
1
8
1
0
2
4
6
0
1.0
L1
L2/3
L4
L5
L6
Spikes (Hz)
Stim
Spon
Stim +LED
Spon+LED
2.0
8
WM
0
4
1µV
2s
L 2/3
L 4
L 5
L 6
1539µm
WM
b
c
Depth from pia (mm)
0
−2
h
0.5
00
1
1
0.5
Control response (p)
Inactivation response (p)
Trial (n)
Time from stim (s)
Time from stim (s)
Fig. 3 Anterograde tracing of callosal projection neurons and effects of optogenetic inhibition. a Coronal section of AAV-YFP injection into binocu
(asterisk). Right, enlarged view of region outlined by box in left panel. Note the red dye injection site for anatomical identification (arrow, same as in
b Coronal V1 section expressing eArchT-YFP (left, scale bar 0.5 mm) and example cell-attached recording from a neuron 1539 µm below pial surface d
periods of spontaneous activity without and with eArchT activation (upper, orange boxes). (Lower) Raster plots from a cell-attached recording sh
responses to visual stimuli without (left) and with eArchT activation (right, orange box). c Pooled data of firing rates before (black) and during activat
eArchT (orange) with recording depth for both spontaneous activity (circles) and stimulus-evoked activity (squares). d Schematic showing ipsilat
population imaging site (objective lens), location of contralateral callosal projecting neurons (arrow), ipsilateral (red) and contralateral (turquoise)
crossed optical pathway targeting lateral geniculate nuclei (LGN, star). Uncrossed pathways in blue. e Average response probabilities from populatio
visually responsive neurons (6 populations with 41 neurons) during binocular (blue), contralateral (turquoise), and ipsilateral stimulation (red) to m
gratings across all eight angles with and without inactivation of vc pathway. f Probability distributions of number of inferred action potentials evoke
stimulus period in a set population of visually responsive neurons (five populations with six neurons each) during binocular (blue), contralateral
(turquoise), ipsilateral (red) stimulation, spontaneous (dark gray), and shuffled (Poisson) spontaneous (light gray). Dashed lines indicate inactivation
pathway. g Probability distributions of number of active neurons per stimulus period. Populations and line conventions as in f. h Diagrams depicting
pathways activated during different conditions in f, g; unbroken lines control, dashed lines vc pathway inactivation. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Direct (turquoise, red, vertical
crossed (turquoise, red, diagonal) visual pathways, LGN (small square), contralateral (large open square) and ipsilateral (large square, green trian
cortex, with callosal pathway (line between large squares) intact or inactivated (yellow cross). Left panel: Binocular stimulation (lower bars). Middle
Contralateral stimulation. Right panel: Ipsilateral stimulation. 1 mm
*
a 0.5 mm
1 mm
*
a 0.5 mm d
LED
Contralate
eye
= LGN
*
*
−2
4
8
1
8
1
0
2
4
6
0
1.0
L1
L2/3
L4
L5
L6
Spikes (Hz)
Stim
Spon
Stim +LED
Spon+LED
2.0
8
WM
0
4
1µV
2s
L 2/3
L 4
L 5
L 6
1539µm
WM
b
c
Depth from pia (mm)
0
−2
Trial (n)
Time from stim (s)
Time from stim (s) 4
0
2
4
6
0
1.0
L1
L2/3
L4
L5
L6
Spikes (Hz)
Stim
Spon
Stim +LED
Spon+LED
2.0
8
WM
c
Depth from pia (mm) d
LED
20X
V1
Contralateral
eye
Ipsilateral
eye
= LGN
*
*
* d c 0
c f
from 10−3
10−2
10−1
100
P
5
10
0
Spikes (n)
f
( ) e
0.5
00
1
1
0.5
Control response (p)
Inactivation response (p)
( ) 2
4
6
10−3
10−2
10−1
100
Active neurons (n)
0
P
g g h
p
(p) h Fig. 3 Anterograde tracing of callosal projection neurons and effects of optogenetic inhibition. a Coronal section of AAV-YFP injection into binocular V1
(asterisk). Right, enlarged view of region outlined by box in left panel. Note the red dye injection site for anatomical identification (arrow, same as in right). b Coronal V1 section expressing eArchT-YFP (left, scale bar 0.5 mm) and example cell-attached recording from a neuron 1539 µm below pial surface during
periods of spontaneous activity without and with eArchT activation (upper, orange boxes). (Lower) Raster plots from a cell-attached recording showing
responses to visual stimuli without (left) and with eArchT activation (right, orange box). c Pooled data of firing rates before (black) and during activation of
eArchT (orange) with recording depth for both spontaneous activity (circles) and stimulus-evoked activity (squares). d Schematic showing ipsilateral
population imaging site (objective lens), location of contralateral callosal projecting neurons (arrow), ipsilateral (red) and contralateral (turquoise) eye
crossed optical pathway targeting lateral geniculate nuclei (LGN, star). Uncrossed pathways in blue. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Average response probability
of visually responsive neurons across all stimulation conditions
was 0.346 ± 0.19, mean ± SD (binocular stimulation 0.406 ± 0.206,
ipsilateral stimulation 0.255 ± 0.177, contralateral stimulation
0.377 ± 0.229,
N = 41
neurons),
and
0.605 ± 0.204
for
the
maximally tuned response angle (binocular stimulation 0.773 ±
0.213, ipsilateral stimulation 0.562 ± 0.264, contralateral stimula-
tion 0.746 ± 0.246, N = 41 neurons). Inactivating the vc pathway NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 e Average response probabilities from populations of
visually responsive neurons (6 populations with 41 neurons) during binocular (blue), contralateral (turquoise), and ipsilateral stimulation (red) to moving
gratings across all eight angles with and without inactivation of vc pathway. f Probability distributions of number of inferred action potentials evoked per
stimulus period in a set population of visually responsive neurons (five populations with six neurons each) during binocular (blue), contralateral
(turquoise), ipsilateral (red) stimulation, spontaneous (dark gray), and shuffled (Poisson) spontaneous (light gray). Dashed lines indicate inactivation of vc
pathway. g Probability distributions of number of active neurons per stimulus period. Populations and line conventions as in f. h Diagrams depicting visual
pathways activated during different conditions in f, g; unbroken lines control, dashed lines vc pathway inactivation. Direct (turquoise, red, vertical) and
crossed (turquoise, red, diagonal) visual pathways, LGN (small square), contralateral (large open square) and ipsilateral (large square, green triangles)
cortex, with callosal pathway (line between large squares) intact or inactivated (yellow cross). Left panel: Binocular stimulation (lower bars). Middle panel:
Contralateral stimulation. Right panel: Ipsilateral stimulation. Fig. 3 Anterograde tracing of callosal projection neurons and effects of optogenetic inhibition. a Coronal section of AAV-YFP injection into binocular V1
(asterisk). Right, enlarged view of region outlined by box in left panel. Note the red dye injection site for anatomical identification (arrow, same as in right). b Coronal V1 section expressing eArchT-YFP (left, scale bar 0.5 mm) and example cell-attached recording from a neuron 1539 µm below pial surface during
periods of spontaneous activity without and with eArchT activation (upper, orange boxes). (Lower) Raster plots from a cell-attached recording showing
responses to visual stimuli without (left) and with eArchT activation (right, orange box). c Pooled data of firing rates before (black) and during activation of
eArchT (orange) with recording depth for both spontaneous activity (circles) and stimulus-evoked activity (squares). d Schematic showing ipsilateral
population imaging site (objective lens), location of contralateral callosal projecting neurons (arrow), ipsilateral (red) and contralateral (turquoise) eye
crossed optical pathway targeting lateral geniculate nuclei (LGN, star). Uncrossed pathways in blue. e Average response probabilities from populations of
visually responsive neurons (6 populations with 41 neurons) during binocular (blue), contralateral (turquoise), and ipsilateral stimulation (red) to moving
gratings across all eight angles with and without inactivation of vc pathway. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 From all tuned neurons (101 neurons, N = 17 animals), 31%
significantly reduced response amplitudes upon silencing the vc
pathway, with 11% of these neurons becoming non-responsive
(not significantly different from spontaneous activity; excluded
from further statistical analysis). While the response amplitudes
at the preferred orientation were significantly reduced (Gauss. fitted
amplitude, control vs. vc pathway inactivated, −1.23 ± 0.69 Hz,
mean ± SD, Wilcoxon signed-rank test P < 0.0001, N = 23 neu-
rons, Supplementary Figs. 6a, 7 and 8), the preferred orientation
was not significantly changed (Supplementary Fig. 6b, absolute
difference in preferred orientation: 7.85 ± 7.94°, mean ± SD, cir-
cular one-sample t-test, P > 0.1, N = 23 neurons). In summary,
while the vc pathway strongly influences stimulus response
strength in a subpopulation of neurons, it has no significant
influence on orientation tuning. Neurons can be dependent or independent of vc pathway. For a
binocular neuron, a reduction in responsiveness upon stimulation
of the ipsilateral eye by inactivating the vc pathway implies that
the pathway must involve the crossed projection through the
contralateral LGN to contralateral V1, and then the vc pathway
from contralateral V1 (Fig. 4f, right panel). It follows that a
reduction in responsiveness upon stimulation of the contralateral
eye implies that the pathway must involve the uncrossed pro-
jection from the contralateral eye through the contralateral LGN
to contralateral V1 and then the vc pathway from contralateral
V1 (Fig. 4f, right panel). By quantifying the relative reduction of
each of the monocular pathways during vc pathway inactivation
compared to controls, each binocular neuron’s reliance on the vc
pathway could be compared. This analysis showed that for these
binocular neurons the contribution made by the vc pathway can
be via either of the monocular (ipsilateral or contralateral)
pathways or both (Fig. 4e, circular markers colored denoting
reduced pathway), suggesting that visually driven suprathreshold
activity can be generated from inputs arising from any of the
potential visual pathways that innervate VCPNs. This was not
specific to binocular neurons, as using the same analysis approach
for monocular neurons revealed a similar range of responses to vc
pathway inactivation (Fig. 4e). For both contralaterally and Vc pathway alters responses in monocular and binocular
neurons. We next asked whether individual monocular and
binocular neurons rely on this pathway for their response prop-
erties. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Stimulus-driven activity from each eye has two potential visual
pathways (crossed vs. uncrossed pathway) to V1, of which one
involves the vc pathway (Fig. 4d). One possibility was that the wide
range of effects of vc pathway inactivation was due to functionally
heterogeneous inputs driving the neurons arising from the different
visual pathways. As both of the monocular pathways can give rise
to either the binocular neurons’ spiking responses or monocular
neurons’ spiking responses, we next separately quantified the
contribution of each pathway for both monocular and binocular
neurons to establish which of the pathways was most affected by vc
inactivation (Fig. 4d). g
First, we ranked all visually responsive neurons according to
the change in their mean stimulus-evoked firing rate caused by vc
pathway inactivation (45 neurons significantly modulated, 26
binocular, 5 ipsilateral, and 14 contralateral (Fig. 4e), same data as
in Fig. 4a), and concurrently quantified for each neuron which of
the two monocular visual pathways was effected the most (Fig. 4e,
color coding denotes effect mainly on contralateral (more
turquoise) or ipsilateral (more red) eye response, black arrow
denotes example in Fig. 4c, gray arrow denotes example in
Supplementary Fig. 10). This analysis showed that the ipsilateral
crossed pathway (ipsi-eye→contra-LGN→contra-V1→ipsi-V1
via vc) reduced most in binocular neurons, especially in neurons
where responses after vc pathway inactivation were indistinguish-
able from spontaneous rates (Fig. 4c, e and Supplementary
Fig. 11a, ipsilateral vs. contralateral stimulus response reduction,
54.08 ± 26.88% vs. 35.95 ± 29.51%, mean ± SD, Mann–Whitney U
test P = 0.036). For this subpopulation of both monocularly and
binocularly responsive neurons, inactivation of the callosal input
resulted in an almost complete cessation of responses to
ipsilateral and contralateral eye stimulations (10 binocularly
responsive, 2 ipsilaterally responsive, and 2 contralaterally
responsive (Fig. 4e), Kruskal–Wallis nonparametric ANOVA
followed by one-tailed Fisher’s test, P > 0.05). It could be expected
that the contribution from the vc pathway forms a continuum
from suprathreshold responses being completely independent of
the vc pathway to them being completely reliant on activation of
the vc pathway (Fig. 4f). We next determined the fraction of
neurons in these different vc pathway-dependent classes. Response amplitude is altered but not preferred orientation. To quantify effects on orientation tuning, tuning curves were
estimated by fitting Gaussian curves to the raw orientation
responses29,30, and the peak angle of the Gaussian fits were
compared between control and vc pathway silencing conditions. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 f Probability distributions of number of inferred action potentials evoked per
stimulus period in a set population of visually responsive neurons (five populations with six neurons each) during binocular (blue), contralateral
(turquoise), ipsilateral (red) stimulation, spontaneous (dark gray), and shuffled (Poisson) spontaneous (light gray). Dashed lines indicate inactivation of vc
pathway. g Probability distributions of number of active neurons per stimulus period. Populations and line conventions as in f. h Diagrams depicting visual
pathways activated during different conditions in f, g; unbroken lines control, dashed lines vc pathway inactivation. Direct (turquoise, red, vertical) and
crossed (turquoise, red, diagonal) visual pathways, LGN (small square), contralateral (large open square) and ipsilateral (large square, green triangles)
cortex, with callosal pathway (line between large squares) intact or inactivated (yellow cross). Left panel: Binocular stimulation (lower bars). Middle panel:
Contralateral stimulation. Right panel: Ipsilateral stimulation. 5 NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 compared the spiking probability distributions based on the
number of active neurons per stimulus trial (N = 5 populations
with six neurons each, Fig. 3g). Both binocular and contralateral
stimulation evoked similar probability distributions of active
neurons per stimulus, whereas ipsilateral stimulation activated
significantly fewer neurons (Fig. 3g; binocular vs. contralateral,
Kolmogorov–Smirnov test P = 0.8532, excluding 0 bin; binocular
vs. ipsilateral; Kolmogorov–Smirnov test P < 0.0001, excluding 0
bin). Binocular stimulation had the highest probability of evoking
one or more neurons per stimulus trial compared to monocular
stimulation of either eye (probability of 0 neurons active:
binocular: 0.0942, contralateral: 0.1386, ipsilateral: 0.2983). For
both binocular and monocular stimuli, vc pathway inactivation
significantly increased the number of trials where 0 neurons were
active and also significantly decreased the probability of observing
activity in groups of neurons of all sizes (binocular LED off vs. on,
Kolmogorov–Smirnov test P < 0.0001; contralateral LED off vs. on, Kolmogorov–Smirnov test P < 0.001; ipsilateral LED off vs. on, Kolmogorov–Smirnov test P < 0.0001). Together, this shows
that both the crossed and uncrossed visual pathways originating
from either eye utilize the vc pathway (Fig. 3h). We next
quantified how this pathway is involved in the suprathreshold
responses of individual neurons and whether individual response
classifications rely on this pathway. (Fig. 4a, b) to reducing visual stimulus responses to not significantly
different to spontaneous activity (Fig. 4c, Supplementary Fig. 9). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 f
0 1 2
0
8
0 1 2
0 1 2
0
8
0 1 2
0 1 2
0 1 2
20%
ΔF/F
2 s
Vc-pathway inactivated
Binocular stim
Contra stim
Ipsi stim
Trial (n)
0 1 2
0
8
0 1 2
0 1 2
20%
ΔF/F
2 s
Binocular stim
Contra stim
Ipsi stim
0 1 2
0
8
0 1 2
0 1 2
b
Vc-pathway inactivated
c
g
0
0.5
1
0
0.1
0.2
0.3
P
VC reduction index
Ipsi-
responsive
only
Bino-
responsive
only
Peak response (Hz)
Contra-
responsive
only
0
1
2
a
e
0
20
40
−0.8
−0.4
0
0.4
Abolished
Mono contralateral
Mono ipsilateral
Binocular
Changed
Reduced
contra
Reduced
ipsi
=
Ranked (n)
Δ
d
Cntrl
Inact
Cntrl
Inact
Cntrl
Inact
Trial (n)
Trial (n)
Trial (n)
Time from stim onset (s)
Time from stim onset (s)
Time from stim onset (s)
Time from stim onset (s)
Fig. 4 Callosal pathway modulates spiking responses in binocular and monocular neurons. a Firing rate changes for maximum response angle from
visually responsive neurons without (control, Cntrl) and with (Inact.) callosal pathway inactivation for contralaterally only (left), ipsilaterally only (middle)
and binocularly (right) responsive neurons. Significant (purple, orange) and non-significant (gray) changes depicted. b Example responses from a neuron
not influenced by vc pathway inactivation. Upper panel, example of Ca2+-transients from each stimulus trial (gray) and resulting average (black) during
binocular (left), contralateral (middle), and ipsilateral eye stimulation (right) for maximum response angle (see “Methods” section), inferred spikes (singles
black, doubles dark blue, triples light blue) from transients for each trial (8 trials). Lower panel, same as in upper panel, but with vc pathway inactivation. c Example responses from a neuron showing a strong vc pathway inactivation effect. Convention same as in b. d Visual pathways activated during control
and vc inactivation conditions, conventions as in Fig. 3h. e Graph depicting amount of significant firing rate change in binocularly (circles), ipsilaterally
(triangles), and contralaterally (squares) responsive neurons during silencing of the callosal pathway (same as purple and orange in a). Neurons ranked
from those that increased firing rates to those that decreased firing to not significantly different from spontaneous activity (open symbols). Symbol color
denotes whether ipsilateral (red) or contralateral (turquoise) response decreased most or both decreased equally (black). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 We first quantified how the activity of individual neurons,
classified as either monocularly responsive (ipsilateral or con-
tralateral) or binocularly responsive (Fig. 1g), was modulated by
the vc pathway (Fig. 4a). Silencing significantly changed stimulus-
evoked firing rates in 30.61% of visually responsive neurons
(45 from 147 responsive neurons, total of 279 neurons recorded,
N = 17 animals, see “Methods” section for details). This change
occurred in both classes of monocularly responsive neurons, as
well as in binocularly responsive neurons (contra-responsive
14/63, ipsi-responsive 5/28, binocular 26/56 neurons, Fig. 4a). This
modulation was not associated with a change in the spontaneous
firing rates between the two conditions (before vs. after con-
tralateral silencing, 0.092 ± 0.25 vs. 0.080 ± 0.27 Hz, median ± SD,
two-sample t-test, P = 0.704). From this analysis, it was clear that vc pathway inactivation had
a range of effects on stimulus-evoked spiking, from ineffective NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Black arrow depicts example
neuron shown in c, gray arrow depicts example neuron shown in Supplementary Fig. 10. f Visual pathways activated during binocular stimulation, showing
different amounts of contribution of the vc pathway, ranging from responses being completely reliant on (right) to being independent of (left) vc pathway
activation. g Probability distribution of the amount of influence of the vc pathway on responses, quantified as the vc reduction index for binocularly (blue),
ipsilaterally (red), and contralaterally (turquoise) responsive neurons. 0 1 2
0
8
0 1 2
0 1 2
20%
ΔF/F
2 s
Binocular stim
Contra stim
Ipsi stim
0 1 2
0
8
0 1 2
0 1 2
Vc-pathway inactivated
c
Trial (n)
Trial (n)
Time from stim onset (s)
Time from stim onset (s) Ipsi-
responsive
only
Bino-
responsive
only
Peak response (Hz)
Contra-
responsive
only
0
1
2
a
Cntrl
Inact
Cntrl
Inact
Cntrl
Inact f
d a c 0 1 2
0
8
0 1 2
0 1 2
Binocular stim
Contra stim
Ipsi stim
b
Trial (n)
Time from stim onset (s) f b 0
20
40
−0.8
−0.4
0
0.4
Abolished
Mono contralateral
Mono ipsilateral
Binocular
Changed
Reduced
contra
Reduced
ipsi
=
Ranked (n)
Δ g
0
0.5
1
0
0.1
0.2
0.3
P
VC reduction index g 0 1 2
0
8
0 1 2
0 1 2
20%
ΔF/F
2 s
Vc-pathway inactivated
Trial (n)
Time from stim onset (s) Time from stim onset (s) Fig. 4 Callosal pathway modulates spiking responses in binocular and monocular neurons. a Firing rate changes fo Fig. 4 Callosal pathway modulates spiking responses in binocular and monocular neurons. a Firing rate changes for maximum response angle from
visually responsive neurons without (control, Cntrl) and with (Inact.) callosal pathway inactivation for contralaterally only (left), ipsilaterally only (middle)
and binocularly (right) responsive neurons. Significant (purple, orange) and non-significant (gray) changes depicted. b Example responses from a neuron
not influenced by vc pathway inactivation. Upper panel, example of Ca2+-transients from each stimulus trial (gray) and resulting average (black) during
binocular (left), contralateral (middle), and ipsilateral eye stimulation (right) for maximum response angle (see “Methods” section), inferred spikes (singles
black, doubles dark blue, triples light blue) from transients for each trial (8 trials). Lower panel, same as in upper panel, but with vc pathway inactivation. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 c Example responses from a neuron showing a strong vc pathway inactivation effect. Convention same as in b. d Visual pathways activated during control
and vc inactivation conditions, conventions as in Fig. 3h. e Graph depicting amount of significant firing rate change in binocularly (circles), ipsilaterally
(triangles), and contralaterally (squares) responsive neurons during silencing of the callosal pathway (same as purple and orange in a). Neurons ranked
from those that increased firing rates to those that decreased firing to not significantly different from spontaneous activity (open symbols). Symbol color
denotes whether ipsilateral (red) or contralateral (turquoise) response decreased most or both decreased equally (black). Black arrow depicts example
neuron shown in c, gray arrow depicts example neuron shown in Supplementary Fig. 10. f Visual pathways activated during binocular stimulation, showing
different amounts of contribution of the vc pathway, ranging from responses being completely reliant on (right) to being independent of (left) vc pathway
activation. g Probability distribution of the amount of influence of the vc pathway on responses, quantified as the vc reduction index for binocularly (blue),
ipsilaterally (red), and contralaterally (turquoise) responsive neurons. Fig. 4 Callosal pathway modulates spiking responses in binocular and monocular neurons. a Firing rate changes for maximum response angle from
visually responsive neurons without (control, Cntrl) and with (Inact.) callosal pathway inactivation for contralaterally only (left), ipsilaterally only (middle)
and binocularly (right) responsive neurons. Significant (purple, orange) and non-significant (gray) changes depicted. b Example responses from a neuron
not influenced by vc pathway inactivation. Upper panel, example of Ca2+-transients from each stimulus trial (gray) and resulting average (black) during
binocular (left), contralateral (middle), and ipsilateral eye stimulation (right) for maximum response angle (see “Methods” section), inferred spikes (singles
black, doubles dark blue, triples light blue) from transients for each trial (8 trials). Lower panel, same as in upper panel, but with vc pathway inactivation. c Example responses from a neuron showing a strong vc pathway inactivation effect. Convention same as in b. d Visual pathways activated during control
and vc inactivation conditions, conventions as in Fig. 3h. e Graph depicting amount of significant firing rate change in binocularly (circles), ipsilaterally
(triangles), and contralaterally (squares) responsive neurons during silencing of the callosal pathway (same as purple and orange in a). Neurons ranked
from those that increased firing rates to those that decreased firing to not significantly different from spontaneous activity (open symbols). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 *
Muscimol
injection
LED
20X
V1
V1
Contralateral eye
Ipsilateral eye
= LGN
*
a
b
MUSC + LED Off
MUSC + LED On
Contra stim
Ipsi stim
1 s
5%
Δ F/F
0
1
2
3
Ipsi stim
Contra stim
LED Off
LED On
MUSC + LED Off
MUSC + LED On
Spon before MUSC
Peak response (Hz)
4
d
e
Neuropil activity
25 μm
c
Fig. 5 Activation of callosal pathway alone from either eye is not
sufficient to drive spiking. a Schematic of experimental design showin necessarily imply that there are neurons wholly reliant on this
pathway for their inputs or subthreshold activity. b
c necessarily imply that there are neurons wholly reliant on this
pathway for their inputs or subthreshold activity. *
Muscimol
injection
LED
20X
V1
V1
Contralateral eye
Ipsilateral eye
= LGN
*
a b
c a Visual responses are abolished by silencing ipsilateral LGN. We
next investigated whether the vc pathway alone was able to
generate spiking activity (Fig. 5a–c). We inactivated the LGN on
one side with muscimol (8.8 mM) and recorded visually evoked
responses in V1 on the same side (N = 4 animals, N = 28 visually
responsive neurons). In a subset of these experiments (N = 2
animals, N = 21 visually responsive neurons), we reversibly
inactivated the vc pathway before LGN inactivation to confirm
that vc inactivation had the same effect as described before
(compare Figs. 5d and 4a). Vc pathway inactivation modulated
visually responsive neurons with the same average reduction as in
the previous experiments (Fig. 5d, Kolmogorov–Smirnov test
P = 0.4741, Supplementary Fig. 12). As in the previous experi-
mental group, a subpopulation of neurons were observed whose
responses to stimulation of the ipsilateral eye were abolished on
vc pathway silencing (Fig. 5d, N = 2 neurons). The inactivation of
ipsilateral LGN abolished visually evoked spiking responses in all
visually responsive neurons (N = 4 animals, N = 28), including
neurons where responses had been reduced to the level of
spontaneous activity on vc pathway silencing. Firing rates in
neurons responsive to ipsilateral eye stimulation were sig-
nificantly reduced upon LGN inactivation (1.585 ± 0.79 Hz vs. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 0.217 ± 0.108 Hz, mean ± SD, paired Wilcoxon signed-rank test
P < 0.0001, N = 10 binocular neurons and five ipsi-monocular
neurons) and in the order of spontaneous rates (0.301 ± 0.135 Hz,
mean ± SD, spontaneous rates before LGN inactivation: paired
Wilcoxon signed-rank test P = 0.018; 0.101 ± 0.09 Hz, mean ± SD,
spontaneous rates after LGN inactivation: paired Wilcoxon signed-
rank test P = 0.0026). Also, firing rates in neurons responsive to
contralateral eye stimulation were significantly reduced upon LGN
inactivation (1.635 ± 0.779 Hz vs. 0.206 ± 0.185 Hz, mean ± SD,
paired Wilcoxon signed-rank test P < 0.0001, N = 10 binocular
neurons and 13 contra-monocular neurons) and not different to
spontaneous firing rates (0.243 ± 0.136 Hz, mean ± SD, before LGN
inactivation, paired Wilcoxon signed-rank test P = 0.307; 0.157 ±
0.123 Hz, mean ± SD, after LGN inactivation, paired Wilcoxon
signed-rank test P = 0.2734). Responses prior to inactivation of
LGN compared to responses after inactivation of LGN are shown
for eight representative binocular neurons (Fig. 5d, ipsilateral eye
stimulation: 1.778 ± 0.703 Hz vs. 0.258 ± 0.094 Hz, mean ± SD,
paired Wilcoxon signed-rank test P = 0.0078; contralateral eye sti-
mulation: 2.068 ± 1.044 Hz vs. 0.24 ± 0.158 Hz, mean ± SD, paired
Wilcoxon signed-rank test P = 0.0078). 0
1
2
3
Ipsi stim
Contra stim
LED Off
LED On
MUSC + LED Off
MUSC + LED On
Spon before MUSC
Peak response (Hz)
4
d d MUSC + LED Off
MUSC + LED On
Contra stim
Ipsi stim
1 s
5%
Δ F/F
e
Neuropil activity
25 μm e e MUSC + LED On 1 s Ipsi stim Contra stim Fig. 5 Activation of callosal pathway alone from either eye is not
sufficient to drive spiking. a Schematic of experimental design showing
inactivation of ipsilateral LGN (muscimol injection) and reversible
inactivation of vc pathway. b Cartoon depicting pathways deactivated
during muscimol injection (upper) for ipsilateral stimulation and for
experiments with muscimol injection and vc pathway inactivation (lower). c Cartoon depicting pathways deactivated during muscimol injection
(upper) for contralateral stimulation and for experiments with muscimol
injection and vc pathway inactivation (lower). d Comparison of spiking
response for maximum response angle during ipsilateral (left, 8 neurons,
ipsilaterally responsive, 2 animals) and contralateral (right, 8 neurons,
contralaterally responsive, 2 animals) only stimulation. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Symbol color
denotes whether ipsilateral (red) or contralateral (turquoise) response decreased most or both decreased equally (black). Black arrow depicts example
neuron shown in c, gray arrow depicts example neuron shown in Supplementary Fig. 10. f Visual pathways activated during binocular stimulation, showing
different amounts of contribution of the vc pathway, ranging from responses being completely reliant on (right) to being independent of (left) vc pathway
activation. g Probability distribution of the amount of influence of the vc pathway on responses, quantified as the vc reduction index for binocularly (blue),
ipsilaterally (red), and contralaterally (turquoise) responsive neurons. suprathreshold activity unaffected by vc inactivation (Fig. 4g). This is consistent with the presence of representatives of all of the
potential anatomical pathways from the eye to the VCPNs within
the total population of monocular neurons in V1. In addition,
although callosally projecting axons have been observed to
accumulate into patches along the lateral edge of V1 in flattened-
cortex histological preparations from rats11, we did not observe
any clustering of neurons whose responses were more strongly
modulated by vc pathway inactivation (Supplementary Fig. 11b). The above analyses suggest that the vc pathway plays a crucial
role in generating spiking responses within its innervation
domain in binocular primary visual cortex, but this does not ipsilaterally responsive neurons, responses to visual stimuli at the
preferred orientation were reduced to spontaneous rates for a
small number of neurons (N = 2 contralaterally responsive neu-
rons, N = 2 ipsilaterally responsive neurons). To more directly
compare the contribution from the vc pathway for monocular
and binocular neurons, we calculated the vc reduction index. This
index was bounded between 0 and 1, with 0 denoting no con-
tribution from the vc pathway and 1 denoting complete reliance
on the vc pathway (see “Methods” section for details). As for the
binocular neurons, the majority of neurons’ monocularly driven
suprathreshold activity arose from a combination of crossed and
uncrossed pathway activation, with a small number of neurons’ TURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 7 7 ARTICLE Discussion
H
h Here, we show that neuronal populations in lateral binocular V1
contain a mixture of projecting, i.e. VCPNs, and non-projecting
neurons, non-VCPNs, that do not show obvious spatial organi-
zation. Functionally, VCPNs can be binocularly or monocularly
responsive to stimulation of either ipsilateral or contralateral eye
and, taken as a population, their functional tuning is not sig-
nificantly different from non-VCPNs. Upon inactivation of the
callosal pathway, spiking responses in a large proportion of
contralateral V1 neurons were modulated, both monocularly and
binocularly responding. The effect of vc pathway inactivation was
marked, and many neurons showed a significant spiking reduc-
tion or, in some cases, a complete extinction of responses to visual
stimuli. We also show that a subpopulation of neurons depends
on both input from callosal projection neurons and simultaneous
input from the ipsilateral LGN for their binocular spiking
responses. In the LGN inactivation experiments, the presence of a visually
evoked neuropil signal is an important indication that the con-
tralateral LGN and callosal pathway are still viable and active in
the presence of ipsilateral LGN inactivation. As the neuropil
signal represents Ca2+-influx into synaptic boutons on axons
labeled with Ca2+-indicators31, this signal probably represents
the activity in the axons of the VCPNs. The finding that the
neuropil signal remaining during VCPN inactivation is abolished
by inactivation of the ipsilateral LGN (Fig. 5) is consistent with
there being only two possible pathways from ipsilateral eye to
visual cortex, the uncrossed projection from the ipsilateral retina
and the crossed projection to the contralateral LGN and visual
cortex via the callosal projection (ipsilateral eye–contralateral
LGN–contralateral visual cortex–ipsilateral visual cortex via cor-
pus callosum). However, as the neuropil response to contralateral
eye stimulation was not abolished with the combination of VCPN
silencing and LGN inactivation, it would seem that there is an
additional anatomical pathway activated by contralateral eye
stimulation. One potential pathway is the retinal projection to the
lateral posterior nucleus of the thalamus, and the latter’s pro-
jection to V1 (refs. 2,37). p
Studies using retrograde tracing or virus-mediated expression
techniques are limited at least to some extent by the total fraction
of the target neuronal population labeled. In the current study,
this applies both to the combined retrograde tracing and
Ca2+-imaging experiments as well as to the optogenetic manip-
ulation of contralateral V1. In retrograde tracing experiments, we
found that around 35% of neurons in layer 2/3 were labeled. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Control (dark blue,
no muscimol), vc pathway inactivation (magenta), LGN inactivation
(purple), and LGN inactivation with vc pathway inactivation (yellow). Spontaneous activity rates (black) shown for each neuron. Ipsilateral
responses not different to spontaneous activity on vc pathway silencing
indicated by orange lines. e Left panel, example field of view with labeled
neuropil ROI (shaded area); right panel, neuropil responses evoked by
stimulation of ipsilateral (left) or contralateral (right) eye recorded during
silencing of ipsilateral LGN without (upper) and with VCPN-inactivation
(lower). Individual (gray) and average (black) responses. Shuffled neuropil
responses (white). Stimulus onset (black arrow head) and offset (gray
arrow head). g
)
To confirm that muscimol injection was confined to ipsilateral
LGN and not causing a more general inactivation of visual
responses,
we
examined
neuropil-related
Ca2+-fluorescence
signals, which should include fluorescence transients in the vc
pathway axons. Bulk loading of cortical tissue is known to
label the neuropil in addition to neurons and astrocytes within
the
loaded
area. Labeled
neuropil
also
displays
strong
fluorescence intensity fluctuations, which have been shown to
be
correlated
with
both
neuronal
intracellular
membrane
potential fluctuations and the electrocorticogram31,32. Further-
more, the neuropil-related Ca2+-signals have also been shown to
originate from labeled axonal structures31. After muscimol
injection, visual stimuli to either the ipsilateral or contralateral
eye evoked small amplitude Ca2+-transients in the neuropil
(Fig. 5e upper). These transients were locked to the visual
stimuli, consistent with the stimulus evoking activity in axons
whose somata of origin lay outside of ipsilateral LGN. These
neuropil transients were not found in a shuffled-data control of NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 8 8 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 possible that this might also be the case for the callosal projection
in other species too. this analysis (Fig. 5e, white traces, two-sample t-test P = 0.304). Optogenetic silencing of contralateral hemisphere, and therefore
the vc pathway axons, abolished the neuropil Ca2+-transients
evoked by stimulation of ipsilateral eye (Ca2+-transients not
significantly different from shuffled data, two-sample t-test P =
0.691, Fig. 5e, left lower). Callosal silencing had little effect on the
neuropil-related Ca2+-transients evoked by stimulation of the
contralateral eye, suggesting that the contralateral eye has a
projection that does not involve the vc pathway or the projection
from the ipsilateral LGN (Ca2+-transients not significantly
different between silencing and non-silencing conditions, two-
sample t-test P = 0.3261 (Fig. 5e, right lower). Discussion
H
h Compared to the unlabeled neurons, the labeled population did
not show significant differences in tuning and response proper-
ties, though the possibility still remains that any unlabeled pro-
jection neurons may have more selective visual response
properties. A similar possibility exists also for the optogenetic
manipulation experiments. However, given that an appreciable
fraction of neurons show significant modulation of responses,
including reduction of evoked responses to the level of sponta-
neous activity, we clearly show that activity in callosally pro-
jecting neurons is an important component of visual responses
for many V1 neurons. The callosal projection in the visual cortex has been observed
in many mammalian species4–10,33–35. Functionally, initial studies
in cats showed that the callosal projection targets the retinoto-
pically matched region of contralateral visual cortex3,4 and,
similar to the findings in the current study, that inhibition of
CPNs by cortical cooling has a variety of consequences for the
visual responses of neurons in the recipient cortical region
including a general reduction in activity36. One key finding in the
current study is the callosal projection in rats is only able to drive
spiking responses in their projection targets when there is
simultaneous activity in the pathway from the ipsilateral LGN. Given that there are broad similarities in callosal anatomy and
function across the different species described above it seems In conclusion, we have shown that the visual callosal pathway,
while contributing substantial input to many neurons and being
responsible for all spiking responses in some neurons, requires
concurrent activation of inputs from the ipsilateral thalamus in
order to drive suprathreshold responses. This suggests a much
greater role of the rodent callosal pathway in cortical processing
than previously thought. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 This strongly
suggests that the vc pathway provides stimulus-driven activity
that is alone unable to drive the ipsilateral neurons to spike but
spiking
activity
is
contingent
upon
co-activation
of
the
ipsilateral LGN. p
Previous studies investigating the role of the vc pathway in
rodents are contradictory, some concluding that the callosal
pathway
contributes
ipsilateral-eye-derived
visually
evoked
activity13,22,23, with others concluding that it does not11,24. Also,
some previous studies found no effect of callosal pathway inac-
tivation on contralateral eye responses23. Our current findings
showing that callosal pathway inactivation has a wide range of
effects on cortical spiking, from no-effect to complete spike
abatement, go some way to reconcile the differences between
seemingly contradictory findings of previous studies. This is most
likely because the present study was able to record simultaneously
from populations of visually responsive neurons across large
spatial areas and reversibly inactivate VCPN somas across all
cortical layers. In the current study, we found no significant influence of
inactivation of the vc pathway on orientation-tuning properties of
V1 neurons. For some neurons, strongly orientation tuned
responses were no longer present in the presence of vc pathway
inactivation (see Supplementary Figs. 7, 8), suggesting that the
orientation tuning was due to the orientation tuning of the pre-
synaptic
callosally
projecting
neurons. For
other
neurons,
strongly tuned responses were reduced in response amplitude, but
without changing the preferred orientation (see Supplementary
Figs. 7, 8). These findings raise the possibility that the axons of
neurons projecting through the vc pathway target contralateral
neurons with similar orientation preference to that of the pro-
jecting neurons, consistent with the findings of a recent study
showing that spines in V1 neurons connecting with axons from
the vc pathway cluster more with spines from local neurons with
similar orientation preference25. This same study also showed
that the callosal-recipient spines have more similar orientation
preference to the soma than non-callosal recipient spines25. All experiments were carried out on male Lister hooded rats (Rattus norvegicus).
For retrograde anatomical tracing experiments, rats (N = 5) were between 93 and NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunicati ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 160 g body weight at the time of tracer injection. For anterograde axonal tracing
experiments, rats (N = 2) were of 55 and 58 g body weights at the time of tracer
injection. For experiments combining imaging and retrograde tracing, rats (N = 3)
were between 135 and 142 g body weight at the time of tracer injection. For
experiments involving imaging or electrophysiology together with V1 inactivation
and/or LGN inactivation, rats (N = 31) were between 137 and 287 g body weight at
the time of the recordings. Experiments were approved by the relevant animal
welfare authorities (Regierungspraesidium Tuebingen and Landesamt für Natur,
Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany). silencing with eArchT and Ca2+-imaging, with the difference that an additional
craniotomy (1 × 1 mm located at Lambda −3 mm, lateral 2 mm) was opened in the
same hemisphere in which Ca2+-imaging was carried out. Through this cra-
niotomy after the completion of the first session involving simultaneous cortical
silencing and Ca2+-imaging, a glass pipette was advanced 3.6 mm into thalamus
(LGN) and a 184 nL bolus of 8.8 mM muscimol (Sigma Aldrich, MO, USA)
injected, after which a second session of Ca2+-imaging was carried out. Electrophysiological recording. Electrophysiological recordings were made using
glass pipettes filled with artificial cerebrospinal fluid (ACSF) with the following
composition (in mM): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH
balanced to 7.2 (300 mOsm/l). Signals were amplified using a multiclamp 700B
amplifier (Molecular Devices, CA, USA), and digitized at 31.25 kHz using a Power
1401 analog to digital (AD) converter (Cambridge Electronic Design, Cambridge,
UK). Timing of visual stimuli was recorded using the same AD converter to allow
analysis of stimulus-related action potential firing. Animal preparation for anatomical tracing experiments. Animals were anes-
thetized with ketamine (200 μg/kg)/medetomidine (100 mg/kg), and body tempera-
ture maintained at 37 °C using a thermal probe and heating pad. Depth of anesthesia
was monitored throughout the procedure via assessment of withdrawal reflexes with
supplemental doses of anesthetic provided where necessary. The head was immo-
bilized using ear and tooth bars and the skin and galea on the dorsal aspect of the
skull opened. ARTICLE A 2 × 2 mm
craniotomy was made, and the underlying dura removed. The exposed cortex was
then stabilized with agar (1.2%, Sigma Aldrich, MO, USA) and a coverslip. Body
temperature was maintained at 37 °C throughout the experiment using a thermal
probe and heating pad, and anesthetic depth monitored throughout via assessment
of withdrawal reflexes with additional anesthetic doses provided where required. Visual stimulation. All visual stimuli were created, controlled, and displayed using
Psychtoolbox software42 executed in Matlab (Mathworks, MA, USA). Visual sti-
muli were presented on a CRT monitor (Sony CPD-G520, resolution: 1600 × 1200,
refresh rate: 60 Hz) having constant gray background (30 candela/m2) that changed
to a full-field stimulus image (mean luminance: 30 candela/m2). The CRT monitor
was placed 48 cm in front of eyes and occupied 45° × 35° (width × height) in visual
space centered in front of the animal’s nose. Animal preparation for cortical silencing and neural recording. Animals were
anesthetized and injected with AAV-eArchT as described above for anatomical
tracing experiments, with the craniotomy (~400 × 400 μm) made over either left or
right visual cortex at lambda −1.3 ± 0.2 mm, lateral 4.1 ± 0.2 mm (mean ± SD). Expression time after injections was between 15 and 24 days. For Ca2+-imaging
experiments, animals were anesthetized with urethane (1.9 g/kg), the skin and galea
on the dorsal aspect of the skull removed, a custom-made metal headplate
implanted over either left or right visual cortex contralateral to the previous AAV-
eArchT injection site, a 2 × 2 mm craniotomy opened and the underlying dura
removed. The underlying cortex was then stabilized with agar and a coverslip. A
craniotomy (3 × 3 mm) was also opened over the previous AAV-eArchT injection
site and a LED (Golden Dragon, 590 nm, OSRAM, Regensburg, Germany) with
attached half-ball lens (S-LAH79, 2 mm diameter, Edmund Optics, NJ, USA)
was mounted over the cortical site, with shielding to prevent the light from escaping
the cortex. The effective power density of the LED at the cortical surface was
12.8 mW/mm2. For electrophysiology experiments to confirm the effectiveness of
eArchT-induced neuronal silencing, animals were prepared as described for imaging
experiments with the exception that the craniotomy (3 × 3 mm) was opened only
over the site of previous AAV-eArchT injection in either left/right visual cortex. ARTICLE For mono-color retrograde tracing experiments, a small (~400 × 400
μm) craniotomy was opened over the right visual cortex (mean ± SD; lambda −2.3 ±
0.2 mm, lateral 3.9 ± 0.2 mm), the underlying dura opened and 115 nL cholera toxin
subunit B (CTB) conjugated to Alexa 594 (Molecular Probes, OR, USA) injected
using a custom-built injection setup. For tri-color retrograde tracing experiments,
three small (~400 × 400 μm) craniotomies were opened over the left visual cortex (rat
1; lambda −1.6, −1.7, and −1.6 mm, lateral 2.0, 2.8 and 4.0 mm, rat 2; lambda −1.5,
−1.4, and −1.6 mm, lateral 2.9, 3.4, and 4.3 mm), small openings in the underlying
dura made in each and one of either CTB conjugated to Alexa Fluor 488, 594, or 647
(all from Molecular Probes, OR, USA) injected into each craniotomy at 0.45 and
1.05 mm below the pia (46 nL at each depth). For anterograde tracing, a single
craniotomy (~400 × 400 μm) was made over the left visual cortex (rat 1; lambda
−1.0 mm, lateral 4.1 mm, rat 2; lambda −1.0 mm, lateral 4.1 mm), a small opening
in the dura made, and an injection of AAV5-CaMKIIα-eArchT3.0-eYFP (UNC
Vector Core, NC, USA) that expresses yellow fluorescent protein was made (injec-
tion depth from pia 0.45 and 1.15 mm, 184 nL at each depth). Following the injec-
tions, the craniotomy was protected with KwikSil (World Precision Instruments, FL,
USA), and the skin incision closed with vicryl sutures. 14–19 days after CTB
injections or 20–22 days after anterograde tracer injections, animals were deeply
anesthetized, perfused transcardially with 4% paraformaldehyde (Roti-histofix 4%,
Carl Roth GmbH, Karlsruhe, Germany) and the brain removed. After postfixation
for at least 12 h, 100 μm-thick coronal sections were cut on a vibratome or a freezing-
microtome and mounted using anti-fade mounting medium (Vectashield, Biozol
Diagnostica Vertrieb GmbH, Eching, Germany). Images of the coronal sections and
fluorescently labeled neurons were subsequently acquired on a conventional fluor-
escence microscope or confocal microscope. Intrinsic imaging of visually related brain activity. Intrinsic imaging was per-
formed as described previously38. In brief, excitation wavelength was 630 ± 30 nm. Visual stimulation consisted of full-field rectangular drifting gratings (width ×
height; 45° × 35°, displayed 48 cm in front of rat’s eyes, 8 drift directions, other
stimulus properties as described below) presented for 5 s with inter-stimulus
interval of 30 s. ARTICLE Image acquisition started 2 s before the onset of each stimulus
presentation and ended 3 s after the offset of stimulus presentation (images were
acquired every 100 ms within this period). The images acquired before the onset of
stimulus presentation were averaged together, with the result used for background
subtraction. Image frames between stimulus onset and offset were averaged and a
contour at the 90 percentile used as indicative of the region activated by the visual
stimulus. To facilitate navigation under multiphoton microscopy and to allow
subsequent alignment of multiphoton data from multiple animals on the basis of
the intrinsic optical signal imaging response (see below for details), we also
acquired an image of the cortical surface vasculature. Ca2+-indicator loading and astrocyte counterstaining. Cortical layer 2/3 neu-
rons were loaded with the Ca2+-indicator Oregon green 488 BAPTA-1 AM
(Thermo Fischer, MA, USA) and astrocytes with sulforhodamine 101 (Sigma
Aldrich, MO, USA) as described previously39,40. Multiphoton microscopy. Multiphoton imaging was performed using a custom-
built, laser scanning multiphoton microscope as described in refs. 39,41. In a subset
of experiments, the galvanometric scanners were replaced with a resonant scanning
system consisting of an 8 kHz resonant scanner on one scan axis (Sutter Instru-
ments, CA, USA) and conventional galvanometric scanner on the other. Excitation
was provided by a Mai Tai laser (Spectra Physics, CA, USA) at 920 nm and images
acquired using Scanimage software (Vidrio Technologies, VA, USA). The objective
lens used for all functional imaging was a Zeiss ×20 1.0 NA objective (Carl Zeiss
AG, Oberkochen, Germany). In experiments using the conventional galvanometric
scanning system, 128 × 64 pixel full frame images were acquired at a frame rate of
18.6 Hz, and in those using the resonant scanning system, 512 × 512 pixel full
frame images were acquired at 29.98 Hz. Animal preparation for anatomical tracing and Ca2+-imaging. For experiments
combining Ca2+-imaging and retrograde tracing of contralateral projection neu-
rons, an injection of Alexa Fluor 594 conjugated CTB was made in the right
hemisphere at lambda −1.7 ± 0.1 mm, lateral 4.3 ± 0.1 mm (mean ± SD) as
described above. After 10–15 days, animals were anesthetized with urethane
(1.9 g/kg) and prepared for in vivo Ca2+-imaging. The skin and galea on the dorsal
aspect of the skull were removed and a custom-made metal headplate implanted
over the left visual cortex (contralateral to the previous CTB injection). Methods All experiments were carried out on male Lister hooded rats (Rattus norvegicus). For retrograde anatomical tracing experiments, rats (N = 5) were between 93 and TURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications 9 9 Preferred orientation ¼ arg ðSÞ
2 The 90th percentile contour
determined from the intrinsic optical signal imaging response data (Supplementary
Fig. 1a) was overlayed first on the image of the cortical vasculature (Supplementary
Fig. 1b). The location of the center of mass was determined with reference to the
surface vasculature pattern, and the corresponding point located manually in
multiphoton images of the surface vasculature above the neurons from which
functional multiphoton imaging data were acquired (Supplementary Fig. 1c). Finally, the location of the individual neurons in the multiphoton image of the
surface vasculature were determined in images within layer 2/3 of z-stacks
containing the images of the surface vasculature (Supplementary Fig. 1c, d). Analysis of neuropil-related Ca2+-fluorescence signal. A region of interest
(ROI) excluding the neuronal somatas and astrocytes, and about 1/5 the total size
of the image was marked to obtain the neuropil-related Ca2+-fluorescence signal. To assess the neuropil signal driven from stimulation of either contralateral or
ipsilateral eye simultaneously with contralateral non-silencing/silencing, the signal
associated with all periods of rectangular grating display (all drift directions and all
repetitions) for each eye and non-silencing/silencing condition was combined, and
compared with the neuropil signal derived from periods of same duration as sti-
mulus but whose occurrence was randomly chosen. As the stimulation periods
occupied a large portion of the imaging duration (42.9%), the procedure of ran-
domly choosing durations was modified so as to assure that shuffling through
different random durations resulted in at least 50% of the chosen durations not
coinciding with stimulation. After the induction of thalamic silencing (MUSC), the
average neuropil signal driven from contralateral eye stimulation and simultaneous
contralateral non-silencing (LED off)/silencing (LED on) was significantly higher
than the average signal associated with randomly chosen durations (Rat 1; MUSC
+ LED off: 3.27 ± 0.25%ΔF/F, mean ± SEM, 1-tailed 2-sample t-test, P < 0.001,
MUSC + LED on: 2.87 ± 0.26%ΔF/F, mean ± SEM, 1-tailed 2-sample t-test, P =
0.004; Rat 2; MUSC + LED off: 2.8 ± 0.19%ΔF/F, mean ± SEM, 2-sample t-test,
P = 0.004, MUSC + LED on: 2.82 ± 0.2%ΔF/F, mean ± SEM, 2-sample t-test, P =
0.003). Preferred orientation ¼ arg ðSÞ
2 This method provides a robust estimate of preferred orientation, a continuous
quantity, when sampling a neuron’s orientation responses with a discrete series of
grating orientations. The estimates obtained are robust to scenarios, such as the
neuron’s actual preferred orientation not falling on one of the grating orientations. p
g
g
g
The orientation selectivity index (OSI) was calculated as described in ref. 46 as
the magnitude of the normalized vector sum (^S) calculated using the formula: ^S ¼
P
k Rkei2θk
P
k Rk
ð3Þ ð3Þ Contralateral-eye bias index (CBI) was calculated as CBI ¼
P
k RContraeye
k
N
P
k RContraeye
k
N
þ
P
k RIpsieye
k
N
ð4Þ
eye ð4Þ P
k RContraeye
k
N
&
P
k RIpsieye
k
N
denotes mean stimulus evoked spike rate during
contralateral and ipsilateral eye stimulations, respectively. N represents number of
drifting directions. Circular statistics was performed using CircStat toolbox47. The vc reduction index was calculated by first adjusting the spike rate of
neurons whose responses were abolished during vc inactivation to zero. The vc
reduction index was then calculated as vc reduction index ¼ Spike rate for peak angle during LEDoff Spike rate for peak angle during LEDon
Spike rate for peak angle during LEDoff vc reduction index ¼ Spike rate for peak angle during LEDoff Spike rate for peak angle during LEDon
Spike rate for peak angle during LEDoff ð5Þ with any values from the above computation that were negative being rounded to
zero. This then results in values ranging from zero to one, with zero representing
activity that is completely unreliant on the vc pathway and one representing activity
that is totally reliant on the vc pathway. For monocular neurons, this value was
calculated only for the responses from the active eye. For binocular neurons, the index
was calculated separately for responses from each eye and combined vc reduction
index calculated as the mean of these. Any negative values were rounded to zero. Multiphoton datasets from different animals were aligned on the basis of the
intrinsic optical signal imaging response to visual stimulation (alignment
procedure shown in Supplementary Fig. 1, intrinsic imaging described above). The
center of mass of the 90th percentile contour determined from the intrinsic
imaging response served as the global reference point, with datasets from different
animals overlayed by alignment of this point. Preferred orientation ¼ arg ðSÞ
2 Whereas for ipsilateral eye stimulation, the average signal with simulta-
neous contralateral non-silencing was significantly higher than the average signal
associated with randomly chosen durations, the average signal with simultaneous
contralateral silencing did not differ from the average signal associated with ran-
domly chosen durations (Rat 1; MUSC + LED off: 2.63 ± 0.22%ΔF/F, mean ± SEM,
1-tailed 2-sample t-test, P = 0.027, MUSC + LED on: 1.95 ± 0.17%ΔF/F, mean ±
SEM, 1-tailed 2-sample t-test, P = 0.854; Rat 2; MUSC + LED off: 2.68 ± 0.19%
ΔF/F, mean ± SEM, 1-tailed 2-sample t-test, P = 0.022, MUSC + LED on: 2.48 ±
0.2%ΔF/F, mean ± SEM, 1-tailed 2-sample t-test, P = 0.185). g
g
y
g
Response probabilities in Fig. 3 and Supplementary Fig. 3a were determined
during the initial 600 ms of the visual stimulus. For the neuronal population
analysis in Fig. 3e, N = 41 neurons were used. To compare population spiking
characteristics for Fig. 3f, g, from these 41 neurons, 5 neuronal populations with 6
neurons each were randomly selected. y
To define responsive neurons, the spike rates resulting from presentations of
orientated grating stimuli and a blank stimulus were subjected to Kruskal–Wallis
nonparametric ANOVA followed by one-tailed Fisher’s test. Monocular responsive
neurons were defined as those whose evoked spike rate during presentation of at
least one drift direction of orientated grating to an eye was significantly higher (P <
0.05) than evoked spike rates from other drift directions presented to the same eye
or spontaneous spike rate. In addition to Kruskal–Wallis nonparametric ANOVA,
also Fisher’s test was used in cases where evoked spike rate of neurons was low
(<1 spike per stimulus presentation) because in such cases the Kruskal–Wallis test
would result in a spurious outcome. In brief, for calculating the contingency table
used in Fisher’s test, the peak orientation (defined below) and blank stimulus
presentations that elicited one or more spikes were marked as being responsive and
the presentations during which no spikes were elicited were marked as being non-
responsive. The nos. of the presentations marked responsive and non-responsive
for peak orientation and blank stimulus were then used in the contingency table for
a one-tailed Fisher’s test (P < 0.05). Preferred orientation ¼ arg ðSÞ
2 Data analysis. Analyses were performed using custom-written routines executed in
Matlab (Mathworks, MA, USA). For quantification of CTB labeling density across
animals, the coronal sections of brains injected with CTB-594 (CTB conjugated to
Alexa 594) were used. The images of coronal sections were acquired using a fixed
exposure time. From the images of coronal sections, the 3D brain volume was
reconstructed and the reconstructed brain volume from each animal was aligned to
rat brain atlas43. The mean CTB labeling density from all animals was then quan-
tified in the co-ordinates of rat brain atlas. This process was carried out through
manually assisted custom-written routine in Matlab and is briefly as follows; the
images of each coronal section were split along the midline and the split images
belonging to one hemisphere were aligned, along the anterior–posterior dimension,
using blood vessels that served as landmarks (blood vessels traversing across sections
provide robust indication of the corresponding locations between two neighboring
sections). The split images belonging to the other hemisphere were aligned similarly. The aligned images now occupying 3D volume were then aligned to rat brain atlas
using posterior end of corpus callosum and dentate gyrus as reference. The back-
ground fluorescence (calculated from part of the section that contained no visible
CTB-594 label) was subtracted for all images and the intensity of labeling across the
entire cortical column was summed. The resulting summed intensity values in each
hemisphere were normalized separately to the maximum of summed intensities in
that hemisphere. The normalized summed intensity was then projected onto the top
view and subsequently from top view images of all animals the mean image was
calculated. Only the CTB-594 labeling in secondary visual mediolateral area (V2ML),
primary visual cortex (V1), and secondary visual lateral area (V2L) was estimated. The width of the labeled band of neurons in medial–lateral dimension was estimated
as the boundary in top view image along that dimension outside which intensity
drops below 2% of maximum value (the width of the band was calculated at a
distance of 2.4 mm anterior from interaural line). Correction of Ca2+-imaging data
for in-frame-motion was performed as described in ref. 44 and analysis of neuronal
Ca2+-transients and detection of putative action-potential-related Ca2+-signals was
performed as described in ref. 26. Rk represents mean spike rate for oriented grating drifting in direction θk. Rk represents mean spike rate for oriented grating drifting in direction θk. ARTICLE Rectangular drifting grating stimuli (spatial frequency 0.05 cycle/degree,
drift speed 40 degrees/s, contrast 96.72%, duration 2.0 s in experiments
combining Ca2+-imaging with CTB tracing and duration 1.5 s in experiments
combining Ca2+-imaging with cortical silencing) were presented at eight angular
orientations and two directions to give 16 separate grating stimuli in total. In
addition, a blank stimulus (same as gray background) of same duration as grating
stimuli was presented, that served as a measure of spontaneous activity. The
stimulus presentations were pseudo-randomized. Automated shutters were used
to exclude visual stimuli from one eye or the other in order to deliver monocular
stimuli, and each grating was presented a total of 8 times to each eye
monocularly, and in a subset of 5 animals also 8 times to both eyes (binocularly). The automated shutters were custom made, and consisted of a neoprene cup to
cover the eye connected via a 8 cm long rod to a servo motor (Supplementary
Fig. 13). The neoprene cup was placed over the eye, pressing slightly into the
surrounding fur, and could be retracted at an angle downwards away from the eye
using the servo motor to allow visual stimulus presentation. The servo motor
position was controlled via the visual stimulus software to allow full computer
control and randomization of monocular or binocular stimulus presentation. Inter-stimulus interval was between 2 and 3 s (randomized across trials) in
experiments combining Ca2+-imaging and CTB tracing and 3–4 s (randomized
across trials) in experiments combining Ca2+-imaging and cortical silencing. In Animal preparation for cortical and thalamic silencing. The procedure for
animal preparation was similar to that described above for combined cortical 10 NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15672-4 Preferred orientation was calculated from the phase angle of vector sum (S), as
described in ref. 45, using the formula: experiments combining Ca2+-imaging, cortical silencing, and thalamic silencing,
the rectangular drifting grating stimuli with only one drift direction were used,
otherwise all other stimuli parameters remained the same as that in experiments
combining Ca2+-imaging and cortical silencing. experiments combining Ca2+-imaging, cortical silencing, and thalamic silencing,
the rectangular drifting grating stimuli with only one drift direction were used,
otherwise all other stimuli parameters remained the same as that in experiments
combining Ca2+-imaging and cortical silencing. S ¼
X
k Rkei2θk
ð1Þ S ¼
X
k Rkei2θk
ð1Þ
Preferred orientation ¼ arg ðSÞ
2
ð2Þ S ¼
X
k Rkei2θk S ¼
X
k Rkei2θk ð1Þ Preferred orientation ¼ arg ðSÞ
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those whose evoked spike rate during presentations to either eye of orientated
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animals. The data presented in Supplementary Fig. 1 is representative of 17 Peak orientation was defined as that orientation of rectangular grating drifting
in a direction that elicited maximum spike rate compared to rectangular drifting
gratings of other orientations. 11 20) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunications ARTICLE ARTICLE Competing interests 23. Zhao, X., Liu, M. & Cang, J. Sublinear binocular integration preserves
orientation selectivity in mouse visual cortex. Nat. Commun. 4, 2088 (2013). Competing interests
The authors declare no competing interests. g
g
p
orientation selectivity in mouse visual cortex. Nat. Commun. 4, 2088 (2013). The authors declare no competing interest NATURE COMMUNICATIONS | (2020) 11:1889 | https://doi.org/10.1038/s41467-020-15672-4 | www.nature.com/naturecommunicatio 12 © The Author(s) 2020 Additional information Additional information Open Access This article is licensed under a Creative Commons
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REPRESENT: REPresentativeness of RESearch data obtained through the ‘General Informed ConsENT’
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BMC medical ethics
| 2,023
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cc-by
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background We assessed potential consent bias in a cohort of > 40,000 adult patients asked by mail after hospitali-
zation to consent to the use of past, present and future clinical and biological data in an ongoing ‘general consent’
program at a large tertiary hospital in Switzerland. Methods In this retrospective cohort study, all adult patients hospitalized between April 2019 and March 2020 were
invited to participate to the general consent program. Demographic and clinical characteristics were extracted from
patients’ electronic health records (EHR). Data of those who provided written consent (signatories) and non-respond-
ers were compared and analyzed with R studio. Results Of 44,819 patients approached, 10,299 (23%) signed the form. Signatories were older (median age 54 [IQR
38–72] vs. 44 years [IQR 32–60], p < .0001), more comorbid (2614/10,299 [25.4%] vs. 4912/28,676 [17.1%] with Charlson
comorbidity index ≤ 4, p < .0001), and more often of Swiss nationality (6592/10,299 [64%] vs. 13,813/28,676 [48.2%],
p < .0001). Results Of 44,819 patients approached, 10,299 (23%) signed the form. Signatories were older (median age 54 [IQR
38–72] vs. 44 years [IQR 32–60], p < .0001), more comorbid (2614/10,299 [25.4%] vs. 4912/28,676 [17.1%] with Charlson
comorbidity index ≤ 4, p < .0001), and more often of Swiss nationality (6592/10,299 [64%] vs. 13,813/28,676 [48.2%],
p < .0001). Conclusions Our results suggest that actively seeking consent creates a bias and compromises the external validity
of data obtained via ‘general consent’ programs. Other options, such as opt-out consent procedures, should be further
assessed. Conclusions Our results suggest that actively seeking consent creates a bias and compromises the external validity
of data obtained via ‘general consent’ programs. Other options, such as opt-out consent procedures, should be further
assessed. Keywords Informed consent, Consent bias, Volunteer bias, External validity, Representativeness Keywords Informed consent, Consent bias, Volunteer bias, External validity, Representativeness REPRESENT: REPresentativeness of RESearch
data obtained through the ‘General Informed
ConsENT’ Cristina Bosmani1*, Sonia Carboni1, Caroline Samer2, Christian Lovis4, Thomas Perneger5, Angela Huttner1,3 and
Bernard Hirschel6 Background Good clinical research practice—and the laws of many
nations—require that patients grant informed consent in
writing before their clinical data or biological material be
used for research purposes. The informed consent form
(ICF) documents the patient’s free will regarding the use
of his/her own personal data; it is thus not only a legal but
also an ethical requirement for most medical research. In
this context, and in an era of increasingly ‘big data’, Swiss
tertiary-care hospitals have collaborated to obtain writ-
ten consent prospectively from both in- and outpatients. Through the ‘General Consent’ program, patients are
approached to consent to the use of their past, present 5 Division of Clinical Epidemiology, Geneva University Hospitals, Geneva,
Switzerland 6 Health Department of the Canton of Geneva, Geneva Cantonal Ethics
Commission, Geneva, Switzerland 6 Health Department of the Canton of Geneva, Geneva Cantonal Ethics
Commission, Geneva, Switzerland Bosmani et al. BMC Medical Ethics (2023) 24:10
https://doi.org/10.1186/s12910-022-00877-7 Bosmani et al. BMC Medical Ethics (2023) 24:10
https://doi.org/10.1186/s12910-022-00877-7 BMC Medical Ethics Open Access Study design, setting, and participantsh y
This single-center retrospective cohort study included
all adult patients (≥ 18 years) who were hospitalized at
the University Hospital of Geneva (HUG) between April
2019 and March 2020, thereafter invited by mail to par-
ticipate in the hospital’s ‘general consent’ program (Addi-
tional file 1), and either provided consent (signatories) or
did not respond (non-responders). Patients who actively
refused the general consent were excluded, in order to
comply with our hospital’s current policy on data protec-
tion. In addition, patients whose invitations to participate
were returned (recipient not found) were also excluded,
as they never received the information and thus were
unable to provide any consent. Demographic and clini-
cal data were extracted from patients’ electronic health
records (EHR).h The HUG is a Swiss university medical network of
eight hospitals, some 2000 beds, > 55,000 annual admis-
sions, and > 1 million treated outpatients per year. The
largest tertiary care center in Switzerland, the HUG
serves a region with > 500,000 international and very
mobile inhabitants. The ‘general consent’ program was
launched at the HUG in 2017. Initially, individual wards Statistical analysis
Th There was no sample size calculation; all adult patients
invited by mail to participate in the ‘general consent’
program (without postal return of the invitation) were
included. Continuous data are presented as the median
with interquartile range and categorical data as frequency
counts and percentages. Mortality through October 2021,
the most recent time point for which data were avail-
able, was assessed. Missing data are reported through-
out. Comparisons between groups were performed with
the Student’s t-test for continuous data and Χ2 or Fish-
er’s exact test for categorical data. Associations with p
values < 0.05 were considered significant (two-sided). Univariable and multivariable logistic regression mod-
els were used to determine associations, if any, between
demographic or clinical baseline factors and general-
consent response. For multivariable logistic regression,
all variables that were significant (p value < 0.05) in uni-
variable analyses were added to the model. Only variables
that were significant were kept in the final model. All
data were analyzed using R language and R studio (4.1.0,
www.R-project.org/), including the packages: ‘ggplot2’,
‘ggpubr’ and ‘comorbidity’. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bosmani et al. BMC Medical Ethics (2023) 24:10 Page 2 of 7 and future data for purely observational analysis, either
in person or by mail. Unfortunately, most patients do not
respond. Patients granting consent are a minority; their
demographic and clinical characteristics may thus differ
significantly from those of non-responders. and clinics were tasked with approaching patients for
general consent during hospitalizations or ambulatory
encounters. Due to uneven levels of engagement, in 2020
the general consent program switched mainly to regular
home mailings of the general consent form to all patients
who had been recently hospitalized (any hospitalization
from April 2019 onwards). The general consent form is
available in French, German, Italian, English, Spanish,
Portuguese, Russian, Arabic, Albanian, Romanian and
Georgian. i
It has been well documented on a small scale that ran-
domized clinical trials (RCTs) exclude patients who are
sicker, more complicated, and whose follow-up is lim-
ited [1, 2]. The external validity of observational studies
is also questionable. Certain demographic and/or clinical
factors may influence the patient’s decision to grant pro-
active, opt-in consent, thus creating a well-documented
‘consent bias’ [3–7]. Age, sex and socioeconomic status
are factors that have been associated with patients’ deci-
sion to grant consent. Disease severity has also been
shown to influence willingness and/or ability to provide
consent [4–7]. Primary and secondary outcomeshf The primary outcome was the difference between signa-
tories and non-responders in three demographic char-
acteristics: age (years), sex (percentage of male versus
female), and country of origin (percentage of Swiss versus
other). Additional outcomes included differences in lan-
guage spoken; country of origin; marital status; religion;
number of hospitalizations; lengths of stay (LOS); and
comorbidity levels (by Charlson comorbidity and Elix-
hauser comorbidity indices). Understanding the representativeness of patients
included in clinical research is important for the medi-
cal community, whose members increasingly rely on gen-
eral-consent programs like that of the Swiss for ongoing
research and policy decisions. We compared socioeco-
nomic, demographic and clinical characteristics of those
who consented versus those who did not in a cohort
of > 40,000 patients of the Geneva University Hospital
invited to provide general consent for the use of their
clinical data in research. Modeling consent In a multivariable analysis including all characteristics
described above, the strongest independent predictors
(p < 0.0001) of granting consent were increasing age,
Swiss nationality, French as first language, not having
any particular religion, being married, a higher comor-
bidity score, and a higher number of hospital encounters
(Table 4). Response and consent rates Some 190,000 patients had one (or more) medical
encounter(s) at the HUG between April 2019 and March
2020. Of these, 44,819 patients (roughly 24%) were hospi-
talized, had a Swiss residential address and thus received Bosmani et al. BMC Medical Ethics (2023) 24:10 Page 3 of 7 Bosmani et al. BMC Medical Ethics Patients mailed
age ≥ 18
Swiss address
hospitalized
n = 44819 (100%)
GC form
returned
n = 13060 (29.1%)
GC form
not returned
n = 31592 (70.5%)
Consent given
n = 10299 (78.9%)
(23% of total)
Consent refused
n = 2761 (21.1%)
Non-valid address
n = 2916 (9.2%)
No response
n = 28676 (90.8%)
(64% of total)
Patient deceased
n = 167 (0.4%)
Patients at the HUG
Apr 19 - Mar 20
n = 187947
Excluded:
age <18
no Swiss address
outpatients
n = 143127
Fig. 1 Flowchart of REPRESENT. About 45,000 patients were mailed
the GC (general consent) form (blue). After excluding refusals and
non-delivered forms (patient deceased or non-valid address), a total
of 38,975 patients were included (light green) were more likely to grant consent (643/9,360 [6.9%] vs
1,391/26,562 [5.2%], p < 0.0001). Patients at the HUG
Apr 19 - Mar 20
n = 187947 Excluded:
age <18
no Swiss address
outpatients
n = 143127 Demographic characteristics of signatories
and non‑responders While signatories were older (median age 54 (IQR
38–72) vs. 46 years (IQR 33–63), p < 0.0001), the over-
all percentage of women in either group did not differ
significantly (5,763/10,299 [56.0%] vs. 16,144/28,676
[56.3%], p = 0.56; Table 1). Among younger patients
(18–40 years), however, signatories were more likely to
be women (1,894/2817 [67.2%] vs. 7,021/12,002 [58.5%],
p < 0.0001), whereas the opposite was observed among
older patients (2,054/4,209 [48.8%] vs. 4,076/7,311
[55.8%], p < 0.0001). Signatories were more often of
Swiss nationality (6,592/10,299 [64.0%] vs 13,813/28,676
[48.2%], p < 0.0001) and French-speaking (7,915/10,299
[76.9%] vs 19,285/28,676 [67.3%], p < 0.0001). Mar-
ried patients were more likely to consent (4,915/10,299
[47.7%] vs. 11,621/28,676 [40.5%], p < 0.0001), as were
patients not practicing any religion (1,455/10,299 [14.1%]
vs. 3,438/28,676 [12%], p < 0.0001). Finally, patients liv-
ing in higher-income townships of the canton of Geneva Consent rates by pathology Consent rates by pathology
Signatories had cancer more frequently than non-
responders (785/10,299 [7.6%] vs. 1193/28,676 [4.2%,
p < 0.0001]), as well as chronic heart failure (592/10,299
[5.7%] vs. 1076/28,676 [3.8%], p < 0.0001), myocardial
infarction (389/10,299 [3.8%] vs. 598/28,676 [2.1%],
p < 0.0001), and rheumatoid disease (129/10,299 [1.3%]
vs. 184/28,676 [0.6%], p < 0.0001). In contrast, only
153/10,299 (1.5%) and 143/10,299 (1.4%) signatories suf-
fered from drug abuse and/or dementia vs. 577/28,676
(2.0%, p < 0.0001) and 587/28,676 (2.0%, p < 0.0001) non-
responders, respectively. Details are shown in Table 3 and
Fig. 2. Fig. 1 Flowchart of REPRESENT. About 45,000 patients were mailed
the GC (general consent) form (blue). After excluding refusals and
non-delivered forms (patient deceased or non-valid address), a total
of 38,975 patients were included (light green) the general consent form by mail (Fig. 1). The overall
response rate from these patients was 13,060/44,819
(29.1%). The majority of responses were positive:
10,299/13,060 (78.9%) respondents granted consent (‘sig-
natories’), while 2,761/13,060 (21.1%) actively refused. In
total, 28,676/44,819 (64.0%) were non-responders, after
exclusion of patients that refused or never received the
consent form (because of death or invalid address). Clinical characteristics of signatories and non‑respondersh The number of patients who died after the general con-
sent form was sent was similar among signatories and
non-responders (203/10,299 [2.0%] vs. 550/28,676
[1.9%], p = 0.77; Table 2). Signatories had significantly
longer LOS (12.7 [IQR 0.8–47.0] vs. 8.8 days [IQR 0.3–
40.6], p < 0.0001). Signatories had more comorbidities:
2,614/10,299 patients (25.4%) vs. 4,912/28,676 (17.1%;
p < 0.0001), had a Charlson comorbidity index score
between 1 and 4, and 3,064 of 10,299 patients (29.8%] vs. 6,457 of 28,676 (22.5%), (p < 0.0001) had an Elixhauser
comorbidity index score between 1 and 4. Patient deceased
n = 167 (0.4%) Religion vs. no religion:
< 0.0001 Discussionh The purpose of a general-consent program is to obtain
and document patients’ consent to the use of their clini-
cal and biological data in the larger effort to strengthen
the evidence base and thus continually improve medical
care. Yet our results demonstrate important, unintended
consequences: actively seeking opt-in consent from
patients—an essential step in guaranteeing their rights
and self-determination—may create a bias itself, thus
potentially compromising the external validity of the data
obtained. In this cohort of > 40,000 patients admitted to hos-
pital in the course of one year, we detected important Bosmani et al. BMC Medical Ethics (2023) 24:10 Page 4 of 7 differences in clinical and demographic characteris-
majority (64%). Consenters are significantly older and
Table 1 Baseline characteristics of patients approached for general consent
Characteristic
Overall
n = 38,975
Signatories
n = 10,299
Χ2/t-test p Value
Sex
Women, n (%)
21,907 (56.2)
26.3
0.56
Men, n (%)
17,068 (43.8)
26.6
Median age, years (IQR)
46 (33–63)
54 (38–72)
< 0.0001
Age groups and sex
18–40 y
Women, n (%)
8915 (60.2)
21.2
< 0.0001
Men, n (%)
5904 (39.8)
15.6
60 + yo
Women, n (%)
6130 (53.2)
33.5
< 0.0001
Men, n (%)
5390 (46.8)
40.0
Country of origin
Switzerland, n (%)
20,405 (52.4)
32.3
Swiss vs. other:
< 0.0001
Other, n (%)
18,454 (47.3)
19.9
Spain, n (%)
1348 (3.5)
22.1
France, n (%)
2174 (5.6)
26.1
Italy, n (%)
1879 (4.8)
26.9
Portugal, n (%)
3211 (8.2)
20.3
N/A, n
116
Religion
Religion, n (%)
19,144(49.1)
26.8
Religion vs. no religion:
< 0.0001
No religion, n (%)
4893 (12.6)
29.7
Refuse to respond, n (%)
1199 (3.1)
28.8
N/A, n
13,739
Marital status
Married, n (%)
16,536 (42.4)
29.7
Married vs. single:
< 0.0001
Single, n (%)
14,461 (37.1)
21.2
Widowed, n (%)
2051 (5.3)
30.6
Other,n
5760
28.6
N/A, n
167
Median household income of the commune of residence
0-100 k CHF, n (%)
5283 (14.7)
23.5
> 200 k vs. 0-100 k:
< 0.0001
100-200 k CHF, n (%)
28,605 (79.6)
26.1
> 200 k CHF, n (%)
2034 (5.7)
31.6
Spoken language
French, n (%)
27,200 (69.8)
29.1
French vs. other:
< 0.0001
Other, translated, n (%)
9227 (23.7)
20.9
Other, not translated n (%)
2120 (5.4)
15.4
N/A, n
429 Table 1 Baseline characteristics of patients approached for general consent majority (64%). Discussionh Consenters are significantly older and
enjoy a higher socioeconomic status. They are more likely
to hold Swiss nationality, speak the dominant language,
and profess no particular religion. Clinically, consenters majority (64%). Consenters are significantly older and
enjoy a higher socioeconomic status. They are more likely
to hold Swiss nationality, speak the dominant language,
and profess no particular religion. Clinically, consenters differences in clinical and demographic characteris-
tics of those who grant consent—and thus, whose data
are being used for myriad ongoing and future research
projects—and those who do not: a silent but massive differences in clinical and demographic characteris-
tics of those who grant consent—and thus, whose data
are being used for myriad ongoing and future research
projects—and those who do not: a silent but massive Bosmani et al. BMC Medical Ethics (2023) 24:10 Page 5 of 7 Table 2 Clinical characteristics
Characteristic
Overall
Signatories
% or median (IQR)
Χ2/t-Test p value
Patient status
Alive, n (%)
38,222 (98.1)
26.4
0.77
Deceased, n (%)
753 (1.9)
27.0
Number of hospital encounters per patient
1–5, n (%)
6–10, n (%)
> 10, n (%)
20,945 (53.7)
10,429 (26.8)
7601 (19.5)
24.7
27.7
29.5
1–5 vs > 10:
< 0.0001
Length of stay (cumulated days per patient),
median (IQR)
50.3 (1.2–304.0)
72.7 (2.8 – 351.0)
< 0.0001
Charlson comorbidity index
Score 0, n (%)
30,969 (79.5)
24.2
Score 0 vs 1 – 4:
< 0.0001
Score 1–4, n (%)
7526 (19.3)
34.7
Score ≥ 5, n (%)
480 (1.2)
36.7
Elixhauser comorbidity index
Score 0, n (%)
26,356 (67.6)
23.6
Score 0 vs 1 – 4
< 0.0001
Score 1–4, n (%)
9521 (24.4)
32.2
Score ≥ 5, n (%)
3098 (7.9)
33.2 Rheumatoid disease
Cancer
Metastatic cancer
Myocardial infarction
Chronic heart failure
Overall
Drug abuse
Dementia
0
10
20
30
40
50
Percentage of consent (%)
Fig. 2 Percentage of consent among patients with selected
diagnoses. Shown in black and as a dashed line the overall percent of
consent of the whole dataset Rheumatoid disease
Cancer
Metastatic cancer
Myocardial infarction
Chronic heart failure
Overall
Drug abuse
Dementia
0
10
20
30
40
50
Percentage of consent (%)
Fig. 2 Percentage of consent among patients with selected
diagnoses. Shown in black and as a dashed line the overall percent of
consent of the whole dataset Table 3 Distribution of selected diagnoses. Discussionh 0, patients without
the selected disease; 1, patients with the disease; OR, odds ratio Table 3 Distribution of selected diagnoses. 0, patients without
the selected disease; 1, patients with the disease; OR, odds ratio
Characteristic
Overall
Signatories (%)
Χ2 p Value
Rheumatoid disease
< .0001
0, n (%)
38,662 (99.2)
26.3
1, n (%)
313 (0.8)
41.2
Cancer
< .0001
0, n (%)
36,997 (94.9)
25.7
1, n (%)
1978 (5.1)
39.7
Metastatic cancer
< .0001
0, n (%)
38,401 (98.5)
26.2
1, n (%)
574 (1.5)
39.5
Myocardial infarction
< .0001
0, n (%)
37,988 (97.5)
26.1
1, n (%)
987 (2.5)
39.4
Chronic heart failure
p < .0001
0, n (%)
37,307 (95.7)
26.0
1, n (%)
1668 (4.3)
35.5
Drug abuse
p < .0001
0, n (%)
38,245 (98.1)
26.5
1, n (%)
730 (1.9)
21.3
Dementia
p < .0001
0, n (%)
38,245 (98.1)
26.6
1, n (%)
730 (1.9)
19.6 Fig. 2 Percentage of consent among patients with selected
diagnoses. Shown in black and as a dashed line the overall percent of
consent of the whole dataset chronic heart disease, but less likely to suffer from drug
abuse or dementia.h This is the largest cohort analyzed to date. In a sys-
tematic review assessing characteristics of patients
approached for access to medical records for 17 differ-
ent prospective observational studies [3], the majority
(66%) of patients granted consent (in stark contrast to are more comorbid, with longer recent lengths of hospi-
tal stay, probably correlating with their older age. They
are significantly more likely to suffer from cancer and Bosmani et al. Discussionh Those granting consent to
access their medical records differed significantly from
non-participants across all 17 different studies, although
in an inconsistent manner [3]. A smaller cohort from Ire-
land published two years later found a selection bias sim-
ilar to what we observe after seeking consent for access
to medical records: consenters were older and socioeco-
nomically more affluent [4]. Other small studies have also
shown that consenters differ from non-consenters, espe-
cially when considering their health status [5–7]. Due
to the recent nature of our data, we are unable to show
differences in survival times after granting consent. Data
from Canadian stroke and New Zealander breast cancer
registries demonstrate that patients granting consent
have lower mortality rates and less advanced disease [5,
7]. It should be noted that there are differences in the tar-
get population of each one of these studies, carried out
in different countries, as well as differences in the way
consent was sought (by mail or in person, with or with-
out follow-up, etc.). Nonetheless, these reports support
the hypothesis that opt-in consent approaches introduce
a bias in observational studies, although the extent and
nature of these biases differ from study to study. Over-
all, our research suggests that, although they constitute
a large percentage of the Geneva population, younger,
more foreign and more underprivileged patients may be
underrepresented in studies derived from the General
Consent program. More longitudinal analyses will need to be performed
to assess whether signatories with comorbidities are in
overall ‘better health’ than non-responders. Further, we
are not currently able to access data from active refus-
ers (those who return the consent form only to refuse
access to their data). This is a population of patients
that is understudied and obviously underrepresented in
current research. Discussionh BMC Medical Ethics (2023) 24:10 Page 6 of 7 Page 6 of 7 Table 4 Independent predictors of granting consent
Odds ratio
95% CI
p-Value
18–40 y, female
0.75
0.69–0.81
< 0.0001
18–40 y, male
0.55
0.50–0.61
< 0.0001
60 + y, female
1.25
1.14–1.36
< 0.0001
60 + y, male
1.57
1.44–1.71
< 0.0001
Nationality—not Swiss
0.63
0.60–0.67
< 0.0001
Practicing a religion
0.80
0.75–0.86
< 0.0001
Unknown religion
0.85
0.79–0.92
< 0.0001
Marital status—single
0.81
0.76–0.86
< 0.0001
Marital status—widowed
0.73
0.66–0.82
< 0.0001
Language—not French
0.76
0.71–0.81
< 0.0001
Number of hospital encoun-
ters—1—5
0.83
0.77–0.88
< 0.0001
Charlson comorbidity index
score—0
0.79
0.63–0.99
0.04
Elixhauser comorbidity index
score—0
1.25
1.10–1.41
< 0.0001
Elixhauser comorbidity index
score—1–4
1.31
1.18–1.45
< 0.0001 Table 4 Independent predictors of granting consent care informed by clinical research that is unbiased and
otherwise methodologically sound. One model may
be a multilateral strategy including increased ethics
and legal oversight (to ensure consistent anonymiza-
tion of data, among other aspects), regular provision of
study information to all patients, and an opt-out pro-
cess that does not require active signatures and return-
ing of forms [8]. For now, the requirement for written
informed consent clearly excludes the clinical experi-
ences of the majority of patients. Prioritizing patient
privacy is critical, but it must be balanced with patients’
other rights, including that of access to well-informed
health care. Our study has limitations. It is retrospective. By defi-
nition, only patients surviving their hospitalization
could be addressed. Importantly, we were unable to test
or choose the method of obtaining informed consent,
here based on a mailing approach. This method could
account for the low participation rate compared to other
studies [3], as patients may forget or misplace the letter,
or be unable to understand the language of the consent
(initially sent in French), thus introducing a bias, as, for
instance, non-responders are largely non-Swiss nationals. The mailing approach has only recently replaced the in-
person approach initially used in the hospital, which was
highly department- and person-dependent. Currently,
Swiss hospitals are developing together a generalized
electronic method to collect consent in the near future. Finally, because there was no follow-up after the mailing,
it is not possible to know the reasons for no-response,
this could add a further bias to our study. our cohort, with only 23%). Conclusions Patients granting consent to the use of their clinical data
for observational study are older, enjoy a higher socio-
economic status, and more likely to suffer from cancer
and chronic heart disease; those with dementia and drug
abuse are underrepresented. In order to increase exter-
nal validity and clinical relevance of current datasets
and thus decrease the probability of misleading or false
research results, alternative strategies should be explored. Public-health policymakers may consider strategies such
as collective, anonymized datasets obtained through
informed, opt-out consent procedures tightly controlled These findings support exploration of other models to
ensure patients’ rights—to both privacy and to health Page 7 of 7 Bosmani et al. BMC Medical Ethics (2023) 24:10 Bosmani et al. BMC Medical Ethics with institutionally mandated ethics oversight, in order
to guarantee anonymity and confidentiality. cohorts: iron-deficiency anaemia and colorectal cancer. Colorectal Dis Off
J Assoc Coloproctology G B Irel. 2011;13(11):e366-373. y
5. Tu JV, Willison DJ, Silver FL, Fang J, Richards JA, Laupacis A, et al. Imprac-
ticability of informed consent in the registry of the canadian stroke
network. N Engl J Med. 2004;350(14):1414–21. Acknowledgements g
We thank Daniel Teixeira from the Information Systems Directorate (DSI) of the
Geneva University Hospitals for data exports and technical assistance, and Dr. Olivier Huber of the Geneva Cantonal Ethics Commission for his creative and
constructive advice. Funding
h
d This study was funded by an institutional grant from the Geneva University
Hospitals’ Medical Directorate and the Research Ethics Committee of the
Canton of Geneva. Additional file 1. General Consent brochure in English. 8. Huttner A, Leibovici L, Theuretzbacher U, Huttner B, Paul M. Closing the
evidence gap in infectious disease: point-of-care randomization and
informed consent. Clin Microbiol Infect. 2017;23(2):73–7. Availability of data and materials The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request. Publisher’s Note
S
i
N
i Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Consent for publication
Not applicable. Not applicable. Author contributions CB, AH and BH conceptualized and designed the study. CB and SC collected
the data. CB performed the data analysis and wrote the manuscript with input
from all authors. All authors participated in critical revision of the draft manu-
script. All authors read and approved the final manuscript. Supplementary Information 6. Macleod U, Watt GC. The impact of consent on observational research:
a comparison of outcomes from consenters and non consenters to an
observational study. BMC Med Res Methodol. 2008;8(1):15. The online version contains supplementary material available at https://doi.
org/10.1186/s12910-022-00877-7. y
7. Elwood JM, Marshall RJ, Tin ST, Barrios MEP, Harvey VJ. Bias in survival
estimates created by a requirement for consent to enter a clinical breast
cancer registry. Cancer Epidemiol. 2019;1(58):178–83. Ethics approval and consent to participate The study was approved by the Cantonal Commission of Research Ethics of
the Canton of Geneva (CCER, BASEC number: 2021-01345). Informed consent
was obtained for signatories before the study, and the need to seek consent
among non-responders was waived by the CCER. All methods were carried
out in accordance with relevant guidelines and regulations. Competing interests p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 26 August 2022 Accepted: 15 December 2022 Received: 26 August 2022 Accepted: 15 December 2022 References 1. Kennedy-Martin T, Curtis S, Faries D, Robinson S, Johnston J. A litera-
ture review on the representativeness of randomized controlled trial
samples and implications for the external validity of trial results. Trials. 2015;3(16):495. 1. Kennedy-Martin T, Curtis S, Faries D, Robinson S, Johnston J. A litera-
ture review on the representativeness of randomized controlled trial
samples and implications for the external validity of trial results. Trials. 2015;3(16):495. •
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At BMC, research is always in progress. Learn more biomedcentral.com/submissions
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Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: 2. Rossel A, Zandberg KP, Albrich WC, Huttner A. How representative is a
point-of-care randomized trial? Clinical outcomes of patients excluded
from a point-of-care randomized controlled trial evaluating antibiotic
duration for Gram-negative bacteraemia: a multicentre prospective
observational cohort study. Clin Microbiol Infect. 2022;28(2):297. 2. Rossel A, Zandberg KP, Albrich WC, Huttner A. How representative is a
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duration for Gram-negative bacteraemia: a multicentre prospective
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Pengaruh Liabilitas dan Ekuitas Terhadap Profitabilitas Pada PT.
Primarindo Asia Infrastructure Tbk
Dwi Urip Wardoyo1, Alifandy Satria Mulawarman2, Asril Naufal Diaz3
1,2,3Universitas Telkom
E-mail: dwiurip@telkomuniversity.ac.id1 , alifandysm@telkomuniveristy.ac.id2,
asrilnd@telkomuniversity ac id3 Dwi Urip Wardoyo1, Alifandy Satria Mulawarman2, Asril Naufal Diaz3
1,2,3Universitas Telkom
E-mail: dwiurip@telkomuniversity.ac.id1 , alifandysm@telkomuniveristy.ac.id2,
asrilnd@telkomuniversity.ac.id3 Abstract: This Study Aims To Determine The Effect Of
Liabilities And Equity On Profitability. Indicators To
Measure Profitability Use Return On Equity (Roe), While
Liabilities Use The Ratio Of Short-Term Debt To Equity
(Stde) And Shareholder Equity Using The Ratio Of
Prorietary Ratio (Pr). This Research Was Conducted At Pt. Primarindo Asia Infrastructure, Tbk. This Type Of Research
Is Associative Research. The Sampling Technique Used Is A
Non Probability Sampling Technique That Is Purposive
Sampling, With The Sample Used Is For 8 Years. The
Analysis Technique Used In This Study Is The Classic
Assumption Test And Multiple Regression Analysis. Based
On The Results Of The Regression Analysis Shows Short-
Term Liabilities, And Capital Alone Has No Influence On
Profitability Either Partially Or Simultaneously. Article History:
Received: 01 Desember 2021
Revised: 15 Desember 2021
Accepted: 30 Desember 2021 Article History:
Received: 01 Desember 2021
Revised: 15 Desember 2021
Accepted: 30 Desember 2021 Keywords: STDE, PR, ROE. Keywords: STDE, PR, ROE. 20 20 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Desember 2021 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Desember 2021 Pengaruh Liabilitas dan Ekuitas Terhadap Profitabilitas Pada PT. Primarindo Asia Infrastructure Tbk
Dwi Urip Wardoyo1, Alifandy Satria Mulawarman2, Asril Naufal Diaz3
1,2,3Universitas Telkom
E-mail: dwiurip@telkomuniversity.ac.id1 , alifandysm@telkomuniveristy.ac.id2,
asrilnd@telkomuniversity.ac.id3
Article History:
Received: 01 Desember 2021
Revised: 15 Desember 2021
Accepted: 30 Desember 2021
Abstract: This Study Aims To Determine The Effect Of
Liabilities And Equity On Profitability. Indicators To
Measure Profitability Use Return On Equity (Roe), While
Liabilities Use The Ratio Of Short-Term Debt To Equity
(Stde) And Shareholder Equity Using The Ratio Of
Prorietary Ratio (Pr). This Research Was Conducted At Pt. Primarindo Asia Infrastructure, Tbk. This Type Of Research
Is Associative Research. The Sampling Technique Used Is A
Non Probability Sampling Technique That Is Purposive
Sampling, With The Sample Used Is For 8 Years. The
Analysis Technique Used In This Study Is The Classic
Assumption Test And Multiple Regression Analysis. Based
On The Results Of The Regression Analysis Shows Short-
Term Liabilities, And Capital Alone Has No Influence On
Profitability Either Partially Or Simultaneously. Keywords: STDE, PR, ROE. PENDAHULUAN Seiring perkembangan jaman perusahaan manufkatur terus mengalami perkembangan dengan
terus menggunakan teknologi terbaru dalam menunjang bisnis mereka. Perusahaan manufaktur
merupakan penunjang bagi perekonomian dunia karena perusahaan manufaktur memiliki keunggulan
tersendiri dibanding sektor lain. pada umumnya perusahaan didirikan agar memperoleh profit atau
laba yang tinggi agar kelangsungaan hidup perusahan terjamin. Prihadi (2010:138-139) menyebutkan profitabilitas adalah kemapuan perusahaan dalam
menghasilkan labai. Semakin tinggi profitabilitas sebuah perusahaan maka semakin bagus kinerja dari
perusahaan tersebut, perusahaan yang memiliki tingkat profitabilitas yang tinggi akan sangat baik bagi
perusahaan. Profitabilitas suatu perusahaan di pengaruhi oleh berbagai faktor salah satunya di
pengaruhi oleh utang jangka pendek. Utang jangka pendek merupakan utang yang jatuh temponya
dalam 1tahun periode, dimana utang jangka pendek ini memiliki bunga yang lebih rendah dari utang
jangka panjang yang bisa meningkatkan profitabilitas suatu perusahaan. Semakin banyak tambahan
utang untuk investasi akan menaikan ROE perusahaanii. (Sudana, 2015:181). 21 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 Ekuitas merupakan modal sendiri yang terus ditingkatkan kesehatannya, terutama untuk memantapkan
struktur modal perusahaan. Modal sendiri merupakan modal yang berasal dari dalam perusahaan. Perusahaan yang memperoleh laba akan menghasilkan kas dari dalam perusahaan sehingga
mengurangi kebutuhan dana yang berasal dari luar perusahaan karena perusahaan mampu
meningkatkan profitabilitasnya. PT. Primarindo Asia Infrastructure Tbk merupakan perusahaan manufaktur yang bergerak di bidang
industri alas kaki. Perusahaan ini selalu melakukan berbagai upaya untuk meningkatkan kinerja
perusahaan guna mendapatkan profit yang baik. Tabel 1 Data Liabilitas, Ekuitas dan Laba PT. Primarindo Asia Infrastructure, Tbk Tahun 2013 – 2018
(dalam satuan rupiah) dikarenakan perusahaan lebih memililih untuk menggunakan dana eksternal
daripada dana internal untuk melakukan kegiatan operasional perusahaan. Tidak hanya itu, laba
perusahaan juga mengalami fluktuasi pada tahun 2015 laba usaha sebesar Rp. 21,82 milyar, kemudian
pada tahun 2016 laba usaha mengalami peningkatan sebesar Rp. 25,11 milyar. Peningkatan laba usaha
karena perusahaan menjual asetnya berupa tanah. Selain itu laba usaha perusahaan mengalami
penurunan seiring dengan penurunan total penjualan. Kemudian pada tahun 2017 laba usaha
mengalami penurunan yaitu sebesar Rp. 17,25 milyar. Penurunan ini disebabkan oleh menurunnya
penjualan, hingga 2018 laba usaha makin menurun, penurunannya Tabel 1. Ekuitas PT. Primarindo Asia
Tahun
Liabilitas
Ekuitas
Laba
2015
301.570.909.687
- 202. 012.514.927
21.828.914.738
2016
189.216.746.183
- 97.175.471.622
25.117.618.621
2017
173.964.702.574
- 84.637.373.721
17.254.663.996
2018
179.038.284.760
- 80.847.643.921
16.758.353.102 Tabel 1. Ekuitas PT. Primarindo Asia Sumber : Annual Report PT. primarindo Asia Infrastructure, Tbk. PENDAHULUAN Berdasarkan tabel diatas dapat dilihat ahwa ekuitas pada PT. Primarindo Asia Infrastructure bernilai
negatif hal ini sebesar Rp. 16,75 milyar. Penurunan ini juga disebabkan oleh menurunnya pendapatan
dari penjualan, yang antara lain disebabkan oleh lemahnya daya beli masyarakat serta persaingan
usahatermasuk dari produk impor yang lebih murah. Dari uaraian masalah diatas mendorong peneliti untuk melakukan penelitian guna untuk
mengetahui dan membuktikan seperti apa “pengaruh liabilitas dan ekuitas pada PT. Primarindo Asia
infrastructure, tbk. Berdasarkan uraian diatas maka rumusan masalah dalam penelitian ini adalah:
pertama, apakah terdapat pengaruh yang signifikan secara parsial liabilitas jangka pendek terhadap
profitabilitas pada PT. primarindo Asia Infrastructure tbk?. kedua, apakah terdapat pengaruh yang
signifikan secara parsial modal sendiri terhadap profitabilitas pada PT. Primarindo Asia Infrastructure
tbk ? ketiga, apakah terdapat pengaruh yang signifikan secara simultanliabilitas jangka pendek dan
modal sendiri terhadap profitabilitas pada PT. Primarindo asia infrastructure Tbk ?. Penelitian ini
bertujuan untuk mengetahui pengaruh yang signifikan secara parsial dan simultan liabilitas jangka
pendek, dan modal sendiri terhadap profitabilitas pada PT. Primarindo Asia Infrastructure tbk. 22 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 a.
Utang jangka pendek Rudianto (2012:47) menyebutkan utang jangka pendek merupakan utang yang memiliki waktu jatuh
tempo kurang dari satu periode akutansi atau satu tahun sejak disusunnya laporan keuangan
perusahaanv. Sudana (2015:203) menjelaskan bahwa utang jangka pendek memiliki cost of capital
atau bunga yang lebih rendah daripada utang jangka panjang. Pengukuran utang jangka pendek pada
penelitian ini menggunakan rumus short therm debt to total equity. Rudianto (2012:277) utang jangka panjang merupakan utang yang jatuh temponya lebih dari satu
periode akutansi. Utang jangka Panjang biasanya timbul karena adanya kebutuhan dana untuk
pembelanjaan investasi jangka Panjang dan bersifat permanen seperti asset tetap dan menaikan jumlah
modal permanenvii. Pengukuran utang jangka Panjang pada penelitian ini menggunakan rumus long
term debt to total equity Liabilitas Menurut Irton (2009). kewajiban (liabilities) merupakan hutang perusahaan masa kini yang
timbul dari peristiwa masa lalu, penyelesaiannya diharapkan mengakibatkan arus keluar dari sumber
daya perusahaan yang mengandung maanfaat ekonomiiii. Samryn (2014:39) kewajiban merupakan
kelompok utang yang masih harus dilunasi kepada pihak ketigaiv. Kewajiban atau utang dapat
diklasifikasikan menjadi dua jenis yaitu : a. Utang jangka pendek LANDASAN TEORI Liabilitas Modal sendiri Riyanto (2015:240) menyebutkan modal sendiri merupakan modal yang berasal dari dalam
perusahaan yang terdiri tiga sumber utama yaitu modal saham, cadangan dan keuntungan. Modal
tersebut terdiri atas saham biasa, saham preferen, akumulasi laba ditahan, dan agio sahamviii. Sudana (2015:152) mengatakan dalam menggunakan modal sendiri perusahaan na (2015:152) mengatakan dalam menggunakan modal sendiri perusahaan Rasio yang digunakan dalam penelitian ini yaitu Return on Equity (ROE) merupakan rasio
yang digunakan untuk mengukur laba bersih setelah pajak dengan modal sendiri. harus mengeluarkan biaya dala menggunakan modal atau cost of capital atas dana yang diperoleh
b
i
d
t
í i
dii
tk
l h
ilik
d li
P
k
d l
di i Rasio yang digunakan dalam penelitian ini yaitu Return on Equity (ROE) merupakan rasio
yang digunakan untuk mengukur laba bersih setelah pajak dengan modal sendiri. yang digunakan untuk mengukur laba bersih setelah pajak dengan modal sendiri. harus mengeluarkan biaya dala menggunakan modal atau cost of capital atas dana yang diperoleh
sebagai pendapatan mínimum yang diisyaratkan oleh pemilik modalix. Pengukuran modal sendiri
pada penelitian ini menggunakan rumus proriety ratio. Menurut brigham dan houston (2006:107) rasio profitabilitas adalah rasio yang digunakan untuk
mengetahui kemampuan perusahaan
dalam menghasilkan laba atau seberapa efektif pengelolaan
perusahaan oleh manajemen.x Menurut kasmir (2008:196), rasio profitabilitas merupakan rasio untuk
menilai kemampuan perusahaan dalam mencari keuntunganxi.Jenis-jenis rasio profitabilitas menurut
(kasmir, 2008:196): a. Net Profit Margin (NPM)
b. Return on Asset (ROA)
c. Earning per Share (EPS)
d. Return on Equity (ROE)
e. Gross Profit Margin (GPM)
f. Return On Investment (ROI)
METODE PENELITIAN a. Net Profit Margin (NPM) a. Net Profit Margin (NPM)
b. Return on Asset (ROA)
c. Earning per Share (EPS)
d. Return on Equity (ROE)
e. Gross Profit Margin (GPM)
f. Return On Investment (ROI)
METODE PENELITIAN f. Return On Investment (ROI) METODE PENELITIAN 23 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 Jenis penelitian ini adalah penelitian asosiatif. Teknik pengambilan sampel yang digunakan
adalah teknik non probability sampling yaitu purposive sampling, dengan sampel yang digunakan
selama 8 tahun. Teknik analisis yang digunakan dalam penelitian ini adalah uji asumsi klasik dan
analisis regresi berganda. H2= Modal sendiri berpengaruh positif terhadap profitabilitas. Pengaruh liabilitas jangka pendek dan modal sendiri terhadap profitabilitas Penelitian silitonga dan ginting (2019) menjelaskan bahwa pendanaan dari luar dan modal
sendiri berpengaruh signifikan terhadap profitabilitas pada perusahaan property dan real estate yang
terdaftar di bursa efek indonesia. H3 = liabilitas jangka pendek dan modal sendiri berpengaruh terhadap prof Uji multikolinearitas Uji multikolinearitas pada pengujian ini menggunakan konsekuensi yang diterima dari nilai
toleransi dan VIF. Biaya toleransi pada pengujian ini menjadi 0,312, yang menjadi di atas 0,1 dan
biaya VIF pada pengujian ini menjadi 3,201, apalagi 10, karena itu tidak ada multikolinearitas dalam
regresi. Pengaruh modal sendiri terhadap profitabilitas Penelitian Prasetyo (2017) menjelaskan bahwa modal sendiri berpengaruh positif dan
signifikan terhadap profitabilitas. Hubungan positif tersebut menunjukan bahwa semakin besar
nilai rasio modal sendiri pada perusahaan property dan real estate akan meningkatkan
profitabilitas dan sebaliknya. H2= Modal sendiri berpengaruh positif terhadap profitabilitas. Uji mormalitas j
Uji normalitas pada penelitian ini menggunakan uji one sample kolmogrov-sminorv (K-S) dan di
dapatkan hasil asymp sig (2-tailed) adalah 0,930 ini berarti informasi terdistribusi secara umum
sebagai hasil dari 0,930 > 0,05. Uji
ltik li
it Uji normalitas pada penelitian ini menggunakan uji one sample kolmogrov-sminorv (K-S) dan di
dapatkan hasil asymp sig (2-tailed) adalah 0,930 ini berarti informasi terdistribusi secara umum
sebagai hasil dari 0,930 > 0,05. Hipotesis Penelitian Pengaruh liabilitas jangka pendek terhadap profitabilitas HASIL DAN PEMBAHASAN Berdasarkan hasil pengolahan data yang dilakukan, diketahui hasil bahwa kewajiban jangka
pendek, dan modal sendiri sama-sama tidak berdampak yang signifikan terhadap profitabilitas. Berikut
adalah hasil pengujian statistik yang dilakukan dalam penelitian ini : j
Penelitian ini telah melewati Uji asumsi klasik yaitu Uji normalitas, Uji multikolinearitas, Uji
heteroksedasitas, dan Uji auto korelasi. Uji mormalitas Penelitian ini telah melewati Uji asumsi klasik yaitu Uji normalitas, Uji multikolinearitas, Uji
heteroksedasitas, dan Uji auto korelasi. Utang jangka pendek berpengaruh positif terhadap profitabilitas H1= Utang jangka pendek berpengaruh positif terhadap profitabilitas Pengaruh liabilitas jangka pendek terhadap profitabilitas Penelitian Prasetyo (2017) menjelaskan bahwa utang jangka pendek berpengaruh positif dan
signifikan terhadap profitabilitas pada perusahaan property dan real estate selama tahun penelitian. Hubungan positif tersebut menunjukan bahwa semakin besar nilai rasio hutang jangka pendek pada
perusahaan property dan real estate akan meningkatkan profitabilitas dan sebaliknya Penelitian Prasetyo (2017) menjelaskan bahwa utang jangka pendek berpengaruh positif dan
signifikan terhadap profitabilitas pada perusahaan property dan real estate selama tahun penelitian. Hubungan positif tersebut menunjukan bahwa semakin besar nilai rasio hutang jangka pendek pada
perusahaan property dan real estate akan meningkatkan profitabilitas dan sebaliknya
H1= Utang jangka pendek berpengaruh positif terhadap profitabilitas
Pengaruh modal sendiri terhadap profitabilitas Uji heteroksedisitas j
Uji heteroksedisitas pada pengamatan ini menggunakan pemeriksaan glejser dan pada
pengamatan ini tidak ada gejala heteroksedisitas. Biaya yang signifikan pada setiap variabel
independen menunjukkan konsekuensi lebih dari 0,05. Biaya besar untuk variabel kewajiban jangka
pendek (X1) adalah 0,127 < 0,05 dan biaya besar untuk modal sendiri 0,55 < 0,05, sehingga 24 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 kesimpulan yang dapat diambil adalah tidak terdapat tanda-tanda heteroskedastisitas dalam hal ini. model regresi. Uji autokorelasi Anova 25
EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022
Model
Sum
of
Squares
Df
Mean
Square
F
Sug
Regressi
on
.024
2
.012
2.358
.190
Residual
.025
5
.005
Total
.049
7
Berdasarkan tabel diatas nilai sig uji F adalah sebesar 0,190 > 0,05. Ini menandakan bahwasanya
ecara bersamaan liabilitas jangka pendek, dan modal sendiri tidak berpengaruh terhadap profitabilitas. 25 Model
Sum
of
Squares
Df
Mean
Square
F
Sug
Regressi
on
.024
2
.012
2.358
.190
Residual
.025
5
.005
Total
.049
7 Berdasarkan tabel diatas nilai sig uji F adalah sebesar 0,190 > 0,05. Ini menandakan bahwasanya
secara bersamaan liabilitas jangka pendek, dan modal sendiri tidak berpengaruh terhadap profitabilitas. Berdasarkan tabel diatas nilai sig uji F adalah sebesar 0,190 > 0,05. Ini menandakan bahwasanya
secara bersamaan liabilitas jangka pendek, dan modal sendiri tidak berpengaruh terhadap profitabilitas. Modal sendiri tidak berpengaruh terhadap proftabilitas Uji signifikasi t menunjukan nilai signifikasi sebesar 0,130 lebih besar dari 0,05. Nilai t-hitung
untuk variabel modal sendiri (X3) adalah sebesar -1,172 Pengujian dua arah α/2 = 0,05/2 = 0,025 dan
derajat bebas (dk) = n-k dengan k adalah jumlah variabel bebas dan terikat, maka (dk) = 8-4 = 4
Sehingga diperoleh nilai t-tabel sebesar 2,776. Nilai t-hitung < dari nilai t-tabel Ho diterima dan Ha
ditolak, dengan demikian dapat dikatakan modal sendiri tidak memiliki pengaruh terhadap
profitabilitas. Liabilitas jangka pendek tidak berpengaruh terhadap profitabilitas Uji signifikasi t menunjukan nilai signifikasi sebesar 0,540 lebih besar dari 0,05, dan nilai
thitung sebesar 0,658 dan Nilai t-hitung untuk variabel utang jangka pendek (X1) adalah sebesar
0,658. Pengujian dua arah α/2 = 0,05/2 = 0,025 dan derajat bebas (dk) = n-k dengan k adalah jumlah
variabel bebas dan terikat, maka (dk) = 8-3 = 5. Sehingga diperoleh nilai t-tabel sebesar 2,571. Nilai t-
hitung < dari nilai t-tabel Ho diterima dan Ha ditolak, Dengan demikian, dapat dikatakan utang jangka
pendek tidak berpengaruh secara signifikan terhadap profitabilitas. Hasil uji statistik t Berdasarkan hasil uji statistik t yang ditunjukan pada tabel 2 Maka dapat disimpulkan sebagai
berikut: berikut: Uji autokorelasi Uji autokorelasi menggunakan uji run test. Berdasarkan hasil keluaran spps diperoleh nilai
asymp.sig (2-tailed) sebesar 0,252 lebih dari > 0,05 sehingga kesimpulan yang dapat diambil adalah
tidak terdapat tanda atau masalah autokorelasi. Hasil Analisis Regresi Linear Berganda Hasil Analisis Regresi Linear Berganda
Tabel 2. Analisis Regresi Linear Berganda
Model
Unstandardized
Coefficients
Standardizes Coefficient
T
Sig. B
Std. Error
Beta
1(Constant) -.039
.278
-.140
.894
STDE
.126
.192
.378
.658
.540
PR
-.056
.092
-.351
-.611
.568
Berdasarkan hasil dari tabel 2, persamaan regresi adalah : Hasil Analisis Regresi Linear Berganda Berdasarkan hasil dari tabel 2, persamaan regresi adalah : Y = a + bX1 + bX2 Y = a + bX1 + bX2 Y = -0,039 + 0,126 X1 – 0,056 X2 Y = -0,039 + 0,126 X1 – 0,056 X2 - Nilai Konstanta (ɑ) = - 0,039 menunjukkan jika nilai liabilitas jangka pendek (X1) dan modal sendiri
(X2) memiliki nilai nol (0) maka ROE (Y) akan turun sebesar 0,039. - Nilai koefisien liabilitas jangka pendek (X1) untuk variabel X1 sebesar 0,126. artinya jika utang
jangka panjang naik sebesar Rp 1 dimana modal sendiri konstan maka profitabilitas pada PT. primarindo Asia Infrastructure, Tbk akan naik sebesar 0,126. - Nilai koefisien liabilitas jangka pendek (X1) untuk variabel X1 sebesar 0,126. artinya jika utang
jangka panjang naik sebesar Rp 1 dimana modal sendiri konstan maka profitabilitas pada PT. primarindo Asia Infrastructure, Tbk akan naik sebesar 0,126. - Nilai koefisien modal sendiri (X2) untuk variabel X2 sebesar -0,056, menunjukkan bahwa modal
sendiri mempunyai hubungan yang berlawanan arah. artinya bahwa setiap kenaikan modal sendiri
(X1) satu satuan maka variabel ROE (Y) akan turun sebesar 0,056. Tabel 3.Model Summary
Model
R
R Square
Adjusted
R
Square
Std. Error of the
Estimate
1
.697a
.485
.280
.07069
a. Predictors: (Constant), PR, STDE
Koefisien korelasi Berdasarkan hasil olah data SPSS nilai koefisien korelasi yaitu sebesar 0,697. Artinya tingkat
keeratan hubungan antara liabilitas jangka pendek dan modal sendiri terhadap profitabilitas pada PT. primarindo Asia Infrastructure, Tbk kuat yaitu sebesar 0,697
Koefisien determinasi Berdasarkan hasil olah data SPSS nilai koefisien determinasi yaitu sebesar 0,458. Artinya
kontribusi pengaruh antara liabilitas jangka pendek dan modal sendiri terhadap profitabilitas pada PT. primarindo Asia Infrastructure, Tbk yaitu sebesar 45,48% sedangkan sisanya 54,2 % dipengaruhi oleh
variabel lain yang tidak diteliti dalam penelitian ini. Hasil uji statistik F Tabel 4. Pengaruh liabilitas jangka pendek terhadap profitabilitas Uji signifikasi parsial (uji t) menunjukan bahwa nilai signifikasi pada variabel liabilitas jangka
pendek adalah lebih besar daripada tingkat level of significant. Sehingga dapat dikatakan variabel
liabilitas jangka pendek tidak memiliki pengaruh terhadap profitabilitas pada PT. Primarindo Asia
Infrastruucture, Tbk selama tahun penelitian. Utang jangka pendek dapat meningkatkan profitabilitas
dengan bunganya yang kecil, beban bunga yang kecil ini nantinya akan meningkatkan laba perusahaan
dan berdampak pada tingginya tingkat profitabilitas yang didapatkan perusahaan. Namun pada
penelitian ini dinyatakan bahwasannya liabilitas tidak terlalu mempengaruhi profitabilitas perusahaan,
dapat dilihat dari hasil penelitian yang tidak signifikan. Hal ini dikarenakan bahwa utang jangka
pendek juga memiliki resiko bagi suatu perusahaan. Utang jangka pendek memiliki biaya bunga yang 26 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 relative berfluktuasi, perusahaan terlalu banyak menggunakan hutang jangka pendek hal ini dapat
menyebabkan kesulitan likuiditas yang akan berdampak pada kebangkrutan. Likuiditas hutang jangka
pendek lebih buruk dibanding hutang jangka panjang. penelitian sesuai dengan penelitian sebelumnya
yaitu penelitian yang dilakukan L ayu (2013) yang meneliti tentang analisis pengaruh penggunaan
dana dari luar perusahaan dan modal sendiri terhadap profitabilitas pada perusahaan Automotive and
components yang Go Public di Bursa Efek Indonesia, menyatakan bahwa utang jangka pendek tidak
berpengaruh terhadap profitabilitas. Pengaruh modal sendiri terhadap profitabilitas Uji signifikasi parsial (uji t) menunjukan bahwa nilai signifikasi pada variabel modal sendiri
adalah lebih besar daripada tingkat level of significant. Sehingga dapat dikatakan variabel modal
sendiri tidak memiliki pengaruh terhadap profitabilitas pada PT. Primarindo Asia Infrastruucture, Tbk
selama tahun penelitian. Setiap perusahaan pasti menyukai dana dari dalam perusahaan karena
memiliki resiko kesulitan keuangan yang kecil daripada dana dari utang. Namun pada penelitian ini
modal sendiri tidak terlalu berpengaruh terhadap profitabilitas dikarenakan perusahaan lebih menyukai
menggunakan dana eksternal daripada dana internal yang menyebabkan modal sendiri tidak terlalu
berpengaruh pada profitabilitas. Peneliitian ini didukung oleh penelitian sebelumnya, penelitian yang
dilakukan oleh Sari (2014) yang meneliti tentang pengaruh penggunaan dana dari luar perusahaan dan
modal sendiri terhadap profitabilitas pada perusahaan property and real estate yang terdaftar di BEI
menyatakan bahwa modal sendiri tidak berpengaruh signifikan terhadap profitabilitas. xiii Pengaruh liabilitas jangka pendek dan modal sendiri terhadap profitabilitas Uji signifikasi (uji F) menunjukan bahwa secara bersama-sama liabilitas jangka pendek dan
modal sendiri tidak berpengaruh terhadap profitabilitas, hal ini dapat dilihat dari hasil pengujian secara
parsial bahwa kedua variabel tersebut tidak memiliki pengaruh terhadap profitabilitas. Penelitian ini
tidak sesuai dengan penelitian sebelumnya, penelitian dari silitonga & ginting (2019) yang meneliti
tentang pengaruh penggunaan dana dari luar perusahaan dan modal sendiri terhadap profitabilitas pada
perusahaan property and real estate yang terdaftar di Bursa Efek Indonesia menyatakan bahwa
pendanaan dari luar (hutang jangka pendek dan hutang jangka panjang) dan modal sendiri berpengaruh
signifikan terhadap profitabilitas. KESIMPULAN Berdasarkan hasil uji hipotesis dan analisis mengenai pengaruh liabilitas dan ekuitas terhadap
profitabilitas pada PT. primarindo Asia Infrastructure Tbk, maka dapat disimpulkan bahwa liabilitas
jangka pendek, dan modal sendiri sama-sama tidak berpengaruh terhadap profitabilitas karena
perusahaan ini lebih menyukai menggunakan dana dari luar perusahaan berupa hutang dalam jangka
waktu yang lama dan menyebabkan resiko yang cukup besar sehingga tidak terlalu berpengaruh
terhadap profitabilitas. p
SARAN Rekomendasi yang dapat diberikan beberapa pihak yaitu bagi perusahaan untuk meningkatkan
profitabilitas perusahaan harus lebih banyak menggunakan dana dari dalam perusahaan agar dapat
mengurangi penggunaan dana dari luar perusahaan yang memilki resiko lebih tinggi. Setiap
perusahaan pasti lebih menyukai penggunaan dana yang berasal dari dalam perusahaan karena
memiliki resiko yang lebih kecil daripada utang. Penggunaan dana dari modal sendiri ini akan sangat 27 EKOMA : Jurnal Ekonomi, Manajemen, Akuntansi
Vol.1, No.1, Januari 2022 baik dan lebih aman, dana yang didapatkan dari investasi atas modal sendiri sengga baik untuk
profitabilitas perusahaan. PT. primarindo Asia Infrastructure Tbk juga harus memperhatikan faktor
eksternal lain yang dapat mempengaruhi profitabilitas perusahaan seperti kondisi ekonomi, tingkat
suku bunga, dan pajak baik dan lebih aman, dana yang didapatkan dari investasi atas modal sendiri sengga baik untuk
profitabilitas perusahaan. PT. primarindo Asia Infrastructure Tbk juga harus memperhatikan faktor
eksternal lain yang dapat mempengaruhi profitabilitas perusahaan seperti kondisi ekonomi, tingkat
suku bunga, dan pajak PENGAKUAN/ACKNOWLEDGEMENTS Ucapan terima kasih disampaikan kepada seluruh pihak yang membantu terselesaikannya artikel
ini. DAFTAR REFERENSI Brigham, E.F. & Houston, J.F. 2014. Dasar-dasar Manajemen Keuangan Terjemahan Yulianto, A.A
2014 (P.A. Ratnaningrum , Ed.) Edisi Sebelas. (Buku 2). Jakarta:erlangga. Irton. 2009. Buku Pegangan Akuntansi. Yogyakarta: Sekolah Tinggi Ilmu Manaj Kasmir. 2008. Bank Dan Lembaga Keuangan Lainnya. Jakarta: Raja Grafindo Persada. L Ayu, R. 2013. Analisis pengaruh pendanaan dari luar perusahaan dan modal sendiri terhadap
profitabilitas pada perusahaan Automotive and components yang Go Public di Bursa Efek
Indonesia. Jurnal akutansi AKUNESA Vol. 1 No. 2. Nadeem, M Et All. 2015. The Effect Leverage On Financial Health Of The Firm: a Study From
Cement Industry Of Pakistan. Industrial Engineering Letters Volume 5 (5): 123-126. Praktito, H., 2019. Pendidikan, Bisnis, Dan Manajemen Menyongsong Era Society 5.0. Cetakan
Pertama, Malang:baskara Media. g
Prihadi, T. 2010. Analisis Laporan Keuangan : Teori Dan Aplikasi. Jakarta Pusat: PPM. 131. Riyanto, B. 2015. Dasar-dasar Pembelanjaan Perusahaan. Cetakan Keempat Belas. Yogyakarta: BPFE. Rudianto B 2012 Pengantar Akutansi: Konsep Dan Teknik Penulisan Laporan Keuangan Jakarta: B. 2015. Dasar-dasar Pembelanjaan Perusahaan. Cetakan Keempat Belas. Yogyaka Riyanto, B. 2015. Dasar-dasar Pembelanjaan Perusahaan. Cetakan Keempat Belas. Yogyakarta: BPFE. Rudianto, B. 2012. Pengantar Akutansi: Konsep Dan Teknik Penulisan Laporan Keuangan. Jakarta: y
j
p
gy
Rudianto, B. 2012. Pengantar Akutansi: Konsep Dan Teknik Penulisan Laporan Keuangan. Jakarta:
Erlangga. Sari,S,S. 2014. Pengaruh pendanaan dari luar perusahaan dan modal sendiri terhadap profitabilitas
pada perusahaan property and real estate yang terdaftar di BEI. Jurnal akutansi Vol. 2 No. 1. Sudana, I.M. 2015. Manajemen Keuangan Perusahaan: Teori Dan Praktik ( N.I.Sallama, Ed). Jakarta:
Erlangga. Syamrin, L.M. 2014. Pengantar Akutansi: Mudah Membuat Jurnal Dengan Pendekatan Siklus
Transaksi. Depok: PT. Rajagrafindo Persada. Thailab, M.K. 2014. Agency theory, capital structure on profitability of energy american firms. International journal of bussines and management invention volume 3 (12):54-61. Vatavu, S. 2015. The Impact Of Capital Structure On Financial Performance In Romanian Listed
Company. Procedia Economics And Finance, 2015 (32): 1314-1322. Yani, F.D. 2016. Hutang Jangka Panjang Dan Profitabilitas Di Bank Syariah: Studi Pada PT Bank
Muamalat Indonesia. Jurnal Manajemen Strategi Bisnis Dan Kewirausahaan, 2016 10(1): 52-63.
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Weibull-Fréchet distribution: A new lifetime distribution with application to gastric cancer data
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Weibull-Fréchet distribution: A new lifetime
distribution with application to gastric cancer
data Marko Chindranata, Siti Nurrohmah*, and Ida Fithriani
Department of Mathematics, Faculty of Mathematics and Natural Sciences (FMIPA),
Universitas Indonesia, Depok 16424, Indonesia Abstract. Lifetime data is a type of data that consists of waiting time until
an event occurs. Some of the events of lifetime data are deaths, occurrence
of a disease, or failure of a machine. The distribution usually used for
modeling lifetime data is the Weibull distribution. However, Weibull
distribution has a limitation in its application: it can only model data with a
monotonic hazard function. Therefore, a method for generalizing the
Weibull distribution is needed so it can model data with a non-monotonic
hazard function. One of those generalizations is the Weibull-Fréchet
distribution
(WFr)
which
was
introduced
by
Afify
in
2016. The WFr distribution has an advantage over the Weibull distribution, due to
its capability in modeling data with unimodal hazard function. The method
used in generating the WFr distribution is the Weibull-G (WG) that were
introduced by Bourguignon in 2014. The WG method combines the
distribution of a Weibull distribution with an arbitrary distribution with a
cumulative distribution function (cdf) G(x) using a function W[G(x)]. The characteristics of WFr distribution discussed include probability density
function (pdf), cumulative distribution function, survival function, hazard
function, and the moment. The hazard function of WFr can be monotonic or
unimodal. The maximum likelihood estimation method is used in estimating
the parameters of the distribution. Finally, lifetime data of gastric cancer
patients is given for illustration purposes. The data is modeled using the WFr
distribution, and both the Weibull and Fréchet distribution for comparison. The model result shows that the WFr distribution is the best distribution for
modeling the lifetime data of gastric cancer patients. The model result shows that the WFr distribution is the best distribution for
modeling the lifetime data of gastric cancer patients. Keywords. Hazard function, maximum likelihood method, unimodal,
Weibull-G 1 Introduction Lifetime data consists of waiting time until an event occurs. Some of the events of lifetime
data are deaths, occurrence of a disease, or failure of a machine [1]. Lifetime data have https://doi.org/10.1051/itmconf/20246101012 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 * Corresponding author: snurrohmah@sci.ui.ac.id * Corresponding author: snurrohmah@sci.ui.ac.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 https://doi.org/10.1051/itmconf/20246101012 positive real value and a skewed distribution pattern. There are two function that are usually
used in modeling lifetime data: survival function and hazard function. Hazard function
usually gives more information of the data than survival function due to its variety of hazard
shapes. The shape of hazard function can be constant, monotonic, bathtub, or unimodal
(inverse-bathtub). positive real value and a skewed distribution pattern. There are two function that are usually
used in modeling lifetime data: survival function and hazard function. Hazard function
usually gives more information of the data than survival function due to its variety of hazard
shapes. The shape of hazard function can be constant, monotonic, bathtub, or unimodal
(inverse-bathtub). Weibull distribution is one of the most popular distribution in modeling lifetime data, due
to its skewed distribution pattern and its monotonic hazard function. However, Weibull
distribution is unable to model data with a non-monotonic hazard function. We consider
Fréchet distribution which is also able to model lifetime data [2] but has a different hazard
function shape that is unimodal. Both Weibull and Fréchet distribution are combined using a
method called Weibull-G (WG) [3]. In generating the cumulative distribution function of a
WG distribution, the cdf of Weibull distribution will be modified using 𝑊𝑊[𝐺𝐺(𝑥𝑥)], which is a
function of the cumulative distribution function with an arbitrary distribution 𝐺𝐺(𝑥𝑥). Some of
other generalizations of Weibull distribution using this method are the Weibull-Uniform,
Weibull-Weibull, Weibull-Burr XII, and Weibull-Normal [3]. Using the WG method, Afify [4] generate the new distribution by using the cdf of Fréchet
distribution for G(x), resulting in the Weibull-Fréchet (WFr) distribution. The WFr
distribution is a generalization of Weibull distribution. This generalization can describe
lifetime data better than the Weibull distribution and its generalizations with WG method,
due to its capability in modeling data with a unimodal hazard shape. This paper explains how to generate the WFr distribution using the WG method, along
with its mathematical properties. Mathematical properties include the shapes of the density
and hazard functions, the moments, and the quantile. The maximum likelihood estimation
(mle) method is used in estimating the parameters of this distribution. * Corresponding author: snurrohmah@sci.ui.ac.id At the end of this paper,
real data modeled using this distribution is presented along with the Weibull and Fréchet
distribution for comparison. For applicative purposes, we use a different data used by Afify,
and use a lifetime data of gastric cancer patient instead. 2.2 Generating Weibull-Fréchet distribution Generating the cdf of The Weibull-Fréchet distribution can be done by substituting G(x) of
Equation (3) with the Fréchet distribution’s cdf. The cdf of WFr is defined as follows: 𝐹𝑥𝑎𝛽𝑏 𝐹𝐹(𝑥𝑥) = 1 −exp −𝑎𝑎ቀexp ቂ൫
𝛼𝛼
𝑥𝑥൯
𝛽𝛽−1 ቃቁ
−𝑏𝑏
൨. The pdf of WFr can be found by finding the derivative of its cdf. Some illustrations on the
pdf of WFr distribution are given on Fig. 1. 𝛼𝛽𝛼𝛽𝑏 𝑓𝑓(𝑥𝑥) = 𝑎𝑎𝑎𝑎𝑎𝑎𝛼𝛼𝛽𝛽𝑥𝑥−𝛽𝛽−1exp ቈ−𝑏𝑏ቀ𝛼𝛼
𝑥𝑥ቁ
𝛽𝛽
ቆ1 −exp ቈ−ቀ𝛼𝛼
𝑥𝑥ቁ
𝛽𝛽
ቇ
−𝑏𝑏−1
exp −𝑎𝑎ቀexp ቂ൫
𝛼𝛼
𝑥𝑥൯
𝛽𝛽−1 ቃቁ
−𝑏𝑏
൨
(4) (4) Survival function of WFr can be formulated by finding the complement of its cdf. Some illustrations on the survival function of WFr distribution are given in Fig. 2. 𝑆𝑥𝑎𝛼𝛽𝑏 Survival function of WFr can be formulated by finding the complement of its cdf. Some illustrations on the survival function of WFr distribution are given in Fig. 2. 𝑆𝑥𝑎𝛼𝛽𝑏 𝑆𝑆(𝑥𝑥) = exp −𝑎𝑎ቀexp ቂ൫
𝛼𝛼
𝑥𝑥൯
𝛽𝛽−1 ቃቁ
−𝑏𝑏
൨. The hazard function of WFr distribution can be found by finding the ratio of its pdf and
survival function. Some illustrations on the survival function of WFr distribution are given
on Fig. 3. 𝑥𝑎𝑎𝑎𝛼𝑥𝑏𝑏 ℎ(𝑥𝑥) = 𝑎𝑎𝑎𝑎𝑎𝑎𝛼𝛼𝛽𝛽𝑥𝑥−𝛽𝛽−1exp ቂ−𝑏𝑏൫
𝛼𝛼
𝑥𝑥൯
𝛽𝛽ቃቀ1 −exp ቂ−൫
𝛼𝛼
𝑥𝑥൯
𝛽𝛽ቃቁ
−𝑏𝑏−1
. The pdf, cdf, survival function, and hazard function of WFr distribution given in the
equations above will be valid for 𝑥𝑥> 0, and its parameters 𝑎𝑎> 0, 𝑏𝑏> 0, 𝛼𝛼> 0, and 𝛽𝛽> 0. 𝑎𝛼 From Fig. 1, we can see that increasing value of parameter 𝑎𝑎 will result in a pdf graph
with a steeper curve, while increasing value of parameter 𝛼𝛼 will result in a pdf graph with a
smoother curve. Altering the values of parameter 𝑏𝑏 or 𝛽𝛽 will result in a pdf graph with
differing skewness. In Fig. 2, increasing the value of parameter 𝑎𝑎 will result in a faster
decrease of survival function graph, while increasing the value of parameter 𝛼𝛼 will have a
slower decrease. Different values of 𝑏𝑏 or 𝛽𝛽 will result in a survival function graph that is
slowly decrease in the start, followed by a steep decrease after certain point of time. For the
hazard function, altering the values of 𝑎𝑎 or 𝛼𝛼 only generate a monotonically increasing hazard
shape, but with a different steepness. 2.1 Weibull-G method The Weibull-G method introduced by Bourguignon [3] is one of the methods used to
generalize The Weibull distribution. This method modifies the cumulative distribution
function of Weibull Distribution with a function 𝑊𝑊[𝐺𝐺(𝑥𝑥)]. Let 𝐺𝐺(𝑥𝑥) be the cdf of arbitrary
distribution, the cdf of WG is defined as follows: 𝐹𝑥𝑎𝑎𝑡𝑒𝑊𝐺𝑥𝑑𝑑𝑥𝑎𝑏 𝐹𝐹(𝑥𝑥) = න
𝑎𝑎𝑎𝑎𝑡𝑡𝑏𝑏−1𝑒𝑒−𝑎𝑎𝑡𝑡𝑏𝑏
𝑊𝑊[𝐺𝐺(𝑥𝑥)]
0
𝑑𝑑𝑑𝑑,
𝑥𝑥> 0, 𝑎𝑎> 0, 𝑏𝑏> 0. (1) 𝑊𝐺𝑥 (1) 𝑊𝐺𝑥 According to Alzaatreh [5], there can be several options in choosing the 𝑊𝑊[𝐺𝐺(𝑥𝑥)]
function for Equation (1). By choosing the 𝑊𝑊[𝐺𝐺(𝑥𝑥)] such as: 𝑊𝐺𝑥𝐺𝑥 𝑊𝑊[𝐺𝐺(𝑥𝑥)] =
𝐺𝐺(𝑥𝑥)
1 −𝐺𝐺(𝑥𝑥),
(2) 𝑊𝐺𝑥 (2) the cdf of WG distribution can be generated by substituting the 𝑊𝑊[𝐺𝐺(𝑥𝑥)] in Equation (2) to
Equation (1), and then by solving the integration part of Equation (1): the cdf of WG distribution can be generated by substituting the 𝑊𝑊[𝐺𝐺(𝑥𝑥)] in Equation (2) to
Equation (1), and then by solving the integration part of Equation (1): 2 2 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 𝐹𝐹(𝑥𝑥) = 1 −exp −𝑎𝑎ቆ
𝐺𝐺(𝑥𝑥)
1 −𝐺𝐺(𝑥𝑥)ቇ
𝑏𝑏
൩,
𝑥𝑥> 0, 𝑎𝑎> 0, 𝑏𝑏> 0. (3) (3) 2.2 Generating Weibull-Fréchet distribution Different value of 𝑏𝑏 or 𝛽𝛽 will generate other shapes of
hazard function such as monotonically decreasing or unimodal. 3 3 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 9th ISCPMS 2023 (a)
(b)
(c)
(d)
Fig. 1. Pdf graph of WFr distribution with (a) varying values of 𝑎𝑎 while other parameters
𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1, (c) varying values
of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while other parameters
𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (b) (a) (b) (a) (c) 𝑏𝛼𝛽𝑏 (d) 𝑎𝛼𝛽 (d) 𝑎𝛼𝛽 (c) Fig. 1. Pdf graph of WFr distribution with (a) varying values of 𝑎𝑎 while other parameters
𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1, (c) varying values
of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while other parameters
𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (a)
(b)
Fig. 2. Survival function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while other
parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (a) 𝑏𝛼𝛽 (b) 𝑎𝑏𝛼𝛽 (b) (a) 𝛼𝛽 Fig. 2. Survival function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while other
parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. 4 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 (c)
(d)
Fig. 2 (continued). Survival function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while
other parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (d) 𝑎𝑏𝛼𝛽 (c) 𝑏𝛼𝛽 (d) (c) 𝑏𝛼𝛽 Fig. 2 (continued). 2.2 Generating Weibull-Fréchet distribution Survival function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while
other parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters a= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (a)
(b)
(c)
(d)
Fig. 3. Hazard function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while other
parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters 𝑎𝑎= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. (b) (a) (b) (a) (d) 𝑎𝑏𝑎𝛼𝛽 (c) 𝑏𝛼𝛽 (d) (c) 𝛼𝛽 Fig. 3. Hazard function graph of the WFr distribution with (a) varying values of 𝑎𝑎 while other
parameters 𝑏𝑏= 𝛼𝛼= 𝛽𝛽= 1, (b) varying values of 𝑏𝑏 while other parameters 𝑎𝑎= 𝛼𝛼= 𝛽𝛽= 1,
(c) varying values of 𝛼𝛼 while other parameters 𝑎𝑎= 𝑏𝑏= 𝛽𝛽= 1, and (d) varying values of 𝛽𝛽 while
other parameters 𝑎𝑎= 𝑏𝑏= 𝛼𝛼= 1. 5 ITM Web of Conferences 61, 01012 (2024) https://doi.org/10.1051/itmconf/20246101012 9th ISCPMS 2023 By applying some Taylor series expansion on the pdf of WFr in Equation (4), the mixture
representation of the WFr pdf is written as: 𝑓𝑥𝑣𝑥 𝑓𝑓(𝑥𝑥) = 𝑣𝑣𝑗𝑗,𝑘𝑘ℎ(𝑘𝑘+1)𝑏𝑏+𝑗𝑗(𝑥𝑥)
∞
𝑗𝑗,𝑘𝑘=0
,
(5) 𝑏𝑎𝑘𝑏 (5) 𝑣𝑣𝑗𝑗,𝑘𝑘=
(−1)𝑘𝑘𝑏𝑏𝑎𝑎𝑘𝑘+1[(𝑘𝑘+ 1)𝑏𝑏+ 1]𝑗𝑗
𝑗𝑗! 𝑘𝑘! [(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
,
(6)
[(𝑘𝑘+ 1)𝑏𝑏+ 1]𝑗𝑗= Γ൫(𝑘𝑘+ 1)𝑏𝑏+ 1 + 𝑗𝑗൯
Γ൫(𝑘𝑘+ 1)𝑏𝑏+ 1൯,
(7) 𝑥𝛼𝑘𝑏𝑗𝛽 (6) (7) and ℎ(𝑘𝑘+1)𝑏𝑏+𝑗𝑗(𝑥𝑥) the pdf of Fréchet distribution with parameters 𝛼𝛼[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
1
𝛽𝛽 and 𝛽𝛽 [4]. Using Equation (5), (6), and (7), we can solve the 𝑟𝑟-th moment of WFr distribution: 𝑟𝑟 𝐸𝐸[𝑋𝑋𝑟𝑟] = 𝑣𝑣𝑗𝑗,𝑘𝑘
∞
𝑗𝑗,𝑘𝑘=0
𝛼𝛼𝑟𝑟[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
𝑟𝑟
𝛽𝛽Γ൬1 −𝑟𝑟
𝛽𝛽൰. (8) (8) The moments (first and second) WFr distribution are derived from Equation (8) by
substituting 𝑟𝑟= 1 and 𝑟𝑟= 2: 𝐸𝑋𝑣𝛼𝑘𝑏𝑗 𝐸𝐸[𝑋𝑋] = 𝑣𝑣𝑗𝑗,𝑘𝑘
∞
𝑗𝑗,𝑘𝑘=0
𝛼𝛼[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
1
𝛽𝛽Γ ൬1 −1
𝛽𝛽൰,
(9)
𝐸𝐸[𝑋𝑋2] = 𝑣𝑣𝑗𝑗,𝑘𝑘
∞
𝑗𝑗,𝑘𝑘=0
𝛼𝛼2[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
2
𝛽𝛽Γ൬1 −2
𝛽𝛽൰. (10) (9) (10) Using Equation (9) and (10), we can get variance of WFr distribution as follows: 𝑉𝑉𝑉𝑉𝑉𝑉(𝑋𝑋) = 𝑣𝑣𝑗𝑗,𝑘𝑘
∞
𝑗𝑗,𝑘𝑘=0
𝛼𝛼2[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
2
𝛽𝛽Γ ൬1 −2
𝛽𝛽൰−ቐ𝑣𝑣𝑗𝑗,𝑘𝑘
∞
𝑗𝑗,𝑘𝑘=0
𝛼𝛼[(𝑘𝑘+ 1)𝑏𝑏+ 𝑗𝑗]
1
𝛽𝛽Γ ൬1 −1
𝛽𝛽൰ቑ
2
. 2.2 Generating Weibull-Fréchet distribution The quantile of WFr distribution is given by: 𝜋𝛼 The quantile of WFr distribution is given by: 𝜋𝛼 𝜋𝜋𝑝𝑝=
𝛼𝛼
ln 1 + −
𝑎𝑎
ln[1 −𝑝𝑝]൨
1
𝑏𝑏൩
1
𝛽𝛽
. https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 2.3 Parameter estimation of Weibull-Fréchet distribution The maximum likelihood estimation (mle) is used in estimating the parameters of WFr
distribution. Let 𝑋𝑋1, 𝑋𝑋2, …, 𝑋𝑋𝑛𝑛 be a random sample from WFr distribution with parameters
𝑎𝑎> 0, 𝑏𝑏> 0, 𝛼𝛼> 0, and 𝛽𝛽> 0. The likelihood function is given by: 𝐿𝐿(𝜽𝜽) = 𝑎𝑎𝑛𝑛𝑏𝑏𝑛𝑛𝛽𝛽𝑛𝑛𝛼𝛼𝑛𝑛𝑛𝑛൭ෑ𝑥𝑥𝑖𝑖
−𝛽𝛽−1
𝑛𝑛
𝑖𝑖=1
൱𝑒𝑒
−𝑏𝑏∑
൬𝛼𝛼
𝑥𝑥𝑖𝑖൰
𝑛𝑛
𝑖𝑖=1
𝛽𝛽
ෑቆ1 −𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖൰
𝛽𝛽
ቇ
𝑛𝑛
𝑖𝑖=1
−𝑏𝑏−1
× exp
⎣
⎢
⎢
⎡
−𝑎𝑎൮
𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖൰
𝛽𝛽
1 −𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖൰
𝛽𝛽൲
𝑏𝑏
⎦
⎥
⎥
⎤
𝑛𝑛
𝑖𝑖=1
. Then the log-likelihood function of WFr distribution: 𝑙𝜽𝑛𝑎𝑏𝛽𝛽𝛼𝛽𝑥𝑏𝛽 Then the log-likelihood function of WFr distribution: 𝑙𝜽𝑛𝑎𝑏𝛽𝛽𝛼𝛽𝑥𝑏𝛽 Then the log-likelihood function of WFr distribution: 𝑙𝜽𝑛𝑎𝑏𝛽𝛽𝛼𝛽 Then the log-likelihood function of WFr distribution: 𝑙𝜽𝑛𝑎𝑏𝛽𝛽𝛼𝛽 𝑙𝑙(𝜽𝜽) = 𝑛𝑛൫ln(𝑎𝑎) + ln(𝑏𝑏) + ln(𝛽𝛽) + 𝛽𝛽ln(𝛼𝛼)൯−(𝛽𝛽+ 1) ∑
ln(𝑥𝑥𝑖𝑖)
𝑛𝑛
𝑖𝑖=1
−𝑏𝑏∑
ቀ
𝛼𝛼
𝑥𝑥𝑖𝑖ቁ
𝛽𝛽
𝑛𝑛
𝑖𝑖=1
−
(𝑏𝑏+ 1) ∑
ln ൭1 −𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖൰
𝛽𝛽
൱
𝑛𝑛
𝑖𝑖=1
−𝑎𝑎∑
ቌ
𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖
൰
𝛽𝛽
1−𝑒𝑒
−൬𝛼𝛼
𝑥𝑥𝑖𝑖
൰
𝛽𝛽ቍ
𝑏𝑏
𝑛𝑛
𝑖𝑖=1
. 𝑠𝛽𝑧𝛽 𝑙𝑙(𝜽𝜽) = 𝑛𝑛൫ln(𝑎𝑎) + ln(𝑏𝑏) + ln(𝛽𝛽) + 𝛽𝛽ln(𝛼𝛼)൯−(𝛽𝛽+ 1) ∑
ln(𝑥𝑥𝑖𝑖)
𝑛𝑛
𝑖𝑖=1
−𝑏𝑏∑
ቀ
𝛼𝛼
𝑥𝑥𝑖𝑖ቁ
𝛽𝛽
𝑛𝑛
𝑖𝑖=1
−𝑏𝑒𝛼𝛽𝑎𝑒𝛼𝑥𝛽𝑏 By letting 𝑠𝑠𝑖𝑖= exp −ቀ
𝛼𝛼
𝑥𝑥𝑖𝑖ቁ
𝛽𝛽
൨ and 𝑧𝑧𝑖𝑖= ቀ
𝛼𝛼
𝑥𝑥𝑖𝑖ቁ
𝛽𝛽
ln ቂ
𝛼𝛼
𝑥𝑥𝑖𝑖ቃ, the partial derivatives of the log-
likelihood function with respect to the parameters are given below: 𝜕𝑛𝑠𝑏𝑛 𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛
𝑎𝑎−൬
𝑠𝑠𝑖𝑖
1 −𝑠𝑠𝑖𝑖
൰
𝑏𝑏
𝑛𝑛
𝑖𝑖=1
,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛
𝑏𝑏−ln(𝑠𝑠𝑖𝑖)
𝑛𝑛
𝑖𝑖=1
−ln(1 −𝑠𝑠𝑖𝑖) −𝑎𝑎൬
𝑠𝑠𝑖𝑖
1 −𝑠𝑠𝑖𝑖
൰
𝑏𝑏
ln ൬
𝑠𝑠𝑖𝑖
1 −𝑠𝑠𝑖𝑖
൰
𝑛𝑛
𝑖𝑖=1
𝑛𝑛
𝑖𝑖=1
,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛𝑛𝑛
𝛼𝛼−𝑏𝑏𝑏𝑏
𝛼𝛼1−𝛽𝛽𝑥𝑥𝑖𝑖
−𝛽𝛽
𝑛𝑛
𝑖𝑖=1
−
(𝑏𝑏+ 1)𝛽𝛽
𝛼𝛼1−𝛽𝛽
𝑠𝑠𝑖𝑖𝑥𝑥𝑖𝑖
−𝛽𝛽
1 −𝑠𝑠𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+ 𝑎𝑎𝑎𝑎𝑎𝑎
𝛼𝛼1−𝛽𝛽
𝑠𝑠𝑖𝑖
−1𝑥𝑥𝑖𝑖
−𝛽𝛽
(𝑠𝑠𝑖𝑖
−1 −1)1+𝑏𝑏
𝑛𝑛
𝑖𝑖=1
,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛
𝛽𝛽+ 𝑛𝑛ln(𝛼𝛼) −ln(𝑥𝑥𝑖𝑖)
𝑛𝑛
𝑖𝑖=1
−𝑏𝑏𝑧𝑧𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+ (𝑏𝑏+ 1) 𝑠𝑠𝑖𝑖𝑧𝑧𝑖𝑖
1 −𝑠𝑠𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+
𝑎𝑎𝑎𝑎𝑠𝑠𝑖𝑖
−1𝑧𝑧𝑖𝑖
(𝑠𝑠𝑖𝑖
−1 −1)1+𝑏𝑏
𝑛𝑛
𝑖𝑖=1
. 𝑎𝑏𝛼𝛽𝑙𝜽𝑙𝜽𝑙𝜽𝑙𝜽 𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛𝑛𝑛
𝛼𝛼−𝑏𝑏𝑏𝑏
𝛼𝛼1−𝛽𝛽𝑥𝑥𝑖𝑖
−𝛽𝛽
𝑛𝑛
𝑖𝑖=1
−
(𝑏𝑏+ 1)𝛽𝛽
𝛼𝛼1−𝛽𝛽
𝑠𝑠𝑖𝑖𝑥𝑥𝑖𝑖
−𝛽𝛽
1 −𝑠𝑠𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+ 𝑎𝑎𝑎𝑎𝑎𝑎
𝛼𝛼1−𝛽𝛽
𝑠𝑠𝑖𝑖
−1𝑥𝑥𝑖𝑖
−𝛽𝛽
(𝑠𝑠𝑖𝑖
−1 −1)1+𝑏𝑏
𝑛𝑛
𝑖𝑖=1
,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 𝑛𝑛
𝛽𝛽+ 𝑛𝑛ln(𝛼𝛼) −ln(𝑥𝑥𝑖𝑖)
𝑛𝑛
𝑖𝑖=1
−𝑏𝑏𝑧𝑧𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+ (𝑏𝑏+ 1) 𝑠𝑠𝑖𝑖𝑧𝑧𝑖𝑖
1 −𝑠𝑠𝑖𝑖
𝑛𝑛
𝑖𝑖=1
+
𝑎𝑎𝑎𝑎𝑠𝑠𝑖𝑖
−1𝑧
(𝑠𝑠𝑖𝑖
−1 −1)
𝑛𝑛
𝑖𝑖=1𝑎𝑏𝛼𝛽 The maximum likelihood estimates of 𝑎𝑎, 𝑏𝑏, 𝛼𝛼, and 𝛽𝛽 are obtained by solving the nonlinear
equations:
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0, and
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0. It’s relatively hard to find
the result analytically, therefore the estimates will be solved using Newton method in by
RStudio version 4.2.2 with nlm() function. The maximum likelihood estimates of 𝑎𝑎, 𝑏𝑏, 𝛼𝛼, and 𝛽𝛽 are obtained by solving the nonlinear
equations:
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0,
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0, and
𝜕𝜕
𝜕𝜕𝜕𝜕𝑙𝑙(𝜽𝜽) = 0. It’s relatively hard to find
the result analytically, therefore the estimates will be solved using Newton method in by
RStudio version 4.2.2 with nlm() function. 7 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 3.1 Data illustration For applicative purposes, real lifetime data is given as an illustration for modeling with the
Weibull-Fréchet distribution. The data in Table 1 is taken from Klein and Moeschberger [1],
which consists of times until gastric cancer patients die (in days). Descriptive Statistics of the
data can be viewed in Table 2. Histogram of the data is given in Fig. 4. It can be seen
graphically that the data is right skewed. Figure 5 gives the Total Time on Test (TTT) plot of
the data. The Total Time on Test is a method to identify the hazard shape of a data, and by
the plot given in Fig. 5 we can see that the data have a unimodal hazard shape. Therefore,
it is possible to assume that Weibull-Fréchet distribution can model gastric cancer data. For comparison purposes, we also model the gastric cancer data with both Weibull and
Fréchet distribution. Parameters generated by the mle method for all the distribution is given
in Table 3. In Fig. 6, some comparison between the pdf and histogram, also for cdf and
survival function to its respective empirical function. Graphically, we can see that the that
the distributions are relatively close with the real data with Weibull-Fréchet distribution is
the closest. Table 1. Time until gastric cancer patients die (in days). 1
63
105
129
182
216
250
262
301
301
342
354
356
358
380
383
388
394
408
460
489
499
523
524
535
562
569
675
676
748
778
786
797
955
968
1000
1245
1271
1420
1551
1694
2363 Table 2. Descriptive statistics of the gastric cancer data. Table 2. Descriptive statistics of the gastric cancer data. Mean
619.6279
Median
489
Mode
301
Variance
231323.1
Skewness
1.546212
Kurtosis
2.495586
Fig. 4. Histogram of gastric cancer data. Fig. 4. Histogram of gastric cancer data. Fig. 4. Histogram of gastric cancer data. 8 8 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 Fig. 5. TTT plot of gastric cancer data. Fig. 5. TTT plot of gastric cancer data. Table 3. Estimated Parameter for gastric cancer data. Distribution
Estimates
𝒂𝒂ෝ
𝒃𝒃
𝜶𝜶ෝ
𝜷𝜷
Weibull
1.27359
0,85806
-
-
Fréchet
-
-
0.33844
1.56958
Weibull-Fréchet
1.27588
0.78096
0.52215
0.98389
(a)
(b)
(c)
Fig. 6. Graphical comparison between data and estimated distribution for (a) pdf with histogram
(b) cdf with empirical cdf, and (c) survival function with empirical survival function. Table 3. 4 Conclusion This paper introduces a new lifetime distribution called Weibull-Fréchet (WFr) distribution
which is a generalization of Weibull distribution. WFr distribution is generated using the
Weibull-G (WG) method and using cumulative distribution function of Fréchet distribution
for G(x). The shape of hazard function of WFr distribution can be unimodal, which is an
improvement from Weibull distribution. The characteristics of WFr distribution such as pdf,
cdf, survival function, hazard function, moment, and quantile can be given explicitly. The mle method is used in estimating the parameters of WFr distribution. Modeling the
illustration data results in WFr distribution to be the best-fit model for gastric cancer data,
compared with both Weibull and Fréchet distribution. 3.1 Data illustration Estimated Parameter for gastric cancer data. Table 3. Estimated Parameter for gastric cancer data. Table 3. Estimated Parameter for gastric cancer data. (b) (a) (b) (a) (c) (c) Fig. 6. Graphical comparison between data and estimated distribution for (a) pdf with histogram,
(b) cdf with empirical cdf, and (c) survival function with empirical survival function. 9 https://doi.org/10.1051/itmconf/20246101012 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 Table 4. Kolmogorov-Smirnov statistics and AIC for the distributions. Distribution
Kolmogorov-Smirnov
Test Statistics
AIC
Weibull
0.18181
521.26625
Fréchet
0.14136
431.93568
Weibull-Fréchet
0.14478
312.10688
Fig. 7. Hazard function of WFr distribution based on gastric cancer data. Table 4. Kolmogorov-Smirnov statistics and AIC for the distributions. Fig. 7. Hazard function of WFr distribution based on gastric cancer data. To get a better conclusion, we will compare The WFr distribution with both Weibull and
Fréchet distribution using Kolmogorov-Smirnov test statistics and the Akaike Information
Criterion (AIC). The AIC is quantified by −2 ln 𝐿𝐿+ 2𝑞𝑞, when ln 𝐿𝐿 is the maximum value of
log-likelihood function and 𝑞𝑞 is the number of parameters of the distribution. The best model
for the data would have the lowest value of AIC. The Kolmogorov-Smirnov statistics and
AIC for all the distributions can be viewed in Table 4. From the results, we can conclude that
the best distribution to model the gastric cancer data is the WFr distribution. It can be seen
in Fig. 7 that the WFr distribution based on gastric cancer data has a unimodal shaped hazard
function. Acknowledgements The authors would like to acknowledge the assistance of all those who contributed to this
paper. Our thanks go to the lecturers for their academic support and consultation to the 10 ITM Web of Conferences 61, 01012 (2024)
9th ISCPMS 2023 https://doi.org/10.1051/itmconf/20246101012 research, and to our families and friends for their unwavering support and encouragement
throughout this endeavour. We would also like to express our sincere gratitude to the
Faculty of Mathematics and Natural Sciences of Universitas Indonesia for providing an
opportunity and making this research possible. research, and to our families and friends for their unwavering support and encouragement
throughout this endeavour. We would also like to express our sincere gratitude to the
Faculty of Mathematics and Natural Sciences of Universitas Indonesia for providing an
opportunity and making this research possible. 4.
A. Z. Afify, H. M. Yousof, G. M. Cordeiro, E. M. M. Ortega, and Z. M. Nofal, J. Appl.
Stat. 43, 2608–2626 (2016). 3.
M. Bourguignon, R. B. Silva, and G. M. Cordeiro, J. Data Sci. 12, 53–68 (2014). References 1. J. P. Klein and M. L. Moeschberger, Survival analysis: techniques for censored and
truncated data (Springer, New York, 2003). 1. J. P. Klein and M. L. Moeschberger, Survival analysis: techniques for censored and
truncated data (Springer, New York, 2003). 2. D. H. Kutal and L. Qian, Stats 1, 176–188 (2018). 2. D. H. Kutal and L. Qian, Stats 1, 176–188 (2018). 3. M. Bourguignon, R. B. Silva, and G. M. Cordeiro, J. Data Sci. 12, 53–68 (2014). 3. M. Bourguignon, R. B. Silva, and G. M. Cordeiro, J. Data Sci. 12, 53–68 (2014). 4. A. Z. Afify, H. M. Yousof, G. M. Cordeiro, E. M. M. Ortega, and Z. M. Nofal, J. Appl. Stat. 43, 2608–2626 (2016). 5. A. Alzaatreh, C. Lee, and F. Famoye, Metron 71, 63–79 (2013). 11 11
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Disease Course Following High Disease Activity Status Revealed Patterns in SLE
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Disease Course Following High Disease Activity Status
Revealed Patterns in SLE
Alberta Hoi
(
alberta.hoi@monash.edu
)
Monash University
https://orcid.org/0000-0002-9416-7383
Rachel Koelmeyer
Monash University Faculty of Medicine Nursing and Health Sciences
Julie Bonin
Monash University Faculty of Medicine Nursing and Health Sciences
Ying Sun
Merck KGaA
Amy Kao
EMD Serono Research and Development Institute
Oliver Gunther
Merck KGaA
Hieu T. Nim
Monash University Faculty of Information Technology
Eric Morand
Monash University Faculty of Medicine Nursing and Health Sciences
Research Article
Keywords: Disease Course, High Disease Activity Status (HDAS), SLE, Lupus Low Disease Activity State (LLDAS)
Posted Date: May 7th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-488123/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at Arthritis Research & Therapy on July 14th, 2021. See the Introduction Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease with potential to cause significant
morbidity and mortality in affected patients. Clinical heterogeneity, in terms of breadth of organ involvement and disease
activity over time, adds to the complexity of disease management and measurement. Tools with which to identify
patients at high risk of adverse outcome are lacking. Recently, we described the concept of High Disease Activity Status
(HDAS), defined as experiencing SLE Disease Activity Index-2000 (SLEDAI-2K) ≥ 10, as a disease severity and prognostic
indicator [1], showing that attainment of HDAS at any time during observation was associated with poorer long-term
outcomes. In addition, in some clinical trials, this SLEDAI-2K cut-off has been associated with better discrimination of
responders to targeted therapy [2–4], suggesting the possibility that such patients may be targeted for treatment
escalation. The natural history of SLE is relapsing and remitting, such that periods of higher disease activity are usually followed by
lower disease activity, either spontaneously or in response to treatment escalation. However, the disease course following
an occurrence of HDAS, and the resolution of such episodes, has not been described. In this study, we examine clinical
associations of different HDAS patterns, and their associated outcomes. Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Arthritis Research & Therapy on July 14th, 2021. See the
published version at https://doi.org/10.1186/s13075-021-02572-1. Page 1/15 Abstract Background: We sought to examine the disease course of High Disease Activity Status (HDAS) patients and their different
disease patterns in a real-world longitudinal cohort. Disease resolution till Lupus Low Disease Activity State (LLDAS) has
been a general treatment goal, but there is limited information on this subset of patients who achieve this. Methods: All consenting patients of the Monash Lupus Cohort who had at least 12 months of observation were included. HDAS was defined as SLEDAI-2K³10 ever, and HDAS episode as the period from the first HDAS clinic visit until attainment
of LLDAS. We examined the associations of different HDAS patterns with the likelihood of damage accrual. Results: Of 342 SLE patients, 151 experienced HDAS at least once, accounting for 298 HDAS episodes. The majority of
HDAS patients (76∙2%) experienced Recurrent HDAS (>1 HDAS visit), and a smaller subset (47∙7%) had Persistent HDAS
(consecutive HDAS visits for longer than 2 months. Recurrent or Persistent HDAS patients were younger at diagnosis and
more likely to experience renal or serositis manifestations; persistent HDAS patients were also more likely to experience
neurological manifestations. Baseline SLEDAI greater than 10 was associated with longer HDAS episodes. Recurrent or
Persistent HDAS were both associated with an increased likelihood of damage accrual. Total duration of HDAS episode
greater than 2 years and experiencing multiple HDAS episodes (>4) were also associated with an increased likelihood of
damage accrual (OR 1∙80, 95%CI 1∙08 - 2∙97, p=0∙02, and OR 3∙31, 95% CI 1∙66-13∙26, p=0∙01 respectively). Conclusion: HDAS episodes have a highly variable course. Recurrent and Persistent HDAS, and longer duration of HDAS
episodes, increased risk of damage accrual. In addition to a major signifier of severity in SLE, its resolution to LLDAS can
determine subsequent outcome in SLE patients. Materials And Methods Study Design, Setting and Participants High Disease Activity Status (HDAS) HDAS was defined as attainment of SLEDAI-2K ≥10 or greater [1]. We classified HDAS patients into Recurrent HDAS,
which refers to those with at least two visits with SLEDAI-2K ≥10, or as Persistent HDAS, which is the subset of Recurrent
HDAS patients who experienced at least one episode of SLEDAI-2K > 10 for at least two consecutive visits over a period of
at least two months. We also defined an HDAS episode as the period from the onset of HDAS until resolution, defined by
attainment of Lupus Low Disease Activity State (LLDAS) [11, 12]. Cohort characteristics At the time of analysis, 406 SLE patients at the Monash Lupus Clinic were enrolled in the ALRB; 342 (84∙2%) had at least
2 clinic visits and had been followed for at least 1 year and were therefore included in the analysis. These patients had a
median (interquartile range, IQR) disease duration of 3∙6 years (0∙7–11∙1 years) at enrolment and a mean (standard
deviation, SD) observation time of 6∙7 years (3∙6 years). The median time-adjusted mean SLEDAI-2K (AMS) of the cohort
was 3∙6 (IQR: 2∙0–5∙3). SLE-related clinical variables Diagnostic assessments and autoantibody positivity were assessed at enrolment. Date of diagnosis refers to when the
diagnosis of SLE was confirmed by a specialist. At each visit, SLE disease activity was measured using the SLEDAI-2K [8]. and using the Physician Global Assessment and SLE Flare Index used in the Safety of Estrogens in Lupus Erythematosus
National Assessment (SELENA) study [9]. Accrual of damage since the onset of SLE was measured annually using the
SLICC-ACR Damage Index (SDI) [10]. Statistical analysis Descriptive statistics were used to describe the characteristics of included patients and the HDAS episodes. Bivariate
tests (e.g., c2 test, Fisher’s exact test, t test and Kruskall-Wallis test) and multinomial logistic regression were used for
group comparisons. Where applicable, unpaired heteroscedastic t tests were used assuming variance of the groups
compared was different. Logistic regression was used to assess the association of different HDAS groups, the number of
HDAS episodes and the duration of HDAS episodes with damage accrual. Other than pathology data, most other
variables had a low level of missing data. Visits with missing SLEDAI-2K data (8∙9% of visits) were excluded when
calculating patterns of HDAS. A p = 0∙05 was set as the threshold for statistical significance. Kaplan Meier survival
analysis was performed to examine the resolution of HDAS episodes in patients at different baseline SLEDAI-2K scores. A
heatmap of HDAS episodes was generated using the image.plot function in the R (version 3.6.3) programming
environment. Study Design, Setting and Participants Study Design, Setting and Participants The Monash Lupus Clinic is a specialist outpatient clinic based at Monash Health in Melbourne, Australia. As a centre of
the Australian Lupus Registry and Biobank (ALRB) [5], observational data are collected prospectively from patients with
SLE. All participants provide written informed consent for their participation and the study was approved by the Monash
Health Human Research Ethics Committee. Data captured includes sociodemographic details, clinical laboratory and
treatment information, and SLE-specific disease activity and damage assessments. SLE patients met either the American Page 2/15 College of Rheumatology (ACR) or the Systemic Lupus International Collaborating Clinics (SLICC) SLE Classification
Criteria prior to enrolment [6, 7]. The current study was limited to data from patients enrolled between April 2007 and July
2019 who had at least 2 clinic visits and at least 1 year of observation. As this was a retrospective analysis of
prospectively collected data and no specific interventions were used, patients received standard of care. College of Rheumatology (ACR) or the Systemic Lupus International Collaborating Clinics (SLICC) SLE Classification
Criteria prior to enrolment [6, 7]. The current study was limited to data from patients enrolled between April 2007 and July
2019 who had at least 2 clinic visits and at least 1 year of observation. As this was a retrospective analysis of
prospectively collected data and no specific interventions were used, patients received standard of care. Patterns of HDAS occurrence Almost half (151/342, 44∙2%) of the participants experienced at least one HDAS visit (SLEDAI-2K ≥ 10); 10∙5% only
experienced HDAS at a single visit, and 33∙6% had Recurrent HDAS (≥ 2 HDAS visits). Of the Recurrent HDAS group,
62∙6% experienced at least one episode of Persistent HDAS. Page 3/15 Table 1 provides descriptive statistics of patient characteristics by HDAS category. One of the main determinants of
whether patients experience Recurrent or Persistent HDAS was their duration of observation, as shown by the higher
duration of observation. We also saw differences with respect to age of diagnosis, ethnicity, and serological activity. There were also higher frequencies of renal involvement, history of serositis, or neurological manifestations in Recurrent
or Persistent HDAS patients (Table 1). To further explore the differences in patient characteristics, we conducted a
multinomial logistic regression analysis (Table 2). The associations of these clinical features with Recurrent or Persistent
HDAS were significantly higher. Recurrent or Persistent HDAS patients were also more likely to be treated with
immunosuppressant (particularly mycophenolate, azathioprine, cyclophosphamide and tacrolimus), and more likely to be
on greater than 15mg per day of prednisolone (Table 1). Table 1 provides descriptive statistics of patient characteristics by HDAS category. One of the main determinants of
whether patients experience Recurrent or Persistent HDAS was their duration of observation, as shown by the higher
duration of observation. We also saw differences with respect to age of diagnosis, ethnicity, and serological activity. There were also higher frequencies of renal involvement, history of serositis, or neurological manifestations in Recurrent
or Persistent HDAS patients (Table 1). To further explore the differences in patient characteristics, we conducted a
multinomial logistic regression analysis (Table 2). The associations of these clinical features with Recurrent or Persistent
HDAS were significantly higher. Recurrent or Persistent HDAS patients were also more likely to be treated with
immunosuppressant (particularly mycophenolate, azathioprine, cyclophosphamide and tacrolimus), and more likely to be
on greater than 15mg per day of prednisolone (Table 1). Page 4/15 Table 1 Table 1
Patient characteristics by HDAS category (n = 342) Patient characteristics by HDAS category (n = 342)
Patient Characteristic
HDAS Patient Category
Evidence of
difference in
characteristic
(p-value)^
never
experienced
HDAS
(n = 191)
one HDAS visit
(n = 36)
Recurrent
but not
Persistent
HDAS
(n = 43)
Persistent HDAS
(n = 72)
Female sex
168
(88∙0%)
30 (83∙3%)
39
(90∙7%)
59 (81∙9%)
0∙46
Asian ethnicity! Patterns of HDAS occurrence 67 (35∙8%)
16 (48∙5%)
26
(61∙9%)
33 (45∙8%)
0∙014
Age at diagnosis (years),
median (IQR)
32 (25, 45)
27 (19, 46∙5)
26 (20,
37)
28∙5 (21, 35)
0∙012
Disease duration at
enrolment (years), median
(IQR)
3 (0∙6,
11∙5)
4∙6 (0∙8, 11∙2)
5∙7 (2,
12∙8)
4∙05 (0∙85, 10∙3)
0∙64
Total patient observation
time (years), median (IQR)
4∙9 (3, 8∙7)
5∙6 (2∙1, 8∙1)
8∙3 (4∙7,
10∙4)
8∙8 (5∙1, 12∙1)
< 0∙001
Categorical observation
time variable
< 5 years
98 (51∙3%)
17 (47∙2%)
12
(27∙9%)
17 (23∙6%)
< 0∙001
5–10 years
62 (32∙5%)
13 (36∙1%)
17
(39∙5%)
22 (30∙6%)
10 + years
31 (16∙2%)
6 (16∙7%)
14
(32∙6%)
33 (45∙8%)
Organ involvement
Skin
126
(66∙0%)
18 (50∙0%)
31
(72∙1%)
45 (62∙5%)
0∙20
Arthritis
134
(70∙2%)
23 (63∙9%)
31
(72∙1%)
44 (61∙1%)
0∙46
Haematological
92 (48∙2%)
21 (58∙3%)
27
(62∙8%)
40 (55∙6%)
0∙26
Renal
43 (22∙5%)
12 (33∙3%)
24
(55∙8%)
50 (69∙4%)
< 0∙001
!89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. #Includes positivity for anti-cardiolipin, anti-beta2GPI or lupus anticoagulant Page 5/15
!89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. #Includes positivity for anti-cardiolipin, anti-beta2GPI or lupus anticoagulant
^Tests used to compare the HDAS categories included the Pearson's chi-squared test, Fisher's exact test and the
Kruskal-Wallis test
*Other immunosuppressants include: Mycophenolate, Azathioprine, Methotrexate, Cyclophosphamide, Tacrolimus,
Leflunomide, Chloroquine, Mercaptopurine & Sulfasalazine Page 5/15
!89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. Patterns of HDAS occurrence #Includes positivity for anti-cardiolipin, anti-beta2GPI or lupus anticoagulant
^Tests used to compare the HDAS categories included the Pearson's chi-squared test, Fisher's exact test and the
Kruskal-Wallis test
*Other immunosuppressants include: Mycophenolate, Azathioprine, Methotrexate, Cyclophosphamide, Tacrolimus,
Leflunomide, Chloroquine, Mercaptopurine & Sulfasalazine Page 5/15 Patient Characteristic
HDAS Patient Category
Evidence of
difference in
characteristic
(p-value)^
never
experienced
HDAS
(n = 191)
one HDAS visit
(n = 36)
Recurrent
but not
Persistent
HDAS
(n = 43)
Persistent HDAS
(n = 72)
Serositis
49 (25∙7%)
11 (30∙6%)
18
(41∙9%)
38 (52∙8%)
< 0∙001
Neurological
16 (8∙4%)
3 (8∙3%)
7 (16∙3%)
19 (26∙4%)
0∙001
Serological profile
Anti_dsDNA
123
(65∙1%)
30 (85∙7%)
42
(97∙7%)
70 (97∙2%)
< 0∙001
Anti_Sm
20 (11∙0%)
8 (22∙9%)
12
(28∙6%)
17 (23∙6%)
0∙007
Anti_Ro
74 (40∙7%)
17 (48∙6%)
26
(61∙9%)
38 (52∙8%)
0∙052
Anti-phospholipid-
autoantibody-positive
94 (49∙2%)
18 (50∙0%)
25
(58∙1%)
42 (58∙3%)
0∙49
Low complement at
baseline#
89 (46∙6%)
24 (66∙7%)
29
(67∙4%)
58 (80∙6%)
< 0∙001
anti-dsDNA-positive and
low complement at
baseline
60 (31∙4%)
20 (55∙6%)
29
(67∙4%)
57 (79∙2%)
< 0∙001
Adjusted mean SLEDAI,
median (IQR)
2∙2 (1∙2,
3∙5)
4∙1 (3∙55,
5∙5)
5∙2 (4∙1, 6∙2)
6∙4
(5∙0,
8∙1)
< 0∙001
Mild/moderate flare rate,
median (IQR) per 100
person-years
0∙3 (0, ∙6)
0∙5 (0∙3,
1∙05)
0∙9 (0∙6, 1∙2)
0∙9
(0∙6,
1∙3)
< 0∙001
Severe flare rate, median
(IQR) per 100 person-
years
0 (0, 0)
0∙1 (0, 0∙4)
0∙2 (0∙1, ∙4)
0∙5
(0∙2,
0∙9)
< 0∙001
Damage accrual over
observation period
51 (26∙7%)
13 (36∙1%)
20 (46∙5%)
48
(66∙7%)
< 0∙001
Treatment received
Hydroxychloroquine
175
(91∙6%)
32
(88∙9%)
43 (100∙0%)
69 (95∙8%)
0∙12
!89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. #Includes positivity for anti cardiolipin anti beta2GPI or lupus anticoagulant Patient Characteristic
HDAS Patient Category !89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. #Includes positivity for anti-cardiolipin, anti-beta2GPI or lupus anticoagulant Patterns of HDAS occurrence #Includes positivity for anti-cardiolipin, anti-beta2GPI or lupus anticoagulant
^Tests used to compare the HDAS categories included the Pearson's chi-squared test, Fisher's exact test and the
Kruskal-Wallis test
*Other immunosuppressants include: Mycophenolate, Azathioprine, Methotrexate, Cyclophosphamide, Tacrolimus,
Leflunomide, Chloroquine, Mercaptopurine & Sulfasalazine Page 6/15 Patient Characteristic
HDAS Patient Category
Evidence of
difference in
characteristic
(p-value)^
never
experienced
HDAS
(n = 191)
one HDAS visit
(n = 36)
Recurrent
but not
Persistent
HDAS
(n = 43)
Persistent HDAS
(n = 72)
Other
immunosuppressant*
119
(62∙3%)
29
(80∙6%)
41 (95∙3%)
72 (100∙0%)
< 0∙001
Hydroxychloroquine/other
immunosuppressant*
183
(95∙8%)
35
(97∙2%)
43 (100∙0%)
72 (100∙0%)
0∙18
Mycophenolate
43 (22∙5%)
15
(41∙7%)
29 (67∙4%)
56 (77∙8%)
< 0∙001
Azathioprine
53 (27∙7%)
15
(41∙7%)
24 (55∙8%)
41 (56∙9%)
< 0∙001
Methotrexate
44 (23∙0%)
9
(25∙0%)
8 (18∙6%)
19 (26∙4%)
0∙81
Cyclophosphamide
4 (2∙1%)
0
(0∙0%)
3 (7∙0%)
17 (23∙6%)
< 0∙001
Tacrolimus
3 (1∙6%)
0
(0∙0%)
4 (9∙3%)
4 (5∙6%)
0∙026
Leflunomide
11 (5∙8%)
1
(2∙8%)
1 (2∙3%)
4 (5∙6%)
0∙73
Prednisolone (any dose)
128
(67∙0%)
32
(88∙9%)
41 (95∙3%)
71 (98∙6%)
< 0∙001
Prednisolone > 15mg per
day
61 (31∙9%)
20
(55∙6%)
34 (79∙1%)
64 (88∙9%)
< 0∙001
!89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. Patient Characteristic
HDAS Patient Category !89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
Strait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
persons of mixed ethnicity. 89∙1% (156) of non-Asians were of Caucasian ethnicity; other ethnicities reported included Aboriginal and/or Torres
trait Islander, African, Egyptian, Latin American, Maori, Middle Eastern, Pacific Islander and Turkish ethnicity and
ersons of mixed ethnicity. Page 7/15 Page 7/15 Page 7/15 Table 2 P
8/15
Clinical Associations that predict HDAS patient categories
Patient Characteristic
HDAS Patient Category! Characteristics of HDAS Episodes. There were 298 HDAS episodes, defined as the period of observation between the first detection of HDAS until attainment
of LLDAS, in 151 patients, observed over a total of 5,193 visits. The duration of HDAS episodes was highly variable, with
a median (IQR) of 147 days (84–294 days) (Fig. 1A). Disease activity at the commencement of an HDAS episode
influenced episode duration. Compared to HDAS episodes where baseline SLEDAI-2K = 10, HDAS episodes with higher
baseline scores (SLEDAI-2K > 10) were significantly longer (mean ± SD; 211 ± 200 vs 280 ± 354 days, p = 0∙032) (Fig. 1B). The median time to resolution (LLDAS) was 5∙5 months vs 5∙1 month (OR 0∙74, 95% CI 0∙12 to 2∙46, p = 0∙014)
(Fig. 1C); and the likelihood of HDA episodes lasting > 2 years was also significantly higher, with odds ratio of 3∙17
(95%CI = 1∙04–9∙6, p = 0∙04). Patterns of HDAS occurrence (Relative Risk, 95% Confidence Interval; p value)
1 HDAS visit
(n = 36)
Recurrent but
not persistent HDAS
(n = 43)
Persistent HDAS
(n = 72)
Asian ethnicity
1∙69 (0∙80–3∙55;
0∙170)
2∙91 (1∙46–5∙81;
0∙002)
1∙52 (0∙87–2∙63;
0∙140)
Total patient observation (years)
0∙99 (0∙89–1∙09;
0∙783)
1∙13 (1∙04–1∙24;
0∙006)
1∙21 (1∙12–1∙30;
<0∙001)
Categorical observation time variable
< 5 years
1∙0 (Not
Applicable)
1∙0 (Not Applicable)
1∙0 (Not Applicable)
5–10 years
1∙21 (0∙55–2∙66;
0∙638)
2∙24 (1∙0–5∙0;
0∙050)
2∙05 (1∙0–4∙15;
0∙048)
10 + years
1∙12 (0∙40–3∙08;
0∙832)
3∙69 (1∙54–8∙81;
0∙003)
6∙14 (3∙01–12∙49;
<0∙001)
Organ involvement by SLICC SLE
Classification Criteria
Skin
0∙52 (0∙25–1∙06;
0∙071)
1∙33 (0∙64–2∙77;
0∙441)
0∙86 (0∙49–1∙51;
0∙599)
Arthritis
0∙75 (0∙36–1∙59;
0∙456)
1∙10 (0∙53–2∙29;
0∙801)
0∙67 (0∙38–1∙18;
0∙163)
Haematological
1∙51 (0∙73–3∙10;
0∙265)
1∙82 (0∙92–3∙59;
0∙086)
1∙35 (0∙78–2∙32;
0∙286)
Renal
1∙72 (0∙80–3∙72;
0∙168)
4∙35 (2∙18–8∙68;
<0∙001)
7∙82 (4∙27–14∙33;
<0∙001)
Serositis
1∙28 (0∙58–2∙78;
0∙541)
2∙09 (1∙05–4∙15;
0∙036)
3∙24 (1∙84–5∙70;
<0∙001)
Neurological
0∙99 (0∙27–3∙60;
0∙993)
2∙13 (0∙82–5∙54;
0∙123)
3∙92 (1∙88–8∙16;
<0∙001)
Serological profile
Anti_dsDNA
3∙22 (1∙19–8∙69;
0∙021)
22∙54 (3∙03–167∙46;
0∙002)
18∙78 (4∙46–79∙03;
<0∙001)
Anti_Sm
2∙40 (0∙96–6∙0;
0∙061)
3∙24 (1∙43–7∙32;
0∙005)
2∙50 (1∙22–5∙12;
0∙012)
Anti_Ro
1∙38 (0∙67–2∙85;
0∙386)
2∙37 (1∙19–4∙73;
0∙014)
1∙63 (0∙94–2∙82;
0∙081)
Anti-phospholipid-autoantibody-positive
1∙03 (0∙51–2∙10;
0∙931)
1∙43 (0∙73–2∙80;
0∙292)
1∙44 (0∙84 − 2∙50;
0∙188)
! Reference category: patients who never experienced HDA (n = 191) Clinical Associations that predict HDAS patient categories ! Reference category: patients who never experienced HDA (n = 191) Page 8/15 Patient Characteristic
HDAS Patient Category! (Relative Risk, 95% Confidence Interval; p value)
1 HDAS visit
(n = 36)
Recurrent but
not persistent HDAS
(n = 43)
Persistent HDAS
(n = 72)
Low complement at baseline
2∙29 (1∙08–4∙85;
0∙030)
2∙37 (1∙18–4∙77;
0∙015)
4∙75 (2∙48–9∙09;
<0∙001)
Anti-dsDNA-positive and low complement
at baseline
2∙73 (1∙32–5∙63;
0∙007)
4∙52 (2∙23–9∙17;
<0∙001)
8∙30 (4∙35–15∙82;
<0∙001)
! Reference category: patients who never experienced HDA (n = 191) HDAS Patient Category! ! Reference category: patients who never experienced HDA (n = 191) Different patterns of HDAS and damage accrual Compared to non-HDAS patients, the risk of damage was markedly increased amongst patients with Recurrent or
Persistent HDAS. When adjusted for observation time, Persistent HDAS patients had a significantly increased likelihood of
damage accrual (OR 3∙7, CI 2∙0–7∙1), p < 0∙001 (Table 3). The duration of an HDAS episode was also strongly
associated with damage accrual (Table 4). This was observed regardless of whether we calculated cumulative exposure
of HDAS episodes for patients or individual HDAS episodes (Table 4). For HDAS Episodes that lasted over 2 years, the
adjusted odd ratio for damage accrual was 1∙80 (95% CI 1∙08–2∙97, p = 0∙02) (Table 4). We also examined the effects of
multiple HDAS episodes on damage accrual and found that only experiencing 4 or more HDAS episodes was associated
with increased damage accrual when adjusting for observation time (OR 3∙31, 95% CI 1∙66 to 13∙26, p = 0∙001 (Table 5). Page 9/15 Page 9/15 Table 3
Risk of damage accrual by HDAS patient category
HDAS Patient Category
Odds of accruing damage
Unadjusted
Adjusted for observation time#
OR (95% CI)
P value
OR (95% CI)
P value
Never experienced HDAS
1
Not
Applicable
1
Not Applicable
One HDAS visit only
1∙6 (0∙7–3∙3)
0∙252
1∙6 (0∙7–3∙6)
0∙267
All Recurrent HDAS
3∙97 (2∙43–
6∙49)
< 0∙001
2∙74 (1∙60–
4∙69)
< 0∙001
Recurrent but not Persistent HDAS
2∙4 (1∙2–4∙7)
0∙012
1∙7 (0∙8–3∙6)
0∙146
At least one episode of Persistent
HDAS
5∙5 (3∙1–9∙9)
< 0∙001
3∙7 (2∙0–7∙1)
< 0∙001
#Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). Table 3 Table 3
Risk of damage accrual by HDAS patient category
Odds of accruing damage #Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). OR = Odds ratio, CI = Confidence Interval *Statistically significant at p < 0∙05 ally significant at p < 0∙05 ervation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years) *Statistically significant at p < 0∙05 Different patterns of HDAS and damage accrual *Statistically significant at p < 0∙05
OR = Odds ratio, CI = Confidence Interval Risk of damage accrual by number of HDAS Episodes
Odds of accruing damage 0
1
Not Applicable
1
Not Applicable
1
1∙35 (0∙72–2∙51)
0∙41
1∙74 (0∙63 − 4∙5)
0∙27
2
2∙27 (1∙13–4∙55)
0∙02 *
1∙09 (0∙48 − 2∙15)
0∙83
3
2∙23 (0∙92 − 5∙41)
0∙08
1∙44 (0∙74 − 2∙58)
0∙23
4 or more
20∙29 (5∙80 − 70∙98)
< 0∙001 *
3∙31 (1∙66 − 13∙26)
0∙01 *
#Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). *Statistically significant at p < 0∙05
OR = Odds ratio, CI = Confidence Interval #Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). Different patterns of HDAS and damage accrual Table 4
Risk of damage accrual by HDAS Episode duration
HDAS Episode duration
Odds of accruing damage
Unadjusted
Adjusted for observation time#
OR (95% CI)
P value
OR (95% CI)
P value
Never experienced HDAS
1
Not Applicable
1
Not Applicable
Cumulative HDAS duration
≥ 1 day and < 1 year
1∙64 (0∙90 − 2∙97)
0∙12
1∙48 (0∙49 − 4∙08)
0∙46
≥ 1 year and < 2 years
1∙70 (0∙76 − 3∙83)
0∙19
1∙27 (0∙60 − 2∙39)
0∙49
2 years or more
5∙55 (2∙88 − 10∙71)
< 0∙001 *
1∙80 (1∙08 − 2∙97)
0∙02 *
Mean duration of HDAS Episode
≥ 1 day and < 1 year
2∙31 (1∙41 − 3∙81)
< 0∙001 *
2∙12 (0∙88 − 5∙00)
0∙09
≥ 1 year and < 2 years
1∙81 (0∙71 − 4∙62)
0∙30
0∙93 (0∙27 − 2∙31)
0∙88
2 years or more
6∙37 (2∙30 − 17∙66)
< 0∙001 *
1∙60 (0∙91 − 2∙67)
0∙08
#Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). *Statistically significant at p < 0∙05 Table 4 #Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). #Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). Page 10/15 Table 5 Table 5 Table 5
Risk of damage accrual by number of HDAS Episodes
Number of HDAS Episodes
Odds of accruing damage
Unadjusted
Adjusted for observation time#
OR (95% CI)
P value
OR (95% CI)
P value
0
1
Not Applicable
1
Not Applicable
1
1∙35 (0∙72–2∙51)
0∙41
1∙74 (0∙63 − 4∙5)
0∙27
2
2∙27 (1∙13–4∙55)
0∙02 *
1∙09 (0∙48 − 2∙15)
0∙83
3
2∙23 (0∙92 − 5∙41)
0∙08
1∙44 (0∙74 − 2∙58)
0∙23
4 or more
20∙29 (5∙80 − 70∙98)
< 0∙001 *
3∙31 (1∙66 − 13∙26)
0∙01 *
#Adjusted for observation time modelled as a categorical variable (< 5 years, 5 - <10 years and 10 + years). Discussion Damage Page 11/15 Page 11/15 accrual was also significantly associated with patients who experienced multiple HDAS episodes (4 or greater), perhaps
suggesting that this was a very select group with poor prognosis. The implication of our findings is that early intervention to control disease activity upon the onset of HDAS is paramount. While a major challenge in SLE management is in prevention of relapse, if disease can be rapidly brought into control, the
risk of damage accrual can be minimised by curtailing the duration of the HDAS episode. The effect of multiple HDAS
episodes on disease outcomes highlights the potential importance of maintenance therapy, particularly in the subset of
patients who are prone to relapse or who have had a prolonged HDAS episode. Other studies report on baseline patient
and disease characteristics as predictors of relapse which can guide clinician decision making [14, 15]. A closer examination of HDAS episodes might also help us design better clinical trials. In our cohort, the median duration
of HDAS episodes was about 5 months, suggesting any interventional trial starting at HDAS and using LLDAS as an
efficacy endpoint should take this into account [3, 16, 17]. Important observation in this context is the longer time to
resolution in patients whose HDAS episode began with a SLEDAI-2K > 10. Currently, most SLE clinical trials are designed
with endpoints at 24 or 48 weeks [18]. If LLDAS is utilised as a primary endpoint, a shorter time to endpoint may allow
greater differentiation between an active treatment arm and placebo when superimposed on standard of care, particularly
if we take into account the baseline SLEDAI-2K. This study has some limitations. Some patients in our cohort entered into an HDAS episode but failed to achieve LLDAS
during the observation period, for reasons such as shorter follow-up period or persistent disease activity. We have chosen
not to include this smaller subset of patients in the current analysis as there may be many reasons for the inability to
achieve LLDAS. Although data were collected prospectively, this analysis is retrospective, and in addition this is a single
centre study, albeit in a multi-ethnic cohort under universal health care. Confirmatory studies in independent cohorts are
required, and we hope that the current findings will stimulate this. Conclusion This study adds to existing evidence that HDAS is a poor prognostic indicator for SLE patients. In addition to previous
findings that ever-attaining high disease activity defined as SLEDAI-2K ≥ 10, this study has demonstrated that Recurrent
or Persistent HDAS, increased duration of HDAS episodes, and multiple repeated HDAS episodes were also associated
with higher damage accrual. Future studies that examine for factors that predict the rapidity of HDAS resolution and
maintenance of LLDAS will be useful. Discussion High Disease Activity Status (HDAS) has recently been confirmed as an important prognostic indicator for severe disease
in SLE [1]. Studies of HDAS patients in real world cohorts can shed light on how disease behaves in this group of patients
which are typically the target population of clinical trials. In this study, we report on a number of disease patterns
following an initial HDAS visit and their association with damage accrual. We observed that the majority of HDAS
patients experience Recurrent HDAS rather than single events, a finding particularly evident with longer periods of
observation suggesting that shorter studies may fail to detect this pattern. Recurrent or Persistent HDAS patients have the
hallmark of a more severe phenotype, with younger at disease onset, more serological activity and more serious organ
involvement including renal disease, serositis and neurological features. We explored the relationship between different patterns of HDAS and damage accrual. Not surprisingly, Recurrent and
Persistent HDAS were associated with increased risk of damage accrual even after adjusting for observation time. Our
data also suggest that the total cumulative period of HDAS greater than 2 years, rather than the number of relapses, had a
greater effect on damage accrual. The attainment of LLDAS is an important treatment goal for people affected with SLE, as it has been associated with
protection from flares and damage accrual.[13] In this study, we analysed the characteristics of HDAS episodes which
were defined by the first attainment of LLDAS after patients experienced HDAS. Our study sheds light on the heterogeneity
of the duration of these HDAS episodes. Longer duration of HDAS episodes and multiple recurrent HDAS episodes were
strong predictors for damage accrual. Many factors can determine the duration of an HDAS episode, but one important
consideration was the baseline disease activity. Higher SLEDAI-2K at the onset of HDAS was associated with longer
HDAS episodes, which in turn was associated with increased likelihood of damage accrual. Monitoring the duration of an HDAS episode may be clinically relevant for planning treatment, as the relationship between
the duration of HDAS episodes and damage appears to uphold whether patients experienced single or multiple HDAS
episodes, implying that strategies aimed at attaining LLDAS more quickly after HDAS could limit damage. Consent for publication All authors approved the final version to be submitted for publication. Author Contributions AH, YS, AK, OG, HN, EM contributed to the study conception and design. AH conducted the literature review. AH and EM
were responsible for data acquisition. AH, RK, JB, HN contributed to the analysis and interpretation of data. All authors
were involved in drafting the article or revising it critically for important intellectual content. Abbreviations ACR American College of Rheumatology Page 12/15 Page 12/15 SDI Systemic lupus erythematosus Damage Index
SELENA Safety of Estrogens in Lupus Erythematosus National Assessment Study
SLE Systemic lupus erythematosus
SLEDAI-2K Systemic lupus erythematosus disease activity index-2000
SLICC Systemic lupus International Collaborating Clinics SDI Systemic lupus erythematosus Damage Index SDI Systemic lupus erythematosus Damage Index SELENA Safety of Estrogens in Lupus Erythematosus National Assessment Study
SLE Systemic lupus erythematosus SLICC Systemic lupus International Collaborating Clinics Funding Merck KGaA (YS, AK, OG) provided specific financial support for this study. Availability of supporting data Ms Koelmeyer and Dr Nim had full access to all of the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis. Ethical Approval and Consent to participate Data collection and storage via the Australian Lupus Registry & Biobank and current study has been approved by Monash
Health Human Research and Ethics Committee Reference 14262A, and was conducted according to the Declaration of
Helsinki. Written informed consent was granted by all study participants. Acknowledgements We thank the patients who consented to the use of their data and samples for research purposes as part of the Australian
Lupus Registry & Biobank. The study was supported by a research grant provided by Merck KGaA. An abstract was
presented at the 2019 American College of Rheumatology Annual Meeting. Competing Interests All authors have no relevant conflicts of interest but we acknowledge funding that was received for the conduct of the
Australia Lupus Registry and Biobank at the Monash Lupus Clinic, in terms of sponsorship and unrestricted educational
grants (AH, RK, JB, HN, EM) from Merck KGaA, GlaxoSmithKline, UCB, and Astra Zeneca. References Page 13/15 1. Koelmeyer R, Nim HT, Nikpour M, Sun YB, Kao A, Guenther O, Morand E, Hoi A: High disease activity status suggests
more severe disease and damage accrual in systemic lupus erythematosus. Lupus Sci Med 2020, 7(1). 2. van Vollenhoven RF, Petri MA, Cervera R, Roth DA, Ji BN, Kleoudis CS, Zhong ZJ, Freimuth W: Belimumab in the
treatment of systemic lupus erythematosus: high disease activity predictors of response. Ann Rheum Dis 2012,
71(8):1343-1349. 3. Morand EF, Isenberg DA, Wallace DJ, Kao AH, Vazquez-Mateo C, Chang P, Pudota K, Aranow C, Merrill JT: Attainment
of treat-to-target endpoints in SLE patients with high disease activity in the atacicept phase 2b ADDRESS II study. Rheumatology (Oxford) 2020. 4. Merrill JT, Wallace DJ, Wax S, Kao A, Fraser PA, Chang P, Isenberg D, Investigators AI: Efficacy and Safety of Atacicept
in Patients With Systemic Lupus Erythematosus: Results of a Twenty-Four-Week, Multicenter, Randomized, Double-
Blind, Placebo-Controlled, Parallel-Arm, Phase IIb Study. Arthritis Rheumatol 2018, 70(2):266-276. 5. O’Neill S, Morand EF, Hoi A: The Australian Lupus Registry and Biobank: a timely initiative. The Medical journal of
Australia 2017, 206(5):194-195. 6. Hochberg MC: Updating the American College of Rheumatology revised criteria for the classification of systemic
lupus erythematosus. Arthritis Rheumatol 1997, 40(9):1725. 7. Petri M, Orbai A-M, Alarcón GS, et al.: Derivation and validation of the Systemic Lupus International Collaborating
Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012, 64:2677-2686. 8. Touma Z, Urowitz MB, Gladman DD: SLEDAI-2K for a 30-day window. Lupus 2010, 19:49-50. Touma Z, Urowitz MB, Gladman DD: SLEDAI-2K for a 30-day window. Lupus 2010, 19:4 9. Buyon JP, Petri MA, Kim MY, et al.: The Effect of Combined Estrogen and Progesterone Hormone Replacement
Therapy on Disease Activity in Systemic Lupus Erythematosus: A Randomized Trial. Ann Intern Med 2005, 142:953-
962. 10. Gladman D, Ginzler E, Goldsmith C, et al.: The Development and Initial Validation of the Systemic Lupus International
Collaborating Clinics/American College of Rheumatology Damage Index for Systemic Lupus Erythematosus. Arthritis
Rheumatol 1996, 39(3):363-369. 10. Gladman D, Ginzler E, Goldsmith C, et al.: The Development and Initial Validation of the Systemic Lupus International
Collaborating Clinics/American College of Rheumatology Damage Index for Systemic Lupus Erythematosus. Arthritis
Rheumatol 1996, 39(3):363-369. 11. References Franklyn K, Lau CS, Navarra SV, Louthrenoo W, Lateef A, Hamijoyo L, Wahono CS, Chen S-L, Jin O, Morton S et al:
Definition and initial validation of a Lupus Low Disease Activity State (LLDAS). Annals of the Rheumatic Diseases
2016, 75(9):1615-1621. 12. Golder V, Kandane-Rathnayake R, Huq, PhD HTN, MD PWL, MD PSFL, MD Y-JJW, MBBS AL, MD SS, MD PSVN et al:
Lupus low disease activity state as a treatment endpoint for systemic lupus erythematosus: a prospective validation
study. The Lancet Rheumatology 2019, 1(2):e95-102. 13. Golder V, Kandane-Rathnayake R, Huq M, Nim H, Louthrenoo W, Luo S, Wu Y, Lateef A, Sockalingam S, Navarra S et
al: Prospective Validation of the Lupus Low Disease Activity State (LLDAS) - a Treat to Target Endpoint for Systemic
Lupus Erythematosus. Lancet Rheumatology 2019. 14. Inês L, Duarte C, Silva RS, Teixeira AS, Fonseca FP, da Silva JA: Identification of clinical predictors of flare in systemic
lupus erythematosus patients: a 24-month prospective cohort study. Rheumatology (Oxford) 2014, 53(1):85-89. 14. Inês L, Duarte C, Silva RS, Teixeira AS, Fonseca FP, da Silva JA: Identification of clinical predictors of flare in systemic
lupus erythematosus patients: a 24-month prospective cohort study. Rheumatology (Oxford) 2014, 53(1):85-89. 15. Petri MA, van Vollenhoven RF, Buyon J, Levy RA, Navarra SV, Cervera R, Zhong ZJ, Freimuth WW, Groups B-aB-S:
Baseline predictors of systemic lupus erythematosus flares: data from the combined placebo groups in the phase III
belimumab trials. Arthritis Rheum 2013, 65(8):2143-2153. 16. Oon S, Huq M, Golder V, Ong PX, Morand EF, Nikpour M: Lupus Low Disease Activity State (LLDAS) discriminates
responders in the BLISS-52 and BLISS-76 phase III trials of belimumab in systemic lupus erythematosus. Ann Rheum
Dis 2019, 78(5):629-633. 16. Oon S, Huq M, Golder V, Ong PX, Morand EF, Nikpour M: Lupus Low Disease Activity State (LLDAS) discriminates
responders in the BLISS-52 and BLISS-76 phase III trials of belimumab in systemic lupus erythematosus. Ann Rheum
Dis 2019, 78(5):629-633. 17. Morand EF, Trasieva T, Berglind A, Illei GG, Tummala R: Lupus Low Disease Activity State (LLDAS) attainment
discriminates responders in a systemic lupus erythematosus trial: post-hoc analysis of the Phase IIb MUSE trial of 17. anifrolumab. Ann Rheum Dis 2018, 77(5):706-713.
18. Mahieu MA, Strand V, Simon LS, Lipsky PE, Ramsey-Goldman R: A critical review of clinical trials in systemic lupus
erythematosus. Lupus 2016, 25(10):1122-1140. References Morand EF, Trasieva T, Berglind A, Illei GG, Tummala R: Lupus Low Disease Activity State (LLDAS) attainment
discriminates responders in a systemic lupus erythematosus trial: post-hoc analysis of the Phase IIb MUSE trial of Page 14/15 Page 14/15 gure 1
) Heatmap of HDAS Episode duration and SLEDAI-2K over time. HDAS Episodes (n=298) are shown as rows, ordere
seline SLEDAI-2K values. (B) Boxplot comparing the duration of HDAS Episodes with baseline SLEDAI-2K >10 vers
seline SLEDIA-2K-10. (C) Effect of baseline SLEDAI-2K on HDAS Episode as shown by Kaplan-Meier survival analy
recovery time of HDAS Episodes between those with baseline SLEDAI-2K >10 and SLEDAI-2K=10 Figure 1 (A) Heatmap of HDAS Episode duration and SLEDAI-2K over time. HDAS Episodes (n=298) are shown as rows, ordered by
baseline SLEDAI-2K values. (B) Boxplot comparing the duration of HDAS Episodes with baseline SLEDAI-2K >10 versus
baseline SLEDIA-2K-10. (C) Effect of baseline SLEDAI-2K on HDAS Episode as shown by Kaplan-Meier survival analysis
of recovery time of HDAS Episodes between those with baseline SLEDAI-2K >10 and SLEDAI-2K=10 Page 15/15
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Generation of megatesla magnetic fields by intense-laser-driven microtube implosions
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports M. Murakami1*, J. J. Honrubia2, K. Weichman3, A. V. Arefiev3 & S. V. Bulanov4,5 M. Murakami1*, J. J. Honrubia2, K. Weichman3, A. V. Arefiev3 & S. V. Bulanov4,5 A microtube implosion driven by ultraintense laser pulses is used to produce ultrahigh magnetic fields. Due to the laser-produced hot electrons with energies of mega-electron volts, cold ions in the inner
wall surface implode towards the central axis. By pre-seeding uniform magnetic fields on the kilotesla
order, the Lorenz force induces the Larmor gyromotion of the imploding ions and electrons. Due to the
resultant collective motion of relativistic charged particles around the central axis, strong spin current
densities of ∼ peta-ampere/cm2 are produced with a few tens of nm size, generating megatesla-order
magnetic fields. The underlying physics and important scaling are revealed by particle simulations
and a simple analytical model. The concept holds promise to open new frontiers in many branches of
fundamental physics and applications in terms of ultrahigh magnetic fields. Laboratory generation of strong magnetic fields have been intensively studied1–18, because such fields may realize
new experimental tools for fundamental studies and support diverse applications. Examples include plasma and
beam physics19–24, astro-25, 26 and solar-physics27, 28, atomic and molecular physics29, and materials science30, 31. Magnetic field reconnection27, 28, generation of collisionless shock19, gamma-ray and pair production32–34, and
fusion application in a strongly magnetized plasma35–38 are receiving increased attention. Although laser−solid
interactions numerically predict magnetic fields ≲ a few hundreds kT18, 21, 23, the highest magnetic field experi-
mentally observed to date is on the kilotesla (kT) order12, 15. y
Here we propose a novel concept called a microtube implosion (MTI), which generates megatesla (MT)
magnetic fields utilizing a structured target and intense laser pulses. Suppose that a long-stretched cylindrical
target contains a coaxial hollow cylindrical space with an inner radius of R0 ∼1 −10 µm (Fig. 1a). Irradiating
the target by ultraintense femtosecond laser pulses with an intensity of IL ∼1019 −1022 Wcm−2 generates hot
electrons with temperatures of Te ∼1− a few 10’s mega-electron volts (MeV) according to the ponderomotive
scaling39. Note that the ponderomotive scaling does not resemble the true dynamics of electrons on a solid surface
with a steep density gradient, where the prerequisites for decoupling the quiver and envelope motion of electrons
do not hold. 1Institute of Laser Engineering, Osaka University, Suita, Osaka 565‑0871, Japan. 2ETSI Aeronáutica y del Espacio,
Universidad Politécnica de Madrid, Madrid, Spain. 3University of California San Diego, 9500 Gilman Drive, La
Jolla, CA 92093‑0411, USA. 4Institute of Physics of the ASCR, ELI-Beamlines, Na Slovance 2, 18221 Prague,
Czech Republic. 5Kansai Photon Science Institute, National Institutes for Quantum and Radiological Science and
Technology, Kizugawa, Kyoto 619‑0215, Japan. *email: murakami‑m@ile.osaka‑u.ac.jp M. Murakami1*, J. J. Honrubia2, K. Weichman3, A. V. Arefiev3 & S. V. Bulanov4,5 Hot electrons ionize the target material to produce a plasma of atomic mass number A and initial
ion density ni0 ∼1023 cm−3 to ionization state Z.hh y
The hot electrons are so energetic that some of them exit the target wall and enter the cavity. Therein the
electron pressure and the electrostatic force balance with each other to form an electron sheath on the plasma/
vacuum interfaces. The surface ions are accelerated inward (implosion, Fig. 1b) through expansion into a vacuum
by the sheath electric field40–44. In the ideal situation where a system has a perfect axial symmetry, the temporal
evolution of the imploding and exploding plasma should also be axially symmetric. In this configuration, a
magnetic field does not evolve. However, if a pre-seeded magnetic field is introduced into the system, an extraor-
dinary magnetic field can be generated at the center with a 2 −3 orders of magnitude larger magnification factor.i y
gi
g
g
g
gi
A uniform magnetic field B0 on the kT order, which is parallel to the cylindrical axis (z-axis), is pre-seeded
by an external laser-plasma device such as a capacitor coil4, 12–14. Using a ns-long laser, such a seed field quickly
rises on the sub-ns time scale and diffuses into the MTI target nearly simultaneously, and then slowly decays
on time scales ≳ 10 ns, which are characterized by impedance of the capacitor-coil. Thus, the lifetime of such
a pre-seeded magnetic field ≳10 ns is much longer than the characteristic time scale of MTI ∼ 100 fs. During 1Institute of Laser Engineering, Osaka University, Suita, Osaka 565‑0871, Japan. 2ETSI Aeronáutica y del Espacio,
Universidad Politécnica de Madrid, Madrid, Spain. 3University of California San Diego, 9500 Gilman Drive, La
Jolla, CA 92093‑0411, USA. 4Institute of Physics of the ASCR, ELI-Beamlines, Na Slovance 2, 18221 Prague,
Czech Republic. 5Kansai Photon Science Institute, National Institutes for Quantum and Radiological Science and
Technology, Kizugawa, Kyoto 619‑0215, Japan. *email: murakami‑m@ile.osaka‑u.ac.jp | https://doi.org/10.1038/s41598-020-73581-4 Scientific Reports | (2020) 10:16653 www.nature.com/scientificreports/ Figure 1. (a) Perspective view of a microtube irradiated by ultraintense laser pulses (laser configuration is just
schematic). Uniform external magnetic field B0 is pre-seeded prior to main laser illumination. (b) Top view of
the inner plasma dynamics. Laser-produced hot electrons drive isothermal expansion of the inner-wall plasma
into vacuum. M. Murakami1*, J. J. Honrubia2, K. Weichman3, A. V. Arefiev3 & S. V. Bulanov4,5 (c) Ultrahigh magnetic field Bc is generated at the center due to the collectively formed currents by
ions and electrons, which are deflected in opposite directions by B0. Figure 1. (a) Perspective view of a microtube irradiated by ultraintense laser pulses (laser configuration
schematic). Uniform external magnetic field B0 is pre-seeded prior to main laser illumination. (b) Top v
the inner plasma dynamics. Laser-produced hot electrons drive isothermal expansion of the inner-wall
into vacuum. (c) Ultrahigh magnetic field Bc is generated at the center due to the collectively formed cu
ions and electrons, which are deflected in opposite directions by B0. Figure 1. (a) Perspective view of a microtube irradiated by ultraintense laser pulses (laser configuration is just
schematic). Uniform external magnetic field B0 is pre-seeded prior to main laser illumination. (b) Top view of
the inner plasma dynamics. Laser-produced hot electrons drive isothermal expansion of the inner-wall plasma
into vacuum. (c) Ultrahigh magnetic field Bc is generated at the center due to the collectively formed currents by
ions and electrons, which are deflected in opposite directions by B0. the implosion, the Lorenz force deflects ions and electrons clockwise and anticlockwise, respectively, gaining
azimuthal momentum, as depicted in Fig. 1c. The ion trajectories draw circles with Larmor radii ∼0.1 −1 mm
for typical laser and target parameters in MTI. In particular, the envelope of the ion paths forms a nanometer-
scale hole at the center (hereafter called the “Larmor hole”). Since electrons are negatively charged, the result-
ant direction of the electron current Jeφ is anticlockwise, which is the same as that of the ion current Jiφ . Then
ultraintense spin currents on the order of 1015 A cm−2 run around the Larmor hole. Consequently, the currents
from the ions and electrons work together to generate MT-order magnetic field Bc at the center. i
Compared with other conventional approaches, the most innovative point of the current concept lies in the
geometrically unique plasma flow. A cylindrically converging flow composed of relativistic electrons and ions,
which are infinitesimally twisted by the pre-seeded magnetic field in opposite directions, can effectively produce
ultrahigh spin currents and consequently, ultrahigh magnetic fields. In addition, the current geometry may be
better suited for many practical purposes.i For over 50 years, researchers have strived to realize high magnetic fields. M. Murakami1*, J. J. Honrubia2, K. Weichman3, A. V. Arefiev3 & S. V. Bulanov4,5 Many approaches have been
employed, including high explosives1, 2, electromagnetic implosions3, 7, high-power lasers10, 11, and Z pinches5, 6. The principal physical mechanism of these works is based on magnetic flux compression (MFC) using hollow
cylindrical structures and pre-seeded magnetic fields. The present scheme also uses a similar physical configura-
tion. However, MTI differs from MFC because the ultrahigh magnetic fields in MTI are generated by the spin
currents induced by collective Larmor gyromotions. Results
T
di The lengths for the unit cell size and full
span of each side of the square domain are 6.25 nm and 10 µm , respectively. Therefore, the whole computational
domain size is 1600 × 1600 mesh2 . The initial inner radius of the microtube is R0 = 3 µm. h
µ
Second, since hot-electron average energy Ehe.av spans the relativistic regime for the parameters of interest,
we use the Maxwell–Jüttner (M–J) distribution46 rather than the Maxwell–Boltzmann (M–B) distribution for
the non-relativistic regime. The M–J distribution defines the hot electron population in terms of the Lorenz fac-
tor γ as f (γ ) =
γ 2β
K2(1/) exp(−γ
) , where β = v/c =
1 −1/γ 2 and = Te/mec2 with Te , me , and c being
the electron temperature, the electron rest mass, and the speed of light, respectively; K2 is the modified Bessel
function of the second kind. The relation between Ehe.av and Te significantly differs between the two distribu-
tions. That is, Ehe.av = 3
2Te for the M–B distribution ( Ehe.av ≪mec2 ) while Ehe.av ≃3Te −mec2 for the M–J
distribution ( Ehe.av ≫mec2).fi ( he.av
e
)
It should be noted that on such an ultrashort timescale as femtoseconds, there is insufficient time for electrons
to be thermalized47, 48. In this sense, employing the M–J distribution, which is characterized by a specific tem-
perature, may not be legitimate. However, high-energy-tail electrons, whose population decreases exponentially
with energy, predominantly influence the energy transport and thus the dynamics of the overall system44. In fact,
both the M–J and M–B distributions have such an exponential dependence in their functional forms. For this
reason, employing the M–J distribution is an acceptable choice. Actually, simulations have confirmed that the
same value of Ehe.av yields a similar result for the implosion dynamics and the generation of the magnetic field
for both energy distributions. Therefore, Ehe.av rather than Te is employed below as a principal parameter. Note
that we later provide another set of simulation results as a proof-of-principle, using more practical conditions
that take the laser−matter interactions into account, where the electron population is not approximated by the
M–J distribution. Results
T
di Two‑dimensional particle simulation. To demonstrate the expected behavior of MTI, we perform 2D
(x, y) PIC simulations using the open-source fully relativistic code EPOCH45. In this first part of EPOCH simula-
tions (v.4.10.17), we employ rather simple and ideal physical conditions to effectively extract the salient features
of the underlying MTI physics. First, the simulation uses the periodic boundary conditions for particles and
fields, where the hollow cylindrical volume is placed at the middle of the square computational domain. This
configuration simulates collective targets with multiple equally spaced microtubes inside a heated material. We https://doi.org/10.1038/s41598-020-73581-4 Scientific Reports | (2020) 10:16653 | www.nature.com/scientificreports/ Figure 2. Temporal evolution of the normalized densities of ions ˜ni = ni/ni0 and electrons ˜ne = ne/ne0 under
charge neutrality ne0 = Zni0 with Z = 6 (fully ionized carbon plasma). Other fixed parameters are R0 = 3 µm ,
ni0 = 1 × 1023 cm−3 (i.e., 2 g cm−3 ), B0 = 4 kT, and Ehe.av = 5 MeV. Dashed curves are obtained by the model
lines for constant velocities (Fig. 4b) and the density given by Eq. (3). Inset shows a magnified view of the
yellow-painted area. Figure 2. Temporal evolution of the normalized densities of ions ˜ni = ni/ni0 and electrons ˜ne = ne/ne0 under
charge neutrality ne0 = Zni0 with Z = 6 (fully ionized carbon plasma). Other fixed parameters are R0 = 3 µm ,
ni0 = 1 × 1023 cm−3 (i.e., 2 g cm−3 ), B0 = 4 kT, and Ehe.av = 5 MeV. Dashed curves are obtained by the model
lines for constant velocities (Fig. 4b) and the density given by Eq. (3). Inset shows a magnified view of the
yellow-painted area. Figure 2. Temporal evolution of the normalized densities of ions ˜ni = ni/ni0 and electrons ˜ne = ne/ne0 under
charge neutrality ne0 = Zni0 with Z = 6 (fully ionized carbon plasma). Other fixed parameters are R0 = 3 µm ,
ni0 = 1 × 1023 cm−3 (i.e., 2 g cm−3 ), B0 = 4 kT, and Ehe.av = 5 MeV. Dashed curves are obtained by the model
lines for constant velocities (Fig. 4b) and the density given by Eq. (3). Inset shows a magnified view of the
yellow-painted area. set 100 particles/cell for carbon ions and 200 particles/cell for electrons. Results
T
di Figure 2 shows the temporal evolution of the normalized densities of ions, ˜ni = ni/ni0 , and electrons,
˜ne = ne/ne0 , under ne0 = Zni0 for a fully ionized carbon plasma with A = 12 and Z = 6 . The solid and dashed
curves indicate the EPOCH results and the model prediction, respectively. The model is described later. Ini-
tially, the inside of the tube is empty, and the remaining volume is filled with uniform ions with Ti = 10 eV and
ni0 = 1 × 1023 cm−3 and uniform electrons with Ehe.av = 5 MeV. The pre-seeded magnetic field B0 = 4 kT is
distributed uniformly over the entire computational domain.t y
p
After launching the plasma expansion into a vacuum at τ = 0 , the implosion phase is observed for a period,
τ ≲70 fs (Fig. 2). The implosion velocity of the innermost ions remains nearly constant at vi ≃6 × 109 cm/s
before the cavity collapse. Macroscopically, ions and electrons in the imploding plasma layer move together and
maintain charge neutrality. The electron sheath thickness at the plasma/vacuum interface is roughly equal to the
local electron Debye length De = (Te/4πnee2)1/2 ∼150 nm, where e denotes the elementary charge, Te ≈1.8
MeV ( Ehe.av = 5 MeV with the M–J distribution), and ne ≈6 × 1021 cm−3 (Fig. 2). Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ Figure 3. Snapshots taken from the dominant period for the magnetic field generation, τ ≈80 −120 fs. Fixed
parameters are the same as those in Fig. 2. (a) Normalized ion density ˜ni = ni/ni0 , (b) normalized electron
density ˜ne = ne/ne0 , (c) azimuthal electron current Jeφ , and (d) magnetic field Bz. Figure 3. Snapshots taken from the dominant period for the magnetic field generation, τ ≈80 −120 fs. Fixed
parameters are the same as those in Fig. 2. (a) Normalized ion density ˜ni = ni/ni0 , (b) normalized electron
density ˜ne = ne/ne0 , (c) azimuthal electron current Jeφ , and (d) magnetic field Bz. Upon cavity collapse, the head group of imploding ions passes the target center at the Larmor hole radius
r = RH and expands outward. Results
T
di The mean-free-path of ion-ion collisions is roughly given by ℓii ∼T2
e /4πniZ2e4 ,
which amounts to ∼ 4 cm ≫R0 under Te = 2 MeV, Z = 6 , and ni = 1023 cm−3 . Hence, these ions collisionlessly
intersect other ions, which are still imploding toward the center. Meanwhile, the central density increases to the
same order as its initial value due to the geometrical accumulation effect (Fig. 2, inset). The Larmor hole radius,
RH ≈20 nm, indicated at the top of the inset corresponds to the analytical prediction at τ = 90 fs (Eq. 5). The
Larmor hole is also seen in the simulation result as the one-humped structure, but the simulated one grows more
quickly than the model and expands outward. This is attributed to the fact that a highly compressed ion sphere
is created at the center and the strong electrostatic field radially pushes the ions outward.i gi
y p
Figure 3 shows snapshots taken from the dominant period of the magnetic field generation, τ ≈80 −120 fs:
(a) the normalized ion density ˜ni = ni/ni0 , (b) the normalized electron density ˜ne = ne/ne0 , (c) the azimuthal
electron current Jeφ , and (d) the magnetic field Bz . Comparing Fig. 3a and b provides insight on how the core
plasma develops. The one-humped structure forms in the central region and oscillates at the ion-plasma fre-
quency ωpi = (4πniZ2e2/mi)1/2 to emit compression waves outward at sound speed cs = (ZTe/mi)1/2 . Applying
the numbers used in Fig. 3 (i.e., ni = 1 × 1023 cm−3 and Te = 1.8 MeV) yields cs ≃9 × 108 cm/s and the cycle
τcyc = 2π/ωpi ≃9 fs ( ν ≡τ −1
cyc ≃110 THz), which agree well with the simulation result. ˙ y
p
cyc
g
According to Ampere’s law, c∇× B = 4πJ + ˙E , the azimuthal current distribution, Jφ = Jeφ + Jiφ ,
directly contributes to the magnetic field Bc generated at the center. The azimuthal electron current density
Jeφ dynamically evolves around the center over the distance approximately equal to the local Debye length
De ∼100 −150 nm (Fig. 3c). According to Faraday’s law, c∇× E = −˙B , when Bc reaches its peak, the dis-
placement current ( ∝∂Eφ/∂t ) becomes substantially small. Results
T
di Under
the self-similar solution, the plasma expands to the right supersonically for x > 0 or ξ > 0 , while the rarefaction
wave propagates to the left at the sound speed cs , corresponding to the path, ξ = −1.h p
p gt
p
p
g
p
The two physical ingredients, collisionless ions and isothermal electrons, provide insight to harness Grevich’s
self-similar solution as a useful approximation and to describe the kinetic behavior of the ions. To accomplish
this, a geometrical modification needs to be added to the self-similar solution. The resultant system behaves
such that individual fluid elements can penetrate each other in cylindrically converging and diverging processes. l
y
y
g
g
g
g
Suppose that an ion with mass mi and ionization state Z is moving at a constant speed vi on the xy-plane
in a uniform magnetic field B0 , which is parallel to the z-axis. The ion draws a circular orbit with a Larmor
radius RL = mivic/ZeB0 , where B0 = |B0| . If the position and velocity of the ion are specified at t = 0 to be
(x, y) = (R0, 0) and (˙x, ˙y) = (−vi, 0) , respectively, the ion moves on the circle, (x −R0)2 + (y −RL)2 = R2
L. L
Here, it is useful to introduce cylindrical coordinates, r =
x2 + y2 and φ = tan−1(y/x) . The ion path s
along its Larmor circle is measured with the distance from the initial point (x, y) = (R0, 0) or with the polar angle
θ = sin−1[(R0 −x)/RL] pivoting around the guiding center (x, y) = (R0, RL) , as illustrated in Fig. 4a. It should
be noted that Fig. 4a is not to scale. The dimensionless coordinate of the self-similar solution is then redefined
in terms of s and θ as ξ = s/cst = RLθ/cst . Note that vi and RL are functions of ξ . Consequently, Grevich’s self-
similar solution for a planar system is reformed for a cylindrical system as (3)
ni = ni0
R0
r e−ξ−1, ni = ni0
R0
r e−ξ−1, (3) (4)
vir, viφ
= (ξ + 1)cs
r
±
r2 −R2
H, RH
, (4) where vir and viφ denote the radial and azimuthal component of the ion velocity, respectively. The reformed set,
Eqs. Results
T
di Analytical model: (a) Schematic explaining the relation between the key parameters (not to scale) Figure 4. Analytical model: (a) Schematic explaining the relation between the key parameters (not to scale). Curve with its coordinate s stands for an ion path, on which the ion implodes at a constant speed as a function
of ξ . In most practical cases, RH(nm) ≪R0(µm) ≪RL(mm) . (b) Radius-time diagram of ξ-contour lines
under the same parameters as in Figs. 2 and 3. Temporal evolution of the density profile shown in Fig. 2 is
directly obtained from this diagram coupled with Eq. (3). The time lag of 12 fs between τ = 0 and t = 0 is fixed
such that the timing of the cavity collapse coincide for both the simulation and the model. Figure 4. Analytical model: (a) Schematic explaining the relation between the key parameters (not to scale). Curve with its coordinate s stands for an ion path, on which the ion implodes at a constant speed as a function
of ξ . In most practical cases, RH(nm) ≪R0(µm) ≪RL(mm) . (b) Radius-time diagram of ξ-contour lines
under the same parameters as in Figs. 2 and 3. Temporal evolution of the density profile shown in Fig. 2 is
directly obtained from this diagram coupled with Eq. (3). The time lag of 12 fs between τ = 0 and t = 0 is fixed
such that the timing of the cavity collapse coincide for both the simulation and the model. (1)
∂ni
∂t + ∂
∂x (nivi) = 0,
(2)
∂vi
∂t + vi
∂vi
∂x = −c2
s
ni
∂ni
∂x , (1) (2)
∂vi
∂t + vi
∂vi
∂x = −c2
s
ni
∂ni
∂x , (2) where ni(x, t) and vi(x, t) are the number density and the velocity of the ions, respectively. Grevich et al.49 found
a self-similar solution to the above system, where the physical quantities are expressed in terms of a single
dimensionless coordinate defined by ξ = x/cst (≥−1) in the forms of ni = ni0e−ξ−1 and vi = (ξ + 1)cs . Results
T
di Then, Bc is given as the sum, Bc = Bce + Bci , where
Bce = (4π/c)
∞
0
Jeφdr and Bci = (4π/c)
∞
0
Jiφdr are the contributions from electrons and ions, respectively. Due to the high mobility of electrons, the effect of electron currents on the magnetic field generation dominates
over that of ion currents. MTI simulations indicate that Bce/Bci ∼3 −4 or equivalently Bc/Bci ∼4 −5 is kept
nearly constant. For example, Bc ≃0.95 MT and Bce ≃0.71 MT are derived at τ = 105 fs from Fig. 3c,d, which
correspond to Bc ≃4Bci. Model. Here, we describe the ion dynamics in terms of a semi-analytical model and demonstrate that it forms
the basis of the whole system. The time origin matters when comparing the model to the simulation results. To
avoid confusion, hereafter, we employ the time variable, t, instead of τ used for the simulation. Suppose that a
planar plasma is held at rest in a half-infinitely stretched region −∞< x ≤0 for t ≤0 , which is composed of
uniform cold ions and hot electrons with densities ni and ne , respectively. We postulate that the plasma is charge
neutral, i.e., Zni = ne . In addition, hot electron temperature Te is assumed to be constant both spatially and tem-
porally due to the high conductivity. Once the boundary between the vacuum and plasma is set free at t = 0 , the
plasma begins to expand into the vacuum. The ion motion is governed by the following hydrodynamic system
describing the mass and momentum conservation as Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ Figure 4. Analytical model: (a) Schematic explaining the relation between the key parameters (not to scale). Curve with its coordinate s stands for an ion path, on which the ion implodes at a constant speed as a function
of ξ . In most practical cases, RH(nm) ≪R0(µm) ≪RL(mm) . (b) Radius-time diagram of ξ-contour lines
under the same parameters as in Figs. 2 and 3. Temporal evolution of the density profile shown in Fig. 2 is
directly obtained from this diagram coupled with Eq. (3). The time lag of 12 fs between τ = 0 and t = 0 is fixed
such that the timing of the cavity collapse coincide for both the simulation and the model. Figure 4. www.nature.com/scientificreports/ (7) The spatial profile of the ion current has a maximum JH(t) at the Larmor hole rim r = RH to spatially decay at
the rate r−2 for r ≥RH.f In Eq. (7), the factor R0/RH explains the cylindrical accumulation effect. Consequently, the ion current
contribution to the central magnetic field follows Bci(t) = (4π/c)
∞
RH Jiφ(r, t)dr = (4π/c)RHJH(t) . The space-
integrated quantity Bci(t) is independent of RH itself. With time t, the numerical factor, (ξc + 1)e−ξc−1 , in Eq. (7)
monotonically increases, and ξc(t) decreases from its initial value ξc(τ = 70 fs) = ξf = 5.5 . Although the factor
asymptotically approaches its maximum, e−1 = 0.37 , with t →∞ or ξc →0 , a cut-off value of ξc exists for the
physical reason described below.ti y
According to the EPOCH simulations, after the cavity collapse at τ = τc(≃70 fs), the magnetic field
grows at the center for a period of 4 −6 ion-oscillations, i.e., τ ∼(4 −6) × τcyc ∼40 −60 fs with the cycle
τcyc = 2π/ωpi ≈ 10 fs for the case in Figs. 2 and 3. This corresponds to τ ∼110 −130 fs or ξc ∼ 3 (Fig. 4b). After the duration τ , the core periodically emits outgoing density waves at a frequency ωpi . These waves carry
a portion of the central plasma energy, as seen in the double-humped structure of the ion density profile for
τ = 110 −115 fs in Fig. 3a. In fact, Bc(t) begins to decay coherently with the first emission of the density wave
(Fig. 3a,d). Since the model does not consider this emission process, we estimate the maximum magnetic field
by limiting the growth at the peak time τpeak = τc + τ . This corresponds to ξc ∼3 . Recalling the observed
constancy, Bc ≃4Bci , leads to its maximum value Bc.max as (8)
Bc.max
1 MT = ≡(Z/6)3/2
(A/12)1/2
ni0
1023 cm−3
R0
3 µm
Ehe.av
6 MeV. (8) Thus, Bc.max is proportional to the total ion flux emitted from the inner surface of the microtube, i.e.,
Bc.max ∝Zni0csR0 (recall cs ∝√ZTe/A). Figure 5 shows the results of about three dozen EPOCH simulations (solid circles) for Bc.max as a function of
defined in Eq. (8). The straight black line denotes the model prediction. www.nature.com/scientificreports/ speed of the ion front, i.e., ˙xf →∞ as ξ →∞ . Assuming that the plasma expands adiabatically, a reasonable
approximation for the dimensionless coordinate of the ion front ξf is obtained such that the plasma front expands
at the speed ˙xf = 2(γ −1)−1cs50, where γ denotes the adiabatic index. Meanwhile, the self-similar solution based
on the isothermal assumption gives the speed of a fluid element at ξ = ξf as ˙xf = (ξf + 1)cs . Equating the two
speeds gives ξf = (3 −γ )/(γ −1) . In particular, the adiabatic index for the relativistic electrons is γ = 4/350,
yielding ξf = 5 . This result well explains ξf ≃5.5 obtained from the simulation (Figs. 2 and 4b). h
Figure 4b shows the r −t diagram obtained from the reformed self-similar system, under the conditions in
Figs. 2 and 3, where the curves correspond to different values of ξ . The time origin of the model corresponding
to τ = 12 fs is chosen such that the cavity-collapse timings coincide with each other on the horizontal axes. This
can be confirmed by the red dashed curve, which shows the trajectory of the innermost ions at an early stage of
implosion according to the EPOCH simulation (Fig. 2). Combining the curves in Fig. 4b and the density profile
given by Eq. (3) leads to the dashed curves in Fig. 2 as the model predictions.h g
y
q ( )
g
p
The Larmor hole radius RH is obtained from the geometrical consideration under RH ≪R0 ≪RL to be
RH ≃R2
0/2RL , which is explicitly rewritten as (5)
RH(t)
1nm ≃43Z
A
B0
1kT
R0
3µm
2 (ξc(t) + 1)cs
109cm/s
−1
, (5) where ξc(t) = R0/cst corresponds to the ions passing by the target center at time t. The azimuthal ion current,
Jiφ = Zeniviφ , is then given with the help of Eqs. (3) and (4) by where ξc(t) = R0/cst corresponds to the ions passing by the target center at time t. The azimuthal ion current,
Jiφ = Zeniviφ , is then given with the help of Eqs. (3) and (4) by (6)
Jiφ(r, t) =
RH
r
2
JH(t),
r ≥RH, (6) (7)
JH(t) =(R0/RH)Zeni0cs(ξc + 1)e−ξc−1. Results
T
di (3) and (4), rigidly satisfies the mass conservation law for a cylindrical geometry such that the factor R0/r
in Eq. (3) explains the geometrical accumulation effect. The minus and plus signs of the double sign in Eq. (4)
correspond to the converging and diverging phases, respectively. The relation between vir and viφ can be under-
stood by considering the simplified physical picture of a single fast ion approaching the center along a straight
line, y = RH , with a constant speed v0 at r = ∞ , i.e., (vir, viφ) = (−v0, 0) . The ion passes the origin (x, y) = (0, 0)
at the shortest distance r = RH , when the velocities replace each other, i.e., (vir, viφ) = (0, v0) . Therefore, the
higher the implosion velocity of an ion, the higher the current and resultant magnetic fields around the center,
i.e., Bz ∝Jiφ = Zev0RH/r. φ
Although Grevich’s solution gives a simple physical picture of plasma expansion into a vacuum, it lacks
important information such as the location of ion front xf . In fact, Grevich’s solution gives an infinite propagation Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Each simulation result corresponds to a
subset with the key parameters, (B0, ni0, R0, Ehe.av) . Their composition is chosen rather randomly over the ranges,
B0 = 1 −10 kT, ni0 = 5 × 1022 −2 × 1023 cm−3 , R0 = 1 −3 µm , and Ehe.av = 5 −15 MeV, while A = 12 and
Z = 6 are fixed. Despite the random choices, the overall simulation results are smoothly linked in a systematic
manner by the dashed curves parameterized by B0 . This demonstrates the physical significance of the parameter
as an essential measure of the magnetic-field generation in the present scheme.h gi
g
p
There is a threshold relation between B0 and such that the simulation results and the model line agree well. For example, for B0 ≳6 kT and ≲2.5 , the model well reproduces the overall behavior of the simulation results. The physical reason why these curves overlap with the model line is as follows. Although the individual trajec-
tories of charged particles are deformed to radially shift outward due to higher B0 and smaller Larmor radius RL
(Fig. 4a), the integrated currents and consequently the magnetic field,
Jiφdr ∝
Jeφdr ∝Bc , are unchanged. With B0 = 4 kT, the difference between the simulation and the model begins to increase for ≳1.6 . Moreo-
ver, for B0 < 3 kT, the behaviors of the simulation results are unpredictable by the model. In particular, with
B0 = 2 kT, the temporal evolution of the system becomes unstable. Although it initially behaves in the reverse
polarity regime ( Bc.max < 0 ), it eventually turns into the forward polarity regime ( Bc.max > 0 ). This phenomenon
is labeled as “polarity switching” in Fig. 5. The polarity switching suddenly occurs on the timescale of several
femtoseconds, when the electron current distribution surrounding the target center evolves very quickly in a
complex manner. One of the potential causes for this phenomenon seems to be the existence of an electron-rich gi
g
p
There is a threshold relation between B0 and such that the simulation results and the model line agree well. For example, for B0 ≳6 kT and ≲2.5 , the model well reproduces the overall behavior of the simulation results. The physical reason why these curves overlap with the model line is as follows. www.nature.com/scientificreports/ Z = 6 and A = 12 are fixed
assuming a fully ionized carbon plasma. Solid circles are EPOCH results, which are linked by the dashed curves
to smoothly guide the readers’ eye. Figure 5. Maximum magnetic field as a function of coupling parameter [Eq. (8)], where
˜n23 = ni0/1023cm−3 , ˜R3 = R0/3µm , and ˜E6 = Ehe.av/6MeV denote normalized values for the initial ion
density, inner radius of the microtube, and average electron energy, respectively. Z = 6 and A = 12 are fixed
assuming a fully ionized carbon plasma. Solid circles are EPOCH results, which are linked by the dashed curves
to smoothly guide the readers’ eye. Figure 5. Maximum magnetic field as a function of coupling parameter [Eq. (8)], where
˜n23 = ni0/1023cm−3 , ˜R3 = R0/3µm , and ˜E6 = Ehe.av/6MeV denote normalized values for the initial ion
density, inner radius of the microtube, and average electron energy, respectively. Z = 6 and A = 12 are fixed
assuming a fully ionized carbon plasma. Solid circles are EPOCH results, which are linked by the dashed curves
to smoothly guide the readers’ eye. space at the center, which is omitted in the model assuming that RH is so large (Eq. 5) that electrons are evacu-
ated from the Larmor hole. Practical simulation with laser‑plasma interaction. In this section, we perform proof-of-princi-
ple EPOCH simulations to demonstrate that strong magnetic fields can still be produced, when the uniform
hot electron population previously assumed by the M–J distribution is replaced with a realistic laser-plasma
interaction45. These simulations reproduce salient features of the MTI process described in the previous sections. Note that although the EPOCH code used here is somewhat customized with the same core modules as recent
releases, the results provided below should be reproducible using the latest release. p
p
g
We consider an isolated target, which consists of an initially cold, fully ionized charge-neutral carbon-electron
plasma with an initial ion density ni0 = 3 × 1022 cm−3 and electron density ne0 = 6ni0 . We have reduced the
target density by a factor of ∼3 relative to the case given in Figs. 2 and 3 to mitigate the computational cost
associated with PIC simulations of the laser−plasma interaction. The target’s outer cross-section is a square with
12-µm-long sides. The inner radius of the microtube is R0 = 3 µm . www.nature.com/scientificreports/ Although the individual trajec-
tories of charged particles are deformed to radially shift outward due to higher B0 and smaller Larmor radius RL
(Fig. 4a), the integrated currents and consequently the magnetic field,
Jiφdr ∝
Jeφdr ∝Bc , are unchanged. With B0 = 4 kT, the difference between the simulation and the model begins to increase for ≳1.6 . Moreo-
ver, for B0 < 3 kT, the behaviors of the simulation results are unpredictable by the model. In particular, with
B0 = 2 kT, the temporal evolution of the system becomes unstable. Although it initially behaves in the reverse gi
g
p
There is a threshold relation between B0 and such that the simulation results and the model line agree well. For example, for B0 ≳6 kT and ≲2.5 , the model well reproduces the overall behavior of the simulation results. The physical reason why these curves overlap with the model line is as follows. Although the individual trajec-
tories of charged particles are deformed to radially shift outward due to higher B0 and smaller Larmor radius RL
(Fig. 4a), the integrated currents and consequently the magnetic field,
Jiφdr ∝
Jeφdr ∝Bc , are unchanged.f g
g
y
gi
φ
φ
g
With B0 = 4 kT, the difference between the simulation and the model begins to increase for ≳1.6 . Moreo-
ver, for B0 < 3 kT, the behaviors of the simulation results are unpredictable by the model. In particular, with
B0 = 2 kT, the temporal evolution of the system becomes unstable. Although it initially behaves in the reverse
polarity regime ( Bc.max < 0 ), it eventually turns into the forward polarity regime ( Bc.max > 0 ). This phenomenon
is labeled as “polarity switching” in Fig. 5. The polarity switching suddenly occurs on the timescale of several
femtoseconds, when the electron current distribution surrounding the target center evolves very quickly in a
complex manner. One of the potential causes for this phenomenon seems to be the existence of an electron-rich Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ Figure 5. Maximum magnetic field as a function of coupling parameter [Eq. (8)], where
˜n23 = ni0/1023cm−3 , ˜R3 = R0/3µm , and ˜E6 = Ehe.av/6MeV denote normalized values for the initial ion
density, inner radius of the microtube, and average electron energy, respectively. www.nature.com/scientificreports/ (a) Blue-line: Laser-produced electron energy spectrum under B0 = 6 kT, normalized
such that
(dN/dE)dE = 1 . Grey dotted line: M-J fit to the spectrum using the ponderomotive temperature
Tp ≡(1 + a2
0)1/2mec2 ≈11 MeV, where a0 ≡|e|E0/mecω for a laser with maximum electric field amplitude
E0 and frequency ω . Green dash-dotted line: M-J fit to the mid-energy part of the spectrum, provided for
comparison. (b) Maximum magnetic field and radius at which the field falls to half its maximum value (r1/2) ,
obtained by azimuthally averaging the magnetic field; t0 is the time when the peaks of the laser pulses reach
either x = 0 or y = 0. Figure 6. Electron energy spectrum and magnetic field growth in laser-driven targets ( L = 0.8 µm , IL = 1021
W/cm2 , τL = 100 fs). (a) Blue-line: Laser-produced electron energy spectrum under B0 = 6 kT, normalized
such that
(dN/dE)dE = 1 . Grey dotted line: M-J fit to the spectrum using the ponderomotive temperature
Tp ≡(1 + a2
0)1/2mec2 ≈11 MeV, where a0 ≡|e|E0/mecω for a laser with maximum electric field amplitude
E0 and frequency ω . Green dash-dotted line: M-J fit to the mid-energy part of the spectrum, provided for
comparison. (b) Maximum magnetic field and radius at which the field falls to half its maximum value (r1/2) ,
obtained by azimuthally averaging the magnetic field; t0 is the time when the peaks of the laser pulses reach
either x = 0 or y = 0. Figure 6. Electron energy spectrum and magnetic field growth in laser-driven targets ( L = 0.8 µm , IL = 1021
W/cm2 , τL = 100 fs). (a) Blue-line: Laser-produced electron energy spectrum under B0 = 6 kT, normalized
such that
(dN/dE)dE = 1 . Grey dotted line: M-J fit to the spectrum using the ponderomotive temperature
Tp ≡(1 + a2
0)1/2mec2 ≈11 MeV, where a0 ≡|e|E0/mecω for a laser with maximum electric field amplitude
E0 and frequency ω . Green dash-dotted line: M-J fit to the mid-energy part of the spectrum, provided for
comparison. (b) Maximum magnetic field and radius at which the field falls to half its maximum value (r1/2) ,
obtained by azimuthally averaging the magnetic field; t0 is the time when the peaks of the laser pulses reach
either x = 0 or y = 0. Figure 7. www.nature.com/scientificreports/ This target is irradiated on each of the four
outer sides by a large-spot (spatially plane wave) laser pulse with a wavelength of L = 0.8 µm , a total duration
( sin2 temporal shape in |E|) of τL = 100 fs, and a peak intensity of IL = 1021 W/cm2 . The pulses are co-timed
such that the peaks of all four pulses interact with the target surface simultaneously. The plasma is modeled with
a resolution of 100 cells/µm and 200 particles/cell for carbon ions and 400 particles/cell for electrons, using
the cubic B-spline particle shape included in EPOCH. The full size of the simulation box is 22 µm × 22 µm.i h
In practical laser-driven configurations, the laser-plasma interaction causes the hot electron population
driving the implosion to depart from the conditions of spatial uniformity and temperature isotropy, which are
assumed in the derivation of the maximum magnetic field given in Eq. (8). The energetic electron spectrum has
multiple energy components and differs from a single-temperature M-J distribution (Fig. 6a). Despite this depar-
ture from the conditions assumed in the previous sections, we still observe strong magnetic field generation with
similar features to the single-temperature case. Due to the spatial non-uniformities, strong magnetic fields with
spatially varying signs can be generated in small regions of the implosion volume even without the presence of
any seed magnetic field. However, the addition of a seed magnetic field ( B0 = 6 kT) increases both the maximum
amplitude of the magnetic field produced and the spatial volume over which it maintains the same sign (Fig. 6b). h
h d
l d
l
l
f h
h
l
h h
d
b p
gi
p
p
g
g
Figure 7 shows the detailed temporal evolution of the physical quantities, which are summarized in Fig. 6b. The magnetic field generation in the laser-driven case occurs in two stages, where the majority of the magnetic
field generation occurs during the first stage. During the implosion, substantial anisotropy in the ion current
crossing through the center of the microtube generates a strong magnetic field around the center ( r < 0.3 µm , Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ Figure 6. Electron energy spectrum and magnetic field growth in laser-driven targets ( L = 0.8 µm , IL = 1021
W/cm2 , τL = 100 fs). Discussionl We here briefly discuss laser requirements for MTI. To achieve MT-order magnetic fields experimentally, a
rough estimate assuming a pulse duration of ∼30 fs suggests that a laser system with a pulse energy of 0.1 −1
kJ and a total power of 10 −100 PW is required. Such high-power laser performance is accessible by today’s laser
technology51, 52. Meanwhile, fundamental studies, including proof-of-principle experiments, should be feasible
using substantially smaller laser systems. Unlike for ultrathin targets with nm-scale thicknesses, MTI targets are
significantly less sensitive to laser contrast due to the micron-thick wall, while beam co-timing should be within
10 − 20 fs for implosions with a timescale of ∼100 fs.i p
Detecting MT-order magnetic fields inside a plasma presents a challenge for conventional techniques that
rely on charged particle sources. In anticipation of achieving ultrahigh magnetic fields, there have been efforts
to develop other techniques to infer the existence of strong B-fields inside a dense plasma in use of, for example,
an XFEL photon beam with Faraday rotation effect17, 20, 24 and spin-polarized neutrons53. f
We roughly estimate the minimum number of beams nB from a uniformity point of view. For simplicity our
discussion here is limited to the cross-sectional dimensions. Nonuniformity of the imploding ion front is directly
influenced by that of hot electron density, which comprises the local electron sheath (Fig. 1b). Hot electrons
produced on the laser-irradiated surface go back and forth between the target surface and the cavity wall with an
in-between distance R = R1 −R0 , where R1 is the initial target radius. This hot electron transport is regarded
as a kind of random-walk diffusion process. The nonuniformity on the outer surface should diminish on the
cavity wall via this diffusion process along the lateral direction. As is well documented, the diffusion distance is
given by Ld ∼
√
N R , where N ∼c t/ R stands for the reflection number between the two surfaces during
the implosion time t . Consequently, nB ≳2πR0/2Ld ∼2 (i.e., two-sided illumination) when employing for
example R0 = 3 µm , R = 2 µm , and t ∼50 fs (Fig. 2). Discussionl It should be noted that controlling both the temporal
and spacial profiles of the incident laser pulses also plays a crucial role to improve the implosion uniformity54, 55.i p
pi
p
p y
p
p
y
In summary, we propose a novel concept called MTI, which produces MT-order magnetic fields using intense
laser pulses. Key physical elements of MTI are imploding ion fluxes with quasi-relativistic speeds and the result-
ant ultrahigh spin currents running around the nanometer-scale Larmor hole at the center. The spin currents are
due to the collective motion of the imploding ions and the accompanying relativistic electrons. The pre-seeded
magnetic field B0 significantly influences the magnetism of the plasma. For example, the forward (reverse)
polarity appears in the domain of higher (lower) B0 . Polarity switching is an extraordinary phenomenon, which
requires further investigation. The scaling law for the maximum magnetic field Bc.max is obtained as a function of
B0 and the total ion flux emitted from the inner surface of microtube . With the realistic laser-plasma interac-
tion taken into account, strong magnetic field generation has been demonstrated as a proof-of-principle of MTI. Received: 16 June 2020; Accepted: 18 September 2020 Received: 16 June 2020; Accepted: 18 September 2020 www.nature.com/scientificreports/ In the presence of the 6-kT seed, the first stage, which includes
the MTI amplification process, generates a ∼100 kT magnetic field over r ≲0.3 µm in approximately 20 fs, while
the second stage amplifies this magnetic field to ∼120 kT over approximately 50 fs and increases the size of the
central spot to a radius of ∼0.5 µm. References 1. Sakharov, A. D. et al. Magnetic cumulation. Sov. Phys. Dokl. 165, 65 (1965).i ,
g
y
,
(
)
2. Fowler, C. M., Garn, W. B. & Caird, R. S. Production of very high magnetic fields by implosion. J. Appl. Phys. 31, 588 (1960).l g
y
2. Fowler, C. M., Garn, W. B. & Caird, R. S. Production of very high magnetic fields by implosion. J. Appl. Phys. 31, 588 (1960
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12. Fujioka, S. et al. www.nature.com/scientificreports/ Detailed temporal evolution of the magnetic field Bz and the normalized ion density ni/ni0 of laser-
driven MTI. Summary plots are given in Fig. 6. (a,b) results with B0 = 6 kT. (c,d) results with B0 = 0 . (a,c)
Magnetic field in the microtube. (b,d) Density of carbon ions during the implosion. The black solid and grey
dashed contours indicate B = 6 kT and B = 0 , respectively. The first time snapshot ( t = t0 + 30 fs) corresponds
to a moment immediately before the cavity collapse. Figure 7. Detailed temporal evolution of the magnetic field Bz and the normalized ion density ni/ni0 of laser-
driven MTI. Summary plots are given in Fig. 6. (a,b) results with B0 = 6 kT. (c,d) results with B0 = 0 . (a,c)
Magnetic field in the microtube. (b,d) Density of carbon ions during the implosion. The black solid and grey
dashed contours indicate B = 6 kT and B = 0 , respectively. The first time snapshot ( t = t0 + 30 fs) corresponds
to a moment immediately before the cavity collapse. Stage 1 in Fig. 7a,c). In the simulations, the magnitude of this magnetic field increases by a factor of ≳2 upon
applying B0 = 6 kT through the MTI process (Fig. 7a). Later, this central magnetic field is further amplified by
electrons undergoing E × B-directed motion as the central ion population explodes outward (Stage 2 in Fig. 7a,c). Unlike Stage 1, which occurs over approximately 30 fs and agrees well with the MTI process described earlier,
Stage 2 can persist for well over 100 fs. Figure 7 does not capture the end of this stage due to the computational
cost of these simulations. However, simulations performed with plastic targets suggest the magnetic field can be https://doi.org/10.1038/s41598-020-73581-4 Scientific Reports | (2020) 10:16653 | www.nature.com/scientificreports/ slowly amplified over hundreds of femtoseconds. In the presence of the 6-kT seed, the first stage, which includes
the MTI amplification process, generates a ∼100 kT magnetic field over r ≲0.3 µm in approximately 20 fs, while
the second stage amplifies this magnetic field to ∼120 kT over approximately 50 fs and increases the size of the
central spot to a radius of ∼0.5 µm. slowly amplified over hundreds of femtoseconds. Received: 16 June 2020; Accepted: 18 September 2020 References & Pitaevskii, L. P. Self-similar moti 49. Grevich, A. V., Pariskaya, L. V. & Pitaevskii, L. P. Self-similar motion of rarefied plasma. Sov. Phys. JETP 22, 449 (196
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M.M. was supported by the Japan Society for the Promotion of Science (JSPS). J.J.H. was supported by the
EUROfusion grant ENR-IFE19.CCFE-01-T001-D001 and the research grant RTI2018-098801-B-I00 of the
Spanish Ministry for Research. J.J.H. thankfully acknowledges the computer resources at MareNostrum and the
technical support provided by the Barcelona HPC of the Spanish Supercomputing Network and the CeSViMa
HPC of the UPM. The work by S.V.B. was supported by the project High Field Initiative (No. CZ.02.1.01/0.0/
0.0/15_003/0000449) from the European Regional Development Fund. The work by K.W. and A.V.A was sup-
ported by the DOE Office of Science under Grant No. DE-SC0018312 and used HPC resources of the Texas
Advanced Computing Center (TACC) at the University of Texas at Austin and the Extreme Science and Engi-
neering Discovery Environment (XSEDE)56, which is supported by National Science Foundation grant number
ACI-1548562. Data collaboration was supported by the SeedMe2 project57 (http://dibbs.seedme.org). Author contributions M.M. conceived the study. Designing and performing the PIC simulations are contributed by J.J.H. on Figs. 2, 3,
and 5, and by K.W. and A.V.A on Figs. 6 and 7. M.M. and S.V.B. developed the analytical model. M.M. wrote the
paper. All authors reviewed the whole work, and approved the manuscript. Competing interests h p
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46. Jüttner, F. Das Maxwellsche Gesetz der Geschwindigkeitsverteilung in der Relativtheorie. Ann. Phys. 339, 856–882 (1911) 47. Bezzerides, B., Gitomer, S. J. & Forslund, D. W. Randomness, Maxwellian distributions, and resonance absorption. Phys. Rev. Lett. 44, 651 (1980). 48. Kluge, T., Bussmann, M., Schramm, U. & Cowan, T. E. Simple scaling equations for electron spectra, currents, and bul
in ultra-intense short-pulse laser-solid interaction. Phys. Plasmas 25, 073106 (2018).i y
49. Grevich, A. V., Pariskaya, L. V. & Pitaevskii, L. P. Self-similar motion of rarefied plasma. Sov. Phys. JETP 22, 449 (1966).h 49. Grevich, A. V., Pariskaya, L. V. & Pitaevskii, L. P. Self-similar motion of rarefied plasma. Sov. Phys. JETP 22, 449 (1966). 50. Landau, L.D. & Lifshitz, E.M. Course in Thoretical Physics, Vol. 6: Fluid Mechanics (Pergamon Press, Oxford, 1959). Grevich, A. V., Pariskaya, L. V. Additional information Correspondence and requests for materials should be addressed to M.M. Correspondence and requests for materials should be addressed to M.M. Scientific Reports | (2020) 10:16653 | https://doi.org/10.1038/s41598-020-73581-4 www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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https://europepmc.org/articles/pmc6477089?pdf=render
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English
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Cellular Immune Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)
|
Frontiers in immunology
| 2,019
|
cc-by
| 12,080
|
Cellular Immune Function in Myalgic
Encephalomyelitis/Chronic Fatigue
Syndrome (ME/CFS) Jacqueline M. Cliff 1*, Elizabeth C. King 1, Ji-Sook Lee 1, Nuno Sepúlveda 1,2,
Asia-Sophia Wolf 1, Caroline Kingdon 3, Erinna Bowman 3, Hazel M. Dockrell 1, Luis Nacul 3,
Eliana Lacerda 3† and Eleanor M. Riley 1†‡ Jacqueline M. Cliff 1*, Elizabeth C. King 1, Ji-Sook Lee 1, Nuno Sepúlveda 1,2,
Asia-Sophia Wolf 1, Caroline Kingdon 3, Erinna Bowman 3, Hazel M. Dockrell 1, Luis Nacul 3,
Eliana Lacerda 3† and Eleanor M. Riley 1†‡ Jacqueline M. Cliff 1*, Elizabeth C. King 1, Ji-Sook Lee 1, Nuno Sepúlveda 1,2,
Asia-Sophia Wolf 1, Caroline Kingdon 3, Erinna Bowman 3, Hazel M. Dockrell 1, Luis Nacul 3,
Eliana Lacerda 3† and Eleanor M. Riley 1†‡ 1 Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical
Medicine, London, United Kingdom, 2 Centre of Statistics and Applications, University of Lisbon, Lisbon, Portugal ,
3 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical
Medicine, London, United Kingdom Keywords: myalgic encephalomyelitis, chronic fatigue syndrome, natural killer cells, T cell differentiation,
MAIT cells, herpes viruses ORIGINAL RESEARCH
published: 16 April 2019
doi: 10.3389/fimmu.2019.00796 Edited by:
Yenan Bryceson,
Karolinska Institute (KI), Sweden Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition
with unknown aetiology, Myalgic encephalomyelitis unclear pathophysiology and with no
diagnostic test or biomarker available. Many patients report their ME/CFS began after
an acute infection, and subsequent increased frequency of infections, such as colds
or influenza, is common. These factors imply an altered immunological status exists in
ME/CFS, in at least a proportion of patients, yet previous studies of peripheral immunity
have been discrepant and inconclusive. The UK ME/CFS Biobank, which has collected
blood samples from nearly 300 clinically-confirmed ME/CFS patients, enables large-scale
studies of immunological function in phenotypically well-characterised participants. In
this study, herpes virus serological status and T cell, B cell, NK cell and monocyte
populations were investigated in 251 ME/CFS patients, including 54 who were severely
affected, and compared with those from 107 healthy participants and with 46 patients
with Multiple Sclerosis. There were no differences in seroprevalence for six human
herpes viruses between ME/CFS and healthy controls, although seroprevalence for the
Epstein-Barr virus was higher in multiple sclerosis patients. Contrary to previous reports,
no significant differences were observed in NK cell numbers, subtype proportions or
in vitro responsiveness between ME/CFS patients and healthy control participants. In
contrast, the T cell compartment was altered in ME/CFS, with increased proportions
of effector memory CD8+ T cells and decreased proportions of terminally differentiated
effector CD8+ T cells. Conversely, there was a significantly increased proportion of
mucosal associated invariant T cells (MAIT) cells, especially in severely affected ME/CFS
patients. These abnormalities demonstrate that an altered immunological state does exist
in ME/CFS, particularly in severely affected people. This may simply reflect ongoing or
recent infection, or may indicate future increased susceptibility to infection. Longitudinal
studies of ME/CFS patients are needed to help to determine cause and effect and thus
any potential benefits of immuno-modulatory treatments for ME/CFS. Reviewed by:
Jakob Theorell,
University of Oxford, United Kingdom
Francisco Borrego,
BioCruces Health Research Institute,
Spain Reviewed by:
Jakob Theorell,
University of Oxford, United Kingdom
Francisco Borrego,
BioCruces Health Research Institute,
Spain *Correspondence:
Jacqueline M. Cliff
jackie.cliff@lshtm.ac.uk *Correspondence:
Jacqueline M. Cliff
jackie.cliff@lshtm.ac.uk *Correspondence:
Jacqueline M. Cliff
jackie.cliff@lshtm.ac.uk †These authors have contributed
equally to this work ‡Present Address:
Eleanor M. Edited by:
Yenan Bryceson,
Karolinska Institute (KI), Sweden Edited by:
Yenan Bryceson,
Karolinska Institute (KI), Sweden Riley,
The Roslin Institute, University of
Edinburgh, Edinburgh,
United Kingdom Specialty section:
This article was submitted to
NK and Innate Lymphoid Cell Biology,
a section of the journal
Frontiers in Immunology Specialty section:
This article was submitted to
NK and Innate Lymphoid Cell Biology,
a section of the journal
Frontiers in Immunology Received: 17 October 2018
Accepted: 26 March 2019
Published: 16 April 2019 Recruitment and Clinical Evaluation Study participants, including PWME, multiple sclerosis (MS)
and non-fatigued healthy controls, were recruited through the
UK National Health Service (NHS) primary and secondary
health care services. In addition, some people with clinically
confirmed severe ME/CFS were identified via support groups
and were invited to participate. All potential participants were
rigorously assessed to ensure that they met the study case
definitions for ME/CFS. Non-fatigued healthy controls were also
recruited by advertisement within Higher Education Institutions
or were friends or family members of PWME. Ethical approval
was granted by the London School of Hygiene & Tropical
Medicine (LSHTM) Ethics Committee (Ref. 6123) and the
National Research Ethics Service (NRES) London-Bloomsbury
Research Ethics Committee (REC ref. 11/10/1760, IRAS ID:
77765). All participants provided written informed consent for
questionnaire, clinical measurement and laboratory test data,
and for samples to be made available for ethically-approved
research, after receiving an extensive information sheet and
consent form, which included an option to withdraw from the
study at any time. There have been a number of reports of inconsistent
abnormalities in natural killer (NK) cell function. NK cells are
large granular lymphocytes that function as innate immune
effectors that, by cytokine production or cytotoxicity, contain
infections (or neoplasias) until an effective adaptive response
can be mounted. Individuals with inherited NK cell disorders
are highly susceptible to systemic herpes virus and other
opportunistic infections (14). Direct NK cell activation follows
interaction with cells which lack major histocompatibility
complex (MHC) Class I ligands for inhibitory NK cell receptors
and/or express stress-induced ligands for NK cell activating
receptors. Indirect NK cell activation occurs following microbial
ligation of pattern recognition receptors on myeloid accessory
cells and is mediated by cytokines (IL-12, IL-15, IL-18, IFN-α)
and by contact-dependent stimuli (15, 16). PWME have been
variously reported to have increased (17, 18), decreased (19,
20) or normal (21–23) numbers of circulating NK (CD16+ or
CD56+, CD3−) cells; their NK cells are reportedly deficient in
Maher et al. (24) or have enhanced expression of Huth et al. (25) the cytotoxic molecule perforin; are less able (or fully able)
to lyse MHC Class 1 deficient target cells (19, 21, 22, 26); and
are defective (or not) in their upregulation of activation markers All participants with ME/CFS or MS had previously received
a confirmed medical diagnosis. Participants were aged between
18 and 60 years. INTRODUCTION after in vitro stimulation (21, 22, 27). Again, the reproducibility
of many of these studies is hampered by their relatively small
size, the diverse clinical presentations of the cases, or the limited
extent of the immunological characterisation in any one study. Myalgic encephalomyelitis (or encephalopathy)/chronic fatigue
syndrome (ME/CFS) is characterised by unexplained persistent
or recurrent incapacitating fatigue for over 6 months leading
to substantial reductions in previous levels of occupational,
educational, social and personal activities (1, 2) and, often, by
moderate or severe physical disability and significant reductions
in quality of life (3, 4). With population prevalence rates
estimated at 0.1–0.4%, ME/CFS has considerable societal and
economic impacts due to its chronic nature, its tendency to affect
young adults in the most productive periods of their lives, and
its impact on family and friends (3, 5–7). The aetiology remains
elusive. Currently, there are no confirmatory diagnostic tests or
specific, evidence-based treatments that are widely accepted and
that consider the heterogeneity of symptoms and their fluctuating
nature. Thus, despite some significant recent advances (8, 9),
ME/CFS continues to be poorly understood. Unfortunately,
many apparently interesting research findings results have not
proven reproducible (10), due in part to the small size and
cross-sectional nature of many studies, and to variability between
studies in the research methods used and their quality. Importantly, only one (23) of these immunological studies
has taken account of the prevalence of human cytomegalovirus
(CMV) infection in cases and controls. CMV infection leaves
a permanent footprint on the immune system including
oligoclonal expansions and terminal differentiation of CD8+ T
cells and expansion of a subset of highly differentiated NKG2C+
NK cells (28); this NK population is further expanded by
subsequent viral infection (28, 29). It remains possible therefore,
that the reported differences in T cell and NK cell phenotype and
functional capacity between PWME and healthy controls may
result from differences in the prevalence of immunomodulatory
viruses such as CMV. Here we report an in-depth analysis
of peripheral blood leucocyte phenotype and function in a
clinically well-defined cohort of moderately and severely affected
ME/CFS cases compared to non-fatigued healthy controls and,
as a control for reduced levels of physical activity, people with
multiple sclerosis. All participants were screened for serological
evidence of human cytomegalovirus (CMV), Epstein–Barr virus
(EBV), herpes simplex virus 1 (HSV1), Herpes simplex virus 2
(HSV2), varicella-zoster virus (VZV), and human herpesvirus
(HHV6) infections. Citation: Cliff JM, King EC, Lee J-S,
Sepúlveda N, Wolf A-S, Kingdon C,
Bowman E, Dockrell HM, Nacul L,
Lacerda E and Riley EM (2019)
Cellular Immune Function in Myalgic
Encephalomyelitis/Chronic Fatigue
Syndrome (ME/CFS). Front. Immunol. 10:796. doi: 10.3389/fimmu.2019.00796 April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org Increased MAIT Cell Frequency in ME/CFS Cliff et al. INTRODUCTION Acute viral infections have frequently been associated with
the onset of ME/CFS (9) but no specific viral aetiology has
been confirmed and there is no consistent association between
persistent or chronic virus infection and ME/CFS. One possibility
is that people with ME/CFS (PWME) are susceptible to
acute viral infections as a consequence of underlying immune
dysfunction. Numerous immune abnormalities, including altered
plasma cytokine profiles, abnormal T lymphocyte activation
and impaired cytotoxic responses, have been described in
individuals with ME/CFS (5, 7), but recent systematic reviews
and
observational
studies
have
failed
to
identify
robust
and reproducible immune biomarkers of disease or disease
severity (11–13). MATERIALS AND METHODS Recruitment and Clinical Evaluation Frontiers in Immunology | www.frontiersin.org Power Calculation We recruited 404 participants comprising 251 with ME/CFS
(54 severely affected and 197 mild/moderate cases), 46 cases
of MS, and 107 healthy controls. Based on our own data on
NK cell responses to cytokine stimulation in HCMV-infected
healthy adults (32) we estimated that the proposed sample size
would provide at least 80% power to detect a 30% difference in
NK cell responses between MS and severely affected cases (the
comparison requiring the highest sample size) and >90% power
to detect a 20% difference in NK cell response between cases and
healthy controls (Epi Info, version 3.5.53). Leucocyte Phenotyping Cryopreserved
PBMCs
were
transported
to
the
research
laboratory on dry ice, and analysed in mixed batches containing
samples across the four clinical groups. PBMC were thawed
by addition of warm RPMI-1640 tissue culture medium
(Invitrogen), washed in RPMI containing 1% foetal bovine
serum (Invitrogen) and counted by trypan blue exclusion. Cells
were resuspended at 4 x 106 cells/ml in complete medium
[RPMI-1640 plus 10% human AB serum (Sigma) and 1%
penicillin, streptomycin/glutamine (Invitrogen)]. Cells were
either used immediately for ex vivo phenotyping or were
placed in culture for functional analysis. For flow cytometry,
cells (200,000/well) were aliquoted into 96 well V-bottom
microtitre plates, stained as described previously (32) in
Cytofix/CytopermTM for intracellular stains, and analysed on an
LSR-II flow cytometer (Becton Dickinson). Antibody staining
panels are shown in Supplementary Tables S2, S3. Data were
collected in FACSDiva v6.1 and analysed using Flowjo v10
(TreeStar) and SPICE (33) software. Herpesviruses Serology (ii) had any vaccinations in the preceding 3 months; (iii) had
a history of acute and chronic infectious diseases such as
hepatitis B and C, tuberculosis, HIV (but not herpes virus or
other retrovirus infection); (iv) another chronic disease such
as cancer, coronary heart disease, or uncontrolled diabetes; (v)
a severe mood disorder; (vi) been pregnant or breastfeeding
in the preceding 12 months; or (vii) were morbidly obese
(BMI ≥40). Once eligibility was confirmed, participants were
invited to a recruiting centre for clinical assessment and blood
sample collection: this included the healthy controls, PWME
and people with MS. Those participants with severe disease and
mobility restrictions were visited at home by a research nurse. Supplementary Table S1 shows the range of clinical variables
including the Fatigue Severity Scale (30) collected from the study
population, some of which were used for assessing eligibility. Participants were not excluded on the basis of minor ailments
such as sore throat, fever/chills, tender glands, flu symptoms, and
or new sensitivity/allergy, to avoid developing an artificial control
group. All participants were part of the UK ME/CFS Biobank
(UKMEB); further details on the method used for selection and
recruitment of participants and processing of data and samples
were described previously (31). Herpesviruses Serology
Plasma concentrations of IgG to herpes simplex virus-1 (HSV
1), HSV 2, Varicella Zoster virus (VZV), human cytomegalovirus
(CMV), Epstein Barr virus (EBV) viral capsid antigen (VCA),
and EBV nuclear antigen 1 (EBNA 1) and human herpes virus
6 (HHV 6) were assayed by commercial quantitative ELISA
[Demeditec Diagnostics, Germany for all viruses, except HHV6
assay from VIDIA (Vestec, Czech Republic)] according to the
manufacturer’s instructions. IgG concentrations were expressed
as arbitrary units per millilitre (U/ml). For HSV1, HSV 2,
VZV, CMV and EBV, samples with IgG concentration ≤8 U/ml
were scored as negative and those with concentration ≥12
U/ml were regarded as positive. Samples with IgG concentration
between 8 and 12 were regarded as equivocal and were re-
tested. Samples were recorded as EBV seropositive if they
gave a positive reaction to EBV-VCA and/or EBNA 1. For
HHV-6, concentrations ≤10.5 U/ml were considered negative,
while concentrations ≥12.5 U/ml were considered positive. Samples giving concentrations between 10.5 and 12.5 U/ml were
considered equivocal and re-tested. Frontiers in Immunology | www.frontiersin.org Sample Collection and Processing,
Including PBMC Freezing Blood samples were collected in sodium heparin (for PBMC) or
in potassium EDTA (for plasma) vacutainers (Becton Dickinson)
and transported within Pathopak containers (in compliance
with UN 3373 regulations), at room temperature (18–25◦C)
protected from direct sunlight and extremes of environmental
temperature. Samples were delivered to the University College
London / Royal Free Hospital (UCL/RFH) BioBank laboratories
for processing and storage within 6 h of collection: the average
time from collection to processing was 2.5 h across all four study
groups. Serum, plasma, and peripheral blood mononuclear cells
(PBMC) were processed and aliquoted in volumes of 200 µl to
2 ml. PBMCs were aliquoted at 5 × 106 cells/ml. Aliquots were
stored in 1 or 2 ml cryotubes, in vapour phase liquid nitrogen at
−180◦C, and shipped to LSHTM for laboratory analysis on dry
ice. Each aliquot was labelled with a 2D bar code corresponding
to a unique 15 digit ISBT 128 (global standard) identifier which
also served to anonymise the samples. All laboratory evaluations
were performed in a blinded manner with samples identified only
by their unique identifier. Recruitment and Clinical Evaluation PWME were reassessed by clinical research
stafffor compliance with the Canadian Consensus (2) and/or
CDC-1994 (“Fukuda”) (1) criteria, which were the study case
definitions, before recruitment into this study. Participants were
excluded if they had (i) taken antiviral medication or drugs
known to alter immune function in the preceding 3 months; April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 2 Increased MAIT Cell Frequency in ME/CFS Cliff et al. Cliff et al. Functional Assays y
Cells
were
aliquoted
(200,000
cells/well)
into
U-bottom
microtitre plates together with one of the following stimuli:
complete medium (negative control), cell stimulation cocktail
(81 nM
PMA/1.34 µM
ionomycin;
eBioscience);
CpG2216
(2.5 µM; Invivogen), IL-12+IL-18 (5 and 50 ng/ml respectively;
Peprotech and R&D, respectively) or MHC Class-I deficient
K562 cells (at a host:target cell ratio of 2:1). In addition,
cells were plated into flat-bottom microtitre plates that had
previously been coated overnight with anti-CD16 antibody
(20 µg/ml, Becton Dickinson) or an isotype control antibody
(20 µg/ml; eBioscience). After incubation at 37◦C in 5% CO2
for 1 h (cell stimulation cocktail) or 15 h (all other stimuli),
brefeldin A (Golgiplug; final concentration 1:1,000; Becton
Dickinson) and monensin (Golgistop, final concentration
1:1,500; Becton Dickinson) were added to each well and plates April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 3 Increased MAIT Cell Frequency in ME/CFS Cliff et al. expected, higher in PWME, with a trend towards higher scores in
those severely affected, followed by people with mild/moderate
ME/CFS, then people with MS and healthy controls presenting
very low levels of fatigue (P < 0.001). Erythrocyte Sedimentation
Rate (ESR), a non-specific marker of inflammation, was slightly
raised in people with mild/moderate ME/CFS, with similar
median values found in all other groups (P = 0.01). Those with
MS were slightly older compared to other study groups, but
distribution by sex, deprivation index, and total white blood cells
and lymphocytes was similar across the groups. were then incubated for a further 3 h before staining for flow
cytometry (as above). For any assays of CD107a expression,
anti-CD107a antibody was added to wells at the start of the
culture period. The 17 h incubation period was selected, based
on our previous reports (34–36), to allow for upregulation of
both IFNγ and CD25, and degranulation via both direct and
indirect mechanisms. Leucocyte Phenotyping Cryopreserved peripheral blood mononuclear cells were thawed
and analysed by flow cytometry; cell gating is shown in Figure 1. A generous leucocyte gate was set to include all live cells and then
a more restrictive lymphocyte gate was set. Within the leucocyte
gate, monocytes were identified by CD14 positivity and CD3
negativity whilst dendritic cells were identified as CD14/CD3
negative and either CD11c positive (myeloid DC) or CD123
positive (plasmacytoid DC). Within the lymphocyte gate, T cells
were gated as CD3+ and either CD4+ or CD8+; B cells as CD19+
and natural killer (NK) cells as CD3 negative/CD56 positive. NK cells were subsequently analysed for expression of NKG2C,
NKp46, and CD57. y
p
The above analysis was extended to control for possible
confounding variables using R statistical software (37). For
flow cytometry data, the percentages of each cell subpopulation
were conveniently transformed into the respective log-odds
[e.g., log %CD4+/(100%–%CD4+)]: the resulting data followed
approximately normal distributions. Multiple linear regression
was used to compare the outcome variables between groups; all
regression models included gender, age at recruitment, analysis
batch and seropositivity for each herpes virus as confounding
factors. To determine the significance of differences between
clinical groups, regression models including group as a variable
were compared with models excluding group using the likelihood
ratio test. The significance level of each individual test was
determined to ensure an overall significance level in a context of
multiple testing. The significance level was pre-specified at 5% for
each separate analysis. In an unadjusted ANOVA analysis, there were significant
differences between the groups in the proportions of live
leucocytes that were monocytes (P = 0.0133), myeloid DCs
(P < 0.0121), and plasmacytoid DCs (P < 0.0001) (Figure 2):
these differences were due to altered frequencies in the multiple
sclerosis patients, and disappeared after adjusting for gender,
age and herpesvirus sero-positivity (not shown). By contrast, the
proportions of leucocytes that were B cells, T cells or NK cells did
not differ significantly among the groups in either the adjusted or
the unadjusted analysis. Serology Plasma samples were tested by quantitative ELISA for six of
the human herpesviruses, HSV-1, HSV-2, VZV, CMV, EBV,
and HHV6. There were no significant differences between the
groups in the proportions of individuals who were seropositive
for any of these viruses (Table 1). However, concentrations
of anti-EBV VCA varied significantly among the groups
(Kruskal-Wallis test: P = 0.016) and this was due to higher
concentrations of anti-EBV VCA antibodies among people with
MS (Supplementary Figure S1). Univariate analyses were conducted using GraphPad Prism
v7.01 unless otherwise stated. Continuous variable datasets were
checked for Normal distribution by D’Agostino & Pearson test,
and analysed by ANOVA or Kruskal-Wallis test for parametric
or non-parametric datasets respectively when comparing clinical
groups, or by Student’s T-test or Mann Whitney test respectively
when comparing the CMV serostatus on the outcome variables. All P-values reported are after correcting for multiple testing
using Tukey’s test for parametric data or Dunn’s test for
non-parametric data. Differences between clinical groups in
herpesvirus seroprevalences were analysed by Chi squared test,
using Stata version 15.1. Receiver Operating Characteristic
(ROC) curve analysis was conducted in GraphPad Prism. Data Analysis The primary data analysis comprised four groups of participants:
people with mild/moderate ME/CFS (ME-M), people severely
affected by ME/CFS (ME-S), healthy controls (C), and people
with multiple sclerosis (MS). All data analyses were carried out
in a blinded manner with groups identified by a code number;
the code was broken only after all analyses were complete. Frontiers in Immunology | www.frontiersin.org RESULTS Within the lymphocyte gate, the proportions of cells that were
either CD4+ T cells or CD8+ T cells (and, as a consequence,
the CD4/CD8 ratio) differed significantly between groups in
the unadjusted analysis (ANOVA P < 0.0001 in each case) and
these differences persisted after adjusting for confounders (P <
0.0001 in each case) (Figure 3). However, these differences were
entirely due to significantly higher proportions of CD4+ T cells,
lower proportions of CD8+ T cells and thus a higher CD4/CD8
ratio in the MS patients; there were no significant differences
in lymphocyte distribution between ME/CFS cases and healthy
controls. Similarly, there were no significant differences between Study Participant Characteristics
The final cohort comprised 251 people with ME/CFS (197
mild/moderate and 54 severe), 46 people with MS and 107
healthy controls. The demographic and clinical characteristics
of the cohort are summarised in Table 1. Symptoms typically
associated with infection or inflammation were more frequent
and more severe among PWME than among those with MS,
although mild symptoms of inflammation were present in nearly
50% of people with MS. Most healthy controls did not present
with any symptoms (P < 0.0001). Fatigue severity scores were, as April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 4 Increased MAIT Cell Frequency in ME/CFS Cliff et al. TABLE 1 | Description of study population, in relation to the following variables–demographic, socio-economic, immune-related symptoms, Fatigue Severity Scores, viral
serology, and baseline routine blood tests results. RESULTS ESR, Erythrocyte sedimentation rate; CMV, Cytomegalovirus; EBV, Epstein–Barr virus [combined results for Viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA)]; HSV1,
Herpes simplex virus 1; HSV2, Herpes simplex virus 2; VZV, varicella-zoster virus; HHV6, Human herpesvirus 6; ME/CFSsa, people with ME/CFS with severe clinical manifestations;
ME/CFSmm, people with ME/CFS with mild/moderate manifestations; MS, people with multiple sclerosis; HC, people recruited as healthy controls. *P-values—from χ2 tests for
categorical variables and Kruskal-Wallis test for continuous variables (which were non-normally distributed). of
CCR7-CD45RA+CD28+CD57+,
CCR7+CD45RA-
CD28+CD57-,
and
CCR7-CD45RA-CD28+CD57-
cells
in people with ME/CFS and an increased proportion of
CCR7+CD45RA+CD28-CD57- cells. of
CCR7-CD45RA+CD28+CD57+,
CCR7+CD45RA-
CD28+CD57-,
and
CCR7-CD45RA-CD28+CD57-
cells
in people with ME/CFS and an increased proportion of
CCR7+CD45RA+CD28-CD57- cells. the groups in the absolute numbers of different leucocyte and
lymphocyte populations (Supplementary Figure S2). More detailed phenotyping of the circulating T cell population
revealed no major differences in the distribution of CD45RA-
and CCR7-defined naïve and memory T cell populations in
univariate analysis (Figure 4). In a more detailed phenotypic
analysis of CD4+ and CD8+ T cell compartments by analysis
of CD28 and CD57 alongside CCR7 and CD45RA, we found
that there were no differences in the CD4+ T cell differentiation
subpopulations
in
people
with
ME/CFS,
although
there
were altered cell population frequencies in people with MS
(Figure 5A). However,
modest
differences
were
observed
in several differentiation populations in the CD8+ T cell
compartment (Figure 5B) with a small increase in the proportion
of EM cells (CCR7-CD45RA-CD28-CD57-) and a reduction in
the proportion of terminally differentiated cytotoxic effector
TEMRA cells (CCR7-CD45RA+CD28–CD57+) in people living
with ME/CFS. There were also small changes in other minor
CD8+ T cell populations, with small reductions in proportions Of greater potential relevance, however, using TCR Vα7.2 and
CD161 to define circulating populations of mucosal associated
invariant T (MAIT) cells, we observed highly significant
differences between the groups in the proportion of T cells that
are MAIT cells and the proportion of MAIT cells that express
CD8 (unadjusted ANOVA, P < 0.001 in both cases) (Figure 6). After adjusting for confounding, the multiple linear regression
analysis revealed that this was due to increased proportions of
MAIT cells (P < 0.001), and particularly of CD8+ MAIT cells, in
people with severe ME/CFS compared to healthy controls (P <
10−5). RESULTS Discrete variables
Category of recruitment
Total
P-value*
ME/CFSsa
ME/CFSmm
MS
HC
(n)
(%)
(n)
(%)
(n)
(%)
(n)
(%)
(n)
(%)
SEX
Male
13
24.1
47
24.0
10
20.4
28
26.2
98
24.1
0.89
Female
41
75.9
149
76.0
39
79.6
79
73.8
308
75.9
IMMUNE-RELATED SYMPTOMS
No symptoms
0
0.0
10
5.1
25
51.0
74
69.1
109
26.9
0.000
Some symptoms
22
40.7
83
42.6
20
40.8
31
29.0
156
38.5
Moderate/severe symptoms
32
59.3
102
52.3
4
8.2
2
1.9
140
34.6
VIRAL SEROLOGY RESULTS
CMV positive
18
33.3
57
29.4
18
45.0
40
37.4
133
33.8
0.40
EBV combined positive
48
88.9
171
88.1
39
95.5
99
93.4
357
90.6
0.42
HSV1 positive
29
53.7
82
42.3
23
57.5
45
42.4
179
45.4
0.49
HSV2 positive
22
40.7
76
39.2
16
40.0
36
33.9
150
38.1
0.12
VZV positive
52
96.3
190
97.9
39
97.5
104
98.1
385
97.7
0.85
HHV6 positive
52
96.3
177
91.2
36
90.0
102
96.2
367
93.1
0.48
Continuous variables
ME/CFSsa
ME/CFSmm
MS
HC
P-value*
Median
IQR
Median
IQR
Median
IQR
Median
IQR
Age (years)
46
32–51
43
34–52
51
32–52
44
32–52
0.002
Index of multiple deprivation (ranking)
18,883
12,463–24,921
16,873
11,177 −23,373
15,853
12,567–21,921
17,02
13,564–25,988
0.29
Fatigue severity scale scores
6.67
6.3–6.89
6.55
6.00–7.00
5.61
3.67–6.44
2.17
1.55–2.67
0.000
BASELINE BLOOD TESTS RESULTS
White blood cells (109/L)
5.88
5.13–7.65
6.10
5.20–6.93
6.01
5.10–7.30
5.99
5.12–6.86
0.93
Lymphocytes (109/L)
1.78
1.57–2.03
1.91
1.60–2.31
1.81
1.49–2.15
1.82
1.52–2.21
0.15
ESR (mm/h)
5
2–10
7
5–12
5
2–8
5
2–8
0.012
ESR, Erythrocyte sedimentation rate; CMV, Cytomegalovirus; EBV, Epstein–Barr virus [combined results for Viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA)]; HSV1,
Herpes simplex virus 1; HSV2, Herpes simplex virus 2; VZV, varicella-zoster virus; HHV6, Human herpesvirus 6; ME/CFSsa, people with ME/CFS with severe clinical manifestations;
ME/CFSmm, people with ME/CFS with mild/moderate manifestations; MS, people with multiple sclerosis; HC, people recruited as healthy controls. *P-values—from χ2 tests for
categorical variables and Kruskal-Wallis test for continuous variables (which were non-normally distributed). TABLE 1 | Description of study population, in relation to the following variables–demographic, socio-economic, immune-related symptoms, Fatigue Severity Scores, viral
serology, and baseline routine blood tests results. T Cell and NK Cell Function total MAIT cell frequency in the T cell population (Figure 6F)
showed a weak capacity to discriminate severely affected ME/CFS
patients from healthy controls, but that the mild/moderately
affected patients were not predicted by this measure. Potentially
more importantly, the proportion of MAIT cells which were
CD8+ (Figure 6G) had a greater capacity to discriminate severe
ME/CFS from health, with an AUC of 0.756. In accordance
with previous reports (38), the vast majority of MAIT cells in
all subjects had an undifferentiated (CD28+ CD57−) phenotype
(Supplementary Figure S3). total MAIT cell frequency in the T cell population (Figure 6F)
showed a weak capacity to discriminate severely affected ME/CFS
patients from healthy controls, but that the mild/moderately
affected patients were not predicted by this measure. Potentially
more importantly, the proportion of MAIT cells which were
CD8+ (Figure 6G) had a greater capacity to discriminate severe
ME/CFS from health, with an AUC of 0.756. In accordance
with previous reports (38), the vast majority of MAIT cells in
all subjects had an undifferentiated (CD28+ CD57−) phenotype
(Supplementary Figure S3). We next compared the ability of T cells and NK cells from
healthy controls, people with MS and PWME to respond to
in vitro stimulation. For assessment of T cell responsiveness,
PBMCs were cultured for 4 h with the mitogenic cocktail of
PMA and ionomycin and then stained for intracellular cytokines. The proportion of cells producing IL-2, IFN-γ or a combination
of IL-2 and IFN-γ was assessed in the CD3+ CD4+ and the
CD3+ CD8+ cell populations but no significant differences were
observed among the groups in either the adjusted or unadjusted
analysis (Figure 8). Looking in more detail at the phenotype of the NK
cell population, we observed no significant differences in
the proportions of CD56 bright cells, in the distribution of
CD57−, CD57+ or CD57++ cells or in the proportions of
cells expressing either NKG2C or NKp46 (Figure 7) or in
the expression of six different killer cell immunoglobulin-
like receptors (KIR) (Supplementary Figure S4) in either the
adjusted or the unadjusted analysis. NK cell function was assessed by culturing PBMCs for 18 h
with IL-12 plus IL-18, with CpG, with MHC Class-I deficient
K562 cells or in plates coated with a cross-linking antibody
to CD16. RESULTS Importantly however, although the overall proportions of
MAIT cells were not higher among people with MS than among
healthy controls, their MAIT cells were also heavily skewed
to the CD8+ subset, indicating that this is not a diagnostic
feature of severe ME/CFS cases. ROC analysis showed that the April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 5 Increased MAIT Cell Frequency in ME/CFS Cliff et al. FIGURE 1 | Leucocyte gating strategy for phenotypic characterisation of PBMC. Thawed PBMC from Biobank participants were stained with labelled antibodies
(Supplementary Table S2), expression data were collected by Flow cytometry and analysed using FlowJo. (A) Single cells were gated based on their forward scatter
height and area, and (B) monocytes within the single cell gate were identified as CD14+. (C) Dendritic cells were identified within the CD3-CD14- cell population as
myeloid (CD11c+) or plasmacytoid (CD123+) DCs. (D) Lymphocytes were identified based on their forward and side scatter area profiles, and B cells were identified
as CD19+ (E), T cells as CD3+ (F), and NK cells as CD56+ (G). Within the NK cell gate, NK cells were characterised further based on NKG2C (H), NKp46 (I), and
CD57 (J) co-expression. FIGURE 1 | Leucocyte gating strategy for phenotypic characterisation of PBMC. Thawed PBMC from Biobank participants were stained with labelled antibodies
(Supplementary Table S2), expression data were collected by Flow cytometry and analysed using FlowJo. (A) Single cells were gated based on their forward scatter
height and area, and (B) monocytes within the single cell gate were identified as CD14+. (C) Dendritic cells were identified within the CD3-CD14- cell population as
myeloid (CD11c+) or plasmacytoid (CD123+) DCs. (D) Lymphocytes were identified based on their forward and side scatter area profiles, and B cells were identified
as CD19+ (E), T cells as CD3+ (F), and NK cells as CD56+ (G). Within the NK cell gate, NK cells were characterised further based on NKG2C (H), NKp46 (I), and
CD57 (J) co-expression Frontiers in Immunology | www.frontiersin.org DISCUSSION between participants with ME/CFS and controls in any of
the responses. In a cohort of 250 well-characterised people living with ME/CFS,
including a number who are severely affected, we have identified
differences in peripheral immune cell phenotype compared to
healthy control individuals. These include highly significantly
enhanced proportions of mucosal associated invariant T cells,
particularly CD8+ MAIT cells, in PWME compared to healthy
controls, and these were further enhanced in individuals who
had severe disease manifestation. We also found an enhancement
in the proportion of effector memory CD8+ T cells, a
reduction in the proportion of terminally differentiated effector
TEMRA cells, and minor modulations in different CD8+ T cell
differentiation subcompartments in PWME. The functional role
and consequence of change within these different CD8+ T cell
subpopulations remains to be determined. We did not find any
evidence for altered function of T cells in PWME. Of note, we did
not find any differences in the proportions of NK cells in PWME,
or in their differentiation status, their expression of KIR receptors
or of activation markers ex vivo or following in vitro stimulation. Based on previous reports of abnormal NK cell function in
ME/CFS (17–19, 24, 25, 27, 40) this was an unexpected finding,
although is consistent with a small number of previous reports of
normal NK cell proportion and function (21–23). In our system,
we cultured PBMC for 18 h to allow time for direct and indirect
cell activation: it is possible that differences in degranulation
or cytokine production may have been observed with a shorter
stimulation period. However, we did find significant differences T Cell and NK Cell Function CD3−/CD56+ NK cells were gated and analysed for
intracellular IFN-γ or for cell surface expression of the activation
marker CD25 or the degranulation marker CD107a (LAMP-
1) (Figure 9). Again, no significant differences were detected April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 6 Increased MAIT Cell Frequency in ME/CFS Cliff et al. FIGURE 2 | Leucocyte populations in peripheral blood from people with ME/CFS, people with MS and healthy controls. PBMC were analysed by flow cytometry, as
described in Figure 1. The proportions of PBMC which were (A) monocytes, (B) myeloid dendritic cells, (C) plasmacytoid dendritic cells, (D) B cells, (E) T cells or
(F) NK cells were calculated and shown for individual study participants. The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis
(MS: n = 46), mild/moderate ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 56), MS (n = 46), ME-M (n = 120),
and ME-S (n = 21) for (B,C). Clinical groups were compared by Kruskal-Wallis test for non-parametric data: *P < 0.05, **P < 0.01, ****P < 0.0001. FIGURE 2 | Leucocyte populations in peripheral blood from people with ME/CFS, people with MS and healthy controls. PBMC were analysed by flow cytometry, as
described in Figure 1. The proportions of PBMC which were (A) monocytes, (B) myeloid dendritic cells, (C) plasmacytoid dendritic cells, (D) B cells, (E) T cells or
(F) NK cells were calculated and shown for individual study participants. The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis
(MS: n = 46), mild/moderate ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 56), MS (n = 46), ME-M (n = 120),
and ME-S (n = 21) for (B,C). Clinical groups were compared by Kruskal-Wallis test for non-parametric data: *P < 0.05, **P < 0.01, ****P < 0.0001. Frontiers in Immunology | www.frontiersin.org Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function Herpesvirus infection, most notably infection with CMV, is
known to affect both the maturational phenotype and the
function of human CD8+ T cells (39) and NK cells (28). We therefore compared lymphocyte phenotype and function
among those subjects who were either seropositive or not for
CMV. Across all the study groups, participants who were CMV-
seropositive had significantly lower proportions of CD4+ and
higher proportions of CD8+ T cells amongst their PBMC
(Supplementary Figure S5). Following stimulation with PMA
and ionomycin, higher proportions of both CD4+ and CD8+
T cells made IFNγ, and more cells were IL-2 and IFNγ double
positive, amongst CMV seropositive individuals. The proportion
of PBMC which were CD3+CD56+ (NKT-like) cells was highly
significantly enhanced amongst CMV-positive participants. In
the NK cell population, more cells were NKG2C+, and there were
decreased proportions of CD56dimCD57intermediate and increased
proportions of CD56dimCD57bright amongst CMV-seropositive
participants. Amongst CMV-seropositive individuals, there was
a decreased in vitro response to IL-12 and IL-18 stimulation,
with significantly reduced CD25, CD107, and IFNγ expression
(Supplementary Figure S5G). April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 7 Increased MAIT Cell Frequency in ME/CFS Cliff et al. FIGURE 3 | T cell subset quantification in PBMC from people with ME/CFS, people with MS and healthy controls. Within the T cell gate (CD3+), the CD4+ and CD8
staining was characterised to calculate the proportion of (A) CD4+ T cells, (B) CD8+ T cells, (C) double positive CD4+CD8+ T cells, and (D) the ratio of CD4+:CD8+
T cells. (E) The proportion of T cells which expressed the γδ TCR within the CD3+ T cell population were determined. Data are from healthy controls (C: n = 107),
multiple sclerosis (MS: n = 46), mild/moderate ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A–D), and from C (n = 56), MS (n = 46),
ME-M (n = 120), and ME-S (n = 21) for (E). Clinical groups were compared by Kruskal-Wallis test for non-parametric data: *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001. FIGURE 3 | T cell subset quantification in PBMC from people with ME/CFS, people with MS and healthy controls. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function Within the T cell gate (CD3+), the CD4+ and CD8
staining was characterised to calculate the proportion of (A) CD4+ T cells, (B) CD8+ T cells, (C) double positive CD4+CD8+ T cells, and (D) the ratio of CD4+:CD8+
T cells. (E) The proportion of T cells which expressed the γδ TCR within the CD3+ T cell population were determined. Data are from healthy controls (C: n = 107),
multiple sclerosis (MS: n = 46), mild/moderate ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A–D), and from C (n = 56), MS (n = 46),
ME-M (n = 120), and ME-S (n = 21) for (E). Clinical groups were compared by Kruskal-Wallis test for non-parametric data: *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0 0001 FIGURE 4 | Delineation of T cell naïve and memory cells in ME/CFS and MS patients and controls. CD4+ (A–E) and CD8+ (F–J) T cells were analysed for CD45RA
and CCR7 co-expression to quantify proportions which were naïve CD45RA+CCR7+ (B,G), effector memory RA (TEMRA) CD45RA+CCR7−(C,H), effector memory
CD45RA−CCR7−(D,I) or central memory CD45RA−CCR7+ T cells. The bars show the Mean and SD. Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and
ME-S (n = 21). Clinical groups were compared by Kruskal-Wallis test for non-parametric data. FIGURE 4 | Delineation of T cell naïve and memory cells in ME/CFS and MS patients and controls. CD4+ (A–E) and CD8+ (F–J) T cells were analysed for CD45RA
and CCR7 co expression to quantify proportions which were naïve CD45RA+CCR7+ (B G) effector memory RA (TEMRA) CD45RA+CCR7−(C H) effector memory FIGURE 4 | Delineation of T cell naïve and memory cells in ME/CFS and MS patients and controls. CD4+ (A–E) and CD8+ (F–J) T cells were analysed for CD45RA
and CCR7 co-expression to quantify proportions which were naïve CD45RA+CCR7+ (B,G), effector memory RA (TEMRA) CD45RA+CCR7−(C,H), effector memory
CD45RA−CCR7−(D,I) or central memory CD45RA−CCR7+ T cells. The bars show the Mean and SD. Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and
ME-S (n = 21). Clinical groups were compared by Kruskal-Wallis test for non-parametric data. April 2019 | Volume 10 | Article 796 8 Frontiers in Immunology | www.frontiersin.org Increased MAIT Cell Frequency in ME/CFS Cliff et al. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function GURE 5 | Proportions of differentiated T cell populations in people with ME/CFS, people with MS and healthy controls. CD4+ (A) and CD8+ (B) T cells were
alysed for CCR7, CD45RA, CD28, and CD57 co-expression to quantify proportions of T cells expressing combinations of these markers using FlowJo and SPICE
tware. The pie charts show the median proportions of each cell type in each clinical group. In the bar and whisker plots, cell populations were compared between
ical groups by Kruskal-Wallis test with Dunn’s correction for multiple comparisons for each cell differentiation subtype, with only significant (P < 0.05) results
own. The colour under each cell phenotype bar chart shows its representation in the pie charts. Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and ME-S
= 21). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. FIGURE 5 | Proportions of differentiated T cell populations in people with ME/CFS, people with MS and healthy controls. CD4+ (A) and CD8+ (B) T cells were
analysed for CCR7, CD45RA, CD28, and CD57 co-expression to quantify proportions of T cells expressing combinations of these markers using FlowJo and SPICE
software. The pie charts show the median proportions of each cell type in each clinical group. In the bar and whisker plots, cell populations were compared between
clinical groups by Kruskal-Wallis test with Dunn’s correction for multiple comparisons for each cell differentiation subtype, with only significant (P < 0.05) results
shown. The colour under each cell phenotype bar chart shows its representation in the pie charts. Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and ME-S
(n = 21). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. April 2019 | Volume 10 | Article 796 9 Frontiers in Immunology | www.frontiersin.org Increased MAIT Cell Frequency in ME/CFS Cliff et al. URE 6 | Mucosal Associated Invariant T cells in peripheral blood of people with ME/CFS, people with MS and healthy controls. By flow cytometry, within the
+γδTCR−T cell population (A), MAIT cells were identified as Vα7.2+CD161++ in PBMC (B). MAIT cells were further characterised based on CD4+ or CD8+
ession (C). (D) The proportion of T cells which were MAIT cells in PBMC from mild-moderate ME/CFS (ME-M), severely affected ME/CFS (ME-S), multiple
osis (MS), and healthy controls (C) is shown. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function (E) The proportion of MAIT cells which were CD8+ is shown for each individual. (A–C) Data from one representative
rely affected ME/CFS patient. (D,E) Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and ME-S (n = 21). Clinical groups were compared by Kruskal-Wallis
for non-parametric data: **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) ROC curve analysis of proportions of the CD3+γδTCR−T cell population which were MAIT
for the different patient groups relative to the Healthy Controls. (G) ROC curve analysis of proportions of MAIT cells which were CD8+ for the different patient
ps relative to the Healthy Controls. AUC, Area Under the Curve. with ME/CFS, people with MS and healthy controls. By flow cytometry, within the
61++ in PBMC (B). MAIT cells were further characterised based on CD4+ or CD8+
om mild-moderate ME/CFS (ME-M), severely affected ME/CFS (ME-S), multiple
ells which were CD8+ is shown for each individual. (A–C) Data from one representative
ME-M (n = 120), and ME-S (n = 21). Clinical groups were compared by Kruskal-Wallis
C curve analysis of proportions of the CD3+γδTCR−T cell population which were MAIT
urve analysis of proportions of MAIT cells which were CD8+ for the different patient FIGURE 6 | Mucosal Associated Invariant T cells in peripheral blood of people with ME/CFS, people with MS and healthy controls. By flow cytometry, within the
CD3+γδTCR−T cell population (A), MAIT cells were identified as Vα7.2+CD161++ in PBMC (B). MAIT cells were further characterised based on CD4+ or CD8+
expression (C). (D) The proportion of T cells which were MAIT cells in PBMC from mild-moderate ME/CFS (ME-M), severely affected ME/CFS (ME-S), multiple
sclerosis (MS), and healthy controls (C) is shown. (E) The proportion of MAIT cells which were CD8+ is shown for each individual. (A–C) Data from one representative
severely affected ME/CFS patient. (D,E) Data are from C (n = 56), MS (n = 46), ME-M (n = 120), and ME-S (n = 21). Clinical groups were compared by Kruskal-Wallis
test for non-parametric data: **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) ROC curve analysis of proportions of the CD3+γδTCR−T cell population which were MAIT
cells for the different patient groups relative to the Healthy Controls. (G) ROC curve analysis of proportions of MAIT cells which were CD8+ for the different patient
groups relative to the Healthy Controls. AUC, Area Under the Curve. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function Subpopulations were defined as CD56bright (A), NKG2C+ (B), NKp46+(C), CD56dimCD57−(D)−,
CD56dimCD57intermediate(E), or CD56dimCD57bright(F) for PBMC from mild/moderately affected ME/CFS (ME-M) or severely affected ME/CFS (ME-S), multiple
sclerosis (MS) patients or healthy control individuals (C). Populations of each type of NK cell were compared across clinical groups by ANOVA, but no significant
differences were observed (P < 0.05). The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis (MS: n = 46), mild/moderate
ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 50), MS (n = 41), ME-M (n = 76), and ME-S (n = 32) for (B,C) g
p p
( ),
( ),
p
( ),
( ) ,
CD56dimCD57intermediate(E), or CD56dimCD57bright(F) for PBMC from mild/moderately affected ME/CFS (ME-M) or severely affected ME/CFS (ME-S), multiple
sclerosis (MS) patients or healthy control individuals (C). Populations of each type of NK cell were compared across clinical groups by ANOVA, but no significant
differences were observed (P < 0.05). The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis (MS: n = 46), mild/moderate
ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 50), MS (n = 41), ME-M (n = 76), and ME-S (n = 32) for (B,C). FIGURE 8 | Production of cytokines by T cells in response to stimulation in vitro. PBMC from mild/moderately affected ME/CFS (ME-M: n = 76), severely affected
ME/CFS (ME-S: n = 32) or multiple sclerosis (MS: n = 41) patients or healthy control (C: n = 50) individuals were cultured in vitro with PMA and ionomycin for 4 h. The
production of IFNγ and IL-2 cytokines was assessed in CD4+ (A–D) and CD8+ (E–H) T cells, with the proportions of cells which produced only IL2 (B,F), both IL2
and IFNγ (C,G) or only IFNγ (D,H) calculated for each study participant. Within the dot plots, the lines show the means and the error bars show ± SD. FIGURE 8 | Production of cytokines by T cells in response to stimulation in vitro. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 10 Increased MAIT Cell Frequency in ME/CFS Cliff et al. FIGURE 7 | Proportions of Natural Killer Cell subsets in PBMC from people with ME/CFS, people with MS and healthy controls. By flow cytometry, NK cells were
defined as CD3−CD56+ as shown in Figure 1. Subpopulations were defined as CD56bright (A), NKG2C+ (B), NKp46+(C), CD56dimCD57−(D)−,
CD56dimCD57intermediate(E), or CD56dimCD57bright(F) for PBMC from mild/moderately affected ME/CFS (ME-M) or severely affected ME/CFS (ME-S), multiple
sclerosis (MS) patients or healthy control individuals (C). Populations of each type of NK cell were compared across clinical groups by ANOVA, but no significant
differences were observed (P < 0.05). The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis (MS: n = 46), mild/moderate
ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 50), MS (n = 41), ME-M (n = 76), and ME-S (n = 32) for (B,C). FIGURE 7 | Proportions of Natural Killer Cell subsets in PBMC from people with ME/CFS, people with MS and healthy controls. By flow cytometry, NK cells were
defined as CD3−CD56+ as shown in Figure 1. Subpopulations were defined as CD56bright (A), NKG2C+ (B), NKp46+(C), CD56dimCD57−(D)−,
CD56dimCD57intermediate(E), or CD56dimCD57bright(F) for PBMC from mild/moderately affected ME/CFS (ME-M) or severely affected ME/CFS (ME-S), multiple
sclerosis (MS) patients or healthy control individuals (C). Populations of each type of NK cell were compared across clinical groups by ANOVA, but no significant
differences were observed (P < 0.05). The bars show the Mean ± SD. Data are from healthy controls (C, n = 107), multiple sclerosis (MS: n = 46), mild/moderate
ME/CFS (ME-M: n = 197), and severely affected ME/CFS (ME-S: n = 54) for (A,D–F) and from C (n = 50), MS (n = 41), ME-M (n = 76), and ME-S (n = 32) for (B,C) oportions of Natural Killer Cell subsets in PBMC from people with ME/CFS, people with MS and healthy controls. By flow cytometry
b i ht
di FIGURE 7 | Proportions of Natural Killer Cell subsets in PBMC from people with ME/CFS, people with MS and healthy controls. By flow cytometry, NK cells were
defined as CD3−CD56+ as shown in Figure 1. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function PBMC from mild/moderately affected ME/CFS (ME-M: n = 76), severely affected
ME/CFS (ME-S: n = 32) or multiple sclerosis (MS: n = 41) patients or healthy control (C: n = 50) individuals were cultured in vitro with PMA and ionomycin for 4 h. The
production of IFNγ and IL-2 cytokines was assessed in CD4+ (A–D) and CD8+ (E–H) T cells, with the proportions of cells which produced only IL2 (B,F), both IL2
and IFNγ (C,G) or only IFNγ (D,H) calculated for each study participant. Within the dot plots, the lines show the means and the error bars show ± SD. effect on NK cells (28) and it is possible that undetermined CMV
status was a confounding factor underlying previous reports of
NK cell dysfunction in ME/CFS. Importantly, the differences in NK cell differentiation status and cytokine production in study
participants across all clinical groups who were CMV-positive,
detectable after the 18 h culture period. CMV has a dramatic April 2019 | Volume 10 | Article 796 Frontiers in Immunology | www.frontiersin.org 11 Increased MAIT Cell Frequency in ME/CFS Cliff et al. FIGURE 9 | Natural Killer Cell function in vitro in people with ME/CFS, people with MS and healthy controls. PBMC were incubated in vitro various stimuli and NK cell
function analysed by flow cytometry. (A) Flow cytometry gating strategy showing NK cell (CD3−CD56+) expression of CD25, IFNγ or CD107a (degranulation marker)
following incubation with or without IL12/IL18 for 18 h, in one representative donor. (B) Expression of CD25, IFNγ, and CD107 in NK cells following IL12/IL18
stimulation of PBMC from mild/moderately affected ME/CFS (ME-M: n = 76), severely affected ME/CFS (ME-S: n = 32) or multiple sclerosis (MS: n = 41) patients or
healthy control (C: n = 50) individuals. CD107a expression on NK cells following 18 h stimulation by (C) cross-linking anti-CD16 monoclonal antibodies, (D) MHC
Class-I deficient K562 cells or (E) CpG. Within the dot plots, the lines show the means and the error bars show ± SD. FIGURE 9 | Natural Killer Cell function in vitro in people with ME/CFS, people with MS and healthy controls. PBMC were incubated in vitro various stimuli and NK cell
function analysed by flow cytometry. (A) Flow cytometry gating strategy showing NK cell (CD3−CD56+) expression of CD25, IFNγ or CD107a (degranulation marker)
following incubation with or without IL12/IL18 for 18 h, in one representative donor. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function (B) Expression of CD25, IFNγ, and CD107 in NK cells following IL12/IL18
stimulation of PBMC from mild/moderately affected ME/CFS (ME-M: n = 76), severely affected ME/CFS (ME-S: n = 32) or multiple sclerosis (MS: n = 41) patients or
healthy control (C: n = 50) individuals. CD107a expression on NK cells following 18 h stimulation by (C) cross-linking anti-CD16 monoclonal antibodies, (D) MHC
Class-I deficient K562 cells or (E) CpG. Within the dot plots, the lines show the means and the error bars show ± SD. PWME resembles a post-exercise state. The more remarkable
finding, however, was the overwhelming predominance of CD8+
cells among the MAIT cell population in severely affected
PWME. Moreover, although overall frequencies of MAIT cells
did not differ between MS patients and healthy controls, their
MAIT cells were also highly enriched for CD8+ cells, confirming
a previous report of high frequencies of CD8+ CD161+ T cells
in MS patients (45). MAIT cells are innate-like T cells which
recognise vitamin B-related antigens from microbes presented
in the context of the MHC Class I-related molecule MR1 (46),
and can also respond to cytokine stimulation. CD8+ MAIT cells
are able to kill infected cells via perforin/granzyme secretion
and therefore have a protective role in microbial infection (47),
but they have recently been implicated in the pathology of
various non-infectious diseases such as cancer (48), colitis (49)
and autoimmune diseases such as rheumatoid arthritis (50) and
type 1 diabetes (51). In MS, these cells are described as pro-
inflammatory and pathogenic. It is possible that the increase
in MAIT cell frequency in some PWME is driven by changes
in the gut microbiome (52) or alternatively could be related to
other, as yet uncharacterised, changes in the immune system. In future studies, the functional phenotype of these CD8+
MAIT cells in PWME could be ascertained, to determine if they
may be contributing to disease pathology. The gut microbiome
in ME/CFS should also be investigated further. Eventually we observed in MAIT cells and CD8+ T cells in PWME were
consistent when CMV serostatus was adjusted for. ME/CFS is often considered to be triggered by an acute viral
infection, and various herpes virus family members have been
implicated (41). Here, we found no difference in the prevalence
of seropositivity for six herpes viruses (CMV, EBV, HSV-1, HSV-
2, VZV, HHV6) between PWME and healthy controls, nor in
the concentration of anti-virus antibodies present. Frontiers in Immunology | www.frontiersin.org DATA AVAILABILITY The datasets generated for this study are available on request to
the corresponding author. The
reason
for
the
altered
frequencies
of
various
intermediately differentiated CD8+ T cells in ME/CFS is
unclear, and is likely to be functionally linked with the
concomitant reduction in effector memory CD8+ T cells (53). As
there was no difference in the frequency of naïve or terminally
differentiated CD8+ T cells, it is possible that in ME/CFS
the cells are rapidly driven through this intermediate stage to
terminal differentiation and are then lost by cell death. The
study design did not allow us to determine the interaction
between the enhanced MAIT cell compartment and the altered
overall T cell differentiation status: this can be addressed in
future studies. The driver behind the faster transition towards
terminal differentiation could be ongoing antigenic stimulation,
possibly due to persistent viral infection or autoimmunity. The
reduced effector memory T cell populations in PWME may in
part explain the increased susceptibility to infection which is
commonly reported in ME/CFS. AUTHOR CONTRIBUTIONS JC, HD, LN, EL, and ER devised the study and obtained funding. EL, EB, LN, and CK designed and implemented the clinical study. CK conducted the clinical assessments and collected samples. JC,
HD, and ER designed and implemented the laboratory studies. EK, J-SL and A-SW conducted the laboratory assays. JC, EK, NS,
EL, and ER analysed the data. JC, NS, LN, EL, and ER wrote the
manuscript. All authors reviewed and approved the final draft of
the manuscript. The proportions of CD4+ and CD8+ T cells were normal in
PWME. However, in MS patients there was a significant skew
towards increased proportions of CD4+ T cells, lower CD8+ T
cells and therefore a raised CD4+/CD8+ T cell ratio. Previously,
there have been mixed reports in PWME, with increased (54) or
decreased (55, 56) CD4+/CD8+ T cell ratios observed, although
heterogeneity is commonly reported and may be confounded by
CMV infection status. Interaction Between Herpesvirus Infection
Status and Lymphocyte Phenotype
and Function Nonetheless,
the possibility remains that herpes viruses may be important
in ME/CFS pathogenesis, that virus reactivation may trigger
a worsening of symptoms, and that measurement of antibody
titres to alternative viral antigens might provide a more relevant
measure (42). Thus, although the seroprevalence of EBV did
not differ among the MS patients, PWME or healthy controls,
MS patients did have significantly higher titres of antibodies
to EBV Viral Capsid Antigen, in line with previous reports
[reviewed by Burnard et al. (43)] and potentially implying recent
virus reactivation. The increased frequency of CD8+ MAIT cells in those
severely affected by ME/CFS observed here has not previously
been reported. Notably, a small number of severely affected
ME/CFS patients had exceedingly high frequencies of these cells. Peripheral MAIT cell frequencies in healthy volunteers have been
reported to increase over 2-fold following acute exercise (44),
suggesting that the peripheral immune phenotype at rest in some April 2019 | Volume 10 | Article 796 12 Cliff et al. Cliff et al. Increased MAIT Cell Frequency in ME/CFS a biomarker signature might be developed for diagnosis of
ME/CFS: in this context the proportion of CD8+ MAIT
cells in peripheral blood had modest discriminatory capacity
alone, but might contribute to a combined factor signature. In this regard, testing of candidate biomarker signatures in
large, independent, longitudinal validation cohorts will be vital,
especially considering historically mixed reports of biological
phenotype in ME/CFS. observations and may provide insight into disease pathogenesis
pathways in ME/CFS by comparison with a more extensively
documented disease. ETHICS STATEMENT This
study
was
carried
out
in
accordance
with
the
recommendations of The London School of Hygiene &
Tropical
Medicine
(LSHTM)
Research
Ethics
Committee
(Ref. 6123) and the National Research Ethics Service (NRES)
London-Bloomsbury Research Ethics Committee (REC ref. 11/10/1760, IRAS ID: 77765), with written informed consent
from all subjects. All subjects gave written informed consent in
accordance with the Declaration of Helsinki. The protocol was
approved by the LSHTM Research Ethics Committee and the
NRES London-Bloomsbury Research Ethics Committee. ACKNOWLEDGMENTS In summary, in a large cohort of very carefully phenotyped
people living with ME/CFS, we have observed significant
differences in peripheral T lymphocyte phenotypes but have
been unable to confirm previous reports of distorted NK cell
phenotype or function. Importantly, we excluded from this
study individuals with evidence of ongoing acute infection,
those with obvious comorbidities and those with a history
of taking immunomodulatory medications. We have also
confirmed that CMV seropositivity has a major impact on the
phenotype and function of NK cells and T cells, underlining
the paramount importance of assessing CMV and other herpes
virus infection status in studies of human immune status. Any of these factors may have confounded the analysis of
previous studies. The most striking finding of this study, the
increased proportion of circulating MAIT cells in people severely
affected by ME/CFS, and the very high proportion of CD8+
MAIT cells in severely affected PWME and in MS patients,
warrants further investigation. This observation also underlines
the importance of including non-fatigued individuals with other
incapacitating illnesses (such as MS patients) in studies of
ME/CFS to control for the physiological effects of reduced levels
of physical activity. Although one might debate whether MS
patients are the most appropriate such group, the very large
immunological database for MS does facilitate benchmarking of We thank all the study participants for their time and energy
and for donating their blood to the UK ME/CFS Biobank
(UKMEB). We also thank the support from the charities The
ME Association, Action for ME, and ME Research UK, as
well as a private donor, who funded the UKMEB start-up
and to the ME Association for continued funding. We thank
Prof. Lauren B. Krupp, who generously granted us permission
to use her validated Fatigue Severity Scale. We thank Steven
Smith for advice on flow cytometry analysis. Research reported
in this paper was mainly supported by the National Institute
of Allergy and Infectious Diseases (NIAID) of the National
Institutes of Health (NIH) under Award Number: R01AI103629. NS acknowledges funding from Fundação para a Ciência e
Tecnologia, Portugal (grant ref. UID/MAT/00006/2013). The
content is solely the responsibility of the authors and does not
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. 45. Annibali V, Ristori G, Angelini DF, Serafini B, Mechelli R, Cannoni S, et al. CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis. Brain. (2011) 134:542–54. doi: 10.1093/brain/awq354 Conflict of Interest Statement: The authors declare that the research was
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cells and MR1-antigen recognition. Curr Opin Immunol. (2017) 46:66–74. doi: 10.1016/j.coi.2017.04.002 Copyright © 2019 Cliff, King, Lee, Sepúlveda, Wolf, Kingdon, Bowman, Dockrell,
Nacul, Lacerda and Riley. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. Copyright © 2019 Cliff, King, Lee, Sepúlveda, Wolf, Kingdon, Bowman, Dockrell,
Nacul, Lacerda and Riley. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
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